(5) “Atomic Absorption Methods Manual”, Vol. 1, Instrumentation
Laboratory, Inc., 1977. (6) Perry, J. H., Ed., “Chemical Engineers’ Handbook”, 4th ed., McGraw-Hill, New York, 1963. (7) Peterson, A. P. G., Gross, E. E., Jr., Eds., “Handbook of Noise Measurement”, 7th ed., Gen Rad, Inc., Concord, Mass., 1974, p 4. ( 8 ) Noller, B. N., Bloom, H., Atmos. Enuiron., 9,505-11 (1975).
(9) Rolfe, G. L., Reinbold, K. A., Eds., “Environmental Contamination by Lead and Other Heavy Metals”, Institute for Environmental Studies, University of Illinois, 1977.
Received for reoiew March 5, 1979. Accepted August 15, 1979. Funding f o r this work was a part of U.S. Environmental Protection Agency Grant No. R804268-01-1.
Accumulation, Elimination, and Metabolism of Dichlorobenzidine in the Bluegill Sunfish Henry T. Appleion and Harish C. Sikka’ Life Sciences Division, Syracuse Research Corporation, Syracuse, N.Y. 13210
The bioconcentration, elimination, and metabolism of 3,3’-dichlorobenzidine (DCB), a suspected human carcinogen, were investigated in bluegill sunfish. [14C]DCBwas rapidly accumulated by the fish from water containing 5 ppb or 0.1 ppm of the chemical. Based on total 14C residues, bioconcentration factors of 495 to 507 were observed in the whole fish with equilibria achieved in 96 to 168 h. The 14Cresidues were distributed in both the edible and nonedible portions. [14C]DCBor its metabolites were not completely eliminated upon transfer of the fish to water free of dichlorobenzidine. The only metabolite detected in the fish was an acid-labile conjugate of DCB, which appears to be an N-glucuronide. The ability of DCB to concentrate in aquatic organisms may present a direct hazard t o human health through consumption of contaminated fish. 3,3’-Dichlorobenzidine (3,3’-dichloro-4,4’-diaminobiphenyl), hereafter referred to as DCB, is widely used as an intermediate in the manufacture of azo pigments. It is of considerable commercial importance; total DCB production in the United States in 1972 was about 4.6 million pounds (1). With current work practices, effluents containing this chemical may be discharged directly into receiving waters. Moreover, the discharge of DCB-pigment wastes into receiving waters constitutes an additional source of DCB contamination in the environment, since free, unreacted DCB is reported to be present in these pigments. DCB is a potent carcinogen in animal species (21, and is regarded by the Occupational Health and Safety Administration (OSHA) as being carcinogenic to man ( 3 ) .Because of the potential hazard presented by DCB, the discharge of DCB into the aquatic environment is of great concern to human health. Human exposure to DCB may occur not only through contamination of drinking water supplies, but also through consumption of fish taken from contaminated waters. The ability of a wide variety of fish species to accumulate high concentrations of certain environmental pollutants by direct uptake from water (bioconcentration) or through food chains (biomagnification) has been well documented and often results in concentrations of the chemical in the fish that are many times greater than found in the aquatic environment ( 4 ) .In several instances, this phenomenon has resulted in the rendering of fish as unsafe for human consumption. DCB possesses a high degree of lipophilicity and a low solubility in water (3.99 ppm at pH 6.9), two properties that are often associated with bioconcentration of chemicals in fish. Because of the potential carcinogenic hazard posed by DCB, the bioconcentration, elimination, and metabolism of DCB 50
Environmental Science & Technology
by the bluegill sunfish were studied as part of our investigations on the environmental fate of DCB. Materials and Methods
Uniformly ring-labeled 3,3’-[14C]dichlorobenzidinedihydrochloride, with a specific activity of 5 mCi/mmol, was obtained from California Bionuclear Corp., Sun Valley, Calif. Radiochemical purity was judged to be greater than 99% by thin-layer chromatography on silica gel 60 (Merck) plates in solvent systems of diethyl ether-hexane (3:1, v/v) and benzene-ethyl acetate-acetic acid (7:3:0.1, v/v). Only one radioactive peak, which cochromatographed with authentic nonlabeled DCB, was detected upon scanning the developed plates with a Nuclear-Chicago Actigraph 111. Nonradioactive DCR-BHCI,provided by Fine Chemicals Division of UpJohn Co., North Haven, Conn., was found to be free of detectable impurities by thin-layer chromatography (TLC) and highpressure liquid chromatography (LC), NMR, and combined gas chromatography-mass spectrometry. All DCB stock solutions were stored refrigerated and protected from light, since DCB was found to be highly photolabile (5). Unless specifically stated, the dihydrochloride of DCB was used in this investigation, and calculations of DCB equivalents were based on the salt form. All other chemicals used were of reagent grade. Radiometric determinations were made with a Model 3255 Packard Tri-Carb liquid scintillation spectrometer, using a phosphor solution containing 5.5 g of PPO (3,5-diphenyloxazole), 0.1 g of POPOP (1,4-bis[2-(5-phenyloxazolyl)]-2benzene), 667 mL of toluene, and 333 mL of Triton X-100. Up to 1 mL of aqueous samples was mixed with 10 mL of the phosphor. Strongly acidic samples were neutralized prior to counting, to prevent quenching. Analysis of DCB solutions by LC was performed with a Waters Associates (Milford, Mass.) liquid chromatograph (Model M6000A) equipped with a UV detector (Schoeffel Instrument Corp., Westwood, N.J., Model GM770). A 4 mm (id.) X 30 cm column packed with p-Bondapak C18 reversed phase medium (Waters Associates) was utilized. DCB was detected by its absorption at 282 nm. The solvent system utilized consisted of acetonitrile-5% glacial acetic acid in water (70:30, v/v). Retention volumes for benzidine and dichlorobenzidine were 6.8 and 3.3 mL, respectively. Five nanograms of DCB was quantifiable in this system a t maximum sensitivity. Bluegills (Lepomis macrochirum Raf.) (2-3 in.) were obtained from the National Fish Hatchery, Orangeburg, S.C., and from a commercial hatchery. The fish were acclimated to laboratory conditions for 30 days under a 14-h light/lO-h dark 0013-936X/80/0914-50$01 .OO/O @ 1980 American Chemical Society
Table 1. 14C Distribution in Bluegills Exposed to 5 ppb of [14C]DCB*2HCI
h
DCB concn In exposure water, ppm
edlble flesh
24 48 72 96 120
0.0038 0.0027 0.0056 0.0034 0.0052
0.062 f O.OIOb 0.394 f 0.036 0.493 f 0.038 0.356 f 0.007 0.587 f 0.015
exposure
time,
ppm of DCB equivalent a head and vlscera
3.796 f 0.027 3.246 f 0.362 3.150 f 0.546 3.285 f 0.001 3.719 f 0.465
whole flsh
1.902 f 0.049 1.815 f 0.086 1.902 f 0.200 1.867 f 0.327 2.180 f 0.090
a The values are the average of duplicate samples per exposure interval, with three fish per sample. The mean water concentration of [14C]DCB.2HCI in the study was 4.1 ppb. f values are standard deviations.
Table II. I4C Distribution in Bluegills Exposed to 0.1 ppm of [14C]DCB*2HCI
h
DCB concn In exposure water, ppm
edlble flesh
24 48 72 96 120 168
0.093 0.105 0.094 0.105 0.1 12 0.105
2.390 f 0.0526 5.333 f 0.586 6.944 f 0.696 17.726 f 0.616 16.142 f 1.886 18.360 f 0.622
exposure
tlme,
ppm of DCB equlvalent a head and vlscera
28.771 f 4.198 54.388 f 7.780 47.737 f 3.097 82.830 f 7.430 81.235 f 3.352 85.776 f 7.413
whole flsh
14.822 f 1.097 29.536 f 1.686 29.154 f 0.640 48.174 f 2.074 56.438 f 0.987 50.978 f 2.198
a The values are the average of two samples per exposure interval, with three fish per sample. The mean water concentration of ['4C]DCB.2HCI in the study was 0.102 pprn. f values are standard deviations.
cycle and fed a pelleted diet. Both dynamic flow and static exposures were conducted at 21 "C in aquaria protected from light, to prevent photodegradation of DCB. The dynamic flow exposure conditions of the study were created by delivering uncontaminated dilution water to a mixing chamber into which a syringe pump delivered a concentrated methanol stock solution of [14C]DCB.The diluted [14C]DCBexposure solution was conveyed from the mixing chamber to the exposure aquarium by gravity flow. The exposure tank consisted of a 15-L glass aquarium fitted with a standpipe drain. The tank was mildly aerated to ensure proper circulation of incoming exposure solution. The concentrations of [14C]DCB used in the dynamic studies, nominally 5 ppb and 0.1 ppm, were maintained by adjusting the concentration of the stock solution and the rate of flow of the syringe pump. In no case did the concentration of methanol in the exposure solution exceed 0.02%. The flow through the exposure tanks was approximately 8 tank volumes per 24 h. The bioconcentration experiments were initiated by allowing the DCB delivery system to operate for 48 h prior to addition of fish to ensure maintenance of a constant chemical concentration. At that time, 70 bluegills (ca. 1 in. in length) were introduced to the system. The DCB concentration in the exposure water was measured routinely by 14C assay, since spot checks of the specific activities of both stock solutions and the exposure medium by LC analysis showed that concentrations of DCB determined by 14Canalyses deviated from LC results by 2% or less. Experiments for measuring the potential metabolism of DCB by fish were also done under static conditions, with loading ratios of 2-3 g/L. In static experiments which exceeded 24 h, the fish were placed in fresh water containing DCB a t each 24-h interval. The renewal of DCB solution minimized the potential for microbial metabolism of DCB in the water bathing the fish, and also prevented deterioration of water quality that might contribute to fish mortality. The exposed fish were removed from the treated water for analysis at appropriate intervals, rinsed with clean water, and sacrificed. The content and distribution of [14C]DCB and
derived materials in exposed fish were measured in the following manner. The fish were dissected into two portions prior to analysis: the head plus viscera fraction (consisting of the head, gills, and internal organs) and the edible flesh fraction (flesh, bones, scales, and skin). After weighing, the tissue fractions were homogenized with methanol ( 5 mL/g fresh weight) in a blender. The slurry was centrifuged and the supernatant was decanted. The residue was reextracted with methanol. After centrifugation, the two extracts were combined, and the amount of 14C in the pooled extract was determined by liquid scintillation counting. The amount of 14C in the tissue residue was determined by solubilization in NCS tissue solubilizer (Amersham Searle Corp.) as described by Sikka et al. (6).The radioactivity in the methanol extract and in the tissue residue was combined to calculate the 14Cconcentration in the fish. The whole fish 14C residues were calculated from the combined values of the edible flesh and head plus viscera fractions. To study the metabolism of DCB by the fish, the methanol extracts of the tissues were analyzed by partition fractionation, TLC, and LC. [14C]DCB and its metabolite(s) in the methanol extracts were fractionated by evaporating the extract to near dryness, dissolving the residues in water, and partitioning the aqueous solution with diethyl ether at various pH values. The amount of radioactivity in each fraction was determined by liquid scintillation counting. The ether extracts were concentrated and analyzed by LC as well as chromatography on thin-layer silica gel plates in the following solvent systems: (i) ether-hexane (3:l) and (ii) benzene-ethyl acetate-acetic acid (7:3:0.1). The chromatograms were scanned for detection of radioactivity in a Nuclear-Chicago Actigraph. The nature of the 14Cremaining in the water bathing the fish during static metabolism studies was determined in the same fashion. Results Bioconcentration of [14C]DCB and Its Metabolites. The results of the uptake studies are given in Tables I and 11. From these results, it is evident that DCB is rapidly and significantly Volume 14, Number 1, January 1980
51
bioconcentrated by bluegills. The apparent equilibrium bioconcentration factors (ppm of DCB in fish/ppm of DCB in water) achieved in the 5-ppb DCB.2HCl (nominal) study were 114,856,and 495 for edible, head and viscera, and whole fish, respectively, based on total 14C residues during the 96- to 120-h period and an average measured water concentration of 4.1 ppb. In comparison, total 14C residue bioconcentration factors for the 0.1-ppm DCB-2HC1 (nominal and measured) study were 170, 814, and 507 for the edible flesh, head and viscera, and whole fish, respectively between 96 and 168 h. The higher concentration of 14C residues in the head and viscera fraction may be due to a higher concentration of nonpolar materials such as lipid in that fraction, compared to the edible flesh. Elimination of [14C]DCB and Its Metabolites. Some of the fish exposed to 0.1 ppm and 5.0 ppb of DCB.2HC1 were transferred to water free of the chemical after equilibration to determine the rate of 14C-residueelimination (depuration). The fish were placed in fresh water, flowing a t a rate to give 12 complete turnovers of water per 24-h period. The fish were periodically sampled and analyzed for 14Ccontent. The depuration of DCB and derived materials is shown in Tables I11 and IV. Although the rate of elimination is rapid initially, DCB levels in the fish appear to be relatively constant in samples taken in the later phase of depuration, particularly in the 0.1-ppm study. Other lipid-soluble chemicals such as lindane and methoprene are bioconcentrated to a large degree but are progressively eliminated after removal to noncontaminated water (7,8). In this investigation, however, a rapid initial rate of elimination was followed by a low or negligible rate, with appreciable residues remaining even after 14 days of exposure to fresh water. This suggests the possibility that an enterohepatic circulation of DCB and metabolite was established or that a portion of the residues is tightly bound to lipoproteins or other substances that are resistant to elimination. Metabolism of Dichlqrobenzidine. Preliminary evidence of metabolism of [14C]DCBby fish was obtained by TLC of the methanol extracts of the edible flesh and the head and viscera fractions of the fish exposed to 0.5 or 2.0 ppm of DCB.2HCl in static systems for up to 120 h. Chromatography in two different solvent systems showed the presence of a t least two 14C materials. One radioactive spot cochromatographed with authentic DCB (Rfof 0.37 in ether-hexane, 3:1, and 0.60 in benzene-ethyl acetate-acetic acid, 7:3:0.1), while the other remained a t the origin, indicating that this material was more polar than DCB. In contrast, only DCB was detected in exposure water by TLC and LC. To characterize further the DCB metabolites in the methanol extracts, the extracts were evaporated to dryness, the residue dissolved in water, and [14C]DCB and its metabolite(s) were fractionated by extracting the aqueous solution with ether at different pH values. In the first step of the fractionation, the pH of the aqueous solution containing the extracted 14Cresidues was adjusted to approximately 11before partitioning with ether. The ether extract was designated as fraction A. The 14Cmaterial which did not partition into ether, constituting a considerable portion of the original 14C, was designated as fraction B. The water-soluble fraction B material also did not partition into ether when the aqueous solutions were acidified to pH 1with HC1. However, when the acidified aqueous solution was further adjusted to pH 11and extracted with ether, 90 to 94% of the 14Cin fraction B was recovered in the ether phase (fraction C). The amount of 14C remaining in water after these treatments was not sufficient for characterization, but may represent another metabolite in addition to the fraction B material. Thin-layer chromatography of fraction C showed that all of the 14Cin this fraction was present as a single compound 52
Environmental Science & Technology
Table 111. Elimination of I4C from Bluegills Exposed to 5 ppb of [14C]DCB.2HCI % of initial ' 4 remaining ~ a
depuration time, h
edible flesh
head and vlscera
whole fish
0 24 72 168 336
100.0 74.2 43.1 30.3 5.2
100.0 86.5 14.9 5.3 7.2
100.0 80.7 16.7 18.7 6.9
Initial 14C residues at time of transfer to DCB-free water were 0.58. 3.72, and 2.18ppm for edible flesh, head and viscera, and whole fish, respectively. Depuration data are the average of duplicate samples per time point with three fish per sample.
Table IV. Elimination of 14C from Bluegills Exposed to 0.1 ppm of [14C]DCB.2HCI % of initial 1% remaining a
depuration time, h
edible flesh
head and viscera
0 24 72 144 216 288
100.0 69.2 42.9 29.2 18.3 18.4
100.0 72.1 63.3 30.4 8.0 6.5
whole fish
100.0 75.0 35.4 32.0 10.7 8.9
a Initial 14C residues at time of transfer to DCB-free water were 18.36,85.78, and 50.98 ppm for edible flesh, head and viscera, and whole fish, respectively. Depuration data are the average of duplicate samples per time point with three fish per sample.
that cochromatographed with authentic [14C]DCB in two solvent systems. The 14C material in fractions A and C was further characterized, both qualitatively and quantitatively, by high-pressure liquid chromatography. The fraction A and C samples showed only one peak, which eluted in the same volume as authentic DCB. In addition, the UV absorbance of the eluted material was scanned with the chromatograph UV detector. The UV spectra of the samples were virtually the same as that of authentic DCB, including identical maximum absorbance a t 282 nm. These results indicate that a highly acid-labile conjugate of DCB (contained in fraction B) comprises a major proportion of the DCB residues contained within the fish. The conjugate was hydrolyzed, forming free DCB, in as little as 30 s at pH 1, but was stable a t pH 9 or higher. The partition behavior of the major metabolite in basic aqueous media indicates that the DCB molecule was probably modified by addition of at least one ionizable acidic group. Mono- or bis(Nsulfate) or N-glucuronide conjugates of DCB would be expected to behave in the observed manner. Because limited amounts of free sulfate are available in vertebrate systems, sulfate conjugates rarely constitute a major portion of metabolism of foreign chemicals (9).In contrast, glucuronidation is well documented as an important metabolic pathway in vertebrates, including fish (10).Furthermore, previous studies of aromatic amine metabolism indicate the occurrence of N-glucuronidation (11).These conjugates appear to be highly labile under mildly acidic conditions (12),and may be formed nonenzymatically ( 1 3 ) .To check this possibility, DCB was reacted with glucuronic acid according to the procedure of Boyland et al. (14). A crystalline product was obtained that was stable in pH 10 media, exhibiting a maximum absorbance a t 285 nm (as opposed to 282 nm for DCB under identical conditions). When the pH was adjusted to 2 with HC1, a
maximum absorbance at 248 nm (corresponding to DCB) rapidly appeared. The acid lability of the synthesized product, producing free DCB, was confirmed by both TLC and LC. Hydrolysis of the conjugated material with bovine liver /3-glucuronidase (P-L Biochemicals, Milwaukee, Wis.) was not pursued in detail due to rapid nonenzymatic hydrolysis at the p H optimum of the enzyme (ca. pH 5) and the lack of enzymatic hydrolysis relative to nonenzymic controls, at pH 7 and 9. The tendency of the conjugate to undergo hydrolysis may explain why the conjugate was not detected in exposure water. The results of the separation of the fish extracts from the dynamic flow uptake and elimination studies into fraction A (DCB) and fraction B (metabolite) materials are given in Tables V and VI. Based on the portion of 14Cresidues that are free DCB, the bioconcentration factors in the 5-ppb (nominal) study were 27.8 for the edible portions and 86.2 for the nonedible tissues, respectively. In the 0.1-ppm study, the bioconcentration value for edible flesh was 114.9 and that for nonedible tissue was 159.3. The differences between the two studies probably reflect the concentration dependence of metabolism, as well as distribution and storage of DCB and its metabolite. A greater portion of the 14Cresidues in the fish in the 5-ppb study is present as metabolite, as compared to the 0.1-ppm study. This might be accounted for by limited amounts of conjugating materials (such as glucuronic acid or uridine diphosphate glucuronic acid). The apparent equilibrium between the relative amounts of DCB and metabolite in the edible and nonedible fractions is probably achieved through a rapid stabilization of several factors including rates of DCB uptake and elimination, and conjugate synthesis, hydrolysis, and elimination, as well as various distributional mechanisms.
Discussion Bioconcentration factors in fish of greater than lo4 have been recorded for such chemicals as DDT ( 1 5 ) and Aroclor 1254 (16),under equilibrium conditions. In comparison, the bioconcentration factors for DCB are rather low. However, the presence of even relatively low concentrations of DCB must be regarded as significant in view of the carcinogenic hazard posed by the chemical. Also, in view of the ease of hydrolysis of the DCB conjugate present in the fish, the conjugated residues should be viewed as the toxicological equivalent of DCB, since rapid hydrolysis to free DCB is likely to occur in the acid medium of the digestive tract after consumption. In addition, the bioconcentration of DCB into edible portions of fish may be underestimated by the conditions of this study. Besides the observed concentration dependence of DCB uptake into fish (edible tissue bioconcentration of total 14C residues of 114-fold from water containing 5 ppb of DCBs2HCl and 170 from water containing 0.1 ppm of D C B S ~ H C which ~), probably is related to the degree of metabolism, it is possible that larger, adult fish may accumulate even greater burdens of DCB than the small fish used in this study. Since larger fish have greater amounts of lipid, a favorable partitioning medium, bioconcentration is often proportionally greater in adults and also in species that are richer in lipid content (17, 18).This would enhance the potential health hazard, since the larger fish are more likely to be consumed by humans. In a study of the fate of a related chemical, benzidine, in bluegills (19), equilibrium bioconcentration factors (based on total I4C residue) of 38-44 in edible flesh were reported. Bioconcentration was 12- to 43-fold higher in the nonedible portions. No evidence of toxicity was seen. Elimination of residues was progressive in the depuration phase of the studies, with 60-73% of the residues in the edible portion and 96% of those in the nonedible portions eliminated after 14days exposure to fresh water. The bioconcentration of
~
_
_
_
_
_
~
Table V. Relative Abundance of DCB and Metabolite in Bluegills Exposed to 5 ppb of [‘4C]DCB2HCI exposure time. h
24 48 72 96 120
O. h abundance a _ ~ -. _ edlble flesh head and viscera fractlon A fraction B fraction A fraction B (DCB) (metabolite) (DCB) (metaboilte)
75.3 41.6 26.9 26.6 22.1
24.7 58.4 73.1 73.4 77.9
12.1 10.4 8.8 11.9 8.2
87.9 89.6 91.2 88.1 91.8
34.3 36.7 37.1 62.7
65.7 63.3 62.9 37.3
17.8 38.8 51.6 46.8
82.2 61.1 48.3 53.2
depuratlon lime, h
24 72 168 336
a Values are the average of duplicate analyses of six fish pooled at each time interval. The data were obtamed from the experiment listed in Tables I and ill.
Table VI. Relative Abundance of DCB and Metabolite in Bluegills Exposed to 0.1 pprn of [‘4C]DCB2HCI exposure the, h
24 48 72 96 120 168
% abundance a head and viscera edlble flesh fraction A fraction B fracllon A fraction B (DCB) (metabolite) (DCB) (metabolite)
72.0 66.2 70.2 77.7 58.7 66.1
28.0 33.8 29.8 22.3 41.3 33.9
25.6 22.5 25.6 16.4 20.7 21.6
74.4 77.5 74.4 83.6 79.3 78.4
21.4 18.9 44.3 49.5 14.5
78.6 81.1 55.7 50.5 85.5
16.9 19.7 35.6 38.9 57.3
83.1 80.3 64.4 61.1 42.7
depuration lime, h
24 72 144 216 288
a Values are the average of duplicate analyses of six fish pooled at each time interval. The data were obtained from the experiment listed in Tables II and IV.
chemicals is well correlated to lipophilicity and water solubility ( 4 ) .This situation may apply to the benzidine series as well, as dichlorobenzidine is much less water soluble and more lipophilic than benzidine, therefore increasing the relative level of bioconcentration and decreasing the degree of depuration. No evidence of significant metabolic alteration of the DCB nucleus, such as ring hydroxylation, was found in this study. Hutzinger et al. (20) did not detect hydroxylation in the metabolism of a series of polychlorinated biphenyls by brook trout, although a hydroxylated product was found to be a minor metabolite of tetrachlorobiphenyl in rainbow trout (21). While microsomal enzymes catalyzing hydroxylation and other oxidative modifications of foreign chemicals are known to occur in fish ( I O ) , in vivo importance of these enzymes may be less in fish than in mammals, due to the much smaller relative size of the liver (the major site of these enzymes) and the lower temperatures involved. In addition, a previous study of DCB fate in mammals (22) failed to reveal important metabolism except for a possible acetylated product. This would suggest that DCB is resistant to mechanisms of metabolism Volume 14, Number 1, January 1980
53
other than conjugation. Our results indicate that conjugated materials comprise a major portion of the 14Cresidues present in the fish. The identity of the acid-labile conjugate was not directly determined because of inability to recover the material intact into nonaqueous media. However, in all aspects examined, the metabolite showed properties similar to the synthetic N-glucuronide of DCB and other aromatic amines ( I I ) , including a free acidic functional group, high lability under acidic conditions, and chromatographic behavior. The ease of chemical synthesis of the glucuronide conjugate may indicate that the reaction proceeds nonenzymatically in vivo. At present, virtually no quantitative information is available on the presence of dichlorobenzidine in various components of the aquatic environment. The results of our investigations emphasize the need for such data, since the potential for exposure to DCB through consumption of contaminated fish now appears to exist. Until the degree of presence of DCB in various aquatic environmental samples (such as fish, water, and sediments) is known, the release of DCB-containing effluents into waterways should be a major cause of concern among governmental health agencies.
(6) Sikka, H., Ford, D., Lynch, R. S., J . Agric. Food Chem., 23,849 (1975). (7) Schimmel, S. C., Patrick, J. M., Jr., Forester, J., J. Towicol.Enuiron. Health, 2,169-78 (1976). (8: Quistad, G . B., Schooley, D. A., Staiger, L. E., Bergot, B. J., Sleight, B. H., Macek, K. J., Pestic. Biochem. Physiol., 6, 523-9 (1976). (9) Mandel, H. C., in “Fundamentals of Drug Metabolism and Drug Disposition”, LaDu, B. N., Mandell, H. G., Way, E. L., Eds., The Williams and Wilkins Co., Baltimore, Md., 1971. (10) Chambers, J. E., Yarbrough, J. D., Comp. Biochem. Physiol., 55C, 77-84 (1976). (11) Shuster, L., Annu. Reu. Biochem., 33,5710596 (1964). (12) Axelrod, J., Inscoe, J. K., Tomkins, G. M., J . Biol. Chem., 232, 835-41 (1958). (13) Bridges, J. W., Williams, R. T., Biochem. J., 83,27P (1962). (14) Boyland, E., Manson, D., Orr, S. F. D.,Biochem. J., 65,417-25 (1957). (15) Hansen, D. J., Wilson, A. J., Jr., Pestic. Monit. J., 4, 51-6 (1970). (16) Hansen, D. J., Parrish, P. R., Lowe, J. L., Wilson, A. J., Jr., Wilson, P. D., Bull. Enuiron. Contam. Toxicol., 6,113-9 (1971). (17) Reinert, R. E., Pestic. Monit. J., 3,233 (1970). (18) Buhler, D. R., Shanks, W. E., J . Fish. Res. Board Can., 27,347 (1970). (19) E G & G, Bionomics, Research Report, Aquatic Toxicology Laboratory, Wareham, Mass., 1975. (20) Hutzinger, O., Nash, D. M., Safe, S., DeFreitas, A. S. W., Nortstrom, R. J., Wildish, D. J., Zitko, V., Science, 178,312-3 (1972). (21) Melancon, M. J., Jr., Lech, J. J., Bull. Enuiron. Contarn. Toxicol., 15,181 (1976). (22) Kellner, H. M., Christ, 0. E., Lotzch, K., Arch. Toxicol., 31,61 (1973).
Literature Cites (1) U.S.Tariff Commission, T. C. Publication 681, U S . Government Printing Office, Washington, D.C., 1974. (2) Pliss, G. B., Acta Unio Znt. Cancrum, 19,499 (1963). (3) Fed. Regist., 38 (85) (1973). (4) Hamelink, J. L., Spacie, A,, Annu. Reu. Pharmacol. Towicol., 17, 167 (1977). (5) Banerjee, S., Sikka, H. C., Gray, R., Kelly, C. M., Enuiron. Sci. Technol., 12, 1425 (1978).
Receiuedfor review May 16,1978. Accepted September 4,1979. This work was supported by Grant No. R 804-584-010from the U.S.Environmental Protection Agency.
Changes in Lead, Zinc, Copper, Dry Weight, and Organic Matter Content of the Forest Floor of White Pine Stands in Central Massachusetts over 16 Years Thomas G. Siccama” and William H. Smith School of Forestry and Environmental Studies, Yale University, New Haven, Conn. 0651 1
Donald L. Mader Department of Forestry and Wildlife Management, University of Massachusetts, Amherst, Mass. 01003
The forest floor has been shown to be an important site of accumulation for a variety of trace elements introduced into forest ecosystems via the atmosphere ( 1 4 ) .Numerous investigations have stressed the significance of the forest floor as a sink for lead (5,6). Our studies of the northern hardwood forest in remote central New Hampshire indicate that lead from the atmosphere is accumulating in the forest floor at a rate of more than 300 g ha-l yeard1 (7,8). Detection of trends in rates of accumulation or loss of trace metals in soils over long periods of time is generally impossible due to the paucity of quantitatively obtained and systematically retained soil samples collected from the same site a t different times. Reported changes in lead in mineral soils have been limited to measurements of changes in concentration (9, IO). To the best of our knowledge, there have been no reports documenting changes in amount as well as concentration of trace elements in the forest floor over extended periods in areas not associated with major local sources of metals such as smelters or highways. A search for preserved soil samples from New England forests revealed a unique opportunity to analyze quantitatively sampled and carefully preserved forest floor material
Lead, zinc, copper, dry weight, and organic matter content of the L, F, and H layers of the forest floor were measured in 10 white pine stands in central Massachusetts in 1962 and again in 1978, thus providing quantitative estimates of these parameters at two points in time 16 years apart.-Forestfloor material retained from a 1962 quantitative study of forest floor weight was compared with samples from the same plots in the same stands collected in 1978. Total lead content increased significantly. Average lead concentration increased in all layers, but the increases were not sta$istically significant primarily due to the dilution effect of the conc,urrent increase in the mass of the forest floor. The observed net increase in lead of 30 mg m-2 year-l is approximately 80% of the estimated total annual input of this element via precipitation in this region during the 16-yearperiod. There were no statistically significant changes in total zinc and copper content of the forest floor. Since the forest floor total dry weight increased significantly (42%), zinc and copper concentfation decreased significantly. The results of this study emphasize the importance of determining both concentration and amount of trace elements in soil studies. 54
Environmental Science & Technology
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0013/936X/80/09
14-54$01.00/0@ 1980 American Chemical Society
