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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.  S i r George Williams University, 1968  M.Sc.  S i r 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 p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y I  further  for  freely  of  this  requirements  for  thesis for  It  financial  The  of  g a i n s h a l l not  U n i v e r s i t y o f B r i t i s h Columbia  e  September 7.1977.  that  this  thesis or  i s understood that copying or p u b l i c a t i o n  ZOOLOGY  2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  for  reference and study.  t h a t p e r m i s s i o n for e x t e n s i v e copying o f  representatives.  Department  t  the  B r i t i s h Columbia, I agree  available  written permission.  a  of  s c h o l a r l y purposes may be granted by the Head of my Department  by h i s  D  fulfilment  the U n i v e r s i t y of  s h a l l make it  agree  in p a r t i a l  be allowed without my  ii  ABSTRACT  This study evaluates the t o x i c i t y , accumulation, chronic e f f e c t s and mode of action of methylmercury on the reproduction of the teleost f i s h , Oryzias l a t i p e s . The median l e t h a l concentration, 96h-LC50, for adult Oryzias was + 8 8 + 9 . 8 pg CH^Hg 11 as determined i n a s t a t i c system.  Residue analysis  by gas chromatography showed that f i s h exposed to 43 to 1000 ug CH^Hg /l +  had tissue levels below 40 ug CH^Hg /g while f i s h exposed to more than +  1000 ug CH^Hg^/l accumulated methylmercury steadily and reached l e v e l s as high as 408.1 ug CH.jHg /g. +  25 ug CH.jHg /g. +  Death seems to occur when tissue l e v e l reaches  Studies on long-term exposure to 0.0, 4,3, 10.7 and 21.5 u  of methylmercury i n 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 i n i n h i b i t i o n of o v i p o s i t i o n .  This observation occurred only oh days when f i s h 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 i n h i b i t i o n +  was observed even on days when f i s h 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 i n i n h i b i t i o n of spawning.  This i n h i b i t i o n was d i r e c t l y related to the  log of exposure concentrations.  At the end of s i x weeks, both male and  female gonads showed reduction i n s i z e ; the females were more s e n s i t i v e . However, h a t c h a b i l i t y of the spawned eggs was not affected by the exposure.  iii Juvenile f i s h were very s e n s i t i v e to methylmercury. of exposure, one-week old juvenile exposed to 0.0, CH Hg /l had mortality rate of 2.2%, +  3  54.3%, 64.9%  Synthetic LH-RH, at concentrations was  4.3,  After two weeks 10.7  and 99.4%  ug  respectively.  of 100 and 1000 ng/g body weight,  e f f e c t i v e i n stimulating ovarian development i n Oryzias.  that the LH-RH (synthesis  and 21.5  This shows  based on structure of porcine LH-RH) has b i o l o -  g i c a l a c t i v i t y i n Oryzias. When exposed to methylmercury, spawning a c t i v i t i e s were i n h i b i t e d . LH i n j e c t i o n s were able to restore the spawning a c t i v i t i e s i n h i b i t e d by the methylmercury treatment, but not LH*-RH. However, histology of the p i t u i t a r y gland showed stimulation of gonadotropic c e l l s by LH-RH i n j e c t i o n with no r e s t o r a t i o n of spawning a c t i v i t i e s . cury- may  This suggests that methylmer-  be blocking the release of gonadotropin.  In v i t r o ovulation was  affected by previous exposure to methylmercury.  Addition of methylmercury d i r e c t l y to the incubation medium further reduced the percentage of i n v i t r o ovulation i n the previously treated f i s h . oocytes from untreated  donor f i s h , the percent  l a t i o n by methylmercury was  i n h i b i t i o n of i n v i t r o ovu-  d i r e c t l y related to the log of doses used.  possible bioassay with i n v i t r o ovulation was  suggested.  A  Among the various  steroids used (progesterone, cortisone, e s t r a d i o l and testosterone), sone was  Using  corti-  the only s t e r o i d e f f e c t i v e i n r e s t o r i n g i n v i t r o ovulation blocked  by the presence of methylmercury i n the incubation medium. E c o l o g i c a l 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 t o x i c i t y tests Long-term exposure Residue analysis Extraction and p u r i f i c a t i o n Gas chromatography RESULTS Acute t o x i c i t y tests Long-term exposure DISCUSSION SECTION I I : 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 s u r v i v a l of juvenile f i s h  ....  34  ....  35  E f f e c t s on oviposition  ....  35  E f f e c t s on gonadal development and spawning  ....  35  E f f e c t s of juvenile f i s h  ....  41  ....  51  RESULTS  DISCUSSION SECTION I I I : MODE OF ACTION OF METHYLMERCURY ON REPRODUCTION  54  INTRODUCTION  55  MATERIALS AND METHODS  61  On ovarian maturation by synthetic LH-RH  ....  61  ....  62  ....  63  On e f f e c t s of LH-RH and LH i n j e c t i o n i n methylmercury treated f i s h On e f f e c t s of methylmercury on i n v i t r o ovulation RESULTS E f f e c t of Synthetic LH-RH on ovarian maturation  66 ....  66  ....  73  E f f e c t s of methylmercury on i n v i t r o ovulation  ....  84  Pre-exposure to methylmercury f o r 6 weeks  ....  84  E f f e c t s of methylmercury on normal oocytes from untreated f i s h  ....  90  E f f e c t s of different steroids on methylmercury i n h i b i t e d ovulation i n v i t r o  ....  90  ....  96  ....  96  E f f e c t s of Synthetic LH-RH and LH i n j e c t i o n s on methylmercury treated f i s h  DISCUSSION E f f e c t of synthetic LH-RH on ovarian maturation  Table of Contents (cont'd)  vi Page  E f f e c t s of synthetic LH-RH and LH i n j e c t i o n s on methlymercury treated f i s h .... E f f e c t of methylmercury on i n v i t r o ovulation  ....  GENEEAL DISCUSSION REFERENCES  99 101 105  ....  109  LIST OF TABLES  Mortality of Oryzias l a t i p e s exposed to d i f f e r e n t concentrations of methylmercury over a 6-week period E f f e c t s of 4-hour exposure to d i f f e r e n t concentrations of methylmercury on the oviposition of Oryzias l a t i p e s Mortality of one-week o l d juvenile Oryzias l a t i p e s exposed to d i f f e r e n t concentrations of methylmercury f o r two weeks S t a t i s t i c a l significance of female gonadosomatic indices among the various treatments . Effects of d i f f e r e n t concentrations of methylmercury on percent i n v i t r o ovulation i n Oryzias previously treated with methylmercury for 6 weeks The e f f e c t s of various concentrations of methylmercury on ovulation i n v i t r o of Oryzias l a t i p e s incubated with 10 ug/ml porcine LH at 1700 hours The effects of methylmercury on ovulation i n v i t r o of Oryzias l a t i p e s incubated with and without LH. (10 yg/ml) commencing at 2200 hours The e f f e c t s of steroids on ovulation i n v i t r o of Oryzias l a t i p e s 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 f i s h to low concentrations of methylmercury, showing the pattern of water flow, heating system, one Mariotte bottle f o r the metering of toxicant and one test-tank  FIGURE 2.  E f f e c t of d i f f e r e n t concentrations of methylmercury on the LT50 of Oryzias l a t i p e s at 23 + 1°C.  FIGURE 3.  Determinations of the median l e t h a l concent r a t i o n (LC50) at 96 hr o f methylmercury f o r Oryzias l a t i p e s  FIGURE 4.  Accumulation of methylmercury i n Oryzias l a t i p e s exposed to d i f f e r e n t concentrations of methylmercury during the acute tests  FIGURE 5.  Accumulation of methylmercury i n Oryzias l a t i p e s under long-exposure to d i f f e r e n t concentrations of methylmercury  FIGURE 6.  Weekly observations, from the 3rd to the 6th week, on spawning and egg-laying of Oryzias l a t i p e s f o r the d i f f e r e n t concentrations of methylmercury  FIGURE 7.  E f f e c t s of d i f f e r e n t concentrations of methylmercury on the t o t a l number of spawning and t o t a l number of eggs l a i d during s i x weeks of exposure  FIGURE 8.  The percent spawning a c t i v i t y of egg-laying a b i l i t y of Orvzias l a t i p e s exposed to d i f f e r ent concentrations of methylmercury  FIGURE 9.  The percent i n h i b i t i o n of spawning i n Oryzias l a t i p e s i n water containing d i f f e r ent concentrations of methylmercury  FIGURE 10.  Gonadosomatic indices of male and female Orvzias l a t i p e s exposed t o d i f f e r e n t concentrations of methylmercury f o r 6 weeks  FIGURE 11.  The percent h a t c h a b i l i t y of eggs exposed to different concentrations of methylmercury  L i s t of Figures (cont'd)  ix  Page 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 i n t e r r e l a t i o n s h i p between environmental cues, hypothalamus, hypophysis, and gonads ...  56  E f f e c t s of d i f f e r e n t dosages of synthetic LH-RH on the gonadosomatic index and the percent d i s t r i b u t i o n of Class I I I oocytes  67  Section of ovary from Oryzias injected with saline twice-a-week f o r 6 weeks at 2 3 + 1 C under 8L/16D  69  Section of ovary from Oryzias injected with 10 ug/g synthetic LH-RH twice-a-week f o r 6 weeks at 23 + 1°C under 8L/16D  69  Section of ovary from Oryzias injected with 100 ng/g synthetic LH-RH twice-a-week f o r 6 weeks at 23 + 1°C under 8L/16D  71  Section of ovary from Oryzias injected with 1000 ng/g synthetic LH-RH twice-a-week f o r 6 weeks at 23 + 1°C under 8L/16D  71  Section of p i t u i t a r y gland from Oryzias i n jected with saline twice-a-week f o r 6 weeks under 23 + 1°C and 8L/16D  74  Section of p i t u i t a r y gland from Oryzias i n jected with 10 ng/g synthetic LH-RH twice-aweek f o r 6 weeks under 23 + 1 C and 8L/16D ...  74  Section of p i t u i t a r y gland from Oryzias i n jected with 100 ng/g synthetic LH-RH twicea-week f o r 6 weeks under 23 + 1 C and 8L/16D  76  Section of p i t u i t a r y gland from Oryzias i n jected with 1000 ng/g synthetic LH-RH twicea-week f o r 6 weeks under 23 + 1 C and 8L/16D  76  Weekly observation on spawning and egglaying of Oryzias f o r the various hormonal treatment i n the presence of methylmercury  78  E f f e c t s of various hormonal treatments on percent spawning, male and female gonadosomat i c indices at the end of 6-week methylmercury exposure  81  L i s t of Figures (cont'd)  FIGURE 24.  FIGURE 25.  FIGURE 26.  FIGURE 27.  FIGURE 28.  x  Section of p i t u i t a r y gland from Oryzias exposed to clean water and injected with saline twicea-week f o r 6 weeks under 23 + 1°C and 16L/8D  85  Section of p i t u i t a r y gland from Oryzias exposed to methylmercury and injected with s a l i n e twice-a-week f o r 6 weeks under 23 + 1°C and 16L/8D  85  .....  Section of p i t u i t a r y gland from Oryzias exposed to methylmercury and injected with l u t e i n i z i n g hormone twice-a-week f o r 6 weeks under 23 + 1°C and L6L/8D  87  Section of p i t u i t a r y gland from Oryzias exposed to methylmercury and injected with l u t e i n i z i n g hormone-releasing hormone twicea week f o r 6 weeks under 23 + 1°C and 16L/8D  87  Percent i n h i b i t i o n of i n v i t r o ovulation by various concentrations of methylmercury i n Incubation media at 1700 hours  .....  92  xi  ACKNOWLEDGEMENTS  I would l i k e to extend my sincere thanks and indebtedness to my supervisor, Dr. W.S.  Hoar, for his i n t e r e s t , advice and encouragement  throughout t h i s study. S p e c i a l thanks are also extended to the members of my committee:  doctoral  Drs, P. Larkin, T. Northcote and H. Kasinsky of the Univer-  s i t y o f B r i t i s h Columbia, Vancouver, Dr. P. O l o f f s of Simon Fraser U n i v e r s i t y , Burnaby and Dr. J , Thompson of Marine Sciences Branch, Environment Canada, V i c t o r i a , f o r t h e i r generous assistance and for reading the t h e s i s . a n a l y s i s i s deeply  The use of Dr. J . Thompson's laboratory f o r residue appreciated.  I am obligated to the v i s i t i n g research fellows: Dr. T.J.  Lam,  Dr. S". Pandey and e s p e c i a l l y Dr. Y. Nagahama f o r many stimulating discussions.  H e l p f u l 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. E i n a n c i a l support for t h i s 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 l e a s t , I would l i k e to express my deepest gratitude to  my wife, Bernadette, for her constant encouragement and patience out t h i s study.  through-  1  GENERAL INTRODUCTION  Since the outbreak of Minamata Disease i n 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 w e l l  documented (Fimreite 1970, Aaronson, 1971; Goldwater, 1971; Nelson 1971; Saha 1972) and the cycling of mercury through the environment has been r e viewed (Gavis and Ferguson 1972) . Poisoning by methylmercury has been demonstrated at a l l l e v e l s i n phylogeny (Skerfving 1972; Clarkson 1972; Katz 1972).  Since the Minamata  episode, studies have shown that f i s h are c l e a r l y s e n s i t i v e to methylmercury (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 p h y s i o l o g i c a l  processes of the animal.  However, some information does exist on the long  term, chronic e f f e c t s of methylmercury i n f i s h ; but information of i t s e f f e c t s on f i s h reproduction i s p a r t i c u l a r l y sparse (Sprague 1971). The present work investigates the t o x i c i t y , accumulation of methylmercury under long and short term exposures and the e f f e c t s of low concentrations of methylmercury on o v i p o s i t i o n , gonadal development, spawning a c t i v i t y , h a t c h a b i l i t y of eggs and s u r v i v a l of hatchlings i n Oryzias l a t i p e s . Since methylmercury has been shown to be a bioaccumulative toxicant, corr e l a t i o n w i l l be made between the amount of accumulated methylmercury i n the  f i s h and i t s e f f e c t s on the reproductive processes,  The l a t t e r part  of t h i s study w i l l examine the mode of action of methylmercury on the  • reproductive physiology i n Oryzias.  .2  The possible e f f e c t s of methylmer-  cury on the endocrine system concerned with reproduction w i l l be i n v e s t i gated.  An attempt w i l l be made to determine whether a blockage  of hor-  mone a c t i v i t y by methylmercury occurs i n the hypothalamic-hypophysealgonadal axis.  This study i s not only of academic i n t e r e s t but might also  suggest a possible remedy f o r f i s h that may be affected s i m i l a r l y i n nature.  Oryzias i s a much hardier f i s h than most other fishes that are of  commercial value, l i k e the salmon.  However, this f i s h i s a convenient  animal f o r laboratory studies on reproduction.  I t i s hoped that the pre-  sent study with Oryzias w i l l suggest some possible e f f e c t s that methylmercury may  have on commercially  important  fishes.  Oryzias o f f e r s several advantages as an experimental animal. small s i z e (30 mm l i m i t e d spaces.  Its  adult) allows handling of large numbers i n r e l a t i v e l y By manipulating temperature and photoperiod, reproductive-  l y mature i n d i v i d u a l s can be obtained throughout the year. induced, occurs d a i l y for 4 to 6 weeks.  Spawning, once  Since Oryzias i s oviparous, the  number of spawnings, number of spawned eggs, h a t c h a b i l i t y of the eggs and mortality of the hatchlings can be e a s i l y quantified. of this f i s h are also i d e a l for the study. 010Q  The spawning habits  Females ovulate d a i l y between  and 0400 hours and o v i p o s i t i o n occurs about two hours a f t e r daybreak;  and the eggs hatch i n about two weeks.  This not only allows studies of  the e f f e c t s of methylmercury on the reproductive physiology of the adults but a l s o , i f desired, on the second generation.  F i n a l l y , there i s much to  be said for working with an animal which has been the subject of so much behavioural, p h y s i o l o g i c a l , 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, F i t z g e r a l d Lyons 1972),  and  In f i s h , mercury may reach l e v e l s 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 i n f i s h i s present as methylmercury (Westtfo 1969). but  The source of methylmercury has not been w e l l determined,  i t has been observed that microorganisms, especially those from s e d i -  ments, can methylate mercury (Fagerstrom and Jernelov 1971; Jensen and Jernelov 1969; Wood e t a l . , 1968).  The methylmercury may then be accu-  mulated to a 'harmful' concentration i n the higher trophic l e v e l s , e.g., f i s h , v i a the food chain (Jernelov and Lann 1971). A maximum permissable l e v e l of mercury i n f i s h of 0.5 yg/g was established by the U.S. Food and Drug Administration i n 1970. At the same time people were cautioned that the human hazard depends l a r g e l y on the quantity of contaminated f i s h eaten (Katz 1972). In mammals, tissue accumulation, d i s t r i b u t i o n and excretion of methylmercury have been documented (Iverson et a l . , 1973; 1974; Casterline J r . and Williams 1972; Skerfving 1974; Ikeda 1973).  Effects of methylmercury  in humans are w e l l known. Methylmercury accumulates i n the central nervous system.  Severe poisoning r e s u l t s i n gross c o n s t r i c t i o n of the v i s u a l  f i e l d , cerebellar ataxia, dysarthria, sensory changes and impairment of hearing.  Memory and i n t e l l i g e n c e are unaffected.  These symptoms are  generally i r r e v e r s i b l e but the motor disturbances may improve a f t e r  4 rehabilitation.  Methylmercury has also the unique property of crossing  the blood-brain b a r r i e r and the p l a c e n t a l - f o e t a l b a r r i e r (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 erythrocytes (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 e t a l . , 1972; Ikeda e t a l . , 1973; Diamond and Sleight  1972;  Barthoud et a l . , 1976; B e r l i n et a l . , 1975); i t also a f f e c t s behaviour (Spyker et a l . , 1972; Hughes 1975) and reproduction (Casterline J r . and Williams 1972, Khera 1973; Skerfving 1974). In b i r d s , methylmercury poisoning impaired reproduction i n the hen (Tejning 1967) and mallard duck (Heinz 1974), and the h a t c h a b i l i t y of hen eggs (Tejning 1967).  Unlike mercuric chloride (Stoewsand e_t a l . ,  1971), eggshell thinning was not observed i n the methylmercury treated ring dove, American k e s t r e l (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 e f f e c t s of toxicants on f i s h reproduction ( Spraque 1971).  Crandall and Goodnight (1962) observed a  delay i n sexual maturity i n the guppy by lead and zinc.  I n h i b i t i o n of  spawning by copper was detected i n fathead minnows (Mount 1968).  Also,  near complete elimination of egg production by zinc was found i n fathead  5 minnows (Brungs 1969).  Resistance of f i s h eggs and f r y to toxicants,  e.g., detergents, zinc, copper and malathion, has been studied and i t seems that eggs are less s e n s i t i v e to toxicants than f r y (Sprague 1971). Methylmercury uptake, d i s t r i b u t i o n and excretion rate have been w e l l studied i n f i s h because of i t s p o t e n t i a l hazard to the f i s 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 i n f i s h include i t s  t o x i c i t y (Akiyama 1970) , i t s e f f e c t s on g i l l metabolism (O'Connor and Fromm 1975; Kendall 1972), blood parameters and erythrocytes and Fromm 1975; Olson and Fromm 1973), kidney  (O'Connor  (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 e f f e c t s of toxicants on f i s h reproduction has been sparse (Sprague 1971).  For organic mercury, the work of Kihlstrtfrn and h i s co-  workers remains a unique piece of information on the sublethal e f f e c t s of phenylmercury on f i s h reproduction KihlstrHm and Hulth 1972) .  (Kihlstrtfm, Lundberg and Hulth 1972;  6  SECTION I THE TOXICITY AND ACCUMULATION OF METHYLMERCURY  7 INTRODUCTION  Mercury and many of I t s compounds have>long been known to be highly ; t o x i c to both plants and animals. The main sources of mercury contamination i n the environment are from industries such as mining, pulp and paper, the manufacture and use of fungicide, p l a s t i c and c h l o r - a l k a l i . Incidences of mercury poisoning and sources of mercury contamination have been w e l l documented (Fimrelte 1970; Aaronson 1971; Goldwater 1971; Nelson 1971; Saha 1972).  The c y c l i n g of mercury through the environment  has been reviewed (Gavis and Ferguson 1972). Though occurring i n several forms, mercury i n f i s h i s mainly present as methylmercury (Westoo 1969).  This i s because methylmercury i s r e a d i l y  absorbed ( a b i l i t y to cross c e l l membranes), i s r e l a t i v e l y r e s i s t a n t to biotransformation, has a low clearance rate and a strong a f f i n i t y f o r prot e i n (Clarkson 1972).  The source of methylmercury has not been well deter-  mined, but i t has been observed that microorganisms,especially those i n 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 l e v e l s , e.g., f i s h and ultimately man (JernelbV and Lann 1971).  Thus, i t  i s worthwhile to investigate the possible e f f e c t s , both l e t h a l and subl e t h a l , of methylmercury i n f i s h . In t h i s section, the t o x i c i t y of methylmercury and i t s accumulation r  i n the tissues of the adult freshwater teleost Oryzias l a t i p e s are investigated.  8 MATERIALS AND METHODS Maintenance of Fish Adult medaka, Oryzias l a t i p e s were obtained v i a air-shipment from Nagoya Aquarium Company, Nagoya C i t y , Japan.  Following a r r i v a l , the  f i s h were d i s t r i b u t e d into 3 5 - l i t r e capacity p l a s t i c tanks equipped with side f i l t e r s and aerated with compressed a i r . City  Dechlorinated Vancouver  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 i n a trough of running cold water (10 + 1°C).  These f i s h were exposed to short photo-  periods of eight hours l i g h t (0900 hr to 1700 hr) alternating with s i x 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  E l e c t r i c L t d . , Toronto). Fish were fed d a i l y  ad l i b i t u m with frozen brine shrimp.  A l l Imported f i s h were subjected  to the above conditions f o r at l e a s t a month before they were slowly adapted to d i f f e r e n t conditions depending on the various experiments. Gravid Fish After this i n i t i a l treatment with low temperature and short photoperiod f o r 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 f i s h to pass out of i t s refractory stage while the warm temperature and long photoperiod induces gonadal development (Yoshioka 1962, 1963).  Fish treated i n this manner were used i n '  acute t o x i c i t y tests and studies of o v i p o s i t i o n .  9  Sexually regressed  fish  For studying gonadal development, f i s h should be sexually regressed at the beginning of the experiment,  Yoshioka  (1962, 1963)  observed  treatment with low temperature (4-15°C) and short photoperiod was  that  (8L/16D)  e f f e c t i v e i n inducing Oryzias l a t i p e s to a sexually regressed s t a t e .  A f t e r this treatment,  f i s h would have passed i t s refractory period and  can e a s i l y be brought to spawning state with warm temperature (18-23°C) and long photoperiod  (16L/8D).  In the present study, treatment  temperature (13 + 1°C) and short photoperiod  of low  (8L/16D) f o r two months was  used to ensure that f i s h were sexually regressed.  Fish i n the regressed  state were used i n a l l long term exposure studies. Acute t o x i c i t y tests T o x i c i t y tests were s t a t i c , conducted i n 1 8 - l i t r e capacity glass aquaria with 10 f i s h per tank.  The f i s h ranged from 2.6  length and weighed 0.25  g; this resulted i n a loading density of  to 0.35  to 3.0  cm i n  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.  F i s h were acclimated to the  test conditions for three days and fed d a i l y with frozen brine shrimp. The test period began on Day 4 when methylmercuric to the tanks and feeding discontinued. seven days and death was movement.  chloride was  f i r s t added  Observations were continued for  defined as a permanent cessation of opercular  A l l f i s h were frozen immediately  A stock solution of methylmercuric  for residue a n a l y s i s .  chloride (1.71 g CH^Hg /l) was +  prepared by dissolving the proper amount of chemical i n d i s t i l l e d water. Appropriate volumes of t h i s stock solution were added to the d i f f e r e n t aquaria to achieve the desired concentrations.  To maintain proper concen-  10 t r a t i o n i n the tanks, about 90% of the solution i n each tank was renewed daily. At  each concentration, the cumulative percentage mortality was  plotted on a probit scale against the s u r v i v a l time i n hours on a l o g a r i t h mic scale, and the median l e t h a l time (LT50) and i t s 95 percent confidence l i m i t s estimated ( L i t c h f i e l d , 1949).  From the e y e - f i t t e d regression  of l o g LT50 against l o g concentration ( L i t c h f i e l d 1949) and by p l o t t i n g the  p a r t i a l mortality at 96 hr ( L i t c h f i e l d and Wilcoxon 1949), the con-  centration required to produce 50 percent mortality (LC50) and i t s 95% confidence l i m i t s at 96 hr were calculated. Long-term exposure Adult medaka weighing 0.25-0.35 g were divided into four groups with 35 f i s h per group.  Each group was exposed i n a flow-through system to  one of four d i f f e r e n t concentrations of methylmercury (0, 4.3, 10.7 and 21.5 yg CH Hg /l) at 23 + 1°C under 16L:8D light-dark regime. +  3  The loading  density i n the tanks was approximately one l i t r e of test solution per gram f i s h and the approximate time f o r 99% replacement of test solution was 3.5 hours.  Fish were fed d a i l y with frozen brine shrimp.  Mortality was  checked each day and f i s h were sampled at 0, 2, 4 and 6 weeks for methylmercury residue determination. The flow-through system i s i l l 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 s t e e l c o i l s 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 s t e e l 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 f o r continuous exposure of f i s h to low concentrations of methylmercury, showing the pattern of water flow, heating system, one Mariotte b o t t l e f o r the metering of toxicant and one test-tank.  12  !3  to mixing buckets situated on top of the test-tanks. The methylmerc u r i c chloride solutions were metered into the mixing buckets by Mariotte b o t t l e s (Leduc 1966).  The mixed solution of water and methyl-  mercury entered the d i f f e r e n t test-tanks by gravity.  The f i n a l  concen-  t r a t i o n s i n the test-tanks were determined by the concentrations of methylmercuric chloride solution i n the Mariotte b o t t l e s .  Test-tanks  used were of translucent, white, polyethylene equipped with p l a s t i c 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 f i b r e g l a s s  screens to prevent f i s h from escaping.  There were four of the above  set-ups f o r d i f f e r e n t methylmercury chloride concentrations of 0.0,  4.3,  10.7 and 21.5 yg CH Hg /l. +  3  Residue analysis Extraction and p u r i f i c a t i o n This i s a modification of the extraction method developed by Newsome i n 1971.  Since weight of i n d i v i d u a l 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 determination) and homogenized i n a Sorval homogenizer f o r 15 min with a solution of 1 N hydrobromic ate was  acid and 2.1 N potassium bromide (40 ml).  The homogen-  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 mide s o l u t i o n .  acid-potassium bro-  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 t h i s combined benzene layer was extracted twice ( 2 x 6  then  ml) with f r e s h l y prepared cysteine acetate s o l u -  14 t i o n (2.0 g cysteine hydro-chloride monhydrate, 4.0 g sodium acetate and 12.5 g anhydrous sodium sulphate i n 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 f i e d 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. Gas  1  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 g l y c o l succinate) on 80-100 mesh Chromosorb W.  For the determination of  methylmercury, t y p i c a l operating temperatures were: i n j e c t i o n port 200°C, oven 170°C and detector 200°C.  The c a r r i e r gas was argon-methane  (95:5);  gas flow was 60 ml/min at 40 p s i .  Under these conditions methylmercury  had a retention time of 150 sec.  Samples and standard were injected as  benzene solutions i n 2,0 u l aliquots. Standards were run daily with each batch of samples and a c a l i b r a t i o n curve was plotted f o r each i n d i v i d u a l batch of samples.  The concentra-  tion of methylmercury i n the samples were calculated as ug CH Hg /g based +  3  on f i s h wet weight.  15  '  RESULTS Nine spiking tests were performed during the entire period of f i s h tissue analysis and the calculated percent recovery varied between 83% to 89% with an average of 86.5 +0.8%.  This f a c t o r (86.5%)  was used to adjust the residual methylmercury (CH^Hg ) concentration +  i n the f i s h samples accordingly. Acute t o x i c i t y tests In the acute t o x i c i t y t e s t s , 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 i n F i g . 2 to 4,  F i s h exposed to lower than 80 ug  CH Hg /l survived f o r more than four days ( F i g . 2) . +  3  The median l e t h a l  concentration, 96 hr-LC50, of methylmercury f o r medaka i s 8 8 + 9 . 8 ug CH Hg /l ( F i g . 3).  Levels of methylmercury accumulated i n f i s h exposed  +  3  to 43 to 1000 yg CH Hg /l varied but remained below 40 yg CH Hg /g ( F i g . +  +  3  3  4) . When f i s h were exposed to concentrations higher than 1000 yg CH^Hg""/! 1  the amount of methylmercury accumulated i n the tissue increased s t e a d i l y + + and reached as high as 408.1 yg CH^Hg /g as i n the case of 42900 yg CH H A 3  exposure ( F i g . 4). Long-term exposure Results are shown i n Table 1 and F i g . 5.  No mortality was observed  i n the control group during the e n t i r e s i x weeks of exposure.  F i s h ex-  posed to 4.3 and 10.7 yg CH Hg /l had one death each during the 4th week +  3  and the 2nd week respectively (Table 1).  At the highest  concentration  (21.5 yg CH Hg / l ) , the f i s h began dying during the second week and mor3  t a l i t y reached a peak during the fourth week of exposure when over 83%  16  Figure 2.  Effects of d i f f e r e n t concentrations of methylmercury on the LT50 of Oryzias l a t i p e s at 23 + 1°C. reported are mean and 95% confidence l i m i t s .  Values  >-  100  96 hrs  50 cc  O ^  10  o  5  in ^  x:  LU  1 05  0.1  J  Q01  l_i QQ5  i—l  Ol  I I 11 III  Q5  '  i  1  EXPOSURE CONCENTRATION  i 1mil  5  i  10  x10  i i l . i i .  50 3  100  ugCH Hg /l +  3  18  Figure 3.  Determination  of the median l e t h a l concentration  (LC50) at 96 hr of methylmercury f o r Oryzias latipes.  19  10  20  40  60 80100  CONCENTRATION Lig C H H ; / 3  g  200  20  Figure 4.  Accumulation  of methylmercury i n Oryzias l a t i p e s  exposed to d i f f e r e n t concentrations of methylmercury during the acute tests (each value represents the average of two determinations on groups of five fish).  1000  _ 500  —  CD  I  • cn  I  <J o> 100 =k  UJ  —-  50 in u  Q CO UJ  3  1 sz  1  CD |  ^  10  ^  5  —  CO CO !—  Im  1 0.01  Q05  LLL Q1  05  1  JL_L  EXPOSURE CONCENTRATION  11 in  LLL  10  x10  J  50  100  ug C H H g / l +  3  M  22  Figure 5.  Accumulation of methylmercury i n Oryzias l a t i p e s under long-exposure to d i f f e r e n t of methylmercury.  concentrations  Each point represents the mean  of two methylmercury determinations on groups of five live  fish.  •  21.5  CH Hg /| +  3  O 10.7 A 4.3  E X P O S U R E TIME (wks) ro  (A)  Table 1.  Mortality of Oryzias l a t i p e s exposed to d i f f e r e n t concentrations of methylmercury over a 6-week period (35 f i s h i n each concentration  Exposure concentration (yg CH Hg+/l) 3  1st wk.  2nd wk.  3rd  wk.  at s t a r t of t e s t ) .  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 f i s h had died.  At the end of the exposure, only three out  of the i n i t i a l 35 f i s h were a l i v e from this group (Table 1). Tissue methylmercury l e v e l of the control remained below 1 yg CH Hg /g (Fig. 5). Both of the 4.3 and 10.7 yg CH Hg /l exposed +  +  3  3  groups accumulated methylmercury steadily during the entire exposure and reached l e v e l s of 19.4 and 30.7 yg CH Hg /g respectively at the end +  3  of the exposure (Fig. 5). A high, probably l e t h a l , l e v e l of methyl4-  mercury (67.1 yg CH Hg /g) was accumulated within two weeks i n f i s h 3  exposed to 21.5 yg CH Hg /l; during this period of two weeks, the +  3  f i s h started dying ( F i g . 5 and Table 1).  At the end of the fourth  week over 83% of the f i s h from this group were dead (Table 1) and the tissue methylmercury l e v e l was 63.4 yg/g.  26  DISCUSSION  The present study has shown that methylmercury i s toxic to the madaka (the 96h-LC50 was 88 + 9.8 yg CH Hg /l). +  3  This value i s high  compared to the 10 yg H g ^ / l (96h-LC50) f o r rainbow trout (Lock 1974) . Thus, the medaka i s at least eight times more tolerant to methylmercury 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 s t e a d i l y suggesting possible damage to the c e l l membranes, e s p e c i a l l y i n g i l l e p i t h e l i a l c e l l s (Rucker and Amend 1969), which would allow a sharp increase i n uptake of methylmercury. A l though the e f f e c t of methylmercury at the molecular l e v e l has not been completely elucidated, i t i s apparent that this compound has a strong a f f i n i t y f o r sulphur, p a r t i c u l a r l y f o r the sulfhydryl group (-SH) i n proteins (Saba 1972).  This chemical binding of methylmercury to pro-  teins i n c e l l membrane may a l t e r the d i s t r i b u t i o n of ions, change e l e c t r i c p o t e n t i a l and thus i n t e r f e r e with movement of f l u i d s across the membrane (Passow et^ a l . , 1961).  Methylmercury enters the f i s 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 f i s h , i t binds t i g h t l y with the sulfhyd r y l group i n proteins, causing a steady increase of methylmercury accumulated.  At concentrations below 1000 yg CH.jHg /l, accumulation of  methylmercury appears variable.  +  Death seems to occur i n the medaka when  27 the tissue l e v e l of methylmercury reaches 25 yg CH.jHg /g (Fig. 4) . +  This value i s quite high when compared with l e v e l s found i n f i s h from uncontaminated areas or from s a l t water (Fimreite and Reynolds 1973; Peterson e t a l . , 1973; Childs and Gaffke 1973).  However, i t i s not  uncommon f o r f i s h from contaminated areas l i k e Clay Lake, Ontario, to reach l e v e l s as high as 20 yg/g of mercury (Fimreite and Reynolds 1973). Unlike many other chemicals, methylmercury can be accumulated to a l e t h a l l e v e l i n f i s h exposed to a low concentration f o r long duration. In the acute t o x i c i t y study, medaka were observed to survive over a week i n 75 yg/1 methylmercury s o l u t i o n ; i n the long-term study, i t was observed that f i s h exposed to 21.5 yg CH,jHg /l solution accumulated a +  l e t h a l l e v e l of methylmercury (over 25 yg CH.jHg /g body weight) and +  started dying a f t e r the second week of exposure. From the foregoing, i t can be seen that the t o x i c i t y and e f f e c t s o f methylmercury depend not only on the exposure concentration but also on the amount of toxicant the f i s h accumulates during the exposure. This r a i s e s serious doubts about the r e l i a b i l i t y of acute t o x i c i t y tests f o r bioaccumulative chemicals l i k e methylmercury.  The use of 96h-  LC50 f o r non-accumulative toxins would be v a l i d but not f o r bioaccumul a t i v e chemicals l i k e most pesticides (Sprague 1969).  The d e f i n i t i o n  of an acceptable sublethal concentration f o r bioaccumulative toxin cannot be some concentration below or a p a r t i c u l a r f r a c t i o n 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 e f f e c t s may occur at r e l a t i v e l y low tissue l e v e l s . To solve this p a r t i c u l a r problem, residue determinations w i l l have to be done concurrently with t o x i c i t y studies.  "Safe" l e v e l s which could  be much lower than the sublethal concentration, w i l l have to be determined with long-term, continuous-flow experiments.  We have only par-  t i a l l y accomplished this and found that f o r medaka, the upper sublethal concentration of methylmercury For  l i e s between 10.7 and 21.5 yg CH.jHg /l. +  chemicals that accumulate i n tissues, exposure to sublethal  concentration of such chemicals w i l l have long-lasting e f f e c t s .  Re-  lease of such chemicals into the environment, even i n minute quantities, w i l l ultimately bring about d r a s t i c changes i n the organisms that come i n contact with them.  Section II examines the e f f e c t s of long-term  exposure to low levels of methylmercury on the reproductive physiology i n the medaka.  SECTION I I CHRONIC EFFECTS OF METHYLMERCURY ON REPRODUCTION  30  INTRODUCTION Very l i t t l e work has been done on the effects of aquatic p o l l u t ants on f i s h reproduction (Sprague 1971). (1962) observed and zinc.  Crandall and Goodnight  a delay i n sexual maturity of guppy treated with lead  Mount (1968) reported i n h i b i t i o n of spawning of fathead  minnows treated with copper, while Brungs (1969) found almost complete elimination of egg production by zinc.  F i s h eggs appear to be less  sensitive than f r y to toxicants such as detergents, zinc, copper, and malathion  (Sprague 1971).  young by phenylmercurie was  A decrease i n number of eggs and  acetate at concentrations as low as 1.0  observed i n zebrafish (Kihlstrom et a l . , 1971).  production was  hatched ug/1  Decreased egg  attributed to the a f f i n i t y of mercurials f o r sulfhydryl  groups thus i n h i b i t i n g 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  acetate; these workers suggested  that this was  phenylmercuric  due to a decrease i n ef-  fects of microorganisms upon the developing eggs and also that low concentrations of mercuric compound may  "stimulate" h a t c h a b i l i t y .  Kihlstrdm's work remains a unique piece of information pertaining to the effects of organic mercury on f i s h reproduction.  However, there  are s t i l l many unanswered questions concerning the e f f e c t s of mercury on f i s h , especially on their reproduction and the mechanism behind these e f f e c t s .  31  This section investigates the e f f e c t s of low  concentrations  of methylmercury on oviposition,,gonadal development, spawning act i v i t y , h a t c h a b i l i t y of eggs and s u r v i v a l of juveniles of Oryzias latipes.  32 MATERIALS AND METHODS On o v i p o s i t i o n Spawning f i s h were used i n this experiment.  To obtain a c t i v e l y  spawning f i s h , Oryzias were maintained i n dechlorinated Vancouver City water f o r over a month at 23 + 1°C under long photoperiod (16L/8D) with the  l i g h t period between 0800 and 2400 hours. The experiments were performed with ten 1 8 - l i t r e capacity glass  aquaria divided into two s e r i e s .  The f i r s t series consisted of f i v e  tanks, each holding a suspended cage (dimension 36 x 20 x 26 cm) made of: f i b r e g l a s s screening.  These cages allowed rapid transfer of f i s h  from one aquarium to another with minimum disturbance.  The second ser-  ies; of f i v e tanks contained various concentrations of methylmercury (Q)„0, 4.3, 8.58, 42.9 and 85.8 pg CH Hg /l) . +  3  Twenty-five pairs of spawning medaka were chosen from the warm temperature and long photoperiod group (see Section I ) . They were d i s t r i b u t e d , 5 males and 5 females per tank, into the cages of the f i r s t s e r i e s of f i v e aquaria.  In the morning before the l i g h t came on, the  caged f i s h were r a p i d l y transferred to f r e s h l y prepared solution of methylmercury  (0.0, 4.3, 8.58, 42.9 and 85.8 yg CH Hg /l) i n the second  s e r i e s of f i v e tanks.  +  3  Spawning was checked at hourly i n t e r v a l s u n t i l  noon at which time the f i s h were returned to clean water i n the f i r s t s e r i e s of f i v e tanks and observation continued for another four hours. Fish were fed d a i l y with frozen brine shrimp during the afternoon while the  f i s h were i n clean water.  The f i s h were exposed to methylmercury  on a l t e r n a t e days only; on other days, the cages were agitated  33 mechanically to simulate the t r a n s f e r . Spawning a c t i v i t y was  Spawning was  checked each day.  recorded as number of females with eggs attached  (number of spawning) and the t o t a l number of eggs c o l l e c t e d i n one tank. On gonadal development and spawning A flow through system as described i n Section I was this part of the experiment.  F i s h used had regressed gonads since they  had been exposed to low temperature (8L/16D) f o r over two months. temperature  employed i n  (13 + 1°C) and short photoperiod  These f i s h were allowed to warm to room  (approximately 23°C) overnight and on the next day were d i s -  tributed to four test-tanks at the rate of 14 males and 20 females per tank, r e s u l t i n g i n 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 Hg /l) +  3  reached i n about 4 hr; the approximate solution was 3.5 hr (Sprague 1969). brine shrimp.  time f o r 99% replacement of test  Fish were fed d a i l y with frozen  Spawning a c t i v i t y was recorded as number of females with  eggs attached and number of eggs c o l l e c t e d each day.  Observations were  continued for s i x weeks. The eggs c o l l e c t e d were allowed to hatch i n disposable p l a s t i c p e t r i dishes containing the same solutions from which they were c o l l e c t e d . At the end of s i x weeks, the f i s h 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 f i s h  There were two experiments performed and since both of them showed s i m i l a r r e s u l t s , the data were pooled. On s u r v i v a l of juvenile f i s h To r a i s e juveniles to maturity i n d i f f e r e n t concentrations of methylmercury, one-week-old j u v e n i l e medaka were collected from untreated parents and d i s t r i b u t e d , approximately 50 to 80 f i s 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. s i m i l a r results and the data were pooled.  Both experiments showed  35  RESULTS E f f e c t s on o v i p o s i t i o n Control f i s h and f i s h experiencing 4-hr exposure of 4.3 yg CH.jHg /l +  on alternate days consistently showed over 60% of o v i p o s i t i o n a c t i v i t y (Table 2).  At a concentration of 8.58 yg CH^Hg*/! the number of spawn-  ings was reduced whenever the f i s h were exposed to the toxic s o l u t i o n ; however, spawning was unaffected on the following day when f i s h were returned to clean water.  A s i m i l a r but more marked e f f e c t 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 (except f o r Day 3) but also affected spawning the next day i n clean water. There seems to be an increased reduction of spawning i n clean water as this group experienced more exposure of the toxic s o l u t i o n . E f f e c t s on gonadal development arid spawning No sign of spawning was observed during the f i r s t two weeks of exposure.  Hence Figure 6 reports only spawning a c t i v i t y during the l a s t  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 f i s h and s i x f i s h died during  the f i r s t and second week, r e s p e c t i v e l y . During the t h i r d week of  exposure spawning was observed i n a l l groups while mortality was observed only at the highest concentration.  During the 3rd, 4th, 5th and  6th week of exposure, treated groups showed a consistent reduction of spawning a c t i v i t y ( F i g . 6); also there seemed to be some r e l a t i o n s h i p between the degree of reduction of spawning a c t i v i t y and the concentrations of .methylmercury.  Figure 7 shows an inverse l i n e a r r e l a t i o n s h i p between  Table 2.  Effects of 4-hour exposure to d i f f e r e n t concentrations of methylmercury on the oviposition of Oryzias l a t i p e s . f i v e females per tank.  P o s i t i v e treatment  Five males and  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 t o t a l number of eggs l a i d were stated within brackets. Day  Treatment  Control  Concentration yg CH Hg /1 8.58 42.9 +  4.3  J  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  +  4  -  5  +  6  -  7  +  8  -  9 10  3(35) 3(30)  3(56) 4(42)  3(37)  4(48)  4(38)  4(50)  3(27)  3(37)  0(0)  2(15) 0(0)  1(8)  4(43)  0(0)  K5)  1(11)  3(42)  5(53)  4(30)  4(38)  4(52)  1(2)  K9)  2(23) 3(27)  3(40)  0(0) 3(29)  3(31)  4(57)  5(43)  2(20)  4(62)  4(39)  + —  3(35)  3(35)  0(0) 1(8)  CO ON  37  Figure 6.  Weekly observations, from the 3rd to the 6th week, on spawning and egg'-laying of Oryzias l a t i p e s f o r the d i f f e r e n t concentrations o f methylmercury. Numbers i n brackets represent number of f i s h i n each group at the end of the week.  600  r  <6&)  •  Net of EGGS  (68)  400  80 (68)  300  60  200  40  100  (55)  0 i  CO  (68)  400  (J) 300  (68)  LU  (T)  80  £z  CL  2 0  100  (46)  CO  o  0  60  300 200  40  (66)  (67)  100  (37)  0  20 0  300  60 (64)  200 100 0  100  60 40 <t  (68)  200  o o  20 0  500  ^  38  U N a o f SPAWNING!100  500  W  120  40 (24)  QO  43  10.7  CONCENTRATION  21.5 ug CiHjHg*/ I  20 0  O o  39  Figure 7.  E f f e c t s of different  concentrations of methylmercury  on the t o t a l number of spawning and t o t a l number of eggs l a i d during s i x weeks of exposure.  40  O  T—  X  28f-  8) 16 o 12  2 al o  o  1  1  3  I I I I II  1  4 5 6 78910  20  30  3 4 5 6 78910  20  20  O ^ 24 X  (/> 2 0 O d>  S o O  < O  1(S  12 8  0  0  i  1  2  CONCENTRATIONS jug C H H g 3  +  / I  41 the t o t a l number of spawnings (number of females with eggs attached) as w e l l as the t o t a l number of eggs c o l l e c t e d over the four weeks and the l o g exposure concentrations.  Considering the control as 100%,  during the four weeks of observation, egg-laying a b i l i t y and spawning a c t i v i t y f o r f i s h exposed to 4.3, 10.7 and 21.5 yg CH^Hg fl were r e spectively 58%, 49% and 10% ( F i g . 8), When transformed  to percent i n -  h i b i t i o n and plotted against l o g exposure concentrations, these values showed a d i r e c t l i n e a r r e l a t i o n s h i p ( F i g . 9). At the end of s i x weeks the gonadosomatic indices of both males and females showed reduction i n r e l a t i o n to the increase i n exposure concentration; females were more s e n s i t i v e than males ( F i g . 10). Spawned eggs were incubated i n the media where they were c o l l e c t e d ; h a t c h a b i l i t y of these eggs bears no r e l a t i o n s h i p to the concentrations o f methylmercury previously used (Fig. 11). The percent h a t c h a b i l i t y f o r the four groups (0.0, 4.3, 10.7 and 21.5 yg CH.jHg /l) d i d not deviate from the control and varied +  between 55% and 68%. E f f e c t s on j u v e n i l e f i s h Mortality occurred i n a l l groups, treated and untreated.  During  the two weeks of exposure, 1-week o l d juveniles exposed to 0.0, 4.3, 10.7 and 21.5 yg CH Hg /l had m o r t a l i t y rates of 2.2%, 54.3%, 64.9% +  3  and 99.4% respectively (Table 3). This shows a high s e n s i t i v i t y of juvenile medaka to methylmercury t o x i c i t y .  42  Figure 8.  The percent spawning a c t i v i t y  and egg-laying  a b i l i t y of Oryzias l a t i p e s exposed to d i f f e r ent concentrations of methylmercury. i s 100 percent.  Control  43  •  0.0  4 . 8 10.7  EGG-LAYING  21.5  CONCENTRATION jjg  CH Hg /l +  3  44  Figure 9.  The percent i n h i b i t i o n of spawning i n Oryzias l a t i p e s i n water containing d i f f e r e n t concentrations of methylmercury.  45  100  O h-  —  A  80 60  <b  CQ  A  40  A  20j  o  1  2  1  —  i  i  i  i  i  11  3 4 5 678910  CONCENTRATION MQ C H H g 3  +  /1  20 30  46  Figure 10.  Gonadosomatic indices of male and female Oryzias l a t i p e s exposed to d i f f e r e n t concentrations  of methylmercury for 6 weeks.  Numbers i n brackets represent sample s i z e ; Asterisk, s t a t i s t i c a l significance ( t - t e s t , P <0.05) compared to control.  47  •  MALE FEMALE  14 1 12 10  CO  O  8 LU  < UJ  0-0  4 . 8 10.7  21.5  CONCENTRATION jug  CH Hg /l 3  +  0  48  Figure 11.  The percent h a t c h a b i l i t y of eggs exposed to d i f f e r e n t concentrations of methylmercury.  49  r  1— _l CD  <  100  80 60 40 20  <  IE  o  o  0  QO  4 . 8 10.7  21.5  CONCENTRATION )jg  CH Hg 3  +  / |  50  Table 3.  Mortality of one-week o l d juvenile Oryzias l a t i p e s exposed to d i f f e r e n t concentrations of methylmercury for two weeks.  Concentration (Ug CH Hg+/l 3  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; i n the presence of a male o v i p o s i t i o n takes place on the same day, soon a f t e r daybreak.  In the present i n v e s t i g a t i o n ,  medaka were exposed to d i f f e r e n t concentrations of methylmercury during t h e i r normal period of o v i p o s i t i o n , i . e . , between 0800 and 1200 hr ( l i g h t period, 0800 to 2400 h r ) .  Control f i s h 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""/! reduced the number of spawning f e 1  males; on return to clean water normal o v i p o s i t i o n occurred the next day. was  This suggests that the concentration of 8.58  yg/1 methylmercury  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 p a r t i a l l y excreted to a l e v e l whereby normal spawning could be achieved on the next day. yg CH Hg /l) i s approximately +  3  yg CH Hg /l, Section 1). +  3  concentration, 42.9 spawning females. oviposition was  This concentration  one-tenth of the 96h-LC50 dose (88 + 9,8  Similar r e s u l t s were observed at a higher  yg CH^Hg"*"/!, with further reduction i n number of However, at the highest concentration, 85.8  yg CH^Hg""/!, 1  abolished a f t e r the t h i r d day, and this e f f e c t was  dent on subsequent days when the f i s h were i n clean water. 4-hour exposure to this high concentration resulted i n an of methylmercury that blocked spawning. bably  (8.58  true for a l l bioaccumulative  than the excretion of the toxin.  evi-  Thus, the accumulation  A similar phenomenon i s pro-  toxins where accumulation  i s faster  52 Kihlstrtfm et a l . (1971) observed a decrease In number of zebraf i s h eggs i n water containing 1 ng or more phenylmercuric gram of water.  acetate per  In the present i n v e s t i g a t i o n using a s i m i l a r range of  concentrations of toxicant as Klhlstro'm's, s i m i l a r r e s u l t s were observed i n Oryzias l a t i p e s exposed continuously to methylmercury for s i x weeks.  Spawning occurred during the t h i r d week of exposure and  continued u n t i l the end of the experiment. spawning f o r f i s h exposed to 4.3,  10.7  The percent i n h i b i t i o n of  and 21.5 yg CH Hg /l was +  3  42,  48  and 88% r e s p e c t i v e l y . Both spawning a c t i v i t y 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), h a t c h a b i l i t y of Oryzias eggs from treated parents was not affected by the exposure to methylmercury. week old f i s h were e s p e c i a l l y sensitive to the toxicant.  One-  This i s often  true f o r other species of f i s h (Sprague 1971) . In rainbow t r o u t , exposure to methylmercury for up to 12 weeks (10 yg Hg^/1) d i d not s i g n i f i c a n t l y a f f e c t the i n v i t r o metabolism of the g i l l or the concentration of plasma e l e c t r o l y t e s (O'Connor and Fromm 1975).  The only deleterious e f f e c t s observed by these workers was  a s i g n i f i c a n t increase i n hematocrit a f t e r 12 weeks exposure.  Oryzias  l a t i p e s i s more "hardy" than the rainbow t r o u t , and yet i n the present i n v e s t i g a t i o n , 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 reproduction i s much more s e n s i t i v e to environmental physiological functions.  Reproduction  of the species and any environmental  p o l l u t i o n than other  i s indispensible to the s u r v i v a l  contamination  that adversely af-  53 fects reproduction w i l l have long l a s t i n g e f f e c t s on the species and the e c o l o g i c a l systems connected with i t . Mercury compounds i n h i b i t mitosis by interacting with s u l f h y d r y l groups (Hughes 1950).  Since Oryzias depends on constant replacement  of new oocytes f o r i t s d a i l y spawning, i n h i b i t i o n of mitosis i s a poss i b l e explanation for the decreased number of eggs spawned i n contaminated water.  Biochemically, methylmercury may i n h i b i t enzyme systems  f o r steroidogenesis by reacting with sulfhydryl groups of the enzymes, thus making them i n a c t i v e .  F i s h reproduction depends on proper b a l -  ance of hormones i n the hypothalamic-hypophysial-gonadal system. i s not known where methylmercury acts.  It  I t may act at the hypothalamic-  hypophysial l e v e l because methylmercury has been observed to accumulate i n large quantities i n the b r a i n , but the p o s s i b i l i t y that I t may also act at the gonadal l e v e l cannot be disregarded. examined i n the following s e c t i o n .  These p o s s i b i l i t i e s are  SECTION I I I MODE OF ACTION OF METHYLMERCURY ON REPRODUCTION  55 INTRODUCTION Previous sections have demonstrated ithat exposure to methylmercury resulted i n reduction of spawning a c t i v i t y l n Oryzias l a t i p e s . However, i t i s not c e r t a i n where methylmercury acts.  This section  attempts to elucidate the action of methylmercury on reproduction at the organ l e v e l . In f i s h , the spawning period corresponds to a season most favourable for the development of the o f f s p r i n g .  The f i s h synchronizes i t s  reproductive physiology with the season by using environmental cues, l i k e 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 i n turn stimulates the hypophysis to produce and r e lease hormone(s) capable of stimulating gonadal development; thus forming the hypothalamic-hypophysial-gonadal axis.  There i s also a feed-  back mechanism i n this system whereby l e v e l s of the hormones are i n proper balance.  F i g . 12 shows such a r e l a t i o n s h i p .  So f a r we have observed the e f f e c t s of methylmercury treatment on the end product of the entire reproductive process.  But i t i s not known  at what point methylmercury acts on this hypothalamic-hypophysial-gonadal axis.  To elucidate t h i s , i t was assumed that methylmercury blocked or  rendered inactive some l i n k i n t h i s system; thus, some hormone was not produced.  I t was further assumed that "replacement therapy" with appro-  p r i a t e hormones would restore reproduction i n methylmercury treated f i s h . In the experiments that follow, d i f f e r e n t hormones from the hypothalamus,  56  Figure 12.  Diagram showing the i n t e r r e l a t i o n s h i p between environmental cues, hypothalamus, hypophysis and gonads.  57  ENVIRONMENTAL CUES temperature photoperiod water chemistry, e t c .  v i a eye, pineal, skin  T  HYPOTHALAMUS  hypothalamic hormones ( r e l e a s i n g 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" i n the methylmercury affected f i s h to determine whether these hormones restore reproductive  activity.  Mammalian l u t e i n i z i n g hormone i s e f f e c t i v e i n inducing maturat i o n and ovulation i n a variety of fishes (Hirose 1971, Goswami and Sundararaj 1972; Donaldson,1973; Hoar 1969), but p i s c i n e hypothalamic hormone, possibly s i m i l a r to mammalian l u t e i n i z i n g hormone-releasing hormones (LH-RH) has not been well studied.  Synthetic l u t e i n i z i n g hor-  mones-releasing hormone (LH-RH), recently synthesized based on the proposed structure of porcine and ovine LH-RH, i s highly e f f e c t i v e i n s t i mulating the release of l u t e i n i z i n g hormone and i n inducing ovulation i n mammals (Schally et a l . , 1973; Humphrey et a l . , 1973; Foxcroft et a l . , 1975) , i n chickens (van Tienhoven and Schally 1972) and i n 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) c e l l s i n the proximal pars d i s t a l i s and induces ovulation i n the goldfish (Lam et_ a l . , 1976) and i n the ayu (Hirose and Ishida 1974). also observed i n the carp  Gonadotropin release by synthetic LH-RH was (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, t r e a t -  ment with mammalian gonadotropin releasing hormone elevated plasma gonadotropin concentration as determined by radioimmunoassay (Crim and Cluett 1974).  However, i t i s not known whether synthetic LH-RH would  stimulate the synthesis and release of gonadotropin(s) i n sexually r e gressed f i s h and subsequently gonadal maturation. observed i n hens (Reeves et a l , , 1973).  This phenomenon was  59 Oryzias l a t i p e s kept under warm temperature  (23 + 1°C) and short  photoperiod (8L/16D) f o r 6 to 12 weeks showed l i t t l e or no development (Chan 1976, Yoshioka 1962,  1963).  gonadal  This system permits  tests to show whether synthetic LH-RH can stimulate a c t i v i t y i n the p i t u i t a r y gonadotropic c e l l s and consequently gonadal maturation.  This  part of the study f i r s t examines the e f f e c t of synthetic LH-RH on o v a r i an development i n the Japanese medaka, Oryzias l a t i p e s . After establishing the effectiveness of synthetic LH-RH on gonadal maturation i n Oryzias, we may  then determine whether hypothalamic  and  hypophysial hormones are capable of restoring reproductive a c t i v i t y i n methylmercury treated f i s h .  By i n j e c t i n g hypothalamic and hypophysial  hormones into f i s h exposed to methylmercury, i t i s possible to determine not only which of the hormones i s e f f e c t i v e i n restoring reproduction i n methylmercury poisoned f i s h but also at what l e v e l of the hypothalamic hypophysial-gonadal axis methylmercury i n h i b i t i o n occurs. examination of the p i t u i t a r y may  Histological  also help to explain the action of  methylmercury on the synthesis and release of gonadotropin(s). to say, an important outcome of this experiment cance since i t may  Needless  i s of e c o l o g i c a l s i g n i f i -  show whether hormonal treatments can restore the spawn-  ing a c t i v i t y i n h i b i t e d by methylmercury when indeed such a contamination does occur i n nature. Hirose (1971) devised a simple i n v i t r o system f o r studying ovulat i o n i n Oryzias.  Using t h i s system, the rate of ovulation at various  s t a r t i n g hours of incubation was  determined  (Hirose & Hirose 1972) and alt  e f f e c t s of various hormones on ovulation were studied  (Hirose 1972a, b,  60 1973).  Similar studies were performed on the Indian c a t f i s h by  Goswami and Sundararaj (1972a, b, 1973, 1974).  Studies by both groups  showed that hypophysial gonadotropic hormone and the corticosteroids were e f f e c t i v e i n inducing ovulation i n v i t r o .  This i n v i t r o system  provides a f a s t , convenient method f o r studying the e f f e c t s of methylmercury on ovulation and may be developed into an e f f e c t i v e bioassay. This part of the study investigates the effects of long term exposure of female f i s h to methylmercury on i n v i t r o ovulation.  The  e f f e c t s of various concentrations of methylmercury on i n v i t r o ovulation of untreated f i s h and the effects of exogenous l u t e i n i z i n g hormone on methylmercury blocked ovulation i n v i t r o of untreated f i 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 l e v e l .  61 MATERIALS AND METHODS On ovarian maturation by synthetic LH-RH In this part of the experiment, only female f i s h were used because they provide better indices for gonadal development fish.  than male  Adult regressed f i s h were obtained by pretreating f i s h with low  temperature (13 + 1°C) and short photoperiod (8L/16D) f o r three months (May 6 to Aug. 6, 1974).  On Aug. 6, 1974, f i 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 d i s t r i b u t e d , 10 to 12 f i s h per tank, into f i v e 2 2 - l i t e r capacity a l l glass aquaria with subgravel f i l t e r s .  The f i s h were maintained under the above con-  ditions for s i x weeks and fed once-a-day with a s l i g h t 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 s a l i n e - i n j e c t e d and acted as a control. Injections were intraperitoneal using a microsyringe (Hamilton) equipped with a 32 gauge hypodermic needle.  The synthetic LH-RH was  i n 0.6% NaCl and the i n j e c t i o n volume was 5 u l per f i s h .  dissolved  Fish were i n -  jected twice a week f o r 6 weeks (Aug. 8 to Sept. 23, 1974). At the end of the experiment, a l l f i s h were s a c r i f i c e d , 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 h i s t o l o g i c a l observations, the ovaries and the p i t u i t a r y glands were fixed i n Bouin's solution, embedded i n p a r a f f i n and sectioned at 5-7  um>  62 Ovarian sections were stained with E r l i c h ' s  hematoxylin-eosin;  aldehyde fuchsin counter-stained with Halmi's was used f o r the p i t u i tary glands.  Oocytes were c l a s s i f i e d into three categories according  to Chan (1976) , and only percent d i s t r i b u t i o n of Class I I I 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 I I I oocytes represents mature or maturing f i s h . On e f f e c t s of LH-RH and LH i n j e c t i o n i n methylmercury treated f i s h This experiment was performed i n a flow-through  system described  i n Section I . Sexually regressed f i s h were obtained by maintaining f i s h at 13 + 1°C under short photoperiod 10, 1974).  (8L/16D) f o r four months (May 11 to Sept.  The f i s 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  t e s t s o l u t i o n per gram f i s h .  one l i t r e of  Acclimation consisted of two days with  running clean water at 23 + 1°C and under long photoperiod a l l four groups.  The same photoperiods  (16L/8D) f o r  and water temperature were used  during the entire experimental period. On the t h i r d 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 f o r 99% replacement of test solution was 3.5 hours (Sprague 1969). fourth group, kept i n clean water, acted as the control.  The  Starting on  the fourth day, the three groups exposed to methylmercury received  63 twice-a-week e i t h e r saline (0.6% NaCl) or l u t e i n i z i n g hormone (10 yg/g body weight; porcine l u t e i n i z i n g hormone, NIH-LH-S8) or synthetic l u t e i n i z i n g hormone-releasing Ayerst Laboratory).  hormone (1 yg/g body weight; AY-24, 031,  The control group received only saline i n j e c t i o n s ,  Injections were performed i n t r a p e r i t o n e a l l y using a micro-syringe (Hamilton) equipped with a 32 gauge hypodermic needle, the hormones were dissolved i n 0.6% NaCl and the i n j e c t i o n volume was 5 y l per f i s h . Exposure to methylmercury and the twice-weekly weeks.  i n j e c t i o n s lasted f o r 6  Fish were fed daily with frozen brine shrimp.  Spawning a c t i v i t y was recorded as number of females with eggs a t tached and t o t a l number of eggs c o l l e c t e d each day. continued f o r 6 weeks.  Observations were  At the end of s i x weeks, the f i s h were k i l l e d ,  weighed, measured and the gonadosomatic indices (GSI) calculated.  For  h i s t o l o g i c a l observation of the p i t u i t a r y glands, the tissues were fixed i n Bouin's f i x a t i v e , embedded i n p a r a f f i n and the sections(6 ym) were stained with aldehyde fuchsin, AF, counter-stained with Halmi's solution.  Analysis of variance was used i n the s t a t i s t i c analyses.  On e f f e c t s of methylmercury on i n v i t r o ovulation A l l f i s h were maintained at 23 + 1°C under long photoperiods 8D; 0800-2400 hour l i g h t ) .  (16L/  Spawning females with eggs attached were  removed each day at noon and kept i n a separate tank u n t i l required for the  experiment. A l l instruments and media were s t e r i l i z e d before use.  was  Each f i s h  swabbed with 70% alcohol and k i l l e d by decapitation. The ovary  was  removed, and placed i n a glass p e t r i dish containing Medium 199 (Hirose  64 1971).  The eggs were i n d i v i d u a l l y separated as free eggs with d i s s e c t -  ing pins.  Only eggs with dismeter greater than 0.8 mm were used.  cubation was carried out at 23 + 1°C.  In-  For each treatment, 10-15 eggs  were placed i n 5 ml of medium i n a watch glass covered with a p e t r i dish.  Incubation started wither at 1700 hours or 2300 h o u r s — t h e two  best times f o r 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 f i s h to methylmercury  was s i m i l a r to that described  i n 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 d i a 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 l u t e i n i z i n g hormone (NIH-LH-S8) and various concentrations of methylmercury i n Medium 199 at 1700 hours and ovulation checked at 1000 hours the following morning.  The experiment was duplicated and since s i m i l a r  results were observed, the data were pooled. Additional experiments were performed using various concentrations of methylmercury  i n the incubation medium with eggs from untreated f i s h .  To study the effects of steroids, progesterone, e s t r a d i o l , cortisone and testosterone, were i n d i v i d u a l l y dissolved i n ethanol: propylene g l y c o l (1:1) and added to the incubation media just p r i o r to incubation, r e s u l t -  65 ing i n a f i n a l concentration of 1 ug/1 s t e r o i d . t i o n was s i m i l a r to that previously described. duplicated and the results were pooled.  Procedure of incubaA l l experiments were  66 RESULTS E f f e c t of Synthetic LH-RH on ovarian maturation The e f f e c t s of synthetic l u t e i n i z i n g hormone-releasing hormone on the gonadosomatic index, percent d i s t r i b u t i o n of Class I I I oocytes and ovulation are shown i n F i g . 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 l e s s ) ; there were no Class I I I oocytes. of  The mean gonadosomatic index (+  t h i s i n i t i a l group was low (1.08 + 0.17).  S.E.)  This value i s character-  i s t i c of sexually regressed f i s h . A f t e r s i x weeks of i n j e c t i o n s , the control group (saline injected) showed only a s l i g h t increase i n GSI (from 1.09  to 1.42) with the ap-  pearance of some Class I I I oocytes i n less than 4% of the f i s h (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 d i s t r i b u t i o n of Class I I I oocytes (Figs, 13, 15, 16, and 17); however, only the two higher doses showed s i g n i f i c a n t l y d i f f e r e n t r e s u l t s from the sal i n e control (P <0.05, t - t e s t ) .  Furthermore, there appeared to be a  l i n e a r relationship between the gonadosomatic index and log of the i n j e c tion doses  ( F i g . 13).  Ovulation was  also observed i n 5 out of the 12  f i s h i n j e c t e d with the highest dose, 1000  ng/g.  The p i t u i t a r y 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 , i d e n t i f i e d as the gonadotrophs (Aoki and Umeura 1970; Kasuga and Takahashi 1971).  The a c t i v i t y of these c e l l s can be e s t i -  mated by t h e i r staining a f f i n i t y with aldehyde fuchsin (AF), and i s r e l a t e d to the changes of reproductive a c t i v i t i e s ; the staining  affinity  67  Figure 13.  E f f e c t s of d i f f e r e n t dosages of synthetic LH-RH on the gonadosomatic index and the percent d i s t r i b u t i o n of Class I I I oocytes.  Numbers i n  brackets represent number o f f i s h i n each group. Values reported are means + standard errors. Asterisk represents s t a t i s t i c a l s i g n i f i c a n c e (P <0.05, t-test) compared to saline injected c o n t r o l , a, ovulation observed.  68  INJECTION DOSES ng/g  69  Figure 14.  Section (7 um) of ovary from Oryzias injected with saline twice-a-week f o r 6 weeks at 23 + 1°C under 8L/16D.  Figure 15.  Hematoxylin-eosin, X 100.  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 f o r 6 weeks under 23 + 1°C and 8L/16D.  Notice the appearance  of Class I I I oocytes with yolk v e s i c l e s as indicated by arrow,  Figure 17.  Hematoxylin-eosin,  X100  Section (7 um) of ovary from Oryzias injected with 1000 ng/g synthetic LH-RH twice-a-week f o r 6 weeks under 23 + 1°C and 8L/16D.  Notice the presence of  Class I I I oocytes with yolk formation as indicated by arrow, and also the enlarged ovarian cavity (C) due to the ovulated oocytes. X100  Hematoxylin-eosin.  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 s a l i n e -  injected control f i s h were not stained with AF, suggesting of these c e l l s ( F i g . 18).  inactivity  Similar r e s u l t s were observed i n the 1 ng/g  injected f i s h and the photomicrograph was omitted here.  As the i n j e c -  tion dose increases, the gonadotropic c e l l s show an increase i n s t a i n a b i l i t y and i n the area stained (Figs. 18, 19, 20 and 21).  These r e -  s u l t s suggest that synthetic LH-RH at doses of 10 to 1000 ng/g were e f f e c t i v e i n stimulating a c t i v i t y of the gonadotropic c e l l s r e s u l t i n g i n ovarian development. E f f e c t s of Synthetic LH-RH and LH i n j e c t i o n s 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 i n j e c t e d with s a l i n e (both methylmercury treated and clean water control) started spawning only during the fourth week of the exposure ( F i g . 22).  Though showing an early s t a r t i n spawning, the methyl-  mercury-exposed LH-RH i n j e c t e d group showed a decline i n spawning a c t i v i t y a f t e r the fourth week, while the methylmercury-exposed LH-injected group showed increasing spawning a c t i v i t y throughout the treatment period.  Methylmercury-exposed s a l i n e - i n j e c t e d f i s h showed spawning only  i n the l a s t three weeks of exposure and these a c t i v i t i e s were lower than that of methylmercury-exposed LH-injected groups.  Though spawning  74  Figure 18.  Section (6 um) of p i t u i t a r y gland from Oryzias injected with saline twice-a-week for 6 weeks under 23 + 1°C and 8L/16D. T, thyrotroph;  P, p r o l a c t i n c e l l ;  G, gonadotroph; N, neurohypo-  physis; S, somatotroph. Aldehyde Fuchsin.  Figure 19.  X410.  Section (6 um) of p i t u i t a r y gland from Oryzias injected with 10 ng/g synthetic LH-RH twice-aweek f o r 6 weeks under 23 + 1°C and 8L/16D,  No*  t i c e the s l i g h t staining of AF i n gonadotrophs, G and increased AF staining i n neurohypophysis, N.  Aldehyde Fuchsin,  X410.  75  76  Figure 20. Section (6 um) of p i t u i t a r y 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 p i t u i t a r y 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 i n 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 i n the presence of methylmercury (10.7  yg/1).  79  120 • saline h^O 100  8 O LJJ  o o  8 0  • o A  -• saline CH^Hg* -o L H Cl-^Hg* •A LH-RH C H H g 3  60 40 20 0  O  <  Q_ 00  o O  2 3 EXPOSURE  r  4 5 (wks)  6  80 occurred l a t e r than i n the hormone i n j e c t e d groups, the clean water saline-Injected control group showed s u b s t a n t i a l l y higher spawning a c t i v i t y than any of the methylmercury exposed groups.  The percent  of spawning a c t i v i t y over the e n t i r e 6-week treatment period of the methylmercury treated groups were less than the clean-water control group ( F i g . 23). Injection of LH was e f f e c t i v e i n restoring part of the spawning a c t i v i t y i n h i b i t e d by methylmercury but not LH-RH. At the end of s i x weeks, the female gonadosomatic indices of the methylmercury treated groups were l e s s than the control (Fig. 23). Male gonadosomatic indices of the d i f f e r e n t groups were variable. Since the female gonadosomatic i n d i c e s provide a better i n d i c a t i o n of the e f f e c t s of the d i f f e r e n t treatment, s t a t i s t i c a l analysis was performed on data c o l l e c t e d from female f i s h only (Table 4 ) . The methylmercury exposed groups were a l l s t a t i s t i c a l l y d i f f e r e n t (P <0.05) from the clean water control group.  Among the methylmercury exposed groups,  the LH-injected group showed s i g n i f i c a n t differences from the s a l i n e i n jected group and the LH-RH i n j e c t e d group, while no s i g n i f i c a n t d i f f e r ence was observed between the s a l i n e injected and LH-RH injected group. The p i t u i t a r y 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 , i d e n t i f i e d as the gonadotrophs, GTH c e l l s (Aoki and Umeura 1970).  The a c t i v i t y of these c e l l s can be estimated by t h e i r staining  a f f i n i t y with aldehyde fuchsin and i s related to the reproductive act i v i t i e s (Kasuga and Takahashi 1971).  The present study showed that  s a l i n e injected clean water control f i s h had high a c t i v i t y i n gonadotrophs and neurosecretion as indicated by more intense staining with AF  81  Figure 23.  E f f e c t s 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 s i z e and the v e r t i c a l bars, standard error.  MALE O  GS I  p  o  p  M  ^  0 )  T  r  p  00  -j  -  O 1  T — i  r  Q O  Q  HK3  Q  Hi CO  Q 3 3 a  loi  i  Q (v)  - i s Q  Q  CO  O  4^  FEMALE  0)  GSI  L  00  09 to  83  Table 4.  S t a t i s t i c a l s i g n i f i c a n c e of female  gonadosomatic  indices among the various treatments by analysis of variance  Clean Water  CH Hg  Saline i n j .  Saline i n j .  Clean Water  P <0.05  Saline i n j . CH Hg  +  3  CH Hg  +  3  CH Hg  +  3  LH i n j .  LH-RH i n j .  P <0.05  P <0.05  P <0.05  n.s.  +  3  Saline i n j . CH Hg  P <0.05  _____  +  3  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 a c t i v i t y was observed i n the  methylmercury-exposed s a l i n e - i n j e c t e d group showing i n h i b i t i o n of GTH c e l l a c t i v i t y by methylmercury.  Methylmercury-exposed LH-injected  f i s h showed moderate a c t i v i t y i n gonadotrophs and neurosecretion while LH-RH injected f i s h showed high a c t i v i t y i n both gonadotrophs and neuro-secretion, suggesting that this hormone was e f f e c t i v e i n stimul a t i n g the a c t i v i t y of the p i t u i t a r y gland (Figs. 24 to 27). E f f e c t s of methylmercury on i n v i t r o ovulation Hirose (1971b) observed that when donor medaka were maintained under a long photoperiod (16L/8D, 0800 to 2400 hr) i n v i t r o ovulation required gonadotropic hormones i f Incubation commenced at 1700 hours but when incubated at 2200 hours, ovulation occurred n a t u r a l l y .  These two  time schedules were used i n the present study depending on the requirements of the i n d i v i d u a l t e s t s . Pre-exposure to methylmercury f o r 6 weeks.  When incubated at 1700  hours, l i t t l e or no ovulation was observed unless LH was present i n the media (Table 5).  This confirms Hirose's observations.  Even without  methylmercury added to the incubation media, pre-exposure of female f i s h to methylmercury f o r 6 weeks reduced the percentage of ovulation i n accordance with the pre-exposure concentrations.  When methylmercury was  added to the incubation media, further reduction i n percent ovulation was observed.  Methylmercury i n the incubation media seems to have an  additive e f f e c t on the concentration of methylmercury to which the f i s h were previously exposed.  85  Figure 24.  Section (6 um) of p i t u i t a r y gland from Oryzias exposed to clean water and injected with saline twice-a-week f o r 6 weeks under 23 + 1°C and 16L/ 8D,  T, thyrotroph;  neurohypophysis.  Note the intense AF s t a i n i n g  i n the gonadotrophs.  Figure 25.  G, gonadotrophs; N,  Aldehyde fuchsin.  X410.  Section (6 um) of p i t u i t a r y gland from Oryzias exposed to methylmercury and injected with saline twice-a-week f o r 6 weeks under 23 + 1°C and 16L/ 8D.  G, gonadotrophs; N, neurohypophysis.  Note  the loss i n AF staining i n the gonadotrophs. Aldehyde fuchsin.  X410.  86  87  Figure 26.  Section (6 um) of p i t u i t a r y gland from Oryzias exposed to methylmercury and injected with l u t e i n i z i n g hormone twice-a-week f o r 6 weeks under 23 + 1°C and 16L/8D.  G,  gonadotrophs; N, neurohypophysis. Note the moderate AF staining i n gonadotrophs.  Figure 27.  Aldehyde fuchsin.  X410.  Section (6 um) of p i t u i t a r y gland from Oryzias exposed to methylmercury and injected with l u t e i n i z i n g hormonereleasing hormone twice-a-week f o r 6 weeks under 23 + 1°C and 16L/8D,  G, gonadotrophs; N, neurohypophysis.  the intense AF staining i n gonadotrophs. fuchsin.  X410.  Aldehyde  Note  88  Table 5.  E f f e c t s o f d i f f e r e n t c o n c e n t r a t i o n s o f m e t h y l m e r c u r y 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.  V a l u e s r e p o r t e d as p e r c e n t o v u l a t i o n ( n o . o f o o c y t e s o v u -  lated/no. o f oocytes used). 6-wk Cone, o f C H ^ g pre-expoincubation media sure to CH Hg +  3  From C o n t r o l  From f i s h exposed to 4.8 ug/1 C H H g  +  3  From f i s h exposed to 10.7 ug/1 CH Hg  3  10 vg/ml LH + C H H g 3  +  +  i n yg/1  Added 0  0  4.8  10.7  21.5  215  480  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)  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)  24.0 (6/25)  33.3 (9/27)  70.0 (14/20)  3  From f i s h exposed to 11.5 ug/1 C H H g  No LH  54.5 (12/22)  20.0 (4/20)  28.9 (8/28)  00 VO  90  E f f e c t s of methylmercury on normal oocytes from untreated f i 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 i n t o percent i n h i -  b i t i o n showed a direct r e l a t i o n s h i p with the logarithm of the doses used ( F i g . 28). E f f e c t s of d i f f e r e n t steroids on methylmercury i n h i b i t e d ovulation in vitro.  Under normal conditions, ovulation occurs n a t u r a l l y when i n -  cubated at 2200 hours.  A p i l o t study was undertaken to determine an  e f f e c t i v e dose of methylmercury whereby ovulation would be i n h i b i t e d at t h i s hour.  Table 7 showed the r e s u l t s of such a study.  Normal oocytes  from untreated f i s h when incubated at 2200 hours ovulated without LH while the addition of methylmercury at concentration of 192 ug/1 and 215 ug/1 was e f f e c t i v e i n 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 f o r an i n v i t r o ovulation study, four steroids (progesterone, cortisone, e s t r a d i o l and testosterone) were tested for t h e i r effectiveness i n restoring methylmercury i n h i b i t e d i n v i t r o ovulation. Table 8 shows the r e s u l t s .  When incubated without  methylmercury at 2200 hours, normal oocytes from untreated f i s h ovulated naturally and this was stimulated by the addition of cortisone. terone, e s t r a d i o l and testosterone had l i t t l e or no e f f e c t .  Proges-  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 s l i g h t l y potent i n inducing a certain degree of ovulation.  91  Table 6.  The effects of various concentrations of methylmercury on ovulation i n v i t r o of Oryzias l a t i p e s incubated with 10 yg/ml porcine LH at 1700  Cone, of CHgHg+ yg/i  No.  hours.  of  oocytes used  No. of  Percent  ovulated eggs  Ovulation  0  25  21  84.0  4,8  38  28  73.7  10.7  26  17  65.4  21.5  33  20  60.6  48.0  35  17  48.6  96.0  45  18  40.0  192.0  51  15  29.4  215.0  40  7  17.5  92  Figure 28.  Percent i n h i b i t i o n of i n v i t r o ovulation by various concentrations of methylmercury i n incubation media at 1700 hours.  93  80 70 60 Z  o  5 0  gQ40  X ± 30 o  o  20 10 J  0  1  I  I I H HL  5  J  iii  I I II I  10  50  100  CONCENTRATION jug C H H g / I +  3  J  I  I I  500  94  Table 7.  The effects of methylmercury on ovulation i n v i t r o of Oryzias l a t i p e s 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  0  25  15  60.0  192.0  10  30  8  26.6  192.0  0  32  8  25.0  215.0  10  34  8  23.5  215.0  0  35  6  17.1  95 Table 8 .  The e f f e c t s of steroids on ovulation i n v i t r o of Oryzias l a t i p e s oocytes incubated with and without  methylmercury  at 2200 hours as the time of incubation  Cone., o f CH Hg  Steroid  +  3  ^S/l  0; 215:  1  Pg/ml  No. of oocytes used  0 0  No. of ovulated eggs  Percen Ovulati  28  15  53.6  34  6  17.6  a  Progesterone  35  22  62.8  215  Progesterone  30  6  20.0  Cortisone  38  32  84.1  Cortisone  41  18  43.9  Estradiol  30  16  53.3  Estradiol  29  5  16.1  Testosterone  30  17  56.6  Testosterone  32  6  18.7  0) 215 0! 215 0' 215  96 DISCUSSION  E f f e c t of synthetic LH-RH on ovarian maturation Results of the present study show that synthetic LH-RH i s e f f e c t i v e i n stimulating the a c t i v i t y of gonadotropic c e l l s i n the p i t u i t a r y as i n dicated by the a f f i n i t y to AF s t a i n i n g (Kasuga and Takahashi 1971), and i n inducing ovarian maturation and ovulation i n Oryzias l a t i p e s maintained at 23 + 1°C under short photoperiods (8L/16D).  This agrees with the r e -  sults obtained i n the ayu (Plecoglossus a l t i v e l i s ) (Hirose and Ishida 1974), g o l d f i s h (Lam et a l . , 1976; Kaul and V o l l r a t h 1974), carp  (Breton  and Weil 1973), and trout (Crim and Cluett 1974). Teleost p i t u i t a r y gonadotropic c e l l s 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 ext r a c t s of goldfish do not have any immunological cross reaction with l a b e l l e d 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 f i s h gonadotropin(s)-releasing  hormone  that may be chemically d i f f e r e n t from mammalian LH-RH but have overlapping biological  activities.  The e f f e c t i v e dose of synthetic LH-RH used, although s i m i l a r to dosages used i n other f i s h studies, was much higher than the e f f e c t i v e dose i n mammals.  This difference could be due to the methods of administration  of the hormone.  Intraperitoneal i n j e c t i o n has been shown to be less e f f e c -  tive than perfusion of the p i t u i t a r y i n s i t u (Vellano et a l . , 1974) or  97  i n t r a c r a n i a l i n j e c t i o n (Lam e t a l . , 1976).  Though not known f o r 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 stimul a t e the p i t u i t a r y at the lower doses.  At a low dose of 10 ng/g, a  s l i g h t increase (though not s t a t i s t i c a l l y s i g n i f i c a n t ) was observed i n gonadosomatic index with no increase i n percent d i s t r i b u t i o n of Class I I I oocytes over the control ( F i g , 13). I t i s possible that low doses only stimulate mild synthesis and release of gonadotropin(s) leading to development of Class 1 and I I oocytes only. Under natural spawning conditions, where male and female f i s h are present together, the gonadosomatic index of female f i s h i s approximatel y 6% (Chan 1976),  In the present study, the GSI was much lower.  This  may possibly be due to the absence of males r e s u l t i n g i n retention of ovulated oocytes i n the ovarian c a v i t y ; these unspawned eggs may have an i n h i b i t o r y effect on the development of the rest of the ovary (Egami and Hosokawa 1973).  Moreover, the e f f e c t of handling the f i s h during  frequent injections cannot be disregarded. In this experiment, s t a i n a b i l i t y with aldehyde fuchsin was used as a c r i t e r i o n of a c t i v i t y i n gonadotropic c e l l s .  Since synthetic LH-RH  i s e f f e c t i v e i n releasing gonadotropin(s) (Hirose and Ishida 1974; Lam et a l . , 1976; Kaul and V o l l r a t h 1974; Breton and Weil 1973), the i n creased AF staining of gonadotropin c e l l s observed i n the present study probably represents the excess of gonadotropin synthesis over i t s release from the p i t u i t a r y .  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) i n the p i t u i t a r y 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 d a i l y throughout the spawning season (Egami and Hosokawa 1973).  Thus i t seems possible  that gonadotropic c e l l a c t i v i t y remains high i n Oryzias throughout the entire spawning season.  The large release of gonadotropin(s) by LH-RH  as observed i n 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 d a i l y ovulation between 0100 and 0400 hr. Of i n t e r e s t also i n the present study i s the increased neurosecretion (increased AF staining of neurohypophysis) associated with increasing doses of synthetic LH-RH (Figs. 18 to 21).  The cause i s not known.  However, i t has been observed that neurohypophysial a c t i v i t y correlates with reproductive a c t i v i t y i n Oryzias (Kasuga and Takahashi 1971), and that neurohypophysial secretions a f f e c t spawning behaviour i n the k i l l i f i s h (Macey, Pickford and Peter 1974) and stimulate a c t i v i t y of the o v i duct and ovarian smooth muscles i n the guppy (Heller 1972).  There are  two possible explanations f o r the stimulation of neurosecretion by synt h e t i c LH-RH: i t i s possible that the stimulation of the p i t u i t a r y by mammalian LH-RH may be mediated through yet another system i n the neurohypophysis, and secondly this stimulation may be the r e s u l t of a feedback by the maturing ovary on the hypothalamus. planations require further research.  These highly speculative ex-  99 In summary, one point seems c l e a r ; a long photoperiod i s essent i a l f o r ovarian development i n the medaka (Yoshioka 1962; 1963; Chan 19760 and t h i s long photoperiod triggers the secretion of a hormone, probably from the hypothalamus  (Peter 1973) , s i m i l a r i n a c t i v i t y to  synthetic LH—RH, which i n turn stimulates the a c t i v i t y o f the gonadot r o p i c c e l l s i n the p i t u i t a r y . E f f e c t s of synthetic LH-RH and LH i n j e c t i o n s on methylmercury treated fish.. Previous experiments demonstrated that a 6-week exposure to 10.7 ug/1 o f methylmercury reduced spawning by about 50% compared t r o l value ( F i g . 8, Section I I ) .  to the con-  In the present study, a further reduc-  t i o n o f spawning by about 20% was probably due to frequent handling during: the twice-^weekly i n j e c t i o n s (see F i g s . 8, 10 and 23). The reduction of spawning a c t i v i t y by methylmercury was p a r t i a l l y prevented by i n j e c t i o n of LH ( F i g . 23) suggesting that the gonads r e mained receptive to LH stimulation while exposed to methylmercury.  Both  hormone i n j e c t e d groups started spawning e a r l i e r than the s a l i n e i n j e c t e d groups suggesting that hormone i n j e c t i o n s 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 gonadotrophiniCs), and synthetic LH-RH has been demonstrated e f f e c t i v e i n doing both. In the present study, however, p i t u i t a r y cytology showed a s l i g h t l y d i f ferent p i c t u r e .  Synthetic LH-RH was e f f e c t i v e i n stimulating gonadotropic  c e l l a c t i v i t y i n methylmercury treated f i s h even though spawning a c t i v i t y was not restored (Fig. 24 to 27).  Previously, i t was suggested that the  100 a c t i v i t y shown by p i t u i t a r y gonadotrophs represents the net r e s u l t between synthesis and release of gonadotropin(s) i n GTH c e l l s . j e c t i o n of synthetic LH-RH into methylmercury  Thus i n -  poisoned f i s h , which r e -  sulted i n stimulation of gonadotropic a c t i v i t y with no increase i n spawning a c t i v i t y , suggests a possible blockage i n the release of gonadotropin^) .  The lowering of r e c e p t i v i t y at the gonadal l e v e l by methyl-  mercury has been ruled out because exogenous LH was e f f e c t i v e i n removing the i n h i b i t i o n of spawning a c t i v i t y by methylmercury  ( F i g . 23).  David and Ramasawmi (1971) observed an increase i n granulation of LH c e l l s i n the langur p i t u i t a r y following cadmium-induced t e s t i c u l a r nec r o s i s suggesting possible blockage i n the release of the hormone. sent findings show similar r e s u l t s with Oryzias. methylmercury  Pre-  Since both cadmium and  have a strong a f f i n i t y for sulfhydryl groups and since i n  both animals the release of gonadotropin were affected, i t seems possible that the action on reproduction f o r these two chemicals are s i m i l a r .  Fur-  thermore, gonadotropin release i n these two animals may w e l l be s i m i l a r , under the influence of s u l f y d r y l group containing compound or enzyme. Since the mechanism c o n t r o l l i n g release of hormones from the p i t u i t a r y i n f i s h i s not known, this suggestion i s highly speculative and requires further i n v e s t i g a t i o n . However, i t i s clear that reproduction may be p a r t i a l l y restored i n methylmercury  poisoned f i s h with l u t e i n i z i n g hormone i n j e c t i o n s .  other words, the gonads of methylmercury  treated f i s h were s t i l l receptive  to exogenous l u t e i n i z i n g hormone stimulation. curred i n nature by methylmercury  In  Reproductive damages i n -  contamination may  thus be remedied  101 p a r t i a l l y by Injecting the appropriate hormone(s). E f f e c t of methylmercury on i n v i t r o ovulation Ovulation i n 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 i s smaller i n diameter than the oocytes, and through which the oocytes i s squeezed out (Asdell 1962).  In Oryzias l a t i p e s , t h i s process can occur i n v i t r o  i n Isolated, i n t a c t f o l l i c l e s  (Hirose 1971).  t h i s process i s not well understood.  The mechanism behind  Pendergrass  (1976) observed an  increase i n microfilaments i n the thecal layer p r i o r to ovulation i n v i t r o and suggested that these microfilaments, being c o n t r a c t i l e , are Involved i n c e l l movement during ovulation.  However, i n v i t r o ovula-  t i o n 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 i s indispensable for both prot e c t i o n and hormonal action (Hirose 1972b). Treatment with methylmercury a f f e c t s the release of gonadotrpin i n the p i t u i t a r y while the ovary remains receptive to stimulation of l u t e i n i z i n g hormone,  Pre-exposure  of f i s h to 21.5 ug/1 of methylmercury for  s i x weeks reduced i n v i t r o ovulation by about 40% even under the stimulation of exogenous LH (Table 5), not known.  The mechanism behind this i n h i b i t i o n i s  I t i s possible that methylmercury affected the enzyme system  f o r s t e r o i d production.  Methylmercury  has a strong a f f i n i t y f o r s u l f y d r y l  groups and the enzymes involved i n steroidogenesis may possess such properties.  Methylmercury  added d i r e c t l y to the incubation media further  102  reduced the percent i n v i t r o ovulation for f i s h previously exposed to the same toxicant (Table 5). system may  This may  be possible because the enzyme  not be completely blocked by methylmercury during the pre-  treatment; thus uncubation with methylmercury i n the i n v i t r o system allowed a more complete i n h i b i t i o n . A dose response was  observed between percent i n h i b i t i o n of i n  v i t r o ovulation and the logarithm of the concentration of methylmercury used.  F i f t y percent i n h i b i t i o n occurs at about 55 ug/1  i n the incubation medium.  of methylmercury  This method provides a f a s t convenient  for testing e f f e c t of a toxicant on reproduction and may  system  well develop  into a bioassay f o r general t o x i c i t y studies. When incubations commenced at 2200 hr or 2400 hr a very high percent of oocytes ovulated n a t u r a l l y without LH (Hirose & Hirose 1972). results were obtained i n the present study incubation media contained 192 ug/1 reaction was may  (Table 7).  or 215 ug/1  Similar  However, i f the  of methylmercury, this  reduced suggesting that methylmercury added to the medium  be i n h i b i t i n g the synthesis, release or action of the hormone(s)  responsible for ovulation.  LH could not be one of the hormones i n question,  since the addition of 10 ug/ml of LH was  not e f f e c t i v e i n increasing the  percent ovulation (Table 7). Corticosteroids are e f f e c t i v e i n inducing ovulation i n Oryzias i n v i t r o (Hirose 1972a, c, 1973), i n Indian c a t f i s h i n vivo and i n v i t r o (Goswami and Sundararaj 1972a, b, and 1974) 1974).  and i n g o l d f i s h i n vivo (Khoo  The present experiment showed similar r e s u l t s .  Cortisone  was  e f f e c t i v e , at least p a r t i a l l y , i n overcoming the i n h i b i t o r y e f f e c t of  103 methylmercury  (Table 8).  Columbo et a l . (1973) demonstrated  corticos-  teroid synthesis i n G i l l i c h t h y s and suggested that endocrine control of ovulation acts by p i t u i t a r y gonadotropin stimulation of the synthesis of corticosteroids i n the ovary.  Sundararaj and Goswami (1974) i n a co-  culture study showed that the i n t e r r e n a l contributes the major portion of corticosteroids f o r ovulation.  However, both of these phenomena have  not been shown f o r other species.  The present study has not c l a r i f i e d  this point.  Since the addition of c o r t i c o s t e r o i d to the media can re-  store some of the ovulation i n h i b i t e d by methylmercury,  i t seems possible  that the f o l l i c u l a r c e l l s of Oryzias are capable of producing some c o r t i costeroids.  Hirose (1972b) also made such a suggestion i n another i n  v i t r o ovulation study with a f o l l i c u l a t e d oocytes.  Presence of methylmer-  cury i n the medium probably blocked corticosteroidogenesis i n the f o l l i c u l a r tissues, while addition of exogenous cortisone was e f f e c t i v e i n indue Ing some ovulation.  When both methylmercury  and cortisone were present,  balance of the two resulted i n a s l i g h t prevention of the i n h i b i t o r y effect of methylmercury  as observed i n the present study.  I t seems possibl  that once c o r t i c o s t e r o i d i s present, even just p r i o r to ovulation, ovulat i o n can ultimately occur. Sex steroids have not been e f f e c t i v e i n inducing ovulation and matur ation (Goswami and Sundararaj 1972b; Hirose 1972a). confirmed t h i s .  The present study  The fact that progesterone was observed to be s l i g h t l y  e f f e c t i v e i n stimulating ovulation and ovarian maturation i n both Oryzias (Hirose 1972b) and the Indian c a t f i s h (Goswami and Sundararaj 1972b) was also observed i n this.study.  However, the addition of methylmercury  p l e t e l y abolished this effect (Table 8).  com-  This suggests that progesterone  104 a precurser of c o r t i c o s t e r o i d s , cannot be converted to c o r t i c o s t e r o i d s because of i n h i b i t i o n of steroidogenesis by methylmercury. foregoing discussion, i t seems that methylmercury may  From the  be a general  b i t o r of enzymes, e s p e c i a l l y enzymes with s u l f y d r y l groups.  inhi-  Since a  majority of proteins possess s u l f y d r y l 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 p i t u i t a r y and gonad, of the hypothalamic-hypophysial-gonadal axis.  levels, How-  ever, i n v i t r o ovulation studies strongly suggested that methylmercury Is a c e l l u l a r i n h i b i t o r of an enzyme system.  This i s probably true be-  cause chemical reactions form the basis of a l l b i o l o g i c a l a c t i v i t i e s .  105 GENERAL DISCUSSION  The median l e t h a l concentration, 96hr-LC50, of methylmercury f o r adult Oryzias l a t i p e s was found to be 8 8 + 9 . 8 yg CH,jHg /l. +  Compared  to rainbow trout, this value i s quite high suggesting that Oryzias i s a much more r e s i s t a n t f i s h . Tissue accumulation of methylmercury increases with exposure time and concentration of the chemical i n the external medium.  Death seems  to occur once tissue methylmercury l e v e l s reached about 25 yg/g as methylmercury.  Such l e v e l s were reached i n two weeks f o r f i s h exposed to 21.5  yg/1 of methylmercury and 6 weeks when exposed to a lower concentration of 10.7 yg CH^Hg"*"/!. However, i n t h e p r e s e n t study, this l e v e l was never reached even at the end of six-weeks exposure to 4.8 yg/1 of methylmercury. Four-hour exposure of spawning f i s h to methylmercury during the f i s h ' s normal oviposition time affected spawning at concentrations equivalent to one-tenth of the medial l e t h a l concentration.  However, this  e f f e c t 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-  t r a t i o n was at the median l e t h a l concentration.  This e f f e c t was  over to the time when the f i s h were returned to clean water.  carried  This short  exposure of 4 hours to the median l e t h a l concentration of methylmercury may have allowed enough accumulation of the chemical to e f f e c t a change i n spawning a c t i v i t y when returned to clean water. occur with bioaccumulative toxicants.  This phenomenon may  I f so, this type of study not only  provides information on the t o x i c i t y but also indicates whether the t o x i 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 e a s i l y quantified, this behavioral response may  be  useful for monitoring environmental changes l i k e water p o l l u t i o n . Long-term exposure to methylmercury at concentrations  approximately  equal to one-eighth of the median l e t h a l concentrations resulted i n about 50% i n h i b i t i o n of reproductive a b i l i t y . one-twentieth  At a lower concentration of about  of the median l e t h a l concentration, 40% i n h i b i t i o n on r e -  productive a b i l i t y was  observed.  These results c l e a r l y showed that repro-  duction i s extremely s e n s i t i v e to environmental contamination,  and since  reproduction i s indispensable for the s u r v i v a l of the species, i t seems reasonable that studies of environmental contaminations  should include  more thorough examinations on reproduction of the species i n question. Sometimes i t may  not be p r a c t i c a l to study larger species l i k e the salmon,  but studies on f i s h l i k e Oryzias may  provide an i n s i g h t into what might  happen i n the more valuable food f i s h e s .  For bioaccumulative  toxicants,  the use of "safe" factors does not seem to hold, since the "sublethal" concentration may  vary with d i f f e r e n t types of bioaccumulative  toxicant  (Sprague 1971). For methylmercury, i t seems that the accumulation  of toxicant by  parent f i s h does not affect the h a t c h a b i l i t y of the eggs. suggest that the toxicant may  This seems to  not have been accumulated i n spawned eggs.  Since no residue analysis was performed on the spawned eggs, this suggestion remains speculative.  The observation that j u v e n i l e f i s h are more  sensitive to methylmercury than adult f i s h agrees with Sprague's reasoning (1971).  107 Synthetic LH-RH at doses of 100 ug/g and 1000 ug/g was e f f e c t i v e i n 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), i n h i b i t i o n of spawning a c t i v i t y was observed i n Oryzias. was not removed by the i n j e c t i o n of synthetic LH-RH.  This i n h i b i t i o n  P i t u i t a r y cytology  revealed high a c t i v i t y i n gonadotrophs as stimulated by the i n j e c t i o n of synthetic LH-RH.  This suggests a possible blockage i n the release of  gonadotropin(s).  P a r t i a l restoration of spawning a c t i v i t y i n methylmer-  cury-treated f i s h by i n j e c t i o n of LH suggests that the gonads were s t i l l receptive to LH. This study demonstrated e f f e c t s o f methylmercury on both the hypophysis and gonads but f a i l e d to show the e f f e c t s of methylmercury at the hypothalamic l e v e l . Ovulation i n v i t r o was used to elucidate further the mode of action of methylmercury at the gonadal l e v e l .  A l o g dose response was observed  i n the percent i n h i b i t i o n of ovulation i n v i t r o .  F i f t y percent i n h i b i t i o n  occurred at concentrations of 55 ug/1 of methylmercury i n the incubation medium.  Once a block developed with methylmercury, l u t e i n i z i n g hormone  was not e f f e c t i v e i n removing this block.  Cortisone was the only steroid  tested that was e f f e c t i v e i n restoring methylmercury-blocked i n v i t r o ovulation.  This observation confirms reports that c o r t i c o s t e r o i d s are  involved i n 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 i n the f o l l i c u l a r layer of the oocytes and thus blocked ovulation while the addition of exogenous c o r t i c o s t e r o i d 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 e f f e c t s on the reproduction i n Oryzias. These physiological  effects are of ecological  i  importance, not only because s u r v i v a l w i l l be reduced but also because other b i o l o g i c a l systems c l o s e l y related to i t may be impaired.  Oryzias  i s more r e s i s t a n t than some other f i s h and, i f methylmercury has such detrimental e f f e c t s on Oryzias, one can expect s u b s t a n t i a l l y greater e f f e c t s of methylmercury on other more " d e l i c a t e " fishes such as salmon. In the present study, methylmercury seems to act at two l e v e l s , the p i t u i t a r y and the gonad. two l e v e l s , "replacement some of these e f f e c t s .  Though i n h i b i t i o n may have occurred at these therapy" seems to be e f f e c t i v e i n overcoming  Reproductive damages occurring i n nature by  methylmercury contamination might thus be p a r t i a l l y remedied by i n j e c t ing the appropriate hormones. c a l functions.  The same may be true f o r other  physiologi-  109 REFERENCES Aaronson, T. Akiyama, A.  1971. 1970.  Mercury i n the environment.  Environ. 13: 16-23.  Acute t o x i c i t y of two organic mercury compounds to  the t e l e o s t , Oryzias l a t i p e s , l n d i f f e r e n t stages of development. B u l l . Jap. Soc. S c i , F i s h . 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.  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