Thresholds of hypoxia for marine biodiversity

Raquel Vaquer-Sunyer* and Carlos M. Duarte

Department of Global Change Research, Instituto Mediterraneo de Estudios Avanzados (Consejo Superior de Investigaciones Cientificas-Universidaddelas Islas Baleares), Esporles (Mallorca) 07190, Spain

Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved August 6, 2008 (received for review April 21, 2008)

Hypoxia is a mounting problem affecting the world’s coastal waters, with severe consequences for marine life, including death and catastrophic changes. Hypoxia is forecast to increase owing to the combined effects of the continued spread of coastal eutrophi- cation and global warming. A broad comparative analysis across a range of contrasting marine benthic organisms showed that hyp- oxia thresholds vary greatly across marine benthic organisms and that the conventional definition of 2 mg O2/liter to designate waters as hypoxic is below the empirical sublethal and lethal O2 thresholds for half of the species tested. These results imply that the number and area of coastal ecosystems affected by hypoxia and the future extent of hypoxia impacts on marine life have been generally underestimated. benthic community ͉ oxygen ͉ coastal ecosystems ͉ eutrophication ͉ impacts Fig. 1. Accumulated number through time of coastal sites where hypoxia has been reported. Exponential growth rate ϭ 5.54% Ϯ 0.23% yearϪ1 (R2 ϭ 0.86, issolved oxygen in coastal waters has changed drastically P Յ 0.01). Dover the past decades, arguably more so than any other ecologically important variable (1, 2), leading to the widespread occurrence of hypoxia. An assessment of the literature shows in the literature (1, 12, 13) are based on limited observations of that the number of coastal sites where hypoxia has been reported Ϫ impacts on organisms (7), and a thorough empirical assessment of has increased with an exponential growth rate of 5.54% year 1 the available experimental evidence is still pending. Whereas the over time [Fig. 1 and supporting information (SI) Table S1]. thresholds of hypoxia proposed in the literature range broadly from Although this growth rate can be partially attributed to an 0.28 mg O2/liter (14) to 4 mg O2/liter (15), most reports (55%) refer increased observational effort, increasing the number of costal to a value of 2 mg O2/l or lower (mean Ϯ SE of thresholds proposed ecosystems monitored and the likelihood of detecting hypoxia in the literature: 2.31 Ϯ 0.10 mg O2/liter; Table S2) used in most therein, this growth also reflects an increase in the prevalence of conventional applications (16). This thresholds refers to the oxygen hypoxia in different types of coastal ecosystems. Multiple reports level for fisheries collapse (12), but there is ample experimental from careful monitoring time series provide evidence for an evidence that a 2-mg O /liter threshold may be inadequate to unambiguous increase in the number of hypoxic zones and their 2 describe the onset of hypoxia impacts for many organisms, which extension, severity, and duration (3–6). This growth is expected experience hypoxia impacts at higher oxygen concentrations (e.g., to continue because the prevalence of hypoxia is forecast to increase further owing to the combined effects of eutrophica- 17). Moreover, the diversity of behavioral and physiologic adapta- tion, leading to the excessive production of organic matter that tions to hypoxia (18) suggests that different taxa are likely to exhibit increases the oxygen demand of coastal ecosystems (7), and the different vulnerability to hypoxia and may have, therefore, different increase in temperature caused by climate change, which en- oxygen thresholds (19), a possibility that is not addressed by the hances the respiratory oxygen demand of the organisms (8), conventional oxygen thresholds in use (cf. Table S2). reduces oxygen solubility (9), and reduces the ventilation of The goal of this article is to examine the variability in oxygen coastal waters by affecting stratification patterns (10). thresholds for hypoxia across benthic organisms and to test for Coastal hypoxia is, thus, emerging as a major threat to coastal the existence of consistent differences among taxa. We do so on ecosystems globally. Hypoxia has been shown to trigger mortality the basis of a comparative analysis of experimentally derived events, resulting in a depletion of metazoans in the ecosystems, oxygen thresholds for lethal and sublethal responses to hypoxia resulting in so-called ‘‘dead zones’’ devoid of fisheries resources, of benthic organisms. We aim to improve our understanding of such as fish, shrimp, and crabs (11, 12). Hypoxia leads to major the levels of hypoxia that cause significant impacts on marine loss in biodiversity and impacts the surviving organisms through benthic communities. This understanding will offer a more sublethal stresses, such as reduced growth and reproduction, rigorous basis on which to establish critical thresholds to pre- physiologic stress, forced migration, reduction of suitable hab- serve fishery resources and to effectively conserve coastal itat, increased vulnerability to predation, and disruption of life cycles (7, 11). Benthic organisms are particularly vulnerable to coastal hypoxia because they live farthest from contact with Author contributions: R.V.-S. and C.M.D. designed research; R.V.-S. performed research; atmospheric oxygen supply and because coastal sediments tend R.V.-S. and C.M.D. analyzed data; and R.V.-S. and C.M.D. wrote the paper. to be depleted in oxygen relative to the overlying water column. The authors declare no conflict of interest. Assessing the thresholds of oxygen at which lethal and sublethal This article is a PNAS Direct Submission. impacts occur is critical to establish the vulnerability of marine Freely available online through the PNAS open access option. organisms to hypoxia and to set management targets to avoid *To whom correspondence should be addressed. E-mail: [email protected]. catastrophic mortality. Hundreds of experiments to determine This article contains supporting information online at www.pnas.org/cgi/content/full/ thresholds of hypoxia for a range of benthic organisms have been 0803833105/DCSupplemental. conducted. However, the oxygen thresholds for hypoxia proposed © 2008 by The National Academy of Sciences of the USA

15452–15457 ͉ PNAS ͉ October 7, 2008 ͉ vol. 105 ͉ no. 40 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803833105 a A Crustacea

Fishes b

Bivalva b

Gastropoda b

0 24 6810

Median Lethal Concentration (mg 02/liter)

B Fishes a

Crustacea b

Mollusca bc

Polychaeta c

Echinodermata c

Cnidaria c 0 2 4 6 8 10 12

Sublethal thresholds (mg O 2/liter)

C Crustacea c Fish c Annelida c Echinoderms b c Cnidaria b c Mollusca b Priapulida a

0.1 0 11001000 10000 Median Lethal Time (h)

Fig. 3. Box plot showing the distributions of oxygen thresholds among taxa for (A)LC50 (mg O2/liter), (B) SCL50 (mg O2/liter), and (C)LT50 (h). The letters indicate the results of the Tukey HSD test, whereby the property examined did not differ significantly for taxa with the same letter.

the existence of a small proportion (10%) of experiments yielding extreme sensitivity to hypoxia, reflected in particularly high oxygen thresholds for hypoxic responses (Ն5mgO2/liter) and short (Յ2 h) lethal times. All relevant thresholds varied significantly across taxa (Fig. 3).

Median Lethal Concentration. Median lethal oxygen concentra- tions (LC50) ranged from 8.6 mg O2/liter for the first larval zoea stage of the crustacean Cancer irroratus (17), the most sensitive

Fig. 2. Cumulative distribution of (A)LC50 (mg O2/liter), (B) SCL50 (mg species tested, to persistent resistance to complete anoxia of the O2/liter), and (C)LT50 (h) for marine benthic communities (Table S3, Table S4, oyster Crassostrea virginica at temperatures of 20°C (20). The and Table S5). The mean Ϯ SE, median Ϯ SE, 90th percentile (10th percentile larval stages of C. irroratus (17) were found to be extremely for LT50), and number of experiments are indicated. vulnerable to hypoxia, with thresholds exceeding the 95th per- centile of the distribution of LC50 values across crustaceans. The mean LC50 (ϮSE) for all organisms tested was found to be 2.05 Ϯ biodiversity as hypoxia continues to rise as a threat to coastal 0.09 mg O2/liter, whereas the median was 1.60 Ϯ 0.12 mg O2/liter, ecosystems. and the coefficient of variation was 78% across experiments, indicative of considerable variability in these thresholds across Results organisms (Fig. 2A). Ninety percent of the experiments showed We found a total of 872 published experiments reporting oxygen LC50 values below 4.59 mg O2/liter (Fig. 2A). ECOLOGY thresholds and/or lethal times for a total of 206 species spanning Some of the variability in median lethal O2 thresholds was the full taxonomic range of benthic metazoans. The examination attributable to differences across groups (ANOVA, F ϭ 10.03, of thresholds for hypoxia derived experimentally revealed the P Ͻ 0.001; Fig. 3A), because crustaceans showed O2 thresholds existence of a broad range of variability, with median lethal and significantly higher than for other taxa (Tukey post hoc honestly sublethal oxygen thresholds and LT50s after exposure to hypoxia significant difference [HSD] test, P Յ 0.05; Fig. 3A). Gastropods ranging over an order of magnitude across experiments (Fig. 2 showed the lowest median lethal oxygen thresholds, although it and Table S3, Table S4, and Table S5). The cumulative distri- did not differ significantly (Tukey post hoc HSD test, P Ն 0.05; butions representing the distribution of oxygen thresholds Fig. 3A) from that of fish and bivalves. We also tested whether present a change in slope near the 90th percentile of the the extent of mobility of the organisms accounted for variability distribution and the 10th percentile of the LT50 (Fig. 2), showing in the experimentally derived median lethal O2 thresholds.

Vaquer-Sunyer and Duarte PNAS ͉ October 7, 2008 ͉ vol. 105 ͉ no. 40 ͉ 15453 Indeed, we found that the median lethal O2 thresholds differed developed stages for any one species, whereas similar effects significantly with the extent of mobility of the organisms tested were not found for lethal or sublethal concentrations. (ANOVA, F ϭ 11.29, P Ͻ 0.001). Two-way ANOVA showed that the degree of mobility (F test, P Յ 0.001), which accounted for Discussion 20% of the variance in median lethal concentrations among The results presented here provide evidence of the broad, order- experiments, was superior to differences among taxa (F test, P Ն of-magnitude variability in the thresholds of oxygen concentrations 0.05) in accounting for variability across experiments. for hypoxia among benthic marine organisms, which cannot be adequately captured by a single, universal threshold. This variability Median Sublethal Concentration. Median sublethal concentration partially derived from significant differences in oxygen thresholds (SLC50) thresholds ranged from 10.2 mg O2/liter for cod, Gadus across taxa. The most sensitive organisms were crustaceans, which morhua, which raises its ventilatory water flow below this showed the highest LC50 and the shortest LT50, whereas fish exhibit concentration (21, 22), to 0.085 mg O2/liter for the burrowing sublethal responses at the highest O2 concentration (Fig. 3). On the shrimp Calocaris macandreae (Thalassinidea), which switches other hand, molluscs, with the lowest LC50, are the organisms most from aerobic to anaerobic metabolism below this threshold (23). tolerant to hypoxia, together with cnidarians, which showed the The mean Ϯ SE SLC50 was 2.61 Ϯ 0.17 mg O2/liter, the median lowest LC50 for sublethal threshold, and priapulids, which showed SLC50 was 2.24 Ϯ 0.21 mg O2/liter, and the coefficient of the longest LT50. variation was 76%, showing important variability in median The differences in oxygen thresholds for hypoxia across taxa sublethal thresholds among experiments (Fig. 2B). Ninety per- probably reflect the broad differences in adaptations to cope cent of the experiments conducted reported median sublethal with low oxygen conditions among benthic organisms, which oxygen concentrations below 5.00 mg O2/liter (Fig. 2B). span a broad range of behavioral and metabolic changes. Mobile As for the median lethal O2 thresholds, some of the variability organisms have the capacity to avoid hypoxic waters and thus in median sublethal O2 thresholds was attributable to differences tend to show comparably high oxygen thresholds. Benthic fish among taxa, which was stronger for sublethal than for lethal have been reported to move to near-surface waters to breathe responses (ANOVA, F ϭ 21.75, P Ͻ 0.001; Fig. 3B). Fish had when bottom waters become hypoxic (26), and crustaceans move significantly higher oxygen thresholds for sublethal responses to shallower areas (27), where these organisms are more vul- (Tukey post hoc HSD test, P Ͻ 0.05; Fig. 3B), which typically nerable to predation. Yet fast-moving organisms (e.g., fish) do involved avoidance of hypoxic waters, depressed activity, shift to not necessarily show higher lethal thresholds than those with oxygen-dependent metabolism, or increased ventilatory water more restrictive mobility (e.g., crustaceans), pointing to differ- flow. Crustaceans also presented significantly higher oxygen ences among taxa independent of their relative mobility. thresholds for sublethal responses than polychaetes, echino- Many benthic organisms (polychaetes, annelids, crustaceans, derms, and cnidarians (Tukey post hoc HSD test, P Ͻ 0.05; Fig. bivalves, priapulids, and anemones) leave their burrows or tubes 3B), which typically involved avoidance of hypoxic waters, re- to move to the sediment surface or reduce their burial depth (28, duced growth, reduced predation rates, lethargy, or decreased 29) in the presence of hypoxia. Some bivalves stretch their activity, among others. Fish and crustaceans, which showed the siphons upward into the water column to reach waters with highest median sublethal O2 thresholds, are also the taxa with higher oxygen concentrations (30). Some echinoderms stand the highest mobility, which confers them some capacity to avoid immobile on their arm tips with the central disk elevated to avoid hypoxic waters. Indeed, two-way ANOVA showed that both the hypoxic bottom water (31), and some gastropods climb differences among taxa and the extent of mobility among structures to reach waters with higher oxygen concentration. organisms were significant (F test, P Յ 0.001), accounting for Metabolic adaptations to cope with hypoxia include depression 52% of the variance in median sublethal concentrations among of activity in the presence of hypoxia, as reported for echino- experiments (F ϭ 11.6, P Յ 0.001). derms (32); reduced feeding activity (e.g., some crustaceans, molluscs, and polychaetes; refs. 33–35); reduced metabolic rates Median Lethal Time. The median lethal time (LT50) upon exposure (e.g., cnidarians; ref. 36) and heartbeat rate (some crustaceans; to acute hypoxia ranged greatly across organisms tested (Fig. ref. 37); and shift to anaerobic metabolism over time scales of 2C), from only 23 min for the flounder Platichthys flesus (19, 24), hours to days, an adaptation widespread among bivalves (38, 39), to more than 32 weeks for the bivalve Astarte borealis at polychaetes (40), oligochaetes (41), echinoderms (42), and the temperatures below 20°C (25). The mean (Ϯ SE) LT50 was mud-shrimp Calocaris macandreae (23), among others. 267.9 Ϯ 22.0 h, the median was 116.7 Ϯ 27.67 h, and the The broad variability in oxygen thresholds shown here is in coefficient of variation was 178% across experiments, indicative contrast with the widespread use of uniform thresholds for of considerable variability in these thresholds across organisms hypoxia in the literature (Table S2). The vast majority of studies (Fig. 2C). Ten percent of the organisms showed LT50 upon and reports continue to use the 2-mg O2/liter convention, exposure to acute hypoxia of less than 6.8 h (Fig. 2C). There were originally derived as the oxygen threshold for fisheries collapse significant differences in LT50 under hypoxia among taxa (12). A total of 43% and 21.5% of the published reports used the (ANOVA, F ϭ 11.12, P Ͻ 0.001; Fig. 3C). In particular, 2-mg O2/liter and the 2-ml/liter (i.e., 2.85 mg O2/liter) threshold, Priapulida, the most tolerant group, had significantly longer LT50 respectively, and a single study (15) used a threshold of 4 mg under hypoxia than other taxonomic groups, and molluscs, the O2/liter (Table S2). A seminal review by Gray et al. (19), which second most tolerant group, also showed significantly longer included experimental studies reporting mortality thresholds LT50 under hypoxia than annelids, fish, and crustaceans, which well above 2 mg O2/liter, concluded that ‘‘mortality occurs where were the most sensitive groups (Fig. 3C). Sessile organisms also concentrations are below 2.0 to 0.5 mg O2/liter’’; and the U.S. had longer LT50 than mobile organisms did (F test, P Յ 0.01). Environmental Protection Agency recommends a threshold of However, two-way ANOVA showed that differences among taxa 2.3 mg O2/liter for juvenile and adult aquatic organism survival (F test, P Յ 0.001), which explained 17% of the variance in LT50, (43). In a recent review, Dı´az and Rosenberg (54) state that were superior to differences in mobility (F test, P Ն 0.05), to “hypoxia occurs when DO falls below 2 ml of O2/liter . . . account for variability among experiments. culminating in mass mortality when DO declines below 0.5 ml of We also found significant ontogenic shifts in survival time, O2/liter.” The results presented here show that the convention- with early stages having survival times, on average, 64% Ϯ 7% ally accepted level of 2 mg O2/liter falls well below the oxygen (H0 linear regression slope ϭ 1, t test, P Ͻ 0.05) of those of more thresholds for the more sensitive taxa.

15454 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803833105 Vaquer-Sunyer and Duarte Whereas the conventional 2 mg O2/liter may signal levels of Table 1. Distribution of thresholds of hypoxia for different hypoxia at which fisheries collapses, the results presented here groups of benthic organisms show that it is inadequate as a threshold to conserve coastal LC50,mg SLC50,mg biodiversity, because significant mortality would have already Organisms O2/liter O2/liter LT50,h been experienced by many species. The frequency distribution of thresholds of hypoxia compiled here (Fig. 2) shows that 61.43% Fish and 42.85% of the species tested here experience substantial Mean Ϯ SE 1.54 Ϯ 0.07 4.41 Ϯ 0.39 59.9 Ϯ 12.3 (Ն50% of the population) mortality and sublethal responses, 90th 2.51 8.09 0.9 percentile respectively, at oxygen thresholds above 2 mg O2/liter. In par- ticular, most fish and crustaceans would be lost before the n 77 34 39 oxygen content of the waters reaches the threshold of 2 mg Crustaceans Mean Ϯ SE 2.45 Ϯ 0.14 3.21 Ϯ 0.28 55.5 Ϯ 12.4 O2/liter for these waters to be considered hypoxic by conven- tional criteria. Indeed, fish and crustaceans are main fishery 90th 5.72 5.0 1.0 percentile resources, so that the 2-mg O2/liter threshold may be too low not only to effectively conserve biodiversity but to conserve fisheries n 168 30 102 resources as well. Gastropods Ϯ Ϯ Currently used thresholds of hypoxia are not conservative Mean SE 0.89 0.11 enough to avoid widespread mortality losses and need be 90th 1.62 critically revised. The frequency distribution of thresholds pre- percentile sented here provides a basis to allow the evaluation of the risk n 12 Bivalves 1.42 Ϯ 0.14 of biodiversity losses with decreasing oxygen concentration, Ϯ thereby considering a range of thresholds for hypoxia, rather Mean SE than a mean value that does not capture the order-of-magnitude 90th 3.43 variability across organisms. For instance, waters with oxygen percentile n 19 concentrations below 4.6 mg O /liter, the 90th percentile of the 2 Molluscs distribution of mean lethal concentrations, would be expected to Mean Ϯ SE 1.99 Ϯ 0.16 412.9 Ϯ 37.3 maintain the population for most, except the 10% most sensitive, 90th 2.83 55.4 species. This oxygen level could thus be considered as a precau- percentile tionary limit to avoid catastrophic mortality events, except for n 28 239 the most sensitive crab species, and effectively conserve marine Annelids biodiversity. Indeed, it is possible to carry this analysis further to Mean Ϯ SE 1.20 Ϯ 0.25 132.2 Ϯ 18.7 consider taxon-specific thresholds of hypoxia, at the 90th per- 90th 1.37 37.8 centile of the distribution of LC50 for the various taxa (Table 1). percentile Taxon-specific approaches help accommodate some of the vari- n 10 43 ability in experimental thresholds and allow the definition of Echinoderms more specific conservation targets in legislative, managerial, and Mean Ϯ SE 1.22 Ϯ 0.22 201.1 Ϯ 44.8 restoration plans. 90th 2.12 33.6 There are important limitations to extrapolate from experi- percentile mentally determined thresholds in controlled, laboratory con- n 823 ditions to the field (1, 44–46), derived from the facts that (i) the Cnidarians oxygen concentrations in the experiments are held constant, Mean Ϯ SE 0.69 Ϯ 0.11 232.5 Ϯ 114.4 whereas they would show variations in nature due to diel cycles 90th 1.43 24 in net community production, including the contribution of the percentile organisms tested themselves, and mixing; (ii) hypoxia often n 19 8 occurs in concert with other stresses in nature, and although Priapulids some experiments addressed thresholds of hypoxia in the pres- Mean Ϯ SE 1512.0 Ϯ 684.0 ence of additional stressors (e.g., high temperature, sulfide), 90th 820.8 most experiments used reduced oxygen as the single treatment percentile variable; and (iii) the experimental evaluation of the role of n 3 mobility in avoiding hypoxia is cumbersome and was directly addressed in only two of the experimental studies reviewed here Shown are mean Ϯ SE, 90th percentile (10th percentile for lethal times), and (47, 48). The alternative approach to estimate oxygen thresholds number of observations of LC50, SLC50, and LT50 for the various groups. for mortality of the various species of benthic organisms in the field is, however, elusive, because this would require an accurate estimate of their population sizes and because, as indicated ratory experiments). Hence, the results derived from laboratory above, oxygen levels fluctuate in ecosystems, rendering it diffi- experiments should be considered conservative. cult to assign observed mortalities to a specific oxygen value. Consideration of the different thresholds of hypoxia among Indeed, the difficulties to resolve oxygen thresholds in the field taxa derived here (Fig. 3 and Table 1) predicts that the sequence ECOLOGY explain why the bulk of the studies conducted to this end, of losses of benthic fauna during hypoxic events should be synthesized here (Table S3 and Table S5), have been conducted initiated by the loss of fish, followed by crustaceans, then worms, under laboratory conditions. These considerations apply not echinoderms, and molluscs as oxygen declines. This prediction is only to oxygen thresholds but to all experiments in toxicology, consistent with the observed sequence of losses of benthic fauna which cannot be appropriately controlled in the field. Most of the during hypoxic events, as reported in the Danish fjords (30) and processes indicated above would lead, however, to the labora- the Baltic Sea (49). The agreement between the sequences of tory-determined oxygen thresholds being below those in the losses of various taxa with hypoxia predicted from laboratory field, except in the case of avoidance for mobile organisms experiments and those observed in coastal areas impacted by (which is, however, addressed as a sublethal response in labo- hypoxia provides additional confidence in the relevance of

Vaquer-Sunyer and Duarte PNAS ͉ October 7, 2008 ͉ vol. 105 ͉ no. 40 ͉ 15455 laboratory experiments. The pattern of recolonization of benthic affected by hypoxia is, thus, likely to be greater than hitherto fauna lost to hypoxia upon subsequent improvement of the realized, and the prospects for future expansion of these areas oxygen conditions differs, however, from the pattern of loss, more disturbing than currently forecasted. Coastal hypoxia is, because recolonization patterns, which are initiated by thus, emerging as a major threat to coastal ecosystems globally. polychaetes (50, 51), are determined by life-history and dispersal The revised thresholds of hypoxia provided here will help better properties of the organisms and not their resistance to hypoxia. protect these ecosystems, conserve their biodiversity, and set The conclusion that oxygen depletion induces significant successful management targets to avoid hypoxia-derived biodi- mortality at critical oxygen thresholds exceeding by 2.3 times the versity losses in coastal waters. 2-mg O2/liter threshold generally used in the literature implies that the present inventory of the number and extent of hypoxic Methods areas in the coastal zone, which uses the occurrence of oxygen We searched the literature for reports of hypoxia on the Web of Science and Scholar Google using the keywords ‘‘hypoxia,’’ ‘‘marine,’’ ‘‘benthic,’’ and levels Յ2mgO2/liter (Fig. 1), represents an underestimate of the coastal areas experiencing mortality of benthic organisms at- ‘‘sea’’ and their combinations to guide the search. This search delivered more tributable to hypoxia. Hence, benthic organisms may be suffer- than 6000 published reports of responses of benthic marine organisms to hypoxia, which were then examined further for the availability of experimen- ing substantial mortality in areas not presently designated as tal assessments of responses to reduced oxygen content. This search delivered hypoxic. The conventional 2-mg O2/liter limit serves to separate a total of 872 experimental assessments examining the distribution of oxygen ‘‘dead zones,’’ depleted of most of the commercially harvested thresholds, involving 206 different species of marine benthic organisms. species, from waters supporting significant benthic animal com- The outcome of experimental assessments, which follow standard toxicity munities. However, it fails to reflect the oxygen threshold at tests, was summarized using the following indicators of oxygen thresholds: which these communities experience hypoxia-derived mortality. LC50 and SLC50, representing the statistically derived O2 concentration at The pace of growth of hypoxia as a major threat to coastal which 50% of the organisms in a given population die or exhibit sublethal biodiversity and associated living resources may be, therefore, responses, respectively, and LT50, representing the statistically derived time greater than hitherto considered. Moreover, there is ample interval at which 50% of a given population dies after exposure to low O2 levels. The vast majority (99.1%) of the experiments designed to assess LT evidence that the oxygen requirements of marine animals are 50 chose Յ2mgO2/liter as experimental conditions, consistent with the wide- even higher in the presence of concurrent stresses, such as high spread acceptance of 2 mg O2/liter as the threshold for hypoxia in the litera- temperature (52) or sulfide concentrations (53), suggesting that ture (Table S2). Yet this choice indicates that the lethal times reported repre- areas under stress are particularly prone to experience hypoxia- sent lethal times under acute hypoxia. Only a few experiments testing species derived catastrophic mortality. These interactions are not con- particularly sensitive to hypoxia (0.87%) used higher experimental O2 condi- sidered in present assessments and classifications but are likely tions. We analyzed these indicators to extract oxygen thresholds conducive to to play a more prominent role in the future as global warming the effective conservation of marine biodiversity. and other mounting stresses in the coastal ocean increase the ANOVA was used to test for differences in oxygen thresholds among sensitivity of benthic organisms to oxygen depletion. Indeed, a taxonomic groups. ANOVA analysis was conducted after checking for normal- ity using the Shapiro-Wilk test and homogeneity of variance using the Levene recent assessment concluded that the area of hypoxia (defined as Յ test. The Tukey post hoc HSD test was used to determine differences between 2mgO2/liter) in Danish coastal waters, one of the countries mean threshold values among taxa (␣ ϭ 0.05). We also classified the species most severely affected by this problem, will more than double tested according to their mobility as ‘‘fast moving’’ (fish and a few mollusks, under the projected temperature increase over the 21st century such as octopus), ‘‘highly mobile’’ (most crustaceans), ‘‘reduced mobility’’ (6), an estimate that needs be revised upwards in light of the (some crustaceans, gastropods, polychaetes, echinoderms, jellyfish, comb fish higher oxygen thresholds for hypoxia proposed here. (ctenophora), priapulids, flatworms, and sipunculida), and ‘‘sesile’’ (anemo- The analysis presented here demonstrates that hypoxia im- nes, bryozoans, and bivalves). ANOVA was used to test for differences in pacts occur at a broad range of oxygen concentrations, including thresholds with mobility, according to the procedures outlined above. Two- way ANOVA was used to test for the combined effect of taxonomic member- oxygen concentrations well above the oxygen thresholds gener- ship and the extent of mobility of the organisms tested on the experimentally ally used to diagnose hypoxia at present. The vulnerability of derived thresholds. coastal ecosystems to hypoxia is, thus, greater than currently recognized, with fish and crustaceans being the most vulnerable ACKNOWLEDGMENTS. This research is a contribution to the Thresholds inte- faunal components. The number and extent of the coastal zones grated project, funded by the European Union 6th Framework Program.

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Vaquer-Sunyer and Duarte www.pnas.org/cgi/content/short/0803833105 4of8 Table S2. Overview of the use of thresholds of hypoxia in the published literature mg O2/liter Reference

0.29 Fiadeiro and Strickland 1968 0.71 Weeks et al 2002 1.00 Vismann 1990 1.14 Normant and Szaniawska 2000 0.5–2 Gray et al. 2002 1.50 Baker and Mann 1994 2.00 Grantham et al. 2004 2.00 Steimle and Sindermann 1978 2.00 Díaz 2001 2.00 Eden et al. 2003 2.00 U.S. EPA Environmental Monitoring and Assessment Program for Estuaries 2.00 Tyson and Pearson 1991 2.00 Engle et al. 1999 2.00 Breitburg 1994 2.00 Condon et al. 2001 2.00 Llanso 1992 2.00 Llanso 1991 2.00 Miller et al. 2002 2.00 Pihl et al. 1991 2.00 Renaud 1986 2.00 Sagasti et al. 2001 2.00 Scavia et al. 2002 2.00 Stickle et al. 1989 2.00 Suzuki 2001 2.00 Tallqvist et al. 1999 2.00 Uzaki et al. 2003 2.00 Yin et al. 2004 2.05 Eriksson and Baden 1997 2.12 Tankersley et al. 2000 2.30 U.S. EPA 2.40 Yokoyama 2002 2.50 Karim et al. 2003 2.80 Ruthenford and Thuesen 2005 2.80 Wu and Or 2005 2.86 Diaz and Rosenberg 1995 2.86 Karlson et al. 2005 2.86 Baden et al. 1990 2.86 Josefson and Hansen 2004 2.86 Widbom 2.86 Marcus et al. 2004 2.86 Rosenberg et al. 1990 2.86 Rosenberg et al. 1991 2.86 Rosenberg et al. 1992 2.86 Rosenberg et al. 2001 2.86 Diaz and Rosenberg 2008 3.40 Kang and Matsuda 1994 3.60 Yanagi 1989 3.60 Sekine et al. 1995 3.60 Karim et al. 2002 4.00 Paerl 2006 2.28 Overall mean

For references, refer to SI References.

Vaquer-Sunyer and Duarte www.pnas.org/cgi/content/short/0803833105 5of8 Table S4. Median sublethal oxygen concentrations of benthic marine organisms reported in experimental assessments. Sublethal threshold

Taxa Group Sp. O2,mgO2/liter Ref.

Algae Eelgrass Zostera marina 2.96 Pedersen et al. 2004 Hydrozoa Anthomedusae Euphysa falmmea 1.73 Rutherford and Thuesen 2005 Hydrozoa Anthomedusae Halitholus sp. 1.34 Rutherford and Thuesen 2005 Hydrozoa Anthomedusae Polyorchis penicillatus 0.43 Rutherford and Thuesen 2005 Hydrozoa Anthomedusae Sarsia sp. 0.53 Rutherford and Thuesen 2005 Hydrozoa Leptomedusae Aequorea victoria 0.38 Rutherford and Thuesen 2005 Hydrozoa Leptomedusae Clytia gregaria 0.63 Rutherford and Thuesen 2005 Hydrozoa Leptomedusae Eutonina indicans 1.00 Rutherford and Thuesen 2005 Hydrozoa Limnomedusae Proboscidactyla flavicirrata 0.49 Rutherford and Thuesen 2005 Hydrozoa Calycophora Mugiaea atlantica 0.22 Rutherford and Thuesen 2005 Hydrozoa Limnomedusae Proboscidactyla flavicirrata 0.55 Rutherford and Thuesen 2005 Hydrozoa Limnomedusae Proboscidactyla flavicirrata 0.43 Rutherford and Thuesen 2005 Hydrozoa Calycophora Muggiaea atlantica 0.19 Rutherford and Thuesen 2005 Hydrozoa Calycophora Muggiaea atlantica 0.24 Rutherford and Thuesen 2005 Scyphozoa Sematostomae Aurelia labiata 0.41 Rutherford and Thuesen 2005 Scyphozoa Sematostomae Cyanea capillata 0.57 Rutherford and Thuesen 2005 Scyphozoa Sematostomae Phacellophora camtschatica 0.40 Rutherford and Thuesen 2005 Anemone Cerianthiopsis americanus 0.71 Diaz, unpublished data Anemone Bunodosoma cavernata 1.43 Ellington 1981 Anemone Metridium senile 1.43 Sassaman and Mangum 1972 Polychaeta Amphictenidae Pectinaria koreni 1.00 Nilsson and Rosenberg 1994 Polychaeta Spionidae Streblospio benedicti 0.57 Llansó 1991 Polychaeta Spionidae Streblospio benedicti 1.00 Llansó 1991 Polychaeta Terebellidae Loimia medusa 1.00 Llansó and Diaz 1994 Polychaeta Capitellidae Capitella sp. 1.14 Warren 1977; Forbes and Lopez 1990 Polychaeta Capitellidae Capitella sp. 1.14 Warren 1977 Polychaeta Paraprionospio pinnata 1.14 Diaz et al. 1992 Polychaeta Scoloplos armiger 0.86 Schöttler and Grieshaber 1988 Polychaeta Spionidae Malacoceros fuliginosus 3.43 Tyson and Pearson 1991 Polychaeta Spionidae Malacoceros fuliginosus 0.71 Tyson and Pearson 1991 Mollusca Bivalva Mysella bidentata 1.00 Ockelmann and Muus 1978, Nilsson and Rosenberg 1994 Mollusca Bivalva Mya arenaria 0.57 Jorgensen 1980 Mollusca Bivalva Abra alba 0.57 Jorgensen 1980 Mollusca Bivalva Cerastoderma edule 0.57 Jorgensen 1980 Mollusca Gastropoda Hydrobia ulvae 0.57 Jorgensen 1980 Mollusca Bivalva Theora fragilis 1.29 Tamai 1996 Mollusca Cephalopoda Octopus vulgaris 2.35 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 1.48 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.66 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 1.84 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 1.26 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.56 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 3.54 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.22 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 3.14 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 1.17 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 1.72 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.51 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.46 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.34 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 1.80 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.82 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.50 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.61 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.48 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.19 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.87 Cerezo and García 2005 Mollusca Cephalopoda Octopus vulgaris 2.62 Cerezo and García 2005 Crustacea Malacostraca Squilla empusa 2.14 Pihl et al. 1991 Crustacea Decapoda Penaeus setiferus 2.14 Renaud 1986 Crustacea Decapoda Penaeus aztecus 2.86 Renaud 1986 Crustacea Decapoda Callinectes sapidus 2.14 Pihl et al. 1991

Vaquer-Sunyer and Duarte www.pnas.org/cgi/content/short/0803833105 6of8 Table S4. (Continued) Sublethal threshold

Taxa Group Sp. O2,mgO2/liter Ref.

Crustacea Decapoda Penaeus aztecus 2.86 Renaud 1986 Crustacea Decapoda Penaeus setiferus 2.86 Renaud 1986 Crustacea Decapoda Carcinus maenas 1.43 Hill et al. 1991 Crustacea Decapoda Munida quadrispina 0.29 Burd and Brinkhurst 1984 Crustacea Decapoda Penaeus schmitti 1.29 MacKay 1974 Crustacea Decapoda Penaeus schmitti 1.71 MacKay 1974 Crustacea Amphipoda Monoporeia affinis 3.90 Johansson 1997 Crustacea Amphipoda Sanduria entomon 1.25 Johansson 1997 Crustacea Decapoda Penaeus schmitti 4.50 Rosas et al. 1997 Crustacea Decapoda Penaeus schmitti 4.50 Rosas et al. 1997 Crustacea Decapoda Penaeus schmitti 4.00 Rosas et al. 1997 Crustacea Decapoda Penaeus schmitti 4.50 Rosas et al. 1997 Crustacea Decapoda Penaeus schmitti 4.50 Rosas et al. 1997 Crustacea Decapoda Penaeus setiferus 5.20 Rosas et al. 1997 Crustacea Decapoda Penaeus setiferus 5.00 Rosas et al. 1997 Crustacea Decapoda Penaeus setiferus 5.00 Rosas et al. 1997 Crustacea Decapoda Penaeus setiferus 5.00 Rosas et al. 1997 Crustacea Decapoda Penaeus setiferus 4.50 Rosas et al. 1997 Crustacea Decapoda Penaeus setiferus 5.00 Rosas et al. 1997 Crustacea Decapoda Penaeus setiferus 4.50 Rosas et al. 1997 Crustacea Decapoda Penaeus setiferus 4.00 Rosas et al. 1997 Crustacea Decapoda Penaeus vannamei 1.91 Seidman and Lawrence 1985 Crustacea Decapoda Penaeus monodon 2.22 Seidman and Lawrence 1985 Crustacea Thalassinidea Calocaris macandreae 0.09 Anderson et al. 1994 Crustacea Decapoda Crangon crangon 2.58 Sandberg et al. 1996 Echinodermata Holothuria forskali 0.86 Astall and Jones 1991 Echinodermata Ophiura albida 0.79 Dethlefsen and von Westernhagen 1983, Baden et al. 1990 Echinodermata Echinocardium cordatum 1.00 Niermann et al.1990, Nilsson and Rosenberg 1994 Echinodermata Amphiura filiformis 1.21 Rosenberg et al. 1991 Echinodermata Amphiura chiaje 0.77 Rosenberg et al. 1991 Echinodermata Amphiuridae Micropholis atra 0.71 Diaz et al. 1992 Echinodermata Ophiura albida 2.00 Baden et al. 1990 Echinodermata Platyasteridae Luidia clathrata 2.40 Diehl et al. 1978 Chordata Osteichthyes Fish 4.00 Karim et al. 2002 Chordata Osteichthyes Rhacochilus vacca 4.56 Webb and Brett 1972 Chordata Osteichthyes Squalus suckleyi 6.69 Lenfant and Johansen 1966 Chordata Osteichthyes Scyliorhinus canicula 4.33 Hughes and Umezawa 1968 Chordata Osteichthyes Callionymus lyra 7.05 Hughes and Umezawa 1968 Chordata Osteichthyes Callionymus lyra 5.63 Hughes and Umezawa 1968 Chordata Osteichthyes Hydrolagus colliel 8.54 Hanson 1967 Chordata Osteichthyes Gadus morhua 10.20 Saunders 1963 Chordata Osteichthyes Oncorhynchus nerka 8.85 Brett 1964 Chordata Osteichthyes Oncorhynchus nerka 6.74 Davis 1973 Chordata Osteichthyes Oncorhynchus nerka 5.07 Randall and Smith 1967 Chordata Osteichthyes Oncorhynchus kisutch 4.50 Whitmore et al. 1960 Chordata Osteichthyes Oncorhynchus kisutch 9.00 Hicks and DeWitt 1971 Chordata Osteichthyes Oncorhynchus kisutch 6.00 Hermann 1958 Chordata Osteichthyes Oncorhynchus tshawytscha 4.50 Whitmore et al. 1960 Chordata Osteichthyes Oncorhynchus tshawytscha 4.50 Whitmore et al. 1960 Chordata Osteichthyes Salmo salar 4.50 Kutty and Saunders 1973 Chordata Osteichthyes Alosa sapidissima 2.75 Chittenden 1973 Chordata Osteichthyes Alosa sapidissima 4.00 Chittenden 1973 Chordata Osteichthyes Esox lucius 3.22 Siefert et al. 1973 Chordata Osteichthyes Fundulus heteroclitus 4.50 Voyer and Hennekey 1972 Chordata Osteichthyes Gadus macrocephalus 2.50 Alderdice and Forrester 1971 Chordata Osteichthyes Diplodus puntazzo 2.14 Cerezo and García 2004 Chordata Osteichthyes Diplodus puntazzo 2.79 Cerezo and García 2004 Chordata Osteichthyes Diplodus puntazzo 2.80 Cerezo and García 2004 Chordata Osteichthyes Diplodus puntazzo 2.70 Cerezo and García 2004 Chordata Osteichthyes Diplodus puntazzo 2.41 Cerezo and García 2004 Chordata Osteichthyes Diplodus puntazzo 2.25 Cerezo and García 2004 Chordata Osteichthyes Diplodus puntazzo 2.91 Cerezo and García 2004

Vaquer-Sunyer and Duarte www.pnas.org/cgi/content/short/0803833105 7of8 Table S4. (Continued) Sublethal threshold

Taxa Group Sp. O2,mgO2/liter Ref.

Chordata Osteichthyes Diplodus puntazzo 2.05 Cerezo and García 2004 Chordata Osteichthyes Diplodus puntazzo 1.94 Cerezo and García 2004 Chordata Osteichthyes Gadus morhua 1.74 Schurmann and Stefenses 1997 Chordata Osteichthyes Gadus morhua 2.17 Schurmann and Stefenses 1997 Chordata Osteichthyes Gadus morhua 2.55 Schurmann and Stefenses 1997

For references, refer to SI References.

Other Supporting Information Files

Table S1 Table S3 Table S5

Vaquer-Sunyer and Duarte www.pnas.org/cgi/content/short/0803833105 8of8