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Chemical sensitivity of the male daphnid, Daphnia magna, induced by exposure to juvenile hormone and its analogs
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Chemosphere 72 (2008) 451–456 www.elsevier.com/locate/chemosphere
Chemical sensitivity of the male daphnid, Daphnia magna, induced by exposure to juvenile hormone and its analogs Takeru Matsumoto a, Erika Ikuno b, Shiro Itoi b,*, Haruo Sugita b b
a Mitsubishi Chemical Safety Institute Ltd., Yokohama, Kanagawa 227-0033, Japan Department of Marine Science and Resources, Nihon University, Fujisawa, Kanagawa 252-8510, Japan
Received 19 December 2007; received in revised form 7 February 2008; accepted 8 February 2008 Available online 2 April 2008
Abstract It was reported that males daphnid Daphnia magna that have been induced by methyl farnesoate exposure exhibit higher tolerance to chemical compounds such as potassium dichromate and pentachlorophenol than females. Male neonates are also known to be induced by exposure to juvenile hormone analogs, such as fenoxycarb and pyriproxyfen. If these analogs can be used to produce male progeny, the biological and physiological studies of daphnid male would be progressed since the effects of these analogs were several hundred times higher than that of methyl farnesoate. Therefore, in the present study, it was investigated that the chemical sensitivity of male neonates induced by exposure to juvenile hormone (methyl farnesoate) and its analogs. The minimum concentrations of methyl farnesoate, fenoxycarb and pyriproxyfen to induce 100% male-reproduction were 200 nM (50 lg/l), 0.23 nM (70 ng/l) and 0.31 nM (100 ng/l), respectively. In addition, no reduction of relative reproduction was observed at the juvenoid concentrations in 24 h exposure producing 100% male progeny. The median effective concentrations (EC50) of potassium dichromate for immobility of male neonates, established by a standardized method for investigating sensitivity to chemicals, were significantly higher (12–29%) than that of females at least after 24 h exposure in all the male neonates induced by juvenoids used in this study (P < 0.05). This study demonstrated that the male daphnids induced by exposure to juvenile hormone and its analogs exhibit similar chemical tolerance. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Acute toxicity test; Fenoxycarb; Methyl farnesoate; Potassium dichromate; Pyriproxyfen; Sex difference
1. Introduction The cladoceran crustacean Daphnia magna, a cyclical parthenogen, are formed asexually as female neonates under favorable environmental conditions. Male neonates appear under unfavorable environmental conditions such as decreasing photoperiod, decreasing food concentration, and increasing population density (Hobaek and Larsson, 1990; Kleiven et al., 1992). These female and male neonates are genetically identical to their mothers. Daphnids enter a sexual phase of reproduction by producing males and sexually responsive females. The female daphnids mate with male counterparts, and the fertilized diapause eggs, encased *
Corresponding author. Tel./fax: +81 466 84 3679. E-mail address: [email protected] (S. Itoi).
0045-6535/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.02.016
in a protective ephippium, are released into the environment. Diapause eggs are highly resistant to factors associated with severe environments such as desiccation and freezing, and can even hatch decades after release (Hebert, 1978). D. magna have a long and productive history in aquatic toxicity testing. This is attributable to several beneficial traits such as the fact that the complete life cycle is spent in water, prolific breeding, ease of handling, and comparatively short longevity (Tatarazako and Oda, 2007). In addition, since daphnids are known to be quite sensitive to a wide range of chemicals, including heavy metals (Tatarazako and Oda, 2007), there have been many studies investigating the sensitivity of females to chemical compounds (Klein, 2000; Yeh and Chen, 2006; Park and Choi, 2007). As a result, this species is highly recommended as a test
452
T. Matsumoto et al. / Chemosphere 72 (2008) 451–456
animal for the OECD test guideline (TG) 202 (OECD, 2004) and TG 211 (OECD, 1998). Although it is phenomenologically well known that male is necessary to produce diapause eggs at gamogenesis phase, little is known concerning the biological and physiological characteristics of male daphnid except for a report of the toxicity of heavy metals to male of D. magna (Saika et al., 2004). The biological and physiological studies of daphnid males require a technique for routinely producing male neonates. Olmstead and LeBlanc (2002) reported that the exposure of daphnid oocytes to the crustacean hormone, methyl farnesoate, during late ovarian development caused the oocytes to develop into male progeny, whereas only females were produced in daphnids that remained unexposed. A similar phenomenon has been observed in other cladoceran taxonomic groups, for example, in the genera Moina and Ceriodaphnia (Oda et al., 2005a). Several studies have demonstrated that the production of male neonates in D. magna can be induced by exposure to not only insect or crustacean juvenile hormones but also pesticides designed as juvenile hormone analogs (Olmstead and LeBlanc, 2002, 2003; Tatarazako et al., 2003; Oda et al., 2005b). However, Tatarazako et al. (2003) and Oda et al. (2005b) found that most of these juvenoids also reduced the total number of neonates considerably in 21 days reproduction tests, based on OECD TG 211 (OECD, 1998). Recently, Oda et al. (2006) exposed females to 5–10 lg/l fenoxycarb or pyriproxyfen for over as short time period (12 h), and found that these treatments induced a production of male neonates, but also a small reduction in the total number of neonates. It was reported that the minimum concentration of methyl farnesoate used over a 24 h exposure period to produce 100% male progeny was 50 lg/l (Ikuno et al., 2008). The report also showed that male daphnids induced by exposure to 100 lg/l methyl farnesoate over a 24 h period exhibited higher tolerance to chemical compounds such as potassium dichromate and pentachlorophenol than the females (Ikuno et al., 2008). Although it is possible for males to be induced by juvenile hormone analogs, as described above, the differences in chemical tolerance between males induced by different endocrine molecules such as juvenile hormone and its analogs has yet to be ascertained. If these analogs can be used to produce male as in the case of methyl farnesoate, male production will be more effective and easier. Therefore, in the present study, the minimum concentration of fenoxycarb and pyriproxyfen to produce 100% males progeny over a 24 h exposure period was determined, and subsequently the sensitivity of these male to potassium dichromate was compared with those of other groups and females. 2. Materials and methods All parts of this study were conducted in the Department of Marine Science and Resources, Nihon University.
2.1. Daphnid culture The D. magna Straus were originally derived from the National Institute for Environmental Studies of Japan (NIES), and then had been maintained at the Mitsubishi Chemical Safety Institute Ltd. for over 10 years. The daphnids were cultured in an incubator at a density of 20 adults in 1 l of the Elendt M4 medium (Elendt and Bias, 1990) at 20 °C with a natural photoperiod. Culture medium was renewed and offspring were removed two times weekly. Cultured daphnids were fed daily with approximately 0.15 ng carbon of microalga Chlorella vulgaris (Super Fresh Chlorella V-12, Chlorella Industry Co., Ltd., Tokyo, Japan) per daphnid. These culture conditions maintained the daphnids in the parthenogenic reproductive phase, with virtually no males produced. 2.2. Chemicals The chemicals used for male offspring reproduction were methyl farnesoate (Echelon Biosciences Inc., Salt Lake, UT, USA), which is a crustacean juvenile hormone (Laufer et al., 1993), fenoxycarb [ethyl 2-(4-phenoxyphenoxy) ethylcarbamate] (Wako Pure Chemical Industries Ltd., Osaka, Japan) and pyriproxyfen [4-phenoxyphenyl (RS)-2-(2-pyridyloxy) propyl ether] (LKT Laboratory Inc., MN, USA). The latter two chemicals are juvenile hormone analogs (Olmstead and LeBlanc, 2003; Tatarazako et al., 2003; Gorr et al., 2006). Potassium dichromate (analytical reagent grade; Wako Pure Chemical Industries Ltd.) was used as the chemical for the toxicity bioassay. Ethanol and dimethylformamide were used as solvents for the preparation of stock solutions of methyl farnesoate and the analogs, respectively, and were obtained from Wako Pure Chemical Industries Ltd. 2.3. Reproduction tests Mature females >13 days old were used for male progeny production in the exposure tests involving methyl farnesoate, fenoxycarb or pyriproxyfen. Daphnids were exposed to various concentrations of methyl farnesoate (4–400 nM, 1–100 lg/l), fenoxycarb (0.03–3.32 nM, 10– 1000 ng/l) or pyriproxyfen (0.03–3.11 nM, 10–1000 ng/l) for 24 h, including the 60–72 h period after the last molt. The same procedures were followed using a solvent control (0.005% v/v ethanol, 0.01% v/v dimethylformamide) and a normal control (Elendt M4 medium only). Glass beakers (500 ml), each containing ten mature D. magna in 500 ml of media were used for each concentration. After 24 h exposure, daphnids were rinsed and transferred to new Elendt M4 medium. Thereafter the medium was not renewed until the next phase of reproduction. All tests were conducted for 5 days in an incubator at a temperature of 20 ± 1 °C with a natural photoperiod. Daphnids were fed daily with approximately 0.15 ng carbon of microalga per daphnid during the experiments. The sex of neonates was identified under a stereoscopic microscope (20–40) using
T. Matsumoto et al. / Chemosphere 72 (2008) 451–456
the morphology of the first antenna as the definitive identifier. Male offspring (%) was defined as the percentage of males in the total number of neonate populations observed at each nominal concentration. The rate of reproduction in the juvenoid-exposure group relative to controls was determined by analyzing the mean number of neonates produced per female in each experiment. 2.4. Acute toxicity test Acute toxicity tests were conducted using potassium dichromate and by referring to OECD TG 202 (OECD, 2004). Male and female neonates were used in these experiments within 24 h of birth and were exposed to various concentrations of the test chemical. Male neonates were induced by exposing mature females (>13 days old) to 100 lg/l methyl farnesoate (400 nM), 500 ng/l fenoxycarb (1.7 nM) or 500 ng/l pyriproxyfen (1.6 nM) over a period of 24 h. Five concentrations of the test chemical (potassium dichromate) were prepared by diluting the stock solution with culture medium. A control containing only Elendt M4 medium was also prepared. Potassium dichromate tests were performed at nominal concentrations of 0.2–1.6 mg/l in a geometric series with a factor of 1.68. Duplicate glass beakers (100 ml), each containing five male or female D. magna neonates in 100 ml of medium, were used at each concentration. The beakers were covered with plastic film to prevent the evaporation of water and test chemical. All tests were conducted in an incubator at a temperature of 20 ± 1 °C with a photoperiod of 16 h light and 8 h dark. No food was provided dur-
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ing the experiments. The experimental endpoint was defined as immobility observed after 24 h and 48 h from the start of exposure. Test results were classified into two categories: mobile or immobile, the latter category including daphnids immobilized for 15 s or dead daphnids. Tests were carried out in 3–6 replicates. Dissolved oxygen and pH in all the experimental Elendt M4 media were 7.9 ± 0.3 mg/l and 7.9 ± 0.4, respectively, at the start of exposure, and 8.4 ± 0.7 mg/l and 7.6 ± 0.2, respectively, after 48 h exposure. 2.5. Statistical analysis Median effective concentration (EC50) values for the acute toxicity tests were calculated based on the nominal concentrations, and estimated by probit, binomial and moving average analysis. The biological variables were tested for homogeneity of variance with the Bartlett’s test. Differences between the controls and the samples induced by juvenoids were calculated by using an analysis of variance (one-way ANOVA) and Dunnett multiple range test. Statistical analysis was performed using stat light (Yukms corp., Tokyo, Japan). 3. Results 3.1. Reproduction tests with juvenile hormone and the analogs The concentration of methyl farnesoate in a 24 h exposure producing 100% males was compared with those of
Fig. 1. Effects of exposure to juvenoids (methyl farnesoate, fenoxycarb and pyriproxyfen) upon male offspring production.
Fig. 2. Effect of methyl farnesoate, fenoxycarb and pyriproxyfen upon relative reproduction in mature female daphnids > 13 days old. Dotted line represents the mean number of neonates per female in the control for each experiment during a 5 days test period.
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fenoxycarb and pyriproxyfen. This showed that the minimum concentration of methyl farnesoate needed to induce 100% male-reproduction was 200 nM (50 lg/l) as reported previously (Ikuno et al., 2008), whereas, those of fenoxycarb and pyriproxyfen were 0.23 nM (70 ng/l) and 0.31 nM (100 ng/l), respectively (Fig. 1). The EC50 values of methyl farnesoate, fenoxycarb and pyriproxyfen were calculated to be 17.6 lg/l (70 nM), 46.9 ng/l (0.16 nM) and 55.7 ng/l (0.17 nM), respectively. Rate of reproduction in the total number of neonate populations relative to the normal rate of reproduction was not dependent on the juvenoid concentrations for 100% male reproduction (Fig. 2). 3.2. Acute toxicity test The results of the acute toxicity test performed with potassium dichromate using females and males (induced by different juvenoids) are shown in Fig. 3. The EC50 of female neonates after 24 h and 48 h exposure were 1.15 ± 0.06 mg/l (3.90 ± 0.19 nM) and 0.77 ± 0.05 mg/l (2.61 ± 0.18 nM), respectively (Fig. 3A), which are similar to our previous report (Ikuno et al., 2008). The EC50 of male neonates induced by methyl farnesoate were 1.48 ± 0.14 mg/l (5.02 ± 0.49 nM) for 24 h exposure and 0.87 ± 0.11 mg/l (2.97 ± 0.39 nM) for 48 h exposure (Fig. 3A). There was a significant difference between male and female neonates in terms of EC50 after 24 h exposure (P < 0.05) but no significant difference between sexes after 48 h exposure. Similar results were observed in the case of the test using fenoxycarb-induced males (Fig. 3B). The EC50 of male and female neonates after 24 h exposure were 1.55 ± 0.12 mg/l (5.27 ± 0.41 nM) and 1.38 ± 0.12 mg/l (4.69 ± 0.40 nM), respectively, with a significant difference at P < 0.05. No significant difference was observed in the EC50 after 48 h exposure between males (1.13 ± 0.17 mg/ l, 3.86 ± 0.59 nM) and females (0.97 ± 0.21 mg/l, 3.29 ± 0.73 nM). In the case of the test using pyriproxyfeninduced males, the EC50 of male and female neonates were 1.43 ± 0.06 mg/l (4.87 ± 0.21 nM) and 1.22 ± 0.09 mg/l (4.14 ± 0.31 nM), respectively, after 24 h exposure, and 1.05 ± 0.06 mg/l (3.57 ± 0.21 nM) and 0.77 ± 0.07 mg/l (2.61 ± 0.22 nM), respectively, after 48 h exposure with significant differences at P < 0.05 (Fig. 3C). 4. Discussion It has been reported that the production of male neonates in D. magna can be induced by exposure to methyl farnesoate, and although methyl farnesoate is rapidly metabolized, the analogs, fenoxycarb and pyriproxyfen, could produce male neonates at the lower concentration rather than methyl farnesoate (Olmstead and LeBlanc, 2002, 2003; Tatarazako et al., 2003; Oda et al., 2005b). Thus, if these analogs could be used as the inducer for male production without biological and physiological effects at least in the case of using the level of methyl farnesoate, these will be used effectively for producing male neonates
Fig. 3. Median effective concentrations (EC50) of potassium dichromate for male and female daphnids. Male neonates were induced by 24 h exposure to methyl farnesoate (A), fenoxycarb (B) and pyriproxyfen (C). EC50 values were derived from an acute immobility test with the three chemical compounds. Open and filled circles represent values for female and male daphnids, respectively. Bars represent means ± standard deviation for 3–6 replicates. Student’s t-test was employed for statistical comparison (* significant at P < 0.05).
on the studies. However, differences in the chemical sensitivity of male daphnids induced by different juvenoids remain unclear. It was revealed that 100% male neonates could be produced by exposing daphnids to 50 lg/l methyl farnesoate based on the nominal concentrations (Ikuno et al., 2008). Therefore, in the present study, the minimum concentrations of fenoxycarb and pyriproxyfen to produce 100% males in a 24 h exposure period were determined to be 70 ng/l and 100 ng/l, respectively, based on the nominal concentrations. These concentrations are 500–700-fold lower than that of methyl farnesoate, based on nominal concentrations, as reported by Tatarazako et al. (2003). Although the concentration of the juvenoids in the test solutions could not be analyzed in this study, the artificial juvenoids (i.e. fenoxycarb and pyriproxyfen) may be more
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stable than their natural counterpart (i.e. methyl farnesoate). Moreover, it was also considered that artificial juvenoids may bind strongly to their receptors and may be more difficult to metabolize in D. magna than their natural counterpart. On the other hand, it has also been reported that although the production of male neonates in D. magna was induced by exposure to juvenoids during a 21 days test period, the total number of neonates was considerably reduced (Tatarazako et al., 2003; Oda et al., 2005a, 2005b, 2006). In the present study, however, there was not observed a reduction in relative reproduction of juvenoids at the concentrations tested in a 24 h exposure during a 5 days test period producing 100% males. Furthermore, it was noted that all of the progeny produced from the mature daphnids after male production were females (data not shown). These results suggest that the method in this study could produce a population consisting of 100% male individuals with minimum physiological effect of juvenoids upon the reproduction. Therefore, this method of exposure may be a very useful tool for the detection of endocrine disruptors acting in a similar manner to juvenoids but with little damage to the test daphnids used. On the other hand, the effects of pyriproxyfen on male production was sometimes unstable, it is remained unclear. In addition, the diapause eggs produced by females and chemical induced males have not been found to be able to hatch in our laboratory. However, it is reported that germ line cells develop in the male embryos produced by exposing to pyriproxyfen almost same as female (Sagawa et al., 2005). Since it might be unfertilized eggs as described by Tatarazako et al. (2003), it would be necessary to compare the characteristics of chemical induced males with those of nature males and to investigate the environmental condition to produce the hatchable diapause eggs. The present study of chemical sensitivity using potassium dichromate showed that the EC50 of male neonates induced by all of the juvenoids used in this study were significantly higher (12–29%) than that of females, at least after 24 h exposure (Fig. 3). Moreover, there was no significant difference among the relative EC50 data of all the juvenoid-induced males when normalized by the EC50 of females from each exposure test, either after 24 and 48 h exposure (data not shown). This result suggests that juvenoid-induced male neonates exhibit no differences in physiological mechanism(s) when compared amongst juvenoids arising from a 24 h exposure with the minimum concentration required for 100% male production. In conclusion, our present study has successfully determined the minimum concentration of juvenile hormone analogs (fenoxycarb and pyriproxyfen) to be used in a 24 h exposure to produce progeny consisting of 100% male individuals. Moreover, all of the male neonates induced by juvenoids exhibited higher tolerance to potassium dichromate than female neonates. Furthermore, because males induced by juvenoids exhibit similar chemical tolerance, it is considered that using the concentrations of juvenoids determined in the present study for the production of males
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does not influence the sensitivity of male neonates induced by different juvenoids. The method of male production established in this study is likely to be highly useful for the screening of chemicals acting in a similar manner to juvenoids and also for studying the ecology of the daphnid male. Furthermore, the present study would contribute to monitoring of the effects of human activities on the ecology through the reproduction role of daphnid in the water environments. Acknowledgements The D. magna NIES strain was kindly provided by Dr. Y. Sugaya, the National Institute for Environmental Studies. We also thank S. Hagino, Sumika Technoservice Corparation, for valuable comment on this manuscript. References Elendt, B.P., Bias, W.R., 1990. Trace nutrient deficiency in Daphnia magna cultured in standard medium for toxicity testing: effects of the optimization of culture conditions on life history parameters of D. magna. Water Res. 24, 1157–1167. Gorr, T.A., Rider, C.V., Wang, H.Y., Olmstead, A.W., LeBlanc, G.A., 2006. A candidate juvenoid hormone receptor cis-element in the Daphnia magna hb2 hemoglobin gene promoter. Mol. Cell. Endocrinol. 247, 91–102. Hebert, P.D.N., 1978. The population biology of Daphnia (Crustacea, Daphnidae). Biol. Rev. 53, 387–426. Hobaek, A., Larsson, P., 1990. Sex determination in Daphnia magna. Ecology 71, 2255–2268. Ikuno, E., Matsumoto, T., Okubo, T., Itoi, S., Sugita, H., 2008. Difference in the sensitivity to chemical compounds between female and male neonates of Daphnia magna. Environ. Toxicol., in press, doi:10.1002/ tox.20403. Klein, B., 2000. Age as a factor influencing results in the acute daphnid test with Daphnia magna Straus. Water Res. 34, 1419–1424. Kleiven, O.T., Larsson, P., Hobaek, A., 1992. Sexual reproduction in Daphnia magna requires three stimuli. Oikos 65, 197–206. Laufer, H., Ahl, J.S.B., Sagi, A., 1993. The role of juvenile hormones in crustacean reproduction. Am. Zool. 33, 365–374. Oda, S., Tatarazako, N., Watanabe, H., Morita, M., Iguchi, T., 2005a. Production of male neonates in 4 cladoceran species exposed to a juvenile hormone analog, fenoxycarb. Chemosphere 60, 74–78. Oda, S., Tatarazako, N., Watanabe, H., Morita, M., Iguchi, T., 2005b. Production of male neonates in Daphnia magna (Cladocera, Crustacea) exposed to juvenile hormones and their analogs. Chemosphere 61, 1168–1174. Oda, S., Tatarazako, N., Watanabe, H., Morita, M., Iguchi, T., 2006. Genetic differences in the production of male neonates in Daphnia magna exposed to juvenile hormone analogs. Chemosphere 63, 1477– 1484. OECD, 1998. Daphnia magna reproduction test. In: OECD Guidelines for Testing of Chemicals, vol. 211. Organization for Economic Cooperation and Development, Paris. OECD, 2004. Daphnia sp., acute immobilization test. In: OECD Guidelines for Testing of Chemicals, vol. 202. Organization for Economic Cooperation and Development, Paris. Olmstead, A.W., LeBlanc, G.A., 2002. Juvenoid hormone methyl farnesoate is a sex determinant in the crustacean Daphnia magna. J. Exp. Zool. 293, 736–739. Olmstead, A.W., LeBlanc, G.A., 2003. Insecticidal juvenile hormone analogs stimulate the production of male offspring in the crustacean Daphnia magna. Environ. Health Perspect. 111, 919–924.
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Park, S.Y., Choi, J., 2007. Cytotoxicity, genotoxicity and ecotoxicity assay using human cell and environmental species for the screening of the risk from pollutant exposure. Environ. Int. 33, 817– 822. Saika, O., Kohayakawa, Y., Fujii, Y., Hara, A., 2004. Sex differences in the acute and long-term toxicity of copper, chromium and zinc to Daphnia magna. Jpn. J. Environ. Toxicol. 7, 105–112. Sagawa, K., Yamagata, H., Shiga, Y., 2005. Exploring embryonic germ line development in the water flea, Daphnia magna, by zinc-fingercontaining VASA as a marker. Gene Exp. Patt. 5, 669–678.
Tatarazako, N., Oda, S., 2007. The water flea Daphnia magna (Crustacea, cladocera) as a test species for screening and evaluation of chemicals with endocrine disrupting effects on crustaceans. Ecotoxicology 16, 197–203. Tatarazako, N., Oda, S., Watanabe, H., Morita, M., Iguchi, T., 2003. Juvenile hormone agonists affect the occurrence of male Daphnia. Chemosphere 53, 827–833. Yeh, H.J., Chen, C.Y., 2006. Toxicity assessment of pesticides to Pseudokirchneriella subcapitata under air-tight test environment. J. Hazard. Mater. 131, 6–12.
Chemosphere 72 (2008) 451–456 www.elsevier.com/locate/chemosphere
Chemical sensitivity of the male daphnid, Daphnia magna, induced by exposure to juvenile hormone and its analogs Takeru Matsumoto a, Erika Ikuno b, Shiro Itoi b,*, Haruo Sugita b b
a Mitsubishi Chemical Safety Institute Ltd., Yokohama, Kanagawa 227-0033, Japan Department of Marine Science and Resources, Nihon University, Fujisawa, Kanagawa 252-8510, Japan
Received 19 December 2007; received in revised form 7 February 2008; accepted 8 February 2008 Available online 2 April 2008
Abstract It was reported that males daphnid Daphnia magna that have been induced by methyl farnesoate exposure exhibit higher tolerance to chemical compounds such as potassium dichromate and pentachlorophenol than females. Male neonates are also known to be induced by exposure to juvenile hormone analogs, such as fenoxycarb and pyriproxyfen. If these analogs can be used to produce male progeny, the biological and physiological studies of daphnid male would be progressed since the effects of these analogs were several hundred times higher than that of methyl farnesoate. Therefore, in the present study, it was investigated that the chemical sensitivity of male neonates induced by exposure to juvenile hormone (methyl farnesoate) and its analogs. The minimum concentrations of methyl farnesoate, fenoxycarb and pyriproxyfen to induce 100% male-reproduction were 200 nM (50 lg/l), 0.23 nM (70 ng/l) and 0.31 nM (100 ng/l), respectively. In addition, no reduction of relative reproduction was observed at the juvenoid concentrations in 24 h exposure producing 100% male progeny. The median effective concentrations (EC50) of potassium dichromate for immobility of male neonates, established by a standardized method for investigating sensitivity to chemicals, were significantly higher (12–29%) than that of females at least after 24 h exposure in all the male neonates induced by juvenoids used in this study (P < 0.05). This study demonstrated that the male daphnids induced by exposure to juvenile hormone and its analogs exhibit similar chemical tolerance. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Acute toxicity test; Fenoxycarb; Methyl farnesoate; Potassium dichromate; Pyriproxyfen; Sex difference
1. Introduction The cladoceran crustacean Daphnia magna, a cyclical parthenogen, are formed asexually as female neonates under favorable environmental conditions. Male neonates appear under unfavorable environmental conditions such as decreasing photoperiod, decreasing food concentration, and increasing population density (Hobaek and Larsson, 1990; Kleiven et al., 1992). These female and male neonates are genetically identical to their mothers. Daphnids enter a sexual phase of reproduction by producing males and sexually responsive females. The female daphnids mate with male counterparts, and the fertilized diapause eggs, encased *
Corresponding author. Tel./fax: +81 466 84 3679. E-mail address: [email protected] (S. Itoi).
0045-6535/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.02.016
in a protective ephippium, are released into the environment. Diapause eggs are highly resistant to factors associated with severe environments such as desiccation and freezing, and can even hatch decades after release (Hebert, 1978). D. magna have a long and productive history in aquatic toxicity testing. This is attributable to several beneficial traits such as the fact that the complete life cycle is spent in water, prolific breeding, ease of handling, and comparatively short longevity (Tatarazako and Oda, 2007). In addition, since daphnids are known to be quite sensitive to a wide range of chemicals, including heavy metals (Tatarazako and Oda, 2007), there have been many studies investigating the sensitivity of females to chemical compounds (Klein, 2000; Yeh and Chen, 2006; Park and Choi, 2007). As a result, this species is highly recommended as a test
452
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animal for the OECD test guideline (TG) 202 (OECD, 2004) and TG 211 (OECD, 1998). Although it is phenomenologically well known that male is necessary to produce diapause eggs at gamogenesis phase, little is known concerning the biological and physiological characteristics of male daphnid except for a report of the toxicity of heavy metals to male of D. magna (Saika et al., 2004). The biological and physiological studies of daphnid males require a technique for routinely producing male neonates. Olmstead and LeBlanc (2002) reported that the exposure of daphnid oocytes to the crustacean hormone, methyl farnesoate, during late ovarian development caused the oocytes to develop into male progeny, whereas only females were produced in daphnids that remained unexposed. A similar phenomenon has been observed in other cladoceran taxonomic groups, for example, in the genera Moina and Ceriodaphnia (Oda et al., 2005a). Several studies have demonstrated that the production of male neonates in D. magna can be induced by exposure to not only insect or crustacean juvenile hormones but also pesticides designed as juvenile hormone analogs (Olmstead and LeBlanc, 2002, 2003; Tatarazako et al., 2003; Oda et al., 2005b). However, Tatarazako et al. (2003) and Oda et al. (2005b) found that most of these juvenoids also reduced the total number of neonates considerably in 21 days reproduction tests, based on OECD TG 211 (OECD, 1998). Recently, Oda et al. (2006) exposed females to 5–10 lg/l fenoxycarb or pyriproxyfen for over as short time period (12 h), and found that these treatments induced a production of male neonates, but also a small reduction in the total number of neonates. It was reported that the minimum concentration of methyl farnesoate used over a 24 h exposure period to produce 100% male progeny was 50 lg/l (Ikuno et al., 2008). The report also showed that male daphnids induced by exposure to 100 lg/l methyl farnesoate over a 24 h period exhibited higher tolerance to chemical compounds such as potassium dichromate and pentachlorophenol than the females (Ikuno et al., 2008). Although it is possible for males to be induced by juvenile hormone analogs, as described above, the differences in chemical tolerance between males induced by different endocrine molecules such as juvenile hormone and its analogs has yet to be ascertained. If these analogs can be used to produce male as in the case of methyl farnesoate, male production will be more effective and easier. Therefore, in the present study, the minimum concentration of fenoxycarb and pyriproxyfen to produce 100% males progeny over a 24 h exposure period was determined, and subsequently the sensitivity of these male to potassium dichromate was compared with those of other groups and females. 2. Materials and methods All parts of this study were conducted in the Department of Marine Science and Resources, Nihon University.
2.1. Daphnid culture The D. magna Straus were originally derived from the National Institute for Environmental Studies of Japan (NIES), and then had been maintained at the Mitsubishi Chemical Safety Institute Ltd. for over 10 years. The daphnids were cultured in an incubator at a density of 20 adults in 1 l of the Elendt M4 medium (Elendt and Bias, 1990) at 20 °C with a natural photoperiod. Culture medium was renewed and offspring were removed two times weekly. Cultured daphnids were fed daily with approximately 0.15 ng carbon of microalga Chlorella vulgaris (Super Fresh Chlorella V-12, Chlorella Industry Co., Ltd., Tokyo, Japan) per daphnid. These culture conditions maintained the daphnids in the parthenogenic reproductive phase, with virtually no males produced. 2.2. Chemicals The chemicals used for male offspring reproduction were methyl farnesoate (Echelon Biosciences Inc., Salt Lake, UT, USA), which is a crustacean juvenile hormone (Laufer et al., 1993), fenoxycarb [ethyl 2-(4-phenoxyphenoxy) ethylcarbamate] (Wako Pure Chemical Industries Ltd., Osaka, Japan) and pyriproxyfen [4-phenoxyphenyl (RS)-2-(2-pyridyloxy) propyl ether] (LKT Laboratory Inc., MN, USA). The latter two chemicals are juvenile hormone analogs (Olmstead and LeBlanc, 2003; Tatarazako et al., 2003; Gorr et al., 2006). Potassium dichromate (analytical reagent grade; Wako Pure Chemical Industries Ltd.) was used as the chemical for the toxicity bioassay. Ethanol and dimethylformamide were used as solvents for the preparation of stock solutions of methyl farnesoate and the analogs, respectively, and were obtained from Wako Pure Chemical Industries Ltd. 2.3. Reproduction tests Mature females >13 days old were used for male progeny production in the exposure tests involving methyl farnesoate, fenoxycarb or pyriproxyfen. Daphnids were exposed to various concentrations of methyl farnesoate (4–400 nM, 1–100 lg/l), fenoxycarb (0.03–3.32 nM, 10– 1000 ng/l) or pyriproxyfen (0.03–3.11 nM, 10–1000 ng/l) for 24 h, including the 60–72 h period after the last molt. The same procedures were followed using a solvent control (0.005% v/v ethanol, 0.01% v/v dimethylformamide) and a normal control (Elendt M4 medium only). Glass beakers (500 ml), each containing ten mature D. magna in 500 ml of media were used for each concentration. After 24 h exposure, daphnids were rinsed and transferred to new Elendt M4 medium. Thereafter the medium was not renewed until the next phase of reproduction. All tests were conducted for 5 days in an incubator at a temperature of 20 ± 1 °C with a natural photoperiod. Daphnids were fed daily with approximately 0.15 ng carbon of microalga per daphnid during the experiments. The sex of neonates was identified under a stereoscopic microscope (20–40) using
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the morphology of the first antenna as the definitive identifier. Male offspring (%) was defined as the percentage of males in the total number of neonate populations observed at each nominal concentration. The rate of reproduction in the juvenoid-exposure group relative to controls was determined by analyzing the mean number of neonates produced per female in each experiment. 2.4. Acute toxicity test Acute toxicity tests were conducted using potassium dichromate and by referring to OECD TG 202 (OECD, 2004). Male and female neonates were used in these experiments within 24 h of birth and were exposed to various concentrations of the test chemical. Male neonates were induced by exposing mature females (>13 days old) to 100 lg/l methyl farnesoate (400 nM), 500 ng/l fenoxycarb (1.7 nM) or 500 ng/l pyriproxyfen (1.6 nM) over a period of 24 h. Five concentrations of the test chemical (potassium dichromate) were prepared by diluting the stock solution with culture medium. A control containing only Elendt M4 medium was also prepared. Potassium dichromate tests were performed at nominal concentrations of 0.2–1.6 mg/l in a geometric series with a factor of 1.68. Duplicate glass beakers (100 ml), each containing five male or female D. magna neonates in 100 ml of medium, were used at each concentration. The beakers were covered with plastic film to prevent the evaporation of water and test chemical. All tests were conducted in an incubator at a temperature of 20 ± 1 °C with a photoperiod of 16 h light and 8 h dark. No food was provided dur-
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ing the experiments. The experimental endpoint was defined as immobility observed after 24 h and 48 h from the start of exposure. Test results were classified into two categories: mobile or immobile, the latter category including daphnids immobilized for 15 s or dead daphnids. Tests were carried out in 3–6 replicates. Dissolved oxygen and pH in all the experimental Elendt M4 media were 7.9 ± 0.3 mg/l and 7.9 ± 0.4, respectively, at the start of exposure, and 8.4 ± 0.7 mg/l and 7.6 ± 0.2, respectively, after 48 h exposure. 2.5. Statistical analysis Median effective concentration (EC50) values for the acute toxicity tests were calculated based on the nominal concentrations, and estimated by probit, binomial and moving average analysis. The biological variables were tested for homogeneity of variance with the Bartlett’s test. Differences between the controls and the samples induced by juvenoids were calculated by using an analysis of variance (one-way ANOVA) and Dunnett multiple range test. Statistical analysis was performed using stat light (Yukms corp., Tokyo, Japan). 3. Results 3.1. Reproduction tests with juvenile hormone and the analogs The concentration of methyl farnesoate in a 24 h exposure producing 100% males was compared with those of
Fig. 1. Effects of exposure to juvenoids (methyl farnesoate, fenoxycarb and pyriproxyfen) upon male offspring production.
Fig. 2. Effect of methyl farnesoate, fenoxycarb and pyriproxyfen upon relative reproduction in mature female daphnids > 13 days old. Dotted line represents the mean number of neonates per female in the control for each experiment during a 5 days test period.
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fenoxycarb and pyriproxyfen. This showed that the minimum concentration of methyl farnesoate needed to induce 100% male-reproduction was 200 nM (50 lg/l) as reported previously (Ikuno et al., 2008), whereas, those of fenoxycarb and pyriproxyfen were 0.23 nM (70 ng/l) and 0.31 nM (100 ng/l), respectively (Fig. 1). The EC50 values of methyl farnesoate, fenoxycarb and pyriproxyfen were calculated to be 17.6 lg/l (70 nM), 46.9 ng/l (0.16 nM) and 55.7 ng/l (0.17 nM), respectively. Rate of reproduction in the total number of neonate populations relative to the normal rate of reproduction was not dependent on the juvenoid concentrations for 100% male reproduction (Fig. 2). 3.2. Acute toxicity test The results of the acute toxicity test performed with potassium dichromate using females and males (induced by different juvenoids) are shown in Fig. 3. The EC50 of female neonates after 24 h and 48 h exposure were 1.15 ± 0.06 mg/l (3.90 ± 0.19 nM) and 0.77 ± 0.05 mg/l (2.61 ± 0.18 nM), respectively (Fig. 3A), which are similar to our previous report (Ikuno et al., 2008). The EC50 of male neonates induced by methyl farnesoate were 1.48 ± 0.14 mg/l (5.02 ± 0.49 nM) for 24 h exposure and 0.87 ± 0.11 mg/l (2.97 ± 0.39 nM) for 48 h exposure (Fig. 3A). There was a significant difference between male and female neonates in terms of EC50 after 24 h exposure (P < 0.05) but no significant difference between sexes after 48 h exposure. Similar results were observed in the case of the test using fenoxycarb-induced males (Fig. 3B). The EC50 of male and female neonates after 24 h exposure were 1.55 ± 0.12 mg/l (5.27 ± 0.41 nM) and 1.38 ± 0.12 mg/l (4.69 ± 0.40 nM), respectively, with a significant difference at P < 0.05. No significant difference was observed in the EC50 after 48 h exposure between males (1.13 ± 0.17 mg/ l, 3.86 ± 0.59 nM) and females (0.97 ± 0.21 mg/l, 3.29 ± 0.73 nM). In the case of the test using pyriproxyfeninduced males, the EC50 of male and female neonates were 1.43 ± 0.06 mg/l (4.87 ± 0.21 nM) and 1.22 ± 0.09 mg/l (4.14 ± 0.31 nM), respectively, after 24 h exposure, and 1.05 ± 0.06 mg/l (3.57 ± 0.21 nM) and 0.77 ± 0.07 mg/l (2.61 ± 0.22 nM), respectively, after 48 h exposure with significant differences at P < 0.05 (Fig. 3C). 4. Discussion It has been reported that the production of male neonates in D. magna can be induced by exposure to methyl farnesoate, and although methyl farnesoate is rapidly metabolized, the analogs, fenoxycarb and pyriproxyfen, could produce male neonates at the lower concentration rather than methyl farnesoate (Olmstead and LeBlanc, 2002, 2003; Tatarazako et al., 2003; Oda et al., 2005b). Thus, if these analogs could be used as the inducer for male production without biological and physiological effects at least in the case of using the level of methyl farnesoate, these will be used effectively for producing male neonates
Fig. 3. Median effective concentrations (EC50) of potassium dichromate for male and female daphnids. Male neonates were induced by 24 h exposure to methyl farnesoate (A), fenoxycarb (B) and pyriproxyfen (C). EC50 values were derived from an acute immobility test with the three chemical compounds. Open and filled circles represent values for female and male daphnids, respectively. Bars represent means ± standard deviation for 3–6 replicates. Student’s t-test was employed for statistical comparison (* significant at P < 0.05).
on the studies. However, differences in the chemical sensitivity of male daphnids induced by different juvenoids remain unclear. It was revealed that 100% male neonates could be produced by exposing daphnids to 50 lg/l methyl farnesoate based on the nominal concentrations (Ikuno et al., 2008). Therefore, in the present study, the minimum concentrations of fenoxycarb and pyriproxyfen to produce 100% males in a 24 h exposure period were determined to be 70 ng/l and 100 ng/l, respectively, based on the nominal concentrations. These concentrations are 500–700-fold lower than that of methyl farnesoate, based on nominal concentrations, as reported by Tatarazako et al. (2003). Although the concentration of the juvenoids in the test solutions could not be analyzed in this study, the artificial juvenoids (i.e. fenoxycarb and pyriproxyfen) may be more
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stable than their natural counterpart (i.e. methyl farnesoate). Moreover, it was also considered that artificial juvenoids may bind strongly to their receptors and may be more difficult to metabolize in D. magna than their natural counterpart. On the other hand, it has also been reported that although the production of male neonates in D. magna was induced by exposure to juvenoids during a 21 days test period, the total number of neonates was considerably reduced (Tatarazako et al., 2003; Oda et al., 2005a, 2005b, 2006). In the present study, however, there was not observed a reduction in relative reproduction of juvenoids at the concentrations tested in a 24 h exposure during a 5 days test period producing 100% males. Furthermore, it was noted that all of the progeny produced from the mature daphnids after male production were females (data not shown). These results suggest that the method in this study could produce a population consisting of 100% male individuals with minimum physiological effect of juvenoids upon the reproduction. Therefore, this method of exposure may be a very useful tool for the detection of endocrine disruptors acting in a similar manner to juvenoids but with little damage to the test daphnids used. On the other hand, the effects of pyriproxyfen on male production was sometimes unstable, it is remained unclear. In addition, the diapause eggs produced by females and chemical induced males have not been found to be able to hatch in our laboratory. However, it is reported that germ line cells develop in the male embryos produced by exposing to pyriproxyfen almost same as female (Sagawa et al., 2005). Since it might be unfertilized eggs as described by Tatarazako et al. (2003), it would be necessary to compare the characteristics of chemical induced males with those of nature males and to investigate the environmental condition to produce the hatchable diapause eggs. The present study of chemical sensitivity using potassium dichromate showed that the EC50 of male neonates induced by all of the juvenoids used in this study were significantly higher (12–29%) than that of females, at least after 24 h exposure (Fig. 3). Moreover, there was no significant difference among the relative EC50 data of all the juvenoid-induced males when normalized by the EC50 of females from each exposure test, either after 24 and 48 h exposure (data not shown). This result suggests that juvenoid-induced male neonates exhibit no differences in physiological mechanism(s) when compared amongst juvenoids arising from a 24 h exposure with the minimum concentration required for 100% male production. In conclusion, our present study has successfully determined the minimum concentration of juvenile hormone analogs (fenoxycarb and pyriproxyfen) to be used in a 24 h exposure to produce progeny consisting of 100% male individuals. Moreover, all of the male neonates induced by juvenoids exhibited higher tolerance to potassium dichromate than female neonates. Furthermore, because males induced by juvenoids exhibit similar chemical tolerance, it is considered that using the concentrations of juvenoids determined in the present study for the production of males
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does not influence the sensitivity of male neonates induced by different juvenoids. The method of male production established in this study is likely to be highly useful for the screening of chemicals acting in a similar manner to juvenoids and also for studying the ecology of the daphnid male. Furthermore, the present study would contribute to monitoring of the effects of human activities on the ecology through the reproduction role of daphnid in the water environments. Acknowledgements The D. magna NIES strain was kindly provided by Dr. Y. Sugaya, the National Institute for Environmental Studies. We also thank S. Hagino, Sumika Technoservice Corparation, for valuable comment on this manuscript. References Elendt, B.P., Bias, W.R., 1990. Trace nutrient deficiency in Daphnia magna cultured in standard medium for toxicity testing: effects of the optimization of culture conditions on life history parameters of D. magna. Water Res. 24, 1157–1167. Gorr, T.A., Rider, C.V., Wang, H.Y., Olmstead, A.W., LeBlanc, G.A., 2006. A candidate juvenoid hormone receptor cis-element in the Daphnia magna hb2 hemoglobin gene promoter. Mol. Cell. Endocrinol. 247, 91–102. Hebert, P.D.N., 1978. The population biology of Daphnia (Crustacea, Daphnidae). Biol. Rev. 53, 387–426. Hobaek, A., Larsson, P., 1990. Sex determination in Daphnia magna. Ecology 71, 2255–2268. Ikuno, E., Matsumoto, T., Okubo, T., Itoi, S., Sugita, H., 2008. Difference in the sensitivity to chemical compounds between female and male neonates of Daphnia magna. Environ. Toxicol., in press, doi:10.1002/ tox.20403. Klein, B., 2000. Age as a factor influencing results in the acute daphnid test with Daphnia magna Straus. Water Res. 34, 1419–1424. Kleiven, O.T., Larsson, P., Hobaek, A., 1992. Sexual reproduction in Daphnia magna requires three stimuli. Oikos 65, 197–206. Laufer, H., Ahl, J.S.B., Sagi, A., 1993. The role of juvenile hormones in crustacean reproduction. Am. Zool. 33, 365–374. Oda, S., Tatarazako, N., Watanabe, H., Morita, M., Iguchi, T., 2005a. Production of male neonates in 4 cladoceran species exposed to a juvenile hormone analog, fenoxycarb. Chemosphere 60, 74–78. Oda, S., Tatarazako, N., Watanabe, H., Morita, M., Iguchi, T., 2005b. Production of male neonates in Daphnia magna (Cladocera, Crustacea) exposed to juvenile hormones and their analogs. Chemosphere 61, 1168–1174. Oda, S., Tatarazako, N., Watanabe, H., Morita, M., Iguchi, T., 2006. Genetic differences in the production of male neonates in Daphnia magna exposed to juvenile hormone analogs. Chemosphere 63, 1477– 1484. OECD, 1998. Daphnia magna reproduction test. In: OECD Guidelines for Testing of Chemicals, vol. 211. Organization for Economic Cooperation and Development, Paris. OECD, 2004. Daphnia sp., acute immobilization test. In: OECD Guidelines for Testing of Chemicals, vol. 202. Organization for Economic Cooperation and Development, Paris. Olmstead, A.W., LeBlanc, G.A., 2002. Juvenoid hormone methyl farnesoate is a sex determinant in the crustacean Daphnia magna. J. Exp. Zool. 293, 736–739. Olmstead, A.W., LeBlanc, G.A., 2003. Insecticidal juvenile hormone analogs stimulate the production of male offspring in the crustacean Daphnia magna. Environ. Health Perspect. 111, 919–924.
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