Threshold effects in genetic toxicity: perspective of chemicals regulation in Germany

In the regulation of chemical substances, it is generally agreed that there are no thresholds for genotoxic effects of chemicals, i.e. , that there are no doses without genotoxic effects. When classifying and labelling chemicals, dangerous properties of chemicals are to be identified. In this context, in general, the mode of action (threshold or not) is not considered for genotoxic substances. In the process of quantitative risk assessment, however, determination of the type of dose-effect relationships is decisive for the outcome and the type of risk management. The presence of a threshold must be justified specifically in each individual case. Inter alia, the following aspects may be discussed in this respect: aneugenic activity, indirect modes of action, extremely steep dose-effect relationships in combination with strong toxicity, specific toxicokinetic conditions which may lead to 'metabolic protection' prior to an attack of DNA. In the practice of the regulation of chemical substances with respect to their genotoxic effects, the discussion of thresholds has played a minor role. For notified new substances, there are, in general, no data available that would allow a reasonable discussion. Concerning substances out of the European programme on existing substances, so far 29 have been assessed in our institute with respect to genetic toxicity. Eight out of these have shown considerable evidence for genotoxicity. For two of them, a possible threshold is discussed: one substance is an aneugen, the other one is metabolised to an endogenic compound with genotoxic potential. In the practice of risk assessment of genotoxic substances, the discussion of the mode of action for genotoxicity is frequently associated with the evaluation of potential carcinogenic effects. Here, tissue-specific genotoxic effects in target organs for carcinogenicity are to be discussed. Moreover, the contribution of genotoxicity to the multifactorial process of tumour development should be assessed.     

An Ecological Risk Assessment of Ammonia in the Aquatic Environment

Ammonia is released in the environment by many industries and other human activities. The major quantifiable sources of ammonia released to aquatic ecosystems across Canada are municipal wastewater treatment plants, at an estimated total quantity of 62,000 tonnes per year. Given the sources of ammonia releases in the environment and the properties of the substance, terrestrial plants and aquatic organisms are potential risk targets. A tiered assessment approach has been used to determine the ecological risk in the aquatic environment from ammonia released in municipal wastewater effluents. The results obtained for two case studies with the probabilistic risk analysis used in the highest tier support the conclusion that the conditions encountered in these two locations can lead to ammonia concentrations capable of producing an adverse ecological impact.     

Utility and relevance of aquatic oligochaetes in Ecological Risk Assessment

Ecological risk assessment (EcoRA) provides both a process and a framework to evaluate the potential for adverse ecological effects occurring as a result of exposure to contaminants or other stressors. EcoRA begins with problem formulation/hazard identification, progresses to effects and exposure assessment, and culminates with risk characterization (an estimate of the incidence and severity of any adverse effects likely to occur). Key components of EcoRA include determining: stressors/contaminants of concern; sensitive, exposed biota; and, appropriate tests and organisms for evaluating effects. Aquatic oligochaetes are not generally used directly in EcoRA because of three major perceptions. First, EcoRA personnel are generally not familiar with or comfortable using this group of organisms. Second, there is believed to be a paucity of widely accepted toxicity tests with these organisms. Third, their taxonomy is considered difficult and uncertain. In fact, aquatic oligochaetes potentially have great utility and relevance to EcoRAs because of factors including: their importance in the aquatic food chain (e.g. prey to fauna including fish and waterfowl; as a vector for contaminant movement through the food chain from bacteria); many species are widely distributed and well studied; representatives include fresh, estuarine and marine species; as a group, they range from sensitive to insensitive over a wide range of environmental insults; they have a long history of use in pollution monitoring and assessment; and, relevant toxicity and biaccumulation tests exist. Toxicity testing under defined conditions is appropriate for problem formulation while more realistic testing for effects assessment (e.g. microcosms) is logistically easier with this group of organisms than with others due to their relatively small size. The importance of aquatic oligochaetes for EcoRA, in particular of sediments, is particularly compelling.     

Testing and Risk Assessment of Persistent and Bioaccumulating Chemical Substances—Improvements within REACH?

In the proposed new European chemicals regulations—the REACH system—the improved control of persistent and bioaccumulating substances is stated as one of the tasks being of particular importance. In this article, the reliability and validity of the scientific basis for identification of persistent (P), bioaccumulating (B), and toxic (T) substances, and for assessing the risks that these substances may pose, are discussed. We have used the European Union risk assessment of pentabromodiphenyl ether, PentaBDE (CAS 32534-81-9) as a study case in the analysis. It is concluded that for PBT substances there is room for development both with regard to test methodology and with regard to risk assessment procedures.     

Lack of micronuclei formation in bone marrow of rats after repeated oral exposure to nickel sulfate hexahydrate

Workplace exposures to mixtures of nickel compounds have been associated with excess respiratory cancer risk. Animal studies with individual nickel compounds indicate that not all nickel substances have the same potency or potential to induce tumors. The bioavailability of nickel ions at critical cellular sites seems to be important to determine the potential of a substance to induce tumors in animals, but much less is understood about the exact nature (genotoxic or non-genotoxic) of the nickel effects. Within many regulatory frameworks (e.g., European Union), substances are classified for mutagenicity based on the available data and this classification will often influence the mode of action assigned to carcinogenic substances and the way in which risk assessment will be conducted. The objective of this study was to evaluate the ability of nickel sulfate hexahydrate to induce micronuclei in polychromatic erythrocytes (PCEs) in rat bone marrow. This study was conducted according to OECD and EU protocol guidelines. In the dose range-finding assays, the maximum tolerated dose was estimated to be 500 mg/kg/day. The doses used in the micronucleus assay were 125, 250, and 500 mg/kg/day. At least 2000 PCEs per animal were analyzed for micronuclei in PCEs. Cytotoxicity was assessed by scoring a minimum of 500 consecutive total polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). Nickel sulfate hexahydrate did not induce statistically significant increases in micronucleated PCEs at any dose examined. The negative results in the present study contribute significantly to the weight of evidence evaluation of the mutagenicity (chromosomal level) of nickel substances. These results are consistent with a non-genotoxic mode of action for soluble nickel that could explain the enhancement of cancer risk seen among refinery workers with mixed exposures and its lack of carcinogenicity in animal studies with single exposures.     

水生动物进口风险评估综述

随着全球水产养殖业的发展,水产动物活体或水产品贸易日益频繁,不可避免地给进口国带来病原风险,造成疾病引入和传播,甚至生物入侵,严重危害水产养殖业的发展。水生动物进口风险评估(IRA),是指从别国或地区进口水生动物活体包括其受精卵、稚鱼、幼鱼、苗种、成体以及商品等的风险分析。风险分析是指对风险事件进行科学、透明、系统分析的一个过程,它由危害识别、风险评估、风险交流和风险管理4个部分构成。目前,常用的风险评估方法有定性风险评估、半定量风险评估和定量风险评估3种。定性风险评估具有灵活性强、适用范围广、易掌握的特点,能够综合各种资料、数据和信息,尤其适合初次风险评估,但容易受评估人员主观因素的影响。定量风险评估可避免主观因素的影响,评估结果准确、可靠,但需要收集大量数据,工作量巨大,评估成本也很高。通常,定性评估结果若能够提供很好的防范措施,则不必进行定量评估。将外来水生病原阻止在引进之初远比引入后根除更加容易。因此,开展水生动物进口风险评估对于阻止水生动物疾病传播和水生态环境破坏具有重要意义,同时也可为各国进行水产贸易提供参考。     

Impact category ecotoxicity and valuation procedure

Within the methodology of LCA, the impact assessment of the impact category ecotoxicity and the valuation step for all categories still pose methodological challenges. A multi-tier approach is a pragmatic solution for the impact assessment of the impact category ecotoxicity. The screening tier only makes use of information concerning substance specific characteristics in order to derive both an identification of critical substances and a comparison of products. Within the detailed tier, critical substances are analyzed in more detail, but still without site-specific information on concentrations, time or region. Valuation combines scientific results with value judgements. Thus, it is not an objective process which can substitute decision making, but instead serves as a base for the interpretation of results. A pragmatic approach combines the results of the impact assessment with a normalization and weighting of the impact categories. Four approaches for deriving general weighting sets (population surveys, expert surveys using the delphi-technique, sustainable development, distance-to-target) are discussed and the first results obtained are presented.     

Classification trees as a tool for predicting cyanobacterial blooms

Nutrient enrichment of aquatic ecosystems caused dramatic increase in the frequency, magnitude and duration of cyanobacterial blooms. Such blooms may cause fish kills, have adverse health effects on humans and contribute to the loss of biodiversity in aquatic ecosystems. Some 50 eutrophic to hypereutrophic ponds from the Brussels Capital Region (Belgium) were studied between 2003 and 2009. A number of the ponds studied are prone to persistent cyanobacterial blooms. Because of the related health concerns and adverse effects on ecological quality of the affected ponds, a tool for assessment of the risk of cyanobacterial bloom occurrence was needed. The data acquired showed that cyanobacteria have threshold relationships with most of the environmental factors that control them. This is negatively reflected on the predictive capacity of conventional statistical methods based on linear relationships. Therefore, classification trees designed for the treatment of complex data and non-linear relationships were used to assess the risk of cyanobacterial bloom occurrence. The main factors determining cyanobacterial bloom development appeared to be phytoplankton biomass, pH and, to a lesser degree, nitrogen availability. These results suggest that to outcompete eukaryotic phytoplankters cyanobacteria need the presence of environmental constraints: carbon limitation, light limitation and nitrogen limitation, for which they developed a number of adaptations. In the absence of constraints, eukaryotic phytoplankters appear to be more competitive. Therefore, prior build up of phytoplankton biomass seems to be essential for cyanobacterial dominance. Classification trees proved to be an efficient tool for the bloom risk assessment and allowed the main factors controlling bloom development to be identified as well as the risk of bloom occurrence corresponding to the conditions determined by these factors to be quantified. The results produced by the classification trees are consistent with those obtained earlier by probabilistic approach to bloom risk assessment. They can facilitate planning management interventions and setting restoration priorities.     

Transabiotic factors in an aqueous environment (a review)

Excretion of metabolites is a characteristic feature of any alive organism. A big group of these products--second metabolites--because of their variability, quantity and physico-biological activity have a special importance in aquatic environment. Exometabolites of some organisms become an important part of environment for the others. The authors discussed the origin and evolution of exometabolites from simple waste products to biologically active substances. Quantitative and qualitative aspects of metabolic excretion by organisms in water conditions are analysed. The data on composition, origin and biological function of some second metabolites of different groups of aquatic organisms are presented. The authors propose a classification of second metabolites according to their functional significance. The role of metabolites and decay products in the development of chemical information streams in hydrobiocoenosis is analysed. Metabolites (soluble organic substances) form a field of chemical information for biotic community. The most important functions of this field are communication and conditioning. The authors emphasize the importance of investigations of chemical bioinformation field in aquatic ecosystems.     

An Ecological Risk Assessment of Hexachlorobutadiene

Hexachlorobutadiene (HCBD) has never been commercially produced in Canada and was imported in the past for use as a solvent. Anthropogenic activity is linked with the entry of this substance into the environment. While current Canadian sources of HCBD involve low-level releases, potentially they can be numerous. Until recently, the most significant point source of HCBD in Canada appeared to be the Cole Drain, which discharges into the St. Clair River at Sarnia, Ontario, and includes outfalls from an industrial landfill and a few industrial companies. HCBD has been detected in Canadian surface waters, sediments, aquatic organisms and, occasionally, air. Considering the properties of the substance, including its persistence and bioaccumulation characteristics, the environmental risk assessment of HCBD was focused on the aquatic environment. The results of a conservative assessment suggest that there is a risk of harmful effects for benthic organisms exposed to sediments contaminated by HCBD in the most contaminated part of the St. Clair River.     

Assessing the ecological values of rivers: an application of a multi-criteria approach to rivers of the South Coast Region,Western Australia

There is a growing awareness that in the light of increased threats, Australia’s freshwater ecosystems of highest value need to be protected. This implies that a consistent and comprehensive system for the classification of freshwater systems in terms of their ecological values is required for conservation planning. Despite earlier steps taken to develop appropriate methodologies for prioritising Australian waterways for protection, there is, as yet, no nationally agreed method for prioritising waterways in Australia based on their ecological values. Using the core criteria of naturalness, representativeness, diversity, rarity and special features, this paper describes the development of a framework of criteria, indicators and measures for the identification of high conservation value aquatic ecosystems, and trials this system by undertaking a comparative assessment of the ecological values of river systems in the South Coast region, Western Australia. A total of 33 river systems located in two aquatic bioregions were successfully ranked according to their ecological value, confirming that the development and implementation of a relatively simple system for assessing ecological values of Australian rivers shows promise, and that core criteria identified previously in the literature provide a good starting point for assessing ecological values of Australian aquatic ecosystems.     

Phenotype Classification of Zebrafish Embryos by Supervised Learning

Zebrafish is increasingly used to assess biological properties of chemical substances and thus is becoming a specific tool for toxicological and pharmacological studies. The effects of chemical substances on embryo survival and development are generally evaluated manually through microscopic observation by an expert and documented by several typical photographs. Here, we present a methodology to automatically classify brightfield images of wildtype zebrafish embryos according to their defects by using an image analysis approach based on supervised machine learning. We show that, compared to manual classification, automatic classification results in 90 to 100% agreement with consensus voting of biological experts in nine out of eleven considered defects in 3 days old zebrafish larvae. Automation of the analysis and classification of zebrafish embryo pictures reduces the workload and time required for the biological expert and increases the reproducibility and objectivity of this classification.     

Olfactory assessment of predation risk in the aquatic environment

The aquatic environment is well suited for the transmission of chemical information. Aquatic animals have evolved highly sensitive receptors for detecting these cues. Here, I review behavioural evidence for the use of chemical cues by aquatic animals for the assessment of predation risk. Chemical cues are released during detection, attack, capture and ingestion of prey. The nature of the cue released depends on the stage of the predation sequence in which cues are released. Predator odours, disturbance pheromones, injury-released chemical cues and dietary cues all convey chemical information to prey Prey use these cues to minimize their probability of being taken on to the next stage of the sequence. The evolution of specialized epidermal alarm substance cells in fishes in the superorder Ostariophysi represent an amplification of this general phenomenon. These cells carry a significant metabolic cost. The cost is offset by the fitness benefit of the chemical attraction of predators. Attempts of piracy by secondary predators interrupt predation events allowing prey an opportunity for escape. In conclusion, chemical cues are widely used by aquatic prey for risk assessment and this has resulted in the evolution of specialized structures among some taxa.     

Trends in Risk Assessment of Chemicals in the European Union

Current legislation in the European Union (EU) requires a risk assessment for industrial chemicals. The underlying procedures and paradigms of such EU risk assessment for new and existing chemicals are explained. The risk assessment is performed according to a harmonised methodology, laid down in the Technical Guidance Documents (TGD). Important new, technical risk assessment aspects covered in a recent revision round of the TGD are highlighted. The most prominent change in the environmental TGD part is the addition of the marine risk assessment, including a Persistent Bioaccumulation and Toxicity (PBT) assessment. In the human health part a significant change is the new data requirement for reproductive toxicity. The performance of both the risk assessment and the risk reduction phase of EU existing chemicals have been evaluated. An important conclusion was that our a priori knowledge on possible risks of chemicals is poor. The European Commission has recently launched a proposal (REACH) for drastically changing the risk management process of industrial chemicals in the EU. Major changes are a shift in responsibility from authorities to industry (including downstream users) for the safe use of chemicals, an acceleration of data collection for ‘non-assessed’ chemicals, and an authorization step for substances of very high concern.     

Development of a new aggregative index to assess potential effect of metals pollution in aquatic sediments

A new aggregative index called modified risk assessment code (mRAC) was developed based on heavy metal toxicity and fractionation in aquatic sediment. Results of the application of mRAC to assess metal pollution in surface sediment samples of Anzali international wetland revealed that the new index led to more precise results than those of other prevalent aggregative indexes, such as modified degree of contamination (mC_d) and ecological risk index (RI). Based on assessment by mRAC, sediments of the study area were at high or very high potential adverse effect levels, compared to using mC_d and RI where sediments were at a moderate or low potential adverse effect level. This is due to mRAC taking both metal toxicity and fractionation into account.     

Refinement and reduction of acute oral toxicity testing: a critical review of the use of cytotoxicity data

Acute oral toxicity testing is still required for the classification and labelling of chemicals, agrochemicals and related formulations. There have been increasing efforts over the last two decades to reduce the number of animals needed for this testing, according to the Three Rs concept. To evaluate the utility of an in vitro cytotoxicity test in our routine testing for acute oral toxicity, we have implemented in our laboratory the neutral red uptake (NRU) method, with Balb/c 3T3 fibroblasts after a 48-hour exposure, which was recommended in ICCVAM Report 07-4519, 2006. Initially, we tested 16 substances that had existing in vivo and in vitro data available, to prove our technical proficiency with the in vitro test. Then, testing was performed with 187 test substances, including a broad variety of chemicals, agrochemicals and formulations. The starting dose for acute oral systemic toxicity assays in rats (LD50) was estimated by using the prediction model presented in the ICCVAM validation study, and subsequently compared to the results obtained by in vivo testing performed according to, or similar to, OECD Test Guideline 423. Comparison of all of the 203 predicted LD50 values that were deduced from the in vitro IC50 values, with the in vivo results from oral toxicity studies in rats, resulted in a low overall concordance of 35%. The in vitro cytotoxicity assay achieved a good concordance of 74%, only for the weakly toxic substances (EU-GHS Cat. 4). However, it must be noted that 71% of the substances tested (i.e. 145/203) were classified as being weakly toxic in vitro. We further analysed the utility of the in vitro test for predicting the starting dose for an in vivo study, and the potential reduction in animal usage that this would engender. In this regard, the prediction by the cytotoxicity test was useful for 59% of the substances. However, the use of a standard starting dose of 300 mg/kg bw by default (without previous cytotoxicity testing) would have been almost as useful (50%). In contrast, the prediction by an experienced toxicologist was correct for 95% of the substances. However, this was only performed for 40% of the substances, mainly those of no to low toxicity. Calculating the theoretical animal numbers needed in several scenarios supported these results. The additional analysis, considering some physicochemical data (solubility, molecular weight, log POW), substance class and mode of action, revealed no specific applicability domains. In summary, the use of the 3T3 NRU cytotoxicity data alone did not sufficiently contribute to refinement and reduction in the acute oral toxicity testing of the substance portfolio tested routinely in our laboratory.     

Workshop on rotifers in ecotoxicology

The aim of the workshop on rotifers in ecotoxicology was to stimulate discussions on new developments in the field. Discussions about the use of biomolecular tools indicate that gene expression analysis with rotifers should be available in the next few years. Such analyses will be a great asset as they enable ecotoxicologists to study molecular mechanisms of toxicity. Rotifers also appear as useful tools in the risk assessment of pharmaceuticals and their metabolites that find their way into aquatic ecosystems because their sensitivity to some of these substances is higher than that of cladocerans and algae. The nature and extent of the impact of potential endocrine disruptors on aquatic invertebrates is another poorly resolved issue for which rotifers are a promising tool. Indeed, rotifers seem to be particularly sensitive to androgenic and anti-antiandrogenic substances, whereas copepods and cladocerans are typically more affected by estrogens and juvenile hormone-like compounds. Besides their usefulness in these emerging fields of aquatic ecotoxicology, it was emphasized that research with rotifers on basic issues like, e.g., toxicant interference with predation, competition, or interspecific and interclonal variation in ecotoxicological tests is still needed. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont & R. Rico-Martínez Advances in Rotifer Research     

A bacterial reporter panel for the detection and classification of antibiotic substances

The ever‐growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy‐based non‐antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.     

Semi-automated classification method addressing marine strategy framework directive (MSFD) zooplankton indicators

Semi-automated classification of zooplankton allows increasing the number of processed samples cost-effectively, albeit with a relatively limited taxonomic accuracy, partly because cost-efficiency trade-off but also due to technological limitations that might be overcome in the future. The present study tests the suitability of using a cost-efficient semi-automated classification methodology as a tool to assess zooplankton indicators for the purpose of the EU Marine Strategy Framework Directive, using samples collected in the Baltic Sea. In this brackish ecosystem the zooplankton individuals are small-bodied and therefore their identification with semi-automated classification is challenging. However, results show that semi-automated zooplankton classification provides a taxonomic classification level that is sufficient for a number of proposed indicators. This analysis also points out weakness of the methodology and proposes already proved solutions based on the latest development of these methodologies applied to zooplankton classification. As proved in the Baltic Sea, complementing manual zooplankton analyses with the semi-automated classification offers new advantages for marine environment assessment.     

Variability in the reproductive performance of beneficial insects in standard laboratory toxicity assays - Implications for hazard classification of pesticides

Nowadays the development of pesticides is not only directed to design compounds with sufficient efficacy against resistant and non-resistant strains of pests as well as with a high margin of safety for man and the environment, but also to allow their potential use in IPM (Integrated Pest Management) programmes. For identification of IPM suitable pesticides, a whole battery of standardised laboratory and semi-field test protocols have been developed. Based on the observed lethal and sublethal effects in those tests, compounds are categorised into different hazard groups which should advise farmers on their suitability for IPM. For a valid classification of compounds on the basis of laboratory test results, threshold values for lethal and sublethal effects have to be developed which reliably differentiate between harmful and safe compounds without bearing the risk of erroneous labelling and/or too frequent requests for higher tier testing. As an evaluation criterion for sublethal effects of pesticides, the reproductive performance of beneficial insects is frequently considered in standard laboratory assays. A preliminary analysis of available data on the reproductive performance of several standard test species elaborated during regulatory testing indicates that this evaluation criterion is subject to high variability. As expected, individual test species differed in their reproductive performance. Aleochara bilineata (Coleop-tera: Staphylinidae) and Orius insidiosus (Heteroptera: Anthocoridae) showed a fairly homogeneous reproductive performance within test series. Based on the observed variability of the reproductive performance in control groups, the average probability of an erroneous labelling of pesticides for these two species was only 4% and 6% (maximum probability: 13% and 19%), respectively, when an adverse effect threshold value of 30% (= actual value of the EU (European Union)) was applied. In contrast, Coccinella septempunctata (Coleoptera: Coccinellidae), Chryso-perla carnea (Neuroptera: Chrysopidae), Aphidius rhopalosiphi (Hymenoptera: Aphidiidae) and Trichogramma cacoeciae (Hymenoptera: Trichogrammatidae) exhibited a high variability in their reproductive performance, giving a mean probability between 25% and 35% (maximum probability: 36–64%) to label a pesticide either false positive or false negative. Besides species-inherent variability, there was an indication that test-inherent factors including parent sex ratio and parent breeding density may have had an influence on the reproductive performance of these insect species. Seasonal influences on the reproductive performance of the beneficial insects in laboratory testing were not evident. Based on our data analyses, there is a significant risk of erroneous classification of pesticides when the reproductive performance is quantitatively assessed following the currently established test protocols and an adverse effects threshold value of 30% is applied. We propose therefore that either the testing procedure for assessing the reproductive performance, or the effect threshold value for this evaluation criterion is reconsidered in the light of the high “species-inherent” variability in the reproductive performance of some beneficial insects.