Fluorescence microscopy utilising Nile Red staining was applied and tested to analyse microplastic particles in soil. Given a large number of analytical methods for measuring plastic particles in environmental and biological matrices, often utilising microscopy/spectroscopy approaches, efforts were made to validate this method for a range of soils and different microplastic (MP) particle types (varying size and morphology).  Eight MP types (including both non-biodegradable and biodegradable plastics) within three size categories (dia. 500-1000 µm, 100-250 µm and 10-150 µm) were spiked into three different agricultural soils (loam-reference soil, clay, and sandy soils) for a comprehensive assessment of the fluorescence microscopy methodology. Each soil (with replicates) was subject to digestion, density separation and filtration (with Nile Red staining) prior to analysis using fluorescence microscopy (GFP filter set, excitation/emission 470/525 nm) and bright filed microscopy for black microplastics. As a tool to shorten the analysis time, a digital image analysis pipeline using Image J was developed, allowing fully automated particle recognition and quantification of MPs in the samples. The main steps for image analysis, including background correction, fluorescent intensity thresholding, watershed segmentation and particle analysis, were optimised, the validation of which showed high accuracy (88% match to true observation) for MP particles on a filter without a soil matrix. To avoid false positive results due to the presence of natural organic particles from the soil matrix, the numbers of microplastics recovered from spiked samples were corrected with those found in the non-spiked soils. Recoveries ranged from 80-90% for MP with sizes from 500-1000 µm regardless of the soil types, whereas those for smaller MP (10-250 µm) varied between different soils and plastic types (e.g., recovery for low-density polyethylene, LDPE, from sand and loam-reference soil were 85% and 90% respectively whereas those for polybutylene adipate terephthalate/polylactic acid, PBAT/PLA, were 60% and 10% respectively). The lowest recovery rates were observed in clayey soil (20% for LDPE and 5% for biodegradable plastics PBAT/PLA). A relationship between the microplastic mass (LDPE and PBAT/PLA fragments) and the corresponding particle number was established in this study, which enabled the conversion between mass and particle number data. Fluorescence microscopy with Nile Red staining and automatic particle recognition software provides a relatively reproducible and accurate technique for plastic particle counts in heterogeneous matrices like soil. Still, selectivity for different polymer types is clearly limited. 

How to cite: Phan Le, Q. N., Halsall, C., Peneva, S., Wridley, O., Amelung, W., Braun, M., Quinton, J., and Surridge, B.: Towards quality-assured measurements of microplastics in soils using fluorescence microscopy, EGU General Assembly 2023, Vienna, Austria, 23–28 Apr 2023, EGU23-13873, https://doi.org/10.5194/egusphere-egu23-13873, 2023.

In Europe, about 0.71 million tonnes of agricultural plastic were intentionally used in 2019. Most widely used were plastic films (about 75%), which are dominated by light density polyethylene (LDPE). Especially LDPE plastic films for mulching covers in direct contact arable soil to increase temperature and reduce evaporation. Thereby, microplastic is detached from the mulch film via mechanical and environmental weathering. Another microplastic pathway in arable soil is the application of sewage sludge. Depending on land use, a 4 to 23 times higher microplastic contamination in soils than in the sea is estimated. Obviously, microplastic input to soils is critically high, but an accurate quantification is still lacking. This is partly caused by challenges in detection and analysis of microplastic in soils. First, it is challenging to extract microplastic from a matrix of organic and inorganic particles of similar size. Second, the well-established spectroscopic methods (e.g., Raman and FTIR) for detecting microplastics in water samples are sensitive to soil organic matter, and they are very time-consuming. Eliminating very stable organic particles (e.g., lignin) from soil samples without affecting the microplastic to be measured is another challenge. Hence, a robust analytical approach to detect microplastic in soils is needed. In this context, we developed a methodological approach that is based on a high-throughput (25 g soil sample) density separation scheme for measurements in a 3D Laser Scanning Confocal Microscope (Keyence VK-X1000, Japan) and subsequently using a Machine-Learning algorithm to classify and analyze microplastic in soil samples. Our aim is to develop a method for a fast screening of microplastic particle numbers in soils while avoiding the use of harmful substances (e.g., ZnCl₂) or prolonged organic carbon destruction. For method development, we contaminate a standard soil (LUFA type 2.1 - sand: 86.6% sand, 9.7% silt, 3.7% clay, 0.58% organic carbon; and LUFA type 2.2 - loamy sand: 72.6% sand, 16.8% silt, 10.7% clay, 1.72% organic carbon) with different concentrations of transparent LDPE microplastic (< 700 µm), LDPE microplastic originating from black mulch film (< 400 µm) and microplastic originating from Bio-degraded black mulch film (< 250 µm). For density separation, three non-toxic, easy to handle mediums were compared for the best microplastic output: distilled water (ρ = 1.0 g/cm³), 26% NaCl solution (ρ = 1.2 g/cm³), and 41% CaCl₂ solution (ρ = 1.4 g/cm³). The separated microplastic plus organic particles and some small mineral particles were scanned using a 3D Laser Scanning Confocal Microscope. For each sample, the 3D Laser Scanning Confocal Microscope generates three different main outputs: color, laser intensity, and surface characteristics. Based on these data outputs, a Machine-Learning algorithm distinguishes between the mineral, organic, and microplastic particles. It was found that color changes of microplastics due to soil contact challenge the classification but can be compensated by surface characteristics that become an essential input parameter for the detection. The presented methodological approach provides an accurate and high-throughput microplastic assessment in soil systems, which is critically needed to understand the boundaries of sustainable plastic application in agriculture.

How to cite: Scheiterlein, T. and Fiener, P.: Microplastic detection in arable soil using a 3D Laser Scanning Confocal Microscope coupled with a Machine-Learning Algorithm, EGU General Assembly 2023, Vienna, Austria, 23–28 Apr 2023, EGU23-4315, https://doi.org/10.5194/egusphere-egu23-4315, 2023.

Plastic is a prominent material finding growing use globally within agricultural supply chains, through cultivational practices to product packaging. Mulch film is one such ‘plasticulture’ application where opaque film is stretched over the soil surface to maintain a humid microclimate at the crops / fruits soil-root zone as well as protecting ungerminated seeds and small crops from pests. However, mulch film represents an unregulated chemical input source to agroecosystems, whose ecotoxicological legacy is yet to be assessed. The first step in addressing this issue is characterising the chemical content of the source material that may later be released. Amidst the polymeric base, manufacturers include a diverse multitude of chemical additives into plastic formulations to aid with manufacturing, improve functionality, retard degradation and alter aesthetics. In most cases, additives are not chemically but physically bound to the polymeric matrix meaning over time they are free to migrate and be released to the surrounding agroecosystem. Once released, these chemicals may cycle between compartments, interacting with biota or entering the food chain through uptake by plant life or livestock. To trace the extend of release and subsequent interaction of chemical additives within ecosystems, the formulation of the film must be determined. An untargeted characterisation approach has to be adopted, as additive packages remain confidential as property of the manufacturers. Microwave assisted solvent extraction and dissolution-precipitation have been implemented for two agriplastic mulch films: synthetic low density polyethylene (LDPE) and biodegradable polylactic acid (PLA; 15 %) / polybutyrate adipateterephthalate (PBAT; 85 %) blend. Extracts were analysed by GC-MS and spectral libraries used to characterise the leachable content of the films. Alternatively, thermal desorption of the additives from the polymeric base and subsequent analysis of the volatiles through py-GC/MS allows for rapid screening of low molecular weight species, with the caveat of loss in quantification accuracy. An array of chemicals have been identified including: plasticisers (phthalates, citrates, adipates), slip agents (fatty amides), antioxidants (hindered phosphates, hindered phenols), antistatics (fatty esters), lubricants (fatty alcohols, alkanes) and transformation products of both additives and polymer. Additive characterisation work is complimented by confirmation of the mulches polymeric compositions or revelation of unreported polymeric components, through techniques such as fourier transform infrared (FTIR) spectroscopy and ¹H nuclear magnetic resonance (NMR). Later investigation seeks to understand the transformation, cycling and fate of these additive targets within agroecosystems at predicted release levels, which are based on leaching studies into water and in-field measurements.

How to cite: Monkley, C., Reay, M., Evershed, R., and Lloyd, C.: Characterising the Chemical Additive Content of Agricultural Plastic Mulch Film, EGU General Assembly 2023, Vienna, Austria, 23–28 Apr 2023, EGU23-9039, https://doi.org/10.5194/egusphere-egu23-9039, 2023.

Non-biodegradable polyethylene mulch films are widely used in agriculture to allow for an extended growing season and to increase crop yields. These mulch films are, however, difficult to completely recollect from the field after use, particularly when they are thin (< 25 µm). Residual mulch film pieces can accumulate in soils over time, thereby negatively impacting soil productivity and possibly turning agricultural soils into sources of plastics to surrounding environments. Mulch films certified as biodegradable in soils promise to be a solution to these problems. While such mulch films are already commercially available, a thorough assessment of the biodegradation dynamics of biodegradable mulch film products in soils in the field is lacking. So far, certification relies exclusively on laboratory soil incubations coupled to respirometric analysis of CO₂ formed from the mulch films during biodegradation. Respirometric analyses are, however, very challenging to implement in field incubation studies. Past studies determining concentrations of biodegradable mulch films in field soils and attempts to follow their biodegradation dynamics in the field have relied on approximate quantification approaches, such as determining the decrease in surface area or gravimetric mass of film pieces recollected by hand. To advance a more robust and quantitative analytical approach for residual mulch film quantification in soils, we present a methodology to solvent extract and quantify the main synthetic polymeric components of commercial biodegradable mulch films, poly(butylene adipate-co-terephthalate) (PBAT) and polylactic acid (PLA), from soil. The methodology is based on exhaustive Soxhlet extraction using chloroform/methanol coupled to quantitative ¹H-NMR of the extracted residual PBAT and PLA. We show full recovery of these polymers added to soils in spike-recovery experiments. Here, we use this approach to assess the biodegradation of two commercial biodegradable mulch films in three Swiss agricultural soils in a multiyear incubation study. These incubations are conducted at three experimental incubation scales: flasks in the laboratory, mesocosms in a greenhouse and the actual field. We statistically compare biodegradation rates and extents between three soils, two tested films across the three incubation scales, as well as differences in the relative rates of biodegradation between PBAT and PLA. Thereby, we assess the transferability of biodegradation results from laboratory incubations to field incubations. Our results highlight variations in biodegradation between soils and polyesters and indicate that laboratory soil incubations show faster biodegradation than measured in the same soil in the field.

How to cite: Arn, S., Wille, F., Cerri, M., Kägi, R., Bucheli, T., Widmer, F., McNeill, K., and Sander, M.: Biodegradation of commercial mulch films in Swiss agricultural soils: Results from combined laboratory, mesocosm and field incubations, EGU General Assembly 2023, Vienna, Austria, 23–28 Apr 2023, EGU23-13542, https://doi.org/10.5194/egusphere-egu23-13542, 2023.

Microplastics are ubiquitous in the environment. While it is well documented that microplastics can be harmful to aquatic and terrestrial life, little is understood about how microplastics affect soil environments. Understanding the processes enhancing or constraining microplastic transport through the environment, including within soils, is essential in order to minimize the negative impacts of microplastic pollution. Soil erosion is thought to be a primary process responsible for translocating microplastics from soils to aquatic environments. This study aims to investigate the processes controlling microplastic movement in response to rainfall and overland flow in laboratory rainfall simulations. Using fluorescent photography, we compared the real- time movement of a natural soil particle, sand, with two types of microplastics of differing densities (linear low-density polyethylene and acrylic) in two size fractions (250-355 μm and 500-600 μm). We quantified the rate and number of microplastic particles transported by overland flow and splash erosion, along with the depth to which the particles migrate into the soil profile. The results from this study represent some of the first empirical data that seek to quantify microplastic surface transport processes. Ultimately, additional research in this area will be required to more accurately estimate the extent to which microplastics are exported from soils and the factors that control this export.

How to cite: Severe, E., Surridge, B., Platel, R., Coogan, M., James, M., Fiener, P., and Quinton, J.: Quantifying the movement of microplastics in soil in response to overland flow and splash erosion, EGU General Assembly 2023, Vienna, Austria, 23–28 Apr 2023, EGU23-13713, https://doi.org/10.5194/egusphere-egu23-13713, 2023.

Micro and macroplastics, produced from plastic mulch film and polytunnels, are contaminants of growing concern in agricultural settings. However, their impact on nitrogen (N) cycling and partitioning in plant-soil-microbial systems, critical to soil health and food security, is poorly understood. The differing impact of conventional plastics (e.g. low density polyethylene; LDPE) and emerging biodegradable plastics on microbially-mediated N transformations is also unclear, especially with accumulation over long timescales. In this mesocosm-scale study, spring barley (Hordeum vulgare L.) was exposed to macro (1 x 1 cm) or microplastic (< 500 μm) produced from LDPE or biodegradable (polylactic acid/polybutylene adipate terephthalate (PLA/PBAT); 15%/85% w/w) plastic mulch at concentrations equivalent to 1 (0.02%), 10 (0.2%) and 20 (0.4%; LDPE only) years of plastic mulch film use. Mesocosms were fertilised with ammonium nitrate (40 kg N ha−1, 20 atom%¹⁵N), and partitioning of ¹⁵N-labelled fertiliser into plant biomass, soil and leachate yielded a partial mass balance. Soil-N partitioning was probed via diffusion of extractable ammonium and nitrate, and compound-specific ¹⁵N-stable isotope analyses of soil microbial protein. Barley chlorophyll content and growth were used to determined effects on plant health. Plant health parameters were not effected by increasing concentrations of micro or macroplastic, however, there were concentration-dependent decreases in plant ¹⁵N uptake. This was linked to increased leached nitrogen for biodegradable and LDPE micro- and macroplastic, due to changes in physical pore flow pathways. This was also observed for total soil ¹⁵N, while varying patterns in soil ¹⁵N partitioning between plastic type, size and concentrations revealed potential complexities of impacts of N cycling for macro and microplastics. Assimilation into soil microbial protein was higher for biodegradable plastics, which we associate with early-stage degradation. Microbial assimilation in the presence of LDPE was a function of abiotic impacts on leaching, with suppression of inorganic N transformations. While micro- and macroplastics altered soil N cycling, the limited impacts on plant health indicated the threshold for negative effects was not reached at agriculturally relevant concentrations during early-stage barley growth. However, changes in soil N cycling and available N will impact nitrogen use efficiency and soil organic matter dynamics. Thus, the differing impacts of conventional and biodegradable macro and microplastics, and effects of accumulation, must be considered in risk assessments for agricultural plastics. 

How to cite: Reay, M., Greenfield, L., Graf, M., Lloyd, C., Evershed, R., Chadwick, D., and Jones, D.: LDPE and biodegradable plastics differentially affect plant-soil nitrogen partitioning and microbial uptake, EGU General Assembly 2023, Vienna, Austria, 23–28 Apr 2023, EGU23-8848, https://doi.org/10.5194/egusphere-egu23-8848, 2023.

Microplastics are emerging pollutant found in many ecosystems including soil. Within them, polyvinyl chloride (PVC) is one of the most toxic polymers and is known for its remarkable resistance to degradation. The recalcitrant nature of PVC and the improper waste disposal could cause serious environmental concerns. In addition, the possible fragmentation and accumulation of small plastic particles in agricultural soils might have impacts on soil chemical and microbiological properties. Based on these considerations, a microcosm experiment was set up to investigate the effects of PVC microplastics (0.021% w/w) on soil chemical properties and soil bacterial and fungal communities at different incubation times (from 3 to 360 days).

Among chemical parameters, soil CO₂ emissions, fluorescein diacetate hydrolysis (FDA), total organic C (TOC), total N, water extractable organic C (WEOC), water extractable N (WEN) and SUVA₂₅₄ were investigated, while the structure of soil microbial communities was studied at different taxonomic levels (phylum and genus) by sequencing bacterial 16S and fungal ITS2 rDNA (Illumina MiSeq). The number and the dimensions of PVC particles were also evaluated after one year of experiment.

The results showed that the presence of PVC particles in soil caused significant (p < 0.05) variations in chemical parameters in short- and medium-term, thus suggesting that the presence of this polymer in soil can affect the turnover of organic matter. In the long period, instead, an increase of the soil enzymatic activity (FDA) was observed.

The analysis of microbiological parameters showed that PVC microplastics significantly affected (p < 0.05) the structure of soil microbial communities changing the abundances of specific bacterial and fungal taxa: Acidobacteria, Actinobacteria, Bacteroides, Candidatus_Saccharibacteria, and Proteobacteria, , among bacteria, and Ascomycota, Basidiomycota, and Mortierellomycota among fungi, suggesting that the impact of this polymer could be taxa-dependent.

A significant (p < 0.05) decrease of the number and dimensions of PVC particles was also detected after one year of incubation, supposing a possible role of microbial community on polymer degradation.

How to cite: Barili, S., Bernetti, A., Sannino, C., Montegiovo, N., Calzoni, E., Cesaretti, A., Pinchuk, I., Pezzolla, D., Turchetti, B., Buzzini, P., Gigliotti, G., and Emiliani, C.: Long term influences of PVC microplastics on soil chemical and microbiological parameters, EGU General Assembly 2023, Vienna, Austria, 23–28 Apr 2023, EGU23-15971, https://doi.org/10.5194/egusphere-egu23-15971, 2023.

Currently, plastic mulch debris represent one of the main sources of microplastic pollution in agricultural soils. However, there is still limited research on plastic and microorganisms' interaction in terrestrial agroecosystems as compared to marine ecosystems. In this study, we have characterized the microbial communities associated with agricultural plastic mulch debris by using culture-dependent, and culture-independent (i.e., high-throughput DNA sequencing) approaches. Weathered plastic mulch debris samples were collected from the topsoil of five agricultural fields in Baza, Granada province in southern Spain, characterized by intensive horticultural production over the last ten years. The bacterial communities from the plastisphere soil (soil tightly adhered to the plastic) as well as the community tightly attached to the plastic surface were assessed by estimating the culturable populations by dilution plating on general media and the total (culturable and non-culturable) populations by NGS analysis of 16S rRNA amplicons. Additionally, all the plastic samples were characterized by FTIR spectroscopy and identified as polyethylene. The results from the culturable approach showed a significantly higher number of colony-forming units in the plastisphere soil than on the plastic surface, revealing some differences among field plots. Furthermore, 16S rRNA amplicon sequencing showed that the bacterial alpha-diversity, as measured by Richness index was higher in the plastisphere soil. Beta diversity Weighted-UniFrac index indicated that the main significant differences in the bacterial communities occurred among field plots, which might be related to the soil type, and/or crop history, and a lower effect of the plastic niche sampled. Some genera such as Arthrobacter, Bacillus, Blastococcus, Kocuria, Nocardioides, Sphingomonas, and Streptomyces were present in high abundance on both plastisphere soil and plastic surface from all the assessed fields. Furthermore, in one of the fields, the genera Polycyclovorans and Bdellovibrio showed significantly higher abundance in the plastic surface than in the plastisphere soil, indicating in this case a selective effect of the plastic for specific bacterial genera. Further research is still needed to better understand the potential impacts of plastic pollution on terrestrial agroecosystems as well as the complex interaction between plastic and microorganisms.

“This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 955334 - SOPLAS.”

How to cite: Macan, G. P. F., Anguita-Maeso, M., and Landa, B. B.: Microbial communities associated with plastic mulch debris in agricultural soils, EGU General Assembly 2023, Vienna, Austria, 23–28 Apr 2023, EGU23-11465, https://doi.org/10.5194/egusphere-egu23-11465, 2023.