to imply t h a t both samples have the same origin, since this is unlikely; however, the analysis shows t h a t the sample was definitely a No. 6 fuel oil. Conclusion
We have recorded infrared spectra of over 50 different petroleum samples; absorptivities of 21 bands between 650 and 1200 cm-1 can be used to characterize the samples. Stored in a computer file, the 21 absorptivities of every known sample can be compared with those of any unknown sample a t a later date. Using the ratios of known to unknown absorptivities, an unknown can be matched to a known in a matter of seconds. The method of analysis presented here is rapid and, for the 50 plus samples we have studied, it provides an unambiguous identification for petroleum products, eliminating the need for adding tracer materials to petroleum products or for other methods of analysis. Ideally, for the method to be used on a worldwide scale, infrared spectra of all possible petroleum samples would have to be measured and the absorptivities of the selected bands stored in a computer file. This could all be accomplished within a relatively short period of time. In addition, to identify samples taken from natural waters, the effects of weathering on the infrared spectra of samples have to be known.
Currently, we are investigating these effects and will report on them shortly. Acknowledgment
The authors wish to express their appreciation to the Gulf Research and Development Co., the Sun Oil Co., and Texaco Inc. for supplying us with a good selection of petroleum samples. In addition, we thank Mark Ahmadjian for collecting field samples, Alfred G. Hopkins for helpful discussions, and Peggy A. Skotnicki for selecting and mixing unknown samples. Literature Cited Adlard, E. R., J. Inst. Petroleurn, 58,63 (1972). Baier. R. E., J . Geophys. Res.. 77,5062 (1972). Cole, R. D., J. Inst. Petroleum, 54, 288 (1968). Kawahara, F. K . , J . Chrornatogr. Sei., 10,629 (1972). Kawahara, F. K.. J. Enuiron. Sci. Technol., 3, 150 (1969) Kawahara, F. K., Ballinger, D. G., Ind. Eng. Chern. Prod. Res. Decelop., 9,553 (1970). Mark. H. B.. Yu. T. C.. Mattson. J. S.. Koloack. R. L.. Enuiron. Sei. Tech., 6,833 (1972). Mattson, J. S., A n a l . C h e m . , 43, 1872 (1971). Mattson, J. S., Mark, H. B., Kolpack, R. L., Schutt, C. E., ibid., 42, 234 (1970).
Receiced for recieu April 2, 1973 Accepted Jul? 26, 1973
Studies on Uptake and Loss of Methylmercury-203 by Bluegills (Lepomis macrochirus Raf.) W. Dickinson Burrows' Associated Water and Air Resources Engineers, Inc., Nashville, Tenn. 37204
Peter A. Krenkel Environmental and Water Resources Engineering Program, Vanderbilt University, Nashville, Tenn. 37235
The uptake of methylmercury-203 directly from water by bluegills was found to be nearly constant after five days a t about 20% per gram of fish per liter of water. Transferred to mercury-free water at 24"C, bluegills exhibited a rapid loss of about 40% of the mercury, followed by a slow loss with a half-time of about five months. Mercury levels in the liver and kidneys were two to seven times higher than whole fish levels, but there was no discernible trend in this ratio with time. The proportion of mercury present as methylmercury in the whole fish remained a t 73 & 10% throughout the course of the experiment. The proportion of methylmercury in the liver and kidneys, however, fell rapidly in the first few weeks after exposure, ultimately leveling off a t about 10%. This suggests that biochemical demethylation is taking place in these organs. The uptake of organic and inorganic mercury compounds has been recorded for a broad range of aquatic organisms. The most comprehensive study is that of Hannerz (1968), who investigated in anatomical detail the ac-
To whom correspondence should be addressed.
cumulation of radioactive mercury compounds by representatives of most of the common aquatic plant and animal phyla, from blue-green algae to cod. The most complete study of methylmercury elimination by fish and other aquatic animals has been carried on by Miettinen (1968, 1969, 1970) and his coworkers (Jarvenpaa et al., 1970, Tillander et al., 1970). Most recently, Giblin and Massaro (1973) have reported the fate of mercury administered intragastrically to rainbow trout as methylmercury. The study reported herein is directly concerned with the assimilation and disposal of mercury by fish exposed to methylmercury in their surroundings, but not their diet. By exposing bluegills to water containing 203HgCH3, we have determined initial uptake rates and have generated a population of fish containing tagged mercury. Using the latter, we have measured the rates of loss of mercury in clean water from the whole fish and various organs. Finally, we have investigated the extent of biochemical transformation of methylmercury in the liver and kidneys. Experimental Procedures
General. Mercury-203 was purchased from New England Nuclear as methylmercury chloride in an aqueous solution containing 1000-2000 pCi/mg of mercury. Analysis by radiographic chromatography and by the benzene Volume 7, Number 13, December 1973
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extraction technique described below showed that the methylmercury solution contained about 5% inorganic z03Hg a t the time of use. For counting large sample, such as whole fish, the Nuclear Chicago Tobor counting system was used. This is a dual, opposed, 3 x 3 in. XaI (Tl) gamma-ray detector system. For small samples, such as fish liver, a Baird Atomic well counter with a ?'-in. diameter chamber was used. Uptake of methylmercury. Fifteen bluegills weighing 2Yz-11 grams each were placed in a 34-liter aquarium containing 20 pCi of 203HgCH3. (The water was not aerated during the uptake experiment, and the terminal pH was 7.5.) After 3 hr and periodically for one week, a fish and a 50-ml water sample were withdrawn and placed in tared 2-02 specimen jars. The jar containing the fish was filled with deionized water, and the fish and water samples were counted. After removal of the slime coat, each fish was placed in a fresh jar of water and recounted. Distribution and Loss of Total Mercury and Methylmercury. Thirteen bluegills, 4-5 in. long were placed in a 60-liter aquarium containing 50 pCi of 203HgCH3. After three days of exposure, the first fish was removed and counted. The remaining 12 fish were transferred to clean water and maintained for 3 months under continuous aeration and filtration, one fish being removed each week. The filter charcoal was changed weekly, and the fish were fed daily. Each fish was scraped and recounted and then was dissected, the liver, kidneys and roe being removed and counted in test tubes under 2 ml of deionized water. The weight of the liver was about 1% and the kidneys 0.1-0.2'70 of the weight of the whole fish. The weight of the roe, 1.2-2.7 grams, was not dependent on the weight of the fish. The dissected fish (less slime, kidneys, liver, and roe) was homogenized with 100-150 ml of 3 N sodium bromide solution and a volume of 1N copper sulfate solution equal in ml to the weight of the fish in grams. The homogenate was rinsed into a 500-ml separatory funnel and shaken for several minutes with 100-150 ml of 3 N sulfuric acid. After standing for 15-20 min, the mixture was extracted with three 50-ml aliquots of benzene. The combined benzene layers were centrifuged, and a weighed aliquot was counted. Liver, kidney, and roe samples were treated similarly, with appropriate reduction in reagent volumes. Rate of Loss of Methylmercury. Twelve bluegills of ca. 3-5 g wt were released in a 34-liter aquarium containing 20 pCi of 203HgCH3. After 96 hr, the fish were removed and placed in individual 4-oz specimen jars filled with water. After being counted, the fish were returned to individual 2-liter tanks. Thereafter the fish were counted every 1 to 2 weeks for three months. After each count, the tanks were scrubbed and filled with clean water. Except as noted, the fish were fed daily. Following the 95-day count, the remaining nine fish were sacrificed and extracted with benzene as above. Results and Discussion
Uptake of Methylmercury. A number of investigators have used small fish, such as guppies, to estimate the methylmercury generated by sediments in situ (Jernelov, 1970, Gillespie, 1972; Langley, 1973). The success of this scheme would appear to require rapid and quantitative uptake of methylmercury by the fish. In the experiments described herein, small bluegills were exposed to water containing about 0.5 pCi/l. in a total concentration of 0.2-0.3 pg/l. of 203HgCH3. Water and fish samples were removed a t intervals and counted. The uptake of methylmercury was neither rapid nor quantitative. In a typical experiment, an initial fish-to-water concentration of 2.2 1128
Environmental Science & Technology
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40
~
$ 30-
B 3
8 20-
lo0
50
100
50
20:
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Figure 1. Projected total uptake of methylmercury-203 by 2.511-gram bluegills at 24°C
DAYS SINCE EXPOSURE
Figure 2. Loss of
mercury-203 by bluegills at 24"C, average of
five g/l. was employed. After five days the maximum uptake by a single fish was 3.8% of the mercury originally present in the water, and the projected total uptake for all fish was 35-40%, as illustrated in Figure 1. (The projected uptake is the activity per gram for each fish multiplied by the weight of all the fish originally present, and divided by the total initial activity of the water.) From this and similar experiments with different fish-to-water ratios, it appears that a fish density of 1 g/l., the maximum recommended for bioassay tests (American Public Health Association, 1971). will achieve no more than 1 6 2 0 % removal of methylmercury in 3-4 days. To accomplish a rapid and near quantitative absorption of methylmercury from water would require a fish density greater than 5 g/l. This in turn would require continuous artificial aeration of the fish tanks, which in our experience results in volatilization of the methylmercury. These observations may be valid only for bluegills, but preliminary investigations by others in this laboratory indicate uptake rates of the same order of magnitude for guppies and other small fish (Taimi, 1973). The maximum value of the concentration factor observed after three days is about 270 for whole bluegills; for pike muscle, Hannerz recorded concentration factors of 100-400 after three days, with a maximum of about 1500 after 30 days. For rainbow trout subjected to constant methylmercury levels in flowing water aquaria, Willford and Reinert (1973) have noted concentration factors as high as 8000 after 90 days. An interesting aspect of the mercury assimilation experiments is that a substantial proportion of the total activity, 16-45%, resides in the slime coat. Concentration in the external mucus has also been noted for inorganic mercury and other heavy metal salts (McKone et al., 1971). The percentage of slime counts for methylmercury shows some inverse dependence on the weight of the fish, as would be expected. Loss of Methylmercury. As in the previous experi-
Table 1. Distribution of *03Hgin Bluegills with Time. T i m e since initial exposure, days
Wt. fish, g
3 10 22 31 38 45 52 59 66 73 80 87 94 a
B a s e d on wet weights.
13.7 17.7 26.2 13.5 21.7 32.9 24.5 27.3 21.3 27.6 32.4 28.4 66 b
CPM/gb fish
27400 12950 9410 9150 6060 6830 7270 6280 5950 5480 5310 3940 3310
CPMlg scraped fish
CPM/g liver
CPM/g fish
CPM/g fish
CPM/g fish
0.75
4.20 7.00 3.12 3.49 5.68 2.19 3.51 3.82 4.50 2.67 3.07 2.54 2.19
4.86 6.97 3.16 6.42 3.93 3.42 3.09 4.06 4.25 2.49 4.75 4.36 3.31
0.95 1.00 1.00 0.96 0.99 0.99 0.96 1.00 1.00
CPMIg r o e
CPM/g kidney
CPM/g fish
1.57
1.42
0.99 1.21
CPM = counts per minute.
ments, 12 small bluegills were exposed to methylmercury203 chloride, each accumulating 0.4-0.5 pCi. Figure 2 is a semilog plot of the average radiomercury content with time for five fish maintained a t 24°C. A rapid loss of about 40% of the mercury is observed, with a biological half-time of 38.5 days, followed by a slow loss with a halftime of 130 days. This is in general agreement with the results of other investigators using different kinds of fish. Jarvenpaa et al.. for example, report that 300-gram pike fed methylmercury perorally lost 5-1070 of the activity in the first few days, and thereafter eliminated mercury with a half-time of 640 f 120 days a t ca. 10°C. Flounder and eel lost substantially more mercury by the fast process, but the half-times for slow elimination were as great or greater than for pike. Willford and Reinert have measured half-times of 2-3 years for elimination of methylmercury from rainbow trout a t 10°C. Because winter mercury levels are alleged to be lower in fish from natural waters (Zeller and Finger, 197l), we stopped feeding one group of four fish after 59 days t o find whether reduction in mercury accompanies loss of fatty tissue. While there is no doubt that the starved fish lost weight, averaging 4.5 grams a t the completion of the experiment contrasted with 8 grams for the others, the starved fish did not demonstrate a n increased elimination rate. Apparently fish do not directly reabsorb the mercury they have excreted. This was demonstrated for one group of five fish by changing the water frequently; the overall mercury elimination rate was no greater t h a n for the control group, for which the water was changed only after counting. This is in agreement with the observation of Hannerz that inorganic mercury is not nearly as readily absorbed from water by fish, and the likelihood t h a t methylmercury is excreted as inorganic mercury (Norseth and Clarkson, 197Ob). Distribution of 203Hg in Bluegills. In a n experiment parallel to the live counts, the course of radioactive methylmercury was followed through 13 larger fish, one of which was sacrificed each week for 13 weeks. Each fish was counted with and without its slime coat; then various organs were removed and counted separately. The results presented in Table I show a uniform decline with time in specific activities for the whole fish and separate organs. Liver and kidney levels are 2-7 times higher than whole fish levels, in agreement with Hannerz, but there is no discernible trend in the ratio for either throughout the course of the experiment. Roe sacs and one gill sample (not reported in Table I ) exhibit about the same specific
Table II. Distribution of Methylmercury-203 in Bluegills l i m e since initial exposure, days
a
Percent total mercury as methylmercury Whole fisha
Liver
Kidney
56.2 28 22 14.3 14.9 18.4 18.4 9.4 10 12.4 12.7 10.2 8.1
36.8 15.3 14.7 12.4 13.7 8.7 8.7 10.0 13.1 13.6 4.2 10.3 10.5
3 10 22 31 63.1 38 83.5 45 71.3 52 75.8 59 71.8 66 82.9 73 69.6 80 68.6 87 78.5 94 67.5 Less liver, kidney a n d roe.
Roe
17.8
9.1
9.0 10.0
activity as the whole fish. For later fish samples, no more than a few percent of the radioactivity was lost on removal of the slime coat, supporting our belief that the initial loss of mercury was due in part to sloughing of the slime layer. Distribution of 203HgCH3 in Bluegills. To determine the ratio of methylmercury to total mercury, the whole fish or partially dissected fish were homogenized in a food blender, digested with hydrobromic acid, and extracted with benzene. When the benzene extract was counted (taking into consideration the organs removed) only 73 f 10% of the mercury in the whole fish was accounted for, as demonstrated in Table 11. Because it has been established that more than 95% of the radioactive methylmercury is extracted from water by this procedure, the last result implies either that methylmercury is incompletely extracted from fish tissue by the Westoo (1966) procedure and its variations, or that methylmercury is converted to some other form by the fish. More noteworthy is the proportion of methylmercury in the organs. Thus, though the ratio of total mercury levels in the liver and whole fish is independent of time, the percentage of mercury as methylmercury falls rapidly in the first few weeks after exposure, as shown in Table I1 and Figure 3. The same is true to a lesser extent for kidney and roe samples. Two explanations present themselves. The first is that inorganic mercury present as a n impurity (ca. 5%) in methylmercury-203 chloride is accumulated in Volume
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of 2-3 years for different species and significantly lower temperatures. It seems likely t h a t half-times for reduction of environmental mercury contamination will be a t least as great, even with the most determined control efforts. Thus, though fish are quite slow in excreting mercury relative to warm-blooded animals, we suspect that any reduction in biological mercury levels will depend on the rate of removal of mercury from the environment (if indeed this can be achieved) and not on the physiology of the organism.
7
Acknowledgments
-0
Figure 3.
50 DAYS SINCE FIRST EXPOSURE
IW
Percent benzene-soluble mercury-203 in bluegills
Invaluable advice and assistance were given by Norman C. Dyer. Department of Nuclear Medicine and Biophysics, Vanderbilt University Medical School, whose radiation counting facilities were used in these experiments. Literature Cited
the liver and kidneys, and excreted a t a lower rate than methylmercury. This would be contrary to the observation of Rothstein and Hayes (1960) that inorganic mercury is not preferentially concentrated in the liver of the rat. For rat kidney, Norseth and Clarkson (19’iOa) have recorded preferential accumulation of a maximum 23% of the injected dose of inorganic mercury per gram of kidney for 170-gram rats. This could account for the level of inorganic mercury observed in fish kidneys only if it is assumed that inorganic and organic mercury are equally absorbed from water, which is not in accord with the data of Hannerz. A more rational explanation, we believe, is that methylmercury is biochemically demethylated in the liver and kidney of the fish, and perhaps in other organs as well. Norseth and Clarkson (197Oa) have demonstrated this biotransformation in the liver of the rat and suggest that demethylation may occur wherever methylmercury accumulates in the body. S u m m a r y and Conclusions
The uptake of methylmercury by fish directly from water may or may not be significant in natural systems. Jernelov (1972) has presented arguments that suggest that 50% of the mercury in Swedish pike comes directly from the water, rather than the food chain. On the other hand, Bishop and Kirsch (1972) found that all of the methylmercury-203 generated in anaerobic sediments remained in the sediments. Heavy industrial contamination of natural waters by methylmercury, such as occurred in Minamata Bay, Japan, may never again be a problem. Accidental spills are always possible, however, and many studies, including this one, leave no doubt that wherever methylmercury enters the water it will be taken up by the indigenous aquatic life, though not necessarily quantitatively. By any determination, the overall process by which fish rid themselves of methylmercury is slow, though a fish removed from a contaminated environment and released in clean water may lose a substantial portion of its burden of mercury in the first few days. The bluegill studies indicate biological half-times for the slow loss of methylmercury of about five months, while others report half-times
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Environmental Science & Technology
Amer. Pub. Health Ass., “Sta;dard Methods for the Examination of Water and Wastewater, 13th ed., p 540, 1971. Bishop, P . , Kirsch, E. J . , 27th Ann. Purdue Indus. Waste Coni., May 1972. Giblin, F. J., Massaro, E . J., Toxicol. Appl. Pharmacol., 21, 81-91 (1973). Gillespie, D. C., J. Fish. Res. Bd. Can., 29,1035-41 (1972). Hannerz. L., Rep. Inst. Freshwater Res. Drottingholm, 48, 120-76 (1968). Jarvenpaa, T . , Tillander, M., Miettinen, J. K., Suom. Kemistilehti, B43,439-42 (1970). Jernelov. A,, in “Environmental Mercury Contamination, ” R. Hartung and B. D. Dinman, Eds., pp 174-7, Ann Arbor Science Pub., Ann Arbor, Mich., 1972. Jernelov, A,, Limnol. Oceanogr., 15,958-60 (1970). Langley, D. G.. J . Water Pollut. Contr. Fed., 45,44-51 (1973). McKone, C. E., Young, R. G., Bache, C. A,, Lisk, D. J., Enciron. Sei. Technol.. 5,1138-1139 (1971) and references cited therein. Miettinen, J . K., Heyraud, M., Keckes, S., F A 0 Tech. Conf. on Marine Pollut. and Its Effects on Living Res. and Fishing, Rome, Italy, December 1970. Miettinen, J. K., Tillander, M., Rissanen, K . , Miettinen, V., Minhk. E.. Torthern Mercurv Svmoosium of Xordforsk. Stockholm, Oct. 1968. Miettinen, J. K., Tillander, M.,Rissanen, K., Miettinen. V., Ohmomo, Y., Proc. 9th Jap. Conf. on Radioisotop., Tokyo, May 1
.
1
__
19fi9
Norseth, T . , Clarkson. T. W..ilrch. Enciron. Health, 21, 717-27 (197Oa). Korseth, T.. Clarkson, T. W., Biochem. Pharm., 19, 2775-83 (1970b). Rothstein, A., Hayes, A. H.. J . Pharmacol. Exp. Ther., 130, 16676 (1960). Taimi, K. I., unpublished research, Vanderbilt University, 1973. Tillander, M . , Miettinen, J. K., Koivisto, I., F A 0 Tech. Conf. on Marine Pollut. and Its Effects on Living Res. and Fishing, Rome, Italy, December 1970. Westoo, G., Acta. Chem. Scand., 20,2131-7 (1966). Willford, W., Reinert. R., Great Lakes Fishery Laboratory. Ann Arbor, Mich., personal communication from W. Willford, 1973. Zeller, H. D., Finger, J . H., Proc. 10th Ann. Enciron. and Water Res. Engrg. Coni., pp 69-99, Vanderbilt Lniv., Kashville. June 1971.
Received f o r recieu April 30, 1973. Accepted August 23, 1973. Paper presented in part at the ICES Incitational Symposium, Chapel Hill, N . C . , September 2972. Work supported by the Sport Fishery Research Foundation, uith cooperative funding from the American Fishing Tackle Manufacturers Association, the John M . Olin Foundation, and the Tennessee Valley Authority; and by the Tennessee Game and Fish Commission.
