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Proceedings Of OMAE-02 21st International Conference on Offshore Mechanics & Arctic Engineering June 23 – 28, Oslo Norway

OMAE 2002/PIPE-28322

REAL-TIME RELIABILITY ASSESSMENT & MANAGEMENT OF MARINE PIPELINES

Robert Bea Charles Smith and Bob Smith University of California at Berkeley U.S. Minerals Management Service Berkeley, California USA Herndon, Virginia USA

Johannes Rosenmoeller, Thomas Beuker, and Bryce Brown ROSEN Pipeline Inspection Lingen, Germany and Houston, Texas, USA

ABSTRACT INTRODUCTION In-line instrumentation information processing procedures Pipeline in-line instrumentation has become a primary have been developed and implemented to permit ‘real-time’ means for gathering detailed data on the current condition of assessment of the reliability characteristics of marine pipelines. pipelines. It would be very desirable for the pipeline owner, The objective of this work is to provide pipeline engineers, operator, and regulator to have a highly automated process to owners and operators with additional useful information that enable preliminary assessment of the reliability of the pipeline can help determine what should be done to help maintain in its current and projected future conditions (Fig. 1) pipelines. This paper describes the real-time RAM (reliability Us e r Specified Data: assessment and management) procedures that have been Pipeline Char act er istics developed and verified with results from laboratory and field e.g. Diameter, Wall Thickness, Ma t er ial St rengt tests to determine the burst pressures of pipelines. These procedures address the detection and accuracy characteristics of put results from in-line or ‘smart pig’ instrumentation, evaluation of the implications of non-detection, and the accuracy of alternative methods that can be used to evaluate the burst Instrument ation Inputs: R eal-Time Calculation of pressures of corroded and dented – gouged pipelines. Input -Line Instrument a tio n Output In addition, processes are described have been developed to Pipeline Defec t Profile In Probability of Failure permit use of the information accumulated from in-line (e g Depth of corrosion instrumentation (pipeline integrity information databases) to make evaluations of the burst pressure characteristics of pipelines that have not or can not be instrumented. Both of these processes are illustrated with applications to Fig. 1: Real-Time RAM process two example pipelines; one for which in-line instrumentation results are available and one for which such information is not Pipeline in-line instrumentation data can provide a large available. amount of data on damage and defects (features) in a pipeline. This data must be properly interpreted before the features can be Keywords: Pipelines, Reliability, Instrumentation characterized. The detection of features varies as a function of

1Copyright © 2002 by ASME the size and geometry of the features, the in-line Insight into the change in the uncertainty associated with instrumentation used, and the characteristics and condition of the pipeline capacity associated with the loss of wall thickness the pipeline. Given results from in-line instrumentation, it is due to corrosion, can be developed by the following: desirable to develop a rapid and realistic evaluation of the effects of the detected features on the pipeline integrity. This ttd©=− evaluation requires and analysis of how the detected features 0 might affect the ability of the pipeline to maintain containment. 10 σ=0.1 σ=0.2 -1 RELIABILITY FORMULATION 10 σ=0.3 The Reliability Assessment and Management (RAM) σ=0.4 10-2 formulation used in this development is based on a probabilistic approach based on Lognormal distributions for -3 10 both pipeline demand and capacity distributions. Such Pf distributions have been shown to provide good approximations 10-4 to the ‘best-fit’ distributions, particularly when the tails of the Lognormal distributions are fitted to the region of the 10-5 distributions that have the greatest influence on the probability of failure. The Lognormal formulation for the probability of 10-6 failure (Pf) is: 246810

  R50   FS ln   50 Pf =−11ΦΦ S50  =− []β σ Fig. 2: Probability of failure as function of central Factor of  ln RS  Safety and total uncertainty   Φ is the Cumulative Normal Distribution for the quantity [•]. t’ is the wall thickness after the corrosion, t is the wall R50 is the median capacity. S50 is the median demand. The ratio thickness before corrosion, and d is the maximum depth of the of R50 to S50 is known as the median or central Factor of Safety corrosion loss. Bars over the variables indicate mean values. (FS50). σlnRS is the standard deviation of the logarithms of the Based on First Order – Second Moment methods, the capacity (R) and demand (S): standard deviation of the wall thickness after corrosion can be expressed as: 22 σσσln RS =+ lnRS ln σσσ=+22 t© t d σlnR is the standard deviation of the capacity and σlnS is the standard deviation of the demand. For coefficients of variation The Coefficient of Variation (COV = V) can be expressed (VX = ratio of standard deviation to mean value of variable X) as: less than about 0.5, the coefficient of variation of a variable is 22 approximately equal to the standard deviation of the logarithm σt© ()Vt+ ( V d ) V == t d of the variable. The quantity in brackets is defined as the Safety t© − Index (β). The Safety Index β is related approximately to Pf as t© td 1≤β≤3): A representative value for the COV of t would be 2%. A Pf ≈ 0.475 exp -(β)1.6 representative value for the COV of d would be Vd = 40%. Fig. 3 summarizes the foregoing developments for a 16-in. The results of this development are summarized in Fig. 2. The (406 mm) diameter pipeline with an initial wall thickness of t probability of failure (loss of containment) is shown as a = 0.5 in. (17 mm) that has an average rate of corrosion of 10 function of the central factor of safety (FS50) and the total mpy (0.010 in. / yr, 0.25 mm / yr). The dashed line shows the uncertainty in the pipeline demands and capacities (σ). Note results for the uncertainties associated with the wall thickness. that the probability of failure can be determined from two The solid line shows the results for the uncertainties that fundamental parameters: the central factor of safety (FS50 = include those of the wall thickness, the prediction of the R50/S50) and the total uncertainty in the demands and capacities corrosion burst pressure, and the variabilities in the maximum (σlnRS=σ). operating pressure. At the time of installation, the pipeline wall thickness TIME DEPENDENT RELIABILITY COV is equal to 2%. But, as time develops, the uncertainties When a pipeline is subjected to active corrosion processes, associated with the wall thickness increase due to the large the probability of failure is a time dependent function that is uncertainties associated with the corrosion rate – maximum dependent on the corroded thickness of the pipeline (tci/e). The depth of corrosion. The solid line that reflects all of the corroded thickness is dependent on the rate of corrosion and the uncertainties converges with the dashed line that represents the time that the pipeline or riser is exposed to corrosion. uncertainties in the remaining wall thickness, until at a time of about 20 years, the total uncertainty is about the same as that of

2Copyright © 2002 by ASME the remaining wall thickness (Vt-d ≈ 25 %). As more time 100 develops, there is a dramatic increase in the COV associated -1 with the remaining wall thickness. These uncertainties are 10 dominated by the uncertainties attributed to the corrosion 10-2 processes. 10-3 10-4 1 Pf(3000psi) 10-5 Pf(5000psi) 10-6 10-7 0.1 (t') Annual Probability of Failure 10-8 (t',Pb,Po) 01020304050 Time - years Uncertainty - Standard Deviation of Logarithms

0.01 Fig. 4: Example pipeline failure rates as function of 00.20.4 0.6 0.8 1 exposure to corrosion Corrosion loss / Nominal thickness = d/t There can be a similar effect on the operating pressure Fig. 3: Uncertainty in pipeline wall thickness and burst demands if there are pressure relief or control mechanisms pressure capacity as a function of the normalized loss in maintained in the pipeline. Such pressure relief or control pipeline wall thickness equipment can act to effectively truncate or limit the probabilities of developing very high unanticipated operating These observations have important ramifications on the pressures (due to surges, slugging, or blockage of the pipeline). probabilities of failure – loss of containment of the pipeline. Pipeline capacity before testing After the ‘life’ of the pipeline is exceeded (e.g. 20 to 25 years), one can expect there to be a rapid and dramatic increase in the uncertainties associated with the corrosion processes. In Pipeline capacity after testing addition, there will be the continued losses in wall thickness. Combined, these two factors will result in a dramatic increase in the probability of failure of a pipeline. Fig. 4 summarizes example results for a 16-in. (406 mm) diameter, 0.5 in (13 mm) wall thickness pipeline that has a maximum operating pressure (MOP) of 5,000 psi (34.5 Mpa). Pressure The COV associated with the MOP is 10%. The pipeline is operated at the maximum pressure, and at 60% of the Proof test pressure maximum operating pressure for a life of 0 to 50 years. The average corrosion rate was taken as 10 mills per year (mpy). For Fig. 5: Effects of proof testing on pipeline capacity the 60% pressured line, during the first 20 years, the annual distribution probability of failure rises from 1E-7 to 5 E-3 per year. After 20 years, the annual probability of failure rises very quickly to This raises the issues associated with pressure testing and values in the range of 0.1 to 1. Perhaps, this helps explain why pressure controls on the computed probabilities of failure. It is the observed pipeline failure rates associated with corrosion in important to note that such distribution truncation the Gulf of Mexico are in the range of 1 E-3 per year. considerations have been omitted from all pipeline reliability based studies and developments that have been reviewed during TRUNCATED DEMAND & CAPACITY DISTRIBUTIONS the past 10 years of research on this topic. Fig. 6 summarizes the results of pipeline proof testing on Real-time RAM analytical models have been developed to the pipeline Safety Index (the probability of loss of allow determination of the effects of user specified truncations β containment is Plc ≈ 10- ) as a function of the ‘level’ of the in pipeline demands, capacities; separately or combined. proof testing pressure factor, K: The effect of pressure testing is to effectively ‘truncate’ the probability distribution of the pipeline burst pressure capacity K = ln (Xp / pb) / σlnpb below the test pressure (Fig. 5). Pressure testing is a form of ‘proof testing’ that can result in an effective increase in the where Xp / pb is the ratio of the test pressure to the median reliability of the pipeline. burst pressure capacity of the pipeline (test pressure deterministic, burst pressure capacity Lognormally distributed) and is the standard deviation of the Logarithms of the pipeline burst pressure capacities. These results have been generated for

3Copyright © 2002 by ASME the case where the uncertainty associated with the maximum corrected wall thickness shown in Fig. 7 was based on these operating / incidental pressures is equal to the uncertainty of the data. pipeline burst pressures and for Safety Indices in the range of β 0.35 = 3 to β = 4.5. For example, if the median burst pressure of the pipeline 0.3 Measured (% t) were 2,000 psi and this had a Coefficient of Variation of 10 %, Corrected (% t) there was a factor of safety on this burst pressure of 2 (f = 0.5) 0.25 (maximum operating pressure = 1,000 psi), and the pipeline was tested to a pressure of 1.25 times the maximum operating 0.2 pressure (Xp = 1,250 psi), the proof testing factor K = -4.7. The results in Fig. 6, indicate that this level of proof testing is 0.15 not effective in changing the pipeline reliability. Even if the pipeline were tested to a pressure that was 1.5 times the 0.1 operating pressure, the change in the Safety Index would be less Measured Corrosion (% t) than 5 %. 0.05 If the test pressure were increased to 75% of the median 0 5000 10000 15000 burst pressure, the Safety Index would be increased by about 25 %. For a Safety Index of β = 3.0 (Pf = 1E-3), these results Distance (m) indicate a β = 3.75 (Pf = 1E-4) after proof testing. Fig. 7: Measured and corrected corrosion readings 1.30 1.4 1.35 1.25 1.3 Pig C 1.20 Pig A οο οο 1.25 ωω ωω

1.15 ββ ββ

1.2 // //

ωω ωω Bias =

1.10 1.15 ββ ββ

1.05 1.1 Safety Index Ratio 1.05

(with proof test / without) 1.00

-4 -3.5 - 3 -2.5 - 2 -1.5 - 1 actual depth / measured 1 50 100 150 200 250 300 Proof Test Factor - K Pit Depth (mils)

Fig. 6: Effects of proof testing on pipeline reliability Fig. 8. Bias in measured corrosion depths Very high levels of proof testing are required before there is Based on using results from inline instrumentation, the any substantial improvement in the pipeline reliability. These results indicate that conventional pressure testing may not be probability of failure can be expressed as: very effective at increasing the burst pressure reliability Pf = PfD + PfND characteristics. Such testing may be effective at disclosing accidental flaws incorporated into the pipeline (e.g. poor where PfD is the probability of failure associated with the welding). detected flaws and PfND is the probability of failure associated with the non-detected flaws.It is important recognize that PROBABILITIES OF DETECTION making evaluations of corrosion rates and wall thicknesses from Fig. 7 shows results from inline Magnetic Flux Leakage the recordings have significant uncertainties/ Fig. 9 shows a (MFL) instrumentation of a 20-in (508 mm) diameter gas line comparison of the Probability of Detection (POD) of corrosion in the Bay of Campeche (Pig C) [1]. The measured and depths (in mils, 50 mils = 1.27 mm) developed by three corrected corrosion expressed as a percentage of the wall different inline MFL instruments. This information was based thickness is shown. on comparing measured results from sections of a pipeline that Fig. 8 summarizes data for two inline MFL instruments in were repeatedly in-line instrumented and then retrieved and the which the in-line data on corrosion defect depths were compared directly measured corrosion depths determined. These are with the corrosion defect depths determined from direct results from three similar MFL in-line instruments. However, measurements on recovered sections of the pipeline that was in- there are significant differences in the POD. This indicates an line instrumented. For this particular condition, both in-line important need to standardize in-line instrumentation and data instruments tend to under estimate the corrosion depth. The interpretation. uncertainties associated with the measured depths ranged from 35% (for 50 mils depths) to 25% (for 200 mils depths). The

4Copyright © 2002 by ASME 1 P[ND] = 1 – P[D] and: 0.8 P[ND] = Σ P[ND | X > xo] P[X > xo] The probability of failure for non-detected flaws is the 0.6 convolution of: Pf = Σ [Pf | X > xo] P[ X ≥ xo | ND ] 0.4 Pig A ND Pig B Fig. 24 shows the probabilities of burst failure (detected Pig C and non-detected) of the pipeline. The majority of the pipeline 0.2 has probabilities of failure of about 1 E-2 per year. However,

Probability of Detection there are two sections that have substantially higher 0 probabilities of failure. One section is a low section in the 100 200 300 400 500 600 700 pipeline where water can accumulate and the other is in the riser Corrosion Depth (mils) section that is subjected to higher temperatures and external corrosion. The probabilities of failure for these two sections are Fig. 9: Probability of detection curves for three in-line 1.7 E-2 and 2.9 E-2 per year, respectively. These two sections instruments of the pipeline would be candidates for replacement. The probability of failure associated with the detected ANALYTICAL MODEL BIAS depth of corrosion can be expressed as: One of the most important parts of a reliability assessment 2 2 0.5 PfD = 1 - Φ{[ln(pB50/pO50)]/[(σ pB+σ pO) ] is the evaluation of the Bias that is associated with various analytical models to determine the capacity of a pipeline. In where pB50 is the 50th percentile (median) burst pressure, pO50 is σ this development, Bias is defined as the ratio of the true or the 50th percentile maximum operating pressure, pB is the measured (actual) loss of containment (LOC) pressure capacity standard deviation of the logarithms of the burst pressure, and σ of a pipeline to the predicted or nominal (e.g. code or guideline pO is the standard deviation of the logarithms of the maximum based) capacity: operating pressures. The pipeline burst pressure is determined True Measured from the RAM PIPE formulation: Bias== B = X Predicted No min al Pbd = 3.2 t SMYS / Do SCF It is important to note that the measured value determined SCF = 1 + 2 (d / R)0.5 from a laboratory experiment is not necessarily equal to the true or actual value that would be present in the field setting. where Pbd is the burst pressure capacity of the corroded Laboratory experiments involve ‘compromises’ that can lead to pipeline, t is the nominal wall thickness (including the important differences between the true or actual pipeline corrosion allowance), Do is the mean diameter (D–t), D is the capacity and that measured in the laboratory. For example, the pipeline outside diameter, SMYS is the specified minimum end closure plates used on laboratory test specimens of yield strength, and SCF is a stress concentration factor that is a pipelines will introduce axial stresses that can act to increase function of the depth of corrosion, d (d≤t), and the pipeline the LOC pressure capacity relative to a segment of the pipeline radius, R. in the field in which there would not be any significant axial The median of the burst pressure is determined from the stresses. medians of the variables. The uncertainty in the burst pressure One important example of the potential differences between is determined from the standard deviations of all of the the true pipeline capacity and the experimentally determine variables: pipeline capacity regards laboratory experiments that are used to 2 2 2 2 2 σ lnpB50 = σ lnS + σ lnt + σ lntc + σ lnD determine the burst pressure capacity of corroded pipelines. To facilitate the laboratory experiments (controlled parameter The probability of a corrosion depth, X, exceeding a lower variations), the corroded features frequently are machined into limit of corrosion depth detectability, xo, is: the pipeline specimen. This machining process can lead to P[ X ≥ xo | ND ] = important differences between actual corroded features and those machined into the specimens; stress concentrations can be very P[ X > xo ] P[ ND | X ≥ xo ] / P[ ND] different; residual stresses imparted by the machining process can be very different; and there can be metallurgical changes P [ X ≥ xo | ND ] is the probability of no detection given X ≥ caused by the machining process. Thus, laboratory results must xo. P [ X > xo ] is the probability that the corrosion depth is be carefully regarded and it must be understood that such greater than the lower limit of detectability. P [ ND | X ≥ xo ] experiments can themselves introduce Bias into the assessment is the probability of non detection given a flaw depth. P [ND] of pipeline reliability. is the probability of non detection across the range of flaw Another important example regards true or ‘measured’ depths where: results that are based on results from analytical models. Such

5Copyright © 2002 by ASME an approach has been used to generate ‘data’ used in several longitudinal defects, circumferential defects, and corrosion recent major reliability based code and guideline developments. patches of varying W/t and d/t. A corrosion patch refers to a The general approach is to use a few high quality physical region where the corrosion covers a relatively large area of pipe laboratory tests to validate or calibrate the analytical model. and the longitudinal and circumferential dimensions were Then the analytical model is used to generate results with the comparable. In some of the pipes, two defects of different sizes model’s parameters being varied to develop experimental data. were introduced and kept far enough apart to eliminate any One colleague has called these “visual experiments.” The interaction. primary problems with this approach concern how the model’s Hopkins and Jones (1992) [4] conducted five vessel burst parameters are varied (e.g. recognition of parameter correlations tests and four pipe ring tests. The pipe diameter were 508 mm. recognized and definition of the parametric ranges), and the The wall thickness was 102 mm. The pipe was made of X52. abilities of the model to incorporate all of the important The defect depth was 40% of the wall thickness. Jones et al physical aspects (e.g. residual stresses, material nonlinearity). (1992) also conducted nine pressurized ring tests. Seven of the The use of analytical models introduces additional uncertainties nine were machined internally over 20% of the circumference, and these additional uncertainties should not be omitted. In one the reduced wall thickness simulating smooth corrosion. All recent case, the analytical models have been calibrated based on specimens were cut from a single pipe of Grade API 5L X60 machined pipeline test sample results. Thus, the analytical with the diameter of 914 mm and wall thickness of 22 mm. models have ‘carried over’ the inherent Bias incorporated into As part of a research project performed at the University of the physical laboratory tests. Waterloo, 13 burst tests of pipes containing internal corrosion In this study, a differentiation has been made between pits were reported by Chouchaoui, et al [5]. In addition, physical laboratory test data and analytical test data. Further, Chouchaoui et al reported the 8 burst tests of pipes containing differentiation has been made between physical laboratory test circumferentially aligned pits and the 8 burst tests of pipes data on specimens from the field and those that are machined or containing longitudinally aligned pits. involve simulated damage and defects. Earlier studies The laboratory test database was used to determine the Bias performed on these databases have clearly indicated potentially in the DNV RP F-101 [6], B31G [7], and RAM PIPE [8] important differences between physical and analytical test data formulations were used to determine the burst pressure bias based Biases and differences between ‘natural’ and simulated (measured burst pressure divided by predicted burst pressure). defects and damage. The results for the 151 physical tests are summarized in Fig. 10 and Fig. 11. These tests included specimens that had corrosion Burst Capacities of Corroded Pipelines depth to thickness ratios in the range of 0 to 1 (Fig. 11). The A test database consisting of 151 burst pressure tests on statistical results from the data summarized in Fig. 10 are corroded pipelines was assembled from tests performed by the summarized in Table 1. American Gas Association [2], NOVA [3], British Gas [4], and Table 1: Bias statistics for three burst pressure the University of Waterloo [5]. The Pipeline Research formulations (d/t = 0 to 1) Committee of the American Gas Association published a report Formulation B mean B V % on the research to reduce the excessive conservatism of the 50 B B31G criterion (Kiefner, et al, 1989)[2] Eightysix (86) test data DNV 99 1.46 1.22 56 were included in the AGA test data. The first 47 tests were used B 31 G 1.71 1.48 54 to develop the B31G criterion, and were full scale tests RAM PIPE 1.01 1.03 22 conducted at Battelle Memorial Institute. The other 39 tests were also full scale and were tests on pipe sections removed The RAM PIPE formulation has the median Bias closest to from service and containing real corrosion. unity and the lowest COV of the Bias. The DNV formulation Two series of burst tests of large diameter pipelines were has a lower Bias than B31G, but the COV of the Bias is about conducted by NOVA during 1986 and 1988 to investigate the the same as for B31G. The B31G mean Bias and COV in Table applicability of the B31G criterion to long longitudinal 1 compares with values of 1.74 and 54 %, respectively, found corrosion defects and long spiral corrosion defects [3]. These by Bai, et al [9]. The burst pressure test data were reanalyzed to pipes were made of grade 414 (X60) steel with an outside include only those tests for d/t = 0.3 to 0.8. The bias statistics diameter of 4064 mm and a wall thickness of 50.8 mm. were relatively insensitive to this partitioning of the data. Longitudinal and spiral corrosion defects were simulated with A last step in the analysis of the physical test database was machined grooves on the outside of the pipe. The first series of to analyze the Bias statistics based on only naturally corroded tests, a total of 13 pipes, were burst. The simulated corrosion specimens. The results are summarized in Fig. 12 and Table 2. defects were 203 mm wide and 20.3 mm deep producing a The Bias statistics for the DNV and B31G formulations were width to thickness ratio (W/t) of 4 and a depth to thickness affected substantially. The results indicate that the machined ratio (d/t) of 0.4. Various lengths and orientations of the specimens develop lower burst pressures than their naturally grooves were studied. Angles of 20, 30, 45 and 90 degrees corroded counterparts. Even though the feature depth and area from the circumferential direction, referred to as the spiral might be the same for machined and natural features, the angle, were used. In some tests, two adjacent grooves were used differences caused by the stress concentrations, residual stresses, to indicate interaction effects. The second series of tests, a total and metallurgical effects cause important differences. of seven pipes, were burst. The defect geometries tested were

6Copyright © 2002 by ASME 10 Table 2. Bias statistics for three burst pressure formulations – naturally corroded tests DNV99 Formulation B mean B 50 V B % B31G DNV 99 2.10 1..83 46 RAM PIPE B 31 G 2.51 2.01 52 RAM PIPE 1.00 1.10 26 1 Burst Capacities of Dented & Gouged Pipelines A database on dented and gouged pipeline tests consisting of 121 tests was assembled from test data published by Battelle Research Corp. and British Gas [10-16] This database was predicted burst pressure organized by the sequence of denting and gouging and type of 0.1

Bias = Measured burst pressure / test performed. Study of this test data lead to the following .01. 1 1 51 02030 50 70809095 99 99.999.99 ≤≤ observations: Percent • Plain denting with smooth shoulders has no Fig. 10: Bias in burst pressure formulations (Lognormal significant effect on burst pressures. Smooth shoulder probability scales) denting is not accompanied by macro or microcracking and the dent is re-formed under increasing internal pressures. 6 • Denting with sharp shoulders can cause macro and micro cracking which can have some effects on burst 5 RAM PIPE pressures and on fatigue life (if there are significant sources B31G of cyclic pressures – straining. The degree of macro and 4 DNV99 micro cracking will be a function of the depth of gouging. Generally, given pressure formed gouging, there will be 3 distortion of the metal and cracking below the primary gouge that is about one half of the depth of the primary 2 gouge. • Gouging can cause macro and micro cracking in 1 addition to the visible gouging and these can have

predicted burst pressure significant effects on burst pressures. In laboratory tests, 0 frequently gouging has been simulated by cutting grooves Bias = measured burst pressure / 00.20.40.6 0.8 1 in the pipe. These grooves can be expected to have less d/t macro and micro cracking beneath the test gouge feature. • The combination of gouging and denting can have Fig. 11. Bias in burst pressure formulations as function of very significant effects on burst pressures. The effects of corrosion depth to wall thickness ratio (d/t) combined gouging and denting is very dependent on the history of how the gouging and denting have been 10 developed. Different combinations have been used in developing laboratory data. In some cases, the pipe is gouged, dented, and pressured to failure. In other cases, the pipe is dented and gouged simultaneously, and then pressured to failure. In a few cases, the pipe is gouged, pressured, and then dented until the pipeline looses 1 containment. These different histories of denting and gouging have important effects on the propagation of DNV99 macro and micro cracks developed during the gouging and B31G denting. It will be very difficult for a single formulation to RAM PIPE be able to adequately address all of the possible

predicted burst pressure combinations of histories and types of gouging and denting.

Bias = measured burst pressure / 0.1 • Gouging is normally accompanied by denting a .01.11510203050708 090959999.999.99 pipeline under pressure. If the pipeline does not loose Percent ≤≤ containment, the reassessment issue is one of determining what the reliability of the pipeline segment is given the Fig. 12: Bias in burst pressure formulations for naturally observed denting and gouging. Addressing this problem corroded test specimens (Lognormal probability scales) requires an understanding of how the pipeline would be expected to perform under increasing pressure demands (loss of containment due to pressure) or under continuing

7Copyright © 2002 by ASME cyclic strains (introduced by external or internal sources). decomposed into sub-systems of a series segments. A series In the case of loss of containment due to pressure, the dent segment is one in which the failure of one of the segments is re-formed under the increasing pressure and the gouge is leads to the failure of the system. propagated during the re-forming. Cracks developed on the A series (weak-link) system fails when any single element shoulders of the dents can also be expected to propagate fails. In probabilistic terms, the probability of failure of a series during the re-forming. system can be expressed in terms of the unions (∪) of the The analyses of the laboratory test database on the loss of probabilities of failure of its N elements as [17]: containment pressure of dented and gouged pipelines was based Pf = ( Pf ) ∪ ( Pf ) ∪ ... ( Pf ) on: system 1 2 N

Pbd = (2 SMTS / SCFDG) (t / D) For a series system comprised of N elements, if the elements have the same strengths and the failures of the where SCF H is the Stress Concentration Factor for the DG elements are independent (ρ = 0), then the probability of failure combined dent and gouge. Two methods were to evaluate the SCF associated with gouging and denting. The first method of the system can be expressed as: (Method 1) was based on separate SCF for the gouging and the Pf = 1 - (1 - Pf ) N dent reformation propagation: system i -1 If Pfi is small, as is usual, then approximately: SCFG = (1 – d/t) Pf ≈ N Pf 3 system i SCFD = 1 +0.2 (H/t) If the N segments of the pipeline are independent and have –1 3 SCFDG = [(1 – d/t) ] [1 +0.2 (H/t) ] different failure probabilities: −−∏N ( ) The second method (Method 2) was based on a single SCF Pf = 11Pfi system = that incorporated the gouge formation and propagation: i 1 -1 SCFDG = {[1 – (d/t) – [16 H/D(1-d/t)]} If the segments are perfectly correlated then: Fig. 13 summarizes results from analysis of the test Pfsystem = maximum (Pfi) database. The dent depths (H) to diameter ratios were in the range H/D = 1.0 % to 3.6 %. The gouge defects had depths (h) There can be a variety of ways in which correlations can be to wall thickness ratios that were h/t = 25%. developed in elements and between the segments that comprise Results of the analyses indicate Method 1 has a median a pipeline system. Important sources of correlations include: • segment to segment strength characteristics correlations, Bias of B50 = 1.2 and a COV of the Bias of VB = 33%. Method 2 has a B = 1.3 and V = 25%. and 50 B • segment to segment failure mode correlations. 2.0 The correlation coefficient, ρ, expresses how strongly the magnitudes of two paired variables, X and Y, are related to each other. The correlation coefficient ranges between positive and negative unity (-1 ≤ ρ ≤ +1). If ρ = 1, they are perfectly correlated, so that knowing X allows one to make perfect predictions of Y. If ρ = 0, they have no correlation, or are 1 ‘independent,’ so that the occurrence of X has no affect on the 0.9 occurrence of Y and the magnitude of X is not related to the 0.8 Method 1 magnitude of Y. Independent random variables are uncorrelated, but uncorrelated random variables (magnitudes not related) are 0.7 Method 2 not in general independent (their occurrences can be related) 0.6 [17]. 0.5 Frequently, the correlation coefficient can be quickly and .01 . 1 1 51 0203050708 0 9 0 9 5 9 9 99.999.99 accurately estimated by plotting the variables on a scattergram

Bias = Measured Pb / Calculated that shows the results of measurements or analyses of the Percent ≤≤ magnitudes of the two variables. Two strongly positively correlated variables will plot with data points that closely lie Fig. 13: Analysis of test database on pipelines with dents along a line that indicates as one variable increases the other and gouges variable increases. Two strongly negatively correlated variables will plot with data points that closely lie along a line that SYSTEMS AND SEGMENTS indicates as one variable increases, the other variable decreases. In development of the formulation for the probability of If the plot does not indicate any systematic variation in the failure, it is important to discriminate between pipeline variables, the general conclusion is that the correlation is very ‘segments’ and ‘systems’. A pipeline system can be low or close to zero.

8Copyright © 2002 by ASME In general, samples of paired pipeline segments are 650 strongly positively correlated; tensile strengths, collapse pressures, and burst pressures show very high degrees of 600 correlation (Figs. 14-16) [18]. These test data were taken from samples of delivered pipeline joints and were not intentionally paired from the same plate or runs of steel. High degrees of 550 correlation of pipe properties were also found by Jaio, et al (1997) for samples of the same pipe steel plate. 500 These results have important implications regarding the ρρ == 00.. 9988 relationship between the reliability of a pipeline system and the reliability of the pipeline system elements and segments. The Test Specimen i 450 probability of failure of the pipeline system will be characterized by the probability of failure of the most likely to 400 fail element – segment that comprises the system. Measured Burst Pressure (MPa) 400 450 500 550 600 650 100 Measured Burst Pressure Test Specimen j 90 Fig. 16: Correlation of measured burst strengths of paired 80 steel pipeline samples from adjacent pipeline segments Correlations can also be developed between the failure 70 modes. A useful expression to determine the approximate correlation coefficient between the probabilities of failure of a Element i 60 ρρ == 00.. 9999 system’s components (or correlation of failure modes) is:

2 50 VS ρfm ≈ Ultimate Tensile Strength 22+ 40 VVRS 40 50 60 70 80 90 100 2 2 where V S and V R are the squared coefficients of variation of Ultimate Tensile Strength the demand (S) and capacity (R), respectively. It is often the Element j case for pipeline systems that the coefficients of variation of the Fig. 14: Correlation of measured ultimate tensile strengths demands are equal to or larger than those of the capacity. Thus, of paired pipeline steel samples from adjacent pipeline the correlation of the probabilities of the failure of the system’s segments segments can be very large, and there is a high degree of correlation between the system’s failure modes. Again, this 40 indicates that the probability of failure of the system can be determined by the probability of failure of the system’s most 35 likely to fail segment. 30 CONCLUSIONS 25 A practical formulation has been developed to allow ‘real- time’ assessments of pipeline likelihoods of LOC (probabilities 20 of failure). This development as involved developing analytical ρρ == 00.. 9999 models to evaluate time effects, Biases introduced by different 15 Specimen i models used to evaluate the LOC pressures, and system versus segment probabilities of failure. Laboratory test data has been 10 used to provide the important parameters for these analytical models. 5 The real-time RAM formulation is a Level 2 approach in 510152025303540

Measured Collapse Pressure (MPa) the general pipeline Inspection, Maintenance, and Repair Measured Collapse Pressure process proposed by Bea, et al [19]. This formulation is Specimen j consistent with the Risk Based Inspection process proposed by Fig. 15: Correlation of measured collapse strengths of Bjornoy, et al [20]. Verification of the real-time RAM LOC paired steel pipeline samples from adjacent pipeline analytical models with field hydro-test to failure data is the segments subject of a companion paper [21]. The ability to develop real-time estimates of the probabilities of LOC can provide the pipeline owner / operator, pipeline engineers, and regulators with useful additional

9Copyright © 2002 by ASME information to help guide their decisions regarding pipeline [10] Kiefner, J.F., Maxey, W.A., Eiber, R.J., and Duffy, A.R., maintenance. 1973, “Failure Stress Levels of Flaws in Pressurized Cylinders,” Progress in Flaw Growth and Fracture ACKNOWLEDGMENTS Toughness Testing, ASTM STP 536, American Society The authors would like to acknowledge the support to for Testing and Materials, New York, NY, pp 120-135. perform this research and the permission to publish these [11] Eiber, R., et alm 1981, The Effects of Dents on the results provided by the U.S. Minerals Management Service and Failure Characteristics of Line Pipe, Battelle Columbus Rosen Inspection Inc. The authors also would like to Report to AGA, NG-18, AGA Catalog No. L51403, acknowledge the analytical and computational assistance Columbus, OH. provided by University of California Berkeley Graduate Student [12] Stephens, D.R., Olson, R.J., and Rosenfeld, M.J., 1991, Researchers Sang Kim, Angus McLelland, and Ziad Nakat. Topical Report on Pipeline Monitoring – Limit State Criteria, Report to Line Pipe Research Supervisory REFERENCES Committee, American Gas Association, Columbus, OH. [1] Lara, L., Matias, J., Heredia, E., and Valle, O., 1998,. [13] Hopkins, P., 1990, A Full Scale Evaluation of the Transitory Criteria for Design and Evaluation of Behaviour of Dents and Defects in Linepipe for the Submarine Pipelines in the Bay of Campeche, First European Pipeline Research Group, British Gas ERS Meeting Report, Instituto Mexicano de Petroleo, Mexico, Report R. 4516, British Gas Research & Technology, DF. Newcastle upon Tyne, UK. [2] Kiefner, J. F., et al, 1989, A Modified Criterion for [14] Hopkins, P., Jones, D. G., and Clyne, A.J.m 1989, The Evaluating the Remaining Strength of Corroded Pipe, Significance of Dents in Transmission Pipelines, British RSTRENG, Project PR 3-805 Pipeline Research Gas plc, Research & Technology, Engineering Research Committee, American Gas Association, Houston, TX. Station, Newcastle upon Tyne, UK. [3] Mok, D.H.B, Pick, R.J., Glover, A.G. and Hoff, R., [15] Shannon, R.W.E., 1974, “The Failure Behaviour of Line 1991, “Bursting of Line Pipe with Long External Pipe Defects,” International Journal of Pressure Vessels & Corrosion,” International Journal of Pressure Vessel & Piping, Applied Science Publishers Ltd, UK. Piping, Vol. 46, Applied Science Publishers Ltd, UK., pp [16] Jones, D.G., 1981, The Significance of Mechanical 159-216. Damage in Pipelines, British Gas Corporation, Research & [4] Hopkins, P., and Jones, D.G, 1992, “A Study of the Technology, Engineering Research Station, Newcastle Behavior of Long and Complex-Shaped Corrosion in upon Tyne, UK. transmission Pipelines,” Proceedings of the Offshore [17] Madsen, H.O., Krenk, S., and Lind, N.C., 1986, Methods of Mechanics and Arctic Engineering Conference, Pipeline Structural Safety, Prentice Hall Inc., Englewood Cliffs, NJ. Symposium, American society of Mechanical Engineers, [18] Bea, R. G., Xu, T., Mousselli, A., Bai, Y., and New York, NY, pp 230-240. Orisamolu, W., 1998, RAM PIPE Project, Risk Assessment [5] Chouchaoui, B. A., et al (1992) “Burst Pressure Prediction and Management Based Allowable Stress Design & Load of Line Pipe Containing Single Corrosion Pits using the and Resistance Factor Design Stress Criteria & Finite Element Methods”, Proceedings 13th International Guidelines for Design and Requalification of Pipelines Offshore Mechanics and Arctic Engineering, Pipeline and Risers in the Bay of Campeche, Mexico, Phase 1, Symposium, American Society of Mechanical Engineers, Reports 1 through 6, Reports to Petroleos Mexicanos and New York, NY, pp 11-19. Instituto Mexicano de Petroleo, Mexico DF. [6] Det Norske Veritas, 2000, Corroded Pipelines, [19] Bea, R. G., Farkas, B, Smith, C., Rosenmoller, J., Recommended Practice RP-F101, Hovik, Norway. Valdes, V., and Valle, O., 1999, “Assessment of Pipeline [7] American Society of Mechanical Engineers (ASME), 1993, Suitability for Service,” Proceedings 9th International Manual for Determining the Remaining Strength of Offshore and Polar Engineering Conference & Exhibition, Corroded Pipelines, Supplement to ANSI / ASME B31G Society of Offshore and Polar Engineers, Golden, CO., pp Code for Pressure Piping, New York, NY. 347-354. [8] Bea, R. G., 2000, “Reliability, Corrosion, and Burst [20] Bjornoy, O.H., Jahre-Nilsen, C., Marley, M.J., and Pressure Capacities of Pipelines,” Proceedings Offshore Willamson, R., 2001, “RBI Planning for Pipelines, Mechanics and Arctic Engineering Conference, Safety and Principles and Benefits,” Proceedings of the Offshore Reliability Symposium, American Society of Mechanical Mechanics and Arctic Engineering Conference, Pipeline Engineers, New York, NY, pp 1-11. Symposium, OMAE/01-PIPE4007, American Society of [9] Bai, Y., Xu, T., and Bea, R.G., 1997, “Reliability-Based Mechanical Engineers, New York, NY., 1-6 Design & Requalification Criteria for Longitudinally [21] Bea, R. G., Smith, C., Smith, R., Rosenmoeller, J., and Corroded Pipelines,” Proceedings of the Seventh Beuker, T., 2002, “Analysis of Field Data from the International Offshore and Polar Engineering Conference, Performance of Offshore Pipelines (POP) Project,” International Society of Offshore and Polar Engineers, Proceedings of the Offshore Mechanics and Arctic Golden, CO, pp 1-12. Engineering Conference, Pipeline Symposium, OMAE/02- PIPE28323, American Society of Mechanical Engineers, New York, NY, pp 1-XX.

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Marine and Petroleum Geology

journal homepage: www.elsevier.com/locate/marpetgeo

Review article Oil and gas wells and their integrity: Implications for shale and unconventional resource exploitation

Richard J. Davies a,*, Sam Almond a, Robert S. Ward b, Robert B. Jackson c,d, Charlotte Adams a, Fred Worrall a, Liam G. Herringshaw a, Jon G. Gluyas a, Mark A. Whitehead e a Durham Energy Institute, Department of Earth Sciences, Durham University, Science Labs, Durham DH1 3LE, UK b Groundwater Science Directorate, British Geological Survey, Keyworth, Nottingham NG12 5GG, UK c School of Earth Sciences, Woods Institute for the Environment, and Precourt Institute for Energy, Stanford University, Stanford, CA 94305, USA d Nicholas School of the Environment, Division of Earth and Ocean Sciences, Duke University, Box 90338, 124 Science Drive, Durham, NC 27708-0338, USA e Ward Hadaway, Sandgate House, 102 Quayside, Newcastle Upon Tyne NE13DX, UK article info abstract

Article history: Data from around the world (Australia, Austria, Bahrain, Brazil, Canada, the Netherlands, Poland, the UK Received 8 December 2013 and the USA) show that more than four million onshore hydrocarbon wells have been drilled globally. Received in revised form Here we assess all the reliable datasets (25) on well barrier and integrity failure in the published liter- 28 February 2014 ature and online. These datasets include production, injection, idle and abandoned wells, both onshore Accepted 1 March 2014 and offshore, exploiting both conventional and unconventional reservoirs. The datasets vary considerably Available online 25 March 2014 in terms of the number of wells examined, their age and their designs. Therefore the percentage of wells that have had some form of well barrier or integrity failure is highly variable (1.9%e75%). Of the 8030 Keywords: Shale wells targeting the Marcellus shale inspected in Pennsylvania between 2005 and 2013, 6.3% of these have Fracking been reported to the authorities for infringements related to well barrier or integrity failure. In a separate Integrity study of 3533 Pennsylvanian wells monitored between 2008 and 2011, there were 85 examples of Barrier cement or casing failures, 4 blowouts and 2 examples of gas venting. In the UK, 2152 hydrocarbon wells Integrity were drilled onshore between 1902 and 2013 mainly targeting conventional reservoirs. UK regulations, Wells like those of other jurisdictions, include reclamation of the well site after well abandonment. As such, there is no visible evidence of 65.2% of these well sites on the land surface today and monitoring is not carried out. The ownership of up to 53% of wells in the UK is unclear; we estimate that between 50 and 100 are orphaned. Of 143 active UK wells that were producing at the end of 2000, one has evidence of a well integrity failure. Ó 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction Some of the shallower strata may contain groundwater used for human consumption or which supports surface water flows and The rapid expansion of shale gas and shale oil exploration and wetland ecosystems. Although it has been routine practice to seal exploitation using hydraulic fracturing techniques has created an wells passing through such layers, they remain a potential source of energy boom in the USA but raised questions regarding the possible fluid mixing in the subsurface and potential contamination (King environmental risks, such as the potential for groundwater and King, 2013). This can occur for many reasons, including poor contamination (e.g. Jackson et al., 2013; Vidic et al., 2013) and well completion practices, the corrosion of steel casing, and the fugitive emissions of hydrocarbons into the atmosphere (e.g. Miller deterioration of cement during production or after well abandon- et al., 2013). ment. Boreholes can then become high-permeability potential Boreholes drilled to explore for and extract hydrocarbons must conduits for both natural and man-made fluids (e.g. Watson and penetrate shallower strata before reaching the target horizons. Bachu, 2009), and vertical pressure gradients in the subsurface can drive movement of fluids along these flow paths. The potential importance of wellbore integrity to the protection of shallow * Corresponding author. Tel.: þ44 1913342346. groundwater has recently been highlighted in research papers and E-mail address: [email protected] (R.J. Davies). reports (e.g. Osborn et al., 2011; The Royal Society & The Royal http://dx.doi.org/10.1016/j.marpetgeo.2014.03.001 0264-8172/Ó 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). 240 R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254

Glossary m3 Cubic Metres mD Milli-Darcies BCF Billion Cubic Feet NOCS Norwegian Offshore Continental Shelf BCM Billion Cubic Metres NY New York BRGM Bureau de Recherches Géologiques et Minières, France PA Pennsylvania BDEP Brazilian Database of Exploration and Production PSA Petroleum Safety Authority, Norway CA California RRC Railroad Commission, Texas

CO2 Carbon Dioxide SCVF Surface Casing Vent Flow CCTV Closed-Circuit Television SINTEF Norwegian Foundation for Scientiﬁc and Industrial DECC Department of Energy and Climate Change, UK Research DEFRA Department of Environment, Food and Rural Affairs, TCF Trillion Cubic Feet UK TCM Trillion Cubic Metres DEP Department of Environmental Protection, USA UK United Kingdom EIA Energy Information Administration, USA UKCS United Kingdom Continental Shelf ERCB Energy Resources Conservation Board, Canada UKOGL United Kingdom Onshore Geophysical Library EUR Estimated Ultimate Recovery UNFCCC United Nations Framework Convention on Climate GM Gas Migration Change GoM Gulf of Mexico US United States IPCC Intergovernmental Panel on Climate Change USA United States of America km2 Square Kilometres WFD Water Framework Directive, Europe M Metres WV West Virginia

Academy of Engineering Report (2012); Jackson et al., 2013; King used to prevent contamination of groundwater, surface water, soils, and King, 2013). In addition to protecting ground and surface wa- rock layers and the atmosphere depend on whether the well is for ters, effective well sealing prevents leakage of methane and other exploration or production, but generally include cement, casing, gases into the atmosphere. This is important as methane is 86 times valves and seals (Fig. 1). Barriers can be nested, so that a well has more effective than CO2 at trapping heat in the atmosphere over a several in place. They can be dynamic (e.g. a valve) or static (e.g. 20-year period and 34 times more effective over a century (IPCC, cement), and may or may not be easily accessible for assessment or 2013). Well barrier and integrity failures can occur during dril- monitoring (see King and King, 2013). ling, production, or after abandonment; in rare examples, including Drilling a well for exploration or production is a multistage in the USA, well leakage has led to explosions at the Earth’s surface process during which the upper parts of a borehole, once drilled, (e.g. Miyazaki, 2009). are sealed with steel casing and cemented into place. Cement was This paper has four aims: 1) to estimate the number of onshore introduced to the petroleum industry as early as 1903, when Frank hydrocarbon wells globally; 2) to explain how onshore wells are Hill of Union Oil Co. poured 50 sacks of Portland cement into a categorised (e.g. producing, abandoned, idle, orphaned) and what well to seal off water-bearing strata (Smith, 1976). Cementing is statistical data are available on the numbers of wells in these now typically carried out by pumping water-cement slurries groups; 3) to document the number of wells that are known to have down the casing to the bottom of the hole, displacing drilling had some form of well barrier and/or integrity failure, placing these ﬂuids from the casing-rock and other annuli, leaving a sheath of numbers in the context of other extractive industries; and 4) to cement to set and harden (Fig. 1). The integrity of these seals is analyse how many onshore wells in the UK can be easily accessed to pressure-tested before the next stage of drilling occurs. Only if the assess for barrier and integrity failure. For well barrier and integrity well passes these pressure tests will drilling continue. If the well failure our approach has been to include all the reliable datasets fails the test, the casing is re-cemented before drilling continues. that are available, rather than de-select any data. This inclusive The sizes and lengths of casing, and the depths at which different approach has the draw-back that the data we present include wells casings are used depend upon the geology, the importance or of different age, of different designs and drilled into different ge- sensitivity of the groundwater that the well penetrates, and the ology. Unsurprisingly there is a signiﬁcant spread in the statistics purpose of the well (Fig. 1). Well completion should follow staton the percentage of wells that have well barrier or integrity failure. utory regulations and/or industry best practice. When a well is The review is largely focused on North America, as it has a long abandoned, cement is normally pumped into the production history of onshore hydrocarbon drilling (including wells drilled for tubing to form a cement plug to seal it. Commonly (e.g. in the UK), shale gas and shale oil) and the UK, which contrasts in having a the top of the well is welded shut. mature offshore drilling industry, but relatively little onshore drilling. It mainly, but not exclusively, covers static well failure (i.e. 1.2. Terminology after drilling operations are completed), and summarises currently available data for regulators, non-government organisations, the The terms ‘well barrier failure’ and ‘well integrity failure’ were public, and the oil and gas industry. differentiated by King and King (2013). They used ‘well integrity failure’ for cases where all well barriers fail, establishing a pathway 1.1. Barrier systems that enables leakage into the surrounding environment (e.g. groundwater, surface water, underground rock layers, soil, atmo- Barriers are containment mechanisms within a well or at the sphere). ‘Well barrier failure’ was used to refer to the failure of well head that are designed to withstand the corrosion, pressures, individual or multiple well barriers (e.g. production tubing, casing, temperatures and exposure times associated with the phases of cement) that has not resulted in a detectable leak into the sur- drilling, production and well abandonment. The types of barriers rounding environment. The same terminology is used in this paper: R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254 241

Figure 1. Schematic diagram of typical well design, showing (A): structure of an exploration well; and (B): a production well. Depths to which different casings are used vary according to geology and pressure regime of drill site. Well diameter exaggerated to show sections more clearly.

‘well integrity failure’ includes cases when gas or fluids are re- A leak can be catastrophic, as seen in cases such as the recent ported to have leaked into soils, rock strata or the atmosphere, and blowout of a Whiting Petroleum Corp oil well (Cherry State 31-16H) ‘well barrier failure’ includes cases where a barrier failure has in North Dakota (North Dakota Department of Health (2014)) and occurred but there is no information that indicates that fluids have rare examples of explosions in urban areas (Chillingar and Endres, leaked out of the well. 2005), or be at sufficiently low rates to be barely detectable. The fluid sources can be hydrocarbon reservoirs (e.g. Macondo, Gulf of 1.3. Routes and driving mechanisms Mexico); non-producing permeable formations (e.g. Marshall and Strahan, 2012); coal seams (e.g. Beckstrom and Boyer, 1993; For a well to leak, there must be a source of fluid (Fig. 2), a Cheung et al., 2010); and biogenic or thermogenic gases from breakdown of one or more well barriers, and a driving force for shallow rock formations (e.g. Traynor and Sladen, 1997; Jackson fluid movement, which could be fluid buoyancy or excess pore et al., 2013). Oil or gas emissions can seep to the surface, though pressure due to subsurface geology (e.g. Watson and Bachu, 2009). leaking methane can be oxidised by processes such as bacterial There are seven subsurface pathways by which leakage typically sulphate reduction (e.g. Van Stempvoort et al., 2005). Well failures occurs (Figs. 3, 4). These pathways include the development of can potentially occur in any type of hydrocarbon borehole, whether channels in the cement, poor removal of the mud cake that forms it is being drilled, producing hydrocarbons, injecting fluid into a during drilling, shrinkage of cement, and the potential for relatively reservoir, or has been abandoned. high cement permeability (e.g. Dusseault et al., 2000). There are Wells can be tested at the surface for well barrier failure and other mechanisms that can operate in specific geological settings. well integrity failure by determining whether or not there is Reservoir compaction during production, for example, can cause pressure in the casing at the surface. This is referred to as sustained shear failure in the rocks and casing above the producing reservoir casing pressure (e.g. Watson and Bachu, 2009), but does not (Marshall and Strahan, 2012; route 7 marked on Fig. 3). Leaking necessarily prove which barrier has failed or its location. Channels wells can also connect with pre-existing geological faults, enabling in cement, which are potential leakage pathways, can be detected leakage to reach the surface (Chillingar and Endres, 2005). A range by running detection equipment down the borehole. Migration of of fluids can leak, for instance formation fluids, water, oil and gas, fluids outside the well is established by inserting a probe into the and they can move through or out of the well bore by advective or soil immediately surrounding the well bore, or by sampling diffusive processes (e.g. Dusseault et al., 2000). Overpressure may groundwater nearby, hydraulically down-gradient of the well. Poor be the driving force for fluid flow (e.g. the Hatfield blow-out near cement barriers can be identified by a number of methods (e.g. Doncaster, UK; Ward et al., 2003), but hydrostatically pressured ultrasonic frequency detection; Johns et al., 2011) and can be successions can also feed leaking wells, with fluids migrating due to repaired in some cases, using cement or pressure-activated sealants buoyancy and diffusion. (e.g. Chivvis et al., 2009). 242 R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254

2. Datasets

This paper draws on a variety of datasets, mostly published, but in some instances sourced from online repositories or national databases, and follows the approach of Davies et al. (2013). In that study, the risk of induced seismicity due to hydraulic fracturing was reviewed, and intentionally included all datasets in the public domain that were considered to be reliable, rather than de- selecting any data (Davies et al., 2013). This inclusive approach has a drawback because well barrier and well integrity failure fre- quencies are probably speciﬁc to the geology, age of wells, and era of well construction (King and King, 2013). A wide range of failure statistics is therefore reported, and although they are presented on a single graph to show the spread of results (Fig. 9), this is not intended to imply that direct comparisons between very different datasets (i.e. size, age of wells, geology) can be made. The sources we used do not report their ﬁndings consistently and it is unclear in some cases whether well barrier failures have led to leaks into groundwater, rock layers, soil or the atmosphere,

Figure 2. Schematic diagram of typical sources of ﬂuid that can leak through a hydrocarbon well. 1 e gas-rich formation such as coal; 2 e non-producing, gas- or oil- bearing permeable formation; 3 e biogenic or thermogenic gas in shallow aquifer; and 4 e oil or gas from an oil or gas reservoir.

Figure 3. Routes for fluid leak in a cemented wellbore. 1 e between cement and surrounding rock formations, 2 e between casing and surrounding cement, 3 e between cement plug and casing or production tubing, 4 e through cement plug, 5 e through the cement between casing and rock formation, 6 e across the cement outside Figure 4. Photographic examples of leak pathways: (a) Corrosion of tubing the casing and then between this cement and the casing, 7 e along a sheared wellbore. (Torbergsen et al., 2012); (b) Cracks in cement (Crook et al., 2003); (c) Corrosion of After Celia et al. (2005) and this paper. casing (Xu et al., 2006). R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254 243 producing a true well integrity failure. To be as clear as possible, Table 1 well barrier and well integrity failure are distinguished in Table 3, Number of hydrocarbon boreholes drilled onshore in selected nation states. quoting directly from the sources used and, where possible, Country Number of wells Source providing additional information on the age of the well and when UK 2152 DECC, 2013 the monitoring was carried out. CanadaeAlberta 316,439 Watson and Bachu (2009) To locate hydrocarbon wells drilled onshore in the UK since Bahrain 750 Sivakumar and Janahi (2004) 1902 (the age of the earliest well recorded by DECC), the United USA 2,581,782 EIA Database Kingdom Onshore Geophysical Library (UKOGL) map of well loca- Austria 1200 Veron (2005) Netherlands 3231 Geological Survey of the Netherlands tions was used (UKOGL, 2013), coupled with satellite imagery from Brazil 21,301 Brazil Database of Exploration Google Earth. A visual inspection and categorisation of the locations and Production (BDEP) was carried out to assess whether the wells have a physical pres- Australia 9903 Geoscience Australia ence at the surface. Pollution incident data were provided by the Poland 7052 Polish Geological Institute Environment Agency (England); these data were used to identify incidents that occurred in close proximity to known well sites. derived a figure of 3.4% well barrier leakage for shale gas production sites in Pennsylvania (219 violations for 6466 wells) between 2008 and 2013. Using the same database, Ingraffea (2012) argued 3. Global well inventory that 211 (6.2%) of 3391 shale gas wells drilled in Pennsylvania in 2011 and 2012 had failed. More recently, Considine et al. (2013) As shale gas and oil exploitation has been carried out primarily identified 2.58% of 3533 individual wells as having some form of onshore to date, the global well inventory in this study reports only barrier or integrity failure. This consisted of 0.17% of wells having the number of hydrocarbon wells drilled onshore, as this provides a experienced blowouts (4 wells), venting or gas migration (2), and more relevant historical context. Data in the public domain were 2.41% having experienced casing or cementing failures. Measurable used, sourced either from published reports or from online datasets concentrations of gas were present at the surface for most wells populated by regulatory authorities. Several comprehensive review with casing or cementing violations. Figure 6 shows a breakdown of papers were also utilised, particularly those addressing the potential the 1144 environmental violations issues for the 3533 wells. of CO2 to leak upwards through wells (e.g. Watson and Bachu, 2009). In this study, the search criteria used to categorise leakage in- A graph of wells drilled per year since the 1930s in Australia, cidents in Pennsylvania followed the approach described by Brazil,the Netherlands, Poland, the UK, and the USA shows that Ingraffea (2012) and are based on code violations reported during some countries, such as the UK, have very modest onshore drilling site inspections. Code violations that would constitute a well failure activity compared to others such as the USA (Fig. 5). Historical data are those likely to result in a significantly increased risk of con- are sparse, so it is difficult to estimate the total number of onshore taminants reaching either the surface or potable water sources. hydrocarbon wells drilled globally, but in the USA alone, at least 2.6 They include: (a) failure to case and cement the well properly; (b) million wells have been drilled since 1949 (EIA database). Former excessive casing seat pressure; (c) failure to case and cement suf- Soviet countries such as Azerbaijan, where many thousands of ficiently to prevent migrations into fresh groundwater; and (d) wells have been drilled, are not included in this study due to a lack insufficient cement and steel casings between the wellbore and the of access to adequate data. Nonetheless, taking into consideration near-surface aquifer to prevent seepage of fluids. Using the Penn- those drilled only in Australia, Austria, Bahrain, Brazil, Canada, the sylvania state database, a well barrier or integrity failure rate of Netherlands, Poland, the UK and the USA, we estimate there are at 6.3% is identified for the years 2005e2013. This includes failures least 4 million onshore hydrocarbon wells (Table 1). noted in inspection reports that were not recorded as a violation, following the methodology of Ingraffea (2012). Without including 4. Well integrity these reports, the failure rate would be 5%. This is higher than the 3.4% well leakage figure reported by Vidic et al. (2013) for the 4.1. Pennsylvania, USA period 2008e2013, and close to the well failure rate of 6.2% reported by Ingraffea (2012). The online database collated by the Department of Environ- mental Protection (DEP) in the US state of Pennsylvania allows oil and gas well records to be searched by various criteria, such as well 4.2. Gulf of Mexico, USA status, operator and drilling date. The unconventional hydrocarbon wells included in that database are those that were drilled to target Data from the US Minerals Management Service show that, of the Marcellus Shale Formation. From these data, Vidic et al. (2013) 15,500 producing, shut in and temporarily abandoned wells in the

Figure 5. Number of wells drilled annually since the 1930s in Australia, Brazil, Netherlands, Poland, the UK and the USA. Sources: DECC, 2013; Geoscience Australia; Geological Survey of the Netherlands; Brazil Database of Exploration and Production (BDEP); EIA, Polish Geological Institute. 244 R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254

4.5. Offshore and onshore UK

For offshore wells on the UKCS, Burton (2005) found that 10% of 6137 wells (operated by 18 companies) had been shut-in (valves at the well head closed) during the last five years as a result of ‘structural integrity issues’. The total number of wells drilled on the UKCS is 9196; exploration boreholes that did not make commercial discoveries were not included in the Burton (2005) study. Onshore, 2152 hydrocarbon wells have been drilled in the UK between 1902 and 2013. Although the onshore sedimentary succession is not thought to be overpressured, hydrocarbons could still migrate upwards because of their buoyancy relative to pore water or the fluid in a borehole (e.g. the Hatfield blow-out near Doncaster, UK; Ward et al., 2003). Pollution incident data were reviewed for all

Figure 6. Breakdown of 1144 notices of violations from 3533 wells in Pennsylvania incidents reported within 1 km of wells in England between 2001 from 2008 to 2011 (after Considine et al., 2013). Red font indicates those related to well and 2013 (the only time period for which data are available). These barrier and integrity failure. (For interpretation of the references to colour in this figure data were filtered for those indicating a release of crude oil to the legend, the reader is referred to the web version of this article.) environment. These incidents were described as pipe failures above or below ground and could be related to the well or pipelines connected to the wells. To act as a control to this data, pollution outer continental shelf of the Gulf of Mexico, 6692 (43%) have incidents within a 5 km radius of the well were also examined to sustained casing pressure on at least one casing annulus (Brufatto assess whether there was a broader issue of hydrocarbon pollution et al., 2003). Of these incidents, 47.1% occurred in the production incidents that should be considered and taken into account. strings, 26.2% in the surface casing, 16.3% in the intermediate cas- The number of wells active prior to the period covered by the ing, and 10.4% in the conductor pipe. pollution records was also calculated. Based on data provided by DECC, 143 onshore oil and gas wells were producing at the start of the year 2000. Between 2000 and 2013, the Environment Agency 4.3. Offshore Norway records nine pollution incidents involving the release of crude oil within 1 km of an oil or gas well (Table 78, Fig. ). The records are not Vignes and Aadnøy (2010) examined 406 wells at 12 Norwegian clear as to whether the incidents were due to well integrity failure, offshore facilities operated by 7 companies. Their dataset included problems with pipework linked to the well, or other non-well producing and injection wells, but not plugged and abandoned related issues. In February 2014, therefore, the present-day opera- wells. Of the 406 wells they examined, 75 (18%) had well barrier tors of the wells at which the nine events occurred were contacted issues. There were 15 different types of barrier that failed, many of (Perenco, IGas, and Humbly Grove Energy Ltd.). The two pollution them mechanical (Fig. 7), including the annulus safety valve, casing, incidents at the Singleton Oil Field (now operated by IGas but cement and wellhead. Issues with cement accounted for 11% of the operated by a different company when the incidents occurred) failures, whilst issues with tubing accounted for 39% of failures. occurred in the early 1990s, and were caused by failure of cement The PSA has also performed analyses of barrier failures and well integrity on the Norwegian continental shelf. Its analysis showed that, in 2008, 24% of 1677 wells were reported to have well barrier failures; in 2009, 24% of 1712 wells had well barrier failures; and in 2010, 26% of 1741 wells had well barrier failures. It is unclear whether the same wells were tested in successive years or whether surveys targeted different wells (Vignes, 2011). A study of 217 wells in 8 offshore fields was also carried out by SINTEF (see Vignes, 2011). Between 11% and 73% of wells had some form of barrier failure, with injectors 2 to 3 times more likely to fail than producers (Vignes, 2011). At the 20th Drilling Conference in Kristiansand, Norway, in 2007, Statoil presented an internal company survey of offshore well integrity (Vignes, 2011). This analysis showed that 20% of 711 wells had integrity failures, issues, or uncertainties (Vignes, 2011). When subdivided into production and injection wells, the survey concluded that 17% of 526 production wells and 29% of 185 injection wells had well barrier failures.

4.4. Onshore Netherlands

The results of an inspection project carried out by the State Supervision of Mines Netherlands were also reported by Vignes (2011). Their inspections, carried out in 2008, included only 31 wells from a total of 1349 development wells from 10 operating companies. Of those wells, 13% (4 of 31) had well barrier problems; Figure 7. Causes of barrier failures for the 75 (of 406) production and injection wells by well type, problems were identiﬁed in 4% of the production wells surveyed in offshore Norway that showed evidence for such failures (from Vignes, (1 of 26) and 60% of the injection wells (3 of 5). 2011). R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254 245

Table 2 Sources of data reporting well barrier and well integrity failures.

Country Region Well location Status Completion date Well type Well numbers Failure statistics Organisation

USA PA X X X X X X Department of Environmental Protection Texas X X X X X RRC Alabama X X X X X Geological Survey of Alabama New York X X X X X New York Department of Environmental Conservation Florida X X X X X Florida Department of Environmental Protection North Dakota X X X X X X North Dakota Oil and Gas Division & North Dakota Department of Environmental Health W. Virginia X X X X X X West Viginia Department of Environmental Protection UK National X X X X DECC Canada Alberta X X X X X X Energy Resources Conservation Board (ERCB) Australia National X X X X X Geoscience Australia France National X X X X BRGM Netherlands National X X X X X Geological Survey of the Netherlands Brazil National X X X X BDEP Norway (offshore) National X X X Norway Offshore Continental Shelf Data Access Portal Poland National X X X X Polish Geological Institute

behind the conductor and the 9 5/8-inch casing. This was identified been careful to provide the exact wording from the published as a result of five groundwater monitoring boreholes installed at source as to the nature of the failure, and to discriminate between the Singleton Oil Field in 1993. The leak was from the well cellar well barrier and well integrity failures. (cement lined cavity in which the well head sits) via the pre- installed conductor and the 9 5/8-inch casing, both of which 5. Orphaned, abandoned or idle wells appear not to have been adequately cemented in-situ in at least one well. A thorough investigation commenced in 1997, including the 5.1. Definitions drilling of a number (>11) of additional boreholes, and the carrying out of tracer tests and CCTV examination under the auspices of, and The terms ‘abandoned’, ‘idle’ and ‘orphaned’ are used to in consultation with, the UK Environment Agency. The leak paths, describe the state of a well that did not locate economic hydro- once identified and verified, were remediated. Monitoring has carbons or a well at the end of its production lifecycle. The USA has continued since that time and the observed pollution levels have the most established and comprehensive definitions of such terms, remained below those set by the Environment Agency as requiring although their meaning can vary at state and federal levels. further action. A review of the various state regulatory practices regarding idle The other seven pollution incidents recorded by the Environ- wells in the USA was conducted by Thomas (2001) and defined idle ment Agency between 2000 and 2013 were not caused by well wells as those not currently being used for production or injection, integrity failure, but due to leaks from pipework linked to the well. but which have not yet been plugged and abandoned. In California, No incidents were reported at the other well sites in the UK that Hesson and Glinzak (2000) and Evans et al. (2003) defined idle were inactive or abandoned. wells as those that have been non-producing and non-injecting for For context, it should be noted that there are natural, high six consecutive months. permeability geological pathways for the migration of buoyant In the USA, the definition of an orphaned well depends largely fluids, which are typically associated with structural features such on the state regulatory body. Thomas (2001) defined orphaned as faults and folds (Selley, 1992). Gas and oil are naturally mobile in wells as those in which the operator has gone out of business or is the UK subsurface: around 200 natural hydrocarbon seeps, mainly insolvent, such that the company that operated the well is no of oil, are known from the onshore UK and some have been used to longer responsible for it. Based on Californian practices, Hesson initiate localised exploitation (Selley, 1992, 2012). A small number (2013) defined orphaned wells as those where the operator is of natural gas seeps from shales were recorded by Selley (2012), defunct, or where the state regulatory body has determined, based with notable occurrences in the Weald Basin of south-east England on certain criteria, that a well is orphaned. Such criteria include a (Selley, 2012, Fig. 5). well having been idle for 25 years or more, without being in compliance with idle well requirements. In Texas, the oil and gas 4.6. Summary of well barrier and integrity failure regulatory body e the RRC e defines orphaned wells as those which have, without permit, been inactive for a year or more. In Penn- For the countries listed (Table 1), publicly available data were sylvania, a 1992 amendment to the 1984 Oil and Gas Act defined an tabulated on well type, well location, completion date, well status, orphaned well as one which was abandoned prior to April 1985, number of wells drilled and whether well barriers and integrity which has not been operated by the present owner, and for which failures had occurred (Table 2). Tabulation of all published and the present owner has received no economic benefit. For the UK online data on well barrier and integrity failure (Table 3, Fig. 9) data in this study, we follow the definition of Thomas (2001) and shows substantial variability in the number of wells that have use ‘orphaned’ to describe wells where the operator is no longer experienced both categories of failure. This probably relates to the solvent. fact that the sizes of the datasets are variable; the included wells were drilled over a period of more than a century, using different 6. USA well designs and technology; were targeting unconventional and conventional hydrocarbons; and were drilled in diverse geological Thirty-two US states have reported data on orphaned oil and gas settings. The most recent dataset from the Marcellus Shale (Penn- wells (IOGCC, 2013). Fifteen of these states account for around sylvania, USA), which includes several thousand wells, has some of 320,000 orphaned wells in total, with w53,000 of these wells the lowest well barrier and failure rates (Fig. 9). In Table 3 we have targeted for plugging (Table 4). The states vary greatly in how they 246 R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254

Table 3 Compilation of published statistics on well barrier and well integrity failure, including information on well age, number of wells included in study, well location, and terminology used to describe nature of well barrier or integrity failures.

Country Location No. Wells studied % Wells with barrier Additional information Published source failure or well integrity failure

USA ONSHORE Operational wells in the >50 75 Well Integrity failures. Leakage based Chillingar and Endres (2005) Santa Fe Springs Oilfield (discovered on the ‘observation of gas bubbles w1921), California, USA seeping to the surface along well casing’. USA ONSHORE Ann Mag Field, South Texas, 18 61 Wells drilled 1998e2011. Well barrier Yuan et al. (2013) USA (wells drilled 1998e2011) failures mainly in shale zones. USA OFFSHORE Gulf of Mexico (wells drilled 15,500 43 Wells drilled w1973e2003. Barrier Brufato et al. (2003) w1973e2003) failure. 26.2% in surface casing. Offshore OFFSHORE Norway, 8 Companies, 193 38 Wells drilled 1970e2011. Well integrity Vignes (2011) Norway Abandoned Wells (wells drilled 1970 and barrier failure. 2 wells with likely e2011) leak to surface. China ONSHORE Kenxi Resevoir, China (dates 160 31.3 Well barrier failure Peng et al. (2007) unknown) China ONSHORE Gudao Resevoir, China (wells 3461 30.4 Wells drilled 1978e1999. Barrier failure Peng et al. (2007) drilled 1978e1999) in oil-bearing layer. Offshore OFFSHORE Norway, 8 Fields (dates 217 25 Wells monitored 1998e2007. Well Randhol and Carlsen (2007) Norway unknown) integrity and barrier failure. 32% leaks occurred at well head. Canada ONSHORE Saskatchewan, Canada (dates 435 22 Wells monitored 1987e1993. Well Erno and Schmitz (1996) unknown) integrity failure: SCVF and GM Offshore OFFSHORE Internal Audit, Location 711 20 Barrier failure Nilsen (2007) Norway Unknown (dates unknown) Offshore OFFSHORE Norway, 12 Offshore 406 18 Wells drilled 1977e2006. Well integrity Vignes and Aadnøy (2010) Norway Facilities (wells drilled 1977e2006) and barrier failure. 1% had well head failure. China ONSHORE Daqing Field, China (wells 6860 16.3 Wells drilled w1980e1999. Barrier Zhongxiao et al. (2000) drilled w1980e1999) failure Bahrain ONSHORE Bahrain (wells drilled 1932 750 13.1 Wells drilled 1932e2004. Failure of Sivakumar and Janahi (2004) e2004) surface casing with some leaks to surface Netherlands ONSHORE Netherlands (dates 31 13 Barrier failure Vignes (2011) unknown) UK OFFSHORE UK Continental Shelf (dates 6137 10 Well integrity and barrier failure. Burton (2005) unknown) USA ONSHORE Marcellus Shale, 8030 6.26 Well reports 2005e2013. Well integrity This study Pennsylvania, USA (wells drilled 1958 and barrier failure. 1.27% leak to e2013) surface. China ONSHORE Gunan Reservoir, China 132 6.1 Barrier failure Peng et al. (2007) (dates unknown) USA ONSHORE Nationwide Gas Storage 6953 6.1 Wells drilled <1965e1988. Well Marlow, 1989 Facilities (<1965e1988) integrity and barrier failure. China ONSHORE Hetan Reservoir, China 128 5.5 Barrier failure Peng et al. (2007) (dates unknown) USA ONSHORE Marcellus Shale, 4602 4.8 Wells drilled 2010e2012. Well barrier Ingraffea (2012) Pennsylvania, USA (wells drilled 2010 and integrity failure. e2012) Canada ONSHORE Alberta, Canada (wells drilled 316,439 4.6 Wells drilled 1910e2004. Monitored Watson and Bachu (2009) 1910e2004) 1970e2004. Well integrity failure: SCVF and GM Indonesia ON/OFFSHORE Malacca Strait (wells 164 4.3 Wells drilled w1980e2010. Both well Calosa and Sadarta (2010) drilledw1980e2004) integrity and barrier failures. Further 41.4% of wells identified as high risk of failure. USA ONSHORE Pennsylvania, USA (wells 6466 3.4 Wells drilled 2005e 2012. Well integrity Vidic et al. (2013) drilled 2008e2013) and barrier issues. Leak to surface in 0.24% wells. China ONSHORE Kenli Resevoir, China (dates 173 2.9 Barrier failure Peng et al. (2007) unknown) USA ONSHORE Marcellus Shale, 3533 2.58 Wells drilled 2008e2011. Well integrity Considine et al. (2013) Pennsylvania, USA (wells drilled 2008 and barrier failure e2011) USA ONSHORE Nationwide CCS/Natural Gas 470 1.9 Well integrity failure. Described as IPCC (2005) Storage Facilities (dates unknown) significant gas loss. R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254 247 treat wells for which they have no data. Two decades ago, the US merged. In the USA such wells are termed orphaned wells. Where EPA estimated that there were at least 1.2 million abandoned oil the company that drilled the well no longer exists, or has been and gas wells in the United States (EPA, 1987); more than 200,000 taken over or merged (up to 53% of UK wells), liability for any well of these wells appear to be unplugged (EPA, 1987). integrity failures that lead to pollution is unclear; in some cases it As the first state to produce oil commercially in the USA, may be that of the landowner. Even if a chain of ownership through Pennsylvania illustrates the difficulty in characterizing abandoned acquisition of prior licensees can be identified, the position is likely and orphaned wells. The state has seen around 325,000 to 400,000 to be more complex as the legal mechanism used for the acquisition oil and gas wells drilled since 1859. As of 2010, the Pennsylvania may not be known. In some instances, it is possible that a company Department of Environmental Protection (DEP) reported 8823 oil was purchased for its assets and the liabilities were left with the and gas wells targeted for plugging (IOGCC 2013). The PA DEP also original entity. reported more than 100,000 orphaned wells, but the precise loca- As a case study, one of the 2152 wells listed by DECC was tion and depth of most of these was unidentified. The number of examined (Fig. 11). Drilled in Sunderland in 2002, the well targeted orphaned wells in Pennsylvania is probably closer to 180,000, being coal mine gas. In February 2014 the company that drilled the well the difference between the conservative estimate of w325,000 was contacted to confirm the status of the well as either abandoned wells drilled in the state and the w140,000 wells listed in the PA or temporarily abandoned (suspended). No gas had been produced DEP database. These wells are mostly a legacy of the first 75e100 due to elevated water levels and the well was temporarily aban- years of oil and gas drilling, before record keeping was common- doned (suspended) in 2002, pending transfer of ownership to the place. In fact, the earliest regulations on well plugging were Coal Authority, for water level monitoring or abandonment. The designed to stop water entering hydrocarbon wells, particularly surrounding land has since been acquired by developers and is during floods, rather than to isolate oil and gas from the currently (February 2014) the site of a new residential housing environment. estate. As of February 2014, the well is now being abandoned Lost wells represent a different classification to abandoned or (DECC, pers. comm.). orphaned wells. States in the USA report that somewhere between 828,000 and 1,060,000 oil and gas wells were drilled prior to a formal regulatory system, most of which have no information available in state databases (IOGCC, 2008). A New York state report in 1994 estimated that, of the 61,000 oil and gas wells drilled to that date, no records existed for 30,000 of them; Bishop (2013) referred to these as ‘forgotten’ rather than abandoned or orphaned wells. The growing number of unplugged wells in New York State illustrates the difficulty of keeping remediation levels commensu- rate with the number of wells being drilled and abandoned (Bishop, 2013). Up to 2010, a total of w75,000 oil and gas wells had been drilled in the state. Eleven thousand wells were still active at that time, leaving 64,000 ‘abandoned’ wells (after Bishop, 2013). Of these, 15,900 had been plugged but 48,000 remained unplugged; thus only 25% of the abandoned wells in 2010 had been plugged, down from 27% in 1994. More importantly, the number of unplugged wells had grown by 13,000 since 1994, when 35,000 such wells existed (Bishop, 2013). This demonstrates that, in at least some regions, the plugging of abandoned wells is not keeping pace with the rate at which wells are being abandoned. Some states have aggressive programmes for plugging abandoned oil and gas wells. Texas has one of the most ambitious, having plugged 41,000 wells between 1991 and 2009 at a cost of w$80 million (IOGCC, 2008). Overall, US states spent w$319 million in recent decades to plug and remediate w72,000 oil and gas wells, at an average cost of w$4500 per well. Based on that unit cost, plugging 150,000 more wells would require $668 million, and plugging all 320,000 wells estimated in Table 4 would cost $1.43 billion. In 2009, the combined balance available in all US state funds for plugging wells was w$2.8 million, many orders of magnitude less than that required to finish the job (IOGCC, 2008).

7. UK

In the UK a total of 2152 hydrocarbon wells were drilled onshore between 1902 and 2013, with a peak in drilling activity during World War II (Fig. 10). Approximately 1000 were drilled by companies that still exist. Approximately 1050 were drilled by companies that were subject to takeovers or mergers. For example, 543 wells were drilled by the D’Arcy company, mainly between 1941 ’ and 1961 and D Arcy is no longer operating. Figure 8. (a) UK map showing locations of wells active in 1999 and crude oil dis- We estimate that between 50 and 100 of the 2152 wells were charges (b) Coincidence of pollution reports with well pads in the Wytch Farm area, drilled by companies that no longer exist and were not bought or southern England. 248 R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254

Many wells have been drilled in areas where there are highly present on the UKOGL map (5 wells); or (c) the wells were listed as productive aquifers (Fig. 12a) and there is a good spatial corre- ‘offshore’ in the DECC onshore well database (9 wells). spondence between potential shale reservoirs and highly produc- The remaining 2024 wells were categorised as follows: tive aquifers (Fig. 12b). In the USA, many shale gas wells have also been drilled where there are active aquifers (King and King, 2013). a. Cleared area of land present, consistent with site being used as well pad; machinery present and site apparently in use; 7.1. Surface identiﬁcation of wells in the UK b. Indications that well had once been present on site, but clearly not active. A surface identiﬁcation study of the 2152 UK onshore hydro- c. No well pad or machinery visible; no indication that well had carbon wells was carried out. 128 wells were not included because: ever been present on site; (a) the wells were younger than the available satellite imagery and so could not be located using this method (114 wells); (b) the wells Of the well sites included in our study (Table 5), 33.7% were were listed in the onshore well database (DECC, 2013) but were not clearly visible (i.e. the well pad and associated equipment could be

Figure 9. Graph of percentage of well barrier and integrity failures reported in 25 different studies around the world, with drilling dates and number of wells in each study. R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254 249

Table 4 abandonment and the cessation of dewatering, groundwater Estimated numbers of orphaned oil and gas wells for each U.S. state reporting at rebound occurs over 10e20 years and has the potential to least 1000 orphaned wells (IOGCC, 2008). Thirty-two of 50 states reported data on contaminate overlying aquifers. This process is driven by the hy- orphaned wells. draulic head in the coal workings exceeding that of the overlying State Orphaned oil or gas wells Orphaned wells targeted aquifer (Younger et al., 2002). In northern England, cessation of by state for plugging pumping for mine dewatering in part of the Durham Coalﬁeld led to Pennsylvania 180,000 8823 pollution of the overlying Magnesian Limestone aquifer, used for New York 44,600 4600 public water supply. As a consequence, this led to the aquifer failing Kansas 30,000 6500 Kentucky 14,880 12,800 an EU Water Framework Directive (WFD) environmental objective Oklahoma 12,000 1685 for groundwater quality (Neymeyer et al., 2007). More broadly, the Ohio 9500 524 2009 River Basin Management Plans, required as part of the Texas 7323 7323 implementation of the EU WFD, reported that 34 out of 304 Tennessee 4053 53 ‘ ’ West Virginia 3999 1385 groundwater bodies in England and Wales had failed good status Illinois 3766 3766 environmental objectives due to groundwater pollution by rising Indiana 3000 756 waters following mine abandonment (including coal and metal Louisiana 2793 2793 mines). In some areas, abandoned mine workings also liberate Missouri 2000 2000 methane, and emissions from abandoned UK coal mines were South Dakota 1288 NA w 3 California 1000 181 estimated to be 14 million m of methane in 2008 (UNFCCC, 2010). Total 320,202 53,189

8.2. Geothermal energy seen; Fig. 13a), 5.5% showed evidence of prior on-site drilling ac- Environmental concerns linked to the exploitation of tivity without the current presence of drilling production, drilling geothermal energy include the mobilisation of contaminants from equipment or a well head (Fig. 13b), and 65.2% were not visible the surrounding rock that could lead to the contamination of (Fig. 13c). For 1.1% of sites it was unclear as to whether a well pad aquifers by geothermal fluids. In the Balcova Geothermal Field in existed. These sites mainly comprise industrial locations where it Turkey, there has been thermal and chemical contamination of the could not be determined visually whether the infrastructure pre- overlying aquifer by elements such as arsenic, antimony and boron. sent was related to a well site. It is likely that the reason that 65.2% Aksoy et al. (2009) recommended that regular inspection and of wells are not visible is that UK regulations state that, after maintenance of geothermal wells should be carried out. abandonment, the well should be sealed and cut and the land Summers et al. (1980) characterised geothermal fluids and reclaimed. investigated the possible sources of well barrier and integrity failure and the potential for contamination. Based on their analysis, 8. Discussion they proposed a methodological framework for identifying groundwater contamination from geothermal energy de- To provide context for the statistics on well barrier failure velopments. Possible sources of well barrier and integrity failure of reviewed above, comparative data are reported from other indus- geothermal wells include loading from the surrounding rock for- trial processes, primarily mining in the UK and geothermal energy mation, mechanical damage during well development, corrosion abstraction. The number of wells that may be required to produce and scaling from geothermal fluids, thermal stress, metal fatigue shale gas is also considered. and failure, and expansion of entrapped fluids (Southon, 2005). The mixing of deep geothermal fluids with shallow groundwa- 8.1. Coal mining ters can occur via natural mechanisms, such as natural upward fluid convection along fault lines (e.g. within the Larderello geothermal There are estimated to be w250,000 lost mining shafts in the UK field, Italy; Bellani et al., 2004), and by anthropogenic activities, (Chambers et al., 2007) and many coal exploration boreholes. including uncontrolled discharges to surface waters, faulty injec- During mine operation, the potential for cross-contamination be- tion procedures (e.g. Los Azufres, Mexico: Birkle and Merkel, 2000), tween mined coal horizons and overlying potable aquifers is rela- and accelerated upward seepage from failed casings within wells tively low due to the fact that mine workings are dewatered (often and boreholes. Casing failures related to inconsistencies in casing at a regional scale, comprising several interconnected pits) to cementation have been cited as one common cause of failure facilitate access by the workforce. However, following mine (Snyder, 1979). The major failures of several geothermal wells on

Figure 10. Graph showing number of hydrocarbon wells drilled in UK per year. 250 R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254

Figure 11. Case study of gas exploration well abandonment in Sunderland, UK: (a) Map of the UK; (b) location of Sunderland; (c) location of new housing estate; (d) photograph of temporarily abandoned (suspended) mine gas exploration borehole on building site of new housing estate (Grid Ref. 438260 557420). Well was completed in 2002 to a depth of 465 m. the island of Milos, Greece, were attributed to thermal stresses on comparison, it has been calculated (Gluyas et al., unpublished data) the well casing that were exacerbated by poor cementation (Chiotis that conventional gas wells in the Rotliegend, which is a gas- and Vrellis, 1995). There is little published literature on failure rates bearing sandstone reservoir in the Southern North Sea, have EURs of geothermal wells, and failure rates are expected to vary due to of between 1 and 100 times more gas per well. the wide range of geological settings from which geothermal energy can be exploited, with volcanically active regions carrying 8.4. Shale exploitation and water contamination higher levels of risk than more tectonically quiescent regions. As shale reservoirs have very low permeability compared to 8.3. Number of wells for shale gas exploitation conventional sandstone or carbonate reservoirs (typically between 3.9 10 6 and 9.63 10 4 mD: Yang and Aplin, 2007), fluid The number of wells that could be drilled to exploit shale gas in movement through and from shales is likely to be extremely slow. Europe depends on various factors, including geological conditions, Therefore the potential for shales at depth to be the source of social acceptance and economics. Based on data from shale gas pollutants in the near-surface environment under natural condi- plays in the USA, the estimated ultimate recovery (EUR) of a shale tions is low. Geological timescales would be required for significant gas well varies from 1.4 BCF (0.0392 BCM) to 5.9 BCF (0.165 BCM) quantities of hydrocarbons to migrate from a shale reservoir that (Table 6; Baihly et al., 2010). If similar recoveries are assumed for has not been artificially hydraulically fractured. wells in European shale plays, between 169 and 714 wells would be The drilling of wells to access gas-bearing shales requires the required for every 1 TCF (0.028 TCM) of total production. In penetration of geological formations close to the surface that will R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254 251

Figure 12. (a) Map of UK showing location of onshore wells drilled for exploration or production and productive aquifers. (b) Map of UK showing location of potential shale gas and Ó oil reservoirs and productive aquifers. Aquifer base map reproduced with the permission of the British Geological Survey. NERC. All rights Reserved.

often contain freshwater. Where there is sufficient permeability Evidence from conventional hydrocarbon fields shows that hy- and storage capacity, these formations will form aquifers (Fig. 12) draulic fracturing due to the injection of fluids can, in very excep- that may be exploited for drinking water or industrial uses, such as tional circumstances, lead to fracture propagation to the surface or agriculture. Even where aquifers are not currently utilised, they near-surface, if it takes place at relatively shallow depths. In the have the potential to be, and therefore require protection. Consid- Tordis Field of offshore Norway, for example, the average rate of eration also needs to be given to protecting groundwater that water injection was 7000 m3 day 1 for 5.5 months (total supports base flow to rivers and wetland ecosystems. Protection is volume ¼ w1,115,000 m3). Hydraulic fractures propagated from a achieved through preventing hazardous pollutants or limiting non- depth of w900 m to the surface through Cenozoic (Tertiary) strata. hazardous pollutants entering groundwater (European The volume of fluid used in these operations, however, was more Commission, 2000). Of the 2152 hydrocarbon wells drilled in the than 120 times greater than that typically used for hydraulic frac- UK, the well heads of 428 (20%) of these are located above highly ture stages in shale gas reservoirs and took place over a time period productive aquifers (likely to be exploited for public water supply) hundreds of times longer. There are several factors in shale fracking and a further 535 (25%) are above moderately productive aquifers, operations, including the relatively low volumes of fluid and the likely to be exploited for both public and private drinking water short pumping times that make the upward propagation of very tall supplies (Fig. 12a). fractures unlikely (Davies et al., 2012). To date, water contamination caused directly by the upward propagation of hydraulic frac- Table 5 tures remains unproven (Davies, 2011), although the possibility Statistics on visibility and accessibility of UK onshore wells. cannot be totally ruled out. As argued by Davies (2011) and Jackson et al. (2013), poor well Number of wells Percentage (out of total of 2024 integrity is a far more likely cause of elevated concentrations of included in study) thermogenic methane in shallow groundwater and water supplies

Visible 682 33.70 than pathways induced solely by hydraulic fracturing. Examples of Not visible 1319 65.17 leaks in shale gas wells have been reported and ﬁnes imposed Unclear 23 1.14 (Roberts, 2010). Number of Wells Percentage on Visible Sites On active sites 626 30.93 8.5. Implications and recommendations Non-active/former/ 112 5.53 derelict sites As with our study, King and King (2013) addressed statistics on Urban 159 7.86 well barrier and integrity failure. They compared the data with that Urban/built over 182 8.99 of other polluting activities in the USA, such as storage tanks, septic 252 R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254

barrier failure. Improved monitoring is crucial for a better understanding of chances of hydrocarbon well barrier and integrity failure and the impact of this. There are examples of good practice. The DEP database for Pennsylvania, USA, was used by Considine et al. (2013) to carry out a detailed breakdown of the types of well infringements and their severity. The Alberta Energy Resources Board (ERCB) database of well integrity failure for 316,439 wells reported by industry dating back to 1910 is not in the public domain, but the data summary is available (Watson and Bachu, 2009). In Alberta wells are checked for well integrity and barrier failure within 60 days of the drill rig being removed (Watson and Bachu, 2009). In the UK there have been a small number of reported pollution incidents associated with active wells and none with inactive abandoned wells. This could therefore indicate that pollution is not a common event, but one should bear in mind that monitoring of abandoned wells does not take place in the UK (or any other jurisdiction that we know of) and less visible pollutants such as methane leaks are unlikely to be reported. It is possible that well integrity failure may be more widespread than the presently limited data show. Surveying the soils above abandoned well sites would help establish if this is the case. In terms of monitoring, abandoned wells could be checked 2e3 months after cement plugging for sustained casing pressure and gas migration. If the well has no evidence for barrier or integrity failure, it could be cut and buried as per regulations. Soils above well sites could be monitored every 5 years for emissions that are above a pre- determined statutory level. As there are 2152 wells in UK at present, only 430 would need to be checked each year. Monitoring could be intensiﬁed or scaled down based upon the results of the ﬁrst complete survey. Monitoring a proportion of future abandoned shale gas and oil wells should also be feasible. A mechanism may need to be established in the UK and/or Europe to fund repairs on orphaned wells, and an ownership or liability survey of existing wells would be timely.

9. Conclusions

Well barrier and integrity failure is a reasonably well- documented problem for conventional hydrocarbon extraction and the data we report show that it is an important issue for unconventional gas wells as well. It is apparent, however, that few data exist in the public domain for the failure rates of onshore wells in Europe. It is also unclear which of the datasets used in this study will be the most appropriate analogues for well barrier and integ- Figure 13. Examples of wells locations taken from UKOGL imaged with Google Earth, illustrating range of surface manifestations of UK onshore wells: (a) cleared area of rity failure rates at shale gas production sites in the UK and Europe. land with appearance of being a maintained well pad; (b) cleared area of land with Only 2 wells in the UK have recorded well integrity failure (Hatfield appearance of poorly maintained and potentially disused well pad. (c) Location of well Blowout and Singleton Oil Field) but this figure is based only on drilling in which no well pad or machinery is visible. data that were publicly available or accessible through UK Envi- ronment Agency and only out of the minority of UK wells which tanks and landfills, and made the point that the number of reports were active. To the best of our knowledge and in line with other of pollution from oil and gas wells was insignificant in comparison. jurisdictions (e.g. Alberta, Canada) abandoned wells in the UK are Nevertheless, for the more than 4 million wells drilled in Australia, sealed with cement, cut below the surface and buried, but are not Austria, Bahrain, Brazil, Canada, Netherlands, Poland, UK and USA subsequently monitored. This number is therefore likely to be an alone, there is scarce published or online data on well integrity or underestimate of the actual number of wells that have experienced integrity failure. A much tighter constraint on the risks and impacts would be obtainable if systematic, long-term monitoring data for Table 6 both active and abandoned well sites were in the public domain. It Estimated Ultimate Recovery (EUR) for 5 shale gas provinces in the USA (from is likely that well barrier failure will occur in a small number of Baihly et al., 2010). wells and this could in some instances lead to some form of envi- Shale play EUR after 30 years (BCF-0.028 BCM) ronmental contamination. Furthermore, it is likely that, in the

Barnett 3.0 future, some wells in the UK and Europe will become orphaned. It is Fayetteville 1.4 important therefore that the appropriate ﬁnancial and monitoring Woodford 1.7 processes are in place, particularly after well abandonment, so that Haynesville 5.9 legacy issues associated with the drilling of wells for shale gas and Eagle Ford 3.8 oil are minimised. R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254 253

Table 7 Crude oil pollution incidents within 1 km of 143 well pads active in UK at start of year 2000.

Environmental impact

Event no. Date reported Lat. Lon. Cause Due to well integrity Air Land Water failure (Y/N)

981998 18/04/2012 51.19415 1.009848 Pipe Failure above ground N No Impact Minor No Impact 639443 08/12/2008 50.93129 0.74344026 Other Y No Impact No Impact Minor 685648 08/06/2009 50.92439 0.73782083 Other Y No Impact No Impact Minor 137932 19/02/2003 50.66674 2.0292232 Accidental spillage N No Impact No Impact No Impact 838199 14/11/2010 50.66655 2.0290391 Pipe failure below ground N Minor Minor No Impact 157014 09/05/2003 50.66737 2.0287566 Control system failure N No Impact No Impact Minor 138317 21/02/2003 50.67028 2.0162917 Pipe failure above ground N No Impact No Impact No Impact 428461 18/08/2006 50.67125 1.9866881 Pipe failure above ground N No Impact No Impact No Impact 8177 07/06/2001 50.68239 1.9825378 Pipe failure below ground N No Impact Minor Minor

Acknowledgements Abandoned Wells. Conference Proceedings, Fourth Annual Conference on Car- bon Capture and Sequestration DOE/NETL, May 2e5. Chambers, J.E., Wilkinson, P.B., Weller, A.L., Meldrum, P.I., Ogilvy, R.D., Caunt, S., We thank Chris Green (GFrac Technologies) and an anonymous 2007. Mineshaft imaging using surface and crosshole 3D electrical resistivity reviewer for their reviews which helped improve the paper. Dr Paul tomography: a case history from East Pennine Coalfield, UK. J. Appl. Geophys. Choate (Choate Technology Services Ltd.) and Dr Will Fleckenstein 62 (4), 324e337. Cheung, K., Klassen, P., Mayer, B., Goodarzi, F., Aravena, R., 2010. Major ion and (Colorado School of Mines) are also thanked for reading and com- isotope geochemistry of fluids and gases from coalbed methane and shallow menting on the manuscript. This research was carried out as part of groundwater wells in Alberta, Canada. Appl. Geochem. 25, 1307e1329. the ReFINE (Researching Fracking in Europe) consortium led by Chillingar, G., Endres, B., 2005. Environmental hazards posed by the Los Angeles Basin urban oilfields: an historical perspective of lessons learned. Environ. Geol. Durham University and funded by the Natural Environment 47, 302e317. Research Council (UK), Total, Shell and Chevron. We thank Alkane Chiotis, E., Vrellis, G., 1995. Analysis of casing failures of deep geothermal wells in Energy, BP, Chevron, Department of Energy and Climate Change, Greece. Geothermics 24, 695e705. Chivvis, R.W., Julian, J.Y., Cary, D.N., 2009. Pressure Activated Sealant Economically Humbly Grove Energy Ltd., IGas, Perenco, Shell, Total for comments. Repairs Casing Leaks on Prudhoe Bay Wells. SPE 120978. The US National Science Foundation (EAR-#1249255) funded some Considine, T.J., Watson, R.W., Considine, N.B., Martin, J.P., 2013. Environmental of the US analyses and the Environment Agency (UK) is thanked for regulation and compliance of Marcellus shale gas drilling. Environ. Geosci. 20, e providing pollution incident data. We are grateful to the Durham 1 16. Crook, R., Kulakofsky, D., Griffith, J., 2003. Tailor lightweight slurry designs to well University Faculty of Science Ethics Committee and the UK conditions and productions plans. World Oil 224 (10). Research Integrity Office for their time in providing advice on Davies, R.J., 2011. Methane contamination of drinking water caused by hydraulic research ethics. We thank the ReFINE Independent Science Board fracturing remains unproven. Proc. Natl. Acad. Sci. 108, E871. Davies, R.J., Mathias, S.A., Moss, J., Hustoft, J., Newport, L., 2012. Hydraulic fractures: (http://www.refine.org.uk/how-we-work/independent-science- how far can they go? Mar. Pet. Geol. 37, 1e6. board) for spending time prioritising the research projects under- Davies, R.J., Foulger, G.R., Bindley, A., Styles, P., 2013. Induced seismicity and hy- e taken by ReFINE. This paper is published with the permission of the draulic fracturing for the recovery of hydrocarbons. Mar. Pet. Geol. 45, 171 185. DECC, 2013. Oil and Gas: Onshore Exploration and Production. Retrieved from. Executive Director of the British Geological Survey and the results https://www.gov.uk/government/uploads/system/uploads/attachment_data/ and conclusions are solely those of the authors. file/200131/Landwells17_May2013.xlsx. Department of Environmental Protection (DEP): Bureau of Oil and Gas Manage- ment, 2000. Pennsylvania’s Plan for Addressing Problem Abandoned Wells and References Orphaned Wells. DEP. 550-0800-001. Dusseault, M., Gray, M., Nawrocki, P., 2000. Why Oilwells Leak: Cement Behavior Aksoy, N., S¸ims¸ ek, C., Gunduz, O., 2009. Groundwater contamination mechanism in and Long-term Consequences. SPE 64733. a geothermal field: a case study of Balcova, Turkey. J. Contam. Hydrol. 103, 13e EIA. Crude Oil and Natural Gas Exploratory and Development Wells. Retrieved 2012, 28. from Energy Information Administration: http://www.eia.gov/dnav/ng/ng_enr_ Baihly, J., Altman, R., Malpani, R., Luo, F., 2010. Shale gas production decline trend wellend_s1_m.htm. comparison over time and basins. Soc. Pet. Eng.. http://dx.doi.org/10.2118/ Energy Resources Conservation Board, Statistical Reports. Retrieved 2013 from 135555-MS. http://www.aer.ca/data-and-publications/statistical-reports/st37. BDEP. Well Data. Retrieved 2013, from National Agency for Oil, Natural Gas and EPA, 1987. Management of Wastes from the Exploration, Development, and Pro- fi Biofuels (ANP), Brazil: http://www.bdep.gov.br/?id¼191. duction of Crude Oil, Natural Gas, and Geothermal Energy. Of ce of Solid Waste Beckstrom, J.A., Boyer, D.G., 1993. Aquifer-Protection considerations of coalbed and Emergency Response, Washington, D.C. http://nepis.epa.gov/Exe/ZyPDF. ¼ methane development in the San Juan Basin. SPE Form. Eval. 8, 71e79. http:// cgi?Dockey 20012D4P.PDF. dx.doi.org/10.2118/21841-PA. Erno, B., Schmitz, R., 1996. Measurements of soil Gas migration around oil and Gas e Bellani, S., Brogi, A., Lazzarotto, A., Liotta, D., Ranalli, G., 2004. Heat flow, deep Wells in the Lloydminster area. J. Can. Pet. Technol. 35, 37 45. temperatures and extensional structures in the Larderello Geothermal Field European Commission, 2000. Directive 2000/60/EC of the European Parliament and fi (Italy): constraints on geothermal fluid flow. J. Volcanol. Geotherm. Res. 132, of the Council establishing a framework for the community action in the eld of 15e29. water policy Directive 2000/60/EC of the European Parliament and of the fi Birkle, P., Merkel, B., 2000. Environmental impact by spill of geothermal fluids at the Council establishing a framework for the Community action in the eld of water geothermal field of Los Azufres, Michoacán, Mexico. Water, Air Soil. Pollut. 124, policy. Off. J. Eur. Commun. L 327/1 - L 327/72. 371e410. Evans, B.L., Sailor, R.P., Santiago, E., 2003. Well Abandonment in the Los Angeles Bishop, R.E., 2013. Historical analysis of oil and gas well plugging in New York: is the Basin. SPE 83443. regulatory system working? New. Solut. 23, 103e116. Florida Department of Environmental Protection, Oil and Gas Data Maps. Retrieved fl Brufatto, C., Cochran, J., Power, L.C.D., El-Zeghaty, S.Z.A.A., Fraboulet, B., Griffin, T., 2013 from http://www.dep.state. .us/water/mines/oil_gas/data.htm. Munk, S., Justus, F., Levine, J., Montgomery, C., Murphy, D., Pfeiffer, J., Geological Society of Alabama, State Oil and Gas Board Databases. Retrieved 2013 Pornpoch, T., Rishmani, L., 2003. From mud to cement e building gas wells. from http://www.gsa.state.al.us/ogb/db_main.html. Schlumb. Oil Field Rev. 15, 62e76. Geological Survey of the Netherlands. Wells. Retrieved 2013, from NL Oil and Gas Bureau de Recherches Géologiques et Minières, French National Oil and Gas Data. Portal: http://www.nlog.nl/nlog/requestData/nlogp/allBor/queryForm? ¼ Accessed 2013 at: www.beph.net/html/beph_sig.htm?idp¼&; menu act. f¼2MST&map¼&x¼500000&y¼2100000&r¼500&langue¼GB. Geoscience Australia. (n.d.). Petroleum Wells. Retrieved 2013, from Australian e Calosa, W.J., Sadarta, B., 2010. Well integrity issues in Malacca Strait contract area. Government Geoscience Australia: http://dbforms.ga.gov.au/www/npm.well. Soc. Pet. Eng.. http://dx.doi.org/10.2118/129083-MS. search. Celia, M.A., Bachu, S., Nordbotten, J.M., Kavetski, D., Gasda, S.E., 2005. Modeling Hesson, B.H., Glinzak, M., 2000. California Division of Oil, Gas and Geothermal Critical Leakage Pathways in a Risk Assessment Framework: Representation of Resources: Idle Well Management Program. SPE 62576. 254 R.J. Davies et al. / Marine and Petroleum Geology 56 (2014) 239e254

Hesson, B.H., 2013. California Department of Conservation/Division of Oil, Gas, and Sivakumar, V.C., Janahi, I., 2004. Salvage of Casing Leak Wells on Artificial Lift in a Geothermal Resources: Orphan Well Program. SPE 165340. Mature Oil Field. SPE. 88747. Ingraffea, A., 2012. Fluid Migration Mechanisms Due to Faulty Well Design and/or Smith, D., 1976. Cementing. Millet the Printer Inc, Dallas. Construction: an Overview and Recent Experiences in the Pennsylvania and Summers, K., Gherini, S., Chen, C., 1980. Methodology to Evaluate the Potential for Marcellus Play. http://www.psehealthyenergy.org/site/view/1057. Ground Water Contamination from Geothermal Fluid Releases, vol. 1. Industrial IOGCC (Interstate Oil and Gas Compact Commission), 2013. Groundwork: Orphaned Environmental Research Laboratory, Office of Research and Development, U.S. Wells Program, Oklahoma City, OK, USA (accessed October 2013). http:// Environmental Protection Agency. groundwork.iogcc.org/topics-index/orphaned-wells/state-progress. Selley, R.C., 1992. Petroleum seepages and impregnations in Great Britain. Mar. Pet. IOGCC (Interstate Oil and Gas Compact Commission), 2008. Protecting Our Coun- Geol. 9, 225e328. try’s Resources: the States’ Case. Orphaned Well Plugging Initiative, Oklahoma Selley, R.C., 2012. UK shale gas: the story so far. Mar. Pet. Geol. 31, 100e109. City, OK, USA. Southon, J.N., 2005. Geothermal well design, construction and failures. In: Pro- IPCC, 2005. Special Report on Carbon Dioxide Capture and Storage, pp. 195e277. ceedings World Geothermal Congress, pp. 24e29. IPCC, 2013. Climate Change 2013; the Physical Science Basis, 714. Snyder, R., 1979. Geothermal well completions: a critical review of downhole Jackson, R.B., Vengosh, A., Darrah, T., Warner, N.R., Down, A., Poreda, R.J., problems and specialized technology needs. In: SPE Annual Technical Confer- Osborn, S.G., Zhao, K., Karr, J.D., 2013. Increased stray gas abundance in a subset ence and Exhibition. of drinking water wells near Marcellus shale gas extraction. Proc. Natl. Acad. Sci. Thomas, K.T., 2001. Produce or Plug? a Summary of Idle and Orphan Well Statistics 110, 11250e11255. and Regulatory Approaches. SPE 68695. Johns, J.E., Aloisio, F., Mayfield, D.R., 2011. Well Integrity Analysis in Gulf of Mexico The Royal Society and The Royal Academy of Engineering, 2012. Shale Gas Extrac- Wells Using Passive Ultrasonic Leak Detection Method. SPE 142076. tion in the UK: a Review of Hydraulic Fracturing. Available. http://royalsociety. King, G.E., King, D.E., 2013. Environmental Risk Arising from Well-construction org/uploadedFiles/Royal_Society_Content/policy/projects/shale-gas/2012-06- Failure e Differences between Barried and Well Failure and Estimates of Fail- 28-Shale-gas.pdf. ure Frequency across Common Well Types, Locations and Well Age. SPE 16142. Torbergsen, H.E., Haga, H.B., Sangesland, S., Aadnøy, B.S., Sæby, J., Johnsen, S., Marshall, M., Strahan, D., 2012. Total foresaw the North Sea gas leak. New. Sci. 214, Rausand, M., Lundeteigen, M.A., 2012. An Introduction to Well Integrity. Nor- 6e7. skOlje&Gass. Available. http://www.norskoljeoggass.no/en/Publica/HSE-and- Marlow, R., 1989. Cement Bonding Characteristics in Gas Wells. SPE 17121. operations/Introduction-to-well-integrity/. Miller, S.M., Wofsy, S.C., Michalak, A.M., Kort, E.A., Andrews, A.E., Biraud, S.C., Traynor, J.J., Sladen, C., 1997. Seepage in Vietnam e onshore and offshore examples. Dlugokencky, E.J., Eluskiewicz, J., Fisher, M.L., Janssens-Maenhout, G., Mar. Pet. Geol. 14, 345e362. Miller, B.R., Miller, J.B., Montzka, S.A., Nehrkorn, T., Sweeney, C., 2013. Anthro- UNFCCC, 2010. Annex I party GHG inventory submissions, united nations frame- pogenic emissions of methane in the United States. Proc. Natl. Acad. Sci. 110, work convention on climate change. 20018e20022. United Kingdom Onshore Geophysical Library (UKOGL), Interactive Map. Accessed Miyazaki, B., 2009. Well Integrity: an Overlooked Source of Risk and Liability for SepeOct 2013 from: http://maps.lynxinfo.co.uk/UKOGL_LIVEV2/main.html. Underground Natural Gas Storage. Lessons Learned from Incidents in the USA. Van Stempvoort, D., Maathuis, H., Jaworski, E., Mayer, B., Rich, K., 2005. Oxidation of In: Geological Society, London, Special Publications, vol. 313, pp. 163e172. fugitive methane in ground water linked to bacterial sulphate reduction. New York Department of Environmental Conservation, Oil and Gas Searchable Ground Water. 43, 187e199. Database. Retrieved 2013 from: http://www.dec.ny.gov/cfmx/extapps/GasOil/ Véron, J., 2005. The Alpine Molasse Basin e review of petroleum geology and search/wells/index.cfm. remaining potential. Bull. für Angew. Geol. 10, 75e86. Neymeyer, A., Williams, R.T., Younger, P.L., 2007. Migration of polluted mine water Vidic, R.D., Brantley, S.L., Vandenbossche, J.M., Yoxtheimer, D., Abad, J.D., 2013. in a public supply aquifer. Q. J. Eng. Geol. Hydrol. 40, 75e84. Impact of shale Gas development on regional water quality. Science 340, Nilsen, L.H., 2007. Brønnintegritet I Statoil og på norsk sokkel. Published at the NPF 6134. 20‘ Kristiansand Conference [Online]. Available. http://www.npf.no/. Vignes, B., Aadnøy, B.S., 2010. Well-integrity issues offshore Norway. SPE 112535. North Dakota Department of Health, Environmental Incident Reports. Accessed 20/ Vignes, B., 2011. Contribution to Well Integrity and Increased Focus on Well Barriers 2/2014 at: http://www.ndhealth.gov/ehs/spills/. from a Life Cycle Aspect (PhD thesis). University of Stavanger. North Dakota Oil and Gas Division, Well Search. (accessed 20.02.14.) at: https:// Ward, J., Chan, A., Ramsay, B., 2003. The Hatfield Moors and Hatfield west Gas www.dmr.nd.gov/oilgas/findwellsvw.asp. (Storage) Fields, south Yorkshire. Geol. Soc. Lond. Memoirs 20, 903e910. Norway Offshore Continental Shelf Data Access Portal, Public Well Statistics. Watson, T., Bachu, S., 2009. Evaluation of the Potential for Gas and CO2 Leakage Retrieved 2013 from http://www.landmarkvspace.com/nocs/wells/stats.aspx. Along Wellbores. SPE 106817. Osborn, S.G., Vengosh, A., Warner, N.R., Jackson, R.B., 2011. Methane contamination West Virginia Department of Environmental Protection, Oil and Gas Production of drinking water accompanying gas-well drilling and hydraulic fracturing. Data. Retrieved 2013 from http://www.dep.wv.gov/oil-and-gas/databaseinfo/ Proc. Natl. Acad. Sci. 108, 8172e8176. Pages/default.aspx. Peng, S., Fu, J., Zhang, J., 2007. Borehole casing failure analysis in unconsolidated Xu, Y., Yang, Q., Li, Q., Chen, B., 2006. The Oil Well Casing’s Anticorrosion and formations: a case study. J. Pet. Sci. Eng. 59, 226e238. Control Technology of Changqing Oil Field. SPE 104445. Polish Geological Institute, Borehole Database, Accessed November 2013 from Yang, Y., Aplin, A.C., 2007. Permeability and petrophysical properties of 30 natural http://otworywiertnicze.pgi.gov.pl/ mudstones. J. Geophys. Res. 112 (B03206), 18. Randhol, P., Carlsen, I.M., 2007. Presentation Assessment of Sustained Well Integrity Younger, P.L., Banwart, S.A., Hedin, R.S., 2002. Mine Water: Hydrology, Pollution, on the Norwegian Continental Shelf. SINTEF Petroleum Research [Online], Remediation, vol. 5. Springer. Available: http://www.co2captureandstorage.info/docs/wellbore/Wellbore% Yuan, Z., Schubert, J., Esteban, U.C., Chantose, P., Teodoriu, C., 2013. Casing Failure 20Presentations/4th%20Mtg/01.pdf. Mechanism and Characterization Under HPHT Conditions in South Texas. SPE RRC. Well Information. Retrieved 2013, from Rail Road Commission of Texas: http:// 16704. www.rrc.state.tx.us/data/wells/index.php. Zhongxiao, L., Yumin, X., Chuanan, Z., 2000. The Repairing Technology of Driving Roberts, J.S., 2010. Testimony of J. Scott Roberts, Deputy Secretary for Mineral Re- Channel on Small Drifting-diameter’s Casing Damage in Daqing Oilfield. SPE sources Management Department of Environmental Protection Before the 60599. House Republican Policy Committee Thursday, May 20. 414 Impacts of ocean acidification on marine fauna and ecosystem processes

Victoria J. Fabry, Brad A. Seibel, Richard A. Feely, and James C. Orr

Fabry, V. J., Seibel, B. A., Feely, R. A., and Orr, J. C. 2008. Impacts of ocean acidiﬁcation on marine fauna and ecosystem processes. – ICES Journal of Marine Science, 65: 414–432.

Oceanic uptake of anthropogenic carbon dioxide (CO2) is altering the seawater chemistry of the world’s oceans with consequences for marine biota. Elevated partial pressure of CO2 (pCO2) is causing the calcium carbonate saturation horizon to shoal in many regions, particularly in high latitudes and regions that intersect with pronounced hypoxic zones. The ability of marine animals, most importantly pteropod molluscs, foraminifera, and some benthic invertebrates, to produce calcareous skeletal structures is directly affected by seawater CO2 chemistry. CO2 influences the physiology of marine organisms as well through acid-base imbalance and reduced oxygen transport capacity. The few studies at relevant pCO2 levels impede our ability to predict future impacts on foodweb dynamics and other ecosystem processes. Here we present new observations, review available data, and identify priorities for future research, based on regions, ecosystems, taxa, and physiological processes believed to be most vulnerable to ocean acidification. We conclude that ocean acidification and the synergistic impacts of other anthropogenic stressors provide great potential for widespread changes to marine ecosystems.

Keywords: anthropogenic CO2, calcification, ecosystem impacts, hypercapnia, ocean acidification, physiological effects, zooplankton. Received 11 July 2007; accepted 14 February 2008 V. J. Fabry: Department of Biological Sciences, California State University San Marcos, San Marcos, CA 92096–0001, USA. B. A. Seibel: Department of Biological Sciences, University of Rhode Island, Kingston RI 02881, USA. R. A. Feely: Pacific Marine Environmental Laboratory, NOAA, Seattle, WA 98115–6349, USA. J. C. Orr: Marine Environmental Laboratories, International Atomic Energy Agency, Monaco MC-98000, Monaco. Correspondence to V. J. Fabry: tel: þ1 760 7504113; fax: þ1 760 7503440; e-mail: [email protected].

Introduction calciﬁcation rates, and via disturbance to acid–base (metabolic)

Rising atmospheric carbon dioxide (CO2) concentration is causing physiology. Recent work indicates that the oceanic uptake of global warming and ocean acidiﬁcation (Caldeira and Wickett, anthropogenic CO2 and the concomitant changes in seawater 2003, 2005; Feely et al., 2004; Orr et al., 2005), which increasingly chemistry have adverse consequences for many calcifying organ- are recognized as important drivers of change in biological systems isms, and may result in changes to biodiversity, trophic inter- (Lovejoy and Hannah, 2005). For at least 650 000 years prior to the actions, and other ecosystem processes (Royal Society, 2005; industrial revolution, atmospheric CO2 concentrations varied Kleypas et al., 2006). Most research has focused on tropical coral between 180 and 300 ppmv (Siegenthaler et al., 2005). As a reefs and planktonic coccolithophores. Little information is avail- result of human activity, today’s atmospheric CO2 concentration able for other important taxa, for processes other than calciﬁca- is 380 ppmv and currently is rising at a rate of 0.5% year21 tion, or for potential ecosystem-level consequences emerging (Forster et al., 2007), which is 100 times faster than any from the oceanic pCO2 levels that are predicted to occur over change during the past 650 000 years (Royal Society, 2005; the next 100 years. Here we discuss the present and projected Siegenthaler et al., 2005). Approximately one-third of the changes in ocean carbonate chemistry, and assess their impacts anthropogenic CO2 produced in the past 200 years has been on pelagic and benthic marine fauna and ecosystem processes. taken up by the oceans (Sabine et al., 2004). The global ocean inven- We exclude corals from this discussion, but note that excellent tory of anthropogenic carbon was 118+19 Pg C in 2004 (Sabine et recent reviews on this topic exist (Langdon and Atkinson, 2005; al., 2004), which can be adjusted upwards to 140 Pg C in 2005 based Guinotte et al., 2006; Kleypas and Langdon, 2006). We highlight on Denman et al. (2007, Table 7.1). Without this ocean sink, the many of the gaps in our knowledge and identify critical questions anthropogenic change in atmospheric CO2 concentration would for future research. be 55% higher than the observed change from 280 to 380 ppmv (Sabine et al., 2004). Although oceanic uptake of anthropogenic The ocean’s inorganic carbon system: present and CO2 will lessen the extent of global warming, the direct effect of future changes CO2 on ocean chemistry may affect marine biota profoundly. The CO2 –carbonate system in seawater Elevated partial pressure of CO2 (pCO2) in seawater (also The inorganic carbon system is one of the most important chemi- known as hypercapnia) can impact marine organisms both via cal equilibria in the ocean and is largely responsible for controlling decreased calcium carbonate (CaCO3) saturation, which affects the pH of seawater. Dissolved inorganic carbon (DIC) exists in

# 2008 International Council for the Exploration of the Sea. Published by Oxford Journals. All rights reserved. For Permissions, please email: [email protected] Impacts of ocean acidiﬁcation on marine fauna and ecosystem processes 415

Figure 2. Atmospheric CO2 concentration projected under the IS92a “business-as-usual” IS92a CO emissions scenario, bounded by Figure 1. Concentrations of carbon species (in units of mmol kg21), 2 the most and least conservative SRES scenarios, B1 and A1F1, pH values, and aragonite and calcite saturation states of average respectively, and projected global average surface seawater pH surface seawater for pCO concentrations (ppmv) during glacial, 2 (modified from Meehl et al. (2007)). preindustrial, present day, two times pre-industrial CO2, and three times pre-industrial CO2. Changes in the inorganic carbon system were computed by assuming equilibrium with atmospheric CO2, 21 21 commonly secreted by marine organisms: assuming PO4 = 0.5 mmol l and Si = 4.8 mmol l , and using the carbonic acid dissolution constants of Mehrbach et al. (1973) as V ¼½ þ2½ 2= ; ð Þ refitted by Dickson and Millero (1987). pH is based on the seawater Ca CO3 Ksp 1 scale. The last column shows the changes from the pre-industrial levels to three times atmospheric CO2 (modified from Feely et al. where the calcium concentration is estimated from the salinity, (2004) and Kleypas et al. (2006)). 22 and [CO3 ] is calculated from DIC and total alkalinity (TA) measurements (Feely et al., 2004). Increasing CO2 concentrations 2 in the atmosphere, and thus in the surface ocean, will continue to seawater in three major forms: bicarbonate ion (HCO3 ), carbon- 22 decrease the [CO22] in the upper ocean, thereby lowering CaCO ate ion (CO3 ), and aqueous carbon dioxide (CO2(aq)), which 3 3 saturation levels by means of the reaction: here also includes carbonic acid (H2CO3). At a pH of 8.2, 2 88% of the carbon is in the form of HCO3 , 11% in the form 22 2 of CO3 , and only 0.5% of the carbon is in the form of dissolved CO2 þ CO3 þ H2O ¼ 2HCO3 : ð2Þ CO2. When CO2 dissolves in seawater, H2CO3 is formed (Figure

1). Most of the H2CO3 quickly dissociates into a hydrogen ion In regions where Varag or Vcal is .1.0, the formation of shells + 2 22 (H ) and HCO3 . A hydrogen ion can then react with a CO3 to and skeletons is favoured. For values ,1.0, seawater is corrosive to form bicarbonate. Therefore, the net effect of adding CO2 to sea- CaCO3 and, in the absence of protective mechanisms (e.g. Corliss 2 + water is to increase the concentrations of H2CO3, HCO3 , and H , and Honjo, 1981; Isaji, 1995), dissolution will begin. Saturation 22 and decrease the concentration of CO3 and lower pH states are generally highest in the tropics and lowest in the high + (pH = 2log[H ]). These reactions are fully reversible, and the latitudes, because the solubility of CaCO3 increases with decreas- basic thermodynamics of these reactions in seawater are well ing temperature and increasing pressure. Consequently, there is known (Millero et al., 2002). The atmospheric CO2 value today significant shoaling of the aragonite saturation horizons in the is 100 ppmv greater than the pre-industrial value (280 ppmv), Pacific, north of 408N, at the equator, and 108N, especially and the average surface ocean pH has dropped by 0.1 unit, towards the east, because of the higher DIC concentrations + which is about a 30% increase in [H ]. Under the IPCC emission relative to TA at shallower depths. These patterns result from scenarios (Houghton et al., 2001), average surface ocean pH could enhanced upwelling that brings deeper waters rich in nutrients decrease by 0.3–0.4 pH units from the pre-industrial values by the and DIC to the upper ocean (Figure 3) and supports high end of this century (Caldeira and Wickett, 2005; Figure 2). animal biomass. As one moves north, the aragonite saturation depth shoals from 1000 m near 308S to 300 m at the equator. Moving farther north, it deepens to 550 m near 308N, then shoals to 100 m north of 508N (Figure 3). In the North Present and future changes in carbonate Pacific, the upward migration of the aragonite saturation saturation horizon from anthropogenic CO2 uptake is currently 1–2 m 21 The reaction of CO2 with seawater reduces the availability of car- year (Feely et al., 2006). bonate ions that are necessary for marine calcifying organisms, Orr et al. (2005) developed model scenarios of future changes such as corals, molluscs, echinoderms, and crustaceans, to in surface ocean carbonate chemistry as a function of changes in produce their CaCO3 shells and skeletons. The extent to which atmospheric CO2, using the IPCC IS92a “business-as-usual” such organisms are affected depends largely upon the CaCO3 sat- CO2 emission scenario, with the median projection of DIC uration state (V), which is the product of the concentrations of changes from 13 ocean models that participated in the 2+ 22 Ca and CO3 , divided by the apparent stoichiometric solubility OCMIP-2 project. Based on their model outputs and global product (Ksp*) for either aragonite or calcite, two types of CaCO3 gridded data (Key et al., 2004), we plotted the projected aragonite 416 V. J. Fabry et al.

Figure 3. Distribution of (a) aragonite saturation; (b) partial pressure of CO2 seawater (pCO2); and (c) dissolved oxygen along the March 2006 P16 N transect along 1528W in the North Pacific. saturation state of the surface oceans for the years 1765, 1994, reach 560 and 900 ppmv, respectively. In the slightly warmer 2050, and 2100 (Figure 4). The model results indicate that, by surface waters of the subpolar North Pacific, aragonite and the time atmospheric CO2 reaches 780 ppmv near the end of calcite undersaturation will occur later, when pCO2 reaches 740 this century under the IPCC IS92a “business-as-usual” CO2 emis- and 1040 ppmv, respectively. The cold waters of the Arctic 22 sion scenario, portions of the Subarctic North Pacific and all of the Ocean are also naturally low in CO3 concentration. Continuing Southern Ocean south of 608S will become undersaturated with research is evaluating how the Arctic Ocean’s changes in carbonate respect to aragonite (Orr et al., 2005). At that point, the global chemistry during the 21st century will differ from those in the 22 average surface water CO3 concentration and aragonite and Southern Ocean (Orr et al., 2006). The warm surface waters of calcite saturation state will be nearly half of what they are today. the tropics and subtropics will not become undersaturated with The aragonite saturation horizons would also shoal from its respect to aragonite or calcite over the range of these projected present average depth of 730 m to the surface in the Southern conditions although, in some regions associated with upwelling, Ocean, from 2600 to 115 m in the North Atlantic, and from shoaling aragonite saturation horizons now impinge on the 140 m to the surface in parts of the North Pacific (Orr et al., depth ranges of many pelagic animals (Feely et al., 2004). 2005). In the cold, high-latitude surface waters typical of polar Priority areas for ocean acidification research are therefore and subpolar regions of the Southern Ocean, aragonite and high-latitude regions, which are projected to experience the calcite undersaturation will occur when seawater pCO2 values greatest changes in carbonate chemistry over decadal to century Impacts of ocean acidification on marine fauna and ecosystem processes 417

Effects of elevated pCO2 on calcification The secretion of CaCO3 skeletal structures is widespread across animal phyla, and evolved independently and repeatedly over geologic time since the late Precambrian period (Knoll, 2003). Protection is one probable advantage of possessing a calcareous skeleton. Additionally, various other biotic and abiotic factors have probably contributed to selection for CaCO3 hard parts in diverse groups of fauna at different times in evolutionary history. Most calcifying organisms investigated to date demonstrate reduced calcification in response to increased pCO2 and decreased 22 [CO3 ], CaCO3 saturation state, and pH (e.g. Gattuso et al., 1998; Langdon et al., 2000, 2003; Riebesell et al., 2000). The majority of work has tested warm-water corals and coccolithophorid algae (Royal Society, 2005; Kleypas et al., 2006). Evidence suggests that the calcification rate in corals is controlled by the CaCO3 saturation state (Gattuso et al., 1998; Langdon et al., 2000, 2003; Marubini et al., 2001, 2003; Leclercq et al., 2002; Ohde and Hossain, 2004; Langdon and Atkinson, 2005; Schneider and Erez, 2006; Silverman et al., 2007), rather than pH or another 2+ parameter of the seawater CO2 system. Because the [Ca ] in the ocean today is approximately constant (depending predominantly 22 on salinity), changes in the [CO3 ] are reflected directly as 2+ changes in the CaCO3 saturation state. In contrast, high [Ca ] in the Cretaceous (Horita et al., 2002) allowed planktonic calcifiers to flourish and large chalk deposits to accumulate (Bown et al., 22 2004), despite CO3 concentrations that were only 25% of the current value (Tyrrell and Zeebe, 2004; Ridgwell and Zeebe, 2005). This observation supports the idea that the CaCO3 2+ 22 saturation state [proportional to the product of Ca and CO3 concentrations as shown in Equation (1)] is the key component of the seawater carbonate system that controls calcification rates.

Holoplankton

The major planktonic CaCO3 producers are the coccolithophores, foraminifera, and euthecosomatous pteropods. These three groups of calcifiers account for nearly all the export flux of CaCO3 from the upper ocean to the deep sea. Planktonic foraminifera and coccolithophores secrete tests or shells made of calcite, whereas pteropods form shells made of aragonite, a metastable polymorph of CaCO3, which is 50% more soluble in seawater than calcite (Mucci, 1983). On a global basis, coccolithophores and foraminifera are thought to produce the majority of pelagic CaCO3 (Schiebel, 2002), while the labile aragonitic shells of pteropods account for a smaller fraction of the total CaCO Figure 4. Surface water aragonite saturation state (V ) for the 3 arag produced by planktonic organisms. The relative contributions of pre-industrial ocean (nominal year 1765), and years 1994, 2050, and 2100. Values for years 1765 and 1994 were computed from the global these three groups of calcifiers can vary substantially on regional gridded data product GLODAP (Key et al., 2004), whereas the and temporal scales, however. There are few concurrent saturation state for years 2050 and 2100 are the median of 13 ocean measurements of the abundances of all three groups, and estimates general circulation models forced under the IPCC’s IS92a of their contributions to global calcification rates are poorly “business-as-usual” CO2 emission scenario (Orr et al., 2005). constrained. Both planktonic foraminifera and euthecosomatous pteropods time-scales. Moreover, coastal regions, which can be greatly are widely distributed in the upper ocean, with highest species impacted by anthropogenic-driven ocean acidification (Doney diversity in tropical and subtropical regions. Shelled pteropods et al., 2007), as well as eutrophication leading to low oxygen can reach densities of 1000s to .10 000 of individuals m23 in zones, are not well-represented in global ocean–atmosphere high-latitude areas (e.g. Bathmann et al., 1991; Pane et al., 2004) coupled models, because of lack of data, complexities of nearshore and are important components of polar and subpolar ecosystems. circulation processes, and too-coarse model resolution. Given the Data are limited on the response of calcification in pteropods and importance of coastal areas to fisheries and other marine resources foraminifera to elevated pCO2 and decreased CaCO3 saturation and services, coastal ecosystems constitute another target region state. Currently, evidence is available for only two of the 50 where research is urgently needed. species of planktonic foraminifera and only one of the 34 418 V. J. Fabry et al. euthecosome species. Although these data suggest that both scenario, there was marked shell dissolution within 48 h (Feely groups reduce calcification in response to ocean acidification, et al., 2004; Orr et al., 2005). In additional experiments, C. pyrami- the small number of species tested precludes the identification of data were placed in 1 l jars, and 45Ca was added to measure shell general trends. Species-specific responses are likely, and it is poss- calcification rates. The jars were then closed and incubated at ible that the calcification rates of some species may not be sensitive 108C for 4–48 h. At the start of this experiment, the seawater to elevated pCO2, as has been found in coccolithophores (Riebesell Varag was 2.4. Forty-eight hours later, respiratory CO2 from et al., 2000; Langer et al., 2006). the actively swimming pteropods had gradually forced the arago- In laboratory experiments with the symbiont-bearing plank- nite saturation state to drop below 1. The 45Ca uptake experiments tonic foraminifera Orbulina universa and Globigerinoides sacculifer, reveal progressively reduced calcification rates in concert with 22 shell mass decreased in response to reduced CO3 concentration decreased aragonite saturation state that resulted from the and calcite saturation state, even though the seawater was supersa- accumulation of metabolic CO2 produced by animals during the turated with respect to calcite (Spero et al., 1997; Bijma et al., 1999, experiment (Figure 5). After 36–48 h, most of the 45Ca that had 2002). When grown in seawater chemistry equivalent to pCO2 of been incorporated into shells had dissolved back into solution. 560 and 740 ppmv, shell mass in these species declined by 4–8 Hence, by 36 h, net dissolution exceeded net calcification, and 6–14%, respectively, compared with the pre-industrial although animals were alive and actively swimming. Scanning elec- pCO2 value. Evidence from microelectrode and culture exper- tron microscopic photographs of experimental shells confirm that 22 iments suggests that elevated pH and CO3 concentration in the dissolution occurred along the leading edge of the shell by 48 h micro-environment immediately adjacent to the shell are critical during incubation in closed jars, but no dissolution was visible in promoting shell growth (Spero and Lea, 1993; Rink et al., in control shells incubated in open jars, where the aragonite satur- 1998; Ko¨hler-Rink and Ku¨hl, 2005). When O. universa is grown ation remained .1 (Figure 6). Based on pteropod oxygen con- under high light, the pH of the micro-environment surrounding sumption rates (Smith and Teal, 1973), it is unlikely that the test can be increased up to 8.8 (0.5 units above ambient sea- animals were oxygen-stressed during these experiments, but water) as a result of CO2 removal during symbiont photosynthesis. future experiments should explore this possibility. Additional In contrast, O. universa grown in the dark has a near-shell micro- experiments are needed to measure calcification directly in euthe- environment pH of 7.9, owing to respiratory CO2 release cosomatous pteropods and foraminifera, as a function of CaCO3 (Ko¨hler-Rink and Ku¨hl, 2005). Because of the large effect of sym- saturation state. It will also be necessary to study the interactive biont photosynthesis on seawater CO2 chemistry at the shell effects of CaCO3 saturation state, temperature, and nutrition for surface, it may be that the impact of ocean acidification on adult multiple species and life stages of these calcareous holoplankton. symbiont-bearing foraminifera will occur primarily during night As pCO2 rises and the CaCO3 saturation state of surface ocean calcification. It is unknown whether foraminifera that do not decreases, euthecosomatous pteropods and foraminifera may possess photosynthetic symbionts are more susceptible to secrete under-calcified or thinner structures (Bijma et al., 1999). 22 reduced CO3 concentration and calcite saturation state than Should the habitat of these organisms approach CaCO3 undersa- those species with symbionts. Similarly, the post-zygote, prolocu- turation, first with respect to aragonite, then with respect to lar ontogenetic stage of foraminifera, during which calcification is calcite, the question of whether net rates of calcification can still weak or absent and symbiotic algae have not yet been acquired exceed dissolution will likely depend on the degree of CaCO3 (Brummer et al., 1987), may be particularly vulnerable to elevated undersaturation and the duration that animals are exposed to pCO2. Because it is currently impossible to maintain the prolocu- undersaturated waters. As yet, no decreases in calcification have lar stage in the laboratory, however, this hypothesis awaits testing. been documented in field populations of either group. However, A positive correlation between foraminiferal shell mass and 22 ambient [CO3 ] is observed in the sedimentary record as a response to known glacial–interglacial changes in atmospheric CO2 of the past 50 000 years (Barker and Elderfield, 2002). However, Barker and Elderfield (2002) demonstrate an increase in Globigerinoides bulloides shell mass from 11 to 19 mg for a 22 21 small change in [CO3 ] from 210 to 250 mmol kg , whereas Bijma et al. (2002) report a much greater increase, from 40 to 22 60 mg in shell mass for O. universa for a change in [CO3 ] from 200 to 600 mmol kg21, and 10 mg increase in shell mass for 22 21 G. sacculifer for a change in [CO3 ] from 100 to 600 mmol kg . Although it appears that culture experiments may underestimate 22 foraminiferal response to altered [CO3 ] compared with the change observed in paleo-reconstructions over glacial-to-interglacial time-scales, temperature and food supply also strongly affect foraminiferal calcification (cf. Barker and Elderfield, 2002; Bijma et al., 2002). Hence, future increases in sea surface temperatures Figure 5. Net calcification (mmol CaCO h21) as a function of shell could lead to higher foraminiferal growth rates. 3 CaCO3 concentration for the Subarctic Pacific euthecosomatous Owing to their highly soluble aragonitic shells, pteropods may pteropod Clio pyramidata. Different sizes of pteropods were be particularly sensitive to ocean acidification. When live Clio pyr- incubated at 108C in closed 1 l jars for 4–48 h. Net calcification rates amidata collected in the Subarctic Pacific were exposed to a level of decreased with time, as the aragonite saturation state of seawater aragonite undersaturation similar to that projected for Southern was progressively reduced owing to respiratory CO2 in sample Ocean surface waters in year 2100 under the IS92a emissions containers. Impacts of ocean acidification on marine fauna and ecosystem processes 419

Figure 6. SEM photographs of the shell of the pteropod Clio pyramidata. (a) Whole shell of an animal incubated for 48 h in a closed container, wherein animal respiration forced the aragonite saturation state ,1; (b) magniﬁed portion of the leading edge of the shell section shown in (a), revealing dissolution of aragonitic rods; (c) magniﬁed leading edge of the shell of a control animal incubated in seawater that remained supersaturated with aragonite for the duration of the experiment.

baseline data on their present-day vertical distributions and calci- benthic calcifying fauna are prominent in nearshore communities fication rates are insufficient to detect possible changes that may and are economically and/or ecologically important. For example, result from ocean acidification. bivalves, such as mussels and oysters, have high commercial value The capacity of euthecosome and foraminifera species to adapt as fisheries and are also important as ecosystem engineers in to progressively acidified ocean waters is not known, but may be coastal areas, providing habitat and other services for a rich diver- related to species’ generation time. If unabated CO2 emissions sity of organisms (Gutie´rrez et al., 2003). Recent work suggests that continue and surface waters of the Southern Ocean and portions benthic adult molluscs and echinoderms are sensitive to changes in of the Subarctic Pacific become undersaturated with respect to ara- seawater carbonate chemistry. In response to an elevated pCO2 gonite by 2100 as projected (Orr et al., 2005), then shelled ptero- level projected to occur under the IS92a emissions scenario pods in these regions would have only 50–150 generations to (740 ppmv in 2100), calcification rates in the mussel Mytilus adapt to corrosive seawater, given that high-latitude pteropods edulis and the Pacific oyster Crassostrea gigas decreased by 25 are thought to have generation times of 0.6–1.5 years and 10%, respectively (Gazeau et al., 2007). When grown for (Kobayashi, 1974; Bathmann et al., 1991; Dadon and de Cidre, more than six months in seawater bubbled with air containing 1992; Gannefors et al., 2005). Generation times for spinose 560 ppmv CO2, a decrease in shell growth was observed in the species of foraminifera are frequently linked with the lunar cycle edible snail Strombus luhuanus, and a reduction in wet weight such that they reproduce every 2–4 weeks; however, non-spinose was reported in both this snail and two species of sea urchins species probably have longer reproductive cycles (Hemleben et al., (Shirayama and Thorton, 2005). 1989). Shorter generation time affords increased opportunities for Early calcifying stages of benthic molluscs and echinoids microevolutionary adaptation. demonstrate a strong response to increased seawater pCO2 and 22 In addition to euthecosomes and foraminifera, other holo- decreased pH, CO3 concentration, and CaCO3 saturation state. plankton calcify during part or all of their life cycles. These In the sea urchins Hemicentrotus pulcherrimum and Echinodetra include the heteropods, visual predators that are found in the epi- mathaei, fertilization success, developmental rates, and larval size pelagic zone of all tropical and subtropical oceans, but which are all decreased with increasing CO2 concentration (Kurihara and absent from high latitudes. Two of the three heteropod families Shirayama, 2004). Abnormal skeletalgenesis of the highly soluble possess aragonitic shells as adults, and species in the third family high-magnesium CaCO3 spicules in urchin larvae was also cast off their larval shells at metamorphosis. Gymnosomes, the observed. Green et al. (2004) found that newly settled juveniles highly specialized predators of eutheocosomatous pteropods, of the hard-shell clam Mercenaria mercenaria revealed substantial possess a large veliger shell, presumed to be aragonite, which is shell dissolution and increased mortality when they were intro- also cast off at metamorphosis. Similarly, pseudothecosomatous duced to surface sediments that were undersaturated with pteropods possess a veliger shell that is discarded at metamorpho- respect to aragonite (Varag 0.3), a level that is typical of near- sis to the gelatinous adult. shore, organic-rich surficial sediments. Within two weeks of settle- ment, the CaCO3 shells were completely dissolved, leaving only the Benthic invertebrates organic matrix of the shell. Nine multicellular invertebrate phyla have benthic representatives The mineralogy and calcification mechanisms of mollusc and with CaCO3 skeletal hard parts (Lowenstam and Weiner, 1989). echinoid larval stages may render them particularly sensitive to These taxa secrete CaCO3 in the form of aragonite, calcite, high- ocean acidification. Although adult gastropods and bivalves magnesium calcite (.5 mole % MgCO3), amorphous CaCO3, secrete aragonite, calcite, or both phases in a diverse array of struc- or a mixture of these CaCO3 phases. Amorphous CaCO3 is less tural patterns, Weiss et al. (2002) suggest that veliger shells of gas- stable than the crystalline phases of CaCO3, and the seawater solu- tropods and bivalves all contain aragonite in similar crystalline bility of high-magnesium calcite is similar to or greater than that ultrastructures, and hence, the mollusc larval shell is highly con- of aragonite (Walter and Morse, 1985; Bischoff et al., 1987). Many served in evolution. Moreover, recent work using infrared 420 V. J. Fabry et al. spectrometry and Raman imaging spectroscopy reveals that larvae is supported by recent work with warm-water reef organisms in of the clam M. mercenaria (adult shell is aragonitic), as well as that which a decreased aragonite saturation state induced the transition of the oyster C. gigas (adult shell is nearly entirely calcitic), form from a CaCO3-dominated system to one dominated by organic amorphous CaCO3 as a transient precursor to aragonite (Weiss algae (Kuffner et al., 2008). et al., 2002). Similarly, during the embryonic development of two species of sea urchins, an amorphous CaCO3 precursor trans- Increased pCO2 and other physiological processes forms to calcite during spicule formation (Beniash et al., 1997; Raz In addition to calcification, a number of other physiological et al., 2003). Because this unstable, transient, amorphous CaCO3 is indices appear to correlate with the capacity for acid–base toler- more soluble than the crystalline minerals of aragonite or calcite, ance, and new data are emerging that test the survival, growth, biomineralization processes that occur during the embryonic development, metabolism, and pH balance of organisms under and larval development of sea urchins, and in gastropod and elevated pCO2. Prior to 1995, most studies used CO2 values that bivalve molluscs, may be particularly vulnerable to ocean were well above what is expected in the future ocean and from con- acidification. venient marine animal models, in order to reveal the fundamental CaCO3 skeletal elements are also present in species of other mechanisms associated with acid–base regulation. Studies con- benthic invertebrates, such as crustaceans, cnidarians, sponges, ducted more recently have tested the physiological response to bryozoans, annelids, brachiopods, and tunicates. Apart from very high CO2 levels as would be associated with purposeful warm-water corals, nothing is known about the effect of elevated sequestration of CO2 in the ocean. There is now a critical need ambient pCO2 on calcification rates in these taxa. Some of these to test the physiological consequences of ocean acidification at animals may use shell dissolution to support acid-based regulation lower pCO2 levels, such as those that are projected to occur over at high internal pCO2, as has been observed in mussels the next century. (Michaelidis et al., 2005) and other organisms. Mechanisms to deal with hypercapnia

Other non-skeletal, calciﬁed secretions of marine fauna When pCO2 levels increase in seawater, dissolved CO2 more readily

In addition to using CaCO3 for strengthening skeletal structures, diffuses across animal surfaces and equilibrates in both intra- and the use of calcium minerals in gravity sensory organs is widespread extracellular spaces. Internal levels rise until a new value is reached among ocean fauna. In the many zooplankton and benthic invert- that is sufficient to restore CO2 excretion against the elevated ebrates that possess statoliths, statocysts, or statocontia, the environmental level. As in seawater, CO2 reacts with internal + mineralogy is indeterminate or is reported to be amorphous body fluids causing H to increase and, therefore, pH to decrease. Ca–Mg–phosphate or gypsum (Lowenstam and Weiner, 1989). Mechanisms available to counteract this acidification are limited In squid and fish, however, the statoliths and otoliths are com- and relatively conserved across animal phyla. The mechanisms posed of aragonite. Gravity sensory organs can have additional are the same as those evolved to deal with metabolically produced functions in some organisms. For example, in planktonic gymno- CO2 and hydrogen ions. They include (i) passive buffering of some snails, statocysts also are actively involved in the motor intra- and extracellular fluids; (ii) transport and exchange of rel- neural programme that underlies search movements for prey evant ions; (iii) transport of CO2 in the blood in those species during hunting behaviour (Levi et al., 2004). Whether mineraliz- that have respiratory pigments; (iv) metabolic suppression to ation of the various types of gravity receptors would be affected wait out periods of elevated CO2 (e.g. Somero, 1985; Truchot, by the changing carbonate chemistry of seawater and, if so, how 1987; Cameron, 1989; Walsh and Milligan, 1989; Hand, 1991; that might impact overall fitness of the organism, are questions Heisler, 1993; Guppy and Withers, 1999; Clairborne et al., 2002; that have not been investigated. Presumably, potential impacts Seibel and Walsh, 2003; Po¨rtner et al., 2004). Those species would depend on the ability of the organisms to regulate the adapted to environments with steep CO2 gradients, such as hydro- acid–base balance in the tissues surrounding those structures. thermal vents or stagnant tide pools, and those species with high Other carbonate secretions of marine fauna include gastroliths, capacity for metabolic production of CO2 have evolved greater mineralized structures formed in the lining of the cardiac capacities for buffering, ion exchange, and CO2 transport (Seibel stomachs of some decapods that serve as storage sites for and Walsh, 2001, 2003). Whether such elevated capacity translates calcium during moult intervals; gastroliths can be calcite, amor- into greater tolerance of chronic ocean acidification remains to phous CaCO3, or calcium phosphate (cf. Lowenstam and be seen. Weiner, 1989). Widespread among marine fish is the intestinal secretion of calcium- and magnesium-rich carbonate complexes, Buffering capacity which are then excreted via the rectum; this process appears to When concentrations are elevated, CO2 readily crosses biological play a critical role in osmoregulation (Walsh et al., 1991; Grosell, membranes and enters the blood and intracellular spaces. 2006; Taylor and Grosell, 2006). Passive buffering is the only mechanism immediately available to limit pH changes within the body. Locomotory muscles of active animals, such as epipelagic fish and cephalopods, have high activi- Consequences of reduced calcification ties of anaerobic metabolic enzymes and, consequently, have high The effects of chronic exposure to increased CO2 on calcifiers, as capacity for buffering pH changes associated with anaerobically well as the long-term implications of reduced calcification rates fuelled burst locomotion (Castellini and Somero, 1981; Seibel within individual species and their ecological communities, are et al., 1997). Organisms with low buffering capacity will experience unknown. Calcification probably serves multiple functions in car- greater fluctuations in intracellular pH during hypercapnia than bonate producers. Decreased calcification would presumably com- others with higher capacity. For example, an increase in seawater promise the fitness of these organisms and could shift the pCO2 sufficient to lower intracellular pH by 0.2 in a sluggish competitive advantage towards non-calcifiers. Such a consequence benthic fish may cause only a 0.02 pH unit drop in an active Impacts of ocean acidification on marine fauna and ecosystem processes 421 epipelagic fish such as tuna (Seibel and Walsh, 2003). Buffering of extracellular fluid is, in some cases, also provided by formation of bicarbonate from dissolution of CaCO3 stores or exoskeletons (shells or tests) as discussed below.

Ion transport In the longer term (hours to days), compensation of acid–base imbalance relies on the ability to transport acid–base equivalent ions across cell membranes. The CO2 that is produced in the cells during routine metabolism is typically hydrated to form bicarbonate and H+, a reaction catalyzed by the enzyme carbonic anhydrase. These hydrogen ions are then buffered in the intracellular space as discussed above, while the bicarbonate is transported out of the cell in exchange for Cl2 via ion transport proteins. Species with ineffective ion transport capacities are poorly equipped for acid–base regulation (Heisler, 1989; Walsh and Milligan, 1989). Low rates of metabolism typically correlate with lower concentrations of ion transport proteins such as Na+/K+ and H+-ATPases (Gibbs and Somero, 1990), suggesting reduced Figure 7. Carbonic anhydrase activity (DpH min g wet mass21)in capacities of acid–base balance. In many cases, the gelatinous gas exchange tissue of benthic macrofauna in habitats with large tissues (e.g. mesoglea) found in diverse zooplankton are distinct fluctuations in environmental pCO2. Bivalves (hatched bars), worms from the muscle tissues that are most metabolically active (grey bars), and crustaceans (open bars). Each bar represents the (Thuesen et al., 2005a, b). Therefore, the active tissues of gelati- mean of several species (note the x-axis is on a log scale). Species are nous animals may have greater CO2 tolerance than would be esti- grouped as ionic regulators (euryhaline species including those from mated, based on rates of whole-animal metabolism. Similarly, hydrothermal vents), shallow-living ionic conformers (those from species not exposed to environmental fluctuations in CO2 may stable shallow-water habitats), and deep-sea conformers. Data for also be ill-equipped to handle ocean acidification. For example, worms and bivalves are from Kochevar and Childress (1996). Data for species from hydrothermal vents have high activities of carbonic crustaceans are from Henry (1984) and unpublished observations anhydrase relative to species from shallower environments. Even (J. Company, pers. comm.). lower still are carbonic anhydrase activities of benthic deep-sea species, far removed from venting water, that experience very little fluctuation in environmental CO2 (Figure 7). and Spicer (1995), demonstrating that sea urchins are unable to Compensation of acidosis via adjustments in ionic composition compensate acidosis resulting from short-term emersion and appears to be a trade-off that is not likely sustainable on longer hypoxia, respectively. time-scales, such as that associated with anthropogenic increases Similarly, the mussel M. edulis compensated both short- and in seawater pCO2. Nevertheless, a survey of species that are more long-term exposure to 1% CO2 ( 10 000 ppmv) by dissolution + or less able to regulate the pH of their internal fluids may be of its shell as indicated by increased Ca levels (Lindinger et al., informative. 1984; Michaelidis et al., 2005). Not surprisingly, long-term exposure resulted in reduced growth and metabolism. A Acid–base regulation via bicarbonate accumulation deep-sea crab measured recently by Pane and Barry (2007) failed A common component of pH compensation in animals is the to accumulate any bicarbonate or control haemolymph pH 2 intracellular accumulation of HCO3 (Walsh and Milligan, 1989; values over 24 h exposure to hypercapnia. This poor performance Po¨rtner and Reipschla¨ger, 1996) that drives an elevation in pH. was attributed to low rates of metabolism, stable environmental A “bicarbonate threshold” is hypothesized (Heisler, 1993; conditions, and reduced oxygen at depth that may have limited Po¨rtner and Reipschla¨ger, 1996), above which the capacity for ion exchange capacity. In short-term experiments, the sipunculid further compensation is limited. Although additional bicarbonate worm Sipunculus nudus demonstrated hypercapnic tolerance, accumulation will always further compensate reduced pH, there with only a 50% elevation in extracellular bicarbonate relative to may be an upper limit beyond which acid–base regulation control levels (Po¨rtner and Reipschla¨ger, 1996). The reduced begins to compromise ionic balance (Cameron and Iwama, metabolic rate observed in this species (40% of control values), 1987). Those species that can tolerate accumulation of bicarbonate under natural, short-term elevation of CO2, allows survival until to levels 4–10 times above control conditions appear generally well-aerated waters return with high tide. However, over the more tolerant of hypercapnia, whereas those with limited bicar- longer term (3–6 week), such metabolic suppression resulted in bonate accumulation may be more vulnerable. 100% mortality (Langenbuch and Po¨rtner, 2004). Miles et al. (2007) recently found incomplete compensation of Species that are more tolerant exhibit greater bicarbonate coelomic fluid pH, despite elevated bicarbonate levels under all accumulation and, consequently, compensate more completely CO2 exposures (pH 6.2–7.4), in an intertidal sea urchin. the acidosis caused by exposure to elevated CO2. For example, Dissolution of the high magnesium calcite test was inferred from exposure of the subtidal crab Necora puber to pCO2 elevations in coelomic Mg2+, and the authors suggested that 10 000 ppmv resulted in haemolymph bicarbonate concen- reductions in surface seawater pH below 7.5 would be severely det- trations more than four times the control levels, in part supplied rimental to this species, and probably other sea urchins as well. by shell dissolution (Spicer et al., 2007). Similarly, the crab These results are consistent with those of Burnett et al. (2002) Cancer magister fully compensated its haemolymph pH over 422 V. J. Fabry et al.

24 h by accumulating more than 12 mM bicarbonate (Pane and interacts with body fluids to produce hydrogen ions that bind to Barry, 2007). Fish appear most tolerant among marine animals. respiratory proteins, altering their affinity for oxygen. That is, The Mediterranean fish Sparus aurata was able to compensate CO2 production causes acidosis that promotes oxygen release at completely both blood plasma pH and intracellular pH in the the tissues, whereas CO2 excretion elevates pH and promotes face of 10 000 ppm pCO2 via elevations in bicarbonate to five oxygen binding at the gills. The sensitivity of oxygen binding to times the control levels. No mortality occurred after ten days of pH is expressed as the Bohr coefficient (DlogP50/DpH, where exposure (Michaelidis et al., 2007). Although feed intake was P50 is the pO2 required to achieve 50% oxygen saturation of the reduced at a seawater pH of 7.25, compensation of internal respiratory protein). The biotic and abiotic factors that contribute acid–base imbalance was accomplished in the sea bass to selective pressure for pH sensitivity are complex and not easily Dicentrarchus labrax via a fivefold elevation in plasma bicarbonate predicted, and the measurement of pH sensitivity and blood (Cecchini et al., 2001). oxygen binding depends on various ionic and organic modulators that vary tremendously from one study to another (Lallier and Mortality Truchot, 1989; Mangum, 1991). All else being equal, however, Mortality occurred in three fish species tested, including yellowtail greater pH sensitivity (a larger Bohr effect) may allow more com- and flounder, only at very high CO2 levels (.50 000 ppmv) after plete release of oxygen in support of high oxygen demand, or from 24 h exposure, and the authors concluded that fish mortality high-affinity respiratory proteins, such as those of species adapted caused by anthropogenic CO2 is never expected in marine to hypoxic environments (Childress and Seibel, 1998; Hourdez environments (Hayashi et al., 2004). Although we believe that and Weber, 2005). this statement is premature, marine fish do appear highly tolerant Po¨rtner and Reipschla¨ger (1996) predicted that species with of CO2 (Kikkawa et al., 2004, 2006). The hatchling stages of some high metabolic rates would be more severely impacted by ocean species appeared fairly sensitive to pH decreases on the order of 0.5 acidification because oxygen binding in their blood is more pH or greater, but high CO2 tolerance developed within a few days of sensitive. Epipelagic squid (e.g. Ommastrephidae, Gonatidae, hatching (Ishimatsu et al., 2004). The relative tolerance of fish and Loliginidae) are hypothesized to be most severely impacted by others may relate to high capacity for internal ion and acid–base the interference of CO2 with oxygen binding at the gills, because regulation via direct proton excretion (Ishimatsu et al., 2004) and their metabolic rates are higher than other aquatic animals an intracellular respiratory protein that results in a high oxygen- (Seibel, 2007; Seibel and Drazen, 2007). Furthermore, oxygen car- carrying capacity and substantial venous oxygen reserve. When rying capacity is constrained in squid relative to active fish compensation of pH fails, mortality of all marine animals increases (Po¨rtner, 1994). As a result, it is necessary for active squid to with the level of CO2 and the duration of exposure (Yamada and utilize all of the oxygen carried in the blood on each pass Ikeda, 1999; Hayashi et al., 2004; Watanabe et al., 2006; Table 1). through the body, even at rest, leaving no venous oxygen reserve. Unloading the entire oxygen store at the tissues requires Metabolic suppression extreme pH sensitivity (Bohr coefficient less than –1.0; Figure 9; If compensation of acid–base imbalance is not achieved, reduced Po¨rtner, 1994). One downside of this adaptation is that an increase pH and elevated pCO2 may depress metabolism in some species in CO2 in the environment will inhibit oxygen binding at the gills. (Hand, 1991; Po¨rtner and Reipschla¨ger, 1996; Guppy and Po¨rtner (1990, 1994) estimates that a reduction in environmental Withers, 1999; Figure 8). Metabolic suppression is considered an seawater pH by as little as 0.15 unit will reduce the scope for adaptive strategy for the survival of short-term hypercapnia and activity in the squid Illex illecebrosus. Recent work demonstrates hypoxia. During periods of environmental oxygen limitation, that elevated pCO2 (1000 ppmv) can create measurable many organisms are able to suppress ATP demand, thereby reductions in the oxygen consumption rate and scope for activity extending the duration of tolerance. In many cases, oxygen limit- of another ommastrephid squid Dosidicus gigas (Figure 8; R. Rosa ation is coincident with internal acid–base disturbance. Metabolic and B. Seibel, unpublished data). However, squid may be excep- suppression is not advantageous, however, under chronic tional both metabolically and in their sensitivity to low pH. We elevations of CO2 (e.g. S. nudus, as cited above; Langenbuch and review the pH sensitivity of oxygen binding in marine animals Po¨rtner, 2004). Metabolic suppression is typically achieved by and find no correlation with metabolic rate or environmental shutting down expensive processes. Chief among these is protein oxygen levels and find no obvious phylogenetic signal (Figure synthesis (Hand, 1991). Reduced protein synthesis, by definition, 9). For example, several metabolically active species have pH will reduce growth and reproductive potential. Although suppres- insensitive respiratory proteins (low Bohr coefficients), while sion of metabolism under short-term experimental conditions is a several others have high pH sensitivity despite low oxygen “sublethal” reversible process, reductions in growth and reproduc- demand. tive output will effectively diminish the survival of the species on longer time-scales. Predicting population and ecosystem responses Table 1 lists the responses of a variety of animals to low pH–high Blood-oxygen binding pCO2 conditions. The data indicate that foraminifera, molluscs, Many marine animals rely on specialized respiratory proteins to and echinoderms demonstrate reduced calcification and some- bind oxygen at respiratory surfaces (e.g. gills) and deliver it to times dissolution of CaCO3 skeletal structures when exposed to 22 the tissues for cellular metabolism. A high gradient from the elevated pCO2 and decreasing pH and CO3 concentration. environment to the blood promotes oxygen binding at the gills, Fertilization rates and early development are also negatively and the gradient from the blood to the metabolizing tissues pro- impacted by high CO2 conditions in a number of groups such as motes its release. However, these gradients alone are often sea urchins, molluscs, and copepods. Significantly, the data are inadequate to facilitate sufficient oxygen saturation and unloading limited with regard to the number of species tested at of the respiratory proteins. CO2 produced by cellular metabolism climate-relevant pCO2 levels. For example, although teleost fish Impacts of ocean acidification on marine fauna and ecosystem processes 423

Table 1. Examples of the response of marine fauna to ocean acidiﬁcation.

Species Description CO2 system Sensitivity Reference parameters Planktonic foraminifera Orbulina universa Symbiont-bearing pCO2 560–780 ppmv 8–14% reduction in shell mass Spero et al. (1997); Bijma et al. (1999, 2002) Globigerinoides Symbiont-bearing pCO2 560–780 ppmv 4–8% reduction in shell mass Bijma et al. (1999, 2002) sacculifer Cnidaria Scyphozoa Jellyfish North Sea seawater Increase in frequency as measured Attrill et al. (2007) Hydrozoa pH drop from 8.3 to by CPR from 1958 to 2000 8.1 Mollusca Clio pyramidata Shelled pteropod Varag ,1 Shell dissolution Feely et al. (2004); Orr et al. (2005); this work Haliotis laevigata Greenlip abalone pH 7.78; pH 7.39 5% and 50% growth reductions Harris et al. (1999). Haliotis rubra Blacklip abalone pH 7.93; pH 7.37 5% and 50% growth reductions Mytilus edulis Mussel pH 7.1 / 10 000 ppmv Shell dissolution Lindinger et al. (1984) pCO2 740 ppmv 25% decrease in calcification rate Gazeau et al. (2007) Crassostrea gigas Oyster pCO2 740 ppmv 10% decrease in calcification rate Mytilus galloprovincialis Mediterranean pH 7.3, 5000 ppmv Reduced metabolism, growth rate Michaelidis et al. (2005) mussel Placopecten Giant scallop pH , 8.0 Decrease in fertilization and embryo Desrosiers et al. (1996) magellanicus development Tivela stultorum Pismo clam pH ,8.5 Decrease in fertilization rates Alvarado-Alvarez et al. (1996) Pinctada fucada Japanese pearl pH 7.7 Shell dissolution, reduced growth Reviewed in Knutzen (1981) martensii oyster pH .7.4 Increasing mortality Mercenaria mercenaria Clam Varag ¼ 0.3 Juvenile shell dissolution leading to Green et al. (2004) increased mortality Illex illecebrosus Epipelagic squid 2000 ppmv Impaired oxygen transport Po¨rtner and Reipschla¨ger (1996) Dosidicus gigas Epipelagic squid 0.1% CO2, 1000 Reduced metabolism/scope for Rosa and Seibel (unpublished) ppmv activity Arthropoda Acartia steueri Copepod 0.2-1%CO2, Decrease in egg hatching success; Kurihara et al. (2004) Acartia erythraea Copepod 2000–10 000 ppmv increase in nauplius mortality rate Copepods Pacific, deep vs. 860–22 000 ppmv Increasing mortality with increasing Watanabe et al. (2006) shallow CO2 CO2 concentration and duration of exposure Euphausia pacifica Krill pH , 7.6 Mortality increased with increasing Yamada and Ikeda (1999) Paraeuchaeta elongata Mesopelagic exposure time and decreasing pH copepod Conchoecia sp. Ostracod Cancer pagurus Crab 1% CO2, 10 000 Reduced thermal tolerance, aerobic Metzger et al. (2007) ppmv scope Chaetognatha Sagitta elegans Chaetognath pH ,7.6 Mortality increased with increasing Yamada and Ikeda (1999) exposure time and decreasing pH Echinodermata Strongylocentrotus Sea urchin pH 6.2–7.3 High sensitivity inferred from lack cf. Burnett et al. (2002) purpuratus of pH regulation and passive Psammechinus miliaris Sea urchin buffering via test dissolution during Spicer (1995); Miles et al. emersion (2007) Hemicentrotus Sea urchin 500–10 000 ppmv Decreased fertilization rates, Kurihara and Shirayama (2004) pulcherrimus impacts larval development Echinometra mathaei Sea urchin Cystechinus sp. Deep-sea urchin pH 7.8 80% mortality under simulated CO2 Barry et al. (2002) sequestration Continued 424 V. J. Fabry et al.

Table 1. Continued

Species Description CO2 system Sensitivity Reference parameters Sipuncula Sipunculus nudus Peanut worm 1% CO2, 10 000 ppmv Metabolic suppression Po¨rtner and Reipschla¨ger (1996) Pronounced mortality in 7-week Langenbuch and Po¨rtner exposure (2004) Vertebrata Scyliorhinus canicula Dogfish pH 7.7 / 0.13%CO2 Increased ventilation Reviewed in Truchot (1987) 7% CO2, 70 000 100% mortality after 72 h Hayashi et al. (2004) ppmv Sillago japonica Japanese whiting 7% CO2, 70 000 Rapid mortality in 1-step exposure Kikkawa et al. (2006) ppmv Paralichthys olivaceus Japanese flounder 5% CO2, 50 000 100% mortality within 48 h Hayashi et al. (2004) ppmv Euthynnus affinis Eastern little tuna 15%CO2, 150 000 100% mortality of eggs after 24 h Kikkawa et al. (2003) ppmv Pagrus major Red sea bream 5%CO2, 50 000 .60% larval mortality after 24 h Ishimatsu et al. (2005) ppmv Seriola quinqueradiata Yellowtail/ 5% CO2, 50 000 ppmv Reduced cardiac output; 100% Ishimatsu et al. (2004) amberjack mortality after 8 h Sparus aurata Mediterranean fish pH 7.3, 5000 ppmv Reduced metabolic capacity Michaelidis et al. (2007) Dicentrarchus labrax Sea bass pH 7.25, 24 mg l–1 Reduced feed intake Cecchini et al. (2001) CO2

marine biota to continuous, long-term exposure to elevated pCO2 or the capacity of these organisms to adapt. Nevertheless, the data in Table 1 clearly demonstrate that elevated pCO2 can adversely impact marine fauna both via decreased carbonate saturation state, which directly affects calciﬁcation rates, and via disturbance to acid–base physiology. We use the available evidence to speculate on possible ecological winners and losers during the 21st century, and to identify priorities for critically needed research.

Species distributions Euthecosomatous pteropods will be first among the major groups of planktonic calcifiers to experience persistent ,1 saturation states in surface waters of their current geographical ranges (Figures 3 and 4). If we assume that these animals are restricted to aragonite-saturated waters, then euthecosomatous pteropod Figure 8. Oxygen consumption rates under elevated CO2 for marine habitat would become increasingly limited, first vertically in the animals as a percentage of control rates (air saturation). Decreases in water column, then latitudinally, by shoaling of the aragonite satur- routine metabolism, an adaptive strategy to short-term hypercapnia, of the squid Dosidicus gigas 1000 ppmv (0.1% at 208C), the ation horizon (Orr et al., 2005). For example, the pteropod C. pyr- pteropod mollusc Limacina helicina antarctica under 789 ppmv amidata is widely distributed and, in the North Pacific, its range (21.868C), the worm Sipunculus nudus and an amphipod Phronima extends to nearly 558N. In other oceans, this species is typically sedentaris under 10 000 ppm (1.0%), and the bivalve Mytilus edulis found at 400–500 m during the day and in surface waters at under 5000 ppmv (0.5%, pH 7.3, 188C) carbon dioxide. (R. Rosa, night (Be´ and Gilmer, 1977). If C. pyramidata has a similar and B. Seibel, unpublished data; Po¨rtner and Reipschla¨ger (1996); pattern of vertical migration in the North Pacific, then this Michaelidis et al. (2005)). species would already be experiencing seawater corrosive to aragonite (V , 1) during part of its diel cycle at 108N and near 508N (Figure 3). When C. pyramidata is transported from coastal waters may appear less sensitive to decreased pH-elevated pCO2 com- via anticyclonic Haida eddies that form along the eastern margin pared with other faunal groups, it must be emphasized that of the Subarctic Pacific and move towards the Alaskan gyre, this there is no information on the response of fish to the pCO2 pteropod can be a dominant member of the eddy zooplankton values that are projected to occur over the next century. community (Mackas and Galbraith, 2002; Tsurumi et al., 2005). Moreover, most empirical evidence comes from short-term exper- Yet, C. pyramidata demonstrated only weak diel vertical migration iments, and we know little or nothing about the response of within an eddy, with most individuals remaining in the upper Impacts of ocean acidification on marine fauna and ecosystem processes 425

undersaturated with respect to aragonite are currently impinging upon the depth ranges of pteropods in several other regions, such as upwelling areas associated with the Benguela Current, western Arabian Sea, and Peru Current, where pteropod abundances can be high (e.g. Be´ and Gilmer, 1977; Fabry, 1990; Boltovskoy et al., 1993; Kalberer et al., 1993; Hitchcock et al., 2002; Mohan et al., 2006). Although future anthropogenically induced reductions in the saturation state of calcite will not be as severe as those for aragonite, species of the calcitic foraminifera may also change their geographic distributions in response to decreased calcite saturation states. As calcite undersaturation is projected to occur 50–100 years subsequent to that of aragonite (Orr et al., 2005), foraminifera could be displaced from high latitudes, where they can be abundant. Changes in species composition could also occur, as has been reported in the California Current in response to anthropogenic warming (Field et al., 2006). Currently, there are few high- quality data on the diel vertical distributions of euthecosomes and foraminifera in these areas to test whether shelled pteropod and foraminiferal populations will shift their depth ranges as the carbonate chemistry of seawater changes. 21 Figure 9. Bohr coefficients (DlogP50DpH ), an indication of the In addition to calcification, other physiological indices indicate sensitivity of oxygen transport to pH, as a function of oxygen general trends that may be useful for predicting species’ vulner- consumption rate at approximate environmental temperature for ability to ocean acidification. Those with low metabolic rates, marine animals from both benthic and pelagic environments. 1. Vampyroteuthis infernalis;2.Glyphocrangon vicara;3.Bythograea including perhaps gelatinous zooplankton and those that experi- thermydron;4.Gnathophausia ingens;5.Octopus vulgaris; ence little natural variation in CO2, appear to demonstrate lesser 6. Coryphanoides armatus;7.Acanthephyra curtirostris; CO2 tolerance. Thus, at first glance, zooplankton inhabiting the 8. Acanthephyra acutifrons;9.Systelaspis debilis; 10. Architeuthis sp., open ocean may appear highly susceptible to ocean acidification, 11. Loligo pealei; 12. Acanthephyra smithi; 13. Loligo vulgaris; 14. given the constancy of the pelagic environment relative to hydro- Oplophorus gracilirostris; 15. Illex illecebrosus; 16. Sepia officinalis; 17. thermal vents or the intertidal zone (Truchot and Duhamel-Jouve, Octopus dofleini; 18. Trematomus bernacchii; 19. Artedidraco sp.; 20. 1980). However, large, vertical gradients in environmental vari- Lolliguncula brevis; 21. Megaleledone senoi. Bohr coefficients from ables, including oxygen, CO2, and pH, exist in the upper 1000 m Grigg (1967); Arp and Childress (1985); Noble et al. (1986); Sanders (Figure 3), and most zooplankton species migrate daily from near- and Childress (1990); Eastman (1993); Bridges (1994); Tamburrini surface waters to depths of 200–700 m. In the expansive regions et al. (1998); Seibel et al. (1999); Lowe et al. (2000); Zielinski et al. (2001). Metabolic rates reviewed in Childress (1995); Seibel and with pronounced oxygen minimum layers (Figure 3c), these diel Drazen (2007)). Metabolic rate for Architeuthis sp. is estimated from migrations expose zooplankton to wide variations in pCO2, activity of citrate synthase. values (Figure 3b) greater than those expected for average surface waters as a result of anthropogenic ocean acidification over the next 100 years (Figure 1). Therefore, although the open 75 m during both day and night (Mackas and Galbraith, 2002). ocean environment has fluctuated historically only over millennial Apart from this Haida eddy study, however, data on the vertical time-scales (Kennett and Ingram, 1995), giving species ample time distribution of C. pyramidata in the Subarctic Pacific are lacking. to adapt (Childress and Seibel, 1998), the effective environment of The euthecosome Limacina helicina is an important high- many species in the open ocean is not constant. latitude species in both the northern and southern hemispheres. Many species migrating into oxygen minimum layers may do In the Arctic Ocean, this species is most abundant between 50– so by suppressing metabolism and supplementing remaining 100 m during winter and in the upper 50 m during summer energy demands with anaerobic metabolic pathways (Childress (Kobayashi, 1974). South of the Antarctic Polar Front, where ara- and Seibel, 1998; Hunt and Seibel, 2000). Anaerobic metabolism gonite undersaturation of the entire water column is projected to itself may exacerbate internal acid–base imbalance (Hochachka occur within the next 50–100 years, this pteropod species com- and Somero, 2002). Therefore, vertically migrating species, like prises nearly all of the of CaCO3 export to the ocean interior those living intertidally or near hydrothermal vents, experience (Collier et al., 2000; Honjo et al., 2000; Accornero et al., 2003). oscillating periods of simultaneous hypoxia and high pCO2 that As aragonite saturation states approach 1 with progressive acid- require specific adaptations for tolerance (Childress and Seibel, ification during the 21st century (Figure 4), we hypothesize that 1998). Those adaptations may make zooplankton in hypoxic shelled pteropod species, such as C. pyramidata and L. helicina, regions more tolerant of elevated pCO2, at least on short time- either will have to adapt to living continuously in seawater under- scales, than those in well-oxygenated regions. This hypothesis is saturated with respect to aragonite or restrict their vertical and supported by the recent work of Watanabe et al. (2006), who latitudinal distributions to warmer, more carbonate-rich regions found greater mortality during short-term exposure to high that remain supersaturated with respect to aragonite. This latter pCO2 in shallow-living and subtropical copepods than in possibility may be limited by the extreme adaptations to low temp- deep-living species in the Subarctic Pacific, where pCO2 is natu- erature that may prevent equatorward movement of polar animals rally much higher. However, Pane and Barry (2007) suggested (Stillman, 2003; Somero, 2005; Seibel et al., 2007). Waters that low oxygen exacerbated internal acid–base imbalance in a 426 V. J. Fabry et al. deep-sea crab. Currently, there is no evidence that adaptation to foraminifera densities decrease in some high-latitude regions, variably hypercapnic environments promotes tolerance of where they are currently abundant (e.g. Subarctic Pacific), chronic ocean acidification such as that expected over the next CaCO3 export to the ocean interior will be reduced, which in century. Furthermore, as warming alters ocean stratification, the turn would decrease their potential to act as ballast in the transport vertical and horizontal extent of the oxygen minimum layer is of organic carbon to the deep sea (Schiebel, 2002). expected to change, and ocean acidification will elevate pCO2 Most of the carnivorous zooplankton and fish (e.g. cod, levels at subsurface depths. Warming is also expected to act syner- pollock, haddock, mackerel) that feed on euthecosomatous ptero- gistically to exacerbate oxygen limitation and further hinder CO2 pods (Ito, 1964; LeBrasseur, 1966; Lalli and Gilmer, 1989) would tolerance (Metzger et al., 2007). be able to switch to other prey types, which could result in Predictive ability of the response of zooplankton populations to greater predation pressure on juvenile fish such as salmon ocean acidification is hampered by a paucity of measurements at (Willette et al., 2001). In contrast, gymnosomes prey exclusively climate-relevant CO2 concentrations (Table 1). In many cases, on shelled pteropods (Lalli and Gilmer, 1989) and would likely no information exists for ecologically important taxa such as lar- shift their geographic distribution in concert with their eutheco- vaceans, salps, amphipods, and euphausiids. Gelatinous zooplank- some prey, assuming both predator and prey are able to overcome ton have not been examined at all for CO2 tolerance. Their low possible thermal tolerance limitations (Seibel et al., 2007). Another metabolic rates may make them highly susceptible; however, specialized predator, the myctophid Centrobrachus brevirostris, they may also exhibit species-specific responses to increased which feeds predominantly on the pteropod C. pyramidata in pCO2, similar to the differential response observed among the Kuroshio waters of the western North Pacific (Watanabe medusae species to hypoxia (Rutherford and Thuesen, 2005; et al., 2002), would also be highly impacted if euthecosomes Thuesen et al., 2005a, b). Attrill et al. (2007) reported a significant were excluded from its habitat in the future; typically, the trade-off correlative relationship between reduced pH and increased fre- in such highly specialized feeding is a reduced ability to diversify quency of medusae, as sampled by the Continuous Plankton when the preferred prey is absent. Recorder for 40 years in the North Sea. The authors suggest that In the North Pacific, the euthecosomes L. helicina and, to a the frequency of medusae in the North Sea will increase over the lesser extent, C. pyramidata can be important prey of juvenile next century as surface water pH values decrease. As yet, no cau- pink salmon (Oncorhynchus gobuscha), which comprise a large sative mechanism linking jellyfish abundance with ocean acidifica- part of the commercial catch of salmon in the North Pacific (cf. tion is known. Armstrong et al., 2005). Pink salmon have a short, two-year life- Fertilization success and early developmental stages of many span, and their recruitment is thought to be affected by diet and faunal groups appear to be particularly vulnerable to elevated availability of prey during their early marine life history. In a three- pCO2 (Table 1). The tolerance of early life stages may impact year study designed to examine the interannual variability of the recruitment success and, ultimately, species abundances and dis- feeding habits of juvenile pink salmon, Armstrong et al. (2005) tributions. If the formation of highly soluble, amorphous CaCO3 found that L. helicina accounted for 60% by weight of the juven- as a transient precursor to crystalline phases in embryonic and ile salmon diet in two of three years, but only 15% in the third larval stages (Beniash et al., 1997; Weiss et al., 2002) is widespread year. Juvenile pink salmon rapidly increase in size during among mollusc and echinoderm species, then these phyla may be summer in the Subarctic Pacific, feeding on progressively larger particularly at risk from progressive ocean acidification in many prey items and switching from L. helicina to the larger C. pyrami- oceanic regions. Additional investigation is needed to determine data by October (Boldt and Haldorson, 2003; Armstrong et al., if ocean acidification-induced mortality of these stages could 2005). In a model study linking oceanic foodwebs to production drive a reorganization of benthic and pelagic communities and and growth rates of pink salmon, Aydin et al. (2005) found that also adversely impact commercially important fisheries. decreased energetic foraging costs for zooplanktivorous juvenile salmon and the ontogenetic diet shift from zooplankton to Trophic dynamics and other ecosystem processes squid were both key factors that strongly influenced the biomass The relative rate of change in surface seawater carbonate ion con- of mature pink salmon. Because euthecosomatous pteropods can centration is greatest in high-latitude regions (Orr et al., 2005; reach swarm densities in near-surface waters during daylight, Figure 4). In polar and subpolar areas, the progressive shoaling either through concentration by eddies (Tsurumi et al., 2005) or of the aragonite saturation horizon and the decreasing calcite sat- life-history traits (Bathmann et al., 1991; Gannefors et al., 2005), uration state of the euphotic zone over future decades will impact visual predators such as juvenile pink salmon may be able to trophic dynamics and other ecosystem processes, including the reduce forage costs by feeding within pteropod patches. Other pre- cycling of CaCO3 and organic matter. Euthecosomatous pteropods liminary model results suggest that a 10% decrease in pteropod are functionally important components of high-latitude ecosys- production could lead to a 20% drop in mature pink salmon tems with the potential to influence phytoplankton stocks body weight (Aydin, pers. comm.). During the ontogenetic diet (Hopkins, 1987), carbon fluxes (Noji et al., 1997; Collier et al., shift of juvenile salmon, the gonatid squid Berrytheuthis anonychus 2000; Honjo et al., 2000), and dimethyl sulphide levels is an important control on adult salmon biomass; availability of (Levasseur et al., 1994) that, in turn, influence global climate this lipid-rich prey species substantially accelerates the growth of through ocean–atmosphere feedback loops. The possible extirpa- both pink and sockeye salmon in bioenergetics models (cf. Aydin tion of euthecosomatous pteropods from the high-latitude regions et al., 2005). Similarly, Gonatus fabricii, among the most abundant would impact the downward organic carbon flux associated with squid species in the North Atlantic, is the most important prey pteropod faecal pellets (Thibault et al., 1999; Collier et al., 2000) item to a number of marine mammals and may be responsible and remove a major source of CaCO3 in such regions (e.g. for their seasonal occurrence in some regions (Bjorke, 2001; Bathmann et al., 1991; Gardner et al., 2000; Honjo et al., 2000; Hooker et al., 2001). Although the respiratory physiology of Accornero et al., 2003; Tsurumi et al., 2005). Similarly, if gonatid squid has not been investigated in detail, their metabolic Impacts of ocean acidification on marine fauna and ecosystem processes 427

rates are high (Seibel, 2007), which may lead to high CO2 sensi- crabs, and lobsters. We also need to test taxa for which there tivity, as described above for ommastrephids (e.g. Po¨rtner and are currently no data available, including medusae, larvaceans, Reipschla¨ger, 1996; Po¨rtner et al., 2004). Ommastrephids are and various crustaceans. Additional experiments should also important components of ecosystems and are important com- examine the interactive effects of seawater CO2 chemistry with mercial fisheries worldwide (Rodhouse and White, 1995; Clarke, temperature, dissolved oxygen, food availability, and other vari- 1996; Nigmatullin et al., 2001). ables that may change as a result of human activities. Ocean acidification could also affect foodwebs and carbon † New approaches (e.g. functional genomics and DNA bar- cycling through bottom-up controls involving pH-dependent spe- coding) and advances in existing technologies (e.g. autonomous ciation of nutrients and metals (Huesemann et al., 2002), which, in chemical sensors and optical plankton samplers) are necessary turn, may alter species composition and rates of primary pro- to investigate the in situ response of organisms that are difficult ductivity. The interactive effects and feedback of changing seawater to maintain in the laboratory, identify sublethal effects of CO2 chemistry with other stressors, such as warming, eutrophica- chronic exposure to elevated pCO2 on marine fauna, and tion, introduced species, and overfishing, may act to alter ecosys- address questions of long-term impacts and potential for adap- tem responses that would otherwise result from only one of these tation over decadal to centennial time-scales. stressors (Schippers et al., 2004; Hutchins et al., 2007). Quantification of these complex ecosystem processes requires † Models are critical to scale up results from manipulative exper- additional empirical data, as well as new modelling efforts, par- iments and field observations to predict ecosystem impacts on ticularly on regional scales. regional and global scales. Although the changes in seawater chemistry that result from the Research needs and conclusions oceanic uptake of anthropogenic CO2 are well characterized over most of the ocean, the biological impacts of ocean acidification Most experimental work on the impacts of ocean acidification on on marine fauna are only beginning to be understood. New tech- marine biota at climate-relevant pCO values has investigated the 2 nologies and advances, as well as integrated, multidisciplinary calcification response of corals and coccolithophores (cf. Kleypas efforts among biologists and chemists, experimentalists and mod- et al., 2006). There is a critical need for information on the sub- ellers will be required to quantify the effects of ocean acidification lethal calcification and energetic responses of a diverse suite of on marine fauna and changes in ecosystem structure and function. zooplankton and micronekton. We need to move forward on Nevertheless, sufficient information exists to state with certainty several fronts in parallel. that deleterious impacts on some marine species are unavoidable, † In sensitive regions and for critical species, we need to track the and that substantial alteration of marine ecosystems is likely over abundances and depth distributions of calcareous and non- the next century. calcifying fauna, measure calcification and metabolic rates of these groups, and relate these data to changes in the CO2 Acknowledgements chemistry of the water column. This requires commitment to This work was supported jointly by the National Science long-term monitoring programmes at appropriate temporal Foundation (NSF grants OCE-0551726 and OPP-0538710 to and spatial scales to detect possible shifts, and distinguish VJF, and OCE-0526493 and OPP-0538479 to BAS) and the between natural variability and anthropogenically induced National Oceanic and Atmospheric Administration (NOAA). We changes. specifically acknowledge programme managers Don Rice, Phil † Using pCO2 levels projected to occur over the next century, Taylor, and Roberta Marinelli of the NSF Chemical and manipulative laboratory experiments are needed to investigate Biological Oceanography Programs and Office of Polar the calcification and dissolution responses, identify physiologi- Programs, respectively; and Kathy Tedesco and Mike Johnson of cal indices useful in predicting CO2 tolerance, determine the the NOAA Climate Change Program for their support. We costs of acid–base regulation, and quantify sensitive energetic thank H. Spero, B. Ho¨nisch, and A. Maas for discussions and com- processes, such as skeletal and tissue growth, reproduction, ments on earlier drafts of this paper. The IAEA is grateful for the and metabolism for critical life stages of key species. support provided to its Marine Environmental Laboratory by the † Mesocosm and field experiments are necessary to quantify eco- Government of the Principality of Monaco. system impacts from ocean acidification that may include forcing from bottom-up controls, changes in foodweb structure, biogeochemical cycling, and feedback mechanisms. References † High-priority areas for research include high-latitude regions, Accornero, A., Manno, C., Esposito, F., and Gambi, M. C. 2003. The which may become undersaturated with respect to aragonite vertical flux of particulate matter in the polynya of Terra Nova Bay. Part II. Biological components. Antarctic Science, 15: as early as 2050, and regions with pronounced oxygen 175–188. minimum layers or coastal hypoxia, which are already charac- Alvarado-Alvarez, R., Gould, M. C., and Stephano, J. L. 1996. terized by high pCO2 and may be particularly at risk, owing Spawning, in vitro maturation, and changes in oocyte electrophysi- to the combined effects of low oxygen with elevated pCO2, ology induced serotonin in Tivela stultorum. Biological Bulletin, warming, and eutrophication. 190: 322–328. Armstrong, J. L., Boldt, J. L., Cross, A. D., Moss, J. H., Davis, N. D., † Target species for investigation in the above regions include Myers, K. W., Walker, R. V., et al. 2005. Distribution, size, and euthecosomatous pteropods, foraminifera, epipelagic squid, interannual, seasonal and diel food habits of northern Gulf of and larval stages and adults of commercially and ecologically Alaska juvenile pink salmon, Oncorhynchus gorbuscha. Deep Sea important benthic invertebrates such as bivalves, sea urchins, Research II, 52: 247–265. 428 V. J. Fabry et al.

Arp, A. J., and Childress, J. J. 1985. Oxygen binding properties of the Burnett, L., Terwilliger, N., Carroll, A., Jorgensen, D., and Scholnick, blood of the deep-sea shrimp, Glyphocrangon vicaria. Physiological D. 2002. Respiratory and acid-base physiology of the purple sea Zoology, 58: 38–45. urchin, Stronglyocentratus purpuratus, during air exposure: pre- Attrill, M. J., Wright, J., and Edwards, M. 2007. Climate-related sence and function of a facultative lung. Biological Bulletin, 203: increases in jellyfish frequency suggest a more gelatinous future 42–50. for the North Sea. Limnology and Oceanography, 52: 480–485. Caldeira, K., and Wickett, M. E. 2003. Anthropogenic carbon and Aydin, K. Y., McFarlane, G. A., King, J. R., Megrey, B. A., and Myer, K. W. ocean pH. Science, 425: 365. 2005. Linking oceanic foodwebs to coastal production and growth Caldeira, K., and Wickett, M. E. 2005. Ocean model predictions of rates of Pacific salmon (Oncorhynchus spp.), using models on chemistry changes from carbon dioxide emissions to the atmos- three scales. Deep Sea Research II, 52: 757–780. phere and ocean. Journal of Geophysical Research, 110: C09S04. Barker, S., and Elderfield, H. 2002. Foraminiferal calcification response doi:10.1029JC002671. to glacial–interglacial changes in atmospheric CO2. Science, 297: Cameron, J. N. 1989. The Respiratory Physiology of Animals. Oxford 833–836. University Press, New York. 353 pp. Barry, J., Seibel, B. A., Drazen, J., Tamburri, M., Lovera, C., and Cameron, J. N., and Iwama, G. K. 1987. Compensation of progressive Brewer, P. 2002. Field experiments on direct ocean CO2 sequestra- hypercapnia in channel catfish and blue crabs. Journal of tion: the response of deep-sea faunal assemblages to CO2 injection Experimental Biology, 133: 183–197. at 3200 m off central California. EOS Transactions of the American Castellini, M. A., and Somero, G. N. 1981. Buffering capacity of ver- Geophysical Union, 83: OS51F-02. tebrate muscle: correlations with potentials for anaerobic function. Bathmann, U. V., Noji, T. T., and von Bodungen, B. 1991. Journal of Comparative Physiology, 143: 191–198. Sedimentation of pteropods in the Norwegian Sea in autumn. Cecchini, S., Saroglia, M., Caricato, G., Terrova, G., and Sileo, L. 2001. Deep Sea Research, 38: 1341–1360. Effects of graded environmental hypercapnia on sea bass Be´, A. W. H., and Gilmer, R. W. 1977. A zoogeographic and taxonomic (Dicentrarchus labrax L.) feed intake and acid-base balance. review of euthecosomatous Pteropoda. In Oceanic Aquaculture Research, 32: 499–502. Micropaleontology, Vol. 1, pp. 733–808. Ed. by A. T. S. Ramsey. Childress, J. J. 1995. Are there physiological and biochemical adap- Academic Press, London. tations of metabolism in deep-sea animals? Trends in Ecology Beniash, E., Aizenber, J., Addai, L., and Weiner, S. 1997. Amorphous and Evolution, 10: 30–36. calcium carbonate transforms into calcite during sea urchin Childress, J. J., and Seibel, B. A. 1998. Life at stable low oxygen levels: larval spicule growth. Proceedings of the Royal Society of adaptations of animals to oceanic oxygen minimum layers. Journal London, Series B, 264: 461–465. of Experimental Biology, 201: 1223–1232. Bijma, J., Honisch, B., and Zeebe, R. E. 2002. Impact of the ocean car- Clairborne, J. B., Edwards, S. L., and Morrison-Shetlar, A. I. 2002. bonate chemistry on living foraminiferal shell weight: comment on Acid-base regulation in fishes: cellular and molecular mechanisms. “Carbonate ion concentration in glacial-age deepwaters of the Journal of Experimental Zoology, 293: 302–319. Caribbean Sea” by W. S. Broecker and E. Clark. Geochemistry, Geophysics, Geosystems, 3: 1064. doi:10.1029/2002GC000388. Clarke, M. R. 1996. The role of cephalopods in the world’s oceans: general conclusions and the future. Philosophical Transactions of Bijma, J., Spero, H. J., and Lea, D. W. 1999. Reassessing foraminiferal the Royal Society of London, Series B–Biological Sciences, 351: stable isotope geochemistry: impact of the oceanic carbonate 1105–1112. systems (experimental results). In Use of Proxies in Paleoceanography: Examples from the South Atlantic, pp. Collier, R., Dymond, J., Honjo, S., Manganini, S., Francois, R., and 489–512. Ed. by G. Fisher, and G. Wefer. Springer-Verlag, Dunbar, R. 2000. The vertical flux of biogenic and lithogenic New York. material in the Ross Sea: moored sediment trap observations Bischoff, W. W., Mackenzie, F. T., and Bishop, F. C. 1987. Stabilities of 1996–1998. Deep Sea Research, 47: 3491–3520. synthetic magnesian calcites in aqueous solution: comparison with Corliss, B. H., and Honjo, S. 1981. Dissolution of deep-sea benthonic biogenic materials. Geochimica et Cosmochimica Acta, 51: foraminifera. Micropaleontology, 27: 356–378. 1413–1424. Dadon, J. R., and de Cidre, L. L. 1992. The reproductive cycle of the Bjorke, H. 2001. Predators of the squid Gonatus fabricii (Lichtenstein) Thecosomatous pteropod Limacina retroversa in the western in the Norwegian Sea. Fisheries Science, 52: 113–120. South Atlantic. Marine Biology, 114: 439–442. Boldt, J., and Haldorson, L. J. 2003. Seasonal and geographic variation Denman, K. L., et al. 2007. Couplings between changes in the climate in juvenile pink salmon diets in the northern Gulf of Alaska and system and biogeochemistry. In Climate Change 2007: The Physical Prince William Sound. Transactions of the American Fisheries Science Basis. Contribution of Working Group I to the Fourth Society, 132: 1035–1052. Assessment Report of the Intergovernmental Panel on Climate Boltovskoy, D., Alder, V. A., and Abelmann, A. 1993. Annual flux of Change, pp. 500–556. Ed. by S. Soloman, D. Qin, M. Manning, Radiolaria and other shelled plankters in the eastern Atlantic at Z. Chen, M. Marquis, K. B. Averyt, M. Tignor, H. L. Miller, et al. 853 m: seasonal variations and polycystine species-specific Cambridge University Press, Cambridge. responses. Deep Sea Research, 40: 1863–1895. Desrosiers, R. R., De´silets, J., and Dube´, F. 1996. Early developmental Bown, P. R., Lees, J. A., and Young, J. R. 2004. Calcareous nanoplank- events following fertilization in the giant scallop Placopecten magel- ton evolution and diversity through time. In From Molecular lanicus. Canadian Journal of Fisheries and Aquatic Sciences, 53: Processes to Global Impact, pp. 481–508. Ed. by H. R. 1382–1392. Thierstein, and J. R. Young. Springer-Verlag, Berlin. Dickson, A. G., and Millero, F. J. 1987. A comparison of the equili- Bridges, C. R. 1994. Bohr and Root effects in cephalopod haemo- brium constants for the dissociation of carbonic acid in seawater cyanins—paradox or pressure in Sepia officinalis? In Physiology media. Deep Sea Research, 34: 1734–1743. of Cephalopod Molluscs: Lifestyle and Performance Adaptations, Doney, S. C., Mahowald, N., Lima, I., Feely, R. A., Mackenzie, F. T., pp. 121–130. Ed. by H. O. Portner, R. K. O’Dor, and D. L. and Lamarque, F. 2007. The impacts of anthropogenic nitrogen MacMillan Gordon and Breach, London. and sulfur deposition on ocean acidification and the inorganic Brummer, G-J. A., Hemleben, C., and Spindler, M. 1987. Ontogeny of carbon system. Proceedings of the National Academy of Sciences extant spinose planktonic foraminifera (Globigerinidae): a concept of the USA, 104: 14 580–14 585. exemplified by Globigerinoides sacculifer (Brady) and G. ruber Eastman, J. T. 1993. Antarctic Fish Biology: Evolution in a Unique (d’Orbigny). Marine Micropaleontology, 12: 357–381. Environment. Academic Press, New York. 322 pp. Impacts of ocean acidification on marine fauna and ecosystem processes 429

Fabry, V. J. 1990. Shell growth rates of pteropod and heteropod mol- Hayashi, M., Kita, J., and Ishimatsu, A. 2004. Acid-base responses to lusks and aragonite production in the open ocean: implications for lethal aquatic hypercapnia in three marine fishes. Marine the marine carbonate system. Journal of Marine Research, 48: Biology, 144: 153–160. 209–222. Heisler, N. 1989. Interactions between gas exchange, metabolism, and Field, D. B., Baumgartner, T. R., Charles, C. D., Ferreira-Bartrina, V., ion transport in animals: an overview. Canadian Journal of and Ohman, M. 2006. Planktonic foraminifera of the California Zoology, 67: 2923–2935. Current reflect 20th-century warming. Science, 311: 63–66. Heisler, N. 1993. Acid-base regulation. In The Physiology of Fishes, pp. Feely, R. A., Sabine, C. L., Byrne, R. H., and Greeley, D. 2006. Direct 343–377. Ed. by D. H. Evans. CRC Press, Boca Raton, FL. evidence for ocean acidification of the North Pacific Ocean. EOS Hemleben, C., Spindler, M., and Anderson, O. R. 1989. Modern Transactions of the American Geophysical Union, 87(52), Fall Planktonic Foraminifera. Springer-Verlag, New York. 363 pp. Meeting Supplement, Abstract OS12B-04. Henry, R. P. 1984. The role of carbonic anhydrase in blood ion and Feely, R. A., Sabine, C. L., Lee, K., Berelson, W., Kleypas, J., Fabry, V. J., acid-base regulation. American Zoologist, 24: 241–251. and Millero, F. J. 2004. Impact of anthropogenic CO on the 2 Hitchcock, G. L., Lane, P., Smith, S., Luo, J., and Ortner, P. B. 2002. CaCO system in the oceans. Science, 305: 362–366. 3 Zooplankton spatial distributions in coastal waters of the northern Forster, P., Ramaswamy, V., Artaxo, P., Berntsen, T., Betts, R., Fahey, Arabian Sea, August, 1995. Deep Sea Research, 49: 2403–2423. D. W., Haywood, J., et al. 2007. Changes in atmospheric constitu- Hochachka, P. W., and Somero, G. N. 2002. Biochemical Adaptation. ents and in radiative forcing. In Climate Change 2007: the Physical Princeton University Press, Princeton, NJ. 466 pp. Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Honjo, S., Francois, R., Manganini, S., Dymond, J., and Collier, R. Change, pp. 129–234. Ed. by S. Solomon, D. Qin, M. Manning, 2000. Particle fluxes to the interior of the Southern Ocean in the Z. Chen, M. Marquis, K. B. Averyt, M. Tignor, et al. Cambridge Western Pacific sector along 1708W. Deep Sea Research II, 47: University Press, Cambridge. 3521–3548. Gannefors, C., Boër, M., Kattner, G., Graeve, M., Eiane, K., Gulliksen, Hooker, S. K., Iverson, S. J., Ostrom, P., and Smith, S. C. 2001. Diet of B., Hop, H., et al. 2005. The Arctic sea butterfly Limacina helicina: northern bottlenose whales inferred from fatty-acid and stable lipids and life strategy. Marine Biology, 147: 169–177. isotope analyses of biopsy samples. Canadian Journal of Zoology, Gardner, W. D., Richardson, M. J., and Smith, W. O. 2000. Seasonal 79: 1442–1454. patterns of water column particulate organic carbon and fluxes Hopkins, T. L. 1987. Midwater food web in McMurdo Sound, Ross in the Ross Sea, Antarctica. Deep Sea Research, 47: 3423–3449. Sea, Antarctica. Marine Biology, 96: 93–106. Gattuso, J-P., Frankignoulle, M., Bourge, I., Romaine, S., and Horita, J., Zimmerman, H., and Holland, H. D. 2002. Chemical evol- Buddemeier, R. W. 1998. Effect of calcium carbonate saturation ution of seawater during the Phanerozoic: implications from the of seawater on coral calcification. Global and Planetary Change, recode of marine evaporates. Geochimica et Cosmochimica Acta, 18: 37–46. 66: 3733–3756. Gazeau, F., Quibler, C., Jansen, J. M., Gattuso, J-P., Middelburg, J. J., Houghton, J. T., Ding, Y., Griggs, D. J., Noguer, M., van der Linden, and Heip, C. H. R. 2007. Impact of elevated CO2 on shellfish calci- P. J., and Xiaosu, D. 2001. Climate change 2001: the scientific fication. Geophysical Research Letters, 34: L07603. doi 10.1029/ basis. Contribution of Working Group I to the Third Assessment 2006GL028554. Report of the Intergovernmental Panel on Climate Change. Gibbs, A. H., and Somero, G. N. 1990. Na+-K+ adenosine triphospha- Cambridge University Press, Cambridge. 944 pp. tase activities in gills of marine teleost fishes, changes with depth, Hourdez, S., and Weber, R. E. 2005. Molecular and functional adap- size and locomotory activity level. Marine Biology, 106: 315–321. tations in deep-sea hemoglobins. Journal of Inorganic Green, M. A., Jones, M. E., Boudreau, C. L., Moore, R. L., and Biochemistry, 99: 130–141. Westman, B. A. 2004. Dissolution mortality of juvenile bivalves Huesemann, M. H., Skillman, A. D., and Crecelius, E. A. 2002. The in coastal marine deposits. Limnology and Oceanography, 49: inhibition of marine nitrification by disposal of carbon dioxide. 727–734. Marine Pollution Bulletin, 44: 142–148. Grigg, G. C. 1967. Some respiratory properties of the blood of four Hunt, J. C., and Seibel, B. A. 2000. Life history of Gonatus onyx species of Antarctic fishes. Comparative Biochemistry and (Teuthoidea: Cephalopoda): ontogenetic changes in habitat, beha- Physiology, 23: 139–148. vior and physiology. Marine Biology, 136: 543–552. Grosell, M. 2006. Intestinal anion exchange in marine fish osmoregu- Hutchins, D. A., Fu, F-X., Zhang, Y., Warner, M. E., Feng, Y., Portune, lation. Journal of Experimental Biology, 209: 2813–2827. K., Berhardt, P. W., et al. 2007. CO2 control of Trichodesmium N2 Guinotte, J. M., Orr, J., Cairns, S., Freiwald, A., Morgan, L., and fixation, photosynthesis, growth rates, and elemental ratios: impli- George, R. 2006. Will human-induced changes in seawater chem- cations for past, present, and future ocean biogeochemistry. istry alter the distribution of deep-sea scleractinian corals? Limnology and Oceanography, 52: 1293–1304. Frontiers in Ecology and the Environment, 4: 141–146. Isaji, S. 1995. Defensive strategies against shell dissolution in bivalves Guppy, M., and Withers, P. 1999. Metabolic depression in animals: inhabiting acidic environments: the case of Geloina (Corbiculidae) physiological perspectives and biochemical generalizations. in mangrove swamps. The Veliger, 38: 235–246. Biological Reviews, 74: 1–40. Ishimatsu, A., Kikkawa, T., Hayashi, M., Lee, K., and Kita, J. 2004. Gutie´rrez, J. L., Jones, C. G., Strayer, D. L., and Iribarne, O. O. 2003. Effects of CO2 on marine fish: larvae and adults. Journal of Mollusks as ecosystem engineers: the role of shell production in Oceanography, 60: 731–741. aquatic habitats. Oikos, 101: 79–90. Ishimatsu, A., Hayashi, M., and Lee, S. 2005. Physiological effects on Hand, S. C. 1991. Metabolic dormancy in aquatic invertebrates. In fishes in a high-CO2 world. Journal of the American Geophysical Advances in Comparative and Environmental Physiology, Vol. 8, Union, 110: C09S09. pp. 1–50. Ed. by R. Gilles. Springer-Verlag, Heidelberg. Ito, J. 1964. Food and feeding habit of Pacific salmon (genus Harris, J. O., Maguire, G. B., Edwards, S. J., and Hindrum, S. M. 1999. Oncorhynchus) in their oceanic life. Bulletin of the Hokkaido Effect of pH on growth rate, oxygen consumption rate, and histo- Regional Fisheries Research Laboratory, 29: 85–97. pathology of gill and kidney tissue for juvenile greenlip abalone, Kalberer, M., Fischer, G., Patzold, J., Donner, B., Segl, M., and Wefer, Halitosis laevigata and blacklip abalone, Halitosis rubra leach. G. 1993. Seasonal sedimentation and stable isotope records of Journal of Shellfish Research, 18: 611–619. pteropods off Cap Blanc. Marine Geology, 113: 305–320. 430 V. J. Fabry et al.

Kennett, J. P., and Ingram, B. L. 1995. A 20 000-year record of ocean Langdon, C., Broecker, W. S., Hammond, D. E., Glenn, E., circulation and climate change from the Santa Barbara basin. Fitzsimmons, K., Nelson, S. G., Pend, T-H., et al. 2003. Effect of Nature, 377: 510–514. elevated CO2 on the community metabolism of an experimental Key, R. M. A., Kozyr, A., Sabine, C. L., Lee, K., Wanninkhof, R., coral reef. Global Biogeochemical Cycles, 17: 1011. doi:10.1029/ Bullister, J. L., Feely, R. A., et al. 2004. A global ocean carbon clima- 2002GB00. tology: results from Global Data Analysis Project (GLODAP). Langdon, C., Takahashi, T., Sweeney, C., Chipman, D., Goddard, J., Global Biogeochemical Cycles, 18: 4031. doi:10.1029/ Marubini, F., Aceves, H., et al. 2000. Effect of calcium carbonate 2004GB002247. saturation state on the calcification rate of an experimental coral reef. Global Biogeochemical Cycles, 14: 639–654. Kikkawa, T., Ishimatsu, A., and Kita, J. 2003. Acute CO2 tolerance during the early developmental stages of four marine teleosts. Langenbuch, M., and Po¨rtner, H. O. 2004. High sensitivity to chroni- Environmental Toxicology, 18: 375–382. cally elevated CO2 levels in a eurybathic marine sipunculid. Aquatic Kikkawa, T., Sata, T., Kita, J., and Ishimatsu, A. 2006. Acute toxicity of Toxicology, 70: 55–61. temporally varying seawater CO2 conditions on juveniles of Langer, G., Geisen, M., Baumann, K-H., Kla¨s, J., Riebesell, U., Thoms, Japanese sillago (Sillago japonica). Marine Pollution Bulletin, 52: S., and Young, J. R. 2006. Species-specific responses of calcifying 621–625. algae to changing seawater carbonate chemistry. Geochemistry, Kikkawa, T., Kita, J., and Ishimatsu, A. 2004. Comparison of the lethal Geophysics, Geosystems, Volume 7, Q09006, doi:10.1029/ 2005GC001127. effect of CO2 and acidification on red sea bream (Pagrus major) during the early development stages. Marine Pollution Bulletin, LeBrasseur, R. J. 1966. Stomach contents of salmon and steelhead 48: 108–110. trout in the northeastern Pacific Ocean. Journal of Fisheries Kleypas, J. A., Feely, R. A., Fabry, V. J., Langdon, C., Sabine, C. L., and Research Board Canada, 23: 85–100. Robbins, L. L. 2006. Impacts of ocean acidification on coral reefs Leclercq, N., Gattuso, J-P., and Jaubert, J. 2002. Primary production, and other marine calcifiers: a guide for future research. Report of respiration, and calcification of a coral reef mesocosm under a workshop held 18–20 April 2005, St Petersburg, FL, sponsored increased CO2 partial pressure. Limnology and Oceanography, by NSF, NOAA, and the US Geological Survey. 88 pp. 47: 558–564. Kleypas, J. A., and Langdon, C. 2006. Coral reefs and changing sea- Levasseur, M., Keller, M. D., Bonneau, E., D’Amours, D., and Bellows, water chemistry. In Coral Reefs and Climate Change: Science and W. K. 1994. Oceanographic basis of a DMS-related Atlantic cod Management, pp. 73–110. Ed. by J. T. Phinney, O. (Gadus morhua) fishery problem: blackberry feed. Canadian Hoegh-Guldberg, J. Kleypas, W. Skirving, and A. Strong, AGU Journal of Fisheries and Aquatic Sciences, 51: 881–889. Monograph Series Coastal Estuarine Studies 61. American Levi, R., Varona, P., Arshavksy, Y. I., Rabinovich, M. I., and Selverston, Geophysical Union, Washington, DC. A. I. 2004. Dual sensory-motor function for a molluskan statocyst Knoll, A. H. 2003. Biomineralization and evolutionary history. In network. Journal of Neurophysioly, 91: 336–345. Biomineralization, Vol. 54, pp. 329–350. Ed. by P. Dove, J. J. De Lindinger, M. I., Lauren, D. J., and McDonald, D. G. 1984. Acid-base Young, and S. Weiner. The Mineralogical Society of America, balance in the sea mussel, Mytilus edulis. III. Effects of environ- Washington, DC. 381 pp. mental hypercapnia on intra- and extracellular acid-base balance. Knutzen, J. 1981. Effects of decreased pH on marine organisms. Marine Biology Letters, 5: 371–381. Marine Pollution Bulletin, 12: 25–29. Lovejoy, T. E., and Hannah, L. 2005. Climate Change and Biodiversity. Yale University Press, New Haven, CT. 418 pp. Kobayashi, H. A. 1974. Growth cycle and related vertical distribution of the thecosomatous pteropod Spiratella (Limacina) helicina in Lowe, T. E., Brill, R. W., and Cousins, K. L. 2000. Blood oxygen- the central Arctic Ocean. Marine Biology, 26: 295–301. binding characteristics of bigeye tuna (Thunnus obesus), a high-energy-demand teleost that is tolerant of low ambient Kochevar, R. E., and Childress, J. J. 1996. Carbonic anhydrase in oxygen. Marine Biology, 136: 1087–1098. deep-sea chemoautotrophic symbioses. Marine Biology, 125: 375–383. Lowenstam, H. A., and Weiner, S. 1989. On Biomineralization. Oxford University Press, Oxford. Ko¨hler-Rink, S., and Ku¨hl, M. 2005. The chemical microenvironment of the symbiotic planktonic foraminifer Orbulina universa. Marine Mackas, D. L., and Galbraith, M. D. 2002. Zooplankton distribution Biology Research, 1: 68–78. and dynamics in a North Pacific eddy of coastal origin: I. Transport and loss of continental margin species. Journal of Kuffner, I. B., Andersson, A. J., Jokiel, P. L., Rodgers, K., and Oceanography, 58: 725–738 Mackenzie, F. T. 2008. Decreased abundance of crustose coralline algae due to ocean acidification. Nature Geoscience, 1: 114. Mangum, C. P. 1991. Salt sensitivity of the hemocyanin of eury- and stenohaline squids. Comparative Biochemistry and Physiology, Kurihara, H., and Shirayama, Y. 2004. Effects of increased atmospheric 99A, 159–161. CO on sea urchin early development. Marine Ecological Press 2 Marubini, F., Barnett, H., Langdon, C., and Atkinson, M. J. 2001. Series, 274: 161–169. Dependence of calcification on light and carbonate ion concen- Kurihara, H., Shinji, S., and Shirayama, Y. 2004. Effects of raised CO2 tration for the hermatypic coral Porites compressa. Marine on the egg production rate and early development of two marine Ecology Progress Series, 220: 153–162. copepods (Acartia steuri and Acartia erythraea). Marine Pollution Marubini, F., Ferrier-Pages, C., and Cuif, J. P. 2003. Suppression of Bulletin, 49: 721–727. skeletal growth in scleractinian corals by decreasing ambient Lalli, C. M., and Gilmer, R. W. 1989. Pelagic Snails: The Biology of carbonate-ion concentration: a cross-family comparison. Holoplanktonic Gastropod Mollusks. Stanford University Press, Proceedings of the Royal Society of London, Series B, 270: Stanford, CA. 179–184. Lallier, F., and Truchot, J. P. 1989. Modulation of haemocyanin Meehl, G. A., Stocker, T. F., Collins, W. D., Friedlingstein, P., oxygen-affinity by L-Lactatge and urate in the prawn, Penaeus japo- Gaye, A. T., Gregory, J. M., Kitoh, A., et al. 2007. Global climate nicus. Journal of Experimental Biology, 147: 133–146. projections. In Climate Change 2007: the Physical Science Basis. Langdon, C., and Atkinson, M. J. 2005. Effect of elevated pCO2 on Contribution of Working Group I to the Fourth Assessment photosynthesis and calcification of corals and interactions with Report of the Intergovernmental Panel on Climate Change. seasonal change in temperature/irradiance and nutrient enrich- pp.747–846. Ed. by S. Solomon, D. Qin, M. Manning, Z. Chen, ment. Journal of Geophysical Research, Oceans, 110: C09S07. M. Marquis, K. B. Averyt, M. Tignor, et al. Cambridge doi:10.1029/2004JC002576. University Press, Cambridge. Impacts of ocean acidification on marine fauna and ecosystem processes 431

Metzger, R., Sartoris, F. J., Langebuch, M., and Po¨rtner, H. O. 2007. Po¨rtner, H. O., and Reipschla¨ger, A. 1996. Ocean disposal of anthro- Influence of elevated CO2 concentrations on thermal tolerance of pogenic CO2: physiological effects on tolerant and intolerant the edible crab, Cancer pagurus. Journal of Thermal Biology, 32: animals. In Ocean Storage of Carbon Dioxide. Workshop 2— 144–151. Environmental Impact, pp. 57–81. Ed. by B. Ormerod, and Michaelidis, B., Ouzounis, C., Paleras, A., and Po¨rtner, H. O. 2005. M. V. Angel. IEA Greenhouse Gas R&D Programme, Effects of long-term moderate hypercapnia on acid-base balance Cheltenham, UK. and growth rate in marine mussels Mytilus galloprovincialis. Po¨rtner, H. O., Langebuch, M., and Reipschla¨ger, A. 2004. Biological Marine Ecology Progress Series, 293: 109–118. impact of elevated ocean CO2 concentration: lessons from Michaelidis, B., Spring, A., and Po¨rtner, H. O. 2007. Effects of long- animal physiology and Earth history. Journal of Oceanography, term acclimation to environmental hypercapnia on extracellular 60: 705–718. acid-base status and metabolic capacity in Mediterranean fish Raz, S., Hamilton, P. C., Wilt, F. H., Weiner, S., and Addadi, L. 2003. Sparus aurata. Marine Biology, 150: 1417–1429. The transient phase of amorphous calcium carbonate in sea urchin Miles, H., Widdicombe, S., Spicer, J. I., and Hall-Spencer, J. 2007. larval spicules: the involvement of proteins and magnesium ions in Effects of anthropogenic seawater acidification on acid-base its formation and stabilization. Advanced Functional Materials, 13: balance in the sea urchin Psammechinus miliaris. Marine 480–486. Pollution Bulletin, 54: 89–96. Ridgwell, A., and Zeebe, R. E. 2005. The role of the global carbonate Millero, F. J., Pierrot, D., Lee, K., Wanninkhof, R., Feely, R. A., Sabine, cycle in the regulation and evolution of the Earth system. Earth C. L., Key, R. M., et al. 2002. Dissociation constants for carbonic and Planetary Science Letters, 234: 299–315. acid determined from field measurements. Deep Sea Research I, Riebesell, U., Zondervan, I., Rost, B., Tortell, P. D., Zeebe, R. E., and 49: 1705–1723. Morel, F. M. M. 2000. Reduced calcification of marine plankton Mohan, R., Verma, K., Mergulhao, L. P., Sinha, D. K., Shanvas, S., and in response to increased atmospheric CO2. Nature, 407: 364–367. Guptha, V. S. 2006. Seasonal variation of ptreropods from the Rink, S., Ku¨hl, M., Bijma, J., and Spero, H. J. 1998. Microsensor Western Arabian Sea sediment trap. Geo-Marine Letters, 26: studies of photosynthesis and respiration in the symbiotic forami- 265–273. nifera Orbulina universa. Marine Biology, 131: 583–595. Mucci, A. 1983. The solubility of calcite and aragonite in seawater at Rodhouse, P. G., and White, M. G. 1995. Cephalopods occupy the eco- various salinities, temperatures and 1 atmosphere total pressure. logical niche of epipelagic fish in the Antarctic Polar Frontal Zone. American Journal of Science, 238: 780–799. Biological Bulletin, 189: 77–80. Nigmatullin, C. M., Nesis, K. N., and Arkhipkin, A. I. 2001. A review of Royal Society. 2005. Ocean acidification due to increasing atmospheric the biology of the jumbo squid Dosidicus gigas (Cephalopoda: carbon dioxide. Policy Document 12/05, The Royal Society, Ommastrephidae). Fisheries Research, 54: 9–19. London. 60 pp. Noble, R. W., Kwiatkowski, L. D., deYoung, A., Davis, B. J., Haedrich, Rutherford, L. D., and Thuesen, E. V. 2005. Metabolic performance R. L., Tam, L. T., and Riggs, F. A. 1986. Functional properties of and survival of medusae in estuarine hypoxia. Marine Ecology hemoglobins from deep-sea fish: correlations with depth distri- Progress Series, 294: 189–200. bution and presence of a swimbladder. Biochimica et Biophysica Sabine, C. L., Feely, R. A., Gruber, N., Key, R. M., Lee, K., Bullister, J. L., Acta, 870: 552–563. et al. 2004. The oceanic sink for CO2. Science, 305: 367–371. Noji, T. T., Bathmann, U. V., von Bodungen, B., Voss, M., Antia, A., Sanders, N. K., and Childress, J. J. 1990. Adaptations to the deep-sea Krumbholz, M., Klein, B., et al. 1997. Clearance of picoplankton- oxygen minimum layer: oxygen binding by the hemocyanin of sized particles and formation of rapidly sinking aggregates by the the bathypelagic mysid, Gnathophausia ingens Dohrn. Biological pteropod, Limacina retroversa. Journal of Plankton Research, 19: Bulletin, 178: 286–294. 863–875. Schiebel, R. 2002. Planktonic foraminiferal sedimentation and the Ohde, S., and Hossain, M. M. M. 2004. Effect of CaCO3 (aragonite) marine calcite budget. Global Biogeochemical Cycles, 16: doi: saturation state of seawater on calcification of Porites coral. 10.1029/2001GB1459. Geochemical Journal, 38: 613–621. Schippers, P., Lu¨rling, M., and Scheffer, M. 2004. Increase of atmos- Orr, J. C., Anderson, L. G., Bates, N. R., Bopp, L., Fabry, V. J., Jones, E., pheric CO2 promotes phytoplankton productivity. Ecology and Swingedouw, D. 2006. Arctic Ocean acidification. EOS Letters, 7: 446–451. Transactions of the American Geophysical Union, 87(36), Ocean Schneider, K., and Erez, J. 2006. The effect of carbonate chemistry on Sciences Meeting Supplement, Abstract OS14B-01. calcification and photosynthesis in the hermatype coral Acropora Orr, J. C., Fabry, V. J., Aumont, O., Bopp, L., Doney, S. C., Feely, R. A., eurystoma. Limnology and Oceanography, 51: 1284–1293. Gnanadesikan, A., et al. 2005. Anthropogenic ocean acidification Seibel, B. A. 2007. On the depth and scale of metabolic rate variation: over the twenty-first century and its impact on calcifying organ- scaling of oxygen consumption and enzymatic activity in the Class isms. Nature, 437: 681–686. Cephalopoda (Mollusca). Journal of Experimental Biology, 210: Pane, E. F., and Barry, J. P. 2007. Extracellular acid-base regulation 1–11. during short-term hypercapnia is effective in a shallow-water Seibel, B. A., Chausson, F., Lallier, F., Childress, J. J., and Zal, F. 1999. crab, but ineffective in a deep-sea crab. Marine Ecology Progress Vampire blood: respiratory physiology of the vampire squid Series, 334: 1–9. (Vampyromorpha: Cephalopoda) in relation to the oxygen Pane, L., Feletti, M., Francomacaro, B., and Mariottini, G. L. 2004. minimum layer. Experimental Biology Online, 4(1): 1–10. Summer coastal zooplankton biomass and copepod community ISSN:1430-3418. structure near the Italian Terra Nova Base (Terra Nova Bay, Ross Seibel, B. A., and Drazen, J. C. 2007. The rates of metabolism in marine Sea, Antarctica). Journal of Plankton Research, 26: 1479–1488. animals: environmental constraints, ecological demands and ener- Po¨rtner, H. O. 1990. Determination of intracellular buffer values after getic opportunities. Philosophical Transactions of the Royal metabolic inhibition by fluoride and nitrilotriacetic acid. Society, Series B – Biological Sciences, 362: 2061–2071. Respiration Physiology, 81: 275–288. Seibel, B. A., Dymowska, A., and Rosenthal, J. 2007. Metabolic temp- Po¨rtner, H. O. 1994. Coordination of metabolism acid-base regulation erature compensation and coevolution of locomotory performance and haemocyanin function in cephalopods. In Physiology of in pteropod molluscs. Integrative and Comparative Biology, 46: Cephalopod Molluscs: Lifestyle and Performance Adaptations, 880–891. pp. 131–148. Ed. by H. O. Portner, R. K. O’Dor, and Seibel, B. A., and Walsh, P. J. 2001. Potential impacts of CO2 injection D. L. MacMillan. Gordon and Breach, London. on deep-sea biota. Science, 294: 319–320. 432 V. J. Fabry et al.

Seibel, B. A., and Walsh, P. J. 2003. Biological impacts of deep-sea importance of algal sinking and mesozooplankton herbivory. carbon dioxide injection inferred from indices of physiological per- Deep Sea Research, 46: 2669–2697. formance. Journal of Experimental Biology, 206: 641–650. Thuesen, E. V., McCullough, K. D., and Childress, J. J. 2005a. Seibel, B. A., Thuesen, E. V., Childress, J. J., and Gorodezky, L. A. 1997. Metabolic enzyme activities in swimming muscle of medusae: is Decline in pelagic cephalopod metabolism with habitat depth the scaling of glycolytic activity related to oxygen availability? reflects differences in locomotory efficiency. Biological Bulletin, Journal of the Marine Biological Association of the UK, 85: 192: 262–278. 603–611. Shiryama, Y., and Thorton, H. 2005. Effect of increased atmospheric Thuesen, E. V., Rutherford, L. D., Brommer, P. L., Garrison, K., CO2 on shallow water marine benthos. Journal of Geophysical Gutowska, M. A., and Towanda, T. 2005b. Intragel oxygen pro- Research, 110: C09S08. doi: 10.1029/2004JC002618. motes hypoxia tolerance of scyphomedusae. Journal of Siegenthaler, U., Stocker, T. F., Monnin, E., Luethi, D., Schwander, J., Experimental Biology, 208: 2475–2482. Stauffer, B., Raynaud, D., et al. 2005. Stable carbon cycle-climate Truchot, J. P. 1987. Comparative aspects of extracellular acid-base relationship during the late Pleistocene. Science, 310: 1313–1317. balance. Springer-Verlag, Berlin. Silverman, J., Lazar, B., and Erez, J. 2007. Effect of aragonite satur- Truchot, J. P., and Duhamel-Jouve, A. 1980. Oxygen and carbon ation, temperature, and nutrients on the community calcification dioxide in the marine intertidal environment: diurnal and tidal rate of a coral reef. Journal of Geophysical Research, 112: changes in rockpools. Respiration Physiology, 39: 241–254. C05004. doi:10.1029/2006JC003770. Tsurumi, M., Mackas, D. L., Whitney, E. A., Dibacco, C., Galbraith, Smith, K. L., and Teal, J. M. 1973. Temperature and pressure effects on M. D., and Wong, C. S. 2005. Pteropods, eddies, carbon flux and respiration of thecosomatous pteropods. Deep Sea Research, 20: climate variability in the Alaska Gyre. Deep Sea Research II, 52: 853–858. 1037–1053. Somero, G. N. 1985. Intracellular pH, buffering substances and pro- Tyrrell, T., and Zeebe, R. E. 2004. History of carbonate ion concen- teins: imidazole protonation and the conservation of protein struc- tration over the last 100 million years. Geochimica et ture and function. In Transport Processes, Iono- and Cosmochimica Acta, 68: 3521–3530. Osmoregulation, pp. 454–468. Ed. by R. Gilles, and M. Walsh, P. J., Blackwelder, P., Gill, K. A., Danulat, E., and Mommsen, Gilles-Baillien. Springer-Verlag, Berlin. T. P. 1991. Carbonate deposits in marine fish intestines: a new Somero, G. N. 2005. Linking biogeography to physiology: evolution- source of biomineralization. Limnology and Oceanography, 36: ary and acclimatory adjustments of thermal limits. Frontiers in 1227–1232. Zoology, 2: 1. doi:10.1186/1742–9994–2-1. Walsh, P. J., and Milligan, C. L. 1989. Coordination of metabolism and Spero, H. J., Bijma, J., Lea, D. W., and Bemis, B. E. 1997. Effect of sea- intracellular acid-base status: ionic regulation and metabolic con- water carbonate concentration on foraminiferal carbon and oxygen sequences. Canadian Journal of Zoology, 67: 2994–3004. isotopes. Nature, 390: 497–500. Walter, L. M., and Morse, J. W. 1985. Magnesian calcite stabilities: a Spero, H. J., and Lea, D. W. 1993. Intraspecific stable isotope variabil- reevaluation. Geochimica et Cosmochimica Acta, 49: 1503–1513. ity in the planktic foraminifera Globigerinoides sacculifer: results Watanabe, H., Kawaguchi, K., and Hayashi, A. 2002. Feeding habits of from laboratory experiments. Marine Micropaleontology, 22: juvenile surface-migratory myctophid fishes (family Myctophidae) 221–234. in the Kuroshio region of the western North Pacific. Marine Spicer, J. I. 1995. Oxygen and acid-base status of the sea urchin Ecology Progress Series, 236: 263–272. Psammechinus miliaris during environmental hypoxia. Marine Watanabe, Y., Yamaguchi, A., Ishida, H., Harimoto, T., Suzuki, S., Biology, 124: 71–76. Sekido, Y., Ikeda, T., et al. 2006. Lethality of increasing CO2 Spicer, J. I., Raffo, A., and Widdicombe, S. 2007. Influence of levels on deep-sea copepods in the western North Pacific. Journal CO2-related seawater acidification on extracellular acid-base of Oceanography, 62: 185–196. balance in the velvet swimming crab, Necora puber. Marine Weiss, I. M., Tuross, N., Addadi, L., and Weiner, S. 2002. Mollusc Biology, 151: 1117–1125. larval shell formation: amorphous calcium carbonate is the precur- Stillman, J. H. 2003. Acclimation capacity underlies susceptibility to sor phase for aragonite. Journal of Experimental Biology, 293: climate change. Science, 301: 65. 478–491. Tamburrini, M., Romano, M., Carratore, V., Kunzmann, A., Coletta, Willette, T. M., Cooney, R. T., Patrick, V., Mason, D. M., Thomas, G. L., M., and di Prisco, G. 1998. The hemoglobins of the Antarctic and Scheel, D. 2001. Fisheries Oceanography. 10: 14–41. fishes Artedidraco orianae and Pogonophryne scotti. Journal of Yamada, Y., and Ikeda, T. 1999. Acute toxicity of lowered pH to some Biological Chemistry, 273: 32 452–32 459. oceanic zooplankton. Plankton Biology and Ecology, 46: 62–67. Taylor, J. R., and Grosell, M. 2006. Evolutionary aspects of intestinal Zielinski, S., Sartoris, F. J., and Po¨rtner, H. O. 2001. Temperature bicarbonate secretion in fish. Comparative Biochemistry and effects on hemocyanin oxygen binding in an Antarctic cephalopod. Physiology, Part A, 143: 423–529. Biological Bulletin, 200: 67–76. Thibault, D., Roy, S., Wong, C. S., and Bishop, J. K. 1999. The downward flux of biogenic material in the NE subarctic Pacific: doi:10.1093/icesjms/fsn048 or collective redistirbution other or collective or means this reposting, of machine, is by photocopy article only anypermitted of with portion the approval The O of This article has been published in Oceanography published been This has article Special Issue Feature

By Richard A. Feely, Scott C. Doney, and Sarah R. Cooley , Volume 4, a quarterly 22, Number The O journal of Ocean Acidification ceanography Society. © 2009 by The O ceanography Society. A ceanography Society. Send all correspondence to:ceanography Society. Send [email protected] Th e O or ll rights reserved. P reserved. ll rights ermission is granted to in teaching copy this and research. for use article R

Present Conditions

and Future Changes 1931, R Box ceanography Society, PO in a High-CO2 World epublication, systemmatic reproduction, ockville, M D 20849-1931, U ockville, S A .

36 Oceanography Vol.22, No.4 Abstract 2005; Orr et al., 2005), with a decrease -1 The uptake of anthropogenic CO2 by the global ocean induces fundamental changes of ~ 0.0018 yr observed over the last in seawater chemistry that could have dramatic impacts on biological ecosystems in quarter century at several open-ocean the upper ocean. Estimates based on the Intergovernmental Panel on Climate Change time-series sites (Bates, 2007; Bates and

(IPCC) business-as-usual emission scenarios suggest that atmospheric CO2 levels Peters, 2007; Santana-Casiano et al., could approach 800 ppm near the end of the century. Corresponding biogeochemical 2007; Dore et al., 2009). By the middle models for the ocean indicate that surface water pH will drop from a pre-industrial of this century, atmospheric CO2 levels value of about 8.2 to about 7.8 in the IPCC A2 scenario by the end of this century, could reach more than 500 ppm, and increasing the ocean’s acidity by about 150% relative to the beginning of the industrial exceed 800 ppm by the end of the era. In contemporary ocean water, elevated CO2 will also cause substantial reductions century (Friedlingstein et al., 2006). in surface water carbonate ion concentrations, in terms of either absolute changes These CO2 levels would result in an or fractional changes relative to pre-industrial levels. For most open-ocean surface additional decrease in surface water waters, aragonite undersaturation occurs when carbonate ion concentrations drop pH of 0.3 nits from current conditions, below approximately 66 µmol kg-1. The model projections indicate that aragonite 0.4 from pre-industrial, by 2100, which undersaturation will start to occur by about 2020 in the Arctic Ocean and 2050 in represents an increase in the ocean’s the Southern Ocean. By 2050, all of the Arctic will be undersaturated with respect to hydrogen ion (H+) concentration by aragonite, and by 2095, all of the Southern Ocean and parts of the North Pacific will 2.5 times relative to the beginning of the be undersaturated. For calcite, undersaturation occurs when carbonate ion concen- industrial era. Results from large-scale -1 tration drops below 42 µmol kg . By 2095, most of the Arctic and some parts of the ocean CO2 surveys and time-series Bering and Chukchi seas will be undersaturated with respect to calcite. However, in studies over the past two decades show most of the other ocean basins, the surface waters will still be saturated with respect to that ocean acidification is a predictable calcite, but at a level greatly reduced from the present. consequence of rising atmospheric CO2 (Feely et al., 2004; Bates and Peters, Introduction and Feely, 2007; Canadell et al., 2007). 2007; Santana-Casiano et al., 2007; Dore Since the beginning of the industrial This absorption has benefited human- et al., 2009; Takahashi et al., 2009) that revolution in the mid-eighteenth kind by significantly curtailing the is independent of the uncertainties and century, the release of carbon dioxide growth of CO2 levels in the atmosphere, outcomes of climate change.

(CO2) from humankind’s combined thereby reducing the global warming Seawater carbonate chemistry is industrial and agricultural activities has realized to date. governed by a series of abiotic chemical resulted in an increase in atmospheric However, when the anthropogenic reactions (CO2 dissolution, acid/base

CO2 concentrations from approximately CO2 is absorbed by seawater, chemical chemistry) and biologically mediated 280 to 387 parts per million (ppm), with reactions occur that reduce seawater reactions (photosynthesis, respiration, 2– as much as 50% of the increase occurring pH, carbonate ion (CO3 ) concen- and CaCO3 precipitation and dissolu- in the last three decades. Earth’s atmo- tration, and saturation states of the tion). The first key reaction occurs when spheric concentration of CO2 is now biologically important CaCO3 minerals CO2 gas from the atmosphere dissolves higher than it has been for more than calcite (Ωca) and aragonite (Ωar) in into seawater: 800,000 years (Lüthi et al., 2008), and a process commonly referred to as CO (atm) CO (aq). (1) it is expected to continue to rise at an “ocean acidification” (Broecker and 2 2 accelerating rate, leading to significant Clarke, 2001; Caldeira and Wickett, This exchange is relatively rapid, with temperature increases in the atmosphere 2003, 2005; Orr et al., 2005; Doney atmosphere-ocean equilibration on a and the surface ocean in the coming et al., 2009; Figure 1). The pH of ocean time scale of several months, so that decades. Over the industrial era, the surface waters has already decreased surface waters in most ocean regions ocean has absorbed about one-quarter of by about 0.1 since the industrial era increase in CO2 concentration from year anthropogenic carbon emissions (Sabine began (Caldeira and Wickett, 2003, to year in proportion to the increased

Oceanography December 2009 37 400 8.40 CO concentration in the atmosphere. (atm CO2)y = 1.811x – 3252.4 2 R2 = 0.95, st err = 0.028 The second set of reactions involves 8.35 the hydration of water to form H CO , 375 LEGEND 2 3

Mauna Loa atmospheric CO2 (ppmv) 8.30 which dissociates in subsequent acid- Aloha seawater pCO2 (µatm) base reactions: Aloha seawater pH 8.25 350 CO2(aq) + H2O H2CO3

2 (2) 8.20 H+ + HCO− 2H+ + CO 2–. pH 3 3 CO 325 8.15 These reactions are very rapid, on time (pCO )y = 1.90x – 3453.96 2 scales of tens of seconds for CO2 hydra- R2 = 0.3431, st err = 0.20 8.10 tion and microseconds for subsequent 300 acid-base reactions (Zeebe and Wolf- 8.05 (pH)y = -0.00188x + 11.842 Gladrow, 2001; Dickson et al., 2007). R2 = 0.289, st err = 0.00022 In most applications, the partitioning 275 8.00 1960 1970 1980 1990 2000 2010 of inorganic carbonate species can be assumed to be in equilibrium. For 4.9 Ωca typical surface ocean conditions, about 5.9 y = -0.012x + 29.615 4.7 90% of the total dissolved inorganic R2 = 0.159, st err = 0.0021 5.7 carbon (DIC) occurs as bicarbonate ions 4.5 – LEGEND (HCO 3) and ~ 9% as carbonate ions ) ) 2– ar Surface Ωca saturation 5.5 ca (CO3 ), with only ~1% remaining as 4.3 Surface Ω saturation ar dissolved CO2(aq) and H2CO3. The third 5.3 key reaction involves the formation and 4.1 dissolution of solid calcium carbonate 5.1 3.9 160°W 158°W 156°W minerals (CaCO3(s)): 23°N Saturation State (Ω Saturation State (Ω Station Aloha 2+ 2– 3.7 22°N 4.9 Ca + CO3 CaCO3(s). (3) Kailua

Honolulu 21°N Kahului Kihei Ω At equilibrium, the calcium concen- 3.5 ar 4.7 20°N Hilo Station Mauna Loa y = -0.00758x + 18.86 tration times the carbonate concen- 2 19°N R = 0.125, st err = 0.0015 3.3 4.5 tration is equal to a constant called 1960 1970 1980 1990 2000 2010 the apparent solubility product Year

Figure 1. (Top) Time series of atmospheric CO2 at Mauna Loa (ppmv) and surface Richard A. Feely (richard.a.feely@ ocean pH and pCO2 (µatm) at Ocean Station Aloha in the subtropical North Pacific noaa.gov) is Senior Scientist, Pacific Ocean (see inset map). Note that the increase in oceanic CO2 over the period of observations is consistent with the atmospheric increase within the statistical limits Marine Environmental Laboratory, of the measurements. (Bottom) Calcite and aragonite saturation data for surface National Oceanic and Atmospheric waters. Mauna Loa data: Pieter Tans, NOAA/ESRL, http://www.esrl.noaa.gov/gmd/ Administration, Seattle, WA, USA. ccgg/trends. HOT/ALOHA data: David Karl, University of Hawaii, http://hahana.soest. hawaii.edu. Updated from Doney et al. (2009). See also Dore et al. (2009). Scott C. Doney is Senior Scientist, Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA, USA. Sarah R. Cooley is Postdoctoral Investigator, Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA, USA.

38 Oceanography Vol.22, No.4 2+ 2– ([Ca ] x [CO3 ] = K'sp). The satu- are characteristic of the pre-industrial horizon is governed primarily by the 2– ration state of calcium carbonate ocean, and therefore decreasing CO3 pressure effect on CaCO3 solubility. The mineral phases is defined by concentrations could decrease calcifica- shells and skeletons of the unprotected 2+ 2– Ω = [Ca ][CO3 ] / K'sp. The apparent tion rates for a number of these species. CaCO3 phases begin to dissolve when the solubility product varies with tempera- Over longer time scales, the ocean’s degrees of saturation of their component ture, salinity, and pressure, and differs capacity for absorbing more CO2 from minerals drop below Ω = 1, so some of among calcium carbonate minerals the atmosphere also depends on the these shells and skeletons dissolve before (e.g., calcite, aragonite, and high-Mg extent of marine carbonate interactions reaching deep sediments below the calcite; Mucci, 1983). The final impor- with CO2 via the dissolution reaction: “saturation horizon,” or the depth where tant reaction is photosynthesis and seawater becomes undersaturated with CO2 + CaCO3 + H2O respiration/decomposition: (5) respect to CaCO3. The locations of the 2HCO− + Ca2+. 3 saturation horizons where Ω = 1 differ CO + H O CH O + O . (4) 2 2 2 2 The increase in total alkalinity (TA) for different carbonate minerals, varying

As CO2 concentrations increase in from this reaction enhances the ocean’s the response of materials composed of seawater, the CO2 gas reacts with water capacity to absorb more CO2 from the aragonite, calcite, or high-magnesium to form H2CO3. Most of the H2CO3 atmosphere (Zeebe and Wolf-Gladrow, carbonate. As the ocean becomes + − dissociates to form a H and a HCO3, 2001). TA is a measure of the excess enriched in anthropogenic CO2, the and most of the resulting H+ reacts with cations (such as Ca2+ and Mg2+) that are locations in the ocean where dissolution 2– − CO3 to produce additional HCO3 ions. balanced by the anions formed by disso- occurs will increase as a function of the

As a result, CO2 dissolution in the ocean ciations of carbonic, boric, and other decrease in CaCO3 saturation state. + increases H (and thus decreases pH) weak acids in seawater. The primary The Ω = 1 chemical threshold is a 2– and decreases CO3 concentrations. contributors to the dissolution reaction useful indicator but not a strict criterion 2– The decrease in CO3 reduces the in Equation 5 are the carbonate shells for biomineralization and dissolution. saturation state of CaCO3, which directly that are produced in the euphotic zone. Some calcifying organisms require affects the ability of some CaCO3- Upon a calcifying organism’s death, its ambient seawater conditions well secreting organisms, such as planktonic carbonate shell falls through the water above saturation, while others may be coccolithophores and pteropods, and invertebrates such as mollusks and corals, to produce their shells or skeletons The uptake of anthropogenic CO2 by (Equation 3, and Doney et al., 2009). This the global ocean induces fundamental is true even though most surface waters in the global ocean are currently super- changes in seawater chemistry that saturated with respect to CaCO (e.g., could have dramatic impacts on biological 3 “ oceanic Ω values of 2–4 for aragonite and ecosystems in the upper ocean. 4–6 for calcite, where Ω = 1 expresses saturation), because of the exceedingly slow abiotic precipitation rates for CaCO3 column and is either deposited in sedi- able to generate or maintain calcified minerals (Table 1). When seawater is ments or dissolved. structures in undersaturated condi- supersaturated with respect to aragonite The saturation states of carbonate tions at a bioenergetic cost.” Similarly, and calcite (saturation levels > 1), calcifi- minerals naturally decrease with depth CaCO3 dissolution can occur in the cation is favored more than dissolution. as total dissolved CO2 increases because water column above the saturation Many organisms have optimal calcium of biological respiration and cold horizon in more acidic microenviron- carbonate precipitation rates at the super- temperatures in deep seawater. Below ments such as marine snow particles and saturated states for these minerals that about 1500 m, the depth of the saturation zooplankton guts.

Oceanography December 2009 39 Data Sets and carbon parameters were measured on called GLODAP), which consists of Modeling Approach all the cruises, but the carbon system about 72,000 sample locations where From 1990 through 1998, carbon system pairs that were measured varied between DIC and TA were collected. The data measurements were made on 99 WOCE/ cruises. The overall accuracy of the are available from the Carbon Dioxide JGOFS (World Ocean Circulation DIC data was ±3 µmol kg-1, and for Information Analysis Center (http:// Experiment/Joint Global Ocean Flux TA, the second most common carbon cdiac.esd.ornl.gov/oceans/).

Study) CO2 survey cruises in the global parameter analyzed, the overall accuracy The National Center for Atmospheric ocean (Key et al., 2004; Sabine et al., was ±5 µmol kg-1. Research (NCAR) Community Climate 2005). These cruises were conducted as The data were corrected for systematic System Model 3.1 (CCSM3) was used a collaborative effort among 15 labora- biases between cruises and maintained to estimate the long-term changes in tories from eight countries. At least two as a unique corrected data set (hereafter the saturation state of seawater with

Table 1. Average concentrations of carbon system parameters and temperature-and-salinity values for surface waters of the major ocean basins based on the Global Ocean Data Analysis Project data set.

2Dissolved 3pH 3Carbonate 1Tempera- Inorganic 2Alkalinity Ocean 1Salinity (Seawater Ion 3Ω 3Ω ture (°C) Carbon (µmol kg-1) ar ca Scale) (µmol kg-1) (µmol kg-1) 4Arctic Ocean 34.639 4.35 2069.7 2299.7 8.231 160.4 2.41 3.82 North of 65°N ± 0.53 ± 3.0 ± 36 ± 27 ± 0.06 ± 18 ± 0.3 ± 0.4 North Atlantic 35.643 19.56 2040.1 2355.8 8.125 222.7 3.47 5.31 60°W to 0° ± 1.37 ± 7.3 ± 38 ± 48 ± 0.06 ± 34 ± 0.6 ± 0.8 0° to 64.5°N South Atlantic 36.022 22.54 2045.4 2379.9 8.102 236.6 3.70 5.63 70°W to 20°E ± 0.79 ± 3.7 ± 25 ± 36 ± 0.03 ± 22 ± 0.4 ± 0.5 0° to 40°S North Pacific 34.051 20.81 1959.3 2255.0 8.105 208.1 3.30 5.04 120°E to 60°W ± 0.86 ± 8.1 ± 42 ± 34 ± 0.06 ± 38 ± 0.7 ± 0.9 0° to 59.5°N South Pacific 35.287 23.29 2003.0 2319.3 8.079 223.4 3.52 5.36 120°E to 70°W ± 0.55 ± 4.3 ± 39 ± 35 ± 0.03 ± 25 ± 0.4 ± 0.6 0° to 40°S North Indian 34.722 28.12 1951.6 2298.2 8.068 244.4 3.94 5.93 20°E to 120°E ± 1.57 ± 0.8 ± 64 ± 68 ± 0.03 ± 12 ± 0.2 ± 0.3 0° to 24.5°N South Indian 35.103 23.46 1983.8 2306.0 8.092 227.0 3.59 5.46 20°E to 120°E ± 0.47 ± 4.3 ± 54 ± 36 ± 0.03 ± 19 ± 0.4 ± 0.5 0° to 40°S Southern Ocean 34.775 16.74 2034.5 2306.9 8.102 193.1 3.02 4.64 South of 40°S ± 0.94 ± 10.5 ± 77 ± 45 ± 0.04 ± 54 ± 0.9 ± 1.3 34.680 16.09 2033.7 2302.7 8.108 190.6 2.98 4.58 All ± 1.02 ± 10.4 ± 75 ± 48 ± 0.05 ± 53 ± 0.9 ± 1.3 Note: the ± values in the table give the standard deviations about the regional means. 1 World Ocean Atlas 2005 data for time period: Annual, http://www.nodc.noaa.gov/OC5/WOA05/pr_woa05.html 2 GLODAP version 1.1 gridded data, http://cdiac.ornl.gov/oceans/glodap/data_files.htm 3 Calculated values using co2sys.xls, http://www.ecy.wa.gov/programs/eap/models/co2sys_ver14.zip 4 Arctic Ocean data (N=70) from GLODAP version 1.1 bottle data, http://cdiac.ornl.gov/oceans/glodap/data_files.htm

40 Oceanography Vol.22, No.4 respect to aragonite and calcite (Doney nodc.noaa.gov/OC5/WOA05/pr_woa05. parameters, GLODAP data were inter- et al., 2006; Steinacher et al., 2009). The html) 1° x 1° gridded temperature and polated to the same 2° x 2° regular modeled decadal mean values were salinity data were used to calculate grid as CCSM3 data. calculated using modeled monthly GLODAP-based carbonate system dissolved inorganic carbon, alkalinity, parameters. For both CCSM3 and Results and Discussion temperature, and salinity output GLODAP-based data sets, carbonate Calcification or dissolution of both from CCSM3, which uses full carbon system parameters were calculated planktonic and benthic calcifying cycle–climate radiative feedbacks and using a standard polynomial solver by organisms commonly depends on the assumes historical fossil fuel emis- Richard Zeebe (University of Hawaii) carbonate ion concentration, often sions and the IPCC Special Report on for Matlab (http://www.soest.hawaii. expressed by the degree of saturation Emissions Scenarios (SRES) noninter- edu/oceanography/faculty/zeebe_files/ of the biominerals aragonite and calcite vention future A2 scenario (Thornton CO2_System_in_Seawater/csys.html) (see Feely et al., 2004, and references et al., 2009). The model’s variable grid and the thermodynamic carbonate therein). Consequently, spatial and (0.9–3.6° latitude x 3.6° longitude) was dissociation constants from Mehrbach temporal changes in saturation state interpolated to a 2° x 2° regular grid et al. (1973) (refit by Lueker et al., 2000), with respect to these mineral phases for analysis. GLODAP 1° x 1° gridded total pH scale, and other dissociation are important for understanding how surface dissolved inorganic carbon and constants reviewed in Zeebe and Wolf- ocean acidification might impact future total alkalinity (http://cdiac.ornl.gov/ Gladrow (2001). Before calculating ecosystems. Figures 2 and 3 show mean 2– oceans/glodap/Glodap_home.htm) and the difference between GLODAP ocean surface pH and CO3 concen- World Ocean Atlas 2005 (http://www. and CCSM-based carbonate system trations for the Pacific, Atlantic, and

Figure 2. (Top and middle rows) National Center for Atmospheric Research Community Climate System Model 3.1 (CCSM3)-modeled decadal mean pH at the sea surface centered around the years 1875, 1995, 2050, and 2095. (Bottom left) Global Ocean Data Analysis Project (GLODAP)-based pH at the sea surface, nominally for 1995. (Bottom right) The difference between the GLODAP-based and CCSM- based 1995 fields. Note the different range of the difference plot. Deep coral reefs are indicated by darker gray dots; shallow-water coral reefs are indicated with lighter gray dots. White areas indicate regions with no data.

Oceanography December 2009 41 Figure 3. (Top and middle rows) CCSM3- 2– modeled decadal mean CO3 at the sea surface centered around the years 1875, 1995, 2050, 2– and 2095. (Bottom left) GLODAP-based CO3 at the sea surface, nominally for 1995. (Bottom right) The difference between the GLODAP- based and CCSM-based 1995 fields. Note the different range of the difference plot. Deep coral reefs are indicated by darker gray dots; shallow-water coral reefs are indicated with lighter gray dots. White areas indicate regions with no data.

2– Indian oceans from the CCSM3 model CO2 (high pH, high CO3 ) during the projections suggest that this will start projections for the decades centered on spring–summer months when produc- to occur at about 2020 in the Arctic 1875, 1995, 2050, and 2095, as well as tivity is strongest and when most of the Ocean (Steinacher et al., 2009) and 2050 the difference between the GLODAP surveys occurred. Because the modeled in the Southern Ocean south of 65°S gridded data and the 1995 model results for 1995 are decadal averages, it is (Orr et al., 2005) in the A2 business-as- results, respectively. Based on the not too surprising that the largest data- usual scenario. By 2050, all of the Arctic GLODAP data set, open-ocean surface model differences occur in these highly will be undersaturated with respect to water pH ranges from about 7.95–8.35 productive subpolar regions. aragonite (Figure 4), and by 2095, all of 2– (mean = 8.11); CO3 concentrations Model projections indicate that the Southern Ocean south of 60°S and range from about 80–300 µmol kg-1 surface ocean pH will drop from a parts of the North Pacific will be under- (mean = 191 µmol kg-1); aragonite pre-industrial average of about 8.2 to saturated. In the Arctic Ocean, the rapid saturation values range from approxi- a mean of about 7.8 in the IPCC A2 decrease in saturation state occurs, in mately 2.4–3.9; and calcite saturation scenario by the end of this century. For part, as a direct result of the decrease in 2– from approximately 3.8–5.9 (Table 1). CO3 , the highest concentrations are TA in surface waters due to the increase GLODAP pH and carbonate ion values in the tropical and subtropical regions, in freshwater inflow from low-alkalinity are higher in highly productive regions particularly on the western side of each river water and from ice melt (Orr of the western North and South Atlantic, basin where the waters are warmer and et al., 2005). For calcite, undersatura- 2– western North and South Pacific, South saltier. For most open-ocean surface tion occurs when CO3 drops below Indian Ocean, Bering Sea, and most of waters, aragonite undersaturation occurs 42 µmol kg-1 (Figure 5). By 2095, most of 2– the Southern Ocean (Figures 2 and 3). when CO3 concentrations drop below the Arctic and some parts of the Bering These regions have a large uptake of approximately 66 µmol kg-1. Model and Chukchi seas will be undersaturated

42 Oceanography Vol.22, No.4 Figure 4. (Top and middle rows) CCSM3- modeled decadal mean Ωar at the sea surface, centered around the years 1875, 1995, 2050, and

2095. (Bottom left) GLODAP-based Ωar at the sea surface, nominally for 1995. (Bottom right) The difference between the GLODAP-based and CCSM-based 1995 fields. Note the different range of the difference plot. Deep coral reefs are indicated by darker gray dots; shallow-water coral reefs are indicated with lighter gray dots. White areas indicate regions with no data.

Figure 5. (Top and middle rows) CCSM3- modeled decadal mean Ωca at the sea surface centered around the years 1875, 1995, 2050, and

2095. (Bottom left) GLODAP-based Ωca at the sea surface, nominally for 1995. (Bottom right) The difference between the GLODAP-based and CCSM-based 1995 fields. Note the different range of the difference plot. Deep coral reefs are indicated by darker gray dots; shallow-water coral reefs are indicated with lighter gray dots. White areas indicate regions with no data.

Oceanography December 2009 43 with respect to calcite. However, in most saturation state profiles for aragonite rapidly in subsurface waters because of of the other ocean basins the surface (solid line) and calcite (dashed line) are the release of CO2, primarily from circu- waters will still be oversaturated with plotted along with depth profiles of pH lation and respiration processes within 2– respect to calcite, but at a level greatly and CO3 concentrations for each of the subsurface water masses. Portions of reduced from the present. the basins (Figure 6). The depth profiles the eastern subtropical Atlantic become 2– In Figure 6, average basin-wide show that both pH and CO3 decrease undersaturated with respect to aragonite between 400 and 1000 m, and all of the Atlantic becomes undersaturated with respect to calcite below a depth of approximately 3000–3500 m (Chung et al., 2004; Feely et al., 2004). In the Indian and Pacific oceans, undersaturation of aragonite begins at much shal- 2– lower depths where the CO3 drops below a value of about 66 µmol kg-1 (depth range of 100–1200 m, depending on location) because the waters are much

older and have acquired more CO2 from the cumulative effects of respiration while they travel along the transport pathway of the deeper water masses. The calcite saturation depths in the Indian and Pacific are generally about 50–200 m deeper than the aragonite saturation depth. The addition of anthropogenic

CO2 over the past 250 years has caused the aragonite saturation horizons to shoal toward the surface in all ocean basins by varying amounts, ranging from about 50–200 m, depending on location (Feely et al., 2004; Orr et al., 2005). Because aragonite and calcite saturation values are highest in warm tropical and subtropical oceans, these regions might also experience the largest absolute changes in saturation state by the end of the century. Figure 7 and Table 2 show the projected absolute and relative changes, or fractional changes, of

2– aragonite state from 1865 to 2095. The Figure 6. Distribution of: (A) pH and (B) CO3 concentration in the Pacific, Atlantic, and Indian oceans. The data are from the World Ocean Circulation Experiment/Joint Global Ocean Flux largest projected decreases in aragonite Study/Ocean Atmosphere Carbon Exchange Study global CO2 survey data (Sabine et al., 2005). saturation between 1865 and 2095 are 2– The lines show the average aragonite (solid line) and calcite (dashed line) saturation CO3 concentration for each of these basins. The color coding shows the latitude bands for the data observed in warm tropical and subtrop- sets (after Feely et al., 2009). ical waters (Figure 7, top). For example,

44 Oceanography Vol.22, No.4 et al., 2009), implying that future low- latitude conditions could stress tropical species by significantly altering the conditions to which they have adapted. On the other hand, the relative 2– changes in CO3 concentrations and aragonite and calcite saturation states are highest in the high latitudes. There, the large changes in carbonate chemistry relative to historical conditions, which are captured in both the large percent changes in modeled carbonate system parameters and the saturation state

changes from supersaturation (Ω > 1)

to undersaturation (Ω < 1), might have large impacts on high-latitude calcifying planktonic and benthic organisms such as pteropods, mussels, and oysters (Fabry et al., 2008; Doney et al., 2009). Whether 2– absolute or relative changes in CO3 concentrations and/or aragonite and calcite saturation state play a controlling role in overall ecosystem changes has yet to be determined. Nevertheless, the striking decreases in aragonite and calcite saturation state projected by the end of this century over the entire ocean clearly indicate that there is a reasonable cause for concern. Figure 7. For the Intergovernmental Panel on Climate Change Special Report on Emissions

Scenarios A2 scenario, (top) the CCSM-modeled decrease in surface Ωar between the decades centered around the years 1875 and 2095 (∆Ωar = Ωar, 1875 – Ωar, 2095), and (bottom) the CCSM- Conclusions modeled percent decrease in surface Ωar between the decades centered around 1875 and 2095 The oceanic uptake of anthropogenic (100 x ∆Ωar, 2095 /Ωar, 1875). Deep coral reefs are indicated by darker gray dots; shallow-water coral reefs are indicated with lighter gray dots. White areas indicate regions with no data. CO2 from the atmosphere is decreasing 2– the pH and lowering the CO3 concen-

tration and CaCO3 saturation states of aragonite and calcite in the upper ocean. in the tropical and subtropical Atlantic, these two extremes. In the tropics and Ocean acidification is expected to result the average decrease in aragonite satura- subtropics, many species of tropical in a pH decrease of ~ 0.3–0.4 units relation state between the present-day levels corals are extremely sensitive to any tive to pre-industrial values by the end of and a 3XCO2-world (~ 840 µatm) is decreases in saturation state (Langdon, this century. Based on the best available -0.81, whereas in the high-latitude North 2002). In these same locations, natural information and model projections, it

Atlantic, it is only -0.39 (Table 2). The variability in the ocean carbonate system appears that as levels of dissolved CO2 temperate latitude differences are gener- is much smaller than forecasted anthro- in seawater rise, the skeletal growth rates ally somewhere in the middle between pogenically driven changes (Cooley of calcium-secreting organisms will be

Oceanography December 2009 45 Table 2. Absolute and relative changes in pH, carbonate ion, and aragonite and calcite saturation states for 2X and 3X pre-industrial CO2 levels at various locations in the Atlantic and Pacific oceans based on National Center for Atmospheric Research Community Climate System Model 3.1 results.

Carbonate fCO ∆ % % % Location 2 pH Δ pH ion Ω Δ Ω Ω Δ Ω µatm Carbonate Change ar ar Change ca ca Change µmol kg-1 North 387 8.040 96.8 1.47 2.34 Atlantic 560 7.893 -0.15 71.2 -25.6 -26.5 1.08 -0.39 -26.5 1.72 -0.62 -26.5 > 50°N 840 7.727 -0.31 49.9 -46.9 -48.4 0.76 -0.71 -48.4 1.21 -1.13 -48.4 North 387 8.032 92.3 1.40 2.24 Pacific 560 7.885 -0.15 67.8 -24.5 -26.5 1.03 -0.37 -26.5 1.65 -0.59 -26.5 > 50°N 840 7.719 -0.31 47.5 -44.8 -48.5 0.72 -0.68 -48.5 1.15 -1.09 -48.5 387 8.055 237.5 3.85 5.81 Tropical 560 7.923 -0.13 187.5 -50.0 -21.0 3.04 -0.81 -21.0 4.59 -1.22 -21.0 Atlantic 840 7.772 -0.28 140.6 -97.0 -40.8 2.28 -1.57 -40.8 3.44 -2.37 -40.8 387 8.035 234.4 3.81 5.71 Tropical 560 7.905 -0.13 185.9 -48.5 -20.7 3.02 -0.79 -20.7 4.53 -1.18 -20.7 Pacific 840 7.755 -0.28 139.9 -94.5 -40.3 2.28 -1.54 -40.3 3.41 -2.30 -40.3 387 8.052 169.3 2.59 4.02 Subpolar 560 7.913 -0.14 129.4 -39.9 -23.6 1.98 -0.61 -23.6 3.07 -0.95 -23.6 Atlantic 840 7.756 -0.30 93.9 -75.3 -44.5 1.44 -1.15 -44.5 2.23 -1.79 -44.5 387 8.038 134.5 2.06 3.24 Subpolar 560 7.896 -0.14 101.3 -33.2 -24.7 1.55 -0.51 -24.7 2.44 -0.80 -24.7 Pacific 840 7.736 -0.30 72.5 -62.0 -46.1 1.11 -0.95 -46.1 1.75 -1.49 -46.1

reduced due to the effects of decreased References Canadell, J.G., C. Le Quéré, M.R. Raupach, 2– Bates, N.R. 2007. Interannual variability of the C.B. Field, E.T. Buitenhuis, P. Ciais, T.J. Conway, ocean CO3 concentrations. If anthropo- oceanic CO2 sink in the subtropical gyre of the N.P. Gillett, R.A. Houghton, and G. Marland. 2007. genic CO2 emissions are not dramatically North Atlantic Ocean over the last 2 decades. Contributions to accelerating atmospheric CO2 reduced in the coming decades, there Journal of Geophysical Research 112, C09013, growth from economic activity, carbon intensity, doi:10.1029/2006JC003759. and efficiency of natural sinks. Proceedings of is the potential for direct and profound Bates, N.R., and A.J. Peters. 2007. The contribution of the National Academy of Sciences of the United impacts on our living marine ecosystems. atmospheric acid deposition to ocean acidification States of America 104(47):18,866–18,870, in the subtropical North Atlantic Ocean. Marine doi:10.1073pnas.0702737104. Chemistry 107(4):547–558. Chung, S.N., G.-H. Park, K. Lee, R.M. Key, F.J. Millero, Acknowledgements Broecker, W., and E. Clarke. 2001. A dramatic Atlantic R.A. Feely, C.L. Sabine, and P.G. Falkowski. 2004. S. Cooley and S. Doney acknowledge dissolution event at the onset of the last glaciation. Postindustrial enhancement of aragonite under- Geochemistry Geophysics Geosystems 2(11):1,065, saturation in the upper tropical and subtropical support from NSF ATM-0628582. doi:10.1029/2001GC000185. Atlantic Ocean: The role of fossil fuel CO2. Richard A. Feely was supported by the Caldeira, K., and M.E. Wickett. 2003. Anthropogenic Limnology and Oceanography 49(2):315–321. carbon and ocean pH. Nature 425(6956):365. Cooley, S.R., H.L. Kite-Powell, and S.C. Doney. NOAA Climate Program under the Caldeira, K., and M.E. Wickett. 2005. Ocean model 2009. Ocean acidification’s potential to alter Office of Climate Observations (Grant predictions of chemistry changes from carbon global marine ecosystem services. Oceanography 22(4):172–181. No. GC04-314 and the Global Carbon dioxide emissions to the atmosphere and ocean. Journal of Geophysical Research 110, C09S04, Dickson, A.G., C.L. Sabine, and J.R. Christian, eds. Cycle Program (Grant No. GC05-288). doi:10.1029/2004JC002671. 2007. Guide to Best Practices for Ocean CO2 Pacific Marine Environmental Lab- Measurements. PICES Special Publication 3, 191 pp. oratory contribution number 3463.

46 Oceanography Vol.22, No.4 Doney, S.C., V.J. Fabry, R.A. Feely, and J.A. Kleypas. Mehrbach, C., C.H. Culberson, J.E. Hawley, and

2009. Ocean acidification: The other 2CO problem. R.M. Pytkowicz, 1973. Measurement of the Annual Reviews of Marine Science 1:169–192. apparent dissociation constants of carbonic Doney, S.C., K. Lindsay, I. Fung, and J. John. 2006. acid at atmospheric pressure. Limnology and Natural variability in a stable 1000 year coupled Oceanography 18:897–907. climate-carbon cycle simulation. Journal of Mucci, A. 1983. The solubility of calcite and aragonite Climate 19(13):3,033–3,054. in seawater at various salinities, temperatures, and Dore, J.E., R. Lukas, D.W. Sadler, M.J. Church, and one atmosphere total pressure. American Journal of D.M. Karl. 2009. Physical and biogeochemical Science 283(7):780–799. modulation of ocean acidification in the central Orr, J.C., V.J. Fabry, O. Aumont, L. Bopp, S.C. Doney, North Pacific. Proceedings of the National R.A. Feely, A. Gnanadesikan, N. Gruber, A. Academy of Sciences of the United States of Ishida, F. Joos, and others. 2005. Anthropogenic America 106(30):12,235–12,240. ocean acidification over the twenty-first century Fabry, V.J., B.A. Seibel, R.A. Feely, and J.C. Orr. 2008. and its impact on calcifying organisms. Nature Impacts of ocean acidification on marine fauna 437(7059):681–686. and ecosystem processes. ICES Journal of Marine Sabine, C.L., and R.A. Feely. 2007. The oceanic sink for Science 65(3):414–432. carbon dioxide. Pp. 31–49 in Greenhouse Gas Sinks. Feely, R.A., J. Orr, V.J. Fabry, J.A. Kleypas, C.L. Sabine, D. Reay, N. Hewitt, J. Grace, and K. Smith, eds, and C. Langdon. 2009. Present and future changes CABI Publishing, Oxfordshire, UK. in seawater chemistry due to ocean acidification. Sabine, C.L., R.M. Key, A. Kozyr, R.A. Feely, R. Section 3 in Carbon Sequestration and Its Role Wanninkhof, F.J. Millero, T.-H. Peny, J.L. in the Global Carbon Cycle. B.J. McPherson and Bullister, and K. Lee. 2005. Global Ocean Data E.T. Sundquist, eds, Geophysical Monograph Analysis Project (GLODAP): Results and Data. Series, Vol. 83, American Geophysical Union, ORNL/CDIAC-15, NDP-083, Carbon Dioxide Washington, DC. Information Analysis Center, Oak Ridge National Feely, R.A., C.L. Sabine, K. Lee, W. Berelson, Laboratory, US Department of Energy, Oak J. Kleypas, V.J. Fabry, and F.J. Millero. 2004. Impact Ridge, TN. Available online at: http://cdiac.

of anthropogenic CO2 on the CaCO3 system in the ornl.gov/oceans/glodap/pubs.htm (accessed oceans. Science 305(5682):362–366, doi:10.1126/ November 13, 2009). science.1097329. Santana-Casiano, J.M., M. Gonzalez-Davila, M.J. Friedlingstein, P., P. Cox, R. Betts, C. Jones, W. von Rueda, O. Llinas, and E.F. Gonzalez-Davila. 2007.

Bloh, V. Brovkin, P. Cadule, S. Doney, M. Eby, The interannual variability of oceanic CO2 param- D. Matthews, and others. 2006. Climate-carbon eters in the Northeast Atlantic subtropical gyre at cycle feedback analysis: Results from the C*MIP the ESTOC site. Global Biogeochemical Cycles 21, model intercomparison. Journal of Climate GB1015, doi:10.1029/2006GB002706. 19(14):3,337–3,353. Steinacher, M., F. Joos, T.L. Frolicher, G.-K. Plattner, Key, R.M., A. Kozyr, C.L. Sabine, K. Lee, R. and S.C. Doney. 2009. Imminent ocean acidifi- Wanninkhof, J.L. Bullister, R.A. Feely, F.J. cation in the Arctic projected with the NCAR Millero, C. Mordy, and T.-H. Peng. 2004. A global coupled carbon cycle-climate model. global ocean carbon climatology: Results from Biogeosciences 6:515–533. Global Data Analysis Project (GLODAP). Takahashi, T., S.C. Sutherland, R. Wanninkhof, Global Biogeochemical Cycles 18, GB4031, C. Sweeney, R.A. Feely, D.W. Chipman, B. Hales, doi:10.1029/2004GB002247. G. Friederich, F. Chavez, C. Sabine, and others. Langdon, C. 2002. Review of experimental evidence 2009. Climatological mean and decadal change

for effects of CO2 on calcification of reef builders. in surface ocean pCO2, and net sea-air CO2 flux Proceedings of the 9th International Coral Reef over the global oceans. Deep Sea Research Part II Symposium 2:1,091–1,098 (Symposium met 56(8–10):554–577. October 23–27, 2000, in Bali, Indonesia). Thornton, P.E., S.C. Doney, K. Lindsay, J.K. Moore, Lueker, T.J., A.G. Dickson, and C.D. Keeling. 2000. N. Mahowald, J.T. Randerson, I. Fung,

Ocean pCO2 calculated from dissolved inorganic J.-F. Lamarque, J.J. Feddema, and Y.-H. Lee. carbon, alkalinity, and equations for K-1 and K-2: 2009. Carbon-nitrogen interactions regulate Validation based on laboratory measurements of climate-carbon cycle feedbacks: Results from an

CO2 in gas and seawater at equilibrium. Marine atmosphere-ocean general circulation model. Chemistry 70(1–3):105–119. Biogeosciences 6:2,099–2,120.

Lüthi, D., M. Le Floch, B. Bereiter, T. Blunier, J.-M. Zeebe, R.E., and D. Wolf-Gladrow. 2001. CO2 in Barnola, U. Siegenthaler, D. Raynaud, J. Jouzel, Seawater: Equilibrium, Kinetics, Isotopes. Elsevier H. Fischer, K. Kawamura, and T.F. Stocker. 2008. Oceanography Series, 65. Elsevier Science, B.V., High-resolution carbon dioxide concentration Amsterdam, 346 pp. record 650,000–800,000 years before present. Nature 453:379–382.

Oceanography December 2009 47

LETTERS PUBLISHED ONLINE: 15 APRIL 2012 | DOI: 10.1038/NCLIMATE1489

Changes in pH at the exterior surface of plankton with ocean acidiﬁcation

Kevin J. Flynn1*, Jerry C. Blackford2, Mark E. Baird3, John A. Raven4, Darren R. Clark2, John Beardall5, Colin Brownlee6, Heiner Fabian1 and Glen L. Wheeler2,6

14–16 + prox Anthropogenically released CO2 is dissolving in the ocean, phytoplankton demonstrate a significant difference in [H ]SW [ +]bulk causing a decrease in bulk-seawater pH (ocean acidification). compared with H SW during photosynthetic carbon uptake. Projections indicate that the pH will drop 0.3 units from its Dispersal and exchange of ions between organisms and the bulk present value by 2100 (ref. 1). However, it is unclear how water is moderated by the diffusive boundary layer whose thickness the growth of plankton is likely to respond. Using simulations increases with size of organism or aggregate (loose colonies). we demonstrate how pH and carbonate chemistry at the Greater differences are also expected with larger surface-area-based exterior surface of marine organisms deviates increasingly metabolic rates. These processes occur in a water column whose from those of the bulk sea water as organism metabolic bulk chemistry also varies, affected by the total metabolic activity, activity and size increases. These deviations will increase in the atmospheric gas exchange and water mass exchanges. Similar future as the buffering capacity of sea water decreases with considerations apply to benthic calcifiers17. decreased pH and as metabolic activity increases with raised Here we explore the influence of organism or aggregate/colony seawater temperatures. We show that many marine plankton size and activity on this phenomenon, and consider potential will experience pH conditions completely outside their recent ecosystem feedback in the context of environmental change. We use historical range. However, ocean acidification is likely to have simulations combining descriptions of dissolved inorganic carbon differing impacts on plankton physiology as taxon-specific (DIC) chemistry7, phytoplankton and their microzooplankton differences in organism size, metabolic activity and growth predators18; Methods are given below with additional details given rates during blooms result in very different microenvironments in Supplementary Methods. In brief, the coupled model describes around the organism. This is an important consideration for changes in total alkalinity, DIC and nutrient concentrations [ +]bulk future studies in ocean acidification as the carbonate chemistry (affecting H SW and DIC equilibria) over a vertically mixed experienced by most planktonic organisms will probably be surface layer with gas exchange at the sea surface and mixing considerably different from that measured in bulk-seawater with bottom water. Near-organism conditions were computed, + prox samples. An understanding of these deviations will assist allowing calculation of [H ]SW . Simulations were carried out for interpretation of the impacts of ocean acidification on plankton steady-state plankton growth and in dynamic conditions simulating of different size and metabolic activity. growth at sea. We present three conditions for comparison. The Since the original report on the ocean-acidification phenomenon historic series assumes an atmospheric partial pressure of CO2 and its potential impact on marine life2, the topic has engendered (p ) of 280 ppm giving a simulated equilibrium bulk-water pH CO2 much research and discussion3. Predictions of ocean acidification of 8.165. p and pH values for extant and future conditions CO2 and its impact have mainly considered large-scale annual mean were 390, 8.03 and 750, 7.76, respectively. As pH is a log events1,3–5. On local scales, strong spatial and seasonal gradients transform, it poorly resolves the ∼2 orders of magnitude of H+ exist in the carbonate system and pH (refs 6–9), together changes experienced by organisms; results are presented as [H+] in 10 + with large diel signals from biological processes . However, sea water ([H ]SW). what ultimately affects organisms is the environment at their Figure 1 shows steady-state relationships between organ- exterior (proximate) surface. The physiological and ecosystem ism/aggregate size (as equivalent spherical diameters, ESDs; x + prox impacts of ocean acidification must thus be determined from axis), net cellular carbon fluxes (C-flux; y axis) and [H ]SW or changes in the proximate acid–base balance, taking into account proximate p ; Supplementary Figs S1 and S2 show proximate CO2 boundary-layer processes. pH and the saturation constant for calcite (calcite). A comparison Regulation of intracellular acid–base balance (pH regulation) between the results shown as [H+] (Fig. 1) and pH (Supplementary is critical for organismal homoeostasis11. Intracellular proton Fig. S1) serves to indicate the confounding influence of the log consumption and production are an inevitable outcome of primary transform (pH scale) in interpreting the consequences of ocean metabolism11–13. Thus, photosynthesis and associated nutrient acidification. Positive C-flux is associated with photosynthesis allied uptake by individual cells decreases their proximate seawater to Redfield assimilation of nitrate-N and phosphate; consumption + + prox + prox H concentration ([H ]SW ) and the action of all cells then of ammonium-N slightly moderates the changes in [H ]SW , [ +]bulk decreases the bulk-water value ( H SW ). Respiration and allied whereas use of silicic acid (diatoms) has very little effect (not nutrient regeneration gives the converse. Studies of marine shown). Negative C-flux with respiration is allied to Redfield-like

1Centre for Sustainable Aquatic Research, Swansea University, Swansea SA2 8PP, UK, 2Plymouth Marine Laboratory, Prospect Place, Plymouth PL1 3DH, UK, 3Plant Functional Biology and Climate Change Cluster, University of Technology Sydney, PO Box 123 Broadway, Sydney, New South Wales 2007, Australia, 4Division of Plant Sciences, University of Dundee at the James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK, 5School of Biological Sciences, Monash University, Clayton, Victoria 3800, Australia, 6Marine Biological Association, Citadel Hill, Plymouth PL1 2PB, UK. *e-mail: [email protected].

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NATURE CLIMATE CHANGE DOI: 10.1038/NCLIMATE1489 LETTERS

Historic Extant Future

30 ) 30

)

–

–1 kg

20 20 20

(nmol (nmol

(nmol kg (nmol

SW ]

SW 10 10 10

]

+ ] –1 –1

+ –1 +

0 0 ) [H

) [H –1 0 [H ) 0 1 –1 1 –1 0 0 1 50 2 50 2 50 2 100 150 100 3 100 3 C-flux (d 150 C-flux (d 3 ESD ( 200 ESD (µ 200 ESD ( 150 C-flux (d µm) m) µm) 200

1,000

1,000 1,000

(ppm)

2 2

(ppm)

(ppm) 500

2 2 Co

2 2 –1 500

500 p –1 Co

Co –1 0 ) –1 p p 0 ) 1 ) –1 0 – 1 1 0 1 50 2 0 2 100 0 2 50 150 3 C-flux (d 50 100 100 3 150 3 C-flux (d 150 C-flux (d ESD (µ 200 200 ESD ( 200 m) ESD (µ µm) m)

| [ +]prox µ Figure 1 Steady-state relationships. Simulated H SW and near-organism pCO2 for organisms or aggregates of ESD up to 200 m growing over a −1 −1 range-positive (photosynthesis) or negative (respiration) C-ﬂux. The full units for C-ﬂux are gC (gC) d . The historic series assumes a pCO2 of 280 ppm, = µ equating here to bulk-water pH 8.165. pCO2 and pH values for extant and future conditions were 390, 8.03 and 750, 7.76, respectively. Values at ESD 1 m approach those in the bulk water. Note the direction of the ESD axis for pCO2 . Versions of these plots showing pH and the saturation constant for calcite are [ +] given in Supplementary Figs S1 and S2. The colour grading in the plots can be gauged by reference to the H SW and pCO2 values in the future plots, as applicable. releases of ammonium and phosphate for a microzooplankter for historic conditions, that for the future is very different (Fig. 2d). feeding on prey of similar C:N:P ratio to itself. Owing to the decreasing buffering capacity of sea water as pH falls, [ +]bulk Near-organism conditions are close to bulk-water conditions for not only are in-bloom variations of H SW greater, but so are the + prox those of small ESD (about <5 µm) and/or with little metabolism diel ranges of [H ]SW and these vary further with size and activity (C- flux); conditions diverge as ESD and metabolic activity increase. (Fig. 2). There is thus an increased risk of organisms experiencing + prox Notably, the range of [H ]SW increases sharply moving from conditions of pH that cross tolerance thresholds for lethality. historic through extant to future conditions. This is because the For smaller organisms/aggregates, future conditions are lowered carbonate-ion concentration under ocean acidification projected to lie always well outside the likely range of decreases the buffering of metabolically driven changes in pH conditions experienced so far (Fig. 2, Supplementary Figs S3–S13). (ref. 19). These organisms have historically experienced conditions Various dynamic simulations were run to examine the potential essentially constrained by the bulk-water chemistry. In contrast, effects of ecosystem feedback during a phytoplankton bloom. larger organisms or those in aggregates experience significant Figure 2 gives an example with other scenarios involving different metabolically driven diel variation in seawater chemistry at the cell nutrient and biomass concentrations (eutrophic to oceanic), or surface. Future substantial algal blooms in eutrophic conditions + prox different phytoplankton groups (harmful algal bloom or calcifiers), are still expected to yield [H ]SW approaching historic bulk-water being shown in Supplementary Figs S3–S13. As ocean acidification conditions (Supplementary Figs S7–S10), as a result of seawater was assumed not to alter plankton physiology (neither autotrophic alkalinization during C-fixation. nor heterotrophic), biomass and bulk nutrient regimes were For calcifiers, the conditions may be quite different, because the same over the simulated period (with its 12:12 h light:dark changes in alkalinity with CaCO3 deposition counter changes diel cycle) under historic, extant and future conditions for in the DIC chemistry and hence these blooms do not drive each oceanographic scenario (see Supplementary Methods). Thus, the same enhanced pH (alkalinization) seen with non-calcifiers (Fig. 2 and Supplementary Fig. S3) a period of mainly nitrate- (Supplementary Figs S3–S4 versus Supplementary Figs S11–S12). supported algal growth (high f-ratio) is followed by N-limitation, For calcifying plankton, values of calcite are much lower throughout then a change to ammonium-supported growth (low f -ratio) as the simulated event for future conditions (Supplementary Figs S2, zooplankton grazing decreases the algal biomass. S11–S12). calcite is little higher for active high-ESD cells/aggregates Over the simulated events (for example, Fig. 2, Supplementary (Supplementary Fig. S2). + prox Figs S3–S4) there were significant changes in bulk-water DIC The variation of [H ]SW predicted by the model (up to 0.4 units [ +]bulk chemistry and hence in H SW , owing to interactions between greater than bulk-water pH) matches that shown in experimental 15–17 [ +]bulk [ +]prox biotic and abiotic processes. The timing and extent of these depend studies . Variation between H SW and H SW will be greater on the scenario, though the patterns are similar (Supplementary if diffusivity is lower than simulated because diffusion rates may Figs S4–S12). Overlain on these bulk changes are additional diel be decreased by cell walls20 and mucus: production of transparent variations in [H+]prox, which are smaller during nutrient-limited extracellular polysaccharide can rise under increased p (ref. 21), SW CO2 growth (for example, Fig. 2, days 6–9). Although the overall plank- though any increased growth under such conditions22 may indicate ton experience under extant conditions is not too dissimilar to that unchanged diffusivity. Notably, in nature cells may form aggregates

NATURE CLIMATE CHANGE | VOL 2 | JULY 2012 | www.nature.com/natureclimatechange 511

LETTERS NATURE CLIMATE CHANGE DOI: 10.1038/NCLIMATE1489

a (mg C m picture of what different types of plankton will experience. Thus, M) Biomass

µ 1.0 Algal C different plankton size classes and activities will result in a diverse 10 DIN [ +]prox Zoo C 0.5 experience of H SW ranges. Already extant populations of at DIP –3

) least the smaller and slower-growing species have been subject 0 0 + prox Nutrients ( to an increased [H ]SW of 50% from historic levels. The lack of 0 5 10 15 20 [ +]prox Time (d) historic experience in changes in H SW may result in smaller b microbial loop organisms (especially those in oligotrophic waters) 20 being more susceptible to ocean acidification. In contrast, larger, ) Bulk –1 Small faster-growing organisms in eutrophic conditions may in the 15 Medium + prox future still experience ranges of [H ]SW overlapping with historic Zoo Future ranges (Supplementary Figs S7–S10). Potentially, larger nuisance

(nmol kg 10

SW organisms (such as Phaeocystis) may thus be better adapted to life ]

+ Extant 5 under a more variable pH regime and with growth enhanced by the

[H Historic higher pCO (ref. 22) at the start of the bloom. 2 + prox Future ranges of [H ]SW for many plankton lie largely if not 0 5 10 15 20 completely outside the window of historic and extant conditions. Time (d) c The operation of pH regulatory mechanisms that rely on passive 20 + +

) Bulk H efflux (such as H channels) may be severely compromised –1 Historic as [H+]prox approaches that of the internal cell environment, 15 Large Extant SW Future requiring significant alterations in trans-plasmalemma membrane Future

(nmol kg 10 potential or induction of alternative pH regulatory mechanisms 13 SW

] to prevent disruption of cellular homeostasis . Although algal

+ Extant

[H 5 growth is sometimes stimulated by higher p (ref. 22), the Historic CO2 lessening of alkalinization of bulk sea water as a driver in algal 6,24 25 10 succession and controller of certain grazers may be replaced 0 5 15 20 [ +]prox Time (d) by encountering (sub)lethal H SW . Furthermore, though larger d phytoplankton and colony/aggregate formers may be better adapted

) 20 to variable pH conditions (as they themselves generate these

–1 Bulk Small over the light:dark cycle), the more extreme future diel range 15 Medium Large may become damaging. Results in Fig. 1 for respiration, together 17 (nmol kg 10 Zoo with works of others , show the scope for such an increase in

SW + prox

] [ ]

+ H . This may be particularly important for organisms that 5 SW

[H form dense accumulations and have high respiration rates (for Historic Extant Future example, dinoflagellates). It would be useful to know how the lethal + prox limits of high [H ]SW for different plankton compare with the [ +]prox Figure 2 | Dynamic relationships. Variation in H SW from simulations extreme conditions indicated by the 95-percentile limits in the for phytoplankton growing alone or in aggregates of ESD 25 µm (small), simulations (Fig. 2d; Supplementary Figs S4, S6, S8, S10, S12, S13). 75 µm (medium) or 150 µm (large) together with zooplankton, under Although our results indicate a clear potential for ocean- historic, extant or future ocean-acidiﬁcation conditions. a, The dynamics of acidification-induced changes in the environmental experience for growth are shown using dissolved inorganic N and P (DIN and DIP, plankton, data linking community- and individually driven changes [ +]prox [ +]bulk [ +]prox respectively). b,c, H SW for different organisms is shown, together with in H SW and H SW back to physiology and viability are lacking. [ +]bulk [ +]prox H SW . d, Variability in H SW over the 20 day period is shown as the These factors affect succession, with potential for changes analogous median (centre rectangle line), 25th and 75th quartiles (lower and upper to those seen in freshwater systems subjected to acidification26. extent of rectangle), errors (whiskers) and the 5th and 95th quartiles (ﬁlled However, coccolithophores seem to be less able than freshwater [ +]bulk circles). The red lines are aligned with the median value for H SW over species to maintain a constant internal pH in response to variable the 20-day period; distributions centred below this line indicate a more external pH (ref. 23), indicating that marine phytoplankton may alkaline experience, whereas distributions above it indicate more acidic possess very different mechanisms of pH homeostasis. The inability experiences. See also Supplementary Table S1 and Fig. S3. to maintain pH homeostasis with increased variability in external pH may have significant consequences for at least certain marine such that the effective size for diffusion may be large even for small phytoplankton groups. As well as the direct impacts of pH and cells. Such aggregates are destroyed by shear forces in sampling carbonate chemistry on the microenvironment around plankton, apparatus and during shaking to homogenize cultures before ex- predicted future changes in the physical parameters of oceanic periments: such methods probably underestimate the effective ESD waters, such as temperature, pressure and turbulence3, may also in phytoplankton communities and hence would underestimate the influence boundary-layer processes. effects of ocean acidification in experiments. Furthermore, changes Finally, although the stability of future phytoplankton succes- + prox in [H ]SW and DIC chemistry may indeed affect physiology, sional events and our ability to predict them depends critically on + prox through changes in ionic states of nutrients, the operation of cellular the sensitivity of plankton to [H ]SW , our work also highlights mechanisms involved in pH homeostasis23 and/or in the require- a concern that experimental artefacts may lead to an underesti- ment for carbon-concentrating mechanisms (CCMs)16. Critically, mation of the effects of ocean acidification (especially for larger we lack information for many groups of plankton in these regards, cells and/or aggregates). emphasis having been placed on diatoms and coccolithophorids. Even given the potential for the simulations to underestimate Methods the changing planktonic experience (both for reasons given above Full details and additional references are supplied in Supplementary Methods. Simulations presented here were achieved by combining a description of DIC and because we have not here considered pH-induced changes chemistry7, using constants recommended by the Ocean Carbon-Cycle Model in physiology), it is clear from our work that changes in bulk- Intercomparison Project, together with mechanistic submodels describing water chemistry alone will probably not give a representative phytoplankton and microzooplankton (as used previously in ref. 18). The model

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NATURE CLIMATE CHANGE DOI: 10.1038/NCLIMATE1489 LETTERS accounted for changes in total alkalinity, DIC, phosphate, nitrate, ammonium 11. Smith, F. A. & Raven, J. A. Intracellular pH and its regulation. Ann. Rev. and silicic acid concentrations (factors affecting pH and DIC equilibria) over a Plant Physiol. 30, 289–311 (1979). vertically mixed surface layer with gas exchange with the atmosphere at the sea 12. Raven, J. A. Exogenous inorganic carbon sources in plant photosynthesis. surface and mixing with water from below the mixed layer. Depth-integrated Biol. Rev. 45, 167–221 (1970). photosynthesis and grazing were simulated. Near-cell concentrations of nutrients, 13. Raven, J. A. Effects on marine algae of changed seawater chemistry with DIC and alkalinity were calculated from modifications of established descriptions27, increasing atmospheric CO2. Biol. Environ. Proc. R. Irish Acad. 111B, as a balance of diffusion between the bulk water and cell surface, against transport 1–17 (2011). rates of metabolites and H+ in or out of organisms. For simplicity, plankton 14. Kühn, S. F. & Köhler-Rink, S. pH effect on the susceptibility to parasitoid (both individuals and aggregates) were considered as spherical and phytoplankton as non-motile with neutral buoyancy. Zooplankton were considered as motile infection in the marine diatom Coscinodiscus spp. (Bacillariophyceae). (see Supplementary Table S1); changes in diffusion owing to boundary-layer Mar. Biol. 154, 109–116 (2008). thinning with motility were calculated27, though these calculations did not 15. Kühn, S. F. & Raven, J. A. Photosynthetic oscillation in individual cells in account for additional impacts of temperature and water pressure. We assumed no the marine diatom Coscinodiscus wailesii (Bacillariophyceae) revealed by physiological effect of external [H+] on organism growth. The mode of operation microsensor measurements. Photosynth. Res. 95, 37–44 (2008). of a CCM in phytoplankton will influence the dynamics of pH and DIC at the cell 16. Milligan, A. J., Mioni, C. E. & Morel, F. M. M. Response of cell surface pH surface. Given that external carbonic anhydrase is (at least effectively) ubiquitous to p and iron limitation in the marine diatom Thalassiosira weissflogii. CO2 among plankton28, we have assumed an effective equilibrium between forms of Mar. Chem. 114, 31–36 (2009). DIC within the integration time step of five minutes. The operation of the CCM 17. Hurd, C. L. et al. Metabolically induced pH fluctuations by some coastal and its influence on pH and DIC around the cell surface is discussed in greater calcifiers exceed projected 22nd century ocean acidification: A mechanism for detail in Supplementary Methods. Diffusivity varies with molecular weight and to differential susceptibility? Glob. Change Biol. 17, 3254–3262 (2011). retain charge balance the faster diffusion of lighter ions (most notably H+) is thus − 18. Mitra, A. & Flynn, K. J. Promotion of harmful algal blooms by zooplankton moderated by that of slower-moving counter ions. Of all of these ions, HCO3 predatory activity. Biol. Lett. 2, 194–197 (2006). is numerically (after H+) the most important diffusing and also has a molecular 19. Hofmann, A. F., Middelburg, J. J., Soetaert, K., Wolf-Gladrow, D. A. & weight of a similar order to others. Accordingly and considering all other vagaries (see discussion concerning experimental artefacts) a common diffusivity constant Meysman, F. J. R. Proton cycling, buffering, and reaction stoichiometry in − natural waters. Mar. Chem. 121, 246–255 (2010). was used based on that expected at the stated salinity and temperature for HCO3 . The value we have used is 1.02×108 µm2 d−1 (1.18×10−9 m2 s−1), as used in ref. 20. Hale, M. S. & Mitchell, J. G. Functional morphology of diatom frustule 29. We have identified three conditions of atmospheric p for comparisons. The microstructures: Hydrodynamic control of Brownian particle diffusion and CO2 historic series assumes an atmospheric p of 280 ppm, giving an equilibrium advection. Aquat. Micro. Ecol. 24, 287–295 (2001). CO2 bulk-water pH under the conditions simulated of 8.165. p and pH values for 21. Engel, A., Thoms, S., Riebesell, U., Richelle-Newall, E. & Zondervan, I. CO2 extant and future conditions were 390, 8.03 and 750, 7.76, respectively30. Various Polysaccaride aggregation as a potential sink of marine dissolved organic scenarios have been trialled (see Supplementary Methods) to give an indication carbon. Nature 428, 929–932 (2002). of the range of experiences possible. Oligotrophic conditions (ultra-low residual 22. Raven, J. A, Beardall, J., Giordano, M. & Maberly, S. C. Algal and aquatic nutrients) would give a range of near-organism experiences even less variable plant carbon concentrating mechanisms in relation to environmental change. than those given here under oceanic, whereas extreme eutrophic would be more Photosynth. Res. 199, 281–296 (2011). variable than those shown under eutrophic. Within the Supplementary Plots we 23. Taylor, A. R., Chrachri, A., Wheeler, G. L., Goddard, H. & Brownlee, C. + ◦ also show how the simulation predicts the conditions in a warmer ( 4 C) ocean, A voltage-gated proton channel underlying pH homeostasis in calcifying together with the magnitude of changes brought about by changes in wind speed coccolithophores. PLoS Biol. 9, e1001085 (2011). (Supplementary Table S1, Figs S10, S12 and S13). To give an indication of the 24. Hansen, P. J., Lundholm, N. & Rost, B. Growth limitation in marine red-tide variation in proximate conditions that an organism may experience under different conditions, we have plotted the median of the model output together with errors, dinoflagellates: Effect of pH versus inorganic carbon limitation. Mar. Ecol. 5th, 25th, 75th and 95th percentiles for each of the dynamic simulations over the Prog. Ser. 224, 63–71 (2007). 20-day period of the simulation. For reference in considering data such as those 25. Smith, M. & Hansen, P. J. Interaction between Mesodinium rubrum and its shown in Fig. 1, we draw attention to the following. For phytoplankton, growing prey concentration, irradiance and pH. Mar. Ecol. Prog. Ser. 338, 61–70 (2007). within a 12:12 h light:dark cycle at a daily growth rate of 1 d−1, a photosynthetic 26. Eriksson, M. O. et al. Predator–prey relations important for biotic changes in rate during daylight exceeding 2 d−1 is required. For microzooplankton, there is an acidified lakes. Ambio 9, 248–249 (1980). inverse relationship between respiration rates and their size; a microzooplankton of 27. Pasciak, W. J. & Gavis, J. Transport limitation of nutrient uptake in − − 200 µm ESD would have a respiration rate of around 0.5 gC (gC) 1 d 1. phytoplankton. Limnol. Oceanogr. 19, 881–888 (1974). 28. Tortell, P. D. et al. Inorganic carbon uptake by Southern Ocean phytoplankton. Received 7 November 2011; accepted 15 March 2012; Limnol. Oceanogr. 53, 1266–1278 (2008). published online 15 April 2012 29. Wolf-Gladrow, D. & Riebesell, U. Diffusion and reactions in the vicinity of plankton: A refined model for inorganic carbon transport. Mar. Chem. 59, References 17–34 (1997). 1. Caldeira, K. & Wickett, M. E. Anthropogenic carbon and ocean pH. Nature 30. Riebesell, U., Fabry, V. J., Hansson, L. & Gattuso, J-P. Guide to Best Practices for 425, 365 (2003). Ocean Acidification Research and Data Reporting (Luxembourg Publications 2. Royal Society of London Ocean Acidification Due to Increased Atmospheric Office of the European Union, 2010). Carbon Dioxide Policy Document 12/05 (The Royal Society of London, 2005). 3. Doney, S. C., Fabrey, V. J., Feely, R. A. & Kleypas, J. A. Ocean acidification: Acknowledgements The other CO2 problem. Annu. Rev. Mar. Sci. 1, 189–192 (2009). 4. Orr, J. C. et al. Anthropogenic ocean acidification over the twenty-first century This work was primarily financially supported by the Natural Environment Research and its impact on calcifying organisms. Nature 437, 681–686 (2005). Council (NERC) NE/F003455/1 to K.J.F., J.C.B. and D.R.C., employing H.F., and to K.J.F. 5. Steinacher, M., Joos, F., Frölicher, T. L., Plattner, G-K. & Doney, S. C. by NERC NE/H01750X/1. C.B. and G.L.W. were financially supported by NERC Oceans Imminent ocean acidification in the Arctic projected with the NCAR global 2025. C.B. was financially supported by NERC NE/E0183191/1, the Biotechnology and coupled carbon cycle-climate model. Biogeosciences 6, 515–533 (2009). Biological Sciences Research Council 226/P15068 and the European Project on Ocean 6. Hinga, K. R. Effects of pH on coastal marine phytoplankton. Mar. Ecol. Prog. Acidification, EC 211384. Ser. 238, 281–300 (2002). Author contributions 7. Blackford, J. C. & Gilbert, F. J. pH variability and CO2 induced acidification in the North Sea. J. Mar. Syst. 64, 229–241 (2007). The model was formulated by K.J.F, M.E.B. and J.C.B.; it was coded and run by K.J.F. All 8. Litt, E. J. et al. Biological control of p at station L4 in the western English authors contributed to design of simulation experiments, analysis and interpretation of CO2 Channel over 3 years. J. Plankton Res. 32, 621–629 (2010). the model output and to writing the paper. 9. Gypens, N., Lacroix, G., Lancelot, C. & Borges, A. V. Seasonal and inter-annual variability of air–sea CO2 fluxes and seawater carbonate chemistry in the Additional information southern North Sea. Prog. Oceanogr. 88, 59–77 (2011). The authors declare no competing financial interests. Supplementary information 10. Santos, I. R., Glud, R. N., Maher, D., Erler, D. & Eyre, B. D. Diel coral reef accompanies this paper on www.nature.com/natureclimatechange. Reprints and acidification driven by porewater advection in permeable carbonate sands, permissions information is available online at www.nature.com/reprints. Correspondence Heron Island, Great Barrier Reef. Geophys. Res. Lett. 38, L03604 (2011). and requests for materials should be addressed to K.J.F.

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Assessment and management of seafood safety and quality Current practices and emerging issues Assessment and management of seafood safety quality

This technical paper compiles the state of knowledge on seafood safety and quality with the aim to provide a succinct yet comprehensive resource book to seafood quality and safety managers, including topics on emerging issues such as new pathogens, the impact of climate change on seafood safety, and the changing regulatory framework. After introductory chapters about world fish production, trade, consumption and nutrition, and about the developments in safety and quality systems, the technical paper provides a detailed review of the hazards causing public health concerns in fish and fish products, covering biological, chemical and physical hazards. This is followed by chapters on seafood spoilage and quality issues; the likely impact of climate change on seafood safety; a detailed coverage of the implementation and certification of seafood safety systems covering risk mitigation and management tools, with a detailed description of the requirements for the implementation of good hygiene practices and good manufacturing practices, the Hazard Analysis and Critical Control Points (HACCP) system, and the monitoring programmes to control biotoxins, pathogenic bacteria and viruses and chemical pollutants; a section on private labelling and certification schemes; details of the international framework covering the World Trade Organization, the Codex Alimentarius Commission, the FAO Code of Conduct for Responsible Fisheries, and the World Organisation for Animal Health; and a presentation of the regulatory frameworks governing seafood trade in the European Union (Member Organization), the United States of America, Japan, Australia and New Zealand.

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5.2 impacts of climate change on harmful algal blooms and seafood safety (Gustaaf Hallegraeff) 5.2.1 Introduction In a strict sense, harmful algal blooms are completely natural phenomena that have occurred throughout recorded history. However, even non-toxic algal blooms can have devastating impacts when they lead to kills of fish and invertebrates by generating anoxic conditions in sheltered bays. Other algal species, although non-toxic to humans, can produce exudates that can cause damage to the delicate gill tissues of fish (raphidophytes Chattonella, Heterosigma, and dinoflagellates Karenia, Karlodinium). Whereas wild fish stocks are free to swim away from problem areas, caged fish in intensive aquaculture operations are trapped and, thus, can suffer devastating mortalities. Of greatest concern to human society are algal species that produce potent neurotoxins that can find their way through shellfish and fish to human consumers where they evoke a variety of gastrointestinal and neurological illnesses. One of the first recorded fatal cases of food poisoning after eating contaminated shellfish happened in 1793, when Captain George Vancouver and his crew landed in British Columbia (Canada) in an area now known as Poison Cove. He noted that, for local Indian tribes, it was taboo to eat shellfish when the seawater became bioluminescent due to algal blooms by the local dinoflagellate Alexandrium catenella/tamarense, which is now known to be a causative organism of PSP. The increase in shellfish farming worldwide is leading to more reports of PSP, DSP (first documented in 1976 in Japan), NSP (reported from the Gulf of Mexico as early as 1844) and ASP (first identified in 1987 in Canada). The explorer Captain James Cook already suffered from the tropical illness of CFP from fish when visiting New Caledonia in 1774. Worldwide, almost 2 000 cases of food poisoning from consumption of contaminated fish or shellfish are reported each year. Some 15 percent of these cases prove fatal. If not controlled, the economic damage through the slump in local consumption and exports of seafood products can be considerable. Whales and porpoises can also become victims when they receive toxins through the food chain via contaminated zooplankton or fish. In the United States of America, poisonings of manatees in Florida via seagrasses and, in California, of pelicans and sea lions via contaminated anchovies have also been reported (Hallegraeff, Anderson and Cembella, 2003). In the past three decades, harmful algal blooms seem to have become more frequent, more intense and more widespread. Four explanations for this apparent increase in algal blooms have been proposed: (i) a greater scientific awareness of toxic species; (ii) the growing utilization of coastal waters for aquaculture; (iii) the stimulation of plankton blooms by domestic, industrial and agricultural wastes and/or unusual climate conditions; and (iv) the transportation of algal cysts either in ships’ ballast water or associated with moving shellfish stocks from one area to another (Hallegraeff, 1993). Few long-term records exist of algal blooms at any single locality; ideally, at least 30 consecutive years of data would be needed. Therefore, whether or not the apparent global increase in harmful algal blooms represents a real increase is a question that will probably not be answered conclusively for some time to come. The growing interest in using coastal waters for aquaculture is leading to a greater awareness of toxic algal species. People responsible for deciding quotas for pollutant loadings of coastal waters, or for managing agriculture and deforestation, should be made aware that one probable outcome of allowing polluting chemicals to seep into the environment will be an increase in harmful algal blooms. In countries that pride themselves on having disease- and pollution-free aquaculture, every effort should be made to quarantine sensitive aquaculture areas against the unintentional introduction of non-indigenous harmful algal species. Nor can any aquaculture industry afford not to monitor for an increasing number of harmful algal species in water and for an Climate change and probable impact on fish safety 175

increasing number of algal toxins in seafood products – using increasingly sophisticated analytical techniques such as LC-MS (see Section 3.2.5). Last, global climate change is adding a new level of uncertainty to many seafood safety monitoring programmes, as are range extensions of harmful algal bloom species through their being transported in ships’ ballast water and as a consequence of increases in sea surface temperatures (Hallegraeff, 2010).

5.2.2 Range extensions by transport in ships’ ballast water Ballast water is seawater that has been pumped into a ship’s hold or dedicated ballast tanks to steady it by making it heavier and thus less likely to roll; the water is released when a ship enters port. Ballast water on cargo vessels was first suggested as a means of dispersing marine plankton more than 100 years ago (Ostenfeld, 1908). However, it was only in the 1980s that the problem sparked considerable interest, after evidence was brought forward that non-indigenous toxic species such as the PSP dinoflagellate Gymnodinium catenatum had been introduced into sensitive aquaculture areas of Australian waters, with disastrous consequences for commercial shellfish farms (McMinn et al., 1997). Similarly, the PSP dinoflagellate Alexandrium catenella, of a diagnostic temperate Asian ribotype, has appeared on French and Spanish Mediterranean coasts in the past two decades (Lilly et al., 2002). Ecosystems disturbed by pollution or climate change are more prone to ballast water invasions (Stachowicz et al., 2002).

5.2.3 Algal bloom range extensions and climate change The dinoflagellate Pyrodinium bahamense is currently confined to tropical, mangrove- fringed coastal waters of the Atlantic and Indo-West Pacific. A survey of cyst fossils (named Polysphaeridium zoharyii) going back to the warmer Eocene 50 million years ago indicates a much wider range of distribution in the past. For example, in the Australasian region at present, the alga is not found farther south than Papua New Guinea, but, some 100 000 years ago, the alga ranged as far south as what is now Sydney Harbour, Australia (McMinn, 1989). There is concern that, with an increased greenhouse effect and warming of the oceans, this species may return to Australian waters (Figure 30). In the tropical Atlantic, in areas such as Bahia Fosforescente in Puerto Rico and Oyster Bay in Jamaica, the glowing red-brown blooms of Pyrodinium are a major tourist attraction. At first considered harmless, Pyrodinium blooms gained a more sinister reputation in 1972 in Papua New Guinea after red-brown water discolorations coincided with the fatal food poisoning (diagnosed as PSP) of three children in a seaside village. Since then, these toxic blooms have apparently spread to Brunei Darussalam and Sabah, Malaysia, (1976), the central (1983) and northern Philippines (1987) and North Maluku, Indonesia. There is strong circumstantial evidence of a coincidence between Pyrodinium blooms and weather perturbations linked to the ENSO (Figure 31). Pyrodinium is thus a serious public health and economic problem for these tropical countries, all of which depend heavily on seafood for protein. In the Philippines alone, Pyrodinium has now been responsible for more than 2 000 human illnesses and 100 deaths resulting from the consumption of contaminated shellfish, sardines and anchovies (Hallegraeff and MacLean, 1989; Azanza and Taylor, 2001). Erickson and Nishitani (1985) reported exceptional PSP episodes by Alexandrium tamarense/catenella in the Pacific Northwest during 7 out of 9 ENSO events between 1941 and 1984. 176 Assessment and management of seafood safety and quality - current practices and emerging issues

Figure 30 Global distribution of Pyrodinium bahamense in recent plankton and the fossil cyst record

Source: Hallegraeff (1993).

Until recently, NSP by the dinoflagellate Karenia brevis was considered to be endemic to the Gulf of Mexico and the east coast of Florida, the United States of America, where red tides had been reported as early as 1844. An unusual feature of NSP is the formation by wave action of toxic aerosols, which can lead to respiratory asthma-like symptoms in humans. In 1987, a major Florida bloom was dispersed by the Gulf Stream northward into the waters of North Carolina, the United States of America, where it has since persisted (Tester et al., 1991; Tester, Geesey and Vukovich, 1993). In early 1993, more than 180 human NSPs were reported from New Zealand. Most likely, this mixed bloom of Karenia mikimotoi and related species was again triggered by the unusual weather conditions at the time, including higher than usual rainfall and lower than usual temperature, which coincided with an El Niño event (Chang et al., 1998; Rhodes et al., 1993). Climate change and probable impact on fish safety 177

Figure 31 Relationship between ENSO events and major toxic Pyrodinium red tides in the western Pacific region for the period 1950–1998

Notes: The Southern Oscillation Index (SOI) values are based on readings taken in Tahiti (French Polynesia), Hawaii (the United States of America) and Darwin (Australia). Arrows indicate years when P. bahamense red tides occurred in the Philippines and Malaysia. Source: Azanza and Taylor (2001).

Ciguatera caused by the benthic dinoflagellate Gambierdiscus toxicus is a tropical food poisoning syndrome well-known in coral reef areas in the Caribbean, Australia and, in particular, French Polynesia (Figure 32). Whereas, in a strict sense, this is a completely natural phenomenon, from being a rare disease two centuries ago, ciguatera has now reached epidemic proportions in French Polynesia. From 1960 to 1984, more than 24 000 patients were reported from this area, which is more than six times the average for the Pacific as a whole. Evidence is accumulating that reef disturbance by hurricanes, military activities and tourist developments (Bagnis, Bennett and Barsinas, 1985), as well as coral bleaching (linked to global warming) and perhaps, in future, increasing coral damage due to ocean acidification (Hoegh-Guldberg, 1999) are increasing the risk of ciguatera (Figure 33). ). Ciguatera dinoflagellates are predicted to become one of the winners from climate change (Tester et al., 2010). In the Australian region, Gambierdiscus dinoflagellates are well-known from the tropical Great Barrier Reef and southwards down to just north of Brisbane. However, in the past five years, this species has exhibited an apparent range extension into southeast Australian seagrass beds as far south as Melbourne, aided by a strengthening of the East Australian Current. In the same region, the red-tide dinoflagellate Noctiluca scintillans (known from the Sydney region as early as 1860) has, since 1994, expanded its range into southern Tasmanian waters, where it has caused problems for the salmonid fish farm industry (McLeod et al., 2012).. In the North Sea, an analogous northward shift of warm-water phytoplankton has occurred as a result of regional climate warming (Hays, Richardson and Robinson, 2005; Edwards and Richardson, 2004; Richardson and Schoeman, 2004). 178 Assessment and management of seafood safety and quality - current practices and emerging issues

Figure 32 Global distribution of ciguatera fish food poisoning

Source: Hallegraeff (1993).

Figure 33 Increase in the reported number of human illnesses of ciguatera in French Polynesia in the period 1960–1984

Note: Increased reef damage by coral bleaching or ocean acidification is predicted to stimulate ciguatera. Source: Bagnis, Bennett and Barsinas (1985).

5.2.4 predicted impact of climate change on phytoplankton abundance Phytoplankton play a central role in several global biogeochemical cycles. Through the process of photosynthesis, they are also a major consumer of carbon dioxide. The ability of the oceans to act as a sink for anthropogenic carbon dioxide largely relies on the conversion of this gas by phytoplankton into particulate organic matter, and subsequent partial loss to the deep ocean (the so-called “biological pump”). Phytoplankton also have important feedback effects on climate. Some species (e.g. Phaeocystis) are producers of dimethylsulphonium propionate, a precursor of dimethylsulphoxide, which in the atmosphere is oxidized into sulphate, which forms Climate change and probable impact on fish safety 179

condensation nuclei for clouds (Charleson et al., 1987). Therefore, phytoplankton can indirectly affect albedo and precipitation and, hence, coastal runoff, salinity, water column stratification and nutrient supply. Increased temperature, enhanced surface stratification, nutrient upwelling, stimulation of photosynthesis by elevated CO2, changes in land runoff and nutrient availability, and altered ocean pH may produce contradictory species- or even strain-specific responses. Complex factor interactions exist, and ecophysiological experiments rarely take into account genetic strain diversity and physiological plasticity. Predicting the impact of global climate change on harmful algal blooms is fraught with uncertainties. However, important lessons can be learned from the dinoflagellate cyst fossil record (Dale, 2001) and from the few long-term data sets available (such as the Continuous Plankton Recorder surveys; Hays, Richardson and Robinson, 2005; Figures 34 and 35). The climate on our planet has been forever changing, from scales of millions of years (glacial to interglacial periods) to short-term oscillations of tens of years (ENSO, and the North Atlantic Oscillation). Even in the past 1 000 years, the planet has gone through episodes much warmer than present (the Medieval Warm Period of 550–1300 AD) or much colder than now (the Little Ice Age 1300–1900 AD). Because of their short generation times and longevity, many phytoplankton can respond to climate change with only a very small time lag. They can spread quickly with moving water masses into climatic conditions that match the requirements of a species in terms of temperature, salinity, land runoff and turbulence.

Figure 34 Decadal anomaly maps (difference between long-term 1960–1989 mean and the period 1990–2002) for four common harmful algal bloom species (from left to right): Prorocentrum, Ceratium furca, Dinophysis and Noctiluca in the North Atlantic

Note: Note the increase in Prorocentrum, Ceratium furca and Dinophysis along the Norwegian coast and increase in Noctiluca in the southern North Sea. Source: Edwards et al. (2008).

5.2.5 impact of global warming and sea surface temperature change Phytoplankton grow over a range of temperatures characteristic of their habitat, and growth rates are usually higher at higher temperature but considerably lower beyond an optimal temperature (Eppley, 1972). Natural populations of phytoplankton are often found at temperatures that are suboptimal for photosynthesis, and it is believed that this is designed to avoid risking abrupt declines in growth associated with the abrupt incidence of warmer temperatures (Li, 1980). Temperature effects on phytoplankton growth and composition are more important in shallow coastal waters, which experience larger temperature fluctuations than oceanic waters. Increasing sea surface temperature may shift the community composition towards species adapted to warmer temperatures, as observed in the temperate North Atlantic (Edwards and Richardson, 2004). Seasonal timing of phytoplankton blooms is now occurring up to 4–5 weeks earlier in the North Sea in relationship to regional climate warming (Figure 35). However, not all trophic levels are responding to the same extent. Where 180 Assessment and management of seafood safety and quality - current practices and emerging issues

zooplankton or fish grazers are differentially affected by ocean warming, this may have cascading impacts on the structure of marine food webs (Figure 36).

Figure 35 Long-term monthly values of “phytoplankton colour” in the central North Sea, 1948–2001

Notes: Circles denote > 2SD above the long-term monthly mean (from Edwards, 2004). Note an apparent shift towards earlier spring and autumn phytoplankton blooms.

Figure 36 Possible pathways for harmful algal bloom formation when the “top-down control” of the food chain is disrupted (e.g. by overfishing)

Note: Differential impacts of climate change on zooplankton or fish grazers can produce similar stimulation of harmful algal blooms. Source: Turner and Graneli (2006). Climate change and probable impact on fish safety 181

5.2.5.1 Sea-level rise, wind and mixed-layer depth Increasing sea surface temperature and water column stratification (shallowing of the mixed layer) can be expected to have a strong impact on phytoplankton because of the resource requirements and temperature ranges to which species are adapted. Wind determines the incidence of upwelling and downwelling, which in turn strongly affect the supply of macronutrients to the surface (recognized as drivers of Gymnodinium catenatum blooms off Spain [Fraga and Bakun, 1990]). Broad changes in ocean circulation such as those comprising the deep-ocean conveyor belt (Figure 37) can cause displacements to existing upwelling areas and associated algal bloom phenomena. Wind-driven currents may also transport phytoplankton away from a region, and affect the size and frequency of formation of mesoscale features such as fronts and eddies. Locally, wind intensity strongly influences the depth and intensity of vertical mixing in the surface layer, thereby affecting phytoplankton access to nutrients, light availability for algal photosynthesis and phytoplankton exposure to potentially harmful UV-B radiation. Finally, winds can influence the supply of iron to the surface ocean through aeolian transport of dust from land to sea, contributing micronutrients such as iron, which can stimulate Karenia brevis blooms off Florida (Walsh and Steidinger, 2001). Extreme climate events such as hurricanes are known to expand the existing distribution of cyst-producing toxic dinoflagellates (e.g. Alexandrium tamarense in New England, the United States of America, after a 1972 hurricane [Anderson, 1997]). Sea-level rise has the potential to increase the extent of continental-shelf areas, providing shallow, stable water columns favouring phytoplankton growth. The proliferation of coccolithophorids in the geological period the Cretaceous has been partially explained on this basis.

Figure 37 Sensitivity of global ocean circulation to sea surface warming

Source: Rahmstorf (2002).

5.2.5.2 Impact of heavy precipitation events and flash floods Changes in the amount or timing of rainfall and river runoff affect the salinity of estuaries and coastal waters. Salinity is relatively constant throughout the year in most oceanic waters and in coastal areas that receive little freshwater input. Coastal phytoplankton is subject to more variation in salinity than phytoplankton in oceanic 182 Assessment and management of seafood safety and quality - current practices and emerging issues

waters. While some species grow well over a wide range of salinities, other species grow best only at salinities that are low (estuarine), intermediate (coastal) or high (oceanic species). Freshwater also modifies the stratification of the water column, thereby affecting nutrient resupply from below. While diatoms seem to be negatively affected by the decrease in nutrient concentrations associated with river discharge, dinoflagellates often benefit as this usually increases stratification and the availability of humic substances (Doblin et al., 2005). PSP dinoflagellate blooms of Gymnodinium catenatum (in Tasmania, Australia [Hallegraeff, McCausland and Brown, 1995]) and Alexandrium tamarense (off Massachusetts, the United States of America [Anderson, 1997]) tend to be closely associated with land runoff events. In Hiroshima Bay, Japan, blooms of the fish-killing raphidophyte Chattonella marina followed typhoon-induced accretion of nutrient-rich land runoff (Kimura, Mizokami and Hashimoto, 1973). Increased temperatures driven by climate change are predicted to lead to enhanced surface stratification, more rapid depletion of surface nutrients and a decrease in replenishment from deep nutrient-rich waters (Figure 38). This in turn will lead to a change in phytoplankton species, with smaller nanoplankton and picoplankton cells with higher surface-area:volume ratios (better able to cope with low nutrient levels) favoured over larger cells. Mixing depth affects sea surface temperature, the supply of light (from above) and nutrients (from below), and affects phytoplankton sinking losses within the surface layer. Climate models predict changes in the mixed-layer depth in response to global warming for large regions of the global ocean. In the North Pacific, decadal-scale climate and mixed-layer variability (Hayward, 1997), and, in the North Atlantic, longer-term changes in wind intensity and stratification since the 1950s have also been related to considerable changes in the phytoplankton community (Richardson and Schoeman, 2004). Similarly, in high-latitude regions with relatively deep mixing and sufficient nutrients, decreasing mixing depth has resulted in higher phytoplankton biomass because of increased light availability. In contrast, in regions with intermediate mixing depth, increased stratification has resulted in decreased phytoplankton biomass owing to reductions in nutrient supply.

Figure 38 Predicted phytoplankton response to increased temperature in ocean surface waters: (a) reduced productivity in the tropics and mid-latitudes caused by reduced nutrient supply; (b) increased productivity at higher latitudes where reduced mixing keeps plankton closer to the well-lit surface layers

Source: Doney (2006). Climate change and probable impact on fish safety 183

5.2.5.3 Ultraviolet radiation Ultraviolet (UV) can negatively affect several physiological processes and cellular structures of phytoplankton, including photosynthesis, nutrient uptake, cell motility and orientation, algal life span, and DNA (Häder, Worrest and Kumar, 1991). Whereas shorter wavelengths generally cause greater damage per dose, inhibition of photosynthesis by ambient UV increases linearly with increasing total dose. In clear oceanic waters, UV-B radiation can reach depths of at least 30 m. Although some phytoplankton may acclimate to, compensate for, or repair damage by, UV, this involves metabolic costs, thereby reducing the energy available for cell growth and division. Raven, Finkel and Irwin (2005) suggest that UV intensity affects the size ratio in phytoplankton communities because small cells are more prone to damaging UV, and have comparatively high metabolic costs to screen it out. Many surface-dwelling red-tide species of raphidophytes and dinoflagellates possess UV-absorbing pigments, which give them a competitive advantage over species lacking such UV protection (Jeffrey et al., 1999).

5.2.5.4 Ocean acidification

It is widely predicted that increasing CO2 will lead to ocean acidification, which can potentially have an adverse impact on calcifying organisms, the most important of which in terms of biomass and carbon sequestration is the coccolithophorid Emiliania huxleyi (Riebesell et al., 2000). Calculations based on CO2 measurements of the surface oceans indicate that uptake by the oceans of approximately half the CO2 produced by fossil-fuel burning has already led to a reduction in surface pH by 0.1 units. Under the current scenario of continuing global CO2 emissions from human activities, average ocean pH is predicted to fall by 0.4 units by 2100 (Orr et al., 2005). Such a pH is lower than has been experienced for millennia and, critically, this rate of change is 100 times faster than ever experienced in the known history of the planet (Royal Society, 2005). Experimental manipulations of pH in Emiliania huxleyi cultures have both produced reduced (Riebesell et al., 2000) and enhanced calcification and growth (Iglesias-Rodriguez et al., 2008). This has been partially attributed to differences in analytical procedures as well as strain-specific responses, while increasingly the potential for adaptive evolution to gradual environmental changes is now also being recognised (Lohbeck, Riebesell and Reusch, 2012). Decreasing pH to< 8.0 has been observed to have a negative effect on nitrification in marine bacteria. Therefore, it could potentially reduce nitrate availability for plankton algae. However, the nitrogen-fixing tropical cyanobacterium Trichodesmium may be a beneficiary of ocean acidification (Hutchins et al., 2007). Decreasing pH has also been found to increase the availability of toxic trace elements such as copper.

Because the relative consumption of HCO3– and CO2 differs between phytoplankton species, changes in their availability may affect phytoplankton on the cellular, population and community level. Most harmful algal bloom species tested thus far lack carbon-concentrating mechanisms and, hence, they may benefit from increased atmospheric CO2, whereas diatom species such as Skeletonema, for which photosynthesis is already CO2-saturated, will remain constant (Beardall and Raven, 2004).

5.2.6 mitigation of the probable impacts on seafood safety Our limited understanding of marine ecosystem responses to multifactorial physicochemical climate drivers, as well as our poor knowledge of the potential of marine microalgae to adapt genetically and phenotypically to the unprecedented pace of current climate change, are emphasized. Some species of harmful algae (e.g. those benefitting from increased water column stratification or increased water temperatures) may become more successful, while others may diminish in areas currently impacted (Hallegraeff, 2010). The greatest problems for human society will be caused by being 184 Assessment and management of seafood safety and quality - current practices and emerging issues

unprepared for significant range extensions of harmful algal bloom species or an increase in algal biotoxin problems in currently poorly monitored areas. While, for example, ciguatera contamination would be expected and monitored for in tropical coral-reef fish, with the apparent range extension of the causative benthic dinoflagellate into warm-temperate seagrass beds of southern Australia, other coastal fisheries could unexpectedly be at risk. Similarly, incidences of increased surface stratification in estuaries or heavy precipitation or extreme storm events are all warning signs that call for increased vigilance in monitoring seafood products for algal biotoxins, even in areas not currently known to be at risk.

Harmful Algae 19 (2012) 133–159

Contents lists available at SciVerse ScienceDirect

Harmful Algae

jo urnal homepage: www.elsevier.com/locate/hal

Harmful algal blooms along the North American west coast region:

History, trends, causes, and impacts

a, b c d b

Alan J. Lewitus *, Rita A. Horner , David A. Caron , Ernesto Garcia-Mendoza , Barbara M. Hickey ,

e f g h i

Matthew Hunter , Daniel D. Huppert , Raphael M. Kudela , Gregg W. Langlois , John L. Largier ,

b j k l,1 m

Evelyn J. Lessard , Raymond RaLonde , J.E. Jack Rensel , Peter G. Strutton , Vera L. Trainer ,

l,2

Jacqueline F. Tweddle

Center for Sponsored Coastal Ocean Research, National Centers for Coastal Ocean Science, National Ocean Service, National Oceanic and Atmospheric Administration, 1305 East West

Highway, Silver Spring, MD 20910, United States

University of Washington, School of Oceanography, Box 357940, Seattle, WA 98195, United States

University of Southern California, Department of Biological Sciences, 3616 Trousdale Ave., Los Angeles, CA 90089, United States

Biological Oceanography Department, Centro de Investigacioń Cientı´fica y de Educacioń Superior de Ensenada, Km 107 carr Tij-Ens, Ensenada, Baja California, Mexico

Oregon Department of Fish and Wildlife, 2001 Marine Drive, Room 120, Astoria, OR 97103, United States

University of Washington, School of Marine Affairs, 3707 Brooklyn Ave. NE, Seattle, WA 98105, United States

University of California Santa Cruz, Ocean Sciences and Institute for Marine Sciences, 1156 High Street, Santa Cruz, CA 95064, United States

California Department of Public Health, Richmond Laboratory Campus, 850 Marina Bay Parkway, G165, Richmond, CA 94804, United States

University of California Davis, Bodega Marine Laboratory, P.O. Box 247, Bodega Bay, CA 94923, United States

University of Alaska Fairbanks, School of Fisheries and Ocean Sciences, Alaska Sea Grant Marine Advisory Program, 1007 W. 3rd Ave #100, Anchorage, AK 99501, United States

Rensel Associates Aquatic Sciences, 4209 234th Street N.E., Arlington, WA 98223, United States

Oregon State University, College of Oceanic and Atmospheric Sciences, 104 COAS Admin Bldg, Corvallis, OR 97331, United States

Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Marine Biotoxin Program, 2725 Montlake Blvd. E., Seattle,

WA 98112, United States

A R T I C L E I N F O A B S T R A C T

Article history: Along the Paciﬁc coast of North America, from Alaska to Mexico, harmful algal blooms (HABs) have

Received 21 November 2010

caused losses to natural resources and coastal economies, and have resulted in human sicknesses and

Received in revised form 23 June 2012

deaths for decades. Recent reports indicate a possible increase in their prevalence and impacts of these

Accepted 23 June 2012

events on living resources over the last 10–15 years. Two types of HABs pose the most signiﬁcant threat

Available online 6 July 2012

to coastal ecosystems in this ‘‘west coast’’ region: dinoﬂagellates of the genera Alexandrium,

Gymnodinium, and Pyrodinium that cause paralytic shellﬁsh poisoning (PSP) and diatoms of the genus

Keywords:

Pseudo-nitzschia that produce domoic acid (DA), the cause of amnesic shellfish poisoning (ASP) in humans. These

Alexandrium

species extend throughout the region, while problems from other HABs (e.g., fish kills linked to raphidophytes or

Domoic acid

Cochlodinium, macroalgal blooms related to invasive species, sea bird deaths caused by surfactant-like proteins

Harmful algal blooms

Heterosigma produced by Akashiwo sanguinea, hepatotoxins from Microcystis, diarrhetic shellfish poisoning from Dinophysis,

Paralytic shellﬁsh poisoning and dinoflagellate-produced yessotoxins) are less prevalent but potentially expanding. This paper presents the state-

Pseudo-nitzschia of-knowledge on HABs along the west coast as a step toward meeting the need for integration of HAB outreach,

research, and management efforts.

Published by Elsevier B.V.

* Corresponding author. Tel.: +1 301 713 3338; fax: +1 301 713 4044.

E-mail addresses: [email protected] (A.J. Lewitus), [email protected] (R.A. Horner), [email protected] (D.A. Caron), [email protected] (E. Garcia-Mendoza),

[email protected] (B.M. Hickey), [email protected] (M. Hunter), [email protected] (D.D. Huppert), [email protected] (R.M. Kudela),

[email protected] (G.W. Langlois), [email protected] (J.L. Largier), [email protected] (E.J. Lessard), [email protected] (R. RaLonde),

[email protected] (J.E. Jack Rensel), [email protected] (P.G. Strutton), [email protected] (V.L. Trainer), [email protected] (J.F. Tweddle).

Current address: Institute for Marine and Antarctic Studies, University of Tasmania, Private Bag 129, Hobart, Tas 7001, Australia.

Current address: NAFC Marine Centre, Port Arthur, Scalloway, Shetland ZE1 0UN, United Kingdom.

1568-9883/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.hal.2012.06.009

134 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

1. Introduction coast HABs will therefore require a regionally integrated approach,

and effective HAB management will depend on interstate and

Harmful algal blooms (HABs) are a global threat to living marine international collaboration and coordination.

resources and human health. These events impact all coastal U.S. Several policy drivers call for a regional approach to addressing

states and large portions of coastal Canada and Mexico (Taylor, marine problems (e.g., U.S. Commission on Ocean Policy, 2004;

1993; Horner et al., 1997; Mudie et al., 2002; Herna´ndez-Becerril NOAA Program Planning and Integration, 2007; NSTC Joint

et al., 2007; Anderson et al., 2008b; Band-Schmidt et al., 2010). Subcommittee on Ocean Science and Technology, 2007; Joint

Harmful algal blooms have had signiﬁcant ecological and Ocean Commission Initiative, 2009). The 2004 Reauthorization of

socioeconomic impacts on Paciﬁc coastal communities of North the Harmful Algal Bloom and Hypoxia Research Control Act also

America for decades, and their prevalence and impacts on living acknowledged the need for a regional approach to HAB research

resources in this west coast region have increased markedly in and response by establishing a procedure for requesting Regional

frequency and geographical distribution over the last 10–15 years Assessments of HABs. The U.S. Commission on Ocean Policy (2004)

(Anderson et al., 2008b; Kudela et al., 2008a; Kahru et al., 2009; and the Pew Oceans Commission (2003) recommended regional

Band-Schmidt et al., 2010; Rensel et al., 2010b; Garcia-Mendoza, ocean governance efforts as an effective mechanism to facilitate

unpubl. data). The HABs that threaten west coast water quality, the regional ecosystem assessment and management. Recognizing this

health of living resources, and the economies of its communities need, the West Coast Governors’ Agreement on Ocean Health

are diverse and often extend beyond jurisdictional boundaries. (WCGA) was established in 2006 as a proactive, regional

Comprehensive understanding of the causes and impacts of west collaboration to protect and manage ocean and coastal resources

Table 1

Reported human illnesses and deaths due to paralytic shellﬁsh poisonings. Additional illnesses are known from all areas, but only those associated with fatalities are reported

here. Dates vary depending on state, country and when monitoring began.

Year Cases Deaths Counties/areas involved Shellﬁsh kind

1799 150+ 100 Sitka, Peril Strait Blue mussels

1934 12 2 Douglas and Admiralty Islands Not known

1944 4 1 Likely Sitka Not known

1947 3 1 Peril Strait Butter clams

1954 8 1 False Pass Blue mussels

1962 27 1 Porpoise Island Littleneck clams

1962 1 1 Hawk Inlet Blue mussels

1962 1 1 Shelter Bay Butter clams

1965 4 1 Hawk Inlet Butter clams

1994 16 1 Kalsin Bay, Kodiak Blue mussels

7,8

1997 9 1 Sturgeon River, Kodiak Butter clams,

littleneck clams

1999 1 Kodiak Not known

2010 5 2 Juneau and Haines Cockles, Dungeness

crab viscera

1793 4 1 Poison Cove Mussels, clams

1942 3 3 Barkley Sound Mussels, clams

1965 4 1 Theodosia Inlet Cockles

1980 7 1 Health Harbor, Gilford Island Butter clams

1942 9 3 Sekiu, Strait of Juan de Fuca Clams, mussels

OR 11

1933 21 1

1903 12 5 Sonoma County California mussels

1927 103 6 Sonoma, Marin, San Mateo Mussels

1929 60 4 Sonoma, Marin, San Mateo Mussels, clams

1936 3 2 Ventura Mussels

1939 76 8 Santa Cruz, Monterey Mussels, clams

1943 20 4 Del Norte, Humboldt Mussels

1944 12 2 San Mateo, Santa Cruz Mussels

1946 3 1 San Mateo Mussels

1948 3 1 San Mateo Mussels

1980 98 2 Sonoma, Marin Mussels, oysters, scallops

1976 7 2 Paciﬁc Mexico

1979–2008 391 24 Paciﬁc Mexico

1979 18 3 Mazatlan Bay, extensive ﬁsh kill Oysters, clams

14,17

1989 99 3 Gulf of Tehuantepec Rocky oysters

2001–2002 600 6 Michoaca´n and Guerrero coasts

2001–2002 101 6 Chiapas, Guerrero coasts

1 2 3 4 5 6 7

Sources: AK: Tikhmenev (1979), Sommer and Meyer (1937), Alaska’s Health (1945), Magnusson et al. (1951), Meyers and Hillian (1955), Orth et al. (1975), Ostasz

8 9 10 11 11

(2001), RaLonde (2001), State of Alaska Epidemiology Bulletin (2010); BC: Chiang (1988); WA: Nishitani and Chew (1988); OR: Nishitani and Chew (1988); CA:

12 13 14 15 16 17

Sommer and Meyer (1937), Price et al. (1991); MX: Saldate-Castan˜eda et al. (1991), Corte´s-Altamirano and Sierra-Beltra´n (2008), Mee et al. (1986), Herna´ndez-

Becerril et al. (2007).

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 135

along the coasts of Washington (WA), Oregon (OR), and California Symptoms of PSP are neurological, onset is rapid and can result in

(CA). Harmful algal blooms were highlighted as needing immedi- paralysis or death through respiratory arrest. Toxicity varies with

ate attention by all three states. shellﬁsh species, and some of the west coast species most likely to

The WCGA called for ‘‘a HAB workshop . . . to reach consensus on be contaminated include mussel species, butter clams (Saxidomus

the present state-of-knowledge and prioritize the information giganteus Deshayes), geoduck clams (Panopea generosa Gould),

needed by decision makers to lessen the impacts of the HAB events razor clams (Siliqua patula Dixon), and Paciﬁc oysters (Crassostrea

on humans and critical marine resources’’ as part of the strategy to gigas Thunberg); see Section 2.2. Several other species reportedly

promote interstate coordination of HAB research and monitoring have also been contaminated, including northern quahogs

efforts (Action Plan for the West Coast Governors’ Agreement on (Mercenaria mercenaria Linnaeus), horse clams (Tresus nuttallii

Ocean Health, 2008). The National Oceanic and Atmospheric Conrad and Tresus capax Gould), Paciﬁc littleneck clams (Protothaca

Administration (NOAA) and the states of CA, OR, and WA convened staminea Conrad), manila clams (Venerupis philippinarum Adams &

the West Coast Regional Harmful Algal Bloom Summit on 10–12 Reeves), varnish clams (Nuttallia obscurata Reeve), purple-hinge

February 2009 in Portland, Oregon, to fulﬁll the WCGA charge. A rock scallops (Hinnites multirugosus Gale) and other scallop species,

White Paper, Harmful Algal Blooms in the West Coast Region: History, cockle species, whelk species, moon snails (Lunatia heros Say),

Trends, and Impacts in California, Oregon, and Washington, was gooseneck barnacles (Pollicipes polymerus Gmelin), Dungeness

developed by the Summit Steering Committee to summarize the crabs (Metacarcinus magister Dana), and spiny lobsters (Panulirus

scope of the HAB problem in this region, in order to provide spp.) (Shumway et al., 1990; Shumway and Cembella, 1993;

background on the state-of-knowledge for Summit attendees. Here, Matter, 1994; Shumway, 1995; Deeds et al., 2008).

we expand on that White Paper, incorporating Summit ﬁndings and Human deaths attributed to PSP date back to 1793 (Table 1),

consensuses, and extending the geographical coverage of HAB when four members of Captain George Vancouver’s Royal Navy

impacts on the west coast to include Alaska (AK), British Columbia crew became sick and one died after eating shellﬁsh from a beach

(BC), the U.S. Paciﬁc coast states, and Mexico. in central BC now called Poison Cove (Quayle, 1969; Fig. 2). The

oldest documented apparent HAB incident in AK occurred in 1799

2. Paralytic shellﬁsh poisoning when the Aleut crew of the Russian fur trader, Alexander Baranof,

became ill after eating blue mussels (Mytilus edulis Linnaeus) in an

2.1. Overview of toxicity, history on the North American west coast area near Sitka, AK, now called Peril Strait (Table 1 and Fig. 1). This

incident resulted in an estimated 100 deaths (Fortuine, 1975).

Paralytic shellﬁsh poisoning is caused by a suite of biotoxins, More recently, a June 2010 incident in southeast AK resulted in ﬁve

collectively called paralytic shellﬁsh toxins (PSTs). Taxa known to illnesses and two deaths, the ﬁrst deaths in AK since 1997.

produce these toxins include species of the dinoﬂagellate genera Elsewhere on the west coast, human poisonings from PSP were

Alexandrium, Gymnodinium, and Pyrodinium. The genus typically apparently common in CA in the last half of the 1800s (Sommer

associated with toxic outbreaks along the U.S. and Canadian west and Meyer, 1937), but the ﬁrst recorded incident occurred in

coasts is Alexandrium, while Gymnodinium and Pyrodinium species Sonoma County, central CA, in 1903, when 12 people became ill

are associated with outbreaks in Mexico (Ochoa et al., 1997). and ﬁve died after eating California mussels (Mytilus californianus

Fig. 1. Alaska coast line showing sites with highest concentrations of paralytic shellfish toxins (number with no units; units are mg/100 g shellfish meat) by shellfish species.

The regulatory limit for paralytic shellﬁsh toxins is 80 mg/100 g.

Data sources: ADHHS-ES database, 1973–2008; http://www.epi.hss.state.ak.us/bulletins/catlist.jsp?cattype=Paralytic+Shellﬁsh+Poisoning+(PSP)), Gessner et al. (1997),

RaLonde (2001).

136 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

Fig. 2. British Columbia coast line with sites of highest concentrations of paralytic shellﬁsh toxins (number with no units; units are mg/100 g shellﬁsh meat) and

domoic acid (ppm).

Data sources: Canadian Food Inspection Agency data, www.inspection.gc.ca, Chiang (1988).

Conrad; Sommer and Meyer, 1937). Paralytic shellﬁsh poisoning distribution in some locations. For example, the frequency and

has been recognized as a serious health risk in CA since 1927 when geographic distribution of associated shellﬁsh closures in Puget

a major outbreak in a multi-county region north and south of San Sound have increased in WA since monitoring ﬁrst began in

Francisco resulted in 102 illnesses and six deaths (Table 1). From the 1940s and 1950s (Trainer et al., 2003) and PST-related

1927 through 1989, PSP related illnesses totaled 511 in CA, shellﬁsh closures have increased on the OR coast from the 1980s

including 32 deaths (Price et al., 1991). The earliest reported PSP (Oregon Department of Fish and Wildlife, ODFW data, http://

poisonings in WA and the only deaths in that state occurred in public.health.oregon.gov/HealthyEnvironments/Recreation/

1942 when one adult and two Native American children died after HarmfulAlgaeBlooms/Pages/index.aspx, Strutton and Tweddle,

eating butter clams and blue mussels collected along the Strait of unpubl. data). Outbreaks have decreased in other locations (e.g.,

Juan de Fuca (Quayle, 1969; Table 1). Three deaths also occurred high PSP levels in Drakes Bay, CA have declined since the 1980s,

further north in Barkley Sound on the West Coast of Vancouver California Department of Public Health, CDPH data, http://

Island, BC (Fig. 2). Additional incidences in BC occurred in 1957 www.cdph.ca.gov/HealthInfo/environhealth/water/Pages/

with 61 cases but no deaths, 1965 with four cases and one death, Shellﬁsh.aspx).

and 1980 with two illnesses and one death. The 1965 incident is

especially signiﬁcant because it was the ﬁrst time that a human 2.2.1. Alaska

death occurred at the same time that shellﬁsh toxicity was Paralytic shellﬁsh toxins are a pervasive problem in AK (Figs. 1

measured, and a bloom of the toxic species, Alexandrium acatenella and 7A). Personal use and subsistence shellﬁsh harvests accounted

(Whedon et Kofoid) Balech was recognized as the causative for 183 conﬁrmed PST illnesses between 1973 and 2008 (Alaska

organism (Prakash and Taylor, 1966; Quayle, 1969). More recent Department of Health and Human Services-Epidemiology Section,

events in WA include 10 illnesses in 1978, ﬁve illnesses in 1998, ADHHS-ES database, 1973–2008; http://www.epi.hss.state.ak.us/

and nine illnesses in 2000, all in Puget Sound (Erickson and bulletins/catlist.jsp?cattype=Paralytic+Shellﬁsh+Poisoning+(PSP)),

Nishitani, 1985; Trainer et al., 2003; Moore et al., 2009). PSP with more than half of the illnesses from consumption of butter

poisonings in OR caused 20 illnesses and one death in Coos Bay in clams that can retain the toxins for more than two years

1933 (Sommer and Meyer, 1935; Halstead, 1965). In Paciﬁc (Shumway, 1990). Blue mussels, cockles (Clinocardium sp. Keen),

Mexico, the first documented report of human shellfish poisoning razor clams, Pacific littleneck clams, and other unknown clams

dates only to 1976, with seven cases and two deaths (Saldate- caused the remaining illnesses. Numbers of reported illnesses may

Castan˜eda et al., 1991). Between 1979 and 2008, 391 poisoning be underestimated by a factor of 10–30, due to underreported

cases were recorded with 24 deaths along the Paciﬁc coast of minor illnesses, inaccurate and incomplete incident recording, and

Mexico. Of these, 34 cases with ﬁve deaths were attributed to misdiagnosis (Gessner and Middaugh, 1995; Gessner and

Gymnodinium catenatum Graham, and 357 cases with 19 deaths McLaughlin, 2008). Rural harvesters are particularly at risk

were attributed to Pyrodinium bahamense var. compressum (Bo¨hm) because they underestimate the potential of illness based on trust

Steidinger, Tester & Taylor (Corte´s-Altamirano and Sierra-Beltra´n, of traditional local knowledge to determine when to consume

2008). shellﬁsh, including use of unreliable environmental cues such as

water color. Furthermore, Alaskans continue to use the myth that

2.2. Trends in prevalence and impacts PSTs occur only in months that do not have an ‘‘r’’ in the spelling;

i.e., May through August, when the reality is that PSTs and illnesses

Outbreaks of Alexandrium spp., and associated shellﬁsh toxicity can occur year-round.

and human illnesses have been a persistent problem along the west The impacts of PSTs on public health are unevenly distributed

coast for decades. Outbreaks may be increasing in frequency and across populations; e.g., compared to other Alaskans, AK Natives

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 137

living in coastal communities are nearly 12 times more likely to The intensity and frequency of blooms also vary among growing

encounter PSTs by consuming untested, subsistence-harvested areas. Baynes Sound on the east coast of Vancouver Island does not

shellﬁsh (Gessner and Schloss, 1996). Sharing subsistence harvest often experience Alexandrium blooms, and the highest level of PST

is common practice among AK Natives and this practice can recorded there between 1994 and 2008 was 430 mg/100 g in

geographically expand the risk of illness far beyond a single California mussels (Canadian Food Inspection Agency data). Areas

community. For example, King Cove, on the Aleutian Peninsula, has such as Barkley Sound on the west coast of Vancouver Island have

a known history of PST illnesses and fatalities, and shellﬁsh from PST events nearly every year. The north and central coasts can have

this region are often shared with Native Americans along the U.S. extensive Alexandrium spp. blooms, and at least one PST event has

west coast and interior AK (Wright et al., 2008). been recorded along the mainland coast each year (Taylor and

The highest PST levels measured to date from various shellﬁsh Harrison, 2002). Paralytic shellﬁsh toxin activity is less common in

species in AK are shown in Fig. 1. The highest level, 20,600 mg/ the areas monitored on the Haida Gwaii (formerly named Queen

100 g shellﬁsh meat, occurred in blue mussels from Kalsin Bay, Charlotte Islands).

Kodiak Island, in late May 1994, and resulted in 16 illnesses Illnesses due to PSP are relatively rare in BC, with none reported

(Ostasz, 2001). Elevated PSTs in Prince William Sound are a rare since 2005 (Canadian Food Inspection Agency data). The low

event because populations of butter clams, Paciﬁc littleneck clams, number of PSP illnesses may be attributed to the biotoxin

and soft shelf clams (Mya arenaria Linnaeus) are depressed due to monitoring program, prompt and effective closures of harvest

the 1964 earthquake, commercial ﬁshery overharvesting of razor areas, and education of the public to the hazards of shellﬁsh

clams, sea otter (Enhydra lutris Linnaeus) predation, and impacts poisoning. The majority of recorded illnesses have been attributed

from the 1989 Exxon Valdez oil spill (Baxter, 1971; Rukuyama et al., to butter clams. As noted previously, these clams can retain toxins

2000; Thomas et al., 2002). High PST levels, up to 7750 mg/100 g, for longer than two years (Quayle, 1969; Kvitek et al., 2008). Due to

exist along the southern shoreline of the Aleutian Peninsula, while the likelihood of high levels of PST in butter clams, this species

the northern shoreline and the Bering Sea have lower levels remains closed for harvest in most areas in BC. The impacts of PST

ranging from 135 to 310 mg/100 g based on Stimpson’s surf clam closures on the commercial shellﬁsh industry are difﬁcult to

(Mactromeris polynyma Stimpson) viscera (Hughes and Nelson, quantify. Anecdotally, the impacts are considered extensive.

1979). Commercial ﬁsheries exist for Paciﬁc littleneck clams in Harvest areas can be shut down for months at a time, resulting

Kachemak Bay, razor clams in lower Cook Inlet, and geoduck clams in signiﬁcant layoffs for staff and harvesters, and markets can be

in southeastern AK, where monitoring occurs during the harvest lost. Butter clams are the preferred shellﬁsh species for Food Social

period. The following shellﬁsh species have shown some record of Ceremonial harvesting for First Nations people on the north coast,

PSTs above the regulatory limit of 80 mg/100 g: blue mussel, butter but it can be difﬁcult to ﬁnd areas that can be opened for their

clam, Stimpson’s surf clam, razor clam, Paciﬁc littleneck clam, harvest because these clams retain toxins for long periods of time.

geoduck clam, scallop species, cockle species, Paciﬁc oyster,

Dungeness crab, Tanner crab (Chionoecetes bairdi Rathbin), and 2.2.3. Washington

snow crab (Chionoecetes opilio Fabricius), particularly in the In Washington (Fig. 3), PST-related closures of recreational

Aleutian area, Kodiak Island, and in the Southeast Alaska region shellﬁsh harvesting have been imposed since an incident

at Juneau and Ketchikan (ADHHS-ES database). Historically, the in 1942 that led to three Native American fatalities on the

toxin appears to constitute a persistent threat to human health, in Strait of Juan de Fuca (Trainer et al., 2003; Table 1). The

particular because while commercially harvested and farmed Washington Department of Health (WDOH; http://www.doh.

products are tested, recreationally harvested shellﬁsh are not wa.gov/CommunityandEnvironment/Shellﬁsh/BiotoxinsIllness

tested. Prevention/Biotoxins.aspx) imposed a harvesting closure at that

Paralytic shellﬁsh toxins also have been measured in a number of time for all bivalve species except razor clams from Dungeness

crab species, including Dungeness, Tanner, snow, hair (Erimacrus Spit to the mouth of the Columbia River from 1 April to 31 October.

isenbeckii Brandt), and red king (Paralithodes camtschaticus Tilesius) The coastal closure is reissued every year, but the Strait of Juan de

crabs (ADHHS-ES database, 1973–2008). Dungeness and Tanner Fuca closures are now regulated by toxin monitoring (F. Cox, pers.

crabs harvested in the Kodiak and Aleutian/Bering Sea ﬁsheries must comm.). Routine monitoring for toxins in commercial shellﬁsh in

be killed, cleaned, and sectioned before being shipped to market. waters north and west of Admiralty Inlet and in Willapa Bay and

Testing of Dungeness crabs in the southeast AK ﬁshery was Grays Harbor began in 1957 following a severe outbreak of PSP in

suspended in 1996 after four years of negative PST tests. BC (Nishitani and Chew, 1988). Washington Department of Health

records indicate that PSP closures occur in these coastal bays on an

2.2.2. British Columbia irregular (sporadic) basis.

In British Columbia, PSTs are the most prevalent biotoxins In the 1950s and 1960s, PSTs occurred in the northern regions of

affecting shellﬁsh growing areas. The frequency and intensity of Puget Sound (e.g., Sequim and Discovery bays), extending

Alexandrium blooms vary from year to year, but blooms are southward during the 1970s and 1980s to the inner Sound

expected each year. Monitoring for PSTs in BC began in 1942, as did (Quayle, 1969; Nishitani and Chew, 1988; Rensel, 1993; Trainer

the ﬁrst formal closures (Quayle, 1969). In 1982, a PST level of et al., 2003; Trainer and Hickey, 2003; Cox et al., 2008). Prior to

30,000 mg/100 g was recorded in California mussels in Work 1978, illnesses due to PSP were not reported in Puget Sound

Channel on the northern BC mainland (Chiang, 1988; Fig. 2). The including Hood Canal, and Whidbey, Central and South basins, but

highest level of PST recorded since 1994 was 10,000 mg/100 g in widespread toxicity occurred in September 1978, beginning in

mussels at Ellen Point on the northeast coast of Vancouver Island in Whidbey Basin and spreading as far south as Des Moines (south of

April 1994 (Fig. 2). Seattle) in the Central Basin (Nishitani and Chew, 1988; Fig. 7B).

In the south coast of BC, PST events usually occur during the Toxin levels in bay mussels (Mytilus trossulus Gould) were as high

months of April to October, but may occur throughout the year as 30,360 mg/100 g shellﬁsh meat (Fig. 3). Ten people reported PSP

(Taylor and Harrison, 2002; Canadian Food Inspection Agency data, symptoms after eating recreationally harvested mussels and pink

www.inspection.gc.ca). Bloom initiation rarely occurs in winter scallops (Chlamys rubida Hinds), but no deaths occurred. The ﬁrst

months. The last widespread closure due to PSTs along the BC shellﬁsh harvest closures in the South Basin occurred in October

coastline occurred in 2008 when most of the main commercial 1988 when toxin levels in Paciﬁc oysters reached 2000 mg (Trainer

growing areas were closed for a portion of the summer. et al., 2003). Since then, repeated closures have occurred in most

138 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

Fig. 3. Washington coast line with sites of highest concentrations of paralytic shellﬁsh toxins (number with no units; units are mg/100 g shellﬁsh meat) and domoic acid

(ppm). Other sites mentioned in the text are also shown.

Data sources: WDOH, http://www.doh.wa.gov/CommunityandEnvironment/Shellﬁsh/BiotoxinsIllnessPrevention/Biotoxins.aspx, Trainer et al. (2003).

years throughout the Puget Sound basins, except south Hood The frequency and duration of PST-related shellﬁsh closures on

Canal, but not always in the same time or place each year (Cox the OR coast increased from 1979 to 1996 (ODFW data, http://

et al., 2008). public.health.oregon.gov/HealthyEnvironments/Recreation/

Ceremonial, subsistence, and commercial harvests by WA tribal HarmfulAlgaeBlooms/Pages/index.aspx, Strutton and Tweddle,

communities have been greatly impacted by PST-associated unpubl. data, Fig. 7C). Twice as many closures occurred from

shellﬁsh closures. The Puyallup, Suquamish, and Jamestown 1990 to 1996 as in all previous years, and most of the recent

S’Klallam tribes have experienced severe economic losses from closures lasted more than 50 days. In the 2000s, the total number

their commercial geoduck ﬁsheries, based on frequent and lengthy of closures, considering the northern and southern coasts

seasonal harvest closures (Wekell and Trainer, 2002). Recalls of separately, was 23, compared with 15 in the 1990s and 6 in the

geoduck related to PST events have cost the tribes about $30,000. 1980s.

Commercial harvesting of Paciﬁc oysters and Dungeness crabs by

the Jamestown S’Klallam Tribe, and of manila clams, Paciﬁc 2.2.5. California

oysters, and basket cockles (Clinocardium nuttallii Conrad) by the Paralytic shellﬁsh poisoning events have occurred along the CA

Lummi Nation have also been signiﬁcantly affected. Subsistence coast (Fig. 5) since before written records were maintained, with

and ceremonial harvesting by the Jamestown S’Klallam Tribe have ‘‘mussel poisoning’’ being recognized by coastal tribes (Meyer

been impacted by PST toxicity of butter, Paciﬁc littleneck, horse, et al., 1928). Paralytic shellﬁsh toxin levels have been highly

and manila clams. Beach closures have also impacted Puyallup variable and unpredictable during the decades that monitoring has

tribal culture by restricting the use of clams for ceremonial dinners been conducted, as has the breadth of geographic range involved

at weddings and funerals. (Price et al., 1991; Langlois, 2001). Despite the temporal and

geographic variability, in every year since 1999, PSP toxins have

2.2.4. Oregon been observed in Drakes Bay along the Marin County coast, north

Irregular monitoring of shellﬁsh for saxitoxins began in OR in of San Francisco (CDPH data).

1958 after high levels of PSTs were reported along the WA coast In general, Alexandrium is absent or constitutes a minor

(Nishitani and Chew, 1988). Changes in monitoring sites, shellﬁsh component of the marine phytoplankton community along the

species monitored, and the possibility that blooms initiated CA coast. This dinoﬂagellate has been observed in approximately

offshore make it difﬁcult to compare the early data with later 3500 of the 24,000 phytoplankton samples collected by the CDPH

values (Nishitani and Chew, 1988). More consistent monitoring monitoring program since 1993. It has comprised less than 10% of

(conducted since 1979 by the Oregon Department of Agriculture, the phytoplankton assemblage in 93% of these samples and 55% of

ODA; http://oregon.gov/ODA/FSD/shellﬁsh_status.shtml) at more the observations have been at <1% relative abundance (CDPH

sites has improved the coverage and has led to frequent closures, data). Visible blooms of Alexandrium are rarely seen along the CA

primarily of razor clam and mussel species shellﬁsheries. Paralytic coast, with only one documented visible event in the past 19 years.

shellﬁsh toxins have severely impacted shellﬁsh harvests at A massive ‘red tide’ due to Alexandrium covered Drakes Bay for a

Clatsop Beach in northern OR (Fig. 4). The severity of a PST- brief period in July 1991 (Langlois, 2001; G. Langlois, pers. comm.).

associated HAB outbreak varies annually between northern and The greatest frequency of Alexandrium observations has been

southern OR coastal areas. In 1992, a PST event affected the central recorded along the Marin County coast, consistent with the general

and northern coast, but not the southern beaches, while in 2001 pattern of PST frequency, followed by sites along the San Luis

PST affected only the southern beaches. Obispo County coast (Langlois, 2001; CDPH data). Each of these

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 139

Fig. 4. Oregon coast line with sites of highest concentrations of paralytic shellﬁsh toxins (number with no units; units are mg/100 g shellﬁsh meat) and domoic acid (ppm).

Data sources: ODA data, http://oregon.gov/ODA/FSD/shellﬁsh_status.shtml, ODFW data, http://public.health.oregon.gov/HealthyEnvironments/Recreation/

HarmfulAlgaeBlooms/Pages/index.aspx, M. Hunter (unpubl. data).

regions experiences more than twice the frequency of Alexandrium contained Alexandrium cells, coinciding with an increase in PSP

observations of any other coastal county. There was an apparent activity in the region (see below). Low abundances of A. catenella

increase in Alexandrium along the Santa Barbara coast beginning in were detected in all seasons from a weekly monitoring program

1999, with the greatest number of observations occurring in 2006; conducted in a small harbor of Santa Monica Bay from 2006 to

15 of 52 phytoplankton samples (29%) collected at Goleta Pier 2009 using a quantitative PCR method (Garneau et al., 2011).

Fig. 5. California coast line with sites of highest concentrations of paralytic shellﬁsh toxins (number with no units; units are mg/100 g shellﬁsh meat) and domoic acid (ppm).

Data sources: CDPH data, http://www.cdph.ca.gov/HealthInfo/environhealth/water/Pages/Shellﬁsh.aspx, Price et al. (1991).

140 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

Highest abundances of the dinoﬂagellate coincided with low but shellﬁsh toxin accumulations are associated with blooms of G.

measurable concentrations of PSTs in the plankton. catenatum and P. bahamense var. compressum. The former species

Data from the CDPH biotoxin monitoring program show that a has been observed from the upper Gulf of California in the north to

major period of PSP activity extended through the 1980s into 1991 Guerrero in the south, while P. bahamense var. compressum is

(Fig. 7D). The highest toxin levels detected in shellﬁsh during this responsible for PSP outbreaks in the southern states of Paciﬁc

time were 26,000 mg/100 g in rock scallops from Sonoma County, Mexico (Ochoa et al., 2002).

associated with the last documented fatality in August 1980, The dinoﬂagellate, G. catenatum, was ﬁrst described from the

16,000 mg/100 g in California mussels from Marin County (July central Gulf of California coast at cell abundances up to 10 /l

1980), 14,000 mg/100 g in California mussels from Humboldt (Graham, 1943), but the ﬁrst documented PSP event occurred near

County (September 1989) and 10,000 mg/100 g in California mussels the mouth of the Gulf in 1979 when three human deaths and an

from Marin County (July 1991) (Price et al., 1991; CDPH data, Fig. 5 extensive ﬁsh kill occurred (Mee et al., 1986, Table 1). Toxin levels

and Table 1). Each of the subsequent 18 years has experienced during that event ranged from <20 to 7640 mg/100 g in the tropical

signiﬁcant levels of PST in shellﬁsh, but these have been well below rocky oyster (Ostrea iridescens Hanley), with cell densities up to

the 1980s maxima and have been restricted in geographic range and 6.6 Â 10 cells/l (Mee et al., 1986, Fig. 6). Additional blooms have

duration (CDPH data, Fig. 7D). During this latter period, the occurred in Bahia Mazatla´ n, Colima, Guerrero, and Oaxaca. Most

concentration of PSTs has exceeded 3000 mg/100 g only once in blooms occur between February and May when the water

California mussels from Marin County (in August 1998). temperature ranges between 17 and 25 8C (Manrique and Molina,

The majority of PSP activity has historically occurred in the 1997; Ga´ rate-Liza´rraga et al., 2004, 2006).

central and northern portions of CA (Price et al., 1991; CDPH data). Blooms of Gymnodinium species have impacted public health.

Despite this general pattern, over the years alert levels of PST have The number of humans affected varies in different reports.

been detected in shellﬁsh from each of the coastal counties (CDPH Herna´ndez-Becerril et al. (2007) mentioned 561 intoxications and

data). In recent years, an increase in PSP activity has been suggested 38 fatalities from 1970 to 2004 related to PSTs of Gymnodinium. In

in some southern CA sites, most notably in commercial shellﬁsh contrast, Corte´s-Altamirano and Sierra-Beltra´n (2008) recognized

growing areas in Santa Barbara and San Diego counties. Sampling only 34 intoxications and ﬁve deaths. No intoxications associated

sites in Santa Barbara experienced alert levels of PST every year with Gymnodinium have been reported recently, which may be

between 2005 and 2008, peaking in 2006 (744 mg/100 g). Prior to attributed to increased attention by Mexican health authorities.

this recent activity and a moderate event in 1998, there had not Periodic bans on harvesting cultured or wild shellﬁsh have been

been alert levels for these toxins in this region since the 1980s. The imposed by health authorities in Mexico since 2004. The majority of

San Diego aquaculture site experienced PST concentrations in those closures were associated with the presence of Gymnodinium

excess of the federal alert level for the ﬁrst time in 2008. The last species (Federal Commission for the Protection against Sanitary

time the alert level was exceeded anywhere in San Diego County Risks, COFEPRIS; http://www.cofepris.gob.mx/AZ/Paginas/Marea%

was during 1985 in a mussel sample from Scripps Pier (La Jolla) and 20Roja/MareaRoja.aspx).

2006 inside San Diego Bay (G. Langlois, pers. comm.). Three major toxic outbreaks of P. bahamense var. compressum

involving human poisoning have been documented on the Paciﬁc

2.2.6. Mexico coast of Mexico (Herna´ndez-Becerril et al., 2007). In November

Paralytic shellﬁsh poisoning is the most important toxic 1989 in the Gulf of Tehuantepec region, three persons died and

syndrome related to HABs in Mexico, and PSTs are the only toxins 99 persons were poisoned as a consequence of a bloom that

associated with human fatalities (Fig. 6 and Table 1). Paralytic reached a maximum abundance of 1.7 Â 10 cells/l (Fig. 6 and

Fig. 6. Mexican coast line with sites of highest concentrations of paralytic shellﬁsh toxins (number with no units; units are mg/100 g shellﬁsh meat) and domoic acid (ppm).

Data sources: COFEPRIS data, http://www.cofepris.gob.mx/AZ/Paginas/Marea%20Roja/MareaRoja.aspx, Garcia-Mendoza et al. (2009), Mee et al. (1986), Saldate-Castan˜eda

et al. (1991). Events described in Herna´ndez-Becerril et al. (2007) and Corte´s-Altamirano and Sierra-Beltra´n (2008).

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 141

Table 1). During this bloom, PST concentrations as high as 811 mg/ Herna´ndez-Becerril et al., 2007), but none have been associated

100 g were recorded in tropical rocky oyster (Saldate-Castan˜eda with toxic events. One species, A. catenella, has been reported in the

et al., 1991). On the Michoaca´n and Guerrero coasts in central phytoplankton assemblage of the Todos Santos Bay region

Mexico, 600 people were affected and six died during a bloom from (northern part of the Baja California peninsula), and resting cysts

November 2001 to February 2002. From November 2001 to August of this species exist in the sediments of the bay (Pen˜a-Manjarrez

2002 on the Chiapas (south) and Guerrero (central) coasts, 101 et al., 2005); however, high densities of A. catenella have not been

persons were poisoned and six died when patches of P. bahamense reported in this region or in other areas of Paciﬁc Mexico.

var. compressum were present in this region. Thirteen other toxic Monitoring of phycotoxins related to HABs started in the 1980s

outbreaks associated with P. bahamense var. compressum were in high risk areas of the Paciﬁc coasts of southern states of Mexico.

documented from 1979 to 2006 but did not result in fatalities Unfortunately, data on the variation of microalgal phycotoxin

(Corte´s-Altamirano and Sierra-Beltra´n, 2008). concentrations were not available until 2001 when a nationwide

Several species of Alexandrium have been reported from Paciﬁc program was implemented by the COFEPRIS (Fig. 7E). A consistent

waters off Mexico (Okolodkov and Garate-Liza´rraga, 2006; monitoring of PSTs started in Baja California, Sonora, and southern

Fig. 7. Time series of paralytic shellﬁsh toxins (mg/100 g shellﬁsh meat) for (A) Alaska, (B) Washington, (C) Oregon, (D) California, and (E) Acapulco, Mexico. The regulatory

alert level of 80 mg/100 g is shown in each graph, and extreme values are annotated for bars exceeding the axis limits. Note that each data set has variable start and end dates.

Data sources are provided in the text and in previous ﬁgures.

142 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

states of the Paciﬁc coast of Mexico. Concentration of toxins in the University of Alaska Southeast and Fairbanks, and NOAA) has

shellﬁsh from Baja California and Sonora were never above the trained shellﬁsh growers, tribal members and volunteers to sample

alert level (‘‘Proyecto Marea Roja’’ of the COFEPRIS). In contrast, the and identify HAB species in coastal areas near Ketchikan, Juneau,

presence of PSTs is a recurrent problem in Chiapas, Oaxaca, and and Kachemak Bay. This program, which began in 2008, will add to

Guerrero. Concentrations above 80 mg/100 g are frequent in the knowledge of HAB species in AK waters.

coastal areas of these states and a maximum concentration of

7309 mg/100 g was detected in the Acapulco area in the winter of 2.3.3. British Columbia

2001. A trend for the presence of PSTs is difﬁcult to assess in the There does not appear to be a link between pollution (or other

Acapulco area. However, toxin outbreaks seemed to diminish in anthropogenic effects) and promotion of Alexandrium blooms in

frequency (number of events above the alert level) but increase in BC. Many fairly unpopulated, remote areas along the BC coast

magnitude (maximum concentration measured in shellﬁsh sam- experience signiﬁcant Alexandrium blooms, while some areas that

ples) from 2003 to 2011. are relatively heavily populated are not as affected by Alexandrium

blooms (Taylor, 1993). In BC, blooms often originate nearshore in

2.3. Factors promoting blooms shallow areas and then spread to larger bodies of water (Taylor

et al., 1994). There is some evidence for increased Alexandrium

2.3.1. Overview: anthropogenic vs. natural factors blooms during El Nin˜o years; however, there have been signiﬁcant

There is little evidence to support anthropogenic factors as blooms also in non El Nin˜o years (Erickson and Nishitani, 1985;

primary promoters of Alexandrium blooms and PSTs in most areas Taylor and Harrison, 2002). There may also be a link between

along the Paciﬁc west coast. In CA, blooms are strongest in the drier warmer water temperatures and increased bloom activity (Yan

seasons and it appears that blooms usually start offshore and move et al., 2003).

onshore when upwelling winds relax (Langlois and Smith, 2001;

Anderson et al., 2008b). Although some blooms start nearshore in 2.3.4. Washington

BC and WA, in general blooms occur during periods of warmer Paralytic shellﬁsh toxin events in Puget Sound are thought to

surface temperatures, which characterize periods of stratiﬁcation originate primarily from local shallow areas of the Sound and not

in upwelling regions. Thus, these PSP events appear related more to from offshore cyst or motile cell populations advected into the

large-scale oceanographic forcing, although there may be potential Sound (Cox et al., 2008). Paralytic shellﬁsh toxin occurrences

inﬂuence from local nutrient inputs when cells reach the shore throughout Puget Sound have been documented since 1978, when

(Anderson et al., 2008a). Presently, the extent and possible role of a bloom of A. catenella spread from the Whidbey Basin through

local nutrient pulses in stimulating blooms of Alexandrium are not central Puget Sound and into the southern extremes of the Sound

known. In WA, little is known about the origin of coastal blooms of (Nishitani and Chew, 1988). A survey in 1981 found motile cells,

Alexandrium, but it is possible that they may also be brought inshore cysts, or low levels of toxin in all areas of the southern Sound

when upwelling winds relax or downwelling winds occur and enter (Nishitani and Chew, 1988). More recently, in a 2005 survey, cysts

coastal bays; e.g., Grays Harbor and Willapa Bay (Roegner et al., were found throughout Puget Sound, with highest abundances in

2002), although this is not always the case (Cox, 2001). The the northern and central regions (Horner et al., 2008, 2011).

relaxation or reversal of upwelling-favorable winds is also likely an Abundance was highest in Quartermaster Harbor, considered a

important mechanism for bringing blooms into contact with the possible ‘‘breeding bay’’ for Alexandrium (Nishitani and Chew,

Oregon coast, and Tweddle et al. (2010) showed that elevated toxin 1984), but little correlation of cyst abundance with physical or

levels were associated with late summer upwelling. Anthropogenic chemical properties of the sediment was found.

nutrient sources are more likely to be relevant in inland waters Blooms of A. catenella in Puget Sound generally occur from late

along the Strait of Juan de Fuca and in Puget Sound (Rensel, 2007), spring through summer (Trainer et al., 2003; Dyhrman et al., 2010).

although with the exception of some shallow bays, the nutrient Like many other dinoﬂagellates, its growth is favored by a stable

source is still more likely upwelled waters. water column and warm temperatures (Nishitani and Chew, 1984),

consistent with the hypothesis that blooms are stimulated by large

2.3.2. Alaska precipitation events followed by warm and calm weather (Erickson

Alaska has a long history of encounters with PSTs that occur and Nishitani, 1985; Determan, 1998). Moore et al. (2009), however,

along much of the Gulf of Alaska coast from the BC border in the did not ﬁnd a correlation between precipitation-induced freshwater

southeast to the Aleutian chain and into the Bering Sea on the west runoff (i.e., elevated Skagit River streamﬂow) and PST events. They

and more northerly coasts. Human health problems persist despite hypothesized that long residence time in surface waters (i.e., low

better understanding of these events, as many coastal residents streamﬂow) would favor PST events because toxin accumulation by

continue to consume potentially toxic shellﬁsh. There is no shellﬁsh would be enhanced. These authors determined that warm

evidence to support anthropogenic factors as promoters of air and water temperatures as well as low streamﬂow conditions

Alexandrium blooms or toxic events in this region. The shoreline preceded exceptional PST events in blue mussels from 1993 to 2007

is long and complex, human populations are remote and widely at four Puget Sound ‘‘hot spots’’ (sites of high PST incidence),

disbursed, and there are many streams, rivers, islands, and extreme Mystery, Discovery, and Sequim bays, and Kingston Marina, all in the

weather events that produce a complex marine ecosystem. The northern and central parts of Puget Sound.

nearshore Alaska Coastal Current, with a seaward boundary near Moore et al. (2008, 2009) also assessed the relationship of large-

the edge of the continental shelf of the Gulf of Alaska, is greatly scale and local climatic factors and PST occurrence in Puget Sound

affected by the local shoreline topography and by freshwater input shellﬁsh. In contrast to previous hypotheses linking large-scale

from fjords and estuaries (Royer, 1979, 1981; Schumacher and climatic variations (e.g., ENSO) with PST events (e.g., Erickson and

Reed, 1980). The North Paciﬁc High in summer allows intrusion of Nishitani, 1985), they found no such correlation. Their statistical

deep, nutrient-rich water into coastal waters resulting in the analyses of a 15-year continuous dataset of mussel toxicity

development of seasonal algal blooms (Horner et al., 1997); indicated that local climatic variability was more important than

however, there has been little historical phytoplankton monitoring large-scale variation in explaining shellﬁsh toxicity in Puget Sound.

with only a few sparse reports of the presence of Alexandrium (as The source(s) of Alexandrium blooms on the open WA coast has

Gonyaulax) (Horner et al., 1997). A new phytoplankton monitoring not been identiﬁed, and little is currently known about its

program (Alaska HAB monitoring program or AHAB, sponsored by occurrence and distribution offshore (F. Cox and R. Horner, pers.

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 143

comm.). If such blooms develop in the Juan de Fuca eddy or any during the fall), and they typically occur during periods of weaker

other offshore region, they are likely to impact the coast only winds following an upwelling event (unpubl. analysis of CDPH

during periods of downwelling winds (storms, which does not data, S. Piedracoba and J. Largier, pers. comm.).

appear to be the case). Blooms occur sporadically in coastal In general, dinoﬂagellate blooms in the central and northern CA

embayments such as Grays Harbor and Willapa Bay, and impact upwelling area are strongest in the fall (A. Paquin, K. Nielsen, and J.

the local shellﬁsh growing areas, but it is not known if these events Largier, pers. comm.), when winds are weaker and near-surface

originate within the bays or are advected from offshore. Razor thermal stratiﬁcation can develop (Largier et al., 1993; Garcia-

clams and mussels on the coastal beaches are sometimes affected Reyes and Largier, 2012). The highest chlorophyll a levels in waters

when shellﬁsh in the embayments are not. off Bodega Bay are observed in the fall (Garcia-Reyes and Largier,

2012), during periods of onshore ﬂow (unpubl. mooring data, E.

2.3.5. Oregon Dever and J. Largier, pers. comm.). During calm periods or

Observations of PSTs indicate a steady increase in frequency southerly winds, warm, low-salinity water that ﬂows out of San

and in both California mussels (M. californianus) and razor clams Francisco Bay can enhance stratiﬁcation and fronts along the Marin

(S. patula) since the inception of the Oregon shellﬁsh toxin and Sonoma coasts (Send et al., 1987; Wing et al., 1998; R. Fontana

sampling program in 1979. This increase was markedly so from and J. Largier, pers. comm.). In addition, southerly/westerly breezes

1992 to 1997 and from 2008 to 2010 (ODA data, http:// in fall lead to weak downwelling conditions, which can concen-

oregon.gov/ODA/FSD/shellﬁsh_status.shtml, M. Hunter, unpubl. trate dinoﬂagellate and other upward-motile plankton near to the

data). Since 2007, Oregon has been monitoring surf zone coast (A. Paquin, K. Nielsen, and J. Largier, pers. comm.),

phytoplankton for the presence of HABs and Alexandrium is particularly along south-facing coasts like those in Drakes Bay

commonly observed in samples, especially from the middle of (unpubl. drifter data and High Frequency-Radar surface current

June through September when the water temperature is >12.5 8C data, J. Largier and C. Halle, pers. comm.). The dinoﬂagellate, A.

(ODFW data at http://bioweb.coas.oregonstate.edu/mocha/ catenella, is a strong swimmer, in part due to the formation of long

odfwdata.html). High levels of saxitoxin in shellﬁsh tissues are chains of cells (Fraga et al., 1988), and it can be expected to be

also often associated with late summer upwelling and higher concentrated in buoyancy fronts and downwelling circulation near

chlorophyll concentrations (Tweddle et al., 2010). Comparing the the coast. From preliminary analysis of PST records, it appears that

tissue PST data with the surf zone Alexandrium data indicates that these events indeed correlate with large-scale oceanographic

a very low abundance of cells can result in elevated levels of tissue events, such as the upwelling-relaxation cycle (B. Keafer, D.

toxins (ODA and ODFW data at http://bioweb.coas.oregonstate. Anderson, and J. Largier, pers. comm.), and during onshore ﬂow.

edu/mocha/odadata.html). These results suggest that blooms are accumulated by interactions

with coastal ﬂows during calm or downwelling periods, whether

2.3.6. California initiated offshore or inshore.

There is no clear evidence to link the occurrence of PST events In an embayment such as Drakes Bay, which is sheltered from

off CA with El Ninõ or La Ninã periods – in fact, the last two major upwelling and receives low-salinity outflow from San Francisco

PST events in CA occurred during opposite conditions: 1989 during Bay, stratiﬁcation is more persistent (Largier, 2004), presumably an

a strong La Nin˜a period and 1991 during a strong El Nin˜o period important factor in the frequent occurrence of high PST levels

(Langlois, 2001). there. High PST concentrations observed in contiguous estuarine

The source of Alexandrium responsible for PST events along the environments (Drakes Estero) occur some days after high

CA coast is in question, but two likely scenarios are possible concentrations are detected on the open coast, consistent with

(Kudela et al., 2005; GEOHAB, 2011). First, this dinoﬂagellate may the scenario that PSP outbreaks initiate on the open coast and are

be transported in offshore warm water masses that can move subsequently transported into estuaries (Langlois, 2001; Banas

onshore under calm conditions. This advection process could et al., 2007; Kimbro et al., 2009). Central San Francisco Bay is not

potentially result in either a quick increase in PSP toxicity if the well suited to the development of dinoﬂagellate blooms owing to

number of transported cells is high, or it may simply provide the strong vertical mixing driven by tides (Cloern et al., 2005), but

cells necessary for a bloom to initiate. Second, resting cysts of coastal blooms could be imported to and spread in the Bay during

Alexandrium in local sediments can, under favorable conditions, calm periods in the dry season.

produce vegetative cells that have the ability to reproduce both The tendency for dinoﬂagellate bloom concentration and

sexually and asexually, resulting in localized ‘‘hot spots’’ of PSP nearshore distributions to occur during weak, southerly, or

toxicity in shellﬁsh. Regardless of the origins of the toxin- westerly winds suggests a tendency for blooms to spread

producing dinoﬂagellates, the general pattern has been for these northward – currents nearshore off northern and central CA are

blooms to be detected ﬁrst along the open coast and in bays (e.g., typically northward and onshore at such times (Largier et al.,

Drakes Bay), followed by transport into enclosed estuaries (e.g., 2006). An apparent northward spread has been observed during

Drakes Estero) (Price et al., 1991; Langlois and Smith, 2001; large-scale outbreaks of PST toxicity, with PST events often

Anderson et al., 2008b). The degree to which coastal phytoplankton initiating near San Francisco and extending north to Point Arena

blooms are found in bays and estuaries depends on the source (Rogers-Bennett et al., 2012) and beyond, as far north as the CA-OR

waters for the bay/estuary (e.g., Banas et al., 2007; Kimbro et al., border (Langlois, 2001), consistent with northward transport of

2009), consistent with the occurrence of HABs in Drakes Estero warm waters and planktonic larvae from Drakes Bay (Send et al.,

following high levels observed in the open waters of Drakes Bay. 1987; Wing et al., 1998). While bloom populations may ﬁnd refuge

The depth of penetration into the bay/estuary is controlled by tidal in bays during brief upwelling events, it appears that bloom

mixing and stratiﬁcation inside the bay (Largier et al., 1997; concentrations dissipate coast-wide during periods of persistent

Kimbro et al., 2009). upwelling, presumably due to the twin negative inﬂuences of

The majority of historical PSP events along the CA coast have offshore Ekman transport and strong vertical mixing due to surface

occurred in central and northern regions (Price et al., 1991). The wind stress (Botsford et al., 2003; Largier et al., 2006).

most common occurrences were in Drakes Bay off the Marin coast Although there is no evidence that nutrients due to land runoff

north of San Francisco (CDPH data), which is sheltered from are a principal factor in triggering or promoting these blooms (Price

upwelling (Largier, 2004). High-PSP events are most likely to occur et al., 1991; Langlois and Smith, 2001; Anderson et al., 2008b), new

either early or late in the upwelling season (i.e., early spring or work on phytoplankton productivity in ammonium-rich outﬂow

144 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

from San Francisco Bay re-opens this question; e.g., the idea that contaminated cultivated blue mussels from Prince Edward Island

elevated ammonium levels preclude nitrate uptake by diatoms, in the Gulf of St. Lawrence (Bates et al., 1989). Since then, however,

allowing dinoﬂagellates to bloom (Wilkerson et al., 2006; Dugdale most of the reported DA events have occurred on the U.S. west

et al., 2007; Glibert, 2010; Glibert et al., 2011). A recent summary of coast. The ﬁrst documented outbreak on the west coast occurred in

nutrient use by harmful algae in upwelling systems, however, 1991, causing the deaths of dozens of brown pelicans (Pelecanus

suggests that while Alexandrium may prefer ammonium as a occidentalis Linnaeus) and Brandt’s cormorants (Phalacrocorax

nitrogen source, chain-forming HABs (including Alexandrium) also penicillatus Brandt) in Monterey Bay, CA (Fritz et al., 1992; Work

are well adapted to use upwelling-derived nitrate (Kudela et al., et al., 1993) and contaminating razor clams and Dungeness crabs in

2010). The relative inﬂuences of upwelled vs. anthropogenic WA, OR, and northern CA (Wekell et al., 1994). In southwest WA

nutrients on Alexandrium bloom properties (e.g., initiation, magni- alone, crab ﬁshing losses were estimated at $7 million. It was

tude, duration) in coastal areas receiving high nutrient loads (e.g., originally thought that 25 human illnesses in WA were attributable

San Francisco Bay) is unresolved. to ASP in the 1991 event (Washington Department of Fish and

Wildlife, 2004). None of these illnesses were ever ofﬁcially

2.3.7. Mexico conﬁrmed and no mortalities occurred (Quick, 1992).

The best studied toxic species present in Mexican coastal After these early ASP events, monitoring efforts and regulations

waters is G. catenatum, but its ecology is still not well understood to prevent harvest of toxin-contaminated shellﬁsh have succeeded

(Band-Schmidt et al., 2010). There is general agreement that HABs in preventing human incidents of ASP, but numerous cases of DA

have increased in recent years (Hernańdez-Becerril et al., 2007; toxicity of finfish, marine mammals, and birds have been

Corte´s-Altamirano and Sierra-Beltra´n, 2008) and G. catenatum- documented (Landsberg, 2002; Shumway et al., 2003; Goldstein

related blooms are no exception (Band-Schmidt et al., 2010); et al., 2008; Fire et al., 2010; Bargu et al., 2012). A number of

however, the increase in G. catenatum blooms along the Mexican shellfish and finfish have been reported as potential vectors of the

Paciﬁc coast does not appear to be related to anthropogenic disease, including razor clams, blue mussels, Paciﬁc littleneck,

activities. For example, extensive aquaculture and agriculture geoduck, and manila clams, Paciﬁc oysters, Dungeness, rock, and

activities are present on the east coast of the Gulf of California, but pelagic red king crab, spiny lobster viscera, Paciﬁc sardines

there is no clear evidence that PST-producing blooms have (Sardinops sagax Jenyns), northern anchovies (Engraulis mordax

increased due to pollution or nutrient runoff from these activities Girard), krill (Euphausia paciﬁca Hansen, Thysanoessa spinifera

(Flores-Trujillo et al., 2009). Blooms of G. catenatum in the Gulf of Holmes), market squid (Loligo opalescens Berry), and benthic

California appear to be related to other environmental forcing invertebrates (Bargu et al., 2002, 2008, 2010, 2012; Landsberg,

factors. It is recognized that blooms occur more frequently in late 2002; Lefebvre et al., 2002; Shumway et al., 2003; Schnetzer et al.,

winter and early spring when upwelling events are present in the 2007; Kvitek et al., 2008; Mazzillo et al., 2010). In addition to

south part of the Gulf (reviewed by Band-Schmidt et al., 2010). commercially harvested species, many other animals can accumu-

Moreover, the paleographic record of absolute and relative late DA, leading to widespread transfer through marine food webs

abundances of the resting cyst of this species (as an indicator of (e.g., Lefebvre et al., 2002; Bargu and Silver, 2003; Bargu et al.,

its abundance in the water column) is correlated to sea surface 2008; Kvitek et al., 2008; Mazzillo et al., 2010). Death or strandings

temperatures (Flores-Trujillo et al., 2009). Therefore, in the have been reported in California sea lions (Zalophus californianus

southern part of the Gulf of California this species seems to Lesson), northern fur seals (Callorhinus ursinus Linnaeus), harbor

respond to interdecadal forcing phenomena, being more abundant porpoises (Phocoena phocoena Linnaeus), common dolphins

in La Nin˜a than in El Nin˜o warm conditions (Flores-Trujillo et al., (Delphinus delphis Linnaeus), sea otters, gray whales (Eschrichtius

2009). robustus Lilljeborg), minke whales (Balaenoptera acutorostrata

Lacepede), brown pelicans, Brandt’s cormorants, ruddy ducks

3. Pseudo-nitzschia spp. and domoic acid poisoning (Oxyura jamaicensis Gmelin), and western grebes (Aechmophorus

occidentalis Lawrence) (Work et al., 1993; Scholin et al., 2000;

3.1. Overview of toxicity, history on North American west coast Landsberg, 2002; Shumway et al., 2003; Goldstein et al., 2008; Fire

et al., 2010; Bargu et al., 2012; D. Caron, pers. obs.).

The genus Pseudo-nitzschia (mostly reported before 1990 as

Nitzschia seriata P.T. Cleve) has been present on the west coast 3.2. Trends in prevalence and impacts

since at least the 1920s (Fryxell et al., 1997). Several species of the

diatom genus, Pseudo-nitzschia, produce domoic acid (DA), a toxin During the last 15 years, numerous blooms of Pseudo-nitzschia

causing amnesic shellﬁsh poisoning (ASP). Of the 12 species of spp. have been reported, and associations between DA and animal

Pseudo-nitzschia known to produce DA, 10 have been reported from deaths and illnesses frequently documented (Scholin et al., 2000;

west coast waters (Horner et al., 1997; Anderson et al., 2008a). The Gulland et al., 2002; Trainer and Hickey, 2003; Goldstein et al.,

species, Pseudo-nitzschia australis Frenguelli and Pseudo-nitzschia 2008). The next exceptional and widespread event after 1991

multiseries (Hasle) Hasle, are most commonly associated with toxic occurred in 1998, when marine mammal deaths attributed to DA

events throughout this region, with Pseudo-nitzschia pseudodeli- were ﬁrst reported (e.g., 81 California sea lions from San Luis

catissima (Hasle) Hasle, and Pseudo-nitzschia cuspidata (Hasle) Obispo to Santa Cruz), and high levels of DA were measured in WA

Hasle also implicated in toxic events in WA waters (Adams et al., and OR razor clams (Adams et al., 2000; Scholin et al., 2000). In CA,

2000; Trainer et al., 2009a). Amnesic shellﬁsh poisoning results in DA outbreaks have occurred in almost every year over the last

gastrointestinal and neurological disorders within 24–48 h of decade (CDPH data, Fig. 8C) and increasingly south of Point

consumption of toxic shellﬁsh by humans, and can be life- Conception. DA levels associated with Pseudo-nitzschia blooms

threatening (Perl et al., 1990; Teitelbaum et al., 1990; Jeffery et al., were exceptionally high in coastal OR and CA in 2010 (CDPH data,

2004; Goldstein et al., 2008; Lefebvre and Robertson, 2010). The ODA and ODFW data at http://bioweb.coas.oregonstate.edu/

disease can lead to short-term memory loss that may become mocha/odadata.html). Off OR, Pseudo-nitzschia cell counts were

permanent. Some symptoms are similar to other diseases and thus as high as 10 cells/l in June 2010, associated with elevated DA

lead to misdiagnoses. levels in razor clams that led to harvesting closures. In CA, DA

Shellﬁsh toxicity due to DA was discovered in 1987 in Canada, levels in Monterey Bay during fall 2010 were exceptionally high in

when three people died and 105 became ill from eating water (dissolved and particulate) and California mussels.

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 145

3.2.1. Alaska locations and times where commercial mollusc harvest and

The AHAB (Alaska HAB) program monitors the occurrence of farming occurred, and the highest DA concentration measured

several HABs including Pseudo-nitzschia blooms in southeast AK was 18.8 ppm. Testing for DA of 4262 commercially harvested

and the Homer region, and is beginning to work with the State of Dungeness crabs showed highest DA concentration of 1.37 ppm in

Alaska to implement screening methods for the measurement of a snow crab harvested in the Aleutian/Bering Sea commercial

DA in recreationally harvested shellfish. The Alaska Department of fishery. A monitoring program, funded by the North Pacific

Environmental Conservation (ADEC) is the only agency responsible Research Board to determine the occurrence and geographical

for marine biotoxin testing in shellﬁsh, with sampling restricted to distribution of DA in shellﬁsh, conducted intensive sampling at

commercially harvested and aquaculture products. The ADEC tests Annette Island and Sea Otter Sound, both near Ketchikan, Sitka

between 600 and 700 samples annually; however, testing is Sound, eastern Prince William Sound, Kachemak Bay, and

primarily conducted on Paciﬁc oysters and geoduck clams, both Unalaska, with additional opportunistic sampling along the entire

poor candidates for monitoring DA. An intensive ADEC study coast (RaLonde and Wright, 2011). DA concentrations were near

between 1992 and 1996 tested for DA in 5123 individual molluscs the undetectable level.

and found measureable levels (ADEC Database, unpubl. data, R. Phytoplankton monitoring in AK has historically concentrated

RaLonde, pers. comm.). The study, however, was limited in scope to on the spring bloom period, primarily to understand the inﬂuence

Fig. 8. Time series of domoic acid toxins (mg/g shellﬁsh meat) for (A) Washington, (B) Oregon, and (C) California. The regulatory alert level of 20 mg/g is shown in each graph.

Note that each data set has variable start and end dates. Data sources are provided in the text and in previous ﬁgures.

146 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

of ocean productivity on marine ﬁsh survival. Because Pseudo- clam beaches have clamming closures due to toxin roughly every

nitzschia is not a major component of the spring bloom, data on the two years (MacFadyen et al., 2005). For toxin to reach coastal

locations, timing, and intensity of Pseudo-nitzschia blooms are rare, beaches, toxic patches must ﬁrst develop, the patches must escape

but see Schandelmeier and Alexander (1981) for early data from the eddy and travel downcoast toward the clamming beaches, and

the Bering Sea. Phytoplankton sampling as part of research the patches must move onto coastal beaches, remaining long

programs in AK waters (GLOBEC, Global Ocean Ecosystem enough to be ingested by the clams. Recent research shows that

Dynamics; BEST, Bering Sea Ecosystem Study) has shown that concentration, as well as onshore transport, occurs during periods

Pseudo-nitzschia is widespread on the Gulf of Alaska and Bering Sea of downwelling winds (storms), whereas escape from the eddy and

shelves and can reach high abundances. For example, in June 2010 travel downcoast occurs during periods of upwelling winds

near St. Lawrence Island, large-sized Pseudo-nitzschia spp. were (MacFadyen and Hickey, 2010).

present at >10 cell/l (E. Lessard, unpubl. data). The ADHHS-ES The relatively few toxic events on Washington’s coastal beaches

database has no record of illness from DA. Since AK has little data in comparison to the regular appearance of DA in the source

from DA monitoring other than screening commercially harvested regions suggests that signiﬁcant impediments to transport occur

product, and because toxin illness is generally underreported, the between source regions and the beaches. One important impedi-

actual impact of DA on human and marine ecosystem health is ment may be the buoyant plume from the Columbia River estuary

unknown (Gessner and Middaugh, 1995). (Hickey et al., 2010). A plume from the Columbia is generated along

the WA coast north of the estuary mouth each time winds on the

3.2.2. British Columbia WA coast switch to a northward direction (a storm period) (Hickey

British Columbia began monitoring for DA after the 1987 et al., 2005). The plume has density fronts along its edges and such

outbreak on the east coast of Canada and currently analyzes fronts can be a barrier to onshore transport of toxic blooms (Hickey

approximately 3000 blue mussel and razor clam samples for DA and Banas, 2003). A new study led by the University of Washington

each year (Canadian Food Inspection Agency data). Domoic acid is (PNWTOX, ‘‘Paciﬁc Northwest Toxins’’) is using numerical models

rarely detected in samples in BC and no conﬁrmed illnesses due to of coastal circulation and also ecosystem variability in association

DA have been reported. The highest level of DA detected since 1994 with previously collected data to determine the effects of the

was 106 ppm on the west coast of Vancouver Island in Port Eliza, Columbia on HABs for both OR and WA coasts.

off Nootka Sound, during March 2002 (Fig. 2). Blooms of Pseudo- Exceptional years of DA-associated beach, razor clam, and

nitzschia have been documented along the west coast of Vancouver Dungeness crab closures in WA include 1991, 1998–1999, 2002–

Island and on the north end of the Haida Gwaii (Forbes and 2003, and 2005 (see Fig. 1 in MacFadyen et al., 2005, Fig. 8A). In

Denman, 1991). 1991, closure of beaches to recreational and commercial razor

On the south coast of BC, only 10 blue mussel samples, all clam and Dungeness crab harvesting resulted in a $15–20 million

collected on the west coast of Vancouver Island, have had revenue loss to ﬁsheries (Anderson, 1995). During 1998–1999

measurable levels of DA above the action level of 20 ppm since (over a year and a half), ﬁshery closures caused Washington’s

1994 (Canadian Food Inspection Agency data). Domoic acid is Quinault tribe to lose all of their razor clam income and a large

rarely seen in samples collected in the Strait of Georgia and the portion of their Dungeness crab income, and the Quileute tribe to

levels have not exceeded 6 ppm. There does not appear to be any lose 50% of their Dungeness crab income (Wekell and Trainer,

regularity to the seasonality of DA detection in monitoring samples 2000). From 2002 to 2003, another prolonged closure period (>1.5

on the south coast (D. Kelly, pers. obs.). years) resulted in a $10.4 million loss in revenue (Wekell and

The highest level of DA detected on the north coast of BC was Trainer, 2002). In 2005, toxic blooms of P. pseudodelicatissima and

37 ppm found in razor clams in August 1995 (Canadian Food P. australis caused signiﬁcant commercial, recreational, and tribal

Inspection Agency data). Detectable levels of DA are found most shellﬁsh harvest losses in Sequim Bay and Penn Cove areas of Puget

years on the Haida Gwaii; however, only during the September Sound, respectively (Trainer et al., 2007). Although DA producing

1995 to May 1996 period were the levels greater than the action blooms of Pseudo-nitzschia species have been known previously in

level of 20 ppm (Fig. 2). Blooms tend to start developing in the Puget Sound (e.g., Hood Canal, Horner et al., 1996), blooms with

spring/summer months on the north coast. toxin levels above the regulatory limit of 20 ppm have been

Populations of Pseudo-nitzschia spp. are usually a minor reported only since 2003 (Bill et al., 2006), causing some concern

component of the phytoplankton community off the southwest that continued escalation/expansion will impact the many

coast of Vancouver Island in late summer, but there is substantial valuable ﬁsheries there (Trainer et al., 2007). The total estimated

variability and species may occur throughout the year (Forbes and impacts of a hypothetical coast-wide seasonal closure of the

Denman, 1991). Species are also found in the Strait of Georgia and recreational razor clam ﬁshery for 2008 was estimated to be

north of Vancouver Island. They are generally found in water $21.9 million, and the income impact of the recreational razor clam

temperatures between 8 and 14 8C and salinity between 30.0 and ﬁshery in WA for 2008 was estimated at $13.5 million (Huppert

32.5 with reduced concentrations of dissolved inorganic nutrients, and Dyson, 2008, see Section 6).

particularly silicate.

3.2.4. Oregon

3.2.3. Washington In addition to Washington’s Juan de Fuca area, other chronic

Toxic Pseudo-nitzschia blooms have caused severe economic sources of toxigenic Pseudo-nitzschia (i.e., ‘‘hot spots’’) include

losses for coastal WA (including tribal) communities from beach Heceta Bank (Trainer et al., 2001; B. Hickey, pers. comm.; P.

and shellﬁsh harvest closures (Fig. 3). Impacted areas span the Strutton, unpubl. data) and south of the Columbia River estuary

coast, with a demonstrated ‘‘hot spot’’ in the Juan de Fuca eddy (Clatsop) areas of OR (Tweddle et al., 2010). Like WA, the 1998 and

area, where Pseudo-nitzschia blooms occur relatively frequently, 2003 events caused beach closures of razor clam harvesting that

associated with high DA concentrations (Trainer et al., 2002; lasted >1.5 years and led to a $4.8 million loss in estimated income

MacFadyen et al., 2005). This area is a chronic upstream source of to coastal communities around Clatsop Beach alone in 2003

toxic Pseudo-nitzschia for the WA coast (Trainer et al., 2002, 2009a; (Tweddle et al., 2010; ODFW data, http://public.health.oregon.gov/

Trainer and Hickey, 2003; Marchetti et al., 2004; MacFadyen et al., HealthyEnvironments/Recreation/HarmfulAlgaeBlooms/Pages/

2005). Results from six cruises conducted over four years suggest index.aspx, Fig. 8B). The impact of DA toxicity on razor clam,

that toxin occurs in the eddy in any 21-d period, although razor mussel species, and Dungeness crab industries appears to be

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 147

increasing in recent years, with exceptionally extensive and Hasle, P. multiseries and Pseudo-nitzschia pungens (Grunow ex

prolonged razor clam and mussel closures occurring from 2002 Cleve) Hasle have been reported in Paciﬁc Mexico (Herna´ndez-

to 2006 (Tweddle et al., 2010, Fig. 8B). The Dungeness crab industry Becerril et al., 2007). Only two DA-associated blooms have been

in OR has never had a closure due to biotoxins, but orders for documented (Fig. 6). Domoic acid was detected in net phytoplank-

evisceration occurred in 1999 when levels of biotoxins in S. patula ton samples and in chocolate clams (Megapitaria squalida Sowerby)

warranted the precaution (ODA data, http://oregon.gov/ODA/FSD/ during a P. fraudulenta bloom in June-July 2006 at La Paz, in the Gulf

shellﬁsh_status.shtml). of California, but the concentration of DA in the clams (0.55 ppm)

was well below the action limit for shellﬁsh (Ga´ rate-Liza´rraga

3.2.5. California et al., 2007). Another toxic bloom occurred in the Todos Santos Bay

Since the 1998 event when DA was ﬁrst linked to sea lion in 2007, in the northern part of the Baja California Peninsula

deaths, toxic blooms and associated mammal and bird illnesses on (Garcia-Mendoza et al., 2009). Here, the maximum DA concentra-

the CA coast have occurred in nearly every year (CDPH data, tion in particulate matter (0.86 ppm) was associated with the

Fig. 8C). For example, toxic Pseudo-nitzschia blooms have been presence of P. australis that reached a maximum abundance of

recorded in the Santa Barbara Channel in every year since 2002, 3.02 Â 10 cells/l (Garcia-Mendoza et al., 2009). Adverse biological

and off Los Angeles in every year since 2003 (Schnetzer et al., 2007; effects related to DA in the environment were not detected during

Anderson et al., 2008a; CDPH data). Blooms of P. australis off these events.

southern CA in 2006 and 2007 were characterized by some of the There are only two incidents where DA toxicity of animals was

highest planktonic DA concentrations measured; e.g., 14.39 mg conﬁrmed in Mexico, both occurring in the Gulf of California

pDA/l on March 17, 2006 just inside and 26.97 mg/l on April 26, (Fig. 6). The deaths of approximately 150 brown pelicans during

2007 just outside the breakwater at Long Beach (A. Schnetzer et al., the winter of 1996 at the tip of the Baja California Peninsula were

unpubl. data, Fig. 5). Since 2003, hundreds of marine mammal and associated with DA toxicity after the animals consumed contami-

bird strandings or deaths from central to southern CA have been nated mackerel (Scomber japonicus Houttuyn; Sierra-Beltra´n et al.,

attributed to DA, and there is evidence that these poisonings are 1997). Another massive mortality of sea birds and marine

increasing. For example, Caron (2008) reported DA toxicity in mammals associated with DA poisoning occurred in the winter

several bird species for the ﬁrst time in 2006–2007. Spring 2007 of 1997 when 766 sea birds (common loon, Gavia immer Brunnich),

was cited as the worst season for marine mammal and bird 168 dolphins, nine sea lions, and four ﬁn whales (Balaenoptera

mortality on the southern CA coast (International Bird Rescue physalus Linnaeus) died. This is the only such event described in an

Research Center, http://www.ibrrc.org/pr_04_25_2007.html). The ofﬁcial technical report issued by SEMARNAP (Former Mexican

link between upper water column blooms and potential exposure Secretary of the Environment and Natural Resources) (SEMARNAP-

of benthic organisms to DA through rapid downward ﬂux has also PROFEPA, 1997). The dead animals were dispersed in several

been demonstrated through toxin measurements in sediment locations on the east coast of the Gulf of California. The responsible

traps from as deep as 500 and 800 m in the Santa Barbara and San species was not identiﬁed.

Pedro Channels, respectively (Schnetzer et al., 2007; Sekula-Wood The limited number of DA ﬁeld measurements or conﬁrmed DA

et al., 2009). poisonings does not necessarily indicate that blooms associated

Domoic acid has been detected in seafood species along the CA with the toxin are unusual in Mexico. Other events consistent with

coast (bivalve shellﬁsh, sardines, anchovies) almost every year DA toxicity have been identiﬁed, for example, mass mortalities of

since the 1991 episode (CDPH data). Concentrations of DA sea mammals and birds in 1995 and 2004 in the Gulf of California;

exceeding the federal public health alert level (20 ppm) have however, no hard data were provided to corroborate the cause of

been detected in seafood species every year between 2000 and mortality (Ochoa et al., 2002). Anecdotal evidence from the

2007, primarily between San Luis Obispo and Los Angeles counties. northern part of the Baja California Peninsula also suggests that

In 2007, a DA concentration of 610 ppm was detected in California some sea lion strandings might have been related to DA toxicity. In

mussels from Santa Barbara, the highest level ever recorded in CA 2002, 87 sea lions were found stranded on beaches from Tijuana to

(Fig. 5). The most persistent problems with elevated DA Ensenada, and it was assumed that DA toxicity was the cause of

concentrations have been in the Santa Barbara-Ventura region, this event (Herna´ ndez-Becerril et al., 2007). Also in 2006, nine

extending offshore to the Channel Islands. corpses and three sea lions with symptoms of DA toxicity were

Based on examination of 715 sea lions with neurological found near Ensenada, Northern Baja California (media report

symptoms collected between 1998 and 2006, Goldstein et al. CP052-06 issued by SEMARNAT-PROFEPA; http://www.profepa.

(2008) identiﬁed two DA syndromes, an acute DA toxicosis and a gob.mx/). These ﬁndings indicate that DA toxicity might be a

chronic epileptic syndrome. Clusters of strandings of acute recurrent phenomenon for the northern part of the Baja California

syndrome cases occurred in 1998 (centered in Monterey Bay), Peninsula. Further evidence suggests that DA occurrence in this

2000, 2001, 2002, and 2005 (centered off San Luis Obispo and Santa region may be on the rise. Domoic acid was not reported in the

Barbara counties). While an increasing trend in acute cases from northern region of the Baja California peninsula before 2007

1998 to 2006 was not found, chronic cases were found to increase (Garcia-Mendoza et al., 2009). Domoic acid content in Paciﬁc

in every year, from four cases in 1999 to 45 cases in 2006. These sardines (S. sagax caerulea Jenyns) collected every two weeks from

data indicate that chronic effects of DA on sea lions have December 2007 to February 2009 was relatively high (>100 ppm

continually increased in recent years. Bargu et al. (2012) also viscera) during winter of 2007–2008 and from July to August of

found evidence for increasing chronic cases over time in their 2008 (G. Cabrales-Talavera and E. Garcia-Mendoza, unpubl. data).

examination of 82 sea lions stranded in Monterey Bay from 2004 to Also, some positive samples were detected in April 2008, but the

2007. concentration was <20 ppm in viscera (G. Cabrales-Talavera and

E. Garcia-Mendoza, unpubl. data). Furthermore, during the last

3.2.6. Mexico week of August and through September of 2010, sardines

Domoic acid detection and the presence of potentially toxic collected near the Todos Santos Bay region had DA concentrations

Pseudo-nitzschia species have been documented in Paciﬁc waters as high as 800 ppm in viscera (E. Garcia-Mendoza, unpubl. data)

off Mexico. Of the 12 species identiﬁed as potential producers of DA and in September of 2011, the ﬁrst ban in the region associated

(Moestrup and Lundholm, 2007), P. australis, Pseudo-nitzschia with the presence of DA in shellﬁsh was implemented by

delicatissima (Cleve) Heiden, Pseudo-nitzschia fraudulenta (Cleve) COFEPRIS.

148 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

3.3. Factors promoting blooms The sparse and widespread population areas preclude the

possibility of much anthropogenic inﬂuence.

3.3.1. Overview: anthropogenic vs. natural factors

At present, there appears to be little evidence to support 3.3.3. British Columbia

anthropogenic nutrient loading as the primary promoter of Pseudo- Species of Pseudo-nitzschia are present and often abundant in all

nitzschia blooms along the west coast of North America. Blooms BC marine waters in summer and fall, with the largest blooms

often occur in offshore areas, e.g., the Juan de Fuca eddy off WA and occurring on the outer continental shelf (Forbes and Denman,

Heceta Bank off OR (Hickey and Banas, 2003; MacFadyen et al., 1991; Taylor and Harrison, 2002). These may be advected into

2005), where anthropogenic inﬂuence is neither expected nor coastal inlets such as Barkley Sound (Taylor and Haigh, 1996).

found. Even in the Southern California Bight, the most highly Further, blooms of Pseudo-nitzschia spp. in BC usually occur ﬁrst in

populated area along the U.S. west coast, Pseudo-nitzschia more southerly regions before more northerly ones (Taylor and

abundances and DA concentrations were higher at offshore Harrison, 2002).

stations and were not associated with higher nutrient concentra-

tions at coastal stations where toxin levels were inversely 3.3.4. Washington

correlated with nutrient levels (Schnetzer et al., 2007). Also, high Blooms of toxic Pseudo-nitzschia on the WA coast in summer

concentrations of the micronutrients Cu and Fe, generally linked to and fall are generally associated with a recent history in the Juan de

anthropogenic activities (e.g., Johnson et al., 2001) were inversely Fuca eddy and a retentive region just offshore of the Strait of Juan

related to DA production in laboratory and ﬁeld studies (Rue and de Fuca (Trainer et al., 2009a), and are more related to physical

Bruland, 2001; Trainer et al., 2009b). Urea might also be a N source forcings than to anthropogenic factors. This region is known for its

for Pseudo-nitzschia and would have an anthropogenic source high concentration of chlorophyll in summer (Hickey and Banas,

(Cochlan et al., 2008). There is no direct evidence, however, that 2008); i.e., the region is favorable to phytoplankton blooms in

Pseudo-nitzschia blooms are related to run-off or eutrophication. general, but Pseudo-nitzschia is typically less than 20% of the

Instead, they are more likely related to large scale physical forcing phytoplankton community by biomass (Trainer et al., 2009a). The

such as upwelling that brings in high quantities of macronutrients factors contributing to phytoplankton blooms in this region

(Kudela et al., 2004; Anderson et al., 2008b; MacFadyen et al., include a steady source of high nutrient waters, a circulation

2008). pattern that favors retention rather than loss, and low grazing

Although anthropogenic sources of nutrients have not been relative to growth rates (MacFadyen et al., 2005, 2008; Foreman

clearly linked to Pseudo-nitzschia blooms on the WA open coast, in et al., 2008; Olson et al., 2008; MacFadyen and Hickey, 2010). As in

smaller, more enclosed areas and embayments in Puget Sound and other portions of the California Current System, the high nutrient

along the Strait of Juan de Fuca, anthropogenic factors might play a waters are derived from large-scale seasonal upwelling of deep

role in promoting blooms. For example, a bloom of P. pseudode- coastal waters. In the Juan de Fuca eddy region however, the

licatissima in Sequim Bay, WA, in September 2005 was attributed in upwelled waters travel up the coastal canyon system into the Strait

part to a pulse of ammonium from an outdated sewage system at a of Juan de Fuca, where they mix and then exit back out as surface

state park (Trainer et al., 2007). A bloom of P. australis occurring at waters into the eddy region, providing as much nitrate as that due

roughly the same time at nearby Penn Cove, WA, was attributed to to traditional upwelling along the entire WA coast (see Table 1 in

water column stratiﬁcation caused by high precipitation, high Hickey and Banas, 2008). In addition, water is upwelled directly

stream ﬂow, and strong local winds (Trainer et al., 2007), similar to into the eddy as the season progresses (MacFadyen et al., 2008).

the causes of a bloom in the same area in 1997 (Trainer et al., 1998). Thus, in contrast to the open WA coast, the nutrient source does

Laboratory studies have demonstrated that several Pseudo- not disappear during periods of wind relaxation or wind direction

nitzschia species are able to use both organic and inorganic reversal to downwelling-favorable. Moreover, nutrients passing

nitrogen sources for growth, and there is often an uptake through the Strait are transported much farther offshore than

preference for ammonium that can be derived from both natural occurs via traditional coastal upwelling (50 km vs. 10 km)

and anthropogenic sources (Howard et al., 2007; Cochlan et al., (Hickey and Banas, 2008). Although the high nutrient concentra-

2008; Kudela et al., 2008a, 2010). Cochlan et al. (2008) suggest that tions favor phytoplankton growth in general, no signiﬁcant

anthropogenic sources such as ammonium and urea may sustain relationship was found between toxin production and macronu-

non-bloom concentrations of Pseudo-nitzschia during periods of trient supply (Trainer et al., 2009a). Signiﬁcant DA concentrations

relaxed upwelling when low nitrate concentrations can be were observed on six 21-d surveys over four years, but the factors

expected within 10–20 km of the coastline (MacFadyen et al., that determine the transition from toxigenic to toxic have not been

2008). The potential remains for anthropogenic sources to promote determined to date (Trainer et al., 2009a; B. Hickey, unpubl. data).

or sustain blooms for inland waters such as Puget Sound and along The timing, frequency, and magnitude of toxic Pseudo-nitzschia

the Strait of Juan de Fuca, or may be linked to the possible spread of blooms impacting the WA coast in summer/fall is therefore largely

toxic HAB species into new areas although proof is still lacking a function of the dynamics of the Juan de Fuca eddy. The eddy

(Anderson et al., 2008b). forms in the spring transition period, increases in spatial extent

throughout the summer, and decreases in the fall. Under the

3.3.2. Alaska typical northerly wind conditions, the eddy is ‘‘leaky’’ to the south,

Species of Pseudo-nitzschia occur along the whole AK coast from but under weak wind conditions or when southerly wind reversals

at least Point Barrow in the north (Bursa, 1963; R. Horner, pers. obs. occur, the eddy is more retentive. These conditions promote

as N. seriata), throughout the Bering Sea (Schandelmeier and maintenance of high abundances of Pseudo-nitzschia and also their

Alexander, 1981 as Nitzschia spp., section Pseudo-nitzschia), and shoreward advection.

into the Gulf of Alaska, including along the Aleutian chain (Cupp, The inﬂuence of wind conditions (timing, speed, direction,

1943) and into south central AK at Port Valdez (Horner et al., 1973), magnitude) on the Juan de Fuca eddy circulatory patterns is a

all as Nitzschia spp. Note that species identiﬁcations may not critical determinant of toxic Pseudo-nitzschia bloom impact, and

always be correct because they were made well before the need to thus is a focus of bloom forecasting models, including the long-

use electron microscopy for this purpose was recognized. The same term effects of climate change (e.g., storminess). In spring, high

oceanographic factors discussed above for PSTs (Section 2.3.2) concentrations of pDA in razor clams are more likely to originate

probably also affect the distribution of Pseudo-nitzschia species. from southern sources, such as Heceta Bank, OR (Hickey et al.,

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 149

2010; see below). This is because regional coastal currents are quantities of macronutrients (Kudela et al., 2004; Anderson et al.,

toward the south in summer/fall, but toward the north in early 2008b; MacFadyen et al., 2008), with outbreaks typically occurring

spring. following upwelling events as nutrients become less available to a

well-developed multi-species phytoplankton bloom (Kudela et al.,

3.3.5. Oregon 2004). The argument for DA events being controlled by large-scale

Analogous to the Juan de Fuca eddy off WA, Oregon’s Heceta oceanic forcing is based on the apparent synchrony of ASP events

Bank is a ‘‘hot spot’’ for DA. It is the dominant bathymetric feature observed in CA. Multiple factors have been shown to trigger the

off the mid-OR coast that enhances retention of highly productive production of DA by Pseudo-nitzschia (cf. reviews by Bates et al.,

waters, and may provide a source of toxic Pseudo-nitzschia blooms 1989; Bates, 1998, 2000; Bates and Trainer, 2006; Trainer et al.,

to the OR nearshore coastal zone and to the southern WA coast in 2008), but the most thoroughly characterized is macronutrient

spring (B. Hickey, pers. comm.). Like the Juan de Fuca eddy, limitation by either phosphate or silicate in cultures (Pan et al.,

southerly winds result in more retention and also shoreward 1996a,b,c). More recently, Anderson et al. (2006) reported a

advection of nutrients and phytoplankton (Barth, 2003; Barth et al., correspondence between limiting Si concentrations indexed by the

3À

2005). The northern OR coast, in particular the Clatsop Beach ratios of Si(OH)4:NO3 and Si(OH)4:PO4 and the concentrations of

region, experiences higher cell counts of Pseudo-nitzschia and Pseudo-nitzschia and particulate DA; however, they concluded that

greater instances of increased DA levels in shellﬁsh, compared to the relationship is complex, with added variability caused by

southern regions of OR (Tweddle et al., 2010). Upwelling effects, mesoscale circulation (see also review by Kudela, 2008).

however, are not greatly different between Clatsop and the A link between ASP events and land runoff has been postulated

Tillamook region immediately to the south. This suggests that the (e.g., that the massive DA event in Monterey Bay in 1998 was

increased toxins at Clatsop may be related to the presence of the triggered by post-El Nin˜o runoff; Scholin et al., 2000), but the

Columbia River plume, possibly a result of enhanced retention in evidence remains circumstantial and the relationship between ASP

this region. High concentrations in summer and fall could also be a and coastal runoff and/or eutrophication remains unclear. In

result of southward advection from the Juan de Fuca eddy. Domoic addition to the possible importance of land-derived macro-

acid contamination of shellﬁsh along the OR coast most closely nutrients, the micro-nutrients in land runoff may be critical.

corresponds to periods of transition from upwelling to down- Recent laboratory and ﬁeld data suggest that Pseudo-nitzschia may

welling (Tweddle et al., 2010) and not to any anthropogenic input. increase toxicity when growing on urea as a nitrogen source

(Howard et al., 2007; Kudela et al., 2008a), a source of N without a

3.3.6. California concomitant source of Si. Urea is primarily from anthropogenic

Pseudo-nitzschia spp. are common in CA waters, but major toxin sources and thus cultural eutrophication may have the unantici-

events occur only at speciﬁc times, although they often occur over pated consequence of both selecting for Pseudo-nitzschia spp. and

large spatial scales (e.g., Southern California Bight to Monterey Bay, promoting toxin production in this organism. In addition, DA

Trainer et al., 2000) and may persist for weeks. Of interest here are production by Pseudo-nitzschia spp. has also been linked to Fe and

not only the factors that control the population bloom, but also the Cu stress. Iron limitation directly modulates Si:N ratios in diatoms,

factors that control the large-scale production of DA. Following and DA may serve as an Fe-acquisition mechanism either directly

Section 3.2.5, the present-day widespread and annually occurring (Rue and Bruland, 2001; Maldonado et al., 2002) or through the

DA problem in CA appears to have only emerged in the last decade. stimulation of a Cu-mediated high afﬁnity transport system (Wells

Speciﬁcally in southern CA, Lange et al. (1994) considered et al., 2005). Anthropogenic changes in runoff amounts and timing,

toxigenic blooms both rare and unusual, but in recent years ASP and Fe or Cu loading (e.g., Johnson et al., 2001; Ladizinsky, 2003)

has become increasingly important (e.g., Trainer et al., 2000; thus may have ampliﬁed effects on coastal waters by triggering or

Anderson et al., 2006; Busse et al., 2006; Schnetzer et al., 2007). suppressing DA outbreaks.

While DA outbreaks are most common in the sheltered waters in

the Southern California Bight (including the Santa Barbara 3.3.7. Mexico

Channel) and Monterey Bay, they also occur along the open coast Water properties associated with wind-driven upwelling were

of central CA (south of Monterey Bay) but are not regularly found in related to the accumulation of P. australis cells in the Todos Santos

the sheltered waters of Drakes Bay (or on the open coast north of Bay region (Garcia-Mendoza et al., 2009). As in more northern

Monterey Bay). Given the retentive and stratiﬁed nature of locations (Trainer et al., 2000; Taylor and Trainer, 2002; Anderson

Monterey Bay and the Santa Barbara Channel, these regions may et al., 2006), the injection of nutrients to upper layers associated

act as source regions comparable with the Juan de Fuca Eddy and with upwelling events appears to promote the formation of toxic

Heceta Bank regions described for WA and OR. Pseudo-nitzschia blooms in the Baja California northern region

The marked shift to DA events in recent years in southern CA (Garcia-Mendoza et al., 2009). Speciﬁcally, it was documented that

may be related to changes in the oceanographic climate. For a high Si(OH)4:N ratio instead of the absolute concentration of each

example, there was a signiﬁcant change in ocean climate in the nutrient was an important factor for the accumulation of P.

eastern Paciﬁc in 1999 as both the PDO and North Paciﬁc Gyre australis (Garcia-Mendoza et al., 2009). Toxic blooms of Pseudo-

Oscillation (NPGO) reversed sign in a manner that would enhance nitzschia have been related to prominent oceanographic features

upwelling effects off central and southern CA. Just as +PDO may such as eddies, fronts, and upwelling events (GEOHAB, 2005). The

correspond with higher DA off cooler OR (see Section 3.3.5), so west coast of Baja California has a number of these mesoscale

ÀPDO and +NPGO may correspond with higher DA off warmer oceanographic features that could offer the conditions that

southern CA (Sekula-Wood et al., 2011). Although this ÀPDO/ encourage the growth of toxigenic diatom species; e.g., upwelling

+NPGO period was interrupted by anomalous years in 2005 and conditions that occur in late spring and in summer in the Mexican

2006, the tendency for cooler conditions has continued to the part of the California Current System (Hickey, 1998; Pe´rez-Brunius

present. The possibility of large-scale ecosystem change since 1999 et al., 2006).

is supported by recent studies that document a dramatic and In summary, the generality emerging from observations of

persistent response in demersal ﬁsh, crab, and shrimp populations Pseudo-nitzschia blooms and outbreaks of DA along the North

in San Francisco Bay (Cloern et al., 2010). American west coast indicates that these diatom species bloom in

As noted in Section 3.3.1, Pseudo-nitzschia blooms are most response to classical upwelling conditions, potentially along with

likely related to large-scale physical forcing that brings in excess numerous other species of diatoms and non-diatom taxa. The

150 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

speciﬁc conditions leading to dominance of Pseudo-nitzschia during Although Heterosigma blooms have been reported on the

these blooms, the factors leading to production of DA, and the CA coast, impacts to ﬁsheries have only been documented in

oceanographic/meteorological conditions leading to exposure of WA and northward. This may be related to the relative lack of

coastal communities to toxic blooms once they have developed are year-round marine ﬁnﬁsh aquaculture facilities in affected CA

less clear, possibly complex, and clearly in need of further study. regions (e.g., San Francisco Bay, Monterey Bay, and Southern

California).

4. Heterosigma, Chattonella, and ﬁsh kills The presence of Heterosigma has been reported from the

Gulf of California (Band-Schmidt et al., 2004), but noxious effects

Blooms of the raphidophyte, Heterosigma akashiwo (Hada) have not been associated with this genus off Mexico; however,

Sournia, have been associated with massive finfish kills in blooms of other raphidophyceans have caused massive fish

temperate waters worldwide and are known for their antagonistic mortalities in the Gulf of California. Speciﬁcally, in April 2003, a

effects on organisms with sizes ranging from bacteria to ﬁsh bloom of Chattonella marina (Subrahmanyan) Hara & Chihara and

(Smayda, 2006). In WA and BC, losses to commercial ﬁsheries, Chattonella cf. ovata Y. Hara & M. Chihara was associated with

particularly aquaculture, have been substantial since the late massive mortalities of ﬁsh, although it was not possible to

1980s, and concurrently wild ﬁsh have been affected (Horner et al., evaluate the magnitude of the impact (Nu´ n˜ez-Va´ zquez et al.,

1997; Taylor and Haigh, 1993; Taylor et al., 1994; Rensel and 2011 and references therein). Benthic fauna were killed in Kun

Whyte, 2003; Rensel et al., 2010b). The mechanism for Heterosigma Kan Bay, Sonora, located on the east coast of the Gulf of

toxicity is not well established. Several modes of toxicity have been California. Another bloom of C. marina during April and May of

proposed and investigated in laboratory settings, including 2006 in a southern location (Sinaloa coast) on the east coast of

production of brevetoxin-like compounds (Khan et al., 1997; the Gulf of California was also associated with mortality of

Keppler et al., 2006), mucus or lectin-like polysaccharides (Pratt, approximately 48–60 tons of ﬁsh (Nu´ n˜ez-Va´ zquez et al., 2011

1966; Chang et al., 1990), reactive oxygen species such as and references therein).

superoxide and hydrogen peroxide (Yang et al., 1995; Twiner Blooms of Heterosigma in inland coastal marine waters of WA

and Trick, 2000) and hemaglutinating and hemolysing compounds are typically associated with summertime warm weather and

(Onoue and Nozouwa, 1989). high river discharge, resulting in brackish salinities and a stable

In coastal marine waters of WA and BC, ﬁnﬁsh aquaculture kills surface layer (Taylor and Haigh, 1993; Rensel, 1995, 2007; Rensel

due to Heterosigma began in 1976 near Lummi Island but did not et al., 2010b). Blooms in WA and BC may be expanding in range

become substantial until the late 1980s (Taylor et al., 1994; Horner and magnitude, and contribution of anthropogenic factors is

et al., 1997; Rensel, 2007; Rensel et al., 2010a,b). A major kill of possible (Anderson et al., 2008b). The role of aquaculture efﬂuent

coho salmon (Oncorhynchus kisutch Walbaum) and chinook salmon in promoting Heterosigma increases has been proposed in other

(Oncorhynchus tshawytscha Walbaum) occurred in Sechelt Inlet, countries (e.g., Scotland; Smayda, 2006), but all commercial

BC, in 1986 resulting in a loss of approximately 1/3 of the salmon ﬁnﬁsh aquaculture sites in WA are sited in non-nutrient sensitive

population and $2.5 million in revenue. In 1989, another massive areas where naturally occurring background concentrations

bloom led to a $4 million loss of caged chinook salmon in BC and and ﬂux of dissolved inorganic nitrogen from the Paciﬁc Ocean

another $4 million of the same species at Cypress Island, WA. is very high compared to the half saturation constants for growth

Aquaculture ﬁsh losses were also severe in central Puget Sound in of this species, implicating other factors, such as light and

1990, when 1.3 million ﬁsh and $5 million revenue were lost (85– advection, in controlling algal bloom dynamics (Rensel, 1991,

100% population losses per pen of Atlantic salmon, Salmo salar 2007; Anderson et al., 2008b). Rensel et al. (2010a,b) showed that

Linnaeus), including an endangered species, wild White River the most frequent and intense blooms of Heterosigma in the

spring chinook (O. tshawytscha Walbaum) brood stock. Wide- region occur in the southern Strait of Georgia, where there are no

spread blooms in 2006 ($2 million loss) and 2007 also caused commercial ﬁsh farms but pronounced inﬂuence of the spring/

substantial losses of farmed Atlantic salmon (S. salar Linnaeus) early summer Fraser River peak discharge that creates ideal

(Rensel, 2007; Rensel et al., 2010b). growth conditions for the alga. Other anthropogenic activities are

Mortalities of wild salmon and marine ﬁsh species associated potential stimulatory factors; for example, sewage efﬂuent spills

with Heterosigma blooms have been documented since 1994 in were correlated with several Heterosigma blooms in poorly

Puget Sound (Hershberger et al., 1997; Horner et al., 1997; Rensel, ﬂushed and nutrient-sensitive central Puget Sound backwaters

2007; Rensel et al., 2010b), particularly in shallow, warm bays (Rensel, 2007; Anderson et al., 2008b). The raphidophyte’s high

where the dead ﬁsh are more visible and likely to ﬂoat and capacity for NH4 uptake, that may originate from many sources,

accumulate on beaches than in the colder main basins (Rensel et al., has been highlighted as an important factor (Anderson et al.,

2010a). More recently, evidence has been found linking Heterosigma 2008b) but the alga is equally adept at using other forms of

blooms in the Strait of Georgia and North Puget Sound to a 2-decade dissolved inorganic nitrogen such as nitrate (Herndon and

decline of a key stock of Fraser River sockeye salmon (Oncorhynchus Cochlan, 2007).

nerka Walbaum), historically the most valuable west coast The ecophysiology of Heterosigma is complex, and numerous

Canadian and U.S. salmon ﬁshery (Rensel et al., 2010a,b). Since behavioral and nutritional adaptive characteristics can contribute

1989, marine survival of Chilko sockeye salmon stock averaged 2.7% to bloom initiation. Niche-deﬁning criteria proposed for Hetero-

in years when juvenile sockeye salmon seawater migration in the sigma include: temperature-regulated excystment, trace-metal

Strait coincided with major Heterosigma blooms vs. 10.9% in years (e.g., Fe) stimulation of growth, stimulation by organic compounds,

with no or minor blooms. Strong correlations were also seen allelopathic deterrence of competitor growth or predator activity,

between major Heterosigma bloom years and young-of-the-year nutrient retrieval via vertical migration, halotolerance, shade

Paciﬁc herring (Clupea pallasii Valenciennes) abundance a few adaptation, and occurrence of different ecotypes within a given

months later. A panel of U.S. and BC ﬁsheries experts assembled by region (Smayda, 1997, 1998; Hallegraeff, 1998; Anderson et al.,

the Paciﬁc Salmon Commission reviewed the evidence (i.e., Rensel 2002; Zhang et al., 2006; Fredrickson et al., 2011). Understanding

et al., 2010a,b) and concluded that at least some years of very poor Heterosigma toxic bloom ecology is further complicated by the

Fraser River sockeye salmon returns, such as 2009, were likely due uncertain knowledge of the species’ mechanism for toxicity (see

to Heterosigma blooms and that future risk necessitates the need to above), hindering management capabilities to predict, prepare for,

further study the issue (Peterman et al., 2010). and respond to these toxic events.

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 151

5. Other HABs Two species of Cochlodinium have widespread distribution on

the west coast but only recently have they been linked to ﬁsh kills.

Other HAB species are widespread along the North American Blooms of Cochlodinium polykrikoides Margelef have been a major

west coast, but historically, bloom formation, toxin production, cause of ﬁsh kills off Japan, China, and Korea, while C. sp., recently

and ecosystem or human health impacts by these species have identiﬁed as Cochlodinium fulvescens Iwataki, Kawami et Matsuoka

been rarely reported. Recent observations suggest possible future using LSU rDNA sequences (Iwataki et al., 2008), was linked to a kill

impacts (discussed below), and west coast regional monitoring of aquacultured Atlantic salmon (S. salar Linnaeus) in BC in 1999–

programs should include emphasis on their detection. Such HAB 2000 (Whyte et al., 2001). More recently, a bloom of Cochlodinium

species include the dinoﬂagellates, Dinophysis spp. (diarrhetic (species not determined, but possibly C. fulvescens, see Iwataki

shellﬁsh poisoning), Cochlodinium spp. (ﬁsh kills, toxic mechanism et al., 2008) that extended over 800 km of CA coastline was linked

unclear), Protoceratium reticulatum (Clapare`de et Lachmann) to a 2004–2005 California mussel mortality event in Monterey Bay

Bu¨ tschli, Lingulodinium polyedrum (Stein) Dodge, and Gonyaulax (Curtiss et al., 2008). Genetic characterization conﬁrmed the

spinifera (Clapare`de et Lachmann) Diesing (yessotoxin producers), presence of C. fulvescens in southern and central CA during this

and Akashiwo sanguinea (K. Hirasaka) G. Hansen & Ø. Moestrup (sea period (Howard et al., 2012). Since the 2004–2005 bloom event,

bird kills due to surfactant-like proteins that coat feathers and Cochlodinium has emerged as a common bloom-forming organism

neutralize water repellency and insulation). Also, in the upper along the CA coastline (Curtiss et al., 2008; Jester et al., 2009;

reaches of some CA estuaries, the cyanobacterium, Microcystis Kudela et al., 2010; Howard et al., 2012; Kudela and Gobler, 2012;

aeruginosa (Ku¨ tzing) Ku¨ tzing, has emerged as a major bloom- CDPH data). Based on nutrient uptake kinetic analyses conducted

former recently and Trichodesmium spp. are listed as potential on samples from a 2006 Monterey Bay Cochlodinium bloom, Kudela

harmful species in Mexico (Herna´ndez-Becerril et al., 2007). Also et al. (2008b) estimated that from 55% (August) to 62% (September)

on the Mexican west coast, problems associated with potentially of N uptake was derived from urea, suggesting a role of cultural

toxic benthic dinoﬂagellates should be considered. There are two eutrophication in the recent increase in bloom prevalence. In

descriptions of ‘ciguatera-like’ intoxications caused by the September 2007, a Cochlodinium bloom cost the Monterey Abalone

consumption of ﬁsh captured at Alijos Rocks located 300 miles Company almost $60,000 worth of abalone (D. Caron, pers. comm.).

from the East coast of Southern Baja California (Lechuga-Deveze Along the Paciﬁc coast of Mexico, Cochlodinium spp. are important

and Sierra-Beltra´n, 1995) and from El Pardito Island in the Gulf of ichthyotoxic species. Two recent reviews mention that since the

California (Heredia-Tapia et al., 2002). beginning of the present decade, ﬁsh mortalities associated with C.

Several species of Dinophysis (e.g., D. acuminata Clapare`de et polykrikoides and Cochlodinium cf. catenatum Okamura are

Lachmann, D. acuta Ehrenberg, D. fortii Pavillard, D. norvegica common phenomena in the central Mexico coastal areas of

Clapare`de et Lachmann, and D. rotundata Clapare`de and Lachmann) Colima, Jalisco, and Nayarit, and in the southern Gulf of California,

that have been shown to produce okadaic acid and cause diarrhetic speciﬁcally in Sinaloa and Baja California Sur coasts (Herna´ndez-

shellﬁsh poisoning (DSP) in other parts of the world are commonly Becerril et al., 2007; Corte´ s-Altamirano and Sierra-Beltra´n, 2008).

found in west coast waters (Herna´ndez-Becerril, 1988; Horner et al., Yessotoxins (YTX) are a new class of lipophilic biotoxins shown

1997; Jester et al., 2009; Trainer et al., 2010). Symptoms of DSP to be tumor promoters in mice but with unknown effects on

include mild to severe gastrointestinal illnesses and, while deaths humans. They are produced by three cosmopolitan, bloom-forming

have not been documented, okadaic acid is known to be a strong dinoﬂagellates, P. reticulatum, L. polyedrum, and G. spinifera, all

tumor promoter (Suganuma et al., 1988; Dominguez et al., 2010). common and sometimes abundant at various times and locations

Okadaic acid was first detected in BC shellfish in 2003 in manila along the Pacific coast. Yessotoxins were detected in west coast

clams at low levels (Canadian Food Inspection Agency data), and in waters, near Grays Harbor, WA, in summer 2004 when P.

Monterey Bay, CA water samples and phytoplankton extracts in reticulatum was abundant (Howard et al., 2008), in isolates of L.

1999, where Dinophysis – primarily D. acuminata – abundance polyedrum and G. spinifera from coastal CA (Armstrong and Kudela,

correlated with inhibition of protein phosphatase activity in an assay 2006; Howard et al., 2008), and in California mussels from Scripps

for okadaic acid (Weber, 2000). Later, Southerland (2008) showed Pier during a bloom of L. polyedrum and from Monterey Bay in 2005

that DSP toxins occurred in sentinel California mussels collected (Howard et al., 2008). One species, P. reticulatum, which is known

from Monterey Bay in 2004–2005. One DSP toxin, okadaic acid, was to have signiﬁcant impacts on the shellﬁsh industry in BC because

positively associated with D. fortii and a second DSP toxin, it reduces the ability of oyster seed to feed (Cassis, 2007), was

dinophysistoxin-1 (DTX-1), also appeared to be produced by documented in bloom abundance in north Puget Sound in 2006

that species. Recently, okadaic acid was linked to three cases and 2008 (Horner et al., 2010; R. Horner, unpubl. obs.). In the Todos

of human DSP illness from Sequim Bay, WA blue mussels (June Santos Bay region (northern Baja California), L. polyedrum blooms

2011) and 62 illnesses from Gorge Harbour, BC blue mussels (August are recurrent phenomena that can cover the whole bay area (Pen˜a-

2011; V. Trainer, pers. comm.). In Mexico, several species of Manjarrez et al., 2005). Production of YTX was not measured

Dinophysis are also present along the Paciﬁc littoral but no cases of during these events; however, due to the magnitude and

DSP have been ofﬁcially recognized (Herna´ndez-Becerril et al., 2007; recurrence of potentially toxic blooms, a phycotoxins monitoring

Corte´s-Altamirano and Sierra-Beltra´n, 2008). Diarrhetic shellﬁsh program started in the region (project FICOTOX funded by the

poisoning toxins have been measured since 2009 in Mexico, but they CONACYT-FORDECYT program; P.I. Garcia-Mendoza) will monitor

were ofﬁcially regulated in 2011 with an action level of 160 mg eq for YTX and other microalgal toxins. An extensive bloom of G.

okadaic acid per kg of shellﬁsh (NORMA Oﬁcial Mexicana, 2011). In spinifera occurred off the WA/BC coasts extending into inland

2010, two sanitary bans associated with the presence of DSP toxins waters of WA and BC in August to September 1990 with shellﬁsh

in cultured oysters (C. gigas Thunberg) were implemented in Baja deaths reported in Barkley Sound, BC due to low oxygen (Taylor

California (COFEPRIS data). The toxins were detected by the mouse and Horner, 1994). Another bloom occurred in northern Puget

bioassay method but the presence of DSP toxins was not conﬁrmed Sound in August 2011, but with no reports of impacts (R. Horner,

by an analytical approach. Toxic effects from these species had not pers. comm.). At the same time, August and September 2011, a

been demonstrated on the west coast until very recently, and toxic bloom occurred on the Sonoma County, CA coast causing massive

effects can be mild and misdiagnosed. British Columbia and WA are mortalities of abalone and sea urchins. The cause of the mortalities

developing more comprehensive DSP monitoring programs (D. Kelly is not known, but low dissolved oxygen was not thought to be a

and J. Borchert, pers. comm.). factor (Rogers-Bennett et al., 2012).

152 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

The death of thousands of seabirds (e.g., surf scoters – Melanitta In addition to nutrient supply, Microcystis has several physio-

perspicillata Linnaeus, white wing scoters – Melanitta deglandi logical characteristics that make it a superior competitor in the Bay

Bonaparte, common murre – Uria aalge Pontoppidan, Paciﬁc loon – Delta. It is a superior algal competitor under elevated pH; like

Gavia paciﬁca Lawrence, northern fulmar – Fulmarus glacialis Egeria, it has highly effective carbon concentrating mechanisms,

Linnaeus, and western grebes) during September and October allowing it to sustain photosynthesis when other algae may

2009 off WA, OR, and CA was attributed to surfactant-like become C-limited (Ja¨hnichen et al., 2007, and references therein,

substances produced by a bloom of A. sanguinea (Jessup et al., Glibert et al., 2011). Like many cyanobacteria HAB formers, it also

2009; Du et al., 2011; Phillips et al., 2011). The dinoﬂagellate- preferentially uses chemically reduced over oxidized nitrogen

produced foam destroys the waterproof layer of feathers that keeps forms (e.g., Glibert et al., 2004). With loads in the Sacramento River

+ À1

seabirds dry, restricting ﬂight and leading to hypothermia. This of efﬂuent NH4 exceeding 14 tons day (Jassby, 2008; Glibert,

bloom extended into Oregon in October to November 2009 and 2010; Glibert et al., 2011), ambient concentrations of NH4 in the

was possibly related to a combination of a prior diatom bloom, a upper Bay Delta (where Microcystis occurs) exceed several mM-N,

stratiﬁed water column with low nutrient concentrations, and an the threshold for inhibition of NO3 uptake, throughout much of

active upwelling event in October. The source of the bloom was the year (e.g., Dugdale et al., 2007). It has also been suggested that

thought to be the WA coast (Du et al., 2011). Microcystis may also have the capability to reduce its P require-

Starting in the late 1990s, massive blooms of the cyanobacteri- ment by lipid substitution, as shown for other cyanobacteria (Van

um M. aeruginosa have recurred in the upper San Francisco Mooy et al., 2009; Glibert et al., 2011). Thus, it can tolerate elevated

(Lehman and Waller, 2003; Lehman et al., 2005, 2008; Moisander N:P ratios, and its dominance under high N:P ratios may also reﬂect

et al., 2009) and Klamath River estuaries, CA (Fetcho, 2007). These the decline in other species that lack such tolerance. As noted by

are not restricted to very low salinities; e.g., M. aeruginosa colonies Glibert (2010), cyanobacteria do not have to grow faster at elevated

were found in salinities as high as 18 during an October 2003 N:P than at lower N:P values to become abundant; they merely

bloom in San Francisco Estuary (Lehman et al., 2005) and M. have to grow faster than competing species groups.

aeruginosa was detected in salinities up to 9.1 in San Francisco Bay Macroalgal blooms have been documented in a number of

Delta waters during August and September 2007 (Moisander et al., Paciﬁc coast estuaries, but data are lacking for many areas (Bricker

2009). A 2004 San Francisco Estuary bloom was associated with et al., 2007). These blooms develop high biomass in shallow water

microcystin detection in the water, zooplankton (mesozooplank- and sea grass habitats, shading other vegetation and negatively

ton including Eurytemora afﬁnis Poppe and Pseudodiaptomus impacting animals through hypoxia formation or possibly by

forbesii Poppe & Richard, amphipods, jellyﬁsh, and worms), and production of toxic secondary metabolites. Examples of macroalgal

clam tissue (Lehman et al., 2008). A massive bloom occurred along blooms from invasive species have been documented recently in

the entire length of the Klamath River in 2005 (Fetcho, 2006, 2007). the San Juan Archipelago of WA (Sargassum muticum (Yendo)

The cyanobacterium is a major concern for the Yurok Tribe because Fensholt; Britton-Simmons, 2004; Klinger et al., 2006), lagoons in

the timing of the bloom coincides with the adult Chinook salmon southern CA (Caulerpa taxifolia (Vahl) C. Ag.; Jousson et al., 2000;

run, which has subsistence and commercial value for this tribal Walters et al., 2006), and Baja California (Ulva spp.; Jorgensen et al.,

ﬁshery. Microcystins were not detected in Chinook salmon livers or 2010; Zertuche-Gonza´lez et al., 2009) that have displaced native

ﬁllets, but trace amounts were detected in one adult steelhead algal species and modiﬁed habitat, leading to losses in living

(Oncorhynchus mykiss Walbaum) liver in 2005, the only year resources and economic costs for eradication.

salmon and steelhead were tested (K. Fetcho, pers. comm.).

Recently, Miller et al. (2010) presented the results of investigations 6. Economics and harmful algal blooms

into the 2007 death of 21 southern sea otters recovered along the

shore of Monterey Bay. The authors conﬁrmed the cause of death Economists have focused on at least three aspects of HABs: (1)

as microcystin intoxication, and provided strong evidence that the the negative regional economic impacts due to HABs; (2) the

toxin was derived from freshwater cyanobacteria transported from economic costs of human illness caused by HABs; and (3) human

eutrophic rivers and accumulated by marine shellﬁsh (i.e., perceptions of risks associated with seafood due to HABs. Each

hepatotoxic shellﬁsh poisoning). research area applies speciﬁc methodologies and concepts, and

An increase in Microcystis abundance and bloom frequency in careful utilization of these economic studies requires a basic

the San Francisco Estuary starting in the late 1990s and escalating understanding of the economic concepts. This section reviews the

over the last decade (Lehman et al., 2005, 2008, 2010) has been concepts and ﬁndings of some published studies.

linked to the inﬂuence of anthropogenic activities on alterations in The economic impacts are usually driven by reduced commer-

food web structure and trophodynamics (Glibert et al., 2011). The cial ﬁshery harvest, reduced aquaculture harvest, and/or reduced

authors used a 30-year data set to examine the relationship participation in and expenditures on marine recreation. As

between nutrient dynamics and ecosystem properties. They found explained by Radtke et al. (1987), a simple estimate of economic

that changes in nutrient loading were linked to a cascade of impact can be calculated using a regional economic model

changes in biogeochemical processes and other ecosystem (typically an Input–Output model) to calculate the ‘‘direct impact’’

properties. Both N and P loading increased from the mid-1980s of reduced ﬁshing or recreational activity, the ‘‘indirect impacts’’

to mid-1990s, but after that, P load reductions (i.e., through caused by reduced purchases of supplies and inputs by the directly

removal of P from laundry detergents and loss of canneries using P impacted sectors, and the ‘‘induced impact’’ resulting from

in their processing; Van Nieuwenhuyse, 2007; Glibert, 2010) decreased purchases of consumer goods and services due to the

without concomitant N reductions led to changes in nutrient combined direct and indirect reductions in regional incomes. The

stoichiometry (increased N:P). The authors concluded that Input–Output (I–O) model is a simple linear model of regional

increased nutrient loads (eutrophication) together with changes economy that documents the aggregate outputs of each economic

in nutrient ratios (stoichiometry) has led to changes in biogeo- sector, the inter-sectoral transactions, and the resulting regional

chemical conditions (e.g., high N, high N:P) and trophic cascades incomes (Miernyk, 1966). The I–O model is used to estimate

(e.g., abundance of the invasive macrophyte, Egeria densa (Planch.) changes in regional income, regional employment, and overall

that increases pH, and the invasive clam, Corbula amurensis gross expenditures in the region. The income and employment

Schrenck that removes macrozooplankton and regenerates nutri- impacts may be relevant to policy, but the gross expenditure does

ents) favoring proliferation of Microcystis. not coincide with the usual economic indicators. Further, the

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 153

income and employment impacts are not valid measures of economic studies on HABs is very few, but this summary suggests

economic beneﬁts or costs. So, the impact analysis is an the types of studies that might contribute to decisions regarding

informative but largely a descriptive measure. monitoring, information dissemination, and control of HABs.

A recent study by Dyson and Huppert (2010) estimated that the

regional impact of a year-long closure of all four WA coast razor 7. Conclusions

clam beaches due to Pseudo-nitzschia blooms would cause an

$11.36 million/year reduction in coastal county incomes due to By virtue of jurisdictional delineation, our summary knowledge

reduced recreational activity, and nearly $2 million/year reduction of HAB distribution, causes, and impacts along the Paciﬁc coasts of

in incomes due to lack of tribal and non-tribal commercial harvest AK through Mexico is based on a collection of disparate sources of

of razor clams. Much lower impacts are estimated for single beach information, with diverse histories of HAB problem recognition

or shorter lasting closures. For example, an annual closure of the and response. Despite this disparity, coastal HAB monitoring and

popular Long Beach peninsula razor clam ﬁshery would have a mitigation has become a management priority throughout the

regional income impact of negative $4.4 million. If the closure were region, reﬂecting a common recognition in each jurisdiction that

for but one clam ﬁshery opening (typically 2–5 days) the negative HABs and their impacts are increasing and can have a profound

income impact would be just $1.4 million (Dyson and Huppert, effect on the health and economies of their coastal communities. It

2010). These are hypothetical impacts that could be used in is tempting to lump HAB trends in this region with worldwide

assessing the effects of avoiding beach closures through better trends of increasing HABs related to eutrophication, but the two

monitoring of HABs. primary types of HABs (dinoﬂagellates causing PSP and Pseudo-

Other economic studies use a simpler approach to assess effects nitzschia spp. producing DA) apparently do not follow this rule

of HABs on commercial landings and value (direct effects). Jin et al. along the North American west coast. They are primarily derived

(2008) estimated a negative relationship between red tides and from offshore waters and carried inshore, where it is possible that

landings of soft shell clams in Maine. They compared annual anthropogenic nutrient sources affect their dynamics, including

landings of northern quahogs, soft shell clams, blue mussels, and increasing magnitude and prolonging duration. The primary

oysters in New England over the period 1990–2005 (with some nutrient drivers for bloom initiation are more consistent with

years missing) to detect whether the extensive red tide bloom in an upwelling source.

2005 was associated with a decline in the ﬁshery. Overall, they Systematic economic assessments of HAB impacts and cost-

estimated total direct impacts of up to $18 million. They also beneﬁt analyses for management strategic planning purposes

showed that imports of shellﬁsh to New England increased during remain important needs for the west coast region. Impacts to the

2005, partly compensating for the loss of local harvests and shellfish industry in the Pacific Northwest region are significant,

reducing the indirect and direct impacts of lost harvest. annually persistent, and well-documented. The effect of Hetero-

In their broad survey of economic effects of HABs, Hoagland et al. sigma on the salmon aquaculture industry is another well-

(2002) include estimates of the cost of human sickness and death recognized economic threat (Rensel et al., 2010b); however, much

caused by shellﬁsh poisoning and ciguatera ﬁsh poisoning in the U.S. of the impact of west coast HABs on coastal communities may not

during 1987–1992. These are rough approximations based on a translate as well to economic losses. The threat to human health is

range of values estimated by others, including $1400 per reported always present, and the June 2010 death of two Alaskans attributed

illness, $1100 per unreported illness (estimated to be 90% of total to PSP is a tragic reminder. The August 2011 discovery of DSP with

illnesses), and $1 million per death. The estimates represent direct human illnesses for the ﬁrst time in BC and WA indicates that

medical expenses and lost work time. Overall, they estimate that the continued vigilance is necessary. Also, DA poisoning has led to

human health costs of shellﬁsh poisoning varied between $11 thousands of sick or dead seals, sea lions, sea otters, dolphins, birds,

thousand and $1.1 million, averaging $400 thousand per year, over and whales along the west coast in the last decade.

the 1987–1992 period. The human health costs of ciguatera ﬁsh Paciﬁc west coast districts of AK, BC, WA, OR, CA, and Mexico

poisoning were estimated for Florida, Hawaii, Puerto Rico, the Virgin each has increased HAB monitoring infrastructure and improved

Islands, Guam, American Samoa, and the Northern Mariana Islands detection and response capabilities in recent years; however,

using rough estimates of $1000 per reported case and $700 per coastal HABs and their impacts frequently traverse state and

unreported case to come up with an overall estimate of $15– federal boundaries, emphasizing the need for effective exchange of

$22 million per year, averaging $19 million annually over 1987– information on HAB ecology and impacts across districts. This

1992. These are rough, ﬁrst-cut estimates which give some paper presents the state of the knowledge of HAB research along

indication of the negative economic effects due to illness. the west coast of North America, as a step toward meeting the need

Whitehead et al. (2003) used a telephone and mail survey to for integration of HAB outreach, research, and management efforts.

investigate the effects of public information about Pﬁesteria

blooms in the mid-Atlantic states on seafood consumer’s percep- Acknowledgments

tions and decisions. They found that survey respondents who

received a brochure about the dangers of Pﬁesteria expressed We thank Deirdre Kelly (Canadian Food Inspection Agency) for

signiﬁcantly increased perception of the health risks; however, her insight and major contributions. We thank Frances Gulland

more than 50% of respondents said that they would not alter their (The Marine Mammal Center) for providing data, Megan Bernhardt

seafood consumption even when ﬁsh kills due to Pﬁesteria were Schatz (School of Oceanography, University of Washington) for

reported. If there were a seafood inspection program to identify providing the maps, Ken Fetcho, Mary Silver, and Pat Glibert for

health risks, the percentage of respondents who would maintain scientiﬁc input, and David L. Graham (BC Canadian Food Inspection

their usual seafood consumption level jumped to about 80%. This Agency) for reviewing the manuscript. We thank Q. Esther Diaz

shows that HABs cause reduced demand for seafood. The authors Carrillo (Directora Ejecutiva de Programas Especiales) for provid-

also estimated that the average seafood consumer would be ing the information of phycotoxins concentration in Mexico

willing to pay $7/year for a seafood inspection program, and that gathered through the ‘‘Proyecto Marea Roja’’ of COFEPRIS. The

the expressed value of seafood consumption increased by $3 per help of Mario Castillo is also acknowledged. We also appreciate the

meal on average if a seafood inspection program is implemented. efforts of Courtney Bogle (NOAA) and David Kidwell (NOAA) in

These examples illustrate the wide range of possible economic communication and logistical support for the West Coast Regional

aspects of HABs and ciguatera ﬁsh poisoning. The number of Harmful Algal Bloom Summit. Sponsors for the Summit were the

154 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

Barth, J.A., Pierce, S.D., Casteleo, R.M., 2005. Time-dependent, wind-driven ﬂow over

NOAA National Centers for Coastal Ocean Science, the Central and

a shallow midshelf submarine bank. Journal of Geophysical Research 110,

Northern California Ocean Observing System, the Southern

C10S05, http://dx.doi.org/10.1029/2004JC002761.

California Coastal Ocean Observing System, Oregon Sea Grant, Bates, S.S., 1998. Ecophysiology and metabolism of ASP toxin production. In:

Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), The Physiological

and the Paciﬁc Coast Shellﬁsh Growers Association. B. Hickey, E.

Ecology of Harmful Algal Blooms. Springer-Verlag, Heidelberg, pp. 405–426.

Lessard, and R. Kudela contributed to this paper through the

Bates, S.S., 2000. Domoic-acid-producing diatoms: another genus added. Journal of

PNWTOX program, funded by NOAA Center for Sponsored Coastal Phycology 36, 978–985.

Bates, S.S., Bird, C.J., Defreitas, A.S.W., Foxall, R., Gilgan, M., Hanic, L.A., Johnson, G.R.,

Ocean Research (CSCOR) awards NA09NOS4780180 (Ecology of

McCulloch, A.W., Dodense, P., Pocklington, R., Quilliam, M.A., Sim, P.G., Smith,

Harmful Algal Blooms Program, ECOHAB) and NA09NOS4780209

J.C., Subba Rao, D.V., Todd, C.D., Walter, J.A., Wright, J.L.C., 1989. Pennate diatom

(ECOHAB), and NSF award OCE0942675. Kudela also contributed Nitzschia pungens as the primary source of domoic acid, a toxin in shellﬁsh from

through the Cal-PreEMPT program funded by NOAA CSCOR award eastern Prince Edward Island, Canada. Canadian Journal of Fisheries and Aquatic

Sciences 46, 1203–1215.

NA04NOS4780239 (Monitoring and Event Response Program,

Bates, S.S., Trainer, V.L., 2006. The ecology of harmful diatoms. In: Grane´li, E., Turner,

MERHAB). V. Trainer contributed to this paper through funding

J. (Eds.), Ecology of Harmful Algae. Heidelberg, Springer-Verlag, pp. 81–93.

to the NOAA West Coast Center for Oceans and Human Health at Baxter, R.E., 1971. Earthquake effects on clams of Prince William Sound. The great

Alaska earthquake of 1964. Biology. National Academy of Science Publication

the Northwest Fisheries Science Center in Seattle, WA. The

No. 1604. National Academy of Sciences, Washington, DC, pp. 238–245.

statements, ﬁndings, conclusions, and recommendations are those

Bill, B.D., Cox, F.H., Horner, R.A., Borchert, J.A., Trainer, V.L., 2006. The ﬁrst closure of

of the participants/authors and do not reﬂect the views of NSF, shellﬁsh harvesting due to domoic acid in Puget Sound, Washington, USA.

African Journal of Marine Science 28, 435–440.

NOAA or the Department of Commerce. This is ECOHAB, MERHAB,

Botsford, L.W., Lawrence, C.A., Dever, E.P., Hastings, A., Largier, J., 2003. Wind

and PNWTOX Contributions #692, #164, and #6.[SS]

strength and biological productivity in upwelling systems: an idealized study.

Fisheries Oceanography 12, 245–259.

Bricker, S., Longstaff, B., Dennison, W., Jones, A., Boicourt, K., Wicks, C., Woerner, J.,

References 2007. Effects of nutrient enrichment in the Nation’s estuaries: a decade of

change. NOAA Coastal Ocean Program Decision Analysis Series No. 26. National

Centers for Coastal Ocean Science, Silver Spring, MD, 322 pp.

Action Plan for the West Coast Governors’ Agreement on Ocean Health, 2008. http://

Britton-Simmons, K.H., 2004. Direct and indirect effects of the introduced alga

westcoastoceans.gov/docs/WCGA_ActionPlan_low-resolution.pdf.

Sargassum muticum on benthic, subtidal communities of Washington State,

Alaska’s Health, 1945. Paralytic Shellﬁsh Poisoning, vol. 3, no. 5. The Department of

USA. Marine Ecology Progress Series 277, 61–78.

Health, U.S. Public Health Service.

Bursa, A., 1963. Phytoplankton in coastal waters of the Arctic Ocean at Point Barrow,

Adams, N.A., Lesoing, A.M., Trainer, V.L., 2000. Environmental inﬂuences on domoic

Alaska. Arctic 16, 239–262.

acid accumulation in razor clams on the Washington coast. Journal of Shellﬁsh

Busse, L., Venrick, E., Antrobus, R., Miller, P., Vigilant, V., Silver, M., Mengelt, C.,

Research 19, 1007–1015.

Mydlarz, L., Pre´zelin, B., 2006. Domoic acid in phytoplankton and ﬁsh in San

Anderson, C.R., Brzezinski, M.A., Washburn, L., Kudela, R.M., 2006. Circulation and

Diego, CA, USA. Harmful Algae 5, 91–101.

environmental conditions during a toxigenic Pseudo-nitzschia australis bloom in

Caron, D.A., 2008. Collaborative HAB research and toxicity on the San Pedro Shelf.

the Santa Barbara Channel, California. Marine Ecology Progress Series 327, 119–

In: Harmful Algal Bloom Monitoring and Alert Program (HABMAP) Working

133.

Group. The Regional Workshop for Harmful Algal Blooms (HABs) in California

Anderson, C.R., Siegel, D.A., Guillocheau, N., Brzezinski, M.A., 2008a. Control on

Coastal Waters. Report #565. Southern California Coastal Water Research

temporal patterns in phytoplankton community structure in the Santa Barbara

Project, Costa Mesa, CA.

Channel, California. Journal of Geophysical Research: Oceans 113, C04038,

Cassis, D., 2007. Temperature, harmful algae, and summer shellﬁsh mortalities in

http://dx.doi.org/10.1029/2007JC004321.

British Columbia. Aquaculture Update 99, 1–4.

Anderson, D.M. (Ed.), 1995. The Ecology and Oceanography of Harmful Algal

Chang, F.H., Anderson, C., Boustead, N.C., 1990. First record of a Heterosigma

Blooms. A National Research Agenda. Woods Hole Oceanographic Institution,

(Raphidophyceae) bloom with associated mortality of cage-reared salmon in

Woods Hole, MA, 66 pp.

Big Glory Bay. New Zealand Journal of Marine and Freshwater Research 24, 461–

Anderson, D.M., Burkholder, J.M., Cochlan, W.P., Glibert, P.M., Gobler, C.J., Heil, C.A., 469.

Kudela, R.M., Parsons, M.L., Rensel, J.E., Townsend, D.W., Trainer, V.L., Vargo,

Chiang, R., 1988. Paralytic shellﬁsh management program in British Columbia,

G.A., 2008b. Harmful algal blooms and eutrophication: examining linkages from

Canada. Journal of Shellﬁsh Research 7, 637–642.

selected coastal regions of the United States. Harmful Algae 8, 39–53.

Cloern, J.E., Hieb, K.A., Jacobson, T., Sanso´ , B., Di Lorenzo, E., Stacey, M.T., Largier, J.L.,

Anderson, D.M., Glibert, P.M., Burkholder, J.M., 2002. Harmful algal blooms and

Meiring, W., Peterson, W.T., Powell, T.M., Winder, M., Jassby, A.D., 2010.

eutrophication: nutrient sources, composition and consequences. Estuaries 25,

Biological communities in San Francisco Bay track large-scale climate forcing

562–584.

over the North Paciﬁc. Geophysical Research Letters 37, L21602, http://

Armstrong, M., Kudela, R., 2006. Evaluation of California isolates of Lingulodinium dx.doi.org/10.1029/2010GL044774.

polyedrum for the production of yessotoxin. African Journal of Marine Science

Cloern, J.E., Schraga, T.S., Lopez, C.B., Knowles, N., Labiosa, R.G., Dugdale, R., 2005.

28, 399–401.

Climate anomalies generate an exceptional dinoﬂagellate bloom in San Fran-

Banas, N.S., Hickey, B.M., Newton, J.A., Ruesink, J.L., 2007. Tidal exchange, bivalve

cisco Bay. Geophysical Research Letters 32, L14608, http://dx.doi.org/10.1029/

grazing, and patterns of primary production in Willapa Bay, Washington, USA. 2005GL023321.

Marine Ecology Progress Series 341, 123–139.

Cochlan, W.P., Herndon, J., Kudela, R.M., 2008. Inorganic and organic nitrogen

Band-Schmidt, C.J., Bustillos-Guzma´n, J.J., Lo´ pez-Corte´s, D.J., Ga´rate-Liza´rraga, I.,

uptake by the toxigenic diatom Pseudo-nitzschia australis (Bacillariophyceae).

Nu´ n˜ez-Va´zquez, E.J., Herna´ndez-Sandoval, F.E., 2010. Ecological and physiolog-

Harmful Algae 8, 111–118.

ical studies of Gymnodinium catenatum in the Mexican Paciﬁc: a review. Marine

Corte´s-Altamirano, R., Sierra-Beltra´n, A.P., 2008. Biotoxins from freshwater and

Drugs 8, 1935–1961, http://dx.doi.org/10.3390/md8061935.

marine harmful algae blooms occurring in Mexico. Toxin Reviews 27, 27–77.

Band-Schmidt, C.J., Morquecho, L., Herna´ndez-Becerril, D.U., Reyes-Salinas, A.,

Cox, A.M., Shull, D.H., Horner, R.A., 2008. Proﬁles of Alexandrium catenella cysts in

Bravo-Sierra, E., 2004. Raphidophyceans on the coasts of Mexico. Hydrobiologia

Puget Sound sediments and the relationship to paralytic shellﬁsh poisoning

515, 79–89.

events. Harmful Algae 7, 379–388.

Bargu, S., Goldstein, T., Roberts, K., Li, C., Gulland, F., 2012. Pseudo-nitzschia blooms,

Cox, F., 2001. The marine toxin problem in Washington State. In: RaLonde, R. (Ed.),

domoic acid, and related California sea lion strandings in Monterey Bay,

Harmful Algal Blooms on the North American West Coast. University of Alaska

California. Marine Mammal Science 28, 237–253.

Sea Grant, AK-SG-0105, pp. 35–37.

Bargu, S., Powell, C.L., Coale, S.L., Busman, M., Doucette, G.J., Silver, M.W., 2002. Krill:

Cupp, E.E., 1943. Marine plankton diatoms of the west coast of North America.

a potential vector for domoic acid in marine food webs. Marine Ecology Progress

Bulletin of the Scripps Institution of Oceanography 5, 1–238.

Series 237, 209–216.

Curtiss, C.C., Langlois, G.W., Busse, L.B., Mazzillo, F., Silver, M.W., 2008. The emer-

Bargu, S., Powell, C.L., Wang, Z., Doucette, G.J., Silver, M.W., 2008. Note on the

gence of Cochlodinium along the California Coast (USA). Harmful Algae 7, 337–

occurrence of Pseudo-nitzschia australis and domoic acid in squid from Mon- 346.

terey Bay, California (USA). Harmful Algae 7, 45–51.

Deeds, J.R., Landsberg, J.H., Etheridge, S.M., Pitcher, G.C., Longan, S.W., 2008.

Bargu, S., Silver, M.W., 2003. Field evidence of krill grazing on the toxic diatom

Non-traditional vectors for paralytic shellﬁsh poisoning. Marine Drugs 6,

genus Pseudo-nitzschia in Monterey Bay, CA. Bulletin of Marine Science 72, 629– 308–348.

638.

Determan, T., 1998. Temporal and spatial distribution of paralytic shellﬁsh

Bargu, S., Silver, M., Goldstein, T., Roberts, K., Gulland, F., 2010. Complexity of

poisoning (PSP) in Puget Sound embayments. In: Puget Sound Action Team,

domoic acid-related sea lion strandings in Monterey Bay, California: foraging

Proceedings of the 1998 Puget Sound Research Conference, Seattle, pp. 627–

patterns, climate events, and toxic blooms. Marine Ecology Progress Series 418, 633.

213–222.

Dominguez, H.J., Paz, B., Daranas, A.H., Norte, M., Franco, J.M., Ferna´ndez, J.J., 2010.

Barth, J.A., 2003. Anomalous southward advection during 2002 in the northern

Dinoﬂagellate polyether within the yessotoxin, pectenotoxin and okadaic acid

California Current: evidence from Lagrangian surface drifters. Geophysical

toxin groups: characterization, analysis and human health implications. Tox-

Research Letters 30, 8019, http://dx.doi.org/10.1029/2003GL017511.

icon 56, 191–217.

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 155

Du, X., Peterson, W., McCulloch, A., Liu, G., 2011. An unusual bloom of the Gessner, B.D., Middaugh, J.P., 1995. Paralytic shellﬁsh poisoning in Alaska: a 20-year

dinoﬂagellate Akashiwo sanguinea off the central Oregon, USA, coast in autumn retrospective analysis. American Journal of Epidemiology 141, 766–770.

2009. Harmful Algae 10, 784–793. Gessner, B.D., Schloss, M.S., 1996. A population-based study of paralytic shellﬁsh

Dugdale, R.C., Wilkerson, F.P., Hogue, V.E., Marchi, A., 2007. The role of ammonium poisoning in Alaska. Alaska Medicine 38, 54–58.

and nitrate in spring bloom development in San Francisco Bay. Estuarine, Glibert, P.M., 2010. Long-term changes in nutrient loading and stoichiometry and

Coastal and Shelf Science 73, 17–29. their relationships with changes in the food web and dominant pelagic ﬁsh

Dyhrman, S.T., Haley, S.T., Borchert, J.A., Lon, B., Kollars, N., Erdner, D.L., 2010. species in the San Francisco Estuary, California. Reviews in Fisheries Science 18,

Parallel analyses of Alexandrium catenella cell concentrations and shellﬁsh 211–232.

toxicity in the Puget Sound. Applied and Environment Microbiology 76, Glibert, P.M., Fullerton, D., Burkholder, J.M., Cornwell, J.C., Kana, T.M., 2011. Eco-

4647–4654. logical stoichiometry, biogeochemical cycling, invasive species, and aquatic

Dyson, K., Huppert, D.D., 2010. Regional economic impacts of razor clam beach food webs, San Francisco Estuary and comparative systems. Reviews in Fisher-

closures due to harmful algal blooms (HABS) on the Paciﬁc coast of Washington. ies Science 19, 358–417.

Harmful Algae 9, 264–271. Glibert, P.M., Heil, C.A., Hollander, D., Revilla, M., Hoare, A., Alexander, J., Murasko, S.,

Erickson, G.M., Nishitani, L., 1985. The possible relationship of El Nin˜o/Southern 2004. Evidence for dissolved organic nitrogen and phosphorus uptake during

Oscillation events to interannual variation in Gonyaulax populations as shown a cyanobacterial bloom in Florida Bay. Marine Ecology Progress Series 280,

by records of shellﬁsh toxicity. In: Wooster, W.S., Fluharty, D.L. (Eds.), El Nin˜o 73–83.

North: Nin˜o Effects in the Eastern Subarctic Paciﬁc Ocean. Washington Sea Goldstein, T., Mazet, J.A.K., Zabka, T.S., Langlois, G., Colegrove, K.M., Silver, M., Bargu,

Grant Program, University of Washington, Seattle, pp. 283–290. S., Van Dolah, F., Leighﬁeld, T., Conrad, P.A., Barakos, J., Williams, D.C., Dennison,

Fetcho, K., 2006. Klamath River Blue-Green Algae Bloom Report Water Year 2005. S., Haulena, M., Gulland, F.M.D., 2008. Novel symptomatology and changing

Yurok Tribe Environmental Program, 17 pp. epidemiology of domoic acid toxicosis in California sea lions (Zalophus califor-

Fetcho, K., 2007. 2006 Klamath River Blue-Green Algae Summary Report. Yurok nianus): an increasing risk to marine mammal health. Proceedings of the Royal

Tribe Environmental Program, 34 pp. Society of London Series B: Biological Sciences 275 (1632), 267–276.

Fire, S.E., Wang, Z.H., Berman, M., Langlois, G.W., Morton, S.L., Sekula-Wood, E., Graham, H.W., 1943. Gymnodinium catenatum, a new dinoﬂagellate from the Gulf of

Benitez-Nelson, C.R., 2010. Trophic transfer of the harmful algal toxin domoic California. Transactions of the American Microscopical Society 62, 259–261.

acid as a cause of death in a minke whale (Balaenoptera acutorostrata) stranding Gulland, F.M.D., Haulena, M., Fauquier, D., Lander, M.E., Zabka, T., Duerr, R.S., 2002.

in southern California. Aquatic Mammals 36, 342–350. Domoic acid toxicity in Californian sea lions (Zalophus californianus): clinical

Flores-Trujillo, J.G., Helenes, J., Herguera, J.C., Orellana-Zepeda, E., 2009. Palynolog- signs, treatment and survival. The Veterinary Record 150, 475–480.

ical record (1483–1994) of Gymnodinium catenatum in Pescadero Basin, south- Hallegraeff, G.M., 1998. Concluding remarks on the autecology of harmful algal

ern Gulf of California, Mexico. Marine Micropaleontology 73, 80–89. blooms. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiolo-

Forbes, J.R., Denman, K.L., 1991. Distribution of Nitzschia pungens in coastal waters gical Ecology of Harmful Algal Blooms. NATO ASI Series, vol. G 41. Springer-

of British Columbia. Canadian Journal of Fisheries and Aquatic Sciences 48, 960– Verlag, New York, pp. 371–378.

967. Halstead, B., 1965. Poisonous and Venomous Animals of the World. Vol. 1. The

Foreman, M.G.G., Wiggins, W., MacFadyen, A., Hickey, B.M., Thomson, R.E., 2008. Invertebrates. U.S. Government Printing Ofﬁce, Washington, DC.

Modeling the generation of Juan de Fuca eddies. Journal of Geophysical Re- Heredia-Tapia, A., Arredondo-Vega, B.O., Nu´ n˜ez-Va´squez, E.J., Yasumoto, T., Yasuda,

search 113 (C03006), http://dx.doi.org/10.1029/2006JC004082. M., Ochoa, J.L., 2002. Isolation of Prorocentrum lima (Syn. Exuviaella lima) and

Fortuine, R., 1975. Paralytic shellfish poisoning in the north Pacific: two historic diarrheic shellfish poisoning (DSP) risk assessment in the Gulf of California,

accounts and implications for today. Alaska Medicine 17, 71–76. Mexico. Toxicon 40, 1121–1127.

Fraga, S., Anderson, D.M., Bravo, I., Reguera, B., Steidinger, K.A., Yentsch, C.M., 1988. Herna´ndez-Becerril, D.U., 1988. Planktonic dinoﬂagellates (except Ceratium and

Influence of upwelling relaxation on dinoflagellates and shellfish toxicity in Rıá Protoperidinium) from the Gulf of California and off the coasts of Baja California.

de Vigo, Spain. Estuarine, Coastal and Shelf Science 27, 349–361. Botanica Marina 31, 423–435.

Fredrickson, K.A., Strom, S.L., Crim, R., Coyne, K., 2011. Interstrain variability in Herna´ndez-Becerril, D.U., Alonso-Rodrı´guez, R., Alvarez-Go´ ngora, C., Baro´ n-Campis,

physiology and genetics of Heterosigma akashiwo (Raphidophyceae) from the S.A., Ceballos-Corona, G., Herrera-Silveira, J., Meave del Castillo, M.E., Jua´rez-

west coast of North America. Journal of Phycology 47, 25–35. Ruiz, N., Merino-Virgilio, F., Morales-Blake, A., Ochoa, J.L., Orellana-Cepeda, E.,

Fritz, L., Quilliam, M.A., Wright, J.L.C., Beale, A., Work, T.M., 1992. An outbreak of Ramirez-Camarena, C., Rodrı´guez-Salvador, R., 2007. Toxic and harmful marine

domoic acid poisoning attributed to the pennate diatom Pseudonitzschia aus- phytoplankton and microalgae (HABs) in Mexican Coasts. Journal of Environ-

tralis. Journal of Phycology 28, 439–442. mental Science and Health Part A 42, 1349–1363.

Fryxell, G.A., Villac, M.C., Shapiro, L.P., 1997. The occurrence of the toxic diatom Herndon, J., Cochlan, W.P., 2007. Nitrogen utilization by the raphidophyte Hetero-

genus Pseudo-nitzschia (Bacillariophyceae) on the West Coast of the USA, 1920– sigma akashiwo: growth and uptake kinetics in laboratory cultures. Harmful

1996: a review. Phycologia 36, 419–437. Algae 6, 260–270.

Ga´rate-Liza´rraga, I., Band-Schmidt, C.J., Lo´ pez-Corte´s, D.J., Bustillos-Guzma´n, J.J., Hershberger, P.K., Rensel, J.E., Postel, J.R., Taub, F.B., 1997. Heterosigma bloom and

Erler, K., 2007. Bloom of Pseudo-nitzschia fraudulenta in Bahı´a de La Paz, Gulf of associated ﬁsh kill. Harmful Algae News 16 1 and 4.

California (June–July 2006). Harmful Algae News 33, 6–7. Hickey, B.M., 1998. Coastal oceanography of western North America from the tip of

Ga´rate-Liza´rraga, I., Bustillos-Guzma´n, J.J., Erler, K., Mun˜eto´ n-Go´ mez, M.S., Luckas, Baja California to Vancouver Is. In: Brink, K.H., Robinson, A.R. (Eds.), The Sea,

B., Tripp-Quezada, A., 2004. Paralytic shellﬁsh toxins in the chocolata clam, vol. 11. Wiley, pp. 345–393.

Megapitaria squalida (Bivalvia: Veneridae), in Bahı´a de La Paz, Gulf of California. Hickey, B.M., Banas, N.S., 2003. Oceanography of the Paciﬁc Northwest coastal ocean

Revista de Biologia Tropical 52, 133–140. and estuaries with application to coastal ecosystems. Estuaries 26, 1010–1031.

Ga´rate-Liza´rraga, I., Bustillos-Guzma´n, J.J., Lo´ pez-Corte´s, D.J., Herna´ndez-Sandoval, Hickey, B.M., Banas, N.S., 2008. Why is the northern end of the California Current

F., Erler, K., Luckas, B., 2006. Paralytic shellﬁsh toxin proﬁles in net phytoplank- System so productive? Oceanography 21, 90–107.

ton samples from Bahı´a Concepcio´ n, Gulf of California, Mexico. Marine Pollution Hickey, B.M., Geier, S., Kachel, N., MacFadyen, A., 2005. A bi-directional river plume:

Bulletin 52, 800–815. the Columbia in summer. Continental Shelf Research 25, 1631–1636.

Garcia-Mendoza, E., Rivas, D., Olivos-Ortiz, A., Almaza´ n-Becerril, A., Castan˜eda- Hickey, B.M., Kudela, R.M., Nash, J.D., Bruland, K.W., Peterson, W.T., MacCready, P.,

Vega, C., Pen˜a-Manjarrez, J.L., 2009. A toxic Pseudo-nitzschia bloom in Lessard, E.J., Jay, D.A., Banas, N.S., Baptista, A.M., Dever, E.P., Kosro, P.M., Kilcher,

Todos Santos Bay, northwestern Baja California, Mexico. Harmful Algae 8, L.K., Horner-Devine, A.R., Zaron, E.D., McCabe, R.M., Peterson, J.O., Orton, P.M.,

493–503. Pan, J., Lohan, M.C., 2010. River inﬂuences on shelf ecosystems: introduction

Garcia-Reyes, M., Largier, J.L., 2012. Seasonality of coastal upwelling off central and and synthesis. Journal of Geophysical Research 115, C00B17, http://dx.doi.org/

northern California: new insights including temporal and spatial variability. 10.1029/2009JC005452.

Journal of Geophysical Research, 117, C03028, http://dx.doi.org/10.1029/ Hoagland, P., Anderson, D.M., Kauro, Y., White, A.M., 2002. The economic effects of

2011JC007629. harmful algal blooms in the United States: estimates, assessment issues, and

Garneau, M.-E` ., Schnetzer, A., Countway, P.D., Jones, A.C., Seubert, E.L., Caron, D.A., information needs. Estuaries 26, 819–837.

2011. Seasonal dynamics of the toxic dinoﬂagellate Alexandrium catenella at Horner, R.A., Baugh, K.A., Paranjpye, R., Adams, N.G., Trainer, V.L., 2010. SoundTox-

Redondo Beach, California, examined by quantitative PCR. Applied and Envi- ins: a monitoring program in Puget Sound, Washington, USA. In: Ho, K.C., Zhou,

ronment Microbiology 77, 7669–7680. M.J., Qi, Y.Z. (Eds.), Proc. 13th International Conference on Harmful Algae.

GEOHAB, 2005. In: Pitcher, G., Moita, T., Trainer, V.L., Kudela, R., Figueiras, P., Probyn, International Society for the Study of Harmful Algae and International Oceano-

T. (Eds.), Global Ecology and Oceanography of Harmful Algal Blooms, GEOHAB graphic Commission of UNESCO, Hong Kong, pp. 220–223.

Core Research Project: HABs in Upwelling Systems. IOC and SCOR, Paris, 82 pp. Horner, R.A., Dick, L.S., Shiels, W.E., 1973. Phytoplankton studies. In: Hood, D.W.,

GEOHAB, 2011. In: McGillicuddy, Jr., D.J., Glibert, P.M., Berdalet, E., Edwards, C., Shiels, W.E., Kelley, E.J. (Eds.), Environmental Studies of Port Valdez. Inst. Mar.

Franks, P., Ross, O. (Eds.), GEOHAB Modeling: A Workshop Report. IOC and Sci., Univ. Alaska Occasional Publ. No. 3, pp. 281–294.

SCOR, Paris and Newark, DE, 85 pp. Horner, R.A., Garrison, D.L., Plumley, F.G., 1997. Harmful algal blooms and red tide

Gessner, B.D., Bell, P., Doucette, G.J., Moczydlowski, E., Poli, M.A., Van Dolah, F., Hall, problems on the U.S. West Coast. Limnology and Oceanography 42, 1076–1088.

S., 1997. Hypertension and identiﬁcation of toxin in human urine and serum Horner, R.A., Greengrove, C.L., Postel, J.R., Gawel, J.E., Davies-Vollum, K.S., Cox, A.M.,

following a cluster of mussel-associated paralytic shellﬁsh poisoning outbreaks. 2011. Spatial distribution of benthic cysts of Alexandrium catenella in surface

Toxicon 35, 711–722. sediments of Puget Sound, Washington, USA. Harmful Algae 11, 96–105.

Gessner, B.D., McLaughlin, J.B., 2008. Epidemiologic impact of toxic episodes: Horner, R.A., Greengrove, C.L., Postel, J.R., Gawel, J.E., Davies-Vollum, K.S., Cox, A.,

neurotoxic toxins. In: Botana, L.M. (Ed.), Seafood and Freshwater Toxins: Hoffer, S., Sorensen, K., Hubert, J., Neville, J., Frost, B.W., 2008. Alexandrium cysts

Pharmacology, Physiology, and Detection. 2nd ed. Food Science and Technolo- in Puget Sound, Washington, USA. In: Moestrup, Ø., et al. (Eds.), Proc. 12th

gy, CRC Press, Boca Raton, pp. 77–104. International Conference on Harmful Algae. International Society for the Study

156 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

of Harmful Algae and International Oceanographic Commission of UNESCO, Kudela, R., Gobler, C., 2012. Harmful dinoﬂagellate blooms caused by Cochlodinium

Copenhagen, pp. 196–199. sp.: global expansion and ecological strategies facilitating bloom formation.

Horner, R.A., Hanson, L., Hatﬁeld, C.L., Newton, J.A., 1996. Domoic acid in Hood Canal, Harmful Algae 14, 71–86.

Washington, USA. In: Yasumoto, T., Oshima, Y., Fukuyo, Y. (Eds.), Harmful and Kudela, R., Lane, J., Cochlan, W., 2008a. The potential role of anthropogenically

Toxic Algal Blooms. International Oceanographic Commission of UNESCO, Paris, derived nitrogen in the growth of harmful algae in California, USA. Harmful

pp. 127–129. Algae 8, 103–110.

Howard, M., Ladizinsky, N., Cochlan, W., Kudela, R., 2007. Nitrogenous preference of Kudela, R., Pitcher, G., Probyn, T., Figueiras, F., Moita, T., Trainer, V., 2005. Harmful

toxigenic Pseudo-nitzschia australis (Bacillariophyceae) from ﬁeld and labora- algal blooms in coastal upwelling systems. Oceanography 18, 185–197.

tory experiments. Harmful Algae 6, 206–217. Kudela, R., Ryan, J., Blakely, M., Lane, J., Peterson, T., 2008b. Linking the physiology

Howard, M.D., Silver, M., Kudela, R.M., 2008. Yessotoxin detected in mussel (Mytilus and ecology of Cochlodinium to better understand harmful algal bloom events: a

californicus) and phytoplankton samples from the U.S. west coast. Harmful comparative approach. Harmful Algae 7, 278–292.

Algae 7, 646–652. Kudela, R.M., Seeyave, S., Cochlan, W.P., 2010. The role of nutrients in regulation and

Howard, M.D.A., Jones, A.C., Schnetzer, A., Countway, P.D., Tomas, C.R., Kudela, R.M., promotion of harmful algal blooms in upwelling systems. Progress in Ocean-

Hayashi, K., Chia, P., Caron, D.A., 2012. Quantitative real-time PCR for Cochlo- ography 85, 122–135.

dinium fulvescens (Dinophyceae), a potentially harmful dinoﬂagellate from Kvitek, R., Goldberg, J.D., Smith, G.J., Doucette, G.J., Silver, M.W., 2008. Domoic

California coastal waters. Journal of Phycology 48, 384–393. acid contamination within eight representative species from the benthic food

Hughes, S.E., Nelson, R.W., 1979. Distribution, Abundance, Quality, and Production web of Monterey Bay, California, USA. Marine Ecology Progress Series 367,

Fisheries Studies of the Surf Clam (Spisula polynema) in the Southeastern Bering 35–47.

Sea, 1978. A Cooperative Industry-Federal-State of Alaska Study. U.S. Depart- Ladizinsky, N., 2003. The inﬂuence of dissolved copper on the production of domoic

ment of Commerce, NOAA; NMFS, Northwest and Alaska Fisheries Center, acid by Pseudo-nitzschia species in Monterey Bay, California: laboratory experi-

Seattle, WA. ments and ﬁeld observations. Masters Thesis. California State University,

Huppert, D.A., Dyson, K., 2008. The Regional Economic Impact of Razor Clam Beach Monterey Bay, 68 pp.

Closures due to Harmful Algal Blooms (HABS) in Washington State. West Coast Landsberg, J.H., 2002. The effects of harmful algal blooms on aquatic organisms.

Center for Oceans and Human Health (WCCOHH) Publication No. 25, 51 pp. Reviews in Fisheries Science 10, 113–390.

Iwataki, M., Kawami, H., Mizushima, K., Mikulski, C.M., Doucette, G.J., Relox Jr., J.R., Lange, C.B., Reid, F.M.H., Vernet, M., 1994. Temporal distribution of the potentially

Anton, A., Fukuyo, Y., Matsuoka, K., 2008. Phylogenetic relationships in the toxic diatom Pseudonitzschia australis at a coastal site in Southern California.

harmful dinoﬂagellate Cochlodinium polykrikoides (Gymnodiniales, Dinophy- Marine Ecology Progress Series 104, 309–312.

ceae) inferred from LSU rDNA sequences. Harmful Algae 7, 271–277. Langlois, G., 2001. Marine biotoxin monitoring in California, 1927–1999. In:

Ja¨hnichen, S., Ihle, T., Petzoldt, T., Benndorf, J., 2007. Impact of inorganic carbon RaLonde, R. (Ed.), Harmful Algal Blooms on the North American West Coast.

availability on microcystin production by Microcystis aeruginosa PCC7806. University of Alaska Sea Grant, AK-SG-0105, Fairbanks, AK, pp. 31–34.

Applied and Environmental Microbiology 73, 6994–7002. Langlois, G., Smith, P., 2001. Phytoplankton. In: Karl, H.A., Chin, J.L., Ueber, E.,

Jassby, A., 2008. Phytoplankton in the upper San Francisco Estuary: recent biomass Stauffer, P.H., Hendley III, J.W. (Eds.), Beyond the Golden Gate – Oceanography,

trends, their causes and their trophic signiﬁcance. San Francisco Estuary and Geology, Biology and Environmental Issues in the Gulf of the Farallones. U.S.

Watershed Science 24 pp, In: http://escholarship.org/uc/item/71h077r1#page-2. Geological Survey Circular 1198, pp. 123–132.

Jeffery, B., Barlow, T., Moizer, K., Paul, S., Boyle, C., 2004. Amnesic shellﬁsh poison. Largier, J., 2004. The importance of retention zones in the dispersal of larvae.

Food and Chemical Toxicology 42, 545–557. American Fisheries Society Symposium 45, 105–122.

Jessup, D.A., Miller, M.A., Ryan, J.P., Nevins, H.M., Kerkering, H.A., 2009. Mass Largier, J.L., Lawrence, C.A., Roughan, M., Kaplan, D.M., Dever, E.P., Dorman, C.E.,

stranding of marine birds caused by a surfactant-producing red tide. PLoS Kudela, R.M., Bollens, S.M., Wilkerson, F.P., Dugdale, R.C., Botsford, L.W., Gar-

ONE 4 (2), e4550, http://dx.doi.org/10.1371/journal.pone.0004550. ﬁeld, N., Kuebel-Cervantes, B., Koracin, D., 2006. WEST: a northern California

Jester, R., Lefebvre, K., Langlois, G., Vigilant, V., Baugh, K., Silver, M.W., 2009. A shift study of the role of wind-driven transport in the productivity of coastal

in the dominant toxin-producing algal species in central California alters plankton communities. Deep Sea Research II 53, 2833–2849.

phycotoxins in food webs. Harmful Algae 8, 291–298. Largier, J.L., Magnell, B.A., Winant, C.D., 1993. Subtidal circulation over the northern

Jin, D., Thunberg, E., Hoagland, P., 2008. Economic impacts of the 2005 red tide event California shelf. Journal of Geophysical Research 98 (C10), 18147–18179.

on commercial shellﬁsheries in New England. Ocean & Coastal Management 51, Largier, J.L., Smith, S.V., Hollibaugh, T., 1997. Seasonally hypersaline estuaries in

420–429. Mediterranean-climate regions. Estuarine, Coastal and Shelf Science 45, 789–

Joint Ocean Commission Initiative, 2009. Changing Oceans, Changing World; Ocean 797.

Priorities for the Obama Administration and Congress. Meridiana Institute, Lechuga-Deveze, C., Sierra-Beltra´n, A.P., 1995. Documented case of ciguatera on the

Washington, DC 20036-5037 38 pp. http://www.jointoceancommission.org/ Mexican Paciﬁc coast. Natural Toxins 3, 415–418.

resource-center/1-Reports/2009-04-07_JOCI_Changing_Oceans,_Changing_ Lefebvre, K.A., Bargu, S., Kieckhefer, T., Silver, M., 2002. From sandabs to blue

World.pdf. whales: the pervasiveness of domoic acid. Toxicon 40, 971–977.

Jorgensen, P., Ibarra-Obando, S.E., Carriquiry, J.D., 2010. Management of natural Lefebvre, K.A., Robertson, A., 2010. Domoic acid and human exposure risks: a

Ulva spp. blooms in San Quintin Bay, Baja California: is it justiﬁed? Journal of review. Toxicon 56, 218–230.

Applied Phycology 22, 549–558. Lehman, P.W., Boyer, G., Hall, C., Waller, S., Gehrts, K., 2005. Distribution and

Johnson, K.S., Chavez, F.P., Elrod, V.A., Fitzwater, S.E., Pennington, J.T., Buck, K.R., toxicity of a new colonial Microcystis aeruginosa bloom in the San Francisco

Walz, P.M., 2001. The annual cycle of iron and the biological response in central Bay Estuary, California. Hydrobiologia 541, 87–99.

California coastal waters. Geophysical Research Letters 28, 1247–1250. Lehman, P.W., Boyer, G., Satchwell, M., Waller, S., 2008. The inﬂuence of environ-

Jousson, O., Pawlowski, J., Zaninetti, L., Zechman, F.W., Dini, F., Di Guiseppe, G., mental conditions on the seasonal variation of Microcystis cell density and

Woodﬁeld, R., Millar, A., Meinesz, A., 2000. Invasive alga reaches California – the microcystins concentration in San Francisco Estuary. Hydrobiologia 600, 187–

alga has been identiﬁed that threatens to smother Californian coastal ecosys- 204.

tems. Nature 408, 157–158. Lehman, P.W., The, S.J., Boyer, G.L., Nobriga, M.L., Bass, E., Hogle, C., 2010. Initial

Kahru, M., Kudela, R.M., Manzano-Sarabia, M., Mitchell, B.G., 2009. Trends in impacts of Microcystis aeruginosa blooms on the aquatic food web in the San

primary production in the California Current detected with satellite data. Francisco Estuary. Hydrobiologia 637, 229–248.

Journal of Geophysical Research 114, C02004, http://dx.doi.org/10.1029/ Lehman, P.W., Waller, S., 2003. Microcystis blooms in the Delta. Interagency Eco-

2008JC004979. logical Program for the San Francisco Estuary Newsletter, vol. 16, pp. 8–19.

Keppler, C.J., Lewitus, A.J., Ringwood, A.H., Hoguet, J., Staton, T., 2006. Sublethal MacFadyen, A., Hickey, B.M., 2010. Generation and evolution of a topographically

cellular effects of short-term raphidophyte and brevetoxin exposures on the linked, mesoscale eddy under steady and variable wind-forcing. Continental

eastern oyster Crassostrea virginica. Marine Ecology Progress Series 312, 141– Shelf Research 30, 1387–1402, http://dx.doi.org/10.1016/j.csr.2010.04.001.

147. MacFadyen, A., Hickey, B.M., Cochlan, W.P., 2008. Inﬂuences of the Juan de Fuca

Khan, S., Arakawa, O., Onoue, Y., 1997. Neurotoxins in a toxic red tide of Heterosigma Eddy on circulation, nutrients, and phytoplankton production in the northern

akashiwo (Raphidophyceae) in Kagoshima Bay, Japan. Aquaculture Research 28, California Current system. Journal of Geophysical Research 113, C08008, http://

9–14. dx.doi.org/10.1029/2007JC004412.

Kimbro, D.L., Largier, J., Grosholz, E.D., 2009. Coastal oceanographic processes MacFadyen, A., Hickey, B.M., Foreman, M.G.G., 2005. Transport of surface waters

inﬂuence the growth and size of a key estuarine species, the Olympia oyster. from the Juan de Fuca eddy region to the Washington coast. Continental Shelf

Limnology and Oceanography 54, 1425–1437. Research 25, 2008–2021.

Klinger, T., Padilla, D.K., Britton-Simmons, K., 2006. Two invaders achieve higher Magnusson, H.W., Carlson, C.J., Chambers, J.S., 1951. The mouse test. Technology

densities in reserves. Aquatic Conservation: Marine and Freshwater Ecosystems Studies on the Alaska Butter Clam. Part 2. Report of the Fisheries Products

16, 301–311. Laboratory, Ketchikan, Alaska (not to be cited without permission).

Kudela, R., 2008. Silicon:nitrogen interactions in the marine environment. In: Maldonado, M.T., Hughes, M.P., Rue, E.L., Wells, M.C., 2002. The effect of Fe and Cu

Capone, D., Bronk, D., Mulholland, M., Carpenter, E. (Eds.), Nitrogen in the on growth and domoic acid production by Pseudo-nitzschia multiseries and

Marine Environment. 2nd ed. Elsevier, New York, pp. 1589–1626. Pseudo-nitzschia australis. Limnology and Oceanography 47, 515–526.

Kudela, R.M., Cochlan, W.P., Roberts, A., 2004. Spatial and temporal patterns of Manrique, F.A., Molina, R.E., 1997. Presencia de mareas rojas en la Bahı´a de

Pseudo-nitzschia spp. in central California related to regional oceanography. In: Bacochibampo, Guaymas, Sonora, Me´xico. Hidrobiologica 7, 81–86.

Steidinger, K.A., Landsberg, J.H., Tomas, C.R., Vargo, G.A. (Eds.), Harmful Algal Marchetti, A., Trainer, V.L., Harrison, P.J., 2004. Environmental conditions and

Blooms 2002. Proc. Xth International Conference on Harmful Algae. Florida Fish phytoplankton dynamics associated with Pseudo-nitzschia abundance and

and Wildlife Conservation Commission and Intergovernmental Oceanographic domoic acid in the Juan de Fuca eddy. Marine Ecology Progress Series 281,

Commission of UNESCO, pp. 347–349. 1–24.

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 157

Matter, A.L., 1994. Paralytic Shellﬁsh Poisoning: Toxin Accumulation in the Marine Pen˜a-Manjarrez, J.L., Helenes, J., Gaxiola-Castro, G., Orellana-Cepeda, E., 2005.

Food Web, with Emphasis on Predatory Snails. Prepared for U.S. EPA, Region 10, Dinoﬂagellate cysts and bloom events at Todos Santos Bay, Baja California,

Water Division, Seattle, WA. EPA 910/R-94-005, 44 pp. Me´xico, 1999–2000. Continental Shelf Research 25, 1375–1393.

Mazzillo, F.F.M., Pomeroy, C., Kuo, J., Ramond, P.T., Prado, R., Silver, M.W., 2010. Pe´rez-Brunius, P., Lopez, M., Pineda, J.P., 2006. Hydrographic conditions near the

Angler exposure to domoic acid via consumption of contaminated ﬁshes. coast of northwestern Baja California: 1997–2004. Continental Shelf Research

Aquatic Biology 9, 1–12. 26, 885–901.

Mee, L.D., Diaz-Gonzalez, G., Espinosa-Damian, M., 1986. Paralitic shellﬁsh poison- Perl, T.M., Be´dard, L., Kosatsky, T., Hockin, J.C., Todd, E.C., Remis, R.S., 1990. An

ing with Gymnodinium catenatum red tide on the Paciﬁc coast of Mexico. Marine outbreak of toxic encephalopathy caused by eating mussels contaminated with

Environment Research 19, 77–92. domoic acid. New England Journal of Medicine 322, 1775–1780.

Meyer, K.F., Sommer, H., Schoenholz, P., 1928. Mussel poisoning. Journal of Preven- Peterman, R.M., Marmorek, D., Beckman, B., Bradford, M., Mantua, N., Riddell, B.E.,

tive Medicine 2, 365–394. Scheuerell, M., Staley, M., Wieckowski, K., Winton, J.R., Wood, C.C., 2010.

Meyers, H.F., Hillian, D.R., 1955. Shellﬁsh poisoning episode in False Pass, Alaska. Synthesis of evidence from a workshop on the decline of Fraser River sockeye,

Public Health Service Report 70, 419–420. June 15–17, 2010. A Report to the Paciﬁc Salmon Commission, Vancouver, BC.

Miernyk, W.H., 1966. The Elements of Input–Output Analysis. Random House, New http://www.psc.org/pubs/FraserSockeyeDeclineWorkshopLinks.pdf.

York. Pew Oceans Commission, 2003. America’s Living Oceans: Charting a Course for Sea

Miller, M.A., Kudela, R.M., Mekebri, A., Crane, D., Oates, S.C., Tinker, M.T., Staedler, Change. Summary Report. Pew Oceans Commission, Arlington, Virginia. http://

M., Miller, W.A., Toy-Choutka, S., Dominik, C., Hardin, D., Langlois, G., Murray, www.pewtrusts.org/uploadedFiles/wwwpewtrustsorg/Reports/Protecting_

M., Ward, K., Jessup, D.A., 2010. Evidence for a novel marine harmful algal ocean_life/POC_Summary.pdf.

bloom: cyanotoxin (microcystin) transfer from land to sea otters. PLoS ONE 5 Phillips, E.M., Zamon, J.E., Nevins, H.M., Gibble, C.M., Duerr, R.S., Kerr, L.H., 2011.

(9), e12576, http://dx.doi.org/10.1371/journal.pone.0012576. Summary of birds killed by a Harmful Algal Bloom along south Washington and

Moestrup, Ø., Lundholm, N., 2007. IOC Taxonomic Reference List of Toxic Plankton north Oregon coasts during October 2009. Northwestern Naturalist 92, 120–

Algae. http://www.bi.ku.dk/ioc/group1.asp. 126.

Moisander, P.H., Lehman, P.W., Ochial, M., Corum, S., 2009. Diversity of Microcystis Prakash, A., Taylor, F.J.R., 1966. A red water bloom of Gonyaulax acatenella in the

aeruginosa in the Klamath River and San Francisco Bay delta, California, USA. Strait of Georgia and its relation to paralytic shellﬁsh toxicity. Journal of the

Aquatic Microbial Ecology 57, 19–31. Fisheries Research Board of Canada 23, 1265–1270.

Moore, S.K., Mantua, N.J., Hickey, B.M., Trainer, V.L., 2009. Recent trends in paralytic Pratt, D.M., 1966. Competition between Skeletonema costatum and Olisthodiscus

shellﬁsh toxins in Puget Sound, relationships to climate, and capacity for luteus in Narragansett Bay and in culture. Limnology and Oceanography 11,

prediction of toxic events. Harmful Algae 8, 463–477. 447–455.

Moore, S.K., Mantua, N.J., Kellogg, J.P., Newton, J.A., 2008. Local and large-scale Price, D.W., Kizer, K.W., Hangsen, K.H., 1991. California’s paralytic shellﬁsh

climate forcing of Puget Sound oceanographic properties on seasonal to inter- poisoning prevention program 1927–1989. Journal of Shellﬁsh Research 10,

decadal timescales. Limnology and Oceanography 53, 1746–1758. 119–145.

Mudie, P.J., Rochon, A., Levac, E., 2002. Palynological records of red tide-producing Quayle, D.B., 1969. Paralytic shellﬁsh poisoning in British Columbia. Bulletin of the

species in Canada: past trends and implications for the future. Palaeogeography, Fisheries Research Board of Canada 168 68 pp.

Palaeoclimatology, Palaeoecology 180, 159–186. Quick, R., 1992. The epidemiology of domoic acid poisoning. In: Proceedings,

Nishitani, L., Chew, K.K., 1984. Recent developments in paralytic shellﬁsh poisoning Domoic Acid Workshop. February 6–8, 1992, San Pedro, CA 20 pp.

research. Aquaculture 39, 317–329. Radtke, H., Dewees, C.M., Smith, F.J., 1987. The ﬁshing industry and Paciﬁc coastal

Nishitani, L., Chew, K., 1988. PSP toxins in the Paciﬁc coast states: monitoring communities: understanding the assessment of economic impacts. Paciﬁc Sea

programs and effects on bivalve industries. Journal of Shellﬁsh Research 7, 653– Grant College Program. University of California. Marine Advisory Program

669. Publication – UCSGMAP-97-1.

NOAA Program Planning and Integration, 2007. Advancing NOAA’s Priorities through RaLonde, R. (Ed.), 2001. Harmful Algal Blooms on the North American West Coast.

Regional Collaboration. http://www.ppi.noaa.gov/Regional_Collaboration/ University of Alaska Sea Grant, AK-SG-0105, Fairbanks.

Regional_Collaboration_Overview_041307.pdf. RaLonde, R., Wright, B., 2011. Using blue mussels as an indicator species for testing

NORMA Oficial Mexicana, 2011. NORMA Oficial Mexicana NOM-242-SSA1-2009, domoic acid toxicity in subsistence bivalve harvest. North Pacific Research

Productos y servicios. Productos de la pesca frescos, refrigerados, congelados y Board Project 821 Final Report. Anchorage, AK, 125 pp.

procesados. Especiﬁcaciones sanitarias y me´todos de prueba. Diario Oﬁcial de la Rensel, J.E., 1991. Phytoplankton and Nutrient Studies Near Salmon Net-Pens at

Federacion, Jueves 10 de febrero de 2011, Mexico, DF. Squaxin Island, Washington. Technical Appendices of Programmatic Environ-

NSTC Joint Subcommittee on Ocean Science and Technology, 2007. Charting the mental Impact Statement: Fish Culture in Floating Net-Pens. Prepared by

Course for Ocean Science in the United States for the Next Decade: An Ocean Parametrix, Battelle Northwest Laboratories and Rensel Associates for the State

Research Priorities Plan and Implementation Strategy. http://ocean.ceq.gov/ of Washington Departments of Ecology, Fisheries and Natural Resources, 161

about/sup_jsost_prioritiesplan.html. pp. and appendices.

Nu´ n˜ez-Va´zquez, E.J., Ga´rate-Lizarraga, I., Band-Schmidt, C.J., Cordero-Tapia, A., Rensel, J.E., 1993. Factors controlling paralytic shellﬁsh poisoning (PSP) in Puget

Lopez-Cortes, D.J., Hernandez Sandoval, F.E., Bustillos-Guzman, J.J., 2011. Im- Sound, Washington. Journal of Shellﬁsh Research 12, 371–376.

pact of harmful algal blooms on wild and cultured animals in the Gulf of Rensel, J.E., 1995. Harmful algal blooms and ﬁnﬁsh resources in Puget Sound. In:

California. Journal of Environment Biology 32, 413–442. Robichaud, E. (Ed.), Puget Sound Research, vol. 1. Puget Sound Water Quality

Ochoa, J.L., Herna´ndez-Becerril, D.U., Lluch-Cota, L., Arredondo-Vega, B.O., Nun˜ez- Authority, Olympia, WA, pp. 422–429.

Va´zquez, E., Heredia-Tapia, A., Pe´rez-Linares, J., Alonso-Rodrı´guez, R., 2002. Rensel, J.E., 2007. Fish Kills from the Harmful Alga Heterosigma akashiwo in Puget

Marine biotoxins and harmful algal blooms in Mexico’s Paciﬁc littoral. In: Sound: Recent Blooms and Review. Prepared by Rensel Associates Aquatic

Taylor, F.J.R., Trainer, V.L. (Eds.), Harmful Algal Blooms in the PICES Region Sciences for the National Oceanic and Atmospheric Administration Center

of the North Paciﬁc. PICES Scientiﬁc Report No. 23, pp. 119–128. for Sponsored Coastal Ocean Research (CSCOR), Washington, DC, 58 pp.

Ochoa, J.L., Sa´nchez-Paz, A., Cruz-Villacorta, A., Nu´ n˜ez-Va´zquez, E., Sierra Beltra´n, A., Rensel, J.E., Haigh, N., Tynan, T., 2010. Summary paper: Fraser River sockeye salmon

1997. Toxic events in the northwest Paciﬁc coastline of Mexico during 1992– marine survival decline and harmful blooms of Heterosigma akashiwo. In:

1995: origin and impacts. Hydrobiologia 352, 195–200. Peterman et al. (Eds.), Synthesis of Evidence from a Workshop on the Decline

Okolodkov, Y.B., Garate-Liza´ rraga, I., 2006. An annotated checklist of dinoﬂagel- of Fraser River Sockeye, June 15–17, 2010. A Report to the Paciﬁc Salmon

lates (Dinophyceae) from the Mexican Paciﬁc. Acta Botanica Mexicana 74, Commission, Vancouver, BC. Appendix C. Session B, 7 pp. http://www.psc.org/

1–154. pubs/FraserSockeyeDeclineWorkshopAppCPt2.pdf.

Olson, M.B., Lessard, E.J., Cochlan, W.P., Trainer, V.L., 2008. Intrinsic growth and Rensel, J.E., Haigh, N., Tynan, T., 2010b. Fraser River sockeye salmon marine survival

microzooplankton grazing on the toxigenic diatom Pseudo-nitzschia spp. from decline and harmful blooms of Heterosigma akashiwo. Harmful Algae 10, 98–

the coastal North Paciﬁc. Limnology and Oceanography 53, 1352–1368. 115.

Onoue, Y., Nozouwa, K., 1989. Separation of toxins from harmful red tides occurring Rensel, J.E., Whyte, J.N.C., 2003. Finﬁsh mariculture and harmful algal blooms. In:

along the coast of Kagoshima prefecture. In: Okaichi, T., Anderson, D.M., Ne- Hallegraeff, G.M., Anderson, D.M., Cembella, A.D. (Eds.), Manual on Harmful

moto, T. (Eds.), Red Tides: Biology, Environmental Science and Toxicology. Marine Microalgae. Monographs on Oceanographic Methodology, vol. 11.

Elsevier Science Publishers, New York, pp. 371–374. UNESCO Publishing, Paris, pp. 693–722.

Orth, D.L., Smelcer, C.S., Feder, H.M., Williams, J., 1975. The Alaska clam ﬁshery: a Roegner, C., Hickey, B.M., Newton, J., Shanks, A., Armstrong, D., 2002. Estuarine-

survey of economic potential. Alaska Sea Grant Report 75-5. nearshore links during a coastal upwelling cycle: plume and bloom intrusions

Ostasz, M., 2001. PSP toxin concentrations in Alaska. In: RaLonde, R. (Ed.), Harmful into Willapa Bay, Washington. Limnology and Oceanography 47, 1033–

Algal Blooms on the North American West Coast. University of Alaska Sea Grant, 1042.

AK-SG-01-05, p. 51. Rogers-Bennett, L., Kudela, R., Nielsen, K., Paquin, A., O’Kelly, C., Langlois, G., Crane,

Pan, Y., Stevenson, R.J., Hill, B.H., Herlihy, A.T., Collins, G.B., 1996a. Using diatoms as D., Moore, J., 2012. Dinoﬂagellate bloom coincides with marine invertebrate

indicators of ecological conditions in lotic systems: a regional assessment. mortalities in northern California. Harmful Algae News, submitted.

Journal of the North American Benthological Society 15, 481–495. Royer, T.C., 1979. On the effect of precipitation and runoff on coastal circulation in

Pan, Y., Subba Rao, D.V., Mann, K.H., 1996b. Acclimation to low light intensity in the Gulf of Alaska. Journal of Physical Oceanography 9, 555–563.

photosynthesis and growth of Pseudo-nitzschia multiseris Hasle, a neurotoxi- Royer, T.C., 1981. Baroclinic transport in the Gulf of Alaska. 2. Freshwater driven

genic diatom. Journal of Plankton Research 18, 1427–1438. coastal current. Journal of Marine Research 39, 251–266.

Pan, Y., Subba Rao, D.V., Mann, K.H., 1996c. Changes in domoic acid production and Rue, E., Bruland, K., 2001. Domoic acid binds iron and copper: a possible role for the

cellular chemical composition of the toxigenic diatom Pseudo-nitzschia multi- toxin produced by the marine diatom Pseudo-nitzschia. Marine Chemistry 76,

series under phosphate limitation. Journal of Phycology 32, 371–381. 127–134.

158 A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159

Rukuyama, A.K., Shigenakaa`, G.W., Hoffa`, R.Z., 2000. Effects of residual Exxon Valdez Taylor, F.J.R., Harrison, P.J., 2002. Harmful algal blooms in western Canadian coastal

oil on intertidal Protothaca staminea: mortality, growth, and bioaccumulation of waters. In: Taylor, F.J.R., Trainer, V.L. (Eds.), Harmful Algal Blooms in the PICES

hydrocarbons in transplanted clams. Marine Pollution Bulletin 40, 1042–1050. Region of the North Paciﬁc. PICES Scientiﬁc Report No. 23. PICES, Sidney, BC, pp.

Saldate-Castan˜eda, O., Va´zquez-Castellanos, J.L., Galva´n, J., Sa´nchez-Anguiano, A., 77–88.

Nazar, A., 1991. Intoxicaciones por toxina paralizante de molusco en Oaxaca. Taylor, F.J.R., Horner, R.A., 1994. Red tides and other problems with harmful algal

Salud Publica de Mexico 33, 240–247. blooms in Paciﬁc Northwest coastal waters. In: Wilson, R.C.H., Beamish, R.J.,

Schandelmeier, L., Alexander, V., 1981. An analysis of the inﬂuence of ice on spring Aitkens, F., Bell, J. (Eds.), Review of the Marine Environment and Biota of Strait of

phytoplankton population structure in the southeast Bering Sea. Limnology and Georgia, Puget Sound and Juan de Fuca Strait: Proceedings of the BC/Washing-

Oceanography 26, 935–943. ton Symposium on the Marine Environment. Canadian Technical Report of

Schnetzer, A., Miller, P.E., Schaffner, R.A., Stauffer, B., Jones, B., Weisberg, S.B., Fisheries and Aquatic Sciences 1948, pp. 175–186.

DiGiacomo, P.M., Berelson, W., Caron, D.A., 2007. Pseudo-nitzschia spp. and Taylor, F.J.R., Trainer, V.L., 2002. Harmful Algal Blooms in the PICES Region of the

domoic acid in the San Pedro Channel and Los Angeles harbor areas of the North Paciﬁc. PICES Sci. Rep. No. 23. PICES, Sidney, BC, 152 pp.

Southern California Bight, 2003–2004. Harmful Algae 6, 372–387. Teitelbaum, J.S., Zatorre, R.J., Carpenter, S., Gendron, D., Evans, A.C., Gjedde, A.,

Scholin, C.A., Gulland, F., Doucette, G.J., Benson, S., Busman, M., Chavez, F.P., Cashman, N.R., 1990. Neurologic sequelae of domoic acid intoxication due to the

Cordarok, J., DeLong, R., De Vogelaere, A., Harvey, J., Haulena, M., Lefebvre, ingestion of contaminated mussels. New England Journal of Medicine 322,

K., Lipscomb, T., Loscutoff, S., Lowenstine, L.J., Marin, R., Miller, P.E., McLellan, 1781–1787.

W.A., Moeller, P.D.R., Powell, C.L., Rowleskk, T., Silvagni, P., Silver, M., Spraker, T., Thomas, D.A., Bodkin, J.L., Fukuyama, A.K., Jewett, S.C., Monson, D.H., O’Clair, C.E.,

Trainer, V.L., Van Dolah, F.M., 2000. Mortality of sea lions along the central VanBlaricom, G.R., 2002. Food limitation and the recovery of sea otters follow-

California coast linked to a toxic diatom bloom. Nature 403, 80–84. ing the ‘Exxon Valdez’ oil spill. Marine Ecology Progress Series 241, 255–270.

Schumacher, J.D., Reed, R.K., 1980. Coastal ﬂow in the northwest Gulf of Alaska: the Tikhmenev, T.A., 1979. In: Pierce, R.A., Donnelly, A.S. (Eds.), A History of the Russian-

Kenai Current. Journal of Geophysical Research 85, 6680–6688. American Company, Vol. 2, Documents. Period 1783–1807 (D. Krenov, Trans.).

Sekula-Wood, E., Benitez-Nelson, C., Morton, S., Thunell, R., Anderson, C., 2011. Limestone Press, Kingston, Ontario.

Pseudo-nitzschia and domoic acid ﬂuxes in Santa Barbara Basin (CA) from 1993 Trainer, V.L., Adams, N.G., Bill, B.D., Anulacion, B.F., Wekell, J.C., 1998. Concentration

to 2008. Harmful Algae 10, 567–575. and dispersal of a Pseudo-nitzschia bloom in Penn Cove, Washington, USA.

Sekula-Wood, E., Schnetzer, A., Benitez-Nelson, C.R., Anderson, C., Berelson, W., Natural Toxins 6, 1–13.

Brzezinksi, M., Burns, J., Caron, D.A., Cetinic, I., Ferry, J., Fitzpatrick, E., Jones, B., Trainer, V.L., Adams, N.G., Bill, B.D., Stehr, C.M., Wekell, J.C., Moeller, P., Busman, M.,

Miller, P.E., Morton, S.L., Schaffner, R., Siegel, D., Thunell, R., 2009. Rapid Woodruff, D., 2000. Domoic acid production near California coastal upwelling

downward transport of the neurotoxin domoic acid in coastal waters. Nature zones, June 1998. Limnology and Oceanography 45, 1818–1833.

Geoscience 2, 272–275. Trainer, V.L., Adams, N.G., Wekell, J.C., 2001. Domoic acid-producing Pseudo-

SEMARNAP-PROFEPA, 1997. Mortandad de mamı´feros marinos cuyos cada´veres nitzschia species off the U.S. west coast associated with toxiﬁcation events.

arribaron a las Costas de Sinaloa. InformeTe´cnico. SEMARNAP, Me´xico. In: Hallegraeff, G.M., Blackburn, S.I., Bolch, C.J., Lewis, R.J. (Eds.), Proceedings of

Send, U., Beardsley, R.C., Winant, C.D., 1987. Relaxation from upwelling in the the Ninth International Conference of Harmful Algal Blooms. Intergovernmen-

Coastal Ocean Dynamics Experiment. Journal of Geophysical Research 92 (C2), tal Oceanographic Commission of UNESCO, pp. 46–48.

1683–1698. Trainer, V.L., Cochlan, W.P., Erickson, A., Bill, B.D., Cox, F.H., Borchert, J.A., 2007.

Shumway, S.E., 1990. A review of the effects of algae blooms on shellﬁsh and Recent domoic acid closures of shellﬁsh harvest areas in Washington State

aquaculture. Journal of the World Aquaculture Society 21, 65–104. inland waterways. Harmful Algae 6, 449–459.

Shumway, S.E., 1995. Phycotoxin-related shellﬁsh poisoning: bivalve molluscs are Trainer, V.L., Eberhart, B.-T.L., Wekell, J.C., Adams, N.G., Hanson, L., Cox, F., Dowell, J.,

not the only vectors. Reviews in Fisheries Science 3, 1–31. 2003. Paralytic shellﬁsh toxins in Puget Sound, Washington State. Journal of

Shumway, S.E., Allen, S.M., Boersma, P.D., 2003. Marine birds and harmful algal Shellﬁsh Research 22, 213–224.

blooms: sporadic victims or under-reported events. Harmful Algae 2, 1–17. Trainer, V.L., Hickey, B.M., 2003. The challenges of forecasting and managing

Shumway, S.E., Barter, J., Sherman-Caswell, S., 1990. Auditing the impact of toxic toxigenic Pseudo-nitzschia blooms on the U.S. west coast. In: Scavia, D.,

algal blooms on oysters. Environmental Auditor 2, 41–56. Valetta-Silver, N. (Eds.), Ecological Forecasting: New Tools for Coastal and

Shumway, S.E., Cembella, A.D., 1993. The impact of toxic algae on scallop culture Marine Ecosystem Management. NOAA Technical Memo, NOS NCCOS 1, pp.

and ﬁsheries. Reviews in Fisheries Science 1, 121–150. 55–60.

Sierra-Beltra´n, A., Palafox-Uribe, M., Grajales-Montiel, L., Cruz-Villacorta, A., Ochoa, Trainer, V.L., Hickey, B.M., Bates, S.S., 2008. Toxic Diatoms. In: Walsh, P.J., Smith,

J.L., 1997. Sea bird mortality at Cabo San Lucas, Mexico: evidence that toxic S.L., Fleming, L.E., Solo-Gabriele, H., Gerwick, W.H. (Eds.), Oceans and

diatom blooms are spreading. Toxicon 35, 447–453. Human Health: Risks and Remedies from the Sea. Elsevier Science Publishers,

Smayda, T.J., 1997. Harmful phytoplankton blooms: their ecophysiology and gen- New York, pp. 219–239.

eral relevance. Limnology and Oceanography 42, 1137–1153. Trainer, V.L., Hickey, B.M., Horner, R.A., 2002. Biological and physical dynamics of

Smayda, T.J., 1998. Ecophysiology and bloom dynamics of Heterosigma akashiwo domoic acid production off the Washington coast. Limnology and Oceanogra-

(Raphidophyceae). In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. phy 47, 1438–1446.

(Eds.), Physiological Ecology of Harmful Algal Blooms. NATO ASI Series, Trainer, V.L., Hickey, B.M., Lessard, E.J., Cochlan, W.P., Trick, G.C., Wells, M.L.,

vol. G 41. Springer-Verlag, New York, pp. 113–131. MacFadyen, A., Moore, S.K., 2009a. Variability of Pseudo-nitzschia and domoic

Smayda, T.J., 2006. Harmful Algal Bloom Communities in Scottish Coastal Waters: acid in the Juan de Fuca eddy region and its adjacent shelves. Limnology and

Relationship to Fish Farming and Regional Comparisons – A Review: Paper Oceanography 54, 289–308.

2006/3. Scottish Executive. www.scotland.gov.uk/Publications/2006/02/ Trainer, V.L., Pitcher, G.C., Reguera, B., Smayda, T.J., 2010. The distribution and

03095327/16. impacts of harmful algal bloom species in eastern boundary upwelling systems.

Sommer, H., Meyer, K.F., 1935. Mussel poisoning. California and Western Medicine Progress in Oceanography 85, 33–52.

42 (6), 423–426. Trainer, V.L., Wells, M.L., Cochlan, W.P., Trick, C.G., Bill, B.D., Baugh, K.A., Beall, B.F.,

Sommer, H., Meyer, K.F., 1937. Paralytic shellﬁsh poisoning. Archives of Pathology Herndon, J., Lundholm, N., 2009b. An ecological study of a massive bloom of

24, 560–598. toxigenic Pseudo-nitzschia cuspidata off the Washington State coast. Limnology

Southerland, C.M., 2008. Diarrhetic shellﬁsh toxins linked to local Dinophysis and Oceanography 54, 1461–1474.

populations in the California coastal waters of Monterey Bay. Masters Thesis Tweddle, J.F., Strutton, P.G., Foley, D.G., O’Higgins, L., Wood, A.M., Scott, B., Everroad,

in Marine Sciences. University of California at Santa Cruz. R.C., Peterson, W., Cannon, D., Hunter, M., Forster, Z., 2010. Relationships among

State of Alaska Epidemiology Bulletin, 2010. Paralytic Shellﬁsh Poisoning in Juneau, upwelling, phytoplankton blooms, and phycotoxins in coastal Oregon shellﬁsh.

Kodiak, and Haines, June 2010. Bulletin No. 17, June 23, 2010. http://www. Marine Ecology Progress Series 405, 131–145.

epi.hss.state.ak.us/bulletins/docs/b2010_17.pdf. Twiner, M.J., Trick, C.G., 2000. Possible physiological mechanisms for production of

Suganuma, M., Fujiki, H., Suguri, H., Yoshizawa, S., Hirota, M., Nakayasu, M., Ojika, hydrogen peroxide by the ichthyotoxic ﬂagellate Heterosigma akashiwo. Journal

M., Wakamatsu, K., Yamada, K., Sugimura, T., 1988. Okadaic acid: an additional of Plankton Research 22, 1961–1975.

non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter. Proceedings U.S. Commission on Ocean Policy, 2004. An Ocean Blueprint for the 21st Century.

of the National Academy of Sciences of the United States of America 85, 1768– Final Report. Washington, DC. http://www.oceancommission.gov/documents/

1771. full_color_rpt/welcome.html.

Taylor, F.J.R., 1993. Current problems with harmful phytoplankton blooms in Van Mooy, B.A.S., Fredricks, H.F., Pedler, B.E., Dyhrman, S.T., Karl, D.M., Koblizek, M.,

British Columbia waters. In: Smayda, T.J., Shimizu, Y. (Eds.), Toxic Phytoplank- Lomas, M.W., Mincer, T.J., Moore, L.R., Moutin, T., Rappe, M.S., Webb, E.A., 2009.

ton Blooms in the Sea. Elsevier Science Publishers, B.V., Amsterdam, pp. 699– Phytoplankton in the ocean use non-phosphorus lipids in response to phos-

703. phorus scarcity. Nature 458, 69–72.

Taylor, F.J.R., Haigh, R., 1993. The ecology of ﬁsh-killing blooms of the chloromonad Van Nieuwenhuyse, E., 2007. Response of summer chlorophyll concentration to

ﬂagellate Heterosigma in the Strait of Georgia and adjacent waters. In: Smayda, reduced total phosphorus concentration in the Rhine River (Netherlands) and

T.J., Shimizu, Y. (Eds.), Toxic Phytoplankton Blooms in the Sea. Elsevier Science the Sacramento-San Joaquin Delta (California, USA). Canadian Journal of Fish-

Publishers, B.V., Amsterdam, pp. 705–710. eries and Aquatic Sciences 64, 1529–1542.

Taylor, F.J.R., Haigh, R., 1996. Spatial and temporal distributions of microplankton Walters, L.J., Brown, K.R., Stam, W.T., Olsen, J.L., 2006. E-Commerce and Caulerpa:

during the summers of 1992–1993 in Barkley Sound, British Columbia. Cana- unregulated dispersal of invasive species. Frontiers in Ecology and the Envi-

dian Journal of Fisheries and Aquatic Sciences 53, 2310–2322. ronment 4, 75–79.

Taylor, F.J.R., Haigh, R., Sutherland, T.F., 1994. Phytoplankton ecology of Sechelt Washington Department of Fish and Wildlife, 2004. Scientists Investigate Marine

Inlet, British Columbia. 11. Potentially harmful species. Marine Ecology Progress Toxin Plaguing Razor Clam Fishery. Fish and Wildlife Science, In: http://

Series 103, 151–164. wdfw.wa.gov/science/articles/razor_clams/index.html.

A.J. Lewitus et al. / Harmful Algae 19 (2012) 133–159 159

Weber, R.J., 2000. The detection of diarrhetic shellﬁsh toxins in Monterey Bay, CA. Work, T.M., Beale, M.A., Fritz, L., Quilliam, M.A., Silver, M., Buck, K., Wright, J.L.C.,

Masters Thesis in Marine Sciences. University of California at Santa Cruz. 1993. Domoic acid intoxication of brown pelicans and cormorants in Santa Cruz,

Wekell, J.C., Gauglitz Jr., E.J., Barnett, H.J., Hatﬁeld, C.L., Eklund, M., 1994. The California. In: Smayda, T.J., Shimizu, Y. (Eds.), Toxic Phytoplankton Blooms in

occurrence of domoic acid in razor clams (Siliqua patula), Dungeness crab the Sea. Elsevier Science Publishers B.V., pp. 643–649.

(Cancer magister), and anchovies (Engraulis mordax). Journal of Shellﬁsh Re- Wright, B.A., RaLonde, R., Gofman, V., Harmon, M., Khanna, K., 2008. Response and

search 13, 587–593. intervention system for climate change induced paralytic shellﬁsh poisoning

Wekell, J.C., Trainer, V.L., 2000. The cost of harmful algal blooms on the West Coast. (PSP) in Aleut communities (paralytic shellﬁsh poison monitoring in Aleut

Red Tides Newsletter, 2nd ed., Autumn 2000. region). North Paciﬁc Research Board Project 644 Final Report. Anchorage,

Wekell, J.C., Trainer, V.L., 2002. Harmful algal blooms and their impacts on West AK, 167 pp.

Coast Tribes. Red Tides Newsletter, 3rd ed., Autumn 2002. Yan, T., Shou, M., Tan, Z., Li, J., Yu, R., Wang, Y., 2003. A survey for paralytic shellﬁsh

Wells, M.L., Trick, C.G., Cochlan, W.P., Hughes, M.P., Trainer, V.L., 2005. Domoic acid: poisoning (PSP) in Vancouver Harbour. Marine Environmental Research 57,

the synergy of iron, copper, and the toxicity of diatoms. Limnology and 137–143.

Oceanography 50, 1908–1917. Yang, C.Z., Albright, L.J., Yousif, A.N., 1995. Oxygen radical-mediated effects of the

Whitehead, J.C., Haab, T.C., Parsons, G.R., 2003. Economic effects of Pﬁesteria. Ocean toxic phytoplankton Heterosigma carterae on juvenile rainbow trout Oncor-

& Coastal Management 46, 845–858. hynchus mykiss. Diseases of Aquatic Organisms 23, 101–108.

Whyte, I., Haigh, N., Ginther, N.G., Keddy, L.J., 2001. First record of blooms of Zertuche-Gonza´lez, J., Camacho-Ibar, V., Pacheco-Ruı´z, I., Cabello-Pasini, A.,

Cochlodinium sp. (Gymnodiniales, Dinophyceae) causing mortality of net-pen Galindo-Bect, L., Guzma´n-Caldero´ n, J., Macias-Carranza, V., Espinoza-Avalos,

reared salmon on the west coast of Canada. Phycologia 40, 298–304. J., 2009. The role of Ulva spp. as a temporary nutrient sink in a coastal lagoon

Wilkerson, F.P., Dugdale, R.C., Hogue, V.E., Marchi, A., 2006. Phytoplankton blooms with oyster cultivation and upwelling inﬂuence. Journal of Applied Phycology

and nitrogen productivity in the San Francisco Bay. Estuaries Coasts 29, 401– 21, 729–736.

416. Zhang, Y., Fu, F-X., Whereat, E., Coyne, K.J., Hutchins, D.A., 2006. Bottom-up controls

Wing, S.R., Botsford, L.W., Ralston, S.V., Largier, J.L., 1998. Meroplanktonic distribu- on a mixed-species HAB assemblage: a comparison of sympatric Chattonella

tion and circulation in a coastal retention zone of the northern California subsalsa and Heterosigma akashiwo (Raphidophytes) isolates from the Delaware

upwelling system. Limnology and Oceanography 43, 1710–1721. Inland Bays. Harmful Algae 5, 310–320.

Harmful Algae 23 (2013) 28–33

Contents lists available at SciVerse ScienceDirect

Harmful Algae

jo urnal homepage: www.elsevier.com/locate/hal

Domoic acid exposure and associated clinical signs and histopathology in

Paciﬁc harbor seals (Phoca vitulina richardii)

a b, c d

Elizabeth A. McHuron , Denise J. Greig *, Kathleen M. Colegrove , Michelle Fleetwood ,

e b a f f

Terry R. Spraker , Frances M.D. Gulland , James T. Harvey , Kathi A. Lefebvre , Elizabeth R. Frame

Moss Landing Marine Laboratories, 8272 Moss Landing Road, Moss Landing, CA 95039, USA

The Marine Mammal Center, 2000 Bunker Road, Fort Cronkhite, Sausalito, CA 94965, USA

Zoological Pathology Program, College of Veterinary Medicine at Urbana-Champaign, LUMC, Bld. 101, Room 0745, 2160 South First Avenue, Maywood, IL 60153, USA

New Hampshire Veterinary Diagnostic Lab, University of New Hampshire, 319 Kendall Hall, 129 Main Street, Durham, NH 03824, USA

Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80526, USA

Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd,

East, Seattle, WA 98112, USA

A R T I C L E I N F O A B S T R A C T

Article history: Domoic acid (DA) is a potent neurotoxin that has caused strandings and mortality of seabirds and marine

Received 27 June 2012

mammals off the California coast. Paciﬁc harbor seals (Phoca vitulina richardii) are an abundant,

Received in revised form 21 December 2012

nearshore species in California; however, DA exposure and toxicosis have not been documented for

Accepted 21 December 2012

harbor seals in this region. To investigate DA exposure in harbor seals, samples were collected from free-

Available online 26 January 2013

ranging and stranded seals off California to assess exposure, clinical signs of toxicosis, and brain lesions

in harbor seals exposed to DA. Domoic acid was detected in 65% (17/26) of urine samples collected from

Keywords:

apparently healthy free-ranging seals, with concentrations of 0.4–11.7 ng/ml. Domoic acid also was

Harmful algal bloom

detected in feces (2.4–2887 ng/g), stomach contents (1.4 ng/g; stranded only), milk (2.2 ng/ml; stranded

Domoic acid

only), amniotic ﬂuid (9.7 ng/ml; free-ranging only), fetal meconium (14.6–39.8 ng/g), and fetal urine

Neurological signs

Mortality (2.0–10.2 ng/ml). Clinical signs indicative of DA toxicosis were observed in two live-stranded seals, and

Pseudo-nitzschia included disorientation, seizures, and uncoordinated movements. Histopathology revealed the presence

Phoca vitulina of brain lesions consistent with DA toxicosis in two live-stranded seals, and one free-ranging seal that

died during capture. Results indicated that harbor seals were exposed to DA, exhibited clinical signs and

histological lesions associated with DA exposure, and that pups were exposed to DA in utero and during

lactation via milk. Future investigation is required to determine the magnitude of impact that DA has on

the health and mortality of harbor seals.

1. Introduction et al., 1993), and marine mammals (Scholin et al., 2000). As

harmful algal blooms are increasing in frequency worldwide (Van

Domoic acid (DA) is a potent neurotoxin produced by some Dolah, 2000), there is an increasing potential for DA poisoning to

diatom species of the genus Pseudo-nitzschia. It is an analog of the impact humans and marine wildlife.

excitatory amino acid glutamate, and is known to affect the Along the California coast, DA poisoning was ﬁrst reported in

gastrointestinal, cardiac, and neurological systems in mammals Brown Pelicans (Pelecanus occidentalis) and Brandt’s Cormorants

and birds through binding to glutamate receptors on cell (Phalacrocorax penicillatus) in Monterey Bay in 1991 (Fritz et al.,

membranes (Bejarano et al., 2008a,b; Costa et al., 2010). Naturally 1992; Work et al., 1993). Since 1991, DA has been the causative

occurring DA poisoning has been observed in humans (amnesic agent in unusual mortality events of California sea lions (Zalophus

shellﬁsh poisoning; Perl et al., 1990), birds (Fritz et al., 1992; Work californianus; Lefebvre et al., 1999; Scholin et al., 2000; Torres de la

Riva et al., 2009), and dolphin species (Torres de la Riva et al.,

2009). Domoic acid toxicosis has been relatively well-studied in

* Corresponding author at: 1436 La Playa Street, San Francisco, CA, USA. California sea lions because DA-related strandings have occurred

Tel.: +1 415 608 8490; fax: +1 415 754 4043. almost annually since 1998, and live-stranded sea lions are

E-mail addresses: [email protected] (E.A. McHuron), [email protected]

accessible for observation and sampling (Bejarano et al., 2008a,b).

(D.J. Greig), [email protected] (K.M. Colegrove), [email protected]

Effects of DA poisoning in California sea lions include neurological

(M. Fleetwood), [email protected] (T.R. Spraker), [email protected]

(Frances M.D. Gulland), [email protected] (J.T. Harvey), symptoms (seizures, head weaving, ataxia, depression, and

[email protected] (K.A. Lefebvre), [email protected] (E.R. Frame). abnormal scratching) associated with brain lesions, reproductive

E.A. McHuron et al. / Harmful Algae 23 (2013) 28–33 29

failure, and degenerative cardiomyopathy (Brodie et al., 2006;

Goldstein et al., 2008, 2009; Zabka et al., 2009).

Domoic acid exposure has not been well-documented in

marine mammals other than California sea lions found along the

California coast, and links between exposure and abnormal

clinical signs and histopathology are limited. Exposure mainly

occurs via ingestion of contaminated food, and potential vectors

include northern anchovies (Engraulis mordax; Lefebvre et al.,

1999), krill (Bargu et al., 2002), and other pelagic and benthic

species (Lefebvre et al., 2002; Kvitek et al., 2008), although in

utero exposure and lactational exposure via milk also are possible

(Brodie et al., 2006). Between 2005 and 2009, approximately half

of northern fur seals (Callorhinus ursinus) that stranded alive along

the central California coast exhibited classic clinical signs of DA

toxicosis (Lefebvre et al., 2010). Domoic acid was identiﬁed as the

primary cause of death in four stranded southern sea otters

(Enhydra lutris nereis), and may be a cause of cardiac disease in this

species (Kreuder et al., 2003, 2005). Domoic acid also was

detected in humpback (Megaptera novaeangliae) and blue

(Balaenoptera musculus) whale fecal samples from Monterey

Bay (Lefebvre et al., 2002), and a stranded minke whale

(Balaenoptera acutorostrata) in southern California (Fire et al.,

2010). The lack of data for some species of marine mammals is

likely a combination of their distribution and life history

characteristics, as some species occur primarily offshore, or

spend their entire life at sea.

The Paciﬁc harbor seal (Phoca vitulina richardii) is a small

phocid that inhabits nearshore environments along the west

coast of North America, and is widely distributed along the

mainland coast, islands, and bays of California. Harbor seals are

opportunistic predators that primarily feed on benthic and

schooling ﬁshes (Harvey et al., 1995; Orr et al., 2004). Despite Fig. 1. Location of free-ranging (circles), died during capture (triangles), and

the fact that harbor seals forage on similar prey and their stranded (squares) harbor seals sampled off central and northern California

between 2009 and 2011. Size of the symbol (regardless of shape) corresponds to the

habitat overlaps with California sea lions, there are no published

number of individuals sampled at any given location.

data on DA exposure or toxicosis in Paciﬁc harbor seals. Hall

and Frame (2010) found that harbor seals in Scotland were

exposed to DA, and suggested that this toxin may play a role in and fetal urine were collected, whereas urine and bile were

regulating their populations. Given that annual DA-producing collected from the two seals from Humboldt Bay.

harmful algal blooms in California coastal waters negatively Samples were collected from ﬁve harbor seals that stranded

impact California sea lions (Bejarano et al., 2008a,b), DA also along the central California coast and were admitted to The Marine

may negatively affect health and cause mortality of harbor seals Mammal Center (TMMC) between 2009 and 2011 (Fig. 1). All seals

in this region. died within 0–48 h of admission, and samples were collected at

The primary objectives of this study were to (1) document DA necropsy for histopathology and DA analysis. All samples for

exposure in free-ranging and stranded harbor seals along the histopathology were ﬁxed in 10% formalin, processed routinely for

California coast, and (2) describe the clinical signs and histopa- histopathology, and stained with H & E before examination.

thology ﬁndings in harbor seals exposed to DA.

2.2. Extraction and quantiﬁcation of domoic acid

2. Methods

Samples analyzed for DA were extracted in 50% aqueous

2.1. Sample collection methanol at a ratio of 4 ml 50% methanol to 1 g sample. Fecal and

stomach content extracts were homogenized for at least 60 s, and

Between June and July 2011, apparently healthy harbor seals urine, milk, and amniotic ﬂuid were sonicated for at least 45 s.

were captured in San Francisco Bay (n = 13), Tomales Bay (n = 8), Extracts were then centrifuged at 10,000 Â g for 20 min at 4 8C. The

and Humboldt Bay (n = 5) with salmon nets, tangle nets, or a supernatant was ﬁltered and samples were stored at 4 8C until

modiﬁed beach seine net (Jeffries et al., 1993; Fig. 1). Animals were analysis, which was typically 1–3 days after extraction. Domoic

sedated with IV diazepam to facilitate handling and sampling (dose acid quantiﬁcation was carried out using Biosense ELISA

rate of 0.2 mg/kg), and urine samples were collected from female (Biosense Laboratories, Bergen, Norway) according to the

seals via free-catch or urethral insertion of a urinary catheter. Age manufacturer’s instructions, with modiﬁcations to the minimum

class was determined based upon length and mass (Bigg, 1969), extract dilutions to avoid matrix effects.

body condition, and date of capture.

Additional samples were collected from three adult female 3. Results

seals that died during capture events in 2010 (n = 1; Elkhorn

Slough) and 2011 (n = 2 of the 5 seals mentioned above; Humboldt 3.1. Domoic acid levels

Bay; Fig. 1). Full necropsies were performed and tissues collected

for histopathology and DA analysis. The animal from Elkhorn Domoic acid was detected in 65% (17/26) of urine samples

Slough was pregnant, and feces, amniotic ﬂuid, fetal meconium, collected from free-ranging seals, with concentrations ranging

30 E.A. McHuron et al. / Harmful Algae 23 (2013) 28–33

urine level (0.8 ng/ml) in this animal. Bile samples (n = 4) did not

contain DA, despite detectable DA in feces of three of these

animals.

3.2. Clinical signs and histopathology

Clinical signs associated with DA in body ﬂuids included

disorientation, seizures, and uncoordinated movements (Table 1).

Histopathology revealed the presence of brain lesions consistent

with DA toxicosis in two live-stranded seals that died following

stranding (HS 2120 and HS 2171), and one free-ranging seal (WHS-

2). HS 2120 had bilateral hippocampal neuronal necrosis primarily

in CA2 through CA4 regions, with lesser involvement of CA1

(Fig. 3A). Other ﬁndings included exudate in the ear canal

associated with ﬁbrinosuppurative meningitis with Gram positive

rods observed on histology, necrosuppurative bronchopneumonia

with Gram negative rods, and rare small foci of myocardial

necrosis. HS 2171 had bilateral hippocampal neuronal loss in CA3

Fig. 2. Number of free-ranging harbor seals (n = 26) with domoic acid (DA; ng/g) in

and CA4 and moderate lymphocytic meningitis. In the left ventricle

urine captured in San Francisco Bay (white), Tomales Bay (gray), and Humboldt Bay

(black) between June and July 2011. Urine from nine seals was below the detectable and septum of the heart, there were rare small foci of subacute

limit (bdl). myocardial necrosis and rare small foci of myocardial ﬁbrosis

(Fig. 3B). WHS-2 had edema and mild neuronal necrosis in the

hippocampus that was most pronounced in the CA3 and CA4

from 0.4 to 11.7 ng/g (Fig. 2). All individuals from Tomales Bay had regions of the hippocampus, and hemorrhage within the brain

detectable levels of DA (8/8), whereas DA was detected only in 46% stem and cerebrum.

(6/13) of seals from San Francisco Bay, and 60% (3/5) of seals from

Humboldt Bay. Domoic acid was not detected in either bile sample 4. Discussion

collected from the two seals that died in Humboldt Bay, despite

detectable levels in urine. To our knowledge, this is the ﬁrst report of DA exposure in

All samples collected from the pregnant seal and her fetus that harbor seals along the west coast of North America, and the ﬁrst

died during capture in Elkhorn Slough (WHS-2 and WHS-2F) tested evidence that harbor seals exhibit clinical signs and histological

positive for DA. The concentration of DA was greatest in feces lesions associated with DA exposure. The detection of DA in

(2887 ng/g), followed by fetal meconium (39.8 ng/g), fetal urine samples from seals in this study likely represented recent exposure

(10.2 ng/ml), and amniotic ﬂuid (9.7 ng/ml). Necropsy revealed (days to weeks) because of relatively short prey retention times

extensive congestion and acute hemorrhage within the brain, and (mean retention time 30 h; Trumble et al., 2003), and because DA

severe acute congestion and hemorrhage with pulmonary edema was rapidly excreted in urine (120 min; intravenous exposure;

in the lungs. The cause of death was likely associated with trauma Suzuki and Hierlihy, 1993) and feces (0–48 h; oral exposure;

to the head of unknown origin and DA toxicosis. Iverson et al., 1989) of laboratory animals following exposure.

Samples were collected from ﬁve stranded harbor seals: four Differences in DA levels among individuals likely were a

stranded alive and one was dead at stranding (premature carcass; consequence of the time between exposure and sampling, the

Table 1). Levels of DA were greatest in fecal samples (1.4–275 ng/ type of sample tested, and the dose ingested.

g), followed by urine (1.8–2.4 ng/ml) and stomach contents Toxic Pseudo-nitzschia spp blooms occur off the California coast

(1.4 ng/g; Table 1). Domoic acid was detected in the one milk every year and DA is routinely detected in seafood species (Lewitus

sample collected (2.2 ng/ml), and the level was greater than the et al., 2012). The detection of DA in samples from free-ranging

Fig. 3. (A) HS 2120 Numerous dead shrunken neurons in the CA2 hippocampal region (examples indicated with arrows), HE (HS 2120). Bar = 50 mm and (B) area of myocardial

necrosis with dead fragmented muscle cells and macrophages, HE (HS 2171). Bar = 50 mm.

E.A. McHuron et al. / Harmful Algae 23 (2013) 28–33 31

Table 1

Domoic acid (DA) concentrations (ng/g or ng/ml for ﬂuids), clinical signs, and pathologic lesions in harbor seals that were examined post mortem. A combination of feces, bile,

urine, aqueous humor (aqueous), stomach contents (stomach), amniotic ﬂuid (amniotic), and milk were collected from male (M) and female (F), fetus, pup (P), subadult (SA),

and adult (A) harbor seals.

Seal ID Sex Age Samples DA Clinical signs Histopathology

HS 1988 F A Feces 2.4 Dull, lethargic Nonsuppurative meningoencephalitis, neuronal

degeneration and edema in hippocampus

HS 1992 F SA Feces 8.2 Disoriented, seizures Fungal encephalitis, myocardial edema

Bile bdl

Urine 1.8

Aqueous bdl

HS 1994 F Fetus Feces 14.6 Premature carcass Brain edema

Bile bdl

Urine 2.0

Stomach 1.4

HS 2120 M A Feces 275 Paralysis, seizures Fibrinosuppurative meningitis with Gram positive rods,

Bile bdl hippocampal neuronal necrosis in CA1-CA4, rare

Urine bdl myocardial necrosis

HS 2171 F A Feces bdl Struggling in the surf, dazed Bilateral hippocampal neuronal loss, lymphocytic

Urine 0.8 and frothing at the mouth meningitis and rare mycocardial necrosis

Bile bdl

Milk 2.2

Pv-062211 F A Urine 0.5 None No signiﬁcant ﬁndings

Bile bdl

Pv-062311 F A Urine 0.4 None No signiﬁcant ﬁndings

Bile bdl

WHS-2 F A Feces 2887 None Neuronal necrosis in hippocampus, cerebral hemorrhage

Amniotic 9.7

WHS-2F M Fetus Feces 39.8 In utero None

Urine 10.2

harbor seals, including WHS-2, did not coincide with the detection it is unclear how exposure levels relate to acute or chronic DA

of DA in any shellﬁsh samples tested by the California Department toxicosis. Although the CA2 hippocampal region is usually not

of Public Health from northern or central California around the affected in sea lions with DA toxicosis (Silvagni et al., 2005), one

time seals were captured (California Department of Public Health, harbor seal in this study did have neuronal necrosis in this region.

http://www.cdph.ca.gov/HealthInfo/environhealth/water/Pages/ Two seals had small areas of myocardial necrosis, which has been

Shellﬁshreports.aspx). Despite this, Psuedo-nitzschia was abundant described in sea lions with DA toxicosis (Silvagni et al., 2005; Zabka

(>50% toxin producing phytoplankton) or present (1–10%) in et al., 2009); however, one of these seals (HS 2120) also had

water samples from the Monterey and San Francisco Bay areas bacterial bronchopneumonia, otitis, and meningitis that could

during the month of and month proceeding collection of samples have resulted in septicemia and hence the myocardial necrosis.

from harbor seals. Domoic acid was detected in one shellﬁsh Exposure to DA not only affects the health of the individual, but

sample from the Santa Cruz Pier during the same month that HS may cause reproductive failure or lasting neurological and

2120 stranded with paralysis and seizures; however, was not behavioral effects to pups exposed in utero or during lactation.

detected in central California around the same time when HS 2171 Maternal transfer of DA has been reported in rats (Maucher and

stranded with symptoms of DA toxicosis. Bloom and toxin data are Ramsdell, 2007) and California sea lions (Brodie et al., 2006;

not always spatially or temporally coincident with stranding data, Goldstein et al., 2009), but this was the ﬁrst report of

but sea lions are likely exposed to DA in their prey throughout the transplacental transfer of DA in harbor seals. The effects of

year (Bargu et al., 2012). We do not have the density of data from prenatal exposure of marine mammals to DA have not been well-

harbor seals that exists for stranded sea lions, but suspect this is studied. Brain edema was the primary lesion found in aborted

true for harbor seals as well. California sea lion pups with measurable DA concentrations,

Free-ranging seals in this study had DA concentrations in urine although the pathogenesis of the brain edema remains unknown

similar to those found in seals from Scotland (Hall and Frame, (Goldstein et al., 2009). Brodie et al. (2006) found that maternal

2010), but concentrations were generally less than those reported levels of DA in urine of California sea lions were at least an order of

for California sea lions that stranded with acute symptoms of DA magnitude greater than levels in fetal urine. Domoic acid was

toxicosis (10–3720 ng/ml; Goldstein et al., 2008). Domoic acid detected in fetal ﬂuids up to a week after the initial stranding,

levels within the range associated with acute toxicosis in sea lions indicating that fetuses were acting as a reservoir for DA (Brodie

were detected in one seal, and the majority of seals had DA et al., 2006). The effects of prenatal DA exposure have been

concentrations in urine within the range associated with chronic investigated in laboratory species, and resulted in hippocampal

effects in California sea lions (2–11 ng/ml; Goldstein et al., 2008). damage, seizure disorders, and persistent behavioral changes

The exposure of harbor seals to DA was not surprising given that (Dakshinamurti et al., 1993; Levin et al., 2005; Tiedeken et al.,

the habitats of harbor seals and California sea lions overlap, and 2005). Postnatal exposure to DA also caused neurobehavioral

that northern anchovies, a DA vector, are an important component changes, but changes were less pervasive than those reported from

of harbor seal diet in this region (Gibble, 2011). prenatal exposure (Levin et al., 2006). In our study, the level of DA

Clinical signs and histological lesions consistent with DA in fetal meconium from WHS-2F was less than in maternal feces,

toxicosis were observed in several seals in this study; however, and the DA level in the fetal urine was similar to levels detected in

32 E.A. McHuron et al. / Harmful Algae 23 (2013) 28–33

Dakshinamurti, K., Sharma, S.K., Sundaram, M., Watanabe, T., 1993. Hippocampal

the urine of California sea lion fetuses that were aborted as a result

changes in developing postnatal mice following intrauterine exposure to

of maternal DA exposure (Brodie et al., 2006). Detection of DA in

domoic acid. Journal of Neuroscience 13, 4486–4495.

amniotic ﬂuid, fetal urine and meconium, and milk indicate that Fire, S.E., Wang, Z., Berman, M., Langlois, G.W., Morton, S.L., Sekula-Wood, E.,

Benitez-Nelson, C.R., 2010. Trophic transfer of the harmful algal toxin domoic

harbor seal pups can be exposed to DA during gestation and

acid as a cause of death in a minke whale (Balaenoptera acutorostrata) stranding

lactation. The effects of this exposure on female reproductive

in southern California. Aquatic Mammals 36, 342–350.

success, and pup development and survival are unknown. Fritz, L., Quilliam, M.A., Wright, J.L.C., Beale, A.M., Work, T.M., 1992. An outbreak of

domoic acid poisoning attributed to the pennate diatom Pseudonitzschia aus-

The apparent increase in DA-producing blooms off California in

tralis. Journal of Phycology 38, 439–442.

recent years highlights the need to understand the role of DA

Gibble, C., 2011. Food Habits of Harbor Seals (Phoca vitulina richardii) in San

toxicosis in the health and population dynamics of marine Francisco Bay, California. M.S. Thesis. San Jose State University. 61 pp.

mammals in this region. Whereas exposure and toxicosis have Goldstein, T., Mazet, J.A.K., Zabka, T.S., Langlois, G., Colegrove, K.M., Silver, M., Bargu,

S., Van Dolah, F., Leighﬁeld, T., Conrad, P.A., Barakos, J., Williams, D.C., Dennison,

been relatively well-studied in California sea lions, this was the

S., Haulena, M., Gulland, F.M.D., 2008. Novel symptomatology and changing

ﬁrst report of exposure in harbor seals from this region. Harbor

epidemiology of domoic acid toxicosis in California sea lions (Zalophus califor-

seals were exposed to levels great enough to cause acute and nianus): an increasing risk to marine mammal health. Proceedings of the Royal

Society B: Biological Sciences 275, 267–276.

chronic effects, and exhibited clinical symptoms similar to those

Goldstein, T., Zabka, T.S., DeLong, R.L., Wheeler, E.A., Ylitalo, G., Bargu, S., Silver, M.,

observed in other species. The presence of mid hippocampal

Leighﬁeld, T., Van Dolah, F., Langlois, G., Sidor, I., Dunn, J.L., Gulland, F.M.D.,

lesions associated with DA toxicosis indicates that this region 2009. The role of domoic acid in abortion and premature parturition of

California sea lions (Zalophus californianus) on San Miguel Island, California.

should be examined in suspected cases of DA toxicosis in harbor

Journal of Wildlife Diseases 45 (1), 91–108.

seals. Domoic acid toxicosis may be a cause of mortality in harbor

Hall, A.J., Frame, E., 2010. Evidence of domoic acid exposure in harbour seals from

seals and a contributing cause of stranding, but the magnitude of Scotland: a potential factor in the decline in abundance? Harmful Algae 9, 489–

493.

the impact remains unknown. Because adult and juvenile harbor

Harvey, J.T., Helm, R.C., Morejohn, G.V., 1995. Food habits of harbor seals inhabiting

seals rarely strand, examining exposure in free-ranging seals, and

Elkhorn Slough, CA. California Fish and Game 81, 1–9.

live- and dead-stranded pups may be a better indication of the role Iverson, F., Truelove, J., Nera, E., Tryphonas, L., Campbell, J., Lok, E., 1989. Domoic

acid poisoning and mussel-associated intoxication: preliminary investigation

that DA plays in the health and mortality of harbor seals off

into the response of mice and rats to toxic mussel extract. Food and Chemical

California. The type of sample used to investigate DA effects in

Toxicology 27, 377–384.

harbor seals will vary with sample collection logistics and project Jeffries, S.J., Brown, R.F., Harvey, J.T., 1993. Techniques for capturing, handling, and

goals. We recommend testing urine and feces whenever practical. marking harbour seals. Aquatic Mammals 19, 21–25.

Kreuder, C., Miller, M.A., Jessup, D.A., Lowenstine, L.J., Harris, M.D., Ames, J.A.,

Feces may be the most appropriate sample for the detection of DA

Carpenter, T.E., Conrad, P.A., Mazet, J.A.K., 2003. Patterns of mortality in

when using less sensitive detection techniques (Lefebvre et al.,

southern sea otters (Enhydra lutris nereis) from 1998–2001. Journal of Wildlife

2010) because it is relatively easy to collect from stranded animals Diseases 39, 495–509.

Kreuder, C., Miller, M.A., Lowenstine, L.J., Conrad, P.A., Carpenter, T.E., Jessup, D.A.,

and DA levels are greater in feces than blood or urine. Urine is

Mazet, J.A.K., 2005. Evaluation of cardiac lesions and risk factors associated with

sometimes easier to collect from free-ranging harbor seals, and

myocarditis and dilated cardiomyopathy in southern sea otters (Enhydra lutris

may maximize the chances of detecting DA exposure because nereis). American Journal of Veterinary Research 66, 289–299.

Kvitek, R.G., Goldber, J.D., Smith, G.J., Doucette, G.J., Silver, M.W., 2008. Domoic acid

excretion of DA in feces may occur more rapidly as a result of toxin-

contamination within eight representative species from the benthic food web of

induced diarrhea (Lefebvre et al., 1999).

Monterey Bay, California, USA. Marine Ecology Progress Series 367, 35–47.

Lefebvre, K.A., Bargu, S., Kieckhefer, T., Silver, M.W., 2002. From sanddabs to blue

Acknowledgements whales: the pervasiveness of domoic acid. Toxicon 40, 971–977.

Lefebvre, K.A., Powell, C.L., Busman, M., Doucette, G.J., Moeller, P.D.R., Silver, J.B.,

Miller, P.E., Hughes, M.P., Singaram, S., Silver, M.W., Tjeerdema, R.S., 1999.

Detection of domoic acid in northern anchovies and California sea lions associ-

We thank the volunteers that assisted in harbor seal captures, the

ated with an unusual mortality event. Natural Toxins 7, 85–92.

staff and volunteers at TMMC, the National Parks Service, US Fish and

Lefebvre, K.A., Roberston, A., Frame, E.R., Colegrove, K.M., Nance, S., Baugh, K.A.,

Wildlife Service, Humboldt Bay NWR Complex, and Don Edwards Wiedenhoft, H., Gulland, F.M.D., 2010. Clinical signs and histopathology asso-

National Wildlife Refuge. We also thank Preston Kendrick for ciated with domoic acid poisoning in northern fur seals (Callorhinus ursinus) and

comparison of toxin detection methods. Harmful Algae 9, 374–383.

assisting with domoic acid analyses. This study was conducted

Levin, E.D., Pizarro, K., Pang, W.G., Harrison, J., Ramsdell, J.S., 2005. Persisting

under permits from the National Marine Fisheries Service (no. 555-

behavioral consequences of prenatal domoic acid exposure in rats. Neurotox-

1870), National Parks Service (PORE-2011-SCI-0003), Don Edwards icology and Teratology 27, 719–725.

Levin, E.D., Pang, W.G., Harrison, J., Williams, P., Petro, A., Ramsdell, J.S., 2006.

National Wildlife Refuge (no. 81640-2011-002), Humboldt Bay NWR

Persistent neurobehavioral effects of early postnatal domoic acid exposure in

Complex (no. 81590-10007), and a Stranding Agreement from The

rats. Neurotoxicology and Teratology 28, 673–680.

Marine Mammal Health and Stranding Response Program. [SS] Lewitus, A.J., Horner, R.A., Caron, D.A., Garcia-Mendoza, E., Hickey, B.M., Hunter, M.,

Huppert, D.D., Kudela, R.M., Langlois, G.W., Largier, J.L., Lessard, E.J., RaLonde, R.,

Rensel, J.E.J., Strutton, P.G., Trainer, V.L., Tweddle, J.F., 2012. Harmful algal

References

blooms along the North American west coast region: history, trends, causes,

and impacts. Harmful Algae 19, 133–159.

Bargu, S., Powell, C.L., Coale, S.L., Busman, M., Doucette, G.J., Silver, M.W., 2002. Krill: Maucher, J.M., Ramsdell, J.S., 2007. Maternal–fetal transfer of domoic acid in rats at

a potential vector for domoic acid in marine food webs. Marine Ecology Progress two gestational time points. Environmental Health Perspectives 115, 1743–1746.

Series 237, 209–216. Orr, A.J., Banks, A.S., Mellman, S., Huber, H.R., DeLong, R.L., Brown, R.F., 2004.

Bargu, S., Goldstein, T., Roberts, K., Li, C., Gulland, F., 2012. Pseudo-nitzschia blooms, Examination of the foraging habits of Paciﬁc harbor seal (Phoca vitulina

domoic acid, and related California sea lion strandings in Monterey Bay, richardsi) to describe their use of the Umpqua River, Oregon, and their predation

California. Marine Mammal Science 28, 237–253. on salmonids. Fishery Bulletin 102, 108–117.

Bejarano, A.C., Gulland, F.M., Goldstein, T., St. Leger, J., Hunter, M., Schwacke, L.H., Perl, T.M., Be´dard, L., Kosatsky, T., Hockin, J.C., Todd, E.C.D., Remis, R.S., 1990. An

Van Dolah, F.M., Rowles, T.K., 2008a. Demographics and spatio-temporal sig- outbreak of toxic encephalopathy caused by eating mussels contaminated with

nature of the biotoxin domoic acid in California sea lion (Zalophus californianus) domoic acid. The New England Journal of Medicine 322, 1775–1780.

stranding records. Marine Mammal Science 24, 899–912. Scholin, C.A., Gulland, F., Doucette, G.J., Benson, S., Busman, M., Chavez, F.P., Cordaro,

Bejarano, A.C., Van Dolah, F.M., Gulland, F.M., Rowles, T.K., Schwacke, L.H., 2008b. J., DeLong, R., De Vogelaere, A., Harvey, J., Haulena, M., Lefebvre, K., Lipscomb, T.,

Production and toxicity of the marine biotoxin domoic acid and its effects on Loscutoff, S., Lowenstine, L.J., Marin III, R., Miller, P.E., McLellan, W.A., Moeller,

wildlife: a review. Human and Ecological Risk Assessment 14, 544–567. P.D.R., Powell, C.L., Rowles, T., Silvagni, P., Silver, M., Spraker, T., Trainer, V., Van

Bigg, M.A., 1969. The harbour seal in British Columbia. Bulletin of the Fisheries Dolah, F.M., 2000. Mortality of sea lions along the central California coast linked

Research Board of Canada 172, 1–33. to a toxic diatom bloom. Nature 403, 80–84.

Brodie, E.C., Gulland, F.M.D., Greig, D.J., Hunter, M., Jaakola, J., St. Leger, J., Leighﬁeld, Suzuki, C.A.M., Hierlihy, S.L., 1993. Renal clearance of domoic acid in the rat. Food

T.A., Van Dolah, F.M., 2006. Domoic acid causes reproductive failure in Cali- and Chemical Toxicology 31, 701–706.

fornia sea lions (Zalophus californianus). Marine Mammal Science 22, 700–707. Silvagni, P.A., Lowenstine, L.J., Spraker, T., Lipscomb, T.P., Gulland, F.M., 2005.

Costa, L.G., Giordano, G., Faustman, E.M., 2010. Domoic acid as a developmental Pathology of domoic acid toxicity in California sea lions (Zalophus californianus).

neurotoxin. NeuroToxicology 31, 409–423. Veterinary Pathology 42, 184–191.

E.A. McHuron et al. / Harmful Algae 23 (2013) 28–33 33

Tiedeken, J.A., Ramsdell, J.S., Ramsdell, A.F., 2005. Developmental toxicity of Van Dolah, F.M., 2000. Marine algal toxins: origins, health effects, and their

domoic acid in zebraﬁsh (Danio rerio). Neurotoxicology and Teratology 27, increased occurrence. Environmental Health Perspectives 108, 133–141.

711–717. Work, T.M., Barr, B., Beale, A.M., Fritz, L., Quilliam, M.A., Wright, J.L.C., 1993.

Torres de la Riva, G., Kreuder Johnson, C., Gulland, F.M.D., Langlois, G.W., Heyning, Epidemiology of domoic acid poisoning in brown pelicans (Pelecanus occiden-

J.E., Rowles, T.K., Mazet, J.A.K., 2009. Association of an unusual marine mammal talis) and Brandt’s cormorants (Phalacrocorax penicillatus) in California. Journal

mortality event with Pseudo-nitzschia spp. blooms along the southern California of Zoo and Wildlife Medicine 24, 54–62.

coastline. Journal of Wildlife Diseases 45, 109–121. Zabka, T.S., Goldstein, T., Cross, C., Mueller, R.W., Kreuder-Johnson, C., Gill, S.,

Trumble, S.J., Barboza, P.S., Castellini, M.A., 2003. Digestive constraints on an aquatic Gulland, F.M.D., 2009. Characterization of a degenerative cardiomyopathy

carnivore: effects of feeding frequency and prey composition on harbor seals. associated with domoic acid toxicity in California sea lions (Zalophus califor-

Journal of Comparative Physiology B 173, 501–509. nianus). Veterinary Pathology 46, 105–119. Energy Policy 55 (2013) 699–705

Contents lists available at SciVerse ScienceDirect

Energy Policy

journal homepage: www.elsevier.com/locate/enpol

Communication The impact of water depth on safety and environmental performance in offshore oil and gas production

Lucija Muehlenbachs a,n, Mark A. Cohen a,b, Todd Gerarden c a Resources for the Future, 1616 P Street NW, Washington, DC 20036, USA b Vanderbilt University, 401—21st Avenue South, Nashville, TN 37205, USA c Harvard Kennedy School, 79 John F. Kennedy Street, Cambridge, MA 02138, USA

HIGHLIGHTS c Analysis of performance indicators for oil production platforms in Gulf of Mexico. c In recent years there have been dramatic increases in the water depths at which offshore oil and gas is extracted. c Self-reported incidents (e.g. blowouts, injuries, spills) increase with water depth. article info abstract

Article history: This paper reports on an empirical analysis of company-reported incidents on oil and gas production Received 19 March 2012 platforms in the Gulf of Mexico between 1996 and 2010. During these years, there was a dramatic Accepted 26 December 2012 increase in the water depths at which offshore oil and gas is extracted. Controlling for platform Available online 17 January 2013 characteristics such as age, quantity of oil and gas produced, and number of producing wells, we ﬁnd Keywords: that incidents (such as blowouts, injuries, and oil spills) are positively correlated with deeper water. Offshore oil and gas Controlling for these and other characteristics, for an average platform, each 100 feet of added depth Oil spills increases the probability of a company-reported incident by 8.5%. While further research into the Safety causal connections between water depth and platform risks is warranted, this study highlights the potential value of increased monitoring of deeper water platforms. & 2012 Elsevier Ltd. All rights reserved.

1. Introduction the Deepwater Horizon oil spill, this work analyzes company- reported incidents and focuses on the variation in outcomes In the aftermath of the Deepwater Horizon oil spill on April 20, across different water depths. 2010, government, industry, and the public sought explanations In the past, high-volume oil spills have been largely associated for the largest offshore oil spill in the history of the petroleum with crude oil transport. Accordingly, the risk analysis literature industry. The United States Government quickly took steps focuses on accidents associated with transport (Epple and intended to reduce the likelihood of a future oil spill of this Visscher, 1984; Stewart and Leschine, 1986; Cohen, 1986, 1987; magnitude by restructuring its regulatory institutions as well as Viladrich-Grau and Groves, 1997; Homan and Steiner, 2008). imposing new technology requirements on industry. However, the literature on offshore oil exploration and produc- To achieve the goal of increased safety through regulatory tion remains relatively underdeveloped. As a result, published modiﬁcations, it is imperative that policymakers and regulators ﬁndings from empirical analysis of offshore oil and gas activity incorporate knowledge of the risk factors contributing to inci- are sometimes inconsistent and even contradictory.1 Further- dents in offshore oil and gas production. To inform regulatory more, the literature to date has used data on incidents that policies, this paper provides an empirical analysis of industry occurred prior to 2000. However, since 2000 there have been activity on offshore production platforms on the Gulf of Mexico dramatic increases in the maximum water depth at which wells Outer Continental Shelf over the period 1996–2010. Motivated by can be drilled offshore. For example, in 2000 the deepest water at which a well was drilled was less than 4000 feet, but in 2010 this

n Corresponding author. Tel.: þ1 202 328 5010; fax: þ1 202 939 3460. E-mail addresses: [email protected] (L. Muehlenbachs), 1 For example, Shultz (1999) ﬁnds water depth decreases the likelihood of [email protected] (M.A. Cohen), incidents but Jablonowski (2007) does not; Iledare et al. (1997) ﬁnd age increases [email protected] (T. Gerarden). the likelihood of reported incidents, but Shultz (1999) does not.

0301-4215/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.enpol.2012.12.074 700 L. Muehlenbachs et al. / Energy Policy 55 (2013) 699–705 was 8000 feet (see Fig. 2). Currently the U.S. Department of the least six well completions or two pieces of production equip- Interior classifies ‘‘deep water’’ as deeper than 1000 feet and a ment). In the analysis here, following BOEMRE’s terminology, new classification of ‘‘ultra-deep,’’ for deeper than 5000 feet, has platforms in water depths less than 1000 feet are considered to be been introduced.2 Yet, for example, Jablonowski (2007) analyzes in shallow water; those between 1000 and 4999 feet are con- data through 1998 and thus considers production in water deeper sidered deepwater; and 5000 feet or more, ultra-deep water. than 400 feet as ‘‘deep water.’’ To our knowledge, no empirical Table 2 shows that platform characteristics vary by water depth: analysis of incidents during oil exploration and development has unlike platforms in shallow waters, all platforms producing from been conducted at water depths beyond 4000 feet. This paper waters between 1000 and 5000 feet are major complexes and are expands the existing literature on predictors of offshore oil manned 24 h a day. Platforms currently in deeper water are also production incidents with a focus on identifying and analyzing younger: platforms are on average 3.2 years old in waters deeper factors relevant to future regulatory decisions in a world of than 5000 feet, 10.03 years old in waters between 1000 and 5000 increasingly deep water oil exploration. feet, and 22.69 years old in water less than 1000 feet (Table 1). We find that platforms in deeper water tend to be younger, more complex, produce more, and are more likely to be active. 2.2. Annual production We also find that deep water platforms have a much higher probability of an incident (such as a spill, accident, or injury) Annual platform-level production and the number of wells reported. We also show that the depth of oil and gas production producing in each year were calculated from monthly well-level has made tremendous leaps over the years. Using recent observa- production data for all wells in the Gulf of Mexico from 1996 to tions of incidents, we find that self-reported incidents increase 2010.5 These data include monthly gas volume, monthly oil with water depth contrary to Jablonowski (2007) and Shultz volume, and days on production. Using information on the date (1999). that each well was drilled, we calculated the total number of Our estimation strategy does not demonstrate that there is a wells at a platform, the average length of time to drill the wells at causal link between water depth and incident or violations but we a platform, the average depth of the wells, and the number of do show that there are statistically significant relationships wells drilled in the year of the observation. In 2009, the average between the variables. In particular, we find that reported platform produced 387 million barrels of oil—ranging from zero incidents are positively related to water depth. In addition, we to 68,302 million barrels. The average shallow water platform find that production volumes, age, complexity, distance to shore, produced 86 million barrels, while the average deep water plat- prior violations as well as the number of platforms managed by form produced 7682 million barrels and the average ultra-deep the operator are all related to self-reported incidents, suggesting water platform produced over 20,000 million barrels in 2009 that enforcement agencies might consider these factors in design- (Table 1). ing targeted inspection programs. 2.3. Lease owners and designated lease operators

2. Background information on offshore platforms and A single lease can have many owners with different percen- production tages of ownership (working interests). A lease may also be divided into different aliquots, or portions, and each aliquot This section provides background information on offshore may have multiple working interests. Data on the lease owner- platforms and production in the Gulf of Mexico. Publicly available ship and the designated operator of a lease were obtained for each data were obtained from the Bureau of Ocean Energy Manage- year from 1996 to 2010.6 Ownership of a lease is on average ment, Regulation and Enforcement (‘‘BOEMRE’’) (Offshore Energy divided among slightly more than two companies, and ranges and Minerals Management, Gulf of Mexico OCS Region, Field from sole ownership to 27 companies. Table 2 shows that the Operations, BOEMRE, 2009)3 and include water depth, age, and average working interest in a lease is lower the deeper the water. measures of size and complexity of platform operations in At any point in time, however, there is only one designated addition to the volume of production by lease operator. operator of the aliquot. The average working interest of the lease operator ranges from 0% to 100% and, again, is lower at greater 2.1. Platform characteristics depth (Table 2). According to these data, there are 25,461 leases assigned in the Gulf of Mexico; however, only 2757 of these leases As of 2010, there were 3020 unique platform ‘‘complexes’’ are associated with platforms (see N in Table 2). remaining in the Gulf of Mexico.4 A platform complex may include more than one structure, and is kept in the government 2.4. Platform operators data set until the entire complex is abandoned or removed. The data set contains information such as the distance to shore, water A platform operator is typically the responsible party in the depth, lease number, location (area and block), whether personnel event of an oil spill. However, in some instances a platform ties in are on board 24 h per day, whether a platform has a heliport, and production from a subsea lease that is miles away and could be the number of beds in the living quarters. There is also an leased to a different operator. Under U.S. government regulations, indicator of whether the platform is considered a ‘‘major com- all three parties (the surface platform operator, the subsea lessee, plex’’ (defined as a platform that has at least one structure with at and the pipeline right-of-way holder) are required to show oil spill financial responsibility. The platform operator, as defined by 2 Throughout this paper we use the term ‘‘deeper’’ as a comparative term that BOEMRE, is either the lease holder or the party designated (and simply means ‘‘increase in water depth’’ while the terms shallow, deep, and ultra- deep are defined as less than 1000 feet, 1000–5000 feet, and greater than 5000 feet, respectively. 5 Monthly Production Data was obtained from: /http://www.data.boem.gov/ 3 BOEMRE was formerly known as the Minerals Management Service (MMS). homepg/pubinfo/freeasci/product/freeprod_ogora.aspS. In addition, borehole data On October 1, 2011, BOEMRE was reorganized and its enforcement arm became was obtained to match production data with platform characteristics: /http:// the Bureau of Safety and Environmental Enforcement (BSEE). www.data.boem.gov/homepg/data_center/well/well.aspS. 4 Platform Masters database and Platform Structures database, /http://www. 6 Lease Ownership & Operator Data, /http://www.data.boem.gov/homepg/ data.boem.gov/homepg/data_center/platform/platform.aspS. data_center/leasing/leasing.aspS. L. Muehlenbachs et al. / Energy Policy 55 (2013) 699–705 701

Table 1 Platform characteristics, by water depth.

Shallow Deep Ultra-deep

Mean s.d. Mean s.d. Mean s.d.

Age (years) 22.69 14.82 10.03 6.69 3.2 2.53 Distance to shore (miles) 33.02 29.09 91.97 45.15 130.4 42.35 Water depth (feet) 102.8 103.5 2900 1230 6427 945.2 Major complex (indicator) 0.48 0.50 1 0 0.9 0.32 Manned 24 h (indicator) 0.20 0.40 1 0 0.9 0.32 Beds in living quarters 13.08 13.49 75.21 48.38 90.88 87.1 Heliport present (indicator) 0.69 0.46 1 0 0.9 0.32 Cranes 0.78 0.72 2.361 0.80 2.25 0.89 Annual oil production (mbbl) 86.19 264.5 7682 9781 20,758 22,844 Annual gas production (mmcf) 906.3 2286 11,716 11,419 64,170 95,398 Inactive (indicator) 0.44 0.50 0 0 0.2 0.42 Producing wells 1.8 3.02 13.31 7.58 7.8 5.73 Wells drilled (2009) 0.48 0.74 0.42 0.62 2.5 1.73 Cumulative wells drilled 4.59 7.34 10.47 16.04 1.8 1.93 Well operations (2009) 1.48 1.02 1.59 1.23 3 2.45 Cumulative well operations 6.61 11.86 25.19 33.95 3.1 3.32 Avg. depth of wells (2009) (feet) 9672 3280 19,145 6573 18,399 2792 Avg. time to drill wells (2009) (days) 26.35 16.16 44.25 22.98 87.81 46.75

Observations 2970 36 10

Note: Data from 2009. mbbl¼1000 barrels; mmcf¼million cubic feet.

Table 2 Lease ownership and designated lease operators, by water depth.

Shallow Deep Ultra-deep

Mean s.d. Mean s.d. Mean s.d.

No. of owners 2.46 2.19 2.26 1.43 2.50 0.84 No. of aliquot portions 1.05 0.28 1 0 1 0 Mean working interest (%) 63.99 33.63 57.76 28.45 43.06 11.08 Min. working interest (%) 55.09 41.55 48.52 34.99 29.86 12.48 Max. working interest (%) 76.14 25.46 69.07 22.48 56.25 18.59 Lease operators working interest (%) 62.49 38.37 50.17 35.57 35.42 31.82

N 2712 39 6

Note: Data on leases with one or more platforms, in 2010.

Table 3 company. For example, Shell Offshore Inc. has 10 subsidiaries in Number of platforms for ﬁrms with deepwater operations, 2010. the Gulf of Mexico (for example, these include, Shell Consolidated Energy Resources Inc., Shell Deepwater Development Inc., Shell Parent company Platforms Oil Company). Thus, all of these subsidiaries were coded as Shell

Shallow Deep Ultra-deep Offshore, Inc. In 2010, 15 companies operated platforms in deep or ultra- ATP Oil & Gas Corporation 37 2 0 deep waters. Table 3 lists these companies with the number of Anadarko Petroleum Corporation 5 5 2 shallow, deep, and ultra-deep platforms they operated in 2010. BHP Billiton Petroleum (Americas) Inc. 3 3 0 These 15 companies, out of a total of 132 companies, operated BP Corporation North America Inc. 27 5 4 Chevron Corporation 352 3 1 29.6% of the platforms in the Gulf of Mexico in 2010. ConocoPhillips Company 0 1 0 Dynamic Offshore Resources NS, LLC 46 1 0 Eni US Operating Co. Inc. 24 2 1 3. Reported incidents Exxon Mobil Corporation 54 1 0 Helix Energy (Energy Resource 99 1 0 Technology GOM, Inc.) Operators and other permit holders are required to report all Hess Corporation 0 1 0 ‘‘incidents’’ to regulatory authorities.7 Prior to 2006, incidents Murphy Exploration & Production Company 0 2 1 were deﬁned to include all serious accidents, fatalities, injuries, Pisces Energy LLC 45 1 0 explosions, and ﬁres. The incident reporting regulations were Shell Offshore Inc. 12 6 1 8 W & T Offshore, Inc. 142 2 0 made more stringent in 2006, requiring companies to report not only serious incidents but also incidents that had the potential to be serious (these include any incident involving structural approved) to operate a portion of a given lease. The history of 7 Incident reporting statistics are available at: /http://www.boemre.gov/ platform operators, received from BOEMRE, was used to deter- incidents/index.htmS. mine the operator of a platform at time t. For purposes of this 8 30 CFR Part 250, Final Rule (FR 19640), Minerals Management Service, U.S. research, each platform operator was matched up with its parent Department of Interior. 702 L. Muehlenbachs et al. / Energy Policy 55 (2013) 699–705

600 Table 4 Annual averages of incidents reported on major complexes, manned 24 h a day by 500 water depth.

400 Shallow Deep Ultra-deep

300 Mean s.d. Mean s.d. Mean s.d.

200 Incident indicator 0.23 0.42 0.74 0.44 0.81 0.39 No. of incidents 0.34 0.78 1.96 2.29 2.47 2.45 Reported Incidents 100 No. of injuries 0.02 0.21 0.042 0.29 0.01 0.10 No. of fatalities 0.004 0.07 0.004 0.07 0 0 Explosion 0.004 0.06 0.003 0.05 0 0 0 Blowout 0.001 0.04 0.001 0.04 0 0 1995 1997 1999 2001 2003 2005 2007 2009 Equipment fail 0.07 0.25 0.22 0.42 0.18 0.39 Year Human error 0.06 0.23 0.18 0.39 0.18 0.39 Accident 0.20 0.40 0.72 0.45 0.79 0.41 Fig. 1. Annual non-weather related incidents reported on offshore production Spill 0.03 0.17 0.05 0.21 0.04 0.19 facilities (1995–2009). Spill volume (barrels) 26.51 159.8 17.54 30.76 0.95 0.36 Weather 0.03 0.18 0.053 0.22 0.05 0.21 Structural damage 0.0006 0.02 0 0 0 0 damage to a facility, injury that led to an evacuation or days away Crane 0.017 0.13 0.15 0.36 0.21 0.41 from work, or property damage exceeding $25,000). The increase Collision 0.002 0.05 0.0043 0.07 0 0 in incident reporting due to this change in regulations is apparent Well control (surface) 0.0001 0.01 0 0 0 0 in Fig. 1, which tracks non-weather related incidents reported on Well control (diverter) 0.0001 0.01 0 0 0 0 Slip, trip, fall 0.01 0.10 0.012 0.11 0.01 0.10 offshore facilities. Including weather related incidents in this Overboard drill fluid 0.0003 0.02 0.001 0.04 0 0 figure obscures the increase in reporting in 2006 (dotted line), Exploration 0.004 0.06 0.026 0.16 0.06 0.23 because 2008, a year with an active hurricane season, had an Development, production 0.18 0.39 0.66 0.47 0.75 0.43 overwhelming number of weather related incidents (576 of 1041 Observations 9976 695 105 reported incidents). Fig. 1 also does not include the year 2010 Note: Annual data from all major complexes that are manned 24 h a day from because we do not have a full 12 months of observations for 1995 to August 2010. Incident indicator equals one if there is one or more incident this year. reported on a platform in a given year and zero otherwise. Averages of the Between 1995 and August 2010, there were 6372 company- indicators of the type of incident (such as accident or spill) are displayed; reported incidents. Data on these incidents include an indicator indicators are zero when no incident occurred in a year. for whether the incident involved a blowout, vessel collision, fire, explosion, collision, injury, fatality, or pollution; whether it was deeper water. Incidents attributed to weather are slightly higher caused by completion equipment, equipment failure, develop- for deeper platforms; deeper platforms are also farther from ment or production operations, exploration operations, human shore, and could be more susceptible to bad weather. In the error, a slip or a trip or a fall, or weather; and whether it involved statistical analysis that follows we only consider non-weather cranes, structural damage, or overboard drilling fluid. Of these related incidents. incidents approximately 4600 were identified as occurring on a production platform (as opposed to a drilling rig, pipeline, etc.). Here we only examine those that occurred on the production 4. Determinants of reported incidents platform. Incidents are common: on average there were .085 incidents We seek to determine the relationship between platform per platform per year from 1995 to 2010. To compare summary characteristics and the probability that a non-weather related statistics of incidents across water depths, we restrict the sample incident is reported in a given calendar year. To do so, we to platforms that are major complexes, manned 24 h a day. Forty- estimate probit models where the dependent variable (Incident) eight percent of platforms in shallow water are categorized as equals 1 if one or more incidents are reported in year t on major complexes and 20% are manned 24 h a day, whereas all platform i and 0 otherwise: deep and ultra-deep platforms are both major complexes and PrðÞ¼Incident Prðb þb ðÞWater Depth þX b þW þy þ e Z0Þ manned 24 h a day. Therefore, only major complexes that are it 0 1 i it 2 D t it manned 24 h a day are used when comparing the shallow, deep, We are interested in estimating the coefficient on water depth and ultra-deep platforms using summary statistics (Table 4). while controlling for other platform and enforcement character-

As shown in Table 4, the average number of incidents for these istics; Xit is a vector of variables describing the characteristics of platforms substantially increases for operations in deeper water: the platform (such as age) and operator (such as their percent compared with platforms at water depths less than 1000 feet, the interest in the lease); WD are district fixed effects that control for average number of incidents increases more than threefold for average occurrence of incidents at each district over time; yt are depths greater than 1000 feet (from 0.23 per platform per year in year fixed effects that control for all time-varying characteristics shallow water compared to 074 and 0.81 in deep and ultra-deep of incident occurrence that are correlated over space (e.g., trends 9 water, respectively). The number of incidents that are reported in reporting frequency); and eit accounts for an unobserved as accidents increases dramatically with water depth. Twenty idiosyncratic error. Across various specifications of a probit percent of shallow water incidents are reported as accidents, regression water depth remains statistically significant and posi- whereas 72–79% are considered to have been ‘‘accidents’’ in tive (Table 5). The first specification includes characteristics of platforms, including age, distance to shore, and whether the platform is considered to be a major complex; the second 9 Platform incident data include exploration activity in addition to production specification adds indicators for production activity levels. The and development. In the statistics below, the probability of an incident from third and fourth specifications add information on lease owner- exploration appears very low; this is because the majority of exploration-related incidents are attributed to drilling rigs (not fixed platforms), which are excluded ship and also include a fixed effect for the year. The results from this analysis. confirm the univariate comparisons in Table 4 showing that the L. Muehlenbachs et al. / Energy Policy 55 (2013) 699–705 703

Table 5 Probit estimates of non-weather related reported incidents.

Speciﬁcations

(1) (2) (3) (4)

Water depth (100 feet) 0.00166nnn 0.00108nnn 0.0012nnn 0.00121nnn (0.000108) (0.000103) (0.000112) (0.000111) Age 0.0000479 0.0000904nnn 0.000105nnn 0.000119nnn (0.0000359) (0.0000336) (0.0000335) (0.0000332) Manned 24 h (indicator) 0.0798nnn 0.0434nnn 0.0422nnn 0.0421nnn (0.00352) (0.00264) (0.00266) (0.00266) Major complex (indicator) 0.0306nnn 0.0178nnn 0.0171nnn 0.0167nnn (0.00149) (0.00124) (0.00124) (0.00122) Distance to shore (miles) 0.000142nnn 0.0000695nnn 0.000065nnn 0.0000665nnn (0.0000172) (0.000015) (0.000015) (0.0000147) YearZ2006 (indicator) 0.0261nnn 0.0296nnn 0.0302nnn 0.0202nnn (0.00134) (0.00136) (0.00139) (0.00283) Inactive (indicator) 0.0207nnn 0.0214nnn 0.021nnn (0.00122) (0.00124) (0.00122) Production (mBOE) 2.17e07 2.84e07n 3.03e07nn (1.38e07) (1.46e07) (1.44e07) Drilling activity (indicator) 0.0317nnn 0.0312nnn 0.0297nnn (0.00278) (0.00279) (0.00272) No. of well operations 0.000432nnn 0.000412nnn 0.000412nnn (0.0000383) (0.0000377) (0.0000372) No. of producing wells 1.25e06 4.21e06 0.0000413 (0.000118) (0.000116) (0.000114) Operator’s % ownership 0.0000158 0.0000117 (9.94e06) (9.81e06) No. of lessees 0.000225 0.000165 (0.00019) (0.000188) District effects Yes Yes Yes Yes Year effects No No No Yes

N 76,595 76,595 74,438 74,438 Pseudo R-squared 0.28314 0.33149 0.33476 0.33724

Dependent variable: whether a non-weather related incident occurred on platform i in year t. Data on non-weather related incidents, 1996–2009. Probit slope derivatives (marginal effects) are reported. Specification includes a constant term (marginal effect not reported). Standard errors are shown in parenthesis. All specifications contain an indicator for the district of jurisdiction, and the last specification contains an indicator for the year. Production is the annual production of oil and gas (where gas is converted to thousand barrels of oil equivalent (mBOE) with 0.178 barrels per thousand cubic feet). n Significant at the 10% level. nn Significant at the 5% level. nnn Significant at the 1% level.

probability of an incident being reported increases with water depth. An increase in the water depth of 100 feet increases reported incidents by 0.108–0.166 percentage points (as seen in the coefficient on Water Depth in Table 5). The predicted increase applies to the average platform—that is, a platform with the same age, annual production, number of producing wells, and number of completions as the average platform in the Gulf. The predicted baseline probability of an incident’s being reported on the average platform is 1.4%; therefore, an increase of 0.12 percentage points is equivalent to an 8.5% increase in the baseline probability. This finding is in contrast to previous studies; Jablonowski (2007) finds that an indicator for water depths deeper than 400 feet is statistically insignificant in determining the likelihood of an incident on drilling rigs between 1990 and 1998, and Shultz (1999) finds the period of 1986–1995 that water depth had a negative effect on the likelihood of accidents. However, as illustrated in Fig. 2, there was a dramatic increase in drilling at the deepest water depths after the mid-1990s with depths continuing to grow throughout the next decade. It is possible that the finding regarding water depth is driven by a lack of industry and operator experience at these new depths. To investigate the temporal effect of learning, an indicator Fig. 2. Maximum and mean platform water depth over time. variable was constructed for platforms that were installed in water 500 feet deeper than all other existing platforms at that were at the time of installation at the leading edge of water depth. time. Although not shown here, we included indicator variables These variables were found to be statistically insignificant as for the first, second, third, fourth, and fifth year of platforms that predictors of company-reported incidents. Another test of the 704 L. Muehlenbachs et al. / Energy Policy 55 (2013) 699–705

Jackson, and Lake Charles) have a lower probability of reported incidents. This is consistent with Jablonowski (2007), who found that location affected the probability of reporting and suggested that spatial variation in inspection characteristics may be the cause. Finally, to account for variation in platforms over time, a year indicator is included in the final specification. When taking time into account, the working interest of the lease operator and the number of firms that have ownership in the lease are not significant in determining incident occurrence.

5. Conclusions

This paper reports on an empirical analysis of performance indicators on offshore oil platforms in the Gulf of Mexico between 1996 and 2010. One of the key findings is that company-reported incidents (such as blowouts, fires, injuries, and pollution) increase with water depth, controlling for platform characteristics such as age, quantity of oil and gas produced, and number of producing wells. Controlling for these and other characteristics, for an average platform, each 100 feet of added depth increases the Fig. 3. Predicted probability of a reported incident, by water depth. probability of a company-reported incident by 8.5%. Pursuant to the OCS Lands Act of 1953 (as amended), all fixed impact of experience on incidents was conducted by constructing production facilities are to be inspected at least once annually for a variable indicating if a platform was in the 99.5th percentile of compliance with safety regulations, accurate recordkeeping, and water depth in a given year (Fig. 2). This variable was also proper maintenance. In addition to this annual inspection, unan- insignificant. Thus, compared with the average platform, ‘‘pio- nounced risk-based inspections are conducted on an irregular neer’’ platforms in deeper waters do not appear to report more basis. Our results suggest targeting deeper water platforms for incidents in the first five years after being installed. inspection may be worthwhile. However, while this paper sheds Platform-level production in deep water is on average much light on the increased risk associated with drilling in deeper higher than production in shallow water (Table 2). Therefore, it is water, it is difficult to draw policy implications without further plausible that the increase in incidents with water depth could be research. For example, one limitation of this study is that avail- correlated with increased production. Including production volume able incident data reflect only those incidents that have been in the regression for reported incidents shows that higher produc- reported or detected. We are unable to rule out the possibility tion does increase the probability of an incident report (in specifica- that there is an unobservable characteristic that is driving more tions (3) and (4)); however, the marginal effect of increasing water reporting in deeper water. Other research has demonstrated the depth is still significant. The marginal effect of water depth also does likelihood of discrepancies between actual and recorded incidents not change substantially when accounting for drilling activity that (Jablonowski, 2007). Shultz (1999) also points out reliability year at the platform (Drilling Activity (Indicator)). On average, plat- concerns and recommends improving ‘‘data acquisition, data forms in deeper water are more complex: they have had more entry, and database management efforts’’ (p. 55) as well as boreholes drilled and more well operations (such as well reentries regularly updating and eliminating errors in the records to and horizontal drills) than platforms in shallow water. However, increase the confidence of any conclusions drawn from even after accounting for the cumulative number of well operations these data. and the current number of producing wells at a platform, water Further statistical analysis of enforcement and noncompliance depth still plays a statistically significant role in determining the data should provide important insights into the causal connec- probability of an incident. tions between inspections and enforcement actions on subse- The predicted annual probability of a reported incident at quent reported incidents and incidents of noncompliance. For different water depths is displayed in Fig. 3. The predicted example, we do not know whether increased enforcement in probability was obtained using estimates from Specification 4 in deeper waters would reduce accidents or spills—or even if they Table 5, holding all other variables in the model at their mean. would, whether the costs would exceed the benefits. These The effects of other variables included in the regression are as important questions are left for future research. expected. Site complexity, approximated by ‘‘major complex’’ status in this analysis, is positively correlated with the probability of an incident, as suggested by Jablonowski (2007) and Iledare et al. (1997). Reported incidents increase as platforms are farther Acknowledgments from the shore or manned 24 h a day. Age also increases the likelihood of a reported incident, supporting the findings of This article incorporates research undertaken by the authors at Iledare et al. (1997); however, Shultz (1999) finds platform age the request of the National Commission on the BP Deepwater to be insignificant. Horizon Oil Spill and Offshore Drilling, but the authors of this In addition, the 2006 policy change that increased stringency article are solely responsible for its content, errors, and opinions. of reporting requirements resulted in a two percentage point Assistance from Madeline Gottlieb, Stefan Staubli, Adam Stern, increase in the probability of an incident’s being reported (as seen and Warren Williamson is gratefully acknowledged. An earlier in the coefficient of the YearZ2006 Indicator). Differences across version of this paper was circulated under the name ‘‘Preliminary jurisdictional districts are also evident: benchmarked against the Empirical Assessment of Offshore Production Platforms in the New Orleans district, all other districts (Houma, Lafayette, Lake Gulf of Mexico’’. L. Muehlenbachs et al. / Energy Policy 55 (2013) 699–705 705

References Jablonowski, C.J., 2007. Employing detection controlled models in health and environmental risk assessment: a case in offshore oil drilling. Human & Ecological Risk Assessment 13, 986–1013. Cohen, M.A., 1986. The costs and beneﬁts of oil spill prevention and enforcement. Offshore Energy and Minerals Management, Gulf of Mexico OCS Region, Field Journal of Environmental Economics and Management 13, 167–188. Operations, BOEMRE, 2009. Facilities Inspections Team Report, March 2, 2009. Cohen, M.A., 1987. Optimal enforcement strategy to prevent oil spills: an Shultz, J., 1999. The Risk of Accidents and Spills at Offshore Production Platforms: application of a principal-agent model with moral hazard. Journal of Law A Statistical Analysis of Risk Factors and the Development of Predictive and Economics 30, 23–51. Epple, D., Visscher, M., 1984. Environmental pollution: modelling occurrence, Models. Doctoral Dissertation. Department of Engineering and Public Policy, detection, and deterrence. Journal of Law & Economics 27, 29–60. Carnegie Mellon University, Pittsburgh. Homan, A.C., Steiner, T., 2008. OPA 90’s impact at reducing oil spills. Marine Policy Stewart, T.R., Leschine, T.M., 1986. Judgment and analysis in oil spill risk 32, 711–718. assessment. Risk Analysis 6, 305–315. Iledare, O.O., et al., 1997. Oil spills, workplace safety and ﬁrm size: evidence from Viladrich-Grau, M., Groves, T., 1997. The oil spill process: the effect of Coast Guard the U.S. Gulf of Mexico OCS. Energy Journal 18, 73–89. monitoring on oil spills. Environmental and Resources Economics 10, 315–339.

Vol 437|29 September 2005|doi:10.1038/nature04095 ARTICLES

Anthropogenic ocean acidiﬁcation over the twenty-ﬁrst century and its impact on calcifying organisms

James C. Orr1, Victoria J. Fabry2, Olivier Aumont3, Laurent Bopp1, Scott C. Doney4, Richard A. Feely5, Anand Gnanadesikan6, Nicolas Gruber7, Akio Ishida8, Fortunat Joos9, Robert M. Key10, Keith Lindsay11, Ernst Maier-Reimer12, Richard Matear13, Patrick Monfray1†, Anne Mouchet14, Raymond G. Najjar15, Gian-Kasper Plattner7,9, Keith B. Rodgers1,16†, Christopher L. Sabine5, Jorge L. Sarmiento10, Reiner Schlitzer17, Richard D. Slater10, Ian J. Totterdell18†, Marie-France Weirig17, Yasuhiro Yamanaka8 & Andrew Yool18

Today’s surface ocean is saturated with respect to calcium carbonate, but increasing atmospheric carbon dioxide concentrations are reducing ocean pH and carbonate ion concentrations, and thus the level of calcium carbonate saturation. Experimental evidence suggests that if these trends continue, key marine organisms—such as corals and some plankton—will have difficulty maintaining their external calcium carbonate skeletons. Here we use 13 models of the ocean–carbon cycle to assess calcium carbonate saturation under the IS92a ‘business-as-usual’ scenario for future emissions of anthropogenic carbon dioxide. In our projections, Southern Ocean surface waters will begin to become undersaturated with respect to aragonite, a metastable form of calcium carbonate, by the year 2050. By 2100, this undersaturation could extend throughout the entire Southern Ocean and into the subarctic Pacific Ocean. When live pteropods were exposed to our predicted level of undersaturation during a two-day shipboard experiment, their aragonite shells showed notable dissolution. Our findings indicate that conditions detrimental to high-latitude ecosystems could develop within decades, not centuries as suggested previously.

Ocean uptake of CO2 will help moderate future climate change, but These rates decline even when surface waters remain supersaturated the associated chemistry, namely hydrolysis of CO2 in seawater, with respect to CaCO3, a condition that previous studies have increases the hydrogen ion concentration [Hþ]. Surface ocean pH predicted will persist for hundreds of years4,8,9. is already 0.1 unit lower than preindustrial values. By the end of the Recent predictions of future changes in surface ocean pH and century, it will become another 0.3–0.4 units lower1,2 under the IS92a carbonate chemistry have primarily focused on global average scenario, which translates to a 100–150% increase in [Hþ]. Simul- conditions1,2,10 or on low latitude regions4, where reef-building corals taneously, aqueous CO2 concentrations [CO2(aq)] will increase and are abundant. Here we focus on future surface and subsurface 22 carbonate ion concentrations ½CO3 will decrease, making it more changes in high latitude regions where planktonic shelled pteropods difficult for marine calcifying organisms to form biogenic calcium are prominent components of the upper-ocean biota in the Southern 11–15 carbonate (CaCO3). Substantial experimental evidence indicates that Ocean, Arctic Ocean and subarctic Pacific Ocean . Recently, it has calcification rates will decrease in low-latitude corals3–5, which form been suggested that the cold surface waters in such regions will begin reefs out of aragonite, and in phytoplankton that form their tests to become undersaturated with respect to aragonite only when 6,7 (shells) out of calcite , the stable form of CaCO3. Calcification rates atmospheric CO2 reaches 1,200 p.p.m.v., more than four times the 22 will decline along with ½CO3 owing to its reaction with increasing preindustrial level (4 £ CO2) of 280 p.p.m.v. (ref. 9). In contrast, our concentrations of anthropogenic CO2 according to the following results suggest that some polar and subpolar surface waters will reaction: become undersaturated at ,2 £ CO2, probably within the next 50 22 2 CO2 þ CO3 þ H2O ! 2HCO3 ð1Þ years.

1Laboratoire des Sciences du Climat et de l’Environnement, UMR CEA-CNRS, CEA Saclay, F-91191 Gif-sur-Yvette, France. 2Department of Biological Sciences, California State University San Marcos, San Marcos, California 92096-0001, USA. 3Laboratoire d’Oceánographie et du Climat: Expe´rimentations et Approches Nume´riques (LOCEAN), Centre IRD de Bretagne, F-29280 Plouzane´, France. 4Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543-1543, USA. 5National Oceanic and Atmospheric Administration (NOAA)/Pacific Marine Environmental Laboratory, Seattle, Washington 98115-6349, USA. 6NOAA/Geophysical Fluid Dynamics Laboratory, Princeton, New Jersey 08542, USA. 7Institute of Geophysics and Planetary Physics, UCLA, Los Angeles, California 90095-4996, USA. 8Frontier Research Center for Global Change, Yokohama 236-0001, Japan. 9Climate and Environmental Physics, Physics Institute, University of Bern, CH-3012 Bern, Switzerland. 10Atmospheric and Oceanic Sciences (AOS) Program, Princeton University, Princeton, New Jersey 08544-0710, USA. 11National Center for Atmospheric Research, Boulder, Colorado 80307-3000, USA. 12Max Planck Institut fu¨r Meteorologie, D-20146 Hamburg, Germany. 13CSIRO Marine Research and Antarctic Climate and Ecosystems CRC, Hobart, Tasmania 7001, Australia. 14Astrophysics and Geophysics Institute, University of Liege, B-4000 Liege, Belgium. 15Department of Meteorology, Pennsylvania State University, University Park, Pennsylvania 16802-5013, USA. 16LOCEAN, Universite´ Pierre et Marie Curie, F-75252 Paris, France. 17Alfred Wegener Institute for Polar and Marine Research, D-27515 Bremerhaven, Germany. 18National Oceanography Centre Southampton, Southampton SO14 3ZH, UK. †Present addresses: Laboratoire d’Etudes en Geóphysique et Oceánographie Spatiales, UMR 5566 CNES- CNRS-IRD-UPS, F-31401 Toulouse, France (P.M.); AOS Program, Princeton University, Princeton, New Jersey 08544-0710, USA (K.B.R.); The Met Office, Hadley Centre, FitzRoy Road, Exeter EX1 3PB, UK (I.J.T.). 681 © 2005 Nature Publishing Group OR2-003844

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22 Changes in carbonate mean surface maps). Changes in ½CO3 and [CO2(aq)] are also We have computed modern-day ocean carbonate chemistry from inextricably linked to changes in other carbonate chemistry variables observed alkalinity and dissolved inorganic carbon (DIC), relying on (Supplementary Fig. S1). 22 data collected during the CO2 Survey of the World Ocean Circulation We also estimated preindustrial ½CO3 from the same data, after Experiment (WOCE) and the Joint Global Ocean Flux Study subtracting data-based estimates of anthropogenic DIC (ref. 17) (JGOFS). These observations are centred around the year 1994, from the modern DIC observations and assuming that preindustrial and have recently been provided as a global-scale, gridded data and modern alkalinity ﬁelds were identical (see Supplementary product GLODAP (ref. 16; see Supplementary Information). Information). Relative to preindustrial conditions, invasion of 22 ½ 22 Modern-day surface ½CO3 varies meridionally by more than a anthropogenic CO2 has already reduced modern surface CO3 by factor of two, from average concentrations in the Southern Ocean of more than 10%, that is, a reduction of 29 mmol kg21 in the tropics 105 mmol kg21 to average concentrations in tropical waters of and 18 mmol kg21 in the Southern Ocean. Nearly identical results 21 22 240 mmol kg (Fig. 1). Low ½CO3 in the Southern Ocean is due were found when, instead of the data-based anthropogenic CO2 to (1) low surface temperatures and CO2-system thermodynamics, estimates, we used simulated anthropogenic CO2,namelythe and (2) large amounts of upwelled deep water, which contain high median from 13 models that participated in the second phase of [CO2(aq)] from organic matter remineralization. These two effects the Ocean Carbon-Cycle Model Intercomparison Project, or reinforce one another, yielding a high positive correlation of present- OCMIP-2 (Fig. 1c). 22 2 day ½CO3 with temperature (for example, R ¼ 0.92 for annual To quantify future changes in carbonate chemistry, we used simulated DIC from ocean models that were forced by two atmospheric CO2 scenarios: the Intergovernmental Panel on Climate Change (IPCC) IS92a ‘continually increasing’ scenario (788 p.p.m.v. in the year 2100) and the IPCC S650 ‘stabilization’ scenario (563 p.p.m.v. in the year 2100) (Fig. 1). Simulated perturbations in DIC relative to 1994 (the GLODAP reference year) were added to the modern DIC data; again, alkalinity was assumed to be constant. To provide a measure of uncertainty, we report model results as the OCMIP median ^ 2j. The median generally outperformed

Figure 1 | Increasing atmospheric CO2 and decreasing surface ocean pH and 22 [CO3 ]. a, Atmospheric CO2 used to force 13 OCMIP models over the industrial period (‘Historical’) and for two future scenarios: IS92a (‘I’ in b and c) and S650 (‘S’ in b and c). b, c, Increases in atmospheric CO2 lead to 22 reductions in surface ocean pH (b) and surface ocean ½CO3 (c). Results are given as global zonal averages for the 1994 data and the preindustrial (‘Preind.’) ocean. The latter were obtained by subtracting data-based anthropogenic DIC (ref. 17) (solid line in grey-shaded area), as well as by subtracting model-based anthropogenic DIC (OCMIP median, dotted line in grey-shaded area; OCMIP range, grey shading). Future results for the year 2100 come from the 1994 data plus the simulated DIC perturbations for the Figure 2 | The aragonite saturation state in the year 2100 as indicated by 22 22 22 two scenarios; results are also shown for the year 2300 with S650 (thick D[CO3 ]A. The D½CO3 A is the in situ ½CO3 minus that for aragonite- dashed line). The small effect of future climate change simulated by the IPSL equilibrated sea water at the same salinity, temperature and pressure. Shown climate–carbon model is added as a perturbation to IS92a in the year 2100 are the OCMIP-2 median concentrations in the year 2100 under scenario (thick dotted line); two other climate–carbon models, PIUB-Bern and IS92a: a, surface map; b, Atlantic; and c, Pacific zonal averages. Thick lines Commonwealth Scientific and Industrial Research Organisation (CSIRO), indicate the aragonite saturation horizon in 1765 (Preind.; white dashed 22 show similar results (Fig. 3a). The thin dashed lines indicating the ½CO3 line), 1994 (white solid line) and 2100 (black solid line for S650; black dashed 22 for sea water in equilibrium with aragonite and calcite are nearly flat, line for IS92a). Positive D½CO3 A indicates supersaturation; negative 22 revealing weak temperature sensitivity. D½CO3 A indicates undersaturation. 682 © 2005 Nature Publishing Group OR2-003845

NATURE|Vol 437|29 September 2005 ARTICLES individual models in OCMIP model–data comparison (Supplemen- simulate that the Southern Ocean’s average aragonite saturation tary Fig. S2). By the year 2100, as atmospheric CO2 reaches horizon will have shoaled from 730 m to 60 m, and that the entire 788 p.p.m.v. under the IS92a scenario, average tropical surface water column in the Weddell Sea will have become undersaturated 22 ^ 21 ½CO3 declines to 149 14 mmol kg . This is a 45% reduction (Fig. 2). In the north, all surface waters remain saturated under relative to preindustrial levels, in agreement with previous predic- the S650 scenario. North of 508 N, the annual average aragonite tions4,8. In the Southern Ocean (all waters south of 608 S), surface saturation horizon shoals from 140 m to 70 m in the Pacific, whereas concentrations dip to 55 ^ 5 mmol kg21, which is 18% below the it shoals by 2,000 m to 610 m in the North Atlantic. Therefore, under threshold where aragonite becomes undersaturated (66 mmol kg21). either scenario the OCMIP models simulated large changes in surface 22 These changes extend well below the sea surface. Throughout the and subsurface ½CO3 : Yet these models account for only the direct Southern Ocean, the entire water column becomes undersaturated geochemical effect of increasing atmospheric CO2 because they were with respect to aragonite. During the twenty-first century, under the all forced with prescribed modern-day climate conditions. 22 IS92a scenario, the Southern Ocean’s aragonite saturation horizon In addition to this direct geochemical effect, ocean ½CO3 is also (the limit between undersaturation and supersaturation) shoals from altered by climate variability and climate change. To quantify the its present average depth of 730 m (Supplementary Fig. S3) all the added effect of future climate change, we analysed results from three way to the surface (Fig. 2). Simultaneously, in a portion of the atmosphere–ocean climate models that each included an ocean subarctic Pacific, the aragonite saturation horizon shoals from carbon-cycle component (see Supplementary Information). These depths of about 120 m to the surface. In the North Atlantic, surface three models agree that twenty-first century climate change will cause 22 waters remain saturated with respect to aragonite, but the aragonite a general increase in surface ocean ½CO3 (Fig. 3), mainly because saturation horizon shoals dramatically; for example, north of 508 Nit most surface waters will be warmer. However, the models also agree 22 shoals from 2,600 m to 115 m. The greater erosion in the North that the magnitude of this increase in ½CO3 is small, typically Atlantic is due to deeper penetration and higher concentrations of counteracting less than 10% of the decrease due to the geochemical anthropogenic CO2, a tendency that is already evident in present-day effect. High-latitude surface waters show the smallest increases in 17,18 19,20 22 data-based estimates and in models (Supplementary Figs S4 ½CO3 ; and even small reductions in some cases. Therefore, our and S5). Less pronounced changes were found for the calcite analysis suggests that physical climate change alone will not sub- 22 saturation horizon. For example, in the year 2100 the average calcite stantially alter high-latitude surface ½CO3 during the twenty-first saturation horizon in the Southern Ocean stays below 2,200 m. century. Nonetheless, in 2100 surface waters of the Weddell Sea become Climate also varies seasonally and interannually, whereas our slightly undersaturated with respect to calcite. previous focus has been on annual changes. To illustrate how climate 22 In the more conservative S650 scenario, the atmosphere reaches variability affects surface ½CO3 ; we used results from an ocean 2 £ CO2 in the year 2100, 50 years later than with the IS92a scenario. carbon-cycle model forced with the daily National Centers for In 2100, Southern Ocean surface waters generally remain slightly Environmental Prediction (NCEP) reanalysis fields21 over 1948– supersaturated with respect to aragonite. However, the models also 2003 (see Supplementary Information). These fields are observa- tionally based and vary on seasonal and interannual timescales. 22 Simulated interannual variability in surface ocean ½CO3 is negligible when compared with the magnitude of the anthropogenic decline (Fig. 3b). Seasonal variability is also negligible except in the 22 ^ 21 high latitudes, where surface ½CO3 varies by about 15 mmol kg

Figure 4 | Key surface carbonate chemistry variables as a function of p . ½ 22 CO2 Shown are both CO3 (solid lines) and [CO2(aq)] (dashed lines) for average surface waters in the tropical ocean (thick lines), the Southern 22 Figure 3 | Climate-induced changes in surface [CO3 ]. a, The twenty-ﬁrst Ocean (thickest lines) and the global ocean (thin lines). Solid and dashed 22 century shift in zonal mean surface ocean ½CO3 due to climate change lines are calculated from the thermodynamic equilibrium approach. For 22 alone, from three atmosphere–ocean climate models—CSIRO-Hobart comparison, open symbols are for ½CO3 from our non-equilibrium,

(short dashed line), IPSL-Paris (long dashed line) and PIUB-Bern (solid model-data approach versus seawater pCO2 (open circles) and atmospheric line)—that each include an ocean carbon-cycle component (see pCO2 (open squares); symbol thickness corresponds with line thickness, Supplementary Information). b, The regional-scale seasonal and which indicates the regions for area-weighted averages. The nearly ﬂat, thin 22 interannual variability is simulated by an ocean carbon-cycle model forced dotted lines indicate the ½CO3 for seawater in equilibrium with aragonite with reanalysed climate forcing. (‘Arag. sat.’) and calcite (‘Calc. sat.’). 683 © 2005 Nature Publishing Group OR2-003846

ARTICLES NATURE|Vol 437|29 September 2005 when averaged over large regions. This is smaller than the twenty- result of changes in overlying marine productivity. However, the first-century’s transient change (for example, ,50 mmol kg21 in the models project a consistent trend, which only worsens the decline in 22 Southern Ocean). However, high-latitude surface waters do become subsurface ½CO3 ; that is, all coupled climate models predict substantially less saturated during winter, because of cooling (result- increased evaporation in the tropics and increased precipitation in 24 ing in higher [CO2(aq)]) and greater upwelling of DIC-enriched the high latitudes . This leads to greater upper ocean stratification in deep water, in agreement with previous observations in the North the high latitudes, which in turn decreases nutrients (but not to zero) Pacific22. Thus, high-latitude undersaturation will be first reached and increases light availability (owing to more shallow mixed layers). during winter. Thus, at 2 £ CO2 there is a 10% local increase in surface-to-deep Our predicted changes may be compared to those found in earlier export of particulate organic carbon (POC) in the Southern Ocean studies, which focused on surface waters in the tropics8 and in the using the Institut Pierre Simon Laplace (IPSL)-Paris model25. Sub- subarctic Pacific22,23. These studies assumed thermodynamic equi- sequent remineralization of this exported POC within the thermo- librium between CO2 in the atmosphere and the surface waters at cline would increase DIC, which would only exacerbate the decrease 22 their in situ alkalinity, temperature and salinity. If, in the equilibrium in high-latitude subsurface ½CO3 : For the twenty-first century, approach, the pCO2 is taken only to represent seawater pCO2 ; then the these uncertainties appear small next to the anthropogenic DIC results agree with our non-equilibrium approach when the sets of invasion (see Supplementary Information). carbonate chemistry constants are identical (Fig. 4). However, The largest uncertainty by far, and the only means to limit the 22 assuming equilibrium with the atmosphere leads to the prediction future decline in ocean ½CO3 ; is the atmospheric CO2 trajectory. To that future undersaturation will occur too soon (at lower atmos- better characterize uncertainty due to CO2 emissions, we compared pheric CO2 levels), mainly because the anthropogenic transient in the the six illustrative IPCC Special Reports on Emission Scenarios ocean actually lags that in the atmosphere. For example, with (SRES) in the reduced complexity, Physics Institute University of the equilibrium approach, we predict that average surface waters in Bern (PIUB)-Bern model. Under the moderate SRES B2 scenario, the Southern Ocean become undersaturated when atmospheric CO2 average Southern Ocean surface waters in that model become under- is 550 p.p.m.v. (in the year 2050 under IS92a), whereas our non- saturated with respect to aragonite when atmospheric CO2 reaches equilibrium approach, which uses models and data, indicates that 600 p.p.m.v. in the year 2100 (Fig. 5). For the three higher-emission undersaturation will occur at 635 p.p.m.v. (in the year 2070). Despite SRES scenarios (A1FI, A2 and A1B), these waters become under- these differences, both approaches indicate that the Southern Ocean saturated sooner (between the years 2058 and 2073); for the two surface waters will probably become undersaturated with respect to lower-emission scenarios (A1T and B1), these waters remain slightly aragonite during this century. Conversely, both of these approaches supersaturated in 2100. Thus, if atmospheric CO2 rises above disagree with a recent assessment9 that used a variant of the standard 600 p.p.m.v., most Southern Ocean surface waters will become thermodynamic equilibrium approach, where an incorrect input undersaturated with respect to aragonite. Yet, even below this level, temperature was used inadvertently. the Southern Ocean’s aragonite saturation horizon will shoal substantially (Fig. 2). For a given atmospheric CO2 scenario, predicted Uncertainties 22 changes in surface ocean ½CO3 are much more certain than the The three coupled climate–carbon models show little effect of climate related changes in climate. The latter depend not only on the model 22 change on surface ½CO3 (compare Fig. 3a to Fig. 1) partly because response to CO2 forcing, but also on poorly constrained physical air–sea CO2 exchange mostly compensates for the changes in surface processes, such as those associated with clouds. DIC caused by changes in marine productivity and circulation. In subsurface waters where such compensation is lacking, these models Ocean CO2 uptake 22 22 could under- or over-predict how much ½CO3 will change as a With higher levels of anthropogenic CO2 and lower surface ½CO3 ; the change in surface ocean DIC per unit change in atmospheric CO2 (mmol kg21 per p.p.m.v.) will be about 60% lower in the year 2100 22 (under IS92a) than it is today. Simultaneously, the CO3 =CO2ðaqÞ ratio will decrease from 4:1 to 1:1 in the Southern Ocean (Fig. 4). These decreases are due to the well-understood anthropogenic reduction in buffer capacity26, already accounted for in ocean carbon-cycle models. On the other hand, reduced export of CaCO3 from the high 22 latitudes would increase surface ½CO3 ; thereby increasing ocean CO2 uptake and decreasing atmospheric CO2. Owing to this effect, ocean CO2 uptake could increase by 6–13 petagrams (Pg) C over the twenty-first century, based on one recent model study27 that incor- 7 porated an empirical, CO2-dependant relationship for calcification . Rates of calcification could decline even further, to zero, if waters actually became undersaturated with respect to both aragonite and calcite. We estimate that the total shutdown of high-latitude aragonite production would lead to, at most, a 0.25 Pg C yr21 increase in 21 ocean CO2 uptake, assuming that 1 Pg C yr of CaCO3 is exported globally28, that up to half of that is aragonite9,29, and that perhaps half of all aragonite is exported from the high latitudes. The actual increase in ocean CO2 uptake could be much lower because the aragonite fraction of the CaCO3 may be only 0.1 based on 30 22 low-latitude sediment traps , and the latitudinal distribution of Figure 5 | Average surface [CO3 ] in the Southern Ocean under various 22 aragonite export is uncertain. Thus, increased CO2 uptake from scenarios. Time series of average surface ½CO3 in the Southern Ocean for the PIUB-Bern reduced complexity model (see Fig. 3 and Supplementary reduced export of aragonite will provide little compensation for Information) under the six illustrative IPCC SRES scenarios. The results for decreases in ocean CO2 uptake due to reductions in buffer capacity. the SRES scenarios A1T and A2 are similar to those for the non-SRES Of greater concern are potential biological impacts due to future scenarios S650 and IS92a, respectively. undersaturation. 684 © 2005 Nature Publishing Group OR2-003847

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Figure 6 | Shell dissolution in a live pteropod. a–d, Shell from a live exposure of aragonitic rods; the prismatic layer (c), which has begun to peel pteropod, Clio pyramidata, collected from the subarctic Paciﬁc and kept in back, increasing the surface area over which dissolution occurs; and the water undersaturated with respect to aragonite for 48 h. The whole shell (a) aperture region (d), which reveals advanced shell dissolution when has superimposed white rectangles that indicate three magniﬁed areas: the compared to a typical C. pyramidata shell not exposed to undersaturated shell surface (b), which reveals etch pits from dissolution and resulting conditions (e).

Biological impacts distributional ranges would then be reduced both within the water The changes in seawater chemistry that we project to occur during column, disrupting vertical migration patterns, and latitudinally, this century could have severe consequences for calcifying organisms, imposing a shift towards lower-latitude surface waters that remain particularly shelled pteropods: the major planktonic producers of supersaturated with respect to aragonite. At present, we do not know aragonite. Pteropod population densities are high in polar and if pteropod species endemic to polar regions could disappear subpolar waters. Yet only five species typically occur in such cold altogether, or if they can make the transition to live in warmer, water regions and, of these, only one or two species are common at carbonate-rich waters at lower latitudes under a different ecosystem. the highest latitudes31. High-latitude pteropods have one or two If pteropods are excluded from polar and subpolar regions, their generations per year12,15,32, form integral components of food predators will be affected immediately. For instance, gymnosomes webs, and are typically found in the upper 300 m where they may are zooplankton that feed exclusively on shelled pteropods33,39. reach densities of hundreds to thousands of individuals per m3 Pteropods also contribute to the diet of diverse carnivorous zoo- (refs 11, 13–15). In the Ross Sea, for example, the prominent plankton, myctophid and nototheniid fishes40–42,NorthPacific subpolar–polar pteropod Limacina helicina sometimes replaces salmon43,44, mackerel, herring, cod and baleen whales45. krill as the dominant zooplankton, and is considered an overall Surface dwelling calcitic plankton, such as foraminifera and indicator of ecosystem health33. In the strongly seasonal high lati- coccolithophorids, may fare better in the short term. However, the tudes, sedimentation pulses of pteropods frequently occur just after beginnings of high-latitude calcite undersaturation will only lag that summer15,34. In the Ross Sea, pteropods account for the majority of for aragonite by 50–100 years. The diverse benthic calcareous the annual export flux of both carbonate and organic carbon34,35. organisms in high-latitude regions may also be threatened, including South of the Antarctic Polar Front, pteropods also dominate the cold-water corals which provide essential fish habitat46. Cold-water export flux of CaCO3 (ref. 36). corals seem much less abundant in the North Pacific than in the Pteropods may be unable to maintain shells in waters that are North Atlantic46, where the aragonite saturation horizon is much undersaturated with respect to aragonite. Data from sediment traps deeper (Fig. 2). Moreover, some important taxa in Arctic and indicate that empty pteropod shells exhibit pitting and partial Antarctic benthic communities secrete magnesian calcite, which dissolution as soon as they fall below the aragonite saturation can be more soluble than aragonite. These include gorgonians46, 22,36,37 47 horizon . In vitro measurements confirm such rapid pteropod coralline red algae and echinoderms (sea urchins) .At2£ CO2, shell dissolution rates38. New experimental evidence suggests that juvenile echinoderms stopped growing and produced more brittle even the shells of live pteropods dissolve rapidly once surface waters and fragile exoskeletons in a subtropical six-month manipulative become undersaturated with respect to aragonite9. Here we show that experiment48. However, the responses of high-latitude calcifiers to 22 when the live subarctic pteropod Clio pyramidata is subjected to a reduced ½CO3 have generally not been studied. Yet experimental level of undersaturation similar to what we predict for Southern evidence from many lower-latitude, shallow-dwelling calcifiers Ocean surface waters in the year 2100 under IS92a, a marked reveals a reduced ability to calcify with a decreasing carbonate 9 dissolution occurs at the growing edge of the shell aperture within saturation state . Given that at 2 £ CO2, calcification rates in some 48 h (Fig. 6). Etch pits formed on the shell surface at the apertural shallow-dwelling calcareous organisms may decline by up to 50% margin (which is typically ,7-mm-thick) as the ,1-mm exterior (ref. 9), some calcifiers could have difficulty surviving long enough (prismatic layer) peeled back (Fig. 6c), exposing the underlying even to experience undersaturation. Certainly, they have not experi- aragonitic rods to dissolution. Fourteen individuals were tested. All enced undersaturation for at least the last 400,000 years49,and of them showed similar dissolution along their growing edge, even probably much longer50. though they all remained alive. If C. pyramidata cannot grow its Changes in high-latitude seawater chemistry that will occur by the protective shell, we would not expect it to survive in waters that end of the century could well alter the structure and biodiversity of become undersaturated with respect to aragonite. polar ecosystems, with impacts on multiple trophic levels. Assessing If the response of other high-latitude pteropod species to aragonite these impacts is impeded by the scarcity of relevant data. undersaturation is similar to that of C. pyramidata, we hypothesize that these pteropods will not be able to adapt quickly enough to live Received 15 June; accepted 29 July 2005. in the undersaturated conditions that will occur over much of the 1. Haugan, P. M. & Drange, H. Effects of CO2 on the ocean environment. Energy high-latitude surface ocean during the twenty-first century. Their Convers. Mgmt 37, 1019–-1022(1996). 685 © 2005 Nature Publishing Group OR2-003848

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2. Brewer, P. G. Ocean chemistry of the fossil fuel CO2 signal: the haline signal of 31. Be´, A. W. H. & Gilmer, R. W. Oceanic Micropaleontology Vol. 1 (ed. Ramsey, A.) “business as usual”. Geophys. Res. Lett. 24, 1367–-1369(1997). 733–-808(Academic, London, 1977). 3. Gattuso, J.-P., Frankignoulle, M., Bourge, I., Romaine, S. & Buddemeier, R. W. 32. Dadon, J. R. & de Cidre, L. L. The reproductive cycle of the Thecosomatous Effect of calcium carbonate saturation of seawater on coral calcification. Glob. pteropod Limacina retroversa in the western South Atlantic. Mar. Biol. 114, Planet. Change 18, 37–-46(1998). 439–-442(1992). 4. Kleypas, J. A. et al. Geochemical consequences of increased atmospheric 33. Seibel, B. A. & Dierssen, H. M. Cascading trophic impacts of reduced biomass carbon dioxide on coral reefs. Science 284, 118–-120(1999). in the Ross Sea, Antarctica: Just the tip of the iceberg? Biol. Bull. 205, 93–-97 5. Langdon, C. et al. Effect of elevated CO2 on the community metabolism of an (2003). experimental coral reef. Glob. Biogeochem. Cycles 17, 1011, doi:10.1029/ 34. Accornero, A., Manno, C., Esposito, F. & Gambi, M. C. The vertical flux of 2002GB001941 (2003). particulate matter in the polyna of Terra Nova Bay. Part II. Biological 6. Riebesell, U. et al. Reduced calcification of marine plankton in response to components. Antarct. Sci. 15, 175–-188(2003). increased atmospheric CO2. Nature 407, 364–-367(2000). 35. Collier, R., Dymond, J., Susumu Honjo, S. M., Francois, R. & Dunbar, R. The 7. Zondervan, I., Zeebe, R., Rost, B. & Riebesell, U. Decreasing marine biogenic vertical flux of biogenic and lithogenic material in the Ross Sea: moored

calcification: A negative feedback on rising atmospheric pCO2 . Glob. sediment trap observations 1996–-1998. Deep-Sea Res. II 47, 3491–-3520 (2000). Biogeochem. Cycles 15, 507–-516 (2001). 36. Honjo, S., Francois, R., Manganini, S., Dymond, J. & Collier, R. Particle fluxes to 8. Broecker, W. S. & Peng, T.-H. Fate of fossil fuel carbon dioxide and the global the interior of the Southern Ocean in the western Pacific sector along 1708 W. carbon budget. Science 206, 409–-418(1979). Deep-Sea Res. II 47, 3521–-3548(2000). 9. Feely, R. A. et al. The impact of anthropogenic CO2 on the CaCO3 system in 37. Betzer, P. R., Byrne, R., Acker, J., Lewis, C. S. & Jolley, R. R. The oceanic the oceans. Science 305, 362–-366(2004). carbonate system: a reassessment of biogenic controls. Science 226, 10. Caldeira, K. & Wickett, M. E. Anthropogenic carbon and ocean pH. Nature 425, 1074–-1077 (1984). 365 (2003). 38. Byrne, R. H., Acker, J. G., Betzer, P. R., Feely, R. A. & Cates, M. H. Water column 11. Urban-Rich, J., Dagg, M. & Peterson, J. Copepod grazing on phytoplankton in dissolution of aragonite in the Pacific Ocean. Nature 312, 321–-326 (1984). the Pacific sector of the Antarctic Polar Front. Deep-Sea Res. II 48, 4223–-4246 39. Lalli, C. M. Structure and function of the buccal apparatus of Clione limacina (2001). (Phipps) with a review of feeding in gymnosomatous pteropods. J. Exp. Mar. 12. Kobayashi, H. A. Growth cycle and related vertical distribution of the Biol. Ecol. 4, 101–-118(1970). thecosomatous pteropod Spiratella “Limacina” helicina in the central Arctic 40. Foster, B. A. & Montgomery, J. C. Planktivory in benthic nototheniid fish in Ocean. Mar. Biol. 26, 295–-301(1974). McMurdo Sound, Antarctica. Environ. Biol. Fish. 36, 313–-318(1993). 13. Pakhomov, E. A., Verheye, H. M., Atkinson, A., Laubscher, R. K. & Taunton- 41. Pakhomov, E., Perissinotto, A. & McQuaid, C. D. Prey composition and daily Clark, J. Structure and grazing impact of the mesozooplankton community rations of myctophid fishes in the Southern Ocean. Mar. Ecol. Prog. Ser. 134, during late summer 1994 near South Georgia, Antarctica. Polar Biol. 18, 1–-14(1996). 180–-192(1997). 42. La Mesa, M., Vacchi, M. & Sertorio, T. Z. Feeding plasticity of Trematomus 14. Fabry, V. J. Aragonite production by pteropod molluscs in the subarctic Pacific. newnesi (Pisces, Nototheniidae) in Terra Nova Bay, Ross Sea, in relation to Deep-Sea Res. I 36, 1735–-1751(1989). environmental conditions. Polar Biol. 23, 38–-45(2000). 15. Bathmann, U., Noji, T. T. & von Bodungen, B. Sedimentation of pteropods in the 43. Willette, T. M. et al. Ecological processes influencing mortality of juvenile pink Norwegian Sea in autumn. Deep-Sea Res. 38, 1341–-1360(1991). salmon (Oncorhynchus gorbuscha) in Prince William Sound, Alaska. Fish. 16. Key, R. M. et al. A global ocean carbon climatology: Results from Global Data Oceanogr. 10, 14–-41(2001). Analysis Project (GLODAP). Glob. Biogeochem. Cycles 18, 4031, doi:10.1029/ 44. Boldt, J. L. & Haldorson, L. J. Seasonal and geographical variation in juvenile 2004GB002247 (2004). pink salmon diets in the Northern Gulf of Alaska and Prince William Sound. 17. Sabine, C. L. et al. The ocean sink for anthropogenic CO2. Science 305, Trans. Am. Fisheries Soc. 132, 1035–-1052(2003). 367–-370(2004). 45. Lalli, C. M. & Gilmer, R. Pelagic Snails (Stanford Univ. Press, Stanford, 1989). 18. Gruber, N. Anthropogenic CO2 in the Atlantic Ocean. Glob. Biogeochem. Cycles 46. Freiwald, A., Fossa˚, J. H., Grehan, A., Koslow, T. & Roberts, J. M. Cold-water 12, 165–-191(1998). Coral Reefs: Out of Sight—No Longer Out of Mind (No. 22 in Biodiversity Series, 19. Sarmiento, J. L., Orr, J. C. & Siegenthaler, U. A perturbation simulation of CO2 UNEP-WCMC, Cambridge, UK, 2004). uptake in an ocean general circulation model. J. Geophys. Res. 97, 3621–-3645 47. Dayton, P. K. in Polar Oceanography, Part B: Chemistry, Biology and Geology (ed. (1992). Smith, W. O.) 631–-685(Academic, San Diego, 1990). 20. Orr, J. C. et al. Estimates of anthropogenic carbon uptake from four 48. Shirayama, Y. & Thornton, H. Effect of increased atmospheric CO2 on shallow- three-dimensional global ocean models. Glob. Biogeochem. Cycles 15, 43–-60 water marine benthos. J. Geophys. Res. 110, C09S09, doi:10.1029/ (2001). 2004JC002561 (2005). 21. Kalnay, E. et al. The NCEP/NCAR 40-year reanalysis project. Bull. Am. 49. Petit, J. R. et al. Climate and atmospheric history of the past 420,000 years Meteorol. Soc. 77, 437–-471(1996). from the Vostok ice core, Antarctica. Nature 399, 429–-436(1999). 22. Feely, R. A. et al. Winter–-summervariations of calcite and aragonite saturation 50. Pearson, P. N. & Palmer, M. R. Middle Eocene seawater pH and atmospheric in the northeast Pacific. Mar. Chem. 25, 227–-241(1988). carbon dioxide concentrations. Science 284, 1824–-1826(1999). 23. Feely, R. A., Byrne, R. H., Betzer, P. R., Gendron, J. F. & Acker, J. G. Factors influencing the degree of saturation of the surface and intermediate waters of Supplementary Information is linked to the online version of the paper at the North Pacific Ocean with respect to aragonite. J. Geophys. Res. 89, www.nature.com/nature. 10631–-10640(1984). 24. Sarmiento, J. L. et al. Response of ocean ecosystems to climate warming. Glob. Acknowledgements We thank M. Gehlen for discussions, and J.-M. Epitalon, Biogeochem. Cycles 18, 3003, doi:10.1029/2003GB002134 (2004). P. Brockmann and the Ferret developers for help with analysis. All but the 25. Bopp, L. et al. Potential impact of climate change of marine export production. climate simulations were made as part of the OCMIP project, which was Glob. Biogeochem. Cycles 15, 81–-99(2001). launched in 1995 by the Global Analysis, Integration and Modelling (GAIM) 26. Sarmiento, J. L., Le Que´re´, C. & Pacala, S. Limiting future atmospheric carbon Task Force of the International Geosphere–Biosphere Programme (IGBP) with dioxide. Glob. Biogeochem. Cycles 9, 121–-137(1995). funding from NASA (National Aeronautics and Space Administration). OCMIP-2 27. Heinze, C. Simulating oceanic CaCO3 export production in the greenhouse. was supported by the European Union Global Ocean Storage of Anthropogenic Geophys. Res. Lett. 31, L16308, doi:10.1029/2004GL020613 (2004). Carbon (EU GOSAC) project and the United States JGOFS Synthesis and 28. Iglesias-Rodriguez, M. D. et al. Representing key phytoplankton functional Modeling Project funded through NASA. The interannual simulation was groups in ocean carbon cycle models: Coccolithophorids. Glob. Biogeochem. supported by the EU Northern Ocean Carbon Exchange Study (NOCES) project, Cycles 16, 1100, doi:10.1029/2001GB001454 (2002). which is part of OCMIP-3. 29. Berner, R. A. in The Fate of Fossil Fuel CO2 in the Oceans (eds Andersen, N. R. & Malahoff, A.) 243–-260(Plenum, New York, 1977). Author Information Reprints and permissions information is available at 30. Fabry, V. J. Shell growth rates of pteropod and heteropod molluscs and npg.nature.com/reprintsandpermissions. The authors declare no competing aragonite production in the open ocean: Implications for the marine carbonate financial interests. Correspondence and requests for materials should be system. J. Mar. Res. 48, 209–-222(1990). addressed to J.C.O. ([email protected]).

Harmful Algae 30 (2013) 37–43

Contents lists available at ScienceDirect

Harmful Algae

jo urnal homepage: www.elsevier.com/locate/hal

High CO2 promotes the production of paralytic shellﬁsh poisoning

toxins by Alexandrium catenella from Southern California waters

a b a b

Avery O. Tatters , Leanne J. Flewelling , Feixue Fu , April A. Granholm ,

David A. Hutchins *

Department of Biological Sciences, University of Southern California, 3616 Trousdale Parkway, Los Angeles, CA 90089, United States

Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 8th Avenue SE, St. Petersburg, FL 33701, United States

A R T I C L E I N F O A B S T R A C T

Article history: In many dinoﬂagellates, cellular toxin levels have been demonstrated to increase when growth is limited by

Received 6 July 2013

essential nutrients such as phosphorus. Despite the recognized importance of nutrient limitation to

Received in revised form 27 August 2013

dinoﬂagellate toxicity, interactions with current and future global environmental change variables have

Accepted 28 August 2013

been relatively unexplored. This is a critical question, as dissolution of anthropogenic CO2 emissions into

seawater is leading to progressively lower pH values, or ocean acidiﬁcation. Sea surface temperatures are

Keywords:

concurrently increasing, a trend that is also projected to continue in the future. We conditioned a clonal

Alexandrium catenella

culture of paralytic shellﬁsh poisoning toxin producing Alexandrium catenella (A-11c) isolated from coastal

Dinoﬂagellate

Southern California to factorial combinations of two temperatures, two pCO2 levels, and two phosphate

Global warming

concentrations for a period of eight months. Interactions between these variables inﬂuenced growth and

Harmful algae

Ocean acidification carbon fixation rates and although these treatments only elicited minor differences in toxin profile, total

Paralytic shellﬁsh poisoning toxins cellular toxicity was dramatically affected. Cells conditioned to high pCO2 (levels projected for year 2075)

Saxitoxin and low phosphate at low temperature (15 8C) were the most toxic, while lower pCO2, higher phosphate

levels, and warmer temperature (19 8C) alleviated this toxicity to varying degrees. Overall increased pCO2

generally led to enhanced potency. Our results suggest that future increased ocean acidiﬁcation may

exacerbate the toxic threat posed by this toxic dinoﬂagellate, especially when combined with nutrient

limitation, but that future warmer temperatures could also offset some of this enhanced toxicity.

1. Introduction saxitoxin (STX) and its derivatives, collectively known as paralytic

shellﬁsh poisoning toxins (PSPs) (Kodama, 2010; Anderson et al.,

Dinoﬂagellates of the class Dinophyceae are prominent 2012). Paralytic shellﬁsh poisoning (PSP) is a potentially life-

members of microalgal communities, particularly in estuaries threatening syndrome that is primarily caused by the consumption

and coastal oceans. Recent observations suggest an increasing of contaminated shellﬁsh. PSPs are heat stable tetrahydropurines

frequency and intensiﬁcation of noxious proliferations or harmful that act as potent, selective muscular and neuronal sodium

blooms caused by some members of this group (Hallegraeff, 1993, channel blockers (Catterall, 1980; Anderson et al., 1990). These

2010). This phenomenon could be related to increased sea surface chemicals (n > 20) are divided into four subgroups (carbamate, N-

temperatures, ocean acidiﬁcation, and their associated interactive sulfo-carbamoyl, decarbamoyl and deoxydecarbamoyl) that are

effects with other environmental drivers like nutrient availability indicative of their potencies, which span orders of magnitude

(Fu et al., 2012). The propensity of some harmful dinoﬂagellates to (Boyer et al., 1987; Hall et al., 1990; Hallegraeff et al., 1995;

opportunistically form dense, sometimes visible blooms coupled Quilliam et al., 2001) (Table 1).

with an ability to synthesize a variety of biologically active and The production of PSPs is inﬂuenced by numerous environ-

often toxic chemicals underscores their importance to human and mental conditions including salinity, light and nutrient concen-

ecosystem health. trations (White, 1978; Hall et al., 1982; Ogata et al., 1987; Boyer

The genus Alexandrium is comprised of over thirty species, of et al., 1987; Parkhill and Cembella, 1999). Although there have

which more than half are conﬁrmed producers of the neurotoxin been considerable efforts to investigate these environmental

inﬂuences on Alexandrium toxicity, surprisingly few genus-wide

correlations were observed. These ﬁndings emphasize the well-

known interspeciﬁc variability and less explored intraspeciﬁc

* Corresponding author. Tel.: +1 213 740 5616.

E-mail address: [email protected] (D.A. Hutchins). variability reported from this genus, as well as from other

38 A.O. Tatters et al. / Harmful Algae 30 (2013) 37–43

Table 1 Table 2

PSP congeners detected in Alexandrium catenella A-11c. Speciﬁc toxicity of each Experimental conditions representing combinations of pCO2, temperature and

compound expressed as mouse units (MU) per mmole of toxin in which 1 MU is a phosphate concentrations employed in this study.

dose sufﬁcient to kill a 20 g male mouse (strain ddy) in 15 min (Oshima, 1995).

Experimental treatments

Compound Potency Compound Speciﬁc toxicity 3

Control replete 380 matm pCO2; 15 8C; 2 mm PO4

STX 2483 C2 239 High temperature-replete 380 matm pCO2; 19 8C; 2 mm PO4

GTX-1 2468 GTX-5 160 High pCO2-replete 750 matm pCO2; 15 8C; 2 mm PO4

neoSTX 2295 C1 15 Greenhouse-replete 750 matm pCO2; 19 8C; 2 mm PO4

GTX-4 1803 Control limited 380 matm pCO2; 15 8C; 0.4 mm PO4

High temperature-limited 380 matm pCO2; 19 8C; 0.4 mm PO4

High pCO2-limited 750 matm pCO2; 15 8C; 0.4 mm PO4

Greenhouse-limited 750 matm pCO2; 19 8C; 0.4 mm PO4

dinoﬂagellates (Anderson et al., 1990; Gribble et al., 2005;

Burkholder and Glibert, 2009; Masseret et al., 2009; Adolf et al.,

2009; Bachvaroff et al., 2009; Tillmann et al., 2009; Kremp et al.,

2012). phosphate-limited treatments at 19 8C, growth rates were

1 1

The buildup of anthropogenic carbon dioxide in the surface 0.07 0.01 day and 0.09 0.01 day for unbubbled and bubbled

ocean is progressively contributing to a phenomenon known as cultures, respectively. This methodology has been employed for

ocean acidification (Sabine et al., 2004; Feely et al., 2008). other ocean acidification experiments focusing on dinoflagellates

Simultaneously, the ‘greenhouse’ effects of atmospheric CO2 (Fu et al., 2008, 2010; Tatters et al., 2013).

increases are leading to the elevation of sea surface temperatures

in many regions (Cane et al., 1997; Xie et al., 2010). The responses 2.2. Experimental conditioning and treatments

of a variety of marine phytoplankton to these two global change

variables are being increasingly examined (Beardall and Raven, The three variable factorial matrix design of the experiment

2004; Hutchins et al., 2009; Hallegraeff, 2010; Fu et al., 2012). used phosphate-replete (2 mm) and phosphate-limited (0.4 mm)

There are, however, only a few reports of how ocean acidiﬁcation cultures, each grown in triplicate under four pCO2/temperature

may affect the toxicity and physiology of harmful algal species (Fu conditions: ‘control’ (15 8C, low pCO2), ‘high pCO2 (15 8C, high

et al., 2008, 2010; Sun et al., 2011; Tatters et al., 2012a, 2013; pCO2), ‘high temperature’ (19 8C, low pCO2), and ‘greenhouse’

Kremp et al., 2012; Flores-Moya et al., 2012; reviewed by Fu et al., (19 8C, high pCO2). This eight-treatment experimental matrix is

2012). Even fewer studies have examined the combined effects of shown in Table 2. To insure that the experimental cultures were

ocean acidiﬁcation and temperature on species within the genus fully acclimated to the temperature, pCO2 and nutrient treatments,

Alexandrium (Kremp et al., 2012; Flores-Moya et al., 2012). The they were maintained under these conditions for a period of eight

interactive effects of ocean acidiﬁcation, temperature and nutrient months prior to the experiments using semi-continuous culture

limitation on toxin-producing Alexandrium have not been docu- methodology (Tatters et al., 2012a). Each ﬂask was diluted to a pre-

mented, even though it is evident that future blooms of PSP- determined in vivo chlorophyll a ﬂuorescence values using

producing species will often be affected by all three of these factors autoclave sterilized L1/20 experimental media on a weekly basis

concurrently. To investigate this, we measured growth and PSP over the conditioning period. The approximate number of

toxin production in an Alexandrium catenella (Whedon and Kofoid) generations for this relatively slow-growing species during this

Balech clone isolated from Southern California coastal waters eight-month time period encompassing all treatments were:

following eight months of conditioning to an experimental matrix control, 14–48; high pCO2, 20–65; high temperature, 25–55; and

of two pCO2 concentrations, two temperatures and two conditions greenhouse 25–63.

of phosphate-availability.

2.3. Cell counts, growth rates and primary production (carbon

2. Materials and methods ﬁxation)

2.1. Sample collection, cell isolation and culture maintenance For cell counts, measured culture volumes were ﬁxed with

acidiﬁed Lugol’s iodine solution, settled in 5 mL settling chambers

The strain of Alexandrium catenella used in this study, and enumerated according to the Utermo¨hl method (Utermo¨hl,

designated clone A-11c, was micropipette isolated from a water 1931) using an Accu-Scope 3032 inverted microscope. Speciﬁc

sample collected at Jalama Beach, Santa Barbara County, growth rates for all treatments were determined in individual

California, USA in March of 2011. This isolate was maintained culture ﬂasks over the eight month conditioning period (data not

in autoclaved L1/20 enriched-seawater growth media minus shown), as well as from triplicate replicates during the ﬁnal two

silicate (Guillard and Hargraves, 1993) at a salinity of 35 on a 12-h dilutions prior to sampling (Fig. 1). Growth rates were calculated

light:12-h dark cycle under a near-saturation photon ﬂuence rate from samples obtained seven days apart (post-dilution and prior to

2 1

of 90 mmol m s of cool white ﬂuorescent illumination at 15 subsequent dilution) using the equation m = ln(Nt/No)/t1 t0

and 19 8C. A subculture was transferred into L1/20 experimental (where N is the number of cells at time t1 and t0 (in days)).

media (replete: 32 mm NO3 and 2 mm PO4 and phosphate- Primary production was measured from triplicate replicates after

3 14

limited: 32 mm NO3 and 0.4 mm PO4 ) within sterile tissue incubation with H CO3 under identical environmental condi-

culture ﬂasks approximately one week after isolation. After tions as described in Fu et al. (2008) and analyzed with a Wallac

one month, aliquots of these cultures were inoculated into System 1400 Liquid Scintillation Counter. A total radioactivity of

experimental media and subject to gentle bubbling in 100 mL 3.7 10 MBq was added to 30 mL of culture.

volumes using commercially prepared air/CO2 mixtures

(Praxair) at two concentrations. A preliminary investigation 2.4. Analytical methods

revealed no signiﬁcant differences in growth rates for unbubbled

cultures compared to gently aerated cultures (p > 0.05). For 2.4.1. PSP toxin detection

phosphate-replete treatments at 15 8C the unbubbled cultures Seven days after the ﬁnal dilution, cells from culture volumes of

1 1

grew at 0.17 0.01 day and bubbled at 0.16 0.02 day . For up to 200 mL from each replicate were collected by gentle vacuum

A.O. Tatters et al. / Harmful Algae 30 (2013) 37–43 39

0.3 CO2 coulometer (UIC) according to King et al. (2011). Experimental

pCO2 (in matm) was calculated using CO2SYS software (Lewis and

Wallace, 1998) using these two measured parameters (Table 3). )

-1

pCO2 concentrations between 260 and 319 ppm are referred to as

yad(et

0.2 ‘low’ and values between 571 and 602 ppm as ‘high’ pCO2.

2.5. Statistics

arht

Differences between speciﬁc growth rates, carbon ﬁxation

worG 0.1

rates, cellular STX equivalents, as well as individual PSP derivatives

and ratios after eight months of conditioning for the temperature,

pCO2 and phosphate combinations were tested by one-way ANOVA

using GraphPad Prism . 0.0 2

3. Results control high CO

high temp. greenhouse

3.1. Semi-continuous growth rates

Fig. 1. Speciﬁc growth rates assessed after eight months of conditioning in

phosphate-replete (shaded gray bars) and phosphate-limited (white open bars)

3.1.1. Phosphate-replete cultures

Alexandrium catenella conditioned to an experimental matrix of four temperature

Semi-continuous growth rates for phosphate-replete cultures

and pCO2 treatments. Values are averages of triplicates with standard deviation

of Alexandrium catenella at 15 8C were signiﬁcantly lower at low

depicted in error bars.

1 1

pCO2 (0.16 0.02 day ) than at high pCO2 (0.24 0.02 day , Fig. 1,

p < 0.001). Growth rates were also signiﬁcantly higher in the

filtration onto 25 mm GF/F filters (Whatman). The filters were greenhouse cultures (19 8C and high pCO2) relative to the high

extracted by homogenization into 1% acetic acid using a Tenbroeck temperature treatment (low pCO2) of 19 8C (0.23 0.02 day versus

tissue grinder and the resulting extracts were ﬁltered (0.45 mm) 0.20 0.01 day , respectively, p < 0.001; Fig. 1). The effects of

prior to analysis. PSPs were detected using high performance liquid temperature in the phosphate-replete treatments at low pCO2

chromatography with ﬂuorescence detection (HPLC-FL) with pre- concentration were signiﬁcant (p < 0.001), but not at high pCO2

column oxidation according to Lawrence et al. (2005). The suite of (p > 0.05).

PSPs constituting the toxin proﬁle of A-11c (Table 1) was identiﬁed

and quantiﬁed by comparison to certiﬁed reference standards from 3.1.2. Phosphate-limited cultures

the National Research Council of Canada, including: saxitoxin Speciﬁc growth rates of phosphate-limited control cultures at

1 1

(STX), neosaxitoxin (neoSTX), gonyautoxins (GTX-1 and 4; GTX-2 15 8C were 0.05 0.01 day at low pCO2 and 0.07 0.01 day at

and 3; GTX-5), C1 and 2, and the decarbamoyl analogs (dcSTX, high pCO2 (Fig. 1). At the high temperature, phosphate-limited

dcNEO, dcGTX-2 and 3). Peaks that were believed to be C3 and C4 cultures grew at an average of 0.09 0.010 div day and under

were observed in all replicates of all treatments. These peaks could greenhouse conditions 0.10 0.013 div day (Fig. 1). For each

not be conﬁrmed or quantiﬁed due to the lack of standards and temperature, the differences between pCO2 treatments were not

represented only a minor fraction of the toxins present. Toxicity in signiﬁcant (p > 0.05), but all pairwise temperature variances

saxitoxin equivalents (STX eq) was calculated using conversion consistently were (p < 0.01). However, the growth rates of the

factors based on the speciﬁc toxicity of each derivative reported by cultures grown under greenhouse conditions were approximately 2-

Oshima (1995). fold higher compared to control conditions. As expected, compared to

phosphate-replete cultures, growth rates of phosphate-limited

2.4.2. Carbonate buffer system characterization treatments were signiﬁcantly lower at each temperature and pCO2

Samples for carbonate chemistry analysis were taken monthly combination (p < 0.001). Compared to pCO2 or temperature, phos-

(data not shown) and at the conclusion of the experiment (Table 3). phate had the most obvious effect on growth rates.

Spectrophotometric pH was determined using a Shimadzu 1800UV

dual-beam spectrophotometer according to Clayton and Byrne 3.2. Carbon ﬁxation/primary production

(1993). Dissolved inorganic carbon was analyzed using a CM5230

The relationship between primary production and pCO2

concentration for both phosphate-replete and phosphate-limit-

Table 3

ed cultures strongly resembled growth (Fig. 2). In general,

Seawater carbonate chemistry system values: spectrophotometric pH and total

regardless of the effect of pCO2 and temperature, the carbon

dissolved inorganic carbon (DIC). Calculated pCO2 values (in matm) were obtained

ﬁxation rates were signiﬁcantly higher under phosphate-replete

from the two measured parameters after eight months of conditioning from all

treatments. pCO2 concentrations between 260 and 319 matm are referred to as ‘low’ than phosphate-limited treatments (p < 0.05, 0.01, 0.001).

and values between 571 and 602 matm as ‘high’ pCO2. Regardless of the temperature, the enrichment of pCO2 did

not stimulate the carbon ﬁxation rates of the phosphate-limited

Treatment pH Total DIC Calculated pCO2

cultures. However, under phosphate-replete conditions, the

Phosphate-replete

carbon ﬁxation rates of the cultures at high pCO2 and

Control replete 8.169 1957 285

greenhouse treatments signiﬁcantly increased relative to the

High temperature-replete 8.135 1986 319

High CO2-replete 7.925 2103 571 control and high temperature treatments, respectively

Greenhouse-replete 7.941 2123 555 (p < 0.05). Under phosphate-replete conditions, there was no

Phosphate-limited signiﬁcant effect of temperature on the carbon ﬁxation rates, but

Control-limited 8.205 1949 261 under phosphate-limitation, an increase of 4 8C resulted in

High temperature-limited 8.199 1976 264

stimulation. Except for the control compared to the greenhouse

High CO2-limited 7.912 2145 603

treatment (p < 0.01), phosphate-limited carbon ﬁxation rates

Greenhouse-replete 7.925 2126 579

were not signiﬁcantly different (p > 0.05).

40 A.O. Tatters et al. / Harmful Algae 30 (2013) 37–43

-11 2.0×10 6 A -1

hr 5 1.5×10-11 ) -1 llecClomxifC -1 l l e

clo 4 1.0×10-11 m

f(qeXTS 3

5.0×10-12 2

1 0 e O 2 us 0 Control High C High temp. Greenho

Fig. 2. Carbon ﬁxation rates assessed after eight months of conditioning in B

phosphate-replete (shaded gray bars) and phosphate-limited (white open bars)

Alexandrium catenella conditioned to an experimental matrix of four temperature

) 40

and pCO2 treatments. Values are averages of triplicates with standard deviation -1

lleclomf(qeXTS

depicted in error bars.

3.3. Cellular saxitoxin equivalents

3.3.1. Phosphate-replete cultures

Phosphate-replete cultures grown at 15 8C had total cellular 10

toxicity quotas that were more than twice as high at high pCO2

(4.64 0.38 fmol STX equivalents cell ) than in the control

1 0

treatment (1.99 0.06 fmol cell , p < 0.001; Fig. 3A). Greenhouse

conditioned cultures also had toxicity quotas that were signiﬁcantly 2

Control High CO High temp. Greenhouse

higher (1.5) compared to the high temperature at low pCO2

1 1

(3.01 0.46 fmol cell versus 2.06 0.14 fmol cell , p < 0.05, 1

Fig. 3. Cellular saxitoxin equivalents (fmol cell ) in cultures of Alexandrium

Fig. 3A). For the phosphate-replete cultures there was no signiﬁcant catenella grown in an experimental matrix of four temperature and pCO2 treatments

difference in STX equivalents per cell for control compared to high under (A) phosphate-replete (shaded gray bars) and (B) phosphate-limited (open

white bars) conditions. Note Y-axis scale differences. All values are the average of

temperature, but toxicity quotas were signiﬁcantly higher in the high

triplicates with standard deviations depicted in error bars.

pCO2 relative to the greenhouse treatment (p < 0.001; Fig. 3A).

3.3.2. Phosphate-limited cultures

Phosphate-limited cells had total toxin contents that were

roughly an order of magnitude higher than those in phosphate-

replete cells in each experimental treatment (Fig. 3A and B).

However, toxicity quotas at 15 8C were again signiﬁcantly elevated

under high pCO2 (44.46 2.13 fmol cell ) compared to the control

treatment at low pCO2 (35.28 3.95 fmol cell , p < 0.01, Fig. 3B).

Likewise, greenhouse conditioned cultures again contained signiﬁ-

cantly more toxin equivalents per cell (17.66 1.19 fmol cell ) than

those at the high temperature grown under low pCO2

(12.66 0.89 fmol cell , p < 0.05, Fig. 3B). At both pCO2 levels, total

cellular toxicity decreased dramatically at the higher temperature

(p < 0.001; Fig. 3B).

3.4. Toxin proﬁles for phosphate-replete treatments

3.4.1. Relative toxin proﬁle of PSP derivatives per replete treatment

For phosphate-replete cultures the relative toxin proﬁle is

depicted for control, high CO2, high temperature and greenhouse

conditioned treatments (Fig. 4A and B). The relative contribution of

GTX-5 was 67.7% (control treatment), 56.4% (high CO2 treatment),

56.3% in the high temperature treatment and 64.7% in the

greenhouse treatment. For STX, the relative composition per

treatment was 4.3% (control), 3.3% (high CO2), 4.5% (high

temperature) and 3.2% (greenhouse). For the C-toxins (C1/2),

composition per treatment was 26.4% (control), 38.4% (high CO2),

Fig. 4. Paralytic shellﬁsh poisoning toxin proﬁle of Alexandrium catenella cultures

38.4% (high temperature) and 29.7% (greenhouse). Regarding

conditioned to an experimental matrix of eight temperature, pCO2 and phosphate

neoSTX, the composition of the control treatment was 1.15%, 0.66% combinations. Total congener abundance representations in fmol cell (Panel A)

at high CO2, 0.99% at high temperature and 0.91% for greenhouse. and carbamate compounds (Panel B).

A.O. Tatters et al. / Harmful Algae 30 (2013) 37–43 41

For GTX1-4, the percent composition for both control and high reduced toxicity in faster growing cells at higher temperature

temperature was below the limit of detection, but for high CO2 and lends further support to this hypothesis, as does our previous work

greenhouse treatments was 1.1% and 1.3%, respectively. with the domoic acid-producing diatom genus Pseudo-nitzschia

Pairwise comparisons of individual PSPs revealed a signiﬁcant (Sun et al., 2011; Tatters et al., 2012a).

difference between control versus high temperature (p < 0.05), but PSPs are carbon and nitrogen rich compounds. Both carboxyla-

not for control versus high CO2 or greenhouse conditions tion by Rubisco as well as nitrate reduction via nitrate reductase

(p > 0.05). For the C-toxins (C1/2), STX and neoSTX there was take place within the plastid, and in microalgae, these events are

no signiﬁcant difference in experimental treatments compared to transcriptionally regulated and controlled by light. Given that both

the control (p > 0.05). Regarding the minor constituents GTX-1 and processes utilize energy created by the light reactions of

4, there were differences between the control compared to high photosynthesis, assimilation of NO3 and CO2 may compete for

CO2 and greenhouse, but the signiﬁcance level cannot be reductant produced during photosynthetic electron transport.

determined due to the fact that the values obtained from control There is evidence that enhancement of carbon assimilation and

and high temperature cultures were below the limit of detection. growth under elevated CO2 is highly dependent on the adequate

Despite the large differences in total STX equivalents (Fig. 3A and supply (Gordillo et al., 2001, 2003) and speciation (Clark and Flynn,

B), the toxin proﬁles (both congeners and ratios) in each treatment 2000) of inorganic nitrogen in some marine algae. These results

of the phosphate-replete cultures (Fig. 4A and B) were virtually suggest the possibility of a positive feedback scenario between

identical to corresponding treatments in phosphate-limited elevated CO2 and nitrate enrichment on algal physiology and

cultures (data not shown). toxicity, which should be investigated in future studies in addition

to the phosphorus availability interactions examined here.

3.4.2. Concentrations of various PSP derivatives per replete treatment Effects from CO2 led to general trends regardless of nutrient

For control cells, the toxin proﬁle (all values reported in status or temperature, suggesting ocean acidiﬁcation conditions

fmol cell ) was: 13.61 (GTX-5), 0.86 (STX), 5.30 (C1/2) and 0.31 (co-varying increased CO2 and decreased pH) signiﬁcantly

(neoSTX). GTX-1 and 4 were either not present or below the limit of impacted the biochemistry of Alexandrium catenella. In the

detection (Fig. 4A and B). On average at high CO2, cells contained nutrient replete treatments, growth rates were signiﬁcantly

29.89 fmol of GTX-5, 1.81 of STX, 20.33 of C1/2, 0.35 of neoSTX and increased at both temperatures by higher CO2. This observation is

0.56 of GTX 1 and 4. At high temperature, the proﬁle consisted of suggestive of alleviation from carbon limitation when other

12.28 fmol (GTX-5), 0.98 (STX), 9.77 (C1/2) and 0.21 (neoSTX) with resources are sufﬁcient. Growth rates were also slightly higher at

no detectable GTX 1 and 4. For the greenhouse treatment, the elevated CO2 in phosphate-limited cells, but these differences

concentrations were 21.25 fmol (GTX-5), 1.17 (STX), 10.76 (C1/2), were not signiﬁcant. Carbon ﬁxation rates generally mirrored the

0.30 (neoSTX) and 0.43 (GTX 1 and 4) (Fig. 4A and B). trends of growth and toxicity (Figs. 2 and 3A,B). As carbon is

Pairwise comparisons of individual toxin derivatives revealed required to biosynthesize these chemicals, additional ﬁxed

that for concentrations of GTX-5, the control treatment was carbon not utilized in growth is likely available to be allocated

signiﬁcantly different compared to both high CO2 and high to secondary metabolite production.

temperature treatments, but not compared to the greenhouse Warmer temperatures stimulated growth at low CO2 (but not

treatment (p < 0.01 and p > 0.05). For STX, the control treatment high CO2) in phosphate-replete cells. In phosphate-limited

was significantly different from the high CO2 treatment cultures, growth was significantly influenced by temperature at

(p < 0.001), but the control was not different from the high both CO2 levels. It has been repeatedly demonstrated that the

temperature and greenhouse treatments (p > 0.05). There was no toxicity of Alexandrium spp. is inversely correlated with tempera-

signiﬁcant difference between treatments regarding concentra- ture (Hall et al., 1982; Anderson et al., 1990; Cembella, 1998;

tions of the minor component neoSTX (p > 0.05). For the C toxins Navarro et al., 2006). In our experiments effects of temperature on

(C1/C2) there was a signiﬁcant difference between the control toxicity were generally as anticipated, except for phosphate-

compared to both high CO2 and greenhouse, but not compared to replete treatments under low CO2 where the difference between

the high temperature treatment (p < 0.001, p < 0.01, and p > 0.05). the two temperatures was not signiﬁcant. In general cultures at the

higher temperature, although growing faster, contained less STX

4. Discussion equivalents per cell (Fig. 3A and B). This could be reﬂective of either

toxin production rates decreasing with increasing temperature or

Despite the environmental and economic importance of ‘growth dilution’, where faster growth promotes partitioning of

harmful bloom-forming marine dinoﬂagellates, few investigations toxin among daughter cells.

have focused on their responses to ocean acidiﬁcation and climate In bloom-forming phytoplankton species, rapid proliferation of

change (Rost et al., 2006; Fu et al., 2010; Tatters et al., 2013; Kremp biomass can often lead to nutrient limitation during later stages of

et al., 2012; Flores-Moya et al., 2012; reviewed by Fu et al., 2008, the bloom. In addition, periodic altered nutrient ratios due to

2012). These gaps in our holistic autecological understanding of anthropogenic loading are common in today’s estuaries and

these organisms are a barrier to accurately predicting their impacts coastal oceans. Thus, future simulations that incorporate nutrient

on marine food webs in the future. In our isolate of Alexandrium limitation in conjunction with global change variables are

catenella, high CO2 led to increased cellular toxin levels regardless environmentally relevant. Phosphate-limitation was previously

of temperature or phosphate status examined. High CO2, demonstrated to increase toxicity in several toxin-producing

phosphate-limited cells conditioned to low temperature were Alexandrium species (Boyer et al., 1987; Jauzein et al., 2010; Tatters

the most toxic both in terms of collective STX equivalents (Fig. 3A), et al., 2012b), as well as in some other toxic dinoﬂagellates (Adolf

absolute concentration of STX per cell (Fig. 4A and B) as well as et al., 2009; Fu et al., 2010). In our experiments the dramatically

total concentrations of PSP toxins per cell. enhanced cellular toxicity we observed under phosphate-limita-

We hypothesize that this effect is due, at least in part, to the tion was expected, but was further exacerbated by ocean

shunting of ‘excess’ ﬁxed carbon through secondary metabolic acidiﬁcation conditions. Increased temperature however sup-

pathways under conditions of high CO2 availability (Fu et al., 2012). pressed toxicity in high-CO2 grown cells, especially in phosphate

It is likely that enhanced net photosynthetic rates, simply due to limited cultures which were on average at least 50% less toxic at

inorganic carbon ‘fertilization’, is a plausible explanation for these the warmer temperature (Fig. 3B). This suggests that under future

ﬁndings. The pronounced effect under phosphate-limitation and ‘‘greenhouse’’ conditions, increases in the toxicity of this strain

42 A.O. Tatters et al. / Harmful Algae 30 (2013) 37–43

driven by high CO2 and/or phosphate-limitation may be partially coupled with the existence of biogeographically isolated popula-

(but not entirely) offset by warmer temperatures. tions of Alexandrium in various marine environments worldwide.

Two other studies have examined the mechanism of cell Temperature imposes biogeographic boundaries and exerts direct

speciﬁc enhancement of toxicity by CO2 in dinoﬂagellates. In a control on cellular metabolic processes (Boyd et al., 2010). The

Chesapeake Bay isolate of the dinoﬂagellate Karlodinium veneﬁcum effects of warming on the physiology and biochemistry of harmful

a dramatic shift was observed in karlotoxin (KmTx) congeners, not dinoﬂagellates including Alexandrium spp. have long been investi-

in the amount of toxin produced, as a function of CO2 (Fu et al., gated, yet the potential synergistic effects of accompanying global

2010). They found that high CO2 promoted the production of the change variables like ocean acidiﬁcation have been inadequately

highly potent KmTx-1, relative to the much less toxic KmTx-2 that explored.

predominated in cells grown at present-day CO2. Some clones of K. Currently, there are wide-reaching impacts from seasonal

veneﬁcum are also known to exclusively produce KmTx-1, 2, or accumulations of PSPs throughout marine food webs in numerous

other derivatives, but the effects of CO2 on the toxin proﬁles of regions around North America, including the Gulf of Maine

these strains are unknown. (Shumway et al., 1988; White et al., 1993), Alaska (Gessner and

In a study by Kremp et al. (2012), only two of eight Schloss, 1996), Washington state (Horner et al., 1997; Moore et al.,

Alexandrium ostenfeldii clones were stimulated by high CO2. 2010), and numerous parts of coastal California (Meyer et al., 1928;

There were few reported differences in total PSPs, but the Sommer and Meyer, 1937; Hall et al., 1990; Jester et al., 2009). The

combined CO2 and temperature treatments led to different objective of this study was to examine the effects of simulated

proportions of PSPs in most clones (Kremp et al., 2012). In our greenhouse ocean conditions on the growth and toxin production

study, there were few changes in the proﬁles of PSPs produced by of a California clone of the wide-ranging species Alexandrium

Alexandrium catenella on a percent basis. One difference was the catenella. This study provides the ﬁrst insight into how this

enhancement of GTX 1 and 4 concentrations at high CO2. The fact important species may respond to impending climate change

that low CO2 led to values below the limit of detection but high impacts. If the responses of our isolate are broadly representative,

CO2 levels were consistently twice this level suggests promotion then it is possible that rising dissolved inorganic carbon

of these compounds with increasing CO2. Although GTX 1 and 4 concentrations may lead to increased cellular toxicity in this

were very minor constituents of the toxin proﬁle, PSP toxin species. Concurrent temperature increases however may offset a

proﬁles of other dinoﬂagellates are dominated by these portion of this enhancement.

derivatives (Franco et al., 1994; Pitcher et al., 2007). As GTX 1 Our ﬁndings demonstrate that Alexandrium catenella responds

and 4 are very toxic derivatives, further investigation is to CO2, but this response depends on other co-occurring variables,

warranted. Although there was little change in composition, emphasizing the need to avoid making general conclusions about

the various treatments certainly modulated total cellular toxicity ocean acidiﬁcation effects until potentially interacting factors

(Fig. 3A and B). There are clearly differences in the ways cells have been fully considered (Boyd and Hutchins, 2012). In

arrived the higher potency levels in these studies. The issue of particular, nutrient limitation has seldom been included in

quantity versus quality of toxin biosynthesis is an important experimental designs of previous ocean acidiﬁcation/warming

facet of examining how these organisms respond to any variable experiments, despite its obvious relevance to phytoplankton

and the interactive outcomes of multiple variables, including growth in the present day and future coastal ocean. Experiments

increasing CO2 and temperature. testing the interactive effects of other variables such as trace

Another recent study grew two clones of Alexandrium minutum metals, vitamins and irradiance should also be considered. In

from the Rio de Vigo, NW Spain at two pH levels (7.5 and 8.0, addition, there is a need to examine the responses of toxic

adjusted with HCl) and two temperatures (20 and 25 8C) for a dinoﬂagellates to medium or long-term environmental changes

duration of 720 days (Flores-Moya et al., 2012). The authors by acclimation versus adaptation (Flores-Moya et al., 2012;

attributed observed deviations in growth and toxicity to mutation, Tatters et al., 2013). Ongoing global change processes such as

and suggested that future blooms of A. minutum could increase in acidiﬁcation and warming that are occurring over timescales of

frequency, but that effects on cellular toxicity were unpredictable years to decades make it critical to address questions about

(Flores-Moya et al., 2012). The ﬁndings of this study relative to evolutionary processes, and their consequences for harmful toxic

growth were in agreement with a study conducted by Hwang and blooms in the future ocean.

Lu (2000), who examined another isolate of A. minutum and

observed more robust growth at pH 7.5 versus 5.5 or 8.5. Siu et al. Acknowledgements

(1997) however reported that optimal pH was around 8.5 for a

clone of Alexandrium catenella from Hong Kong. More recently, This study was funded by USC Sea Grant, NSF OCE-0962309 and

Kremp et al. (2012) examined eight strains of Alexandrium the Florida Fish and Wildlife Conservation Commission.[SS]

ostenfeldii from the North Sea grown at 20 and 24 8C in batch

culture under CO2 concentrations of 385 and 750 ppm. The authors References

reported a variety of responses from the different clones, but

Adolf, J.E., Bachvaroff, T.R., Place, A.R., 2009. Environmental modulation of karlo-

overall there was a trend of increasing STX concentrations with

toxin levels in strains of the cosmopolitan dinoﬂagellate Karlodinium veneﬁcum

increasing pCO and increasing temperature individually, as well

2 (Dinophyceae). J. Phycol. 45, 176–192.

as with these variables in concert (Kremp et al., 2012). However, Anderson, D.M., Kulis, D.M., Sullivan, J.J., Hall, S., Lee, C., 1990. Dynamics of

physiology of saxitoxin production by the dinoﬂagellates Alexandrium spp.

these various studies are difﬁcult to directly compare with ours not

Mar. Biol. 104, 511–524.

only because different species were used, but also because they

Anderson, D.M., Alpermann, T.J., Cembella, A.D., Collos, Y., Masseret, E., Montresor,

employed different experimental techniques (acid additions M., 2012. The globally distributed genus Alexandrium: multifaceted roles in

marine ecosystems and impacts on human health. Harmful Algae 14, 10–35.

versus CO2 bubbling, batch versus semi-continuous culturing

Bachvaroff, T.R., Adolf, J.E., Place, A.R., 2009. Strain Variation in Karlodinium vene-

and toxin detection methods, etc.). Increased cellular toxicity

ﬁcum (Dinophyceae): toxin proﬁles, pigments and growth characteristics for

appears to be a general effect of ocean acidiﬁcation on our A. strains grown under common garden conditions. J. Phycol. 45, 137–153.

Beardall, J., Raven, J.A., 2004. The potential effects of global climate change on

catenella A-11c isolate, but the effects on other strains may differ.

microalgal photosynthesis, growth and ecology. Phycologia 43 (1) 26–40.

Aside from these methodological uncertainties, it is no surprise

Boyd, P.W., Strzepek, R., Fu, F.-X., Hutchins, D.A., 2010. Environmental control of

to ﬁnd non-uniform responses to environmental stressors given open ocean phytoplankton groups: now and in the future. Limnol. Oceanogr. 55,

1353–1376. the sheer number of species within this wide-ranging genus,

A.O. Tatters et al. / Harmful Algae 30 (2013) 37–43 43

Boyd, P.W., Hutchins, D.A., 2012. Understanding the responses of ocean biota to a California, USA. Mar. Biol. 156 (3) 493–504, http://dx.doi.org/10.1007/s00227-

complex matrix of cumulative anthropogenic change. Mar. Ecol. Prog. Ser. 470, 008-1103-z.

125–135, http://dx.doi.org/10.3354/meps10121. King, A.L., San˜udo-Wilhelmy, S.A., Leblanc, K., Hutchins, D.A., Fu, F.-X., 2011. CO2 and

Boyer, G.L., Sullivan, J.J., Anderson, R.J., Harrison, P.J., Taylor, F.J.R., 1987. Effects of vitamin B12 interactions determine bioactive trace metal requirements of a

nutrient limitation on toxic production and composition in the marine dinoﬂa- subarctic Paciﬁc diatom. Intl. Soc. Microb. Ecol. J. 5 (8) 1388–1396.

gellate Protogonyaulax tamarensis. Mar. Biol. 96, 123–128. Kodama, M., 2010. Paralytic shellﬁsh toxins: biochemistry and origin. Aqua BioSci.

Burkholder, J.M., Glibert, P.M., 2009. The importance of intraspeciﬁc variability in Monogr. 3 (1) 1–38.

harmful algae. Preface to a collection of topical papers. Harmful Algae 8 (5) Kremp, A., Godhe, A., Egardt, J., Dupont, S., Suikkanen, S., Casabianca, S., Penna, A.,

744–745. 2012. Intraspeciﬁc variability in the response of bloom-forming marine micro-

Cane, M.A., Clement, A.C., Kaplan, A., Kushnir, Y., Pozdnyakov, D., Seager, R., Zebiak, algae to changed climate conditions. Ecol. Evol. 2 (6) 1195–1207, http://

S.E., Murtugudde, R., 1997. Twentieth-century sea surface temperature trends. dx.doi.org/10.1002/ece3.245.

Science 275 (5302) 957–960, http://dx.doi.org/10.1126/science.275.5302.957. Lewis, E., Wallace, D.W.R., 1998. Program developed for CO2 system calculations,

Catterall, W.A., 1980. Neurotoxins that act on voltage-sensitive sodium channels in Carbon Dioxide Information Analysis Center. Report ORNL/CDIAC-105. Oak

excitable membranes. Annu. Rev. Pharmacool. Toxicol. 20, 15–43. Ridge National Laboratory, Oak Ridge, Tennessee, USA.

Cembella, A.D., 1998. Ecophysiology and metabolism of paralytic shellﬁsh toxins in Lawrence, J.F., Niedzwiadek, B., Menard, C., 2005. Quantitative determination of

marine microalgae. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), paralytic shellﬁsh poisoning toxins in shellﬁsh using prechromatographic

Physiological Ecology of Harmful Algal Blooms. Springer, Berlin, pp. 381–403. oxidation and liquid chromatography with ﬂuorescence detection: collabora-

Clayton, T.D., Byrne, R.H., 1993. Spectrophotometric seawater pH measurements: tive study. J. AOAC Int. 88 (6) 1714–1732.

total hydrogen ion concentration scale calibration of m-cresol purple and at-sea Masseret, E., Grzebyk, D., Nagai, S., Genovesi, B., Lasserre, B., et al., 2009. Unex-

results. Deep-Sea Res. Pt. I 40, 2115–2129, http://dx.doi.org/10.1016/0967- pected genetic diversity among and within populations of the toxic dinoﬂagel-

0637(93)90048-8. late Alexandrium catenella as revealed by nuclear microsatellite markers. Appl.

Clark, D.R., Flynn, K.J., 2000. The relationship between the dissolved inorganic Environ. Microb. 75, 2037–2045.

carbon concentration and growth rate in marine phytoplankton. Proc. R. Soc. Meyer, K.F., Sommer, H., Schoenholz, P., 1928. Mussel poisoning. J. Prevent. Med. 2,

B: Biol. Sci. 267, 953–959. 365–394.

Feely, R.A., Sabine, C.L., Hernandez-Ayon, J.M., Ianson, D., Hales, B., 2008. Evidence Moore, S.K., Mantua, N.J., Hickey, B.M., Trainer, V.L., 2010. The relative inﬂuences of

for upwelling of corrosive ‘‘acidified’’ water onto the continental shelf. Science El Ninõ-Southern Oscillation and Pacific Decadal Oscillation on paralytic shell-

320, 1490–1492. fish toxin accumulation in northwest Pacific shellfish. Limnol. Oceanogr. 55 (6)

Flores-Moya, A., Rouco, M., Garcia-Sanchez, M.J., Garcia-Balboa, C., Gonzalez, R., 2262–2274, http://dx.doi.org/10.4319/lo.2010.55.6.2262.

Costas, E., Lopez-Rodas, V., 2012. Effects of adaptation, chance, and history on Navarro, J.M., Munoz, M.G., Contreras, A.M., 2006. Temperature as a factor regulat-

the evolution of the toxic dinoﬂagellate Alexandrium minutum under selection ing growth and toxin content in the dinoﬂagellate Alexandrium catenella.

of increased temperature and acidiﬁcation. Ecol. Evol. 2 (6) 1251–1259, http:// Harmful Algae 5, 762–769.

dx.doi.org/10.1002/ece3.198. Ogata, T., Ishimaru, T., Kodama, M., 1987. Effect of water temperature and light

Franco, J.M., Ferna´ndez, P., Reguera, B., 1994. Toxin proﬁles of natural populations intensity on growth rate and toxicity change in Protogonyaulax tamarensis. Mar.

and cultures of Alexandrium minutum Halim from Galician (Spain) coastal Biol. 95, 217–220.

waters. J. Appl. Phycol. 6, 275–279. Oshima, Y., 1995. Post-column derivation liquid chromatographic method for

Fu, F.-X., Zhang, Y., Warner, M.E., Feng, Y., Hutchins, D.A., 2008. A comparison of paralytic shellﬁsh toxins. J. AOAC Int. 78 (2) 528–532.

future increased CO2 and temperature effects on sympatric Heterosigma aka- Parkhill, J.P., Cembella, A.D., 1999. Effect of salinity, light and temperature and

shiwo and Prorocentrum minimum. Harmful Algae 7, 76–90. inorganic nitrogen in growth and toxicgenicity of the marine dinoﬂagellate

Fu, F.-X., Place, A.R., Garcia, N.S., Hutchins, D.A., 2010. CO2 and phosphate availabili- Alexandrium tamarense from northeastern Canada. J. Plank. Res. 21 (5)

ty control the toxicity of harmful bloom dinoﬂagellate Karlodinium veneﬁcum. 939–955.

Aquat. Microb. Ecol. 59, 55–65, http://dx.doi.org/10.3354/ame01396. Pitcher, G.C., Cembella, A.D., Joyce, L.B., Larsen, J., Probyn, T.A., Ruiz Sebastia´n, C.,

Fu, F.-X., Tatters, A.O., Hutchins, D.A., 2012. Global change and the future of harmful 2007. The dinoﬂagellate Alexandrium minutum in Cape Town harbour (South

algal blooms in the ocean. Mar. Ecol. Prog. Ser. 470, 207–233, http://dx.doi.org/ Africa): bloom characteristics, phylogenetic analysis and toxin composition.

10.3354/meps10047. Harmful Algae 6 (6) 823–836.

Gessner, B.D., Schloss, M.S., 1996. A population-based study of paralytic shellﬁsh Quilliam, M.A., Dell’Aversano, C., Hess, P., 2001. Analysis of PSP toxins by liquid

poisoning in Alaska. Alaska Med. 38 (2) 54–58. chromatography–mass spectrometry. In: Book of Abstract. Second Internation-

Gordillo, F.J.L., Niell, F.X., Figueroa, F.L., 2001. Non-photosynthetic enhancement of al Conference on Harmful Algae Management and Mitigation, 83, 12–16

growth by high CO2 level in the nitrophilic seaweed Ulva rigida C. Agardh November, Qingdao, China.

(Chlorophyta). Planta 213, 64–70. Rost, B., Richter, K., Riebesell, U., Hansen, P.J., 2006. Inorganic carbon acquisition in

Gordillo, F.J.L., Jimenez, C., Figueroa, F.L., Niell, F.X., 2003. Inﬂuence of elevated CO2 red tide dinoﬂagellates. Plant Cell Environ. 29, 810–822.

and nitrogen supply on the carbon assimilation performance and cell compo- Sabine, C.L., Feely, R.A., Gruber, N., Key, R.M., Lee, K., Bullister, J.L., et al., 2004. The

sition of the unicellular alga Dunaliella viridis. Physiol. Plant. 119, 513–518. oceanic sink for anthropogenic CO2. Science 305, 367–371.

Gribble, K.E., Keafer, B.A., Quilliam, M.A., Cembella, A.D., Kulis, D.M., Manahan, A., Siu, G.K.Y., Young, M.L.C., Chan, D.K.O., 1997. Environmental and nutritional factors

Anderson, D.M., 2005. Distribution and toxicity of Alexandrium ostenfeldii which regulate population dynamics and toxin production in the dinoﬂagellate

(Dinophyceae) in Gulf of Maine, USA. Deep-Sea Res. Pt. II 52, 2745–2763. Alexandrium catenella. Hydrobiologia 352, 117–140.

Guillard, R., Hargraves, P.E., 1993. Stichochrysis immobilis is a diatom, not a chryso- Sommer, H., Meyer, K.F., 1937. Paralytic shellﬁsh poisoning. Arch. Pathol. 24,

phyte. Phycologia 32, 234–236. 560–598.

Hall, S., Reichardt, P.B., Neve, R.A., Boyer, G.L., Wichman, C.F., Schroes, H.K., 1982. Sun, J., Hutchins, D.A., Feng, Y., Seubert, E.L., Caron, D.A., Fu, F.-X., 2011. Effects of

Studies on the origin and nature of toxicity in Alaskan bivalves: toxins from changing pCO2 and phosphate availability on domoic acid production and

Protogonyaulax of the Northeast Paciﬁc. J. Shellﬁsh Res. 2 (1) 119. physiology of the marine harmful bloom diatom Pseudo-nitzschia multiseries.

Hall, S., Strichartz, G., Moczydlowski, E., Ravindran, A., Reichart, P.B., 1990. The Limnol. Oceanogr. 56 (3) 829–840.

saxitoxins: sources, chemistry and pharmacology. In: Hall, S., Strichartz, G. Tatters, A.O., Fu, F.-X., Hutchins, D.A., 2012a. High CO2 and silicate limitation

(Eds.), Marine Toxins: Origin, Structure and Molecular Pharmacology, vol. 418. synergistically increase the toxicity of Pseudo-nitzschia fraudulenta. PLoS ONE

Am. Chem. Soc. Symp. Ser, pp. 29–65. 7 (2) e32116, http://dx.doi.org/10.1371/journal.pone.0032116.

Hallegraeff, G.M., 1993. A review of harmful algal blooms and their apparent global Tatters, A.O., Van Wagoner, R.M., Wright, J.L.C., Tomas, C.R., 2012b. Regulation of

increase. Phycologia 32, 79–99. spiroimine neurotoxins and hemolytic activity in laboratory cultures of the

Hallegraeff, G.M., McCausland, M.A., Brown, R.K., 1995. Early warning of toxic dinoﬂagellate Alexandrium peruvianum (Balech & Mendiola) Balech & Tan-

dinoﬂagellate blooms of Gymnodinium catenatum in southern Tasmanian gen. Harmful Algae 19, 160–168, http://dx.doi.org/10.1016/j.hal.2012.07.

waters. J. Plankt. Res. 17, 1163–1176. 002.

Hallegraeff, G.M., 2010. Ocean climate change, phytoplankton community Tatters, A.O., Schnetzer, A., Fu, F.-X., Lie, A.A.Y., Caron, D.A., Hutchins, D.A., 2013.

responses, and harmful algal blooms: a formidable predictive challenge. J. Short-versus long-term responses to changing CO2 in a coastal dinoﬂagellate

Phycol. 46 (2) 220–235. bloom: implications for interspeciﬁc competitive interactions and community

Horner, R.A., Garrison, D.L., Plumley, F.G., 1997. Harmful algal blooms and red tide structure. Evolution 67 (7) 1879–1891, http://dx.doi.org/10.1111/evo.12029.

problems on the U.S. West Coast. Limnol. Oceanogr. 42 (5 (Pt 2)) 1076–1088, Tillmann, U., Alpermann, T.L., da Puriﬁcacao, R.C., Krock, B., Cembella, A., 2009.

The ecology and oceanography of harmful algal blooms. Intra-population clonal variability in allelochemical potency of the toxigenic

Hutchins, D.A., Mulholland, M.R., Fu, F.-X., 2009. Nutrient cycles and marine dinoﬂagellate Alexandrium tamarense. Harmful Algae 8, 759–769.

microbes in a CO2-enriched ocean. Oceanography 22, 128–145. Utermo¨hl, H., 1931. Neue Wege in der quantitativen Erfassung des Planktons (Mit

Hwang, D.F., Lu, Y.H., 2000. Inﬂuence of environmental and nutritional factors on besonderer Beriicksichtigung des Ultraplanktons). Verh. Int. Verein. Limnol. 5,

growth, toxicity, and toxin proﬁle of dinoﬂagellate Alexandrium minutum. 567–596.

Toxicon 38, 1491–1503. White, A.W., 1978. Salinity effects on growth and toxin content of Gonyaulax

Jauzein, C., Labry, C., Youenou, A., Que´re´, J., Delmas, D., Collos, Y., 2010. Growth and excavata, a marine dinoﬂagellate causing paralytic shellﬁsh poisoning. J. Phycol.

phosphorus uptake by the toxic dinoﬂagellate Alexandrium catenella (Dinophy- 14, 475–479.

ceae) in response to phosphate limitation. J. Phycol. 46 (5) 926–936. Xie, S.-P., Deser, C., Vecchi, G.A., Ma, J., Teng, H., Wittenberg, A.T., 2010. Global

Jester, R., Baugh, K.A., Lefebvre, K.A., 2009. Presence of Alexandrium catenella and warming pattern formation: sea surface temperature and rainfall. J. Climate 23,

paralytic shellfish toxins in finish, shellfish and rock crabs in Monterey Bay, 966–986, http://dx.doi.org/10.1175/2009JCLI3329.1. Critical Review

pubs.acs.org/est

A Critical Review of the Risks to Water Resources from Unconventional Shale Gas Development and Hydraulic Fracturing in the United States † † ‡ § ̂∥ † Avner Vengosh,*, Robert B. Jackson, , Nathaniel Warner, Thomas H. Darrah, and Andrew Kondash † Division of Earth and Ocean Sciences, Nicholas School of the Environment, Duke University, Durham, North Carolina 27708, United States ‡ School of Earth Sciences, Woods Institute for the Environment, and Precourt Institute for Energy, Stanford University, Stanford, California 94305, United States § Department of Earth Sciences, Dartmouth College, Hanover, New Hampshire 03755, United States ∥ ̂School of Earth Sciences, The Ohio State University, Columbus, Ohio 43210, United States

*S Supporting Information

ABSTRACT: The rapid rise of shale gas development through horizontal drilling and high volume hydraulic fracturing has expanded the extraction of hydrocarbon resources in the U.S. The rise of shale gas development has triggered an intense public debate regarding the potential environmental and human health effects from hydraulic fracturing. This paper provides a critical review of the potential risks that shale gas operations pose to water resources, with an emphasis on case studies mostly from the U.S. Four potential risks for water resources are identified: (1) the contamination of shallow aquifers with fugitive hydrocarbon gases (i.e., stray gas contamination), which can also potentially lead to the salinization of shallow groundwater through leaking natural gas wells and subsurface flow; (2) the contamination of surface water and shallow groundwater from spills, leaks, and/or the disposal of inadequately treated shale gas wastewater; (3) the accumulation of toxic and radioactive elements in soil or stream sediments near disposal or spill sites; and (4) the overextraction of water resources for high-volume hydraulic fracturing that could induce water shortages or conflicts with other water users, particularly in water-scarce areas. Analysis of published data (through January 2014) reveals evidence for stray gas contamination, surface water impacts in areas of intensive shale gas development, and the accumulation of radium isotopes in some disposal and spill sites. The direct contamination of shallow groundwater from hydraulic fracturing fluids and deep formation waters by hydraulic fracturing itself, however, remains controversial.

1. INTRODUCTION conventional oil and gas wells in that state (2012 data; Figure 6 Production from unconventiognal natural gas reservoirs has 3). At the end of 2012, the Marcellus Shale (29%), Haynesville substantially expanded through the advent of horizontal drilling Shale (23%), and Barnett Shale (17%) dominated production and high-volume hydraulic fracturing (Figure 1). These of natural gas (primarily methane, ethane, and propane) from technological advances have opened vast new energy sources, shales in the U.S., with the remaining 31% of total shale gas such as low-permeability organic-rich shale formations and production contributed by more than a dozen basins (Figure “tight-sand” reservoirs, altering the domestic energy landscape 1). Oil and hydrocarbon condensates are also targeted in − in the United States.1 3 The total production of natural gas has numerous basins, including the Barnett, Eagle Ford, Utica-Point increased by more than 30% during the past decade. In 2012, Pleasant, and Bakken. 4 unconventional shale gas and tight sand productions were Future energy forecasts suggest that U.S. unconventional respectively accounting for 34% and 24% of the total natural natural gas production from shale formations will double by gas production in the U.S. (0.68 trillion m3).4 The increase in energy production has been broadly Special Issue: Understanding the Risks of Unconventional Shale Gas distributed across the United States (Figure 2) and densely Development distributed within speciﬁc shale plays (Figure 3). Unconven- tional hydrocarbon extraction from organic-rich shale for- Received: November 18, 2013 mations is now active in more than 15 plays in the U.S. In PA Revised: February 9, 2014 alone, 7234 shale gas wells were drilled into the Marcellus Accepted: February 18, 2014 Formation5 in addition to the 34 376 actively producing

2035 and generate ∼50% of the total domestic natural gas production.4 Similarly, U.S. domestic oil production from unconventional shale formations is projected to increase by as much as 15% over the next several decades.7 Unconventional extraction (horizontal drilling and high volume hydraulic fracturing) for shale gas has already expanded to Canada8 and will soon be launched on a global scale, with significant reservoirs in South America, northern and southern Africa, Europe, China,9,10 and Australia.11,12 The current global estimate of natural gas reserves in unconventional shale is approximately 716 trillion m3 (2.53 × 1013 Mcf).11,12 Despite the large resource potentials and economic benefits, the rapid expansion of shale gas development in the U.S. has triggered an intense public debate over the possible environmental and human health implications of the unconventional energy development. Some primary concerns include air pollution, greenhouse gas emissions, radiation, and ground- − water and surface water contamination.1,3,13 36 These concerns have been heightened because the 2005 Energy Policy Act exempts hydraulic fracturing operations from the Safe Drinking Figure 1. Evolution of the volume of natural gas production from Water Act (SDWA). The only exception to the exemption is different unconventional shale plays in the U.S. Data from the U.S. the injection of diesel fuel. Additionally, because environmental 4 Energy Information Administration. oversight for most oil and gas operations is conducted by state rather federal agencies, the regulation, monitoring, and enforcement of various environmental contamination issues

Figure 2. Map of unconventional shale plays in the U.S. and Canada, based on data from the U.S. Energy Information Administration.4

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Figure 3. Map of active unconventional (yellow) and conventional (purple) oil and gas wells in Pennsylvania and West Virginia. Note areas of coexisting conventional and unconventional development (e.g., southwestern PA and WV) relative to areas of exclusively unconventional development (e.g., northeastern PA). Well locations were obtained from the West Virginia Geological Survey (http://www.wvgs.wvnet.edu/) and the Pennsylvania Department of Environmental Protection’s oil and gas reporting Web site (https://www.paoilandgasreporting.state.pa.us/ publicreports/Modules/Welcome/Welcome.aspx). The background topographic map, Marcellus Formation outline, and state boundaries were downloaded from http://www.pasda.psu.edu/ and the Carnegie Museum of Natural History.148 related to unconventional shale gas development are highly the Interior recommends monitoring if water contains more − variable throughout the U.S.37 39 than 10 mg/L (∼14 cc/L) of methane and immediate action if This paper provides an overview and synopsis of recent concentrations rise above 28 mg/L. Several states have defined investigations (updated to January 2014) into one set of a lower threshold (e.g., 7 mg/L in PA), from which household possible environmental impacts from unconventional shale gas utilization of methane-rich groundwater is not recommended. development: the potential risks to water resources. We identify Stray gas migration in shallow aquifers can potentially occur four potential modes of water resource degradation that are by the release of gas-phase hydrocarbons through leaking illustrated schematically in Figure 4 and include (1) shallow casings or along the well annulus, from abandoned oil and gas aquifers contaminated by fugitive natural gas (i.e., stray gas wells, or potentially along existing or incipient faults or contamination) from leaking shale gas and conventional oil and fractures40 with target or adjacent stratigraphic formations gas wells, potentially followed by water contamination from following hydraulic fracturing and drilling (Figure 4).27 The fl hydraulic fracturing uids and/or formation waters from the latter mechanism poses a long-term risk to shallow ground- deep formations; (2) surface water contamination from spills, water aquifers. Microseismic data suggest that the deformation leaks, and the disposal of inadequately treated wastewater or and fractures developed following hydraulic fracturing typically fl hydraulic fracturing uids; (3) accumulation of toxic and extend less than 600 m above well perforations, suggesting that radioactive elements in soil and the sediments of rivers and ffi fl fracture propagation is insu cient to reach drinking-water lakes exposed to wastewater or uids used in hydraulic aquifers in most situations.41 This assertion is supported by fracturing; and (4) the overuse of water resources, which can noble gas data from northeastern PA,42 yet stray gas migration compete with other water uses such as agriculture in water- through fractures and faults is considered a potential limited environments. mechanism for groundwater contamination.40 Across the northeastern Appalachian Basin in PA, the 2. GROUNDWATER CONTAMINATION majority of shallow groundwater had detectable, naturally 2.1. Stray Gas Contamination. Elevated levels of methane occurring methane with thermogenic stable-isotope fingerprints δ13 − δ2 − 27−29,42,43 fi and other aliphatic hydrocarbons such as ethane and propane in (e.g., C CH4 and H CH4). These ndings shallow drinking water wells pose a potential flammability or imply that the high methane in shallow aquifers from this explosion hazard to homes with private domestic wells. The region is predominantly thermogenic in origin.28,29,42,43 In saturation level of methane in near-surface groundwater is northeastern PA, however, a subset of shallow drinking water about ∼28 mg/L (∼40 cc/L) and thus the U.S. Department of wells consistently showed elevated methane, ethane, and

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intermediate layers (e.g., Upper Devonian Formations) in others. In cases where the composition of stray gas is consistent with the target shale formation, it is likely that the occurrence of fugitive gas in shallow aquifers is caused by leaky, failing, or improperly installed casings in the natural gas wells. In other cases, hydrocarbon and noble gas data also indicated that fugitive gas from intermediate formations apparently flowed up through the outside of the well annulus and then leaked into the overlying shallow aquifers.27,29,42 In these cases, the isotopic signatures and hydrocarbon ratios matched the gases in intermediate formations rather than Marcellus shale production gases. In sum, the combined evidence of hydrocarbon stable isotopes, molecular hydrocarbon ratios, and helium geochemistry indicate that stray gas contamination occurs in a subset of wells <1km from drilling in northeastern PA. In contrast to these reports, other investigators22,43,44 have suggested that higher methane concentrations in shallow Figure 4. Schematic illustration (not to scale) of possible modes of groundwater were natural and could be explained by topo- water impacts associated with shale gas development reviewed in this graphic factors associated with groundwater discharge zones. paper: (1) overuse of water that could lead to depletion and water- Geochemical data do suggest that some natural gas migrated to quality degradation particularly in water-scarce areas; (2) surface water shallow aquifers in northeastern PA through geologic time. and shallow groundwater contamination from spills and leaks of However, these characteristics occur in areas with higher wastewater storage and open pits near drilling; (3) disposal of hydraulic connectivity between the deep and shallow inadequately treated wastewater to local streams and accumulation of formations.34,42 A recent analysis showed that topography contaminant residues in disposal sites; (4) leaks of storage ponds that was indeed a statistically significant factor in some cases but did are used for deep-well injection; (5) shallow aquifer contamination by not explain the variations in methane and ethane concen- stray gas that originated from the target shale gas formation through 29 leaking well casing. The stray gas contamination can potentially be trations with respect to distance to gas wells. followed by salt and chemical contamination from hydraulic fracturing Additional evidence for stray gas contamination because of fluids and/or formational waters; (6) shallow aquifer contamination by poor well construction is provided by the isotopic composition stray gas through leaking of conventional oil and gas wells casing; (7) of surface casing vent flow (SCV). Integrating the δ13C data of − shallow aquifer contamination by stray gas that originated from methane (C1), ethane (C2), and propane (C3)45 47 showed intermediate geological formations through annulus leaking of either that stray gas contamination associated with conventional oil shale gas or conventional oil and gas wells; (8) shallow aquifer fl fl wells in Alberta, Canada re ected methane sourced from contamination through abandoned oil and gas wells; (9) ow of gas intermediate formations leaking into shallow aquifers and not and saline water directly from deep formation waters to shallow from the production formations such as the Lower Cretaceous aquifers; and (10) shallow aquifer contamination through leaking of 48 49 injection wells. Mannville Group. Jackson et al. (2013) listed several other case studies that demonstrate evidence for stray gas contamination. While such studies have shown evidence for methane, ethane, and propane contamination associated with propane concentrations (i.e., relatively low hydrocarbon ratios conventional oil production48,50 and coal bed methane,45 51 (C1/C2)) and relatively enriched thermogenic carbon isotope Muehlenbachs (2013) also showed direct evidence for SCV fingerprints in groundwater exclusively <1 km from shale gas leakage from intermediate zones in newly completed and drilling sites. A subset of samples with evidence for stray gas hydraulically stimulated horizontal shale gas wells in the contamination display isotopic reversals (Δ13C= Montney and Horn River areas of northeastern British δ13 −δ13 51 CH4 C2H6 > 0) and proportions of methane, ethane Columbia, Canada. Methane leaking from the annulus of and propane that were consistent with Marcellus production conventional oil and gas wells was also demonstrated in PA.52,53 gases from the region, while some other wells had natural gas Combined, these studies suggest that stray gas contamination compositions consistent with production gases in conventional can result from either natural gas leaking up through the well wells from the overlying Upper Devonian formations.27,29 New annulus, typically from shallower (intermediate) formations, or evaluations of the helium content29 and noble gas geo- through poorly constructed or failing well casings from the chemistry42 in these samples further supports a distinction shale target formations. between naturally occurring “background” hydrocarbon gases The migration of natural gas to the surface through the and groundwater with stray gas contamination in wells located production casing and/or well annulus is “a common near (<1 km) shale gas drilling sites. “Background” gases occurrence in the petroleum industry”51 and can affect a large typically had lower proportions of ethane and propane and fraction of conventional wells. Among the 15 000 production elevated helium concentrations that reflect the history of oil wells tested from the Gulf of Mexico, 43% have reported natural gas migration from the Marcellus source rock to the cement damage after setting that leads to sustained casing Upper Devonian reservoir rocks throughout geological pressure (SCP). These effects increased with time; whereas time.29,42 Thus, the combination of gas geochemical finger- 30% reported damage during the first 5 years after drilling, the printing suggests that stray gas groundwater contamination, percentage increased to 50% after 20 years.54 Likewise, the BP where it occurs, is sourced from the target shale formations Deepwater Horizon oil spill was partly attributed to the fact (i.e., the Marcellus Formation) in some cases, and from that “cement at the well bottom had failed to seal off

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XXXX, XXX, XXX−XXX Environmental Science & Technology Critical Review hydrocarbons”.55 In PA the overall reports of cementing, casing, drinking water wells was associated with an increase of methane and well construction violations total 3% of all shale gas wells.22 with a thermogenic isotopic fingerprint, both of which were However a closer look at the distribution of violations shows associated with an increase in the number of conventional oil large variations in percentage with time (before and after 2009), and gas wells.62 The fraction of drinking water wells that had space, and type of wells.5,56 In particular, the percentage of well chloride concentrations >250 mg/L (EPA threshold for violations was much higher in northeastern and central counties drinking water) in groundwater from Garfield County doubled in PA (10−50%).5 Consequently, reports of stray gas between 2002 (4%) and 2005 (8%), with chloride up to 3000 contamination in areas of unconventional shale gas develop- mg/L in drinking water wells.62 The parallel rise in salinity and ment in the northeastern Appalachian Basin (U.S.) and methane with a thermogenic isotope signature in Garfield Montney and Horn River Basins (Canada) may be associated County could reflect either migration from leaking oil and gas with leaking of oil and gas wells. wells or contamination from unlined surface impoundments.62 In contrast to the results from the Marcellus, Montney, and Overall, the geochemical composition of the salinized ground- Horn River Basins, the Fayetteville Shale in north-central water in such scenarios would mimic the composition of either Arkansas showed no evidence of methane contamination in the formation water in the production formations34 or the groundwater. Studies in this area showed low methane fluids in the shallower or intermediate units (that typically have fi 36,57 concentrations with a mostly biogenic isotopic ngerprint. adifferent water chemistry). While there might be evidence for The authors hypothesized the potential for stray gas water contamination in some areas of conventional oil and gas contamination likely depends on both well integrity and local exploration, groundwater sites in areas affected by stray gas geology, including the extent of local fracture systems that contamination near shale gas sites in northeastern PA have not fl 36 provide ow paths for potential gas migration. to our knowledge shown signs of salinization induced directly In addition to groundwater, surface waters could serve as an by leaking natural gas wells.27,29,34 Unlike other areas in PA, indicator of regional migration from unconventional shale gas northeastern PA was developed recently and almost exclusively development. To date, streams in areas of shale gas drilling for shale gas (Figure 3), with few legacy wells reported in the have not shown systematic evidence of methane contamination. area. Thus, any water contamination in this area attributable to A new methodology for stream-gas sampling as a reconnais- natural gas extraction would be related to current shale gas sance tool for evaluating natural and anthropogenic methane operations rather than to older legacy wells. Therefore leakage from natural gas reservoirs into surface waters was conclusions regarding contamination from saline water and recently demonstrated using inorganic and gas geochemical hydraulic fracturing fluids flow are restricted in both space and tracers and could be applied more widely in areas of oil and gas time and further studies are needed to address this question. development.59 A second mode of groundwater contamination that could 2.2. Groundwater Contamination with Salts or Other evolve from stray gas contamination is oxidation of fugitive Dissolved Constituents. The presence of fugitive gas in methane via bacterial sulfate reduction.50 Evidence for shallow drinking water wells could potentially lead to dissimilatory bacterial sulfate reduction of fugitive methane salinization and other changes of water quality in three possible near conventional oil wells in Alberta, Canada, includes sulfide ways. First, the leaking of natural gas can be associated with the 13 fl fl generation and C-depleted bicarbonate, with lower residual ow of hydraulic fracturing uids and saline formation waters to 50 overlying shallow aquifers. Given the buoyancy of gas, the flow sulfate concentrations relative to the regional groundwater. rate of denser saline water would be substantially slower than Bacterial sulfate reduction reactions due to the presence of the flow of natural gas, and would depend on both the pressure fugitive methane could trigger other processes such as reductive gradients and hydraulic connectivity between the overpressur- dissolution of oxides in the aquifer that would mobilize redox- ized annulus or leaking sites on the wells and the overlying sensitive elements such as manganese, iron, and arsenic from aquifers.53 the aquifer matrix and further reduce groundwater quality. Low An EPA study60 near the town of Pavillion, Wyoming found levels of arsenic and other contaminants, recorded in some drinking water aquifers in TX, were suggested to be linked to water contamination in two shallow monitoring wells. Although 63 this initial study was questioned for adequate sampling contamination from the underlying Barnett Shale, although protocols,22 a follow up study by the U.S. Geological Survey evidence for a direct link to the Barnett remains uncertain. confirmed elevated levels of specific conductance (1500 mS/ A third hypothetical mode of shallow groundwater cm), pH (10−11), methane (25−27 mg/L), ethane, and contamination associated with the presence of stray gas propane.61 However, the mechanisms that caused the apparent contamination is the formation of toxic trihalomethanes contamination of the shallow groundwater in this area are still (THMs), typically co-occurring with high concentrations of under investigation (i.e., contamination from surface ponds or halogens in the saline waters. THMs are compounds with subsurface leaking cement from shale gas wells). halogen atoms (e.g., Cl, Br, or I) substituted for hydrogens in The ability to trace and identify contamination from shale gas the methane molecule. The formation of THMs were exploration is limited because of the relatively short time frame previously recorded in untreated groundwater in the U.S., since the beginning of large-scale shale gas exploration in early- unrelated to shale gas activities, but associated with agricultural 2000s compared to typical groundwater flow rates (i.e., contamination of shallow aquifers.64,65 Numerous studies have decades). However, an evaluation of water contamination demonstrated that the presence of halogens together with associated with conventional oil and gas exploration provides a organic matter in source waters can trigger the formation of much longer time frame for evaluating possible groundwater THMs, specifically in chlorinated drinking water (see contamination. Possible evidence of long-term (2000−2007) references in Section 2.1). However, no data has to our increases in the salinity of groundwater associated with knowledge been reported for the presence of THMs in conventional oil and gas drilling was reported from Garfield groundwater associated with stray gas contamination from shale County, CO. There, a rise of chloride concentrations in gas wells.

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Figure 5. Map of Pennsylvania with density of unconventional well drilling and occurrence of reported environmental violations. Warm colors (red) represent areas of higher density of unconventional well drilling while cooler colors (blue) are areas of lower drilling density. Unconventional wells with reported violations of a release to the environment are shown by yellow dots. Violations include discharge of industrial waste to streams, drill cuttings, oil, brine and/or silt, discharged without a permit, and polluting substances discharged to waterways. Data on violations was obtained from http://www.fractracker.org/downloads/. Unconventional well density was derived from unconventional well data points obtained from the Pennsylvania Department of Environmental Protection’s oil and gas reporting Web site (https://www.paoilandgasreporting.state.pa.us/ publicreports/Modules/Welcome/Welcome.aspx). Background maps, the Marcellus Formation, and state boundaries were downloaded from http://www.pasda.psu.edu/ and the Carnegie Museum of Natural History148

In addition to the effects of poor oil and gas-well integrity, Devonian aquifers in northeastern PA. In this area of PA, which shallow aquifers could potentially be contaminated by the had little oil and gas drilling prior to recent shale gas migration of deep hypersaline water or hydraulic fracturing development (Figure 3), the presence of elevated salts was fluids through conductive faults or fractures.15,34 The potential recorded in a subset of domestic wells during the 1980s.34 upward flow of fluids from the impermeable shale formations is These findings indicate that the salinization phenomenon is not 34 highly debated; one model has suggested that the advective related to recent unconventional drilling in shales. The preferential flow through fractures could allow the transport of presence of naturally occurring saline groundwater in areas of contaminants from the fractured shale to overlying aquifers in a shale gas development in the Appalachian Basin poses relatively short time of six years or less.15 Other studies have challenges for quantifying contamination from active shale gas − disputed this model,66 68 suggesting that the upward flow rate development, including the ability to distinguish naturally of brines would typically be fairly low because of the low occurring groundwater salinization from anthropogenic sources permeability of rocks overlying the shale formations, low of groundwater pollution. upward hydraulic gradients, and the high density of fluids.41,69,70 The hydraulic conductivity along a fault zone 3. SURFACE WATER CONTAMINATION ff seems to have an important e ect on the potential for upward Few studies have analyzed the major chemical constituents in fl 40 migration of hydraulic fracturing uid or underlying brines. injected hydraulic fracturing fluids (although considerable fl fl Evidence for cross-formational uid ow of deep saline information is available on the Web site www.fracfocus.org). groundwater into overlying shallow aquifers, independent of oil Based on the available information, hydraulic fracturing fluids and gas operations, was demonstrated in the Devonian oil- include water (either fresh water or reused hydraulic fracturing 71 bearing formations in southwestern Ontario, Canada, east- water), proppants (sand, metabasalt, or synthetic chemicals 72 central Michigan Basin, Ogallala Aquifer, Southern High added to “prop” incipient fractures open), acids (e.g., Plains, Texas,73,74 and shallow aquifers overlying the Marcellus hydrochloric acid), additives to adjust fracturing fluid viscosity Shale in northeastern PA.34 The latter case appears to be an (guar gum, borate compounds), viscosity reducers (ammonium example of a naturally occurring process in which deep-seated persulfate), corrosion inhibitors (isopropanol, acetaldehyde), Middle Devonian Marcellus-like saline waters (deterimined by iron precipitation control (citric acid), biocides (glutaralde- Br/Cl and 87Sr/86Sr ratios) migrated upward to shallow Upper hyde), oxygen scavengers (ammonium bisulfite), scale inhib-

F dx.doi.org/10.1021/es405118y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Critical Review itors (e.g., acrylic and carboxylic polymers), and friction leaks and spills of flowback and produced water associated with reducers (surfactants, ethylene glycol, polyacryla- shale gas operations (e.g., overflow or breaching of surface pits, − mide).22,49,75 77 Based on different hazardous components of insufficient pit lining, onsite spills); (2) direct, unauthorized, or hydraulic fracturing fluid additives used in wells from the illegal disposal of untreated wastewater from shale gas Marcellus Shale, it was suggested that sodium hydroxide, 4,4- operations; and (3) inadequate treatment and discharge of dimethyl, oxazolidine, and hydrochloric acid would be good shale gas wastewater (e.g., treatment at plants not sufficiently indicators to monitor water contamination upon a leak or a spill designed to remove halogens, radionuclides, or heavy metals). of hydraulic fracturing fluids.78 The first mode of impact from spills and leakage typically More information is available on the inorganic chemistry of occurs near drilling locations. Figure 5 presents the locations of the “flowback” fluids (fluids that return to the surface after the sites where violations related to spills and leaks associated with hydraulic fracturing process is complete) and produced waters shale gas operations have been reported in PA (data on (fluids that are extracted together with the natural gas during violations was obtained from http://www.fractracker.org/ production). Flowback water is a mixture of the injected downloads/). The occurrence and frequency of the spills and hydraulic fracturing fluids and the natural fluids within the leaks appear to coincide with the density of shale gas drilling,92 formation (e.g., brine). In some cases the injected fluid contains as demonstrated by the co-occurrence of Marcellus unconven- recycled flowback water from one or more different drill sites. tional well density in northeastern and western PA (Figure 5). About 10−40% of the volume of the injected fracturing fluids An analysis shows that the number of reported violations returns to the surface after hydraulic fracturing, and the flow increases in areas closer to higher (>0.5 well per km2) shale gas rates of flowback water slow with time to levels of 2−8m3/day drilling density, and the frequency of violations per shale gas during the production stage of a shale gas well.75 The typical well doubles in areas of higher drilling density (Supporting salinity of flowback water increases with time after hydraulic Information Figure S1). One of the unique features of the fracturing due to an increasing proportion of the formation unconventional energy production of low permeable shale and water mixing with injection fluids.79 Produced waters are tight sand formations is the rapid decrease of the natural gas typically composed of naturally occurring hypersaline formation production, up to 85% during the first three years of water, and can also contain oil, bitumen, and hydrocarbon operation.93,94 In order to overcome this decline in production, condensates with high concentrations of total dissolved organic unconventional wells are drilled at high rates, and over 20 000 carbon (up to 5500 mg/L), in addition to the added organic wells have been constructed since the mid 2000s through the chemicals that were reported in flowback waters (e.g., solvents, U.S. The rapid growth and intensity of unconventional drilling − biocides, scale inhibitors).34,35,49,75,79,80 86 In most flowback could lead to a higher probability of surface spills or leaks and and produced waters, the concentrations of toxic elements such potential stray gas contamination of adjacent drinking water as barium, strontium, and radioactive radium are positively wells. correlated with the salinity.34,49,79,81,82 Some flowback and Spills or leaks of hydraulic fracturing and flowback fluids can produced waters, such as those found in the Marcellus shale, pollute soil, surface water, and shallow groundwater with are also enriched in arsenic and selenium87 that are associated organics, salts, metals, and other constituents. A survey of with the high metal contents in shale rock.88 The correlation of surface spills from storage and production facilities at active toxic and radioactive elements with salinity suggests that many well sites in Weld County, Colorado that produces both of the potential water quality issues associated with wastewaters methane gas and crude oil, showed elevated levels of benzene, from unconventional shale gas development may be attribut- toluene, ethylbenzene, and xylene (BTEX) components in able to the geochemistry of the brines within the shale affected groundwater. Following remediation of the spills, a formations. The total dissolved salts (TDS) content of significant reduction (84%) was observed in BTEX levels in the produced water ranges from below seawater (25 000 mg/L) affected wells.95 As mentioned earlier, an EPA study60 in to 7 times more saline than seawater, depending on the shale Pavillion, Wyoming found increased concentrations of benzene, formation. For example, the Fayetteville (25 000 mg/L), xylenes, gasoline range organics, diesel range organics, hydro- Barnett (60 000 mg/L), Woodford (110 000−120 000 mg/L), carbons, and high pH in two shallow monitoring wells.60 The Haynesville (110 000−120 000 mg/L), and Marcellus (up to U.S. Geological Survey conducted a follow up study and found 180 000 mg/L) shale formations vary by nearly an order of similar elevated levels of specific conductance (1500 mS/cm), magnitude in TDS values.83 The salinity, strontium, and barium pH (10−11), methane (25−27 mg/L), ethane and propane, yet contents vary geographically within formations as shown for the low levels of organic compounds.61 The shallow groundwater Marcellus shale.79,82 The volume and the salinity of flowback contamination was linked in part to surface pits used for the waters also vary spatially among shale gas wells. 75 storage/disposal of drilling wastes and produced and flowback In some cases the flowback and produced waters are stored waters.60 Similarly, leaks, spills, and releases of hypersaline in ponds near the drilling sites. The salinity variations of the flowback and produced waters are expected to impact the wastewater generate chemical stratification within the ponds inorganic quality of surface water because these brines contain that is also associated with anoxic conditions of the bottom highly elevated concentrations of salts (Cl, Br), alkaline earth waters in the ponds.89,90 The high salinity and temperature of elements (e.g., Ba, Sr), metalloids (e.g., Se, As), and the flowback water and the anoxic conditions control the radionuclides (e.g., Ra). microbial community in these storage ponds by increasing the A second mode of contamination would be disposal of proportion of halotolerant and anaerobic bacteria spe- untreated wastewater from shale gas operations. A joint U.S. − cies.76,77,89 91 Geological Survey and U.S. Fish and Wildlife Service study Given that produced waters have much higher salinities than showed that the unauthorized disposal of hydraulic fracturing surface waters (typically TDS ≪ 1000 mg/L), even small fluids to Acorn Fork Creek in southeastern Kentucky in May inputs can impact freshwater quality. We consider three and June 2007 likely caused the widespread death or distress of potential modes of impacts on surface water: (1) surface aquatic species.96 Likewise, an experimental release of hydraulic

G dx.doi.org/10.1021/es405118y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Critical Review fracturing fluids in a forest in WV has shown severe damage and management in the Marcellus and other unconventional shale mortality to ground vegetation over a very short time (10 days). gas basins, especially in areas where surface water discharge for Over a longer time (two years), about half of the trees were dust and ice control is still common. dead and sodium and chloride increased by 50 fold in the soil.97 The increasing volume and the potential environmental A third mode of surface water contamination can occur impacts associated with wastewater treatment have increased through improper handling and disposal of hydraulic fracturing the demand for deep well injection sites, catalyzed the fluids and associated wastewater. These types of environmental development of new suitable treatment processes, and led to releases may occur through the disposal of inadequately treated the reuse and recycling of a larger fraction of the wastewater. In wastewater. In the U.S., wastewater from unconventional many states (e.g., Texas), deep injection is the most commonly energy production is managed in various ways; wastewater is applied wastewater management practice, although reuse and sometimes recycled for subsequent hydraulic fracturing recycling is becoming increasingly common during the last operations, injected into deep injection wells, or treated. The several years.102 However, each of these wastewater manage- treatment options include publicly owned treatment works ment methods has environmental risks. For example, the (POTWs), municipal wastewater treatment plants (WWTP), injection of high volumes of wastewater into deep disposal wells − or commercially operated industrial wastewater treatment may induce seismicity and earthquakes,115 122,123 and ground- plants. In addition to these wastewater management techniques, water near injection wells may become contaminated by some states allow operators to spread the fluids onto roads for cement failure or issues of injection well integrity.124 In − dust suppression or deicing.23,35,39,98 102 addition, many of the injection wells are associated with storage The disposal of inadequately treated wastewater from shale ponds that could also pose environmental risks upon leakage gas operations may contaminate surface waters at the disposal from improper lining and management. sites.23,35,99 Effluent discharge from treatment sites in PA were characterized by high levels of salinity (TDS up to 120 000 mg/ 4. THE ENVIRONMENTAL LEGACY OF CHEMICAL L), toxic metals (e.g., strontium, barium), radioactive elements RESIDUES IN AREAS OF DISPOSAL AND LEAKS (radium isotopes), and organic constituents such as benzene Over time, metals, salts, and organics may build up in 23 and toluene. For example, chlorine concentrations were sediments, scales, and soil near wastewater disposal and/or elevated 6000-fold above stream background levels at the point spill sites. Each respective compound has a fixed solubility and of wastewater effluent discharge into surface water at a reactivity (e.g., adsorption), the latter commonly described by treatment facility, while bromide was enriched by up to the distribution coefficient (K ) that varies as a function of pH, 35 d 12 000-fold. In spite of significant dilution, bromide levels in Eh, temperature, and the occurrence of other components in downstream streamwater (∼2 km) were still elevated (16-fold) the water. As a result, the physicochemical conditions of surface 35 above background stream levels. waters and the distribution coefficients of each compound will Such bromide enrichment in waterways has important determine how it interacts with particulate matter (e.g., implications for downstream municipal water treatment plants, colloidal particles) or river sediments. Ultimately, these given the potential formation of carcinogenic THMs in properties will determine the long-term environmental fate of 103−111 chlorinated drinking water. As the volume of wastewater such reactive contaminants; reactive constituents would be treatment from unconventional energy development has adsorbed onto soil, stream, or pond sediments and potentially expanded, bromide concentrations downstream from waste- pose long-term environmental and health risks. 112 water disposal sites along the Monongahela River in PA, and Marcellus wastewaters contain elevated levels of naturally THM concentrations, especially of brominated species, occurring radionuclides (NORM) in the form of radium 113 increased in municipal drinking water in Pittsburgh, PA. isotopes.34,35,81,85 The elevated radium levels in Marcellus Both sources of contamination were linked directly to the brines is due to the mobilization of radium from uranium-rich disposal and ineffective removal of bromide from wastewater source rocks into the liquid phase under high salinity and 112−114 from shale gas development. In spite of a 2011 ban on reducing conditions.85 Disposal of the NORM-rich Marcellus the disposal of shale gas wastewater to streams in PA, evidence waste fluids to freshwater streams or ponds would cause radium 35 for the Marcellus wastewater disposal based on isotopic ratios adsorption onto the stream sediments in disposal and/or spill and elevated Br levels collected from the Clarion River after sites because radium adsorption is inversely correlated with 113 − May 2011 was suggested to reflect either illegal dumping or salinity.125 128 Disposal of treated wastewater originating from incomplete implementation of the ban where a portion of both conventional and unconventional oil and gas production unconventional wastewater is still being transferred to brine in western Pennsylvania has caused radium accumulation on 113 treatment facilities. stream sediments downstream of a disposal site from a brine In several disposal sites examined in PA, the wastewater treatment facility.35 The radium accumulated in the stream effluent had Marcellus-like geochemical fingerprints such as sediments had 228Ra/226Ra ratios identical to those of the 99 high TDS, low SO4/Cl ratio and distinctive Br/Cl, Marcellus brines, thus linking this accumulation directly to the 228Ra/226Ra, and 87Sr/86Sr ratios.35 These geochemical param- disposal of unconventional shale gas wastewater. The level of eters suggest that at least some of the stream contamination in radioactivity found in sediments at one brine-treatment western Pennsylvania was related to wastewater disposal from discharge site exceeded the management regulations in the shale gas operations, in addition to the legacy disposal of U.S. for a licensed radioactive waste disposal facility.35 Elevated wastewater from conventional oil and gas activities on longer NORM levels were also found in soils near roads associated (decades) time scales. The potential formation of THMs in with road spreading of conventional oil and gas brines for bromide-rich water is not restricted to shale gas operations, and deicing129 and on pond bottom sediments associated with a could also result from disposal of wastewaters from conven- spill from hydraulic fracturing activities.58 High NORM levels tional oil and gas or coalbed methane operations. Overall, more were recorded also in soil and sludge from reserve pits used in data is needed to evaluate the impact of wastewater unconventional natural gas mining.130 In addition to the high γ

H dx.doi.org/10.1021/es405118y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Critical Review

Table 1. Water Use and Wastewater Production Per Shale Gas Well in Diﬀerent Shale Gas Basins in the U.S. Based on Previous a Reports and on Data Compiled in This Study

basin water use per well (m3) wastewater per well (m3) source Horn River Basin (British Columbia, Canada) 50 000 Johnson and Johnson (2012)136 Marcellus Shale, PA (<2010) 7700−38 000 Kargbo et al. (2010)1 Marcellus Shale, PA (2008−2011) 11 500−19 000 5200 Lutz et al. (2013) 101 Marcellus Shale, PA (2012) 3500 this study Woodford Shale, OK 16 000 Murray (2013)135 Barnett Shale, TX 10 600 Nicot and Scanlon (2012)134 Haynesville Shale, TX 21 500 Nicot and Scanlon (2012)134 Eagle Ford, TX 16 100 Nicot and Scanlon (2012)134 Niobrara, CO (2012) 13 000 4000 this study aCalculations for water use and wastewater volume per well for Marcellus Shale in PA were made for 2012 data retrieved from PA Department of Environmental Protection’s oil and gas reporting website (https://www.paoilandgasreporting.state.pa.us/publicreports/Modules/Welcome/ Welcome.aspx) and for the Niobrara Shale in CO were taken from the Colorado Oil and Gas Conservation Commission (http://cogcc.state.co. us/).

Figure 6. Map of the baseline water stress conditions139 in relation to shale play locations across the U.S. Note that many of the western shale plays are associated with high to severe baseline water stress, which reflects the ratio between water withdrawals and availability.139 radiation associated with radionuclides from the 226Ra decay solids from brine treatment facilities132 will sometimes have series (214Pb, 214Bi, 210Pb) and 232Th- decay series (228Ra, 228Th, similar high levels of radioactivity. We conclude that reactive 208Tl), elevated beta radiation was observed, up to 50 000 Bq/ residuals in brines, such as metals and radioactive elements, can kg.130 accumulate in river and lake sediments and could pose long- These results highlight the risks associated with the disposal term environmental and health effects by slowly releasing toxic or spill of NORM-rich flowback and produced waters; even if elements and radiation in the impacted areas. the disposal is within regulations, the high volumes of wastewater can lead to a buildup of radium, and radiation can 5. WATER SCARCITY AND SHALE GAS pose substantial environmental and health risks. Likewise, DEVELOPMENT radium-bearing barite is a common constituent of scale and Evaluations of the water footprint for shale gas development sludge deposits that are associated with conventional oil and gas have been based on the amount of water used for drilling and − exploration.130 133 Elevated radium levels were recorded in soil hydraulic fracturing. Reports of the water consumption for and pipe-scale near oil production sites in the U.S., with 226Ra shale gas development from the Marcellus,101 Barnett, activity up to ∼490 000 Bq/kg.131 We expect that solid wastes Haynesville, Eagle Ford,134 Woodford Shale,135 and Horn from drilling and soil near shale gas drilling sites as well as River in British Columbia8,49,136 showed that water use varies

I dx.doi.org/10.1021/es405118y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Critical Review from 8000 to 100 000 m3 (2−13 million gallons) per geochemical techniques such as major and trace elements, δ18O unconventional well (Table 1). and δ2H in water and δ13C in DIC, methane concentration, and δ13 − δ2 − Total water use for shale gas development overall is relatively stable isotopes of methane ( C CH4, H CH4)for low compared to other water withdrawal sources, such as adequate characterization of the chemical and isotopic cooling water for thermoelectric-power generation, which composition of regional aquifers in areas of shale gas consists ∼40% of total freshwater withdrawals in the development. The baseline data, followed by data generation U.S.137,134,135 Based on two independent reports,6,138 the from continuous monitoring and production gas chemistry total number of shale gas wells in the U.S. was about 20 000 in should become accessible to the scientific community and will 2012. That number of wells accounts for an overall be used to evaluate cases where water contamination may (cumulative) water footprint (based on an average of 15 000 occur. Third, transparency and data sharing, including full m3 used per well) of ∼300 × 106 m3.Adifferent study has disclosure of all hydraulic fracturing chemicals, are critical for suggested a similar volume of water use of ∼250 × 106 m3.139 establishing an open and scientific discussion that could Assuming that 10% of the water used for thermoelectric-power alleviate potential legal and social conflicts. generation is lost through evaporation137 (∼27.8 × 109 m3 out With respect to wastewater management, enforcing zero of 278 × 109 m3 per year), the total water volume that has been discharge policy for untreated wastewater and establishing consumed during the past decade for hydraulic fracturing adequate treatment technologies could prevent surface water (∼300 × 106 m3) was only ∼1% of that annual water loss from contamination. Currently two types of wastewater treatment cooling thermoelectric-power generation. are used; thermal evaporation/distillation and brine treatment 100,144 However, in geographic areas with drier climates and/or through lime and Na2SO4 addition. While thermal higher aquifer consumption, such as Texas, Colorado, and evaporation/distillation removes all dissolved salts in the California, groundwater exploitation for hydraulic fracturing wastewater, brine treatment with lime and Na2SO4 addition can lead to local water shortages134 and subsequent degradation only removes metals such as barium and NORM but does not of water quality. Even in wet areas, variations in the stream remove halogens (chloride and bromide)23,35 In order to flows can induce water shortage upon water extraction for reduce the potential formation of THM compounds in hydraulic fracturing.140 In small to moderate streams in the downstream drinking water facilities, additional remediation Susquehanna River Basin of northern Appalachian Basin, water technologies, such as complete desalination,145 should be withdrawals for hydraulic fracturing can exceed the natural introduced for removal of the dissolved salts to levels flows, particularly during low-flow periods.37 Likewise, water acceptable for healthy stream ecology (e.g., TDS <500 mg/ use for hydraulic fracturing in southern Alberta, Canada has L). Likewise, reduction of the radioactivity from wastewater and become limited because the river waters is already allocated for safe disposal of NORM-rich solid wastes and/or solid residues other users, and in other locations in British Columbia, the from treatment of wastewater is critical in preventing variability in stream discharge is a limiting factor for contamination and accumulation of residual radioactive 8 withdrawals for shale gas exploration. In addition to detailed materials.35 Since disposal of wastewater through deep-well 134,135 water balance evaluations in several basins, nearly half of injection is not always possible and large volume of injection the shale gas wells in the U.S. were developed in basins with could induce seismicity in some areas, developing remediation 139 high water scarcity, particularly in Texas and Colorado. The technologies for adequate treatment and safe disposal of overlap of the shale plays with water basins where water wastewater is critical for protecting waterways. 139 withdrawal exceeds 40% of the annual replenishment is Finally, the possible limitation of fresh water resources for illustrated in Figure 6 and consistent with the exceptional 2013 shale gas development could be mitigated by either using drought conditions in western U.S. (SI Figure S2). Alternative alternative water resources that would substitute for fresh water 8 water sources, such as brackish to saline groundwater, treated or using other types of liquids (e.g., gel) for hydraulic domestic wastewater, and/or acid mine drainage in the fracturing. The beneficial use of marginal waters (i.e., water 141−143 Appalachian Basin should be considered as potential with low quality that cannot be used for the domestic or alternatives for drilling and hydraulic fracturing in these areas. agricultural sectors) is that in many cases such water is Likewise, the increased reuse of shale gas wastewater for discharged and can harm the environment; using these water 75 hydraulic fracturing could mitigate the water gap. Overall the sources for hydraulic fracturing could therefore have multiple expected rise in unconventional wells will increase the water use advantages. For example, the use of acid mine drainage (AMD) and possibly the water footprint in the U.S. for hydraulic fracturing could mitigate the current AMD discharge and contamination of numerous waterways in eastern 6. POSSIBLE SOLUTIONS U.Sly. Experimental blending of AMD and Marcellus flowback Given the different risks to water resources that are associated waters has shown that the blending causes the formation of Sr- with shale gas development in the U.S., we consider several Barite salts that in turn capture some of the contaminants in plausible solutions that could mitigate some of the identified both fluids (e.g., sulfate and iron in AMD, barium, strontium, problems. Previous studies have identified stray gas contami- and radium in flowback waters).143 In the Horn River Basin of nation particularly in drinking water wells located less than 1 British Columbia, Canada, saline groundwater with TDS of up 27,29 km from drilling sites. Enforcing a safe zone of 1 km to 30 000 mg/L is treated to remove H2S and other gases and between new installed shale gas sites and already existing used for hydraulic fracturing.8 The current upper limit for drinking water wells could reduce the risk of stray gas salinity (for adjusting to friction reducers) in hydraulic contamination in drinking water wells in these areas. Second, fracturing fluids is about 25 000 mg/L, although a salt-tolerant the debate whether the occurrence of natural gas in drinking and water-based friction reducer has been developed to enable water is naturally occurring or related directly to contamination recycling of even higher saline wastewater for hydraulic through leaking from shale gas wells could be addressed by fracturing.146 Consequently, utilization of saline, mineralized, mandatory baseline monitoring that would include modern and other types of marginal waters should be considered for

J dx.doi.org/10.1021/es405118y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Critical Review hydraulic fracturing and drilling, particularly in areas highly alleviate the impact of shale-gas development on water water scarcity. Recycling shale gas wastewater with marginal resources. waters can generate both a new water source for hydraulic fracturing and mitigate the environmental effects associated ■ ASSOCIATED CONTENT with the wastewater disposal. Likewise, oil and gas produced fi *S Supporting Information waters can be bene cially used upon adequate treatment and fi management147 Supplement gures on the association of water spill violations to shale gas well density and water scarcity are presented in 7. CONCLUSIONS Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Our survey of the literature has identified four plausible risks to water resources associated with shale gas development and hydraulic fracturing, as illustrated in Figure 4. The first risk is ■ AUTHOR INFORMATION contamination of shallow aquifers in areas adjacent to shale gas Corresponding Author development through stray gas leaking from improperly *Phone: 919-681-8050; fax: 919-684-5833; e-mail: vengosh@ constructed or failing gas wells. Over a longer-time scale, the duke.edu. quality of groundwater in aquifers where stray gas contami- Notes nation has been identified could potentially be impacted by Notes. The authors declare no competing financial interest. both leaking of saline water and hydraulic fracturing fluids from shale gas wells and by secondary processes induced by the high content of methane in the groundwater (i.e., sulfate reduction). ■ ACKNOWLEDGMENTS Thus, evidence of stray gas contamination could be indicative The Nicholas School of Environment at Duke University, the of future water quality degradation, similar to that observed in National Science Foundation (EAR-1249255), and the Park some conventional oil and gas fields. The second risk is Foundation supported some parts of the research described in contamination of water resources in areas of shale gas this study. development and/or waste management by spills, leaks, or disposal of hydraulic fracturing fluids and inadequately treated ■ REFERENCES wastewaters. The third risk is accumulation of metals and radioactive elements on stream, river and lake sediments in (1) Kargbo, D. M.; Wilhelm, R. G.; Campbell, D. J. Natural gas plays in the Marcellus Shale: challenges and potential opportunities. Environ. wastewater disposal or spill sites, posing an additional long- Sci. Technol. 2010, 44 (15), 5679−5684. term impact by slowly releasing toxic elements and radiation to (2) Kerr, R. A. Natural gas from shale bursts onto the scene. Science the environment in the impacted areas. The fourth risk is the 2010, 328 (5986), 1624−1626. water footprint through withdrawals of valuable fresh water (3) Schnoor, J. L. Shale gas and hydrofracturing. Environ. Sci. Technol. from dry areas and overexploitation of limited or diminished 2012, 46 (9), 4686−4686. water resources for shale gas development. (4) Annual Energy Outlook 2013; United States Energy Information Much of the debate on the possibility of water contamination Administration, 2013. is related to the availability of baseline water chemistry data in (5) Brantley, S. L.; Yoxtheimer, D.; Arjmand, S.; Grieve, P.; Vidic, R.; aquifers before shale gas development. Yet full baseline data is Pollak, J.; Llewellyn, G. T.; Abad, J.; Simon, C. Water resource impacts often unavailable, given the lack of systematic and component- during unconventional shale gas development: The Pennsylvania fi Experience. Int. J. Coal Geol. 2014 (http://dx.doi.org/10.1016/j.coal. speci c monitoring of private wells and surface water systems 2013.12.017). across the U.S. Developing novel geochemical and isotopic (6) Ridlington, E.; Rumpler, J. Fracking by the Numbers: Key Impacts tracers that would confirm or refute evidence for contamination of Dirty Drilling at the State and National Level; Environment America can help fill this data gap. The study of water contamination is Research & Policy Center, 2013; p 46. often based on the characterization of water quality in a (7) Annual Energy Outlook 2014 Early Release Overview; United States regional aquifer and/or surface water away from contamination Energy Information Agency, 2013. sites, rather than monitoring water quality changes through (8) Rivard, C.; Lavoie, D.; Lefebvre, R.; Sejourné ,́ S.; Lamontagne, time. Retrospective studies of water contamination associated C.; Duchesne, M. An overview of Canadian shale gas production and with shale gas development should therefore include a environmental concerns. Int. J. Coal Geol., 2013, in press (http://dx. comprehensive investigation of the hydrology, hydrogeology, doi.org/10.1016/j.coal.2013.12.004). (9) Hu, D.; Xu, S. Opportunity, challenges and policy choices for water chemistry, and isotopic tracers for delineating the sources China on the development of shale gas. Energy Policy. 2013, 60,21− and mechanisms of water contamination in questioned areas. 26. Finally, more studies are needed across a broader geographic (10) Xingang, Z.; Jiaoli, K.; Bei, L. Focus on the development of shale area, particularly because many shale gas developments occur in gas in China, Based on SWOT analysis. Renewable Sustainable Energy areas that have been historically exploited for conventional oil Rev. 2013, 21, 603−613. and gas (e.g., PA, WV, CO, TX, and in the future also CA). (11) Boyer, C.; Clark, B.; Jochen, V.; Lewis, R.; Miller, C. K. Shale Most of the scientific publications thus far have addressed water gas: A global resource. Oilfield Rev 2011, 23,28−39. issues in the Appalachian Basin, whereas information for many (12) World Energy Outlook 2012; International Energy Agency (IEA): other basins is limited or not available. Future research should Paris, France, 2012. include studies from other basins in order to overcome these (13) Howarth, R.; Santoro, R.; Ingraffea, A. Methane and the greenhouse-gas footprint of natural gas from shale formations. Clim. gaps and determine the overall risks to water resources from Change 2011, 106 (4), 679−690. shale-gas development. Importantly, many of the risks (14) Howarth, R. W.; Ingraffea, A.; Engelder, T. Natural gas: Should identified in the literature thus far appear possible to mitigate fracking stop? Nature 2011, 477 (7364), 271−275. with increased engineering controls during well construction (15) Myers, T. Potential contaminant pathways from hydraulically and alternative water-management or water-disposal options to fractured shale to aquifers. Ground Water. 2012, 50 (6), 872−882.

K dx.doi.org/10.1021/es405118y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Critical Review

(16) Walton, J.; Woocay, A. Environmental issues related to (33) Tutuncu, A. Environmental challenges of gas and oil shale enhanced production of natural gas by hydraulic fracturing. J. Green stimulation. In SEG Technical Program Expanded Abstracts 2011; pp Build. 2013, 8 (1), 62−71. 3707−3709. (17) Eaton, T. T. Science-based decision-making on complex issues: (34) Warner, N. R.; Jackson, R. B.; Darrah, T. H.; Osborn, S. G.; Marcellus shale gas hydrofracking and New York City water supply. Down, A.; Zhao, K.; White, A.; Vengosh, A. Geochemical evidence for Sci. Total Environ. 2013, 461−462, 158−169. possible natural migration of Marcellus Formation brine to shallow (18) Rozell, D. J.; Reaven, S. J. Water pollution risk associated with aquifers in Pennsylvania. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (30), natural gas extraction from the Marcellus Shale. Risk Anal. 2012, 32 11961−11966. (8), 1382−1393. (35) Warner, N. R.; Christie, C. A.; Jackson, R. B.; Vengosh, A. (19) Vengosh, A.; Warner, N. R.; Jackson, R. B.; Darrah, T. The Impacts of shale gas wastewater disposal on water quality in western effects of shale gas exploration and hydraulic fractruing on the quality Pennsylvania. Environ. Sci. Technol. 2013, 47 (20), 11849−11857. of water resources in the United States. Procedia Earth Planet. Sci. (36) Warner, N. R.; Kresse, T. M.; Hays, P. D.; Down, A.; Karr, J. D.; 2013, 7, 863−866. Jackson, R. B.; Vengosh, A. Geochemical and isotopic variations in (20) Hatzenbuhler, H.; Centner, T. J. Regulation of water pollution shallow groundwater in areas of the Fayetteville Shale development, from hydraulic fracturing in horizontally-drilled wells in the Marcellus north-central Arkansas. Appl. Geochem. 2013, 35, 207−220. − Shale Region, USA. Water. 2012, 4 (4), 983 994. (37) Rahm, B. G.; Riha, S. J. Toward strategic management of shale (21) Schmidt, C. W. Blind rush? Shale gas boom proceeds amid gas development: Regional, collective impacts on water resources. − human health questions. Environ. Health Perspect. 2011, 119, a348 Environ. Sci. Policy. 2012, 17,12−23. a353. (38) Rahm, D. Regulating hydraulic fracturing in shale gas plays: The (22) Vidic, R. D.; Brantley, S. L.; Vandenbossche, J. M.; Yoxtheimer, case of Texas. Energ. Policy 2011, 39 (5), 2974−2981. D.; Abad, J. D. Impact of shale gas development on regional water (39) Rahm, B. G.; Bates, J. T.; Bertoia, L. R.; Galford, A. E.; quality. Science 2013, 340, 6134. Yoxtheimer, D. A.; Riha, S. J. Wastewater management and Marcellus (23) Ferrar, K. J.; Michanowicz, D. R.; Christen, C. L.; Mulcahy, N.; Shale gas development: Trends, drivers, and planning implications. J. Malone, S. L.; Sharma, R. K. Assessment of effluent contaminants from Environ. Manage. 2013, 120, 105−113. three facilities discharging Marcellus Shale wastewater to surface − (40) Kissinger, A.; Helmig, R.; Ebigbo, A.; Class, H.; Lange, T.; waters in Pennsylvania. Environ. Sci. Technol. 2013, 47 (7), 3472 Sauter, M.; Heitfeld, M.; Klünker, J.; Jahnke, W. Hydraulic fracturing 3481. in unconventional gas reservoirs: risks in the geological system, part 2. (24) Dale, A. T.; Khanna, V.; Vidic, R. D.; Bilec, M. M. Process based Environ. Earth Sci. 2013, 70, 3855−3873. life-cycle assessment of natural gas from the Marcellus Shale. Environ. (41) Flewelling, S. A.; Tymchak, M. P.; Warpinski, N. Hydraulic Sci. Technol. 2013, 47 (10), 5459−5466. fracture height limits and fault interactions in tight oil and gas (25) Pacsi, A. P.; Alhajeri, N. S.; Zavala-Araiza, D.; Webster, M. D.; formations. Geophys. Res. Lett. 2013, 40 (14), 3602−3606. Allen, D. T. Regional air quality impacts of increased natural gas (42) Darrah, T. H.; Vengosh, A.; Jackson, R. B.; Warner, N. R.; production and use in Texas. Environ. Sci. Technol. 2013, 47 (7), Poreda, R., Constraining the Source and Migration of Natural Gas in 3521−3527. Shallow Aquifers within Active Shale Gas Production Zone: Insights from (26) Olmstead, S. M.; Muehlenbachs, L. A.; Shih, J. S.; Chu, Z. Y.; Integrating Noble Gas and Hydrocarbon Isotope Geochemistry; Geo- Krupnick, A. J. Shale gas development impacts on surface water quality in Pennsylvania. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (13), 4962− logical Society America Annual Meeting: Charlotte, NC, 6 November 4967. 2012. (27) Osborn, S. G.; Vengosh, A.; Warner, N. R.; Jackson, R. B. (43) Molofsky, L. J.; Connor, J. A.; Wylie, A. S.; Wagner, T.; Farhat, S. K. Evaluation of methane sources in groundwater in northeastern Methane contamination of drinking water accompanying gas-well − drilling and hydraulic fracturing. Proc. Natl. Acad. Sci. U. S. A. 2011, Pennsylvania. Groundwater 2013, 51 (3), 333 349. 108, 8172−8176. (44) Molofsky, L. J.; Connor, J. A.; Farhat, S. K.; Wylie, A. S.; Wagner, T. Methane in Pennsylvania water wells unrelated to (28) Osborn, S. G.; Vengosh, A.; Warner, N. R.; Jackson, R. B. Reply − to Saba and Orzechowski and Schon: Methane contamination of Marcellus shale fracturing. Oil Gas J. 2011, September, 54 67. drinking water accompanying gas-well drilling and hydraulic fracturing. (45) Tilley, B.; McLellan, S.; Hiebert, S.; Quartero, B.; Veilleux, B.; Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (37), E665−E666. Muehlenbachs, K. Gas isotope reversals in fractured gas reservoirs of (29) Jackson, R. B.; Vengosh, A.; Darrah, T. H.; Warner, N. R.; the western Canadian Foothills: Mature shale gases in disguise. AAPG − Down, A.; Poreda, R. J.; Osborn, S. G.; Zhao, K.; Karr, J. D. Increased Bull. 2011, 95 (8), 1399 1422. stray gas abundance in a subset of drinking water wells near Marcellus (46) Tilley, B.; Muehlenbachs, K. Gas maturity and alteration shale gas extraction. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (28), systematics across the Western Canada Sedimentary Basin from four − 11250−11255. mud gas isotope depth profiles. Org. Geochem. 2006, 37 (12), 1857 (30) Jackson, R. B.; Osborn, S. G.; Vengosh, A.; Warner, N. R. Reply 1868. to Davies: Hydraulic fracturing remains a possible mechanism for (47) Tilley, B.; Muehlenbachs, K. Isotope reversals and universal observed methane contamination of drinking water. Proc. Natl. Acad. stages and trends of gas maturation in sealed, self-contained petroleum Sci. U. S. A. 2011, 108 (43), E872. systems. Chem. Geol. 2013, 339, 194−204. (31) Allen, D. T.; Torres, V. M.; Thomas, J.; Sullivan, D. W.; (48) Rowe, D.; Muehlenbachs, K. Isotopic fingerprints of shallow Harrison, M.; Hendler, A.; Herndon, S. C.; Kolb, C. E.; Fraser, M. P.; gasesintheWesternCanadiansedimentary basin: tools for Hill, A. D.; Lamb, B. K.; Miskimins, J.; Sawyer, R. E.; Seinfeld, J. H. remediation of leaking heavy oil wells. Org. Geochem. 1999, 30 (8A), Measurements of methane emissions at natural gas production sites in 861−871. the United States. Proc. Natl. Acad. Sci. U. S. A. 2013, DOI: 10.1073/ (49) Jackson, R. E.; Gorody, A. W.; Mayer, B.; Roy, J. W.; Ryan, M. pnas.1304880110. C.; Van Stempvoort, D. R. Groundwater protection and unconven- (32) Petron, G.; Frost, G.; Miller, B. R.; Hirsch, A. I.; Montzka, S. A.; tional gas extraction: the critical need for field-based hydrogeological Karion, A.; Trainer, M.; Sweeney, C.; Andrews, A. E.; Miller, L.; research. Ground Water. 2013, 51 (4), 488−510. Kofler, J.; Bar-Ilan, A.; Dlugokencky, E. J.; Patrick, L.; Moore, C. T.; (50) Van Stempvoort, D.; Maathuis, H.; Jaworski, E.; Mayer, B.; Rich, Ryerson, T. B.; Siso, C.; Kolodzey, W.; Lang, P. M.; Conway, T.; K. Oxidation of fugitive methane in ground water linked to bacterial Novelli, P.; Masarie, K.; Hall, B.; Guenther, D.; Kitzis, D.; Miller, J.; sulfate reduction. Ground Water. 2005, 43 (2), 187−199. Welsh, D.; Wolfe, D.; Neff, W.; Tans, P. Hydrocarbon emissions (51) Muehlenbachs, K. Identifying the Sources of Fugitive Methane characterization in the Colorado Front Range: A pilot study. J. Associated with Shale Gas Development; Resources For the Future: Geophys. Res., Atmos. 2012, 117. Washington, DC, 2011.

L dx.doi.org/10.1021/es405118y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Critical Review

(52) Harrison, S. S. Evaluating system for ground-water contami- (73) Mehta, S.; Fryar, A. E.; Banner, J. L. Controls on the regional- nation hazards due to gas-well drilling on the glaciated Appalachian scale salinization of the Ogallala aquifer, Southern High Plains, Texas, Plateau. Ground Water. 1983, 21 (6), 689−700. USA. Appl. Geochem. 2000, 15 (6), 849−864. (53) Harrison, S. S. Contamination of aquifers by overpressuring the (74) Mehta, S.; Fryar, A. E.; Brady, R. M.; Morin, R. H. Modeling annulus of oil and gas wells. Ground Water. 1985, 23 (3), 317−324. regional salinization of the Ogallala aquifer, Southern High Plains, TX, (54) Brufatto, C.; Cochran, J.; Conn, L.; Ower, D. From mud to USA. J. Hydrol. 2000, 238 (1−2), 44−64. cementBuilding gas wells. Oil Field Review2003, Autum, 62−76. (75)Gregory,K.B.;Vidic,R.D.;Dzombak,D.A.Water (55) Graham, B.; Reilly, W. K.; Boesch, D. F.; Garcia, T. D.; Murray, management challenges associated with the production of shale gas C. A.; F., U. Deep Water: The Gulf Oil Disaster and the Future of by hydraulic fracturing. Elements 2011, 7 (3), 181−186. Offshore Drilling; National Commission on the BP Deepwater Horizon (76) Struchtemeyer, C. G.; Elshahed, M. S. Bacterial communities Oil Spill and Offshore Drilling, 2011. associated with hydraulic fracturing fluids in thermogenic natural gas (56) Ingraffea, A. R.; Burchell, A.; Howarth, R.; Colborn, T.; Wilson, wells in North Central Texas, USA. FEMS Microbiol. Ecol. 2012, 81 W.; Doe, P.; Wood, M. C. Hydrocarbon Development from Shale: A (1), 13−25. set of important, unsolved problems. In American Geophysical Union (77) Struchtemeyer, C. G.; Morrison, M. D.; Elshahed, M. S. A Fall Meeting; San Fransisco, CA, December 9−13,2013. critical assessment of the efficacy of biocides used during the hydraulic (57) Kresse, T. M.; Warner, N. R.; Hays, P. D.; Down, A.; Vengosh, fracturing process in shale natural gas wells. Int. Biodeterior. Biodegrad. − A.; Jackson, R. B. Shallow Groundwater Quality and Geochemistry in the 2012, 71,15 21. Fayetteville Shale Gas-Production Area, North-Central Arkansas, 2011: ; (78) Aminto, A.; Olson, M. S. Four-compartment partition model of USGS: 2012. 31. hazardous components in hydraulic fracturing fluid additives. J. Nat. − (58) Warner, N. R.; Jackson, R. B.; Vengosh, A. Tracing the Legacy of Gas Sci. Eng. 2012, 7,16 21. Accidental Spills and Releases of Marcellus Wastewater in Pennsylvania; (79) Barbot, E.; Vidic, N. S.; Gregory, K. B.; Vidic, R. D. Spatial and Geological Society of America: Denver, CO, 27−30 October 2013. temporal correlation of water quality parameters of produced waters (59) Heilweil, V. M.; Stolp, B. J.; Kimball, B. A.; Susong, D. D.; from devonian-age shale following hydraulic fracturing. Environ. Sci. − Marston, T. M.; Gardner, P. M. A stream-based methane monitoring Technol. 2013, 47 (6), 2562 2569. approach for evaluating groundwater impacts associated with (80) Orem, W.; Tatu, C.; Varonka, M.; Lerch, H.; Bates, A.; Engle, unconventional gas development. Ground Water. 2013, 51 (4), 511− M.; Crosby, L.; McIntosh, J. Organic substances in produced and 524. formation water from unconventional natural gas extraction in coal (60) DiGiulio, D. C.; Wilkin, R. T.; Miller, C.; Oberley, G. and shale. Int. J. Coal Geol. 2014 (http://dx.doi.org/10.1016/j.coal. Investigation of Ground Water Contamination near Pavillion, Wyoming 2014.01.003). (81) Haluszczak, L. O.; Rose, A. W.; Kump, L. R. Geochemical (Draft Report); U.S. Environmental Protection Agency, 2011. (61) Wright, P. R.; McMahon, P. B.; Mueller, D. K.; Clark, M. L. evaluation of flowback brine from Marcellus gas wells in Pennsylvania, USA. Appl. Geochem. 2013, 28,55−61. Groundwater-Quality and Quality-Control Data for Two Monitoring (82) Chapman, E. C.; Capo, R. C.; Stewart, B. W.; Kirby, C. S.; Wells near Pavillion, Wyoming,; U.S. Geological Survey, 2012; p 26. Hammack, R. W.; Schroeder, K. T.; Edenborn, H. M. Geochemical (62) Thyne, T. Review of Phase II Hydrogeologic Study (Prepared for and strontium isotope characterization of produced waters from Garfield County), 2008. marcellus shale natural gas extraction. Environ. Sci. Technol. 2012, 46 (63) Fontenot, B. E.; Hunt, L. R.; Hildenbrand, Z. L.; Carlton, D. D., (6), 3545−3553. Jr; Oka, H.; Walton, J. L.; Hopkins, D.; Osorio, A.; Bjorndal, B.; Hu, (83) Osborn, S. G.; McIntosh, J. C.; Hanor, J. S.; Biddulph, D. Q. H.; Schug, K. A. An evaluation of water quality in private drinking Iodine-129, 87Sr/86Sr, and trace element geochemistry of Northern water wells near natural gas extraction sites in the Barnett Shale − Appalachian Basin brines: Evidence for basinal-scale fluid migration formation. Environ. Sci. Technol. 2013, 47 (17), 10032 10040. and clay mineral diagenesis. Am. J. Sci. 2012, 312, 263−287. (64) Carter, J. M.; Lapham, W. W.; Zogorski, J. S. Occurrence of (84) Engle, M. A.; Rowan, E. L. Interpretation of Na-Cl-Br volatile organic compounds in aquifers of the United States. J. Am. − systematics in sedimentary basin brines: Comparison of concentration, Water Resour. As. 2008, 44 (2), 399 416. element ratio, and isometric log-ratio approaches. Math. Geosci. 2013, (65) Carter, J. M.; Moran, M. J.; Zogorski, J. S.; Price, C. V. Factors 45 (1), 87−101. associated with sources, transport, and fate of chloroform and three (85) Rowan, E. L.; Engle, M. A.; Kirby, C. S.; Kraemer, T. F. Radium other trihalomethanes in untreated groundwater used for drinking Content of Oil- And Gas-Field Produced Waters in the Northern − water. Environ. Sci. Technol. 2012, 46 (15), 8189 8197. Appalachian Basin (USA)Summary and Discussion of Data: U.S. (66) Cohen, H. A.; Parratt, T.; Andrews, C. B. Potential contaminant Geological Survey Scientific Investigations Report 2011−5135; U.S. pathways from hydraulically fractured shale to aquifers. Ground Water. Geological Survey, 2011. − 2013, 51 (3), 317 319. (86) Engle, M. A.; Rowan, E. L. Geochemical evolution of produced (67) Myers, T. Potential contaminant pathways from hydraulically waters from hydraulic fracturing of the Marcellus Shale, northern ’ fractured shale to aquifers author s reply. Ground Water. 2013, 51 (3), Appalachian Basin: A multivariate compositional data analysis 319−321. approach. Int. J. Coal Geol. 2013 (http://dx.doi.org/10.1016/j.coal. (68) Saiers, J. E.; Barth, E. Potential contaminant pathways from 2013.11.010). hydraulically fractured shale aquifers. Ground Water. 2012, 50 (6), (87) Balaba, R. S.; Smart, R. B. Total arsenic and selenium analysis in 826−828. Marcellus shale, high-salinity water, and hydrofracture flowback (69) Engelder, T. Capillary tension and imbibition sequester frack wastewater. Chemosphere 2012, 89 (11), 1437−1442. fluid in Marcellus gas shale. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (88) Chermak, J. A.; Schreiber, M. E. Mineralogy and trace element (52), E3625−E3625. geochemistry of gas shales in the United States: Environmental (70) Flewelling, S. A.; Sharma, M. Constraints on upward migration implications. Int. J. Coal Geol. 2013 DOI: 10.1016/j.coal.2013.12.005. of hydraulic fracturing fluid and brine. Ground Water 2013, 52 (1), 9− (89) Morali Mohan, A. M.; Hartsock, A.; Bibby, K. J.; Hammack, R. 19. W.; Vidic, R. D.; Gregory, K. B. Microbial community changes in (71) Weaver, T. R.; Frape, S. K.; Cherry, J. A. Recent cross- hydraulic fracturing fluids and produced water from shale gas formational fluid flow and mixing in the shallow Michigan Basin. Geol. extraction. Environ. Sci. Technol. 2013, 47, 13141−13150. Soc. Am. Bull. 1995, 107 (6), 697−707. (90) Mohan, A. M.; Hartsock, A.; Hammack, R. W.; Vidic, R. D.; (72) Long, D. T.; Wilson, T. P.; Takacs, M. J.; Rezabek, D. H. Stable- Gregory, K. B. Microbial communities in flowback water impound- isotope geochemistry of saline near-surface ground water: East-central ments from hydraulic fracturing for recovery of shale gas. FEMS Michigan basin. Geol. Soc. Am. Bull. 1988, 100 (10), 1568−1577. Microbiol. Ecol. 2013, 86 (3), 567−580.

M dx.doi.org/10.1021/es405118y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Critical Review

(91) Davis, J. P.; Struchtemeyer, C. G.; Elshahed, M. S. Bacterial for disinfection byproduct mixtures health effects research: US EPA’s Communities Associated with Production Facilities of Two Newly four lab study. Environ. Sci. Technol. 2010, 44 (19), 7184−7192. Drilled Thermogenic Natural Gas Wells in the Barnett Shale (Texas, (111) Richardson, S. D.; Plewa, M. J.; Wagner, E. D.; Schoeny, R.; USA). Microb. Ecol. 2012, 64 (4), 942−954. DeMarini, D. M. Occurrence, genotoxicity, and carcinogenicity of (92) Entrekin, S.; Evans-White, M.; Johnson, B.; Hagenbuch, E. regulated and emerging disinfection by-products in drinking water: A Rapid expansion of natural gas development poses a threat to surface review and roadmap for research. Mutat. Res., Rev. Mutat. Res. 2007, waters. Front. Ecol. Environ. 2011, 9 (9), 503−511. 636 (1−3), 178−242. (93) Hughes, J. D. In Tight oil: A solution to U.S. import dependence?, (112) Wilson, J.; Vanbriesen, J. Source water changes and energy Geological Society of America, Abstracts with Programs: Denver, CO, extraction activities in the Monongahela River, 2009−2012. Environ. 2013; p 398. Sci. Technol. 2013, 47, 12575−12582. (94) Hensen, J. In It is more than a simple bell curve., Geological (113) States, S.; Cyprych, G.; Stoner, M.; Wydra, F.; Kuchta, J.; Society of America, Abstracts with Programs: Denver, CO, 2013; p Monnell, J.; Casson, L. Marcellus Shale drilling and brominated THMs 399. in Pittsburgh, Pa., drinking water. J. Am. Water Works Ass. 2013, 105 (95) Gross, S. A.; Avens, H. J.; Banducci, A. M.; Sahmel, J.; Panko, J. (8), 53−54. M.; Tvermoes, B. E. Analysis of BTEX groundwater concentrations (114) Hladik, M. L.; Focazio, M. J.; Engle, M. Discharges of from surface spills associated with hydraulic fracturing operations. J. produced waters from oil and gas extraction via wastewater treatment Air Waste Manage. Assoc. 2013, 63 (4), 424−432. plants are sources of disinfection by-products to receiving streams. Sci. − (96) Papoulias, D. M.; Velasco, A. L. Histopathological analysis of Total Environ. 2014, 466, 1085 1093. fish from Acorn Fork Creek, Kentucky exposed to hydraulic fracturing (115) Ellsworth, W. L. Injection-induced earthquakes. Science 2013, fluid releases. Southeastern Naturalist 2013, 12,90−111. 341 (6142), 142. (97) Adams, M. B. Land application of hydrofracturing fluids (116) Rutqvist, J.; Rinaldi, A. P.; Cappa, F.; Moridis, G. J. Modeling damages a deciduous forest stand in West Virginia. J. Environ. Qual. of fault reactivation and induced seismicity during hydraulic fracturing − 2011, 40 (4), 1340−1344. of shale-gas reservoirs. J. Petrol. Sci. Eng. 2013, 107,31 44. (98) Wilson, J. M.; VanBriesen, J. M. Oil and gas produced water (117) Frohlich, C. Two-year survey comparing earthquake activity and injection-well locations in the Barnett Shale, Texas. Proc. Natl. management and surface drinking water sources in Pennsylvania. − Environ. Pract. 2012, 14 (4), 288−300. Acad. Sci. U. S. A. 2012, 109 (35), 13934 13938. (99) Wilson, J. M.; Wang, Y.; VanBriesen, J. M. Sources of high total (118) Frohlich, C.; Hayward, C.; Stump, B.; Potter, E. The Dallas- Fort Worth Earthquake Sequence: October 2008 through May 2009. dissolved solids to drinking water supply in southwestern Pennsylva- − nia. J. Environ. Eng. 2013, DOI: 10.1061/(ASCE)EE.1943- Bull. Seismol. Soc. Am. 2011, 101 (1), 327 340. 7870.0000733. (119) Horton, S. Disposal of hydrofracking waste fluid by injection (100) Veil, J. A. Water Management Technologies Used by Marcellus into subsurface aquifers triggers earthquake swarm in central Arkansas 2012 Shale Gas Producers; prepared for US Department of Energy Oﬃce of with potential for damaging earthquake. Seismol. Res. Lett. , 83 (2), 250−260. Fossil Fuel Energy, National Energy Technology Laboratory, 2010. (120) Gan, W.; Frohlich, C. Gas injection may have triggered (101) Lutz, B. D.; Lewis, A. N.; Doyle, M. W. Generation, transport, earthquakes in the Cogdell oil field, Texas. Proc. Natl. Acad. Sci. U. S. A. and disposal of wastewater associated with Marcellus Shale gas 2013, 110 (47), 18786−18791. development. Water Resour. Res. 2013, 49 (2), 647−656. (121) Keranen, K. M.; Savage, H. M.; Abers, G. A.; Cochran, E. S. (102) Maloney, K. O.; Yoxtheimer, D. A. Production and disposal of Potentially induced earthquakes in Oklahoma, USA: Links between waste materials from gas and oil extraction from the Marcellus Shale wastewater injection and the 2011 M-w 5.7 earthquake sequence. play in Pennsylvania. Environ. Pract. December 2012, 14 (4), 278-287. Geology 2013, 41 (6), 699−702. (103) Chen, B. Y.; Westerhoff, P. Predicting disinfection by-product − (122) van der Elst, N. J.; Savage, H. M.; Keranen, K. M.; Abers, G. A. formation potential in water. Water Res. 2010, 44 (13), 3755 3762. Enhanced Remote Earthquake Triggering at Fluid-Injection Sites in (104) Chowdhury, S.; Champagne, P.; McLellan, P. J. Factorial the Midwestern United States. Science 2013, 341 (6142), 164−167. analysis of trihalomethanes formation in drinking water. Water Environ. − (123) Llenos, A. L.; Michael, A. J. Modeling earthquake rate changes Res. 2010, 82 (6), 556 566. in Oklahoma and Arkansas: Possible signatures of induced seismicity. (105) Chowdhury, S.; Champagne, P.; McLellan, P. J. Investigating Bull. Seismol. Soc. Am. 2013, 103 (5), 2850−2861. effects of bromide ions on trihalomethanes and developing model for (124) Yuan, Z.; Gardoni, P.; Schubert, J.; Teodoriu, C. Cement predicting bromodichloromethane in drinking water. Water Res. 2010, − failure probability analysis in water injection well. J. Petrol. Sci. Eng. 44 (7), 2349 2359. 2013, 107,45−49. (106) Xue, S.; Zhao, Q. L.; Wei, L. L.; Jia, T. Effect of bromide ion on (125) Krishnaswami, S.; Bhushan, R.; Baskaran, M. Radium isotopes isolated fractions of dissolved organic matter in secondary effluent and 222Rn in shallow brines, Kharaghoda (India). Chem. Geol. 1991, − during chlorination. J. Hazard. Mater. 2008, 157,25 33. 87 (2), 125−136. (107) Pan, Y.; Zhang, X. R. Four groups of new aromatic halogenated (126) Webster, I. T.; Hancock, G. J.; Murray, A. S. Modelling the disinfection byproducts: Effect of bromide concentration on their effect of salinity on radium desorption from sediments. Geochim. formation and speciation in chlorinated drinking water. Environ. Sci. Cosmochim. Acta 1995, 59 (12), 2469−2476. − Technol. 2013, 47 (3), 1265 1273. (127) Sturchio, N. C.; Banner, J. L.; Binz, C. M.; Heraty, L. B.; (108) Jones, D. B.; Saglam, A.; Song, H. C.; Karanfil, T. The impact Musgrove, M. Radium geochemistry of ground waters in Paleozoic of bromide/iodide concentration and ratio on iodinated trihalo- carbonate aquifers, midcontinent, USA. Appl. Geochem. 2001, 16 (1), methane formation and speciation. Water Res. 2012, 46 (1), 11−20. 109−122. (109) Kitis, M.; Yigita, N. O.; Harmana, B. I.; Muhammetoglu, H.; (128) Vinson, D. S.; Vengosh, A.; Hirschfeld, D.; Dwyer, G. S. Muhammetoglu, A.; Karadirek, I. E.; Demirel, I.; Ozdenc, T.; Palancic, Relationships between radium and radon occurrence and hydro- I. Occurrence of trihalomethanes in chlorinated groundwaters with chemistry in fresh groundwater from fractured crystalline rocks, North very low natural organic matter and bromide concentrations. Environ. Carolina (USA). Chem. Geol. 2009, 260 (3−4), 159−171. Forensics 2010, 11 (3), 264−274. (129) Skalak, K. J.; Engle, M. A.; Rowan, E. L.; Jolly, G. D.; Conko, (110) Pressman, J. G.; Richardson, S. D.; Speth, T. F.; Miltner, R. J.; K. M.; Benthem, A. J.; Kraemer, T. F. Surface disposal of produced Narotsky, M. G.; Hunter, E. S.; Rice, G. E.; Teuschler, L. K.; waters in western and southwestern Pennsylvania: Potential for McDonald, A.; Parvez, S.; Krasner, S. W.; Weinberg, H. S.; McKague, accumulation of alkali-earth elements in sediments. Int. J. Coal Geol. A. B.; Parrett, C. J.; Bodin, N.; Chinn, R.; Lee, C. F.; Simmons, J. E. (130) Rich, A. L.; Crosby, E. C. Analysis of reserve pit sludge from Concentration, chlorination, and chemical analysis of drinking water unconventional natural gas hydraulic fracturing and drilling operations

N dx.doi.org/10.1021/es405118y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Critical Review for the presence of technologically enhanced naturally occurring radioactive material (TENORM). New Solutions 2013, 23 (1), 117− 135. (131) Zielinski, R. A.; Otton, J. K.; Budahn, J. R. Use of radium isotopes to determine the age and origin of radioactive Barite at oilfield production sites. Environ. Pollut. 2001, 113 (3), 299−309. (132) Crabtree, M.; Eslinger, D.; Fletcher, P.; Miller, M.; Johnson, A.; King, G. Fighting scaleRemoval and prevention. Oilfield Rev. 1999, 11,35−45. (133) Swann, C.; Matthews, J.; Ericksen, R.; Kuszmaul, J. Evaluations of Radionuclides of Uranium, Thorium, And Radium Associated with Produced Fluids, Precipitates, And Sludges from Oil, Gas, And Oilﬁeld Brine Injection wells in Mississippi University; MS:Mississippi Mineral Resources Institute/Department of Pharmacology/Department of Geology and Geological Engineering, University of Mississippi, 2004; p 83. (134) Nicot, J. P.; Scanlon, B. R. Water use for shale-gas production in Texas, U.S. Environ. Sci. Technol. 2012, 46 (6), 3580−3586. (135) Murray, K. E. State-scale perspective on water use and production associated with oil and gas operations, Oklahoma, U.S. Environ. Sci. Technol. 2013, 47 (9), 4918−4925. (136) Johnson, E. G.; Johnson, L. A. Hydraulic Fracture Water Usage in Northeast British Columbia: Locations, Volumes, And Trends; Geoscience Reports: Victoria, BC, Canada, 2012. (137) Kenny, J. F.; Barber, N. L.; Hutson, S. S.; Linsey, K. S.; Lovelace, J. K.; Maupin, M. A. Estimated Use of Water in the United States in 2005; U.S. Geological Survey Circular 1344. Reston, VA, 2009. 52. (138) FracFocus FracFocus 2.0. http://fracfocus.org/ (accessed 10/ 17/2013). (139) Freyman, M.; Salmon, R. Hydraulic Fracturing and Water Stress: Growing Competitive Pressures for Water; CERES: Boston, MA, 2013; p 12. (140) Mitchell, A. L.; Small, M.; Casman, E. A. Surface water withdrawals for Marcellus Shale gas development: Performance of alternative regulatory approaches in the Upper Ohio River Basin. Environ. Sci. Technol. 2013, 47 (22), 12669−12678. (141) He, C.; Zhang, T.; Vidic, R. D. Use of abandoned mine drainage for the development of unconventional gas resources. Disrupt. Sci. Technol. 2013, 1, 169−176. (142) Yoxtheimer, D.; Blumsack, S.; Murphy, T. The Decision to use acidic coal mine drainage for hydraulic fracturing of unconventional shale-gas wells. Environ. Pract. December 2012, 14 (4), 301−-307. (143) Kondash, A. J.; Warner, N. R.; Lahav, O.; Vengosh, A. Radium and barium removal through blending hydraulic fracturing fluids with acid mine drainage. Environ. Sci. Technol. 2014, 48 (2), 1334−1342. (144) Veil, J. A. White Paper on SPE Summit on Hydraulic Fracturing; Society of Petroleum Engineers: Houston TX, 2011. (145) Hickenbottom, K. L.; Hancock, N. T.; Hutchings, N. R.; Appleton, E. W.; Beaudry, E. G.; Xu, P.; Cath, T. Y. Forward osmosis treatment of drilling mud and fracturing wastewater from oil and gas operations. Desalination 2013, 312,60−66. (146) Ferguson, M. L.; Eichelberger, P. B.; Qiu; Roell, R. L. Innovative Friction Reducer Provides Improved Performance and Greater Flexibility in Recycling Highly Mineralized Produced Brines; Society of Petroleum Engineers (SPE), 2013. (147) Guerra, K.; Dahm, K.; Dundorf, S. Oil and Gas Produced Water Management and Beneﬁcial Use in the Western United States; U.S. Department of the Interior, Bureau of Reclamation, Technical Service Center, Water and Environmental Resources Division, Water Treatment Engineering Research Group: Denver, CO, 2011; p 113. (148) Carnegie Museum of Natural History Pennsylvania Unconven- tional Natural Gas Wells Geodatabase; Pittsburgh, PA: Carnegie Museum of Natural History, 2000−2013.

O dx.doi.org/10.1021/es405118y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX RESEARCH ARTICLE Adrenal Gland and Lung Lesions in Gulf of Mexico Common Bottlenose Dolphins (Tursiops truncatus) Found Dead following the Deepwater Horizon Oil Spill

Stephanie Venn-Watson1*, Kathleen M. Colegrove2, Jenny Litz3, Michael Kinsel2, Karen Terio2, Jeremiah Saliki4, Spencer Fire5,6, Ruth Carmichael7, Connie Chevis8, Wendy Hatchett8, Jonathan Pitchford8, Mandy Tumlin9, Cara Field10, Suzanne Smith10, Ruth Ewing3, Deborah Fauquier11, Gretchen Lovewell12, Heidi Whitehead13, David Rotstein14, Wayne McFee15, Erin Fougeres16, Teri Rowles11

1 National Marine Mammal Foundation, San Diego, California, United States of America, 2 University of Illinois, Zoological Pathology Program, Maywood, Illinois, United States of America, 3 National Marine Fisheries Service, Southeast Fisheries Science Center, Miami, Florida, United States of America, 4 Athens Veterinary Diagnostic Laboratory College of Veterinary Medicine, University of Georgia, Athens, Georgia, United States of America, 5 NOAA National Ocean Service, Marine Biotoxins Program, Charleston, South OPEN ACCESS Carolina, United States of America, 6 Florida Institute of Technology Department of Biological Sciences, Melbourne, Florida, United States of America, 7 Dauphin Island Sea Lab and University of South Alabama, Citation: Venn-Watson S, Colegrove KM, Litz J, Dauphin Island, Alabama, United States of America, 8 Institute for Marine Mammal Studies, Gulfport, Kinsel M, Terio K, Saliki J, et al. (2015) Adrenal Gland Mississippi, United States of America, 9 Louisiana Department of Wildlife and Fisheries, Baton Rouge, and Lung Lesions in Gulf of Mexico Common Louisiana, United States of America, 10 Audubon Aquarium of the Americas, New Orleans, Louisiana, Bottlenose Dolphins (Tursiops truncatus) Found United States of America, 11 National Marine Fisheries Service, Office of Protected Resources, Silver Spring, Maryland, United States of America, 12 Mote Marine Laboratory, Sarasota, Florida, United States of Dead following the Deepwater Horizon Oil Spill. PLoS America, 13 Texas Marine Mammal Stranding Network, Galveston, Texas, United States of America, ONE 10(5): e0126538. doi:10.1371/journal. 14 Marine Mammal Pathology Services, Olney, Maryland, United States of America, 15 National Centers for pone.0126538 Coastal Ocean Science, National Ocean Service, Charleston, South Carolina, United States of America, Academic Editor: Cheryl S. Rosenfeld, University of 16 National Marine Fisheries Service, Southeast Regional Office, St. Petersburg, Florida, United States of America Missouri, UNITED STATES

Received: December 22, 2014 * [email protected]

Accepted: March 30, 2015 Published: May 20, 2015 Abstract Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, A northern Gulf of Mexico (GoM) cetacean unusual mortality event (UME) involving primari- transmitted, modified, built upon, or otherwise used ly bottlenose dolphins (Tursiops truncatus) in Louisiana, Mississippi, and Alabama began in by anyone for any lawful purpose. The work is made February 2010 and continued into 2014. Overlapping in time and space with this UME was available under the Creative Commons CC0 public domain dedication. the Deepwater Horizon (DWH) oil spill, which was proposed as a contributing cause of adrenal disease, lung disease, and poor health in live dolphins examined during 2011 in Bara- Data Availability Statement: All relevant data are available within the paper and Supporting Information taria Bay, Louisiana. To assess potential contributing factors and causes of deaths for files. stranded UME dolphins from June 2010 through December 2012, lung and adrenal gland Funding: This study was funded by the Deepwater tissues were histologically evaluated from 46 fresh dead non-perinatal carcasses that Horizon National Resource Damage Assessment stranded in Louisiana (including 22 from Barataria Bay), Mississippi, and Alabama. UME being conducted cooperatively among the National dolphins were tested for evidence of biotoxicosis, morbillivirus infection, and brucellosis. Oceanic and Atmospheric Administration (NOAA) (www.noaa.gov), other Federal and State Trustees, Results were compared to up to 106 fresh dead stranded dolphins from outside the UME and BP; the Contingency Fund of the Working Group area or prior to the DWH spill. UME dolphins were more likely to have primary bacterial for Marine Mammal Unusual Mortality Events pneumonia (22% compared to 2% in non-UME dolphins, P = .003) and thin adrenal cortices (WGMMUME)(http://www.nmfs.noaa.gov/pr/health/

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mmume/history.htm); National Fish and Wildlife (33% compared to 7% in non-UME dolphins, P = .003). In 70% of UME dolphins with prima- ’ Foundation (www.nfwf.org); and NOAAs Marine ry bacterial pneumonia, the condition either caused or contributed significantly to death. Mammal Health and Stranding Response Network (http://www.nmfs.noaa.gov/pr/health/). NOAA and the Brucellosis and morbillivirus infections were detected in 7% and 11% of UME dolphins, re- WGMMUME aided in reviewing the study design and spectively, and biotoxin levels were low or below the detection limit, indicating that these manuscript. BP funders had no role in study design, were not primary causes of the current UME. The rare, life-threatening, and chronic adrenal data collection and analysis, decision to publish, or gland and lung diseases identified in stranded UME dolphins are consistent with exposure preparation of the manuscript. to petroleum compounds as seen in other mammals. Exposure of dolphins to elevated pe- Competing Interests: This work was part of the troleum compounds present in coastal GoM waters during and after the DWH oil spill is Deepwater Horizon National Resource Damage Assessment being conducted cooperatively among proposed as a cause of adrenal and lung disease and as a contributor to increased NOAA, other Federal and State Trustees, and BP. dolphin deaths. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Introduction A large, multi-year cetacean unusual mortality event (UME) has been ongoing in the northern Gulf of Mexico (GoM) since February 2010, continuing into 2014 [1]. This event has involved predominantly (87%) common bottlenose dolphins (Tursiops truncatus) (hereafter referred to as ‘dolphins’) stranded in Louisiana, Mississippi, and Alabama [2]. The UME coincided with the Deepwater Horizon (DWH) oil spill, the largest marine-based spill in U.S. history [3]. Dur- ing and following the DWH oil spill, significantly elevated polycyclic aromatic hydrocarbon (PAH) levels attributed to this spill were detected in coastal GoM waters, including Louisiana, Mississippi, and Alabama [4]. These locations coincided with the states most impacted by the ongoing UME since the DWH oil spill [2]. Dolphin strandings, however, were elevated during March and April before the spill, necessitating an investigative approach including numerous potential causes [1,2,5]. Combined oil exposure, an unusually cold winter during 2011, and fresh water infusions have been proposed as potential causes contributing to this UME [6]. Barataria Bay, Louisiana was one of the heaviest oiled coastal areas from the DWH oil spill, including visualized oiling from the spill encompassing 40 km and 366,000 m2 of Barataria Bay’s shoreline lasting in decreasing amounts for at least 2 years [7–10]. The presence of increased coastal PAH levels associated with the DWH oil spill, especially near Grand Isle, Loui- siana in Barataria Bay have been confirmed [4]. Further, within the time period of January 2010 to June 2013, the longest lasting cluster of dolphin strandings throughout the northern GoM was in Barataria Bay (August 2010 through 2011) [2]. During the DWH oil spill and response period, numerous dolphins, including dolphins in Barataria Bay, were observed swimming through visibly oiled waters and feeding in areas of surface, subsurface, and sediment oiling [11]. Due to the extensive oiling in Barataria Bay, health assessments were conducted on live dolphins in this area during the summer of 2011 [11]. Barataria Bay dolphins had a high prevalence of moderate to severe lung disease and blood value changes indicative of hypoadrenocorticism; specific blood changes included low serum cortisol, aldosterone, and glucose, and high neutrophil counts [11]. Nearly half (48%) of Barataria Bay dolphins were given a guarded to grave prognosis for long-term survival [11]. The DWH oil spill was proposed as a contributor to adrenal gland and lung disease in live Barataria Bay dolphins. Previous to the ongoing event, there have been ten dolphin GoM UMEs since 1991, as well as one large die-off during 1990 that occurred before the UME declaration process [1, 12–15]. The majority (82%) of previous dolphin GoM events had brevetoxicosis or morbillivirus as confirmed or suspected causes [1]. While brevetoxicosis events do not leave a histologic

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signature in affected dolphins, brevetoxicosis-related events are often associated with known algal blooms and deaths that appear to be acute in otherwise healthy-looking dolphins [15]. In prior events classified as brevetoxicosis-related, 50% or more sampled dolphins were positive for brevetoxin with most at high concentrations [15]. Similarly, past UMEs that have been attributed to morbillivirus involved successful detection of morbillivirus in greater than 60% of dolphins tested. [13,16]. There is evidence that Brucella, which is commonly found in marine mammals worldwide, can cause disease in cetaceans, including bottlenose dolphins [17–21]. As such, there was a need to evaluate all of these potentially important diseases as playing contributing or leading roles in the ongoing UME. To assess contributing factors and causes of deaths for stranded UME dolphins following the DWH oil spill, tissues were histologically evaluated from 46 carcasses that stranded in Louisiana, Mississippi, and Alabama, including 22 from Barataria Bay, from June 2010 through December 2012. Perinatal dolphins, stranded dolphins that were less than 115 cm in body length that likely died during late-term pregnancy or shortly after birth, were excluded from this study. On the basis of the live dolphin health assessment findings from Barataria Bay, this study included a focused evaluation of adrenal, lung, and liver lesions with the expectation that if stranded dolphins had been impacted by the DWH oil, they would have lesions consistent with the clinical evidence indicating lung disease and hypoadrenocorticism found in live dolphins. Other potential causes of and contributors to dolphin deaths were investigated, including the presence of histologic lesions and diagnostic test results consistent with brevetoxicosis, morbillivirus infections, and brucellosis. Results were compared to a reference group of fresh dead dolphins from North Carolina, South Carolina, Texas, and the Gulf coast of Florida that stranded prior to or remote from the UME and DWH oil spill timeframes and geographic location.

Materials and Methods Tissues and data from bottlenose dolphins that stranded dead or stranded and died were used in this study in coordination with NOAA’s Marine Mammal Health and Stranding Response Program (MMHSRP, T. Rowles) and NOAA’s Southeastern United States Marine Mammal Stranding Network. Only samples from stranded dolphins were included in this study. No animal was killed for the purposes of this study.

UME dolphins This study includes common bottlenose dolphins (Tursiops truncatus) with a total body length greater than or equal to 115 cm that stranded in Louisiana, Mississippi, or Alabama from June 2010 through December 2012. Dolphins with a total body length less than 115 cm were likely either near-term pregnancy losses or died soon after birth. Carcasses included in this study were either characterized as stranding fresh dead by the responding stranding network personnel or stranded alive then died, had archived tissues available for histologic evaluations, and had tissues not compromised by decomposition based on histologic evaluations by a pathologist. Fresh dead dolphins were the focus of this study to enable the most reliable histologic evaluations which could be compared to other diagnostic test results. Blood-based clinical pathology data were not available from dead, stranded dolphins as a direct comparison to the indices of hypoadrenocorticism detected in the previously published, live dolphin health assessment.

Reference dolphins A total of 106 fresh dead reference dolphins, each with a total body length greater than or equal to 115 cm and an existing histopathology report, were compared to UME dolphins in this

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Table 1. Total number, stranding dates, and locations of 152 freshly dead stranded non-perinatal (greater than or equal to 115 cm in total body length) common bottlenose dolphins (Tursiops truncatus) from the ongoing northern Gulf of Mexico cetacean Unusual Mortality Event (UME) and reference groups.

Study group Number Observed stranding dates State or Parish UME dolphins 46 JUN 2010—DEC 2012 AL (n = 6), LA (n = 28), MS (n = 12) Barataria Bay, Louisiana UME dolphin subset 22 SEP 2010—OCT 2012 Jefferson (n = 16), Plaquemines (n = 3), Lafourche (n = 3) Reference dolphins 106 APR 1996—AUG 2012 FL (n = 49), NC (n = 2), SC (n = 47), TX (n = 8) References dolphins—standardly scored subset 51 FEB 2002—JUL 2009 FL (n = 35), NC (n = 2), SC (n = 6), TX (n = 8)

Standardly scored subset references had both histopathology reports and archived tissues available for evaluation. doi:10.1371/journal.pone.0126538.t001

study [22]. Of these, 51 (48%) reference dolphins had archived tissues available for standardized assessment by a common pathologist (K. Colegrove) heretofore called the ‘standardly scored reference subset’. Reference dolphins for this study stranded in North Carolina and South Carolina from 1996 through 2012, or from Texas or the Gulf coast of Florida from 2002 through 2009 (Table 1). Histologic lesion comparisons from reference dolphins were made to the UME dolphins using both the full reference group and the standardly scored reference subset group. It is possible that some of the reference dolphins may have been part of historical UMEs, specifically brevetoxin UMEs in Florida. Potential brevetoxicosis reference dolphins were not excluded from this study to ensure there was no bias against this as a cause of the ongoing UME.

Sample collection and processing for histologic examination Post mortem examinations of UME and reference dolphins were conducted by members of NOAA’s Southeastern United States Marine Mammal Stranding Network either in the field or at institutional facilities. Stranding demographic, temporal and spatial data were collected using a standardized Level-A data record (NOAA Form 89–864 (rev.2007) OMB No. 0648– 0178) according to approved NOAA protocols at the time of data collection. Level A data used for this study were the most recently available when extracted from the NOAA’s MMHSRP database during September 2014. Tissue samples from most major organs of stranded dolphins were collected in 10% neutral buffered formalin, many of which included adrenal glands, bladder, brain, eye, heart, kidney, liver, lung, lymph node(s), muscle, pancreas, reproductive organs, skin, small and large intestines, spinal cord, spleen, thymus, tongue, and trachea. Tissues were processed routinely, em- bedded in paraffin, sectioned at 5 μm, and stained with either hematoxylin and eosin (HE) (UME dolphins, South Carolina reference dolphins, and Gulf coast of Florida reference dolphins) or hematoxylin, phloxine, and saffron (HPS) (Texas reference dolphins). While this study focused on lung, adrenal gland, liver, lymph nodes and brain to test our hypothesis, full sets of tissues from the UME cases, including the above organs, were evaluated by a pathologist and used for the cause of death determination. Among the 10 UME dolphins with primary bacterial pneumonia, gram stains were completed on all dolphins, and Ziehl Nielson acid fast stains were completed on lung sections of four dolphins. Not all tissues for all dolphins were available for both histologic and other diagnostic tests (e.g. there may have been formalin-fixed lung for histologic evaluation, but no frozen lung for morbillivirus molecular diagnostics). As such, the total numbers of tissues varied throughout the study based upon their availability for each investigation.

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Histologic grading Targeted evaluation of lesions in select organs (adrenal gland, brain, lung, lymph node, spleen, and liver) from UME dolphins was performed by a board certified veterinary pathologist (K. Colegrove) through evaluation of HE or HPS stained slides. To standardize comparisons among tissues, the same evaluation was completed on the reference dolphins by the same pathologist (K. Colegrove) through evaluation of stained slides (n = 51, standardly scored reference set) or thorough interpretation of lesion descriptions with or without images from histopathologic reports (n = 55) written by other pathologists. To reduce bias, severity grades were carried out blindly without knowledge of the animal identification number. Adrenal gland. The adrenal gland was evaluated quantitatively by measurement of corticomedullary ratios (thickness of cortex divided by thickness of medulla). Corticomedullary ratios were determined similarly to previously published methods on mid-sagittal or mid-cross sections of adrenal glands from 36 UME and 44 reference dolphins using an Olympus BX-41 microscope and Olympus DP-BSW Version 03.02 measuring software [23]. Corticomedullary ratios in dolphins remain constant along the length of the adrenal gland, indicating that ratios calculated on mid- sagittal or mid-cross sections are adequately representative of the entire adrenal gland [24]. Three corticomedullary ratios were obtained from three different representative sites along the section and averaged. For most UME and reference dolphins, only one appropriate mid-sagittal or mid-cross section of either right or left adrenal gland was available for evaluation. Adrenal corticomedullary ratios from 44 reference dolphins were used to statistically define ‘thin’ and ‘thick’ adrenal gland cortices. Thin was defined as a corticomedullary ratio less than the 10th quantile among reference dolphins (0.447), and thick was a corticomedullary ratio greater than the reference’s90th quantile (0.838). UME and reference dolphins were excluded from evaluation of corticomedullary ratios when mid-sagittal sections of adrenal glands were not present or sections had been cut in a way that precluded precise measuring of cortex or medulla thickness. Corticomedullary ratios were obtained from both the left and right adrenal glands of ten reference dolphins, and using a Fisher’s exact test, measurements were comparable between the left and right sides (P = 0.85). Presence or absence of vacuolation of corticocytes in the zona fasciculata was also evaluated. Lung. Lung inflammation severity was graded as mild, moderate, or severe based on extent and number of inflammatory cells. Mild lesions were those in which there were small and often multifocal accumulations of small numbers of inflammatory cells with minimal disruption of parenchyma. Moderate lesions were multifocal to more widespread, had moderate accumulations of inflammatory cells, and moderate disruption of parenchyma. Severe lesions had regional to diffuse accumulations of large numbers of inflammatory cells that often completely obscured or disrupted parenchyma. The distribution of the inflammation was defined as bronchopneumonia, bronchointerstitial pneumonia, or interstitial pneumonia. The types of inflammatory cells present within the inflammatory lesions were denoted as neutrophilic, eosinophilic, lymphoplasmacytic and granulomatous and were not mutually exclusive. Lesions were evaluated for the visual presence of bacterial and fungal organisms. The cause of inflammation was defined as primary bacterial if bacterial organisms were visualized and associated with lesions and there was no concurrent evidence of lungworm, fungal or viral infection contributing to the inflammation. Inflammation was defined as primary lungworm if lesions were consistent with dolphin lungworm infection as previously described [25] and there was no concurrent or secondary bacterial, fungal, or viral infection. Inflammation was defined as primary fungal or viral-associated if the pathogen was visualized without other concurrent pathogens. Mixed infections were those in which there were multiple infectious agents (e.g. bacteria and lungworm) associated with the inflammatory lesions.

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Pulmonary fibrosis was graded based on extent and frequency of lesions. Mild lesions were those in which lesions had a patchy distribution (less than 30% of the parenchyma was affected), and alveolar septae and/or perivascular connective tissue were mildly thickened by increased amounts of collagenous matrix. Moderate lesions were those which had a more widespread distribution (30–60% of parenchyma affected), alveolar septae and/or perivascular connective tissue moderately thickened by increased amounts of collagenous matrix, and/or thick bands of fibrous connective tissue with moderate disruption of pulmonary parenchyma. Severe lesions had large areas of parenchyma affected (greater than 60%), marked thickening of alveolar septae and/or perivascular connective tissue by increased amounts of collagenous matrix and large areas in which normal architecture was completely obliterated by fibrous tissue. Angiomatosis was defined as a proliferation of small thick-walled vessels, similar to lesions previously described at a prevalence of 46% in dolphins stranded in Texas from 1991 to 1996 [26]. Lungworm-associated pulmonary granulomas, when present, were defined as active or chronic similarly to what has been previously described in GoM dolphins [25]. Other histologic evaluations. Degree of inflammation in the central nervous system was subjectively graded as mild, moderate, or severe based on extent and number of inflammatory cells and frequency of inflammatory foci in tissues collected from the brain or spinal cord. Lymphoid depletion was evaluated as present or absent in lymph nodes and the spleen. Thy- mus was not available for examination in most cases, thus was not scored. Fibrosis, inflammation, necrosis, lipidosis, hemosiderosis, and biliary hyperplasia were evaluated in the liver. To assess nutritional status, presence or absence of thin epicardial adipose tissue, pancreatic zymogen depletion, and retained gastric superficial epithelial cell layers was determined in UME dolphins. The presence or absence of suspected morbillivirus infections or neurobrucellosis was determined based on available diagnostic tests and the presence or absence of representative histologic lesions in pulmonary, lymphoid, and central nervous system tissues similar to lesions previously described [16, 17, 27].

Cause of death Causes of death for UME and reference dolphins were acquired from original histopathology reports generated by a veterinary pathologist. Causes of death were based on histologic findings, gross necropsy reports, and ancillary test results. To ease comparisons among groups in the current study, causes of death were grouped into nine general categories: infectious, unknown—poor body condition, unknown—body condition not noted as likely contributor, trauma, organ failure, food obstruction/fish spine injury, maternal separation, and neoplasia. Cause of death was listed as multifactorial if more than one primary cause of death was noted by the pathologist. The prevalence of cause of death categories were determined for all UME dolphins, the Barataria Bay UME dolphin subset, UME dolphins with thin adrenal gland cortices, UME dolphins with primary bacterial pneumonia, all reference dolphins, and the standardly scored reference dolphin subset.

Diagnostic tests Morbillivirus, marine biotoxin, and Brucella tests were conducted on UME dolphins when appropriate tissues were available. Lung or lung-associated lymph node tissues, frozen and stored at -80°C, were tested for dolphin morbillivirus using a previously validated PCR assay [16]. Liver, feces, or gastric samples were analyzed for brevetoxin using an enzyme-linked immuno- sorbent assay (ELISA) and/or liquid chromatography—mass spectrometry (LC-MS), and analyzed for the presence of domoic acid by tandem mass spectrometry coupled with LC-MS/MS (MS) [12, 28, 29]. A Brucella PCR assay was used to test for Brucella in multiple tissues

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including lung, lung-associated lymph node, reproductive tract and/or central nervous system tissues frozen and stored at -80°C. Numbers and types of tissues tested varied by individual and were based upon tissues that were collected and available for testing. Brucella DNA was extracted from tissue samples using a commercially available kit (DNeasy, Qiagen) following the manufacturer’s instructions. Samples were tested for Brucella DNA using a semi-quantitative real-time PCR with standard reagents (Gene Expression Master Mix, Applied Biosystems) and a minor groove binding probe that cross-reacts with known terrestrial and marine Brucella species (University of Illinois, Zoological Pathology Program Molecular Diagnostic Laboratory). Presence of Brucella DNA was confirmed in all equivocal and positive samples by PCR amplifi- cation and sequencing of a 135 bp fragment of the Brucella 16SrRNA gene and/or sections of the outer membrane protein (OMP) 2 gene according to published protocols [30]. Nucleotide sequences were determined in both directions on an automated sequencer (Applied Biosystems 3730XL). As a follow up to UME dolphins in which bacterial pathogens were suspected in the lung, lung samples from five dolphins were tested for the presence of bacterial pathogen DNA using a commercially available kit (MicroSeq 500 16S rDNA Bacterial Identification System, Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions and compared nucleotide sequences using the MicroSeq ID Analysis Software. Lung samples from UME dolphins were also tested for the presence of Nocardia DNA using previously published protocols [31]. Necropsy culture swabs and tissues were also collected for classical microbial culture techniques. Isolated bacteria from lesions that were not believed to be due to overgrowth contamination, based upon the pathologists’ interpretation of tissues, were reported.

Data management A Microsoft Excel datasheet was used to enter the study data in a standardized way into an ana- lyzable format. Variables in the datasheet included the unique animal field identifier, total body length (cm), sex, and state, county, and date of observed dolphin stranding. Diagnostic test results, including samples tested for morbillivirus, Brucella, and biotoxins were included. Histology variables described in the section above were entered into the database using standardized terms.

Statistics Data were analyzed using World Programming System (WPS 3.1) (World Programming Ltd., United Kingdom). The following four study groups were used for analysis: all UME dolphins, Barataria Bay subset UME dolphins, all reference dolphins, and the standardly scored subset reference dolphins. Descriptive statistics included stranding observation dates and state, sex, and body length. Body lengths and sex distribution were compared between UME and reference dolphins using a Wilcoxon rank-sum test and chi-square test, respectively. Throughout the UME and reference dolphin comparisons, sample sizes varied based upon information available, tissues collected, and tests conducted on individual UME and reference dolphins. The prevalence of targeted histologic lesions and causes of death, described above, were compared between UME and reference dolphins using chi-square tests and odds ratios or Fish- er’s exact tests (if compared categories had less than or equal to five values). To further assess the potential influence of sex and body length on those lesions with significant differences between standardly scored cases and references, a general linear model was used which included sex and body length as covariates and dummy, binary variables for the presence or absence of lesions. Significance was defined as a P value less than 0.05.

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Results UME and reference dolphin demographics Forty-six fresh dead dolphins that stranded between June 2010 and December 2012 were included in the study (Table 1). A total of 106 reference dolphins were fresh dead and had tissues and/or histologic reports available, including dolphins that stranded along the Gulf coast of Florida (2002–2009), North Carolina (2004), South Carolina (1996–2012) and Texas (2003– 2005). There were no significant differences in body length or sex when comparing GoM UME and reference dolphins (Table 2). Using a general linear model, body length and sex were re- confirmed as non-significant covariates for lesions with significant differences between UME and reference dolphins. Accordingly, potential physiologic influences of age and sex did not confound interpretation.

Adrenal gland Among UME dolphins in which appropriate adrenal gland tissue was available for evaluation, one-third (12/36) had a thin adrenal gland cortex and low corticomedullary ratio (less than 0.447), including 9 (50%) Barataria Bay dolphins (Table 2). UME dolphins were more likely to have a thin adrenal gland cortex compared to reference dolphins (33% versus 7%, P = 0.003, Table 2). Adrenal glands with a thin cortex had reduced numbers of cells with only a thin band of cortex remaining (Fig 1). In most cases, distinction between the zona fasciculata and reticu- laris was difficult. Although cells in the zona fasciculata region appeared reduced in number, it was not possible to distinguish whether the zona glomerulosa and zona reticularus were also affected. In the most severely affected cases, corticocytes were small, though cell size varied greatly within and among the cortex of all individuals. Some dolphins without a thin cortex had small corticocytes. There was no evidence of hemorrhage, necrosis, or fibrosis in affected adrenal glands. The size of the adrenal medulla was within normal limits for all dolphins. The proportion of stranded dolphins with thin adrenal gland cortices was similarly high by year (June–December 2010 = 3/9, 33%, 2011 = 4/15, 27%, 2012 = 5/12, 42%) (Fig 2). Of UME dolphins with a thin cortex, 4 (33%) had a concurrent primary bacterial pneumonia. None of the thin adrenal cortex UME dolphins had evidence of morbillivirus infection, and one case had Brucella meningoencephalitis. None of the UME dolphins with a thin cortex had depleted epicardial adipose tissue, an indicator of advanced, poor nutritional state. The most common noted cause of death among dolphins with a thin adrenal cortex was unknown death not attributed to poor body condition (42%). Among all UME dolphins, mild adrenal corticocyte vacuolation was noted in three (7%) dolphins, and adrenal gland inflammation was noted in two (4%); one of these dolphins had intralesional bacteria associated with disseminated infection. There was no significant difference in the prevalence of corticocyte vacuolation between UME and reference dolphins (7% versus 12%, P = 0.49). No other pathogens were noted in any other UME dolphin adrenal glands.

Lung Of the UME dolphins, 22% had a primary bacterial pneumonia. In 7 (70%) of UME dolphins with primary bacterial pneumonia, the condition either caused or contributed significantly to death (Tables 3 and 4). The prevalence of primary bacterial pneumonia was higher than reference dolphins (2%) (P = 0.003). Of the 10 UME dolphins with primary bacterial pneumonia, 5 (50%) had severe pneumonia. Overall, UME dolphins had a higher prevalence of severe pneumonia compared to standardly scored references (16% versus 2%, P = 0.03). Primary bacterial

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Table 2. Prevalence comparisons of lesions detected using histologic examination for fresh dead, stranded common bottlenose dolphins (Tur- siops truncatus) among 1) UME cases from Louisiana, Mississippi, and Alabama, 2) Barataria Bay, Louisiana UME subset, 3) all reference dolphins, and 4) slide-reviewed and standardly scored reference dolphin subset.

Description UME case Reference dolphins P value (UME v. UME case dolphin Reference dolphins All Standardly scored standardly scored subset Barataria Bay dolphins All (n = 46) subset (n = 51) reference dolphins) only (n = 22) (n = 106) Body length (cm) 213 ± 37 225 ± 40 0.06 215 ± 39 211 ± 42 Female 19/46 (41%) 22/50 (44%) 0.79 9/22 (41%) 47/105 (45%) Adrenal gland cortex Adrenal gland cortex 0.55 ± 0.23 0.60 ± 0.15 0.33 0.51 ± 0.22 0.60 ± 0.15 (average C:M ± SD) Thin adrenal gland 12/36 (33%) 3/44 (7%) 0.003 9/18 (50%)1 3/44 (7%) cortex (C:M < 0.447) Thick adrenal gland 3/36 (8%) 4/44 (9%) 0.91 1/18 (6%) 4/44 (9%) cortex (C:M > 0.838) Splenic lymphoid and lymph nodes Splenic lymphoid 10/41 (24%) 3/42 (7%) 0.03 4/21 (19%) 13/73 (18%) depletion Lymph node depletion 10/40 (25%) 0/42 (0%) < 0.0001 4/20 (20%)1 13/80 (16%) Splenic lymphoid & 5/35 (14%) 0/35 (0%) 0.05 1/19 (5%) 9/56 (16%) lymph node depletion Reactive lymph nodes 17/39 (44%) 11/36 (32%) 0.25 9/20 (45%) 20/67 (30%) Liver Abnormal liver tissue 34/42 (81%) 40/45 (89%) 0.30 19/21 (90%) 76/94 (81%) Fibrosis 29/42 (69%) 30/45 (67%) 0.82 17/21 (81%) 48/94 (51%) Necrosis 2/42 (5%) 1/45 (2%) 0.61 2/21 (10%) 2/94 (2%) Hepatitis 15/42 (36%) 23/45 (51%) 0.15 8/21 (38%) 41/94 (44%) Hemosiderosis 13/42 (31%) 12/45 (27%) 0.61 8/21 (38%) 17/94 (18%) Lipid deposition 5/42 (12%) 8/45 (18%) 0.44 4/21 (19%) 19/94 (20%) Hepatobiliary 11/42 (26%) 12/45 (27%) 1.00 6/21 (29%) 24/94 (26%) hypertrophy Central nervous system Abnormal CNS tissue 9/45 (20%) 13/44 (30%) 0.30 3/21 (14%) 32/85 (38%)2 Encephalitis3 9/45 (20%) 9/44 (20%) 0.96 3/21 (14%) 16/85 (19%) Bacterial 5/45 (11%) 4/44 (9%) 1.00 2/21 (10%) 7/85 (8%) Fungal 2/45 (4%) 1/44 (2%) 0.51 0 (0%) 3/85 (4%) Parasitic 1/45 (2%) 2/44 (5%) 0.62 0 (0%) 4/85 (5%) Viral 1/45 (2%) 5/44 (11%) 0.11 0 (0%)1 5/85 (6%)2 Brucella PCR-positive 2/33 (6%) 0/2 NA 1/14 (7%) 1/13 (8%) CNS tissue Morbillivirus PCR- 5/33 (15%) Not tested NA 1/14 (7%) Not tested positive CNS tissue

Denominators varied based upon information available, tissues collected from, and tests conducted on individual UME and reference dolphins. 1Barataria Bay, Louisiana UME dolphin subset values different (P < 0.05) than standardly scored reference dolphins. 2UME dolphin values different (P < 0.05) than full reference group. 3Represents encephalitis, meningitis, or meningoencephalitis. C:M = adrenal corticomedullary ratio. CNS = central nervous system.

doi:10.1371/journal.pone.0126538.t002

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Fig 1. Hematoxalin and eosin stained sections of adrenal glands from common bottlenose dolphins (Tursiops truncatus) C = cortex; M = medulla. A. Normal adrenal gland from a subadult male dolphin that stranded fresh dead along the west coast of Florida in 2003. Bar = 1 mm. B. Adrenal gland from a subadult male dolphin that stranded in Barataria Bay, Louisiana in 2011. Note the thin adrenal cortex, especially in the fasciculata region. Bar = 1mm. doi:10.1371/journal.pone.0126538.g001

pneumonia was most common in UME dolphins from June to December 2010 and was less prevalent during 2011 and 2012 (2010 = 4/10, 40%, 2011 = 3/20, 15%, 2012 = 3/16, 19%) (Fig 2). All UME years examined, however, had a higher prevalence of primary bacterial pneumonia compared to reference dolphins (Fig 2, line indicating prevalence among standardly scored

Fig 2. Prevalence of key histologic lesions among fresh dead, stranded common bottlenose dolphins (Tursiops truncatus) by year in Louisiana, Mississippi & Alabama: June 2010 to December 2012. The gray line represents the percentage of lesion prevalence among standardly scored reference dolphins. All years had prevalence of lesions greater than the reference group (P > 0.05). doi:10.1371/journal.pone.0126538.g002

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reference dolphins). Six (60%) UME dolphins with primary bacterial pneumonia had bronchopneumonia. UME dolphins overall had a higher prevalence of inflammatory lung infiltrate characterized as granulomatous compared to the standardly scored reference dolphins (78% versus 57%, P = 0.03, Table 3).

Table 3. Prevalence comparisons of lung lesions detected using histologic examination for fresh dead, stranded common bottlenose dolphins (Tursiops truncatus) among UME dolphins from 1) Louisiana, Mississippi, and Alabama, 2) slide-reviewed and standardly scored reference dolphin subset, 3) Barataria Bay, Louisiana UME dolphin subset, 4) and all reference dolphins.

Description UME case Reference dolphins P value (UME v. UME case dolphin Reference dolphins All Standardly scored standardly scored subset Barataria Bay dolphins All (n = 46) subset (n = 51) reference dolphins) only (n = 22) (n = 106) Abnormal lung 46 (100%) 51 (100%) 1.00 22 (100%) 104 (98%) Pneumonia 44 (96%) 43 (84%) 0.07 20 (91%) 88 (83%)2 Severe 7 (15%) 1 (2%) 0.03 1 (5%) 6/86 (7%) Distribution: Bronchopneumonia 30 (65%) 23 (45%) 0.05 16 (73%)1 33/80 (41%)2 Interstitial pneumonia 4 (9%) 5 (10%) 0.57 2 (9%) 26 (33%) Bronchointerstitial 10 (22%) 15 (29%) 0.39 2 (9%) 21 (26%) Inflammatory infiltrate: Granulomatous 36 (78%) 29 (57%) 0.03 18 (82%) 37/73 (51%)2 Lymphoplasmacytic 26 (57%) 23 (45%) 0.26 13 (59%) 37 (51%) Eosinophilic 13 (28%) 13 (25%) 0.76 7 (32%) 27 (37%) Suppurative 25 (54%) 18 (35%) 0.06 12 (55%) 19 (26%)2 Cause: Primary lungworm 24 (52%) 31 (61%) 0.14 13 (59%) 65/84 (77%)2 Primary bacterial 10 (22%) 1 (2%) 0.003 4 (18%)1 2 (2%)2 Primary viral 4 (9%) 1 (2%) 0.19 1 (5%) 1 (1%) Primary fungal 0 (0%) 1 (2%) NA 0 (0%) 1 (1%) Primary protozoal 0 (0%) 0 (0%) NA 0 (0%) 1 (1%) Mixed 4 (9%) 10 (20%) 0.16 2 (9%) 14 (17%) Aspiration and secondary 1 (2%) 0 (0%) NA 0 (0%) 0 (0%) bacterial Pathogens identified: Lungworm 19 (41%) 37 (73%) 0.002 10 (45%)1 66/106 (62%)2 Bacteria 15 (33%) 8 (16%) 0.05 6 (27%) 14/102 (14%)2 Virus 4 (9%) 1 (2%) 0.18 1 (5%) 2/102 (2%) Fungus 3 (7%) 3 (6%) 0.71 0 (0%) 3/102 (3%) Fibrosis 40 (87%) 40 (78%) 0.27 20 (91%) 57 (54%)2 Moderate to severe 16 (35%) 17 (33%) 0.89 7 (32%) 30 (28%) Angiomatosis 24 (52%) 31 (61%) 0.39 12 (55%) 46/59 (78%)2 Granuloma 16 (35%) 19 (37%) 0.80 9 (41%) 31 (29%) Active 7 (15%) 5 (10%) 0.42 3 (14%) 14 (13%) Chronic 9 (20%) 13 (25%) 0.68 6 (27%) 16 (15%) Lung or lung-associated lymph 2/38 (5%) 1/12 (8%) 0.58 1/20 (5%) 3/37 (8%) node Brucella PCR-positive Lung morbillivirus PCR- 6/43 (14%) Not tested NA 2/20 (10%) Not tested positive

Denominators varied based upon information available, tissues collected from, and tests conducted on individual UME and reference dolphins. 1Barataria Bay, Louisiana UME dolphin subset values different (P < 0.05) than standardly scored references; 2All reference dolphin values different (P < 0.05) than all UME dolphins

doi:10.1371/journal.pone.0126538.t003

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Table 4. Prevalence comparisons of causes of death for fresh dead, stranded common bottlenose dolphins (Tursiops truncatus) among 1) unusual mortality event cases from Louisiana, Mississippi, and Alabama, 2) slide-reviewed and standardly scored reference dolphin subset, 3) Barataria Bay, Louisiana UME dolphin subset, and 4) all reference dolphins.

Description UME case References P value (UME v. UME cases Thin adrenal Primary bacterial dolphins all standardly scored standardly scored Barataria Bay cortex cases pneumonia cases (n = 46) set (n = 36) reference dolphins) (n = 22) (n = 12) (n = 10) Infection 18 (39%) 5 (14%) 0.01 6 (27%) 3 (25%) 7 (70%)* Unknown 13 (28%) 9 (25%) 0.84 9 (41%) 5 (42%) 0 Unknown—poor 3 (7%) 3 (8%) 0.54 2 (9%) 0 0 body condition Unknown—body 10 (22%) 6 (17%) 0.57 7 (32%) 5 (42%) 0 condition not noted likely contributor Trauma 8 (17%) 10 (28%) 0.26 3 (14%) 1 (8%) 0 Multifactorial 4 (9%) 2 (6%) 0.69 2 (9%) 2 (17%) 3 (30%) Organ failure 2 (4%) 0 NA 1 (5%) 1 (8%) 0 Food obstruction/spine 1 (2%) 8 (22%) 0.005 1 (5%) 0 0 injury Maternal separation 0 2 (6%) NA 0 0 0 Neoplasia 0 0 NA 0 0 0

Denominators varied based upon information available, tissues collected from, and tests conducted on individual UME and reference dolphins. *Values different (P < 0.05) than reference dolphins. doi:10.1371/journal.pone.0126538.t004

In UME dolphins with primary bacterial pneumonia, bronchioles and alveolar spaces were often filled with viable and necrotic neutrophils (i.e. suppurative) and fewer macrophages. Bac- teria were extracellular or intracellular within neutrophils and macrophages. In some cases, there was extensive necrosis. In the most severe cases, large accumulations of inflammatory cells completely obscured normal pulmonary parenchyma. Inflammatory cells and bacteria were encircled by dense bands of fibrous connective tissue and there were multifocal abscesses (Fig 3). In several cases, inflammatory cells surrounded bacterial colonies that were bordered by radiating, bright eosinophilic club-shaped material (Splendore-Hoeppli material). Among UME dolphins with primary bacterial pneumonia, bacteria within lung lesions included coccobacilli (n = 3), cocci (n = 2), and gram positive acid fast negative filamentous bacteria (n = 2). Pulmonary Escherichia coli, Staphylococcus aureus, and Streptococcus spp. Group G infections were confirmed via bacterial culture of lung tissue in UME cases. Staphylococcus aureus and Streptococcus spp. infections were associated with sepsis. Another UME dolphin with primary bacterial pneumonia had concurrent PCR-confirmed Brucella meningoencephalitis, although Brucella PCR on lung tissue was negative. Among UME dolphins overall, two dolphin lungs were positive for Brucella sp. via PCR. Histologically, these dolphins did not have lung lesions consistent with active brucellosis. Addi- tional bacterial PCR assays used in other primary bacterial pneumonia cases were unsuccessful in amplifying any single pathogenic bacterial species from affected lung tested, likely due to postmortem bacterial overgrowth. Of UME dolphins with primary bacterial pneumonia and had a measurable adrenal gland, 4n/9 (44%) had a thin adrenal gland cortex. One of 10 (10%) UME dolphins with primary bacterial pneumonia and known cardiac histologic evaluation had depleted cardiac adipose tissue, 3 of 6 (50%) had zymogen depletion, and 2 of 6 (33%) had retained gastric epithelium. Other common pulmonary lesions among UME and reference dolphins included lungworm pneumonia, pulmonary fibrosis, and angiomatosis. The most common cause of pneumonia

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Fig 3. Hematoxalin and eosin (HE) stained section of lung from an adult female common bottlenose dolphin (Tursiops truncatus) that stranded in Barataria Bay, Louisiana in December 2011 with primary bacterial pneumonia. Airspaces and septae are obscured by accumulations of neutrophils and fibrous connective tissue that surround large bacterial colonies (arrows). Bar = 500 μm. doi:10.1371/journal.pone.0126538.g003

amongst both UME and reference dolphins was primary lungworm infection (Table 3). Refer- ence dolphins, however, were more likely to have this common cause of pneumonia than the UME dolphins (62% versus 41%, respectively, P = 0.005).

Lymphoid tissues UME dolphins had a higher prevalence of lymphoid depletion in either or both the spleen or lymph node than reference dolphins (Table 2). When lymphoid depletion was present in either the lymph nodes or the spleen, both B lymphocyte (lymphoid follicles in the spleen and lymph node and medullary cords of the lymph node) and T lymphocyte (periarterior lymphoid sheaths in the spleen and the paracortex of the lymph nodes) regions were affected in the majority of the UME cases. Affected lymphoid follicles had a thin mantle zone often depleted of small lymphocytes. Of the five UME cases with both splenic and lymph node depletion, three died from morbillivirus infections, one died from non-Brucella bacterial meningoencephalitis, and another died from generalized debilitation associated with poor body condition.

Central nervous system Meningitis or meningoencephalitis were equally common in UME and reference dolphins (20% and 30%, respectively) (Table 2). Central nervous system (CNS) inflammation among UME dolphins was characterized as mild (n = 1), moderate (n = 5), and severe (n = 3). The most common pathogen category associated with meningoencephalitis was bacterial (5, 11% of UME dolphins), which was similar to the reference dolphins (4, 9%) (Table 2). Three (7%) UME dolphins had Brucella-associated meningoencephalitis. Two of the UME dolphins with encephalitis or meningoencephalitis had a thin adrenal gland cortex, and one had a primary bacterial pneumonia.

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Fig 4. Hematoxylin and eosin (HE) stained section of liver from a juvenile dolphin that stranded fresh dead in Barataria Bay in July 2012. There is severe centrilobular hepatocellular vacuolation, necrosis, and loss with lobular collapse (arrow). Star indicates a portal triad. Arrow head indicates unaffected liver parenchyma for comparison. Bar = 500μm. doi:10.1371/journal.pone.0126538.g004 Liver The prevalence of hepatic lesions, including fibrosis, necrosis, hepatitis, hemosiderosis, lipid deposition, and hepatobiliary hypertrophy, was similar among UME and reference dolphins (Table 2). The prevalence of moderate to severe hepatic fibrosis (31% versus 36%, P = 0.65) and inflammation (2% versus 7%, P = 0.62) were also comparable between the UME and reference dolphins. Fibrosis and inflammation had a periportal distribution in the majority of the dolphins from both groups, and lesions were most often mild to moderate in severity. Two UME cases were identified with centrilobular hepatocellular vacuolation, degeneration, necrosis, and loss with stromal collapse (Fig 4). Both of these dolphins stranded in Barataria Bay, one in June 2011 and one in July 2012. The June 2011 stranded dolphin was found live and externally oiled. The July 2012 stranded dolphin had evidence of jaundice on gross examination consistent with significant liver disease.

Causes of death The most common cause of death for UME dolphins was infectious disease (39%), and UME dolphins were four times more likely to die from infectious causes than the reference dolphins (14%, P = 0.01) (Table 4). The most common causes of death for Barataria Bay dolphins were infectious disease (27%) and unknown, not attributed to poor body condition (32%) (Table 4). An unknown cause of death not attributed to poor body condition was most common in UME dolphins with a thin adrenal gland cortex (42%, 5/12). Only three of 19 (16%) UME dolphins that died from infectious causes (and had available adrenal gland for measurement) had a thin adrenal gland cortex; of all 19 UME dolphins that died from infectious causes, eight (42%) had a primary bacterial pneumonia, and five (26%) died from morbillivirus infections.

Nutritional status Of UME dolphins in which appropriate tissues were available to asses nutritional status, 2/42 (5%) had thin epicardial adipose tissue indicative of emaciation. There were also indicators of

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inanition by pancreatic zymogen depletion and retained superficial gastric epithelium in 5/28 (18%) and 7/27 (26%) of dolphins, respectively.

Morbillivirus, Brucella, and marine biotoxins A total of 6/43 (14%) lung and 5/33 (15%) CNS samples from UME dolphins were positive for morbillivirus using PCR, with a total of 8 (19%) UME dolphins with tissues positive for morbillivirus. Five of these eight UME cases had relevant histologic lesions in tissue from which morbillivirus was identified. Five UME morbillivirus PCR positive cases stranded in 2011 and three in 2012 in multiple states. Most (6 of 8) UME dolphins with morbillivirus infections were either calves, juveniles, or subadults. Lesions noted in morbillivirus-affected dolphins included splenic and lymph node lymphoid depletion, syncytial cells, pneumonia, and lymphocytic encephalitis. Two cases had concurrent fungal infections. Three cases of brucellosis, all with moderate to severe Brucella-associated lymphoplasmacytic meningoencephalitis and confirmed identification of Brucella using PCR or culture, were identified among UME dolphins. The prevalence of brucellosis based upon histopathology and ancillary testing in UME dolphins (6%) was similar to those in the standardly scored subsets (2/42, 5%, P = 0.68). There were also no differences in the prevalence of Brucella detection using PCR on lung or lung-associated lymph node between UME and reference dolphins (5% and 8%, respectively, P = 0.58, Table 3). All UME dolphins with brucellosis were males. Fecal, gastric content, or liver samples from twenty-eight of the UME dolphins included in this study were tested for brevetoxin and/or domoic acid. Of 26 UME dolphins tested for brevetoxin, one dolphin had detectible levels of brevetoxin by ELISA (2 ng/g) but was negative by LC-MS. Of 27 UME dolphins tested for domoic acid, three were positive at low concentrations (all were 8 ng/g). All positive results were consistent with background exposures [12].

Discussion To our knowledge, adrenal cortical atrophy as found in this study has not been previously described in free-ranging cetaceans, including bottlenose dolphins previously studied in the northern GoM [32]. The normal corticomedullary ratio of dolphin adrenal glands has been determined to be approximately 1:1 [24]. Thus, the discovered high prevalence of adrenal cortical atrophy in dolphins stranding during the ongoing GoM UME may be part of a syndrome that has not been previously reported in dolphins during mortality events. The prevalence of adrenal cortical atrophy identified in this study is consistent with the high prevalence (approximately 50%) of live Barataria Bay dolphins with evidence of hypoadrenocorticism assessed during 2011, including a relatively high proportion of dolphins with low blood cortisol, aldosterone, and glucose [11]. Follow up evaluation of adrenal glands from stranded dolphins in subsequent years will help to determine the persistence of adrenal insufficiency observed relative to the timing of the UME and the concurrent DWH oil spill. There are a number of different causes of adrenal insufficiency in mammals, including autoimmune disease, metastatic neoplasia, fungal infections, stress, trauma, miliary tuberculosis, corticosteroid toxicity, and contaminant exposure [33]. Additionally, infection with phocine herpesvirus-1 has been demonstrated to cause adrenal cortical necrosis in marine mammals [34]. In the current study, only 2 of 46 UME dolphins had inflammation in the adrenal gland, and with the exception of one case with a disseminated bacterial infection, neither infectious agents nor neoplasia were identified in UME dolphin adrenal glands. Further, there was no histologic evidence of autoimmune adrenalitis or neoplasia in any UME dolphin adrenal glands, indicating that adrenal cortical atrophy in UME dolphins was not due to direct infection of the adrenal gland, autoimmune disease, or neoplasia.

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In humans, chronic demand on the adrenal glands, including chronic illness, has been postulated to lead to cortical thinning and potential adrenal exhaustion associated with lipid depletion of the fasciculata cells [35,36]. Previous evaluations of adrenal glands from stranded GoM dolphins from Texas with both acute and chronic disease have been conducted, but no cases of adrenal cortical atrophy were identified [32]. Instead, adrenal glands of dolphins dying from chronic disease (likely chronically stressed individuals) were significantly heavier, and corticomedullary ratios were significantly higher than those dying from acute disease or acute trauma. Findings from Clark et al. (2006) suggest that adrenal gland enlargement and cortical hyperplasia are common responses to chronic stress and disease in bottlenose dolphins, similar to that noted in other cetaceans and other mammalian species [24, 32, 37–39]. Of the 12 UME dolphins with a thin adrenal gland cortex, one-third had primary bacterial pneumonia, leaving the majority of adrenal cortex cases without evidence of active or chronic infections. Further, none of the UME dolphins with a thin adrenal gland cortex had depleted cardiac adipose tissue, indicating that UME dolphins were not in an advanced, debilitated nutritional state. These results do not support general infection or chronic poor body condition as underlying causes of adrenal gland cortex depletion. Although the effects of polycyclic aromatic hydrocarbon (PAHs) on the hypothalamus-pi- tuitary-adrenal (HPA) axis are poorly understood, the adrenal gland is reported to be the most common endocrine organ to exhibit lesions with exposure to toxigenic chemicals [40, 41]. In general, mechanisms of direct adrenal toxicity include impaired steroidogenesis, activation of toxins by cytochrome p450 enzymes generating reactive oxygen metabolites, DNA damage, and exogenous steroid action [42]. The adrenal gland can be a significant site for metabolism of PAHs, thus increasing the adrenal gland to exposure from these contaminants and their metabolites [43]. Several studies have shown that PAHs or oil can affect the HPA axis and adrenal gland function. Hypoadrenocorticism has been reported in mink fed either bunker C or artificially weathered fuel oil [44,45]. In these mink, adrenal cortical hypertrophy with vacuolation of corticocytes was detected histologically. These studies, however, did not monitor changes in response to higher level exposure and/or over longer periods of time. Chemicals that induce adrenal cortical vacuolar degeneration can lead to loss of adrenocortical cells due to necrosis and adrenal cortical atrophy. It is possible PAHs may act in a similar fashion [42]. Naphtha- lene, a common PAH associated with crude oil, reduced plasma corticosterone in mallard ducks following ingestion of petroleum-contaminated food, and a similar acute decrease in cortisol was detected in exposed eels [46, 47]. House sparrows exposed orally to 1% crude oil from the GoM exhibited decreases in cortisol in response to stressors or to adrenocorticotropin hormone injection [48]. Mammalian exposure to PAHs can greatly increase hepatic metabolism of other compounds (e.g. 7,12-dimethylbenz(α)anthracene), which in turn can cause targeted and severe injury to the adrenal gland, including necrosis and hemorrhage [49–51]. Removal of the inciting chemical, if the adrenal cortical injury is not too advanced, may result in regained function and resolved lesions characterized by fibrosis, atrophy, nodular regeneration or calcification [42, 47, 51]. Ultrastructural analysis can be beneficial in helping to identify direct toxic damage. Unfortunately, optimally fixed, minimally autolyzed tissue from affected dolphin adrenal glands was not available for ultrastructural analysis. The lack of adrenal lesions beyond cortical atrophy suggests, however, that potential chemical effects may be higher in the HPA axis [52]. During and following the DWH oil spill, significantly elevated PAH levels were detected in the coastal GoM waters, including Louisiana, Mississippi, and Alabama [53]. These locations coincide with the states most impacted by the ongoing UME since the DWH oil spill [1]. Thus, northern GoM dolphins’ exposures to DWH spill-associated PAHs, especially in Louisiana and

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Mississippi, may account for the observed effects on adrenal function found in both live and dead dolphins [11]. Given the lack of evidence of alternative causes of adrenal cortical atrophy and the high prevalence of this lesion among stranded dolphins following the DWH oil spill, the leading hypothesis is that exposure to contaminants from the DWH oil spill led to chronic injury of the adrenal gland cortex at least through 2012. Chronic adrenal insufficiency (CAI) is a life-threatening disease that can lead to adrenal crisis and death in mammals [53]. Adrenal crises in people with CAI are triggered by infections, fever, major pain, psychological distress, heat, and pregnancy [54]. Cold temperatures can also increase the risk of death among animals with CAI. Angora goats with a genetically-driven high incidence of primary CAI lack proper cortisol and glucose response and, as such, are susceptible to die-offs from cold stress [54, 55]. GoM dolphins were exposed to colder than normal temperatures during early 2011, and if those dolphins from the UME had pre-existing CAI, they may have been at higher risk of cold stress-related deaths [6]. This hypothesis is further supported in that dolphins have a compensatory adrenal response in cold temperatures, including increased cortisol levels, presumably to help generate metabolic heat [56]. Adrenal crisis may have been the cause of death for many of the UME dolphins with adrenal cortical atrophy following stress events to which a healthy dolphin could have otherwise adapted. In addition to the cold weather during 2011, adrenal crisis could have also been precipitated by late-term pregnancies and infections, including bacterial pneumonia [57]. Compared to reference dolphins, UME dolphins were more likely to have a primary bacterial pneumonia. Many of these pneumonias were much more severe than bacterial pneumonias in the reference dolphins. These findings are consistent with the high prevalence of moderate to severe lung disease detected in live Barataria Bay dolphins [11]. During the DWH oil spill and response period, numerous dolphins, including dolphins in Barataria Bay, were observed swimming through visibly oiled waters and feeding in areas of surface, subsurface, and sediment oiling [11]. As mentioned, the presence of increased coastal PAH levels associated with the DWH oil spill, especially near Grand Isle, Louisiana in Barataria Bay, have been confirmed, indicating an increased risk of inhaled PAHs in dolphins [4]. Given that the dolphin's blowhole is at the surface of the water, chemicals, including volatile PAHs, could have been readily inhaled. In other animals, inhaled PAHs can irritate airways, denude mucosal surfaces, and cause peribronchial inflammation and systemic toxicity [58]. Damaged epithelium and cilia, in turn, can severely impair immune defenses. In other animals, the severity of chemical inhalation injury is dependent on breathing patterns, in which deep breaths increase injury to tissues deeper within the lung [59]. This pattern is of particular importance given the dolphin's respiratory anatomy and physiology. While humans exchange approximately 10 to 20% of air with each breath, dolphins exchange 75 to 90% of deep lung air [60–63]. They also lack nasal turbinates and cilia to filter the air prior to reaching the lungs, and have deep inhalations followed by a breath hold that provides potential for more prolonged contact and exchange between air-borne particulates and the blood [60–63]. All of these factors would likely amplify the effects of inhaled chemical irritants in dolphins compared to observations and studies involving other mammals. The severe bacterial pneumonias found in UME dolphins could represent a chronic sequelae to hydrocarbon inhalation or aspiration, or have been secondary to PAH induced immune system compromise. The most common sequela to hydrocarbon inhalation and ingestion in humans and animals are aspiration pneumonia and pneumonia often involving the bronchioles [64–68]. Inhaled hydrocarbon vapors or aspirated hydrocarbons may cause necrosis of bronchial and bronchiolar epithelium, and pneumocyte and alveolar septal necrosis which leads to inflammation and secondary infection [64–66]. During the 2007 firestorm in San Diego, dolphins and people living in San Diego Bay area were exposed to high levels of PAHs

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[69, 70]. The month following the fires, these dolphins demonstrated decreased absolute and percent neutrophils [70]. This change indicated that dolphins exposed to PAHs through inhalation may have had a compromised immune response and an increased risk of acquiring bacterial pneumonia. In addition to inhalation risks, hydrocarbon ingestion can lead to gastrointestinal mucosal irritation, vomiting or regurgitation, and resultant aspiration pneumonia. Cattle that ingest petroleum develop bacterial pneumonia due to chemical-induced regurgitation and/or aspiration [67,68]. Correspondingly, based on histologic examination, one dolphin that stranded during June 2010 in Mississippi during the DWH oil spill had suspected aspiration pneumonia, secondary bacterial infection, ulcerative tracheitis, and ulcerative gastritis with edema. Both the tracheal and gastric lesions, although non-specific, could have resulted from mucosal irritation, such as may occur with toxin ingestion. Though there was no difference in prevalence of liver lesions when comparing UME and reference dolphins in this study, two UME dolphins had similar severe centrilobular liver lesions characterized by hepatocyte loss, necrosis and vacuolation that could potentially be associated with toxin exposure. Both of these dolphins stranded in Barataria Bay. Hepatocellular vacuolation, degeneration and necrosis have been associated with exposure to crude oil and benzo[a]pyrene (BaP) [71 72]. Hepatotoxic liver injury may occur due to xenobiotic metabolism of substances producing injurious metabolites and lesions most often occur in the centrilobular zones where hepatocytes have the highest concentration of cytochrome p450 enzymes [71]. Other rule-outs for centrilobular degeneration and necrosis include hypoxia, severe or precipitous anemia (e.g. hemolytic anemia), chronic heart disease, or circulatory failure associated with septic shock [72, 73]. There was no other evidence of hypoxia, hemolytic anemia or heart disease in either of the affected dolphins and lesions were more chronic than would be expected secondary to acute hypoxia or shock. Some oiled sea otters that died following the Exxon Valdez oil spill had centrilobular hepatic necrosis, though whether the lesions were due to direct toxic insult or secondary to anemia is unclear [74]. Biliary or periportal inflammation and fibrosis secondary to infection by the trematode Campula spp. are common hepatic lesions noted in a number of cetacean species, and periportal lesions noted in both UME and reference dolphins were consistent with the chronic sequelae of biliary trematode infection [75]. This study did not support that previously documented or suspected contributing factors for GoM dolphin UMEs were primary contributors to the ongoing UME among non-perinatal dolphins. All UME dolphins in this case study had biotoxin levels that were below detectable levels except for one with low levels [12]. Relatively few morbillivirus cases were identified among UME cases. In previous known dolphin morbillivirus-associated die-offs, more than 60% of cases tested positive for the virus when using a similar PCR assay [14,16]. Exposure to morbillivirus has been documented in GoM dolphins, and the cases identified in 2011 and 2012 may represent exposure to the virus in a small number of susceptible individuals in the population [76]. Similarly, there were too few brucellosis cases in this study to explain the ongoing UME, with only two cases that had Brucella identified in the lung, demonstrating that Brucella was not the driver for increased primary bacterial pneumonia. Despite global reports of Brucella infections in marine mammals, there have been, to date, no documented brucellosis epizootics in cetaceans [17]. Due to the long duration and large scope of the ongoing UME, there may be multiple factors affecting the health of dolphins by region through time. Aside from the DWH oil spill, there were two relatively smaller oil spills that occurred in and around Barataria Bay during this study’s timeframe. Specifically, the T/V Pere Ana C spill in Mud Lake, Louisiana on July 27, 2010 (approximately 7,000 gallons spilled) and the Cedyco Manilla Village Spill in Bayou Dupont, Louisiana which occurred on September 11, 2011 (approximately 10,500 gallons)

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[77,78]. In comparison, however, the DHW oil spill released approximately 126,000,000 gallons and was visible across 40 km and 366,000 m2 of Barataria Bay’s shoreline in decreasing amounts over time for at least 2 years, demonstrating the higher magnitude of the DWH oil spill’s likely impact compared to other spills [7–10]. The Gulf of Mexico has historically had documented dead zones with episodes of seasonal hypoxia associated with nutrient loading from the Mississippi River watershed [79,80]. The geographic area and clusters of dolphin stranding identified from the current UME, however, were not limited to single seasons or specific dead zone hotspots [2]. Further, dead zones are often associated with fish die-offs and habitat loss; the lack of emaciation as the primary contributor to the deaths of dolphins in this study supports that the primary driver of this UME was not loss of prey [80]. The lack of baseline diagnostic and histologic data on fresh stranded dolphins prior to 2010 in the UME area, as well as during the pre-DWH oil spill period, paired with an assumption that stranded dolphins should have similar lesion prevalence regardless of location, are limitations in this study. The surprisingly high number of assessed lesions that were not significantly different between the UME and reference dolphins, however, increased the confidence that the study groups were indeed comparable. Continual assessment of trends and changing disease states over time are needed, however, to better understand the potential roles of multiple contributing factors to dolphin mortality during the ongoing UME. In summary, UME dolphins had a high prevalence of thin adrenal gland cortices (especially in Barataria Bay dolphins) and primary bacterial pneumonia. These findings are consistent with endocrinologic and pulmonary-based observations of live bottlenose dolphins from health assessments in Barataria Bay during 2011 [11]. Previously documented or suspected contributing factors for GoM UMEs (marine biotoxins, morbillivirus, and brucellosis) were not supported by this study as contributors to the ongoing UME. Due to the timing and nature of the detected lesions, we hypothesize that contaminants from the DWH oil spill contributed to the high numbers of dolphin deaths within this oil spill’s footprint during the northern GoM UME following the DWH oil spill. Direct causes of death likely included: 1) affected adrenal gland cortices, causing chronic adrenal insufficiency, 2) increased susceptibility to life-threatening adrenal crises, especially when challenged with pregnancy, cold temperatures, and infections, and 3) increased susceptibility to primary bacterial pneumonia, possibly due to inhalation injury, aspiration of oil, or perturbations in immune function.

Supporting Information S1 Table. Raw demographic, histologic, and diagnostic data from a case-reference study (June 2010–December 2012) investigating the potential cause(s) of increased bottlenose dolphin (Tursiops truncatus) deaths in the northern Gulf of Mexico following the Deepwa- ter Horizon oil spill. (XLSX)

Acknowledgments This work could not have been conducted without the efforts of the Marine Mammal Stranding Network, including personnel from those agencies working on the current northern GoM UME: Louisiana Department of Wildlife and Fisheries (especially staff from the Fisheries Re- search Lab in Grande Isle), Audubon Aquarium of the Americas, Institute for Marine Mammal Studies, Dauphin Island Sea Lab, Emerald Coast Wildlife Refuge, and Gulf World Marine Park, as well as, Micah Brodsky, Jeremy Hartley, Michelle Kelley, Courtney Nelson Seely, Bob

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MacLean, and Sandi Maillian. The authors acknowledge support from the northern GoM UME Investigative Team and the past and present members of the Working Group for Marine Mammal Unusual Mortality Events. In addition, we thank the following individuals who helped coordinate stranding response, manage or validate regional stranding data, or helped by reviewing or contributing to portions of this manuscript: Laura Aichinger Dias, Laura Engleby, Sandra Horton, Blair Mase-Guthrie, Lauren Noble, Gina Rappucci, Lori Schwacke, Elizabeth Stratton, Cynthia Smith, Sabrina Stevens, and Fran Van Dolah. This work was part of the Deep- water Horizon NRDA being conducted cooperatively among NOAA, other Federal and State Trustees, and BP.

Author Contributions Conceived and designed the experiments: SVW KMC JL KT JS DF EF TR. Performed the experiments: SVW KMC JL MK KT JS SF RC CC WH JP MT CF SS RE DF GL HW DR WM. Analyzed the data: SVW KMC JL KT. Contributed reagents/materials/analysis tools: SVW KMC JL SF RC CC WH JP MT CF SS DF GL HW DR EF. Wrote the paper: SVW KMC JL KT JS SF RC CC WH JP MT CF RE DF DR WM EF TR.

References 1. Litz J, Baran M, Bowen-Stevens S, Carmichael R, Colegrove K, Garrison L, et al. Review of historical unusual mortality events (UMEs) in the Gulf of Mexico (1990–2009): providing context for the multi-year northern Gulf of Mexico cetacean UME declared in 2010. Dis Aqua Org. 2014; 112: 161–175. 2. Venn-Watson S, Garrison L, Litz J, Fougeres E, Mase B, Rappucci G, et al. Demographic clusters identified within the northern Gulf of Mexico common bottlenose dolphin (Tursiops truncatus) unusual mortality event: January 2010–June 2013. PLOS ONE. 2015; 10: e0117248. doi: 10.1371/journal.pone. 0117248 PMID: 25671657 3. McNutt MK, Chu S, Lubcheno J, Hunter T, Dreyfus G, Murawski SA, et al. Applications of science and engineering to quantify and control the Deepwater Horizon oil spill. PNAS. 2012; 109:20222–20228. doi: 10.1073/pnas.1214389109 PMID: 23213225 4. Allan SE, Smith BW, Anderson KA. Impact of the Deepwater Horizon oil spill on bioavailable polycyclic aromatic hydrocarbons in the Gulf of Mexico coastal waters. Environ Sci Technol. 2012; 46: 2033–2039. doi: 10.1021/es202942q PMID: 22321043 5. Antonia FJ, Mendes RS, Thomaz SM. Identifying and modeling patterns of tetrapod vertebrate mortality rates in the Gulf of Mexico oil spill. Aqua Toxicol. 2011; 105: 177–179. 6. Carmichael RH, Graham WM, Allen A, Worthy G, Howden S. Were multiple stressors a 'perfect storm' for northern Gulf of Mexico bottlenose dolphins (Tursiops truncatus) in 2011? PLOS ONE. 2012; 7: e41155. doi: 10.1371/journal.pone.0041155 PMID: 22815950 7. Michel J, Owens EH, Zengel S, Graham A, Nixon Z, Allard T, et al. Extent and degree of shoreline oiling: Deepwater Horizon oil spill, Gulf of Mexico, USA. PLOS ONE. 2013; doi: 10.1371/journal.pone. 0065087 8. Lin Q, Mendelssohn IA. Impacts and recovery of the Deepwater Horizon oil spill on vegetation structure and function of coastal salt marshes in the Northern Gulf of Mexico. Environ Sci Technol. 2012; 46: 3737–3743. doi: 10.1021/es203552p PMID: 22369124 9. Silliman BR, van de Koppel J, McCoy MW, Diller J, Kasozi GN, Earl K, et al. Degradation and resilience in Louisiana salt marshes after the BP—Deepwater Horizon oil spill. PNAS. 2012; 109: 1123–11239. 10. Kokaly RF, Couvillion BR, Holloway JM, Roberts DA, Ustin SL et al. Spectroscopic remote sensing of the distribution and persistence of oil from the Deepwater Horizon spill in Barataria Bay marhes. Re- mote Sens Environ. 2013; 129: 210–230. 11. Schwacke LH, Smith CR, Townsend FI, Wells RS, Hart LB, Balmer BC, et al. Health of common bottlenose dolphins (Tursiops truncatus) in Barataria Bay, Louisiana, following the Deepwater Horizon oil spill. Environ Sci Tech. 2014; 48: 93–103. doi: 10.1021/es403610f PMID: 24350796 12. Fire SE, Wang Z, Byrd M, Whitehead HR, Paternoster J, Morton SL. Co-occurrence of multiple classes of harmful algal toxins in bottlenose dolphins (Tursiops truncatus) stranding during an unusual mortality event in Texas, USA. Harmful Algae. 2011; 10: 330–336.

PLOS ONE | DOI:10.1371/journal.pone.0126538 May 20, 2015 20 / 23 Adrenal and Lung Lesions in Gulf Dolphins

13. Krafft A, Lichy JH, Lipscomb TP, Klaunberg BA, Kennedy S, Taubenberger JK. Postmortem diagnosis of morbillivirus infection in bottlenose dolphins (Tursiops truncatus) in the Atlantic and Gulf of Mexico epizootics by polymerase chain reaction-based assay. J Wildl Dis. 1995; 31: 410–415. PMID: 8592367 14. Lipscomb TP, Kennedy S, Moffett D, Krafft A, Klaunberg BA, Lichy JH, et al. Morbillivirus epizootic in bottlenose dolphins of the Gulf of Mexico. J Vet Diagn Invest. 1996; 8: 283–290. PMID: 8844569 15. Twiner M J, Flewelling LJ, Fire SE, Bowen-Stevens SR, Gaydos JK, Johnson CK, et al. Comparative analysis of three brevetoxin-associated bottlenose dolphin (Tursiops truncatus) mortality events in the Florida panhandle region (USA). PLOS ONE. 2012; 7: 1–19. 16. Schulman FY, Lipscomb TP, Moffett D, Krafft AE, Lichy JH, Tsai MM, et al. Histologic, immunohisto- chemical and polymerase chain reaction studies of bottlenose dolphins from the 1987–1988 United States Atlantic coast epizootic. Vet Pathol. 1997; 34: 288–295. PMID: 9240837 17. Hernandez-Mora G, Palacios-Alfaro JD, Gonzalez-Barrientos R. Wildlife reservoirs of brucellosis: Bru- cella in aquatic environments. Rev Sci Tech Off Int Epi. 2013; 32: 89–103. PMID: 23837368 18. Cassle S, Johnson S, Lutmerding B, Jensen E. Pulmonary brucellosis in an Atlantic bottlenose dolphin (Tursiops truncatus) (2009) In Proceedings of the 40th Conference of the International Association of Aquatic Animal Medicine: 2–6 May 2009. San Antonio. 1999:129. 19. Dagleish MP, Barley J, Howie FE, Reid RJ, Herman J, Foster G. Isolation of Brucella species from a diseased atlanto-occipital joint of an Atlantic white-sided dolphin (Lagenorhynchus acutus). Vet Rec. 2007; 160: 876–6. PMID: 17586794 20. Davison NJ, Cranwell MP, Perrett LL, Dawson CE, Deaville R, Stubberfield EJ, et al. Meningoencepha- litis associated with Brucella species in a live-stranded striped dolphin (Stenella coeruleoalba) in southwest England. Vet Rec. 2009; 165: 86–9. PMID: 19617615 21. Miller WG, Adams LG, Ficht TA, Cheville NF, Payeur JP, Harley DR, et al. Brucella-induced abortions and infection in bottlenose dolphins (Tursiops truncatus). J Zoo Wildl Med 1999; 30: 100–110. PMID: 10367651 22. Hoffman RJ. History, goals and achievements of the regional marine mammal stranding network. pp 7–17. In: Reynolds J.E. and Odell D.K. (eds.) Marine Mammal Strandings in the United States. NOAA Technical Report NMFS 98. 1991;157 pp. 23. Terio KA, Marker L, Munson L. Evidence for chronic stress in captive but not free-ranging cheetahs (Acinonyx jubatus) based on adrenal morphology and function. J Wild Dis. 2004; 40: 259– 266. PMID: 15362825 24. Clark LS, Pfeiffer DC, Cowan DF. Morphology and histology of the Atlantic bottlenose dolphin adrenal gland with emphasis on the medulla. Anat Histol Embryol. 2005; 34: 132–140. PMID: 15771677 25. Fauquier DA, Kinsel MJ, Dailey MD, Sutton GE, Stolen MK, Wells RS, et al. Prevalence and pathology of lungworm infection in bottlenose dolphins Tursiops truncatus from southwest Florida. Dis Aqua Org 2009; 88: 85–90. 26. Turnbull BS, Cowan DF. Angiomatosis, a newly recognized disease in Atlantic bottlenose dolphins (Tursiops truncatus) in the Gulf of Mexico. Vet Path. 1999; 36: 1–28. 27. Sierra E, Sanchez S, Saliki JT, Blas-Machado U, Arbelo M, Zucca D, Fernandez A. Etiologic agents associated with non-suppurative meningoencephalitis in stranded cetaceans: a retrospective study in the Canary Islands. J Clin Microbiol. 2014; doi: 10.1128/JCM.02906-13 28. Fire SE, Flewelling LJ, Wang Z, Naar J, Henry MS, Pierce MS, et al. Florida red tide and brevetoxins: association and exposure in live resident bottlenose dolphins (Tursiops truncatus) in the eastern Gulf of Mexico, U.S.A. Mar Mamm Sci; 2008; 24: 831–844. 29. Maucher JM, Briggs L, Podmore C, Ramsdell JS. Optimization of blood collection card method/ enzyme-linked immunoassay for monitoring exposure of bottlenose dolphin to brevetoxin-producing red tides. Environ Sci Technol. 2007; 41: 563–567. PMID: 17310722 30. Ficht TA, Husseinen HS, Derr J, Bearden SW. Species-specific sequences at the omp2 locus of Bru- cella type strains. Int J Syst Bact 1996; 46: 329–31. PMID: 8573514 31. St. Leger JA, Begeman L, Fleetwood M, Frasca S Jr, Garner MM, Lair S, et al. Comparative pathology of nocardiosis in marine mammals. Vet Pathol. 2009; 46: 299–308. doi: 10.1354/vp.46-2-299 PMID: 19261643 32. Clark LS, Cowan DF, Pfeiffer DC. Morphological changes in the Atlantic bottlenose dolphin (Tursiops truncatus) adrenal gland associated with chronic stress. J Comp Pathol. 2006; 135: 208–216. PMID: 17067620 33. Oelkers W. Adrenal insufficiency. N Engl J Med. 1996; 335: 1206–1212. PMID: 8815944 34. Gulland FMD, Haulena M, Lowenstine LJ, Munro C, Graham PA, Bauman J, et al. Adrenal function in wild and rehabilitated Pacific harbor seals (Phoca vitulina richardii) and in seals with phocine herpesvirus-associated adrenal necrosis. Mar Mamm Sci. 2006; 15:810–827.

PLOS ONE | DOI:10.1371/journal.pone.0126538 May 20, 2015 21 / 23 Adrenal and Lung Lesions in Gulf Dolphins

35. Zwemer RL. A study of adrenal cortex morphology. Am J Path. 1936; 12: 107–111. PMID: 19970248 36. Sasano H, Nakamura Y, Moriya T, Suzuki T. Adrenal Cortex. In Endocrine Pathology: Differential Diag- nosis and Molecular Advances, Lloyd R.V. (ed). Springer Science + Business Media, LLC 2010. pp 261–262. 37. Kuiken T, Hofle U, Bennett PM, Allchin CR, Kirkwood JK, Baker JR, et al. Adrenal cortical hyperplasia, disease, and chlorinated hydrocarbons in the harbor porpoise (Phocoena phocoena). Mar Poll Bull. 1993; 26: 440–446. 38. Lair S, Beland P, De Guise S, Martineau D. Adrenal hyperplastic and degenerative changes in beluga whales. J Wildl Dis. 1997. 33: 430–437. PMID: 9249687 39. Rideout BA, Gause GE, Benirschke K, Lasley BL. Stress-induced change and their relationship to reproductive failure in captive nine-banded armadillos (Dasypus novemcinctus). Zoo Bio. 1985; 4: 129–137. 40. Ribelin WE. The effects of drugs and chemicals upon the structure of the adrenal gland. Fund Appl Tox- icol. 1984; 4: 105–119. 41. Nichols JW. Breen M, Denver RJ, Distefano JJ, Edwards JS, Hoke RA, et al. Predicting chemical impacts on vertebrate endocrine systems. Environ Toxicol Chem. 2011; 30:39–51. doi: 10.1002/etc.376 PMID: 20963851 42. Rosol T. Adrenal gland: structure, function, and mechanisms of toxicity. Toxicol Path. 2001; 29: 41–48. PMID: 11215683 43. Greiner JW, Kramer RE, Robinson DA, Canady WJ, Colby HD. Interaction of aromatic hydrocarbons and drugs with adrenal microsomal cytochrome P-450 and the guinea pig. Biochem Pharm. 1976; 25: 951–955. PMID: 1267839 44. Mohr FC, Lasley B, Bursian S. Chronic oral exposure to bunker C fuel oil causes adrenal insufficiency in ranch mink (Mustela vison). Arch Environ Contam Toxicol; 2008; 54: 337–347. PMID: 17763884 45. Mohr FC, Lasley B, Bursian S. Fuel oil-induced adrenal hypertrophy in ranch mink (Mustela vison): effects of sex, fuel oil weathering, and response to adrenocorticotrophic hormone. J Wildl Dis. 2010; 46: 103–110. PMID: 20090023 46. Gorsline J, Holmes WN. Adrenocortical function and hepatic naphthalene metabolism in mallard ducks (Anas platyrhynchos) consuming petroleum distallates. Environ Res. 1982; 28: 139–146. PMID: 7106069 47. Teles M, Pacheco M, Santos MA. Anguilla Anguilla L. liver ethoxyresorufin-deethlylation, glutathione S- tranferase, erthrocytic nuclear abnormalities, endocrine responses to naphthalene and BI-naphthofla- vone. Ecotox Environ Safety 2003; 55: 98–107. PMID: 12706398 48. Lattin CR, Ngai HM, Romero LM. Evaluating the stress response as a bioindicator of sublethal effects of crude oil exposure in wild house sparrows (Passer domesticus) PLOS ONE. 2014; 9: e102106. doi: 10.1371/journal.pone.0102106 PMID: 25029334 49. Bond B, Orr JW. The effects of a single dose of 7,12-Dimethylbenz(α)anthracene on the epidermis and hair follicles of mice, with notes on concurrent changes in the ovaries and adrenals. Br J Cancer. 1969; 23: 188–196. PMID: 5768435 50. Huggins C, Morii S. Selective adrenal necrosis and apoplexy induced by 7,12—Dimethylben(α)antra- cene. J Exptl Med. 1961; 114: 741–760. 51. Levin W, Conney AH. Stimulatory effect of polycyclic hydrocarbons and aromatic azo derivatives on the metabolism of 7,12-Dimethylbenz(α)anthracene. Cancer Res. 1967; 27:1931. PMID: 4169331 52. Bornstein SR, Chrousos GP. Clinical review 104: adrenocorticotropin (ACTH)- and non-ACTH- mediated regulation of the adrenal cortex: neural and immune inputs. J Clin Endocrinol Metab. 1999; 84: 1729–1736. PMID: 10323408 53. Arlt W, Allolio B. Adrenal insufficiency. Lancet. 2003; 361: 1881–1893. PMID: 12788587 54. Hahner S, Loeffler M, Bleicken B, Drechsler C, Milovanovic D, Fassnacht, et al. Epidemiology of adrenal crisis in chronic adrenal insufficiency: the need for new prevention strategies. Eur J Endocrinol. 2009; 162: 597–602. doi: 10.1530/EJE-09-0884 PMID: 19955259 55. Engelbrecht Y, Herselman T, Louw A, Swart P. Investigation of the primary cause of hypoadrenocorticism in South African angora goats (Capra aegagrus): a comparison with Boer goats (Capra hircus) and Merino sheep (Ovis aries). J Anim Sci. 2000; 78: 371–379. PMID: 10709928 56. Houser DS, Yeates LC, Crocker DE. Cold stress induces an adrenocortical response in bottlenose dolphins (Tursiops truncatus). J Zoo Wildlf Med. 2011; 42: 565–571. PMID: 22204049 57. Hills G, Venning EH, Dohan FC, Webster GD, Richardson EM. Pregnancy and adrenocortical function: endocrine studies of pregnancy occurring in two adrenal-deficient women. J Clin Invest. 1954; 33: 1466–1480. PMID: 13211801

PLOS ONE | DOI:10.1371/journal.pone.0126538 May 20, 2015 22 / 23 Adrenal and Lung Lesions in Gulf Dolphins

58. Demling RH. Smoke inhalation lung injury: an update. Eplasty. 2008; 8: e27. PMID: 18552974 59. Lalonde C, Demling R, Brian J, Blanchard J. Smoke inhalation injury in sheep is caused by the particle phase not the gas phase. J Appl Physiol. 1994; 77:15–22. PMID: 7961227 60. Green RF. Observations of the anatomy of some cetaceans and pinnipeds. In: Ridgway SH, editor. Mammals of the sea: biology and medicine. Springfield, IL: Charles C. Thomas Publishers. 1972. p.247–297. 61. Irving L, Scholander PF, Grinnell SW. The respiration of the porpoise, Tursiops truncatus. J Cell Comp Physiol. 1941; 17: 145–168. 62. Ridgway SH, Scronce BL, Kanwisher J. Respiration and deep diving in the bottlenose porpoise. Sci- ence. 1969; 166: 1651–1654. PMID: 5360592 63. Ridgway SH, Ed. Mammals of the Sea: Respiration System. Charles C. Thomas, Springfield, Illinois. 1972. pp 260–264. 64. Azeez OM, Akhigbe RE, Anigbogu CN. Exposure to petroleum hydrocarbon: implications in lung lipid peroxidation and antioxidant defense system in rat. Toxicol Int. 2012; 19: 306–309. doi: 10.4103/0971- 6580.103678 PMID: 23293471 65. Coppock RW, Mostrom MS, Stair EL, Semalulu SS. Toxicopathology of oilfield poisoning in cattle: A review. Vet Human Toxicol. 1996; 38: 36–42. PMID: 8825749 66. Eade NR, Taussig LM, Marks MI. Hydrocarbon pneumonitis. Pediatrics. 1974; 54: 351–356. PMID: 4606628 67. Rowe LD, Dooahite JW, Camp BJ. Toxicity of two crude oils and kerosene to cattle. JAVMA. 1951; 162: 61–66. 68. Edwards WC. Toxicology of oil field wastes. Hazards to livestock associated with the petroleum industry. Food Animal Prac. 1989; 5: 363–374. PMID: 2667711 69. Bytnerowicz A, Cayan D, Riggan P, Schilling S, Dawson P, Tyree M, et al. Analysis of the effects of combustion emissions and Santa Ana winds on ambient ozone during the October 2007 southern Cali- fornia wildfires. Atmosph Environ. 2010; 44: 678–687. 70. Venn-Watson S, Smith CR, Jensen ED, Rowles T. Assessing the potential health impacts of the 2003 and 2007 firestorms on bottlenose dolphins (Tursiops truncatus) in San Diego Bay. Inhal Toxicol. 2013; 25: 481–491. doi: 10.3109/08958378.2013.804611 PMID: 23905966 71. Agamy E. Histopathological changes in the livers of rabbit fish (Siganus canaliculatus) following exposure to crude oil and dispersed oil. Toxicol Pathol. 2012; 40: 1128–1140. doi: 10.1177/ 0192623312448936 PMID: 22659245 72. Pancheco M, Santos MA. Biotransformation, genotoxic, and histopathologic effects of environmental contaminants in European eel (Anguilla Anguilla L.). Ecotoxicol and Environ Safety. 2002; 53:331–347. PMID: 12485576 73. Cullen JM. Liver. Biliary system, and exocrine pancreas, In Pathologic Basis of Veterinary Diseases, 4th ed, editors McGavin MD and Zachary JF. Mosby Elsevier, St. Louis Missouri. 2007. pp 393–462. 74. Lipscomb TP, Harris RK, Moeller RB, Pletcher JM, Haebler RJ, Ballachey BE. Histopathologic lesions in sea otters exposed to crude oil. Vet Pathol. 1993; 30: 1–11. PMID: 8442322 75. Jaber JR, Arbelo M, Andrada M, Hidalgo M, Gomez-Villamandos JC, Van Den Ingh T, et al. Hepatic lesions in cetaceans in cetaceans stranded in the canary islands. Vet Pathol. 2004; 41: 147–153. PMID: 15017028 76. Duignan P, House C, Odell DK, Wells RS, Hansen LJ, Walsh MT, et al. Morbillivirus infection in bottlenose dolphins: Evidence for recurrent epizootics in the western Atlantic and Gulf of Mexico 1996. Ma- rine Mamm Sci. 1996; 12: 499–515. 77. National Oceanic and Atmospheric Administration. T/V Pere Ana C. Available: http://incidentnews. noaa.gov/incident/8241. Accessed 2010 Jul 27. 78. National Oceanic and Atmospheric Administration. Cedyco Manila Village Spill. Available: http:// incidentnews.noaa.gov/incident/8358. Accessed 2011 Sep 11. 79. Turner RE, Rabalais NN, Justic D. Gulf of Mexico hypoxia: Alternate states and a legacy. Environ Sci Technol. 2008; 42: 2323–2327. PMID: 18504960 80. Diaz RJ, Rosenberg R. Spreading dead zones and consequences for marine ecosystems. Science. 2008; 321: 926–929. doi: 10.1126/science.1156401 PMID: 18703733

PLOS ONE | DOI:10.1371/journal.pone.0126538 May 20, 2015 23 / 23