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Detection, Occurrence, and Fate of
Emerging Contaminants in
Agricultural Environments
Daniel D. Snow1, Shannon L. Bartelt-Hunt2, Samuel E. Saunders3 and David A. Cassada4 ‘Emerging' contaminants are any synthetic or steroid hormones and other endocrine-disrupting naturally-occurring chemical or microorganism not commonly monitored in the environment. These "new" disinfection byproducts, new pesticides and pesticide contaminants are of interest because of their potential to metabolites, and naturally-occurring algal toxins. Whether enter the environment and cause known or suspected or not a chemical or other contaminant becomes an adverse ecological or human health effects. Release of ‘emerging' contaminant depends on the availability of emerging chemical or biological contaminants to the analytical methods for its detection in the environment. environment has occurred for quite some time, but methods The analysis of these typically polar contaminants in environmental matrices (water, wastewater, soils and concentrations have only recently become available. sediments) is particularly challenging because of the low Recent studies of emerging contaminants are detection limits required, the complex nature of the directed at understanding the environmental fate and effects samples, and difficulty in separating these compounds from of surfactants, antibiotics and other pharmaceuticals, interferences. New extraction and clean-up techniques, coupled with improvements in instrumental technologies provide the needed sensitivity and specificity for accurate 1Assistant Professor and Director, School of Natural Resources, 202 Water Science Laboratory, University of Nebraska-Lincoln, Lincoln, NE 68583-0844; Tel. 402-472-7539; Fax. 402-472-9599; email: Methods utilizing mass spectrometry are fast 2Assistant Professor, Department of Civil Engineering, 203B Peter Kiewit Institute, University of Nebraska-Lincoln, Omaha, NE 68182- 0178, Tel. (402) 554-3868; Fax. (402) 554-3288; email: becoming the standard for analysis of emerging sbartelt2@unl.edu 3 contaminants. Liquid chromatography with mass Graduate Research Assistant, Department of Civil Engineering, University of Nebraska-Lincoln Department of Civil Engineering, 203B Peter Kiewit Institute, University of Nebraska-Lincoln Omaha, NE spectrometry (LC/MS) is especially useful for detection of 68182-0178, Tel. (402) 554-3868; Fax. (402) 554-3288; email: 4Chemist, School of Natural Resources, 202 Water Sciences Laboratory, these compounds because of their chemical properties. University of Nebraska-Lincoln, Lincoln, NE 68583-0844; Tel. 402- 472-7539; Fax. 402-472-9599; email: dcassada1@unl.edu When gas chromatography/mass spectrometry (GC/MS) is Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
used for compound detection, these contaminants must Agricultural systems utilize waste management practices often be quantitatively derivatized and converted to a more that may contribute to the potential for release of associated volatile substance prior to analysis. With both GC and LC contaminants to the environment such as spray irrigation technologies, as well as more conventional techniques, (Goel et al. 2006), liquid and biosolids application to crop selective and quantitative extraction is paramount for fields (Kinney et al. 2006), runoff and subsurface transport accurate and sensitive detection in environmental samples. from crops and confined animal-feeding operations Of particular interest and importance are (CAFOs) (Davis et al. 2006; Jindal et al. 2006), animal emerging contaminants in agricultural systems, which can waste and carcass disposal (i.e. landfilling and composting) be responsible for significant contaminant loadings to the (Rapp et al. 2006), and aquaculture (Cabello 2006). environment. For example, veterinary antibiotics account The objective of this paper is to review the for about half of all antibiotics used in the United States literature published in 2006 evaluating the detection, fate, (Qiang et al. 2006). The environmental occurrence and and occurrence of emerging contaminants, with a particular effects of veterinary pharmaceuticals such as antibiotics focus on emerging contaminants in agricultural systems. and steroid hormones is of particular concern in agricultural Relevant contaminants are EDCs (particularly hormones areas with a high population of livestock. Antibiotics have been widely-used for disease prevention, treatment and for pharmaceuticals associated with wastewater, and prions. growth promotion in livestock since the 1960s. The Studies on pesticides and flame retardants are not reviewed potential for proliferation of antibiotic resistant bacteria unless they were evaluated in the same study. because of this practice is unknown but undergoing Analytical Methods for Emerging Contaminants
investigation in many areas. Natural steroid hormones are Hormones and Anabolic Steroids. Methods for
excreted in livestock waste, while both synthetic and analysis of natural and synthetic steroid hormones continue natural hormones are used as growth promoters particularly to be developed and have been the subject of numerous in the cattle industry. Because hormones are integrally investigations over the past year. This group of emerging linked to reproductive functions, their occurrence and contaminants are generally slightly soluble in water, impact to aquatic organisms such as fish are also under lipophilic, and highly sorptive. Efficient extraction and purification methods are required in order to separate these Important agricultural contaminants include compounds from interferences and complex matrices to EDCs and antibiotics, as well as antibiotic resistance genes which they can become tightly bound. Detection limits and prions, which garnered increased attention in 2006. required for accurate environmental measurements of Many of these contaminants, including prions and steroid hormones are in the low ng/L to even pg/L range, veterinary antibiotics, are unique to agriculture. Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
and typically require the use of technologically advanced YES assay indicated that 27 out of 29 samples showed an instrumentation. In addition to the free hormones, steroid estrogenic response higher than 10%, and samples from conjugates or metabolites may also be detected and stations located in inner coastal waters showed higher quantified in order to fully assess the environmental fate of estrogenic activities than stations located farther from steroid hormones and steroid-like growth promoters shore. Assay results and instrumental analysis showed associated with concentrated animal feeding operations. significant differences, possibly due to the presence of anti- Because of the complexity of the sample matrices and the estrogenic compounds and/or the estrogenic activity of difficulties associated with instrumental methods, some unidentified contaminants. Based on the results, however, studies use bioassays to screen samples and identify those the main contributors to the overall estrogenic activity were with estrogenic activity prior to analysis with more believed to be synthetic and natural hormones. conventional techniques. Beck and Radke (2006) measured steroids, Beck et al. (2006) utilized a yeast estrogenic sterols and inorganics in a wastewater treatment plant assay (YES) to screen samples from coastal waters of the aeration system to determine the potential for atmospheric Baltic Sea impacted by sewage treatment plant effluent. exposure. Sterols are steroid alcohols and include Fifty liter samples were extracted in the field with 4-gram cholesterol and its metabolites such as coprostanol, which polymeric solid phase extraction (SPE) cartridges from five constitute as much as 60% of sterols in human waste. different locations in northeastern Germany. Cartridges Wastewater and aerosol samples were collected from a were stored for up to eight days, washed and eluted with treatment plant in Bayreuth, Germany. Fifty milliliter 200 milliliters of 80/20 acetone/methanol, purified on wastewater samples were filtered, spiked with internal silica, and concentrated to a final volume of 400 L. The standard, and passed through C18 and aminopropyl SPE extract was split with one portion used for the YES assay cartridges in tandem. Steroids and sterols were eluted from and another analyzed by LC-tandem mass spectrometry. both cartridges using a mixture of dichloromethane and The YES assay uses yeast cells transcripted with human methanol. The eluate was evaporated, redissolved in estrogen receptor genes and the production of b- hexane, and compounds were derivatized using Sylon BFT galactosidase in these cells when exposed to estrogenic bis(trimethylsilyl) substances. A chromogenic substrate is measured trifluroacetamide and trimethylchlorosilane). Derivatives colorimetrically. Standards were prepared from estradiol were separated and quantified using gas chromatography- and the results expressed as estradiol equivalents. LC- mass spectrometry (GC/MS). Recovery from wastewater tandem MS analysis utilized a reverse phase column and samples fortified with known concentrations of target triple quadrupole mass spectrometer, electrospray compounds averaged between 34 and 99%. Aerosol ionization in both negative and positive ion modes. The particles were fortified with internal standards, extracted Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
with a mixture of hexane and acetone, purified with silica and methanol and eluted with methanol. The two gel, derivatized and analyzed as the wastewater extracts. trenbolone metabolites were analyzed by HPLC with Wastewater concentrations of coprostanol and cholesterol fluorescence detection and by derivatization followed by ranged from to 30 to 180 µg/L in waste water and to 400– GC/MS. Androgenic activity indicated by the CV-1 assay 5000 pg/m3 in aerosol particles. Estrone and 17 -estradiol was indicated in most samples collected near the cattle were about 165 pg /m3 in aerosol particles, and 0.5 to 4.5 feeding operation. Trace levels of both 17 - and 17 - µg/L in wastewater, with no clear correlation between trenbolone were detected in several samples by HPLC and aerosol and wastewater concentrations. confirmed by GC/MS. Androgenic activity could not be Chen et al (2006) studied leaching of 17 - directly correlated to trenbolone metabolite levels. estradiol and estrone, as well as two alkylphenols, and Farre et al (2006) evaluated four commercially- compared different concentration methods for analyzing available immunoassays (ELISA) for detection of estrogens these compounds in small effluent volumes from soil in wastewater, river water and groundwater and compared columns. Lyophilization, solid phase extraction, and results to analysis by LC tandem mass spectrometry with a solvent extraction were evaluated for concentrating these triple quadrupole instrument. The extraction efficiency of compounds as was liquid chromatography mass two concentration methods was evaluated– one using a C18 spectrometry and GC/MS both with ion trap SPE with clean-up using aminopropyl (NH2) cartridge and instrumentation. LC-MS-MS with lyophilization provided another with a C18 SPE cartridge. Recoveries from the best recovery and precision in fortified samples of two fortified reagent water were slightly better for C18 estrogen hormones, while solvent extraction and GC/MS extraction alone, though both methods provided method were preferred for alkylphenol analysis. Soil satisfactory results. ELISA results for the extracts column samples were extracted with acetone after leaching correlated well with the results of the LC tandem MS experiments to measure sorbed fractions. results for all sample types in both fortified and unfortified Durhan et al (2006) screened and analyzed samples indicating that ELISA can be a useful screening samples of river water adjacent to a cattle feeding operation tool in environmental studies of estrogens. for metabolites of the anabolic steroid trenbolone. Samples Steroids in water samples are typically were screen for androgenic activity using a CV-1 assay concentrated using commercially-available solid phase with cells transfected with human androgen receptor vector. extraction (SPE) cartridges. Garcia-Prieto et al. (2006) Luminescence of the culture medium was calibrated using describe a method for pre-concentrating estrogens SPE solutions of 17 - and 17 -trenbolone. Filtered 1-liter water cartridges modified with hemimicelles and admicelles of samples were extracted using C18 reverse phase SPE two surfactants - sodium dodecyl sulfate (SDS) and cartridges. Cartridges were rinsed with a mixture of water cetyltrimethyl ammonium bromide. The use of more Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
selective sorbents may simplify sample handling and extraction (SBSE) coupled with "dual derivatization" reduce potential for co-extracting interferences. The GC/MS analysis. Polydimethylsiloxane coated stir bar surfactant-modified SPE cartridges provided quantitative extraction is a semi-automated method for concentrating retention of estrogenic steroids for sample volumes up to organics from aqueous samples prior to analysis by thermal 0.5 liters and could be eluted with as little as 2 milliliters of desorption gas chromatography. The authors improved the methanol. Detection limits for the pre-concentration extraction efficiency of this method by the addition of method using HPLC with fluorescence and UV detection potassium carbonate and acetic acid anhydride directly to a were estimated at near 0.02 to 0.10 g/L. Samples of raw 10 milliliter water sample immediately prior to extraction. and treated wastewater and from effluent-impacted river In situ acylation and extraction of estradiol in water was water from several locations in Spain were analyzed by this performed by stirring the sample at 120oC over 120 method and traces of 17 -estradiol, estrone and ethynyl minutes. The sorbent-coated stir-bar was removed form the estridiol were found at concentrations up to 0.20 g/L. sample, dried, and placed in a glass capillary containing Gomez et al. (2006) describe a method for BSTFA (N,o-bis(trimethylsilyl) trifluoroacetamide). The concentrating 17 -estradiol, estrone, ethynylestradiol, and capillary containing the stir bar was heated in a thermal desethylstilbestrol from wastewater using an automated off- desorption inlet on a GC/MS system where the second line extraction system with polymeric solid phase derivatization reaction occurred as the compound was extraction cartridges. An ion trap GC/MS was used in desorbed and swept onto the column. Using 13C3-labeled selected reaction monitoring mode and steroids were estradiol as the internal standard, detection limits were analyzed without derivatization. Sample volumes ranged estimated at 0.5 ng/L, compared to 100 ng/L for from 100 to 200 milliliters, cartridges were washed with underivatized estradiol. The method was used to analyze reagent water, eluted with 8 milliliters of ethyl acetate and samples of river water where 17 -estradiol was detected at the eluate evaporated to 500 L. Large volume (8 L) injection and MS/MS detection reportedly permitted Swartz et al. (2006) analyzed samples of ground detection limits in the range of 2-20 ng/L. Samples of raw water impacted by septic system leachate on Cape Cod, and treated effluent were collected from two wastewater MA. Samples were analyzed for estrogens and conjugates, treatment plants over a 12-month period and analyzed by as well as several other types of wastewater contaminants the method. Desethylstilbestrol, estrone and bisphenol A including caffeine, EDTA, and alkylphenols by previously were detected at concentrations up to 1.7 g/L and published methods using isotope dilution GC/MS and appeared to be consistent with previous investigations. LC/MS. Caffeine and its dealkalated metabolites were Kawaguchi et al. (2006) describe a novel method analyzed by HPLC-florescence. for analysis of 17 -estradiol in water using stir bar sorptive Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
Concentrations of estrogens were highest in limits ranged from 20-350 ng/L for fortified lake water. samples collected from the suboxic to anoxic portion of the Samples of tap water, lake water and treated sewage were plume nearest the source, and were similar to those analyzed by the method. observed in samples collected from the septic systems. Steroids are relatively difficult to ionize for Alkylphenols were much higher, while caffeine and its detection by LC/MS, though new ionization mechanisms metabolite were much lower in ground water than the may help increase ionization efficiency. Yamamoto et al. septic system. Estradiol sulfate was the only conjugate (2006) compared the sensitivity of LC tandem MS using detected in the ground water, and estimated to be only a electrospray ionization (ESI) with atmospheric pressure minor fraction of the estrogens present. photoionization (APPI) for analysis of free and conjugated Selective extraction methods can be used to forms of androgenic and estrogenic steroid hormones. enhance sensitivity of steroid analysis using electrospray Comparison of transition ion intensities indicated that APPI ionization LC/MS. Watabe et al. (2006) describe the produce a much higher signal for detection of free development and application of molecular imprint hormones, especially testosterone and androstenedione, polymers (MIP) for extraction of 17 -estradiol in river though electrospray ionization was better for steroid water. Three types of MIP sorbents were prepared with and conjugates. River and estuarine water samples (2-L) without a surface modifier and used to pre-concentrate 17 - impacted by sewage effluent were extracted using estradiol for HPLC and LC/MS analysis. The surface graphitized carbon SPE cartridges and free steroids eluted modified MIP provided the highest level of selectivity for with a mixture of dichloromethane and methanol, while 17 -estradiol. Based on the variability of results for spiked conjugated forms of steroids were eluted with 5mM river water, detection limits for 17 -estradiol was estimated tetramethylammonium chloride in dichloromethane and methanol. Florisil cartridges were used for cleanup of free Wen et al. (2006) describe a method using in- steroid extracts. Deuterated internal standards were added tube solid phase microextraction (SPME) with HPLC and and eluates evaporated to dryness and redissolved in 200 fluorescence detection for automated analysis of 17 - µL of mobile phase. Method detection limits for 10 free estradiol, estriol, ethynyl estradriol, and bisphenol A. An steroids by APPI and 6 conjugated steroids ranged from acrylamide polymer monolith synthesized in a length of 0.06 ng/L for testosterone to 12 ng/L for estriol 3- tubing was used as the sorbent. Samples were filtered and glucoronide. Androgenic steroid concentrations were lower adjusted to pH 6 immediately prior to injection into the than estrogens. Estrone, estrone 3-sulfate, and SPME tube. Detection limits in the range of 6-100 ng/L androstenedione were most frequently detected in nearly all were estimated from the variability of water samples samples at maximum concentrations of 51, 5.1, and 6.4 fortified with the target compounds, and quantification ng/L, respectively. Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
Yang et al. (2006) describe a fully automated out that the savings in extraction time, cost, and waste method for analysis of steroid hormones and alkylphenols coupled with the decrease in matrix effects makes the using immersion SPME on-fiber derivatization GC/MS. SPME method a viable technique in the analysis of Trimethylsilyl derivatives of steroids and akylphenols wastewater for sulfonamide antibiotics. were prepared by immersing a polyacrylate fiber in a vial Batt et al. (2006) studied the impact of a confined of derivatization reagent immediately prior to immersing animal feeding operation (CAFO) in Idaho on groundwater and extraction of a sample on a SPME autosampler. from six nearby wells. Two sulfonamide antibiotics, Extraction and desorption conditions were optimized to sulfamethazine and sulfadimethoxine, were found in all maximize the response of all target compounds. Detection wells at concentrations up to 0.22 µg/l for sulfamethazine limits ranged from 4 to 413 ng/L. The method was used to and 0.068 µg/l for sulfadimethoxine using SPE with quantify octylphenol, nonylphenol, diethylstilbestrol, LC/MS/MS analysis. Elevated nitrate concencentrations estrone, 17 -estradiol, and testosterone in river water and above the maximum contaminant level (MCL) set by the US Environmental Protection Agency were observed in Pharmaceuticals and Antibiotics. Abuin et al. (2006)
three of the samples while another sample contained an describe a method for the analysis of five macrolides in elevated ammonium concentration. Isotopic ratio mass river water using LC/MS and LC/MS/MS with solid phase spectrometric analysis of the samples indicated δ15N values extraction (SPE) concentration via Oasis HLB cartridges. consistent with an animal waste source. Detection limits for each macrolide were about 1 ng/L for a Brown et al. (2006) investigated the efficiency of 250 ml sample with recoveries at spike levels of 25 and 125 a wastewater treatment plant (WWTP) in New Mexico to ng/L between 85-115% for all analytes except for process antibiotic containing effluent from hospitals, azithromycin ( 70%). Comparison of results from single residential facilities, and dairies, and in municipal quadrupole and triple quadrupole analyses is presented. wastewater. Eleven antibiotics from several classes of Balakrishnan et al. (2006) use a solid phase compounds were analyzed by SPE LC/MS/MS and microextraction (SPME) method to minimize matrix effects evaluated for concentrations in the raw wastewater, treated from wastewater collected from sewage treatment plants in effluent, and river water both upstream and downstream of the analysis of sulfonamide antibiotics. Optimized the WWTP discharge. Detection of at least one antibiotic conditions using a carbowax/divinylbenzene SPME fiber was observed in 58% of the raw effluent samples while for sorption time, solution salinity, pH, and desorption time 25% had three or more antibiotics present. High levels of are presented. While detection limits for the SPME method ofloxacin (up to 35,500 ng/L) found in hospital and were considerably higher (1000 to 20000 times) than those residential effluents could be of concern. Concentrations of obtained from a comparable SPE method, the authors point antibiotics at the WWTP were reduced by 20% to 77% in Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
the treatment process. No antibiotics were observed in A multi-residue method for the analysis of 29 upstream river water while only one antibiotic multi-class pharmaceuticals in surface water and (sulfamethoxazole at 300 ng/L) was found downstream. wastewater using SPE LC/MS/MS with electrospray Cha et al. (2006) present a method using SPE ionization is described by Gros et al. (2006). A recovery LC/MS/MS with electrospray ionization to analyze β- comparison study between different sorbents was lactam antibiotics in surface water and urban wastewater in performed to optimize the method. The best recoveries for Colorado. Detection limits for each antibiotic were all analytes were obtained using an Oasis HLB sorbent between 8-10 ng/L in surface water, 13-18 ng/L in the without pH adjustment with most analytes recovered at influent and 8-15 ng/L in the effluent from a wastewater levels greater than 60%. Detection limits ranged from 1 to treatment plant. Recoveries from fortified samples were 30 ng/L for surface water (500 mL sample) and 3 to 160 generally above 70% with standard deviations below 10%, ng/L for wastewater (100 – 200 mL sample). Suitability of however amoxicillin was poorly recovered and was not different compensation techniques for observed matrix quantified in the water matrices studied. Matrix effects effects suppression from the wastewater samples was also were found to be minimal. Analysis of surface water and wastewater indicate minimal contamination from β-lactam Karthikeyan et al. (2006) describe a method for the analysis of antibiotics in surface and ground water Davis et al. (2006) studied the impact of originating from WWTPs in Wisconsin. Analytes were antibiotic (sulfonamide, tetracycline, and macrolide) analyzed by LC/ESI-MS/MS after SPE extraction with contaminated runoff from agricultural fields. Antibiotics tandem HLB and MCX sorbents. Limits of quantitation for were analyzed by LC/MS/MS from sediment and runoff a 500 mL sample were between 0.02 and 0.05 µg/L for generated by simulated rain events after application of the ground water and 0.05 and 0.2 µg/L for surface water. Six antibiotics to various types of soil. Monensin was found to different antibiotics were detected at various WWTP have the highest runoff concentration as well as the highest surface water sites at concentrations <1.3 µg/L with two absolute loss during the study while erythromycin had the antibiotics (sulfamethoxazole and tetracycline) detected in highest sediment concentration. The lowest absolute loss ground water wells adjacent to the WWTPs. was observed for tetracycline and chlortetracycline which Sulfonamide antibiotics were analyzed by Perret also had low runoff concentrations. The authors speculate et al. (2006) using polymeric SPE and LC/MS/MS. that erosion control practices will be effective in Recoveries exceeded 87% for all analytes with limits of minimizing antibiotic losses from fields for those quantification between 0.005 and 0.021 µg/L for surface antibiotics with high partitioning coefficients. water samples. Optimized parameters for the extraction, separation, and analysis steps of the method are given. Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
Application of the method for river and lake surface water concentrations lower than 10 ng/L, but a maximum and mineral and municipal drinking water samples from concentration of 54 ng/L was observed for pipemidic acid. Italy is also presented. Sulfonamide antibiotics were Confirmation of positive detections of pipemidic acid in the detected in 3 of 4 river samples at concentration levels up samples was discussed and illustrated by increasing the to 0.4 µg/L and in 4 samples of bottled mineral water with number of ion transitions collected for the analyte. a maximum concentration of 0.08 µg/L. Samples from lake Application of using a quadrupole-time-of-flight (Q-TOF) surface water and municipal drinking water did not have mass spectrometer for confirmation was limited in this detectable levels of sulfonamides. study due to its lower sensitivity and the low concentrations Pozo et al. (2006) discuss both successes and of analytes observed. pitfalls related to the confirmation of pesticides and Stoob et al. (2006) present a method for the antibiotics in environmental samples using triple analysis of sulfonamide antibiotics and their acetyl quadrupole (QqQ) and hybrid quadrupole-time-of-flight conjugates in soil samples using a pressurized liquid (Q-TOF) instrumentation. A minimum of two daughter-ion extraction (PLE) technique. Optimized parameters for the transitions is needed in analyses with QqQ to minimize the extraction with acetonitrile:water (0.1 M tris) (15:85) and occurrence of false positives. Q-TOF analysis can provide extract workup are given which result in detection limits of accurate mass determination for the detection, but still can between 5 and 15 µg/kg for a 4 g soil sample. The produce false positives, especially at low concentration extraction temperature used was critical in minimizing ion levels. Application to real world samples is presented to suppression in the mass spectrometer due to matrix effects illustrate the different problems which can occur in the but was offset by increased extraction efficiency. Isotope confirmation process. dilution internal standards helped to compensate for An in-line SPE/LC/MS/MS method is described decreases in sensitivity in favor of higher extraction yields. by Pozo et al. (2006) to obtain low pptr detections limits for Illicit drugs and metabolites were analyzed by the analysis of 10 quinolone and 6 penicillin antibiotics. A Castiglioni et al. (2006) using LC/MS/MS. Mixed 9.8 mL sample is extracted through a C-1 cartridge which reversed-phase/cation-exchange cartridges (Oasis-MCX) is then eluted directly into the HPLC/MS/MS for separation were used to concentrate the analytes from acidified and detection with a triple quadrupole mass spectrometer samples obtained from WWTP influents and effluents in (QqQ). Detection limits of 0.4 to 4.3 ng/L were obtained Switzerland and Italy. Quantification for each analyte was with recoveries between 74% and 123%. Application of performed by isotope dilution with the corresponding the method to surface and ground water samples resulted in deuterated analog as internal standard. Detection limits detections of several quinolone antibiotics in the surface were in the low nanogram-per-liter range with recoveries water samples. These detections were usually at higher than 80%. Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
Acidic drugs and caffeine were the focus of a Algal Toxins. The occurrence of freshwater
method developed by Verenitch et al. (2006) using SPE cyanotoxins has been the subject of many recent extraction with C-18 and HLB cartridges and an ion-trap investigations and may be related to agricultural inputs GC/MS/MS for analysis. The acidic drugs were derivatized affecting water quality. Billam et al. (2006) present results by methylation for the analysis while caffeine was analyzed of a 1-year study of two lakes in western Texas. Seasonal directly. Instrument detection limits of 0.5–20 ng/mL were variations in microcystin-LR contamination were examined obtained for all analytes with method detection limits by using enzyme-linked immunosorbent (ELISA) and ranging from 0.1 ng/L to 20 ng/L. The method was applied protein phosphatase inhibition assays and compared to tap water and effluents from WWTPs with recoveries between seasons and between lakes. The detection limits ranging from 55 to 128% depending on type of water were 0.15 µg/L for the ELISA assay and approximately 5 sample and analyte. ng/L for the more sensitive phosphatase inhibition assay. An analytical method for the detection of neutral Microcystin-LR was detected in both lakes for all samples and basic pharmaceuticals in drinking water, surface water, with the highest concentrations occurring during the spring. and wastewater is described by Vieno et al. (2006). Concentration comparisons between the two assay methods Samples are extracted using polymeric HLB cartridges and were correlated by a correlation coefficient of 0.82 with analyzed by LC/MS/MS. Detection limits ranged from 0.2 concentrations obtained by the ELISA assay greater than to 8.4 ng/L for drinking water (1000 mL), from 0.5 to 24 those obtained by the phosphatase inhibition assay by a ng/L for surface water (500 mL), from 1.4 to 29 ng/L for WWTP effluent water (200 mL ), and from 3.5 to 163 ng/L Bogialli et al. (2006) describe a method for the for WWTP influent water (100 mL sample). Ion analysis of microcystins and cylindrospermopsin in lake suppression due to matrix effects was examined for the water using LC/MS/MS. The microcystins were extracted WWTP water samples and discussed. The reduced with a Carbograph 4 SPE cartridge resulting in recoveries recoveries of carbamazepine, ofloxacin, and the beta between 93 and 103% and limits of quantification between blockers (acebutolol, atenolol, metoprolol and sotalol) 2 and 9 ng/L (500 mL sample). Direct injection analysis studied were attributed to reduction in signal due to ion for cylindrospermopsin from a 0.5 mL sample gave a limit suppression. Two other analytes, ciprofloxacin and of quantification of 300 ng/L. Survey samples from a lake norfloxacin, were not extracted efficiently and had reduced in Italy also detected demethylated metabolites of recoveries for all sample matrices. Application of the microcystin- RR and microcystin-LR using parent ion method to WWTP in Finland is also presented and The extraction of microcystins from soil and lake sediment was the basis of a method developed by Chen et Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
al. (2006). Optimized parameters for the extraction are Fate and Occurrence of Emerging Contaminants
given which include extraction solvent, pH, extraction Steroids and other Endocrine Disruptors.
approaches, and number of extractions. The microcystins Endocrine-disrupting compounds (EDCs) have been were analyzed from soil and sediment samples from previously identified in both municipal and agricultural Chinese lakes by LC/UV analysis. Detection limits of systems. Recent evidence indicates that the magnitude of approximately 0.1 µg/g were obtained. This extraction EDC loading into the environment by agricultural systems method could also be applied with more sensitive trace may exceed that of municipal systems. In 2006, a case analysis methods such as LC/MS and ELISA. study was performed to evaluate the potential EDC loading Li et al. (2006) describe a LC/MS/MS method for by farm animals in the U.K. (Johnson et al. 2006). In this the monitoring of microcystin-LR in lake water. An study, the normalized estrogen loading (measured as total instrument detection limit of 2 pg on column was achieved mass of estrogenic compounds/cow/d) attributable to cattle, by using the doubly-charged microcystin-LR precursor in pigs, and poultry by both urinary and fecal routes was MRM mode to obtain a higher intensity daughter ion than determined. This data was then used to evaluate the that obtained by use of the singly-charged precursor. relative contribution of each species to the total estrogen Method detection limits for lake water samples (5 mL) load as well as to compare agricultural and municipal were 2 ng/L using nodularin as an internal standard. The loading to the environment. This study determined that a method was used to monitor microcystin-LR levels in 6 normalized dairy cow and a normalized pig excrete two Chinese reservoirs. orders of magnitude and one order of magnitude more Zhao et al. (2006) present a solid-phase steroid estrogens, respectively, than a normalized human. microextraction (SPME) method procedure for sample Based on these results, it was determined that the farm preparation for the analysis of microcysins in lake water by animal population in the UK excretes approximately four use of microbore LC/Q-TOF MS. Optimized parameters times more estrogens than the human population. for fiber type and extraction and elution variables are The presence of various hormones and other presented along with instrumental parameters. Detection endocrine-disrupting compounds in agricultural effluents limits were at sub-µg/L levels for a 12 mL sample, was detected in multiple studies, with a particular focus on comparable to other methods which typically use 500 mL synthetic and natural estrogen compounds. A survey of of sample. Recoveries for microcystin-RR and municipal sewage and animal waste effluent in New microcystin-LR were greater than 86% and 71%, Zealand was performed by Sarmah et al (2006). In this study, animal waste effluents were collected from seven dairy, one goat, and one swine farm. Both solid and liquid animal wastes were collected, as well as samples from Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
water used to clean milking and animal storage areas. The EDCs in influent samples taken in October 2003 were samples were analyzed for both synthetic and natural similar, however the 17 -estradiol and equol concentrations estrogen compounds including 17 -estradiol, 17 -estradiol, increased by 500 ng/L and 160,000 ng/L, respectively. estriol, estrone, and 17 -ethynlestradiol. Significant inter- Lorenzen et al. (2006) also evaluated the toxicity of liquid site variability in dairy farm effluents were observed. For swine waste and determined that a wide range of both example concentrations of 17 -estradiol and 17 -estradiol natural and synthetic estrogens were present. The ranged from no detect (ND) to 1028 ng/L, and ND to 331 estrogenic activity in the sample was dominated by equol, a ng/L, respectively. At all agriculture sites sampled, the phytoestrogen, as well as natural steroidal hormones synthetic estrogen 17 -ethynlestradiol and estriol, a natural including estradiol and estrone. hormone derivative, was not detected. The highest In addition to direct sampling of livestock concentrations of 17 -estradiol, and its derivative, estrone, effluents, EDCs have been evaluated in receiving waters were detected in dairy farm effluent when compared to impacted by agricultural activity. A study performed by effluents collected from goat and swine farms. The total Shappell (2006), evaluated the estrogenic activity using an estrogen load from dairy farms ranged from 60 to E-screen bioassay in samples from various environments approximately 4000 ng/L. By comparison, swine and goat impacted by municipal effluents and farming activities. In effluents were found to contain a total estrogen load of 46 this study, grab samples from wetlands and ponds were ng/L and 376 ng/L, respectively. The authors noted that collected from waterbodies surrounded by agriculturally- sites where higher concentrations of estrogen was detected active areas to evaluate the impact of land use on estrogenic corresponded to larger farms with greater numbers of activity. Land uses evaluated included livestock (cattle animals. The presence of endocrine-active compounds alfalfa/soybean, including natural and synthetic estrogens and wheat/soybeans, and wheat/sugarbeets. Samples were also phytoestrogens was measured in swine farm effluent. In collected from agriculturally-inactive areas in the this study, influent to a pilot-scale swine waste treatment Conservation Reserve Program (CRP). Results indicated facility was sampled to capture seasonal variability in EDC that observed estrogenic activity was not significantly concentrations (Furuichi et al. 2006). In samples collected different based on land use and that variability within sites in July 2003, estrogenic substances including 17 -estradiol, was greater than the difference between mean values 17 -estradiol, estrone, estriol, 4-t-octylphenol, 4- observed for individual land uses. Interestingly, the CRP nonylphenol, bisphenol A, genistein and equol were land use sites had estrogenic activity similar to the detected at concentrations of 650, 1000, 5200, 2200, 120, livestock-impacted sites. It should be noted, however, that 2300, 110, <0.4, and 940,000 ng/L, respectively. Little the livestock sites sampled in this study did not represent a variability was observed with season. Concentrations of concentrated animal feeding operation (CAFO). The Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
author indicates that these observed results could be a result effluent was analyzed by liquid-scintillation counting. of waterfowl that are commonly present at CRP sites, as Transport of the two hormones was compared with results these animals have been previously-determined to have from a chloride tracer. Based on chloride tracer data, higher levels of circulating estradiol than mammals or preferential flow paths were detected within the humans. In another study, the presence of estradiol and undisturbed soil sample. Peak hormone concentrations testosterone was evaluated in soil and runoff from areas were detected at the same time as the chloride tracer, where poultry litter was land-applied (Jenkins et al. 2006). indicating that the hormones were also transported through Average concentrations of estradiol and testosterone in the the column by preferential flow paths. On average, 27% poultry litter applied to the watersheds were 3.1 mg/ha and and 42% of 17 -estradiol and testosterone, respectively, 0.08 mg/ha, respectively, and the authors noted that these were recovered in the column effluent. Peak hormone concentrations were lower than those measured in previous concentrations in column effluent ranged from 0.006 to studies where poultry litter was land applied. The average 0.123 µg/L for 17 -estradiol, which is within the range concentration of testosterone and estradiol in the upper 5 previously reported to cause endocrine disruption in fish. cm of soil was 1.1 to 72.4 ng/kg, and 65.3 to 636 ng/kg, Peak concentrations of testosterone ranged from 0.031 to 0.378 µg/L. Sorption experiments were also performed on Numerous studies have also investigated the fate bulk soil samples obtained from the same locations as the of EDCs in agricultural systems. Both estrogenic and undisturbed cores. Soils were samples at two depths, 0-10 androgenic compounds were found to sorb appreciably to cm and 20-30 cm. Batch sorption experiments were soil. In addition, studies have demonstrated the ability for performed with initial hormone concentrations ranging EDCs to be biodegraded in the environment, specifically from 0.001 to 1 µg/mL. Results from these indicated that under aerobic conditions. Khanal et al. (2006) provided a both hormones were strongly sorbed to soils, and data were review of the fate, transport and biodegradation of natural fit with a Freundlich isotherm. Values of the Freundlich estrogens in the environment. In addition, there has been exponent determined for both compounds were less than 1, limited evaluation of the transport of EDCs in the indicating that available sorption sites decreased with subsurface. Transport of 17 -estradiol and testosterone in increasing aqueous hormone concentration. Average soil columns was evaluated by Sangsupan et al (2006). In Freundlich coefficient (Kf) values for estradiol were 36.9 this study, undisturbed soil cores (15 cm diameter and 32 µg1-n mLn g-1 and 25.7 µg1-n mLn g-1 for the 0-10 cm and cm in length) were taken from agricultural areas subjected 20-30 cm soil depth, respectively. Average Kf values were to both no-till and conventional tilling practices. 26.7 µg1-n mLn g-1 and 14.0 µg1-n mLn g-1 for the 0-10 cm Radiolabelled 17 -estradiol and testosterone were and 20-30 cm soil depth, respectively, indicating that for a introduced into the top of the saturated soil sample, and the given soil type, 17 -estradiol was sorbed more strongly Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
than testosterone. Hildebrand et al (2006) also evaluated sorbed most strongly to the silty clay and clay loam soils. sorption of estrone, 17 -estradiol, and 17 -ethynlestradiol In general, less desorption was observed from the fine- to four agricultural soils. Batch sorption experiments were textured soils. Similarly, Loffredo and Senesi (2006) performed for all three compounds with initial estrogen evaluated the sorption behavior of four EDCs: bisphenol concentrations ranging from 10 to 1000 ng/mL. Initial A, octylphenol, 17 -ethynlestradiol, and 17 -estradiol on kinetic experiments indicated that a 24-hr equilibration two sandy soils. Initial aqueous concentrations used in this time was sufficient to ensure equilibrium conditions were study were 1 to 40 mg/L for bisphenol A, 1 to 20 mg/L for met. During the course of the batch sorption experiments, 17 -ethynlestradiol and 17 -estradiol, and 0.1 to 5 mg/L 17 -estradiol conversion to estrone was observed. The for octylphenol, which are significantly higher than authors evaluated two chemical inhibitors (0.5 g mercuric concentrations detected in environmental samples. chloride/kg soil and 0.2 g sodium azide/kg soil) and Experimental data were fit to sorption isotherm models and autoclaving soil at 121˚C for 30 minutes. It was it was determined that a liner model provided the best fit to determined that autoclaving was the most effective bisphenol A sorption data. 17 -ethynlestradiol sorption treatment for minimizing 17 -estradiol conversion. Batch data were fit with either a Freundlich isotherm model, sorption data to each of the four soils were fit with a octylphenol data were fit with either a linear or Freundlich Freundlich isotherm. In all experiments, estrone was isotherm, and data for 17 -estradiol were fit to a Langmuir sorbed more strongly to soil than either 17 -estradiol or isotherm. Desorption tests were also performed for each 17 -ethynlestradiol. The Freundlich exponent for estrone compound. In these experiments, a known initial was less than 1, indicating that sorption sites were saturated concentration of EDC was equilibrated with soil for 24 with increasing estrogen concentration. Sorption of 17 - hours. Following equilibration, the aqueous phase was ethynlestradiol was nearly linear, with Freundlich replaced with distilled water every 24 hours, and the exponents close to one. Freundlich exponents determined concentration of EDC in the aqueous phase was monitored for sorption of 17 -estradiol to each of the four soils were as a function of time. Desorption of 17 -estradiol could greater than 1, indicating that sorption increased with not be quantified as it was apparently degraded during the increasing estrogen concentration. Based on these results, experiments. Unquantifiable degradation products were the authors hypothesized that different mechanisms could observed during HPLC analysis. It was determined that be responsible for the observed sorption behavior. bisphenol A sorption to the soils evaluated is generally Desorption of the three estrogens was also evaluated for the reversible with complete desorption occurring in 3-4 d. four soils. In general, all compounds were found to desorb Octylphenol and 17 -ethynlestradiol were more tightly from the soils, with an increase in desorption as the initial bound to soil with only limited desorption observed. estrogen concentration decreased. 17 -ethynlestradiol was Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
Several studies have also evaluated EDC of moisture content and temperature. Although the mineralization in agricultural soils. Lucas and Jones (2006) experiments were conducted under aerobic conditions, little evaluated the biodegradation of 17 -estradiol and estrone degradation of 17 -estradiol, estrone and testosterone was in manure-amended soils. In this study, radiolabelled observed. In general, an increase in mineralization was hormones were added to a sample of soil obtained from associated with increasing water content and decreasing three agricultural grassland areas that had been used for temperature. At water potentials of -12 and -24 mPa, less sheep grazing. The hormones were added to the soil than <2% of testosterone was mineralized at 45˚C. Up to sample in three different solvent phases: water, artificial 27% of testosterone was mineralized at these water urine and natural sheep urine). The degree of hormone potentials when the temperature was decreased to 25˚C. In mineralization was found to be highly dependent on the soil general, mineralization of estradiol and estrone was type and the solvent matrix. There were no distinct trends negligible and was <1% in all treatments, except at 25˚C observed, and the calculated half-lives for the hormones in and -12 MPa. At this treatment, 5.9% and 7.8% of soil ranged from 5 to 25 d. In addition, hormones were estradiol and estrone, respectively, were mineralized. applied to soils that had been amended with cattle and Based on these results, the authors conclude that poultry sheep manure of varying ages (7 d to 2 yrs). Generally, litter will constitute a viable source of EDCs as treatments half-lives for hormones in manure-amended soil (t1/2 = 1-9 that are commonly observed in a poultry house did not d) were shorter than the unamended soils. In contrast, result in hormone mineralization. Lorenzen et al. (2006) evaluated the mineralization of Antibiotics and Other Pharmaceuticals. Sarmah et al.
estradiol, estrone, ethnylestradiol and 4-nonylphenol in soil (2006) reviewed the global use, exposure pathways, microcosms and observed very rapid degradation under occurrence, fate and effects of veterinary antibiotics in the aerobic conditions. All studies were found to be non- environment. This article provides a good introduction to persistant, with half-lives ranging from a few hours to days. the current state of agricultural practice and research related Because these compounds were observed to be more stable to veterinary antibiotics in the environment. Antibiotics are in sterilized soils, degradation is assumed to be used in cattle, swine, and poultry operations at therapeutic microbially-mediated. In addition, the authors found that levels to fight disease and at sub-therapeutic levels to aerobic conditions enhance degradation as the EDCs prevent infections and promote growth (McEwen 2006). evaluated were more persistence in anaerobic systems. Aquaculture also makes extensive use of antibiotics. One study was also performed to evaluate the Cabello (2006) reviews the use of antibiotics in aquaculture biodegradability of poultry litter during waste stockpiling. and the potential risks to human and animal health through Hemmings and Hartel (2006) investigated the increases in antibiotic resistance and direct contact by biodegradation of hormones in poultry litter as a function industry workers and consumers . Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
The use of antibiotics in agriculture greatly varies antibiotics (Allaire et al. 2006; Carlson and Mabury 2006). by country, from widespread use in the United States (U.S.) Batt et al. (2006) found sulfonamides from 0.076 to 0.22 and Canada to limited, therapeutic use in Scandinavia and µg/L in former drinking water wells impacted by a nearby Japan (Sarmah et al. 2006). The most common veterinary antibiotics in use, both globally and in the U.S., are River surface water in Southern Ontario affected ionophores (i.e. monensin, salinomycin), tetracyclines (i.e. by agricultural run-off was found to have a number of chlortetracycline, oxytetracycline), pharmaceuticals, including monensin (6.2-1172 ng/L) and a sulfonamides (i.e. sulfadiazine, sulfadimidine), and number of sulfonamides (0.2-408 ng/L) (Lissemore et al. macrolides (i.e. tylosin) (Sarmah et al. 2006). There is 2006). Samples were collected over an 8-month period and growing concern and evidence that antibiotics in the various parameters were used to predict antibiotic environment might lead to an increase in resistant bacteria concentration. Dissolved organic carbon was correlated to (McEwen 2006; Sarmah et al. 2006). A number of studies monensin concentrations, while nitrate and phosphate ion investigated the occurrence and fate of antibiotic-resistant concentrations did not. genes and bacteria in the environment. Three studies investigated antibiotics and Jindal et al. (2006) investigated antibiotic use and antibiotic resistance along the Cache la Poudre River in resistance in swine farm waste treatment systems. The Northern Colorado. These studies sampled from five authors used polymerase chain-reaction (PCR) and culture- distinct locations: one pristine and the others affected by based methods to detect tetracycline and macrolide varying urban and agricultural activities. Kim and Carlson resistance at a number of Illinois CAFOs with varying (2006) developed a SPE HPLC-MS/MS method to detect antibiotic use practices. Resistance to both antibiotic three ionophore antibiotics, monensin, salinomycin, and classes was found on all farms, including an organic farm narasin, and found one or more in surface water and with no current antibiotic usage and was detected in feed sediments at all sampling sites except the pristine buildings, solids-settling basins, lagoons, and soil samples. environment. The concentrations varied significantly In another study, tetracycline resistance was also detected between time points and between some sampling sites and in groundwater impacted by swine CAFOs (Mackie et al. were much higher in sediment than in water. Pei et al. 2006). Four common tetracycline resistance genes were (2006) found tetracyclines (45.7-399.1 µg/L total) and found in wells nearby two separate swine farms in Illinois. sulfonamides (3.5-22.4 µg /L total) at all sites except the However, only trace amounts of tetracyclines (0.08-0.4 pristine site. Using PCR methods, the authors detected µg/L) were detected and in a minority of wells. This agrees sulfonamide and tetracycline resistance genes at all sites, with other 2006 studies indicating a high affinity for with gene concentrations significantly higher at the sorption and low mobility in soil for tetracycline and other agricultural & urban influenced sites than at the pristine site Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
when normalized to the 16S gene copy number. Finally, tests with increasing manure content in amended soil and Pruden et al. (2006) detected tetracycline and sulfonamide with increasing moisture content (Wang et al. 2006b). resistance genes in nearby dairy lagoon water and irrigation Dilution of more contaminated manure with less- ditches with the statistically significant relationship: lagoon contaminated manure and storage of manure prior to water gene quantity > irrigation ditch water > agriculturally application are both recommended to diminish impacted river sediments. sulfadimethoxine contamination (Wang et al. 2006a; Wang Lab-scale tests showed that chlortetracycline and tylosin sorb quickly (< 10 min & < 3 hr respectively) to Tetracycline and sulfonamide resistance was sandy loam and clay (Allaire et al. 2006). Soil sterilization investigated over time in soil microcosms and field tests did not affect sorption kinetics over the studied 24 hrs, (Schmitt et al. 2006). Resistance diversity in collected suggesting that 24hr sorption studies are adequate to fully Swiss soil samples was high even before manure sorb antibiotics without significant degradation by application, while German samples were much lower. After microorganisms. A 38-day field study found first-order manure application, resistance diversity stayed at high dissipation half-lives in soil between 3.3 and 24 days for levels for the complete growing season. Gene tylosin, chlortetracycline, and monensin, indicating the quantification (via quantitative-PCR) was not performed. antimicrobials were not highly mobile (Carlson and Davis et al (2006) considered veterinary Mabury 2006). Only chlortetracycline was found below 25 antibiotic transport via runoff and soil loss in field-scale cm of depth after surface application. Manure application studies. Simulated rainfall events yielded runoff rates and significantly increased the tylosin dissipation rate (but not pseudo-partitioning coefficients. Monensin had the highest chlortetracycline or monensin). In a 120-day lab study of runoff loss, while tetracyclines had the lowest. Since six antibiotics in soil, Schlüsener and Bester (2006) found tylosin and erythromycin had the highest affinity for first order half-lives ranging from 5 days (salinomycin) to sediment transport, the authors conclude that erosion 27 days (oleandomycin) with R² values >0.91. control practices could be used to reduce tetracycline, A modified first-order degradation model was tylosin, and erythromycin losses but would not be effective suggested for sulfadimethoxine in manure and manure- for monensin or for the sulfonamides tested. amended soil (Wang et al. 2006a; Wang et al. 2006b). In Wastewater
treatment
microbial
community effects. Recent studies have begun to examine
sulfadimethoxine much faster than sterile manure, and the treatment methods for eliminating antibiotics, with a particular focus on human drinking water and wastewater sulfadimethoxine concentration (Wang et al. 2006a). treatment plants (WWTPs) (see Chamberlain and Adams Sulfadimethoxine degradation was accelerated in lab-scale 2006 and others). One 2006 study focused on the treatment Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
of swine wastewater with free chlorine (Qiang et al. 2006). in the environment has been identified as an area of Influent and effluent samples from various stages of a emerging concern, as a horizontal transmission pathway CAFO wastewater system were treated with free chlorine has been identified for certain prion diseases, including or monochloramine. A dose close to breakpoint was chronic wasting disease (CWD). required to achieve complete removal of antibiotics; A number of studies have evaluated prion however, a 100 mg/L dose was found to effectively treat degradation in the environment and prion sorption to soil. the polishing lagoon wastewater in an aerated system. It has been shown that PrPSc is degraded by certain The effects of veterinary antibiotics on microbial microbial proteinases, although pre-heating and the communities remain an important focus of environmental presence of a detergent was required for complete digestion microbiology. Briefly, Zielezny et al. (2006) found that (Suzuki et al. 2006). Pre-treatment by heating, detergents, sulfadiazine significantly affected soil respiration in the or alkaline pH (pH 12) is required prior to enzyme reaction presence of glucose while chlortetracycline did not. in order to reduce PrPSc infectivity. Microbiological However, chlortetracycline did inhibit growth in disk tests. consortia taken from the rumen and colon of cattle could Oxytetracycline affected functional diversity, evenness, degrade PrPSc to undetectable levels within 20 hours under average well color development, and decreased substrate anaerobic conditions at 37˚C, but prion infectivity was not utilization (Kong et al. 2006). Effects were more evaluated (Scherbel et al. 2006). An in situ study also pronounced with the addition of copper (Cu). Chun et al. demonstrates the potential for environmental degradation of (2006) found chlortetracycline and tylosin can decrease the PrPSc. PrPSc was shown to be degraded in soil below lamb stimulatory effect of 17 -estradiol in certain soils. carcasses in a field burial site, and PrPSc degradation was Fate of Prions. Prion diseases, also called
correlated to an increase in proteolytic activity and transmissible spongiform encephalopathies (TSEs) are a substrate induced respiration (Rapp et al. 2006), however group of fatal neurodegenerative diseases that impact a PrPSc infectivity was not specifically quantified in this number of mammalian species and include bovine spongiform encepalopathy or ‘mad cow' disease, scrapie of Studies evaluating prion sorption to soils and clay sheep and goats, chronic wasting disease (CWD) of deer, minerals have focused on batch experiments evaluating the elk and moose, and Creutzfeldt-Jakob disease in humans. interaction between various prion strains and whole soils The etiological agent in TSEs (denoted PrPSc) is an and/or clay minerals such as montmorillonite (Mte) and abnormally-folded conformer of a normal cellular prion kaolinite. Results indicate strong uptake of PrPC and PrPSc protein (PrPC). It is known that prions are long-lived in the by all soils, clay minerals and sand, and notably avid environment and unusually resistant to most inactivation uptake of PrPSc by Mte clay particles was observed in treatments for conventional pathogens. Recently, prion fate several studies (Johnson et al. 2006; Rigou et al. 2006). Water Environment Research, Volume 79, Number 10—Copyright 2007 Water Environment Federation
Subsequent extraction of PrPSc from Mte is difficult and samples. Analyst, 131, 407-414.
saline buffers and chaotrophic agents are ineffective at Balakrishnan, V. K.; Terry, K. A.; Toito, J. (2006) Determination removing prions from Mte and soil surfaces, suggesting of sulfonamide antibiotics in wastewater: A comparison of solid phase microextraction and solid phase irreversible binding under normal environmental conditions extraction methods. J. Chromatogr., A, 1131 (1-2), 1-
(Johnson et al. 2006; Leita et al. 2006; Rigou et al. 2006). PrPSc extracted from Mte surfaces exhibits a lower Barber, L. B.; Keefe, S. H.; Antweiler, R. C.; Taylor, H. E.; Wass, molecular mass than the starting material (Johnson et al. R. D. (2006) Accumulation of Contaminants in Fish 2006). Using an antibody directed at amino acids 23-27 on from Wastewater Treatment Wetlands. Environ. Sci. the N-terminus of the protein, Johnson et al. (2006) was Technol., 40 (2), 603-611.
able to show that some or all of the epitope of this antibody Batt, A. L.; Snow, D. D.; Aga, D. S. (2006) Occurrence of could not be detected on the desorbed protein. Sorption sulfonamide antimicrobials in private water wells in Washington County, Idaho, USA. Chemosphere, 64
experiments conducted with only the C-terminal portion of (11), 1963-1971. the prion protein also exhibited strong uptake, but showed Beck, I.-C.; Bruhn, R.; Gandrass, J. (2006) Analysis of estrogenic no further reduction in molecular mass upon desorption activity in coastal surface waters of the Baltic Sea using (Johnson et al. 2006). Based on these results, the N- the yeast estrogen screen. Chemosphere, 63 (11), 1870-
terminus of PrPSc has been implicated in the mechanism for prion sorption to soil surfaces. In addition, hamsters that Beck, M.; Radke, M. (2006) Determination of sterols, estrogens were intracerebrally inoculated with Mte-bound PrPSc and inorganic ions in waste water and size-segregated exhibited clinical signs (Johnson et al. 2006), providing aerosol particles emitted from waste water treatment. Chemosphere, 64 (7), 1134-1140.
some evidence that Mte-bound PrPSc may remain Billam, M.; Tang, L.; Cai, Q.; Mukhi, S.; Guan, H.; Wang, P.; Wang, Z.; Theodorakis, C. W.; Kendall, R. J.; Wang, References
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Antibiotic resistance is ancient Vanessa M. D'Costa1,2*, Christine E. King3,4*, Lindsay Kalan1,2, Mariya Morar1,2, Wilson W. L. Sung4, Carsten Schwarz3,Duane Froese5, Grant Zazula6, Fabrice Calmels5, Regis Debruyne7, G. Brian Golding4, Hendrik N. Poinar1,3,4 & Gerard D. Wright1,2 The discovery of antibiotics more than 70 years ago initiated a with high concentrations of Escherichia coli harbouring the gfp (green

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