The scientific community has invested effort into standardising methodologies for the regulatory ecotoxicity testing of engineered nanomaterials (ENMs), but the practical requirements for ...bioaccumulation testing of ENMs have been given less attention. A strategy for a tiered approach to bioaccumulation testing of ENMs using fish is proposed, with recommendations for its implementation by regulatory agencies. The strategy recognises that testing the many shapes, sizes and chemistries of ENMs as new substances
in vivo
would be an unrealistic workload. The approach therefore includes grouping/read-across methods and tools to screen out ENMs of negligible/low bioaccumulation potential. The strategy proposes reductions of animal use for
in vivo
testing and with greater consideration of
in vitro
methods. The first tier uses dissolution in water or lipids and particle settling rates as environmental chemistry triggers for 'ENMs of concern'. The first tier also involves a weight of evidence from these tests, plus using existing data sets from selected literature that meet data quality criteria for ENMs. Tier 2 involves new data generation using
in silico
models now being validated for ENMs, including QSARs and systems biology tools. Tier 2 also includes using existing experimental data, and an option to collect new data. These data can be on soils/sediments, microbial degradation, and bioaccumulation studies on invertebrates or fish cell lines. In tier 3, an
in chemico
digestibility assay simulating the gut lumen of fish is proposed to identify the bioaccessible fractions from an oral exposure to ENMs. If the digestibility assay is positive, then
in vitro
gut sacs from rainbow trout can be used to confirm accumulation by the gut mucosa. Only if both these tests in tier 3 are positive would the work proceed to the final
in vivo
test (tier 4) which is essentially the OECD TG 305 method for dietary bioaccumulation testing using fish, with some caveats and recommendations for ENMs. These include considerations of terminology, how to prepare contaminated food for dietary exposures, the additional controls and endpoints for ENMs, measuring ENMs in food and tissues to confirm the exposure, and the limitations of any subsequent calculation of the bioaccumulation potential.
The scientific community has invested effort into standardising methodologies for the regulatory ecotoxicity testing of engineered nanomaterials (ENMs), but the practical requirements for bioaccumulation testing of ENMs have been given less attention.
In the conventional cultivation of potatoes, weed control and the control of potato late blight caused by
Phytophthora infestans are carried out by the application of herbicides and fungicides. We ...investigated the impacts of the herbicides metribuzin and linuron and the fungicide fluazinam on soil microbiota in microcosms, in mesocosms and in the field. The toxicity of each pesticide in solution was assessed using the luminescent bacteria test and in soil by a solid phase modification. In microcosm tests, the microbial activity and biomass were estimated by measuring several soil enzyme activities together with soil ATP content. In the mesocosm tests, the separate addition of each pesticide and the simultaneous use of all the pesticides were investigated. We monitored the impacts on ten different soil enzyme activities and measured soil toxicity with the luminescent bacteria test separately in the 5
cm top layer and in the layer from 5 to about 20
cm below the surface. During one season, the impact of the use of pesticides was monitored in the field in plots receiving pesticides for the third consecutive year and in control plots cultivated without the use of pesticides for the 3 preceding years. The pesticide concentrations were monitored in each experiment. The luminescent bacteria toxicity test revealed a strong inhibition by fluazinam. In microcosms the herbicides increased several enzyme activities but metribuzin inhibited luminescence in the bacterial test. Fluazinam was highly toxic in microcosms. In the mesocosm with combined use of pesticides, decreased activities of some enzymes were observed first in the surface soil and at harvesting also deeper in the soil. In the mesocosm experiment with separate use of pesticides, less impacts were observed. In the field experiment the pesticides decreased seven enzyme activities, when calculated per soil fresh weight but activities of four enzyme decreases if calculated per soil organic matter. Controlling weeds by herbicides decreased weed growth and the decreases in enzyme activities were interpreted to be due to the lack of the stimulating impact of weed roots. A strong inhibition in the soil toxicity test and continuing bioavailability of fluazinam were detected throughout the experiments even after winter in the field.
Heterogenous oily waste from an old dumping site was composted in three windrows constructed from different proportions of waste, sewage sludge, and bark. The objectives of this pilot study were to ...examine the usefulness of composting as a treatment method for this particular waste and to study decontamination in the composting process by using a battery of toxicity tests. Five samples from the windrow having intermediate oil concentrations were tested with toxicity tests based on microbes (Pseudomonas putida growth inhibition test, ToxiChromotest, MetPLATE, and three different modifications of a luminescent bacterial test), enzyme inhibition (reverse electron transport), plants (duckweed growth inhibition and red clover seed germination), and soil animals (Folsomia candida, Enchytraeus albidus, and Enchytraeus sp.). The luminescent bacterial tests were used as prescreening tests. Chemical analyses of samples were carried out simultaneously. Both toxicity and oil concentration, including those of polyaromatic hydrocarbons (PAHs), were reduced during composting and soil quality improved significantly. The total oil hydrocarbon concentration decreased from 90,000 to 19,000 mg/kg, measured with the IR method, in 4 months, and from 86,000 to 1400 mg/kg, measured with GC method. The concentration of PAHs decreased from 135 to 23.5 mg/kg. During the fourth month of composting (stabilization stage), the proportion of the heaviest oil fractions (asphaltenes) became dominant. Toxicity varied between different samples and between different bioassays; however, the first sample was significantly more toxic than the others, and most of the tests revealed a decrease in toxicity during the composting process.
The Organisation for Economic Co-operation and Development (OECD) Test Guidelines Programme is involved in the international harmonisation and validation of test methods to evaluate effects of ...chemicals, including potential endocrine active substances. To meet their existing and foreseen regulatory needs in this area, OECD member countries have encouraged the development of test methods and their emergence at the OECD level. Validation activities are underway in countries and industry to ascertain the relevance and reliability of these tests to enable future regulatory acceptance. This includes work on development and (sexual) reproduction of aquatic invertebrates. What is the importance of mechanistic information in regulating chemicals, and how to address the issue of possible endocrine disruption in invertebrates while integrating these tests in a regulatory scheme are the current questions faced by the OECD countries.
This review paper reports the consensus of a technical workshop hosted by the European network, NanoImpactNet (NIN). The workshop aimed to review the collective experience of working at the bench ...with manufactured nanomaterials (MNMs), and to recommend modifications to existing experimental methods and OECD protocols. Current procedures for cleaning glassware are appropriate for most MNMs, although interference with electrodes may occur. Maintaining exposure is more difficult with MNMs compared to conventional chemicals. A metal salt control is recommended for experiments with metallic MNMs that may release free metal ions. Dispersing agents should be avoided, but if they must be used, then natural or synthetic dispersing agents are possible, and dispersion controls essential. Time constraints and technology gaps indicate that full characterisation of test media
during
ecotoxicity tests is currently not practical. Details of electron microscopy, dark-field microscopy, a range of spectroscopic methods (EDX, XRD, XANES, EXAFS), light scattering techniques (DLS, SLS) and chromatography are discussed. The development of user-friendly software to predict particle behaviour in test media according to DLVO theory is in progress, and simple optical methods are available to estimate the settling behaviour of suspensions
during
experiments. However, for soil matrices such simple approaches may not be applicable. Alternatively, a Critical Body Residue approach may be taken in which body concentrations in organisms are related to effects, and toxicity thresholds derived. For microbial assays, the cell wall is a formidable barrier to MNMs and end points that rely on the test substance penetrating the cell may be insensitive. Instead assays based on the cell envelope should be developed for MNMs. In algal growth tests, the abiotic factors that promote particle aggregation in the media (e.g. ionic strength) are also important in providing nutrients, and manipulation of the media to control the dispersion may also inhibit growth. Controls to quantify shading effects, and precise details of lighting regimes, shaking or mixing should be reported in algal tests. Photosynthesis may be more sensitive than traditional growth end points for algae and plants. Tests with invertebrates should consider non-chemical toxicity from particle adherence to the organisms. The use of semi-static exposure methods with fish can reduce the logistical issues of waste water disposal and facilitate aspects of animal husbandry relevant to MMNs. There are concerns that the existing bioaccumulation tests are conceptually flawed for MNMs and that new test(s) are required. In vitro testing strategies, as exemplified by genotoxicity assays, can be modified for MNMs, but the risk of false negatives in some assays is highlighted. In conclusion, most protocols will require some modifications and recommendations are made to aid the researcher at the bench.
The estimation of biodegradation rates is an important source of uncertainty in chemical risk assessment. The existing OECD tests for ready biodegradability have been developed to devise screening ...methods to determine whether a chemical is potentially easily biodegradable, rather than to predict the actual rate, of biodegradation in the environment. However, risk assessment needs degradation rates. In practice these rates are often estimated (default values) from ready biodegradability tests. These tests have many compromising arbitrary features compared to the situation in the real environment. One important difference is the concentration of the chemical. In wastewater treatment or in the environment many chemicals are present at ng
l
−1 to μg
l
−1 levels whereas in the tests the concentrations exceed 10–400 mg carbon per litre. These different concentrations of the chemical will lead to different growth kinetics and hence different biodegradation rates. At high concentrations the chemical, if it is degradable, can serve as a primary substrate and competent microorganisms will grow exponentially, resulting in a sigmoid biodegradation curve. At low environmental concentrations the chemical does not serve as a primary substrate, and therefore does not support significant growth of the degraders, and the substrate has a linear biodegradation rate. In this study the biodegradation rates of two reference chemicals, aniline and 4-chloroaniline, were compared in a standard method and in more realistic conditions at low concentrations, using
14C-labelled substances and different sources of inocula. Biomass evolution during the tests was monitored by adenosine triphosphate measurement and also on the basis of the residual
14C-activity in the particulate matter. The results partly support the thesis that low concentrations lead to different biodegradation kinetics compared to the concentrations used in the standard tests. Furthermore the biodegradation rates of the chemicals studied, particularly of 4-chloroaniline, in Finnish natural waters appeared to be lower than those reported in some other countries.
Leaching tests, CEN prEN 12457-2, CEN PrEN 12457-3, and NEN 7349, were conducted for varnish residue and urea resin waste, two industrial wastes containing organic chemicals. The leaching test ...eluates were analyzed for solvent concentrations and total organic carbon. Aqueous leaching tests were found to be suitable for both chemical and biological testing. Ecotoxicity was assessed by luminescent bacteria, plant root growth, reverse electron transport, and ToxiChromopad. The eluates were highly toxic but the toxicity decreased in later stages of the multistep leaching test (NEN 7349). Urea resin eluates were significantly less toxic than varnish residue eluates. The solid wastes markedly inhibited plant seed germination before and after the leaching test (CEN prEN 12457-3). The solid wastes were not biologically degradable in the standard test (ISO 14593), whereas the eluates from the CEN prEN 12457-3 test degraded slowly.
Effects of two pesticides, dimethoate, an insecticide, and benomyl, a fungicide, applied singly or together, on soil organisms and plant growth were studied in microcosms containing agricultural soil ...and indigenous soil fauna together with introduced invertebrates and barley. Dimethoate reduced soil microarthropod populations and the reduction was stronger in the upper than in the lower soil layer. The collembolan community structure was affected by both pesticides. Populations of microarthropods in pesticide-treated microcosms recovered during the experiment but the community structures remained differentiated. Total numbers of enchytraeids and nematodes were not affected by either of the pesticides. The pesticides affected microbial biomass (ATP content) to some extent, but did not affect CO
2 production, soil mineral N or barley growth. It can be concluded that although pesticides had transient effects on microorganisms and, possibly, some microbivorous animals, their influence on nutrient dynamics was negligible and they did not affect plant growth indirectly.
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