Detection, Occurrence and Fate of

Emerging Contaminants in Agricultural

Environments
Daniel D. Snow1*, David A. Cassada2, Shannon L. Bartelt–Hunt3, Xu Li4,
Yun Zhang5, Yuping Zhang5, Qi Yuan5 and J. Brett Sallach5

ABSTRACT: A review of the literature published in KEYWORDS: pharmaceuticals, steroid hormones,

2011 including 70 papers on topics ranging from analytical analytical methods, water and wastewater, agricultural

methods, to occurrence and environmental fate of emerging environment.

contaminants in agricultural environments is presented.

This review is divided into the following sections: doi: 10.2175/106143012X13407275694635

Introduction, Analytical Methods for Emerging

Contaminants, Passive Samplers and Screening Methods, INTRODUCTION

Steroids in Agricultural Environments, Veterinary Nutrients, sediments, and pesticides are well

Pharmaceuticals in Agricultural Environments, Antibiotic known and extensively studied contaminants impacting

Resistance Genes, and Prions as Emerging Contaminants. water quality in agricultural environments. These groups of

contaminants typically occur at easily measured

concentrations in surface run–off in agricultural

______________________________________________________ watersheds. Nutrients, especially nitrogen, and pesticides

1
Research Associate Professor and Laboratory Director, Nebraska
Water Center, part of the Robert B. Dougherty Water for Food have also been shown to impact ground water quality in
Institute, 202 Water Sciences Laboratory, University of Nebraska,
Lincoln, NE 68583–0844; Tel. 402–472–7539; Fax. 402–472–9599; areas susceptible to contamination. Less well–known are
email: dsnow1@unl.edu
2
Chemist, Nebraska Water Center, part of the Robert B. Dougherty environmental impacts of newer classes of contaminants
Water for Food Institute, 202 Water Sciences Laboratory, University
of Nebraska–Lincoln, Lincoln, NE 68583–0844; Tel. 402–472–7539; such as pharmaceuticals, steroids, antibiotic–resistance
Fax. 402–472–9599; email: dcassada1@unl.edu
3
Associate Professor, Department of Civil Engineering, 203B Peter genes and prion proteins. These “emerging” contaminants
Kiewit Institute, University of Nebraska–Lincoln, Omaha, NE
68182–0178, Tel. (402) 554–3868; Fax. (402) 554–3288; email:
sbartelt2@unl.edu clearly have potential to enter the environment and cause
4
Assistant Professor, Department of Civil Engineering University of
known or suspected adverse ecological or human health
Nebraska–Lincoln 844 N. 16th St., N117 SEC Link, Lincoln, NE
68588–6105; xli4@unl.edu
5 effects. Release of these contaminants to the environment
Graduate Research Assistant, Department of Civil Engineering,
University of Nebraska–Lincoln Department of Civil Engineering,
Department of Civil Engineering University of Nebraska–Lincoln, has occurred for quite some time, but methods for their
844 N. 16th St., N117 SEC Link, Lincoln, NE 68588–6105

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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
detection at environmentally–relevant concentrations have hormones in animal manure or agricultural soil. The

only recently become available. method consisted of two parts: inverse and integrated

Evaluating the environmental fate and effects of clean–up pressurized liquid extraction; and gas

emerging contaminants includes compounds such as chromatography–tandem mass spectrometry (GC–MS/MS).

surfactants, antibiotics and other pharmaceuticals, steroid In pressurized liquid extraction (PLE), the samples were

hormones and other endocrine–disrupting compounds flushed with heptane to avoid the interference of undesired

(EDCs), fire retardants, sunscreens, disinfection matrix and then dealt with internal clean–up PLE to elute

byproducts, new pesticides and pesticide metabolites, and the steroids. A two step solid phase clean–up (aminopropyl

naturally–occurring algal toxins. Detection of these and silica gel) was then used to further purified the PLE

contaminants in environmental matrices (water, extracts. There was a one hour derivatization before the

wastewater, soils and sediments) is particularly challenging samples were analyzed with GC–MS/MS. The absolute

because of the low detection limits required, the complex PLE recoveries in this study ranged from 67 to 107 % from

nature of the samples, and difficulty in separating these 0.5 g manure spiked with 50 ng. The development and

compounds from interferences. New extraction and clean– implementation of a method for simultaneous detection of

up techniques, coupled with improvements in instrumental five sulfonamides, three tetracyclines, and one macrolide

technologies provide the needed sensitivity and specificity was accomplished by Pan et al. (2011). Using this method,

for accurate measurement. 126 samples from 21 concentrated animal feeding

The objective of this paper is to review the operations (CAFOs) were analyzed. Concentrations

literature published in 2011 evaluating the detection, fate, reached as high as 764.4 mg/kg (chlortetracycline) and the

and occurrence of emerging contaminants, with a particular tetracyclines had detection frequencies between 84.9–

focus on emerging contaminants in agricultural systems. 96.8%, 0.8–51.6 % for sulfonamides, and 4.8% for the

Relevant contaminants are EDCs (particularly hormones macrolide antibiotic. It was concluded that CAFOs manure

and anabolic steroids), antibiotics and other may act as a non–specific source of antibiotics in farmlands

pharmaceuticals associated with wastewater, antibiotic and aquatic environments.

resistance genes in bacteria and prions. Studies on Sixteen compounds including antibiotics,

pesticides and flame retardants are not reviewed unless they hormones, analgesics, stimulants, antiepileptics, and X–ray

were evaluated in the same study. contrast media were analyzed from 31 water treatment

Analytical Methods for Emerging Contaminants facilities in the study by Wang et al. (2011). Method

Steroid Hormone and Pharmaceutical detection limits (MDLs) were determined in reagent water

Analysis. Hansen et al. (2011) developed a novel and municipal tap water ranging from 0.1 to 9.9 ng/L.

analytical method for simultaneous detection of ten steroid While most concentrations were below the MDL, caffeine
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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
was detected at the highest concentration 224 ng/L. Wang extraction parameters using Discovery DSC–18Lt

et al. (2011) also noted seasonal differences, with higher cartridges resulted in detection limits of 3.0–5.4 ng/L for

concentrations in the summer than winter. Due to water each analyte. Results from application of the method to

volume differences, the total loadings into receiving waters samples from river water near wastewater treatment plants

were similar in both seasons. (WWTP) are given.

Avbersek et al. (2011) combined trimethylsilyl Lucci et al. (2011) describe a method for clean–

derivatization GC–MS analysis with the ER–Calux® up and preconcentration of natural and synthetic estrogens

estrogenicity assay into a single protocol to determine using molecular imprinted polymers (MIPs) as a selective

steroid estrogen concentrations and total estrogenic solid phase extraction (SPE) medium. Recoveries of >85%

potential (E2 equivalents, EEQ) from the same sample were obtained for natural estrogens while synthetic

extract. Optimization of the dual method required estrogens had recoveries of >48%. Detection limits ranged

introduction of a solvent (ethyl acetate) compatible with from 4.5 to 9.8 ng/L for spiked river and tap water samples.

each phase of analysis. Correlation of results obtained from Comparisons of MIP results to those obtained using

spiked and unspiked waste water samples (effluent and commercial C–18 SPE cartridges are discussed.

influent) using each method was given with r2 > 0.92. Trinh et al. (2011) describe a derivatization

Detection limits of 0.68 ng(EEQ)/L were obtained for the method for the gas chromatography mass spectrometry

estrogenicity assay and 0.1–1.4 ng/L for GC–MS. (GC/MS) analysis of estrogens and androgens in aqueous

Iparraguirre et al. (2011) report on a method samples. Isotope labelled internal standards were used for

using stir bar solid phase extraction coupled with in–tube 8 of the 12 analytes which provided improved accuracy for

derivatization and thermal desorption GC–MS for analysis the analysis. Detection limits of 1–5 ng/L were obtained in

of endocrine disruptor analytes in estuaries and wastewater various aqueous matrices. Results from application of the

effluents and influents. Detection limits of 0.8–74 ng/L method to surface waters and effluent wastewaters are

were obtained from extraction of 100 mL water samples. discussed.

Results from optimization studies of various extraction and Zhang et al. (2011) report on the use of a mixed–

derivatization parameters are given along with mode cation exchange (MCX) SPE method for

environmental results obtained from effluent and influent determination of 31 endrocrine–disruptor compounds in

wastewater using the optimized analytical method. surface waters with optimized detection by LC–MS/MS in

A capillary liquid chromatography–tandem mass positive and negative electrospray modes. Detection limits

spectrometry (cLC–MS–MS) method has been reported by obtained from 1000 mL samples were 0.2–1.9 ng/L for all

Kozlik et al. (2011) for the separation and detection of analytes with recoveries of 84.4–103.0%. Results are

estrogens in water samples. Optimization of solid phase

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reported from application of the method to surface waters water fortified with 40 mg/L sulfadiazine and provided

in Shanghai, China. comparable results to samples analyzed by LC–MS/MS.

A method for accessing estrogenic activity in Steroids in Agricultural Environments

surface waters and sediment has been reported by Zhao et Occurrence and Fate of Steroid Hormones.

al. (2011) combining recombinant yeast estrogen screening Bevacqua et al. (2011) compared the occurrence of steroid

with GC–MS in negative chemical ionization (NCI) mode. hormones in municipal biosolids and poultry litter. The

Water samples were extracted using Oasis HLB SPE analyzed steroids included estrone, 17β–estradiol, estriol,

cartridges and sequentially eluted with methanol and 17α–ethinylestradiol, E1–sulfate, E2–3–sulfate, E2–17–

dichloromethane. Sediment samples were ultrasonically sulfate, testosterone, and progesterone. The results showed

extracted with ethyl acetate. Results obtained from river that estrone and progesterone were detected at levels of 2.5

water indicated good correlation between the bioassay and to 21.7 ng/g dry weight and 2.5 to 470 ng/g dry weight

chemical analysis (R2 > 0.91). respectively in the samples of limed biosolids collected at a

Passive Samplers and Screening Methods wastewater treatment plant over a four year period. The

Bartelt-Hunt et al. (2011) improved the average concentrations of estrone, E1–sulfate and

application of polar organic chemical integrative samplers progesterone in poultry litter collected from 12 mid–

(POCIS) for steroid detection in surface waters receiving Atlantic farms were 41.4, 19.2, and 63.4 ng/g dry weight,

wastewater treatment effluent and agricultural runoff by respectively.

determining the chemical–specific uptake rates of 16 Gall et al. (2011) analyzed nine steroid hormones

steroids and metabolites, including 11 compounds for in samples collected from subsurface tile drains and a

which uptake rates were not previously available. The receiving ditch at a working farm in the Midwestern United

uptake rates of these steroidal compounds were determined States where animal wastes are applied to agricultural

at 25oC under flowing conditions ranging from 0.19 to 0.65 fields. More than 64% of the samples collected at each

L/d. Uptake rates for pharmaceuticals were also reported. sampling site contained hormones, among which estrone

Almeida et al. (2011) describe a method for rapid was detected the most frequently and estriol the least. The

automated in–situ monitoring of relatively high synthetic androgens were detected in < 15 % of samples,

concentrations of sulfadiazine used in aquaculture with which was less frequently than natural androgens

potentiometric sensor based on a tubular electrode and PVC testosterone and androstendione. The highest concentration

membrane. The detection limit using the optimized flow of synthetic androgens (168 ng/L) in subsurface tile drains

injection method with the sensor was estimated at 3 µM was observed during a snowmelt period. In the receiving

(~750 µg/L) and allowed repeatable detection of surface ditch, the highest concentrations of total estrogens and

natural androgens were 87 ng/L and 52 ng/L respectively,

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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
both of which were observed in June, the early larger possibility that 17α–estradiol will leached from

development period of many aquatic species. agricultural fields. Based on the results, it was deduced that

By exposing female fathead minnows to the stereoselective sorption of these two isomers might be

sediment samples collected from three sites within two associated with aromatic interactions and H–bonding,

agriculturally intense Nebraska watersheds (Bow Creek and though the sorption appeared to be driven primarily by

the Elkhorn River), Sellin Jeffries et al. (2011) found that hydrophobic forces.

the hepatic mRNA expression of two estrogen responsive Bera et al. (2011) investigated the sorption of

genes, estrogen receptor α (ERα) and vitellogenin (Vtg) 17β–estradiol and testosterone in soil without and with

decreased significantly, which indicated the bioavailability poultry litter. When 3H–estradiol and 14C–testosterone were

of some anti–estrogenic compounds present in the sediment applied to the soil separately without poultry litter, the

or water matrix. Besides, the lipophilic extracts of the sorption of estradiol to soil was stable from 2 to 24 h and

sediments were able to induce significant reductions in the then decreased to 72 h, while testosterone in soil increased

estrogenic reporter activity. Correspondingly, various from 2 to 48 h and then kept relatively constant thereafter.

steroid hormones were detected in the sediments, including This difference may result from different transformation

those applied very often in the local beef cattle feedlots (i.e. rates of the two hormones with different solid–phase

β–trenbolone, α–zearalanol and α–zearalenol). These affinity. The maximum sorption coefficients (Kd) for the

results suggest that agriculturally utilized steroid hormones two steroids were 20.2 and 19.6 mL•g–1 respectively.

are able to enter the surrounding watersheds and potentially When applied together, Kd of 14C–testosterone decreased to
3
result in the defeminization of aquatic organisms. It is 12.5 mL•g–1 and H–estradiol 7.4 mL•g–1. It was

possible that the sediment acts as a potential source of anti– speculated that the competition between the two steroids

estrogenic compounds. and their metabolites for sorption sites might be responsible

Mashtare et al. (2011) investigated the sorption of for the decrease of sorption. Besides, it was also found that

17α–estradiol and 17β–estradiol on seven surface soils of poultry litter had different impact on the sorption of the two

various soil properties, avoiding the impact of steroids to soil: estradiol sorption increased but testosterone

biodegradation. The results showed that the sorption of decreased. The reason that resulted in this difference might

estradiol was well correlated to the organic carbon (OC) be poultry litter slowed down the transformation of the

content of soils, and the average log organic carbon– parent hormones.

normalized distribution coefficients (log Koc, L•kgoc–1) Mansell et al. (2011) investigated the release and

were 2.97±0.13 for 17α–estradiol and 3.14±0.16 for 17β– transport of six endogenous steroids from a steer feedlot

estradiol. Typically, the sorption of 17α–estradiol was on during simulated storm events. Both soil and runoff

average 50% less than 17β–estradiol, which indicated samples were collected and analyzed for 17α–estradiol,
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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
17β–estradiol, estrone, testosterone, androstenedione, and Rhodobacter, among which Sphingomonas taking up over

progesterone. It was found that only 17α–estradiol, 46% of the total DNA sequence was the dominant species

testosterone, and progesterone were present in fresh and Rhodobacter was the second most abundant. Besides,

manure, however, 17β–estradiol, estrone, and the mineralization pathway of the testosterone was

androstenedione appeared in the surficial soil after two elucidated based on the degradation products analysis. The

weeks. 17α–estradiol concentration in the surficial soil products identified included androstenedione,

decreased by about 25%, meanwhile an equivalent of androstadienedione, and dehydrotestosterone for sure, as

estrone and 17β–estradiol increased. For an additional well as 9α–hydroxy– testosterone, 9α–

aging period of 7 days for the soils, the concentrations of hydroxyandrostadienedione or 3–hydroxy–9,10–

estrogen and testosterone didn’t change significantly, while secoandrosta–1,3,5 (10)–triene–9,17–dione, and 9α–

androstenedione concentrations obviously decreased and hydroxydehydrotestosterone or 9α–hydroxy–

progesterone concentrations increased. After a simulated androstenedione by inference. Within 8 days of incubation,
14 14
storm event, the estrogen concentrations in the soil 49–68% of the C–testosterone was mineralized to CO2

remained almost the same, while the concentrations of and the reaction followed pseudo–first– order reaction

androgens and progesterone decreased by approximately kinetics with half–lives of 10–143 h.

85%. Except 17β–estradiol, the other five steroids in the Khunjar et al. (2011) have evaluated the relative

runoff were present in both the filtered and particle– roles of ammonia oxidizing and heterotrophic bacteria

associated phases to a level above thresholds for biological during the transformation of 17α–ethinylestradiol. It was

responses. There was less 17α–estradiol and testosterone, found that ammonia oxidizing bacteria was only able to

but more estrone, androstenedione, and progesterone transform 17α–ethinylestradiol, whereas heterotrophs were

detected in the runoff from aged plots than from unaged capable of mineralizing it as well as its metabolites

plots. transformed by ammonia oxidizing bacteria. However, the

Yang et al. (2011) revealed that Proteobacteria transformation rate by ammonia oxidizing bacteria was five

played an important role in aerobic degradation and times greater than that by heterotrophs. The removal of

mineralization of testosterone in manure treatment or land 17α–ethinylestradiol was significantly slowed down if

application. They enriched testosterone degrading culture ammonia oxidizing bacteria were inhibited, which further

from swine manure by using testosterone as the sole carbon proved its important role in transforming ethinylestradiol.

and energy source. Six genera of the bacteria were Besides, two major metabolites were found in ammonia

identified from the enriched culture based on DNA oxidizing, heterotrophic, and their mixed culture; one of

sequencing results: Acinetobacter, Brevundimonas, them, sulfo–ethinylestradiol, was largely resistant to further

Comamonas, Sphingomonas, Stenotrophomonas, and biodegradation.

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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
 Writer et al. (2011)b compared the Mao et al. (2011) provided an insight to an

biodegradation and attenuation of 17β–estradiol, estrone, enzymatic reaction that played an important role in the

17α–ethynylestradiol in three different environmental transformation of steroid hormones under natural

matrices: biofilm, sediment and water. The results showed conditions. The kinetic behaviors of lignin peroxidase

that biofilm played an important role in the attenuation of (LiP)–mediated reactions for estone, estradiol, estriol, and

steroidal compounds in surface waters due to both ethinylestradiol weve investigated with the absence and

biodegradation and sorption processes. The sorption rates presence of veratryl alcohol (VA), a chemical produced

to both biofilm and sediment was greater (<1 h) than along with LiP by certain fungi which has been proved to

biodegradation (50% mineralization at >185), which significantly enhance the reaction efficiency. Then the

potentially resulted in accumulation in biofilm and quantitative structure activity relationship (QSAR)

sediment. In addition, 17β–estradiol was biodegraded more equations were established by correlating the kinetic

effectively in sediment than in the biofilm and water parameters with structural features of LiP/substrate binding

matrices. Biodegradation of 17α–ethynylestradiol by the complexes. The result indicated that binding distance

sediment occurred at later time intervals (70 and 185 d) and between a phenolic proton of substrate and δN of HIS47’s

was not observed in the biofilm or water matrices. imidazole ring was very important in adjusting substrate

Another study by Writer et al. (2011a) focused on reactivity toward LiP with and without VA.

the sorption of 17β–estradiol and 17α–ethynylestradiol to Factors influencing steroid transformation.

biofilm colonized on artificial substrata in surface waters Lee et al. (2011) evaluated the impacts of humic acid at

by batch experiment. It was found that the sorption was different concentrations (0, 10, 30, 50 mg C/L) on the

linearly correlated with organic matter content, and the biodegradation of 17β–estradiol in aqueous phase based on

dominant sorption mechanism might be hydrophobic batch experiments. Besides 17β–estradiol, estrone was also

partition. The partition coefficients (Kom, L•kg–1) for the analyzed for each sample and estrogenicity associated with

two steroids were 102.5–2.8 L•kg–1 and 102.5–2.9 L•kg–1, 17β–estradiol removal was estimated with E–screen

respectively. This suggested that steroids could readily sorb bioassay. It was found that the biodegradation rate of 17β–

to and accumulate in biofilms, which was a potential estradiol and the formation rate of estrone significantly

exposure pathway for organisms in higher trophic levels. decreased with the increase of humic acid concentration,

Though various environmental conditions resulted in while the increasing humic acid level resulted in the higher

differences in the composition of periphyton and sorption of 17β–estradiol from 6% to 22%.

heterotrophic bacteria of the biofilm, they did not Correspondingly, the solution without humic acid showed

significantly affect the sorptive properties of the biofilm. the highest estrogenicity which represented the highest

17β–estradiol removal, and the presence of humic acid

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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
showed slightly higher estrogenicity. Overall, 17β–estradiol River and analyzed for 22 antibiotics and seasonal

biodegradation mainly accounted for its removal in variances between June and December. Antibiotic classes

aqueous phase, and the biodegradation was enhanced with included tetracyclines, chloramphenicols, macrolids,

the sorption significantly decreased at various HA fluoroquinoles, sulfonamides, and trimethoprim. Jiang et

concentrations. In addition, the presence of nutrients al. (2011) found that dominant species varied seasonally

enhanced the biodegradation of 17β–estradiol, which and that veterinary antibiotics dominated in suburban

suggested that the level of nutrients in natural waters sampling sites. All but 4 of the 22 compounds were

should be taken into account in estimating estrogenicity of detected with frequencies ranging from 5.3 % to 100%.

environmental samples due to complex biodegradation and Hoa et al. (2011) examined the detection of

sorption processes. antibiotics in commonly used in agricultural systems as

Homklin et al. (2011) evaluated the well as urban environments and correlated the results with

biotransformatoion of 17α–methyltestosterone (MT), an the presence of antibiotic resistant bacteria in these

anabolic androgenic steroid, in sediment with different environments. In their study, they identified numerous

electron acceptors. The results showed that the half–life of sulfonamides and common macrolides. They found

MT under aerobic and sulfate–reducing conditions was 3.8 macrolides, including erythromycin (154–2246 ng/l) and

d and 5.3 d, respectively, and the androgenic activity could clarithromycin (2.8–778 ng/l), were most common in city

eventually disappearance. Under methanogenic condition, canal sites. Sulfamethazine (475–6662 ng/l) was dominant

the androgenic activity could persist 45 d or longer in pig farm ponds and sulfamethoxazole (612–4330 ng/l)

although transformation of MT did occur. Besides, MT was dominant in city canal and aquaculture sites. The

transformed slowly under iron(III)–reducing condition and study showed specific contamination patterns, but

was hardly transformed with nitrate as electron acceptors. It concluded that antibiotic–resistant bacteria occurred not

was inferred that the methyl group at the C–17 position only within contaminated sites but also less contaminated

might account for the hindrance of transformation of MT sites.

under nitrate–reducing condition. As a result, MT and its The detection of 21 antibiotics from six different

metabolites with androgenicity could potentially classes in coastal waters was investigated by Zou et al.

accumulate in the iron(III)–reducing, nitrate–reducing and (2011). Water samples were taken from the Bahai Bay, six

methanogenic sediments. contributing rivers, and three aquaculture breeding sites.

Veterinary Pharmaceuticals in Agricultural Zou et al. (2011) concluded that areas in the north Bahai

Environments Bay region, characterized by high density human activity,

Fate and Occurrence. Jiang et al. (2011) contributed to environmental exposure. The higher

collected water samples from 19 sites along the Huangpu concentrations in the river samples than bay samples
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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
revealed the importance of river discharge as an antibiotic handling a variety of waste sources including households,

source posing ecological risk to the bay. livestock farms, hospitals, and pharmaceutical

Seventeen common human and veterinary manufacturing. They characterized the dominant

antibiotics in four classes (fluoroquinolones, tetracycline, pharmaceutical contaminant in the influent waste from the

sulfonamides, and macrolides) were investigated by Zhou different sources. Antibiotics dominated the livestock farm

et al. (2011)in three rivers in northern China. The study source. Concentrations detected in waste effluent was less

revealed higher concentrations in river sediment of one consistent between sites and additional variability coming

river (Hai River) than the other two, with norfloxazin, from sampling periods.

ofloxacin, ciprofloxacin, and oxytetracycline being the Wei et al. (2011) investigated the prevalence of

most commonly observed at concentrations up to 5770, ten veterinary antibiotics in animal wastewater and surface

1290, 653, and 652 ng/g, respectively. Sources of exposure water around large–scale livestock and poultry farms in

were determined to be large cities, feedlots, and fish ponds. Jiangsu Province of China. All ten compounds investigated

This data was strongly correlated with physicochemical were detected in the animal wastewater, eight antibiotics

properties (TOC, texture, and pH) of the river sediment. were detected in pond water, while nine of the ten were

This suggests that sediment properties are important factors detected in animal farm–effluent and river water samples.

influencing the distribution of antibiotics in aquatic The most frequently detected compounds were

environments. sulfamethazine (75%), oxytetracycline (64%), tetracycline

The occurrence of human pharmaceuticals, (60%), sulfadiazine (55%), and sulfamethoxazole (51%).

disinfection by–products and other wastewater The highest concentrations of the compounds were found in

contaminants in irrigation water was investigated by the animal wastewaters. Detected concentrations of

Calderan–Preciado et al. (2011). The use of reclaimed specific antibiotics were related to animal species revealing

wastewater for irrigation is becoming increasingly a high spatial variation.

important source for crop production, though may The occurrence of veterinary pharmaceuticals in

inadvertently introduce contaminants to food crops. lagoons and in groundwater adjacent to confined cattle and

Twenty–six chemicals, including pharmaceuticals, swine feeding operations was evaluated by Bartelt–Hunt et

pesticides, estrogens, antioxidants, and disinfection by– al. (2011a). Pharmaceuticals detected in lagoons at cattle

products were detected in samples of wastewater, river facilities included monensin, erthyromycin, and

water receiving effluent and irrigation water. sulfathiazole. Erthromycin and monensin were also

Occurrence and distribution of pharmaceuticals detected in groundwater underlying cattle feeding

in waste treatment facilities was studied by Sim et al. operations at concentrations ranging from <100 ng/L to

(2011). Sim et al. (2011) examined treatment facilities 2300 ng/L. There were fewer detections of
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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
pharmaceuticals in groundwater samples obtained from decreasing pH. Although, sulfonamides were found to be

swine facilities and antibiotics detected in groundwater at highly mobile, in the presence of manure increased their

these facilities included ractopamine, erythromycin, and attenuation. This attenuation was most likely caused by

sulfamethazine. The lower concentrations of antibiotics microbial transformation and irreversible sorption to the

detected at swine facilities was attributed to the greater soil matrix.

depth to water at these facilities relative to the cattle Biodegradation of sulfamethoxazole (SMX) was

facilities. examined using water–sediment batch experiments by Xu

Finally, Fatta–Kassinos et al. (2011) reviewed the et al. (2011). The influence of temperature, presence of

risks associated with xenobiotics in wastewater reuse for humic acids, and initial antibiotic concentration on

agricultural purposes. This review covered regulatory degradation was studied using a first–order decay model.

framework, specific chemicals of concern, uptake by plants The results showed that increased temperature and humic

and impacts to groundwater, accumulation in soils, and acid concentrations increased degradation of SMX, while

release of antibiotic resistance genes through wastewater initial antibiotic concentration had little effect. Further

reuse. biological analysis revealed the prevalence of Bacillus

Fate and Transformation of Antibiotics. Lin et firmus and Bacillus cereus implicating their role in the co–

al. (2011) examined the effect of vegetative buffers using metabolism mechanism of SMX biodegradation. This

simulated run–off losses of veterinary antibiotics and publication highlights an effort to explore better solutions

herbicides from row crops fertilized. Tylosin, for SMX removal from environmental sources to mitigate

sulfamethazine and enrofloxicin were applied to test plots public health risks.

on grassed buffer strips and subjected to simulated rainfall. Antibiotic Resistance Genes. Heuer and co–

Antibiotic concentrations in run–off were measured using workers investigated the accumulation of sulfonamide

enzyme–linked immunosorbent assays (ELISAs). Grass resistance genes in soil after repeated applications of swine

widths of 4 to 8 m reduced dissolved sulfamethazine manure containing sulfadiazine (SDZ) (Heuer et al. 2011).

transport in the surface runoff by more than 70%. Tall Manure with and without SDZ was land applied to two

fescue was overall most effective at reducing dissolved agricultural soils and the sulfonamide resistance genes sul1

tylosin and enrofloxacin transport in the runoff (>75%). and sul2, which code for dihydropteroate synthases

The effects of manure and pH were examined by insensitive to sulfonamides, were monitored 60 days after

Strauss et al. (2011), where they tested solute displacement each manure application. Results show that the resistance

of sulfadimethoxine, sulfamethazine, and sulfamethoxazole genes increased by up to 3 orders of magnitude in soil

at different pH (5, 6.5, 8.5) with and without manure. They receiving SDZ–containing manure than in soils receiving

found that recovery of sulfonamides decreased with SDZ–free manure. It was suggested that elevated sul1 and
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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
sul2 levels in soil were due to the selective pressure exerted chloramphenicol and tetracycline, 1 to >256 µg/mL for

by bioavailable SDZ in soil. nalidixic acid and 12 to >256 µg/mL for penicillin.

Meyer et al. (2011) compared run–off losses of However, no significant correlation was found between

pesticides and pharmaceuticals, and steroid hormones in antimicrobial resistance and dairy farm age.

two small watersheds in Luxemborg. Ibuprofen, Another study described the antibiotic resistance

sulfamethoxazole, diclofenac, and estrone were detected in profiles of E. coli isolates from swine manure, lagoon

storm run–off and related to other water quality effluent, and soils that received the lagoon effluent (Graves

measurements, though the locations of source areas could et al. 2011). A total of 616 E. coli isolates were obtained

not be established. from manure, lagoon and soil on a swine farm in North

Veach and Bernot (2011) report on a study of Carolina. 60.6% to 91.3% of the isolates harbored genes

pharmaceutical levels in 2 streams in central Indiana with coding resistance mechanism against

both agricultural and suburban land use. Twelve streptomycin/spectinomycin (aadA/strA and strB),

pharmaceuticals were detected including acetaminophen, tetracycline (tetA and tetB), and sulfonamide (sul1). The

caffeine, carbamazepine, cotinine, N,N–diethyl–meta– authors concluded that the occurrence of specific antibiotic

toluamide (DEET), gemfibrozil, ibuprofen, resistance genes (ARGs) varied with seasons as well as the

sulfadimethoxine, sulfamethazine, sulfamethoxazole, environmental conditions of the locations on the swine

triclosan, and trimethoprim. Concentrations of farm.

pharmaceuticals were comparable and increased during the To understand the environmental impact of

winter at both locations and decreased in spring and antibiotic application in plant agriculture, Walsh and co–

summer. Pharmaceutical levels were not correlated with workers developed multiplex quantitative PCR (qPCR)

nitrogen isotope analysis of sediment, though the δ15N of assays for streptomycin (strA, strB, aadA and insertion

sediment was higher in the urban influenced watershed. sequence IS1133) and tetracycline (tetB, tetM and tetW)

Correlation was sought between the antimicrobial resistance genes and applied them on orchard soil samples

resistance profiles of Escherichia coli isolates and the age (Walsh et al. 2011). Among these genes, strB, aadA, tetB

of dairy facilities (Jones et al. 2011). E. coli was isolated and tetM were detected most frequently. After application

from the topsoil of 11 dairy farms with varying ages (0.5 – of swine manure, the relative concentration of tetB and

24 years) and the minimum inhibitory concentrations streptomycin resistance genes in soil decreased, while the

(MICs) of the isolates to antimicrobials were determined quantity of tetM and tetW either remained at the same level

using E–test strips and gradient plate method. The MICs of or increased.

the isolates ranged from 0.75 to >256 µg/mL for The occurrence of resistance to heavy metals and

antibiotics were reported for Pseudomonas isolates from

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river water and agricultural soils irrigated with wastewater methicillin resistance gene (mecA) in 71 phage DNA

and groundwater (Malik and Aleem 2011). This study samples from livestock fecal wastes/wastewater using

showed that the majority of the Pseudomonas isolates qPCR. They reported that the abundance of ARGs in the

exhibited resistance to multiple heavy metals and phage samples ranged from 103 to 104 copies/g of slurry or

antibiotics. For example, among the 48 Pseudomonas mL of wastewater for blaTEM, 102 to 103 copies/g or mL

isolates from wastewater–irrigated soil, 87.5% were for blaCTX–M, and 101 to 103 copies/g or mL for mecA.

resistant to sulfadiazine, 79.1% resistant to both ampicillin The authors suggested in addition to selective pressure due

and erythromycin, and 20.8% co–resistant to eight heavy to environmental residue of antibiotics, the ARGs on

metals. In comparison, lower percentages of the isolates bacteriophages could also contribute to the proliferation of

from groundwater–irrigated soil exhibited resistance to antibiotic resistance in the environment.

heavy metals and antibiotics. Munir and Xagoraraki studied the fate of ARGs

Torres–Cortes and co–workers identified and (tetW, tetO, and sul1) in soil after land application of

characterized novel antibiotic resistance genes in soil manure and biosolids at two experimental sites (Munir and

(Torres-Cortes et al. 2011). A functional metagenomic Xagoraraki 2011). The levels of ARGs in manure and

approach was used to search for novel resistance genes in biosolids ranged from 108 to 1010 copies/g, much higher

three soil samples by constructing metagenomic libraries than the background levels of 104–106 copies/g in

and isolating resistant clones. By using this approach, 11 untreated soil. Over a four–month period after manure

novel antibiotic resistance genes were identified: 3 and/or biosolids were land applied at the two sites, the

conferring resistance to ampicillin, 2 to gentamicin, 2 to ARGs in soil appeared to increase at one site but not the

chloramphenicol, and 4 to trimethoprim. This study other. It was suggested that the differences of the fate of

indicates that soil bacteria are a reservoir of antibiotic ARGs at the two sites might be affected by environmental

resistance genes. In addition, the authors found that a conditions and soil types.

reductase belonging to the dihydrofolate reductase group Lanthier and co–workers characterized the

conferred resistance to trimethoprim, demonstrating the antibiotic resistance profile of enterococci species in an

advantage of functional metagenomics in discovering novel agriculture intensive drainage basin (Lanthier et al. 2011).

antibiotic resistance functions of enzymes. The authors isolated 1558 enterococci isolates from the

Bacteriophages could be an environmental vector South Nation River watershed over a 3–year period, and

for the horizontal transfer of antibiotic resistance genes in determined their virulence and antibiotic resistance using

the environment (Colomer-Lluch et al. 2011). Colomer– PCR and culture methods, respectively. Results showed

Lluch and co–workers quantified two β–lactam antibiotics that 28.5% of the isolates were resistant to lincomycin

resistance genes (blaTEM and blaCTX–M) and one while less than 2% to ciprofloxacin and vancomycin, two
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category I antibiotics. The authors concluded that the significant correlation was observed between quartz

chance of dispersion of antibiotic resistance via water borne attachment and selected genes encoding attachment factors.

enterococci in an agricultural drainage basin is small. Prions as Emerging Contaminants

Graham led a study to investigate the The prion, or PrPSc, is believed to be the

contribution of various environmental pollutants (i.e., infectious agent responsible for prion diseases which are

antibiotic, heavy metals, and organic pollutants) to the also called transmissible spongiform encephalopathies

emergence of antibiotic resistance in the environment (TSEs). In recent years, prions have gained increasing

(Graham et al. 2011). The authors collected samples from attention from the public as an emerging contaminant due

8 locations along the Almendares River in western Havana, to its fast and facile spread among cervids (chronic wasting

Cuba, where industrial pollution widely exists but disease, CWD), ovines (scrapie), and bovines (mad cow

antibiotics are only used sparingly. High levels of ARGs disease, or bovine spongiform encephalopathy, BSE) and

(>10–4 copies/16S rRNA gene) were detected in water and also because of the relationship between consumption of

sediment of the river, which passes through various land infected cattle and incidence of human prion diseases such

uses such as urban agriculture and industrial zones. as Creutzfeldt–Jakob disease (CJD) and others. It is very

Statistical analysis indicated that certain ARGs (tetM, tetO, likely that soil serves as an important environmental

tetQ, tetW, blaOXA) were correlated to Cu and ampicillin. reservoir for prions, and soil is though to play an important

Although the study does not give a definitive answer on the role in indirect transmission of prion diseases among

relative influence of each pollutant on ARG occurrence, it animals. Direct contact between animals is another major

showed that unregulated pollution has the potential of route for prion disease transmission in that infectious prions

affecting ARGs. have been detected in saliva, urine, and blood as well as in

The association between antibiotic resistance and the central nervous system (CNS).

bacterial attachment was investigated for agricultural E. There are many studies in 2011 that provided

coli isolates (Liu et al. 2011). Liu and co–workers more information on the detection of prion in vivo, the

examined 203 E. coli isolates from swine facilities and development of prions after uptake, and the fate and

found that bacterial attachment to quartz was positively transport of prions in the environment. These results would

related to combined resistance to 6 antibiotics (i.e., be useful for prion disease control and prevention of

amoxicillin, streptomycin, tetracycline, sulfamethazin, environment contamination by prions.

tylosin and chlortetracycline) but negatively related to Prion Detection Methods. Currently, the most

combined resistance to 3 other antibiotics (i.e., nalidixic popular TSEs diagnostic techniques are based on the

acid, kanamycin, and neomycin). Surprisingly, no immunodetection of PrPSc by Western blotting (WB),

enzyme–linked immunosorbent assay (ELISA), and

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immunohistochemistry (IHC). They have been applied to a detection of PrPSc from PASA in urine by combining of

number of studies identifying the existence of prions SOFIA and PMCA indicated an alternate for antemortem

resistant to proteolysis. However, they are not sufficiently diagnosis of prion disease.

sensitive to detect prions at extremely low levels. Although widely used in prion detection, PMCA

This problem has been solved partially by the detection of human prions in accessible body fluids was

development of a more sensitive method called protein unsuccessful. For this reason, a highly sensitive assay

misfolding cyclic amplification (PMCA). It is an in–vitro named ‘real–time QUIC’ (RT–QUIC) for the detection of

technique simulating the conversion and replication of prion in human cerebrospinal fluid (CSF) was established

cellular prion protein (PrPC) to its infectious isoform (Atarashi et al. 2011). This method was a combination of

(PrPSc) by repeated incubation and sonication of prion seed QUIC technology and thioflavin T (ThT) fluorescence dye

and uninfected substrate. This new technique had then been to monitor amyloid formation which possibly minimized

used widely in the detection of prions in various tissues and the time necessary for the detection of protease–resistant

excreta which contain trace amount of prions that were recombinant PrP. RT–QUIC had been successfully applied

unable to be detected by traditional methods (Garza et al. to the assessment of more than 200 CSF specimens from

2011; Haley et al. 2011; Rubenstein et al. 2011; Terry et al. Japanese and Australian patients with a more than 80%

2011). accuracy for the correct diagnosis of CJD. This method

Higher efficiency of PrPSc replication was could also used for other prions and was able to quantify

reported by Gonzalez-Montalban et al. (2011) by PASA when combined with an end–point dilution of

modifying the original PMCA technique. The addition of samples.

small amount of Teflon beads was found to increase the Occurrence of Prions. Prions have been

conversion of PrPC to PrPSc from ~10% to up to 100% for detected in the excretions, blood, and birthing matter of

selected prion strains. The sensitivity of prion detection in CWD positive cervids and scrapie positive animal models,

one PMCA round (24 hours) was observed to increase by 2 environmental surfaces accessible for infected ovine

to 3 orders of magnitude. animals, and natural aqueous environment. To identify the

Based on the application of PMCA, Rubenstein et source(s) of infectious prions in the excreta, a study

al. (2011) provided the first report on prion disease– conducted by Haley et al. (2011) analyzed the tissues

associated seeding activity (PASA) detection of PrPSc proximate to saliva, urine, and fecal production of 27

from the urine of naturally or preclinical prion–diseased white–tailed deer exposed to CWD positive/negative

ovine or cervids. Detection was achieved by using the sources by serial PMCA. PrPCWD–generating activity was

surround optical fiber immunoassay (SOFIA) to measure detected in a range of tissues and was highest in the

the products of limited serial PMCA. This newly developed salivary gland, urinary bladder, and distal intestinal tract.
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Deer with highest levels of PrPCWD amplification in the agents. Okada et al. (2011) analyzed 28 calves exposed to

brain had higher and more widely disseminated prion 5 g of homogenized brainstems confirmed as BSD agents.

amplification in excretory tissues. Different results for the detection of prion were found in

Improvements on prion detection from ovine different places in the continuous Peyer’s patch and the

animals were also made. Based on the previous results of CNS, providing important information for understanding

detection of prions from excreta, blood, and birthing matter the pathogenesis of BSE. The other study investigated the

of animal models, Terry et al. (2011) identified the occurrence and distribution of disease–associated prion in

existence of prion in the faeces of sheep naturally infected three different parts in the small intestine of cattle orally

with classical scrapie by using serial PMCA along with two exposed to a 1 g or 100 g dose of a titrated BSE brainstem

extraction methods. 7 of 15 sheep in clinical phase and 14 homogenate Stack et al. (2011). Results were relative to the

of 14 sheep in preclinical phase showed amplification of exposure dose and age, and suggested a very low BSE risk

PrPSc in their feaces, demonstrating that prion could be from food products containing the jejunum and duodenum

shedded in faeces from a naturally infected host and was a of cattle slaughtered for human consumption.

likely source of prion contamination in the environment. To identify the first uptake and replication sites

Besides faeces, prions were also successfully of prion in the gut, Kujala et al. (2011) reported the

detected in the liver of sheep naturally infected with scrapie trafficking of prion toward Peyer’s patches of wild type and

and challenged with BSE, at both clinical and preclinical PrP–deficient mice using high–resolution

stages of the disease. The result showed that PrPSc immunofluorescence and cryo–immunogold electron

accumulated in the liver of 89% of sheep naturally infected microscopy. At 7–21 day post feeding (dpf), increased PrP

with scrapie and 100% of sheep challenged with BSE labeling was observed on the plasma membranes of

(Everest et al. 2011). Moreover, in utero vertical follicular dendritic cells (FDCs) in germinal centres of

transmission of scrapie has also been studied by Garza et Peyer’s patches from wild type mice, identifying FDCs as

al. (2011). Six fetuses from three natural infected pregnant the first sites of PrP conversion and replication.

ewes in an advanced clinical stage of scrapie were Fate and Transport of Prions. Prions can enter

analyzed. For each fetus, samples from amniotic fluid, the environment through decomposition of infected animal

brain, spleen, ileo–cecal valve and retropharyngeal lymph carcasses or shedding from pre–clinical or clinical host

node showed amplification after PMCA, suggesting the animals, and thus will enter the soil matrix or the aqueous

possibility of in utero transmission of scrapie in natural environment. While prions can bind to soils, the interaction

infected sheep. of prions with soil was found to be dependent on the

Two studies from Europe and Japan respectively solvent, aging time, and soil types.

reported detection of prion in cattle orally exposed to BSE

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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
 Saunders et al. (2011)c investigated hyper (HY) al. 2011a; Miles Miles et al. 2011b). Different temperature

strain of transmissible mink encephalopathy (TME) ranges were utilized to treat prions in water and biosolids

adsorption to soil minerals in different aqueous solutions for different incubation time periods, and a scrapie cell

including phosphate buffered saline (PBS), sodium assay was performed to determine prion infectivity and

chloride, calcium chloride, and deionized water. Samples quantity. Extraction of prions from biosolids was used with

incubated at different time periods were also studied with 4 M urea at 80°C for 10 minutes followed by a membrane

WB and PMCA for replication ability analysis. They found centrifugation to reduce the concentration of urea.

that adsorption solution chemistry could affect prion Reduction of infectious prions was reported for prions both

replication and protease–resistance, especially after in water and Class B biosolids, with higher reduction in

incubation periods of 30 d or longer. However, soil bound biosolids.

prions could remain a risk for prion disease transmission While a lot of effort devoted by researchers to the

after months in the environment. investigation of fate and transport of prions in soil and

In addition to the previously mentioned factors, water, concerns on the transmission of prions by aerosols

soil type was suggested to be an important factor on the arose (Stitz and Aguzzi 2011). Aerosols can originate very

behavior of soil bound prions (Saunders et al. 2011b). easily in a broad variety of experimental and natural

When adsorbed to soil and soil minerals, HY TME yielded environmental conditions. They are a virtually unavoidable

a greater–than–one–log decrease in PMCA replication consequence of the handling of fluids thus complete

efficiency when binding to silty clay loam soil (SCL). prevention of the generation of aerosols is very difficult.

Clay– and organic surface– bound HY exhibited Although there is no strong evidence indicating that prions

significantly lower replication efficiencies compared with are airborne under natural circumstances, a relative study

sand–bound HY. Furthermore, infectivity of SCL–HY was has reported that mouse scrapie can be efficiently

decreased by a 1.3–log in titer comparing to unbound transmitted via aerosols (Haybaeck et al. 2011), leading to

control. These data suggested that dominant soil types in a re–thinking on prion biosafety guidelines in research and

local sites may be a significant determinant in the diagnostic laboratories due to the potential risk of airborne

environmental transmission of prion diseases. And an prion transmission.

analysis from Walter et al. (2011) indicated that 1% Degradation and Inactivation of Prions. Prions

increase in the clay–sized particle content in soils within are known to be quite resistant to the conventional

the approximate home range of an individual deer increased inactivation methods such as proteases, heat, radiation, and

its odds of infection by up to 8.9%. formalin treatments, although their infectivity can be

Miles et al. evaluated the effect of temperature on reduced by such treatments. Efficient inactivation options

the fate of prions in water and Class B biosolids (Miles et may involve incineration, autoclaving, and/or harsh
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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
chemical treatments which are not practical for most providing a more practical option for the reduction of the

environmental applications. Therefore, more applicable risk of prion disease transmission via soil or other

degradation and inactivation methods in situ for prions environmental surfaces.

were explored. Other than application of mild treatment by

One of the related studies was conducted by Piro enzyme, two more studies focused on the degradation of

et al. (2011) who assumed that photodegradation of prions with more environmentally friendly methods.

incorporated polyanions would alter prion infectivity Johnson et al. (2011) found that acetone extracts of three

because polyanions were indicated as a possible cellular lichen species (Parmelia sulcata, etc) had the ability to

cofactor necessary for the formation of infectious prions in degrade prion protein from TSE–infected hamsters, mice,

vitro. To test this hypothesis, synthetic prions were and deer. Reduction of PrPTSE was reported to be more

produced using a photocleavable, 100–base oligonucleotide than two logs measured by immunoblots and PMCA.

(PC–oligo). In serial PMCA reactions, PC–oligo was Additionally, reduced levels in PrPTSE–enriched preps or

incorporated into physical complexes with PrPSc molecules infected brain homogenates were also found following

resistant to benzonase digestion and then exposed to long exposure to freshly–collected P. sulcata or an aqueous

wave ultraviolet light (315 nm) which induced degradation extract of the lichen. Lichen is ubiquitous in the

of PC–oligo into 5 base fragments. However, the bioassay environment and has strong survival ability in challenging

in hamsters indicated that light–induced photolysis of ecological niches, its inactivation ability for prions suggests

incorporated PC–oligo did not alter the infectivity of in a possible source for agents to degrade prions. Observation

vitro–generated prions, which also meant that intact of proteolysis of a recombinant prion in a lamb brain–

polyanions were not required for the maintenance of prion amended loamy soil has been reported by Rapp et al.

infectivity. (2011). In this study, a 15N–labelled recombinant PrP

Saunders et al. investigated the inactivation (rPrP) was added to soil in which microbial biomass and

efficiency of soil bound prion with a commercially soil proteolytic activity had been increased by either

purchased prionzyme (Saunders et al. 2011a). HY TME simultaneous or prior amendment with lam brain. The

was adsorbed to a variety of soil and soil minerals at majority of the recovered rPrP–N was associated with the

preferred conditions and then was treated with the soil particles, which drew the conclusion that high content

prionzyme under environmentally relevant conditions of animal organic matter with the sorption properties of

(22°C, pH 7.4). The amplification efficiency of treated soil prion protein and stimulation of the soil microbial

samples was compared to controls of known infectious component were required to predict prion transmission in

titer. Results suggested 104– to > 106–fold decreases in soil.

soil bound prion infectivity following enzyme treatment,

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Water Environment Research, Volume 84, Number 10—Copyright © 2012 Water Environment Federation
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