The Influence of Thermal Stress on Cadmium Uptake in Arctic Charr (Salvelinus alpinus) and Its Effects on Indicators of Fish Health and Condition, with Implications for Climate Change
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe presented research is scientifically valuable since it evidently shows the negative influence of elevated temperature on Cd uptake and antioxidant system in Arctic charr. The topic is actual from point of view of climate changing, especially in cold-climate regions where Arctic charr is a common species. In whole, the experiments are clear and logical, methods and protocols are correct and thoroughly described. The results well presented, and conclusions are corresponded to findings. In contrast, there is some inappropriate statistics application, i.e., linear regression models, and interpretation of the results obtained with this statistical method. Detailed recommendation is given in Comments List.
Comments List
1st highlight is not good and should be changed/removed. A highlight must be informative with relation to the study’ field and finding.
Line 81-83 Specific information on experimental protocol and conditions (temperature values, cadmium concentrations) must be moved to methodical section.
Line 95-114 This paragraph needs to be extensively reformulated. It should tell why and what indicators of the stressor should be considered; what combined effects of stressors on fish condition may take place. At the end, aims and goals of the study must be exactly formulated. All circumstances of the planned experiments and statistical analyses of the data must be removed to Methods.
Line 226 Lipids were not assayed, hence, reword the title of 2.3.
Line 268-270 The phrase on PCA results presentation is trivial and is recommended to delete.
Table 1. Correct or explain data presentation. For instance, fork length for 6C-Cd equals to 165±12 mm. What is 126, 197(101)? What for is repetition of sample size for each parameter? Did you measure length for some individuals, but miss to measure their weight or HSI?
Fig.1. Be consistent in use of µg or ug in this Figure and throughout.
Table 2, line 355-372 Although some significant relationships between tissue cadmium concentrations and biometric variables were found, it must be noted that all values of coefficient determination are low, and below than 0,5. This means that Cd accumulation explains only little part of variability of all the studied biometric values. Hence, it is recommended to stress this negligible effect or remove this part of statistical estimations. Moreover, linear model statistics is not a correct choice of analysis for separate four experimental treatments (two temperature and two Cd levels). If the several temperature/cadmium levels were tested, and all of them were included in a total linear regression model, one may evaluate an effect of cadmium accumulation on biometric parameters.
Line 382, paragraph 3.3. When describe effect of cadmium and temperature on SOD, CAT and LDH activities, not only significance of the differences should be considered, but also magnitude of the effect. For instance, CAT activities among four experimental sets ranged only in some percents, while LDH activities varied almost two-fold.
Line 415-421 The same comment as it for line 355-372 concerns an attempt to find a relation between Cd tissue concentrations and the enzyme activities in four experimental treatments.
Figure 3, the legend. Add “in three tissues” after Cd concentrations.
Line 471-472 The thermal stress may be different for various fish species according to their native thermal adaptations. This fact is not concerned in the current paragraph.
Line 549-550 I recommend to give a figure with relation of concentration of Cd with fish body size with statistical data. From Table 4 it is obvious that no statistically significant correlation between these data, and only slight one between kidney Cd and folk length.
Line 585-586 Please, explain the term “Critical Thermal Maximums (CTMax)”. Is this term applied to fish species or to an enzyme? It is difficult to imagine that temperature of 16C or even up to 25 C will promote protein denaturation and degradation.
Line 600 What is “anaerobic capacities in Arctic charr”?
Line 616-617 The thesis “temperature appears sufficient to promote Cd uptake in dorsal muscle tissues” is not supported by the obtained data (See Fig.1).
Author Response
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Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors, I read with interest your paper, The influence of thermal stress on cadmium uptake in Arctic charr (Salvelinus alpinus) and its effects on indicators of fish health and condition, with implications for climate change, but the current form did not convince me to recommend the article for publication.
- additional experiment needed (e.g. behavioral assays, GPx, MDA, protein carbonyl content, histopathology and immunochemistry, etc. )
- the Cd concentration and the two temperature selected must better justified.
- more experimental details would be useful for readers.
- the mechanism underlying the observed effects must be developed.
- the novelty of the study must be highlighted.
Author Response
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Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript “The Influence of Thermal Stress on Cadmium Uptake in Arctic charr (Salvelinus alpinus) and Its Effects on Indicators of Fish Health and Condition, with Implications for Climate Change” is devoted to the combined effect of thermal stress and Cd contamination on the physiology of Arctic charr. The manuscript is well-written, the methods are appropriate and thoroughly described, the results are both detailed and well-illustrated. However, I have several comments regarding the experimental design:
- According to line 146, the optimal temperature for this species is 11°C ± 1.0°C. Yet, the experimental design only included a lower (6°C) and a higher (16°C). Why was the optimal temperature not included?
- Cadmium typically occurs in the marine environment at ultra-trace levels that rarely exceed 20 ng/kg. In this work, only one concentration of Cd (3000 ng/kg), which is considerably higher than even polluted water levels. How can the findings be applied to environmental studies, given that the concentration surpasses typical thresholds?
Minor comment:
The graphical abstract is somewhat misleading, as it suggests that each experimental tank contained only a single animal.
Author Response
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Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe current form did not convince me to recommend the article for publication in Environments.
Additional experiments are necessary to validate the findings and enhance the paper's overall quality.
(i) The Cd concentration should be determined also in the stomach/intestine, gills and brain to assess the extent of Cd accumulation by fish tissue. Besides detoxifying organs, the CNS is also particularly vulnerable to heavy metals, inducing neurotoxicity and exploratory impairments.
(ii) The concentrations of essential elements in the tissues should be measured to have a complete picture of the fish's health status under the Cd and Thermal stress.
(iii) SOD and CAT are two important enzymes for scavenging ROS and useful indicators for oxidative stress, but to investigate the extent of oxidative damage in tissues, additional experiments are required, including the assessment of MDA level, GPX activity and protein carbonyl content.
(iv) To provide cellular and molecular insights into tissue changes induced by Cd and thermal stress, histological analysis and immunohistochemistry approaches should be also performed. (v) Fish were acclimatized for 14 days at11°C ± 1.0°C, then a high temperature treatment of 16°C ± 1.0°C and a cold temperature treatment of 6°C ± 1.0°C were used for experiments. An additional lot should have been included, a control group for thermal stress maintained at 11°C ± 1.0°C.
Figure 1 Cd dorsal muscle (ug/kg) concentration in the 16C treatment is significantly greater than for 16C treatment in the presence of Cd, an additional control lot (11°C ± 1.0°C) would help to clarify the observed results.
Author Response
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Reviewer 3 Report
Comments and Suggestions for AuthorsAll previously raised issues have been satisfactorily addressed. My only remaining suggestion is to reformat the newly added tables and figures so that their font size and overall layout match those of Table 1.
Author Response
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Round 3
Reviewer 2 Report
Comments and Suggestions for AuthorsNo additional comments to address.
