A Real-Time, Non-Invasive Technique for Visualizing the Effects of Acid Mine Drainage (AMD) on Soybean

Round 1

Reviewer 1 Report

I feel good about the paper, a result based on the research team's experience in bOCT.

If you revise a few things, it will make the reader feel better. For example, It would be better to reduce the number of siginificant figures shown in Tables 1 and 2. 

There is not any results in the treatment of 80ML/l AMD. So I think it is not reasonable to mark it as zero in the figure 5.

Overall, bOCT covers the two-dimensional temperature difference of seeds, and enzyme activities and MDA concentration to prove this are the results from the analysis of the entire seed. Therefore, it can be thought that the results of bOCT can indirectly indicate the physiological activity of seeds. I think this should be addressed in context.

Author Response

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Reviewer 2 Report

OCT and mainly bOCT are interesting methods to analyze rapid changes in the morphology and physiology of the object of interest. In this sense, this paper provides data that fit to expectations and support the results obtained with traditional methods.

However, there are some aspects to improve.

 In Materials and Methods, a “simulated AMD” formed only by FeSO₄·7H₂O (pH 2.7-3.0) is described. The Fe concentrations used are 24 and 48 mg/l. And therefore, those of sulfates are 41 and 92 mg/l. In a real AMD environment, Fe concentrations are frequently higher than 120 mg/l (even as high as 3500 mg/l) and those of sulfate range between 400-10000 mg/l, which explains the high conductivity of the water affected by AMD. Furthermore, the environment is very oxidizing because the Fe³⁺/Fe²⁺ ratio may be very high. Moreover, there is a complex community of bacteria that affect the Fe/S cycles. Therefore, it seems excessive to refer to the medium used as "simulated AMD". There are many specific publications describing the composition of AMD in different locations that have not been cited.

The control only includes distilled water (pH is not reported). The results cannot be clearly explained because it is not possible to discern between the effects of pH, iron and sulfate. For example, to conclude that “the low pH of AMD is responsible for the inhibition of shoot and root length in soybean seedlings” (at the end of Discussion), an experiment with water at pH ≈ 3.0 in the absence of iron would be necessary. Other parameters as conductivity or redox potential have not been considered.

Furthermore, how is it possible to measure the iron uptake in the control when it only contains distilled water (Table 2)? Clarification is needed.

In summary, the article can be published after correcting certain excesses and clarifying some methodological aspects.

Author Response

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