Potential nitrous oxide production by marine shellfish in response to warming and nutrient enrichment

Marine Pollution Bulletin 146 (2019) 236–246

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Potential nitrous oxide production by marine shellfish in response to warming and nutrient enrichment

T

Gárate M.a, Moseman-Valtierra S.b, , Moen A.c ⁎

a

Mass Audubon, 500 Walk Hill St, Boston, MA 02126 Department of Biological Sciences, 120 Flagg Road, Kingston, RI 02881 c URI Diving Safety Program, 215 South Ferry Road, Narragansett, RI 02882 b

ARTICLE INFO

ABSTRACT

Keywords: Nitrification Denitrification Greenhouse gas Mytilus edulis Mercenaria mercenaria Crassostrea virginica

Bivalves facilitate microbial nitrogen cycling, which can produce nitrous oxide (N2O), a potent greenhouse gas. Potential N2O production by three marine bivalves (Mytilus edulis, Mercenaria mercenaria and Crassostrea virginica) was measured in the laboratory including responses to nitrogen (N) loading and/or warming over shortterms (up to 14 or 28 days). N additions (targeting 100 μM-N ammonium nitrate) or warming (22 °C) individually and in combination were applied with experimental controls (20 μM-N, 19 °C). N2O production rates were higher with N additions for all species, but warming lacked significant direct effects. Ammonium and nitrate concentrations varied but were consistent with nitrification as a potential N2O source for all bivalves. Highest N2O emissions (7.5 nmol N2O g−1 h−1) were from M. edulis under hypoxic conditions coincident with a drop in pH. Macro-epifauna on M. edulis did not significantly alter N2O production. Thus, under short-term hypoxic conditions, micro-organisms in M. edulis guts may be a particularly significant source of N2O.

1. Introduction Bivalves significantly influence nitrogen (N) cycling in aquatic ecosystems (Kellogg et al., 2014; Reitsma et al., 2017; Stief, 2013). Filter-feeding by bivalves promotes strong benthic-pelagic coupling by removing phytoplankton biomass from the water column (Carmichael et al., 2012; Kellogg et al., 2014; Rose et al., 2014) and by enriching marine sediments via excretion of ammonium and biodeposition (Stief, 2013). Reef-building bivalves also enhance the surface area for N-cycling microbes to grow on their shells, potentially removing or producing N through nitrification, denitrification or coupled processes (Heisterkamp et al., 2013; Svenningsen et al., 2012). Further, bivalves ingest microorganisms including denitrifiers (microbes that reduce reactive nitrate to inert N2 gas) that can remain metabolically active in the anoxic gut micro-environment. However, under reduced conditions such as those found within animal guts, the denitrification pathway may terminate early with nitrous oxide (N2O) (Miller et al., 1986; Stief et al., 2009), a potent greenhouse gas and agent of stratosphere ozone depletion (Stief et al., 2009). Globally significant sources of N2O include coastal ecosystems with high amounts of bioavailable N, such as estuaries (Bange, 2006; Seitzinger and Kroeze, 1998). High levels of reactive N are known to

⁎

increase N2O emissions from sediments and water columns in a range of environments (Corredor et al., 1999; Kroeze and Seitzinger, 1998; Muñoz-Hincapié et al., 2002; Seitzinger et al., 1983) due to plentiful substrates for microbial metabolism. In eutrophic coastal ecosystems, bivalve shellfish experience high N concentrations from anthropogenic inputs via fertilizer run off and/or sewage effluent (Gruber and Galloway, 2008). Similarly, in aquaculture operations, shellfish may be exposed to high dissolved concentrations of nitrogen in places or periods of urea build-up, such as in densely packed and/or poorly flushed cages (Jackson et al., 2003; Lunstrum et al., 2018). N2O is produced through multiple microbial pathways including the first step of nitrification (the oxidation of ammonium to nitrate) (Goreau et al., 1980) and in denitrification and nitrifier denitrification (Wrage et al., 2001; Zumft, 1997). Bivalves can indirectly stimulate the production of N2O within those coastal ecosystems by excreting reactive N in urea and biodeposits, (Heisterkamp et al., 2010; Stief et al., 2009; Svenningsen et al., 2012), or they can be direct sources of N2O via the microbes they harbor on their shells or in their guts (Stief et al., 2009). Temperature may also significantly impact N2O emissions from shellfish and their microbial associates. With climate change, the ocean is generally projected to increase by 2–3 °C by 2100 (IPCC, 2007). Microbial activities increase with warmer temperatures (Boulêtreau

Corresponding author. E-mail address: [email protected] (S. Moseman-Valtierra).

https://doi.org/10.1016/j.marpolbul.2019.06.025 Received 21 September 2018; Received in revised form 10 June 2019; Accepted 11 June 2019 Available online 20 June 2019 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.

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et al., 2012; Kroeze and Seitzinger, 1998). Also, warming significantly affects bivalve health and fitness in ways that may affect their emissions of N2O. For example, warming laboratory experiments, with temperatures between 18 and 28 °C, have been found to decrease growth rates of juvenile bivalves (Talmage and Gobler, 2011) increase mortality rates (Malham et al., 2009), reduce immune response (Mackenzie et al., 2014), and increase respiration rates (Dickinson et al., 2012; Dove and Sammut, 2007; Mackenzie et al., 2014; Matoo et al., 2013). Warmer waters increase rates of shellfish filter-feeding, due to increased oxygen demand (Kittner, 2005), which may increase ingestion rates of microorganisms and may potentially increase uptake rates for fixed N into bivalve guts. In addition to environmental influences, rates of N2O production may differ among shellfish species due to distinctions in their habitats, behavior, and size. N2O production from aquatic invertebrates seems thus far to be positively related to biomass (Heisterkamp et al., 2010; Stief et al., 2009; Stief and Eller, 2006; Stief and Schramm, 2010) because larger gut sizes can accommodate more active and abundant microbial consortia. Filter-feeders ingest greater quantities of bacteria compared to carnivores, shredders, and grazers (Stief et al., 2009) and those that filter-feed close to the sediment-water interface may consume more bacteria than others due to higher amounts of suspended detritus (Kach and Ward, 2008). Microbial biofilms on the exterior of bivalve shells seem to account for a significant portion of overall N2O production, but the extent seems to vary among species. For example, approximately 25% of the N2O production associated with the zebra mussel, Dreissena polymorpha, was attributed to shell biofilm microbiota (Svenningsen et al., 2012). Alternatively, nearly all N2O production from the blue mussel, Mytilus edulis, came from its shell biofilm, potentially due to the high lysozyme activity in their gut (Heisterkamp et al., 2013). Moreover, reef-building shellfish such as M. edulis frequently harbor mixed macro-epifauna assemblages, which have not been tested for potential roles in N cycling and/or N2O production. The goals of this study were to: (1) examine the effect of N addition and/or warming on potential N2O production and biomass of three coastal bivalves (Mytilus edulis, Mercenaria mercenaria and Crassostrea virginica); (2) test relationships between shellfish biomass and N2O production rates; and to (3) to estimate the contribution of macroepifauna on M. edulis shells to N2O production. To test for potential interactions and synergisms between nitrogen and temperature effects, this two-factor laboratory study (Experiment 1) was performed with the following treatments: (1) N addition (targeting 100 μM-N ammonium nitrate), (2) warming (22 °C), (3) N addition + warming and (4) no N addition or warming (control, 20 μM-N, 19 °C). Potential N2O production rates were assayed after an immediate (< 1 day) exposure and short-term (14 or 28 day) exposures to these conditions in the laboratory. We hypothesized that both N additions and warming treatments would significantly increase the N2O emissions rates from each species and that N2O production rates would be greater after longer exposure periods. Lastly, hypothesized that the contribution of macro-epifauna on M. edulis would increase overall N2O production rates (Experiment 2). For both experiments, we examined relationships of N2O production rates to water quality parameters (pH, DO, dissolved inorganic nitrogen) and the physiological status (condition index) of the three bivalve species.

−71.438985) during the early spring season of 2015. Narragansett Bay has a well-documented, historic gradient in anthropogenic N loads (Deacutis, 2008; Oczkowski et al., 2008) and these collection sites were selected for low nutrient levels. Water temperatures of the Narragansett Bay historically ranged from 1 °C to 23 °C annually in the middle and lower bay, and winter temperatures have increased by an average of 2.2° since 1960 (Nixon et al., 2009). To characterize water quality at each collection site, seawater was collected in centrifuge tubes (50 mL) at the surface and at the depth of each bivalve sampling site (ranging from 3 to 8 m). Salinity was determined in situ using a handheld refractometer, and temperature, pH and dissolved oxygen (DO) were measured in the same samples using a Thermo Scientific Orion Star A326 pH/Dissolved Oxygen Portable Multiparameter Meter. To determine dissolved inorganic nitrogen (DIN) concentrations, a subsample of the water was filtered using disposable syringe filters (Advantec; 0.45 μm; sterile) and frozen (−17 °C) at the time of collection and later analyzed for ammonia and nitrate using a micro-segmented continuous flow nutrient analyzer (Astoria Analyzer, model 303a). Immediately after collection, organisms were transported to environmental temperature-controlled chambers (Holman Engineering) at the Marine Sciences Research Facility (MSRF) at the University of Rhode Island Bay Campus. They were acclimated in a series of 16 identical glass aquaria (Fig. 1) to which they were randomly assigned and kept in aerated, unfiltered seawater at ambient water temperature (19 °C) for at least 12 h before start of the incubations. 2.2. Experiment 1: nitrogen and warming manipulations Nitrogen and warming manipulations (n = 4) were applied to each of the shellfish species. Based on approximate field abundances in New England waters (Schulte et al., 2009; Tam and Scrosati, 2011), 3 adult individuals were used per aquarium for M. edulis and C. virginica and one adult of the larger M. mercenaria was used per aquarium (Thelen and Thiet, 2009). Individual bivalves were randomly assigned to aquaria and aquaria were randomly assigned to one of the 4 treatments. These experiments were maintained for 28 days, however, assays ended after 14 days and replication for M. edulis was reduced to 3 replicates by the end of the experiment due to unanticipated mortality (detailed further below). The 28 day target was intended to be sufficient to test physiological effects of exposure to altered nutrient and temperature conditions during typically active seasons for these species. For N addition treatments (hereafter referred to as “+N”), each aquarium received a pulse of ammonium nitrate after the acclimation period to increase the overall DIN concentration to 100 μM-N. This N

2. Methods 2.1. Field collection For this study, M. edulis, M. mercenaria and C. virginica individuals were collected from subtidal Narragansett Bay, Rhode Island, USA via scuba diving. M. mercenaria and M. edulis individuals were collected in Conimicut Point (41.720467, −71.365083) and Narragansett (41.492317, −71.419060), respectively, during the late summer 2014. C. virginica was collected in North Kingstown (41.566948,

Fig. 1. Invertebrate mesocosm diagram. 237

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level was selected in order to target maximal total N concentrations in the bay that were previously reported (Oviatt et al., 2002). N additions were made by injecting concentrated stock solutions (in filtered sea water) immediately prior to sealing the aquarium for each of the N2O production assays (detailed below). A magnetic stir bar inside each tank (Fig. 1) was used and gently driven at 40 rotations per minute by a magnetic carousel in order to homogenize the water (Figueiredo-Barros et al., 2009). All aquaria were maintained in the dark (except during sampling and experimental preparations) to limit DIN uptake by any active phytoplankton in the seawater. Unfiltered seawater, pumped directly from Narragansett Bay, was used throughout Experiment 1 in order to maintain natural microbial populations as a food source similar to that in situ. Seawater was replaced (with appropriate N levels and water temperatures) every other day in order to maintain adequate food supply for the organisms and in attempt to control ammonia accumulation from bivalve excretion. To track changes in DIN concentrations during N2O production assays, water was sampled from each aquarium (40 mL) at the beginning and end of each of the 5 h assays, filtered (0.45 μm) and frozen for subsequent DIN analysis. Temperatures were manipulated by placing the aquaria into one of two environmentally controlled chambers. Control treatments (hereafter referred to as “−W”) were kept at 19 °C based on average summer temperatures in the bay over the past decade (Nixon et al., 2009). Aquaria receiving the warming treatment (hereafter referred to as “+W” for warming) were maintained at 22 °C based on the average projected increase of ocean surface temperatures in New England by 2100 (Mora et al., 2013) and were monitored with HOBO pendant temperature loggers (Onset Inc., Bourne, MA).

2.4. N2O production rates Nitrous oxide concentrations were determined in samples via a gas chromatograph (Shimadzu GC 2014) equipped with an electron capture detector (325 °C). Ultra-high purity helium as a carrier gas and a 5% methane mixture (balance argon) was used as a makeup gas with a flow rate of 2.5 mL min−1. A series of Hayesep stainless steel columns had a flow rate of 25 mL min−1 and a temperature of 80 °C based on specifications of the manufacturer (Shimadzu). Specialty gas standards (Airgas, Billerica, MA) were used to construct standard curves. The detection limit of the GC is 0.1 ppm N2O. The linear change in N2O concentrations over 5 h was determined when calculating the rate of N2O production in each aquarium. For each water sample (in the gas tight syringes), N2O concentrations were calculated as the sum of the gas phase and dissolved phases of the sampled seawater according to the following equation:

Cw = K 0 x PVwp +

(

x PVhs RT ) −1

)/V

wp ]

where Cw is the dissolved concentra-

tion of N2O (nmol L ), K0 is the solubility coefficient for N2O (mol L−1 atm−1), x′ is the dry gas mole fraction of N2O in the sample headspace (ppb), P is the atmospheric pressure (1 atm), Vwp is the volume of water phase (mL), Vhs is the volume of the headspace (mL), R is the gas constant (L atm K−1 mol−1), and T is temperature upon equilibration (K) (Walter et al., 2005; Weiss and Price, 1980). 2.5. Experiment 2: macro-epifauna contributions to M. edulis N2O production M. edulis was selected for tests of epifauna contributions to N2O production in Summer 2015 because it harbored the most visible macro-epifaunal communities, dominated by Semibalanus spp. and Crepidula fornicate. For this experiment, M. edulis individuals were obtained from a seawater out-take pipe at the MSRF and acclimated overnight at 19 °C. Experimental protocols, including animal maintenance and nitrogen addition procedures, were the same as Experiment 1. One exception is that, for this experiment, all mesocosms were filled with filtered seawater (0.2 μm, filtered twice) to minimize the influence of any N-transforming microbes in the water column and limit variability within experimental treatments. Individuals were randomly assigned to each aquarium (identical to those in Experiment 1) in a two-factor design with the following treatments: (1) no macroepifauna (experimentally removed), (2) no macro-epifauna + N addition (at the same target levels as Experiment 1, 100 μM-N), (3) intact macro-epifauna (not removed), (4) intact macro-epifauna + N addition. In the “no macro-epifauna” treatments, visible epifauna were gently removed from M. edulis individuals using a knife. Four M. edulis individuals were used per aquarium. As a handling control, mussels assigned to the “macro-epifauna” treatment were handled similarly but not scraped with a knife. All aquaria were maintained in the environmental control chambers at 19 °C. The duration of this experiment was the same as “immediate exposures” in Experiment 1 and N2O production was determined on day 1 only (within five hours of the experimental manipulations). Potential N2O production was determined as described for Experiment 1. For aquaria with N addition treatments, water samples were collected from each aquarium to confirm DIN levels immediately after N additions.

2.3. Assays of potential N2O production Potential N2O production rates were determined for bivalve in each aquarium over immediate (day 1) and short term periods (after 28-days for C. virginica and M. mercenaria; 14-days for M. edulis) using five hourlong incubations. These time periods will hereafter be referred to as “immediate” and “short-term”, respectively. During measurements of N2O production rates, silicon and fiberglass gas tight tops equipped with inflow and outflow sampling ports with closing valves were sealed on top of each aquarium (10.1 L, Fig. 1). Water samples (35 mL) were taken from each aquarium with 60 mL nylon syringes equipped with stopcock valves (Cole Parmer) immediately after sealing the aquaria with the gas tight cover by first establishing a gentle siphon in the outflow sampling port, flushing out the first 35 mL in the syringe with water from the aquaria, and then collecting the next 35 mL of water in the syringes (T0). The same procedure was repeated after three and five hours. Dissolved N2O samples for the M. mercenaria and M. edulis shortterm assays were preserved with 1 mL of 50% w/v zinc chloride in gas tight septum bottles and stored at room temperature until further analysis (approximately 3 weeks after collection). Water samples for the M. mercenaria and M. edulis short-term assays were equilibrated with ultra-high purity helium (Moseman-Valtierra et al., 2015) at the time of analysis. All other samples were equilibrated within fifteen minutes of collection. After equilibration, water was eliminated from syringes ensuring to maintain gas phase in syringe and stored in syringes with stopcock valves at 4 °C until further analysis. Gas samples were analyzed within 48 h of equilibration (details below). The “short-term” time period was shorter for M. edulis (14-days) than the other bivalve species (28-days) due to mortality of several individuals during the course of the experiment. Individuals were pooled together within the same treatment to maintain 3 individuals per aquaria. This resulted in use of three replicate aquaria per treatment rather than four for the other species at the final time point (14 days). Each bivalve species was tested separately in series using the same aquaria which were cleaned with freshwater and bleach between uses.

2.6. Water quality parameters For both experiments, water pH and DO within each aquarium were measured before and after the five-hour periods of each N2O production assay by inserting pH/DO meter into aquaria (cleaned with 70% ethanol). However, DO was not measured for either of the M. 238

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mercenaria experiments or for the M. edulis short-term exposure due logistical constraints. 20 mL of water was collected from each aquarium at T0 and T5 and analyzed for ammonium and nitrate following the same protocol as described above (field collection).

3.2. Impacts of N addition and warming on N2O production by bivalves As hypothesized, highest potential N2O production rates were generally found in the +N treatments for all bivalves. However, the potential N2O production rates observed under N enrichment for M. edulis were highest in general. They were more than an order of magnitude greater than those of C. virginica (Figs. 2, 4) and 4 to 5 times greater than for M. mercenaria in immediate and short term +N exposures, respectively (Figs. 2, 3). Biomass-normalized N2O production rates were 30 and 5 times higher for M. edulis than for C. virginica and M. mercenaria respectively (Figs. 2–4). Individual species responses are detailed separately below: Mytilus edulis M. edulis incubations under short-term exposures (14 days) had the highest potential N2O production rates observed in this study, exceeding 200 nmol ind−1 h−1 (Fig. 2A). Potential N2O production rates were significantly increased by +N treatments (F1,20 = 30.6, p < 0.0001, Table 2) and were approximately 5 times greater than controls (-N-W) for both immediate and short-term exposure periods. In contrast, warming did not affect N2O production rates, nor was there any significant nitrogen X warming interaction (Fig. 2A, Table 2). N2O production was approximately 5 times higher in the short-term exposure compared to the immediate exposure, with a significant exposure X nitrogen treatment interaction (F1,20 = 15.6, p < 0.001, Fig. 2A, Table 2). When N2O production rates were normalized per wet mass (g) for M. edulis, +N similarly had a significant (F3,8 = 6.9, p = 0.03) positive effect on N2O production rates while +W or the interaction of nitrogen X warming did not (Fig. 2B, Table 3). For M. edulis, DIN concentrations of the aquaria changed in different ways for each treatment during the (5 h) closed periods for gas production assays (Table 4). M. edulis was the only bivalve species to have higher than expected DIN values, even in the controls (Figs. 5–6). Surprisingly, initial (T0) NH4+ concentrations were approximately 4 times higher in all M. edulis treatments than in aquaria for the other species during immediate exposure (Fig. 5A). During the 5 h N2O production assays, NH4+ concentrations decreased in the +N-W and –N +W treatments; this was true for both immediate and short-term exposure periods (Fig. 5A). However, NH4+ values in the other treatments (-N-W and +N+W) increased over these time periods. In the +N treatment only, initial and final NH4+ concentrations (both T0 and T5 combined) were negatively related to N2O concentrations in the M. edulis aquaria (R2 = 0.54, p = 0.04). Initial (T0) NO3− concentrations were greater in the +N treatments than controls as expected (Fig. 6A). However, the change in NO3− concentrations during N2O incubations did not differ significantly between treatment (p = 0.09) or exposure period (p = 0.89). Interestingly, NO3− increased between T0 and T5, while NH4+ decreased in +W treatments, indicating nitrification activity (Fig. 6A). The pH dropped significantly during the N2O incubation assays (5 h) across all treatments and DO reached hypoxic levels (< 3 mg L−1) in immediate exposure treatments (Table 5). M. edulis had the greatest pH drop among species tested in this study. As DO decreased, N2O increased (R2 = 0.51, p < 0.01) for M. edulis, but no relationship was found between pH and N2O nor among pH and treatments. Mercenaria mercenaria Potential N2O production rates of M. mercenaria significantly varied with N additions, warming and exposure (Fig. 3A, Table 2). However, interactions among these factors were also significant (Fig. 3, Table 2). The largest rates of N2O production (biomass-normalized and per individual) were found for M. mercenaria in the +N-W treatment which also displayed the only evident increase (approximately 5-fold) between immediate and short-term exposures (Fig. 3B, Tables 3, 4). During M. mercenaria incubations for N2O production, T0 NH4+ (Fig. 5B) and NO3− concentrations (Fig. 6B) were higher in treatments with N additions as expected for both immediate and short-term

2.7. Invertebrate condition index After incubations, all bivalves, including mortalities, were frozen (−17 °C) and later analyzed to determine their condition index (CI). They were shucked using a scalpel and wet weights of soft tissues and shells were determined on an analytical balance. Organisms were then placed in a drying oven at 70 °C for 48 h (when a constant weight was reached) and re-weighed. The following formula was used to calculate CI (Crosby and Gale, 1990):

CI =

dry soft tissue (g) total wet weight of bivalve (g) dry shell weight (g)

1000

2.8. Statistical analysis A mixed effects model ANOVA was used to determine the statistical significance of differences in potential N2O production rates (normalized per individual) among treatments for Experiment 1 (factor 1 = nitrogen, factor 2 = warming, factor 3 = exposure period) for each bivalve species. Fixed effects were nitrogen, warming, and exposure; the tanks used for the assays are the random effects to account for repeated measures of the design. Due to potential gas loss from two syringes, two outliers from the M. mercenaria data set were excluded (one from each exposure period). N2O production by M. mercenaria was log-transformed to achieve equal variance among treatments. A twofactor ANOVA (factor 1 = nitrogen, factor 2 = warming) was used to compare biomass-normalized N2O production rates in the final (shortterm) exposure period and condition indices among treatments for the three bivalve species. Mortality rates (applicable only to M. edulis in Experiment 1) were also compared between treatments with a twofactor ANOVA. A mixed effects model (factor 1 = nitrogen, factor 2 = warming, factor 3 = exposure period) was applied to test for differences among treatments during Experiment 1 in changes in the ammonium, nitrate, pH and DO by the end of the closed N2O incubations (T5-T0). For Experiment 2 (epifaunal removal), a two-factor ANOVA was used to test for significant differences in N2O production rates of M. edulis (factor 1 = epifauna presence, factor 2 = nitrogen). For all ANOVAs, assumptions of equal variance and normality were tested using the Bartlett test and Shapiro-Wilk test, respectively. All statistical analyses were performed with JMP 10.0 software and significance levels of α = 0.05 were used. Linear regressions were performed to test relationships between N2O concentrations and water quality parameters at T0 and T5 (ammonium, nitrate, pH, DO). Regressions were performed using all treatments and exposures per species. 3. Results 3.1. Field site characterization Surface water ammonia (NH4+) concentrations ranged between 25.0 and 31.5 μM among collection sites, while bottom water concentrations ranged from 22.6 to 24.4 μM (Table 1). Surface and bottom water nitrate (NO3−) were below 2.3 μM (Table 1). Temperatures ranged between 12 and 23 °C and were generally similar between depths, with Conimicut Point having the highest temperatures and North Kingstown having the lowest (Table 1). Salinity ranged 28–33 ppt and was similar among sites and depths, which were similar to our lab conditions (Table 1). 239

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Table 1 Water quality parameters of field sites at time of invertebrate collection for Experiment 1. Site

Conimicut Point Narragansett North Kingstown

Depth

Surface Bottom Surface Bottom Surface Bottom

Ammonia

Nitrate

(μM-N)

(μM-N)

31.54 22.72 25.00 24.36 26.46 22.63

1.78 2.27 0.30 1.58 0.59 1.28

pH

DO (mg L

8.02 7.69 7.9 7.8 8.1 7.9

7.52 4.15 7.19 5.93 7.19 6.32

−1

)

Temperature

Salinity

(°C)

(ppt)

22.73 20.54 21.87 21.63 15.17 12.34

28.42 32.67 30.46 29.98 30.78 31.01

Fig. 2. Mytilus edulis N2O production for Experiment 1 in A) immediate exposure (1-day) and short-term exposure (28-days), normalized per individual, and B) shortterm exposure, biomass normalized. Error bars show standard error.

Fig. 3. Mercenaria mercenaria N2O production for Experiment 1 in A) immediate exposure (1-day) and short-term exposure (28-days), normalized per individual, and B) short-term exposure, biomass normalized. Error bars show standard error.

exposures (Table 4). Both NH4+ and NO3− concentrations increased in all treatments during N2O production assays (Figs. 5B, 6B). The increases in NO3− concentrations between T0 and T5 were significantly larger for +N-W than other treatments (F1,8 = 11.92, p < 0.01) and for the immediate than the short-term exposure periods (F1,8 = 31.75, p < 0.001). The increases in NH4+ concentrations during these assays varied between treatments but there were significant exposure X nitrogen or exposure X warming interactions (Table 4). For M. mercenaria incubations, pH decreased (Table 5) during the N2O

production assay and the change differed significantly depending on exposure (F1,56 = 80.1, p < 0.0001), warming (F1,56 = 13.8, < 0.001), and the interaction of warming X exposure (F1,56 = 9.7, p < 0.0001). However, the DO of aquaria remained above hypoxic levels, contrary to those of M. edulis (Table 5). Crassostrea virginica C. virginica was unique among the bivalves for showing N2O consumption, and this was observed in unamended treatments, but like the other species net production of N2O was observed and highest in +N 240

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Fig. 4. Crassostrea virginica N2O production (and consumption) for Experiment 1 in A) immediate exposure (1-day) and short-term exposure (28-days), normalized per individual, and B) short-term exposure, biomass normalized. Error bars show standard error. Table 2 Summary of mixed model analysis for impacts of Experiment 1 on N2O production rates (nmol ind−1 h−1) for the 3 bivalves. *Statistical significance at α = 0.05. Parameter

M. edulis

N W N*W Exposure Exposure*N Exposure*W Exposure*N*W

M. mercenaria

C. virginica

F-ratio

p-Value

F-ratio

p-Value

F-ratio

p-Value

F1,20 = 30.6 F1,20 = 0.1 F1,20 = 1.7 F1,20 = 62.2 F1,20 = 15.6 F1,20 = 0.2 F1,20 = 1.2

< 0.0001* 0.83 0.20 < 0.0001* < 0.001* 0.68 0.28

F1,22 = 113.6 F1,22 = 7.4 F1,22 = 3.4 F1,22 = 8.8 F1,22 = 13.9 F1,22 = 13.0 F1,22 = 19.0

< 0.0001* 0.01* 0.08 0.01* < 0.01* < 0.01* < 0.001*

F1,24 = 6.6 F1,24 = 3.6 F1,24 = 2.3 F1,24 = 3.6 F1,24 = 0.5 F1,24 = 18.2 F1,24 = 1.6

0.02* 0.07 0.14 0.07 0.49 < 0.001* 0.22

treatments (Fig. 4A). Rates of N2O consumption (−5.5 ± 1.3 nmol N2O ind−1 h−1 in the -N-W treatment) were similar in magnitude to the largest N2O emissions (8 nmol N2O ind−1 h−1 in the short-term +N-W treatment (Fig. 4A, Table 2). However, there were significant interactions between exposure X warming both on a perindividual and per-mass basis (Fig. 4, Table 2, Table 3). For C. virginica incubations changes in NH4+ concentrations during the N2O assays significantly varied between treatments and exposure periods (Table 4). The treatment with highest N2O production (+N-W) had noticeably higher NH4+ concentrations than all other treatments during the short term exposures. During gas assays after the short-term

Table 3 Summary of two-factor ANOVA analysis for impacts of Experiment 1 on shortterm biomass normalized N2O production rates (nmol g−1 h−1) for the 3 bivalves. *Statistical significance at α = 0.05. Treatment

N W N*W

M. edulis

M. mercenaria

C. virginica

F-ratio

p-Value

F-ratio

p-Value

F-ratio

p-Value

F3,8 = 6.9 F3,8 = 0.3 F3,8 = 6.1

0.03* 0.15 0.48

F3,12 = 11.6 F3,12 = 3.5 F3,12 = 3.7

0.005* 0.08 0.08

F3,12 = 10.0 F3,12 = 5.0 F3,12 = 7.8

0.01* 0.05* 0.02*

Table 4 Results of mixed model analysis for change in ammonium concentrations between the beginning (T0) and end (T5) of each 5-h incubation in Experiment 1. *Statistical significance at α = 0.05. Parameter

Ammonium change (T5-T0) N W N*W Exposure Exposure*N Exposure*W Exposure*N*W

M. edulis

M. mercenaria

C. virginica

F-ratio

p-Value

F-ratio

p-Value

F-ratio

p-Value

F1,8 = 5.31 F1,8 = 5.17 F1,8 = 6.82 F1,8 = 12.80 F1,8 = 2.72 F1,8 = 1.15 F1,8 = 1.80

0.05* 0.05* 0.03* 0.007* 0.14 0.31 0.22

F1,8 = 6.42 F1,8 = 12.28 F1,8 = 7.25 F1,8 = 2.34 F1,8 = 14.24 F1,8 = 6.39 F1,8 = 2.97

0.04* 0.01* 0.03* 0.16 0.01* 0.04* 0.12

F1,14 = 0.86 F1,14 = 5.28 F1,14 = 0.64 F1,14 = 16.20 F1,14 = 4.61 F1,14 = 39.68 F1,14 = 6.57

0.37 0.04* 0.44 0.001* 0.05* < 0.0001* 0.02*

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Fig. 5. Ammonium concentrations for Experiment 1 at start (light grey, T0) and end (dark bars, Tf) of each incubation for the three bivalve species: (A) Mytilus edulis; (B) Mercenaria mercenaria; (C) Crassostrea virginica treatments. Standard error bars shown. N = Nitrogen addition, W = Warming, NW=Nitrogen + warming. Light bar = initial (T0) and dark bar = final (T5).

exposure, average NH4+ concentrations decreased for the +N-W treatments and controls (-N-W) but increased for the warming treatments (the –N+W and +N+W) (Fig. 5C). For C. virginica, NH4+ concentrations (both T0 and T5) were negatively related to N2O concentrations in the +N-W treatments (R2 = 0.47, p = 0.02). This indicates the high N2O production under N addition may have been fueled by nitrification and this relationship was not observed with warming. There was an overall 2-fold decrease in NH4+ concentrations between immediate and short-term periods across treatments, particularly in the N amended treatments, indicating active nitrogen cycling occurred (Fig. 5C). While ammonium was declining generally, concentrations of NO3− were displaying a 2–5 fold increase between exposure periods and were particularly high in the short-term +N-W treatment where N2O production had been highest. (Fig. 6C). During gas assays, declines in NO3− were largest in both treatments with nitrogen addition (F1,14 = 22.68, p < 0.001), and varied with exposure period (F1,14 = 26.1, p < 0.001) with significant interactions between exposure and nitrogen (F1,14 = 14.53, p = 0.002). For C. virginica, pH and DO decreased between T0 and T5 of assay for both exposure periods but were generally high for all species in Experiment 1 (Table 5). Nitrogen (F1,56 = 5.3, p = 0.03), warming (F1,56 = 5.4, p = 0.02) and warming X exposure (F1,56 = 50.8, p < 0.0001) significantly negatively affected the drop in pH (Table 5). The decline in DO levels was significantly greater with longer exposure (F1,56 = 271.9, p < 0.0001) and warming (F1,56 = 75.0, p < 0.0001).

3.3. Physical condition of bivalves M. edulis individuals had a 31% mortality rate over the short-term 14-day incubation period in Experiment 1. During this period, there was no significant difference in mortality rates per treatment (F3,12 = 0.04, p = 0.99). The other species did not experience mortality during this study, nor was there any mortality for M. edulis in Experiment 2. M. mercenaria had the highest condition index (CI) among the three species while C. virginica had the lowest (Fig. 8). For C. virginica, CI was significantly lower among individuals in the warming treatments (Fig. 8, F3,44 = 0.67, p < 0.01). There was a significantly interaction between nitrogen X warming for the CI of M. edulis such that the CI of mussels in the +N + W treatment were highest while +N-W or –N + W treatments had average CIs lower than controls (Fig. 8, F3,44 = 2.12, p = 0.02). 3.4. Experiment 2: macro-epifaunal impacts on M. edulis N2O production Similarly to Experiment 1, N addition treatments significantly increased N2O production (F3,10 = 6.7, p = 0.03). However, there was no significant impact of the presence of macro-epifauna (F3,10 = 2.1, p = 0.18) nor the interaction of N X Epifauna (F3,10 = 0.03, p = 0.87). In Experiment 2, M. edulis showed lower overall N2O production rates (Fig. 7) than M. edulis in Experiment 1 (Fig. 2). M. edulis in the +Epifauna +N treatment had the highest average N2O production rates when normalized per wet mass, at

Fig. 6. Nitrate concentrations for Experiment 1 at start (T0) and end (Tf) of each incubation for the three bivalve species: (A) Mytilus edulis; (B) Mercenaria mercenaria; (C) Crassostrea virginica. N = Nitrogen addition, W = Warming, NW = Nitrogen + warming. Light bar = initial (T0) and dark bar = final (T5).

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Table 5 Average pH and dissolved oxygen (DO) values at the start (T0) and end (T5) of each experimental incubation period. Experiment

Species

Exposure

DO (mg L−1)

pH T0

1

M. edulis

1

M. mercenaria

1

C. virginica

2

M. edulis

Immediate Short-term Immediate Short-term Immediate Short-term

8.0 7.8 8.1 7.9 8.0 8.0 7.9

T5 ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.1

7.3 7.3 7.7 7.8 7.4 8.1 7.6

T0 ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.1

9.1 ± N/A N/A N/A 9.2 ± 9.2 ± 9.3 ±

T5 0.04

0.10 0.10 0.04

2.7 ± N/A N/A N/A 7.3 ± 8.1 ± 7.6 ±

0.19

0.15 0.11 0.13

highest emissions were likely related to the most severe hypoxic conditions and physiological stress than to characteristics of the host species per se. The highest N2O emissions (over 14 days) may have been due to artificially high ammonium levels that were observed in the laboratory mesocosms, which may have increased nitrification rates, as has been previously described in laboratory experiments with shellfish (Kellogg et al., 2013). Based on declines in ammonium and increases in nitrate, the +N+W and –N+W treatments were ones which display evidence of this potential artifact. The lower N2O rates found in the immediate exposure conditions may therefore be more representative of field conditions. 4.1. Key roles of nitrogen availability and oxygen in N2O production by M. edulis Notable differences in the magnitude of increased N2O emissions between bivalve species reflected unique water chemistry conditions which are linked to physiological activities of the animals. For M. edulis in Experiment 1, surprisingly high NH4+ concentrations during M. edulis assays (~50–250 μM NH4+ across all treatments) in the immediate exposure (Fig. 5A) can be attributed to high rates of excretion, which then increased substrates available for microbial communities that could produce N2O. Heisterkamp et al. (2013) directly measured excretion rates of M. edulis by examining NH4+ accumulation and concluded that M. edulis N2O production is sustained by their own NH4+ excretions through tightly coupled nitrification-denitrification. Yet, the highest overall N2O production rates in M. edulis were shown in the 14-day short-term exposure across all treatments (Fig. 2). During the N2O assays of M. edulis for Experiment 1, significant declines in NH4+ concentrations (Fig. 5A) and increases in NO3− (Fig. 6A) between T0 and T5 suggest that nitrification (potentially an artifact of high initial ammonium levels) fueled at least some of the observed N2O production, particularly during the short-term exposure. Oxygen availability also plays a key role, however, in mediating N2O production. In Experiment 1 the highest N2O production rates by M. edulis occurred along with drastic decreases in DO and pH concentrations during the five-hour immediate N2O assay period (Table 5). These transient hypoxic conditions were not observed for other species or Experiment 2, suggesting that filtration and/or respiration rates were high for these mussels and that their behaviors were causing unusually rapid consumption of oxygen and production of CO2. This large drop in DO from an average of ~9 mg L−1 to 2.5 mg L−1 between T0 and T5 was exceptional among all bivalves assayed. The decrease in M. edulis DO in Experiment 1 at T5 was about 3 times greater than mussels assayed in Experiment 2. The source population of M. edulis in Experiment 1 may have been in poor health, as they displayed a relatively high mortality rate (25%) over the 14 days following the initial hypoxic assays. Although the reasons for those initial drops in pH and DO are unclear, there may have been feedbacks such that hypoxia and low pH during the incubation induced higher metabolic demands, higher respiration rates and possibly higher filtration rates from this source population of M. edulis (Riisgard and Randløv, 1981). The drop in pH may have been

Fig. 7. Average N2O production rates from M. edulis in Experiment 2 exposed to: Control = no macro-epifauna, no N addition (white bars); +N = macroepifauna removed, N addition (light grey); Epifauna = macro-epifauna present, no N addition (dark grey); Epifauna+ N = macro-epifauna present, N addition (black). Error bars show standard error.

0.15 nmol N2O g−1 h−1. Average production rates normalized per wet mass for the remaining treatments were as follows: control (-epifauna -N) = 0.02 nmol N2O g−1 h−1, +N treatment = 0.11 nmol N2O g−1 h−1, and +Epifauna treatment = 0.05 nmol N2O g−1 h−1. Similarly to Experiment 1, pH (t13 = −7.3, p < 0.01) and DO levels (t13 = −13.6, p < 0.01) did significantly drop between the start and end of the N2O assays (Table 5). However, DO concentrations did not reach hypoxic levels, in contrast to Experiment 1. N2O production rates from all treatments showed a weak negative relationship with pH (R2 = 0.37, p = 0.02) as well as DO (R2 = 0.33, p = 0.03). 4. Discussion This study revealed that three prominent bivalves may be sources of N2O when exposed to high levels of fixed N, and displayed a switch from sink to source in one species (C. virginica, Fig. 4) with N addition, but showed surprising differences among species. The increase of N2O production in response to N addition is expected, as more N is available for various metabolic pathways of microbes associated with the bivalve guts and shells. However, the large differences in the direction and magnitude of N2O emissions among species (Figs. 2, 3, 4) reveals that aspects of shellfish biology (size, filtration activity, respiration rates) play a key role. M. edulis produced the highest mass-specific N2O emissions reaching 7.5 nmol N2O g−1 h−1. However, notable differences in rates of N2O production by M. edulis from two different source populations and sampling times (in Experiments 1 and 2) suggest that 243

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caused by both the increase in dissolved CO2 from respiration by the mussels as well as from nitrification which consumes NH3, a contributor to seawater alkalinity. We hypothesize that nitrification was a key pathway of N2O production from M. edulis (see below) and recognize that it may have been in part an artifact. Thedrop in pH levels may in part be due to increased nitrification rates. The significant negative relationships between N2O concentrations and DO concentrations from M. edulis (Experiment 1 and 2), whereby as DO decreased, N2O increased, support that hypoxic conditions favor high N2O concentrations. However, hypoxia and low pH were experienced during the 5 h assays across all treatments, whereas only N addition treatments had significantly higher N2O emissions. Thus, the combination of hypoxic conditions and increased N (as both ammonium and nitrate) were necessary for the high N2O production rates from M. edulis. The negative relationship of N2O concentrations to NH4+ concentrations among M. edulis with N additions could reflect low oxygen availability or some other related constraint on nitrification.

observed in N2O production by the two groups of M. edulis employed in our studies, and they highlight an underexplored potential link between health of shellfish and changes in their nutrient transformation efficiency. 4.3. N2O consumption by C. virginica indicated complete denitrification C. virginica was unique for displaying N2O consumption (a decrease in concentrations over the 5 h assay period) and showed the lowest overall N2O production rates in this study (0.5–2.8 nmol N2O ind−1 h−1) which is the same order of magnitude as other aquatic bivalves (Heisterkamp et al., 2010; Stief et al., 2009, p. 200; Svenningsen et al., 2012). The consumption rates of N2O were generally low as well and were observed in the -N-W and +N-W treatments after immediate exposures only. N2O consumption occurs in cases of complete denitrification and may have occurred in anoxic microenvironments, such as the bivalve gut. Low N2O production by C. virginica may be explained by the time of collection and experimentation. The C. virginica experiment was conducted during the early Spring when phytoplankton concentration in the water is higher, as opposed to the mid and late summer of M. mercenaria and M. edulis, respectively, when abundances are lower (Sundbäck et al., 2000). The phytoplankton density in the unfiltered aquarium water may have been large enough to compete with the nitrogen-transforming microbes for fixed N (Vieillard and Fulweiler, 2014), though this should have been minimal as we performed this study in the dark to minimize the influence of phytoplankton. Further, C. virginica seemed to have lower excretion rates to feed coupled nitrification-denitrification than other bivalves since there no significant NH4+ changes by the end of the 5 h incubation for all treatments and exposure times (Fig. 5C).

4.2. Role of bivalve health and sources of intraspecific variation in N2O production The notable mortality rates of M. edulis in Experiment 1 seem to conflict with the relatively high condition indices which were well within the range of the other bivalves in our study (Fig. 8). Possibly these indices, though commonly employed in ecological studies (Capelle et al., 2014; Lander et al., 2012; Lee, 1996), are not sufficient to detect short term and acute physiological stress that may have affected the M. edulis individuals. CI were not measured for the M. edulis individuals in Experiment 2,which did not show such drastic low DO concentrations during the same N2O assay periods and were processed with identical methods. This highlights the validity of the methods employed and the role of intra-specific variation in potential N2O production. However, the difference in field source population, collection times (late summer for Experiment 1 and early spring for Experiment 2), and filtered vs unfiltered water may be significant contributors to the differences in health of the bivalves and subsequent N2O production. Decreased health of M. edulis has been documented between June–August post spawning by several biomarkers such as increased heartrate and decreased feeding (Hagger et al., 2010). Food availability may also be a significant contributor, due to higher plankton abundances in the Spring compared to the late Summer (Borkman and Smayda, 2009; Sundbäck et al., 2000), thus the mussels in Experiment 2 may potentially have had lower physiological stress levels. Further, Experiment 1 used unfiltered seawater, which may have introduced disease agents; however, Experiment 2 used filtered seawater, thereby reducing possibility of potential pathogens in the aquaria. All of these factors may have contributed to the differences

4.4. Nitrification as a likely source of N2O production by bivalves Nitrification may have been the source of N2O production for M. edulis, C. virginica and M. mercenaria. M. edulis showed highest overall N2O production in the short-term exposure, which also had decrease of NH4+ (Fig. 5A) and an increase of NO3− (Fig. 6A) between exposure times, showing evidence for potential nitrification as a pathway to N2O. Although DO levels were low for this species, perhaps there were microsites of oxygen on the mussel shell to allow for nitrification to occur. For C. virginica, there was a 2-fold decrease in NH4+ (Fig. 5C) and 3fold increase in NO3− (Fig. 6C) between immediate and short-term exposure in the treatment with highest N2O production. Additionally, there was a significant negative relationship between N2O concentrations and NH4+ concentrations in that treatment (+N-W) for C. virginica, which also indicated nitrification. M. mercenaria showed similar patterns, where the +N-W treatment after 28-days in the short-term

Fig. 8. Condition index ratio per species for Experiment 1. Error bars show standard error. N = Nitrogen added (light grey), W = Warming (dark grey), NW = Nitrogen added + Warming (black), controls (white). 244

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exposure showed highest N2O production (Fig. 3). Within this experiment, there was evidence of nitrification due to the decrease in NO3− between exposure times (Fig. 6B). Heisterkamp et al. (2013) also showed nitrification to be an important pathway in N2O production from bivalves, particularly in the shell biofilms. We did not test for shell biofilm N2O production in this experiment, but it may be that external microbial consortia are the main contributors to N2O production related to these bivalves, rather than in the gut microbes, and that a longer exposure to high N conditions increases microbial abundance and activity. Our experiments also were not designed to test for roles of biodeposition by bivalves in sediments which may increase denitrification and/or regeneration in marine ecosystems.

of bacteria (McHenery and Birkbeck, 1985); therefore, the rates described associated with M. edulis in the epifauna removal experiment (Fig. 7) are likely produced from N2O producing microbes already present within the gut of the organism before the start of this experiment as well as potential microbial biofilms on their shells. 5. Conclusion Our study illustrates that N loading increases N2O emissions from bivalve shellfish (M. edulis, M. mercenaria, C. virginica) while temperature has little direct effect over short terms. Potential N2O emissions from bivalves differed between species; M. edulis produced the highest rates of N2O, which we believe to be due to a combination of high NH4+ production and the induction of hypoxic conditions that were related to high respiration rates and possibly physiological stress of one source population. Nitrification likely played a key role in N2O production from these bivalves based on patterns of NH4+ consumption and NO3− production over the short-term duration of these experiments, and it may have been artificially increased in the case of some treatments for M. edulis. We did not find that macro-epifauna on M. edulis significantly contribute to N2O production and thus microbial communities in guts and on shells are major sources. Notably, N2O emissions from all bivalves increased with exposure period to N loading and these animals may therefore be contributing to N2O emissions in degraded, eutrophic ecosystems. Since marine bivalves are abundant in coastal systems where the combination of eutrophic, warming and low DO often coincide, their potential contribution to N2O emissions from benthic systems warrants further investigation.

4.5. Warming impact on bivalve N2O production Our findings indicate that warming had little direct impact on N2O emissions associated with these bivalves, even when condition indices decreased (C. virginica, Fig. 8) and that temperature's effects are interactive with other factors in complex ways. For M. mercenaria, warming impacts on N2O production rates were significant but complicated by the interaction of exposure X warming and exposure X warming X nitrogen (Fig. 3, Table 2). Similarly, for N2O production by C. virginica, there were interactive effects between warming and exposure period (Figs. 2–4, Table 2). Denitrification rates are typically dependent on both temperature and NO3− availability (Stief and Schramm, 2010) among other factors and possibly higher rates of complete denitrification under the combination of higher nitrogen and warming with a longer duration of exposure led to lower N2O production rates (Fig. 4). Warming effects were not significant for M. edulis, possibly because of exceptionally high DIN concentrations and low DO which likely trumped any warming impacts, although these factors are tightly linked in the environment. No known studies have thus far examined warming impact on N2O production from bivalve species, though studies have examined warming impacts on bivalve health and growth and have shown decreased shell growth, increased mortality, and lowered immune response (Hiebenthal et al., 2012; Lannig et al., 2006; Mackenzie et al., 2014; Matoo et al., 2013). The duration of these studies were 3–6 months, and we recognize that warming has more substantial effects on shellfish health and thereby likely also on their N-transformation rates over longer time periods.

Acknowledgements This research and part of M. Gárate's graduate stipend was supported by the National Science Foundation (NSF OCE-1225825) awarded to Moseman-Valtierra. M. Gárate was also supported by a NSF Graduate Research Fellowship and part of her dive equipment was provided by the American Academy of Underwater Sciences Hollis Dive Gear Award. A. Moen was supported by a NSF-funded EPSCoR Summer Undergraduate Research Fellowship. The authors would like to thank Stephen Granger for boat access, Ed Baker for use of the NSF-EPSCoRfunded Marine Sciences Research Facility and assistance with the animal maintenance, Linda Green for nutrient analysis, the Art Gold lab for access to an additional gas chromatograph, and Dr. Dawn Cardace for sharing lab space and supplies. We would also like to thank Robert Ventura, who was supported by a NSF Summer Undergraduate Research Fellowship in Oceanography and helped tremendously with initial methods development and experimental trials. Further thanks to Moseman-Valtierra lab for excellent assistance: Dr. Rose Martin, Jesse Iacono, Dennis Conetta, Dr. Elizabeth Brannon, Ryan Quinn and Heather Chan.

4.6. Macro-epifauna contribution to M. edulis N2O production Contrary to our hypothesis, results of Experiment 2 (Fig. 7) suggest that macro-epifauna organisms did not significantly contribute to N2O production of M. edulis in this population. Shell microbial biofilms may have contributed to N2O production, explaining the discrepancy between our results and past research that has found shell biota to be significant contributors (Heisterkamp et al., 2013, 2010; Svenningsen et al., 2012). M. edulis individuals from the epifaunal removal experiment (Fig. 7) had lower N2O production when compared to Experiment 1 (Fig. 2), and the former were more consistent with previous production rates of M. edulis (Heisterkamp et al., 2010; Stief and Schramm, 2010). They are also on the same order of magnitude as C. virginica biomass-normalized N2O production rates, while about 2 times less than M. mercenaria biomass-normalized rates. Apart from hypoxic conditions for M. edulis in Experiment 1, we hypothesize that the difference between these experiments is due to the filtered vs. unfiltered water used, respectively. N2O production in the epifauna (with and without N addition, Fig. 7) treatment was ~3 times lower than immediate exposure control results for M. edulis in Experiment 1 (Fig. 2). Filtration of the water column may have significantly reduced the abundances of bacteria (including N2O producers) that M. edulis could normally ingest from the water column. M. edulis is an efficient filter feeder that ingests large amounts

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