## 4 Discussion
In addition, endpoints such as monitoring of the heart rate and behavioral changes, or analyses of selected biomarkers (e.g., indicators of oxidative stress, gene expression), are recorded for comprehensive evaluation [24; 25; 35; 58]. [PERSON] et al. [72] carried out a comparative evaluation of fish acute toxicity tests and embryonic toxicity tests for 143 different substances. They concluded that these embryonic toxicity tests are a potential alternative to classic acute toxicity tests and provide strong scientific results. Further, [PERSON] et al. [73] studied the toxicity of five different antidepressant drugs on embryo-larval development and metamorphosis success in the Pacific oyster. They reported that embryotoxicity tests were more sensitive than the metamorphosis bioassay for most of the molecules tested. Moreover, embryonic toxicity tests also offer great technical benefits, such as their short duration and lower financial costs, and their requirement of a smaller volume of tested solutions. Finally, yet importantly, embryos are not considered experimental animals according to European legislation. Although a cocktail of pollutants is present in the aquatic ecosystem, only limited ecotoxicological studies deal with combined toxicity. From this point of view, the aim of the present study was to evaluate the embryotoxicity of two frequently used antidepressants (fluoxetine hydrochloride and citalopram hydrochloride) as both single substances and as a mixture. Additionally, we compared the sensitivity of two aquatic species embryos--zebrafish, as a model of fish organisms, and the African clawed frog, as a model of amphibian organisms. Mortality is one of the traditional endpoints of toxicity studies and its monitoring is usually carried out in all types of toxicity tests, whether acute or chronic toxicity, and using different stages of tested individuals (e.g., embryo, larvae, juvenile, adult). In general, the mortality risk is very low or close to zero for environmentally relevant doses of tested chemicals. On the other hand, the toxicity of these low doses can be manifested by other sublethal endpoints, especially after chronic exposure. In our research, the zebrafish embryos were much more resistant to the tested antidepressants than the embryos of the African clawed frog and survived the exposure to concentrations many times higher than the environmentally relevant levels. No significant differences were observed between the cumulative mortality of the zebrafish embryos in all the experimental groups and to the relevant control group at the same time of exposure. By contrast, a 100% mortality of African clawed frog embryos was recorded in the highest tested concentrations of both antidepressants as single substances at 72 hpf. Similarly, [PERSON] et al. [23] studied the effects of tricyclic antidepressants (amnitriptyline, nortripyline, clomipramine) on the embryo-larval developmental stages of the common carp (_Cyprinus carpio_). The combination of all three antidepressants at the highest tested concentration (500 ug/L) caused a 100% cumulative mortality at 6 days post-fertilization. A cumulative mortality of almost 100% was also found at the same concentration in the experimental groups exposed to amnitriptyline and clomipramine as single substances, but at 8 and 9 days post-fertilization, respectively. The authors reported that the cardiotoxicity of these types of antidepressants seemed to be the main reason for the higher mortality. Furthermore, a statistically significant increase in the cumulative mortality of zebrafish embryos was also observed in a similar study, in which the embryotoxicity of amnitriptyline was evaluated. A total of 47% of dead embryos was found in an experimental group exposed to 3000 ug/L. In contrast to our study, the authors observed no significant differences in cumulative mortality in the toxicity test on amphibian Western clawed frog (_Xenopus tropicalis_) embryos after amnitriptyline exposure [24]. Furthermore, [PERSON] et al.