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Trace elements (Zn, Cu, Cd) in fish from rearing ponds of Emilia-Romagna region (Italy)
the Science of the
Total E n v i r o n m e n t inlo the En',a~*menl
ELSEVIER
I n d its R e l l t m n s h i p ~ i l h ~11~
The Science of the Total Environment 141 (1994) 139-146
Trace elements (Zn, Cu, Cd) in fish from rearing ponds of Emilia-Romagna region (Italy) E. C a r p e n e
,,a
,B.
•
G u m i e r o b, G . F e d n z z l
.a
, R. Serra a
aDepartment oJ Biochemistry ( Veterinary Biochemistry Section). Belmeloro St. 8/2. hDepartment of Evolutionary and E.~perimental Biology S. Giacomo St 0.9, University of Bologna, Bologna, Italy
(Received 30 March 1992: accepted 8 August 1992}
Abstract
Significant differences in Zn and Cu content were found between different organs. Zinc reaches its maximum, mean value (929/~g/g dry wt), in the kidney of the common carp and consistently has a high value (276-552 #g/g dry wt) in the ovary of all the species examined; Cu is at its maximum (21 #g/g dry wt) in the liver of goldfish. Zinc concentration in brain tissue is rather constant and is probably well regulated because of a functional role in this organ. Cadmium was only found in the liver and kidney, being approximately four times higher in the latter. Seasonal variations in trace element contents have been demonstrated in some organs of catfish; in the ovary, zinc concentrations could be linked with the reproductive cycle, while in the muscle with a growth cycle. Key words: Zinc; Copper; Cadmium; Fish
1. Introduction
Fish can obtain their trace elements, either directly from the water through the gills or indirectly from food through the alimentary tract (Willis and Sunda, 1984; Hardy et al., 1987; Spry and Wood, 1988). The mechanisms of transport of metals across the membrane barriers have yet to be fully examined. Although zinc and copper are essential to fish (Ogino and Yang, 1978; Knox et al., 1984), high concentrations may be toxic (Waldichuck, 1974; Crespo, 1984; El-Domiaty, 1987). In contrast cadmium is considered only for * Corresponding author.
its negative effects (Crespo et al., 1986). The availability of these elements can be influenced by many environmental factors (Zitko and Carson, 1977; Chakoumakos et al., 1979) and by composition of diet (Hardy and Shearer, 1985); as a consequence the physiological requirements of the essential elements are difficult to determine. The analysis of metal content in different tissues of fish can provide further information on trace element requirements and on their toxicity (Badsha and Goldspink, 1982; Carpen6 et al., 1990). In an area close to Bologna there are several small ponds which now are utilized for rearing fish. The water in these ponds comes mainly from irrigation channels and in part from artesian wells,
0048-9697/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved. SSDI 0048-9697(92J03522-4
E. Carpenk et al/Sci. Total Environ. 141 (1994) 139-146
140
the latter are considered unpolluted, but data on metal concentrations are lacking. Most of the reared fish belong to Carassius auratus (goldfish) and to Ictalurus melas (catfish), other species such as Cyprinus carpio (common carp) are occasionally present. They are semi-intensively and intensively reared and fed with artificial pelleted diets, containing different amounts of copper and zinc. The aim of this study is to determine the metal content (Zn, Cu, Cd) in tissues of fish reared in different ponds, in order to provide information about the influence of water and diets on the tissue metal composition. The results could be useful for an eventual improvement of diet formulation (Zn, Cu). The cadmium concentration was also calculated so that the levels of this potentially harmful element could be determined and fish could be used as indicators of pollution by heavy metals. Moreover, in one species (catfish) the three elements were analysed over a period of I year, to observe if there were seasonal variations, which could be related to fish biology. 2. Materials and methods
2.1. Animals Fish, including specimens of catfish (Ictalurus melas), goldfish ( Carassius auratus) and common carp (Ciprinus carpio) were caught with seine nets during 1989-1990 in different ponds of EmiliaRomagna region. They were measured (fork length) and their length was, respectively: 17.9 ± 2.8 cm (n = 125) for catfish, 9.1 + 4.0 cm (n = 30) for goldfish and 29.9 ± 9.4 cm (n = 25) for common carp. The number and size range was based on the availability of fish in the catch, since fish were obtained directly from the pond. Fish were separated into pools of five specimens and organs (brain, liver, kidney, muscle and ovary) were sampled from each pool and kept lyophilised until analysed. Only for catfish was it possible to provide a complete set of samples to cover the period of 1 year. Catfish were collected during the following five different periods: June-July, AugustSeptember, October-November, January-April and May.
2.2. Pon& Fish were reared in 10 different ponds which we
numbered from 1 to 6 for catfish and from 7 to 10 for goldfish and common carp. The surface areas ranged from a minimum of 2755 m 2 to a maximum of 26 000 m 2. Water was obtained from irrigating channels and only in one case from an artesian well (pond 1). Water temperature ranged between I°C in winter and 30°C in summer; oxygen concentrations fluctuated, falling during the night close to 1-2 mg/litre and, during the day they increased beyond the saturation levels. From spring to fall the water was rather turbid due to continuous phytoplankton blooms.
2.3. Feeding offish Fish were fed with commercial pelleted food of different brands. The catfish (data from food labels) were fed with diets that contained Zn and Cu in the range of 50-80 and 2.5-150 #g/g dry weight, respectively, cadmium was not determined. For the other two species it was not possible to identify the brand of food given to the fish and hence trace element concentrations, because farmers did not adopt the same diet throughout the year.
2.4. Metal analysis Samples of lyophilized tissues from each separated pool were weighed and ashed overnight in platinum dishes at 480°C; the white ash was dissolved in 1 N HC1 and analysed for zinc, copper and cadmium by flame atomic absorption spectrophotometry (AAS) using an Instrumentation Laboratories model IL-11 spectrophotometer. The accuracy of the method was evaluated by calibration with international standards (IAEA): MA-A2 (fish); MA-M-1 (oyster): MA-A-1 (copepod); MA-M-2 (mussels). The concentrations ( > 80%) found with the method used in this study fell in the confidence interval given by IAEA.
2.5. Statistical analysis The data (ug/g dry wt) presented in the tables are mean values + S.D. The error bars in the figures are standard deviations of the mean. The one- and two-way analysis of variance (ANOVA) was used to test significant differences, with P = 0.05 chosen as the minimum level of significance. Regression analysis between fish length and metal concentrations was computed.
141
E. Carpen~ et al/Sci. Total Environ. 141 (1994) 139-146 Table 1 Mean concentration of zinc 4- S.D. (t~g/g dry wt.) in tissues and organs of catfish, goldfish and carp Fish species
White muscle
Brain
Liver
Kidney
Ovary
lctalurus melas Carassius auratus Ciprinus carpio
22.6 ± 15.6 43.6 ± 5.9 28.3 4- 4.7
56.8 + 8.7 64.4 + 2.3 62 4- 18
86 4- 22 78 4- 43 333 4- 142
106 4- 14 218 + 67 929 4- 197
276 4- 100 304 + 35 552 4- 289
3. Results The mean Zn content and relative standard deviation in tissues of the catfish, the goldfish and the common carp are reported in Table 1. Significant differences (P < 0.001) were found between the different tissues of each species and when the same tissue was compared between the different species (P < 0.001); only the brain and ovary were exceptions.
Kidneys and ovaries contain the highest concentrations of Zn in all the three species, reaching a maximum of 929 and 552/~g/g dry weight in the common carp, 218 and 304 #g/g in goldfish, 106 and 276/zg/g in catfish. The liver is also rich in zinc and again it is at its highest level in the common carp (333 #g/g), whereas in catfish and goldfish it is 86 and 78/~g/g, respectively. The lowest concentrations of zinc were found in the muscle of catfish (22.6 ~g/g), carp (28.3 /zg/g) and goldfish (43.6
Table 2 Mean concentration of copper 4- S.D. (/~g/g dry wt.) in tissues and organs of catfish, goldfish and carp Fish species
White muscle
Brain
Liver
Kidney
Ovary
lctalurus melas Carassius auratus Ciprinus carpio
1.70 + 0.79 2.71 4- 0.94 1.41 4- 0.73
4.8 + 1.6 8.0 4- 3.0 4.64 + 0.60
22.4 4- 9.8 21 4- 19 18.8 4- 9.5
6.7 4- 2.3 10.4 4- 1.6 5.8 4- 1.0
6.2 4- 1.2 7.1 4- 3.2 3.4 + 1.1
Table 3 Zinc:copper ratio in tissues of catfish, goldfish and carp Fish species
Pond number
White muscle
Brain
Liver
Ictalurus melas
1 2 3 4 5 6
15.3 13.0 14.4 11.7 12.9 12.5
11.5 11.0 14.0 11.7 11.4 10.9
3.7 3.3 4.2 2.3 4.0 4.6
Carassius auratus Ciprinus carpio
(7 - 10) 4
15.0 20.0
8.0 13.0
3.7 17.7
Kidney
Ovary
18.6 15.8 16.5 13.5 16.6 15.5
46.8. 32.1 41.1 51.2 39.6 45.3
21.1 160.4
42.8 164.3
142
E. Carpenk et al/Sci. Total Environ. 141 (1994) 139-146
Table 4 Mean concentration of cadmium ± S.D. (/,tg/g dry wt.) in tissues of catfish, goldfish and carp Fish species
Liver
Kidney
lctalurus melas Carassius auratus Ciprinus carpio
0.30 + 0.15 0.34 + 0.27 0.37 + 0.15
1.36 + 0.83 1.46 -4- 0.94 1.21 + 0.64
/~g/g). Brain values were rather constant varying between 56.8 and 64.4/~g/g in catfish and goldfish, respectively. Copper concentrations are reported in Table 2; significant differences (P < 0.001) were found between the tissues (intra- and inter-species). Copper has generally lower concentrations than those for zinc and has the highest concentrations in the liver (18.8-22.4 /zg/g), other tissues vary between a minimum of 1.41 /~g/g in the muscle of the common carp to a maximum of 10.4 in the kidney of goldfish. The Zn:Cu ratio varies between a minimum of 2.3 in the liver of the catfish to a maximum of 164.3 in the ovary of the common carp and is very constant in catfish caught from the six different ponds. The data on the brain for all the species, were found to be rather uniform with values of: 10.9-14.0 ttg/g (catfish), 13.0 ~tg/g (carp) and 8.0 /zg/g (goldfish) (Table 3). Table 4 shows the cadmium content in the kidney and liver; in other organs the metal was below
the detection limit of 0.05 #g/g dry weight. Cadmium concentrations in the kidney are higher with respect to the liver and similar values were found between the species: 1.21-1.46/~g/g for kidney and 0.30-0.47/~g/g for liver. The origin of water and metal concentration (Zn, Cu) of diets utilized in the rearing ponds (only for catfish) is shown in Table 5. Concentrations of zinc, copper and cadmium from the catfish caught in the different ponds (Table 6) failed to-show any association between the trace element concentrations in tissues and the origin of water and the metal concentrations of diets. Seasonal variations of zinc, copper and cadmium were only studied for the catfish and are shown in Figs. 1-3, respectively. Zinc concentrations (Fig. 1) show significant differences in the muscle (P < 0.01), reaching a maximum in OctoberNovember, whereas in the ovary, (P < 0.01) there was a continuous increase starting from June to July until January to April. Zinc levels in brain were more constant. In Fig. 2 the Cu profiles of the different tissues are reported and again the muscle shows significant differences (P < 0.001) with a minimum in August-September, which is also repeated in the kidney (P < 0.005), in the ovary (P < 0.05) and in the brain (P < 0.01). The concentration of cadmium in catfish was only above the detection limit 0.05/~g/g in the kidney and liver, in the latter there were significant variations (P < 0.05) with a minimum in October-November (Fig. 3).
Table 5 Integrations a of zinc and copper (mg/kg) in the artificial diets fed to catfish in different ponds Pond number
Zinc
Copper
1 2 3 4 5 6
50 50 28 50 50 80
150 150 5 10 10 2,5
(artesian well) (channel: 'Canale Emiliano Romagnolo') (channel: 'Savena abbandonato') (channel: 'Navile') (channel: 'Canal Chiaro') (channel: 'Canal Dosolo')
aData obtained from the label of the company. The origin of water utilized in the rearing ponds is reported in brackets.
143
E. Carpenb et al/Sci. Total Environ. 141 (1994) 139-146 Table 6 Mean concentrations of zinc, copper and cadmium ± S.D. 0zg/g dry wt) in tissues of catfish in six different ponds Zinc Pond 1 Muscle Liver Kidney Ovary Brain
22.7 99.1 106 346 56.2
Copper
± 8.7 ± 8.3 ± 11 ± 142 ± 4.9
1.48 26.6 5.7 7.40 4.9
± ± ± ± ±
Cadmium
0.43 8.4 1.5 0.97 2.3
found among tissues of the fish, investigated in this paper, clearly indicate (Tables 1, 2 and 4), that each tissue has a characteristic metabolism for these trace elements. For Zn these results are not surprising, zinc is involved in many biochemical processes; Zn enzymes are rather common. The 40
0.34 ± 0.16 1.24 ± 0.69
3"0
20
Pond 2 Muscle Liver Kidney Ovary Brain
21.9 97 108.4 209 50.8
± ± ± 4±
1.2 15 9.4 71 5.8
1.68 30 6.9 6.5 4.6
± ± ± ± ±
0.60 14 2.4 1.4 1.0
Pond 3 Muscle Liver Kidney Ovary Brain
21.2 77.6 100 228 59.0
± ± ± ± ±
4.9 8.3 15 50 2.0
1.47 18.6 6.0 5.5 4.2
± ± 4± ±
0.67 8.2 2.1 1.1 1.2
10
80
60
40
0.29 ± 0.15 1.84 ± 0.94
20 f
E-
Pond 4 Muscle Liver Kidney Ovary Brain
MUSCLE
0.37 ± 0.17 2.0 ± 1.2
23.3 70 115 304 58.2
± ± ± ± ±
5.6 31 17 70 7.4
2.0 18.5 8.5 5.9 5.0
± ± ± ± ±
1.2 6.8 3.0 1.0 1.3
i
i
i
l
I
i
L
120 100
0.29 ± 0.18 1.15 ± 0.54
-,3
80 60
v
E
40
,5
20
o
Pond 5 Muscle Liver Kidney Ovary Brain
20.5 96 95.0 221 52.0
± ± ± ± ±
1.1 12 4.0 102 3.1
1.58 24.1 5.7 5.6 4.6
± ± ± ± ±
0.70 9.2 1.6 1.0 1.3
N
0.32 ± 0.04 1.27 ± 0.35
15o
lo0 KIDNEY 50
Pond 6 Muscle Liver Kidney Ovary Brain
24.8 76 105.3 289 62.2
± ± ± ± ±
4.2 13 5.2 27 4.0
1.98 16.6 6.81 6.39 5.7
± ± ± ± ±
0.47 3.7 0.37 0.31 1.7
i
0.19 ± 0.06 0.73 ± 0.07
I
500 400 300 209
The regression analysis between fish length and metal content gave negative results.
lOO
J u n'-Ju, lly " 1989
4. Discussion The significant differences in metal contents,
Aug-Seo" (Time)
Oct~Nov
Jan'-Apt
May
1990
Fig. 1. Seasonal variations of zinc (#g/g dry wt) in different tissues of catfish. The vertical bars are standard deviations of the mean values.
144
E. CarpenO et al/Sci. Total Environ. 141 (1994) 139-146
0,6
,.c o-I
"t3
0,4
0,2
2,5 o
2,0
E E (D
KIDNEY
Jun-July 1989
40 30
Aug-See Oct-Nov (Time)
Jan-Apt 1990
May
Fig. 3. Seasonal variations of cadmium (#g/g dry wt) in kidney and liver of catfish. The vertical bars are standard deviations of the mean values.
"o r~
1,0 0,5
BRAIN
J~
1,5
20
1
(.9
KIDNEY
OVARY
Joo'-Ju,y 1989
Aog'-Se, oot-;~o.
J..-A,,
(Time)
1990
M.'y
Fig. 2. Seasonal variations of copper (#g/g dry wt) in different tissues of catfish. The vertical bars are standard deviations of the mean values.
Zn:Cu ratios are always > 1 (Table 3), while in brain and muscle the Zn:Cu ratios are rather constant compared to liver and kidney, which are variable. Zn:Cu ratios of catfish brain, notwithstanding that fish are kept in different ponds
and fed with different diets, only vary between 10.9 and 14.0 and similar ratios are presented by the common carp, 13.0, and the goldfish, 8.0. This organ seems to be able to efficiently regulate the concentrations of the two metals. Zinc concentrations in fish brain are relatively high compared to muscle values and could be associated with an important physiological role. Recent studies have shown that in the mammalian central nervous system the abundance of Zn is related to the modulation of synaptic transmission (Xie and Smart, 1991). Moreover a zinc metallothionein has been isolated from a rat's brain after intracerebroventricular administration of Zn and Cu (Ebadi et al., 1989). Similar concentrations were found previously in fish, but in two predatory species (trout and sea bass) where Zn:Cu ratios in brain were 3.7 and 1.4-2.0, respectively, the change was due mostly to an increase in copper rather than a decrease in Zn (Carpen6 et al., 1990). The rather high levels of Zn found in the ovary, are probably linked to a functional role (storage of essential elements for the developing embryo). The high values for Zn, Cu and Cd found in the liver and kidney are likely linked to the synthesis of metallothionein that can be easily induced by these
E. Carpen6 et al / Sci. Total Environ. 141 (1994) 139-146
elements in fish. In goldfish a cadmium-thionein, experimentally induced by intraperitoneal injection of cadmium with doses of 0.5-2.0 mg Cd/kg body weight, has been recently isolated and purified (Carpen6 and Vas~k, 1989). The most evident differences among the three species studied, in relation to metal content, are the high values of Zn found in the kidney and liver of the common carp. The amounts of zinc and copper in liver can provide a useful parameter for the evaluation of a balanced diet, as these elements are stored in this organ when they are available in excess. Differences among species can, however, exist; Overnell et al. (1988) reported that there is an apparent lack of an effect of supplementary dietary zinc on the zinc metabolism and some type of regulation must be involved. In the common carp the dietary zinc affects zinc concentrations in the tissues (Jeng and Sun, 1981). The ratio of trace elements between liver and kidney can also depend on metallothionein which is firstly synthesized in the liver and then transferred to the kidney, where it is accumulated reaching values which can exceed those of the liver (Webb, 1987). In this respect the presence of cadmium in the liver and kidney can be a useful environmental indicator for cadmium exposure: a subacute longterm exposure will cause higher renal levels respect to the liver; in contrast higher hepatic concentrations, respect to the kidney, will be present in acute intoxication. Channel catfish, Ictalurus punctatus can accumulate cadmium, in the liver and kidney, from very dilute solutions, (10 9 M) whereas in the muscle the metal was detectable after 7 weeks of exposure to concentrations of 10 4 M (Bentley, 1991). Seasonal variations, (Figs. 1-3) only studied in the catfish, showed a peak of Zn (32.7 ~.g/g) in the muscle and a corresponding maximum concentration of 395 >g/g of the same metal in the ovary, both occurring during the fall. In the muscle the zinc peak could be due to a growth cycle; for the grey mullet a growth cycle is indicated by the appearance of new muscle fibers at the beginning of September (Carpen6 and Veggetti, 1981). Several Zn-enzymes are involved in protein synthesis and gene expression is regulated by Zn-finger proteins
145
(Klug and Rhodes, 1987), all these phenomena will probably increase the zinc content. In the ovary the variation can be linked with reproduction, for example in catfish the spawning period is between May and June. A decrease of Zn, Cu, Fe, V and Cd during the reproduction period is reported for Mullus barbatus which ends after the deposition of eggs (Lafaurie et al., 1980); then metals increase again for a new cycle. In the ovary of the marine teleost Blennius polis, Cu and Cd show a rapid reduction during April and early May (the spawning period starts in March and extends until July) (Shackley et al., 1981). We conclude from our data that each tissue has its own metabolism as regard to trace elements; changes in water quality and diets are not always associated with changes in metal concentrations in fish tissues. The amounts of Cd in liver and kidney tissues can be a useful indicator for levels of environmental Cd pollution. Seasonal variations are linked to cyclic biological processes which are present in fish. 5. References Badsha, K.S. and C.R. Goldspink, 1982. Preliminary observations on the heavy metal content of four species of freshwater fish in NW England. J. Fish Biol., 21: 251-267. Bentley, P.J. 1991. Accumulation of cadmium by channel catfish (Ictalurus punctatus): influx from environmental solutions. Comp. Biochem. Physiol., 99C: 527-529. Carpen6, E. and A. Veggetti, 1981. Increase in the lateralis muscle (white portion) of Mugilidae (pisces, Teleostei). Experientia, 37: 191-193. Carpen& E. and M. Vasfik, 1989. Hepatic metallothionein from goldfish tCarassius auratus). Comp. Biochem. Physiol., 92B: 463-468. Carpene, E., O. Canani, G.P. Serrazaneni, G. Fedrizzi and P. Cortesi, 1990. Zinc and copper in fish from natural waters and rearing ponds in Northern Italy. J. Fish Biol., 37: 293-299. Chakoumakos, C., R.C. Russo and R.V. Thurston, 1979. Toxicity of copper to cut-throat trout (Salmo clarki) under different conditions of alkalility, pH and hardness. Environ. Sci. Technol., 13: 213-219. Crespo, S. 1984. An in vitro study of the effects of zinc on osmoregulatory processes. Mar. Pollut. Bull., 15: 341-342. Crespo, S., G. Nonnotte, D.A. Colin, C. Leary, L. Nonnotte and A. Aubree, 1986. Morphological and functional alterations induced in trout intestine by dietary cadmium and lead. J. Fish Biol., 28: 69-80. Ebadi, M., V.K., Paliwal, T. Takahashi and P.L. lversen, 1989.
146 Zinc metallothionein in mammalian brains. In: D. Winge and D. Hamer (Eds), Metal Ion Homeostasis: Molecular Biology and Chemistry. A.R. Liss, Inc., New York, pp. 257-267. EI-Domiaty, N.A. 1987. Stress response of juvenile Clarias lazera elicited by copper. Comp. Biochem. Physiol., 88: 259-262. Hardy, R.W., C.V. Sullivan and A.M. Koziol, 1987. Absorption, body distribution and excretion of dietary zinc by rainbow trout (Salmo gairdneri). Fish Physiol. Biochem., 3: 133-143. Hardy, R.W. and K.D. Shearer, 1985. Effect of dietary calcium phosphate and zinc supplementation on whole body zinc concentration of rainbow trout (Salmo gairdneri). Can. J. Fish. Aquat. Sci., 42: 181-184. Jeng, S.S. and L.T. Sun, 1981. Effects of dietary zinc levels on zinc concentration in tissues of common carp. J. Nutr., I 11: 134-140. Klug, A. and D. Rhodes, 1987. "Zinc fingers": a novel protein motif for nucleic acid recognition. Tibs, 12: 464-469. Knox, D., C.B. Cowey and J.W. Adron, 1984. Effects of dietary zinc intake upon copper metabolism in rainbow trout (Salmo gairdneri). Aquaculture, 40: 199-207. Lafaurie, M., P. Miramand, J.C. Guary and S.W. Fowler, 1980. Variation des concentrations de Cu, Fe, Zn, Mn, Cd, et V dans les principaux organes de Mullus barbatus Linn au cours du cycle sexuel. In the Proceedings of: Workshop on Pollution of the Mediterranean. Cagliari. C.I.E.S.M. Monaco, pp. 373-376. Ogino, C. and G.Y. Yang, 1978. Requirement of rainbow trout for dietary zinc. Bull. Jpn. Soc. Sci. Fish, 44: 1015-1018.
E. Carpenk et al/ Sci. Total Environ. 141 (1994) 139-146 Overnell, J., T.C. Fletcher and R. Mclntosh, 1988. The apparent lack of effect of supplementary dietary zinc on zinc metabolism and metallothionein concentrations in the turbot, Scophthalmus maximus (L.). J. Fish Biol., 33: 563-570. Shackley, S.E., P.E. King and S.M. Gordon, 1981. Vitellogenesis and trace metals in a marine teleost. J. Fish Biol., 18: 349-352. Spry, D.J. and C.M. Wood, 1988. Zinc influx across the isolated, perfused head preparation of the rainbow trout (Salmo gairdneri) in hard and soft water. Can. J. Fish. Aquat. Sci., 45: 2206-2215. Syed, M.A. and T.L. Coombs, 1982. copper metabolism in the plaice, Pleuronectes platessa (L.). J. Exp. Mar. Biol. Ecol., 18: 281-296. Waldichuck, M. 1974. Some biological concerns in heavy metal pollution. In: F.J. Vernberg and W.B. Vernberg (Eds), Pollution and Physiology of Marine Organisms. Academic Press, London-New York, pp. 1-57. Webb, M. 1987. Toxilogical significance of metallothionein. In: J.H.R. K/igi and Y. Kojima (Eds), Metallothionein II. Birkh/iuser Verlag, Basel, pp. 109-134. Willis, J.N. and W.G. Sunda, 1984. Relative contributions of food and water in the accumulation of zinc by two species of marine fish. Mar. Biol., 80: 273-279. Xie, X. and T.G. Smart, 1991. A physiological role for endogenous zinc in rat hippocampal synaptic neurotransmission. Nature, 349: 521-524. Zitko, V. and W.G. Carson, 1977. Seasonal and developmental variation in the lethality of zinc to juvenile atlantic salmon (Salmo salar). J. Fish. Res. Board Can., 34: 139-141.
Total E n v i r o n m e n t inlo the En',a~*menl
ELSEVIER
I n d its R e l l t m n s h i p ~ i l h ~11~
The Science of the Total Environment 141 (1994) 139-146
Trace elements (Zn, Cu, Cd) in fish from rearing ponds of Emilia-Romagna region (Italy) E. C a r p e n e
,,a
,B.
•
G u m i e r o b, G . F e d n z z l
.a
, R. Serra a
aDepartment oJ Biochemistry ( Veterinary Biochemistry Section). Belmeloro St. 8/2. hDepartment of Evolutionary and E.~perimental Biology S. Giacomo St 0.9, University of Bologna, Bologna, Italy
(Received 30 March 1992: accepted 8 August 1992}
Abstract
Significant differences in Zn and Cu content were found between different organs. Zinc reaches its maximum, mean value (929/~g/g dry wt), in the kidney of the common carp and consistently has a high value (276-552 #g/g dry wt) in the ovary of all the species examined; Cu is at its maximum (21 #g/g dry wt) in the liver of goldfish. Zinc concentration in brain tissue is rather constant and is probably well regulated because of a functional role in this organ. Cadmium was only found in the liver and kidney, being approximately four times higher in the latter. Seasonal variations in trace element contents have been demonstrated in some organs of catfish; in the ovary, zinc concentrations could be linked with the reproductive cycle, while in the muscle with a growth cycle. Key words: Zinc; Copper; Cadmium; Fish
1. Introduction
Fish can obtain their trace elements, either directly from the water through the gills or indirectly from food through the alimentary tract (Willis and Sunda, 1984; Hardy et al., 1987; Spry and Wood, 1988). The mechanisms of transport of metals across the membrane barriers have yet to be fully examined. Although zinc and copper are essential to fish (Ogino and Yang, 1978; Knox et al., 1984), high concentrations may be toxic (Waldichuck, 1974; Crespo, 1984; El-Domiaty, 1987). In contrast cadmium is considered only for * Corresponding author.
its negative effects (Crespo et al., 1986). The availability of these elements can be influenced by many environmental factors (Zitko and Carson, 1977; Chakoumakos et al., 1979) and by composition of diet (Hardy and Shearer, 1985); as a consequence the physiological requirements of the essential elements are difficult to determine. The analysis of metal content in different tissues of fish can provide further information on trace element requirements and on their toxicity (Badsha and Goldspink, 1982; Carpen6 et al., 1990). In an area close to Bologna there are several small ponds which now are utilized for rearing fish. The water in these ponds comes mainly from irrigation channels and in part from artesian wells,
0048-9697/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved. SSDI 0048-9697(92J03522-4
E. Carpenk et al/Sci. Total Environ. 141 (1994) 139-146
140
the latter are considered unpolluted, but data on metal concentrations are lacking. Most of the reared fish belong to Carassius auratus (goldfish) and to Ictalurus melas (catfish), other species such as Cyprinus carpio (common carp) are occasionally present. They are semi-intensively and intensively reared and fed with artificial pelleted diets, containing different amounts of copper and zinc. The aim of this study is to determine the metal content (Zn, Cu, Cd) in tissues of fish reared in different ponds, in order to provide information about the influence of water and diets on the tissue metal composition. The results could be useful for an eventual improvement of diet formulation (Zn, Cu). The cadmium concentration was also calculated so that the levels of this potentially harmful element could be determined and fish could be used as indicators of pollution by heavy metals. Moreover, in one species (catfish) the three elements were analysed over a period of I year, to observe if there were seasonal variations, which could be related to fish biology. 2. Materials and methods
2.1. Animals Fish, including specimens of catfish (Ictalurus melas), goldfish ( Carassius auratus) and common carp (Ciprinus carpio) were caught with seine nets during 1989-1990 in different ponds of EmiliaRomagna region. They were measured (fork length) and their length was, respectively: 17.9 ± 2.8 cm (n = 125) for catfish, 9.1 + 4.0 cm (n = 30) for goldfish and 29.9 ± 9.4 cm (n = 25) for common carp. The number and size range was based on the availability of fish in the catch, since fish were obtained directly from the pond. Fish were separated into pools of five specimens and organs (brain, liver, kidney, muscle and ovary) were sampled from each pool and kept lyophilised until analysed. Only for catfish was it possible to provide a complete set of samples to cover the period of 1 year. Catfish were collected during the following five different periods: June-July, AugustSeptember, October-November, January-April and May.
2.2. Pon& Fish were reared in 10 different ponds which we
numbered from 1 to 6 for catfish and from 7 to 10 for goldfish and common carp. The surface areas ranged from a minimum of 2755 m 2 to a maximum of 26 000 m 2. Water was obtained from irrigating channels and only in one case from an artesian well (pond 1). Water temperature ranged between I°C in winter and 30°C in summer; oxygen concentrations fluctuated, falling during the night close to 1-2 mg/litre and, during the day they increased beyond the saturation levels. From spring to fall the water was rather turbid due to continuous phytoplankton blooms.
2.3. Feeding offish Fish were fed with commercial pelleted food of different brands. The catfish (data from food labels) were fed with diets that contained Zn and Cu in the range of 50-80 and 2.5-150 #g/g dry weight, respectively, cadmium was not determined. For the other two species it was not possible to identify the brand of food given to the fish and hence trace element concentrations, because farmers did not adopt the same diet throughout the year.
2.4. Metal analysis Samples of lyophilized tissues from each separated pool were weighed and ashed overnight in platinum dishes at 480°C; the white ash was dissolved in 1 N HC1 and analysed for zinc, copper and cadmium by flame atomic absorption spectrophotometry (AAS) using an Instrumentation Laboratories model IL-11 spectrophotometer. The accuracy of the method was evaluated by calibration with international standards (IAEA): MA-A2 (fish); MA-M-1 (oyster): MA-A-1 (copepod); MA-M-2 (mussels). The concentrations ( > 80%) found with the method used in this study fell in the confidence interval given by IAEA.
2.5. Statistical analysis The data (ug/g dry wt) presented in the tables are mean values + S.D. The error bars in the figures are standard deviations of the mean. The one- and two-way analysis of variance (ANOVA) was used to test significant differences, with P = 0.05 chosen as the minimum level of significance. Regression analysis between fish length and metal concentrations was computed.
141
E. Carpen~ et al/Sci. Total Environ. 141 (1994) 139-146 Table 1 Mean concentration of zinc 4- S.D. (t~g/g dry wt.) in tissues and organs of catfish, goldfish and carp Fish species
White muscle
Brain
Liver
Kidney
Ovary
lctalurus melas Carassius auratus Ciprinus carpio
22.6 ± 15.6 43.6 ± 5.9 28.3 4- 4.7
56.8 + 8.7 64.4 + 2.3 62 4- 18
86 4- 22 78 4- 43 333 4- 142
106 4- 14 218 + 67 929 4- 197
276 4- 100 304 + 35 552 4- 289
3. Results The mean Zn content and relative standard deviation in tissues of the catfish, the goldfish and the common carp are reported in Table 1. Significant differences (P < 0.001) were found between the different tissues of each species and when the same tissue was compared between the different species (P < 0.001); only the brain and ovary were exceptions.
Kidneys and ovaries contain the highest concentrations of Zn in all the three species, reaching a maximum of 929 and 552/~g/g dry weight in the common carp, 218 and 304 #g/g in goldfish, 106 and 276/zg/g in catfish. The liver is also rich in zinc and again it is at its highest level in the common carp (333 #g/g), whereas in catfish and goldfish it is 86 and 78/~g/g, respectively. The lowest concentrations of zinc were found in the muscle of catfish (22.6 ~g/g), carp (28.3 /zg/g) and goldfish (43.6
Table 2 Mean concentration of copper 4- S.D. (/~g/g dry wt.) in tissues and organs of catfish, goldfish and carp Fish species
White muscle
Brain
Liver
Kidney
Ovary
lctalurus melas Carassius auratus Ciprinus carpio
1.70 + 0.79 2.71 4- 0.94 1.41 4- 0.73
4.8 + 1.6 8.0 4- 3.0 4.64 + 0.60
22.4 4- 9.8 21 4- 19 18.8 4- 9.5
6.7 4- 2.3 10.4 4- 1.6 5.8 4- 1.0
6.2 4- 1.2 7.1 4- 3.2 3.4 + 1.1
Table 3 Zinc:copper ratio in tissues of catfish, goldfish and carp Fish species
Pond number
White muscle
Brain
Liver
Ictalurus melas
1 2 3 4 5 6
15.3 13.0 14.4 11.7 12.9 12.5
11.5 11.0 14.0 11.7 11.4 10.9
3.7 3.3 4.2 2.3 4.0 4.6
Carassius auratus Ciprinus carpio
(7 - 10) 4
15.0 20.0
8.0 13.0
3.7 17.7
Kidney
Ovary
18.6 15.8 16.5 13.5 16.6 15.5
46.8. 32.1 41.1 51.2 39.6 45.3
21.1 160.4
42.8 164.3
142
E. Carpenk et al/Sci. Total Environ. 141 (1994) 139-146
Table 4 Mean concentration of cadmium ± S.D. (/,tg/g dry wt.) in tissues of catfish, goldfish and carp Fish species
Liver
Kidney
lctalurus melas Carassius auratus Ciprinus carpio
0.30 + 0.15 0.34 + 0.27 0.37 + 0.15
1.36 + 0.83 1.46 -4- 0.94 1.21 + 0.64
/~g/g). Brain values were rather constant varying between 56.8 and 64.4/~g/g in catfish and goldfish, respectively. Copper concentrations are reported in Table 2; significant differences (P < 0.001) were found between the tissues (intra- and inter-species). Copper has generally lower concentrations than those for zinc and has the highest concentrations in the liver (18.8-22.4 /zg/g), other tissues vary between a minimum of 1.41 /~g/g in the muscle of the common carp to a maximum of 10.4 in the kidney of goldfish. The Zn:Cu ratio varies between a minimum of 2.3 in the liver of the catfish to a maximum of 164.3 in the ovary of the common carp and is very constant in catfish caught from the six different ponds. The data on the brain for all the species, were found to be rather uniform with values of: 10.9-14.0 ttg/g (catfish), 13.0 ~tg/g (carp) and 8.0 /zg/g (goldfish) (Table 3). Table 4 shows the cadmium content in the kidney and liver; in other organs the metal was below
the detection limit of 0.05 #g/g dry weight. Cadmium concentrations in the kidney are higher with respect to the liver and similar values were found between the species: 1.21-1.46/~g/g for kidney and 0.30-0.47/~g/g for liver. The origin of water and metal concentration (Zn, Cu) of diets utilized in the rearing ponds (only for catfish) is shown in Table 5. Concentrations of zinc, copper and cadmium from the catfish caught in the different ponds (Table 6) failed to-show any association between the trace element concentrations in tissues and the origin of water and the metal concentrations of diets. Seasonal variations of zinc, copper and cadmium were only studied for the catfish and are shown in Figs. 1-3, respectively. Zinc concentrations (Fig. 1) show significant differences in the muscle (P < 0.01), reaching a maximum in OctoberNovember, whereas in the ovary, (P < 0.01) there was a continuous increase starting from June to July until January to April. Zinc levels in brain were more constant. In Fig. 2 the Cu profiles of the different tissues are reported and again the muscle shows significant differences (P < 0.001) with a minimum in August-September, which is also repeated in the kidney (P < 0.005), in the ovary (P < 0.05) and in the brain (P < 0.01). The concentration of cadmium in catfish was only above the detection limit 0.05/~g/g in the kidney and liver, in the latter there were significant variations (P < 0.05) with a minimum in October-November (Fig. 3).
Table 5 Integrations a of zinc and copper (mg/kg) in the artificial diets fed to catfish in different ponds Pond number
Zinc
Copper
1 2 3 4 5 6
50 50 28 50 50 80
150 150 5 10 10 2,5
(artesian well) (channel: 'Canale Emiliano Romagnolo') (channel: 'Savena abbandonato') (channel: 'Navile') (channel: 'Canal Chiaro') (channel: 'Canal Dosolo')
aData obtained from the label of the company. The origin of water utilized in the rearing ponds is reported in brackets.
143
E. Carpenb et al/Sci. Total Environ. 141 (1994) 139-146 Table 6 Mean concentrations of zinc, copper and cadmium ± S.D. 0zg/g dry wt) in tissues of catfish in six different ponds Zinc Pond 1 Muscle Liver Kidney Ovary Brain
22.7 99.1 106 346 56.2
Copper
± 8.7 ± 8.3 ± 11 ± 142 ± 4.9
1.48 26.6 5.7 7.40 4.9
± ± ± ± ±
Cadmium
0.43 8.4 1.5 0.97 2.3
found among tissues of the fish, investigated in this paper, clearly indicate (Tables 1, 2 and 4), that each tissue has a characteristic metabolism for these trace elements. For Zn these results are not surprising, zinc is involved in many biochemical processes; Zn enzymes are rather common. The 40
0.34 ± 0.16 1.24 ± 0.69
3"0
20
Pond 2 Muscle Liver Kidney Ovary Brain
21.9 97 108.4 209 50.8
± ± ± 4±
1.2 15 9.4 71 5.8
1.68 30 6.9 6.5 4.6
± ± ± ± ±
0.60 14 2.4 1.4 1.0
Pond 3 Muscle Liver Kidney Ovary Brain
21.2 77.6 100 228 59.0
± ± ± ± ±
4.9 8.3 15 50 2.0
1.47 18.6 6.0 5.5 4.2
± ± 4± ±
0.67 8.2 2.1 1.1 1.2
10
80
60
40
0.29 ± 0.15 1.84 ± 0.94
20 f
E-
Pond 4 Muscle Liver Kidney Ovary Brain
MUSCLE
0.37 ± 0.17 2.0 ± 1.2
23.3 70 115 304 58.2
± ± ± ± ±
5.6 31 17 70 7.4
2.0 18.5 8.5 5.9 5.0
± ± ± ± ±
1.2 6.8 3.0 1.0 1.3
i
i
i
l
I
i
L
120 100
0.29 ± 0.18 1.15 ± 0.54
-,3
80 60
v
E
40
,5
20
o
Pond 5 Muscle Liver Kidney Ovary Brain
20.5 96 95.0 221 52.0
± ± ± ± ±
1.1 12 4.0 102 3.1
1.58 24.1 5.7 5.6 4.6
± ± ± ± ±
0.70 9.2 1.6 1.0 1.3
N
0.32 ± 0.04 1.27 ± 0.35
15o
lo0 KIDNEY 50
Pond 6 Muscle Liver Kidney Ovary Brain
24.8 76 105.3 289 62.2
± ± ± ± ±
4.2 13 5.2 27 4.0
1.98 16.6 6.81 6.39 5.7
± ± ± ± ±
0.47 3.7 0.37 0.31 1.7
i
0.19 ± 0.06 0.73 ± 0.07
I
500 400 300 209
The regression analysis between fish length and metal content gave negative results.
lOO
J u n'-Ju, lly " 1989
4. Discussion The significant differences in metal contents,
Aug-Seo" (Time)
Oct~Nov
Jan'-Apt
May
1990
Fig. 1. Seasonal variations of zinc (#g/g dry wt) in different tissues of catfish. The vertical bars are standard deviations of the mean values.
144
E. CarpenO et al/Sci. Total Environ. 141 (1994) 139-146
0,6
,.c o-I
"t3
0,4
0,2
2,5 o
2,0
E E (D
KIDNEY
Jun-July 1989
40 30
Aug-See Oct-Nov (Time)
Jan-Apt 1990
May
Fig. 3. Seasonal variations of cadmium (#g/g dry wt) in kidney and liver of catfish. The vertical bars are standard deviations of the mean values.
"o r~
1,0 0,5
BRAIN
J~
1,5
20
1
(.9
KIDNEY
OVARY
Joo'-Ju,y 1989
Aog'-Se, oot-;~o.
J..-A,,
(Time)
1990
M.'y
Fig. 2. Seasonal variations of copper (#g/g dry wt) in different tissues of catfish. The vertical bars are standard deviations of the mean values.
Zn:Cu ratios are always > 1 (Table 3), while in brain and muscle the Zn:Cu ratios are rather constant compared to liver and kidney, which are variable. Zn:Cu ratios of catfish brain, notwithstanding that fish are kept in different ponds
and fed with different diets, only vary between 10.9 and 14.0 and similar ratios are presented by the common carp, 13.0, and the goldfish, 8.0. This organ seems to be able to efficiently regulate the concentrations of the two metals. Zinc concentrations in fish brain are relatively high compared to muscle values and could be associated with an important physiological role. Recent studies have shown that in the mammalian central nervous system the abundance of Zn is related to the modulation of synaptic transmission (Xie and Smart, 1991). Moreover a zinc metallothionein has been isolated from a rat's brain after intracerebroventricular administration of Zn and Cu (Ebadi et al., 1989). Similar concentrations were found previously in fish, but in two predatory species (trout and sea bass) where Zn:Cu ratios in brain were 3.7 and 1.4-2.0, respectively, the change was due mostly to an increase in copper rather than a decrease in Zn (Carpen6 et al., 1990). The rather high levels of Zn found in the ovary, are probably linked to a functional role (storage of essential elements for the developing embryo). The high values for Zn, Cu and Cd found in the liver and kidney are likely linked to the synthesis of metallothionein that can be easily induced by these
E. Carpen6 et al / Sci. Total Environ. 141 (1994) 139-146
elements in fish. In goldfish a cadmium-thionein, experimentally induced by intraperitoneal injection of cadmium with doses of 0.5-2.0 mg Cd/kg body weight, has been recently isolated and purified (Carpen6 and Vas~k, 1989). The most evident differences among the three species studied, in relation to metal content, are the high values of Zn found in the kidney and liver of the common carp. The amounts of zinc and copper in liver can provide a useful parameter for the evaluation of a balanced diet, as these elements are stored in this organ when they are available in excess. Differences among species can, however, exist; Overnell et al. (1988) reported that there is an apparent lack of an effect of supplementary dietary zinc on the zinc metabolism and some type of regulation must be involved. In the common carp the dietary zinc affects zinc concentrations in the tissues (Jeng and Sun, 1981). The ratio of trace elements between liver and kidney can also depend on metallothionein which is firstly synthesized in the liver and then transferred to the kidney, where it is accumulated reaching values which can exceed those of the liver (Webb, 1987). In this respect the presence of cadmium in the liver and kidney can be a useful environmental indicator for cadmium exposure: a subacute longterm exposure will cause higher renal levels respect to the liver; in contrast higher hepatic concentrations, respect to the kidney, will be present in acute intoxication. Channel catfish, Ictalurus punctatus can accumulate cadmium, in the liver and kidney, from very dilute solutions, (10 9 M) whereas in the muscle the metal was detectable after 7 weeks of exposure to concentrations of 10 4 M (Bentley, 1991). Seasonal variations, (Figs. 1-3) only studied in the catfish, showed a peak of Zn (32.7 ~.g/g) in the muscle and a corresponding maximum concentration of 395 >g/g of the same metal in the ovary, both occurring during the fall. In the muscle the zinc peak could be due to a growth cycle; for the grey mullet a growth cycle is indicated by the appearance of new muscle fibers at the beginning of September (Carpen6 and Veggetti, 1981). Several Zn-enzymes are involved in protein synthesis and gene expression is regulated by Zn-finger proteins
145
(Klug and Rhodes, 1987), all these phenomena will probably increase the zinc content. In the ovary the variation can be linked with reproduction, for example in catfish the spawning period is between May and June. A decrease of Zn, Cu, Fe, V and Cd during the reproduction period is reported for Mullus barbatus which ends after the deposition of eggs (Lafaurie et al., 1980); then metals increase again for a new cycle. In the ovary of the marine teleost Blennius polis, Cu and Cd show a rapid reduction during April and early May (the spawning period starts in March and extends until July) (Shackley et al., 1981). We conclude from our data that each tissue has its own metabolism as regard to trace elements; changes in water quality and diets are not always associated with changes in metal concentrations in fish tissues. The amounts of Cd in liver and kidney tissues can be a useful indicator for levels of environmental Cd pollution. Seasonal variations are linked to cyclic biological processes which are present in fish. 5. References Badsha, K.S. and C.R. Goldspink, 1982. Preliminary observations on the heavy metal content of four species of freshwater fish in NW England. J. Fish Biol., 21: 251-267. Bentley, P.J. 1991. Accumulation of cadmium by channel catfish (Ictalurus punctatus): influx from environmental solutions. Comp. Biochem. Physiol., 99C: 527-529. Carpen6, E. and A. Veggetti, 1981. Increase in the lateralis muscle (white portion) of Mugilidae (pisces, Teleostei). Experientia, 37: 191-193. Carpen& E. and M. Vasfik, 1989. Hepatic metallothionein from goldfish tCarassius auratus). Comp. Biochem. Physiol., 92B: 463-468. Carpene, E., O. Canani, G.P. Serrazaneni, G. Fedrizzi and P. Cortesi, 1990. Zinc and copper in fish from natural waters and rearing ponds in Northern Italy. J. Fish Biol., 37: 293-299. Chakoumakos, C., R.C. Russo and R.V. Thurston, 1979. Toxicity of copper to cut-throat trout (Salmo clarki) under different conditions of alkalility, pH and hardness. Environ. Sci. Technol., 13: 213-219. Crespo, S. 1984. An in vitro study of the effects of zinc on osmoregulatory processes. Mar. Pollut. Bull., 15: 341-342. Crespo, S., G. Nonnotte, D.A. Colin, C. Leary, L. Nonnotte and A. Aubree, 1986. Morphological and functional alterations induced in trout intestine by dietary cadmium and lead. J. Fish Biol., 28: 69-80. Ebadi, M., V.K., Paliwal, T. Takahashi and P.L. lversen, 1989.
146 Zinc metallothionein in mammalian brains. In: D. Winge and D. Hamer (Eds), Metal Ion Homeostasis: Molecular Biology and Chemistry. A.R. Liss, Inc., New York, pp. 257-267. EI-Domiaty, N.A. 1987. Stress response of juvenile Clarias lazera elicited by copper. Comp. Biochem. Physiol., 88: 259-262. Hardy, R.W., C.V. Sullivan and A.M. Koziol, 1987. Absorption, body distribution and excretion of dietary zinc by rainbow trout (Salmo gairdneri). Fish Physiol. Biochem., 3: 133-143. Hardy, R.W. and K.D. Shearer, 1985. Effect of dietary calcium phosphate and zinc supplementation on whole body zinc concentration of rainbow trout (Salmo gairdneri). Can. J. Fish. Aquat. Sci., 42: 181-184. Jeng, S.S. and L.T. Sun, 1981. Effects of dietary zinc levels on zinc concentration in tissues of common carp. J. Nutr., I 11: 134-140. Klug, A. and D. Rhodes, 1987. "Zinc fingers": a novel protein motif for nucleic acid recognition. Tibs, 12: 464-469. Knox, D., C.B. Cowey and J.W. Adron, 1984. Effects of dietary zinc intake upon copper metabolism in rainbow trout (Salmo gairdneri). Aquaculture, 40: 199-207. Lafaurie, M., P. Miramand, J.C. Guary and S.W. Fowler, 1980. Variation des concentrations de Cu, Fe, Zn, Mn, Cd, et V dans les principaux organes de Mullus barbatus Linn au cours du cycle sexuel. In the Proceedings of: Workshop on Pollution of the Mediterranean. Cagliari. C.I.E.S.M. Monaco, pp. 373-376. Ogino, C. and G.Y. Yang, 1978. Requirement of rainbow trout for dietary zinc. Bull. Jpn. Soc. Sci. Fish, 44: 1015-1018.
E. Carpenk et al/ Sci. Total Environ. 141 (1994) 139-146 Overnell, J., T.C. Fletcher and R. Mclntosh, 1988. The apparent lack of effect of supplementary dietary zinc on zinc metabolism and metallothionein concentrations in the turbot, Scophthalmus maximus (L.). J. Fish Biol., 33: 563-570. Shackley, S.E., P.E. King and S.M. Gordon, 1981. Vitellogenesis and trace metals in a marine teleost. J. Fish Biol., 18: 349-352. Spry, D.J. and C.M. Wood, 1988. Zinc influx across the isolated, perfused head preparation of the rainbow trout (Salmo gairdneri) in hard and soft water. Can. J. Fish. Aquat. Sci., 45: 2206-2215. Syed, M.A. and T.L. Coombs, 1982. copper metabolism in the plaice, Pleuronectes platessa (L.). J. Exp. Mar. Biol. Ecol., 18: 281-296. Waldichuck, M. 1974. Some biological concerns in heavy metal pollution. In: F.J. Vernberg and W.B. Vernberg (Eds), Pollution and Physiology of Marine Organisms. Academic Press, London-New York, pp. 1-57. Webb, M. 1987. Toxilogical significance of metallothionein. In: J.H.R. K/igi and Y. Kojima (Eds), Metallothionein II. Birkh/iuser Verlag, Basel, pp. 109-134. Willis, J.N. and W.G. Sunda, 1984. Relative contributions of food and water in the accumulation of zinc by two species of marine fish. Mar. Biol., 80: 273-279. Xie, X. and T.G. Smart, 1991. A physiological role for endogenous zinc in rat hippocampal synaptic neurotransmission. Nature, 349: 521-524. Zitko, V. and W.G. Carson, 1977. Seasonal and developmental variation in the lethality of zinc to juvenile atlantic salmon (Salmo salar). J. Fish. Res. Board Can., 34: 139-141.