## 4 Discussion
### A. tonsa Findings and Other Existing Research  
Our study's consistent maintenance of environmental parameters aligns with the rigorous standards required in the _A. tonsa_ bioassay, thereby validating our findings. The laboratory was actively engaged in simultaneously testing, developing specific research methodologies for culturing and studying _Skeletonema costatum_ (Mediophyceae) and golden grey mullet _Chelon auratus_ ([PERSON], 1810) as reference species for ecotoxicity tests in the Black Sea [21]. By including multiple trophic levels, from primary producers like phytoplankton (such as _Skeletonema costatum_) to higher-level consumers like the golden grey mullet _Chelon auratus_, researchers can better evaluate the ecological risks and predict the overall impact of contaminants on biodiversity and ecosystem health in environments like the Black Sea. Copepod growth and reproductive success, including egg production rates and egg viability or hatching success, are often seen as a response to food availability (typically measured in terms of carbon, C), with higher rates corresponding to an increased food supply [70; 71]. The findings of this study align with and extend previous research on the dietary preferences and nutritional needs of _A. tonsa_. Prior studies have highlighted the importance of diet quality on the physiological performance of copepods, indicating that nutritional content directly influences survival, growth, and reproductive success [70; 71; 72; 73; 74]. Our results demonstrated that _A. tonsa_ fed with _R. baltica_ exhibited a significantly higher survival rate compared to those fed with _I. galbana_ and a mixed algal diet. These findings are consistent with another study, which reported that diets rich in essential fatty acids, such as those found in _R. baltica_, enhance copep survival [75]. The high survival rate observed in this study underscores the superior nutritional quality of _R. baltica_. While previous studies have established the general importance of algal quality in copep diets, our study provides specific evidence for the benefits of _R. baltica_. For instance, the research by [PERSON] et al. [76] showed that copepods fed with algae rich in essential fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exhibited improved physiological performance [77]. _R. baltica_ is known for its high EPA content, which likely contributed to the positive outcomes observed in our study. Additionally, several studies [78; 79; 80] demonstrated the crucial role of carotenoids in enhancing copep reproductive success, _R. baltica_ containing high levels of carotenoids [81]. Research involving the cultivation of copepods, such as _A. tonsa_, faces several challenges that must be addressed. These challenges include the significant resource and labor demands for maintaining cultures, the increased risk of contamination, and the potential decline in genetic diversity over time [82; 83; 84; 85]. Furthermore, copepods in artificial culture environments may undergo stress, leading to altered behavior and physiology, which can affect the validity of experimental results [66; 82; 84; 86; 87]. These considerations are essential when designing studies that utilize cultured copep populations. Additionally, purchasing eggs can be expensive, subject to supply chain issues, and may lack genetic diversity and environmental context. The health status of commercially sourced eggs can also vary. Balancing these factors against the study's specific objectives is crucial for making informed decisions. Future research on the use of copepods, such as _A. tonsa_, in toxicity testing is important for advancing our understanding of environmental pollutants and their impacts on marine ecosystems, particularly in the Black Sea. One promising direction involves developing standardized protocols for using copepods in toxicity assays to ensure reproducibility and comparability of results across various studies and laboratories [88].