Diana S. Aga

Potential ecological and human health impacts of antibiotics and antibiotic-resistant bacteria from wastewater treatment plants.

The occurrence of antibiotics and other pharmaceuticals in the environment has become an increasing public concern as recent environmental monitoring activities reveal the presence of a broad range of persistent pharmaceuticals in soil and water. Studies show that municipal wastewater treatment plants (WWTPs) are important point sources of antibiotics and antibiotic-resistant bacteria in the environment. The fate of antibiotics and other pharmaceuticals in WWTPs is greatly influenced by the design and operation of treatment systems. Because knowledge on the fate of antibiotics and resistant bacteria in WWTPs is important in estimating their potential impacts on ecology and human health, investigations on occurrence, treatment, and observed effects are reviewed in this article. In addition, human health risk assessment protocols for antibiotic and resistant bacteria are described. Although data on other pharmaceutical compounds are also presented, discussion is focused on antibiotics in the environment because of the potential link to increased emergence of resistance among pathogenic bacteria. The applications of modern analytical methods that facilitate the identification of novel transformation products of pharmaceuticals in environmental matrices are also included to illustrate that the disappearance of the parent pharmaceuticals in WWTPs does not necessarily equate to their complete removal.

Humic Acid-Induced Silver Nanoparticle Formation Under Environmentally Relevant Conditions

The formation of silver nanoparticles (AgNPs) via reduction of silver ions (Ag(+)) in the presence of humic acids (HAs) under various environmentally relevant conditions is described. HAs tested originated from the Suwannee River (SUW), and included samples of three sedimentary HAs (SHAs), and five soils obtained across the state of Florida. The time required to form AgNPs varied depending upon the type and concentration of HA, as well as temperature. SUW and all three SHAs reduced Ag(+) at 22 °C. However, none of the soil HAs formed absorbance-detectable AgNPs at room temperature when allowed to react for a period of 25 days, at which time experiments were halted. The appearance of the characteristic surface plasmon resonance (SPR) of AgNPs was observed by ultraviolet-visible spectroscopy in as few as 2-4 days at 22 °C for SHAs and SUW. An elevated temperature of 90 °C resulted in the accelerated appearance of the SPR within 90 min for SUW and all SHAs. The formation of AgNPs at 90 °C was usually complete within 3 h. Transmission electron microscopy and atomic force microscopy images showed that the AgNPs formed were typically spherical and had a broad size distribution. Dynamic light scattering also revealed polydisperse particle size distributions. HAs appeared to colloidally stabilize AgNPs based on lack of any significant change in the spectral characteristics over a period of two months. The results suggest the potential for direct formation of AgNPs under environmental conditions from Ag(+) sources, implying that not all AgNPs observed in natural waters today may be of anthropogenic origin.

Evaluating the biodegradability of sulfamethazine, sulfamethoxazole, sulfathiazole, and trimethoprim at different stages of sewage treatment

The aerobic biodegradability of four antimicrobials (sulfamethazine, sulfamethoxazole, sulfathiazole, and trimethoprim) was investigated in sewage collected at four treatment stages (primary treatment, activated sludge treatment, aerobic nitrification process, and after disinfection of treated sewage) of a municipal sewage treatment plant. The biodegradability tests were conducted in aerated batch reactors by spiking the sewage with 20 microg/L of each of the test substance. Concentration profiles of the assayed compounds were monitored during a 54-d period using liquid chromatography/electrospray ionization/mass spectrometry. Substantial differences in the degradation curves were observed between trimethoprim and the three sulfonamides. The behavior of the latter was characterized by a general biodegradability in the primary and secondary treatment. The highest degradation rates were obtained in the sewage from the activated sludge treatment, where no adaptation phase was observed. On the other hand, the onset of biodegradation in the sewage from the primary treatment was preceded by a lag phase ranging from 10 to 15 d. In contrast, trimethoprim displayed high resistance to microbial degradation in the sewage from the primary treatment and the activated sludge treatment. However, primary degradation of this compound was completed within only 3 d in the sewage from the nitrification process.

Pharmaceutical metabolites in the environment: analytical challenges and ecological risks.

The occurrence of human and veterinary pharmaceuticals in the environment has been a subject of concern for the past decade because many of these emerging contaminants have been shown to persist in soil and water. Although recent studies indicate that pharmaceutical contaminants can pose long-term ecological risks, many of the investigations regarding risk assessment have only considered the ecotoxicity of the parent drug, with very little attention given to the potential contributions that metabolites may have. The scarcity of available environmental data on the human metabolites excreted into the environment or the microbial metabolites formed during environmental biodegradation of pharmaceutical residues can be attributed to the difficulty in analyzing trace amounts of previously unknown compounds in complex sample matrices. However, with the advent of highly sensitive and powerful analytical instrumentations that have become available commercially, it is likely that an increased number of pharmaceutical metabolites will be identified and included in environmental risk assessment. The present study will present a critical review of available literature on pharmaceutical metabolites, primarily focusing on their analysis and toxicological significance. It is also intended to provide an overview on the recent advances in analytical tools and strategies to facilitate metabolite identification in environmental samples. This review aims to provide insight on what future directions might be taken to help scientists in this challenging task of enhancing the available data on the fate, behavior, and ecotoxicity of pharmaceutical metabolites in the environment.

Elucidating the Relative Roles of Ammonia Oxidizing and Heterotrophic Bacteria during the Biotransformation of 17α-Ethinylestradiol and Trimethoprim

The biological fate of 17α-ethinylestradiol (EE2; 500 ng/L to 1 mg/L) and trimethoprim (TMP; 1 μg/L to 1 mg/L) was evaluated with flow through reactors containing an ammonia oxidizing bacterial (AOB) culture, two enriched heterotrophic cultures devoid of nitrifier activity, and nitrifying activated sludge (NAS) cultures. AOBs biotransformed EE2 but not TMP, whereas heterotrophs mineralized EE2, biotransformed TMP, and mineralized EE2-derived metabolites generated by AOBs. Kinetic bioassays showed that AOBs biotransformed EE2 five times faster than heterotrophs. The basal expression of heterotrophic dioxygenase enzymes was sufficient to achieve the high degree of transformation observed at EE2 and TMP concentrations ≤ 1 mg/L, and enhanced enzyme expression was not necessary. The importance of AOBs in removing EE2 and TMP was evaluated further by performing NAS experiments at lower feed concentrations (500-1000 ng/L). EE2 removal slowed markedly after AOBs were inhibited, while TMP removal was not affected by AOB inhibition. Two key EE2 metabolites formed by AOB and heterotrophic laboratory-scale chemostats were also found in independent laboratory-scale mixed culture bioreactors; one of these, sulfo-EE2, was largely resistant to further biodegradation. AOBs and heterotrophs may cooperatively enhance the reliability of treatment systems where efficient removal of EE2 is desired.

Application of ELISA in determining the fate of tetracyclines in land-applied livestock wastes.

The potential use of a class-specific enzyme-linked immunosorbent assay (ELISA) in studying the occurrence and fate of tetracyclines in the environment was evaluated. Several manure samples collected from hog lagoons and cattle feedlots were screened for the presence of tetracycline residues using ELISA. The levels varied from less than the detection limit (0.5 parts per billion) to 200 parts per million. The degradation of tetracyclines in soil-applied manure was followed using ELISA to measure the decline in tetracycline concentrations. Low levels of tetracyclines remained detectable in soil for up to 28 days. The ELISA procedure also proved useful in determining the leaching potential of tetracyclines in undisturbed soil columns and in the analysis of total tetracyclines in manure, soil, and water. Based on the cross-reactivity of the antibodies employed, this ELISA method can be an important screening tool for the presence of other tetracycline compounds, such as chlortetracycline and oxytetracycline. The ELISA method also detects the epimers of tetracyclines and the corresponding dehydration by-products, anhydrotetracyclines. Analysis of selected manure extracts by liquid chromatography with mass spectrometry (LC-MS) showed lower concentrations of total tetracyclines compared to the values obtained by ELISA, indicating the presence of other structurally related compounds or transformation products of tetracyclines being detected by ELISA in the samples. Because analysis of manure and soil samples by LC-MS requires extensive clean-up procedures, ELISA provides an alternative method for conducting environmental fate and transport studies of antibiotics.

Enhanced biodegradation of carbamazepine after UV/H2O2 advanced oxidation.

Carbamazepine is one of the most persistent pharmaceutical compounds in wastewater effluents due to its resistance to biodegradation-based conventional treatment. Advanced oxidation can efficiently degrade carbamazepine, but the toxicity and persistence of the oxidation products may be more relevant than the parent. This study sets out to determine whether the products of advanced oxidation of carbamazepine can be biotransformed and ultimately mineralized by developing a novel methodology to assess these sequential treatment processes. The methodology traces the transformation products of the (14)C-labeled carbamazepine during UV/hydrogen peroxide advanced oxidation and subsequent biotransformation by mixed, undefined cultures using liquid scintillation counting and liquid chromatography with radioactivity, mass spectrometry, and UV detectors. The results show that the oxidation byproducts of carbamazepine containing a hydroxyl or carbonyl group can be fully mineralized by a mixed bacterial inoculum. A tertiary treatment approach that includes oxidation and biotransformation has the potential to synergistically mineralize persistent pharmaceutical compounds in wastewater treatment plant effluents. The methodology developed for this study can be applied to assess the mineralization potential of other persistent organic contaminants.

Investigating uptake of water-dispersible CdSe/ZnS quantum dot nanoparticles by Arabidopsis thaliana plants

Interest on the environmental impacts of engineered nanomaterials has rapidly increased over the past years because it is expected that these materials will eventually be released into the environment. The present work investigates the potential root uptake of water-dispersible CdSe/ZnS quantum dots (QDs) by the model plant species, Arabidopsis thaliana. Experiments revealed that Arabidopsis exposed to QDs that are dispersed in Hoaglands solution for 1-7 days did not internalize intact QDs. Analysis of Cd and Se concentrations in roots and leaves by inductively-coupled plasma mass spectrometry indicated that Cd and Se from QD-treated plants were not translocated into the leaves, and remained in the root system of Arabidopsis. Furthermore, fluorescence microscopy showed strong evidence that the QDs were generally on the outside surfaces of the roots, where the amount of QDs adsorbed is dependent on the stability of the QDs in suspension. Despite no evidence of nanoparticle internalization, the ratio of reduced glutathione levels (GSH) relative to the oxidized glutathione (GSSG) in plants decreased when plants were exposed to QD dispersions containing humic acids, suggesting that QDs caused oxidative stress on the plant at this condition.

A systematic investigation to optimize simultaneous extraction and liquid chromatography tandem mass spectrometry analysis of estrogens and their conjugated metabolites in milk

In this study, the simultaneous extraction of estrone (E1), 17beta-estradiol (E2), estriol (E3), ethinylestradiol (EE2), and their glucuronated and sulfated metabolites in milk was optimized using solid-phase extraction (SPE). The aim of this research was to analyze estrogens and their conjugated metabolites by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in a single run, without the need to perform enzymatic cleavage and derivatization. Two SPE cartridges in tandem were used, consisting of sorbents based on the hydrophilic-lipophilic balance and amine-functionalized packing materials. To monitor analyte loss at every step of the SPE procedure (14)C-labeled E2 was spiked into the milk sample and the radioactivity was monitored at all stages of the SPE. In addition, non-radiolabeled standards of estrogens and metabolites were used to optimize solvent systems for the SPE and LC-MS/MS. The optimized method described in this paper can achieve recoveries ranging from 72% to 117% for the free estrogens (E1, E2, E3, and EE2), and 62% to 112% for seven conjugated metabolites. The three doubly conjugated, highly polar metabolites included in this study gave lower recoveries (< or = 43%) due to poor retention in SPE. Finally, commercial milk samples were analyzed for the presence of estrogens and their conjugated metabolites. Estrone (concentration range: 23-67 ng/L) was found to be the major free estrogen present in all milk samples. Estradiol was consistently observed in milk, but the concentrations were below the limit of detection (LOD of 10 ng/L), and no estriol and ethinylestradiol were detected. Several conjugated estrogen metabolites were identified, 17beta-estradiol-3-glucuronide (71-289 ng/L), estrone-3-sulfate (60-240 ng/L), 17beta-estradiol-3,17beta-sulfate (< LOD to 30 ng/L), and estrone-3-glucuronide (< LOQ of 25 ng/L). This method proved efficient in the simultaneous analysis of estrogens and their metabolites in milk.

Assessing antibiotic sorption in soil: a literature review and new case studies on sulfonamides and macrolides

The increased use of veterinary antibiotics in modern agriculture for therapeutic uses and growth promotion has raised concern regarding the environmental impacts of antibiotic residues in soil and water. The mobility and transport of antibiotics in the environment depends on their sorption behavior, which is typically predicted by extrapolating from an experimentally determined soil-water distribution coefficient (Kd). Accurate determination of Kd values is important in order to better predict the environmental fate of antibiotics. In this paper, we examine different analytical approaches in assessing Kd of two major classes of veterinary antibiotics (sulfonamides and macrolides) and compare the existing literature data with experimental data obtained in our laboratory. While environmental parameters such as soil pH and organic matter content are the most significant factors that affect the sorption of antibiotics in soil, it is important to consider the concentrations used, the analytical method employed, and the transformations that can occur when determining Kd values. Application of solid phase extraction and liquid chromatography/mass spectrometry can facilitate accurate determination of Kd at environmentally relevant concentrations. Because the bioavailability of antibiotics in soil depends on their sorption behavior, it is important to examine current practices in assessing their mobility in soil.