Nitrogen source tracing in the Wye River Complex
Nitrogen source tracing in the Wye River Complex
Prepared for:
Midshore Riverkeeper Conservancy
Prepared by:
Dr Simon Costanzo
Integration and Application Network
University of Maryland center for Environmental Science
Nitrogen source tracing in the Wye River Complex
1 INTRODUCTION ................................................................................................................................ 3 1.1 OVERVIEW ..................................................................................................................................... 3 1.2 SOURCING NITROGEN IN THE WYE RIVER COMPLEX. ....................................................................... 4 2 METHODOLOGY ............................................................................................................................... 6 2.1 SITE LOCATIONS ............................................................................................................................ 6 2.2 OYSTER INCUBATION ..................................................................................................................... 8 2.3 SAMPLE ANALYSIS ......................................................................................................................... 9 3 RESULTS AND DISCUSSION ......................................................................................................... 10 4 REFERENCES ................................................................................................................................. 12 APPENDIX 1 PHOTOS OF SITE LOCATIONS ..................................................................................... 13 APPENDIX 2 PROTOCOL FOR PREPARING ORGANIC MATTER FOR STABLE ISOTOPE
ANALYSIS .............................................................................................................................................. 17 Nitrogen source tracing in the Wye River Complex
1 Introduction
1.1
Overview
The Wye River Complex (WRC) is part of the Upper Eastern Shore Tributary Strategy Basin and drains
into Eastern Bay that joins the Chesapeake Bay. The Wye River Complex consists of the Wye East,
Wye Main Stem and Wye Narrows, has an approximate watershed area of 53 square miles, and has a
length of approximately 9 mi both from south to north and from southwest to northeast.
Routine water quality monitoring by the Maryland Department of the Environment (MDE) has revealed
unacceptable levels of fecal coliform bacteria (often indicating contamination with the fecal material of
humans or other animals) in the shellfish harvesting areas of the WRC prompting numerous closures
over the past decade. Shellfish, such as oysters and clams, are filter feeders and filter the water around
them for microorganisms, phytoplankton, detritus, and inorganic particles a food source. As shellfish
are often eaten raw or partially cooked, the presence of elevated bacteria in the water column can
result in contaminated shellfish and a human health hazard if consumed.
The source of the elevated bacteria in the WRC has yet to be identified. The Wye River Watershed is
comprised of large homes on septic systems; farms that include cattle, sheep, emu; cropland,
woodland, and rural communities, which are all potentially contributing factors to bacterial inputs
impacting water quality in the Wye River. In addition, there is a large population of wild geese in the
vicinity. These factors, combined limited tidal flushing, are believed to have contributed to elevated
fecal coliform levels in the Wye River.
The objective of this study was to use an alternative method to trace the source of elevated bacteria
levels in the Wye River Complex via the use of stable nitrogen isotopes (δ15N). δ15N in the tissue of
aquatic organisms can be used to distinguish between chemically synthesized agricultural fertilizers
and human or animal wastes, the latter being associated with high bacterial loads. Oysters have been
shown to integrate the different sources of nitrogen in the water column over time (Fertig et al., 2010),
providing a benefit over direct measurements of the water column, which only provide an instantaneous
measurement.
Determination of nitrogen source by measuring δ15N is possible, because natural sources and synthetic
fertilizers are ‘‘fixed’’ from atmospheric N2 (0%) and have correspondingly low δ15N values: generally -4
to +4% (Bateman and Kelly, 2007; Hubner, 1986; Macko and Ostrom, 1994; Vitoria et al.,
Nitrogen source tracing in the Wye River Complex
2004). More positive δ15N values are indicative of human waste, animal waste, or both types of nitrogen
sources, but these cannot be distinguished from one another because they are due to the same
process of fractionation. This approach was tested in the Wye River Complex to further focus
investigations into the source of elevated bacterial levels identified in this system.
1.2
Sourcing nitrogen in the Wye River Complex.
The nitrogen cycle is the process by which nitrogen is converted between its various chemical forms
(Figure 1-1). This transformation can be carried out through both biological and physical processes.
The majority of Earth's atmosphere (78%) is nitrogen gas (N2), making it the largest pool of nitrogen.
However, atmospheric nitrogen has limited availability for biological use, leading to a scarcity of usable
nitrogen in many types of ecosystems. Human activities such as fossil fuel combustion, use of artificial
nitrogen fertilizers, and release of nitrogen in wastewater have dramatically altered the global nitrogen
cycle.
N2#
Figure 1-1 The varied molecular forms of inorganic nitrogen (blue atom) as nitrogen gas (N2), Nitrate (NO3-), nitrite (NO2-)
and ammonia (NH4+).
There are two naturally occurring forms of the nitrogen atom (Figure 1-2). The common form that
contains seven protons and seven neutrons is referred to as nitrogen 14 and is expressed as 14N. A
heavier form that contains an extra neutron is called nitrogen 15 and is expressed as 15N.
Nitrogen source tracing in the Wye River Complex
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Figure 1-2 Nitrogen exists naturally in two isotopic forms. The common form contains seven protons and seven neutrons ( N)
15
and the heavier form that contains an extra neutron ( N).
The various sources of nitrogen pollution to coastal ecosystems often have distinguishable 15N:14N
ratios (referred to as δ15N), thereby providing a means to identify the source of pollution (Heaton, 1986).
For example, nitrogen fertilizer and sewage-derived nitrogen have distinct differences in their δ15N
signatures. The main method of nitrate and ammonium fertilizer production is by industrial fixation of
atmospheric nitrogen, resulting in products that have δ15N values close to zero (Figure 1-3).
However in animal or sewage waste, nitrogen is excreted mainly in the form of urea, which when
hydrolyzed, produces a temporary rise in pH. The more basic conditions favor conversion to ammonia,
which is easily lost by volatilization to the atmosphere. Fractionation during this process results in the
ammonia, which is lost from the system, being depleted in 15N. The remaining ammonium, now
correspondingly enriched in 15N, is subsequently converted to 15N-enriched nitrate, which is more
readily leached and dispersed by water (Heaton,1986) (Figure 1-3). The elevated δ15N signature of
treated sewage (δ15N = 10%) therefore distinguishes it from other nitrogen sources entering marine
ecosystems (cf. fertilizer nitrogen δ15N = 0%) (Heaton, 1986).
Nitrogen source tracing in the Wye River Complex
Atmospheric,
nitrogen,
Atmospheric,
fixa2on,
14N,
15N,
Vola2liza2on,
Industrial,
fixa2on,
Denitrifica2on,
Biological,,
fixa2on,
Ammonium,
Nitrite,
Nitrate,
Figure 1-3 The nitrogen cycle highlighting sources of bioavilable nitrogen to the environment and demonstrating the process of
15
N enrichment through fractionation, volatilzation and denitrification.
Aquatic organisms reflect their exposure to elevated nitrogen through increases in tissue nitrogen
content (%N), and to different nitrogen sources through variations in tissue δ15N over a given timeframe
(Costanzo et al., 2001; Peterson and Fry, 1987). Assessing the variations of δ15N organisms such as
shellfish provides a technique for detecting and sourcing biologically available nitrogen entering the
Wye River Complex.
2 Methodology
2.1
Site locations
A total of 16 locations were identified throughout the WRC, ranging in distribution from the confluence
of the river system with Eastern Bay to the upper navigable limits of the system (Figure 2-1).
Nitrogen source tracing in the Wye River Complex
Figure 2-1 Sampling locations for oyster deployment within the within the Wye River Complex.
Nitrogen source tracing in the Wye River Complex
Table 2-1 Locations of oyster deployment.
Location Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8 Site 9 Site 10 Site 11 Site 12 Site 13 Site 14 Site 15 Site 16 Latitude 38.8451 38.8605 38.8710 38.8878 38.9051 38.9189 38.9427 38.9695 38.9102 38.8895 38.9183 38.8885 38.8837 38.8778 38.8656 38.8557 Longitude -­‐76.2056 -­‐76.1982 -­‐76.1960 -­‐76.1887 -­‐76.1696 -­‐76.1657 -­‐76.1568 -­‐76.1620 -­‐76.1358 -­‐76.1148 -­‐76.1142 -­‐76.0822 -­‐76.1298 -­‐76.1618 -­‐76.1631 -­‐76.1800 A photo library of sampling locations can be found in Appendix 1.
2.2
Oyster incubation
Oysters (Crassostrea virginica) from the Midshore Riverkeeper Conservancy Oyster Restoration
Project (spat sourced from the University of Maryland Center for Environmental Science Oyster
Hatchery) were used for this project. Oysters of uniform age and size and grown at Site 2, close to the
river mouth, were placed in a plastic mesh bag (Figure 2-2) and suspended 1 m below the low water
mark for 90 days (08/23/2013 – 10/21/2013). Each mesh bag contained 3 oyster replicates and were
periodically cleaned of fouling throughout the incubation period to ensure adequate flow through of
water.
Nitrogen source tracing in the Wye River Complex
Figure 2-2 Oysters were incubated in a plastic mesh bag.
2.3
Sample analysis
All oyster samples were stored frozen prior to overnight courier to, and analysis for nitrogen content
and stable isotope analysis, by the Odum School of Ecology, University of Georgia (refer to Appendix 1
and Appendix 2 for detailed methodology) (stylized methodology as per Figure 2-3).
o
Figure 2-3 Sample analysis includes drying of plant samples at 60 C for 24-48 hours, grinding and analysis in a mass
spectrometer.
Nitrogen source tracing in the Wye River Complex
3 Results and Discussion
δ15N values of whole oyster tissue following the 90-day incubation in the Wye River Complex ranged
from 7.6 ‰ – 12.0 ‰ suggesting the entire system is influenced to varying degrees by elevated δ15N
values, characteristic of animal and/or human waste. Overall the highest value obtained was at Site 15
in Shaw Bay, whereas lowest values were recorded in the upper reaches of Wye East (Sites 10 and
12) and at the mouth of the river at Site 1. Values were generally uniform along the length of the Wye
River (~10 ‰).
Despite generally widespread elevation of δ15N values, variability did exist between sites and highest
values were more prevalent in the lower and western reaches of the system (highlighted by the white
dotted circle in Figure 3-1), rather than at the extreme upper reaches as may be expected due to poorer
tidal flushing in these areas. This suggests the source of elevated δ15N is originating towards the
western and lower watershed rather than upstream in the Wye East. Other than a prevalence of
residential properties along the western peninsula (Bennett Point) of the Wye River, most of the other
adjoining land in the immediate watershed appears from satellite imagery to be agricultural (including
the Wye Research and Education Center along Wye Narrows). This close proximity between most of
the elevated δ15N values and residential properties warrants further investigation into the possibility of
failing septic systems in the region.
It should be noted, however, that oysters did originate from Site 2 and likely had elevated levels at the
beginning of the study. Despite this, oysters have shown a response to varied conditions through
increases and decreases in δ15N values over the study period (compared to the value recorded at Site
2) indicating the trends observed and reported remain valid.
Future work
Additional works that would further refine the source of elevated δ15N values would include a more
targeted survey in the region highlighted by the white dotted circle in Figure 3-1, potentially using
aquatic plants as indicators for ease of collection and processing. This combined with an assessment of
septic failures for residential properties along the Wye River would further elucidate the as yet unknown
source of elevated bacteria levels being identified in the system.
Nitrogen source tracing in the Wye River Complex
10.3#
10.0#
10.7#
10.8#
8.6#
11.3#
7.9#
10.1#
10.4#
7.6#
11.7#
11.3#
10.8#
12.0#
11.1#
8.8#
15
Figure 3-1 δ N values (‰ or parts per thousand) of whole oyster tissue following 90 day incubation. White dotted line
encompasses the values above 10 ‰.
Nitrogen source tracing in the Wye River Complex
4 References
Bateman, A.S. and Kelly, S.D., 2007. Fertilizer nitrogen isotope signatures. Isotopes in Environmental
and Health Studies, 43, 237–247.
Costanzo, S. D., O'Donohue, M. J., Dennison, W. C., Loneragan N. R. and Thomas, M. T. (2001). A
new approach for detecting and mapping sewage impacts. Marine Pollution Bulletin, 42: 149-156.
Fertig, B., Carruthers, T.J.B., Dennison, W.C., Fertig, E.J., Altabet, M.A., 2010. Eastern
oyster (Crassostrea virginica) δ15N as a bioindicator of nitrogen sources: observations and modeling.
Mar. Poll. Bull. 60: 1288-1298.
Heaton, T. H. E. (1986). Isotopic studies of nitrogen pollution in the hydrosphere and atmosphere: A
review. Chemical Geology: 59, 87-102.
Hubner, H., 1986. Isotope effects of nitrogen in the soil and biosphere. In: Fritz, P., Fontes, J.C. (eds.),
Handbook of Environmental Isotope Geochemistry. The Terrestrial Environment, Vol. 2B. Amsterdam:
Elsevier, pp. 361–425.
Macko, S.A. and Ostrom, N.E., 1994. Pollutions studies using nitrogen isotopes. In: Lajtha, K.,
Michener, R.M. (eds.), Stable Isotopes in Ecology and Environmental Science. Oxford, UK: Blackwell,
pp. 45–62.
Peterson, B. I. and Fry, B. (1987). Stable isotopes in ecosystem studies. Annual Review of Ecology and
Systematics: 18, 293-320.
Vitoria, L.; Otero, N.; Soler, A., and Canals, A. 2004. Fertilizer characterization: isotopic data (N, S, O,
C, and Sr). Environmental Science & Technology, 38, 3254–3262.
Nitrogen source tracing in the Wye River Complex
Appendix 1 Photos of site locations
Site 1
Site 3
Site 2
Site 4
Nitrogen source tracing in the Wye River Complex
Site 5
Site 6
Site 8
Site 7
Nitrogen source tracing in the Wye River Complex
Site 9
Site 11
Site 10
Site 12
Nitrogen source tracing in the Wye River Complex
Site 13
Site 14
Site 15
Site 16
Nitrogen source tracing in the Wye River Complex
Appendix 2 Protocol for preparing organic matter for stable
isotope analysis
Laboratory Equipment and Materials:
Wiley Mill with 40- or 60-mesh screen (or equivalent)
Microbalance accurate to 0.001 mg
Scintillation vials
Encapsulating tins (holder optional)
96 well microliter plates for storing encapsulated samples
DRY & GRIND
1. Dry organic matter samples at 60 C for at least 48 hrs
2. Grind sample to a uniform fine-grained (flour-like) texture using a Wiley mill with 40- or 60-mesh
screen (or equivalent)
3. Clean the mill with ethanol between samples
ENCAPSULATE & WEIGH
4. Place tin capsule on balance accurate to 0.001 mg and tare
5. Clean all surfaces and utensils by wiping with ethanol. It is best to work over an area that is
white (a piece of white plastic tape can be placed down on the table) so that you can see if any
material is spilled
6. Place tared tin capsule on bench and carefully add material using small spatula using the
weighing criteria below
7. Place capsule back on balance (using forceps) to record added dry mass (or remove and add
more material if mass is too low) according to weighing notes below
8. Remove tin capsule from balance and place on clean white surface to crimp tin down to small
packet (fold over top to close, then crimp sides down so that all dimensions are < 2 mm)
9. Check to see that there is no leakage of material by dropping tin packet (from a height of 2
inches) onto white surface
10. Tin packet is then placed in one well location (use 96-well microliter plates) and the well location
(e.g., A1…A12, B1…B12, etc), sample type, sampling station, and dry mass is recorded using
the same tared balance
Nitrogen source tracing in the Wye River Complex
ANALYSIS
11. Combust at 1100C and deliver gases via continuous-flow for analysis of del13C, del15N,
percentage carbon and percentage nitrogen using an isotope ratio mass spectrometer (IRMS)
12. Determine C:N ratio from percentage element weight
WEIGHING NOTES:
Because organic material will generally contain plenty of C for detection by the mass
spectrophotometer, it is more important to assure that there is enough N in each type of sample when
analyzing for C and N simultaneously. Most labs have an upper limit (300 mg N) and a lower limit
(about 20-25 mg N) for 15N samples (optimum is 100 mg N), different masses of material must be
weighed out for different sample types (different %N content):
SAMPLE TYPE (%N CONTENT)
WEIGHT
Sediment (0.5 to 0.15 %N):
15 – 30 mg
Leaves (0.5 to 2 %N):
6 – 8 mg
Roots/Stems (0.4 to 1.3 %N)
5 – 12 mg
Wood (0.3 to 2 %N):
10 – 12 mg
Fine organic material (FOM) or soil (0.3 to 2 %N):
10 – 12 mg
Suspended organic material (SOM) (0.5 to 3 %N):
5 – 7 mg
Biofilm (0.5 to 3 %N):
5 – 7 mg
Plant grains (1.5 to 3.5 %N):
3 – 7 mg
Grass/algae (1.5 to 5 %N):
3 – 5 mg
Animal, Fish & Invertebrates (~10 %N):
0.6 – 1.7 mg
ENRICHED SAMPLES
•
If samples are from a stable isotope labeling experiment (e.g., 15NH4 uptake in plants), process
samples in order of least enriched to most enriched (background/control samples of all types
first, then least enriched to most enriched of each type; for example, it is expected that wood
would be first because they are expected to have the least 15N and fast-assimilating tissue like
algae last because they would likely have the highest 15N values).
•
The background samples of all types should be grouped together first in the well plates (e.g.,
first 1 or 2 columns of wells - locations A1 through A12 and B1 through B12) and then the 15N-
Nitrogen source tracing in the Wye River Complex
enriched samples from least to most enriched. This helps to avoid carry-over effects with the
mass spectrometer. Generally, the differences in δ15N will be greater between sample types
than with increasing distance from the labeling source. The order of 15N enrichment for sample
types in streams, for example, is likely to be wood < FOM < leaves < SOM < biofilm < algae, so
you would group them in this order in the microplates.
.
SAMPLES ON FILTERS
•
For samples retained on GFF filters (FBOM, SPOM, epilithon), carefully remove only organic
material from filter to add to tin.
•
For samples that do not have a thick “cake” on filter (epilithon, SPOM), be as careful as possible
to minimize inclusion of filter fibers within the sample (e.g., use a sharp scalpel or small knife for
scraping material from filters).
•
If it is not possible to scrape material from filter without inclusion of filter material in the sample,
then it may be possible to encapsulate the entire filter, but record only the dry mass of material
on the filter (subtract the filter tare mass) as the sample dry mass. The well plates with samples
can then be stored for up to several months before shipment to the 15N analytical lab.
References:
Mulholland et al. 2004. LINX II STREAM 15N EXPERIMENT PROTOCOLS
http://www.faculty.biol.vt.edu/webster/linx/linx2proto-rev5.pdf
UC Davis Stable Isotope Facility:
http://stableisotopefacility.ucdavis.edu/sample-weight-calculator.html