# Shift in benthic assemblages and organisms' diet under salmon farms: community structure and stable isotope analyses.

Myriam D Callier, Sébastien Lefebvre, Mary K Dunagan, Marie-Paule Bataillé, Jennifer Coughlan, Tasman P Crowe

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Myriam D Callier, Sébastien Lefebvre, Mary K Dunagan, Marie-Paule Bataillé, Jennifer Coughlan, et al. Shift in benthic assemblages and organisms' diet under salmon farms: community structure and stable isotope analyses. Marine Ecology Progress Series, 2013, 483, pp.153-167. 10.3354/meps10251 hal-00913479

HAL Id: hal-00913479

https://hal.science/hal-00913479

Submitted on 10 May 2021

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
# Shift in benthic assemblages and organisms' diet at salmon farms: community structure and stable isotope analyses

Myriam D. Callier<fnref n="1" />,<fnref n="2" />,*, Sébastien Lefebvre<fnref n="3" />, Mary K. Dunagan<fnref n="2" />, Marie-Paule Bataille<fnref n="4" />, Jennifer Coughlan<fnref n="2" />, Tasman P. Crowe<fnref n="2" />

**ABSTRACT:** The extent of the influence of salmon farming on the environment and on the uptake of particulate and dissolved effluents by benthic organisms was assessed using community structure and stable isotope analyses. Sediment cores were collected in 2 directions: perpendicular and parallel to the main residual current, 0, 25 and 200 m from 2 salmon farms (Millstone and Cranford) located in Mulroy Bay, Ireland. In addition, artificial substrates were placed for 2 mo at 1 m depth 0, 25 and 200 m from one farm to trace the uptake of farm-related nutrients by fouling organisms. The extent of measurable change in benthic communities (abundance, diversity, structure, trophic composition) depended on residual current direction. Intraspecific variation in isotopic values in benthic invertebrates was mostly explained by distance from cages. Organisms collected at impacted sites exhibited a shift in isotopic composition towards that of farm wastes. A shift in δ¹³C was observed in several invertebrates, including the polychaetes *Malacoceros fuliginosus* and *Nephtys hombergii*, Nematoda and the anemone *Anthopleura balii*. Fouling communities collected on artificial structures, mainly composed of the tunicate *Asciidiella aspersa*, showed higher δ¹⁵N values at fish cage sites compared to sites 200 m away. The study demonstrated that fish effluents were assimilated and became food sources for several organisms.

**KEY WORDS:** Aquaculture · Organic matter · Stable isotope · Community structure · Diet shift · Trophic structure

Resale or republication not permitted without written consent of the publisher

## INTRODUCTION

Aquaculture now contributes almost 50% of the global aquatic food supply. As the demand for aquatic food is increasing, the sustainable development of aquaculture is crucial to contribute to food safety, community development and to reduce overexploitation of natural aquatic resources (FAO 2010). Today, most aquaculture activities are located in coastal areas, where anthropogenic pressures are already high. A better understanding of aquaculture–envi-

ronment interactions is therefore needed to determine the environmental carrying capacity of coastal areas for on-going and future aquaculture operations (McKindsey et al. 2006).

Benthic marine food webs are an important component of coastal ecosystems as they contribute to organic matter recycling, linking the pelagic and benthic compartments through various processes (e.g. feeding and bioturbation) (Herman et al. 1999). However, little is known about how changes in biodiversity affect these processes and the extent to which

<footnote n="1">¹Ifremer, Département Ressources Biologiques et Environnement, Unité Biologie des Organismes Marins Exploités, Station de Palavas, UMR 5119 ECOSYM, Chemin de Maguelone, 34250 Palavas-les-Flots, France</footnote>

<footnote n="2">²School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland</footnote>

<footnote n="3">³Université de Lille 1 sciences et technologies, Station Marine de Wimereux, UMR CNRS 8187 LOG Laboratoire d'Océanologie et Géosciences, 28 av. Foch, 62930 Wimereux, France</footnote>

<footnote n="4">⁴UMR INRA 950 EVA, Institut de Biologie Fondamentale et Appliquée, Université de Caen, 14032 Caen, France</footnote>

<footnote>*Email: myriam.callier@ifremer.fr</footnote>
ecosystems can absorb and recover from recurrent stresses (Hughes et al. 2005). The introduction of cultured species into the ecosystem may influence trophic interactions by locally modifying the sources of natural food (Lefebvre et al. 2009). One of the predominant environmental impacts related to fish farming is the accumulation of faeces and uneaten food under cages that may lead to organic enrichment of the sea bottom and water column. Organic enrichment may have profound effects on both pelagic and benthic communities (including macrofauna, meiofauna, phytoplankton, seagrasses and bacteria) by modifying: (1) the physico-chemical characteristics of the sediment or water (e.g. decrease in oxygen, change in nutrient ratio, sulphate reduction leading to sulphide accumulation; see review by Hargrave 2005) and (2) the quality and quantity of food sources for auto- and heterotrophic organisms (Dubois et al. 2007a). Most of the waste from aquaculture (70% of nitrogen waste for fish farms; Lefebvre et al. 2001) is released into the water column as dissolved nutrients. The remainder (between 5 and 30% of nitrogen waste) is released as particulate organic matter (POM). POM may be ingested by consumers, such as heterotrophic bacteria, invertebrates and fish, and the dissolved component may enter the food web by being assimilated by autotrophs such as cyanobacteria, phytoplankton and macroalgae (Pitta et al. 2009). Although numerous studies have documented the effects of organic enrichment due to fish farming on biodiversity and community structure (see review by Hargrave 2005), little has been done to understand the assimilation of fish waste into the biota and its effects on trophic interactions between organisms and the food web (Sarà et al. 2004). Shifts in benthic communities along a gradient of organic enrichment may be caused by the loss of sensitive species due to change in environmental conditions and/or the enhancement of opportunistic species, which may benefit from these novel food sources.

Stable isotope analysis is increasingly being used to study the pathways of organic matter in food webs and to determine the contributions of various food sources to organisms' diets (Dubois et al. 2007a). In fish aquaculture studies, natural stable isotopes can be efficient tracers if the particulate waste (faeces and uneaten food) present intrinsic characteristics that clearly differ from the natural environment (Kutti et al. 2007a) and if the local environment is not influenced by other terrestrial inputs (Vizzini & Mazzola 2004, Dolenec et al. 2007). Nutrients and organic material derived from fish waste are generally enriched in ¹⁵N and depleted in ¹³C (Mazzola & Sarà

2001). Cultured carnivorous fish, such as salmon, are high-level predators and are thus fed with animal products such as fish meal, fish oil and crustacean meal. The flesh and wastes of farmed fish are usually enriched in ¹⁵N because of trophic step fractionation (natural enrichment of heavy isotopes along the trophic web) (Yokoyama et al. 2009). In addition, δ¹³C may be used as a tracer (Yokoyama & Ihihi 2007) as feed pellets are not only composed of fish, but of terrestrial vegetables depleted in ¹³C (such as wheat and soya bean). Moreover, due to isotopic discrimination against ¹²C during feeding, lighter ¹²C is preferentially excreted with respect to the heavier ¹³C isotope (Michener & Schell 1994). Benthic filter-feeders are able to feed on material derived from fish faeces (Lefebvre et al. 2000), and the use of natural δ¹³C and δ¹⁵N tracers has shown that POM from aquaculture wastes may be incorporated into the food chain by both suspension-feeders (Lojen et al. 2005) and deposit-feeders that process sediments (Dubois et al. 2007a). Dolenec et al. (2007) created a geochemical map of δ¹⁵N values in POM and selected invertebrates, and were able to trace the dispersal of ¹⁵N loading generated by aquaculture and sewage and its subsequent incorporation into the adjacent biota. Vizzini & Mazzola (2006) have suggested that benthic sessile species are better indicators of point-source pollution than nectobenthic ones (fish around cages) which disperse incorporated wastes over larger spatial scales. Some studies have also shown that species changed their trophic behaviour directly because of shellfish aquaculture particulate wastes (Dubois et al. 2007a). From a management perspective, there is a need to evaluate the efficiency of quantitative and qualitative indices such as stable isotopes to detect the extent of influence of fish farms on the environment.

The objective of the study was to evaluate the fate of aquaculture-related organic loading by combining analyses of community structure and δ¹³C and δ¹⁵N isotopic signatures. Specifically, we assessed: (1) the spatial variation in macrofaunal diversity, abundance, community structure and the biotic index M-AMBI under and around 2 salmon farms to evaluate the extent of salmon farm impacts; (2) the inter- and intraspecific variation in isotopic values of invertebrates at impacted and non-impacted sites to determine whether species were benefiting from salmon waste as a food source; and (3) the transfer of particulate and dissolved nutrients generated by aquaculture into naturally occurring benthic species and fouling organisms growing on artificial structures, using natural isotopic (δ¹³C and δ¹⁵N) abundance.
# MATERIALS AND METHODS

## Study sites and sampling design

The study sites were located in Mulroy Bay—a fully marine, fjordic inlet, situated on the northern coast of Co. Donegal, Ireland (Fig. 1). Production levels of Atlantic salmon within Mulroy Bay are ~800 to 900 t yr⁻¹. The study was done at 2 farms, Millstone (M) and Cranford (C), to assess the generality of any observed patterns. It was expected that the extent of influence of the farms would be greater in the direction of the main water current due to the dispersion of farm waste, as shown by previous studies using a hydrodynamic model such as DEPOMOD (Cromey et al. 2002). To test this hypothesis, sampling was done along 2 separate transects perpendicular (T1) and parallel (T2) to the main water current direction, at distances of 0, 25 and 200 m from the fish cages (12 stations in total). The effects related to the accumulation of faeces and uneaten food under cages are generally limited to within 200 m of the farm (Mayor et al. 2010). Field effects are usually difficult to detect due to the dispersion and dilution of dissolved nutrients by currents (Hargrave 2003, Modica et al. 2006). In the present study, we focused on the near-field effects of fish farms on the benthic environment and

fouling communities, known as integrators of environmental variation. Water depth in the sampling area ranged from 8 to 10 m. The study was done in the summer of 2009, when the effects of local organic enrichment related to salmon farming operation were likely to have been maximal due to increased salmon production rates on the farms, organic matter deposition and increased water temperature, which may enhance mineralisation rates and thus benthic oxygen demand. We sampled potential sources of particulate nutrients (salmon feed pellets, salmon faeces, cage deposit, macroalgae), the water column and sediments (POM and sedimentary organic matter [SOM]) and the potential consumers (benthic organisms, fouling organisms).

## Potential sources of particulate nutrients

Salmon feed pellets were composed of fish meal, fish oil, wheat, soya bean, crustacean meal and wheat gluten (30% oil, 37% protein, 9% ash, 1% fibre, based on the manufacturer's description). Samples of feed pellets (ca. 200 g) were randomly collected at the farm in March 2009, June 2009 and May 2010 to evaluate the average feed pellet isotopic ratios and the seasonal variation between stocks. Salmon at both farms were fed with the same feed pellet stock. To determine the composition of salmon faeces, 2 salmon were sacrificed in March and July 2009 to collect their gut contents. Faeces were rinsed with filtered seawater to remove mucus as it may influence the isotopic ratios. Isotopic analyses were performed on both faeces with mucus and rinsed faeces. Data from March 2009 could not be used as the fish had been starved for several days before the sampling, which may have modified the faeces stable isotope ratios. To determine cage deposit composition (including uneaten feed pellets, salmon faeces plus faeces of fouling organisms and any other organic material drifting down from the farm or the surrounding areas), 2 sediment traps were placed under 2 random salmon cages for 24 h. Although faeces and deposit values should be interpreted with care as they represent only a snapshot measure, they are valuable data and may be interpreted in relation to the feed pellet composition. Macroalgae, including *Ulva* sp., *Fucus* sp. and *Laminaria* sp. were collected by hand on the rocky shore in Mulroy Bay at sites not influenced by the farms (>2 km away), as macroalgae settling to the bottom may be a source of organic matter for benthic invertebrates.

<figure><img src="image_3.png" /><figcaption>Fig. 1. Locations of the 2 farms studied: Millstone and Cranford, in Mulroy Bay, Co. Donegal, Ireland. Sampling sites were located 0 m (■), 25 (□) and 200 m (○) from the salmon cages (open symbols enclosed in dotted rectangles), in 2 directions: perpendicular (T1) and parallel (T2) to the main water current direction. Map of Mulroy Bay adapted from Telfor & Robinson (2003). Macroalgae were collected at sites located > 2 km from the salmon farms (C1 and C2)</figcaption></figure>
## Benthic community characteristics

In July 2009, sediment samples (n = 3) were collected by divers using a PVC corer (11 cm in diameter) at each station to determine characteristics of the benthic community (total abundance, diversity, structure, trophic composition). Samples were gently sieved through a 500 µm mesh. Organisms were sorted, identified, cleaned of epibionts and kept alive for 24 h in filtered seawater to evacuate their gut content before isotopic analysis (see 'Stable isotope analyses'). Infaunal specimens were stored in a freezer at -20°C until analysed. The remaining material was fixed in a 5% formaldehyde-saline solution and then stored in 70% ethanol. Identification was done to the lowest taxonomic level possible, usually to species. Sites were characterised in terms of total abundance, diversity (number of species per site [S] and Shannon-Wiener diversity index [H']) and multivariate community structure (see 'Statistical analysis'). Species were separated into trophic groups according to the classification available in the literature (Fauchald & Jumars 1979, Word 1990). The AZTI marine biotic index (M-AMBI) was used to evaluate the ecological quality of the macrofaunal communities. This index was developed in Europe and is now used worldwide (Muxika et al. 2007, Callier et al. 2008), thus showing its wide applicability. Each identified species is classified into 1 of 5 ecological groups, based on their sensitivity/tolerance to pollution (Borja & Muxika 2005). The organisms thus classified were used to calculate the AMBI biotic index (software AMBI 5.0). Subsequently, the AMBI index was combined with richness and Shannon Wiener index to give the multivariate biotic index M-AMBI (Muxika et al. 2007) and to determine the ecological quality status (high, good, moderate, poor or bad status). In the present study, sampling stations with a good/high status were considered as 'non-impacted' and poor/bad status as 'impacted'.

The same specimens collected for the analysis of benthic communities were used for stable isotope analysis. To ensure an adequate biomass of each infaunal species was collected and to sample epibenthic species, a second sampling was done 4 wk later by collecting 2 grab samples at the same 12 stations. In the laboratory, the organisms were cleaned of epibionts and kept alive for several hours in filtered seawater from the sampling areas to evacuate their gut contents. For abundant and small species, individuals from the same replicate were combined, as the present study was focussing on small-spatial

scale differences within a species related to aquaculture effluents and not on differences between individuals of a single species.

## Fouling organisms

To evaluate whether farm-derived nutrients (dissolved and particulate) were assimilated by the fouling communities, artificial structures (stone tiles attached to plastic mesh cylinders suspended from a buoy) were deployed at 1 m water depth at Millstone, 0, 25 and 200 m along the T1 transect for 71 d (from 3 April 2009 to 23 June 2009). At each distance, 3 structures covered with 6 tiles were placed. Early colonising species were collected 2 mo after deployment. At a distance of 200 m, 2 structures were lost. For this station, variation was therefore calculated on the 6 tiles from the 1 remaining structure. Tiles were frozen separately, and fouling organisms growing on the inner and outer sides of each tile were then sorted and pooled per tile for isotopic analyses. They were cleaned carefully to remove epibionts before drying and grinding.

## Sediment OM and water POM

Sediment samples (n = 2) were collected at each station by divers using a cut-off 30 ml plastic syringe. Total carbon and nitrogen content and isotopic composition of the surficial (0 to 1 cm) and deeper (1 to 5 cm) sediment layers were analysed separately.

Water POM was collected at each station (4–1 water sample) at 8 m depth. Extra water samples were collected at 1 m depth along the transect (Millstone-T1) where artificial structures were deployed. After collection, water samples were prefiltered through a 200 µm mesh to remove the largest particles, then filtered onto pre-weighed, pre-combusted (450°C, 4 h) Whatmann GF/C filters. Filters were then freeze-dried until subsequent stable isotope analysis.

## Stable isotope analyses

Before isotopic analysis, sediment samples, GF/C filter and sampled calcareous organisms were divided into 2 subsamples. Subsamples of sediment and calcareous organisms were treated with 1 N HCl (12 h) to remove inorganic carbon and were used for ¹³C analyses. These were then rinsed with distilled
water to remove the acid. GF/C filters were treated
with concentrated HCl flumes to remove carbonates
(Lorrain et al. 2003). Subsamples for isotopic nitrogen
analyses were not decarbonated because this has
been reported to affect the δ¹⁵N and total organic N
values (Ryba & Burgess 2002).

Freeze-dried, powdered and homogenised sam-
ples were analysed using a CHN elemental analyser
(EA 3000, EuroVector, Université de Caen Basse-
Normandie) for particulate organic carbon and par-
ticulate nitrogen to calculate their C/N atomic ratios.
Analytical precision for the experimental procedure
was estimated to be better than 0.01 % dry weight for
C and 0.03 % dry weight for N. The resultant gas of
elemental analyses was introduced into an isotope
ratio mass spectrometer (IRMS) (IsoPrime, Elemen-
tar) to determine ¹⁵N/¹⁴N and ¹³C/¹²C ratios. Stable
isotopic values are expressed as the relative differ-
ences (in ‰) between the samples and the conven-
tional standards, Pee Dee Belemnite (PDB) for carbon
and atmospheric N₂ for nitrogen, according to the
following equation: δ = [(Rsample/Rstandard)−1] × 1000,
where δ is ¹³C or ¹⁵N relative abundance (in ‰)
regarding the conventional standard and R is the
¹³C/¹²C or ¹⁵N/¹⁴N ratio. The internal standard was
the USGS 40 of the International Atomic Energy
Agency (δ¹³C = −26.2; δ¹⁵N = −4.5). The typical preci-
sion in analyses was ±0.25‰ for carbon and ±0.40‰
for nitrogen isotope ratios.

The carbon and nitrogen isotopic compositions of
consumer tissues are thus functions of the ratios of
¹³C to ¹²C (δ¹³C) and ¹⁵N to ¹⁴N (δ¹⁵N) of each assimili-
lated food source. A comparison between stable iso-
topic compositions of several consumers to estimate
their trophic relationships must be made under the
strong assumption that trophic step fractionation is
accurately estimated for each species. However, very
few values of trophic step fractionation are available
for marine invertebrates and, as done in most ecolo-
gical studies using isotopic approaches (e.g. Lefebvre
et al. 2009), we assumed that the fractionation values
were the same for all species, sites and seasons.
Trophic step fractionation values of 1 for δ¹³C and 3.4
for δ¹⁵N were chosen. Trophic levels (TL) were calcu-
lated using primary consumers. Long-lived primary
consumers, such as mussels, satisfy these criteria in
aquatic food webs and can provide an appropriate
baseline to quantify the trophic position of secondary
consumers. We used the equation TLconsumer = 2 +
(δ¹⁵Nconsumer – δ¹⁵Nbase)/3.4 (van Oevelen et al. 2009),
with δ¹⁵Nbase = 5.1 corresponding to the *Mytilus
edulis* value collected on pontoons at the entrance to
Mulroy Bay (Fig. 1).

Statistical analysis

Variations in univariate indices (abundance, diversity, sediment %C total and %N total, and sediment stable isotope values) were evaluated using ANOVA followed by Tukey's multiple comparison tests with SYSTAT. The normality and homogeneity of variance were tested using the Kolmogorov-Smirnov test and Cochran's test, respectively. ANOVA factors were: farm (2 levels, fixed: Millstone and Cranford), direction (2 levels, fixed: T1 perpendicular and T2 parallel) and distance (3 levels, fixed: 0, 25 and 200 m from the farm), n = 3. Non-parametric multivariate analyses of community structure, including multi-dimensional scaling were performed using PRIMER (Clarke & Warwick 1994). Analyses were done using individual cores as replicates. Variation in benthic community structure was evaluated using PERMANOVA (a distance-based nonparametric multivariate analogue of ANOVA) (Anderson & Ter Braak 2003). The level of similarity and the contribution of each species to the total similarity among samples were determined using SIMPER. Data were square-root-transformed for all multivariate analyses to reduce the influence of the dominant taxa, and similarity matrices were based on the Bray-Curtis index of similarity. Based on community analysis, 2 clusters were defined, one containing sites impacted by the salmon farm, the other one containing sites not impacted. For invertebrate stable isotope analysis, the difference between isotopic composition of the species collected both at impacted and non-impacted sites was analysed through ANOVA.

RESULTS

Potential source of particulate nutrients in the
vicinity of salmon farms

Although feed pellet composition was similar
throughout the year, significant variation in nitrogen
and carbon isotopic composition was measured in
fish feed pellets between the 3 sampling dates
(Table 1; δ¹⁵N: March 2009 ≠ June 2009 ≠ May 2010,
p < 0.001; δ¹³C: March 2009 ≠ June 2009 = May 2010,
p = 0.002). Compared to other potential sources of
organic matter in the environment, fish feed pellets
were depleted in ¹³C and relatively enriched in ¹⁵N
(Table 1). Salmon faeces presented similar values for
δ¹⁵N, but lower values for δ¹³C compared to the
average (±SD) feed pellet composition (8.8 ± 1.2‰,
-23.0 ± 0.5‰). Isotopic values of cage deposits were
<table><caption>Table 1. Mean (±SD) stable isotopic values (δ¹⁵N and δ¹³C), %N, %C and C/N ratio of salmon farm organic matter and macroalgae. Salmon feed pellets were collected from the feed stock at Millstone and Cranford (same feed used at both farms). Salmon faeces were collected from gutted salmons, and cage deposits from sediment traps deployed beneath Millstone cages for 24 h. Macroalgae were collected in Mulroy Bay at sites located >2 km from the farms (C1 for *Fucus* sp., and C2 for *Laminaria* sp. and *Ulva* spp.; see Fig. 1). n = number of samples</caption><thead><tr><th></th><th>Collection date</th><th>n</th><th>δ<sup>15</sup>N</th><th>%N</th><th>δ<sup>13</sup>C</th><th>%C</th><th>C/N</th></tr></thead><tbody><tr><td colspan="8"><strong>Farm organic matter</strong></td></tr><tr><td>Feed pellet</td><td>Mar 2009</td><td>4</td><td>8.5 ± 0.3</td><td>7.3 ± 0.3</td><td>-23.5 ± 0.1</td><td>51.6 ± 1.2</td><td>7.1 ± 0.3</td></tr><tr><td>Feed pellet</td><td>Jun 2009</td><td>4</td><td>7.9 ± 0.2</td><td>6.4 ± 0.3</td><td>-22.6 ± 0.2</td><td>54.6 ± 1.0</td><td>8.5 ± 0.6</td></tr><tr><td>Feed pellet</td><td>May 2010</td><td>2</td><td>10.2 ± 0.3</td><td>7.0 ± 0.5</td><td>-22.9 ± 0.4</td><td>51.6 ± 0.4</td><td>7.3 ± 0.6</td></tr><tr><td>Faeces</td><td>Jul 2009</td><td>3</td><td>7.9 ± 0.1</td><td>3.1 ± 1.5</td><td>-21.7 ± 0.2</td><td>27.5 ± 0.1</td><td>10.0 ± 3.7</td></tr><tr><td>Faeces rinsed</td><td>Jul 2009</td><td>2</td><td>8.6 ± 0.5</td><td>2.1 ± 0.0</td><td>-20.3 ± 0.0</td><td>23.2 ± 0.5</td><td>11.3 ± 0.0</td></tr><tr><td>Cage deposits</td><td>Jul 2009</td><td>4</td><td>6.4 ± 0.8</td><td>1.8 ± 0.2</td><td>-22.3 ± 0.1</td><td>23.2 ± 3.0</td><td>12.9 ± 0.6</td></tr><tr><td colspan="8"><strong>Macroalgae</strong></td></tr><tr><td><em>Fucus</em> sp.</td><td></td><td>2</td><td>6.5 ± 1.2</td><td>1.3 ± 0.0</td><td>-16.7 ± 0.1</td><td>32.5 ± 0.4</td><td>24.5 ± 0.2</td></tr><tr><td><em>Laminaria</em> sp.</td><td></td><td>2</td><td>4.4 ± 0.3</td><td>0.8 ± 0.1</td><td>-19.4 ± 0.3</td><td>25.5 ± 0.5</td><td>32.0 ± 3.2</td></tr><tr><td><em>Ulva lactuca</em></td><td></td><td>1</td><td>4.7</td><td>3.2</td><td>-18.2</td><td>30.2</td><td>9.4</td></tr><tr><td>Enteromorpha (<em>Ulva</em>) sp.</td><td></td><td>1</td><td>6.3</td><td>1.8</td><td>-18.8</td><td>27.2</td><td>15.5</td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>Fig. 2. δ¹⁵N, %N, δ¹³C, %C and the C/N ratio (mean ± SD, n = 2) of the sediment organic matter (2 layers) and of the water particular organic matter (POM) (at 2 depths) sampled at the salmon farms Millstone (M) and Cranford (C) in Aug 2009. Sediment and water samples were collected at 3 stations (0, 25 and 200 m from the farms) along each of 2 transects (perpendicular [T1] and parallel [T2] to the current). Horizontal lines over bars indicate groups that are not significantly different (Tukey's pairwise comparison, p > 0.05). When no lines are shown, there are no significant differences. Note some y-axes do not start at 0</figcaption></figure>
intermediate between salmon feed pellets, faeces and algae for δ¹³C, but depleted in ¹⁵N, probably because cage deposits may include salmon faeces, uneaten feed pellets as well as faeces from fouling organisms and macroalgae detritus. Algae (*Fucus* sp., *Laminaria* sp., *Ulva* spp.) sampled in Mulroy Bay at sites located >2 km from the farms (Fig. 1) were enriched in ¹³C and depleted in ¹⁵N compared to farm sources of organic matter (OM).

## Sediment OM and water POM analysis

Overall, no clear pattern was observed in either sediment or water characteristics (Fig. 2). Sediment δ¹⁵N, %N, δ¹³C, %C and C/N did not differ among distances (0, 25 and 200 m), directions (parallel vs. perpendicular to water current) or depths (0 to 1 cm vs. 1 to 5 cm). Average (±SD) sediment %C, %N and C/N were higher at Cranford farm (2.5 ± 0.6%, 0.4 ± 0.1% and 6.7 ± 0.7, respectively) than at Millstone farm (0.7 ± 0.5%, 0.1 ± 0.1% and 5.6 ± 0.9, respectively) (p < 0.005, Fig. 2). Averaged for the 2 farms, δ¹⁵N and δ¹³C were 6.5 ± 0.4‰ and -21.2 ± 0.5‰, respectively. Although variation in water POM isotopic values did not differ significantly in relation to farm, distance, direction, or depth, slightly higher δ¹⁵N were observed at Millstone under the cages (0 m) compared to 25 and 200 m away from them (Fig. 2). The C/N of water POM (maximum: 10.6 ± 4) was also slightly higher at the Millstone 0 m station.

## Benthic community

A total of 63 macrofaunal species were identified at Millstone and Cranford farms. At both farms, a lower

<figure><img src="image_5.png" /><figcaption>Fig. 3. Number of benthic species, total abundance and Shannon diversity index (H') per core sample (mean ± SD, n = 3, corer surface = 105.7 cm²) at Millstone (M) and Cranford (C) farms, at 3 stations (0, 25 and 200 m from the farms) along each of 2 transects (perpendicular [T1] and parallel [T2] to the current) in July 2009. See Table 2 for ANOVA results. Significant differences among groups as identified by Student-Newman-Keuls pairwise contrasts are indicated by different letters above bars</figcaption></figure>

<table><caption>Table 2. ANOVA results for benthic macrofaunal characteristics (number of species, total abundance, Shannon-Wiener diversity [H']) observed at 2 salmon farms. Fixed factors were farm (FA; Millstone and Cranford), direction (DIR; perpendicular and parallel to the main water current) and distance (DIS; 0, 25 and 200 m from the salmon cages). **Bold**: significant (p ≤ 0.05)</caption><thead><tr><th rowspan="2">Sources of variations</th><th rowspan="2">df</th><th colspan="3">Number of species</th><th colspan="3">Total abundance</th><th colspan="3">H'</th></tr><tr><th>MS</th><th>F</th><th>p</th><th>MS</th><th>F</th><th>p</th><th>MS</th><th>F</th><th>p</th></tr></thead><tbody><tr><td>FA</td><td>1</td><td>0.028</td><td>0</td><td>0.945</td><td>20164</td><td>6.27</td><td><strong>0.019</strong></td><td>0.009</td><td>0.12</td><td>0.733</td></tr><tr><td>DIR</td><td>1</td><td>8.028</td><td>1.41</td><td>0.247</td><td>2272.11</td><td>0.71</td><td>0.409</td><td>0.826</td><td>11.06</td><td><strong>0.003</strong></td></tr><tr><td>DIS</td><td>2</td><td>214.750</td><td>37.71</td><td><strong>&lt;0.001</strong></td><td>27346.08</td><td>8.5</td><td><strong>0.002</strong></td><td>3.893</td><td>52.11</td><td><strong>&lt;0.001</strong></td></tr><tr><td>FA × DIR</td><td>1</td><td>42.250</td><td>7.42</td><td><strong>0.012</strong></td><td>3680.44</td><td>1.14</td><td>0.299</td><td>0.335</td><td>4.48</td><td><strong>0.045</strong></td></tr><tr><td>FA × DIS</td><td>2</td><td>6.194</td><td>1.09</td><td>0.353</td><td>22481.58</td><td>6.99</td><td><strong>0.004</strong></td><td>0.102</td><td>1.37</td><td>0.273</td></tr><tr><td>DIR × DIS</td><td>2</td><td>45.986</td><td>8.05</td><td><strong>0.002</strong></td><td>5095.03</td><td>1.58</td><td>0.226</td><td>1.128</td><td>15.1</td><td><strong>&lt;0.001</strong></td></tr><tr><td>FA × DIR × DIS</td><td>2</td><td>1.083</td><td>0.19</td><td>0.828</td><td>8172.53</td><td>2.54</td><td>0.099</td><td>0.723</td><td>4.84</td><td><strong>0.017</strong></td></tr><tr><td>Residual</td><td>24</td><td>5.694</td><td></td><td></td><td>3218.16</td><td></td><td></td><td>1.793</td><td></td><td></td></tr></tbody></table>
diversity (number of species and Shannon $H'$) was observed under the cages (0 m stations) compared to the stations 200 m from the cages in both water current directions (Fig. 3). Diversity at 25 m was either similar to diversity at 0 m along the dominant current direction transect or similar to 200 m sites along the transect perpendicular to the dominant water current (Table 2). Therefore, at both farms, the extent of the effect on diversity was <25 m when perpendicular to the main water current and >25 m but <200 m along the main current direction. Macrofaunal abundances did not vary significantly with distance from cages (Fig. 3), except under Cranford cages (C-T1-0 m), which exhibited a particularly high abundance of *Capitella* sp.

Community structure analysis, based on abundance, revealed 2 distinct clusters (Fig. 4; PERM-ANOVA, $p < 0.05$). The first cluster included the 0 m stations and 25 m T2 stations (i.e. along the main current direction) at both Millstone and Cranford farms. SIMPER analysis indicated that Cluster 1 was characterised by 3 dominant species: *Capitella capitata*, *Malacoceros fuliginosus* and *Tubificoides* sp., with particularly high abundance of *C. capitata* under Cranford cages (up to 252 ind. core⁻¹, corresponding to 26 517 ind. m⁻²) and high abundance of *M. fuliginosus* under Millstone cages (66 ind. core⁻¹, corresponding to 6945 ind. m⁻²). The second cluster included the 200 m stations

and the 25 m T1 stations (i.e. perpendicular to the main water current direction). Contrary to Cluster 1, Cluster 2 was characterised by very low abundance of *C. capitata* and *M. fuliginosus*, and a more diverse community. Furthermore Cluster 2 exhibited subdivisions between stations: The communities located at 25 m from Millstone and 200 m from Millstone and Cranford were characterised by the presence of Malценidae, *Heteromastus filiformis*, *Lumbrineris gracilis*, the bivalve *Tellimya ferruginosa* and the holothurid *Leptosynapta inhaerens*. Dissimilarity between the Cranford 25 m station (T1) and the other stations of Cluster 2 was largely related to the presence of *Galathowenia oculata* and *Polydora* sp. in samples from the former station.

<figure><img src="image_6.png" /><figcaption>Fig. 4. Non-metric multi-dimensional scaling illustrating variation in benthic community structure (using square-root-transformed abundance data) near 2 salmon farms: Millstone (M) and Cranford (C). Benthic samples were collected in July 2009 at 3 stations (0, 25 and 200 m from the farms) along 2 transects (perpendicular [1] and parallel [2] to the current)</figcaption></figure>

## Trophic groups and M-AMBI

Of the 63 species observed, 32 were surface/subsurface deposit-feeders (e.g. *Capitella* sp., *Malacoceros fuliginosus*), 9 were suspension-feeders (e.g. *Tellimya ferruginosa*) and 12 were carnivores (e.g.

<table><caption>Table 3. Trophic group composition of benthic communities, biotic index classification (AMBI and M-AMBI) and ecological status at Millstone (M) and Cranford (C) farms. Samples were collected at 3 stations (0, 25 and 200 m from the farms) along each of 2 transects (perpendicular T1 and parallel T2 to the current) in July 2009. Df: deposit-feeder; Sf: suspension-feeders; Car: carnivores; H: herbivores; O: omnivores. The trophic group of a few species was undetermined (max. 7% per station). AMBI and M-AMBI are based on the sensitivity/tolerance level of species to organic enrichment. (–) no species found belonging to that trophic group</caption><thead><tr><th rowspan="2">Station</th><th colspan="5">Abundance (%)</th><th colspan="3">Biotic index</th></tr><tr><th>Df</th><th>Sf</th><th>Car</th><th>H</th><th>O</th><th>AMBI</th><th>M-AMBI</th><th>Status</th></tr></thead><tbody><tr><td>M-T1-0</td><td>99 ± 1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>6.0</td><td>0.2</td><td>Poor</td></tr><tr><td>M-T1-25</td><td>88 ± 10</td><td>9 ± 10</td><td>2 ± 2</td><td>-</td><td>0 ± 1</td><td>1.1</td><td>0.9</td><td>High</td></tr><tr><td>M-T1-200</td><td>65 ± 22</td><td>25 ± 20</td><td>9 ± 3</td><td>1 ± 1</td><td>0 ± 1</td><td>3.4</td><td>0.9</td><td>High</td></tr><tr><td>M-T2-0</td><td>99 ± 1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>6.0</td><td>0.1</td><td>Bad</td></tr><tr><td>M-T2-25</td><td>95 ± 5</td><td>3 ± 6</td><td>2 ± 3</td><td>-</td><td>-</td><td>5.8</td><td>0.1</td><td>Bad</td></tr><tr><td>M-T2-200</td><td>45 ± 7</td><td>44 ± 3</td><td>9 ± 11</td><td>-</td><td>2 ± 2</td><td>1.4</td><td>1.0</td><td>High</td></tr><tr><td>C-T1-0</td><td>100 ± 0</td><td>0 ± 0</td><td>-</td><td>-</td><td>-</td><td>6.0</td><td>0.2</td><td>Bad</td></tr><tr><td>C-T1-25</td><td>67 ± 28</td><td>7 ± 5</td><td>19 ± 19</td><td>-</td><td>-</td><td>3.2</td><td>0.7</td><td>Good</td></tr><tr><td>C-T1-200</td><td>65 ± 7</td><td>5 ± 5</td><td>29 ± 9</td><td>-</td><td>-</td><td>1.5</td><td>0.9</td><td>High</td></tr><tr><td>C-T2-0</td><td>95 ± 6</td><td>0 ± 1</td><td>1 ± 1</td><td>-</td><td>-</td><td>5.8</td><td>0.3</td><td>Poor</td></tr><tr><td>C-T2-25</td><td>99 ± 1</td><td>0 ± 1</td><td>0 ± 0</td><td>-</td><td>-</td><td>5.9</td><td>0.3</td><td>Poor</td></tr><tr><td>C-T2-200</td><td>40 ± 13</td><td>40 ± 9</td><td>18 ± 9</td><td>-</td><td>1 ± 1</td><td>1.4</td><td>0.9</td><td>High</td></tr></tbody></table>
*Lumbrineris gracilis, Ophiocomina nigra* (Table 3). Benthic communities of Cluster 1 (i.e. 0 m-T1, 0 m-T2, 25 m-T2 at both farms) were almost exclusively dominated by deposit-feeders (95 to 100%). The benthic status at those stations was classified as poor/bad by M-AMBI (Table 3). Stations in Cluster 2 were characterised by more diverse trophic groups, with the proportion of suspension feeders varying from 5 to 44% and carnivores from 2 to 29% (Table 3). The benthic status of the Cluster 2 station was classified as good/high by M-AMBI.

Based on the benthic community analysis, 2 conditions were observed at Millstone and Cranford farms: communities that were highly impacted by the salmon farms and communities that were not or only slightly impacted by the farms. We hypothesized that species assimilating salmon waste as a food source will present different isotopic signatures at impacted versus non-impacted sites.

<figure><img src="image_7.png" /><figcaption>Fig. 5. (a) Interspecific and (b) intraspecific variation of mean (±SD, only shown in [a]) stable isotopic ratios in benthic species sampled at sites impacted (poor/bad biotic index status; see Table 3 for site status) or non-impacted (good/high status) by salmon waste at Millstone (M) and Cranford (C) farms. Red polygons represent the farm waste isotopic values for fish feed pellets collected in March 2009, June 2009 and May 2010 (FO<sub>m9</sub>, FO<sub>j9</sub> and FO<sub>m10</sub>, respectively); deposits collected beneath cages (Dep); faeces with mucus (Fa); and faeces without mucus (Fa<sub>r</sub>)</figcaption></figure>

## Inter- and intraspecific variations in isotopic signatures

Interspecific variations in isotopic signature of the benthic organisms confirmed the trophic position of species within the community (Fig. 5a). At non-impacted stations, at Millstone and Cranford, respectively, Maldanidae (δ<sup>15</sup>N: 8.7‰ and 8.6‰), *Pherusa plumosa* (δ<sup>15</sup>N: 8.2‰ and 8.3‰) and *Malacoceros fuliginosus* (δ<sup>15</sup>N: 8.1‰ in Millstone) presented lower δ<sup>15</sup>N, relative to the anemone *Anthopleura balii* (δ<sup>15</sup>N: 9.3‰ and 9.5‰), the polychaetes *Nephtys hombergii* (δ<sup>15</sup>N: 9.9‰ and 10.7‰) and *Lumbrineris gracilis* (δ<sup>15</sup>N: 11.2‰ in Cranford), the ophiuroids *Ophiocomina nigra* (δ<sup>15</sup>N: 10.4‰ in Millstone) and the holothurids *Leptosynapta inhaerens* (δ<sup>15</sup>N: 9.8‰ and 10.3‰). Nematoda had a similarly high δ<sup>15</sup>N (10.6‰ in Cranford).

At impacted stations, the 2 dominant deposit-feeders, *Capitella capitata* (δ<sup>15</sup>N: 7.1‰ and 7.8‰) and *Malacoceros fuliginosus* (δ<sup>15</sup>N: 7.7‰ and 8.0‰) presented low δ<sup>15</sup>N as did the polychaetes *Pherusa plumosa* (δ<sup>15</sup>N: 7.5‰ in Cranford) and *Anthopleura balii* (δ<sup>15</sup>N: 7.5‰ in Millstone) (Fig. 5a). The polychaetes *Nephtys hombergii* (δ<sup>15</sup>N: 9.0‰ and 9.8‰) and *Lumbrineris gracilis* (δ<sup>15</sup>N: 11.1‰ in Cranford), the ophiuroids *Ophiocomina nigra* (δ<sup>15</sup>N: 9.6‰ in Millstone), the holothurids *Leptosynapta inhaerens* (δ<sup>15</sup>N: 8.6‰ in Millstone) and the Nematoda (δ<sup>15</sup>N: 10.1‰ and 11.0‰) were enriched in δ<sup>15</sup>N compared to *Capitella* sp. and *M. fuliginosus*.

Intraspecific variation observed for several species between impacted (poor/bad benthic status) and non-impacted stations (good/high benthic status) (Fig. 5b) allowed us to detect variation in the assimilation of food by these species. Among these species, a clear shift in isotopic values towards the farm waste values was observed in *Malacoceros fuliginosus*, *Nephtys hombergii*, *Ophiocomina nigra* and Nematoda. Those species collected at impacted stations were depleted in ¹³C relative to specifics collected at non-impacted stations (variation of 2‰ in δ¹³C). Slight interspecific variations in δ¹⁵N (<1‰) were observed for species
sampled at impacted vs. non-impacted sites; this variation was more pronounced for *N. hombergii* (Fig. 5b). *Pherusa plumosa* and *Leptosynapta inhaerens* had lower δ¹⁵N at impacted than at non-impacted sites but were the only species with higher δ¹³C. No variation was observed for *Lumbrinereis gracilis*.

Intraspecific variation in isotopic values was also observed for fouling communities on sampling tiles (Fig. 6). After 2 mo, the early colonizing community was dominated in terms of biomass by the solitary tunicate *Asciidiella aspersa*, colonial tunicate *Diplosoma* sp. and filamentous alga *Enteromorpha* sp. *Mytilus edulis*, hydroids and gastropod larvae were also present, but in very low abundance. The tunicates *A. aspersa* and *Diplosoma* sp. showed relatively little variation (~1‰ δ¹³C) (Fig. 6). *A. aspersa* and the colonial tunicates at the 0 m station were enriched in ¹⁵N (δ¹⁵N mean ± SD: 8.2 ± 0.5‰) relative to those at the 200 m station (δ¹⁵N: 7.6 ± 0.1‰), while no significant difference in isotopic values was observed for fouling algae (Fig. 6).

## DISCUSSION

We observed a gradient of organic enrichment around the studied fish farms such that benthic communities were strongly impacted by the farms (low diversity, dominance of opportunistic species) at some distances and orientations, whereas other communities were not or were only slightly impacted by the farms (higher diversity, presence of sensitive species). Stable isotope analysis supports the hypothesis that fish farm effluents contributed to the diets of several benthic and fouling organisms present in the vicinity of the farms.

### Potential source of nutrients

Isotopic analysis (δ¹⁵N, δ¹³C) was used to track the fate of salmon-farm-related products (e.g. fish feed and waste) in the environment to determine if waste was used as a new source of food by adjacent biota (Yokoyama et al. 2006). Variation in isotopic signatures of the food sources was sufficient to identify variation in the organisms' diets. Effluent derived from salmon cages presented an isotopic composition that was distinct from other potential food sources (e.g. algae), and the point-source nature of the fish farm effluent reduced the confounding effects of mixed nutrient sources (Costanzo et al. 2003). As expected, fish feed pellets and faeces were enriched in ¹⁵N compared to

<figure><img src="image_8.png" /><figcaption>Fig. 6. Intraspecific variation of mean (±SD, n = 3) stable isotope signatures of fouling organisms collected on suspended structures in June 2009 (6 tiles fixed on 3 structures per distance) placed 0 m (black), 25 m (grey) and 200 m (white) from cages, at Millstone salmon farm, perpendicular to the main water current direction. Data are pooled per structure (see 'Materials and methods') for the solitary tunicate *Asciidiella aspersa*, colonial tunicate *Diplosoma* sp. and filamentous alga *Enteromorpha* sp.</figcaption></figure>

other organic matter sources such as macroalgae, as they were composed of fish and crustacean meal, and depleted in ¹³C, probably due to the presence of terrestrial vegetables (see 'Materials and methods') and fish oil in their composition (Yokoyama et al. 2006). Deposits collected within sediment traps placed under fish cages were depleted in ¹⁵N compared to feed pellets and faeces. This suggests that deposits were probably composed of a mixture of uneaten feed pellets, faeces and other material which may include faeces from fouling organisms, macroalgae etc. settling from the cages. However, caution should be taken with the interpretation of the deposit isotopic values, as these values only reflected 24 h of deposition.

High temporal variation was observed in the isotopic values of the fish feed pellets (δ¹⁵N varied from 7.9‰ in 2009 to 10.2‰ in 2010). Although food composition was similar, the fish meal and oil composing the pellets may have varied through time, as sources may differ over time, depending on wild fish supply. It is therefore important to consider seasonal variation in the stable isotope composition of fish feed pellets, as this may also change the isotopic value of both faeces and dissolved inorganic nitrogen. That being said, in the present study, fish feed pellets collected in March 2009 and June 2009, just before the organisms were sampled, presented similar isotopic compositions and were distinct from other food sources. Additionally, both farms were using the same pellet stock, which allows the findings from the 2 farms to be compared.
## Fate of aquaculture waste in the environment

Water column and sediment isotopic composition did not vary with distance from the fish cages (except for the water POM at 1 m depth). However, significant variation in isotopic values was observed in benthic organisms (see next subsection). This suggests a rapid incorporation of fresh settling material by the local biota, thus preventing the accumulation of POM in the sediment. This observation is in accordance with the observations made by Kon et al. (2009), who showed that, even though no changes could be found in the isotopic composition of the SOM, shrimp farming waste induced a food availability shift to aquaculture feed in benthic animals (fiddler crabs *Uca anulipes*, *U. forcipata*, and *U. bengali*). Kon et al. (2009) suggested that biological accumulation, i.e. the assimilation of organic matter by animals, is a better indicator than sediments. The lack of a clear sediment isotopic signal suggests that the proportion of labile OM in fish wastes was high. Lefebvre et al. (2000) showed that fish faeces were assimilated by suspension feeders with an absorption efficiency of 60%. The low C/N ratios measured in fish feed pellets (7.9) confirmed the great nutritional value of fish waste.

## Benthic community characteristics and diet

Abundance, diversity and community structure

At both studied farms, similar trends were observed in terms of variation in macrofaunal diversity and structure. The extent of measurable change in benthic communities was a function of residual current direction. Along the transect parallel to the main current direction, communities at 0 and 25 m sites were more similar to each other than those observed at 200 m, suggesting that the extent of influence was between 25 and 200 m. Along the transects perpendicular to the residual currents, the communities at a distance of 25 m were more similar to those at 200 m than to those directly under the farms. The current data combined with previous observations made at the Millstone and Cranford farms (Telfor & Robinson 2003) suggest that the extent of influence in this direction was <25 m, which is consistent with previous studies considering the impacts of fish farming on benthic communities (Mazzola et al. 2000, Nickell et al. 2003, Kutti et al. 2007b).

Changes in benthic community structure and decreased diversity were observed under fish cages. Low diversity is consistent with the general pattern of

community changes due to organic enrichment, as increasingly anoxic sediment favours the proliferation of organisms that can tolerate such conditions (Pearson & Rosenberg 1978, Diaz & Rosenberg 1995, Holmer et al. 2005). Indeed, increased sediment oxygen demand may lead to anoxia and higher sulphide concentration that may not be tolerated by certain species (Findlay et al. 1995, Pearson & Black 2001, Kinoshita et al. 2008). At Millstone farm, no difference in total abundance was observed among distances, but at Cranford, the abundance of invertebrates was increased beneath the cages relative to other distances, indicating that tolerant species benefited from localized conditions, probably in response to increased food availability derived from the salmon cages (faeces, uneaten feed pellets, fouling), with a reduction of predators and competitors that are sensitive to high organic loading. Indeed, communities under salmon cages were dominated by deposit-feeders, whereas stations further away from the farm were dominated by carnivores and suspension-feeders, which are sensitive to organic loading. Species able to assimilate this new source of food and to tolerate new habitat conditions may have outcompeted species that were normally dominant.

## Interspecific stable isotope variation

Interspecific variations in stable isotope values reveal the trophic position of each species composing the macrobenthic communities. At non-impacted sites, communities were diverse and were represented by species within a wide range of trophic levels, including deposit-feeders, suspension-feeders and predators. The lowest trophic level was represented by polychaete deposit-feeders of the family Maldaniidae, *Pherusa plumosa* and *Malacoceros fuliginosus*. Species representing higher trophic levels included the anemone *Anthopleura balii*, the predatory polychaetes *Nephtys hombergii* and *Lumbrineris gracilis* and the ophiuroid *Ophiocomina nigra* (higher $\delta^{15}$N). Under the influence of salmon cages, communities were almost exclusively dominated by deposit-feeders that are able to thrive under high organic loading (Pearson & Black 2001). The polychaetes *Capitella capitata* and *M. fuliginosus* became dominant and presented the lowest trophic level of the macrofaunal community (relatively low $\delta^{15}$N). *C. capitata* and *M. fuliginosus* are known to be tolerant to high organic loading (Cardell et al. 1999, Lindqvist et al. 2009). Part of their diet was probably composed of fish farm waste (as shown by the intraspecific variation in
distance of the substrates, the fouling community
probably feeds on fresh fish farm effluent, whereas
the benthic community further away may feed on
altered faecal material, thus potentially modifying
the δ¹⁵N due to bacterial activity. Algae potentially
assimilating the inorganic dissolved nutrients pre-
sented no clear difference in isotopic values between
stations. This may be due to the faster dispersion of
dissolved nutrients relative to POM (Lin & Fong 2008).

CONCLUSION

Our study showed small-scale influences of salmon
farms on benthic and fouling communities, with pos-
sible effects on trophic structure. Stable isotope com-
position was shown to be a powerful tool for tracing
the fate of fish waste into the biota, allowing the
assimilation of salmon waste by benthic species to be
detected and thus showing that they shifted their
diets to include this nutrient input. The study high-
lights the need to sample multiple sites in order to
consider both inter- and intraspecific variability and
to better determine the fate of aquaculture waste in
the structure of the food web. Sedimentary and foul-
ing organisms—potential sinks for fish effluents—
play an important role in the carrying capacity of
ecosystems for aquaculture. Adapted husbandry
practices, such as integrated multi-trophic aquacul-
ture, could promote biomass of species able to assim-
ilate fish waste and, therefore, dampen environ-
mental impacts (Cook et al. 2006). However, further
experiments are needed to confirm that adjacent
biota can significantly reduce nutrient concentrations
in the water column and sediments surrounding
fish cages and effectively prevent aquaculture en-
closures from organically enriching the surrounding
environment.

Acknowledgements. We thank University College Dublin and University of Caen and especially the MARBEE team for their precious help during field work. Thanks to Francis O'Beirn (Marine Institute) and Grainne O'Brien (Bord Iascáigh Mhara) for advice and assistance. Thanks to Marine Harvest and Catherine McManus who kindly provided access to the farms. Thanks also to all the Mulroy Bay salmon farmers and divers who helped with the work. The manuscript has been improved thanks to the comments of Dr. C. W. McKindsey and 3 anonymous reviewers. This paper has been published as part of the SIMBIOSYS project (2007-B-CD-1-S1), part of the Science, Technology, Research and Innovation for the Environment (STRIVE) Programme 2007–2013. The programme is financed by the Irish Government under the National Development Plan 2007–2013; it is administered on behalf of the Department of

the Environment, Heritage and Local Government by the
Environmental Protection Agency which has the statutory
function of co-ordinating and promoting environmental
research.

LITERATURE CITED

Anderson MJ, Ter Braak CJF (2003) Permutation tests for multi-factorial analysis of variance. J Statist Comput Simulation 73:85-113

Black KD (2001) Environmental impacts of aquaculture. Sheffield Academic Press, Sheffield

► Bolam SG, Fernandes TF, Read P, Raffaelli D (2000) Effects of macroalgal mats on intertidal sandflats: an experimental study. J Exp Mar Biol Ecol 249:123-137

► Borja A, Muxika I (2005) Guidelines for the use of AMBI (AZTI's Marine Biotic Index) in the assessment of the benthic ecological quality. Mar Pollut Bull 50:787-789

Callier MD, McKindsey CW, Desrosiers G (2007) Multi-scale spatial variations in benthic sediment geochemistry and macrofaunal communities under a suspended mussel culture. Mar Ecol Prog Ser 348:103-115

► Callier MD, McKindsey CW, Desrosiers G (2008) Evaluation of indicators used to detect mussel farm influence on the benthos: two case studies in the Magdalen Islands, eastern Canada. Aquaculture 278:77-88

► Cardell MJ, Sarda R, Romero J (1999) Spatial changes in sublittoral soft-bottom polychaete assemblages due to river inputs and sewage discharges. Acta Oecol Int J Ecol 20:343-351

Clarke KR, Warwick RM (1994) Change in marine commu-
nities: an approach to statistical analysis and interpreta-
tion. Plymouth Marine Laboratory, Plymouth

► Cook EJ, Black KD, Sayer MDJ, Cromey CJ and others (2006) The influence of caged mariculture on the early development of sublittoral fouling communities: a pan-European study. ICES J Mar Sci 63:637-649

Costanzo S, O'Donohue M, Dennison W (2003) Assessing the seasonal influence of sewage and agricultural nutrient inputs in a subtropical river estuary. Estuar Coast Shelf Sci 26:857-865

► Cromey CJ, Nickell TD, Black KD (2002) Depomod—mod-
eling the deposition and the biological effects of waste
solids from marine cage farms. Aquaculture 214:211-239

► Davy SK, Turner JR (2003) Early development and acquisi-
tion of zooxanthellae in the temperate symbiotic sea
anemone *Anthopleura balii* (Cocks). Biol Bull 205:66-72
Diaz RJ, Rosenberg R (1995) Marine benthic hypoxia: a
review of its ecological effects and the behavioural
responses of benthic macrofauna. Oceanogr Mar Biol
Annu Rev 33:245-303

► Dolenec T, Lojen S, Kniewald G, Dolenee M, Rogan N (2007) Nitrogen stable isotope composition as a tracer of fish farming in invertebrates *Aplysina aerophoba*, *Balanus perforatus* and *Anemonia sulcata* in central Adriatic. Aquaculture 262:237-249

► Dubois S, Marin-Leal JC, Ropert M, Lefebvre S (2007a) Effects of oyster farming on macrofaunal assemblages associated with *Lanice conchilega* tubeworm populations: a trophic analysis using natural stable isotopes. Aquaculture 271:336-349

► Dubois S, Orvain F, Marin-Leal JC, Ropert M, Lefebvre S (2007b) Small-scale spatial variability of food partitioning between cultivated oysters and associated suspending
sion-feeding species, as revealed by stable isotopes. Mar Ecol Prog Ser 336:151–160

FAO (Food and Agriculture Organization) (2010) The state of world fisheries and aquaculture 2010. FAO, Rome

Fauchald K, Jumars PA (1979) The diet of worms: a study of polychaete feeding guilds. Oceanogr Mar Biol Annu Rev 17:193–284

► Findlay RH, Walting L, Mayer LM (1995) Environmental impact of salmon net-pen culture on Maine marine benthic communities: a case study. Estuaries 18:145–179

► Franco MA, Soetaert K, Costa MJ, Vincx M, Vanaverbeke J (2008) Uptake of phytodetritus by meiobenthos using $^{13}$C labelled diatoms and *Phaeocystis* in two contrasting sediments from the North Sea. J Exp Mar Biol Ecol 362:1–8

Hargrave BT (2003) Far-field environmental effects of marine finfish aquaculture. In: A scientific review of the potential environmental effects of aquaculture in aquatic ecosystems. Can Tech Rep Fish Aquat Sci 2450:1–35

Hargrave BT (ed) (2005) Environmental effects of marine finfish aquaculture. The handbook of environmental chemistry, Vol 5. Water pollution, Part M. Springer, Berlin

Herman MJ, Middelburg JJ, Van de Koppel J, Heip CHR (1999) Ecology of estuarine macrobenthos. In: Advances in ecological research, Vol 29. Academic Press, San Diego, CA, p 195–240

Holmer M, Wildish DJ, Hargrave B (2005) Organic enrichment from marine finfish aquaculture and effects on sediment biogeochemical processes. In: Hargrave BT (ed) The handbook of environmental chemistry, Vol 5. Water pollution, Part M. Springer, Berlin, p 181–206

► Hughes TP, Bellwood DR, Folke C, Steneck RS, Wilson J (2005) New paradigms for supporting the resilience of marine ecosystems. Trends Ecol Evol 20:380–386

► Kinoshita K, Tamaki S, Yoshioka M, Srithonguthai S and others (2008) Bioremediation of organically enriched sediment deposited below fish farms with artificially mass-cultured colonies of a deposit-feeding polychaete *Capitella* sp. I. Fish Sci 74:77–87

► Kon K, Kawakubo N, Aoki JI, Tongnunui P, Hayashizaki KI, Kurokura H (2009) Effect of shrimp farming organic waste on food availability for deposit feeder crabs in a mangrove estuary, based on stable isotope analysis. Fish Sci 75:715–722

► Kullman MA, Kidd KA, Podemski CL, Paterson MJ, Blanchfield PJ (2009) Assimilation of freshwater salmonid aquaculture waste by native aquatic biota. Can J Fish Aquat Sci 66:1965–1975

► Kutti T, Ervik A, Hansen PK (2007a) Effects of organic effluents from a salmon farm on a fjord system. I. Vertical export and dispersal processes. Aquaculture 262: 367–381

► Kutti T, Hansen PK, Ervik A, Hoisaeter T, Johannessen P (2007b) Effects of organic effluents from a salmon farm on a fjord system. II. Temporal and spatial patterns in infauna community composition. Aquaculture 262: 355–366

► Lefebvre S, Barille L, Clerc M (2000) Pacific oyster (*Cras-sostrea gigas*) feeding responses to a fish-farm effluent. Aquaculture 187:185–198

► Lefebvre S, Bacher C, Meuret A, Hussenot J (2001) Modeling approach of nitrogen and phosphorus exchanges at the sediment-water interface of an intensive fishpond system. Aquaculture 195:279–297

► Lefebvre S, Marin Leal JC, Dubois S, Orvain F and others (2009) Seasonal dynamics of trophic relationships among

co-occurring suspension-feeders in two shellfish culture dominated ecosystems. Estuar Coast Shelf Sci 82: 415–425

► Lin DT, Fong P (2008) Macroalgal bioindicators (growth, tissue N, $\delta^{15}$N) detect nutrient enrichment from shrimp farm effluent entering Opunohu Bay, Moorea, French Polynesia. Mar Pollut Bull 56:245–249

► Lindqvist S, Norling K, Hulth S (2009) Biogeochemistry in highly reduced mussel farm sediments during macrofaunal recolonization by *Amphiura filiformis* and *Nephtys* sp. Mar Environ Res 67:136–145

► Lojen S, Spanier E, Tsemel A, Katz T, Eden N, Angel DL (2005) $\delta^{15}$N as a natural tracer of particulate nitrogen effluents released from marine aquaculture. Mar Biol 148:87–96

► Lorrain A, Savoye N, Chauvaud L, Paulet YM, Naulet N (2003) Decarbonation and preservation method for the analysis of organic C and N contents and stable isotope ratios of low-carbonated suspended particulate material. Anal Chim Acta 491:125–133

► Mayor DJ, Zuur AF, Solan M, Paton GI, Killham K (2010) Factors affecting benthic impacts at Scottish fish farms. Environ Sci Technol 44:2079–2084

► Mazzola A, Sarà G (2001) The effect of fish farming organic waste on food availability for bivalve molluscs (Gaeta Gulf, Central Tyrrhenian, Mediterranean): stable carbon isotopic analysis. Aquaculture 192:361–379

► Mazzola A, Mirto S, La Rosa T, Fabiano M, Danovaro R (2000) Fish-farming effects on benthic community structure in coastal sediments: analysis of meiofaunal recovery. ICES J Mar Sci 57:1454–1461

► McKindsey CW, Thetmeyer H, Landry T, Silvert W (2006) Review of recent carrying capacity models for bivalve culture and recommendations for research and management. Aquaculture 261:451–462

Michener RH, Schell DM (1994) Stable isotope ratios as tracers in marine aquatic food webs. In: Lajtha K, Michener RH (eds) Stable isotopes in ecology and environmental sciences. Blackwell Scientific, Oxford, p 138–157

► Modica A, Scilipoti D, La Torre R, Manganaro A, Sarà G (2006) The effect of mariculture facilities on biochemical features of suspended organic matter (southern Tyrrhenian, Mediterranean). Estuar Coast Shelf Sci 66:177–184

► Muxika I, Borja À, Bald J (2007) Using historical data, expert judgement and multivariate analysis in assessing reference conditions and benthic ecological status, according to the European Water Framework Directive. Mar Pollut Bull 55:16–29

► Nickell LA, Black KD, Hughes DJ, Overnell J and others (2003) Bioturbation, sediment fluxes and benthic community structure around a salmon cage farm in Loch Creran, Scotland. J Exp Mar Biol Ecol 285/286:221–233

Pearson TH, Black KD (2001) The environmental impacts of marine fish cage culture. In: Black KD (ed) Environmental impacts of aquaculture. Sheffield Academic Press, Sheffield, p 1–32

Pearson TH, Rosenberg R (1978) Macrobenthic succession in relation to organic enrichment and pollution of the marine environment. Oceanogr Mar Biol Annu Rev 16: 229–311

► Pinnegar JK, Polunin NVC (2000) Contributions of stable isotope data to elucidating food webs of Mediterranean rocky littoral fishes. Oecologia 122:399–409

► Pitta P, Tsapakis M, Apostolaki ET, Tsagaraki T, Holmer M, Karakassis I (2009) 'Ghost nutrients' from fish farms are
transferred up the food web by phytoplankton grazers.
Mar Ecol Prog Ser 374:1-6

► Post DM (2002) Using stable isotopes to estimate trophic position: models, methods, and assumptions. Ecology 83: 703–718

Rensel JE, Forster JRM (2007) Beneficial environmental effects of marine finfish mariculture. NOAA National Marine Fisheries Service, Office of Oceanic and Atmospheric Research, Washington, DC

► Ryba SA, Burgess RM (2002) Effects of sample preparation on the measurement of organic carbon, hydrogen, nitrogen, sulfur, and oxygen concentrations in marine sediments. Chemosphere 48:139–147

► Sarà G, Scilipoti D, Mazzola A, Modica A (2004) Effects of fish farming waste to sedimentary and particulate organic matter in a southern Mediterranean area (Gulf of Castellammare, Sicily): a multiple stable isotope study (δ¹³C and δ¹⁵N). Aquaculture 234:199–213

Telfor T, Robinson K (2003) Environmental quality and carrying capacity for aquaculture in Mulroy Bay, Co. Donegal. Marine Environment and Health Series, No. 9. http://oar.marine.ie/handle/10793/1/browse?type=author&order=ASC&rpp=20&value=Telfor%2C+T (accessed April 2012)

► Tsutsumi H (1990) Population persistence of *Capitella* sp. (Polychaeta; Capitellidae) on a mud flat subject to environmental disturbance by organic enrichment. Mar Ecol Prog Ser 63:147–156

► Tsutsumi H, Fukunaga S, Fujita N, Sumida M (1990) Relationship between growth of *Capitella* sp. and organic enrichment of the sediment. Mar Ecol Prog Ser 63: 157–162

Tsutsumi H, Kinoshita K, Srithongouthai S, Sato A and others (2005) Treatment of the organically enriched sediment below the fish farm with the biological activities of artificially mass-cultured colonies of a small deposit feeding polychaete, *Capitella* sp. I. Benth Res 60:25–38

► van Oevelen D, Soetaert K, Franco MA, Moodley L, van Ijzerloo L, Vincx M, Vanaverbeke J (2009) Organic matter input and processing in two contrasting North Sea sediments: insights from stable isotope and biomass data. Mar Ecol Prog Ser 380:19–32

► Vizzini S, Mazzola A (2004) Stable isotope evidence for the environmental impact of a land-based fish farm in the western Mediterranean. Mar Pollut Bull 49:61–70

► Vizzini S, Mazzola A (2006) The effects of anthropogenic organic matter inputs on stable carbon and nitrogen isotopes in organisms from different trophic levels in a southern Mediterranean coastal area. Sci Total Environ 368:723–731

Word JQ (1990) The infaunal trophic index, a functional approach to benthic community analyses. PhD dissertation, the University of Washington, Seattle, WA

► Yokoyama H, Ishihi Y (2007) Variation in food sources of the macrobenthos along a land-sea transect: a stable isotope study. Mar Ecol Prog Ser 346:127–141

► Yokoyama H, Abo K, Ishihi Y (2006) Quantifying aquaculture-derived organic matter in the sediment in and around a coastal fish farm using stable carbon and nitrogen isotope ratios. Aquaculture 254:411–425

► Yokoyama H, Takashi T, Ishihi Y, Abo K (2009) Effects of restricted feeding on growth of red sea bream and sedimentation of aquaculture wastes. Aquaculture 286: 80–88

Editorial responsibility: Jana Davis,
Annapolis, Maryland, USA

Submitted: December 13, 2011; Accepted: January 7, 2013

Proofs received from author(s): May 7, 2013