' V - I r’ -. ID . . .Q. I. - 'Ifi/ ‘: I . ~. . .’ ' .' ’f 1") :- . I .‘u ' T" . . O 'y‘.’ . ‘. _ _ . ~o . ." '.. “(LI-,3, IN VITRD GILL ’M ‘ AND BLOOD PARAM 'vw‘ww v‘v—‘v—v-‘VV‘vu-y- ww—v vwm‘w . ‘V‘i" wvv --- ‘ ‘w—‘v ‘vvtv\wwv' ~‘>‘.\‘—Vvfi" . RAINBOW TROUT (SALMD GAIRDNERI) - _ _ AFTER CHRONIC EXPOSURE Io METHYL MEcumc CHLORIDE .,.'4‘ '..“.§\.“".‘.".t' esis for the Degree of M. S. ICHIGAN STATE UNIVERSITY DANIEL V. O'CONNOR 1974 .. . v o. . I ‘ " n ' I - I _. . r- ' . ' ‘. . I o - O .' c ‘ ' I j .‘ ’ . ‘f- .' , ' . . . . ’ . a n n '1 ’ ‘ ‘ . v a I (" v . ' - . . —. . "' 0 I’ ‘r -' ’ ‘ . ‘rn ' .J - '- . . ' " ' .‘ol. - . ' , y 0 -av ‘ . . _ o . ‘4'. _ .1 . .u,‘ 1 . ‘ .. .. Iv v . ' .t , .yr: 5 ’a_.f r' ’ o '0’ ' ' I x "' ' . ‘ " I ma ’. . - -v. . " ' . ' ' v J .' " . 4""! ' ‘0' .' .I’ ‘ 3‘ 0~ 0‘ '. . I U ”' ’.' "' ' J' "' ‘, ' , . .f.'y , v/ '7‘ . 'c..ff-ad'.' ".fifif q ' o ‘ ' ‘. . 0.. .‘ '"l. ,.‘ .‘o ‘ ’2 r ‘4 5 Di '2', ‘. " . 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' ':;"l "- ."'040 "";"I".. -- ‘ LI B RA R Y Michigan State University L1. .h'l'" QNERG av E HUAB & SUNS' a! 1 3! 809K BINDERY INC. *8 “A RY BINDERS Imam" unit-u... ABSTRACT £11 VITRO GILL METABOLISM AND BLOOD PARAMETERS OF RAINBOW TROUT (SALMO GAIRDNERI) AFTER CHRONIC EXPOSURE TO METHYL MERCURIC CHLORIDE 3? Daniel V. O'Connor Olson (1972) reported the primary pathway for the uptake of methyl mercury in fish.was through the gill and that chronic exposure (0.3 ug Hg/liter for 4 and 8 weeks) resulted in chloride cell degeneration and epithelial cell vacuolization as visualized with scanning electron microscopy. The present investigation was conducted to determine if chronic exposure to methyl mercury would also affect the metabolism and physiological function (plasma electrolyte regulation) of the gill. Accordingly, rainbow trout were exposed toJIDug Hg/liter administered as methyl mercuric chloride and data were obtained for fish after 4, 8, and 12 weeks exposure. The in zigrg_oxygen consumption of gill fila— ments in 10% and 100% phosphate buffered saline (PBS), hematocrit, and plasma electrolyte concentrations (Na+, K+, Cl—, Mg++, Ca++) were determined for control and mercury exposed fish. Results indicate there is no significant difference in oxygen consumption between control and mercury treated fish in 10% or 100% PBS, however, both groups show a higher rate of oxygen consumption in 100% PBS than in 10% PBS. Although ++ plasma levels of Na+, K+, and Mg did fluctuate during the course of the experiment, no significant difference in the concentration of plasma Daniel V. O'Connor electrolytes was seen between control and experimental fish. The only difference observed between control and experimental rainbow trout appeared in values for hematocrit where mercury treated fish showed a marked increase in hematocrit after 12 weeks exposure. Other investi- gators have reported histological and/or morphological changes in the gill as a result of chronic exposure to methyl mercury. Results from the present investigation, however, indicate that chronic exposure to methyl mercury does not alter the metabolism of the gill or affect its role in plasma electrolyte regulation. INLVITRO GILL METABOLISM AND BLOOD PARAMETERS OF RAINBOW TROUT (SALMO GAIRDNERI) AFTER CHRONIC EXPOSURE TO METHYL MERCURIC CHLORIDE By Daniel V; O'Connor A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1974 H; q C129“ \ DEDICATION To my parents Mr. and Mrs. Virgil O'Connor from a grateful son 11 ACKNOWLEDGMENTS The author wishes to express his sincere thanks to Dr. P. 0. Fromm for his support, patience, and aid in the preparation of this thesis. I would also like to express my appreciation to Ken Olson, Harold Bergman, and Dick Walker for advice given throughout the laboratory work and in preparation of this thesis. The author is grateful to Maribetthills for typing all the rough drafts and to Dr. J. R. Hoffert for the photographic work. I would also like to express my sincere gratitude to Esther Brenke for her invaluable assistance throughout this work. The author is indebted to the Environmental Protection Agency for support of this work through Grant R-801034. iii TABLE OF CONTENTS LIST OF TABLES O O O O O O O O O O O O O O O O O 0 LIST OF FIGURES O O O O O O O D O O O O O O O O O O 0 INTRODUCTION AND LITERATURE REVIEW . . . . . Methyl Mercury - Mode of Entry into Fish. . . . The Gill - Its Role in Electrolyte Homeostasis. The Effects of Mercury and Other Heavy Metals . RESMRCH RATIOW 0 O O O O O O O O I O O O O O O O MTmIALS AND METHODS O O O O O O O O O O O O O O O 0 Experiment 1 O O O O O O O O O O O O O O O O O 0 Experiment 2 O O O O O I O O O O O O O O O O O 0 Procedure for Blood Samples . . . . . . . . . . 02 Consumption of Gill Tissue . . . . . . . . . Experiment 3. O O I O O O O O O O O O O O O O O Stat istics O O O O O O O O O O O O O O O O O O 0 RESULTS 0 O O O O O O O O I O O O O O O O O O O O O 0 Experiment 1 (Grayling Hatchery Fish) . . Experiment 2 (4 Weeks Exposure to 10 ug Hg/liter Adminis- tered as CHfiHgCl). . . . . . . . . . Experiment 3 (4, 8, and 12 Weeks Exposure to 10 Administered as CH3HgC1) . . . . . . . . . Plasma Electrolytes . . . . . . . . . . . ' DISCUSSION 0 O O O O O O O O O O 0 O O I O O O O 0 Blood Parameters. . . . . . . . . . . . . . . . Gill Metabolism and Active Transport. . . . . Starvation. O O O O O O O O O O O O O O O O 0 Temperature C O O O O O O O O O O O O O 100% and 10% Phosphate Buffered Saline (PBS). . SWY AND CONCLUS IONS O O O O O O O O O O O O O O 0 APPENDIX 0 o o o o o o o o o o o o o o o o o o o o o LITEMTURE CITED 0 O O O O O O O O O O O O 0 iv pg Hg/liter Page vi V§U 10 10 11 ll 12 14 14 15 15 15 18 25 39 40 42 44 45 45 46 47 48 LI ST OF TABLES TABLE Page 1. The solubility of oxygen in 10/ and 100/ phosphate bUffered saline. 0 O O O O O O O I O O O O O 0 O O O O O 13 FIGURE 1A. 13. 5A. 5B. 6A. 63. 7A. LIST OF FIGURES A model for the chloride cell activity in fresh.water teleosts. O O O O O O O O O O O O O O O O O O O I O O O A model for the chloride cell activity in salt water teleosts. O O O O C O O O O O O O O O 0 O O O O O C I 0 Plasma electrolyte concentrations for 18 rainbow trout (1.3—3.6 Kg, Experiment 1) from the Graying hatchery. . The effect of 100% and 10% phosphate buffered saline on the oxygen consumption of gill filaments (Experiment 2, 17°C) from control and experimental rainbow trout after 4 weeks exposure to 10 pg Hg/liter administered as CH HgCl . . . . . . . . . . . . . . . . . . . . . . . . 3 The plasma electrolyte concentration of control and experimental rainbow trout (Experiment 2) after 4 weeks exposure to 10 ug Hg/liter administered as CHBHgCl. . . The effect of 100% and 10% phosphate buffered saline on the 0 consumption of gill filaments from rainbow trout exposed to 10 pg Hg/liter administered as CHBHgCI for 4, 8, and 12 weeks (Experiment 3, 12°C) . . . . . . . The effect of 100% and 10% phosphate buffered saline on the 0 consumption of gill filaments (4, 8, and 12 0 weeks; from control rainbow trout (Experiment 3, 12 C). 0 consumption of gill filaments (Experiment 3, 12°C) from control and experimental rainbow trout in 10% phosphate buffered saline after 4, 8, and 12 weeks exposure to 10 ug Hg/liter administered as CHBHgCl. . . 0 consumption of gill filaments (Experiment 3, 12°C) from control and experimental rainbow trout in 100% phosphate buffered saline after 4, 8, and 12 weeks exposure to 10 pg Hg/liter administered as CHBHgCI. . . The effect of temperature on the 0 consumption of gill filaments from control and mercury treated fish in 100% phosphate buffered saline after 4 weeks exposure to 10 ug Hg/liter administered as CH3HgC1. . . . . . . . . vi Page 17 2O 22 24 24 27 27 29 LIST OF FIGURES--Continued FIGURE 73. 10. 11. 12. 13. 14. The effect of temperature on the 0 consumption of gill filaments from control and mercury treated fish in 10% phosphate buffered saline~after 4 weeks exposure to 10 pg Hg/liter administered as CH3HgC1. . . . . . . . . Plasma concentration of Na+ for control and experimen- tal rainbow trout after 4, 8, and 12 weeks exposure to 10 pg Hg/liter administered as CH3HgCl (Experiment 3) . Plasma concentration of K+ for control and experimental rainbow trout after 4, 8, and 12 weeks exposure to 10 pg Hg/liter administered as CH3HgCl (Experiment 3) . Plasma concentration of Mg++ for control and experi- mental rainbow trout after 4, 8, and 12 weeks exposure to 10 pg Hg/liter administered as CHBHgCl (Experiment 3). . . . . . . . . . . . . . . . . . . . . . . . . . . Plasma concentrations of Ca++ for control and experi- mental rainbow trout after 4, 8, and 12 weeks exposure to 10 pg Hg/liter administered as CHBHgCl (Experiment 3). . . . . . . . . . . . . . . . . . . . . . . . . . . Plasma concentration of C1- for control and experi- mental rainbow trout after 4, 8, and 12 weeks exposure to 10 pg Hg/liter administered as CH3HgC1 (Experiment 3). . . . . . . . . . . . . . . . . . . . . . . . . . Hematocrits for control and experimental rainbow trout after 4, 8, and 12 weeks exposure to 10 pg Hg/liter administered as CH3HgC1 (Experiment 3). . . . . . . . . A comparison of plasma electrolyte concentrations (Na+, Cl', Ca++) between rainbow trout sampled in February, 1973 (Experiment 2) and in June-August, 1973 (Experiment 3) after 4 weeks exposure to 10 pg Hg/liter administered as CH3HgCl . . . . . . . . . . . . . . . . vii Page 29 31 31 34 34 36 36 38 INTRODUCTION AND LITERATURE REVIEW Through the indiscriminate use of mercurials in agriculture and industry the United States is presently releasing over 600 tons of mercury per year into the environment (Anonymous, 1970). In view of the well-known tragedies at Minamata and Niigata and the recent numerous reports of widespread mercury contamination among aquatic organisms, justification for the continued use of mercurials represents one of the great paradoxes of our time. Much of the mercury released into the environment is eventually deposited on the bottoms of lakes and rivers in the inorganic form. Inorganic mercury represents a rather stable deposit and if it remained unchanged it would not present a critical health problem or contaminate the water and aquatic animals living therein. However, Jensen and Jernelov (1969) and Wood £5 31. (1968) have recently shown that inorganic mercury can be methylated by microorganisms and by methanogenic bacteria present in the sludge and sediments of rivers and lakes. L3fr3th (1969) points out that natural methylation of mercury produces dimethyl mer- cury which is both volatile and lipophilic. It decomposes into methyl mercury at slightly acidic pH and also forms complexes with anionic groups. This mobilization of mercury by microorganisms from the sedi- ment in rivers and lakes makes the organic form of mercury available. for assimilation by all aquatic organisms. Furthermore, the lipophilic nature of dimethyl mercury facilitates its translocation across biological membranes; methyl mercury is very soluble in aqueous media and is readily accumulated by aquatic organisms. Whether fish accumulate mercury from the environment in the organic or inorganic form has been a point of discussion for many years. But the ubiquitous distribution of methanogenic bacteria and microorganisms capable of methylating inorganic mercury and recent reports that 90-95% of mercury found in fish is methyl mercury (WestEES 1967; Smith 3531;. 1971) seems to support the theory that fish accumulate mercury in the methylated form. Pollution of our waterways with mercurials is not only of current concern; it will also affect succeeding generations as the large pool of mercury already in our lakes and streams is methylated by micro- organisms and bacteria and assimilated by food chain organisms. It has been estimated that it will take from 50 to 100 years to rid our lakes of mercury providing no further pollution with mercurials occurs. Giblin and Massaro (1972) have recently shown that rainbow trout given a single intragastric dose of methyl mercury (0.5 mg Hg/Kg body wt) had a half retention time of 780 days. Miettinen 25 31. (1970) reported half-retention times of 267 to 1,000 days for methyl mercury in fish, crabs, and molluscs. This extensive retention time of mercury threatens these species as a viable source of food. The effects of chronic mercury exposure on reproduction, general physiology, and genetics of fish and other aquatic species has yet to be fully elucidated. Methyl Mercury - Mode of Entry into Fish If we assume that the primary form of mercury accumulated by fish is methyl mercury, the major avenue of entry into the fish becomes of primary concern in determining possible pathological effects. Knoll (1962) has shown that the heavy metal, chromium, is unable to penetrate the skin of rainbow trout and it is a well-established fact that the skin of fresh water teleosts is relatively impermeable to both water and ions (Motais gtugl, 1969; Maetz 1971). These reports would seem to preclude the skin as a major site of uptake for methyl mercury. The rapid absorption of methyl mercury in the intestinal tract of mammals (humans, rats, cats, and monkeys) has been reviewed by Friberg and Vostal (1972). Little work has been done on fish with the excep- tion of Miettinen‘ggugl. (1969) who found that some of the mercury in- jected perorally is retained by fish for long periods of time but much is regurgitated by the fish shortly after administration. Under normal conditions in the environment fish absorb mercury through the gut as a result of ingesting mercury contaminated food chain organisms. However, in the present investigation fish were starved for the entire length of the experiment. Maetz (1970) points out that fresh water teleosts drink very little water and Shehadeh and Gordon (1969) have shown that rainbow trout in fresh water do not drink, thus the gut is most likely not a major avenue of uptake for methyl mercury. Although no conclusive information is available for the exact pathway of methyl mercury uptake by fish, the evidence seems to point to the gill as the most feasible site because of its role in electrolyte regulation (Smith 1930; Krogh 1937). The gill is also the site for the respiratory exchange of gases in fish and a large amount of water must flow over the gills to meet the oxygen demand of fish due to the low solubility of oxygen in water (Rahn 1966). Consequently, the gills are bathed with large amounts of water containing soluble pollutants such as methyl mercury. Hannerz (1968) and Rucker and Amend (1969) have shown that gill tissue is able to concentrate mercury up to several thousand times that found in the media. Olson (1972) reported that rain- bow trout after esophageal ligation were able to concentrate methyl mercuric chloride and mercuric chloride at the same rate as control fish (without esophageal ligation). The Gill - Its Role in Electrolyte Homeostasis The gills of fish consist of four branchial arches supported by cartilaginous rods on either side of the buccal cavity. Two rows of paired filaments extend perpendicular from each arch and many small leaf-like structures called secondary lamellae, extend from both sides of each filament. At the base of the secondary_lamellae are found the chloride cells which are thought to be the actual site of active ion transport in the gill. The diffusional distances from water to blood are small and facilitate the movement of ions and transfer of respirav tory gases. Figure 1 shows a model for salt transport by chloride cells in fresh water and salt water teleosts (Maetz, 1971). FIGURE lA.--A model for the chloride cell activity in fresh water teleosts. The external side of the chloride cell is exposed to fresh water and Na+ is activel. transported into the cell and exchanged for NH + or ' .Cl' is actively pumped into the cell in exchange for HCO' from H20 and CO in the presence of carbonic +anhygrase. On the internal side of the chloride cell Na+ is pumped into the blood and exchanged for K+ which passively dif- fuses back into the blood. An electrogenic Cl‘ pump translocates Cl" from the cell into the blood and NH3 and COZ diffuse passively from the blood into the chloride cell. FIGURE 1B.--A.model for the chloride cell activity in salt water teleosts. The Na+/K+ linked pump and the electrogenic Cl' pump are located on the external or salt water side of the cell. The K+ that is pumped in diffuses back out passively. The CI' /HCO3' pump and the Na +/H+ pump are located on the internal or blood side of the cell where Na+ and Cl' from the blood is exchanged for H+ and HCO3' from the chloride cell. The source of the H+ and HCO3’ is from the 002 that diffuses passively into the cell from the blood. Q and -—-’ = passive diffusion '<:::::) = electrogenic pump . “and % 8 exchange or linked pump int. Na‘ 1.. NH3 0T0. HCOB- 0 Cl- .......... .p FIGURE 1A C H 4 )\H {ammo Iacid / FIGURE 1B (“(303)0—L02m201deonination 002 K45 Cl- The Effects of Mercury and Other Heavy Metals Much of the initial work on heavy metal toxicity involved the determination of lethal concentrations of various heavy metals and their salts on fish. IMore recently the trend is toward elucidating the bio- chemical and physiological effects of heavy metals. Indicative of the deleterious effects of heavy metals on bio— chemical and physiological parameters Jackim 25H213~Cl970)~have shown the inhibitory effect of lead, copper, mercury, beryllium, cadmium, and silver on five liver enzymes in the killifish. Skidmore (1970) found that rainbow trout exposed to zinc sulphate showed a seven-fold increase in oxygen utilization, a six-fold increase in gill ventilation volume, and a 50% reduction in heart rate. The concentration of sodium, potassium, calcium, and magnesium in the blood was unaffected but there was significant damage to the gill epithelium. Bilinski and Jonas (1973) reported similar damage to gill epithelium when fish were exposed to lO—SM CdCl2 for 48 hours. Cadmium and copper also caused a 50% reduction in the oxidation of lactate by the gills. Olson (1972) also found highly vacuolated epithelial cells and chloride cell degeneration in rainbow trout exposed to 0.3 pg Hg/liter administered as methyl mercuric chloride for periods of 4 and 8 weeks. A review by Webb (1966) vividly points out the wide range of ef— fects mercurials can have on an organism. HgCl2 is fairly soluble in both aqueous and lipid solutions while organic mercurials are highly soluble in lipids which facilitates their rapid movement across bio— logical membranes. Mercurials bind preferentially to thiol groups or the sulfhydryl (SH) groups of proteins. Changes in the tertiary structure of proteins as a result of mercurials binding to SH groups greatly affects their activity and can completely inhibit an enzyme. This enzyme inhibition can be either competitive or non-competitive. In some cases mercurials have been shown to stimulate the activity of enzymes. Mercurials readily complex with purines and pyrimidines and are able to precipitate adenosine monophosphate, guanine monophos- phate, and inosine monophosphate. Mercurials also attack nucleic acids and affect the dimer/monomer ratio and the aggregation of mono- mers. They can also increase or decrease the affinity of hemoglobin for oxygen depending on the type of hemoglobin and the physiological conditions (pH, etc.) present. Mercurials can cause the dissociation of enzyme-cofactor and enzyme-coenzyme complexes and can inhibit ATPase activity, glycolysis, electron transport, oxidative phosphory- lation, as well as affecting protein and lipid biosynthesis. RESEARCH RATIONALE It was the intent of this experiment to determine if the chloride cell degeneration and epithelial cell vacuolation associated with chronic exposure to methyl mercuric chloride (Olson, 1972) could be correlated to an impairment of the physiological functions of the gill in electrolyte regulation. The role of the gill in electrolyte regula- tion for fresh water teleosts can be considered as two-fold; the chloride cell functions as the site for the active absorption of ions from the external media while the rest of the gilI serves as a barrier to the passive diffusion of ions out of the gill. It was felt that if mer- cury exerted an inhibitory effect on electrolyte regulation it would be manifest in the following ways: first, chloride cell degeneration might be associated with a reduced capacity to actively transport ions and result in lower plasma electrolyte concentrations; secondly, if surface epithelial cells represent a rate limiting barrier to the passive diffusion of ions out of the;gill, then vacuolation of these cells might increase their permeability and contribute to a greater“ efflux of plasma electrolytes and result in lower plasma ion levels, and finally, active transport is an energy consuming process and the inhibitory action of mercurials on glycolysis, electron transport and oxidative phosphorylation might affect the oxygen consumption of the gill. MATERIALS AND METHODS Experiment 1 Rainbow trout (Salmo gairdneri) weighing 1.3-3.6 Kg were captured from the breeding stock of the Michigan Department of Natural Resources hatchery at Grayling, Michigan and anesthetized with MS-222. Blood samples (3.0—4.5 ml) were taken immediately from the ventral side of the hemal arch approximately one inch posterior to the anal fin using a syringe fitted with a 1 1/2 inch 21 gauge needle and rinsed with ammonia heparin. All samples were then centrifuged for 10 minutes (at a setting of 4) on an International Equipment Company (IEC) clinical centrifuge (Needham Heights, Mass.). The plasma was then drawn off and immediately frozen in a dry ice/alcohol bath and transported to Michigan State Uni- versity in a styrofoam cooler containing dry ice. A 0.1 m1 sample of plasma was diluted with 1.9 ml lithium chloride and Na+ and K+ concentra- tions determined on a Beckman Flame Photometer, Model 105 (Beckman Instruments, Fullerton, Cal.). Samples (0.2 m1) of plasma diluted with 20 ml of lanthanum oxide were used to determine Mg++ and Ca++ concentra- tions on a Perkin-Elmer Atomic Absorption Spectrophotometer, Model 290-B (Norwalk, Conn.). Plasma chloride concentrations were measured with a Buchler Chloridometer (Buchler Instruments Inc., Fort Lee, N.J.). All plasma electrolyte determinations were done in duplicate. 10 11 Experiment.2 Rainbow trout weighing 120-250 g were obtained from‘theflMichigan Department of Natural Resources, Grayling, Michigan and maintained in 300 liter fiberglass tanks at 12:10C with a 14 hour daily photoperiod. The tanks were aerated with charcoal filtered compressed air and there was a constant flow of filtered (dechlorinated) water through the tanks. The fish were fed commercial salmon pellets three times a week before the start of the experiment (Ewos 159 SaLmon Pellets, Astra—Ewos, .S3dert31je, Sweden). Experimental design involved exposing a group of starved fish to 10 pg Hg/liter administered as methyl mercuric chloride from a gravity feed system while another group of starved fish served as controls. A 4 liter bottle containing a concentrated solution of methyl mercuric chloride was placed approximately 6 feet above the tank holding the experimental fish and a piece of polyethylene tubing was used to intro— duce the methyl mercury into the tank. The flow rate of the concentra- ted mercury solution into the tank was correlated to the flow of water through the tank to maintain a concentration of 10 pg Hg/liter admin— istered as methyl mercuric chloride. At the end of four weeks the fish were killed and assayed for hematocrit, 0 consumption of gill tissue, 2 protein content of gill sample, and plasma electrolyte concentration (Na+, K+, c1“, MgH, CaH). Procedure for Blood Samples After capture as much blood as possible (2.4-4.5 ml) was drawn from the hemal arch (see Experiment 1) of each unanesthetized fish 12 within a limited time span (2—4 min.). Each fish was then decapfitated and the head placed in 500 m1 of Mammalian Ringer solution (Appendix) containing sodium heparin. The incision for decapitation was made to insure the integrity of the heart; whose continued pumping in the heparinized Ringer solution facilitated the removal of any blood remain- ing in the branchial arches. A small sample of blood was removed from the syringe using a microhematocrit capillary tube (American Hospital Supply Co., Miami, Fla.) and centrifuged for three minutes on an IEC MiCrohematocrit Centrifuge (Model MB). Hematocrits were determined on an IEC Circular Microcapillary Tube Reader. ~Plasma'electrolyte concen— trations were determined as in Experiment 1. .92 Consumption of Gill Tissue The second and third branchial arches from the right side of the head were removed and put in separate 200 m1 flasks containing 100% phosphate buffered saline (Grand Island Biological Co., Grand Island, N.Y.) while the second and third arches from the left side were put in a flask with 10% phosphate buffered saline (PBS). The 100% PBS (compo— sition given in Appendix) had a pH of 7.4 and an osmotic concentration of 285-290 mOsm/Kg, whereas the 10% PBS had a pH of 7.38 and an osmotic concentration of 28-30 mOsm/Kg. A sample of filaments (60—80 mg) was then cut from the middle of each arch and suspended in 3‘m1 of 10% or 100% PBS and the oxygen consumption measured polarographically with a Y.S.I. Model 53 biological oxygen monitor (Yellow Springs Instrument Co., Yellow Springs, Ohio). The 02 consumption was recorded with a 13 Beckman 10 inch strip chart recorder. The temperature of the sample was maintained at 1710.500 with a constant temperature water bath (Haake 70, Polyscience Corp., Evanston, 111.). Calculation of oxygen consumption was based on the solubility of oxygen in 10% and 100% PBS at 17°C (Table l) and the percent of initial consumed during a 10 minute period. The oxygen consumption is expressed as 3 pl 02 consumed per hour per mg protein. TABLE l.--The solubility of oxygen in 10% and 100% phosphate buffered saline. Data given as ml 02 dissolved per ml of fluid (as determined using the Winkler procedure) when air is at 1 atmosphere pressure. 12°C 17°C 102 PBS 0.0421ip.002(15) 0.033319.001(12) 100% PBS 0.040919.003(15) 0.031419.001(12) iis . E. (N) Following 0 consumption measurements the 3 ml sample of PBS 2 containing the gill tissue was sonified for 2.5 minutes with a sonifier cell disruptor (Heat Systems Co., Melville, N.Y.) to remove as much tissue as possible from the cartilaginous skeleton. A modified Lowry method (Oyama and Eagle 1956) was then used to determine the amount of protein in each sample. Protein standards were prepared from an 800 pg/ml standard (Dade Reagents Inc., Miami, Fla., Lot No. PRS-406). 14 Experiment 3 Both control and experimental fish.were starved for the entire experiment. Experimental fish were exposed to 10 pg Hg/liter admin— istered as methyl mercuric chloride for 4, 8, and 12 weeks. Protocols for determining hematocrit, 0 consumption of gill tissue, protein 2 content of gill sample, and plasma electrolyte concentration (Na+, KI, 01-, Mg++, Ca++) were the same as in Experiment 2, with the exception that 02 consumptions of gill filaments from this experiment were done at 12°C. Values for dissolved oxygen in 10% and 100% PBS at 12°C are shown in Table l, on the preceding page. Statistics One way analyses of variance were used for all comparisons and Tukey's w-procedure (Sokal and Rohlf 1969) was used for multiple comparisons among means. All data are expressed as mean i_S.E. A P level < 0.05 was considered significant. RESULTS Experiment 1 jGraylingpflatchery Fishl The plasma electrolyte concentrations of blood samples taken from 18 rainbow trout (l.3—3.6 Kg) at the Grayling hatchery in February, 1973 are shown in Figure 2. These samples were obtained to establish some normal range of values for hatchery reared rainbow trout. However, these fish were considerably older and larger than fish used in subse- quent experiments which may be one reason for the significant difference in plasma electrolyte concentrations between Experiment 1 and those observed in Experiment 2 and Experiment 3. The difference in protocol between Experiment 2 and Experiment 3 is as follows. Fish in Experiment 2 were exposed to 10 ug Hg/liter administered as methyl mercuric chloride for a period of 4 weeks during February, 1973 and the 0 consumption of gill filaments was determined 2 at 17°C. Fish in Experiment 3 were exposed to 10 ug Hg/liter adminis- tered as methyl mercuric chloride for periods of 4, 8, and 12 weeks during June-August, 1973 and the 0 consumption of gill filaments was 2 determined at 12°C. Experiment 2 (4 Weeks Exposure to 10 pg Hg/liter Administered as ChaHgC1) Fifteen rainbow trout were exposed to water containing 10 pg Hg/ liter administered as methyl mercuric chloride for a period of 4 weeks. The oxygen consumption of gill filaments from experimental and control 15 l6 .m.m H coma mum wouuoam .mdoaumuuaooaoo +mz was IHo you mumfinwuo pawwm .mfloaumuuamosoo ++mo use 1.»: .+M How oumaflouo amen Samson—m: wsfifinmuu oau Scum AH usmfiuomnm .wM o.mlm..3 uaouu sonafimu ma How meowuwuucooaoo oumflouuomam mammamll.m 95on 17 (1/baw) 'SONOO vwsvw éééfigéfiéqg _' 4- p H 12- 4! H {To _ 14“... +9 1'23 1 + 12" E15: .2 a s: s:- s :3 <2- (‘I/baW) 'soNoo vwsvw FIGURE 2 18 fish was determined in 100% PBS and 10% PBS to ascertain if the salt concentration of the bathing media had any effect on oxygen consumption. Results indicate that there were no significant differences in 0 con- 2 sumption between samples from control and mercury exposed fish determined in 10% or 100% PBS, however, both groups show a higher rate of oxygen consumption in 100% than in 10% PBS (Figure 3). Similarly, no change in the concentration of plasma electrolytes was seen in trout exposed to methyl mercury for 4 weeks (Figure 4). Hematocrits for mercury treated fish were significantly higher (P<<0.05) than values for control fish (mercury treated 15.211.6(15); control 13.Qil.l(11). Experiment 3 (4, 8piand 12 Weeks Exposure to 10 pg galliter Administered as CHaHgClz To determine if a longer period of exposure to methyl mercury would affect gill metabolism or plasma electrolyte levels, rainbow trout were exposed to 10 ug Hg/liter administered as methyl mercuric chloride and data were obtained for fish after 4, 8, and 12 weeks exposure. Values for the oxygen consumption of gill filaments from this experiment were similar to those obtained in Experiment 2. Gill filaments run in 100% PBS had significantly higher rates of oxygen consumption than those run in 10% PBS. This effect was observed in all control and experi- mental fish assayed after 4, 8, and 12 weeks exposure to methyl mercury (Figure 5A and SB). As in Experiment 2, paired comparisons indicate that there were no significant differences in the oxygen consumption of gill filaments from control and experimental fish throughout the entire 12 weeks of the experiment. Gill oxygen consumption of mercury treated 19 FIGURE 3.--The effect of 100% and 10% phosphate buffered saline on the oxygen consumption of gill filaments (Experiment 2, 17 C) from control and experimental rainbow trout after 4 weeks exposure to 10 pg Hg/liter administered as CH3HgC1. Plotted are N value and mean : S.E. Means not underlined by the same line are significantly different (P< 0.05): 10% PBS 10% PBS 100% PBS 100%PBS control mercury mercury control 18.684 19.649 22.255 22.996 02 CONSUMPTION (pl/hr/mg PROTEIN) 3o 20 [:l I5 ll: L IOO% PBS FIGURE 3 CONTROL MERCURY I L IO% PBS 21 .Hm>oH mo.ouvm on» um moamofimaemfiw 0: 303m ouxaouuomam some now msomfiumeaoo wouwmm .msowumuuemosoo +mzmxswnao wow mumaflvuo uzmfiu manoeumuusmo Iaoo ++mo new ++wz .+M you mumawuuo amen .m.m Hugues use enam> 2 one wouuoam .Hommmmu mm woumumwafiavm umufia\wm m: OH on mummomxw wxmm3 e Houmm AN unmawuwaxmv usouu Bonafimu Housmsaumewo mam Houuaoo mo aoHumuuaooaoo oumaouuomam «Human m£HII.¢ mmaon 22 (‘l/balN) 'ONOO VWSV‘Id 0 £9 '0' O z '— 0 We '0 0% .J>- -95: on: m: _ l-0 : 20: a» on: 2 02 d ..... * x '1 o no 0' O O- 0- (‘I/baW) 'DNOC) vwsvfld 23 FIGURE 5A.--The effect of 100% and 10% phosphate buffered saline on the 02 consumption of gill filaments from rainbow trout exposed to 10 ug Hg/liter administered as CH3HgC1 for 4, 8, and 12 weeks (Experiment 3, 12°C). Plotted are N value and mean :_S.E. Significance for paired compari- sons P< 0.001 = ***. FIGURE SB.--The effect of 100% and 10% phosphate buffered saline on the 02 consumption of gill filaments (4, 8, and 12 weeks) from control rainbow trout (Experiment 3, 12°C). Plotted are N value and mean :_S.E. Significance for paired comparisons P<10.001 = ***. O2 CONSUMPTION O2 CONSUMPTION 24 3O El IOO% PBS 2 IO% PBS LIJ mt an m +- 20- _ a {- O. 0‘ 5 IO- 35 2 .-.;.;.;.;.;.;.;. IIIIIIIIII 3 l2 21 |2§§|2 8 s l J L J l J 4 8 12 TIME (WEEKS) A 30* III 100% P88 LIJ S 201 D: O. O’ E IO« 1:- \ 3 TIME (WEEKS) FIGURE 5B 25 fish in Experiment 2 (Figure 3), is significantly higher than values for experimental fish exposed to the same methyl mercury concentration for the same length of time (4 weeks) in Experiment 3 (Figure 5A and SB). Gill metabolism of both experimental and control groups measured in 10% PBS showed a significant decrease between the fourth and eighth week of exposure (Figure 6A), however, no significant change was noted throughout the entire 12 weeks for gill filaments determined in 100% PBS (Figure 6B). All oxygen consumption measurements in Experiment 3 were determined at 12°C while those in Experiment 2 were measured at 17°C. This temperature differential is probably responsible for the increased rate of oxygen consumption observed in Experiment 2 tissues. Gill metabolism of bofih control and experimental fish was affected in a similar manner by temperature. The differences in oxygen consumption of gill filaments between Experiments 2 and 3 are shown in Figure 7A (for those tissues measured in 100% PBS) and Figure 7B (for those measured in 10% PBS). Plasma Electrolytes Plasma sodium in control fish remained relatively constant at 140 mEq/l throughout Experiment 3 but sodium levels for experimental fish were variable. Plasma sodium in fish exposed to methyl mercury for 8 weeks was higher and those exposed 12 weeks were lower than values for fish exposed only 4 weeks (Figure 8). Plasma potassium levels in both control and mercury treated fish decreased significantly between the fourth and eighth week of exposure to a level that remained rather stable for the remainder of the experiment (Figure 9). Figure 6A.--0 26 consumption of gill filaments (Experiment 3, 12°C) from control and experimental rainbow trout in 10% phosphate buffered saline after 4, 8, and 12 weeks exposure to 10 pg Hg/liter administered as CH3HgC1. Plotted are N value and mean i S.E. Means not under- lined by the same line are significantly different (P < 0.05): control control control 8 weeks 12 weeks 4 weeks 8.744 9.780 14.132 mercury mercury mercury 8 weeks 12 weeks 4 weeks 8.648 9.575 13.130 Figure 6B.--O consumption of gill filaments (Experiment 3, 12°C) from control and experimental rainbow trout in 100% phosphate buffered saline after 4, 8, and 12 weeks exposure to 10 pg Hg/liter administered as CH HgCl. Plotted are N value and mean :_S.E. Means no under— lined by the same line are significantly different (P< 0.05): control control control 8 weeks 12 weeks 4 weeks 15.568 16.470 17.353 mercury mercury mercury 8 weeks 12 weeks 4 weeks 14.481 17.272 18.535 02 CONSUMPTION 27 .1 so E] CONTROL 2:: gfifl¢ g E MERCURY g: a 2 m . 3 °' 2 § 8 § 3 8 ON L l l—J L——-‘ 4 8 :2 TIME (WEEKS) FIGURE 6A 2 30 CI CONTROL 5 MERCURY *5 20 a: 0. O 5 IO E 3. I2 I2‘ 3 Li. ‘1 l L__———-—-' L""--J 8 TIME (WEEKS) FIGURE 6B 28 FIGURE 7A.-~The effect of temperature on the 02 consumption of gill filaments from control and mercury treated fish in 100% phosphate buffered saline after 4 weeks exposure to 10 pg Hg/liter administered as CH3HgCl. Filaments from Experiment 2 were measured in 100% phosphate buffered saline at 17°C and filaments from Experiment 3 were measured in 100% phosphate buffered saline at 12°C. Plotted are N value and mean i S.E. Means not under- lined by the same line are significantly different (P< 0.05): Expt. 3 Expt. 3 Expt. 2 Expt. 2 control mercury mercury control 17.352 18.535 22.255 22.996 FIGURE 7B.--The effect of temperature on the 02 consumption of gill filaments from control and mercury treated fish in 10% phosphate buffered saline after 4 weeks exposure to 10 pg Hg/liter administered as CH3HgCl. Filaments from Experiment 2 were measured in 10% phosphate buffered saline at 17°C and filaments from Experiment 3 were measured in 107. phosphate buffered saline at 12°C. Plotted are N value and mean : S.E. Means not under- lined by the same line are significantly different (P < 0.05): Expt. 3 Expt. 3 Expt. 2 Expt. 2 mercury control control mercury 13.130 14.132 18.381 19.649 CONSUMPTION (pl/hr/mg PROTEIN) 02 O2 CONSUMPTION (pl/hr/mg PROTEIN) 301 _, N C? IO‘ 29 El CONTROL MERCURY OI 9 N 9 9 T7°C 2 one pmuuoam .uemsfipoexo some now esonm Oman mum swam Houueoo pom mosam> .Huwmmmo mm pmumumaflafipm HmuHH\mm m: CH ou musmomxm execs q poems Am unwafipmexmv mnmfl .umsw:¢rmssh a“ was AN usmsfipwmxmv mmmH .kumspnmm ea woaefimm usouu Bonafimp ammsumn A++wo .IHU .+mzv mdoaumuunmosoo mumaopuomam mammam mo somuueesoo