Dietary copper exposure in the African walking catfish, Clarias gariepinus: Transient osmoregulatory disturbances and oxidative stress

Aquatic Toxicology 83 (2007) 62–72

Dietary copper exposure in the African walking catfish, Clarias gariepinus: Transient osmoregulatory disturbances and oxidative stress I. Hoyle, B.J. Shaw, R.D. Handy ∗ Ecotoxicology and Stress Biology Research Group, School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, United Kingdom Received 19 January 2007; received in revised form 19 March 2007; accepted 20 March 2007

Abstract There are few dietary Copper (Cu) toxicity studies on warm water fish. We used the African walking catfish (Clarias gariepinus) to perform the first in vivo dietary Cu toxicity study on this species. We measured end points normally associated with metal toxicity (nutritional performance, haematology, histology, tissue Cu, Na+ , and K+ , Na+ K+ -ATPase activity) and add to the limited data on oxidative stress during dietary Cu exposure (thiobarbituric acid reactive substances or TBARS, and total glutathione). Clarias gariepinus were fed to satiation on a Cu-loaded diet (1500 mg Cu kg−1 dw feed), or a control diet (15 mg Cu kg−1 dw feed), for 30 days. Dietary copper exposure caused elevated Cu concentrations in the intestine (20 fold), liver (5 fold) and gills (4 fold) of Cu-exposed fish compared to controls after 30 days (ANOVA, P < 0.05). Copper-exposed fish showed a reduction in food intake and specific growth rate (SGR), but only very modest reductions in mean body mass at the end of the experiment (the latter not statistically significant). There were no treatment-dependent effects on food conversion ratio or proximate composition, and only transient disturbances to tissue electrolytes and Na+ K+ -ATPase activity. Haematology was normal throughout the experiment. Cu-exposed fish showed an increase in TBARS in the gill (1.5 fold) and intestine (2 fold increase) compared to the controls (ANOVA or Kruskal–Wallis, P < 0.05). Total glutathione content in the intestine of Cu-exposed fish doubled by the end of the experiment compared to controls, reaching 12.7 ± 2.85 ␮mol g−1 wet weight (mean ± S.E.M., n = 6, Student’s t-test, P < 0.05). The liver showed some glycogen depletion consistent with reduced food intake, but no overt pathologies in the gills, liver or intestine were observed. © 2007 Elsevier B.V. All rights reserved. Keywords: Dietary copper; Clarias gariepinus; Na+ K+ -ATPase; TBARS; Glutathione; Oxidative stress

1. Introduction There is now a considerable literature on dietary copper (Cu) exposure in fish (reviews, Handy, 1996; Clearwater et al., 2002; Handy et al., 2005) and laboratory studies have addressed several issues including Cu metabolism (Grosell et al., 1998), growth and survival (Clearwater et al., 2002), physiological and behavioural effects (Handy et al., 1999; Kamunde et al., 2001; Campbell et al., 2005), Cu levels for aqua feeds (Berntssen et al., 1999a,b), and interactions of dietary salt and aqueous Cu exposure (Kjoss et al., 2005; Niyogi et al., 2006). However, most of this ecotoxicological research has been on temperate water species, especially trout and salmon. There are only a few reports of dietary Cu exposure in warm water species of fish.

∗

Corresponding author. Tel.: +44 1752 232900; fax: +44 1752 232970. E-mail address: [email protected] (R.D. Handy).

0166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2007.03.014

The nutritional Cu requirements of a few warm-water fish have been suggested (e.g. Oreochromis hybrids, about 4 mg Cu kg−1 feed, Shiau and Ning, 2003). The nutritional Cu requirements of African walking catfish (Clarias gariepinus, Burchell, 1822) are uncertain, but the fish are healthy and grow on typical aqua feeds containing around 10 mg Cu kg−1 , and their general nutritional requirements are broadly similar to other teleosts (Baker et al., 1997). The toxicological information on warm-water species is mostly limited to studies on growth performance. Dietary Cu levels of around 16 mg kg−1 food depress growth in the channel catfish, Ictalurus punctatus (Murai et al., 1981), and Nile tilapia fed 1500 mg Cu kg−1 dry weight (dw) of feed for 42 days showed decreased growth rate with postexposure hepatic lipidosis (Shaw and Handy, 2006). Clarias gariepinus remains an important aquacultural species in Africa and Asia (Chimatiro, 1998; De Silva et al., 2006), and are grown in areas heavily contaminated from copper mines (Syakalima et al., 2001). This has prompted studies on aqueous Cu toxicity to African walking catfish (e.g. Kotze et al., 1999; Van Vuren

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et al., 1994), but apart from some in vitro studies on the gut (Handy et al., 2000, 2002), dietary Cu toxicity has not been systematically investigated in this species from an ecotoxicological perspective. The overall aim of this study was to add to the sparse literature on dietary Cu toxicity to warm-water species of fish by overviewing the toxic effects of dietary Cu in vivo to the African walking catfish. The study also contributes to the limited literature demonstrating oxidative stress during dietary Cu exposure in fish (e.g. Baker et al., 1998; Berntssen et al., 2000) and partly compliments existing in vitro data on the intestinal Cu uptake models in this species (Handy et al., 2000, 2002). 2. Materials and methods 2.1. Experimental design African walking catfish weighing 89.1 ± 20.4 g (mean ± S.E.M., n = 360) were obtained from Sparsholt College (Winchester, Hampshire, UK) and held for 3 weeks in stock aquaria supplied with recirculating, aerated Plymouth tap water (see Section 2.7 below for water quality) and fed to satiation on a commercial fish food (standard No. 40, Skretting, Northwich, Cheshire, UK). Fish were then graded into six 250 l experimental aquaria (65 fish in each tank) supplied with recirculating Plymouth tap water (filtered by a 1000 l biofilter) flowing at a rate of 8 l min−1 in each tank. The pre-filters on the biofilter were cleaned daily, and the system had an automated trickle feed of clean fresh water to replace incidental water lost from the system (e.g. by evaporation or splashing). Fish were held on a 12L:12D photoperiod throughout. Initially all fish were fed on the control diet, with no added copper, for 1 month to ensure feeding had resumed in the experimental tanks. Thereafter, three tanks were randomly selected and the fish in them were fed a Cu-supplemented diet for 30 days (1500 mg Cu kg−1 food) while the other tanks remained on a normal control diet (15 mg Cu kg−1 food). The level of Cu for the supplemented diet was selected to be above threshold values for reduced growth in teleosts (Handy et al., 1999; Clearwater et al., 2002), but was not expected to produce lethal toxicity or abolish feeding (Handy, 1996; Baker et al., 1998). Fish were given 2 feeds a day to satiation at 10 am and 3 pm, and fed for 30 days on the appropriate diet. Fish were sampled from the tanks every 10 days for metal analysis, haematology and biochemistry (see below). Care was taken to ensure that all food pellets added to the tanks were eaten. Tanks were fed randomly to minimise tank bias effects on feeding. The self-cleaning design of the tanks removed faecal waste quickly. Nonetheless, water samples were taken to assess potential Cu leaching from expelled faecal material. Aqueous Cu levels remained low throughout the experiment (see below). 2.2. Diet formulation The control diet was prepared from raw ingredients using the following formulation (in g kg−1 dry weight): fishmeal 560; marine fish oil 35 (cod liver oil); vegetable oil 20; vitamins

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40; minerals 40; cornstarch 210; dextrin 75; carboxymethylcellulose 20. The Cu supplemented diet was prepared using the same formulation except that 5.9 g kg−1 of cornstarch was omitted to compensate for the mass of copper sulphate added to the food. A Cu-supplement of 5.9 g of CuSO4 ·5H2 O dissolved in 500 ml of water was added to the blended dry ingredients. The resulting paste was extruded through a Hobart food mixer and air dried at 40 ◦ C for 96 h. Pellets were stored in air-tight containers until required. Copper content of the food was confirmed by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Perkin-Elmer Liberty 200 AES with Varian software) and was (mean ± S.E.M., n = 6) 15.62 ± 1.95 and 1495 ± 4.6 mg kg−1 dw for control and Cu-supplemented diets, respectively (equivalent to 0.25 and 23.5 mmol Cu kg−1 feed, respectively). The moisture, protein, lipid, and ash content (% dry matter, ± S.E.M., n = 6) were: 8 ± 0.19, 39 ± 0.11, 13 ± 0.14, and 10 ± 0.12% for the control diet, compared to 10 ± 0.12, 37 ± 0.36, 13 ± 0.05 and 9 ± 0.05% for the copper diet. 2.3. Growth and nutritional performance To establish whether exposure to dietary copper influenced nutritional performance several parameters were measured as previously described (Handy et al., 1999). Briefly, food intake was calculated from the amount of food fed to each tank per day (by weighing food containers before and after feeding to calculate food eaten). Condition factor (K = weight (g)/alengthb (cm), where a = 0.0032 and b = 3.214 for African catfish, Britton and Harper, 2006) was calculated for each treatment from individual fish weights at each sampling time in the experiment. Specific growth rate (SGR = (Ln final weight − Ln initial weight/number of days) × 100) and food conversion ratio (FCR = dry feed fed/wet weight gained) were calculated from the mean weights of the fish. The serial sacrifice and removal of fish from the tanks during the experiment prevented these nutritional parameters from being calculated using cumulative biomass per tank. Thus, SGR was calculated from the mean gain in body weight over the course of the whole experiment. The proximate composition of the carcass was determined for 6 fish taken at random from the tanks 1 day before the Cu diet was introduced (initial fish), and from a further 6 fish per treatment at the end of the experiment. Apparent net Cu retention for each treatment was calculated from the whole body Cu concentrations and ingested Cu (apparent net Cu retention = (final carcass metal content − initial carcass metal content/metal consumed) × 100%). 2.4. Tissue metal analysis and haematology Every 10 days, 14 fish were collected from each treatment (3–4 fish/tank for each analysis) for tissue metal analysis and biochemistry (7 fish for ion analysis, 7 for tissue Na+ K+ -ATPase). Fish were sacrificed by a sharp blow to the head and the brain pithed in accordance with ethical approval. For all procedures, tissues were collected with ion-clean dissection instruments and acid-washed glassware was used. Tissues for metal analysis were

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dissected in the order of gill, liver and then intestine, rinsed in deionised water (fish were also not fed on the morning of sampling to minimise material in the gut) and stored in −20 ◦ C until digested for metal analysis according to Handy et al. (2000). Briefly, tissues were oven dried, digested in 5 ml of concentrated Aristar grade nitric acid at 50 ◦ C for 2 h, then diluted to 20 ml with deionised water prior to Cu, K+ and Na+ analysis using ICP-AES (as above). Detection limits for 0.1 g of tissue, ICPAES were (in ␮mol l−1 ): K+ , 3.5; Na+ , 1.48; Cu, 0.096. Samples were matrix matched with standards and spike recovery tests as described in Handy et al. (2000). At the end of the feeding trial, an additional 6 fish from each treatment were anaesthetised with unbuffered MS 222 and whole blood was collected from the caudal vein into lithium-heprinised syringes. Haematocrit was determined immediately (in duplicate), and a 20 ␮l aliquot of blood was also mixed with 0.98 ml of Dacie’s fluid in preparation for red cell counts (Handy and Depledge, 1999). 2.5. Biochemistry Gills and intestines were dissected from 7 fish/treatment, rapidly frozen in liquid nitrogen, and stored at −80 ◦ C for subsequent biochemistry. Tissues (0.5 g) were homogenised in 5 volumes (2.5 ml) of ice-cold isotonic buffer (in mmol l−1 , 300 sucrose, 0.1 EDTA (ethylenedinitrilo-tetraacetic acid), 20 HEPES (4-(2-hydroxyethyl)-1-piperazineerganesulfonic acid), and adjusted to pH 7.8 with Tris(2-amino-2-hydroxylmethyl1,3-propanediol)). Tissues were homogenised on ice using a free standing homogeniser (Cat X520D with a T6 shaft, Bennett & Co., Weston Super-Mare) at medium speed (3 × 10 s bursts), and then frozen in 0.5 ml aliquots at −80 ◦ C until required for analysis. Three assays were performed (see below), Na+ K+ -ATPase to aid interpretation of any effects on osmoregulation, as well as total glutathione and thiobarbituric acid reactive substances (TBARS) to assess oxidative stress. The Na+ K+ -ATPase assay was derived from Bonting et al. (1961) with modifications for fish tissue by Silva et al. (1977) and is based on the liberation of inorganic phosphate from ATP in the presence/absence of K+ (the K+ containing buffer in mmol l−1 : 100 NaCl, 10 KCl, 5 MgCl2 , 5 Na2 ATP, 30 HEPES, pH 7.4; the K+ -free buffer: as above without KCl, plus 1.0 mmol l−1 ouabain). Briefly, after optimising incubation times and reagent volumes, 15 ␮l of homogenate (in triplicate for each sample) and 400 ␮l of each assay buffer (with/without K+ ) were incubated at 37 ◦ C for 10 min, and the reaction then stopped by the addition of 1 ml of cold trichloroacetic acid. One milliliter of colour reagent was added to each tube (9.6% w/v FeSO4 ·6H2 O, 1.15% w/v ammonium heptamolybdate dissolved in 0.66 M H2 SO4 ), and colour allowed to develop for 20 min at room temperature. Absorbances were then measured at 630 nm on a Dynex MRX plate reader against 0–0.5 mmol l−1 phosphate standards. Total glutathione was determined according to Owens and Belcher (1965). Briefly, 20 ␮l of gill or intestine tissue homogenate (in triplicate) was added to a microplate well containing 20 ␮l of 10 mmol l−1 DTNB (5,5 -dithiobis(2-nitrobenzoic acid)), 260 ␮l of assay buffer (100 mmol l−1

K2 HPO4 , 5 mmol l−1 EDTA, pH 7.5), and 20 ␮l of 2 U ml−1 glutathione reductase (Sigma chemicals, Poole, UK). After mixing for 1 min, the reaction was started by adding 20 ␮l of 3.63 mmol l−1 NADPH. The change in absorbance of the samples over approximately 15 min was recorded at 412 nm (Dynex MRX) and calibrated (in triplicate) against reduced glutathione standards (0–200 ␮mol l−1 GSH). Total lipid peroxidation products were measured by TBARS according to Camejo et al. (1998). Briefly, 40 ␮l of homogenised tissue (as above) was added to a well of a 96-well microplate (in triplicate), containing 1 mmol l−1 butylated hydroxytoluene(2,6-di-O-tert-butyl-4-methylphenol), and the final volume was made up to 190 ␮l with 1 mmol l−1 phosphate buffered saline (adjusted to pH 7.4). Following this, 50% (w/v) trichloroacetic acid (TCA) and 1.3% (w/v) thiobarbituric acid (TBA) (dissolved in 0.3% (w/v) NaOH), were added, and the plate incubated at 60 ◦ C for 60 min and then cooled on ice. Absorbances were recorded (in triplicate) first at 530 nm and then at 630 nm (OptimaxTM tunable microplate reader, Molecular Devices) to normalize samples for any turbidity prior to reading against 1,1,3,3-tetraethoxypropane standards (0.5–25 nmol ml−1 ). 2.6. Histology Six catfish were taken at random from each treatment at the final time point for histological examinations, and were carefully euthanized with an over dose of anaesthetic (buffered MS 222). Samples of gill, liver and intestine were carefully dissected as described above. Tissues samples were fixed in buffered formal saline and processed for wax sectioning. Wax sections were cut at 7 ␮m and subsequently stained with Mallory’s trichrome (Handy et al., 1999). Gill injuries were quantified by manually counting the number of injuries observed in each slide. Two primary filaments were selected from the middle of the second gill arch from each fish, and at least 100 secondary lamellae were counted in each specimen. 2.7. Water quality Water samples were collected on a weekly basis. In addition, random checks were made between weekly sampling. Water samples were collected in ion-clean tubes (13 ml) and acidified with one drop of 6 M nitric acid and stored at 4 ◦ C until analysed. Water quality was (in mmol l−1 , means ± S.E.M., n = 10): Ca, 0.42 ± 0.12; Mg, 0.13 ± 0.003; Na, 0.32 ± 0.006; K, 0.09 ± 0.003; Cu, <0.0000001 (6 ␮g l−1 ); Zn, <0.0000001; total ammonia 0.02 ± 0.01; pH 6.9 ± 0.14; dissolved oxygen, 80% ± 3; temperature 26 ◦ C ± 0.27. Trace metals were determined using ICP-AES (as above). All pH measurements were performed using a low conductivity pH electrode (Russell, Auchtermuchty, Scotland). Dissolved oxygen was measured using a portable oxygen meter (OXI 96, WTW, Weilheim, Germany). There were no statistical differences between the water quality in the control or treatment tanks for any parameter (P > 0.05, paired t-test).

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2.8. Statistical analysis All statistical analyses were carried out using Statgraphics 4.0 (at 95% confidence limits) as earlier described in Handy et al. (2000). Briefly, after checking for kurtosis, skewness and unequal variances (Bartlett’s test), data were tested for treatment or time effects by one-way analysis of variance (ANOVA) followed by a Fisher’s 95% least-squared difference (LSD) multiple-range test. When ANOVA could not be applied, the non-parametric Kruskall–Wallis test was used and post hoc differences located by notched box and whisker plots. In some of the biochemistry and haematology, treatment effects at the end of the experiment were explored using a 2-tailed Student’s t-test. 3. Results 3.1. Copper accumulation Exposure to copper via the oral route was confirmed by large increases in the copper content of the intestine and liver compared to fish on the control diet (Kruskall–Wallis, P < 0.05), while contamination in the gills remained low (Fig. 1). Over the entire experiment, Cu levels in the intestine and liver increased 20 fold and 4.7 fold, respectively, compared to controls (Fig. 1). Copper levels in the gills of control and Cu-fed fish showed a small (but statistically significant) decrease in Cu content at day 10 compared to the initial fish, but this quickly recovered (Fig. 1). Copper concentrations in the gills of Cu-fed fish were about 0.3 ␮mol g−1 dw after day 20 of exposure, and remained significantly higher (Kruskall–Wallis, P < 0.05) than the Cu levels in the gills of the control (or initial fish) for the rest of the experiment. 3.2. Growth and nutritional performance No tank effects within treatment were observed in the experiment, and overall, fish from both treatments gained body weight during the experiment (Fig. 2) without mortality. The Cu-fed fish did show a slight trend towards a lower mean body weights compared to the controls but this was not statistically significant (ANOVA P > 0.05). However, the trend is apparently consistent with the slightly lower cumulative food intake of the Cu-fed fish (Fig. 2). Food regurgitation was not observed. FCR was similar in both groups of fish, and no statistical differences in carcass proximate composition were observed between treatments at the end of the experiment (Table 1, ANOVA, P > 0.05). Whole body Cu concentration did increase significantly in Cu-fed fish compared to controls (Table 1). Calculated apparent Cu retention from whole body Cu and the total ingested Cu, gave estimates of 355% for control fish and 8.9% for Cu-fed fish. 3.3. Haematology and tissue electrolytes There were no treatment-dependant changes in the red cell counts, whole blood haemoglobin concentration, or haematocrit after 30 days of dietary Cu exposure (unpaired, 2-tailed t-tests, P > 0.05). Red cell counts were 2.16 ± 0.3 and

Fig. 1. Copper accumulation in the (A) intestine, (B) liver, and (C) gill of African walking catfish fed either a control diet (open circles, 15 mg Cu kg−1 feed) or a Cu-contaminated diet (filled circles, 1500 mg Cu kg−1 feed) over 30 days. Data are means ± S.E.M., n = 7–9 fish. * Significant difference from the control diet within time point, different letters indicate significant time effect within treatment (Kruskall–Wallis, P < 0.05). Note: time zero is for one group of the initial fish collected immediately prior to starting the experimental diets (grey circle).

2.11 ± 0.02 × 106 mm3 for control and exposed fish respectively (mean ± S.E.M., n = 6). Haemoglobin content of whole blood was 8.33 ± 0.69 and 7.95 ± 1.7 g dl−1 for control and exposed fish, respectively (mean ± S.E.M., n = 6). Haematocrits were 30 ± 2.2% and 32 ± 3.9% for control and exposed fish, respectively (mean ± S.E.M., n = 6). There were no statistically significant differences between treatments for tissue electrolytes or moisture content (Table 2), except for a transient decrease in tissue K+ in the liver (ANOVA, P < 0.05) and a rise in moisture content of the intestine (Kruskall–Wallis, P < 0.05) at day 20. There were some time effects within treatment, mainly in the liver, which showed a progressive increase in tissue Na+ and K+ , and declining moisture content compared to the initial fish (Table 2). The intestine also showed a general time-dependent decrease in moisture content, but no Cu-related effect (Table 2).

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Table 1 The effect of dietary copper on nutritional performance of African walking catfish fed either a control diet or a Cu-contaminated diet over 30 days Control diet (15 mg Cu kg−1 feed)

Parameter Initial mean weight (g) Final mean weight (g) Initial condition factor Final condition factor SGR (% day−1 ) FCR Final whole body [Cu] (␮mol g−1 dw)

109.45 ± 27.8 (27)

165.7 ± 55.6 (33)

73.5 16.4 5.4 3.3

± ± ± ±

144.48 ± 43.39 (33)

0.93 ± 0.03 (27)

0.95 ± 0.01 (33) 1.38 0.61 ± 0.07 (3) 0.82 ± 0.01 (6)

Proximate carcass compositiona Moisture (%) Protein (%) Lipid (%) Ash (%)

Copper diet (1500 mg Cu kg−1 feed)

0.96 ± 0.01 (33) 0.93 0.68 ± 0.09 (3) 1.28 ± 0.04* (6)

0.5 (6) 0.5 (6) 0.4 (6) 0.1 (6)

72.6 15.8 6.2 3.3

± ± ± ±

0.8 (6) 0.3 (6) 0.6 (6) 0.1 (6)

Data are means ± S.E.M. (n fish/treatment), except food conversion ratio (FCR) which is a mean of triplicate tanks/treatment for the entire experiment. Condition factor is in arbitrary units derived from the equation reported in the methods. Specific growth rate (SGR) is calculated from the mean change in fish weight for each treatment over the entire 30 days. * Significant increase in whole body copper (Kruskall–Wallis, P = 0.0014). a Values are for fish at the end of the experiment expressed as a percentage of dry matter. Proximate carcass composition of initial fish for moisture, protein, lipid and ash, respectively, were (%, means ± S.E.M. n = 6): 73.7 ± 0.3; 15.9 ± 0.5; 5.8 ± 0.6; 3.1 ± 0.1. No significant differences were observed between treatments for proximate composition or body size (ANOVA, P > 0.05).

3.4. Biochemistry: Na+ K+ -ATPase, total glutathione and TBARS Na+ K+ -ATPase activities in crude homogenates of gill and intestine were also assessed (Fig. 3). There were no statistical

differences in Na+ K+ -ATPase activities between treatments by the end of the experiment (day 30, ANOVA, P > 0.05). Na+ K+ ATPase activities in the gill at day 30 were 2.5 ± 0.63 and 3.0 ± 0.73 ␮mol Pi mg−1 protein h−1 (mean ± S.E.M., n = 7) for control and Cu-exposed, respectively. Similarly for

Table 2 The effect of dietary copper on tissue electrolytes and moisture content of African walking catfish fed either a control diet (15 mg Cu kg−1 feed) or a Cu-contaminated diet (1500 mg Cu kg−1 feed) over 30 days Tissue/electrolyte (␮mol g−1 dw)

Treatment

Time (days) 0 (Initial fish)

10

20

30

Control Copper

115.4 ± 25.3

124.6 ± 14.0 123.0 ± 7.9

140.8 ± 15.3 145.2 ± 12.7

117.6 ± 12.4 121.9 ± 7.9

Liver

Control Copper

33.1 ± 3.2a

74.8 ± 5.8b 75.8 ± 7.9b

Gill

Control Copper

214.9 ± 16.9

242.8 ± 18.0 227.2 ± 16.4

251.9 ± 26.4 238.1 ± 10.6

224.8 ± 12.4 232.6 ± 4.1

Control Copper

286.7 ± 67.9

334.5 ± 32.3 305.3 ± 11.4

303.5 ± 21.2 314.7 ± 6.7

271.8 ± 14.4 290.6 ± 18.0

177.7 ± 12.8b 179.6 ± 14.7b

205.4 ± 18.1b 170.4 ± 9.2*,b

203.6 ± 12.7b 197.2 ± 11.3b

128.3 ± 16.0 121.6 ± 6.3

144.1 ± 14.1 133.9 ± 4.8

128.8 ± 7.7 132.2 ± 3.8

Na+

Tissue Intestine

Tissue K+ Intestine Liver

Control Copper

Gill

Control Copper

Tissue moisture (%) Intestine

87.7 ± 13.4a 125.4 ± 9.0

89.9 ± 10.5c 84.0 ± 9.3b

90.4 ± 10.6c 93.2 ± 6.4b

Control Copper

82.4 ± 1.9a

80.4 ± 0.5a 80.3 ± 0.6b

79.3 ± 1.6a 84.1 ± 0.7a,*

77.4 ± 0.5b 79.3 ± 1.5b*

Liver

Control Copper

62.0 ± 1.3a

66.3 ± 1.0a 66.5 ± 1.1b

66.9 ± 0.5b 68.4 ± 1.5b

71.1 ± 0.9c 69.4 ± 0.9c

Gill

Control Copper

76.3 ± 1.5a

76.5 ± 0.8a 79.4 ± 2.2a

75.5 ± 1.0a 80.0 ± 0.8a

76.1 ± 0.9a 75.8 ± 0.6b

Data are means ± S.E.M., n = 7–9 fish. * Significant difference from the control diet within time point. Different superscript letters a, b, c indicate significant time effect within treatment (Kruskall–Wallis or ANOVA, P < 0.05). Where no letters are shown, no time-effect within treatment occurred. Note: time zero is for one group of the initial fish collected immediately prior to starting the experimental diets.

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Fig. 2. Weight gain (A) and cumulative food intake (B) of African walking catfish fed a control diet (open symbols, 15 mg Cu kg−1 feed) or a Cu-contaminated diet (filled symbols, 1500 mg Cu kg−1 feed) for 30 days. In panel (A) data are means ± S.E.M., n = 21–27 fish. # Significant difference in fish weight compared to initial fish (Kruskall–Wallis, P < 0.05), no treatment-effects were observed in fish weight. In panel (B) cumulative food intake is the sum of food eaten/treatment (mean of triplicate tanks) up to each time point.

intestine, Na+ K+ -ATPase activities were 3.5 ± 0.81 and 4.1 ± 0.35 ␮mol Pi mg−1 protein h−1 (mean ± S.E.M., n = 7) for control and Cu-exposed fish, respectively. However, there was a transient 45% decrease in Na+ K+ -ATPase activity in the intestine of Cu-exposed fish compared to controls at day 20 (statistically significant, Student’s t-test, P < 0.05), but this treatment-effect was lost by the end of the experiment (Fig. 3). Total glutathione content in the intestine of Cu-fed fish showed a statistically significant rise (203% increase, Student’s t-test, P = 0.04) compared to controls at the end of the experiment (Fig. 4). Gill tissue also showed a trend of increasing total glutathione levels (73% increase, Fig. 4), although this was not statistically significant (Student’s t-test, P = 0.095). Fish fed excess dietary Cu showed a progressive increase in tissue lipid peroxidation compared to controls, as measured by the TBARS assay (Fig. 5). The treatment effect was statistically significant from day 20 onwards, in both gill and intestine (gill, ANOVA, P = 0.005; intestine, Kruskall–Wallis, P = 0.012). At the end of the experiment, Cu-fed fish had TBARS in the gills of 2.85 ± 0.28 compared to 1.83 ± 0.18 ␮mol mg−1 protein (mean ± S.E., n = 6) in the controls. Background levels of TBARS in the intestine were much lower than gill tissue (Fig. 5A

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Fig. 3. Na+ K+ -ATPase activity in the (A) gill and (B) intestine of African walking catfish fed either a control diet (open circles, 15 mg Cu kg−1 feed) or a Cu-contaminated diet (filled circles, 1500 mg Cu kg−1 feed) over 30 days. Data are means ± S.E.M., n = 6 fish. No overall time or treatment effects were observed by ANOVA or Kruskall–Wallis (P > 0.05). However, the appropriate post hoc analysis (e.g. multiple range test for ANOVA) found some differences in individual data points (also confirmed by separate t-tests at those data points). * Significant difference from the control diet within time point. # Significantly different from initial fish (time zero, grey circle).

and B), but the intestine of Cu-fed fish showed the same effect. At the end of the experiment TBARS in the intestine were 0.78 ± 0.016 and 0.39 ± 0.08 ␮mol mg−1 protein in Cu-fed and control fish, respectively. 3.5. Histology Overall there were no overt toxicological effects of dietary Cu exposure on the gills, liver or intestine of the fish at the end of the experiment (Fig. 6). The gills were examined mainly to confirm the route of exposure by showing that the fish did not have pathologies normally associated with aqueous metal expo-

Fig. 4. Total glutathione concentration in the gill and intestine of African walking catfish fed either a control diet (open bars, 15 mg Cu kg−1 feed) or a Cu-contaminated diet (filled bars, 1500 mg Cu kg−1 feed) after 30 days. Data are means ± S.E.M., n = 6–7 fish, expressed per mg wet weight (ww) of tissue. * Significant difference from the control diet (Student’s t-tests, P = 0.095 for gill, P = 0.048 for intestine).

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rhage, peri-venule injuries, or abnormal lipidosis. Five out of the six control fish examined had hepatocytes with much larger opaque areas within the cells (apparent glycogen stores that do not take the stain) compared to the livers from all the Cu-fed fish (Fig. 6C and D). This relative reduction in apparent glycogen content in the livers of Cu-fed fish is consistent with their lower cumulative food intake compared to controls. Overt lipidosis was absent from all the fish examined. However, 5 out of 6 livers examined from the Cu-fed fish had occasional foci of lipidosis (probably not pathological but related to glycogen depletion), and this also was observed in the one control fish examined with a lean liver. In 2 out of 6 livers from the Cu-fed fish, a rare incidence of individual cells in the early stages of nuclear atrophy was observed (one or two cells/field of view, sub-clinical) and this was not observed in the controls. 4. Discussion

Fig. 5. Thiobarbituric acid reactive substances (TBARS) in the (A) gill and (B) intestine of African walking catfish fed either a control diet (open circles, 15 mg Cu kg−1 feed) or a Cu-contaminated diet (filled circles, 1500 mg Cu kg−1 feed) over 30 days. Data are means ± S.E.M., n = 7 fish. * Significant difference from the control diet within time point, different letters indicate significant time effect within treatment (by ANOVA or Kruskall–Wallis, P < 0.05). Note: time zero is for one group of the initial fish collected immediately prior to starting the experimental diets (grey circle).

sure. The gills of all fish showed normal histology with uniform secondary lamellae without overt oedema, epithelial lifting, or mucous cell discharge. The background incidence of lesions on the secondary lamellae were <2% in both control and Cufed fish. The proportions of secondary lamellae with oedema were (mean percentage ± S.E.M., n = 6 fish): 1.75 ± 0.49 and 0.29 ± 0.12% of secondary lamellae for control and Cu-fed fish respectively (not significantly different, t-test, P = 0.26). The proportions of secondary lamellae with swollen tips were (mean percentage ± S.E.M., n = 6 fish): 0.93 ± 0.38 and 1.43 ± 0.32% of secondary lamellae for control and Cu-fed fish, respectively (not significantly different, t-test, P = 0.68). One control fish showed one foci of two fused secondary lamellae, and another control fish showed one aneurism at the base of one secondary lamellae. Swollen, granular or completely discharged mucocytes were not observed in the gills of any of the fish. Haemorrhage was not observed in the gills of any of the fish. In the intestine, no pathologies were observed in the serosa, muscularis or mucosa (zero incidence in all fish examined). The intestinal villi were intact without erosion of the tips of the villi, and with a uniform mucosal epithelium and normal goblet/mucus cells (Fig. 6E and F). There was no evidence of fusion of villi, vacuolation of the villi or the sub-mucosa, and fibrosis was not observed. There was no evidence of inflammation, aneurisms or haemorrhage in the muscularis and the serosa appeared normal without evidence of fibrosis or adhesions. The liver showed normal gross histology with no signs of haemor-

This study gives new data on the chronic dietary toxicity of Cu to African walking catfish in vivo. Overall we show that the fish accumulate Cu in the intestine and liver with only marginal reductions in growth rate (not statistically significant) that are associated with reduced food intake, and reflect some depletion of hepatic glycogen stores. Changes in tissue electrolytes, Na+ K+ -ATPase activity, and lipid peroxidation (TBARS) suggest these fish show transient disturbances to osmoregulation, and suffer some oxidative stress. Elevation of the glutathione pool in the intestine may help to preserve physiological integrity of the gut. 4.1. Copper accumulation Copper levels in the tissues of control fish in this study (1 ␮mol g−1 dw or less, Fig. 1) are broadly similar to previous reports for catfish (intestine, 0.3 ␮mol g−1 dw, Handy et al., 2002; gill and liver 0.1–2 ␮mol g−1 dw, AvenantOldewage and Marx, 2000), and other teleosts such as tilapia (<0.2 ␮mol g−1 dw in gill and intestine, Shaw and Handy, 2006), and rainbow trout held in similar water quality (<2 ␮mol g−1 dw or much less, Handy et al., 1999). The Cu accumulation pattern also reflected the route of exposure with large increases in the Cu content of the liver and intestine (Fig. 1), and is consistent with previous reports on teleost fish (e.g. Nile tilapia, Shaw and Handy, 2006; rainbow trout, Handy et al., 1999; Kamunde and Wood, 2003; Atlantic salmon, Berntssen et al., 1999a). Notably, Cu levels in the intestine of Cu-exposed fish start to plateau after 20 days of exposure, suggesting that the gut tissue was reaching saturation with Cu (Fig. 1). Most of this Cu (about 90%, Handy et al., 2000) is in the gut mucosa, and export from the gut cells to the blood is the rate-limiting step in Cu uptake across the intestine (Handy et al., 2000). However, at the same time (after day 20), Cu levels in the liver also showed a rapid rise (Fig. 1). This suggests that there was a lag-time between Cu overwhelming the gastro-intestinal barrier and subsequent Cu accumulation in the liver. If the exposure had continued beyond 30 days, it is possible that the liver Cu levels in the catfish might eventually reach similar levels to the gut (as observed in Nile tilapia, Shaw

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Fig. 6. Sections of the gills (panels A and B), liver (panels C and D) and intestine (panels E and F) from African walking catfish fed either a control diet (left panels, 15 mg Cu kg−1 feed) or a Cu-contaminated diet (right panels, 1500 mg Cu kg−1 feed) over 30 days. The gills and intestine were normal. Note: the hepatocytes in the livers of Cu-exposed fish show some reduction in glycogen content compared to the controls. Sections were 7 ␮m thick and stained with Mallory’s trichrome.

and Handy, 2006). This is contrary to the rainbow trout where liver Cu levels usually soon exceed those in the gut tissue during dietary exposure (e.g. Handy et al., 1999). However the trout is an unusual vertebrate animal with respect to the body distribution of Cu, having one of the highest proportions of Cu in its liver relative to other organs in the animal kingdom (e.g. 12% of whole body Cu in the liver, Handy, 1992). The gills also showed a small increase in Cu content during exposure (Fig. 1), even though waterborne Cu levels remained low and gill morphology was normal. This effect has been previously reported with

elevated dietary Cu in fish (e.g. Shaw and Handy, 2006; Handy, 1996; Kamunde et al., 2001) and reflects systemic Cu in the gill tissue, and distribution of some dietary Cu to the gill from the gut. The apparent net Cu retention in the whole carcass was about 8.9% in Cu-exposed fish, which is broadly similar to other fish fed with excess Cu (Atlantic salmon, 1–2%, Berntssen et al., 1999b) and consistent with the low Cu absorption efficiency reported in perfused intestines of Clarias during Cu exposure (3–6% or less, Handy et al., 2000). These perfused intestine

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studies also indicate a maximum Cu uptake rate across the gut of about 2.2 ␮mol g−1 h−1 (Handy et al., 2000), and this can easily explain the net increase in whole body Cu observed in the Cu-fed fish in vivo (about 0.46 ␮mol g−1 increase over controls, Table 1, or about 66 ␮mol fish−1 in 30 days). Interestingly, the control fish had an apparent net retention of about 355% which suggests the control fish were obtaining most of their Cu from sources other than the food. Cu levels in the water remained normal and fish did not regurgitate food, so with the typical Cu inclusion of a teleost commercial diet formulation, the control fish in our experiment may get two-thirds or more of their basic Cu requirements by uptake from the water. 4.2. Growth and nutritional performance African walking catfish fed excess dietary Cu showed only marginal reductions in body weight during dietary Cu exposure (not statistically significant, Fig. 2), but overall this did cause a decrease in SGR in the Cu-fed fish compared to controls over the entire experiment (Table 1). The reduction in SGR is probably explained by reduced food intake during Cu exposure (Fig. 2), which is also consistent with depletion of glycogen stores in the liver without pathology (Fig. 6). Poor absorption of the major nutrients is unlikely given that FCR, condition factor, proximate composition, and intestinal morphology were similar in both control and treated fish (Table 1, Fig. 6). These findings are similar to the response of Nile tilapia fed 2000 Cu mg kg−1 food (Shaw and Handy, 2006), which showed a reduction in growth associated with reduced food intake. 4.3. Haematology, tissue electrolytes and Na+ K+ -ATPase There were no treatment differences in haematology, and only transient changes in tissue electrolyte levels (Table 2) which suggest that African walking catfish did not suffer any major osmotic disturbances. This is consistent with previous reports on trout (Handy et al., 1999) and tilapia (Shaw and Handy, 2006). The haemoglobin and HCT ranges we report for African walking catfish (6–8 g dl−1 and 28–36%, respectively) are within the ranges reported for this species. Adham et al. (2000) give values for haemoglobin of 7–9 g dl−1 in aquaculture, and Gabriel et al. (2004) reports haemoglobins of 6–9 g dl−1 and haematocrits of 22–26% in wild African catfish. The transient changes observed in tissue electrolytes and moisture content at day 20 (Table 2) suggest that fish were able to maintain osmotic balance most of the time, but had short periods where small adjustments in osmoregulation were made. This is reflected in the Na+ K+ -ATPase activity which was stable, apart from a transient change in Cu-fed fish at day 20 (Fig. 3). Transient change in Na+ K+ -ATPase activity have been reported during aqueous Cu exposures (e.g. toadfish intestine, Grosell et al., 2004). The cause of the Na+ K+ -ATPase transient in our experiment is uncertain, but TBARS started to rise at the same time (Fig. 5), and the Na+ pump is modulated by oxidative stress (Huang et al., 1995). We are not aware of previous measurements of Na+ K+ -ATPase activity in Clarias gariepinus, but the values we report for gill and intestine (2–4 ␮mol Pi mg−1 protein h−1 )

are typical of tissue homogenates from fish (e.g. perch gills, 2–7 ␮mol Pi mg−1 protein h−1 , Alam and Frankel, 2006; intestine of Atlantic salmon, 1–6 ␮mol Pi mg−1 protein h−1 , Veillette and Young, 2005). 4.4. Oxidative stress There are only a few reports of dietary Cu-dependent oxidative stress in fish, although the cell biology of Cu-induced oxidative stress is relatively well known from mammalian studies (Stohs and Bagchi, 1995; Letelier et al., 2005). At least two report shows that high levels of dietary Cu can cause lipid peroxidation in fish (grey mullet, fed 2400 mg Cu kg−1 feed, Baker et al., 1998; Atlantic salmon, fed 691 mg Cu kg−1 feed, Berntssen et al., 2000), and hepatic fatty change during dietary Cu exposure (rainbow trout, fed 500 mg Cu kg−1 feed, Handy et al., 1999) could be partly attributed to oxidative stress. In addition, pre exposure to aqueous Cu is known to exacerbate oxidative stress responses in fish (Ahmad et al., 2005). However, few experiments have measured physiological responses to dietary Cu exposure (e.g. ionic regulation), and oxidative stress parameters (TBARS, glutathione) at the same time. The TBARS assay measures the presence of lipid peroxides, and therefore an increase in TBARS gives an approximation of oxidative stress. The values we report (Fig. 5) are broadly similar to previous measurements in fish (e.g. trout intestine, about 0.5 nmol mg−1 protein, Carriquiriborde et al., 2004). The progressive rise in TBARS in both the gills and the intestine suggest some lipid peroxidation during Cu exposure. Notably the time profiles of TBARS in the gill and intestine are almost identical (Fig. 5), and this coincidence might suggest the oxidative stress is rapidly mediated via the blood to cause simultaneous effects in different tissues. However, this idea is not supported by the haematology which remained normal throughout, and there is no pathology in the liver that could be associated with overt oxidative stress. Overall the rises in tissue TBARS during Cu-exposure did not cause toxic effects in the tissues, since no major disturbances to osmoregulation (Table 2) and only transient change in Na+ K+ -ATPase were observed (Fig. 3). This is consistent with the absence of overt pathology (e.g. no oedema, no epithelial erosion or large areas of necrosis) in the gill or intestine (Fig. 6). Together these observations suggest that anti-oxidant defences may be protecting the tissue. The total glutathione pool is one of the early chemical defences against oxygen radicals in cells (Ferreira et al., 1993), and by the end of the experiment the total glutathione pool in the intestine had doubled, as well as showing an increasing trend in the gill (Fig. 4). We therefore suggest the increase in total glutathione (Fig. 4) is primarily a protective response to oxidative stress. Glutathione is also an important Cu-carrier and is the main initial chelator of Cu in cells (Ferreira et al., 1993; Ferruzza et al., 2000). However, we do not think the rise in total glutathione is primarily associated with Cu regulation. There are only a few reports on Cu-dependent glutathione induction/depletion in fish (none on Clarias gariepnus), but these reports from aqueous Cu exposures show limited or no changes in glutathione levels even when tissue Cu levels increase (Laur´en and McDonald,

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1987; Parvez and Raisuddin, 2006). Berntssen et al. (2000) even showed a fall in total glutathione in the intestine and liver, when tissue Cu levels were rising in Atlantic salmon fed excess dietary Cu. The levels of glutathione observed in the intestine of Cu-exposed fish (12.7 ␮mol g−1 ww tissue, Fig. 4) would only account for two thirds of the intestinal Cu, even in the unlikely theoretical event that glutathione was chelating all the Cu. The total glutathione in the gill (Fig. 4) greatly exceeds the tissue Cu content (Fig. 1), so Cu regulation can only be a small role for the tissue glutathione in our experiment. 4.5. Conclusions and perspectives on dietary Cu exposure in warm water versus temperate species of fish In this study, African walking catfish showed Cu accumulation in the intestine and liver, as well as reduced SGR and food intake during dietary Cu exposure. At the biochemical level the fish show only small transient osmoregulatory disturbance, but elevated TBARS indicates some Cu-induced oxidative stress. This was followed by a rise in total glutathione that may offer some protection from oxidative stress and help avoid organ pathology. But is this response typical of warm water and temperate species of fish? The target organs for dietary Cu in African walking catfish (e.g. mainly intestine and liver) are the same as other warm water (e.g. Nile tilapia, Shaw and Handy, 2006) and temperate species (e.g. rainbow trout and Atlantic salmon, Handy et al., 1999; Kamunde and Wood, 2003; Berntssen et al., 1999a). Apparent net Cu retention is the same order of magnitude as temperate species (e.g. 8.9% retention in this study, compared to 1–2% in Atlantic salmon, Berntssen et al., 1999b). Small differences in Cu retention between species could be attributed to changes in absorption efficiency with dose (Handy et al., 2000), or differences in the feed formulations used for each species that would inevitably cause subtle changes in Cu bioavailability. Growth rate effects of dietary Cu also seem to be broadly similar in warm water and temperate species. Daily ingested dose can be used to compare the growth effects of excess dietary Cu between species (see Clearwater et al., 2002 for discussion). In our study the exposure dose was about 15 mg Cu kg−1 bw day−1 (calculated from a dietary Cu level of 1495 mg Cu kg−1 dw feed and the actual food ingested), and given the marginal changes in body weight (Fig. 1), our data suggests we are probably around the threshold dose for growth effects in this species. The dose causing clear growth effects in Nile tilapia is 25 mg Cu kg−1 bw day−1 (Shaw and Handy, 2006), and 35–45 mg Cu kg−1 bw day−1 for rainbow trout (Clearwater et al., 2002), suggesting African walking catfish have broadly similar tolerances to other warm and temperate species of teleosts. The general absence of major haematological or osmoregulatory disburbances during dietary Cu exposure is also emerging as a common feature of studies on different species of fish (Handy et al., 2005). However, it is premature to extend these arguments to the underlying biochemical and molecular responses of temperate and warm water species; mainly because there are so few measurements giving the details of time or dose effects of Cu on Na+ K+ -ATPase, TBARS, glutathione, etc. in the latter. We argue

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above that background values for these biochemical parameters in African walking catfish are similar to other teleosts, and while there is a logic for biochemical change during Cu exposure in the African walking catfish, more work is needed to confirm if this species and the responses of Nile tilapia (Shaw and Handy, 2006) are representative of all warm water fish at the biochemical level. Acknowledgements This study was partly supported by a grant from the Leverhulme Trust to R. Handy. Technical support from Michael Hockings and Robert Serwata is appreciated. References Adham, K.G., Hashem, H.O., Abu-shabana, M.B., Kamel, A.H., 2000. Vitamin C deficiency in the catfish Clarias gariepinus. Aquacult. Nutr. 6, 129–139. Ahmad, I., Oliveira, M., Pacheco, M., Santos, M.A., 2005. Angilla anguilla L. oxidative stress biomarkers responses to copper exposure with or without ␤-naphthoflavone pre-exposure. Chemosphere 61, 267–275. Alam, M., Frankel, T.L., 2006. Gill ATPase activities of silver perch, Bidyanus bidyanus (Mitchell), and golden perch, Macquaria ambigua (Richardson): effects of environmental salt and ammonia. Aquaculture 251, 118–133. Avenant-Oldewage, A., Marx, H.M., 2000. Bioaccumulation of chromium, copper and iron in the organs and tissues of Clarias gariepinus in the Olifants River, Kruger National Park, Water SA, 26, 569–582. Baker, R.T.M., Martin, P., Davies, S.J., 1997. Ingestion of sub-lethal levels of iron sulphate by African catfish effects growth and tissue lipid peroxidation. Aquat. Toxicol. 40, 51–61. Baker, R.T.M., Handy, R.D., Davies, S.J., Snook, J.C., 1998. Chronic dietary exposure to copper affects growth, tissue lipid peroxidation, and metal composition of the grey mullet Chelon labrosus. Mar. Environ. Res. 45, 357–365. Berntssen, M.H.G., Hylland, K., Wendelaar Bonga, S.E., Maage, A., 1999a. Toxic levels of dietary copper in Atlantic salmon (Salmo salar L.) parr. Aquat. Toxicol. 46, 87–99. Berntssen, M.H.G., Lundebye, A.K., Maage, A., 1999b. Effects of elevated dietary copper concentrations on growth, feed utilisation and nutritional status of Atlantic salmon (Salmo salar L.) fry. Aquaculture 174, 167–181. Berntssen, M.H.G., Lundebye, A.K., Hamre, K., 2000. Tissue lipid peroxidative responses in Atlantic salmon (Salmo salar L.) parr fed high levels of dietary copper and cadmium. Fish Physiol. Biochem. 23, 35–48. Bonting, S.L., Simon, K.A., Hawkins, N.M., 1961. Studies on Na-K-ATPase I. Quantitative distribution in several tissues of the cat. Arch. Biochem. Biophys. 95, 416–423. Britton, J.R., Harper, D.M., 2006. Length-weight relationships of fish species in the freshwater rift valley lakes of Kenya. J. Appl. Ichthyol. 22, 334–336. Camejo, G., Wallin, B., Enoj¨arvi, M., 1998. Analysis of oxidation and antioxidants using microtiter plates. In: Armstrong, D. (Ed.), Free Radical and Antioxidant Protocols. Methods in Molecular Biology, vol. 108. Humana Press Inc., Totawa, NJ, pp. 377–386. Campbell, H.A., Handy, R.D., Sims, D.W., 2005. Shifts in a fish’s resource holding power during a contact paired interaction: the influence of a coppercontaminated diet in rainbow trout. Physiol. Biochem. Zool. 78, 706–714. Carriquiriborde, P., Handy, R.D., Davies, S.J., 2004. Physiological modulation of iron metabolism in rainbow trout (Oncorhynchus mykiss) fed low and high iron diets. J. Exp. Biol. 207, 75–86. Chimatiro, S.K., 1998. Aquaculture production and the potential for food safety hazards in sub-Saharan Africa: with special reference to Malawi. Int. J. Food. Sci. Tech. 33, 169–176. Clearwater, S.J., Farag, A.M., Meyer, J.S., 2002. Bioavailability and toxicity of dietborne copper and zinc to fish. Comp. Biochem. Physiol. 132C, 269–313. De Silva, S.S., Nguyen, T.T.T., Abery, N.W., Amarasinghe, U.S., 2006. An evaluation of the role and impacts of alien finfish in Asian inland aquaculture. Aquacult. Res. 37, 1–17.

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