Alan S. Kolok

QUANTITATIVE EVALUATION OF LABORATORY UPTAKE RATES FOR PESTICIDES, PHARMACEUTICALS, AND STEROID HORMONES USING POCIS

Polar organic chemical integrative samplers (POCIS) are useful in monitoring for a wide range of chemicals in aquatic systems; however, a lack of available uptake rate data for compounds of environmental interest is one limitation in the application of these samplers to environmental studies. In this study, laboratory calibration experiments were conducted with POCIS for 65 compounds at 25°C under flowing conditions to determine chemical-specific uptake rates (R(s)). Experimental uptake rates measured in this study ranged from 0.034 to 1.33 L/d, and uptake rates were determined for 36 compounds with no previously reported values. Experimentally determined uptake rates were applied to data obtained from POCIS samplers deployed downstream of three wastewater treatment plant (WWTP) effluent discharges and in four surface waters influenced by agricultural runoff. Time-weighted average concentrations for atrazine and metolachlor determined using uptake rates generated in this study compare well with results from composited grab sampling previously conducted in agricultural watersheds in Nebraska, USA.

The Environmental Impact of Growth-Promoting Compounds Employed by the United States Beef Cattle Industry: History, Current Knowledge, and Future Directions

The current state of knowledge regarding the environmental impact of growth-promoting compounds associated with the U.S. beef cattle industry is extensive in some areas but virtually nonexistent in others. The compounds administered to the cattle are quite well understood, as are bovine metabolism and excretion. If the sex and age of the cattle on the feedlot are known, the metabolites excreted by the cattle should be predictable with a great deal of accuracy. The fate, transport, and biological effects of growth-promoting compounds are just beginning to be studied. Most of the research conducted on the fate and transport of growth-promoting compounds has focused on 17beta-E2; however, much of this research was not conducted using feedlot runoff or manure. Studies are needed that focus specifically on manures and runoff from experimental or commercial feedlots. To date, the degree to which growth-promoting compounds are released from feedlots in a bioavailable form remains a point of speculation. The environmental fate and transport of TBA, P, and MGA have not been well studied. Comparisons between the fate and transport of T and 17beta-E2, however, make it clear that compounds with similar structure may behave very differently once released into the environment. Considering that 17beta-E2 is a naturally occurring estrogen and that TBA is a nonaromatizable androgen, it is not surprising that these compounds directly impact the reproductive physiology of fishes. The effects of these two compounds have been well documented, as has been described here; however, the effects of P and MGA exposures have gone largely uninvestigated. This is a serious critical gap in our knowledge base because progestogins play an important role in sex steroid synthesis and reproduction. Clearly, additional research on the consequences of exposures to P and MGA is warranted. The majority of research investigating the effects of 17beta-E2 and TBA metabolites on fish has been conducted in the laboratory and has typically focused on continuous, pharmacological exposures to single compounds. These exposures may not bear much similarity to environmentally relevant exposures, and as such may offer little information regarding biological effects seen in nature. Cattle feedlot runoff is likely to contain a suite of growth-promoting compounds rather than any single compound. Clearly, deciphering the biological effects of exposure to complex mixtures containing androgenic, estrogenic, and progestogenic compounds will remain an important area of study for the next few years. A second complexity associated with the biological runoff from cattle feedlots is the discontinuous nature of the release. It is likely that inadvertent entry of growth-promoting compounds will follow spring snowmelt or rainstorm events. These events will result in intermittent, pulsed exposures to high concentrations of these compounds interspersed by long-term exposures to lower concentrations. The effects of exposure timing and duration should be considered to generate a clearer understanding of the biological consequences of exposures to growth-promoting compounds. To date, a very limited number of studies (only one!) have sought to determine whether fish living in waterways receiving runoff from cattle feedlots are adversely affected by growth-promoting compounds associated with the runoff. Clearly, more field studies need to be conducted before a relationship between cattle feedlot effluent and biological consequences can be elucidated.

The anti-estrogenic activity of sediments from agriculturally intense watersheds: assessment using in vivo and in vitro assays.

The goal of the current study was to determine whether sediments from agriculturally intense watersheds can act as a potential source of anti-estrogenic endocrine-disrupting compounds. The specific objectives of the current study were to determine (1) whether female fathead minnows (Pimephales promelas) experience alterations in endocrine function when exposed to sediments collected from agriculturally intense watersheds and (2) if these sediments display anti-estrogenic activity in an in vitro assay. In addition, sediment samples were analyzed for the presence of steroid hormones and pesticides associated with local agricultural practices. To accomplish this, sediments and water were collected from three sites within two agriculturally intense Nebraska watersheds (Bow Creek and the Elkhorn River). In 2009, minnows were exposed to sediment and/or water collected from the two Bow Creek sites (East Bow Creek and the Confluence) in the laboratory, while in 2010, minnows were exposed to sediment and/or water from East Bow Creek, the Confluence and the Elkhorn River. Following the 7-day exposure period, the hepatic mRNA expression of two-estrogen responsive genes, estrogen receptor α (ERα) and vitellogenin (Vtg) was determined. In 2009, females exposed to Confluence sediments, in the presence of laboratory water or Confluence water, experienced significant reductions in ERα expression relative to unexposed and Confluence water-exposed females. The defeminization of these females suggests the presence of a biologically available anti-estrogenic compound in sediments collected from this site. In 2010, sediments were assessed for anti-estrogenic activity on days 0 and 7 of the exposure period using a 4-h yeast estrogen screen. Lipophilic extracts (LEs) of day 0 sediments collected from the Confluence and the Elkhorn River induced significant reductions in the estrogenic reporter activity of treated yeast cultures suggesting the presence of a lipophilic anti-estrogenic compound in these extracts. Chemical analysis revealed the presence of a variety of steroid hormones, including those associated with the production of beef cattle (i.e. β-trenbolone, α-zearalanol and α-zearalenol), in sediments indicating that compounds utilized by local beef cattle operations are capable of entering nearby watersheds. Overall, the results of this study indicate that an environmentally relevant anti-estrogenic compound is present in sediments from agriculturally intense watersheds and that this compound is bioavailable to fish. Furthermore, the presence of steroid hormones in sediments from these watersheds provides evidence indicating that steroids are capable of sorbing to sediments.

Reductions in hepatic vitellogenin and estrogen receptor alpha expression by sediments from an agriculturally impacted waterway

Previous studies have reported alterations in the endocrine function of fathead minnows (Pimephales promelas) collected and deployed in the Elkhorn River. The goal of the current study was to determine whether sediment from the Elkhorn River watershed could act as a source of endocrine-active compounds. To accomplish this, four aquaria containing sexually mature fathead minnows and polar organic chemical integrative samplers (POCIS) were established. The aquaria contained either: (1) laboratory water only, (2) Elkhorn River water only, (3) laboratory water and Elkhorn River sediment or (4) Elkhorn River water and Elkhorn River sediment. Steroid hormones were not detected in the extracts of POCIS or sediment. Pesticides were detected in POCIS extracts from tanks containing Elkhorn River water, but were not detected in the extracts of sediment or POCIS suspended in the tank containing laboratory water and Elkhorn River sediment suggesting that sediments do not act as a significant source of the 14 steroid hormones or 24 pesticides that were analyzed for in the current study. The hepatic mRNA expression of vitellogenin (vtg) and estrogen receptor alpha (ERalpha) in fathead minnows from each group was assessed. Female minnows exposed simultaneously to sediment and water collected from the Elkhorn River experienced defeminization as indicated by significant reductions in both vtg and ERalpha expression. Significant reductions in vtg mRNA expression were also observed in females exposed to laboratory water and Elkhorn River sediment, but not in females exposed to Elkhorn River water only. This finding suggests that exposures to sediments, rather than water, collected from the Elkhorn River lead to the defeminization of females. However, the compound(s) responsible for this effect have yet to be determined.

Agrichemicals in nebraska, USA, watersheds: Occurrence and endocrine effects

The objective of the present study was to determine the occurrence and endocrine effects of agrichemicals in four Nebraska, USA, watersheds--the Elkhorn, Platte, Niobrara, and Dismal rivers. Land use in the Elkhorn River and Platte River watersheds is characterized by intense agriculture, including row crop and beef cattle production. In contrast, land within the Niobrara River and Dismal River watersheds consists primarily of grasslands. Polar organic chemical integrative samplers (POCIS) and caged fathead minnows were deployed at a site within each watershed for 7 d. The POCIS were analyzed for pesticides and hormones, while the caged minnows were analyzed for the expression of estrogen- and androgen-responsive genes. Amounts of pesticides recovered in POCIS extracts from the Elkhorn and Platte rivers were higher than those recovered from the Niobrara and Dismal rivers. Furthermore, female minnows deployed in the Elkhorn River experienced significant reductions in expression of two estrogen-responsive genes (vitellogenin and estrogen receptor α) relative to females deployed at the other sites, indicating alterations in endocrine function. However, the defeminization of these females could not be definitely linked to any of the agrichemicals detected in the POCIS recovered from the Elkhorn River.

Copper tolerance in fathead minnows: II. Maternal transfer

Female fathead minnows (Pimephales promelas) exposed to copper (Cu) maternally transfer Cu tolerance to their larval offspring. Larvae produced after female parents received a sublethal 5-d, 100 microg/L Cu exposure had significantly greater survivorship in potentially lethal Cu solutions than larvae produced before those females were exposed to Cu.

The spring runoff in nebraska's (USA) Elkhorn River watershed and its impact on two sentinel organisms

The objectives of the present study were to determine 1) if temporal variability influenced the toxicity of Elkhorn River, Nebraska, USA, water, and 2) if the toxic effect was consistent between 2 sentinel organisms, the fathead minnow (Pimephales promelas) and the northern leopard frog (Rana pipiens). During spring 2012, atrazine indicator strips were used to document the occurrence of agrichemical pulses in the Elkhorn River. Polar organic chemical integrative samplers (POCIS) were deployed for 14 d during both a pulse and a postpulse period as indicated by the atrazine strips. Pesticide concentrations detected in the POCIS extracts ranged from 1.6-fold to 281-fold higher during the pulse period compared to the postpulse period. Fish and frog bioassays were conducted for 7 d, and hepatic mRNA expression of vitellogenin (VTG) and estrogen receptor alpha (ERα) was determined by quantitative real-time polymerase chain reaction. Compared with lab water controls, fish exposed to water collected during an agrichemical pulse experienced significant reductions in VTG and ERα, whereas exposed female frogs did not. Male leopard frogs, in contrast, experienced significant increases in the expression of ERα, whereas pulse-exposed male minnows did not. The significant effects observed following agrichemical pulse exposure demonstrate that episodic agrichemical runoff adversely impacts sentinel organisms, and that the adverse impacts observed depend on the sex and species of the sentinel organism.

CADMIUM EXPOSURES DURING EARLY DEVELOPMENT: DO THEY LEAD TO REPRODUCTIVE IMPAIRMENT IN FATHEAD MINNOWS?

The primary objective of this study was to determine whether cadmium (Cd) exposures during embryonic and larval development alter the reproductive performance, reproductive physiology, and sex ratio of fathead minnows (Pimephales promelas). Embryonic exposures were conducted by exposing adult female fathead minnows to 0, 25, or 100 microg/L Cd for 8 d prior to breeding. Larval exposures were conducted by exposing the larvae to waterborne Cd at 0, 12.5, 25, or 50 microg/L from 8 to 29 d posthatch (the time period associated with female sexual differentiation). Minnows from each exposure period were raised to maturity, at which time their reproductive success, secondary sexual characteristics, gonadosomatic index (GSI), plasma 11-ketotestosterone (11-KT) concentrations, and sex ratios were assessed. Results from the embryonic study reveal that Cd exposures alter the secondary sexual characteristics of male fathead minnows but do not alter reproductive performance, GSI, 11-KT concentrations, or sex ratios. Larval exposures, during the period of female sexual differentiation, significantly reduce the frequency of adult spawning and increase clutch size but do not alter fecundity, secondary sexual characteristics, GSI, or 11-KT. Subtle alterations in sex ratio were observed, indicating that larval Cd exposures may increase the proportion of females in an exposed population.

Occurrence and endocrine effects of agrichemicals in a small Nebraska, USA, watershed.

The Bow Creek watershed (Nebraska, USA) is dominated by the production of beef cattle and row crops; therefore, surface waters are likely to receive runoff containing steroid hormones and pesticides. The goal of the present study was to determine the occurrence and endocrine effects of agrichemicals in this watershed. To accomplish this, four sites within the watershed-Pearl, Bow, and East Bow Creeks and a site at the confluence with the Missouri River-were selected. In June of 2008, polar organic chemical integrative samplers (POCIS) were deployed at each site, whereas in June of 2009, water and sediment samples were collected. Caged fathead minnows (Pimephales promelas) were deployed at all of the selected sites in both years. Analysis of these samples revealed that steroid hormones were not present; however, pesticides were present in POCIS extracts and water samples. In general, the amount of pesticides was higher in POCIS retrieved from Pearl and Bow Creeks than in POCIS from East Bow Creek and the confluence. This variation between sites appeared to be related to row crop density, as row crop land cover surrounding the Pearl and Bow Creek sites was higher than that surrounding the East Bow and confluence sites. To determine the endocrine effects of agrichemicals within this watershed, the hepatic mRNA expression of vitellogenin and estrogen receptor α (ERα), as well as the gonadal expression of P450 aromatase A, was determined for the caged minnows. Females deployed at East Bow Creek and the confluence experienced decreases in the expression of ERα, suggesting that these females had been defeminized; however, this defeminization could not be attributed to any of the pesticides detected at these sites.

Empowering Citizen Scientists: The Strength of Many in Monitoring Biologically Active Environmental Contaminants

Citizen scientists can play a vital role in the first-tier screening of chemical contaminants in aquatic environments. For this to occur, the citizen scientists must be motivated and convinced to coalesce into functioning but loosely structured groups. Furthermore, the data they collect will have to be inexpensive to gather but also meaningful at fairly high levels of discrimination. Finally, the data from individual citizen scientists will have to be integrated into a large, professionally managed, and comprehensive database. We contend that all of the above criteria are already met, and in fact, other environment-related disciplines are already putting citizen scientists into action. Citizen scientists represent a collective workforce that can amass large data sets at minimal effort or cost, which can then be analyzed in an aggregate and used as a first-tier screening tool in environmental assessment.