Document XRegJzNg8V1XYwZZQnqXyL9dG

Complete Statements of the four EPA Witnesses Received May 28, 1974 Registered Mail - Return Receipt Requested MOMS 203407 ' ^v r<prw'"-,,`.l " jmup&i.w pji RHV , \ ` ; , \ 1 t i I ; , I ' \ ! . | ; [' f` \' t j t i i l 1 MAY 18 1974 STATEMEW7 OF TWOMAS W. DUKE My name is Thomas V, Puke. 7 am the director ol the Environmental Protection Agency'6 Gutl Breeze. Environmental Reieo^cA Laboratory at Gull Breeze, Elorida. 7 have directed research activities at this laboratory since 1 961. Tke mission oi the Gull breeze Laboratory is to study the ellects ol toxic organics on marine organisms. My educational background is as lottou 7 received my 8.5. degree in Zoology loom Texas ASM University in 7953, my M.S. degree in Oceanography Irom Texas AIM Uni versity in I960 and my Pk.V. in Oceanography Irom Texas A I M University in 1962. A list ol my scientilic publi cations is appended to this testimony. My testimony wilt be taken Irom completed and ongoing research, as well as pertinent results ol related studies in the literature. My testimony is concerned with the routes, rates and reservoirs ol Aroclor 1254 in Escambia Bay, Florida and with the ellects ol various Arodtors on marine organisms as determined in the laboratory. The loliouUng exhibits are included in my testimony: 1. Vuke, T. W., J. 7. Lowe, and A. J. Wilson, Jr. 1970. A Polychlorinated Biphenyl {Aroctor(&12S4) in the Water, Sediment, and Biota ol Escambia Bay, Florida. Bulletin ol Environmental - Contamination and Toxicology, Volume $, Mo. t, pages 171-ISO. . HONS 203408 2 2. Ninmo, P. R,, P. 3. Hanten, 3. A. Couch, N. R. Cooley, P. R. PaKKish, and 3. I. Lowe. 1974. Toxiaity o{ AkocIok^ 12S4 and Its Phyticlogical Activity in Several Estuarine Organisms. Archives Environmental Contamination and Toxi cology. (In press) 3. Mrmo, V. R., P. V. Wilton, R. R. Blackman and A. J. Wilson, 3k. 1971, Polychlorinated Biphenyl Abtorbed (rom Sediments by fiddler Crabt and Pink Shrimp. Nature, Volume 231, No. 5297, pageA 50-52. 4. Wilson, A. 3., 3k. 1972, Etcambia Bay PCB Survey. Unpublished data. 5. Nimo, P. R., R. R. Blackman, A. 3. Wilton, 3k. S> and 3. forester. 1971. Toxicity and PittKibution oi AkocIok 1254 in the Pink Shrimp Penaeut duoraruni. Marine Biology, Volume 11, No. 3, pages 191-197. 6. Nirmo, P. R,, 3, forester, P. T. Heitmullen and G. H. Cook. 1974, Accumulation o( AkocIok 1254 in Gnatt ShKimp (Palaemoneiet pugio} in LaboKatoKy and field txpotuA.es. Bulletin oj Environmental Contamination and Toxicology. (In pKest) 1. Staii, Ouli BKeeze tnviKonmental Research LaboKatoKy. 1969-1974. AkocIok S> 1254 in Etcambia Bay Syttem. Unpubtithed data, S. PKoceedingt. 1971. Second Session oi Coherence in the Matter oi Pollution oi the Interstate Waters oi Etcambia River Batin (Alabama-TloKida) and the Intrastate PoKtiont oi the Etcambia Batin and Bay within the State oi Florida. Second Sestion. MON$ 203409 3 9. Proceeding*. 1972. ThiAd Session oi ConieAence in the Natter. oi Pollution ^ the Interstate WateA* oi Eicambla Riven Basin and the Jntra*tate Portion* o{ the Escambia 8asin and Bay within the State oi florida, ThUd Senior. 10. Guti Breeze Environmental Research Laboratory. 1969-1974. Aroctoris)l254 in Selected Oy*teA* iron Escambia Bay, ftorida, Unpublished data. 11. Cooley, N. R., J. M. Kellner, JA, and J. forester. 1973. The Polychlorinated Biphenyls, Aroclor*124S and 1260> EHect on and Accumulation by Tetrahymena pi/rljormis. Journal oj Protozoology, Volume 20, No. 3, pages 443-445. 12. Lowe, Jack 1. 1969. Guti Breeze Environmental () Research Laboratory - Bioas*ay Screening Test on Arcelor 124l{<U,fjnputtiAhed data. 73. Cooley, W. R., Kellner, J. M., Jr, and J. Forester, 1972. NiAex and Aroclor12$4i EHect on and Accumulation by Tetsahymena py/Ujonmis Strain W. JouAnal oj Protozoology, Volume 19, Ho. 4, pages 636-631. 14. Wildish, V. J, 1972, PolychloAinated Biphenyls {PCS) in Sea Water and TheiA EHect on Reproduction oi GamaAus oceanicus. Bulletin oj EnviAomental Contamination and Toxicology, Volume 7, No. 2/3, pages 732-737. 75. Wildish, V. J. and V. litko, 1971. Uptake oi Poly chloAinated Biphenyls inom Sea WateA by OcomaAus oceanicus. Marine Biology, Volume 9, No. 3, pages 273-273. HONS 203410 4 16, Parrish, P. R. 1972. Giill Breeze Environmental Research Laboratory - &cotU4<i/ Screening Test on Arodors 1242 and 1254, Unpubtcihed da^a, 77. BouJUjiun, A, W. 1974. Growth Responses o(5 btua>Une. Bacteria to Mirex, Heptacklar and Polychlorinated Biphenyl*. Unpublished manuscript. It. Kinter, U, B,, L. S, Menkens, R. H. Janicki and A. M. Guarino, 7972, Studies on the Mechanism ol Toxicity 0(5 WT and Polychlorinated Biphenyls {PCB'i)j Disruption 0(5 Osmoregulation in Marine Pish. Environmental. Health Perspectives, Volume 7, pages 769-773, 79. Hansen, V. J., P. R. Parrish and J, Porester. 7974. Aroclor^^1016t Toxicity to and Uptake by Estuarine Animals. Environmental Research. {In press) 10. Stall, Environmental Protection Agency** Gull Breeze Environmental Research Laboratory. 1974. Toxicity ol Aroclors 1260, 1254, 1248, 1242 and 1016 to Salt Water Organisms in 48and 96-hour Bioassays. Unpublished summary. 21. Lome, J. I., P. R. Parrish, J. M. Patrick, Jr. and J. Forester. 1972. EHeots ol the Polychlorinated Biphenyl Aroclor()l254 on the American Oyster Crassostrea Virginiai. Marine Biology, Volume 17, No. 5, pages 208-214. 22. Parrish, Patrick R. 1974. Aroclor1254, WT and WV, and Vieldrint Accumulation and Loss by American Oysters (Crassostrea virginica) Exposed Continuously lor 56 Weeks, Proceedings ol National Sketllisherie*. {In press) HONS 203411 5 23, Couch, John A. 1974, free and Occluded Virus, Similar to Baculovirus, in Hepatopancreas oi Pink ShAimp. Nature, Volume 147, Wo, .5433, pages 229-237. 24, MOmo, V, R. and L, H. BahneA. 7973. Physiological Consequences 0|$ Polychlorinated Biphenyl and Salinity-sires6 in Pemeid Shrimp. Symposium on Pollution and the Physio logical Ecology oi Estuarine and Coastal Water Organisms, November 14-17, 7973, Hobcaw Barony, South Carolina, 25, Wildish, V, J, 1970, The Toxicity oi Polychlorinated Biphenyls (PCS) in Sea Water to Grammanus Cceanicus, Bulletin oj Environmental Contamination and Toxicology, Volume 5, No, 3, pages 202-204, 26, Hansen, P. J.( P, R. Parrish, J. I, Lowe, A. J. Wilson, Jr. and P, P. Wilson. 1971, Chronic Toxicity, Uptake, and Retention oi Arodorl2S4 in Two Estuarine fishes. Bulletin oj Environmental Contamination and Toxicology, Volume 6, No. 2, pages 773-779. 27, ScKUmel, Steven C., Vavid J. Hansen, and Jerrold / forester. 1974. Eiiects oi Arocion1t$4 on Laboratory- reared Embryos and fry oi Sheeps head tUnnom iCyprinodon varlcgatus). Transactions oj the American fisheries Society, (In press) 21. Hansen, P. J,, $. C. ScKUmel and J. forester, 1974. Aroclor12S4 in Eggs oi Sheepshead Minnows t EHect on ferti lization Success and Survival oi Embryos and fry. Proceedings oj the Southeastern Game and fish Comission, (In HONS 203412 6 29. Johansson, N., S. Jensen and M. Olsson. 1970. PCB - Indication* oi Ejects on Pish. In "PCB Conference, Wenner-Gren Center," September 29, 1970, pagea 59-6*. National Environmental Protection Board, Stockholm, 30. Hansen, Vavid J. 1974. Aroclor12$4t Effect on Veveloping Estuarine Animal Cormunities in the Laboratory. Contribution* to Karine Science, (Jn press) 31. Hansen, V. J., S. C. Schimet and J. Forester. 1974. Effects of Aroclor1016 on Embryo, Pry, Juvenile and Adult Sheepshead Minnow (Cyprinodon variegabus). Manuscript approved by Gull Breeze Environmental Research Laboratory. ' 32. Staff, Environmental Protection Agency'* Gull Breeze Environmental Research Laboratory. 1974. Toxicity of Arcelor* 1234 and 1016 to Salt Water Organisms in Plou)- through Bioassays Exceeding 96-hour Vuration. Unpublished summary. HONS 203413 7 ROUTES, RATES AW RESERVOIRS OF AROCLORJ254 IN ESCAMBIA BAy, FLORIPA We detected the polychlorinated biphenyl, Aroctor'' 1254, in the wate*., sediment and biota oi Escambia Say in 1969 {Exhibit I) and continue to iotlow the movement and accumulation o{ this chemical within the bay system. Only one AouAce oi the chemical, the Monsanto chemical plant on the Escambia Riven, hot been located at this time. The' Aroclor reportedly entered the plant's eUluent through accidental leakage oi a heat exchange ituid. The leakage was stopped aiter the company wcu notiiied oi the appearance oi PCB6 in the bay system. However, a small amount oi this chemical continue/, to enter the System, possibly ^tom teaching irom contaminated sediments into the plant's eiituent ditch. Several condusions have been reached irom this study: (I) Much oi the chemical entering the water oi Escambia River appeared in particulate iorm in the water samples (f) Arocto$2S4 was distributed irom the point oi entry in the Escambia River to sediment and biota in the Escambia Say system by physical or biological trans port (3) Some oi the organisms containing residues oi Aaoc/oa 1254 are used as seaiood by man U) Although the quantity oi the Aroclor in the Escambia River was reduced drastically, residues continue NONS 203414 8 to appear In oysters approximately 8 mites down stream. Exhibit 1 summarizes the .history oh the. discovery oh 1254 concentrations in water, Pediment and biota hAom September 1969 to Vecember 1971 ahter the leakage mi itopped. By comparison, ample/, oh sediment/, had high concentrations ranging haom non-detectable to 50.0 mg/kg [day uieight). In the initial survey (1970) oh sediments hAom the river and bay, concentrations were high at the source oh the pollutant (Exhibit 5, Eig. 1; see below the industry) and also at the mouth oh the river (Exhibit 5, Eig. I). OtheA surveys (1970-1972) show that concentrations oh Aroco$?2$4 in sediments $*om thAee locations in the river and bay appear to decrease with time (Exhibit 2, Fig. 3). Also, the concentrations in sediment strata appear to decrease with depth. While the number oh samples was inadequate hr absolute comparison, these data indicate a trend. Data hrom tiie latest survey (1972) taken in the channel (Exhibit 4, page 4, see Transect A-middle) indicate that in November, 1972, the sediments above the trestle still had considerable amounts oh Aroclo^254. Concentrations ranged hrom 0.16 to 9.0 mg/kg (dry. weight). The sediment reservoir oh Aroclo 254 is probably a source oh this chemical to the biota oh the bay because residues continued to appear ahter the amount in the water n4 reduced drastically. For exrnple, the initial survey oh biota HONS 203415 9 (ExfUbi 1, TabEe I) showed biota, with level* a* high a* 12.0 mg/kg. In surveys conducted In 1969-70, shrimp collected ar {/torn the original source had whole body concentrations a* high a* 14 mg/kg wet weight [Exhibit 2, Tig. 4). Concentration* oi Arodor lound in *pecie* collected in tsaambia and Ea*t Bay* are ihown in Exhibit 2, Tig. S. The biota in East Bay are about 35 kilometer* from the original AouA.ce o the chemical, and yet have detectable level* o{ the chemical. There i* alio a higher concentration oi Aroclor^^12$4 in higher trophic level* in both bait and E*cambia Bay** biota which could implicate a iood chain transfer loom *ediment to large animal*. Three separate *tudie* have been conducted to *tudy the capacity oi benthic animal* to accumulate ArocJtor^^ 1294 Irom contaminated sediment*. The iirst o{ these [Exhibit 3) showed that shrimp placed on various naturally-occurring substrates containing this PC8 accumulated the chemical In proportion to the concentration o{ the chemical in the sediment. The second study showed that penaeld shrimp [Penaeus duonarum), placed on Moctor12 54-contaminated sediments, in upper Escambia. Bay, accumulated the material [Exhibit 5, page 197). Oaoaa shrimp, Palaemonetes pugio, exposed ior 3 month* to sediments in the bay accumulated Aroclo^^2S4 to about 0.4 mg/kg whole, body [Exhibit 6) in the third study. HONS 203416 1 JO Additional data on the level* o^ AroclorLly 12 54 in the water oi the Escambia Bay system ate 4ho*t in Exhibit 7, We analyzed both filleted and uniiltered sample* oi surface and bottom Oaten and monitoted water irom the new outloll that the industry constructed in 1971. Vuplicate sample* were also analyzed periodically, and in genetal, thete was good agteement. The water column ms i<Utly homogeneous judging irom the consistency oi sutiace to bottom sample*. But in iilteted sample* oi water, little Aroclor(&12S4 m* recoverable. This obsetvation ptobably means that most oi the chemical was attached to suspended particulates in the tivet water. Othet evidence oi this is shorn, in Exhibit t, Fig. 1/ Exhibit 9, Fig. 1, where mteA* in Escambia Bay show only trace levels [0.3 to 0.03 Mg!ty, whereas the levels in the sediments ate relatively high [Exhibit t, Fig. 2; Exhibit 9, Fig. 2). The Aroclofc^ 1254 concentrations irom a new outialt constructed by the industrial plant in 1971 are listed in Exhibit 7. Levels oi this PCB continue to be above 0,03 Mgfl but well below those iound in the initial surveys (1969). Some Aroclor(S)l234 is now iound in the river above the industry and we assume this is due to the eiiects oi high tides and winds moving surffl.ce waters slightly up the river. . We originally discovered Aroelor'*/12$4 in the bay in oysters irom Vevil's Point Light and we have continued to HONS 203417 n monitox thete mollutkt [Exhibit 10). The incxeatet in Axocloxfyl2$4 Icvelt in Hay to July each yuan one. pxobably due to the inornate in egg matt which could have a high affinity to that chemicalt becaute oi a high lipid content. The levelt dxop thoxtty aitex the oyttext 4pawn. This phenomenon aJUo it obtexved in the laboxatoxy. T the data ixom VeviVt Point Light axe analyzed by linear xegxettion analytic it it projected that thete molLutkt would aeach the cO.Olxgfl level by 1915. Thit pxediction attumet that no none chemical it put into the tyttem and that cJUmate, hydxologic o>i othex iactoxt aemain the tame. . EFFECTS OF AROCLORS OH MARINE ORGANISMS At the time Axc'clcx12$4 wat ditcovexed in Eteambia Bay, ettentially no data exitted on the eiiectt o thit chemical on othex PC81 on matilne otiganitmt. Since it it difficult and tometimet impottible to determine toxic eiieett oi tpeciiic chemicalt in the natuxal envixonment became oi the pxetence oi othex chemicalt and ituctuating envixonmental conditiont, we conducted tpeciiic toxicity tett with Axoclcx(^ 1t$4 and othex Axocloxt unde/1 controlled conditiont in the laboxatoxy, In oxdex to make ouX bioatt ay inioemotion at meaningiul at pottibte, we conducted mott oi ouX tettt with indigencut oxganitmt (tom Eteambia Bay ok timilax axeat, and utilized itowing teatoatex when pottible. The toxicity oi the tett chemicalt wat detexmined iox tpeciiic maxine oxganitmt iox a given pexiod oi time. Vetailed tett conditiont axe given in the exhibitt. HONS 203418 12 Residue. analyses were conducted where possible to determine ii the animals concentrated the chemical from the woten. in which they were tested. This is an important aspect oi these bioassays because Arocloi 12S4 occurred in animats ilom Escambia Bay that one used by man ioi iood. Obviously, these animals could stive as a vectoi tfo* the movement oi Aiocloi 1264 ^iom site apptcea-tcon to humans. Ji residues in marine oiganisms were to exceed levels deemed halmul oi human consumption, the iisheiy resource could not be harvested and would be adversely aected. Consequently, the well-being oi commercial isheAmen and those with lelated occupations would be adversely aected. The fallowing conclusions were leached iiom taboiatoiy bioassay experiments * (1) All oi the Aioctois tested are acutely toxic {oi certain estuarine organism. [2] Bioassays tasting longer than 96 hours demonstrate that short-team or acute tests underestimate toxi cides oi some oi the Arodtors. (3} Bioassays lasting over 96 hours indicate that Aioctor1254 is toxic to comerdalty valuable shrimps at concentrations less than 1 Hgtl and its toxicity is greatest to young shrimp and to shrimp that are subjected to stress. U) fishes, particularly sheeps head minnows, are extremely sensitive to Aioclor12$4. Concentrations HONS 203419 J3 about 0.1 Mg/t in wateA are lethal to fry and affect reproduction of this fish. About 7 H$/g in eggs of the. fish is Lethal to fry hatching in water free of this chemical, (5) The acute on shoot-term toxicity of Anoclo*(& 1016 to estuarine organisms is similar to other Arodors but it appeals less toxic to Wishes in long exposures than does Anodor1254. 810ASSAVS OF U HOURS OR LESS DURATION MoctoA 1260s Acute bioassays lasting as long as 96 hours show that Anode* 1260 is toxic to a vide variety of animals. Growth of the ciliate, Tetrakymena pyriformis. was reduced aftvt 96 houfis in growth media containing ,1,000^{g/l of Arodor$&1260 [Exhibit II). ?inish were apparently un- >. aected and lot of the pink shrimp died in lOQcjg/L of the PCS [Exhibit 12) during a 4i-hour test. Oysters, however, were sensitive to Arodor1260 with growth diminished by 44i in 10/[g/l and 52! in 100/0/1 in a 96-hour test. Arodor 1254: Acute bioassays indicate that Arodor 1254 may be more toxic to some estuarine organisms than Arodof{& 1260, Growth rates of the ciliate protozoan, T. pyrifornis, were significantly reduced by 96 hours exposure to Istg/l (part * tfb per billion) of Arodor^ 1254 in 10 ml of growth medium /) [Exhibit 13). A concentration o$ lOOjygll of Arodor^ 1254 in 4$-hour flowing water bioassays appeared to have no e^ect on MOMS 203420 u juvenile piniith, lagodon Khomboidet, but kitted J00| o the juvenile pink tbiimp, Penaeut duoKanum. Shell gKowth o oytteKA, Ouuio6tnea viA.girU.cji, expoted ion 96 homu wot completely inhibited by IOOtfg/1 and decKzated 41% oven that oi unexpoted contaolt by lO/fg/l oj AKodU>i& 1254 {Exhibit IIJ. Ml ouk tpeciet accumulated AKocton. > 1254 with quantitiei in oyAteAA, pinUh, and pink A bump cxpoied to 10j{q/1 in exce&i o{ 1 J{g/g {pant pen million). The AaocIoa(3^1254 it toxic to the amphipod, GarmaKut Oceanian, {Exhibit M). The amphipod alio accwmlatet AKocJbOK <>1, 254 inom mtex {Exhibit 15). AkocIok 124S: The toxicity o AKoctod^124t to T. pynijonmiA {Exhibit 2) it about 1,000 tbnei leAA than that o{ A\oclou 1254. JtA toxicity to Ah/iimp, oyAteAA, and |oinith wcla AimiloA to that oi AKocto*f^1254 {Exhibit 12). Motion. 1242: Bioattayt lotting 96 houAA demonttKate that thU PCB it toxic to oyAteAA and tbumpi at concentnationA oi lOOajgfl ok leAA {Exhibit 16). ThU PCB, like the othent tetted, had no appaKent eiiect on piniith. AkocIok'-^1242 InhibitA the gnowth oi certain Attaint o tituaKine baetetia (Exhibit 17). AkocIok 122U In Atatic bioaAAoyA lotting 96 houKA, 10| oi the killiiiAh, Fundulut heteKodUtut. expoted to 25,000 Mg!I o Mocto>$)l221 died {Exhibit II). Ml contaot and 1,500 M$H expoted iiih tuKvived the 96-houa expefument. Lethal concentnationt o AkocIok^I221 did decKeate the ability o thit killiiiAh to otmoKegulate. The otmolaAity and Na concentKation o the AeKum toat continently incKeated in ith expoted HONS 203421 15 to Ltthal levels of Aroclor^&i221, Aroclor 1016t Bioassays touting H hours show that Aroctor1016, tiki other PCBi, is acutely toxic to many estuarine organisms at concentrations of IOOjq/1 or less {Exhibit 19). About lO^glt um estimated to be lethal to 501 oj the oysters, brom shrimp {Ptnams aztexus) and grass shrimp {Valaemonetes pugio) in flowing water bLoassays. Only III of the pinfish exposed to IOOjtg/1 died in the 96-hour exposure. Alt four of these species accumulated the PC8 with the quantities accumulated being related to concentra tions in the test water and apparently not to species of animal. Aroclor &1016 inhibits the growth of certain strains of estuarine bacteria (Exhibit 17), Vata on the acute toxicity PC84 tested is summarized in Exhibit 20. 8JCASSAYS EXCEEDING 95 HOURS DURATION Aroclor 12S4t Hotlusca - Young oysters were continuously exposed to Aroclor It$4 for 24 or 30 weeks in flowing, unfiltered sea water (Exhibit 21). Growth rates (height and in water weights) were significantly reduced in oysters exposed for 24 weeks to Suglt (3.9stgft measured), but apparently were not affected by 30 weeks exposure to 1 xfg/1 (0,64 ^g!I measured). Survival of oysters was not affected by these concentrations of the chemical. Oysters accumulated as muck as 101,000 X the concentration in the test water. Concentration was 4.0.3 Mg fg in Sj^g/t exposed oysters after 2t weeks of depuration in PCB-^ee HONS 203422 16 water anddO.iM.glg in Itig/l exposed oyAtetA alter 12 weekA ol deputation. OyAtetA exposed to 5^gJt lor 24 weekA displayed genital tiAAue alterationA in the veAicular connective tiAAue around the digestive diverticula o{ the hepatopancteoA. In anothet experiment, oyAtetA were expoied continuouAly, with. no Aignilicant mortality, lot 56 week* to O.OIxtgfl ol (t) AtoclotK~'1254 and Akowed a concenttation lactot ol 165,000 [Exhibit 22). Maximum concenttation ol 1.6Sxeg/g occurred in oyAtetA expoied lot eight week*. VattetnA ol accumulation and Ioaa were AeaAonal. KeAidueA decreoAed 4$) to $11 in eatly July and late Octobet, apparently oa a teAult ol Apawning, and incteaoed lolloping theie petiodA. Thi& data indicateA that tile-hatoty ol oyAtetA muAt be known when evaluating teAidue data Itom monitoting ptogtamA. CtuAtacea - blue ctabA. Juvenile blue ctabi, CaUUnecteA AapiduA, expoAed lot 20 dayA to 5/(git (3.5 to lA^glt (T) meoAuted) ol Aroctov^1254 were apparently not allected (Exhibit I), Average concentration in live crabA mu 23jc^glg. The Atoclot1254 mu Alow to ItuAh Itom ctabA. Alter one week in PCB~lree water concentrationA in a Aecond group ol live crabA averaged 22x{g/g end alter 4 weekA Aix ctabA contained 11^g/g. tiddler crabA. Tiddler crabA, Uca pugilator. were expoAed J&, to Atoolot'--y1254 contaminated Aediment ltom tAcaabia bay. CD Elotida to determine il the ctabA would accumulate the Atoclot HONS 203423 17 1254 (xom the Aexiimtnt (Ex.lu.btt 3). Fiddtex cxabA accumulated Axoctox In quantities dixtctly related to the. amount* in sediment*. The chemical was accumulated by ingestion o\ by it* teaching into the -watex. fink Ahximp. Pink Ahximp wexe expoAed ion 20 dayi to 54\g/t o( Axoctox(E)l254 (3.S to 4.2xtg/l measuxed) in (towing un(ittexed watex {Exhibit !) Seventy-tioo pexcent (It o( 2$) died du/Ung the expoAuxe, no contxots died. The (Out Ahximp died on the tenth day o( expoAuxe, and a (ew died each day during the next 10 dayA. Sevexal died duxing molting. SymptomA o( insecticide poisoning wexe not exhibited by dying Ahximp. A AexieA 0($ additional bioasAayA using pink Ahximp Ahornd that Axoctox1254 is di((exentially toxic to juvenile and adult Ahximp (Exhibit 5). In (towing wcutex bioaSAayA, a measuxed ooncentxation o( Axoctox o( 0.94 Mg!t kitted 511 o( the juvenile Ahximp (2.5 to 3.2 cm) within IS dayA. ExpoAuxe to l.Suglt {measuxed concentxation) (ox 35 dayA xeAutted in a moXtatity o( 50t o( a gxoup o( (9.5 to 12,5 cm) adult Ahximp. / Vying Ahximp exhibited no appaxent outwaxd AymptomA o( poisoning. Uoxtality was delayed, they died at the Kate o( one ox two pex day and seemed moAt Auxceptible duxing molting. Shximp obtained the PCB (xom watex and (ood and concentxated it in the gxeatest amounts in the hepatopancxea*. Shximp placed in PC8-(xee watex toAt about 401 o( theix total body buxden o( PCB within 5 weeks. J( Axoctox1254 caused Ahximp moxtaJUty in the environment, it would be unlikely that it could be obAexved because? HONS 203424 It (1) Ahnimp would die. ovca an extended periled oh time, (2) dying AhrUmp would decompose aapidly ok be eaten by pKedatoKA, and (3) dead Ahnimp would not be visible, became unlike ii&h, they do not iloat on the AuAhace oh the wateA. Pathological examination*) of, exposed pink Ahnimp denominated that, even ih theAe weAt no outmKd AymptomA oh poiAoning, theAe weAe attenatlonA at the cellutaA and AubcellulaA level. The hepatopancAeoA o{ Ahnimp expoAed to AaocIok12$4 did exhibit dnamatic ti&Aue change* which. inctude*> the pneAence oh py/iamidal oAyAtalloidA in the nuclei oh epithelial cellA [Exhibit 2). Along with the pneAence oh thiA cAyAtaltoid weAe xod-Ahaped, *ee [non-occluded) and occluded viAonA which appeaK Aimiian to the BaculoviAuA gAoup oh inveAtebnate viAuAe* [Exhibit 17). Moc2o*(B)l2$4 may haoilltate the tnanAmi*Alon oa enhance the expKe&Aion oh latent viAal inhectionA, BAown Ahnimp - adult bKown Ahnimp (Penaeu* aztecua) weAe oa AenAitive to Knaelcn 1254 oa adult fink AhnImp [Exhibit 24). ThKttAfg/l wo* lethal to both AhAimpA within 30 daya. Baowh Ahnimp expoAed to a Aublethal quantity oh the AaocIoaHS4 hoa A even dayA died ih the Aalinity oh the teAt wateA decAeaAed gradually in t houru {AOm 30%* to and 7%*. Salinity change& aucii oa thiA occuA dunlng tidal cycle*. OAtnonegulation Aeemed impaired became concentnation* oh moAt majoA ion* in Atna oh KB-expoAed AhAimp became Aignihicantty le*A oa the HONS 203425 19 ambient salinity decreased. This was reitected as a decrease in the Mm oi the major ions [t.g. Ha, Ca, Mg, K, Cu, and C) and pcMicularty in the sodium, chloride, and calcium ions. Grass shrimp - grass shrimp, Palaemonetts pugio, were expoAed to measured concentrations oi Aroclor(&12$4 hanging fam 0.17 to 12.Sj^gft ion 7 oh 16 dajfA {Exhibit 6). A concentration oi 4.0 tigft produced significant mortality but mentality oi l.lMgll expoAed shrimp wxa not signiiicantly diHehent from that oi control shrimp. Shrimp expoAed ion A even dayA to inom 0.11 to 9.1/{g/l contained Aroclor in amounts hanging from 5,200 to 11,000 X the measured concentration in the test water. Grass shrimp accumulated the Aroctor 1254 in even increasing amounts in expoAuA.es lasting as long as 55 days. Anphipod - the amphipod, Geomarus Oceanians, was exposed to nominal concentrations oi ArocJtor^1254 oi inom 1 to 10,000 jygft ioh up to 50 days (Exhibit 25). Aroctor1254 waa lethal at concentAotionA as low as lOatgll. Some Gamarus had severely nechosed branchiae. Branchial nechosis was ound in some animals exposed to 1 Mg!I oi the Aroctorl2$4. ' fishes - Spot. In thn.ee separate How-through bioassays, 5 gft oi Aroctor 1254 {measured concentration with * 20t oi nominal) was lethal to juvenile spot [ Leiostomus xanthunus) exposed in itowing water ion. 20, 26 or 45 days (Exhibit 26]. Spot exposed to 1 jygfl showed no apparent eiitets. Aifrcted HONS 203426 20 spot usually ceased ietdbtg, became. emaciated, and developed flagged bins and lesions on the body. In some instances, 6&h continued to die even though they wene place in PC8~bnee waten. Ahoclon1254 was accumulated by exposed 6pot utith amount* nelated to duAation and extent expo*uhe and not appahently to mortality. Spot exposed to 1 #gll o$ the Anoctoh?^ 1254 boh. 56 days hapidly stoned this' chemical, maximum level* wine attained in 14 to 26 daya. Maximum amount Atohed whole bi*h was 57,000 times that in the test waten. Abteh 64 days in PC8~bnee waten, 39% ob the Ahoclonl254 hemained in the bish. Pinbi*h - in two sepanate bioaAAays tasting 14 oh 35 days, 5xfg/l ob Ahoeloh1254 was lethal to juvenile pinbish [Exhibit 26), Abbtcted bish usually developed ngus-tike lesions on the body, especially abound the mouth. Concentha- in) tion ob the Anoclon^ 1254 in whole pinbish exposed bon 35 days to 5x/gfl was 109Aglg (21,600 X the amount in the test waten). Sheepskead minnows. Sheepskead minnows ahe the most sensitive estuahine onganism to AhodU>f$ill54 that we have tested {Exhibit 27). Jntenmittent blow bioassay* lasting thhtt weeks wehe conducted with embhyo, b^Hr juvenile and adult bish. Newly hatched byiy west the most sensitive tib^-stage tested. Signibicant bhy mentality occo/tned in 0.3 ogll [0.16.*sg/l measuhed) and 0.1 uglt [0.06 n%ft measuhed] had no opponent % e^ec. Tny did not die immediately upon hatching but wehe kilted oven most ob the loo weeks botlowing hatching. Embhyos HONS 20342? and juveniles oi this iish were aiiected by loqgll (3,5^-gft meo*uAed) but this concentration was not lethal to adults. Many fay, juveniie and adult iish developed fan-rot, Arociorl254 in egg* adult sheepshead minnows exposed in bioassays to as little as O.iMgfl [0.14 Mgfl /o') measured] oi the Aro cloves 1254 can decrease survival oi embryos and fay that a/it maintained in water faee oi this ckejrUcal [Exhibit 28]. Fertilization Success was unimpouxed by concentration* in eggs a* high a* 201 **gfg but survival oi embryo* and fay too* signiiicantly reduced. Usually, fay faom egg* containing I.Os/gfg or moAe began dying 24-48 hour* ufaer hatching. Some p*e6iroinaAy research indicates that PCS* in egg6 . mag decrease iertility and survival in early stages oi embryonic development in Atlantic salmon, SaJbno solar [Exhibit 29], Communities oi Estuarine Organisms: A/ioclorl2S4 aifaeted the composition oi communities oi estuarine animals that developed irom planktonic larvae in salt water that faowed through 10 control aquaria, and 10 aquaria contaminated with 0.1, 1, or 10*/yfl oi the Aroctor) I 1254 [Exhibit 30). Communities that developed in control aquaria and aquaria that received O.lMg/l oi PCB in water ior lour months were dominated 0751) by arthropods, primarily the qmphipod Corophim volutator. In aquaria receiving 1 and 10 Mg!I, the number oi arthropods decreased and the number oi r chordates, primarily the tunicate Holguta mahattensis, increased; over 75% OjJ the animals in lO^gfl aquaria were tunicate*, numbers oi phyla, species, and individuals [particularly HONS 203428 n amphipods, bxyozoans, cxabs, and moltusks] toexe decxeased, but thexe ms no apparent eiitct on the abundance o annelids, bwchipods, coelentexates, echinodexms ok nemexteans. The Shannon-Weavex Index o& species divexsity ms not aliened by Axoctox 1254. Axoclox 1016 - Piniish. The chxonic toxicity and uptake oi Axoclox 1016 by piniish ms tested in tM.ee sepaxate ilow-thxough bioassays lasting 42 days {Exhibit 79). Uoxtality oi piniish exposed to il^gll \15ug/l measuxed in one expeximent and 21 Mgfl in the second] ok IOQ&qH (59 MQH measuned} ms signiiicantty gKeaten than moKtatities oi unexposed piniish, Uoxtality oi lOMgft (about 7jtgfl measuned) exposed iish ms negligible. Colon oi most dying iish dankened, they stopped ieeding and they ^uun ennaticatly uuth thein head inclined dommxd. ViOicutty in swimming pnogKessed until the iish smm upside down Just puioK to death. Pathological examination oi IlMgll exposed iish accented several liven and pancxeatic altexoticns that distinguished them piom contxot iish. Concentxaticns oi Axoctox1016 stoxed by the piniish exposed iox 42 days xanged ixom 11,000 to 24,000 times the nominal concentration in the test mteK. Concentxaticns in piniish exposed to 1 Mgll iox 56 days incxeased iox thxee ox ioux weeks and then stablized. When placed in PC8-*ee mtex iox 56 days, concentxaticns {tsglg} in these iish decxeased by 511. HONS Z03429 n Shtepshead minnow * The chronic toxicity oh A*odor 10U to h>iyt juvenile and adult shttp&htxd minnow wtu investigated in intermittent flow tUoassays lasting it days {Exhibit 31). Fry, juvenile, and adult hizh were apparentty not ajjecXerf by nominal concentrations oh 0.1, 0.32, 1.0, 3.2 on IOctg/1 but died in 32 and 1004g)l oh this chemical. Chemical analyses oh the test water showed about 4mq!1 in the 1049/1 aquaria, 13 Mg/1 in 32*g/l aquaria and At My!I /r) in the aquatic, contaminated with 100 ttgll oh Aroclor^lOU. Responses to poisoning included darkened body coloration, uncoordinated swbming, cessation oh heeding, lesions on ' the body oj many juveniles and adults, and death. Skeepshead minnows accumulated the PCS in proportion to its concentration in the test water. Fry contained 2,500 to 8,100 X the nominal concentration in the test water, adults 4,700 to 14,000 X and juveniles 10,000 to 34,000 X. Eggs j*om exposed adults were hertitized and placed in PC8-jA.ee water. As much as llng/g oh the PCB in these eggs apparently did not ajject survival / oh embryos and hMf h* weeks. Jnhomotion on hl*>w-through bioassays tasting longer than 96 hours is summarized in Exhibit 32. HONS 203430 DUKC, Thomas Wade Page 1 of 3 Published Works: Duke, Thomas W. 1961. Availability of sediment-sorbed radionuclides to the biota. Ph.D. Dissertation. Texas A i H University. With Don Hood and Bernadette Stevenson. 1961. Measurement of toxicity of organic wastes to marine organisms. J. Water Pollution Control Federation 9: 982-996. Duke, Thomas W. 1962. Observations on the movement of radionuclides in selected marine environments. Bull. Ecological Soc. of America 43: 120 (Abstract). Duke, Thomas W., E. R. Ibert, and K. M. Rae. 1963. Availability of sediment-sorbed material tt> the biota. In First National Symposium on Radioecology, V. Schultz and W. Element (eds.), Reinhold Publishing Company, pp. 171-175. Duke, Thomas W, 1964. Use of radioisotopes in marine biological research. Proceedings of the Second Oak Ridge Radioisotope Conference. 1). S. Atomic Energy Commission T1D-7689, pp. 69-72. Duke, Thomas W. 1965. Biogeochemcial cycling of radionuclides in the estuarine environment. Proceedings of the 17th Annual Conference Southeastern Association of Game and Fish Commission, pp. 315-323. Duke, Thomas W., James N. Willis, and Thomas J. Price, 1965. Cycling of zinc in experimental marine environments. Association of South eastern Biologists Bulletin 12: 45 (Abstract). Duke, Thomas W., J. H. Willis, and T. J. Price. 1966. Cycling of trace elements in the estuarine environment. I. Movement end distribution of zinc 65 and stable zinc in experimental ponds. Chesapeake Science 71 1-10. Duke, Thomas W., J. P. Baptist, and D. E. Hobs. 1966. Bioaccumulation of radioactive gold aa a sediment tracer in the estuarine environment. U.S. Fish and Wildlife Service, Fishery Bulletin 65(2): 427-436. Duke, Thomas W. 1967. Possible routes of zinc 65 from an estuarine environment to man. J. Water Pollution Control Federation 39(4): 536-542. Duko, Thomas W., James N. Willis, and Douglas A. Wolfe. 1967. Studies of the exchange of trace elements between estuarine sediments and water.. Association of Southeastern Biologists Bulletin 14(2): 27 (Abstract). MOMS 203631 DUF.F., Thomas Wade Page 2 of 3 Published workBt (continued) Duke, T. W., J. N. WilliB, T. J. Price, and K. Fischler. 1967. In|uence of environmental factors on the concentration of ZVi'* by an experimental community. Proceedings of the Second National Symposium on Radioecology held in Ann Arbor, Michigan, May 15-17, 1967. Duke, T.W., and T.R. Rice. 1967. Cycling of nutrients in estuaries. Proceedings of the Gulf and Caribbean Fisheries Institute, 19th Annual Session, pp. 59-67. . Duke, Thomas W. 1966. Salt Water Radioecology. Wildlife In North Carolina 32(5)s 4-6. Duke, Thomas W., James N. Willis, and Douglas A. Wolfe. 1968. A technique for studying the exchange of trace elements between estuarine sediments and water. Limnology and Oceanography 13(3): 541-545. - With Kenneth R. Tenore, and Donald B. Horton. 1968. Effects of bottom substrate on the brackish water bivalve Rangla cuneata. Chesapeake Science 9(4): 238-248. With T.R. Rice. 1969. Radioactivity in the Sea. In Encyclopedia of Marine Resources, Frank Firth (ed.), Van Noatrand Reinhold, pp. 566-569. With D.W. Hayne, and T.J. Sheets. 1969. Pesticides in Estuaries. In, H.T. Odum, B.J. Copeland and Elisabeth McMahan. Coastal ecological systems of the United States. (A Report to the Federal Water Pollution Control Administration. Institute of Marine Science, University of North Carolina, Morehead City, N.C.). Duke, Thomas W. 1970. Estuarine Pesticide Research--Bureau of Commercial Fisheries. Proceedings of the Gulf and Caribbean Fisheries Institute, Tvnaty-second Annual Session, November, 1969, pp. 146-153. Duke, T.W., J.I. Lowe, and A.J. Wilson, Jr. 1970. A polychlorinated biphenyl (Aroclor1254) in the water, sediment, and biota of Eacambia Bay, Florida. Bulletin of Environmental Contamination 6 Toxicology 5(2): 171-180, 1970. Duke, Thomas W. 1970. Effects of pesticides on estuarine organisms, Marine Pollution Bulletin 1(8): 126, August, 1970. HONS 203432 _ l>UKL, Thomas Wade Page 3 of 3 Published works: (continued) With T.R. Rice, J.P. Baptist, and F.A. Cross. 1970. Potential hazarda from radioactive pollution of the estuary. Proceedings of the PAO Technical Conference on Marine Pollution and Its Effects on the Living Resources and Fishing, Rone, Italy, 9-1B December 1970. * With Ford A. Cross and James N. Willis. 1970. Bldgeochealatry of trace elements in a coastal plain estuary: Distribution of manganese, iron, and einc in sediments, water, and polychaetous worms. Chesapeake Science, 11(4): 221-234. With J. 1. Lowe, P. R. Parrish. A. J. Wilson, Jr., and P. W. Wilson. 1971. Effects of airex on selected estuarine organisms. Trans* actions of the 36th Worth American Wildlife and Natural Resources Conference, March 7-10, 1971, Portland, Oregon, pp. 171-186. Duke, Thomas W. and A. J. Wilson, Jr. 1971. Chlorinated hydrocarbons in livers of fishes from the Northeastern Pacific Ocean. Pesticides Monitoring Journal 5(2): 228-232. With D. L. Coppage. 1971. Effects of pesticides in estuaries along the Gulf and Southeast Atlantic Coasts. Proceedings of the 2nd Gulf Coast Conference on Mosquito Suppression and Wildlife Management, New Orleans, Louisiana, October 20-22, 1971, pp. 24-31. With C. G. Bookhout, A. J. Wilson, Jr., and J. 1. Lowe. 1972. Effects of airex on the larval development of two ersbs. Water, Air, and Soil Pollution 1: 165-180. With Borthwick, P. W,, A. J. Wilson, Jr., J. 1. Lowe, J. M. Patrick, Jr., and J. C. Oberheu. 1973, Accumulation and movement of mirex in selected estuaries of South Carolina, 1969-71. Pesticides Monitoring Journal, 7(1): 6-26. HONS 203433 MAY 2 8 1974 fc,+> - TO XICm OF AROCLOR(f^t 1254 AND IT ` PHYSIOLOGICAL ACTIVITY IN SEVERAL ESTUARINE ORGANISMS 1 ^ - D, R. NLmmo, 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) * "In this paper, Aroclov and PCB are used interchangeably for Aroclor 1254. Arocloris a registered trademark of the Monsanto Company, St. Louis, Mj. Reference to commercial products does not constitute endorse ment by the Environmental Protection Agency. ^Contribution No. 162, Gulf Breeze Environmental Research Laboratory. HONS 203434 Abstract 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 (ETA). Monitoring of the estuary indi, ' cates 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 parts-per--billion concentrations In water* With a ciliate protozoan Tetrahymena pyrlformls W. shrimps Penaeus duorarum, P. aztecus and Palaemonetes pualo. and a fish, Fundulus slmllis. affected at or near 1.0 ppb._Tissue ^concentrations ofAroclor I254_similar_ to those found in natural populations of shrimps from the contaminated i estuary were successfully duplicated iu laboratory experiments. Shrimps also concentrated 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 vlrginjca. developed abnormal infiltration of leukocytes in the connective tissue, spot, Lelostomus xanthurus, developed fatty changes in their livers, and shrimp, Penaeus duorarum. developed crystalloides in hepatopancreatic nuclei. / . MOHS 203435 Ij*y E* \ n H j---* * . . . .. _............ Introduction . ............ Polychlorinated biphenyl (PCB) residues were found in water, sedlI. | sent and biota of Escambia Bay, Florida, in April 1969 ( Duke et_ aJL. I 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, Gulf Breeze, Florida. In this report, we present data on the chemical in water, sediments and biota of Escambia j Bay and adjacent areas, review toxicological data and present some physio logical-pathological information. Experimental methods and materials used as veil as chemical analyses are given elsewhere (Duke et. al. 1970; Cooley et al. 1972; Hansen et al. 1971; Kimao et al. 1971a; Lowe et al. 1972). PCB in Escambia Bay Unlike other studies in which PCBs have been found in the environ ment, the chemical in Escambia Bay apparently came from a single pointsource. Figure 1 is a comparison of chromatogram* of (a) an Aroclor 1254 standard, (b) Aroclor 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 concentrations in both oyster samples were similar and both samples and standard were analyzed on the some chroma tograph. Earlier, it was established by mass spectroscopy that the PCB in Escambia Bay was Aroclor 1254 (Niomo elt jal. 1971a) and in the com parison presented here the similarity is apparent. In laboratory studies with Aroclor 1254, the oysters were continuously exposed to approximately 10 parts per trillion for 72 weeks and the similarity of tissue residues to the PCB found in oysters from Escambia Bay indicates that the chemical HONS 203436 * in the Bay has changed little with time.' -- -- Monitoring data for the period September 1969 through December 1971 are presented in Table 1. Concentrations of Aroclor in unfiltered water .. ., samples from Escambia River.downstream averaged 0.6 parta-per-billion. Aroclor was found in 641! of the water samples from the River and in 271 of the water samples from Escambia Bay. Average concentration in sedi-. ment samples from the Bay was 2.3 parts-per-million (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. * Residues of Aroclor in sediment samples from a survey taken in February 1970 (Nimmo et al. 1971b)aTe listed in Figure 2. The samples were taken from the upper strata (e. g-, upper 10 inches) by corer or by dredge. In this survey, the maximum residue (61 ppm) observed In the River was a found at the outfall from the industry; the maximum in Bscambia Bay - (30 ppm) was found near the mouth of the River. * - The amounts of PCB in sediment samples from several locations within the River and Bay have decreased in the ensuing months. The number of samples was inadequate for absolute comparison, but the data indicate a trend. A decrease Is especially noticeable in the December 1970 and October 1971 surveys (Fig. 3), in which sediment samples were taken with a corer at three locations in the Bay. Cenerally, residues in the 1971 survey were about one-tenth the 1970 values, except one sample taken be low the trestle in the surface stream. PCB in the lower strata (4-12 inches) in the 1971 survey was non-detectable, except at the outfall of the industry. Later, we examined a deeper core -- to 24 inches -- taken HONS 203437 ' , above the trestle but found no residues. Cores taken in a 1972 survey generally Indicated less FCB than In 1971. Whole-body residues of Aroclor 1254 found in Bhrlmps from Escambia . ** 9i Bay and contiguous waters daring 1969/1970 are shown, in Fig. 4. Each datum represents a composite sample of at least 5 individuals. We show these data to indicate the dispersal of 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. - 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. 5). In the survey of October, 1971, we found no detectable PCB In the sea grasses. Spartins sp. and Bostera marina. The mollusk, Nerltina recllvata. contained 0.49 ppm. Of two crustaceans, blue crabs, Calllnectes sapldus. had the greater residues (6.9 ppm). Among fishes, one might expect sand eeatrout, Cynoscion arenarius. and Atlantic cutlassfish, Trichiurus lepturns, to have the highest residues because these species are predators; Instead, the highest residue (10 ppm) was found in silversides, Menidia berylllna, a species whose diet consists mainly of plankton. In Figure 5, 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 kilometers from the original source of the chemical. PCB residues in HONS 203438 species from Escambia Bay were 5 to 10 times greater than those found in East Bay but the data clearly show that the chemical was found in species V captured distant from the original source of the PCB. . '' *_ * * ' Toxicity of Arodor 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. Population growth in test-tube cultures of .the ciliate protozoan, Tetrabyaeoa nyr iforals W was reduced, sign If irantly by exposure to 1 ppb Aroclor 1254 (Table 2). Reduction was measured as effect on population - growth rate and on population density at 96 hours. Growth rate was esti mated as the quantity b. of the least squares estimate of the line jr " a+bx for the exponential growth phase of the population growth curve. These clliates 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 1 ppb Aroclor. Because the dilates can accumulate PCB from a culture medium, they could.be a step in the transport of the chemical into aquatic food weba under natural conditions* l ' 'i - . Bioassays were conducted in flowing water to determine the toxicity of Aroclor 1254 to a mollusk, three crustaceans and three fishes (Table 3). Growth in oysters, Crassostres virginica, exposed to 5 ppb for 24 weeks was significantly reduced, but growth in oysters exposed to 1 ppb for 30 weeks was not. Earlier work showed that hydrocarbon compounds inhibited shell deposition significantly at concentrations of 0.1 to 0.5 njrj HONS 203439 in short-term tests (Butler, 1966). In tests lasting about 2 weeks, various shrimps, Pcnaeus duorarum, Palaemonetes burIo, and Penaeus oztecus. were killed by exposure to 0.9, 1.3, and 1.4 ppb, respectively. In tests lasting 2 weeks or longer this chemical was lethal to longnose killlflsh. Fundulus similis, at 1 ppb and plnfish, Lagodon rhomboides. and spot, Leiostomus xanthurua, at 5 ppb. Test anlmala exposed for over one week accumulated the PCB from the vatar and concentrations ranged ' from about 10^* in crustaceans and fishes to 10^ in oysters over exposure concentrations. Acute toxicity testa did not ahow the true senaitivlty of marine species to this compound. In comparison to short-term tests lasting 48 hours, Aroclor In chronic bioassays lasting one week or more proved to be 100 times more toxic. * Mortalities were "delayed" aa they usually did not bagin until after one week of exposure and continued to occur after the anlmals were removed from the totclmt. This delayed mortality mas similar to that observed with the Insecticide mirex <bowe et al. 1971). Groms signs of poisoning varied with species. Fishes typically developed *- hemorrhagic lesions on the body, ragged fins, and stopped feeding. '*' , r Shrimps became lethargic and aide stopped feeding, thereby mimicking the effects of low concentrations of DOT (1, 1, 1, Trichloro -- 2, 2 - bis (p-chlorophenyl) ethane) (Nlmmo and Blackman, 1972). Shrimps appear to be most susceptible to the chotical during molting, as previously noted by Duke et al. (1970) and Wildish (1970). i '' HONS 203440 .. . ' ________ _ 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 waa stated by Sodergren et al. (1972) after a study of the accumulation of DDT and PCB by a crustacean. If we could produce In the laboratory tissue distributions of PCB similar to those found in the field, some insight might be gained into its mode of entry and concentrations present in nature. We began a study by determining PCB residues of shrimp from Escambia and Pensacola Bays (Fig. 6). We administered PCB at three concentrations (0.2, 0.68 and 43 ppm) in food (Pig' 7). PCB was added at 3.0 ppb to seawater filtered through gravel and charcoal and 3.0 ppb was added to unflltered seawater (Fig. 8). PCB was added to unflltered seawater at 3.5 and 0.2 ppb (Pig. 9) Analytical methods for PCB were those of Kimmo et al. (1971a). The results of the field surveys and laboratory studies are expressed as the "rela tive concentration" (Figs. 4, 7, 8, 9): .< ppm In a single tissue or organ RELATIVE CONCENTRATION - x 10Q ppm In ell tissues or organa 4. *. . > The proportion of Aroclor found in tissues of shrimp exposed to 0.2 ppb In water In the laboratory was nearest to that found in shrimp which were exposed naturally in the bays (Fig. 9). We assume that shrimp In the laboratory experiments obtained most of the chemical from the water but HONS 203441 some may have been consumed in detritus that was in the test aquaria. The distribution of PCB in shrimp fed 0.68 ppm in food and distribution in shrimp from the bays were similar (Fig. 7). 'Even though we varied the time of exposure and conducted the tests at what^we- considered realistic concentrations, we cannot distinguish the main pathway of PCB into shrimp from these experiments. However, we believe that concentrations of PCB available to shrimp in Escambia and Pensacola Bays were low (e. g., <1.0 ppb in water; <1.0 ppn in food). In order to determine if there was a concentration below which shrimp could not accumulate the cbemfcaXT'we tasted^several hundrpd grass shrimp, Palaemonetes puftio, at 0.04, 0.09 and 0.62 ppb of Aroclor 1254. The tents were conducted in flowing-vater aquaria as before, witb one modifi cation. Each tank had a false floor of nylon screen to hold the animals above the detritus. We believe the shrimp obtained more chemical through s absorption from tha water than In previous experiments. Within tbe test concentrations, no threshold level existed below which shrimp did not accumulate the chemical (Fig. 10). Concentrations pro duced in tne shrimp (whole-body) were about 0.2, 1.0 and 10 ppm, respect ively' and these were reached between the third and fifth weeks of ex posure. 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 after four weeks. HONS 203442 - * Pathology in estuarine organisms j 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 ^Dalgren et al. 1972; , -* ' Fbe, 1972; Norback and Allen, 1972) and a single study on fishes (Couch, 1972). Structural changes found in tissues of oysters, fiah, and shrimp exposed to Aroclor 1254 are characterized below. Oysters exposed to 5.0 ppb Aroclor 1254 for up to 6 months showed several major tissue changes. Normal structural pattern of oyster vesicular connective tissue (parenchyma) i* seen in Fig. 11, and the _ irregular and broken pattafn representative of altered tissue from ex posed animals Is seen in Fig, -12. In exposed oysters, an abnormal in filtration of leukocytes mas found in the vesicular connective tissue r (Figs. 13, 14). Sections of digestive glands of normal control oysters (Fig. 15) can be compared to those in exposed oysters (Fig. 16). The * epithelial .of distal digestive tubules of exposed oysters have undergone atrophy and surround enlarged lumioa. 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 eatuarine fish, exposed for two weeks or longer to 5.0 ppb Aroclor, showed fetty changes in their livers. Normal liver tissue has regular distribution of hepatic cells and typical nuclei (Fig. 17). Note the regularity in the orientation of liver cords, cells and uniform scattering of nuclei. In Intermediate stages of liver pathogenesis in experimental fish, there are axtreme fatty changes characterized by HONS 203443 presence of large vacuoles within hepatocytes and disorientation of liver cord distribution (Fig. 18). Figure 19 shows an advanced stage of patho genesis in a moribund fish. Note the presence of intracellular PAS-posirive bodies (ceroid) and congestion of blood sinuses, ohd! 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. 23). Figure 24 shows a cross section through one of these tubules. In the hepatopancreas of exposed shrimp, pyramidal crystalloids of various sizes were found as inclusion bodies In the nuclei of epithelial' cells (Figs. 24, 25). Free crystalloids are shown in Fig. 26. Ue know of no other report of the occurrence of precisely shaped crystalloids in hepatopan creatic tissue of crustaceans. We have routinely studied unfixed, fresh exposed shrimp and have found crystalloids in squashes of the tissue (See Fig. 21, 22). . Epithelial cells of the hepatopancreas from shrimp which were exposed to 3 ppb Aroclor for at least 30 days are shown in Figure 20. 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. Eventually, the growth of the crystalloid inclusion dis torts and ruptures the nuclear membrane. Several nuclear membranes and Inclosed crystalloids are Indicated in Figure 20. The crystalloids are bistochcmically 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 isain hepatopan creatic ducts. MONS 203444 -- References 1 Butler, P. A.: Pesticides in the marine environment. J. Appl. Ecol. 3 (suppl.), 253 (1966). ' Cooley, H. R., J. M. Keltner, Jr., and J. Forester^ Mirex and Aroclor . * 1254: Effect on and accumulation by Tetrahymena pyrlformis W. J. Protozool. 19(4), 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-j_ R-J.-_ Linden, and C._ W. Carlson: Polychlorinated biphenyls: their effects on penned pheasants. Environ. Health Perspect. 1, 89 (1972). 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, Contain. Toxicol. 5, 171 (1970). 4 Friend, M., and D. 0. Trainer: Polychlorinated biphenyl: Interaction with duck hepatitis virus. Science 17, 1314 (1970). tiansan, D. J., P. R. Parrish, J. I. Lowe, A. J. Wilson, Jr., and P. DWilson: Chronic toxicity, uptake, and retention of Aroclor ^1254 in two estuarine fishes. Bull. Environ. Contain. Toxicol. 6, 113 (1971). Love, J. I., P. R. Parrish, J. M. Patrick, Jr., and J. Forester: Effects of the polychlorinated biphenyl Aroclor 1254 on the oyster Crassostrea virglnics. Mar. Biol. 11(3), 209 (1972). MOMS 203445 These crystalloids were found in individual shrimp before moribundity or death. In certain exposures, crystalloids have been found in up to 80Z of the survivors but the incidence is very low over time until about 75X 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 con taminated areas in nature. Several possibilities exist ss to the origin of the crystalloid in clusions. 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 virus* and were produced under PCB stress. In reference to this suggestion. Friend and Trainer (1970) showed that PCB enhanced the pathogenic effects of hepatitis virus in -ducks. \ *0ne of us (J. C.) as a result of electron microscope studies has recently found rod-shaped virus-like particles occluded within the crystalloid inclusion bodies. This matter is presently under study. HONS 203446 Lowe, J. 1., P. R. Parrish, A. J. Wilson, Jr., P. D. Wilson and T. W, Duke: Effects of mirex on selected estuarine organisms. Trans. 36th N. Ab. Wlldl. Hat. 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(3), 547 (1972). ___ , R. R. Blackman, A. J. Wilson, Jr., and J. Forester: Toxicity and distribution of Aroclor ^ 1254 in the pink shrimp Penaeus duorarum. Mar. Biol. 11(3), 191 (1971a). - ____, ?. D. Wilson,- R. R. .Blackman and A. J. Wilson,-Jr.: Polychlorinated biphenyl absorbed from sediments by fiddler crabs and pink shrimp. Nature 231 (5297), 50 (1971b). Rorback, D. H., and J. R. Allen: Chlorinated aromatic hydrocarbon in duced 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. Follut. 3, 25 (1972). V06, J. C.: Toxicology of PCBb for mammals and for birds. Environ. Health Perspect. 1, 105 (1972). ' Wildish, D. J.: The toxicity of polychlorinated biphenyls (PCB) in sea water to Cammarus oceanicus. Bull. Environ. Contam. Toxicol. 5, 202 (1970). MONS 203447 Legend for Figures Figure 1- Chromatograms of an Aroclor 1254 standard, Aroclor 1254 in oysters from a 72-veek chronic exposure in the laboratory and PCB in oysters from Escambia Bay- '* y . Table 1- Concentration of Aroclor 1254 in water, sediment, and biota, September 1969 through December 1971. Figure 2. Collecting stations in Pensacola Estuary, 24 February 1970. Residues found in sediments at each station are givun in parts-permillion (ppm). N. D. not detected. (After Nlmoo et al., 1971b) Figure 3. Comparison of concentrations of Aroclor 1254 in .cores taken 10 months spart in upper Escambia Bay and River. Concentrations given in 1971 above and below trestle are averages of 2 cores each. Figure 4. Residues of Aroclor 1254 in shrimp (whole body) from Escambia Bay and contiguous waters during 1969/1970. Each datum represents a composite sample of at least-5 individuals. Figure 5. Comparison of concentrations of Aroclor 1254 found in species collected in Escambia (left side) and East Bays (right side): seagrasses, Spartlna sp. and Zostera marina; Rangla clams, Rangia cuneata: olive nerite, Kerlflna reclivata; brown and white shrimp, Pcnaeus aztecus and P. setlferus; blue crabs, Calllnectes sapldus; hay anchovy, Anchon mltchllll; sea catfish and gafftopsall catfish, Arius fells and Barge marinus; tidewater silversides, menIdla beryllina; silver perch Bairdiella chrysura: sand seatrout, Cynoscion arenarlus; spotted seatrout , Cynoscion ncbulosus: spot, leiostomus xanthurus; Atlantic croaker, Micropogon undulatus; hogchoker, Trinectes maculatus; HONS 203446 and Atlantic cutlassfish, Trlchiurus lepturus. Tabic 2. Effect of Aroclor 1254 on population growth of Tetrahyaena pyrifonnls W. (After Cooley et_ al., 1972) __ Table 3. _4 Chronic toxicity of Aroclor 1254 to estuarine animals. ** *% Figure 6. Distribution of Aroclor 1254 in tissi&s of shrimp expressed as relative concentration (I). The grey area represents the range of f . concentrations in four composite samples of shrimp from different locations In the Pensacola estuary on different datas. Figure 7. Distribution of Aroclor 1254 in tissues of shrimp fed Aroclor 1254-contamlnated diets. Figure 8. Distribution of Aroclor 1254 in tissues of shrimp exposed to Aroclor 12S4 in filtered end unfiltered seawater. Figure 9. Distribution of Aroclor 1254 In tissues of shrimp exposed to the chemical In f lowing-^water aquaria. Figure 10. Aroclor 1254: Uptake and depuration in grass shrimp exposed to 0.04, 0.09 and 0.62 ppb in water. Figure 11. Normal vesicular, connective tissue {parenchyma) from control oyster. Note uniform cell patterns and distribution of leukocytes. noo. Figure 12. Vesicular connective tissue from oyster exposed to PCB for six months. Note loss of uniform cell distribution and infiltration by many leukocytes. X100, Figure 13. Normal vesicular connective tissue from control oyster. X450. Figure 14. Tissue from exposed oyster. Note many leukocytes and degener ation of vesicular connective tissue adjacent to gut epithelium. X450. HONS 203449 Figure 15. Normal digestive gland tubules From control oyster. . , ^ _ . . ,_.* --.** w v **.->- i - -*n '^s , , .- m k w - *- * * a- - .--< Note the thick epithells, which form normal triradiate luuina. - X450. (from Love t al.. 1972) . Figure 16. Digestive gland tubules of -oyster exposed to PCB. Note atrophy (thinning) of tubule epithelium and enlarged, abnormal lumen of tubule. 14 5Q. Figure 17,. Liver parenchyma of normal spot (Lagodon rhomboidee) from control tank. Mote uniform orientation of bepatocytes. HOOD. Figure IS. Liver parenchyma of spot exposed to PCS for several weeks, intermediate-pathogenesi*-.- Note large, mmooth-edged vecueles indice- tlve of abnormal fatty-change in bepatocytes. XI000. Figure 19. Liver parenchyma of spot exposed to PCB until moribund, ad vanced pathogenesis. Mote large vacuplea, amorphous Inclusions and sinusoidal congestion. X1000. . Figure 20. Hepetopancress (digestive gland) tubule from pink shrimp ex posed to PCB. Note triangular crystalloids in some hypertrophied nuclei (M); also, normal nuclei (N) with large, prominent endosomes. (Feulgen reaction* DMA appears black in ptotomicrograph)- XI000. Figure 21. Fteab squash of hepatopencxeaa from exposed shrimp. Note two crystalloids in tenter. I10O0. a .. _ Figure 22. .Mingle, large crystalloid from fresh squash of hepatopancreas of exposed shrimp. X1000. . -Figure 25. Longitudinal section of hepatopancreatic duct with branching tUbules. In exposed shrimp, the crystalloids appear in the tubule eplthelia nearer the main hepatopancreatic ducts. X100CL . HONS 203450 Figure 24. CroSs-section of hepatopancreas tubule from exposed shrimp. Note crystalloids "in :several epithelial' nuclei; also, normal nuclei with conspicuous endosomes. 1450. > a Figure 25. Intermediate sire crystalloid within hypertrophied epithelial ` *,, * nucleus. X1000. * *" Figure 26. Pathologic effect of crystalloid in PCB-exposed shrimp. Note rupture of cells and nuclei releasing the crystalloids. XI000. ? 1 1 MOMS 203451 Figure 1. Chromatogram of an Aroclor 1254 standard, Arodor 1254 in oysters from a 72-weefc chronic exposure in the laboratory and PCB in oysters fro* Escambia Bay. ,,. >_ HONS 203452 1i I t HONS 203453 Table 1. Concentration of Aroclor 1254 in water, sediment, and biota, September 1969 through December 1971. v Location Escambia River, Fla, Escambia Bay, Fla. Escambia Bay, Fla. Escambia Bay, Fla Escambia Bay, Fla. Type r Samples Total ... ... Number Positive , Percent . Concentration Average Range - . PP" - .. PP" _ ....... _ Water 67 6* 0.0006 ND- 0.0086 Water Sediment 37 27 1 ND 1 36 . 78 2.33 Invertebratea Fishes 101 17 92 ! 0.81 | 100 1 3.99 ND- 0.00007 ND-30. ND- 6.9 0.29-20. ND Non-detectablet Water, <0,00003 ppm; Sediment or Biota, i^O.Ol ppm j i HONS 203454 i  Figure 2, Collecting stations in Pensacola Estuary, 24 February 1970- Residue* found In sediments at each station, are given in parts per million (ppra). H. D. * not detected. (After Niurno et al., 6). *' MOMS 203455 HONS 203456 > I f II I ' j -l \ Figure 3. i, 1 Comparison of concentrations of Aroclor 1234 in cores taken lO months apart in upper 8Scanib}a Bay and Fiver. Concentrations given in 1971 above and helm* trestle are averages of 2 cores each. \ t i HONS 203457 SEDIMENT CORE CONCENTRATIONS (ppm) OF AROCLOR c Figure 4. Kesldoe* of Aroclor 1254 in shrimp (tfholi body) ftoa ~t f - Esceatbi* jay And contiguous ^etafe during 1969/1970. Such i datum represents composite temple of at least 5 individuals. HONS 203459 ^. `I RESIDUES OF AROCLOR 1254 (ppm) Figure 5, Comparison at concentrations of Afoclor 1254 found in species collected in Escambia (left aide) and East Beys (right aide); seagraoses, SpartlnS sp, and Zostera marina; Rangia clams, Rang!a cuneata; olive norite, Nerltlna recllvata; brown and white shrimp, Penaeus artecus and P. setiferus; blue crabs, Calllnactes sapldus'; bay anchovy, Ancboa mitchllli; sea catfish and gafftopsail catfish, Arius felis and Barge marlnu3; tidewater silversides, menldla'beryllina,; silver perch J Balrdlalle chrysura; Sand seatrout, Cynoaclon arenarins; spotted seatrout, Cynoscion nebulosus; spot, Leiostomus xanthuvus; Atlantic croaker, Micropogon undulatua; hogchoker, Trinectes tnaculatus; and Atlantic cutlass fish. Trichiurus lepturua. HONS 203461 ) ND ND 0.4 9 wd : 0. 9 8' 6.90 3.00 3.80 10.00 4.5 0' 1.50 -- 1.80 1.60 1.30 2.90 AROCLOR 1254 I 1i SPARTINA 1 ZOSTERA j OLIVE NERITE | RANGIA j .. _ i PENAE1D shrimp ; BLUE CRABS BAY ANCHOVY CATFISH. TIDEWATER SILVERSIDES SILVER PERCH SAND StATROUT SPOTTED SEATROUT SPOT ATLANTIC CROAKERy HOGCHOXER ATLANTIC CUTLASSFISH ND (pp ND ND ND TRACK 0.46 0.68 0.58 0.95 0.4 8 4ft __ __ 0.1 2 TRACE TRACE ND 0.72 HONS 203462 Table 2. Effect of Arocloif 1254 bn population growth of Tutrahyncna pyrlforais. W. * * -- -- 9 ' Toxicant Mean Growth Rate* (vg/litar) ~b 0 0.1 1.0 10.0 '\ 0.0212 0.0203 0.0195 0.0199 ' Difference ;i x ii i. 1 ** 1i -a* . r' 1 -6* Mean Population Density (absorbance) ` 1.044 0.984 0.936 0.954 ^After Cooley el al.(1972). 1 f_ F(3,15) * 6.00 (P>0.01); bP(3,15) - 23.001 (P>0.005). *Mean* of 6 replicate experiments. { - Difference X -6 -iob - 9b MOWS 203463 Table 3 Chronic toxicity of Aroclor1254 to estuarine animals.' Test Animals Pink Shrimp Longnose Kill!fish Grass Shrimp Brown Shrimp Pinfish Spot Eastern Oyster Concentrations (Range) Minimum Affecting r* ------------- r-ppb (vg/l)--------------------------- 0.6 - 19.0 0.9 1.0 - 100.0 0.2 - 12.5 . M 1.3 0.1 - 1.4 5.0 1.0 - 5.0 - 1.4 5.0 5.0 1.0 - 5.0 5.0 Controls did not exceed 25% mortality. Toxicity In oysters was measured by reduced shell growth. *1-30 weeks in flowing water. MONS 203464 "Figure 6. Distribution of Aroclor 1254 in tlssume of shrimp expressed I1 - as relative concentration (7,). The grey are* represents the range of concentrations in four composite samples of shrimp from different locations In the Pensacola estuary on different dates. MIMS 203465 r e l a t iv e c o n c e n t r a t io n (%) DISTRIBUTION OF AROCLOR 1254 , IN TISSUES OF SHRIMP i HONS 203466 Figure 7. Distribution of Aroelor 1254 in titsuee of shrimp fed Aroelor 1254- contaminated diets. - i t \ i * - - ' ' i \i i i I .! < MOMS 203467 1 DISTRIBUTION OF ARQCLOR 1254 . IN TISSUES OF SHRIMP 80 Pink shrimp fed spot (43 ppm whole body)for 16 days 60 Pink shrimp fed spot(fiold-captured, 0*2 ppm whole body) for 16 days Pink shrimp fed croaker (0.66 ppm in muscle) for 50 days R E LA TIV E C O N C E N T R A T IO N (%) I HEPATO- I VENTRAL I DIGESTIVE I HEART | GUIS PANCREAS NERVE 1 TTBRAACrtT 1 > IABDOMINAL I EXO- I 1 MMUiKSCrtLFE >' CSKKEtLirEmTONN 1 HONS 203468 \ i Ii r, Figure 8. Distribution of Aroclor 1254 In tlesueo of fthrio? exposed fto , 4. 1 Aroclor 1254 In ufcfiltored nod anfllterod COsMotor. i - I .. - . .. _ r - * -' - MOWS 203469 DISTRIBUTION OF AROCLOK 1254 : IN TISSUES OF SHRIMP 80 r e l a t iv e c o n c e n t r a t io n ( x) 60 m.Pink shrimp exposed to 3Oppb Aroclor In filtered water for K> days 40 - MtHWM Pink shrimp exposed to 3.0 ppb Aroclor in unfiitered water for TO days I 20 - ........ A\ ; V.___/ l/.-r. \\\ \r-J t f ` J * . -\W.V ,'A ..... - Sh\ \- HEPATO- I VENTRAL | WOE STIVE | HEART I GILLS PANCREAS ' NERVE 1 TTBRAACrTr 1 l |ABDOMINAL I EXO I MMUtSicCnLeE I1 eSKvEeLnETON HONS 203470 r i I I I !. Figure 9. Distributibn of Aroelor 1234 the chemical in fl caring-water aquaria. tissues o shrimp exposed to i i t HONS 203471 R E L A T IV E C O N C E N T R A T IO N (%) DISTRIBUTION OF AROCLOR 1254 IN TISSUES OF SHRIMP 80 60 Mita Pink shrimp exposed to 0.2 ppb Aroclor in water for 50 days .. Pink shrimp exposed to 3.5 ppb . Aroclor in wator for 35 days 40 20 - ......ss. I HEPATO- I VENTRAL | DIGESTIVE | HEART ` 1 PANCREAS 1 NERVE 1 TRACT 1 GILLS ........... (ABDOMINAL I EXO- 1 MUSCLE 1 SKEL ETON HONS 203472 Figure 10. Aroclor 1254: Uptake and depuration in grass shrinp exposed to ' .f 0.04, 0.09 suid 0.62ppb in water. , i I MONS 203473 AROCLOR 1254: UPTAKE AMD DEPURATION IN GRASS SHRIMP WHOLE BODY (ppm ) WEEKS HONS 203*74 Figure 11. Normal vesicular connective tissue (parenchyma) from control oyster. Note uniform cell distribution and paucity o leukocytes. X100. Figure 12. Vesicular connective tissue from oyster exposed to PCB for six months. Note loss of uniform cell distribution and infiltration by leukocytes. X100. . '~ Figure 13, Normal vesicular connective tissue from control oyster. X450. " Figure 14. Tissue from exposed oyster. Note many leukocytes and degenerating of vesicular connective tissue adjacent to- gut epithelium. XA50. Figure 15. Normal digestive gland tubules from control oyster. Note the thick epithelia which form normal trlradlate lumina- X450. (fro.- Lo-./e et al,, 5), ' Figure 16 Digestive gland tubules of oyster exposed to PCB. Note atrophy of tubule epithelium and enlarged, abnormal lumen of tubule. X450. HONS 203475 [ 1 Figure 17, Liver parenchyma of normal apot (Lagodon rhonboides) from control tank. Note uniform orientation of bepatocytes. X1003, * -\ Figure 18. Liver parenchyma of spot exposed to PCB for several weeks, '..intermediate pathogenesis. Note large, smooth-edged vacuoles indicative m '`of abnormal fatty-change in bepatocytes. X1000. Figure 19. Liver parenchyma of apot exposed to PCB until moribund, advanced * * 1> pathogenesis. Note large vacuoles, amorphous inclusions and sinusoidal I congestion. X1000. `- . Figure 20. Hepatopancreas (digestive gland) tubule from pink shrimp exposed , to PCB. Note triangular crystalloids in soma hypertrophied nuclei (R); also, normal nuclei (N) with large, prominent endosomes. (Feulgen reaction; DNA. appears black in photomicrograph). XL000. . i' '' HONS 203477 * / . ' 1* ' 14 W' | i vi- ^ :. +***1"" -M (V*- rfriy *. 1$ . _ . A Vi> > ,> 4 -^^'rtSL^- rt MONS 203478 Figure 21. Freeh squash of hepatopancreas from exposed shrimp. Note two crystalloids in center. X1Q00, ' *' ' i* Figure 22. Single, large crystalloid from fresh squash of hepatopancreas ef exposed shrimp. XI000. ' , . .......................... ................... .. .................. ............... ........ Figure 23. Longitudinal section of hepatopancreatic duct with branching tubules. In axposed shrimp, the crystalloids appear in the tubule 1. epithelia nearer the main hepatopancreatic ducts. HQO. Figure 24. Cross-section of hepatopancreas tubule from exposed shrimp. Note crystalloids lo several epithelial nuclei; also, normal nuclei with large do some*. X&SO. '' Figure 25. Intermediate else crystalloid within hypertrophied epithelial nucleus. XI000. Figure 26. Pathologic effect of crystalloid In ?CB-exposed shrimp. Note rupture of cells and nuclei releasing the crystalloids. X1000. HONS 203479 HONS 203480 / ACCUMULATION OF AROCLOR'"-'' 1254 IN CRASS SHRIMP (Palaemonetes pu%lo) IN LABORATORY AND FIELD EXPOSURES i* 4 - by * * - * D. R. tfianoo, J. Forester, P. T. Eeltmul3.tr end C. E. Cook U. S. ENVIRONMENTAL PROTECTION AGENCY gulf breeze environmental research laboratory SABINE ISLAND, GULF BREEZE, FLORIDA 32561 9 ''1 f . HONS 2 03481. Results of several experiments Indicate that aquatic inverte brates accumulate total body concentrations of polychlorinated bi phenyls (PCB) thousands of times greater than that of the sur- ' rounding water. For example, Sanders an^ Chandler (1) showed that fresh water insects and crustaceans rapidly (1 day) accumulated PCB (Aroclor 1254) up to 24,000 times greater than the concentration in the water. Results of similar exposures conducted with estuarine animals showed oysters concentrating 85,000 (2), shrimp 10,000 (3) and fish 30,000 (4) times the amount of PCB in the water. Although Sanders and Chandler stated that PCBs entering the aquatic environment are below concentrations acutely toxic to in vertebrates (1), we have noted that most of the accumulation studies conducted thus far by the investigators cited in the para graph above have been at concentrations of 1,0 yg/l and above, l.e., concentrations demonstratively toxic to teat animals. Little is known about accumulation in marine invertebrates at extremely low concentrations, and with ona exception (3) no one to our knowledge has placed PCB-free animala In a natural environment ' known to have PCBs and followed accumulation with time. Ve report here the results-of* several experiments on chronic toxicity of Aroclor 1254 to Palasmonetes puglo, an estuarine gra&r shrimp, as well as concentration and loss of the compound from the animals with time. Ve also exposed grass shrimp for up to 3 months to Aroclor 1254-contaminatad ssdimants in Escambia Bay, near Pensacola, Florida. ' ~ .- ^ Gulf Braeze Environmental Research Laboratory Contribution No. 170 . 2 Associate Laboratory of the National Environmental Research Center, Corvallis, Oregon HONS 203482 -if T " > > V_>M.'Xr4 ; f. ' ' i#-* 'J I ^ T ' METHODS ACT MATERIALS With one exception, *11 laboratory experiments were conduct**! in 30-ml chambers supplied with flowing water from Santa Ros* Soeua Three sets of 5 chambers each received test coocentrat ionsj the fourth set was a control. Each chamber contained 4-10 shrlap, number depending on the size of the animals. Water flowed co*. tinuausly through each chamber at rate of 1.0 1/hr. Aracler 1254, dissolved in polyethylene glycol (mol. wt. 200), was metered into each mixing tank with, a syringe pump before the vat* eatery the test chamber. An equal amount of solvent was added t $e water flowing to controls. David J. Hansen of this laboacrry, fouol the eeneltlvity of a marine fish to Arodor 1254 m*frnd -g changed When he varied concentrations of polyethylene g$mff'seed to deliver the toxicant (personal communication). The flchp fed dally a commercial molly-flake diet (<0.02 mg/kg 'ArwomhlxiHg^ compounds). -, The experiment to determine the concentration end the tissuae of P. puglo vaa also conducted in a flawing****." ' system. We constructed 18-liter aquaria with false- flume ti nylmt screen (1/4-inch mesh) to hold shrimp above the 4salha broaght In with tile weter or produced by the animals. Sfeset^ fication was intended to prevent the animals from natijgrbae particles with adsorbed Arodor 1254. Coneaquently, we seme* that shrimp obtained more of the chemical from the vaoetlBssw* - sorption through tha gills rather than from Ingestion ef<m~> > tanfaated detritus. The-shrimp were not fed during tVli imps i meat. f, .v Concentrations of Arodor 1254 In tissues fey gas diXiietopaphy were detarmlned using pooled temples of at laser JTt shrimp each (S). JL* punlo were exposed to Arodor 1254--contaiBlaamsfeaf^y t In eppsr Escaabie Bay from November 1971 to February HfiS* ., shrimp in specially-constructed cages (5) were etposedSmct to tiie sediments. Average concentration of Arodor' US*db rf uppermost two Inches of sediment In November'1971 was 5A'*~:r' (dry weight). RESULTS OF LABORATORY EXPOSURES Teste conducted in flowing water showed- tjt puf. o.anfimt s/.r. cs{Bible to Aroclor 1254 (Table 1). In a 7-Ba^F expose^, ABTt dlaar 9.1 gg/l, but significant mortality dlft nor aexr nr:' o ant 0.62 pg/i. In the second aeries of tests *lasti , 4.ftsad 12.5 yg/t were toxic, but siguificafit*wnrtaLlg;id'-:.:.- *0Ns 203483 / '1 *\ t ,1 1 . it t i CONTROL 7 4(0 * 20) . - * 6.17 7 8(0 - 40) ` 0.62 7 4(0 - 20) , t.Jt .7 60120 - 80)*** ' 16 -- 25(0 - SO) 40(0 - 100) 4.0 16 * 45(25 - 50)*** 12.1 . * *> 16 t ' 35(50 - 75)*** 0.1 1.3 5.4 <5.0 <0.1 18.0 27.0 46.0 ` 7600 8700 7100 14000 6700 3700 * , *A1L exposure* were conducted'in flowing seawatert salinity and temperature ranges were 22 to 2t& and 17 to 28* C. **5 replicates per concentration: at least 4 shrimp per repli cation, - - . .^ ***Sigaifleant at P >0.05. . 'f HONS 203464 -** Test Cone. (pg/1) TABLE 2 ACCUMULATION OF AROCLOR 1254 T Falaenonetes Juglo* 1 J "- ' Body Cone* (mg/kg) -. ` Concentration Factor 0.17 0.62 1.0 2.3 2.7 3.2 3.2 5.2 5.3 5.3 9.1 1.3 5.4 3.2 25.0 19.0 15.0 " v 26.0 * 29.0 16.0 30.0 65.0 . . - 7600 ' 8700 3200 11000 ` 7000 4800 'V8100 5600 3000 - 5700 7100 ' - *7-day exposures conducted in flowing seawater at salinity and temperature ranges of 22 to 28 and 17 to 28* C. . ? 't T '> HONS 2034B5 .-S3 '. ./ ( ' .. occur i*n 1*3<k yg/tXae - f ^ f --%, "* v, , -i * At the conclusion of several one-week exposures tQ a' range of concentrations (0.17 to 9.1 ug/i), surviving shrimp from each ex posure were analyzed for whole-body residues. Ambient concen tration of toxicant in the water and resultant residues in the shrimp were correlated (r~0.91, Table 2). In some cases, duplicate test concentrations produced biological accumulations that differed by a factor of 2, Concentration factors ranged from 3,000 to 11,000. These rsages were similar to those found In tests using penaeld shrimp (3) but were soaewhet lower then those found by Sanders and Chandler (1), In tests using several Invertebrate species in fresh water. ., There appeared to be no threshold below which levels of the chemical added to water failed to produce residues In the tissues - (Table 3) In our tests. Whole--body concentrations produced after 5 weeks exposure to 0.04, 0.09 and 0.62 pg/l ranged from 200 to 26,000 times the concentrations in the test water. Concentrations' did not reach equilibrium and from 60 to 90 percent of the -Aroclor 1254 was lost from tbs shrimp within 4 weeks efter exposure to the chemical was stopped. Test coecentratione of the chemical were not significantly toxic to shrimp. Although accumulation increased with Increasing concentration of lOxlcant in this test, this was not observed in earlier studies (see Tables 1 and 2). Implications ere to be discussed elsewhere* .. 1 '^ - RESULTS CT FIELD EXPOSURES ' ( ' \* Average whole-body residue of Arodor 1254 In P. puglo after 1 month was 0.41 mg/kg (0.34 to 0.57); after 3 months, 0.42 mg/kg (0.37 to 0.50). There was no evidence that significant mortality occurred during the exposures of grass shrimp to contaminated ssdlacQts. ' .. _ DISCUSSION 'Concentrations of Arodor 1254 in P. pngio. after exposure to contaminated sediments for 3 months was equivalent to a laboratory-exposure of 0.09 vg/l In water for 2 weeks (Table 3). We expected residues to be higher in caged shrimp since ve had found that fiddler crabs exposed In the laboratory accumulated residues equal to or.greater than (wet-weight basis) that of the contaminated substratum (dry-weight basis) after 30 daftrs'*(6) . Concentrations of Arodor 1254 in caged shrimp exposed to con taminated sediments appeared to reach a plateau, but this was not the case In laboratory exposures (Table 3) where an equilibrium HONS 203486 ii l ) / * TABLE 3 ACCUMULATION OF AROCLOR 1254 IN Palaemonete* pqgln WITH TIME AFTER EXPOSURES TO THE CHEMICAL IN HATER AT THREE CONCENTRATIONS (gg/l) (Each value represent* a composite sample of 10 animals) Length of TVpoaure (hr/ day*) Control Body Cone. . Cone, Factor * (mg/kg) 0.04 Body Cone. Cone. Factor Cng/kg) 0. 09 Body Cone. Cone. Factor ' (mg/kg) 0. 62 Body Cacc. Cone. Faaor (mg/kg) 0, 1* 2 3 :4 '* 0.1 -- -- -- *- * *; *< *i i8 ' '-- * ; 12 ; : 16 --- :-- A ;* -24/1 * -- - * 36 / 1.5 , 48 / 2 , > ; 72 / 3 { 96 / 4 '154/6.5 336 / 14 ' ., . ;' ' -- -- ____ 0.1 __ -- '' * * A A n A . 504 / 21 0.1 * 672 / 28 0.14 *i 840 / 35 : 0.10 : * t j 1 0,1 0.1 o.i * *. * 0.1 o.i * :* r 0.1 A 0.1 A 0.1 0.1 * 0.1 A A 0.1 . 0.1 .* 0.1 A :* A 0.1 1590 0,13 3250 0.15 3750 . 0.17 4250 0.21 5250 PCB - STOPPED 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.10 0.14 0,15 ` 0.33 0.43 0.45 1.57 . 0.75 .7 A A h * A A A A A A 1100 1560 1670 3670 4780 5000 17400 8330 . - ; l 0.1 0.1 0.1 0.1 0.1 0,12 0,14 0.26 0.20 0.20 0.37 0.58 0.40 1.28 7.40 6.67 10.82 . 16.48 A A A A A 190 230 420 320 470 600 930 650 2060 11930 10900 1745.* 26566 ' : 1 HONS 203487 . r table 3 (Continued) 1176 / 49 1312 / 63 0.1 0.15 * * ' 0,1 * 0.1 * * Magnification factor not calculated, 0.12 0.13 ft * 3.24 1.64 ft U 'i I \ ! V; ,,* HONS 203488 V (H / was not reached. Therefore, we believe that shrimp ex posed to the sediments might have obtained PCB from the water or food singly, but shrimp exposed to Aroclor In the laboratory obtained chemical from two sources, water end food. It might also be more available In the laboratory than the field due to the carrier. In earlier laboratory studies with penaeld shrimp, both water and food appeared to be sources (3)'- ' " f* " No significant mortality was observed In caged shrimp and none would be predicted since residues produced In the field were similar to those found In shrimp after laboratory exposures which caused no death. - Penaeld shrimp spend only a fraction of their life cycle`In an estuary, moving Into oceanic waters after reaching maturity (7), but grass shrimp are endemic in estuaries.- Therefore, In relation to tine of exposure we would expect grass shrimp to > accumulate a pollutant from a contaminated estuary to a greater degree than penaeld shrimps, nevsrtbeless, this Is not true. In August 1968, penseld shrimps (Penaeua duorarua, P. setiferus. and P. aztecus) collected during a survey of Escambia Bay, Florida, had whole-body residues of Aroclor 1254 as high as 14.0 mg/kg (3). In that survey and. In subsequent collections, P. puglo had a maximum residue of only 1.4 mg/kg. Lower residues In P. puglo from Escambia Bay may be due to amounts of PCB in bay sediments and bahavlorlal patterns of the animals. We noted earlier (3) that residues In species of penaeld shrimp were related to higher concentrations of Aroclor 1254 In sediments that predominate In upper Escambia Bay. Wa found that penaald shrimp, as adults, usually ara captured In deeper waters and burrow Into silty or sandy substratas. In contrast, grass chrlmp usually do not burrow, rather are found along shallow sandy beaches and grass beds, where thiy obtain food that Is relatively uncontaalnSted with PCB. HONS 203489 T y 7 . = ' : . , J { , . * REFERENCES t " ' ' 1. SANDERS, H. 0., and CHANDLER, J. H., Bull, Ealron. Contain, and Toxicol., 7, 257 (1972). 2. LOWE, J. I., PARRISH, P. R,, PATRICE, *d* M., and FORESTER, J., Mar. Biol. (Berl.), 17, 209 (1972).'' 3. N3MM0, D. R,, BLACKMAN, R. R., WILSON, JR., A. J, and FORESTER, J., Mar. Biol. (Berl.), 11, 191 (1971). 4. HANSEN, D. J., PARRISH, P. R., LOWE, J. I., WILSON, JR., A. J. and WILSON, P. D., Bull. Environ. Contain, and Toxicol., 6,- 113 (1971). 5. HEITMDLLER, P. T., and NTMMD, D. R., Prog. Fish-Cult., 34, 120 (1972). 6. NIMMO, D. R., WILSON, P. D., BLACKMAN, R. R. and WILSON, JR., A. J., Nature, 231(1971). 7. PEREZ FAHFANTE, I., U.S. Fish Wildl. Serv., Fish. Bull., 67, 461 (1969). , . .. HONS 203490 f ljMBBgi3ff:gigKEaggJBJd'agw.yigaM. n-<ffainireis`arnKawnaw> n I. , ' -i *a I -a -.i 4 Mm 4 -+I -4 % REFERENCES 1. SANDERS, H. 0-, end CHANDLER, J. H., Bull. Eoiron. Coatam. end Toxicol., 7, 257 (1972). /. 2. LOWE, J. I., PARRISH, F. R., PATRICK,'J* H., and FORESTER, J., Mar. Biol. (Berl.), 17, 209 (1972)." 3. NIMMO, D. R., BLACKMAN, R. R.. WILSON, JR., A. J. and FORESTER, J., Mar. Biol. (Berl.), 11, 191 (1971). A. HANSEN, D. J., PARRISH, P. R., LOWE, J. I., WILSON, JR., A. J. and WILSON, F. D-, Bull. Environ. Conran, and Toxicol., 6, 113 (1971). 5. HEITMDLLER, P. T., and NIHMO, D. R., Prog. Fiah-Cult., 34, 120 (1972). ___ 6. NZMMO, D. R., WILSON, P. D., BLACKMAN, R. R. and WILSON, JR., A. J., Nature, 231 (1971). 7. PEREZ FAKFANTE, I., U.S. Fith Wildl. Serv., Flah. Bull., 67, 461 (1969). , . HONS 203491 ' - L 7~J~T- - 6 Aroclor1016: Toxicity to and "Uptake by Estuarine Animals^/ *_ * * ' D. J. Hansen, P. R. Parrish and J. Forester 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) ---------- \ If Contribution Ho. 172, Gulf Breeze Environmental Research Laboratory. \ @ ^ Registered trademark, Monsanto Company, St. Louis, HO. Mention of commercial products or trade names does not constitute endorse ment by the Environmental Protection Agency. HONS 203492 - 'T.r*, T~ l* Aroelar 1016: Toxicity to Estuarine Animals 0. S. Environmental Protection Agency Culf Breeze Environmental Research Laboratory -' *1 Sabine Island, Gulf Breeze, Florida- 32561 MOMS 203493  Aroclor 1016: Toxicity to Estuarine Animals --- Hansen ABSTRACT * *. * Bioassays were conducted to determine the acute toxlcities of the polychlorinated biphenyl (PCB) Aroclor 1016 la flowing sea water to American oysters (Crassostrea vlrglnlca). brown shrimp (Penaeus aztecus), grass shrimp (Palaemonetes puglo) and plnfish (Lagodon rhomboides) and to determine its chronic toxicity to and uptake and retention by pinfish. Acute 96-hour EC50/q were: oysters, 10.2 pg/i; brown shrimp, 10.5 ug/f; grass shrimp, 12.5 Ug/l. ttre'PCB was not toxic "to pinflsh at 100 ug/l for 96 hours but significant mortality occurred when plnfish were exposed to .' 32 Tig/1 o,f tAroclor 1016 for A2 days. Plnfish exposed to 1 yg/ for . V!rt * * *- m 56 day* mfccuraulated the chemical vlth maximum concentrations attained in whole-fish by 21 to 28 days. Maximum whole-body residue (wet - weigh O' was 17,000 x the nominal concentration in test water. Tissue alterations such as severe vacuolation in the pancreatic exocrine tissue surrounding the portal veins occurred in plnfish exposed to 32 pg/t of Aroclor 1016 for 42 days. HONS 203494 Aroclor 1016: Hansen Toxicity to Estuarine Animals j V 1 Folychlorinated biphenyls (PCB's) have been used industrially for over forty years (Broadhurst, 1972), and tecently there has been concern about their environmental impact.^ Because of this concern, manufacture and sale of most PCB's was discontinued and I sales restricted for uses that are not likely to produce environnental contamination. A new FCB, Aroclor 1016, is now manufactured in the United States for sale to capacitor manufacturers as a substitute for all other PCB's. This new ?CB is similar to Aroclor 1242, except that amounts of isomers containing five or more chlorine ew atoms per biphenyl group have been considerably reduced. Domestic 6 sales of Aroclor 1016 Increased from about 3.3 x 10 pounds in 1971 to 6 2/ 20.9 x 10 pounds is 1972 (W.B. Papageotge, personal communication- ). Cur study was conducted to determine the acute toxicity of Aroclor 1015 to the American oyster (Crassostrea virglnica), brows shrimp (Peaaaus aitecus), grass ehrlmo (Palaamocetes pugio). and pinfish (Lagodon rhomooides) and to determine its chronic toricJty to and uptake and retention by pinfish. / -- 1). F. Papageorge, Monsanto Industrial Chemicals Company. 800 N. Lindbergh Boulevard, St. Louis, MO 63166, USA. HONS 20349S Aroclor 1016: Hansen Toxicity to Estuarine Animals i " -. i - -- " " . * MATERIALS AUD METHODS , . ' Test animals ` Test animals were collected near the Culf Breeze Laboratory snd ^- ` acclimated to laboratory conditions for at least seven days before exposure. If mortality exceeded 12 In the 43 hours immediately preceding the test, or if abnormal behavior was observed during acclimation, the animals were not used. Oysters tested vere from 35 to 55 mm in height; brown shrimp, 11 to 26 mm rostrum-telson length; grass shrimp, 20 to 32 mn rostrum-telson length; and pinfish 27 to 84 mm standard length. Animals vere not fed during acute toxicity tests but they could obtain plankton from the unfiltered sea water* In the chronic exposures and the uptake and retention test, pinflsh were fed commercial fish food that contained no detectable PCB (<'01 ug/gV ( Acute end chronic tests . Acute toxicity to Aroclor 1016 was determined by exposing ten individual animals to 1, 10 or 100 yg/1 for 96 hours in each of two 201 aquaria. Each experiment was conducted twice. The PCB was dissolved in acetone or polyethylene glycol 200 and metered at 30 or 0.1 ml/hr, respectively, into unfiltered sea water that entered each aquarium at 75 l/hr. Two control aquaria received the same quantitieo of water and solvent. Temperature aud salinity of the water flowing into cqoaria in replicated tests vere siniltr (+^10%). Chronic toxicity of Aroclor 1016 to pinflsh was determined in three experiments, each lasting 42 days. In each experiment, 50 fish were HONS 203496 Aroclor 1016r Toxicity to Estuarine Animals Hansen | " . placed in each 901 aquarium that received 140 /hr o sea water. The PCS, dissolved in polyethylene glycol, was metered into the *t water at 0.083 ml/hr in the- first two experiments and 1.04 ral/hr in the third experiment. Control aquaria received the same quantity of water and solvent. The same exposure techniques that were used in the first two chronic toxicity experiments were used to determine: (1) the rate of uptake and retention of Aroclor 1016 in pinflsh exposed to 1 pg/l for 56 days fttid (2) the rate of depuration of Aroclor 1016 by pinflsh in PCB-free water for 56 days. Effect of Aroclor 1016 was assessed by measuring percentage reduc tion In shell growth of exposed oysters as compared to control oysters (Butler, 1962), by determining mortality in shrimps and fish, and by pathological examination of chronically exposed fish. Klstopatholoaicel examination Dr. J. A. Couch, pathoblologist at this laboratory, examined viscera from live pinflsh from the third 42-day exposure. Viscera were fixed either in 10Z neutral buffered formalin or Is Devidson's fixative. Those fixed in Davidson's were stored in 70Z ethyl alcohol until processed for paraffin sections (7u) and stained with Harris hematoxylin, and eosln (HHE) or Periodic Acid Schifr's (PAS). Viscera fixed in 10Z neutral buffered formalin were processed for frozen sections (I2p) and stained with oil Red 0 and hematoxylin. HONS 203497 Aioclor 10161 loxicity to Lstuarine Ari.mal3 Hanson 7 Chemical analyses ' Concentrations of Aroclor 1016 In water and animals were determined by electron capture gas chromatography. Unfllt&red vater samples from each *' concentration were analyzed once during the 96-hour exposures and weekly during longer exposures. Concentrations in animals that survived the 96-hour exposures were determined as whole-body residues. At the conclusion of each chronic exposure, surviving pinfish were dissected and FCB residues in flesh, flesh and scaleless skin, and remaining tissue determined. Residues in all tissues were succeed to compute concentrations of Aroclor 1016 in whole fish. The same . procedure was followed In the uptake and retention study, except fish ware removed for analysis at selected Intervals during exposure and depuration. Also, at the end of the 56-day exposure, brain, gills, heart and liver were removed from exposed fish for residue analysis.' All fish samples were composites of 10 Individuals. Tissue samples that weighed more than 5 g were prepared for analysis by mixing them with anhydrous sodium sulfate in a blender. The mixture was extracted for 4 hours with petroleum ether in a / Soxhlet apparatus. Extracts were concentrated to approximately 10 ml and transferred in 3- to 4-ml portions to a 400 x 20 mo chromatographic column that contained 76 ml of unactiviated Florisll. After each portion settled in the column, vacuum was applied until all solvent was evaporated. This was repeated with three 5 ml HONS 203498 /roclor 1016; Toxicity to Estuarine Aiii-aJ'; Hcr.seo _` ' '' ' t' . j / * -_ - * -- " ''* * - --s # -- #* -- -1 ^ -r .r _ , . - . <k. . *, ,, ^ rinses. The residue va3 eluted from the column with 70 ml of s 9:1 mixture (v/v) of acetonitrile end distilled water. The eluate .t was evaporated to dryness and the residue transferred to a Floris 11 column (lulls* et al., 1963) with petroleum ether. Aroclor 1016 was eluted with 6 percent ethyl ether in petroleum ether. Tissue.samples that weighed less than 5 g were analyzed by a nodi-* flcation of the micro method described in the Pesticide Analytical Manual, Volume XIX (U.S. Food and Drug Administration, 1970). The samples were weighed into a size 23 Ouall tissue grinder*and extracted three times with 5 ml portions of acetonitrile. The acetonitrile was flooded with 15 ml of 2% (v/v) sodium sulfate in distilled water end extracted with three 5 ml portions of hexane. The hexane was evaporated to approximately one ml and transferred to a 9 x 200 xn v Chrcmaflex column with a 50 ml reservoir that contained 3.3 g of Florisil topped with 3.3 g of anhydrous sodium sulfite. Aroclor 1016 was eluted with 20 ml of 5Z ethyl ether In hexane and adjusted to cn appropriate volume for analysis. . Water samples were extracted with petroleum ether, dried with anhydrous sodium sulfate and adjusted to an appropriate volume for analysis. All samples were analysed by electron capture gas chromatography using a 15 x 3.2 ten glass column packed with 2% 0V-101 on 100-120 Gas Chrom Q. Nitrogen flow rate was 25 ml/min, the oven temperature i t MOHS 203499 Aroclor 1016: Toxicity to Estuarine Animals Hansen - was 190 C, and the injector and detector temperature was 210 C. Aroclor 1016 was quantitated by comparing the total height of all peaks in the sample with the total height of all peaks in a standard of known concentration. Recoveries were greater than 80Z; data were not adjusted for recovery. All tissue residues were determined on a wet weight basis. - - -- - RESULTS AND DISCUSSION - -- -- - Acute (96-hr) exposure . Aroclor 1016 was acutely toxic to the estuarine organisms tested - (Tables 1 and 2). , Shell growth in oysters was inhibited greatly by exposure'to 100 yg/. for 96 hours. Sensitivities of brown shrimp and grass shrimp were similar and piftfish was the least sensitive species. Acute toxicitles of Aroclor 1016 to oysters, brown shrimp and pinfish were similar to that of Aroclor 1242 to these species2/ and Aroclor 1254 to oysters, pink shrimp (Penaeus duorarun) and pinfish (Duke st el., 1970). All animals accumulated Aroclor 1016 (Table 1). The quantities accumulated depended on the exposure concentrations and not on species. Vhole-body concentrations in live animals ranged from 440 to 4,200 x the nominal concentration in test water and 1,200 to 6,700 x the measured concentration in tent water. 3/ P. R. Parrish, unpublished data. MQNS 203500 Aroelor 1016: Hansen Toxicity to Estuarine Animals ' - I' t ]0 Chronic (42-day) exposure ' Toxicity of Aroclor 1016 to juvenile piofisfx was greater in testa lasting six weeks than in 96-hour exposures! Pinfish seemed unaffected by 10 ug/t or less, but died in concentrations of 32 and 100 yg/i (Table 3). Mortality began in the second week of exposure. Fifty-percent mortality did not occur in the third exposure, for example, until the 33rd day at 32 ygft and the 18th day at 100 yg/l. Delayed mortality of pinfish was also observed with odor 1254 (Kansan et al., 1971), - Host of the fish that died in the 42-day exposure exhibited symp toms of poisoning, such as changed appearance and behavior. Initially, their color darkened, they stopped feeding, and they swam erratically with bodies Inclined downward. Difficulty in swimming progressed until the fish cvam with their tills and dorsal fins breaking the water surface. Finally, the fish lost equilibrium, swam upside down, and died. Affected fish became vulnerable to attack by other pir.fish in the tank. In the third experiment, the majority of dying fish exposed to 32 and 100 yg/l lost scales, skin and, finally, flesh in front of the dorsal fin forming lesions sometimes as deep as the neural spine. This condition did not occur in fish exposed to 32 yg/% in the second experiment. Kepatocytes in liver sections of thirteen control fish showed no unusual characteristics when stained with 1EHE and PAS or oil Red 0. The hepatocytes from six fish demonstrated only a moderate PAS-positive HONS 203501 Aroclor 1016: Toxicity to Estuarine Animals Hansen ^ reaction, Indicating noderate-to-light glycogen reserves. In seven other control fish,.liver sections stalled with oil Red 0 also shoved a "broad range of lipid patterns. Structure `of livers of control fish was normal, being tubulosinusoidal in nature, with dis seminated pancreas prominent along th.e course of the portal vein (Fig. 1). " " . Eight fish exposed to 10 pg/i. of Arotlor 1016 for 42 days showed no pathologic microscopic visceral characteristics distinguishable from^control fish. Half of the fish samples were paraffin-processed; half were prepared for frozen sections. Pinfish exposed to 32 yg/fc of Aroclor 1016 had several liver and pancreatic alterations that distinguished them from control fish and fish exposed to 10 pg/l. Tissues from eight fish were examined; half were paraffin-processed and half were studied as frozen sections. Hepatocytes appeared slightly enlarged end core basophilic than in the control fish (Fig. 2). Kormal liver cord orientation was some what eltered and PAS-positive granules accumulated at the edge of the pancreatic acinar tissue in the Uver (Fig. 3) . Less lipid material existed in the livers of fish exposed to 32 iig/l than in control fish. This contrasts with the heavy, abnormal accumulation bf lipid in the livers of spot exposed to 6 ug/i of Aroclor 1254 for 30 days (Couch, 1973). Tha moat remarkable alteration in the pinfisVi was the occurrence of severe vacuolation in the pancreatic exocrine tissue surrounding the portal veins (Figs. 2,3). This HONS 203502 Aroclor 1016: Toxicity to Estuarine Animals Hansen ,> ]7 . 'i vecuolatlon was distinguishable from normal secretory vacuoles and deposits In pancreatic tissue from control fish because those in 0 exposed fish were small, abundant, end contained no secretory . i granules (Fig. 1). ' .* After the first exposure, 16 pinflsh From each control or Aroclor 1016-contaminated aquarium were held in PCB-free water and the salinity reduced to determine if ability of pinflsh tq survive * osmotic stress had been Impaired by exposure to sublethal concen trations of Arodox 1016. (Aroclor 1254 reduced the ability of * pink* shrimp, Penaeus duorana, to withstand stress of decreased salinity--D. R, Hirano, personal communication.--4/ ) Salinity was lowered from an initial salinity of 26 o/oo by 50X on each of four consecutive days (14, 7, 3.5 and 1.8 o/oo). None of ths fish died until the salinity fell bellow 2 o/oo.',At that salinity, mortalities i of control and FCB-exposeS fish ware similar. Pinflsh exposed for 42 days to concentrations of Aroclor 1016 between 0.1 and 32 pg/t stored the chemical In proportion to the concentration in test water (Table 3). Concentrations in whole fish renged from 11,000 to 24,000 x the nominal concentration in the test water and 14,000 to 55,000 x the measured concentration in test water, vbaraa:-. hJ 0. R. Klmao, Gulf Breeze Environmental Research Laboratory, Gulf Breeze, Florida 32561. > J HONS 203503 Aroclor 1016: Toxicity to Fstuarine Animals Hansen 13 the concentration factor in spot (Leiostomus xanthurus) exposed for 42 days to 1 pg/i of Aroclor 1254 was 30,000 x the nominal concentration (Hansen al., 1971) . ' ^ ,, The concentration of Axoclor 1016 in edible tissue was less than that in whole fish. Concentrations in whole fish averaged 2.1 times those in flesh and scaleless skin end 2.8 times those in flesh. Pinfish exposed to 1 pg/l of Aroclor 1016 stored quantities that exceeded the Pood and Drug Administration's provisional action- level for all PCB's (5 pg/g) in edible tissues (Table 3). Chronic (56-day) exposure Pinfi3h exposed to 1 pg/i of Aroclor 1016 for 56 days accumulated the chemical, maximum concentrations in'whole fish being attained in 21 to 28 days (Table 4). In a similar study with Aroclor 1254, * wj- whole-body concentrations of spot stabilise at about the sane tine, 14 to 28 days (Hansen at al., 1971). Maximum whole-body residue in pinfish was 17,000 x the nominal concentration in test water. Increases ir. concentrations in edible tissues were also rapid (Table 4), but nay hot have reached a maximum even after 56 days of exposure. The quantity of Aroclor 1016 accumulated by pinfish differed in the various tissues and organs. Fish exposed to 1 yg/t for eight weeks Accumulated 17 pg/g whole-body residue. Concentrations (uj/n) in other tissues or organs were: gills, 23; skin, 19; liver, 16; brain, 8.7; muscle, 5.9; end remaining tissues, 22. Aroclor 1016 was lost from the tissues after pinfish were placed i HOMS 203504 Aroclor 1016: Toxicity to Estuarine Animals Hanaen .... in PCB-free water (Table A). After 56 days of depuration, concentra- * tions in whole fish decreased by 51Z. In an earlier study, spot which had accumulated Aroclor 1254 lost 6671 after 56^dayb in PCB-free water (Hansen et al. 1971). . _, We examined chromatograms to compare the proportions of nine peaks of Aroclor 1016 in reference standards (Pig. 4) with those peaks from ** water and pinfish tissue samples (Table 5). Chromato grams of start- dards, tissue spikes and water samples were similar, while chromato grams^ of standards and tissue samples were dissimilar. Chromatograms of Aroclor 1016 from different pinfish tissues (flesh, flesh and akin, and rest) were similar. Chromatograms from ell tissues had smaller early eluting peaks, numbers 1, 2 and 3: Reduction in early eluting peaks of Aroclor 1254. from shrimp and fish tissue was noted by liiaso et al. (1971), The "relative proportions of the nice peaks found in chromatograms fxoa pinfish tissues early In the 56-day exposure, late in the exposure and throughout the 56-day depuration period were 4f similar. We do not know whether differences between chromatograms from reference standards and chromatograms from tissue samples reflect alterations in Aroclor 1016 molecules, differential solu bility, or other factors. It is unlikely, however, that the PC3 molecules were altered because no change in relative proportions of peaks was noted throughout the 112-day experiment. The potential environmental hazard of a chemical is dependent upon its likelihood of entering the environment and its potential HONS 203505 Arcelor 1016: Toxicity to Estuarine Animals E'anaen 15 j hazard to organisms. Arodor 1016 is similar to other FCB's in its toxicity to and uptake and retention by estuarine animals. *i - Therefore, its substitution for other PCB's reduces environmental hazard only if the policy of restricting sales for uses not likely to produce environmental contamination is continued* ACKNOWLEDGEMENTS . We thank Dr. J. A. Couch for hlstopathologlcal examination of the picfish, S. S. Fosa for preparing illustrations, and the Monsanto Company for providing Arodor 1016. * 1 MONS 203506 Arnclor 1016: Torlcity to Estuarina Animals- hausen i LITERATURE CITED Broadhurst, M. G. 1972- Use and repleceability of PCB'a. Environ. Health Perspect. 2: 81*102. * ' ' Butler, P. A. 1962. Reaction of 6eme estuarine nollusks to environ mental factors. In; Biological problems in water pollution. Third Seminar. U. S. Dept, of Haalth, Education and Welfare, Public Health Serv. Publ. No. 999-VF-25, 1965: 92-104. Couch, J. A. 1973. Pathologic effects of pesticides and related chemicals on the livers of fishes. Proc. Pish. Path. Symp. APIP. Univ. Vis. Press. In press. Duke, T. V., J. I. Lowe and A. J. Wilson, Jr. 1970. A polychlorinated biphenyl (Axoelor 1254In the water, sediment and biota of Escatdbia Bay, Florida. Bull. Environ. Contain. Toxicol. 5(2); 171-180. % Hansen, D. J., P. R. Parrish, J. 1. Lowe, A. J. Vilaon, Jr. and P, D. Wilson. 1971, Chronic toxicity, uptake and retention of Arodor1254 in two estuarine fishes. Bull. Environ. Conten. Toxicol. 6(2): 113-119. tiimmo, D, R,, R. R. Blackman, A, J. Wilson, Jr. and J. Forester. 1971. Toxicity and distribution of Aroclor ^ 1254 in pink shrirp, Peneeus duorerua. har, Biol. (Berl.) 11(3): 190-197. Mills, P. A., J, F. Onley and R, A, Caither. 1963. Rapid method for chlorinated pesticide residues in non-fatty foods. J, Ar.soc. Agric. Chen. 46(2): 1SS-191. "r HONS 203507 Aroclor 1D16; Toxicity to Estuarine Animals Fansen U. S. Food and Drug Administration, 1970. Pesticide Analytical Manual. U. S. Dept, of Health, Education and Welfare. 17 HONS 203506 Aroclor 1016: Toxicity to Estuarine Animals Hansen . TABLE 1 Acute toxicity to and uptake of Aroclor 1016 by.American oysters - *' (Crassostrea virginlca), brown shrimp (Fenaeus aztecus) . grass shrimp fPalacmonetes pugio), and pinflsh (Lagodon rhomboides) in $ 96-hour exposures. Effect Is expressed as percent reduction in shell growth in oysters and death in shrimps and fish. Whole body residues are from animals alive at end of exposure period. SPECIES C. vlrEinica P. a7tecus. P. puftio . L. rhomboides TEST CONCENTRATION (u?./i> Nominal Measured Control ip* 1 0.6 10 7.2 100 58; Control 1 10 100 ND 0.9 8.9 33. Control 1 10 100 ND 0.4 9.4 38. Control 1 10 100 ND 0.8 6.9 56. EFFECT (Z> WHOLE-BODY ' RESIDUE (wet weight, ng/g 0 NDX 10 4.0 38 32. 93 95 0 ND 8 3.8 43 42. 100 - 8 NO 33 1.1 38 22. 93 44. 2 NO 5 2.2 0 . 21 . 18 65. *ND - not detectable; <0.05 yg/l in water; <0.1 pg/g in tissue. HONS 203509 Aroclor 1016: Toxicity to Estuarine Animals Hansen TABLE 2 Acute toxicity of Aroclor 1016 to American oysters (Crassostrea - i yirginica). brown shrimp (Penacus aztecus) and'grass shrimp (Palaemonetes pugio). Effect is expressed as percent reduction in shell growth in oysters and death in shrimps. C..virginlca P. aztecus P. pugio 96-II0UR EC50 Nominal 10.2 10.5 , 12.5 temperature SALINITY (C) f oo Mean Range Mean Range 30 25-32 29 26-31 31 29-32 29 28-30 30 29-32 29 25-30 HONS 203510 Aroclor 1016: Toxicity to Ectu.irine AnInals Hanser 20 TABLE 3 . * Toxicity and uptake of Aroclor 1016 by pinfish (Lagodon rhonboidas) i exposed for 42 days In three separate expericertts.' TEST CONCENTRATION ('ft/?.) Nominal - Measured Control 0.1 1.0 10.0 NI)i/ 0.1 0.8 3.0 MORTALITY Z 36 16 48 38 CONCENTRATION IN FISH (ur/r. vet weight) Flesh Flesh & skin Whole fish ND - . ND 0.7 0.8 5.1 . 6.3 60. 90. ND 2.4 11. * 166. Control 1.0 3.2 10.0 32.0 ND 0.9 2.5 7.0 13. 12 16 16 28,, 0.5 4.0 34. 63. 140. 0.6 6.0 39. 76. 180. 0.5 17. 65. 170. 620. Control 10.0 32.0f 100.ol/ 13) 6.8 21. 59. 6 5o|{ 50*/ * ND 23. 30. 38. ND 49. 48. 72. ND 111. 106. 205. A/ NO not detectable: <0.05 vg/i In vater; <0.1 yg/g in tissue. 2/ -- Mortality significantly greater than in control ish*a -0.01. -- Exposure terminated and tlssuas analyzed vhen 50% of the fish died: 33 days at 32 ygft and 18 days at 100 yg/i. HONS 203511 ^roclor 1016: Toxicity to Estuarine Animals Vansen - *" TABLE 4 . ' -ar`-s-''' I * Concentrations of Aroclor 1016 C vs/s vet weight) in pinflsh (Lac;odon rhoDboides) exposed toi .1 ygl1 of this PCB . - ' sample consisted of tissues from ten fish. Each i DAYS OF EXPOSURE Flesh CONCENTRATION Flesh and skin 0 3 0.8 7 1.6 , >14 2.3 ... 21 : - 3.2 .* `i 28 42 . J 3.0 1 4.0 's 36 - 5.9 ND 0.9 ?-4 3.3 4.3 ` 6.8 ` 6.0 8.3 Whole fish ND x:6 3.9 6.5 9.7 25. 17. ' 17. Depuration 14 28 , 56 4.1 3.5 2.2 7.8 6.6 3.9 A/ HD - not detectable; <0.1 yg/g. I 13.5 . 9.3 ` ' 6.6 '- * y HONS 203512 Aroclor 1016: Toxicity to Estuarine' Animals Hansen t 22 ' 1 . TABLE 5 Percentages of the nine measured peaks from chromatograms of Aroclor ' * 1016 reference standards and from tissues of pjnflsh exposed to 1 %! of Aroclor 1016 for 56 days and than held in PCB-free vater for 56 days. PERCENTAGE OF PEAK*- C2ROMATO- ___________________________________________________________ GRAMS 1 - _... 2 . ,, 3 4 . 5 6 7 B - 9 % Reference standard 12.4 11.3 10.0 23.5 13.3 6.4 8.1 8.8 6.2 8 All tissues through 14 days of exposure 2.4 5.3 1.8 39.6 7.7 10.3 12.1 9.4 11.4 7 All tissues, days 21 56 of exposure 2.5 5.0 2.1 33.2 7.8 11.9 11.3 10.3 15.9 12 All tissues during 56-day depuration 1.0 1.7 0.3 38.2 2.8 15.0 12.4 11.9 16.7 7 11 Determined, as peak height x 100. sum of nine peaks MONS 203513 .i.-roelor 3 016-. Toi'icity to Estuarine Anlrals Kensen LECEND FOR FIGURES 35TT 23 FIG. 1. Section of normal liver from control'pinfish. The vacolues in hapatocytes are results of extraction1 of lipid and/or glycogen during paraffin processing. Note the normal pan creatic exocrine tissue containing normal secretory deposite (arrows). (X450). * FIG. 2. Section of liver fron pinfish exposed to 32 Ug/1 Aroclor 1016 ' for 42 days. The hapatocytes appear relatively-dense with Dore prominent nuclei than in Fig. 1. Kote the lack, of lipid or glycogen vacuoles in hapatocytes. Small, abnormal vacuoles in pancreatic exocrine tissue (arrows) distinguish fish exposed to 32 pg/ from control fish. (430). % FIG. 3. Plgaent cl a position (?A5-positivs) between pancreatic exocrine tissue and live parenchyma (arrow). Hots nature of small vacuoles in pancreatic tissue. This tissue 13 fron fish exposed to 32 vg/1 Aroclor 1016. (X10Q0). HONS 203514 ` 7: v*.-> \ . ' * r --- -* - :< 4 ' > - . *. "ScJ *V ,, V , - -- > -"> ,, ~ . J. *"*<*-." --i _r *= : % t*> a A| *o c- ;.-.vrv nS^t^'V co r l- *'"' .v -,'S.vV'*' ' w**> Aroclor 1016: Toxicity to Estuarine Animals Kanseo i . FIGURE 4 , Chromatogram of nine peaks of Aroclor 1016 reference standard. >i Operating conditions: gas flow nitrogen 25 mlfarJtn; injection and detector temperature 210 C; oven temperature 190 C; electron capture detector; 152.4 x 0.32 ca glass column packed with 2Z 0V-101 on 100-120 Gas Chrora Q. 24 HONS 203516 -fe;- 4i *. * HONS 03517 I ,, ...... . .I \ Aroclor 1254, DDT and DDD, and Dieldrin: . Accumulation and Loss by ._ - .- American Oysters (Crassostrea virglnlca) ' 1 Exposed Continuously for 56 Week* . 'i , - Patrick R, Farcisti ................. 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) . Separate populations of oysters were exposed continuously for 56 weeks to 0.01 pg/1 of Aroclorv-x1254, p,p'-DDT and DDD, or dieldrin .it and sampled at 8-week intervals for residues. Maximum concentrations based on body weight (pg/g) occurred after 8 weeks of exposure, but maximum concentrations based on* absolute amount of toxicant accumulated (jig) occurred after 56 weeks of exposure. After 8 weeks, average whole-body residues (wet weight) from five oysters analyzed individually were: Aroclor 1254, 1.65 pg/g, 4.0 pg; DDT (and metabolites DDD and DDE), 0.46 pg/g, 1.0 pg; and dieldrin, 0.08 pg/g, 0.2 ug. After 56 * Contribution No. l)4, Gulf Breeze Environmental Research Laboratory. (^Registered trademark, Monsanto Company, St. Louis, MO. Mention of coirmerclal products or trade names does not constitute endorsement by the Environmental Protection Agency. HONS 203518 r / weeks, residues were: Aroclor 1254, 0.89 pg/g, *25.7 pg; DDT and ' metabolites, 0.37 pg/g, 7.0 yg; and dleldrln, 0.03, pg/g, 0.6 pg. - ** Seasonal patterns of accumulation and loss of the three toxicants were similar. Residues based on body weight (pg/g) decreased 452 to 812 in early July and late October, apparently as the result of spawning, and increased following these periods. This shows that the life history of oysters must be considered when evaluating residue data from monitoring programs. Growth rate,(height and ln-vater weight) of exposed oysters was not different from that of control oysters (Student's t-tast; a " 0.01). Mortality was not algnlfleant In any group. ' I HONS 203519 '2i- ,^ r--. jr> ^--w--r*- -- I` PHYSIOLOGICAL CONSEOHEN'CES OF POLYCI-LORIttATED BIPFF.rrYL- AND SALINITY-STRESS IN PENAEID SHRIHP / fey \> D. R. Niraro and L. R. Bahner - - * i.j------P--0" Paper to be presented at a symposium entitled "Pollution end the Physiological Ecology of tfaJEvarine end Coastal b'ater Orgnols-.s" November 14-17, 1^73 at Hobcaw Barony, South Carolina ` * 'i'l * Contribution No. 393, Gulf Breete Environmental Research Laboratory. t 'i I HONS 203520 ^ww 'at a- INTRODUCTION lI , \ ' '` ' Estuaries are dynanic environrants and animals that live in - then nust be able to cope with change. Soce dynamic natural fac tors are temperature, salinity, currents, hydrostatic pressure, and oxygen or carbon dioxide concentrations. .Unfortunately, - * * domestic sewage (nutrients), oils, industrial chemicals, pesti- ^ cides, metals, or altered temperatures are an influence In estuaries. Unfortunately, the combined affects of the natural and tan-intro duced factors on estuarine organises are largely unknown. Excep tions are the estuaries which receive undue amounts of pollutants that far all practical purposes are biologically dead. In contrast, wen interactions occur gradually and in combination, the effect could be the death of assemblages of organises or an entire estuary without our being aware of the trend. Therefore, the problem facing us today is understanding and predicting the interactions of pollu- a tanta and natural stresses. \ Xt Is coacan knowledge that the commercial shrimps along the Gulf Coast undertake distinct auryhallne migrations. After adult nrrin? spawn in the open Gulf from spring to fall, the post-rysids end juveniles migrate into the fresher waters of bays where they grow rapidly to adulthood before returning to the Gulf. Obviously, these stages of shrir.? isust be able to adjust to the changing salinities encountered in tha estuary and this adjustment requires physiological facilities which'must operate at optimum. One group of chemicals introduced by nan that has recently been the concern of many ecologists is the PCEs, or polychlorinated HONS 203521 ' GO biphenyls. In 1969, a PCB, identified ns Arcelor 1254 was discovered as a contaminant in water, sediment, and fauna 1 of r.scambia Bay, Florida (Duke, Lowe and Wilson, 1970). An early survey indicated that whole body residues of the chemical in feral shrimp were as high as 14 mg/kg whole body (Niunro et al., 1971a). Subsequent toxicity tests on juvenile pink shrimp (Penaeus '" duorarum) revealed that about l.Oog/i in the water would kill % 50H within 15 days (Niaico et al., 1971b). ' ** While conducting bioassays at our laboratory w*.noted on several occasions that salinity appeared to affect toxicity. * In one instance, ve conducted a chronic exposure In which adult ' pink shrimp, were exposed to a sublethal concentration of the chemical (about l.Oyg/t ). The purpose of the test was to determine if structural damage night occur in gill tissues. On the 27th da^ of exposure at vhich time we had recorded no previous deaths from the PCB, the salinity of .the incoming water decreased oo ` from 20v/oo to 11 /Qo within 4 hours due to rain, tides and wind. The reault was death of ten experimental shrimp before the o salinity had returned to 20 /co During the next two days, the salinity was lowered again by aberrant tides and climatic condi tions and store experimental, but not control, shrimp died. We, therefore, beesne interested in the possible interaction of Aroclor 1254 end environmental stress, particularly the effuct of PCB on the ability of shrimp to regulate osr.oticallv and ionically at reduced salinities. i' t MOMS 203522 MATERIALS A>T> METHODS Adult brown shrimp (Penr.eu3 aztccus), 11.5 - 13.7 cm, rostrum to telson, were captured near Culf Shores, Alabama and used in ouw Gtudi.es. Approximately equal numbers of both sexes were used and the methods of exposure to the Aroclor 1254 were'similar to those * * reported previously (Niir.no et al., 1971a). The modifications were - (1) ambient salinity was maintained at 30 1 /00 and temperature, at 25 + 2 C, and (2) the exposures to the chemical were "sublethal" and lasted but 7 days* Three-ys/JL was chose a as the test concentration i because previous tests with adult brown shrimp as veil as adult pink shrimp (jP. duorarum), demonstrated that this concentration would causa 50 'foo mortality within 30 day3. i Following exposure to PCB, equal numbers of PCB~exposei and "control" shrimp were transferred to separate aquaria at ambient temperature and salinity (Tig. 1). While temperature was kept constant, the salinity in each equariun vis gradually lowered during 8 hours to a`predetermined level. Sine* the possibility existed of physiological stress from handling or inherent in the experimental design, both PCB-expos-id ard control shrimo ware analysed for osmotic and ionic concentrations after going through the procedure without external salinity change (30 /oo). Fot the first group, the salinity was maintained at 30 /oo fot 8 hours; the second, salinity vas lovared from 30 /oo to 22 /oo; the third, from 30 /oo to 10 /oo, and the fourth, from 30 /oo to 7 /oo. Although there was a time differential between groups of shrimp, end therefore, a possible MOMS 203523 CONTROL SHRIMP Ctrrier only * 1t Held for 7 days in flowing water at 30+1 /oo salinity EXPERIMENTAL SH?.T;f? Aroclor 1254 (3 wg/t) + catiici --:___ ------------------------------- Salinity stress-* flowing water with the capacity of controlled flux of B-hr duration 1-Z.! * Blood sample drawn fran pericardial sinus, placed In glass tube, allowed to clot and oseotic concen- . tratlon determined I, * - . - --...... Shrln? saved for resid-*.enalysis (GLC) Seruai separated from clot, placed In assail'crucible, ashad at 480 C for 6 hours and ash dissolved in 2B V4 / Delamin&uion of cations (Atonic absorption) Determination of chloride (Couloaetric titration) FICL7.E 1. FLO'.V SHEET FOR EXPERIMENTAL PROCEDURE HONS 203524 '*- J 1 II Table 1. ^ ' I. WHOLE BODY CONCENTRATIONS 0f AROCLOR 1254 IN FCB-EXPOSED SHRIMP* * oo o SALINITY AVERAGE irR/ksc RANGE 30 9.6 3.0-13.7 22 7.8 1.9-18.8 .1 0 7.1 * 3.9-14.0 7 . 8.5 2.8-15.0 . Concentration of Aroclor 1254 In the test water was 3 ppb; length of exposure,_7 days. Control shrimp had less than 0.1 ppm of Aroclor 1254. _ MOMS 203525 difference in test animals due to a slight loss of PCB; analyses I for the chemical revealed no significant difference in whole body ** % concentrations among groups (Table 1). As in earlier studies, there was a wide range in individual concentrations of PCB (Nimno at al., 1971b). ' *. * '* Samples of whole blood (hemolyraph) for osmotic concentration were removed from each shrimp by pericardial puncture. A ground- glas3 syringe (1ml), fitted with a 022 gauge stainless-stttel needle was Inserted into the animal at an oblique engle to obtain at lease 0.3 ml of blood from each animal. The blood was immediately transferred to an osmometer tube and the osciotic concentration "" determined with a Fisk Model G-66 osmometer. Since the blood clotted quickly, it was difficult to determine whether we measured osmotic concentration on whole blood or on serum, but on analyz ing them separately, ue found no significant difference In their osmotic concentrations. Replicate determinations of 10 separate t aliquots of pooled sera from sevsral shrimp yielded a standard error of 1.2 (mean concentration- 629 mOs). , Analyses of ions was performed on ashed sera. To prepare the Sample, the clot contained in the osmometer tube was squeezed with a small glass rod, the clot was removed and exectly 0.2 ml of the serum was transferred to a small crucible. The crucible was placed In en oven and the contents ashed at 480 C for 6 hr, cooled end the ash was dissolved in 2H H SO . Analysis of chloride was performed 24 t with a Buchlcx-Cotlove Chloridometer and cations were determined on a Model 403 Perkin-Elmer atomic absorption spectrophotone ter ................. _ . _ . HOMS 208526 equipped with a deuterium arc-background corrector and' an HGA-70 heated graphite atomizer. Cations in standard solutions vere in the same proportions as those in the sera. As a check on our methods, an analysis of a single aliquot of serum by emission and atomic absorp tion yielded Identical results for the eleraeAt; potassium. Five replicates of pooled sera from several shrimp yielded a standard- error of 0.24 mEq/1 for Cl (mean " 257 oEq/l). Replicate analyses on seram aliquots yielded standard errors in mSq/1 of 8.26 for Fa (mean 324.9), of 0.04 for Mg (mean 16.0), of 0.07 for K (mean__ 8.1), of 0.19 for Ca (mean * 4.74), and of 0.10 for Cu (mean - 2.84). Hie 95% confidence interval vaa used to evaluatq significance as _ of differences in the data. The 95% Intervals are Indicated in the ' graphs by vertical bars on each datum and are listed in each tabic. It ia aometimes difficult to relate "osmolality" or "osmotic con centration" to the environment. For the purpose of opr discussion of the marine environment, we expressed the concentration of the ' environment as salinity. The relationship between oOs and salinity 1$ Indicated along the X axis In Figure 2. . * Concentrations of Aroclor 1254 ware determined on individual shrimp by gas chromatography, using procedures summarized earlier (Nimmo et al., 1971b). RESULTS The most significant result of this study vas the discovery thnt a sublethal concentration of Aroclor 1254 at constant salin ity for 7 days heear.a lethal when shrimp vara subjected to a MONS 20352? |W lrf1 SSRUM (m Os) ENVIRONMENT HONS 203528 FIGURE 2 OSMOTIC CONCENTRATION t SERUM-ENVIRONMENT IN PENAEUS AZTECUS (after McFarland & ,Lce, 1?G3). Our study ' covered the range of 7 /oo to 30 /oo salinity. / HONS 203529 l gradual decrease in salinity over an 8-hr period. Usually the shrimp exhibited increased swimming activity while the salinity changed front 30 /oo to 20 /oo, and this was the only behavioral aberration observed. Deaths In experlmental shrimp began at a salinity of about 12-13 /oo and at 10 /00 and 7 /bo, when * m 8 hours had elapsed, mortality of experimental shrimp was nearly,50Z. Khan 5071 of the experimental shrimp had become moribund or had died, living PCB-e:cposed shrimp were taken for analyses of osmotic con centrations and ion determinations on sera. - The results of these analyses indicated that concentrations of most major Ions In the sera of FCB-expo3ed shrlnp became significantly less as the ambient salinity decreased, the sum of major ior.s (e.g. ' Na, Ca, Wg, K, Cu, and Cl) being 187. less after ambient salinity reached 10 /oo or 7 /oo (Fig. 3). .Of this total, sodium was 162 less (Fig* 4), chloride, 197 (Fig. 5) and caldtan, 257 (Fig. 6), There was some indication that magnesium decreased, but not significantly (Table '2). No apparent differences in potassium (Table 3), or copper (Table-A) were noted. , Data for iron(T_bla 3) are not included in the totals in Fig. 3 because we could not distinguish the divalent from the trlvalent form. ' Despite significant alterations in the major ion complement or in some major ions, osmotic contentration was not significantly affected by PCB and salinity stress (Table 6). Seemingly, osmotic O, o. pressure was lets in rC3-cxposed shrimp at 10 too or 7 too HONS 203530 ^OOr f? Lr-.* i P 1175 i 950 725 -JL 00*r' 10 SALINITY (%Q) 30 MONS 203531 FIGURE 3. TOTAL IONS IN BROWN SHRIMP SERUM IN RELATION TO SALINITY * f MOWS 203532 'W S O K JU .-U (n ii'a /] ) f FIGURE 4. SERUM SODIUM IN BROWN SHRIMP IN RELATION TO SALINITY HONS 203534 NOWS 203535 FIGURE 5. SERUM CHLORIDE IN BROWN SHRIMP IN RELATION TO SALINITY HONS 203536 SGRSJiV) CAS.CJUJV1 (mSr/J) I MONS 203537 I \ FIGURE 6 SERl'M CALCIUM IN BROWN SHRIMP IN RELATION TO SALINITY t i / * *Q*S 20353a Table 2 AVERACE SERUM CONCENTRATIONS OF MAGNESIUM IN BROWN SHRIMP IN RELATION TO SALINITY SALINITY CONTROL nEq/1 957. CONFIDENCE EXPERIMENTAL INTERVAL . 95% CONTIDENCE INTERVAL oa o*- 30 20.1 16.7--23.5 22 16.3 14.2--18.4 10 14.8 12.4--17,2 7 15.0 11.7--18.3 17.3 14.6 13.0 11.5 12.7--21.8 12.3--16.9 11.0--15.0 8.9--14.2 NOWS 203539 * L \ < Table 3. AVERAGE SERUM CONCENTRATIONS OF POTASSIUM IN BROWN SHRIMP IN RELATION TO SALINITY ' SALINITY CONTROL /oo nEa/l 95Z CONFIDENCE INTERVAL EXPERIMENTAL 957. CONFIDENCE INTERVAL 30 14.2 22 11.7 10 10.4 7 9.6 13.5--14.9 11.1--12.3 9.8--11.2 8.4--10.7* 14.7 12.1 10.1 10.0 - 13.6--15.7 " 11.2--12.9 8.7--11.4 8.4--11.6 / HONS 203540 j Table 4. . _ * * ' AVERAGE SERUM CONCENTRATIONS OF TOPPER IN BROWN SHRUIP IN RELATION TO SALINITY SALINITY CONTROL /oo mEq/1 952 CONFIDENCE EXPERIMENTAL INTERVAL 95% CONFIDENCE INTERVAL 30 4.7 3.6--5.9 22 4.0 3.4--4.6 10 5.2 4.0--6.4 7 4.8 3.9--S.7 5.3 5.9 4* 6 5.7 4.4--6.3 * 4.8--7.0 3.8--5.3 5.0--6.4 HONS 203541 Table 5. AVERAGE SERUM CONCENTRATIONS OF WON IN BROUN SHRIMP IN RELATION TO SALINITY SALINITY CONTROL /oe nM/1 95% CONFIDENCE EXPERIMENTAL INTERVAL 95% CONFIDENCE ItNTERVAL' 30 .23 .17--.29 22 .28 .15--.35 10 .20 .15--.25 7 .24 .14-- .33 .31 .15--.48 .18 ." .14--.22 .26 .17--.35 .33 .24--.41 HONS 203542  . . t ' Table 6. AVERAGE SERUM 05N0TIC CONCENTRATIONS IN BROVT,' SHRIIt? IN RELATION TO SALINITY SALINITY '_________________________ tilLLIOSMOLES CONTROL 95% CONFIDENCE EXPERIMENTAL 7oo INTERVAL 95% CONFIDENCE INTERVAL 30 749 728--771 - . 752 22 687 665--703 688 10 551 SOS--593 523 7 547 521--572 516 726--779 653--718 500--546 495--537 HONS 203543 0MP4tWnW\fJ salinity but Individual variation was too groat to chow a signifi cant difference from controls. DISCUSSION .* Knowledge of interactions between toxic 'compounds and envlron- - mantel factors is essential for predicting their effects on ecosys tems or species. Examples of this need have been demonstrated in both fresh and marine investigations. In fresh water, low-levs 1 chronic exposure of the darter (Etheostona nigmaj'to dieldrin greatly effected its ability to survive thermal stress (Silbergeld, 1973), The sublethal effects of mercury on fiddler crahe (Uca pugilator) reduced survival times when crabs were placed under temperature and salinity stress (Vernberg and Verabarg, 1972). Mortality of fiddler crabs previously exposed to cadmium was great est at high temperatures and law salinltes (O'Hara, 1973). In our studies Aroclor (^TD 1254 possibly interfered with the adenosine -triphosphatase (ATPase) activity in gills of shrirp. ATPas* activity is associated with actlva ion transport (Tanaka, Sakamoto add Sakamoto, 1971), Polychlorinated Insecticides and the related polychlorinated biphenyls have bsen shown by several "in vitro" essays to inhibit ATPase in the tissues of fishes (Davis and Tedcmeyer, 1971e; 197ib; Davis, Friedhoff and '..'edameyer, 1972: Cutkor ct al., 1972; Yap et al., 1971) and in the nerves of lobsters (Natsun ura and Narahashi, 1971). The need for greater.-efficiency or c.ipacir. of ATPase in marine organisms can be inferred from the results of HONS 203544 PEeiler and Kirschncr (1972), v.ho should that gill ATPase activity of rainbow trout adapted to salt water was greater than in fish adapted to fresh water. . Polychlorinated hydrocarbons have interferred with either *. osmo- or ionic-regulation in aquatic animal's (fcisler and Edmunds, 1966; Grant and Mehrle, 1970; Kinter et al., 1972; TCirmo and Blackman, 1972). Tha physiological relationship of ionic effects to that of ATPase activity was first reported by Writer et el. (1972), who postulated that lipophilic agents such as DDT and PCBs, might interact with the phospholipid-activating components of the lipoprotein enzyme. The effect of dieldrin on ion movement in the narvoua system of cockroaches shewed that dieldrin inhibited bind ing of calcium to tne phospholipid moiety of the snzyme, thus inhib iting the movement of calcium across the nerve membrane (liayashi and Matsunura, 1957). Calcium salts, in fresh water greatly in creased the ability of marina and euryhaline animals to survive in that medium (Black, 1957). Toxic symptoms of DDT poisoning ir. fr*3h water fish could be alleviat'd by the addition of cal cium salts (Keffler, 1972). In our studies calcium was lowered 257. in the sera of PCB-e:cposed shrimp at 7 /oo salinity, and it may be that this reduction vas responsible in part for tha observed decrease in sodiuw, chloride, ar.d other ions. HONS 203545 I l t Field observations of juvenile and subadult bro-m shrimp Ly \ several investIgators Indicate that those shrimp tolerate a wide Targe of salinities: v Salinity 0.22 - 0.36 /oo 0-1.0 /oo 0 - 2.0 /oo 69 /oo 'Location ,, "" St. Lucie estuary, Fla. ` Observer Cunter and Hall (1963) Mobile Bay, Ala. Loesch (In Cunter at al. 1964) Choctawhatchee Bay, Fla. Hitcoo (unpubl. data) Laguna Madre, Texas (195?) Cunter, Christmas, and Ktllebrew (1964), found that in Texas . bays, young bro'^r shrirp were most abundant at 10 to 30 /00>vith greater abundance above 20 /oo. Zein-Eldin end Aldrich (1965) found that postlarval brown shrina withstood a wide range of salinity- temperature combinations. . ` It is evident that adult shrimp are osmoreguleters at all but extremes of salinity (Fig. 2). Williams (1950) gives the isosrotic point of shrimp herolyr.ph at 26.5 C/oo (783 nOs), as conpared to our ealculation of 23.4 /00 (694 m.Os) in control shrimp. Nevertheless, there was no significant diffarer.ee in FC3-exposed and control shrimp (Table 6). Obviously, e slight change in environmental osmotic pressure would not be critical to the osmoregulatory ability of the animals et isosroticity, but was critical in the dilute environment. Therefore, tht salinities where the PCE exerted its MQNS 203546 greatest effect in the laboratory (as judged from mortality of shrimp) were well within the range in which brown shrirp occur in nature. . Although no appreciable difference occurred ^ln osmotic concen tration, there was significant diiference in majonions In the sera of - / ' PCB-exposed shrimp (Fig. 3). McFarland and Lee (1963) found that ,, the point of convergence of the total ions in the hesrolymph of feral shrimp to that of the environment was 27 /oo (800 IDO3). In our study, by extrapolation, convergence occurred at 34 /oo`{1000 tr.Os) in controls, whereas in PC3-exposad shrimp showed no convergence and total Ions paralleled unity. * In surveys conducted soon after PCB was first discovered in the Pensacola estuary the distribution of shrimp in relation to salinity seemed to be related tha amount of the chemical in the animals (Nieto et al., 1971b). Of three species captured, brown shrimp had the highest whole-body residue's (14 ppm) although most sarnies were lower. Beamingly, a concentration of 14 ppm PCB in feral shrimp would have been lethal if the animals ware subjected to salihity stress such as imposed by our experimental procedure. V'e have unpublished data that suggast existence of a threshold in average whole-body concentration of PCB (5.6 to 7.8 mg/kg) in oink shrimp (P. tluoraru") that would be lethal when superimposed "1 on salinity stress caused by our procedure. However, a recent survey of feral shrimp from the Pensacola estuary showed that young adult shrimp now have only a fraction of PCB concentrations found in 1969/70 ""W *035<7 periods. For example, a sample taken from Escadbla Bay in Auguat, 1973 had a whole-body concentration of'only 0.1 trg/kg. Future studies should include research on interaction of PCB and salinity on juvenile and postlarval ^hrinp since chronic ,, ' toxicity tests have shown that these stages were nore suscept- , ible to the chemical (Kitano et al., 1971a). In addition, studies by Dana Beth- Tyler-Schroeder, of the Gulf Breeze laboratory, have showti the susceptibility of larvae of grass shrimp TFalaenonetes puglo) to the PCBs, Aroclors 1016 and 1242, decreases with age (personal communication). Also, the "in vivo" effect of PCBs on* * ATPa&e activity in ehrinp should be fully Investigated. t* i MOMS 203548 LITERATI fRE CITED Black, V. S. 1957. Excretion and osmoregulation. In Physiology f Fishes, pn. 3 63-199, ed, by M, F.. Brown. Kew York; Academic Press. / Cutlaomp, T.. K., Yap, H. F., Desal&h, It., and<Koch, R. B. 1972. The sensitivity of fish ATPases to polychlorinated biphenyls. Environ, Health Perspect., 1; 165-168. Davis, F. W., Friedhoff, J. H., and Wedexeyer, C. A. 1972. Organo- chlorine insecticide, herbicide and polychlorinated biphenyl (PCS) inhibition of Na, K - ATPase in rainbow trout. Bull. Environ. - - Content. Toxicol., 8: 69-72. ----------- - - Davis, P. W. and Wedeneyer, G, A. 1971a. Inhibition by organo- chlorire pesticides of I'a, K - activated adenosine triphospha tase activity in the brain of rainbow trout. Proc, West. Pharmacol. Soc., 14: 47. Davis, P. V. and Wedeoeyer, G. A. 1971b. Ita, 1C -- activated ATPase inhibition in rainbow troutt a site for orger.ochlorine -pesticide toxicity, Coro. Biochan, Physiol,, 40B: 823-927, Duke, T. W., Lowe, J. 1., and Wilson, Jr., A. J. 1970. A poly chlorinated biphenyl (Aroclor 1254) in tha water, sediment, and biota of Fscanoia Bay, Florida. Bull. Environ. Contan. and Toxicol., 5: 171-180. Eisler, R. and Edmunds, P. H. 1966, Effects of endrin on blood and tissue chemistry of a marine fish. Trans.-Am. Fish. Soc., 95: 153-159. MOMS 203549 1 Grant, B, T. and Tlehrle, P. M. 1970. Chronic endrin poisoning in I goldfish, Carasslus auratus. J. Pish. Res. Board Canada 27: 2225-2232. * Gunter, C. and Hall, C. E. 1963. Biological investigations of the St. Lucie estuary (Florida) in connection *dth Lake Okeechobee , * '' ' discharges through the St. Lucie canal. Culf Rea. Pep. 1: 189-307. Cunter, C., Christmas, J. V., and Killabrew, R. 1964. Some relations of salinity to population distributions of aotile estuarine organisms, with special reference to penaeld shrimp. Ecology 45: 181-185. - Hayashl, K. and Hatsumura, M. 1967. Insecticide node of action: * e> * Effect of dleldrln on ion movement in the nervous system of . Perloleneta r.nrlc?na and B1 a telle gem?nlca cockroaches. J.Agrlc. Food Chen., 15: 622-6.27. Keffler, L. R. 1972. A study of the Influence of calcium on the effects of DDT on fishes. Ph.H. dissertation, Dnlv. Hiss., 131 p. Xir.ter, LT. B., ITsrker.s, L. S., Janicki, R. H., and Guarioo, A. M. 1972. Studies on the mechanism of toxicity of DDT and ooly- . chlorinated biphenyls (PC3s): Disruption of osmoregulation in marine fish. Environ. Health Perspect. 1: 169-173. Hatsumure, F. and Harahashi, T. 1971. ATPase inhibition and electrophysiologleal change caused by DDT and related neuro _ active agents in lobster nerve. Rioehem. Pharmacol. 20: 825-837. HONS 203550 i McFarland, N. V. find l.ee, P.. P. 1963. Osmotic and Ionic con centrations of penaeidean shrimps of the Texa* coast. Bull. Mar. Set. CulE Carlhb. 13: *191--417. Ninm.o, I). R. snd Blackman, R. R. 1972. Effects of DDT on cations In the hepatopancreas of penaeld shrimp/ .Trans. Am. Fish. Soc. . * ' 101: 547-549. * Nimmo, D. R,, Blackman, P,. R., Wilson, Jr., A. J. and Forester, J. 1971a. Toxicity and distribution of Aroclor 1254 in the pink shrimp, Penaeus duoraruo. Mar. Biol., 11: 19i-197. Nimmo, D. R., Wilson, P. D., Blacknan, R. and Wilson, Jr., A. J. 1971b. Polychlorinated biphenyl absorbed from sediments by * fiddler crahs and pink shrimp. Nature, (London) 231: 50-52. O'liara, J. 1973. the influence of temperature and salinity on the toxicity of cadmium to the fiddler crab. Pea pugllator. V. S. Fish, ttildl. Serv. Fish. Bull., 71: 149-153. Pfeiler, E. and Kirschner, L. B. 1972. Studies on gill ATPase of rainbow trout (Salno gairdnerl). Biochim. Biopbys. Acta, 282: 301-310. Silbergeld, F. K. 1973. Pieldric: Effects of chronic sublethal , exposure on adaptation to thermal stress in freshwater fish. Environ. Sci. Tectmol., 7: 846-849. Simmons, E. G. 1957. An ecological survey of the upper Laguna Hadre of Texas. Publ. Inst. Har. Sci., Univ. Tex. 4: 156-200 Tanaka, R., Sakamoto, T. and Sakamoto, Y, 1971-. Mechanism of lipid activation of Na, K, Mg - activated adenosine triphosphn tase and K, Mr - activated phosphatase of bovine cerebral * J 'i i' Uir - . * lwte * *> * . y^v Ofi TVS'** T~~- r** ,i* 1 , HONS 203551 `t*. cortex. I J. Nrtnbranc. Biol. 4: i 42--51.^ Vernberg, W. B. and Vernberg, J. 1972. The dynergistlc effects of temperature, salinity and mercury on survival and metabolism of the adult fiddler crab, Hca puellator-.' U. S. Fish. Vildl. Serv. Fish. Bull., 70: 415-420. - *. * Williams, A. B. 1960. The influence of temperature on osmotic , regulation in two species of estuarine shrimps (Penaeus). Biol. Bull.' (Woods Foie), 117: 560-571. Yap, H. H. , Vesalah, D., Cutkorap, L. K. and Koch, R. R. 1971. Sensitivity of fish ATFases to polychlorinated biphenyls. Fature (London), 233: 61-62. v Zein-Eldin, Z. P. and Aldrich, H. V. 1965. ' * Growth and survival of postlarval Per.aeus astacus under controlled conditions of temperature and salinity. Biol_ Bull. (Woods Vole) 129: 199-216. . HONS 203552 '1 Effects of Aroclor 1254 on Lnborr.tory-tt'jnred ' ` 1_, br>os and Try of She::;'she ad Minnows (Cyprinodem verierntus)^ Steven C. Sehlncel, David J. Fansen and Jcrtold Forester (3) U. S. Environmental Protection Agen-cJ' Cul Freere Environmental Research laboratory . Sabine Island, Culf breeze, Florida 325S1 (Associate Laboratory of the Eationei Environmental Research Center, Corvallis, Oregon) "` Contribution Ho. 173, Cul5C hreasa Environmental P.esearch Laboratory. ^ ibjj.istered tradaaarlc, Monsanto Co., St. Louts, Mo- Mention, of commercial products does not constitute endorsement by the T-avironnental Protection . ''uncy. HONS 203553 ` ABSTRACT . i Stcvun C. . iic.himel ' . i.t tLEicially fertilized shcepshead oiucow (Cvruripodor*. variegates) cgg3 v^r': c.'po'.ed to logarithmic concentrations of Aroclor 1255 <10.0 to 0.1 yg/l) in ssaiatcr avcrzgias 30 C and 24 /oo in tn in remittent-flaw bioassay. Fertilization was not affected but significantly fewer eabryos developed in the 10.0 pg./l and fewer fry survived in concentrations greater than 0.1 V'J 1. Try were more susceptible to Aroclor 1254 than were Juveniles or adults. Shscpshead cinrov eggs were artificially fertilized and maintained at temperatures fren IS to 35 C and in salinities, frea 0 to 35 /oo to datamba efficient culture conditions. Fertilization was not affected by the * te=p?ruturc end salinity ranges chosen, but hatching success was greatest ;.<*'=0.01) ct a temperature range of 24 to .35 C end e salinity range, of 15 to 30 o/oo. , M0NS 203554 If. nfODUCTIOM (Fur 1 -t , 1973), end tin; occurrence of one, Aroclor 1254( in nearby I'scnuMa Bn> ami its aculc LoxiciLy to .estuarine animals hea been documented (Duke ct b\ . , 1970), Hausen et al. (1971) found 5 pg/t of Aroclor 1254 to'dc to the juvenile estuarine iLsnas, pir.fls'n (Lagodon rhonboldes) end spot I '* ' * (Lciostonuy xnnthurus), in 14 to'45 day bioassays, tie are unaware of ----- 1----------------------------------- data un the effects of this PCB on embryonic and larval stages of estuarine I fichen. .. The sheepshead minnow (Cyorinodon variegates) is found in brackish waters fron Cape Cod, Massachusetts to Brownsville, Te::cs (Hildebrand, 1917). It Is important in estuarine food chains as a voracious omnivore and as food for pjrcdetoib ^such as croakers (mcro-po^on. unduiatus) and spotted seatrout (Cynoscion. r.ebulosus) (Darnell, 1958). The size, hardiness, high fecundity, and gar.Eratio-i time of the sheepshead ninzov cakes it a nearly ideal labora tory test fish. , : In this paper, we determined water temperatures and salinities suitable for the culture of embryos end fry of the sheepshead minnow end observed the effect of Aroclor 1254 in water on the various life stages of this fl,. ' ",. kT-"' ~ i`jy "i jt!,, ___ HONS 203555 t ML'i'UODS A.ND MATERIALS Steven C, Schinral Temperature ar.d Srlirlty Studies fc^arly identical methods vere used in. the tenoeratura and salinity cco^ri-- 1 .' " rents to fertilize eggs. Adult fish were acclimated for one week in salt water averaging 25 C and 20 o/oo. The following week, female fish were given three intraperitoaeal injections of 50 I.U. human chorionic goan- trophic hormone to induce egg maturation. Over 701 of the fish produced * '^ viable eggs. gS3 were manually stripped and deposited in AO nl of 1 `* filtered seawater. Only large, round, clear eggs were selected for fertilisation. Testes from 7-10 acclimated males were excised, macerated in 2! rl of`filtered seawater end mined with eggs in a 100 nl beaker, allowing 30 minutes fer fertilization. Seawater conditions^used! for fertilization in the temperature test vasy25 -C and 20 0/00 salinity while the salinity test conditions were 30 C and the test salinity. Each. experiment lasted as long as was required for embryos to hatch plus an. . *\ ' '' . additional two weeks to determine survival of fry. - The effect of temperature on hatching was inve3tigatsd by withdrawing 25 egpo from t,p,e 100 ml beaker with a ^rLde--bore pipette and placing then re two, 1.* dishes of 25 C, 20 /oo seawater. Each oE the dishes ware partially submerged ir. a bath at 15, 20, 25, 30, or 40 C in one temperature test ar.i 22, ?i, 26 or 2S C in another. One hour later, when temperatures in the dishes equalled the bath temperature, fertilization was confirmed micro scopically. * The criterion for fertilization was cleavage. Dishes rare checked drily during the 3-week static tests and dead or non-fortile cf, ;% a:*d larvae were removed. The criteria for eebryo mortality was ........................................................... ....... - HONS 203556 t Steven C, Schirnral .. in op'.r.iz, white, fungal growth and for the larvae a white coloration of the trunk musculature. Tcapcrasures ver-a monitored continuously and. were within 1 C of the desired temperatures. Salinity alterations due to i 4* evaporation in both the salinity and temperature ,tcst*i were cocpensated 1 , * r- ' for daily by addition of distilled water. .. i Fertilization, procedures for the salinity study were identical to those of the temperature study-except that the number of eggs in each salinity varied from 35-75* depending on egg availability. Filtered seawater from Santa Rosa Sound* Florida was diluted to give salinities of 0* 5, 10* 15, 20, 25, 30 and 35 /oo. Tha 30 and 35 /oo concentrations ware attained by ceding Rila''-'salts to tha water froa the Sound.' Salinities, checked daily with a TS refractoa2ter, were within 2 /oo of the desired level. ' *' r * Aroclor 1254 Exposure ' An Aroclor 1254, intermittent-flow bioassay was accomplishad using culture car.ditionW determined in the temperature and salinity tests- ni t '*,* r`` Eggs were fertilized in control water and placed in Fetri dishes ~ four V.. dishes (20 eggs per dish) in each PCS concentration and control. Fertility V23 confirmed alter one hour. Temperatures in both tests averaged 29 C (range 27-31 C) and salinity averaged 24 /oo (range 1^-32 o/oo). The salinity fluctuated With that of Santa Rosa Sound. The toxicant dosing 4 system used in thlc test vuS a modification of the apparatus of Brungs and .* -*r Mour-t (1970) . In our systea Lha toxicant and carrier were injected into the delivery'tuba leading to each exposure tank (Figure 1). Our apparatus $ Registered Crndcnark, Rila Products, Teaneck, R.J. Rvp.iMorcd trademark, American Optical Corp., Buffalo, E. Y. HONS 203557 Stevt-n C,, Sc.hic=eL ` ailo.ed us to retain the advantages of the Mount and Erungn (1967) do-in?, , spp?ratuE and to select any concentration of toxicant while 'maintaining the saj-e concentration of carrier in each toxicant concentration. jeauater used in this bioassay was pimped from JJfmta Rosa Round into a constant bead box in the laboratory. An oscillating' pump .(1A) , regulated by a rheostat, pumped water from the head box through a 20 p --pore polypronyleca 1' filter end into the compartments in the dosing apparatus. Artur all con-- partntats were filled, the self-starting siphon in tfce~last compartment enptied into a receiving bucket (IB). The weight oE the bucket being filled operated two micro--switches. One switch (la) shut off tha cs cilia ting pun? and the other (le) activated a solenoid (IE) that raised the lever of the injection apparatus (IT). Tnis rising lever was connected to gears that . forced tha barrels of six 50-nl syringes (1G) equal distances. Kiting bottles (IB) received Injections from a control syringe containing the carrier (polyethylene glycol 200) . Stock solutions of the carrier' and G.i,-Aroclor 125*4 were mixed to give concentrations 6.32, 1.0,- 3.2 end 10.0 -* - -p3ft of Aroclor per.litsr of water. Tha water then flowed to thi distribution. 'i hoxa3 (Figure 2) containing one large and four small cocpaxtnects, each with a standpip? or siphon. Each compartment filled completely during each acd the siphon del.itvveerread water to tha aquarium in which adults ar.d ***** . ` ` * * "* , ` ' P juvenile fxsh ware held, Hater renair.irg in the four small compartments flowed slovly out through submerged holes in the standpipes into each of four containers holding eggs or fry. Each container received 100 cl ot "water per'cycle. The number of cycles per day ranged from 100 to 140 ap.d veio sufficient to replace voter in etch egg"container nt least three time; each day. HONS 203556 I l Steven C. Kciticac-il iii.lc effects of polyethylene glycol 200 in this.tcsc (7.4`Dg/i) ware not l . ic ipatcd because (1) VL v/v polyechylsce glycol was not lethal to j-.-vonile sheepsheod minnows "in 96 hour tests and (2) iu earlier tests at this laboratory, toxicity of 5 pg/i of Aroclor 1254 to pinfish. vas not i changed by an increasing solvent concentration, oue^hundred ties* (0.1 to 10 eg/1) (Hansen, unpublished data). Chemical Methods Aroclor 1254 concentrations in test and control water were determined * weekly by gas chromatography. Methods were the sane as those of kicno et cl. (1971), except that an 07-101 colurm was used end all peak heights were avereged for PCS quantification. Measured PCB concentrations in test water were 'typically 35Z to 601 of nominal concentrationsj control water contained no ^detectable PC3 (<.03 pg/l). Recovery efficiency of Aroclor 1254 was greater than 60Z. Measured concentrations were not corrected for percentage recovery. 1 *. ' Dissolved^'QX-y^en- was determined vaekiy using . the .modified Winkler cethcd (Strickland end Parson*, 19SS). Concentrations seemed adequate _And above 507, saturation.. . , , Statistical Zlithods . . . j'' * --~--:-- ........................ Ttv Chi-square (x^) test was used in the statistical analysis of the data. In temperature end salinity studies the maxitcun positive response wei compared viith all other responses to determine the most efficient culture conditions. Pata in the two temperature experiments were ar.alyre'd. scpnratqly. ` - - ------- -* .- >IHI HONS 203559 .1 PXSULTS AND DISCUSSION Steven C. Schinmel Temperature Study -j Temperature determined the nuaber of days required for hatching (tiica-to- hstch) and affected the srrvival of embryos and fry of the sheepshead cinno-J A (Trblc 1). Sheepshead minnow eggs hatched at water tempira-turns froa 22-40 C *" * t but none hatched at lover temperatures. Hatching success vas greatest at temperatures ranging fron 24-35 C. Try survival did cot differ in. the range of 22 to 35 C. The longest tins-to-hatch was 15 days at 22 C, and. decreased rapidly, leveling off at about 4 days at temperatures ranging from 30 to 40 C. Salinity Study ,, Salinity affected survival of embryos and fry but did not affect tima-to- hatch (Table 2). Survival to hatching was greater at salinities from. 15 _ to 30 o/oo and fry survival did not differ in that range. Under natural conditions, adult end juvenile shaapshaad ainnowa occur in a wide salinity range (SIxpson, 1956). Our data show that adbryos end fry also can. Aroclor 1254 effected the survival of both embryos and fry of the sheapsnaad minnow, but had no effect on tine-to-hatch. Ecbryos devalboad and hatched at all PCB concentrations, but hatching success at 10 pg/2, was s i g p.i f I c an tIy--1e s ' th an' that - of-coh trbl -'eggs I ~'"Af ter the`eggs hatched, ' survival of fry was significantly less than control fry at all PCS concentrations except 0.1 pg/i. Mortality increased with as increasing concentration of PCB,. Three-^jeek LC50 in exposed embryos and fry vas estimated at 0*53. hz/i (S.E. "y._0.Vg/i) .. Try did not die immediately i HONS 203560 IA--' ... hatching but ';~'rc billed o^cr no-.w oT toe tvo-ves-h period ot pocL ' t h.^tdi exposure. llany of the dying fish developed fin rot as previously , described in other PCB exposed fishes (ilsnsen et al., 1971). Aroelor 1254 was more toxic to fry of sheepshead miutio-.m that it was to juveniles, adults or to the fertilization of eggs (Table 4). In three week | . ^` exposures to the same concentrations of this PCB, mortality of the juveniles !* iras significantly greater (twenty-four percent) in the 10p.g/" concentration. Adult fish were not killed in a three-uaek exposure ut became lethargic and exhibited fin rot. The concentration of PCB in the fijecoapared to that in. the water (concentration factor) was nearly identical with that of adult fish. Concentration factors in fry ranged from 1,6 to 3.2 x llA and those f jr adults ranged from 1.1 to 3.2 x 10^. A twenty-four hour static test in i 10.9pg/^ PCB showed no inhibition, jof fertilization of aheapshead ninrew egg;. The greater sensitivity of early life stages of this fish to Aroelor 12J4 stresses the need for bioassays designed to assess the effects of such chemical pollutanto. on" these- stagey. ' 1V" ' * " HONS 203561 ACta:0tflXDCE:L2ITS - . ' < .1 - -* ' w w We Lhank Cary Cook and Dermis Knight for,,cheraical analyses of water srrrplc: from the Aroelor 1254 test and Steve Foss for preparing the illustratices * l - . 3 -s -1 ftr* . Ux -V**" * ---- MONS 203562 * i _ - J , .. Literature Cited. ^1 . F.rngs, V. A. and D, 1. Mount. 1970. A water delivery system for smell fish holding tanks*. Trans. Arar.' Fish. Soc. 99(4): 799-802. ' Butler, P. A. 1973. Organochlorlnc residues in estuarine mollusks. Festic. Nonit. J. (In Tress). , - ^ * ' \" Darnell, R. M. 1958. Food habits of fishes and larger invertebrates of Lake For-tchartrain, Louisiana, an estuarine community. Fubl. Inst. Mar. ScL. tTniv. T. 5:354-416. . ._ Duka, T. W., J. I. Lowa, and A. J. Wilson, Jr., 1970. A polychlorinated ' (9 ' ' Biphenyl (Arodor 1254 ^ ) in the water, sediment, and biota of Escambia Bay, Florida. Bull. Environ. Contan. Toxicol. 6, 171--180, %fc ` " I'snsen, D. J., F. R. Parrish, J. I. Lowe, A. J. Wilson, Jr. and P. D. Wilson. 1971. Chronic toxicity, uptake, and retention of Arodor 1254 in two estuarine fishes. Bull. Environ.'.Co'ntam. Toxicol. 6, 113-119. Hildebrand, S. F. 1919. . Notes on the life history of the minnows (C^V-s:a * * affinis and Cyorinodon varisyatua. Rep. U.'S. Coma. Pish., 1917: 1-14. fount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus far fish toxicology studies. Water Res. 1, 21-29. Lluoo, D.. K., R. R. Biackaua, A. J. Wilson, Jr. and J. Forester. 1971. (S Toxicity and distribution of Arodor1254 in the pir.k. shrimp Penaaus duorarua. Mar. Biol. (Berl.) 11(3) 191-197. Simpson, D. G. and G. Gunter, 1956. Notes on -habitats, systematic characters end life histories of Texas salt water cyprinodontes. Tulane Stud. Zool. 4, 115-134. . - St rlcV.lcnd, J. D.. II. and T. R. Farsons. 1962. A practical handbook of se?.- v*iir analysis. Fish res. Board Can, Bull. 167: 21--26. MOMS 203563 i FIGURE 1. Define cppnrntU3i A-o a dilating pump; a-pump mlcro-8witeh; B-mixing 4 bottles; C-vacuu-,1 lines; D-rcc'civing bucket; 2-solenoid; e-solenoid micro-switch; F-ln^cctor lever apparatus; G-5Qcc. syringe; H-toxicant delivery tubing; I-water delivery tube. 1 MONS 203564 4- I * ' f V f i 1 FIGURE 2. Distribution apparatus! J-clistribution box; K-ceg/larvac trays: L-Pctri dishes; H-siphcn. ' MOWS 203565 I I I ] Jlf Vi w II *w ** ^ | *. Cor two vceks following hatching t>f ahcenohcatl minnova~fC?nrinotlQtrVAfiCf^TTEual. t ' ' ' "" . Temperatures varied + 0,5 C; ealinity averaged 20 o/oo, + 2 o/oo. t\ ' Temperature CO Numbor ., Tertile % Hatching Days Survival $ 7, ' Fry ` Survival ' l 15 20 22 24 25 ' 50 92 50 98 50 100 ' 1 50 W:1 50 98 * * , ii 1 i i i No' hatch \ 0ft* . i o** j No hatch 15,0 k 9,0 J o** *. o** 28.0**. ICO 50,0 , 100 | H ' 7.0 . 83<7 . . SO 26 20 30 ' 35 40 '- 25* . 92 " 50 . 50 901 ; .. 94 ' ` 50 94 ; 50 94 / *i | ' j^ r j i *i 7,0 ` 6,0 '4.5 4 4,0 4.0 * 78.0 55,5 * . * 82,9 70,2 ! 100 *r " P2 90 ICO j * 1 ` 8,5** . 50 . * * i .< * Loss of 23 eggs due to cplllage, . ki< Significantly lcsa than the * -t greatest fertility or survival (x";a 0.01). HONS 203566 i Table 2,--Effect of salinity on fertility, tinn-to-hetch, hatch success ai'.d survival of fry ' ;i . for two weeks following hatching of Bbcepchcad minnow ;(Cyprinodon vrrfe,>ratus). N . Temperature 30 + 2 C; sclinlty varied +1 o/oo, Salinity o/oo 1 0 5 10 ' 15 20 25 30 35 JETJL Number Fertllc Z 60 35 75 35 75 35 . '75'.. ,35 35 46 35' 51 44 54 49 37 Da ye f Hitching Survival Lr* - Significantly lens than the greatest fertility or survival (x^SP 0.01), i? i. __1 _ .>4 * * Try Survival l 0 50 * 89 92 10Q 90* 93; f} 50:i I * V.' HONS 203567 Table 3,--Effect of Arcelor 1254^on fcrtillbyi timc-to-hatch, hatch oucce39 and survival of fry for two weeks following hatching of oheepohead minnow (Cyprinodon vnriegntus), Temperature 0 averaged 30 C Orange 27-31 C); salinity averaged 24 o/oo (range 16-32 o/oo). 1i Concentration fitft/*) Nominal Measured , * Number Fertile t . Days a Hatching Survival . 7. , ' Survival . %< \ Control o,i ;* i <0,03 lig/1 ,0.06 ' 160; i 1 86 ' \'7,Q j 86 , v . 6.5 il 79 ; 89 _j 69 ' 95 ' 0.32 ' * * ' 0.1(3 160 86 7.0 73 . ' 62* i l.o 3.2 j.; 10.0 ' \ 0,3(3 .;1.04 r ' 3,48 . ' '-'f ` 160 j ' ' '* ' < i (f-j 1160 7 ' /'I ' 9i!- ` i. f . 85. - 91 7.0 . 82 '-V' ", ** . j 7.0 ,... 75 ' J* ' 6,5 ' 1 ` -57*-. ! '* ' , * ' t --< r- i--..**------------------- 63* ' 40* fl* *V. (I 2 . * Significantly different from control fish Cx a 0.01> HONS 203568 i WU*, V Table 4. --Relative susceptibility of various lift ctOf.cs of oheepshcad minnowo (Cynrlnodcn variegatM*-.) to Arodor 1254 -in a flow-through system, -Criteria dre infertility of eggs and death of embryos, larvae, juveniles,.and adults, } ?* r'. - * ; Life Stage Egg fertilization* a - Snbryoa Larvae Juveniles - , 1 Adulta * 24-hour atatie teat. / .* Exposure- 't Concentration (Pg/i) _ - (days) J. Minimum ' Maxir.u-n JAffecting Not affecting J .. 1> 6.4 " * 1 *' * (' i' ` 10.0 . a ` 7 *4 / . 10.0 i 3.2 . 21 . 21* "21 1 ` J11 11 0.32 1 . HO O r 1* l* 4 0.1 - 3,2 10.0 ' ' -1) ' j 4 * ' ' _ 1 0 t tt '* . jv- l'St ' 4 ' ! hons 203569 ( i AROCLOR 1254 It! ECCS OF SHEEPSKEAD MINNOWS: EFFECT ON FERTILIZATION SUCCESS AND SURVIVAL OF EMBRYOS AliD FRY 1 David J. F.ansen,' Steven C. Schirrcel and Jetrold Tore3ter U. S. Environmental Protection Agency - Gulf Breeze Er.viroman.tal Research Laboratory ; ** Sabine Island, Gulf Breeze, Florida 32561 (Associate Laboratory of the National Environmental Research Center, Corvallis, Oregon) ^ Contribution No. 177, Culf Breeze Environmental Research Laboratory Registered trademark, Monsanto Coupary, St* Louis, Ho. Mention of commercial products or trade names does not constitute endorsement by the Environmental Protection Agency. , HONS 203570 *- 3 A^ -, . _. ,jf ^ t _ 7f / ~ r* abstract , . ' ............................. *-l The effect of the polychlorinated biphenyl (PCB), Aroclor 1254, in eggs of the sheepshead minnow, Cyprlnodon variegates. on fertilization success and survival of embryos and fry,was investigated. Adult fish were exposed for four weeks to 0.1, 0.32, 1.0, 3.2 or 10.0 pg/t of PCB, then injected twice with 50 IU of human chorionic gonadotrophin to stimulate egg production. The aggs wara fertilized, placed ia PC3- frea flowing seawater and observed for mortality. Fertilization success was unimpaired by concentrations in eggs .as high as 201 pg/g but survival of embryos and fry was reduced. Usually, fry from eggs ' containing 7.0 pg/g or core began dying 24-48 hours after hatching. " If this PCB affects other species similarly, then populations of . fish that presently have comparable concentrations in their eggs may be endangered. . INTRODUCTION Polychlorinated biphenyls (PCB's) have bees found frequently in estuarine organisms from cany states (Butler, 1973) and la an estuary near the Gulf Breeze Laboratory (Duka at el., 1970). PCB's in sea water are toxic to and accumulated by juvenile shrimp, crabs, oysters , and fishes (Niarao, et el., 1971; Love, et el., 1972 end Hansen, et al., 1971). The relationship between the amount accumulated by fish and subsequent effects is poorly understood. However, PCB's m eggs may decrease fertility and survival in early stages of embryonic development in Atlantic Salmon, Salop sal.ir (Johanns3on et al., I 1970), and PCB's have been Implicated in poor reproductive success of striped bass, Morone saxatilis (Anonymous, 1971). Because HONS 203571 reproductive success with both fishes varied, the exact relation * - - '* - \ ship of success to concentration of PCB in eggs remains unclear. *- Our study was conducted to determine the effect of one PCB, Aroclor 1254, on fertilization success a eggs of sheepshead minnows, .Cyorinodon vartstatus, and on survival of embryos and fry. Aroclor 1254 * ' was selected because we found eggs from striped bass that exhibited decreased reproductive success contained a PCB whose chromatograms closely resembled Aroclor 1254. Sheepshead minnows were selected because they can be readily exposed in the laboratory and reproductive success Is excellent. ` iV ' t' Test fish" . ' _ MATERIALS AND METHODS ------------ ---------- " ' Adult sheepshead minnows vara seined from ponds on laboratory grounds >'a and acclimated to laboratory conditions'for four days before exposure. During acclimation, mortality was lass than IT and no abnormal be havior wsb observed. Females averaged 42.5 cm standard length, range 35-52 es, and talas averaged 42.8 cm, range 35-52 tm. Curing accli mation and exposure, fish were fed commercial fish, food that contained no detectable PCB (<0.01 ug/g)* ' Adult exposure , We exposed 20 female and 10 mals fish In aquaria containing none, 0.1, 0.32, 1.0, or 3.2 ug/i of Aroclor 1254 and exposed 25 females ar.d 15 males to 10 pg/l for four weeks in an intermittent-flow bioassay. The apparatus used was a modification of that-of Brungs and Mount (197^) In our modification, Aroclor 1254 and carrier, polyethylene glycol 2C0, were injected Into seawater each time the apparatus cycled. Each cycle HONS 203572 siphoned water to six 80t test aquatia. The. injection device was - I- operated by a solenoid that raised a lever each cycle turning gears on six injectors and pushing the plungers of six 50cc syringes. Each of the approximately 150 daily cycles delivered 1.5i of filtered 30C seawater, 11 y| of carrier and appropriate amounts of PCB to ' * each aquarium. Water - and carrier without PCB were delivered to the control aquarium. Salinity of water averaged 17 o/oo, range 5 to 28 o/oo. ., Egg fertilisation, embryo and fry survival The effect of Arodor 1254 in eggs was determined by enhancing egg '^ production in exposed fish by honsonal injection, fertilizing the eggs artificially and monitoring their development in flowing FCB-free seawater. Female sheepshead minnows were Injected intraperitoneally with a 50 I.U. human chorifimic gonadotrophic hormone on exposure-days .25-and 27.'-On day 28, -eggs-were "Stripped manually from five females from each aquarium and those from each female placed in individual beakers containing 40 ml of filtered 30C seawater. Ninety-three of 96 females that survived produced eggs. Eggs from a female were fertilized with excised macerated testes from a male from the same aquarium. In addition, eggs from five control fish and two fish surviving exposure to 10 yg/l PCB were fertilized by males exposed to 1,0 jjg/l. TWeoty-five eggs from each fish were placed In Petri dishes to which a nine cm high collar of 500y nitex ns3h . was glued. Dishes were submerged 7 cm in the 801 aquarium which received approximately 2251 of filtered PCB-free seawater per day; 2 Manufactured by Ceorge Frazer, 4528 Pitt Street, Duluth, Minn. 55E0^ HONS 203573 average salinity was 16 o/oo, range 10 - 27 o/oo. Success of fertilization was confirmed by checking microscopically for cleavage 1.5 hours after fertilization. Thereafter, dishea were checked daily to determine survival of embryos and fry* Dishes remained in the *a aquarium for 34 days. Fry were fed brine,shrimp nauplii or dry commercial fish food daily. _ - Chemical analyses Concentrations of Aroclor 1254 la water, eggs and fish were determined by electron capture gas-chronatograpby. Unfiltered water samples from each aquarium ware analyzed weekly during the four--week exposures of adults. At tha and of the adult exposure, concentrations were determined in the fertilized eggs from each fish and in surviving adult males and females. Also, fry that hatched from these eggs and survived for four weeks in PCB-fres* water were analyzed for Aroclor 1254 content. Analytical methods for water* eggs and fish were the same as those of ITirao et al: (1971), except than an 0V-1Q1 column .T was used and all peak heights ware summed for PCB quantification. P.ecovary efficiency of Aroclor 1154 exceeded 80%. Pleasured con- centraCions were not corrected for percentage recovery. Statistical analysis Probit analysis was used to determine whether in,creasing con centration of PCB in eggs Increased the effect on fertilization -. 3 success and on survival of embryos and fry. The x test for Independent samples was used to compare'data for eggs from individual unexposed end exposed fish. Differences were considered real at 2 a* 0.05 for problt analysis and e 0.01 for y tests. * HONS 203574 - - ` ' ' RESULTS AND DISCUSSION Arodor 125A in water vas toxic to and accumulated by adult sheeps- head minnows exposed for four weeks (Table 1). Kbrtality of fish was negligible, except in the aquarium receiving 10 yg/f. Dying fish in this aquarium typically became lethargic, ceased feeding, and soma developed fin rot. Fish accumulated the PCS in direct proportion to the concentration in the water and concentrations in fish ranged from 15,000 to 30,000 X the nominal concentration in the water. Concentrations in males and females were similar. Concentrations of the chemical In eggs from exposed adult fish were proportional to ifc the concentrations in the fish and concentrations in female fish were 1.8 to 2.3 times greater than the concentrations in their eggs. The PCD exposure apparently did not alter the percentage of females producing eggs or their fecundity. Fewer embryos and fry from eggs of exposed fish survived than did embryo* fry froo eggs of control fish (Table 2). The percentage of the eggs fertilized was not affected, but survival of embryos to hatching vas less in eggs from fish exposed to 10 yg/JL. Survival yate of fry in the first week following hatching vas less in eggs from fiah exposed to 0.32 to 10.0 yg/f. than in eggs from unexposed fish. The estimated LC50 vas 6.1 yg/g; 95 percent confidence licit equals 3.5 to 11.8 yg/g. Fry typically began to die one or two day3 after hatching, about the time they started feeding. If fry sur vived tha first week, there seemed to be no additional cortalitic3 MONS 203575 related to FCB during three weeks of additional observation. Con- r centrations of Aroclor 1254 in surviving fry were similar, 0.26 0.56 Ug/g, end not'proportional to concentrations in eggs. Embryo survival decreased at the highest concentration of PC3 in eggs and fry survival decreased with increasing concentration of TCB in eggs. The amount in eggs was critical*because it va3 the sole source of PCB for the embryos and fry reared in PCB-free water. PCB in milt was probably not critical to fertility and survival because when eggs from control fish were fertilized with milt from either control or 1.0 pg/i exposed males, survival rate of embryos end fry vta not altered (Table 2). Survival rate of fry hatched . ..from eggs containing 7.0 pg/g or core of FCB was significantly less than the lowest survival rate of eggs from any of the five control fish (Table 3). If the^f>act of PCB in eggs of othar fishes Is similar to that found with sheepshead minnows -- and we have no data to support this view -- then variations in published information concerning the..chemicals relation to spawning success could be explained. Atlantic salmon eggs containing up to 1.9 pg/g of PCB had decreased fertility and survival of early embryos, but survival of late embryos and sac fry was unimpaired (Johannsson et el., 1970)'; Chesapeake Bay striped bass eggs containing FCB's had decreased fertility and survival of newly hatched fry (Anonymous, 1971). Our analysis of eggs from eleven striped bass from the Eastern shore of Chesapeake Bay showed that the eggs contained about 2.5 to 8.7 Pg/3 of a PCB resembling Aroclor 1254. Because concentrations of HONS 203576 PCU in eggs of sheepshead minnows as high as 201 yg/g' were not accompanied by decreased fertility and only minimal embryo mortality, it seems unlikely that decreased fertility and embryo survival in Atlantic salmon and striped bass could be related solely to FCB in their eggs. Diminished survival of newly hatdhed striped bass fry, however, could be PCB-related since concentrations in their eggs were similar to those in sheepshead minnow eggs which produced fry whose survival was poor. LITERATURE CITED " Anonymous. 1971. The striper -- this century's dinosaur. Stripers Unlimited 1971 Directory and Guidebook, pp. 11--62. Brungs, V. A. and D. 1, Mount. 1970. A water delivery system for small fish-holding tacks.' Trans. An. Fish. Soc. 99(4): 799-802. Butler, P. A. 1973. Organochlorine residues In estuerine ooHusks, 1965-1972. National Pesticide Monitoring Program. Pestic. Moult. J. 6(4): 238-362. '. ' Duke, T. W., J. I. Lowe, end A. J. Wilson, Jr. 1970. A poly chlorinated biphenyl (Arodor 1254) in the water, sediment and biota of Escambia Bay, Florida. Bull Environ. Contan. To:d,col. . 5(2): 171-180. Pansen, D.-iJl, P. R. Parrish, J. I. Lowe, A. J. Wilson, Jr. and P. D. Wilson. 1971. Chronic toxicity, uptake and retention of Aroclor 1254 is two estuarine fishes. Bull. Environ. Contain. Toxicol. 6(2): 113-119. Johannsson, Nils, S. Jensen and M. Olsson. 1970. .PCB-indicators of effects on fish. In "PCB Conference, Venner-Gren Center", HONS 20357? Sept. 29, 1970. pp. 59-68. Natl. Environ. Prot. Bd'., Stockholn. Lowe, J. 1., P. R. Parrish, J. M.Patrick, Jr. and J. Forester. 1972 Effects of the polychlorinated biphenyl Aroclor1254 on the American oyster (Crassostrea vlrglnica) : Mar. Biol. (Berl.) 17(3) 209-214. * * '* Nicnao, D. R., R. F.. Blackman, A. J. Wilson, Jr. and J. Forester. 1971. Toxicity and distribution of Aroclor1254 in the ptnlc shrimp (Penaeus duorarua). Mar. Biol. (Berl.) 11(3): 191-197. HONS 203578 Table 1. Toxicity and uptake of Aroclor1254 by adult sheepahead minnows (Cyprlnodon variegatus) exposed for 23 days in an Internittent-Elou bioassay. Thirty fish were tested per concentration. Residue analyses are for a talnlnwn of 7 male and 17 fenale fish and eggs'from 5 fish. \> TEST CONCENTRATION Mf ORTALITY (us/.i)________ Z MenuInal Measured CONCENTRATION IN FISH , wet we UhO Males Fenales FEMALES CRAVED % Control HD* 7 0.64 0.47 0.52 100 0.1 0.09 13 2.5 1.9 0.88 89 0.32 ' ' 0.14 1.0 0.39 ` ' "7" ` 9.7 10 -- 9*3 25. 5.1 7' ioo ' 11. 100 3.2 10.0 1.1, 5.6 3 *49. 49. 27. 95 ------ * -- 170 R* 94 100 average FECUNDITY No. 97 121 110 127 152 133 * HD not dcectaM,<.0.03 vg/t. ** Sggs frow two fish. HONS 203579 - -- - ** <4l Piffirf'i'aiHiJ-y' - - -- fW* - - - - *' s Tabic 2. Success of fertili .ration of eggs from sheepshead minnows cnpoasd to ( > ' Aroelor ^ 1254 for four weeks, survival of embryos from fertile eggs until hatching and survival of hatched fry. Eggs are from five fish per con centration (except two fish from 10 ug/). Percentages are in parentheses. CONCENTRATION `. , EGGS * " ` FRY SciiTt Eggs Tested Fertile Hatched Survival Erooaura (yg/D Control Average ______________ 1 (ug/g) * 0.52 125 125 (100) 116 (93) Weak 1 Veeks 2,3.4 111 (95) 106 (96) 0.1 0.32 . 0.63 . 125 5.1 126 120 (96) 120 (95) 106 (88) 107 (80) 103 (97) 98 (95) `82 (77)* 76 (93) 1,0 11. ' 126 121. (96) 118 (98) 31 (26)* 26 (96) 3.2 27. 126 118 (94) 100 (85) 23 (23)* 19 (83) 10.0 170. 50 46 (92) 33 (72)* 0 (0)* 0-- Control 2 and 1.0 ,-- 128 119 (93)-'" 113 (95) \ 112 (99) 111 (99) )^LLA_____ . 2 * Significantly less than control hatching or one week fry survivals (* : *- < 0.01) **'.'* .. . HONS 203580 Table 3. Comparison of concentration of Aroclor^* 1254 in eggs (wet weight) I from sheepshead minnows exposed to the PCB for four weeks and success of ,' * - ' - *: -**'' ' fertilization of eggs and survival of embryos and fry. ,, Concentration ia Egga Cyg/g) 0.41 0.44 0.45 0.53 0.57 0.76 0.84 0.91 0.98 ' 1.1 3.7 4.1 5.4 5.4 7.0 7.1 V 10.8 ------------ ECGS Tested Fertile Hatched .FRY Week 1 Weeks 2,3,4 Adults from Aauaria - (yg/i) 25 25 23 23 22 25 25 24 19 19 25 25 25 25 24 * 25 25 24 24 23 Control II . . Mt. -____ _ n 25 24 19 IS 18 0.1 25 25 20. 20 18 Control 15 "23 21 21 19 25 25 . 25 ' 23 23 25 23 17 17 ' 15 25 25 24 24 23, 0.1 H It II 25 23 22 " 26 26 23 25 24 19 25 24 22 . 25 * 23 21 26 23 ' 22 25 * 24 23 25 25 25 21 20 22 21 13 12 21 19 5- 4 22 7. 7 85 0.32 11 It It VI 1.0 II . It HONS 203581 Table 3. (Continued) - ' 13*' Concentration In Eggs EGGS - Tested Fertile Hatched (yg/s) 13.2 13.3 23.6 25.7 27.9 28.6 28.7 145. 201. 25 25 ' 25 ' 25 25 25 26 25 - 25 25 25 24 23 23 " " 23 24 16 25 ' 23 25 25 21 13 23 . 15 ' 23 18 $ * ..FRY Week 1 Weeks 2,3,4 9 '5 4" 6 0 5 8 0 0 7 5 A. 6 0 3, 6 0 0 Adults from Aquaria Cj-g/0 1.0 II 3.2" " tl tl It ' Tl 10.0 n HONS 203502 AHOCLOH.1254: EFFECT ON COMPOSITION 0" DEVELOPING ESTUATINT '' ( ANIMAL COMIUMITIES IN THE LABORATORY* I '* S David J. Hansen U.S. Environmental Protection Agency Culf Breeze Environmental Research laboratory */ Associate Laboratory of the National Environmental Research Center, Corvallis, Oregon , Culf Breeze, Florida, U.S.A. 32561 Running page haad: PCB Affects Estuarina Coramnitle3' * Contribution No. 164, Gulf-Breeze 'Environmental Research Laboratory Registered trademark, Monsanto Company. St. Louis, Missouri, V.R.A. Mention of comarcial product or trade names does not constitute endorsement by the Environmental Protection Agency. , HONS 203583 AJB SIP-ACT Aroclor^ 1254, a polychlorinated biphenyl, affected thel composition of communities of estuarine animals that developed fron planktonic larvae in salt water that flowed through-10 control * aquaria and 10 aquaria contaminated xrith 0.1, 1 or 10 *a V * of this PCB. Communities that developed in control "aquaria and aquaria that received 0,1 pg/i of PCB in water for four months were dominated (>757) by arthropods, primarily the amphlpod Coronhlua volutator. In aquaria receiving 1 and 10 pg/i, the -number of arthropods decreased and the number of chordates, primarily the tunicate Nolgula manhattensls, increased; over 757 of the aninals .. In 10 pg/i aquaria were tunicates. Numbers of phyla, species, and V . individuals (particularly amphipods, bryosoaas, crabs, and nollusUo) were decreased in this PC3, but there was no apparent effect on the abundance of imnelids, -benchipods, coaler.terates, echinoderms or nemerteana. The Shaanon-rWaaver index of species diversity'va3 not altered by Aroclor 1254. t HONS -203584 INTRODUCTION Polychlorinated biphenyls (PCEs) have been manufactured for various uses (Erocdhurst, 1972) for over 40 yuarr, but their occurrence in aquatic ecosystens was not ccmfiroad until 1966 (Anonymous, 1965). Since then, PCEs have been detected in estuarine - ' ' . organisms from 5 of 15 df the coastal United States (P. A. Butler,* 1973). One FC3, Aroclor 1254, was discovered, in. the water, sediment end biota of Escambia Bay, Florida (Duke et al., 1970). Chronic and acute toxicity experiments conducted at the Gulf Breeze Laboratory have established that Aroclor 1254 is to:-:ic to some estuarine organisms. A concentration of 100 yg/t of Aroclor was acutely toxic (48 to 96 hours) to pink, shrimp, Pcnaeus duorarvn., and oysters, Crassostrea virglnica, but not to pinfish, Lagodor. rhooboldas" (Duke et al. ,-`1970)Chronic-toxicity-ves up to 100 times greater'than acute toxicity. In exposures lasting more than two . v iwj Jt* of- Aroelor^.-254'killed* pink"shriicpjt'(Iii.5t=j9'-epy;al"^y -; - y 7 Vi'..*---.-*- 77'id. Z-.- "r" ' 1971a), whereas 5 yg/i killed pinfish and spot, Lsiostovus xarthuns (Hansen et al., 1971) and significantly reduced oyster growth rate (Parrish et al., 1972), but it was not lethal to blue crabs, Calllnectes sap 1 du3'(Duke et al., 1970). Aroclor 1254 is, therefore, toxic to certain estuarine species exposed separately. However, its effect on communities of estuarine animals is not known. This study reports experiments that determined the effect of this chcriccl on development of estuarine, animal communities in the laboratory, , . HONS 203565 I MATERIALS MID METHODS' i1 * ' I investigated the-effect of Aroclor 125A on development of ' estuarine com/Eonities by comparing the number, species, and diversity of animals that grew from planktonic larvae in appara tuses continuously contaminated with 0.1, 1 or 10 yg/i of PCB for four months, 18 May to 25 September 3.970, with animals from, an identical apparatus that was not contaminated, - The apparatus used in this investigation lb illustrated in Fig. 1 (only one of four identical apparatuses is shown). Sea water with its natural component of plankton was pumped from the estuary adjacent to the laboratory into the primary constant head box. Salinity of the * , . water ranged from 10 to 34 /oo (average, 29.7 /oo) end temperature t, ranged from 22 to 33 C (averaga,^28,5 C) . In contaminated appara- * ' ' _ " ' "" " , .Cases Arod^'r-1254 .was added-to \;ater after it was siphoned, at the rate of 2,300 thl/nin, from the primary into the secondary constant ^heail/box-prthb-'con.trol-apparartus-xecaived:the'saire,flow of water, t'eter then flowed from the secondary constant head box to,each of 10 adjacent aquaria-lQ replicates for each treatment. (Treatment in cludes control cad contaminated apparatuses), Flow rate' through each aquarlun was maintained at 200 ml/min each by adjusting the height of a 2.7 Her diameter hole in each of the 10 standpipes in the secondary * constant head box. Each aquarium ves 44.cm long, 9 cm wide and 14 cm high. Water depth in each aquarium vas maintained at 8 cm. PCB-f rec. .sand was placed in _ecch aquarium to a depth of 6 ca. HONS 203586 Figure 1 -- Apparatus used to test the effect of Aroclor 1254 on composition of estuarine animal communities. *. *` i HONS 203580 Planktonic larva? colonised this a and ar.d tcua valla e the aquarU, '` llic siphon and constant hsad boxes were cleaned weekly so that all ' * aquaria received planktonic larvae from a common source-the in coming water. Uater leaving each aquariun flowed through a V-shaped i opening and into a corron drain for the apparatus. Aroclor 1254, dissolved in polyethylene glycol 200, was metered by a syringe pump into the water as it entered the secondary constant head box of each .experimental apparatus. The sane amount of poly- . ethylene glycol (2 ml/day, 0.68 ng/f) was netered into the control apparatus* Solvant-induced effect was not expected because: (1) . .. polyethylene glycol 200 did not affect development of two species a ' , of crabs at this concentration {Epifanio, 1971) (2) concentrations * up to 12 (v/v) were not lethal to grass shrinp, Halnemonates nunio, . jar .nhaepahaad .minnowa, - Cvprlnodon *variegatus, in 96 hours in ' ** static tests {Hansen, unpublished data) and (3) the toxicity _nf S jij/i of Axoclor 1254 tQ-brown. shrico.. Penaeus aztpeus'. and pin-. **.-- i Ji--?'Jifi","'' , v --- \V - fish wa? not increased by increasing the concentration of solvent up to 100 times (0.1 to 10.0 mg/i) (Hansen, unpublished data). Concentrations of Aroclor in test water and sediment were determined by gas chromatography. Methods of analysis for vater are described by Kimco et al. (1971a) end for sediment by hicro et al. (1971b), except chat an 0V-101 column was used. Recovery'rates were above 702, but data in this report do not include a correction factor for recovery. Vater from the secondary constant hood box of each HONS 203589 ,-ipparatu3 was analyzed Culca a month (Table 1). Copcenrr.irica:; * throughout this box were uniform; water froa each standpipe, analyzed once during the 10 wg/*, 'exposure, averaged 7.9 ^g/* (range 6.8 - 8.7) Sediment cores from 4 of 10 aquaria from each apparatus were analyzed at the end of the exposure (Table 1). - *. t At the end of the four-month exposure, cnimals were scraped from the sides uf the aquaria and the contents of the aquaria siphoned into a 1 mm cash sieve. During the lest three months of the experiment, animals that left or were lost from the aquaria or were cleaned from the secondary constant head box and tha drainage area were also collected in a 1 run mesh sieve. Animals retained by the sieve were placed in flngerbawla of seawater relaxed with KgCl, *> - ` preserved in 507. isopropanol and identified. To determine effect of Aroclor 1254 in each treatment, an index jjf species dJ.varsj.ty as ,*<ell *s tha nueber and percent odcuren.ee of various species in each treatment {contaminated and control-aquaria) "' were cuinpared'T' A species-diversity^indexTprovidas a-ntraarical rzear.3 - of assessing community structure that is independent of sample size, expresses the relative importance of each species and Is dimensionless . .* K Modifications of the Shannon-#*aver (1963) formula, H' - i p^logp^. 1-1 w* here pj la the proportion of the ifch species in* the collection and s the number of species, have been used in freshwater (Wilhc, and Dorris, 1966) and saltwater (Bechtel and Copeland, 1970) to assess affects of pollution on natural communities. In unpolluted HONS 203590 areas, many species of animals are abundant pud diversity is high, but pollution can decrease diversity by tasking a few species very abundant and all others rare. Xn this study, the Shannon-Weaver 0 formula (log ) vas used to determine the usefulness of the species 2 -. diversity index in assessing the effect of Jtroclor 1254 on cocnuoity structure in laboratory experiments.' Pooled data from each Arodor concentration and control ware 'j , compared statistically using the y test for independent samples. Data from each of the 10 aquaria receiving one treatment were com pared with data from 10 aquaria receiving a different treatment ___ using the Maim-Whitney 'XJ" test (Slegal, 1956). '0 considered real at a - 0.01. % Differences were cT. .. . - ;..KJSGLXS . - _. * . A large number and variety of animals were found in all aquaria J^r.Qm-.nine^phyla, ,2?. were taollushs V '"7 V y\-- ,T .. . .- ' (15 pslecypods and 12 gastropods), 23 annelids, 6 arthropods, 3 chordates, "2 coaleuterstes, 2 echinoderms, 2 nemertear.s, 1 brnchiopod and 1 hryosoan. Arthropods ware most abundant (3,842) of the 5,897 animals followed by chordates (1.302), annelids ((48)* mollusks (219) and animals ,-f roc, other phyla (86). Ihe two most abundant animals were the attphipod, Coroohlum volutator (3.770), and the tunicate. " . 1a 1 j Tolgula manhattensxs , (1,164) . . Spe**cies con-position and ahuadan.ee of individual species varied MOMS 203591 among the 40 aquaria. The number of species ,ln each aquarium ranged from 5 to 23 (average-13). Annelids and mollusks were present in all aquaria. Animals from other phyla vere present in from 1 to 36 #, aquaria (Table 3) . Seventeen species" 'were found in all treatments *| ` . and 26 vere in only one. . *` Aroclor 1254 prevented animals of certain phyla from colonizing (Teble 3). Although nine phyla vere found, the number of phyla represented in any aquarium ranged from three to seven. The number of phyla in the control, 0.1 and 1 yg/i aquaria averaged 5.7, 5.4 * - .. + and 5.7, respectively. Fewer phyla, average 4.1, vere in aquaria contaminated with 10 yg/i of Aroclor because fewer aquaria contained arthropods, and none contained bryozoans. The total number of species found in each apparatus ranged. ' from .25 .to 32 but-the percentage of species in each phylum vas similar,in all four treatments (Table 4). In each apparatus, more ."specie5**35T^Go~Irlusks".war's-found"thcn'speciea from any-other phylum. - The relative^numbers of tolluscan species vere similar for all treatments (40 - 44 percent). The number of annelid species vas only slightly less (29 * 35 percent). Although fever arthropods and chordates vere found, the relativa numbers of each were similar In all treatments. '' The number of species In each aquarium of en apparatus vas -altered by Aroclor 1254 (Tables 2 & 5). The total number-of species-and-the number of species from each phylum in the ten HONS 203592 f 1* control* 0.1 and 1 Pg/X contaminated aquaria were similar. How ever, there were significantly fewer species and the species com position differed in 'the ten aquaria contaminated "by 10 Hg/ of the PCB. The greatest shifts in species composition were found in i i >. arthropods, hryozoans, and nollusks. Although' there were si gni * > ficant reductions in the number of molluscan species in the 10 Vg/ aquaria, tnere was no difference in the gastropod - pelecypod ratio. < The total number of animals in each aquarium did not differ significantly among the four treatments; whereas the number and percentage occurrence of species was markedly different (Tables 2 S (). Arthropods (primarily the tube-dwelling smphipod, Corophlun volutator) were the dominant animals in the control (76 percent) and 0.1 pg/x TCB (64 percent) aquaria. In the:,e aquaria, chordates (primarily Kolgala nar.hattensis) uera - > secondarily-abundant. -Arthropods were .also -abundant ,(55T?ercent)`. in equarla that received 1 pg/i hut a significant decrease in their abundance and an increase in abundance (31 percent) of chordates occurred. 'Doru.aance was different in aquaria cohtaal- nated by 10 pgft* 30 percent of the animals were chordates. This difference fron comuni ties dominated by arthropods in control aquaria and in aquaria contaminated by 0.1 pg/f of Aroclor 1254 to communities dominated by chordates in. aquaria receiving the highest concentration of this PCB was the. cost striking FCE-. " HONS 203593 induced effect in this expericent- The abundance of animals of other phyla^ Although less striking, was also altered by PCB. There vere more mollusks in the control aquaria than in treated aquaria*, but the percentage * of their occurrence was not different in the PCB environments. \ *i Colonies of the encrusting bryozoan, Heobranipora tenuis, were not counted, and therefore their numbers are not adequately represented in Table 6. However, their exclusion from tha ten * < aquaria contaminated with 10 Vg/1 was significant. Abundance of polychaetes was not altered by any of the three concentrations of PCB. ._ .......... ........................................... - ------- .. - The Shannon-Veaver (1963) index of species diversity calculated for each aquarium did not differ among the control and three contaminated apparatuses (Table 7). Species diversity is a - -T*, ' .. ' function -of "two -components, -richness '(nuribers of species (Table 5) end equltablllty Dr relative number of each species (Table 7) `(lioyd aixdrGhelardi, 1964)'r"Xn my studyspecies diversity is n'ot correlated (r - 0.094) with richness of species, but Is correlated (r 0.S82) with relative abundance of each species: J' eclculated diversity t maximum diversity (Pielou, 1966). (Maximum diversity is defined as species diversity where all species are equally abundant.) Equitability did not differ between treatcents because communities in this study vere usually dominated by one species and were rot rich in species. Therefore, the effect of Aroclor was not on species diversity but on species composition. HONS 203594 Conclusions based on abundance and diversity of anirala collected at tbe end of the four-ronth exposure were corroborated by the abundance end diversity of animals that migrated from arid were washed from each apparatus during th^ exposure (Table 8). 1' *1 * The total number of animals and species fropi aquaria with 10 pg/l was taarkedly lower than those from the other aquaria. Arthro pods were abundant in collections froa the effluents of control aquaria and aquaria with. 0.1 and 1 jjg/l but rare froa the aquaria with 10 jifc/i.. Thu effect of Aroclor on crabs collected fron the aquaria could not be assessed at the end of the exposure because only two were found In all aquaria. However, presence of exo-- slfeltons in seven of 10 control, six of 10 0.1 yg/l and eight of v. 10 lyg/l aquaria and absence of exoskaltons in the 10 yg/t aquaria i strongly suggests that crabs were sensitive to highest concentration, of Aroclor 1254. This sensitivity was substantiated by collections of crabs- froa the effluents of the equaria. Eurrpap-opeas sp., `Weopanopg sp., Mnnlxa ?p., and portunids were abundant in effluents of control, 0.1 and 1 ps/i aquaria but absent from the effluent of the 10 jis/i aquaria. Tha bryozoar,, R. tenuis, was absent in the effluent froa the .10 jig/jl aquaria, but present in the effluent from the other aqurria. Hollushs were rost abundant in effluent from tbe 10 control aquaria. - HONS 203595 ' DISCUSSION AND CONCLUSIONS ' The polychlorinated biphenyl, Aroclor 1254, Influenced the l composition of animal coicnunItie3 that developed from planktonic * *m r , * larvae in sea water which entered the test aquaria. The primary influence was that the dominant species in*the control aquaria was the amphipod, C. volutator, and .the dominant species in aquaria receiving 10 pg/i, PCB was the tunicate, M. manhattensis. Also, there were fewer .species and the number of animals was markedly but not significantly fewer in the 10 pg/f aquaria.' The abundance of arthropods, chordates, bryozoans and mollusks differed signi- i' ` * *? ~ ' 'v ' flcaatly but abundance o annelids, brachiopods, coelenterates, *. echlnodenaa or nenarteans apparently did not differ. ' differences in coimrunity structure that were apparent at the love sc Concentration jCQ.,1 Pg/l) hecamemore pronounced-as .the SI ' ** M < ***>* ' ' w mr r-_. ,* ^ * T '" " -- - concentration of Aroclor 1254 increased to 10 pg/t. Control aquaria ware dominated by arthropods (76 percent), with lesser numbers of * ''animals-from.eight other-phyla.* Aquaria: tori Cami-r.e ted by-0.1, 1 or 10 pg/2 contained fewer nollusks than did control aquaria; however, the percentage occurrence of mollusks was not altered by the PCD. 4 Aquaria receiving 1 pg/l or 10 pg/l had -more tunlcates and fewer arthropods than did control aquaria. Aquaria receiving 10 pg/l were dominated by tunicates (60 percent) with lesser numbers of -five other phyla; only 12 of the animals were arthropods. Animals most-reduced in nunbers at-the highest PCB concentration included HONS 203596 the nraphipod, JC. volutntor; the xanthid crabs, Eurypnnoneus dcpressu3 and Keopanbp tc>:ana; the bryoroan, K. tenuis; the gastropod, Bittium varlum; and the peleeypods, Ahrn aagnalig end Telllna altcrnata. , t `Few of these changes could have been predicted from in * formation from current literature, because only one species in this study had been challenged previously with PC3 and only a few are phylogentical-ly related to previously challenged species. Arthropods, particularly anphipods and crabs, were sensitive to Arodor 1254 in this experiment and in the experiments hy Micro et al. (1971a) in which pink shrimp were killed by 1 yg/l. w - Juvenile blue crabs appeared -resistant to 5 yg/i (Duke et al., I, 1970). In my experiment, the numbers of xanthid crabs was reduced`by 10 yg/i indicating that larval stages nay be parti cularly sensitive to PCBs., Sensivity of crab larvae has been shown with the insecticides dieldrin (Epifenio-, 1971) and mirex (uookhoOt et. al., 1972). Aroclor 1254 va3 lethal to tuo. estuarine fishes at 5 yg/t C^tnsan et. al., 1971) but in this study iovar chordates (tunicates) seamed unaffected and were cost nsuniant in the PCB-stressed communities. Lethal effects of PCBs on nollusks are not known. However, growth of oysters was reduced significantly without mortality by 5 yg/f, of Aroclor 1254 (Parrish et al-, 1972). In my experiment fewer nollusks occurred in all exposure norscen- trations possibly because the larval stages are sensitive or because HONS 203597 of factors other than the presence of Aroclor. Studies on proto zoans (Cooley, Keltner, and Forester, 1972) provided the only other data X am aware of on the sensitivity of estuarine animals of other phyla totPCBs. / iv The Shannon-Weaver species diversity index-has been used as * .' . * ' an indicator of the effects of sons types of pollution on animal communities In estuaries, but in this experiment, the index was not decreased even though composition of the communities were *4 greatly altered. This species diversity index did not decrease as the concentration of FCB Increased because the index was proportional to the relative number of each species present in tha aquaria, and communities Chat developed at each treatment con-- * . " centra cion vera dominated by one species of animal. If Aroclor 1254 affected tha composition of established communities in an estuary as It did the developing communities In this experiment, * tais^spaciaa- div&rsi'ty^Lui^i- tau^'^ not, he .used to estimate effects cf this pollutant in tba emriromacat. ' ' " * One purpose of this experiment was to determine whether the effect of a toxicant on developing estuarine animal communities can be investigated in ths laboratory. Preliminary experiments at this laboratory indicated, that the structure of coiir.unities of organisms setting in aquaria was altered by presence of the (r) 2 insecticide Dursban.Viy (J. I, Lowe, personal communication ). Hy analysis of his data indicated that replicate aquaria were *" * ' n necessary to separate the effects of a toxicant from the effects "* * . . ,,* * '' r - ** . HONS 203598 I r.mriw m if * TV. of an efficient predator or from the effects of an animal vith `' \- great reproductive capacity. The use of ten replicate aquaria for each treatment in ny experiment readily separated the eEfect of Aroclor 1254 from other factors that night influence community structure. My experiment also showed that^small aquaria can be . ' used, provided that larger anlnals *caa emigrate before they drastically affect comunity structure and thus mask effects of the toxicant. Emigrating anlnals must be caught and enumerated * -` _ '* so the effects on them can be assessed. ,,* 1 Mr. Jack J. Lows, EivATOtmanfl Protection Agency, Culf Rreez; Fla. 32561 " Registered trademark: 1* Dow Chemical Co. . - . HONS 203599 AC KKOWL EDCEMENTS ' _ I am grateful to Mr. Edward Matthews, who helped during the four-month experiment, and to Mr. Johruiie Knight, who prepared water and sediment samples for chemical analysis. I am parti- i cularly indebted to Dr. * Kelson R. C.ooley, * because identification * 1 of the numerous species of animals would have been nost difficult without his help. 1ITEEATURE CITED Anonymous. 1966. Report of a new chemical hazard, Kew Sci. 32, 612. aa Bechtel. T. J, and B. J. Copeland. , 1970. .. ' Tish species diversity indices as indicators of pollution in Galveston Bay, Texas. . -* - ,, Contrib. Mar. Sci. 151 103-133. ' BooVhout, C. G,, A. J. Wilson, Jr., T. W. Dulce and J. I. Lowe. r * TTTl9727"Effeetsnof- Kite^WA' JJjd^ecvalrderelopisettt~of-rtma:.----- ------ crabs. Water, Air, and Soil Pollut. 1; 165-180. - Lroedhurst, K. G. 1972. Use and replaceablllty of ?CBs. Environ. Health Perspectives, 2; 81-102. A" .- * ' Butler, P. A. 197-3. Orgar.ochlorine residues in estuarine mollusks - 1965-1972. Pestic. Konit. J. 6(4). ' Cooley, l|. R,, J, M. Keltner, Jr. and J. -Porester. 1972. Kircx and . Aroclor1254: Effect on and accumulation by Tetrahyirena . _ pyrifomis.strain U. .J.. protozool. 19.: 63G-63S*, HONS 203600 1 Duke, T. W., J. I- Lowe and A. J. Wilson, Jr> 1970. A polychlori nated biphenyl (Aroclor 1254 > in the water * sediment and biota of Escambia Eay, Florida, Bull. Environ. Content. Toxicol. 5, 171-180. , Epifanio, C. E. 1971. '- ` * Effects of dleldrinrin seawater on the development of two species of crab larvae, Leptodius florldanus and Psnopeus herbstil. Mar. Biol. {Berlin) 11(4): 356-362. . '.. ' *' . Baasen, D. J., P, R. Parrish, J. 1, Lowe, A. J. Wilson, Jr. and P. D, Wilson, 1971. Chronic toxicity, uptake and retention. of Aroclorl254 in two estuarine fishes. Bull. Environ. " Coataa. Toxicol. 6, 113-119 (1971). *. .... Lloyd, M. and R. J. Ghelardi. ,1964. A table for calculating the v . cgultability component of-Species-diversity. J. Anin. Ecol. 33: 217-225. ' ' - Kimmo, D. R., R. R. Blackman,- A. J. Wilson, Jr. and J. Forester. 1971a. Toxicity and distribution of Aroclor ^^1254 in the pin'.. shrimp Penceus duoratua. Mar. Biol. (Berlin) 11(3): 191-197. Micro, D. R., P. D. Wilson, R. R. Blackman and A. J. Vi 1 sen, Jr. 1971b. Polychlorinated biphenyl absorbed from sediments by fiddler crabs and pink shrimp. Mature, London 231: 50-52. Parrish, P. P-., J. 1. Lowe, A* J. Wilson, Jr. and. J. M. Patrick, Jr. .. 1972. Effects of Aroclor ^ 1254, a PCB, on oysters. Crassostree i HONS 203601 virglnica (Blvalvio: Protobranchia*. Ostreidae).' ASS Bull. 19(2): 90. ^' i ** " -` t ' Pielou, E. C. 1966. The measure of diversity in different types of jbioloaical collections. J. Theor.-Biol. 13: 131-144. Shannon, C. E. and W. Weaver. 1963. The mathematical theory of communication. University of Illinois Press, Urbana. Slegal, S. 1955. Won parametric statiestics for the behavioral *^ ,sciences. 312 p. McGraw-Hill Book Co., Inc. New York. ` * ** Villui, J. L. and T. D. Dorris. 1963. Biological parameters for vater quality criteria. BloScience 18(6): 477-481. k *w* * - - " h. r* uersi > '-w . a . i i , , r i HONS 203602 Table 1. Range and average concentration of Aroclor'O' 1254 in water and sediment fron experimental and control apparatuses. ` Water was analyzed twice monthly and sediment was analyzed at the end of the four month experiment. Limit of quanti- * .. fication was 0.1 vg/* in'water an 0.015 mg/kg (dry weight) .t , , In sediment. Correction for recovery (>70%) is not included. Concentration in water, Nominal v 'Measured Average Range Concentration In sediment, vzfz Measured Average Range Control 0.L 1.0. 10.0 None <0.1 0.6 6,7 <0.1 - 0.1 0.48 - 0.72 5.2 - 7.-B Nona 0.1 0.36 2.0 - . 0.05 - 0.18 0.25 - 0.42 1.5 - 2.5 rT HONS 203603 -- 'ctr. - ren control aquaria cm l ram nqcarla 'cntarCnated for four -ont^s %_tti trorl^- . .sci Cor eac- treotnont, Nuiber of animals and number o: aruaria' from which they irre collected arc Control . " oTl'yg/jt Animals Aquaria ' -Animals Aquaria Aroclor 1254 1 gg/t ' ' 10 vg/l " Animals Aquaria Animal* Aquaria r --- - t'Ca re:.! is 8 , Crc e.la capitat.h. ' 34 Clr- - - 'l.s sp. 1 nrh-t sp. 0 l j. f ~`.u clantbus 6 JC. . otu.* icoln Hot" -c-eotes Cl Ilforris 8 0 ; 'Tele culvcrl 1 e- - i crel 5 oarv-pedata 1 1'v't sa i--'.z.>re~ 1 C rstuj e itfernciensls 0 __-s j.ccrrca 8 1 etc* s t'js h^-irpodut 0 ` cor ~j_z, tear r.u-rlca 1 -Lis. *r'" ~'roe:eri 23 ol. .era sa. 2 L'^z'lzzz - n *" br-jyx i 1 i;1.' t_^ ^ hrrrtMctl G L tp- -r r 0 -ore-Ctia- sp. 6 - i:; --tiiirJ sp. j! 2 ;p, 12 1 5 10 1 0 5 4 0 1 1 1 0 6 0 1 10 2 1 .1 4 0 J 2 1 11 30 0 0 S 13 0 0 0 0 0 11 0 1 35 1 2 0 7 0 0 0 0 A 0 0 0 .G 9 0 0 0 0 0 7 0 1 10 1 1 0 4 c 0 0 0 Total /olrals 22 121 34 43 2 1 1 1 C 1 2 116 4 3 1 15 3 2 *0f$ 203604 fi CCo r;ai;cd) c! ' ii.' /; < t ; .U: yA' t '' <i 7AX0V l r Vtnrcpoda , 1` I ' ' Esiar.us Sp, J | . Caar^llr sp. ' I , ' Corcahatn volutatQT ' \eoagrofi ttxana ( { '..poretia * 1 t!ec-?od larva*, , , j ! urldcr.E. *p, PyenocwldM, unldsnt. [' i . '` j Bjrtchiopod* I i | Glottldla pyraaidaca I J ; Qiordata J i . ; Bet trlchobraitchua lvia-ls ; . | _ i '>T^r:t-icstone csrlbaegn ! i | j J ydp.la sandattcnsls i. Coolentarata I- * lopir-.adusae*, unident, ? 2oar:baria, unldent, op. Control Anlnalt Aanarla 0 1338 2 4 0 10 2 0 0 0 100 2 91 5 0 r ' o,i vg/t Anlaala Aauarla 4 Aroclor 1254 1 ug/l Aniaala Aauarla io ugA Anir.ali Aquaria 2] 2 13 10 736 0. 0 0' 0 oi 0 6 3' . 1 f 21 3' 10 0 O, 2' . 0 3 0 l 1 0 '3 0 1 0 .1 ; 0` 1 44 00 3, 3 Total Aniaala 19 14 3770 2 1 4 32 i 1 134 3 ir 24 ,1 5 ` t HONS 203605 \_ TASQy ~ 111 | i E<hlr.od*mCa _ | 1 Kcri^oila elornaea ' ' A.-- 'iCMti* SP I--------- " . Oetcprocta * j ygnbranlrora tamila* ) Molluscs ! Acr*- aeoeil la . : I^t oaavrla ' /i-ac1'.; craisl-rata i 1''~J ' tn^sv^.'sg , * -~f * - cca:aca j , ' > ? ' 11:-jt v.-.ri'.jr ; [ > Ccccv-i oulthclluct i 1 ; C-js~?strea vlrutn.lce i ' Crao :-jia fermcata ' , 1 im'--1 h1' ip'ircysl I' La ;v-tartfi:r Kortoai ' a i1--, - I .alira j t I'aaara frjallm ' ' yit:t 11a lenatn , ' I'ajit'lus JTariaanaa j ' 11. dcnisst-s Control Anlnal Aquaria ** 1 1 1 0,1 yg/l Animal Anuarla IT 41 00 Aroclor 1254 1 Vg/l Acl*al Aquaria 13 6 00 WvtJl Anlttal Aauarla 09 00 6S 6 44 00 15 8 10 22 64 54 16 7 00 43 00 00 95 21 0 .0 22 11 11 11 4` 0 4 1 4 2 5 0 0 '3 1 1` 0 0 1 6 3 0 4 '1 4 2 4 0 0 3 4, 1 0 0 1 5 0 0 3 1 7 0 4 0 1 2 1/ 11 1 1 2 5 0 0 3 1 4 0" 3 o1 2 1 1 1 1 1 ,0 6 0 '6 1 1 0 4 3 0 4 0 0 0 1' 0 0 6 0 5 1 1 0 4 3 0 3 0 0 0 1 0 Total Ar.lrala l 54 1 31 20 2 19 6 26 :2 L 17 13 *I t 16 4 2 =7 3 >3 :4 MONS 203606 I (Continued) TAXON ' Holla*ce (CentUuad) [ , fell-la lateralis . 1 k <* I *a lateral!* fciSfiiiB alt'js * II 0;tre~ equcstrl* (tetus:1 canaliculata : Riaso'-na catcabyana jjfojelus divisus j Telliaa altemata (Eelicaeea, unidant. bp. | Gastropoda, unident. ap. Neaortirea - . , 111 . ! 1 Ocratrdla dorsal! j ` j Unlacr.tifled sp( Control Anlaal Aquaria 53 74 11 00 11 11 11 00 91 1X 21 Colonies: Counted ns on* nic*l, .!!' \ *1- ; MI i:! > :|! * !{i | ! i-1 Antwal Aquaria ; Arcelor 1254 "r 1 ugft 1 10 pg/i Aniaal Aauarla Ardal Amiarlt Total Antral* t 15 1 1 2 1 1 2 20 1 3 2 X NONS 203607 i v* -r ^5=r=r 1 1 M__ __ ___ * ` . .. ,, V .^4 iKy< *1 y*\V< -- * ' ' '* Table 1. Huaber of control aadex.pertiMntDl aquaria that contained " - - anluols/bf p^luBr-Tan aquaria uere u**d for each control ^ ' *' f> 1 -- end contamlnatad apparatus, Expatirantal aquaria ware contaminated continuously vith Arotlor 1154 for four oonthc, t '. .. . ,, 1! . . Fhylum r . ,, - Control Aquaria . Aroclor 1234 Aquaria 0.1 ppb 1.0 ppb 10.0 ppb - io -- Arthropods ',, . . 10 " *q Inaddopait I Chordata Ok ' a ' ' tS -46 . Coelaxrarats 1 7 - 3 Bcjhi&odarsata * 3. Ectoprocta . .Mr * Bollaaca . | -. a > a* ' 10 RwrtM 3' i ' af * 10 . . 10 10 ' in- 10 4 00 0 10 f 10 ' 4' 7 4 0 `( 4 . 0 10 IO 10 0 .o Total pbyla In apparatus Average number of phyla par aqbariua * S.T 7 5.4 1, s.* ' 4.1 t- , . A* a.* ^ -a K l HONS 203606 ---T - - I -- --------r~ -a. n- . I. Table 4. Number of tpeclat, by phylu*, that developed Iron planktonic larraa In emtrol , apparatoa <Ad In cpparatuaai eorttanlnated continuoutly for four ootxtht with 0,1, /' ' 1 or 10 yr/t of Arocler 1234, Each apparatni emulated of 10 aquaria. Ttron Control Number Percent 0.1vtil Arcelor 1234 ' l.pB/t ............10 yg/t ffueber Percent Number Percent Number Percent Annelida Arthropod* 10 >34.6 S* 10 2,4 14 32.6 8 32.0 3 3.8 *4 11,8 4 9.3 4 16.0 Chordata 3 i 3-*., ` 3 , 8.8 -X. 4.6 f Hollueea 21 ' Jfi * `40a4 * 14 41.2 19 44.2 10 40.0 Other phyla 7 13.4 3 8.8 4 ( 9,3 * L #i'l a! 4.0 1 TOTAL 32 100.0 t t 34 100.0 43 100.0 23 100.0 jI moms z3610 I l I !'i : ! ji I Ta':lc 6, !i!M!: ! . \* Average number par aquarian and avorogc percent frequency par aquarium of anlnala( bp pbplue (range In parenthese*), that ' , , developed from planktonic larva* In 10 control aquaria and 10 aquaria.that for four month# Tocolvad 0.1, 1 or 10 pg/l o:f i! ! I Aroclor 1234, , ?4ylun Control i fuirber Percentage i.l vt/l Number Porcrntoxa < Aroclor 1234 ; 2 p*/i i Number ' Pdrcentaxe `, . - 10 yg/t , , , Number ' PereciraRo > * Acrelida . 11.6 (3-23) 6.3 (1.0-21:4) U.9 (4-21) 3.8 a. 3-42.8) 12.8 (7-22) 9.0 (3,9-38,1) 8,5 (5-16) 12,5 (5,3-66.7' Arthropod# ! 134.6 (6-406) 73.8 (12.7-94.0) 170.9 (14-$2) 03.6 (28.6-96.2) 78.0 (8-199) 54,9 (20^5-06,9) . 0.7 (0-4) ; ; lil (0-2.8) ,! jChordata 19.3 (0-112) 10.9 (0-64.7) 14.4 (1-32) . 7.0 (0.6-19.4) 43.7 (0-130) 30.8 (0-39.1) 32.8 (3-160) ' 80.4 (2J.0-E8.C) Kol'-uren ' 10.1 (3-18) 3.7 (0.7-20.6) 4.4 (1-10) 2.2 (0.8-12.2) ! 4.4 a-9) S.l (1.3-33.3) 3.0 (1-6) ; 4,6.d',9-9,0) f j' l Othsr phyla 2.0 (0-4) 1.1 (0-7.1) 2.7 fO-11) v 1!4 (0-6.3) 3.2 (1-2) 2.2 (0.9-7.7) 4 ) .:; ' t 0.7 (0-1) *! l.'O (01-3.6) t TOTAL 177,6 (14-432) 100,0 204.3 (49-589) 100.0 142.1 (24-239) 100^0 '. 63.7 (12-186) 100.0 , !!. :'.j i: HONS 203611 !I ( I ii i!? r:i-. ' Table 7. Shanr.on-Voy*r index'of ftpeelei dlvtnity end Index of ipeclet rlehn'aa la the ten control aquaria and ten'aquaria r>\ ! :. i eontaniaated with 0,1, 1 or 10 iig/t of Aroclor 1234. ` i "i 1. -i Spades diveraity Equi.ability <J ) Naaa i.so 0.47 r J* Aroclor 1254 ,1 Control Ran*o ' Scd. Srror i ""j i 1 0,1 ug)t latt* std. 4 Kaaa Error 0.37-2,83 0.24 1.42 1 0.36-3,17 0.26 2.07 1.0 0j/t Ktnga Std.4 Error . 0.94-3.26 0.20 0.14-0.89 0.07 0.38 ' 0,10-0.81 0.07 0.35 0,23-0.86 0.06 I r Naaa l ;io.o we/t . j *; ; l-. linnga st3d. ;J tj ;; !- r ' Ei'ror *. j 1 ' 1.62 0.33 1.03-2.19 0*13 t i | - ** t 0.26-0.94 o.o6 : 1 HONS 203612 ( > t 1, <i 'iI ! Tnblt*B.~ Specie* and total rtuff'bcr of an tea Is collected froo the ~**--- effluents of 10 control tquaiU atjd 10 aquaria contanI.nac.cd ^ r*'* fT" fo*r four ontha vith 0.1,1 or .10 Mt/t Areetor UH. Taxon -*' r . Control 0.1'ug^t I tf.fl 10 tt/> Annelida " ' : ett -. gupoTnetus lUnthat. I. protullcola yeanthgs gucclnea , " Total ^ Arthropod* . ' r- v'tV. *) *.*!' *. J ,-I. 6 i 1 1 ,2 .) 0 1 1 1 0 0 0 0 Balanm ap. XO ` * 2 Cearella apCllbanarls tricolor 0 A 010 Qprophlyjs TOlntator 7 13 32 l;am>sE.Qtaa3 dayman* V ' *. . 5 baooaaopa tar.tna v> * 3 raaurtiaTonglcgrpoa i00 Wnnlats chaatoptara _ ... . _ j,_. - *- 0 Ppogabla ffinis i 1 .0 poespod soea, cidint. op. 0 0 1 Tortucldaa, wildcat. ap. t 1 0 pTCCogotvldaa, wildeat. ap,' 0 0 3 Total . 55 30 Chordata -> . 1 0 0 1 0 0 0 0 0 0 0 0 2 goatrlehobranchua allsUrls 3 6 0 graochloatoo* carlbaaua .0 i Wolculn eanhattsnals 72 W3 " 304 Total 'n . 103 ' 53s ' 0 0 35 35 Coclaatarata Lsptoxadoaa**, voidant. *p. . \ tchloodanaata '. 1. k -----Baadpholla alongata - 1 .O' Ettoprocta . . - - -- - Hwftrcalpor* tenuis* _ - Jid1' v -1, . ,, -1 ' i* . , 3` i 1 0 0 Molluca ' . - Anadata ovalia ' A. ttansvetsa . llttluo alternate g. varlvm . Crmmtrta vlrfinlca borldcllc ohsmre tacvlcardlm" aortwit * Wltrella luneta 1 n 0 3 2 17 --1 1 0 1 0 0 7 .. ' 0 0* 4 0 1 1 2 O 7 0 1 0 0 1 l 0 2 HONS 203613 Table 6, (Continued) Mollusc* (Continued) Tlusculus lateralis ' ilbus Tnftelu* 4ivl*u* Eolldace*. unldcat. ap. Total . .1 i 0 `2 1 0 '1 - 2 1. 1 38 , T ' 1 0 0 1 12 0 0 0 3 8 TOTALS:: Animals ----------- r-------- ' 156 '156 Species '*7 . 1B *Colosiii: Counted as oae animal. 375 20 - 47 10 HONS 203614 * f $ > / ' ^: it Fi^vrc. 16, Total Ion Cufrrcnt Chro^atosras ? of the Folar .Titian,, Fraction (Silicic Acid) < of a Grocn Say Trc-i lUcract MONS 203777 \