Embryotoxicity of Selective Serotonin Reuptake Inhibitors—Comparative Sensitivity of Zebrafish (Danio rerio) and African Clawed Frog (Xenopus laevis) Embryos
Round 1
Reviewer 1 Report
The manuscript entitled “Embryotoxicity of selective serotonin reuptake inhibitors – comparative sensitivity of zebrafish (Danio rerio) and African clawed frog (Xenopus laevis) embryos” is an important study was to assess the embryotoxicity of fluoxetine hydrochloride and citalopram hydrochloride on early life stages of zebrafish (Danio rerio) and African clawed frog (Xenopus laevis). The embryos were exposed to various concentrations of the individual antidepressants and their mixtures for 96 hours. The tested levels included both environmentally relevant and higher concentrations for the evaluation of dose-dependent effects. This study demonstrated that even environmentaly relevant concentrations of these psychiatric drugs influenced zebrafish embryos, which has been proven by a significant increase (p < 0.01) in the heart rate after the fluoxetine hydrochloride exposure and in the hatching rate after the exposure to a combination of both antidepressants, and thus revealed a potential risk to aquatic life of these organisms.
The introduction is relevant but must include new references. The discussion, in the light of results and knowledge, is relevant.
Your manuscript will not be accepted unless both the technical and grammatical revisions have been made successfully.
Based on these comments, I recommend a moderate revision of analytical aspects of this manuscript before final decision about its acceptance.
Moderate comment:
Introduction:
L33-105: Rewrite this part with new references it is a very old research (Part P. et al.; 2010 and Schiffelers, M.J.W.A. et al.; 2014)
Recent study (Fazio F. et al., 2019) have shown a new analysis technique important in recognizing cells morphology through to Flow cytometry
Francesco Fazio, Concetta Saoca, Gregorio Costa, Alessandro Zumbo, Giuseppe Piccione, Vincenzo Parrino - Flow cytometry and automatic blood cell analysis in striped bass Morone saxatilis (Walbaum, 1792): A new hematological approach, Aquaculture 513 - 734398 (2019).
L77-82: Please insert this sentence: Some authors (Parrino et al., 2018 – 2021) have highlighted the effect of anthropogenic.
Reference:
Parrino V. et al., (2018 and 2021) - Comparative study of haematology of two teleost fish (Mugil cephalus and Carassius auratus) from different environments and feeding habits. The European Zoological Journal, 85:1,194-200.
Parrino V., De Marco G., Minutoli R., Lo Paro G., Giannetto A., Cappello T., De Plano L. M., Cecchini S., & Fazio F. - Effects of pesticides on Chelon labrosus (Risso, 1827) evaluated by enzymatic activities along the north eastern Sicilian coastlines (Italy). The European Zoological Journal, vol. 88:1, p. 540–548; - ISSN:https://doi.org/10.1080/24750263.2021.1905090; (2021).
Recent study (Acar Ü., et al., 2018) and (Osman S.K., et al., 2020) and (Fazio F., et al., 2021) showed the role of supplementation on growth performance, some hematological:
Acar Ü. et al., (2018) - Effects of Different Levels of Pomegranate Seed Oil on Some Blood Parameters and Disease Resistance Against Yersinia ruckeri in Rainbow Trout. Frontiers in Physiology, vol. 9, p. 1-7.
Osman S.K., Parrino V., Ümit A., Sevdan Y., Lo Paro G., Fazio F. – Effect of Monterey cypress (Cupressus macrocarpa Hartw) leaf essential oil as a dietary supplement on growth performance and haematological and biochemical parameters of common carp (Cyprinus carpio L.). doi:10.2478/aoas-2020-0041. p. 1-33; Annals of Animal Science – ISSN: 1642-3402; (2020).
Francesco Fazio, Gregorio Costa, Giuseppe Piccione, Claudia Giannetto, Vincenzo Parrino, Francesca Arfuso - Innovative approach for haematological analysis in Gobius niger (Linnaeus, 1758) and Mugil cephalus (Linnaeus, 1758): Useful model in fish preventive medicine. Aquaculture Research.; 00:1–6 DOI: 10.1111/are.15629; (2021).
- Materials and Methods
2.1. Chemicals and Materials
Can authors describe monitoring of heart rate in detail? Why did you choose different time for zebrafish and frog?
L117-118: Authors stated: The lowest concentrations of the individual psychiatric drugs were chosen with respect to environmentally relevant levels in surface waters“
Why did authors choose different number of eggs for toxicity test on zebrafish and frog?
All sampled of zebrafish (Danio rerio) and African clawed frog (Xenopus laevis) were clinically healthy?
Please described the absence of lesion.
Statistical:
The statistical analysis used is appropriate.
Results:
The authors should be reduce this part, please the results shown in figures.
Discussion:
This part is very long, reduce it and discuss only the obtained results.
Reference missing (please insert some references).
Add clearly how this research should be useful in the future in indicating that hatching is a critical period of the embryo development and therefore its monitoring is an important indicator of embryotoxicity after exposure to pharmaceuticals, pesticides or emerging pollutants.
Author Response
reviewer 1:
Point 1: The introduction is relevant but must include new references. The discussion, in the light of results and knowledge, is relevant. L33-105: Rewrite this part with new references it is a very old research (Part et al. 2010 and Schiffelers et al. 2014).
Response 1: We would like to thank the reviewer for thoroughly reviewing our manuscript. I agree that some of the references are already dated. However, in case of the two mentioned papers (Part et al. 2010; Schiffelers et al. 2014), we prefer to keep them in our manuscript because they are closely related to the topic. According to reviewer suggestions, we have also included some more recent references in our manuscript. However, we did not include all of the suggested references because after studying them, we concluded that not all of them are completely related to the topic of our manuscript. For example, some of the proposed references are narrowly focused on hematological analysis, which was not performed in our study.
Point 2: L117-118: Authors stated: The lowest concentrations of the individual psychiatric drugs were chosen with respect to environmentally relevant levels in surface waters“
Response 2: We added new references which we used for choosing of these environmentally relevant concentrations.
Point 3: Can authors describe monitoring of heart rate in detail? Why did you choose different time for zebrafish and frog?
Response 3: Monitoring of heart rate was performed for zebrafish and African clawed frog at 48 and 56 hpf, respectively. Different time was chosen due to transparence of embryos. Embryos of African clawed frog was not so good transparent at 48 hpf, for this reason we choose different time. But all frog embryos were monitored at the same time. As we mentioned in our manuscript, observation was performed manually by visual inspection a and it was repeated three times for each embryo. The number of beats was counted for 30 seconds, and the result was expressed as the number of beats per minute. Embryo monitoring was performed by experienced examiners who were blind to the treatments. Detail information about monitoring were added in the manuscript.
Point 4: Why did authors choose different number of eggs for toxicity test on zebrafish and frog?
Response 4: Different number of eggs for toxicity tests on zebrafish and frogs were chosen due to technical reason. In the case of zebrafish, we had a higher number of embryos from producer, so a higher number was used in this toxicity test. However, number of embryos used for both tests is sufficient to perform statistical analysis and obtain relevant results.
Point 5: All sampled of zebrafish (Danio rerio) and African clawed frog (Xenopus laevis) were clinically healthy? Please described the absence of lesion.
Response 5: For our experiment, fertilized eggs from Mendel University in Brno (Czech Republic) and Masaryk university in Brno (Czech Repbulic) were used. Both producers have certified commercial breeding. Before we used eggs in our toxicity test, we checked quality and stage of embryos using stereomicrocope. Only fertilized eggs maximally at the 16-cell stage without any obvious abnormalities were selected for the embryonic toxicity test. Absence of lesion was monitored using stereomicroscope as it is stated in our manuscript. All of these information are mentioned in our manuscript.
Point 6: Results - The authors should be reduce this part, please the results shown in figures.
Response 6: We tried to reduced some parts of this section. Reviewer suggested to show results only in figures. However, we believe that the results presented in the table are more informative and clearer. We therefore prefer to keep them in their current form.
Point 7: Discussion – This part is very long, reduce it and discuss only the obtained results. Reference missing (please insert some references). Add clearly how this research should be useful in the future in indicating that hatching is a critical period of the embryo development and therefore its monitoring is an important indi cator of embryotoxicity after exposure to pharmaceuticals, pesticides or emerging pollutants.
Response 7: We tried to improve discussion, add new references and discuss our results more in detail.
Reviewer 2 Report
The paper presents an embryotoxicity study on selective serotonin reuptake inhibitors (fluoxetine hydrochloride and citalopram hydrochloride in particular). Early life stages of zebrafish (Danio rerio) and African clawed frog (Xenopus laevis) embryos exposed to these antidepressants were investigated. I find the topic timely as antidepressant consumption increases globally along with their presence in the environment. The manuscript is well-written, the methods are adequately described, the results and conclusions are properly supported by the presented data. Although I can support the publication of the paper, I have some mostly minor points to address first:
- The authors use the term “in environmentally relevant concentration range” many times, but they do not clearly define it. There is one sentence in the introduction, that states, that in surface waters antidepressant concentrations can reach 0.1-100 ng/l concentrations. Based on this, only 4/17 of total tested concentrations can be considered as “environmentally relevant” (0.1ug/l for F, 0.01 and 0.1 ug/l for C, and ‘low’ for F+C). Please clearly define the environmentally relevant concentration range for the two tested antidepressants, and also be specific about these concentrations in the discussion, conclusion sections.
- There are some inconsistencies in the presented data, which are not referred to or properly discussed in the text. For example, in Table 4, malformations in the African clawed frog embryos. The exposure to individual antidepressants resulted in a statistically significant difference compared to the control group only in two cases: F - 1000 ug/l and C 100 ug/l. Surprisingly, the combined exposure to F+C only resulted in a statistically significant difference at the lowest concentration. The highest concentration of combined exposure, which contains exact same amount as the two mentioned cases of single exposure yielded the lowest percentage of malformations. A similar trend is visible for the zebrafish embryos in Table 3. The highest combined concentration yielded the lowest percentage, while the individual F exposure at the same 1000 ug/l concentration resulted in a significant difference. Could the authors reflect on this?
- Please add a scalebar to the microscopy images of Fig. 4.
- There are some typos in the text, I also suggest a detailed language check. E.g. “hunderds”, page 2, line 69.
Author Response
Point 1: The authors use the term “in environmentally relevant concentration range” many times, but they do not clearly define it. There is one sentence in the introduction, that states, that in surface waters antidepressant concentrations can reach 0.1-100 ng/l concentrations. Based on this, only 4/17 of total tested concentrations can be considered as “environmentally relevant” (0.1ug/l for F, 0.01 and 0.1 ug/l for C, and ‘low’ for F+C). Please clearly define the environmentally relevant concentration range for the two tested antidepressants, and also be specific about these concentrations in the discussion, conclusion sections.
Response 1: The environmentally relevant concentrations chosen were a compromise of data reported in the literature. In general, for fluoxetine, the detected concentrations were slightly higher than for citalopram, so the environmentally relevant concentrations were chosen differently. We also added new references which we used for choosing of these environmentally relevant concentrations.
Point 2: There are some inconsistencies in the presented data, which are not referred to or properly discussed in the text. For example, in Table 4, malformations in the African clawed frog embryos. The exposure to individual antidepressants resulted in a statistically significant difference compared to the control group only in two cases: F - 1000 ug/l and C 100 ug/l. Surprisingly, the combined exposure to F+C only resulted in a statistically significant difference at the lowest concentration. The highest concentration of combined exposure, which contains exact same amount as the two mentioned cases of single exposure yielded the lowest percentage of malformations. A similar trend is visible for the zebrafish embryos in Table 3. The highest combined concentration yielded the lowest percentage, while the individual F exposure at the same 1000 ug/l concentration resulted in a significant difference. Could the authors reflect on this?
Response 2: Thank you for your very valuable comments on our results. As the reviewer correctly states, at first view some of our results appear inconsistent. The surprising finding in the group exposed to the lowest concentration of antidepressant mixture may indicate a possible drug interaction. Unfortunately, this phenomenon is not described in the literature and for us it is another topic for future research. This issue is also discussed in the discussion section.
Point 3: Please add a scalebar to the microscopy images of Fig. 4.
Response 3: Scalebars were added in all figures.
Point 4: There are some typos in the text, I also suggest a detailed language check. E.g. “hunderds”, page 2, line 69.
Response 4: Thank you for your very valuable comment. We have re-corrected the English language and hope that everything will be fine now.
Reviewer 3 Report
Here Blahova and colleagues compared the embryotoxicity of two popular representative selective serotonin reuptake inhibitors, fluoxetine hydrochloride and citalopram hydrochloride, in early life stages of Zebrafish and African clawed frog. And they found that African clawed frog is more sensitive than Zebrafish when exposure to the highest concentrations of these 2 drugs resulted in total mortality. This work is informative that worth publishing within Applied Sciences readership. However, there are some minor concerns to be addressed before acceptance for publication.
Line 14: “consumption” is not suitable here, recommending switch with “prescription”.
Line 148: The heart rate observation was performed manually by visual inspection and it was repeated three times for each embryo. Please clarify that the experienced examiners are blind to the treatments.
Line 132 and 156: All the commercial materials should be cited with reference if there are published papers using the same eggs. These details will benefit the interested readers.
In the final discussion sections, the authors should necessarily discuss other aquatic model organisms and parameters that widely applied in the neurotoxicity field, especially the embryotoxicity. And the significance of the current study should be emphasized with future application perspectives.
Author Response
Point 1: Line 14: “consumption” is not suitable here, recommending switch with “prescription”.
Response 1: As recommended by the reviewer, we have made the appropriate change.
Point 2: Line 148: The heart rate observation was performed manually by visual inspection and it was repeated three times for each embryo. Please clarify that the experienced examiners are blind to the treatments.
Response 2: We verify that examiners who performed monitoring of heart rate were blind to the treatment. This statment was added to the manuscript.
Point 3: Line 132 and 156: All the commercial materials should be cited with reference if there are published papers using the same eggs. These details will benefit the interested readers.
Response 3: We added references for zebrafish eggs because we used these eggs from same commercial producer many times in our previous studies. Unfortunately, in case of African clawed frog, we do not have any previous study with this producer, because we started this new colaboration now.
Point 4: In the final discussion sections, the authors should necessarily discuss other aquatic model organisms and parameters that widely applied in the neurotoxicity field, especially the embryotoxicity. And the significance of the current study should be emphasized with future application perspectives.
Response 4: This chapter has been expanded to include discussion with other organisms.
Reviewer 4 Report
The manuscript from Blahova et al. describe embryotoxic effects of psychoactive compounds fluoxetine and citalopram in zebrafish and african-clawed frog embryos and larvae. I found the study interesting, of good quality and especially using also environmentally relevant concentrations relevant. I have only few comments on the manuscript.
Specific comments:
1) In the case of analysis of zebrafish heart rate, even the lowest concentration of fluoxetine and fluoxetine+citalopram are having an effect. I recommend authors to test even lower concentrations to find level where effects are no more seen. This would be useful in future risk assessment and estimation of "safety levels" for release of these compounds to the environment. If experimentation is not possible, it would be important to discuss this.
2) I recommend the authors to use statistical analysis to determine if combinational effects of fluoxetine and citalopram are additive, synergistic or antagonistic. This analysis could be done on selected phenotypes. Superficially, it seems to me that effects are synergistic but it would be important to test this also using formal statistical analysis. There are different options for this, but one easy solution (2-way ANOVA) is discussed in "Slinker BK. The statistics of synergism. J Mol Cell Cardiol. 1998 Apr;30(4):723-31. doi: 10.1006/jmcc.1998.0655. PMID: 9602421."
Author Response
Point 1: In the case of analysis of zebrafish heart rate, even the lowest concentration of fluoxetine and fluoxetine+citalopram are having an effect. I recommend authors to test even lower concentrations to find level where effects are no more seen. This would be useful in future risk assessment and estimation of "safety levels" for release of these compounds to the environment. If experimentation is not possible, it would be important to discuss this.
Response 1: Thank you for your very valuable comments. We ourselves are aware that these findings that even the lowest concentration significantly affects the observed indices is very important for us. Since the topic of antidepressant toxicity has been a long-standing concern of our department and it will continue in the future, these findings were inspiring for us to design further studies. At this moment, it is not possible to conduct another experiment and add to the existing results.
Point 2: I recommend the authors to use statistical analysis to determine if combinational effects of fluoxetine and citalopram are additive, synergistic or antagonistic. This analysis could be done on selected phenotypes. Superficially, it seems to me that effects are synergistic but it would be important to test this also using formal statistical analysis. There are different options for this, but one easy solution (2-way ANOVA) is discussed in "Slinker BK. The statistics of synergism. J Mol Cell Cardiol. 1998 Apr;30(4):723-31. doi: 10.1006/jmcc.1998.0655. PMID: 9602421."
Response 2: I quite clearly agree with comment of reviewer that the study of synergistic and antagonistic effects would be very important and will improve our study. It might also explain some of the inconsistencies ou the presented results. The proposed use of two-factor analysis of variance is the right solution, but the problem is that analysis of variance can be used for quantitative continuous data. In our study, we primarily analyzed qualitative data (e.g., mortality, hatching) where application of this method is not possible.
