Inputs of anthropogenic nitrogen influence isotopic ...

MPB-07153; No of Pages 7 Marine Pollution Bulletin xxx (2015) xxx–xxx

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Inputs of anthropogenic nitrogen influence isotopic composition and trophic structure in SE Australian estuaries Debashish Mazumder a,⁎, Neil Saintilan b, Brendan Alderson c, Suzanne Hollins a a b c

Australian Nuclear Science and Technology Organisation, Locked Bag 2001 Kirrawee DC, NSW 2232, Australia Department of Environmental Sciences, Macquarie University, Sydney, NSW 2109, Australia Cardno, PO Box 19, St Leonards, NSW 1590, Australia

a r t i c l e

i n f o

Article history: Received 2 April 2015 Received in revised form 31 August 2015 Accepted 31 August 2015 Available online xxxx Keywords: Estuaries Anthropogenic N Trophic linkages Stable isotopes

a b s t r a c t Urban development in coastal settings has increased the input of nitrogen into estuaries globally, in many cases changing the composition of estuarine ecosystems. By focussing on three adjacent estuaries with a gradient of anthropogenic N loadings, we used stable isotopes of N and C to test for changes due to increased anthropogenic N input on the structure of some key trophic linkages in estuaries. We found a consistent enrichment in δ15N corresponding to increased anthropogenic N at the three ecosystem levels studied: fine benthic organic matter, grazing invertebrate, and planktivorous fish. The degree of enrichment in δ15N between fine benthic organic matter and the grapsid crab Parasesarma erythrodactyla was identical across the three sites. The glassfish Ambassis jacksoniensis showed lower levels of enrichment compared to basal food sources at the higher N-loaded sites, suggesting a possible effect of anthropogenic N in decreasing food-chain length in these estuaries. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Anthropogenic influences on nutrient inputs into estuaries, in particular the elevation of nitrogen levels, have the potential to alter the structure and function of estuarine ecosystems. The accumulation of biomass in most coastal systems is nitrogen (N) limited (Rabalais, 2002). The nitrogen enrichment in the coastal water is increasing as a result of increasing anthropogenic activities (Fry et al., 2003) and globally several large estuarine and nearshore systems have been subject to eutrophication and hypoxia (Rabalais, 2002). In a global review, Leavitt et al. (2006) report a continued trend in the period following 1950 of increases in N and P leading to oxygen depletion and alterations to primary productivity, as a direct result of point and nonpoint pollution. One effect of elevated N in estuarine waters is a shifting of the production base from macrophytes to microphytic algae. Macreadie et al. (2012) demonstrated a shift in C to N ratios of organic carbon, consistent with a transition from macrophyte to algal carbon sources in estuarine cores from Botany Bay, Australia. The period of transition corresponded to catchment development and alterations to nutrient concentrations following European colonisation and catchment development. Such changes have the potential to alter the diet of benthic and intertidal herbivores, and Macreadie et al. (2012) called for further work to determine how increases in microalgae in Botany Bay have affected food web dynamics and ecosystem function within the Bay. ⁎ Corresponding author. E-mail address: [email protected] (D. Mazumder).

The trophic structure of benthic communities in Botany Bay has been extensively studied using stable isotopes of carbon and nitrogen (Mazumder et al., 2011; Saintilan and Mazumder, 2010). Generally crabs graze within narrowly defined feeding ranges (Guest et al., 2004), which allows the contrasting carbon isotope signatures of C3 and C4 saltmarsh plants, growing in mosaics, to be used to identify the relative contribution of macrophyte carbon in crab diets (Guest and Connolly, 2005). These studies have demonstrated a mixture of microphytobenthos and macrophyte detritus contributing to the diet of grapsid crabs, both at Botany Bay (Saintilan and Mazumder, 2010), and at nearby Brisbane Water (Alderson et al., 2013). Grapsid crabs play a keystone role in estuarine ecosystems in SE Australia, releasing larvae into the ebbing spring tide waters which provides an important source of nutrition for small fish (Mazumder et al., 2006) with a trophic relay traceable to commercially important predatory species (Mazumder et al., 2011). Shifts in the productivity base are therefore likely to show flow-on effects through the entire ecosystem. Stable nitrogen isotope analysis has emerged as an effective way of exploring the uptake of anthropogenic N in estuarine ecosystems. Inputs of nitrogen in catchments increase the amount of N cycling in soils, a process that results in enrichment of the heavier 15N isotope (Fry et al., 2003). Nitrate from human wastewater is also enriched in 15N as a result of de-nitrification and volatilization of ammonia (Kendall et al., 2007), with δ15N values typically in the range of +10‰ to + 20‰ (Hoffman et al., 2012). Surface and groundwater bearing atmospherically derived N typically support background δ15N signatures of +2‰ to +8‰ (McClelland et al., 1997).

http://dx.doi.org/10.1016/j.marpolbul.2015.08.047 0025-326X/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Mazumder, D., et al., Inputs of anthropogenic nitrogen influence isotopic composition and trophic structure in SE Australian estuaries, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.047

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D. Mazumder et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

This alteration to the δ15N signatures resulting from anthropogenic nitrogen inputs has been detected in an extensive range of ecosystem components. The nitrogen isotope ratios of primary producers reflect that of their nitrogen source, though fractionated (differential use of 15 N and 14N) during uptake. Consumers typically show a + 3‰ to + 5‰ enrichment in δ15N with respect to their diet (De Niro and Epstein, 1981; Minagawa and Wada, 1984). Elevated levels of δ15N have been identified in sediment and suspended particulate organic matter (Kohzu et al., 2009); macroalgae and macrophytes (Cole et al., 2004; Kaldy, 2011); bivalves (Oczkowski et al., 2008); macroinvertebrates and zooplankton (Peterson et al., 2007), and fish (Bannon and Roman, 2008; Hoffman et al., 2012). The δ15N values of nitrogen from animal waste, agricultural fertilisers and urban effluent sources are isotopically distinct from each other (Peterson, 1999; Costanzo et al., 2001, 2003), therefore the use of δ15N provides the advantage of integrating, through the relatively long absorption time (3–5 weeks) water quality fluctuations occurring over the short term. However, the approach may not detect very high ammonium inputs from sewage (Fry et al., 2003). Multi-estuary comparisons using gradients of N input have demonstrated the utility of δ15N as a potential indicator of N loading. McClelland et al. (1997) sampled a range of trophic levels in a series of estuaries in Cape Cod (USA) in which nitrogen loads varied from 2 to 467 kg N ha−1yr−1, and found that anthropogenic N was detectable even at relatively low loadings. Hoffman et al. (2012) compared larval fish δ15N across three watersheds spanning a large population density gradient in Lake Superior (USA), finding that δ15N increased with N concentration, reflecting higher anthropogenic inputs. Fry et al. (2003), compared N loading with δ15N in components of the estuarine biota in four West-Coast American systems, none low impact in respect to nitrogen loading, finding that macroalgae provided a good indicator of anthropogenic N. Several issues remain unresolved in relation to the impacts of nitrogen loading in the estuarine environment. Although numerous studies have demonstrated significant uptake of anthropogenic N in tissues of estuarine organisms, it is unclear in many cases whether this amounts to an environmental problem such that ecosystem structure and function are disrupted, and if so, at what thresholds of N loading these changes occur (Rabalais, 2002). The exploration of ecosystem-level effects of elevated anthropogenic N requires consideration of trophic linkages using multiple stable isotope tracers across impact gradients. In this study, we use stable isotopes of C and N in three estuarine settings that span a range of N loadings to explore the effect of elevated anthropogenic N on ecosystem trophic structure. Because δ15N is fractionated between consumer and prey by predictable amounts the measure can be used to determine trophic position, where the signature of basal δ15N is known. The dietary source is identified using δ13C, which has a lower degree of fractionation between diet and consumer (De Niro and Epstein, 1978). We utilise a well-studied trophic link between benthic detrital sources of carbon and nitrogen, and the dietary carbon source and trophic position of grazing crabs of the species Parasesarma erythrodactyla, and the glassfish Ambassis jacksoniensis, known to predate on the larvae of P. erythrodactyla, amongst other zooplankton sources. Three adjacent estuaries in the Sydney region, SE Australia, provide contrasting levels of anthropogenic N input while supporting common species and habitats. Our hypothesis is that the gradient of anthropogenic nitrogen input will correspond with a gradient of δ15N values at all trophic levels, but that the trophic position and dependencies of organisms will be otherwise unchanged. 2. Methods 2.1. Site selection Samples were collected from the three SE Australian estuaries with variable anthropogenic nitrogen inputs (Fig. 1). Brisbane Water is

situated on the northern entrance of Broken Bay, and the intertidal wetlands form on flood-tidal deltaic sands. The primary hydrological input is oceanic tidal water with no major rivers contributing sediment or water. This site is the least disturbed of the three chosen, with the lowest level of catchment clearance, lowest population density, and lowest percentage increase in anthropogenic N against estimated predevelopment levels (Table 1). The mangrove community at the site is dominated by Avicennia marina, fronted at the seaward edge by Zostera seagrass meadows. Botany Bay represents an intermediate level of anthropogenic modification amongst the three sites, in percentage clearance, and population density. Though current N inputs are lower than Brisbane Water, the Bay receives inputs from the Georges River, the major tributary, which contains a relatively high level of anthropogenic N (Table 1). Water clarity at the sampling site of Towra Point, Botany Bay is sufficient to support the seagrasses Posidonia australis and other species in the families Zosteraceae and Hydrocharitaceae. The mangrove community is dominated by A. marina, and the wetland consists of a diverse saltmarsh assemblage in the upper intertidal zone. In the late 1970s Towra Point was declared a nature reserve and in the mid-1980s its adjacent waters were declared an aquatic reserve. Homebush Bay, within the Parramatta River estuary, is the site of highest anthropogenic disturbance with the highest population density, highest level of clearance and highest levels of local anthropogenic nitrogen input of the three sites. The wetlands are situated on estuarine silts approximately 20 km from the ocean. From the late 1920s until the mid-1980s, the eastern shore of Homebush Bay was the site of various industrial facilities at which timber preservatives, tar-based products, herbicides, pesticides, chlorine gas and plastics were produced (Alexander, 2002). Some of the by-product material was disposed of on site, and contaminated spoil was used to reclaim land for the expansion of adjacent industrial facilities (Birch et al., 2007). A National Dioxin Study identified the highest dioxin and furan concentrations in Australia to be from Homebush Bay (Mueller et al., 2004). The industrial sites on the east side of the bay have been progressively replaced with residential apartments, with conditions of approval requiring the dredging, removal and treatment of contaminated soil. In the upper portion of the bay, brickworks and an abattoir operated for many decades but were replaced in the late 1990s to provide the site for the Sydney 2000 Olympics. Two small creeks, channelized and hence vastly modified from their natural condition, enter Homebush Bay from the south. Mangrove (A. marina) and saltmarsh are present, but seagrass is not, presumably due to reduced water clarity at this location. There were no sewage treatment facilities in the vicinity of any of the three sampling sites. 2.2. Sampling and analytical methods Three components of the food chain: sediment organic matter (SOM), burrowing crabs (P. erythrodactyla) and estuarine glassfish (A. jacksoniensis) were collected from saltmarsh and mangrove habitats of the three estuaries. Sample replication was determined using the prior power analysis of Mazumder et al. (2008). Sediment organic matter is the likely base of the food chain, and was collected from saltmarsh/mangrove habitats of the three estuarine ecosystems (n = 9–17). Sediment organic matter includes variety of living and non-living sources including microalgae (Nils, 2003). Sediment scrapings were collected from the top 1 cm of surface sediment (Melville and Connolly, 2003) and the samples were washed through 4-mm, 1-mm, 0.5-mm, and 0.25-mm sieves following the method of (Loneragan et al., 1997). Material elutriated from the finest sizefractions (b0.25 mm) were analysed for isotopic values. As stable isotopes used in trophic ecology rely on C fixed in the organic form (Mateo et al., 2008), acid-washing of sediment samples was performed prior to analysis. To avoid bias introduced by inorganic C, a small volume of 1 M hydrochloric acid was added to sediment sub-samples and left

Please cite this article as: Mazumder, D., et al., Inputs of anthropogenic nitrogen influence isotopic composition and trophic structure in SE Australian estuaries, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.047

D. Mazumder et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

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Fig. 1. Sampling sites from the three SE Australian estuaries with variable anthropogenic nitrogen inputs indicated by triangles.

for 3 h to remove the inorganic carbon present in the sediment (Polunin et al., 2001). The samples were then rinsed with distilled water, dried and ground to a fine powder. Since this acid-washing process results in enrichment of δ15N (Pinnegar and Polunin, 1999), untreated sediment sub-samples were analysed for δ15N. The dominant shore crab species P. erythrodactyla is a benthic grazer with a diet consisting of fine benthic material on the sediment surface (Guest and Connolly, 2005; Saintilan and Mazumder, 2010). Adult P. erythrodactyla were collected by hand from mangrove habitats at the three sites and frozen immediately for laboratory processing. White muscle tissue from crabs was sampled following the removal of exoskeleton. Glassfish (A. jacksoniensis) is a small fish (adult length ranges 43–44 mm; Mazumder et al., 2011) that is common to estuaries Table 1 Anthropogenic modification metrics for each study site, including Total Nitrogen (TN) loading in 2012. Georges River drains into Botany Bay and nitrogen loading at the point of sampling is likely to be influenced by this source. Source: NSW Office of Environment and Heritage, Australia (http://www.environment. nsw.gov.au).

Catchment area (km2) % catchment cleared Population Population density (no/km2) TN (t yr−1) % increase TN on pre-development Flushing time (days)

Georges River

Botany Bay

Parramatta River

Brisbane Water

450,940 59 851,544 900.3 450,940 870.6 62.5

18,566 61.5 110,990 1210.5 18,466 824.7 39.9

67,918 86.1 652,176 2460 67,918 397 17.3

39,025 52.1 100,969 644.4 39,025 188 24.6

along the southeast coast and on the basis of limited evidence (SPCC, 1981), appears to be consumed by a number of higher order predators. In turn, A. jacksoniensis is a carnivore, preying on crab larvae (Mazumder et al., 2006, 2011). Adult glassfish were collected from the saltmarsh and mangrove habitats using fyke nets and frozen instantly for laboratory processing. White muscle tissue from the dorsal region of glassfish was collected for isotopic analysis. Lipid extractions were not undertaken as delipidation from muscle tissue appears to lead to only small (b1‰) isotope shifts in δ13C and δ15N values (Sotiropoulos et al., 2004). Muscle tissue samples were dried at 60 °C for 72 h and then ground to a fine powder with mortar and pestle. After processing, samples were loaded in tin capsules and were analysed with a continuous flow isotope ratio mass spectrometer (CF-IRMS), model Delta V Plus (Thermo Scientific Corporation, U.S.A.), interfaced with an elemental analyser (Thermo Fisher Flash 2000 HT EA, Thermo Electron Corporation, U.S.A.). The data are reported relative to IAEA secondary standards that have been certified relative to VPDB for carbon and air for nitrogen. A two point calibration is employed to normalise the data, utilising standards that bracket the samples being analysed. Two quality control references were also included in each run. Results are accurate to 1% for both C% and N% and ±0.3 per mill for δ13C and δ15N. Stable isotope values were reported in delta (δ) units in parts per thousand (‰) relative to the international standard and determined as follows:  Xð‰Þ ¼

 Rsample −1  1000 Rstandard

where X = δ13C or δ15N, and R = 13C/12C or 15N/14N, respectively.

Please cite this article as: Mazumder, D., et al., Inputs of anthropogenic nitrogen influence isotopic composition and trophic structure in SE Australian estuaries, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.047

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D. Mazumder et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

2.3. Statistical methods

3. Results

Multifactorial permutational analysis of variance (PERMANOVA, Anderson et al. (2008) was used to investigate the difference between estuaries for δ13C and δ15N values of food chain components (i.e., SOM, crab and glassfish). The analyses were univariate and were based on Euclidean distance matrices calculated by PRIMER v6 for Windows (PRIMER-E Ltd., Plymouth, UK) using untransformed data. The food chain components analysed included δ13C and δ15N for SOM (sediment organic matter), P. erythrodactyla and A. jacksoniensis. The advantages of PERMANOVA approach over traditional ANOVA are that there is no assumption that the data are normally distributed and the test is not affected by unbalanced designs (Anderson et al., 2008). For the factor site, pairwise comparisons of disturbance levels (low impact, moderate and high nitrogen input sites) were performed to test whether significant differences between sites for isotopic values of food chain components were apparent. Trophic position of species was calculated using the formula reported by (Post, 2002):

Similar patterns of food chain structure were found in all three sites. The δ13C values of crabs were close to the SOM signature, suggesting this to be the common source of crab dietary carbon at all sites. The δ15N enrichment between SOM and P. erythrodactyla was 2.2‰ to 2.8‰ across the three sites, positioning crabs less than one trophic level above SOM in all cases (Fig. 2). If the standard enrichment of 3.4‰ is to be applied, suggesting either additional, depleted food sources or an uncommonly low level of enrichment for this trophic link. The small carnivorous fish A. jacksoniensis was consistently placed above the herbivorous crabs in food-chain structure all three sites. The δ15N enrichment between P. erythrodactyla and A. jacksoniensis though smaller for Parramatta River (4.9‰) and Botany Bay (6.2‰), was somewhat higher (8.1‰) at Brisbane Water. Trophic positions of P. erythrodactyla were similar across sites, ranging from 1.6 to 1.8, whereas trophic positions of A. jacksoniensis were similar at Botany Bay and Parramatta River sites and slightly higher (~0.6) in the Brisbane Water site (Fig. 2). The δ15N values of all components increased with increasing N loading, with a 1.9‰ increase in δ15N between Brisbane Water and Botany Bay for SOM, and a 4.2‰ increase between Brisbane Water and Parramatta River for SOM. These differences were promulgated through the ecosystems. δ15N values of P. erythrodactyla and A. jacksoniensis increased 2.5‰ and 0.7‰ respectively from Brisbane Water to Botany Bay and 4.9‰ and 1.6‰ respectively from Brisbane Water to Sydney Harbour. Carbon stable isotope ratios confirm the importance of sediment organic matter as the basal carbon source to the dominant herbivores (P. erythrodactyla) and carnivores (A. jacksoniensis) at all sites. While

 TPO ¼ λ þ δ15 Norganism −δ15 Nbase of

food web

 =3:4

where λ is the trophic level of the organism used to estimate δ 15 N base of food web . In this study, sediment organic matter (SOM) from study sites were identified as the likely base trophic resource (i.e. trophic level = 1, Kelleway et al. (2010). We applied commonly used trophic enrichment factor 3.4‰ (Post, 2002) to estimate trophic position.

Fig. 2. Stable carbon and nitrogen bi-plots showing food chain connectivity amongst three ecosystem level samples in the three studied sites with variable anthropogenic N inputs. a) Brisbane Water = low impact site, b) Botany Bay = moderate site and c) Parramatta River = high impacted site. Individual and mean values ± sd of samples were shown in the figures.

Please cite this article as: Mazumder, D., et al., Inputs of anthropogenic nitrogen influence isotopic composition and trophic structure in SE Australian estuaries, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.047

D. Mazumder et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

δ13C values varied between sites (being more depleted at the Parramatta River site), this positioning of consumer δ13C in relation to SOM δ13C did not change, being slightly fractionated between trophic levels, as would be expected (0 to + 1‰ fractionation between levels for δ13C; De Niro and Epstein, 1978; Peterson and Fry, 1987). PERMANOVA results found that sites were significantly different (P b 0.05) for both isotopes in SOM, P. erythrodactyla and A. jacksoniensis (Table 2). Comparison of the isotopic (δ13C and δ15N) values of samples between sites generally showed a significant difference (P b 0.05) in both isotopes in all samples types at all sites; the exceptions were the δ13C values of P. erythrodactyla samples from Brisbane Water (low impact) and Botany Bay (moderate) for which no difference was found (Table 3).

Table 3 Pairwise comparisons between levels (i.e., Brisbane Water: low impacted site, Botany Bay: moderately impacted site and Parramatta River: high impacted site) based on δ13C and δ15N isotope values of three food chain components. Significant differences between sites are determined by P b 0.05. δ13C

δ15N

Samples

Groups

t

P (Perm)

t

P (Perm)

SOM

Low, moderate Low, high Moderate, high Low, moderate Low, high Moderate, High Low, moderate Low, high Moderate, high

2.7364 8.4502 3.2972 0.20807 13.03 8.6201 4.6139 8.2161 6.8352

0.0136 0.0001 0.0076 0.8377 0.0001 0.0001 0.0001 0.0001 0.0001

7.61 13.5 12.1 10.8 18 9.42 2.37 6.25 4.24

0.0001 0.0001 0.0002 0.0001 0.0001 0.0001 0.0257 0.0001 0.0001

P. erythrodactyla

A. jacksoniensis

4. Discussion Degradation in the water quality of estuaries is a global trend (Smith et al., 1997), and commonly moves through two phases: firstly a pulse of sediment and nutrient resulting from deforestation and altered land use, and then the ongoing elevation of nitrogen and phosphorus loading from point and non-point sources (Lotze et al., 2006). Elevated N levels in estuarine waters promotes the growth of algae, potentially shifting the production base of estuarine food webs from macrophytic to microalgal sources (Elliot and Quintino, 2007). This is either because of the prevalence of microalgae or the replacement of seagrass due to poor water quality (Cole et al., 2004). The increase in N loading in estuaries globally has occurred primarily in the last halfcentury (Lotze et al., 2006), and is strongly associated with human population pressure. In temperate Australia, estuaries in their natural state are characterised by low levels of nitrogen, resulting from the character of soil vegetation and traditional land-use in the catchment (Specht and Specht, 1999). As a result, settlement and population growth following European colonisation and the introduction of fertilisers have led to profound alterations to the nutrient dynamics of Australian estuaries (Roy et al., 2001). Focussing seagrass habitat adjacent to one of our sampling sites, Macreadie et al. (2012) demonstrated a shift in C:N ratio of organic material within Botany Bay coincident with industrial and residential development within the catchment. They suggested that a trend towards algal rather than macrophyte sources might have flow-on effects in estuarine food chains. We tested this hypothesis across a gradient of N-impacted estuaries in the Sydney region, utilising stable isotopes of carbon and nitrogen in a well-documented trophic link between sediment organic material, grazing crabs and ambassids, the numerically dominant zooplanktivorous fish in the region. The Ambassids we analysed migrate between the seagrass beds and the adjacent mangrove and saltmarsh over the tidal cycle, as do several other fish species (Saintilan et al., 2007), feeding primarily on material exported from the mangrove and saltmarsh (Mazumder et al., 2006). This model of trophic association between brachuryan larvae, and feeding by Ambassids on lunar cycles finds common application across

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multiple estuaries in eastern Australia (Hollingsworth and Connolly, 2006; Platell and Freewater, 2009). Given the numerical importance of Ambassids in the estuarine environment, we expect that any changes in the trophic position or function of the species would be propagated through the estuarine ecosystem. Our findings concord with previous studies have shown that fine benthic organic material, comprising plant detritus and microphytobenthos, is the primary source of nutrition for grazing crabs in the region (Saintilan and Mazumder, 2010; Alderson et al., 2013). Trends in the δ15N values of fine benthic organic material, the dominant source of dietary carbon to grazing invertebrates, closely followed the trends in the level of anthropogenic N input across the three estuaries studied. These were reflected in the δ15N signatures of the grazing grapsid crab P. erythrodactyla, which were fractionated by 2.2–2.8‰ at each site. This level of δ15N fractionation falls within reported ranges between 0.7‰ to 5.2‰ for invertebrates (Caut et al., 2009). Crab δ13C signatures also followed differences in SOM δ13C between estuaries. Crabs appear tightly coupled with SOM and the link is unaffected by elevated levels of anthropogenic N inputs between sites. The δ15N of muscle tissue of the glassfish A. jacksoniensis was enriched approximately 5.5‰ compared to P. erythrodactyla at the Botany Bay and Parramatta River sites, and 8.1‰ at the Brisbane Water site. The estimated trophic position of the species changed in association with the gradient in anthropogenic N inputs between estuaries, being 3.3 at the most heavily impacted site, 3.6 at the site of intermediate impact, and 4.0 at the least impacted site. A. jacksoniensis is a small carnivore, not directly preying upon adult P. erythrodactyla but preying on zooplankton including P. erythrodactyla larvae (Mazumder et al., 2006, 2011), but also caridean decapods, amphipods, sergestid decapods and algae (McPhee et al., 2015a,2015b). The species can shift between diet sources depending on availability, and has been demonstrated to do so over a spring tidal cycle (McPhee et al., 2015a,b). We postulate that differences in trophic position of the species in relation to basal food sources may relate to the availability of decapod

Table 2 Univariate PERMANOVA results for δ13C and δ15N values of three food chain components sampled from three sites (i.e., Brisbane Water, Botany Bay and Parramatta River) in NSW, Australia. Significant differences amongst sites for isotopic values are determined by P b 0.05. Samples

SOM

P. erythrodactyla

A. jacksoniensis

δ13C

δ15N

Source of variation

df

SS

MS

Pseudo-F

P (Perm)

Source of variation

df

SS

MS

Pseudo-F

P (Perm)

Site Residual Total Site Residual Total Site Residual Total

2 27 29 2 40 42 2 38 40

90 56 146 148 48.2 196 142 53.9 196

44.977 2.0759

21.667

0.0001

0.0001

0.0001

84.116 0.42469

198.07

0.0001

71.151 1.4178

50.183

0.0001

89.884 9.1727 99.056 168.23 16.988 185.22 16.843 16.091 32.934

132.29

61.447

2 27 29 2 40 42 2 38 40

44.942 0.33973

74.099 1.2059

Site Residual Total Site Residual Total Site Residual Total

8.4214 0.42344

19.888

0.0001

Please cite this article as: Mazumder, D., et al., Inputs of anthropogenic nitrogen influence isotopic composition and trophic structure in SE Australian estuaries, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.047

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and algal food sources between sites, with the elevated δ15N signatures of the larval zooplankton counterbalanced by a higher availability of algae at the N enriched sites. Further research on variation in the diet of this species along N pollution gradients would help elucidate this. We found no evidence to support the contention that elevated N shifted the source of dietary carbon of grazing crabs from benthic macrophyte-derived sources to microphytos. We found microphytobenthos was the dominant source of dietary carbon for crabs grazing beneath mangrove at the least impacted site, as documented in Alderson et al. (2013). The δ13C value of microalgae in Brisbane Water has previously been measured as approximately − 22‰ (Alderson et al., 2013) and the δ13C values of macrophytes in Botany Bay have been found to range from approximately −15‰ (Sporobolus virginicus saltmarsh) to − 27‰ (Mazumder et al., 2011). The more depleted δ13C values at the Parramatta River site, the most impacted of the three, suggests a higher contribution of macrophyte carbon. This is the site most heavily impacted by N pollution, though the results may reflect the relatively greater distance from the estuary entrance, and presumably lower concentrations of marine diatoms. Crabs seem to feed on fine detrital material of macrophyte or algal origin depending on availability within their narrow feeding range, and make an important contribution to the diet of glassfish, most probably through the export of larvae (Mazumder et al., 2011). Our results provide further evidence of the utility of stable N isotopes in monitoring the uptake of anthropogenic N in estuarine ecosystems. The organisms chosen are easily sampled, and suitable statistical power is provided by a modest number of replicates in most settings (Mazumder et al., 2008). The stable N isotope ratios of tissues collected across the three sites reflected the degree of anthropogenic modification and nitrogen pollution in the estuaries, and corresponded to population density within the catchments, in common with North American studies (Bannon and Roman, 2008; Cole et al., 2004). Acknowledgements Samples were collected under scientific license SL100432. Barbora Neklapilova (ANSTO) and Scott Allchin (ANSTO) assisted with sample preparation and analysis. Stuart Hankin (ANSTO) for drawing Fig. 1. Dr Swapan Paul (SOPA) is thanked for assisting in the collection of samples from Homebush Bay. Office of Environment and Heritage, NSW provided access to the Towra Point sites, and data on N loadings and anthropogenic modification to the estuaries. Comments from anonymous reviewers allowed us to improve the quality of the completed manuscript. Animal ethics approval was granted by the Director General's Animal Ethics Committee of the Department of Primary Industries, project number 02/3477 and samples collected from Brisbane Water were collected under NSW Scientific Collection Permit P07/0122-2.0. References Alderson, B., Mazumder, D., Saintilan, N., Zimmerman, K., Mulry, P., 2013. Application of isotope mixing models to discriminate dietary sources over small-scale patches in saltmarsh. Mar. Ecol. Prog. Ser. 487, 113–122. Alexander, M., 2002. Environmental Impact Statement; the Remediation of the Lednez site, Rhodes Peninsula and Homebush Bay—Summary. Parsons Brincherhoff, Australia. Anderson, M.J., Gorley, R.N., Clarke, K.R., 2008. PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods. Plymouth, UK. Bannon, R.O., Roman, C.T., 2008. Using stable isotopes to monitor anthropogenic nitrogen inputs to estuaries. Ecol. Appl. 18, 22–30. Birch, G.F., Harrington, C., Symons, R.K., Hunt, J.W., 2007. The source and distribution of polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofurans in sediments of Port Jackson, Australia. Mar. Pollut. Bull. 54, 295–308. Caut, S., Angulo, E., Courchamp, F., 2009. Variation in discrimination factors (Δ15N and Δ13C): the effect of diet isotopic values and applications for diet reconstruction. J. Appl. Ecol. 46, 443–453. Cole, M.L., Valiela, I., Kroeger, K.D., Tomasky, G.L., Cebrian, J., Wigand, C., McKinney, R.A., Grady, S.P., Carvalho da Silva, M.H., 2004. Assessment of a δ15N isotopic method to indicate anthropogenic eutrophication in aquatic ecosystems. J. Environ. Qual. 33, 124–132.

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Please cite this article as: Mazumder, D., et al., Inputs of anthropogenic nitrogen influence isotopic composition and trophic structure in SE Australian estuaries, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.047