Document 5vobO9akzjGoq82xeKR2qGkD

E. P. Wheeler toxicity oFjfllHBHgiiifr AND ITS PHYSIOLOGICAL ACTIVITY IN SEVERAL ESTUARINE ORGANISMS D. R. nimmo, D. J. Hansen, j. a. couch, n. r. Cooley, P. R. PARRISH and J. I. LOWE U. S. Environmental Protection Agency Gulf Breeze Environmental Research Laboratory Sabine Island, Gulf Breeze, Florida 32561 (Associate Laboratory of the National Environmental Research Center, Corvallis, Oregon) The occurrence of high concentrations of a PCB (Aroclor 1254) in the Pensacola estuary prompted field and laboratory studies by the Gulf Breeze Environmental Research Laboratory (EPA). Monitoring of the estuary indicates the chemical is present in all components-particularly in sediments and fishes. Residues appear to be diminishing in sediments. Toxicity tests show estuarine species sensitive at ppb concentrations in water, with a ciliate protozoan (Tctrahymena pyriforrnis W), shrimps (Penaeus duorarum, P. aztecus, and Palaemonetes pugio), and a fish (Fundulus sitnilis), affected at or near 1.0 ppb. Tissue concentrations of Aroclor 1254 similar to those found in natural populations of shrimps from the contaminated estuary were successfully duplicated in laboratory experiments. Shrimps also con centrated the PCB from very low concentrations (0.04 ppb) in the water. Three estuarine species demonstrated pathologic changes at tissue and cellular level after chronic exposure to the chemical. Oysters (Crassostrea virgitiica) developed abnormal infiltration of leukocytes in the connective tissue, spot (Leiostomus xanthurus) developed fatty changes in their livers, and shrimp (Penaeus duorarum) developed crystalloids in hepatopancreatic nuclei. Polychlorinated biphenyl (PCB) residues were found in water, sediments and biota of Escambia Bay, Florida, in April 1969 (Duke et al. 1970). Subsequently, investigation Into the effects of this chemical on estuarine organisms has been a major research goal of the Gulf Breeze Environmental Research Laboratory. In this report, we present data on the chemical in water, sediments and biota of Escambia Bay and adjacent areas, review toxicological data and present some physiological-pathological information. Experimental methods and materials used as well as chemical analyses are given elsewhere (Duke et al. 1970, Cooley ctal. 1972, Hansen era/. 1971, Nummo era/. 1971a, Lowe era/. 1972). In thi* paper, Aroclor and PCB are used interchangeably for Aroclor 1254. Aroclor is a registered trademark of the Monsanto Company, St. Louis, Mo. Reference to commercial products does not constitute endorsement by the Environmental Protection Agency. Contribution No. 162, Gulf Breeze Environmental Research Laboratory. Archives of Envrionmentnl Contamination and Toxicology, Vol. 3, No. t, 197J, I97S by Springer-Verlag New York Inc. 22 DSW 033750 STLCOPCB4017712 rY MS Y, I in the Pensacola re Environmental * the chemical is Icsiducs appear to s sensitive at ppb ta pyriformis W), ipo), and a fish ations of Arocior the contaminated shrimps also conthe water. Three titular level after eloped abnormal omus xanihurus) crum) developed ents and biota of tiy, investigation research goal of present data on tnt areas, review >n. Experimental here (Duke et al. a a!. 1972). or* is * registered noducts does not Toxic action of Arocior 1254 in estuarine organisms 23 PCB in Escambia Bay Unlike other studies in which PCBs have been found in the environment, the chemical in Escambia Bay apparently came from a single point-source and has been identified as Arocior 1254. Figure 1 is a comparison of chromatograms of (a) an Arocior 1254 standard, (b) Arocior 1254 in oysters from a 72-week chronic exposure to the chemical in the laboratory, and (c) PCB isolated from oysters taken from Escambia Bay. PCB con centrations in both oyster samples were similar and both samples and standard were analyzed on the same chromatograph. In laboratory studies with Arocior 1254, the oysters were continuously exposed to approximately ten ppt for 72 weeks and the similarity of tissue residues to the PCB found in oysters from Escambia Bay indicates that the chemical in the bay has changed little with time. Monitoring data for the period September 1969 through December 1971 are presented in Table I. Average concentrations in water are given by assuming non-detcctable levels as Fig. I. Chromatograms of an Arocior 1254 standard, Arocior 1254 in oysters from a 72week chronic exposure in the laboratory and PCB in oysters from Escambia Bay. DSW 033751 STLCOPCB4017713 24 D. R. Nimmo et al. zero. Concentrations of Aroclor in unfiltered water samples from Escambia River down stream averaged 0.6 ppb. Aroclor was found in 64% of the water samples from the <ivcr and in 27%of the watersamples from Escambia Bay. Thcaverage concentration in sediment samples from the bay was 2.3 ppm. PCB in 101 samples of invertebrates, predominately mollusks and crustaceans, averaged 0.8 ppm. Fishes from the bay had five times as much PCB in their tissues as did invertebrates. Aroclor in sediment samples from a survey taken in February 1970 were all in the ppm range (Nimmo et al. 1971b). The samples were taken from the upper strata (e. g.. ..-.'pet ten in.) by corer or by dredge. In this survey, tKe maximum residue (61 ppm) observed m the river was found at the outfall from the industry; the maximum in Escambia Bay (30 ppm) was found near the mouth of the river. The amounts of PCB in sediment samples from several locations within the ri' c and bay have decreased in the ensuing months. The number of samples was inadequa (or absolute comparison, but the data indicate a trend. A decrease is especially notices the December 1970 and October 1971 surveys (Fig. 2), in which sediment sa ,-;?s were taken with a corer at three locations in the bay. Generally, residues in the survey were about one-tenth the 1970 values, except one sample taken below the in the surface stream. PCB in the lower strata (4-12 inches) in the 1971 surer w a* non-detectable, except at the outfall of the industry. Later, we examined a core-to 24 inches-taken above the trestle but found no residues. Cores taken in i survey generally indicated less PCB than in 1971. Whole-body residues of Aroclor 1254 found in shrimps from Escambia Bay ar c tiguous waters during 1969/1970 are shown in Figure 3. Each datum represents i . --posite sample of at least five individuals. We show these data to indicate the disper- _ Table I. Concentration ofAroclor 1254 in water, sediment, and biota, September 1969 through December 1971, in Escambia Bay, Florida Type Samples Total No. Positive % Concentration Average (ppm) Range (ppm Water Water Sediment Invertebrates Fishes 67 37 56 101 17 64 0.0006 ND- 0.008c 27 ND ND- 0.0000~ 78 2.33 ND-30. 92 0.81 ND- 6 9 100 3.99 0.29-20. ND Non-detectable: Water, < 0.00003 ppm; sediment or biota, < 0.01 ppm. mmmmm OSW 033752 STLCOPCB4017714 ambia River down* nples from the river itration in sediment lies, predominately i five times as much were all In the ppm strata (e. g., upper I ppm) observed in 1 in Escambia Bay ithin the river and was Inadequate for clally noticeable in sediment samples sidues in the 1971 n below the trestle 1971 survey was xamined a deeper es taken in a 1972 nbia Bay and conrepresents a com ite the dispersal of and biota, Florida ------------------- lion_____________ Range (ppm) SD- 0.0086 VD- 0.00007 SD-30. N'D- 6.9 i:9-:o. 01 ppm. ) I j i 1 I Toxic action of Aroclor 1254 in estuarine organisms 25 the chemical in an estuarine environment from an apparent point source by either biological or physical transport. Although the material was originally localized in the sediments of upper Escambia Bay, shrimps captured in lower Pensacola Bay also contained significant amounts of the PCB. In contrast, shrimps from adjacent bays such as Perdido or Choctawhatchee did not have detectable levels of PCBs. The amounts of Aroclor in biota from the estuary remain relatively high and the latest survey showed amounts generally increasing at higher trophic levels (Fig. 4). In the survey of October 1971, we found no detectable PCB in the sea grasses,Spartina sp. and Zostera marina. The moilusk, Neritina reclivata, contained 0.49 ppm. Of two crustaceans, blue crabs {Callinectes sapidus) had the greater residues (6.9 ppm). Among fishes, one might expect sand seatrout (Cynoscion arenarius) and Atlantic cutlassfish (Trichiurus Upturns) to have the highest residues because these species are predators; instead, the highest residue (10 ppm) was found in silversides (Menidia beryllina), a species whose diet consists mainly of plankton. Above trestle Fig. 2. Comparison of concentrations of Aroclor 1254 in cores taken 10 months apart in upper Escambia Bay and River. Concentrations given in 1971 above and below trestle are averages of two cores each. DSW 033753 STLCOPCB4017715 26 D. R. Nimmo ct at. In Figure 4, PCB residues are compared in the same species or in species occupying similar trophic levels and captured on the same day in Escambia and East Bays. The collecting site in East Bay is about 35 km from the original source of the chemical. PCB residues in species from Escambia Bay were five to ten times greater than those found in East Bay but the data clearly show that the chemical was found in species captured dis tant from the original source of the PCB. Toxicity of Aroclor 1254 to estuarine organisms Laboratory research on the toxicity of PCB to estuarine organisms began immediately after the chemical was discovered in the Bay. Animals from several trophic levels have been tested. . Fig. 3. Residues of Aroclor 1254 in shrimp (whole body) from Escambia Bay and con tiguous waters during 1969/1970. Each datum represents a composite sample of at least five individuals. . DSw 033754 STLCOPCB4017716 in species occupying '-'J and East Bays. The ct of the chemical. PCB ' than those found in in species captured dis- amsms trru began immediately traJ trophic levels have j 1 i tt Eaet Bay obit Bay and con i sample of at least Toxic action of Aroclor 1254 in estuarine organisms 27 Population growth in test-tube cultures of the ciliate protozoan, Tetrahymena pyriformis W, was reduced significantly by exposure to one ppb of Aroclor 1254 (Table II). Reduction was measured as effect on population growth rate and on popula tion density at 96 hr. Growth rate was estimated as the quantity b of the least squares estimate of the line y - a + bx for the exponetial growth phase of the population growth curve. These ciliates accumulated the PCB from the test media during the exposures. Cells contained a maximum of 60 ppb (dry-weight basis) of PCB when grown for seven days in medium that contained one ppb of Aroclor. Because the ciliates can accumulate PCB from a culture medium, they could be a step in the transport of the chemical into aquatic food webs under natural conditions. Bioassays were conducted in flowing water to determine the toxicity of Aroclor 1254 to a mollusk, three crustaceans and three fishes (Table III). Growth in oysters (Crassostrea TTf-" ' * Escambia Bay East Bay (ppm) ND . ND Spartina Zottera ND (ppm) NO 0.49 NO ' \ Oliva Narite Rangia , ND ! ND 0.98 Panaeid Shrimp Trace 6.90 ''j Slue Crabs 3.00 . Bay Anchovy 0.46 0.68 3.80 > Cattish 0.58 10.00 ' Tidewater silversides 0.95 4.50 , Silver perch 0.48 , 1.50 . - ->J Sand seatrout Spotted seatrout 0.12 I'80' * 1.60 A 1.30 2.90 . . Spot Atlantic croaker Hogchoker ^>-Atlantic cutlasitish Trace ____ - -Trace NO . _ 1 >k` n Fig. 4. Comparison of concentrations of Aroclor 1254 found in species collected in Escambia (left side) and East Bays (right side): scagrasses, Spartina sp. and Zosiera marina; Rangia clams, Rangia cuneala: olive nerite, Neritina reclivata\ brown and white shrimp, Penaeus oztecus and P. sctiferus\ blue crabs. Callinectes 5pulus. bay anchovy, Anchoa mitchilii\ sea catfish and gafftopsai! catfish, Arius felts and Bagre marinus: tide water silversides, Mertidia beryllina-, silver perch Bairdiclla chrysura\ sand seatrout, Cynoscion arenarius\ spotted seatrout, Cynoscion nebulosus', spot, l.eiostomus xanthurus: Atlantic croaker, Micropogon undulatus; hogchoker, Trinectes maculatus\ and Atlantic cutlassfish, Trichiums leplurus. DSW 033755 mm ix STLCOPCB4017717 28 D. R. Nimmo et al. virginica), exposed to five ppb for 24 weeks was significantly reduced, but growth in oysters exposed to one ppb for 30 weeks was not. Earlier work showed that hydrocarbon compounds inhibited shell deposition significantly at concentrations of 0.1 to 0.5 ppm in-short-term tests (Butler 1966). In tests lasting about two weeks, various shrimps Table II. Effect of Aroclor 1254 on population growth of Tetrahymena pyriformis IV Toxicant Gig/liter) 0 0.1 1.0 10.0 Mean growth rateb b 0.0212 0.0203 0.0195 0.0199 Difference (%) -4 - 8C -6' Mean population density** (absorbance) 1.044 0.984 0.936 0.954 Difference (%) -6 - 10 - 9d After Cooley et al. (1972). bMeans of 6 replicate experiments. eF(3, 15) = 6.00 (P < 0.01). dF(3, 15)= 23.001 (P< 0.005). Table III. Chronic toxicity ofAroclor 1254 to estuarine animals in flowing water, 1 to 30 weeks * Test animals Pink shrimp Longnose killifish Grass shrimp Brown shrimp Pin fish Spot . Eastern oyster Concentration, ppb (/ig/1) Range Minimum affecting 0.6- 19.0 1.0-100.0 0.2- 12.5 0.1- 1.4 5.0 1.0- 5.0 1.0- 5.0 0.9 1.0 1.3 1.4 5.0 5.0 5.0 Controls did not exceed 25% mortality. Toxicity in oysters was measured by reduced shell growth. DSW 033756 STLCOPCB4017718 MU duced, but growth in wed that hydrocarbon )ns of 0.1 to 0.5 ppm eeks, various shrimps >vth of ion Difference ') (%) -6 - 10* - 9d i inimals 1) .n affecting 3.9 1.0 1.3 1.4 0 <0 ;.o i measured by Toxic action of Aroclor I 254 in estuarine organisms 29 {Penaeus duorarum, Penaeus aztecus, and Palaemoneies pugio) were killed by exposure to 0.9, 1.4, and 4.0 ppb, respectively. In tests lasting two weeks or longer this chemical was lethal tolongnosekillifish(/ri/m/t//u.ssimilis)at 1.0 ppb, and pinfish (Lagadon rhomboides) and spot (Leiostomus xanthurus) at 5.0 ppb. Test animals exposed for over one week accumulated the PCB from the water; concentrations ranged from about 104 in crustaceans and fishes to 10s in oysters over water concentrations. Acute toxicity tests did not show the true sensitivity of marine species to this com pound. In comparison to short-term tests lasting 48 hr, Aroclor in chronic bioassays lasting one week or more proved to be 100 times more toxic. Mortalities were ``delayed'' as they usually did not begin until after one week of exposure and continued to occur after the animals were removed from the toxicant. This delayed mortality was similar to that observed with the insecticide mirex (Lowe el al. 1971). Gross signs of poisoning varied with species. Fishes typically developed hemorrhagic lesions on the body, ragged fins, and stopped feeding. Shrimps became lethargic and also stopped feeding, thereby mimicking the effects of low concentrations of DDT 11,1,1trichJorO'2,2-br'j(p-chlorophenyl)ethane] (Nimmo and Blackman 1972). Shrimps appear to be most susceptible to the chemical during molting, as previously noted by Duke el al. (1970) and Wildish (1970). Accumulation of Aroclor 1254 by shrimp The pathway by which organisms obtain toxicants or the actual effects observed in the laboratory under controlled conditions may or may not approximate those obtained under field conditions. The pathway appears to be an open question in the field of aquatic toxicology and the need for such research was slated by Sodergren et al. (1972) after a study of the accumulation of DDT and PCB by a crustacean. If we could produce tissue distributions of PCB in shrimp in the laboratory similar to those found in the field, some insight migh' be gained into its mode of entry and con centrations in food or water in nature. We began a study by determining PCB residues of shrimp from Escambia and Pensacola Bays (Fig. 5). We administered PCB at three con centrations (0.2, 0.68 and 43 ppm) in food in aquaria with flowing PCB-free seawater (Fig. 6). PCB was added at 3.0 ppb to seawater filtered through gravel and charcoal and 3.0 ppb was added to unfiltered seawater (Fig. 7). PCB was added to unfiltered seawater at 3.5 and 0.2 ppb (Fig. 8). Analytical methods for PCB were those of Nimmo era/. (1971a). The results of the field surveys and laboratory studies are expressed as the "relative con centration" (Fig. 5,6, 7, 8): ppm in a single tissue or organ Relative concentration =--------------------------------------------- X 100. ppm in all tissues or organs The proportion of Aroclor found in tissues of shrimp exposed in the laboratory to 0.2 ppb in water was nearest to that found in feral shrimp captured in the bays (Fig. 8) and concentrations were within the ranges found in shrimp from the field. Thus, we believe DSW 033757 STLCOPCB4017719 r 30 D. R. Nimmo et at. that shrimp from the laboratory exposures or feral shrimp from the bays probably ob tained most of the chemical from water. Shrimp could have absorbed PCB from the water column near the sediment-water interface or directly from interstitial water of the sub strate. However, this suggestion does not exclude the possibility that shrimp obtained some of the chemical from food, as was the case observed in Fig. 6. We believe the rate of Aroclor loss from shrimp was low and insufficient to affect the localization of Aroclor in the tissues of shrimp because in earlier experiments, we found that in the hepatopancreas only half of the chemical was lost in 17 days and in the re maining tissues, Aroclor content remained constant for five weeks (Nimmo et at. 1971a). The shrimp used in the laboratory exposures and those captured in the bays are all benthic species-either feeding on sediments or burrowing directly in them. It seems reasonable to suppose that adsorption of PCB directly through the gills from contaminated sediments would be greater because the animals are continually moving PCB-contaminated water through them. The results of the laboratory studies lead us to believe that concen trations of PCB available to shrimp in Escambia and Pensacola Bays were low (e.g., <1,0 ppb in water; <1.0 ppm in food). To determine if there was a concentration below which shrimp could not accumulate the chemical, we tested several hundred grass shrimp (Palaemonetes pugio) at 0.04,0.09 %7 9 | tX \ Natural populations (range of 4 samples) tc -- K i\ s \ I\ ' *\ rTMT-- | Hepato- | Ventral | Digestive | Heart j Gilts pancreas nerve tract | Abdominal | Exo muscle skeleton Fig. 5. Distribution of Aroclor 1254 in tissues of shrimp expressed as relative concentra tion (%). The grey area represents the range of concentrations in four composite samples of shrimp from different locations in the Pensacola estuary on different dates. DSW 033758 STLCOPCB4017720 the bays probably obed PCB from the water itial water of the subthat shrimp obtained sufficient to affect the xperiments, we found 17 days and in the re[Nimmo et al. 1971 a). ed in the bays are all ly in them. It seems Is from contaminated ng PCB-contaminated 0 believe that concenswere low (e.g.,<1.0 rould not accumulate pugio) at 0.04,0.09 ; 1 j ; * njlitiom umplei) Toxic action of Aroclor 1254 in estuarine organisms 31 Fig. 6. Distribution of Aroclor 1254 in tissues of shrimp fed Aroclor 1254-contaminated diets. The grey area is as in Fig. 5. lominat | Exoda skeleton relative concentracomposite samples t dates. Fig. 7. Distribution of Aroclor 1254 in tissues of shrimp exposed to Aroclor 1254 in filtered and unfiltered seawater. The grey area is as in Fig. 5. DSW 033759 HP STLCOPCB4017721 32 D. R. Nimmocra/. Fig. 8. Distribution of Aroclor 1254 in tissues of shrimp exposed to the chemical in nowing-water aquaria. The grey area is as in Fig. 5. Fig. 9. Aroclor 1254: Uptake and depuration in grass shrimp exposed to 0.04, 0.09 and 0.62 ppb in water. W QSW 033760 STLCOPCB4017722 Toxic action of Aroclor 1254 in estuarine organisms 33 r y^-- 't tfr'rizTv' rv.- * -'/ V. V ' 'A ` a ^TS ';s,r^:v^ Ati; ./ / V~" 4"' j ... a, >*^v ,, - :: " .. 9wt /*' k / . y v * ^ *#,. i `lA: V/'-j . v"> rr*w*., v#? "V-A';':' .v ;/r *^ -o . . . - ,*fJS tr'V v?V\v iSv ` ed to 0.04, 0.09 and I Fig. 10. (A) Normal vesicular connective tissue (parenchyma) from control oyster. Note uniform cell patterns and distribution of leukocytes. XI00. (B) Vesicular connective tissue from oyster exposed to PCB for six months. Note loss of uniform cell distribution and infiltration by many leukocytes. XI00. (C) Normal vesicular connective tissue from control oyster. X450. (D)Tissue from exposed oyster. Note many leukocytes and degener ation of vesicular connective tissue adjacent to gut epithelium. X450. (E) Normal digestive gland tubules from control oyster. Note the thick epilhelia which form normal triradiate lumina. X4S0. (F) Digestive gland tubules of oyster exposed to PCB. Note atrophy (thinning) of tubule epithelium and enlarged, abnormal lumen of tubule X450. DSW 033761 STLCOPCB4017723 34 D. R. Nimmo ft al. Fig. 11. (A) Liver parenchyma of norma! spot (Lagodon rhomboides) front control tank. Note uniform orientation of hepatocytes. X1000. (B) Liver parenchyma of spot exposed to PCB for several weeks, intermediate pathogenesis. Note large, smooth-edged vacuoles indicative of abnormal fatty-change in hepatocytes. XI000. (C) Liver parenchyma of spot exposed to PCB until moribund, advanced pathogenesis. Note large vacuoles, amor- DSW 033762 STLCOPCB4017724 STLCOPCB4017725 36 D. R. Nimmo el al. Fi. 12. (A) Fresh squash of hepatopancreas from exposed shrimp. Note two crystalloids in center. X1000. (B) Single, large crystalloid from fresh squash of hepatopancreas of exposed shrimp. XI000. (C) Longitudinal section of hepatopancreatic duct with branching tubules. In exposed shrimp, the crystalloids appear in the tubule epithelia nearer the main hepatopancreatic ducts. XI000. (D) Cross-section of hepatopancreas tubule from exposed shrimp. Note crystalloids in several epithelial nuclei; also, normal nuclei with conspicuous endosomes. X450. (E) Intermediate size crystalloid within hypertrophied epithelial nucleus. X1000. (F) Pathologic effect of crystalloid in PCBexposed shrimp. Note rupture of cells and nuclei releasing the crystalloids. XI000. 033?^ DSW STLCOPCB4017726 Vote two crystalloids ?f hepatopancreas of increatic duct with the tubule epiihelia n of hepatopancreas nuclei; also, normal e crystalloid within crystalloid in PCBloids. XI000, Toxic action of Aroclor I 254 in estuarine organisms 37 and 0.62 ppb of Aroclor 1254. The tests were conducted in flowing-water aquaria as before, with one modification. Each tank had a false floor of nylon screen to hold the an imals above the detritus. We believe the shrimp obtained more chemical through absorp tion from the water than in previous experiments. Within the test concentrations, no threshold level existed below which shrimp did not accumulate the chemical (Fig. 9). Concentrations produced in the shrimp (whole-body) were about 0.2, 1.0 and 10 ppm, respectively, and these were reached between the third and Fifth weeks of exposure. Concentrations in the shrimp did not reach equilibrium during the five-week exposure but the rate of accumulation decreased with time. When transferred to PCB-free water, the shrimp lost most of the chemical in four weeks. Pathology in estuarine organisms To date, toxicology of PCBs with respect to histopathological effects in estuarine organisms has been little studied. Published sources of information are from studies on mammals and birds (Dahlgrene/rrf. 1972, Vos 1972, Norback and Allen 1972) and a single study on fishes (Couch 1972). Structural changes found in tissues of oysters, fish, and shrimp exposed to Aroclor 1254 are characterized below. Oysters exposed to 5.0 ppb of Aroclor 1254 for up to six months showed several major tissue changes. Norma] structural pattern of oyster vesicular connective tissue (parenchyma) is seen in Figure 10A, and the irregular and broken pattern representative of altered tissue from exposed animals is seen in Figure 10B. In exposed oysters, an abnormal infiltration of leukocytes was found in the vesicular connective tissue (Fig. 10C and 10D). Sections of digestive glands of normal control oysters (Fig. 10E) can be compared to those in exposed oysters (Fig. 10F). The epithelia of distal digestive tubules of exposed oysters have undergone atrophy and surround enlarged lumina. Oysters that were removed from Aroclor-contaminated water and allowed to live in natural water for several weeks demonstrated partial or complete tissue recovery. Spot, an estuarine fish, exposed for two weeks or longer to 5.0 ppb of Aroclor, showed fatty changes in their livers. Normal liver tissue has regular distribution of hepatic cells and typical nuclei (Fig. 11 A). Note the regularity in the orientation of liver cords, cells and uniform scattering of nuclei. In intermediate stages of liver pathogenesis in experi mental fish, there are extreme fatty changes characterized by the presence of large vacuoles within hepatocytes and disorientation of liver cord distribution (Fig. 1IB). Figure 11C shows an advanced stage of pathogenesis in a moribund fish. Note the presence of intracellular PAS-positive bodies (ceroid) and congestion of blood sinuses, and severe vacuolation. Probably the most dramatic tissue change associated with chronic PCB exposure was observed in shrimp. The normal hepatopancreas, or digestive gland, of shrimp is a tightly packed organ of small elongated tubules (Fig. 12C). Figure 12D shows a cross section through one of these tubules. In the hepatopancreas of exposed shrimp, pyramidal DSW 033765 STLCOPCB4017727 38 D. R. Nitnmot'/rrA crystalloids of various sizes were found as inclusion bodies in the nuclei of epithelial cells (Fig. I 2D and 1 2E). Free crystalloids are shown in Figure I 2F. We know of no other report of the occurrence of precisely shaped crystalloids in hepatopancreatic tissue of crustaceans. We have routinely studied unfixed, fresh exposed shrimp and have found crystalloids in squashes of the tissue (See Fig. 12A and I2B). Epithelial cells of the hcpatopuncreas from shrimp which were exposed to three ppb Aroclor for at least 30 days are shown in Figure 1 ID. Exposed shrimp that do not have crystalloids have no conspicuous pathologic tissue signs. In those that have the crystalloids, hypertrophy of the affected nucleus results. F.ventually, the growth of the crystalloid inclusion distorts and ruptures the nuclear membrane. Several nuclear membranes and inclosed crystalloids are indicated in Figure 11D. The crystalloids arc histochemically positive for protein. They occur in widely separated nuclei but may also appear in clusters of adjacent nuclei and are most abundant in epithelial cells of tubules proximal to the main hepatopancreatic ducts. These crystalloids were found in individual shrimp before moribundity or death. In certain exposures, crystalloids have been found in up to 80% of the survivors but the incidence is very low over time until about 75% of the test animals have died. Crystalloids were found more often in larger shrimp than in juveniles. At present, we are attempting to establish whether crystalloids occur in individuals from other localities when exposed to PCB, in other species of shrimp, or in shrimp from contaminated areas in nature. Several possibilities exist as to the origin of the crystalloid inclusions. One suggestion was that they may be the result of sequestering of some normal or abnormal metabolite. Another possibility is that they represent a material produced by a virus2 and weic pro duced under PCB stress. In reference to this suggestion, Friend and Trainer (1970) showed that PCB enhanced the pathogenic effects of hepatitis virus in ducks. References Butler, P. A.: Pesticides in the marine environment: J. Appl. Ecol. 3 (suppl.), 253 (1966). Cooley, N. R., J. M. Kellner, Jr., and J. Forester: Mirex and Aroclor 1254: Effect on and accumulation by Tetrahymempyriformis W. J. Protozool. 19, 636 (1972). Couch, J. A.: Histopathologic effects of pesticides and related chemicals on the livers of fishes. Proc. Fish Disease Symposium. Armed Forces Inst. Path., Univ. of Wisconsin Press (In press) (1972). Dahlgren.R. B., R. L. Linden, andC. W. Carlson: Polycltlorinated biphenyls: Their effects on penned pheasants. Environ. Health Perspect. 1,89 (1972). 20nc of us (J. C.) as a result of eleclron microscope studies has recently found rod-shaped virus-like panicles occluded within the crystalloid inclusion bodies. This matter is currently under study. OSW 033766 STLCOPCB4017728 e nuclei of epithelial cells know of no other report atic tissue of crustaceans, ave found crystalloids in re exposed to three ppb shrimp that do not have tat have the crystalloids, owth of the crystalloid nuclear membranes and >ids are histochemically I y also appear in clusters tubules proximal to the aribundity or death. In f the survivors but the have died. Crystalloids t. we are attempting to ilities when exposed to eas in nature. usions. One suggestion abnormal metabolite, a virus2 and were projfrainer (1970) showed i (suppl.), 253(1966). lot 1254: Effect on . 19.636(1972). micals on the livers of h., Univ. of Wisconsin 'henyls: Their effects Toxic action of Aroclor 1254 in estuarine organisms 39 Duke,T. W.,J. I. Lowe,and A. J. Wilson, Jr.: A polychlorinated biphenyl (Aroclor 1254) in the water, sediment, and biota of Escambia Bay, Florida. Bull. Environ. Contam. Toxicol. 5, 171 (1970). Friend, M., and D. 0. Trainer: Polychlorinated biphenyl: Interaction with duck hepatitis virus. Science 17, 1314(1970). Hansen, D. J., P. R. Parrish, J. I. Lowe, A. J. Wilson, Jr., and P. D. Wilson: Chronic toxicity, uptake, and retention of Aroclor 1254 in two estuarine fishes. Bull. Environ. Contam. Toxicol. 6, 113 (1971). Lowe, J. 1., P. R. Parrish, J. M. Patrick, Jr., and J. Forester: Effects of the polychlorinated biphenyl Aroclor 1254 on the oyster Crassostrea virginica. Mar. Biol. 11,209 (1972). Lowe, J. L, P. R. Parrish, A. J. Wilson, Jr., P. D. Wilson, and T. W. Duke: Effects of miicx on selected estuarine organisms.Trans. 36th N. Am. Wildl. Nat. Resour. Conf., p. 171 (1971). Nimmo, D. R., and R. R. Blackman: Effects of DDT on cations in the hepatopancreas of penaeid shrimp. Trans. Am. Fish. Soc. 101,547 (1972). Nimmo, D. R., R. R. Blackman, A. J. Wilson, Jr., and J. Forester: Toxicity and distribu tion of Aroclor 1254 in the pink shrimp Penaeus duorarum. Mar. Biol. 11, 191 (1971a). Nimmo, D. R., P. D. Wilson, R. R. Blackman, and A. J. Wilson, Jr.: Polychlorinated biphenyl absorbed from sediments by fiddler crabs and pink shrimp. Nature 231, 50 (1971b). Norback, D. H., and J. R. Allen: Chlorinated aromatic hydrocarbon induced modifications of the hepatic endoplasmic reticulum: Concentric membrane arrays. Environ. Health Perspect. 1, 137 (1972). Sodergren, A., Bj. Svensson, and S. Ulfstrand: DDT and PCB in South Swedish streams. Environ. Pollut. 3, 25 (1972). Vos, J. G.: Toxicology of PCBs for mammals and for birds. Environ. Health Perspect. 1, 105 (1972). Wildish, D. J.: The toxicity of polychlorinated biphenyls (PCB) in sea water to Gammarus Oceanian. Bull. Environ. Contam. Toxicol. 5, 202 (1970). Manuscript received December 12, 1973; accepted April 12, 1974 xl rod-shaped virus-like 'tly under study. DSW 033767 STLCOPCB4017729