Do trout farm effluents affect Atlantic salmon smolts? Preliminary studies using caged salmon smolts

Aquaculture 362–363 (2012) 209–215

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Do trout farm effluents affect Atlantic salmon smolts? Preliminary studies using caged salmon smolts C.P. Waring a, A. Moore b,⁎, J.H. Best a, 1, N. Crooks a, L.E. Crooks a a b

University of Portsmouth, School of Biological Sciences, King Henry Building, King Henry I Street, Portsmouth, Hampshire, UK, PO1 2DY The Centre for Environment, Fisheries and Aquaculture Science, Lowestoft Laboratory, Pakefield Road, Lowestoft, Suffolk, UK, NR33 0HT

a r t i c l e

i n f o

Article history: Received 22 October 2009 Received in revised form 8 June 2011 Accepted 30 November 2011 Available online 8 December 2011 Keywords: Salmon Smolts Aquaculture

a b s t r a c t Although many studies have shown that trout farm effluents can affect water quality and macro-invertebrate populations downstream of the farm, few studies have investigated effects on fish. Previous work has suggested that trout farm effluents can affect salmonid parr and embryos but there is no data as to whether they affect salmonid smolts. In this experiment, Atlantic salmon smolts were caged upstream and downstream of trout farm effluents for 3 days on two UK Rivers and compared against hatchery controls in two years (2005–2006). No persistent effects on plasma osmolality, plasma sodium, plasma chloride, condition factor and hepato-somatic indices were observed, although there were variations in responses between years. No effects of the effluents were observed on gill Na+K+ATPase activity or plasma thyroid hormones. There was evidence that fish placed downstream of the fish farms had modified plasma potassium regulation, although the relative influence of the fish farm effluents on the physiology compared to other compounds in the river has not been determined. Only one of the smolts (5%) caged downstream of the effluents died and there was little effect on plasma ionoregulatory and osmoregulatory indices, but mortalities increased when the smolts were given a 48 h seawater challenge. However, it is possible that other compounds within the river in addition to the fish farm effluents may have influenced the survival of the smolts. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

1. Introduction Over the last few decades there has been a significant increase in the development of intensive in-river aquaculture for the production of salmonids in a number of English rivers. However, there have been surprisingly few studies examining the effects of these facilities, and particularly their effluents, on wild salmonid populations residing downstream of the fish farms. Where studies have been carried out in other countries, effluents from fish farms have been implicated in changes to the abundance and survival of juvenile salmonids. For instance Prévost (1999) found that rainbow trout farms in Brittany had significant effects on wild juvenile Atlantic salmon (Salmo salar). Two trout farms caused a significant reduction in salmon parr abundance that was evident for several kilometres downstream of the effluent. Dumas et al. (2007) examined the effects of fish farm effluents in the Pyrenees on the egg-to-fry survival rates of implanted brown trout (Salmo trutta) eggs in artificial redds placed upstream and downstream of the effluents. They found that the egg-to-fry survival rates were lower in the redds placed downstream of the effluents compared to upstream and that the effluents also caused a delay in development of yolk-sac fry. ⁎ Corresponding author. Tel.: + 44 1502 52 4212. E-mail address: [email protected] (A. Moore). 1 Present address: Scottish Environment Protection Agency.

Studies on fish farm effluents have shown that they may modify a range of water quality parameters downstream of the facility. For instance there are often increases in the total ammonia-nitrogen content (Bergheim et al., 1984; Boaventura et al., 1997; Foy and Rosell, 1991; Kelly et al., 1994; Korzeniewski et al., 1982; Selong and Helfrich, 1998), increases levels of phosphorus and phosphates (Boaventura et al., 1997; Foy and Rosell, 1991; Korzeniewski et al., 1982; Trojanowski, 1990; Villanueva et al., 2000; ) and significantly less oxygen (Bergheim and Selmer-Olsen, 1978; Kelly and Karpinski, 1994; Korzeniewski and Salata, 1982; Korzeniewski et al., 1982, 1985) compared to sites upstream of the fish farms. Research has also indicated that fish farm effluents may also affect non-salmonid fish species. Oberdorff and Porcher (1994) examined the effects of trout farm effluents on the fish communities of some streams in Brittany. They found that, generally, downstream of the trout farm effluents most fish species increased their density and biomass. However, some pollution-intolerant species disappeared (Cottus gobio), whereas other pollution-tolerant species (Rutilus rutilis) increased. The authors suggested that the major reasons for these effects were from nutrient enrichment and that fish excreta and uneaten food were the main sources of pollution from the trout farm. Fish farm effluents have also been shown to affect bacterial populations (Brown and Goulder, 1996; Carr and Goulder, 1990) and the diatom and periphyton community downstream of the effluent (Szluha, 1974; Villanueva et al., 2000). Freshwater fish farm effluents

0044-8486/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.11.046

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can stimulate the growth of phytoplankton such as Selenastrum and macrophytes such as Ranunculus, which is thought to be due to the increased phosphorus released by the trout farm effluents. Trout farm effluents can also affect benthic macro-invertebrates. Camargo (1992) found that macro-invertebrate species richness and diversity was reduced downstream of a trout farm in Spain. Amphipods, plecopterans and planarians were the macro invertebrates most affected. Camargo and Gonzalo (2007) re-surveyed this site and found that in the intervening twenty years the trout farm effluent was still impacting macro-invertebrates downstream of the effluent. Selong and Helfrich (1998) also examined the effects of trout farm effluents on river invertebrates. They found that macro-invertebrate richness and abundance of sensitive taxa (mayflies, stoneflies, and caddis flies) were reduced downstream, and pollution-tolerant non-insect taxa (isopods and gastropods) increased. Loch et al. (1996) examined the effects of trout farm effluents on macro invertebrates in a North Carolina stream. Higher abundances of pollution-tolerant forms (oligochaetes, bivalves, chironomids, simulids) and fewer stoneflies and caddis flies were found downstream of the trout farm. More recent studies in Turkey, Germany and the USA examining the effects of trout farm effluents on macro-invertebrate richness downstream of the farms have concluded that the effluents had significant effects (Kirkagac et al., 2009; Roberts et al., 2009; Sindilariu et al., 2009) due to altered water quality. In a number of river systems, wild migrating salmon smolts may be exposed to diluted effluents from a number of trout farms. However, there is no data as yet as to what impact, if any, trout farm effluents can have on smolting Atlantic salmon. It is likely that any impact would also depend upon the farming management practices within the river catchment, specific ecological characteristics of the site, location along the river and any other additional point source and diffuse contamination from industry and intensive agriculture. In this study, the effects of trout farm effluents on caged Atlantic salmon smolts in two U.K. chalk rivers were investigated. In particular, the effects on osmoregulation and osmoregulatory ability after salinity challenge were examined. It has been shown previously that Atlantic salmon smolts are sensitive to stress to a greater extent than parr (Carey and McCormick, 1998). In particular, plasma ion concentrations in were found to be more responsive to stressors than in parr (Monette and McCormick, 2008). Since salmon smolts require a well developed osmoregulatory ability in order to survive the transition to seawater, the study concentrated on osmoregulatory and ionoregulatory parameters in the smolts caged upstream and downstream of the trout farm effluents. 2. Materials and methods Two trout farm sites were examined in this study. One site was on the River Test, Hampshire (UK) and one site was on the River Avon, Wiltshire (UK). The River Test site was a commercial rainbow trout (Oncorhynchus mykiss) farm producing fish for the table. The River Avon site was a brown trout (Salmo trutta) hatchery producing fish primarily for stocking. We are unable to report further details (e.g. tonnages) because of data protection. Atlantic salmon smolts (Length: 160–200 mm; Weight: 40–60 g) were obtained from Yorkshire Salmon, Skipton, North Yorkshire in late March of 2005 and 2006. The smolts were all silvery in appearance and had the morphological characteristics of migratory smolts. The peak migratory period of this salmon stock is in April (Lower and Moore, 2007). They were brought to a hatchery site on the River Test and kept in 2 m 2 fibreglass tanks with flow-through borehole water (temperature 12–13 °C, pH 7.66, Dissolved Oxygen 7.68 mg/l). Other aspects of the water quality of the borehole water used are reported by Greig et al. (2005). They were left at least 3 weeks to recover from the transportation and fed Skretting Excel 30, 3 mm commercial pellet twice a day to satiation.

After three weeks, some of the smolts were removed and transported to the field sites. The remaining smolts which were used as controls continued to be fed Skretting Excel 30, 3 mm commercial pellet twice a day to satiation. The experiments were conducted by comparing sites close to the effluent discharge points of two farms with reference (control) sites. It was not possible to place the controls upstream of the farms in either the River Test or Avon for biosecurity reasons, but both rivers comprise a number of interconnected channels, and control cages could therefore be located in parallel channels close to the farm sites. On the River Test, part of the river divides into two (Channels 1 and 2) about 900–1000 m upstream of the fish farm and these rejoin about 200 m downstream of the farm. Water supplying the fish farm is abstracted from and returned to Channel 1, and fish were caged in the effluent stream from the farm (referred to as “downstream”) before it rejoins Channel 1. The ‘upstream’ reference site was located in Channel 2, a short distance upstream of where it rejoins Channel 1. Channel 1 passes immediately beside a small industrial site before it reaches the farm abstraction point, whereas the Channel 2 flows 50–200 m further to the west. There was therefore potential for differential inputs of contaminants into the two Channels other than from the farm, although there were no records of such events occurring during the study. At the experimental site on the River Avon, a channel splits from the main river approximately 250 m upstream of the farm, and the majority of the flow from this channel passes through the farm ponds. A further channel splits from the main river 125 m further downstream and receives the effluent from the fish farm before rejoining the main river. The cages were all placed in the second channel, with the control cages (referred to as “upstream”) and the treatment cages (referred to as “downstream”) being placed about 40 m upstream and 60 m downstream respectively of the point where the fish farm effluent entered the channel. Both channels pass through similar farmland, but there was potential for the upstream and downstream cages to receive different contaminants from sources other than the farm, although there were no records of such events occurring during the study. The cages (each of 310l) were constructed from galvanised stainless steel with an inner layer of 1 cm 2 plastic meshing. At each location, 2–4 cages were placed in the river and each cage held 7–10 smolts. The smolts were left in the cages without being fed. Water pH, conductivity and dissolved oxygen concentrations were measured in situ at the field sites with a portable multimeter (Multi 340 SET 1 meter). Water samples were also filtered through preweighed 47 mm glass fibre filters (Fisherbrand) in order to measure suspended solid levels. The filters were dried to constant weight at 40 °C and reweighed. Total Ammonia-Nitrogen concentrations were also measured in the water samples using a method based upon that of Solorzano (1969). The experiment was run over two seasons. In each season, the smolts were caged in the rivers in early May. In 2005, the smolts (means ± SEM: Length: 187.2 ± 1.3 mm, weight 55.1 ± 1.3 g) were left in the cages for three days (n = 20 at each site) before being sampled. In addition, smolts from the hatchery borehole water were sampled at the same time and were designated as controls. The fish were anaesthetised with 0.4 ml 1 − 1 2-phenoxyethanol and humanely killed (striking of the cranium, with destruction of the brain before return to consciousness). The length (cm) and weight (g) of each fish was recorded. Blood was sampled from the caudal blood vessels using lithium heparinised needles and syringes and a white muscle sample was removed from between the lateral line and dorsal fin. The muscle samples were wrapped in tin foil, kept on ice at the field site, and then stored at −20 °C. The liver was removed from each fish and weighed (mg). The blood was kept on ice until return to the lab (1–2 hours later) where the blood was centrifuged (5000 g for 10 minutes at 4 °C) and the plasma was then stored at

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−20 °C. In 2006, the smolts (Length: 167.6 ± 1.0 mm, Weight 46.6 ± 0.9 g) were again left in their cages at each field site for 3 days (n = 20–22 at each site). Half of the fish were sampled on site, and also sampled from the hatchery borehole water, as described above. However, in addition to blood and muscle tissue sampling, the uppermost hemi-branch of the first gill arch was removed from each fish and the gill samples were stored in SEID buffer solution and snap frozen in liquid nitrogen. They were subsequently stored at −80 °C for up to 8 weeks. The remaining smolts were then transported in their river water to the Institute of Marine Sciences at the University of Portsmouth where they were given a 48 h seawater challenge. In addition, some smolts that had been kept in the hatchery borehole water were given a seawater challenge test at the same time. The transportation time from the hatchery site and the field sites to the marine laboratory was 60–90 min. They were kept in 100 litre tanks with flow through (2 l min − 1) seawater (13–14 °C: 35 ppt salinity). Mortality was recorded over the 48 h and then the surviving fish were sampled as described above. 2.1. Analysis Gill Na +K +ATPase activity was measured according to Schrock et al. (1994). Muscle water content was determined by drying 100 mg pieces of muscle at 85 °C until constant weight was achieved. Plasma osmolality was measured using freezing point depression (Löser Osmometer), plasma chloride by electrochemical titration (Jenway Chloride Meter). Plasma sodium and potassium concentrations were measured by flame photometry (Jenway Flame Photometer) after being diluted with Reverse Osmosis water. Plasma thyroxine (T4) and triiodothyronine (T3) concentrations were measured using the methods described by Lower and Moore (2007). In order to screen for the presence of heptotoxicants we used the Hepato-Somatic Index as a fairly crude marker. Hepato-Somatic Index (HSI) was calculated by expressing the liver weight as a percentage of the body weight. Fulton's Condition Factor was calculated using the equation [Weight(g)/Length (cm) 3] * 100. Most of the data were analysed using two-way ANOVA followed with year (i.e. 2005 or 2006) and location (i.e. hatchery, upstream, downstream) as factors. If significance was indicated, Holm-Sidak tests were used as the multiple range test. However, plasma T4 and T3 concentrations and gill Na +K +-ATPase activity were only measured in 2006 and so these data were analysed by 1-way ANOVA or if normality failed a Kruskal-Wallis tests. In all cases, significance was accepted if P b 0.05. Dunn's Method was used after KruskalWallis tests indicated significance. Muscle water data were arc-sine transformed before analyses with ANOVA. Means ± SEM are reported. 3. Results 3.1. Water quality measurements at the field sites Table 1 shows the physico-chemical parameters measured at the upstream and downstream sites of the trout farm effluents on the Rivers Test and Avon. Water temperature, conductivity, dissolved

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oxygen and pH did not differ significantly between the upstream and downstream sites. At the trout farm on the River Test, suspended solids were significantly higher (P = 0.041) downstream of the effluent compared to upstream. There was no significant difference in suspended solids between the upstream and downstream sites on the River Avon. At both the trout farms, water total-ammonia nitrogen concentrations were significantly (P b 0.001) higher at the site downstream of the effluent compared to the upstream site. 3.2. Caging at the trout farm sites In 2005, survival was 100% at all of the sites after 3 days of being caged. In 2006, there was again 100% survival of the smolts caged for 3 days at the upstream and downstream site on the River Avon. In 2006 there was 100% survival of the smolts caged upstream of the trout farm effluent on the River Test. There was 95% survival of the smolts caged downstream of the effluent on the River Test. Table 2 shows the data measured from the smolts from the hatchery and the caging sites from the years 2005 and 2006. Smolt condition factor was found to vary significantly by location (P = 0.011) and by year (P b 0.001). The interaction between year and location was also significant (P b 0.001). The Condition Factor of the smolts in 2006 was significantly higher than that measured in the 2005 smolts. The overall Condition Factors of the smolts caged upstream and downstream of the trout farm effluent in the River Avon were significantly lower than those of the hatchery smolts and this was especially marked in 2005. The Condition Factors of smolts caged in the River Test sites did not differ significantly to those of the hatchery controls. In no case was there a significant difference between the Condition Factors of smolts caged downstream of a trout farm effluent compared to their upstream counterparts. The HSI of the salmon smolts was also found to be significantly affected by location (P = 0.005) and also by year (P b 0.001). The interaction between location and year was also significant (P = 0.018). The HSI was significantly higher in the 2006 smolts compared to those used in 2005. The HSI of smolts caged downstream of the trout farm effluents on the River Test and the River Avon were significantly higher than those of the hatchery smolts but this was not consistent between years for a particular river. There were no differences between the HSI of smolts caged downstream of a trout farm effluent compared to their upstream counterparts. Smolt plasma osmolality was found to be significantly affected by location (P = 0.002) and year (P = 0.001) but there was no significant interaction between location and year (P = 0.170). Plasma osmolality measured in the smolts was higher in 2005 (323.3 ± 3.4 mOsm kg H2O − 1) compared to 2006 (306.1 ± 3.9 mOsm kg H2O − 1). There was few significant location differences in the plasma osmolality measured in the smolts after being caged for 3 days. Smolts caged downstream of the effluent at the River Test site had a significantly higher plasma osmolality than the smolts caged downstream of the effluent at the River Avon site. Smolts caged downstream of the effluent at the River Avon site had a significantly lower plasma osmolality compared to the smolts sampled at the hatchery. Plasma sodium concentrations were not significantly affected by location (P = 0.472) but

Table 1 The physico-chemical characteristics of the water sampled from the field sites. Data represents means ± SEM. n = 5–8 per site. * = P b 0.05 compared to the upstream site. River Test

Temperature (°C) pH Conductivity (μS) Dissolved oxygen (mg/l) Suspended solids (mg/l) Total ammonia-nitrogen (mg/l)

River Avon

Upstream

Downstream

Upstream

Downstream

13.52 ± 0.70 7.98 ± 0.14 562 ± 8 8.17 ± 0.47 4.75 ± 1.43 0.04 ± 0.01

13.38 ± 0.68 7.77 ± 0.16 559 ± 8 7.69 ± 0.38 8.25 ± 1.34* 0.12 ± 0.04*

13.1 ± 1.0 7.70 ± 0.12 603 ± 10 7.51 ± 0.38 3.90 ± 1.16 0.05 ± 0.01

13.2 ± 0.5 7.74 ± 0.14 617 ± 10 7.26 ± 0.45 4.32 ± 1.39 0.13 ± 0.07*

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Table 2 Condition factor, hepato-somatic index (HSI:%), plasma osmolality (mOsm kg H2O− 1), sodium (mM), chloride (mM), potassium (mM), and muscle water (% water) in smolts caged Upstream or Downstream of the trout farm effluents on the River Test or Avon sites for 3 days in 2005 or 2006. Data represents means ± standard errors. N = 10–20 per group. * = P b 0.05 compared to the hatchery smolts for that year. a = P b 0.05 compared to its upstream counterpart in that year. b = P b 0.05 compared to the 2005 value for that site. Hatchery

2005 Season Condition Factor HSI Osmolality Sodium Chloride Potassium Muscle Water 2006 Season Condition Factor HSI Osmolality Sodium Chloride Potassium Muscle Water

River Test

River Avon

Upstream

Downstream

Upstream

Downstream

0.97 ± 0.02 0.75 ± 0.07 336.9 ± 5.6 123.0 ± 2.8 122.4 ± 2.5 3.88 ± 0.20 77.0 ± 0.2

0.79 ± 0.02* 1.06 ± 0.08* 314.9 ± 9.9 109.3 ± 4.9 113.1 ± 5.0 2.60 ± 0.1* 76.5 ± 0.5

0.83 ± 0.02* 1.09 ± 0.07* 341.6 ± 11.6 127.3 ± 7.1 111.3 ± 5.0 0.93 ± 0.1* 76.5 ± 0.3

0.78 ± 0.02* 0.94 ± 0.07 310.5 ± 7.6 111.0 ± 4.7 102.6 ± 3.8* 1.67 ± 0.17* 78.1 ± 0.3

0.79 ± 0.02* 1.10 ± 0.07* 312.4 ± 7.4 118.4 ± 3.9 111.3 ± 4.7 1.0 ± 0.2*a 77.6 ± 0.3

0.93 ± 0.02 1.31 ± 0.08b 311.9 ± 6.3 136.3 ± 3.4 128.8 ± 5.3 2.21 ± 0.07b 77.0 ± 0.3

1.02 ± 0.02*b 1.18 ± 0.08 320.6 ± 6.9 140.1 ± 0.5 135.2 ± 3.4b 0.51 ± 0.03* 77.1 ± 0.1

0.99 ± 0.02b 1.36 ± 0.08b 311.4 ± 8.5 139.0 ± 1.0 124.3 ± 5.2b 0.88 ± 0.15* 77.4 ± 0.4

0.98 ± 0.02b 1.49 ± 0.08b 297.7 ± 5.6 139.8 ± 0.4 122.5 ± 4.1b 2.11 ± 0.26b 78.1 ± 0.7

1.00 ± 0.02b 1.44 ± 0.08b 288.7 ± 6.5 139.2 ± 1.2 105.7 ± 5.4* 4.01 ± 0.38*ab 78.2 ± 0.3

there was a significant difference between years (P b 0.001). The interaction between location and year was not significantly different (P = 0.155). Plasma sodium concentrations in the smolts sampled in 2006 (138.9 ± 1.9 mmol l − 1) were significantly higher than those measured in 2005 (117.6 ± 1.7 mmol l − 1). Plasma chloride concentrations were much more variable. There was a significant effect of location (F = 5.318, P b 0.001), year (P b 0.001) and the interaction between location and year was also significant (P = 0.02). The major difference in plasma chloride concentrations was between years. Plasma chloride concentrations were higher in the smolts sampled in 2006 (123.3 ± 2.2 mmol l − 1) compared to 2005 (112.1 ± 1.9 mmol l − 1). Overall, plasma chloride concentrations were significantly lower in the smolts sampled upstream and downstream of the trout farm effluent on the River Avon compared to the smolts sampled at the hatchery. However, this was not consistent between the years. There were no differences between the smolts caged at the River Test sites between the upstream and downstream sites and also compared to the hatchery smolts. Plasma potassium concentrations in the smolts were significantly affected by location (P b 0.001) and year (P b 0.001). The interaction between year and location was also statistically significant (P b 0.001). Plasma potassium concentrations in smolts sampled in 2006 were significantly higher compared to those sampled in 2005. In the smolts caged in the River Test, both upstream and downstream of the trout farm effluent, plasma potassium concentrations were significantly lower than the hatchery smolts in both seasons. However, in both years there was no significant difference in the plasma potassium concentrations in the smolts caged downstream compared to upstream of the trout farm effluent at the River Test site. At the River Avon site, plasma potassium concentrations were significantly lower than in the hatchery smolts in the fish caged both upstream and downstream of the trout farm effluent in one year but this was not evident in the following year. In the smolts caged downstream of the effluent, plasma potassium concentrations were significantly lower compared to the hatchery smolts in 2005 but in 2006 they were significantly higher than the hatchery smolts. However, in both years plasma potassium concentrations were significantly different in the smolts caged downstream of the River Avon trout farm effluent compared to the smolts caged upstream of the effluent. In 2005, plasma potassium concentrations were significantly lower in the smolts caged downstream of the effluent compared to their counterparts caged upstream whereas in 2006 plasma potassium concentrations were significantly higher in the smolts caged downstream compared to their upstream counterparts.

Muscle water contents were significantly affected by location (F = 5.343, P b 0.001) but not by year (P = 0.103). The interaction between year and location was also not statistically significant (P = 0.689). Most of the inter-location differences were between smolts caged at the river Test sites compared to those at the River Avon sites. Muscle water contents were lower in the smolts caged in the River Test compared to those on the River Avon. There were no significant differences in muscle water contents between smolts caged downstream compared to upstream of a trout farm effluent in either River. In 2006 only, gill Na +K +ATPase activities and plasma T4 and T3 concentrations were also measured in the smolts sampled at the field sites (Table 3). There was no significant difference in the gill Na +K +ATPase activities in smolts sampled from the borehole water hatchery and those caged upstream and downstream of the trout farm effluents in the Rivers Test and Avon for 3 days (P = 0.183). Similarly, there was no difference in plasma T4 (P = 0.205) and plasma T3 (P = 0.408) concentrations in these smolts. 3.3. 2006 seawater challenge All of the smolts transferred to seawater from the borehole water hatchery survived the 48 h challenge (Fig. 1). Similarly, all of the smolts that had been caged in the River Test upstream of the trout farm effluent survived the 48 h seawater challenge. However, 27.3% of the smolts that had been caged downstream of the River Test trout farm effluent died during the 48 h in seawater. Of the smolts that were caged upstream of the River Avon trout farm effluent, 27.3% of them died during the 48 h in seawater. However, 60% of the smolts that had been caged downstream of the River Avon trout farm effluent died during the 48 h seawater challenge. In all cases, the mortalities occurred within the first 24 h of the seawater

Table 3 Plasma thyroxine (T4: ng/ml), triiodothyronine (T3: ng/ml) concentrations and gill Na+-K+ATPase activity (μM Pi/mg protein/hour) in smolts caged Upstream or Downstream of the trout farm effluent at the River Test or Avon sites for 3 days. Data represents means ± standard errors. N = 10–11 per group. Hatchery

Na+-K+ ATPase T4 T3

35.6 ± 1.5 3.2 ± 1.2 4.1 ± 1.4

River Test

River Avon

Upstream

Downstream

Upstream

Downstream

36.5 ± 2.3 3.0 ± 0.8 5.4 ± 1.0

39.0 ± 1.5 4.7 ± 1.8 5.2 ± 0.9

32.4 ± 2.4 1.6 ± 0.7 4.9 ± 1.3

35.9 ± 3.6 3.6 ± 1.4 2.5 ± 0.7

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Fig. 1. Mortality rates in salmon smolts after a 48 h seawater challenge after being caged at sites upstream or downstream of trout farm effluents in the Rivers Avon or Test compared to those held in a hatchery.

challenge. Table 4 shows the data obtained from the surviving smolts after the 48 h seawater challenge. Across the smolts from the different exposure groups there were no significant differences in Condition Factor (P = 0.497) or HSI (P = 0.148) in the surviving smolts sampled after the 48 h seawater challenge. Similarly, there were no significant differences in gill Na +K +ATPase activity (P = 0.297), muscle water contents (P = 0.060), plasma osmolality (P = 0.769) and plasma chloride concentration (P = 0.913). There was a significant difference in plasma sodium concentrations (P = 0.016) but consistent significant differences between groups could not be identified. There were significant effects of exposure on plasma potassium concentrations (P b 0.001). Smolts that had been caged downstream or upstream of the trout farm effluent on the River Avon had a significantly higher plasma potassium concentration compared to the hatchery borehole water smolts after seawater challenge. Smolts that had been caged downstream of the trout farm effluent on the River Test also had a significantly higher plasma potassium concentration compared to the hatchery control smolts after the seawater challenge. For both Rivers, there were no significant differences in plasma potassium concentrations after seawater challenge between smolts that had been caged upstream or downstream of the trout farm effluents. There were no significant effects on plasma T4 (P = 0.132) or plasma T3 (P = 0.620) concentration in the smolts after their seawater challenge. 4. Discussion On both rivers, the total ammonia-nitrogen concentrations were significantly higher at the site downstream of the effluent compared to the upstream site. Downstream of the trout farm on the River Test the suspended solid levels were also significantly elevated compared to the upstream site. These findings are consistent with the findings of other studies (Bergheim et al., 1984; Boaventura et al., 1997; Foy and Rosell, 1991; Kelly et al., 1994; Korzeniewski et al., 1982; Selong and Helfrich, 1998), but the measured concentrations of total ammonia-nitrogen and suspended solids are not within the

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ranges expected to be acutely lethal to salmonids. There was no difference in the levels of dissolved oxygen between the upstream and downstream sites, whereas some other studies have found significantly lower dissolved oxygen concentrations in water sampled downstream of fish farms (Bergheim and Selmer-Olsen, 1978; Kelly and Karpinski, 1994; Korzeniewski and Salata, 1982; Korzeniewski et al., 1982, 1985). In 2005, all the salmon smolts survived the 3-day caging in the rivers at both the upstream and downstream sites. In 2006, a single smolt died (5% mortality) after being caged for 3 days within the effluent downstream of the trout farm on the River Test. Therefore, there was no indication that any of the effluents from the trout farms were acutely toxic to Atlantic salmon smolts after short term exposure in freshwater. Although Prévost (1999) had found that long term exposure to trout farm effluents in Brittany reduced salmon parr abundance downstream of the effluents compared to upstream, previous studies on the migratory behaviour of wild salmon smolts suggests that the fish would reside for significantly shorter periods within the effluents during their seaward emigration (Moore et al., 1995, 1998). Therefore, the exposure of the smolts to the effluents for 3 days is based on the actual migration patterns of wild salmon and is considered to be a realistic exposure period. Both fish farms were located low down the river catchments and water passing these sites had therefore flowed through both urban areas and agricultural areas (e.g. arable and livestock farming). Therefore, the caging sites upstream of the two fish farms would not necessarily be pristine and free of contaminants. Both rivers receive point source contamination from a range of other sources and so differentiating the actual effects of the effluents alone would be difficult. For this reason hatchery reared smolts, which had not previously been exposed to a wide range of freshwater contaminants were chosen for the laboratory control fish. In both 2005 and 2006 salmon smolts were obtained from the same supplier at the same time of year and came from the same stock, and the caging experiments were undertaken at the same time of year with virtually identical water temperatures. All the smolts used were silvery in appearance and had lost their parr marks. However, there were marked differences between the 2005 and the 2006 smolts in most of the parameters measured (e.g. condition factor, HSI, plasma osmolality, plasma sodium concentration, plasma chloride concentration). This suggests that the smolts used in the different years were at different stages in the smoltification process. In 2006 the results of the salinity challenge experiments clearly indicate that the smolts used in this year had undergone smoltification and were ready for seawater transfer. In 2005, the physiological data suggest that the smolts were not at a similar smoltification status. For example, in 2005 the smolts caged upstream and downstream of the trout farm effluents in the River Test and River Avon had significantly lower condition factors compared to the hatchery smolts. There were no significant differences between the downstream caged smolts compared to the upstream caged smolts. In 2006, the only significant difference in the condition factor was

Table 4 Plasma osmolality (mOsm kg H2O− 1), sodium (mM), chloride (mM), potassium (mM), thyroxine (T4: ng/ml), triiodothyronine (T3: ng/ml) concentrations and muscle water (% water) and gill Na+-K+ATPase activity (μM Pi/mg protein/hour) in smolts caged Upstream or Downstream of the trout farm effluent at the River Test or Avon sites for 3 days and then given a 48 hour seawater challenge. Data represents means ± standard errors. N = 4–10 per group.* = P b 0.05 compared to the hatchery smolts. Hatchery

Osmolality Sodium Chloride Potassium T4 T3 Muscle water Na+-K+ ATPase

357.3 ± 7.8 156.1 ± 5.6 168.9 ± 4.1 2.4 ± 0.1 6.3 ± 1.9 6.1 ± 1.4 76.8 ± 0.1 35.8 ± 1.4

River Test

River Avon

Upstream

Downstream.

Upstream

Downstream.

353.1 ± 9.9 142.8 ± 2.2 166.0 ± 4.9 4.2 ± 0.4 4.4 ± 2.4 5.9 ± 1.8 76.0 ± 0.4 33.3 ± 1.2

365.2 ± 25.3 143.9 ± 0.8 172.6 ± 13.4 5.7 ± 0.5* 2.4 ± 1.2 3.2 ± 1.5 74.7 ± 0.7 31.1 ± 1.9

343.4 ± 6.2 150.3 ± 2.0 164.4 ± 1.5 6.1 ± 0.5* 5.4 ± 1.7 5.3 ± 1.4 76.0 ± 0.5 33.5 ± 1.8

373.8 ± 35.7 154.2 ± 0.6 166.8 ± 11.2 6.8 ± 0.8* 1.7 ± 0.6 3.9 ± 1.9 74.7 ± 1.6 32.3 ± 2.2

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between the smolts caged upstream of the effluent on the River Test compared to the hatchery smolts, but in this case the condition factor was significantly higher than the hatchery smolts. Similar inconsistent effects between years and sites were evident for HSI and also for plasma chloride concentrations. In addition, there was no evidence that trout farm effluents affected gill Na +K +ATPase activity or thyroid hormones such as T4 and T3 in either year. These parameters are also key processes in salmon smoltification and are required for the successful transfer to seawater (McCormick et al., 1998). The only effect of the fish farm effluents observed between years and between sites was on plasma potassium concentrations. In both 2005 and 2006, plasma potassium concentrations were significantly lower in the smolts caged upstream and downstream of the trout farm effluent in the River Test compared to the hatchery smolts. However, there was no significant difference between the smolts caged downstream compared to upstream. An identical pattern was evident in 2006. The plasma potassium concentrations measured in these smolts were remarkably low. If these low concentrations persisted then mortalities may be expected to occur. At the River Avon site, plasma potassium concentrations were significantly different between upstream and downstream caged smolts. In 2005, the concentrations were significantly lower in the downstream caged smolts whereas in 2006 they were significantly higher. The data suggests that trout farm effluents can affect plasma potassium regulation but the mechanism(s) involved appear to be complex. However, as there was also evidence that the potassium levels were modified in fish caged upstream of the fish farm on the River Avon but not in the hatchery smolts, the possibility of stress resulting from the caging of the smolts affecting potassium concentrations should not be ruled out. Measurements of plasma cortisol and/or glucose would also have been very informative. In 2006 all of the smolts transferred from the hatchery to seawater survived for 48 h demonstrating that the smolts were physiologically ready for seawater entry. Indeed, some of the hatchery smolts transferred to seawater were not sampled after 48 h and were kept for another 30 days in the seawater tanks. They fed readily and grew well. All of the smolts caged at the upstream site on the River Test also survived the 48 h seawater transfer. At the River Avon site, 27% of the smolts caged upstream of the trout farm effluent died during the 48 h seawater transfer. It is becoming clear that there are compounds that can be present in freshwater, such as pesticides, that are sublethal and have few physiological effects on the smolts whilst in freshwater but can cause mortalities after seawater transfer (Nieves-Puigdoller et al., 2007; Waring and Moore, 2004). However, on both rivers, smolts caged downstream of the trout farm effluents had higher mortality rates than their counterparts caged upstream of the effluents. This suggests that the pre-exposure to the effluents for 3 days impaired the ability of the smolts to transfer successfully to seawater, although other contaminants within the river may also have influenced survival. In the smolts that survived transfer to seawater, there were no significant effects on plasma osmolality, plasma sodium concentrations, plasma chloride concentrations, and muscle water contents. This suggests that the trout farm effluents did not impact on tissue water regulation or on the major monovalent ions contributing to plasma osmolality. Equally, there was no effect on plasma thyroid hormone concentrations or on gill Na +K +ATPase activity. Therefore, from the parameters measured it is not possible to discern the precise cause of death. For the River Avon smolts, plasma potassium concentrations after 48 h in seawater were significantly higher in the smolts that had been caged both upstream and downstream of the effluents but again there was no difference between the smolts caged downstream compared to upstream of the effluent. It is interesting that significantly elevated plasma potassium concentrations after seawater transfer were only found in smolts from locations where mortalities occurred. The results indicate that the disturbance in plasma potassium regulation

in the smolts caged downstream of the farms which was apparent whilst they were in freshwater also appear to occur when the smolts were transferred to seawater. However, it also appears that there may be other compounds in the rivers that can affect potassium regulation, which may be independent of fish farm effluents. In conclusion, there was little evidence of persistent effects of trout farm effluents on the osmoregulatory and ionoregulatory abilities of Atlantic salmon smolts whilst exposed in freshwater for 3 days. The data suggests that plasma potassium regulation may be affected by trout farm effluents, but further work is required to investigate this in more detail, particularly the influence of other unidentified compounds from other sources within the river. In particular, sampling at different times of exposure may determine whether the plasma potassium response to the effluents is multi-phasic. The effects on fish placed downstream of the fish farms became much more evident when they were transferred to seawater. The data suggests that trout farm effluents may affect the subsequent marine survival of salmon smolts, although once again it is not clear to what extent this may have been influenced by compounds from other sources within the river. The data from the present study on salmonid smolts and that of Dumas et al. (2007) on salmonid eggs and Prévost (1999) on salmonid parr suggest that trout farm effluents may impact wild salmonid populations and should be investigated further. Acknowledgements We thank Defra for funding this research and Lucia Privitera for carrying out the Na +K +ATPase activity measurements. References Bergheim, A., Hustveit, H., Kittelsen, A., Selmer-Olsen, A.R., 1984. Estimated pollution loadings from Norwegian fish farms. II. Investigations 1980–1981. Aquaculture 36, 157–168. Bergheim, A., Selmer-Olsen, A.R., 1978. River pollution from a large trout farm in Norway. Aquaculture 14, 267–270. Boaventura, R., Pedro, A.M., Coimbra, J., Lencastre, E., 1997. Trout farm effluents: characterization and impact on the receiving streams. Environmental Pollution 95, 379–387. Brown, S.E., Goulder, R., 1996. Extracellular-enzyme activity in trout-farm effluents and a recipient river. Aquaculture Research 27, 895–901. Camargo, J.A., 1992. Temporal and spatial variations in dominance, diversity and biotic indices along a limestone stream receiving a trout farm effluent. Water, Air, and Soil Pollution 63, 343–359. Camargo, J.A., Gonzalo, C., 2007. Physicochemical and biological changes downstream from a trout farm outlet: comparing 1986 and 2006 sampling surveys. Limnetica 26, 405–414. Carey, J.B., McCormick, S.D., 1998. Atlantic salmon smolts are more responsive to an acute handling and confinement stress than parr. Aquaculture 168, 237–253. Carr, O.J., Goulder, R., 1990. Fish-farm effluents in rivers - I. Effects on bacterial populations and alkaline phosphatase activity. Water Research 5, 631–638. Dumas, J., Bassenave, J.G., Jarry, M., Barriere, L., Glise, S., 2007. Effects of fish farm effluents on egg-to-fry development and survival of brown trout in artificial redds. Journal of Fish Biology 70, 1734–1758. Foy, R.H., Rosell, R., 1991. Loadings of nitrogen and phosphorus from a Northern Ireland fish farm. Aquaculture 96, 17–30. Greig, S.M., Sear, D.A., Smallman, D., Carling, P.A., 2005. Impact of clay particles on the cutaneous exchange of oxygen across the chorion of Atlantic salmon eggs. Journal of Fish Biology 66, 1681–1691. Kelly, L.A., Bergheim, A., Hennessy, M.M., 1994. Predicting output of ammonium from fish farms. Water Research 28, 1403–1405. Kelly, L.A., Karpinski, A.W., 1994. Monitoring BOD outputs from land-based fish farms. Journal of Applied Ichthyology 10, 368–372. Kirkagac, M.U., Pulatsu, S., Topcu, A., 2009. Trout farm effluent effects on water sediment quality and benthos. Clean-Soil, Air, and Water 37, 386–391. Korzeniewski, K., Banat, Z., Moczulska, A., 1982. Changes in water of the Uniesc and Skotowa rivers caused by intensive trout culture. Polish Archives of Hydrobiology 29, 683–691. Korzeniewski, K., Salata, W., 1982. Effect if intensive trout culture on chemistry of Lake Letowo waters. Polish Archives of Hydrobiology 29, 633–657. Korzeniewski, K., Trojanowski, J., Trojanowska, C., 1985. Hydrochemical study of Lake Szcytno Male with trout cage culture. Pol. Arch. Hydro. 32, 157–174. Loch, D.D., West, J.L., Perlmutter, D.G., 1996. The effect of trout farm effluents on the taxa richness of benthic macroinvertebrates. Aquaculture 147, 37–55. Lower, N., Moore, A., 2007. The impact of a brominated flame retardant on smoltification and olfactory function in Atlantic salmon (Salmo salar L) smolts. Marine and Freshwater Behaviour and Physiology 40, 267–284.

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