Reproduction, larval growth, and reproductive development in African clawed frogs (Xenopus laevis) exposed to atrazine.

Reproductive success and development of F2 offspring from F1 adult African clawed frogs (Xenopus laevis) exposed to atrazine throughout larval development and as sexually mature adults was examined. Larval X. laevis were exposed to one of four nominal concentrations of atrazine (0, 1, 10, 25 microg atrazine/l) beginning 96 hr after fertilization and continuing through two years post-metamorphosis. Clutch size and survival of offspring were used as measurement endpoints to gauge reproductive success of the F1 frogs. Larval survivorship and time to metamorphosis were used to gauge developmental success of the F2 offspring from atrazine-exposed frogs. Testes in F1 and F2 frogs were examined for incidence of anomalies, such as testicular ovarian follicles, and sex ratios in F2 offspring were investigated to determine if exposure to atrazine caused trans-generational effects (effects on F2 individuals due to exposure of F1 individuals). There were no effects of any of the studied concentrations of atrazine on clutch size of F1 frogs. There were also no effects on hatching success or time to metamorphosis. Sex ratios did not differ between F2 offspring among treatments. There was no evidence to suggest a transgenerational effect of atrazine on spawning success or reproductive development of X. laevis. This is consistent with the presence of robust populations of X. laevis in areas where they are exposed to atrazine that has been used for several decades for weed control in production of corn. Our observations also are consistent with the results of most other studies of frogs where no effects were found to be associated with exposure to atrazine. Our data do not support the hypothesis that atrazine significantly affects reproductive fitness and development of frogs.

Chemical contaminants, health indicators, and reproductive biomarker responses in fish from the Colorado River and its tributaries.

Common carp (Cyprinus carpio), black bass (Micropterus spp.), and channel catfish (Ictalurus punctatus) were collected from 14 sites in the Colorado River Basin (CRB) to document spatial trends in accumulative contaminants, health indicators, and reproductive biomarkers. Organochlorine residues, 2,3,7,8-tetrachlorodibenzo-p-dioxin-like activity (TCDD-EQ), and elemental contaminants were measured in composite samples of whole fish, grouped by species and gender, from each site. Selenium (Se) and mercury (Hg) concentrations in fish were elevated throughout the CRB, and pesticide concentrations were greatest in fish from agricultural areas in the Lower Colorado River and Gila River. Selenium concentrations exceeded toxicity thresholds for fish (>1.0 microg/g ww) at all CRB sites except the Gila River at Hayden, Arizona. Mercury concentrations were elevated (>0.1 microg/g ww) in fish from the Yampa River at Lay, Colorado; the Green River at Ouray National Wildlife Refuge (NWR), Utah and San Rafael, Utah; the San Juan River at Hogback Diversion, New Mexico; and the Colorado River at Gold Bar Canyon, Utah, Needles, California, and Imperial Dam, Arizona. Concentrations of p,p'-DDE were relatively high in fish from the Gila River at Arlington, Arizona (>1.0 microg/g ww) and Phoenix, Arizona (>0.5 microg/g ww). Concentrations of other formerly used pesticides including toxaphene, total chlordanes, and dieldrin were also greatest at these two sites but did not exceed toxicity thresholds. Currently used pesticides such as Dacthal, endosulfan, gamma-HCH, and methoxychlor were also greatest in fish from the Gila River downstream of Phoenix. Total polychlorinated biphenyls (PCBs; >0.11 microg/g ww) and TCDD-EQs (>5 pg/g ww) exceeded wildlife guidelines in fish from the Gila River at Phoenix. Hepatic ethoxyresorufin O-deethylase (EROD) activity was also relatively high in carp from the Gila River at Phoenix and in bass from the Green River at Ouray NWR. Fish from some sites showed evidence of contaminant exposure as indicated by fish health indicators and reproductive biomarker results. Multiple health indicators including altered body and organ weights and high health assessment index scores may be associated with elevated Se concentrations in fish from the Colorado River at Loma, Colorado and Needles. Although grossly visible external or internal lesions were found on most fish from some sites, histopathological analysis determined many of these to be inflammatory responses associated with parasites. Edema, exophthalmos, and cataracts were noted in fish from sites with elevated Se concentrations. Intersex fish were found at seven of 14 sites and included smallmouth bass (M. dolomieu), largemouth bass (M. salmoides), catfish, and carp and may indicate exposure to endocrine disrupting compounds. A high proportion of smallmouth bass from the Yampa River at Lay (70%) was intersex but the cause of this condition is unknown. Male carp, bass, and catfish with low concentrations of vitellogenin were common in the CRB. Comparatively high vitellogenin concentrations (>0.2 mg/mL) were measured in male bass from the Green River at Ouray NWR and the Colorado River at Imperial Dam and indicate exposure to estrogenic or anti-androgenic chemicals. Anomalous reproductive biomarkers including low GSI and gonadal abnormalities (calcifications, edema, and parasites) observed in fish downstream of Phoenix are likely related to the poor water-quality of the Gila River in this area.

Topical dose delivery in the reptilian egg treatment model.

Developing assays to detect endocrine-mediated toxicity from in ovo or in utero exposure is a current challenge in regulatory toxicology. Some species of reptiles exhibiting a unique mode of sex determination, in which the incubation temperature during a critical period determines gonadal sex, have been explored as an in ovo model to screen environmental contaminants for endocrine effects. We critically review published egg-exposure studies and conclude that data regarding the pharmacokinetics of topically applied substances are insufficient to validate dose-response relationships for the effects of chemicals on in ovo endocrine function or gender determination in reptiles. The insufficiencies in these data largely result from methodological failures, including lack of measurement verification, failure to investigate and control extraneous factors affecting the measurements, and lack of independent replication of results. Considerable additional research will be necessary to alleviate these methodological inadequacies. Given the current status of the data, topical treatment of reptilian eggs cannot be considered to be a valid means of establishing causal relationships between chemical treatment and biological outcome.

Dose verification after topical treatment of alligator (Alligator mississippiensis) eggs.

Numerous studies have used temperature-dependent sex determination in reptilian eggs to investigate potential developmental effects of exogenously applied substances. However, few studies have measured the dose carried across the eggshell. We report embryonic mortality and internal egg concentrations determined by gas chromatography-mass spectrometry two weeks after exposure of American alligator (Alligator mississippiensis) eggs to chlorinated organic pesticides via injection or topical application. Puncturing the eggshell for injection produced high mortality compared with unpunctured controls; therefore, further evaluation of this method was abandoned. Although higher than controls, mortality was much lower in eggs treated topically than in those injected. Transfer of chemicals across the eggshell was very low, highly variable, and did not correlate with the applied dose after topical application. These results are consistent with previous reports in the literature, casting doubt on whether a reproducible internal dose can be achieved in reptilian eggs by topical treatment.

Influence of seasonality and exposure on the accumulation and reproductive effects of p,p'-dichlorodiphenyldichloroethane and dieldrin in largemouth bass.

Two studies investigated the accumulation and reproductive effects of p,p'-dichlorodiphenyldichloroethane (DDE) and dieldrin over 30 or 120 d of oral exposure in captive Florida, USA, largemouth bass (Micropterus salmoides floridanus). The 30-d exposures were conducted during the peak reproductive season, and the 120-d study was conducted to simulate exposure throughout the ovarian cycle. Whole body chemical residue concentrations were similar, regardless of exposure duration, for the medium and high feed concentrations of either chemical; however, the low-dose residue concentrations were much lower, yet similar to natural exposures. No clear dose-response relationships were identified between chemical dose and morphological (length, weight, hepatosomatic index) or reproductive endpoints (sex steroid concentration, gonadosomatic index, percentage of fry hatching). Reproductive parameters were variable within treatment groups, indicating that circulating sex steroids and percent hatch endpoints have high natural variability among fish of the same age and reproductive stage. However, in general there was a decrease in plasma estradiol and 11-ketotestosterone for female and male fish, respectively, that were exposed to dieldrin. Overall, results suggest that exposure throughout ovarian (follicular) development to either DDE or dieldrin alone does not result in the depressed endocrine status and poor reproductive success reported in highly organochlorine pesticide-contaminated environments in Central Florida, USA.

Dietary exposure of largemouth bass to OCPs changes expression of genes important for reproduction.

Dieldrin and p,p'-DDE are ubiquitous contaminants known to act as endocrine disruptors, causing impaired development and reproduction in fish and wildlife. In order to elucidate the mechanisms by which dieldrin and p,p'-DDE cause endocrine disruption in largemouth bass (Micropterus salmoides), fish were exposed subchronically through the diet to both contaminants. Following 120 days of exposure, p,p'-DDE decreased estradiol in females, but increased 11-ketotestosterone in both sexes. Dieldrin on the other hand, decreased estradiol and 11-ketotestosterone in both sexes. Both pesticides also altered steady state mRNA expression levels of a set of genes chosen to represent three possible mechanisms of endocrine disruption: (1) direct interaction with soluble sex steroid receptors, (2) biosynthesis of endogenous sex hormones, and (3) metabolism of endogenous hormones. p,p'-DDE acted as a weak estrogen, increasing the expression of vitellogenin and estrogen receptor alpha in the liver. p,p'-DDE also altered the expression of genes involved in the synthesis of endogenous hormones as well as their metabolism. Dieldrin, on the other hand, only altered expression of vitellogenin and not estrogen receptor alpha. Dieldrin also altered the expression of genes involved in hormone synthesis and metabolism, and it dramatically lowered plasma hormone levels. Both pesticides targeted expression of genes involved in all three modes of action, suggesting that they each have multiple modes of action.

Necropsy findings in American alligator late-stage embryos and hatchlings from northcentral Florida lakes contaminated with organochlorine pesticides.

Increased American alligator (Alligator mississippiensis) embryo and neonatal mortality has been reported from several northcentral Florida lakes contaminated with old-use organochlorine pesticides (OCPs). However, a clear relationship among these contaminants and egg viability has not been established, suggesting the involvement of additional factors in these mortalities. Thus, the main objective of this study was to determine the ultimate cause of mortality of American alligator late-stage embryos and hatchlings through the conduction of detailed pathological examinations, and to evaluate better the role of OCPs in these mortalities. Between 2000 and 2001, 236 dead alligators were necropsied at or near hatching (after approximately 65 days of artificial incubation and up to 1 mo of age posthatch). Dead animals were collected from 18 clutches ranging in viability from 0% to 95%. Total OCP concentrations in yolk ranged from approximately 100 to 52,000 microg/kg, wet weight. The most common gross findings were generalized edema (34%) and organ hyperemia (29%), followed by severe emaciation (14%) and gross deformities (3%). Histopathologic examination revealed lesions in 35% of the animals, with over half of the cases being pneumonia, pulmonary edema, and atelectasis. Within and across clutches, dead embryos and hatchlings compared with their live cohorts were significantly smaller and lighter. Although alterations in growth and development were not related to yolk OCPs, there was an increase in prevalence of histologic lesions in clutches with high OCPs. Overall, these results indicate that general growth retardation and respiratory abnormalities were a major contributing factor in observed mortalities and that contaminants may increase the susceptibility of animals to developing certain pathologic conditions.

Absorption of p,p'-dichlorodiphenyldichloroethylene and dieldrin in largemouth bass from a 60-d slow-release pellet and detection using a novel enzyme-linked immunosorbent assay method for blood plasma.

This work describes the uptake of two organochlorine pesticides from slow-release pellets by largemouth bass and the utility of a blood plasma enzyme-linked immunosorbent assay (ELISA) method for exposure verification. We measured blood and tissue levels by gas chromatography/mass spectrometry and by a novel ELISA method, and present a critical comparison of the results.

An evaluation of biomarkers of reproductive function and potential contaminant effects in Florida largemouth bass ( Micropterus salmoidesfloridanus) sampled from the St. Johns River.

The objective of this study was to describe and compare several reproductive parameters for Florida largemouth bass (Micropterus salmoides floridanus) inhabiting the St. Johns River and exposed to different types and/or degrees of contamination. Welaka was selected as the reference site in this study because of its low urban and agricultural development, Palatka is in close proximity to a paper mill plant, the Green Cove site is influenced by marine shipping activities and Julington Creek site receives discharges of domestic wastewater and storm water runoff from recreational boating marinas. For this study, bass were sampled both prior to (September 1996) and during the spawning season (February 1997). In order to characterize chemical exposure, bass livers were analyzed for up to 90 trace organics and 11 trace metal contaminants. Reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads and plasma concentrations of vitellogenin (VTG), 17beta-estradiol (E2) and 11-ketotestosterone (11-KT). In general, the sum of organic chemicals was highest in livers from Palatka bass and bass from Green Cove and Julington Creek had higher hepatic concentrations of low molecular polycyclic aromatic hydrocarbons and polychlorinated biphenyls when compared to fish from Welaka. Metals were more variable across sites, with highest mean concentrations found in bass from either Julington Creek (Ag, As, Cr, Cu, Zn) or Welaka (Cd, Hg, Pb, Se, Tn). Female bass from Palatka and Green Cove had lower concentrations of E2, VTG and lower GSI in relation to Welaka. Males from Palatka and Green Cove showed comparable declines in 11-KT in relation to males from Julington Creek and GSI were decreased only in Palatka males. These results indicate a geographical trend in reproductive effects, with changes being most pronounced at the site closest to the paper mill (Palatka) and decreasing as the St. Johns River flows downstream. Since reproductive alterations were most evident in bass sampled from the site closest to the paper mill discharge, it is possible that exposure to these effluents might explain at least some of the results reported here. However, the presence of reproductive alterations in fish sampled at a considerable distance from the mill discharge (Green Cove, 40 km) would suggest exposure to chemicals released from sources other than the paper mill plant. It is clear that additional studies are needed to evaluate the potential impact of these reproductive changes in populations of Florida largemouth bass inhabiting the St. Johns River.

Interactive effects of p,p'-dichlorodiphenyldichloroethylene and methoxychlor on hormone synthesis in largemouth bass ovarian cultures.

p,p'-Dichlorodiphenyldichloroethylene (DDE) and methoxychlor were tested alone and in combination to assess the similarity of their actions on hormone synthesis in gonadal tissue from largemouth bass (Micropterus salmoides floridanus), a species whose reproductive fitness has relevance to ecosystem health in Florida (USA). Gonads were harvested from adult female bass (age, two to three years) during the peak reproductive season (January-May), minced, and incubated in culture medium with or without test agents for 48 h. Duplicates of each treatment were performed in each of three experiments using tissue from a different female. Both 17beta-estradiol and testosterone were measured in aliquots of culture medium by validated radioimmunoassay procedures. Dose-response relationships of individual agents were characterized over a 6-log concentration range (1 X 10(-2) to 1 X 10(4) ppb). Both DDE and methoxychlor, tested individually, produced a dose-dependent decrease in testosterone levels. 17beta-Estradiol levels were unaffected. Mixtures of the agents were tested at all concentration combinations of 0.01, 1, 100, and 10,000 ppb in culture medium. Statistical tests indicated that of 16 dose combinations tested, 15 were antagonistic, and only 1 was additive based on the Loewe additivity model of no interaction. These results imply that methoxychlor and DDE inhibit testosterone production by different mechanisms in bass ovaries.

Predicting maternal body burdens of organochlorine pesticides from eggs and evidence of maternal transfer in Alligator mississippiensis.

Few data exist regarding maternal-embryonal transfer of organochlorine pesticides (OCPs) in reptiles. The objective of the present study was to evaluate maternal transfer of OCPs in American alligators (Alligator mississippiensis) from low-, intermediate-, and high-OCP-exposure sites. Overall, total OCP burdens ranged from less than 0.8 ppb in blood to more than 44,000 ppb in abdominal adipose tissue (wet wt concentrations). Lipid-adjusted ratios of maternal adipose burdens (total OCPs) to yolk burdens were close to one (0.94 +/- 0.31:1), suggesting that animals were in steady state and that OCPs in eggs originated from adipose lipids. In contrast, lipid-adjusted muscle and liver OCP burdens were greater than yolk OCP burdens, suggesting that lipids in muscle were not utilized during oogenesis and that nonlipid liver tissue sequesters OCPs. Predictive equations were derived for several tissues and several OCP analytes with r2 values ranging from 0.40 to 0.99 (p < 0.05). We suggest that yolk burdens are predictive of maternal tissue burdens for certain tissues and OCPs and that certain OCPs are maternally transferred in the American alligator. Furthermore, we suggest that future studies should investigate the applicability of these predictive equations for assessing maternal exposure in other crocodilian species.

Effects of pulp and paper mill effluents on reproductive success of largemouth bass.

This study evaluated the effects of bleached and unbleached kraft mill effluent on reproductive success of largemouth bass (Micropterus salmoides). Bass were exposed to effluent concentrations (0, 10, 20, 40, or 80%) for 28 and 56 d. Parameters measured included hepatosomatic index (HSI) and gonadosomatic index (GSI) and plasma concentrations of 17beta-estradiol (E2), 11-ketotestosterone (11-KT), and vitellogenin (VTG). At the end of the 56-d period, bass were moved to hatchery ponds to evaluate spawning success. Spawning mats with eggs either were brought indoors for evaluation of fecundities, hatchabilities, and egg and fry size (measured at age 3 d), or were left in ponds and fry number and size recorded (average age of 14 d). Effluent exposure was verified by measuring resin acids (isopimaric, abietic. and dehydroabietic acids) in bile. Compared to controls, exposed bass had greater concentrations of resin acids in bile. In general, exposed females had lower concentrations of E2 and VTG (> or = 20% effluent), whereas males had lower concentrations of 11-KT (> or = 20% effluent) and increased E2 (> or = 20% effluent). The HSI values increased in females (> or = 10% effluent), and GSI values decreased in both sexes (> or = 40% effluent). Fecundity, egg size, and hatchability did not differ across treatments, but an increase in the frequency of fry abnormalities and a decrease in fry weights was observed at effluent exposures of 40% and higher. However, results from the pond study, revealed a significant reduction in fry growth and survival (> or = 10%). This decline may have been caused by an increased frequency of deformities, in conjunction with alterations of growth. These changes could have resulted from alterations in egg quality because of failure of parental reproductive systems, from acute embryo toxicity after translocation of contaminants from the mother to the developing embryo, or from both.

Selected resin acids in effluent and receiving waters derived from a bleached and unbleached kraft pulp and paper mill.

Water samples were collected on three dates at 24 sites influenced by effluent from Georgia-Pacific's Palatka Pulp and Paper Mill Operation, a bleached and unbleached kraft mill near Palatka, Florida, USA. The sampling sites were located within the mill retention ponds, Rice Creek, and the St. John's River. Samples were analyzed by gas chromatography-mass spectrometry for abietic, dehydroabietic, and isopimaric acids, all of which are potentially toxic by-products of pulp production. Isopimaric acid concentrations greater than 12 mg/L were measured at the mill's effluent outfall but were less than 20 microg/L at the end of Rice Creek. This result indicates that the waters of Rice Creek provide dilution or conditions conducive for degradation or sorption of these compounds. Large differences in resin acid concentrations were observed between sampling events. In two sampling events, the maximum observed concentrations were less than 2 mg/L for each analyte. In a third sampling event, all of the compounds were detected at concentrations greater than 10 mg/L. Data from the three sample dates showed that resin acid concentrations were below 20 microg/L before the confluence of Rice Creek and the St. John's River in all cases.

Achieving environmentally relevant organochlorine pesticide concentrations in eggs through maternal exposure in Alligator mississippiensis.

Alligator mississippiensis eggs from organochlorine pesticide (OCP) contaminated sites in Florida exhibit high rates of embryonic mortality compared to reference sites (P < 0.05). The objective of the present study was to use captive adult alligators to test the hypotheses that maternal exposure to OCPs results in increased OCP concentrations in eggs, and that increased exposure is associated with increased embryonic mortality. A total of 24 adult alligators (8 males and 16 females) were housed in eight pens. Eight females in four pens were dosed with a mixture of p,p'-DDE, toxaphene, dieldrin, and chlordane at a rate of 0.2+/-0.01 mg/kg/day for 274+/-8 days. Treated females produced eggs containing higher OCP concentrations (12,814+/-813 ng/g yolk) than controls (38+/-4 ng/g yolk). Eggs of treated females exhibited decreased viability (13+/-22%) as compared to controls (45+/-20%). Results indicated that 0.6% of administered OCPs were maternally transferred to the eggs of American alligators, and that maternal exposure is associated with decreased egg/embryo viability in this species.

Organochlorine pesticides and thiamine in eggs of largemouth bass and American alligators and their relationship with early life-stage mortality.

Thiamine deficiency has been linked to early mortality syndrome in salmonids in the Great Lakes. This study was conducted to compare thiamine concentrations in American alligators (Alligator mississippiensis) and Florida largemouth bass (Micropterus salmoides floridanus) eggs from sites with high embryo mortality and high exposure to organochlorine pesticides (OCPs) (Lakes Apopka and Griffin, and Emeralda Marsh, Florida, USA) to those from sites that have historically exhibited low embryo mortality and low OCPs (Lakes Woodruff and Orange, Florida). During June-July 2000, 20 alligator clutches were collected from these sites, artificially incubated, and monitored for embryo mortality. Thiamine and OCPs were measured in one egg/clutch. During February 2002, 10 adult female bass were collected from Emeralda Marsh and Lake Woodruff and mature ovaries analyzed for thiamine and OCP concentrations. Although ovaries from the Emeralda Marsh bass contained almost 1,000-fold more OCPs compared with the reference site, Lake Woodruff, there were no differences in thiamine concentrations between sites (11,710 vs. 11,857 pmol/g). In contrast, alligator eggs from the reference site had five times the amount of thiamine compared with the contaminated sites (3,123 vs. 617 pmol/g). Similarly, clutches with >55% hatch rates had significantly higher concentrations of thiamine compared with clutches with <54% hatch rates (1,119 vs. 201 pmol/g). These results suggest that thiamine deficiency might be playing an important role in alligator embryo survival but not in reproductive failure and recruitment of largemouth bass. The cause(s) of this thiamine deficiency are unknown but might be related to differences in the nutritional value of prey items across the sites studied and/or to the presence of high concentration of contaminants in eggs.

Detection by microsatellite analysis of early embryonic mortality in an alligator population in Florida.

In the 1980s, alligators (Alligator mississippiensis) of Lake Apopka (Florida, USA) underwent a population decline associated with decreased egg viability, effects that have been associated with endocrine-disrupting, persistent organochlorine pesticides. It is currently unknown whether the decreased egg viability is due to fertilization failure or early embryonic death. Therefore, we conducted a preliminary study to evaluate the use of microsatellite DNA loci to determine the fertilization status of nonviable eggs. Using microsatellite analysis, we compared genotypes from blasto-disks and embryos with the genotypes from females trapped at the nest. Four of five nonviable egg samples tested yielded evidence of fertilization. No evidence of unfertilized eggs was obtained, but amplifiable DNA could not be obtained from one entirely nonviable clutch. Thus, we demonstrate that early embryonic mortality in alligators can be detected by microsatellite analysis, but also suggest substantial effort is needed to improve the recovery of DNA and amplification of alligator microsatellite loci.

Fathead minnow response to broad-range exposure of ß-sitosterol concentrations during life-cycle testing.

The ß-sitosterol concentration in pulp and paper mill effluents is typically greater than that of other phytosterols and has been shown to cause a variety of effects in fish. The authors exposed fathead minnow (Pimephales promelas) to low (22 ± 0.93 µg/L), medium-low (70 ± 2.1 µg/L), medium-high (237 ± 5.5 µg/L), and high (745 ± 16.2 µg/L) concentrations of ß-sitosterol as well as negative (water), positive (ethynyl estradiol, 16 ± 0.58 ng/L), and carrier (0.6 mL/L acetone) controls. Fish were monitored over a full life cycle for population-level endpoints including growth and survival, reproductive endpoints (e.g. fecundity, sex steroids and vitellogenin, gonado-/hepatosomatic indices, and gonad histology). No significant differences were seen in fish growth, mortality, or reproduction with ß-sitosterol exposure, although a trend for lower egg production in ß-sitosterol exposures relative to the water control may be related to the acetone carrier. All ethynyl estradiol-exposed fish were smaller, showed female characteristics, and did not spawn. Sex steroid and vitellogenin were highly variable with no detectable treatment-related differences. Gonadal tissue showed no ß-sitosterol-related differences in reproductive development and spawning capability, although most ethynyl estradiol-exposed males had ovarian tissue and were not spawning-capable. The results indicate that ß-sitosterol exposure had little apparent impact on fathead minnow survival, growth, and reproduction even at concentrations >10 times that of typical effluents, although small sample size and variability precluded fully evaluating treatment responses on sex steroids and vitellogenin.

Nutrient and organochlorine pesticide concentrations in American alligator eggs and their associations with clutch viability.

Since the early 1900s, the lakes of the Ocklawaha basin in central Florida have experienced ecological degradation due to anthropogenic development. One species affected by this degradation is the American alligator Alligator mississippiensis, which has suffered from poor clutch viability and embryo mortality. Although some studies indicate that organochlorine pesticides (OCPs) may be involved, OCPs do not account for all of the variation seen in hatch rates. Indeed, nutrition and non-OCP contaminants have been associated with developmental problems in fish and birds. Our study evaluated embryo mortality in alligators at reference and OCP-contaminated sites as a function of exposure to OCPs, polychlorinated biphenyls (PCBs), and polyaromatic hydrocarbons (PAHs), along with egg nutrients (Zn, Se, and vitamins A, E, and B1). The four-pronged study consisted of a case-control cohort study, an expanded field study, a topical egg treatment thiamine amelioration experiment, and a topical egg treatment thiamine antagonist experiment. The results from the two field studies suggested that the total thiamine levels in the eggs were positively associated with clutch viability and negatively associated with the lipid content and certain OCPs measured in egg yolks. In addition, PCBs, PAHs, Zn, Se, and vitamins A and E were not found to be associated with the observed clutch viability defects. The thiamine levels in the eggs explained 38% of the variation in clutch survival in the case-control cohort study and 27% in the expanded field study. The topical egg treatment experiments were successful in elevating the thiamine concentrations in the albumin but not the yolk. No significant differences were noted among treatment groups in either egg treatment experiment with respect to clutch survival. In summary, thiamine egg concentrations explain some of the variation in the clutch viability of free-ranging alligators, but the cause-effect relationships are still unclear.

Changes in reproductive biomarkers in an endangered fish species (bonytail chub, Gila elegans) exposed to low levels of organic wastewater compounds in a controlled experiment.

In arid regions of the southwestern United States, municipal wastewater treatment plants commonly discharge treated effluent directly into streams that would otherwise be dry most of the year. A better understanding is needed of how effluent-dependent waters (EDWs) differ from more natural aquatic ecosystems and the ecological effect of low levels of environmentally persistent organic wastewater compounds (OWCs) with distance from the pollutant source. In a controlled experiment, we found 26 compounds common to municipal effluent in treatment raceways all at concentrations <1.0 microg/L. Male bonytail chub (Gila elegans) in tanks containing municipal effluent had significantly lower levels of 11-ketotestosterone (p=0.021) yet higher levels of 17beta-estradiol (p=0.002) and vitellogenin (p=0.036) compared to control male fish. Female bonytail chub in treatment tanks had significantly lower concentrations of 17beta-estradiol than control females (p=0.001). The normally inverse relationship between primary male and female sex hormones, expected in un-impaired fish, was greatly decreased in treatment (r=0.00) versus control (r=-0.66) female fish. We found a similar, but not as significant, trend between treatment (r=-0.45) and control (r=-0.82) male fish. Measures of fish condition showed no significant differences between male or female fish housed in effluent or clean water. Inter-sex condition did not occur and testicular and ovarian cells appeared normal for the respective developmental stage and we observed no morphological alteration in fish. The population-level impacts of these findings are uncertain. Studies examining the long-term, generational and behavioral effects to aquatic organisms chronically exposed to low levels of OWC mixtures are needed.

Parental exposure to pesticides and poor clutch viability in American alligators.

In central Florida, alligators (Alligator mississippiensis) inhabiting lakes contaminated with organochlorine pesticides (OCPs) produce eggs that have high OCP concentrations and low clutch viability (proportion of eggs in a clutch that yield a live hatchling) compared to those from less contaminated lakes (reference lakes). However, a clear dose-response relationship has not been established between OCPs and poor clutch viability. In order to better elucidate a cause and effect relationship between OCP exposure and clutch viability, we conducted concurrent field and laboratory studies. Our field study reaffirmed that eggs of wild alligators from OCP-contaminated lakes and wetlands continue to have lower clutch viability and higher OCP burdens than eggs from reference lakes. Our field study also demonstrated that OCP egg burdens were strongly correlated with clutch viability for some of the OCP-contaminated sites, but not all. To better test causal relationships, a parental exposure study was conducted using captive adult alligators. Our laboratory study demonstrated that dietary exposure of captive alligators to an ecologically relevant OCP mixture caused alligators to produce eggs with higher OCP burdens and reduced clutch viability, as compared to the captive-control population. The experimentally induced egg burdens and clutch viability reductions were similar to those of wild alligators from OCP-contaminated sites. Our field and laboratory results suggest parental OCP exposure may be contributing to low clutch viability in wild alligators inhabiting OCP-contaminated habitats, raising some concern for endangered crocodilians living in OCP-contaminated habitats.