Developing Surrogate Markers for Predicting Antibiotic Resistance "Hot Spots" in Rivers Where Limited Data Are Available.

Pinpointing environmental antibiotic resistance (AR) hot spots in low-and middle-income countries (LMICs) is hindered by a lack of available and comparable AR monitoring data relevant to such settings. Addressing this problem, we performed a comprehensive spatial and seasonal assessment of water quality and AR conditions in a Malaysian river catchment to identify potential "simple" surrogates that mirror elevated AR. We screened for resistant coliforms, 22 antibiotics, 287 AR genes and integrons, and routine water quality parameters, covering absolute concentrations and mass loadings. To understand relationships, we introduced standardized "effect sizes" (Cohen's D) for AR monitoring to improve comparability of field studies. Overall, water quality generally declined and environmental AR levels increased as one moved down the catchment without major seasonal variations, except total antibiotic concentrations that were higher in the dry season (Cohen's D > 0.8, P < 0.05). Among simple surrogates, dissolved oxygen (DO) most strongly correlated (inversely) with total AR gene concentrations (Spearman's ρ 0.81, P < 0.05). We suspect this results from minimally treated sewage inputs, which also contain AR bacteria and genes, depleting DO in the most impacted reaches. Thus, although DO is not a measure of AR, lower DO levels reflect wastewater inputs, flagging possible AR hot spots. DO measurement is inexpensive, already monitored in many catchments, and exists in many numerical water quality models (e.g., oxygen sag curves). Therefore, we propose combining DO data and prospective modeling to guide local interventions, especially in LMIC rivers with limited data.

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.

Scientific concepts and methods for moving persistence assessments into the 21st century.

The evaluation of a chemical substance's persistence is key to understanding its environmental fate, exposure concentration, and, ultimately, environmental risk. Traditional biodegradation test methods were developed many years ago for soluble, nonvolatile, single-constituent test substances, which do not represent the wide range of manufactured chemical substances. In addition, the Organisation for Economic Co-operation and Development (OECD) screening and simulation test methods do not fully reflect the environmental conditions into which substances are released and, therefore, estimates of chemical degradation half-lives can be very uncertain and may misrepresent real environmental processes. In this paper, we address the challenges and limitations facing current test methods and the scientific advances that are helping to both understand and provide solutions to them. Some of these advancements include the following: (1) robust methods that provide a deeper understanding of microbial composition, diversity, and abundance to ensure consistency and/or interpret variability between tests; (2) benchmarking tools and reference substances that aid in persistence evaluations through comparison against substances with well-quantified degradation profiles; (3) analytical methods that allow quantification for parent and metabolites at environmentally relevant concentrations, and inform on test substance bioavailability, biochemical pathways, rates of primary versus overall degradation, and rates of metabolite formation and decay; (4) modeling tools that predict the likelihood of microbial biotransformation, as well as biochemical pathways; and (5) modeling approaches that allow for derivation of more generally applicable biotransformation rate constants, by accounting for physical and/or chemical processes and test system design when evaluating test data. We also identify that, while such advancements could improve the certainty and accuracy of persistence assessments, the mechanisms and processes by which they are translated into regulatory practice and development of new OECD test guidelines need improving and accelerating. Where uncertainty remains, holistic weight of evidence approaches may be required to accurately assess the persistence of chemicals. Integr Environ Assess Manag 2022;18:1454-1487. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Moving persistence assessments into the 21st century: A role for weight-of-evidence and overall persistence.

Assessing the persistence of chemicals in the environment is a key element in existing regulatory frameworks to protect human health and ecosystems. Persistence in the environment depends on many fate processes, including abiotic and biotic transformations and physical partitioning, which depend on substances' physicochemical properties and environmental conditions. A main challenge in persistence assessment is that existing frameworks rely on simplistic and reductionist evaluation schemes that may lead substances to be falsely assessed as persistent or the other way around-to be falsely assessed as nonpersistent. Those evaluation schemes typically assess persistence against degradation half-lives determined in single-compartment simulation tests or against degradation levels measured in stringent screening tests. Most of the available test methods, however, do not apply to all types of substances, especially substances that are poorly soluble, complex in composition, highly sorptive, or volatile. In addition, the currently applied half-life criteria are derived mainly from a few legacy persistent organic pollutants, which do not represent the large diversity of substances entering the environment. Persistence assessment would undoubtedly benefit from the development of more flexible and holistic evaluation schemes including new concepts and methods. A weight-of-evidence (WoE) approach incorporating multiple influencing factors is needed to account for chemical fate and transformation in the whole environment so as to assess overall persistence. The present paper's aim is to begin to develop an integrated assessment framework that combines multimedia approaches to organize and interpret data using a clear WoE approach to allow for a more consistent, transparent, and thorough assessment of persistence. Integr Environ Assess Manag 2022;18:868-887. © 2021 ExxonMobil Biomedical Sciences, Inc. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Improved quantitative microbiome profiling for environmental antibiotic resistance surveillance.

BACKGROUND: Understanding environmental microbiomes and antibiotic resistance (AR) is hindered by over reliance on relative abundance data from next-generation sequencing. Relative data limits our ability to quantify changes in microbiomes and resistomes over space and time because sequencing depth is not considered and makes data less suitable for Quantitative Microbial Risk Assessments (QMRA), critical in quantifying environmental AR exposure and transmission risks. RESULTS: Here we combine quantitative microbiome profiling (QMP; parallelization of amplicon sequencing and 16S rRNA qPCR to estimate cell counts) and absolute resistome profiling (based on high-throughput qPCR) to quantify AR along an anthropogenically impacted river. We show QMP overcomes biases caused by relative taxa abundance data and show the benefits of using unified Hill number diversities to describe environmental microbial communities. Our approach overcomes weaknesses in previous methods and shows Hill numbers are better for QMP in diversity characterisation. CONCLUSIONS: Methods here can be adapted for any microbiome and resistome research question, but especially providing more quantitative data for QMRA and other environmental applications.

Increased cell numbers improve marine biodegradation tests for persistence assessment.

Currently available OECD biodegradation screening tests (BSTs) are not particularly suited for persistence screening. Their duration can be much less than international half-life thresholds for persistence and they are variable and stringent, therefore prone to false negatives. The present study extended test durations beyond 28 days and increased biomass concentrations for marine BSTs to better represent the microbial diversity inherent in the sampled environment. For this so-called environmentally relevant BST (erBST) marine cell concentrations were nominally increased 100-fold by tangential flow filtration. The marine erBST was validated against a standard BST using five 14C labeled reference compounds with a range of biodegradation potentials (aniline, 4-fluorophenol, 4-nitrophenol, 4-chloroaniline and pentachlorophenol) in a modified OECD 301B test. A full mass balance was collated to follow chemical fate in the tests. The erBST was more accurate and less variable than the comparator BST in assigning the reference compounds to their expected biodegradation classifications (non-persistent or potentially persistent). According to the REACH non-persistence criterion of ≥60% biodegradation over 60 days, the erBST correctly classified 60% of chemical replicates according to their expected biodegradation classification and had a coefficient of variation of 21% between replicates. In contrast, the BST correctly assessed 40% of reference chemicals in regards to their expected biodegradation classification with a coefficient of variation of 36%. All non-persistent chemicals showed increased degradation in the erBST, except for 4-chloroaniline, which did not degrade in either BST or erBST. Both tests showed no false positive results, correctly classifying the negative control pentachlorophenol as potentially persistent. Next, it is recommended to further validate the marine erBST in an inter-laboratory study incorporating different seawater sources to fully assess its variability and reliability.

Improving the biodegradability in seawater test (OECD 306).

Growth and extensive urbanisation of the human population has been accompanied by increased manufacture and use of chemical compounds. To classify the fate and behaviour of these compounds in the environment, a series of international standardised biodegradation screening tests (BSTs) were developed over 30 years ago. In recent years, regulatory emphasis (e.g. REACH) has shifted from measuring biodegradation towards prioritisations based on chemical persistence. In their current guise, BSTs are ineffective as screens for persistence. The marine BST OECD 306 in particular is prone to high levels of variation and produces a large number of fails, many of which can be considered false negatives. An ECETOC funded two-day workshop of academia, industry and regulatory bodies was held in 2015 to discuss improvements to the marine BSTs based on previous research findings from the Cefic LRI ECO11 project and other foregoing studies. During this workshop, methodological improvements to the OECD 306 test were discussed, in addition to clarifying guidance on testing and interpretation of results obtained from marine BSTs (such as pass criteria, lag phases, freshwater read across and complex substances). Methodologically: (i) increasing bacterial cell concentrations to better represent the bacterial diversity inherent in the sampled environments; and (ii) increasing test durations to investigate extended lag phases observed in marine assessments, were recommended to be validated in a multi-institutional ring test.