The submitted Master’s thesis deals with the ecotoxicological assessment of trifluoroacetate (TFA), a highly persistent and mobile substance of increasing environmental relevance. The aim of the thesis was to provide additional data on the acute ecotoxicity of TFA to freshwater organisms and to investigate its potential effects on sediment microbial processes and carbon cycling. The topic is timely and environmentally relevant, particularly with respect to the increasing occurrence of TFA in aquatic systems and the still limited knowledge regarding its ecotoxicological effects.
The theoretical part provides a general overview of freshwater ecosystems, the occurrence of PFAS and TFA, their fate in the environment, and available ecotoxicological data. The theoretical part is supported by 109 relevant references, demonstrating a relatively good orientation in the topic. However, some parts remain rather descriptive, and the logical connection between individual statements could be clearer.
The experimental part combines standardized acute toxicity tests with two organisms representing different trophic levels, Desmodesmus subspicatus and Daphnia magna, with a 70-day anoxic freshwater sediment incubation experiment. The sediment experiment included repeated measurements of CH₄ and CO₂ production and subsequent statistical evaluation of the obtained data, thereby extending the assessment from organism-level toxicity to a functional ecosystem-level endpoint associated with sediment microbial carbon cycling. The use of linear mixed-effects models, together with the additional evaluation of gas ratios and methanogenesis dynamics, provides a more comprehensive evaluation of the experimental data.
The experimental part has some limitations. Due to problems with the assignment of RNA samples, the effects of TFA on sediment microbial communities could not be directly assessed and this objective was addressed only indirectly through the evaluation of microbial carbon cycling processes. In addition, TFA concentrations were not analytically verified in the acute toxicity tests, which were therefore evaluated using nominal NaTFA concentrations.
The results are generally presented clearly and the main findings are adequately highlighted. No clear concentration-dependent effects were observed in the acute toxicity tests or for methane production and methanogenesis dynamics in the sediment experiment. However, significant treatment effects on CO₂ concentrations were found at two treatment levels.
In the discussion, the student adequately compares his findings with the published literature and generally maintains an appropriately cautious interpretation where statistically significant effects were not observed. Some interpretations, particularly those concerning changes in measured TFA concentrations and possible degradation or sorption processes, remain somewhat speculative, although alternative explanations and the limitations of the experimental design are appropriately acknowledged.
The conclusions are consistent with the obtained results and adequately summarize the main findings of the study. The student clearly indicates which of the formulated hypotheses were or were not supported and appropriately points out that the absence of clear effects under the tested conditions does not exclude possible effects under chronic exposure or other ecological conditions. The need for further research is therefore appropriately emphasized.
A weaker aspect of the thesis is its scientific writing and formal presentation. The submitted version still contains some linguistic, stylistic, and formal shortcomings. For a Master’s thesis, greater attention should have been paid to final language editing and formal consistency.
Overall, the thesis addresses a relevant topic and contains a substantial experimental component and useful original data. At the same time, the missing microbial community data, the lack of analytical verification of exposure concentrations in the acute tests, some limitations in the interpretation of the results, and deficiencies in scientific writing and formal presentation reduce the overall quality of the thesis.
I recommend the submitted Master’s thesis for defence and propose the evaluation good / C (2.0).

The thesis focuses on a highly relevant topic and is generally of good scientific quality; however, several shortcomings have been identified.
The hypothesis predicting increased methane production following TFA exposure is mainly based on studies investigating antibiotics, which have a different mode of action and therefore could have been supported by more appropriate literature. Another methodological limitation is the lack of analytical verification of exposure concentrations in the acute toxicity tests, as the evaluation was based solely on nominal concentrations.
The most significant weakness of the study is the failure to assess changes in microbial communities due to incorrect sample labeling. Consequently, one of the original objectives of the thesis could not be achieved, which considerably limited the interpretation of the sediment experiment. In addition, some parts of the discussion contain speculative explanations of possible TFA effects that are not directly supported by experimental evidence obtained in this study.
Furthermore, the discussion section focuses more on summarizing the literature than on critically evaluating the author's own results. A more detailed assessment of the environmental relevance of the observed effects in relation to the actual environmental TFA concentrations would have strengthened this study. From a formal perspective, the thesis is written in clear scientific English and has a well-organized structure. However, minor language inaccuracies, inconsistencies in formatting, and occasional issues with the presentation of units and concentrations are present throughout.
These comments do not substantially reduce the overall scientific value of this study. Nevertheless, together with the limitations of the experimental work, they justify the proposed overall grade of C (good quality). Topics for thesis defence:

1. Why do you think TFA had no significant effect on methane production in the sediment experiment?
2. What additional ecotoxicological endpoints would you recommend for assessing the long-term effects of TFA?
3. Why did you use both Daphnia magna and Desmodesmus subspicatus in your study?