Zebrafish Larval Melanophores Respond to Electromagnetic Fields Exposure

Round 1

Reviewer 1 Report (Previous Reviewer 3)

I am satisfied with the changes the authors made to the manuscript and I have no further comments or concerns.

Reviewer 2 Report (Previous Reviewer 1)

SUMMARY

The authors deal with the issue of the influence of magnetic and electromagnetic fields on a group of zebrafish (Danio rerio) embryos. A selected group of embryos received radiation of different frequencies and intensities. The applied frequencies ranged from extremely low frequency to ultra-high frequency with different applied magnetic field intensities. For each frequency, a group of ten embryos was selected and monitored for five days of exposure to external fields. Changes in body pigmentation were observed in zebrafish embryos and larvae in terms of the total number, area, and morphology of melanophores. The authors declare that this research can contribute to the evaluation of the role of magnetic radiation with implications from current medical devices to the use of communication using 5G networks.

POSITIVE ASPECTS

1. Based on a literature review, the authors point out the issues associated with an interaction between living beings and various external electromagnetic fields with frequencies from 0 Hz up to several GHz.
2. The authors prepared experimental setups for generating a magnetic field (static, extremely low frequency) and an electromagnetic field (low frequency, very high frequency, and ultra-high frequency) to expose living organisms (zebrafish embryos) to these fields.
3. The authors evaluated the effects of external fields generated by experimental setups on zebrafish melanophores by statistical analysis using an unpaired t-test.
4. The authors follow up on previous experiments on zebrafish embryos exposed to both strong static and low alternating magnetic fields.
5. The authors aim to continue researching the effects of the magnetic field in the zebrafish model in future works.

CONCLUSION

The revised article overall well addressed the review comments and suggestions. I recommend accept it for publication.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

SUMMARY

The authors deal with the issue of the influence of magnetic and electromagnetic fields on a group of zebrafish (Danio rerio) embryos. A selected group of embryos received radiation of different frequencies and intensities. The applied frequencies ranged from extremely low frequency to ultra-high frequency with different applied magnetic field intensities. For each frequency, a group of ten embryos was selected and monitored for five days of exposure to external fields. Changes in body pigmentation were observed in zebrafish embryos and larvae in terms of the total number, area, and morphology of melanophores. The authors declare that this research can contribute to the evaluation of the role of magnetic radiation with implications from current medical devices to the use of communication using 5G networks.

POSITIVE ASPECTS

1. Based on a literature review, the authors point out the issues associated with an interaction between living beings and various external electromagnetic fields with frequencies from 0 Hz up to several GHz.
2. The authors prepared experimental setups for generating a magnetic field (static, extremely low frequency) and an electromagnetic field (low frequency, very high frequency, and ultra-high frequency) to expose living organisms (zebrafish embryos) to these fields.
3. The authors evaluated the effects of external fields generated by experimental setups on zebrafish melanophores by statistical analysis using an unpaired t-test.
4. The authors follow up on previous experiments on zebrafish embryos exposed to both strong static and low alternating magnetic fields.
5. The authors aim to continue researching the effects of the magnetic field in the zebrafish model in future works.

ISSUES

The presented work is useful but has some issues that need to be removed. I have a few comments that can be used to improve the article.

Minor issues
1. Symbols of units are always in roman (upright) type according to ISO 80000-1: 2009 standard. Correct all text accordingly, including Abstract, tables, and figures.
2. Arabic numerals are written in upright type see, for example, the description of Figure 1. It also applies to indices in equations and the marks of physical quantities and superscripts. Correct accordingly throughout the text.
3. Signs of physical quantities need to write in italics according to ISO 31-4 (ISO 80000-5: 2007). Corrections need to be done throughout the article, including tables and figures.
4. The authors use solid circles to indicate nodes in the scheme in Figure 3. However, in some nodes, solid circles are missing, in some parts of the scheme solid circles are redundant. Correct the wiring diagram in Figure 3 to make it clear where the nodes are and are not.
5. It is not explained what the designations I_B and V_out mean on line 127. Add the meaning of the designations I_B and V_out to the text.
6. The authors used the unknown word “orizzontal” in line 139.
7. The reference to formula (2) is missing. Add a missing reference to formula (2).
8. According to the ISO guidelines, no spaces should be used after plus, minus, or plus-or-minus signs that designate positive or negative values, e.g., 266.9 ±19.6 and not 266.9±19.6 (Table 1). Correct accordingly throughout the text.
9. Use a decimal point instead of a decimal comma according to the recommended magazine template. Correct accordingly throughout the text.
10. Table 1 lists the markers for Morphological alterations, but the text does not comment on the significance of the markers. Add to the manuscript the meaning of the signs used in Table 1 in the column marked as Morphological alterations.

Major issues
1. The diameter of the permanent magnet used in the experiment is smaller than the diameter of the Petri dish. At the edges of a permanent magnet, there is a strong magnetic field gradient that cannot be neglected. To ensure a homogeneous magnetic field, a permanent magnet with a larger diameter than the diameter of the Petri dish must be used. How do the authors guarantee that the examined Danio rerio embryos were permanently in a homogeneous magnetic field and were not located near the edges of the permanent magnet? Otherwise, when the Danio rerio could be in the region of a strong gradient magnetic field near the edges of the permanent magnet during the experiment, it would be appropriate to repeat the experiment. Add to the manuscript a comment on the mentioned arguments.
2. According to the circuit diagram in Figure 3, it is clear that the transistors used are bipolar. Therefore, the use of the term gate is not correct. Correct the term gate to the correct one according to the type of transistors used.

REMARKS

After substituting the parameters into equation (2), R0 = 58.6 ohm, which is 17.2 percent more than the normalized impedance of 50 ohms. It would be appropriate to indicate how much power was reflected back from the antenna to the amplifier because this affects the radiated power from the antenna and thus also the intensity of the electric and magnetic field.

RECOMMENDATIONS

1. The article title does not describe the essence and goal of the experimental work of the authors. I recommend that the authors revise the title of the article.
2. The constants ε0 and μ0 constants given on lines 158 to 159 are relevant for vacuum. Add a missing note to the manuscript.
3. In Table 1, it is not appropriate to combine Total count with percentages, but for percentages, I recommend adding another column in Table 1.
4. I recommend moving Table 2 to the end of the Materials and Methods section.

QUESTIONS

I have four questions for the authors of the article.
1. What physical unit are the authors referring to in lines 161 to 162 and 177 (apostrophe + Greek letter omega)?
2. The authors state in line 162 that the load is 50 ohms and consists of four 200 ohm parallel resistors. What type of resistors are these? Are these resistors non-inductive? The self-inductance of the resistors affects the total load impedance and the transmitted power. Complete the missing data in the manuscript regarding the resistors used for the load.
3. What was the height of the water column in the Petri dish in which the embryos were placed? Did the authors maintain the same water column height in the Petri dishes? Specify the height of the water column in which the embryos were placed during the experiment. Indicate whether the same height of the water column was always observed in the Petri dishes during the experiment.
4. Was the photoperiod (14 h light : 10 h darkness) maintained even during the five days of field exposure? Add to the manuscript information regarding the photoperiod during the five-day experiment.

CONCLUSION

I find this article helpful. Regretfully, the paper cannot be accepted in its present form. The authors of the present article have to correct the issues.

Reviewer 2 Report

In the manuscript entitled "Suitable Devices for Studying Zebrafish Embryos Exposure to Static and Variable Electromagnetic Fields, the authors extend their previous work to define the effects of different magnetic fields on zebrafish melanophores. 

 

Figure 7 shows images of zebrafish. Image quality needs improvement, but even with this resolution, it is evident that not all fish were oriented in the same way (compare 7c - lateral view, 7b - ventral view and 7F - tilted lateral view). As pigment cells are not evenly distributed, the orientation of the zebrafish larvae impacts the number of pigment cells that can be observed. Hence no reliable data could have been gathered using images like those presented in Fig 7.

 

The authors state that within 12 hpf, embryos were divided into groups. Half a day in a zebrafish’s early life is a relatively long and essential period during which many developmental changes occur. During this period, oxygen consumption grows, and too high egg density can impact embryo growth. What happened with the eggs that were not divided into groups soon after fertilization? Where are they sorted or left unattended? When was the stress applied?

 

The authors state that the embryos were divided into groups within 12 hpf (line 209) and treatment lasted 5 days (line 214). The authors should carefully explain the experimental design. My concerns are that 0-12hpf plus 5 days = 5 to 5,5 days (>120hpf, which is the limit for experiments that do not require ethical permits). From the current description, it is unclear if all fish were exposed to the magnetic field at the same stage of development and for an equally long time or whether each group was treated differently. Moreover, it may look as if some zebrafish past 120 hpf were used without ethical permits. The authors should state whether this experiment was repeated (if so, how many times) or is a one-time observation on 10 fish.

 

In addition, the manuscript contains small but essential gaps/errors. For example, the correct DOI number for the reference [5] should be 10.1088/1748-0221/15/05/C05056 and not 10.1088/1748-022/15/05/C05056. The name of the line which was used is missing, and so are the water parameters at which adult fish were kept. Which commercial feed was used?

 

The fish part in its current form cannot be recommended for publication.

Reviewer 3 Report

Manuscript ID: applsci-1809523

Title: Suitable Devices for Studying Zebrafish Embryos Exposure to Static and Variable Electromagnetic Fields

 

General Comments:

The manuscript discusses the impact of electromagnetic fields on skin pigmentation. The paper requires significant editing for writing and grammar before it is ready to be published, and though the topic is appropriate for the journal there is not sufficient data included in the manuscript to be a publication. I have these additional concerns that should be addressed prior to publication

1.     The introduction discusses magnetic fields in general but does not clearly define scenarios when these exposures are likely to be encountered by fish, what how environmental doses compare to the doses used in the study, and why this work is important.

2.     Figures 1-6 are all method diagrams that could be included as supplemental information and there is really only 1 figure and 1 table that presents any data. This is not sufficient to stand alone as a research paper.

Specific Comments:

Line 196 – what strain of wildtype zebrafish were used?

Line 202- what stage were the embryos when they were collected/ how long after spawning were embryos collected?

Figure 7 – why is the static exposure (panel B) imaged from above and the rest are imaged from the side? What is the scaling on all these images (they appear to be different).