Assessment of the suitability of standard soil assays to study the biodegradation of polymer microparticles.

Biodegradable polymers are increasingly suggested as alternatives for conventional polymers, particularly in agricultural applications where soil is the end-of-life environment. Complete environmental biodegradation is required to ensure that persistent micro and nano plastics do not form, hence reproducible and accurate standard tests for measuring biodegradation are critical. Polymer biodegradation in the environment is measured using standard test methods, with the International Organisation for Standardisation (ISO) test guideline ISO 17556:2019 being applicable for the soil environment. The respirometric endpoints of biological oxygen demand and carbon dioxide (CO2) evolution are used to indirectly measure polymer biodegradation. However, the suitability of respirometric endpoints for measuring polymer biodegradation is uncertain because they were developed using the principles of small molecule biodegradation and lack extensive testing on high molecular weight, insoluble polymers. Further, respirometric endpoints only measure the fraction of a polymer that is completely mineralised, and do not provide information on the environmental fate of the non-mineralised polymer fraction which includes the bioassimilation of the polymer by soil microbes or residual polymer that persists in the soil. The non-mineralised polymer fraction is important to determine, as there are concerns that incomplete biodegradation could lead to persistent micro and nano plastic pollution, with potentially negative impacts for soils. This research investigated the suitability of respirometric tests to measure the biodegradation of the polymer microcrystalline cellulose in both natural and standard (i.e. artificial) soils and identified the potential causes of poor reproducibility within the ISO 17556:2019 test method. Additionally, the suitability of soil microplastic extraction methods to identify non-mineralised residual polymers, were investigated, to be used alongside the respirometric methods to develop a greater understanding of the environmental fate of polymers. The reproducibility of the standard test was found to be poor, both between soils and between respirometric measurement endpoints. Significant variation within key soil parameters such as soil water content and nutrient availability are permitted, which contributes to widely varying test conditions. Thus, a key recommendation is that reducing the acceptable range of variability for test parameters would likely improve test reproducibility. Additionally, the use of respirometric endpoints means that the measurement of oxygen demand or evolved CO2 cannot be directly attributed to the polymer. Soil microplastic extraction methods were considered as a potential method to identify residual polymers but were found to induce degradation in the biodegradable polymers polylactic acid (PLA) and polyhydroxybutyrate (PHB), therefore this was not considered an appropriate method for further identification of the residual polymer. Pyrolysis gas chromatography-mass spectrometry (Py-GC-MS) was also investigated and shown to be a useful analytical method for directly identifying polymers in soil, that could provide insights into the degradation mechanisms of polymers. This work also highlighted how additional analysis alongside respirometric techniques can provide insights regarding the assumptions of respirometric methods. For example, Py-GC-MS analysis showed no evidence of PLA degradation, despite an increase in measured CO2, which provides evidence to suggest that part of the measured CO2 can be soil derived whereas respirometric techniques assume all measured CO2 is due to polymer biodegradation. Overall, it is important to continually develop methodologies that can accurately determine the environmental fate of biodegradable polymers to ensure better regulation and minimise environmental risk.