The impact of oysters on the fate of nitrogen inputs to estuarine sediments

Ruby An

The University of Chicago

Chicago, IL 60637

Advisor: Anne Giblin

The Ecosystems Center

Marine Biological Laboratory

Woods Hole, MA 02543

Semester in Environmental Science

Class of 2015

ABSTRACT

Nitrogen pollution from anthropogenic sources is a major threat facing estuaries in the Falmouth

area with severe ecological and economic consequences. Oyster aquaculture is a novel water

quality management strategy that could potentially be implemented in Falmouth estuaries.

Oysters can play an important role in estuarine nitrogen cycle processes by consuming

phytoplankton blooms stimulated by anthropogenic nitrogen inputs. Some studies suggest oysters

may also increase the denitrification potential of estuarine sediments; however, this is

complicated by the ecosystem-level affects of eutrophication on oxygen and nitrate

concentrations. To investigate how oysters alter estuarine nitrogen cycling in the context of

historical eutrophication, I collected sediment cores from two estuaries in the Falmouth area,

Little Pond (highly eutrophic) and Waquoit Bay (less eutrophic). I cultured N15 labeled

phytoplankton and added it to sediment cores with and without oysters from each site. I measured

oxygen, ammonium, and nitrate concentrations, calculated fluxes over a 15-day incubation

period, and used stable isotope analysis to track nitrogen fate. Results indicate that oysters

incorporate about 40% of nitrogen inputs into their tissues and decrease the relative amount of

nitrogen that ends up in sediments by over 50%. Within the timeframe of this project, oysters did

not increase denitrification potential or significantly alter final nitrogen concentrations in the

water column. A discrepancy in initial oxygen exposure likely neutralized short-term differences

in site response; however, overall data suggests that biogeochemical context determined by site

history, in particular oxygen availability, plays a critical role in determining the impact of oyster

aquaculture on estuarine nitrogen cycling.

Key words: oysters, Falmouth estuaries, sediments, nitrogen cycle, stable isotope analysis

INTRODUCTION

Nutrient pollution from anthropogenic sources is a major threat facing estuaries in the

Falmouth area and worldwide. Excess anthropogenic nitrogen entering coastal watersheds from

sources such as septic wastewater and lawn fertilizers stimulates large algal blooms in historically

N-limited estuarine ecosystems. Algal blooms shade out native eelgrass beds that serve as

important habitats for many ecologically and economically valuable species; furthermore, as algal

blooms decompose, they can cause severe hypoxic events that suffocate benthic organisms and

lead to massive fish kills. The widespread degradation of ecosystem services that results from

eutrophic conditions has negative consequences on an ecosystem-level that includes the local

Falmouth community.

Oyster (Crassotrea virginica) aquaculture as a water quality management strategy has

been employed at locations including New York Harbor, Chesepeake Bay, and as of 2013 at

Little Pond, Falmouth, MA (Billion Oyster Project, Oyster Recovery Project, Little Pond

Shellfish Demonstration Project). Oysters are extremely effective filter feeders and can feed on

algae blooms stimulated by excess nitrogen pollution (Team, 2007). While the extent to which

oyster aquaculture can save our estuaries is a controversial subject (Pomeroy et al. 2007), oysters

play an important role in many estuarine nitrogen cycle processes. Oysters incorporate nitrogen

from algae they consume into their own tissues, deposit it to the sediment as feces or

pseudofeces, or excrete it to the water column as ammonia (Fig. 1). Some studies suggest that

oysters not only remove nitrogen through phytoplankton consumption and incorporation into

their own tissues, but may also increase the denitrification potential of estuarine sediments thus

increasing loss of nitrogen from estuaries as N2 gas (Kellog et al. 2013). This possible

relationship is complicated by the fact that microbial denitrification is tightly coupled to

nitrification (Jenkins and Kemp, 1984), a process that requires sufficiently aerobic conditions

uncommon in eutrophic estuaries.

In this context, the goal of my project was to investigate how the presence of oysters alters

the fate of anthropogenic nitrogen inputs to estuarine sediments. I examined the estuarine

nitrogen cycle by quantifying oxygen, nitrate and ammonium fluxes and tracking the relative fate

of N15 labeled organic matter inputs to sediment cores incubated for 15 days. I used stable

isotope analysis to determine whether nitrogen inputs ended up in the sediment, water column,

incorporated into oyster tissue, or denitrified as N2 gas. I collected sediment cores from two

estuaries in the Falmouth area that historically experience different levels of nitrogen pollution to

determine whether the history of eutrophication altered estuarine sediment responses to oyster

aquaculture.

Site Background

I collected sediment cores from Little Pond and Waquoit Bay. Situated in a highly developed

watershed, Little Pond experiences substantial nitrogen runoff from downtown Falmouth,

Falmouth mall, and surrounding residential homes. As a national research reserve located in a

less-developed watershed, Waquoit Bay experiences less nitrogen runoff and organic matter

loading than Little Pond but has still suffered from the effects of eutrophication (Bowen et al.

2001). In her 2015 SES Project, Em Stone found that Little Pond sediment was 24.2% organic

matter by weight and Waquoit Bay sediment was 15.9% organic matter by weight. Both these

sites are locations where oyster aquaculture occurs. Caged oysters were deployed in Little Pond

beginning in 2013 as part of the Little Pond Shellfish Demonstration project with the goal of

improving water quality. Waquoit Bay is home to Washburn Island Oysters, a commercial

aquaculture business. Little Pond cores were collected on 11/15 from the middle of the pond near

Narragansett Street on (Fig. 2 – left). Waquoit Bay cores were collected three days later on 11/18

(due to 11/15 equipment malfunction) from the center of the head of the bay (Fig. 2 – right).

METHODS

Experimental Design

We collected six sediment cores per site and subjected them to three treatments (two cores

per treatment): control (CTRL), N15 organic matter addition (OM), and N15 organic matter

addition with oysters (OM+OYST). We used sediment core tubes that were 9.5 cm in diameter

and 30 cm in height. During collection, we aimed for a sediment height of 10 cm. Post-collection,

cores were incubated in room-temperature seawater (16-20 °C), bubbled with air to maintain

oxygen levels, and kept well mixed with magnetic stir bars (Fig. S2). Water level was maintained

at about 2 cm below the top of the core tube, except during oxygen flux measurements.

Treatments and sampling consisted of initial flux measurements followed by a 15-day

incubation period. Once all cores were collected, the overlying water in each core was drained

and replaced with 25 mm GF/F filtered site water (collected at the same location as the cores).

This allowed us to measure initial oxygen, nitrate, and ammonium fluxes from the sediment.

After these measurements, oysters were placed in each of the OM+OYST cores. Core treatments

and subsequent sampling occurred over the next 15 days. N15 organic matter was added daily to

both OM and OM+OYST cores on days 1-6. On day 7, oysters were removed and all cores were

closed up for oxygen and N2 flux measurements (Post OM+ flux). Subsequently, all cores were

incubated under the same conditions until day 15 on which a final oxygen and N2 flux was

performed.

N15 Algae Aquaculture

For the N15 organic matter additions, we cultured 22 L of the microalgae Isochyysis

galbana (T.Iso) in F/2 growth media enriched approximately 20% with additional 15N. To do so,

40 mL of 100 mM 15NO3 was added to 18 L of F/2 media and inoculated with about 4L of an

existing T.Iso culture (>3 million cells/mL). This culture was placed under constant light for six

days prior to the start of experimental treatments.

The OM addition process required a concentration of algal cells to avoid adding large

amounts of N15 from the growth media, which would obscure the N15 signal from the organic

matter. Since T.Iso cells were too small to be filtered (4-6 µm), 50 mL Falcon tubes were used to

centrifuge down 45 mL of algal mixture at a time and excess liquid poured off, resulting in a

pellet of organic matter. This process was repeated three times, such that each tube contained the

concentrated algae from 4 x 45 mL of algal mixture. To add to the cores, this algal pellet was

frozen for 30 minutes to kill algal cells, thawed, resuspended and rinsed out with about 10 mL of

site water. Two tubes, the equivalent of 360 mL of algal mixture, were added per core per day.

This was roughly calculated as the amount of organic matter equivalent to 3% the dry weight of

the oysters. Since algal cell concentration likely varied within the culture over the six day period,

an additional two tubes each containing 45mL of algal mixture were centrifuged and frozen per

day to track the total amount of N15 added.

Oysters

Oysters were obtained with the help of Falmouth resident Sia Karplus from an oyster reef

in West Falmouth Harbor near Chappaquoit bridge. Sia Karplus started the reef from oyster spat

on shells as part of ongoing efforts by the town of Falmouth to improve water quality. These

oysters were collected 10 days prior to the start of experimental treatments. In the interim, oysters

were kept in running seawater and fed a mixture of diatoms and flagellates. Three oysters of

equal size distribution were added to each OM+OYSTR core in hanging baskets constructed from

plastic netting to simulate caged oyster aquaculture (Fig. S1). After oysters were taken out of the

cores on day 7, they were fed non-enriched phytoplankton several times to clear their guts before

they were frozen on day 9.

Sampling

To measure oxygen fluxes, tubing and air stones for core aeration were removed and the

cores closed with specialized tops to prevent air from entering the core water. Oxygen

concentrations were measured using WTW probes and recorded in each core every couple hours

until the concentration dropped below 5 mg O2/L. Time intervals varied, as some cores took

overnight to reach desired O2 concentrations. After initial oxygen flux measurements, core tops

were removed and aeration resumed. After the day 7 oxygen flux (Post OM+), core tops were left

on with some headspace and aeration resumed.

To measure nitrate and ammonium fluxes, water samples were collected and filtered using

25 mm GF/F swinnex filters and frozen for later analysis. The total liquid volume removed from

each core (~33 mL per sample due to rinsing) was replaced with 25 mm GF/F filtered site water.

Water samples for initial NH4 and NO3 fluxes were taken at four time points over the same time

period as the initial oxygen flux measurements. Water samples for NH4 and NO3 fluxes over the

15-day treatment incubation were taken daily (with the exception of days 8-10 over Thanksgiving

break).

Following the 15-day incubation period, we analyzed the oysters, sediment, core water

samples, and N2 water samples for N15 enrichment. Oysters were unfrozen and shucked. Oyster

soft tissue was combined per core, cut up, oven dried overnight, and ground to a powder. The top

4 cm of sediment from each core was sectioned off into metal tins, mixed, air-dried overnight and

by oven, and homogenize by hammering in Ziploc bags. Wet and dry weights were recorded for

both oysters and sediments. 100 mL GFF filtered water samples were taken on day 7 and day 15

to determine N15 enrichment of dissolved inorganic nitrogen. These water samples were

combined per core. Devarda’s alloy was used to perform ammonium diffusions. We took water

samples for N2 concentration and N15 enrichment at the end of the day 7 and day 15 oxygen flux

measurements. These samples were stored underwater in the same incubating bath as the cores to

maintain temperature. To measure N2 enrichment, we used a mass inlet membrane spectrometry

(MIMS). OM addition aliquots (12 tubes) used to measure N15 additions were combined into two

replicates of total addition over days 1-6 and analyzed for N15 content.

Nutrient and Data Analysis

Nitrate concentrations were determined using the Lachat method adapted from Wood et

al. 1967. Ammonium concentrations were determined using a modification of the method from

Strickland and Parsons (1972) based on the phenol-hypochloric method from Solarazano (1969).

Ammonium samples for days 1-15 were diluted 5:1. Core water volume calculated from water

height and core diameter was used to convert concentrations to molar amounts.

I used R to perform all data processing and statistical analysis. Nutrient fluxes were

calculated as a linear regression of collected concentration data and times. Fluxes with R2 values

less than 0.6 were omitted from further analysis. Week 1 nitrate and ammonium fluxes were

calculated using concentrations from days 1-6. Week 2 fluxes were calculated using

concentrations from days 12-15. Factorial design ANOVA was used to determine the statistical

significance of flux differences by treatment, site, and treatment:site interaction. N15 enrichment

above natural levels for sediment and water samples was calculated using the controls as

reference. Background oyster N15 levels were taken from previous SES data (Waquoit Bay

oyster).

Methodological Exceptions

On day 6, an extra 180 mL of algal mixture was added by accident to both Little Pond

OM+OYST cores. The magnetic stir bar in one of the Waquoit Bay OM cores (WB4) consistently

became stuck, resulting in reduced mixing and elevated oxygen concentration measurements

throughout the experiment. During oxygen flux measurements, the top of one of the Little Pond

control cores consistently leaked in air, likely elevating O2 concentration measurements.

Ammonium data for a Waquoit Bay OM+OYST cores (WB1) time point 4 was removed from

flux calculations as air entered the sample stream during analysis, artificially elevating the

reading.

RESULTS

Initial oxygen flux measurements showed some variation in cores; however differences by

treatment assignment and by site were not significant (Table 1.1). The average initial O2 flux was

-10.13 mgO2/d for Little Pond cores and -9.31 mgO2/d for Waquoit Bay cores. A week of OM

additions increased oxygen uptake: during the day 7 O2 flux, both OM and OM+OYST cores had

much greater O2 fluxes whereas CTRL fluxes were fairly similar to initial measurements (Fig. 5).

In general, OM cores had a slightly higher O2 uptake than OM+OYST cores. Final oxygen flux

measurements on day 15 were lower than on day 7, but still higher in OM and OM+OYST than

CTRL cores (Fig. 4).

Initial fluxes for both ammonium (NH4) and nitrate (NO3) varied with no significant

difference by treatment assignment or site (Table 1.2-3). Initial NH4 fluxes when significant (R2

> 0.6) were positive and averaged 15.1 µmol/d for Little Pond cores and 11.7 µmol/d for Waquoit

Bay cores. During Week 1, NH4 concentrations increased in all cores, approaching 300 µM in

OM+OYST cores (Fig. S2). Week 1 NH4 fluxes were significantly different by treatment

(p<0.0001) but not site (Table 1.2). OM core fluxes were higher than CTRL cores; OM+OYST

core fluxes were on average 35 µmol/d higher than OM core fluxes (Fig. 5). Week 2 NH4

concentrations exhibited much more variation: notably, OM+OYST and Waquoit Bay OM cores

showed significant negative NH4 fluxes (Fig. 5).

Initial NO3 fluxes were negative in all cores and averaged -7.74 for Little Pond cores and

-4.67 for Waquoit Bay cores. NO3 concentrations were quite low during Week 1, on average only

3.34 µM, but began increasing over the last three days of the experiment (Fig. S3). For most

cores linear regression did not describe the majority of variation (R2<0.6) and for those it did,

fluxes were small and negative. In the last three days of Week 2 (days 12-15), all cores exhibited

significant positive nitrate fluxes (Fig. 6). CTRL cores had higher NO3 fluxes than OM and

OM+OYST cores. In Waquoit Bay cores, fluxes exhibited more variation but were on average

higher than Little Pond cores (Fig. 6).

Stable isotope analysis indicated significant enrichment of oysters, sediment, and water

samples but no enrichment of N2 compared to controls and background values. OM additions

were highly enriched (on average ∂15

N=16000). However, estimates for total N15 added as

organic matter per core had a large standard deviation, 20.3 ± 3.3 µmol/core (22.0 ± 0.4 for Little

Pond OM+OYST cores). Total recovered OM N15 per core (sum of sediment, water, oysters

when present) was on average 22.7 µmol, within one standard deviation of average total N15

addition (Fig. 7). Core replicates by treatment and site were nearly identical and sites were very

similar (Fig. S4). In OM cores, about 60% of N15 was found in the sediment and 40% in the

water. In OM+OYST cores, about 25% of N15 was found in the sediment, 35% in the water, and

40% in the oysters (Fig. 8).

DISCUSSION

As expected, nutrient fluxes indicated that adding organic matter to simulate eutrophic

conditions increased sediment core oxygen uptake. Lower oxygen uptake by sediment in

OM+OYST cores suggests oysters decreased the amount of organic matter readily available to be

oxidized either by incorporating it into their tissues or repackaging it as biodeposits. This was

corroborated by visual observations that water in OM+OYST cores was much clearer than in OM

cores. It is important to note that while total oxygen uptake by sediments in OM+OYST cores was

lower, this flux was measured with oysters removed. Based on oyster respiration rates of 0.1-0.5

mgO2/hr/ind, oyster respiration per core (3 oysters) would have been in the range of 7.2-36.0

mgO2/d (Uline 2013). Thus, total oxygen uptake in OM+OYST cores would likely have been

significantly greater than in OM and CTRL cores. These calculations suggest that oyster

aquaculture has the potential to further stress estuaries already experiencing hypoxic conditions.

While one would anticipate that oxygen fluxes would be distinct by site that Little Pond

sediment cores would have greater oxygen uptake rates due to higher organic matter content,

oxygen flux data was surprisingly uniform between sites. This result was likely explained by the

three-day difference in sediment core collection time. Since Little Pond cores were collected

three days before Waquoit Bay cores and incubated with aeration, the additional exposure to

oxygen rich conditions (~8 mgO2/L) likely oxidized much of the readily available organic matter

before treatments began. This highlights the importance of oxygen availability in regulating

subsequent sedimentary nitrogen cycle processes (Fig. 1) and explains the relative lack of site-

specific variation in the nitrogen flux data and stable isotope analysis.

The major difference between sites occurred in nitrate flux measurements at the very end

of the incubation period. Large negative ammonium fluxes coupled with positive nitrate fluxes in

Waquoit bay OM and OM+OYST cores suggesting that nitrification begins to occur during the

last three days of the incubation period (Fig. 5-6, S3). Data from Little Pond cores fits the same

pattern, but is much less conclusive and total nitrate concentrations remain lower. This implies

that over longer time periods, site history of eutrophication and oxygen availability may condition

the future response of the sedimentary nitrogen cycle. Since denitrification is tightly coupled to

nitrification, this suggests that the effect of oysters on long-term denitrification potential may be

dependent on site history; however, data from this 15-day experiment cannot be conclusive.

The lack of any evidence for denitrification of N15 enriched inputs is not surprising in the

context of the high oxygen uptake and low nitrate concentrations, which were the baseline

conditions of this experiment until the last three days. Without sufficient oxygen to oxidize

ammonium to nitrate, nitrate conditions were simply too low for denitrification to occur.

Although oysters did not increase denitrification potential, they did act as effective filter feeders,

incorporating 40% of added nitrogen into their tissues. This decreased the relative amount of

nitrogen that ended up in the sediment from 60% to 25%, but did not significantly alter water

column concentrations, likely due to the fact that oyster actively excreted ammonia while

metabolizing. These results indicate that oysters that care must be taken to consider nitrogen

recycling and oxygen availability in the context of site history when considering oyster

aquaculture as a water quality management strategy.

ACKNOWLEDGEMENTS

This project would not have been possible without the help of many. In particular, I would like to

thank my adviser Anne Giblin for her incredible patience, perspective, and support. Special

thanks are due to Dave Bailey and the Lindell lab, Sia Karplus, Tyler Messerschmidt, Sam

Kelsey, Jane Tucker, Sam Kelsey, Marshall Otter, and the fantastic SES TA team of 2015.

LITERATURE CITED

Bowen, Jennifer L., and Ivan Valiela. "The ecological effects of urbanization of coastal

watersheds: historical increases in nitrogen loads and eutrophication of Waquoit Bay

estuaries." Canadian journal of fisheries and aquatic sciences 58.8 (2001): 1489-1500.

Hoellein, Timothy J., and Chester B. Zarnoch. "Effect of eastern oysters (Crassostrea virginica)

on sediment carbon and nitrogen dynamics in an urban estuary." Ecological Applications 24.2

(2014): 271-286.

Jenkins, Mark C., and W. Michael Kemp. "The coupling of nitrification and denitrification in two

estuarine sediments1, 2." Limnology and Oceanography29.3 (1984): 609-619.

Kellogg, M. Lisa, et al. "Denitrification and nutrient assimilation on a restored oyster reef." Mar

Ecol Prog Ser 480 (2013): 1-19.

Mortazavi, Behzad, et al. "Evaluating the impact of oyster (Crassostrea virginica) gardening on

sediment nitrogen cycling in a subtropical estuary."Bulletin of Marine Science 91.3 (2015): 323-

341.

Pomeroy, Lawrence R., Christopher F. D'Elia, and Linda C. Schaffner. "Limits to top-down

control of phytoplankton by oysters in Chesapeake Bay." (2006): 301.

Solarzano L. 1969.” Determination of ammonium in natural waters by phenol hypochloric

method.” Limnological Oceanography 14:799-800.

Strickland JDH and Parsons TR. 1972. A practical handbook of Seawater Analysis. Ottawa,

Fisheries Research Board of Canada. 2nd Ed.

Team, Eastern Oyster Biological. "Status review of the eastern oyster (Crassostrea

virginica)." Report to the National Marine Fisheries Service, Northeast Regional Office (2007).

Wood, Elwyn Devere, F. A. J. Armstrong, and Francis A. Richards. "Determination of nitrate in

sea water by cadmium-copper reduction to nitrite." Journal of the Marine Biological Association

of the United Kingdom47.01 (1967): 23-31.

FIGURES

Figure 1. Oyster N-cycle diagram from Kellog et al. 2013.

Figure 2. Little Pond (left) and Waquoit Bay (right) sediment core collection sites, circled in red.

Figure 3. Aerial view of experimental set-up of sediment core incubations.

Figure 4. Initial, Post OM+, and Final oxygen fluxes for all twelve cores by site and treatment.

Figure 5. Initial, Week 1, and Week 2 ammonium fluxes.

Figure 6. Initial, Week 1, and Week 2 nitrate fluxes.

Figure 7. N15 mass balance stacked bar graph showing the location of N15 recovered.

Figure 8. Pie charts exhibiting relative fate of recoverd N15.

SUPPLEMENTARY FIGURES

Figure S1. Oysters added per core and basket mechanism.

Figure S2. NH4 concentration time series (days 1-15).

Figure S3. NO3 concentration time series (days 1-15).

Figure S4. N15 recovery separated by site and location.

TABLES

Table 1. p-values from Factorial ANOVA analysis of nutrient fluxes .

Table 2. Mass balance of recovered N15 per core (µmol).

Table 3. Relative recovery of N15 per core (%).

Figure 1. Oyster N-cycle diagram from Kellog et al. 2013.

Figure 2. Little Pond (left) and Waquoit Bay (right) sediment core collection sites, circled in red.

Figure 3. Aerial view of experimental set-up of sediment core incubations. This picture shows

the six Little Pond cores, with treatments in rows (OM+OYST, OM, CTRL from back to front)

and replicates in columns. The cylindrical apparatus in the center is hooked up to a battery that

causes the central disc to spin, rotating the white magnetic stir bars in each core keeping them

well mixed. Waquoit Bay cores were set up identically in another tub.

Figure 4. Initial, Post OM+, and Final oxygen fluxes for all twelve cores by site and treatment.

Error bars show the replicates. The Post OM+ flux was taken on day 7. The Final flux was taken

on day 15.

Figure 5. Initial, Week 1, and Week 2 ammonium fluxes. Missing values indicate linear

regressions with R2 < 0.6. Week 1 was calculated as the flux from days 1-7. Week 2 was

calculated as the flux from days 12-15.

Figure 6. Initial, Week 1, and Week 2 nitrate fluxes. Missing values indicate linear regressions

with R2 < 0.6. Week 1 was calculated as the flux from days 1-7. Week 2 was calculated as the

flux from days 12-15.

Figure 7. N15 mass balance. The dark green line is the average estimate for total N15 OM added.

The light green lines indicate one standard deviation above and below. The yellow line indicates

the upper standard deviation for the Little Pond OM+OYST cores (see Methods).

Figure 8. Pie charts exhibiting relative fate of recovered N15.

Figure S1. Oysters added to OM+OYST cores. All three oysters per core were placed in

makeshift plastic netting baskets like those shown in row three above.

Oysters

Figure S2. Ammonium concentration timeseries (days 1-15).

Figure S3. Nitrate concentration timeseries (days 1-15).

Figure S4. N15 recovery separated by site and location.

TABLES

Table 1.1.

O2 p-values

Initial Post OM+ Final

Treatment 0.319 0.001 0.015

Site 0.380 0.326 0.414

Treatment:Site 0.115 0.094 0.321

Table 1.2.

NH4 p-values

Initial Week 1 Week 2

Treatment 0.470 5.34*10-5

0.063

Site 0.564 0.073 0.396

Treatment:Site 0.175 0.867 NA

Table 1.3.

NO3 p-values

Initial Week 1 Week 2

Treatment 0.934 0.677 0.245

Site 0.158 0.305 0.144

Treatment:Site 0.111 0.257 0.578

Table 1. p-values from factorial ANOVA analysis of O2, NO3, NH4 fluxes.

MASS BALANCE PER CORE

Core

Oysters

(µmol

N15)

Sediments

(µmol N15)

Water

(µmol

N15)

Recovered

(µmol N15)

OM+

(µmol

N15)

Discrepancy

%

WB1 9.55 6.54 8.03 24.12 20.32 15.76%

WB2 9.80 5.29 7.19 22.29 20.32 8.84%

WB3 -- 12.79 8.29 21.08 20.32 3.62%

WB4 -- 12.25 8.18 20.43 20.32 0.57%

LP1 9.16 5.76 8.03 22.95 22.01 4.10%

LP2 11.44 5.07 8.09 24.59 22.01 10.51%

LP3 -- 14.09 9.13 23.22 20.32 12.50%

LP4 -- 13.21 9.88 23.09 20.32 12.00%

Table 2. Mass balance of recovered N15 per core.

RELATIVE RECOVERY

Site Treatment Oysters Sediment Water

WB1 OM+Oysters 39.6% 27.1% 33.3%

WB2 OM+Oysters 44.0% 23.7% 32.3%

WB3 OM 0.0% 60.7% 39.3%

WB4 OM 0.0% 59.9% 40.1%

LP1 OM+Oysters 39.9% 25.1% 35.0%

LP2 OM+Oysters 46.5% 20.6% 32.9%

LP3 OM 0.0% 60.7% 39.3%

LP4 OM 0.00% 57.20% 42.80%

Table 3. Relative recovery of N15 per core.