Biodegradability of Disposable Surgical Face Masks Littered into Soil Systems during the COVID 19 Pandemic—A First Approach Using Microcosms
Round 1
Reviewer 1 Report
Dear Authors, I would like to congratulate you for selecting the most current topic possible and for analytical and statistical job you have done. Applied methods are very well chosen and sophisticated. You have found very good publications to discuss your results. As a Reviewer I found just one very small mistake line 347 after abbreviation sp dot should stand. As non-native English speaker I cannot evaluate the level of English language but text I well readable, tables and figures are very clear and I cannot say anything else that I got excellent manuscript to review and I accept publication as it is.
Author Response
Dear Editor, dear Reviewer:
Thanks a lot for your comments and your judgement. The small error in line 347 was changed according to the suggestion
Reviewer 2 Report
The research content is impressive and associated with realistic problems which are prevailing in this current scenario. The quality of the paper is good and the overall interpretation is well managed. Furthermore, this is the initial study in-depth study is required to gain an insight into the research.
Author Response
Dear Reviewer, thanks a lot for your comment which is greatly appreciated.
Reviewer 3 Report
The manuscript describes and experiment in which soil and soil-mask component mixtures were prepared and the production of CO2 measured over 7, 31 and 178 days. NMR spectral measurements have also been carried out to determine whether any structural changes have occurred in this incubation time. Considering the COVID 19 pandemic and the use of masks throughout, the results of the experiments with respect to mask biodegradability are of interest.
From a language perspective, the manuscript would benefit from further proof reading and also contains some awkward phrasing. The introduction sets the scene well, but for instance, lines 47-52 has no citations and is not well written.
From an experimental perspective I would like to see the methods section be significantly improved, in particular lines 130 -157 I found difficult to follow in terms of what was being added to each microcosm particularly around the addition of organic carbon to each vessel.
- It is unclear in the test microcosms what is used as a liquid source, as distilled water is used in the controls?
- What is MOC? this is undefined.
- How was OC of the inner/outer/combined sections made the same?
- I would like to see perhaps a supplementary or main figure which shows the experimental set up, and perhaps the narrative of the methods would be improved through being written more systematically.
- Why was 25degC chosen? is this a typical soil temperature.
Although I appreciate the authors cannot go back and repeat experiments, there are European standard test methods for assessing biodegradability which include specific mineral media compositions, it is unclear from the manuscript where the organisms in the biodegradation are obtaining other key nutrients such as phosphorus and other trace elements. If these are from the soil, I appreciate that a reference is provided for the soil but again perhaps a supplementary table with this information. The concerns around the experimental set up contribute to my concerns/misunderstandings around the results.
Based on the results obtained in which a significant increase in C consumption is observed, if the test experiments have had water added to them and additional organic carbon, then how to do the authors know that the masks are being degraded and that the additional carbon consumption is not due to the promotion of biofilms on the surface of the mask material? The mask may be acting as a surface for biofilm formation, where there is evidence to suggest that organisms existing in a biofilm exhibit greater degradation rates than planktonic cells. The authors state that there were slight changes to the NMR signals but could this be due to the formation of biofilm along the fibres? Understanding total carbon in the microcosms is imperative, as there was no difference seen in the individual mask layers and the complete mask in terms of CO2 output, which may further suggest that biofilms are increasing C degradation rather than the mask acting as a C source itself. There is insufficient explanation of the methods or consideration of the results for me to agree with the authors that the mask itself is degrading.
In terms of the visual differences discussed, do the authors still have the samples to provide images?
From a results perspective, the paper has lots of tables, could the authors perhaps combine these into a more substantial figure, or at least in some cases consider alternative displays?
Overall, I think the topic of the article is interesting since there is a significant volume of mask bearing waste already produced, and in the process of being created. It may be that my concerns can be explained through a better description of the methodology but in its present form it is unclear whether the results obtained are true.
.
Author Response
First I want to thank the reviewer for the helpful comments and careful reading of the manuscript. Please find below the answers to the comments:
Line 47-52: This estimation is not from the literature. It was done by us using the numbers of Prat et al and Benson et al. The weight of the masks was measured in our laboratory. Accordingly we changed the sentence to:
Using the numbers of Prata et al. and Benson et al. [3,4] the following rough estimation can be done. If during the peak times of the pandemic just 1% of the discarded single use face masks with an average weight of 4 g (measured in our laboratory) had been littered improperly to the soil, an additional 136 tons of plastic waste would have entered the soil environment per day.
Reviewer:
From an experimental perspective I would like to see the methods section be significantly improved, in particular lines 130 -157 I found difficult to follow in terms of what was being added to each microcosm particularly around the addition of organic carbon to each vessel.
- It is unclear in the test microcosms what is used as a liquid source, as distilled water is used in the controls?
Answer: The soils were humified with aqua dest to a soil humidity of 75% of the maximal water retention capacity of the soil. This value was determined prior to the experiment. It is a very common approach in soil incubation experiments. The control was a humified soil without any addition of mask material (as described in line 148: ..As a control, beakers were filled with only 20g of soil without mask material). In addition to the soil incubates, vessels were filled with 10 ml destilled water and 1 ml inoculated water but without soil (line 168). Thus these vessles were not control but additional. For a better understanding, we included a graphical scheme of the experimental design (Figure 2).
Thus, in general there was no liquid media used in this experiment (except the above mentioned extra vessels)
Reviewer:
- What is MOC? this is undefined.
Answer: This is the organic C contained by the masks (determined by elemental analysis). Now we include ((mask organic carbon: MOC)
Reviewer:
- How was OC of the inner/outer/combined sections made the same?
Answer: We added the following explanation: The carbon concentration of the mask was determined via elemental analysis and was the same for each layer. Therefore, addition of 250 mg of mask pieces with a C content of 860 mg g-1 added another 215 mg of organic C (mask organic carbon: MOC) ......
Reviewer:
- I would like to see perhaps a supplementary or main figure which shows the experimental set up, and perhaps the narrative of the methods would be improved through being written more systematically.
Answer: a scheme, explaining the experimental approach is already given with the graphical abstract. However, a more detailed scheme is now added (Figure 2)
Reviewer:
- Why was 25degC chosen? is this a typical soil temperature.
Answer: No, soil temperatures vary as does the outside temperature. We used 25C since this is commonly used as standard room temperature in laboratory experiments.
Reviewer:
Although.... it is unclear from the manuscript where the organisms in the biodegradation are obtaining other key nutrients such as phosphorus and other trace elements. If these are from the soil, I appreciate that a reference is provided for the soil but again perhaps a supplementary table with this information......
Answer: We agree that for future experiments a more detailed analysis of the nutrients of the soil may be helpful for optimizing the degradation of mask material. However, the present study was performed as a first approach to see if masks are degraded in soils. After the positive feedback, we started more detailed studies on the identification of the microorganism and a future study will approach the nutrient availabilty. However, for the present experiment we do not have the P data of the soil.
Reviewer:
Based on the results obtained in which a significant increase in C consumption is observed, if the test experiments have had water added to them and additional organic carbon, then how to do the authors know that the masks are being degraded and that the additional carbon consumption is not due to the promotion of biofilms on the surface of the mask material?
Answer: The formation of biofilms on the mask is very likely - either on the mask material or on soil material... However, the respective microorganism still must have a C-source to be formed and to survive. What we found is that more CO2 was formed. The respective C must come either from the soil organic matter or from the organic C of the mask (inorganic carbonate was not present). The NMR studies showed that no preferential degradation of any organic group of the soil organic matter occurred which allows the conclusion that the metabolization of the soil organic material was not affected by the mask material or the additional microorganism. As a consequence, the additional CO2 must derive from the degradation of the organic C of the mask. I am sure (but do not have a proof) that the formation of a biofilm helped with the degradation of this MOC. Actually, in a present study, we could observe a kind of biofilm on mask material that was incubated only with a nutrient solution.
Reviewer:
The authors state that there were slight changes to the NMR signals but could this be due to the formation of biofilm along the fibres?
Answer: Microorganism have a defined composition of their cell material. This comprises lipids, proteins, carbohydrates etc. Their C results in a typical NMR spectrum with signals of each C with an intensity which is proportional to the concentration of the respective C in the samples. Thus, if the amount of microorganism forming a biofilm on the fibres is high enough to affect the chemical composition of the sample, NMR signals of carboxylic C, O-alkyl C and N-alkyl C should also be visible. The missing of these signals indicate that the contribution of microbial biomass to the fibres is > 1% and the changes of the intensity distribution is due to chemical changes of the fibres.
Reviewer:
Understanding total carbon in the microcosms is imperative, as there was no difference seen in the individual mask layers and the complete mask in terms of CO2 output, which may further suggest that biofilms are increasing C degradation rather than the mask acting as a C source itself. There is insufficient explanation of the methods or consideration of the results for me to agree with the authors that the mask itself is degrading.
Answer: As mentioned above, the only sources of C is the soil organic C and the mask C. There is no other C source which can be used by the microorganism for degradation. Here it doesn`t matter if the microorganism are single or act as biofilm. They must feed on something to produce a stoichiometric amount of CO2.
The reviewer state that biofilms increase the degradation. Which is true but this more of degradation must have a C-source, which in our approach is either soil organic matter or mask organic matter. There is no other C-source. Here it should also be noted that the different components of soil organic matter have different degration rates - some are easily degraded (carbohydrates, hemicellulose, amino acids etc.) others (lignin, some lipids etc) are more difficult to be microbiologically attacked. This would lead to a selective enrichment of material that needs more effort to be degraded (i.e. lignin) and thus to a change in the intensity distribution of the respective NMR spectra. The latter we did not observe.
Reviewer:
In terms of the visual differences discussed, do the authors still have the samples to provide images?
Answer: We separated the masks from the soil for the NMR measurement. For homogenization we had to tear the fibers appart before filling them into the NMR rotor. However, this was easier with the central mask after 6 months of incubation. Accordingly, the following sentence was included (286)
....After 6 months, on the other hand, in particular the center masks showed clear signs of physical deterioration, which was also indicated by the fact that it was more easy to tear the fibers apart with tweezers than the original mask material. ....
Reviewer:
From a results perspective, the paper has lots of tables, could the authors perhaps combine these into a more substantial figure, or at least in some cases consider alternative displays?
Answer: We had already thought about it but realized that combining the tables makes the paper more confusing and difficult to follow. Therefore, we prefer to leave it how it is.
Reviewer:
Overall, I think the topic of the article is interesting since there is a significant volume of mask bearing waste already produced, and in the process of being created. It may be that my concerns can be explained through a better description of the methodology but in its present form it is unclear whether the results obtained are true.
Answer: Thanks for this encouraging comment. I think a main problem of understanding is the fact that the experiment was performed in soil substrate and not in a nutrient solution. We hope that the additional scheme in Figure 2 could clearify some of the doubts.
Round 2
Reviewer 3 Report
The methods section and inclusion of figures significantly improves the level of understanding of the manuscript and how experiments were set up.
Figure 3- NMR spectra, the 100-200 ppm region is missing from the fresh mask layer, this makes this figure difficult to compare, in addition, this figure very much suggests that nothing has happened to that masks in that the structure is still very much intact, i.e what is its value to the main finding of ‘masks were degraded’ the NMR shows that the mask that was left, was still the same. It may be simply that this figure is removed as it does not support the findings or provide any value to the manuscript in its current state. If anything they NMR data indicates how difficult PP is to biodegrade.
At least some of the tables could be presented in alternative formats (x,y scatter). If the tables are to retained, a figure comparing and contrasting the at least the most pertinent facets of the mask layers would significantly improve the overall understanding with the manuscript. In its present form, it is laborious to read.,
Line 275. No evidence is supplied to support this data. The masks ‘sticking together’ with soil does not constitute evidence of degradation. Figure 1 is a prime example of this where in 18months at oct21, there is almost no evidence of degradation that can be seen visually.
Overall the manuscript could do with a thorough proof reading for English language as it feels conversational in places.
.Lines 47-52 (now 52-54) are still poorly written.
Line 176 lid? or lit
Line 270, 'up by 24% and added up to 53 to 58mg' this sentence needs revising as it doesn't make sense? An increase of 24% from 53 would not be 58, or do you mean that there was an increase, and that increase ranged from 53 - 58mg? if the latter I would also expect to see some consideration of error
Line 271. Delete the word hardly
Author Response
Dear Reviewer: Thanks for the comments. Most of the doubts you expressed seem to be due to a misunderstanding of the NMR technique or how the results should be interpreted. We hope that we were able to clarify some of the doubts in the following explanations.
Figure 3- NMR spectra, the 100-200 ppm region is missing from the fresh mask layer, this makes this figure difficult to compare....
Answer: Polypropylene gives only three NMR resonance lines and in the field of NMR spectroscopy it is usual to limit the size of the spectra according to the information which can be obtained. If I present the total spectrum, the chemical shift range would be from 1000 to -1000 ppm, which does not make a lot of sence.
In order to be able to compare the spectra, the chemical shifts of the different peaks are indicated on the top of the respective signal. I am not sure but it may have been misunderstood that in 13C NMR spectroscopy, the unit ppm corresponds to the chemical shift of the resonance line which is specific for each 13C in a molecule. In contrast to mass spectrometry, a signal DOES NOT correspond to a complete molecule but only to the specific carbon. The intensity - thus the hight of the signal - is the relative proportion of this C to the total C of the sample. However, for clarification I introduced the following sentence in the Figure description:
...The chemical shift of the different resonance lines are indicated by numbers above the signal. No signals were expected from polypropylene nor were identified at chemical shifts downfield of 100 ppm. Therefore, only the region between 0 and 100 ppm is shown. However, since degradation may lead to carboxyl C giving rise to resonance lines in the region between 225 and 160 ppm, the spectra of the degraded masks are the downfield scale was expanded to 250 ppm, although no additional signals were detectable in this region.
Reviewer:
...in addition, this figure very much suggests that nothing has happened to that masks in that the structure is still very much intact, i.e what is its value to the main finding of ‘masks were degraded’ the NMR shows that the mask that was left, was still the same. It may be simply that this figure is removed as it does not support the findings or provide any value to the manuscript in its current state. If anything they NMR data indicates how difficult PP is to biodegrade.
Answer: Here the reviewer is completely wrong. Figure 3 has to be seen together with Table 1 showing that the three signals change their relative intensity, thus their contribution to the total C of the sample. As explained in the text, this leads to a decrease of the relative contribution of -CH- with a concomitant relative increase of -CH3. This is best explained with an shorten of the polypropylene chains. The only way how this can occur is by degradation. From this point of view, Figure 3 together with Table 1 are extremly important for demonstrating our results.
Reviewer:
At least some of the tables could be presented in alternative formats (x,y scatter). If the tables are to retained, a figure comparing and contrasting the at least the most pertinent facets of the mask layers would significantly improve the overall understanding with the manuscript. In its present form, it is laborious to read.,
Answer: I am not sure, but I have the impression that reviewer 3 did misunderstand the nature of NMR data. None of the tables can be presented as x,y graphs since they are not functions but integrations of NMR signals (Table 1), NMR relaxation times (Table 2) and respective claculations using those numbers. There is no x value nor is there a y value. Comparably, Table 3,4 give single values, none of which can be presented as a simple graph. Trying to transfer those data to a figure will make the reading even more complicated and would not allow to present for example results of the statistical analysis. Therefore, since no improvement can be expected with the suggestion and for clarity reason it is more appropriate to leave the tables as they are.
Reviewer:
Line 275. No evidence is supplied to support this data. The masks ‘sticking together’ with soil does not constitute evidence of degradation. Figure 1 is a prime example of this where in 18months at oct21, there is almost no evidence of degradation that can be seen visually.
Answer: I am a bit astonished about those comments. Looking at Figure 1c) which is the picture taken Oct 22, 21, there is a clear degradation indicated. There are holes in the mask and residues of the inner and center mask are visible. The latter can only be if the blue mask has suffered degradation. The soil is clearly visible as well as plant material is mixed in with the mask material. Please note that this photo was NOT taken 18 months after the first picture but only 10 months latter. Please note further that in figure 1d, almost all of the mask material is gone. Only a thin lateral part stayed.
The second reasoning of the reviewer, that sticking of the mask to the soil material does not provide evidence for degradation is completely correct. It only indicates that there are some physical interactions between the soil and the mask material. We never claimed that this is an indication for degradation.
See our wording in line 281:--- but most of the cuts stuck together with the soil material suggesting that at least some interactions (now included: between mask and soil material) occurred.
I am not completely sure what is meant by refering to line 275. I assume that the reviewer does not believe that the mask addition to the soil did increase the CO2 production. However, if organic materials are biodegraded, its C is converted into CO2. Thus, only more CO2 is produced if more organic material has been converted. Now, if two samples have the same amount of soil organic matter, both will produce the same amount of CO2. If one sample has an additional input of organic matter - in our case the mask - and produces more CO2, the only source of this additional CO2 can be the additional mask.
In our case, the first sample without mask produced 47 mg released C (corresponds to 171 mg CO2). The sample with soil and mask produced 53mg (complete mask) and 58 mg (single mask layers) (corresponds to 194 to 212 mg CO2) which is with 6 to 11 mg C definitively more than in the only soil sample. Thus there is no other conclusion that this additional CO2-C derives from the added masks. If 47 mg CO2-C are 100% of the C-loss of the only soil samples, the additional 6 to 11 mg correspond to 11 to 24% of the total loss of the only soil sample which is too high for being explained with a priming effect.
Reviewer: Improvement of English
Answer: A native English colleague had a closer look on the manuscript and had some minor suggestions for improving the English. The respective suggestions were performed and are indicated in the text.
Reviewer:
Lines 47-52 (now 52-54) are still poorly written.
Answer: We changed the part to:
...As such, they jeopardize the health of waste collectors or litter pickers, but also members of the public. In particular playing children who first come across the litter can be endangered. Animals which unintentional feed on their residues may become malnourished as their stomachs are filled up with plastic residues without providing nutrition....
Reviewer:
..Line 176 lid? or lit
Answer: changed to lid
Reviewer:
Line 270, 'up by 24% and added up to 53 to 58mg' this sentence needs revising as it doesn't make sense? An increase of 24% from 53 would not be 58, or do you mean that there was an increase, and that increase ranged from 53 - 58mg? if the latter I would also expect to see some consideration of error
Answer: We did not express it well. The numbers included the values for the complete masks, which was not indicated in the text. To avoid this we changed the sentence as follows:
...Addition of mask cuts of the single layers to the soil significantly increased CO2-C release after 1 month by 24% and added up to 58 mg. s....
The respective standard deviations are given in Table 3.
Reviewer:
Line 271. Delete the word hardly
Answer: We changed the word hardly to not. Removing the word hardly would change the meaning of the sentence
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
Polypropylene is chemically very stable, and so are other olefin polymers. For this reason, strong nitric and sulforic acids are stored in polyolefin bottles. Since PET is a polymer made by condensation polymerization, there is a high possibility of decomposition by hydrolysis. But biodegradation of polyolefins can take thousands of years. However, the authors saw a reduction of several percent for the biodegradation of pp.
Among polyolefins, it has been reported that LDPE is much more biodegradable to a few micrograms than HDPE. Even HDPE, which is relatively more stable than LDPE, has a much slower rate of degradation. PP is thermally and chemically superior to HDPE (higher crystallinity and stronger intermolecular bonding). Therefore, there is much less literature on PP biodegradation. However, the biodegradation rate of PP observed by the authors is too fast. It is very difficult to observe several percent pp biodegradation. It is comparable to PLA, a biodegradable plastic, which have relatively slow biodegrdation (needing composting condition) among biodegrdable plastics
From the co2 generation data, we cannot be sure of biodegradation for mask (PP). Because of the "priming effect". This is because the added pp can accelerate the decomposition rate of soil som.
Author Response
We are aware that PP represents a polymer that is not easily degraded. However, it is well known that it is not UV-resistant and as such it is likely that it is at least desintegraded during its exposure to sunlight. In order to show that this is possible we included photos obtained from a mask found on a fallow and "monitored" during its natural degradation. After one year not much of the mask remained. However, the question whether the remains were transported into the close by ant nest, mixed with the soil or transported by other means to other places cannot be answered with the information we have.
On the other hand, the controlled conditions of our microcosm allow the conclusion that microbial degradation can occur. As we have stated in the introduction, we are not the first and only scientist that were able to show that PP can be degraded. Please have a closer look at the papers of Arkatkar et al., or Jeon and Kim. Thus, I think the conclusion of the reviewer that PP cannot be degraded by microorganism is a bit like "it cannot- be thus it is not". The NMR spectra show that chemical alterations take place, thus chemical reworking of the mask material must have occurred during incubation. Purely chemical or physical processes are unlikely since this - as the reviewer states - requires drastic treatment. This was not applied.
With respect to priming. Well, the amount of additional CO2 which was produced in the microcosm of the soil/mask mixture is too high to be solely explainable by priming. And priming cannot explain the chemical changes of the face mask. This effect would only change the chemical nature of the native soil organic matter.
With respect to the degradation rate. Here it has to be born in mind that the experiment was performed under "optimal" conditions with respect to temperature or moisture. I would not expect the same MRTs under natural conditions. However, the 6 to 10% C loss of the mask observed in our study are still smaller as the 13 to 20% observed by Jeon and Kim for the degradation of high and low molecular weight PP by Stenotrophomonas panacihumi. Thus, our degradation efficiency is even lower as that reported already in the literature. I think, if several authors have observed microbial degradation of PP, there is no reason why masks should be resistant to such processes. I think our results clearly show that much more research is needed to understand the fate of plastics in soils. In addition it has to be born in mind that PP, PE or PET are chemically different.
Reviewer 2 Report
The manuscript is a timely contribution that is probably going to create a lot of public interest, but also scrutiny. Therefore, it is important to avoid simplifications. The general message is important and relevant and, although this can only be a first step in deciphering the consequences of the effect on surgical PP masks in soil, it definitively deserves to be published. The fact that the CO2 release from the mask cuts mixed into soil is higher than from the two individual components is definitively interesting.
I have two concerns about the manuscript:
Are you sure that NMR spectroscopy is an adequate method to detect changes in the composition of the masks? If so, please explain the specific advantages of the method that make it superior to other methods as FTIR spectroscopy. This is especially important as no large changes were detected using NMR spectroscopy.
Is it possible relate the shifts in chemical composition / functional groups to CO2 evolution?
L 66-68: Isn’t it far-fetched to expect masks to be worked into the subsoil? Maybe you could describe the expected pathways of mask incorporation into the soil? In case you agree that this is far-fetched, how about deleting this passage. The incubation took place in topsoil.
L 97: Isn’t it more realistic to assume an effect comparable to plastic, not microplastic?
L 120-121: I understand that cutting the mask into pieces is required for such an experiment. But exactly this step, the fragmentation of the mask, is unlikely in real soils as no soil fauna that would do this exists.
L 269: This should be Fig. 2, not 1.
Author Response
Answers to the comments of Reviewer 2:
First we want to thank the reviewer for carefully reading the manuscript and the suggestions to improve the manuscript:
Considering that both reviewers were concerned about the degradability of face mask material under natural conditions, we included photos obtained by chance from a unintentionally deposited (most likely lost during walking) mask on a fallow close to the house of the first author. These photos were taken during a time frame of about 1 year and clearly show that desintegration of mask material is possible under natural conditions and that at least a part of the remains are incoporated into the soil. With this photos we hope we can answer some of the questions of the reviewer which most likely are also concerns of some readers of the journal.
reviewer:
Are you sure that NMR spectroscopy is an adequate method to detect changes in the composition of the masks? If so, please explain the specific advantages of the method that make it superior to other methods as FTIR spectroscopy. This is especially important as no large changes were detected using NMR spectroscopy.
Answer:
Yes, we are sure that solid-state NMR is an adequate technique for the analysis of chemical alterations of mask material during degradation. If the correct acquistion parameters are used, this technique is quantitative. A big advantage is that it is non-degradative, thus no extractions that could alter the chemistry are necessary.
The critical acquistition parameters are the contact time and T1H. Both parameters were determined prior to analysis and correctly adjusted. Thus, the obtained NMR data can be considered to be quantitative.
With respect to IR spectroscopy, I would not say that NMR is superior in general. It is another technique. However, the big advantage of NMR is that it is quantitative and a spectrum reflects an intensity distribution which is proportional to the distribution of different spins (here carbons) in the sample. For more information about advantages and disadvantages of solid-state NMR in soil sciences a reference is included (Knicker, 2011)
