Comrmentaries Reproductive Toxins and Alligator Abnormalities at Lake Apopka, Florida Jan C. Semenza,1,2 Paige E. Tolbert,2 3 Carol H. Rubin,2 Louis J Guillette Jr.,4 and Richard J. Jackson2 1Epidemic Intelligence Service, Epidemiology Program Office, and 2National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341 USA; 3Rollins School of Public Health, Emory University, Atlanta, GA 30322 USA; and 4Department of Zoology, University of Florida, Gainesville, FL 32611 USA The alligator population at Lake Apopka in central Florid ld l b 90 and 1987. Endocrine-disrupting chemical and spe DD olite have been impit. ed in the alligtors' rpodut Te DD ab li s is based la o observation of elevated co n s ofhp-DE SndADD i aligator egg obt edfom Lake Apopka in 1984 and 1985. In th oin me n w drw a twone tocides that are estoductive topin -i hua ib and ethylene dibromide (EIB), which coild also 'have pL a r in the reprductive ilure observed in ia fiom L A - pok in t e 1980K w alig DBCTh DDT, EDB, environmentd estogen, nem Atocide petdes rptile. Environ Heda/ Poep 105:1030-1032 (1997). hhp:llehisnie/snnih.gv Developmental abnormalities of the gonad and abnormal sex hormone concentrations have been documented in juvenile alligators from Lake Apopka (1). Recently, it has been shown that many of the environmen- tal chemicals found in alligator plasma or eggs bind the alligator estrogen and/or progesterone receptors in vitro (2). Guillette et al. (1) suggested that the reproductive failure at Lake Apopka could have been related to general agricultural pollution and to a spill from a nearby pesticide manufac- turing facility. From 1957 to 1981, the facility (Tower Chemical Co.) manufac- tured and stored both chlorinated and organophosphate insecticides as well as a copper-salt-based fungicide at a site 1.5 miles from Lake Apopka. Wastewater from the manufacturing process was discharged into an unlined pond, and chemicals were burned or buried on site. During a heavy rain in 1980, the percolation pond over- flowed and acidic wastewater discharged into a marsh that drains into Lake Apopka. DDT and other chemicals contaminated the lake during this extensive spill. The area surrounding the chemical company's plant was declared an EPA Superfund site in 1983. DDT and other pesticides have also entered the lake as a result of extensive agri- cultural activity surrounding the lake, pri- marily activity in orange groves and veg- etable muck farms. Because of the endocrine-disruptive potential of DDT's degradation products DDE and DDD, they have been the prime suspects in the repro- ductive abnormalities of the alligators (1). During a review of sampling reports and inventory documents, we became aware of two other reproductive toxins, 1,2-dibro- mo-3-chloropropane (DBCP) which is known by the trade name Nemagon, and ethylene dibromide (EDB), that could also have contributed to a decline of the alliga- tor population. DBCP, a well-established human reproductive toxin, was first report- ed to be associated with male sterility at a California pesticide-formulation plant in 1977 (3). The EPA banned DBCP from all U.S. crops in 1985, and it was subse- quently replaced by EDB. Since 1948, EDB has been used extensively as a fumi- gant for grain, fruit, and vegetable infesta- tions. EDB has also been shown to be a reproductive toxin and to damage sperm and decrease fertility in humans (4). Both of these chemicals were present in the Lake Apopka environment at elevated concen- trations according to historic monitoring records described below. Dibromochloropropane In addition to being contaminated by DDT and its metabolites, the soil, sedi- ment, and surface water at the Superfund site also contained high levels of DBCP following the spill. The method of waste disposal used by the company was to burn and bury waste in an area of approximately 58,200 ft2, from which up to 70 drums were excavated during clean-up efforts. Analytical results from the 1980 EPA (5) and the 1981 Florida Department of Environmental Regulation (FDER) (6) sampling investigations, as well as analyses of material uncovered during the 1983 clean-up of the burn/burial site, confirm that pesticide waste was disposed of at that site. DBCP was found at concentrations as high as 1,340,000 ppb in surface soil sam- ples at the burn/burial site (Table 1) (5-7). In 1983, portions of the burn/burial area were removed by the EPA. DBCP was also found in sediment samples collected from the wastewater pond by the EPA and the FDER in 1980, 1981, and 1983 in the range of 162,000-165,000 ppb (Table 1) (5-7). Subsequently, the pond was drained and sediment, to an average depth of 2 ft, was removed from an area estimated to be 30,100 ft2. However, the remaining lower sediment layers of the former wastewater pond remain a potential source of contami- nants, subject to groundwater migration. Surface water samples taken in 1983 from the lagoon and a stream off-site contained only low levels of DBCP contamination (<0.041 ppb) (7). This finding is consistent with modeling data using the Henry's law constant, which indicates a half-life of 13.5 hr for evaporation from a model river 1-m deep (8). The physicochemical properties of DBCP are very different from those of DDT in that DBCP is very volatile and does not bioaccumulate (9,10). A volatiliza- tion half-life of 8 days can be calculated from a model pond by using a three-com- partment EXAMS model (11). The high environmental mobility of DBCP in soil and water (12) may have contributed to the fact that it was not detected in subsequent samples and that the EPA decided not to include it on the short list for long-term monitoring. The route of exposure for the alligators living in this environment might have occurred through both oral and dermal exposure. Animal studies in rats show that DBCP is rapidly absorbed from the gas- trointestinal tract after oral administration by gavage when water is used as a vehicle (13). Dermal absorption is not as well docu- mented in animal studies, but it is support- ed by the observation that death occurred following a 24-hr dermal exposure (14). Occupational exposures to DBCP probably occurred through both inhalation and der- mal exposure; thus, the contribution of der- mal absorption to reproductive health out- comes cannot easily be determined. The testicular abnormalities in alligators from Lake Apopka are similar to those in pesticide workers exposed to DBCP in that the seminiferous tubules are the affected tar- get tissues. However, whereas alligator testes Address correspondence to J.C. Semenza, University of California Irvine, College of Medicine, 224 Irvine Hall, Irvine CA 92697-7550 USA. We would like to thank Mark McClanahan for helpful discussions and Philip Beane for his assis- tance with the graphics. Received 28 April 1997; accepted 12 June 1997. Volume 105, Number 10, October 1997 * Environmental Health Perspectives 1 030 Commentaries * Reproductive toxins at Lake Apopka, Florida show poorly organized seminiferous tubules with bar-shaped cellular structures (1), the seminiferous tubules from humans exposed to DBCP are devoid of spermatogenic cells altogether (15). Moreover, the mechanism by which these effects occur may be differ- ent. Guillette et al. (16) have proposed that the developmental abnormalities of the gonad reported in alligators is due to orga- nizational modifications; that is, the devel- opment of the testis has been altered such that the molecular and/or cellular functions of this organ are modified. Thus, the changes observed are permanent and not reversible. In contrast, the observed testicu- lar abnormalities in pesticide workers are activational modifications affecting the same organ and apparently similar cell types-the spermatogonia. Among pesti- cide workers diagnosed with oligospermia, normal testicular functioning was restored once the workers were removed from the source of DBCP because their testes were organizationally normal. It is possible that the depressed clutch viability and reduced neonatal survival among these alligators (1) parallel the increase in spontaneous abor- tions observed among humans following exposure to DBCP (17); that is, exposure of alligators to DBCP following the spill may have modified male reproductive function- ing dramatically by an activational mecha- nism, resulting in poor recruitment and thus a population decline. A reduction in male alligator reproductive ability in con- cert with altered female reproductive func- tioning and elevated persistent, biomagni- fled contaminants in eggs (such as DDT metabolites) would lead to the dramatic population decline and egg/juvenile mortal- ity reported (16,18). Extensive monitoring initiated in 1983 by the Florida Department of Environmental Protection (FDEP) and Florida Department of Health and Rehabilitative Services (FHRS) failed to reveal DBCP contamination in ground water in wells surrounding the lake (C. Cosper, personal communication; A. Reich, personal communication). However, given the levels found in the remaining pond, it is almost certain that DBCP entered Lake Apopka during the 1980 spill. Ethylene Dibromide Sampling of private wells initiated in 1984 in the vicinity of Lake Apopka by the FHRS found another reproductive toxin, EDB, at concentrations exceeding the Florida maxi- mum contaminant limit (MCL) of 0.02 ppb. The majority of sampling by the FHRS in this area was conducted in the southwest corner of the lake where the Superfund site is located. More than 80 wells were found to have EDB levels above Table 1. Concentrations of 1,2-dibromo-3-chloropropane (DBCP) detected in samples taken by the EPA and the Florida Department of Environmental Regulation at the Superfund site near Lake Apopka Site Concentration of DBCP (ppb) Reference Surface soil samples from burn/burial area (0-1 ft) 1,337,792 (5) Surface soil samples from burn/burial area (1.0 ft) 10-1,500 (7) Surface soil samples from burn/burial area (2 in) 10.0-750 (7) Sediment samples from waste water pond (1 ft) 162,000-165,000 (5-7) Sediment samples from waste water pond (2 ft) 200,000-300,000 (7) Surface water samples from the unnamed stream <0.041 (7) * ~~~~~~~~~~ ~ ~ MSuperfund site - i> i;*;;x, g ~~~~~~Negative EDB * ~jw - .0 . - (ti; Z s @EDB 1.0ppb ;ff -e 4 . it ' . t ' - . t Figure 1. Private well sampling and ethylene dibromide (EDB) testing in the vicinity of Lake Apopka (insert), Florida. The Florida maximum contaminant limit is 0.02 ppb (pg/I), with remediation of wells occur- ring at levels greater than 0.025 ppb. Data were obtained from the Florida Department of Health and Rehabilitative Services (unpublished results). the MCL; 29 of these wells were contami- nated with levels greater than 1.0 ppb (Fig. 1). EDB was a commonly used agricultural nematocide in Florida; therefore, the cont- amination is likely to have originated from agricultural use rather than the spill. Because the EDB-contaminated wells are located upstream from the Lake Apopka aquifer, which regionally flows toward the northeast, cross-contamination of the lake could have occurred. In field experiments, EDB has been detected in soil 19 years after its last known application (19). In aquifers, where volatization does not occur, the half- life for EDB undergoing uncatalyzed hydrolysis is 6 years (20). Like DBCP, EDB is very volatile and does not bioaccumulate (21,22); EDB can be absorbed via the der- mal or oral routes (23). In humans, EDB is known to be associated with decreased sperm count, an altered percentage of viable and motile sperm, and an increase in certain types of morphological abnormalities of sperm (4,24). If this compound was present in the lake in the early 1980s, which is very likely, and given its use in agricultural activ- ities and presence in samples from wells sur- rounding the lake, it could also have con- tributed to the reproductive problems that led to a decline in the alligator population. However, to our knowledge, no monitoring of EDB at the Superfund site was conduct- ed on water, muck, or biological samples from the lake during the early 1980s. Therefore, historic inferences cannot be drawn from current monitoring efforts. Conclusion Although DDT, its metabolites, and other persistent bioaccumulated pesticides contin- ue to be prime suspects in the egg and embryonic abnormalities reported, the alli- gator population decline that occurred in the early 1980s at Lake Apopka could have involved two other potent reproductive tox- ins as well. DBCP and EDB, two docu- mented reproductive toxins in humans, were observed in elevated concentrations at the Environmental Health Perspectives * Volume 105, Number 10, October 1997 1031 Commentaries - Semenza et al. Superfund site (DBCP) and in well water (EDB) and could have affected alligators at Lake Apopka. The findings discussed above indicate a complex exposure scenario in which the eti- ology of the reproductive failure cannot be reconstructed with certainty due to the his- toric nature of the event. REFERENCES 1. Guillette U Jr, Gross TS, Masson GR, Matter JM, Percival HF, Woodward AR. Developmental abnormalities of the gonad and abnormal sex hor- mone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environ Health Perspect 102:680-688 (1994). 2. Vonier PM, Crain DA, McLachlan JA, Guillette U Jr, Arnold SF. Interaction of environmental chemi- cals with the estrogen and progesterone receptors from the oviduct of the American alligator. Environ Health Perspect 104:1318-1322 (1996). 3. Whorton MD, Krauss RM, Marshall S, Milby TH. Infertility in male pesticide workers. Lancet 2:1259-1261 (1977). 4. Schrader SM, Turner TW, Ratcliffe JM. The effects of ethylene dibromide on semen quality: a comparison of short-term and chronic exposure. Reprod Toxicol 2:191-198 (1988). 5. U.S. EPA. Investigation Report, Tower Chemical Company and Surrounding Area, Clermont, Florida. Athens, GA:U.S. Environmental Protection Agency, 1980. 6. FDER. Tower Chemical Company Investigation, Lake County, Florida, Project Files. File Reference T-15.E.5. Orlando, FL:Florida Department of Environmental Regulation, 1981. 7. U.S. EPA. Superfund Cleanup, Tower Chemical Company Site, Clermont, Florida. Athens, GA:U.S. Environmental Protection Agency, 1983. 8. Lyman WJ, Reehl WF, Rosenblatt DH, eds. Handbook of Chemical Property Estimation Methods. New York:McGraw-Hill, 1982. 9. Bysshe SE. Bioconcentration factor in aquatic organisms. In: Handbook of Chemical Property Estimation Methods (Lyman WJ, Reehl WF, Rosenblatt DH, eds). New York: McGraw-Hill, Book Co., 1982;5-5. 10. Munnecke DE, VanGundy SD. Movement of fiumigants in soil, dosage responses, and differential effects. Annu Rev Phytopathol 17:405-429 (1979). 11. U.S. EPA. Exposure Analysis Modeling System: Reference Manual for EXAMS II. EPA/600/3- 85/038. Athens, GA:U.S. Environmental Protection Agency, 1985. 12. Hodges LR, Lear B. Persistence and movement of DBCP in three types of soil. Soil Sci 118:127-130 (1974). 13. Gingell R, Beatty PW, Mitschke HR, Page AC, Sawin VL, Putcha L, Kramer WG. Toxicokinetics of 1,2-dibromo-3-chloropropane (DBCP) in the rat. Toxicol Appl Pharmocol 91:386-394 (1987). 14. Torkelson TR, Sadek SE, Rowe VK. Toxicologic investigations of 1,2-dibromo-3-chloropropane. Toxicol Appl Pharmacol 3:545-559 (1961). 15. Biava CG, Smuckler EA, Whorton MD. The tes- ticular morphology of individuals exposed to dibromochloropropane (DBCP). Exp Mol Pathol 29:448-458 (1978). 16. Guillette LJ Jr, Crain DA, Rooney AA, Pickford DB. Organization versus activation: the role of endocrine-disrupting contaminants (EDCs) dur- ing embryonic development in wildlife. Environ Health Perspect 103(suppl 7):157-164 (1995). 17. Kharrazi M, Potashnik G, Goldsmith JR. Reproductive effects of dibromochloropropane. Isr J Med Sci 16:403-406 (1980). 18. Woodward AR, Percival HF, Jennings ML, Moore CT. Low clutch viability of American alligators on Lake Apopka. Florida Sci 56:52-63 (1993). 19. Steinberg SM, Pignatello JJ, Sawhney BL. Persistence of 1,2-dibromoethane in soil: entrap- ment in intraparticle micropores. Environ Sci Technol 21:1201-1208 (1987). 20. Barbash JE, Reinhard M. Abiotic dehalogenation of 1,2-dichloroethane and 1,2-dibromoethane in aqueous solution containing hydrogen sulfide. Environ Sci Technol 23:1349-1358 (1989). 21. Eisenreich SJ, Looney BB, Thorton JD. Airborne organic contaminants in the Great Lakes ecosys- tem. Environ Sci Technol 15:30-38 (1981). 22. Kawasaki M. Experiences with the test scheme under the chemical control law of Japan: an approach to structure-activity correlations. Ecotoxicol Environ Saf 4:444-454 (1980). 23. Alexeeff GV, Kilgore WW, Li MY. Ethylene dibromide: toxicology and risk assessment. Rev Environ Contain Toxicol 112:49-122 (1990). 24. Ratcliffe JM, Schrader SM, Steenland K, Clapp DE, Turner T, Hornung RW. Semen quality in papaya workers with long term exposure to ethyl- ene dibromide. Br J Ind Med 44:317-326 (1987). 1032 Volume 105, Number 10, October 1997 * Environmental Health Perspectives