PHYTOPLANKTON COMMUNITIES IN THE WASTEWATER PLUME OF THE LOWER SACRAMENTO RIVER
A Thesis submitted to the faculty of / A s San Francisco State University In partial fulfillment of The requirements for The Degree B iol •TV3 Master of Science
In
Biology: Marine Biology
by
Nicole Mayu Travis
San Francisco, California
August 2015 Copyright by Nicole Mayu Travis 2015 CERTIFICATION OF APPROVAL
I certify that I have read “Phytoplankton communities in the wastewater plume of the lower Sacramento River” by Nicole Mayu Travis, and that in my opinion this work meets the criteria for approving a thesis submitted in partial fulfillment of the requirement for the degree Master of Science in Biology: Marine Biology at San Francisco State
University.
Frances P. Wilkerson, Ph.D. Research Professor, RTC
Alexander E. Parker, Ph.D. Assistant Professor The California Maritime Academy
Edward J. Carpenter, Ph.D. \ Professor, Department of Biology PHYTOPLANKTON COMMUNITIES IN THE WASTEWATER PLUME OF THE LOWER SACRAMENTO RIVER
Nicole Mayu Travis San Francisco, California 2015
As a highly urbanized ecosystem, the San Francisco Estuary (SFE) has many wastewater treatment facilities discharging large volumes of high nutrient effluent into the estuary.
Phytoplankton primary production can be depressed in the presence of ammonium-rich effluent, and ammonium tolerant phytoplankton species can become successful. The direct response of phytoplankton metabolic processes to anthropogenic nitrogen may play a key role in bottom-up structuring phytoplankton species composition and abundance in the lower Sacramento River, CA.
I certify that the Abstract is a correct representation of the content of this thesis.
Chair, Thesis Committee Date ACKNOWLEDGEMENTS
This work was completed with the support and advice of many amazing scientists. Thank you to my committee members Dr. Frances Wilkerson, Dr. Alexander Parker and Dr.
Edward Carpenter for their patient and enthusiastic teaching, many hours of paper editing, and their continuing advice on being a scientist. This would not have been possible without the help and moral support of my ParWilkerDale colleagues, Shannon
Strong, Allison Johnson, Jamie Lee, Tricia Lee, Sarah Blaser and Ned Antell. Ned, thank you for rocking the mass spec and autoanalyzer for me! Shannon, thank you for my carpooling lessons in music trivia. Thanks to Dr. Dick Dugdale for scientific critiques and caffeinated inspiration. Much of the field work would not have been possible without
Dr. Tamara Kraus, the USGS Water Science Center group, Dr. Timothy Mussen and the
Sacramento Regional Sanitation District, who enabled a large part of the field work through collaboration during the larger lagrangian study.
Much appreciation for the funding sources supporting this project along the way: COAST
Student Research Award, SFSU IRA Supplies Award, ARCS Scholarship, USGS/IEP
#G13AC00335, SFCWA #14-22 and Delta Stewardship Council #2038.
Special thank you to my Billy for keeping my spirits up with hugs! TABLE OF CONTENTS
List of Tables...... vii
List of Figures...... viii
List of Appendices...... ix
1.0 Introduction...... 1
2.0 Materials and Methods...... 6
2.1. Site Description...... 6
2.2. Experimental Design...... 6
2.3. Detailed Methods and Analysis...... 10
2.4. Calculations and Statistical Analyses...... 14
3.0 Results...... 16
3.1. In Situ May 2014 River Manipulations...... 16
3.2. River Enclosures...... 22
3.3. Additional Replicated Enclosures...... 29
4.0 Discussion...... 34
5.0 References...... ^...... 61
6.0 Figures and Tables ...... 71
7.0 Appendices...... 83 LIST OF TABLES
Table Page
1. Experimental enclosure treatments...... 78 2. Mean river conditions for May 2014 in situ manipulation...... 79 3. Mean river uptake rates for May 2014 in situ manipulation...... 80 4. Summary of enclosures experiments...... 81 5. TO and T96 comparisons for replicated enclosures...... 82
vii LIST OF FIGURES
Figures Page
1 . Map of lower Sacramento River study site...... 71 2. In situ river manipulation ratio of TRC:SAT uptake rates...... 72 3. River enclosure nutrient and uptake rates...... 73 4. River enclosure chlorophyll growth data...... 74 5. Replicated enclosure nutrient data...... 75
6 . Replicated enclosure chlorophyll growth and cell counts...... 76 7. Replicated enclosure flow cytometric size class groups...... 77
viii LIST OF APPENDICES
Appendix Page
Appendix 1. Results from in situ river manipulation in October 2013...... 83 1.1 Figure Appendix Nutrient and chlorophyll data...... 83 1.2 Figure Appendix Nitrogen uptake rates...... 84 1.3 Appendix Nutrient and chlorophyll summary table...... 85
1.4 Appendix Results...... 86 Appendix 2. Results from river enclosures carried out in October 2013...... 89 2.1 Figure Appendix Nutrient, chlorophyll and uptake rate data...... 89
2 .2 Appendix Results...... 90 Appendix 3. Results from in situ river manipulation in May 2014...... 93 3.1 Figure Appendix Nutrient and chlorophyll data...... 93 3.2 Figure Appendix Nitrogen and carbon uptake rates, f-ratios...... 94 3.3 Figure Appendix Dissolved inorganic carbon...... 95 Appendix 4. River uptakes from October 2013 and May 2014...... 96
4.1 Figure Appendix Trace uptake rates across NH4 concentration...... 96 Appendix 5. Extra replicated enclosures...... 97 5.1 Figure Appendix Nutrient data...... 97 5.2 Figure Appendix Chlorophyll and cell counts...... 98 5.3 Figure Appendix Fluorescence of particle size classes...... 99 5.4 Figure Appendix Results...... 100 Appendix 6-9. Data tables 2013...... 102 Appendix 10-14. Data tables for 2014...... 106 Appendix 15. Data from additional enclosures...... 113 1
1.0 Introduction
Nutrient loading into aquatic systems is increasing as human population size continues to rise across the globe. Densely populated urban areas bring runoff from cities, factories, roads and agriculture, as well as necessitating wastewater treatment facilities that typically discharge their nutrient-rich effluent into local waterways. Nutrient enrichment of estuary ecosystems can have a wide range of negative impacts on the environment that result from cultural eutrophication (an increase in organic matter production resulting from anthropogenic activity, e.g. (Fisher et al. 2006) such as toxic algal blooms (Heisler et al. 2008), loss of important habitat including eelgrass beds (Short & Burdick 1996), loss of biodiversity (Hautier et al. 2009), exotic species invasion (Cohen & Carlton 1998,
Chase & Knight 2006) and oxygen depletion (Diaz 2001). Symptoms of cultural eutrophication from wastewater have been observed worldwide in estuaries and coastal embayments, e.g. (Scheldt Estuary, Wascana Creek, Delaware Bay, California coast,
Hong Kong Bay; Maclsaac et al. 1979, Yoshiyama & Sharp 2006, Cox et al. 2009,
Waiser et al. 2011, Xu et al. 2012). Nutrient enrichment may be detrimental to ecosystem function and services, and can significantly affect the economics of local fisheries and aquacultures, and recreational use of coastal areas (Dodds et al. 2008).
The San Francisco Estuary (SFE) has been described as a nutrient-replete estuary (Cloem
& Jassby 2012), that is supplied largely by anthropogenic nutrient sources. It receives wastewater effluent from multiple wastewater treatment facilities in its northern freshwater tributaries including the Sacramento River and these facilities have been discharging nutrient-rich water into the estuary for decades. Generally, increased nitrogen load is thought to fuel increased phytoplankton productivity. However, high nitrogen inputs do not necessarily cause algal bloom events in all systems, a phenomenon that has been described as a high-nutrient low-chlorophyll (Cloem 2001) or high-nutrient low- growth system (Sharp 2001). High turbidity in the SFE makes the ecosystem light-limited
(Cole and Cloem 1984), thus imparting potential resilience to high nutrient loads and eutrophication. The SFE has seen significant changes in the natural phytoplankton bloom cycles and a long term decrease in total chlorophyll-a (chl-a), a proxy of primary production (Cole & Cloem 1984, Jassby et al. 2002). Recent studies of the SFE show either a breakdown in some of this resilience (i.e. potential for cultural eutrophication -
Jassby 2008) or other signs of nutrient impacted ecosystem function in the SFE (Sommer et al. 2007, Dugdale et al. 2007, Glibert 2010, Parker et al. 2012a) and some chemical forms of nitrogen may be inhibitory to productivity in some phytoplankton (Wilkerson et al. 2006, Dugdale et al. 2007, Parker et al. 2012a).
The tidally-influenced lower Sacramento River, from the cities of Sacramento to Isleton, is strongly affected by effluent discharged by the Sacramento Regional County Sanitation
District wastewater treatment facility (SWTF). The SWTF serves 1.3 million people in the City of Sacramento and surrounding communities and produces effluent discharge of
141 million gallons per day (mgd) on average of secondarily treated (ammonium (NH4)- rich) municipal wastewater effluent to the Sacramento River. SWTF is scheduled to transition to advanced secondary treatment by 2021. This will result in conversion of NH4-rich effluent to nitrate (N0 3 )-rich effluent as a consequence of microbial nitrification methods, and a reduction in total nitrogen load by 20-30% (max. monthly river concentration 7.6-12.2|wM NH4 and 51 |_iM NO3). The series of changes in effluent treatment and subsequent N and P discharge to the Sacramento River will undoubtedly result in changes in nutrient concentrations and relative amounts of ammonium versus nitrate in the dissolved inorganic nitrogen (DIN) pool. Less clear is the potential influence that these changes in wastewater management practices may have on microbial communities within the Sacramento River and downstream in the SFE.
Analysis of ~25 years of data from long-term monitoring programs in the SFE showed significant correlation between historical nutrient loads, and changes in the local food web over time (Glibert et al. 2011). When the SWTF came online in the early 1980s, there was a significant rise in NH4 input into the estuary. The pre-1980s NC>3-based food webs were dominated by diatoms in the phytoplankton assemblage (Ball & Arthur 1979), while effluent-influenced NFL-based food webs saw higher proportions of flagellates, cryptophytes and cyanobacteria (Lehman 2004, Glibert et al. 2011, Kress et al. 2012).
Missing from these previous studies is information on the response of picoplankton to nitrogen availability. Picoplankton abundance varies within the SFE (Kimmerer et al.
2012), and can comprise up to 15% to 30% of the total phytoplankton biomass during spring blooms (Ning et al. 2000, Lidstrom 2009). Only phytoplankton cells larger than
~5 jxm are easily available to copepod grazers (Berggreen et al. 1988). As a nutritious food source for juvenile fish, low diatom biomass may contribute to declining 4
zooplankton and consequently fish abundances in the SFE (Brett & Mtiller-Navarra 1997,
Muller-Solger et al. 2002).
Uptake of NH4 and NO3 by phytoplankton varies across time and location within the SFE
(Wilkerson et al. 2006). NH4 uptake rates throughout the year are higher than for NO3 in the northern SFE. The spring phytoplankton bloom is correlated with high NO3 uptake rates (Dugdale et al. 2007, Wilkerson et al. 2015) while a fall bloom correlates with high
NFLt-uptake rates (Wilkerson et al. 2006). NH4 appears to inhibit algal uptake of NO3, with complete inhibition occurring at NH4 concentrations >4|j.M NH4 (Parker et al.
2012b, Dugdale et al. 2007). Measurements taken along a transect of the Sacramento
River showed nitrogen concentrations increasing significantly just downstream of the
SWTF effluent discharge site (Parker et al. 2012a) and phytoplankton nitrogen-uptake shifting from NO3 and NH4 upstream of the SWTF to exclusively NH4 immediately downstream. Laboratory enclosure experiments verified that N(>3-uptake will only occur when NH4 concentrations are reduced to below ~4(jM NH4 (Parker et al. 2012b).
The aim of this study was to investigate the relationship between chemical form of DIN
(NH4 or NO3) and phytoplankton in the Sacramento River above and below the SWTF discharge point, using context from a larger collaborative in situ river manipulation study and corresponding experimental enclosures. Withholding effluent discharge into the river offered a large-scale perspective on potential nutrient processes and patterns in the river in situ, while amendment experiments using effluent, NH4CI or KNO3 were used to directly link nitrogen form and concentration to resulting changes in phytoplankton dynamics. The larger study, led by the USGS Water Science Center (T. Kraus, B.
Bergamaschi), has further details on the lagrangian river experiments, however components useful for interpretation of enclosures are included in the appendix.
The specific objectives were to investigate the river phytoplankton response following 1)
NH4 removal in situ and in enclosures, 2) amendment with NH4 or effluent-based NH4 to establish whether observed effects on phytoplankton were caused by NH4 or some other contaminant present in effluent and 3) with KNO3 enrichment. Comparison between NH4 and NO3 supported phytoplankton growth contributes to understanding the ecological impacts of projected management changes from secondary to advanced-secondary (and later tertiary) wastewater treatment. 6
2.0 Material and methods
2.1 Site description
The Sacramento River is a main freshwater tributary of the San Francisco Bay Delta system in California. The stretch of river between Sacramento and Isleton, CA is the original winding river route, and connects with the deep-water ship channel just past
Isleton, CA (Fig. 1). This section of river has a few minor connecting sloughs, and freshwater is actively managed through the Sacramento River for agriculture and fisheries, which can influence the timing and magnitudes of water moving in and out of the system. The SWTF outfall is located just south of the Freeport Bridge (RM 46.32), and effluent is discharged through a 300ft diffuser pipe running perpendicular to river flow across the river bottom. Discharge volume and rate is modulated by facilities management based on tides and river flow in order to maintain nutrient loads below permitted US Environmental Protection Agency regulated concentrations. Current discharge averages of 14 tons of NH4-rich effluent per day into the river, and scheduled upgrades to the wastewater treatment facility are planned (CRWQCB 2013).
2.2 Experimental design
2.2.I In situ river manipulation
This project involved connecting landscape-scale manipulations of in situ river conditions with more controlled study of natural phytoplankton communities using 10-L experimental enclosures. Effluent discharge from the SWTF into the Sacramento River 7
near Freeport (lat = 38.454167, Ion = 121.5) was diverted into holding basins for approximately 15 hours on 31 May, 2014 (Same experiment conducted in October 2013, see Appendix 1, 2). The 15 hour effluent stoppages created a segment of effluent-free river, and offered a unique comparison of in situ river processes and water quality within adjacent river segments of two different conditions: ambient, high nutrient, effluent- influenced and experimental, low nutrient, effluent-free conditions.
An ambient river segment and an effluent-free river segment were sampled in a lagrangian manner over five days down river from upstream of the town of Freeport down to Isleton. Collaborators at the USGS used multiple techniques to track the water parcels each day, including flow rate estimations from nearby monitoring buoys, drifters deployed during the day and optical measurements (CDOM, specific conductance) capable of differentiating effluent-influenced water (pers. comm., B. Bergamaschi).
Correct locations within and outside of the experimental river sections were later confirmed using laboratory measurements of elevated or reduced NH4 concentration.
Water parcels were sampled for analyses of inorganic nutrient concentrations including ammonium (NH4), nitrate (NO3), nitrite (NO2), phosphate (PO4), silicate (Si(OH)4), and dissolved inorganic carbon (DIC). Phytoplankton biomass was estimated using chl-a, and samples for phytoplankton microscopic identification were preserved with Lugols solution. Samples were also collected and incubated for dual-labeled l3C/l5N primary production and nitrogen uptake measurements. The effluent-free river segment was tracked using a laboratory “houseboat” that floated within the effluent-free parcel. A 8
smaller “rover” boat was used to sample water from the ambient effluent-containing segment of river, and samples were returned to the laboratory “houseboat” for processing.
A composite sample of effluent from the wastewater treatment plant was obtained from the discharge stoppage time period. The effluent sample was analyzed for inorganic nutrient constituents, and frozen for use during the enclosure nutrient addition experiments.
2.2.2 River manipulation enclosures
Concurrent with in situ river sampling and manipulation (i.e. whole river effluent stoppage), river water was collected from ~1 m depth into 10-L clear low density polyethylene enclosures and monitored for at least 96 hours to assess phytoplankton growth dynamics of natural river assemblages in the absence of benthic processes and mixing with external water inputs (Dugdale et al. 2007). Ten enclosures treatments were established and monitored during the 2014 river manipulation. Experiments used source water from both the ambient and effluent-free river segments and included nutrient amendments (Table 1). Nutrient supplements included liquid solutions of ammonium chloride (NH4CI), potassium nitrate (KNO3) and phosphate (P0 4 )(Sigma). Effluent additions were made from a SWTF composite effluent sample obtained directly from the
SWTF, which corresponded to the effluent stream that was discharged into the ambient river segment of the May 2014 in situ river manipulation (May 31st). The composite sample contained 2.5 mM NH4, 18.4juM NO3, 1.6p.M NO2 and 73.1|aM PO4. (Three 9
enclosures also conducted in October 2013, see Appendix 3,4). Nutrient additions to each experimental enclosure were calculated to match concentration in ambient river water, or equivalent across comparisons. Source water for each enclosure corresponded directly with a morning in situ river sampling time point, providing extra information on the conditions of the source water for each enclosure. Once treatment additions were established, enclosures were mixed gently by inverting and a starting (TO) sample set was collected from each enclosure. Enclosures were incubated on the surface of the river in a floating corral covered with window screening to reduce photosynthetically active radiation (PAR) levels by 50%, mimicking average water column light. Enclosures were incubated directly in the river alongside the floating houseboat laboratory for the first 5 days of the river cruise, and finished incubating in temperature controlled outdoor tanks at the Romberg Tiburon Center (RTC) in Tiburon, CA after the end of the river cruise.
Daily sampling, for the same parameters as the in situ river segments, continued for 96-
120 hours (5-6 days) each morning at ~10 am.
2.2.3 Additional laboratory enclosure experiments
On two occasions during 2013 and 2014, additional triplicate enclosure experiments were conducted (Table 1) that included amendments of NH4CI or KNO3 to compare with amendments of effluent composite obtained during the river manipulation. Enclosures were filled with source water (i.e. natural phytoplankton community) ~ 6 .8 km upriver from the SWTF effluent discharge location on an ebb tide to ensure minimal effluent- influence. Near-surface water was collected with a triple-rinsed bucket off the dock at 10
Stan’s Yolo Marina (SYM, Fig. 1), and six 10-L enclosures were triple-rinsed and filled.
Experimental additions of nutrients were made within six to 12 hours of sample collection before initial (TO) samples were collected. Enclosures were incubated in temperature-controlled outdoor water tanks at the RTC set to approximate ambient river temperatures, and covered with window screening to reduce PAR by 50%. Samples were collected each morning over 120 hours (6 days) for dissolved inorganic nutrients (NH4,
NO3, PO4, Si(OH)4, NO2, and DIC) and chl-a. Samples for phytoplankton enumeration were collected at TO and T96 in 250-ml glass amber bottles and preserved with Lugols solution. Cell number and size spectra of the phytoplankton cells were measured daily using a Cytobuoy flow cytometer. No isotopic tracer uptake rate incubations were conducted for these additional enclosures.
2.3. Detailed analytical methods
2.3.1 Nutrient concentrations
For all nutrient analyses the sample water was first filtered through a 25-mm Whatman
GF/F filter using an acid-washed 50-ml high density polyethylene (HDPE) syringe. 25-ml samples for NH4 analysis were stored in 50-ml conical centrifuge tubes at -20°C.
Colorimetric analysis of NH4 concentration was conducted using the approach of
(Solorzano 1969) using a 10-cm path length cell on a Hewlett Packard diode array spectrophotometer. 20 ml of filtered sample was also collected for analysis of NO3, PO4,
Si(OH)4, N 0 2 and was stored in 25-ml HDPE scintillation vials at -20°C. These samples 11
were analyzed on a Bran and Luebbe AutoAnalyzer II with MT-19 manifold chemistry module, following Bran and Luebbe Inc. (Bran Luebbe Inc. 1999a, b, c) for all chemistries with additional information from (Whitledge et al. 1981) for nitrogen analyses. Dissolved inorganic carbon samples were collected in 20-ml glass vials and preserved with 200pL of 5% w/v HgCh (Sharp et al. 2009) and analyzed on a Licor DIC analyzer (Model 6252) with acid-sparging and non-dispersive infrared analysis (Parker et al. 2006).
2.3.2 Chl-a concentration
A 100-ml water sample was filtered onto a 25-mm Whatman GF/F filter (nominal pore size 0.7-pm) under minimal light. Filters were stored at -20°C until analysis. Samples were extracted at -20°C in 8 ml of 90% v/v acetone over 24 hours, and in vitro fluorometric analysis of chl-a pigment was performed using a Turner Designs Model 10 fluorometer (Arar & Collins 1992). Phaeophytin was also measured in each sample by acidifying with 10% hydrochloric acid, and used to correct chl-a values (Holm-Hansen &
Riemann 1978). Calibration was conducted with commercially available chl-a standards from Turner Designs. Additionally, a 25-mm diameter 5.0pm Nucleopore pore-sized polycarbonate filters were used to select for the > 5 pm size fraction of the phytoplankton community.
2.3.3 Phytoplankton identification: 12
Samples for phytoplankton identification were collected in 250-ml glass amber bottles and preserved with 1 ml Lugols iodine solution. For microscope investigation, 27 mis of preserved sample was settled in the dark for approximately 24 hours using a clear plastic
Utermohl settling chamber. Phytoplankton were counted and identified on an Olympus
1X85 microscope. Live whole water samples were also observed using a 1-ml Sedgwick
Rafter chamber, to help qualitatively identify phytoplankton groups and species using coloring, movement and fragile morphologies. Additionally, epifluorescence microscopy was used to qualitatively detect phycocyanin and phycoerythrin-containing cells using a green excitation filter. Photographs were taken of each identified species, and each view frame counted.
A minimum of 400 cells were counted for each sample, and at least 100 of the dominant cell type. Species were consolidated into the following functional groupings: pennate diatoms, centric diatoms, picoplankton, green algae, cryptophytes, and other cells.
2.3.4 Carbon and nitrogen uptake rates
During the 2014 river transect, water column phytoplankton carbon uptake and nitrogen uptake rates were measured using addition of l3C/15N tracer isotopes (Legendre &
Gosselin 1997, Parker 2005). Sample water was collected at each time point in two clear
160-ml polycarbonate bottles, with one bottle receiving HI3C0 3 and I5NH4C1 and the second bottle receiving HI3C0 3 and KI5N0 3 . Nitrogen additions were estimated to be
-10% of ambient river concentrations (“trace” addition) or -50% of ambient river (-90% 13
of effluent-free river) concentrations (“saturating” addition). Incubation bottles were kept in ambient river water alongside the boat in a floating corral, and covered with window screening to reduce ambient light to -50% of surface PAR. Incubations lasted for 24 hours, so that samples could be started at different times throughout the day and receive similar total irradiance.
At the end of the incubation, samples were filtered onto combusted (450°C for 4 hours)
25-mm Whatman GF/F filters and stored at -20°C. Samples were dried and pelletized in tin before being analyzed on a PDZ Europa 20/20 gas chromatograph - mass spectrometer for particulate carbon (POC), particulate nitrogen (PON) and l3C and l5N enrichment. As calculated in (Dugdale & Wilkerson 1986), nutrient uptake rates are reported as p (pmol L"1 d"1) and V (biomass-specific uptake, d'1). Dilution of NH4 may have occurred due to bacterial remineralization and zooplankton excretion, which can lead to an underestimate of NH4 uptake rate (Dugdale and Wilkerson 1986).
2.3.5 Flow cytometry
Cell counts and size spectra analysis were conducted using a CytoSense Flow Cytometer and the CytoClus 2 analysis software (Dubelaar et al. 1989, 1998). Live phytoplankton samples were introduced into the CytoBuoy at a flow rate of 4.89 ml s' 1 with a maximum run time set to 10 min or 10000 particles counted, whichever was reached first. Single particles were analyzed for forward scatter, side scatter and three specific wavelengths of pigment. Length was automatically calculated based on an internally calibrated forward 14
scatter to length ratio, and reported in microns by the CytoSifit software (confirmed by bead calibration)(CytoBuoy b.v.). Flow cytometry data were not collected during the in situ river cruises because live samples could not be promptly returned to the lab. Flow cytometric counts were conducted in the laboratory at the termination of each river enclosure experiment, and daily for the whole time course of the additional laboratory enclosure experiments. Flow cytometry was not run for the in situ river manipulations.
2.4 Calculations and Statistical Analyses
Statistical comparisons of mean river constituents were done using a Welch’s unequal variance t-test to account for different samples size. Standard deviations are calculated for mean values presented. Error bars on graphs are reported using 95 percent confidence intervals.
Using the uptake data, an f-ratio can be calculated to understand the proportion of production being generated by growth on NO3 substrate compared to the total DIN pool.
Fratio = (pN03 )/(pN03 + pNH4). The ratio of trace addition:saturated isotope addition (TRC:SAT) uptake rates were calculated using biomass specific rates (VNH4 and VNO3) for samples that had rates > 0.02 d'1.
The AChl: ADIN ratio was calculated by finding the difference in chl-a between TO and the time at maximum chl-a (AChl). The same time period was used to calculate change in
DIN (ADIN). 15
Chlorophyll-a data was plotted over time and fit to an exponential function C2 = C1 e plt, where C = Chi concentration, t = time. The growth rate (|i) was used to calculate divisions per day (d'1), where divisions per day (d_1) = The time interval used to fit the exponential function was from TO, and included all consequent time points during exponential growth phase. Additional estimates of growth rate were obtained from the trace N-uptake rates. Both trace VNH4 and VNO3 were summed to get total N uptake
(VNT), and used as a proxy for growth (|li) assuming steady state conditions, no detrital
PON, and N as the limiting substrate. These assumptions are acknowledged to not necessarily be met, but this approach provides an independent estimate of growth for comparison with those obtained from changes in chl-a, and discussion about the limitations of this approach are useful in thinking about cell physiology in response to changing nutrient environments. Divisions per day was again calculated as divisions per day (d -1) = 16
3.0 Results
3.1 In situ river manipulations May 2014
3.1.1 River conditions
During the May 2014 river manipulation experiment, the California Water Year
Hydrologic Classification was determined as “critical” (http://cdec.water.ca.gov/cgi- progs/iodir/wsihist); i.e. extreme drought condition. Average tidally filtered river flow during the 5 day lagrangian river manipulation was 215.6 m3 s"1 as measured at Freeport,
CA USGS data buoy (http://nwis.waterdata.usgs.gov/ca/nwis). Due to low water flow conditions and tidal influence, river flow was negative (upstream) four times during flood tides. Mean river temperature in May 2014 was 21.8°C ± 0.5 (s.d.) in all in situ river samples, with upstream samples being slightly cooler than downstream (Table 2 ).
3.1.2 River nutrients
In the manipulation experiment, the average DIN upstream of the effluent discharge site was low (<3pM), with slightly more NO3 than NH4. Mean NH4 concentration was 0.88 ±
0.42|-iM, and mean NO3 was 1.23 ± 1.45|nM (Table 2).
The influence of SWTF effluent discharge was seen clearly as a large increase in NH4 concentration (>50|aM) in the river during ambient conditions, highlighting an effluent stream rich in both NH4. The highest NH4 concentration seen in the ambient river samples from May 2014 was 58.8|iM and averaged 50.63 ± 5.65|jM. Average.NO3 was 17
nearly 3-fold higher downstream of the effluent discharge (6.1 ± 3.55pM.) but still low compared to NH4 concentrations. DIN was low in the effluent-free segment during the
2014 manipulation but was higher than concentrations measured upstream of the effluent discharge. Average NH4 concentration was 3.24 ± 2.85pM, and average NO3 concentration was 2.88 ± 1.02pM. Mean NH4 in all three river conditions (upstream, ambient and effluent-free) were statistically different from one another (all P<0.001). For
NO3, ambient and effluent-free conditions statistically the same, but the upstream average was different (Table 2). (See Appendix 5, 6 )
3.1.3 Chlorophyll, DIC and particulate organic matter
Chlorophyll-a was highest upstream of the effluent discharge site, but decreased below 5 pg L' 1 once past the effluent discharge site in both ambient and manipulated river segments. Average upstream chl-a was 10.8 ± 5.3 pg L'1, while downstream averages in the ambient and effluent-free river were 4.7 ± 1.8 and 3.6 ± 0.9 pg L'1, respectively
(Table 2). The upstream chl-a average was statistically higher than the other river segments (P=0.004). Ambient and effluent-free river segment chl-a averages were not significantly different.
Average DIC in the river ranged between 752-1175pM. DIC in the ambient river was highest, with an average of 1064 ± 74pM. In the effluent-free river condition, the averages DIC was 8 8 6 ± HOpM. The ambient river and the effluent-free river had statistically different DIC averages (P=0.01). Upstream average DIC was 989 ± 97pM, 18
and was not significantly different from either ambient or effluent-free conditions.
Average POC and PON were not statistically different between river conditions. POC
averages ranged from 93-115|uM and were not statistically different from one another.
PON averages ranged between 6 -8 |^M and were also not statistically different from one
another. (Table 2 )
3.1.4 N uptake rates
Upstream samples showed similar biomass-specific trace uptake rates (VN) for NH4 and
NO3 (Table 3). Mean VNH4 and VNO3 were 0.17 ± 0.14 d' 1 and 0.18 ± 0.05 d'1,
respectively (Table 3). Downstream in both river segments VNH4 increased to higher
than upstream values, with ambient river average VNH4 of 0.51 ± 0.07 d' 1 and effluent-
free average VNH4 of 0.36 ± 0.1 d' 1 (Table 3). Mean VNO3 in the ambient river segment
was near zero. Some NO3 uptake still occurred in the effluent-free river (0.13 ± 0.11 d'1),
but was statistically distinct from the upstream and ambient VNO3. The sum of the N
uptake (VNT, i.e. VNH4 + VNO3) was not statistically different between the river
conditions, with upstream VNT of 0.36 ±0.17 d"1, ambient VNT of 0.51 ± 0.07 d' 1 and
effluent-free VNT of 0.48 ± 0.08 d"1 (Table 3).
For saturating uptake measurements in which more N isotope was added to the
incubations, the patterns were slightly different. There was no difference in VsatNFU between each river condition (Table 3), with an upstream rate of 0.46 ± 0.05 d"1, an ambient rate of 0.46 ± 0.06 d"1, and an effluent-free rate of 0.43 ± 0.06 d'1. The difference 19
between upstream and ambient VsatN 0 3 (0.33 ± 0.09 vs 0.01 ± 0.005) was statistically significant (P<0.05). Additionally, the VsatN0 3 measurements were statistically different between ambient and effluent-free river conditions (0.01 ± 0.005 d' 1 vs 0.12 ± 0.07 d’1), which was not true in the trace uptake rates potentially from larger variance in trace measurements (Table 3). Summed saturated N uptake in the upstream river was the highest (0.8 ± 0 .1 d'1), and was statistically different from both ambient or effluent-free conditions (0.48 ± 0.06 d''and 0.55 ± 0.09 d'1, respectively). In the ambient river and effluent-free river, measurements of VsatNH4 and VsatNOs were similar between trace and saturating methods. However, upstream the saturating measurements for VsatNH} and V sa tN 0 3 were higher than the trace measurements.
The ratios of TRC:SAT VN were plotted against travel time downriver for the effluent- free river segment The difference in trace and saturating V N measurements was largest in the upstream section of river preceding the start of both the ambient and effluent free river sections. This was reflected in the TRC:SAT measurements that were generally less than 1 for all upstream sampling points (Fig. 2A, 2B). Within all the upstream samples
(combined from both effluent-free and ambient river sections), the average TRC:SAT
VN 0 3 was 0.615 ± 0.31 d' 1 and the average TRC:SAT VNH4 was 0.374 ± 0.33 d' 1 (Table
3). In the effluent-free river (Fig. 2A), once the river moved past the effluent discharge site the TRC:SAT increased to near 1 for both VNO3 and VNH4. Average TRC:SAT for
VNO3 was 0.952 ± 0.43 d' 1 and the average for TRC:SAT VNH4 was 0.819 ± 0.17 d' 1
(Table 3). However, within the ambient river (containing elevated effluent) the TRC:SAT 20
VNO3 declined from upstream to 0.34 0.26 d '1, while the TRC:SAT V N H 4 increased
from upstream to close to 1 (1.09 ± 0.09 d'')(Table 3).
Nitrogen transport (i.e. rho (p) uptake) values showed similar trends as the V uptake
rates. Upstream trace PNH4 and PNO3 were 0.91 ± 0.57 and 1.18 ± 0.24pMol L' 1 d'1, respectively. The upstream pNFLj rate was statistically lower than in either ambient or
effluent-free conditions (2.39 ± 0.36 and 1.64 ± 0.31pMol L' 1 d'1, respectively). For
PNO3 however, the upstream rate was higher than ambient and effluent-free conditions
(0.03 ± 0.02 and 0.66 ± 0.51|amol L’ 1 d'1, respectively). Comparison of sum pN uptake
(pNT, i.e PNH4 + PNO3) showed no statistical difference between each river condition
(Table 3).
Saturating pN values were higher than trace values for mean upstream PsatNF^ and
PsatNC>3. Upstream saturating pN values were 2.75 ± 0.69pMol L' 1 d' 1 forNFLt and 2.55 ±
1,06|iMol L' 1 d’ 1 for N O 3, which were higher than rates seen in the ambient and effluent- free river conditions downriver. Saturating PNH4 rates in ambient and effluent-free conditions (2.52 ± 0.6 and 1.74 ± 0.17pMol L' 1 d'1) were similar to the corresponding trace PNH4 rates, suggesting the NH 4 concentrations in the sampled water were already saturating for uptake prior to isotope addition. The pSatN0 3 was low in the ambient and effluent-free river (0.09 ± 0.02 and 0.65 ± 0.44, respectively) compared with upstream.
The PsatN T was highest in the upstream samples (5.3 ± 1.62pMol L' 1 d'1) and was 21
statistically different from ambient and effluent-free pNT (2.61 ± 0.59 and 2.39 ±
0.55pMol L' 1 d '1, respectively).
3.1.5 C uptake rates
Biomass specific carbon uptake measured under trace nitrogen addition (V trcC ) was higher in the upstream samples (0.25 ± 0.06 d'1), but was not statistically different from the ambient and effluent-free VtrcC rates (0.19 ± 0.04 and 0.21 ± 0.08).
When VC was measured during N-saturated isotope addition (V satC), V satC was again highest in the upstream samples (0.3 ± 0.08 d'1), and was statistically different from the ambient and effluent-free river conditions (0.19 ± 0.05 and 0.19 ± 0.08 d'1) which is a similar trend seen in the VtrcC measurements (Table 3).
Rho carbon uptake rates (pC) show a similar pattern to the VC rates, with highest values in the upstream samples, and a decrease in both the ambient and effluent-free river conditions. Carbon uptake ( p t r c C ) upstream was 23.04 ± 5.75|aMol L' 1 d"1, statistically higher than either the ambient or effluent-free river conditions (16.56 ± 5.09 and 16.4 ±
6.18pMol L' 1 d"1, respectively). For p s a t C , rates were highest in upstream samples (29.39
± 11.26pMol L' 1 d '1) and were statistically different from the ambient and effluent-free
P s a tC values (17.39 ± 6.38 and 15.47 ± 6.45pMol L' 1 d’1, respectively). 22
3.1.6 Growth rate estimates
Using the N-uptake rate as a proxy for potential phytoplankton growth, potential doubling times were calculated. Based on these calculations, phytoplankton should double in 1.9 days, 1.4 days and 1.4 days, for the upstream, ambient river and the effluent-free river respectively.
3.2 Enclosure studies conducted during May 2014 river manipulation experiment
In this series of enclosure experiments, nitrogen additions were made to ambient
(effluent-containing) river source water or effluent-free source water. Effluent-free water was amended with effluent, KNO3 and/or NH4CI solution (Table 1). In general, DIN declined over time in all enclosure experiments and chl-a increased.
3.2.1 Ambient enclosure
In the ambient enclosure (containing effluent), initial DIN was at 54.4pM, with 47.7|aM
NH4, 6.1|iM NO3 and 0.7|aM NO2 (Fig. 3A). Ammonium declined after 17 hours, and was depleted to <0.5pM by 72 hours when drawdown of NO3 began. NO3 was depleted to <0.5(iM by 89 hours. Saturated VNH4 uptake increased over the first 24 hours from
0.46 to 0.63 d' 1 and then declined to 0.06 d' 1 by 96 hours. Nitrate uptake remained at 0.01 d' 1 for the first 48 hours, but increased to 0.25 d' 1 at 72 hours when NH4 was depleted to
0.3|aM. Carbon uptake began at 0.28 d' 1 and reached a peak of 0.89 d' 1 at 48 hours and declined thereafter (Fig. 3A). F-ratio was near zero for the first 48 hours then increased steadily to 0.84 (not shown). 23
Chl-a concentration was 3.0 pg L’ 1 at the start of the experiment (TO) and increased to a maximum of 54.2 jug L' 1 at 96 hours (Fig. 4A). The >5 (am fraction of chl-a started at 1.6
pg L’ 1 at TO, and increased to a maximum of 50.5 pg L' 1 at 72 hours. The AChkADIN was 0.9, calculated from a change of 46.4 pg L’ 1 chl-a over the first 72 hours. Phosphate concentration at TO was 2.8pM and declined to near zero (0.19pM) by 72 hours (Fig.
4A). The DIN:P was 19 at TO, increased sharply to 45.4 at 65 hours at the time of highest chl-a, but then declined sharply after 65 hours and was nearly zero by 89 hours. DIN:P of the source effluent in 2014 was 34. Algal cell division per day calculated from change in chl-a during the first 72 hours was 1.45 d'1, while divisions per day based upon N-uptake rate was 0.93 d' 1 (Table 4).
3.2.2 - Ambient with KNO3 addition enclosure
When additional KNO3 (~30pM) was added to ambient source water (containing effluent), total DIN increased to 83pM with 47.7pM NH4, 34.7pM NO3 and 0.7pM NO2
(Fig. 3D). Drawdown of NH4 was similar to the ambient enclosure without added NO3
(Fig. 3A), where NH4 declined to zero by 72 hours in both enclosures. The elevated nitrate concentrations were not drawn down during the first 72 hours, but began to decline after 89 hours once NH4 concentrations were depleted to <0.5pM. Even though
NO3 had been added, saturated nitrogen uptake rates were similar to the ambient enclosure, where NH4 uptake rate increased over the first 24 hours from 0.39 to 0.64 d' 1 and then declined to 0.04 d’ 1 by 96 hours. Nitrate uptake was 0.1 d' 1 until NH4 concentration was reduced to 0 .3 5 pM at 72 hours, and NO3 uptake increased to 0.25 d'1. 24
Carbon uptake (VC) began at 0.23 d' 1 and increased to a peak of 0.63 d’ 1 at 48 hours. F- ratio (not shown) was near zero for the first 48 hours then increased to 0.84 by 96 hours.
With added NO3, chl-a reached a maximum concentrations of 53.4 pg L’ 1 at 72 hours from a starting concentration of 2.6 pg L''(Fig. 4D). The chl-a was again dominated by large cells. The >5 pm fraction of chl-a increased from 1.7 pg L' 1 at TO to a maximum of
52.4 jug L' 1 at 72 hours. AChl:ADIN (Table 4) was 1.03 calculated at peak chl-a.
Phosphate concentration was 2.9pM at TO (Fig. 4D), and declined to near zero by 72 hours. The DIN:P showed a similar trend to the ambient source water enclosure with slightly higher values, where DIN:P began at 28.6, had a sharp peak to 180.5 at 72 hours and declined sharply after 72 hours to 23.3. Using the chl-a increase from TO to 72 hours, the divisions per day were 1.55 d'1. When calculated from N-uptake rates, divisions per day were 0.95 d' 1 (Table 4).
3.2.3 - Effluent-free enclosure
In the effluent-free enclosure, total DIN was low at 3.4pM, (1.4pM NH4, 1.9pM NO3 and
0.05pM NO2) and both nitrogen sources (NH4 and NO3) were drawn down below 0.5pM within the first 24 hours (Fig. 3B) and stayed <0.5pM for the remainder of the experiment. Saturated nitrogen uptake rates were highest at TO (VNH4 0.52 d’ 1 and VNO3
0.48 d"1) and declined over 96 hours. Although NH4 uptake was higher (max at 0.54 d’1), uptake of NO3 still occurred over the whole time course (always >0.28 d '1). Carbon 25
uptake (VC) began at 0.34 d' 1 and peaked at 24 hours reaching 0.48 d’1. F-ratio ranged between 0.4 and 0.6 for the duration of the experiment.
Starting chl-a was 6.8 pg L' 1 and >5pm chlorophyll was 2.4 pg L*1. The maximum chl-a reached was 8.8 pg L' 1 after 24 hours (Fig. 4B). The >5pm chl-a fraction was also low, reaching a maximum of 4.4 pg L' 1 at 24 hours. The change in chl-a was only 2.2 over the first 24 hours, with a AChkADIN of 0.72. Phosphate started at 0.7pM, and declined thereafter. The DIN:P was maximal at TO (4.6) and declined to near zero within 24 hours.
Cell divisions per day calculated from chl-a data was 0.23 d'1, while calculated divisions per day based on N-uptake rate was 1.45 d' 1 (Table 4). However, since chl-a did not accumulate past 24 hours, the exponential curve fit was poor (r2=0.23; Table 4).
3.2.4 - Effluent-free with KNO3 addition enclosure
When additional KNO3 was added to effluent-free source water (Fig. 3E), the total DIN at the start of the enclosure experiment was 31pM (1.7pM NH4, 29.1pM NO3, and
0.1 pM NO2). Nitrate was drawn down by 72 hours. Saturated nitrogen uptake rates for
NH4 and NO3 both increased over the first 24 hours, from 0.38 to 0.51 d' 1 and 0.3 to 0.6 d'1, respectively. Ammonium uptake declined to 0.09 d' 1 by the end of the experiment and VNO3 declined to 0.12 d‘\ Carbon uptake at TO was 0.37 d-1, peaked at 0.49 by 24 hours and declined slowly thereafter. F-ratio had two peaks at 24 hours and 96 hours and ranged between 0.33 to 0.62. 26
More chl-a accumulated in the KNO3 addition compared to the effluent-free enclosure, reaching a maximum of 43 pg L' 1 at 89 hours (Fig. 4E). The >5pm chlorophyll increased from 2.2 pg L' 1 to 29.2 pg L' 1 by 89 hours. The AChl:ADIN ratio was 1.19, with a change in chl-a of 36.4 over 89 hours. Phosphate was similar to the effluent-free source water enclosure, starting at 0.8pM and declining quickly to near zero by 41 hours. DIN:P ratio started at 40.3, increased rapidly near 41 hours to a peak at 68.3, and declined to <2 by 89 hours. Divisions per day were 0.84 d' 1 when calculated from the chl-a increase in the first
89 hours. When using N-uptake as a proxy for p, the divisions per day were 1.59 d"1
(Table 4).
3.2.5 - Effluent-free with effluent-NH4 addition enclosure
In the enclosure where effluent (~50pM NH4) was manually added to effluent-free source water, the total DIN was 48.4pM with 46.1pM NH4, 2.2pM NO3, and 0.1 pM NO2 (Fig.
3C). The depletion of NH4 over time was similar to the ambient enclosure (effluent- influenced river water, Fig. 3A) with NH4 concentrations dropping below 0.5pM by 72 hours. Nitrate concentrations were initially low (2.2pM), but not drawn down until NH4 concentration reached 0.37pM at 72 hours, then NO3 was depleted to zero rapidly.
Nitrogen uptake rates were also similar to the ambient enclosure, with VNH4 > VNO3 while NH4 was available during the first 72 hours. An increase in NH4 uptake from 0.42 to 0.66 d’ 1 occurred in the first 24 hours, and then uptake declined to 0.06 d' 1 by 96 hours.
Nitrate uptake was <0.03 d' 1 for the first 48 hours, and increased to 0.23 d' 1 at 72 hours corresponding to when NH4 concentration declined to 0.37pM. Carbon uptake rate (VC) 27
started at 0.37 d"1 and reached at a peak of 0.49 d' 1 by 24 hours. F-ratio remained near zero for the first 48 hours, and gradually increased to 0.8 by 96 hours.
Chl-a also behaved similarly to the ambient river source water enclosure (effluent- influenced, Fig. 3 A). Chl-a started at 4.1 pg L' 1 and increased to maximum of 49.4 pg L' 1 by 72 hours (Fig. 4C). Change in chl-a over the first 72 hours was 45.3, and the
AChl:ADIN was 0.94. Phosphate started higher than the effluent-free enclosure (2.1pM) because effluent has elevated PO4 as well as elevated NH4. Phosphate declined to near zero by 65 hours. The DIN:P started at 22.7, had a maximum at 48 hours of 34.8 and declined quickly to zero by 72 hours in a similar shape to the ambient enclosure. Division per day based on chl-a increases were 1.39 d’1, while estimates using N-uptake rates gave a divisions per day of 0.96 d' 1 (Table 4).
3.2.6 - Effluent-free with KNO3 and effluent-NH4 additions enclosure
When both effluent (~50pM NH4) and KNO3 (30pM) were added to effluent-free source water, the total DIN was the highest of all enclosures at 79.5pM with NH4 50.3pM, NO3
29.1pM and NO2 0.1 pM (Fig. 3F). As in the effluent addition enclosure (Fig. 3C), NH4 was depleted to <0.5pM by 72 hours. Nitrate was elevated, but was not drawn down until around 72 hours. Uptake rates for NH4 increased over the first 24 hours from 0.46 to 0.74 d"1, and declined to 0.05 d' 1 after 96 hours. Nitrate uptake was below 0.02 d' 1 for the first
48 hours, and increased to 0.25 d' 1 at 72 hours corresponding to when NH4 concentration dropped to 0.5pM. Carbon uptake started at 0.34 d' 1 and reached a peak at 48 hours of 28
0.78 d'1. The f-ratio was near zero for the first 48 hours then increased steadily to 0.8 by
96 hours.
When both effluent (~50pM NH4) and KNO3 (30pM) were added to effluent-free river source water, the highest amount of chlorophyll in all May enclosures was seen at 60.5 pg L' 1 by 72 hours (Fig. 4F). The >5pm chlorophyll reached a maximum of 63.8 pg L' 1 at
89 hours from 2.2 pg L' 1 at TO. The AChl:ADIN was 1.04 (Table 4), with a change in chl- a of 54.3 over 72 hours. Initial phosphate was 2.4pM (Fig. 4F) and declined to zero by 48 hours. The DIN:P started at 32.7, reached a sharp maximum at 144.6 at 72 hours and declined rapidly afterwards. The DIN:P pattern was very similar to the ambient river water with NO3 addition. Divisions per day from the chi-a estimation method were 1.1 d'
', while the N-uptake method gave a divisions per day value of 1.09 d’ 1 (Table 4).
3.2.7 - Effluent-free with NH4 CI and KNO3 additions enclosure
Finally an enclosure treatment was carried out in which effluent-free source water was amended with NH4C1 (NOT effluent-NH4) and KNO3. When NH4C1 (50pM) and KNO3
(30pM) were added to effluent-free source water, the total DIN started at 72.1 pM (Fig.
3G). Ammonium declined from 42.6pM over the 96 hours experiment but never fell below 4.8pM. Nitrate was un-used, starting the experiment at 29.5 and ending at 28pM.
Ammonium uptake increased from 0.54 to 0.71 d' 1 over the first 24 hours, and declined to
0.02 d' 1 by the end of 96 hours. Nitrate uptake remained below 0.03 d '1. Carbon uptake 29
was 0.43 d' 1 at TO, and reached a peak of 0.5 d' 1 by 24 hours. The f-ratio was low for the first 48 hours then rose to a peak at 0.54 by 96 hours.
The peak chi-a was seen at 65 hours reaching 35.5 pg L"1 (Fig. 4G). The >5 pm chlorophyll began at 2.4 pg L' 1 and reached a maximum of 33.3 pg L_l at 89 hours.
Change in chl-a to the maximum at 65 hours was 30.9, and the AChkADIN was 1.1.
Phosphate level started lower than any effluent-influenced enclosures (0.9pM), and decreased to 0.2pM by 41 hours. The DIN:P started at 76, reached a peak at 72 hours of
234.4 and a second peak at 227.4 before declining. Divisions per day were 1.17 d’ 1 when estimated from chl-a accumulation, while N-uptake rates gave a divisions per day estimate of 1.04 d '1.
3.3 Enclosure studies replicated in incubation tanks
3.3.1- October 2014 - NH4 CI + PO4 additions vs Effluent-NH4 addition
In October, two enclosure treatments were compared using starting river source water from just upstream of the SWTF discharge site, at SYM. One set of enclosures received addition of both NH4CI and PO4, while the second treatment received addition of effluent-NFLt in order to compare growth without differing PO4 limitation (Fig. 5A vs
5B). The NH4CI and PO4 enclosure had a starting total DIN of 50.2pM, with 46.7pM
NH4, 3.4pM NO3, 0.09pM NO2 and 2.8pM PO4. The effluent enclosure had a starting
DIN of 47.6pM, composed of 44.8pM NH4, 2.7pM NO3, 0.09pM NO2 and 2.6pM PO4.
Change in DIN was minimal in the first 24 hours, but then both treatments drew down the 30
NH4 thereafter. The NH4CI and PO4 enclosures depleted NH4 to zero by 120 hours, and the effluent enclosures depleted NH4 to zero at 168 hours. The available NO3 was not used in the first 120 hours in the NH4CI and PO4 enclosures, but was depleted quickly to zero at 168 hours. In the effluent treatment, the NO3 was not used in the first 96 hours, but was depleted to zero by 120 hours. Nitrite in NH4CI and PO4 enclosure treatments was consistently near 0.08pM for the first 120 hours, and then declined to zero at 168 hours. Nitrite in the effluent treatment was near 0.09pM for the first 48 hours, then increased slightly to 0.14pM at 96 hours, and declined to 0.04pM by 120 hours.
Phosphate started similarly at 2.8 and 2.6pM in the NH4CI and PO4 vs effluent enclosures, respectively (Fig. 5A, 5B). Both enclosures depleted the available PO4 by 96 hours, although the NH4CI and PO4 addition enclosure had an anomalous increase in PO4 from 0 to 24 hours. Chl-a concentration started at 1.2 pg L' 1 and 1.1 pg L"1 in the NH4CI and PO4 enclosure vs the effluent enclosure (Fig. 6 A, 6 B), respectively. Peak chl-a was reached in both treatments at 120 hours, up to 32.2 pg L' 1 and 29.4 pg L' 1 in the NH4CI and PO4 enclosure vs the effluent enclosure, respectively. At T96 there was no significant difference in average chl-a concentrations between treatments (P=0.15)(Table 5, Fig. 6 A vs 6 B). The mean AChl:ADIN ratio was 0.7 in the NH4CI and PO4 addition, and 0.6 in the effluent addition (P=0.1). Divisions per day were 1.11 d' 1 in the NH4CI and PO4 addition, and 1.22 d' 1 in the effluent addition (P=0.08)(Table 4).
Flow cytometry counts at TO were not statistically different (P=0.89) between treatments at 3.1 x 106 and 3.02 x 106. After 96 hours, the dominant size class did not change, but 31
remained the 2-3 |am size class (Fig. 7B). Total cell counts were not statistically different at T96 (Table 5). In all three pigment wavelengths (phycoerythrin, phycocyanin and chl- a), the effluent NF^-containing treatment had a larger total cell count, although not statistically different (Appendix 5.3). The largest increase in cells occurred in the phycocyanin pigment range (6 6 8 - 601nm). Phytoplankton functional groups after 96 hours of growth were similar between the NH4+PO4 treatment and the effluent treatment
(Table 5).
The starting phytoplankton community seen in the lugols preserved samples for both treatments was dominated by small picoplankton cells (2-4|j.m size range) making up 44-
45% of the total cell count in both treatments. After 96 hours, the picoplankton community was still the dominant phytoplankton group by percent of total count but had increased up to 85-89% of the total count (Table 5). The cryptophye group was the second largest category at the start of the enclosures, with 29% in the NH4+PO4 treatment and 21% in the effluent treatment. After 96 hours the cryptophytes made up 3% pf the total cell count. The pennate diatom group, the cryptophyte group and the other group all declined from TO to 96 hours. Centric diatoms increased in percent of total count, but only from 1% to 3% of the total cell count.
3.3.2- April 2015 - KNO3 + PO4 additions vs Effluent-NH4 addition
In April, effluent growth was compared to growth when amended with both KNO3 and
PO4 (Fig. 5C vs 5D). Total starting DIN was 50.7|aM in the KNO3 and PO4 addition 32
enclosures, and 57.7|^M in the effluent enclosures. Change in DIN was minimal in the first 24 hours, but then both treatments drew down the DIN thereafter. In the KNO3 and
PO4 addition, DIN declined to 3.5(aM by the end of the experiment at 120 hours. NH4 started at 2(iM, and was depleted to <0.5|_iM by 48 hours, while NO3 continued to be used. As the dominant source of DIN, NO3 started at 48.6|aM and declined to 2.6|aM by
120 hours. The effluent enclosure depleted the DIN to l^M by 120 hours. NO3 in the effluent enclosures was not used in the first 96 hours (remained ~ 2.5|iM), but was depleted to 0.3 at 120 hours. Nitrite in the KNO3 and PO4 addition started at 0.15|iM and increased over the experiment to a maximum of 0.47|iM by 120 hours. Nitrite in the effluent enclosure was fairly constant at 0.16(J.M, with a slight dip at 120 hours to
0.11 pM. PO4 was depleted in both enclosures by 96 hours.
Chl-a started at 4.1 (ag L' 1 and 6.2 pg L' 1 in the KNO3 and PO4 vs effluent enclosures, respectively (Fig. 6 C, 6 D). Chl-a increased over time in both enclosures, reaching a peak at 120 hours in both the KNO3 and PO4 enclosure and the effluent enclosure (Fig. 6 C vs
6 D). Maximum chl-a was 23.5 pg L"1 in the KNO3 and PO4 enclosure, and 33.8 pg L’ 1 in the effluent enclosure. At T96 there was no significant difference in average chl-a concentrations between treatments (P=0.3)(Table 5). Both enclosures show a dip in chl-a concentration at the 96 hour timepoint. Variance in chl-a concentration across replicates was higher than previous enclosures. The mean AChl:ADIN was 0.49 in the KNO3 and
PO4 addition, and 0.74 in the effluent addition (P=0.006). Divisions per day were 0.76 d’ 1 in the KNO3 and PO4 addition, and 0.86 d' 1 in the effluent addition (P=0.45)(Table 4). 33
At TO the cell concentrations counted by flow cytometry were already different with 7.57 x 106 cells L' 1 in the NO3+PO4 and vs 1.01 x 107 cells L' 1 in the effluent treatments
(PO.Ol). After 96 hours, the total cell counts were no longer statistically different 6.5 x
107 vs 8.2 x 107 (P=0.42), but the effluent treatment had higher counts than the NO3 and
PO4 enclosures (Table 5). The starting size class distribution (Fig. 7C, 7D) had the highest counts in the 2-3 size range, but high counts were also seen in cells ranging from 3-8|im, which is different from the other replicated enclosure experiments (Fig.
7D). Similar to the NH4+PO4 experiment, all three of the pigment wavelengths showed higher total cell counts within the effluent-NF^ treatments (Appendix 5.3).
At the start of the experiment, the NO3+PO4 and effluent treatments already had slightly different phytoplankton communities (Table 5). The dominant group in the NO3+PO4 treatment was the centric diatoms, while the dominant group in the effluent treatment was the green algae. At the end of 96 hours, the dominant phytoplankton group had changed to picoplankton group in both treatments. 34
4.0 Discussion
4.1 Overview
Upgrades to the SWTF in accordance with their new permit will result in a decrease in the total amount of DIN discharged into the lower Sacramento River and the form of nitrogen will be predominantly NO3 (CRWQCB 2013). These future changes in river
nitrogen conditions have the potential to impact phytoplankton dynamics as nitrogen is a
critical nutrient for phytoplankton growth (Ryther & Dunstan 1971). Our large-scale river
manipulation experiments in combination with controlled enclosure experiments show
dramatic changes in nutrient concentrations and forms but little evidence of change in the
phytoplankton processes with the reduction of total DIN, as chl-a did not change in the
effluent-free river, phytoplankton species were not altered by the form of nitrogen
provided, and total nitrogen uptake was not drastically changed despite large changes in
nitrogen concentration.
Phytoplankton grown in enclosures were able to use any of the three nitrogen sources
provided (effluent-NH4, NH4CI, or KNO3), although NH4 was utilized prior to NO3.
Enclosure experiments highlighted that NH4 concentrations greater than ~l-4pM inhibit
the uptake of NO3, consistent with previous results from the SFE (Parker et al. 2012b,
Wilkerson et al. 2006, Dugdale et al. 2007). Ammonium concentrations in the ambient
(effluent-containing) river under present day wastewater operations exceed this inhibition
threshold (Parker et al. 2012a, Foe et al. 2010). Phytoplankton in the enclosures used the 35
available DIN to grow to peak chl-a, generally approximating the proportion of 1 (iM N yielding 1 jag L' 1 chl-a (Wilkerson et al. 2015). In enclosure experiments with no additional N, the low DIN was fully utilized resulting in accumulation of low chl-a biomass accumulation as phytoplankton became nitrogen limited within 24 hours. Results from these enclosures stood in contrast to observations made in situ in the Sacramento
River, where chl-a was higher upstream and lower in either downstream condition. Low
TRC:SAT rates of VN suggest that upstream locations were nitrogen limited, while downstream locations were saturated for VNH4 in both the ambient and effluent-free river.
In contrast to Parker et al. (2012b), enclosure experiments that compared phytoplankton growth on NO3 compared to growth on NH4 suggest there is no difference in ability to grow, or achieve peak biomass on either substrate. Phytoplankton functional groups were different at the end of 96 hours of growth, and as suggested by others (Berg et al. 2003,
Glibert et al. 2011) N form appeared to influence phytoplankton composition with a higher proportion of centric diatoms observed in the NO3 + PO4 treatment and higher proportions of picoplankton in the effluent treatment. However the starting communities were already different, complicating end point comparisons. Growth differences on
NH4CI compared to effluent-NH4 were not apparent in terms of chl-a accumulation, VN, or phytoplankton functional groups suggesting there was no toxicity effect at the concentrations tested. However, in an October 2013 enclosure of ambient river water 36
containing ~80|iM NH4 the VNH4 rates were depressed for the first 48 hours of the time course.
Overall the enclosures show an existing potential for phytoplankton growth on any nitrogen form seen as accumulation of chi-a. This potential for growth was not realized in the river, even when DIN was replete, suggesting there are other controls on accumulation of chl-a biomass besides nitrogen. If nitrogen is not utilized within this stretch of river, the overall consequence of elevated SWTF discharge into the river may be seen farther downstream where environmental conditions may be more optimal for uptake of the large DIN pool. Currently effluent-NFU is advected downstream into the northern estuary, frequently at concentrations high enough to inhibit VNO3 (Dugdale et al. 2007). A reduction in effluent-NFLt and conversion to effluent-N0 3 in the Sacramento
River, may result in reduction of NH4 inhibition of VNO3 down into the northern estuary, and a potential for increased frequency and magnitude of phytoplankton blooms
(Wilkerson et al. 2015).
4.2 Effluent-free experimental river conditions compared to ambient river
The large scale river manipulation (diverting effluent discharge for 15 hours) resulted in effluent-free river conditions that may be comparable to river conditions after the SWTF upgrades in 2021. The effluent-free river had significantly lower average NH4 and total
' i - v - § | i ^ DIN, however NH4 was not ilBi completely removed from the system. Within the effluent- free river section, residual NH4 averaged ~3.2|aM (much lower than ambient river 37
conditions ~50pM). There are a number of adjacent side sloughs where effluent-NIT} could have remained unflushed during the 15 hours effluent hold. River flow was also low during this drought year, which may have contributed to the presence of residual
NH4. There may also be other unknown sources of NH4 into this stretch of river, including metabolic processes converting NO3 to NH4.
The lagrangian method of tracking a water parcel was useful to follow processes that were occurring through time as a section of river moved downstream (Scherwass et al.
2010). However, there were likely many external interactions that could have influenced the processes measured in the water column such as interaction with the benthos, river flow velocities, tidal backwash, contributions from and loss to side sloughs, and difficulty tracking a water mass that is not visibly distinct. However based on NH4 concentrations as a tracer of the effluent, this study was able to follow the experimental effluent-free river more easily than the ambient river.
The large reduction in river DIN had little impact on the total chl-a biomass seen downstream of the SWTF. Average chl-a was not statistically different between the ambient river conditions and the experimental effluent-free river conditions during this study. The chl-a remained low in both river conditions (with or without effluent), indicating that the presence of elevated nutrients (i.e. effluent-NHt) was likely not controlling the abundance of chl-a along this stretch of river. Chl-a measurements taken through the same stretch of ambient river in April and May 2009 by Parker et al. (2012a) were similar to averages from this study (2.4 ± 0.6 and 3.2 ± 0.4 ^g L'1, compared to 4.7 38
± 1.8 (j.g L' 1 in May 2014). However, average chl-a sampled from sites upstream of the
SWTF discharge had closer to ~10pg L , significantly higher than either downstream condition.
The lack of change in phytoplankton biomass (chl-a) may have been because our lagrangian sampling was not continued for long enough to see changes in phytoplankton * biomass, or more likely, a result of conditions of the river such as dilution, phytoplankton settling, and mixing into low light conditions. This study did not attempt to measure the impact of zooplankton grazing pressure on phytoplankton biomass, which may play a role in limiting chl-a accumulation. In addition to zooplankton, benthic grazers (e.g.
Corbicula) may also contribute to loss of chl-a (Chervin et al. 1981, Cohen et al. 1984).
Low chl-a fits with the general understanding that cultural eutrophication symptoms (e.g. excessive algal blooms) are not seen in the SFE in spite of elevated nutrients, in part because of light limitation due to high turbidity (Alpine & Cloern 1988). It may also be that flow in the river contributes to insufficient residence time for phytoplankton nutrient uptake to overcome washout, as described by Dugdale et al. (2012).
Low chl-a is not a new phenomenon in this stretch of river, as Ball & Arthur (1979) showed that average chl-a concentration at HOOD (-12 km downstream from the effluent pipe, lat = 38.36861, long = 121.52139) from 1968-1974 was < 2 ng L'1.
Interestingly, 1977 was the only year in the Ball & Arthur (1979) data set where chl-a accumulated significantly, and it reached 40 |ig L' 1 and stayed above 10 (ig L' 1 for 3 39
months (April -June 1977). The year 1977 was a drought year in California, and the low river flows (ie high residence times) were cited as the reason for the large standing stock of chl-a. Water velocities and residence time in streams and rivers can influence primary productivity (Odum 1956, Soballe & Kimmel 1987, Reddy et al. 2015), and when phytoplankton doubling is slower than the residence time in the northern SF delta, phytoplankton biomass can be lost to advection downstream (Dugdale et al. 2012, Liu & de Swart 2015).
Although the current study also measured chl-a during the spring of a critically dry water year in 2014, we did not see accumulation of an anomalous drought-year spring chl-a bloom, as observed in March 2014 in the northern estuary and upstream in the
Sacramento River by Glibert et al. (2014). The 1977 Sacramento Valley fresh water runoff was 1.94 million acre-feet (maf), while 2014 runoff was 2.6 maf, which is the lowest volume of runoff since the 1977 drought year. Perhaps the residence time in 2014 was still too short to produce a chl-a bloom in the mid-river, but another difference is that the wastewater treatment facility (SWTF) was not present in 1977, as it came online in
1983. Effluent toxicity and elevated-NH4 from treated wastewater can cause depression in primary productivity (Welch 2002, Yoshiyama & Sharp 2006, Waiser et al. 2011), and multiple factors may be acting to keep chl-a low in this stretch of river. 40
4.3 Changes in nitrogen uptake physiology in situ
Even though total chl-a biomass remained low, there was evidence of a physiological response to the reduction of total DIN in the N uptake physiology. Nitrogen uptake in the ambient river (~50pM effluent-NRt) was dominated by VNH4, while VNO3 was suppressed to effectively zero. This NH4 inhibition of VNO3 has been well documented
(Collos et al. 1989, Dortch 1990, Cochlan & Harrison 1991), and has been seen in this stretch of Sacramento river previously (Parker et al. 2012a). During the May 2014 experimental manipulation in the effluent-free section of river, the reduction of NH4 alleviated the effect of NH4 inhibition and allowed for recovery of VNO3.
Elevated NH4 levels have been implicated as a cause of degraded phytoplankton productivity in estuary systems (Parker et al. 2012, Yoshiyama and Sharp 2006, Dugdale et al. 2007, 2012, Wilkerson et al. 2006, 2015, Glibert 2010, Waiser et al. 2011,
Livingston et al. 2002) when the major DIN pool is NO3 and is inaccessible for growth.
However increased NH4 has also been shown to stimulate phytoplankton growth in natural assemblages and cultures (Thompson et al. 1989, Tada et al. 2009). Esparza et al.
(2014) showed that the high NH4 water of a shallow lagoon near a wastewater treatment facility fueled growth and accumulation of chl-a. In this study no change was detected in the chl-a concentrations, but chl-a can take days to increase, perhaps longer than the river was monitored during this study. Differences seen in N-uptake between ambient and effluent-free conditions indicate a potential changes in the metabolism and physiology that can occur over hours. Exposure to effluent occurs almost instantaneously as the river 41
passes the outfall diffuser, but response in the phytoplankton community occurs at a
variety of timescales, from minutes to upregulate N-transporters, to generations to shift
community composition. The distinct change from upstream community N-metabolism
(both VNH4 and VNO3) to downstream effluent-influenced N-metabolism (VNH4 only)
was seen across a timespan as small as six hours in 2014 (Appendix 3.2).
The reduction of VNO3 to near zero was documented in the Sacramento River at NH4
concentrations near 4|aM or lower (Parker et al. 2012), and downstream in San Francisco
Bay (Dugdale et al. 2007). The inhibitory effects of NH4 on VNO3 over a range of
concentrations have been shown under various environmental and experimental
conditions (Berman et al. 1984, Pennock 1987, references in Dortch 1990). Inhibition of
NH4 on VNO3 is difficult to distinguish from preference for NH4 in field conditions
where both nitrogen forms are naturally occurring (Dortch 1990). Many species have
unique levels of inhibition response to NH4, or have varying responses under different
environmental conditions. There may also be some situation and species that experience
NO3 inhibition of VNH4 (Dortch & Conway 1984), which could play a larger role in the
ecosystem if the dominant DIN pool in the future river is NO3. Turning off the effluent-
NH4 decreased effluent-NH4 concentrations enough to allow some NO3 uptake, VNO3, to
occur downstream. However, total N-uptake (VNT) in the ambient river vs the effluent-
free river were not significantly different (0.51 vs 0.48 d’1). The proportion of VNH4 vs
VNO3 contributing to the sum VNT was different, with ambient river dominated by
VNH4, while the effluent-free river VNT was -36% VNO3 and 64% VNH4. Although 42
VNT is similar in both river segments, phytoplankton biomass will ultimately be limited by total available DIN (if it is the limiting nutrient).
Previous studies have seen a depression primary production within the effluent-plume extending downstream from the SWTF discharge site in the Sacramento mainstem
(Parker 2012a). The current study did not see the same depression in carbon uptake in the effluent-containing river compared to the upstream river when measured in the trace nitrogen uptake incubations; VtrcC values were not statistically different between upstream and ambient (with effluent) river conditions (Table 3, Appendix 6 ). Although the averages between upstream conditions and ambient conditions were not different, the trend moving downstream was a decrease in VC (Appendix 6 ) which may hint at potential toxic or inhibitory effect of NH4 or effluent downstream. VC values from the incubations done with saturating N isotope additions resulted in an increase in VC in the upstream samples, indicating that the upstream river is likely limited for nitrogen which subsequently limits VC.
Comparison of trace vs saturating uptake rates gives an estimate of potential for phytoplankton to increase their uptake with increased nutrients, and indicate a potential
Vmax for uptake (Dugdale & Wilkerson, 1986). The ratios of trace to saturating uptakes
(TRC:SAT) gives a good indication of whether the river is replete with nitrogen, or may be nitrogen limited. When TRC:SAT is low (large difference between trace and saturating uptake) it suggests that the addition of extra nitrogen isotope increased the uptake rate, and the in situ uptake is a nitrogen limited condition. When TRC:SAT 43
approaches 1 (no difference between trace and saturating uptake rates) it indicates that the phytoplankton where already at their Vmax for N uptake since the addition of
saturating N isotope did not increase the uptake rates above trace N isotope additions.
Uptake within the ambient river is already at a maximum with respect to nitrogen. Figure
2 shows TRC:SAT ratios well below 1 in the upstream river, indicating that the upstream river is nitrogen limited. This suggests that the effluent-free river is saturated for VNO3
even at average NO3 concentrations of 2.88pM. When effluent is in the river (ambient), the NH4 TRC:SAT ratio is ~1, meaning NH4 is saturated (~50pM NH4). TRC:SAT for
NO3 however is low, which may be a consequence of NH4 inhibition on NO3 uptake,
since there is NO3 available (~6 pM).
The effluent-free river condition is interesting, because it has much lower total DIN than
the ambient river, so it would make sense that the ambient river is closer to maximum
nitrogen uptake. TRC:SAT ratios for NO3 are near 1 with 2.9pM NO3 in the effluent-free
river and no indication of NH4 inhibition on VNO3 occurring since NH4 is low (~3.2pM).
The TRC:SAT ratios for NH4 are slightly lower than 1, suggesting 3.2pM NH4 in situ is
not quite enough to be replete and there is potential for higher uptake rates to occur if
more nitrogen is provided.
4.4 Potential nitrogen uptake physiology in enclosures
Enclosure experiments filled with effluent-free or ambient (i.e. with effluent) river water
showed a similar pattern of N uptake as observed during the in situ river experiment. 44
Each nitrogen form tested (effluent-NH4, NO3, and NH4CI) was drawn down over time
and appeared to be used by phytoplankton to support their growth. Not surprisingly, the effluent-free enclosures with lower total DIN became nitrogen limited several days before the ambient or nutrient amended enclosures and reached much lower peak chl-a
concentration. Under experimental conditions of 50% surface PAR, phytoplankton in
enclosures were unlikely to be light-limited. This is in contrast to in situ conditions where
phytoplankton experience a well-mixed and deep river, and are more likely to be light-
limited (Parker et al. 2012b, Kimmerer et al. 2012). Within enclosures, phytoplankton
experience physical conditions that are unlike the river in situ, with no sinking to the
benthos, and no input or export by advection. These important differences between
enclosures and the river in situ must be considered when interpreting these results, and
comparison between enclosures may provide the most meaningful insights.
The patterns of NH4 drawdown in all enclosure follow a similar pattern of slow decline in
the first 24 hours and rapid drawdown near 48 hours (Fig. 3, Fig. 5). This is similar to
other enclosures conducted using water from the SFE (Parker et al. 2012b, Wilkerson et
al. 2015) where peak drawdown occurs near 48-72 hours. The VNH4 and VNO3 appear to
follow the idealized sequence for enclosure experiments presented in Parker et al.
(2012b) where peak VNH4 is earlier than VNO3 when both NH4 and NO3 are present. As
noted by others, the sequence of NH4 uptake followed by NO3 uptake is likely due to
NH4 inhibition of VNO3. Parker et al. (2012b) found that peak VC was linked to peak
VNO3. Interestingly, VC does not follow the idealized pattern, where VC peaks at the 45
same time as peak VNO3. This correspondence between VNO3 and VC only occurred in the effluent-free and effluent-free+N0 3 enclosures (Fig.3B, 3C) where NH4 substrate was not available. In the other enclosures with elevated NH4, the peak VC does not aligned with peak VNO3, but aligns closer with VNH4. Peak VC often occurred 24 hours after peak VNH4 (Fig. 3A, 3C-D, 3F-G). This may indicate that surge NH4 uptake occurred, with carbon uptake following after (Conway & Harrison 1977). However, the two ambient enclosures experienced effluent in the river for -24 hours in situ before being sampled for the enclosure experiment, yet still show evidence of surge uptake. This suggests that surge NH4 uptake takes place once water has been placed in enclosure conditions and may be linked to the increased light availability or decreased flow.
Nitrogen uptake rates in the enclosures showed a delayed uptake response in which N uptake initially is low, but increased after 24 hours. This was observed for both VNH4 and VNO3, and suggests there is an acclimation period for phytoplankton after being enclosed which may be relevant when considering potential surge uptake in enclosures.
While initial VNH4 (0.46 d '1) in the enclosure was similar to river average NH4 uptake, peak N uptake rates in the enclosures were higher. For example, the ambient river VNH4 averaged 0.46 ± 0.06 d"1, while the corresponding ambient enclosure had a peak VNH4 of
0.63 d’1, a nearly 40% increase in NH4 uptake. The increase in NH4 uptake within the enclosure may be the response to light acclimation, as cells are initially exposed to constant 50% surface light during incubation. 46
All effluent-containing enclosures depleted PO4 by 72 hours, and other enclosures depleted PO4 earlier because they were not amended with effluent-based PO4 (Fig. 4). In the replicated experiments, PO4 was added in addition to nitrogen amendments and PO4 was depleted at a similar time (96 hrs) across all treatments. Phosphate limitation has been shown to control chl-a biomass in the San Joaquin river system (Van Nieuwenhuyse
2007), and N:P ratio can also impact growth and community structure (Elser et al. 1990,
Glibert et al. 2011). In the effluent-free river, PO4 concentrations were reduced by 50% from ambient conditions, while DIN was reduced by 90% thus altering the N:P within the river. PO4 limitation may be the reason that enclosures with +70(iM DIN did not achieve a peak chl-a biomass of 70 \xg L' 1 (Fig.3D, 3F, 3G). In the future river, with reduced DIN in the pointsource effluent, the N:P ratios may be lower with N becoming the primary limiting nutrient (Anderson et al. 2002, Van Nieuwenhuyse 2007). N:P can be a selective force for phytoplankton community composition (Bulgakov & Levich 1999, Glibert
2010), where high N:P may lead to more green algae and low N:P can select for cyanophytes.
In these enclosure experiments, when two sources of nitrogen were present, NH4 was drawn down first and subsequently NO3 drawdown began once NH4 was below ~4|aM
(Fig. 3B-F). When NH4 concentration remained >4|^M, no NO3 drawdown occurred (Fig.
3G). The NH4 inhibitory effect on VNO3 was seen in enclosures where NH4 concentration was high, and VNO3 rebounded once NH4 was depleted over time in enclosures with low starting NH4. In contrast to the river in situ, enclosures reached much 47
higher peak chl-a biomass when any form of DIN was provided, but was limited by the total DIN available.
There was a much larger dynamic range in VN values over the 120 hour time course of the enclosures as phytoplankton switched from VNH4 to VNO3, which is different from the measurements taken over the 5 day in situ river experiment where VN values were relatively consistent within river conditions. This is likely an enclosure effect, where the conditions within the enclosure vary more widely in terms of nutrient concentration due to the small volume, whereas the river is large enough to remain relatively homogenous over the study area.
4.5 Growth rates and chl-a biomass in the phytoplankton community
Other studies have also found that addition of wastewater to an aquatic system stimulates phytoplankton growth (Esparza et al. 2014). In this study enclosures filled with water containing effluent-NIHU were able to build similar amounts of chl-a to enclosures amended with NH4CI. The in situ river showed no difference in chl-a between the experimental effluent-free conditions and ambient conditions (remaining below 5 pg L"1), yet the enclosure corresponding to the ambient river (containing effluent) was able to grow up to ~50pg L' 1 chl-a in 72 hours (Fig. 3A). A similar discrepancy between field chl-a growth and enclosure growth was seen in the work of Kudela et al. (2015) in the coastal waters offshore of Huntington Beach, CA near the discharge outfall for the
Orange County Sanitation District. A planned diversion of treated effluent created a new 48
plume of NH4-rich effluent (42^iM within the plume), yet there was no change in phytoplankton biomass.
In all the enclosure experiments, chl-a increased, with the maximum limited by the total initial DIN that was provided. This was especially obvious in enclosures using low-DIN effluent-free river water, which did not accumulate more than 5 ng L' 1 chl-a. Comparing the enclosures with the in situ river data shows the potential for phytoplankton growth under treatment conditions, and illustrates how innately different the enclosure is from the real environment. Even though the starting source water was identical in both scenarios, the outcome in terms of chlorophyll accumulation was different since phytoplankton in enclosures have increased light availability, less grazing pressure especially from the benthos and no loss to sinking. However enclosure experiments are useful for understanding the uptake physiology of the phytoplankton that can occur with different amendments such as added effluent vs no-effluent.
The duration of the May 2014 lagrangian study was five days, but the amount of time actually spent downstream of the effluent discharge pipeline was only -72-85 hours
(three days). This length of time is very similar to the enclosure experiments run using the same source river water. Phytoplankton growth rates in the river were 0.74 and 0.7 divisions per day in the ambient river and effluent-free river, respectively, calculated using total (NO3+NH4) I5N-uptake rates in 2014 (V jr c N T = 0.51 d"1 and 0.48 d’1). These growth rates suggest that any potential change in chl-a should have been observable within the study period (3 days), yet no increase was observed. Alpine & Cloem (1988) 49
observed phytoplankton growth rates of 0 .2 -0 .5 divisions per day in the northern estuary, and in this same stretch of river the VNT data from Parker et al. (2012a) suggests growth rates of 0.27 and 0.36 divisions per day. In the corresponding enclosures for ambient river water and effluent-free river water (Table 4), the growth rates calculated using uptake of total N (VsatNT = 0.65 d' 1 and 1.0 d'1) gave divisions per day of 0.93 in the ambient enclosure and 1.45 in the effluent free enclosure. In enclosures, growth rates calculated from chl-a accumulation showed the same patterns as rates estimated from
VNT. The enclosure growth rates were faster than those calculated for the river, but enclosures likely had higher light and no dilution effect from a flowing river. Growth rates estimated from chl-a accumulation and N-uptake rates were not significantly different from those with added NH4CI. In other rivers, zones of low chl-a accumulation have been correlated with the presence and high abundance of corbicula clams (Cohen et al. 1984). Clam abundance was not measured in this study, but their presence along this stretch of river was observed.
Species composition of estuarine phytoplankton has been shown to change in response to environmental factors, including the presence of elevated NH4 (Lehman 2004, Heil et al.
2007, Glibert 2010, Kress et al. 2012, Ma et al. 2014). In the enclosure experiments, there was no obvious difference in the phytoplankton groups after 96 hours whether grown on
NH4CI or effluent (Table 5). The most dominant group by proportion of total count was generally the small picoplankton in both fall 2013 and spring 2014. Previous studies investigating phytoplankton communities in the lower Sacramento River found a 50
dominance of cryptophytes and small flagellates (Kress et al. 2012), and other studies hypothesize these functional groups to correspond with elevated NH4 conditions (Glibert
2010).
In river systems, flow rate and water depth have been shown to impact species composition (Leland 2003). Bahnwart et al. (1998) found that deeper, low velocity water led to higher abundance of diatoms and cryptophytes, while shallower fast -moving water selects for chlorophytes. In enclosures from May 2014, the size fractionated chl-a showed high correlation between the >5 pm fraction and the total chl-a, suggesting that the community was predominantly >5pm sized cells. This makes senses considering smaller phytoplankton with higher surface area to volume ratios are thought to be more competitive for nutrients, yet more susceptible to grazing (Munk & Riley 1952).
Phytoplankton functional groups showed a seasonal difference, seen in the starting community of phytoplankton at the same river location between October 2014 and April
2015. In the fall, the phytoplankton community was dominated by 2-3pm cells, while the spring phytoplankton community has a wider range of size classes present and slightly higher starting chl-a (Fig. 7A vs 1C). Variance in community composition across seasons and years was also seen in Kress et al. (2012) where flagellates and chlorophytes dominated the assemblage near Stan’s Yolo Marina (GRC station) in April and August
2010, while diatoms dominated in April 2011. At the end of 96 hours of growth in the fall enclosure experiment, we did not see a clear difference in the phytoplankton functional groups present in the NH4 + PO4 treatments compared to the effluent addition treatments. 51
Both treatments had 84-89% of the total count consisting of picoplankton cells (2-3 (jM cells) and 3.1% centric diatoms. This finding does not match previous studies on phytoplankton community composition in the SFE that found correlation between increased cryptophytes and declines in diatoms in response to elevated effluent concentrations in situ (Glibert 2010). However, enclosure studies are different than in situ measurements because enclosure conditions may be biased towards r-strategist species
(Davis 1982). Lehman (2000) suggested that phytoplankton community varies with
“water year”, where wet, cold years are dominated by diatoms and warmer, dry years have more flagellates. During the dry “water years” of this study, the phytoplankton communities were not dominated by flagellates, however flagellates can be difficult distinguish in Lugols preserved samples. There was no increase in cyanobacterial cells with addition of NH4 or effluent, which is counter to other findings where cyanobacteria increased.
In the spring experiment comparing NO3 + PO4 and effluent, the composition at the end of 96 hours of growth appeared to be different, however composition was already different at TO so it is unclear if this was a treatment effect. In both treatments, the dominant functional group was the small picoplankton. Overall percentages were smaller than in the fall, with 41% picoplankton in the NO3 + PO4 treatment and 62% picoplankton in the effluent treatment (Table 5). The centric diatoms were also much higher at the end of 96 hours, with the NO3 + PO4 treatment having 31% centric diatoms by total count, and the effluent treatment having 11% centric diatoms. This is consistent 52
with findings in the effluent-N0 3 dominated river system of the San Joaquin River, where dominance by diatoms has been found (Leland 2003, Kress et al. 2012). In enclosure experiments with NO3 addition, Glibert et al. (2014) saw an increase in fucoxanthin containing cells which is indicative of an increase in diatoms. Other research in marine environments has also shown that the diatom functional group has the fastest growth rates in response to nitrate enrichment (Latasa et al. 1997, Schliiter 1998). However, studies in the Chesapeake Bay have shown that spring diatom blooms decline when water temperatures increase above 18°C possibly due to physiological limits on NO3 uptake
(Glibert et al. 1995, Lomas & Glibert 2000). With warmer and drier conditions in the
Sacramento region, we may see phytoplankton species more suited for warm temperatures and elevated NH4.
4.6 Signs ofNHdinhibition and effluent toxicity
Potential toxicity of the effluent stream in the Sacramento River has been discussed as a potential cause of depressed primary productivity (Parker et al. 2010, 2012). Toxicity effects of NH4 have been shown to have a variable effect on growth rate across different phytoplankton groups, with chlorophytes being most tolerant to high NH4 followed by cyanophytes (Collos & Harrison 2014). This toxicity is attributed to the unionized and lipid soluble NH3 form, which is a higher percentage of the total NH3/NH4 pool when pH increases (Erickson 1985) and can be toxic due to destabilization of membranes (Glibert et al. 2015). Ammonium toxicity may not occur until extremely high concentrations that are rarely experienced in the natural environment (lOOspM), yet NH4 can still have 53
negative impacts on growth even at lower concentrations. In Maclsaac et al. (1979) an inhibition of VNH4 was seen in effluent concentrations of >10|^M NH4, and an inhibition of VC at >100fiM effluent-NR}. This study did not see effluent toxicity effect in the enclosures where effluent was added at 50pM NH4, which is comparable to the river average seen during this study (50.6|aM), and slightly higher than the yearly average of
32.8(aM NH4 seen in the lower Sacramento River from 2009-2010 (Foe et al. 2010).
Interestingly, +100|aM NH4 concentrations are occasionally seen in the lower Sacramento
River, such as in October 2013. During the October 2013 river manipulation, the average
NH4 concentration in the ambient river was 83.4, with some samples reaching ~100|jM
(Appendix 1.1 A). These extreme high concentrations are likely due to a combination of low flow conditions and SWTF discharge operations, since the SWTF must discharge only during downstream flow, only to permitted concentrations and has limited storage capacity. The lower Sacramento River is tidally influenced, and when reversals in river flow occur, discharge of effluent must be withheld until the river returns to downstream flow. To prevent overflow of effluent in SWTF holding basins, higher volumes of effluent must be released to compensate for periods of discharge stoppage.
Effluent not only has the potential toxicity of elevated NH4, but also contains other constituents that may be toxic to algal growth, such as pharmaceuticals etc. In order to differentiate between direct NH4 toxicity and toxicity of another unidentified constituent of the effluent stream, enclosures were conducted with either an NH4CI solution or with effluent-NFLt. A similar strategy was carried out in Maclsaac et al. (1979) using effluent 54
addition experiments, concluding that phytoplankton in effluent additions had lower
VNH4 compared to those in NH4CI additions at concentrations higher than 1 0 |aM. Below
10|iM Maclsaac et al. (1979) found no difference in VNH4 between growth on NH4CI or effluent-NH4. Parker et al. (2010) also did a series of preliminary experimental additions using effluent and NH4CI, and found reduction in primary productivity in the effluent supplemented enclosures upwards of 60|iM, but unclear results in the NH4CI additions.
When enclosures were conducted using ambient river water from October 2013, containing +80|iM effluent-NH4 (Appendix 2.1 A), the VNH4 rates were depressed compared to rates measured in the NH4CI addition, or to effluent-containing enclosures from 2014 (Fig. 3). This may indicate a concentration threshold for observation of toxicity effects from effluent that is higher than the concentrations tested in the 2014 enclosures (50|nM effluent-NlL). The VNH4 rates from samples collected in October
2013 were lower than those from May 2014, and when plotted against NH4 concentration, showed a declining trend in VNH4 at NH4 concentrations greater than
~60|iM (Appendix 4). NH4 inhibition of VNH4 can occur when high cellular NH4 concentrations inhibit NH4 transporters (Glibert et al. 2015). However, it is still unclear from this data whether inhibition is from effluent-NH4, or from another effluent toxin, since we did not test NH4CI at concentrations higher than 50|aM. Some studies suggest that byproducts of the chlorination process in wastewater treatment can inhibit phytoplankton growth for 3 days or longer, although these studies were in coastal waters
(Eppley et al. 1976, Kudela et al. 2015). 55
Comparison of phytoplankton growth on NH4CI vs effluent-NH4 can be made from two enclosures from May 2014 (Fig. 3F, 3G). Initial total DIN concentrations were similar
(79.5 vs 72.1pM), but maximum chl-a that was reached was different, 60.6 |ag L"1 with the effluent addition and 35.5 |ig L"1 with the NH4CI addition (Fig. 4F, 4G). The discrepancy in max chl-a could be because in the NH4CI addition experiment available
PO4 was depleted by 48 hours, whereas the effluent treatment was not PO4 depleted until
65 hours because it started with almost twice as much PO4. Comparing the chl-a accumulation that occurred before PO4 limitation and nearing stationary growth phase, the calculated growth rates when grown on effluent-NFLi compared to NH4CI were not different (1.1 d' 1 and 1.17 d '1) (Table 4). A similar lack of difference was seen between growth rates calculated using peak N-uptake rates (1.09 d’ 1 vs 1.04 d'1). Removing PO4 limitation, by experimentally adding PO4, appears to alleviate the discrepancy in peak chl-a, as seen in the enclosures experiments from October 2014 (Fig. 5A vs 5B).
Overall, NH4CI vs effluent addition enclosures were similar in terms of AChkADIN ratios (i.e. increase in chlorophyll relative to decrease in DIN). Calculated growth rates
(divisions d’1) using either chl-a accumulation or the biomass specific N-uptake rates were not different between NH4CI and effluent enclosures, indicating no detrimental effect of another unknown constituent in the effluent. However the enclosure grown on
NH4 vs NO3 were only tested at a single total DIN concentration, so this is more of a comparison of differential N-metabolism. Although there were no bulk differences in chl- a or N-metabolism between NH4CI and effluent-NH4 in enclosures, there is still potential 56
for more long term impacts, as tolerance thresholds of specific phytoplankton groups may select for different community compositions in the presence of high effluent-NfL loads
(Collos and Harrison 2014). The trace VNH4 vs saturating VNH4 in the river shows a hint of inhibition with higher NH4, where the saturating ambient VNH4 rate is lower than the trace uptake rate (Table 3) because the -100% addition meant ~90pM NH4 in the sample.
The preferential uptake of NH4 is usually attributed to the energetic favorability of a more reduced N form, with less cost in transporting across membranes and no need to reduce the NO3 to NO2 to NH4 in the cell. NH4 and NO3 are often found together in the field, and
interactions between the two nitrogen sources are well studied. NH4 inhibits NO3 uptake
through down-regulation of NO3 transporters and suppression of nitrate reductase activity
which can occur in a matter of hours (Vergara et al. 1998). Glibert et al. (2015) suggests
that the negative impacts of elevated NH4 on phytoplankton growth could be caused by
failure of cellular balancing of energy flow and redox within an algal cell. Not only does
elevated NH4 inhibit NO3 uptake, but it also reduces the dissimilatory NO3 pathway,
which helps cells dissipate excess energy produced in the photosynthetic pathways
(Lomas & Glibert 1999, Parker & Armbrust 2005). Additionally, growth on NH4 has
been shown to increase photorespiratory response to environmental stressors, especially
in diatoms (Parker & Armbrust 2005). There are many signaling pathways that help
phytoplankton balance their metabolic budgets based on light, temperature, nutrient
availability and other environmental stresses (Parker & Armbrust 2005). NO3 is generally
a positive signal for N metabolism, as NO3 substrate increases NR activity is shifted up to 57
assimilate more NO3. NH4 is a negative signal to the ammonium transporter gene expression, which may be why high concentrations of NH4 show NIHLj-uptake inhibition.
Growth difference between NO3 and either NH4 form (as NH4CI or within effluent) were minimal in this study. Enclosures from April 2015 grown on NO3+PO4 vs effluent-NH4 showed no statistical difference in total chl-a grown, total number of cells, cell size classes, divisions per day or AChl increase: ADIN drawdown. Recent comparison of local phytoplankton growth on NO3 vs NH4 found that when grown in low light, growth on
NO3 built 50% more total chl-a per cell (Glibert et al. 2014), that was apparently in fucoxanthin-containing cells (diatoms). Previous studies of phytoplankton dynamics in the northern estuary have shown that chl-a blooms exceeding 10 pg L"1 were associated with increased NO3 uptake and higher rates of carbon production (Wilkerson et al. 2006,
Parker et al. 2012a). However, the current study did not find any difference in chl-a per cell, likely because enclosures were grown in relatively high light.
4.7 Future river scenario
In combination, the river manipulation experiment and the corresponding enclosures suggest that future river conditions, with upgrades to the SWTF, will not show much change in total chl-a biomass within the stretch of river studied here, even with a large decrease in DIN and effluent-NHi. Although the reduction in NH4 may alleviate inhibition of VNO3 and allow phytoplankton to utilize existing NO3 pools, the VNT values are similar in the effluent-free and ambient conditions and VC values are also 58
similar. The fact that chl-a accumulated in experimental enclosures suggests there is potential for phytoplankton growth in the river if other limiting conditions were to
change. With nitrification technologies added to the SWTF process, NO3 will likely be the primary form of DIN in the future river, but enclosures suggest little difference in
phytoplankton growth dynamics between NO3 versus NH4.
Comparison of river phytoplankton communities provided with NO3 compared to those
with NH4 or effluent, did not show clear community shifts over the 96 hr time course. By
2021, the SWTF plans to reduce NH4 discharge limits into the river by -90%, and total
DIN will be reduced by 20-30% based on new permit regulations (CRWQCB 2013). This
means the future river will likely receive more NO3 in addition to upstream sources (the
SWTF permit max -51 pM), and the NFLt-plume in the river below the discharge site will
likely be ~5pM NH4 average (permit max 7-12pM). This is close to the residual levels of
NH4 we observed in the effluent-free river segment, where NO3 uptake inhibition was
slightly alleviated. In agreement, the effluent-free enclosure had low NH4, allowing for
NO3 uptake to also occur.
This suggests that post-2021, NO3 uptake will occur alongside NH4 uptake in this stretch
of river. However, if NH4 concentrations are not sufficiently reduced, there is potential
that VNO3 will continue to be inhibited. Overall, this study suggests that an upgrade to
the SWTF, that will result in NO3 dominated discharge and lower total DIN, may not
affect total chl-a, or total N-uptake (NH4 + NO3) by phytoplankton along this stretch of
the lower Sacramento River. However, there will be repercussions further downstream if 59
the increased NO3 discharge from the SWTF is effectively transported untouched out to the Bay, past the range of this study. Rivers contaminated with wastewater may have lower retention of nutrients, and productivity can become decoupled from the light availability and not fulfill potential for growth (Marti et al. 2004). Nutrients will likely be exported downstream into the northern estuary, especially during higher flow regimes. A phytoplankton bloom model for Suisun Bay shows that flow rates at Freeport, CA near
O 1 the SWTF discharge site need to reach 800 m s' in order to stimulate a bloom in Suisun
Bay (Dugdale et al. 2012).
4.4 Conclusions
In conclusion, the low chl-a abundance in the lower Sacramento River was not significantly changed by the temporary stoppage of SWTF effluent discharge, although
NH4 reduction did alleviate NH4 inhibition of VNO3, and allow phytoplankton to utilize
NO3. Both effluent-NFLt and NH4CI were usable for phytoplankton growth in enclosures filled with river water incubated with high light availability, low mixing/export/sinking and no benthic influence, suggesting that at 50|iM NH4, effluent did not have a more detrimental effect on phytoplankton than NH4C1. However, uptake rates in the October
2013 river where NH4 was >80|aM showed depression of VNH4 (Appendix 8 ). Growth differences between NO3 and either NH4 form were not apparent in enclosure experiments, suggesting NH4 itself is not toxic or inhibitory at the concentrations tested
in enclosure (<50|iM) and that N0 3 -rich effluent from an upgraded SWTF might not alter phytoplankton biomass immediately. This study suggests that the future lower 60
Sacramento River, with upgraded effluent treatment reducing total DIN and converting to NO3, will likely remain a low chl-a habitat like the historic chl-a records in the northern estuary indicate. However, drought conditions (i.e. increased residence time) in combination with N 0 3 -dominated DIN pools may alter potential phytoplankton growth dynamics farther downstream in the northern SFE. 61
5.0 References
Alpine A, Cloern J (1988) Phytoplankton growth rates in a light-limited environment, San Francisco Bay. Marine ecology progress series Oldendorf 44:167-173
Anderson DM, Glibert PM, Burkholder JM (2002) Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences. Estuaries 25:704-726
Arar E, Collins G (1992) In vitro determination of chlorophyll a and phaeophytin a in marine and freshwater phytoplankton by fluorescence—USEPA Method 445.0. USEPA methods for determination of chemical substances in marine and estuarine environmental samples Cincinnati, OH
Bahnwart M, Hubener T, Schubert H (1998) Downstream changes in phytoplankton composition and biomass in a lowland river-lake system (Warnow River, Germany). Hydrobiologia 391:99—111
Ball M, Arthur J (1979) Planktonic chlorophyll dynamics in the northern San Francisco Bay and Delta. San Francisco Bay: The Urbanized Estuary Pacific Division, American Association for the Advancement of Science, San Francisco, Califomia:265-285
Berg G, Balode M, Purina I, Bekere S, Bechemin C, Maestrini S (2003) Plankton community composition in relation to availability and uptake of oxidized and reduced nitrogen. Aquatic Microbial Ecology 30:263-274
Berggreen U, Hansen B, Kiorboe T (1988) Food size spectra, ingestion and growth of the copepodAcartia tonsa during development: Implications for determination of copepod production. Marine biology 99:341-352
Berman T, Sherr B, Sherr E, Wynne D, McCarthy JJ (1984) The characteristics of ammonium and nitrate uptake by phytoplankton in Lake Kinneretl. Limnology and Oceanography 29:287-297
Bran Luebbe Inc. (1999a) Bran Luebbe Autoanalyzer Applications: Auto Analyzer Method No. G-177-96 Silicate in water and seawater. Bran Luebbe, Inc., Buffalo Grove, IL
Bran Luebbe Inc. (1999b) Bran Luebbe Autoanalyzer Applications: AutoAnalyzer Method No. G-175-96 Phosphate in Water and Seawater. Bran Luebbe, Inc., Buffalo Grove, IL 62
Bran Luebbe Inc. (1999c) Bran Luebbe Autoanalyzer Applications: AutoAnalyzer Method No. G-172-96 Nitrate and Nitrite in Water and Seawater. Bran Luebbe, Inc., Buffalo Grove, IL
Brett M, Muller-Navarra D (1997) The role of highly unsaturated fatty acids in aquatic foodweb processes. Freshwater Biology 38:483-499
Bulgakov N, Levich A (1999) The nitrogen: Phosphorus ratio as a factor regulating phytoplankton community structure: Nutrient ratios. Archiv fur hydrobiologie 146:3-22
Chase J, Knight T (2006) Effects of eutrophication and snails on Eurasian watermilfoil (Myriophyllum spicatum) invasion. Biological Invasions 8:1643-1649
Chervin MB, Malone TC, Neale PJ (1981) Interactions between suspended organic matter and copepod grazing in the plume of the Hudson River. Estuarine, Coastal and Shelf Science 13:169-183
Cloern J (2001) Our evolving conceptual model of the coastal eutrophication problem. Marine ecology progress series 210:223-253
Cloern J, Jassby A (2012) Drivers of change in estuarine-coastal ecosystems: Discoveries from four decades of study in San Francisco Bay. Reviews of Geophysics 50:n/a- n/a
Cochlan W, Harrison P (1991) Inhibition of nitrate uptake by ammonium and urea in the eucaryotic picoflagellate Micromonas pusilla (Butcher) Manton et Parke. Journal of Experimental Marine Biology and Ecology 153:143-152
Cohen AN, Carlton JT (1998) Accelerating invasion rate in a highly invaded estuary. Science 279:555-558
Cohen RR, Dresler PV, Phillips EJ, Cory RL (1984) The effect of the Asiatic clam, Corbicula fluminea, on phytoplankton of the Potomac River, Maryland. Limnology and Oceanography 29:170-180
Cole B, Cloern J (1984) Significance of biomass and light availability to phytoplankton productivity in San Francisco Bay. Marine ecology progress series Oldendorf 17:15-24
Collos Y, Harrison P (2014) Acclimation and toxicity of high ammonium concentrations to unicellular algae. Marine Pollution Bulletin 80:8 - 23 63
Collos Y, Maestrini S, Robert J-M (1989) High long-term nitrate uptake by oyster-pond microalgae in the presence of high ammonium concentrations. Limnology and Oceanography 34:957-964
Conway H, Harrison P (1977) Marine diatoms grown in chemostats under silicate or ammonium limitation. IV. Transient response of Chaetoceros debilis, Skeletonema costatum, and Thalassiosira gravida to a single addition of the limiting nutrient. Marine Biology 43:33—43
Cox T, Maris T, Soetaert K, Conley D, Damme S, Meire P, Middelburg J, Vos M, Struyf E (2009) A macro-tidal freshwater ecosystem recovering from hypereutrophication: the Schelde case study. Biogeosciences 6
CRWQCB (2013) ORDER R5-2013-0124 “Wastewater discharge requirements.”
CytoBuoy b.v. CytoBuoy CytoSense. www.cytobuoy.com
Davis CO (1982) The importance of understanding phytoplankton life strategies in the design of enclosure experiments. In: Marine mesocosms. Springer, p 323-332
Diaz R (2001) Overview of hypoxia around the world. Journal of environmental quality 30:275-281
Dodds W, Bouska W, Eitzmann J, Pilger T, Pitts K, Riley A, Schloesser J, Thombrugh D (2008) Eutrophication of US freshwaters: analysis of potential economic damages. Environmental Science & Technology 43:12-19
Dortch Q (1990) The interaction between ammonium and nitrate uptake in phytoplankton. Marine ecology progress series Oldendorf 61:183-201
Dortch Q, Conway H (1984) Interactions between nitrate and ammonium uptake: variation with growth rate, nitrogen source and species. Marine Biology 79:151- 164
Dubelaar GBJ, Groenewegen AC, Stokdijk W, Den Engh GJ Van, Visser JWM (1989) Optical plankton analyser: A flow cytometer for plankton analysis, II: Specifications. Cytometry 10:529-539
Dubelaar G, Tangen K, Gerritzen P, Beeker A, Jonker R (1998) CytoBuoy: In situ optical scanning of individual particles with a buoy mounted flow cytometer. In: Third European Marine Science and Technology Conference, Lisbon.p 23-27 64
Dugdale R, Wilkerson F (1986) The use of 15N to measure nitrogen uptake in eutrophic oceans; experimental considerations 1, 2. Limnology and Oceanography 31:673- 689
Dugdale RC, Wilkerson FP, Hogue VE, Marchi A (2007) The role of ammonium and nitrate in spring bloom development in San Francisco Bay. Estuarine, Coastal and Shelf Science 73:17-29
Dugdale R, Wilkerson F, Parker A, Marchi A, Taberski K (2012) Anthropogenic ammonium impacts spring phytoplankton blooms in the San Francisco Estuary: the cause of blooms in 2000 and 2010. Estuarine and Coastal Shelf Science Submitted for publication
Elser JJ, Marzolf ER, Goldman CR (1990) Phosphorus and nitrogen limitation of phytoplankton growth in the freshwaters of North America: a review and critique of experimental enrichments. Canadian Journal of fisheries and aquatic sciences 47:1468-1477
Eppley R, Renger E, Williams P (1976) Chlorine reactions with seawater constituents and the inhibition of photosynthesis of natural marine phytoplankton. Estuarine and Coastal Marine Science 4:147-161
Erickson RJ (1985) An evaluation of mathematical models for the effects of pH and temperature on ammonia toxicity to aquatic organisms. Water Research 19:1047— 1058
Esparza ML, Farrell AE, Craig DJ, Swanson C, Dhaliwal BS, Berg GM (2014) Impact of atypical ammonium concentrations on phytoplankton abundance and composition in fresh versus estuarine waters. AQUATIC BIOLOGY 21:191-204
Fisher T, Hagy III J, Boynton W, Williams M, others (2006) Cultural eutrophication in the Choptank and Patuxent estuaries of Chesapeake Bay. Limnology and Oceanography 51:435^147
Foe C, Ballard A, Fong S (2010) Nutrient Concentrations and Biological Effects in the Sacramento-San Joaquin Delta. Central Valley Regional Water Quality Control Board
Glibert PM (2010) Long-term changes in nutrient loading and stoichiometry and their relationships with changes in the food web and dominant pelagic fish species in the San Francisco Estuary, California. Reviews in Fisheries Science 18:211-232 65
Glibert PM, Conley DJ, Fisher TR, Harding Jr LW, Malone T (1995) Dynamics of the 1990 winter/spring bloom in Chesapeake Bay. Marine Ecology-Progress Series 122:27-43
Glibert PM, Dugdale RC, Wilkerson F, Parker AE, Alexander J, Antell E, Blaser S, Johnson A, Lee J, Lee T, others (2014) Major-but rare-spring blooms in 2014 in San Francisco Bay Delta, California, a result of the long-term drought, increased residence time, and altered nutrient loads and forms. Journal of Experimental Marine Biology and Ecology 460:8-18
Glibert PM, Fullerton D, Burkholder JM, Cornwell JC, Kana TM (2011) Ecological stoichiometry, biogeochemical cycling, invasive species, and aquatic food webs: San Francisco estuary and comparative systems. Reviews in Fisheries Science 19:358^117
Glibert PM, Wilkerson FP, Dugdale RC, Parker AE, Alexander J, Blaser S, Murasko S (2014) Phytoplankton communities from San Francisco Bay Delta respond differently to oxidized and reduced nitrogen substrates—even under conditions that would otherwise suggest nitrogen sufficiency. Frontiers in Marine Science 1:17
Glibert P, Wilkerson F, Dugdale R, Raven J, Dupont C, Leavitt P, Parker A, Burkholder JM, Kana TM (2015) Pluses and minuses of ammonium and nitrate uptake and assimilation by phytoplankton and implications for productivity and community composition, with emphasis on nitrogen-enriched conditions. Limnology and Oceanography in review
Hautier Y, Niklaus PA, Hector A (2009) Competition for light causes plant biodiversity loss after eutrophication. Science 324:636-638
Heil CA, Revilla M, Glibert PM, Murasko S (2007) Nutrient quality drives differential phytoplankton community composition on the southwest Florida shelf. Limnology and Oceanography 52:1067-1078
Heisler J, Glibert PM, Burkholder JM, Anderson DM, Cochlan W, Dennison WC, Dortch Q, Gobler CJ, Heil CA, Humphries E, others (2008) Eutrophication and harmful algal blooms: a scientific consensus. Harmful algae 8:3-13
Holm-Hansen O, Riemann B (1978) Chlorophyll a determination: improvements in methodology. Oikos:438^147 66
Jassby AD, Cloem JE, Cole BE (2002) Annual primary production: patterns and mechanisms of change in a nutrient-rich tidal ecosystem. Limnology and Oceanography 47:698-712
Kimmerer WJ, Parker AE, Lidstrom UE, Carpenter EJ (2012) Short-term and interannual variability in primary production in the low-salinity zone of the San Francisco Estuary. Estuaries and Coasts 35:913-929
Kress, E, Parker, AE, Wilkerson, FP, Dugdale, RC (2012) Assessing phytoplankton communities in the Sacramento and San Joaquin Rivers using microscopic and indirect analytical approaches. Interagency Ecological Program Newsletter 25
Kudela RM, Lucas AJ, Hayashi K, Howard M, McLaughlin K (2015) Death from below: Investigation of inhibitory factors in bloom development during a wastewater effluent diversion. Estuarine, Coastal and Shelf Science
Latasa M, Landry MR, Louise S, Bidigare RR (1997) Pigment specific growth and grazing rates of phytoplankton in the central Equatorial Pacific. Limnology and Oceanography 42:289-298
Legendre L, Gosselin M (1997) Estimation of N or C uptake rates by phytoplankton using 15N or 13C: revisiting the usual computation formulae. Journal of Plankton Research 19:263-271
Lehman P (2000) The influence of climate on phytoplankton community biomass in San Francisco Bay Estuary. Limnology and Oceanography 45:580-590
Lehman P (2004) The influence of climate on mechanistic pathways that affect lower food web production in northern San Francisco Bay Estuary. Estuaries 27:311- 324
Leland HV (2003) The influence of water depth and flow regime on phytoplankton biomass and community structure in a shallow, lowland river. Hydrobiologia 506:247-255
Lidstrom UE (2009) Primary production, biomass and species composition of phytoplankton in the low salinity zone of the northern San Francisco Estuary. San Francisco State University
Liu B, Swart HE de (2015) Impact of river discharge on phytoplankton bloom dynamics in eutrophic estuaries: A model study. Journal of Marine Systems 67
Livingston RJ, Prasad AK, Niu X, McGlynn SE (2002) Effects of ammonia in pulp mill effluents on estuarine phytoplankton assemblages: field descriptive and experimental results. Aquatic Botany 74:343 - 367
Lomas M, Glibert P (1999) Interactions between NH+ 4 and NO- 3 uptake and assimilation: comparison of diatoms and dinoflagellates at several growth temperatures. Marine Biology 133:541-551
Lomas MW, Glibert PM (2000) Comparisons of nitrate uptake, storage, and reduction in marine diatoms and flagellates. Journal of Phycology 36:903-913
Ma Y, Li G, Li J, Zhou H, Jiang B (2014) Seasonal succession of phytoplankton community and its relationship with environmental factors of North Temperate Zone water of the Zhalong Wetland, in China. Ecotoxicology 23:618 - 625
Maclsaac J, Dugdale R, Huntsman S, Conway H (1979) The effect of sewage on uptake of inorganic nitrogen and carbon by natural populations of marine phytoplankton. Journal of Marine Research 37
Marti E, Aumatell J, Gode L, Poch M, Sabater F (2004) Nutrient retention efficiency in streams receiving inputs from wastewater treatment plants. Journal of Environmental Quality 33:285-293
Miiller-Solger AB, Jassby AD, Miiller-Navarra DC (2002) Nutritional quality of food resources for zooplankton (Daphnia) in a tidal freshwater system (Sacramento- San Joaquin River Delta). Limnology and Oceanography 47:1468-1476
Munk WH, Riley GA (1952) Absorption of Nutrients by Aquatic Plants.
Nieuwenhuyse EE Van (2007) Response of summer chlorophyll concentration to reduced total phosphorus concentration in the Rhine River (Netherlands) and the Sacramento-San Joaquin Delta (California, USA). Canadian Journal of Fisheries and Aquatic Sciences 64:1529-1542
Ning X, Cloern JE, Cole BE (2000) Spatial and temporal variability of picocyanobacteria Synechococcus sp. in San Francisco Bay. Limnology and oceanography 45:695- 702
Odum HT (1956) Primary Production in Flowing Watersl. Limnology and oceanography 1:102-117
Parker AE (2005) Differential supply of autochthonous organic carbon and nitrogen to the microbial loop in the Delaware Estuary. Estuaries 28:856-867 68
Parker MS, Armbrust E (2005) Synergistic effects of light, temperature, and nitrogen source on transcription of genes for carbon and nitrogen metabolism in the centric diatom Thalassiosira pseudonana (Bacillariophyceae). Journal of Phycology 41:1142-1153
Parker AE, Dugdale RC, Wilkerson FP (2012a) Elevated ammonium concentrations from wastewater discharge depress primary productivity in the Sacramento River and the northern San Francisco Estuary. Marine Pollution Bulletin 64:574-586
Parker A, Fuller J, Dugdale R (2006) Estimating dissolved inorganic carbon concentrations from salinity in San Francisco Bay for use in 14C-primary production studies. Interagency Ecological Program for the San Francisco Estuary 19:17-22
Parker AE, Hogue VE, Wilkerson FP, Dugdale RC (2012b) The effect of inorganic nitrogen speciation on primary production in the San Francisco Estuary. Estuarine, Coastal and Shelf Science 104:91-101
Parker AE, Marchi AM, Davidson-Drexel J, Dugdale RC, Wilkerson FP (2010) Effect of Ammonium and Wastewater Effluent on Riverine Phytoplankton in the Sacramento River, CA. Final Report to the State Water Resources Control Board 73Pps
Pennock JR (1987) Temporal and spatial variability in phytoplankton ammonium and nitrate uptake in the Delaware Estuary. Estuarine, Coastal and Shelf Science 24:841 -8 5 7
Reddy N, Naidu S, Sridevi B, Venkataramana V, Kumar B, DileepKumar M, Bandyopadhyay D, Sarma V, Acharyya T, Bharathi M, others (2015) Reduced river discharge intensifies phytoplankton bloom in Godavari estuary, India.
Ryther JH, Dunstan WM (1971) Nitrogen, phosphorus, and eutrophication in the coastal marine environment. Science 171:1008-1013
Scherwass A, Bergfeld T, Schol A, Weitere M, Arndt H (2010) Changes in the plankton community along the length of the River Rhine: Lagrangian sampling during a spring situation. Journal of Plankton Research 32:491-502
Schluter L (1998) The influence of nutrient addition on growth rates of phytoplankton groups, and microzooplankton grazing rates in a mesocosm experiment. Journal of Experimental Marine Biology and Ecology 228:53-71
Sharp JH (2001) Marine and aquatic communities, stress from eutrophication. Encyclopedia of Biodiversity 4:1-11
# 69
Sharp JH, Yoshiyama K, Parker AE, Schwartz MC, Curless SE, Beauregard AY, Ossolinski JE, Davis AR (2009) A biogeochemical view of estuarine eutrophication: seasonal and spatial trends and correlations in the Delaware Estuary. Estuaries and Coasts 32:1023-1043
Short FT, Burdick DM (1996) Quantifying eelgrass habitat loss in relation to housing development and nitrogen loading in Waquoit Bay, Massachusetts. Estuaries 19:730-739
Soballe DM, Kimmel BL (1987) A Large-Scale Comparison of Factors Influencing Phytoplankton Abundance in Rivers, Lakes, and Impoundments. Ecology 6 8 :pp. 1943-1954
Solorzano L, others (1969) Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol Oceanogr 14:799-801
Sommer T, Armor C, Baxter R, Breuer R, Brown L, Chotkowski M, Culberson S, Feyrer F, Gingras M, Herbold B, others (2007) The collapse of pelagic fishes in the upper San Francisco Estuary: El colapso de los peces pelagicos en la cabecera del Estuario San Francisco. Fisheries 32:270-277
Tada K, Suksomjit M, Ichimi K, Funaki Y, Montani S, Yamada M, Harrison PJ (2009) Diatoms grow faster using ammonium in rapidly flushed eutrophic Dokai Bay, Japan. Journal of oceanography 65:885-891
Thompson PA, Levasseur ME, Harrison PJ (1989) Light-limited growth on ammonium vs. nitrate: What is the advantage for marine phytoplankton? Limnology and oceanography 34:1014-1024
Vergara JJ, Berges JA, Falkowski PG (1998) Diel periodicity of nitrate reductase activity and protein levels in the marine diatom Thalassiosira weissflogii (Bacillariophyceae). Journal of Phycology 34:952-961
Waiser MJ, Tumber V, Holm J (2011) Effluent-dominated streams. Part 1: Presence and effects of excess nitrogen and phosphorus in Wascana Creek, Saskatchewan, Canada. Environmental Toxicology and Chemistry 30:496—507
Welch EB (2002) Ecological Effects of Waste Water: Applied limnology and pollutant effects. CRC Press
Whitledge TE, Malloy SC, Patton CJ, Wirick CD (1981) Automated nutrient analyses in seawater. Brookhaven National Lab., Upton, NY (USA) 70
Wilkerson FP, Dugdale RC, Hogue VE, Marchi A (2006) Phytoplankton blooms and nitrogen productivity in San Francisco Bay. Estuaries and Coasts 29:401-416
Wilkerson F, Dugdale R, Parker A, Blaser S, Pimenta A (2015) Nutrient uptake and primary productivity in an urban estuary: using rate measurements to evaluate phytoplankton response to different hydrological and nutrient conditions. Aquatic Ecology: 1-23
Xu J, Glibert PM, Liu H, Yin K, Yuan X, Chfen M, Harrison PJ (2012) Nitrogen sources and rates of phytoplankton uptake in different regions of Hong Kong waters in summer. Estuaries and coasts 35:559-571
Yoshiyama K, Sharp JH (2006) Phytoplankton response to nutrient enrichment in an urbanized estuary: Apparent inhibition of primary production by overeutrophication. Limnology and Oceanography 51:424—434 6.0 Figures and Tables
Sacramento- San Joaquin x Delta
Sacramento
Stan's Ybk? NORTH ^Freeport \ Marina NOT TO SCALE Sacramento RM46.32 Regjona| Sanitation District Outfall SWTF
M(CMA)d
Dead Morv*
Kyt*
$01 *0«6 Rio Vista C*?v*l Ranch Isleton tract
Figure 1. Study site on the lower Sacramento River, CA. Outfall of the Sacramento Regional Sanitation District (SWTF) is marked with a star near Freeport, CA. 72
B
1.8 TRCSAT N03 V 1.6 i TRC:SAT NH4 V 1.4 saturated 1:1 1.2
< - - — — • 0.4 0.2 .n.d. n d n d 0 I , d. § to cn o m UD oo rsi fNI rsi rsi rsi rsi rsi Elapsed time (hours) Figure 2. Ratio of biomass specific uptake rates using trace or saturating levels of l5N 0 3 or l5NH4 from the May 2014 in situ river manipulation experiment. A) ratios for each lagrangian sampling time in the effluent-free experimental river, B) ratios over time in the ambient river that contains typical effluent concentrations. Dashed line indicates 1:1 ratio where river is saturated with respect to nitrogen uptake. Star at Ohr is effluent discharge location, n.d. indicates no data collected. 73 A Ambient D Ambient + N 0 3 1.2 1 N03 1.2 i 60 15NH4 15N03 0.8 40 *4 avg 13 C 0.8 40 !>. •oa > *o 0.4 > 20 0.4 20 0 Effluent-free E Effluent-free + N 0 3 0.8 30 C Effluent-free + effluent F Effluent-free + effluent + N 03 0.8 60 1 i 60 Elapsed time (hours) Effluent-free + NH4Cl + NO, Figure 3. Enclosures from the May 2014 in situ river G manipulation experiment with ambient river source water 0.8 (A, D) and effluent-free river source water (B,E,C,F,G). Experimental nitrogen additions included +30|iM KNO 3 • (D, E), +50|iM effluent-NH4 (C), +30^M KN03 an d . + 50 jiM effluent-NH4 (F) and +30 jliM K N 0 3 and + 50fiM ' NH 4 C1(G). Background nutrient concentrations plotted 0.2 |iM or N03 NH4 NH4orN03MM or N03|iM NH4 or N03|JifV! NH4 versus elapsed time for NH4 and NO^ (shaded dark grey and striped). Saturating biomass specific (V) uptake rates for NH4, NO^ (triangles and diamonds) and trace carbon Elapsed time (hours) uptake (line). 74 A Ambient D Ambient + N 0 3 60 3 Ar "d Ar "d /r »d r "Od wrt i/yri »od lAlri "Od 2 lAlrt "Od §20 1 0 C Effluent-free + effluent F Effluent-free + effluent + N 03 0 50 100 Elapsed time (Hours) G Effluent-free + NH4C1 + NO, Figure 4. Enclosures from the May 2014 in situ 60 3 river manipulation experiment. Enclosures included ambient river source water (A, D) and effluent-free river source water (B,E,D,F,G). Experimental nitrogen additions included +30|iM KN03 (D, E), +50^M effluent-NH4 (C), +30|liM KN03 and +50^M effluent-NH4 (F) and +30jliM K N 03 and + 50|iiM NH4 Cl (G). Total chl-a (closed circles), <5jnm chl-a (open circles) 0 50 100 and PO, (asterisks) plotted versus elapsed time. Elapsed time (Hours) \i N\ enclosures using naturally effluent-free upstream river source water from Stan’s from water source river upstream effluent-free naturally using enclosures 5. Figure Yolo Marina (SYM) near the Garcia Bend sampling station. Mean Mean station. sampling Bend Garcia the near (SYM) Marina Yolo P04 or N02 P04 or N02 mM sym+no C A A SYM 4 8 2 6 120 96 72 48 24 I Nutrient concentration versus elapsed time in additional replicated replicated additional in time elapsed versus concentration Nutrient Elapsed time (hours) time Elapsed + NH4 + PO4 + NH4 + ” 1 3 , +pc >4 M*. -M • N02 N03 NH4 P04 April 2015 April 40 60 20 October 2014 October t o’ f 0 a a 40 60 20 2 2 Z Z 2 o O a Z o £ ju 3 s s § ? o O & o N T 2 D 2 B 4 2 SYM + Effluent SYM + Effluent + SYM 2 4 7 9 120 96 72 48 24 0 24 Elapsed time (hours) time Elapsed ‘S. 872 48 I ± JL 95% Cl (n=3). Cl 95% 96 120 60 40 20 40 h 60 20 z O 75 NH4orN03yM effluent-free upstream river source water from Stan’s Yolo Marina (SYM) near the the near (SYM) Marina YoloStan’s from water source river upstream effluent-free Figure aca ed apig tto. oa cla sld ie ad lw yoer cell cytometry flow and (n=3). naturally 95%CI ± line) Mean enclosure. using (solid 120hr experimental the chl-a over line) enclosures Total(dashed count station. replicated sampling Bend additional Garcia in time elapsed versus 1) L Cell count xlO7 cells L1 Cell count xlO'cells L1 16 12 16 12 0 4 8 A A SYM+NO C SYM + NH4C1+P04 SYM 6 . Chlorophyll concentration (|j.g L'1) and number of fluorescing cells (cells (cells cells fluorescing of (|j.gnumber L'1)and concentration Chlorophyll . 4 8 2 6 120 96 72 48 24 -Chl-a Elapsed time (hours) time Elapsed »Chl-a • Ceil count lpe tm (hours) time Elapsed Cell count 3 +PO 4 coe 2014 October 60 30 60 April 2015 April 5 o D SYM + Effluent Effluent + SYM D B 6 ; 16 16 SYM + Effluent Effluent + SYM Elapsed time (hours) time Elapsed Elapsed time (hours) time Elapsed “Cell count 60 30 0 g 30 60 76 9 77 October 2014 A to B T9 6 3.E+03 4.E+04 2.E+03 NH4 + P04 * Effluent 2.E+04 l.E+03 O.E+OO ■ ■ ■ O.E+OO 4 ■ ■ i - i . . y y y y y y y y y -/ y y / / / y \ April 2015 TO D 196 3.E+03 N03 + P04 (Effluent 4.E+04 2.E+03 2.E+04 l.E+03 O.E+OO ( i O.E+OO i 111 i /V v<<> /O v<^ ^ v<<> o- ^ ^ * *> v* ^ sr^ v^ '° j r J * j ? JF ^ - r « Figure 7. Number of fluorescing cells of different size classes observed at TO and T96 in additional replicated enclosures using naturally effluent-free upstream river source water from Stan’s Yolo Marina (SYM) near the Garcia Bend sampling station. Size classes counted using forward scatter from a CytoBuoy flow cytometer. Error bars are 95% confidence intervals (n=3). c Table. 1 Summary of enclosure experiments by date, including source water and nutrient additions. SYM = Stan’s Yolo Marina. Fijju re Date Source water Treatment Additions A May-14 Ambient %A D May-14 Ambient 30piM N 0 3 uCD 3 B May-14 Effluent-free O u c E May-14 Effluent-free 30pM N 0 3 lu (D.(D OJ C May-14 Effluent-free 50nM Effluent > cn F May-14 Effluent-free SOiiM Effluent 30piM N 0 3 G May-14 Effluent-free 50pM NH4CI 30|iM NOs A Oct-14 Upstream - SYM 50pM NH4CI 1.7|iM P04 B Oct-14 Upstream - SYM 50p.M Effluent c © . © . © e C Apr-15 Upstream - SYM 50pM N03 1.7nM P04 D Apr-15 Upstream - SYM 50|iM Effluent OO .. - Table. 2 Summary of mean river conditions during the 2014 river manipulation in the combined upstream samples from both river segments, in the downstream ambient river and the downstream experimental effluent- free river. Mean ± sd. Welch’s t-test for statistical significance (p<0.05). Temp T-Test NH4 T-Test NO3 T-Test NO 2 T-Test Chi -a T-Test DIC T-Test POC T-Test PON T-Test C pM }iM piM UgL'1 }iM samples (n) Upstream 7-8 21.4 ±0.45 a 0.88 ±0.42 a 1.23 ±1.45 a 0.05 ±0.01 a 10.8 ±5.3 a 989 ±97 a, b 115 ±22.6 a 8 ±2.1 a Ambient 4-7 22.1 ±0.68 b 50.63 ±5.65 b 6.1 ±3.55 b 0.9 ±0.83 b 4.7 ±1.8 b 1064 ±74 a 99 ±10.9 a 7 ±1.6 a Effluent-free 7-8 21.9 ±0.25 b 3.24 ±2.85 c 2.88 ±1.02 b 0.19 ±0.17 b 3.6 ±0.9 b 886 ±110 b 93 ±10.2 a 6 ±0.9 a -J uptake are also given. Mean ± sd. Welch’s t-test for statistical significance (p<0.05). significance statistical for Welch’ssd.± t-test Mean given. also are uptake Table. 3Table. Uptake Rho Biomass specific V carbon and (VNT) rates nitrogen Sum (p). volume per rates anduptake (V) rates specific biomass for shown (nmol L 1 d 1± SO) ( d ’ iS O ) ffluent-free E EffJuent-free U pstream 7 0.17 t 0.14 t 0.17 7 pstream U U pstream 7 0.91 ± 0.57 ± 0.91 7 pstream U e t ien b m A bient m A Uptake rates from the May 2014 river manipulation experiment. Saturating and trace uptake rates are rates uptake trace and Saturating experiment. manipulation river 2014May the from rates Uptake mples p am s M 4 .9±03 b 0.36 ± 2.39 4 7 7 m e a n t SD t n a e m .1±00 b 0.07 ± 0.51 1.64 ± 0.31 ± 1.64 .6±01c 0.1 ± 0.36 NH atwc ,6±05 c 0.51 ± 0,66 c | .8±00 a 0>05 ± 0.18 a a m e a n i SO i n a e m .3+01 .1i .8a04 +00 .3 .6a01 .7c01 .8b05 00 .5 .3b089±0. 7 .1 0 ± 0.819 b 0.43 ± 0.952 b 0.09 i 0.55 b 0.08 ± 0.19 c 0.07 ± 0.12 a 0.06 ± 0.43 a 0.02 ± 0.03 0.08 + 0.48 a 0.08 i 0.21 b 0.11 + 0.13 1.18 + 0.24 + 1.18 0.004 ± 0.004 NOsiac 0.003 b 30 57 .9 .7a 0.77 + 2.09 b a 5.75 i 23.04 a 2 m e a n i SD i n a e m 65 .9a24 i .8a 0.38 i 2.42 a 5.09 1 16.56 0.19 + 0.04 + 0.19 0.06 + 0.25 16.4 + 6.18 + 16.4 Carbon t » c a 2.29 2.29 a a 0.36 ± 0.17 a 0.46 + 0.05 + 0.46 a a 0.17 ± 0.36 a 1 SD i n a e m 0.51 ± 0.07 ± 0.51 sumjNOj* sumjNOj* NH«) TRC t 03 a 0.32 a | a +S S a ± SD ± ean m SD i n a e m SO + ean m .6 .6a00 +005b01 05b04 ±00 .4±02 a 0.26 ± 0.34 b 0.06 ± 0.48 b 5 .0 0 ± 0.19 b 0.005 + 0.01 a 0.06 + 0.46 .5 .9a 0.69 ± 2.75 1.74 + 0-17 + 1.74 .2±06ab00 00 73 +63 ,1 .9b 0.59 + 2,61 b 6.38 + 17.39 b 0.02 i 0.09 a,b 0.6 ± 2.52 NH* sat .3+00 3+0. 8+0. .1 .1a,b 0.31 i 0.615 a .1 0 + .8 0 a 8 .0 0 + .3 0 a 0.09 + 0.33 a b 1 .5 .4c1,7+64 .9+05 b 0.55 + 2.39 b 6.45 + 15,47 c 0.44 + 0.65 2.55 2.55 NO i ssat .6a29. 26a 6 .2 1 1 1 9 .3 9 2 a 1.06 | C arbon arbon C sat I m e a n i SD i n a e m sum {NOj ♦sum . 62a 2 .6 1 ± 5.3 NH*)SAT 1 Omeant SD t n a e m SO i n a e m TRC.-SAT NO* 2 .7 i 0.33 i 0.374 .9 t 0.09 t 1.094 TRC:SAT NHi .1 13a,b 3 .1 0 ± 1.017 b | .3 .9b 0.19 ± 1.135 c .8±01 a 0.17 ± 0.88 a SD i n a e m TRC:SAT Carbon | o oo Table. 4 Summary of 7 enclosures conducted during the 2014 river experiment, and 4 additional replicated enclosure experiments. Max V and Max p (maximum V and r observed in the enclosure) and time to reach r SAT SAT v ' Max V for C, NO^, and NH^. Initial N 0 3 and NH^ and time to NO^ exhaustion and NH4 < 1 pM also provided. Growth rates calculated from chl-a accumulation rates. Means and 96%CI provided for replicated experiments. River Enclosures SYM Replicated Enclosures effluent-free , ambient + . , effluent-free effluent-free _ effluent-free ambient effluent-free + effluent + NH4+PO4 Effluent NO3+PO4 Effluent no3 + N03 + effluent isj03 + NHdCI * N°3 Chi at start (pigL 3.03 2.59 6.81 6.68 4.16 6.31 4.54 1.19 ±0.14 1.14 ±0.22 4.13± 1.3 6.24+3.28 Chi^JngL1) 49.42 53.41 8.83 49.42 43.04 60.58 35.47 32.16+1.37 29.42 ±0.34 23.53 ±4.70 33.85 ±18.5 Time to Chlmax (hrs) 72 72 24 89 72 72 65 120 120 120 120 Cht:DlN at Chln^ 0.90 1.03 0.72 1.19 0.94 1.04 1.10 0.70 0.60 0.49 0.74 Divisions per day (n/ln2) 1.45 1.97 0.23 0.84 1.39 1.10 1.17 1.11 1.22 0.76 0.86 Initial NH4 (hM) 47.69 47.69 1.44 1.74 46.11 50.27 42.57 46.65 + 1.37 44.80 ±2.17 1.96 ±0.20 54.98 ± 2.14 Max VMTNH4(hr*) 0.63 0.64 0.54 0.51 0.66 0.74 0.71 n.d. n.d. n.d. n.d. Time to max V NH4 24 24 72 24 24 24 24 n.d. n.d. n.d. n.d. Max p NH4 14.72 16.51 4.57 5.89 13.98 13.58 8.90 n.d. n.d. n.d. n.d. Time to max p NHd 48 48 24 72 72 72 24 n.d. n.d. n.d. n.d. Initial N03(jiM) 6.065 34.654 1.89 29.11 2.20 29.09 29.48 3.43 ±0.71 2.69 ±0.01 48.61 ±0.29 2.60 ±0.07 Max N03 (hr 0.29 0.25 0.48 0.59 0.24 0.25 0.03 n.d. n.d. n.d. n.d. Time to max V N03 96 96 24 24 96 72 72 n.d. n.d. n.d. n.d. Max p NOs 11.31 12.02 4.29 7.48 9.32 8.63 1.03 n.d. n.d. n.d. n.d. Time to max p NOB 96 96 24 24 72 72 72 n.d. n.d. n.d. n.d. Initial DIC (ixM) 1201.2 1101.7 936 996 1043 993.3525 998.41 n.d. n.d. n.d. n.d. Max V C (hr1} 0.89 0.63 0.50 0.49 0.67 0.78 0.50 n.d. n.d. n.d. n.d. Time to max V C 48 48 24 24 48 48 48 n.d. n.d. n.d. n.d. Max rho C 201.64 166.70 75.66 80.41 190.27 185.36 85.73 n.d. n.d. n.d. n.d. Time to max rho C 48 96 24 24 48 48 72 n.d. n.d. n.d. n.d. ADIC:ADINT96 9 6 39 6 9 4 8 n.d. n.d. n.d. n.d. Table. 5 Replicated enclosures with starting source water from upstream of the effluent discharge site near Stan’s Yolo Marina (SYM). Data from T96 for Chl-a, cell counts, and phytoplankton functional groups. Chi total flow cytometry cell court manual cell count P .n n a.e0 adorns centric Ds atoms % Pscoplanktcn % Green algae % Cryptophytes% Other % tug I * ) (cel Is I"1) (ceils L*4! TO T96 TO T96 TO T96 TO T96 TO T96 TO T96 TO T95 TO T96 TO T96 mean * SO P value mean *50 Pvalue mean + SD P value mean * SO P value Oct-14NH4*POi 1.19*0.14 072 24.5±1.7 0.92 3.10E*0Sil.iaE-«35 0.89 5 58E+0713.98E+06 0.72 3 53E-KJ5 3.14£*07 11% 0% 1% 3% 44% 85% 4% 8% 29% 3% 13% 1% Oct-14 Effluent 1.14±0.22 2 4 .H 6 .1 3.02£*O6±7.G4£*0S S.92E+G7 + 1.38E+G7 4.18£*05 3.28E+07 9% 0% 1% 3% 4S% 89% 10% 5% 21% 3% 14% 0% ^pr-lSNOr^PO-i Apr-15 Effluent 6.24±3.28 11.3*5.6 1.01E-»07±4.30E-KS5 8.16£-»0714 36E4C7 1.26£*36 1 35E*07 5% 3% 19% 10% 24% 62% 28% 19% 16% 6% 7% 0% 83 7.0 Appendices APPENDIX 1: Results from in situ river manipulation experiment in October 2013 October 2013 Ambient C Effluent-free 100 75 5 a. 2a 50 25 0 iChl-a D 50 -DIN:P 1 U=L Elapsed time (hours) Elapsed time (hours) Figure Appendix 1.1. Nutrient, chlorophyll-a concentrations measured during the in situ river manipulation experiment in October 2013 from the ambient river segment (A,B) or the effluent-free river segment (C,D). Effluent discharge pipeline plotted as TO h elapsed time (dashed line). 84 October 2013 Ambient C Effluent-free 0.6 0.6 15N03 * 15N03 • 15NH4 ■ 15NH4 0.4 — f-ratio 4 0.4 - f-ratio *o o > .2 ^ u 0.5 w u 0 .5 1 p £ P 0.2 0.2 11 Elapsed time (hours) Elapsed time (hours) B Ambient Effluent-free 0.6 » 15N03 * 15N03 ■ 15NH4 * 15NH4 -f-ratio — f-ratio 0.4 0.4 T? > 0.5 ? > 0.5 «*- < 0.2 0.2 i Elapsed time (hours) Elapsed time (hours) Figure Appendix 1.2. Nitrogen uptake rates and f-ratios measured during the in situ river manipulation experiment in October 2013 from the ambient river segment (A,B) or the effluent-free river segment (C,D). Trace uptake rates are in the upper two panels, and saturating rates are in the lower two panels. Effluent discharge pipeline plotted as TO h elapsed time (dashed line). Appendix 1.3. Average river conditions for in situ river experiment in October 2013. Mean ± sd. Welch’s t-test used for statistical analysis (P<0.05). t* 1* U 175 i* o © o & -a o C |iM mM UM Ugl*2 }iM \xM ptM samples (n) Upstream 7 16.49 ±0.19 b 0.56 ±0.15 a 3.19 ±0.49 a 0.08 ±0.004 a 7.74 ±3.47 a n.d. n.d. 4.78 ±1.18 a Ambient 10-13 16.95 ±0.32 a 83.35 ±11.04 b 10.13 ±7.01 b 0.61 ±0.41 b 3.05 ±1.63 b n.d. n.d. 4.01 ±1.01 a,b Effluent-free 8-14 16.47 ±0.34 b 3.51 ±2.35 c 6.31 ±3.24 b 0.21 ±0.11 c 3.4 ± 1.11 b n.d. n.d. 3.45 ±0.6 b 86 Appendix 1.4. October 2013 river conditions results During the October 2013 river manipulation experiment, the California Water Year Hydrologic Classification was “dry” for the Sacramento region (http://cdec.water.ca.gov/cgi-progs/iodir/wsihist). Average tidally filtered river flow during the 5 day lagrangian river manipulation was 197.8 m3 s' 1 as measured at the Freeport, CA USGS data buoy (http://nwis.waterdata.usgs.gov/ca/nwis). Due to low water flow conditions and tidal influence, river flow was negative (upstream) three times at flood tides during the 6 day study period. Mean river temperature was 16.6°C ± 0.4 across all samples taken during the October cruise. Mean specific conductivity (pS/cm) ranged from 119.1 to 160.0 and was lowest upstream of the effluent discharge, and highest in the ambient river segment. Maximum level of fluorescence of dissolved organic matter (FDOM) was seen in the in the ambient river segment at 11.5 quinine sulfate equivalents (QSE), and averaged 9.24 ± 2.5 in the ambient river segment. (Appendix 1.3) October 2013 river nutrients Upstream of the effluent discharge site (RM46.32), the average DIN was less than 3.8pM in both the ambient and effluent-free river segments (Appendix 1A, D). The DIN pool was dominated by NO3, with a mean NO3 concentration of 3.19 ± 0.49pM, mean NH4 concentration of 0.56 ± 0.15pM and mean NO2 concentration of 0.08 ± <0.001 pM. Phosphate concentration was 0.8 ± 0.05pM. The influence of the Sanitation District effluent discharge at RM46.32 was seen clearly during the in situ sampling on the river cruises as an abrupt increase in NH4 concentration during ambient conditions that remained elevated for the duration of the study period (Appendix 1.1 A) and averaged 83.3 ± 11 pM. The elevated NH4 signal was tracked by a corresponding increase in PO4 levels (Appendix 1.1 A) averaging 3.91 ± 0.83pM, which is indicative of an effluent stream rich in both NH4 and PO4. The composite effluent 87 sample obtained from the treatment facility in during the effluent stoppage in 2013 had 2203 pM NH4, 6 pM N 0 3, and 87.7pM P 0 4 (DIN:P of 25.2). The highest NH4 concentration seen in the ambient river segment was 101.3[J.M which was 6 hours from the effluent discharge site. Average NO3 and NO2 in the ambient river segment were higher than upstream at 10.13 ± 7.01pM and 0.61 ± 0.41 pM respectively. During the effluent-free in situ river manipulation when the effluent discharge was stopped for 15 hours, total DIN in the river was drastically reduced (Appendix 1.1C). The effluent-free river segment retained low total DIN over the course of the 6 days, averaging 3.51 ± 2.35pM NH4, 6.31 ± 3.25pM NO3 and 0.21 ± 0.1 lpM NO2. Statistical analysis of average river segment nutrients showed that average NH4 and PO4 concentrations in the three river conditions (upstream, ambient and effluent-free) were statistically different from each other (all P<0.0004). Average NO3 upstream was different from both ambient and effluent-free river averages (P<0.0039). October 2013 river chlorophyll, N- uptake rates and growth Chl-a concentration was highest upstream of the effluent discharge site, but remained below 5 pg L' 1 downriver of RM46.32 in both river segments (Appendix 1 .IB, 1.1D). Average upstream chl-a was 7.74 ± 3.47 pg L'1. Both the ambient river segment and effluent-free river segments had lower average chl-a concentrations than upstream, 3.05 ± 1.63 pg L’ 1 and 3.4 ± 1.11 pg L"1, respectively, and were statistically not different from each other (P=0.52). Biomass -specific nitrogen uptake rates (trace) in the upstream samples in 2013 had similar average uptake rates for VNH4 and VNO3. Mean upstream VNH4 was 0.22 ± 0.09 d’ 1 while the mean VNO3 was 0.18 ± 0.1 d‘\ Downstream of the effluent discharge site, both river segments had higher VNH4 uptake rates than VNO3. Mean VNH4 and VNO3 downstream in the ambient river segment were 0.31 ± 0.09 d' 1 and 0.01 ± 0.02 d"1, respectively. Mean uptake rates in the effluent-free river segment for NH4 and NO3 were 0.23 ±0.12 d' 1 and 0.07 ± 0.1 d'1, respectively. The mean VNH4 were not significantly 88 different between any of the three river conditions. Mean VNO3 uptake rates upstream were significantly higher than the ambient river segments (P=0.0029), but not different from the effluent-free segment (P=0.036). Mean total N uptake rate (sum of trace VNO3 and VNH4) was slightly higher in the upstream samples, but none of the river conditions were significantly different from each other. We can use average N-uptake rates as a proxy for potential growth rate (p) for each river condition by assuming steady state growth and that N is the limiting nutrient. Using p, a doubling time can be estimated. Based on this calculation, phytoplankton should double upstream, in the ambient river and the effluent-free river in 1.7 days, 2.1 days and 2.0 days respectively. The expected chl-a increases associated with these doubling time calculations was not seen in October 2013. 89 APPENDIX 2: Results from river enclosures carried out in October 2013 B Effluent-free 0 6 NOS 1.5 NH4 N02 20 a. A 15NH4 . 0.4 ♦ 1SN03 2 Z =L D O o 15 Z z 2.5 1 10 0.2 C Effluent-free + NH4CI 0.6 0 0 50 o 50 100 Elapsed time (hours) Elapsed tim e (hours) Figure Appendix 2.1. Nutrient and chlorophyll-^ concentrations, saturating N uptake, and N:P ratio in in situ enclosure experiments conducted in Oct. 2013 using; A, D river water containing effluent; B, E effluent-free river water; C, F effluent-free source water with 40|iM NH4C1 addition. 90 Appendix 2.2 - Enclosure studies conducted during river manipulation experiment October 2013 River Enclosures During the October cruise, water was collected in the experimental effluent-free segment of river for an effluent-free enclosure (Appendix 2. IB) and an NH4C 1 addition enclosure (Appendix 2.1C). Source water was also collected from the ambient river, which had elevated levels of effluent-based NH4 already in it from normal effluent discharge into the river (Appendix 2.1 A). Total NH4 in the ambient river was higher than expected (mean 83.4 ± 11 pM), so additions of NH4 in the NH4CI addition are lower than the natural river. Ambient enclosure The enclosure filled with ambient river water (containing effluent NH4) started with 87.2pM total DIN at TO, with 83.7pM NH4, 3.4pM NO3 and 0.13pM NO2 (Appendix 2.1 A). Ammonium was never depleted to zero over the experiment, but declined continuously to 6 8 pM by the end of the 120 hour experiment. Nitrate increased by lpM after 100 hours up to 4 .4 pM. Nitrite declined over the experiment. Saturated rates of nitrogen uptake were dominated by NH4 uptake, while NO3 uptake was less than 0.005 d' '. NH4 uptake rate started at 0.22 d"1 and increased continuously to a maximum of 0.52 d' 1 by 96 hours (Appendix 2.1 A). Chl-a in the ambient enclosure at TO was 2.9 jug L'1, decreased to near zero at 50 hours and then increased to 22.3 jug L"1 at the last measurement taken at 100 hours (Appendix 2.ID). The change in chl-a from TO to maximum chl-a at T100 was 19.42 pg L"1, and using the AD IN over the same time period (TO-lOOhr) the AChl: ADIN was 0.79. Phosphate ranged between 3.4-5.3pM. DIN:P ratio ranged from 16.5-24.5. DIN:P of the sampled source effluent was 25. The divisions per day of the phytoplankton community was 0.74 d’ 1 calculated using the p extracted from an exponential curve fit to the first 99 91 hours of the chl-a data plotted over time. Similar calculation of growth rate using the sum of the trace N-uptake rates resulted in 0.75 d"1. Effluent-free enclosure At the start of the 2013 effluent-free enclosure (filled with river water collected after the effluent discharge had been turned off), NO3 was the dominant nitrogen source (3pM) while ammonium was 0.74pM, and NO2 was 0.1 pM (Appendix 2.IB). Total DIN was low, starting at 3.9pM. Available NH4 declined to ~0pM by 30 hours, and NO3 was depleted to ~0 pM by 50 hours. Saturated values of nitrogen uptake were highest for NH4 (0.31 ± 0.02 d'1), but VNO3 (0.23 ± 0.05 d'1) still occurred over the whole time course. NO3 uptake was 0.16 d"1 at TO, reached a maximum at 0.3 d' 1 after 22 hours, and declined continuously to 0.19 d' 1 over the remaining 3 days. Starting chl-a (2.5 pg L'1) increased to a maximum after 80 hours to 6 .6 pg L' 1 then declined slightly (Appendix 2.IE). Change in chl-a from TO to maximum chl-a at T80 was 4.1, and the AChl: ADIN ratio was 1.07. Phosphate declined from 0.96 to 0.37pM over 100 hours. DIN:P was <5 over the whole experiment. Divisions per day calculated from chl-a data was 0.67 d '1, and 0.93 d"1 calculated from the sum N-uptake. Effluent-free with NH4Cl addition enclosure In the NH4CI addition enclosure, starting total DIN was 38.7pM, with 35.2pM NH4, 2.9pM NO3 and 0.1 pM NO2 (Appendix 2.1C). Ammonium was never depleted to <0.5pM over the experiment, but declined continuously to reach 19.6pM NH4 after 100 hours. Nitrate only changed slightly over 100 hours, and was 3.1 pM at the end of the experiment. Similar to the ambient enclosure, saturated nitrogen uptake was dominated by NH4 uptake while NO3 uptake remained below 0.009 d'1. Ammonium uptake at TO (Appendix 2.1C) was greater than the effluent containing enclosure (Appendix 2.1 A) 0.45 d"1, with a maximum at 45 hours of 0.50 d' 1 and final uptake rate of 0.31 d' 1 by 94 hours. Chl-a was 3.0 pg L' 1 at TO, and increased to 18.1 pg L' 1 by 100 hours (Appendix 92 2.IF). Change in chl-a from TO to maximum chl-a at T94 was 16.1, and the AChl: ADIN was 1.07. Phosphate was 0.85pM at TO and declined to 0.23 pM by 100 hours. DIN:P ratio ranged from ~45-132 and showed an increasing trend over time. Divisions per day from the exponential equation fit from the chl-a data was 0.78 d'1, and 0.73 d' 1 calculated from the N-uptake rates. 93 APPENDIX 3: Results from in situ river manipulation experiment and enclosures carried out in May 2014 May 2014 Ambient D Effluent-free 100 I *M«* MMM i M i l i t mm I.--. Vi CO K Chl-a i Chl-a -DIN:P »DiN:P Elapsed time (hours) Elapsed time (hours) Figure Appendix 3.1. Nutrient, chlorophyll-# concentrations measured during the in situ river manipulation experiment in May 2014 from the ambient river segment (A,B) or the effluent-free river segment (C,D). Effluent discharge pipeline plotted as TO h elapsed time (dashed line). Approximate physical location are marked (SYM=Stan’s Yolo Marina, CLK=Clarksburg, CRT=Cortland, WGR=Walnut Grove, ISL=Isleton). 94 May 2014 Ambient Effluent-free C it 15N03 » 15NH4 15N03 — f ratio 0.6 m 15NH4 0.6 • avg 13C - f ratio • avg 13C 0.4 0.4 o ”o “ L 0.5 *8 u> 0.5 e I 0.2 0.2 1 |l i i i * - 1 m t 0 j * un ro o (N Eiapsed time (hours) Elapsed time (hours) Figure Appendix 3.2. Nitrogen uptake rates, carbon uptakes and f-ratios measured during the in situ river manipulation experiment in May 2014 from the ambient river segment (A,B) or the effluent-free river segment (C,D). Trace uptake rates are in the upper two panels, and saturating rates are in the lower two panels. Carbon uptakes are all measured with saturating additions. Effluent discharge pipeline plotted as TO h elapsed time (dashed line). 95 effluent-free "Hi effluent-free + N03 effluent-free + effluent 1400 — — effluent-free + eff + N03 effluent-free + NH4CI + N03 1200 ******* ambient 1000 — — ambient + N03 S 800 Q5 600 400 200 0 20 40 60 80 100 120 Elapsed Time (hours) ♦ effluent-free ■ effluent-free + N03 100 k effluent-free + effluent 90 X X effluent-free + eff + N03 80 I effluent-free + NH4CI + N03 # ambient 70 ambient + N03 60 50 40 30 20 x 10 0 % 20 40 60 80 100 120 Elapsed Time (hours) Figure Appendix 3.3. Ratio of change on DIC to change in DIN from TO. Data from the May 2014 in situ river enclosures. APPENDIX 4: River data from both 2013 and 2014 in situ river manipulations. river situ in 2014 and 2013 both from data River 4: APPENDIX May 2014 and October 2013 data from the lower Sacramento River. Sacramento lower the from data 2013 October and 2014 May iueApni . A Al VNH All A) 4.1 Appendix Figure n situ in V N 0 3 B A ie mnplto ars a ag o bevd NH observed of range a across manipulation river H uM NH4 4 tae dt n B VNO B) and data (trace) 21 Tae 15NH4 Trace 2013 ♦ 4 concentrations. Includes Includes concentrations. 3 (trace) data from the the from data (trace) 96 97 APPENDIX 5: Extra data from replicated enclosures experiments with SYM source water. August 2014 A SYM + NH4CI C SYM + Effluent 4 r 50 4 i r 50 0 24 48 72 96 120 0 24 48 72 96 Elapsed tim e (hours) Elapsed tim e (hours) July 2014 B SYM + N03 D SYM + Effluent '/////A N03 60 4 NH4 pM pM pM or N03 NH4 NO3 NO3 NH4 or NH4 24 48 72 96 0 24 48 72 96 120 Elapsed tim e (hours) Elapsed tim e (hours) Figure Appendix 5.1. Nutrient data for additional enclosures in triplicate without PO4 supplement. in triplicate without PO without triplicate in Figure Appendix 5.2. Chl-a, cell counts and DIN:P data for additional enclosures enclosures additional for data DIN:P and counts cell Chl-a, 5.2. Appendix Figure Cell count xlO? 16 B B A A sym SYM+ NH4 + kno cell count 3 4 supplement. Error bars are 95% Cl. 95% are bars Error supplement. 2S[ r : 100f* 125 r loo 150 200 0 50 uy 2014 July 75 uut 2014 August z, O 2 a. 0 § i L 5 z CL ? <30 a & l SYM + Effluent D c 16 SYM + Effluent —^—Chla D^;P C e l t c o u n t count 0 50 100 150 2 0 25 50 75 1(X) 125 a) Chl-o |ig L'* or DIN:P Chl-o Mg I > or DIN :P 98 99 A to E T96 Effluent Effluent 4.E+05 3.E+05 -j 3.E+05 s 2.E+0S w October 2014 B TO F T96 2.E+04 NH. + PO, Effluent 4.E+05 NH. + PO Effluent IE *04 3.E+05 1.E+04 ^ 3.E+05 g l.E+04 J. 2.E+0S = B-E+03 v 6.E+03 g 2.E+05 4.E+03 l.E+OS 2.E+03 5.E+04 O.E+OO 0.E+00 less than greater greater greater less than greater Sum than Sum than Sum than Sum Sum than Sum July 2Q U c TO G T96 7.E+03 n o 3 Effluent 6.£+05 NO, Effluent 6.E+03 1 5.E+05 S.E+03 1 - 4.E+05 E 4.E+03 I > 3.E+03 = 3E+05 u 2.E+03 " 2.E+0S l.E+03 ! m m l.E+OS O.E+OO m fliH O.E+OO less than greater less than greater less than greater less than greater Sum than Sum Sum than Sum Sum than Sum Sum than Sum April 2015 TO H T96 4.£+04 N0.+ PO, Effluent 4.E+05 n o 3+ p o 4 Effluent 4.E+04 3.E+0S Phycoerythrin (cryptos) 3. £+04 | 3.E+0S Phycocyanin (cyano)l s Chl-a 1 3.E+04 | 2.E+0S 2.E+04 t 2.E+0S 2.E+04 | l.E+04 j l.E+OS S.Ef03 ■ ■ § I _ — j — m less than greater than less than greater than less than greater than less than greater than Sum Sum Sum Sum Sum Sum Sum Sum Figure Appendix 5.3. Additional enclosures, flow cytometric fluorescence for two size groups at TO and T96. 100 Appendix 5.4. - Phosphate limited replicated enclosures August 2014 Experiment - NH4 CI addition vs Effluent-NH4 addition In August, an enclosure experiment was run comparing growth on NH4CI vs effluent (Appendix 11A, 11C). Starting DIN was 46.6pM and 45.1 pM in the NH4CI addition and effluent addition, respectively. Change in DIN was minimal in the first 24 hours, but then both treatments drew down the NH4 thereafter. DIN was drawn down in the NH4CI addition to 3.6 pg L"1 at 120 hours, and did not reach zero. The NO3 was not depleted, but remained above 1.1 pM for the whole experiment. The DIN in the effluent addition was depleted to zero over 96 hours. The NO3 remained near 1,3pM until 72 hours, then was drawn down to near zero. Nitrite in both enclosure treatments was always below 0.05pM. In the NH4CI addition, PO4 was less than half the PO4 in the effluent addition enclosure (0.96pM and 2.5pM, respectively)(Appendix 11 A, 11C). In the NH4CI addition, available PO4 was depleted to near zero by 48 hours, while PO4 was not depleted to near zero until 72 hours in the effluent addition. Chl-a started at 1.1 pg L' 1 and 1.3 pg L' 1 in the NH4 and effluent enclosures, respectively. Peak chl-a occurred at 96 hours in both treatments, with the NH4 enclosure reaching 45.7pg L' 1 and the effluent enclosure reaching 55.5 pg L 1. At T96 there was no significant difference in average chl-a concentrations between treatments (P=0.04). The mean AChkADIN ratio was 1.45 in the NH4CI addition, and 1.22 in the effluent addition (P=0.08). Divisions per day calculated using chl-a accumulation was 1.9 in the NH4CI addition, and 1.69 in the effluent addition (P=0.31). Flow cytometry counts showed starting cell concentrations of 1.32 xlOA6 and 1.3 x 10A6 (P=0.78), dominated by the 2-3pm size group. After 96 hours, the total cell counts were not significantly different at 9.0 xlOA7 and 9.9 xlOA7 (P=0.32). The cell size distribution changed to be dominated by 3-4pm sized cells with increases in all size classes at 96 hours. 101 July 2014 Experiment - KNO3 addition vs Effluent-NH4 addition The first set of triplicate enclosures from July 2014 compared growth on 30|aM NO3 with growth on effluent obtained during the May 2014 river cruise (Appendix 1 IB, 1 ID). Total DIN was 51^M and 44|iM in the NO3 addition enclosure and effluent addition enclosure, respectively. Change in DIN was minimal in the first 24 hours, but then both treatments drew down the DIN thereafter. In the NO3 addition, DIN was depleted over the course of 120 hours, but did not reach zero (8.3^M at 120 hours). There was a minimal amount of NH4 in this enclosure, starting at 3.5|aM and declining over the experiment. Total DIN in the effluent addition was depleted to zero by 96 hours. The NO3 in the effluent addition began at 1.9|^M, but did not begin declining until 72 hours. Nitrite in the KNO3 addition started at 0.13|iM but increased gradually to a peak of 0.48|iM at 96 hours. Nitrite in the effluent treatment started at 0.12|iM but declined to zero by 96 hours. Phosphate was more than twice as abundant in the effluent addition (2.8|iM PO4) than the NO3 addition enclosure (1.1 (J.M PO4) (Appendix 11B, 11D). IntheN0 3 addition, PO4 was depleted to zero by 72 hours, but was not depleted to zero until 96 hours in the effluent addition. Starting chlorophyll was 1.4 ± 0 . 5s d Mg L' 1 in the six July enclosures. Chlorophyll reached a maximum after 96 hours, \ig L’ 1 in the NO3 addition and 60|^g L' ‘in the effluent addition. At T96 there was no significant difference in average chl-a concentrations between treatments (P=0.05). The mean AChl:ADIN ratio was 1.23 in the KNO3 addition, and 1.34 in the effluent addition (P=0.59). Divisions per day calculated using chl-a accumulation was 1.64 d"1 in the KNO3 addition and 1.5 d’ 1 in the effluent addition (P=0.47). Flow cytometric cell counts had 1.33 xlO6 and 1.35 x 106 cells/L at the start of each treatment, and were not statistically different (P= 0.71). Size classes were similar between treatments at TO, with the majority of cells being in the 2-3 |nm size range. After 96 hours, the size distribution shifted upward in average size, and the dominant cell size was 3-4|am with increases in all size classes. Appendix Elapsed River Time n o 3+n o 2 Sample label Project Treatm ent Date.Time Latitude Longitude Mile N otes Hours n h 4 (uM) P 0 4 Si04 n o 2 Chi a T Phaeo T Chi a F Phaeo F n o 3 Total DIN DIC Sample 2 LAG 13-2 180 10/24/13 10:16 38.59899 -121.54838 approx E.T -35.00 0.424885 3.976 0.783 300.26 0.081 15.14522 -5.28406 n.d. n.d. 3.895 4.400885 n.d. Sample 7 LAG 13-2 clean (above) 10/25/13 9:07 38.53892 -121.51452 56 -18.88 0.605665 2.808 0.801 272.53 0.085 7.971169 -1.9449 n.d. n.d. 2723 3.413665 n.d. Sample 10 LAG13-2 clean (above) 10/25/13 13:51 38.51112 -121.55457 52.3 -14.15 0.707119 2.713 0.84 261.89 0.09 5.360359 -0.59274 n.d. n.d. 2.623 3.420119 n.d. Sample 12 LAG13-2 clean (above) 10/25/13 16:00 38.48795 -121.55263 50.57 -12.00 0.790243 2.832 0.74 273.07 0.082 7.063061 -0.47508 n.d. n.d. 2.75 3.622243 n.d. 6 Sample 15 LAG 13-2 clean 10/26/13 9:00 38.41685 -121.52247 42.35 5.00 1.36561 3.22 0.874 269.08 0.105 4.036035 1.165002 n.d. n.d. 3.115 4.58561 n.d. of. Table Sample 17 LAG13-2 clean 10/26/13 12:10 38.41331 -121.52108 42.12 8.17 1.215492 3.258 0.886 103.53 0.125 3.342341 1.07854 n.d. n.d. 3.133 4.473492 n.d. Sample 19 LAG 13-2 clean 10/26/13 14:16 38.41059 -121.51928 41.9 10.27 0.802573 3.087 0.829 259.59 0.077 2.9009 0.653141 n.d. n.d. 3.01 3.889573 n.d. Sample 20 LAG13-2 clean 10/26/13 16:55 38.38255 -121.51952 39.8 12.92 1.849266 3.372 0.909 269.79 0.125 4.918918 -0.41135 n.d. n.d. 3.247 5.221266 n.d. Sample 21 LAG 13-2 clean 10/27/13 9:30 38.29126 -121.56104 31.6 29.50 4.332285 4.347 0.952 269.39 0.181 3.531531 0.88935 n.d. n.d. 4.166 8.679285 n.d. Sample 23 LAG13-2 clean 10/27/13 12:30 38.30465 -121.57320 32.64 32.50 1.21874 3.534 0.923 266.08 0.107 3.468468 0.518994 n.d. n.d. 3.427 4.75274 n.d. Sample 25 LAG13-2 clean 10/27/13 14:30 38.31480 -121.57830 33.35 34.50 7.789497 5.432 1.185 262.35 0.242 2963963 0.156659 n.d. n.d. 5.19 13.2215 n.d. in situ Sample 27 LAG 13-2 clean 10/27/13 16:55 38.30302 -121.57135 32.48 36.92 7.018818 7.683 1.346 267.77 0.207 5.423422 -1.95606 n.d. n.d. 7.476 14.70182 n.d. Sample 29 LAG 13-2 clean 10/28/13 9:38 38.23844 -121.53240 25.86 53.63 2.084819 6.432 1.158 263.44 0.151 4.288287 0.219278 n.d. n.d. 6.281 8.516819 n.d. Sample 31 LAG 13-2 clean 10/28/13 12:00 38.23930 -121.52175 26.45 56.00 2.360116 8.656 1.242 262.25 0.158 3.985585 6.697344 n.d. n.d. 8.498 11.01612 n.d. ie ntin dt fo 2013. from data nutrient river Sample 35 LAG 13-2 clean 10/29/13 9:07 38.17369 -121.64950 15.38 77.12 5.180142 13.972 1.304 260.66 0.412 1.765765 2.048328 n.d. n.d. 13.56 19.15214 n.d. Sample 37 LAG 13-2 clean 10/29/13 12:10 38.16378 -121.61624 17.38 80.17 4.921304 9.42 1.248 261.12 0.309 1.576576 1.804098 n.d. n.d. 9.111 14.3413 n.d. Sample 38 LAG 13-2 clean 10/29/13 14:30 38.16900 -121.59647 18.58 82.50 2.954916 9.814 1.088 261.3 0.35 2144144 1.23653 n.d. n.d. 9.464 12.76892 n.d. Sample 39 LAG 13-2 clean 10/29/1317:27 38.16333 -121.60982 17.74 85.45 6.095565 9.009 1.25 264.7 0.347 3.279278 -0.50539 n.d. n.d. 8.662 15.10457 n.d. Sample 3 LAG13-2 WW (above) 10/24/13 11:20 38.54250 -121.51210 56.3 -18.67 0.402481 3.545 0.742 274.19 0.083 4.729729 2.898458 n.d. n.d. 3.462 3.947481 n.d. Sample 4 LAG 13-2 WW (above) 10/24/13 15:30 38.52612 -121.52909 54.62 -14.50 0.505917 3.553 0.83 276.04 0.08 7.572611 -0.9985 n.d. n.d. 3.473 4.058917 n.d. Sample 5 LAG13-2 WW (above) 10/24/13 17:30 38.51620 -121.54420 52.98 -12.50 0.450153 3.494 0.883 266.73 0.09 6.306305 0.455043 n.d. n.d. 3.404 3.944153 n.d. Sample 9 LAG13-2 WW 10/25/13 12:20 38.43476 -121.521% 44.2 6.33 101.2796 4.384 4.676 292.7 0.163 2.837837 0.196101 n.d. n.d. 4.221 105.6636 n.d. Sample 11 LAG13-2 WW 10/25/13 15:08 38.41837 -121.52405 42.49 9.13 92.00391 4.733 4.358 292.98 0.175 7.25225 -0.92432 n.d. n.d. 4.558 96.73691 n.d. Sample 13 LAG13-2 WW 10/25/13 17:40 38.38769 -121.51482 40.23 11.67 94.52513 4.859 4.253 284.95 0.21 4.162161 -1.04154 n.d. n.d. 4.649 99.38413 n.d. Sample 14 LAG 13-2 WW 10/26/1311:10 38.33205 -121.57156 34.64 29.17 63.32913 6.722 3.71 286.7 0.294 2.081081 1.386277 n.d. n.d. 6.428 70.05113 n.d. Sample 16 LAG13-2 WW 10/26/13 14:00 38.33582 -121.56689 35 32.00 76.98667 7.004 3.896 289.39 0.457 3.090089 -0.74962 n.d. n.d. 6.547 83.99067 n.d. Sample 18 LAG 13-2 WW 10/26/13 16:00 38.32308 -121.57735 33.95 34.00 76.49124 7.468 3.995 282.96 0.45 1.954954 0.645564 n.d. n.d. 7.018 83.95924 n.d. Sample 22 LAG13-2 WW 10/27/13 10:20 38.21727 -121.55731 22.88 5233 86.24583 12.973 4.076 279.9 0.807 2.144144 0.80311 n.d. n.d. 12166 99.21883 n.d. Sample 24 LAG13-2 WW 10/27/1313:54 38.24002 -121.54053 25.43 55.90 82.72273 13.802 4.112 273.77 0.878 4.288287 0.566014 n.d. n.d. 12.924 96.52473 n.d. Sample 26 LAG13-2 WW 10/27/1317:00 38.23976 -121.53835 25.55 59.00 85.04578 15.546 4.115 270.82 0.918 2.333333 0.440553 n.d. n.d. 14.628 100.5918 n.d. Sample 28 LAG 13-2 WW 10/28/13 10:45 38.17144 -121.59430 18.8 76.75 71.21208 28.75 4.757 277.36 1.035 2.522522 -0.09537 n.d. n.d. 27.715 99.96208 n.d. Sample 30 LAG 13-2 WW 10/28/13 12:53 38.18683 -121.58158 20.06 78.88 77.22338 9.428 1.985 227.35 1.161 1.576576 0.157103 n.d. n.d. 8.267 86.65138 n.d. Sample 32 LAG 13-2 WW 10/28/13 16:36 38.18576 -121.58336 19.94 82.60 77.99406 19.99 2.34 276.65 1.277 1.072072 3.782229 n.d. n.d. 18.713 97.98406 n.d. Sample 6 LAG 13-2 WW 10/25/13 9:20 38.43472 -121.51725 44.47 3.33 98.47762 3.971 4.534 274.49 0.132 4.288287 2.299692 n.d. n.d. 3.839 102.4486 n.d. Sample 1 LAG 13-2 Am Riv 10/24/13 8:50 38.59971 -121.50632 approx E.T. na 1.157416 1.35 0.17 154.53 0.034 1.255459 0.716774 n.d. n.d. 1.316 2.507416 n.d. Sample 8 LAG 13-2 above (+) 10/25/1311:11 38.45552 -121.50183 46.4 not parcel na 0.854759 2931 0.887 267.08 0.087 2.648648 1.858917 n.d. n.d. 2.844 3.785759 n.d. Sample 33 LAG 13-2 clean* 10/28/13 14:58 38.24628 -121.51142 27.25 contam wit 58.97 18.16997 11.129 2.185 267.06 0.244 6.369368 -1.51507 n.d. n.d. 10.885 29.29897 n.d. Sample 34 LAG13-2 clean* 10/28/13 17:00 38.23910 -121.52650 26.2 contam wit 61.00 24.36018 15.264 1.335 269.54 0.283 6.054053 -0.85302 n.d. n.d. 14.981 39.62418 n.d. Sample 36 LAG 13-2 MID 10/29/13 10:18 from upstn 85.00 92.96726 19.946 4.674 275.54 0.896 4.477476 -0.31665 n.d. n.d. 19.05 k 112.9133 n.d. Sample 40 LAG13-2 blank 10/29/13 17:27 85.45 0.034602 0 0.004 0.71 0 n.d. n.d. n.d. n.d. 0 0.034602 n.d. Sample 41 LAG13-2 effluent 10/28/13 0:00 na 2203.068 7.133 87.7 575.49 1.18 n.d. n.d. n.d. n.d. 5.953 2210.201 n.d. Sample 15d LAG13-2 clean (dup) 10/26/13 9:10 dup 5.17 1.216593 2.587 0.703 269.13 0.104 3.279278 1.575022 n.d. n.d. 2.483 3.803593 n.d. K>o pedx . al of Table 7. Appendix Elapsed TRACE 15N03 15N03_13 15N03_13 15NH4 15NH4 15NH4JL3 15NH4_13 avg Rho f ratio River Time 15N03 Rho 15N03 PON M- C Rho ng- 15N03_13 C POC w - Rho Hg- 15NH4 PONug- C Rho Jig- 15NH4_13 C POC H£- C avg POC avg PON Sample label Project Treatment Date.Time Mile Notes Hours V/d at/l ■t/L/d CV/d «t/L •t/L/d V/d «t/l •t/L/d CV/d at/L Sample 2 LAG13-2 180 10/24/13 10:16 approx E.T -35.00 2.325685 0.328731 8.42381 n.d. n.d. n.d. 0.96833 0.352216 3.31619 n.d. n.d. n.d. n.d. 0.706033 n.d. 5.87 Sample 7 LAG13-2 clean (above) 10/25/13 9:07 56 -18.88 0.245432 0.080812 3.164762 n.d. n.d. n.d. 1.110197 0.28867 4.479524 n.d. n.d. n.d. n.d. 0.181047 n.d. 3.822143 Sample 10 IAG13-2 clean (above) 10/25/13 13:51 52.3 -14.15 0.674184 0.15967 4.58619 n.d. n.d. n.d. 0.968267 0.255953 4.328095 n.d. n.d. n.d. n.d. 0.410474 n.d. 4.457143 Sample 12 LAG13-2 clean (above) 10/25/13 16:00 50.57 -12.00 0.210842 0.05214 4.151905 n.d. n.d. n.d. 0.588877 0.225009 2.944286 n.d. n.d. n.d. n.d. 0.263645 n.d. 3.548095 Sample 15 LAG13-2 clean 10/26/13 9:00 42.35 5.00 0.021264 0.006831 3.123333 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 3.123333 Sample 17 LAG13-2 clean 10/26/13 12:10 42.12 8.17 0.308006 0.080433 3.989524 n.d. n.d. n.d. 1.19188 0.381674 3.828571 n.d. n.d. n.d. n.d. 0.205353 n.d. 3.909048 Sample 19 LAG13-2 clean 10/26/13 14:16 41.9 10.27 0.028857 0.008451 3.429048 n.d. n.d. n.d. 0.867209 0.250461 3.949524 n.d. n.d. n.d. n.d. 0.032204 n.d. 3.689286 Sample 20 LAG13-2 dean 10/26/13 1655 39.8 12.92 0.046342 0.013436 3.472381 n.d. n.d. n.d. 0.690743 0.249337 3.158095 n.d. n.d. n.d. n.d. 0.062871 n.d. 3.315238 Sample 21 LAG13-2 clean 10/27/13 9:30 31.6 2950 0.068435 0.022749 3.042857 n.d. n.d. n.d. 1.130374 0.314924 4.241429 n.d. n.d. n.d. n.d. 0.057086 n.d. 3.642143 Sample 23 LAG13-2 clean 10/27/13 12:30 32.64 32.50 0.230005 0.056205 4.210476 n.d. n.d. n.d. 0.55522 0.206089 3 n.d. n.d. n.d. n.d. 0.292917 n.d. 3.605238 Sample 25 LAG13-2 clean 10/27/13 14:30 33.35 34.50 0.025889 0.009461 2.749524 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 2.749524 Sample 27 IAG13-2 clean 10/27/13 1655 32.48 36.92 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. in situ Sample 29 LAG13-2 clean 10/28/13 9:38 25.86 53.63 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Sample 31 LAG13-2 clean 10/28/1312:00 26.45 56.00 n.d. n.d. n.d. n.d. n.d. n.d. 0.371252 0.165133 2.449048 n.d. n.d. n.d. n.d. n.d. n.d. 2.449048 Sample 35 LAG13-2 clean 10/29/13 9:07 15.38 77.12 0.048996 0.012136 4.061905 n.d. n.d. n.d. 1.678 0.434396 4.87619 n.d. n.d. n.d. n.d. 0.028371 n.d. 4.469048 Sample 37 LAG13-2 clean 10/29/13 12:10 17.38 80.17 1.054233 0.282908 4.326667 n.d. n.d. n.d. 0.473281 0.089614 5.528571 n.d. n.d. n.d. n.d. 0.690163 n.d. 4.927619 2013. October from uptakes Trace river Sample 38 LAG13-2 clean 10/29/13 14:30 18.58 82.50 1.070375 0.227512 5.3 n.d. n.d. n.d. 0.69434 0.135998 5.47619 n.d. n.d. n.d. n.d. 0.606542 n.d. 5.388095 Sample 39 LAG13-2 clean 10/29/13 17:27 17.74 85.45 -0.02328 -0.00719 3.224144 n.d. n.d. n.d. 0.217845 0.062822 3.58 n.d. n.d. n.d. n.d. -0.11964 n.d. 3.402072 Sample 3 LAG13-2 WW (above) 10/24/13 11:20 56.3 -18.67 0.827725 0.202907 4.534762 n.d. n.d. n.d. 0.703292 0.150301 5.057143 n.d. n.d. n.d. n.d. 0540637 n.d. 4.795952 Sample 4 IAG13-2 WW (above) 10/24/13 15:30 54.62 -14.50 0.939685 0.224888 4.700476 n.d. n.d. n.d. 0.68651 0.154623 4.809524 n.d. n.d. n.d. n.d. 0.577843 n.d. 4.755 Sample 5 LAG13-2 WW (above) 10/24/13 17:30 52.98 -12.50 0.961417 0.238887 4.561905 n.d. n.d. n.d. 0.274184 0.114432 2.540952 n.d. n.d. n.d. n.d. 0.778097 n.d. 3.551429 Sample 9 IAG13-2 WW 10/25/13 12:20 44.2 6.33 0.012918 0.002112 6.12381 n.d. n.d. n.d. 1.307511 0.265081 5.671429 n.d. n.d. n.d. n.d. 0.009783 n.d. 5.897619 Sample 11 LAG13-2 WW 10/25/13 15:08 42.49 9.13 0.014373 0.002416 5.957143 n.d. n.d. n.d. 1.57979 0.320795 5.838095 n.d. n.d. n.d. n.d. 0.009016 n.d. 5.897619 Sample 13 IAG13-2 WW 10/25/13 17:40 40.23 11.67 0.021632 0.003924 5.52381 n.d. n.d. n.d. 1.449599 0.472518 3.955238 n.d. n.d. n.d. n.d. 0.014703 n.d. 4.739524 Sample 14 LAG13-2 WW 10/26/13 11:10 34.64 29.17 0.039367 0.009645 4.101429 n.d. n.d. n.d. 1.374958 0.374511 4.483333 n.d. n.d. n.d. n.d. 0.027834 n.d. 4.292381 Sample 16 LAG13-2 WW 10/26/13 14:00 35 32.00 0.011979 0.002845 4.217143 n.d. n.d. n.d. 1.100983 0.298441 4.32 n.d. n.d. n.d. n.d. 0.010763 n.d. 4.268571 Sample 18 LAG13-2 WW 10/26/13 16:00 33.95 34.00 0.046811 0.01419 3.322381 n.d. n.d. n.d. 5.952052 -1.33228 4.463333 n.d. n.d. n.d. n.d. 0.007803 n.d. 3.892857 Sample 22 LAG13-2 WW 10/27/13 10:20 22.88 52.33 0.04058 0.013892 2.941429 n.d. n.d. n.d. 1.028583 0.329697 3.716667 n.d. n.d. n.d. n.d. 0.037955 n.d. 3.329048 Sample 24 LAG13-2 WW 10/27/13 13:54 25.43 55.90 0.027696 0.007145 3.890476 n.d. n.d. n.d. 45.62593 0 4.03381 n.d. n.d. n.d. n.d. 0.000607 n.d. 3.962143 Sample 26 IAG13-2 WW 10/27/13 17:00 25.55 59.00 0.022122 0.005804 3.822381 n.d. n.d. n.d. 0.636402 0.310925 2.413333 n.d. n.d. n.d. n.d. 0.033594 n.d. 3.117857 Sample 28 LAG13-2 WW 10/28/13 10:45 18.8 76.75 0.163134 0.073069 2.317143 n.d. n.d. n.d. 0.829018 0.331221 2.984286 n.d. n.d. n.d. n.d. 0.164425 n.d. 2.650714 Sample 30 LAG13-2 WW 10/28/13 12.53 20.06 78.88 0.012187 0.00428 2.853333 n.d. n.d. n.d. 0.347049 0.112659 3.26381 n.d. n.d. n.d. n.d. 0.033926 n.d. 3.058571 Sample 32 LAG13-2 WW 10/28/13 16:36 19.94 82.60 0.038318 0.013011 2.964286 n.d. n.d. n.d. 2.108233 8.930449 2.232857 n.d. n.d. n.d. n.d. 0.017851 n.d. 2.598571 Sample 6 LAG13-2 WW 10/25/13 9:20 44.47 3.33 0.031406 0.006881 4.58 n.d. n.d. n.d. 1.362534 0.303524 5.271429 n.d. n.d. n.d. n.d. 0.022531 n.d. 4.925714 Sample 1 LAG13-2 Am Riv 10/24/13 8:50 approx E.T. na 0.038952 0.011788 3.32381 n.d. n.d. n.d. 0.445052 0.054117 8.452381 n.d. n.d. n.d. n.d. 0.080478 n.d. 5.888095 Sample 8 LAG13-2 above FB {+) 10/25/13 11:11 46.4 not parcel na 0.415401 0.111131 3.957143 n.d. n.d. n.d. 0.754792 0.170061 4.847619 n.d. n.d. n.d. n.d. 0.354985 n.d. 4.402381 Sample 33 LAG13-2 clean* 10/28/13 14:58 27.25 contam wil 58.97 0.904825 0.206472 4.880952 n.d. n.d. n.d. 0.8372% 0.190963 4.842857 n.d. n.d. n.d. n.d. 0.519381 n.d. 4.861905 Sample 34 LAG13-2 clean* 10/28/13 17:00 26.2 contam wil 61.00 0.054539 0.012254 4.478095 n.d. n.d. n.d. 1.321419 0.406886 4.037619 n.d. n.d. n.d. n.d. 0.039637 n.d. 4.257857 Sample 36 LAG13-2 ♦ o 104 Appendix 8 . Table of in situ river Saturating uptakes from October 2013. Tj •d “d “d •d ■d Tj ■d Tj •d -d -d ~d “d "d Tj Tj Tj Tj •d -d "d "d "d “d "d Tj Tj TJ “d T3 TJ "d TJ “d Tj Tj -d “d -d 8 d d c d d d d C d C c d d c c c c C Q. c d d d d d d d d d d d d d d d d d d d d c d d 60 I t'- VO ro «a- n- 00 cn rs tH § Tj Tj Tj u^ >c Tj Tj Tj Tj g ■d Tj -d o 18 CT> s 1 i s 3 s 5 in ? 3 s a 3 s g 1 C C C 3 I d d d d d d d 2 If) 1 s a LD I S s » s 8 i m d § cn a g a o o o § o o o i o o s o o d o o o o o o o o o o o o o o o o o o o o o o o o d o 0 Tj -D “d TJ “d ■d Tj Tj Tj •d Tj ■d Tj Tj Tj Tj Tj Tj Tj ■d -d Tj Tj Tj “d Tj Tj Tj “d ■d Tj Tj Tj Tj Tj Tj -d Tj -d d C c c d c d C c c d c d c c C C C d d C d d d d d d d d d d d d d d d d d d d d d f o D) > IV *d "d “d “d "d TJ -d Tj Tj Tj -6 “d -d Tj TJ Tj Tj Tj Tj Tj ■d -d Tj TJ Tj Tj Tj Tj Tj Tj Tj -d -d “d TJ “d Tj Tj -d Tj -d c d d d d c d C C C d c d c C c c C C C C d d d d d d d d d d d d d d d d d d d d c H. Tj T> “d *d Tj Tj •d Tj Tj ■d Tj -q Tj Tj Tj Tj Tj Tj Tj Tj "d ■d Tj Tj Tj Tj Tj T3 Tj Tj Tj Tj Tj Tj Tj Tj -d Tj -d 1 C d d d C C d C C c d c d C C d d C C C C d d d d d d d d d d d d d d d d d d d d d 5 1 > Tj ■D TJ Tj TJ "d -d -d TJ ■d Tj ■d Tj Tj Tj Tj T3 ■d T3 ■d "d "d *d Tj Tj Tj Tj Tj Tj TJ -d "d “d TJ Tj T3 •d Tj -d C d C C C c d c C C d C d C C C C C d d d d d d d C d d d d d d d d d d d d d d d d i l l a u % cn CT> IL 2 u 8 “d tj ~d “d Tj Tj “d Tj Tj Tj T3 ■d Tj ■d •d Tj Tj Tj “d Tj Tj Tj Tj Tj Tj Tj “d Tj Tj Tj Tj Tj -d ■d Tj Tj TJ Tj -d Tj ■d c c d d C d c C C C C d C d d c d d d d C d d d d d d d d d C d d d d d d d d d C d 1 ? iH U “d tj “d TJ TJ “d Tj Tj Tj Tj Tj -d Tj ■d -d "d *d ~6 ~o “d ■d TJ “d T3 ■d TJ Tj Tj Tj TJ Tj Tj -d ■d Tj Tj -d -d -d ~d -d d d d C C c C C C C C d C d d c c d d d d d d d C d d d d d d d d d d d d d d d d d s ' l s SSS cn cr> cn 00 cn cn cn cn CM In 2 s Tj Tj Tj VO s 8 Tj Tj -d Tj -d 8 3 S 3 S UD 9 3 VD 3 e S S rS jg r- C C d § d d d d c S JN 1 1C a 1 s s B s 5 f < 3 3S S in § s j U3 1 vq a ? cn § 3 VO 1 3 2 s r>i in in in s ! 00 tH 8 m 8S Tj Tj Tj S 3 s s Tj Tj VO -d ■d s VD S5 C d d | 2 £ 1 i 8 1 £ a 8 1 I s § d d d d d § o d & s § S 1 i s S o 8 i § 1 § 8 s a I 8 fN 1 HU o O o o o d o o o o d o o o o o o o o o o o d o o o o d o o o o co ro rN rH u> a a s Tj Tj Tj 3 T3 Tj s -d Tj Co I 8 a » a s s § s S e 8 S « if) C C C VDS d d d d d 83 1 » oj 1 s a o o r\j 8 s o l 1 o I o 8 1 | 1 § s 1 o o s o o 1 8 § I 8 8 1 8 o d o d d o d d o d o o H o o o o o o o d o d d d d o rH gj CO o ro ro 8 §8 » 8 s g s s VO 8 a VO 5? S 3 8 8 s 8 (C 8 c c cn g 8 § 00 vo oi vjd 00 s 3 W 3 a a s si »? S P: § s a rH a 3 In in ffi S s VO H3 2 E lapsed H ours T im e H i i "s CL ro E E 3 01 8 o Q. 1 5 a a c E CL I Q. CL o g 9 z ro c § 8 T3 00 up 00 00 00 cn 00 m £ ffj 3 s !S s 3 s ro § 5 00 8 S $ vo s 2 s 3 * s? J l s? 3 3 2 PI a S 3 s si § si rN »« S a 1 tc. Z VD o o O o o o in 8 8 1/1 s 8 ° IS 8 8 ° PM s ° s 8 s 8 8 s !S S a ? a 8 O "a? *aT > > I '57 o o o > 'a -a J9 0 O o 3 (TJ 1 -Q JS JQ > c 3L c c c c c c c c c c c c C c c C II c i 03 ro n> «l ro 1 ro ro TO ro | | | i | | i ro C 3 _aj _a> JU 0 ro £ 8 _0J JV _a; J3! _0J -Sf JM E •V; tc u < 01 rM fN CM fN fN rM I 3 3 3 3 3 3 3 3 3 3 3 a a a a a a a a a a a a 3 3 3 3 3 3 a a a 3 a a a a 3 3 a 3 3 o a o O o o 13 o e? e? o <5 o CJ w o e> o o 15 C3 o o o d 13 o t CL § 3 3 5 § < § 3 § 3 5 3 5 5 3 3 5 5 3 § § < 5 s § 5 5 3 3 5 5 3 § 3 *3 0 O OJ vo 00 vo o vo -2 a a rM S8 "fr to o> tH vo 00 S § 2 a» 0) 1V (V V 01 IV a; 0J Appendix 9. All data from October 2013 river enclosures. I o o> rv I a VO lD 5 i s S 3 a a UJ 1 cn § 8 173 £ q 1 . s 7 9 15 2 » » to 1 fN . o a 00 °0 o 1 9 8 . 7 8 7 | 1 9 .7 4 9 8 1 o fN 2 4.4076 78.71861 130 18.2 4 5 8 2 | 55 16.9 6 2 1 4 ) 99.532551 2 1 .2 5 1 5 l | 2 0 .902631 16.5 2 8 8 8 | o o r-i s a s a a 1 2 2 .6 6 2 3 7 1 DIN:P ra tio 1 1 0.9569771 I j j 3.1840561 | 0.254562| CT> 1 0.241433|oo | 45.778321 | 45.85984| | 44.459181 1 00 I 115.03831 I 21.511371 1 20.819221 g in TD i? S a $ C a H 1 s a 0 5 3 o 0 O 0 0 0 0 1 o o o o o o 0 d 0 d 0 0 0 f f r a tio rH JsT 8 oS h* T3 s » a 00 s C i 1 i 5 > § s » s uS I 2 * uS 00 vd i< a Oj o> cr> -d * a a o c s 1 a HI 1 s 1 0 0 5 o o o o o d O O 0 0 0 0 I 5 NH4 V /d <7» h IN " o ’o CT| » a fN 2 s lO S § § g 1 g i 1 1 O 1 $ 1 £ § S l vo 1 8 cr» % & 1 8 o I rH a s s rH X o o s a * z o o o o o o o o o o S $ S a S3 1 i s 00 s 1 s S3 a vS o o LO cn O q So 1C 1C s 3 ss s vo ts VO § m 00 1 5 LTI s 1 i 1 1 1 1 s o 1 5 s s 1 i 00 0 i 3 s 8 00 § 1 vd s s £ § 1 fN » 3 1 s a S I a o\ s E la p se d H o u rs T im e o o o o O o o o o O 0 0 O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 c c c o o o | l I % N o te s £ s 5 . £ s £ s o o 0 0 3 8 s a s g !S 8 8 a a S a s s 8 8 a s g s s S a ss 8 8 oi Od 00 00 oi 00 1 a rH 1 s 10/ 25/13 16 : l l | D a te .T im e I I 10/ 28/13 12:3o| 1 10/ 25/13 11: 111 1 10/ 25/13 16 :I l l 10/ | 25/13 18:3 0 | | 10/ 25/13 l l : l l | T e a tm e n t N a m eTr ** **• X X X X X X X X X X X X X X z z z z z z z z z z z z z z c c c c c c c c c c c c C s 5 5 5 2 2 5 5 5 5 5 ro to TO to TO TO | | | | f | t s i 1 a> a> d> 0) a> V V QJ 0) V i» 3 J t J -Q T3 “u u u u u u u T3 u T3 u i i i § i i i i i i i § § i fN fN fN P r o je c t 5 5 3 S 3 5 3 § 3 5 3 3 3 3 3 3 3 3 3 3 1 s 5 § 5 5 5 5 3 3 3 3 3 3 3 3 3 3 3 3 5 1 rH c c c c c c c c c c c c c c c c c c c c. c c c C c C c c c C C c c c c C c c c £ Jo Zrt Jo 5^ 5^ Ji tr> 0^ 5? | fN ro ^r ^r 'J- u T5 ! c C c c c c c c c c c c c c c c c c C e c c c c c c c c c c c c c c C C c iS UJ UJ LU £ UJ 1 UJ £ UJ £ 106 Appendix 10. Table of in situ river nutrient data from May 2014. 0 o 11 in I u 1 6 H S' 5 ro z o R CT> LO n m xi ro ro n vd SS Q 8 R 3 LO c 3 1 ^ IS 1 8 S 1 2 i 8 VD § o s £ t-H § 00 0.373146 0.373146 1175.8 3.126906 975.8 14.22933 14.22933 838.6 4.329837 4.329837 946.3 2.087668 911.9 1.046291 1030.6 6.671435mMMMi 2.652807 2.652807 932.5 m 6.886646 752.0 58.27184 58.27184 1031.3 £ d cri 61.366511 1131.1 I £ i ts 32.29404 in 1169.6 O LO S3 fN 1 1 1.0555791 891.0 1 4.9370391 1 1067.5 1 | 3.3708041 881.8 1 ' 56.055031 1117.6 | 72.431931 r 965.0 1 ' 2488.393 % 6.4182811 1 939.0 1 rsi ro cn fv PO 00 d; cn I 5 o § 3 R VD s rv CM 8 8 ro § £ 8 8 § o VD 1 ro n 8 954 1 8 i-'. s § I n.d. . 3 o 0 fN 6.7l| 0 CN 18.39[ 1.7071 o o o ro ro cri 0 rsi 3 iri » fN 4.0951 rsi 4 6.69ll rsi cri s z in I fN a -O xi xi uO ■d ■q -d xi xi xi xi s ■6 -6 ro C c c a c n.a. c c c c c c c (N S s LO g cn 004992 LO 0.78136 . o 0.656851 1.203505 2.040683 1.551845 1.890557 1.144418| 1 0.595335 1.654351| 0.7362811 1.015132J o d 0.8191541 Phaeo Phaeo F 1 1 1.40299 1 0.55763111 [ 1.9702691 06 0 1 0160060 00 VD X> xi "d rvcn ■D -d ■d d xi xi xi £ g n.a. d c c a C c c c c c c c n.d.| r s i 5 VD% 00 6.1378| i 1.917117 0.882883 1.7536571 0.9333331 6.217512 5.978377 2.550774| 1.26126l| 3.3478911 PO fN pri 2.7101981 ChlaF 1 2.2319271 1 1.3117111 1 1 1.16036 00 “d xi lO -d 8 ■d -d xi d xi xi n.a. c c c s n.d.| c c c c c c S 3 s vq rsi 3 -0.601351 -0.2861l| -1.13602 -5.53333 -0.63196| 1.763989 0.566014 2.709269| 1.6226881 3.717834| 0.277728| d PhaeoT | | 2.457461 j | | -9.07247 | 16.52825 4.7879081 1 -0.16043 rv | -1.70426 | 1.1745551 oo I 3.970974| PO (N xi xi xi cn PS xi 8 -d d -d xi •d xi s 00 LO n.a. c c PO cri rsi LO S c rri c c c c c c n.d.| § s o 00 PO a 4.288287 2.8696211 3.666738| 3.027026| R 00 vb iri 4.792792| ChlaT | | 20.72504 | | 3.783783 1 1 15.94234 I I 4.666665j | 2.5507741 | 11.55821 | 4.918918| | 7.567566| 1 7 3 9 9 rv oo CN ro zo CQOT rsi 0 LO rN 0 s “d I o 3 o i O § s rv 0 § LO c 0 .0 3 1 0 .0 3 3 0 .0 7 9 0 .0 9 2 0 .0 4 9 0 .1 8 6 0 .2 4 6 0 .3 4 4 1.604) 0 .0 2 7 0 .0 5 4 | 0.4961 0 .0 7 3 | 0 .1 6 8 | 0 .1 8 4 0 .5 9 8 | 0 .9 0 1 0.163 o o o o o o o 0 O 0 0.0421 o z oo fN rv VD $ r>- cn rv o rv 9 xi I o LO % c r ts 2 6 6 .6 | 1 4 8 .2 7 | 2 6 5 .5 9 ] 2 6 8 .0 6 | 3 07.471 2 7 7 .4 6 | 2 2 2 .9 6 | 253.941 i i 8 3 CN CN r 3 3 6 2 7 .8 l| o | | 273.01 I 259.2l| | 257.98| | 264.66| 1 265.441 | 3 0 6 .3 6 | [ 248.021 | 3| 1 4 .6 6 | 2[ 5 6 .0 l| | 2 6 8 .9 3 | 2 6 8 .3 5 | | 2 6 0 .6 2 | | 2 4 7 .0 6 | LD (XI VD cn CN CN 00 rsi v ro LO rsi s cn lD rv g CN O lO xi iv i 8! oo CN CN rv § fN 8 3 5 8 § 3 rv o 3 2 2.86 * c 1.016 I 1.044 0.744 1.819| 2.89l| o o o o 0 PO rsi N cri 0.032| ro cri d IV 0 o CL LO VD r^ oo f ! s ro xi 3 fN s | c I LO § if) £ rri 0.823101 2.593646 3.837617 1.350807| 1.069043| 58.80451| 52.98984| 44.130611 48.638031 51.371351 28.83704| 2.556157| 2468.417| Z o o 67.43593| 0.553674| O 85.23362| | | 0.312921 | 1.650906 | 1.377008 | 2.023837 1 1.898829 | 1.842804 [ 11.00033 [ 3.651435 | 2.5432811 | | 0.981668| | 1.036391 | 0.5782911 > be E N otes behind georgiana [approx [approx E.T. [georgiana < CD CD CD o m VD o cn VD cn rv cn O cn ro 00 fN J ro m c c c c C C c PO LO CN fri cS fN 5 cri 2 ri VD s LO 3 3 fn cr? 2 r 3 s § $ in 28.3 I «Q s River Mile | 1 00 1 V c c C c c c c x> 1 rv 5 LO LO f -121.52047 -121.55387 -121.51651 -121.51327 -121.52955 -121.52936 -121.53957 -121.52918 -121.50115 -121.52494 -121.50480 -121.51868 -121.54263 -121.54263 | -121.51897 | 5 -121.58585 1 1 1 | | -121.54870 | I I I I | | I | -121.584611[ | -121.595611 -121.51838 I | | -121.51463 | I -121.54650 | | -121.53168| | -121.51408 | | -121.57784 | | -121.55701 -121.50290I | | | | | 1 j CD CTJ cu c c c c c c c n ro r3 1 00 00 38.57036 38.59843 38.53437 38.51212 38.47455 38.45098 38.42610 38.35882 38.35548 38.56984 38.46919 38.40342 38.23840 38.23840 38.18435| 38.137281 38.53843 38.32055 38.32055 38.28806 38.25840 3 8 .2 4 0 6 2 | 3 8 .2 5 8 9 5 ro 38.51581 Latitude * s ro | | I | | | | 38.27446|| | |38.19144 1 1 1 3 s T r-i 12-251 'T ro 6/1/14 9:20| 6/2/14 9:40| 6/3/14 9:30| 6/4/14 8:40| 6/2/14 9:101 5/31/14 9:40| 6/1/14 6/1/14 15:05| 6/2/14 15:40| 6/3/14 13:lo| 6/2/14 12:30| 6/3/14 16:20| 6/4/1410:45 5/30/14 9:55| 5/31/14 9:30 6/1/14 10:30| 6/1/14 13:lo|6/1/14 15:lol 6/4/14 13:10| 0 vo 6/2/14 11:00] 6/2/14 10:00| 6/2/14 10:50| 6/3/14 15:00 6/1/14 10:12| 5/30/14 12:20 5/31/14 12:20| 5/31/14 15:lo| 5/30/14 12:20| 5/30/14 15:20| 5/31/14 13:20|5/31/14 15:20| 5/30/14 14:10| 5/30/14 11:55| Date.Time M/V\ AAM | £ above above (180) clean clean clean (above) clean clean clean clean clean WW (above) WW (above) [ clean Treatm ent clean WW (above) WW (above) | WW (mixing?) [ | WW WW WW blank above (Am above (Am Riv)[ WW clean WW effluent clean clean (a b o v e )] above [clean [clean |clean |clean (above) |clean (above) jclean (above) | 4 4 ^r 2 4 2 3 s tH 14-1 1 3 3 3 3 3 (J ID KD u 3 3 3 3 3 3 3 3 3 3 3 3 13 3 Project 3 14-1 LAG LAG |LAG14-1 |LAG14-1 | 3 3 ILAG14-1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 ! ! I 3 3 3 3 3 3 3 3 | | 3 3 3 3 3 | | | | | 2 5 ■0 14 16 1.5 1.5 24 24 19 | 29 29 | c c c .2 QJ 0 D 3 ro Station Station 22 Station 26 Station Station 6 Station 8 Station 12 Station Station 20 Station 25 Sample label Station Station 27 Station 3 Effluent Station Station 28 Station 4 Station 5 Station 7 Station 15 Station 17 Blank Station Station 10 Station 13 z Z | Station Station | Istationll | |s ta tio n l8 | | Station 1 Station | 1 I |station23 | | 1 1 | 1 1 | Station | 9 | 1 | [Station Station | Station | [station 2 1 1 Nutrient| Gratj 1 1 jstation 107 Appendix 11. Table of in situ river Trace uptakes from May 2014. J 4 Mi III—II l l l l l l l ■BHinnn ■Mill ■ ill. 3 s i n I i pedx 2 Tbe of 12.Table Appendix SAT 15N03 1SNOB 15N03_13 15N03_13 15N03_13 Elapsed 15NH4 15NH4 1SNH4_13 1SNH4_13 15N03 V/d PONn*- C Rho y*- CV/d C POC |if- avg Rho River Time Rho 15NH4 PON w- C Rho JIJ- 15NH4_13 C POC ng- f ratio avg PO C avg PON Rho pe at/L •t/L/d •t/L C ■t/L/d V/d at/L/d CV/d «t/l Sam ple label Project Treatment Date.Time M ile Notes Hours at/L/d •t/L Station 1 IAG14-1 above (180) 5/30/14 12:20 63.0 approx E.T. -35.00 4.208715 0.432262 12.27619 51.24111 0.409178 155.8889 3.485311 0.43117 10.18571 38.75532 0.308588 147.8889 44.99822 0.547011 151.8889 11.23095 Station 6 IAG14-1 clean (above) 5/31/14 9:40 58.5 -26.08 2.538051 0.395641 7.92381 21.23468 0.212932 111.4444 2.340839 0.432828 6.819048 19.34079 0.209829 102.8333 20.28774 0.520211 107.1389 7.371429 Station 8 1AG14-1 clean (above) 5/31/1412:20 55.6 -23.42 Station 11 LAG14-1 clean (above) 5/31/14 15:10 52.4 -20.58 2.095517 0 303584 8.104762 31.23417 0.338497 110.4444 2.273838 0.476778 6.161905 35.96553 0.416601 107.8889 33.59985 0.479594 109.1667 7.133333 Station 12 LAG14-1 clean (above) 6/1/14 9:20 48.0 -2.42 1.304633 0.243892 6.080952 14.20339 0.19284 81.44444 2.138518 0.574168 5.080952 21.16407 0.29215 84.55556 17.68373 0.378907 83 5.580952 Station 14 LAG14-1 clean 6/1/1412:25 46.1 Station 16 LAG14-1 clean 6/1/1415:05 43.1 3.33 1.197237 0.196277 6.757143 24.23199 0.272352 102.7222 1.662723 0.359302 5.604762 22.79432 0.272591 96.55556 23.51315 0.41862 99.63889 6.180952 Station 18 LAG14-1 clean 6/2/14 9:40 37.9 21.92 0.770163 0.131002 6.290476 14.57381 0.170422 93.44444 1.657073 0.445216 4.73 14.66987 0.198309 82.05556 14.62184 0.3173 87.75 5.510238 Station 20 LAG14-1 clean 6/2/1412:30 37.6 Station 22 LAG14-1 clean 6/2/14 15:40 34.8 27.92 1.212168 0.196279 6.842857 23.39853 0.258731 103.6667 2.109995 0.455167 5.92381 26.5356 0.317308 99 24.96707 0.364873 101.3333 6.383333 Station 23 LAG14-1 clean 6/3/149:30 29.9 45.75 0.22677 0.042687 5.428571 12.62196 0.154207 88.66667 1.729151 0.502729 4.512857 11.96536 0.155098 83.61111 12.29366 0.11594 86.13889 4.970714 Station 25 LAG 14-1 clean 6/3/1413:10 26.1 Station 26 LAG14-1 clean 6/3/14 16:20 20.7 52.58 0.287875 0.04337 6.785714 7.089117 0.06265 116.8333 1.747465 0.337321 6.204762 7.14625 0.072268 102.6111 7.117684 0.141438 109.7222 6.495238 situin Station 27 LAG 14-1 clean 6/4/14 8:40 19.9 68.92 0.666277 0.155756 4.641905 11.54286 0.149071 83.72222 1.712907 0.46107 4.77619 13.02803 0.16677 85.27778 12.28544 0.280044 84.5 4.709048 Station 28 LAG 14-1 dean 6/4/14 10:45 georgiana 71.00 0.188774 0.051167 3.786667 12.66295 0.167371 82.55556 1.583487 0.465572 4.374286 14.333 0.186106 84.88889 13.49798 0.106516 83.72222 4.080476 Station 3 LAG14-1 WW (above) 5/30/149:55 58.4 -25.08 3.360491 0.423093 9.966667 30.45567 0.281472 125.5556 3.797737 0.478405 10.27143 35.11258 0.345869 122.0556 32.78413 0.469458 123.8056 10.11905 Station 4 LAG14-1 WW (above) 5/30/14 12:20 56.0 2014. May from uptakes Saturating river Station 5 LAG14-1 WW (above) 5/30/1415:20 52.9 -19.67 2.952081 0.305503 11.37143 39.36189 0.340441 138.7222 3.031474 0.446081 8.647619 40.05945 0.409128 122.0556 39.71067 0.493366 130.3889 10.00952 Station 7 LAG14-1 WW (above) 5/31/149:30 47.4 -1.50 1.381034 0.223462 6.947619 16.43903 0.179583 100.5 2.173347 0.407947 6.628571 16.90459 0.194423 96.22222 16.67181 0.388544 98.36111 6.788095 Station 9 LAG14-1 WW (mixing?) 5/31/1413:20 43.3 Station 10 LAG14-1 WW 5/31/1415:20 41.3 4.33 0.071371 0.008338 8.595238 25.58647 0.261057 112.3333 3.415707 0.443902 9.747619 25.91484 0.257246 115.2222 25.75065 0.020467 113.7778 9.171429 Station 13 LAG14-1 WW 6/1/1410:30 33.8 23.50 0.088548 0.014464 6.166667 13.27291 0.159007 90.66667 2.114536 0.444706 6.042857 13.56 0.162883 90.61111 13.41645 0.040193 90.63889 6.104762 Station 15 IAG14-1 WW 6/1/14 13:10 31.2 Station 17 LAG14-1 WW 6/1/14 15:10 28.3 28.17 0.106052 0.01439 I 7.42381 20.01859[ 0.2235951 100.833311 2.295382|I 0.4134l| 6.952381| 17.68649| 0.1956151 100.27781il8.85254| 0.044162 [ 100.555611 7.188095| Station 21 LAG14-1 WW 6/2/1414:00 15.4 Station 24 IAG14-1 WW 6/3/1411:20 georgiana 72.33 0.106486 0.019416 5.538095 12.74661 0.148426 92.72222 2.239185 0.551008 5.471429 10.33209 0.123467 89.16667 11.53935 0.045397 90.94444 5.504762 Station 29 LAG14-1 WW+ 6/4/14 13:10 25.3 behind 50.00 0.107958 0.01341 8.104762 24.87153 0.250305 113.3333 3.010501 0.631961 6.704762 21.56537 0.230532 105.5556 23.21845 0.034619 109.4444 7.404762 Station 19 LAG14-1 +WW* 6/2/1411:00 na 0.100897 0.015951 6.37619 21.39585 0.246104 98.94444 2.505349 0.453653 7.047619 20.45499 0.250641 93.11111 20.92542 0.038714 96.02778 6.711905 Station 2 LAG14-1 above (Am Riv) 5/30/1414:10 60.5 Am Riv -30.00 0.255578 0.064042 4122857 3.052271 0 041719 74.72222 0.685902 0.211908 3.61619 2.934962 0.04341 69.11111 2.993617 0.271464 71.91667 3.869524 Blank LAG14-1 blank 6/2/14 9:10 na na Nutrient Grak LAG14-1 WW 6/2/14 10:00 na 1 47.00 Nutrient Grat LAG14-1 clean 6/2/1410:50 na 2 23.08 Nutrient Grat LAG14-1 WW 6/3/1415:00 na 3 25.00 Effluent LAG 14-1 effluent na na Station 12 du LAG 14-1 clean (above) 6/1/14 10:12 na -1.55 Station 1.5 LAG 14-1 above 5/30/14 11:55 na -40.001 ooo 109 Appendix 13. Enclosure nutrient data from May 2014 river enclosures. g rH 01 LD fN ID ro 00 cr> g c n h . ro ID LO cn 00 ID oo CM fN a o 00 o ■d g lT) ro rsi fN o rsi s fN ° fN s $ 8 01 S 5 o d o s 00 s r^- o s § 8 3 0 r * 00 8 8 o o o o o o d vd s 3 o d ' rsi o o o o o i cn 01 00 ID ID 1 fs! CM 3 fN ! i ID ID cn cn ON cn VO oo oo f cn CM Oi IN -a- r* cn $ -d rsj s 0 s O 8 o 00 s S d s s 8 1 rv o £ s 1 S 8 ! rH 3 s i g § fN no 1 fN ts 1 r-i 5 S in 1 1 00 rI 1 S j 1 cn S 8 n j rsi 1 100 cn fN CM CM I (N P3 00 in ro rsi CM 00 s £ o S o o o S oo ro VX> ro f o Z d o o o o o o o o d d o c 8? cn o o o o S § 3 CM o O o o r* CM o CM o r* fN s ts g * § CM 5 tfl P i s X f § ts s 8 g CM § ts P i 8 3 ° * 3 E la p s e d H o u rs T im e 'T *ar rH 3 3 3 ro ro l? t s 3 s LO LO I J 3 3 3 3 rM 3 r Jf 3 ID" JJ ST s s $ ID“ to iB - i s- ^ s s S S s s ; b - S5- id " S J § s D a t e .T im e oo oo ro ro 00 00 0 0 O O o z z Z Z z zO oz oz ZO zo 00 ro ro + + + + + + + + O O O O O o O O O o c 3 4 : 5 t 5= 3= 5C 5 i 5C z z z Z Z z z z z z ; J a» 0) 0) Q) V a> HI 'o i 0) 'a ; 01 01 o; 0J + + -f- + + + + -t- + + + + + + + + + + + + + + + + + c c c c c C c c c c c c c c c c c c C 1- c c c C c c c c c c c c c c c c c c ro (U ro ro ro a> 0J ro ro ro ro ro ro ro roro ro ro ro a> _a> T r e a t m e n t u u u u u u o ” J u u U “u u u u rH t ; 5 o 00 o 00 ID o> rsi o 5 IN S 3 o 8 1 LT) LT) ID | 8 VD 2 PI 83 3 8 o 9 S m s S 1 ot m S VO i 1 $ cr> 1 o 8 S z ?! 3 fH s S 1 rs1 f< 00 oo o 1 d rH § « d d LD o d a <3 s i VD Ln s s d d VD 00 rsi z s a 8 P? w £ rsi s 8 S3 rH S o tH £ i a I £ s s Ln rsi a i 1 i 1 5 00 rC r< ro 8 S ? s ID CTl 1 LT) ID O 00 l $ o CTi 8 S VD 00 ro rsi d LD VO VD s d § d o Pf VO VO 3 rn I if) 5 35 o o H fS s H S8 s s o VD oo o ID r- •b TJ -6 ■D -d “d “d -d -d -d xl ■d •d -d -d § o d d c if! c c c c S c c 1 c c 8 c 8 d d d VO S VO s 8 ID CO 8 rri s j s » fN fN § s l S 3 i 8 s a <1 o 3 oS a o VO o o ■q s -D -6 ■d jC ■d "d “d -d ■d $ ■d ■d -d ■d Pf -d •d s § S d C c c d c 1 c c c c c d d d d 0 z = • 1 S 9 O 1 S 8 fN 1 $ 5 5 2 rsi oo s ro ro m 1 o> 00 ro cri oo <1 <3 3 K r< d CM rsi fN 00 CT -d §8 *d •D -d "d *d ■d xi -d -d -d •d ■d ■d o * S fS 8 c d c C c c c c c c d ID d c d d i § VD a I 3 s a 1 s 3 VO oo rH m 00 1 s l r>. -6 -d a "b S -d *u S3 ■d 00 -d s ■d a ■d ■d ■d s -d a ■d ro oo c r< d d c c O c oo c ID c (N c c o d d c d c d I ID £ « m rsi 8 r* VO vo VO rH u s r>s 1 S fN rsi 00 00 fN 01 m 00 o 8 LT) s r3 rsi P! m s in s i fN 8 s oo o § 5 S vb o i rsi fN vo 8 VO 3 § s § rsi 3 00 d VD rri vb d o o d d 2 s i R s s R 3 a ? S s s? S Pi s 6 * z <71 vo O) rsi ID 8 on 8 s § S jq a § LD 3 R 8 LD S 8 1 s I i 1 S 1 8 1 g 1 £ o E 1 g § o 3 1 VO rH 9 o 1 i vo s 1 E o 3 o o d rri rsi CL d o o 00 d ro rri o 00 00 vo a a § 8 s S a a a a 8 2 £ 3 <3- a to I S & ID § 1 § i 3 fN 1 1 2 8 2 rri s S 0 0 0 0 O o 0 0 o O z z z z z z z z z z + + + + + + § 5 § 5 § 1 1 1 1 X £ 1 1 X X z 2 z z z z z z z z O O O O O o O o O O + + + + + + + z z z z z z z z z z c c c c c c c c c c + + + + + + re rere re re re re re -S ii 0) JO) _o; « JU T r e a tm e nu t rH rH rH C 4 3 4 4 vr 4 4 « 2 2 s 3 i 5 JT 2 4 4 4 o' 3 y? ID 3 13 ID 3 o C5 o 3 3 3 3 3 5 5 5 3 1 § 5 i § 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Appendix 14. Enclosure uptake data from May 2014 river enclosures. oc JO “O “O VO 2 fN 9 c c cr> in s 2 o 1 i r^ LOS 3 rH 0 8 % s m 00 S ? VO o> s r\l 1C oo K aj 1 § r* 4444 in S CTi CT> rsi , s S S fN 1 R 200.0833 110 3 fN s 112.77781 15NH4_1 3C POC 3C ug-at/L rH O 1 102.77781 in § a? PJ fN 3 P! rH m r^ o o o o o 8 o o rC So LO 8 vo o vo VO d r5 0.417263 0.387216 0.31398l| 0.526182) o o O 0 d O 0.727484| 0 15NH4_1 3CV/d 1 0.3272141 1 in o> 00 VO in 2 S3 o 8 0 § ? in O rH oo L/S fN vo vo 8 1 vo 3 8 vo vo ^r ai fN 0 ct! 137.246l| 180.59961 £ VO VO (T) 29.873931 i 15NH4_1 Hg-at/L/d 3C Rho 3C 00 1 28.190271 I 71.38822 00 So S8 PI s 2 g 8 VO K oo 00 1 in 8 s s 3 tH 9.414286 36.60714 25.221431 41.32857 48.814291 lO a S a> 48.3857l| 15NH4 si | | 12.19286 I § s IS s vo 3 o o o o o 2 0 S 8 1 inr^ vo t9% 8 O 1 0.382682 0.308303 0.283721 0.088595 0.410507 0.46424l| o O 0 0 O 0.737358 d 15NH4 0 V/d I I 1 1 I CTJ J In Pj 8 2 s O ar*"* 3 8943 o> O 80702 . 1 2 00 1 tH . 1 2.175185 5.201481 2 £ § NT rH 12.42422 1 1 13.580971 15NH4 SAT oc ro 1 1 1 I 1 3.3513121 1 -£ONSTT- a! s rC vo VO oo s 00 00 m o cn cr» 1 in 1 3 a fN O cn 1 s 111.38891 fN i 107.22221 ft ug-at/L l 3CP0C i 1 1 o 00 00 cr» a lH a CTj d o o o o 00 i § In rN 3 A 0.2851151 0.370204 0.708799| 0.829689| 0.4905781 o o o o 0 0 O O 0.40228l| 15N03_1 3C V/d 3C | | I 0.676575| 3 cn 2 o P? o in 00 S 2 CTlr5 CTt O§ s 8 s i s jjj oo £ § 33.31906 149.0506| s VO 1 in 99.60537 rH i 15N03_1 9 Hg-at/L/d 3C Rho 3C oo t-H cn 00 cn CTJ m s 'T 00 8 o 00 a cn o 0 rH ms vos 00 g VO 00 17.50714| 18.54286 22.97143| 24.2357l| 8.0952381 44.757141 40.97857 00 15N03 PON ug- PON at/L I I § rH S g fN tH d d d d £ 1 § 00 3 £ rH§ s 0 S 3 0 1 O 0 d O 0.010738| 0.242447| o o o o 0 d O 0 0.16717l| 1SN03 V/d | | 0.594393| I 0.0107531 VO 00 1 a 8 s In I I 1 *o 5^ § i 1 I 00 VO «H 1.812053 2.855294 1.1611121 0.086572| 9.321443 0 00 0.112712| 0.397495 d 00 15N03 SAT £ o r* o rsi O 0 s 5 3 ts $ 8 § (N «Sf § 2 £ 8 5 vo 8 8 s § § 5 9 2 8 8 s s Elapsed Hours Time *r 01 r-t rH tH 2 s E § (N 3* > £ kD s vo P § § 6 o UD VO VD VOVOVO VO vo- VO- kD VO' VO VD U) VO' vo vo VO* vo VO VD vo 1 VD vo U3 ro Q Name Q Q Q Q Q o Q Q Q 0 O O 0 O O 0 O N03 N03 N03 N03 N03 2 N03 Z 2 2 + + + + + + + + O O O O O o o o O $: It !t 5t !t N03 N03 eff eff eff eff eff eff eff eff eff eff eff eff 2 2 2 2 2 2 2 2 z V 0) 0) a> V (V 0) eff + + + + + + + + + + + + + + + + + -f + + + + + + + c c C c c c c c c c C C C C c c c c c c c c C c c c 03 ro ro ro rT3 03 ro ro ro ro -2 -S _a; V _0» V j? V 0) .2 .2 V 0) J£ Treatment clean clean clean clean clean clean clean clean clean clean clean u u clean clean clean [clean [clean dean r-i rH rH rH rH rH rH Project 5 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Appendix 14 cont. Enclosure uptake data from May 2014 river experiment 0.52465 0.60062 8 0.55814 o 0.98243 0.433333 0.433831 0.651163 0.747986 0.960279 0.949367 0.767165 1.020825 1.177215 1.210066 0.541714 0.755052 1.414846 0.822222 0.770501 0.662123 o o o o d 0.963126 o >5 : CHI T oo VO 00 0.427328 o o o 0.417587 m1? o o o o o o o o o oo rsi (J\ 00 in r» cr> rN H fC Ch i V£> i 3 £ r>* IS CT1 00 r^ 1 r* r—i S s o> 1 O ro r< g o s rsi ft? S3 00 o3 s S 3 s avg avg Rho C o no ro Sn to fN In a S3 lO 2 cn ig in 1 oo 1 o 00 in s s s i 436.6667 § l 1 15N H 4 1 ug-at/L 3C P O C vo in oo §§ fS m s rH m rH o g rH 00 s LO m o 1 1 o 0.843861 0.462781 o o o o o o o o o o 0.508351| 15N H 4_1 3C 3C V/d VD in rH 3 s fQ s rH 1C s 2 CTl in 00 8 s ' i > | £n 1 § £ ec ro s o 8 jo 00 £ cn 00 I S3 s i $ 3 a a a i r* OJ *3- s c! rH s CT> in s rH 00 tn 8 rv ^3- cn s 00 00 in 5 Elapsed Hours T im e <3- 'J- *)• KJ- rH rH s rH rH rH > £ I?r lo § Sr in > s VO VD U3 VO VO § S vS" u? ^D VO- VO vo VO" § vo $ v3" D ate.Tim e N am e I X X XXXXX XX v X V O o o 0 0 O 0 0 0 0 z 2 z z z z z z z z + + 4* + + + + U s 3 § 5 X X X X 1 X X 1 1 1 z z z z z z z z z z O O O o o Oo OOo + + + + + + z z z z z z z z z z c c c c c c C c + + + + + + + + + + nj ro (D cro c a> QJ 0) 01 Project 5355555 53 3 S S535S 5 35555335553 113 Appendix 15. Additional replicated enclosure data Elapsed N03+N0 P ro je ct T rea tm e n t Nam e D ate.T im e NH4 P0 4 Si 04 N02 C h la T N03 FC Tim e Hours 2 (u M ) NMT1 clean + N03 A 07/23/14 0 3.52 47.46 1.05 261.06 0.13 1.26 47.33 1.33E+06 NMT1 clean + N03 A 07/24/14 24 1.08 46.23 0.93 259.09 0.18 2.19 46.06 3.50E+06 NMT1 clean + N03 A 07/25/14 48 1.20 40.71 0.57 250.07 0.27 12.11 40.44 1.90E+07 NMT1 clean + N03 A 07/26/14 72 0.50 27.98 0.17 228.78 0.44 31.62 27.54 6.08E+07 NMT1 clean + N03 A 07/27/14 96 0.49 20.07 0.17 213.27 0.49 44.37 19.58 7.98E+07 NMT1 clean + N03 A 07/28/14 120 0.84 7.50 0.17 172.59 0.43 42.11 7.07 5.30E+07 NMT1 clean + eff B 07/23/14 0 41.93 2.02 2.77 265.45 0.12 1.59 1.89 1.35E+06 NMT1 clean + e ff B 07/24/14 24 40.50 1.99 2.64 269.52 0.15 1.90 1.84 2.76E+06 NMT1 clean + e ff B 07/25/14 48 33.55 2.12 2.25 253.59 0.18 10.34 1.94 1.98E+07 NMT1 clean + e ff B 07/26/14 72 10.87 2.19 0.54 223.22 0.18 39.32 2.02 7.81E+07 NMT1 clean +eff B 07/27/14 96 0.42 0.21 0.12 182.57 0.00 60.05 0.21 1.31E+08 NMT1 clean + eff B 07/28/14 120 0.52 0.25 0.12 157.39 0.00 41.98 0.25 8.50E+07 NMT2 clean+ NH4CI A 08/20/14 0 45.35 1.27 0.96 266.52 0.02 1.13 1.25 1.32E+06 NMT2 clean + NH4CI A 08/21/14 24 44.94 1.26 0.84 261.20 0.02 4.05 1.24 4.81E+06 NMT2 clean + NH4CI A 08/22/14 48 34.99 1.20 0.30 283.04 0.02 18.60 1.18 2.42E+07 NMT2 clean + NH4CI A 08/23/14 72 16.69 1.30 0.20 252.80 0.02 42.91 1.27 6.86E+07 NMT2 clean + NH4CI A 08/24/14 96 7.38 1.15 0.19 245.15 0.03 45.70 1.12 9.03E+07 NMT2 clean + NH4CI A 08/25/14 120 3.61 1.19 0.23 215.26 0.05 42.65 1.14 1.20E+08 NMT2 clean + eff B 08/20/14 0 43.42 1.64 2.60 325.45 0.05 1.30 1.59 1.30E+06 NMT2 clean + eff B 08/21/14 24 41.77 1.62 2.52 309.83 0.04 3.93 1.57 5.19E+06 NMT2 clean +eff B 08/22/14 48 33.50 1.53 1.96 352.70 0.05 13.49 1.49 2.23E+07 NMT2 clean + eff B 08/23/14 72 7.36 1.34 0.31 293.61 0.05 43.57 1.30 7.16E+07 NMT2 clean + eff B 08/24/14 96 0.45 0.01 0.21 225.14 0.01 55.53 0.00 9.85E+07 NMT2 clean + eff B 08/25/14 120 0.42 0.01 0.25 225.68 0.01 36.53 0.00 7.52E+07 NMT3 50uM NHCI + 1.7uM P04 A 10/17/14 0 46.65 3.52 2.80 0.09 1.20 3.43 3.10E+06 NMT3 50uM NHCI + 1.7uM P04 A 10/18/14 24 45.43 4.11 3.73 240.98 0.08 2.69 4.03 4.69E+06 NMT3 50uM NHCI + 1.7uM P04 A 10/19/14 48 41.75 2.76 2.76 267.66 0.08 5.71 2.68 1.11E+07 NMT3 50uM NHCI + 1.7uM P04 A 10/20/14 72 32.55 2.37 1.27 269.98 0.06 12.85 2.30 2.66E+07 NMT3 50uM NHCI + 1.7uM P04 A 10/21/14 96 16.87 2.38 0.21 252.09 0.08 24.51 2.31 5.58E+07 NMT3 50uM NHCI + 1.7uM P04 A 10/22/14 120 3.86 2.00 0.12 229.38 0.08 32.16 1.92 7.91E+07 NMT3 50uM NHCI + 1.7uM P04 A 10/23/14 168 0.49 0.03 0.17 197.87 0.01 26.97 0.01 3.70E+07 NMT3 50uM Effluent B 10/17/14 0 44.80 2.78 2.59 308.33 0.09 1.15 2.69 3.02E+06 NMT3 50uM E fflu e n t B 10/18/14 24 45.15 2.79 2.56 287.88 0.09 1.74 2.70 5.40E+06 NMT3 50uM Effluent B 10/19/14 48 39.75 2.79 2.29 278.31 0.09 6.14 2.69 1.02E+07 NMT3 50uM Effluent B 10/20/14 72 29.67 3.24 1.51 283.30 0.10 13.14 3.13 2.67E+07 NMT3 50uM E fflu e n t B 10/21/14 96 9.45 3.32 0.38 303.56 0.14 24.12 3.18 5.92E+07 NMT3 50uM Effluent B 10/22/14 120 0.29 0.00 0.17 277.31 0.05 29.42 -0.05 9.91E+07 NMT3 50uM Effluent B 10/23/14 168 0.33 0.00 0.16 232.91 0.04 17.94 -0.04 2.61E+07 NMT4 50uM N03+ 1.7uM P04 A 04/05/15 0 1.96 48.76 2.26 261.33 0.15 4.13 48.61 7.57E+06 NMT4 50uM N03+ 1.7uM P04 A 04/06/15 24 1.54 48.43 2.29 259.67 0.17 4.06 48.26 8.64E+06 NMT4 50uM N03+ 1.7uM P04 A 04/07/15 48 0.41 43.06 1.82 253.33 0.22 9.15 42.84 1.95E+07 NMT4 50uM N03+ 1.7uMP04 A 04/08/15 72 0.35 33.01 1.01 235.67 0.33 19.71 32.67 3.28E+07 NMT4 50uM N03+ 1.7uMP04 A 04/09/15 96 0.41 17.81 0.25 211.00 0.47 19.53 17.34 5.59E+07 NMT4 50uM N03+ 1.7uMP04 A 04/10/15 120 0.43 3.07 0.16 180.33 0.47 23.53 2.60 6.19E+07 NMT4 50uM Effluent B 04/05/15 0 54.98 2.76 2.58 260.33 0.16 6.24 2.60 1.01E+07 NMT4 50uM Effluent B | 04/06/15 24 54.96 2.73 2.51 266.67 0.16 3.10 2.56 8.92E+06 NMT4 50uM Effluent B 04/07/15 48 45.75 2.71 2.08 260.67 0.16 12.93 2.55 2.17E+07 NMT4 50uM Effluent B 04/08/15 72 28.71 2.69 0.82 240.00 0.16 26.77 2.53 5.52E+07 NMT4 50uM Effluent B 04/09/15 96 6.23 2.41 0.18 205.67 0.16 11.33 2.25 8.16E+07 NMT4 50uM Effluent B 04/10/15 120 0.63 0.38 0.17 180.00 0.11 33.85 0.27 6.93E+07