Ernest E. Smith

Effects of Atrazine on Fish, Amphibians, and Aquatic Reptiles: A Critical Review

The herbicide atrazine is widely used in agriculture for the production of corn and other crops. Because of its physical and chemical properties, atrazine is found in small concentrations in surface waters—habitats for some species. A number of reports on the effects of atrazine on aquatic vertebrates, mostly amphibians, have been published, yet there is inconsistency in the effects reported, and inconsistency between studies in different laboratories. We have brought the results and conclusions of all of the relevant laboratory and field studies together in this critical review and assessed causality using procedures for the identification of causative agents of disease and ecoepidemiology derived from Kochs postulates and the Bradford–Hill guidelines. Based on a weight of evidence analysis of all of the data, the central theory that environmentally relevant concentrations of atrazine affect reproduction and/or reproductive development in fish, amphibians, and reptiles is not supported by the vast majority of observations. The same conclusions also hold for the supporting theories such as induction of aromatase, the enzyme that converts testosterone to estradiol. For other responses, such as immune function, stress endocrinology, parasitism, or population-level effects, there are no indications of effects or there is such a paucity of good data that definitive conclusions cannot be made.

EFFECTS OF ATRAZINE ON METAMORPHOSIS, GROWTH, AND GONADAL DEVELOPMENT IN THE GREEN FROG (Rana clamitans)

Embryos of the green frog (Rana clamitans) were collected from the field and exposed to 1 of 6 water-borne treatments for 273 d (mid July 2001 to mid April 2002). The treatments were 0, 10, or 25 μg/L atrazine, 0.005% ethanol (EtOH), or 0.1 mg/L estradiol or dihydrotestosterone carried in 0.005% EtOH. Treatments were applied in a static renewal system with a 50% test solution replacement approximately every 3 d. Following the exposure period, tadpoles were reared in freshwater until metamorphosis or until study termination (at d 506). Time to initiate and complete metamorphosis, stage-specific mortality, length and weight at metamorphosis, and gross morphology and histology of the gonads were examined. At environmentally relevant concentrations, atrazine did not consistently affect growth or metamorphosis. Compared to controls, the length of the larval period was greater in tadpoles exposed to 10 μg/L atrazine. However, the length of the larval period was not markedly different between tadpoles in the control and 25 μg/L atrazine treatments. Neither gross gonadal morphology nor histopathology of the gonads in postmetamorphic frogs was significantly altered in response to atrazine exposure. This study provides evidence that environmentally relevant concentrations of atrazine do not adversely affect the growth or reproductive development of R. clamitans.

PLASMA SEX STEROID CONCENTRATIONS AND GONADAL AROMATASE ACTIVITIES IN AFRICAN CLAWED FROGS (XENOPUS LAEVIS) FROM SOUTH AFRICA

Adult African clawed frogs (Xenopus laevis) were collected from a corn-growing region (CGR) and a non-corn-growing region (NCGR) with different exposure profiles for atrazine and related triazines. Physical, chemical, and biological parameters from the catchment areas were also measured. Frogs were surveyed for possible effects of exposure to triazine herbicides on plasma testosterone (T) and estradiol (E2) titers, gonadal aromatase activity, and gonad growth (GSI). Concentrations of both T and E2 varied among locations and were correlated to some accessory factors, such as pH, several ions, and metals. Greatest median plasma T concentrations (males: 19 ng/ml; females: 16 ng/ml) occurred in frogs inhabiting NCGR as compared to those from the CGR (males: 4 ng/ml; females: 1 ng/ml). Median E2 concentrations were also greater in frogs collected from the NCGR (males: 3 ng/ml; females: 28 ng/ml) than those in frogs from the CGR (males: 2 ng/ml; females: 5 ng/ml). Because some exposure to agricultural chemicals at both regions occurred, as did simultaneous exposures to multiple chemicals, a regression analysis was employed. Negative correlations were observed between plasma T concentrations and concentrations of atrazine, deisopropylatrazine, deethylatrazine, and tertbuthylazine in females and between T and diaminochlorotriazine in males. Estradiol in females exhibited a significant negative correlation with atrazine and deethylatrazine. No correlations were observed between gonadal aromatase activity or GSI and any of the agricultural chemicals measured. Median aromatase activities in ovaries varied among sampling sites ranging from 7 to >3000 times greater than those in males when measurable. Testicular aromatase activity was below the detection limit of the assay in male frogs at most of the sites. Although exposure to agricultural inputs did not affect aromatase activities, effects of atrazine or coapplied pesticides on sex steroid homeostasis cannot be excluded at this point.

Assessment of laryngeal muscle and testicular cell types in Xenopus laevis (Anura Pipidae) inhabiting maize and non-maize growing areas of South Africa

Abstract We tested the hypothesis that adult African clawed frogs (Xenopus laevis) inhabiting water bodies in maize‐growing areas (MGA) of South Africa would exhibit differences in testicular structure compared to frogs from water bodies in non‐maize‐growing areas (NMGA) in the same locale. Adults of both sexes were collected during the autumn of 2002 in South Africa, and stereological analytical techniques were used to quantify the distribution of testicular cell types. In addition, total laryngeal mass was used as a gauge of secondary sex differences in animals from MGA and NMGA study sites. Evaluation of the total laryngeal mass revealed that there were no statistically significant differences between X. laevis of the same sex from the NMGA and MGA sites. Mean percent fractional‐volume values for seminiferous tubule distribution of testicular cell types of mature X. laevis, ranged from 3–4% for spermatogonia, 26–28% for spermatocytes, 54–57% for spermatozoa, and 14–15% for other cells types. The mean percent volume for blood vessels ranged from 0.3–0.4%. These values did not differ significantly between frogs from NMGA and MGA areas. Collectively, these data demonstrated no differences in gonadal and laryngeal development in X. laevis collected in South Africa from MGA and NMGA areas and that there is little evidence for an effect of agricultural chemicals used in maize production functioning as endocrine disrupters in this species. Screening of X. laevis testes revealed a small incidence of Stage 1 testicular oocytes in adult male frogs collected from the NMGA (3%) and MGA (2%).

Population structure of the African Clawed Frog (Xenopus laevis) in maize‐growing areas with atrazine application versus non‐maize‐growing areas in South Africa

Abstract The herbicide atrazine has been suggested to cause gonadal deformities in frogs and could possibly impact on reproduction. Since the early 1960s, atrazine has been used in large amounts in maize production areas of South Africa. These areas overlap with populations of the African Clawed Frog (Xenopus laevis) that has a wide distribution in southern Africa and is found in most water‐bodies including those where atrazine residues are detected. The aim of this study was to compare various attributes of individual‐ and population‐level responses of X laevis from maize‐growing and non‐maize‐growing areas. Xenopus laevis were studied in three reference and five maize‐growing sites. Sex ratio, snout‐vent length, body‐mass and age profiles were found to be similar for populations in maize‐growing and non‐maize‐growing areas. Our mark‐recapture data indicated that all sites had robust populations. There were no significant relationships between exposure to atrazine and any of the parameters investigated in populations of X. laevis.

Effects of chronic 2,4,6,-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene exposure on developing bullfrog (Rana catesbeiana) tadpoles.

Chronic aqueous exposures were conducted using bullfrog (Rana catesbeiana) tadpoles (8 d old) exposed to TNT (0-4 mg/L), 2,4-DNT (0-4 mg/L), and 2,6-DNT (0-8 mg/L) for 90 d. Survival of tadpoles examined using Cox proportional hazard models was reduced at all concentrations tested. Percent of abnormal swimming and other morphological abnormalities after sublethal exposure to TNT, 2,4-DNT, and 2,6-DNT at 2 mg/L were also evaluated. The effects of TNT, 2,4-DNT, and 2,6-DNT on wet body mass, snout vent length (SVL), and developmental stage of surviving tadpoles were examined. Only 2,4-DNT did not have a significant effect on body mass or SVL, but all three compounds tested had significant effects on survival. Long-term continuous exposure to these compounds at concentrations of 0.25 mg/L could lead to significant changes in growth and survival of larval amphibians.

REPRODUCTIVE EFFECTS OF HEXAHYDRO-1,3,5-TRINITROSO-1,3,5-TRIAZINE IN DEER MICE (PEROMYSCUS MANICULATUS ) DURING A CONTROLLED EXPOSURE STUDY

Contamination with hexahydro-1,3,5-trinitro-1,3,5-triazine (Royal Demolition Explosive [RDX]) has been identified at areas of explosive manufacturing, processing, storage, and usage. Thus, the potential exists for exposure to N-nitroso compounds, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine, hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine, and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX), formed via anaerobic transformation of RDX. Following exposure, reproductive toxicity of TNX was evaluated in three consecutive litters of deer mice (Peromyscus maniculatus). Hexahydro-1,3,5-trinitroso-1,3,5-triazine was administered ad libitum via drinking water at four doses: 0 (control), 1, 10, and 100 microg/L. Endpoints investigated included reproductive success, offspring survival, offspring weight gain, offspring organ weights, and liver TNX residues. Data from the present study indicate that TNX bioaccumulates in the liver and is associated with postpartum mortality, dose-dependent decrease in body weight from birth to weaning, and decrease in kidney weight of deer mice offspring.

Effects of in utero and lactational ammonium perchlorate exposure on thyroid gland histology and thyroid and sex hormones in developing deer mice (peromyscus maniculatus) through postnatal day 21.

Thyroid gland hormone levels and histology and sex hormone levels in developing deer mice ( Peromyscus maniculatus ) were measured following in utero and lactational exposure to ammonium perchlorate (AP), a component of rocket fuel and a thyroid toxicant. Breeding pairs were dosed continuously with 0, 1 n M , 1 µ M , or 1 m M concentrations of AP in drinking water from the time of cohabitation until pups from the third litter were weaned. Pups from the second litter were used for evaluation in this study. The active (colloid-containing) thyroid follicle number per unit area was significantly different between treatment groups. The 1-n M and 1-m M treatment groups had significantly fewer active follicles per unit area than did controls. The 1-m M treatment group also had significantly fewer active follicles than the 1-µ M and the 1-n M treatment groups. Total T 4 concentrations were significantly increased in the 1-n M and 1-µ M groups compared to the controls. No significant difference was observed in total T 3 concentrations. None of the 1-m M plasma had concentrations of total testosterone above the detection limit, and only one of the 1-µ M samples was above the detection limit of the assay. All estradiol concentrations were below the detection limits of the assay. In contrast to the situation in adult rodents, it appears that AP increases thyroid hormone production in developing deer mice and produces variable effects with increasing concentrations.

In utero and lactational exposure to ammonium perchlorate in drinking water: Effects on developing deer mice at postnatal day 21

The effects of in utero and lactational exposure to ammonium perchlorate (AP), a component of rocket fuel and a thyroid toxicant, on developing deer mice ( Peromyscus maniculatus ) were evaluated. Breeding pairs were dosed continuously with 0, 1 n M , 1 µ M , or 1 m M AP in drinking water, from cohabitation until pups were euthanized at postnatal day (PND) 21. Pups from the second litter were used for evaluation in this study. No significant differences were observed in any analysis performed when litter means were used in statistical analysis. All reported significant differences occurred when statistical analysis was performed on individual pup data. Body weights were significantly different between treatments at PND 5 and PND 20, with the 1-µ M body weights being lower than that of controls. Body weight and liver weight in the 1-m M group were significantly higher than the 1-µ M weights at PND 21 when analyzed by analysis of variance (ANOVA). However, there were no significant differences in liver weights when analyzed with body weight as the covariate. Heart weights were significantly different between males and females. Male heart weights in the 1-µ M and 1-m M groups were significantly lower than in controls when analyzed by analysis of covariance (ANCOVA) with body weight as the covariate. Litter size and survival percentage were not significantly different among treatments. Although significant differences were observed only when the individual pup was used as the experimental unit, these data suggest that AP exposure at different concentrations may variably alter body weight and male heart weight during mammalian development.

An intrinsic CRF signaling system within the optic tectum

Previous work indicates that CRF administration inhibits visually guided feeding in amphibians. We used the African clawed frog Xenopus laevis to examine the hypothesis that CRF acts as a neurotransmitter in the optic tectum, the major brain area integrating the visual and premotor pathways regulating visually guided feeding in anurans. Reverse transcriptase PCR revealed that cells in the optic tectum express mRNA for CRF and the CRF R1 receptor but not the CRF R2 receptor. Radioligand binding studies indicated that specific binding of [(125)I]-Tyr-oCRF to tectal cell membranes can be displaced by the CRF R1 antagonists antalarmin or NBI-27914. CRF increased the expression of mRNA encoding regulator of G-protein signaling 2 (rgs2) in tectal explants and this effect was blocked by antalarmin. CRF had no effect on basal glutamate or gamma-aminobutyric acid (GABA) secretion but inhibited secretion of norepinephrine from tectal explants, an effect that completely blocked by antalarmin. Using a homologous radioimmunoassay we determined that CRF release from tectal explants in vitro was potassium- and calcium-dependent. Basal and depolarization-induced CRF secretion was greater from optic tectum than hypothalamus/thalamus, telencephalon, or brainstem. We concluded that the optic tectum possesses a CRF signaling system that may be involved in modulating communication between sensory and motor pathways involved in food intake.