(20) Gordon, G. E., Director, “Study of the Emissions from Major Air Pollution Sources and Their Atmospheric Interactions, First Year Progress Report,” Nov. 1, 1972-Oct. 31, 1973, University of Maryland, College Park, Md. (21) Lee. R. E.. Jr.. von Lehmden. D. J.. J . Air Pollut. Contr. . Ass., 23 (lo), 853 (1973). (22) Toca, F. M., “Lead and Cadmium Distribution in the Particulate Effluent from a Coal Fired Boiler.” Ph.D. Dissertation, U. of Iowa, July’ 1972; see also Diss Abstr I n t , 33, 3156B (1973). (23) Sparks, C. J., Oak Ridge Xational Laboratory, “Analysis of Particles Collected on a Brinks Impactor,” private communication, 1973. (24) Nicholls, G. D., in “Coal and Coal Bearing Strata,” D. Merchison and P. Stanlev Westoll. Eds.., DD 269-307. Oliver and Boyd, London, 1.968. (25) Gluskoter, H. J., Lindahl. P. C., Science, 181.264-6 (1973) (26) Hulett, L. D., Oak Ridge National Laboratory, “Analysis of Individual Fly Ash Particles,” personal communication, 1973. (27) Hulett, L. D., ibid, ”The Chemical State of Sulfur in Fly Ash,” personal communication, 1973. . I
(28) Butcher, S. S., Charlson, R. J., “An Introduction to Air Chemistry,” Chap. 9, Academic Press, New York, N.Y., 1972. (29) Gladney, E. S., Gordon, G. E., Zoller, W. H., “Abnormally Enriched Trace Elements in the Atmosphere,” Seventh Annual Conference on Trace Substances in Environmental Health, U. of Missouri, Columbia, Mo., June 1973. (30) Hodgeman, C . D., Ed.-in-chief, “Handbook of Chemistry and Phvsics.” D 2429. 44th ed.. The Chemical Rubber Publishing Co.,‘Clevelkd, Ohio, 1963. ’ (31) Pillav, K. K . S., Thomas, C . C., Jr.. J . Radioanal. Chem.. 7, 107-18 (1971). (32) Muller, R. O., “Spectrochemical Analysis of X-ray Fluorescence,” pp 72-86, Plenum Press, New York, N.Y., 1972. (33) von Lehmden, D. J., Jungers, R. H., Lee, R. E., Jr., Anal. Chem., 46 (2), 239 (1974). Received for review November 5, 1973 Accepted August 11, 1974. Work supported by research grants NSF-GH33634 and NSF-GI31605-IES-7. Mention of commercial products is for identification only and does not constitute endorsement by any agency of the U.S. Government
Partition Coefficient to Measure Bioconcentration Potential of Organic Chemicals in Fish W. Brock Neely,*9’ Dean R . Branson,* and Gary E. Blau3 T h e Dow Chemical Co., Midland, M i c h . 48640
T h e bioconcentration of several chemicals in trout muscle was found to follow a straight line relationship with partition coefficient. Bioconcentration in this paper is defined as the ratio of the concentration of the chemical between trout muscle and the exposure water measured at equilibrium. Partition coefficient has the usual meaning in that it is the ratio of the equilibrium concentration of the chemical between a nonpolar and polar solvent (in this case, n-octanol and water were the two solvents used). T h e relationship was established by measuring the bioconcentration in trout of a variety of chemicals over a wide range of partition coefficients. An equation of the straight line of best fit was determined and used to predict the bioconcentration of other chemicals from their partition coeffic Lents. The predicted values agreed with the experimental values in the literature.
T h e ability of some chemicals t o move through the food chain resulting in higher and higher concentrations a t each trophic level has been termed biomagnification or bioconcentration ( I ) . T h e widespread distributions of DDT (2, 3 ) and the polychlorinated biphenyls (PCBs) ( 4 ) have become classic examples of such movement. From an environmental point of view this phenomenon becomes important when the acute toxicity of the agent is low and the physiological effects go unnoticed until the chronic effects become evident. Due t o the insidious nature of the
1 Ac-Organics Product DeDartment. The Dow Chemical Co.. Midlind, Mich. 48640 2 Waste Control Laboratory, The Dow Chemical Co., Midland, Mich. 48640 3 Computations Research Laboratory, The Dow Chemical Co., Midland. Mich. 48640
bioconcentration effect, by the time chronic effects are noted, corrective action such as terminating the addition of the chemical t o the ecosystem, may not take hold soon enough t o alleviate the situation before irreparable damage has been done. For this reason prior knowledge of the bioconcentration potential of new or existing chemicals is desired. T h e importance of bioconcentration is also recognized by the Environmental Protection Agency (EPA) . For example, the ability of a material t o build u p in the environment has become one of the proposed criteria that this regulatory agency is using in establishing toxic pollutant effluent standards (5). In spite of t h e complexity of the reactions involved in the biomagnification process, we felt it important t o see if a simple relationship could be established between the physicochemical properties of a chemical and its ability to bioconcentrate. It was our belief t h a t the partition coefficient would be the most logical parameter to examine in this connection. If a simple relationship could be established it would be of great benefit in planning the future direction of any development work on a new chemical and in directing research efforts to determine the ultimate fate and distribution of others. Materials a n d Methods Chemicals. T h e following chemicals, representing a wide range of partition coefficients, were evaluated: 1,1,2,2-tetrachloroethylene, hexachlorobenzene, 2,2’,4,4‘tetrachlorobiphenyl, 2-biphenylyl phenyl ether, diphenyl ether, carbon tetrachloride, and p-dichlorobenzene. All materials were examined for purity by means of gas chromatography and found t o be >99% pure. Bioconcentration Factor in Fish. The method described by Branson et al. (6) was used to determine the bioconcentration factor in rainbow trout (Salmo gairdneri Richardson). This method is based on determining the ratio of the concentration of the chemical in trout muscle Volume8. Number 13, December 1974
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to the exposure water under steady state conditions. The trout were 4-5 in. in length and weighed 8-10 grams. They were fed Purina 22 Trout Chow three times each day a t a rate to ensure vigorous feeding. A photo period of 16 hr of daylight was maintained in the laboratory. Lake Huron water was dechlorinated by passage through activated carbon and cooled by refrigeration to 53-55°C. The analysis of the water before filtration was made according to standard criteria (7) and is shown in Table I. Essentially there are three parts to the procedure; First, the concentration of chemical in the fish muscle is determined at various time periods during the uptake portion of the experiment. This is done by random sampling and sacrificing the fish from the bath. Second, concentrations are determined in a n analogous fashion during the clearance phase when the fish are placed in fresh water. Third, the kinetic rate constants k l and k z describing the rate of uptake and clearance of chemical from the fish are estimated from the concentration time data via a nonlinear parameter estimation procedure (8). The ratio of these two estimates provides a n estimate of the bioconcentration factor a t steady state. The test equipment consisted of five aquaria, a constant temperature water bath and two proportional dilutors. Each dilutor as described by Mount and Brungs (9) was constructed for delivery of two chemical concentrations each a n order of magnitude apart. Partition Coefficient. The partition coefficient of the chemical between n-octanol and water was either taken from the tabulation of Leo et al. ( I O ) or calculated using the additivity principles as described by Hansch et al. ( 1 1 ) and illustrated below. The solvent system of n-octano1 and water was used primarily because of the large accumulated data base available. A partition coefficient between n-octanol and water may be calculated using Equation 1. log Px = S substituent constants log P h (1)
+
where log Px = the log of the partition coefficient to base 10 of the chemical in question log Ph = the log of the partition coefficient to base 10 of the parent structure Substituent constants were obtained from the listing of Leo (10) and represent the contribution of each group to the parent structures t h a t give
Px While the comparison between the calculated and experimental values have been shown by Hansch et al. (11) to be good, it should be remembered t h a t the calculation is still an estimate. The various substituent constants used in this paper are shown in Table 11. Examples of the comparison between experimental and calculated values may be found in the many references of Hansch (only two have been cited in this paper).
Results The results of measuring the uptake and clearance rates of the various chemicals are shown in Table 111. The values of the bioconcentration factor (log k l / k z ) are shown in Table IV. The 95% confidence intervals for these factors were calculated by a Bayesian estimation procedure. The logarithm of the partition coefficients are also given in Table IV. The values indicated were obtained in the following manner: 1. Tetrachloroethylene. An experimental value of 2.29 was obtained experimentally for trichloroethylene (10). These authors also indicated that a chlorine attached to a double bond is somewhere between an aliphatic and an aromatic chlorine. Consequently a value of 0.55 was added to 2.29. 1114
Environmental Science & Technology
Table I. Chemical Composition of Lake Huron Water Used in Bioconcentration Studies Property
Value
PH Total dissolved solids Chloride Calci u m Magnesium Phosphate Organic nitrogen Ammonia nitrogen
8 150 p p m 16 P P ~ 27 PPm 7 PPm 30 PPb Nil 0.1 ppm
Table II. List of Substituent Constants Used for Calculating Partition Coefficients (10) for Complete Listing Substituent constant
Group
Aromatic 0.7
CI on benzene CI on phenol
0.73 1.04 0.98 2.13
ortho meta para
Benzene Phenol
1.46 Aliphatic
Chlorine
0.39
Table 111. Results of Measuring Uptake and Clearance of Various Chemicals in Trout Muscle. Chemical in exposure water
1,1,2,2-Tetrachloroethylene Carbon tetrachloride p-Dichlorobenzene Diphenyl oxide Diphenyl 2- Bi p he nylyl phenylether Hexachlorobenzene 2,2',4,4'-Tetrachlorodiphenyl oxide
Uptake rate, kl, hr-1
Clearance rate, k2, h r -I
6ioco.ncentratlon, kifkz
3.3232~0.45
0 . 0 8 2 3 4 ~0.030
3 9 . 6 3 ~5.5
4,054Z 0.83
0.2294Z 0.025
17.71: 2.4
5.670 4Z 0.425 5 . 4 9 9 i 0.722 6,794Z 0.52
0.0264 k 0.00157 0 . 0 2 8 0 i 0.0042 0.01552C 0.0012
215 i 21 1 9 6 k 39 438; 48
8 . 0 6 d ~0.715
0.0146C 0.0025
5524Z 107
1 8 . 7 6 i 0.78
0.002384Z 0.0004
7880 i 350
12.24Z 0.05
0.00099 c 0.0002
12400 4Z 2290
These are t h e c o m b i n e d results of t w o separate experiments o n each chemical a t t w o different exposure levels. a
Table IV. Bioconcentration Factor in Trout and Partition Coefficients of Chemicals Studied Chemical
Log partition coeff
1,1,2,2-Tetrachloro. 2.88 ethylene Carbdn tetrachloride 2.64 p-Dichlorobenzene 3.38 4.20 Diphenyl oxide 4.09 Diphenyl 2-Biphenyl phenyl ether 5.55 Hexachlorobenzene 6.18 2,2',4,4'-Tetrachloro7.62 diphenyl oxide .~ " Figures in parenthesis are nonsymmetrical 95%
Log bioconcn factor
1.59 ( 1 . 4 - 1 . 7 4 ) O 1.24 (1.16-1.30) 2.33 (2.32-2.39) 2.29 (2.23-2.34) 2.64 (2.59-2.68) 2.74 (2.64-2.81) 3.89 (3.80-4.07) 4.09 (4.00-4.16) confidence limits.
2. The values for carbon tetrachloride, p-dichlorobenzene, diphenyl, and diphenyl oxide were obtained experimentally (10). 3. 2-Biphenylyl phenyl ether. T o diphenyl was added a value of 1.46 for phenol giving a value of 5.55. 4. Hexachlorobenzene. Four chlorines (2.8) were added top-dichlorobenzene (3.38) to give 6.18 for this material. 5 . 2,2’,4,4’-Tetrachloro diphenyl ether. Values of 2 x (0.73 and 0.98) was added to diphenyl oxide to give a final result of 7.62. The straight line of best fit was drawn through the points of partition coefficient and bioconcentration factor and is shown in Figure 1 with the equation for the line given in Equation 2. log (bioconcn. factor) = 0.542 log (partition coeff)
+ 0.124
95%
Confidence Reglo”
0
4
(2)
A multiple correlation coefficient of 0.948 and a standard error of 0.342 were obtained from the regression. An F test indicated a confidence level of 0.999. The 95% confidence region for the line is also shown in Figure 1.
I 5
i
3
I
I
6
7
I 8
I 9
Log P a r t j t l o l i Coefficient
Figure 1. Linear regression between logarithms of partition coefficient and bioconcentration of various chemicals in trout muscle Numbers refer to chemicals listed in Table I V
~~
Discussion As can be seen from Figure 1 and the resulting statistics, a straight line can be used to represent the relationship between partition coefficient and bioconcentration factor. The 95% confidence limits on the values of bioconcentration factor predicted by the straight line are larger for values of the partition coefficient further removed from the mean value of the partition coefficient used to construct the styaight line of best fit. Consequently, less confidence must be placed on any predictions from partition coefficients that fall outside the range of the data in Figure 1. With this in mind, it is instructive to see how good Equation 2 is for predicting the bioconcentration factor in fish. The values presented in Table V were taken from unpublished work a t The Dow Chemical Co. as well as from the literature (12). In both cases the experimental procedure was slightly different and the fish species was mosquito fish ( G a m b u s i a affinia). I t is rather striking, in view of these differences that such a close agreement between the experimental and calculated bioconcentration factor was observed. In interpreting the bioconcentration factor for chloropyrifos or any chemical it must be remembered that metabolism for the agent may be a very active process (23). Consequently, the total amount of material in the ecosystem is constantly being reduced while the ratio between the fish and the environment will remain relatively fixed. The large standard deviation associated with the calculated value for pyridinol in Table V illustrates the dangers of departing too far from the mean of the regression line. Since the experimental bioconcentration factor for DDT was 5.23 ( 1 4 ) and outside the region of the present regression, no attempt was made to make a prediction based on partition coefficient. As more data is generated it will be important to modify the least squares parameter estimates of the straight line and recalculate the confidence regions. However, the present study does allow an investigator to begin rating
Table V. Use of Regression Equation 2 for Predicting Bioconcentration Factor Log (bioconcn factor)
Log
Chemical
(partition coeff)
Calcd
Exptl
Endrin C h lorpyrif osa 3,5,6*Trichloropyrid ino1
5.60 4.82d
3.47 + 0.989 2.87 = 0.963b
3.17 2.67
1.350 0.88 = 1.13g6 0.49 Calculated. *Standard deviation calculated from Draper a n d Smith ( 8 ) . 0 0-diethyl 0-(3,5,6-trichloro-2-pyridyl)phosphorothioate. Ex p eri m e nta I .’
the potential of new materials to concentrate. By the matching of this potential with the intended end use, an early judgment decision can be made as to the possible long-term environmental problems that may be faced.
Literature Cited (1) Kenaga, E. E., Residue Rev., 44,73 (1972). (2) Burnett. R.. Science. 174.606 (1971). ( 3 ) N a t u r e , 240,219 (1972). (4) Gustafson, C. G.. Enuiron Sci Technol , 4.814 (1970). (5) Quarks, J., Fed. Regist., 38,24342 (1973). (6) Branson, D. R., Blau, G. E., Alexander, H. C., Thielen, D. R., Neelv, W. B.. W a t e r Pollut. Contr. A s s . , in Dress (1974). (7) “Standard Methods,” 13th ed., p 33, American Public Health Assoc., New York, N.Y., 1971. (8)Draper, N . R., Smith, H.. ‘‘Amlied Regression Analvsis.” John Wiley & Sons, Inc., New York; N.Y., 1966. (9) Mount, D. I., Brungs, W. A., WaterRes., 1,21 (1967). (10) Leo, A., Hansch, C., Elkins, D., Chem. Rev., 71,525 (1971). (11) Hansch, C., Leo, A., Nikaitani, D., J. Org. Chem., 37, 3090 (1972). (12) Ferguson, D. E., Ludke, J . L., Murphy, G . G., T r a n s . A m e r . Fisheries Soc., 95, 335 (1966). (13) Smith, G. N., Watson. B. S., Fisher, F. S., J . Econ. E n t o mol., 59, 1464 (1966). (14) Hamelink, J . L., Waybrant, R. C., Ball, R. C., T r a n s . A m e r . Fisheries Soc., 100, 207 (1971).
Received f o r review J a n u a r y 17, 1974. Accepted A u g u s t 19, 1974
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