Nano-TiO2 Phototoxicity in Fresh and Seawater: Daphnia magna and Artemia sp. as Proxies
, Carlos Barata Martí 1 and M. Silvia Diaz-Cruz 1,*Round 1
Reviewer 1 Report
The publication is scientifically valuable for two reasons:
- because the paper discusses not only the influence of nano-TiO2 on the aquatic environment, but also the influence of the aquatic environment on the mechanism of action of nano-TiO2.
- because the research covers a wide range of nano-TiO2 concentrations from 0.000018, through 0.01, 0.1, 1, to 10 mg/L used in biological research at a time relevant for photochemical processes.
In this perspective, the work is innovative.
The publication does not contain any research or discussion of a very important problem related to aquatic toxicity, namely after what time in the aquatic environment (experimental conditions) nano-TiO2 loses its toxic properties. This problem is important for the treatment of wastewater containing nano-TiO2. If nano-TiO2 loses its activity over time, the wastewater with nano-TiO2 should be collected in sedimentation tanks for a certain period of time (?) and then released to the environment.
The second problem that should be discussed in the paper is influence of size of TiO2 (micro or nano) on toxicity on aquatic environment.
What is the decay time of biological activity of the tested material nano-TiO2 under the conditions of the experiment?
Author Response
Dear Reviewer,
Thank you for the time taken to review this article below are answered to each of the points:
- Since TiO2 nanoparticles are metallic nanoparticles, they are not biodegradable so it will not lose its toxic properties but will persist in the environment. The nTiO2 form aggregates in the aqueous environment, some aggregates can sediment and remain immobilized, while others are dispersed and will be more mobile, bioavailable and toxic, and the interaction with other particles and organic matter, etc., is also supposed to alter their dynamic properties. and therefore toxic. Until now, it has not been possible to predict the environmental or biological impacts of nTiO2 due to the complexity of the marine aquatic ecosystem, since in freshwater the behaviour is different than in saltwater in terms of the aggregates that may form. It is known that in conventional treatment plants the removal of these nanoparticles is not carried out due to the small size that escapes from filters and membranes and actually the removal of nTiO2 by means of sedimentation is not viable, but in pilot plants, it is not feasible. It has been observed that the removal of the nTiO2 can be achieved through ultrafiltration, achieving a 95% removal of this nanomaterial.
- The size and shape of the nTiO2 can have a negative influence, since being very small, aquatic organisms may take nTiO2 as food, in addition to the nanosize interactions with other chemical/physical factors are greater they can cause increased toxicity or adverse effects in different marine species. Regarding the biological decomposition time, as already stated in point number 1, there cannot be decomposition because it is an inert material, and simply observe the dispersion or sedimentation, it must be taken into account that the nTiO2 are in contact with other particles that influence the aggregation behaviour, therefore, the concentration of nTiO2 is another important factor that governs the behaviour of this material in aqueous media, in our study sedimentation in freshwater was 10 to 15 hours, with greater stability than in saltwater, that the sedimentation time ranged from 20 to 24 hours, this taking into account the range of concentrations.
Reviewer 2 Report
The study describes a straightforward investigation of nTiO2 toxicity in two indicator species. Interestingly, in the absence of solar radiation an EC50 was found in Daphnia but not Artemia. The presence of solar radiation increased the EC50 for Daphnia, but lowered it for Artemia. The authors convincingly document interactive effects between organic compounds, solar radiation, and nTiO2 on Artemia. They also provide compelling evidence for the importance of exposure times.
A deficiency of the study was that the full combination of organics-solar radiation-nTiO2 was only studied in Artemia. The reasons for this should be noted in the text
Specific comments:
Section 2.1. Define EC50.
It is not sufficient to note that you used a website. The website could disappear, or the EC50 algorithm could change. Describe the algorithm used in sufficient detail that someone could recreate the calculations without needing to access the website.
Section 3.1, 3.2: Why was Daphnia mortality recorded after 48 hrs and Artemia after 72 hrs?
Line 344: This is confusing to me. Daphnia’s 48-hr EC50 more than doubled when irradiation was included. That would seem to indicate reduced toxicity. Please clarify.
Figure 3: move the legend so that it is not confused for actual data points.
Figure 4. Error in figure caption says this shows Daphnia response.
Table 2. Fix typos. “Concentration”
Line 352: Fix typos “organic a notorious increment”
Author Response
Dear Reviewer,
Thank you for the corrections, here are my answers and the proper corrections were made in the Manuscript
Specific comments:
- Section 2.1. Define EC50.
It is not sufficient to note that you used a website. The website could disappear, or the EC50 algorithm could change. Describe the algorithm used in sufficient detail that someone could recreate the calculations without needing to access the website.
A. To determine the EC50 values, the mortality of the D. magna and A. sp, during exposure to the tested compounds, was followed data normalization to the control mean percentage using Abbot's formula ((2004) Abbott's Formula. In Encyclopedia of Entomology. Springer, Dordrecht.) Afterwards, the percentage values were log-transformed using Quest Graph™ EC50 Calculator (https://www.aatbio.com/tools/ec50-calculator/), and AAT Bioquest, Inc, 11 Nov. 2019.
2. Section 3.1, 3.2: Why was Daphnia mortality recorded after 48 hrs and Artemia after 72 hrs?
A.The toxicity immobilization tests were carried out according to the guidelines of the Organization for Economic Co-operation and Development, OECD for Daphnia magna indicated to obtain the EC50 in 48 h, and for Artemia sp indicate 96 h to obtain this value.
3. Line 344: This is confusing to me. Daphnia’s 48-hr EC50 more than doubled when irradiation was included. That would seem to indicate reduced toxicity. Please clarify.
A. Done. The proper correction was made in the Manuscript
4. Figure 3: move the legend so that it is not confused for actual data points.
A. Done. The proper correction was made in the Manuscript
5. Figure 4. Error in figure caption says this shows Daphnia
A. Done. The proper correction was made in the Manuscript
6. Table 2. Fix typos. “Concentration”
A. Done. The proper correction was made in the Manuscript
7. Line 352: Fix typos “organic a notorious increment”
A. The proper correction was made in the Manuscript. Exposure to irradiation, nTiO2 and a mixture of organic UV filters, a notorious increment of the immobilization was observed.
Reviewer 3 Report
The subject is interesting, the experimental design seems to be correct, and the results seem to be sound.
Unfortunately, the manuscript is very poorly written: it is not clearly shown why the experiments were carried out, and the results are inadequately presented and discussed. This may be because of the language usage and writing style: I think the manuscript needs a thorough copy-editing by someone with the appropriate linguistic expertise.
Overall, the manuscript may contain good science but in its present form, it is not acceptable.
To mention a few problematic points:
1) I marked many inappropriate expressions and convoluted sentences in the manuscript.
2) The authors in the Summary and the Conclusions sections need to state what is new and original in their work.
3) I find the experimental part most problematic: the experiments are not described in detail (see comments and questions in the manuscript). The organisms studied are not described correctly, it is not known how many of them were studied in one experiment, how many times the experiments were repeated, what kind of control experiments were carried out etc.
4) There are no STD bars shown on the figures, it is not known which data are significantly different.
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My itemized summary of the work:
Clarity of objectives: good
Quality of methods/correctness of mathematics: poor
Quality of data: medium
Validity of assumptions and analyses: poor
Extent to which the interpretations/conclusions are supported by the data: poor
Overall significance of this work: impossible to tell
Comments for author File: 
Comments.pdf
Author Response
Dear Reviewer,
Thank you for the time taken to review this article below are answered to each of the points:
1) I marked many inappropriate expressions and convoluted sentences in the manuscript.
A. All the corrections were made properly in the Manuscript. A native Englishman reviewed the manuscript to improve quality.
2) The authors in the Summary and the Conclusions sections need to state what is new and original in their work.
A. The statement of the innovation is explained in the Conclusions section already.
This work shows several bioassays with combinations of experimental circumstances, it covers from the smallest concentration of 0.000018 mg/L to a higher concentration of 100 mg/L, and it was demonstrated that the prolonged UV radiation exposure time as 24 and 48-h, might increase the phototoxicity of the nTiO2. Also, this study has played an important role, exposing Artemia sp to nTiO2 combined with the most toxic UV organic filters, and then exposing to SSR, preceding subsequent investigations for the hazardous joint effects when physical and chemical UV filters are combined.
3) I find the experimental part most problematic: the experiments are not described in detail (see comments and questions in the manuscript). The organisms studied are not described correctly, it is not known how many of them were studied in one experiment, how many times the experiments were repeated, what kind of control experiments were carried out, etc.
A. We improve the explanations of the Experimental section as recommended by the reviewer.
4) There are no STD bars shown on the figures, it is not known which data are significantly different.
A. We change some figures for the sake of clarity.
- In Figure 4, where the bioassay with Artemia sp, the chart shows the increment of the immobilization with three replicates per Mix, the upper and lower bounds of the box represent the percentages of immobilization.
- In Figure 3 and 5, the charts were improved and now it can appreciate with two bars that shows the low and high immobilization at 24 and 48 h, a big difference on the immobilization percentage can be seen when the exposure time is prolonged
Round 2
Reviewer 1 Report
The authors of the publication, having read the comments of the reviewers, introduced a number of changes in the text. After the changes introduced, the publication presents valuable and substantive scientific material.
I recommend publication in this form for edition in this journal.
Author Response
Dear Reviewer 1,
We properly made all the corrections in the Manuscript,
Thank you and Regards,
Reviewer 2 Report
I appreciate the authors' detailed and thoughtful responses to reviewer comments and the additional detail added in the Methods and Discussion sections. I think the manuscript is much improved in its current form and I recommend it for publication.
Author Response
Dear Reviewer 2,
We properly made all the corrections in the Manuscript,
Thank you and Regards,
Reviewer 3 Report
The authors almost completely tended to the fundamental questions from the review and modified the original copy altogether. The new version of the paper is publishable in its present form.
One final comment: it would be highly beneficial from the point of soundness if error bars would be added to Figures 1 and 2 of the manuscript.
Author Response
Dear Reviewer 2,
We properly made all the corrections in the Manuscript, and we made the relevant changes as you suggested in figure 1 and 2.
Thank you and Regards,
