Jessica K. Leet

A review of studies on androgen and estrogen exposure in fish early life stages: effects on gene and hormonal control of sexual differentiation.

Teleost fish are unique among vertebrates in that phenotypic sex or onset of sex inversion can be easily manipulated by hormonal treatments. In recent years, researchers have begun reporting concentrations of synthetic and natural hormones in the environment. Although concentrations are very low (in the parts per trillion to low parts per billion), they are still of concern because of the high potency of synthetic hormones and the enhanced susceptibility of teleost fishes, especially early life stages, to hormonal exposures. In this review, we will focus on sex differentiation in teleost fishes and how these processes in fish early life stages may be impacted by environmental hormones which are known to contaminate aquatic environments. We will start by reviewing information on sources and concentrations of hormones in the environment and continue by summarizing the state of knowledge of sex differentiation in teleost gonochoristic fishes, including information on genes involved (e.g. cyp19, dmrt1, sox9 and foxl2). We will end our review with a summary of studies that have examined the effects of androgens and estrogens on fish sex differentiation after exposure of fish embryos and larvae and with ideas for future research. Copyright

Assessing impacts of land-applied manure from concentrated animal feeding operations on fish populations and communities.

Concentrated animal feeding operation (CAFO) manure is a cost-effective fertilizer. In the Midwest, networks of subsurface tile-drains expedite transport of animal hormones and nutrients from land-applied CAFO manure to adjacent waterways. The objective of this study was to evaluate impacts of land-applied CAFO manure on fish populations and communities. Water chemistry including hormone, pesticide, and nutrient concentrations was characterized from study sites along with fish assemblage structure, growth, and endocrine disruption assessed in selected fish species. Although most CAFO water samples had hormone concentrations <1 ng/L, equivalent concentrations for 17β-E2 and 17α-TB peaked at >30 ng/L each during the period of spawning, hatching, and development for resident fishes. CAFO sites had lower fish species richness, and fishes exhibited faster somatic growth and lower reproductive condition compared to individuals from the reference site. Fathead minnows (Pimephales promelas) exposed to CAFO ditchwater during early developmental stages exhibited significantly skewed sex ratios toward males. Maximum observed hormone concentrations were well above the lowest observable effect concentrations for these hormones; however, complexities at the field scale make it difficult to directly relate hormone concentration and impacts on fish. Complicating factors include the consistent presence of pesticides and nutrients, and the difference in temperature and stream architecture of the CAFO-impacted ditches compared to the reference site (e.g., channelization, bottom substrate, shallow pools, and riparian cover).

Sex-Specific Gonadal and Gene Expression Changes throughout Development in Fathead Minnow

Although fathead minnows (Pimephales promelas) are commonly used as a model fish in endocrine disruption studies, past studies have not characterized sex-specific baseline expression of genes involved in sex differentiation during development in this species. Using a sex-linked DNA marker to verify gender, we evaluated the expression over time of genes involved in sex differentiation (dmrt1, cyp19a, cyp17, star, esr1, ar) in developing fathead minnows (10-45 days post hatch). Evaluation of these molecular markers in combination with gender identification help us to better understand the mechanisms regulating sex differentiation in fathead minnows and how endocrine-disrupting chemicals may alter these processes.

Tris(1,3-dichloro-2-propyl)phosphate Induces Genome-Wide Hypomethylation within Early Zebrafish Embryos

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is a high-production volume organophosphate-based plasticizer and flame retardant widely used within the United States. Using zebrafish as a model, the objectives of this study were to determine whether (1) TDCIPP inhibits DNA methyltransferase (DNMT) within embryonic nuclear extracts; (2) uptake of TDCIPP from 0.75 h postfertilization (hpf, 2-cell) to 2 hpf (64-cell) or 6 hpf (shield stage) leads to impacts on the early embryonic DNA methylome; and (3) TDCIPP-induced impacts on cytosine methylation are localized to CpG islands within intergenic regions. Within this study, 5-azacytidine (5-azaC, a DNMT inhibitor) was used as a positive control. Although 5-azaC significantly inhibited zebrafish DNMT, TDCIPP did not affect DNMT activity in vitro at concentrations as high as 500 μM. However, rapid embryonic uptake of 5-azaC and TDCIPP from 0.75 to 2 hpf resulted in chemical- and chromosome-specific alterations in cytosine methylation at 2 hpf. Moreover, TDCIPP exposure predominantly resulted in hypomethylation of positions outside of CpG islands and within intragenic (exon) regions of the zebrafish genome. Overall, these findings provide the foundation for monitoring DNA methylation dynamics within zebrafish as well as identifying potential associations among TDCIPP exposure, adverse health outcomes, and DNA methylation status within human populations.

Reproductive physiology in eastern snapping turtles (Chelydra serpentina) exposed to runoff from a concentrated animal feeding operation.

Abstract The eastern snapping turtle (Chelydra serpentina) is widely distributed throughout the eastern and central US and is a useful model organism to study land-use impacts on water quality. We compared the reproductive condition of turtles from a pond impacted by runoff from land applied with animal manure from a concentrated animal feeding operation (CAFO) relative to animals from a control pond. Turtles from the CAFO site were heavier and had higher plasma concentrations of vitellogenin (VTG, mean±SE; females; 859±115 vs. 401±127 ng/mL from controls) and testosterone (T, males; 39±7.0 vs. 3.8±6.9 ng/mL from controls). No VTG was detected in males. Body mass was positively correlated with VTG and T. Our results suggest that nutrient pollution of the CAFO pond indirectly resulted in higher plasma VTG in females and T in males because of an increase in body mass. The population-level consequences of these effects are not clear, but could result in females producing larger clutches.

Improving Waste Management Strategies for Small Livestock Farms

I recent years, there has been growing public concern over the environmental and human health issues associated with livestock production. In particular, water quality is typically considered the issue of greatest concern because of potential impacts on drinking water, as well as aquatic ecosystems. Contaminants from animal waste generated from livestock operations can be transported into surface and groundwater through various routes, such as leaking storage lagoons, runoff from land application, and flooding. These contaminants include nutrients, pathogens, heavy metals, pharmaceuticals, and natural/synthetic hormones. Although large regulated livestock farms dominate national discussions and debates, smaller unregulated livestock farms may also contribute to potential adverse impacts on water quality, particularly at the local and regional scale. Therefore, we argue that incentives and programs for smaller farms are needed to improve waste management and enhance water quality. Animal feeding operations (AFOs) have become the norm for producing livestock. AFOs are defined by the United States Environmental Protection Agency (U.S. EPA) as operations that are 1) “stabled or confined and fed or maintained for a total of 45 days or more in any 12-month period” and 2) have no vegetation grown within the operation lot during a normal growing season. Concentrated animal feeding operations (CAFOs) are AFOs defined as medium or large operations by species-specific animal capacity thresholds or small operations when pollutant discharge exceeds criteria established by the Clean Water Act. Any CAFO that has potential to release waste is required to obtain a permit through the National Pollution Discharge Elimination System (NPDES) and develop a Nutrient Management Plan (NMP). Similar to historic trends in U.S. crop production, from the 1980s to late 1990s livestock operations were increasingly consolidated to greater numbers of larger operations. However, this trend toward an increase in large CAFOs halted and, over the past decade, the number of large livestock operations has remained steady. CAFOs account for only ∼15% of AFOs, with the remainder (∼85%) represented by small, unregulated AFOs across all species of livestock. For example, ∼97% of cattle farms have less than 500 head and, for beef alone, the majority of production (>50%) occurs within small AFOs. However, these small operations often lack the resources to efficiently manage animal waste, leading to greater potential for unintended adverse impacts on environmental health. While there is a recognized need for better understanding CAFO-induced water quality impacts and improving CAFO regulations, currently there is little attention paid to the potential impact of smaller, unregulated livestock farms. When CAFO-generated animal waste is stored and land-applied under controlled conditions, it can be utilized as an efficient fertilizer for nearby agricultural fields. Although the farming community generally values environmental stewardship, smaller livestock operations face many challenges when managing animal waste, including a lack of (1) sufficient land for fertilizer application, (2) adequate storage capacity for animal waste, (3) transportation for excess waste, and (4) advanced technology to effectively assess and monitor soil nutrient levels and demands. As smaller AFOs are not required to develop NMPs and apply for NPDES permits, these operations likely have practices that impair water quality and are noncompliant with CAFO regulations, such as animals grazing directly in adjacent bodies of water, overapplication of animal waste to crop fields, and inadequate protection of manure piles. While there is a public perception that large “factory” farms adversely impact water quality, the reality is that larger CAFOs, unlike smaller, unregulated AFOs, have more available resources and financial incentives to develop and comply with efficient NMPs. For this reason, improved incentives and programs are needed to encourage smaller AFOs, those not regulated by the U.S. EPA, to improve waste management, especially given that these farms contribute the large majority of livestock production. Although some financial support is available to smaller AFOs via the United States Department of Agriculture’s Environmental Quality Incentives Program (EQIP), there is a need for more structured support and incentives for small livestock farms to use best management practices (BMPs). In our opinion, two potential solutions may encourage better management of waste among small livestock farms. First, tax