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The uptake and loss of dissolved cadmium by the stickleback, Gasterosteus aculeatus L.
ECOTOXICOLOGY
AND ENVIRONMENTAL
SAFETY
14,88-96 (1987)
The Uptake and Loss of Dissolved Cadmium by the Stickleback, Gasterosteus aculeatus L. J. A. 0. ORONSAYE’ Queen Elizabeth College, Campden Hill Road, London WS, England Received May 13. I986 The whole-body uptake of cadmium by the three-spitted stickleback (Gasterosteus aculeatus) has been measured after exposure of fish to 2.5 and 5.0 mg @+/liter hard water and equal amounts of 2.0 mg Cd*+ plus 2 mg Zn*+/liter and 4.0 mg Cd” plus 4.0 mg Zn’+/liter hard water, respectively. Fish absorbed and retained cadmium while the uptake and accumulation of zinc was depressed. The loss of absorbed cadmium was quicker in fish previously exposed to cadmium plus zinc solutions used together. This phenomenon is discussed in relation to the ability of fish to excrete absorbed cadmium. @1987 Academic press, IUC.
1. INTRODUCTION In all cases where fish have been exposed to cadmium in solution, it has been found that the metal is absorbed and retained (Coombs, 1974). Such absorption is probably due to passive diffusion (Bryan, 1976) down a gradient between the mucus coating the gill on the one hand, and the internal organs on the other. Difhtsion is presumably responsible for the uptake of zinc by the eggs of coho salmon (Oncorhynchus kisutch) (Wedemeyer, 1968), and by the adults of numerous fish species (Skidmore, 1964), and also for the uptake of lead by the spotted wolffish (Anarchichas miner) (Bollingberg and Johansen, 1979). So uptake of cadmium is also likely to be due to simple diffusion. Mount and Stephan (1967) studied the uptake and accumulation of cadmium by bluegill (Lepomis macrochirus). They reported that when fish were exposed to a high concentration (10 mg/liter of cadmium) the gills were the major site of uptake. The latter authors found that cadmium accumulated in L. macrochirus and that, within 30 to 60 days, an equilibrium was established between the concentrations of cadmium in the water and in the fish tissue. Sephar (1967) exposed flagtish (Jo&nellufloridae) to cadmium in water of 44 mg/liter hardness as CaC03 at 25’C. These fish were shown to accumulate more cadmium over longer exposure periods and in higher concentrations of the metal. (The fish were exposed to sublethal doses of 1.73 1 mg/liter of cadmium). Spehar ( 1976) observed that “cadmium uptake increased with increasing exposure concentration but levelled off at 16 pg/liter,” and suggested that a possible equilibrium was reached between cadmium concentrations in the water and in the tissue. Tafanelli and Summerfelt ( 1975) introduced cadmium intraperitoneally into goldfish (Carussius auralus). These authors injected 6 mg Cd2’/kg wet weight and ob’ Present address: Department of Fisheries, Faculty of Agriculture, University of Benin, P.M.B. 1154, Benin City, Nigeria. 0147-6513187 $3.00 C@yri&t 8 1987 by Academic PUSS,Inc. AU rights of n5pmduction in any form rcsemed.
88
UPTAKE
OF CADMIUM
BY Gusterosteusuculeatus
89
served that the kidney concentrated an amount of cadmium up to 400 mg/kg (in terms of dry weight of the organ). No value was reported for the gills in this fish. The kidneys of fish have a role in the excretion of absorbed cadmium. When rainbow trout (Salvo guirdneri) were transferred to cadmium-free water after previous exposure to 0.0048 mg/liter cadmium for time periods up to 30 weeks, Kumada et al. ( 1972) observed that the concentration of cadmium in the whole body of the fish fell by about 60% in 10 weeks. The study by Calamari and Marchetti (1977) also on S. guirdneri appears to confirm the results obtained by Kumada et al. (1972) on that species. Calamari and Marchetti (1977) observed that the concentration of cadmium (in fish previously exposed to 50 pg mg/liter for 120 days) in the gills fell by 50% after 50 days. It can be assumed from the latter result that, in recovering fish, a movement of cadmium from other organs to the kidney caused the persistently high kidney residue levels. Cadmium often occurs in the environment along with other heavy metals. The uptake of cadmium together with other heavy metals, principally zinc, has been studied. Eisler and Gardner ( 1973) for example, reported that the uptake of zinc by mummichog (Fundzdus heteroclitus)was reduced in the presence of cadmium. The fish were exposed to l-10 mg Cd2+/liter along with 36 or 60 mg Zn2+/liter in synthetic sea water. In the present study, using Gusterosteus, the uptake of cadmium has been investigated using solutions of cadmium alone or solutions of cadmium along with equimolar concentrations of zinc. The loss of cadmium from fish after previous exposure to these metals (cadmium or cadmium plus zinc) has also been studied. 2. METHODS Fish in batches of 25 were exposed to either 2.50 or 5.0 mg Cd2+/liter tap water (with a hardness of 299.0 mg CaCOJiter). A second uptake experiment involved two mixed solutions of cadmium and zinc, 2 mg Cd’+ plus 2 mg Zn’+/liter, and 4 mg Cd2’ plus 4 mg Zn’+/liter. To avoid uptake by the gut, fish were not fed during exposure to cadmium. Lengths of exposure ranged between 1 and 10 days. In experiments on the loss of metals, fish were exposed to either 2.1 or 4.0 mg Cd’+/liter. Other experiments used mixed solutions of cadmium and zinc: 2.0 mg Cd2+ plus 2.0 mg Zn2+/liter, and 4.0 mg Cd2+ plus 4.0 mg Zn’+/liter. Exposure times ranged between 8.5 and 10 days. Surviving fish were allowed to recover in uncontaminated water for periods between 1 and 2 1 days. Only recovering fish were fed (a daily ration of freezedried Tubifx sp.). All experiments were carried out in the continuous-flow system (Oronsaye and Brafield, 1984). Experimental conditions were similar. A control experiment (also using unfed fish) was run. The details of experiments are given in Table 1. The experimental tanks, the tubing of the continuous flow system, and the 50-ml Pyrex beakers used for “wet digestion” of fish (according to the method of Leonard, 197 l), were cleaned with Divo-lab to remove ions from their surfaces. (The beakers had been previously soaked overnight in 50% nitric acid and rinsed in deionized water.) After being placed in uncontaminated water for 5 min to remove any cadmium or zinc adhering to their body surfaces, fish were removed at intervals and their body loads of cadmium (or zinc) were measured. Fish were pithed and the dried with filter paper. Because of their small sizes fish were placed in pairs in the beakers. The wet weights and, after drying in an oven, the dry weights of fish were recorded.
90
J. A. 0. ORONSAYE TABLE 1 DETAILSOFEXPERIMENTS
Exposure times
Test solution concentrations
(days)
Cadmium uptake 2.5 mg @+/liter
Recovery times (&YS)
l-10
-
l-10 l-9
-
10
24-504 24-408
10
24-m 24-312
Wet weight of fish
k) 0.3-0.6
5.0 mg @+/liter
Cadmium uptake in the presence of zinc 2.0 mg Cd*+ + 2.0 mg Zn’+/liter 4.0 mg Cd” + 4.0 mg Zn’+/liter
Loss of absorbed cadmium after exposure to 2.1 mg Cd2+/liter 4.0 mg Cd2’/liter Loss of absorbed cadmium and zinc after exposure to 2.0 mg Cd” + 2.0 mg Zn’+/liter 4.1 mg Cd” + 4.1 mg Zn’+/liter
0.4-0.7
0.2-0.6
0.2-0.5 8.5
Fish were small with wet weights between 0.2 and 0.7 g and dry weights between 2.05 and 0.17 g. Fish were not ashed and, as recommended by Leonard ( 197 l), digestion involved a 4: 1 mixture of nitric acid and perchloric acid. Beakers containing the specimens (whole fish) were placed on a hot plate with a fume hood, and with water flushing the hood. Eight milliliters of 70% nitric acid and 2 ml of 70% perchloric acid (analytical grade) were added to each beaker. The hot plate was maintained at 80 + 5°C during digestion, which lasted between 26 and 29 hr. When fats appeared in solution during this digestion, generally indicated by a color change from pale yellow to brown, additional nitric acid (2 ml) was added to each sample. Evaporation was continued until the white fumes of perchloric acid ceased. Each residue (which was now white) was allowed to cool in a desiccator before being dissolved in 10 ml deionized water. The amounts of either cadmium or zinc in the sample (and in the test solutions) were measured by an atomic absorption spectrophotometer, previously calibrated. Levels of cadmium and zinc found in fish tissue are shown in Figs. 1 to 7. All experiments were run in tap water, at 12 + 2”C, and two fish generated each point shown in the graphs. 3. RESULTS
Uptakeof Cadmium Fish that died during the experiments were not analyzed for cadmium residue in the tissues. F. heteroclitus(Eisler, 1974) and G. aculeatus(Pascoe and Mattey, 1977) have been shown to accumulate cadmium after death, so analyses which could have resulted in very high cadmium concentrations in tissues were avoided. Results are expressed in terms of wet weight of fish, not dry weight. Brafield and Matthiessen
UPTAKE
OF CADMIUM
EXPOSURE
BY Gasterosteus
TIME
acuieutus
91
(days)
FIG. 1. Whole-body tissue levels of cadmium in fish exposed to 2.5 (W) or 5.0 (0) mg Cd*+/liter.
(1976) presented their results on oxygen consumption of G. aculeutus in terms of wet weight, though dry weight is often used and is perhaps preferable. The mean concentration of cadmium in whole-body preparations of eight unexposed G. aculeatus was 0.0 1 mg Cd2+/g wet weight, with a range of 0.0093 to 0.0 106 mg/g. Fish exposed to 2.50 mg Cd2+/liter for 24 hr showed an increase in body cadmium, 0.1653 mg Cd’+/g, which represents a total (about 0.175 mg/g) nearly 18 times the normal mean value. A longer exposure of hsh (up to 10 days) to 2.50 mg Cd2’/liter resulted in an erratic decline in body load (Fig. 1). On the other hand, fish exposed to 5.0 mg Cd2’/liter failed to reduce the internal concentration of cadmium. As is shown in Fig. 1, the internal concentration of cadmium showed a high increase with time. After a lo-day exposure, body burden was 52 times the normal level (0.5241 mg/g wet wt compared to 0.0 1 mg/g wet wt). Uptake of Cadmium in the Presence of Zinc The mean concentration of zinc in whole-body preparations of eight unexposed fish was 0.0601 mg Zn2+/g wet weight, with a range of 0.0544 to 0.0702 mg/g. Fish exposed to 2 mg Cd2’ plus 2 mg Zn’+/liter built up high cadmium tissue levels: 30, 25, and 20 times normal tissue levels after 6, 8, and 10 days exposure, respectively. Figure 2 shows that there was also an initial influx of zinc but that the level fell from twice the normal tissue level to a near normal level after 3 days. At the close of the experiment ( 10 days) the zinc residue was at the normal tissue concentration for this metal, while cadmium retained in fish was about 20 times the normal tissue level of cadmium. When the concentration of the test solution was doubled, however, fish did not appear to concentrate zinc at any stage (Fig. 3), although cadmium accumulated to levels 16 and 22 times that of normal fish after 3 and 7 days, respectively. Although the levels of metals retained in fish are lower than those found when fish were in a solution half as concentrated (Fig. 2) more fish died during the experiments using 4 mg Cd2+ plus 4 mg Zn2+/liter. Loss ofAbsorbed Cadmium When fish exposed to either 2.0 or 4.0 mg Cd2+/liter for 10 days were allowed to recover in cadmium-free water, approximately 50% of the absorbed metal was lost
92
J. A. 0. ORONSAYE
NWlll~l cadmium Jtis.ue level
-----3
-.-
EXPOSURE FIG.
-.
J
Normal zinc tiollue level Normal caclmlum
TIME (days)
2. Whole-body tissue levels of cadmium (M) and zinc (0) in fish exposed to 2 mg Cd*+ + 2 mg Zn*+/
liter. FIG. 3. Whole-body tissue levels of cadmium (a) and zinc (0) in fish exposed to 4 mg of Cd” + 4 mg Znz+/liter.
from the body after 3 days. Figures 4 and 5 indicate that increasing the recovery period beyond 2 1 days could result in all previously absorbed metal being lost from the body, although the rate of efflux slows with time. Fish in both experiments still held approximately 15% of the absorbed cadmium at the close of the experiment. Loss ofAbsorbed Cadmium and Zinc During the uptake experiments in which G. aculeatus were exposed to solutions of both cadmium and zinc, it was observed that zinc was not concentrated by the fish.
0.2 m
-0
.,4 3
---L
s
E
‘--‘---.,c 1
3
5
7 9 RECOVERY
11 TIME
13 15 (days)
17
19
. 21
.lOO
FIG. 4. Loss of cadmium from the tissue of fish while in clean water after a previous exposure to 2 mg Cd*‘/liter for 10 days.
UPTAKE
OF CADMIUM
RECOVERY
93
BY Gusterosteus uculeutus
TIME
(days)
FIG. 5. Loss of cadmium from the tissue of fish while in clean water afkr a previous exposure to 4 mg Cd*+/liter for 10 days.
When these fish were allowed to recover in uncontaminated water, after either 10 days of exposure to 2 mg Cd*+ plus 2 mg Zn*+/liter or 8.5 days of exposure to 4 mg Cd*+ plus 4 mg Zn*+/liter, the concentration of cadmium in whole fish fell by about 50% after 24 hr. Figures 6 and 7 show that the rate of loss of cadmium from the fish was slow, perhaps slower than when fish previously exposed to only cadmium solutions were recovering. However, at the close of experiments, after 2 1 days (Fig. 6) and 13 days (Fig. 7), fish held about 12.5 and 20% of the original metal levels, respectively. 4. DISCUSSION The whole-body digests of Gasterosteus were examined for cadmium and zinc. A mean concentration of approximately 0.0 1 mg Cd*+/g wet wt was obtained for eight unexposed normal fish. Since cadmium is not normally found in tissue (unlike zinc it has no biochemical function), it is assumed that fish acquired this metal from the collection site. (Bottom sediments in the site of fish collection contained about 2 ppm Cd*+, on a dry weight basis (Oronsaye, 198 I).) The mean concentration of zinc found in the whole-body digests from the eight unexposed normal Gasterosteus was 0.060 1 mg/g wet weight. This is similar to the 0.072 mg/g wet wt of whole-body digests obtained by Matthiessen (1973). When Gasterosteus were exposed to either 2.5 or 5.0 mg Cd*+/liter for 10 days, the fish absorbed and retained the metal. These fish are able to reduce the concentration
1 . 1
3
5
7 9 RECOVERY
11 13 15 TIME (days)
17
19
’ 21
hl
FIG. 6. Loss of cadmium from the tissue of fish while in clean water after a previous exposure to 2 mg Cd*+ + 2 mg Zt?+/liter for 10 days.
94
J. A. 0. ORONSAYE
1
3 5 RECOVERY
7
9 TIME
11
13
. 15
100
(days)
FIG. 7. Loss of cadmium from the tissue of fish while in clean water after a previous exposure to 4 mg Cd*+ + 4 mg Zn’+/liter for 8.5 days.
of the metal absorbed (Fig. 1). This phenomenon was reported by Matthiessen and Brafield (1977) when Gusterosteus were exposed to solutions containing zinc, and has been “explained” by the fact that these fish are euryhaline and so adapted to sudden influx of ions. Other species of fish such as S. guirdneri (Kumada et al., 1972), absorb and accumulate cadmium. In the higher concentration of 5.0 mg C$+/liter uptake experiment which lasted for 10 days, more cadmium (in mg/g wet wt of fish) was retained. This observation appears to agree with those of Eisler (1974) on F. heteroclitus and Pascoe and Mattey (1977) on G. aculeutus, both fish being exposed to cadmium. The latter authors found that Gusterosteus exposed to cadmium (in water with a hardness of 103-l 11 mg CaCOJliter) increased the body load of the metal with increasing external concentration (0.90 cLg/g fresh weight at 0.001 mg Cd2+/liter to 5 1.O &g fresh weight at 100 mg Cd”/liter). Exposure of the fish to cadmium was reported to have lasted 79 days, at which time about 80% of the fish in each test concentration had died. The fish analyzed by Pascoe and Mattey ( 1977) had died during the experiments, but were removed as soon as possible after death. It appears that the excretion of cadmium by Gasterosteus exposed to 5.0 mg Cd2+/liter was poor when compared with the lower concentration of 2.5 mg Cd”/ liter. It is likely that cadmium diffusing into the fish after the sixth day reached a level high enough to inhibit the excretion of the metal, presumably through poisoning of the gill ion excretory cells. This suggestion may explain why the fish exposed to 5.0 mg Cd”/liter built up a body load of a 0.54 mg Cd2’/g wet weight, which is approximately 50 times the normal tissue level of the metal, whereas in 2.5 mg Cd2+/liter, fish accumulated only 0.07 mg Cd2+/g, approximately 7.5 times the normal tissue level, over the same period of time ( 10 days). In the experiments involving exposure to cadmium and zinc together, the accumulation of zinc by Gasterosteus was reduced by the presence of cadmium (Figs. 2 and 3). A similar result was obtained by Eisler and Gardner ( 1973), for when they exposed F. heteroclitus to solutions of both cadmium and zinc, the whole-body uptake of zinc was inhibited by the cadmium. Furthermore, the Gasterosteus used in my experiments (involving cquimolar concentrations of either 2 mg Cd2’ plus 2 mg Zn’+/liter or 4 mg Cd” plus 4 mg Zn2+/liter) retained a lower body load of cadmium over an 8-day test period, when compared to the fish exposed to 5.0 mg Cd”/liter alone. Since it is shown that cadmium and zinc may bind to the same metallothionein (Coombs, 1974), it can be assumed that the internal competition between these met-
UPTAKE
OF
CADMIUM
BY
Gusterosteus aculeatus
95
als for binding sites probably reduced the concentration of cadmium retained by the fish. Gusterosteus appears to lose cadmium quicker during recovery from exposure to cadmium and zinc together. The internal concentrations of the absorbed cadmium fell by 50% within 1 day (24 hr) in recovering fish previously exposed to 2 mg Cd” plus 2 mg Zn’+/liter, whereas 50% was lost after 3 days in the fish that were exposed to either 2.0 or 4.0 mg Cd”/liter. It is likely that the assumed competition by cadmium and zinc may leave a substantial amount of cadmium in the ionic state. It is conceivable that such unbound cadmium will be rapidly lost from the fish that are allowed to recover in clean water. 5. CONCLUSIONS This study has confirmed that Gasterosteus absorbs, accumulates, and excretes cadmium. That cadmium depresses the uptake of zinc when present in conjunction was also demonstrated. The latter appears to shed some light on the mode of action of cadmium to fish. Since zinc is an essential element in biochemical systems, its displacement will induce a state of zinc element deficiency. This phenomenon can result in the death of fish through poisoning by cadmium, a nonessential element now present in the protein molecules. It is conceivable, therefore, that even though cadmium may be lost from fish after exposure to cadmium and zinc used together, the bound cadmium not lost from the fish may continue to displace more zinc and eventually kill fish through cadmium poisoning. ACKNOWLEDGMENT I am grateful to Dr. A. E. Brafield for his encouragement, criticism, and supervision of this study.
REFERENCES BOLLINGBERG, H. J., AND JOHANSEN, P. (1979). Lead in spotted wolfish, Anarchichas minor, near a leadzinc mine in Correnland. J. Fish Res. Board Canad. 36,1023-1028. BRAFIELD, A. E., AND MAT~HIESSEN, P. (1976). Oxygen consumption by sticklebacks (Gasterosteus aculeatus L.) exposed to zinc. J. Fish Biol. 9,359-370. BRYAN, G. W. (1976). Some aspects of heavy metal tolerance in aquatic organisms. In E$ects ofPollutant on Aquatic Organisms (A. P. M. Lockwood, Ed.), pp. 7-34. Cambridge Univ. Press, London. CALAMARI, D., AND MARCHETII, R. (1977). Intluence of water hardness on cadmium toxicity to S&no guirdnert, Richardson. Paper presented to the International Workshop on Structure and Function of Fish Gills, 26-29 September 1977, Messina (mimeo). COOMBS, T. L. (1974). The significance of multielement analysis. In Metal Polhdions Studies. Ecological Toxicolo@al Research. NATO Science Committee Conference, London, Vol. 7, pp. 187- 195. EISLER, R. (1974). Radiocadmium exchange with seawater by Fund&s heterochtus (L.) (Pisces: Cyprinodontidae). J. Fish Bioi. 6,60 l-6 12. EISLER, R., AND GARDNER, G. R. (1973). Acute toxicology to an estuarine teleost of mixtures ofcadmium, copper, and zinc salts. J Fish Biol. 5,13 1- 142. KUMADA, H., K~MURA, S., YOKOTE, M., AND MATIDA, Y. (1972). Acute and chronic toxicity, uptake and retention of cadmium in freshwater organisms. Bull. Freshwater Fish Res. Lab. 22(2). LEONARD, E. N. (197 l).lThe determination of copper in fish tissue by atomic absorption spectrophotometry. At. Absorpt. Newsl. 10,84-85. MATTHIESSEN, P. (1973). Zinc Uptake and Its Efects on the Oxygen Consumption and Gilt Ultrastructure of the Stickleback Gasterosteus aculeatus (L.). Ph.D. thesis, University of London.
96
J. A. 0. ORONSAYE
MATTHIEGSEN, P., AND BRAFIELD, A. E. (1977). Uptake and loss of dissolved zinc by the stickleback Gasterosteus aculeatus (L.). J. Fish Biol. 10,399-4 10. MOUNT, D. I., AND STEPHEN, C. E. (1967). A method for detecting cadmium poisoning in fish. J. Wildl. Manage. 31,168-172. ORONSAYE, J. A. 0. (198 1). The Toxicity of Cadmium to the Stickleback Gasterosteus aculeatus L. and Its Effect on the Gills and Kidneys. Ph.D. thesis, University of London. ORONSAYE, J. A. O., AND BRAFIELD, A. E. (1984). The effect of dissolved cadmium on the chloride cells of the gills of the stickleback, Gasterosteus aculeatus L. J. Fish Biol. 25,253-258. PA-E, D., AND MAITEY, D. L. (1977). Studies on the toxicity of cadmium to the three-spined stickleback. Gasterosteus aculeatus (L.). J Fish Biol. 11,207-2 15. SKIDMORE, J. F. (1964). Toxicity of zinc compounds to aquatic animals with special reference to fish. Q. Rev. Biol. 39,227-248. SPEHAR, R. L. (1976). Cadmium and zinc toxicity to flag&h, Jordanella floridae. J. Fish. Res. Board Canad. 33,1939-1945. TAFANELLI, R., AND SUMMERFELT, R. C. ( 1975). Cadmium induced histopathologicrd changes in goldfish. In The Pathology ofFishes. (W. E. Ribelin and G. Migaki, Eds.), pp. 6 13-645. Univ. of Wisconsin Press. WEDEMEYER, G. (1968). Uptake and distribution of Zn6’ in the coho salmon egg (Oncorhynchus kistuch). Comp. B&hem. Physiol. 26,271-279.
AND ENVIRONMENTAL
SAFETY
14,88-96 (1987)
The Uptake and Loss of Dissolved Cadmium by the Stickleback, Gasterosteus aculeatus L. J. A. 0. ORONSAYE’ Queen Elizabeth College, Campden Hill Road, London WS, England Received May 13. I986 The whole-body uptake of cadmium by the three-spitted stickleback (Gasterosteus aculeatus) has been measured after exposure of fish to 2.5 and 5.0 mg @+/liter hard water and equal amounts of 2.0 mg Cd*+ plus 2 mg Zn*+/liter and 4.0 mg Cd” plus 4.0 mg Zn’+/liter hard water, respectively. Fish absorbed and retained cadmium while the uptake and accumulation of zinc was depressed. The loss of absorbed cadmium was quicker in fish previously exposed to cadmium plus zinc solutions used together. This phenomenon is discussed in relation to the ability of fish to excrete absorbed cadmium. @1987 Academic press, IUC.
1. INTRODUCTION In all cases where fish have been exposed to cadmium in solution, it has been found that the metal is absorbed and retained (Coombs, 1974). Such absorption is probably due to passive diffusion (Bryan, 1976) down a gradient between the mucus coating the gill on the one hand, and the internal organs on the other. Difhtsion is presumably responsible for the uptake of zinc by the eggs of coho salmon (Oncorhynchus kisutch) (Wedemeyer, 1968), and by the adults of numerous fish species (Skidmore, 1964), and also for the uptake of lead by the spotted wolffish (Anarchichas miner) (Bollingberg and Johansen, 1979). So uptake of cadmium is also likely to be due to simple diffusion. Mount and Stephan (1967) studied the uptake and accumulation of cadmium by bluegill (Lepomis macrochirus). They reported that when fish were exposed to a high concentration (10 mg/liter of cadmium) the gills were the major site of uptake. The latter authors found that cadmium accumulated in L. macrochirus and that, within 30 to 60 days, an equilibrium was established between the concentrations of cadmium in the water and in the fish tissue. Sephar (1967) exposed flagtish (Jo&nellufloridae) to cadmium in water of 44 mg/liter hardness as CaC03 at 25’C. These fish were shown to accumulate more cadmium over longer exposure periods and in higher concentrations of the metal. (The fish were exposed to sublethal doses of 1.73 1 mg/liter of cadmium). Spehar ( 1976) observed that “cadmium uptake increased with increasing exposure concentration but levelled off at 16 pg/liter,” and suggested that a possible equilibrium was reached between cadmium concentrations in the water and in the tissue. Tafanelli and Summerfelt ( 1975) introduced cadmium intraperitoneally into goldfish (Carussius auralus). These authors injected 6 mg Cd2’/kg wet weight and ob’ Present address: Department of Fisheries, Faculty of Agriculture, University of Benin, P.M.B. 1154, Benin City, Nigeria. 0147-6513187 $3.00 C@yri&t 8 1987 by Academic PUSS,Inc. AU rights of n5pmduction in any form rcsemed.
88
UPTAKE
OF CADMIUM
BY Gusterosteusuculeatus
89
served that the kidney concentrated an amount of cadmium up to 400 mg/kg (in terms of dry weight of the organ). No value was reported for the gills in this fish. The kidneys of fish have a role in the excretion of absorbed cadmium. When rainbow trout (Salvo guirdneri) were transferred to cadmium-free water after previous exposure to 0.0048 mg/liter cadmium for time periods up to 30 weeks, Kumada et al. ( 1972) observed that the concentration of cadmium in the whole body of the fish fell by about 60% in 10 weeks. The study by Calamari and Marchetti (1977) also on S. guirdneri appears to confirm the results obtained by Kumada et al. (1972) on that species. Calamari and Marchetti (1977) observed that the concentration of cadmium (in fish previously exposed to 50 pg mg/liter for 120 days) in the gills fell by 50% after 50 days. It can be assumed from the latter result that, in recovering fish, a movement of cadmium from other organs to the kidney caused the persistently high kidney residue levels. Cadmium often occurs in the environment along with other heavy metals. The uptake of cadmium together with other heavy metals, principally zinc, has been studied. Eisler and Gardner ( 1973) for example, reported that the uptake of zinc by mummichog (Fundzdus heteroclitus)was reduced in the presence of cadmium. The fish were exposed to l-10 mg Cd2+/liter along with 36 or 60 mg Zn2+/liter in synthetic sea water. In the present study, using Gusterosteus, the uptake of cadmium has been investigated using solutions of cadmium alone or solutions of cadmium along with equimolar concentrations of zinc. The loss of cadmium from fish after previous exposure to these metals (cadmium or cadmium plus zinc) has also been studied. 2. METHODS Fish in batches of 25 were exposed to either 2.50 or 5.0 mg Cd2+/liter tap water (with a hardness of 299.0 mg CaCOJiter). A second uptake experiment involved two mixed solutions of cadmium and zinc, 2 mg Cd’+ plus 2 mg Zn’+/liter, and 4 mg Cd2’ plus 4 mg Zn’+/liter. To avoid uptake by the gut, fish were not fed during exposure to cadmium. Lengths of exposure ranged between 1 and 10 days. In experiments on the loss of metals, fish were exposed to either 2.1 or 4.0 mg Cd’+/liter. Other experiments used mixed solutions of cadmium and zinc: 2.0 mg Cd2+ plus 2.0 mg Zn2+/liter, and 4.0 mg Cd2+ plus 4.0 mg Zn’+/liter. Exposure times ranged between 8.5 and 10 days. Surviving fish were allowed to recover in uncontaminated water for periods between 1 and 2 1 days. Only recovering fish were fed (a daily ration of freezedried Tubifx sp.). All experiments were carried out in the continuous-flow system (Oronsaye and Brafield, 1984). Experimental conditions were similar. A control experiment (also using unfed fish) was run. The details of experiments are given in Table 1. The experimental tanks, the tubing of the continuous flow system, and the 50-ml Pyrex beakers used for “wet digestion” of fish (according to the method of Leonard, 197 l), were cleaned with Divo-lab to remove ions from their surfaces. (The beakers had been previously soaked overnight in 50% nitric acid and rinsed in deionized water.) After being placed in uncontaminated water for 5 min to remove any cadmium or zinc adhering to their body surfaces, fish were removed at intervals and their body loads of cadmium (or zinc) were measured. Fish were pithed and the dried with filter paper. Because of their small sizes fish were placed in pairs in the beakers. The wet weights and, after drying in an oven, the dry weights of fish were recorded.
90
J. A. 0. ORONSAYE TABLE 1 DETAILSOFEXPERIMENTS
Exposure times
Test solution concentrations
(days)
Cadmium uptake 2.5 mg @+/liter
Recovery times (&YS)
l-10
-
l-10 l-9
-
10
24-504 24-408
10
24-m 24-312
Wet weight of fish
k) 0.3-0.6
5.0 mg @+/liter
Cadmium uptake in the presence of zinc 2.0 mg Cd*+ + 2.0 mg Zn’+/liter 4.0 mg Cd” + 4.0 mg Zn’+/liter
Loss of absorbed cadmium after exposure to 2.1 mg Cd2+/liter 4.0 mg Cd2’/liter Loss of absorbed cadmium and zinc after exposure to 2.0 mg Cd” + 2.0 mg Zn’+/liter 4.1 mg Cd” + 4.1 mg Zn’+/liter
0.4-0.7
0.2-0.6
0.2-0.5 8.5
Fish were small with wet weights between 0.2 and 0.7 g and dry weights between 2.05 and 0.17 g. Fish were not ashed and, as recommended by Leonard ( 197 l), digestion involved a 4: 1 mixture of nitric acid and perchloric acid. Beakers containing the specimens (whole fish) were placed on a hot plate with a fume hood, and with water flushing the hood. Eight milliliters of 70% nitric acid and 2 ml of 70% perchloric acid (analytical grade) were added to each beaker. The hot plate was maintained at 80 + 5°C during digestion, which lasted between 26 and 29 hr. When fats appeared in solution during this digestion, generally indicated by a color change from pale yellow to brown, additional nitric acid (2 ml) was added to each sample. Evaporation was continued until the white fumes of perchloric acid ceased. Each residue (which was now white) was allowed to cool in a desiccator before being dissolved in 10 ml deionized water. The amounts of either cadmium or zinc in the sample (and in the test solutions) were measured by an atomic absorption spectrophotometer, previously calibrated. Levels of cadmium and zinc found in fish tissue are shown in Figs. 1 to 7. All experiments were run in tap water, at 12 + 2”C, and two fish generated each point shown in the graphs. 3. RESULTS
Uptakeof Cadmium Fish that died during the experiments were not analyzed for cadmium residue in the tissues. F. heteroclitus(Eisler, 1974) and G. aculeatus(Pascoe and Mattey, 1977) have been shown to accumulate cadmium after death, so analyses which could have resulted in very high cadmium concentrations in tissues were avoided. Results are expressed in terms of wet weight of fish, not dry weight. Brafield and Matthiessen
UPTAKE
OF CADMIUM
EXPOSURE
BY Gasterosteus
TIME
acuieutus
91
(days)
FIG. 1. Whole-body tissue levels of cadmium in fish exposed to 2.5 (W) or 5.0 (0) mg Cd*+/liter.
(1976) presented their results on oxygen consumption of G. aculeutus in terms of wet weight, though dry weight is often used and is perhaps preferable. The mean concentration of cadmium in whole-body preparations of eight unexposed G. aculeatus was 0.0 1 mg Cd2+/g wet weight, with a range of 0.0093 to 0.0 106 mg/g. Fish exposed to 2.50 mg Cd2+/liter for 24 hr showed an increase in body cadmium, 0.1653 mg Cd’+/g, which represents a total (about 0.175 mg/g) nearly 18 times the normal mean value. A longer exposure of hsh (up to 10 days) to 2.50 mg Cd2’/liter resulted in an erratic decline in body load (Fig. 1). On the other hand, fish exposed to 5.0 mg Cd2’/liter failed to reduce the internal concentration of cadmium. As is shown in Fig. 1, the internal concentration of cadmium showed a high increase with time. After a lo-day exposure, body burden was 52 times the normal level (0.5241 mg/g wet wt compared to 0.0 1 mg/g wet wt). Uptake of Cadmium in the Presence of Zinc The mean concentration of zinc in whole-body preparations of eight unexposed fish was 0.0601 mg Zn2+/g wet weight, with a range of 0.0544 to 0.0702 mg/g. Fish exposed to 2 mg Cd2’ plus 2 mg Zn’+/liter built up high cadmium tissue levels: 30, 25, and 20 times normal tissue levels after 6, 8, and 10 days exposure, respectively. Figure 2 shows that there was also an initial influx of zinc but that the level fell from twice the normal tissue level to a near normal level after 3 days. At the close of the experiment ( 10 days) the zinc residue was at the normal tissue concentration for this metal, while cadmium retained in fish was about 20 times the normal tissue level of cadmium. When the concentration of the test solution was doubled, however, fish did not appear to concentrate zinc at any stage (Fig. 3), although cadmium accumulated to levels 16 and 22 times that of normal fish after 3 and 7 days, respectively. Although the levels of metals retained in fish are lower than those found when fish were in a solution half as concentrated (Fig. 2) more fish died during the experiments using 4 mg Cd2+ plus 4 mg Zn2+/liter. Loss ofAbsorbed Cadmium When fish exposed to either 2.0 or 4.0 mg Cd2+/liter for 10 days were allowed to recover in cadmium-free water, approximately 50% of the absorbed metal was lost
92
J. A. 0. ORONSAYE
NWlll~l cadmium Jtis.ue level
-----3
-.-
EXPOSURE FIG.
-.
J
Normal zinc tiollue level Normal caclmlum
TIME (days)
2. Whole-body tissue levels of cadmium (M) and zinc (0) in fish exposed to 2 mg Cd*+ + 2 mg Zn*+/
liter. FIG. 3. Whole-body tissue levels of cadmium (a) and zinc (0) in fish exposed to 4 mg of Cd” + 4 mg Znz+/liter.
from the body after 3 days. Figures 4 and 5 indicate that increasing the recovery period beyond 2 1 days could result in all previously absorbed metal being lost from the body, although the rate of efflux slows with time. Fish in both experiments still held approximately 15% of the absorbed cadmium at the close of the experiment. Loss ofAbsorbed Cadmium and Zinc During the uptake experiments in which G. aculeatus were exposed to solutions of both cadmium and zinc, it was observed that zinc was not concentrated by the fish.
0.2 m
-0
.,4 3
---L
s
E
‘--‘---.,c 1
3
5
7 9 RECOVERY
11 TIME
13 15 (days)
17
19
. 21
.lOO
FIG. 4. Loss of cadmium from the tissue of fish while in clean water after a previous exposure to 2 mg Cd*‘/liter for 10 days.
UPTAKE
OF CADMIUM
RECOVERY
93
BY Gusterosteus uculeutus
TIME
(days)
FIG. 5. Loss of cadmium from the tissue of fish while in clean water afkr a previous exposure to 4 mg Cd*+/liter for 10 days.
When these fish were allowed to recover in uncontaminated water, after either 10 days of exposure to 2 mg Cd*+ plus 2 mg Zn*+/liter or 8.5 days of exposure to 4 mg Cd*+ plus 4 mg Zn*+/liter, the concentration of cadmium in whole fish fell by about 50% after 24 hr. Figures 6 and 7 show that the rate of loss of cadmium from the fish was slow, perhaps slower than when fish previously exposed to only cadmium solutions were recovering. However, at the close of experiments, after 2 1 days (Fig. 6) and 13 days (Fig. 7), fish held about 12.5 and 20% of the original metal levels, respectively. 4. DISCUSSION The whole-body digests of Gasterosteus were examined for cadmium and zinc. A mean concentration of approximately 0.0 1 mg Cd*+/g wet wt was obtained for eight unexposed normal fish. Since cadmium is not normally found in tissue (unlike zinc it has no biochemical function), it is assumed that fish acquired this metal from the collection site. (Bottom sediments in the site of fish collection contained about 2 ppm Cd*+, on a dry weight basis (Oronsaye, 198 I).) The mean concentration of zinc found in the whole-body digests from the eight unexposed normal Gasterosteus was 0.060 1 mg/g wet weight. This is similar to the 0.072 mg/g wet wt of whole-body digests obtained by Matthiessen (1973). When Gasterosteus were exposed to either 2.5 or 5.0 mg Cd*+/liter for 10 days, the fish absorbed and retained the metal. These fish are able to reduce the concentration
1 . 1
3
5
7 9 RECOVERY
11 13 15 TIME (days)
17
19
’ 21
hl
FIG. 6. Loss of cadmium from the tissue of fish while in clean water after a previous exposure to 2 mg Cd*+ + 2 mg Zt?+/liter for 10 days.
94
J. A. 0. ORONSAYE
1
3 5 RECOVERY
7
9 TIME
11
13
. 15
100
(days)
FIG. 7. Loss of cadmium from the tissue of fish while in clean water after a previous exposure to 4 mg Cd*+ + 4 mg Zn’+/liter for 8.5 days.
of the metal absorbed (Fig. 1). This phenomenon was reported by Matthiessen and Brafield (1977) when Gusterosteus were exposed to solutions containing zinc, and has been “explained” by the fact that these fish are euryhaline and so adapted to sudden influx of ions. Other species of fish such as S. guirdneri (Kumada et al., 1972), absorb and accumulate cadmium. In the higher concentration of 5.0 mg C$+/liter uptake experiment which lasted for 10 days, more cadmium (in mg/g wet wt of fish) was retained. This observation appears to agree with those of Eisler (1974) on F. heteroclitus and Pascoe and Mattey (1977) on G. aculeutus, both fish being exposed to cadmium. The latter authors found that Gusterosteus exposed to cadmium (in water with a hardness of 103-l 11 mg CaCOJliter) increased the body load of the metal with increasing external concentration (0.90 cLg/g fresh weight at 0.001 mg Cd2+/liter to 5 1.O &g fresh weight at 100 mg Cd”/liter). Exposure of the fish to cadmium was reported to have lasted 79 days, at which time about 80% of the fish in each test concentration had died. The fish analyzed by Pascoe and Mattey ( 1977) had died during the experiments, but were removed as soon as possible after death. It appears that the excretion of cadmium by Gasterosteus exposed to 5.0 mg Cd2+/liter was poor when compared with the lower concentration of 2.5 mg Cd”/ liter. It is likely that cadmium diffusing into the fish after the sixth day reached a level high enough to inhibit the excretion of the metal, presumably through poisoning of the gill ion excretory cells. This suggestion may explain why the fish exposed to 5.0 mg Cd”/liter built up a body load of a 0.54 mg Cd2’/g wet weight, which is approximately 50 times the normal tissue level of the metal, whereas in 2.5 mg Cd2+/liter, fish accumulated only 0.07 mg Cd2+/g, approximately 7.5 times the normal tissue level, over the same period of time ( 10 days). In the experiments involving exposure to cadmium and zinc together, the accumulation of zinc by Gasterosteus was reduced by the presence of cadmium (Figs. 2 and 3). A similar result was obtained by Eisler and Gardner ( 1973), for when they exposed F. heteroclitus to solutions of both cadmium and zinc, the whole-body uptake of zinc was inhibited by the cadmium. Furthermore, the Gasterosteus used in my experiments (involving cquimolar concentrations of either 2 mg Cd2’ plus 2 mg Zn’+/liter or 4 mg Cd” plus 4 mg Zn2+/liter) retained a lower body load of cadmium over an 8-day test period, when compared to the fish exposed to 5.0 mg Cd”/liter alone. Since it is shown that cadmium and zinc may bind to the same metallothionein (Coombs, 1974), it can be assumed that the internal competition between these met-
UPTAKE
OF
CADMIUM
BY
Gusterosteus aculeatus
95
als for binding sites probably reduced the concentration of cadmium retained by the fish. Gusterosteus appears to lose cadmium quicker during recovery from exposure to cadmium and zinc together. The internal concentrations of the absorbed cadmium fell by 50% within 1 day (24 hr) in recovering fish previously exposed to 2 mg Cd” plus 2 mg Zn’+/liter, whereas 50% was lost after 3 days in the fish that were exposed to either 2.0 or 4.0 mg Cd”/liter. It is likely that the assumed competition by cadmium and zinc may leave a substantial amount of cadmium in the ionic state. It is conceivable that such unbound cadmium will be rapidly lost from the fish that are allowed to recover in clean water. 5. CONCLUSIONS This study has confirmed that Gasterosteus absorbs, accumulates, and excretes cadmium. That cadmium depresses the uptake of zinc when present in conjunction was also demonstrated. The latter appears to shed some light on the mode of action of cadmium to fish. Since zinc is an essential element in biochemical systems, its displacement will induce a state of zinc element deficiency. This phenomenon can result in the death of fish through poisoning by cadmium, a nonessential element now present in the protein molecules. It is conceivable, therefore, that even though cadmium may be lost from fish after exposure to cadmium and zinc used together, the bound cadmium not lost from the fish may continue to displace more zinc and eventually kill fish through cadmium poisoning. ACKNOWLEDGMENT I am grateful to Dr. A. E. Brafield for his encouragement, criticism, and supervision of this study.
REFERENCES BOLLINGBERG, H. J., AND JOHANSEN, P. (1979). Lead in spotted wolfish, Anarchichas minor, near a leadzinc mine in Correnland. J. Fish Res. Board Canad. 36,1023-1028. BRAFIELD, A. E., AND MAT~HIESSEN, P. (1976). Oxygen consumption by sticklebacks (Gasterosteus aculeatus L.) exposed to zinc. J. Fish Biol. 9,359-370. BRYAN, G. W. (1976). Some aspects of heavy metal tolerance in aquatic organisms. In E$ects ofPollutant on Aquatic Organisms (A. P. M. Lockwood, Ed.), pp. 7-34. Cambridge Univ. Press, London. CALAMARI, D., AND MARCHETII, R. (1977). Intluence of water hardness on cadmium toxicity to S&no guirdnert, Richardson. Paper presented to the International Workshop on Structure and Function of Fish Gills, 26-29 September 1977, Messina (mimeo). COOMBS, T. L. (1974). The significance of multielement analysis. In Metal Polhdions Studies. Ecological Toxicolo@al Research. NATO Science Committee Conference, London, Vol. 7, pp. 187- 195. EISLER, R. (1974). Radiocadmium exchange with seawater by Fund&s heterochtus (L.) (Pisces: Cyprinodontidae). J. Fish Bioi. 6,60 l-6 12. EISLER, R., AND GARDNER, G. R. (1973). Acute toxicology to an estuarine teleost of mixtures ofcadmium, copper, and zinc salts. J Fish Biol. 5,13 1- 142. KUMADA, H., K~MURA, S., YOKOTE, M., AND MATIDA, Y. (1972). Acute and chronic toxicity, uptake and retention of cadmium in freshwater organisms. Bull. Freshwater Fish Res. Lab. 22(2). LEONARD, E. N. (197 l).lThe determination of copper in fish tissue by atomic absorption spectrophotometry. At. Absorpt. Newsl. 10,84-85. MATTHIESSEN, P. (1973). Zinc Uptake and Its Efects on the Oxygen Consumption and Gilt Ultrastructure of the Stickleback Gasterosteus aculeatus (L.). Ph.D. thesis, University of London.
96
J. A. 0. ORONSAYE
MATTHIEGSEN, P., AND BRAFIELD, A. E. (1977). Uptake and loss of dissolved zinc by the stickleback Gasterosteus aculeatus (L.). J. Fish Biol. 10,399-4 10. MOUNT, D. I., AND STEPHEN, C. E. (1967). A method for detecting cadmium poisoning in fish. J. Wildl. Manage. 31,168-172. ORONSAYE, J. A. 0. (198 1). The Toxicity of Cadmium to the Stickleback Gasterosteus aculeatus L. and Its Effect on the Gills and Kidneys. Ph.D. thesis, University of London. ORONSAYE, J. A. O., AND BRAFIELD, A. E. (1984). The effect of dissolved cadmium on the chloride cells of the gills of the stickleback, Gasterosteus aculeatus L. J. Fish Biol. 25,253-258. PA-E, D., AND MAITEY, D. L. (1977). Studies on the toxicity of cadmium to the three-spined stickleback. Gasterosteus aculeatus (L.). J Fish Biol. 11,207-2 15. SKIDMORE, J. F. (1964). Toxicity of zinc compounds to aquatic animals with special reference to fish. Q. Rev. Biol. 39,227-248. SPEHAR, R. L. (1976). Cadmium and zinc toxicity to flag&h, Jordanella floridae. J. Fish. Res. Board Canad. 33,1939-1945. TAFANELLI, R., AND SUMMERFELT, R. C. ( 1975). Cadmium induced histopathologicrd changes in goldfish. In The Pathology ofFishes. (W. E. Ribelin and G. Migaki, Eds.), pp. 6 13-645. Univ. of Wisconsin Press. WEDEMEYER, G. (1968). Uptake and distribution of Zn6’ in the coho salmon egg (Oncorhynchus kistuch). Comp. B&hem. Physiol. 26,271-279.