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1.  Standard inocula preparations reduce the bacterial diversity and reliability of regulatory biodegradation tests 
OECD ready biodegradability tests have been central to understanding the biodegradation of chemicals from a regulatory perspective for many decades. They are not fit for contemporary prioritisation of chemicals based on persistence, however, due to the low concentration of inocula used, short duration and high variability between tests. Two OECD standard inoculum pretreatment methods (settlement and filtration) were investigated to observe their effect on the probability of biodegradation and associated changes in bacterial community structure and diversity of inocula sourced from the activated sludge process of wastewater treatment plants. Both settlement and filtration were shown to dramatically and significantly reduce the probability and increase the variability of biodegradation of 4-nitrophenol compared to the use of unprocessed inocula. These differences were associated with a significant hundred-fold reduction in cell numbers and solids content and a significant shift in bacterial community structure that was sometimes accompanied by significant reductions in detectable operational taxonomic unit richness and evenness. The natural variation (between different environments) and variation due to differential selection of bacterial communities (by different pretreatment methods) is offered as an explanation for the historical high variability in standard OECD ready biodegradability tests.
Electronic supplementary material
The online version of this article (doi:10.1007/s11356-013-2064-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4133024  PMID: 24043502
Biodegradation; Persistence; Diversity; Bacterial community structure; 4-Nitrophenol; REACH
2.  Human Health Risk Assessment (HHRA) for Environmental Development and Transfer of Antibiotic Resistance 
Environmental Health Perspectives  2013;121(9):993-1001.
Background: Only recently has the environment been clearly implicated in the risk of antibiotic resistance to clinical outcome, but to date there have been few documented approaches to formally assess these risks.
Objective: We examined possible approaches and sought to identify research needs to enable human health risk assessments (HHRA) that focus on the role of the environment in the failure of antibiotic treatment caused by antibiotic-resistant pathogens.
Methods: The authors participated in a workshop held 4–8 March 2012 in Québec, Canada, to define the scope and objectives of an environmental assessment of antibiotic-resistance risks to human health. We focused on key elements of environmental-resistance-development “hot spots,” exposure assessment (unrelated to food), and dose response to characterize risks that may improve antibiotic-resistance management options.
Discussion: Various novel aspects to traditional risk assessments were identified to enable an assessment of environmental antibiotic resistance. These include a) accounting for an added selective pressure on the environmental resistome that, over time, allows for development of antibiotic-resistant bacteria (ARB); b) identifying and describing rates of horizontal gene transfer (HGT) in the relevant environmental “hot spot” compartments; and c) modifying traditional dose–response approaches to address doses of ARB for various health outcomes and pathways.
Conclusions: We propose that environmental aspects of antibiotic-resistance development be included in the processes of any HHRA addressing ARB. Because of limited available data, a multicriteria decision analysis approach would be a useful way to undertake an HHRA of environmental antibiotic resistance that informs risk managers.
Citation: Ashbolt NJ, Amézquita A, Backhaus T, Borriello P, Brandt KK, Collignon P, Coors A, Finley R, Gaze WH, Heberer T, Lawrence JR, Larsson DG, McEwen SA, Ryan JJ, Schönfeld J, Silley P, Snape JR, Van den Eede C, Topp E. 2013. Human health risk assessment (HHRA) for environmental development and transfer of antibiotic resistance. Environ Health Perspect 121:993–1001;
PMCID: PMC3764079  PMID: 23838256
3.  Management Options for Reducing the Release of Antibiotics and Antibiotic Resistance Genes to the Environment 
Environmental Health Perspectives  2013;121(8):878-885.
Background: There is growing concern worldwide about the role of polluted soil and water environments in the development and dissemination of antibiotic resistance.
Objective: Our aim in this study was to identify management options for reducing the spread of antibiotics and antibiotic-resistance determinants via environmental pathways, with the ultimate goal of extending the useful life span of antibiotics. We also examined incentives and disincentives for action.
Methods: We focused on management options with respect to limiting agricultural sources; treatment of domestic, hospital, and industrial wastewater; and aquaculture.
Discussion: We identified several options, such as nutrient management, runoff control, and infrastructure upgrades. Where appropriate, a cross-section of examples from various regions of the world is provided. The importance of monitoring and validating effectiveness of management strategies is also highlighted. Finally, we describe a case study in Sweden that illustrates the critical role of communication to engage stakeholders and promote action.
Conclusions: Environmental releases of antibiotics and antibiotic-resistant bacteria can in many cases be reduced at little or no cost. Some management options are synergistic with existing policies and goals. The anticipated benefit is an extended useful life span for current and future antibiotics. Although risk reductions are often difficult to quantify, the severity of accelerating worldwide morbidity and mortality rates associated with antibiotic resistance strongly indicate the need for action.
PMCID: PMC3734499  PMID: 23735422
agriculture; antibiotic manufacturing; antibiotic resistance; aquaculture; livestock; manure management; policy; wastewater treatment
4.  Implementing Ecopharmacovigilance in Practice: Challenges and Potential Opportunities 
Drug Safety  2013;36(7):533-546.
Ecopharmacovigilance (EPV) is a developing science and it is currently very unclear what it might mean in practice. We have performed a comparison between pharmacovigilance (PV) and EPV and have identified that there are similarities, but also some important differences that must be considered before any practical implementation of EPV. The biggest difference and greatest challenge concerns signal detection in the environment and the difficulty of identifying cause and effect. We reflect on the dramatic vulture decline in Asia, which was caused by the veterinary use of diclofenac, versus the relative difficulty in identifying the specific causes of intersex fish in European rivers. We explore what EPV might mean in practice and have identified that there are some practical measures that can be taken to assess environmental risks across product life cycle, particularly after launch of a new drug, to ensure that our risk assessments and scientific understanding of pharmaceuticals in the environment remain scientifically and ecologically relevant. These include:Tracking environmental risks after launch of the product, via literature monitoring for emerging data on exposure and effectsUsing Environmental Risk Management Plans (ERMPs) as a centralized resource to assess and manage the risks of a drug throughout its life cycleFurther research, testing or monitoring in the environment when a risk is identifiedKeeping a global EPV perspectiveIncreasing transparency and availability of environmental data for medicinal products.These measures will help to ensure that any significant environmental issues associated with pharmaceuticals in the environment (PIE) are identified in a timely way, and can be managed appropriately.
PMCID: PMC3691479  PMID: 23620169
5.  Pharmaceuticals and Personal Care Products in the Environment: What Are the Big Questions? 
Environmental Health Perspectives  2012;120(9):1221-1229.
Background: Over the past 10–15 years, a substantial amount of work has been done by the scientific, regulatory, and business communities to elucidate the effects and risks of pharmaceuticals and personal care products (PPCPs) in the environment.
Objective: This review was undertaken to identify key outstanding issues regarding the effects of PPCPs on human and ecological health in order to ensure that future resources will be focused on the most important areas.
Data sources: To better understand and manage the risks of PPCPs in the environment, we used the “key question” approach to identify the principle issues that need to be addressed. Initially, questions were solicited from academic, government, and business communities around the world. A list of 101 questions was then discussed at an international expert workshop, and a top-20 list was developed. Following the workshop, workshop attendees ranked the 20 questions by importance.
Data synthesis: The top 20 priority questions fell into seven categories: a) prioritization of substances for assessment, b) pathways of exposure, c) bioavailability and uptake, d) effects characterization, e) risk and relative risk, f ) antibiotic resistance, and g) risk management.
Conclusions: A large body of information is now available on PPCPs in the environment. This exercise prioritized the most critical questions to aid in development of future research programs on the topic.
PMCID: PMC3440110  PMID: 22647657
antibiotic resistance; ecotoxicity; exposure assessment; health effects; personal care products; pharmaceuticals; prioritization; risk assessment; risk management
6.  Integrating Omic Technologies into Aquatic Ecological Risk Assessment and Environmental Monitoring: Hurdles, Achievements, and Future Outlook 
In this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics—Moving into Regulation and Monitoring, held 21–23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada).
The consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets.
Participants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data.
The participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.
PMCID: PMC2831950  PMID: 20056575
environment; environmental monitoring; fish; metabolomics; microarray; regulatory toxicology; transcriptomics
7.  Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI project 
Nature biotechnology  2008;26(8):889-896.
The Minimum Information for Biological and Biomedical Investigations (MIBBI) project provides a resource for those exploring the range of extant minimum information checklists and fosters coordinated development of such checklists.
PMCID: PMC2771753  PMID: 18688244
9.  Meeting Report: Validation of Toxicogenomics-Based Test Systems: ECVAM–ICCVAM/NICEATM Considerations for Regulatory Use 
Environmental Health Perspectives  2005;114(3):420-429.
This is the report of the first workshop “Validation of Toxicogenomics-Based Test Systems” held 11–12 December 2003 in Ispra, Italy. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and organized jointly by ECVAM, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). The primary aim of the workshop was for participants to discuss and define principles applicable to the validation of toxicogenomics platforms as well as validation of specific toxicologic test methods that incorporate toxicogenomics technologies. The workshop was viewed as an opportunity for initiating a dialogue between technologic experts, regulators, and the principal validation bodies and for identifying those factors to which the validation process would be applicable. It was felt that to do so now, as the technology is evolving and associated challenges are identified, would be a basis for the future validation of the technology when it reaches the appropriate stage. Because of the complexity of the issue, different aspects of the validation of toxicogenomics-based test methods were covered. The three focus areas include a) biologic validation of toxicogenomics-based test methods for regulatory decision making, b) technical and bioinformatics aspects related to validation, and c) validation issues as they relate to regulatory acceptance and use of toxicogenomics-based test methods. In this report we summarize the discussions and describe in detail the recommendations for future direction and priorities.
PMCID: PMC1392237  PMID: 16507466
acceptance; alternatives; biomarker; predictive test; regulatory use; standardization; toxicogenomics; toxicology; validation
10.  Development of a Rapid Assay for Determining the Relative Abundance of Bacteria 
Applied and Environmental Microbiology  2005;71(12):8481-8490.
A sandwich hybridization assay for high-throughput, rapid, simple, and inexpensive quantification of specific microbial populations was evaluated. The assay is based on the hybridization of a target rRNA with differentially labeled capture and detector probes. Betaproteobacterial ammonia-oxidizing bacteria (AOB) were selected as the target group for the study, since they represent a phylogenetically coherent group of organisms that perform a well-defined geochemical function in natural and engineered environments. Reagent concentrations, probe combinations, and washing, blocking, and hybridization conditions were optimized to improve signal and reduce background. The detection limits for the optimized RNA assay were equivalent to approximately 103 to 104 and 104 to 105 bacterial cells, respectively, for E. coli rRNA and RNA extracted from activated sludge, by using probes targeting the majority of bacteria. Furthermore, the RNA assay had good specificity, permitted discrimination of rRNA sequences that differed by a 2-bp mismatch in the probe target region, and could distinguish the sizes of AOB populations in nitrifying and nonnitrifying wastewater treatment plants.
PMCID: PMC1317421  PMID: 16332838
11.  eGenomics: Genomes and the Environment 
Comparative and Functional Genomics  2005;6(7-8):357-362.
PMCID: PMC2447498  PMID: 18629203
12.  Simultaneous Fluorescent Gram Staining and Activity Assessment of Activated Sludge Bacteria 
Applied and Environmental Microbiology  2002;68(10):4772-4779.
Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.
PMCID: PMC126428  PMID: 12324319
13.  Bioinformatics and Data Management Support for Environmental Genomics 
PLoS Biology  2005;3(8):e297.
The UK Natural Environment Research Council has funded the creation of a dedicated bioinformatics centre as part of a £26m Environmental Genomics initiative.
PMCID: PMC1187860  PMID: 16089508

Results 1-13 (13)