Can biochemical methods determine if salmonids feed and thrive after escaping from aquaculture cages

CAN BIOCHEMICAL METHODS DETERMINE IF SALMONIDS FEED AND THRIVE AFTER ESCAPING FROM AQUACULTURE CAGES? A PILOT STUDY

Kรกtya G. Abrantes, Jayson M. Semmens, Jeremy M. Lyle & Peter D. Nichols

August 2010 NRM Cradle Coast Project CCCPR24006

1

Abrantes, K.G., 1Semmens, J.M., 1Lyle, J.M., 2Nichols, P.D.

Spatial management of reef fisheries and ecosystems: Understanding the importance of movement.

1

Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Private Bag 49, Hobart, Tasmania 7001, Australia 2

CSIRO Marine and Atmospheric Research, Wealth from Oceans Flagship, GPO Box 1538, Hobart, Tasmania 7001, Australia ďƒ“ The Tasmanian Aquaculture and Fisheries Institute, University of Tasmania and Fisheries Research, CSIRO and NRM Craddle Coast 2010.

This work is copyright. Except as permitted under the Copyright Act 1968 (Cth), no part of this publication may be reproduced by any process, electronic or otherwise, without the specific written permission of the copyright owners.

Information may not be stored electronically in any form whatsoever without such permission.

Disclaimer The authors do not warrant that the information in this document is free from errors or omissions. The authors do not accept any form of liability, be it contractual, tortious, or otherwise, for the contents of this document or for any consequences arising from its use or any reliance placed upon it. The information, opinions and advice contained in this document may not relate, or be relevant, to a readerâ€&#x;s particular circumstance. Opinions expressed by the authors are the individual opinions expressed by those persons and are not necessarily those of the Tasmanian Aquaculture and Fisheries Institute or the University of Tasmania or CSIRO or NRM Cradle Coast.

Enquires should be directed to: Dr Jayson Semmens Tasmanian Aquaculture and Fisheries Institute University of Tasmania Private Bag 49, Hobart, Tasmania 7001 E-mail: Jayson.Semmens@utas.edu.au Ph. (03) 6227 7277 Fax (03) 6227 8035

Fate of escaped salmonids

Table of Contents 1. Executive summary .........................................................................................................2 2. Introduction .....................................................................................................................4 3. Methods ...........................................................................................................................8 3.1 Study Area .................................................................................................................8 3.2. Animal collection .....................................................................................................8 3.3 Stomach content analysis.........................................................................................11 3.4 Fish condition ..........................................................................................................11 3.5 Stable isotope analysis .............................................................................................13 3.6 Fatty acid analysis ...................................................................................................16 4. Results ...........................................................................................................................19 4.1 Stomach contents analysis .......................................................................................19 4.2 Body condition ........................................................................................................19 4.3 Stable isotope analysis .............................................................................................22 4.4 Fatty acid analysis ...................................................................................................27 5. Discussion .....................................................................................................................41 6. Conclusions ...................................................................................................................49 7. Acknowledgements .......................................................................................................49 8. References .....................................................................................................................50

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1. Executive summary Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss were introduced to Australia in the late 1800s as sportfish for recreational anglers. Today, Tasmania produces approx. 30000 tones of head-on, gutted salmonids per annum for human consumption. Fish are grown-out in protected high density sea cages, but occasional losses are unavoidable and fish do escape to the natural environment. Salmonids are opportunistic carnivores and therefore their introduction in large numbers in Australian coastal systems is likely to have significant negative impacts on the local ecology if they survive and thrive after escaping.

In this study, a multi-faceted approach, including a novel use of the established biochemical techniques stable isotope and fatty acid analyses, was used to determine if salmonids feed on native fauna and thrive after escaping from aquaculture cages in Macquarie Harbour, a large semi-enclosed harbour in western Tasmania.

Stomach content analysis indicated that Atlantic salmon do not feed on native fauna as most stomachs were either empty or contained non-nutritious food like twigs and myrtle leaves. For rainbow trout, the proportion of empty stomachs was much lower than for Atlantic salmon (38 vs. 79%), and a large proportion (~20%) of stomachs analysed contained feed pellets, including stomachs from fish collected ~10 km away from the closest fish farms. This indicates that rainbow trout can move away from fish farms, but return to feed on the highly nutritious pellets. About a fifth of the stomachs analysed contained native fauna, including one fish, indicating that rainbow trout escapees have the capability to capture and consume mobile species.

Different body condition indices were also used, and indicate that salmonids loose condition after escaping. For both Atlantic salmon and rainbow trout, Fultonâ€&#x;s K, viscerosomatic index and muscle lipid content were good condition indicators. Visceral fat was also a good condition indicator for rainbow trout. However, the hepatosomatic index (HSI) was not a useful measure of fish condition, as escapees often had similar or higher HSI values than caged animals, and caged animals had HSI values similar to the wild brown trout.

For stable isotope analysis, only one Atlantic salmon had a chemical signature that suggested feeding on native fauna, probably a combination of small fish and grapsid crabs.

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This individual was also separated in fatty acid composition, with both biochemical techniques corroborating that it had been feeding significantly on native species. One second Atlantic salmon and one rainbow trout escapee were also separated from the rest of salmonids based on their fatty acid composition. This second separated Atlantic salmon was in very poor condition and had stable isotopic composition similar to the caged salmonids, suggesting that the differences in fatty acid compositions were a result of starvation, and not of a change in diet. The rainbow trout, however, was in good condition, suggesting that it was thriving, and that the difference in fatty acid composition was a result of a difference in diet.

Hence, there was evidence of successful feeding on native fauna for only two fish, one Atlantic salmon (7.7% of total Atlantic salmon) and one rainbow trout (2.6% of total) and results indicate that, in general, escapees do not switch to feed on native fauna. However, only a limited number of escapees were analysed and therefore results can not be considered as conclusive. Moreover, these results are only valid for Macquarie Harbour and the situation in other parts of Tasmania where the native invertebrate and fish fauna is more abundant and diverse, may be different. Further studies should be conducted in other systems where salmonid farms occur, so that a more conclusive assessment of the fate and impacts of salmon escapees in Tasmania can be drawn.

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2. Introduction Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, are native to the North Atlantic and North Pacific respectively, and were introduced to Australia in the late 1800s as sportfish for recreational anglers. Commercial farming of salmonids commenced in Australia in the mid-1980s and today Tasmania produces approx. 30000 tones of headon, gutted salmonids per annum, with Atlantic salmon accounting for the majority of production (ca. 90%). The industry has significant economic benefits, with current annual production valued at over AUD$300M. Salmonids are typically grown-out in sea cages, which allow high density fish rearing and low overhead costs compared to equivalent landbased facilities.

Although cages are generally protected by an outer net to reduce stock loss from predation by seals and sharks, losses are unavoidable and fish frequently escape into the natural environment. Escapes occur as large pulses or through small leakages, a consequence of human error, net breakages, or from natural causes such as predator (seals, sharks) or storm damage to cages (Gausen & Moen 1991, McKinnell et al. 1997). In Tasmania, it is estimated that 2-3% of stocked fish is lost due to low-level leakage (DPIW 2006).

However, large-scale losses also occur, generally the result of large net tears or storms. By law, these large-scale losses (>1000 individuals) have to be reported to DPIPWE. Between 2000 and 2006, over 250000 escaped salmonids were reported (DPIW 2006), a figure that is likely to be conservative. Although exact numbers and frequency of escapes are not known, these are sufficiently large to provide an impetus for significant targeted recreational fishing activity.

Salmonids are opportunistic carnivores, feeding on small crustaceans, mesopelagic fish and squid (Jacobsen & Hansen 2001, Haugland et al. 2006). If large numbers of escaped individuals survive and thrive, this is likely to have significant negative impacts on the local ecology (Gross 1998, Simon & Townsend 2003). Although cross-breeding with wild populations is not a problem in Tasmania, as salmonids are introduced species, negative impacts on native fauna may be important, either by direct predation or by competition for food or habitat, disturbance of spawning beds or by transfer of diseases or parasites into wild populations (Heggberget et al. 1996, Gross 1998).

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Breeding populations of rainbow trout have been established in Australia (Ward et al.

2003), New Zealand (Crowl et al. 1992), Argentina and Chile (Soto et al. 2006), but colonization success is generally low and highly variable with location (Fausch et al. 2001).

Atlantic salmon, however, is a poor colonizer (Hough & Naylor 1992), and despite several attempts of introduction, no self sustaining populations have been established outside its natural range, with the exception of some landlocked systems in Argentina and New Zealand (MacCrimmon & Gots 1979).

Although Atlantic salmon escapees survive in their natural range of distribution (e.g. Carr et al. 1997, Lacroix et al. 2005), and in the North Pacific (McKinnell et al. 1997, Volpe et al. 2000), feeding on the same food sources as their wild counterparts, this does not seem to be the case in the Southern Hemisphere. In a study conducted in Chile, escapees did not thrive and continued feeding to some extent on feed pellets that float from the cages (Soto et al. 2001). Similarly, escapees in Macquarie Harbour, western Tasmania, did not switch to feed on native fauna, and continued feeding on feed pellets (Steer & Lyle 2003). However, these two studies based their conclusions on results from stomach content and body condition analyses, despite the observation that naturally occurring wild salmonids frequently exhibit a high incidence of empty stomachs (Jacobsen & Hansen 2001) and that their body condition tends to be lower than that of caged individuals (Thorstad et al. 1997, Johnston et al. 2006). Hence, there is still no definitive answer regarding the fate of salmonid escapees in Tasmania.

Stomach content analysis is an important method of diet analysis, providing crucial information on prey taxonomy and size composition, especially for carnivorous species such as salmonids, which ingest their prey whole. However, this method does not provide reliable quantitative results as it can lead to bias due to differences in detectability, quantifiability and digestibility among food sources (CrĂŠach et al. 1997, ReĂąones et al.

2002). Moreover, it can confirm the diet in an animal only at a specific time. Regurgitation and post-mortem digestion of stomach contents is also frequent, and the presence of unidentifiable, highly decomposed material can make identifications challenging (Haywood 1995). This is further limited by the fact that escapees frequently have high proportions of empty stomachs, even in their native range (e.g. Scottish waters, Hislop & Webb 1992).

Biochemical techniques such as stable isotope and fatty acid analysis can be more useful to determine if escaped salmonids switch diet to feed on native fauna. These two approaches TAFI Report Page 5

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provide information on the material that is assimilated and incorporated into an organisms‟ tissues and not just what is ingested, reflect the diet over relatively long periods (weeks to months), allow the identification of food sources not easily detectable by gut contents analysis, and generally require a reduced number of samples when compared to stomach content analysis.

Stable isotope analysis measures the ratio of heavy to light isotopes of an element. The stable isotope signatures of carbon (δ13C) and nitrogen (δ15N) are generally used in trophic studies because they undergo little (δ13C) or predictable (δ15N) change as they are passed on to consumers. Hence, the isotopic signature of a consumer is a reflection of its food source.

The fractionation of dietary C and N from food source to consumers generally results in δ13C increases of ~0.5-1.0‰ (DeNiro & Epstein 1978), and δ15N increases of 2-5‰ (DeNiro & Epstein 1981). The analysis of δ13C generally provides information on the base of the food web because different food sources can have distinct δ13C, and especially because of the relatively small fractionation from food source to consumer. On the other hand, δ15N is generally used as an indicator of trophic position as it undergoes a much higher fractionation between trophic levels. The analysis of both elements combined can lead to more precise characterization of diets and should also be useful to detect any shift in diet away from feed pellets in escapees. This will however only be possible if the isotopic composition of artificial feed pellets differs from that of potential native prey (e.g. fish, small crustaceans). In this study, δ15N analysis should be particularly informative as salmonid feed generally contains fish meal and fish oil and is therefore more enriched in 15

N than local primary producers.

As with stable isotopes, the fatty composition of a consumer is also a reflection of its food sources, as fatty acids with 14 or more carbons are deposited in consumer tissues in a relatively unmodified manner (Budge et al. 2006). Moreover, because animals can not synthesise polyunsaturated fatty acids (PUFA) de novo, some can be used as tracers or biomarkers, to trace the origin of the food to a specific source (Dalsgaard & John 2004).

The use of individual fatty acids as tracers is, however, only possible if certain fatty acids can be traced to only one of the possible food sources (e.g. Iverson 1993). Fatty acid analysis has been previously used to separate farmed from wild salmonids (Martinez et al.

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2009) and, as with stable isotope analysis, will be especially useful if the fatty acid composition of feed pellets is different to that of native organisms.

In this pilot study conducted in Macquarie Harbour, western Tasmania, a multi-faceted approach including body condition and stomach content, was used to assess if stable isotope and fatty acid analyses are useful for determining if salmonids feed and thrive after escaping from aquaculture cages. Possible trophic interactions with native species were also investigated.

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3. Methods 3.1 Study Area This study was conducted in Macquarie Harbour, western Tasmania, Australia (Fig. 1).

This is a semi-enclosed ~32 km long and ~8 km wide estuarine system, with an average depth of 20 m and an area of ~276 km2. Freshwater inputs from two river systems and a narrow opening to the sea result in limited water circulation and strong stratification in the water column. Typically, the surface waters are brackish, the middle layer is of intermediate salinity, the water is characterised by low dissolved oxygen, and the deep layer is essentially seawater. The substrate is mostly sandy-mud, with gravel, pebbles and rocks along the margins. Small, sparse seagrass patches occur in the shallow areas around the margins. Macroalgae occur in very low abundance and only in a narrow band along the margins, attached to rocks and pebbles. The waters have a dark brown coloration due to the high concentration of tannins and other humic leachates that enter the harbour from the Gordon River and surrounding area. Consequently, aquatic productivity is very low (Edgar & Cresswell 1991, Edgar et al. 1999). The area is surrounded mainly by National Park, with only one small populated area of Strahan in the northwest margin (population: ~700).

The environmental conditions of the harbour have been described by Carpenter et al.

(1991).

3.2. Animal collection Macquarie Harbour was sampled for Atlantic salmon and rainbow trout escapees at two sites: Swan Basin and Table Head (Fig. 1). Swan Basin, about 10 km from the closest fish farms, is a shallow (max 4 m) and relatively protected area in the north-west part of the Harbour. Table Head is located closer (~1 km) to the farm leases. The two sites were selected for being relatively protected from strong winds, hence facilitating fieldwork, and for being at different distances from the farm leases. Sampling was conducted in November 2008 (spring) and April 2009 (autumn) to identify possible differences in feeding activity between the colder and warmer months of the year and to maximise the number of escapees collected. Each sampling trip consisted of three days and two nights of intensive fishing TAFI Report Page 8

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using six multi-panel monofilament gillnets, each comprised of three 30-50 m long nets of different mesh sizes (50 to 200 mm). Nets were set from the shore perpendicular to the waterline, and soak times varied between 6 and 12 h.

Fig. 1. Map showing Macquarie Harbour and the two sampling sites, Swan Basin and Table Head.

- approximate location of the aquaculture leases; - approximate area netted.

All salmonids collected were retained, including the wild brown trout (Salmo trutta), a species introduced to Tasmania in the mid 1800s for the recreational fishery. Additional muscle and stomach samples were obtained from catches taken by recreational fishers in Swan Basin. Upon capture, fish were stunned with a blow in the head and their brains spiked to produce a fast death (Ethics Approval A0010272, University of Tasmania). Each fish was tagged with an individual number so that each muscle/stomach sample could later be linked to a particular individual. Immediately after death, a small (~3 g) piece of muscle tissue was excised from a standardized location just below the dorsal fin, labelled, sealed in TAFI Report Page 9

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aluminium foil and placed in liquid nitrogen. Muscle samples were removed from a standardized location because different parts of the animal can have different lipid content (Aursand et al. 1994, Einen et al. 1998, Regost et al. 2003). Samples were kept in liquid nitrogen until transported to the laboratory, where they were stored at -20°C until analyses.

Bodies were immediately placed in ice slurry to prevent further decomposition of stomach contents. Caged Atlantic salmon and rainbow trout and artificial feed pellets were also obtained from the Tassal (Atlantic salmon) and Petuna (rainbow trout) fish farms in November 2008, to provide information on the stable isotope and fatty acid composition of caged animals and their feed.

In addition to caged and escaped salmonids, native fish and invertebrate species were also sampled for stable isotope analysis, to give an indication of what the stable isotope composition of salmonids could be in case of diet switch from artificial feed to native fauna. Carnivorous fish species, considered as potential competitors, were caught in the same gillnets as salmonids and included the red cod (Pseudophycis bachus), juvenile blue grenadier (Macruronus novaezelandiae), Australian salmon (Arripis trutta), as well as wild brown trout (Salmo trutta). Potential prey, including small fish (<100 mm TL), crustaceans and bivalves, were also collected. These included crustaceans such as grapsid crabs (Paragrapsus gaimardii) and caridean shrimps (Macrobrachium sp. and an unidentified species), and small fish including jollytails (Galaxias sp.), silverfish (Leptatherina presbyteroides), pilchards (Sardinops sagax), short-headed hardyheads (Kestrastherina brevirostris), two unidentified Gobiidae species and small juveniles (100 mm TL) of greenback flounder (Rhombosolea tapirina), yellow-eye mullet (Aldrichetta forsteri) and Australian salmon (Arripis trutta).

Small fish, crabs and shrimps were collected with seine nets and fish traps. Isopods and mussels were picked from between the rocks and pebbles along the shore. Amphipods were collected from the substrate by sieving the surface sediment through a 500 Οm sieve and picking animals with tweezers. Potential prey were killed by immersion in ice slurry and placed in liquid nitrogen until transport to the laboratory. All samples were kept in liquid nitrogen until transported to the laboratory, where they were stored at -20°C until analyses.

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Primary producers were also collected to obtain the baseline δ15N for the food web at Macquarie Harbour. This information is necessary to accurately determine if salmonid escapees feed after they escape, as it provides a point of comparison with the δ15N of artificial feed pellets. At each site, green leaves of the seagrass Zostera muelleri were collected from 3-5 individual plants and pooled for analysis. Phytoplankton was also collected at each site with a 125 μm plankton net. Epilithic microalgae, mainly filamentous green algae, diatoms and cyanobacteria, were collected at both sites by scraping 5-10 well separated (>5 m) greenish pebbles with a scalpel. The scrapped material was then passed through a 125 μm sieve, washed, and collected on a 5 μm GF/F Whatman filter. Brown and red epiliths were also collected. In the laboratory, these algae were carefully washed with distilled water, and all possible debris and other contaminants removed under a dissecting microscope. For each algae group, material collected at each site was pooled for analysis.

Potential fish competitors and the grapsid crab Paragrapsus gaimardii were collected in both sampling periods, while primary producers, other invertebrates and small fish were only collected in April 2009.

3.3 Stomach content analysis Stomachs of all salmonids were dissected and contents sorted and identified. Food items were placed into one of the following categories: artificial feed pellets, fish, crustaceans (caridean shrimps and grapsid crabs), bivalves (blue mussel Mytilus edulis), algae/seagrass, leaves/twigs, sand/pebbles, and “other” (other non-nutritious material). For each species, the presence of each food type was recorded and the diet was summarised as the frequency of occurrence of each food type, i.e. percentage of total individuals that contained a particular food (Hyslop 1980).

3.4 Fish condition In the laboratory, animals were measured to the nearest 0.5 cm and carcasses, viscera and liver weighed to the nearest 0.1 g. Body condition was compared between caged and escaped salmonids using a range of condition indexes. Fulton‟s K, an index based on the length-weight relationship, was calculated as: K = (100 x GW) x TL-3, where GW is gutted TAFI Report Page 11

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weight (g) and TL is total length (cm). Liver weight was used to calculate the hepatosomatic index (HSI), which gives an indication of condition by measuring the proportion of liver to body weight: HSI = LW/GW x 100 (LW = liver weight in g). The viscerosomatic index (VSI) was also calculated based on the proportion of viscera (VW, in g) to total body weight (BW, in g): VSI = VW/BW x 100. Viscera consisted of the digestive tract and all other internal organs, including visceral fat. Because rainbow trout frequently had high quantities of visceral fat (VF), this was carefully separated from the stomach, intestine and other internal organs, and weighed to calculate the proportion of visceral fat to body weight: %VF = VF/BW x 100.

Body condition was also measured based on the lipid content in white muscle (% wet weight). Prior to lipid extraction, any parts of the sample exposed to air were trimmed to remove oxidised tissue, and between 1.0 and 1.5 g of white muscle was weighed. Samples were extracted in 250 ml separatory funnels by a modification of the Bligh & Dyer (1959) method, using a chloroform:methanol:water solvent solution (10:20:8 ml). Tissues were extracted overnight in a single phase, after which chloroform and saline water (10:10 ml) was added to break phase, and allowed to separate for 2 h (final solvent ratio: 1:1:0.9, by vol. chloroform:methanol:water). The lower chloroform phase, containing lipid, was then collected and concentrated under vacuum using a rotary evaporator at 40°C. Lipid was then transferred into a pre-weighed 1.5 ml vial and blown down under a stream of nitrogen until constant weight to give the total lipid extract (TLE). This was used to calculate the total lipid content of each sample (% wet mass).

Data analysis Fultonâ&#x20AC;&#x;s K and VSI were compared between species (Atlantic salmon and rainbow trout) and living conditions (caged vs. escaped) with two-way analyses of variance (ANOVAs), after the assumptions of normality and homogeneity of variances were met. For HSI and muscle lipid content, due to the high variability found for escaped rainbow trout, separate one-way ANOVAs were run, one for each species. Although no data transformation was necessary for Atlantic salmon, data for rainbow trout had to be transformed to meet the assumption of homogeneity of variances (square-root for HSI and inverse for lipid content).

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For rainbow trout, %VF was also compared between caged and escaped individuals with a one-way ANOVA. All analyses were done in STATISTICA v.7.

Since condition of escaped salmonids is lower than that of caged individuals (Thorstad et al. 1997), it is not possible to determine if escapees are thriving or wasting away simply by comparing condition between caged and escaped fish. Ideally, comparisons should be made between wild and escaped individuals but because there are no wild populations of Atlantic salmon and it is not known if there are wild rainbow trout populations in Tasmania, condition indexes of the wild brow trout, Salmo trutta, were used.

3.5 Stable isotope analysis All stable isotope samples were processed within a week of collection. For salmonids and large fish, each individual was analysed separately to allow the detection of differences in diet between individuals. To reduce the intraspecific variability in isotopic values of potential prey, 2-3 individuals were pooled per sample for small fish, and 3-5 for decapod crustaceans. For small invertebrates (peracarid crustaceans and blue mussels), 10-30 individuals were combined in each sample to obtain enough dry material for analysis.

Whenever possible, only white muscle tissue was used since it is less variable in δ13C and δ15N than other tissue types (Pinnegar & Polunin 1999, Yokoyama et al. 2005). Tissue was excised from the trunk behind the pectoral fin in small fish, from claws and legs in crabs, from the abdominal muscle in shrimps and the adductor muscle in blue muscles. Because of their small size, peracarid crustaceans were processed whole but were not acid washed, as ecologically significant shifts and higher variability in δ15N have been reported for acid treated samples (Bunn et al. 1995).

Because lipids are depleted in

13

C in relation to proteins and carbohydrates, tissue lipid

content affects its δ13C values (DeNiro & Epstein 1977, McConnaughey & McRoy 1979).

This can lead to ecologically significant inaccuracies when conclusions are based on δ13C results of lipid rich samples. Since salmonids are typically very rich in lipids, lipids were chemically removed from samples before δ13C analysis to reduce contents to consistent low levels. Lipids were also removed from the artificial feed pellets, since due to isotopic routing (Schwarcz 1991) the isotopic signature of a tissue is a reflection of the particular TAFI Report Page 13

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nutrients from which it is formed (Gannes et al. 1997, Bearhop et al. 2002), and not of the overall diet (e.g. diet protein is routed to the protein component of the tissue).

Lipids were removed with a chloroform:methanol:water solvent solution (1:2:0.8 by vol.) as described above for the analysis of muscle lipid content. Because lipid extraction can cause a fractionation in δ15N (Pinnegar & Polunin 1999, Sotiropoulos et al. 2004), δ15N analysis was conducted on untreated samples. Hence, each muscle sample was divided into two pieces prior to analysis, and analysed separately for δ15N and δ13C. Due to financial constraints, separate analyses were not conducted for species other than salmonids. Instead, δ13C values were mathematically normalized based on the carbon-to-nitrogen (C:N) ratios using the equation: δ13Cnormalised = δ13Cuntreated - 3.32 + 0.99 x C:N (Post 2007), where δ13Cuntreated corresponds to the measured, original δ13C. This is the best correction for lipid content in aquatic animals (Post et al. 2007). Lipids from aquatic producers tissues were also not removed and δ13C values not corrected as aquatic producers had very low % carbon (<40%) and no relationship between lipid content or C:N ratios and δ13C is present for producers with % carbon <40% (i.e. all aquatic producers; Post et al. 2007).

Samples were dried to a constant weight at 60°C, homogenized into a fine powder with a mortar and pestle and the carbon and nitrogen isotopic composition and concentration were measured with an Isoprime mass spectrometer coupled with an element analyser. Results are expressed as per mil (‰) deviations from the standards, as defined by the equation: δ13C, δ15N = [(Rsample / Rreference) - 1] x 103, where R =

13

C/12C for carbon and

15

N/14N for

nitrogen. Primary standards were ANU cane sucrose for δ13C and IAEA-305A for δ15N, which were calibrated against PD Belemnite for δ13C and ambient air for δ15N. Duplicates were run every 12th sample and two standards were also run after every 12 samples. Results had a precision (1 SD) of ± 0.1‰ for both δ13C and δ15N.

Data analysis Values for δ13C and δ15N were compared between caged and escaped animals with a oneway ANOVA to identify differences in isotopic profiles likely to be a result of changes in diet after escapement. This was done for Atlantic salmon and rainbow trout separately.

Here, data from escapees captured at both sites and in both sampling trips was pooled.

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δ13C and δ15N of primary producers and native fish and invertebrates was graphed to give an indication of the stable isotopic composition escaped salmonids would have if they successfully fed on native fauna. Artificial feed pellets and caged salmonids were also plotted to allow the visual comparison of the signatures of local and artificial sources. For native species, stable isotope results of individuals collected at both sites and on both sampling occasions were combined. This was only done after verifying, with a two-way ANOVA, that there were no spatial or temporal differences in δ13C or δ15N for the two species, the grapsid crab, Paragrapsus gaimardii, and red cod, Pseudophycis bachus, which were collected at both sites and both sampling occasions. The stable isotope composition of each individual escapee was graphically compared to that of local species and artificial feed pellets so that differences in isotopic composition could be identified and related to differences in diet and feeding success.

Since δ15N has been found to increase with starvation (Hobson et al. 1993, Adams & Sterner 2000), lipid content of escapees was correlated with δ15N values. A positive relationship could mean that any changes in δ15N could be a result of starvation, and not of a change in diet.

The importance of local producers and artificial feed pellets was also determined for escaped salmonids and for native species. For native species, the concentration-weighed linear mixing model (Phillips & Koch 2002) was used, while considering algae, seagrass and artificial pellets as potential sources. Aquatic algae (phytoplankton, epiliths and brown and red filamentous algae) were grouped to minimize the number of sources and hence simplify the solution (Phillips et al. 2005). Fractionation values of 1‰ for δ13C and 3‰ for δ15N were used as these values have been found to be appropriate estimates when analysis is conducted on non-acid treated white muscle tissue (McCutchan et al. 2003). This model was only run for fish, shrimps and crabs, as δ15N trophic fractionation can be very variable for invertebrates of low trophic level, especially when analysed whole (Vander Zanden & Rasmussen 1999, McCutchan et al. 2003). Among invertebrates, grapsid crabs were considered to be of trophic level 2 and caridean shrimps 2.5. For fish, a trophic level of 2.5 was used for the phytodetritivorous mullet (Aldrichetta forsteri), 3 for the greenback flounder (Rhombosolea tapirina) and small fish prey (Galaxias sp., Leptatherina presbyteroides, Sardinops sagax, Kestrastherina brevirostris and small juveniles of TAFI Report Page 15

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Rhombosolea tapirina, Aldrichetta forsteri and A. trutta) and 3.5 for carnivorous species (Pseudophycis bachus, Macruronus novaezelandiae, Arripis trutta and Salmo trutta).

For escaped salmonids, the importance of the different food sources (small fish, invertebrates and feed pellets) was assessed by superimposing the adjusted isotopic values of consumers (corrected for fractionation), onto the isotopic values of potential food sources. The similarity between corrected isotope values of individual salmonids and the isotopic composition of potential food sources was considered as indicative of the importance of the respective source to the salmonid diet. A mixing model was not run for salmonid escapees because different fish and crustacean prey species often had different isotope composition.

3.6 Fatty acid analysis The fatty acid composition of artificial feed pellets, caged and escaped salmonids and native species was analysed by gas chromatography. Lipids were obtained as described in Section 2.4. Fatty acid analyses were conducted on aliquots of the total lipid extracts, which were treated with a solution of methanol:hydrochloric acid:chloroform (10:1:1 by vol) at 100°C for 2 h to produce fatty acid methyl esters (FAME). After cooling, FAME were separated by addition of 1 ml water, and extracted three times with a 1.8 ml of 4:1 v/v hexane:chloroform solution. FAME were analysed using a Agilent Technologies 7890A GC (Palo Al Alto, California, USA) equipped with an Equity™-1 fused silica capillary column (15 m x 0.1 mm i.d., 0.1 μm film thickness), a FID, a split/splitless injector and an Agilent Technologies 7683B Series auto sampler and injector. Helium was the carrier gas, and inlet pressure was maintained at 425 kPa. Samples were injected in splitless mode with an oven temperature of 120°C, and temperature was increased to 250°C at 10°C/min and then to 270°C at 3°C/min. Peaks were quantified with Agilent Technologies ChemStation software (Palo Alto, California, USA). Individual fatty acids were identified by mass spectral data and by comparing retention times with those obtained for authentic and laboratory standards. Results are expressed as percentage of the total fatty acids.

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Fate of escaped salmonids

Data analysis To explain the extent to which animal fatty acid composition depends on species (all species analysed, including native fish and crustaceans), site of collection (Table Head, Swan Basin and cages) and time of collection (November 2008 and April 2009), a classification and regression tree (CART) (De'ath & Fabricius 2000) was constructed using the TREES package on S-PLUS 2000ÂŽ (MathSoft, Cambridge, MA, USA). CART analysis is a non-parametric method especially useful in cases of large number of variables, and is therefore an appropriate method for analysis of fatty acid data (Smith et al. 1997). The model was based on percentage of total fatty acids, and the size of the tree was selected by cross validation, based on the 1-SE rule. A second CART was used to group the different species based on their fatty acid composition, i.e. while considering fatty acids as explanatory variables and species as the response variables. All identified fatty acids were considered in the CART analyses.

The fatty acid profiles of feed pellets, caged and escaped salmonids and native fish and crustaceans were also compared using principal component analysis (PCA) in PRIMER v.6 (Plymouth Marine Laboratory, UK). Only fatty acids that contributed a mean higher than 1% for any of the species were included. The proportion of saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) was also considered in this analysis. Data was firstly arcsine transformed, as appropriate for the analysis of percentage data that includes a wide range of values, including values close to 0 and 100% (Zar 1999). The fatty acids responsible for the separation of organisms were identified, and the diet of escapees was determined by comparing the principal component scores with those from caged salmonids and native fish. The fatty acid composition of Atlantic salmon analysed by CSIRO in 2002 (Mooney et al. 2002, Nichols et al. 2002) was also included in the PCA. Additionally, a PCA considering only Atlantic salmon and rainbow trout was also constructed to identify the fatty acids responsible for differences between caged and each particular escaped individual.

Changes in fatty acid composition with time spent in the wild For escaped Atlantic salmon and rainbow trout, changes in fatty acid composition with time spent in the wild were explored by regression analysis. Lipid content (% wet weight) was considered as a surrogate for time spent in the wild and each individual fatty acid was TAFI Report Page 17

Fate of escaped salmonids

considered individually in the regression. Moreover, since there were differences in MUFA and PUFA contents as well as the ω6:ω3 and EPA:DHA ratios between caged salmonids and native species, the relationships between lipid content and these sums and ratios were also analysed by regression analysis. This was performed for Atlantic salmon and rainbow trout separately. The relationship between the proportion of fatty acids generally present in feed based on vegetable oil (18:1ω9c and 18:2ω6) and the proportion of fatty acids characteristic of marine food webs (20:5ω3 and 22:6ω3), was also analysed for each species.

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Fate of escaped salmonids

4. Results In total, five escaped Atlantic salmon, 38 escaped rainbow trout and nine wild brown trout were collected. Muscle and stomach samples from eight additional Atlantic salmon, nine rainbow trout and three brown trout were obtained from recreational fishers during the sample period. Three escaped rainbow trout collected in November 2008 and one Atlantic salmon collected in April 2009 had well developed eggs.

4.1 Stomach contents analysis The stomach contents of 47 escaped rainbow trout, 13 escaped Atlantic salmon and 12 wild brown trout were analysed (Table 1). For rainbow trout, 63.2% of stomachs were empty or contained non-nutritious material such as filamentous algae, seagrass, myrtle leaves, twigs, pebbles or sand. The remaining 36.8% contained nutritious material such as artificial feed pellets (21%) or native animals (23.7%), including one individual that had consumed a ~10 cm fish. From individuals that fed on artificial feed pellets, half were collected from Swan Basin, ~10 km away from the farm lease. No nutritious material was identified in Atlantic salmon stomachs. Most (79%) were empty, and three contained high quantities of myrtle leaves (Table 1). Brown trout diet was composed mainly of fish (58%), crabs (17%), and one stomach contained high quantities of artificial feed pellets.

Table 1. Diet, in percentage occurrence, of the escaped rainbow trout, Atlantic salmon and of wild brown trout collected in Macquarie Harbour. Data was pooled for both sites and sampling periods.

Species

21.4

Leaves/ twigs 31.0

Pebbles/ sand 19.0

0.0

21.4

0.0

7.1

0.0

0.0

0.0

0.0

Empty

Pellets

Fish

Crust

Biv

Alg/Sg

O. mykiss (n = 47)

38.1

21.4

2.4

14.3

9.5

S. salar (n = 13)

78.6

0.0

0.0

0.0

0.0

S. trutta (n = 12)

16.7

8.3

58.3

16.7

0.0

Other 7.1

4.2 Body condition Biometric characteristics of caged and escaped salmonids are presented in Table 2. Due to logistic constraints, size and weight data was not recorded for the samples obtained from recreational fishers.

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Fate of escaped salmonids

Table 2. Biometric parameters of caged and escaped Atlantic salmon and rainbow trout and wild brown trout. For size and weight, values indicate range; for Fulton‟s K, HSI, VSI, %VF and muscle lipid content, mean ± SE is presented. For lipid content, numbers in brackets correspond to number of samples since lipid content was also measured in fish captured by recreational fishermen.

Total length (mm) Gutted weight (g) Fulton‟s K HSI VSI %VF Muscle lipid content (%)

Caged Atl. salmon (n = 4) 555-640 2130-2720 1.1 ± 0.1 1.0 ± 0.1 8.8 ± 0.4 0.1 ± 0.0 15.4 ± 1.3

Caged R. trout (n = 5) 510-565 2310-3510 1.6 ± 0.1 0.8 ± 0.1 14.6 ± 1.1 11.1 ± 0.8 21.2 ± 1.3

Escaped Atl. salmon (n = 5) 510-690 905-3000 0.9 ± 0.1 1.0 ± 0.3 4.5 ± 0.5 0.1 ± 0.0 5.0 ± 1.1 (13)

Escaped R. trout (n = 38) 335-570 580-2725 1.4 ± 0.0 1.4 ± 0.1 12.9 ± 0.6 5.8 ± 0.5 12.0 ± 1.2 (39)

Brown trout (n = 9) 350-560 470-1880 1.1 ± 0.0 0.8 ± 0.1 5.2 ± 0.4 0.2 ± 0.1 3.3 ± 0.8 (12)

There was an effect of living conditions (caged vs. escaped) (F1,48 = 5.640, p = 0.0218) and species (F1,48 = 27.383, p< 0.0000) on Fulton‟s K, but no interaction between the two factors (F1,48 = 0.0185, p = 0.8924). Escapees had lower K values than caged individuals, and rainbow trout had higher values than Atlantic salmon (Table 2, Fig. 2). Fulton‟s K of wild brown trout was lower than that of both caged and escaped rainbow trout; similar to that of caged Atlantic salmon, and within the range of Atlantic salmon escapees, although some escapees had lower values (Table 2, Fig. 2).

For the viscerosomatic index (VSI), there was also a significant effect of living conditions (F1,48 = 6.520, p = 0.0143) and species (F1,48 = 43.394, p< 0.0000), but no interaction between the two factors (F1,48 = 2.2385, p = 0.1419). As with Fulton‟s K, escapees had lower VSI values than caged animals, and rainbow trout higher values than Atlantic salmon (Table 2, Fig. 2). Brown trout had VSI similar to escaped Atlantic salmon and lower than in other salmonid groups (Fig. 2).

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Fate of escaped salmonids

2.5

5 n = 38 n=4

1.5

4

n = 38

n=5

n=9

n=5

1.0

HSI (%)

Fulton's K

2.0

3 2

n=5 n=5

0.5

1

0.0

0

25

30 n = 38

n = 39 n=4

25

20

n=9

n=4

n=4

% lipid

VSI (%)

20 15 n=5

10

n=9

n=5 n = 13

15 10

n = 12

n=5

5 0

5

Caged AS

Caged Escaped Escaped RT AS RT

BT

0

Caged AS

Caged Escaped Escaped RT AS RT

BT

Fig. 2. Box plots showing the median (line within the boxes), interquartile ranges (boxes), 10 th and 90th percentiles (whiskers) and outliers ( ) of Fultonâ&#x20AC;&#x;s K, HSI, VSI and % lipid (wet weight) in muscle of caged and escaped Atlantic salmon (AS) and rainbow trout (RT), and wild brown trout (BT). n = number of individuals included in the analyses.

Rainbow trout had much more visceral fat than Atlantic salmon or brown trout (Table 2) and, among rainbow trout, caged individuals had more visceral fat (10-14% body weight) than escapees (1-10%) (Table 2) (F1,41 = 15.824, p = 0.0003).

Regarding the hepatosomatic index (HSI), caged and escaped Atlantic salmon had similar values (F1,9 = 0.0058, p = 0.9413; Table 2, Fig. 2), although escapees had slightly higher variability than caged individuals (Table 2, Fig. 2). For rainbow trout, escapees had highly variable HSI (Table 2, Fig. 2), with livers ranging from 0.2% to 4.2% body weight, a value over five times higher than the mean found for caged individuals (0.8%). Although the ANOVA did not find significant differences between caged and escaped rainbow trout (F1,41 = 2.3256, p = 0.1353), it was possible to observe that escapees had slightly higher HSI (Table 2, Fig. 2), and five individuals (13%) had HSI much higher than caged individuals (>2). In general, HSI values of caged Atlantic salmon, caged rainbow trout and TAFI Report Page 21

Fate of escaped salmonids

escaped Atlantic salmon were similar, and also similar to values of wild brown trout (Fig. 2).

Escaped Atlantic salmon and rainbow trout had highly variable muscle lipid content (Fig. 2), most likely a result of different times spent in the wild. Lipid content was lower in wild brown trout (Fig. 2). For both species, caged individuals had much higher mean lipid content than escapees and than wild brown trout (Fig. 2). The difference between caged and escaped individuals was significant for Atlantic salmon (F1,16 = 10.894, p = 0.0045). For rainbow trout, the variability in lipid content was much higher in escapees, and included very high values, close to caged individuals, and very low values, close to the wild brown trout (Fig. 2). Given this high variability, no significant differences in fat content were detected for rainbow trout (F1,41 = 2.9900, p = 0.0913).

For rainbow trout, there was a significant relationship between muscle lipid content (% wet weight) and Fultons„ K (K = 1.24 + %lipid x 0.02, R2 = 0.252, F1,36 = 9.4509, p = 0.0047) and lipid content and %VF (K = 3.10 + %lipid x 0.31, R2 = 0.405, F1,36 = 18.4039, p = 0.0002) but not between lipid content and HSI (p = 0.1101). No significant relationships between muscle lipid content and other condition indexes occurred for Atlantic salmon.

4.3 Stable isotope analysis Stable isotope composition of artificial feed and farmed salmonids Atlantic salmon and rainbow trout feed pellets had similar δ13C and δ15N values (Table 3).

Caged Atlantic salmon was 1.0‰ more enriched in δ13C and 4.0‰ more enriched in δ15N than feed, and for caged rainbow trout the difference was 1.2‰ for δ13C and 3.0‰ for δ15N. Among escaped salmonids, δ13C and δ15N were similar for individuals collected at both sites and sampling seasons (Table 3). There were also no significant differences in δ13C or δ15N between farmed and escaped animals for either species (Atlantic salmon: F1,15 = 1.0988, p = 0.3111 for δ13C and F1,15 = 0.5680, p = 0.4627 for δ15N; rainbow trout: F1,42 = 0.2946, p = 0.5901 for δ13C and F1,42 = 1.7375, p = 0.1944 for δ15N), suggesting that animals continue feeding on pellets after escaping.

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Fate of escaped salmonids Table 3. Carbon and nitrogen isotopic composition (mean ± SE) of artificial feed pellets and caged and escaped salmonids in Macquarie Harbour. SB = Swan Basin; TH = Table Head.

Species/location Atlantic salmon pellets Rainbow trout pellets Atlantic salmon Farm Swan Basin Table Head Rainbow trout Farm Swan Basin Table Head

Season -

n 3 3

δ13C (‰) -19.4 ± 0.2 -20.3 ± 0.2

δ15N (‰) 7.4 ± 0.2 7.8 ± 0.2

November 08 April 09 November 08 April 09

4 4 7 1 1

-18.4 ± 0.1 -18.7 ± 0.0 -18.8 ± 0.3 -18.5 -18.8

11.4 ± 0.1 11.4 ± 0.2 12.0 ± 0.5 11.0 10.8

November 08 April 09 November 08

5 13 5 21

-19.1 ± 0.2 -19.0 ± 0.2 -19.5 ± 0.1 -18.8 ± 0.1

10.8 ± 0.2 10.8 ± 0.2 11.4 ± 0.4 11.1 ± 0.2

Test for a spatial or temporal variability in δ13C or δ15N in native animals δ13C and δ15N of the native grapsid crab, Paragrapsus gaimardii, and the red cod, Pseudophycis bachus, were compared with a two-way ANOVA to test for the presence of spatial or temporal variability in stable isotope signatures. There was no effect of site (F1,5 = 0.382, p = 0.5642 for δ13C; F1,5 = 0.229, p = 0.6526 for δ15N) or time of collection (F1,5 = 0.645, p = 0.4597 for δ13C, F1,5 = 0.365, p = 0.5720 for δ15N) on grapsid stable isotope composition. Similarly for the red cod, δ13C and δ15N signatures were similar between sites (F1,12 = 2.345, p = 0.1516 for δ13C; F1,5 = 0.200, p = 0.6624 for δ15N) and times of collection (F1,12 = 0.771, p = 0.3972 for δ13C; F1,12 = 3.879, p = 0.0724 for δ15N). Given the absence of temporal or spatial differences in δ13C or δ15N, results from animals caught during both field seasons were combined for each species in the analyses.

Stable isotope composition of local organisms The carbon stable isotopic composition of local primary producers varied from -27.0‰ for epilithic green microalgae to -12.5‰ for the seagrass Zostera muelleri (Fig. 3a).

Phytoplankton (δ13C = -26.5 to -26.1‰) and algae (red filamentous algae: -24.5 to -23.9‰; brown filamentous algae: -23.9 to -23.3‰) had relatively low δ13C, close to epilithic green algae (Fig. 3a). Regarding δ15N, local primary producers had close values, averaging 4.1 ± 0.5 (± SE) (Fig. 3a). Thus, δ15N of local producers was on average ~3‰ lower than rainbow trout and Atlantic salmon feed.

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Fate of escaped salmonids

Invertebrates ranged in δ13C from -23.7 to -20.1‰ and fish from -23.3 to 19.2‰ (Fig. 3a).

Potential competitors had very close δ13C and δ15N (ranges: δ13C = 1.5‰; δ15N = 0.7‰ (Fig. 3a), suggesting very similar diets. On the other hand, yellow-eye mullet (A. forsteri), a phytodetritivore, had stable isotopic signatures similar to caged Atlantic salmon and rainbow trout (Fig. 3a), suggesting that this species relies, at least to some extent, on food pellets that float away from the cages.

Detailed analysis of escaped salmonids δ13C of escaped Atlantic salmon varied between -23.0 and -18.2‰, although 12 out of the 13 individuals had δ13C higher than -18.8‰. Only one animal had a very low δ13C, with carbon and nitrogen isotopic composition similar to the blue grenadier and the most

13

C-

depleted brown trout (Fig. 3b), indicating feeding on similar sources. Escaped rainbow trout had a lower variability in δ13C, ranging from -19.8 to -17.9‰ (Fig. 3b).

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Fate of escaped salmonids

14

a) Escaped AS Caged AS

12

Escaped RT

10

δ15N (‰)

Caged RT RT Feed

8

AS Feed

6 Epiliths

4

Seagrass Phytopl Fil algae

2 0 -30 14

-28

-26

-24

-22

-20

-18

-16

-14

-12

b) BG AustS

12

RC

δ15N (‰)

Caged AS

10

Caged RT

RT Food AS Food

8

6 -24

-23

-22

-21

-20

-19

-18

-17

13

δ C (‰)

Fig. 3. Carbon and nitrogen stable isotope composition (mean ± SE) of organisms collected in Macquarie Harbour. a) All native organisms analysed, including primary producers, artificial feed pellets, caged and escaped Atlantic salmon (AS) and rainbow trout (RT), and native invertebrates (white symbols) and fish (grey symbols). Producers: Epil ma = epilithic microalgae; Fil algae = red and brown filamentous algae; Phytopl = phytoplankton; Seagrass is Zostera muelleri. Invertebrates: - blue mussel Mytilus edulis; - isopods and amphipods; - decapod crustaceans. Fish: potential prey; - benthivores and detritivores; - carnivores (potential competitors), - wild brown trout. Data for November 2008 and April 2009, and for Swan Basin and Table Head is pooled. b) Isotopic results of each individual escaped Atlantic salmon ( ), rainbow trout ( ), and wild brown trout ( ) in relation to artificial feed pellets, caged individuals, and the three native carnivores: juvenile blue grenadier (BG), Australian salmon (AustS) and red cod (RC). Note the differences in scale in the x and y axes.

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Fate of escaped salmonids

δ15N varied between 10.5 and 13.2‰ for escaped Atlantic salmon, and between 9.8 and 12.7‰ for rainbow trout. There was no relationship between muscle lipid content (i.e. fish condition, a proxy for amount of time since escapement) and δ15N values of Atlantic salmon or rainbow trout (p >0.05; Fig. 4), suggesting that the differences in δ15N between individuals (Fig. 3b) were not the result of starvation. For both species, the variability in δ15N was, however, higher for individuals of poorer condition (Fig. 4). For example, for animals with muscle lipid content lower than 5%, δ15N varied between 10.5‰ and 13.7‰ for Atlantic salmon and between 9.2‰ and 12.5‰ for rainbow trout (Fig. 4), in both cases differences greater than 3‰, the mean value of δ15N trophic fractionation.

14

δ15N (‰) (‰)

13

12

11

10

9 0

5

10

15

20

25

30

35

% lipid

Fig 4. Plot of lipid content (% wet weight) and δ15N values for escaped Atlantic salmon ( ) and rainbow trout ( ). No relationship was observed.

Importance of local producers and artificial feed pellets to consumers Corrected isotopic values of most native species fell close to algae, indicating a dependence on these sources (Fig. 5a). However, the phytodetritivore mullet (A. forsteri) fell closer to artificial feed, suggesting that this species also relies on nutrition from pellets (Fig. 5a). The concentration-dependent mixing model indicates that feed pellets contribute ~62% and algae ~38% to the mullet, while seagrass was not an important contributor. Feed pellets could also be of minor importance for species such as pilchards, hardyheads and caridean TAFI Report Page 26

Fate of escaped salmonids

shrimps, which had high corrected δ15N. However, since the mixing model was calculated using the average isotopic values of the different types of algae, and given the high δ15N of epilithic microalgae (5.7‰), it is possible that feed pellets were not at all important for these species. Seagrass was not an important contributor to animals in the area.

For escaped salmonids, the importance of food pellets and local prey (small fish and crustaceans) was estimated based on the similarity between corrected isotopic values of salmonids and the original signatures of food sources. Most Atlantic salmon and rainbow trout escapees fell close to artificial feed pellets (Fig. 5b). One Atlantic salmon, however, had lower corrected δ13C, closer to small fish like the hardyheads, silverfish and pilchard, although corrected δ15N was lower than these species. For wild brown trout, different individuals were well separated in corrected δ13C, ranging from -24.3 to -19.4‰ (Fig. 5b), indicating that some feed on native species, some on artificial feed pellets, and some on a mixture of the two sources.

4.4 Fatty acid analysis A total of 57 individual fatty acids were identified. Feed pellets were very rich in 18:1ω9c (oleic acid; 26-31%) and 16:0 (palmitic acid; 20-21%) (Table 4). Caged and escaped Atlantic salmon and rainbow trout were also very rich in 18:1ω9c (up to 35%), which occurred in much higher proportions than in native species, and also contained more 16:1ω7c (palmitoleic acid) and 18:2ω6 (linoleic acid) than native species (Table 4). In contrast, native fish were much richer in 22:6ω3 (docosahexaenoic acid, DHA) and were also slightly more enriched in 20:4ω6 (arachidonic acid) and 20:5ω3 (eicosapentaenoic acid, EPA) (Table 4, Fig. 6). High variability in 18:1ω9c and 22:6ω3 composition was also observed in native species (Fig. 6). This was due to the influence of greenback flounder, for which there was a high intraspecific variability in percentage composition of these fatty acids (see Table 4) inferring that individuals relied differentially on different food sources.

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Fate of escaped salmonids

8

a) Native species YEM

7

δ15N (‰)

Salmonid feed

6 Pil HH

Car

5

100%

4

Seagrass

25%

50%

75%

Algae

3 -28

13

-26

-22

-20

-18

-16

-14

-12

b) Salmonids HH

12

SF

YEM Pil Gb

11

δ15N (‰)

-24

AuS

GFl

10

Carid

Jollyt

Mcr

9 Crb

8 Salmonid feed

7 6 -25

-24

-23

-22

-21

-20

-19

-18

δ13C (‰)

Fig. 5 Corrected δ13C and δ15N values of consumers (grey symbols) and original values of potential sources (black symbols). a) Native fish and decapod crustaceans with algae (phytoplankton, red and brown filamentous algae and epiliths), seagrass and artificial feed, showing the mixing triangle for the concentration-weighed model. Isolines represent the relative contribution of the three sources to consumer diets, in 25% steps. b) Escaped Atlantic salmon ( ), escaped rainbow trout ( ) and wild brown trout ( ) with artificial feed and small fish and crustacean prey. AuS = Australian salmon; BG = blue grenadier; Car = caridean shrimps; Crb = grapsid crabs; Gb = unidentified Goby; GFl = greenback flounder; HH = short-headed hardyhead; Jollyt = jollytail; Mcr = Macrobrachium sp.; Pil = pilchard; SF = silverfish; YEM = yellow-eye mullet.

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Fate of escaped salmonids

Table 4. Percentage fatty acid composition (mean ± SE) and lipid content of feed pellets, caged and escaped Atlantic salmon and rainbow trout, wild brown trout and native fish and crustaceans collected in Macquarie Harbour. Crustacean prey, and includes grapsid crabs (n = 2, each composed by 5 individuals) and caridean shrimps (n = 1, composed by 10 individuals). Fish prey includes galaxids (n = 1), hardyheads (n = 1), and juveniles of greenback flounder (n = 2), yellow-eyed mullet (n = 1) and Australian salmon (n = 1), with each replicate composed of 3-5 individuals.

Atl. salm. R. trout Caged Caged Escaped Escaped Brown feed feed Atl. salm. R. trout Atl. salm. R. trout trout Fatty acid (n = 3) (n = 3) (n = 4) (n = 5) (n = 13) (n = 39) (n = 12) 14:0 15:0 16:0 18:0 22:0 Σ SFA

2.0 ± 0.5 0.3 ± 0.0 21.4 ± 0.1 5.6 ± 0.2 0.1 ± 0.0 29.8 ± 0.5

2.0 ± 0.2 0.3 ± 0.0 19.7 ± 0.1 5.4 ± 0.2 0.2 ± 0.0 28.1 ± 0.1

2.3 ± 0.2 0.1 ± 0.1 17.7 ± 0.4 4.9 ± 0.2 0.0 ± 0.0 25.9 ± 0.3

1.6 ± 0.1 0.2 ± 0.0 16.4 ± 0.6 4.5 ± 0.1 0.1 ± 0.0 23.3 ± 0.1

1.7 ± 0.2 0.2 ± 0.0 17.9 ± 0.8 5.8 ± 0.4 0.1 ± 0.0 26.1 ± 0.9

1.6 ± 0.1 0.2 ± 0.0 18.4 ± 0.4 5.3 ± 0.1 0.0 ± 0.0 26.1 ± 0.4

1.5 ± 0.2 0.4 ± 0.0 21.8 ± 0.7 5.3 ± 0.3 0.1 ± 0.0 29.7 ± 0.8

16:1ω7c 17:1ω8c+a17:0 18:1ω9c 18:1ω7c 20:1ω9+11c 20:1ω7c 24:1ω9c Σ MUFA

7.1 ± 0.1 0.3 ± 0.0 30.1 ± 0.4 3.3 ± 0.0 1.5 ± 0.1 0.2 ± 0.0 0.3 ± 0.0 42.8 ± 0.4

7.5 ± 0.2 0.3 ± 0.0 27.1 ± 0.6 3.3 ± 0.0 0.9 ± 0.1 0.2 ± 0.0 0.2 ± 0.0 39.6 ± 0.6

7.5 ± 0.3 0.2 ± 0.1 26.0 ± 0.6 4.0 ± 0.0 1.5 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 41.0 ± 1.0

8.3 ± 0.3 0.3 ± 0.0 33.6 ± 0.8 3.7 ± 0.1 1.7 ± 0.1 0.2 ± 0.0 0.1 ± 0.0 49.1 ± 0.5

6.5 ± 0.5 0.3 ± 0.1 27.1 ± 0.9 3.6 ± 0.3 1.8 ± 0.1 0.3 ± 0.0 0.5 ± 0.1 40.1 ± 1.4

8.0 ± 0.2 0.3 ± 0.0 30.9 ± 0.5 3.9 ± 0.1 1.6 ± 0.02 0.2 ± 0.0 0.2 ± 0.0 45.8 ± 0.5

4.6 ± 0.4 0.4 ± 0.1 20.8 ± 1.8 2.9 ± 1.2 1.1 ± 0.2 0.2 ± 0.1 1.1 ± 0.0 31.1 ± 2.1

18:4ω3 18:2ω6 18:3ω3 20:4ω6 (ARA) 20:5ω3 (EPA) 20:3ω6 20:4ω3 20:2ω6 21:5ω3 22:5ω6 22:6ω3 (DHA) 22:4ω6 22:5ω3 Σ PUFA

0.8 ± 0.0 9.0 ± 0.1 0.8 ± 0.0 0.6 ± 0.0 6.4 ± 0.3 0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 3.7 ± 0.2 0.0 ± 0.0 1.0 ± 0.1 23.1 ± 0.7

1.2 ± 0.1 9.2 ± 0.0 1.0 ± 0.0 0.8 ± 0.0 9.1 ± 0.3 0.2 ± 0.0 0.3 ± 0.1 0.2 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 3.9 ± 0.1 0.0 ± 0.0 1.1 ± 0.0 27.6 ± 0.6

1.3 ± 0.1 7.6 ± 0.0 0.7 ± 0.0 0.8 ± 0.0 7.6 ± 0.3 0.4 ± 0.1 0.2 ± 0.0 0.3 ± 0.1 0.6 ± 0.1 0.1 ± 0.0 7.7 ± 0.8 0.1 ± 0.0 3.2 ± 0.0 32.4 ± 0.9

0.9 ± 0.0 8.7 ± 0.6 0.7 ± 0.1 0.6 ± 0.0 4.3 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 0.3 ± 0.0 0.2 ± 0.0 6.8 ± 0.1 0.1 ± 0.0 2.0 ± 0.1 32.4 ± 0.9

1.0 ± 0.1 7.2 ± 0.5 0.7 ± 0.1 1.2 ± 0.3 6.8 ± 0.5 0.3 ± 0.1 0.4 ± 0.1 0.5 ± 0.1 0.3 ± 0.0 0.2 ± 0.0 8.8 ± 1.2 0.1 ± 0.0 2.9 ± 0.2 30.5 ± 0.3

0.9 ± 0.0 7.9 ± 0.1 0.6 ± 0.0 0.7 ± 0.0 5.1 ± 0.1 0.3 ± 0.0 0.4 ± 0.0 0.4 ± 0.0 0.3 ± 0.0 0.1 ± 0.0 7.6 ± 0.2 0.1 ± 0.0 1.8 ± 0.1 27.2 ± 0.4

0.8 ± ±0.1 2.5 ± 0.4 1.1 ± 0.2 1.4 ± 0.3 5.2 ± 0.4 0.1 ± 0.0 1.0 ± 0.2 0.3 ± 0.0 0.2 ± 0.0 0.3 ± 0.0 21.7 ± 2.4 0.3 ± 0.1 2.4 ± 0.2 37.3 ± 2.5

Others 11.4 ± 0.2 12.0 ± 0.1 11.2 ± 0.6 11.2 ± 0.6 10.5 ± 0.6 10.6 ± 0.3 7.5 ± 0.6 ω6/ω3 0.8 ± 0.2 0.6 ± 0.0 0.4 ± 0.0 0.4 ± 0.2 0.5 ± 0.0 0.6 ± 0.0 0.2 ± 0.0 EPA/DHA 1.7 ± 0.0 2.3 ± 0.0 0.9 ± 0.1 1.0 ± 0.0 0.9 ± 0.1 0.7 ± 0.0 0.3 ± 0.0 Lipid content 27.9 ± 0.4 25.4 ± 0.5 15.4 ± 1.3 21.2 ± 1.3 5.0 ± 1.1 12.0 ± 1.2 3.3 ± 0.8 (% wet weight) SFA = saturated fatty acids, MUFA = monounsaturated fatty acids, PUFA = polyunsaturated fatty acids. *Others: 14:1ω5c, i15:0, 15:1ω6c, C16PUFAs, i16:0, 16:1ω9c, 16:1ω7t, 16:1ω5c, Br17:1, 17:1, 18:3ω6, i18:0, 18:1ω7t, 18:1ω5c, 18:1, 19:1s, 20:1ω5c, 20:0, 22:1ω11c, 22:1ω9c, 22:1ω7c, 24:0, 24:1ω9c, 24:1ω11c, 24:5ω3, 24:6ω3, MBrFA:1, MBrFA.

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Fate of escaped salmonids

Table 4 (cont). Percentage fatty acid composition (mean ± SE) and lipid content of feed pellets, caged and escaped Atlantic salmon and rainbow trout, wild brown trout and native fish and crustaceans collected in Macquarie Harbour. Crustacean prey, and includes grapsid crabs (n = 2, each composed by 5 individuals) and caridean shrimps (n = 1, composed by 10 individuals). Fish prey includes galaxids (n = 1), hardyheads (n = 1), and juveniles of greenback flounder (n = 2), yellow-eyed mullet (n = 1) and Australian salmon (n = 1), with each replicate composed of 3-5 individuals.

Red cod Blue Austr.

Y-eyed Greenb Fish prey Crust. (n = 7) grenadier salmon mullet flounder (n = 7) prey Fatty acid (n = 1) (n = 3) (n = 7) (n = 4) (n = 3) 14:0 15:0 16:0 18:0 22:0 Σ SFA

0.0 ± 0.0 0.1 ± 0.1 20.2 ± 0.7 7.6 ± 0.4 0.0 ± 0.0 28.6 ± 0.7

0.0 0.0 18.6 7.3 3.3 30.0

0.3 ± 0.2 0.2 ± 0.1 20.8 ± 0.5 8.5 ± 0.4 0.0 ± 0.0 31.4 ± 0.8

1.0 ± 0.2 0.2 ± 0.1 21.1 ± 0.5 6.5 ± 0.7 0.0 ± 0.0 27.0 ± 0.4

0.9 ± 0.5 0.2 ± 0.1 16.4 ± 0.8 8.1 ± 1.4 0.0 ± 0.0 26.4 ± 0.5

0.2 ± 0.1 0.1 ± 0.0 17.3 ± 0.8 6.7 ± 0.5 0.1 ± 0.1 25.8 ± 1.0

0.0 ± 0.0 0.0 ± 0.0 11.0 ± 3.7 8.2 ± 0.2 0.6 ± 0.3 21.5 ± 2.5

16:1ω7c 17:1ω8c+a17:0 18:1ω9c 18:1ω7c 20:1ω9+11c 20:1ω7c 24:1ω9c Σ MUFA

2.1 ± 0.2 0.2 ± 0.1 11.7 ± 0.6 3.1 ± 0.2 0.2 ± 0.1 0.0 ± 0.0 1.2 ± 0.2 19.2 ± 0.5

1.8 0.0 9.0 3.1 0.0 0.0 1.8 17.4

2.5 ± 0.7 0.3 ± 0.2 15.9 ± 3.3 3.2 ± 0.4 0.5 ± 0.3 0.0 ± 0.0 1.5 ± 0.3 25.0 ± 3.8

6.7 ± 1.0 0.5 ± 0.1 13.3 ± 0.5 4.6 ± 0.3 0.6 ± 0.2 0.9 ± 0.2 0.6 ± 0.1 28.7 ± 4.5

5.3 ± 0.9 0.6 ± 0.3 21.5 ± 3.5 6.3 ± 0.2 1.2 ± 0.4 0.3 ± 0.2 0.4 ± 0.2 36.6 ± 4.5

2.9 ± 0.9 0.3 ± 0.2 11.0 ± 0.4 3.4 ± 0.4 1.1 ± 0.3 0.5 ± 0.2 1.3 ± 0.2 21.5 ± 1.2

2.2 ± 0.6 1.0 ± 0.5 12.2 ± 1.8 4.9 ± 0.1 0.0 ± 0.0 0.3 ± 0.2 0.0 ± 0.0 21.0 ± 2.1

18:4ω3 18:2ω6 18:3ω3 20:4ω6 (ARA) 20:5ω3 (EPA) 20:3ω6 20:4ω3 20:2ω6 21:5ω3 22:5ω6 22:6ω3 (DHA) 22:4ω6 22:5ω3 Σ PUFA

0.1 ± 0.1 2.2 ± 0.4 0.2 ± 0.1 5.7 ± 0.4 9.6 ± 0.6 0.0 ± 0.0 0.1 ± 0.1 0.1 ± 0.1 0.0 ± 0.0 0.2 ± 0.1 30.5 ± 1.1 0.6 ± 0.2 2.9 ± 0.3 52.2 ± 0.9

1.1 1.5 0.9 2.2 6.1 0.0 0.0 0.0 0.0 0.8 36.0 0.0 1.1 51.1

0.1 ± 0.1 3.2 ± 1.1 0.1 ± 0.1 2.6 ± 0.5 7.7 ± 0.8 0.0 ± 0.0 0.3 ± 0.1 0.2 ± 0.1 0.1 ± 0.1 0.7 ± 0.1 26.0 ± 5.5 0.3 ± 0.2 3.0 ± 0.8 44.1 ± 4.5

1.1 ± 0.2 1.9 ± 0.3 0.9 ± 0.3 4.4 ± 0.5 12.8 ± 0.8 0.2 ± 0.1 0.4 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 0.3 ± 0.1 13.3 ± 0.8 0.3 ± 0.1 4.1 ± 0.3 41.0 ± 1.4

0.9 ± 0.4 3.2 ± 0.5 0.6 ± 0.4 5.8 ± 1.1 9.5 ± 1.3 0.0 ± 0.0 0.4 ± 0.2 0.1 ± 0.1 0.0 ± 0.0 0.4 ± 0.2 9.9 ± 1.9 0.7 ± 0.3 4.8 ± 0.9 36.6 ± 4.7

0.9 ± 0.3 2.4 ± 0.6 0.5 ± 0.2 3.1 ± 0.8 11.8 ± 1.9 0.0 ± 0.0 0.8 ± 0.3 0.3 ± 0.1 0.2 ± 0.1 0.5 ± 0.1 28.3 ± 3.6 0.2 ± 0.1 3.4 ± 0.9 52.3 ± 1.6

0.2 ± 0.1 3.2 ± 0.5 0.4 ± 0.4 6.5 ± 0.1 27.8 ±1.2 0.1 ± 0.1 0.2 ± 0.1 0.5 ± 0.5 0.0 ± 0.0 0.6 ± 0.4 14.5 ± 0.7 0.0 ± 0.0 2.1 ± 1.2 55.5 ± 1.5

Others ω6/ω3 EPA/DHA Lipid content (% wet weight)

4.1 ± 0.3 0.2 ± 0.0 0.3 ± 0.0 0.5 ± 0.0

4.3 0.1 0.2 0.7

4.7 ± 0.7 0.2 ± 0.0 0.3 ± 0.1 1.0 ± 0.1

10.4 ± 1.2 0.2 ± 0.0 1.0 ± 0.1 1.1 ± 0.1

7.2 ± 1.5 0.4 ± 0.1 1.0 ± 0.1 1.3 ± 0.2

5.8 ± 1.1 0.2 ± 0.0 0.5 ± 0.1 1.3 ± 0.2

4.2 ± 0.5 0.2 ± 0.0 1.9 ± 0.1 0.7 ± 0.1

SFA = saturated fatty acids, MUFA = monounsaturated fatty acids, PUFA = polyunsaturated fatty acids. *Others: 14:1ω5c, i15:0, 15:1ω6c, C16PUFAs, i16:0, 16:1ω9c, 16:1ω7t, 16:1ω5c, Br17:1, 17:1, 18:3ω6, i18:0, 18:1ω7t, 18:1ω5c, 18:1, 19:1s, 20:1ω5c, 20:0, 22:1ω11c, 22:1ω9c, 22:1ω7c, 24:0, 24:1ω9c, 24:1ω11c, 24:5ω3, 24:6ω3, MBrFA:1, MBrFA.

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Fate of escaped salmonids

For native species, the major fatty acids were 22:6ω3, accounting for up to 36‰ for the blue grenadier, followed by 16:0, 18:1ω9c and 20:5ω3 (Table 4), and together constituted over 55% of the total fatty acids in all species. Artificial feed pellets and caged and escaped Atlantic salmon and rainbow trout also had much higher ω6/ω3 and EPA/DHA ratios than native animals (Table 4). Given the large differences in 18:1ω9c and 22:6ω3 content and ω6/ω3 and EPA/DHA ratios between caged salmonids and native animal species (Table 4), these parameters can be used as biomarkers to identify the source of nutrition for Atlantic salmon and rainbow trout escapees.

40

Oleic acid (%)

30

20

10

0 50

DHA (%)

40 30 20 10 0 CAS

CRT

EAS

ERT

Nat

Fig. 6. Box plots showing the median (line within the boxes), interquartile ranges (boxes), 10th and 90th percentiles (whiskers) and outliers ( ) of the percentage of oleic acid (18:1ω9c) and DHA (22:6ω3) in lipid of caged and escaped Atlantic salmon (CAS and EAS), rainbow trout (CRT and ERT) and native fish species (Nat; based on the average for each species).

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Fate of escaped salmonids

Caged and escaped Atlantic salmon and rainbow trout were also richer in monounsaturated fatty acids (mean = 40 to 49%) than brown trout and native fish, and had lower proportions of polyunsaturated fatty acids (PUFA) than these species (Table 4, Fig. 7). The proportion of saturated fatty acids was similar for all groups (Table 4, Fig. 7).

60 50

%

40 30 20 10 0 Caged AS

Caged RT

Escaped AS

Escaped RT

Brown trout

Native fish

Fig. 7. Percentage composition (mean Âą SE) of saturated ( ), monounsaturated ( ) and polyunsaturated ( ) fatty acids in caged and escaped Atlantic salmon (AS), rainbow trout (RT), brown trout and native fish from Macquarie Harbour.

Classification and Regression Tree Analysis Based on the overall fatty acid composition, a CART with species and site (Swan Basin, Cages and Table Head) and time (November 2008, April 2009) of collection considered as explanatory variables resulted in a model explaining 64% of variability (Fig. 8). This model indicates that fatty acid composition depends mainly on species, with all native species and wild brown trout separated from both caged and escaped Atlantic salmon and rainbow trout. Hence, the fatty acid composition of escaped Atlantic salmon and rainbow trout is similar to that of caged animals, indicating that, in general, escaped fish do not switch to feed on native fauna, and continue feeding on artificial pellets that float from the aquaculture cages, or do not feed and starve. A second split, also based on species and explaining much less of the variability, separated benthic species such as crustaceans TAFI Report Page 32

Fate of escaped salmonids

(grapsid crabs and caridean shrimps), mullets and flounders, from carnivorous species (Fig. 8). No effect of site or time of collection was detected.

Species: Native species, BT Caged and escaped AS and RT

Species: small fish and carnivores Crustaceans, mullets and flounders (n = 61)

(n = 26)

(n = 18)

R2 = 64%

Fig. 8. Three-leaf classification and regression tree explaining animal fatty acid composition based on species, site and time of collection. Numbers in brackets correspond to number of samples. AS = Atlantic salmon; BT = brown trout; RT = rainbow trout.

A second CART model, built to group species according to their fatty acid composition, explained 82% of the variability and separated animals in five groups. In this CART, The first split, explaining most of the variability, separated animals into two main groups based on the percentage of 18:1ω9c (Fig. 9). This separated native species (18:1ω9c <22.8%) from all caged and most escaped Atlantic salmon and rainbow trout, as well as seven brown trout (18:1ω9c >22.8%), again indicating that escaped animals are not feeding on native sources. Among the salmonid group, a second split separated wild brown trout based on the 22:6ω3 (DHA) content, as wild brown trout had more DHA (>11.6%) than Atlantic salmon and rainbow trout (Fig. 9). Among native species, animals were also further divided based on the 22:6ω3 composition. In carnivorous fish and small fish prey, this fatty acid constituted more than 23.2% of the total fatty acids, while in benthic feeders (yellow-eye mullet, greenback flounder, grapsid crabs and caridean shrimps) this fatty acid was less TAFI Report Page 33

Fate of escaped salmonids

abundant (Fig. 9). A fourth subdivision separated benthic fish from benthic crustaceans, based on 20:5ω3.

18:1ω9c>22.8%

22:6ω3<11.6%

Caged and escaped Atl salmon and Rainbow trout (n = 61)

22:6ω3>23.2%

Brown trout (n = 9)

20:5ω3<21.4% Carnivorous fish and small fish prey (n = 18)

Flounders and mullets (n = 14)

Crustaceans (n = 3)

R2 = 82%

Fig. 9. Multivariate classification and regression tree showing the five homogeneous groups defined based on differences in fatty acid composition. The fatty acid responsible for each split is indicated at each node, including the value that separates the two branches.

Overall, 83.8% of animals were correctly classified into their groups: farmed salmonids (caged and escaped Atlantic salmon and rainbow trout; 95.1% correct classifications), brown trout (71%), carnivorous fish and small fish prey (i.e. all native fish species with the exception of benthic flounders and mullets; 77.8%), benthic fish species (flounders and mullets; 66.7%) and crustaceans (100%). The remaining 16.2% of individuals that were not correctly classified into their groups included one brown trout and two greenback flounder that were placed in the farmed salmonid group; one Atlantic salmon and one Australian salmon that were placed in the brown trout group; four brown trout placed in the group of TAFI Report Page 34

Fate of escaped salmonids

carnivorous fish and small fish prey; and one escaped rainbow trout, one escaped Atlantic salmon and one brown trout that were placed in the benthic fish group. Brown trout were well spread between the different leaves of the CART, suggesting that different individuals specialize in feeding on different sources.

Principal Component Analysis The different animal groups appeared well separated in the PCA (Fig. 10). The first component of the PCA, explaining 73.2% of variability, was positively correlated with 18:1ω9c (oleic acid) and sum of MUFA and negatively correlated with 22:6ω3 (DHA) and sum of PUFA. The second component, explaining 9.6% of the variability, was positively correlated with 20:5ω3 (EPA) and negatively correlated with 22:6ω3.

Artificial feed pellets, caged salmonids and most escaped Atlantic salmon and rainbow trout formed a very tight group in the PCA, and were well separated from native species (Fig. 10). This separation was explained by the higher content of oleic acid and MUFA, and lower percentages of DHA and PUFA in relation to native species. Among native species, and in agreement with the CART analysis (Fig. 9), EPA and DHA and AA separated carnivorous fish and small fish prey from benthic species such as flounders, mullets and decapod crustaceans (Fig. 10). The first group had lower PC2 scores, indicating higher percentages of DHA, while benthic species were richer in EPA and 20:4ω6 (arachidonic acid) (Fig. 10). In comparison, Atlantic salmon fed pellets containing higher levels of fish oil and fish meal and analysed by CSIRO in 2002 (Mooney et al. 2002, Nichols et al. 2002) fell between artificial pellets and native carnivorous fish (Fig. 10).

As with stable isotopes, the fatty acid composition of brown trout also differed greatly between individual fish. Brown trout were well scattered along PC1, with one falling close to farmed salmonids, others falling close to carnivorous species, and most in between. This indicates that different individuals used different food sources, with some feeding mainly on feed pellets that escape from the aquaculture cages, others feed primarily on native species, while others feed on both native fauna and feed pellets. Note that the individual that fell in the caged salmonid group was also placed in that group by the CART analysis (Fig. 10), and was one of the two fish collected at Table Head, close to the fish farms.

Benthic species, namely the yellow-eye mullet and greenback flounder, were also well separated in PC1 (Fig. 10), meaning that different individuals, even within the same species TAFI Report Page 35

Fate of escaped salmonids

relied on different food sources. Two greenback flounder caught at Table Head had fatty acid composition similar to farmed salmonids (Fig. 10), indicating that they relied significantly on artificial feed pellets for nutrition. However, apart from these two greenback flounder, there was no pattern in the variation of fatty acid signatures between Swan Basin and Table Head for native species (data not shown), i.e. animals collected closer to fish farms were not more similar to caged fish than those collected further away from the farms.

Two escaped Atlantic salmon (15% of total; a and b in Figs. 10 and 11) and one escaped rainbow trout (c in Figs. 10 and 11) were separated from the salmonid group. This could either mean that these individuals had switched to feeding on native fauna or, alternatively, that the changes in fatty acid composition were a result of differential metabolism of fatty acids as the animals wasted away. For Atlantic salmon, the two separated individuals were those that had the lowest fat content (3.2% for individual a and 0.8% for individual b), suggesting that differences in fatty acid composition were linked to decreased fish condition. However, a 3.2% lipid content, although much lower than caged and most escaped Atlantic salmon, was within the range found for the wild brown trout (see Fig. 2).

Individual a was separated because it was richer in 16:0 and saturated fatty acids, and also slightly richer in the ω3 fatty acids 20:5ω3 and 22:6ω3 (EPA and DHA) (Fig. 11). It was also the fish most separated in δ13C from all other farmed salmonids, with δ13C values much lower than other farmed salmonids, and close to blue grenadier and most wild brown trout (see Fig. 3); this individual placed in the brown trout group in the CART model (Fig. 9). These data indicates that individual a had fed successfully on sources other than artificial feed pellets.

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Fate of escaped salmonids

0.4

0.3

PC2 (9.6%)

0.2

0.1

0.0

b -0.1

c -0.2

a

-0.3 -0.4

-0.2

0.0

0.2

0.4

0.6

20:5ω3

20:4ω6

PC2 (9.6%)

Sum PUFA Sum MUFA 0.7

18:1ω9c

16:0

22:6ω3

PC1 (73.2%)

Fig. 10. Scores (top) and factor loadings (bottom) of principal component analysis (PCA) showing the first two components based on fatty acid data of feed pellets and animals. Arrows indicate the fatty acids that contribute most to the respective PC, along with the strength and direction of influence.

- Atlantic salmon feed pellets; - caged Atlantic salmon; - escaped Atlantic salmon; - rainbow trout feed pellets; - caged rainbow trout; - escaped rainbow trout; caged Atlantic salmon analysed in 2002 (see text); - brown trout; - grapsid crabs and caridean shrimps; - detritivorous and benthivorous fish (greenback flounder, yellow-eye mullet); carnivorous fish (red cod, blue grenadier, Australian salmon); - potential fish prey (hardyhead, Galaxias sp., small juveniles of yellow-eye mullet, flounder and Australian salmon). a, b - two Atlantic salmon separated from the Atlantic salmon group (3.2% and 0.8 and lipid content respectively); c – rainbow trout separated from the rainbow trout group (17.1% lipid content) (see text for details).

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Fate of escaped salmonids

0.6

0.4

18:1ω7c

22:5ω3 Sum PUFA

PC2 (23.3%)

0.2

Sum MUFA

0.0

22:6ω3 20:5ω3

18:1ω9c

b -0.2

a c 16:0 Sum SFA

-0.4

-0.6 -0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

PC1 (29.6%)

Fig. 11. Scores and factor loadings of the first two components of PCA based on fatty acid data of artificial feed pellets and caged and escaped Atlantic salmon and rainbow trout. - Atlantic salmon feed pellets; - caged Atlantic salmon; - escaped Atlantic salmon; - rainbow trout feed pellets; - caged rainbow trout; - escaped rainbow trout. a, b - two Atlantic salmon that separated from the Atlantic salmon group (3.2% and 0.8 and lipid content respectively); c – rainbow trout separated from the rainbow trout group (17.1% lipid content) (see text for details).

The individual in poorest condition (lipid content = 0.8%, Fulton‟s K = 0.6, HSI = 0.8; individual b in Figs. 10 and 11), was separated from other farmed salmonids due to the higher percentage of EPA and DHA (Fig. 11). This individual had a DHA level of 16%, about two times higher than other Atlantic salmon. It also had the lowest δ15N (see Fig. 5), but was similar in δ13C to caged and most escaped salmonids (-18.4‰).

Unlike with the separated Atlantic salmon, the separated rainbow trout (c in Figs. 10 and 11), was in very good condition, with a fat content of 17.1% (Fulton‟s K = 1.7, HSI = 1.1, VSI = 11.2, %VF = 5.3) suggesting that this individual escaped a significant time before capture (evident from the difference in fatty acid composition) and also that it had been consuming native food (evident from the separated fatty acid composition). However, this individual had also fed significantly on feed pellets, as its fatty acid composition was still closer to farmed salmonids than to native fish (Fig. 10), and its stable isotope composition TAFI Report Page 38

Fate of escaped salmonids

was similar to caged fish (δ13C = -19.1‰; δ15N = 10.3‰). Sum of SFA and 16:0 were responsible for the separation of this individual from the rest of the group (Fig. 11).

To test for changes in fatty acid composition with time spent in the wild, the relationships between lipid content and each individual fatty acid were analysed using regression analysis. For Atlantic salmon, there was a significant positive relationship between lipid content and 14:0 and 16:1ω7c (Table 5), meaning that the proportion of these fatty acids decreased with time spent in the wild. There was also a negative relationship between 18:0, 18:4ω6, 20:4ω3 and 22:6ω3, meaning that the relative levels of these fatty acids increased as lipid content decreased, i.e. with time spent in the wild. For rainbow trout, the relationships were only significant for three fatty acids: 16:1ω7 (positive relationship), 20:4ω6 and 22:6ω3 (negative relationships), and a positive relationship between lipid content and the EPA/DHA ratio was also observed (Table 5). There were no significant relationships between lipid content and any other fatty acids, or between lipid content and sum of SFA, sum of MUFA, or ω6 and ω3 PUFA. For Atlantic salmon, there was also a significant negative relationship between the sum of 18:1ω9c and 18:2ω6, the fatty acids present at high relative levels in feed based on vegetable oils (and/or animals feeding on vegetable oils), and 20:5ω3 and 22:6ω3, fatty acids characteristic of marine organisms (Fig. 12). However, this was not observed for rainbow trout (Fig. 12).

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Fate of escaped salmonids

Table 5. Linear regression equations and diagnostic statistics for the relationships between lipid content (wet weight) and relative levels of individual fatty acids. Only significant relationships are presented.

Regression equation

Atlantic salmon (1-19% fat content) 14:0 18:0 16:1ω7c 18:4ω6 20:4ω3 22:6ω3 (DHA) Rainbow trout (4-27% fat content) 16:1ω7c 20:4ω6 (AA) 22:6ω3 (DHA) EPA/DHA ratio

Significance level (p-values)

Variance explained (R2)

%FA = 0.08 x % lipid + 1.09 %FA = -0.15 x % lipid + 6.86 %FA = 0.19 x % lipid + 5.02 %FA = -0.17 x % lipid + 1.68 %FA = -0.04 x % lipid + 0.77 %FA = -0.35 x % lipid + 11.57

0.0004 0.0069 0.0007 0.0287 0.0200 0.0325

0.575 0.395 0.544 0.281 0.311 0.270

%FA = 0.069 x % lipid + 7.090 %FA = -0.010 x %lipid + 0.868 %FA = -0.11 x %lipid + 8.87 EPA/DHA = 0.01 x %lipid + 0.21

<0.0000 <0.0000 <0.0000 0.0003

0.351 0.383 0.353 0.283

30

EPA + DHA

25

20

15

10

5

0 25

30

35

40

45

50

55

18:1ω9c + 18:2ω6

Fig 12. Relationship between content of 18:1ω9c and 18:2ω6 (oleic and linoleic acid) and EPA and DHA for Atlantic salmon ( ) and rainbow trout ( ). Salmon: (18:1+18:2) = -0.76 x (EPA+DHA) + 44.43; R2 = 0.782, p <0.0001; Rainbow trout: relationship was not significant (p = 0.3047).

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Fate of escaped salmonids

5. Discussion The stable isotope and fatty acid composition of escaped Atlantic salmon and rainbow trout was similar to that of caged animals and significantly different to that of native species.

This successful novel application of these biochemical techniques, coupled with the fact that the condition of escaped individuals was lower than that of caged animals, indicates that, in general, escapees do not thrive and do not feed on native fauna after escaping from aquaculture cages in Macquarie Harbour. However, this study included a limited number of escapees, and so results are only preliminary.

Gut content analysis For Atlantic salmon, no native fauna or artificial feed pellets were identified in the stomachs, and most were either empty or contained non-nutritious food like twigs and myrtle leaves. However, only 13 stomachs were sampled, and so results are not conclusive.

Steer & Lyle (2003), in a study conducted in the same system in 2003, analysed a much larger number of Atlantic salmon escapees (200) and found that ~60% were empty, while 14% contained feed pellets and 4% invertebrates or fish. Similarly, ~15% of Atlantic salmon escapees in the Inner Seas, southern Chile, also had pellets in their stomachs (Soto et al. 2001). However, unlike in Macquarie Harbour, fish and crustaceans occurred in a high proportion of stomachs (over 10% for each group) in that study. This discrepancy in results could be a result of higher prey availability in the southern Chile systems.

Macquarie Harbour has a very low invertebrate and fish species diversity (Edgar et al.

1999, Edgar et al. 2000), and this may explain the lower feeding success of escapees in this system. However, 70% of wild brown trout stomachs analysed in the present study contained fish that had been consumed whole, meaning that these prey are readily available, and thus it is not likely that the differences observed are solely the result of low prey diversity and availability.

For rainbow trout, the proportion of empty stomachs was much lower than for Atlantic salmon (38 vs. 79%). A large proportion (~20%) of stomachs analysed contained feed pellets, which is consistent with the results of Steer & Lyle (2003). Of the fish examined in the present study, about half were collected in Swan Basin, ~10 km away from the closest fish farms, indicating that rainbow trout can move away from fish farms, but return to feed TAFI Report Page 41

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on the highly nutritious pellets. Non-nutritious plant material was also consumed, a result that has been found for escapees in other areas (e.g. Rikardsen & Sandring 2006). About a fifth of the stomachs analysed contained native fauna, including one fish, indicating that rainbow trout escapees have the capability to capture and consume mobile species.

Different results are reported for southern Chile, where rainbow trout escapees fed mostly on crustaceans (over 30% of stomachs) and fish (~15%; Soto et al. 2001).

Salmonids, like most fish species, need a certain period of time to adjust to differences in taste, smell, texture and behaviour of a new food source. This can explain the reduced feeding on native species, and the return of some individuals to the proximity of cages to feed on pellets. The dispersal of rainbow trout escapees throughout a relatively large area, and their return to the farm to feed on feed pellets has been previously reported for other areas (Bridger et al. 2001, Blanchfield et al. 2009). Movement and feeding patterns have also been found to differ between individuals, with fish that remain close to cages having higher survival rates (Blanchfield et al. 2009). Atlantic salmon escapees, however, do not seem to return to the proximity of the farms, and animals rapidly disperse away from the cages (Whoriskey et al. 2006).

Our study site, Macquarie Harbour, has a very low species diversity, density and biomass due to the nutrient poor and tannin-stained waters that limit primary production (Edgar et al. 1999, Edgar & Barrett 2002). Since salmonids are visual predators with reduced prey detection and predation capabilities in low light conditions (Vogel & Beauchamp 1999, Mazur & Beauchamp 2003), it is likely that escapees do not feed on native fauna due to a combination of several factors, including the low light levels that limit predation success, the low prey availability in the local environment, and the availability of abundant, easy to obtain, nutrient rich and highly palatable feed pellets in the area.

The high proportion of empty stomachs is however not sufficient to determine if salmonid escapees feed and thrive in Macquarie Harbour. In fact, carnivorous fish generally have high proportions of empty stomachs, especially those that eat their prey whole (Arrington et al. 2002) and high proportions of empty stomachs also occur in areas where salmonids are known to thrive (e.g. Fraser 1987, Hislop & Webb 1992, Jacobsen & Hansen 2001).

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Body condition The different body condition indices indicate that salmonids loose condition after escaping.

This was anticipated, as caged individuals are regularly fed highly nutritious pellets to satiation. After escaping, individuals need time to learn to feed on live prey and hence body condition decreases (Soto et al. 2001). Nevertheless, even if thriving, body condition of escapees will be lower than that of caged animals, and even in their native range, wild salmonids always have lower condition than their farmed counterparts (Thorstad et al.

1997, Morris et al. 2003, Johnston et al. 2006). The body condition of most escapees captured in this study was higher (possibly indicating a more recent escape) or within the range of wild brown trout, and of salmonids thriving in their native ranges (Thorstad et al.

1997, Jacobsen & Hansen 2001, Haddix & Budy 2005). There was only one Atlantic salmon in very poor condition, with 0.8% fat content (Fulton‟s K = 0.6).

For both Atlantic salmon and rainbow trout, Fulton‟s K, VSI and muscle lipid content were good condition indicators. Visceral fat was also a good condition indicator for rainbow trout, where it accounted for up to 14% of total body weight in caged individuals, while leaner escapees had much less visceral fat (<1%). However, the hepatosomatic index (HSI) was not a useful measure of fish condition, as escapees often had similar or higher HSI values than caged animals, and caged animals had HSI values similar to the wild brown trout (Fig. 2). Although HSI is generally considered as a good condition indicator for bony fish, a deficiency in essential fatty acids can lead to a degeneration of liver, where livers become fatty, pale and swollen, leading to high HSI (Castell et al. 1972, Watanabe 1982).

In agreement with results from the present study, very high HSI values due to poor diet or starvation have previously been reported for rainbow trout (Kim & Kaushik 1992, Krogdahl et al. 2004), but this does not seem to occur in Atlantic salmon (Hemre et al.

1995, Krogdahl et al. 2004). Therefore, for rainbow trout, higher HSI can be a result of a better condition, but can also indicate nutrient deficiency and starvation (high liver weight relative to body weight).

In salmonids, the perivisceral and visceral adipose tissue and muscle are preferential sites of lipid deposition (Frøyland et al. 1998, Figueiredo-Silva et al. 2005, Quillet et al. 2007), while liver is not an important site of storage (Nassour & Léger 1989). On the other hand, in times of fasting or starvation, muscle fat content is mobilized first followed by viscera, and liver fat is mobilized last (Sheridan 1988, Kiessling et al. 1991, Einen et al. 1998).

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Hence, different parts of the body respond differently and at different rates to variations in condition, leading to poor correlations between the different condition indexes. This is not, however, a disadvantage as information regarding the timing of escapements can be gained by the analysis of different indices. For example, high muscle lipid content, high K and very high HSI would indicate that the animal has escaped relatively recently, has nutrient deficiency and is probably starving. In this example, the sole analysis of muscle lipid content and K would have led to erroneous conclusions regarding the fate of those two animals.

There is presently no information on the time required for the differences in condition to be noticeable in the temperature conditions of Tasmanian waters. At ~2-6°C, two months are enough to detect differences due to starvation in muscle in Atlantic salmon (Einen et al.

1998). In Tasmania, salmonids are farmed in waters that can reach up to 19°C. Since metabolic, growth and fat deposition rates increase with water temperature (Beck & Gropp 1995, Cho & Bureau 1995, Ruyter et al. 2006), it is likely that differences can be noticeable in less than two months.

Biochemical analyses In agreement with stomach content results, stable isotope and fatty acid analyses indicated that, in the Macquarie Harbour environment, escapees do not switch to feed on native fauna. For stable isotope analysis, only one Atlantic salmon had a δ13C value that indicated feeding on native fauna, probably a combination of small fish and grapsid crabs (see Fig. 4a). This individual was also separated by its fatty acid composition (individual a Fig. 10), with both biochemical techniques corroborating that it had been feeding significantly on native species. Two other Atlantic salmon also had relatively high δ15N, with values that could result from feeding on native species such as caridean shrimps and galaxiids (see Fig. 4b). However, given the relatively small difference between δ15N of feed pellets and local sources, and because of the natural variability in δ15N trophic fractionation (McCutchan et al. 2003, Vanderklift & Ponsard 2003) and the increase in δ15N with starvation (Hobson et al. 1993, Doucett et al. 1999, Olive et al. 2003), it is not possible to confirm that these two individuals have been feeding on native fauna based on stable isotope analysis alone.

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Nevertheless, δ15N values were not correlated to lipid content for either species, meaning that the high δ15N values were not a result of starvation. It is possible that the individuals had different feeding success, making any relationship difficult to detect.

In the present study, pellet δ15N was not as high as expected and, consequently, the difference in δ15N between pellets and local producers was small, making results difficult to interpret. Salmonid aquaculture has until recently traditionally used feeds based on wild caught fish. Due to the higher trophic position of these fish, feed pellets had high δ15N values and δ15N analysis has therefore been useful to identify the input of artificial feed into local food webs (Yamada et al. 2003, Yokoyama et al. 2006) and to distinguish wild from farmed salmonids (Dempson & Power 2004, Molkentin et al. 2007). However, for various reasons, a large part of the diet has been replaced by vegetable protein and oils and/or poultry, beef or pork by-products. Since these ingredients have lower δ15N than wild fish, the differences in δ15N between artificial feed and the baseline of most aquatic food webs are not as pronounced as in the past, when fish meal and fish oil comprised the bulk of the feed. Hence δ15N is, in this instance, no longer as useful to identify differences in diet.

For fatty acid analysis, however, a good separation between caged salmonids and native species occurred, due in part to the lower content of fish meal and fish oil, and higher content of vegetable and/or terrestrial animal oil and meal in the artificial feed. Vegetable oils are generally rich in linoleic acid (18:2ω6), oleic acid (18:1ω9c) and other MUFA, while fish oil is rich in the long chain PUFA of the ω3 family, particularly EPA, DHA and DPA. Hence, these fatty acids and the ω6/ω3 PUFA ratio could be used as lipid biomarkers to identify the importance of feed pellets and native fish for escaped animals. Had the artificial feed been based solely on fish meal and fish oil, then its fatty acid composition would have been closer to that of native fauna, and it is possible that fatty acid analysis would not have led to conclusive results. For example, Atlantic salmon analysed in 2002 by CSIRO (Mooney et al. 2002), when fish meal and fish oil content in artificial feed was higher, fell closer to native fish (see Fig.10).

Three escapees (two Atlantic salmon and one rainbow trout) were separated from the rest of salmonids based on their fatty acid composition. Differences in fatty acid composition could mean that escapees were able to switch to feed on native fauna, or alternatively that they were starving and that the changes in fatty acid composition were a result of TAFI Report Page 45

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differential mobilization of the different fatty acids. A change in fatty acid composition due to fasting or starvation has been reported for several fish species, and has been found to vary among tissue types (Kiessling & Kiessling 1993, Einen et al. 1998).

In salmonid muscle, MUFA are preferentially mobilized during starvation (Jezierska et al.

1982, Kiessling & Kiessling 1993, Einen et al. 1998), while essential PUFA are preferentially retained or spared (Tidwell et al. 1992, Kiessling & Kiessling 1993, Kiessling et al. 2001). For example, DHA appeared in higher relative levels in muscle of caged animals than in food (see Table 4), meaning that it was preferentially retained. Hence, the higher DHA levels found in the two escaped Atlantic salmon could be a result of starvation, and not a result of a diet switch to marine fauna. One of the separated Atlantic salmon escapees (individual b, Figs. 10 and 11) also had the lowest MUFA content, which again indicates starvation. It will, however, be important to conduct starvation experiments to identify the nature of changes in fatty acid composition with starvation under the temperature conditions of Tasmanian waters.

In contrast to Atlantic salmon, the separated rainbow trout had relatively high MUFA content, suggesting that it was not starving, and that the difference in fatty acid composition was a result of consuming native fish. This individual, corresponding to 2.6% of total individuals analysed, was in good condition and did not have a high HSI value (1.1) (indicator of nutrient deficiency), again indicating that it was not starving. Its isotopic signatures, however, were similar to caged individuals, showing that, and in this case, fatty acid analysis was more useful to identify a change in diet.

All rainbow trout with very HSI values (13% of total individuals analysed) were well within the salmonid group in the PCA, indicating that for these fish, the similarity in fatty acid composition was a result of starvation. However, for most individuals (~85.5%), the similarity in fatty acid composition was a result of feeding on feed pellets from the cages.

This again demonstrates the value of using different approaches to clarify the sources of feed and feeding success of escapees.

For both stable isotope (Hesslein et al. 1993, Gorokhova & Hansson 1999) and fatty acid (Regost et al. 2003, Robin et al. 2003) analysis, a certain time period under different food TAFI Report Page 46

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conditions is necessary for the animals‟ tissues to reflect the signatures of a new food source. However, there is presently no information on how long it takes for the stable isotopic composition of escapees to change after a shift in diet, as isotopic turnover varies with both growth and metabolic rate. Changes in stable isotope signatures will be slower than changes in fatty acid signatures because stable isotopes mainly reflect the composition of protein, which has a lower turnover rate compared to lipid, especially in large fish with slow growth (note that lipids were removed from δ13C samples). Moreover, due to isotopic routing (Schwarcz 1991), different nutrients in food are directed to similar compartments in the consumer. For example, lipids in food can be used mostly as energy or incorporated as lipid in a consumer, while dietary amino acids will be used to synthesize protein. This can explain the lack of relationship found between δ15N and lipid content. Hence, since protein, carbohydrates and lipids have different turnover rates, stable isotope and fatty acid analysis can provide complementary information on diet. For example, the large (4.6‰) difference in δ13C between the most

13

C-depleted Atlantic salmon escapee and caged individuals

indicates that this animal had been feeding in the wild for a relatively long period, but its fatty acid composition indicates that most of the lipid was derived from artificial pellets, which are very rich (up to 30%) in lipid.

Changes in fish fatty acid profiles after a change in diet can be detectable within 2-6 weeks for large fish (Skonberg et al. 1994, Kirsch et al. 1998, Jobling et al. 2002). However, it is not known how long it takes signatures to fully equilibrate and reflect the new food sources.

Moreover, the extent of these changes will depend on differences in fatty acid composition between the old and new diets, differences in food quality, feeding rates, and on whether there is a total or only a partial change in diet.

Incorporation of pellet material into the local food web Both stable isotope and fatty acid analysis suggest that there was some incorporation of material from feed pellets into the local food web, particularly for the benthivorous greenback flounder, the phytodetritivorous yellow-eye mullet and caridean shrimps. Some wild brown trout also had stable isotope and fatty acid composition similar to caged salmonids, and feed pellets were observed in the stomachs of wild brown trout (Table 1) and Australian salmon (pers. obs). Moreover, the levels of oleic acid in some Australian salmon and some greenback flounder individuals were relatively high, between 20 and TAFI Report Page 47

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30%, higher than the average (13%) for temperate Australian fish (Nichols, unpubl. data).

Although macroalgae and seagrass are also rich in this fatty acid (Alfaro et al. 2006, Crawley et al. 2009), it is unlikely that these were the primary sources responsible for these high relative levels, given the low availability of these producers. These high levels are more likely to result from a direct incorporation of material from uneaten pellets that disperse away from the cages, or from the transport of faecal material.

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6. Conclusions The combined use of traditional approaches to the analysis of fish diet (stomach content analysis) and condition with the novel use of biochemical techniques (stable isotope and fatty acid analyses) was applied to determine if salmonid escapees feed and thrive in Macquarie Harbour. Results of this pilot study indicate that, in general, escapees do not feed on native fauna. There was evidence of successful feeding on native fauna for only two fish, one Atlantic salmon (7.7% of total) and one rainbow trout (2.6% of total).

However, only a limited number of escapees were analysed, and therefore results can not be considered as conclusive. Moreover, results are only valid for Macquarie Harbour, and the situation in other parts of Tasmania where the native invertebrate and fish fauna is more abundant and diverse, may be different. Further studies should be conducted in other systems where salmonid farms occur, so that a more conclusive assessment of the fate and impacts of salmon escapees in Tasmania can be drawn.

7. Acknowledgements We thank Graeme Ewing and Adam Barnett for their help in the field. Zack Wingfield from Tassal Pty Ltd for providing farmed Atlantic salmon and Barry McClure from Petuna Seafarms for the farmed rainbow trout. Justin (Jungle) Clarke, Matt Gaby, Andrew Blenkhorn and Doug Keep, recreational fishers who willingly provided extra muscle and stomach samples from their catch and greatly contributed to improve this study. Research was conducted under the General Fisheries Permit no. 7068 (Department of Primary Industries, Parks, Water and Environment, Tasmania) and all research procedures reported received the approval from the Animal Ethics Committee, University of Tasmania (Ethics Approval A0010272). This project was supported by Cradle Coast NRM through funding from the Australian Governmentâ&#x20AC;&#x;s Natural Heritage Trust.

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