Polylactic Acid-Based Microplastic Particles Induced Oxidative Damage in Brain and Gills of Goldfish Carassius auratus

Round 1

Reviewer 1 Report (New Reviewer)

water-2410307

Polylactic acid-based microplastic particles induced oxidative damage in brain and gills of goldfish Carrasius auratus

The manuscript describes the accumulation effects in the organism of the golden caracuda (Carrasius auratus) of two different types of bioplastic particles originating from compostable polylactic acid (PLA)-based shopping bags and cups, while petroleum-based polyamide microparticles were investigated.

The study was conducted to evaluate the potential effects of tapping microplastic particles based on polylactic acid in the organism of the golden carp /Carrasius auratus/ and the induction of oxidative stress leading to lesions in the brain and gills of the fish.

The study investigated the effect of 96-hour exposure of goldfish Carassius auratus to two different types of bioplastic particles, derived from compostable polylactic acid (PLA)-based shopping bags and cups, and petroleum-based polyamide microparticles. All particles were studied both in their pure form and after exposure to UV-degradation in laboratory conditions.

Based on these results, this study shows that commercial (indicatively compostable) PLA bioplastic induces significant LPO (oxidative damage) in goldfish brain and/or gills, unlike bioplastic shopping bag particles and petroleum-based polyamide particles . UV-degradation of articles of all types investigated had no significant effect on the level of LPO in this experiment compared to non-UV-exposed particles.

In the first part, called "Summary", the authors explain the production and use of bioplastics, which are modern alternatives to petroleum-based plastics. The environmental hazard of petroleum-based plastics is increasingly appreciated for many types of organisms (mostly aquatic) and at different levels (cellular, molecular, whole organism) due to their persistence in the environment, their uptake by organisms, and the associated toxic/physiological effects (eg tissue damage, inflammation, false saturation). Research suggests that the dangers of bioplastics - an increasingly marketed green alternative to petroleum-based plastics - are significantly less well-studied. But data is already available indicating toxicity and problematic degradability of bioplastics.

The section also describes data from research on the biodegradability of different types of biodegradable plastics. Of five biodegradable polyesters (PLA, poly(lactic-co-glycolic acid) - PLGA, polycaprolactone - PCL, poly (3-hydroxybutyrate) - PHB, Ecoflex) exposed to fresh and sea water for a year, only PLGA degraded completely, PHB shows only surface erosion, and PLA and PCL show no signs of degradation.

It is also described that bioplastics can be toxic to living objects and should be carefully studied for their ecotoxicity before policy makers decide on the beneficial use of bioplastics.

Information is also provided on bioindicators that have been successfully used to assess the toxicity of environmental pollutants, including metals, pesticides, pharmaceuticals, and plastic particles. The reaction of organisms to the ingestion of pollutants (including plastics) leads to the formation of oxygen radicals (ROS)q which can target nucleic acids, proteins and lipids of cells, damaging their structure and function.

The use of the golden carp as a pollution biomarker and post-exposure study with two types of plastic materials – bioplastics versus petroleum-based plastics that can cause oxidative damage (LPO) in fish is described. To better address the environmental impact of plastics, two states of each type of plastic were considered: before and after simulated environmental degradation (exposure to artificial UV radiation).

The Materials and Methods section contains a detailed description of the two types of plastic material: bioplastic and leum-based petroplastic used in the study. The bioplastic material was obtained from two products/items sold in supermarkets: a fruit and vegetable bag (BioBag brand) and a cup (Bag and Cup). The bag is made from generation raw materials, with a higher content of renewable sources, according to the description on the brand's website (https://biobagworld.com/products/retail-2/). The cup is made of PLA as specified by the manufacturer. Both products had the "composted" logo in accordance with the European standard EN 13432 and therefore both materials are declared to be fully biodegradable. The particles used were in two different states: untreated (before exposure to UV irradiation) and UV-degraded (after UV irradiation to simulate photodegradation).

The conduct of the experiment in the study, in which fish of the species Carrasius auratus were used, is also described in detail. The plastic particles used in the experiments are labeled as follows: Bag-V (first condition from the bioplastic bag), Bag-UV (irradiated condition of the bag), Cup-V (untreated condition of the bioplastic cup), Cup-UV (irradiated condition of the glass), PA-V (first state of petroleum based polyamide) and PA-UV (irradiated state of polyamide). Data are also given on the method of rearing, feeding and euthanizing the fish by removing the brain and gills. Sample processing and assays for determination of LPO and total protein are described. The authors also describe the statistical processing of the data.

Section 3. "Results" details the validity of the test and the impact of bioplastics exposure on the biomarker of oxidative damage (lipid peroxidation). Levels of LPO (nmol TBARS per mg protein) in the brain or gills of the fish in the experimental groups were significantly different in most cases compared to the control group. Significant differences in LPO levels among groups exposed to pure plastic were as follows: in the brain, Cup-V > Bag-V (44 vs. 26 nmol/mg, respectively) and Cup-V > PA-V (26 vs. 13 nmol /mg, respectively); in gills, Cup-V > Bag-V (119 vs. 52 nmol/mg, respectively).

It was also reported that significant differences in fish brain LPO levels among groups exposed to UV-degraded plastic (bag, cup and PA) were as follows: in brain Bag-UV > Cup-UV (52 vs 14 nmol/ mg, resp. entirely) and Bag-UV > PA-UV (14 vs. 6 nmol/mg, resp.). However, the LPO level in Bag-UV was not significantly different from that in the control.

Significant differences were noted in the brain and gills only between the pristine and UV-rated PLA Cup groups exposed to untreated and UV-degraded particles. Exposure to virgin Cup resulted in significantly higher LPO in both organs compared to exposure to UV-degraded Cup: in the brain, Cup-V > Bag-V (44 vs. 14 nmol/mg, respectively), in the gills, Cup -V > Bag-V (119 vs. 48 nmol/mg, resp. whole). In a comparison of lipid peroxidation in the brain and gills of fish exposed to different types of plastic particles studied no significant differences were found between the two organs, but in fact the evidence for LPO in the gills was greater than in the brain.

In the Discussion section, it was shown that oxidative damage in the brain and gills after exposure to Virgin PLA-based glass was the most prominent among the exposures, significantly exceeding that of the control. Apparently, the PLA bioplastic particles had a greater power to compromise the cell's defense system, compared to the petroleum-based PA or Bag bioplastic. The exact mechanisms of the effects of plastic particles on organisms (DNA damage, neurotoxicity, reproductive impairment, oxidative stress, histopathological changes) remain largely unknown.

The section also notes that although C. auratus is selective in its food selection (avoids ingestion of Bag particles), accidental ingestion of solid PLA cup particles from the contaminated environment may have occurred and this may trigger the defense system of the cell. However, mechanical abrasion caused by particle edges can also lead to a biomarker response. The LPO levels and potency of PLA cup particles to cause LPO in the gills and brain of goldfish during such a short exposure time (48 h) indicates the urgent need for further studies.

It is also discussed that exposure to untreated Bag and PA particles causes a low (physiological) level of LPO, apparently not a threat to fish, in contrast to the effect of untreated PLA particles from cups. Although without elucidating the causal relationship behind this effect, our results suggest that bioplastics (perceived by the public as environmentally safe) may harm aquatic organisms and should be studied as thoroughly as possible for the potential adverse effect on local communities.

In general, the changes occurring in the plastic polymer matrix after UV-irradiation are expected to lead to a more pronounced effect of UV-degraded particles on the fish of the experimental groups compared to the control group. However, although brain LPO level after Bag-UV exposure significantly exceeded that of PA-UV and Cup-UV exposure, there was no significant oxidative damage resulting from exposure to UV-degraded particles: LPO levels in both organs after exposure to UV-degraded particles of all species studied remained within the physiological ranges of LPO.

Levels of LPO observed after exposure to clean plastic of all types were higher than those observed after exposure to UV-degraded counterparts. Evidence for a greater degree of LPO in the gills is generally greater than in the brain. Such a change in the level of LPO in the gills probably.

  In the Conclusion section, it is noted that in a 96-hour exposure of goldfish C. auratus to bioplastic and petroleum-based plastic particles (both untreated and UV-degraded), it was found that the particles originating from the commercial mesh (indicative of compost) PLA bioplastic cup are clearly more harmful than particles originating from bioplastic shopping bag and petroleum based polyamide. PLA particles cause significant oxidative damage to the brain and/or gills of goldfish.

1. Overall opinion

The manuscript may be of potential interest to researchers, biochemists, and ecologists, but needs thorough revision before publication to ensure better structure and flow. The text should be revised and organized. A detailed discussion of the results is lacking. More authors need to be cited to support the results obtained.

The English format needs revision regarding word forms and typos in the text.

2. Comments

The summary is too short. The summary does not contain sufficient information on the analytical methods used to determine lipid peroxidation levels in the brain and gills of fish. More data on the biodegradability of the different types of biodegradable plastics are also needed.

A more detailed discussion of the results related to levels of lipid peroxidation in golden carp exposed to pure plastic particles of all species and the application of UV-degraded counterparts is needed. The obtained results should be compared with more similar studies.

The conclusion is short and does not contain numerical data regarding the obtained values of the investigated indicators of the fish.

Figure 2 caption indicates number of brain and gill samples n=3. For what reason the number of samples is so small as it is not enough for good statistics!?

Position 7 of the references should align with the others.

Position 12 is fully underlined. The underline should be removed.

The references are missing justification on both sides and there is underlining in places that need to be removed.

References on lines 475, 602, and 606 are missing information about the specified document, such as pages, document link, etc.

The English format needs revision regarding word forms and typos in the text.

Author Response

We thank the reviewer for her time and comments. Our detailed responses are in the attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

In my opinion, it is a practical article but it needs to be considered the following comments before publishing.

The result in abstract part must be extended.

All abbreviations were defined when used for the first time in the manuscript.

Introduction: This work lack novelty to be considered as an original in this paper. At the end of the introduction, the necessity and novelty of the work must be expressed well. The manuscript does not illustrate great attention and activity in the field. The purpose of the study is not well expressed.

The environmental hazard of petroleum based plastics has been increasingly evaluated for many species (mostly aquatic) and at ------- [3-12]: avoid self-citations, better to provide original references, most recent one rather bunch of references

For example, bioplastic made from polylactic acid (PLA) accounted for 21% of global bioplastic production in 2022 and its production is expected to rise to 38% by 2027 (https://www.european bioplastics.org/market/). This is not proper, link should go in reference section, provide number of reference here only

The last paragraphs of the introduction just provide the purpose of the study without any references.

Add more instrumental characterization in the manuscript.

Materials and methods including experiments: Why and how the said parameters were selected for this work. More specific details are needed to be added with use of latest reference.

QA/QC information has to be included in manuscript.

Results: better to have some data in tables in this manuscript.

Good to have a separate section for the mechanism of the process

Future scope of this study can be added as well as social impact can also be discussed in this paper.

Conclusions: State main findings only

References are not unified and systematic.

Author Response

We thank the reviewer for her time and comments. Our detailed responses are in the attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

The manuscript is out of the scope of the journal

Author Response

We thank the reviewer for her time and comment. However we cannot agree to it. Our work is exactly within the topics of Journal.

Round 2

Reviewer 1 Report (New Reviewer)

water-2410307

Polylactic acid-based microplastic particles induced oxidative damage in brain and gills of goldfish Carrasius auratus

1. Overall opinion

The manuscript may be of potential interest to researchers, biochemists and ecologists, and has been revised by the authors for better structure and flow.

The discussion of the results is more detailed than the previous version. More authors are cited in support of obtained results.

The English format has been revised for the most part in terms of word forms and typographical errors in the text.

2. Comments

The abstract has been revised, adding information on analytical methods used to determine levels of lipid peroxidation in fish brain and gills. Also added data for

the biodegradability of different types of biodegradable plastics.

I understand that it is difficult to find such a study for comparison. The discussion has been improved.

Added the important information about the findings resulting from your research.

I understand that you adhere to the European ethical principle to minimize the use of vertebrate animals in scientific research (Directive 2010/63/EU) and the OECD protocol on fish numbers and replicates (7 fish per tank, no replicate).

Position 7 of the references is matched.

Removed highlighting at position 12.

The general appearance of the references has been corrected

For reference 602, the missing data has been added.

Reference 606 has been replaced by the appropriate reference.

The English format has been revised for the most part in terms of word forms and typographical errors in the text.

Reviewer 2 Report (New Reviewer)

The authors have made sufficient modifications, and I suggest that this paper be accepted without further modification.

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

Overall:

1. In this study, only 9 fish were cultured per treatment. The number of fish seemed to be a bit low.

2. The biomarker of this study was too single, and only TBARs were measured.

3. The liver is an important metabolic organ, but unfortunately the author did not test it.

4. The experimental period is only 96 hours, which seemed a bit short.

 

Title: PLA should provide the full name.

 

L149 Why not set 3 replicates. Biology experiments require at least 3 replicates for statistical analysis.

 

L158 When the fish had been euthanized, tissue samples should be taken quickly rather than frozen at -80 °C.

 

L183-189 Water temperature, pH, total dissolved solids and oxygen content should be described in the section of Materials and methods.

 

In Figure 3, 4 and 5, they are actually a repetition of the data in Figure 2.

 

L261-262 Is the adverse effects of PLA Cup caused by their relatively rigid edges or toxicity?

No.

Reviewer 2 Report

See attached file.

Comments for author File: Comments.pdf

English language is fine.

Reviewer 3 Report

The manuscript is not in the Journal's style, moreover, an English language revision should be performed.

The introduction section needs a huge improvement in its first part, better arguing the plastic/bioplastics part. In the present form, there is a disequilibrium with the biomarkers part, even from a reference point of view. References support and arguments are needed in focal points such as lines: 25; 28; 32; 44; 47; 50.

Moreover, even a small introduction to the study model should be provided.

From the material and methods section, lines 133-135 and 143-148 it is not clear if the plastics particles were counted before administration and if they were replaced with water exchanges. 

The results section does not show evidence of ingestion or contact of microplastics with model organisms, has this occurred? This is an essential point of the study. Moreover, considering the reported sedimentation of the plastics on the tank's bottom, the ingestion with consequences in the gastrointestinal tract would be the main goal of the study, that instead it was not considered. Effects on gills and the brain, without evidence of an interaction between plastics and fish organs, could result in a bias. Gut and liver should be considered, in this case, the study results are incomplete. Ehit this experimental design, you need a more appropriate study model such as a filtration crustacean species, just for example.

Consequently, the discussion section is so basic and also in this case poor of adequate references and comparisons. MPs' interactions with gills were too synthetically treated and no possible way of connecting with the brain come up from the arguing. Comparing your not standardized and in any case big-sized particles with 1-5 standard experimental microspheres is not scientifically valid.

 

Best regards

The Reviewer

 

 

 

Writing needs a moderate revision made by an expert.