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The β-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 β-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 β-sitosterol exposure, although a trend for lower egg production in β-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 β-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 β-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. Environ Toxicol Chem 2014;33:458–467. © 2013 SETAC

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.

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.

In recent years, there has been an increased use of the measurement of sex steroid hormone levels in the blood of animals exposed to chemicals as an indicator of reproductive impairment or an alteration in endocrine function. Although levels of hormones are often compared among animals and laboratories, there has been no study to examine the between-laboratory variability in actual steroid measurements. Therefore, we initiated a study with white sucker collected from a site receiving pulp mill effluent, previously documented as having reduced steroid levels, to address this issue. Samples of plasma and media from in vitro gonadal incubations were delivered to eight outside laboratories with the ability to measure steroid hormones. These laboratories ranged from well-established fish endocrine laboratories to wildlife toxicology laboratories, which have recently implemented the methods to measure steroid hormones. In this study, we have considered both the absolute measure of steroid content between laboratories as well as the ability to discriminate between reference and exposed populations as important criteria when evaluating the utility of these measures. Of the eight outside laboratories conducting the analyses, six detected identical site differences in circulating levels of testosterone and 17beta-estradiol to those documented by our Burlington laboratory (ON, Canada). However, the absolute value of the steroid hormones measured in the plasma varied significantly (plasma testosterone 0.6-23.1 ng/ml, 17beta-estradiol 77.6-1782.7 pg/ml) with coefficients of variation of 70.4% and 60.3% respectively. Similar results were demonstrated for the measurement of steroid hormones in media following in vitro gonadal incubation. Although there was a fair amount of variability in the absolute measure of steroid hormone levels, we would predict a far greater coherence of interlaboratory results through the sharing of reagents and the use of a common methodology between laboratories. These results are very promising, providing evidence for the inclusion of steroid hormones in monitoring endocrine disruption in wildlife species.

The β-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 β-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 β-sitosterol exposure, although a trend for lower egg production in β-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 β-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 β-sitosterol exposure had little apparent impact on fathead minnow survival, growth, and reproduction even at concentrations10 times that of typical effluents, although small sample size and variability precluded fully evaluating treatment responses on sex steroids and vitellogenin.

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.

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.

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.

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.

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.

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.

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.

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.