Review of 'Plastic agriculture using worms: Augmenting polystyrene consumption and using frass for plant growth towards a zero-waste circular economy.'

Review

“Plastic agriculture using worms: Augmenting polystyrene consumption and using frass for plant growth towards a zero waste circular economy”

Darius Wen-Shuo Koh et al

Recommendation

Major revision required within 60 days or paper will be rejected.

Any revised paper will be subject to further review as it will be a substantially different paper.

Reasons for recommendation

Overview provided here. Detailed points provided in the attached copy of a pdf of the paper.

Overview:

I think the paper seeks to determine whether:

1. “worms” (actually insect larvae, as the authors acknowledge) can consume and breakdown a recalcitrant waste product – polystyrene.
2. Frass, from larvae fed on this material could, as part of a more circular economy, be used in plant production.

The paper is within scope of the journal in that in covers a number of disciplinary areas – waste management, sustainability (circular economy), biology (animal and plant), chemical analysis in a broadly ecotoxicological context.

The paper addresses an important topic and may represent an early study designed to help develop  more detailed studies in future (if so, this must be stated) but to be published it needs substantial revision in the areas set out below:

Throughout the paper attention needs to be paid to this general point: Such complex studies need a very clear set of aims and objectives and well-considered experimental designs and protocols if they are to succeed. The paper does not set out these aims, objectives, designs and protocols at all clearly and does not explain why, when such a rich set of information is potentially available from the studies, such a limited set of data is presented, very little of which has been subject to an appropriate statistical analysis. More detail is provided on the comments in the text of the paper which has been returned to the authors direct.

All the points mentioned here and on the text of the paper need to be addressed in the revision.

Methodology

The paper must include a rationale for and a description of the experimental design for both the studies on “worms” and those on cacti. These descriptions must include an outline of experimental conditions (for example the temperature at which studies were conducted) sufficient to enable the study to be reproduced by other workers, a description of the nature of any control and treatment samples, together with some information on the consistency with which treatments were applied (e.g. what media were used to rear the worms used in the study; and what time was allowed for this media to pass through the worms before the polystyrene study began so as to minimise its influence on the results; the weights of materials included in each group of worms; how many worms were used in each test sample; by how much did any of these factors vary; how any changes were measured – for example did this involve batches of worms or measures on individuals).

Although innovative use of materials for studies is welcomed, it seems odd to state that wine glasses were used to grow cacti without stating why this choice was made. Linked to this was a passing mention of an office environment. Were these studies not conducted in a laboratory equipped for plant growth studies? If not, then there may be questions about the suitability of the data derived from the cacti experiments. More information is required about how the cacti were raised as this could influence how they reacted in the experiment. For example, were they produced for the experiment in/on other growth media or were they grown on the experimental media only? Likewise, the sources of some of the materials would appear to be retail outlets. If so, information on how the authors ensured consistency between such commercial batches is required (such information is also relevant of course to materials purchased form more standard laboratory suppliers). This is important as there are many different kinds of bran for example.

Data presentation

Data presented in the Tables refers mostly to changes in variables in larvae or plants and, puzzlingly, there is little data about the way in which the larvae breakdown the polystyrene material. This is a major weakness. It is unclear how much polystyrene material was actually consumed and whether this led to simple physical fragmentation of the material or its real chemical breakdown to simpler chemical components. Simple fragmentation of the waste might not represent a real advance in the management of polystyrene waste. This is because merely breaking the waste into smaller physical components could just move issues from one part of the environment to another and create new risks and issues without really reducing previous ones.

Absolute and not just change data is required in the Tables if the reader is to be able to judge the value of the work (for example, data is needed on how much “worms” grew, how much cacti grew – just giving percentage change data is inadequate). It is important to establish how the larvae responded to consuming polystyrene in this study and how this was affected by the additions of other material (again how much other material, such as bran on sugar, was used needs to be stated clearly).

Data analysis has used a mixed selection of statistical tests and the choice of tests has not been justified. Some of the tests are demonstrably inappropriate. For example, a one sided T-test is employed when it is clear that the data show a two-sided test would have been appropriate. The use of p values is at least on one occasion inappropriate. Very surprisingly, there is little or no indication of the number of samples used in each of the experimental groups. Without this information the validity of the statistics cannot be judged and thus the validity of the studies.

The data on cacti growth seems to be presented in a partial fashion. Photographs are presented that are difficult to interpret without supporting quantitative data or at least some more descriptive information that a reader could interpret with respect to the qualitative information (e.g. on rooting).

The GCMS studies on the chemical composition of some elements of the various pieces of experimental work leaves a good deal of detail out and the choice of diagrams is set out without justification. No attempt seems to have been made to determine which of the chemicals listed in one of the supplementary tables is actually present in the samples analysed and no listing or other information is provided as to the differences between experimental control and treatment groups. This makes it very difficult for a reader to understand how the consumption of polystyrene by the worms might impact a circular economy waste cycle. If such an approach to managing polystyrene waste is to be effective, then the ability of the worms to break down the polystyrene chemically needs to be clearly demonstrated. It is not sufficient in such studies to simply present sample traces without making it clear how representative these are of the overall study results. If the worms are simple breaking down the polystyrene physically then it is important to know this. The GCMS studies could have provided vital information on this very important element of the study.

Discussion/interpretation of results

This is very limited and it is not clear what the authors believe their study has demonstrated and this arises because this complex study appears to have been undertaken with a number of aims in mind not all of which have been accounted for in the study design. A separate section of the paper is required that interprets the results of these studies. Several of the result appear to be “negative” in nature and whilst the presentation of such results is welcome it is important that their meaning for the circular economy potential of worms and polystyrene waste processing is explained.

Detailed points per line

Line 138 - How were additives removed from PS before weighing?

Line 139 - Mortality rate required. There will be missing data if this information is not included.

Line 142 - Were these therefore just the control samples that were used? If so why were the samples from treatments that had additives not investigated?

Line 143 - Why is this reference made to an office environment. Were these studies not conducted in a laboratory with a controlled environment?

Line 144 - Can the authors explain why wineglasses were used to grow cacti?

Line 149 - The last sentence seems to contradict the first sentence. Please clarify

Line 152 - What is a technical replicate?

Line 160 - What does this mean? The extent to which samples were diluted at this stage could have a substantial effect on the results.

Line 163 - Please say why this was done.

Line 166 - Please say why this temperature pattern was used.

Line 170 - If other additives were used please say which ones. It is otherwise not clear because of the phrase "such as".

Line 176 - How much is “small amounts"? This is important to know in the interests of reproducibility.

Line 180 - What does "outperform" mean? Does this refer to PS consumption or to some other characteristics of the worms?

Line 181 - Is the paper then testing the efficacy of additives or the fact that these insect larvae eat PS? This is not clear in the paper from this section.

Line 182 - when were worms weighed? Please indicate when this happened in the experimental protocol.

Line 184 - The experimental design used in the study design is very unclear. Some basic details are needed. Some of what is required is suggested in the overview of this review.

Line 191 – Line 192 - What type of bran was used (for example, the plant the bran was derived from needs to be stated)? Was it the same batch throughout the studies? Did the bran used for the cacti differ from the bran used for the larvae?

Line 193 - Again, because the experimental design Lacks sufficient detail it is difficult to interpret these results. Much greater clarity is required on, say, the total numbers of cacti used in the experiment so that the reader can understand how clear (or not) the result is.

Fig 1: shows that the authors had access to very rich sources of information, such as the growth rates of larvae (length and weight data), rates of consumption of polystyrene, generation of frass, various aspects of plant growth etc. It is a pity that the opportunity that such a rich source of data provided has not been exploited fully. Also an opportunity seems to have been missed to properly determine whether the larvae have actually simple broken the polystyrene down physically (basically they have simply ground the material into smaller units) or whether the larvae have actually decomposed the polystyrene molecules into simpler chemical components. The GCMS data, as presented, fails to do this.

Fig 2: shows the results from this study are very variable. Many reviewers would say the statistical analysis is wrong. With this kind of experimental design the best statistical analysis is some form of analysis of variance especially as the underlying variability in these results is very high. A p value of 0.1 is of little or no interest if it arises from a one-sided T-test especially when the choice of a one-sided test is inappropriate as the results themselves demonstrate. One-sided statistical tests should be used with great caution and cannot really ever be used in circumstances where responses could be higher or lower than controls. The use of a one-sided test needs a great deal of justification. If a two sided test were use these result might not be significant in the normal sense of that word.

Fig 4: What is a “technical” replicate? What does technical mean in this sense? Please explain this term. It is clear some rooting occurred in B (right photo) was this detail recorded or not?

Fig 5: Because the experimental design of the study is unclear the source of the samples subject to GCMS analysis is unclear. More information on the samples used for GCMS analysis is needed.

Table S1: This table should not be in supplementary information. The data here suggest that only in one instance did worms gain weight. Weight loss occurred in all other groups suggesting the worms were not thriving. Is this not a problem for their use in a circular economy?

Table S2: The data in this table cannot be judged by a reviewer unless absolute heights are provided. Just supplying change data is insufficient. Other tables have the same problem. The table seems to suggest that the only significant result is from the group where most cacti died. This factor needs accounting for in some additional manner. Also, the Table is difficult to interpret as it is not possible to use a growth media that is a control and a treatment in the same analysis. Were any cacti grown on a standard growth medium? This could have been a real control.

Table S3: This table should not be in supplementary information and again is in any case inadequate as the authors do not indicate in which samples these chemicals were found.