Practical considerations for conducting ecotoxicity test methods with manufactured nanomaterials: what have we learnt so far?

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.

Biodegradation of chemicals in a standardized test and in environmental conditions.

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 microg 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.

Developing Test Guidelines on invertebrate development and reproduction for the assessment of chemicals, including potential endocrine active substances- the OECD perspective.

The Organisation for Economic Co-operation and Development (OECD) Test Guidelines Programme is involved in the international harmonization 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.

Toxicity of industrial wastes and waste leaching test eluates containing organic compounds.

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.