Linda S. Lee

Agricultural Contributions of Antimicrobials and Hormones on Soil and Water Quality

Detection of many emerging chemicals of concern, including antimicrobials and steroid hormones, in the environment has increased in the past decade with the advancement of analytical techniques. There are several potential sources of these inputs, including municipal wastewater discharge, municipal biosolids, pharmaceutical production, and agriculture‐related activities. However, the heavy use of antibiotics in the livestock industry and the dramatic shift in recent years toward more highly concentrated animal feeding operations (CAFOs), thus a concomitant increase in the volume of animal wastes per unit of land, has drawn attention to the role of animal waste‐borne antimicrobials, antibiotic‐resistant bacteria, and steroid hormones on ecosystem and human health. Antimicrobials, although frequently detected, are typically present in water at concentrations in orders of magnitude below what would be considered inhibitory to most biota. Most antibiotics have a high affinity for soil and sediment, thus residual soil concentrations are usually much higher than noted in water but still often below concentrations of concern. The focal point with antibiotic use in animal production is the development of antibiotic‐resistant bacteria. Although there is a growing body of evidence of the presence of numerous antibiotic‐resistant genes in animal wastes, in soils where wastes are land applied, and in water bodies receiving runoff from manure‐amended fields or discharges from aquacultures, conclusive evidence of animal‐derived antibiotic‐resistant pathogens compromising human health is lacking. In contrast to antibiotics, hormones and related chemicals can cause significant biological responses at very low concentrations. CAFO discharges will include a variety of estrogens, natural and synthetic androgens and progesterones, and phytoestrogens associated with animal feed. Measurable concentrations of many of these hormones have been detected in soil, and ground and surface waters receiving runoff from fields fertilized with animal manure and downstream from farm animal operations. Overall, hormones appear to be moderately to highly sorbed and to dissipate quickly in an aerobic soil environment, but quantitative information on hormone persistence in manure‐applied fields and subsequent effects of hormone loads from CAFOs to the aquatic environment is lacking. Research directed toward evaluating the facilitated transport processes with regards to antimicrobial and hormone inputs from manure‐amended fields is in its infancy. With the advances in analytical techniques and what has already been learned with regards to transport of nutrients (nitrogen, phosphorus, and carbon) and pesticides from agricultural fields, a reasonable evaluation of CAFOs and associated activities (land application of animal wastes) should be forthcoming in the next decade. Meanwhile, implementation of management practices that optimize reduction in already regulated nutrient releases from CAFOs should also help to minimize the release of antimicrobials and hormones.

Hormone Discharges from a Midwest Tile-Drained Agroecosystem Receiving Animal Wastes

Manure is increasingly being viewed as a threat to aquatic ecosystems due to the introduction of natural and synthetic hormones from land application to agricultural fields. In the Midwestern United States, where most agricultural fields are tile-drained, there is little known about hormone release from fields receiving animal wastes. To this end, seven sampling stations (four in subsurface tile drains and three in the receiving ditch network) were installed at a Midwest farm where various types of animal wastes (beef, dairy, and poultry lagoon effluent, dairy solids, and subsurface injection of swine manure) are applied to agricultural fields. Water flow was continuously monitored and samples were collected for hormone analysis during storm events and baseline flow for a 15 month study period. The compounds analyzed included the natural hormones 17α- and 17β-estradiol, estrone, estriol, testosterone, and androstenedione and the synthetic androgens 17α- and 17β-trenbolone and trendione. Hormones were detected in at least 64% of the samples collected at each station, with estrone being detected the most frequently and estriol the least. Testosterone and androstendione were detected more frequently than synthetic androgens, which were detected in fewer than 15% of samples. Hormone concentrations in subsurface tile drains increased during effluent irrigation and storm events. Hormones also appeared to persist over the winter, with increased concentrations coinciding with early thaws and snowmelt from fields amended with manure solids. The highest concentration of synthetic androgens (168 ng/L) observed coincided with a snowmelt. The highest concentrations of hormones in the ditch waters (87 ng/L for total estrogens and 52 ng/L for natural androgens) were observed in June, which coincides with the early life stage development period of many aquatic species in the Midwest.

Sorption and degradation in soils of veterinary ionophore antibiotics: Monensin and lasalocid

Monensin and lasalocid are polyether ionophores commonly used in the beef and poultry industries for the prevention of coccidial infections and promotion of growth. These ionophores can exhibit higher toxicity than many other antibiotics; thus, evaluating their fate in the environments associated with concentrated feed operations is important. Sorption of monensin and lasalocid was measured in eight soils of varying physiochemical composition. Organic carbon-normalized sorption coefficients (log Koc) ranged from 2.1 to 3.8 for monensin and from 2.9 to 4.2 for lasalocid and were inversely correlated to equilibrium soil-solution pH. Degradation of lasalocid and monensin in two contrasting soils with and without manure amendment was measured in moist soils at 23 degrees C and 0.03 MPa moisture potential. The half-life of both compounds in the fresh nonsterile soils was less than 4 d, for which monensin degraded slightly faster than lasalocid. Fresh liquid manure amendments did not significantly alter degradation of either compound. Based on parallel 60Co-sterilized soil experiments, some abiotic degradation of monensin was apparent, whereas lasalocid only degraded in the presence of microbes. Analysis of beef-derived lagoon effluent used for irrigation confirmed that monensin can be present at low-ppb to low-ppm concentrations in the aqueous and suspended solids fractions, respectively; however, subsequent analysis of drainage water in a nearby ditch suggested that attenuation by soil after land application will greatly reduce the amount entering surface waters.

Ciprofloxacin sorption by dissolved organic carbon from reference and bio-waste materials

Amphoteric pharmaceuticals are released to the environment in treated wastewater, medicated aquaculture feed, and through land disposal of livestock wastes and municipal biosolids. These wastes are rich in dissolved organic carbon (DOC), which may bind and potentially enhance pharmaceutical transport in the environment. To assess this potential, the antimicrobial ciprofloxacin (CIP) was chosen as a model amphoteric pharmaceutical to quantify DOC-water sorption coefficients (K(doc), L kg(-1)DOC) for four reference humic materials with varying chemical properties, digested and undigested municipal biosolids, treated municipal wastewater, and beef lagoon wastewater. For reference humic materials, the predominance of sorption by cation exchange was exemplified by decreasing sorption with increasing pH and ionic strength, as well as highly predictable relationships between the sorbed CIP concentration normalized to the DOC charge versus the free aqueous concentration of the CIP cation. In contrast to humic materials, however, sorption to DOC from digested biosolids was low and showed no pH dependence, suggesting that additional weaker mechanisms contributed to binding interactions. CIP had no quantifiable affinity for DOC from undigested biosolids, treated wastewater, or beef lagoon wastewater. With K(doc) values of approximately 10(4)L kg(-1) estimated for CIP concentrations reported in biosolids ( approximately 20-60 microg kg(-1)wwt), the DOC measured for digested biosolids in this study (40 mg L(-1)) could increase CIP mobility by up to approximately 15%, thus DOC-facilitated transport should be considered in environmental fate assessments for amphoteric pharmaceuticals. However, the potential for waste-derived DOC to enhance CIP transport would have been greatly overestimated using information derived only from reference humic materials.

Stereoselective sorption by agricultural soils and liquid-liquid partitioning of trenbolone (17α and 17β) and trendione.

Trenbolone acetate (TBA) is a synthetic anabolic hormone used for growth promotion in beef cattle, which excrete primarily 17alpha-trenbolone along with small amounts of 17beta-trenbolone and trendione. To aid in predicting transport of manure-borne TBA metabolites, multiconcentration sorption isotherms for 17alpha- and 17beta-trenbolone and trendione were generated with five autoclaved-sterilized soils that represented a range in soil properties. Hormone concentrations were measured independently in solution and soil phases, and quantified using liquid chromatography with electrospray mass spectrometry. In addition, partition coefficients between apolar hexane and water (K(hw)) and bipolar octanol and water (K(ow)) were measured for the three androgens to better ascertain the mechanisms that may be responsible for the sorption differences observed between isomers. In all five soils, trendione sorbed the most, and 17alpha- and 17beta-trenbolone isomers exhibited different sorption magnitudes. 17beta- trenbolone consistently sorbed a factor of 2 more than 17alpha-trenbolone. For all three androgens, sorption is proportional to the soil organic carbon (OC) content with average log OC-normalized distribution coefficients (log K(oc), L/kg OC) of 2.77 +/- 0.12 for 17alpha-trenbolone, 3.08 +/- 0.1 for 17beta-trenbolone and 3.38 +/- 0.19 for trendione, which suggests the dominance of hydrophobic partitioning. However, differences in K(hw) values between 17alpha- and 17beta-trenbolone were small indicating differences are not simply due to differences in aqueous activity. In contrast, similarly different K(ow) and K(oc) values for the two isomers indicate the likely contribution of H-bonding to stereoselective sorption.

Sorption of tylosin A, D, and A‐aldol and degradation of tylosin a in soils

Heightened concerns regarding the potential impact on soil and water quality of veterinary antibiotics warrant a better understanding of the environmental fate of antibiotics in soil. Sorption of the macrolides tylosin A (TA), tylosin D, and TA-aldol was measured in several soils and evaluated with respect to soil pH, organic matter content, percentage clay, and cation-exchange capacity (CEC). Tylosin and related compounds exhibit similar sorption characteristics and generally are strongly sorbed, with sorption being well and positively correlated to surface area, clay content, and CEC. Sorption coefficients normalized by CEC were within a narrow range (10(4.1+/-0.21 L/molc) for all but one soil; however, good extraction recoveries with only methanol for most soils suggested that hydrophobic processes also contribute to sorption. Aerobic degradation of TA over a three-month period in two freshly collected agricultural soils and 60Co-irradiated soils indicated that both abiotic and microbial processes contribute to TA transformation. The abiotic process was much slower and dominated in the first two weeks, followed by rapid microbial degradation within 3 d. Three primary degradation products were identified using liquid chromatography with full-scan mass spectrometry, with unconfirmed identifications of TA having the aldehyde group oxidized to an acid (m/z = 932) in both soils and tyslosin B (m/z = 772) as well as tylosin B having the aldehyde group oxidized to an acid (m/z = 788) in the sandy soil.

Soil temperature and moisture effects on the persistence of synthetic androgen 17α-trenbolone, 17β-trenbolone and trendione

Trenbolone acetate (TBA) is a synthetic androgenic steroid hormone administered as a subcutaneous implant for growth promotion in beef cattle. The primary metabolite excreted in manure from implanted cattle is 17alpha-trenbolone with lesser amounts of 17beta-trenbolone and trendione also present. At 22 degrees C and favorable moisture conditions in a controlled laboratory environment, trenbolone degrades to trendione in a few hours; however, these conditions are often not what exist in the field. Therefore, aerobic degradation rates of 17alpha-trenbolone, 17beta-trenbolone and trendione were determined in a sandy soil and silty clay loam under a range of temperature and water availability combinations that may be expected in the field. A first-order exponential decay model was used to estimate rates and generally resulted in good model fits to the data. Degradation rates decreased with decreasing water availability (i.e., more negative soil matric potential) and decreasing temperature. However, when water availability was substantially reduced (-1.0MPa), hotter temperatures (35 degrees C) significantly reduced trenbolone degradation rates. Once temperature was low enough to limit microbial activity, no further changes were observed with decreasing matric potential. Trendione also exhibited similar moisture and temperature dependent degradation, but persisted longer than the parent trenbolone. The latter was discussed in light of extracellular versus intracellular enzymatic degradation and sorption. Half lives at colder temperatures (5 degrees C) even under favorable moisture conditions were 2-3d for the trenbolone isomers and approached 10d for trendione.

Assessing impacts of land-applied manure from concentrated animal feeding operations on fish populations and communities.

Concentrated animal feeding operation (CAFO) manure is a cost-effective fertilizer. In the Midwest, networks of subsurface tile-drains expedite transport of animal hormones and nutrients from land-applied CAFO manure to adjacent waterways. The objective of this study was to evaluate impacts of land-applied CAFO manure on fish populations and communities. Water chemistry including hormone, pesticide, and nutrient concentrations was characterized from study sites along with fish assemblage structure, growth, and endocrine disruption assessed in selected fish species. Although most CAFO water samples had hormone concentrations <1 ng/L, equivalent concentrations for 17β-E2 and 17α-TB peaked at >30 ng/L each during the period of spawning, hatching, and development for resident fishes. CAFO sites had lower fish species richness, and fishes exhibited faster somatic growth and lower reproductive condition compared to individuals from the reference site. Fathead minnows (Pimephales promelas) exposed to CAFO ditchwater during early developmental stages exhibited significantly skewed sex ratios toward males. Maximum observed hormone concentrations were well above the lowest observable effect concentrations for these hormones; however, complexities at the field scale make it difficult to directly relate hormone concentration and impacts on fish. Complicating factors include the consistent presence of pesticides and nutrients, and the difference in temperature and stream architecture of the CAFO-impacted ditches compared to the reference site (e.g., channelization, bottom substrate, shallow pools, and riparian cover).

Aerobic Soil Biodegradation of 8:2 Fluorotelomer Stearate Monoester

A laboratory investigation on the biotransformation of 8:2 fluorotelomer stearate monoester (8:2 FTS) in aerobic soils was conducted by monitoring the loss of 8:2 FTS, production of 8:2 fluorotelomer alcohol (8:2 FTOH) and stearic acid, which would be released by cleavage of the ester linkage, and subsequent degradation products from FTOH for 80 d. Soil microcosms were extracted with ethyl acetate followed by two heated 90/10 v/v acetonitrile/200 mM NaOH extractions. 8:2 FTS was degraded with an observed half-life (t(1/2)) of 10.3 d. The rate of 8:2 FTS biotransformation substantially decreased after 20 d with 22% of 8:2 FTS still remaining on day 80. No biotransformation of 8:2 FTS occurred in autoclaved soil controls, which remained sterile with 102 ± 6% recovery, through day 20. 8:2 FTOH was generated with cleavage of the ester linkage of 8:2 FTS followed by a rapid decline (t(1/2) ~ 2 d) due to subsequent biodegradation. All the expected 8:2 FTOH degradation products were detected including 8:2 fluorotelomer unsaturated and saturated carboxylic acids, 7:2s FTOH, 7:3 acid, and three perfluoroalkyl carboxylic acids with the most prominent being perfluorooctanoic acid (PFOA). PFOA consistently increased over time reaching 1.7 ± 0.07 mol % by day 80. Although cleavage of the ester linkage was evidenced by 8:2 FTOH production, an associated trend in stearic acid concentrations was not clear because of complex fatty acid metabolism dynamics in soil. Further analysis of mass spectrometry fragmentation patterns and chromatography supported the conclusion that hydrolysis of the ester linkage is predominantly the first step in the degradation of 8:2 FTS with the ultimate formation of terminal products such as PFOA.

Evaluating stereoselective sorption by soils of 17α-estradiol and 17β-estradiol.

The application of manure and biosolids onto agricultural land has increased the risk of estrogenic exposure to aquatic systems. Both αE2 and βE2 have been routinely detected in surface and ground waters with higher concentrations reported near concentrated animal feeding operations and agricultural fields. Although movement through the soil to a water body is highly dependent on hormone-soil interactions, to date, only the interaction of βE2 with soils has been characterized despite αE2 often being the more common form excreted by livestock such as beef cattle and dairy. In predicting the transport of estradiol, sorption characteristics for the stereoisomers have been assumed to be the same. To evaluate this assumption, sorption of αE2 and βE2 was measured on seven surface soils representing a range in soil properties. Soils were autoclave-sterilized to minimize loss due to biotransformation, and both solution and soil phase concentrations were measured. Overall, E2 sorption is best correlated to soil organic carbon (OC) with an average log OC-normalized distribution coefficient (logKoc, L kgoc(-1)) of 2.97±0.13 for αE2 and 3.14±0.16 for βE2 with βE2 consistently exhibiting higher sorption than αE2 with the highest β/α sorption ratio of 1.9. Assuming that the two isomers sorb the same is not a conservative decision making approach. The lower sorption affinity of αE2 increases the likelihood that it will be leached from agricultural fields.