A growing number of contaminants of emerging concern (CECs) are found routinely in permitted discharges and their receiving waters. For the few CECs for which analytical methods exist, these methods are still largely in development and only some are routinely performed by commercial services laboratories. As the development and manufacture of chemicals presents an ever changing landscape, those CECs that are produced in high volumes and/or that are capable of being discharged via treated municipal or industrial wastewater effluent or stormwater runoff represent a moving target for environmental quality managers tasked with assessing and/or mitigating their potential for impact.

The CECs of most concern are those which may be potent at trace concentrations (parts per trillion range) and work as endocrine disruptors. Their presence in waterbodies may be harmful to aquatic biota inhabiting these locations. Such endocrine disrupting chemicals can interact directly with soluble hormone receptors or can interfere with the natural synthesis or metabolism of endogenous hormones and thereby impede normal function of these processes in exposed organisms. Most attention has been focused on chemicals which act as estrogens or androgens or their antagonists. Estrogens are important in brain development and programming of tissue differentiation at early time points during development (Feist and Schreck 1996; Lassiter and Linney 2007; Mandiki et al. 2005; Remage-Healey and Bass 2007; Tomy et al. 2009; Vetillard et al. 2006; Zhang et al. 2008).

In our own work, exposure of fathead minnows to concentrations of ethinylestradiol (EE2) at 2 ng/L induced pericardial/yolk sac edema (Johns et al. 2009). The estrogenic mycotoxin zearalenone (exposure range of 2-50 ng/L) also resulted in myocardial edema (Johns et al. 2009). In addition, we analyzed a limited set of gene expression changes including Vtg, which was up-regulated by the two estrogens, steroidogenic acute regulatory (StAR) protein, insulin-like growth factor 1 (IGF-1) and growth hormone (GH) which were also altered. Thus, these genes in target fish species would be viewed as critically important to include in future studies of responses to estrogenic CECs at the molecular level.

Concurrently, novel in vitro methods based on receptor binding or transactivation have been developed that are extremely sensitive to target chemicals acting with the same mode of action, including the potent endocrine disrupting CECs described above. Work is being performed to adapt these in vitro bioassays for water quality assessment and monitoring purposes. Few studies, however, link results from such in vitro assays with higher order in vivo effects which result in adversity for survival, growth, reproduction, or susceptibility to disease.

The goal of this project is to establish quantitative linkages between the in vitro receptor-based assays and traditional endpoints of adversity in a sensitive estuarine fish model, the common silverside (Menidia beryllina) which is an established EPA model for estuarine toxicity. As a demonstration, we will focus on estrogenic responses of selected chemicals of interest first in lab exposures (Year 1) followed by exposure to field-collected wastewater treatment plan (WWTP) effluent and estuarine and marine receiving waters (Year 2).

Study Plan
This study will test estrogenic chemicals that were recently recommended for monitoring in California’s receiving waters by the State’s Science Advisory Panel for CECs (Anderson et al. 2012), e.g. estrone (E1), bisphenol A (BPA), 4-nonylphenol (4-NP) and galaxolide (HHCB). Traditional in vivo endpoints for early life stages of silversides (M. beryllina) will include: development, growth, and survival and for juveniles: growth, survival and biochemical endpoints such as plasma vitellogenin and hormone concentrations (Vtg, E2 and T) and hepatic gene expression for at least 5 genes per life stage. We will index estrogen equivalency concentrations required for altering higher order endpoints with biochemical responses within the fish and responses obtained with commercially available estrogen receptor (ER) transactivation assays. These linkages will enable the use of in vitro assays as measures of both exposure and effect. The concentrations required for both in vivo and in vitro assays will be quantified to determine reference concentrations above which effects may be expected.

Programs and Focus Areas: 
Clean Water Program
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