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UBC Theses and Dissertations

The biological half-life of inorganic mercury in the Dungeness crab Cancer magister Dana Sloan, John Peter 1974

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T H E B I O L O G I C A L H A L F - L I F E -OF I N O R G A N I C M E R C U R Y IN T H E D U N G E N E S S C R A B , Cancer magister D A N A by John Peter Sloan B . S c , Universi ty of B r i t i s h Columbia, 1971 A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in the Department of Zoology We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F BRITISH C O L U M B I A A p r i l 1974 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g ree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada A B S T R A C T The biological half - l i fe of inorganic mercury in the Dungeness crab, Cancer magister Dana, was determined experimentally to be about 25 days. Crabs were exposed to mercury solutions, sacr if iced after varying periods of time, and mercury determinations of whole body homogenates made with an atomic absorption spectrophotometer. The simple and widely used negative exponential equation for calculating biological half - l i fe was not entirely adequate for describing the mercury elimination process . A better description was obtained using a non-linear least-squares fit of an equation describing elimination at different speeds f r o m two types of t issues. A further model allowed for recycling of mercury that was eliminated, and gave marginally better descriptions in some cases. i i i T A B L E O F C O N T E N T S Page A B S T R A C T i i T A B L E O F C O N T E N T S i i i L I S T O F F I G U R E S iv L I S T O F T A B L E S v A C K N O W L E D G M E N T S v i i I N T R O D U C T I O N 1 M E T H O D S 3 A . General and Pilot Tests 3 B. Holding and Dosing 8 C . M e r c u r y Removal by Precipitation 11 D . Dissect ion and Weighing 11 E . Digestion 12 F . Determination 14 R E S U L T S 17 Biological H a l f - T i m e . 17 The Adequacy of Simple Negative Exponential Clear ing Rates 23 DISCUSSION 28 L I T E R A T U R E C I T E D 31 A P P E N D I X 33 iv LIST OF FIGURES Figure Page 1 Relation between log body content of Hg (ng/g) and time in crabs exposed to Hg solutions. A to E, experiments 1 to 5 respectively 20 2 Relation between log body content of Hg (ng/g) and log time in crabs exposed to Hg solutions. A to E, experi-ments 1 to 5 respectively 21 V LIST OF TABLES Table Page IA Pilot test. Mercury content (|ig/g) of Hg in wet weight of crab tissue. Dose 480 Lig/g for two hours, freeze dry and wet weight procedures, gill and muscle analyzed separately. A and B are replicate tissue samples 4 IB Pilot test. Mercury content (Lig/g) of Hg in wet weight of crab tissue. Dose 580 |ig/g for two hours first animals, three hours for last two animals. A and B are replicate tissue samples, repeated observations are replicate spectrophotometer determinations 5 IC Pilot test. Mercury content (Lig/g) of Hg in wet weight of crab tissue. Dose 300 Lig/g for two hours. A and B, replicate tissues, repeated observations are replicate determinations 6 ID Pilot test. Mercury content (Lig/g) of Hg in wet weight of crab tissue. Dose 68 Lig/g for two hours. A and B, replicate tissues; repeated observations are replicate determinations 7 II Results of standardization tests on mercury content of crab tissues 16 III Dosing procedures in experiments on retention of Hg in crabs 18 IV Body content Hg (|Jg/g) in crab tissue homogenates at time (hours) after dose 19 V Experimentally determined biological half-life of - -inorganic mercury in Cancer magister 22 vi Table Page VI Probability that there is no significant departure from linearity in regression 24 VII Estimated parameters for concurrent processes model (Equation 2) 26 VIII Estimated parameters for concurrent processes model with recycling (Equation 3) 27 A C K N O W L E D G M E N T S The research reported here was done at the F e d e r a l Department of the Environment Pacif ic Environment Institute to partially fulfil the requirements for a Master of Science degree f r o m the Department of Zoology, Universi ty of B r i t i s h Columbia. The staff at P . E . I . , especially D r . John Davis and M e s s r s , J . With and R. Harbo, gave indispensably pract ical assistance and encouragement. D r . J . A . Thompson, my supervisor at P . E . I . , is responsible for the topic of my project, it direct supervision, the maintenance and direction of the laboratory in which I worked, and for underwriting the entire project financially f r o m his r e s e a r c h grant. He bore the extra burden of a novice graduate student f r o m another f ield with, at times, incredible patience and understanding. D r . P . A . L a r k i n , as my M a s t e r ' s program supervisor, looked after my personal financial support and administrative arrangements. He arranged for the teaching assistantship at U . B . C . that paid my rent. His unfailingly positive point of view always seems to alleviate the most awkward situations I was able to manufacture. Both he and D r . Thompson generally provided me with everything I needed to spend time learning, including advice, and it is understood that their experience and teaching ability are expressed in any parts of this report that are worthwhile. Its shortcomings are a l l my own. I N T R O D U C T I O N 1 Man's awareness of the effects of industrial wastes on ecosystems has recently resulted in surveys of freshwater and marine fishes for mercury and other toxic pollutants. In particular, it was discovered in Canada that f i sh caught downstream f r o m c h l o r - a l k a l i plants which utilize mobile cathods contained elevated concentrations of mercury (Wobeser, et al . , 1970; F imrei te , 1970; F i m r e i t e , et al . , 1971). A c h l o r - a l k a l i plant located on the Canadian west coast at the head of Howe Sound, B . C . , uses about 275,000 pounds of mercury annually for electrolytic decomposition of brine . The chlorine and sodium hydroxide produced are then supplied to nearby pulp m i l l s . In early 1970, specimens of various marine fishes and invertebrates were obtained f r o m Howe Sound near the plant site, and Dungeness crab, Cancer magister, were found to contain up to 13ppm mercury (Bligh, 1971). Flounder (Citharichthys spp.) taken f r o m the same area contained over one ppm of the metal . A s a result of these findings, fishing in this area was banned. In October, 1972, levels of up to 3.3ppm in C. magister were sti l l present. Because crabs are fa i r ly omnivorous bottom-feeders, and because they also support a valuable West Coast f ishery (1971 wholesale value in B . C . , $946,000), they have been included as one of several species used at the Pacif ic Environment Institute (Federal Department of the Environment), West Vancouver, B . C . , to study mercury bio-accumulation. Par t of this p r o g r a m is directed toward determining mercury levels in water, bottom sediments, and organisms in Howe Sound; levels which must be presumed to be the result (wholly or in large part) of waste output f r o m the c h l o r -alkali plant. In the past five years, a number of studies of bio-accumulation of mercury by freshwater and marine organisms have been reported, with the bulk of the literature dealing with freshwater organisms (Hannerz, 1968; Rucker and Amend, 1969; Uthe, 1972). Of particular interest to researchers 2 is determination of rates of accumulation and biological half - l ives of different chemical forms of m e r c u r y . It is now well known that the p r e -dominant m e r c u r i a l found in freshwater f ish is methyl mercury; its existence in lake water has been demonstrated by Chau and Saitoh (1973) and in estuariune sediments by Andren and H a r r i s s (1973). Thus, it is assumed that methylmercury, which possesses an affinity for proteins, is obtained via the food chain. Bacter ial conversion has been shown to be the pathway of origin of methylmercury (Jensen and Jernelov, 1969; Wood et a l . , 1968), though this does not obviate the need to look into other, possibly non-biological , mechanisms. In saline waters Anfalt, et al . , (1968) have shown that the prevailing m e r c u r i a l species is the complex anion ( H g C l ^ - , and since the existence of the methylmercurie cation or dimethyl mercury in seawater has yet to be conclusively demonstrated, it must be assumed that the important f o r m of mercury , exclusive of feeding, to which organisms are exposed, is inorganic. The exploration of this aqueous solution avenue of contact with the metal is the purpose of this study of the biological half - l i fe of inorganic mercury . Experiments were designed to explore three problems: (1) the clearing rate (biological half-time) of inorganic mercury f r o m C . magister following a brief exposure to relatively high concentrations of the ion; (2) the relation of clearing rate to concentration and duration of dose; and (3) evaluation of other possible circumstances that might lead to further understanding of the physiology of mercury clearing in these organisms. A pilot experiment on.LD^Q estimates for m e r c u r y toxicity is reported in Appendix 1. 3 M E T H O D S A . General and Pi lo t Tests Crabs were bought c o m m e r c i a l l y at Crescent Beach, B . C . , where the natural ly-occurr ing level of m e r c u r y is low (<200ppb). Injection of mercury via the soft joints of crabs was rejected mainly because it was an unnatural way of introducing the mercury, and exposure to mercury dissolved in seawater was chosen as a direct, simple method, s imilar to the situation in a polluted natural environment. Measurement of mercury was done using an atomic absorption spectrophotometer, though it gives only total mercury, and necessitated kil l ing animals . Testing blood samples was not possible because of instrument limitations. It was not known how much mercury crabs would assimilate, or what effects the metal would have on them. Accordingly , pilot tests were run on a few specimens using smal l indoor aquaria. Various amounts of mercury were tried, always for a two-hour period. During the tests several other possibilit ies were considered, including separate analysis of various body parts, wet weight analysis versus dry weight analysis, and evaluation of the variabili ty among crabs as a guide to suitable sample size for the main experiments. It seemed advisable to take more frequent readings immediately after dosing, since this was when fastest loss was expected (two readings in the f i r s t day, another the second day, a fourth the fourth day, and so on). The pilot tests (Table I) suggested a range of dosing of f r o m 10-500ppm digrams per g r a m or parts per mil l ion mercury) . This amount seemed to give readable levels of mercury in the animals, but not enough to cause any apparent i l l effects. TABLE IA. Pilot test. Mercury content (\lg/g) of Hg in wet weight of crab tissue. Dose 480 (ig/g for two hours, freeze dry and wet weight procedures, gill and muscle analyzed separately. A and B are replicate tissue samples. Wet Freeze Dry Animal Muscle Gill Muscle Gill 1. Control 98 43 353 658 2. Immediately after , 0 _c_ ' _. . 3 272 1, 879 2, 052 21,014 dosing 3. 12 hours (A) 1,198 17, 475 after dosing (B) 1, 86l 15,233 4. 24 hours after (A) 2, 140 819 - -dosing (B) 721 1Z, 245 5. 12 days after (A) 679 15,466 dosing (B) 601 12, 878 TABLE IB. Pi lot test. M e r c u r y content (|Jg/g) of Hg in wet weight of crab tissues. Dose 580 Lig/g for two hours f i rs t animals, three hours for last two animals . A and B are replicate tissue samples, repeated observations are replicate spectrophotometer determinations. Homogenate of Remainder A n i m a l Muscle G i l l of Soft Tissues 1. Control A 250 400 B 784 735 C 1, 650 - -1,500 116 hours A 626 30,200 2, 745 after dosing 1, 011 31, 200 2, 725 B 1, 024 33, 631 1, 937 1, 170 - 2, 015 C - 40, 444 -. - 43, 333 -12 days A 556 38, 945 2, 516 after dosing - 38, 721 3, 104 B 115 49, 873 5, 322 461 46, 202 4, 298 C 2, 560 37, 248 3, 063 2, 006 33, 075 2, 698 6 T A B L E I C . P i l o t t e s t . M e r c u r y c o n t e n t ftig/g) of H g i n w e t w e i g h t of c r a b t i s s u e s . D o s e 300 Ug / g f o r two h o u r s . A a n d B , r e p l i c a t e t i s s u e s , r e p e a t e d o b s e r v a t i o n s a r e r e p l i c a t e d e t e r m i n a t i o n s . H o m o g e n a t e of R e m a i n d e r W h o l e B o d y A n i m a l ; M u s c l e G i l l of S o f t T i s s u e s H o m o g e n a t e 1. C o n t r o l A - - - 11 22 B - - - 19 28 C - - 26 - - 26 I m m e d i a t e l y A 13 11. 142 18 a f t e r d o s i n g 13 11, 781 22 B 34 12, 621 20 42 12,439 16 6 h o u r s A 2 1, 736 17 a f t e r d o s i n g 4 - 19 B 12 1, 830 20 8 1, 786 23 18 h o u r s A 159 3, 869 1, 266 a f t e r d o s i n g . 130 4, 020 1, 396 B - 3. 985 1, 273 - 3, 985 1. 599 5. 48 h o u r s A - 679 a f t e r d o s i n g - - - 673 B - - - 568 529 6. 120 h o u r s A - ' - - 278 a f t e r d o s i n g - - - 256 B - - 228 - - - 209 C - - - 222 • - - 247 7 T A B L E I D . P i l o t t e s t . M e r c u r y c o n t e n t (|Jg/g) of H g i n w e t w e i g h t of c r a b t i s s u e s . D o s e 68 ( i g / g f o r two h o u r s . A a n d B , r e p l i c a t e t i s s u e s ; r e p e a t e d o b s e r v a t i o n s a r e r e p l i c a t e d e t e r m i n a t i o n s . H o m o g e n a t e of R e m a i n d e r W h o l e B o d y A n i m a l M u s c l e G i l l ° f Sott. T i s s u e H o m o g e n a t e 1. C o n t r o l A - 100 100 B - - - 39 39 C - - - 26 40 D • - - - 22 22 2. I m m e d i a t e l y A a f t e r d o s i n g 75 B 22 14 3. 6 h o u r s A 13 a f t e r d o s i n g 11 B 17 4. 18 h o u r s A 796 a f t e r d o s i n g 8 56 B 4 7 9 450 5. 48 h o u r s A a f t e r d o s i n g B C D 6. 120 h o u r s A a f t e r d o s i n g B C 4, 575 5 4, 907 3 4, 270 4, 479 3,120 28 3, 262 26 2, 596 37 2,912 26 5,427 75 5,888 76 7,143 3 7, 143 37 670 693 628 628 592 566 555 555 397 339 355 368 304 294 8 Using homogenized wet crab tissue, it was difficult to control the exact amount of seawater in the blender. Crab tissue is extremely soft, and when the organs are dissected out of the shell, a certain amount of the material forms a soup with water contained in the shell . The analysis of separate tissues f r o m every animal was given up after two pilot tests when it became obvious that it would be too time consuming. One sample analysis usually took 8-12 hours, and only about half of that was free time while the samples were sitting undergoing reactions. Although some steps were as easily done on forty samples as on twenty, there were several " r a t e - l i m i t i n g " steps where each sample had to be handled separately, e.g., weighing tissue samples and running the spectrophotometer. F r e e z e - d r y i n g seems to concentrate mercury by about a factor of ten, indicating the high water content in crab tissue. In one instance in which separate tissues were analyzed (test 1), there was an inconclusive difference between g i l l and muscle concentrations. G i l l concentration in f reshly-dosed animals, on the other hand, was relatively large (a factor of f r o m ten to a hundred over muscles, and slightly less, on the average, over remaining tissue). Comparing average mercury contents for tests 3 and 4, it might be suspected that a large dose collects heavily in gi l l tissue, but does not accelerate m e r c u r y accumulation in muscle over a small dose. B . Holding and Dosing F i v e clearing rate experiments were performed during the winter and spring months of 1972. Mature specimens of C . magister were obtained c o m m e r c i a l l y f r o m Boundary Bay in the extreme south-west corner of B r i t i s h Columbia. F o r each experiment, sixty crabs were maintained in captivity, exposed simultaneously in a tank to inorganic 9 m e r c u r y dissolved in aquarium water, returned to unpolluted seawater in the original aquarium, and then analyzed for total mercury content in groups of five (one as a control) at various time intervals after exposure. Several large (1.8m dia. , 1.2m depth) cy l indr ica l f ibreglass aquaria were used to accomplish this. E a c h aquarium used for holding animals before and after dosing was fitted with a central double concentric standpipe with outlets in the outer pipe at the bottom (for m a x i m u m circulation), and length of the inner pipe (approximately 0.5m) set to maintain constant depth. Water taken f r o m B u r r a r d Inlet entered f r o m a pipe trained obliquely to the aquarium at a rate thatrprovided a complete water change every 10 to 15 minutes. Water flow also caused a slow, clockwise circulation in each aquarium; aeration was supplemented by an airstone. During the experi-ments, salinity and temperature showed only smal l seasonal variations. When dosing was done, a closed system was maintained in which the outlet at the centre of a separate aquarium and another near its p e r i -meter were each fitted with a rubber hose through which dosing water was drained, and recycled over the b r i m of the aquarium by two electric pumps. A l l crabs were left undisturbed throughout the program and were fed to satiation with h e r r i n g . A standard procedure was followed throughout the p r o g r a m . The basic routine was simple: (1) maintain about sixty crabs in captivity for a week, which was presumably sufficient to acclimatize them to the aquarium and to any conditions of water that may have differed f r o m Boundary Bay; (2) mark ten of these as controls, not to be exposed to mercury ; (3) place the remaining fifty for two hours in a separate aquarium containing a measured amount of dissolved mercury; (4) remove treated crabs and return them to the holding aquarium; (5) capture, at specified time intervals following exposure, four dosed and one control animal; (6) k i l l , and dissect a l l tissue f r o m these five; (7) digest tissue samples for 10 a n a l / s i s ; (8) measure total mercury concentration on an atomic absorp-tion spectrophotometer. Dosing was always done early on a Saturday morning, and the weekend used to do the f irst two or three sets of mercury determinations. E a c h dosing aquarium had two outlets at the bottom, hooked up by a rubber hose to two electric water pumps. The output lines f r o m the pumps fed over the r i m of the aquarium so that water would circulate around the tank. When two aquaria were used, the hoses crossed, functioning via their four pumps as a single system. Clamps on one of the hoses regulated any flow discrepancy. The dosing tank contained 1930 l i t res of seawater. A calculated amount of mercury salt (HgNO-j) was dissolved in a few l i t res (usually three) of warmed seawater, and then added to the water circulating in the aquarium, letting it mix for about one-half hour. Just p r i o r to dosing time 50 experimental crabs were carefully piled in a large plastic tub beside the dosing aquarium. At the start of dosing time, crabs were put quickly into the dosing tank, carefully enough to avoid disturbance and fighting, and this took about three minutes. Next, a smal l sample of aquarium water was taken, to which 6N, H_.SC) was added. (The acid stops loss of mercury to the air and sides 2 4 of the plastic container.) Additional samples were taken every half-hour to monitor the dose. Toward the end of dosing (within three minutes), the experimental crabs were caught and returned to the circulating holding aquarium, which took about six to eight minutes. Next, four 0-time experimental crabs and one control were selected and caught, and a f inal water sample was taken; then the pumps were shut off and a mercury-precipi tat ing mixture was added to the aquarium. 11 . C . M e r c u r y removal by precipitation About 30g each, of FefNO-^-j and Na2S were used to co-precipitate m e r c u r i c ion in the exposure water before it was discharged to B u r r a r d Inlet. After two days settling, it was possible to siphon off the 2000 Z of clean seawater, and then wash the precipitate into buckets through the plug hole. These buckets could then be left to settle some more and the precipitate could finally be fi l tered and dried. Siphoned seawater was tested on the spectrophotometer, which indicated total mercury below 0.5|ig/g. Dosing water always showed mercury loss . F r o m the level calcu-lated f r o m the weighed amount added, the start of each dosing period, total m e r c u r y dropped f a i r l y steadily. In establishing a dose for each experiment the mean of five readings was taken. Ranges of readings were: Experiment 1: 10-15|ig/g; Experiment 2: 443-557|jg/g; Experiment 3: 410-460Llg/g; Experiment 4: 482-541|ig/g; Experiment 5: 7-12|ig/g. D . Dissect ion and Weighing Crabs were ki l led by hitting them with a hammer handle at the apex of the triangular ventral panel. After measuring the length of each crab and counting its l imbs, soft tissue was removed -(except the muscle f r o m the outer two leg segments) and placed in a Waring blender. A special 100ml plastic bottle attachment was used for this since it was an ideal size (later, a Sorval l stainless steel blender was used that worked well). Between each use, the bottle was r insed with HNO3 and dist i l led water. The homogenate was poured into weighing boats and covered and marked. Weighing developed to a routine using plastic disposable straw-type syringes . About a g r a m of homogenate was added to a weighed test tube, and weighed by difference. 12 E . Digestion Digestion of tissue for total mercury determination was done following the method of A r m s t r o n g and Uthe (1971). The development of the method reported here to the point where it was giving satisfactory results , was perhaps the most formidable problem encountered in the project . Basically , the digestion involves two steps, one using sulfuric and nitric acid, and a second oxidation step using permanganate or peroxide. F o r tissue-bound mercury , certain manipulations are needed to get the mercury into its free atomic state, H g ° . M e r c u r y in tissue is usually +2 found bound to proteins. In this f o r m it is a divalent cation, Hg , and is usually attached by a covalent bond to protein sulfur, or to one or two methyl groups as methyl or dimethyl m e r c u r y . A strong oxidizing agent (8ml of concentrated H2SO4 and 2ml of concentrated HN.O3 per sample) was used as a f i r s t step in breaking these bonds. Concentrated acid, over time, with heat, yields free divalent mercury , some methyl and proteinated mercury, and a solution of proteoses and amino acids. After thorough acid digestion (one hour) this tissue had become a clear solution. The proteinated and methyl m e r c u r y can be broken down further by using a second oxidizing agent (6 per cent M n O ^ , and later 50 per cent H2O2) which, with incubation for several hours, wil l break up nearly all + 2 of these compounds so that nearly a l l the mercury exists as free Hg During permanganate (MnC>4 ) oxidation, manganese undergoes reduction to M n G ^ (MnlV) which appears as a brown precipitate. Before reduction of the mercury , it is f i rs t necessary to remove the precipitate by further reduction to soluble Mn(II). Reduction is best obtained by addition of a solution of an hydroxylamine salt or hydrogen peroxide. 13 Once this step is finished, as shown by the last bit of sludge being cleared, adding a couple of drops of the original Mn04 w i l l return the +2 solution to being safely "oxidizing" , and preserve the mercury as Hg , but none of the sludge of the intermediate M n O ^ is produced. When H ^ O ^ is used, no sludge is produced. F o r measuring mercury quantity in this solution, either a flame is +2 used to atomize the Hg , or, in the f lameless technique a strong reductant is added. To accomplish this, an addition funnel full of reductant is fitted to the vessel containing the sample solution, and closed underneath with a stopcock. In this way, an airtight system can be set up so that air or nitrogen can be introduced. A reaction vessel containing the solution with the reductant funnel above it, s t i l l closed, is included in the airtight system so that the air or nitrogen wil l bubble into the solution. A magnetic s t i r rer is placed under the reaction vessel , which opens at the top both to the closed reductant funnel and to a second a i r l ine . This second airl ine leads to a glass c e l l which has been lined up in the spectrophotometer beam, and which is open via a mercury trap containing nitr ic acid, to the atmosphere. To run a determination, some reductant is f i r s t released into the reaction vessel , and the s t i r rer is started. At this point, the reduction to H g ° takes place, but the system is airtight and static, so that no volatile H g ° escapes. After the reaction is complete, air or nitrogen is bubbled through the sample vessel at a constant rate, and a fraction of the total H g ° is swept out of solution and up into the cel l where it is exposed to the spectrophotometer beam. The gas flow drives the H g ° on into the trap, and finally clears the system after the H g ° has been released and recorded. 14 F . Determination Total mercury was determined by flameless atomic absorption spectrophotometry, using a variation of a method described by Hatch and Ott (1968). The glass c e l l (quartz windows) used was a cylinder, 15cm long and 22mm in diameter. Standards were made up daily f r o m a 100ppm solution of mercuric chloride in 6N, H C l . Some problems were encountered in achieving satisfactory recovery of mercury, but the results reported here were taken using a method that gave 70-90 per cent recovery . The weighed amount of tissue, in solution, was made up to a known volume, so that the concentration of mercury read by the spectrophotometer (against standards) would be meaningful. The spectrophotometer delivery apparatus was fitted with special 50ml pear-shaped sample vessels , and in the early tests the sample was made up to 100ml in volumetric flasks and then 50ml was pipetted into sample vessels . Later , 50ml digestion tubes were used to make up to volume, and poured directly into the sample vessels . Although the quantity in the sample vessel was a c r i t i c a l issue in the same way as the volume of the air tubes, it did not seem to affect results when the vessels got 49+ ml , poured out of the tubes, instead of 50+_.02 ml f r o m the pipettes. The concentration was accurate in either case. Reduction of mercury in the reaction vessel was done with 2ml of S n C l ^ i with the s t i r rer run for sixty second. The c a r r i e r gas was research grade nitrogen, delivered using a needle valve at 300ml/min. A plastic drying tube was fitted onto the gas line just before the glass cel l ; this tube contained magnesium perchlorate and was changed once in 20 to 30 determinations. Standardization of the spectrophotometer was done f r o m a stock of lOOOppm HgQ.2 in 6N, H C l . Once a week a substock of lOOppm was made f r o m the lOOppm solution, in 0.2 N H C l . It has to be assumed, and is 15 almost certain, that these solutions, when fresh, were good to _+ the tolerance marked on the volumetric glassware. The results reported here are all quantities measured against external standards. This means that they must al l be low, compared to the r e a l mercury contents of the crabs, since some mercury wi l l have been lost in preparation of the samples. The information here reported was used for measuring biological half- t ime, and the f o r m of the line relationship between time and body content. Neither of these is affected by inaccurate results, as long as the results are consistently inaccurate, by the same proportion in samples of different content. F r o m running internal standards and per cent recoveries , late in the project, and also having samples analyzed at another laboratory, the results probably represented 40 to 60 per cent of the rea l mercury contents. Table II shows per cent recovery results, and also a comparison of some values using external standards, with some internal standards on the same samples. F r o m these numbers it is theoretically possible to rework all the previous results so that they would be expressed in terms (1) of internal standards, and (2) corrected for per cent recovery . The mean per cent recovery of about 77 per cent, shown in part B of Table II, is meaningful only in terms of percentage recoveries reported in the majority of analytical chemistry literature that deals with tissue analysis . Percentage recovery is there (e.g., A r m s t r o n g and Uthe, 1970) treated as that per cent of a spiked blank which is shown in a spiked  tissue sample. In other words, a standard solution is added to a sample of tissue, as in the third procedure described previously, and the recovery compared to an internal standard, or reagent blank with standard solution. T A B L E II. Results of standardization tests on mercury content of crab tissues. A. Comparison of internal and external standards (from test 5). Experiment/ Internal Standard Animal External Standard 5/1 .335 5/2 .710 5/3 .640 5/4 .532 5/5 .680 Mean .579 Standard Deviation .152 B. Percent recovery, relative to external standards and internal standards. Percent Percent Experiment/ Recovery Recovery Animal (External) (Internal) 2/7 34.0 58.7 3/1 45.0 77.7 3/7 56.0 96.7 3/8 48.4 83.5 3/9 31.3 54.0 3/9 57.4 99.1 4/2 33.9 58.5 4/3 30.7 53.0 4/6 58.3 100.0 4/7 50.2 86.7 Mean 76.7 Standard Deviation 19.3 Comparison of external standards to internal standard, and to spiked tissue. » Internal Standard Spiked Tissue (as fraction of (as fraction of Animal External Standard) External Standard) 1 .910 .522 2 .868 .785 3 .923 .614 4 .843 .810 5 .843 .586 6 .873 .560 7 .991 .881 8 .872 .564 Mean .881 Standard Deviation . 564 17 There could be other factors than poor technique involved in low percentage recover ies . Perhaps the matrix effect of tissue on release of m e r c u r y causes tissue sample standards always to be low relative to inorganic blank standards. R E S U L T S F i v e experiments were conducted, the circumstances being described in Table III. The data in Table IV show measured body content of inorganic mercury at various times following dosing. When graphed, these data array themselves as scattered points which indicate a drop in body content with time (Figures 1 and 2). A n a l y s i s of the data addressed itself to two questions: (1) what, roughly, is the time required for clearing of half of the mercury taken up at dosing (biological half- t ime) ; and (2) is the assumption of a simple negative exponential clearing rate a valid one and, if not, what other mathematical model would more adequately describe the way in which inorganic mercury is cleared f r o m C . magister? A third question of whether clearing rate varies with concentration or duration of dose may be answered, somewhat inconclusively, by refer r ing to Table III. Biological H a l f - T i m e The data indicate that the f i rs t question may be answered as shown in Table V . Half - t imes were calculated using the formula where T ^ is biological half - t ime; 2 18 T A B L E III. Dosing procedures in experiments on retention of Hg in c r ab s. Experiments Dose Hg (lig/g) , Remarks 1 12 Hg ( N 0 3 ) 2 Cages used 478 Hg ( N O s ) 2 Cages used 433 Hg O (in acid solution) No cages 500 Hg ( N O s ) 2 No cages 5 7-2 Hg ( N 0 3 ) 2 No cages T A B L E I V . B o d y c o n t e n t H g (ug/g) i n c r a b t i s s u e h o m o g e n a t e s at t i m e ( h o u r s ) a f t e r d o s e . E x p e r i m e n t 1 E x p e r i m e n t 2 E x p e r i m e n t 3 E x p e r i m e n t 4 E x p e r i m e n t 5 T i m e B o d y C o n t e n t ( h o u r s ) (4 a n i m a l s ; Ug/g) 1 512, 479, 445, 12 27 5, 362, 226. 36 275, 225, 268, 144 195, 239, 171. 816 U 5 . 229. 162, m e a n c o n t r o l : 64(+ 22) 1 532, 718. 724. 12 788, 426, 548, 24 547, 420. 364, 48 303, 249, 249, 552 147, 121. 169, 912 120, 180, 263, 1536 186. 127. 200, m e a n c o n t r o l : 78 ( + 13) 1 665. 1871. 655, 12 1020, 724. 521, 24 834, 804, 652, 48 649, 677. 687. 72 753, 670, 772, 96 314. 397. 526. 168 422, 416, 546, 384 387, 439. 440, 576 329, 204, 3 54, 744 388, 306. 152, m e a n c o n t r o l : 99 (+ 14 1 617, 980, 512, 18 565, 550, 507, 42 502, 686, 501, 72 473, 598, 624, 165 550, 343, 584, 264 381, 499. 307, 4 0 8 482. . 475, 3 58, 840 314, 207. 374, m e a n c o n t r o l : 74 (+ 36 1 269, 375. 247, 17.2 318, 370, 308, 41.8 221, 224, 176, 94.6 152, 204, 197, 144 172, 156, 174, 672 .119. 131. 156. S t a n d a r d M e a n D e v i a t i o n 305 435 91 329, 298 59 271 260 23 199 201 28 161 172 40 511 621 115 666 607 155 466 4 4 9 77 221 256 40 206 161 30 318 254 69 147 161 84 741 983 593 770 759 205 555 711 189 626 660 27 704 725 46 434 418 87 657 510 114 348 404 44 259 287 68 282 282 97 981 ~ 758 228 820 611 141 451 535 103 567 566 65 460 4 8 4 107 321 377 87 473 447 59 268 291 70 296 - 2 9 7 • 55 286 321 35 271 223 38 196 175 29 205 175 22 174 145 24 m e a n c o n t r o l : 53 (+ 21) 20 Figure 1. Relation between log body content of Hg (ng/g) and time in crabs exposed to Hg solutions. A to E , experiments 1 to 5 respectively. 2 1 TIME. (HOURS) 5 10 30 100 1000 T I M E (HOURS) Figure 2 . Relation between log body content of Hg crabs exposed to Hg solutions. A to E , respectively. (ng/g) and log time in experiments 1 to 5 22 T A B L E V. Experimentally determined biological half-life of inorganic mercury in Cancer magister. Arithmetic Experiment plot Semi-log plot (days) (days) 1 27.1 34.5 2 32.9 25.8 3 18.3 15.4 4 24.4 26.7 5 21.7 21.7 Average 24.9 Standard Deviation 5.5 24.8 7.0 23 R is the rate of release, as determined by a least-squares line of best fit to the data; C Q is the init ial body content after dosing, as determined by the Y-intercept of the line of best fit; B is the average background body content, i .e. , the mean body content of al l control animals. The Adequacy of Simple Negative Exponential Clearing Rates The second question (whether a simple negative exponential relationship is qualitatively adequate for describing the release of mercury f r o m C_. magister), was approached by evaluation of linear and non-linear components of least-squares lines of best fit. This procedure was used to evaluate as well as the negative exponential line form, both an arithmetic time versus arithmetic body content line form, and a log time versus log content f o r m . The graphs of two of these transformations are shown in F igures 1 and 2. Table VI shows the results of this analysis expressed as probabilities that the line of best fit is an adequate description of the relationship between time and body content. The inadequacy of any of the three simple line forms in describing the relationship led to the idea that the clearing rate might be the result of multiple events, but that these events might not in themselves be complex. Specifically, it seemed possible that two or more sources of mercury existed in the animal and that the clearing rate f r o m each source might be different. Further , the relatively fast initial clearing rates and relatively slow subsequent ones suggested two concurrent processes, each with a simple negative exponential clearing rate. Equation (2) shows a model conceived to test this idea. 24 . T A B L E VI . Probabil i ty that there is no significant departure f r o m linearity in regress ion . (Legend: axes are indicated as follows: A : arithmetic body content versus arithmetic time B: logarithmic body content vs . arithmetic time C : logari thmic body content vs . logarithmic time) A x e s Experiment Probabili ty A 1 .00043 2 .00001 3 .15201 4 .09665 5 .00013 B 1 .00128 2 .00005 3 .10295 4 .18025 5 .00029 C 1 .93413 2 .01884 3 .02484 4 .15076 5 .00710 25 H S T = H g 0 ( P l e " K l T + ( 1 - P l ) e " K 2 T ) ( 2 ) where H g Q is init ial body content Hg; P j is the proportion of the total Hg in the f i rs t concurrent process ; is the rate of release f r o m the f i rs t concurrent process ; K 2 is the rate of release f r o m the second concurrent process . A program (MARQD = Marquardt, 1963) to estimate parameters for non-linear systems such as this one was applied, for equation (2), to the data f r o m a l l five experiments. Table VII shows the best parameters derived by th is program, and the per cent variance explained by each set of parameters . Except for the data of experiment 4, the least-squares fit for equation (2) explained significantly more of the total variabili ty than simple linear regression with either ari th-log or log-log axes. There thus seems to be reason to believe that the process of clearing is more complex than is suggested by simple linear models. A modification to equation (2), which would allow a proportion Q of the mercury released by the f i r s t concurrent process to be recycled into the second concurrent process and then released at a rate, K£, (equation (3)) was also tested. H g T = H g Q P i e " K l T + ( l - P l ) e " K 2 T + Q P 1 ) l - e " K l T ) e " K 2 T _ (3) The parameters derived for this model by two calls to the M A R Q D program, are shown in Table VIII. It is clear, f r o m the large residual variabili ty for the simple negative exponential relationship of body content to time, that values for biological half- t ime (calculated using that line form) are useful only as crude approximations. TABLE VII. Estimated parameters for concurrent processes mod (Equation 2) Percent variance Experiment P K K explained J. X w 1 .51481 .00040 .09044 77.69 2 .74733 .02696 .00003 81.35 3 .65209 .00128 .06527 53.34 4 convergence not achieved 5 .43778 .00006 .01408 73.12 27 T A B L E VIII. Experiment 1 Estimated parameters for concurrent processes model with recycling (Equation 3) Percent Variance Explained C a l l 1 2 1 2 1 2 1 2 .51480 37.830* .83745 .83768 .65207 .65209 .01169 .01234 .00040 .00023 .00084 .00084 .00128 .00128 .06732 .06730 .09017 .000 58 .00559 .00559 .06460 .06467 .00077 .00077 .58392 1.8443* 20.487* 20.478* .27364 .26844 23.407* 22.124* 1 .45727 .00000 .01055 959.67* 2 C O N V E R G E N C E N O T A C H I E V E D 77.69 59.86 83.92 83.92 53.34 53.35 59.09 59.09 73.17 * indicates nonsense estimate 28 The concurrent processes models (equations (2) and (3)) also remain unsubstantiated by the statistical tests applied to them, although a fa i r ly large percentage of the variation is explained by those models in many cases. The recycling model showed nonsense values for best estimates of some of its parameters, and without independent evidence as a basis for constraining some of the variables, it was not possible to evaluate the validity of the model assumption. DISCUSSION The results indicate that Cancer magister assimilates environ-mental inorganic mercury so that its body content approaches or surpasses that of the surrounding water. Whole body levels of between 100 and 600 (ig/g (up to 1000 if method inaccuracies are allowed for) are found after a two-hour exposure to mercury , and these body levels appear to exist even when the dose is appreciably reduced (see Experiments 1 and 5). Litt le experimental data exist dealing with comparable exposure times to inorganic mercury in other species, but levels found in the environment in f ish (Wobeser, et a l . , 1970; F i m r e i t e , 1970) are comparable both to environmental levels in crabs (Bligh, 1971) and to my control levels (all between 50 and 150 lig/g). It was found in these experiments, as might be expected f r o m the negative exponential clearing rate concept, that at the end of most experiments residual mercury remained well above control levels . Clearing rates are also s i m i l a r to those found in other species (Rucker and Amend, 1969). It is important, though, to distinguish between work on inorganic mercury where clearing rates produce a half-time of the order of a few weeks to a few months, and work on methylated mercury , in which as much as two to seven years may be required for halt the compound to be c leared. M e r c u r y levels, both organic and inorganic, as found even near to sources of pollution in matural waters, are far below any used in this 29 study. The residual level in natural ly-occurr ing animals of about 50 to 150 l ig/g results f r o m water levels of total mercury about one-tenth of those found in the animals . This indicates, of course, that long- term exposure tends to increase mercury concentration in t issues. The relevance of this study is perhaps then less in its quantitative application to present natural ly-occurr ing levels of mercury, than in its conclusions that long- term effects result even f r o m brief mercury exposure and in the implication that, far l o n g e r - t e r m effects might be expected f r o m the more toxic organic mercurials and f r o m chronic expo sure. The mathematical models of clearing rates were conceived to explain the results on the following basis : it was thought that two concurrent systems could exist in one or more of several physical situations. Release of mercury f r o m two different tissues (e.g., l iver and muscle) might occur at different but intr insical ly consistent rates, perhaps due to differences in protein content in the t issues. Further models could be almost infinitely extended to include other tissues. F o r example, two chemical forms of mercury might express their different affinities for tissue in different rates of release. In either case, m e r c u r y could leave the pool of one faster con-current process to enter that of the slower one. It is conceivable that the slower process, whether a tissue or a f o r m of mercury, might hold a greater amount of mercury at some time after dosing than it does i m m e -diately following exposure. F inal ly , another hypothesis (not tested by a model here), that would_ explain a fast initial clearing rate and a slow later one, is degenerative pathological change. Destruction, or blocking of enzyme pathways, in excretory organs could lead to progressively reduced ability to eliminate free mercury, which would then recycle into the organism. - • 30 The doubts raised by the results obtained here as to the validity of the widely-used negative exponential "biological h a l f - t i m e " in C . magister has led to the speculation that s imilar doubts could be raised if other species and other foreign materials were examined closely. It seems reasonable to assume that the interface of l iving tissue and non-l iving, external material , may often be complicated by variation in form, whether of the tissue or the material , or by pathological changes caused by the mater ia l . It may also be that in studying the mechanisms of uptake, binding, and release, where these are of interest, hypotheses could be conveniently tested with models and line f o r m analysis s i m i l a r to those used here. 31 L I T E R A T U R E C I T E D Andren, A . W . , and R . C . H a r r i s s . 1973. Methylmercury in estuarine sediments. Nature, 245:256-257. Anfalt, T . , D . Dryssen, E . Ivanova and D . Jagner. 1968. The state of divalent mercury in natural waters. Svensk kemisk tidskrift 80: 340-342. A r m s t r o n g , F . A . J , and J . F . Uthe. 1972. Semi-automated determination of mercury in f ish tissue. Atomic Absorption Newsletter 10:101-104. Bligh, E . G . 1971. M e r c u r y levels in Canadian f i s h . Royal Society of Canada International Symposium on M e r c u r y in M a n ' s Environment, Ottawa, p. 73. Chau, Y - K and H . Saitoh. 1970. Determination of submicrogram quantities of mercury in lake water. Environ . S c i . & Technol. , 4:839. F i m r e i t e , N . 1970. M e r c u r y uses in Canada and their possible hazards as sources of mercury contamination. Environ . Pollut . 1:119-131. F i m r e i t e , N . , W . N . Holsworth, J . A . Keith, P . A . Pearce and I . M . Gruchy. 1971. M e r c u r y in f ish and fish-eating birds near sites of industrial contamination in Canada. Canad. F i e l d - N a t . 85:212-220. Hannerz, L . 1968. Experimental investigations on the accumulation of mercury in water organisms. Rept. Inst. F reshw. Res. , Drottningholm. 48:120-175. Hatch, W . R . and W . L . Ott. 1968. Determination of s u b - m i c r o g r a m quantities of m e r c u r y by atomic absorption spectrophotometry. A n a l . C h e m . 40:2085. 32 Jensen, S. and A . Jernelov. 1969. Biological methylation of mercury in the aquatic environment. Nature (G.B.) 223:5207. Marquardt, D . W . 1963. A n algorithm for least-squares estimation of non-linear parameters . J . Soc. Ind. A p p l . Math. 11:431-441. Rucker , R . R . and D . F . A m e n d . 1969. Absorption and retention of organic mercur ia ls by rainbow trout and chinook and sockeye salmon. P r o g . F i s h . Cult . 31:197-201. Uthe, J . E . (ed.) MS 1972. M e r c u r y in the aquatic environment: a summary of r e s e a r c h c a r r i e d out by the Freshwater Institute 1970-1971. F i s h . R e s . B d . Canada MS Rept. Series 1167: 163pp. Wobeser, G . , N . O . Nielsen and R . H . Dunlop. 1970. M e r c u r y concentra-tions in tissues of f ish f r o m the Saskatchewan R i v e r . J . F i s h . Res . B d . Canada 27:830-834. Wood, J . M . , F . S . Kennedy and C . G . Rosen. 1968. The synthesis of methyl -mercury compounds by extracts of methanogenic bacteria . Science 220:173-4. 33 A P P E N D I X - L D Experiment 50 Par t of my Biology 101 teaching duties was running a two-week elective p r o g r a m for some of the students, and I took the opportunity to test for LD50 of Hg (i.e., the dose of a toxic material required to k i l l half of a population). S m a l l shore crabs were used rather than C . magister, because of expense and convenience of handling. The elective group made a f ield trip to a local beach and collected about 300 Cancer productus, Hemigrapsus  oregonensis, and Hemigrapsus nudis, each weighing between one and two grams. The species were mixed and the male-female ratio was approxi-mately equal in each tank. Four students were responsible for each of nine tests. E a c h test was set up to show how many crabs a given dose would k i l l , with doses increasing in eight steps f r o m 10.4 to 3400ppb mercury, and one control tank. Each 40 litre tank was f i l led to 28 l i tres with seawater, hooked up to an air supply, and c o m m e r c i a l f i s h food was added. The water was dosed f r o m a roughly-measured solution of mercury, and the m e r c u r y concentrations resulting were determined. The crabs were added immediately after the mercury . The experiment was complicated by the fact that (1) it took some time for even the strongest dose to k i l l crabs, and (2) the mercury solu-tions decayed. The decaying solutions were monitored three t i m e s - - o n the second, fifth and sixteenth days. On the sixteenth day there was no measurable mercury in any of the tanks. The average of the f i rs t two readings was considered to be a guess of the average dose over the period of the experiment. Table A - l shows the average doses and the estimated times required to k i l l half the crabs at each dose. Most of the estimates were perforce extrapolated. 34 T A B L E A - I Dose (|ig/ml) 0 9 43 73 500 725 1300 1200 3250 T i m e (days) 370 144 28 16 11.2 (not completed) 9.8 5.3 4.3 These results seemed to be internally consistent. Appl ied to the half-time experiment, they suggest the possibility of damage to the crabs, especially at the higher doses. 


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