# Key Properties for the Toxicity Classification of Chemicals:
## 4 Chemical's Toxicity Assessment Studies Aligned with Regulatory Requirements
### Ecotoxicological Information
In the case of protozoa, _Tetralumena pyriformis_[99], _Stentor coeruleus_, _Stylonychia lemnea_[100], or _Paramecium caudatum_[101], among others, are used. These tests are quite popular among the scientific community because these biomodels are more sensitive than vertebrate biomodels. In general, amphibians are not specifically considered in risk assessment because it is assumed that they are covered by the toxicity studies for aquatic invertebrates, fish, mammals, or birds. However, many endpoints have been analyzed using amphibians such as mortality, growth rates, behavior, or time spend feeding. These tests are usually conducted towards embryos and larvae [102]. Experimental tests with amphibians are related to absorption, toxicity, hormonal studies, and analysis of insecticides and their metabolites [103]. Some species that have been used include: _Xenopus laevis_[104; 105; 106], _Bufo americus_[107; 108; 109], _Rana pipiens_[109; 110; 111], _Plethodon glutinosus_[112], _Bufo woodhousii_[113], _Pristimantis taeniatus_[114], _Rana temporaria_[115; 116; 117], or _Ambystoma gracile_[118; 119] and _Ambystoma tigrinum_[120; 121] among others. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{4}{c}{**Short-Term or Acute Biomodels (53\%)**} & \multicolumn{4}{c}{**Long-Term or Chronic Biomodels (47\%)**} \\ \hline
**Aquatic** & **Terrestrial Quality** & **Air Quality** & **Aquatic** & **Terrestrial Quality** & **Air Quality** \\
**Quality (80\%)** & **(14\%)** & **(6\%)** & **Quality (87\%)** & **(10\%)** & **(3\%)** \\ \hline \hline \multirow{4}{*}{Amphibians (3\%)} & \multirow{4}{*}{Sediments (10\%)} & \multirow{4}{*}{Eames (2\%)} & \multirow{4}{*}{Microorganisms (2\%)} & \multirow{4}{*}{Birds or eggs (81\%)} \\  & & & & & \\  & & & & & \\  & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Ecotoxicological studies found in the literature (WOS on 9 September 2022). On the other hand, mollusks are also good biomodels to analyze aquatic contamination. Some studies related to oxidative stress, immunotoxicity, neurotoxicity, or genotoxicity have been carried out [122]. Some of the used species are: _Corbicula flumina_[123; 124], _Ruditapes decussatus_[125], _Ruditapes philippinarum_[126; 127], _Sapharca subcrenata_[128], or _Crassostrea virginica_[129; 130]. Rotifers are abundant in aquatic medium, and they are very useful to evaluate the full life cycle and population level effects when they are in contact with chemicals [131; 132]. In general, acute, and chronic toxicity is analyzed and additionally, effects on the feeding behavior and reproduction parameters can be also studied. Some species used for toxicity tests are: _Proales similis_[133], _Brachionus ibericus_[133], _Brachionus calyciforus_[134], _Brachionus plattilis_[135], or _Brachionus koreanus_[136]. Regarding terrestrial biomodels, plants, microorganisms, earthworms, sediments, and mites are normally used. The terrestrial plant tests have been carried out in different species such as _Zea mays_, _Allium tuberosum_, _Solanum lycopersicum_, _Lactuca sativa_, _Glycine max_[137], _Mucuna aterrima_, _Blassica alaracea_[138], _Tripleuospermum inodorum_, _Stellaria media_[139], _Brassica rapa_[140], or _Allium ccpa_[141]. These species have been used for toxicity studies and determining vegetative and reproductive endpoints [142]. In the case of earthworms, toxic profiles, reproductive toxicity analysis, or even histopathological effects can be evaluated; the main species used are: _Eisenia andrei_[143; 144], _Eisenia ftida_[145; 146; 147], _Dendrobaena veneta_[148], _Eudrilus eugeniae_[149], _Pontosoclex corethrurus_[150], _Aporrectodea caliginosa_[151], and _Eudrilus eugeniae_[151] among others. The studies of sediment toxicology began to increase in popularity from the 1970s onwards, driven by growing environmental concerns and the ability of sediments to influence ecosystems [152]. Most sediment tests consist of acute toxicity experiments [153].