a Van Dorn sampler were made at two depths, 5 m and 16 m. Two or three dissolved gas sampling bottles were filled with water from each of the casts with the Van Dorn sampler. The dissolved gas concentrations in each sampling bottle were determined three or four times. These data, therefore, indicate a measure of the variability encountered in analysis of one sample bottle and from taking separate samples at a particular depth. The range of values was greater for separate samples from one depth than for several measurements from one sample bottle. With one exception, the mean values for both nitrogen and methane concentrations from all sample bottles from a given depth were judged to be statistically equivalent at the 95% confidence level according to the t test (Huntsberger, 1961). The exception, the nitrogen concentrations for Van Dorn cast number one, was higher than those of casts numbers two and three. The variations in dissolved methane concentrations were slightly greater than those in the nitrogen determinations. In all cases, however, the precision of the analyses was within +7% of the average and generally within f5 %. Conclusions
The data gathered by this study indicate that collection and storage of water samples in the dissolved gas sampling bottles is a method that maintains dissolved nitrogen and methane
concentrations constant during storage. The overall method of collection, filling, storing, and analyzing is precise to within +7 % or less. The sampling bottles offer a convenient means of filling and transferring the water sample to the gas stripping chamber. When this method of sample collection and storage is used, the largest source of analytical error for this study was apparently the instrumentation. Literature Cited
Huntsberger, D. V., “Elements of Statistical Inference,” Allyn and Bacon, Boston, Mass., 1961. Hydro Products, OEC, Seahorse 2 (4) (1967). Swinnerton. J. W.. Linnenbom. V. J., Cheek, C. H., Anal. Chem. 34, 483 (1962a). Swinnerton, J. W., Linnenbon, V. T., Cheek, C. H., ibid. 34. 1509 (1962b). Weimer, W. C. ‘“Some Considerations of the Chemical Limnology of Lake Mary, Vilas County, Wisconsin,” M.S. Thesis, Water Chemistry Program, University of Wisconsin, Madison, Wis., 1970. Received for review Jan. 22, 1971. Accepted April 6, 1971. This investigation was supported by a National Defense Education Act Title IV Fellowship, Federal Water Pollution Control Administration training Grant 5TI- WP-184, and the University of Wisconsin Engineering Experiment Station, Civil Engineering Dept., and Water Resources Center.
Rapid Uptake of Mercuric Ion by Goldfish Colin E. McKone,‘ Roger G. Young, Carl A. Bache, and Donald J. Lisk2 Department of Entomology, Cornel1 University, Ithaca, N. Y 14850 Experimental
Mercuric ion has been shown to be rapidly absorbed from water by goldfish. The extent of absorption is dependent on mercury concentration and time of exposure. As has been previously shown with other metals, mercury was found to concentrate initially in the external mucus secreted by the fish. The presence of mercury in water appeared to stimulate this secretion.
R
ecent high levels of mercury in fish and the environment have been well publicized (Klein and Goldberg, 1970; Chem. Eng. News, 1970b; Chem. Eng. News 1970a; Znd. Res., 1970; Parker, 1970; Zwerdling, 1970). Microbiological methylation of mercury and the resultant presence in fish of highly toxic methylmercuric salts (Westoo, 1969; Westoo and Rydalv, 1969) are of particular concern. Since mercuric ion is one of the major forms of mercury as an initial industrial pollutant, we found it interesting to study the rate of absorption of this ion by fish.
1 Present address : Weed Research Organization, Agricultural Research Council, Begbroke Hill, Savoy Lane, Yarnton, Oxford, England. 2 To whom correspondence should be addressed.
1138 Environmental Science & Technology
Goldfish (Carassius auratus) were used for this study. In the first experiment, 50 fish ranging in size from about 4 to 6 cm and weighing between 1 and 3.5 grams were placed in a 57-liter aquarium with 50 liters of distilled water containing 0.25 ppm of mercury as mercuric chloride. The temperature was maintained at 21OC. The water volume was sufficiently large to obviate the need for aeration. Two grams of Strike fish food (Agway, Inc., Ithaca, N. Y.) was added daily, and was rapidly and totally consumed. At specific intervals fish were removed in groups of four, adhering water was removed by blotting and each was freeze-dried and mechanically ground and mixed. One gram of each sample was pelleted and burned in a modified Schoniger flask (Gutenmann and Lisk, 1960). Mercury was determined colorimetrically as the dithizonate. The method was sensitive to about 0.25 ppm of mercury in fish. With this method, the recovery of 0.5 ppm of mercury added to two samples of fish as mercuric chloride was 102 and 108%. Apparent mercury in unexposed goldfish was less than 0.25 ppm. Results Figure 1 illustrates the rapid uptake of mercury by the fish as a function of time. In preliminary experiments it was found that goldfish died within 4 hr when exposed to 1 ppm of mercury as mercuric chloride. The fish exhibited no outward signs of toxicity during the test period in the 0.25 ppm solution used.
In a second experiment, goldfish were individually exposed to lower mercury concentrations. Each of three fish was placed alone in 1 liter of solution at a given mercury concentration and sampled after 81 hr and analyzed. Table I lists the extent of mercury absorption by individual fish at different mercury concentrations. It was first observed by Carpenter (1927, 1930) and is now generally accepted that heavy metals such as lead may exert their toxic effect on fish by precipitating or coagulating the normal mucus secreted by the gills and skin. The resultant surface plugging and thus interference with respiration, secretion of waste products, and salt balance may then be fatal. Carpenter (1927) detected no lead in the bodies of fish killed in solutions of lead nitrate and then externally extracted with acetic acid to remove mucus. The acetic acid washings, however, contained most of the lead to which the fish was initially exposed. A heavy accumulation of mucus formed on the external surfaces of the goldfish which had been exposed to 1 ppm of mercury for 3 hr. It was of interest, therefore, to determine the proportion of “absorbed” mercury which may have been present in the accumulated mucus. In the third experiment, five goldfish were exposed to 1 ppm of mercury as mercuric chloride in 2 liters of distilled water for 3 hr. Similarly, five fish were placed in 2 liters of distilled water for 3 hr to serve as controls. Copious production of mucus was observed on the surface of the fish exposed to mercuric chloride during this time, some of which sloughed off. The exposed fish were removed and thoroughly rinsed with a vigorous stream of distilled water to remove mucus. The mercuric chloride and distilled water rinse solutions were centrifuged, the mucus was collected and thoroughly washed with water to remove adhering mercuric chloride solution. The mucus was dried, combusted, and mercury was determined as described (Gutenmann and Lisk, 1960). The exposed goldfish were then externally extracted by immersing in 100 ml of 80% ethanol in water (in which the mucus was soluble) for 15 min to remove any remaining mucus and this solution was evaporated on filter paper and similarly combusted and analyzed. Finally, the extracted exposed fish as well as the unextracted control fish were individually lyophilized and combusted for the determination of mercury. Of the total mercury (213 pg) found in the five exposed fish, 79.3% was in the mucus which sloughed off and was then removed by the water washing and alcohol extraction. Conclusion
In the first experiment in which 50 fish were in contact with 50 liters of 0.25 ppm of mercury, the concentration of mercury absorbed reached levels in the range of 40 to 50 ppm after 100 hr. This was considerably more than the 3 to 4 ppm levels of mercury absorbed by the fish individually exposed to 0.1 ppm of mercury for 81 hr in the second experiment. There are probably many reasons for this difference. The higher mercury level in water and the longer time of exposure would increase the final concentration absorbed. Also, the higher mercury concentration in water would induce a greater production of mucus in the exposed fish and perhaps therefore increase the efficiency of absorption of mercury. This is evident from the data in the third experiment in which the concentration of mercury absorbed by the fish was about 39.5 ppm when the five fish were exposed to 1 ppm of mercury for 3 hr. In conclusion, it is apparent that mercuric ion is rapidly
Figure 1. Concentration of mercury absorbed by goldfish from a solution containing 0.25 ppm of mercury as mercuric chloride as a function of time
Table I. Mercury Absorbed by Individual Goldfish Exposed for 81 Hr to 1 Liter of Mercuric Chloride Solution at the Concentrations Indicated Mercury absorbed by individual fish, Mercury in water, PPm
PPm
0.001 0.01 0.1
0.25, 0.25, 0.25 0.48, 0 . 5 4 , 0 . 5 8 4.6, 3.2, 3.4
taken up from water by goldfish. The magnitude of this reaction is affected by exposure time and concentration. Other factors known to influence the toxicity of heavy metals to fish may also affect this uptake, these include water hardness, temperature, pH, volume, the associated heavy metal anion, and the presence of other heavy metal ions (Doudoroff and Katz, 1953). Mercuric ion in water, apparently as certain other heavy metals, is rapidly absorbed into the mucus externally secreted by fish and its presence appears to stimulate this secretion. These findings may be significant since Jernelov (1970) has reported that bacterial methylation of mercury occurs when mercuric ion is incubated with dead fish but only if the mucus is present. Work is underway to determine the kinetics of this methylation reaction and the fate of the methylated product(s) in fish. Literature Cited Carpenter, K. E., Brit. J. Exp. Biol. 4, 378 (1927). Carpenter, K. E.,J. Exp. Zool.56,407 (1930). Doudoroff, P., Katz, M., Sewage Ind. Wastes25,802 (1953). Gutenmann, W. H., Lisk, D. J., J . Agr. Food Chem. 8, 306 (1960). Jernelov, A., Presentation at International Conference on Environmental Mercury Contamination, University of Michigan, Ann Arbor, Mich., Sept. 30,1970. Klein. D. H.. Goldberg. E. D.. ENVIRON. SCI. TECHNOL. 4. 765(1970).’ “Mercury: High Levels in Foods,” Chern. Eng. News 48 (42). 8 (1970a). “Mercury Menace Prompts Firm to Offer Test Data,” Ind. Res. p 25, (October 1970). “Mercury Stirs More Pollution Concern,” Chem. Eng. News 48 (26) 36 (1970b). Parker, C. E., Conseraationisr pp 6-9, (August-September 1970). Westoo, G., Var. Foeda21,138 (1969). Westoo, G., Rydalv, M., ibid., 20 (1969). Zwerdling, D. New Republic 17, (Aug. 1,1970). Received for reaiew March 18, 1971. Accepted June 21, 1971. Volume 5, Number 11, November 1971 1139
