Northeastern Naturalist Vol. 22, No. 1 C. Wigand, H. Smith, C. Spears, B. Keith, R. McKinney, M. Chintala, and K. Raposa 2015 1 2015 NORTHEASTERN NATURALIST 22(1):1–9 Relationships of Modeled Nitrogen Loads with Marsh Fish in the Narragansett Bay Estuary, Rhode Island Cathleen Wigand1,*, Heather Smith1, Cassius Spears1, Brandon Keith1, Richard McKinney1, Marnita Chintala1, and Kenneth Raposa2 Abstract - The human population and associated watershed development has risen steadily since the 1850s in Rhode Island. With these increases, human-derived wastewater has also risen dramatically, resulting in increasing nitrogen loads to estuarine systems. In this study, we examined relationships of modeled watershed nitrogen loads of 6 coastal subwatersheds of varying land development with the stable nitrogen isotope ratio (δ15N) of salt marsh fish and larvae. There was a significant positive relationship (r = +0.97, P < 0.05) between the watershed modeled percent wastewater and δ15N in Fundulus heteroclitus L. (Common Mummichog), and significantly higher (P < 0.05) δ15N in fish larvae collected from developed mainland marsh sites compared to less-developed island marsh sites. Our results support earlier published findings that fish in coastal marshes are assimilating nitrogen derived from watershed wastewater sources. Furthermore, there was an inverse relationship (P = 0.05) between the modeled percentage of human wastewater and mummichog size. The increasing loads of watershed nitrogen entering into coastal salt marshes are a concern because it is unclear how well salt marsh ecosystems can continue to assimilate high nitrogen inputs especially when also subjected to a warming climate. Introduction Fish in salt marshes are known to be hardy and opportunistic since they are adapted to a life in often harsh conditions (Bigelow and Schroeder 1953). Salt marsh fish live in water with rapidly changing salt and tide levels and, in recent times, increasing loads of anthropogenic nutrients. The human population in Rhode Island has risen steadily since the 1850s, and with this increase, human-derived wastewater has also risen dramatically (Nixon et al. 2008). While some studies report that increased nutrient inputs into coastal habitats may increase primary production and food resources available for some coastal marsh fish (Nixon 1992, Oczkowski et al. 2009, Tober et al. 2000), others report adverse effects of high nutrient loading due to low-oxygen events, sometimes even resulting in fishkills and mass mortality of marine invertebrates (Deacutis 2008; Diaz and Rosenberg 1995, 2001). Longterm nutrient loadings have also been implicated in marsh loss (Deegan et al. 2012, Wigand et al. 2014). Researchers have found direct relationships between the rise in the human population, coastal watershed land development, and the stable nitrogen isotope ratios of (δ15N) in Spartina alterniflora, Loisel (Smooth Cordgrass), 1USEPA, National Health and Environmental Effects Research Laboratory, Atlantic Ecology Division, Narragansett, RI 02882. 2Narragansett Bay National Estuarine Research Reserve, 55 South Reserve Drive, Prudence Island, RI 02872. *Corresponding author - Wigand.Cathleen@epa.gov. Manuscript Editor: Hunter J. Carrick Northeastern Naturalist 2 C. Wigand, H. Smith, C. Spears, B. Keith, R. McKinney, M. Chintala, and K. Raposa 2015 Vol. 22, No. 1 Geukensia demissa, Dillwyn (Ribbed Mussel), and Fundulus heteroclitus, L. (Common Mummichog) in tidal wetlands (e.g., Bannon and Roman 2008, Cole et al. 2004, McClelland and Valiela 1998, McKinney et al. 2001). These relationships may result from elevated δ15N values characteristic of human-derived wastewater. In this study, we examined the stable nitrogen isotope ratios, abundance, and size of small fish in 6 subwatersheds of the Narragansett Bay Estuary (RI) along a gradient of high to low nitrogen loadings. In addition, the δ15N of larval fish were measured from 3 developed mainland sites and 3 less-developed island sites to see if there was an anthropogenic signal in the larvae from the developed watersheds. We examine and report the relationships of the fish δ15N, size, and abundance with the modeled watershed nitrogen loadings. Site Description and Methods. Marsh fish were sampled twice at each site in the summer of 2005 in 6 Narragansett Bay subestuaries, which had varying watershed land use and estimated nitrogen loads (Table 1, Fig. 1). Seines were cast from shore (i.e., quarter circle haul) during an ebb tide using a 6-m-long seine (3-mm mesh). Larval fish were collected by pit traps (plexiglass trays: 28 x 18 x 4 cm) set flush with the soil surface in the high marsh (mixed meadow of Spartina patens (Aiton) Muhl [Saltmeadow Cordgrass] and Distichlis spicata (L.) Greene [Saltgrass]) at 3 developed mainland sites (Bissel Cove, Old Mill Cove, and Passeonkquis Cove) and at 3 less-developed coastal island sites (Fox Hill Pond, Jenny Marsh, and Providence Point) (Fig. 1). Pit traps were placed in 2 parallel lines of 5 traps each, a few meters apart, coincident with flooding of the high marsh at each site. The pit traps were sampled on spring tides around the full and new moons on 7 dates between 21 June–17 September. Estimates of the watershed nitrogen loads entering the salt marshes were previously reported (Wigand et al. 2003) and calculated using (1) 1995 aerial photography (1:24,000 scale) showing land use and land cover and (2) a landbased nutrient loading model (Valiela et al. 1997). The nutrient-loading model (NLM: NLOAD homepage, http://nload.mbl.edu) was developed and verified for Table 1. Land use and modeled nitrogen loads for six Narragansett Bay subwatersheds. Ag. = agricultural; Ind. and comm. = industrial and commercial; Recr. = recreational; Natural lands include inland wetlands, forests, and brush lands; and Resid. = residential. Site locations are shown in Figure 1: Jenny Marsh (JEN), Sapowet (SAP), Mary Donovan (DON), Bissel Cove (BIS), Watchemoket (WAT), and Apponaug Cove (APP). % land use and land cover Modeled watershed nitrogen loads Ind. and Natural Nitrogen load % wastewater Location Ag. comm. Recr. lands Resid. (Kg N ha-1 y-1) nitrogen Jenny Marsh 0.0 0.0 0.0 81.3 4.0 2.5 20.3 Sapowet 34.7 0.4 0.1 49.0 13.8 183.3 21.7 Mary Donovan 20.0 0.5 0.1 57.2 10.0 412.6 25.7 Bissel Cove 4.7 1.9 1.2 55.7 22.1 3183.1 68.5 Watchemoket 0.0 9.5 13.9 3.8 56.0 6727.0 82.0 Apponaug Cove 3.1 12.3 3.8 22.7 43.3 11407.2 83.3 Northeastern Naturalist Vol. 22, No. 1 C. Wigand, H. Smith, C. Spears, B. Keith, R. McKinney, M. Chintala, and K. Raposa 2015 3 Cape Cod, MA (Valiela et al. 1997, 2000) and has additionally been verified for Barnegat Bay, NJ (Bowen et al. 2007a, b). Each of the components required to estimate the nitrogen loading in the NLM are subject to uncertainty, and Collins et Figure 1. Location of subwatersheds (n = 6) used to model nitrogen loadings, and locations of mainland (n = 3) and island (n = 3) pit-trap sites where we sampled fish larvae. We cast seines in open water in marshes adjacent to the 6 subwatersheds. Northeastern Naturalist 4 C. Wigand, H. Smith, C. Spears, B. Keith, R. McKinney, M. Chintala, and K. Raposa 2015 Vol. 22, No. 1 al. (2000) calculated a 13% uncertainty in the loading estimates of the NLM using a bootstrap resampling method. The NLM has more recently been used to successfully estimate nitrogen loading to 74 subwatersheds in southern New England (Latimer and Charpentier 2010) and 33 subwatersheds of the Great South Bay, NY (Kinney and Valiela 2011). The NLM estimates nitrogen loads from fertilizer application, human wastewater, and atmospheric deposition to 4 land-use types (i.e., natural vegetation, turf, agricultural land, and impervious surface). The model accounts for attenuation as the nitrogen passes through the watershed surface and subsurface zones and calculates the net input of nitrogen as the sum of the inputs minus losses in various land-use types. The estimate of the nitrogen load to the marsh is then calculated as the sum of the net loads determined from the NLM from fertilizer, human wastewater, and atmospheric deposition, We randomly selected 10 seine-collected mummichogs from each site and froze them for δ15N analysis. We removed their guts and heads, dried the remaining portions, and ground and homogenized them with a mortar and pestle. We treated larval samples similarly except we did not remove their guts and heads. We used continuous- flow isotope-ratio mass spectrometry to measure the δ15N of the samples, and known differences in nitrogen isotope ratios to examine if there was an anthropogenic nitrogen signal in the fish samples. Compared with estimates of atmospheric deposition of nitrogen (δ15N of +2 to +8 ‰, Kreitler and Jones 1975) and fertilizer (δ15N of -3 to +3 ‰; Freyer and Aly 1974, Macko and Ostrom 1994), nitrogen derived from wastewater (δ15N of +10 to +22 ‰; Aravena et al. 1993, Kreitler and Jones 1975) is relatively enriched in 15N and has a higher stable nitrogen isotope ratio. Therefore, human-derived nitrogen inputs would result in an enriched δ15N in fish when the percentage of watershed wastewater increases. Results and Discussion. We found a significant relationship (r = +0.97, P < 0.05) between the watershed modeled wastewater nitrogen loads (Table 1) and the δ15N in Common Mummichog (Fig. 2). The varying δ15N of the fish collected from 6 subestuaries in Narragansett Bay having high to low watershed nitrogen loads (Table 2) support the premise proposed by others (e.g., Cole et al. 2004, McClelland and Valiela 1998) that watershed nitrogen was entering into salt marshes and being assimilated by biota. The larval fish collected from the developed mainland sites had significantly higher δ15N (13.2 ± 1.3 ‰) than those collected from the less-developed island marshes (9.9 ± 0.16 ‰) (P < 0.05, using a two-tailed t-test on log transformed data), further supporting the hypothesis that the watershed loads of human-derived nitrogen from developed areas are being assimilated by marsh fish. In contrast, Pruell et al. (2006) inferred that the changes in stable nitrogen isotope ratio in biota among 3 Narragansett Bay subestuaries might be due to the differences in distance from point sources of sewage outfall from the head of the bay. Previously, the stable nitrogen isotope ratios of Smooth Cordgrass (Wigand et al. 2001) and Ribbed Mussels (McKinney et al. 2001) were measured in 10 Narragansett Bay salt marshes. Similar to the relationship we found with Common Northeastern Naturalist Vol. 22, No. 1 C. Wigand, H. Smith, C. Spears, B. Keith, R. McKinney, M. Chintala, and K. Raposa 2015 5 Figure 2. Relationships of the stable nitrogen isotope ratios of Common Mummichog, Ribbed Mussel, and Smooth Cordgrass with the wastewater fraction of the modeled nitrogen loads. The Ribbed Mussel and Smooth Cordgrass results were first reported in McKinney et al. (2001) and Wigand et al. (2001), respectively. Table 2. The stable nitrogen isotope ratios (δ15N), abundance, and size of Commmon Mummichogs from 6 subestuaries with varying modeled watershed nitrogen loads. Location of sites are shown in Figure 1, and watershed nitrogen loads are provided in Table 1. Site δ15NA Abundance (total #)B Size (mm)C Jenny Marsh 9.1 161.5 63.9 Sapowet 9.0 81.0 57.8 Mary Donovan Marsh 9.2 101.0 44.3 Bissel Cove 11.8 88.5 29.1 Watchemoket Cove 14.7 21.5 33.0 Apponaug Cove 13.7 421.5 41.7 Among-site mean 11.3 145.8 45.0 Among-site SE 1.0 58.1 5.6 ACommon Mummichog stable nitrogen isotope ratio from sample of 1 0 fish. BAverage # mummichogs in 2 seine catches. CAverage size of 10 mummichogs. Northeastern Naturalist 6 C. Wigand, H. Smith, C. Spears, B. Keith, R. McKinney, M. Chintala, and K. Raposa 2015 Vol. 22, No. 1 Mummichog in this study, the stable nitrogen isotopic ratios of Smooth Cordgrass and Ribbed Mussels also significantly increased with increasing percent wastewater nitrogen loads estimated from the watershed NLM model. When the δ15N of all 3 organisms among the Narragansett Bay sites are considered together, it is apparent that the stable nitrogen isotope ratios of the Common Mummichog and Ribbed Mussel are higher than the Smooth Cordgrass ratios (Fig. 2). Since stable nitrogen isotopic ratios increase in species further up the food chain, it is not a surprise that the fish and mussels would have a higher signal than the plants at the base of the food chain. The fish are omnivores, and their stable nitrogen isotope ratios are elevated because of the consumption of small mollusks and crustaceans in addition to plant detritus. Ribbed Mussels are filter-feeders and consume plant detritus, phytoplankton, bacteria, and other particulate matter. McClelland et al. (1997) demonstrated trophic transfer using stable nitrogen isotope ratios in biota in 2 Waquoit Bay, MA, sites (i.e., Sage Lot Pond, Childs River). In the seines, we primarily caught juvenile Common Mummichog, but there were also small numbers of Menidia menidia, L. (Atlantic Silverside) and Cyprinidon variegatus, Lacépède (Sheepshead Minnow). In addition to fish species, the seines also captured small shrimp, crabs, and Ulva lactuca, L. (Sea Lettuce). The most (422) and least (22) numbers of mummichogs were collected from the 2 most urbanized sites with the greatest modeled wastewater nitrogen loads, Apponaug Cove and Watchemoket Cove, respectively. Average mummichog size among the marsh sites was 45 ± 6 mm, and average abundance among sites was 146 ± 58 fish (Table 2). There was an inverse trend (r = -0.81, P = 0.05) between the watershed modeled percent wastewater nitrogen and mummichog size, and a positive trend (r = +0.73, P = 0.10) of the watershed modeled total nitrogen load and mummichog abundance in Narragansett Bay. LaBrecque et al. (1996) and Tober et al. (2000) reported significantly greater abundances but no difference in growth rates of Common Mummichogs in subestuaries with greater nitrogen loadings in Waquoit Bay, MA. Our seining study was of short duration and only 6 marshes in Narragansett Bay were sampled, so we propose that future long-term and more detailed studies might better elucidate the relationships of fish abundance, size, and growth with watershed nitrogen loadings in a number of different urban estuaries. Urbanization and the associated cultural eutrophication (especially the overenrichment of nitrogen) is known to cause reduced system oxygen levels and macrofauna declines (Deacutis 20008; Diaz and Rosenberg 1995, 2001), plant species shifts on the salt marsh landscape (Bertness et al. 2002, Silliman and Bertness 2004, Wigand et al. 2003), and marsh-bank deterioration (Deegan et al. 2012). The increasing loads of watershed nitrogen entering into New England salt marshes are a concern, because it is unclear how well salt marsh ecosystems and fish in particular can assimilate these inputs, especially when also subjected to the additional stress of a warming climate (Nixon et al. 2004). Because salt marshes are located between the coastal sea and the uplands, and provide numerous ecosystem services, such as fish habitat, flood abatement, and water-quality maintenance, it is important to monitor and understand watershed nitrogen effects on coastal systems. Northeastern Naturalist Vol. 22, No. 1 C. Wigand, H. Smith, C. Spears, B. Keith, R. McKinney, M. Chintala, and K. Raposa 2015 7 Watershed nutrient sources are likely some of the most appropriate for managing estuarine water-quality conditions because these sources are most amenable to watershed-scale remedial action (Latimer and Charpentier 2010). Acknowledgments Heather Smith, Cassius Spears, and Brandon Keith were sponsored through the EPA/ University of Rhode Island Environmental Sciences Research and Training Opportunities Program (RTOP), a multi-institutional cooperative agreement (CT8 3045). During the summer of 2005, they conducted research with mentor scientists, Cathleen Wigand, Richard McKinney, and Marnita Chintala of the US Environmental Protection Agency, Narragansett Laboratory. Kenneth Raposa is the Research Coordinator at the Narragansett Bay National Estuarine Research Reserve, where 2 of our sampling sites were located. Special thanks to Kim Anderson and Darryl Keith for coordinating RTOP and connecting the students with the EPA mentors. We are also grateful to the GIS expertise of Mike Charpentier in determining the land use for the watersheds described in this study. Mention of trade names or commercial products does not constitute endorsement or recommendation for use by the US EPA. This report, contribution number AED #07-056, has been technically reviewed by the US EPA’s Office of Research and Development, National Health and Environmental Effects Research Laboratory, Atlantic Ecology Division, Narragansett, RI, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency. Literature Cited Aravena, R.M., M.L. Evans, and J.A. Cherry. 1993. Stable isotopes of oxygen and nitrogen in source identification of nitrate from septic systems. Ground Water 31:180–186. Bannon, R.O., and C.T Roman, C. 2008. 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