Finding A Niche for Soil Microbial Toxicity Tests in Ecological Risk Assessment

Soil microbial toxicity tests are seldom used in ecological risk assessments or in the development of regulatory criteria in the U.S. The primary reason is the lack of an explicit connection between these tests and assessment end-points. Soil microorganisms have three potential roles with respect to ecological assessment endpoints: properties of microbial communities may be end-points; microbial responses may be used to estimate effects on plant production; and microbial responses may be used as surrogates for responses of higher organisms. Rates of microbial processes are important to ecosystem function, and thus should be valued by regulatory agencies. However, the definition of the microbial assessment endpoint is often an impediment to its use in risk assessment. Decreases in rates are not always undesirable. Processes in a nutrient cycle are particularly difficult to define as endpoints, because what constitutes an adverse effect on a process is dependent on the rates of others. Microbial tests may be used as evidence in an assessment of plant production, but the dependence of plants on microbial processes is rarely considered. As assessment endpoints are better defined in the future, microbial ecologists and toxicologists should be provided with more direction for developing appropriate microbial tests.     

The distinct properties of natural and GM cry insecticidal proteins

The Cry toxins are a family of crystal-forming proteins produced by the bacterium Bacillus thuringiensis. Their mode of action is thought to be to create pores that disrupt the gut epithelial membranes of juvenile insects. These pores allow pathogen entry into the hemocoel, thereby killing the insect. Genes encoding a spectrum of Cry toxins, including Cry mutants, Cry chimaeras and other Cry derivatives, are used commercially to enhance insect resistance in genetically modified (GM) crops. In most countries of the world, such GM crops are regulated and must be assessed for human and environmental safety. However, such risk assessments often do not test the GM crop or its tissues directly. Instead, assessments rely primarily on historical information from naturally occurring Cry proteins and on data collected on Cry proteins (called ‘surrogates’) purified from laboratory strains of bacteria engineered to express Cry protein. However, neither surrogates nor naturally occurring Cry proteins are identical to the proteins to which humans or other nontarget organisms are exposed by the production and consumption of GM plants. To-date there has been no systematic survey of these differences. This review fills this knowledge gap with respect to the most commonly grown GM Cry-containing crops approved for international use. Having described the specific differences between natural, surrogate and GM Cry proteins this review assesses these differences for their potential to undermine the reliability of risk assessments. Lastly, we make specific recommendations for improving risk assessments.     

Contamination of Groundwater Systems in the US and Canada by Enteric Pathogens, 1990–2013: A Review and Pooled-Analysis

Background

Up to 150 million North Americans currently use a groundwater system as their principal drinking water source. These systems are a potential source of exposure to enteric pathogens, contributing to the burden of waterborne disease. Waterborne disease outbreaks have been associated with US and Canadian groundwater systems over the past two decades. However, to date, this literature has not been reviewed in a comprehensive manner.

Methods and Principal Findings

A combined review and pooled-analysis approach was used to investigate groundwater contamination in Canada and the US from 1990 to 2013; fifty-five studies met eligibility criteria. Four study types were identified. It was found that study location affects study design, sample rate and studied pathogen category. Approximately 15% (316/2210) of samples from Canadian and US groundwater sources were positive for enteric pathogens, with no difference observed based on system type. Knowledge gaps exist, particularly in exposure assessment for attributing disease to groundwater supplies. Furthermore, there is a lack of consistency in risk factor reporting (local hydrogeology, well type, well use, etc). The widespread use of fecal indicator organisms in reported studies does not inform the assessment of human health risks associated with groundwater supplies.

Conclusions

This review illustrates how groundwater study design and location are critical for subsequent data interpretation and use. Knowledge gaps exist related to data on bacterial, viral and protozoan pathogen prevalence in Canadian and US groundwater systems, as well as a need for standardized approaches for reporting study design and results. Fecal indicators are examined as a surrogate for health risk assessments; caution is advised in their widespread use. Study findings may be useful during suspected waterborne outbreaks linked with a groundwater supply to identify the likely etiological agent and potential transport pathway.     

Characterising microbial protein test substances and establishing their equivalence with plant-produced proteins for use in risk assessments of transgenic crops

Most commercial transgenic crops are genetically engineered to produce new proteins. Studies to assess the risks to human and animal health, and to the environment, from the use of these crops require grams of the transgenic proteins. It is often extremely difficult to produce sufficient purified transgenic protein from the crop. Nevertheless, ample protein of acceptable purity may be produced by over-expressing the protein in microbes such as Escherichia coli. When using microbial proteins in a study for risk assessment, it is essential that their suitability as surrogates for the plant-produced transgenic proteins is established; that is, the proteins are equivalent for the purposes of the study. Equivalence does not imply that the plant and microbial proteins are identical, but that the microbial protein is sufficiently similar biochemically and functionally to the plant protein such that studies using the microbial protein provide reliable information for risk assessment of the transgenic crop. Equivalence is a judgement based on a weight of evidence from comparisons of relevant properties of the microbial and plant proteins, including activity, molecular weight, amino acid sequence, glycosylation and immuno-reactivity. We describe a typical set of methods used to compare proteins in regulatory risk assessments for transgenic crops, and discuss how risk assessors may use comparisons of proteins to judge equivalence.     

Impact of surrogate selection on risk assessment for total petroleum hydrocarbons

Environmental contamination involving total petroleum hydrocarbons (TPH) is being investigated and remediated at underground storage tanks, tank farms, pipelines, and refineries across the country. Human health and environmental risk play a significant role in decision making at these sites. However, risk assessment for sites contaminated with petroleum products typically is complicated by inadequate information about the composition of TPH present at the site and the physical and chemical properties and toxicity of the components. To address these data gaps, risk assessors can select surrogate compounds to represent the movement of TPH in the environment at the site and toxicity of TPH present at the site. This article illustrates the potential impact of choice of surrogates on risk estimates, which in turn affect remediation costs.     

Quantification of the presence and activity of specific microorganisms in nature

Traditional techniques for assessment of microbial numbers and activity generally lack the specificity required for risk assessment following environmental release of genetically engineered microbial inocula. Immunological and molecular-based techniques, such as DNA probing and genetic tagging, were initially used to determine the presence or absence of microorganisms in environmental samples. Increasingly they are being developed for quantification of populations of specific organisms, either indigenous or introduced, in the environment. In addition, they are being used to quantify the activity of particular organisms or groups of organisms, greatly extending the range of techniques available to the microbial ecologist. This article reviews the use of traditional techniques for the quantification of microbial population size and activity and the application of molecular techniques, including DNA probing, genetic marking, use of fluorescent probes, and quantitative PCR, in combination with advanced cell detection techniques such as confocal laser scanning microscopy and flow cytometry.     

Dynamic role and importance of surrogate species for assessing potential adverse environmental impacts of genetically engineered insect-resistant plants on non-target organisms

Surrogate species have a long history of use in research and regulatory settings to understand the potentially harmful effects of toxic substances including pesticides. More recently, surrogate species have been used to evaluate the potential effects of proteins contained in genetically engineered insect resistant (GEIR) crops. Species commonly used in GEIR crop testing include beneficial organisms such as honeybees, arthropod predators, and parasitoids. The choice of appropriate surrogates is influenced by scientific factors such as the knowledge of the mode of action and the spectrum of activity as well as societal factors such as protection goals that assign value to certain ecosystem services such as pollination or pest control. The primary reasons for using surrogates include the inability to test all possible organisms, the restrictions on using certain organisms in testing (e.g., rare, threatened, or endangered species), and the ability to achieve greater sensitivity and statistical power by using laboratory testing of certain species. The acceptance of surrogate species data can allow results from one region to be applied or “transported” for use in another region. On the basis of over a decade of using surrogate species to evaluate potential effects of GEIR crops, it appears that the current surrogates have worked well to predict effects of GEIR crops that have been developed (Carstens et al. GM Crops Food 5:1–5, 2014), and it is expected that they should work well to predict effects of future GEIR crops based on similar technologies.     

A Quantitative Prioritisation of Human and Domestic Animal Pathogens in Europe

Disease or pathogen risk prioritisations aid understanding of infectious agent impact within surveillance or mitigation and biosecurity work, but take significant development. Previous work has shown the H-(Hirsch-)index as an alternative proxy. We present a weighted risk analysis describing infectious pathogen impact for human health (human pathogens) and well-being (domestic animal pathogens) using an objective, evidence-based, repeatable approach; the H-index. This study established the highest H-index European pathogens. Commonalities amongst pathogens not included in previous surveillance or risk analyses were examined. Differences between host types (humans/animals/zoonotic) in pathogen H-indices were explored as a One Health impact indicator. Finally, the acceptability of the H-index proxy for animal pathogen impact was examined by comparison with other measures. 57 pathogens appeared solely in the top 100 highest H-indices (1) human or (2) animal pathogens list, and 43 occurred in both. Of human pathogens, 66 were zoonotic and 67 were emerging, compared to 67 and 57 for animals. There were statistically significant differences between H-indices for host types (humans, animal, zoonotic), and there was limited evidence that H-indices are a reasonable proxy for animal pathogen impact. This work addresses measures outlined by the European Commission to strengthen climate change resilience and biosecurity for infectious diseases. The results include a quantitative evaluation of infectious pathogen impact, and suggest greater impacts of human-only compared to zoonotic pathogens or scientific under-representation of zoonoses. The outputs separate high and low impact pathogens, and should be combined with other risk assessment methods relying on expert opinion or qualitative data for priority setting, or could be used to prioritise diseases for which formal risk assessments are not possible because of data gaps.     

The use of meta-analysis in food contact materials risk assessment

Abstract

As materials intended to be brought into contact with food, food contact materials (FCMs) – including plastics, paper or inks – can transfer their constituents to food under normal or foreseeable use, including direct or indirect food contact. The safety of FCMs in the EU is evaluated by the European Food Safety Authority (EFSA) using risk assessment rules. Results of independent, health-based chemical risk assessments are crucial for the decision-making process to authorize the use of substances in FCMs. However, the risk assessment approach used in the EU has several shortcomings that need to be improved in order to ensure consumer health protection from exposure arising from FCMs. This article presents the use of meta-analysis as a useful tool in chronic risk assessment for substances migrating from FCMs. Meta-analysis can be used for the review and summary of research of FCMs safety in order to provide a more accurate assessment of the impact of exposure with increased statistical power, thus providing more reliable data for risk assessment. The article explains a common methodology of conducting a meta-analysis based on meta-analysis of the dose-effect relationship of cadmium for benchmark dose evaluations performed by EFSA.     

What Can Be Done to Ensure the Usefulness of Epidemiologic Data for Risk Assessment Purposes?

Epidemiologic studies can play a central role in risk assessments. They are used in all risk assessment phases: hazard identification, dose-response, and exposure assessment. Epidemiologic studies have often been the first to show that a particular environmental exposure is a hazard to health. They have numerous advantages with respect to other sources of data which are used in risk assessments, the most important being that they do not require the assumption that they are generalizable to humans. For this reason, fewer and lower uncertainty factors may be appropriate in risk characterization based on epidemiologic studies. Unfortunately, epidemiologic studies have numerous problems, the most important being that the exposures are often not precisely measured. This article presents in detail the advantages of and problems with epidemiologic studies. It discusses two approaches to ensure their usefulness, biomarkers and an ordinance which requires baseline and subsequent surveillance of possible exposures and health effects from newly sited potentially polluting facilities. Biomarkers are biochemical measures of exposure, susceptibility factors, or preclinical pathological changes. Biomarkers are a way of dealing with the problems of poor measures, differential susceptibility and lack of early measures of disease occurrence that inherent in many environmental epidemiologic studies. The advantages of biomarkers is they can provide objective information on exposure days, months or even years later and evidence of pathology perhaps years earlier. The ordinance makes possible the use of a powerful epidemiologic study design, the prospective cohort study, where confounder(s) are best measured, and exposures, pathological changes, and health effects can be detected as soon as possible.     

Improving biodiversity surrogates for conservation assessment: A test of methods and the value of targeted biological surveys

Aim

Conservation assessment and planning across extensive regions rely on the use of mapped or modelled surrogates because direct field‐based inventories of biodiversity rarely provide complete spatial coverage. Surrogates are assumed to represent spatial patterns in the distribution of biodiversity, yet the validity of this assumption is rarely evaluated. Here, we use data from new biological surveys targeting poorly known taxonomic groups across sparsely surveyed landscapes to test: (1) the performance of established and novel surrogates; and (2) the value of targeted survey data in further improving surrogate effectiveness.

Location

Continental Australia.

Methods

Surrogates were derived from either mapped land classifications (bioregions, vegetation types), or models of spatial turnover in biodiversity composition. Models were derived by linking best‐available biological observations to high‐resolution mapped climate, terrain and soil attributes using generalized dissimilarity modelling (GDM). The performance of surrogates was evaluated using survey data for eight biological groups collected as part of the Bush Blitz programme ( http://bushblitz.org ). For the GDM‐based surrogates, within‐ and cross‐taxon performance was first evaluated for models fitted to biological data available prior to Bush Blitz, and then for models enhanced through the addition of the Bush Blitz data.

Results

All of the tested surrogates performed significantly better than random across all eight biological groups. GDM‐based surrogates performed over 10% better on average than the best performing combination of mapped land classifications. The addition of Bush Blitz targeted data in GDM‐based surrogates led to further improvements in surrogate performance.

Main conclusions

Our results support continued investment in targeted biological survey programmes to enhance the performance of surrogates and ensure that surrogates represent a wider breadth of biodiversity. The strong performance of compositional turnover modelling, relative to mapped land classifications, suggests that this surrogate strategy deserves greater consideration in future conservation assessments and has potential for use in continental‐scale monitoring of biodiversity.

     

Current Perspectives on Issues in Risk Assessment Methods

The Office of Research and Development (ORD) of the U.S. Environmental Protection Agency was reorganized in 1995 to follow the risk assessment paradigm developed by the National Research Council. With the reorganization, a number of different research strategies and plans were developed on problem-driven topics (e.g., arsenic, particulate matter, and microbial pathogens/disinfectant byproducts) as well as on core research (e.g., ecological research, human health risk assessment research, and pollution prevention research). The human health risk assessments research strategy, which is currently under development, addresses a variety of issues which affect all human health risk assessments. These include mechanism of action, variation in response, aggregate risk, and effect on public health. While all of the issues that have been identified are important, the challenge to ORD is to identify which will have the greatest impact on improving risk assessment so that resources can be most strategically applied.     

Framework for the Integration of Health and Ecological Risk Assessment

The World Health Organization's International Programme on Chemical Safety (IPCS), the Organization for Economic Cooperation and Development (OECD), and the U.S. Environmental Protection Agency have developed a collaborative partnership to foster integration of assessment approaches for human health and ecological risks. This paper presents the framework developed by that group. Integration provides coherent expressions of assessment results, incorporates the interdependence of humans and the environment, uses sentinel organisms, and improves the efficiency and quality of assessments relative to independent human health and ecological risk assessments. The paper describes how integration can occur within each component of risk assessment, and communicates the benefits of integration at each point. The goal of this effort is to promote the use of this internationally accepted guidance as a basis for harmonization of risk assessment.     

Evaluating the risk of releasing genetically engineered organisms

This article considers the question of a priori assessment of the safety of releasing recombinant DNA engineered organisms. Now and for the foreseeable future, decisions to release such an organism must be based on the results of limited, case-by-case risk assessment studies. The criteria calling for the termination of release programs must be agreed upon in advance of these studies. There is no justification for excluding classes of release organisms from risk assessment. Theory is useful in suggesting a hierarchy of risks, raising the questions that have to be addressed in case-by-case risk assessment and providing protocols for the standardization and execution of these studies. We do not believe that theory can be used to argue categorically for or against the safety of specific releases of recombinant DNA engineered organisms.     

NESTED AUTOMATED RIBOSOMAL INTERGENIC SPACER ANALYSIS: A RAPID AND ACCURATE METHOD FOR COMPARISON OF BACTERIAL COMMUNITY COMPOSITION

Nested automated ribosomal intergenic spacer analysis (ARISA) was used to examine the community structure of epilithic biofilms in freshwater streams experiencing different levels of human impact. This molecular fingerprinting technique generated reproducible profiles of bacterial community structure that varied significantly between stream sites. Nested ARISA was determined to be a cost-effective, high-throughput approach to assess bacterial community composition from very small sample volumes, requiring little sampling effort and without the need for taxonomic identification of individual organisms. In combination with multidimensional scaling, nested ARISA provides a rapid and sensitive method to carry out complex analyses of bacterial community structure.

PRACTICAL APPLICATIONS

Nested automated ribosomal intergenic spacer analysis (ARISA) provides a high-throughput molecular method with which to screen large numbers of environmental samples for differences in microbial community structure. This sensitive approach benefits assessments from small sample volumes or environments exhibiting reduced microbial biomass (both aquatic and terrestrial). Differences in bacterial community structure (obtained from ARISA profiles) could be used to characterize the impact of anthropogenic disturbance on freshwater systems, analogous to the current use of macroinvertebrate indicators of freshwater ecological health.     

Quantitative risk assessment of Listeria monocytogenes in ready-to-eat foods: the FAO/WHO approach

Quantitative microbiological risk assessment is a very new and unique scientific approach able to link, for the first time, data from food (in the farm-to-fork continuum) and the various data on human disease to provide a clear estimation of the impact of contaminated food on human public health. The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) have recently launched risk assessment studies of a number of pathogen-food commodity combinations (Salmonella in eggs and in broiler chickens, Listeria monocytogenes in ready-to-eat foods, Campylobacter in broiler chickens, Vibrio in seafood) to be used to lower the risk associated with these food-borne diseases and ensure fair practices in the international trade of food. The FAO/WHO Listeria risk assessment was undertaken in part to determine how previously developed risk assessments done at the national level could be adapted or expanded to address concerns related to L. monocytogenes in ready-to-eat foods at an international level. In addition, after initiation of the risk assessment, the risk assessors were asked by the Codex Committee on Food to consider three specific questions related to ready-to-eat foods in general, which are: (1). estimate the risk for consumers in different susceptible populations groups (elderly, infants, pregnant women and immunocompromised patients) relative to the general population; (2). estimate the risk for L. monocytogenes in foods that support growth and foods that do not support growth under specific storage and shelf-life conditions; (3). estimate the risk from L. monocytogenes in food when the number of organisms ranges from absence in 25 g to 1000 colonies forming units per gram or milliliter, or does not exceed specified levels at the point of consumption. To achieve these goals, new dose-response relationships and exposure assessments for ready-to-eat foods were developed. Preliminary data indicate that eliminating the higher dose levels at the time of consumption has a large impact on the number of predicted cases.     

The Concept of Data Utility in Health Risk Assessment: A Multi-Disciplinary Perspective

Human and ecological health risk assessments and the decisions that stem from them require the acquisition and analysis of data. In agencies that are responsible for health risk decision-making, data (and/or opinions/judgments) are obtained from sources such as scientific literature, analytical and process measurements, expert elicitation, inspection findings, and public and private research institutions. Although the particulars of conducting health risk assessments of given disciplines may be dramatically different, a common concern is the subjective nature of judging data utility. Often risk assessors are limited to available data that may not be completely appropriate to address the question being asked. Data utility refers to the ability of available data to support a risk-based decision for a particular risk assessment. This article familiarizes the audience with the concept of data utility and is intended to raise the awareness of data collectors (e.g., researchers), risk assessors, and risk managers to data utility issues in health risk assessments so data collection and use will be improved. In order to emphasize the cross-cutting nature of data utility, the discussion has not been organized into a classical partitioning of risk assessment concerns as being either human health- or ecological health-oriented, as per the U.S. Environmental Protection Agency's Superfund Program.     

The Contribution of Systematic Review and Meta-Analysis Methods to Human Health Risk Assessment: Neurobehavioral Effects of Manganese

Current methods of human health risk assessment may lack transparency in respect of identification, review, and synthesis of potentially relevant human and animal evidence. The nature, degree, and source of uncertainties are often unclear. This article aims to demonstrate the contribution that systematic review and meta-analysis methods can make to providing more structured, transparent, and systematic risk assessments. We focus on disparities between five risk assessments for neurobehavioral effects of manganese, and then illustrate advantages of a systematic approach. Fifty-five human epidemiological studies and 37 animal experiments were identified. Where appropriate, meta-analysis methods demonstrated consistent adverse effects associated with manganese exposure across species. In particular, there was reduced activity in subjects exposed to manganese, although exposed rats tended to be more active than controls. Limitations of exposure measurement and reporting restricted use of more quantitative methods of evidence synthesis. From a methodological viewpoint, we conclude that systematic review and meta-analysis methods can contribute to a more systematic and transparent human health risk assessment making more efficient use of available evidence, compared to current methods of risk assessment. More complex methods could encompass further differences between relevant studies and so further improve the risk assessment process.     

Safety and risk assessment for the human superorganism

This editorial on safety evaluation and risk assessment for the human superorganism introduces a series of papers arguing for a fundamental shift in how we approach human health risk assessment. In this series emphasis is placed on the risk of infectious disease. Our 21^stst century understanding of human biology is that, as holobionts, we possess a majority of microbial cells and genes. In fact, our microbes fundamentally affect our interactions with the external environment, metabolism, physiology, and risk of both pathology and disease. As holobionts, we require our microbial partners for us to be both complete and healthy. Using the 20^th century understanding of human biology, we failed to capture the effects of the microbiome in evaluating health risks. But 21^st century risk assessments such as those associated with exposure to specific microbial pathogens need to include the human microbiome. Microbiome status will be central in determining the health risks for both individuals and populations.     

Species Risk Assessment of Microscopic Exotic Organisms Associated with Forest-Related Commodities and Goods

Recent increases in international trade have increased the cost to control and eradicate exotic species. Although many species are under quarantine control for agriculture, forestry, and public health, most species invisible to the naked eye are ignored because of the lack of both specialized assessors and risk assessments. We developed a species risk assessment particularly adapted to fungi, nematodes, and mites, that might be unintentionally introduced with exotic forest products and become threats to terrestrial ecosystems. We developed our assessment with reference to existing risk assessments for exotic organisms, including their ecological features such as phoresy and parasitism. We then tested our assessment with well-known organisms and assessed the risks of organisms unintentionally introduced into Japan. The assessment demonstrated scientifically acceptable scores for each organism. We suggest quarantine control of risk pathways as a practical approach for controlling unintentionally introduced organisms that are invisible to the naked eye.