Various types of toxicity classification systems have been elaborated by scientists in different countries, with the aim of attributing a hazard score to polluted environments or toxic wastewaters or of ranking them in accordance with increasing levels of toxicity. All these systems are based on batteries of standard acute toxicity tests (several of them including chronic assays as well) and are therefore dependent on the culturing and maintenance of live stocks of test organisms. Most systems require performance of the bioassays on dilution series of the original samples, for subsequent calculation of L(E)C50 or threshold toxicity values. Given the complexity and costs of these toxicity measurements, they can only be applied in well-equipped and highly specialized laboratories, and none of the classification methods so far has found general acceptance at the international level. The development of microbiotests that are independent of continuous culturing of live organisms has stimulated international collaboration. Coordinated at Ghent University, Belgium, collaboration by research groups from 10 countries in central and eastern Europe resulted in an alternative toxicity classification system that was easier to apply and substantially more cost effective than any of the earlier methods. This new system was developed and applied in the framework of a cooperation agreement between the Flemish community in Belgium and central and eastern Europe. The toxicity classification system is based on a battery of (culture-independent) microbiotests and is particularly suited for routine monitoring. It indeed only requires testing on undiluted samples of natural waters or wastewaters discharged into the aquatic environment, except for wastewaters that demonstrate more than 50% effect. The scoring system ranks the waters or wastewaters in 5 classes of increasing hazard/toxicity, with calculation of a weight factor for the concerned hazard/toxicity class. The new classification system was applied during 2000 by the participating laboratories on samples of river water, groundwaters, drinking waters, mine waters, sediment pore waters, industrial effluents, soil leachates, and waste dump leachates and was found to be easy to apply and reliable.

Cylindrospermopsis raciborskii is a planktonic freshwater cyanobacterium that has become increasingly prevalent in tropical and temperate water bodies world-wide. This species is of concern from a water-quality perspective because of its known ability to produce toxins that can affect the health of humans and other animals. This study investigates genetic variation between strains of C. raciborskii isolated from freshwater rivers and reservoirs in Australia, Brazil, Germany, Hungary, Portugal and the USA. Strains were first characterized by analysis of their 16S rRNA gene nucleotide sequences and were found to have a sequence divergence of 99.1%. A phylogenetic tree, constructed using the 16S rRNA gene sequences showed that strains grouped into Australian, European and North/South American phylotypes. To investigate further the observed separation of strains into geographically distinct groups, we applied a cyanobacterium-specific short tandem repeat sequence technique, HIP1. An electrophoretic comparison of the HIP1 polymerase chain reaction products showed clear distinctions between the C. raciborskii strains. A phylogenetic tree, based on the repeat element banding patterns, also revealed three distinct groups of C. raciborskii strains. The first group consisted of strains from the USA and Brazil; the second comprised European strains from Germany, Hungary and Portugal; and the third were strains from Australia. In general, between-country variation was greater than within-country variation, indicating that this fingerprinting technique can successfully distinguish C. raciborskii strains taken from different global locations. The relationship between toxicity and the observed HIP1 polymerase chain reaction fingerprint profiles was less clear, although it is interesting to note that of the strains analysed in this study, only Australian strains are known to produce cylindrospermopsin and only Brazilian strains have been reported to produce paralytic shellfish poisoning toxins.

EXECUTIVE SUMMARYOne of the most internationally used bioassays for toxicity screening of chemicals and for toxicity monitoring of effluents and contaminated waters is the acute toxicity test with daphnid crustaceans, and in particular that performed with Daphnia magna. Standard methods have been developed for this assay that were gradually endorsed by national and international organisations dealing with toxicity testing procedures, in view of its application within a regulatory framework. As for all toxicity tests, the organisms used for the acute D. magna assay have to be obtained from live stocks which are cultured in the laboratory on live food (micro-algae). Unsurprisingly the various standard protocols of this particular assay differ -at least to a certain extent -with regard to the test organism culturing conditions. In addition, some technical aspects of the toxicity test such as the effect criterion (mortality of immobility), the exposure time, the type of dilution water, etc., also vary from one standard to another. Although this particular assay is currently used in many countries, the technical and biological problems inherent in year-round culturing and availability of the biological material and the culturing/maintenance costs of live stocks restrict its application to a limited number of highly specialised laboratories. This fundamental bottleneck in toxicity testing triggered investigations which brought forward the concept of "microbiotests" or "small-scale" toxicity tests. "Culture/maintenance free" aquatic microbiotests with species of different phylogenetic groups were developed in the early 1990s at the Laboratory for Environmental Toxicology and Aquatic Ecology at the Ghent University in Belgium. These assays which were given the generic name "Toxkits", are unique in that they employ dormant stages ("cryptobiotic eggs") of the test species, which can be stored for long periods of time and "hatched" at the time of performance of the assays. One of these microbiotests is the Daphtoxkit F magna, which is currently used in many laboratories worldwide for research as well as for toxicity monitoring purposes. The microbiotest technology has several advantages in comparison to the "traditional" tests based on laboratory cultures, especially its independence of the stock culturing burden. However, the acceptance (or possible non-acceptance) of performing assays with test organisms obtained from "dormant eggs" should be clearly dictated by the "sensitivity" and "precision" criteria of the former assays in comparison to the latter. The first part of this review therefore thoroughly reviews the scientific literature and of data obtained from various laboratories for assays performed with either D. magna test organisms obtained from lab cultures or hatched from dormant eggs. Attention has focused on data of quality control tests performed on reference chemicals, and in particular on potassium dichromate (K 2 Cr 2 O 7 ) for which an acceptability range of 0.6-2.1 mg·L -1 has been set in ISO standard 6...

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