Progress 03/20/01 to 08/01/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? 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. The affect of BACs on 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 regarding frog malformations 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. 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 because of a lack of knowledge regarding the health risks to people, wildlife, and aquatic ecosystems posed by these contaminates. Additionally, the significance of this research results from a lack of knowledge about their presence in the food or food products we consume. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? 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. 2. List the milestones (indicators of progress) from your Project Plan. Objective 1 (Determine BACs in fresh water) Year 1 (FY 2001): Construct frog/FETAX facility. Train personnel. Water and sediment sample collection. Year 2 (FY 2002): Complete FETAX analysis of water samples. FETAX screening of extracts/fractions. Establish collaborations to conduct functional assays. Screening for estrogenic, retinoic acid, AHR, and androgenic activity in MN water samples completed. Establish screening procedures for biomarkers. Year 4 (FY 2004): Identify BACs from affected sites. Establish and complete screening for thyroid agonist/antagonist activity. Complete analysis of tissue samples for biomarkers. Prepare antibodies against suspected BACs. Year 5 (FY 2005): Complete FETAX based metabolic activation system (MAS) to study BAC
metabolites. Identify biomarker profiles that may be used for determining BAC exposure. Design ELISA tests for BACs. Design immuno test strips for BACs. Objective 2 [Determine impact of agricultural activity (manure) on BACs in water] Year 1 (FY 2001): Acquire samples from various pristine, agricultural, municipal settings. Year 2 (FY 2002): Complete estrogen and androgen receptor-based assays. Initiate identification of BACs. Complete FETAX assays. Complete quantitation of estradiol and testosterone in waters using EIAs. Year 4 (FY 2004): Complete identification of BACs. Acquire water samples from outside the U. S. Initiate half-life study of estradiol and testosterone in composted manure & soil. Year 5 (FY 2005): Generate antibodies to BACs for EIA or ELISA development. Initiate study on movement of identified BACs into surface and groundwater. Complete half-life study of estradiol and testosterone in composted manure and soil. 3a List the milestones that were
scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Complete FETAX based metabolic activation system (MAS) to study BAC metabolites. Identify biomarker profiles that may be used for determining BAC exposure. Milestone Not Met Redirection of Research focus due to change in priorities 2. Complete half-life study of estradiol and testosterone in composted manure and soil. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? This CRIS 5442-32000-010-00D has been terminated. 4a What was the single most significant accomplishment this past year? Factors effecting fate and transport of estrogens in soil. When the natural female hormone, estradiol, which is found in animal manures, was applied to soil-filled columns, we found that
estradiol and estrone (estadiol degradation product) were the predominant compounds found to elute from the column. The amount and timing of the estrogens eluting from the column showed that if the organic content of the soil increased, more of the estradiol was absorbed to the soil. Further, we observed that if soil organic content increased the time to achieve complete estrogen-soil binding also increased. The absorption of estradiol or estrone to soil was also studied by mixing soil, water and estradiol or estrone in vials then sampling the amount of hormone remaining in water over time. It was found that a constant amount of absorption of both estrogens to soil was observed with time until a time point was reached when no more estrogen absorbed to the soil. For estradiol and estrone these time points were 5 and 24hours, respectively, which may help explain why hormones are consistently detected at low concentrations in the environment despite their strong soil absorption and
ease of degradation. 4b List other significant accomplishments, if any. Atrazine inhibits phosphodiesterase in pig heart, lung, and brain. We found that atrazine, one of the most widely used herbicides in the U.S. , inhibited the important signal transduction enzyme phosphodiesterase in pig heart, lung, and brain, but not kidney and liver. There are over 50 different types of phosphodiesterase distributed among all tissues and in various subcellular locations. To determine which phosphodiesterases from tissues and subcellular locations were inhibited by atrazine exist, we fractionated tissues into cell cytosol (liquid inside), cell membranes and other cellular organelles. Only phosphodiesterase from heart cytosol was inhibited by atrazine. These studies are important because claims have been made that atrazine disrupts the endocrine system which could affect the proper maturation of animals. Previously, we showed that endocrine disruption did not occur by atrazine directly
binding to the estradiol/estrogen receptor, but instead atrazine inhibited phosphodiesterase. Atrazine inhibition of phosphodiesterase may be the mechanism by which maturation is disrupted. 4c List any significant activities that support special target populations. Swine wastewater was evaluated for estrogenic activity and Kjeldahl nitrogen (TKN) as it was sequentially treated by a two stage anaerobic lagoon and a constructed wetland system over three seasons. Estrogenic activity of water from the manure pit was ~900 picomolar estradiol equivalents (pM E2 Eq) in both spring and summer. The E2 Eqs dropped dramatically in the primary lagoons to 1% of pit values (12 to 14 pM). The estrogenic activity of lagoon wastewater was so low as it entered the wetlands in these seasons (~3 pM E2 Eq) that little to no change in activity was found across the wetlands. In the cooler temperatures of November, the E2 Eq entering the wetlands was ~100 pM, yet ~94% of the activity was removed by the
wetlands, while the TKN was reduced by ~75% for wetlands. These data indicate that wetlands are useful in reducing both N and estrogenic activity from animal waste. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The research reported is conducted under National Program 108, Food Safety and directly contributes to research Component 1.1, Pathogens, Toxins, and Chemical Contaminates Preharvest . Specifically involving research on the metabolism, transport, sites of deposition and excretion of chemical residues. Research activities are further related to ARS Strategic Plan Goal 3, Enhance protection and safety of the Nation's agricultural and food supply; Objective 3.1, Provide science-based knowledge on the safe production, storage, processing, and handling of plant and animal products and on the detection and control of toxin- producing bacteria and fungi parasites, mycotoxins, chemical residues, and plant toxins so as
to assist regulatory agencies and the food industry in reducing the incidence of foodborne illnesses. We determined the transport and fate of the naturally-occurring hormones, i.e. testosterone and estradiol and the antibiotics, sulfadimethoxine, sulfamethazine, and sulfaquinoxaline, used in animal feed in various soils. These hormones and drugs are excreted in manure and applied to soil as fertilizer. Our research showed that only limited amounts of testosterone and estradiol were transported through soils, while virtually all the sulfa antibiotics were transported through soils. The impact of these hormones and sulfas moving through soil to ground water needs to be evaluated in terms of potential toxicity and detrimental developmental outcomes in the environment. Mammals naturally produce and excrete the potent hormones 17beta- estradiol and testosterone. The purpose of this study was to determine if composting reduced or eliminated these hormones. Initial average estradiol
concentration was 92 ng/g (ppb, on a dry weight basis) with 83% degraded (16 ppb) after 175 days. Initial testosterone compost concentrations were greater than estradiol, i.e. 212 ng/g and were degraded to 13 ppb during composting. The results of the study demonstrated that composting of poultry manure was an effective means of reducing, but not eliminating, the introduction of the potent hormones, estradiol and testosterone, into the environment. A laboratory scale composter and was used to study the fate of estradiol and testosterone in manure. Results showed that virtually all of the estradiol was degraded in the composter to estrone(another estrogenic hormone); only 0.3% was degraded to CO2, and testosterone was significantly degraded to CO2 (27% of the applied radiolabel). The impact of this study is that the hormones in manure can be degraded by the composting process, which may effectively remediate the hormones so that their impact on the environment/groundwater is limited.
We found that atrazine inhibits the phosphodiesterase enzyme, metabolites of atrazine inhibited phos phodiesterase with a potency 50 to 500-fold less than atrazine, and neither atrazine or its metabolites bind to the estrogen receptor. Because phosphodiesterase controls cellular signaling and is key enzyme involved in the onset of sexual maturation and the estrous cycle, atrazine inhibition of this enzyme in a signaling pathway may adversely affect reproduction. The impact is an important new concept that an endocrine disrupting-like process is possible when a chemical inhibits an enzyme in the cellular signaling pathway, which can affect life processes much like hormones. Field studies indicated malformed northern leopard frogs in the MN region were associated with infection by the flat worm parasite Ribeiroia ondatrae, and not associated with contamination of water by agrichemicals. Surveys of northern leopard frogs in the western MN and eastern ND region indicated presence of
this parasite and associated malformations are rare. Sites used for agricultural use were not found to have higher rates of frog malformation than those pristine sites studies. These findings suggest the scientific community should investigate factors influencing incidence of Ribeiroia ondatrae. The estrogenic activity of various surface waters were assessed to provide a context for evaluating surface and ground waters from agricultural sites. The E-Screen assay used is more than 100 times more sensitive than LC MS-MS, which is already capable of detecting estradiol at pg/ul or parts per quadrillion. Estrogenic activity in wetlands and ponds varied independent of land use (less than 1 X 10 -12 M estradiol equivalents, E2 Eq ), river samples were typically at least 10 fold lower, with slightly higher values found downstream from municipal wastewater treatment effluent release sites, and highest concentrations found in winter (~ 2 X 10 -13 M E2 Eq), but values were still 40 times
lower than estrogenic activity of wastewater effluents and 400-4000 times lower than circulating estradiol levels in women. 6. 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 and durability of the technology products? Presentations by N. Shappell of seminar Estrogenic activity in the environment: municipal wastewater effluent, river, ponds and wetlands to stake holders (Fargo Water and Wastewater Treatment Plant Operators 5/04; and ND Water and Pollution Control Conference 10/04); Sigma Xi chapter, Tifton, Ga (audience ARS/U. of GA); USDA-ARS Coastal Plains Soil, Water and Plant Research Center). As a result of presentations, collaborations were established between the Coastal Plains ARS unit, our unit and North Carolina Agricultural and Technical State University. 7. List your most important
publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Science News, 2004, vol. 165, pp. 10 and 29, When testosterone gets down and dirty.
Impacts
(N/A)
Publications
- Roberge, M.T., Hakk, H., Larsen, G.L. 2004. Atrazine and its metabolites are non-competitive inhibitors of phosphodiesterase. [abstract] 4th SETAC (Society of Environmental Toxicology and Analytical Chemistry) World Congress, Portland OR, November 14-18, 2004.
- Casey, F., Larsen, G.L., Hakk, H., Simunek, J. 2004. Fate and transport of testosterone in agricultural soils. Environmental Science and Technology 38:790-798.
- Jaehoon, L., Casey, F., Hakk, H., Larsen, G.L. 2004. Sorption and mobility of sulfamethazine in various soils: a laboratory study. [abstract] American Society of Agronomy, Crop Science Society of America and Soil Science Society of America Annual Meeting, Seattle, WA, Nov. 1-4, 2004.
- Roberge, M.T., Hakk, H., Larsen, G.L. 2004. Atrazine is a competitive inhibitor of phosphodiesterase but does not affect the estrogen receptor. Toxicology Letters 154/1-2:61-68.
- Casey, F., Lee, J., Simunek, J., Larsen, G.L., Hakk, H. 2005. Sorption, mobility and transformation of estrogenic hormones in natural soil. Journal of Environmental Quality 34:1372-1379.
- Hakk, Heldur, Millner, Patricia, Larsen, Gerald. 2005. Decrease in water- soluble 17beta-estradiol and testosterone in composted poultry manure with time. Journal of Environmental Quality 34:943-950.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? 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. The affect of BACs on 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 regarding frog malformations 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. 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 because of a lack of knowledge regarding the health risks to people, wildlife, and aquatic ecosystems posed by these contaminates. Additionally, the significance of this research results from a lack of knowledge about their presence in the food or food products we consume. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? 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. 2. List the milestones (indicators of progress) from your Project Plan. Objective 1 (Determine BACs in fresh water) Year 1 (FY 2001): Construct frog/FETAX facility. Train personnel. Water and sediment sample collection. Year 2 (FY 2002): Complete FETAX analysis of water samples. FETAX screening of extracts/fractions. Establish collaborations to conduct functional assays. Screening for estrogenic, retinoic acid, AHR, and androgenic activity in MN water samples completed. Establish screening procedures for biomarkers. Year 4 (FY 2004): Identify BACs from affected sites. Establish and complete screening for thyroid agonist/antagonist activity. Complete analysis of tissue samples for biomarkers. Prepare antibodies against suspected BACs. Year 5 (FY 2005): Complete FETAX based metabolic activation system (MAS) to study BAC
metabolites. Identify biomarker profiles that may be used for determining BAC exposure. Design ELISA tests for BACs. Design immuno test strips for BACs. Objective 2 [Determine impact of agricultural activity (manure) on BACs in water] Year 1 (FY 2001): Acquire samples from various pristine, agricultural, municipal settings. Year 2 (FY 2002): Complete estrogen and androgen receptor-based assays. Initiate identification of BACs. Complete FETAX assays. Complete quantitation of estradiol and testosterone in waters using EIAs. Year 4 (FY 2004): Complete identification of BACs. Acquire water samples from outside the U. S. Initiate half-life study of estradiol and testosterone in composted manure & soil. Year 5 (FY 2005): Generate antibodies to BACs for EIA or ELISA development. Initiate study on movement of identified BACs into surface and groundwater. Complete half-life study of estradiol and testosterone in composted manure and soil. 3. Milestones: A. Year 4 (FY 2004)
milestones. Objective 1: Identify BACs from affected sites. This part of the milestone was not met, because reports of frog malformations have virtually stopped, the parasite Ribeiroia ondatrae has been found to be the only agent to cause the malformations observed in nature, and no BACs could be found which affect frog development. However, cell culture techniques for the assessment of estrogenicity (E-screen) and toxicity and Tecan florescence polarization spectrophotometer using various receptors (alpha and beta estradiol and testosterone receptors) have been incorporated into our Research Program as a screen for BACs. Because Ribeiroia has been shown to cause the frog malformations the following parts of Objective 1 milestones were not continued: Complete analysis of tissue samples for biomarkers. However, phosphodiesterase has been found to be significantly inhibited by atrazine (see below 4a) and can be a biomarker for this pathway. Prepare antibodies against suspected
BACs. Complete identification of BACs. Acquire water samples from outside the U. S. Objective 2. [Determine impact of agricultural activity (manure) on BACs in water] This milestone was to initiate a half-life study of estradiol and testosterone in composted manure & soil. The soil work has been completed and the composing part of the study is on going and will be completed within the year. B. List the milestones that you expect to address over the next 3 years. What do you expect to accomplish, year by year, under each milestone? During FY 2005 we plan to: 1) develop a liquid chromatography/mass spectrometry method for quantitation of extractable estradiol and testosterone in manure and compost; 2) Measure the levels of sulfonamide antibiotics released into the environment (groundwater and surface water) from a farm setting where animals are being administered the antibiotics as growth promoters; 3) determine the effect of exposure of 0, 1, 10, and 25ppb atrazine on northern
leopard frogs; and 4) validate the quantitation of estrogens using E-screen cell line as compared to ELISA and LS/MS/MS methods. During FY2006 we plan to: 1) study of the fate and transport of two common anabolic steroids administered to cattle as growth promoters, i.e. trenbolone acetate and melengestrol acetate; 2) study the fate and transport of estradiol and testosterone in the B-horizon of soils. This portion of the soil column represents the subsoil, void of organic matter; 3) conduct a histological evaluation of a subset of frog reproductive tracts from frogs exposed to atrazine; and 4) evaluate estrogenic and antibiotic activity from waste of different swine waste management systems. During FY2007 we plan to: 1) determine the degradation rate during composting of sulfa drugs used in production agriculture; 2) determine the degradation rate during composting of estradiol; 3) determine fate and transport of estradiol and testosterone in undisturbed soil columns; 4) evaluate
estrogenic activity of dairy waste; and 5) evaluate estrogenic activity of animal waste treated with commercial additives. 4. What were the most significant accomplishments this past year? A. We found that atrazine inhibits the phosphodiesterase enzyme, metabolites of atrazine inhibited phosphodiesterase with a potency 50 to 500-fold less than atrazine, and neither atrazine or it's metabolites bind to the estrogen receptor. Because phosphodiesterase controls cellular signaling and is key enzyme involved in the onset of sexual maturation and the estrous cycle, atrazine inhibition of this enzyme in a signaling pathway may adversely affect reproduction. The impact is an important new concept that an endocrine disrupting-like process is possible when a chemical inhibits an enzyme in the cellular signaling pathway, which can affect life processes much like hormones. B. A survey of northern leopard frogs (Rana pipiens) was conducted in the rural region of eastern North Dakota and
western Minnesota to determine the prevalence of malformed frogs and frog parasites. The flat worm Ribeiroia ondatrae in frogs is of particular interest, because the presence of Ribeiroia cysts in metamorphosed frogs is the only factor found associated with frog malformations in nature. Frogs were examined from twenty U.S. sites, in addition to two Minnesota sites with histories of high malformation frequencies in R. pipiens, for parasite profiles and frog body condition relative to surrounding land use. Frequency of both R. ondatrae infection and frog malformations were very low. Infection frequencies of seven other species of trematodes ranged from 32 - 80%. No correlations were found between parasite abundance, land use, and malformations of R. pipiens from these sites. C. Estrogenic activity of regional water samples from wetlands and ponds at locations involved in various agricultural land uses, from three river sites over four seasons, and from municipal wastewater
effluent held in storage lagoons were evaluated. An estrogen-responsive cell line was used to determine estradiol equivalents of water samples extracted by solid-phase extraction. Estrogenic activity in surrounding wetlands and ponds varied independent of land use and were less than 1 X 10 -12 M E2 Eq or < 0.3 parts per trillion (ppt). Estrogenic activity of river samples were typically at least 10 fold lower, with slightly higher values found downstream from municipal wastewater treatment effluent release sites, and highest concentrations found in winter ( 2 X 10 -13 M E2 Eq or 0.05 ppt estradiol equivalents), but values were still 40 times lower than estrogenic activity of wastewater effluents and 400-4000 times lower than circulating estradiol levels in women. During the summer and fall a toxic factor was found in river samples upstream from municipal wastewater release sites. The timing of toxicity did not correlate to the presence of elevated fecal coliforms. Estrogenic
activity in wastewater effluent from lagoons decreased over time with an apparent T 1/2 of 8 days for one lagoon. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. We determined the transport and fate of the naturally-occurring hormones, i.e. testosterone and estradiol and the antibiotics, sulfadimethoxine, sulfamethazine, and sulfaquinoxaline, used in animal feed in various soils. These hormones and drugs are excreted in manure and applied to soil as fertilizer. Our research showed that only limited amounts of testosterone and estradiol were transported through soils, while virtually all the sulfa antibiotics were transported through soils. The impact of these hormones and sulfas moving through soil to ground water needs to be evaluated in terms of potential toxicity and detrimental developmental outcomes in the environment. Mammals naturally produce and excrete the potent hormones 17beta- estradiol and testosterone.
The purpose of this study was to determine if composting reduced or eliminated these hormones. Initial average estradiol concentration was 92 ng/g (ppb, on a dry weight basis) with 83% degraded (16 ppb) after 175 days. Initial testosterone compost concentrations were greater than estradiol, i.e. 212 ng/g and were degraded to 13 ppb during composting. The results of the study demonstrated that composting of poultry manure was an effective means of reducing, but not eliminating, the introduction of the potent hormones, estradiol and testosterone, into the environment. A laboratory scale composter and was used to study the fate of estradiol and testosterone in manure. Results showed that virtually all of the estradiol was degraded in the composter to estrone(another estrogenic hormone); only 0.3% was degraded to CO2, and testosterone was significantly degraded to CO2 (27% of the applied radiolabel). The impact of this study is that the hormones in manure can be degraded by the
composting process, which may effectively remediate the hormones so that their impact on the environment/groundwater is limited. 6. 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 and durability of the technology products? Transport and fate of estradiol and testosterone in soil to county and water resource officials from North Dakota. This technology is currently available to livestock producers. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Gave progress report on our findings on the transport and fate of estradiol and testosterone in soil at the North Dakota Water Resources Workshop, Devils Lake, Fargo and Bismarck, ND in March 2004.
Impacts
(N/A)
Publications
- Shappell, N.W. 2003. Decay of estrogenic activity in municipal wastewater lagoons. Society of Environmental Toxicology and Chemistry Asia/Pacific & Australasian Society for Ecotoxicology Meeting, Sept. 28-Oct. 2, 2003, Christchurch, New Zealand.
- Casey, F., Simunek, J., Hakk, H., Larsen, G.L. 2003. Sorption and mobility of testosterone in agricultural soils. American Society of Agronomy-Crop Science Societies of America-Soil Science Society of America, November 2-6, 2003, Denver, CO.
- Garber, Eric A.E., Erb, Judith L., Magner, Joseph, and Larsen, Gerald. 2004. Low levels of sodium and potassium in the water from wetlands in Minnesota that contained malformed frogs affect the rate of Xenopus development.Environmental Monitoring and Assessment. 90:45-64.
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Progress 10/01/02 to 09/30/03
Outputs
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 regarding frog malformations 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. Additionally, the 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 Program Component(s) to which it has been assigned? 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 2003 year: A laboratory scale composter and radiolabeled steroid hormones were used to study the fate of estradiol and testosterone in manure after the composting operation by the Animal Metabolism-Agricultural Chemicals Research Unit. Results showed that virtually all of the estradiol was degraded in the composter to estrone(another estrogenic hormone)with only 0.3% degraded to CO2. In a similar composting study, testosterone was significantly degraded to CO2 (27% of the applied radiolabel). The impact of this study is that the active hormones in manure can be degraded by the composting process which may effectively remediate the hormones so that their impact on the environment/groundwater is limited. B. Other Significant
Accomplishment(s), if any: 1. Hormone levels in native waters of northwest Minnesota, representing recreation, agriculture, and pristine environments were determined by the Animal Metabolism-Agricultural Chemicals Research Unit. ELISA kits specific for estradiol and testosterone were used to detect these hormones. Most sites were void of any detectable hormones, even those agricultural sites where cattle were in close proximity and, in a few instances near municipal treatment plants where estradiol levels were 4-5 times background. Measurable testosterone levels were observed at nearly every site tested but at very low levels; these data determine baseline levels of these hormones in recreation, agriculture, and pristine environments. 2. Estrogenic activity of water from three sites on the Red River surrounding the Fargo-Moorhead area were sampled seasonally by the Animal Metabolism-Agricultural Chemicals Research Unit. Results indicate very low estrogenic activity in Red River
water; the highest value was 2 X 10- 13 M or 0.05 ppt estradiol equivalents (40 times lower than estrogenic activity of wastewater effluents and 400-4000 times lower than circulating estradiol levels in women). Anti-estrogenic and/or toxic factors were found to be present in fall and summer samples, indicating the need for further evaluation of toxic factors in river water. 3.Estrogenic activity of wastewater held in municipal lagoons was monitored over a three month period using the MCF7 cell line in a modified Escreen by the Animal Metabolism-Agricultural Chemicals Research Unit. At the completion of lagoon filling, wastewater had 24 X 10-13 M estradiol equivalents and fell to 6 X 10-13 M after 84 days, with a half-life of eight days. These results indicate substantial decay of estrogenic activity of wastewater in municipal lagoons and have implications for manure lagoons used on concentrated animal feed lot operations. 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. 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? During FY 2004 we plan to: 1) collaborate with another ARS laboratory equipped with a lab composter using radiolabelled hormones, which will allow us to determine the metabolic fate of estradiol and testosterone during the composting
process; 2) validate methods to detect hormones in environmental water samples by three different analytical methods: hormone receptor assays, ELISA kits, LC-MS, and a estrogen-receptor specific cell line.; 3)measure the degradation of excreted radiolabeled sulfonamide antibiotics in a laboratory scale composter.4) Work at the Milnor hog farm site determine the fate and transport of estradiol and testosterone in a field setting. Manure, compost and lagoon waste will be spread unto fields at agronomic rates, and constructed lysimeters and wells below ground will collect leachate. Leachate will be analyzed for the hormones by way of specific ELISA kits, LC-MS and an estrogen- responsive cell line; and 5)Red River water samples will be assessed in the FETAX assay to determine if organismal effects are present. During FY 2005 we plan to: 1) complete FETAX analysis of water samples; 2) develop a robust gas chromatography/mass spectrometry method for quantitation of extractable estradiol
and testosterone in manure and compost; 3) Measure the levels of sulfonamide antibiotics released into the environment(groundwater and surface water) from a farm setting where animals are being administered the antibiotics as growth promoters; and 4) determine the effect of observe exposer of northern leopard frogs to 0, 1, 10, and 25ppb atrazine. During FY2006 we plan to: 1)Study of the fate and transport of two common anabolic steroids administered to cattle as growth promoters, i.e. trenbolone acetate and melengestrol acetate; 2) Study the fate and transport of estradiol and testosterone in the B- horizon of soils. This portion of the soil column represents the subsoil, void of organic matter; and 3)conduct a histological evaluation of a subset of frog reproductive tracts from frogs exposed to atrazine. 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 and durability of the technology products? Our studies on degradation of estrogens by composting, transport and fate of estrogens and veterinary drug in soils, and the developmental delay in frogs caused by estrogenic compounds and low mineral media associated with waters from MN lakes containing frog malformations have been reported at international meetings to scientists from government, industry, and academia. These were the first reports of these topics. Gerald Larsen presented a lecture entitled, 'It's not easy being green, seeking the cause/s of frog malformation' to a Watershed Heroes Core4 Conference and National Ecoteam Competition sponsored by the American Farm Bureau, June 19, 2003 in St. Peter, MN
Impacts
(N/A)
Publications
- Shappell, N.W. 2003. Assessment of estrogenic activity and toxicity of Red River water. [abstract]. 1st International Water Conference, Water Science Decision-Making, April 23-24, 2003, Moorhead, MN.
- Casey, F.X.M, Larsen, G., Hakk, H., Simunek, J. Fate and transport of 17beta-estradiol in soil - water systems. Environmental Science and Technology. 2003. v. 37. p. 2400-2409.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Question 1: 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 and much of its interaction with the environment occurs through its skin. However, very little scientific research regarding frog malformations 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. Additionally, the 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 Program Component(s) to which it has been assigned? This research is covered under the Drug Residues and Environmental Contaminants National Program Action Plan (National Program #108). 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, the food chain, or food animals. 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2001 year: The degradation rate of estradiol and testosterone during composting of manure was determined by the Animal Metabolism-Agricultural Chemicals Research Unit with the collaboration of Patricia Millner, USDA-ARS, Beltsville, MD. Initial concentrations of testosterone and estradiol in chicken manure averaged 212 and 92 ppb, respectively, but fell gradually over 19 weeks to 13 ppb for testosterone and 16 ppb for estradiol. The rate of degradation of testosterone was 3X that of estradiol, probably due to the aromaticity of estradiol. This work suggests that composting may be an environmentally friendly technology suitable for
reducing the concentrations of these endogenous hormones at concentrated animal operation facilities and may prevent their transport into surface or groundwater systems. B. Other Significant Accomplishment(s), if any: 1. 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 regarding 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. In addition, developmental delay was observed when the Northern leopard frog (Rana pipiens) were exposed separately to genestein, estradiol, and diethylstilbesterol in high and low sodium/potassium rearing water. 2. Studies were continued to determine the transport and fate of testosterone and estradiol in manure once applied on four soil
types (loam, sandy loam, clay loam, and silt loam)in a column or sand column. Other studies on estradiol showed that most of the estradiol is bound to soil and this binding was associated with particle size and organic matter. Estradiol was found not to be adsorbed by sand and limited (0-3%) amounts of estradiol were transported through of the soils except for silt loam (21%) and sand (82%). A field lysimeter experiment indicated that estradiol was transported through 2.3 m of soil and was present in concentrations ranging from 30-525 ng L-1, which are concentrations 30 to 500 X greater than levels that are known to cause abnormal hormone (vitellogenin) production in male fish. 3. 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 is available. The thyroid system is important in frog development and controls metamorphosis. Rearing conditions for the embryos and the techniques for extraction of intestinal RNA with minimal trauma have been developed. 4. Techniques were established for identifying parasites and establishing cell culture techniques for the assessment of estrogenicity in water samples. The parasite associated with limb malformation in frogs was present at only two of 20 sites monitored, and at very low intensities with no correlation found between land use, parasite burden, and frog health. Water from these sites were assessed for estrogenic activity and preliminary analyses have been completed. More than 50% of the sites had detectable levels of estrogenic activity, and water from some sites were either toxic or prevented the
"normal" induced estradiol growth response. 5.Transport and fate of Sulfadimethoxine, sulfamethazine, and sulfaquinoxaline in various soils were studied using column experiments applied at concentrations estimated to be present in manures as applied in the field. Each compound was each individually tested in sandy loam columns and was readily transported through and eluted from the columns with recoveries in the eluates of 95.4%, 93.2%, and 76.9%, respectively. Sulfamethazine was individually applied to sand, silt loam, loam, and clay loam columns, was also readily transported through and eluted from the columns with recoveries in the eluates of 99.7%, 85.9%, 82.6%, and 68. 9%, respectively. These studies indicated that sulfa-drugs, and/or their metabolites are not adsorbed to organic or mineral soil fractions, which increases the chance of sulfa drug mobility in the soil and chances of ground water contamination. 5. Describe your 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. 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? During FY 2003 we plan to: 1)identify BACs from affected sites and from fractions shown to have activity in the various assays described above; 2) establish screening for thyroid agonists, antagonists and Xenopus laevis mRNA; 3) obtain functional
bioassays for estrogenicity and androgenicity; 4) continue studies relating to the environmental fate of by-products associated with hog farming; 5) complete a survey of Midwest agriculture, pristine environments, and municipal waterways; and 6) study the biology involved with frog malformations resulting from the parasite, Ribeiroia. During FY 2004 we plan to: 1) collaborate with another ARS laboratory equipped with a lab composter using radiolabelled hormones, which will allow us to determine the metabolic fate of estradiol and testosterone during the composting process; 2) utilize functional bioassays for determining the overall estrogenicity or androgenicity of water samples or manure extracts; 3) study the role played by chemical exposure, rearing in low sodium and potassium media, and estrogen exposure on the susceptibility of frogs to malformation by the amphibian parasite Ribeiroia; 4) measure hormone levels in soil, manure, compost, and ground water derived from swine
operations; and 5) characterize biologically active components of environmental water. During FY 2005 we plan to: 1) complete FETAX analysis of water samples; 2) develop a robust gas chromatography/mass spectrometry method for quantitation of extractable estradiol and testosterone in manure and compost; 3) expand studies of wetlands associated with the occurrence of malformed frogs and known to contain the parasite, Ribeiroia; and 4)conduct a histological evaluation of a subset of frog reproductive tracts (used in parasite and health assessment) will also be performed, and conduct mineral characterization of the water from the sites known to contain parasites. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Our studies on degradation of estrogens by composting, transport and fate of estrogens
and veterinary drug in soils, and the developmental delay in frogs caused by estrogenic compounds and low mineral media associated with waters from MN lakes containing frog malformations have been reported at international meetings to scientists from government, industry, and academia. These were the first reports of these topics. Composting information and technology are essentially now available to the farmer. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) Article written about Dr. Heldur Hakk's work on degradation of hormones in manure. "Composting cuts manure's toxic legacy." Science News. v. 160. p. 285.
Impacts
(N/A)
Publications
- Larsen, G., Casey, F., Magelky, B., Pfaff, C., Hakk, H. Sorption, mobility, and fate of 17-ss estradiol and testosterone in loam soil and sand. Proceedings of the 2nd International Conference on Pharmaceuticals and Endocrine Disrupting Chemicals in Water. 2001. p. 138-143.
- Larsen, G., Casey, F., Bergman, A., Hakk, H., Garber, E. Sorption, mobility and fate of tetrabromobisphenol A (TBBPA) in loam soil and sand. Proceedings of the 2nd International Conference on Pharmaceuticals and Endocrine Disrupting Chemicals in Water. 2001. p. 144-151.
- Hakk, H., Millner, P., Larsen, G. Fate of the endogenous hormones 17ss- estradiol and testosterone in composted manure. Proceedings of the 2nd International Conference on Pharmaceuticals and Endocrine Disrupting Chemicals in Water. 2001. p. 128-137.
- Garber, E.A.E., Erb, J.L., McGarvey, A.M., Larsen, G.L., Magner, J. Mineral deficiency, estrogenic activity, and occurrence of malformed frogs in Minnesota. Proceedings of the 2nd International Conference on Pharmaceuticals and Endocrine Disrupting Chemicals in Water. 2001. p. 218- 232.
- Erb, J.L., Garber, E.A.E., Downward IV, J.G., Priuska, E.M., Wittliff, J.L. , Magner, J. Data from an estrogen receptor-based biosensor correlates with evidence of frog malformation and demonstrates a differential response of hER- & to beneficial and harmful estrogenic compounds. Proceedings of the 2nd International Conference on Pharmaceuticals and Endocrine Disrupting Chemicals in Water. 2001. p. 203-217.
- Garber, E.A.E., McGarvey, A.M., Larsen, G.L., Erb, J.L., Kirkpatrick, H. The effect of 17-ss-estradiol, diethylstilbestrol (DES), and genistein on Xenopus laevis and Rana pipiens tadpole development. e. Hormone. 2001. Abstract No. 47.
- Larsen, G.L., Casey, F.X., Magelky, B.K., Pfaff, C.M., Hakk, H., Smith, D. J. Sorption, mobility and fate of sulfadimethoxine, sulfamethoxine, and sulfaquinoxaline in various soil types. SETAC Europe 12th Annual Meeting. 2002. Abstract No. 46-05.
- Gackle, N.G., Shappell, N.W., Grier, J. Assessment of parasite abundance and health of native frogs relative to surrounding land use in ND and MN. American Society of Ichthyologists & Herpetologists National Meeting. 2002. Abstract No. 48.
- Shappell, N.W., Gackle, N.J., Canfield, J. Malformation, mortality and parasites in Northern Leopard Frogs in MN and ND. MN Water 2002: Working Together in a Climate of Change to Manage Minnesota's Water Resources Conference. 2002. Abstract p. 28.
- Shappell, N.W., E.A.E. Garber, Gackle, N.J, Canfield, J., Larsen, G. L. Malformation versus mortality, a study of Northern Leopard Frogs development in situ. MN Water 2002: Working Together in a Climate of Change to Manage Minnesota's Water Resources Conference. 2002. Abstract p. 28.
- Davis, M.A., Fernholz, P.L., McGarvey, A.M., Garber, E.A., Larsen, G.L. Preliminary Study: Rana pipiens showed developmental delay and lower weights when reared in water which contained low sodium and potassium concentrations and in the presence of either estradiol, diethylstilbesterol, or genistein. MN Water 2002: Working Together in a Climate of Change to Manage Minnesota's Water Resources Conference. 2002. Abstract No. 19.
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Progress 10/01/00 to 09/30/01
Outputs
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 National Program 108, Food Safety. 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: To determine the transport and fate of naturally occurring hormones in manure once applied on soil or sand. The Animal Metabolism-Agricultural Chemicals Research Unit, in collaboration with Francis Casey, North Dakota State University Soil Science Department, conducted studies in which tes- tosterone & estradiol were each applied to a silt loam soil or sand column. Most of the testosterone and estradiol (80% and 96%, respectively) was sorbed to the top five centimeters of soil; at least two metabolites were found in this sorbed material but most of the testerone and estradiol (87% and 85%, respectively) was not sorbed on a similar sand column. These results describe the fate and transport of these estrogens in manure in a silt loam soil and sand after a manure application. 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?
During FY 2002 we plan to: 1) complete FETAX analysis of water samples; 2) develop screening assays for BACs based on estrogen, androgen, progesterone, and glucocorticoid receptors; 3) construct a soil column in the laboratory and using radiolabeled hormones measure the transport rate and fate of these substances in soil; 4) develop a robust gas chromatography/mass spectrometry method for quantitating extractable estradiol and testosterone in manure and compost; 5) measure hormone levels in soil, manure, compost, and ground water derived from swine operations; and 6) expand studies of wetlands associated with the occurrence of malformed frogs and include other sites outside of Minnesota that have been shown not to have the parasite, Ribeiroia. During FY 2003 we plan to: 1)identify BACs from affected sites and from fractions shown to have activity in the various assays described above; 2) establish screening for thyroid agonists, antagonists and Xenopus laevis mRNA; 3) obtain
functional bioassays for estrogenicity and androgenicity; 4) continue studies relating to the environmental fate of by-products associated with hog farming; 5) complete a survey of Midwest agriculture, pristine environments, and municipal waterways; and 6) measure hormone levels in compost derived from cattle manure. During FY 2004 we plan to: 1) collaborate with another ARS laboratory equipped with a lab composter using radiolabelled hormones, which will allow us to determine the metabolic fate of estradiol and testosterone during the composting process; 2) develop or have access to a functional bioassay for determining the overall estrogenicity or androgenicity of water samples or manure extracts; and 3) expand our bioassay facility to include the use of Zebra fish.
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.
Impacts
(N/A)
Publications
- Erb, J. L., Garber, E., Priuska, E., Wittliff, J., Downward, J. Use of a fiber optic biosensor to demonstrate the presence of an estrogen mimicking substance in pond water hosting malformed frogs. McLachlan, J.A., Guillette, L.J., Iguchi, Taisen, editors. New York Academy of Sciences. Environmental Hormones: The Scientific Basis of Endocrine Disruption. 2001. Abstract #33.
- Garber, E.A.E., Erb, J.L., Downward, J. G., Priuska, E. M., Wittliff, J. L., Feng, W., Magner, J., Larsen, G.L. Biosensor, ELISA, and frog embryo teratogenesis assay: Xenopus (FETAX) analysis of water associated with frog malformations in Minnesota. Proceedings of Photonic Detection and Intervention Technologies for Safe Food. 2000. v.4206. p. 147-158.
- Garber, E.A.E., Erb, J.L., Wittliff, J.L., Magner, J., Larsen, G. Biosensor, ELISA, and FETAX analysis of water associated with frog malformations in Minnesota. Photonics East. 2000. Abstract #4206-36.
- Garber, E. A. E., Larsen, G. L., Magner, J. FETAX and ELISA analysis of water associated with frog malformations in Minnesota. 2000. 21st Annual Meeting of the Society of Environmental Toxicology and Chemistry. Abstract #PWP048. p. 254-255.
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