THE EFFECT OF COPPER EXPOSURE DURING EMBRYONIC DEVELOPMENT ON DEFORMATIONS OF NEWLY HATCHED COMMON CARP LARVAE, AND FURTHER CONSEQUENCES

Katarzyna Ługowska, Małgorzata Witeska

ABSTRACT

The study was done on common carp larvae, the embryonic development of which took place in clean tap water (control – K group) or at 0.2 mgˇdm³ of copper (Cu group). The experiments were carried out until the 20 day from hatching. Among newly hatched larvae four types of body malformations were distinguished: A – curvature of the spine, B – C-shaped larva, E – deformed yolk sac, G – shortened body. These deformations are not copper-specific, and were observed also in fish exposed to other heavy metals. Deformations that impair larval locomotion must adversely affect feeding efficiency, thus would reduce larval survival. That was confirmed by the results concerning survival of larvae fed Artemia sp. nauplii. Only the larvae able to take up exogenous food survived until the end of the experiment. Copper exposure affected survival of larvae. Starved normal larvae from control group survived 19 days and started to die from the 13^th day. The deformed larvae from Cu

Key words: copper, embryonic development, fish, deformations..

INTRODUCTION

Among newly hatched larvae, even under optimum conditions, various body deformations may occur. Usually, only a small percent of larvae show such deformations [1, 8, 21, 24, 28]. However, any adverse environmental factors may induce an increase in their frequency. Various data indicate that environmental conditions such as: temperature below or over the optimum range [14, 16, 26], pH changes [7,10], or nitrate [15] may increase frequency of body deformations in newly hatched larvae. Water contamination with heavy metals also considerably increases frequency of deformed larvae (review: 11).

Most authors observed various curvatures of the spine [2, 4, 5, 21, 22, 23, 27]. Munkittrick, Dixon [20] classified spine curvatures of Catostomus commersoni larvae after copper and zinc exposure as: corkscrew, L-shape, C-shape, two-headed, shortened body. Jezierska et al. [9] developed a catalog of various body deformations of common carp larvae exposed in embryonic period to cadmium, copper and lead. The authors distinguished following types of body malformations: A – curvature of the spine, B – C-shaped larva, C – deformed skull, D – deformed eyes, E – deformed yolk sac, F – cardiac edema, G – shortened body, H – twin form, and further divided them into subtypes in which particular malformations were accompanied by the other ones. Similar catalog for grass carp was developed by Ługowska et al. [17]. However, no data are available on further performance of deformed larvae.

The aim of the present study was to evaluate the percentage, and types of body deformations in common carp exposed to copper during embryonic period, and how they may affect further larval development.

MATERIALS AND METHODS

The study was done on common carp newly hatched larvae, the embryonic development of which took place at 0.2 mgˇdm³ of copper (Cu group) or in clean tap water (control – K group). Copper solution was made using CuSO₄·5H₂O. The experiments were done in 3 series.

Newly hatched larvae were counted and inspected. The hatching rate was calculated as a percentage of hatched larvae per initial number of incubated eggs. Among freshly hatched larvae, the share of deformed ones was evaluated, and malformations were classified using the catalog [9].

For 20 days from hatching, the larvae were reared under control conditions: dechlorinated tap water, temperature 22ą2°C, oxygen saturation about 80%, pH 7.8, hardness 230 mgˇdm³ as CaCO₃.

In all series, the larvae were reared separately in the 50 ml glass vessels of 7.5 cm diameter. Each vessel was individually marked in order to identify each larva. During the experiments, the fish were starved. Their morphology was examined daily, and mortality was recorded.

The observations were made using a thick slide with a concave chamber. The chamber was filled with clean water, in which a larva was carefully placed with a wide-tube dropper, and observed using the binocular (magnification 1.5x1.6) connected with the camera, computer image analysis system MultiScan and video recorder (super VHS). The observations were recorded in photographs and films.

In series III, the fed (twice a day with Artemia sp. nauplii), and starved larvae were reared in the 100 ml glass vessels, at the density of 20 fish per vessel. Normal and deformed larvae from K group, and deformed ones from Cu group were reared separately. Survival was recorded daily.

RESULTS AND DISCUSSION

Reduction of hatching rate, and numerous body deformations were the most obvious effects of metal exposure. Hatching rates in the control groups were 90, 71, and 77%, while in Cu groups: 60, 38, and 40%, respectively. In all series, the percentage of deformed larvae were high in Cu groups, sometimes exceeding 60%.

The obtained results show the differences among the series but the effect of metal was distinct. Copper exerted toxic influence upon the embryos despite the protective egg shell. Metal enters the egg mainly during the process of swelling [25], which may result in developmental anomalies, deformations, and even death of embryos (review: 11). Another stage at which the embryos are very susceptible to intoxication is hatching when the egg shell breaks and larva becomes directly exposed to environmental factors. At that time, and soon after hatching, an increased mortality is often observed, and new deformations may occur.

An increase in body deformation rate induced by exposures to various heavy metals were reported by many authors (review: 11). Copper-induced malformations were observed by various authors[2, 3, 9, 12, 16, 18, 19, 22, 25, 29].

In the present study, a detailed individual classification of deformed larvae was done, according to the catalog [9] (Table 1). In 13 larvae, vertebral deformations were found, quite commonly occurring also under optimum conditions. In other 13 individuals, body shortening was observed, and in 19 fish – the changes in yolk sac shape. These deformations are not copper-specific but were observed also in fish exposed to other heavy metals. Various vertebral curvatures (scoliosis, curling of entire body) were described in lead-treated fish [23]. Heisinger and Green [4] reported tail curvature downwards (C-shaped body) in Oryzias latipes larvae subjected to mercury. According to Weiss and Weiss [27], 40% of lead-exposed Fundulus heteroclitus larvae showed lordosis. Sarnowski and Jezierska [21] reported edema of anterior body part, vertebral curvatures, and tail shortening in common carp larvae subjected to lead exposure.

Body deformations affect swimming, and often considerably impair locomotion. The examples of such abnormal larval movements are shown in Film 1. First larva (A type) showed only circular movements, the second, C-shaped (B type) was able only to weak tail movements, and the third larva (E type) was very poorly active, and unable to swim straight.

Deformations that impair larval locomotion must adversely affect feeding efficiency, thus would reduce larval survival, even in metal-free environment.

That was confirmed by the results concerning survival of larvae fed Artemia sp. nauplii (Fig. 1). Normal larvae from the control group, reared under optimum conditions, and provided with food ad libitum showed very high survival rate, while deformed ones (from both, control and Cu exposures) kept dying over entire experimental period. Only 6 deformed individuals from the control, and 2 from Cu-exposed group survived until the end of the experiment. To survive, they must have taken up exogenous food, thus it seems that their deformations did not seriously impair feeding abilities.

Survival of starved larvae is shown in Figure 2. Most of the control larvae survived 13 days post hatching utilizing their yolk sac materials. Before the 20 day, all larvae quickly died. These results are similar to those obtained by Jówko et al. [13], who observed that at 22°C most of the starved carp larvae died between the 11 and 15 day post hatching, when probably all yolk sac nutrients were used up. It is in accordance with the data concerning the so called “ point of no return”, the moment when the larvae irreversibly lose ability of feeding, and die even if provided with food. In common carp larvae the no return point occurs about the 9 day post hatching [6] which indicates that starvation-induced mortality of common carp larvae takes place after that time.

The deformed larvae from Cu exposure gradually died from the beginning of the experiment, only 2 (series I, III), and 1 (series III) larvae survived until the 13 day, and all died before the 16 day. It seems that mortality was induced by some internal disturbances related to deformations.

Detailed analysis of deformations (Fig. 3) provides more information on this issue. All the larvae showing G-type deformation died first, and only 2 of them partly recovered. Similarly, also E-type larvae died soon after hatching, and only 2 fish in series III completely recovered, and survived until 10 and 15 day. Both, G, and E-type larvae showed very complex deformations, often including several changes in various body parts. These changes were probably accompanied by deep internal disturbances, and developed at very early, and very sensitive developmental stages (until 24 hours post fertilization). They did not cause death of embryos but resulted in serious larval anomalies that made further development impossible.

On the contrary, B deformation was reversible, and B-type larvae survived until the 8 (series I), and 12 (series II) day post hatching. Also most A-type larvae, showing only vertebral deformations gradually recovered, and these fish showed the longest survival.

Such an example of gradual recovery in 5 days post hatching is shown in Figure 4. This fish initially showed lateral abdominal vertebral curvature. The curvature started to decrease already on the first day post hatching, and disappeared until the 5 day when the larva looked quite normally.

CONCLUSIONS

These data show that mortality was directly related to the type of deformation. In some strongly deformed fish internal disturbances probably occurred, inducing early death, while in less deformed fish (with vertebral curvatures only) deformations were reversible, and recovery was possible.

Thus, it seems that fed deformed larvae that survived until the end of the experiment were those that recovered, and were able to feed actively.

In the literature no data were found on the possibility of recovery from larval body deformations, but this issue seems interesting, and needs further investigation.

REFERENCES

1. Benoit D. A., Holcombe G. W., 1978. Toxic effects of zinc on fathead minnows Pimephales promelas in soft water. J. Fish Biol. 13, 701-708.

2. Bieniarz K., Epler P., Sokołowska-Mikołajczyk M., Popek W., 1997. Reproduction of fish in conditions disadvantageously altered with the salts of zinc and copper. Arch. Pol. Fish. 5, 21-30.

3. Giles M. A., Klaverkamp J. F., 1982. The acute toxicity of vanadium and copper to eyed eggs of rainbow trout (Salmo gairdneri). Water. Res. 16, 885-889.

4. Heisinger J. F., Green W., 1975. Mercuric chloride uptake by eggs of the ricefish and resulting teratogenic effects. Bull. Environ. Contam. Toxicol. 14, 665-673.

5. Holcombe G. W., Benoit D. A., Leonard E. N., McKim J. M., 1976. Long-term effects of lead exposure on three generations of brook trout (Salvelinus fontinalis). J. Fish. Res. Board Can. 33, 1731-1741.

6. Jezierska B., Jówko G., Korwin-Kossakowski M., 1985. Wpływ pokarmu na wylęg karpia (Cyprinus carpio L.) [The effect of food on common carp (Cyprinus carpio L.) larvae]. Zesz. Nauk. WSR-P Siedl., Zootechnika 7, 187-203.

7. Jezierska B., 1993. Wpływ aluminium na ryby w wodach zakwaszonych [The effect of aluminum on fish in acidic waters]. Zesz. Nauk. WSR-P Siedl., Zootechnika 32, 205-215.

8. Jezierska B., Słomińska I., 1997. The effect of copper on common carp (Cyprinus carpio L.) during embryonic and postembryonic development. Pol. Arch. Hydrobiol. 44, 261-272.

9. Jezierska B., Ługowska K., Witeska M., Sarnowski P. 2000: Malformations of newly hatched common carp larvae. Electron. J. Pol. Agric. Univ., Fisheries 3 (2) http:// www.ejpau.media.pl/series/volume3/issue2/fisheries/art-01./pdf

10. Jezierska B., Witeska M., 1995. The influence of pH on embryonic development of common carp (Cyprinus carpio L.). Arch. Ryb. Pol. 3 (1), 84-94.

11. Jezierska B. Witeska M., 2001. Metal Toxicity to Fish. Wydaw. Akad. Podl. Siedl. 318.

12. Jezierska B., Słomińska I., 1997. The effect of copper on common carp (Cyprinus carpio L.) during embryonic and postembryonic development. Pol. Arch. Hydrobiol. 44, 261-272.

13. Jówko G., Korwin-Kossakowski M., Jezierska B., 1981. Wpływ wybranych czynników środowiska na larwy karpia (Cyprinus carpio L.) [The effect of some environmental factors on common carp (Cyprinus carpio L.) larvae]. Rocz. Nauk Rol., H, 99, 25-37.

14. Kokurewicz B., Kowalewski M., Witkowski A., 1978. Wpływ temperatury na rozwój zarodkowy lipienia europejskiego [The effect of temperature on grayling embryonic development]. Gospod. Ryb. 2, 6-8.

15. Korwin-Kossakowski M., Myszkowski L., Kazuń K., 1996. Acute toxicity of nitrate to grass carp (Ctenopharyngodon idella (Val.)) larvae. Pol. Arch. Hydrobiol. 43, 465-468.

16. Ługowska K., Jezierska B., 2000. Effect of copper and lead on common carp embryos and larvae at two temperatures. Folia Univ. Agric. Stetin., Piscaria 205 (26), 29-38.

17. Ługowska K., Jezierska B., Witeska M., 2001. Deformations of newly hatched grass carp larvae. Folia Univ. Agric. Stetin., Piscaria 218 (28), 21-28.

18. McKim J. M., Benoit D. A., 1971. Effects of long-term exposures to copper on survival, growth, and reproduction of brook trout (Salvelinus fontinalis). J. Fish. Res. Board. Can. 28, 655-662.

19. Miś J., Bieniarz K., Epler P., Sokołowska-Mikołajczyk M., Chyb J., 1995. Incubation of fertilized common carp (Cyprinus carpio L.) eggs in different concentrations of copper. Pol. Arch. Hydrobiol. 42, 269-276.

20. Munkittrick K. R., Dixon D. G., 1989. Effects of natural exposure to copper and zinc on eggs and larval copper tolerance in white sucker (Catostomus commersoni). Ecotoxicol. Environ. Saf. 18, 15-26.

21. Sarnowski P., Jezierska B., 1999. The effect of lead exposure on grass carp spermatozoa and developing embryos. Heavy Metals in the Environment: an Integrated Approach, Vilnius, 304-308.

22. Słomińska I., 1998. Wrażliwość stadiów młodocianych karpia (Cyprinus carpio L.) na toksyczne działanie ołowiu i miedzi [Sensitivity of common carp (Cyprinus carpio L.) juveniles to lead and copper toxicity]. PhD Thesis. Institute of Inland Fisheries, Olsztyn.

23. Stouthart A. J. H. X., Spanings F. A. T., Lock R. A. C., Wendelaar Bonga S. E., 1994. Effects of low water pH on lead toxicity to early life stages of the common carp (Cyprinus carpio). Aquat. Toxicol. 30, 137-151.

24. Stouthart A. J. H. X., Spanings F. A. T., Lock R. A. C., Wendelaar Bonga S. E., 1995. Effects of water pH on chromium toxicity to early life stage of the common carp (Cyprinus carpio). Aquat. Toxicol. 32, 31-42.

25. Stouthart A. J. H. X., Haans J. L. M., Lock A. C., Wendelaar Bonga S. E., 1996. Effects of water pH on copper toxicity to early life stages of the common carp (Cyprinus carpio). Environ. Toxicol. Chem. 15, 376-383.

26. Tatarko K. I., 1965. Vlijanie temperatury na embrionalnoje razvitie prudovo karpa [The effect of temperature on embryonic develpment of common carp]. Gidrobiol. Zh. 1, 62-66.

27. Weis J. S., Weis P., 1977. Effects of heavy metals on development of the killifish, Fundulus heteroclitus. J. Fish. Biol. 11, 49-54.

28. Witeska M., Jezierska B., Chaber J., 1995. The influence of cadmium on common carp embryos and larvae. Aquaculture 129, 129-132.

29. Yulin W., Hongru Z., Lanying H., 1990. Effects of heavy metals on embryos and larvae of flat fish Paralichthys olivaceus. Oceanol. Limnol. Sin. 21, 386-392.

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed ‘Discussions’ in each series and hyperlinked to the article.