Wastewater nitrogen and trace metal uptake by biota on a high-energy rocky shore detected using stable isotopes

MPB-07119; No of Pages 8 Marine Pollution Bulletin xxx (2015) xxx–xxx

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Wastewater nitrogen and trace metal uptake by biota on a high-energy rocky shore detected using stable isotopes Joanne M. Oakes ⁎, Bradley D. Eyre Centre for Coastal Biogeochemistry Research, Southern Cross University, Lismore, NSW 2480, Australia

a r t i c l e

i n f o

Article history: Received 31 October 2014 Received in revised form 12 August 2015 Accepted 16 August 2015 Available online xxxx Keywords: Stable isotopes Rocky shore Sewage Gastropod Algae Australia

a b s t r a c t On high-energy rocky shores receiving treated wastewater, impacts are difficult to distinguish against a highly variable background and are localised due to rapid dilution. We demonstrate that nitrogen stable isotope values (δ15N) of rocky shore biota are highly sensitive to wastewater inputs. For macroalgae (Ulva lactuca and Endarachne binghamiae), grazing snails (Bembicium nanum and Nerita atramentosa), and predatory snails (Morula marginalba), δ15N was enriched near a wastewater outfall and declined with distance, returning to background levels within 290 m. Any of these species therefore indicates the extent of influence of wastewater, allowing identification of an appropriate scale for studies of ecosystem impacts. For M. marginalba, significant regressions between δ15N and tissue copper, manganese, and zinc concentrations indicate a possible wastewater source for these metals. This suggests that δ15N is a proxy for exposure to wastewater contaminants, and may help to attribute variations in rocky shore communities to wastewater impacts. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Sewage treatment plants are a major source of anthropogenic input to coastal environments (Costanzo et al., 2001). Depending on the level of treatment, the wastewater released from sewage treatment plants may contain a variety of contaminants, including nutrients, particulate organic matter, and trace metals (Costanzo et al., 2001; Cabral-Oliveira et al., 2015). This has led to concern regarding the impact of treated wastewater on receiving environments. Wastewater is often discharged to estuaries and coastal environments and a number of studies have assessed its impact on marine communities. These have shown that the effect of wastewater can be species-specific. Whereas more tolerant species may demonstrate increased productivity (Cabral-Oliveira et al., 2014) and recruitment (Bellgrove et al., 1997), the health of more sensitive species and/or individuals may be adversely impacted (e.g., Schlacher et al., 2007), causing them to become less dominant (López-Gappa et al., 1990). These changes can be reflected in altered population structure (Hindell and Quinn, 2000) and diversity (Fairweather, 1990; López-Gappa et al., 1990), and the species composition and biomass of communities in the receiving environment (Littler and Murray, 1975; López-Gappa et al., 1990; Bellgrove et al., 1997). The impacts of wastewater on marine communities are more difficult to detect where treated wastewater is discharged to high-energy coastal environments such as rocky shores. Due to rapid dilution and ⁎ Corresponding author. E-mail address: [email protected] (J.M. Oakes).

dissipation of wastewater in these environments, impacts can be highly localised (e.g., Fairweather, 1990), and/or difficult to detect against a background of high environmental and community variability (Smith, 1994; O'Connor, 2013). Sampling at an inappropriate scale can lead to an inability to identify impacts or incorrect assessment of impacts (Bishop et al., 2002). To counteract these limitations, it has been suggested that studies aiming to determine wastewater impacts on rocky shores should sample at multiple scales (Bishop et al., 2002), and/or include multiple control sites (Fairweather, 1990; Hindell and Quinn, 2000; Cabral-Oliveira et al., 2015). However, where the ability to adopt these recommendations is limited by the additional effort and cost involved and/or availability of suitable controls, it would be valuable to ascertain, a priori, the extent of influence of the wastewater plume, and the likely exposure of rocky shore communities to treated wastewater and its contaminants. A relatively inexpensive, simple method to detect the distribution of anthropogenic inputs within aquatic environments is the analysis of stable isotopes (Oakes et al., 2010). In the case of wastewater, preferential removal of 14N via ammonia volatilisation during secondary and tertiary treatment leads to wastewater-derived nitrogen typically having a nitrogen isotope signature (δ15N) that is relatively enriched in the rare, heavy isotope, 15N (Heaton, 1986; Costanzo et al., 2001). This is reflected in enriched δ15N values for nutrients within discharged wastewater, which is readily assimilated by primary producers (Costanzo et al., 2001; Cole et al., 2004). Consumers that feed upon these primary producers also acquire an enriched δ15N ‘wastewater signal’, and this is then passed to higher consumers via trophic transfer (e.g., Connolly et al., 2013). Fractionation usually results in further

http://dx.doi.org/10.1016/j.marpolbul.2015.08.013 0025-326X/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Oakes, J.M., Eyre, B.D., Wastewater nitrogen and trace metal uptake by biota on a high-energy rocky shore detected using stable isotopes, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.013

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J.M. Oakes, B.D. Eyre / Marine Pollution Bulletin xxx (2015) xxx–xxx

enrichment of δ15N values with each trophic transfer (Minagawa and Wada, 1984; McCutchan et al., 2003). Depending on tissue turnover time, analysis of δ15N values of flora and fauna gives a temporallyintegrated indication of wastewater nitrogen inputs, compared to analysis of δ15N within water column nutrients, which can vary rapidly with changes in discharge rate and environmental conditions (Hood et al., 2014). Stable nitrogen isotope analysis has previously been used to investigate wastewater nitrogen uptake by biota in a variety of aquatic environments but, to our knowledge, has not previously been applied in a high-energy, rocky shore environment receiving wastewater input. In the current study, we aimed to determine (1) if δ15N values could be used to detect wastewater input in a high-energy rocky shore environment (sensitivity), and (2) how well δ15N values of species across multiple trophic levels reflected wastewater input (suitability of species as indicators). Based on this, we further aimed to determine (3) the extent of influence of wastewater input to a rocky shore environment, and (4) if variations in wastewater exposure (based on δ15N values) explained variations in trace metal concentrations within biota (i.e., if stable isotopes are a proxy for the spread of other contaminants). 2. Materials and methods 2.1. Study site The study was done in mid-2009 in northern New South Wales, Australia. Impacted sites were located in the intertidal zone approximately 10 m, 40 m, 60 m, 100 m, 150 m, and 290 m north of an outfall pipe which discharges secondary-treated effluent from the Lennox

Head Wastewater Treatment Plant just below low tide level at the base of a cliff at Skennars Head (28°49′28″S, 153°36′25″E; Fig. 1). Longshore drift is north. A generally flat basaltic intertidal platform approximately 1700 m south of the outfall (Flat Rock) was selected as the non-impacted control site (Fig. 1). The study sites are exposed to the open ocean and are subject to high wave energy, depending on tidal height. At the time of the study, treated wastewater from the Lennox Head Wastewater Treatment Plant had average concentrations of ammonium, nitrate, total nitrogen, and total phosphorous of 35.7, 149.9, 285.6 and 164.6 μmol L−1, respectively (ranges of 0.7–464.1, 6.4–359.8, 128.5–778.2, and 51.6–298.6 μmol L−1). Data regarding metal concentrations in treated wastewater are not available for the time when the study was done, or for all metals. However, over 12 months from September 2007 to 2008 average (± standard error) concentrations for some metals dissolved in the treated wastewater were as follows: cadmium b0.001 ± b0.001 mg L−1, chromium b0.01 ± b0.001 mg L−1, copper 0.02 ± b0.01 mg L−1, iron 0.10 ± 0.02 mg L−1, lead b0.01 ± 0.00 mg L−1, zinc 0.01 ± b 0.01 mg L−1, boron 0.15 ± 0.02 mg L−1, copper 0.02 ± 0.01 mg L−1, calcium 42.6 ± 1.5 mg L−1, magnesium 25.2 ± 1.2 mg L−1, potassium 19.9 ± 0.8 mg L−1, sodium 192.1 ± 10.0 mg L−1, and sulphate 84.5 ± 4.4 mg L− 1. Some treated wastewater from the treatment plant is re-used locally, with the volume re-used determined by weather conditions (more during dry periods) and the surplus discharged to the ocean at a rate of up to 100 L s− 1. Daily flow from the outfall varies with weather conditions. For example, under similar operating conditions over 6 months from December 2007 to May 2008, the daily flow ranged from 4.4 to 1036.6 ML d−1 (average 45.4 ± 12.7 ML d−1).

Fig. 1. Sampling locations at Skennars Head. Inset shows control site at Flat Rock and area encompassed by main map (defined by square). Approximate location of the wastewater outfall is indicated by an arrow.

Please cite this article as: Oakes, J.M., Eyre, B.D., Wastewater nitrogen and trace metal uptake by biota on a high-energy rocky shore detected using stable isotopes, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.013

J.M. Oakes, B.D. Eyre / Marine Pollution Bulletin xxx (2015) xxx–xxx

2.2. Sample collection Samples of rocky shore biota were collected to represent three trophic levels on the rocky shore. These were two species of macroalgae (trophic level 1; Ulva lactuca (Chlorophyceae) and Endarachne binghamiae (Phaeophyceae)), two species of grazing gastropod (trophic level 2; Bembicium nanum and Nerita atramentosa), and one species of predatory gastropod (trophic level 3; Morula marginalba). Where possible, five replicate samples of each of these species were collected at each site. For animal samples, five individuals from within each of five haphazardly selected ~1 m2 plots were collected and pooled to provide sufficient material for laboratory analyses. Due to variations in distribution and abundance, only one sample of E. binghamiae was collected at the site 100 m from the outfall, and only four replicate samples of U. lactuca were collected 10 m from the outfall. B. nanum was not present at the site 40 m from the outfall.

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Principal components analysis was used for each species to assess relationships among metals and δ15N and determine which metals displayed similar spatial trends to δ15 N values. Where δ15 N and a metal both contributed significantly to a component, and the direction of this contribution was the same (i.e., both had a positive or negative association with the component), there was considered to be a strong positive association between δ15N and that metal (i.e., metal concentration increased with δ15N enrichment). In these cases, linear regression analysis was used to determine if the variations in metal concentration were significantly correlated with variations in δ15N (significance level = 0.05). Values for the control site and the site 290 m from the outfall were excluded from regression analyses, as δ15N values indicated that there was no wastewater influence at these sites. 3. Results 3.1. Nitrogen isotope ratios without wastewater exposure

2.3. Laboratory analyses Gastropod shells were cracked using a plastic-wrapped hammer, avoiding metal contamination. The entire body of the gastropods was then removed using plastic tweezers and rinsed in Milli-Q to remove any shell fragments. Algae were gently scraped clean of debris and attached fauna and flora and rinsed using Milli-Q. All samples were lyophilised, then homogenised using a mortar and pestle. Subsamples of dried, homogenised plant and animal material were weighed and sealed into tin capsules for δ15N analysis using an Elemental Analyser (Eurovector EA 3000) interfaced with a continuous flow–isotope ratio mass spectrometer (Thermo Delta V Plus). Stable isotope ratios are expressed in standard delta notation, calculated as follows: δ15 N ¼




 Rsample =Rstandard −1  1000

where R is 15N/14N. Values are reported in units of per mil (‰). Reproducibility was ±0.2‰. Following removal of material for isotope analysis, the remaining sample material was analysed for silver, aluminium, arsenic, cadmium, chromium, copper, iron, manganese, nickel, lead, selenium, zinc, mercury, vanadium, boron, silicon, cobalt, molybdenum, barium, calcium, magnesium, potassium, sodium, bromide, and sulphur concentrations. Samples were digested in HNO3 and HCl and trace metal concentrations were determined using inductively coupled plasma mass-spectrometry (ICP-MS) according to standard American Public Health Association methods (APHA 3120 ICP-MS; APHA, 2005). Results are expressed as dry weights (μg g−1 of dried tissue). 2.4. Data analysis For each species, a one-way Analysis of Variance (ANOVA) was used to look for significant differences in δ15N values among sites (significance level = 0.05). Levene's test was used to test for homogeneity of variances. Data were transformed (log(x + 1)) to improve homogeneity where Levene's test returned a significant result. Where significant differences were detected, post-hoc Tukey tests were used to determine which of the seven sampling sites were different. Linear regressions were used to describe the gradient of depuration of δ15N values from the outfall. These regressions excluded δ15N values for the control site. Values for the site 290 m from the outfall, which did not exhibit a wastewater signal, were also excluded. This was intended to avoid unfairly reducing the magnitude of regression gradients, as it was unknown at what distance between the sites at 150 m and 290 m from the outfall δ15N values returned to control levels. An analysis of covariance (ANCOVA) was used to test if the slopes of the regressions, or their elevations, were similar (significance level = 0.05). Post-hoc Tukey tests were used to determine which elevations were different from one another.

Values of δ15N for algae and snails at the control site confirmed the assumed trophic levels of the species collected, with ~2‰ enrichment in 15N with each trophic level. Both algal species had similar δ15N values (E. binghamiae, 7.0 ± 0.5‰; U. lactuca 7.4 ± 1.4‰). Values of δ15N were also similar for the two grazing snails, but were 1.5‰ to 2.5‰ more enriched than for algae (B. nanum, 9.0 ± 0.4‰; N. atramentosa, 9.6 ± 0.6‰). The most enriched δ15N values at the control site were for the predatory snail M. marginalba (10.8 ± 0.6‰). 3.2. Wastewater nitrogen distribution There was evidence of wastewater nitrogen assimilation across the three trophic levels studied. There were significant differences in δ15N values across sites for both species of macroalgae (E. binghamiae, ANOVA: F6,18 = 241.01, p b 0.001; U. lactuca, ANOVA: F6,27 = 182.48, p b 0.001), both grazing snail species (B. nanum, ANOVA: F5,24 = 499.209, p b 0.001; N. atramentosa, ANOVA: F7,27 = 21.529, p b 0.001), and the predatory snail (M. marginalba, ANOVA: F6,28 = 69.543, p b 0.001) (Fig. 2). For all species, δ15N values were significantly enriched in 15N at the site nearest the outfall (10 m) compared to the control site. This was the site where the most enriched δ15N values were detected for all species except E. binghamiae, for which values 40 m and 60 m from the outfall were the most enriched (Fig. 2). For all species, δ15N values generally declined with distance from the outfall, δ15N values 290 m from the outfall were statistically indistinct from control δ15N values, and there was a significant linear regression between δ15N and distance from the outfall (from 10 m to 150 m, p ≤ 0.001 for all species except N. atramentosa, for which p = 0.012; Fig. 2). Distance from the outfall accounted for 25% and 52% of the variation in δ15N values for N. atramentosa and E. binghamiae, respectively, and for 71–77% of the variation in δ15N for the remaining species. The gradient of δ15N depuration (slope of the regressions) was similar across species (ANCOVA: F4,102 = 0.26, p = 0.90), with δ15N values becoming depleted by ~ 0.04‰ m−1 (common (pooled) regression slope estimate). This equates to a change of 1‰ in δ15N over 25 m. However, the elevations of the regressions differed significantly among species (ANCOVA: F4,106 = 30.48, p b 0.001). In contrast to variations in δ15N among species at the control site, post-hoc Tukey tests revealed that at wastewater-impacted sites δ15N values were more 15N-enriched at lower trophic levels. This indicates that assimilation of wastewater nitrogen decreased with trophic level. Values of δ15N for the two species of macroalgae at the wastewater-impacted sites were similar, but were significantly enriched compared to values for the snail species. M. marginalba δ15N values were significantly depleted compared to those of B. nanum. Values for N. atramentosa were intermediate to, and not statistically different from, those of M. marginalba and B. nanum. Reflecting this, the difference between control δ15N values and the most enriched δ15N values (Δδ15N) was 15.1 and

Please cite this article as: Oakes, J.M., Eyre, B.D., Wastewater nitrogen and trace metal uptake by biota on a high-energy rocky shore detected using stable isotopes, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.013

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Fig. 2. δ15N values (mean ± S.E.) of a) Endarachne binghamiae, b) Ulva lactuca, c) Bembicium nanum, d) Nerita atramentosa, and e) Morula marginalba at different distances from the Skennars Head wastewater outfall. Letters beneath each error bar show Tukey results, where letters which are the same indicate no significant difference. Note: For E. binghamiae, the site 100 m from the outfall was not included in Tukey tests due to lack of replication (- = not determined).

16.4‰ for U. lactuca and E. binghamiae (trophic level 1), 11.4 and 19.9‰ for B. nanum and N. atramentosa (trophic level 2), and 7.1 for M. marginalba (trophic level 3). This equates to an increase in the atom % 15N for the three trophic levels of approximately 0.006%, 0.004%, and 0.003%, respectively.

3.3. Trace metal distribution and link to wastewater inputs Principal components analysis showed that 71.3%, 57.3%, 55.4%, 63.3%, and 53.7% of the total variation within the data for each species could be explained using three (U. lactuca, N. atramentosa) or four components (E. binghamiae, B. nanum, M. marginalba). Only nine strong positive associations between δ15N and metals were detected and tested via linear regression analysis. Values of δ15N were positively associated with silver and molybdenum for U. lactuca (component 3); copper, silver, cadmium, and mercury for B. nanum (component 3); and magnesium, copper, and zinc for M. marginalba (component 1) (Table 1). Linear regressions between metal concentration and δ15N across wastewater-impacted sites were only statistically significant in the case of M. marginalba. For this species, concentrations of copper (F1,23 = 31.776, p b 0.001, R2 = 0.58), zinc (F1,23 = 9.963, p = 0.004, R2 = 0.30) and magnesium (F1,23 = 18.397, p b 0.001, R2 = 0.44) increased significantly with δ15N values, by approximately 17 μg g− 1, 49 μg g− 1, and 518 μg g− 1, respectively, with each 1‰ increase in δ15N (Fig. 3).

4. Discussion In the current study we demonstrated that (1) rocky shore biota at three trophic levels assimilated wastewater derived nitrogen, (2) there was greater incorporation of wastewater nitrogen by species at lower trophic levels, (3) the wastewater δ15N ‘signal’ reduced with distance from the outfall, at a similar rate across all species, (4) the influence of wastewater nitrogen on the rocky shore was highly localised (detected only within 290 m of the outfall), and (5) uptake of wastewater nitrogen correlated with variations in copper, zinc, and magnesium concentrations in the predatory snail M. marginalba. 4.1. Sensitivity of stable isotopes to detect wastewater nitrogen in highly variable environments There was a roughly linear decline in δ15N values of all species with increasing distance from the wastewater outfall, reflecting a relatively simple mixing pattern at the study site, with greater dilution of wastewater further from the outfall. Furthermore, the gradient of δ15N depuration was similar across species and trophic levels. These patterns of wastewater nitrogen assimilation were not particularly unexpected, but the ability of stable nitrogen isotopes to detect the input of wastewater into the high-energy environment studied is remarkable. The rapid dilution of wastewater nitrogen at the site, indicative of this high energy, is evident in the gradient of δ15N depuration with distance

Please cite this article as: Oakes, J.M., Eyre, B.D., Wastewater nitrogen and trace metal uptake by biota on a high-energy rocky shore detected using stable isotopes, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.013

Parameter

Algae

Snails

E. binghamiae

δ15N Silver Arsenic Lead Cadmium Chromium Copper Manganese Nickel Selenium Zinc Mercury Iron Aluminium Vanadium Boron Silicon Cobalt Molybdenum Barium Calcium Magnesium Potassium Sodium Bromide Sulfur

U. lactuca

B. nanum

N. atramentosa

M. marginalba

C1

C2

C3

C4

C1

C2

C3

C1

C2

C3

C4

C1

C2

C3

C1

C2

C3

C4

−0.799 0.688 0.164 0.550 0.710 0.731 0.140 −0.020 0.756 0.507 −0.016 −0.118 0.937 0.879 0.908 0.152 0.802 0.323 0.647 −0.140 −0.035 0.129 −0.386 −0.303 0.529 −0.091

0.116 −0.091 −0.176 −0.260 −0.183 0.094 0.151 0.664 0.161 0.577 −0.088 0.636 0.100 0.063 0.189 0.684 0.054 0.214 0.000 −0.141 0.256 0.438 −0.067 0.791 0.644 0.934

−0.241 −0.282 0.645 −0.525 0.199 0.189 0.086 −0.345 −0.322 0.036 −0.403 0.549 0.040 0.066 −0.134 0.035 0.163 −0.763 −0.149 −0.845 −0.799 −0.080 0.058 −0.102 −0.041 −0.004

0.331 −0.031 0.318 −0.134 −0.035 0.427 0.872 0.193 −0.036 0.121 0.833 0.129 0.014 −0.114 0.027 0.034 0.279 0.168 −0.069 0.063 0.204 −0.296 0.719 −0.434 −0.172 0.133

−0.549 0.246 0.227 0.369 0.353 0.391 −0.514 0.334 0.015 −0.326 −0.176 0.055 0.729 0.681 0.771 −0.124 0.217 0.382 0.209 0.532 0.710 0.675 0.009 −0.871 −0.551 0.073

0.034 0.186 0.032 0.534 −0.099 0.661 0.532 0.045 0.592 0.847 −0.093 0.860 0.358 0.316 −0.051 0.531 0.807 −0.025 −0.038 0.359 −0.027 0.075 −0.053 0.223 0.678 0.818

−0.730 −0.642 0.706 −0.348 0.049 0.109 0.067 −0.537 0.636 −0.133 −0.019 −0.102 −0.230 −0.212 −0.328 −0.318 −0.187 −0.400 −0.600 −0.365 0.173 0.633 0.216 −0.032 0.027 0.362

0.185 −0.152 0.542 0.072 0.249 0.037 −0.202 0.200 0.091 0.889 0.700 0.266 −0.061 0.210 0.100 0.773 0.781 0.059 −0.091 −0.035 0.077 0.138 0.368 0.829 0.899 0.330

−0.155 0.095 0.215 0.297 −0.291 −0.077 0.204 0.576 0.081 −0.064 −0.061 −0.134 0.930 0.777 0.897 0.228 0.230 0.597 0.237 0.750 −0.307 −0.346 0.127 −0.010 −0.163 −0.008

−0.705 −0.889 0.020 0.409 −0.682 0.017 −0.905 −0.484 −0.200 −0.016 −0.461 −0.680 0.053 0.386 −0.001 0.247 −0.116 −0.030 −0.370 −0.206 −0.065 −0.049 −0.444 0.060 0.087 −0.268

−0.041 −0.058 0.446 0.114 −0.069 0.842 −0.042 0.133 0.835 −0.028 −0.150 0.227 0.076 −0.102 −0.047 0.239 −0.211 −0.062 0.192 0.108 0.240 0.347 −0.529 −0.018 0.062 −0.563

−0.097 −0.019 −0.199 0.261 0.110 0.761 0.368 0.830 0.549 0.403 0.074 −0.134 0.862 0.844 0.946 0.684 0.523 0.812 0.066 0.443 −0.115 0.729 0.522 0.623 0.626 0.165

−0.629 0.736 0.823 0.374 0.690 0.403 −0.102 −0.252 0.280 0.337 0.005 0.343 −0.094 −0.019 0.101 −0.352 0.152 −0.193 −0.031 −0.392 0.133 −0.049 −0.029 0.016 −0.010 0.290

−0.366 −0.114 −0.310 0.510 0.020 0.075 −0.482 −0.032 0.375 −0.662 −0.722 −0.123 0.229 0.232 0.021 −0.064 −0.317 0.132 0.524 −0.190 0.048 −0.300 −0.311 −0.387 −0.231 −0.764

−0.726 0.010 0.387 −0.451 0.101 −0.052 −0.752 −0.177 −0.074 0.427 −0.653 −0.117 −0.148 −0.190 0.104 0.228 −0.163 0.212 −0.321 −0.083 0.566 −0.829 −0.225 0.358 0.218 −0.181

0.118 0.047 0.107 −0.055 0.249 0.262 −0.065 0.945 0.156 0.417 0.137 0.774 0.897 −0.017 0.362 −0.148 −0.353 −0.067 0.099 0.004 0.264 −0.003 −0.401 0.174 −0.131 −0.577

−0.240 −0.149 −0.306 0.183 −0.367 0.706 −0.390 0.105 0.752 0.190 0.114 0.114 0.166 0.328 −0.088 0.839 0.130 −0.287 −0.202 0.017 −0.127 0.169 0.173 0.691 0.361 0.094

−0.450 0.801 0.374 0.499 −0.252 −0.165 0.128 −0.025 −0.099 0.169 −0.126 −0.006 0.096 0.149 0.764 0.145 0.458 0.506 0.135 0.645 0.094 0.071 −0.311 −0.218 0.520 0.078

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Please cite this article as: Oakes, J.M., Eyre, B.D., Wastewater nitrogen and trace metal uptake by biota on a high-energy rocky shore detected using stable isotopes, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.013

Table 1 Principal components analysis of metal concentrations and δ15N values measured in algae and snails, indicating the contribution of each parameter to the identified components. C1 to C4 = components 1 to 4. The association of a parameter with a component was considered significant for absolute values N0.6 (highlighted in bold).

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Fig. 3. Significant linear regressions between δ15N values and concentrations of a) copper, b) zinc, and c) magnesium in the predatory snail Morula marginalba.

from the outfall, which is far greater than has been reported previously (1‰ depletion with every 25 m of increased distance from the outfall, compared to the next greatest reported rate of 1‰ every 1.33 km; Connolly et al., 2013). This demonstrates the power of stable isotopes to distinguish wastewater nitrogen inputs in highly variable, highenergy environments. Despite the clear potential for nitrogen stable isotopes to be sensitive indicators of wastewater inputs, in some cases biota show no apparent gradient of wastewater nitrogen assimilation with increasing distance from a point source, even in more sheltered environments (e.g., Tucker et al., 1999; Gartner et al., 2002). A number of factors determine whether δ15N values of biota reflect a gradient of wastewater nitrogen inputs to their environment. Clearly, a wastewater ‘signal’ can only be detected in biota where (1) the δ 15N of wastewater is distinct from that of other available nitrogen sources, (2) wastewater nitrogen forms a substantial part of the nitrogen available for assimilation, and (3) the target species assimilates wastewater nitrogen via direct uptake or trophic transfer. A gradient in δ15N values with distance from the outfall will occur where there is (4) conservative mixing of wastewater with ocean water, creating a gradient of exposure. However, this gradient will only be reflected within (5) biota that are sessile, relatively immobile, or display site fidelity (at least when feeding) and where (6) the nitrogen source for the species considered is consistent across sampling sites. In the current study, all species considered were either sessile (algae) or relatively immobile (snails) and the assimilation pathway for nitrogen was consistent across locations; wastewater-derived nitrogen within DIN was assimilated by algae and passed to higher consumers via trophic transfer. The δ15N values of biota therefore indicate levels

of exposure to wastewater nitrogen. The clear gradient in δ15 N values of biota, reflecting their exposure to wastewater nitrogen, suggests that treated wastewater flows north from the outfall along the rocky shore for up to 290 m, becoming more diluted with ocean water at increasing distances from the outfall. This distinct pattern is notable for such a dynamic, high-energy environment. The current study is also notable for the consistency of patterns in wastewater δ15N ‘signal’ depuration with distance across multiple trophic levels. Similar patterns across trophic levels have been reported in only one previous study (Connolly et al., 2013). In both cases, this suggests that the ultimate source of nitrogen and its entry pathway is similar across the species considered. The δ15N values of all species therefore show the same pattern in exposure to the wastewater N source. Furthermore, consistent patterns in δ15N across species indicate that the input of wastewater to the receiving environment has been sustained and the species considered are therefore at equilibrium with the wastewater nitrogen source. Where wastewater input varies over time, there are likely to be considerable differences in patterns of δ15N due to variations in tissue turnover rates of the species considered. Species with a rapid turnover rate will reflect more recent inputs of wastewater, whereas those with a slower turnover rate will integrate longer term variations in wastewater nitrogen inputs. When applied appropriately, analysis of δ15N values in biota exposed to wastewater nitrogen should indicate the exposure of the rocky shore environment to wastewater inputs. Previous studies have highlighted the need to consider sampling scale in studying rocky shores (Bishop et al., 2002), and impacts of wastewater can be hard to detect, or to distinguish from natural variability in these environments (O'Connor, 2013). It is relatively easy and inexpensive to obtain values of δ15N, and this data will clearly be useful in high-energy environments, including rocky shores, for identifying the extent of influence of wastewater inputs and defining an appropriate scale of study. Variations in δ15N values represent variations in the exposure of organisms to wastewater material, and can therefore be used to relate contaminant exposure to ecological and biological responses of the rocky shore biota and communities. The current study suggests that macroalgae are most suited to identifying inputs of dissolved components of wastewater, as they demonstrated the greatest assimilation of wastewater dissolved inorganic N, and are sessile, and easily collected and processed. However, similar patterns in δ15N for the snails studied, combined with their low mobility, suggests that these are also a suitable indicator of wastewater inputs, as has been observed for other gastropods (Vermeulen et al., 2011). The turnover time and location in the tidal zone of biota should, however, be considered in selection of a suitable study species. Given the high sensitivity of δ15N in rocky shore biota suggested by the current study, a further application of δ15N values in high-energy environments would be the investigation of water mixing patterns, provided that there are inputs of nitrogen (deliberate or otherwise) with a distinct δ15N value. 4.2. Assimilation of wastewater nitrogen by rocky shore biota The uptake of wastewater nitrogen by rocky shore biota was not surprising; the nitrogen discharged at Skennars Head was primarily in dissolved form, which is readily assimilated by algae (Cole et al., 2004) and passed to consumers via trophic transfer (Connolly et al., 2013). However, the greater assimilation of wastewater nitrogen at lower trophic levels is more interesting. Within a closed food chain, direct transfer of wastewater nitrogen would be expected to result in similar assimilation of nitrogen at each trophic level, with fractionation leading to an enrichment of δ15N values with each trophic level (McCutchan et al., 2003). This is in direct contrast to our observations and highlights that, although the species collected in the current study represent different trophic levels within the rocky shore community, they do not represent a closed food chain. The grazers considered feed upon

Please cite this article as: Oakes, J.M., Eyre, B.D., Wastewater nitrogen and trace metal uptake by biota on a high-energy rocky shore detected using stable isotopes, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.013

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microalgae (Underwood, 1984), rather than the macroalgae that were sampled, and although M. marginalba can prey upon N. atramentosa and B. nanum (Fairweather and Underwood, 1991), the species also consumes various other gastropods as well as filter-feeding bivalves and barnacles (Fairweather et al., 1984). Differences in wastewater nitrogen assimilation may reflect a combination of species-specific tissue turnover times and temporal variations in the quality and quantity of discharged wastewater. For example, due to their rapid tissue turnover time, δ15N values for macroalgae would reflect the availability and δ15N values of wastewater dissolved inorganic nitrogen over a more recent time period than would δ15N values of any of the snail species. The greater assimilation of wastewater nitrogen by algae may therefore indicate recent increases in the quantity of discharged N. However, it is likely that differences in assimilation also relate to species-specific differences in availability and uptake of wastewater N. The macroalgae occurred in the low intertidal, whereas the snails were collected from the mid- to high-tide level. Given the limited mobility of the species considered, the algae upon which the snails graze would also occur in this region. Differences in tidal inundation may therefore have led to differences in the exposure to wastewater nitrogen of the macroalgae collected from the low intertidal, and the microalgae upon which snails graze in the mid- to high-intertidal zone. Although microalgae have a greater affinity for dissolved inorganic nitrogen uptake than macroalgae due to their greater surface area to volume ratio (Hein et al., 1995), their exposure to wastewater nitrogen is likely to be lower than that of macroalgae lower on the shore due to differences in immersion periods. Given that the mid- to highintertidal zone is inundated only at high tide, algae growing in this zone would be exposed to wastewater that is more diluted with ocean water, and their wastewater δ15N would consequently be dampened. This dampened wastewater ‘signal’ would be propagated through trophic transfer to the grazing snails that feed upon them, providing an explanation for the depletion of δ15N values in grazing snails compared to the primary producers (macroalgae) that were sampled. Although predators that feed upon the grazing snails that were sampled would be expected to have a slightly more enriched δ15N value, due to fractionation during trophic transfer, δ15N values for M. marginalba were more depleted. Given that M. marginalba were collected from the same zone as the grazing snails, this depletion of δ15N values suggests that M. marginalba had an alternative primary source of N. This is likely to be depleted, non-wastewater derived nitrogen, and may reflect the partial feeding of M. marginalba upon filter feeders such as barnacles (Fairweather et al., 1984), which would consume particulate material containing nitrogen that is not derived from wastewater. 4.3. Identifying inputs of wastewater-derived metals using stable nitrogen isotopes Organisms in the marine environment take up both essential and non-essential metals into their tissues (Rainbow, 2002). These can be accumulated within tissues and/or biomagnified through trophic transfer and, depending on concentration, can be toxic to marine life (Stankovic et al., 2014). A variety of factors, including differences in physiology and physicochemical state of organisms (Taylor and Maher, 2006) and variations in environmental conditions (Wang and Fisher, 1999), can affect metal uptake, accumulation, and excretion. This can make it difficult to tease out the effects of exposure to metals, particularly in highly variable environments. Therefore, although it is not surprising that significant differences were detected among individuals of the same species collected across sites, as well as among species, these differences are not necessarily attributable to uptake of wastewater-derived contaminants. Gastropods, however, can be sensitive indicators of metal contamination (e.g., B. nanum accumulates copper, selenium, zinc, and cadmium; Gay and Maher, 2003; Taylor and Maher, 2006), with metal accumulation in response to inputs

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over-riding other influences on metal concentrations (Taylor and Maher, 2006). Inputs of wastewater-derived metals at the study site could therefore be reflected in differences in tissue concentrations of rocky shore species corresponding with variations in exposure to wastewater inputs. Determining the exposure of biota to wastewater inputs in a rocky shore environment is not necessarily straightforward. High variability in environmental conditions at locations adjacent to the outfall will affect water mixing, and potentially the immersion times of individuals due to differences in their location within the intertidal zone at each site. For example, each species occupies a broad range within the tidal zone, but the position of individuals within that zone will be determined, at least in part, by the availability of suitable habitat. Values of δ15N have previously been used as a proxy for exposure of mobile fish to wastewater pollution, allowing health effects to be attributed to wastewater-derived contaminants (Schlacher et al., 2007). Given the remarkable sensitivity of δ15N values to reveal patterns in wastewater nitrogen assimilation demonstrated in the current study, it should also be possible to use variations in δ15N as a proxy for exposure of rocky shore biota to wastewater and its contaminants, including metals. In the current study, there was evidence that M. marginalba takes up wastewater-derived copper, zinc, and magnesium. Variations in exposure of M. marginalba to wastewater inputs, based on δ15N values, accounted for 58%, 30% and 44%, respectively, of the variation in tissue concentrations of these trace metals. Similar trends were not observed for lower trophic levels, however. This may indicate that M. marginalba acquires and bioaccumulates these metals directly from its environment (i.e., not via trophic transfer). Alternatively, M. marginalba may biomagnify wastewater-derived metals obtained via trophic transfer. In this case, the inability to detect a wastewater influence on metal concentrations in grazing snails and macroalgae may reflect lower tissue concentrations of wastewater-derived metals. For species at lower trophic levels, this would make it difficult to distinguish variations in concentration due to wastewater exposure from variations due to other factors. We may also have simply not collected the prey species from which M. marginalba obtains wastewater-derived metals. Regardless, the significant relationship between exposure (δ15N values) and metal concentration for M. marginalba demonstrates the potential application of δ15N values to link variations in communities (e.g., contaminant loads and ecosystem function) on rocky shores and other high-energy environments to wastewater exposure. 5. Conclusion This study is the first, of which we are aware, to use δ15N values to trace wastewater uptake by rocky shore biota exposed to wastewater input and has shown that: 1) Stable isotopes of nitrogen are highly sensitive and are effective for establishing the extent of influence of wastewater inputs within high-energy, rocky shore environments. This could be useful for establishing appropriate scales for study of ecosystem impacts. 2) Macroalgae, grazing snails, and predatory snails showed similar patterns of uptake of wastewater-derived nitrogen and are useful indicators of wastewater input. 3) Using stable isotopes of nitrogen as a proxy for wastewater nitrogen could be useful for attributing variations in rocky shore communities to wastewater impacts. Acknowledgements We thank S. Turner and D. Maher for assistance with the field work and M. Carvalho for the isotope analyses. The project was funded by Ballina Shire Council. J. Oakes was financially supported by an Australian Research Council Discovery Early Career Researcher Award (DE120101290).

Please cite this article as: Oakes, J.M., Eyre, B.D., Wastewater nitrogen and trace metal uptake by biota on a high-energy rocky shore detected using stable isotopes, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.013

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References APHA, 2005. Standard Methods for the Examination of Water and Wastewater. 21st ed. American Public Health Association (APHA), Washington, DC. Bellgrove, A., Clayton, M.N., Quinn, G.P., 1997. Effects of secondarily treated sewage effluent on intertidal macroalgal recruitment processes. Mar. Freshw. Res. 48, 136–146. Bishop, M.J., Underwood, A.J., Archambault, P., 2002. Sewage and environmental impacts on rocky shores: necessity of identifying relevant spatial scales. Mar. Ecol. Prog. Ser. 236, 121–128. Cabral-Oliveira, J., Mendes, S., Marnahão, P., Pardal, M.A., 2014. Effects of sewage pollution on the structure of rocky shore macroinvertebrate assemblages. Hydrobiologia 726, 271–283. Cabral-Oliveira, J., Pratas, J., Mendes, S., Pardal, M.A., 2015. Trace elements in edible rocky shore species: effect of sewage discharges and human health risk implications. Hum. Ecol. Risk. Assess. 21, 135–145. 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. Connolly, R.M., Gorman, D., Hindell, J.S., Kildea, T.N., Schlacher, T.A., 2013. High congruence of isotope sewage signals in multiple marine taxa. Mar. Pollut. Bull. 71, 152–158. Costanzo, S.D., O'Donohue, M.J., Dennison, W.C., Loneragan, N.R., Thomas, M., 2001. A new approach for detecting and mapping sewage impacts. Mar. Pollut. Bull. 42, 149–156. Fairweather, P.G., 1990. Sewage and the biota on seashores: assessment of impact in relation to natural variability. Environ. Monit. Assess. 14, 197–210. Fairweather, P.G., Underwood, A.J., 1991. Experimental removals of a rocky intertidal predator: variations within two habitats in the effects on prey. J. Exp. Mar. Biol. Ecol. 154, 29–75. Fairweather, P.G., Underwood, A.J., Moran, M.J., 1984. Preliminary investigations of predation by the whelk Morula marginalba. Mar. Ecol. Prog. Ser. 17, 143–156. Gartner, A., Lavery, P., Smit, A.J., 2002. Use of δ15N signatures of different functional forms of macroalgae and filter-feeders to reveal temporal and spatial patterns in sewage dispersal. Mar. Ecol. Prog. Ser. 235, 63–73. Gay, D., Maher, W., 2003. Natural variation of copper, zinc, cadmium and selenium concentrations in Bembicium nanum and their potential use as a biomonitor of trace metals. Water Res. 37, 2173–2185. Heaton, T.H.E., 1986. Isotopic studies of nitrogen pollution in the hydrosphere and atmosphere: a review. Chem. Geol. 59, 87–102. Hein, M., Pedersen, M.F., Sand-Jensen, K., 1995. Size-dependent nitrogen uptake in microand macroalgae. Mar. Ecol. Prog. Ser. 118, 247–253. Hindell, J.S., Quinn, G.P., 2000. Effect of sewage effluent on the population structure of Brachidontes rostratus (Mytilidae) on a temperate intertidal rocky shore. Mar. Freshw. Res. 51, 543–551.

Hood, J.L.A., Taylor, W.D., Schiff, S.L., 2014. Examining the fate of WWTP effluent nitrogen − 15 15 using δ15N-NH+ 4 , δ N-NO3 and δ N of submersed macrophytes. Aquat. Sci. 76, 243–258. Littler, M.M., Murray, S.N., 1975. Impact of sewage on the distribution, abundance and community structure of rocky intertidal macro-organisms. Mar. Biol. 30, 277–291. López-Gappa, J.J., Tablado, A., Magaldi, N.H., 1990. Influence of sewage pollution on a rocky intertidal community dominated by the mytilid Brachidontes rodriguezi. Mar. Ecol. Prog. Ser. 63, 163–175. McCutchan, J.H., Lewis, W.M., Kendall, C., McGrath, C.C., 2003. Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur. Oikos 102, 378–390. Minagawa, M., Wada, E., 1984. Stepwise enrichment of 15N along food chains: further evidence and the relation between δ15N and animal age. Geochim. Cosmochim. Acta 48, 1135–1140. O'Connor, N.E., 2013. Impacts of sewage outfalls on rocky shores: Incorporating scale, biotic assemblage structure and variability into monitoring tools. Ecol. Indic. 29, 501–509. Oakes, J.M., Eyre, B.D., Ross, D.J., Turner, S.D., 2010. Stable isotopes trace estuarine transformations of carbon and nitrogen from primary- and secondary-treated paper and pulp mill effluent. Environ. Sci. Technol. 44, 7411–7417. Rainbow, P.S., 2002. Trace metal concentrations in aquatic invertebrates: why and so what? Environ. Pollut. 120, 497–507. Schlacher, T.A., Mondon, J.A., Connolly, R.M., 2007. Estuarine fish health assessment: evidence of wastewater impacts based on nitrogen isotopes and histopathology. Mar. Pollut. Bull. 54, 1762–1776. Smith, S.D.A., 1994. Impact of domestic sewage effluent versus natural background variability: an example from Jervis Bay, New South Wales. Aust. J. Mar. Freshwat. Res. 45, 1045–1064. Stankovic, S., Kalaba, P., Stankovic, A.R., 2014. Biota as toxic metal indicators. Environ. Chem. Lett. 12, 63–84. Taylor, A., Maher, W., 2006. The use of two marine gastropods, Austrocochlea constricta and Bembicium auratum as biomonitors of zinc, cadmium, and copper exposure: effect of tissue distribution, gender, reproductive state, and temporal variation. J. Coast. Res. 22, 298–306. Tucker, J., Sheats, N., Giblin, A.E., Hopkinson, C.S., Montoya, J.P., 1999. Using stable isotopes to trace sewage-derived material through Boston Harbor and Massachusetts Bay. Mar. Environ. Res. 48, 353–375. Underwood, A., 1984. Microalgal food and the growth of the intertidal gastropods Nerita atramentosa Reeve and Bembicium nanum (Lamarck) at four heights on a shore. J. Exp. Mar. Biol. Ecol. 79, 277–291. Vermeulen, S., Sturaro, N., Gobert, S., Bouquegneau, J.M., Lepoint, G., 2011. Potential early indicators of anthropogenically derived nutrients: a multiscale stable isotope analysis. Mar. Ecol. Prog. Ser. 422, 9–22. Wang, W.-X., Fisher, N.S., 1999. Delineating metal accumulation pathways for marine invertebrates. Sci. Total Environ. 237–238, 459–472.

Please cite this article as: Oakes, J.M., Eyre, B.D., Wastewater nitrogen and trace metal uptake by biota on a high-energy rocky shore detected using stable isotopes, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.08.013