Arch. Environ. Contam. Toxicol. 52, 535–540 (2007) DOI: 10.1007/s00244-006-0222-0
Cadmium Exposures in Fathead Minnows: Are There Sex-Specific Differences in Mortality, Reproductive Success, and Cd Accumulation? Marlo K. Sellin, Tess M. Eidem, Alan S. Kolok Department of Biology, University of Nebraska at Omaha, 6001 Dodge Street, Omaha, Nebraska 68182-0040, USA
Received: 6 October 2006 /Accepted: 10 November 2006
Abstract. The primary goal of this experiment was to determine whether cadmium (Cd) exposure has sex-specific effects on the reproductive success of fathead minnows as measured by time to first spawn, spawning frequency, clutch size, fecundity, fertilization success, hatching success, and offspring mortality to 2 d post hatch. Prior to breeding, minnows were either exposed to 50 lg/L Cd or sham exposed for 21 d. After exposures, minnows were paired (male · female) into one of four breeding groups—control · control (C · C), control · exposed (C · E), exposed · control (E · C) or exposed · exposed (E · E). Pairs of minnows were subjected to a 21-d breeding study during which the reproductive parameters mentioned above were measured. During the breeding study, minnows in the E · E pairs had significantly higher mortality than minnows in the C · C pairs; however, the mortality of minnows in the C · E and E · C did not differ from that of C · C pairs. Presumably, behavioral alterations in both males and females exposed to Cd accounted for the increased mortality in the E · E group. The results of the breeding study did not reveal any significant differences among any of the reproductive parameters measured with the exception of offspring mortality. Offspring from C · E pairs did not differ from offspring from C · C pairs with regard to mortality; however, offspring from pairs containing exposed males (E · C and E · E) had significantly higher mortality than offspring from C · C pairs suggesting that paternal exposure to Cd leads to an increase in offspring mortality.
It is not uncommon for the toxicity of a compound to be different between males and females (Keitt et al. 2004). For example, some studies have identified sex-specific differences in mortality due to cadmium (Cd). In fathead minnows (Pimephales promelas) exposed to 50 lg/L Cd for 21 d, males experience significantly greater mortality than females, with male mortality at 74% and female mortality at 32% (Sellin and
Correspondence to: Marlo K. Sellin; email:
[email protected] .edu
Kolok 2006a). In contrast, a study by Hatakeyama and Yasuno (1986) found that female guppies (Poecillia reticulata) were more sensitive to Cd exposures than males were. Specifically, upon exposure to dietary Cd, a significant increase in female mortality occurred, whereas male mortality remained unchanged. Exposure to Cd not only leads to sex-specific differences in mortality, but also to sex-specific alterations in physiological function. For example, in Japanese medaka (Oryzias latipes), exposure to Cd causes a significant increase in female 17bestradiol concentrations, but has no effect on male estradiol concentrations, suggesting that with regard to this endpoint, females are more sensitive than males (Foran et al. 2002). In addition, the results of several studies have shown that the testes are particularly sensitive to Cd. Tilton et al. (2003) demonstrated that exposure to Cd in Japanese medaka causes a significant reduction in testicular gonadosomatic index (GSI), but does not alter ovarian GSI. Furthermore, exposure to 10 mg/L Cd increases caspase-3 gene expression, an indicator of apoptosis, in the testes of black gobies (Gobius niger) (Migliarini et al. 2005). Whether or not this was also the case for ovarian tissue was not determined. Cadmium not only alters the physiological response of organisms, but can also alter reproductive success. Specifically, when both male and female fathead minnows were exposed to 50 lg/L Cd while spawning, a significant reduction in spawning frequency, and subsequently fecundity, occurred (Sellin and Kolok 2006a). Similarly, male and female pairs of fathead minnows that are exposed to Cd as larvae also experience a significant reduction in spawning frequency and fertilization success (Sellin and Kolok 2006b). Although these studies provide valuable information on the effects of Cd on reproduction, they do not determine whether the observed reduction in reproductive success is due to alterations in the reproductive physiology of males, females, or both. The primary goal of this experiment was to determine whether exposure to Cd has sex-specific effects on various reproductive parameters including time to first spawn, spawning frequency, clutch size, fecundity, fertilization success, hatching success, and offspring mortality. The secondary goal of this study was to determine whether any sex-specific differences in reproduction are related to sex-specific differences in whole-body Cd concentrations.
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Materials and Methods
Cd Analysis
Fish Maintenance
During the Cd exposures, a water exchange (>30% of total volume) was conducted daily. To replenish the experimental tanks, the appropriate volume of a stock solution of CdCl2 was pipetted into each tank. Water samples (125 ml) were collected daily from each aquarium. Each water sample was acidified with nitric acid to a pH of 0.05 in both cases; Figure 3). In contrast, the mortality of minnows in the E · E group was significantly greater than that of minnows in the C · C group (chi-square test, p < 0.041). No significant differences between male and female mortality during the breeding period were detected (chi-square test, p = 0.70).
Discussion Reproductive Success Exposures to Cd prior to spawning did not adversely affect spawning frequency, clutch size, fecundity, fertilization success, or hatching success. Findings from a study by Sellin and Kolok (2006a) support some of these findings. Specifically, fathead minnows exposed to 50 lg/L Cd while spawning did not experience any alterations in clutch size, fertilization success, or hatching success. However, contrary to the results of the current study, Sellin and Kolok (2006a) demonstrated that minnows exposed to 50 lg/L Cd while spawning experienced a significant reduction in spawning frequency and fecundity relative to controls. It is possible that alterations in spawning frequency and fecundity were not detected in this study because the Cd exposures were terminated before breeding began. Furthermore, minnows did not spawn immediately after the initiation of the breeding study, but rather it took an average of between 8 and 10 d for the first spawning event to occur. It is possible that during this time, the effects of the Cd exposures had attenuated. Offspring mortality was significantly higher among larvae produced by E · E pairs than larvae produced by C · C pairs, indicating that parental exposure to Cd adversely affects the
Larval Cd Concentrations There were no significant differences in the Cd concentrations of larvae produced by any of the breeding groups. The larval Cd concentrations of larvae produced by E · C, C · E, and E · E pairs were similar to those of larvae produced by C · C pairs, indicating that Cd was not transferred from either the males or the females to their offspring. Although the lack of paternal transfer is not surprising, the lack of maternal transfer is of interest because previous studies have shown that Cd is maternally transferred. Sellin and Kolok (2006b) demonstrated that females exposed to 100 lg/L Cd for 8 d produce larvae with significantly higher Cd concentrations than unexposed females; however, the same did not hold true for females exposed to 50 lg/L. It appears as if there is a minimum threshold concentration that must be reached before females begin to maternally transfer Cd to their young. It has been hypothesized by Sellin and Kolok (2006b) that at lower concentrations, females do not transfer Cd to their larvae because it is bound by a metal-binding protein such as metallothionein. Presumably, once a threshold concentration is reached, the female would begin to maternally transfer the Cd to her offspring.
Adult Whole-Body Cd Concentrations Immediately after the exposure period (day 0 of the breeding study), Cd-exposed minnows had significantly higher wholebody Cd concentrations than unexposed minnows. Several other studies have also documented that Cd-exposed fish have higher whole-body Cd concentrations than unexposed fish (Hollis et al. 1999; McGeer et al. 2000). A comparison of whole-body Cd concentrations among Cd-exposed minnows showed that immediately after the exposure period (day 0), minnows have significantly higher whole-body Cd concentrations than after the breeding period (day 21). Furthermore, after 21 d, the whole-body Cd concentrations of exposed minnows had returned to control levels. This finding indicates that minnows significantly reduce their whole-body Cd burden during a 21-d depuration period. Other studies have provided evidence indicating that Cd elimination occurs relatively quickly. For example, Glynn et al. (1992) found that the halftime of 109Cd in the whole bodies of juvenile minnows (Phoxinus phoxinus) was 9.7 d.
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Cadmium Exposures in Fathead Minnows
The fairly rapid elimination of whole-body Cd helps to put the results regarding breeding success into perspective. Our initial hypothesis was that Cd-exposed minnows would have a lower postexposure breeding success than unexposed minnows. If fathead minnows are capable of depurating the Cd during the first 8–10 d of the breeding study, a time frame coinciding with the average time to first spawn, this may very well explain the lack of significant differences in the reproductive parameters measured. The ability to expose one member of the breeding pair to Cd while maintaining the other in control conditions would eliminate the depuration period; however, this is not technically feasible. Significant differences in male and female whole-body Cd concentrations were detected, suggesting that whole-body Cd concentrations are sex-specific. In all cases, females had significantly higher whole-body Cd concentrations than males, indicating that females have either higher accumulation rates than males or lower clearance rates than males. Further investigation is required to determine exactly why male and female whole-body Cd concentrations differ from each other.
Adult Mortality The results of this study show that during an exposure period of 21 d, minnows exposed to 50 lg/L Cd have significantly higher mortality than unexposed minnows. Several other studies have demonstrated the lethality of Cd exposure (Benoit et al. 1976; Middaugh and Dean 1977). However, the mortality of minnows during the exposure was not as high as originally anticipated. In a previous study, fathead minnows exposed to 50 lg/L Cd experienced a mortality of 75% (Sellin and Kolok 2006a). In this study, minnows exposed to the same dose experienced only 9% mortality. In the Sellin and Kolok (2006a) study, minnows were exposed to Cd while spawning, whereas in the current study, minnows were exposed to Cd while being held in same-sex aquaria. When the results of these studies are taken together, it appears that spawning increases the lethality of Cd to fathead minnows. During the 21-d breeding period, E · E pairs of minnows had significantly higher mortality than C · C pairs; however, the mortality of E · C and C · E pairs did not differ from that of C · C pairs. This suggests that the pairing of fish influences mortality, because mortality increases significantly when two Cd-exposed individuals are paired, but remains unchanged when a Cd-exposed individual is paired with an unexposed individual. This finding was unexpected and somewhat peculiar because mortality does not appear to depend solely upon exposure history. It is possible that the observed increase in mortality among E · E pairs is related to alterations in both male and female behaviors. Studies have shown that exposure to Cd can lead to hyperactivity (Ellgaard et al. 1978; Peterson et al. 1983), and indeed, during the exposure period of this study, it was observed that male Cd-exposed minnows exhibited hyperactive behaviors. It is possible that in breeding males hyperactivity manifests itself by leading to increased aggressiveness toward female mates. Although unexposed females may be able to escape the aggressive attacks from males, it is possible that Cd-exposed females are unable to do so because studies have shown that exposure to Cd inhibits
escape behaviors in fishes (Faucher et al. 2006). The combination of aggressive males being paired with females with an impaired escape response may lead to an increased mortality for both sexes. During both the exposure period and the breeding period, no significant differences between male and female mortality were detected. The results from whole-body Cd analysis showed that Cd-exposed females had significantly higher Cd concentrations than males. If whole-body Cd concentrations are directly correlated with mortality, it would be predicted that females would have higher mortality than males. However, this was not the case, suggesting that mortality does not depend entirely upon Cd accumulation.
Acknowledgments. This study was partially supported by an Environmental Protection Agency Greater Research Opportunities Fellowship (#91636301-0) given to M.K.S. and by NIH grant # 1 P20 RR16469 from the BRIN Program of the National Center for Research Resources. Special thanks to Debbie Akerly for her assistance with fish care and maintenance.
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