Ecotoxicity of 2,4-Dichlorophenol to Microsorium pteropus by High Spatial Resolution Mapping of Stoma Oxygen Emission

Round 1

Reviewer 1 Report

Revision of manuscript #water-2922091

Journal: Water (MDPI)

Manuscript title: “Ecotoxicity of 2,4-dichlorophenol to Microsorium pteropus by High Spatial Resolution Mapping of Stoma Oxygen Emission”

By Zhong & Zhang

Corresponding author: Daoyong Zhang, [email protected]; Tel./fax: +86 991 7885446

General comments.

Overall, this manuscript appears well written and the experimental phase well conducted. However. the main problem is probably linked to the fact that the authors sometimes provide few truly convincing and supporting elements to the experimentation they propose. In addition, currently ecotoxicological investigations are increasingly directed towards the study of synergistic effects in the presence of multiple contaminants or environmental factors such as variations in temperature, pH, etc. In this sense, the authors should make an additional effort to try to provide a supporting theoretical basis for any future studies, alternatively, the present study probably needs a greater number of observations. Having said this, further specific comments follow.

Specific comments.

L44-46: “Photosynthesis organ of plants is highly sensitive to the toxicity of organic pollutants. The toxic effects of pollutants on photosynthesis are usually probed by chlorophyll fluorescence and oxygen evolution rate”. Although the concept is not incorrect, the authors should explain it in more detail, allowing the reader to better understand the main focus of the text, also considering the fact that this sentence represents the incipit of the introduction.

L46-49: “Measurement of photosynthetic oxygen evolution rate of plants under stress, which directly indicate the toxicity of pollutants to the oxygen evolution complex, is performed by a Clark oxygen electrode in a thermostatic closed chamber at a constant temperature.” Several references should be included here.

L49-52: “This conventional electrochemical method, however, cannot probe oxygen emission at a single stoma level and cannot give the spatial distribution of oxygen concentration in and around the stoma, which is sometimes important for understanding the responses of plants to environmental stresses.” Again, a reference should be included supporting the sentence.

L84-87: “2,4-dichlorophenol (2,4-DCP) has been listed by the U.S. Environment Protection Agency as a priority control pollutant because of its potential carcinogenicity and its high toxicity even at a low concentration. 2,4-DCP mainly arising from the extensively use of pesticides, herbicides and fungicides.”   It seems that the main purpose of the following manuscript is essentially methodological; from this point of view, the authors should however better justify the choice of the chemical substance selected as a model for their tests, offering valid arguments that corroborate the environmental relevance of the toxic substance in question.

L88-91: “M. pteropus is a common higher freshwater aquatic plant which has high demand in ornamentation and often use as a water pollution indicator. M. pteropus was chosen as the test material because previous research has shown that its multiple sites in PSII and PSI was highly sensitive to mercury exposure.” Following the previous comment, also in this case the authors should provide convincing arguments to support the choice of the biological model used.

Aims in general: Although the general aim of the work is sufficiently clear, the authors should however provide more elements that highlight its actual relevance, at least on a theoretical level.

L110-112: “The nominal concentration level gradients for 2,4-DCP treatment are set to 0.00, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, 100.0, and 250.0 mg L-1”. The authors should provide more information regarding the selected concentration range, arguing the reasons and describing its environmental relevance and consistency.

L112-113: “The actual concentration of 2, 4-DCP in tap-water was measured by high performance liquid chromatography (HPLC, 890-0203 HITACHI, Hitachi, Japan)”. A brief description of the HPLC method should be included here.

L115. “2, 4-DCP concentration in the tap-water was under the detection limit.” What the detection limit was?

Figure 3: text labels inside the figure was sometimes not fully readable due the little dimension, it should be fixed.

L343-346: “Literatures show that photosynthetic oxygen evolution rate (Reflecting photosynthetic rate) determined by the conventional oxygraph respirometers is a reliable indicator to quantify the inhibition of photosynthesis by pollutants”. Considering the relevance of this point, some examples from the literature should be described and discussed here.

L352-355: “However, the above methods cannot provide spatial distribution of the bioindicators. CLSM can acquire images, but the spatial resolution is at best submicrometer resolution and in most cases cannot provide accurate concentration information of the bioindicators”. The authors here should underline what in their opinion is the relevance linked to obtaining a spatial resolution of the result and in addition they should determine and quantify the uncertainty (error) of this type of result.

L374: “These limited pioneering studies demonstrated…” What do the authors mean by "limited"? This is not clear.

L387-389: “Before its wide application to ecotoxicology, the protocols to ensure reliability of SECM needs to be checked with more higher plants and photosynthesis microbes exposed to more pollutants (heavy metals, saline-alkali, and compound contaminants).” A further aspect that in my opinion remains unexplored is related to the sensitivity of the test. Can the authors establish or hypothesize that the concentration range used overlaps with an environmentally relevant range? Is there information regarding responses with different chemicals or mixtures, especially in relation to the range of environmental use? A comment should be included here.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this study，nanoscale electrochemical mapping of photosynthetic oxygen evolution of aquatic plants was conducted by SECM (scanning electrochemical microscopy). The information of oxygen emission at a single stoma level and the spatial distribution of oxygen concentration in and around the stoma can be provided by SECM. It was validated by the confocal laser scanning microscopy, the traditional Clark oxygen electrode method and chlorophyll fluorescence technique. The results are useful to conduct toxicity test for the plant. There are several issues need to be fixed before accepted.

1.       line68, before the “[8]”, something is missed.

2.       Line312, this information is not necessary to be here, it should be put in the note part of the title of figure.

3.       line343-352，this sentence is too long. Simple sentences are preferred.

The expression of language is fine. Minor editing of English language might be required. some sentences need to be simpilified.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have applied changes to the text based on previous requests, I have no further comments to provide.