detection of wastewater plumes from the 15n isotopic ... · & valiela, 1997). west falmouth...
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Detection of Wastewater Plumes from the 15
N Isotopic Composition of
Groundwater, Algae and Bivalves in West Falmouth Harbor
Kara Marie Annoni
University of Minnesota Duluth
Duluth, MN
Advisor: Dr. Kenneth Foreman
The Ecosystems Center: The Marine Biological Laboratory
Semester in Environmental Science (SES)
Woods Hole, MA
2012
Abstract:
Nutrient loading to coastal and estuarine waters poses a threat to the
structure and function of biotic communities within the ecosystem. It is important
to locate and diagnose the sources of excess nitrogen input in order to mitigate
chronic detrimental responses. In this study, we aim to locate sources of nitrogen
inputs via organismal uptake of the 15
N isotope tracer. We focus on δ15
N and
nitrate concentrations within groundwater, Mya arenaria and Ulva lactuca at 8
different sites along the West Falmouth Harbor shoreline. Specifically, we
attempt to support the hypothesis suggesting that a main source of nutrient
loading is due to wastewater contamination of groundwater within the watershed
via septic systems and wastewater treatment facilities. 5 Mya arenaria were
collected at each shoreline site and combined to create 1 composite sample per
site. Ulva lactuca was also collected and compared to potential nitrogen source
signals. Nutrient and isotopic analyses revealed increasing trends of nitrogen
signals at sites 1-3. Results from this study suggest that excess nutrients from
wastewater may affect the biotic factors of an ecosystem. δ15
N revealed
correlations with groundwater samples that had higher nitrate concentrations
suggesting that the excess nutrients may be coming from wastewater. Data also
suggests that groundwater that is affected by wastewater inputs, can be localized
and detected within Mya arenaria.
Introduction:
In southeastern Massachusetts, West Falmouth Harbor (WFH) is
experiencing excessive nitrogen loading through groundwater inputs (Bowles
et.al., 2007). As a response, eutrophication of coastal environments can occur in
addition to macro-algae blooms (Costello & Kenworthy, 2011), which affects the
biological function and environmental quality of an ecosystem. Short and long
term anoxia may increase due to excessive decomposition of overly produced
organic matter, which in turn results from nitrogen loading. Widespread losses
of biota due to physiological stress can occur (Holmer & Laursen, 2002), causing
changes in abundance of consumers and producers within the environment.
Furthermore, nitrogen loading contributes to the loss of tourism, cleanliness,
recreational and commercial uses of West Falmouth Harbor.
Many anthropogenic sources of nitrogen have been identified using N
stable isotope ratios, which include fertilizers, atmospheric deposition, and
wastewater from septic systems and wastewater treatment facilities (McClelland
& Valiela, 1997). West Falmouth Harbor is susceptible to nitrogen loading
primarily because of its sandy unconsolidated aquifers (McClelland & Valiela,
1997). Because of this characteristic, nitrogen leaches into the watershed,
contaminating the groundwater, which eventually contributes excess nitrogen
input to the seep.
In 2007, the largest source of nitrogen loading into West Falmouth Harbor
originated from wastewater treatment facilities (60%) and septic systems (20%);
both caused by anthropogenic activity (Bowles et al., 2007). Because of its
location within the watershed of West Falmouth Harbor, the Falmouth
Wastewater Treatment Plant (FWTP) was suspect to be a major source of nitrogen
loading into the estuary. In 2006, the FWTP switched management from primary
to tertiary systems, which decreased the concentration of nitrogen within
wastewater from 35mg/L to 5mg/L. The wastewater is then released into a
leaching field. Before the switch, treated wastewater was disposed of by spray
irrigation (Jordan, Knute, & Fry, 1997). The spray methods lead to higher
concentrations of nitrogen leaching into the watershed, eventually seeping into
West Falmouth Harbor. It is important to note that the rate at which groundwater
travels through the WFH watershed, originating from the wastewater treatment
facility, is approximately 1 foot per year (Jordan, Knute, & Fry, 1997). The
amount of time needed for groundwater to reach the seep from the wastewater
treatment facility is approximately 10 years (Thoms, Giblin & Foreman, 2003).
Therefore, the present nitrogen loading in West Falmouth Harbor originates from
nitrogen sources in 2002.
In order to monitor or diagnose the current condition of the ecosystem, it
is important to determine the localized areas of nitrogen input, and the sources of
nitrogen loading responsible for the increase or decrease of biota. Therefore, we
aim to detect spatial variation of wastewater inputs to WFH by measuring
organismal uptake of the isotope tracer 15
N.
To assess the spatial variation and localized areas of enriched groundwater
input, we will utilize Ulva lactuca and Mya arenaria ability to assimilate
nitrogen. Ulva lactuca is a macro-algae in which studies suggest may be used as a
“non-discriminatory bio-integrator” of nitrogen (Cohen and Fong, 2005). Mya
arenaria is a filter-feeding soft-bodied clam. Through the use of isotopic
analysis, we know that fractionation in consumers discriminate against lighter
isotopes when the element of 15
N is passed to higher trophic levels, increasing the
15
N value. Therefore, organisms that are heavy in 15
N will become heavier;
essentially you are what you eat (McClelland & Valiela, 1997). The thought is
that if either organism exhibits an increased 15
N value, it may be due to nitrogen
loading. If the organisms resemble the same 15
N value as wastewater, it may
suggest that the organism is receiving nutrients from groundwater contaminated
by wastewater or resources influenced by wastewater. The wastewater 15
N value
of Falmouth Wastewater Treatment Plant ranged between +8 to +11 parts per
thousand in the winter. In the summer, del 15
N values were as high as +40 parts
per thousand but an average of +13 to +19 parts per thousand due to increased
human activity. These numbers were calculated from the year that the wastewater
was incorporated into the groundwater within the WFH watershed (Jordan, Knute
& Fry, 1997). Because the nitrogen loading to WFH was originally sourced in
2002, these values deem as a reference until 2016 (when the management of the
wastewater treatment facility was upgraded in 2006). Additionally, we will assess
wastewater inputs in regards to dissolved inorganic nitrogen, ammonium and
nitrate, to provide insight to the natural recycling of nitrogen.
Field Methods
Groundwater Sites (G1, G2, … G9)
We examined the nitrogen isotope signature of groundwater, ulva and
mussels at 8 sites along the eastern shoreline of West Falmouth Harbor during the
month of November 2012 (Figure 4). At each site, we collected ulva (preferably
attached to a substrate), 5 mussels, and 250 mL of groundwater. Groundwater
was drawn from wells at different depths, using a hydro-lab pump. For each well
collection, the groundwater was pumped 5 times the well casing volume to purge
the well, ensuring a representative sample was collected. The sample was then
filtered through a swinnex filter holder and 25mm GF/F ashed filter to remove
particulates. Samples of wastewater were collected from the Falmouth
Wastewater Treatment Plant.
Harbor Sites (H1, H2, … H7)
Seven harbor sites were chosen to examine the ability to trace wastewater
contamination through organismal isotopic composition (Figure 4). Ulva was
collected from Waquoit Bay and incubated for one week with running seawater,
provided by the Marine Biological Laboratory. At each site, we launched 1
mooring with 5 samples of incubated Ulva as a bioassay for nitrogen. Each
mooring was comprised of 1 cinderblock, rope with a length to accommodate tide
cycles, 1 buoy, and 5 mesh bags encasing the ulva. Wet weight measurements
were taken before the ulva was introduced to West Falmouth Harbor, and a
composite control sample was prepared for isotopic analysis. Overall, there were
7 deployed moorings and 35 mesh bags containing ulva (5 per mooring). The
moorings were collected after one week.
Laboratory Methods
Nutrient Analyses
Nutrient analyses (NO3-,NH4
+, PO4
3-) were carried out for each groundwater
collection, and analyzed separately. Protocols used for nitrate, ammonium, and
phosphate analysis were adapted from Wood et. al. 1967, Solarzano 1969, and
Murphy et. al. 1962 respectively.
Each groundwater sample tested for nitrate required the use of a Lachat
Flow Injection Analyzer (FIA). Nitrate standards and samples were incubated for
2 hours at room temperature in darkness after reagent additions. Samples and
standards tested for ammonium were treated with phenol, sodium nitroprusside
and oxidizing reagents respectively, vortexing the test tube between each addition.
Samples and standards tested for phosphate were treated with the PO43-
mixed
reagent, vortexed and incubated for at least one hour. Both phosphate and
ammonium samples were analyzed with a Shimadzu 1601 spectrophometer with
wavelength settings at 885nm, and 640 nm respectively.
Ulva and Mussel Samples
Ulva samples were rinsed carefully and quickly (as to not lyse cells) with
deionized water to reduce contamination. Ulva at the ground sites were dried and
analyzed for 15
N separately by site. The 5 ulva samples attached to each mooring
were combined by site, totaling 7 composite samples. Wet weights were
measured before drying.
Each mussel sample (5 per site) was scrubbed with deionized water to
reduce contamination. Within the mussel, the adductor muscle was an “easy to
locate” body tissue in which I prepared for isotopic analysis by dissecting using a
scalpel and tweezers, cleaning using 10% HCL, and rinsing with deionized water.
The five mussel samples collected at each ground site were combined to create
one composite sample per site. The mussel and ulva samples were dried at 60
degrees Fahrenheit.
Ulva samples were homogenized using the Wig-L-Bug and mussel
samples were coarsely ground in a glass vial using a glass rod. Dr. Marshall Otter
carried out isotopic analysis at the MBL Starr Stable Isotope Laboratory. For
more information concerning the stable isotope methods please visit the following
website: http://dryas.mbl.edu/silab/
Diffusion Protocol
Groundwater samples were analyzed separately by site and well. To
prepare groundwater samples for isotopic analysis, I used diffusion protocols
adapted from Holmes, R.M., et al. 1998 and Sigman, et al. 1997.
Thirty-seven filter packs were made using 10 mm GF/D ashed filters, and
25mm teflon filter tape. Each 10mm GF/D filter was placed upon the teflon filter
tape and 25 μL of 2M H2SO4 was pipetted onto the filter. The tape was then
folded over the filter making a “teflon tape sandwich” and an empty plastic scint
vial was centered over the filter and pressed down to seal the edges of the teflon
tape creating a visibly thinner ring around the GF/D filter. This enabled the filter
pack to float atop the groundwater sample during incubation without being
contaminated by the sample. Filter packs were immediately transferred to a clean,
closed, storage bottle until ready to incubate.
Because we wanted to examine the isotopic values of both nitrate and
ammonium, we did not boil the sample with MgO to remove ammonia. In order
to collect 10μM of DIN per filter, the amount of sample to analyze was
determined based on previous nitrate concentrations collected at the same sites in
October of 2012. The total amount of sample plus deionized water equaled 150
mL. Using a 250 mL square nalgene HDPE bottle, materials were added in the
respective order: DI water, 7g ashed NaCl, 1g ashed MgO, filter pack, sample,
and 0.3g Devardas Alloy. The bottles were capped tightly and quickly to reduce
the loss of nitrate/ammonium. Two blanks were made (150 mL of DI) and three
standards (100μM, 50μM, and 25μM) were made using 100 uM KNO3 stock
solution. The 37 diffusion bottles were then placed in a shaker table held at a
constant temperature of 38 degrees Celsius. After 7 days of incubation, the filter
packs were removed and dried in a desiccator for 2 days before isotopic analysis.
Results
Nitrate concentrations within the groundwater ranged from 53 (μM) to
274.33 (μM). This was a much larger scale compared to ammonium and
phosphate concentrations (Figure 5). The largest concentration of nitrate was
found in groundwater sites 2-4, which correlates with analyzed nitrate
concentrations in 2006 (Figure 13). Suspended ulva also exhibited high
concentrations of nitrate within harbor sites 1,2,3, and 4 (Figure 6). The N:P ratio
of groundwater exhibited increased values at sites 2 through 4 (Figure 7). All
values of N:P ratio exceeded the Redfield Ratio of 16:1. Likewise, the δ15
N of
groundwater displayed similar trends with increased values at groundwater sites 1
through 3 (Figure 8). Ribbed mussels were found to have δ15
N values ranging
from 10.4 to 12.4 and ulva 7.1 – 10.8. Groundwater δ15
N values in comparison
with δ15
N of ulva and ribbed mussels exhibited slightly higher values at sites 1
and 2 (Figure 9). Ulva suspended in the harbor sites increased in δ15
N values
compared to the control sample (5.3 o/oo), although the values did not exhibit
critical observational differences between sites (Figure 10). As in Figure 11, the
δ15
N and δ
13C
are compared in terms of ulva and ribbed mussel, in which ulva
indicates lower values of the δ15
N and δ
13C
than the ribbed mussel.
Compared to
spartina (-13.8, 3.9) and plankton (-20.5, 8.5) values of δ13
C and δ
15N of ribbed
mussels within West Falmouth Harbor displayed higher values of δ15
N and
differed in δ13
C (Figure 12). Sites 1 and 2 revealed the highest value of δ15
N.
Discussion
There was no general pattern in ammonium and phosphate concentrations
within groundwater. This was expected due to the general recycling
characteristics of nitrogen. Sites 1 through 4 exhibited the highest concentration
of groundwater input in reference to thermal imaging (Figure 4). Because these
sites exhibited high concentration of nitrate, it is likely that the excess nitrogen is
introduced into the system by groundwater. It is important to note that high
concentrations of nitrate correlate with high values of δ15
N of groundwater
(10.16-13.4 o/oo) at the same groundwater sites; suggestive that excess nutrients
is due to groundwater that is contaminated with wastewater. Overall, this
suggests that the localized groundwater is contaminated with excess nitrogen,
possibly in the form of nitrate; wastewater contains nitrate as it is leached into the
watershed (Jordan et al, 1997). The trend of high nitrogen concentrations was
also exhibited by the C:N ratio of the ulva suspended in the harbor at harbor sites
1,2, and 3 which were similarly located near ground sites 1,2,3 and 4 (Figure 6).
The N:P ratio revealed similar trends at ground sites 2,3 and 4 furthering
evidence that the groundwater input is enriched in nitrogen in those areas (Figure
7). All values of the N:P ratio exceeded the Redfield ratio of 16:1. Most coastal
systems are nitrogen limited and the presence of high N:P ratios exceeding the
Redfield ratio may suggest that the excess nitrogen is creating a phosphorous
limited system.
Comparison of data found within this study and data collected from
previous experiments show correlation in location of increased nitrate
concentration and high values of δ15
N (Figures 8 & 13), furthering the support of
groundwater contaminated with wastewater. Likely, the mussel and ulva would
exhibit similarly increased isotopic values because the data suggests that
wastewater is present within the seep; essentially assimilation of nitrogen within
each organism is influenced by the excess nutrients and will possess an isotopic
value resembling the material taken in. Again, at sites 1,2, and 3 mussel and ulva
samples from the ground sites showed an increased δ15
N value suggesting that as
groundwater δ15
N increases, so does the δ15
N in ribbed mussel and ulva (Figure
9). The suspended algae at the harbor sites had increased δ15
N values compared
to the control algae, yet there was no variation between sites, which could be due
to the placement of the mooring (not close enough to the shoreline to exhibit full
effects of groundwater), although there was evidence of excess nitrogen due to the
C:N ratios – which did exhibit increases of nitrogen at sites 1,2, and 3.
The δ13
C and δ15
N of mussels and ulva both exhibit high values of δ15
N
but different values of δ13
C. This data can support the assumption that additional
nutrients added to the estuary affects organisms within different food webs
(Figure 11). Figure 12 describes the relationship between δ15
N values of mussels
and isotope values of which mussels would acquire their usual nitrogen content
(Fry & Peterson, 1985). According to a study carried out in the Great Sippiwisset
Marsh (GSM), nitrogen can increase with a dieting shift from spartina to
plankton, but in this case there are higher δ15
N values from samples taken in the
Mashapaquit Creek marsh sites, with similar abiotic and biotic factors as the GSM
(ground sites 1,2 and 3), suggesting that their excess nutrients is coming from a
different source rather than diet. This source, according to previous data and data
collected for this study, may be coming from groundwater inputs that have been
contaminated with wastewater leeching into the watershed from septic and the
wastewater treatment plant.
Overall, the data collected in this experiment provides supporting evidence
that excess nutrients from wastewater may affect the whole ecosystem due to the
evidence of elevated δ15
N values. The characteristics of West Falmouth Harbor
and the geological attributes of the watershed suggest that a large source of excess
nutrients may originate from the wastewater treatment plant and seep into the
estuary. Data suggests that groundwater, which is affected by wastewater inputs,
can be localized and detected within ribbed mussels.
Acknowledgements
I would like to thank, my advisor Dr. Kenneth Foreman, Richard
McHorney, and Carrie Harries for the many hours spent helping me to perfect
protocols, collecting and analyzing data. I would also like to thank Alice Carter
for the time spent in West Falmouth Harbor on very cold days deploying
moorings and collecting and analyzing data. Thank you to Marshall Otter of the
MBL Starr Stable Isotope Laboratory who contributed his expertise enabling me
to analyze my data. Thank you to Suzanne Thomas for lending a helping hand
with the diffusion protocol. The Semester in Environmental Science at the
Marine Biological Laboratory was one of the best experiences of my life, and I
want to thank all the people who made the experience worthwhile. This project
was funded by the MBL Semester in Environmental Science Program.
Figure 1: Display of the groundwater watershed leading to the West Falmouth
Harbor, Massachusetts. An orange shape, east of the harbor, represents the
Falmouth Wastewater Treatment Plant.
Figure 2: The Massachusetts Department of Environmental Protection measured
the amount of nitrogen loading within the West Falmouth Harbor in 2007. Most
nitrogen input is due to wastewater treatment facilities (WWTP) and septic
systems. The wastewater treatment facilities account for approximately 60% of
nitrogen being loaded into the seep.
70
50
30
10
Figure 3: Records of the total dissolved nitrogen, total nitrogen, and flow of
wastewater through the Falmouth Wastewater Treatment Plant. In 2006, the
treatment plant changed management from primary to tertiary hence the decreased
concentration of total nitrogen.
Figure 4: Thermal image of West Falmouth Harbor. Dark areas represent
groundwater input and experimental sites are displayed. Orange marks indicate
groundwater sites from which groundwater, ribbed mussel and ulva samples were
collected. The blue marks indicate harbor sites where moorings were deployed; 5
samples of incubated ulva were attached to each mooring in order to act as a
bioassay of nitrogen.
Figure 5: Nutrient values were taken from groundwater during November 2012.
Wells were located along the eastern shoreline of West Falmouth Harbor,
Massachusetts. Concentrations of nitrate show increasing trends at sites 2,3, and
4.
Co
nce
ntr
ati
on
(μ
M)
Groundwater Sites
0.0
0.5
1.0
1.5
2.0
0 2 4 6 8 10
PO
43
-
-2
0
2
4
6
NH
4+
0
100
200
300
400
NO
3-
Figure 6: Carbon to nitrogen ratio of ulva suspended within the harbor. Ulva was
incubated for one week within the MBL laboratories before introduced to West
Falmouth Harbor to act as bioassay of nitrogen. More nitrogen per carbon was
present at sites 1-3.
R² = 0.867
0
2
4
6
8
10
12
14
16
18
20
0 1 2 3 4 5 6 7
C:N
Ra
tio
(μ
M)
Harbor Sites
Figure 7: Nitrogen per phosphorous was greater at sites 1-4. All values exceed
the Redfield Ratio. Data was collected from West Falmouth Harbor in
Massachusetts during November of 2012.
Figure 8: Groundwater samples were collected from the eastern shoreline of West
Falmouth Harbor in Massachusetts. The isotopic composition of the groundwater
samples exhibit increased trends of δ15
N at sites 1-3. Each site corresponds to the
groundwater sites depicted in Figure 4.
10
100
1000
10000
0 1 2 3 4 5 6 7 8 9
N:P
Ra
tio
(u
M)
Sites
N:P Groundwater
Redfield Ratio 16:1
0
4
8
12
16
20
0 2 4 6 8 10
δN
15
(o
/oo
vs.
AIR
)
Sites
Figure 9: δ
15N concentrations were taken of ulva (Ulva lactuca) and ribbed
mussels (Mya arenaria) collected from groundwater sites within West Falmouth
Harbor in Massachusetts. At sites 1-3, both mussels and ulva exhibited higher
δ15
N values.
Figure 10: The δ15
N of incubated Ulva lactuca increased from 5.3 o/oo to an
average of 8.1 o/oo. Ulva was originally collected from Waquoit Bay Estuaries
and incubated for one week with running seawater from MBL facilities. Samples
4
5
6
7
8
9
10
11
12
13
4 6 8 10 12 14
δ1
5N
(o
/oo
vs.
AIR
)
Groundwater δ15N (o/oo vs. AIR)
Ulva Ribbed Mussel
0
2
4
6
8
10
0 1 2 3 4 5 6 7
d1
5N
(o
/o
o v
s. A
IR)
Harbor Sites
Suspended Algae d15N vs. sites
Suspended Ulva
Control
were attached to moorings and deployed in the West Falmouth Harbor for one
week during November of 2012.
Figure 11: δ
15N and δ
13C values of ribbed mussels and ulva collected at the
groundwater sites display increased values of δ15
N and differences in δ13
C values.
Samples were collected in November of 2012 from West Falmouth Harbor in
Massachusetts.
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
-25.0-20.0-15.0-10.0
δ1
5N
(o/o
o v
s. A
IR)
δ13C (o/oo vs. PDB)
Ulva
Mussel
Figure 12: δ
15N and δ
13C of ribbed mussel collected from groundwater sites in
West Falmouth Harbor (WFH) are compared to the δ15
N and δ13
C of spartina and
plankton collected from the Great Sippiwisset Marsh (Fry & Peterson 1985).
Note that the ribbed mussel values do not resemble that of either spartina or
plankton at groundwater site 1 and 2. Sites 1 and 2 within WFH exhibit similar
abiotic and biotic factors as the Great Swippiwisset Marsh.
G1&2 G3
G4 G5
G7
G8 G9
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
-21.0-19.0-17.0-15.0-13.0
d1
5N
(o
/oo
vs.
AIR
)
d13C (o/oo vs. PDB)
Mussel WFH
Spartina SIP
Plankton SIP
Figure 13: Cross section of West Falmouth Harbor well sites along the eastern
shore. The colored graph indicates the sites in relation to nitrate concentration
and depth. The bottom graph indicates groundwater sites in relation to δ15
N and
distance between sites. According to Foreman and McHorney, sites 1,2,3 and 4
have the highest amount of nitrate, and sites 3, 2 and 1 are most influenced by
wastewater input.
G4
G3
G5
G7
G8
G2 G1
G9
G9 G8 G7 G5 G4 G3 G2 G1
-5
-10
-15
0 1 2 3 4 5 6 7 8 9 10 11 12
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