Ronald J. Kendall

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.

Preliminary assessment of perchlorate in ecological receptors at the Longhorn Army Ammunition Plant (LHAAP), Karnack, Texas.

There have been increasing human health and ecological concerns about ionic perchlorate (ClO4) since it was detected in drinking water sources in 1997. Perchlorate is known to affect thyroid function, causing subsequent hormone disruption and potential perturbations of metabolic activities. According to current estimates, perchlorate is found in the surface or groundwater of 14 states, including Texas. Longhorn Army Ammunition Plant, located in east central Texas, was a facility historically associated with perchlorate-containing propellants and rocket motors. Subsequently, perchlorate contamination in ground and surface waters at the facility has been reported. Soil, sediment, water, vegetation, and animal tissue samples were collected from several locations within the plant for a preliminary site assessment of perchlorate contamination. Perchlorate concentrations ranged from 555–5,557,000 ppb in vegetation, 811–2038 ppb in aquatic insects, below detection limits (ND) to 207 ppb in fish, ND-580 ppb in frogs, and ND–2328 ppb in mammals.Consistent with our hypothesis, aquatic organisms inhabiting perchlorate-contaminated surface water bodies contained detectable concentrations of perchlorate. Additionally, terrestrial organisms were exposed through pathways not necessarily related to contaminated surface waters. Therefore, these data demonstrate that aquatic and terrestrial species are exposed to perchlorate in the environment. To our knowledge, this represents the first incidence of perchlorate exposure among wild animals reported in the scientific literature.

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.

EVIDENCE OF AN OXYSPIRURA PETROWI EPIZOOTIC IN NORTHERN BOBWHITES (COLINUS VIRGINIANUS), TEXAS, USA

Abstract We captured 36 Northern Bobwhites (Colinus virginianus) in Mitchell County, Texas in June–September 2013, and examined them for the eyeworm Oxyspirura petrowi. We recovered 334 eyeworms from 28 of 29 adult bobwhites (97%); infections ranged from 1–40 worms and mean (±SD) abundance of 11.9±13.0. Three of seven juveniles were infected, and those infected had one eyeworm each. Prevalence of eyeworms was similar among months. However, mean abundance of eyeworms peaked in July and August (3.3±2.1, 13.5±15.0, and 16.9±15.5), and decreased in September (6.3±3.0). We suggest that several previous studies may underreport prevalence and abundance because in those studies only the eye surface and nictitating membrane were examined, and not eye-associated tissue, ducts, glands, or sinuses.

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%).

Chlorpyrifos: Ecotoxicological Risk Assessment for Birds and Mammals in Corn Agroecosystems

A tiered risk assessment was conducted for the use of granular and liquid formulations of chlorpyrifos in corn agroecosystems in the U.S. The initial screening Tier I assessment suggested that under high-exposure scenarios the granular and some spray formulations present potential hazards to birds. Higher tiered probabilistic risk assessments were conducted separately for the granular and liquid formulations. The probabilistic assessment indicated that risk to birds from exposure to granular formulation is small and that this route of exposure would not be a significant source of mortality. Similarly, the assessment of potential exposure of birds to food items contaminated with chlorpyrifos showed that the risk from exposure via food was small, even if it was assumed that birds feed only on the treated fields. Although they have potentially greater sensitivity to chlorpyrifos, effects in nestling birds consuming food items from fields treated with granular chlorpyrifos were negligible. However, risks to young birds may be greater where the major source of food is from fields treated with liquid formulations of chlorpyrifos. A review of field studies showed that wildlife mortality incidents associated with use of either granular or liquid formulations of chlorpyrifos are not widely apparent in agroecosystems. Based on the multiple lines of evidence, we conclude that the presumption that chlorpyrifos use in corn agroecosystems will result in extensive mortality of terrestrial wildlife, particularly birds and mammals, is not supported by the scientific evidence.

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.

Organochlorine contaminant assessment in great blue herons using traditional and nonlethal monitoring techniques

A nonlethal method is discussed for the evaluation of contaminant concentrations in whole eggs. Concentrations of pentachlorobenzene, hexachlorobenzene, DDE, and the PCB congeners, BZ-60, BZ-118, BZ-138, BZ-180, and BZ-170 were quantified in tissue samples from great blue herons (Ardea herodias). All tissues within whole eggs from two colonies were homogenized together and analysed for these chlorinated contaminants. Contents and chorio-allantoic membranes (CAMs) of additional whole eggs were separated and analysed. Contaminant distributions were determined for the CAM and contents of whole eggs from the same colonies. CAM tissues remaining in hatched eggs were also analysed for comparative purposes. Utilizing the distributions derived for contaminants between CAM and egg contents and the chemical concentrations determined in CAMs from hatched eggs, contaminant burdens in whole eggs were calculated. This process produced concentration estimates that described actual, whole egg burdens of chlorinated contaminants within a factor of 2 (1.03-3.7). Contaminant burdens in eggs from the two colonies were also statistically different for DDE and total PCB concentrations.

Human and ecological risk assessment of a crop protection chemical: a case study with the azole fungicide epoxiconazole

Abstract Conventional risk assessments for crop protection chemicals compare the potential for causing toxicity (hazard identification) to anticipated exposure. New regulatory approaches have been proposed that would exclude exposure assessment and just focus on hazard identification based on endocrine disruption. This review comprises a critical analysis of hazard, focusing on the relative sensitivity of endocrine and non-endocrine endpoints, using a class of crop protection chemicals, the azole fungicides. These were selected because they are widely used on important crops (e.g. grains) and thereby can contact target and non-target plants and enter the food chain of humans and wildlife. Inhibition of lanosterol 14α-demethylase (CYP51) mediates the antifungal effect. Inhibition of other CYPs, such as aromatase (CYP19), can lead to numerous toxicological effects, which are also evident from high dose human exposures to therapeutic azoles. Because of its widespread use and substantial database, epoxiconazole was selected as a representative azole fungicide. Our critical analysis concluded that anticipated human exposure to epoxiconazole would yield a margin of safety of at least three orders of magnitude for reproductive effects observed in laboratory rodent studies that are postulated to be endocrine-driven (i.e. fetal resorptions). The most sensitive ecological species is the aquatic plant Lemna (duckweed), for which the margin of safety is less protective than for human health. For humans and wildlife, endocrine disruption is not the most sensitive endpoint. It is concluded that conventional risk assessment, considering anticipated exposure levels, will be protective of both human and ecological health. Although the toxic mechanisms of other azole compounds may be similar, large differences in potency will require a case-by-case risk assessment