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Chronic effects of methylmercury on the reproduction of the teleost fish, Oryzias latipes Chan, Kenneth Ka-Sing 1977

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CHRONIC EFFECTS OF METHYLMERCURY ON THE REPRODUCTION OF THE TELEOST FISH, QRYZIAS LATIPES by KENNETH KA-SING CHAN B.Sc. Sir George Williams University, 1968 M.Sc. Sir George Williams University, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1977 © Kenneth Ka-Sing Chan, 1977 In present ing th is thes is in p a r t i a l fu l f i lment o f the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f r ee ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for s c h o l a r l y purposes may be granted by the Head of my Department or by his representa t ives . It is understood that copying or p u b l i c a t i o n of th is thes is for f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of ZOOLOGY The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e September 7.1977. i i ABSTRACT This study evaluates the toxicity, accumulation, chronic effects and mode of action of methylmercury on the reproduction of the teleost fish, Oryzias latipes. The median lethal concentration, 96h-LC50, for adult Oryzias was + 88+9.8 pg CH^Hg 11 as determined in a static system. Residue analysis by gas chromatography showed that fish exposed to 43 to 1000 ug CH^Hg+/l had tissue levels below 40 ug CH^Hg+/g while fish exposed to more than 1000 ug CH^Hg^/l accumulated methylmercury steadily and reached levels as high as 408.1 ug CH.jHg+/g. Death seems to occur when tissue level reaches 25 ug CH.jHg+/g. Studies on long-term exposure to 0.0, 4,3, 10.7 and 21.5 u of methylmercury in a flow-through system confirmed this observation. Four-hour exposure of 8.5 and 42.9 yg/1 of methylmercury on alternate days during the fish's normal oviposition period resulted in inhibition of oviposition. This observation occurred only oh days when fish were ex-posed to methylmercury but not on days when f i s h were returned to clean water. However, at a concentration of 85 Pg CH^ Hg+/1, complete inhibition was observed even on days when fish were returned to clean water. High rates of accumulation with low rates of excretion of methylmercury were suggested explanations for these observations. Six-week exposure to 4.3, 10.7 and 21.5 Vg/1 of methylmercury resulted in inhibition of spawning. This inhibition was directly related to the log of exposure concentrations. At the end of six weeks, both male and female gonads showed reduction in size; the females were more sensitive. However, hatchability of the spawned eggs was not affected by the exposure. i i i Juvenile fish were very sensitive to methylmercury. After two weeks of exposure, one-week old juvenile exposed to 0.0, 4.3, 10.7 and 21.5 ug CH 3Hg +/l had mortality rate of 2.2%, 54.3%, 64.9% and 99.4% respectively. Synthetic LH-RH, at concentrations of 100 and 1000 ng/g body weight, was effective in stimulating ovarian development in Oryzias. This shows that the LH-RH (synthesis based on structure of porcine LH-RH) has biolo-gical a c t i v i t y in Oryzias. When exposed to methylmercury, spawning act i v i t i e s were inhibited. LH injections were able to restore the spawning a c t i v i t i e s inhibited by the methylmercury treatment, but not LH*-RH. However, histology of the p i -tuitary gland showed stimulation of gonadotropic cells by LH-RH injection with no restoration of spawning a c t i v i t i e s . This suggests that methylmer-cury- may be blocking the release of gonadotropin. In vitro ovulation was affected by previous exposure to methylmercury. Addition of methylmercury directly to the incubation medium further reduced the percentage of i n vitro ovulation i n the previously treated f i s h . Using oocytes from untreated donor f i s h , the percent inhibition of in vitro ovu-lation by methylmercury was directly related to the log of doses used. A possible bioassay with in vitro ovulation was suggested. Among the various steroids used (progesterone, cortisone, estradiol and testosterone), c o r t i -sone was the only steroid effective i n restoring i n vitro ovulation blocked by the presence of methylmercury in the incubation medium. Ecological implications of these findings are: discussed. TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ACKNOWLEDGEMENTS GENERAL INTRODUCTION LITERATURE REVIEW SECTION I: THE TOXICITY AND ACCUMULATION OF METHYLMERCURY INTRODUCTION MATERIALS AND METHODS Maintenance of Fish Gravid Fish Sexually regressed fish Acute toxicity tests Long-term exposure Residue analysis Extraction and purification Gas chromatography RESULTS Acute toxicity tests Long-term exposure DISCUSSION SECTION II: CHRONIC EFFECTS OF METHYLMERCURY ON REPRODUCTION INTRODUCTION Table of Contents (cont'd) v Page MATERIALS AND METHODS 32 On oviposition .... 32 On gonadal development and spawning .... 33 On survival of juvenile fish .... 34 RESULTS .... 35 Effects on oviposition .... 35 Effects on gonadal development and spawning .... 35 Effects of juvenile fish .... 41 DISCUSSION .... 51 SECTION III: MODE OF ACTION OF METHYLMERCURY ON REPRODUCTION 54 INTRODUCTION 55 MATERIALS AND METHODS 61 On ovarian maturation by synthetic LH-RH .... 61 On effects of LH-RH and LH injection in methylmercury treated fish .... 62 On effects of methylmercury on i n vitro ovulation .... 63 RESULTS 66 Effect of Synthetic LH-RH on ovarian maturation .... 66 Effects of Synthetic LH-RH and LH injections on methylmercury treated f i s h .... 73 Effects of methylmercury on in vit r o ovulation .... 84 Pre-exposure to methylmercury for 6 weeks .... 84 Effects of methylmercury on normal oocytes from untreated fish .... 90 Effects of different steroids on methylmercury inhibited ovulation in vitro .... 90 DISCUSSION .... 96 Effect of synthetic LH-RH on ovarian maturation .... 96 Table of Contents (cont'd) v i Page Effects of synthetic LH-RH and LH injections on methly-mercury treated fish .... 99 Effect of methylmercury on in vitro ovulation .... 101 GENEEAL DISCUSSION 105 REFERENCES .... 109 LIST OF TABLES Mortality of Oryzias latipes exposed to different concentrations of methylmercury over a 6-week period Effects of 4-hour exposure to different concentrations of methylmercury on the oviposition of Oryzias latipes Mortality of one-week old juvenile Oryzias latipes exposed to different concentrations of methylmercury for two weeks Sta t i s t i c a l significance of female gonado-somatic indices among the various treatments . Effects of different concentrations of methyl-mercury on percent in vitro ovulation in Oryzias previously treated with methylmercury for 6 weeks The effects of various concentrations of methylmercury on ovulation in vitro of Oryzias latipes incubated with 10 ug/ml porcine LH at 1700 hours The effects of methylmercury on ovulation in vitro of Oryzias latipes incubated with and without LH. (10 yg/ml) commencing at 2200 hours The effects of steroids on ovulation in vitro of Oryzias latipes oocytes incubated with and without methylmercury at 2200 hours as the time of incubation LIST OF FIGURES FIGURE 1. Diagram of the experimental apparatus used for continuous exposure of fish to low con-centrations of methylmercury, showing the pattern of water flow, heating system, one Mariotte bottle for the metering of toxicant and one test-tank FIGURE 2. Effect of different concentrations of methyl-mercury on the LT50 of Oryzias latipes at 23 + 1°C. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. FIGURE 11. Determinations of the median lethal concen-tration (LC50) at 96 hr of methylmercury for Oryzias latipes Accumulation of methylmercury i n Oryzias  latipes exposed to different concentrations of methylmercury during the acute tests Accumulation of methylmercury i n Oryzias  latipes under long-exposure to different concentrations of methylmercury Weekly observations, from the 3rd to the 6th week, on spawning and egg-laying of Oryzias  latipes for the different concentrations of methylmercury Effects of different concentrations of methylmercury on the total number of spawn-ing and total number of eggs l a i d during six weeks of exposure The percent spawning activity of egg-laying a b i l i t y of Orvzias latipes exposed to differ-ent concentrations of methylmercury The percent inhibition of spawning in Oryzias latipes in water containing di f f e r -ent concentrations of methylmercury Gonadosomatic indices of male and female Orvzias latipes exposed to different con-centrations of methylmercury for 6 weeks The percent hatchability of eggs exposed to different concentrations of methylmer-cury List of Figures (cont'd) ix FIGURE 12. FIGURE 13. FIGURE 14. FIGURE 15. FIGURE 16. FIGURE 17. FIGURE 18. FIGURE 19. FIGURE 20. FIGURE 21. FIGURE 22. FIGURE 23. Diagram showing the interrelationship between environmental cues, hypothalamus, hypophysis, and gonads ... Effects of different dosages of synthetic LH-RH on the gonadosomatic index and the percent distribution of Class III oocytes Section of ovary from Oryzias injected with saline twice-a-week for 6 weeks at 2 3 + 1 C under 8L/16D Section of ovary from Oryzias injected with 10 ug/g synthetic LH-RH twice-a-week for 6 weeks at 23 + 1°C under 8L/16D Section of ovary from Oryzias injected with 100 ng/g synthetic LH-RH twice-a-week for 6 weeks at 23 + 1°C under 8L/16D Section of ovary from Oryzias injected with 1000 ng/g synthetic LH-RH twice-a-week for 6 weeks at 23 + 1°C under 8L/16D Section of pituitary gland from Oryzias i n -jected with saline twice-a-week for 6 weeks under 23 + 1°C and 8L/16D Section of pituitary gland from Oryzias i n -jected with 10 ng/g synthetic LH-RH twice-a-week for 6 weeks under 23 + 1 C and 8L/16D ... Section of pituitary gland from Oryzias i n -jected with 100 ng/g synthetic LH-RH twice-a-week for 6 weeks under 23 + 1 C and 8L/16D Section of pituitary gland from Oryzias i n -jected with 1000 ng/g synthetic LH-RH twice-a-week for 6 weeks under 23 + 1 C and 8L/16D Weekly observation on spawning and egg-laying of Oryzias for the various hormonal treatment in the presence of methylmercury Effects of various hormonal treatments on percent spawning, male and female gonadosoma-t i c indices at the end of 6-week methylmer-cury exposure Page 56 67 69 69 71 71 74 74 76 76 78 81 List of Figures (cont'd) x FIGURE 24. Section of pituitary gland from Oryzias exposed to clean water and injected with saline twice-a-week for 6 weeks under 23 + 1°C and 16L/8D 85 FIGURE 25. Section of pituitary gland from Oryzias exposed to methylmercury and injected with saline twice-a-week for 6 weeks under 23 + 1°C and 16L/8D ..... 85 FIGURE 26. Section of pituitary gland from Oryzias exposed to methylmercury and injected with luteinizing hormone twice-a-week for 6 weeks under 23 + 1°C and L6L/8D 87 FIGURE 27. Section of pituitary gland from Oryzias exposed to methylmercury and injected with luteinizing hormone-releasing hormone twice-a week for 6 weeks under 23 + 1°C and 16L/8D 87 FIGURE 28. Percent inhibition of in vitro ovulation by various concentrations of methylmercury in Incubation media at 1700 hours ..... 92 x i ACKNOWLEDGEMENTS I would like to extend my sincere thanks and indebtedness to my supervisor, Dr. W.S. Hoar, for his interest, advice and encouragement throughout this study. Special thanks are also extended to the members of my doctoral committee: Drs, P. Larkin, T. Northcote and H. Kasinsky of the Univer-s i t y of B r i t i s h Columbia, Vancouver, Dr. P. Oloffs of Simon Fraser University, Burnaby and Dr. J, Thompson of Marine Sciences Branch, Environment Canada, Victoria, for their generous assistance and for reading the thesis. The use of Dr. J. Thompson's laboratory for residue analysis i s deeply appreciated. I am obligated to the v i s i t i n g research fellows: Dr. T.J. Lam, Dr. S". Pandey and especially Dr. Y. Nagahama for many stimulating discus-sions. Helpful advice and encouragement from fellow graduate students: Dr. K. Khoo, Dr. N. Stacey, Dr. W. Marshall, Mr. J.-G. Godin, Mr. R. Neuman and Ms. M. Hurlburt, throughout the entire study are also deeply appreciated. Einancial support for this research was from the National Research Council of Canada through grants-in-aid to Dr. W.S. Hoar and a post-graduate scholarship from the National Research Council of Canada and a B.C. Summer Fellowship to myself. Last but not least, I would like to express my deepest gratitude to my wife, Bernadette, for her constant encouragement and patience through-out this study. 1 GENERAL INTRODUCTION Since the outbreak of Minamata Disease in Japan, methylmercury has been recognized as one of the most hazardous environmental pollutants (Katsuki et a l . , 1957, Takuomi, 1961; Takeuchi et a l . , 1962, Okinaka et a l . 1964). The sources of many contaminating incidences have been well documented (Fimreite 1970, Aaronson, 1971; Goldwater, 1971; Nelson 1971; Saha 1972) and the cycling of mercury through the environment has been re-viewed (Gavis and Ferguson 1972) . Poisoning by methylmercury has been demonstrated at a l l levels i n phylogeny (Skerfving 1972; Clarkson 1972; Katz 1972). Since the Minamata episode, studies have shown that f i s h are clearly sensitive to methyl-mercury (see Literature Review). Accumulation of methylmercury i n f i s h i s very high, sometimes as high as 5000 times over the environment (Johnels et_ a l . , 1967), while excretion i s very slow (Burrows and Krenkell 1973). This high accumulation i n f i s h probably affects the physiological processes of the animal. However, some information does exist on the long term, chronic effects of methylmercury i n f i s h ; but information of i t s effects on fish reproduction i s particularly sparse (Sprague 1971). The present work investigates the toxicity, accumulation of methyl-mercury under long and short term exposures and the effects of low concen-trations of methylmercury on oviposition, gonadal development, spawning -activity, hatchability of eggs and survival of hatchlings in Oryzias latipes. Since methylmercury has been shown to be a bioaccumulative toxicant, cor-relation w i l l be made between the amount of accumulated methylmercury in the fish and i t s effects on the reproductive processes, The latter part of this study w i l l examine the mode of action of methylmercury on the • .2 reproductive physiology i n Oryzias. The possible effects of methylmer-cury on the endocrine system concerned with reproduction w i l l be investi-gated. An attempt w i l l be made to determine whether a blockage of hor-mone acti v i t y by methylmercury occurs i n the hypothalamic-hypophyseal-gonadal axis. This study is not only of academic interest but might also suggest a possible remedy for f i s h that may be affected similarly i n na-ture. Oryzias is a much hardier fi s h than most other fishes that are of commercial value, like the salmon. However, this fish i s a convenient animal for laboratory studies on reproduction. It is hoped that the pre-sent study with Oryzias w i l l suggest some possible effects that methyl-mercury may have on commercially important fishes. Oryzias offers several advantages as an experimental animal. Its small size (30 mm adult) allows handling of large numbers in relatively limited spaces. By manipulating temperature and photoperiod, reproductive-ly mature individuals can be obtained throughout the year. Spawning, once induced, occurs daily for 4 to 6 weeks. Since Oryzias i s oviparous, the number of spawnings, number of spawned eggs, hatchability of the eggs and mortality of the hatchlings can be easily quantified. The spawning habits of this fish are also ideal for the study. Females ovulate daily between 010Q and 0400 hours and oviposition occurs about two hours after daybreak; and the eggs hatch in about two weeks. This not only allows studies of the effects of methylmercury on the reproductive physiology of the adults but also, i f desired, on the second generation. Finally, there i s much to be said for working with an animal which has been the subject of so much behavioural, physiological, genetical and endocrinological research. 3 LITERATURE REVIEW Levels of mercury In natural waters are generally low except i n contaminated areas (Voege 1971, Gavis and Ferguson 1972, Fitzgerald and Lyons 1972), In fish, mercury may reach levels above 25 yg/g when collected from contaminated areas while specimens from uncontaminated areas are generally below 1.0 yg/g , but frequently above 0.2 yg/g, the maximum natural background concentration (Fimreite and Reynolds 1973) . It appears that most of the mercury in fish is present as methylmercury (Westtfo 1969). The source of methylmercury has not been well determined, but i t has been observed that microorganisms, especially those from sedi-ments, can methylate mercury (Fagerstrom and Jernelov 1971; Jensen and Jernelov 1969; Wood et a l . , 1968). The methylmercury may then be accu-mulated to a 'harmful' concentration in the higher trophic levels, e.g., fi s h , via the food chain (Jernelov and Lann 1971). A maximum permissable level of mercury in fish of 0.5 yg/g was es-tablished by the U.S. Food and Drug Administration in 1970. At the same time people were cautioned that the human hazard depends largely on the quantity of contaminated fish eaten (Katz 1972). In mammals, tissue accumulation, distribution and excretion of methyl-mercury have been documented (Iverson et a l . , 1973; 1974; Casterline Jr. and Williams 1972; Skerfving 1974; Ikeda 1973). Effects of methylmercury in humans are well known. Methylmercury accumulates i n the central ner-vous system. Severe poisoning results in gross constriction of the visual f i e l d , cerebellar ataxia, dysarthria, sensory changes and impairment of hearing. Memory and intelligence are unaffected. These symptoms are generally irreversible but the motor disturbances may improve after 4 rehabilitation. Methylmercury has also the unique property of crossing the blood-brain barrier and the placental-foetal barrier (Clarkson 1972). Skerfving e_t a l . (1970) observed chromosome breakage i n leukocytes from humans with elevated blood mercury concentrations. Experimental studies •with other mammals have shown that methylmercury interacts with erythro-cytes (White and Rothstein 1973; Mykkanen and Ganther 1974), damages l i v e r (Chang and Yamaguchi 1974; Desnoyers and Chang 1975a, b; Lucier et a l . , 1972), kidney (Hirsch 1971, Chang and Sprecher 1976; Fowler e_t a l . , 1974) and the central nervous system (Kim 1971, Herigstad et a l . , 1972, Albanus et a l . , 1972; Ikeda et a l . , 1973; Diamond and Sleight 1972; Barthoud et a l . , 1976; Berlin et a l . , 1975); i t also affects behaviour (Spyker et a l . , 1972; Hughes 1975) and reproduction (Casterline Jr. and Williams 1972, Khera 1973; Skerfving 1974). In birds, methylmercury poisoning impaired reproduction in the hen (Tejning 1967) and mallard duck (Heinz 1974), and the hatchability of hen eggs (Tejning 1967). Unlike mercuric chloride (Stoewsand e_t a l . , 1971), eggshell thinning was not observed in the methylmercury treated ring dove, American kestrel (Peakdall and Liner 1972) and mallard duck (Heinz 1974). However, methylmercury was shown to be both embro-lethal and teratogenic to early chick embryogenesis (Gilani 1975) and also to affect mallard duckling behaviour (Heinz 1975). Very l i t t l e work has been done on the effects of toxicants on fi s h reproduction ( Spraque 1971). Crandall and Goodnight (1962) observed a delay in sexual maturity in the guppy by lead and zinc. Inhibition of spawning by copper was detected in fathead minnows (Mount 1968). Also, near complete elimination of egg production by zinc was found i n fathead 5 minnows (Brungs 1969). Resistance of fis h eggs and fry to toxicants, e.g., detergents, zinc, copper and malathion, has been studied and i t seems that eggs are less sensitive to toxicants than fry (Sprague 1971). Methylmercury uptake, distribution and excretion rate have been well studied in fish because of i t s potential hazard to the fis h eating population (JarvenpHH et a l . , 1970; Olson and Fromm 1973; Miettinen ejt a l . , 1969; Olson et a l . , 1973; Freeman and Home 1973; Rucher and Amend 1969; Burrows 1973). Other studies of methylmercury in f i s h include i t s toxicity (Akiyama 1970) , i t s effects on g i l l metabolism (O'Connor and Fromm 1975; Kendall 1972), blood parameters and erythrocytes (O'Connor and Fromm 1975; Olson and Fromm 1973), kidney (Kendall 1972; Matsumura e_t a l . , 1975) and f e r t i l i z a t i o n of eggs (Mclntyre 1973). Generally, stu-dies on effects of toxicants on fis h reproduction has been sparse (Sprague 1971). For organic mercury, the work of Kihlstrtfrn and his co-workers remains a unique piece of information on the sublethal effects of phenylmercury on fish reproduction (Kihlstrtfm, Lundberg and Hulth 1972; KihlstrHm and Hulth 1972) . 6 SECTION I THE TOXICITY AND ACCUMULATION OF METHYLMERCURY 7 INTRODUCTION Mercury and many of Its compounds have>long been known to be highly ; toxic to both plants and animals. The main sources of mercury contamina-tion in the environment are from industries such as mining, pulp and paper, the manufacture and use of fungicide, plastic and chlor-alkali. Incidences of mercury poisoning and sources of mercury contamination have been well documented (Fimrelte 1970; Aaronson 1971; Goldwater 1971; Nelson 1971; Saha 1972). The cycling of mercury through the environment has been reviewed (Gavis and Ferguson 1972). Though occurring in several forms, mercury in fish i s mainly present as methylmercury (Westoo 1969). This i s because methylmercury i s readily absorbed (ability to cross c e l l membranes), i s relatively resistant to biotransformation, has a low clearance rate and a strong a f f i n i t y for pro-tein (Clarkson 1972). The source of methylmercury has not been well deter-mined, but i t has been observed that microorganisms,especially those in sediments, can methylate mercury (Fagerstrom and JernelBv 1971, Jensen and JernelOv 1964; Wood et a l . , 1968). This methylmercury may then be accumu-lated v i a the food chain to a harmful concentration i n the higher trophic levels, e.g., fish and ultimately man (JernelbV and Lann 1971). Thus, i t is worthwhile to investigate the possible effects, both lethal and sub-lethal, of methylmercury in fish. In this section, the toxicity of methylmercury and i t s accumulation r in the tissues of the adult freshwater teleost Oryzias latipes are inves-tigated. 8 MATERIALS AND METHODS Maintenance of Fish Adult medaka, Oryzias latipes were obtained via air-shipment from Nagoya Aquarium Company, Nagoya City, Japan. Following a r r i v a l , the fish were distributed into 35-litre capacity plastic tanks equipped with side f i l t e r s and aerated with compressed a i r . Dechlorinated Vancouver City water was used (hardness 2-10 mg CaCO^/l, pH 6.8-7,0). Temperature of the water was maintained at 13 + 1°C by standing the tanks in a trough of running cold water (10 + 1°C). These fish were exposed to short photo-periods of eight hours light (0900 hr to 1700 hr) alternating with six-teen hours darkness (8L/16D). Light was provided by fluorescent lamps suspended 60 cm above the tanks and the photoperiod regulated by time clocks (Intermatic, Marr Electric Ltd., Toronto). Fish were fed daily ad libitum with frozen brine shrimp. A l l Imported fi s h were subjected to the above conditions for at least a month before they were slowly adap-ted to different conditions depending on the various experiments. Gravid Fish After this i n i t i a l treatment with low temperature and short photo-period for a month, a group of medaka was slowly adapted to and kept at a warm temperature (23 + 1°C) and under long photoperiod (16L/8D). After about three weeks of such treatment most of the f i s h become gravid and spawning occurs i n the fourth week. The cold temperature and short photo-period pretreatment allows the fish to pass out of i t s refractory stage while the warm temperature and long photoperiod induces gonadal develop-ment (Yoshioka 1962, 1963). Fish treated in this manner were used in' acute toxicity tests and studies of oviposition. 9 Sexually regressed fi s h For studying gonadal development, f i s h should be sexually regressed at the beginning of the experiment, Yoshioka (1962, 1963) observed that treatment with low temperature (4-15°C) and short photoperiod (8L/16D) was effective in inducing Oryzias latipes to a sexually regressed state. After this treatment, fi s h would have passed i t s refractory period and can easily be brought to spawning state with warm temperature (18-23°C) and long photoperiod (16L/8D). In the present study, treatment of low temperature (13 + 1°C) and short photoperiod (8L/16D) for two months was used to ensure that fi s h were sexually regressed. Fish i n the regressed state were used i n a l l long term exposure studies. Acute toxicity tests Toxicity tests were st a t i c , conducted i n 18-litre capacity glass aquaria with 10 fish per tank. The f i s h ranged from 2.6 to 3.0 cm in length and weighed 0.25 to 0.35 g; this resulted i n a loading density of 6 l i t r e s test solution per gram of f i s h . Test water was dechlorinated Vancouver City water warmed to 23 + 1°C. Fish were acclimated to the test conditions for three days and fed daily with frozen brine shrimp. The test period began on Day 4 when methylmercuric chloride was f i r s t added to the tanks and feeding discontinued. Observations were continued for seven days and death was defined as a permanent cessation of opercular movement. A l l fish were frozen immediately for residue analysis. A stock solution of methylmercuric chloride (1.71 g CH^Hg+/l) was prepared by dissolving the proper amount of chemical in d i s t i l l e d water. Appropriate volumes of this stock solution were added to the different aquaria to achieve the desired concentrations. To maintain proper concen-10 tration in the tanks, about 90% of the solution in each tank was renewed daily. At each concentration, the cumulative percentage mortality was plotted on a probit scale against the survival time in hours on a logarith-mic scale, and the median lethal time (LT50) and i t s 95 percent confidence limits estimated (Litchfield, 1949). From the eye-fitted regression of log LT50 against log concentration (Litchfield 1949) and by plotting the partial mortality at 96 hr (Litchfield and Wilcoxon 1949), the con-centration required to produce 50 percent mortality (LC50) and i t s 95% confidence limits at 96 hr were calculated. Long-term exposure Adult medaka weighing 0.25-0.35 g were divided into four groups with 35 fi s h per group. Each group was exposed i n a flow-through system to one of four different concentrations of methylmercury (0, 4.3, 10.7 and 21.5 yg CH 3Hg +/l) at 23 + 1°C under 16L:8D light-dark regime. The loading density i n the tanks was approximately one l i t r e of test solution per gram fish and the approximate time for 99% replacement of test solution was 3.5 hours. Fish were fed daily with frozen brine shrimp. Mortality was checked each day and fish were sampled at 0, 2, 4 and 6 weeks for methyl-mercury residue determination. The flow-through system is ill u s t r a t e d i n Fig. 1. Dechlorinated Vancouver City water was f i r s t heated to approximately 18°C by stainless steel coils carrying flowing hot water. The 18°C water was then pumped to a head tank and further heated to and maintained at 23 + 1°C with two 1000-watt stainless steel immersion heaters. The warmed water was then delivered at a constant rate (regulated by flowmeters; Jencons, England) 11 Figure 1. Diagram of the experimental apparatus used for con-tinuous exposure of f i s h to low concentrations of methylmercury, showing the pattern of water flow, heating system, one Mariotte bottle for the metering of toxicant and one test-tank. 12 !3 to mixing buckets situated on top of the test-tanks. The methylmer-curic chloride solutions were metered into the mixing buckets by Mariotte bottles (Leduc 1966). The mixed solution of water and methyl-mercury entered the different test-tanks by gravity. The f i n a l concen-trations in the test-tanks were determined by the concentrations of methylmercuric chloride solution in the Mariotte bottles. Test-tanks used were of translucent, white, polyethylene equipped with plastic outlets (2 cm I.D.) on one side of the tank positioned to maintain a water volume of about 9 l i t r e s . The outlets were f i t t e d with fibreglass screens to prevent fish from escaping. There were four of the above set-ups for different methylmercury chloride concentrations of 0.0, 4.3, 10.7 and 21.5 yg CH 3Hg +/l. Residue analysis Extraction and purification This is a modification of the extraction method developed by Newsome in 1971. Since weight of individual f i s h was small (0.2 to 0.3 g per f i s h ) , f i s h samples were pooled (approximately 1.5 to 2.5 g per determina-tion) and homogenized in a Sorval homogenizer for 15 min with a solution of 1 N hydrobromic acid and 2.1 N potassium bromide (40 ml). The homogen-ate was f i l t e r e d through glass wool under gravity on a Buchner funnel and washed with a further portion (40 ml) of hydrobromic acid-potassium bro-mide solution. The f i l t r a t e s were pooled and extracted three times (3 x 50 ml) with benzene (nanograde, Caledon). The benzene layers were combined and a portion (100 ml) of this combined benzene layer was then extracted twice ( 2 x 6 ml) with freshly prepared cysteine acetate solu-14 tion (2.0 g cysteine hydro-chloride monhydrate, 4.0 g sodium acetate and 12.5 g anhydrous sodium sulphate in 100 ml d i s t i l l e d water). An aliquot (8 ml) of the combined cysteine layer was recovered and a c i d i -fied with 1 ml of 48% hydrobromic acid. The mercury was then extracted with benzene (either 5 ml or 9 ml) and the benzene extract subjected to analysis by gas chromatography. 1 Gas chromatography. Gas chromatography was performed on a Hewlett-Packard Model 7620 63 Gas Chromatograph f i t t e d with a Ni f o i l electron capture detector. The glass column was 182 cm X 4 mm and packed with 10% DEGS (diethylene glycol succinate) on 80-100 mesh Chromosorb W. For the determination of methylmercury, typical operating temperatures were: injection port 200°C, oven 170°C and detector 200°C. The carrier gas was argon-methane (95:5); gas flow was 60 ml/min at 40 psi. Under these conditions methylmercury had a retention time of 150 sec. Samples and standard were injected as benzene solutions i n 2,0 ul aliquots. Standards were run daily with each batch of samples and a calibration curve was plotted for each individual batch of samples. The concentra-tion of methylmercury in the samples were calculated as ug CH3Hg+/g based on fish wet weight. 15 ' RESULTS Nine spiking tests were performed during the entire period of fi s h tissue analysis and the calculated percent recovery varied be-tween 83% to 89% with an average of 86.5 +0.8%. This factor (86.5%) was used to adjust the residual methylmercury (CH^Hg+) concentration in the fish samples accordingly. Acute toxicity tests In the acute toxicity tests, no mortality occurred i n the control group and i t was concluded that any mortality observed i n the treated groups was due to the effects of the methylmercury. Results are shown in Fig. 2 to 4, Fish exposed to lower than 80 ug CH 3Hg +/l survived for more than four days (Fig. 2) . The median lethal concentration, 96 hr-LC50, of methylmercury for medaka i s 88+9.8 ug CH 3Hg +/l (Fig. 3). Levels of methylmercury accumulated in fish exposed to 43 to 1000 yg CH 3Hg +/l varied but remained below 40 yg CH3Hg+/g (Fig. 4) . When fish were exposed to concentrations higher than 1000 yg CH^ Hg"1"/! the amount of methylmercury accumulated in the tissue increased steadily + + and reached as high as 408.1 yg CH^ Hg /g as in the case of 42900 yg CH3H A exposure (Fig. 4). Long-term exposure Results are shown in Table 1 and Fig. 5. No mortality was observed in the control group during the entire six weeks of exposure. Fish ex-posed to 4.3 and 10.7 yg CH 3Hg +/l had one death each during the 4th week and the 2nd week respectively (Table 1). At the highest concentration (21.5 yg CH3Hg / l ) , the fish began dying during the second week and mor-t a l i t y reached a peak during the fourth week of exposure when over 83% 16 Figure 2. Effects of different concentrations of methylmercury on the LT50 of Oryzias latipes at 23 + 1°C. Values reported are mean and 95% confidence limits. >- 100 50 cc O ^ 10 o 5 in ^ 1 x: LU 05 0.1 96 hrs J l_i i — l I I 11 III ' i i 1 m i l i i i l . i i . Q01 QQ5 Ol Q5 1 5 10 5 0 100 EXPOSURE CONCENTRATION x 1 0 3 u g C H 3 H g + / l 18 Figure 3. Determination of the median lethal concentration (LC50) at 96 hr of methylmercury for Oryzias  latipes. 19 10 20 4 0 60 80100 200 CONCENTRATION Lig C H 3 H g ; / 20 Figure 4. Accumulation of methylmercury in Oryzias latipes exposed to different concentrations of methylmer-cury during the acute tests (each value represents the average of two determinations on groups of five f i s h ) . 1000 _ 500 — C D I • cn I <J o> 100 — -=k -UJ 50 in Q u 1 CO 3 1 UJ sz CD | ^ 10 ^ 5 — CO CO !— 1 Im LLL JL_L LLL 11 in 0.01 Q05 Q1 05 1 EXPOSURE CONCENTRATION 10 50 100 x 1 0 J ug C H 3 H g + / l M 22 Figure 5. Accumulation of methylmercury i n Oryzias latipes under long-exposure to different concentrations of methylmercury. Each point represents the mean of two methylmercury determinations on groups of five live f i s h . • 21.5 C H 3 H g + / | O 10.7 A 4.3 EXPOSURE TIME (wks) r o (A) Table 1. Mortality of Oryzias latipes exposed to different con-centrations of methylmercury over a 6-week period (35 fish in each concentration at start of test). Exposure concentration (yg CH3Hg+/l) 1st wk. 2nd wk. 3rd wk. 4th wk. 5th wk. 6th wk. 0 0 0 0 0 0 0 4.3 0 0 0 1 0 0 10.7 0 1 0 0 0 0 21.5 0 1 7 21 3 0 -P-25 of the fish had died. At the end of the exposure, only three out of the i n i t i a l 35 fish were alive from this group (Table 1). Tissue methylmercury level of the control remained below 1 yg CH3Hg+/g (Fig. 5). Both of the 4.3 and 10.7 yg CH 3Hg +/l exposed groups accumulated methylmercury steadily during the entire exposure and reached levels of 19.4 and 30.7 yg CH3Hg+/g respectively at the end of the exposure (Fig. 5). A high, probably lethal, level of methyl-4-mercury (67.1 yg CH3Hg /g) was accumulated within two weeks in fis h exposed to 21.5 yg CH 3Hg +/l; during this period of two weeks, the fish started dying (Fig. 5 and Table 1). At the end of the fourth week over 83% of the fis h from this group were dead (Table 1) and the tissue methylmercury level was 63.4 yg/g. 26 DISCUSSION The present study has shown that methylmercury is toxic to the madaka (the 96h-LC50 was 88 + 9.8 yg CH 3Hg +/l). This value i s high compared to the 10 yg H g ^ / l (96h-LC50) for rainbow trout (Lock 1974) . Thus, the medaka i s at least eight times more tolerant to methylmer-cury than the rainbow trout; salmonids have frequently been observed to be more sensitive to toxicants than many other fishes (Jones 1964). At concentrations above 1000 yg CH^ Hg / l , the rate of mercury ac-cumulation increases steadily suggesting possible damage to the c e l l membranes, especially in g i l l epithelial cells (Rucker and Amend 1969), which would allow a sharp increase i n uptake of methylmercury. A l -though the effect of methylmercury at the molecular level has not been completely elucidated, i t is apparent that this compound has a strong a f f i n i t y for sulphur, particularly for the sulfhydryl group (-SH) in proteins (Saba 1972). This chemical binding of methylmercury to pro-teins in c e l l membrane may alter the distribution of ions, change elec-t r i c potential and thus interfere with movement of fluids across the membrane (Passow et^ a l . , 1961). Methylmercury enters the fis h mainly through the g i l l s , although some may be absorbed through the skin (Olson et a l , , 1973); once inside the fi s h , i t binds tightly with the sulfhy-dryl group in proteins, causing a steady increase of methylmercury ac-cumulated. At concentrations below 1000 yg CH.jHg+/l, accumulation of methylmercury appears variable. Death seems to occur in the medaka when 27 the tissue level of methylmercury reaches 25 yg CH.jHg+/g (Fig. 4) . This value i s quite high when compared with levels found in fish from uncontaminated areas or from salt water (Fimreite and Reynolds 1973; Peterson et a l . , 1973; Childs and Gaffke 1973). However, i t is not uncommon for fi s h from contaminated areas like Clay Lake, Ontario, to reach levels as high as 20 yg/g of mercury (Fimreite and Reynolds 1973). Unlike many other chemicals, methylmercury can be accumulated to a lethal level in fish exposed to a low concentration for long duration. In the acute toxicity study, medaka were observed to survive over a week i n 75 yg/1 methylmercury solution; i n the long-term study, i t was observed that fish exposed to 21.5 yg CH,jHg+/l solution accumulated a lethal level of methylmercury (over 25 yg CH.jHg+/g body weight) and started dying after the second week of exposure. From the foregoing, i t can be seen that the toxicity and effects of methylmercury depend not only on the exposure concentration but also on the amount of toxicant the fish accumulates during the exposure. This raises serious doubts about the r e l i a b i l i t y of acute toxicity tests for bioaccumulative chemicals like methylmercury. The use of 96h-LC50 for non-accumulative toxins would be valid but not for bioaccumu-lative chemicals like most pesticides (Sprague 1969). The definition of an acceptable sublethal concentration for bioaccumulative toxin can-not be some concentration below or a particular fraction of the 96h-LC50. Sublethal concentrations, where uptake of toxin equals i t s excretion without causing death, cannot be regarded as 'safe' concentration be-28 cause sublethal effects may occur at relatively low tissue levels. To solve this particular problem, residue determinations w i l l have to be done concurrently with toxicity studies. "Safe" levels which could be much lower than the sublethal concentration, w i l l have to be deter-mined with long-term, continuous-flow experiments. We have only par-t i a l l y accomplished this and found that for medaka, the upper sublethal concentration of methylmercury l i e s between 10.7 and 21.5 yg CH.jHg+/l. For chemicals that accumulate i n tissues, exposure to sublethal concentration of such chemicals w i l l have long-lasting effects. Re-lease of such chemicals into the environment, even i n minute quantities, w i l l ultimately bring about drastic changes i n the organisms that come in contact with them. Section II examines the effects of long-term exposure to low levels of methylmercury on the reproductive physiology in the medaka. SECTION II CHRONIC EFFECTS OF METHYLMERCURY ON REPRODUCTION 30 INTRODUCTION Very l i t t l e work has been done on the effects of aquatic pollut-ants on fish reproduction (Sprague 1971). Crandall and Goodnight (1962) observed a delay in sexual maturity of guppy treated with lead and zinc. Mount (1968) reported inhibition of spawning of fathead minnows treated with copper, while Brungs (1969) found almost complete elimination of egg production by zinc. Fish eggs appear to be less sensitive than fry to toxicants such as detergents, zinc, copper, and malathion (Sprague 1971). A decrease i n number of eggs and hatched young by phenylmercurie acetate at concentrations as low as 1.0 ug/1 was observed i n zebrafish (Kihlstrom et a l . , 1971). Decreased egg production was attributed to the a f f i n i t y of mercurials for sulfhydryl groups thus inhibiting mitosis, enzyme reactions and protein hormones necessary for egg production and egg laying. In another study by KihlstrHm and Hulth (1972) , increased frequency of hatching of zebrafish eggs was reported i n water containing 10 to 20 ng/g phenylmercuric acetate; these workers suggested that this was due to a decrease i n ef-fects of microorganisms upon the developing eggs and also that low con-centrations of mercuric compound may "stimulate" hatchability. Kihlstrdm's work remains a unique piece of information pertaining to the effects of organic mercury on fish reproduction. However, there are s t i l l many unanswered questions concerning the effects of mercury on f i s h , especially on their reproduction and the mechanism behind these effects. 31 This section investigates the effects of low concentrations of methylmercury on oviposition,,gonadal development, spawning ac-t i v i t y , hatchability of eggs and survival of juveniles of Oryzias  latipes. 32 MATERIALS AND METHODS On oviposition Spawning fish were used i n this experiment. To obtain actively spawning f i s h , Oryzias were maintained i n dechlorinated Vancouver City water for over a month at 23 + 1°C under long photoperiod (16L/8D) with the light period between 0800 and 2400 hours. The experiments were performed with ten 18-litre capacity glass aquaria divided into two series. The f i r s t series consisted of five tanks, each holding a suspended cage (dimension 36 x 20 x 26 cm) made of: fibreglass screening. These cages allowed rapid transfer of fis h from one aquarium to another with minimum disturbance. The second ser-ies; of five tanks contained various concentrations of methylmercury (Q)„0, 4.3, 8.58, 42.9 and 85.8 pg CH 3Hg +/l) . Twenty-five pairs of spawning medaka were chosen from the warm temperature and long photoperiod group (see Section I ) . They were dis-tributed, 5 males and 5 females per tank, into the cages of the f i r s t series of five aquaria. In the morning before the light came on, the caged f i s h were rapidly transferred to freshly prepared solution of methylmercury (0.0, 4.3, 8.58, 42.9 and 85.8 yg CH 3Hg +/l) in the second series of five tanks. Spawning was checked at hourly intervals u n t i l noon at which time the fis h were returned to clean water in the f i r s t series of five tanks and observation continued for another four hours. Fish were fed daily with frozen brine shrimp during the afternoon while the f i s h were in clean water. The fish were exposed to methylmercury on alternate days only; on other days, the cages were agitated 33 mechanically to simulate the transfer. Spawning was checked each day. Spawning activity was recorded as number of females with eggs attached (number of spawning) and the total number of eggs collected i n one tank. On gonadal development and spawning A flow through system as described i n Section I was employed in this part of the experiment. Fish used had regressed gonads since they had been exposed to low temperature (13 + 1°C) and short photoperiod (8L/16D) for over two months. These fish were allowed to warm to room temperature (approximately 23°C) overnight and on the next day were dis-tributed to four test-tanks at the rate of 14 males and 20 females per tank, resulting in a loading density of approximately one l i t r e of test solution per gram f i s h . The acclimation period consisted of four days with running warm water (23 + 1°C) and long photoperiod (16L/8D, 800 to 2400 hours) but no methylmercuric chloride added. On the f i f t h day, methylmercuric chloride solutions were metered into the test-tanks and the desired concentrations (0.0, 4,3, 10.7 and 21.5 yg CH 3Hg +/l) reached i n about 4 hr; the approximate time for 99% replacement of test solution was 3.5 hr (Sprague 1969). Fish were fed daily with frozen brine shrimp. Spawning activity was recorded as number of females with eggs attached and number of eggs collected each day. Observations were continued for six weeks. The eggs collected were allowed to hatch i n disposable plastic petri dishes containing the same solutions from which they were collected. At the end of six weeks, the fish were k i l l e d , weighed, measured and the gonadosomatic indices calculated by the following formula: 34 wt. of gonad Gonadosomatic Index = X 100 wt. of fish There were two experiments performed and since both of them showed similar results, the data were pooled. On survival of juvenile fish To raise juveniles to maturity in different concentrations of methylmercury, one-week-old juvenile medaka were collected from un-treated parents and distributed, approximately 50 to 80 fis h per tank, into four test-tanks containing 0.0, 4.3, 10.7, and 21.5 yg CH.jHg+/l. However, high mortality occurred i n a l l treated groups within the f i r s t week and consequently the experiment was terminated i n two weeks. Duplicate experiments were performed. Both experiments showed similar results and the data were pooled. 35 RESULTS Effects on oviposition Control fi s h and fish experiencing 4-hr exposure of 4.3 yg CH.jHg+/l on alternate days consistently showed over 60% of oviposition activity (Table 2). At a concentration of 8.58 yg CH^Hg*/! the number of spawn-ings was reduced whenever the fi s h were exposed to the toxic solution; however, spawning was unaffected on the following day when f i s h were returned to clean water. A similar but more marked effect was observed In the group exposed to 42.9 yg CH^Hg / l . Exposure to the highest con-centration, 85 yg CH^Hg^/l, not only prohibited spawning completely (ex-cept for Day 3) but also affected spawning the next day i n clean water. There seems to be an increased reduction of spawning in clean water as this group experienced more exposure of the toxic solution. Effects on gonadal development arid spawning No sign of spawning was observed during the f i r s t two weeks of ex-posure. Hence Figure 6 reports only spawning activity during the last four weeks of exposure. The f i s h fed normally but there was mortality i n the 21.5 yg CH^ Hg"*"/! exposed group; two fi s h and six f i s h died dur-ing the f i r s t and second week, respectively. During the third week of exposure spawning was observed i n a l l groups while mortality was ob-served only at the highest concentration. During the 3rd, 4th, 5th and 6th week of exposure, treated groups showed a consistent reduction of spawning activity (Fig. 6); also there seemed to be some relationship be-tween the degree of reduction of spawning activity and the concentrations of .methylmercury. Figure 7 shows an inverse linear relationship between Table 2. Effects of 4-hour exposure to different concentrations of methyl-mercury on the oviposition of Oryzias latipes. Five males and five females per tank. Positive treatment represents presence of the 4 -hour exposure to the toxic solution on that day; negative treatment represents no such exposure. Numbers without brackets represent number of spawning females while the total number of eggs laid were stated within brackets. Day Treatment Control 4.3 Concentration yg 8.58 CH Hg+/1 J 42.9 85.8 1 - 5(62) 5(76) 5(59) 5(65) 5(69) 2 - 4(44) 4(49) 3(31) 4(41) 4(53) 3 + 3(35) 3(35) 2(20) 0(0) 1(2) 4 - 3(30) 3(56) 3(31) 3(29) 4(30) 5 + 4(42) 4(62) 2(23) K 9 ) 0(0) 6 - 3(37) 4(57) 3(27) 4(38) 3(27) 7 + 4(39) 4(48) 1(11) K5) 0(0) 8 - 5(43) 3(40) 4(52) 3(37) 2(15) 9 + 5(53) 3(42) 1(8) 0(0) 0(0) 10 — 4(38) 4(50) 4(43) 3(35) 1(8) CO ON 37 Figure 6. Weekly observations, from the 3rd to the 6th week, on spawning and egg'-laying of Oryzias latipes for the different concentrations of methylmercury. Numbers in brackets represent number of fish i n each group at the end of the week. 600 500 400 300 200 100 0 i 500 W 400 (J) 300 LU 200 ^ 100 0 300 200 100 0 300 200 100 0 r <6&) o o (68) (68) (68) (67) • Net of EGGS U N a o f SPAWNING! (68) (68) (66) (64) (55) (46) (37) (24) QO 43 10.7 21.5 CONCENTRATION ug CiHjHg*/ I 120 38 100 80 60 40 20 0 CO 100 (T) 80 £z 60 40 <t CL 2 0 CO o 60 40 20 0 60 40 20 0 O o 39 Figure 7. Effects of different concentrations of methylmercury on the total number of spawning and total number of eggs l a i d during six weeks of exposure. 40 O T — X 28f-8) 16 o 12 2 al o o 1 O ^ 24 X (/> 2 0 O S 1 ( S d> o O 12 8 < O 0 i 1 I I I I I I 3 4 5 6 78910 1 20 30 0 1 2 3 4 5 6 78910 20 20 CONCENTRATIONS jug C H 3 H g + / I 41 the total number of spawnings (number of females with eggs attached) as well as the total number of eggs collected over the four weeks and the log exposure concentrations. Considering the control as 100%, during the four weeks of observation, egg-laying a b i l i t y and spawning activity for fish exposed to 4.3, 10.7 and 21.5 yg CH^ Hg fl were re-spectively 58%, 49% and 10% (Fig. 8), When transformed to percent i n -hibition and plotted against log exposure concentrations, these values showed a direct linear relationship (Fig. 9). At the end of six weeks the gonadosomatic indices of both males and females showed reduction i n relation to the increase i n exposure concentration; females were more sensitive than males (Fig. 10). Spawned eggs were incubated in the media where they were collected; hatchability of these eggs bears no relationship to the concentrations of methylmercury previously used (Fig. 11). The percent hatchability for the four groups (0.0, 4.3, 10.7 and 21.5 yg CH.jHg+/l) did not deviate from the control and varied between 55% and 68%. Effects on juvenile f i s h Mortality occurred in a l l groups, treated and untreated. During the two weeks of exposure, 1-week old juveniles exposed to 0.0, 4.3, 10.7 and 21.5 yg CH 3Hg +/l had mortality rates of 2.2%, 54.3%, 64.9% and 99.4% respectively (Table 3). This shows a high sensitivity of juvenile medaka to methylmercury toxicity. 42 Figure 8. The percent spawning activity and egg-laying a b i l i t y of Oryzias latipes exposed to d i f f e r -ent concentrations of methylmercury. Control i s 100 percent. 43 • EGG-LAYING 0 . 0 4 . 8 1 0 . 7 2 1 . 5 CONCENTRATION j jg C H 3 H g + / l 44 Figure 9. The percent inhibition of spawning in Oryzias  latipes in water containing different concen-trations of methylmercury. 45 100 8 0 O h-— 6 0 CQ 4 0 2 0 -o 1 A <b A A j 1 — i i i i i 11 2 3 4 5 678910 20 CONCENTRATION MQ C H 3 H g + /1 30 46 Figure 10. Gonadosomatic indices of male and female Oryzias latipes exposed to different con-centrations of methylmercury for 6 weeks. Numbers in brackets represent sample size; Asterisk, s t a t i s t i c a l significance (t-test, P <0.05) compared to control. 47 • MALE FEMALE 1 0 - 0 4 . 8 1 0 . 7 2 1 . 5 CONCENTRATION jug C H 3 H g + / l 1 4 1 2 CO 10 O 8 0 LU < UJ 48 Figure 11. The percent hatchability of eggs exposed to different concentrations of methylmercury. 49 r 100 1— _ l 8 0 CD 6 0 < 4 0 2 0 < 0 I E o o Q O 4 . 8 1 0 . 7 2 1 . 5 C O N C E N T R A T I O N )jg C H 3 H g + / | 50 Table 3. Mortality of one-week old juvenile Oryzias latipes exposed to different concentrations of methylmercury for two weeks. Concentration (Ug CH3Hg+/l 0.0 4.3 10.7 21.5 Actual Mortality 3/134 57/105 100/154 157/158 % Mortality 2.24 54.28 64.93 99.37 51 DISCUSSION Under normal conditions, female Oryzias ovulate between 0100 and 0400 hr; in the presence of a male oviposition takes place on the same day, soon after daybreak. In the present investigation, medaka were exposed to different concentrations of methylmercury during their normal period of oviposition, i.e., between 0800 and 1200 hr (light period, 0800 to 2400 hr). Control fish and f i s h ex-posed to 4.3 yg CH^ Hg / l were not affected but exposure to a higher concentration of 8,58 yg CH^ Hg"1"/! reduced the number of spawning fe-males; on return to clean water normal oviposition occurred the next day. This suggests that the concentration of 8.58 yg/1 methylmercury was strong enough to induce a stress that reduced the number of spawn-ing females. Although there may be some accumulation of methylmercury this seems to have been par t i a l l y excreted to a level whereby normal spawning could be achieved on the next day. This concentration (8.58 yg CH3Hg+/l) i s approximately one-tenth of the 96h-LC50 dose (88 + 9,8 yg CH 3Hg +/l, Section 1). Similar results were observed at a higher concentration, 42.9 yg CH^ Hg"*"/!, with further reduction in number of spawning females. However, at the highest concentration, 85.8 yg CH^ Hg"1"/!, oviposition was abolished after the third day, and this effect was evi-dent on subsequent days when the fi s h were in clean water. Thus, the 4-hour exposure to this high concentration resulted in an accumulation of methylmercury that blocked spawning. A similar phenomenon i s pro-bably true for a l l bioaccumulative toxins where accumulation is faster than the excretion of the toxin. 52 Kihlstrtfm et a l . (1971) observed a decrease In number of zebra-f i s h eggs in water containing 1 ng or more phenylmercuric acetate per gram of water. In the present investigation using a similar range of concentrations of toxicant as Klhlstro'm's, similar results were ob-served in Oryzias latipes exposed continuously to methylmercury for six weeks. Spawning occurred during the third week of exposure and continued u n t i l the end of the experiment. The percent inhibition of spawning for f i s h exposed to 4.3, 10.7 and 21.5 yg CH 3Hg +/l was 42, 48 and 88% respectively. Both spawning activity and egg-laying a b i l i t y were related to the log of exposure concentrations. Unlike that of zebrafish (KihlstrHm and Hulth 1972), hatchability of Oryzias eggs from treated parents was not affected by the exposure to methylmercury. One-week old fi s h were especially sensitive to the toxicant. This i s often true for other species of fi s h (Sprague 1971) . In rainbow trout, exposure to methylmercury for up to 12 weeks (10 yg Hg^/1) did not significantly affect the in vitro metabolism of the g i l l or the concentration of plasma electrolytes (O'Connor and Fromm 1975). The only deleterious effects observed by these workers was a significant increase i n hematocrit after 12 weeks exposure. Oryzias  latipes i s more "hardy" than the rainbow trout, and yet i n the present investigation, the reproduction of Oryzias was adversely affected by even lower concentrations of methylmercury than that used on the rainbow trout by O'Connor and Fromm (1975); these findings suggest that repro-duction is much more sensitive to environmental pollution than other physiological functions. Reproduction is indispensible to the survival of the species and any environmental contamination that adversely af-53 fects reproduction w i l l have long lasting effects on the species and the ecological systems connected with i t . Mercury compounds inhibit mitosis by interacting with sulfhydryl groups (Hughes 1950). Since Oryzias depends on constant replacement of new oocytes for i t s daily spawning, inhibition of mitosis is a pos-sible explanation for the decreased number of eggs spawned in contam-inated water. Biochemically, methylmercury may inhibit enzyme systems for steroidogenesis by reacting with sulfhydryl groups of the enzymes, thus making them inactive. Fish reproduction depends on proper bal-ance of hormones in the hypothalamic-hypophysial-gonadal system. It is not known where methylmercury acts. It may act at the hypothalamic-hypophysial level because methylmercury has been observed to accumulate in large quantities in the b rain, but the possi b i l i t y that It may also act at the gonadal level cannot be disregarded. These p o s s i b i l i t i e s are examined in the following section. SECTION III MODE OF ACTION OF METHYLMERCURY ON REPRODUCTION 55 INTRODUCTION Previous sections have demonstrated ithat exposure to methyl-mercury resulted i n reduction of spawning activity ln Oryzias latipes. However, i t is not certain where methylmercury acts. This section attempts to elucidate the action of methylmercury on reproduction at the organ level. In f i s h , the spawning period corresponds to a season most favour-able for the development of the offspring. The fish synchronizes i t s reproductive physiology with the season by using environmental cues, like photoperiod and temperature. These environmental cues are trans-mitted to the hypothalamus v i a the eye, the pineal and possibly the skin. The hypothalamus in turn stimulates the hypophysis to produce and re-lease hormone(s) capable of stimulating gonadal development; thus form-ing the hypothalamic-hypophysial-gonadal axis. There i s also a feed-back mechanism i n this system whereby levels of the hormones are i n proper balance. Fig. 12 shows such a relationship. So far we have observed the effects of methylmercury treatment on the end product of the entire reproductive process. But i t is not known at what point methylmercury acts on this hypothalamic-hypophysial-gonadal axis. To elucidate this, i t was assumed that methylmercury blocked or rendered inactive some link in this system; thus, some hormone was not produced. It was further assumed that "replacement therapy" with appro-priate hormones would restore reproduction in methylmercury treated f i s h . In the experiments that follow, different hormones from the hypothalamus, 56 Figure 12. Diagram showing the interrelationship between environmental cues, hypothalamus, hypophysis and gonads. 57 ENVIRONMENTAL CUES temperature photoperiod water chemistry, etc. v i a eye, pine a l , s k i n T HYPOTHALAMUS hypothalamic hormones (releasing or i n h i b i t i n g hormones) HYPOPHYSIS gonadotropln(s) GONADS gonadal development steroidogenesis ( ? ) 58 hypophysis and gonads are "replaced" in the methylmercury affected fi s h to determine whether these hormones restore reproductive activity. Mammalian luteinizing hormone is effective in inducing matura-tion and ovulation i n a variety of fishes (Hirose 1971, Goswami and Sundararaj 1972; Donaldson,1973; Hoar 1969), but piscine hypothalamic hormone, possibly similar to mammalian luteinizing hormone-releasing hormones (LH-RH) has not been well studied. Synthetic luteinizing hor-mones-releasing hormone (LH-RH), recently synthesized based on the pro-posed structure of porcine and ovine LH-RH, is highly effective i n s t i -mulating the release of luteinizing hormone and in inducing ovulation in mammals (Schally et a l . , 1973; Humphrey et a l . , 1973; Foxcroft et a l . , 1975) , in chickens (van Tienhoven and Schally 1972) and in amphibian (Thornton 1974; Mazzi et al.,1974; Vellano et a l . , 1974). In teleost fishes, administration of synthetic LH-RH causes release of secretory granules from the gonadotropic (GtH) cells in the proximal pars dist a l i s and induces ovulation i n the goldfish (Lam et_ a l . , 1976) and in the ayu (Hirose and Ishida 1974). Gonadotropin release by synthetic LH-RH was also observed in the carp (Breton and Weil 1973) but the reaction was less than that e l i c i t e d by carp hypothalamic extracts. In trout, treat-ment with mammalian gonadotropin releasing hormone elevated plasma gonadotropin concentration as determined by radioimmunoassay (Crim and Cluett 1974). However, i t is not known whether synthetic LH-RH would stimulate the synthesis and release of gonadotropin(s) i n sexually re-gressed fish and subsequently gonadal maturation. This phenomenon was observed in hens (Reeves et a l , , 1973). 59 Oryzias latipes kept under warm temperature (23 + 1°C) and short photoperiod (8L/16D) for 6 to 12 weeks showed l i t t l e or no gonadal development (Chan 1976, Yoshioka 1962, 1963). This system permits tests to show whether synthetic LH-RH can stimulate activity in the pituitary gonadotropic cells and consequently gonadal maturation. This part of the study f i r s t examines the effect of synthetic LH-RH on ovari-an development in the Japanese medaka, Oryzias latipes. After establishing the effectiveness of synthetic LH-RH on gonadal maturation in Oryzias, we may then determine whether hypothalamic and hypophysial hormones are capable of restoring reproductive activity in methylmercury treated f i s h . By injecting hypothalamic and hypophysial hormones into fish exposed to methylmercury, i t is possible to determine not only which of the hormones is effective i n restoring reproduction in methylmercury poisoned fish but also at what level of the hypothalamic hypophysial-gonadal axis methylmercury inhibition occurs. Histological examination of the pituitary may also help to explain the action of methylmercury on the synthesis and release of gonadotropin(s). Needless to say, an important outcome of this experiment is of ecological s i g n i f i -cance since i t may show whether hormonal treatments can restore the spawn-ing activity inhibited by methylmercury when indeed such a contamination does occur i n nature. Hirose (1971) devised a simple i n vitro system for studying ovula-tion i n Oryzias. Using this system, the rate of ovulation at various starting hours of incubation was determined (Hirose & Hirose 1972) and alt effects of various hormones on ovulation were studied (Hirose 1972a, b, 60 1973). Similar studies were performed on the Indian catfish by Goswami and Sundararaj (1972a, b, 1973, 1974). Studies by both groups showed that hypophysial gonadotropic hormone and the corticosteroids were effective in inducing ovulation in vitro. This in vitro system provides a fast, convenient method for studying the effects of methyl-mercury on ovulation and may be developed into an effective bioassay. This part of the study investigates the effects of long term ex-posure of female fish to methylmercury on in vitro ovulation. The effects of various concentrations of methylmercury on i n vitro ovula-tion of untreated fish and the effects of exogenous luteinizing hormone on methylmercury blocked ovulation in vitro of untreated fi s h w i l l also be examined. The effects of various steroids on methylmercury affected ovulation w i l l be investigated, thereby some postulations can be made on the effects of methylmercury on the ovary level. 61 MATERIALS AND METHODS On ovarian maturation by synthetic LH-RH In this part of the experiment, only female fish were used be-cause they provide better indices for gonadal development than male fish. Adult regressed fish were obtained by pretreating fish with low temperature (13 + 1°C) and short photoperiod (8L/16D) for three months (May 6 to Aug. 6, 1974). On Aug. 6, 1974, fi s h weighing about 0.3 g each were warmed to room temperature (23 + 1°C) overnight under short photoperiod (8L/16D = 0800 to 1700 hours); they were distributed, 10 to 12 fish per tank, into five 22-liter capacity a l l glass aquaria with subgravel f i l t e r s . The fish were maintained under the above con-ditions for six weeks and fed once-a-day with a slight excess of frozen brine shrimp during the morning. Four groups received injections of synthetic LH-RH (AY-24, 031. Ayerst Laboratory) at doses of 1, 10, 100 and 1000 ng/g body weight respectively; the f i f t h group was saline-injected and acted as a control. Injections were intraperitoneal using a microsyringe (Hamilton) equipped with a 32 gauge hypodermic needle. The synthetic LH-RH was dissolved in 0.6% NaCl and the injection volume was 5 ul per f i s h . Fish were i n -jected twice a week for 6 weeks (Aug. 8 to Sept. 23, 1974). At the end of the experiment, a l l fish were sacrificed, body weights and gonad weights were recorded to the nearest 0.2 mg and the gonadosomatic index (GSI = gonad weight X 100/body weight) calculated. For histological observations, the ovaries and the pituitary glands were fixed in Bouin's solution, embedded in paraffin and sectioned at 5-7 um> 62 Ovarian sections were stained with Erlich's hematoxylin-eosin; aldehyde fuchsin counter-stained with Halmi's was used for the p i t u i -tary glands. Oocytes were classified into three categories according to Chan (1976) , and only percent distribution of Class III oocytes (number of oocytes with yolk formation x 100/total number of oocytes i n a mid-section of the ovary) was calculated because appearance of Glass III oocytes represents mature or maturing fi s h . On effects of LH-RH and LH injection in methylmercury treated fi s h This experiment was performed in a flow-through system described in Section I. Sexually regressed fish were obtained by maintaining fish at 13 + 1°C under short photoperiod (8L/16D) for four months (May 11 to Sept. 10, 1974). The fis h weighing 0.25 to 0.30 g were warmed to 23 + 1°C overnight and divided Into four groups, 15 males and 15 females i n each group. Loading density of the tanks was approximately one l i t r e of test solution per gram fis h . Acclimation consisted of two days with running clean water at 23 + 1°C and under long photoperiod (16L/8D) for a l l four groups. The same photoperiods and water temperature were used during the entire experimental period. On the third day, methylmercuric chloride solution was added to three of the four groups and the desired concentration of 10.7 ug/1 as methylmercury was reached i n about four hours; the approximate time for 99% replacement of test solution was 3.5 hours (Sprague 1969). The fourth group, kept in clean water, acted as the control. Starting on the fourth day, the three groups exposed to methylmercury received 63 twice-a-week either saline (0.6% NaCl) or luteinizing hormone (10 yg/g body weight; porcine luteinizing hormone, NIH-LH-S8) or synthetic l u -teinizing hormone-releasing hormone (1 yg/g body weight; AY-24, 031, Ayerst Laboratory). The control group received only saline injections, Injections were performed intraperitoneally using a micro-syringe (Hamilton) equipped with a 32 gauge hypodermic needle, the hormones were dissolved in 0.6% NaCl and the injection volume was 5 yl per f i s h . Exposure to methylmercury and the twice-weekly injections lasted for 6 weeks. Fish were fed daily with frozen brine shrimp. Spawning activity was recorded as number of females with eggs at-tached and total number of eggs collected each day. Observations were continued for 6 weeks. At the end of six weeks, the fi s h were k i l l e d , weighed, measured and the gonadosomatic indices (GSI) calculated. For histological observation of the pituitary glands, the tissues were fixed i n Bouin's fixative, embedded in paraffin and the sections(6 ym) were stained with aldehyde fuchsin, AF, counter-stained with Halmi's solu-tion. Analysis of variance was used in the s t a t i s t i c analyses. On effects of methylmercury on in vitro ovulation A l l f i s h were maintained at 23 + 1°C under long photoperiods (16L/ 8D; 0800-2400 hour l i g h t ) . Spawning females with eggs attached were removed each day at noon and kept in a separate tank until required for the experiment. A l l instruments and media were ste r i l i z e d before use. Each fish was swabbed with 70% alcohol and k i l l e d by decapitation. The ovary was removed, and placed in a glass petri dish containing Medium 199 (Hirose 64 1971). The eggs were individually separated as free eggs with dissect-ing pins. Only eggs with dismeter greater than 0.8 mm were used. In-cubation was carried out at 23 + 1°C. For each treatment, 10-15 eggs were placed in 5 ml of medium in a watch glass covered with a petri dish. Incubation started wither at 1700 hours or 2300 hours—the two best times for commencing incubation with or without LH (Hirose and Hirose 1972). Percent ovulation was recorded at 1000 hours the following morning with the help of a dissecting microscope. Ovulated eggs were characterized by their detachment from the f o l l i c l e s and presence of filaments around the ovulated eggs (Hirose 1971). Normal ovulation was confirmed by a r t i f i c i a l f e r t i l i z a t i o n and observation of development. Pre-exposure of fish to methylmercury was similar to that described in Section I. At the end of the exposure period (6 weeks), a l l female f i s h were k i l l e d , the ovaries were dissected and a l l oocytes with dia-meters greater than 0.8 mm from the same treatment group were pooled. The eggs were then incubated with 0 or 10 ug/ml porcine luteinizing hormone (NIH-LH-S8) and various concentrations of methylmercury in Medium 199 at 1700 hours and ovulation checked at 1000 hours the following morning. The experiment was duplicated and since similar results were observed, the data were pooled. Additional experiments were performed using various concentrations of methylmercury in the incubation medium with eggs from untreated fi s h . To study the effects of steroids, progesterone, estradiol, cortisone and testosterone, were individually dissolved i n ethanol: propylene glycol (1:1) and added to the incubation media just prior to incubation, result-65 ing i n a f i n a l concentration of 1 ug/1 steroid. Procedure of incuba-tion was similar to that previously described. A l l experiments were duplicated and the results were pooled. 66 RESULTS Effect of Synthetic LH-RH on ovarian maturation The effects of synthetic luteinizing hormone-releasing hormone on the gonadosomatic index, percent distribution of Class III oocytes and ovulation are shown in Fig. 13, The ovary at the i n i t i a t i o n of this study was mostly composed of small oocytes (diameter 0.1 mm or less); there were no Class III oocytes. The mean gonadosomatic index (+ S.E.) of this i n i t i a l group was low (1.08 + 0.17). This value is character-i s t i c of sexually regressed f i s h . After six weeks of injections, the control group (saline injected) showed only a slight increase i n GSI (from 1.09 to 1.42) with the ap-pearance of some Class III oocytes in less than 4% of the fish (Figs. 13 and 14). Groups injected with synthetic LH-RH, with the exception of 1 ng/g, showed an increase i n both gonadosomatic index and percent dis-tribution of Class III oocytes (Figs, 13, 15, 16, and 17); however, only the two higher doses showed significantly different results from the sa-line control (P <0.05, t-test). Furthermore, there appeared to be a linear relationship between the gonadosomatic index and log of the injec-tion doses (Fig. 13). Ovulation was also observed in 5 out of the 12 f i s h injected with the highest dose, 1000 ng/g. The pituitary gland of Oryzias has already been described and the globular basophils, located at the most ventral portion of the proximal pars d i s t a l i s , identified as the gonadotrophs (Aoki and Umeura 1970; Kasuga and Takahashi 1971). The activity of these cells can be e s t i -mated by their staining a f f i n i t y with aldehyde fuchsin (AF), and is re-lated to the changes of reproductive a c t i v i t i e s ; the staining a f f i n i t y 67 Figure 13. Effects of different dosages of synthetic LH-RH on the gonadosomatic index and the percent dis-tribution of Class III oocytes. Numbers i n brackets represent number of fis h in each group. Values reported are means + standard errors. Asterisk represents s t a t i s t i c a l significance (P <0.05, t-test) compared to saline injected control, a, ovulation observed. 68 INJECTION DOSES ng/g 69 Figure 14. Section (7 um) of ovary from Oryzias injected with saline twice-a-week for 6 weeks at 23 + 1°C under 8L/16D. Hematoxylin-eosin, X 100. Figure 15. Section (7 um) of ovary from Oryzias injected with 10 ng/g synthetic LH-RH twice-a-week for 6 weeks at 23 + 1°C under 8L/16D. Hematoxylin-eosin. X 100 70 71 Figure 16. Section (7 um) of ovary from Oryzias injected with 100 ng/g synthetic LH-RH twice-a--week for 6 weeks under 23 + 1°C and 8L/16D. Notice the appearance of Class III oocytes with yolk vesicles as indicated by arrow, Hematoxylin-eosin, X100 Figure 17. Section (7 um) of ovary from Oryzias injected with 1000 ng/g synthetic LH-RH twice-a-week for 6 weeks under 23 + 1°C and 8L/16D. Notice the presence of Class III oocytes with yolk formation as indicated by arrow, and also the enlarged ovarian cavity (C) due to the ovulated oocytes. Hematoxylin-eosin. X100 72 73 i s highest during the spawning season from May to August, less evident during the post-spawning period from September to October and absent during the resting period from November to February (Kasuga and Takahashi 1971). In the present study, the gonadotrophs of the saline-injected control fish were not stained with AF, suggesting inactivity of these cells (Fig. 18). Similar results were observed i n the 1 ng/g injected fish and the photomicrograph was omitted here. As the injec-tion dose increases, the gonadotropic cells show an increase in stain-a b i l i t y and in the area stained (Figs. 18, 19, 20 and 21). These re-sults suggest that synthetic LH-RH at doses of 10 to 1000 ng/g were effective i n stimulating ac t i v i t y of the gonadotropic cells resulting i n ovarian development. Effects of Synthetic LH-RH and LH injections on methylmercury treated  fish Spawning was f i r s t observed i n groups injected with hormones (either LH or syn. LH-RH) during the second week of the methylmercury exposure; while groups injected with saline (both methylmercury treated and clean water control) started spawning only during the fourth week of the ex-posure (Fig. 22). Though showing an early start in spawning, the methyl-mercury-exposed LH-RH injected group showed a decline in spawning a c t i -vity after the fourth week, while the methylmercury-exposed LH-injected group showed increasing spawning activity throughout the treatment period. Methylmercury-exposed saline-injected fish showed spawning only in the last three weeks of exposure and these activities were lower than that of methylmercury-exposed LH-injected groups. Though spawning 74 Figure 18. Section (6 um) of pituitary gland from Oryzias injected with saline twice-a-week for 6 weeks under 23 + 1°C and 8L/16D. P, prolactin c e l l ; T, thyrotroph; G, gonadotroph; N, neurohypo-physis; S, somatotroph. Aldehyde Fuchsin. X410. Figure 19. Section (6 um) of pituitary gland from Oryzias injected with 10 ng/g synthetic LH-RH twice-a-week for 6 weeks under 23 + 1°C and 8L/16D, No* tice the slight staining of AF in gonadotrophs, G and increased AF staining in neurohypophysis, N. Aldehyde Fuchsin, X410. 75 76 Figure 20. Section (6 um) of pituitary gland from Oryzias i n -jected with 100 ng/g synthetic LH-RH twice-a-week for 6 weeks under 23 + 1°C and 8L/16D. Note the increased AF staining i n both gonadotrophs, G and neurohypophysis, N. Aldehyde Fuchsin. X410. Figure 21. Section (6 urn) of pituitary gland from Oryzias i n -jected with 1000 ng/g synthetic LH-RH twice-a-week for 6 weeks under 23 + 1°C and 8L/16D. Note the further increase of AF staining in both gonadotrophs, G and neurohypophysis, N. Aldehyde Fuchsin. X410, 77 78 Figure 22. Weekly observation on spawning and egg-laying of Oryzias for the various hormonal treatment in the presence of methylmercury (10.7 yg/1). 79 120 100 8 8 0 O LJJ 60 o o • o A • saline h^O -• saline CH^Hg* -o LH Cl-^Hg* •A LH-RH CH 3 Hg 40 20 0 O < Q_ 00 o O 2 3 E X P O S U R E r 4 5 (wks) 6 80 occurred later than in the hormone injected groups, the clean water saline-Injected control group showed substantially higher spawning activity than any of the methylmercury exposed groups. The percent of spawning activity over the entire 6-week treatment period of the methylmercury treated groups were less than the clean-water control group (Fig. 23). Injection of LH was effective in restoring part of the spawning activity inhibited by methylmercury but not LH-RH. At the end of six weeks, the female gonadosomatic indices of the methylmercury treated groups were less than the control (Fig. 23). Male gonadosomatic indices of the different groups were variable. Since the female gonadosomatic indices provide a better indication of the effects of the different treatment, s t a t i s t i c a l analysis was per-formed on data collected from female f i s h only (Table 4). The methyl-mercury exposed groups were a l l s t a t i s t i c a l l y different (P <0.05) from the clean water control group. Among the methylmercury exposed groups, the LH-injected group showed significant differences from the saline i n -jected group and the LH-RH injected group, while no significant d i f f e r -ence was observed between the saline injected and LH-RH injected group. The pituitary gland of Oryzias has been described and the globular basophils, located at the most ventral portion of the proximal pars d i s t a l i s , identified as the gonadotrophs, GTH cells (Aoki and Umeura 1970). The activity of these cells can be estimated by their staining a f f i n i t y with aldehyde fuchsin and i s related to the reproductive ac-t i v i t i e s (Kasuga and Takahashi 1971). The present study showed that saline injected clean water control f i s h had high activity i n gonadotrophs and neurosecretion as indicated by more intense staining with AF 81 Figure 23. Effects of various hormonal treatments on percent spawning, male and female gonadosomatic indices at the end of 6-weeks methylmercury exposure. Numbers i n brackets represent sample size and the ver t i c a l bars, standard error. M A L E GS I O Q O Q Q i O p o p p -j -M ^ 0 ) 00 O T r T — i 1 r H K 3 H i C O l o i - i s 4^ 0) Q 3 3 a Q (v) Q Q CO L 00 F E M A L E GSI 09 t o 83 Table 4. Statistical significance of female gonadosomatic indices among the various treatments by analysis of variance Clean Water Saline i n j . CH3Hg+ Saline i n j . CH3Hg+ LH i n j . CH3Hg+ LH-RH i n j . Clean Water Saline i n j . P <0.05 P <0.05 P <0.05 CH3Hg+ Saline i n j . P <0.05 _____ P <0.05 n.s. CH3Hg+ LH i n j . P <0.05 P <0.05 P <0.05 84 (Figs. 24, 25, 26 and 27). Very l i t t l e activity was observed i n the methylmercury-exposed saline-injected group showing inhibition of GTH c e l l activity by methylmercury. Methylmercury-exposed LH-injected fis h showed moderate activity in gonadotrophs and neurosecretion while LH-RH injected f i s h showed high activity in both gonadotrophs and neuro-secretion, suggesting that this hormone was effective in stimu-lating the activity of the pituitary gland (Figs. 24 to 27). Effects of methylmercury on i n vi t r o ovulation Hirose (1971b) observed that when donor medaka were maintained under a long photoperiod (16L/8D, 0800 to 2400 hr) in vitro ovulation required gonadotropic hormones i f Incubation commenced at 1700 hours but when incubated at 2200 hours, ovulation occurred naturally. These two time schedules were used i n the present study depending on the require-ments of the individual tests. Pre-exposure to methylmercury for 6 weeks. When incubated at 1700 hours, l i t t l e or no ovulation was observed unless LH was present in the media (Table 5). This confirms Hirose's observations. Even without methylmercury added to the incubation media, pre-exposure of female fi s h to methylmercury for 6 weeks reduced the percentage of ovulation in accordance with the pre-exposure concentrations. When methylmercury was added to the incubation media, further reduction in percent ovulation was observed. Methylmercury i n the incubation media seems to have an additive effect on the concentration of methylmercury to which the fish were previously exposed. 85 Figure 24. Section (6 um) of pituitary gland from Oryzias exposed to clean water and injected with saline twice-a-week for 6 weeks under 23 + 1°C and 16L/ 8D, T, thyrotroph; G, gonadotrophs; N, neurohypophysis. Note the intense AF staining in the gonadotrophs. Aldehyde fuchsin. X410. Figure 25. Section (6 um) of pituitary gland from Oryzias exposed to methylmercury and injected with saline twice-a-week for 6 weeks under 23 + 1°C and 16L/ 8D. G, gonadotrophs; N, neurohypophysis. Note the loss in AF staining in the gonadotrophs. Aldehyde fuchsin. X410. 86 87 Figure 26. Section (6 um) of pituitary gland from Oryzias exposed to methylmercury and injected with luteinizing hormone twice-a-week for 6 weeks under 23 + 1°C and 16L/8D. G, gonadotrophs; N, neurohypophysis. Note the moderate AF staining in gonadotrophs. Aldehyde fuchsin. X410. Figure 27. Section (6 um) of pituitary gland from Oryzias exposed to methylmercury and injected with luteinizing hormone-releasing hormone twice-a-week for 6 weeks under 23 + 1°C and 16L/8D, G, gonadotrophs; N, neurohypophysis. Note the intense AF staining i n gonadotrophs. Aldehyde fuchsin. X410. 88 Table 5. E f f e c t s of d i f f e r e n t concentrations of methylmercury on i n v i t r o o v u l a t i o n i n Oryzias p r e v i o u s l y t r e a t e d w i t h methylmercury f o r 6 weeks. Values reported as percent o v u l a t i o n (no. of oocytes ovu-lated/no. of oocytes used). 6-wk Cone, of CH^g pre-expo- i n c u b a t i o n sure to CH 3Hg + media No LH Added 0 0 10 vg/ml LH + CH 3Hg + i n yg/1 4.8 10.7 21.5 215 480 From C o n t r o l 3.3 (1/30) 83.3 (20/24) 76.9 (30/39) 70.1 (22/30) 53.8 (21/40) 27.3 (9/33) 19.1 (8/42) From f i s h exposed to 4.8 ug/1 CH 3Hg + 5.5 (2/36) 73.7 (28/38) 68.4 (20/36) 55.5 (20/36) 50.0 (16/32) 28.1 (9/32) 26.6 (8/30) From f i s h exposed to 10.7 ug/1 CH3Hg 70.0 (14/20) 24.0 (6/25) 33.3 (9/27) 20.0 (4/20) From f i s h exposed to 11.5 ug/1 CH 3Hg + 54.5 (12/22) 28.9 (8/28) 0 0 VO 90 Effects of methylmercury on normal oocytes from untreated fi s h . Normal oocytes from untreated f i s h incubated i n methylmercury solutions showed a reduced percentage of ovulation (Table 6). More than 50% re-duction of ovulation was observed i n oocytes exposed to 48 ug/1 of methylmercury. Percent observed ovulation converted into percent i n h i -bition showed a direct relationship with the logarithm of the doses used (Fig. 28). Effects of different steroids on methylmercury inhibited ovulation in v i t r o . Under normal conditions, ovulation occurs naturally when i n -cubated at 2200 hours. A p i l o t study was undertaken to determine an effective dose of methylmercury whereby ovulation would be inhibited at this hour. Table 7 showed the results of such a study. Normal oocytes from untreated fish when incubated at 2200 hours ovulated without LH while the addition of methylmercury at concentration of 192 ug/1 and 215 ug/1 was effective in reducing not only this reaction but also that induced by the addition of 10 ug/ml LH to the incubation media. Using the above modified system for an in vitro ovulation study, four steroids (progesterone, cortisone, estradiol and testosterone) were tested for their effectiveness in restoring methylmercury inhibited in vitro ovulation. Table 8 shows the results. When incubated without methylmercury at 2200 hours, normal oocytes from untreated fi s h ovulated naturally and this was stimulated by the addition of cortisone. Proges-terone, estradiol and testosterone had l i t t l e or no effect. When methyl-mercury at concentrations of 215 ug/1 was present i n the incubation media, ovulation was reduced i n a l l cases with the exception of cortisone which remained slightly potent i n inducing a certain degree of ovulation. 91 Table 6. The effects of various concentrations of methylmercury on ovulation i n vitro of Oryzias latipes incubated with 10 yg/ml porcine LH at 1700 hours. Cone, of CHgHg yg/i + No. of oocytes used No. of ovulated eggs Percent Ovulation 0 4,8 10.7 21.5 48.0 96.0 192.0 215.0 25 38 26 33 35 45 51 40 21 28 17 20 17 18 15 7 84.0 73.7 65.4 60.6 48.6 40.0 29.4 17.5 92 Figure 28. Percent inhibition of i n vitro ovulation by various concentrations of methylmercury i n incubation media at 1700 hours. 9 3 8 0 7 0 6 0 Z o 5 0 g Q 4 0 X ± 3 0 o o 2 0 1 0 J I I I H H L J I I I I I i i i J I I I 0 1 5 10 50 100 CONCENTRATION jug C H 3 H g + / I 5 0 0 94 Table 7. The effects of methylmercury on ovulation in vitro of Oryzias latipes incubated with and without LH (10 yg/ml) commencing at 2200 hours. Cone, of CHgHg ug/1 LH yg/ml No. of oocytes used No. of ovulated eggs Percent Ovulation 0 192.0 192.0 215.0 215.0 0 10 0 10 0 25 30 32 34 35 15 8 8 8 6 60.0 26.6 25.0 23.5 17.1 95 Table 8 . The effects of steroids on ovulation i n vitro of Oryzias latipes oocytes incubated with and without methylmercury at 2200 hours as the time of incubation Cone., of CH 3Hg + Steroid No. of No. of Percen ^ S / l 1 Pg/ml oocytes used ovulated eggs Ovulati 0; 0 28 15 53.6 215: 0 34 6 17.6 a Progesterone 35 22 62.8 215 Progesterone 30 6 20.0 0) Cortisone 38 32 84.1 215 Cortisone 41 18 43.9 0! Estradiol 30 16 53.3 215 Estradiol 29 5 16.1 0' Testosterone 30 17 56.6 215 Testosterone 32 6 18.7 96 DISCUSSION Effect of synthetic LH-RH on ovarian maturation Results of the present study show that synthetic LH-RH is effective in stimulating the activity of gonadotropic cells in the pituitary as i n -dicated by the af f i n i t y to AF staining (Kasuga and Takahashi 1971), and in inducing ovarian maturation and ovulation in Oryzias latipes maintained at 23 + 1°C under short photoperiods (8L/16D). This agrees with the re-sults obtained i n the ayu (Plecoglossus a l t i v e l i s ) (Hirose and Ishida 1974), goldfish (Lam et a l . , 1976; Kaul and Vollrath 1974), carp (Breton and Weil 1973), and trout (Crim and Cluett 1974). Teleost pituitary gonadotropic cells appear to be under the control of a releasing hormone from the hypothalamus (Peter 1970). However, radioimmunoassay studies of Deery (1974) have shown that hypothalamic ex-tracts of goldfish do not have any immunological cross reaction with l a -belled synthetic LH-RH while rat hypothalamic extracts do. Also, Breton and Weil (1973) found that carp hypothalamic extract i s more potent than synthetic LH-RH i n stimulating release of gonadotropin(s). These obser-vations suggest the existence of a fish gonadotropin(s)-releasing hormone that may be chemically different from mammalian LH-RH but have overlapping biological a c t i v i t i e s . The effective dose of synthetic LH-RH used, although similar to do-sages used i n other fish studies, was much higher than the effective dose in mammals. This difference could be due to the methods of administration of the hormone. Intraperitoneal injection has been shown to be less effec-tive than perfusion of the pituitary in situ (Vellano et a l . , 1974) or 97 intracranial injection (Lam et a l . , 1976). Though not known for f i s h , LH-RH has a very short h a l f - l i f e i n mammals (Redding e_t a l . , 1973) ; hence, i n this study, twice-a-week may not be frequent enough to stimu-late the pituitary at the lower doses. At a low dose of 10 ng/g, a slight increase (though not s t a t i s t i c a l l y significant) was observed in gonadosomatic index with no increase i n percent distribution of Class III oocytes over the control (Fig, 13). It is possible that low doses only stimulate mild synthesis and release of gonadotropin(s) leading to development of Class 1 and II oocytes only. Under natural spawning conditions, where male and female fi s h are present together, the gonadosomatic index of female fish i s approximate-ly 6% (Chan 1976), In the present study, the GSI was much lower. This may possibly be due to the absence of males resulting in retention of ovulated oocytes in the ovarian cavity; these unspawned eggs may have an inhibitory effect on the development of the rest of the ovary (Egami and Hosokawa 1973). Moreover, the effect of handling the fish during frequent injections cannot be disregarded. In this experiment, stainability with aldehyde fuchsin was used as a criterion of activity in gonadotropic c e l l s . Since synthetic LH-RH is effective in releasing gonadotropin(s) (Hirose and Ishida 1974; Lam et a l . , 1976; Kaul and Vollrath 1974; Breton and Weil 1973), the i n -creased AF staining of gonadotropin cells observed i n the present study probably represents the excess of gonadotropin synthesis over i t s release from the pituitary. Ovarian maturation probably depends on a high rate of synthesis of gonadotropin(s) with a low but sustained release of 98 hormone(s) as observed i n the present study while ovulation depends on a large release (surge) of the accumulated gonadotropin(s) in the pituitary as observed In goldfish (Lam et a l . , 1976) and ayu (Hirose and Ishida 1974). Under favourable conditions of long photoperiod, warm temperatures and good supply of food, Oryzias spawns daily throughout the spawning season (Egami and Hosokawa 1973). Thus i t seems possible that gonadotropic c e l l activity remains high in Oryzias throughout the entire spawning season. The large release of gonadotropin(s) by LH-RH as observed in goldfish (Lam e_t a l . , 1976) and ayu (Hirose and Ishida 1974) w i l l probably be observed i n Oryzias only during i t s daily ovula-tion between 0100 and 0400 hr. Of interest also in the present study is the increased neurosecre-tion (increased AF staining of neurohypophysis) associated with increas-ing doses of synthetic LH-RH (Figs. 18 to 21). The cause is not known. However, i t has been observed that neurohypophysial activity correlates with reproductive activity in Oryzias (Kasuga and Takahashi 1971), and that neurohypophysial secretions affect spawning behaviour i n the k i l l i -f i s h (Macey, Pickford and Peter 1974) and stimulate activity of the ovi-duct and ovarian smooth muscles in the guppy (Heller 1972). There are two possible explanations for the stimulation of neurosecretion by syn-thetic LH-RH: i t is possible that the stimulation of the pituitary by mammalian LH-RH may be mediated through yet another system in the neuro-hypophysis, and secondly this stimulation may be the result of a feedback by the maturing ovary on the hypothalamus. These highly speculative ex-planations require further research. 99 In summary, one point seems clear; a long photoperiod i s essen-t i a l for ovarian development i n the medaka (Yoshioka 1962; 1963; Chan 19760 and this long photoperiod triggers the secretion of a hormone, probably from the hypothalamus (Peter 1973) , similar i n activity to synthetic LH—RH, which i n turn stimulates the activity of the gonado-tropic c e l l s i n the pituitary. Effects of synthetic LH-RH and LH injections on methylmercury treated  fish.. Previous experiments demonstrated that a 6-week exposure to 10.7 ug/1 of methylmercury reduced spawning by about 50% compared to the con-t r o l value (Fig. 8, Section II). In the present study, a further reduc-tion of spawning by about 20% was probably due to frequent handling dur-ing: the twice-^weekly injections (see Figs. 8, 10 and 23). The reduction of spawning activity by methylmercury was part i a l l y prevented by injection of LH (Fig. 23) suggesting that the gonads re-mained receptive to LH stimulation while exposed to methylmercury. Both hormone injected groups started spawning earlier than the saline injected groups suggesting that hormone injections accelerated gonadal development and that the f i s h may not have accumulated enough methylmercury during the f i r s t two weeks of exposure to block reproduction. Gonadal maturation depends on the synthesis and release of gonadotro-phiniCs), and synthetic LH-RH has been demonstrated effective in doing both. In the present study, however, pituitary cytology showed a slightly d i f -ferent picture. Synthetic LH-RH was effective i n stimulating gonadotropic c e l l a c t i v i t y i n methylmercury treated fish even though spawning activity was not restored (Fig. 24 to 27). Previously, i t was suggested that the 100 activity shown by pituitary gonadotrophs represents the net result be-tween synthesis and release of gonadotropin(s) i n GTH c e l l s . Thus i n -jection of synthetic LH-RH into methylmercury poisoned f i s h , which re-sulted i n stimulation of gonadotropic activity with no increase i n spawning activity, suggests a possible blockage in the release of gona-dotropin^) . The lowering of receptivity at the gonadal level by methyl-mercury has been ruled out because exogenous LH was effective in remov-ing the inhibition of spawning activity by methylmercury (Fig. 23). David and Ramasawmi (1971) observed an increase in granulation of LH cells in the langur pituitary following cadmium-induced testicular ne-crosis suggesting possible blockage in the release of the hormone. Pre-sent findings show similar results with Oryzias. Since both cadmium and methylmercury have a strong a f f i n i t y for sulfhydryl groups and since in both animals the release of gonadotropin were affected, i t seems possible that the action on reproduction for these two chemicals are similar. Fur-thermore, gonadotropin release i n these two animals may well be similar, under the influence of sulfydryl group containing compound or enzyme. Since the mechanism controlling release of hormones from the pituitary i n f i s h i s not known, this suggestion is highly speculative and requires further investigation. However, i t is clear that reproduction may be p a r t i a l l y restored in methylmercury poisoned fish with luteinizing hormone injections. In other words, the gonads of methylmercury treated f i s h were s t i l l receptive to exogenous luteinizing hormone stimulation. Reproductive damages i n -curred in nature by methylmercury contamination may thus be remedied 101 p a r t i a l l y by Injecting the appropriate hormone(s). Effect of methylmercury on in vitro ovulation Ovulation in non-mammalian vertebrates has been described as a process whereby f o l l i c u l a r layers Immediately surrounding the apex of the oocytes are dissociated, forming a rupture which is smaller in diameter than the oocytes, and through which the oocytes is squeezed out (Asdell 1962). In Oryzias latipes, this process can occur in vitro i n Isolated, intact f o l l i c l e s (Hirose 1971). The mechanism behind this process is not well understood. Pendergrass (1976) observed an increase i n microfilaments i n the thecal layer prior to ovulation in vitro and suggested that these microfilaments, being contractile, are Involved i n c e l l movement during ovulation. However, i n vitro ovula-tion i n Oryzias seems to be under the control of several gonadotropic hormones (Hirose 1971, 1972c, Hirose and Donaldson 1972) and steroids (Hirose 1972a) and the f o l l i c u l a r envelope is indispensable for both pro-tection and hormonal action (Hirose 1972b). Treatment with methylmercury affects the release of gonadotrpin in the pituitary while the ovary remains receptive to stimulation of lutein-izing hormone, Pre-exposure of fi s h to 21.5 ug/1 of methylmercury for six weeks reduced in vitro ovulation by about 40% even under the stimula-tion of exogenous LH (Table 5), The mechanism behind this inhibition is not known. It is possible that methylmercury affected the enzyme system for steroid production. Methylmercury has a strong a f f i n i t y for sulfydryl groups and the enzymes involved in steroidogenesis may possess such pro-perties. Methylmercury added directly to the incubation media further 102 reduced the percent in vitro ovulation for fi s h previously exposed to the same toxicant (Table 5). This may be possible because the enzyme system may not be completely blocked by methylmercury during the pre-treatment; thus uncubation with methylmercury in the in vitro system allowed a more complete inhibition. A dose response was observed between percent inhibition of in vitro ovulation and the logarithm of the concentration of methylmercury used. F i f t y percent inhibition occurs at about 55 ug/1 of methylmercury in the incubation medium. This method provides a fast convenient system for testing effect of a toxicant on reproduction and may well develop into a bioassay for general toxicity studies. When incubations commenced at 2200 hr or 2400 hr a very high percent of oocytes ovulated naturally without LH (Hirose & Hirose 1972). Similar results were obtained in the present study (Table 7). However, i f the incubation media contained 192 ug/1 or 215 ug/1 of methylmercury, this reaction was reduced suggesting that methylmercury added to the medium may be inhibiting the synthesis, release or action of the hormone(s) responsible for ovulation. LH could not be one of the hormones in question, since the addition of 10 ug/ml of LH was not effective in increasing the percent ovulation (Table 7). Corticosteroids are effective in inducing ovulation in Oryzias in  vitro (Hirose 1972a, c, 1973), in Indian catfish in vivo and in vitro (Goswami and Sundararaj 1972a, b, and 1974) and in goldfish in vivo (Khoo 1974). The present experiment showed similar results. Cortisone was effective, at least partially, in overcoming the inhibitory effect of 103 methylmercury (Table 8). Columbo et a l . (1973) demonstrated corticos-teroid synthesis in Gillichthys and suggested that endocrine control of ovulation acts by pituitary gonadotropin stimulation of the synthesis of corticosteroids in the ovary. Sundararaj and Goswami (1974) in a co-culture study showed that the interrenal contributes the major portion of corticosteroids for ovulation. However, both of these phenomena have not been shown for other species. The present study has not c l a r i f i e d this point. Since the addition of corticosteroid to the media can re-store some of the ovulation inhibited by methylmercury, i t seems possible that the f o l l i c u l a r cells of Oryzias are capable of producing some c o r t i -costeroids. Hirose (1972b) also made such a suggestion in another in vitro ovulation study with afolliculated oocytes. Presence of methylmer-cury i n the medium probably blocked corticosteroidogenesis in the f o l l i c u l a r tissues, while addition of exogenous cortisone was effective in indue Ing some ovulation. When both methylmercury and cortisone were present, balance of the two resulted in a slight prevention of the inhibitory ef-fect of methylmercury as observed i n the present study. It seems possibl that once corticosteroid i s present, even just prior to ovulation, ovula-tion can ultimately occur. Sex steroids have not been effective in inducing ovulation and matur ation (Goswami and Sundararaj 1972b; Hirose 1972a). The present study confirmed this. The fact that progesterone was observed to be slightly effective i n stimulating ovulation and ovarian maturation in both Oryzias (Hirose 1972b) and the Indian catfish (Goswami and Sundararaj 1972b) was also observed i n this.study. However, the addition of methylmercury com-pletely abolished this effect (Table 8). This suggests that progesterone 104 a precurser of corticosteroids, cannot be converted to corticosteroids because of inhibition of steroidogenesis by methylmercury. From the foregoing discussion, i t seems that methylmercury may be a general i n h i -bitor of enzymes, especially enzymes with sulfydryl groups. Since a majority of proteins possess sulfydryl groups, methylmercury remains a very potent toxicant for a l l l i v i n g organisms. In this section, we have shown that methylmercury acted at two levels, pituitary and gonad, of the hypothalamic-hypophysial-gonadal axis. How-ever, i n vitro ovulation studies strongly suggested that methylmercury Is a cellular inhibitor of an enzyme system. This is probably true be-cause chemical reactions form the basis of a l l biological a c t i v i t i e s . 105 GENERAL DISCUSSION The median lethal concentration, 96hr-LC50, of methylmercury for adult Oryzias latipes was found to be 88+9.8 yg CH,jHg+/l. Compared to rainbow trout, this value is quite high suggesting that Oryzias i s a much more resistant f i s h . Tissue accumulation of methylmercury increases with exposure time and concentration of the chemical in the external medium. Death seems to occur once tissue methylmercury levels reached about 25 yg/g as methyl-mercury. Such levels were reached i n two weeks for fish exposed to 21.5 yg/1 of methylmercury and 6 weeks when exposed to a lower concentration of 10.7 yg CH^ Hg"*"/!. However, in thepresent study, this level was never reached even at the end of six-weeks exposure to 4.8 yg/1 of methylmer-cury. Four-hour exposure of spawning fish to methylmercury during the fish's normal oviposition time affected spawning at concentrations equi-valent to one-tenth of the medial lethal concentration. However, this effect was not carried over to the time when the f i s h were returned to clean water. Oviposition was completely abolished when exposure concen-tration was at the median lethal concentration. This effect was carried over to the time when the fish were returned to clean water. This short exposure of 4 hours to the median lethal concentration of methylmercury may have allowed enough accumulation of the chemical to effect a change in spawning activity when returned to clean water. This phenomenon may occur with bioaccumulative toxicants. If so, this type of study not only provides information on the toxicity but also indicates whether the toxi-cant i n question i s bioaccumulative. This suggestion i s hypothetical and 106 requires further investigations. Since Oryzias has a fixed pattern of spawning which is easily quantified, this behavioral response may be useful for monitoring environmental changes like water pollution. Long-term exposure to methylmercury at concentrations approximately equal to one-eighth of the median lethal concentrations resulted in about 50% inhibition of reproductive a b i l i t y . At a lower concentration of about one-twentieth of the median lethal concentration, 40% inhibition on re-productive a b i l i t y was observed. These results clearly showed that repro-duction is extremely sensitive to environmental contamination, and since reproduction is indispensable for the survival of the species, i t seems reasonable that studies of environmental contaminations should include more thorough examinations on reproduction of the species in question. Sometimes i t may not be practical to study larger species lik e the salmon, but studies on fi s h l i k e Oryzias may provide an insight into what might happen i n the more valuable food fishes. For bioaccumulative toxicants, the use of "safe" factors does not seem to hold, since the "sublethal" concentration may vary with different types of bioaccumulative toxicant (Sprague 1971). For methylmercury, i t seems that the accumulation of toxicant by parent fish does not affect the hatchability of the eggs. This seems to suggest that the toxicant may not have been accumulated in spawned eggs. Since no residue analysis was performed on the spawned eggs, this suggest-ion remains speculative. The observation that juvenile fish are more sensitive to methylmercury than adult fi s h agrees with Sprague's reason-ing (1971). 107 Synthetic LH-RH at doses of 100 ug/g and 1000 ug/g was effective in inducing ovarian development at warm temperatures (23 + 1°C) and short photoperiods (8L/16D), When exposed to methylmercury (10.7 ug/1) even at warm temperatures (23 + 1°C) and long photoperiods (16L/8D), inhibition of spawning activity was observed i n Oryzias. This inhibition was not removed by the injection of synthetic LH-RH. Pituitary cytology revealed high activity in gonadotrophs as stimulated by the injection of synthetic LH-RH. This suggests a possible blockage in the release of gonadotropin(s). Partial restoration of spawning activity i n methylmer-cury-treated fish by injection of LH suggests that the gonads were s t i l l receptive to LH. This study demonstrated effects of methylmercury on both the hypophysis and gonads but failed to show the effects of methyl-mercury at the hypothalamic level. Ovulation in vitro was used to elucidate further the mode of action of methylmercury at the gonadal level. A log dose response was observed i n the percent inhibition of ovulation in vitro. Fifty percent inhibition occurred at concentrations of 55 ug/1 of methylmercury in the incubation medium. Once a block developed with methylmercury, luteinizing hormone was not effective in removing this block. Cortisone was the only steroid tested that was effective in restoring methylmercury-blocked in vitro ovulation. This observation confirms reports that corticosteroids are involved in ovulation, and that i n Oryzias corticosteroids may be produced by the f o l l i c u l a r layer of the oocytes. Methylmercury may have blocked the synthesis of corticosteroids in the f o l l i c u l a r layer of the oocytes and thus blocked ovulation while the addition of exogenous corticosteroid to the medium restored some ovulation i n the methylmercury treated oocytes. 108 The present study has shown that methylmercury, at concentrations as low as 4.8 to 21.5 ug/1 as CH^ Hg"*", has detrimental effects on the reproduction i n Oryzias. These physiological effects are of ecological i importance, not only because survival w i l l be reduced but also because other biological systems closely related to i t may be impaired. Oryzias i s more resistant than some other fish and, i f methylmercury has such detrimental effects on Oryzias, one can expect substantially greater effects of methylmercury on other more "delicate" fishes such as salmon. In the present study, methylmercury seems to act at two levels, the pituitary and the gonad. Though inhibition may have occurred at these two levels, "replacement therapy" seems to be effective i n overcoming some of these effects. Reproductive damages occurring i n nature by methylmercury contamination might thus be pa r t i a l l y remedied by inject-ing the appropriate hormones. The same may be true for other physiologi-cal functions. 109 REFERENCES Aaronson, T. 1971. Mercury i n the environment. Environ. 13: 16-23. Akiyama, A. 1970. Acute toxicity of two organic mercury compounds to the teleost, Oryzias latipes, l n different stages of development. Bull. Jap. Soc. Sci, Fish. 36: 563-570. Albanus, L., L. Frankenburg, C. Grant, U. Van H-artman, A. Jernelov, G. Nordberg, H. Rydalv, A. Schutz, and S. Skerfving, 1972. Toxicity for cats of methylmercury i n contaminated fi s h from Swedish lakes and of methylmercury hydroxide added to fi s h . En-vironmental Research 5: 425-442. Aoki, K. and H. Umeura. 1970. Cell types in the pituitary of the medaka, Oryzias latipes. Endocrinol. Japon. 17: 45-55. Asdell, S. 1962. Mechanism of ovulation. In 'The Ovary', pp. 436-449. Ed. S. Zuckerman, Academic Press, New York. Berlin, M., C.A. Grant, J. Hellberg, J. Hellstrtim and A. Schutz. 1975. Neurotoxicity of methylmercury i n squirrel monkeys. Arch. Environ. Health 30: 340-348. Berthoud, H.R., E.H. Garman and B. Weiss. 1976. Food intake, body weight, and brain histopathology i n mice following chronic methylmercury treatment. Toxicology and Applied Pharmacology 36: 19-30. Breton, B., and C. Weil. 1973. Effects du LH/FSH-RH synthetique et d'extraits hypothalamus de carpe sur le se"cre"tion d'hormone gona-dotrope in vivo chez l a carpe (Cyprinus carpio L.) CR. Acad. Sci. Ser. D., 277: 2061-2064. Brungs, W.A. 1969. Chronic toxicity of zinc to the fathead minnow (Pimephales promelas, Rafinesque). Trans. Am. Fish. Soc. 98: 272-279. 110 Burrows, W.D. and P.A. Krenkel. 1973. Studies on uptake and loss of methylmercury-203 by Bluegills (Lepomis macrochlrus Ref.) Environ. Sci. Tech. 7: 1127-1130. Caster l i n e , J.L. Jr., and CH. Williams. 1972. Elimination pattern of methylmercury from blood and brain of rats (Dams and offspring) after delivery, following oral administration of i t s chloride salt during gestation. B u l l . Environ. Contam. Toxicol. 7(5): 292-295. Chan, K.K. 1976. Presence of a photosensitive daily rhythm in the fe-male medaka, Oryzias latipes. Can. J. Zool. 54: 852-856. Chang, L.W. and J.A. Sprecher. 1976. Degenerative changes in the neo-natal kidney following in-Utero exposure to methylmercury. En-vironmental Research 11: 392-406. Chang, L.W. and S. Yamaguchi. 1974. Ultrastructure changes i n the l i v e r after long-term diet of mercury contaminated tuna. Environmental Research 7: 133-148. Childs, E.A. and J.N. Gaffke. 1973. Mercury content of Oregon ground-fi s h . Fishery Bulletin 71: 713-717. Clarkson, T.W. 1972. Recent advances in the toxicology of mercury with emphasis on the alkyl mercurials. C.R.C. C r i t i c a l Reviews in Toxicology 1: 203-234. Colombo, L., N.A. Bern, J. Pieprzyk and D.W. Johnson. 1973. Biosynthe-sis of 11-deoxycorticoids by teleost ovaries and discussion of their possible role in oocyte maturation and ovulation. Gen. Comp.. Endocrinol. 21: 168-178. " 111 Crandall, CA. and Goodnight, C J . 1962. Effects of sublethal concen-tration of several toxicants on growth of the common guppy, Lebistes reticulatus. Limnol. Oceanogr. 7: 233-239. Crim, L.W. and D.J. Cluett. 1974. Elevation of plasma gonadotropin con-centration i n response to mammalian gonadotropin releasing hormone (GRH) treatment i n the male brown trout as determined by radioimmu-noassay. Endocrin. Res. Commun. 1: 101-110. David, G.F.K. and L.S. Ramaswami. 1971. Changes observed in the FSH and LH cells of the adenohypophysis of Presbtis entellus entellus f o l -lowing cadmium induced testicular necrosis. Experentia 27: 342-343. Deery, D.J. 1974. Determination by radioimmunoassay of the luteinizing hormone-releasing hormone (LHRH) content of the hypothalamus of the rat and some lower vertebrates. Gen. Comp. Endocrinol. 24: 280-285. Desnoyers, P.A. and L.W. Chang. 1975a. Dltrastructural changes i n rat hepatocytes following acute methylmercury intoxication. Environ-mental Research 9: 223-239. Desnoyers, P.A. and L.W. Chang. 1975b. Ultrastructural changes in the l i v e r after chronic exposure to methylmercury. Environmental Re-search 10: 59-75. Diamond, S.S. and Sleight, S.D. 1972. Acute and subchronic methylmercury toxicosis i n the rat. Toxicol. Appl. Pharmacol. 23: 197-207. Donaldson, E.M. 1973. Reproductive endocrinology of fishes. Amer. Zool. 13: 909-927. Egami, N. and K. Hosokawa. 1973. Responses of the gonads to environmen-t a l changes in the f i s h , Oryzias latipes. In 'Responses of fi s h to Environmental Changes'. Ed. W. Chavin, pp. 279-301. Charles C. Thomas, Publisher. Fagerstrom, T. and A. Jernelov. 1971. Formation of methyl-mercury from pure mercuric sulphide i n aerobic organic sediment. Water Research 5: 121. Fimreite, N. 1970. Mercury use i n Canada and their possible hazard as sources of mercury contamination. Environ. Pollution 1: 119-131. Fimreite, N. and L.M. Reynolds. 1973, Mercury contamination of f i s h in Northwestern Ontario, J. Wildl. Manage. 37: 62-68. Fitzgerald, W.F. and W.B. Lyons. 1973. Organic mercury i n Coastal waters. Nature 242: 452-453. Fowler, B.A., H.W. Brown, G.W. Lucier and M.E. Beard. 1974. Mercury up-take by renal lysosomes of rats ingesting methylmercury hydroxide. Ultrastructural observations and energy dispersive X-ray analysis. Arch. Pathology 98: 297-301. Foxcroft, G.R., J.W, Hamilton and A.V. Nalbandov. 1975. Ovulation and luteal function i n the rabbit i n response to injection or infusion of synthetic gonadotropin-releasing hormone (Gm-LH). Biol. Reprod. 12: 284-288. Freeman, H.C. and D.A. Home. 1973. Total mercury and methylmercury content of the American eel (Anguilla rostrata). J. Fish. Res. Bd. Can. 30: 454-456. 113 Gavis, J. and J.F. Ferguson. 1972. The cycling of mercury through the environment. Water Research 6: 989-1008. Gilani, S.H. 1975. Congenital abnormalities i n methylmercury poisoning. Environmental Research 9: 128-134. Goldwater, L.J. 1971, Mercury in the environment. Sci. Am. 224: 15-21. Goswami, S.V. and B.I. Sundararaj. 1972a. Temporal effects of ovine l u -teinizing hormone and desoxycorticosterone acetate on maturation and ovulation of oocytes of the catfish, Heteropneustes f o s s i l i s , (Bloch): An in vivo and in vitro study. J. Exp. Zool. 178: 457-466. Goswami, S.V. and B.I. Sundararaj. 1972b. In vitro maturation and ovula-tion of the catfish, Heteropneustes f o s s i l i s (Bloch): Effects of mammalian hypophyseal hormones, Catfish pituitary homogenate, steroid precursors and metabolites, and gonadal and adrenocortical steroids. J. Exp. Zool. 178: 467-478, Goswami, S.V. and B.I, Sundararaj. 1973. Effects of actinomycin D, mitomycin C puromycin, and cycloheximide on desoxycorticosterone-induced in vitro maturation i n oocytes of the catfish, Heteropneutes  f o s s i l i s (Bloch). J. Exp. Zool. 185: 327-332. Goswami, S.V. and B.I. Sundararaj. 1974. Effects of C^g, C^g, and steroids on i n vitro maturation of oocytes of the catfish, Heteropneustes f o s s i l i s (Bloch). Gen. Comp. Endocrinol. 23: 282-285. Heller, H. 1972. The effects of neurohypophysial hormones on the female reproductive tract of lower vertebrates. Gen. Comp. Endocrinol. Suppl. 3: 703-714. 114 Heinz, G. 1975. Effects of methylmercury on approach and avoidance behavior of mallard ducklings. Bull. Environ. Contamin. Toxicol. 13: 554-564. Heinz, G. 1974. Effects of low dietary levels of methylmercury on mallard reproduction. Bull. Environ., Contamin. Toxicol. 11: 386-392. Herigstad, R.R., CK. Whitehaid and N. Beyer. 1972. Chronic methylmer-cury toxicosis in calves. J. Am. Vet. Med. Ass. 160: 173-182. Hirose, K. 1971. Biological study on ovulation in vitro of f i s h - I. Effects of pituitary and chorionic gonadotropins on ovulation in  vitro of medaka, Oryzias latipes. Bu l l . Jap. SOCJ Sci. Fish. 37: 585-591. , - _ Hirose, K. 1972a. Biological study on ovulation i n v i t r o of f i s h - IV. Induction of in_ vitro ovulation i n Oryzias latipes oocytes using steroids. Bull. Jap. Soc. Sci. Fish. 38: 457-461. Hirose, K. 1972b. Biological study on ovulation in vitro of f i s h - V. Induction of i n vitro ovulation in Oryzias latipes removed from their f o l l i c u l a r tissues. Bull. Jap. Soc. Sci. Fish. 38: 1081-1096. Hirose, K. 1972c Effectiveness of duration of exposure to hydrocorti-sone and HCG on ovulation i n vitro in Oryzias latipes. Bu l l . Jap. Soc. Sci. Fish. 38: 869. Hirose, K. and H. Hirose. 1972. Biological study on ovulation in vitro of f i s h . II. Differences of ovulation rates in Oryzias latipes a t the various starting hours of incubation. Bull. Jap. Soc. Sci. Fish. 38: 33-42. 115 Hirose, K. 1973. Biological study on ovulation in vitro of f i s h - VI. Effects of metopirone (SU-4885) on salmon gonadotropin- and cortisol-induced in vitro ovulation in Oryzias latipes. Bull. Jap. Soc. Sci. Fish. 39: 765-769. Hirose, K. and E.M. Donaldson. 1972, Biological study on ovulation in vitro of fish - III. The induction of in vitro ovulation of Oryzias latipes oocytes using salmon pituitary gonadotropin. Bull. Jap. Soc. Sci. Fish. 38: 97-100, Hirose, K. and R. Ishida. 1974. Ineuction of ovulation i n the ayu, Plecoglossus a l t i v e l l s , with LH-releasing hormone (LH-RH). Bull. Jap. Soc. Sci. Fish. 40: 1235-1240. Hirsch, G.H. 1971. Inhibition of renal organic ion transport by methyl-mercury. Environmental Physiology 1: 51-54. Hoar, W.S. 1969. Reproduction, In "Fish Physiology", vol. 3 (W.S. Hoar and D.J. Randall, eds.), Academic Press, New York. Hughes, A.F.W. 1950. The effect of inhibitory substances on c e l l d i v i -sion. A study of l i v i n g cells in tissue cultures. Quart. Microsc. Sci. 91: 251-278. Hughes, R., R. Belser and C.W. Brett. 1975. Behavioral impairment pro-duced by exposure to subclinical amounts of methylmercury chloride. Environmental Research 10: 54-58. Humphrey, R.R., W.C. Dermody, H.O. Brink, F.B. Bousley, N.H. Schottin, F. Sekowski, J.W. Vaitkus, H.T. Veloso and J.R. Reed. 1973. Induction of luteinizing hormone (LH) release and ovulation in rats, hamsters and rabbits by synthetic luteinizing hormone-re-leasing factor (LRF). Endocrinology 92: 1515-1526. 116 Ikeda, Y., M. Tobe, K. Kobayashi, S. Suzuki, Y. Kawasaki and H. Yonemaru. 1973. Long-term toxicity study of methylmercurie chloride i n monkeys. Toxicology 1: 361-375. Iverson, „F., R.H. Downie, C. Paul and Trenholm, H.L. 1973. Methylmer-cury: Acute toxicity, tissue distribution and decay profiles in the guinea pig. Toxicol. Appl. Pharmacol. 24: 545-554. Iverson, F., R.H. Downie, H.L. Trenholm and C. Paul. 1974. Accumula-tion and tissue distribution of mercury i n the Guinea Pig during subacute administration of methylmercury. Toxicology and Applied Pharmacology 27: 60-69. Jarvenpaa, T., M. Tillander and J.K. Miettinen. 1970. Methylmercury: half-time of elimination i n flounder, pike and eel. F.A.O. Technical Conference on marine pollution and i t s effect on l i v -ing resources and fishing. Jensen, S,, and A. Jernelov* 1969. Biological methylation of mercury in aquatic organisms. Nature 223: 753-754. Jernelov, A. and H. Lann. 1971. Mercury accumulation i n food chain. Oikos 22: 403-406. Johnels, A.G., T. Westermack, W., Berg, P.I., Persson and B. Sjostrand. 1967. Pike (Essox lucius L.) and some other aquatic organisms in Sweden as indicators of mercury contamination i n the environment. Oikes 18: 303-333. Jones, J.R. 1964. Fish and river pollution. 203 pp. Butterworths, London. Kasuga, S. and H. Takahashi. 1971, The preoptico-hypophysial neuro-secretory system of the medaka, Oryzias latipes, and i t s changes i n relation to the annual reproductive cycle under natural condi-tions. Bull. Fac. Fish. Hokkaido Univ. 21: 259-268. Katsuki, S., S. Hirai , and T. Terao. 1957. On the disease of central nervous system in Minamata District with unknown etiology, with special references to the c l i n i c a l observation. Kumamoto Igakkai Zasshi 31: 110-212. Katz, A. 1972. Mercury pollution: The making of an environmental c r i s i s . C.R.C. C r i t i c a l Reviews in Environmental Control 2: 517-534. Kaul, S., and L. Vollrath. 1974. The goldfish pituitary. I. Cytology, Cell Tiss. Res. 154: 211-230. Kendall, M.W. 1975, Acute effects of methylmercury toxicity in channel catfish, (Ictalurus punctatus) kidney. Bull, Environ, Contamin. Toxicol. 13: 570-578. Khera, K.S. 1973. Reproductive capability of male rats and mice treated with methyl mercury. Toxicol. Appl. Pharmacol. 24: 167-177. Khoo, K.H. 1974. Steroidogenesis and the role of steroids in the endo-crine control of oogenesis and vitellogenesis in the goldfish, Carassius auratus. Ph.D. Thesis, the University of B r i t i s h Columbia, 123 pp. Kilhstrdm, J.E., C, Lundberg, and L, Hulth, 1971. Number of eggs and young produced by zebrafish (Brachydanio rerio, Ham-Buch) spawn-ing in water containing small amounts of phenylmercuric acetate. Environ. Res. 4: 355-359. 118 KilhstrMm, J.E. and L. Hulth. 1972. The effect of phenylmercuric ace-tate upon the frequency of hatching of eggs from the zebrafish. Bull. Environ. Contem. Toxicol. 7: 111-114. Kim, S.U. 1971. Neurotoxic effects of alkyl mercury compound on myeli-nating cultures of mouse cerebellum. Exp. Neurology 32: 237-246. Lam, T.J., S. Pandey, Y. Nagahama, and W.S. Hoar. 1976. Effects of syn-thetic luteinizing hormone-releasing hormone (LH-RH) on ovulation and pituitary cytology of the goldfish, Carassius auratus. Can. J. Zool. 54: 816-824. Leduc, G. 1966. Une bouteille a debit constant pour volumes de liquides. La Naturaliste Canadiene 93: 61-64. Lit c h f i e l d , J.T. 1949. A method for rapid graphic solution of time per-cent effect curves. J. Pharmac, Exp. Ther. 97: 399-408. Lit c h f i e l d , J.T. and F. Wilcoxon. 1949. A simplified method of evaluat-ing dose effect experiments. J. Pharmac. Exp. Ther. 96: 99-113. Lock, R.A.C. 1974. Methylmercury uptake from water and food by aquatic organisms from different trophic levels. M.Sc. Thesis. Simon Fraser University, 42 pp. Lucier, G., 0. McDaniel, P. Brubaker and R. Klein. 1972. Effects of methylmercury hydroxide on rat l i v e r microsomal enzymes. Chem. Biol. Interact. 4: 265-280. Macey, M.J., G.E. Pickford and R.E. Peter. 1974. Forebrain localiza-tion of the spawning reflex response to exogenous neurohypophysial hormones i n the k i l l i f i s h , Fundulus heteroclitus. J. Exp. Zool. 190: 269-280. 119 Matsumura, F., F. Gotch Doherty, K. Furukawa and G.M. Boush. 1975. 203 Incorporation of Hg into methylmercury in fish l i v e r : Studies on biochemical mechanisms in vitro. Environmental Research 10: 224-235. Mazzi, V., C. Vellano, D. Colluci and A. Merlo. 1974. Gonadotropin stimulation by chronic administrations of synthetic luteinizing hormone-releasing hormone in hypophysectomized pituitary grafted male newts. Gen. Comp. Endocrinol. 24: 1-9. Mclntyre, J.D. 1973. Toxicity of methylmercury for steelhead trout sperm. Bull. Environ. Contamin. Toxicol. 9: 98-99. Miettinen, J.K., M. Tillander, K. Rissanen et a l . 1969. Distribution and excretion of rate of phenyl- and methylmercury nitrate i n fi s h , mussels, molluscs and crayfish. Proc. Jap. Conf. Radioisotop. pp. 474-478. Mount, D.I. 1968. Chronic toxicity of copper to fathead minnows (Pimephales promelas, Rafinesque). Water Research 2: 213-233. Mykkanen, H.M. and H,E. Ganther. 1974. Effect of mercury on erythrocyte glutathione reductase activity. In vivo and i n vitro studies. Bull. Environ. Contamin. Toxicol. 12: 10-16. Nelson, N. 1971. Study group on mercury hazards. Environ. Res. 4: 1-69. Newsome, W.H. 1971. Determination of methylmercury in fish and in cereal grain products. J. Agr. Food Chem. 19: 567-569. Okinaka, S., M. Yoshikawa, T. Mozai, T. Mizune, T. Tercio, H. Watanshe, K, Ogiharo, J. Harai, Y. Yashino, T. Inose, S. Azar and H. Tsuda. 1964. Encephalomyelopathy due to an organic mercury compound . Neurology 4: 68-86. 120 Olson, K.R., H.L. Bergman and P.O. Fromm. 1973. Uptake of methylmercur-i c chloride and mercuric chloride by trout: A study of uptake path-ways into the whole animal and uptake by erythrocytes in vitro J. Fish. Res. Bd. Canada 30: 1293-1299. Olson, K.H., and P.O. Fromm. 1973. Mercury uptake and ion distribution in g i l l s of rainbow trout (Salmo gairdne.rii): tissue scans with an electron microscope. J. Fish. Res. Bd. Can. 30: 1575-1578. O'Connor, D.V. and P.O. Fromm. 1975. The effect of methylmercury on g i l l metabolism and blood parameters of rainbow trout. Bull. Environ. Contamin. Toxicol. 13: 406-411. Passow, H.A., A. Rothstein and T.W. Clarkson, 1961. The general pharma-cology of heavy metals. Pharmacol. Rev. 13: 1885. Peakall, D.B. and Lincer, J.L. 1972. Methyl mercury: Its effect on egg-shell thickness. Bull. Environ. Contamin. Toxicol. 8(2): 89-90. Pendergrass, P. and P. Schroeder. 1976. The ultrastructure of the thecal c e l l of the teleost, Oryzias latipes, during ovulation in vitro. J. Reprod. Fert. 47: 229-233. Peter, R.E. 1970. Hypothalamic control of thyroid gland activity and gonadal activity in goldfish. Gen. Comp. Endocrinol. 14: 334-356. Peter, R. 1973. Neuroendocrinology of teleosts. Am. Zool. 13: 743-757. Peterson, C.L., W.L. Klawe and G.D. Sharp. 1973. Mercury in tunas: A Review. Fishery Bulletin 71: 603-613. Redding, T.W., A.J. Kastin, D. Gonzales-Barcena, D.H. Coy, D.S. Schalch and A.W. Schally. 1973. The h a l f - l i f e metabolism and excretion of t r i t i a t e d luteinizing hormone-releasing hormone (LH-RH) in man. J. Clin. Endocrinol. Meta. 37: 626-631. 121 Reevs, J.J., P.C. Harrison, and J.M. Casey. 1973. Ovarian development in hens treated with synthetic (porcine) luteinizing hormone-releatiing hormone/follicle stimulating hormone releasing hormone (LH-RH/FSH-RH). Poult. Sci. 52: 1883-1886. Rucker, R.R. and D.F. Amend. 1969. Absorption and retention of organic mercurials by rainbow trout and chinook and sockeye salmon. Prog. Fish Culturist 31: 197-201 Saha, J.G. 1972. Significance of mercury in the environment. Residues Review 42: 103-164. Schally, A.V., A. Arimura and A.J. Kastin. 1973. Hypothalamic regula-tory hormones. Science 179: 341-350. Skerfving, S., K. Marsson and J. Lindstein. 1970. Chromosome breakage i n human exposed to methyl mercury through fish consumption. Aech, Environ. Health. 21: 133-139. Skerfving, S. 1972. Mercury in fi s h . Some toxicological considerations. Fd. Cosmet. Toxicol. 10: 545-556. Skerfving, S i 1974. Methylmercury exposure, mercury levels i n blood and hair, and health status i n Swedes consuming contaminated fi s h . Toxicology 2: 3-23, Sprague, J.B. 1969. Measurement of pollutant toxicity to fish - I. Bioassay methods for acute toxicity. Water Research 3: 793-821. Sprague, J.B. 1971. Measurement of pollutant toxicity to fi s h . III. Sublethal effects and "safe" concentrations. Water Research 5: 245-266. Spyker, J.M., S,B, Sparber and A.M. Goldberg. 1972. Subtle consequences of methylmercury exposure: behavioral deviation i n offspring of treated mothers.. Science 177: 621-623. 122 Storwsand, G.S., J.L. Anderson, W.H. Gutenmen, L.A. Bache and D.J. Llsk. 1971. Eggshell thinning in Japanese quail fed mercuric chloride. Science, 173: 1030-1031. Sundararaj, B.I. and S.V. Goswami. 1974. Effects of ovine luteinizing hormone and porcine adrenocorticotropin on maturation of oocytes of the catfish, Heteropneustes f o s s i l i s (Bloch), in Ovary-Inter-renal co-culture. Gen. Comp. Endocrinol. 23: 276-281. Takeuchi, T., N. Morikawa, H. Matsumoto, and Y. Shiraishi. 1962. A pathological study of Minamata disease in Japan. Acta Neuro-pathol. 2: 40-57. Tajning, S. 1967, Biological effects of methylmercury dicyandiamide-treated grain in the domestic fowl, Gallus gallus L. Oikos Suppl. 8: 1-116. Thornton, V.F. 1974. Hypothalamic control of gonadotropin release in amphibian: Evidence from studies of gonadotropin release in_ vitro and in vivo. Gen. Comp, Endocrinol. 23: 294-301. Tokuomi, G. 1961. Minamata disease, an unusual neurological disorder occuring i n Minamata, Japan. Kumamoto Med. J. 14: 47-64. Van Tienhoven, A., and A.V. Schally. 1972. Mammalian luteinizing hor-mone-releasing hormone induces ovulation in the domestic fowl. Gen. Comp. Endocrinol. 19: 594-595. Vellano, C , A. Bona, V. Mazzi and D. Colluci. 1974. The effect of synthetic luteinizing hormone releasing hormone on ovulation in the crested newt. Gen. Comp. Endocrinol. 24: 338-340. 123 Voege, F.A. 1971. Levels of mercury contamination in water and i t s boundaries, p. 107-117. In "Mercury i n Man's Environment". Proc. Symposium from Royal Society of Canada. 1971. Ottawa, i Canada. West88, G. 1969. Mercury and methylmercury levels in some animal food products. Var. Foeda, 7: 137-154. White, J.F. and A. Rothstein. 1973. The interaction of methylmercury with erythrocytes. Toxicology and Applied Pharmacology 26: 370-384. Wood, J.M., E.S. Kennedy and CG. Rosen. 1968. Synthesis of methyl-mercury compounds by extracts of a methanogenic bacterium. Nature 220: 173-174. Yoshioka, H. 1962. On the effects of environmental factors upon the reproduction of fishes. The effects of daylength on the reproduc-tion, of the Japanese k i l l i f i s h , Oryzias latipes. Bull. Fac. Fish, Hokkaido Univ. 13: 123-136. Yoshioka, H. 1963. On the effects of enviornmental factors upon the reproduction of fishes. 2. Effects of short and long daylength on Oryzias latipes, during the spawning season. Bull, Fac. Fish. Hokkaido Univ. 14: 137-171. 

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