The Toxic Effects of Lindane via the Impact of pH and Dissolved Organic Matter on Microcystis aeruginosa

Round 1

Reviewer 1 Report

In the article presented for review, it is shown the toxic effects of Lindane (γ-BHC) on Microcystis aeruginosa.  The research was carried out in laboratory conditions. Total protein levels, as well as malondialdehyde (MDA) levels and superoxide dismutase (SOD) enzyme activity, in algal cells, were determined after exposure to different concentrations of Lindane. The bioaccumulation of Lindane, as well as the influence of pH and dissolved organic matter (DOM), on the toxic effects was also evaluated in algal cells. The growth of M. aeruginosa was inhibited by Lindane treatment (96 h), resulting in 50% of maximal effect (EC50) concentration of 442 μg/L. In addition the lowest observed effect concentration (LOEC) was found to be 120 μg/L, the no observed effect concentration (NOEC) 60μg/L, and the maximum acceptable toxicant concentration (MATC) 85 μg/L. It was also very important to determine the metabolites of Lindane degradation. Since Lindane is a very dangerous substance, the article presents very important research, especially on environmental issues.

However, I have a few comments:

1. Is it known how long Lindane stays in the environment. Can it be determined by citing publications on the subject. Please complete your introduction with this information.
2. BG 11 medium - please describe.
3. Were algae cells washed with PBS before and after fixation? Please clarify this.
4. LOEC value (lines 145-147)? The value determined is not similar to the US EPA ECOTOX database reported value (300 μg / L). It needs to be described differently.
5. Please enter the designations in figure 2 (a, b, c).
6. Please complete the description under Figure 3 so that it is the same as the markings in the Figure 3.

The publication is a valuable source of information, especially for environmental research, and forms the basis for further research.

Thank you for considering my opinion. I encourage the authors to continue working on improving the manuscript.

Author Response

Reviewer# 1

However, I have a few comments:

1. Is it known how long Lindane stays in the environment? Can it be determined by citing publications on the subject? Please complete your introduction with this information.

Thank you very much for your comments, we are very sorry that we did not sufficiently explained our introduction. In response to this comment, we have added sufficient information regarding introduction.

Lindane is categorized as persistent organic pollutant and its concentrated form persists in aquatic environment. However, a significant amount of Lindane is also found in soil sediments and in air vapors.

Lindane is highly persistent due to presence of its high lipid content and is highly toxic as it migrate to long distances and spread contamination by effecting environment. While the reported half-life for lindane in water is 2292 days and in soil is 708 [1].

Lindane enters in food chain and accumulate in adipose tissues of living organisms and is observed highly toxic for aquatic organisms. While in case of mammals and birds, it has been found moderately toxic. The presence of high lipid content and its solubility increase its toxic effects for both animals and humans [2].

1. BG 11 medium - please describe.

Thank you for your comments and pointing out this section. In response to this section we have tried to make these paragraphs more clear and simple by explaining BG 11 media.

BG-11 media is used as universal media for optimum growth and cultivation of blue green algae and their derivatives. In those areas where growth of cyanobacterial algae is not visible through naked eye, BG-11 media is convenient to use for isolation of such cyanobacteria from rivers and lakes. For preparation of BG-11 media, 1.627 gram BG-11 is suspended in one liter of distilled water.

1. Were algae cells washed with PBS before and after fixation? Please clarify this.

Thank you for your comments.

Phosphate buffer saline is used for washing before and after fixation of cells. This saline helps to maintain constant pH in all the cells. This saline is used for washing of algal cells because it act as isotonic and non-toxic solution for most cells. For washing of algal cells, pH is maintained around 7 to 7.6 that maintain osmotic balance between cells to keep them healthy. 

1. LOEC value (lines 145-147)? The value determined is not similar to the US EPA ECOTOX database reported value (300 μg / L). It needs to be described differently.

      Thank you for your comments.

      Lowest observed effect concentration (LOEC) was found to be 120 μg/L, the no observed    effect                

    concentration (NOEC) 60 μg/L, and the maximum acceptable toxicant concentration (MATC) 85 μg/L.   

    This software US EPA ECOTOX database is used to compare our results with available reported value 

    (300μg/L) and     LOEC value is observed same as reported in database (US EPA, 2013).

1. Please enter the designations in figure 2 (a, b, c).

    Response Thank you very much for your comments, we are very sorry that we did not sufficiently explained our added figures. In response to this comment, we have added designations regarding figures respectively.

     A, B and C is added in figure 2.

     (A) Protein content (B) SOD activities, (C) MDA content

1. Please complete the description under Figure 3 so that it is the same as the markings in the Figure 3.

Response: Thank you very much for your comments, we are very sorry that we did not sufficiently explained our added figures. In response to this comment, we have added complete description in figure 3 respectively. Figure 3 shows effect of Lindane on the cellular ultrastructure of M. aeruginosa (7000×). A, B, C, D, E, F: ultrastructure of M. aeruginosa in 0, 50, 89, 158, 281 and 500 μg/L Lindane, respectively; 1-cell growth, 2-cell division. (A) shows cyanophycin granules, nucleoid and phosphate granules (B) shows thylakoid and phosphate granules (C) shows thylakoid, phosphate granules and nucleoid (D) shows Cyanophycin, thylakoid and phosphate granules (E) shows cytoplasm, thylakoid and cell wall (F) shows cell wall and cytoplasm

Reviewer 2 Report

In this work the toxic effects of Lindane on Microcystis aeruginosa and the influence of pH and DOM dependent effects of Lindane on Microcystis aeruginosa are described. Authors determined the total protein levels, as well as malondialdehyde levels and superoxide dismutase enzyme activity in algal cells after exposure to different concentrations of Lindane. It was shown that the toxic effects of Lindane decreased as pH increased and as concentrations of HA increased. Finally, the ultrastructure images of M. aeruginosa showed that increasing concentrations of Lindane destroyed cellular structures. This destruction resulted in the loss of photosynthesis and  reproduction eventually leading to cell death. The article looks like a short communication and may be published after major revision.

Notes:

1. Authors should not use abbreviations like “DOM” in the Title of the article.
2. Which buffer solutions were used by authors for carrying out the experiments in various pH (pH 5, 7, 9) conditions (section 2.6. The influence of pH and DOM)? It should be noted.
3. The “A”, “B”, “C” should be presented on the Figure 2. Also the descriptions to axis should be increased.
4. The relevance of the study and the novelty of this work should be written more detailed from the ecological point of view in the Introduction.

Author Response

Reviewer# 2

1. Authors should not use abbreviations like “DOM” in the Title of the article.

Thank you for this comment and correction is followed.

DOM abbreviation is used to describe dissolved organic matter. Abbreviations should not be used in the title of the article. So, suggested titles are these:

1. The influence of pH, dissolved organic matter and toxic effects of Lindane on Microcystis aeruginosa
2. The toxic effects of Lindane via impact of pH and dissolved organic matter on Microcystis aeruginosa

1. Which buffer solutions were used by authors for carrying out the experiments in various pH (pH 5, 7, 9) conditions (section 2.6. The influence of pH and DOM)? It should be noted.

 Thank you for your comment and pointing out this section. In response to this section, we have addressed it     in this way. Phosphate buffer saline (PBS) is used as buffer solution to maintain pH around 7 to 7.4 in this research. While alkaline solutions such as phosphate buffers are used to maintain basic pH around 7 and 9 for algal cells.

3. The “A”, “B”, “C” should be presented on the Figure 2. Also the descriptions to axis should be increased.

Thank you very much for your comments, we are very sorry that we did not sufficiently explained our added figures. In response to this comment, we have added designations regarding figures respectively.

         A, B and C is added in figure 2.

(A) Protein content (B) SOD activities (C) MDA content

1. The relevance of the study and the novelty of this work should be written more detailed from the ecological point of view in the Introduction.

Thank you very much for your comments, we are very sorry that we did not sufficiently explain our introduction from ecological point of view. In response to this comment, we have added sufficient information in introduction part. The toxic effects of Lindane (γ-BHC) on Microcystis aeruginosa were especially studied because of its persistence in aquatic ecosystems. From ecological point of view, lindane is studied to inhibit Microcystis aeruginosa as it has potential to restrict its growth. This study is possible by means of different lab experiments under different lab conditions. This study is a novel approach as it shows satisfied results to inhibit growth of algae in aquatic ecosystems. Inhibition of freshwater algae from direct exposure of lindane concentration and exposure time ultimately favors our research. This study provides a complete approach to inhibit growth of algae under different parameters. However, further investigations are still required to determine toxic effects of lindane on algal cells [3].

Reviewer 3 Report

This important and interesting work presents the results of studying the effect of Lindane on cyanobacteria M. aeruginosa cells. The study was conducted carefully and competently.

There are several questions and comments that are aimed at improving the text of the manuscript. They are listed below.
1) Figure 2. Please add "A, B, C" in the figure.
2) The Title of the manuscript should be changed. Try not to use abbreviations in the Title or decipher them at the first mention.
3) Can a decrease in the activity of SOD be associated with a decrease in the content of cellular proteins?
4) Why does the activity of SOD decrease with an increase in oxidative stress?

Author Response

Reviewer# 3

There are several questions and comments that are aimed at improving the text of the manuscript. They are listed below.

1) Figure 2. Please add "A, B, C" in the figure.

Thank you very much for your comments, we are very sorry that we did not sufficiently explained our added figures. In response to this comment, we have added designations regarding figures respectively.

A, B and C is added in figure 2.

• Protein content (B) SOD activities (C) MDA content

2) The Title of the manuscript should be changed. Try not to use abbreviations in the Title or decipher them at the first mention.

Thank you for this comment and correction is followed.

DOM abbreviation is used to describe dissolved organic matter. Abbreviations should not be used in the title of the article. So, suggested titles are these:

1. The influence of pH, dissolved organic matter and toxic effects of Lindane on Microcystis aeruginosa
2. The toxic effects of Lindane via impact of pH and dissolved organic matter on Microcystis  aeruginosa

3) Can a decrease in the activity of SOD be associated with a decrease in the content of cellular proteins?

Thank you for your comments and pointing out this section. In response to this section we have tried our best to explain this section.Yes, decrease in the activity of superoxide dismutases is associated with decrease in the contents of cellular proteins. Under abiotic stresses, enhancement of ROS is mainly responsible for oxidative damage in algal cells.  In such conditions, increasing level of ROS produce harmful effects in various cellular components including cell membrane and contents of proteins and lipids. To combat such oxidative stresses, algal cells produce SOD that function as antioxidants for suppression of ROS.

4) Why does the activity of SOD decrease with an increase in oxidative stress?

Thank you for your comment and pointing out this section. In response to this section, we have addressed it in this way. Superoxide dismutases constitute important part in signaling pathways by producing ROS (reactive oxygen species)   lower in number. The activity of SODs decrease with increase in oxidative stress to maintain balance present between production and suppression of ROS. In algal cells antioxidant enzymes such as superoxide dismutases help to cope such oxidative damage.

Round 2

Reviewer 2 Report

In this work the toxic effects of Lindane via impact of pH and dissolved organic matter on Microcystis aeruginosa were described. Total protein levels, as well as malondialdehyde (MDA) levels and superoxide dismutase (SOD) enzyme activity, in algal cells, were determined after exposure to different concentrations of Lindane. It was shown that the toxic effects decreased with increasing pH and humic acid concentrations. Ultrastructure cell images were used to depict Lindane induced apoptosis.

Authors have corrected all comments in the paper and quite clearly answered to the questions. I hope these corrections improved the paper and the revised version corresponds to high standards of Water. After careful consideration, I think that this article may be published in this view.

Author Response

Dear Reviewer,
Thank you so much for the critical evaluation of our manuscript. We have addressed the comments one by one and are shown in track changes in the main text and a response letter has been attached.

Author Response File: Author Response.docx