Effect of methylmercury on egg and juvenile viability in two populations of killifish Fundulus heteroclitus

ENVIRONMENTAL RESEARCH 44, 272-278 (1987)

Effect of Methylmercury on Egg and Juvenile Viability in Two Populations of Killifish Fundulus heteroclitus ABUT.

K H A N AND J U D I T H S. W E I S

Department of Biological Sciences, Rutgers University, Newark, New Jersey 07102 Received December 17, 1986 Kiliifish (Fundulus heteroclitus) eggs from a polluted creek (Piles Creek (PC)) and a relatively pristine estuary in Long Island (LI) were exposed for 20 main to various concentrations of naethylnaercuric chloride (MeHg) prior to combination with untreated sperm. PC killifish eggs showed a higher LCs0 value (1.7 nag/liter) than LI eggs (0.7 nag/liter). PC eggs that were fertilized by nontreated sperm after exposure to 1.0 or 2.5 rag/liter meHg and then placed in clean sea water (15 parts per thousand) for 1 week showed a 5 and 7% malformations of the embryos, respectively. However, exposure of LI eggs to 1.0 rag/liter prior to fertilization caused 32% malformations of the embryos, and at 2.5 nag/liter a/naost all the embryos died. The data indicate that LI killifish eggs are less tolerant to meHg than PC eggs, This is in keeping with previous data on embryonic tolerance to naeHg in these two populations. However, 96-hr LCs0 values of juvenile fish (25-45 mna standard length) did not differ between these two populations. © 1987AcademicPress, Inc.

INTRODUCTION The fertilization process is a critical stage in the life cycle of animals. Direct effects of pollutants on gamete survival may prevent successful fertilization. While effects of metals on the development and survival of fertilized eggs of fish have been examined by numerous authors (Kihlstrom et al., 1971; McKim et al., 1976; Weis and Weis, 1977a, b), little information is available on the effect of metals on gametes prior to fertilization. McIntyre (1973) exposed steelhead trout (Salmo gairdneri) sperm to various concentrations of methylmercury (meHg) for 30 rain before they were combined with untreated eggs. The results indicated that concentrations greater than 1.0 rag/liter markedly reduced sperm viability. Billard and Roubaud (1985) reported that 1.0 rag/liter mercuric chloride (Hg) exposure (40 rain) of rainbow trout (S. gairdneri) sperm caused a significant reduction of fertilization success, but comparable exposure of eggs prior to fertilization did not have any effect on fertilization success. Shaw and Brown (1971) found no effect of copper (1.0 rag/liter) and nickel (1.0 mg/liter) exposure to rainbow trout (S. gairdneri) eggs. Rosenthal and Alderdice (1976) did not see any effect of cadmium (10.0 rag/liter) e x p o s u r e on fertilization of herring (Clupea harengus harengus) eggs. However, delayed effects of exposure become noticeable when fully developed eggs began to die prior to hatching. Deleterious effects of mercury on fish reproduction have been discussed by numerous authors. The sexual development of fathead minnow (Pimephales promelas) was arrested at 0.25 rag/liter methylmercury exposure (U.S. EPA, 1972). Kihlstrom et al. (1971) observed that zebrafish (Brachydanio rerio) that spawned in 1.0 rag/liter phenylmercuric acetate produced fewer eggs. McKim et al. (1976) 272 0013-9351/87 $3.00 Copyright © 1987 by Academic Press, lnc, All rights of reproduction in any form reserved.

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reported that methylmercury (0.93 ~g/liter) affected the growth, survival, and reproduction of brook trout (Salvelinus fontinalis). Ram and Sathyanesan (1983) found that 0.01 rag/liter mercuric chloride exposure of Chana punctatus for 6 months caused reduction in oocyte development. The killifish (Fundulus heteroclitus) is a common estuarine species that has continued to survive in some of the highly polluted estuaries of New Jersey. Weis et al. (1981) reported that killifish embryos from Piles Creek (PC), a tributary of Arthur Kill, an area heavily impacted by metal and oil pollution, in Linden, New Jersey, were less susceptible to methylmercury-induced abnormalities than embryos from a relatively nonpolluted area in Long Island (LI), New York. Mercury concentration in PC sediment was 10.3 ppm (Koepp et al., 1977), whereas in LI sediment mercury concentration was not detectable (Weis and Weis, 1983), Khan and Weis (1987) found 11.23, 5.78,623.47, and 627.78 ppm of Hg, Cd, Cu, and Zn, respecively in PC sediment; the concentrations in LI sediment were within normal ranges (0, 0.46, 41.00, and 49.40 ppm of Hg, Cd, Cu, and Zn, respectively). Khan and Weis (1986) reported that exposure of F. heteroclitus sperm to either 0.05 mg/liter meHg or 0.05 rag/liter Hg for 1 rain caused a significant reduction of fertilization in fish from a clean estuary in Southampton, Long Island, New York. However, meHg exposure of PC killifish sperm for 2 min did not have any effects on fertilization success. Comparable exposure of PC sperm to Hg caused a significant reduction in fertilization success. Thus, PC killifish sperm are resistant to meHg but not Hg. We (Khan and Weis, 1987) have found that prior exposure (20 rain) of PC and LI killifish eggs to 0.05 ppm meHg before fertilization had no effect on fertilization success. Since we saw no effect of 0.05 ppm meHg on fertilization success, in order to find if any population differences in resistance existed, we used higher concentrations of meHg in the present study. This paper reports on the effect of meHg exposure of eggs prior to fertilization in killifish from PC and Southampton, Long Island, New York. This paper also reports of the effects of various concentrations of meHg on juvenile killifish (25-45 mm standard length) of these two populations. MATERIALS AND METHODS Adult killifish were caught by using minnow traps during the summer of 1986 from Piles Creek, Linden, New Jersey, and Southampton, Long Island, New York. The water salinity in these two areas ranged from 20 to 25 parts per thousand (ppt). Fish were brought back into the lab and acclimated 3-5 days in 20 ppt artificial seawater ("Instant Ocean," Carolina Biological Supply Co.) and a light:dark cycle of 14:10 hr. Males and females were kept in separate tanks to prevent spawning in the tank. Sperm and eggs were obtained by stripping the gravid fish. PC and LI eggs were stripped into 5 ml of 15 ppt seawater with 0, 0.5, 1.0, 2.5, and 5.0 mg/liter meHg (98% purity, ICN Pharmaceuticals, Plainview, NY) for 20 min prior to combination with sperm. After 20 rain, eggs were washed with 15 ppt seawater two to three times to remove the contaminants, and then untreated newly stripped sperm were added. After 2 rain, eggs were again washed to remove the excess sperm, and then left in 100 ml of 15 ppt seawater for develop-

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merit. After 120-150 min, eggs were checked under dissecting microscope for cleavage, which was the sign that fertilization had taken place. Each experiment was done by using the eggs and sperm of a single female and a single male and was replicated five to eight times. After a week of incubation at 24°C in 15 ppt seawater, the embryos were examined for malformations of the head, cardiovascular system, and skeletal system (Weis and Weis, 1982). To find out the tolerance of juvenile killifish (25-45 mm standard length), fish from each population were tested for acute response to meHg. For this test, 50-80 fish from each population were treated with 0, 0.025, 0.05, 0.075, 0.20, 0.25, 0.50, and 1.0 mg/liter meHg for 96 hr. All treatments were diluted to the appropriate concentration by mixing 20 ppt seawater with known stock solution. Five fish were held in a container in 2 liters of test solution. The polystyrene containers holding the fish were covered with clear plastic film. Fish were not fed during the 96-hr test period, but they were checked daily, and dead individuals were removed. Data were analyzed by using Probit analysis (Huber, 1984) and X2 (Zar, 1984). RESULTS

PC killifish eggs were less susceptible to meHg than LI killifish eggs. Probit analyses gave 20-rain LCs0 values of 1.7 rag/liter meHg for PC (95% confidence limits of 1.4-1.8 rag/liter) and 0.7 rag/liter meHg for LI (95% confidence limits of 0.57-0.72 rag/liter) killifish eggs, respectively. Thus, the LCs0 value of PC killifish eggs was significantly higher than L1 eggs. Exposure of PC killifish eggs to 0.5 rag/liter for 20 rain prior to insemination did not affect mean percentage fertilization success or embryonic development (Fig. 1). However, exposure of PC eggs to 1.0 rag/liter meHg caused a significant re-

~. 10o +

80

~

r~

60

;~ 40

N 20

o o

0.5

1.0

2.5

5.0

METHYLMERCURY (PPM) FIG. 1. Effect of m e t h y l m e r c u r y on m e a n % fertilization s u c c e s s of Piles Creek and L o n g Island Killifish eggs (exposure o f eggs to m e H g prior to fertilization).

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duction in mean percentage fertilization s u c c e s s (X 2 = 7.63, P < 0.05) (Table 1). Delayed effects on embryos were also noticed at 1.0 and 2.5 mg/liter. These eggs produced 5 and 7% abnormal embryos, respectively (Table 2). These abnormalities consisted of craniofacial, cardiovascular, and skeletal malformations. On the other hand, exposure of LI killifish eggs prior to fertilization to 0.5 rag/liter meHg caused a significant reduction in mean fertilization success (X2 = 11.0, P < 0.05) (Fig. 1, Table 1), but did not effect embryonic development. Exposure to 1.0 rag/liter meHg reduced mean percentage fertilization success by 70% and caused 32% embryonic malformations in those that were fertilized (Table 2). However, almost all embryos (91%) died after 2.5 rag/liter meHg treatment of eggs. These data indicated that LI killifish eggs were less tolerant to meHg than PC eggs. Probit analyses on the juvenile fish gave 96-hr LCs0 values of 0.21 (95% confidence limits of 0.1-0.23 mg/liter) and 0.19 (95% confidence limits of 0.17-0.21 rag/liter) rag/liter meHg for PC and LI killifish, respectively. Therefore PC eggs showed more tolerance to meHg; juvenile killifish from the two sites did not show any difference in meHg tolerance. DISCUSSION

The present study has reported the effects of egg exposure to meHg on fertilization success, and how this effect differs between two populations. PC kiUifish eggs showed more tolerance to meHg than LI killifish eggs. This is in keeping with earlier work done with embryos from these two populations. Weis et al. (1981) reported that killifish embryos from PC were less susceptible to craniofacial, skeletal, and cardiovascular defects caused by meHg than were conspecific embryos from a nonpolluted area. In those studies, exposure began after fertilization and the beginning of cleavage. In the present study, eggs were exposed prior to fertilization only. TABLE 1 EFFECT OF METHYLMERCURY ON FERTILIZATION SUCCESS OF PC AND LI KILLIFISH EGGS PC

LI

MeHg (ppm)

Total No. eggs

% Fertilized

Total No. eggs

% Fertilized

0 0.5 1.0 2.5 5.0

369 531 488 610 352

95 90 ~ 72 C 31 0

328 410 664 581 320

90 57 b 30 a 2 0

a N o significant difference b e t w e e n 0 and 0.5 ppm. b Significant difference b e t w e e n 0 and 0.5 ppm. c Significant difference b e t w e e n 0 and 1.0 ppm. Significant difference b e t w e e n 0 and 1.0 ppm.

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TABLE 2 ABNORMALITIES IN EMBRYOS AS A RESULT OF MeHg EXPOSURE (20 min) PRIOR TO FERTILIZATION PC

LI

MeHg (ppm)

Total No. E m b r y o s

% Abnormal

Total No. embryos

0 0.5 1.0 2.5

237 238 281 129

0.8 a 1.68 ~ 5a 7~

108 216 161 All dead

% Abnormal 0.9 b,c

1.85 b 32 c

a No significant difference. b No significant difference. c Significant difference.

There have been a number of studies addressing the question of development of metal tolerance in chronically exposed populations. Bryan and Hummerstone (1971) reported that the polychaete worm (Nereis diversicolor) from a chronically polluted area with copper developed a tolerance to copper. They also reported that this tolerance was not readily lost nor readily induced in individuals of the same species from a less polluted area, implying genetically based tolerance. Moraitou-Apostolopoulou et al. (1979) compared two populations of copepods (Acartia clausi) from differentially cadmium-polluted areas and found that copepods from chronically polluted sites lived longer in 0.1 and 1.0 fxg/liter cadmium than the ones from the clean area. Likewise, Gadkari and Marathe (1983) studied the effect of cadmium and lead on fish (Lebistes reticulatus) from two different habitats. The fish from polluted areas were more resistant to cadmium and lead than those from clean areas. Callahan and Weis (1983) found increased resistance to meHg in fiddler crabs (Uca pugnax) from PC than crabs collected from a clean area. Although PC eggs were more tolerant to meHg than LI eggs, this tolerance was no longer present in the PC juvenile fish, in which there was no difference in 96-hr LCs0 values at PC vs LI juveniles. It may be that tolerance to metals is limited to early life stages. Moraitiou-Apostolopoulou et al. (1982) found that small shrimp (Palaemon elegans) (24-27 mm) from a polluted estuary were more tolerant to Cd and Cr than those from a less polluted area. However, larger shrimp (27-31 mm) did not show any differences in resistance to either Cd or to Cr. Rahel (1981) reported that adult shiners from zinc-polluted streams were less tolerant and appeared to be under stress. Renna (1982) and Weis et al. (1987) found that PC adult killifish were less tolerant to meHg in terms of fin regeneration and survival than those from an unstressed population. Luoma (1977) stated that greater tolerance to a toxicant in a population from one area, as compared to another area, is direct evidence that the toxicant is exerting selective pressure at the first site. PC killifish eggs showed more tolerance to meHg. This is in agreement with earlier data on this population. Weis et al. (1982a) showed that meHg was less

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toxic for PC than LI embryos, and Khan and Weis (1986) showed meHg tolerance in sperm from this population. The evidence indicates that this population has developed a resistance to meHg but only in the gametes and embryonic stages. ACKNOWLEDGMENTS This research was supported by a Biomedical Research support grant and funds from the Center for Coastal and Environmental Studies. We express our appreciation to Ajay Viswambharan, Lissane D'Andrea, and Nelson Herreira of the Department of Biological Sciences for their technical assistance.

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