## 5 Conclusions  
This study successfully established a validated protocol for testing chemical effluents using _Acartia tonsa_ as a model organism, specifically employing the reference toxicant 3,5-dichlorophenol. By standardizing procedures for culturing, maintaining, and testing _A. tonsa_, the research provides a scientifically robust method for assessing the ecotoxicity of pollutants. This approach contributes to a deeper understanding of the impacts of pollutants on marine ecosystems, which is crucial for informing conservation and management strategies, particularly in sensitive environments like the Black Sea. The standardized protocol ensures reliable and comparable results, enhancing the sensitivity and accuracy of ecotoxicity assessments. Future research should focus on refining these protocols to account for specific environmental conditions and expanding their application to a wider range of pollutants. Utilizing the data generated from _A. tonsa_ ecotoxicity tests can inform environmental policies, promote sustainable product innovation, and guide conservation efforts. By broadening the use of _A. tonsa_ in toxicity testing, a more comprehensive understanding of the ecological impacts of various pollutants can be achieved. In light of these findings, the following recommendations are proposed to enhance the effectiveness and scope of ecotoxicity testing using _A. tonsa_:  
* continue refining and validating _A. tonsa_ testing protocols to ensure they account for specific environmental conditions, enhancing sensitivity and accuracy in ecotoxicity assessments;expand the use of _A. tonsa_ in toxicity testing to include a wider range of pollutants, providing a more comprehensive understanding of their ecological impacts;
* utilize the data generated from _A. tonsa_ ecotoxicity tests to inform environmental policies, promote sustainable product innovation, and guide conservation efforts;
* conduct comparative studies with other marine organisms to improve the ecological relevance and robustness of toxicity assessments. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app14219861/s1](https://www.mdpi.com/article/10.3390/app14219861/s1)]([https://www.mdpi.com/article/10.3390/app14219861/s1](https://www.mdpi.com/article/10.3390/app14219861/s1)), Figure S1. _Acartia tonsa_ eggs (original photo--OlympusSZX10 stereomicroscope, 4 xzoom), Figure S2. Inoculation of _A. tonsa_ eggs (original photo), Figure S3. Cultures of _A. tonsa_ transferred to dilution water (original photo), Figure S4. _Chryptomans bulleta_ (original photo), Figure S5. _C. bulleta_ microalgae cultures (original photo), Figure S6. Analysis of _C. bulleta_ under the microscope (left) using the hemocytometer (right) (original photos), Figure S7. Copepods transfer into the test chamber (original photo), Figure S8. Monitoring of parameters for copep culture (original photo), Figure S9. Light intensity check before the start of the experiment (original photo), Table S1. Walnes medium composition (per 1 L), Table S2. Dilution series of the test substances, Table S3. Water quality parameters, Table S4. Nutrient concentrations and maximum admissible levels, Table S5. Concentrations of proganochlorine pesticides and maximum admissible levels, Table S7. Concentrations of polycyclic aromatic hydrocarbons (PAHs) and maximum admissible levels, Table S8. Water quality measurements before and after the experiment. Conceptualization, E.B., L.L., G.H. and V.C. ; methodology, E.B., L.L., G.H. and V.C. ; software, E.B., L.L. and V.C. ; validation, E.B., L.L. and V.C. ; formal analysis, E.B., L.L. and V.C., investigation, E.B., L.L., G.H. and V.C. ; resources, E.B., L.L., G.H. and V.C. ; data curation, E.B., L.L., G.H. and V.C. ; writing--E.B. and L.L. ; writing--E.B., L.L. and V.C. ; visualization, E.B., L.L., G.H. and V.C. ; supervision, L.L. and V.C. ; project administration, L.L. and V.C. ; funding acquisition, V.C. All authors have read and agreed to the published version of the manuscript.