1a. Objectives (from AD-416)
All animals produce natural hormones in the healthy function and regulation of their endocrine system. When a hormone is eliminated from the body and gets into the environment, it can act upon other animals as an endocrine disrupting compound by binding to and activating receptors within hormone-responsive organs. In sensitive organisms this can occur at the parts per trillion level, and risks include sex reversals and cancer. The potential for livestock farms to serve as sources of hormones to the environment has been recognized for many years. To guide the objectives of this proposal, the following research questions (RQ) were formulated to evaluate the respective testable hypotheses (TH): RQ 1) Does dissolved organic carbon and particulates from lagoon waste or soil enhance the mobility of estrogens in mineral soil and thus increase the potential for off-site movement? TH 1) If the movement of estrogens is related to the presence of lagoon- or soil-derived dissolved organic carbon and particulates, then estrogen in the presence of high amounts of soil/lagoon dissolved organic carbon will increase their mobility compared to aqueous free estrogens. RQ 2) Does dissolved organic carbon and particulates of lagoon waste or soil offer estrogenic hormones protection from microbial degradation and increase their longevities in the soil or manure storage facilities? TH 2) If estrogenic hormones are bound to dissolved organic carbon or particulates and not easily accessible to microbial processes, then hormone in the presence of high amounts of dissolved organic carbon or particulates will increase persistence in the environment compared to free aqueous hormone. RQ 3) Do estrogen conjugates (i.e. polar metabolites) promote the mobility or persistence of estrogens in soil? TH 3) If conjugated hormones are more water soluble than their de-conjugated forms, then they are also more mobile in the soil and can potentially migrate to areas of reduced microbial degradation. RQ 4) Do estrogens and their conjugates from lagoon waste applied to fields move into groundwater and into surface waters? TH 4) If the mobility of estrogens in soil is dependant on hormone conjugation, the presence of dissolved organic carbon or particulates, and the low retention time in upper soil layers, then application of manures on tile drainage fields will increase the presence of hormone/hormone conjugates in ground and surface waters. To answer these research questions and test the hypotheses, the following experimental objectives were formed: Objective 1 – Determine the sorption characteristics of estradiol, estradiol glucuronide and sulfate conjugates with particulates originating from natural organic matter from soil and from animal manure lagoon liquors (laboratory); Objective 2 – Determine the degradation of the parent compounds estradiol, estradiol glucuronide and sulfate conjugates in the presence of these particulates and soil (laboratory); and Objective 3 – Determine how land application strategies (surface applied vs. injection) for lagoon liquor influence the fate and transport of estradiol in a field setting.
1b. Approach (from AD-416)
Objective 1: Degradation of hormones is mediated primarily by biological activity and can lead to misinterpretation of observed physical/chemical soil absorption processes. In order to identify these processes experimentally, the experiments for this objective will be conducted under sterile conditions. Four hormone concentrations and nine time points will be used and sampled. Qualititative and quantitative assessment of hormone degradation and soil absorption will be made by chromatographic and spectrometric methods, and absorption curves and models can then be determined. Objective 2: High and low organic soils will be spiked with radioactive hormone conjugates with and without lagoon waste under aerobic/anaerobic and sterile/non-sterile conditions to measure biological degradation in soil. Mass balances, qualitative assessment of hormone fate, and separation of hormone compartments into the various soil fractions under these conditions will be determined. Objective 3: Well-defined field-scale Discovery Farms will be used to measure the transport of hormones when injected into soil and followed by uncontrolled and controlled drainage (rainfall). Both well water and soil extracts will be analyzed for hormone content across two growing seasons.
3. Progress Report
Steroid hormones are potent agonists of estrogen and androgen receptors, and when exposed exogenously can alter an organism’s normal hormone balance. This can lead to reproductive abnormalities, such as feminization of male organisms or vice versa. A major source of the introduction of steroid hormones into the environment is from animal waste, especially from concentrated animal feeding operations. Despite the constant detection of hormones in the environment, our previous laboratory data suggested that these hormones should not be mobile in the environment, and that movement into groundwater must be governed by more than simple rainfall transport from upper soil layers to lower. Therefore, mechanisms need to be discerned as to how hormones enter and persist in the environment. We have discovered two mechanisms that may help to explain hormone transport. 1. Flushed swine manure wastewaters are composed of two compartments, i.e. the dissolved aqueous compartment and the particulate compartment. We developed a method to quantify the particulate compartment based on selective filtration. We then conducted studies with radiolabeled estradiol (the principle female steroid hormone) spiked into flushed swine manure wastewater to measure the amount of the hormone in each of the two compartments. The amount present in the particulate-bound (colloidal) compartment was considered environmentally mobile. Upwards of 20% of the applied estradiol dose was associated with colloid particles. Results have provided a putative mechanism of for the introduction of this endocrine disruptor into the environment. A question to be addressed in the future is whether these mobile, estradiol-ladened colloid particles ever release the estradiol, or if the estradiol-colloid complex is biologically active. 2. Since steroid hormones are excreted to a large extent as water-soluble conjugates of sugar and acid molecules, this has been hypothesized as the means by which these hormones move in the environment. We also conducted laboratory soil/water batch studies to determine contribution of conjugates in the transport of steroid hormones. Preliminary data showed that a window exists for estradiol sulfate, an endogenous urinary metabolite of estradiol, to move through the environment before soil bacteria degrade it. However, degradation did not result in release of parent compound over the 2 week length of the study. Results indicate that whereas parent compound is poorly transported in soil/water systems, a mechanism of conjugate transport has been discovered which may explain the long-range transport of these endocrine disrupting compounds in the environment, although, under the conditions studied, does not explain how the potent parent compound itself is frequently detected in the environment. These results are very significant to understand the fate and transport behavior of these compounds, and provide the basis needed to devise means to reduce their environmental release.