Impact of Redox Conditions on Antibiotic Resistance Conjugative Gene Transfer Frequency and Plasmid Fate in Wastewater Ecosystems.

Wastewater is a common pathway for the spread of antibiotic resistance (AR) genes and bacteria into the environment. Biological treatment can mitigate this path, but horizontal gene transfer (HGT) between bacteria also occurs in such processes, although the influence of bioreactor habitat and ecology on HGT frequency is not well understood. Here, we quantified how oxidation-reduction (redox) conditions impact the fate of a Green fluorescent protein (Gfp)-tagged AR plasmid (pRP4-gfp) within an E. coli host (EcoFJ1) in the liquid phase and biofilms in bioreactors. Replicate reactors treating domestic wastewater were operated under stable aerobic (+195 ± 25 mV), anoxic (-15 ± 50 mV), and anaerobic (-195 ± 15 mV) conditions, and flow cytometry and selective plating were used to quantify donor strain, EcoFJ1(pRP4-gfp), and putative transconjugants over time. Plasmid pRP4-gfp-bearing cells disappeared rapidly in aerobic ecosystems (∼2.0 log reduction after 72 h), especially in the liquid phase. In contrast, EcoFJ1(pRP4-gfp) and putative transconjugants persisted much longer in anaerobic biofilms (∼1.0 log reduction, after 72 h). Plasmid transfer frequencies were also higher under anaerobic conditions. In parallel, protozoan abundances were over 20 times higher in aerobic reactors relative to anaerobic reactors, and protozoa numbers significantly inversely correlated with pRP4-gfp signals across all reactors (p < 0.05). Taken together, observed HGT frequency and plasmid retention are impacted by habitat conditions and trophic effects, especially oxygen conditions and apparent predation. New aerobic bioreactor designs are needed, ideally employing passive aeration to save energy, to minimize resistance HGT in biological wastewater treatment processes.

Multi-laboratory Validation of a New Marine Biodegradation Screening Test for Chemical Persistence Assessment.

Current biodegradation screening tests are not specifically designed for persistence assessment of chemicals, often show high inter- and intra-test variability, and often give false negative biodegradation results. Based on previous studies and recommendations, an international ring test involving 13 laboratories validated a new test method for marine biodegradation with a focus on improving the reliability of screening to determine the environmental degradation potential of chemicals. The new method incorporated increased bacterial cell concentrations to better represent the microbial diversity; a chemical is likely to be exposed in the sampled environments and ran beyond 60 days, which is the half-life threshold for chemical persistence in the marine environment. The new test provided a more reliable and less variable characterization of the biodegradation behavior of five reference chemicals (sodium benzoate, triethanolamine, 4-nitrophenol, anionic polyacrylamide, and pentachlorophenol), with respect to REACH and OSPAR persistence thresholds, than the current OECD 306 test. The proposed new method provides a cost-effective screening test for non-persistence that could streamline chemical regulation and reduce the cost and animal welfare implications of further higher tier testing.

ECOdrug: a database connecting drugs and conservation of their targets across species.

Pharmaceuticals are designed to interact with specific molecular targets in humans and these targets generally have orthologs in other species. This provides opportunities for the drug discovery community to use alternative model species for drug development. It also means, however, there is potential for mode of action related effects in non-target wildlife species as many pharmaceuticals reach the environment through patient use and manufacturing wastes. Acquiring insight in drug target ortholog predictions across species and taxonomic groups has proven difficult because of the lack of an optimal strategy and because necessary information is spread across multiple and diverse sources and platforms. We introduce a new research platform tool, ECOdrug, that reliably connects drugs to their protein targets across divergent species. It harmonizes ortholog predictions from multiple sources via a simple user interface underpinning critical applications for a wide range of studies in pharmacology, ecotoxicology and comparative evolutionary biology. ECOdrug can be used to identify species with drug targets and identify drugs that interact with those targets. As such, it can be applied to support intelligent targeted drug safety testing by ensuring appropriate and relevant species are selected in ecological risk assessments. ECOdrug is freely accessible and available at: http://www.ecodrug.org.

Spatially Explicit Large-Scale Environmental Risk Assessment of Pharmaceuticals in Surface Water in China.

With improving healthcare and an aging population, the consumption of human pharmaceuticals in China has been increasing dramatically. Environmental risks posed by many active pharmaceutical ingredients (APIs) are still unknown. This study used a spatially explicit dilution-factor methodology to model predicted environmental concentrations (PECs) of 11 human-use APIs in surface water for a preliminary environmental risk assessment (ERA). Median PECs in surface water across China range between 0.01 and 8.0 × 103 ng/L for the different APIs, under a moderate patient use scenario. Higher environmental risks of APIs in surface water are in regions with high water stress, e.g., northern China. Levonorgestrel, estradiol, ethinyl estradiol and abiraterone acetate were predicted to potentially pose a high or moderate environmental risk in China if consumption levels reach those in Europe. Relative risks of these four APIs have the potential to be among those chemicals with the highest impact on surface water in China when compared to the risks associated with other regulated chemicals, including triclosan and some standard water quality parameters including BOD5 (5-day biological oxygen demand), COD (chemical oxygen demand), Cu, Zn, and Hg and linear alkylbenzene sulfonate. This method could support the regulation of this category of chemicals and risk mitigation strategies in China.

Application of prioritization approaches to optimize environmental monitoring and testing of pharmaceuticals.

Pharmaceuticals are ubiquitous in the natural environment with concentrations expected to rise as human population increases. Environmental risk assessments are available for a small portion of pharmaceuticals in use, raising concerns over the potential risks posed by other drugs that have little or no data. With >1900 active pharmaceutical ingredients in use, it would be a major task to test all of the compounds with little or no data. Desk-based prioritization studies provide a potential solution by identifying those substances that are likely to pose the greatest risk to the environment and which, therefore, need to be considered a priority for further study. The aim of this review was to (1) provide an overview of different prioritization exercises performed for pharmaceuticals in the environment and the results obtained; and (2) propose a new holistic risk-based prioritization framework for drugs in the environment. The suggested models to underpin this framework are discussed in terms of validity and applicability. The availability of data required to run the models was assessed and data gaps identified. The implementation of this framework may harmonize pharmaceutical prioritization efforts and ensure that, in the future, experimental resources are focused on molecules, endpoints, and environmental compartments that are biologically relevant.

High Throughput Biodegradation-Screening Test To Prioritize and Evaluate Chemical Biodegradability.

Comprehensive assessment of environmental biodegradability of pollutants is limited by the use of low throughput systems. These are epitomized by the Organisation for Economic Cooperation and Development (OECD) Ready Biodegradability Tests (RBTs), where one sample from an environment may be used to assess a chemical's ability to readily biodegrade or persist universally in that environment. This neglects the considerable spatial and temporal microbial variation inherent in any environment. Inaccurate designations of biodegradability or persistence can occur as a result. RBTs are central in assessing the biodegradation fate of chemicals and inferring exposure concentrations in environmental risk assessments. We developed a colorimetric assay for the reliable quantification of suitable aromatic compounds in a high throughput biodegradation screening test (HT-BST). The HT-BST accurately differentiated and prioritized a range of structurally diverse aromatic compounds on the basis of their assigned relative biodegradabilities and quantitative structure-activity relationship (QSAR) model outputs. Approximately 20 000 individual biodegradation tests were performed, returning analogous results to conventional RBTs. The effect of substituent group structure and position on biodegradation potential demonstrated a significant correlation (P < 0.05) with Hammett's constant for substituents on position 3 of the phenol ring. The HT-BST may facilitate the rapid screening of 100 000 chemicals reportedly manufactured in Europe and reduce the need for higher-tier fate and effects tests.

Environmentally Relevant Inoculum Concentrations Improve the Reliability of Persistent Assessments in Biodegradation Screening Tests.

Standard OECD biodegradation screening tests (BSTs) have not evolved at the same rate as regulatory concerns, which now place an increased emphasis on environmental persistence. Consequently, many chemicals are falsely assigned as being potentially persistent based on results from BSTs. The present study increased test duration and increased inoculum concentrations to more environmentally relevant levels to assess their impact on biodegradation outcome and intratest replicate variability for chemicals with known environmental persistence. Chemicals were assigned to potential persistence categories based on existing degradation data. These more environmentally relevant BSTs (erBSTs) improved the reliability of persistence assignment by reducing the high variability associated with these tests and the occurrence of failures at low inoculum concentrations due to the exclusion of specific degraders. Environmental fate was determined using a reference set of 14C-labeled compounds with a range of potential environmental persistences, and full mass balance data were collated. The erBST correctly assigned five reference chemicals of known biodegradabilities to their appropriate persistence category in contrast to a standard OECD Ready Biodegradation Test (RBTs, P < 0.05). The erBST was significantly more reproducible than an OECD RBT (ANOVA, P < 0.05), with more consistent rates and extent of biodegradation observed in the erBST.

Fluoxetine Exhibits Pharmacological Effects and Trait-Based Sensitivity in a Marine Worm.

Global production of pharmacologically active compounds exceeds 100 000 tons annually, a proportion of which enters aquatic environments through patient use, improper medicine disposal, and production. These compounds are designed to have mode-of-action (MoA) effects on specific biological pathways, with potential to impact nontarget species. Here, we used MoA and trait-based approaches to quantify uptake and biological effects of fluoxetine, a selective serotonin reuptake inhibitor, in filter and deposit feeding marine worms (Hediste diversicolor). Worms exposed to 10 µg L(-1), accumulated fluoxetine with a body burden over 270 times greater than exposure concentrations, resulting in ∼10% increased coelomic fluid serotonin, a pharmacological effect. Observed effects included weight loss (up to 2% at 500 µg L(-1)), decreased feeding rate (68% at 500 µg L(-1)), and altered metabolism (oxygen consumption, ammonia excretion, and O/N from 10 µg L(-1)). Bioconcentration of fluoxetine was dependent on route of uptake, with filter feeding worms experiencing up to 130 times greater body burden ratios and increased magnitudes of effects than deposit feeders, a trait-based sensitivity likely as a consequence of fluoxetine partitioning to sediment. This study highlights how novel approaches such as MoA and trait-based methods can supplement environmental risk assessments of pharmaceuticals.

Refinement of biodegradation tests methodologies and the proposed utility of new microbial ecology techniques.

Society's reliance upon chemicals over the last few decades has led to their increased production, application and release into the environment. Determination of chemical persistence is crucial for risk assessment and management of chemicals. Current established OECD biodegradation guidelines enable testing of chemicals under laboratory conditions but with an incomplete consideration of factors that can impact on chemical persistence in the environment. The suite of OECD biodegradation tests do not characterise microbial inoculum and often provide little insight into pathways of degradation. The present review considers limitations with the current OECD biodegradation tests and highlights novel scientific approaches to chemical fate studies. We demonstrate how the incorporation of molecular microbial ecology methods (i.e., 'omics') may improve the underlying mechanistic understanding of biodegradation processes, and enable better extrapolation of data from laboratory based test systems to the relevant environment, which would potentially improve chemical risk assessment and decision making. We outline future challenges for relevant stakeholders to modernise OECD biodegradation tests and put the 'bio' back into biodegradation.