Field research for the authorisation of pesticides.

On request of the Dutch government a committee of the Health Council of the Netherlands has reviewed the role that results of field research in its broadest sense (i.e., including multi-species toxicity tests in the laboratory, research on model ecosystems et cetera) can play in ecotoxicological risk assessment for the authorisation of pesticides. The Committee believes that field research can provide valuable additional data about the exposure of non-target organisms and the resultant effects at population, community and ecosystem level. However, it frequently is unclear how these data might be used in reaching a decision about authorisation. To solve this problem, it is necessary to specify what is understood by "unacceptable damage". Both more clearly formulated protection goals of the government and a better understanding of the ecological significance of effects are needed to clarify this. Furthermore, the Committee points out that the statistical power of field trials must be sufficient to allow for the detection of changes that might be regarded as ecologically relevant. Finally, it recommends keeping a finger on the pulse in relation to authorised pesticides by monitoring their presence in environmental compartments and by investigating their role in suddenly occurring mortality among conspicuous animal species, such as birds, fish and honeybees. This kind of research forms a safety net for substances that have been wrongly authorised.

In vivo detection of waste water and industrial effluent genotoxicity: use of the Newt Micronucleus Test (Jaylet Test).

The genotoxic potential of various waste waters has been evaluated in a micronucleus test using amphibian larvae. Genotoxicity was detected after dilution, in waste water from tanneries and from various petrochemical industries. Further studies have shown that sample treatment used for in vitro testing may affect the genotoxic response. Sterilization by gamma irradiation lowered genotoxic activity. Furthermore, microfiltration of effluent and extraction of organic micropollutants on XAD-4 resins, lead to the preparation of extracts which are not fully representative of the initial water sample. Testing of concentrates, as required for in vitro studies, will limit the scope of a survey to that part of the organic matter that can be recovered by concentration techniques. Many of the problems encountered in in vitro genotoxicity studies of waters, may be circumvented with direct testing on aquatic organisms. Thus, there is no need to concentrate or sterilise a sample. The tests can be carried out with intact animals, thus taking into account uptake and elimination, internal transport and metabolism. Finally, in vivo test-systems, such as the Newt Micronucleus Test, are more relevant to eukaryotes than bacterial assays and are suitable to assess the real impact of genotoxins discharged in the aquatic environment.

In vivo detection of genotoxicity in waste water from a wheat and rye straw paper pulp factory.

The genotoxicity of waste water from a wheat and rye straw paper pulp mill was investigated by in vivo genotoxicity tests using micronuclei and sister chromatid exchange as endpoints. Micronuclei were studied in mussels (Mytilus edulis) and sister chromatid exchange in fish (Nothobranchius rachowi). The paper mill uses chlorine dioxide for bleaching, and the bleaching effluent as well as the combined effluent, i.e. the mixture of all waste water streams, were both tested. Both effluents induced micronuclei and sister chromatid exchanges, although the presence of toxic substances could mask the expression of genotoxicity in some cases for both test systems. The study revealed that genotoxins are produced in the chlorine dioxide bleaching process as well as in the pulping process, indicating also genotoxic activity of non-chlorinated compounds. In contrast to previous studies in which mutagenicity was determined with bacterial assays, genotoxins were only associated with chlorinated organics from bleaching with chlorine and failed in detecting genotoxins in chlorine dioxide bleaching effluents. Aquatic in vivo genotoxicity tests are sensitive and efficient systems and seem to be a promising tool in effluent testing.

QSARs in Netherlands water quality management policies.

QSARs are a useful tool for predicting the potential toxic effects of compounds for which no data are available. Within strictly defined limits, QSARs can be applied to assess the potential impact of a spill, to evaluate ecotoxicological effects and environmental fate of organics in waste water and to set priorities for water quality criteria. For a wider application, there is a need for 'worst case' SARs providing a 'safer' estimate of toxicity than QSARs with an optimum fit, which might underestimate toxicity.

Mutagenicity testing of water with fish: a step forward to a reliable assay.

A step forward has been set towards the realization of a reliable "sister-chromatid" exchange (SCE) assay with fish for the detection of mutagens in water. This test can detect mutagenic contaminants in water without prior concentration steps. A healthy breeding stock of the tropical fish Nothobranchius rachowi has been set up in our laboratory. This fish is particularly suited for the SCE assay because of its low number of chromosomes. The major improvement consisted of the development of a reliable staining technique for the differentiation of sister-chromatids. The assay can now be used for research purposes. With a few improvements it can become a rapid low-cost test for the screening of effluents on contamination with DNA-damaging agents.

The influence of water treatment processes on the presence of organic surrogates and mutagenic compounds in water.

The effects of granular activated carbon filtration and of the combination of ozonation and GAC filtration on the quality of Rhine water were studied in a pilot plant. The scope of the study was to compare both systems in relation to the removal of organic contaminants in water, and to the reduction of the side effects of chlorination. The water quality was measured with organic surrogate parameters (organohalogen, -nitrogen, -phosphorus and -sulphur) and in bacterial mutagenicity assays. In this particular setting, the combination of ozonation and GAC filtration was superior in all points to GAC filtration alone. The effects of ozonation are sometimes quite different, depending on the type of water treated. Its positive influence should be confirmed in a local situation. As GAC treatment causes a shift towards formation of more brominated THM after chlorination, special attention was given to this item. A higher inorganic bromide/DOC ratio resulted in higher brominated THM concentrations after chlorination. However, the mutagens formed during chlorination in presence of more inorganic bromide could be inactivated more easily by rat liver homogenate than in the normal setting. The results of this study confirmed earlier findings stating a negative influence of chlorination on water quality.

Some factors affecting optimal differential staining of sister-chromatids in vivo in the fish Nothobranchius rachowi.

In the past few years, routine studies of SCE induction in vivo in fish have been hampered by unreliable SCD techniques. This paper presents a number of modifications of the SCD technique in vivo in Nothobranchius rachowi. Major improvements were obtained by BrdU incorporation from aqueous solutions, short intervals between preparation and staining of slides and post-treatment with HCl. These improvements resulted in a highly reliable SCD procedure in Nothobranchius with a low level base-line SCE frequency (0.90 SCE/metaphase, 0.059 SCE/chromosome). Further research is now directed at gathering additional data on base-line SCE frequencies, establishing the sensitivity of the assay for aqueous solutions of known mutagens, and defining an experimental set-up for optimal statistical evaluation.

Toxicological assessment of river water quality in bioassays with fish.

A series of bioassays with fish was developed in order to evaluate toxicological aspects of polluted rivers in The Netherlands. A long term exposition of trout to riverwater under standardized conditions enables the detection of pathological effects such as growth retardation, liver and kidney enlargement and changes in clinical blood parameters. Bioaccumulation of heavy metals and organochlorine compounds can also be measured. Embryo-larval tests with trout were less suitable, because of yearly variations in egg quality. In the near future, sister chromatid exchange (SCE) assays in vivo with Nothobranchius may become available for the detection of mutagenic effects. It was possible to measure trends in toxicological quality of Rhinewater with these tests. However extrapolation of results to ecosystems and tracing of the causes of changes occurring in waterquality are still problematic.

Induction of sister-chromatid exchanges in fish exposed to Rhine water.

The induction was studied of sister-chromatid exchanges in gill and testis of fish exposed to Rhine water. The eastern mudminnow, Umbra pygmaea, was chosen because the usefulness of this species with a karyotype of 22 large chromosomes had been demonstrated in cytogenetic mutagenicity testing. Fish exposed to Rhine water for 3 and 11 days showed a 2-fold and 3-fold higher SCE rate resp., compared with fish exposed to ground water of drinking water quality. There was no difference in SCE rate between gill and testicular cells before and after exposure. GLC/MS analysis indicated that different classes of chemical compounds may be involved in the cytogenetic effects found in fish. The mudminnow has proved to be a useful fish species for biological monitoring of mutagens in polluted waters. The SCE test may be a suitable test system to prescreen their mutagenic potential.

Ultrastructure and lipid content of the liver of the zebrafish, Brachydanio rerio, related to vitellogenin synthesis.

The female zebrafish is capable of producing mature eggs on the fifth day of each reproductive cycle. During this five-day period the ultrastructure of hepatocytes undergoes several changes. The number of nuclear pores increases rapidly during spawning, followed by a proliferation of RER within 24 h. Two days after spawning, glycogen has disappeared and the liver contains large amounts of lipids. The lipid droplets are closely surrounded by elongated mitochondria. Golgi complexes are abundant, secreting dense bodies. Four days after spawning the hepatocytes tend to regain their pre-spawning appearance. It is suggested that the changes in the hepatocytes, which coincide with special phases of ovarian activity, are related to vitellogenin synthesis. Steroids, especially estradiol-17beta, may trigger this process in the liver.