The Toxicity of Polyester Fibers in Xenopuslaevis

Round 1

Reviewer 1 Report

Authors investigated the toxicity of polyester microfibers released during clothes washing and drying. The authors state that the main aim of the paper is to evaluate mortality and potential adverse effects of Pets on the digestive system and swimming behavior of the in Xenopus laevis larvae.
The design of the experiment set this way, unfortunately, makes it impossible to notice variability in the phenomena that the authors investigated. Although the experiments were performed in triplicates, the experimental animals were taken at random from all replicates, and the authors did not provide information on the variability between individual replicates. Also, this made it impossible to obtain data on variability later during the statistical analysis.

I find the paper interesting, but there are improvements to be made. Specific details are listed below:

2. Materials and Methods

2.2. Animals and experimental design

150-151 Authors state that “…dead embryos were removed”. How many dead embryos were there? Please provide survival percentage per concentration. Were there some significant differences in mortality between the replicates?

2.4. Statistical analysis

L190 What is endpoint of this EC50 value?

3. Results

L194 Authors state that no mortality occurred in controls and PEF treated groups. However, in L150-L151 authors stated that dead embryos were removed? Please clarify.

L200-L201 Again, what was the endpoint for EC50 96h? Beside explanation I find that authors should provide dose-response curve in the results section.

3.2. Effects of PEFs on swimming behavior

In general, I find that results on swimming behavior should be put in a table or synthesized in some way, as it would be easier to track the results. Graph on the regression analysis should also be provided in this section.

Author Response

Reviewer 1

Authors investigated the toxicity of polyester microfibers released during clothes washing and drying. The authors state that the main aim of the paper is to evaluate mortality and potential adverse effects of Pets on the digestive system and swimming behavior of the Xenopus laevis larvae.

The design of the experiment set this way, unfortunately, makes it impossible to notice variability in the phenomena that the authors investigated. Although the experiments were performed in triplicates, the experimental animals were taken at random from all replicates, and the authors did not provide information on the variability between individual replicates. Also, this made it impossible to obtain data on variability later during the statistical analysis.

 

Response to the Reviewer:

Exposures were run in triplicates since it is not unusual that some of the Xenopus sp. embryos may die during the early developmental phases as this stage is the most sensitive of the whole amphibian development. During the exposure, the Petri dish in which embryos were developing might be contaminated by the substances released by the degenerating embryo(s), reducing the number of those reaching the free-swimming larva stage. Because the planned behavioural and morphological analyses needed a sufficient number of larvae, mainly for light and electron microscopy studies, we kept batches separate from each other. However, as noticed by the Reviewer (but also as clearly stated in the manuscript: L152-153 for the behavioural analyses, L166-167 for the histological analyses, and L.173-174 for the electron microscopy studies), at the end of the test, we pooled the replicates. The variability among replicates of the same concentration was considered only for mortality and the developmental stage reached at the end of the test. Since both resulted the same not only among replicates but also among concentrations (0% mortality and NF 46 as developmental stage), we pooled the replicates. The differences among concentrations in the PEF-induced abnormal gut coiling were observed only later when the microscopic analysis was performed. For this reason, we specified throughout the manuscript that we pooled embryos from each concentration, and we never reported and neither compared percentages from the batches of the same exposure concentration. Comparisons were made between concentration vs concentration and concentration vs control. As noticed by the Reviewer, we did not provide any data about variability among the three replicates, but this was because we were aware that our experimental plan did not allow this kind of information.

 

I find the paper interesting, but there are improvements to be made. Specific details are listed below:

 

2. Materials and Methods

 

2.2. Animals and experimental design

 

L150-151 Authors state that “…dead embryos were removed”. How many dead embryos were there? Please provide survival percentage per concentration. Were there some significant differences in mortality between the replicates?

 

Response to the Reviewer:

As reported in the first line of the Results Section, no mortality was recorded in controls and in PEF treated group, thus no survival percentage per concentration has been reported and no comparison among the different replicates has been made. In the Materials and Methods (line 150-151), we wrote that every day dead embryos were removed since the check for viability was made daily and, if present, dead specimens removed. According to the criticism, we modified the sentence in the material and methods with:

“Each day, all solutions were renewed, and embryos checked for viability.”

 

2.4. Statistical analysis

 

L190 What is endpoint of this EC50 value?

 

Response to the Reviewer:

L190 We specified the endpoint of the EC₅₀ modifying the sentence in the Statistical analysis Section:

“EC₅₀ value at 96 h of the abnormal gut coiling was calculated using Log-probit regression analysis. Log regression was preferred because of the best fit (z_slope = 4.76; P<0.001***).

 

3. Results

 

L194 Authors state that no mortality occurred in controls and PEF treated groups. However, in L150-L151 authors stated that dead embryos were removed? Please clarify.

 

Response to the Reviewer:

See comments above

 

L200-L201 Again, what was the endpoint for EC50 96h? Beside explanation I find that authors should provide dose-response curve in the results section.

 

Response to the Reviewer:

An explanation about the considered EC₅₀ endpoint has been added in the Materials and Methods (L190, see above) and in the Results Section (L200-201). A new Figure of the Log-probit regression has been added to the revised version of this manuscript. We calculated the EC₅₀ value even if it was estimated only with few points because of the good fit of the regression and to propose an indicative value for further and more detailed studies.

 

3.2. Effects of PEFs on swimming behavior

 

In general, I find that results on swimming behavior should be put in a table or synthesized in some way, as it would be easier to track the results. Graph on the regression analysis should also be provided in this section.

 

Response to the Reviewer:

As suggested by the Reviewer, a new table (Table 1) with the results of the swimming behaviour has been added to the manuscript. Also, the graphs of the data distribution and regression are now available as the new Figure 8 with its related test as reported below:

 

Table 1. Number (N) and percentage (%) of larvae which spent 0-10 sec, >10-20 sec, and >20-30 sec in the inner or outer part of the Petri dish (Total time); those which did not move for 0-10 sec, >10-20 sec and >20-30 sec in the inner and outer part of the Petri dish (Immobilization time), and those which travelled for a total distance of 0-100, >100-200 and >200 mm in the two sectors (Distance travelled).

Treatment	Total time					Immobilization time					Distance travelled
	Time	Inner part		Outer part		Time	Inner part		Outer part		Distance	Inner part		Outer part
	sec	N	%	N	%	sec	N	%	N	%	mm	N	%	N	%
CTRL	0-10	19	95	0	0	0-10	20	100	9	45	0-100	20	100	12	60
	>10-20	1	5	1	5.0	>10-20	0	0	4	20	>100-200	0	0	1	5.0
	>20-30	0	0	19	95	>20-30	0	0	7	35	>200	0	0	7	35
	Total	20	100	20	100	Total	20	100	20	100	Total	20	100	20	100
1 μg mL-1	0-10	20	100	0	0	0-10	20	100	5	25	0-100	20	100	12	60
	>10-20	0	0	0	0	>10-20	0	0	7	35	>100-200	0	0	3	15
	>20-30	0	0	20	100	>20-30	0	0	8	40	>200	0	0	5	25
	Total	20	100	20	100	Total	20	100	20	100	Total	20	100	20	100
10 μg mL-1	0-10	17	85	0	0	0-10	20	100	5	25	0-100	18	90	14	70
	>10-20	3	15	3	15	>10-20	0	0	5	25	>100-200	2	10	3	15
	>20-30	0	0	17	85	>20-30	0	0	10	50	>200	0	25	3	15
	Total	20	100	20	100	Total	20	100	20	100	Total	20	100	20	100
50 μg mL-1	0-10	18	90	0	0	0-10	19	95	4	20	0-100	20	100	17	85
	>10-20	2	10	2	10	>10-20	1	5	6	30	>100-200	0	0	2	10
	>20-30	0	0	18	90	>20-30	0	0	10	50	>200	0	0	1	5
	Total	20	100	20	100	Total	20	100	20	100	Total	20	100	20	100
Total	0-10	74	92.5	0	0	0-10	79	98.7	23	28.8	0-100	78	97.5	55	68.8
	>10-20	6	7.5	6	7.5	>10-20	1	1.3	22	27.5	>100-200	2	2.5	9	11.2
	>20-30	0	0	74	92.5	>20-30	0	0	35	43.7	>200	4	10.5	16	20
	Total	80	100	80	100	Total	80	100	80	100	Total	38	100	80	100

 

 

 

Box-plot analysis of the distribution of the mean swimming speed of each larva in the inner and outer sector of the Petri dish is shown in Figure 8. The regression analysis of the log transformed data confirmed a significant decreasing trend of the mean speed vs PEF concentration (p = 0.008**), despite the wide variability of the data. Also Kruskal-Wallis test showed a significant difference of the mean speed in relation to the PEF concentration (χ² = 9.2; d.f. = 3; p = 0.027*).

Figure 8. Box-plot of the mean swimming speed of each larva in the inner and outer sector of the Petri dish in relation to the PEF concentration (A), and linear regression of the Log transformed mean swimming speed of each larva in the inner and outer sector of the petri in relation to the Log transformed PEF concentration (B); regression equation and parameters: y = -0.169x + 0.42; n= 118; R² = 0.06; p = 0.008**).

 

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The research concerns of analysis the effect on Xenopuslaevis larva development of polyester fibers (PEFs), taken from a drier machine in which 100% polyester fabrics, were dried after washing. The presented results show that the gastrointestinal tract (GIT), was the most affected system by PEFs, which modified the normal shape of the intestine with an EC₅₀ 96h value of 6.3 μg/mL. The fibers to pressed against the digestive epithelium, deforming the normal architecture of the gut. No other damages were registered. The results were summarized on the basis  of  experiments, possessing a certain degree of innovation. However article needed to be improved.

1. Line: 35 “Microplastic fibers (MPFs) derive from various sources” – could you give some examples.
2. Should attach SEM images and EDS analysis of PET fibers because the obtained results no confirmed.
3. The larvae were exposed only for 96h and the duration of effective PEFs ingestion was only for about 40h. Why was the exposure time not extended? (the results would be more interesting)

Author Response

Reviewer 2

The research concerns of analysis the effect on Xenopus laevis larva development of polyester fibers (PEFs), taken from a drier machine in which 100% polyester fabrics, were dried after washing. The presented results show that the gastrointestinal tract (GIT), was the most affected system by PEFs, which modified the normal shape of the intestine with an EC₅₀ 96h value of 6.3 μg/mL. The fibers to pressed against the digestive epithelium, deforming the normal architecture of the gut. No other damages were registered. The results were summarized on the basis of experiments, possessing a certain degree of innovation. However, article needed to be improved.

 

1. Line: 35 “Microplastic fibers (MPFs) derive from various sources” – could you give some examples.

 

Response to the Reviewer:

We modified the sentence specifying the sources of PEFs, also adding a new citation to a very recent review (Rebelein et al., 2021. Microplastic fibers - Underestimated threat to aquatic organisms? Science of the Total Environment, 777: 146045).

 

The new sentence is:

“Microplastic fibers (MPFs) derive from various sources: from clothes mostly made of polyethylene terephthalate, namely polyester, through a direct pathway from clothing to water courses or via the atmosphere [9-10]. They are known to enter the environment during the production process,laundering the end products, or due to disintegration of textiles and non-laundering fabrics such as flags, sails, furniture, carpets, or mattresses. Household and office dust, but also abrasions of insulating materials from construction sites represent other known sources for PEFs [11 and references within].”

 

2. Should attach SEM images and EDS analysis of PET fibers because the obtained results no confirmed.

 

Response to the Reviewer:

A new figure with an image by SEM of PEFs and two spectra of two different fibers was added to the manuscript. The following sentence was added.

 

 

“Figure 1 shows an example of two spectra from PEF fibers used for the exposure experiment. The obtained experimental C:O atomic ratios (71.5 : 28.5 and 71.9 : 28.1) resulted very close to the stoichiometric PET ratio (71.4:28.6) and close to the experimental C:O ratio of PET measured with the same instrument [29]”

 

3. The larvae were exposed only for 96h and the duration of effective PEFs ingestion was only for about 40h. Why was the exposure time not extended? (the results would be more interesting)

 

Response to the Reviewer:

The authors agree with the Reviewer#2 that it would be very interesting to explore the effects of PEFs for a longer time, but according to the EU directive 2010/63/EU (European Union, 2010), the last not independently feeding and, therefore not normed larval form, was NF stage 47, the stage immediately following our final stage. Going further is certainly possible, but an authorization is needed. At the moment, we are not allowed to extend our trial.

 

European Union, 2010. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific Purposes. Off. J. Eur. Union L27633.

 

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

This is a valuable paper: well designed and carried out research, well-written.

I would suggest summarizing the discussion in for of a table/infographic/bullet points/etc because being robust it is difficult to read. In my opinion, it would make this paper better, however, it already can be published as it is.

Author Response

Reviewer 3

 

This is a valuable paper: well designed and carried out research, well-written.

I would suggest summarizing the discussion in for of a table/infographic/bullet points/etc because being robust it is difficult to read. In my opinion, it would make this paper better, however, it already can be published as it is.

 

Response to the Reviewer:

The discussion is now divided into four paragraphs to facilitate reading:

 

• The digestive system: the main target for PEFs
• PEFs in the gut: physical effects
• Side effects due to PEFs
• Future perspectives

 

Author Response File: Author Response.docx

Reviewer 4 Report

The article submitted by Bacchetta et al. describes the effect of polyester fibres released from a domestic tumble dryer after washing of 100% polyester fabrics on the early development of Xenopus larvae.

 

In addition to toxicity, malformation and gut histology, abnormalities in swimming behavior have been reported. Fibres were characterized by light microscopy, scanning microscopy and energy dispersive x-ray spectroscopy and relevant concentrations were tested. 

Description of experiments and presentation in general is good but data on larvae length (growth as indication for growth retardation) and positive controls (heavy metal salts, such as Cd, Pb, Ni etc.) are missing.

Minor: spelling error in abstract “drier”

Author Response

Reviewer 4

 

The article submitted by Bacchetta et al. describes the effect of polyester fibres released from a domestic tumble dryer after washing of 100% polyester fabrics on the early development of Xenopus larvae.

In addition to toxicity, malformation and gut histology, abnormalities in swimming behavior have been reported. Fibres were characterized by light microscopy, scanning microscopy and energy dispersive x-ray spectroscopy and relevant concentrations were tested. 

Description of experiments and presentation in general is good but data on larvae length (growth as indication for growth retardation) and positive controls (heavy metal salts, such as Cd, Pb, Ni etc.) are missing.

 

Response to the Reviewer:

According to the Reviewer, no data about the size of larvae at the end of the experiment were provided, even if an indicator of their possible growth retardation was considered looking at their developmental stage at the end of the exposure. As already specified in the answer to Reviewer#1, no differences in larval size were noticed among replicates and treatments. However this point was clarified by the following new sentence at the beginning of the Results Section:

 

" No mortality was recorded among replicates during the exposure and at the end of the test, and all larvae from controls and PEF-exposed groups reached the NF stage 46 [28].”

 

Regarding the positive control, we agree with the Reviewer that it is an important tool for toxicity evaluation, but for PEFs and MPs in general, they are not defined yet. The suggested heavy metals or other possibly toxic chemicals can be suitable for chemical toxicity, but not for chemically inert substances such as PEFs, which resulted in having mainly physical effects on the gastrointestinal tract of the larvae. In the future, just PEFs, once better defined, and natural polymer fibers such as cotton or wool might be assumed as reference positive control for gastrointestinal occlusion effects.

 

Minor: spelling error in abstract “drier”

 

Response to the Reviewer:

L14 Corrected, thank you

 

Author Response File: Author Response.docx

Reviewer 5 Report

In the manuscript Water-1469537 by Bacchetta et al, authors have examined the adverse effects in an amphibian model, Xenopus laevis, against exposure to the polyester fibers (PEFs). They first collected PEF samples and did characterization. Thereafter, authors examined mortality and organ system malformations in the exposed animals using whole organism imaging, histological staining and scanning electron microscopy. They also observed behavioral defect in the exposed animals. Exposed animals exhibited accumulation of PEFs, gastrointestinal malformations and impaired mobility at highest concentration used. This study suggests the potential hazardous effect of polyester fibers to the aquatic animals. Experiments are well conducted. I have following points to improve the manuscript.

 

1. In abstract section, Line 11-12. How does a commercial standard microplastic is different from environmental plastic? Do authors imply that there is different composition of the microplastics? One can think of having environmental microplastic content as result of commercial standard usage only. Please rephrase or change accordingly.

 

2. Line 21-23. Authors mentioned above that no mortality was observed even though exposed larvae had GI tract defects. Therefore, “threatening growth and survival” might be too strong to conclude. Please rephrase.  

 

 

3. Introduction section. Line 67. It would be helpful to mention the rationale for selecting the 1, 10 and 50ug/mL concentrations here.

 

 

4. Result section, 3.2. Swimming behavior readout data should be presented either as graph plots or tables with the mean±SD values. It helps readers/researchers for the better assessment in their experiments.

 

 

5. Did authors examine any signature for cytotoxicity?

 

Author Response

Reviewer 5

 

In the manuscript Water-1469537 by Bacchetta et al, authors have examined the adverse effects in an amphibian model, Xenopus laevis, against exposure to the polyester fibers (PEFs). They first collected PEF samples and did characterization. Thereafter, authors examined mortality and organ system malformations in the exposed animals using whole organism imaging, histological staining and scanning electron microscopy. They also observed behavioral defect in the exposed animals. Exposed animals exhibited accumulation of PEFs, gastrointestinal malformations and impaired mobility at highest concentration used. This study suggests the potential hazardous effect of polyester fibers to the aquatic animals. Experiments are well conducted. I have following points to improve the manuscript.

 

1. In abstract section, Line 11-12. How does a commercial standard microplastic is different from environmental plastic? Do authors imply that there is different composition of the microplastics? One can think of having environmental microplastic content as result of commercial standard usage only. Please rephrase or change accordingly.

 

Response to the Reviewer:

According to the Reviewer, we modified the sentence in the Abstract at L11-13 with:

“Most of the exposure experiments for determining microplastic toxicological effects were performed with a microplastic varying little in shape and size (often purchased microplastic beads), but few studies deal with non-homogeneous samples.”

 

1. Line 21-23. Authors mentioned above that no mortality was observed even though exposed larvae had GI tract defects. Therefore, “threatening growth and survival” might be too strong to conclude. Please rephrase.  

 

Response to the Reviewer:

We modified the final sentence of the Abstract, removing the last portion of the sentence.

 

1. Introduction section. Line 67. It would be helpful to mention the rationale for selecting the 1, 10 and 50ug/mL concentrations here.

 

Response to the Reviewer:

An indication for the selected concentrations was added.

 

“Since no previous studies of microplastic fiber effects on amphibian larvae were available so far, three different increasing concentrations with a wide range were selected: 1, 10, and 50 mg mL^-1.”

 

 

1. Result section, 3.2. Swimming behavior readout data should be presented either as graph plots or tables with the mean±SD values. It helps readers/researchers for the better assessment in their experiments.

 

Response to the Reviewer:

Following the Reviewer’s suggestion and in agreement with the Reviewer#1, we added a new Table (Table 1) summarizing the behavioural data, and a new Figure (Figure 8).

 

Table 1. Number (N) and percentage (%) of larvae which spent 0-10 sec, >10-20 sec, and >20-30 sec in the inner or outer part of the Petri dish (Total time); those which did not move for 0-10 sec, >10-20 sec and >20-30 sec in the inner and outer part of the Petri dish (Immobilization time), and those which travelled for a total distance of 0-100, >100-200 and >200 mm in the two sectors (Distance travelled).

Treatment	Total time					Immobilization time					Distance travelled
	Time	Inner part		Outer part		Time	Inner part		Outer part		Distance	Inner part		Outer part
	sec	N	%	N	%	sec	N	%	N	%	mm	N	%	N	%
CTRL	0-10	19	95	0	0	0-10	20	100	9	45	0-100	20	100	12	60
	>10-20	1	5	1	5.0	>10-20	0	0	4	20	>100-200	0	0	1	5.0
	>20-30	0	0	19	95	>20-30	0	0	7	35	>200	0	0	7	35
	Total	20	100	20	100	Total	20	100	20	100	Total	20	100	20	100
1 μg mL-1	0-10	20	100	0	0	0-10	20	100	5	25	0-100	20	100	12	60
	>10-20	0	0	0	0	>10-20	0	0	7	35	>100-200	0	0	3	15
	>20-30	0	0	20	100	>20-30	0	0	8	40	>200	0	0	5	25
	Total	20	100	20	100	Total	20	100	20	100	Total	20	100	20	100
10 μg mL-1	0-10	17	85	0	0	0-10	20	100	5	25	0-100	18	90	14	70
	>10-20	3	15	3	15	>10-20	0	0	5	25	>100-200	2	10	3	15
	>20-30	0	0	17	85	>20-30	0	0	10	50	>200	0	25	3	15
	Total	20	100	20	100	Total	20	100	20	100	Total	20	100	20	100
50 μg mL-1	0-10	18	90	0	0	0-10	19	95	4	20	0-100	20	100	17	85
	>10-20	2	10	2	10	>10-20	1	5	6	30	>100-200	0	0	2	10
	>20-30	0	0	18	90	>20-30	0	0	10	50	>200	0	0	1	5
	Total	20	100	20	100	Total	20	100	20	100	Total	20	100	20	100
Total	0-10	74	92.5	0	0	0-10	79	98.7	23	28.8	0-100	78	97.5	55	68.8
	>10-20	6	7.5	6	7.5	>10-20	1	1.3	22	27.5	>100-200	2	2.5	9	11.2
	>20-30	0	0	74	92.5	>20-30	0	0	35	43.7	>200	4	10.5	16	20
	Total	80	100	80	100	Total	80	100	80	100	Total	38	100	80	100

 

 

 

Box-plot analysis of the distribution of the mean swimming speed of each larva in the inner and outer sector of the Petri dish is shown in Figure 8. The regression analysis of the log transformed data confirmed a significant decreasing trend of the mean speed vs PEF concentration (p = 0.008**), despite the wide variability of the data. Also Kruskal-Wallis test showed a significant difference of the mean speed in relation to the PEF concentration (χ² = 9.2; d.f. = 3; p = 0.027*).

Figure 8. Box-plot of the mean swimming speed of each larva in the inner and outer sector of the Petri dish in relation to the PEF concentration (A), and linear regression of the Log transformed mean swimming speed of each larva in the inner and outer sector of the petri in relation to the Log transformed PEF concentration (B); regression equation and parameters: y = -0.169x + 0.42; n= 118; R² = 0.06; p = 0.008**).

 

 

 

 

1. Did authors examine any signature for cytotoxicity?

 

 

Response to the Reviewer:

We did not specifically analyse cytotoxicity effects. However, as reported in the manuscript (L238-240), no signs of cytotoxicity were noticed by light microscopy. Also, the ultrastructural analyses by SEM did not evidence particular signs of cytotoxicity (L276-278).

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 4 Report

The authors addressed my comments.

Reviewer 5 Report

Authors revised manuscript satisfactorily.