1

Response greenhouse gases (CO2, CH4 and N2O) emissions to increased salinity and

nitrogen inputs along a marsh restoration gradient

Arianna N. Cocallas, Beloit College, Beloit, WI 53511

Anne E. Giblin PhD, Christine McCarthy, and Katherine Klammer

Abstract

There is strong interest in understanding the dynamics of greenhouse gas emissions from

salt marshes, as policy makers are interested in using them in and effort to mitigate climate

change. The effects of salt marsh restoration on greenhouse gas emissions are not well

understood and require further research. Here, I used sediment cores collect from an impacted

salt marsh determine the emissions of carbon dioxide, methane and nitrous oxide over a two

week laboratory experiment with increased salinity levels modeling effects of restoration.

Carbon dioxide emissions increased substantially in the treatment intended to show the effect of

salt marsh restoration. Methane and nitrous oxide emissions were highly variable, but showed

increased emissions with a nitrogen addition. This project clearly demonstrated that salt marsh

soil processes are affected restoration.

Key Words

Salt marshes, Restoration, Greenhouse Gases, Carbon Dioxide, Methane and Nitrous Oxide

Introduction

There is great interest in understanding the dynamics greenhouse gas emissions from salt

marshes around the world including the study of carbon sequestration, methane emissions and

nitrous oxide release. Salt marshes have a high rate of carbon sequestration in fact salt marshes

store similar amounts of carbon dioxide equivalence per hectare as tropical rainforests (Mitsh et

al. 2013, Pendleton et. al. 2012). Even though salt marshes do not cover as much area as tropical

2

forests policy makers are interested in conserving and protecting salt marshes as a tool for carbon

offsets that can be purchased (IPCC 2014). Salt marshes are also important in a variety of areas,

providing many ecosystem services such as habitat for many animal and plant species as well as

erosion and storm protection for coastal communities (Pendleton et. al. 2012).

Salt marsh restoration can come in many forms, but normally areas of marshes at some

point in the past have been impounded or diked meaning the normal tidal cycle of salt water

either is completely cut off from this impacted area, or the area receives limited amounts of salt

water (CITAION). The cut off from tidal circulation will either saw the area to “go fresh” or see

much lower overall salinity levels or drain down, as area is not longer receiving sufficient

supplies of seawater to maintain its previous water table and salinity level. It is necessary to

explore how the reintroduction of a regular tidal flow will affect the greenhouse gas emissions

from restored salt marshes.

We do not fully understand the dynamics of carbon dioxide, methane and nitrous oxide

release from salt marshes. The release of carbon dioxide from a restored salt marsh will depend

on the amount of available carbon for respiration. Methane emissions are tend to decrease as

salinity increases, so as a salt marsh is restored one would expect to see methane emissions

decrease (Bartlett et. al. 1987). As well, some salt marshes are near agriculture or residential

areas, which increase the impact of nitrogen loading so it is important to assess how additional

nitrogen, might affect these systems (Valiela and Cole 2002). Since carbon dioxide, methane and

nitrous oxide are all greenhouse gases, understanding their emission dynamics is key in

determining whether a salt marsh can be considered a net source or sink of greenhouse gases to

the atmosphere (IPCC 2014).

3

The goal of the project was to model the short-term response of how carbon dioxide,

methane and nitrous oxide emissions change in an impacted salt marsh as the tidal flow is

restored and seeks to answer the following questions:

1. How do carbon dioxide, methane and nitrous oxide emissions vary across a salinity gradient

in a salt marsh as a model of the short-term response to restoration?

2. How do carbon dioxide, methane and nitrous oxide emissions vary across a salinity gradient

in a salt marsh as affected by the addition of nitrate as a model of the short-term response to

restoration near densely populated areas and the impact of agriculture?

Sampling site description

This project will use the restoration gradient in the salt marshes on Long Creek in East

Sandwhich, Cape Cod, MA at the East Sandwich Game Farm as a model. The section of the salt

marsh we sampled from contains three distinct sections (Figure 1). The impacted salt marsh site

has been partially cut off from the regular salt rich tidal flows by a train track. The restored site

was once cut-off from the regular tidal flows, but since 2006 has been restored to allow more

regular levels of tidal flow into this site. The unimpacted site has not been impounded; seawater

has always been able to reaching this area uninhibited by human construction. Using sediment

cores collected from the impacted site, I modeled the short-term responses greenhouse gas

emissions as salinity levels increase across the cores to mimic the process of salt marsh

restoration in a laboratory setting.

Methods:

Sediment core preparation and maintenance

On November 7th, I collected sixteen sediment cores from the salt marsh within the East

Sandwich Game Farm on Long Creek in East Sandwich, Massachusetts. Twelve sediment cores

4

were retrieved from the impacted salt marsh (Figure 1). Four cores were collected from the un-

impacted area of the salt marsh (Figure 1).

This procedure resulted in sediment cores that were approximately twenty-centimeters in

depth and were taken using circular PVC pipe (7cm diameter and 30 cm deep). Once returned to

the laboratory setting cores were capped with specially constructed caps that allowed for the

water within the cores to be drained periodically and sealed with on the bottom end of the core.

They were then suspended using an apparatus designed to allow water samples to be collected

from the bottom of the cores. The tops of the cores where covered with aluminum foil to prevent

light from affecting them (Figure 14).

From November 7th to 10

th, I allowed the sediment cores adjusted to the laboratory

setting. On November 11th, initial water treatments were applied to the cores. Water treatments

consisted of 100 mL of either freshwater (DI), 3ppt seawater, or 30ppt seawater with either no

nitrate or 100umol/L nitrate added. Duplicate cores were treated with the same treatment

resulting in eight different treatments as cores were collected from two sites (Table 2). Water

treatments were applied on November 11th

, 13th, 18

th, 20

th and 22

nd to mimic periodic tidal flow

that would normally be seen in a salt marsh setting (Table 1). These water samples were

collected for further analysis by collaborators. When a water treatment was applied the tubing

was opened and allowed to drain while the water was replaced with the new water from the next

treatment. The sediment cores from the impacted site drained easily normally within twenty

minutes while the cores from the un-impacted site were very slow to sometimes taking as long as

8 hours to drain the previous set of 100 ml of water. Because these cores were so slow to drain

sometime standing water was still on the surface of the core while flux measurements were take.

5

The experiment lasted for two weeks with the cores dismantled on November 24th as the soil

from the cores was used for further analysis.

Flux Measurements: Carbon Dioxide, Methane and Nitrous Oxide

Greenhouse gas flux measurements for carbon dioxide, methane were taken

approximately 18-20 hours after a water treatment on November 12th, 19

th and 23

rd (Table 1).

Flux measurements for nitrous oxide were taken on November 14th, 20 hours after a water

treatment was applied and 42-44 hours after other water treatments on November 20th and 24

th

(Table 1). Greenhouse flux measurements were taken using the following standard procedure.

The only difference in the sampling method between methane and carbon dioxide as opposed to

nitrous oxide was that flux measurements were taken over a 30 minute time period for methane

and carbon dioxide and a two-hour time period for nitrous oxide.

Before flux measurements were taken, each of the 48 syringes were filled with 20mL of

N2 gas and then sealed with Lauer valves and rubber bands. Each core was then capped and

checked that it was sealed tightly. For carbon dioxide and methane sampling times occurred at

10, 20 and 30 minutes and for nitrous oxide 30, 60 and 120 minutes after the core was capped. At

each sampling time, the N2 gas from a single syringe was injected into the headspace of the

sediment core and allowed to mix, by pulling air in and out with the syringe creating pressurized

space in the core headspace where the gas had adequately mixed so a representative gas sample

was taken.

The each gas concentration was determined by gas chromatography. The change in

concentration over the thirty-minute period or two-hour period was used to determine the gas

flux or change in concentration over time. Each individual flux was calculated using the ideal gas

law.

6

Lignin Assay

An additional set of sediment cores was taken on November 5th, so various analysis could

be performed while the experiment was in progress. I determined the lignin content of the

sediment core samples using an acid digest modified from a TAPPI method. I weighed, dried,

and re-weighed a subsample of each sediment core to determine the water content of each

sample. I ground each sample in the WiglBug grinder and placed 0.500g +/- 0.010g into the

bottom of a glass tube. To breakdown the organic molecules, I added 3mL of 12 M H2SO4 to

each tube, and incubate them, in a 30-degree water bath for one hour, gently swirling the tubes to

mix them every 15 minutes. I diluted the solutions to 0.4 H2SO4 and slowly increase the

temperature to 60 degrees over the course of 3 hours. Then I increased the temperature to 70

degrees for two hours. To measure the mass of the remaining solids, I filtered the contents of

each tube through a pre-ashed, pre-weighed glass fiber filter (GFF) and dried the GFF in a 60

degree drying oven over night. I weighed the filters, then ash them in a 450 muffle furnace for

four hours and then weighed them again. To calculate the mass of lignin in the sample, I used the

following formula: Mass of lignin = mass of crucible and sample before ashing – mass of

crucible and sample after ashing.

Sable Isotope Analysis

For methods regarding stable isotope analysis please see Kat Klammer’s paper Nitrogen

Cycling in a Restored Salt Marsh.

Results

Soil Carbon Content

Stable carbon isotope analysis revealed that the impacted site cores had d13C

isotope values of about -28 at 5cm, 10cm, 15cm and 20cm sampling depths. Unimpacted

7

site cores had carbon isotope values on average near -18 at 5cm, 10cm, 15cm and 20cm

sampling depths. Restored site cores showed isotope values at 5cm, 10cm and 15cm near -

19 while the 20cm isotope values were near -25. (Figure 2)

Isotope analysis also revealed that percent total carbon in the top 5cm was 24.3% in

the impacted site, 7.6% at the restored site and 7.4% at the unimpacted site (Table 3). The

lignin assay showed that the lignin content of each of the percent total carbon was 62.6%

lignin at the impacted site, 63% lignin at the restored site and 68.4% at the unimpacted site

(Table 3).

Carbon Dioxide Flux Measurements

The carbon dioxide flux measurements were averaged between duplicate cores and

all three sampling time points since the flux measurements stayed fairly consistent among

the three sampling time points (Table 4). The lowest flux measurement came from the

impacted fresh treatment and the highest from the impacted 30ppt treatment (Table 4.) All

the nitrate treatments saw decreases from their perspective controls (Table 4, Figure 4).

Higher carbon dioxide flux measurements were observed from the impacted 30ppt

treatment when compared to the unimpacted 30ppt treatment (Table 4, Figure 1.) Higher

carbon dioxide flux measurements were found in the impacted drained fresh treatment

when compared to the impacted fresh treatment (Figure 6).

Methane Flux Measurements

The methane flux measurements were variable between each sampling date between

treatments. Averaged duplicates with standard error from each sampling date are noted

(Table 5). Across the impacted fresh, impacted 3ppt, and impacted 30ppt treatments there

was no clear trends as the results were highly variable (Figure 7). The most consistent

8

results came from the impacted 30ppt treatment with low emissions overall. The impacted

fresh treatment had typically lower flux results than the impacted fresh + nitrate treatment

(Figure 8). There was no clear difference between methane emissions from the impacted

30ppt and the impacted 30ppt + nitrate (Figure 9).

Nitrous Oxide Measurements

The nitrous oxide measurements were highly variable between duplicate cores and the

three sampling days. Results from each sampling show averaged flux with standard error

(Table 6). The nitrous oxide measurements were consistently higher in the impacted

drained fresh than the impacted fresh treatment to a high degree of error. One of the

impacted drained fresh cores produced much more nitrous oxide, which is the main

controller in this trend. Nitrous oxide measurements across the three sampling times were

highly variable between the impacted fresh, impacted 3ppt and impacted 30ppt treatments

producing no clear trends (Figure 11). Across the three sampling times the impacted fresh

+ nitrate treatment clearly produced higher nitrous oxide fluxes than its comparison

control the impacted fresh treatment (Figure 12). In the impacted 30ppt treatment and the

impacted 30ppt + nitrate treatment did not produced a clear trend but on average the

impacted 30ppt + nitrate treatment did produce less nitrous oxide than the impacted fresh

+ nitrate treatment (Table 13).

Discussion

Soil Carbon Content

Through the stable carbon isotope analysis of the initial sediment cores I was able to

use the carbon isotope signal as an indication of the source of the carbon and depth as a

proxy for time. I determined that the impacted site has been dominated by Phragmites for

9

quite come time as we see that the cores have a consistent signal between -27 and -28 all

the way to the deepest depths (Choi et. al. 2005) (Figure 2). I also found that the

unimpacted site has its entire depth profile has consistent signal near -19 which matches

the signal for Spartina (Currin et. al. 1995). Because Phragmites prefers water with lower

level salinity and Spartina exists in areas with higher salinities this also confirms the idea

that the sites have contained the species of plant that we would expect for its perspective

level of restoration (Choi et. al. 2005). The most important take away from the carbon

stable isotope results is the shift in the signal for the cores from the restored site, showing

that they were once much more dominated by Phragmites and over time as salinity levels

increased and the plant community changed back towards the native and dominating salt

marsh species Spartina. This confirms that the site has undergone restoration as seen

through the shift in the plant community (Figure 2). However, an important additional

analysis would be to determine the age of the perspective cores at each depth in order to

further compare the affects of restoration overtime on the plant community. It would have

been better to collect cores with greater depths to get a better sense of how the plant

community has changed over time as well run more than just duplicate cores through the

isotope analysis to capture more of the spatial variability within each sampling site.

Isotope analysis also revealed that percent total carbon in the top 5cm was 24.3% in

the impacted site, 7.6% at the restored site and 7.4% at the unimpacted site (Figure 3). The

highest carbon content was found at the impacted site, but in order to determine how much

labile carbon, or easily decomposed carbon that was available for respiration and emission

as carbon dioxide it was important to perform a lignin assay. All the sites showed relatively

similar lignin content with 62.6% lignin at the impacted site, 63% lignin at the restored site

10

and 68.4% at the unimpacted site (Table 3). Because the lignin content was so similar

between the three sites and lignin is a form of carbon calculated within the percent total

carbon calculation, we then know that there is more available for respiration at the

impacted site than the restored and unimpacted sites.

Carbon Dioxide Flux

The impacted fresh treatment had the lowest carbon dioxide flux measurement

with a slight increase to the impacted 3ppt treatment. This is an expected result since the

treatments are not extremely different, but they are also likely extremely similar because

the cores were producing similar salinity levels in the outflow from the cores (Klammer

2014). The impacted site we collected the cores from is not entirely cut off from tidal

influences even though it is extremely reduced (Figure 1). This suggests that there were

similar amounts of sulfate available as an electron acceptor between the cores. The

impacted 30ppt treatment, which was intended to model the effects of restoration as tidal

flows are restored and salinity levels in the water increase, saw much higher carbon

dioxide fluxes due to the increased availability of sulfate for the sulfate reducing bacteria,

which produce carbon dioxide through respiration (Weston et, al. 2011). The impacted

30ppt treatment also saw increased carbon dioxide emission when compared to the

unimpacted 30ppt treatment or the natural salt marsh. The difference in the carbon dioxide

emissions can likely be attributed to the difference in the available carbon content between

the two sites with the impacted site containing much more available carbon than the

unimpacted site (Figure 5). The nitrate treatments, which represented a model of nitrogen

loading from residential housing or urban centers, saw much lower carbon dioxide

emissions that their perspective controls (Figure 4). While I would have expected the

11

nitrate addition to cause increased carbon dioxide emissions, since nitrate is an extremely

favorable energetically available compound, the opposite trend. One possible explanation

for this result is the microbes were using the available nitrate to build biomass instead of

using it as an electron acceptor and respiring more. The impacted drained treatment had

slightly higher carbon dioxide treatments in comparison to the impacted fresh treatment,

however the emissions were still much lower than the impacted 30ppt treatment (Figure 6,

Table 4).

Methane Flux

The methane flux measurements were marked by high variability between each

sampling date and between treatments. Across the impacted fresh, impacted 3ppt and

impacted 30ppt the results produced no clear trends, even though we would have expected

to see the highest methane flux measurements from the fresh treatment and decrease with

an increase in salinity (Figure 7). These results are likely explained by the duration of the

experiment, as it was too short to achieve the treatment salinity levels within the sediment

cores. The impacted fresh treatment did not see high methane fluxes because the sediment

cores were still being flushed of the salt already contained within the cores. As expected

there were consistently low methane fluxes from the impacted 30ppt treatment, since the

sulfate reducing bacteria were likely able to out compete the methanogens for available

resources within the sediment cores (Poffenbarger et. al. 2011). It has been shown that

increased nitrate will cause larger methane fluxes and these results seem to support that

idea (Liu and Greaver 2009). Over the course of the three sampling times the methane fluxes

were higher in the impacted fresh + nitrate than the impacted fresh treatment. One of the

impacted fresh + nitrate cores was producing much more methane than the other and

12

perhaps if the experiment ran for a longer period both cores would have equalized and

produced higher methane fluxes from the impacted fresh + nitrate treatment (Figure 8).

The impacted 30ppt + nitrate treatment also produced higher methane fluxes than the

impacted 30ppt treatment further supporting this idea (Figure 9).

Nitrous Oxide

The nitrous oxide fluxes were also marked by high variability between the each

individual core as well as across the treatments. The impacted drained fresh treatment was

consistently higher than the impacted fresh treatment to a high degree of error, because

one of the sediment cores within the impacted drained fresh treatment had extremely high

nitrous oxide fluxes in comparison (Figure 10). There was no clear trend between the

impacted fresh, impacted 3ppt and impacted 30ppt treatments, which were also highly

variable (Figure 11). However, these results are consistent with literature in that there is

no clear affect of salinity on nitrous oxide emissions (Moseman-Valtierra et. al. 2011). The

most consistent trend with the nitrous oxide fluxes was between the impacted fresh and

impacted fresh + nitrate treatments. Across all of the samplings the impacted fresh +

nitrate treatment produced higher nitrous oxide fluxes than the impacted fresh treatment

(Figure 12). Nitrous oxide is produced from both nitrification and denitrification, but

nitrate was added to these cores so it is likely that the additional produced nitrous oxide is

likely from denitrification. In comparison, the nitrous oxide fluxes from the impacted 30ppt

and impacted 30ppt + nitrate did not show a clear trend and were very similar across the

three sampling points (Figure 13).

Conclusion

13

As a model of restoration, I demonstrated that restoring the tidal flow to an

impacted salt marsh would affect the soil microbial processes. If impacted salt marshes are

restored to high salinity levels they will likely see higher carbon dioxide emissions at least

initially as shown with the sediment cores. While the results did not clearly depict this

trend it has been established that less methane is emitted as salinity level increases with

time the treatments would have likely shown this trend if their internal salinity levels had

more time to equilibrate to their treatment salinity levels. The results indicated higher

methane fluxes and nitrous oxide emissions with nitrate addition. It is important to note

that these trends will undoubtedly be influenced by the affect of plants within natural salt

marsh, as they will mitigate some of the carbon dioxide release as well as take up some of

the additional nitrate. Since soil microbial processes were affected this only warrants

further study in the field and lab, to gain a full understand of how greenhouse gas

emissions in salt marshes will be affected by restoration.

Acknowledgements

Many thanks to: Anne Giblin for mentoring me throughout the duration of this project

and providing numerous support throughout all of the field work, sampling and data analysis;

Klammer and Christine McCarthy my collaborators for their effort and work to make this project

a success; Rich McHorney and Jane Tucker for teaching me how to operate the gas

chromatography machine and the SES TAs Fiona Jevon, Tyler Messerschmidt, and Nick Barrett

for their endless willingness to answer my questions and provide support to us all.

References Bartlett, K. B., R.C. Harriss, and D. I. Sebacher. 1987. Methane Emissions along a Salt

Marsh Salinity Gradient. Biogeochemistry 4: 183-202.

14

Choi, W.J., H.M. Ro, and S.X. Chang. 2005. Carbon isotope composition of Phragmites

australis in a constructed saline wetland. Aquatic Botany. 82: 27-38.

Currin, C.A., Newell, S.Y., and Paerl, H.W. 1995. The role of standing dead Spartina

alterniflora and benthic microalgae in salt marsh food webs: considerations

based on multiple stable isotope analysis. Marine Ecology Progress Series 121:99-

116.

IPCC (2014) 2013 Supplement to the 2006 IPCC Guidelines for National

Greenhouse Gas Inventories: Wetlands. In: Hiraishi T, Krug T, Tanabe K, Srivastava N,

Baasansuren J, Fukuda M, Troxler TG (eds) Intergovernmental Panel on Climate Change,

Geneva

Klammer, K., The effects of salt marsh restoration on nutrient retention and nitrogen

cycling. 2014.

Liu, L. and T.L. Greaver. 2009. A review of nitrogen enrichment effects on three

biogenic GHGs: the CO2 sink may be largely offset by stimulated N2O and

CH4 emissions. Ecology Letters. 12: 1103-1117

Mitsch W. J., Bernal B., Nahlik A. M., Mander U., Zhang L., Anderson C. J. and,

Jørgensen SE, Brix H (2013) Wetlands, carbon, and climate change. Landsc Ecol

28:583–597

Moseman-Valtierra, S., et al. “Short-term nitrogen additions can shift a coastal wetland

from a sink to a source of N2O” Atmospheric Environment 45 (2011): 4390-4397. Print.

15

Pendleton, L., Donato, D.C., Murray, BC., Crooks, S., Jenkins, WA., Sifleet, S., Craft,

C., Fourqurean, JW., Kauffman, JB., Marbà, N., Megonigal, P., Pidgeon, E., Herr, D.,

Gordon, D., Baldera, A. 2012. Estimating Global ‘Blue Carbon’ Emissions from

Conversion and Degradation of Vegetated Coastal Ecosystems. PLOS ONE. 7:1-7

Poffenbarger, H.J., B.A. Needleman and J.P. Megonigal. 2011. Salinity influence on

methane emissions from tidal marshes. Society of Wetland Scientists. 31: 831-842

Weston, N. B., M. A. Vile, S. C. Neubauer, and D. J. Velinsky. 2011. Accelerated

microbial organic matter mineralization following salt-water intrusion into tidal

freshwater marsh soils. Biogeochemistry 102:135-151.

Valiela, I. and M.L. Cole. 2002. Comparative Evidence that Salt Marshes and Mangroves

May Protect Seagrass Meadows from Land-derived Nitrogen Loads. Ecosystems. 5:92-

102.

ASSa

16

Table 1. An overview of the project calendar during the month of November 2014.

Sun Mon Tue Wed Thu Fri Sat 2 3

4

5

Initial core

collection

6

7

Experimental core

collections

Core incubation

set-up

8

9

10

11

(Day 1)

Water Treatment

12

(Day 2)

CO2 Flux

CH4 Flux

13

(Day 3)

Water Treatment

14

(Day 4)

N2O Flux

15

(Day 5)

16

(Day 6)

17

(Day 7)

18

(Day 8)

Water Treatment

19

(Day 9)

CO2 Flux

CH4 Flux

20

(Day 10)

N2O Flux

Water Treatment

21

(Day 11)

22

(Day 12)

Water

Treatment

23

(Day 13)

CH4 Flux

CO2 Flux

24

(Day 14)

N2O Flux

25

26

27

28

29

17

Core Collection Site Impacted Impacted Impacted Unimpacted

Salinity Level 0 ppt

Freshwater

3 ppt

Seawater

30 ppt

Seawater

30 ppt

Seawater

Drained Core Yes/No No No --

No nitrate CO2, CH4,

N2O Flux (additional set of cores

with draining

manipulation)

CO2, CH4,

N2O Flux

CO2, CH4,

N2O Flux

CO2, CH4,

N2O Flux

Nitrate added CO2, CH4,

N2O Flux

--

CO2, CH4,

N2O Flux

CO2, CH4,

N2O Flux

Table 2. Project experimental design.

18

Core Collection Site % Total Carbon in Top 5 cm % Lignin in Top 5 cm

Impacted 24.3 62.58

Restored 7.63 63.07

Unimpacted 7.39 68.44

Table 3. Percent total carbon and percent lignin in the top 5 cm at the impacted, restored and unimpacted sites.

19

Core Collection Site Treatment Type Average CO2 Flux

(umol m-2

d-1

) ± SE

Impacted Drained Fresh 198,799 ± 18,343

Impacted Fresh 132,198 ± 13,521

Impacted Fresh + Nitrate 103,092 ± 12,061

Impacted 3ppt 142,059 ± 28,803

Impacted 30ppt 336,058 ± 48,903

Impacted 30ppt + Nitrate 164,444 ± 19,595

Unimpacted 30ppt 230,504 ± 62,418

Unimpacted 30ppt + Nitrate 137,935 ± 28,629

Table 4. Average carbon dioxide flux (umol m-2 d-1) measurements from three samplings across all of the experimental treatments.

Table 5. Methane flux measurements (umol m-2 d-1) from the three sampling dates across all experimental treatments.

20

CH4 Flux (umol m-2

d-1

) ± SE

Core Collection

Site

Treatment Type Nov. 12th

Nov. 19th

Nov. 23rd

Impacted Drained Fresh 499 ± 479 242 ± 172 168 ± 28

Impacted Fresh 137 ± 54 293 ± 102 195 ± 30

Impacted Fresh + Nitrate 150 ± 47 698 ± 639 1,030 ± 959

Impacted 3ppt 497 ± 326 401 ±182 213 ± 182

Impacted 30ppt 166 ± 72 174 ± 143 182 ± 91

Impacted 30ppt + Nitrate 1,089 246 ± 202 326 ± 91

Unimpacted 30ppt 1,637 1,942 ± 1,811 1,744 ± 125

Unimpacted 30ppt + Nitrate 4,348 ± 3858 10,244 ± 9,556 12,711 ± 5,845

Table 6. Nitrous oxide flux measurements (umol m-2 d-1) from the three sampling dates across all experimental treatments.

21

N2O Flux (umol m-2

d-1

) ± SE

Core

Collection Site

Treatment Type Nov. 14th

Nov. 20th

Nov. 24th

Impacted Drained Fresh 17 ± 16 45 ± 43 57 ± 54

Impacted Fresh 3 ± 2 5 ± 3 9 ± 3

Impacted Fresh + Nitrate 49 ± 13 33 ± 16 44 ± 32

Impacted 3ppt No Data 4 ± 3 44 ± 1

Impacted 30ppt 127 ± 124 25 ± 19 26 ± 17

Impacted 30ppt + Nitrate 9 63 ± 54 19 ± 7

Unimpacted 30ppt 2 8 ± 1 27 ± 24

Unimpacted 30ppt + Nitrate 63 ± 56 28 ± 23 49 ± 34

22

Figure 1. The Long Creek Marsh, on the East Sandwich Game Farm in East Sandwich, MA with

sampling locations and surface water salinity levels at the time of sampling noted.

23

0

2

4

6

8

10

12

14

16

18

20

-30-25-20-15

Dep

th (

cm)

𝛿13C

Impacted

Restored

Unimpacted

Treatment Type:

Figure 2. Initial stable carbon isotope sediment core depth profiles from impacted (blue), restored (red) and unimpacted (green) sites.

24

0

5

10

15

20

25

30

�

% T

ota

l C

arbon

in

Top 5

(cm

)

Impacted

Restored

Un-impacted

Treatment Type:

Figure 3. Percent total carbon in the top 5 cm from impacted (blue), restored (red) and unimpacted (green) sites.

25

0

50,000

100,000

150,000

200,000

250,000

300,000

350,000

400,000

�

Aver

age

CO

2 F

lux

(um

ol

m-2

d-1

)

30ppt

Fresh

Fresh + Nitrate

30ppt

30ppt + Nitrate

Treatment Type:

Figure 4. Average carbon dioxide flux measurements from unimpacted 30ppt (orange), impacted fresh (blue), impacted fresh + nitrate (light green), impacted 30ppt (purple) and impacted 30ppt + nitrate (dark green) treatments.

26

0

50,000

100,000

150,000

200,000

250,000

300,000

350,000

400,000

�

Aver

age

CO

2 F

lux

(um

ol

m-2

d-1

) 30ppt

Fresh

3ppt

30ppt

Treatment Type:

Figure 5. Average carbon dioxide flux measurements from unimpacted 30ppt treatment (orange), impacted fresh (blue), impacted 3ppt (red) and impacted 30ppt (purple) treatments.

27

0

50,000

100,000

150,000

200,000

250,000

300,000

350,000

400,000

450,000A

ver

age

CO

2 F

lux

(um

ol

m-2

d-1

)

Fresh

Drained Fresh

Treatment Type:

Figure 6. Average carbon dioxide flux measurements between impacted fresh (blue) and drained fresh (purple) treatments.

28

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12 14

CH

4 F

lux (

um

ol

m-2

d-1

)

Time (Days)

Fresh

3ppt

30ppt

Treatment Type:

Figure 7. Methane flux measurements among the three sampling points from the impacted fresh (blue), impacted 3ppt (red), and impacted 30ppt (purple) treatments.

29

0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

2,000

0 2 4 6 8 10 12 14

CH

4 F

lux (

um

ol

m-2

d-1

)

Time (Days)

Fresh

Fresh + Nitrate

Treatment Type:

Figure 8. Methane flux measurements from the fresh (blue) and fresh + nitrate (light green) treatments.

30

0

200

400

600

800

1,000

1,200

0 2 4 6 8 10 12 14

CH

4 F

lux (

um

ol

m-2

d-1

)

Time (Days)

30ppt

30ppt + Nitrate

Treatment Type:

Figure 9. Methane flux measurements from the impacted 30ppt (purple) and impacted 30ppt + nitrate (dark green) treatments.

31

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14

N2O

Flu

x (

um

ol

m-2

d-1

)

Time (Days)

Drained Fresh

Fresh

Treatment Type:

Figure 10. Nitrous oxide measurements from the impacted drained fresh (purple) and impacted fresh (blue) treatments.

32

0

20

40

60

80

100

120

140

160

180

200

0 2 4 6 8 10 12 14

N2O

Flu

x (

um

ol

m-2

d-1

)

Time (Days)

Fresh

3ppt

30ppt

Treatment Type:

Figure 11. Nitrous oxide measurements from the impacted fresh (blue), impacted 3ppt (red) and impacted 30ppt (purple) treatments.

33

0

10

20

30

40

50

60

70

80

0 2 4 6 8 10 12 14

N2O

Flu

x (

um

ol

m-2

d-1

)

Time (Days)

Fresh

Fresh + Nitrate

Treatment Type:

Figure 12. Nitrous oxide measurements from the impacted fresh (blue) and impacted fresh + nitrate (light green) treatments.

34

0

50

100

150

200

250

300

0 2 4 6 8 10 12 14

N2O

Flu

x (

um

ol

m-2

d-1

)

Time (Days)

30ppt

30ppt + Nitrate

Treatment Type:

Figure 13. Nitrous oxide measurements from the impacted 30ppt (purple) and 30ppt + nitrate (green) treatments.

35

Figure 14. Experimental core set-up with water sampling being taken.