Identify and quantitate biologically active compounds (i.e. toxins, mycotoxins, alkaloids, etc.) in water, which may occur from a variety of sources, such as algae, unknown sources associated with amphibian declines and deformities, animal feeding operations or municipal sewage treatment plants.
Through collaboration with other laboratories, water or food known to contain a biologically active compound(s) (i.e. toxins, pharmaceuticals, estrogenic substances, etc.) will be analyzed to characterize the nature of the biologically active compound(s). Initially, this may involve developing an assay for the biologically active compound, such as a bioassay. Subsequent, separation techniques will be utilized to purify the biologically active compound. Once isolated, immuno, instrumental, or analytical analyses will be developed to quantitate the biologically active compound in water or food.
PROGRESS: 2000/10 TO 2001/09
TECT FOOD AND THE ENVIRONMENT 1. What major problem or issue is being resolved and how are you resolving it? Identify and quantify biologically active compounds (BACs; e.g. toxins, mycotoxins, phytoestrogens, estrogens, or other endocrine disrupting materials) in fresh water sources that affect food safety, agricultural activity and the food chain. Areas of focus are the identification of hormonally active agents, drugs, and other naturally occurring compounds from a variety of known or unknown sources. These compounds may be associated with amphibian declines and deformities or may result from animal feeding operations or municipal sewage treatment plants. We have obtained water samples from lakes in Minnesota and Vermont in which deformed frogs have been found. Additional samples will be obtained from manure composting and stockpiling sites and sites near municipal sewage treatment plants. Lake samples will be studied to determine possible agents responsible for the frog malformations. Water samples from manure handling areas and surrounding areas will be studied for estrogen levels to determine estrogen load on the environment. Levels of pharmaceuticals and pesticides in the environment will also be determined. 2. How serious is the problem? Why does it matter? The seriousness of BACs in the environment has not yet been fully determined but has been targeted by the U.S. government as a priority. Interest in frogs is partially due to their role as indicators, "sentinel species," of environmental health. This classification as an ecological indicator is partially based on the fact that they live both in water and on land. However, very little scientific research has been reported in peer reviewed literature. Steroidal hormones, such as estradiol and testosterone, from human and animal sources are constantly entering the environment and appear to be resistant to environmental degradation. Good evidence also exists that estrogenic chemicals are present in the aquatic environment at concentrations high enough to adversely affect aquatic organisms. This research is significant due to a lack of knowledge regarding the health risks to people, wildlife, and aquatic ecosystems posed by these contaminates. Additional significance of this research results from a lack of knowledge about their presence in the food or food products we consume. 3. How does it relate to the National Program(s) and National Component(s)? This research is covered under the Drug Residues and Environmental Contaminants National Program Action Plan. The research is focused on BACs in water, which may occur from a variety of known or unknown sources and which may become contaminants in animal and human food. BACs may result from the degradation of environmental or natural components, the use of animal drugs or chemicals, or the transformation and persistence of hazardous chemicals or environmental pollutants. A particular concern is the ability of BACs to find their way into the environment, food animals, or the food chain. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2001 year: The transport and fate of testosterone and estradiol were conducted by application of these naturally-occurring estrogens to columns packed with loam soil (Glyndon silt loom) or sand. Once applied, neither of the estrogens could be removed from the soil column with water. Analysis of the soil sections showed that 80% and 96% of the testosterone and estradiol, respectively, remained in the top five centimeters of soil. Organic solvents were used to extract the soil from these estrogen columns. Testosterone and estradiol appeared to be converted into at least two metabolites in the soil. At least 50% of the testosterone and 70% of the estradiol were nonextractable from the soil after sequential organic solvent extraction. In contrast, most of the testosterone and estradiol (87% and 85%, respectively) was removed from a similar column filled with sand. Testosterone appeared to be intact but estradiol was changed to something different in the sand column. These results show that these estrogens are strongly absorbed by loam soil and are changed in the soil matrix but are transported and not absorbed in sand. B. Other Significant Accomplishment(s), if any: 1. Last year Biosensor and ELISA studies initiated a correlation between the presence of estrogenic compounds, not explainable by the presence of estradiol, and various wetlands shown to contain malformed frogs. These results were confirmed by fluorescence polarization (in collaboration with H. Kirkpatrick of Pan Vera, Madison, WI). Seasonal and yearly fluctuations in the estrogenic activity were also examined. Because environmental conditions (i.e. mineral content) may influence the biological activity of hormonally active agents, analyses were conducted on the mineral content of several wetlands over a two-year period and compared with FETAX and survey data with regard to the incidence of malformed frogs. Correlations between low levels of sodium or potassium and developmental delay in Xenopus laevis in the FETAX bioassay were observed. A delay in embryo development could lengthen exposure to hormonally active agents and, thereby, alter the window of susceptibility to chemical and biological agents. Laboratory experiments conducted using the native species, northern leopard frogs (Rana pipiens), also showed a developmental delay when sodium and potassium levels were low or estrogen was present. Chemical analyses of water samples collected at various wetlands that contained malformed frogs have ruled out the involvement of phthalates and ten commonly-used pesticides. 2. The FETAX bioassay has been modified to include an extended incubation period to facilitate the detection of teratogens which could be masked by factors that delay development. In addition, the development of methodologies to use Xenopus laevis as a bioassay for the detection of hormonally active agents that affect the expression of genes associated with the thyroid, retanoic acid, and estrogen receptors during the later stages of tadpole development through metamorphosis has been initiated. Rearing conditions for the embryos and the techniques for extraction of intestinal RNA with minimal trauma have been developed. The effects of pulsed exposure to key hormonal antagonists will be initiated shortly. 3. Techniques were established for identifying parasites and establishing cell culture techniques for the assessment of estrogenicity in water samples. Field studies were initiated to examine the affect of land use (specifically agricultural practices) on estrogenicity in waters and parasite burden in frogs. Along with water samples, frogs from sites in ND and MN are being collected and examined for physical abnormalities (including sex organs) and parasite burden (quantitation and characterization). Snail prevalence at sites is being evaluated, with specific attention paid to the presence of Ramshorn snails, known to shed the parasite riberoia, found associated with frog malformations. In the lab, parasite shedding by snails is being evaluated. 4. Estradiol and testosterone, eliminated in large amounts in the feces and urine of agricultural animals, are among the most potent estrogenic and androgenic substances known. Composting is an effective means of degrading organic substances and stabilizing them. We obtained chicken-layer manure from a commercial producer and conducted a 12-week composting study. Analysis of the composted manure for levels of estradiol and testosterone was performed with enzyme immunoassay kits at weekly time intervals. Our results showed that both hormones could be degraded under composting conditions, but that complete degradation did not occur. The rate of degradation of testosterone was 3X that of estradiol, probably due to the aromaticity of estradiol. A pair of soil amendment products created from cattle and swine manure also showed relatively high levels of estradiol and testosterone. The conclusions of this study were that composting reduced, but did not eliminate, introduction of these potent endocrine disruptors into the environment. In another study, we collected surface water samples from multiple agricultural, municipal, and pristine environments and analyzed them for estradiol and testosterone. This provided us with information on background levels for both hormones in the environment. Levels of these two hormones were very low in pristine and agricultural settings. In some municipal settings, levels of estradiol and testosterone were found to be 5-9 times that of the limit of detection for the enzyme immunoassay kits. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. Animal wastes contain significant quantities of the endogenous sex hormones, 17 beta-estradiol and testosterone. These wastes are a potentially significant source of hormones in the environment because they are directly applied to land. Livestock manure has been shown to be a source of environmental contamination for these hormones. Our hypothesis was that the levels of these hormones could be reduced or maybe even eliminated by the environmentally friendly process of aerobic composting. Composting is a beneficial waste management option that stabilizes organic by-products, such as manure, reduces its weight, destroys pathogens and weed seeds, and produces a low odor soil conditioner that also has some fertilizer value. Using commercial enzyme immunoassay kits to measure estradiol and testosterone levels in compost showed that hormones were broken down by aerobic microorganisms. Maintenance of high temperatures (>140 degrees F.) during composting resulted in more rapid degradation of both hormones than did composting conducted below 130 degrees F. Testosterone was more readily degraded than estradiol. Because these hormones are water soluble up to the parts per million range, they can be transported to surface waters through rainfall events or to groundwater systems by transport through the soil column. The impact of these hormones in soil and ground water needs to be evaluated in terms of potential toxicity and detrimental developmental outcomes in the environment; however, composting shows promise as an alternative animal waste management tool to reduce the environmental input from agricultural sources. 6. What do you expect to accomplish, year by year, over the next 3 years? CRIS project terminated. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product? Our studies on determination of the presence of estrogenic compounds in water associated with frog malformation from MN using FETAX analysis and an evanescent field fluorometry-based biosensor has been reported at international meetings to scientists from government, industry, and academia. These were the first reports of these topics. 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below) None.