- Email: [email protected]
Effects of elevated dietary copper concentrations on growth, feed utilisation and nutritional status of Atlantic salmon (Salmo salar L.) fry
Aquaculture 174 Ž1999. 167–181
Effects of elevated dietary copper concentrations on growth, feed utilisation and nutritional status of Atlantic salmon žSalmo salar L. / fry Marc H.G. Berntssen ) , Anne-Kathrine Lundebye, Amund Maage Institute of Nutrition, Directorate of Fisheries, P.O. Box 185, N-5002 Bergen, Norway Accepted 7 January 1999
Abstract The present experiment was conducted to study effects of elevated dietary Cu and establish upper limits of Cu in fish feed. Atlantic salmon fry were reared for 3 months on experimental diets containing either 5 Žcontrol., 35, 500, 700, 900, or 1750 mg Cu kgy1, provided as CuSO4 P 5H 2 O. Dietary Cr2 O 3 was included Ž1%. in all experimental diets for the last two weeks in order to assess apparent digestibility of both major food components and availability of minerals ŽCu, Zn and Se.. Growth was significantly Ž P - 0.01. reduced after 3 months at dietary Cu concentrations of G 500 mg Cu kgy1 compared to the 35 mg Cu kgy1 group. Similarly, whole-body content of protein, glycogen and Se were significantly reduced at these dietary Cu concentrations compared to controls. Apparent digestibility Ž%. of the major food components and availability of Zn and Se did not differ among dietary treatments. However, apparent availability of dietary Cu was significantly reduced in fish fed experimental diets containing F 900 mg kgy1 compared to controls. Whole-body minus intestine Cu concentrations were only significantly increased in fish fed dietary Cu concentrations G 900 mg Cu kgy1 compared to controls, indicating a strong intestinal regulating capacity of dietary Cu uptake. Dietary Cu concentrations of 500 mg Cu kgy1 and above caused toxic responses in Atlantic salmon fry as concluded from the antagonistic interaction with Se, and reduced growth and whole-body energy stores Žprotein and glycogen.. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Cu concentration; Feed utilisation; Salmo salar L.
)
Corresponding author. Tel: q47-55-23-8138; Fax: q47-55-23-8095; E-mail: [email protected]
0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 0 1 5 - 0
168
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
1. Introduction Since copper is an essential element for all organisms including fish, it is often supplemented to commercial feeds ŽLorentzen et al., 1998. in concentrations which may reach the upper limit of toxicity ŽTacon and de Silva, 1983.. Supplementation of Cu to fish feed is therefore a balance between fulfilling the Cu requirement and avoiding Cu toxicity ŽMertz, 1993.. To establish the upper limits of dietary Cu supplementation, growth, feed utilisation, hepatic enzyme activity and tissue mineral accumulation have been used as toxicity parameters Že.g., Lanno et al., 1985a,b; Julshamn et al., 1988.. However, little information exits on the nutritional status of fish exposed to elevated dietary copper concentrations. This includes both whole-body composition Žlipid, protein and carbohydrate. and mineral status, which are parameters of interest in aquaculture. Tissue deposition of lipid, protein, carbohydrate and minerals is dependent on feed intake, metabolic use and intestinal absorption, and these factors can all be influenced by elevated dietary Cu concentrations. Lanno et al. Ž1985b. reported reduced feed intake in rainbow trout exposed to elevated dietary Cu concentrations, although body composition was not measured in that study. DeBoeck et al. Ž1997. reported decreased liver and muscle protein, lipid and glycogen content in the common carp exposed to waterborne Cu despite normal feed consumption. Increased metabolic expenditure for detoxification and maintenance of homeostasis was assumed to cause this change in energy stores. Since elevated dietary Cu concentrations are known to cause an onset of adaptive responses in Atlantic salmon ŽBerntssen et al., 1998., depletion of energy stores due to increased metabolic rate may also occur simultaneously. Impaired intestinal absorption may be particularly important because the intestine is the main target organ of dietary metals ŽHandy, 1992.. Mercury has been reported to reduce the intestinal absorption of glycine in rainbow trout ŽPeres et al., 1976. and carbohydrate and amino acids in toad fish ŽFarmanfarmaian et al., 1985.. Furthermore, mineral interactions at the intestinal level have been suggested to reduce the absorption of essential elements changing the mineral status of the fish Že.g., Hilton, 1989.. An earlier study on Atlantic salmon parr exposed to elevated dietary Cu showed an onset of histological Žintestinal cell proliferation and apoptosis. and biochemical Žintestinal and hepatic metallothionein. responses ŽBerntssen et al., 1998.. The aim of the present study was to investigate if elevated dietary Cu concentrations could also effect the nutritional status of Atlantic salmon fry by measuring mineral, lipid, protein and glycogen deposition and absorption. In addition, the effects of dietary Cu exposure on growth and feed conversion were investigated in Atlantic salmon fry, which is a phase in the life cycle characterised by a very high growth rate ŽAustreng et al., 1987.. 2. Material and methods 2.1. Diet preparation A fish feed based on fish meal as the protein source was used ŽTable 1., containing 200 mg kgy1 ethoxyquin as an antioxidant. Wheat was added as the carbohydrate source, capelin oil provided lipids, and gelatin was added as a binder. In addition, ground squid was added to enhance palatability of the experimental diets. Diets were
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
169
Table 1 Composition of the basal diet g kgy1 Fish meal ŽNorse LT94, Norway. Capelin oil ŽNorsalmoil, SSF, Norway. Wheat meal ŽCODRICO, Netherlands. Gelatin ŽTORO, Norway. Squid Žfreshly minced. Mineral mix a Vitamin mix b
573 119 168 28 95 9 9
a
The mineral mix provided Žmg kgy1 diet. as follows: Zn Žas ZnSO4 )7H 2 O., 68; Fe Žas FeSO4 )7H 2 O., 34; and Mn Žas MnSO4 )H 2 O., 13. b The vitamin mix provided Žmg kgy1 diet. as follows: retinyl palmitate, 5; cholecalciferol, 4.8; alpha-tocopheryl acetate, 100; menadione, 5; thiamin, 10; riboflavin, 20; pyridoxine, 10; pantothenic acid, 40; niacin, 170; biotin, 1.2; vitamin B12 , 0.02; inositol, 450; ascorbic acid Žcoated., 1000; and folic acid, 5.
supplemented with either 5, 35, 500, 700, 900 or 1750 mg Cu per kg dry diet, using CuSO4 P 5H 2 O. The copper sulphate was dissolved in 500 ml acidified water and mixed well with the other feed ingredients before pelleting. The diets were cold pelleted Ž2.5 mm diameter. after adding approximately 12% water and then dried. The dry 2.5 mm pellets were crumbled and sieved into batches of 0.6–1.0 and 1.0–1.5 mm and stored at y208C until use. Final Cu concentrations in the experimental diets Ž n s 9. were found to be: 7.2 " 0.4, 37 " 1, 467 " 10, 638 " 5, 868 " 7 and 1780 " 50 mg kgy1 DW, respectively. Concentrations of other essential elements in the diets were: Zn, 137.1 " 3.2 and Se, 1.4 " 0.2 mgrkg DW. Proximate analyses of the diets indicated a crude protein content of 54.8%, crude lipid 21.0%, ash 7.6%, and calculated carbohydrates 16.6% which contained 81 mg gy1 digestible carbohydrate. 2.2. Experimental set up The experiment was performed at the aquaculture station of the Institute of Marine Research in Matre, Western Norway. Atlantic salmon Ž Salmo salar . fry bred locally at this station were used. At the beginning of the experiment, weight, length Žfork-tail. and condition factor of the fish were Žmean " SD., 0.9 " 0.2 g, 4.4 " 0.3 cm and 1.1 " 0.1 g cmy3 , respectively Ž n s 900.. The fish had previously been fed a commercial salmon diet ŽSkretting, Norway. with a copper concentration of 10 mg kgy1 as shown by analysis. Eighteen separate tanks Ž1.5 = 1.5 = 0.5 m. were each stocked with approximately 1000 fry. Prior to the experiment, fish were fed the control diet for 3 weeks to acclimate. Each of the six experimental diets were fed to fish in triplicate tanks for 12 weeks. Fish were reared under constant light during the exposure period and fed by automatic feeders Žfor 5 s, every 7 min, 24 h per day. according to the growth tables of Austreng et al. Ž1987. with a daily rate of 3.5% of biomass for the first 8 weeks and 3.2% of the biomass for the last 4 weeks. A feed conversion of 1 gram wet mass gain per gram of dry feed fed was assumed. The feed particle size was gradually increased during the experiment. After 0, 6, 8 and 10 weeks, the fish were bulk weighed and the
170
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
amount of feed given was adjusted in accordance with the biomass. During the last 2 weeks the fish were fed the same experimental diets supplemented with Cr2 O 3 Ž1%. as an external indicator to estimate apparent digestibility and availability of the various feed constituents ŽLied et al., 1982.. Water temperature was measured daily and varied from 12.8 to 13.48C during the experimental period. Before each bulk weighing, water samples Ž50 ml. were taken from each exposure tank for Cu analysis. The fish were kept in a flow–through system with freshwater supplemented with 0.5% seawater from a local well to buffer the soft, acidic freshwater which resulted in the following water quality: pH 6.5, Ca 11.4 " 7.5 mg ly1 and Cu 0.6 " 0.2 mg ly1 . The water flow to the tanks was 12.4 l miny1 , oxygen concentrations were measured weekly and were never below 8 mg ly1 . 2.3. Biological sampling Fish were bulk weighed at the start of the experiment and after 6, 8, 10, and 12 weeks of exposure. At each sampling point, 100 fish were removed from each tank and transferred to aerated 30 l containers with static water Žchanged daily. and starved for 4 days to remove their intestinal content. Afterwards, these fish were sacrificed in tricaine methanesulfonate ŽMS-222.. Fifty of the sampled fish were pooled and frozen for subsequent whole-body analyses. The 50 remaining fish were used to measure weight Žg. and fork length Žcm. in order to determine the condition factor ŽK-factors 100ŽweightŽfork length.y3 .. Bulk weights were used to determine growth inhibition Ž%., gross feed conversion efficiency ŽGFCE, %.. After 6 and 10 weeks of exposure, an extra 100 fish from each container were removed and killed immediately to compensate for increased fish density in the containers. At the end of the experiment, an additional 46 un-starved fish from each container were sacrificed and a pooled stripped faecal sample was taken from 40 fish by pressing the abdomen from the ventral fins to the anus ŽAustreng, 1978.. The gastrointestinal tract Žfrom the oesophagus to the rectum. was dissected out from the remaining six fish. The six fish from each container were pooled and frozen for later Cu analyses of whole-body minus the gastrointestinal tract. The pooled faeces and whole-body samples were freeze dried Žuntil successive daily weighing was unchanged., homogenised and used for mineral ŽCu, Zn and Se., lipid, protein, ash, and glycogen analyses. 2.4. Composition analyses Dry matter content of samples was determined gravimetrically after freeze drying. Crude protein content in whole fish, faeces and feed was analysed by adding 5 ml H 2 SO4 Ž95–97% Merck. and two Kjeldahl tablets ŽThompson and Capper. to 40 mg of freeze-dried material. This mixture was digested at 3808C for 1.5 h, subsequently cooled to room temperature and diluted to 75 ml with Milli-Q water. The ammonium content ŽNHq . 4 in the sample solution was determined by an automated colorimetric method as described by Crooke and Simpson Ž1971., crude protein was estimated as N = 6.25. Casein ŽSigma, C-8654. was used as reference material. The method was found satisfactory within the 95% confidence limit. Digestible carbohydrate in feed and faeces
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
171
and glycogen in sample material was determined using an enzymatic method after Hemre et al. Ž1989. modified from Murat and Serfaty Ž1974. and Holm et al. Ž1986.. Starch in 0.5 g freeze-dried material was hydrolysed with the heat stable enzymes amylase Ž30 min at 808C. ŽTermamyl-120L; Novo-Industries, Denmark. and amyloglucosidase Ž30 min at 608C. ŽEC 3.2.1.3.; Boehringer.. Glucose was subsequently measured spectrophotometrically as NADPH at 340 nm after a hexokinase reaction in an automated analyser. Total lipid in whole fish and feed Ž1 g. was determined gravimetrically after extraction with ethyl acetate. 2.5. Trace element analyses For chromium analyses, 50 mg of feed or faecal sample were digested by the Kjeldahl method as described above. For other element analyses, 2 ml HNO 3 Ž65%, Suprapur, Merck. and 0.5 ml H 2 O 2 Ž30%, ISO, Merck. were added to samples of whole fish, feed Ž200 mg. or faeces Ž50 mg. and digested under pressure in a micro-wave oven ŽMilestone MLS-1200 MEGA. for 17 min Ž1 min at 250 W, 1 min at 0 W, 5 min at 250 W, 5 min at 400 W, and 5 min at 650 W., and subsequently diluted to 25 ml with Milli-Q water. Chromium, copper and zinc concentrations were analysed by flame atomic absorption on a Perkin-Elmer 3300 AAS Žthree replicates per sample.. For copper analyses, this instrument was equipped with a high sensitivity nebulizer Ždetection limit 0.005 mg Cu ly1 .. Se concentrations were analysed by cold vapour atomic absorption ŽPerkin-Elmer 3300 AAS., with the use of a standard addition curve to compensate for phosphorous interaction. Water samples were acidified with nitric acid Ž65% HNO 3 , Suprapur, Merck. in a final concentration of 5.2% Ž2 ml HNO 3 added to 25 ml sample. and analysed for total Cu by inductively coupled plasma mass spectrometry ŽPerkin-Elmer Elan 5000.. Accuracy and precision of the element analyses with each set of samples were controlled by analyses of dogfish muscle tissue DORM-1 ŽNRCC.. The methods were found satisfactory within the 95% confidence limit. 2.6. Calculations and statistics Specific daily growth rate ŽSGR. was calculated according to the formula of Houde and Schekter Ž1981.: SGR s Ž e g y 1.)100%, where g s ŽlnŽW2 . y lnŽW1 ..)Ž t 2 y t 1 .y1 and W2 and W1 are weights on day t 2 and t 1 , respectively. Gross feed conversion efficiency ŽGFCE. was calculated and modified after Kinghorn Ž1983. with the formula: GFCEs ŽW2 y W1 .)Cy1 , where W2 and W1 are weights at sampling times t 2 and t 1 , respectively, and C denotes the dry weight of feed fed in the sample period. Apparent digestibility ŽAD. and mineral availability ŽAA. was calculated after Maynard and Loosli Ž1969. using the formula: ADrAA s 100 y ŽCrd )CX f .) ŽCrf )CX d .y1 )100 where d is diet, f is faeces, Cr is chromium content and CX is nutrient content. All statistics were performed using the program STATISTICAe ŽStatsoft, USA, 1993.. A Kolmogrov–Smirnov test was used to assess normality of distribution ŽZar, 1984.. Data which were found not to be normally distributed were log transformed. The
172
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
homogeneity of variances was tested using Levene F-test ŽZar, 1984.. Differences in mineral, protein, lipid, glycogen, ash content, digestibility, and growth among dietary treatments at different time points were assessed by one-way analysis of variance ŽANOVA.. To account for the variance among the experimental tanks within one dietary treatment, differences in individual length, weight, K-factor, and Cu water content among dietary treatments on different dates were assessed by two-way analysis of variance Žnested ANOVA. ŽZar, 1984. with the Žrandom. experimental tanks nested in their dietary exposure groups. Significance was tested using Tukey’s HSD test Ž P - 0.01 and P - 0.05. ŽSokal and Rohlf, 1981.. Pearson’s correlation coefficients were calculated to evaluate the relationships between growth inhibition and exposure time for each dietary exposure group ŽZar, 1984.. A series of models were tested to find the best fit. 3. Results 3.1. Water chemistry, growth, K-factor, GFCE, and mortality A non-significant Ž P ) 0.05. increase in total copper concentrations was observed in the water from tanks in which fish were fed with the Cu-supplemented diets compared to the tanks in which fish were fed the control diet. Concentrations of Cu in the water were 0.6 " 0.3, 0.6 " 0.2, 1.2 " 0.6, 1.6 " 0.8, 1.6 " 0.5, 1.4 " 0.4 mg ly1 Ž n s 6. for control and 35, 500, 700, 900, 1750 groups, respectively. After 12 weeks of dietary exposure, individual weight was significantly reduced Ž P - 0.01. in Atlantic salmon fed diets containing G 900 mg Cu kgy1 compared to control fish. However, individual weight was also significantly reduced in fish fed diets containing G 500 mg Cu kgy1 compared to the 35 mg Cu kgy1 diet ŽTable 2.. Growth inhibition Ž%. compared to fish fed 35 mg Cu kgy1 reached a plateau after 12 weeks of exposure in fish fed diets containing 500 and 700 mg Cu kgy1 Ž22 and 27%, respectively., whereas in the 900 and 1750 mg Cu kgy1 diet groups the growth inhibition was still exponential at the end of the experimental period Ž37 and 45%, respectively. ŽFig. 1..
Table 2 Individual weight, K-factor Ž ns150., bulk gross feed conversion efficiency ŽGFCE. Ž ns 3., and mortality of Atlantic salmon fry after 12 weeks of dietary Cu exposure Žmean"SD. 5 ŽControl.
Cu added to feed Žmg kgy1 .1 35
Weight 3 Žg. 10.3"0.9 a,b 11.9"1.5a y3 . Ž K-factor g cm 1.3"0.1a,b 1.3"0.2 a GFCE Žg gy1 . 0.9"0.1a 0.9"0.1a Ž . Mortality % 3.6"0.3 3.8"0.3 1
ANOVA2
500
700
900
1750
9.1"0.5 b 1.3"0.1a,b 0.8"0.1a,b 3.5"1.2
8.7"0.3 b,c 1.3"0.1a 0.7"0.1a,b 3.4"0.6
7.5"1.2 c,d 1.2"0.1b,c 0.5"0.0.2 b,c 4.3"0.1
6.6"0.7 d 1.2"0.1c 0.4"0.1c 4.8"0.7
Cu concentration in unsupplemented feed was 2.2"0.3 mg kgy1 . Values in rows with the same superscripts are not significantly different Ž P ) 0.05.. 3 Fish were weighed in bulk and counted. 4 n.s.s Non significant. 2
P - 0.05 P - 0.05 P - 0.05 n.s.4
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
173
Fig. 1. Relative growth inhibition Ž%. of Atlantic salmon fry exposed to elevated dietary Cu concentrations for 12 weeks compared to those fed 35 mg Cu kgy1 .
Condition factor was significantly reduced at 1750 mg Cu kgy1 compared to controls, however compared to the 35 mg Cu group condition factor was reduced at G 900 mg Cu kgy1 ŽTable 2.. Gross feed conversion efficiency was significantly Ž P - 0.01. reduced in fish fed on diets containing G 900 mg Cu kgy1 compared to
Table 3 Apparent digestibility of lipid, protein, glycogen, zinc and selenium, zinc and selenium content Žmg kgy1 DW., and apparent Cu retention in whole Atlantic salmon fry after 12 weeks of dietary Cu exposure Žmean"SD, ns 3. 5 ŽControl.
Cu added to feed Žmg kgy1 .1 35
500
700
ANOVA2 900
1750
Digestibility (%) Lipid Protein Glycogen
89.3"3.5 86.2"2.2 96.2"5.1
91.4"4.5 88.0"2.3 92.3"7.1
92.7"5.5 86.1"0.6 94.5"5.2
88.3"4.5 86.4"1.1 92.9"9.2
87.5"2.5 86.2"0.4 92.3"3.4
90.4"6.8 84.7"2.6 91.5"5.8
n.s. n.s. n.s.
AÕailability (%) Zinc Selenium
28.4"1.2 54.0"6.3
36.4"12.3 30.8"6.7 56.2"11.0 47.1"7.1
30.2"8.8 43.9"6.4
31.1"1.1 58.2"4.3
35.7"7.5 56.7"3.4
n.s. n.s.
Content (mg r kg) Zinc 172.8"5.5 168.3"6.5 139.1"9.5 147.8"17.6 151.1"9.3 155.1"10.1 n.s. Selenium 1.02"0.02 a 0.99"0.15a 0.79"0.03 b 0.87"0.08 a 0.81"0.03 b 0.78"0.032 b P - 0.05 3 Ž . Cu retention % 25.1"4.0 a 5.7"1.2 b 1.10"0.3 c 1.1"0.1c 1.5"0.1b,c 2.2"0.5 b,c P - 0.05 1
Cu concentration in unsupplemented feed was 2.2"0.3 mg kgy1 . Values in rows with the same superscripts are not significantly different Ž P ) 0.05.. 3 Apparent Cu retentions Žfinal total carcass Cu contentyinitial carcass Cu content.)100rtotal Cu fed. Carcasss whole fishygastro-intestinal tract. 2
174
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
Fig. 2. Apparent digestibility Ž%. of Cu Žbars, left axis., and Cu content in whole fish and whole fish minus intestine Žlines, right axis. in Atlantic salmon fry exposed to elevated dietary Cu concentrations for 12 weeks. Error bars are the SD, values significantly different ŽANOVA, Tukey’s t-tests. from the controls at dietary Cu treatments are indicated with asterisks Ž) for P - 0.05 and )) for P - 0.01, ns 3..
controls and the 35 mg Cu kgy1 group after 12 weeks of exposure. No food refusal was observed in any of the experimental groups during the experimental period. Mortality that occurred during the experimental period was low and did not differ among dietary treatments ŽTable 2.. 3.2. Apparent digestibility, Cu retention, and whole-body content of minerals and major compounds After 12 weeks of exposure, no significant differences Ž P ) 0.05. were observed in the digestibility of dietary lipid, protein, glycogen, and availability of Zn and Se between dietary treatments ŽTable 3.. Apparent availability of Cu was significantly Ž P - 0.05. reduced Ž65%. in diets containing G 35 and F 900 mg Cu kgy1 compared to controls ŽFig. 2.. Apparent Cu retention was significantly reduced Žmax. 96%. in diets containing G 35 Cu kgy1 compared to controls ŽTable 3.. After 12 weeks of exposure, no significant differences were observed in whole-body Zn content among dietary treatments. Se content was significantly reduced in fish reared on test diets of 500 mg Cu kgy1 compared to controls ŽTable 2.. Whole-body Cu content and whole-body Cu content minus intestine increased significantly in fish fed test diets containing G 500 and G 900 mg Cu kgy1 , respectively, compared to controls ŽFig. 2.. Fig. 3. Lipid ŽA. and protein ŽB. content Ž% DW., and glycogen ŽC. concentrations Žmg gy1 DW. in Atlantic salmon fry after 0, 6, 10, and 12 weeks of exposure to elevated dietary Cu concentrations. Error bars are the SD, values significantly different ŽANOVA, Tukey’s t-tests. from the controls at dietary Cu treatment are indicated with asterisks Ž) for P - 0.05 and )) for P - 0.01, ns 3..
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
175
After 6 weeks of exposure, whole-body lipid content was significantly reduced in fish fed diets containing G 900 mg Cu kgy1 compared to controls. No significant differ-
176
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
ences in lipid content were observed after 10 and 12 weeks of exposure among the dietary treatments ŽFig. 3A.. After 10 weeks of exposure, whole-protein protein content was significantly reduced in fish reared on diets containing G 500 mg Cu kgy1 . After 12 weeks of exposure, only the whole-body protein content of the 1750 mg Cu kgy1 group was significantly lower than the controls ŽFig. 3B.. After 12 weeks of exposure, glycogen concentrations were significantly lower in fish fed diets containing G 500 mg Cu kgy1 compared to the controls ŽFig. 3C.. 4. Discussion 4.1. Growth The lower Žnon-significant. growth in 5 mg Cu group Žcontrol. compared to the 35 mg Cu group Ž13% after 3 months. could indicate Cu deficiency in the control fish, despite that the dietary Cu concentration in the control diet Ž7.2 mg Cu kgy1 . should meet the suggested dietary Cu requirement for Atlantic salmon of 5–10 mg Cu kgy1 ŽLorentzen et al., 1998.. Earlier studies on the Zn requirement of Atlantic salmon fry suggested a higher Zn requirement in fry with a higher growth rate ŽMaage et al., 1991.. Hence, the requirements of essential dietary minerals may be dependent on their growth rate, with a higher mineral requirement in faster growing fish. Since the fry stage is a phase in the life cycle with one of the fastest relative growth rates ŽAustreng et al., 1987., the dietary Cu concentration in the control diet may have been sub-optimal to the actual Cu requirement for this life stage, resulting in an impaired growth. In the present study, fish fed at the 35 mg Cu kgy1 diet will therefore be considered as the reference group in growth responses. The non-significant increase in waterborne copper in the groups fed diets containing 500 mg Cu kgy1 and more compared to the controls Ž1.2 " 0.3 vs. 0.6 " 0.3 mg Cu ly1 ., indicates Cu leakage from faeces andror pellets. Marr et al. Ž1996. did not find a significant growth inhibition or whole-body copper accumulation in rainbow trout fry reared at 2.2 mg Cu ly1 for 60 days. Furthermore, in the present study, a significant Ž P - 0.05. difference was observed in growth inhibition and whole-body Cu accumulation between fish fed diets containing 700 mg Cu kgy1 and 1750 mg Cu kgy1 , whereas there was no difference in the waterborne Cu concentrations between these groups Ž1.6 " 0.3 vs. 1.4 " 0.4 mg Cu P ly1 .. It is therefore unlikely that the leakage of Cu from feed andror faeces to water had an affect on growth or whole-body Cu contents in the present study. Lanno et al. Ž1985a. reported a significantly lower individual body weight in rainbow trout when exposed to 730 mg Cu kgy1 for 16 weeks. In the present experiment, growth in Atlantic salmon was already significantly reduced at a dietary Cu concentration of 500 mg Cu kgy1 compared to the reference group. Thus, Atlantic salmon may be more sensitive to elevated dietary Cu concentrations than rainbow trout. However, differences in the duration of the experimental period, husbandry, and life stage of the fish make it difficult to compare these studies. In the present study, gross feed conversion efficiency and condition factor were less responsive parameters to elevated dietary Cu exposure than growth response; both
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
177
GFCE and K-factor were only significantly reduced in fish exposed to 900 mg Cu kgy1 . This is in contrast to an earlier study which found a simultaneous reduction in growth, K-factor and feed efficiency in rainbow trout exposed to 730 mg Cu kgy1 for 16 weeks ŽLanno et al., 1985a.. Reduced condition factor may be a result of either reduced feed intake, or increased metabolic expenditure. For technical reasons, feed spillage was not quantified in the present study. However, feed rejection was not observed and this is in agreement with Lanno et al. Ž1985a. who also did not observe feed refusal in rainbow trout reared on diets containing up to 1550 mg Cu kgy1 . 4.2. Mineral status and aÕailability Dietary Se, Cu and Zn availability in fish fed the control diet were 54, 64 and 28%, respectively. Bell and Cowey Ž1989. found a selenium digestibility of 46.6% in Atlantic salmon reared on a fish meal based diet. Sugiura et al. Ž1998. reported Cu and Zn availability in coho salmon Ž60 and 61%, respectively. and rainbow trout Ž20 and 5%, respectively.. These authors suggest that differences in mineral availability between these two species is not only genetically based. Rearing conditions, e.g., feeding frequency, feed composition, water temperature and water quality greatly contribute to mineral absorption. Since rearing conditions, use of indicator, or faeces sampling techniques are rarely the same in different studies, it is difficult to compare mineral availability between studies. The significant reduction in Cu availability, Cu retention, and the lack of whole-body minus intestine Cu accumulation in fish fed up to 900 mg Cu kgy1 , indicates a strong regulatory capacity in dietary Cu uptake in Atlantic salmon. The present findings are supported by Sugiura et al. Ž1998. who observed a negative correlation between dietary Cu concentrations and Cu availability in coho salmon fed practical diets. In addition, Julshamn et al. Ž1988. also found a markedly low Cu retention Ž1.3%. in rainbow trout fed on diets containing 990 mg Cu kgy1 . In the present study, the intestine contained approximately 50% of the whole-body Cu burden in fish reared on diets containing Cu concentrations of 500 and 700 mg kgy1 . Similarly, Handy Ž1992. reported that 53% of the Cu body burden was restricted to the intestine in rainbow trout exposed to 200 mg Cu kgy1 . The strong intestinal Cu accumulation suggests that dietary Cu uptake is regulated by retention of excess Cu in the intestinal tissue. Even though elevated dietary Cu is suggested to have an antagonistic effect on intestinal Zn uptake by competing for binding sites on proteins involved in intestinal trace mineral transport ŽCousins, 1985., no significant differences were found in Zn availability among dietary treatments. Hence, no significant reduction in whole-body Zn concentrations was observed. However, a study on rainbow trout reared on diets containing 800 mg Cu kgy1 reported an increase in whole-body Zn concentrations ŽLanno et al., 1985b.. The authors attributed this to the production of specific storage proteins Že.g., metallothioneins. induced by dietary Cu exposure. In a previous study, increased levels of intestinal and hepatic metallothionein were found in Atlantic salmon exposed to 700 mg Cu kgy1 ŽBerntssen et al., 1998.. However, the aforementioned mechanism did not appear to affect whole-body Zn in the present study.
178
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
Whole-body Se concentrations were significantly reduced in fish exposed to dietary Cu concentrations of 500 mg Cu kgy1 . Similarly, Lorentzen et al. Ž1998. reported a negative correlation between tissue Se concentrations and increased dietary Cu concentrations in Atlantic salmon. The formation of insoluble Cu–Se complexes in the intestinal lumen reducing bioavailability of dietary Se, or the excretion of Cu–Se complexes from the liver through the bile are mechanisms suggested by these authors which may cause reduced liver Se concentrations at elevated dietary Cu concentrations. In the present study, the formation of insoluble Cu–Se complexes did not appear to occur since dietary Se availability was not affected by increased Cu concentrations in the diets. 4.3. Deposition of energy stores and digestibility In the present study, apparent digestibility of protein Ž86%. and lipid Ž89%. were in the range of expected digestibility in salmonids reared on commercial diets ŽJobling, 1994.. Carbohydrate digestibility Ž96.5%., which was analysed as total glucose after enzymatic degradation, was comparable with glucose digestibility in tilapia fed a practical diet containing Cr2 O 3 Ž0.5%. as indicator ŽShiau and Liang, 1995.. Surprisingly, no difference in digestibility of the major feed components was found in Atlantic salmon exposed to elevated dietary Cu concentrations. A previous study on Atlantic salmon parr fed diets containing 700 mg Cu kgy1 for one month showed histological changes in the small intestine Žincreased cell proliferation and regulated cell death. ŽBerntssen et al., 1998., which have been suggested to cause malabsorption of nutrients from the intestinal tract in rats ŽIkuno et al., 1995.. In addition, Farmanfarmaian et al. Ž1985. reported reduced intestinal absorption of carbohydrates and amino acids in toad fish exposed to waterborne mercury chloride. In the present study, practical diets were used in which copper may complex with other feed components ŽCousins, 1985., which may reduce the direct toxic effects of dietary Cu on intestinal ingestion and absorption. Studies on digestibility of major feed components in metal exposed fish are scarce. However, DeSilva et al. Ž1997. also found no effects on protein digestibility in goldfish exposed to sublethal concentrations of waterborne Cd. Secondary stress responses such as depletion of glycogen tissue reserves, lipolysis, inhibition of protein synthesis and catabolism of muscle protein occur typically in sublethal exposed fish ŽJobling, 1994.. In the present study, from the three analysed energy stores, lipid content was first significantly reduced Žafter 6 weeks., followed by protein Žafter 10 weeks. and glycogen Žafter 12 weeks.. The reduction in lipid content was induced at a higher dietary Cu concentration Ž900 mg Cu kgy1 . than both protein and glycogen Ž500 mg Cu kgy1 ., and recovered completely to control levels after 10 weeks of exposure. Similarly, Lett et al. Ž1976. and DeBoeck et al. Ž1997. found a weak, initial depression in lipid stores and subsequent recovery in rainbow trout and common carp, respectively, exposed to waterborne Cu. At the same time as protein contents recovered Žafter 12 weeks., glycogen concentrations decreased significantly at 500 mg Cu kgy1 . DeBoeck et al. Ž1997. also reported reduced protein and glycogen levels in waterborne Cu stressed fish, however, these
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
179
authors found that glycogen was depleted first followed by recovery, and subsequent protein reduction. These authors suggest that first glycogen stores are depleted as glucose source followed by protein breakdown and subsequent gluconeogenesis from amino acids to maintain glucose levels. In the present study, protein catabolism appears to be the primary source for release of stored energy in dietary Cu stressed fish, later taken over by mobilisation of glycogen.
5. Conclusion The observed growth inhibition in the control group indicates a dietary Cu requirement in the fast growing Atlantic salmon fry which is higher than previously suggested in literature Ž) 7.2 mg Cu kgy1 vs. 5–10 mg Cu kgy1 .. The intestine appears to play an important role in reducing the Cu influx at elevated dietary Cu concentrations, thereby maintaining stable whole-body Cu concentrations in fish exposed to dietary Cu concentrations up to 180 times their presumed requirement level Ž5 vs. 900 mg Cu kgy1 .. Apparent availability and retention of dietary Cu is reduced Ž65 and 77%, respectively. at relatively low dietary Cu concentrations Ž35 mg Cu kgy1 .. Therefore, based on the efficiency of Cu uptake from the feed, the recommended upper limit for Cu supplementation could be set at 35 mg Cu kgy1 . However, based on traditionally dietary responses Žreduced growth, depletion of body energy stores, and Se antagonism. it seems possible to permit up to 500 mg Cu kgy1 .
Acknowledgements We thank Prof. Dr. S.E. Wendelaar Bonga, University of Nijmegen, for giving valuable comments on the manuscript. The authors wish to thank J. Sleirer for her excellent practical assistance. We are grateful to B. Engen Solli for her assistance on selenium analysis. Cu water samples were analysed at Alex Stewart Environmental Services. This study was partly financed by the Norwegian Research Council.
References Austreng, E., 1978. Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture 13, 265–272. Austreng, E., Storebakken, T., Asgard, T., 1987. Growth rate estimates for cultured Atlantic salmon and rainbow trout. Aquaculture 60, 157–160. Bell, J.G., Cowey, C.B., 1989. Digestibility and bioavailability of dietary selenium from fishmeal, selenite, selenomethionine and selenocystine in Atlantic salmon Ž Salmo salar .. Aquaculture 81, 61–68. Berntssen, M.H.G., Hylland, K., Wendelaar Bonga, S.E., Maage, A., 1998. Toxic levels of dietary Cu in Atlantic salmon Ž Salmo salar L.. pre-smolt. Aquatic Toxicol., in press. Cousins, R.J., 1985. Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65, 238–308. Crooke, W.M., Simpson, W.E., 1971. Determination of ammonium in Kjeldahl digest of crops by an automated procedure. J. Sci. Food Agric. 22, 9–10.
180
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
DeBoeck, G., Vlaeminck, A., Blust, R., 1997. Effects of sublethal copper exposure on copper accumulation, food consumption, growth, energy stores, and nucleic acid content in common carp. Arch. Environ. Contam. Toxicol. 33, 415–422. DeSilva, S.S., Deng, D.F., Rajendram, V., 1997. Digestibility in goldfish fed with and without chromic oxide and exposed to sublethal concentrations of cadmium. Aquacult. Nutr. 3, 109–114. Farmanfarmaian, A., Socci, R., Sun, L., Iannaccone, V., Pugleise, K., 1985. Interactions and distribution of mercury compounds in the intestine of fish—implications for aquaculture. J. World Maricult. Soc. 16, 473–483. Handy, R.D., 1992. The assessment of episodic metal pollution: II. The effects of cadmium and copper enriched diets on tissue contamination analysis in rainbow trout Ž Oncorhynchus mykiss .. Arch. Environ. Contam. Toxicol. 22, 82–87. Hemre, G.I., Lie, Ø., Lied, E., Lambertsen, G., 1989. Starch as an energy source in feed for cod Ž Gadus morhua.: digestibility and retention. Aquaculture 80, 261–270. Hilton, J.W., 1989. The interactions of vitamins, minerals and diet composition in the diet of fish. Aquaculture 79, 223–244. Holm, J., Bjorck, I., Drews, A., Asp, N.G., 1986. A rapid method for the analysis of starch. Starke 38, ¨ ¨ 224–226. Houde, E.D., Schekter, R.C., 1981. Growth rate, rations and cohort consumption of marine fish larvae in relation to prey concentrations. Rapp. P.V. Reun. Cons. Int. Explor. Mer. 178, 441–453. Ikuno, N., Soda, H., Watanabe, M., Oka, M., 1995. Irinotecan ŽCPT-11. and characteristic mucosal changes in the mouse ileum and cecum. J. Natl. Cancer Inst. 87, 1976–1983. Jobling, M., 1994. Environmental stressors. In: Jobling, M. ŽEd.., Fish Bioenergetics. Chapman and Hall, London, pp. 285–288. Julshamn, K., Andersen, K.J., Ringdal, O., Brenna, J., 1988. Effect of dietary copper on hepatic concentration and subcellular distribution of copper and zinc in the rainbow trout Ž Salmo gairdneri .. Aquaculture 73, 143–155. Kinghorn, B., 1983. Genetic variation in food conversion efficiency and growth in rainbow trout. Aquaculture 32, 141–155. Lanno, R.P., Slinger, S.J., Hilton, J.W., 1985a. Effects of ascorbic acid on dietary copper in rainbow trout Ž Salmo gairdneri Richardson.. Aquaculture 49, 269–287. Lanno, R.P., Slinger, S.J., Hilton, J.W., 1985b. Maximum tolerable and toxicity levels of dietary copper in rainbow trout Ž Salmo gairdneri Richardson.. Aquaculture 49, 257–268. Lett, P.F., Farmer, G.J.H., Beamish, F.W.H., 1976. Effect of copper on some aspects of the bioenergetics of rainbow trout Ž Salmo gairdneri .. J. Fish. Res. Board Can. 33, 1335–1342. Lied, E., Julshamn, K., Braekkan, O.R., 1982. Determination of protein digestibility in Atlantic cod Ž Gadus morhua. with internal and external indicators. Can. J. Fish. Aquatic Sci. 39, 854–861. Lorentzen, M., Maage, A., Julshamn, K., 1998. Supplementing copper to a fish meal based diet fed to Atlantic salmon parr affects liver copper and selenium concentrations. Aquacult. Nutr. 4, 67–77. Maage, A., Lorentzen, M., Bjornevik, M., Julshamn, K. Zinc requirement of Atlantic salmon Ž Salmo salar . fry during start feeding. In: Kaushik, S.J., Luquet, P. ŽEds.., Fish Nutrition in Practice. INRA Editions 61, Paris, France, 1991, pp. 873–882. Marr, J.C.A., Lipton, J., Cacela, D., Hansen, J.A., Bergman, H.L., Meyer, J.S., Hogstrand, C., 1996. Relationships between copper exposure duration, tissue copper concentration, and rainbow trout growth. Aquatic Toxicol. 36, 17–30. Maynard, L.A., Loosli, J.K., 1969. Animal Nutrition, 6th edn. McGraw-Hill, New York, p. 613. Mertz, W., 1993. Essential trace metals: new definitions based on new paradigms. Nutr. Rev. 51, 287–295. Murat, J.C., Serfaty, A., 1974. Simple enzymatic determination of polysaccharide Žglycogen. content of animal tissue. Clin. Chem. 20, 1576–1577. Peres, G., Tritar, B., Boge, G., Rigal, A., 1976. Preliminary data concerning the effects of methyl mercury on the in vitro intestinal absorption of glycine 14C by rainbow trout Ž Salmo gairdnerii R... Ann. Inst. Michel. Pacha. 9, 11–21. Shiau, S.Y., Liang, H.S., 1995. Carbohydrate utilisation and digestibility by tilapia Ž Oreochromis niloticus= O. aureus ., are affected by chromic oxide inclusion in the diet. J. Nutr. 125, 976–982. Sokal, R.R., Rohlf, F.J., 1981. Biometry, 2nd edn. W.H. Freeman, New York.
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
181
Sugiura, S.H.G., Dong, F.M., Rathbone, C.K., Hardy, R.W., 1998. Apparent protein digestibility and mineral availability’s in various feed ingredients for salmonid feeds. Aquaculture 159, 177–202. Tacon, A.G.J., de Silva, S.S., 1983. Mineral composition of some commercial fish feeds available in Europe. Aquaculture 31, 11–20. Zar, J.H., 1984. Biostatistical Analysis, 2nd edn. Prentice-Hall, Englewood Cliffs, NJ.
Effects of elevated dietary copper concentrations on growth, feed utilisation and nutritional status of Atlantic salmon žSalmo salar L. / fry Marc H.G. Berntssen ) , Anne-Kathrine Lundebye, Amund Maage Institute of Nutrition, Directorate of Fisheries, P.O. Box 185, N-5002 Bergen, Norway Accepted 7 January 1999
Abstract The present experiment was conducted to study effects of elevated dietary Cu and establish upper limits of Cu in fish feed. Atlantic salmon fry were reared for 3 months on experimental diets containing either 5 Žcontrol., 35, 500, 700, 900, or 1750 mg Cu kgy1, provided as CuSO4 P 5H 2 O. Dietary Cr2 O 3 was included Ž1%. in all experimental diets for the last two weeks in order to assess apparent digestibility of both major food components and availability of minerals ŽCu, Zn and Se.. Growth was significantly Ž P - 0.01. reduced after 3 months at dietary Cu concentrations of G 500 mg Cu kgy1 compared to the 35 mg Cu kgy1 group. Similarly, whole-body content of protein, glycogen and Se were significantly reduced at these dietary Cu concentrations compared to controls. Apparent digestibility Ž%. of the major food components and availability of Zn and Se did not differ among dietary treatments. However, apparent availability of dietary Cu was significantly reduced in fish fed experimental diets containing F 900 mg kgy1 compared to controls. Whole-body minus intestine Cu concentrations were only significantly increased in fish fed dietary Cu concentrations G 900 mg Cu kgy1 compared to controls, indicating a strong intestinal regulating capacity of dietary Cu uptake. Dietary Cu concentrations of 500 mg Cu kgy1 and above caused toxic responses in Atlantic salmon fry as concluded from the antagonistic interaction with Se, and reduced growth and whole-body energy stores Žprotein and glycogen.. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Cu concentration; Feed utilisation; Salmo salar L.
)
Corresponding author. Tel: q47-55-23-8138; Fax: q47-55-23-8095; E-mail: [email protected]
0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 0 1 5 - 0
168
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
1. Introduction Since copper is an essential element for all organisms including fish, it is often supplemented to commercial feeds ŽLorentzen et al., 1998. in concentrations which may reach the upper limit of toxicity ŽTacon and de Silva, 1983.. Supplementation of Cu to fish feed is therefore a balance between fulfilling the Cu requirement and avoiding Cu toxicity ŽMertz, 1993.. To establish the upper limits of dietary Cu supplementation, growth, feed utilisation, hepatic enzyme activity and tissue mineral accumulation have been used as toxicity parameters Že.g., Lanno et al., 1985a,b; Julshamn et al., 1988.. However, little information exits on the nutritional status of fish exposed to elevated dietary copper concentrations. This includes both whole-body composition Žlipid, protein and carbohydrate. and mineral status, which are parameters of interest in aquaculture. Tissue deposition of lipid, protein, carbohydrate and minerals is dependent on feed intake, metabolic use and intestinal absorption, and these factors can all be influenced by elevated dietary Cu concentrations. Lanno et al. Ž1985b. reported reduced feed intake in rainbow trout exposed to elevated dietary Cu concentrations, although body composition was not measured in that study. DeBoeck et al. Ž1997. reported decreased liver and muscle protein, lipid and glycogen content in the common carp exposed to waterborne Cu despite normal feed consumption. Increased metabolic expenditure for detoxification and maintenance of homeostasis was assumed to cause this change in energy stores. Since elevated dietary Cu concentrations are known to cause an onset of adaptive responses in Atlantic salmon ŽBerntssen et al., 1998., depletion of energy stores due to increased metabolic rate may also occur simultaneously. Impaired intestinal absorption may be particularly important because the intestine is the main target organ of dietary metals ŽHandy, 1992.. Mercury has been reported to reduce the intestinal absorption of glycine in rainbow trout ŽPeres et al., 1976. and carbohydrate and amino acids in toad fish ŽFarmanfarmaian et al., 1985.. Furthermore, mineral interactions at the intestinal level have been suggested to reduce the absorption of essential elements changing the mineral status of the fish Že.g., Hilton, 1989.. An earlier study on Atlantic salmon parr exposed to elevated dietary Cu showed an onset of histological Žintestinal cell proliferation and apoptosis. and biochemical Žintestinal and hepatic metallothionein. responses ŽBerntssen et al., 1998.. The aim of the present study was to investigate if elevated dietary Cu concentrations could also effect the nutritional status of Atlantic salmon fry by measuring mineral, lipid, protein and glycogen deposition and absorption. In addition, the effects of dietary Cu exposure on growth and feed conversion were investigated in Atlantic salmon fry, which is a phase in the life cycle characterised by a very high growth rate ŽAustreng et al., 1987.. 2. Material and methods 2.1. Diet preparation A fish feed based on fish meal as the protein source was used ŽTable 1., containing 200 mg kgy1 ethoxyquin as an antioxidant. Wheat was added as the carbohydrate source, capelin oil provided lipids, and gelatin was added as a binder. In addition, ground squid was added to enhance palatability of the experimental diets. Diets were
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
169
Table 1 Composition of the basal diet g kgy1 Fish meal ŽNorse LT94, Norway. Capelin oil ŽNorsalmoil, SSF, Norway. Wheat meal ŽCODRICO, Netherlands. Gelatin ŽTORO, Norway. Squid Žfreshly minced. Mineral mix a Vitamin mix b
573 119 168 28 95 9 9
a
The mineral mix provided Žmg kgy1 diet. as follows: Zn Žas ZnSO4 )7H 2 O., 68; Fe Žas FeSO4 )7H 2 O., 34; and Mn Žas MnSO4 )H 2 O., 13. b The vitamin mix provided Žmg kgy1 diet. as follows: retinyl palmitate, 5; cholecalciferol, 4.8; alpha-tocopheryl acetate, 100; menadione, 5; thiamin, 10; riboflavin, 20; pyridoxine, 10; pantothenic acid, 40; niacin, 170; biotin, 1.2; vitamin B12 , 0.02; inositol, 450; ascorbic acid Žcoated., 1000; and folic acid, 5.
supplemented with either 5, 35, 500, 700, 900 or 1750 mg Cu per kg dry diet, using CuSO4 P 5H 2 O. The copper sulphate was dissolved in 500 ml acidified water and mixed well with the other feed ingredients before pelleting. The diets were cold pelleted Ž2.5 mm diameter. after adding approximately 12% water and then dried. The dry 2.5 mm pellets were crumbled and sieved into batches of 0.6–1.0 and 1.0–1.5 mm and stored at y208C until use. Final Cu concentrations in the experimental diets Ž n s 9. were found to be: 7.2 " 0.4, 37 " 1, 467 " 10, 638 " 5, 868 " 7 and 1780 " 50 mg kgy1 DW, respectively. Concentrations of other essential elements in the diets were: Zn, 137.1 " 3.2 and Se, 1.4 " 0.2 mgrkg DW. Proximate analyses of the diets indicated a crude protein content of 54.8%, crude lipid 21.0%, ash 7.6%, and calculated carbohydrates 16.6% which contained 81 mg gy1 digestible carbohydrate. 2.2. Experimental set up The experiment was performed at the aquaculture station of the Institute of Marine Research in Matre, Western Norway. Atlantic salmon Ž Salmo salar . fry bred locally at this station were used. At the beginning of the experiment, weight, length Žfork-tail. and condition factor of the fish were Žmean " SD., 0.9 " 0.2 g, 4.4 " 0.3 cm and 1.1 " 0.1 g cmy3 , respectively Ž n s 900.. The fish had previously been fed a commercial salmon diet ŽSkretting, Norway. with a copper concentration of 10 mg kgy1 as shown by analysis. Eighteen separate tanks Ž1.5 = 1.5 = 0.5 m. were each stocked with approximately 1000 fry. Prior to the experiment, fish were fed the control diet for 3 weeks to acclimate. Each of the six experimental diets were fed to fish in triplicate tanks for 12 weeks. Fish were reared under constant light during the exposure period and fed by automatic feeders Žfor 5 s, every 7 min, 24 h per day. according to the growth tables of Austreng et al. Ž1987. with a daily rate of 3.5% of biomass for the first 8 weeks and 3.2% of the biomass for the last 4 weeks. A feed conversion of 1 gram wet mass gain per gram of dry feed fed was assumed. The feed particle size was gradually increased during the experiment. After 0, 6, 8 and 10 weeks, the fish were bulk weighed and the
170
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
amount of feed given was adjusted in accordance with the biomass. During the last 2 weeks the fish were fed the same experimental diets supplemented with Cr2 O 3 Ž1%. as an external indicator to estimate apparent digestibility and availability of the various feed constituents ŽLied et al., 1982.. Water temperature was measured daily and varied from 12.8 to 13.48C during the experimental period. Before each bulk weighing, water samples Ž50 ml. were taken from each exposure tank for Cu analysis. The fish were kept in a flow–through system with freshwater supplemented with 0.5% seawater from a local well to buffer the soft, acidic freshwater which resulted in the following water quality: pH 6.5, Ca 11.4 " 7.5 mg ly1 and Cu 0.6 " 0.2 mg ly1 . The water flow to the tanks was 12.4 l miny1 , oxygen concentrations were measured weekly and were never below 8 mg ly1 . 2.3. Biological sampling Fish were bulk weighed at the start of the experiment and after 6, 8, 10, and 12 weeks of exposure. At each sampling point, 100 fish were removed from each tank and transferred to aerated 30 l containers with static water Žchanged daily. and starved for 4 days to remove their intestinal content. Afterwards, these fish were sacrificed in tricaine methanesulfonate ŽMS-222.. Fifty of the sampled fish were pooled and frozen for subsequent whole-body analyses. The 50 remaining fish were used to measure weight Žg. and fork length Žcm. in order to determine the condition factor ŽK-factors 100ŽweightŽfork length.y3 .. Bulk weights were used to determine growth inhibition Ž%., gross feed conversion efficiency ŽGFCE, %.. After 6 and 10 weeks of exposure, an extra 100 fish from each container were removed and killed immediately to compensate for increased fish density in the containers. At the end of the experiment, an additional 46 un-starved fish from each container were sacrificed and a pooled stripped faecal sample was taken from 40 fish by pressing the abdomen from the ventral fins to the anus ŽAustreng, 1978.. The gastrointestinal tract Žfrom the oesophagus to the rectum. was dissected out from the remaining six fish. The six fish from each container were pooled and frozen for later Cu analyses of whole-body minus the gastrointestinal tract. The pooled faeces and whole-body samples were freeze dried Žuntil successive daily weighing was unchanged., homogenised and used for mineral ŽCu, Zn and Se., lipid, protein, ash, and glycogen analyses. 2.4. Composition analyses Dry matter content of samples was determined gravimetrically after freeze drying. Crude protein content in whole fish, faeces and feed was analysed by adding 5 ml H 2 SO4 Ž95–97% Merck. and two Kjeldahl tablets ŽThompson and Capper. to 40 mg of freeze-dried material. This mixture was digested at 3808C for 1.5 h, subsequently cooled to room temperature and diluted to 75 ml with Milli-Q water. The ammonium content ŽNHq . 4 in the sample solution was determined by an automated colorimetric method as described by Crooke and Simpson Ž1971., crude protein was estimated as N = 6.25. Casein ŽSigma, C-8654. was used as reference material. The method was found satisfactory within the 95% confidence limit. Digestible carbohydrate in feed and faeces
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
171
and glycogen in sample material was determined using an enzymatic method after Hemre et al. Ž1989. modified from Murat and Serfaty Ž1974. and Holm et al. Ž1986.. Starch in 0.5 g freeze-dried material was hydrolysed with the heat stable enzymes amylase Ž30 min at 808C. ŽTermamyl-120L; Novo-Industries, Denmark. and amyloglucosidase Ž30 min at 608C. ŽEC 3.2.1.3.; Boehringer.. Glucose was subsequently measured spectrophotometrically as NADPH at 340 nm after a hexokinase reaction in an automated analyser. Total lipid in whole fish and feed Ž1 g. was determined gravimetrically after extraction with ethyl acetate. 2.5. Trace element analyses For chromium analyses, 50 mg of feed or faecal sample were digested by the Kjeldahl method as described above. For other element analyses, 2 ml HNO 3 Ž65%, Suprapur, Merck. and 0.5 ml H 2 O 2 Ž30%, ISO, Merck. were added to samples of whole fish, feed Ž200 mg. or faeces Ž50 mg. and digested under pressure in a micro-wave oven ŽMilestone MLS-1200 MEGA. for 17 min Ž1 min at 250 W, 1 min at 0 W, 5 min at 250 W, 5 min at 400 W, and 5 min at 650 W., and subsequently diluted to 25 ml with Milli-Q water. Chromium, copper and zinc concentrations were analysed by flame atomic absorption on a Perkin-Elmer 3300 AAS Žthree replicates per sample.. For copper analyses, this instrument was equipped with a high sensitivity nebulizer Ždetection limit 0.005 mg Cu ly1 .. Se concentrations were analysed by cold vapour atomic absorption ŽPerkin-Elmer 3300 AAS., with the use of a standard addition curve to compensate for phosphorous interaction. Water samples were acidified with nitric acid Ž65% HNO 3 , Suprapur, Merck. in a final concentration of 5.2% Ž2 ml HNO 3 added to 25 ml sample. and analysed for total Cu by inductively coupled plasma mass spectrometry ŽPerkin-Elmer Elan 5000.. Accuracy and precision of the element analyses with each set of samples were controlled by analyses of dogfish muscle tissue DORM-1 ŽNRCC.. The methods were found satisfactory within the 95% confidence limit. 2.6. Calculations and statistics Specific daily growth rate ŽSGR. was calculated according to the formula of Houde and Schekter Ž1981.: SGR s Ž e g y 1.)100%, where g s ŽlnŽW2 . y lnŽW1 ..)Ž t 2 y t 1 .y1 and W2 and W1 are weights on day t 2 and t 1 , respectively. Gross feed conversion efficiency ŽGFCE. was calculated and modified after Kinghorn Ž1983. with the formula: GFCEs ŽW2 y W1 .)Cy1 , where W2 and W1 are weights at sampling times t 2 and t 1 , respectively, and C denotes the dry weight of feed fed in the sample period. Apparent digestibility ŽAD. and mineral availability ŽAA. was calculated after Maynard and Loosli Ž1969. using the formula: ADrAA s 100 y ŽCrd )CX f .) ŽCrf )CX d .y1 )100 where d is diet, f is faeces, Cr is chromium content and CX is nutrient content. All statistics were performed using the program STATISTICAe ŽStatsoft, USA, 1993.. A Kolmogrov–Smirnov test was used to assess normality of distribution ŽZar, 1984.. Data which were found not to be normally distributed were log transformed. The
172
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
homogeneity of variances was tested using Levene F-test ŽZar, 1984.. Differences in mineral, protein, lipid, glycogen, ash content, digestibility, and growth among dietary treatments at different time points were assessed by one-way analysis of variance ŽANOVA.. To account for the variance among the experimental tanks within one dietary treatment, differences in individual length, weight, K-factor, and Cu water content among dietary treatments on different dates were assessed by two-way analysis of variance Žnested ANOVA. ŽZar, 1984. with the Žrandom. experimental tanks nested in their dietary exposure groups. Significance was tested using Tukey’s HSD test Ž P - 0.01 and P - 0.05. ŽSokal and Rohlf, 1981.. Pearson’s correlation coefficients were calculated to evaluate the relationships between growth inhibition and exposure time for each dietary exposure group ŽZar, 1984.. A series of models were tested to find the best fit. 3. Results 3.1. Water chemistry, growth, K-factor, GFCE, and mortality A non-significant Ž P ) 0.05. increase in total copper concentrations was observed in the water from tanks in which fish were fed with the Cu-supplemented diets compared to the tanks in which fish were fed the control diet. Concentrations of Cu in the water were 0.6 " 0.3, 0.6 " 0.2, 1.2 " 0.6, 1.6 " 0.8, 1.6 " 0.5, 1.4 " 0.4 mg ly1 Ž n s 6. for control and 35, 500, 700, 900, 1750 groups, respectively. After 12 weeks of dietary exposure, individual weight was significantly reduced Ž P - 0.01. in Atlantic salmon fed diets containing G 900 mg Cu kgy1 compared to control fish. However, individual weight was also significantly reduced in fish fed diets containing G 500 mg Cu kgy1 compared to the 35 mg Cu kgy1 diet ŽTable 2.. Growth inhibition Ž%. compared to fish fed 35 mg Cu kgy1 reached a plateau after 12 weeks of exposure in fish fed diets containing 500 and 700 mg Cu kgy1 Ž22 and 27%, respectively., whereas in the 900 and 1750 mg Cu kgy1 diet groups the growth inhibition was still exponential at the end of the experimental period Ž37 and 45%, respectively. ŽFig. 1..
Table 2 Individual weight, K-factor Ž ns150., bulk gross feed conversion efficiency ŽGFCE. Ž ns 3., and mortality of Atlantic salmon fry after 12 weeks of dietary Cu exposure Žmean"SD. 5 ŽControl.
Cu added to feed Žmg kgy1 .1 35
Weight 3 Žg. 10.3"0.9 a,b 11.9"1.5a y3 . Ž K-factor g cm 1.3"0.1a,b 1.3"0.2 a GFCE Žg gy1 . 0.9"0.1a 0.9"0.1a Ž . Mortality % 3.6"0.3 3.8"0.3 1
ANOVA2
500
700
900
1750
9.1"0.5 b 1.3"0.1a,b 0.8"0.1a,b 3.5"1.2
8.7"0.3 b,c 1.3"0.1a 0.7"0.1a,b 3.4"0.6
7.5"1.2 c,d 1.2"0.1b,c 0.5"0.0.2 b,c 4.3"0.1
6.6"0.7 d 1.2"0.1c 0.4"0.1c 4.8"0.7
Cu concentration in unsupplemented feed was 2.2"0.3 mg kgy1 . Values in rows with the same superscripts are not significantly different Ž P ) 0.05.. 3 Fish were weighed in bulk and counted. 4 n.s.s Non significant. 2
P - 0.05 P - 0.05 P - 0.05 n.s.4
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
173
Fig. 1. Relative growth inhibition Ž%. of Atlantic salmon fry exposed to elevated dietary Cu concentrations for 12 weeks compared to those fed 35 mg Cu kgy1 .
Condition factor was significantly reduced at 1750 mg Cu kgy1 compared to controls, however compared to the 35 mg Cu group condition factor was reduced at G 900 mg Cu kgy1 ŽTable 2.. Gross feed conversion efficiency was significantly Ž P - 0.01. reduced in fish fed on diets containing G 900 mg Cu kgy1 compared to
Table 3 Apparent digestibility of lipid, protein, glycogen, zinc and selenium, zinc and selenium content Žmg kgy1 DW., and apparent Cu retention in whole Atlantic salmon fry after 12 weeks of dietary Cu exposure Žmean"SD, ns 3. 5 ŽControl.
Cu added to feed Žmg kgy1 .1 35
500
700
ANOVA2 900
1750
Digestibility (%) Lipid Protein Glycogen
89.3"3.5 86.2"2.2 96.2"5.1
91.4"4.5 88.0"2.3 92.3"7.1
92.7"5.5 86.1"0.6 94.5"5.2
88.3"4.5 86.4"1.1 92.9"9.2
87.5"2.5 86.2"0.4 92.3"3.4
90.4"6.8 84.7"2.6 91.5"5.8
n.s. n.s. n.s.
AÕailability (%) Zinc Selenium
28.4"1.2 54.0"6.3
36.4"12.3 30.8"6.7 56.2"11.0 47.1"7.1
30.2"8.8 43.9"6.4
31.1"1.1 58.2"4.3
35.7"7.5 56.7"3.4
n.s. n.s.
Content (mg r kg) Zinc 172.8"5.5 168.3"6.5 139.1"9.5 147.8"17.6 151.1"9.3 155.1"10.1 n.s. Selenium 1.02"0.02 a 0.99"0.15a 0.79"0.03 b 0.87"0.08 a 0.81"0.03 b 0.78"0.032 b P - 0.05 3 Ž . Cu retention % 25.1"4.0 a 5.7"1.2 b 1.10"0.3 c 1.1"0.1c 1.5"0.1b,c 2.2"0.5 b,c P - 0.05 1
Cu concentration in unsupplemented feed was 2.2"0.3 mg kgy1 . Values in rows with the same superscripts are not significantly different Ž P ) 0.05.. 3 Apparent Cu retentions Žfinal total carcass Cu contentyinitial carcass Cu content.)100rtotal Cu fed. Carcasss whole fishygastro-intestinal tract. 2
174
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
Fig. 2. Apparent digestibility Ž%. of Cu Žbars, left axis., and Cu content in whole fish and whole fish minus intestine Žlines, right axis. in Atlantic salmon fry exposed to elevated dietary Cu concentrations for 12 weeks. Error bars are the SD, values significantly different ŽANOVA, Tukey’s t-tests. from the controls at dietary Cu treatments are indicated with asterisks Ž) for P - 0.05 and )) for P - 0.01, ns 3..
controls and the 35 mg Cu kgy1 group after 12 weeks of exposure. No food refusal was observed in any of the experimental groups during the experimental period. Mortality that occurred during the experimental period was low and did not differ among dietary treatments ŽTable 2.. 3.2. Apparent digestibility, Cu retention, and whole-body content of minerals and major compounds After 12 weeks of exposure, no significant differences Ž P ) 0.05. were observed in the digestibility of dietary lipid, protein, glycogen, and availability of Zn and Se between dietary treatments ŽTable 3.. Apparent availability of Cu was significantly Ž P - 0.05. reduced Ž65%. in diets containing G 35 and F 900 mg Cu kgy1 compared to controls ŽFig. 2.. Apparent Cu retention was significantly reduced Žmax. 96%. in diets containing G 35 Cu kgy1 compared to controls ŽTable 3.. After 12 weeks of exposure, no significant differences were observed in whole-body Zn content among dietary treatments. Se content was significantly reduced in fish reared on test diets of 500 mg Cu kgy1 compared to controls ŽTable 2.. Whole-body Cu content and whole-body Cu content minus intestine increased significantly in fish fed test diets containing G 500 and G 900 mg Cu kgy1 , respectively, compared to controls ŽFig. 2.. Fig. 3. Lipid ŽA. and protein ŽB. content Ž% DW., and glycogen ŽC. concentrations Žmg gy1 DW. in Atlantic salmon fry after 0, 6, 10, and 12 weeks of exposure to elevated dietary Cu concentrations. Error bars are the SD, values significantly different ŽANOVA, Tukey’s t-tests. from the controls at dietary Cu treatment are indicated with asterisks Ž) for P - 0.05 and )) for P - 0.01, ns 3..
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
175
After 6 weeks of exposure, whole-body lipid content was significantly reduced in fish fed diets containing G 900 mg Cu kgy1 compared to controls. No significant differ-
176
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
ences in lipid content were observed after 10 and 12 weeks of exposure among the dietary treatments ŽFig. 3A.. After 10 weeks of exposure, whole-protein protein content was significantly reduced in fish reared on diets containing G 500 mg Cu kgy1 . After 12 weeks of exposure, only the whole-body protein content of the 1750 mg Cu kgy1 group was significantly lower than the controls ŽFig. 3B.. After 12 weeks of exposure, glycogen concentrations were significantly lower in fish fed diets containing G 500 mg Cu kgy1 compared to the controls ŽFig. 3C.. 4. Discussion 4.1. Growth The lower Žnon-significant. growth in 5 mg Cu group Žcontrol. compared to the 35 mg Cu group Ž13% after 3 months. could indicate Cu deficiency in the control fish, despite that the dietary Cu concentration in the control diet Ž7.2 mg Cu kgy1 . should meet the suggested dietary Cu requirement for Atlantic salmon of 5–10 mg Cu kgy1 ŽLorentzen et al., 1998.. Earlier studies on the Zn requirement of Atlantic salmon fry suggested a higher Zn requirement in fry with a higher growth rate ŽMaage et al., 1991.. Hence, the requirements of essential dietary minerals may be dependent on their growth rate, with a higher mineral requirement in faster growing fish. Since the fry stage is a phase in the life cycle with one of the fastest relative growth rates ŽAustreng et al., 1987., the dietary Cu concentration in the control diet may have been sub-optimal to the actual Cu requirement for this life stage, resulting in an impaired growth. In the present study, fish fed at the 35 mg Cu kgy1 diet will therefore be considered as the reference group in growth responses. The non-significant increase in waterborne copper in the groups fed diets containing 500 mg Cu kgy1 and more compared to the controls Ž1.2 " 0.3 vs. 0.6 " 0.3 mg Cu ly1 ., indicates Cu leakage from faeces andror pellets. Marr et al. Ž1996. did not find a significant growth inhibition or whole-body copper accumulation in rainbow trout fry reared at 2.2 mg Cu ly1 for 60 days. Furthermore, in the present study, a significant Ž P - 0.05. difference was observed in growth inhibition and whole-body Cu accumulation between fish fed diets containing 700 mg Cu kgy1 and 1750 mg Cu kgy1 , whereas there was no difference in the waterborne Cu concentrations between these groups Ž1.6 " 0.3 vs. 1.4 " 0.4 mg Cu P ly1 .. It is therefore unlikely that the leakage of Cu from feed andror faeces to water had an affect on growth or whole-body Cu contents in the present study. Lanno et al. Ž1985a. reported a significantly lower individual body weight in rainbow trout when exposed to 730 mg Cu kgy1 for 16 weeks. In the present experiment, growth in Atlantic salmon was already significantly reduced at a dietary Cu concentration of 500 mg Cu kgy1 compared to the reference group. Thus, Atlantic salmon may be more sensitive to elevated dietary Cu concentrations than rainbow trout. However, differences in the duration of the experimental period, husbandry, and life stage of the fish make it difficult to compare these studies. In the present study, gross feed conversion efficiency and condition factor were less responsive parameters to elevated dietary Cu exposure than growth response; both
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
177
GFCE and K-factor were only significantly reduced in fish exposed to 900 mg Cu kgy1 . This is in contrast to an earlier study which found a simultaneous reduction in growth, K-factor and feed efficiency in rainbow trout exposed to 730 mg Cu kgy1 for 16 weeks ŽLanno et al., 1985a.. Reduced condition factor may be a result of either reduced feed intake, or increased metabolic expenditure. For technical reasons, feed spillage was not quantified in the present study. However, feed rejection was not observed and this is in agreement with Lanno et al. Ž1985a. who also did not observe feed refusal in rainbow trout reared on diets containing up to 1550 mg Cu kgy1 . 4.2. Mineral status and aÕailability Dietary Se, Cu and Zn availability in fish fed the control diet were 54, 64 and 28%, respectively. Bell and Cowey Ž1989. found a selenium digestibility of 46.6% in Atlantic salmon reared on a fish meal based diet. Sugiura et al. Ž1998. reported Cu and Zn availability in coho salmon Ž60 and 61%, respectively. and rainbow trout Ž20 and 5%, respectively.. These authors suggest that differences in mineral availability between these two species is not only genetically based. Rearing conditions, e.g., feeding frequency, feed composition, water temperature and water quality greatly contribute to mineral absorption. Since rearing conditions, use of indicator, or faeces sampling techniques are rarely the same in different studies, it is difficult to compare mineral availability between studies. The significant reduction in Cu availability, Cu retention, and the lack of whole-body minus intestine Cu accumulation in fish fed up to 900 mg Cu kgy1 , indicates a strong regulatory capacity in dietary Cu uptake in Atlantic salmon. The present findings are supported by Sugiura et al. Ž1998. who observed a negative correlation between dietary Cu concentrations and Cu availability in coho salmon fed practical diets. In addition, Julshamn et al. Ž1988. also found a markedly low Cu retention Ž1.3%. in rainbow trout fed on diets containing 990 mg Cu kgy1 . In the present study, the intestine contained approximately 50% of the whole-body Cu burden in fish reared on diets containing Cu concentrations of 500 and 700 mg kgy1 . Similarly, Handy Ž1992. reported that 53% of the Cu body burden was restricted to the intestine in rainbow trout exposed to 200 mg Cu kgy1 . The strong intestinal Cu accumulation suggests that dietary Cu uptake is regulated by retention of excess Cu in the intestinal tissue. Even though elevated dietary Cu is suggested to have an antagonistic effect on intestinal Zn uptake by competing for binding sites on proteins involved in intestinal trace mineral transport ŽCousins, 1985., no significant differences were found in Zn availability among dietary treatments. Hence, no significant reduction in whole-body Zn concentrations was observed. However, a study on rainbow trout reared on diets containing 800 mg Cu kgy1 reported an increase in whole-body Zn concentrations ŽLanno et al., 1985b.. The authors attributed this to the production of specific storage proteins Že.g., metallothioneins. induced by dietary Cu exposure. In a previous study, increased levels of intestinal and hepatic metallothionein were found in Atlantic salmon exposed to 700 mg Cu kgy1 ŽBerntssen et al., 1998.. However, the aforementioned mechanism did not appear to affect whole-body Zn in the present study.
178
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
Whole-body Se concentrations were significantly reduced in fish exposed to dietary Cu concentrations of 500 mg Cu kgy1 . Similarly, Lorentzen et al. Ž1998. reported a negative correlation between tissue Se concentrations and increased dietary Cu concentrations in Atlantic salmon. The formation of insoluble Cu–Se complexes in the intestinal lumen reducing bioavailability of dietary Se, or the excretion of Cu–Se complexes from the liver through the bile are mechanisms suggested by these authors which may cause reduced liver Se concentrations at elevated dietary Cu concentrations. In the present study, the formation of insoluble Cu–Se complexes did not appear to occur since dietary Se availability was not affected by increased Cu concentrations in the diets. 4.3. Deposition of energy stores and digestibility In the present study, apparent digestibility of protein Ž86%. and lipid Ž89%. were in the range of expected digestibility in salmonids reared on commercial diets ŽJobling, 1994.. Carbohydrate digestibility Ž96.5%., which was analysed as total glucose after enzymatic degradation, was comparable with glucose digestibility in tilapia fed a practical diet containing Cr2 O 3 Ž0.5%. as indicator ŽShiau and Liang, 1995.. Surprisingly, no difference in digestibility of the major feed components was found in Atlantic salmon exposed to elevated dietary Cu concentrations. A previous study on Atlantic salmon parr fed diets containing 700 mg Cu kgy1 for one month showed histological changes in the small intestine Žincreased cell proliferation and regulated cell death. ŽBerntssen et al., 1998., which have been suggested to cause malabsorption of nutrients from the intestinal tract in rats ŽIkuno et al., 1995.. In addition, Farmanfarmaian et al. Ž1985. reported reduced intestinal absorption of carbohydrates and amino acids in toad fish exposed to waterborne mercury chloride. In the present study, practical diets were used in which copper may complex with other feed components ŽCousins, 1985., which may reduce the direct toxic effects of dietary Cu on intestinal ingestion and absorption. Studies on digestibility of major feed components in metal exposed fish are scarce. However, DeSilva et al. Ž1997. also found no effects on protein digestibility in goldfish exposed to sublethal concentrations of waterborne Cd. Secondary stress responses such as depletion of glycogen tissue reserves, lipolysis, inhibition of protein synthesis and catabolism of muscle protein occur typically in sublethal exposed fish ŽJobling, 1994.. In the present study, from the three analysed energy stores, lipid content was first significantly reduced Žafter 6 weeks., followed by protein Žafter 10 weeks. and glycogen Žafter 12 weeks.. The reduction in lipid content was induced at a higher dietary Cu concentration Ž900 mg Cu kgy1 . than both protein and glycogen Ž500 mg Cu kgy1 ., and recovered completely to control levels after 10 weeks of exposure. Similarly, Lett et al. Ž1976. and DeBoeck et al. Ž1997. found a weak, initial depression in lipid stores and subsequent recovery in rainbow trout and common carp, respectively, exposed to waterborne Cu. At the same time as protein contents recovered Žafter 12 weeks., glycogen concentrations decreased significantly at 500 mg Cu kgy1 . DeBoeck et al. Ž1997. also reported reduced protein and glycogen levels in waterborne Cu stressed fish, however, these
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
179
authors found that glycogen was depleted first followed by recovery, and subsequent protein reduction. These authors suggest that first glycogen stores are depleted as glucose source followed by protein breakdown and subsequent gluconeogenesis from amino acids to maintain glucose levels. In the present study, protein catabolism appears to be the primary source for release of stored energy in dietary Cu stressed fish, later taken over by mobilisation of glycogen.
5. Conclusion The observed growth inhibition in the control group indicates a dietary Cu requirement in the fast growing Atlantic salmon fry which is higher than previously suggested in literature Ž) 7.2 mg Cu kgy1 vs. 5–10 mg Cu kgy1 .. The intestine appears to play an important role in reducing the Cu influx at elevated dietary Cu concentrations, thereby maintaining stable whole-body Cu concentrations in fish exposed to dietary Cu concentrations up to 180 times their presumed requirement level Ž5 vs. 900 mg Cu kgy1 .. Apparent availability and retention of dietary Cu is reduced Ž65 and 77%, respectively. at relatively low dietary Cu concentrations Ž35 mg Cu kgy1 .. Therefore, based on the efficiency of Cu uptake from the feed, the recommended upper limit for Cu supplementation could be set at 35 mg Cu kgy1 . However, based on traditionally dietary responses Žreduced growth, depletion of body energy stores, and Se antagonism. it seems possible to permit up to 500 mg Cu kgy1 .
Acknowledgements We thank Prof. Dr. S.E. Wendelaar Bonga, University of Nijmegen, for giving valuable comments on the manuscript. The authors wish to thank J. Sleirer for her excellent practical assistance. We are grateful to B. Engen Solli for her assistance on selenium analysis. Cu water samples were analysed at Alex Stewart Environmental Services. This study was partly financed by the Norwegian Research Council.
References Austreng, E., 1978. Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture 13, 265–272. Austreng, E., Storebakken, T., Asgard, T., 1987. Growth rate estimates for cultured Atlantic salmon and rainbow trout. Aquaculture 60, 157–160. Bell, J.G., Cowey, C.B., 1989. Digestibility and bioavailability of dietary selenium from fishmeal, selenite, selenomethionine and selenocystine in Atlantic salmon Ž Salmo salar .. Aquaculture 81, 61–68. Berntssen, M.H.G., Hylland, K., Wendelaar Bonga, S.E., Maage, A., 1998. Toxic levels of dietary Cu in Atlantic salmon Ž Salmo salar L.. pre-smolt. Aquatic Toxicol., in press. Cousins, R.J., 1985. Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65, 238–308. Crooke, W.M., Simpson, W.E., 1971. Determination of ammonium in Kjeldahl digest of crops by an automated procedure. J. Sci. Food Agric. 22, 9–10.
180
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
DeBoeck, G., Vlaeminck, A., Blust, R., 1997. Effects of sublethal copper exposure on copper accumulation, food consumption, growth, energy stores, and nucleic acid content in common carp. Arch. Environ. Contam. Toxicol. 33, 415–422. DeSilva, S.S., Deng, D.F., Rajendram, V., 1997. Digestibility in goldfish fed with and without chromic oxide and exposed to sublethal concentrations of cadmium. Aquacult. Nutr. 3, 109–114. Farmanfarmaian, A., Socci, R., Sun, L., Iannaccone, V., Pugleise, K., 1985. Interactions and distribution of mercury compounds in the intestine of fish—implications for aquaculture. J. World Maricult. Soc. 16, 473–483. Handy, R.D., 1992. The assessment of episodic metal pollution: II. The effects of cadmium and copper enriched diets on tissue contamination analysis in rainbow trout Ž Oncorhynchus mykiss .. Arch. Environ. Contam. Toxicol. 22, 82–87. Hemre, G.I., Lie, Ø., Lied, E., Lambertsen, G., 1989. Starch as an energy source in feed for cod Ž Gadus morhua.: digestibility and retention. Aquaculture 80, 261–270. Hilton, J.W., 1989. The interactions of vitamins, minerals and diet composition in the diet of fish. Aquaculture 79, 223–244. Holm, J., Bjorck, I., Drews, A., Asp, N.G., 1986. A rapid method for the analysis of starch. Starke 38, ¨ ¨ 224–226. Houde, E.D., Schekter, R.C., 1981. Growth rate, rations and cohort consumption of marine fish larvae in relation to prey concentrations. Rapp. P.V. Reun. Cons. Int. Explor. Mer. 178, 441–453. Ikuno, N., Soda, H., Watanabe, M., Oka, M., 1995. Irinotecan ŽCPT-11. and characteristic mucosal changes in the mouse ileum and cecum. J. Natl. Cancer Inst. 87, 1976–1983. Jobling, M., 1994. Environmental stressors. In: Jobling, M. ŽEd.., Fish Bioenergetics. Chapman and Hall, London, pp. 285–288. Julshamn, K., Andersen, K.J., Ringdal, O., Brenna, J., 1988. Effect of dietary copper on hepatic concentration and subcellular distribution of copper and zinc in the rainbow trout Ž Salmo gairdneri .. Aquaculture 73, 143–155. Kinghorn, B., 1983. Genetic variation in food conversion efficiency and growth in rainbow trout. Aquaculture 32, 141–155. Lanno, R.P., Slinger, S.J., Hilton, J.W., 1985a. Effects of ascorbic acid on dietary copper in rainbow trout Ž Salmo gairdneri Richardson.. Aquaculture 49, 269–287. Lanno, R.P., Slinger, S.J., Hilton, J.W., 1985b. Maximum tolerable and toxicity levels of dietary copper in rainbow trout Ž Salmo gairdneri Richardson.. Aquaculture 49, 257–268. Lett, P.F., Farmer, G.J.H., Beamish, F.W.H., 1976. Effect of copper on some aspects of the bioenergetics of rainbow trout Ž Salmo gairdneri .. J. Fish. Res. Board Can. 33, 1335–1342. Lied, E., Julshamn, K., Braekkan, O.R., 1982. Determination of protein digestibility in Atlantic cod Ž Gadus morhua. with internal and external indicators. Can. J. Fish. Aquatic Sci. 39, 854–861. Lorentzen, M., Maage, A., Julshamn, K., 1998. Supplementing copper to a fish meal based diet fed to Atlantic salmon parr affects liver copper and selenium concentrations. Aquacult. Nutr. 4, 67–77. Maage, A., Lorentzen, M., Bjornevik, M., Julshamn, K. Zinc requirement of Atlantic salmon Ž Salmo salar . fry during start feeding. In: Kaushik, S.J., Luquet, P. ŽEds.., Fish Nutrition in Practice. INRA Editions 61, Paris, France, 1991, pp. 873–882. Marr, J.C.A., Lipton, J., Cacela, D., Hansen, J.A., Bergman, H.L., Meyer, J.S., Hogstrand, C., 1996. Relationships between copper exposure duration, tissue copper concentration, and rainbow trout growth. Aquatic Toxicol. 36, 17–30. Maynard, L.A., Loosli, J.K., 1969. Animal Nutrition, 6th edn. McGraw-Hill, New York, p. 613. Mertz, W., 1993. Essential trace metals: new definitions based on new paradigms. Nutr. Rev. 51, 287–295. Murat, J.C., Serfaty, A., 1974. Simple enzymatic determination of polysaccharide Žglycogen. content of animal tissue. Clin. Chem. 20, 1576–1577. Peres, G., Tritar, B., Boge, G., Rigal, A., 1976. Preliminary data concerning the effects of methyl mercury on the in vitro intestinal absorption of glycine 14C by rainbow trout Ž Salmo gairdnerii R... Ann. Inst. Michel. Pacha. 9, 11–21. Shiau, S.Y., Liang, H.S., 1995. Carbohydrate utilisation and digestibility by tilapia Ž Oreochromis niloticus= O. aureus ., are affected by chromic oxide inclusion in the diet. J. Nutr. 125, 976–982. Sokal, R.R., Rohlf, F.J., 1981. Biometry, 2nd edn. W.H. Freeman, New York.
M.H.G. Berntssen et al.r Aquaculture 174 (1999) 167–181
181
Sugiura, S.H.G., Dong, F.M., Rathbone, C.K., Hardy, R.W., 1998. Apparent protein digestibility and mineral availability’s in various feed ingredients for salmonid feeds. Aquaculture 159, 177–202. Tacon, A.G.J., de Silva, S.S., 1983. Mineral composition of some commercial fish feeds available in Europe. Aquaculture 31, 11–20. Zar, J.H., 1984. Biostatistical Analysis, 2nd edn. Prentice-Hall, Englewood Cliffs, NJ.