# Key Properties for the Toxicity Classification of Chemicals:
## 3 Chemical Properties and Safety Assessment Tests Required under the REACH
### Ecotoxicological Information  
Most chemical waste, both industrial and domestic, ends up in the environment. Thus, it is essential to develop regulations that minimize the impact in the environment derived from the use of chemical substances and demand minimum requirements to ensure environmental safety. It is important to determine all environmental characteristics and subsequently analyze abiotic and biotic factors [14]. In this sense, abiotic factors are related to non-living physicochemical properties such as air, soil, water, sunlight, or minerals. On the contrary, biotic factors are living microorganisms living in ecosystems (animals, plants, fungi, etc.) With this in mind, REACH uses different tests with the aim to study the toxicity of different chemicals, their behavior in the environment and, additionally, how biotic and abiotic factors can be modified. The use of these tests depends on the tones per year of each substance marketed in the European Union. In Table 1, the ecotoxicological tests recommended by REACH are presented. These tests can be divided into a) biomodels; b) degradation (biotic, abiotic, and ready biodegradability), fate, and behavior in the environment (adsorption or desorption and bioaccumulation in aquatic species). Biomodels are widely used in ecotoxicology because the use of some indicators, generally from different trophic levels, can provide valuable information on how chemicals behave in the environment [15; 16]. REACH is mainly focused on aquatic and terrestrial toxicity. For evaluating the environmental impact, several biomodels are proposed. For instance, algae are used to represent the primary producers (first step in the food chain). The OECD TG 201 (freshwater alga and cyanobacteria, growth inhibition test) is recommended. The aim of this bioassay it to determine the effects of a chemical on microalgae or cyanobacteria exposed to the chemical for 72 h. Then, the growth inhibition is analyzed. The limit concentration corresponds to 100 mg/L [17]. Another required biomodel is invertebrates such as crustaceans, _Daphnia magna status_, classified as primary consumers. REACH recommends carrying out the OECD TG 202 (acute immobilization test). In this test, chemicals are in contact with young daphnids aged less than 24 h. Immobilization is observed at 24 h and 48 h and compared with the control [18]. Finally, vertebrates (normally fish) are widely known as secondary consumers. For the evaluation of chemical toxicity towards fishes, it is recommended to use the OECD TG 203 (fish acute toxicity test). In this test, one or more species can be used, and fishes are exposed to the chemical for 96 h and mortalities are analyzed at 24 h, 48 h, 72 h, and 96 h. After this period, a lethal concentration 50% (LC50), which is the concentration of a chemical expected to produce an effect in 50% of test organisms, value is obtained [19]. For terrestrial studies, invertebrates, plants, and sediment organisms are used. In the case of plants, the OECD 208 terrestrial plant test: seedling emergence and seedling growth test, is usually used. This assay can evaluate the effects of a chemical on seedling emergence and early growth of higher plants. At the end of the assay, measurements are visual based on seeding emergence, biomass concentrations, shoot height, and different visual effects on different parts of the plant. Values of NOEC, which are defined as no observed effect concentration or the LOEC, determine the lowest observed effect concentration [20]. In the case of earthworms, the recommended tests are OECD 207: earthworm, acute toxicity tests and OECD 222: earthworm reproduction test (_Eisenia fetida_/_Eisenia andrei_). In the first one, the recommended species is _Eisenia fetida_ and the test consists of putting the worms in contact with the chemical in an artificial soil and analyzing the results after 7 and 14 days of exposition [21]; in the second one, the main aim is to analyze the reproductive output in _Eisenia fetida_ or _Eisenia andrei_ in a 4-week period.