Environ. Sci. Technol. 1984, 18, 705-713
from other tests, they were consistent over the range of conditions tested in this study. Acknowledgments The efforts of Todd Snyder, Wallace Kistler, Bob Hammond, Steve Hall, and Terry Hammond in executing the test program are gratefully acknowledged. The guidance and direction of EPA Project Officer Leslie Sparks are appreciated. Literature Cited (1) Bush, P. V.; Pontius, D. H. In ”Third Symposium on the Transfer and Utilization of Particulate Control Technology: Control of Emissions from Coal Fired Boilers”; U.S. Environmental Protection Agency: Washington, DC, July 1982; Vol. I, EFA-600/9-82-005a.
(2) Bush, P. V.; Pontius, D. H., presented at the Fourth Symposium on the Transfer and Utilization of Particulate Control Technology, Houston, TX, Oct 1982. (3) Mosley, R. B.; Anderson, M. H.; McDonald, J. R. “A Mathematical Model of Electrostatic Precipitation”. Feb 1980, EPA-600/7-80-034 (NTIS PB80-190994) (Revision 2). (4) Gooch, J. P.;Marchant, G. H., Jr. Palo Alto, CA, Aug 1978, EPRI Report RP-792, Vol. 3. ( 5 ) Fed. Reg. 1976, 41, 187, 42020. (6) Johnson, J. W.; Clinard, G. I.; Felix, L. G.; McCain, J. D. “A Computer-Based Cascade Impactor Data Reduction System”. EPA-600/7-78-042 (NTIS PB-285433). Received for review October 13,1983. Accepted March 29,1984. This work was funded under EPA Cooperative Agreement CR808973-01.
Chemical Pollutants in Sediments and Diseases of Bottom-Dwelling Fish in Puget Sound, Washington Donald C. Mallns,” Bruce B. McCain, Donald W. Brown, Sin-Lam Chan, Mark S. Myers, John T. Landahl, Patty G. Prohaska, Andrew J. Friedman, Linda D. Rhodes, Douglas 0. Burrows, William D. Groniund, and Harold 0. Hodglns Environmental Conservatlon Division, Northwest and Alaska Fisheries Center, National Marine Fisheries Service, Seattle, Washington 98 112
A 4-year multidisciplinary study was conducted on relationships between pollutants and diseases of fish in Puget Sound, WA. In this study, high concentrations of a large number of anthropogenic chemicals were found in certain Puget Sound sediments. For example, over 900 individual organic compounds were detected in sediment from one urban bay (Commencement Bay), and evidence was obtained for the presence of numerous additional compounds. Many of the chemicals accumulated in bottom-dwellingfish, and high levels of certain toxic chemicals in the urban bays were linked to serious diseases (e.g., liver carcinomas) of English sole (Parophrys vetulus) and other demersal fish species. I t was concluded that application of the approach used would reveal comparable serious pollution-related health problems in marine biota in other urban coastal areas of the world. Introduction Estuaries and other coastal waters receive many of the approximately 70 000 synthetic chemicals in commercial use (I). These chemicals-which enter coastal waters from a number of sources, including spills, dumping operations, urban runoff, and municipal and industrial waste discharges-consist of materials such as petroleum hydrocarbons, polychlorinated biphenyls (PCBs) and other chlorinated compounds, pesticides, and metals. Many of the organic compounds are subjected to chemical and biological transformations in the environment which result in a host of new compounds (2,3),but little is known about the toxicity of most of the chemicals or their transformation products. Numerous studies have shown that sediments are major reservoirs for pollutants. For example, surface sediment from the Hudson-Raritan Estuary near Newtown Creek in New York contained 180 ppm (all concentrations in this paper are on a dry weight basis) of aromatic hydrocarbons (AHs), including 5.6 and 1.3 ppm of the carcinogens benz[a]anthracene and benzo[a]pyrene (4). Higher con-
centrations of these carcinogens, 11000 and 9000 ppm, respectively, were found in surface sediments from the estuary of the Elizabeth River, VA (5). Further, the mean concentration of PCBs in sediments near a Los Angeles (Palos Verdes) sewage outfall was 3.4 ppm (6),and mean concentrations of lead, mercury, and cadmium in Newark Bay sediments varied from 7 to 400 ppm (7). Fish accumulate many of the chemicals from polluted aquatic environments. MacLeod et al. ( 4 ) found PCBs, chlorinated hydrocarbon pesticides, and AHs in fish from the New York Bight area; PCBs ranged from 13 to 54 ppm in the skeletal muscle of striped bass (Morone saxatilis) from the Hudson River. Gossett et al. (8) reported, similarly, that livers from halibut (species not given), Dover sole (Microstomus pacificus), and Pacific sanddab (Citharichthys sordidus) from the Palos Verdes area contained PCBs (3.2-12 ppm), DDE (78-270 ppm), DDT (0.69-3.5 ppm), and hexachlorobenzene (HCB; 0.02-0.1 ppm). Young (9) and Sherwood (10) concluded, however, from studies of the Southern California Bight and the New York Bight, that metal concentrations generally were not elevated in demersal fish from urban compared to nonurban coastal marine environments. A number of pathological conditions have been observed in marine fish from polluted coastal waters and estuaries. Fin erosion was found in a variety of species from the Southern California Bight, the New York Bight, and the Duwamish Waterway in Seattle (11-13). In most cases, the highest prevalences of this disease were in fish from areas with highly contaminated bottom sediments. Liver neoplasms have been reported in fish from areas with highly contaminated sediments: in Atlantic hagfish (Myxine glutinosa) from a fjord in Sweden (14),in Atlantic tomcod (Microgadus tomcod) from the Hudson River estuary (15),and in two species of bottom fish (English sole, Parophrys vetulus, and starry flounder, Platichthys stellatus) from Puget Sound, WA (16,17). Liver neoplasms have also been reported in a freshwater bottomdwelling fish species, the brown bullhead (Ictalurus ne-
Not subject to U.S. Copyright. Published 1984 by the American Chemical Society
Environ. Sci. Technol., Vol. 18, No. 9, 1984
705
Table I. Chemicals Analyzed in Sediment and Biota from Puget Sound aromatic hydrocarbons acenaphthene anthracene benz[a]anthracene benzo[a]pyrene benzo[e]pyrene benzofluoranthene biphenyl chrysene dibenz[a,h]anthracene 2,6-dimethylnaphthalene 3,6-dimethylphenanthrene fluoranthene fluorene indan isopropylbenzene 1-methylnaphthalene 2-methylnaphthalene I-methylphenanthrene naphthalene n-propylbenzene perylene phenanthrene pyrene 1,2,3,4-tetramethylbenzene 2,3,5-trimethylnaphthalene
chlorinated organic compounds aldrin a-chlordane m,p’-DDD o,p’-DDD p,p’-DDD o,p’-DDE p,p’-DDE o,p’-DDT p,p’-DDT dichlorobiphenyls dichlorobutadienes heptachlor heptachlorobiphenyls hexachlorobenzene hexachlorobiphenyls hexachlorobutadiene lindane nonachlorobiphenyls octachlorobiphenyls pentachlorobiphenyls pentachlorobutadienes tetrachlorobiphenyls tetrachlorobutadienes trans-nonachlor trichlorobiphenyls trichlorobutadienes
bulosus) from polluted sites in a number of rivers associated with the Great Lakes (18-20). Etiological relationships between specific chemical pollutants and diseases of fish have been studied, but only to a very limited extent. An understanding of causeand-effect relationships between xenobiotics and fish diseases still remains elusive. In an attempt to increase the understanding of such relationships, and to increase knowledge of the impacta of human population centers and related activities on adjacent marine environments, we conducted a 4-year multidisciplinary study (1979-1982) of pollutants and fish diseases in Puget Sound (Figure 1). Approximately 2 million people live in the Puget Sound area. Although many areas of Puget Sound are still considered to be relatively pristine, the eastern shore is heavily industrialized. Tacoma has a large smelter and several chemical plants, Seattle has a number of heavy industries and manufacturing plants, and Everett and Tacoma have wood products industries. In addition, Commencement Bay (Tacoma) was recently designated by the US. Environmental Protection Agency as one of the top 10 worst hazardous waste sites in the U S . Materials and Methods
Sediments and bottom-dwelling fish [English sole, rock sole (Lepidopsetta bilineata), and Pacific staghorn sculpin (Leptocottusarmatus)]were collected from various urban and nonurban embaymenta and analyzed for AHs, PCBs, chlorinated pesticides, other chlorinated organic compounds, and metals (Table I). The term “CAHs” represents the summed concentrations of individual compounds. Sediment and tissue samples were analyzed for organic compounds by using solvent extraction, column chromatography, and capillary column gas chromatography with mass spectrometry, flame ionization, and electron capture detectors (21, 22). Samples for metal analyses were digested in hot acid and analyzed either by atomic absorption spectrophotometry or by inductively coupled argon plasma emission spectroscopy (23). Individuals of each of the three target fish species were also examined for pathological conditions. The fish were 706
Environ. Sci. Technol., Vol. 18, No. 9, 1984
metals and other elements aluminum antimony arsenic barium beryllium bismuth boron cadmium calcium chromium cobalt copper gallium germanium iron lead lithium magnesium manganese
mercury molybdenum nickel phosphorus potassium scandium selenium silicon silver sodium strontium tin titanium tungsten vanadium yttrium zinc zirconium
measured for length and weight and examined for externally visible abnormalities and tissue samples from major organs (liver, kidney, spleen, gastrointestinal tract, gall bladder, gills, heart, and gonad), and grossly visible lesions were excised and placed in Dietrich’ fixative (24). The tissue samples were processes for paraffin embedding, sectioned at 5 pm, and stained with Mayer’s hematoxylin and eosin-phloxine for light microscopy (25). The urban embayments investigated were Elliott Bay, Commencement Bay, Budd Inlet (Olympia),Sinclair Inlet (Bremerton), and Port Gardner (Everett). The nonurban embayments studied were Case Inlet, Port Madison, and Port Susan, all within Puget Sound, and Discovery Bay, just outside the northern entrance to Puget Sound. The sampling stations in each embayment were grouped into subareas (A-S). Each smaller embayment consisted of a subarea containing two or more sampling stations; however, the two largest embayments (Commencement and Elliott Bays) each had six subareas, with a subarea containing one to six stations (Figure 1). A variety of statistical and mathematical methods were used to analyze the chemistry and fish disease data and evaluate possible relationships between the two types of data. To determine whether the prevalence of each type of disease diagnosed in each of the three fish species was statistically uniform among the subareas, the G test for heterogeneity was performed (26). In cases where the prevalence patterns were significantly heterogeneous, the data sets were submitted to simultaneous comparisons for homogeneity to identify which subareas were statistically indistinguishable, or homogeneous, on the basis of lesion prevalence. Cluster analysis and factor analysis of chemistry data were performed by using the programs of Wishart (27). Spearman’s rank correlation (26) was used to compare prevalences of disease types at the individual sampling stations with the total concentrations of sediment-associated xenobiotics in the groups of chemicals derived by factor analysis. Significance levels for the G test for heterogeneity and Spearman’s rank correlation procedure were adjusted according to the number of statistical tests performed for
taneous comparison analysis because one test for each lesion type (a total of four tests) was performed for each target species. The significance level of p 5 0.0031 was used for all bivariate Spearman's rank correlations, because 16 comparisons were made for each species (i-e.,4 hepatic lesion types X 4 groups of xenobiotics = 16). This method of adjusting for the number of bivariate statistical tests performed, which utilizes a Bonferroni inequality, is simple but highly conservative (30); hence, some of the results with significance levels higher than the adjusted significance value would also be regarded as statistically significant by a less conservative method.
Results
;y
nt
Seattle
7.
L
6.
Flgure I. Map of Pug& Sound showing locatlons of 43 sampling stations. The stations were grouped into subareas identified by the letters A-S. The followlng subareaslstations were in Commencement and Elliott Bays: Cl27; Dl26; El24 and 25; Fl19-23; 0115-18; Hf28; 3/13 and 14; Kll-4 and 1 0 Lf5,6, and 8; M f l l ; Nf7 and 9; and 0112.
each data set. When multiple comparison tests are performed on the same set of data, the probability of finding significant differences increases with the number of tests (28). Therefore, the significance level for individual tests was adjusted by dividing the overall significance level for each analysis (0.05 in the case of our study) by the number of testa performed (29). For example, the significance level of 0.0125 was selected for each heterogeneity and simul-
Chemicals in Sediments and Tissues. A large number of chemicals were present in Puget Sound sediments. For example, in Commencement Bay sediment more than 900 individual organic compounds were detected-more than 500 AHs and hundreds of chlorinated hydrocarbons, as well as various bromine-, sulfur-, nitrogen-, and oxygen-containing compounds. Evidence for many additional compounds was also obtained, but the numbers and identities of these compounds have not been fully determined because of the complexity of the chemical mixtures. Mean concentrations of CAHs (Figure 2) in sediment for four of the major urban embayments (Commencement Bay, Elliott Bay, Sinclair Inlet, and Port Gardner) were as much as 46 times the mean concentration (0.28 ppm) of CAHs in sediments from the nonurban embayments (Case Inlet, Port Madmon, Port Susan, and Discovery Bay); however, within the urban areas, the values varied greatly among individual stations (e.g., 0.15-63 ppm in Elliott Bay). In the Elliott Bay area, the highest concentrations of CAHs were found in sediments from station 5 and station 12 (Figure 1); both stations receive municipal and industrial waste and urban runoff (31). Sediments from station 5 also had the highest mean concentrations of the carcinogens benz[a]anthracene (7.6 ppm) and benzo[a]pyrene (2.4 pprn); the mean concentrations of these compounds from the nonurban areas were 0.022 and 0.014 ppm, respectively. Chlorinated organic compounds-including PCBs, HCB, a variety of chlorinated butadienes (CBDs), and pesticides-were found in almost every sediment sample. The mean concentrations of PCBs, CBDs, and HCB, respectively,were the following: from Elliott Bay, 0.38,0.01, and 0.0002 ppm; Commencement Bay, 0.27, 1.6, and 0.07 ppm; Sinclair Inlet, 0.13, 0.06, and 0.0003 ppm; eastern Port Gardner, 0.08,0.02, and 0.0004 ppm. PCBs, CBDs, and HCB in nonurban sediments averaged 0.005,0.003, and 0.0001 ppm, respectively. Thus, the concentrations of PCBs were usually substantially higher in the major urban embaymenta than in the nonurban embayments and the concentrations of CBDs and HCB were far higher in Commencement Bay than in the other embayments. Hundreds of other halogenated compounds, presently unidentified, were also detected in sediments from Commencement Bay. The concentrations of PCBs, CBDs, and HCB varied markedly within an urban embayment. For example, in Commencement Bay, concentrations of PCBs ranged from
