White clover growth responses to soil microplastics derived from conventional and biodegradable agricultural mulch films

Experimental design/sampling regime
Two commercially available mulch films were selected composed of conventional polyethylene (PE) (12 µm thickness) and a polylactic acid/polybutylene adipate terephthalate (PLA/PBAT) blend (15 µm thickness). Both mulch films were prepared by first cutting into 1.5 cm² fragments before processing into microplastics. The milled material was size fractionated using stainless steel sieves and shaker to retain particles ranging from 250 to 2000 μm. Microplastics were 'dry-dosed' into a locally sourced, pre-sieved (4mm) clay loam soil. Depending on dosage, 1000 mg (0.1%) or 2000 mg (0.2%) of microplastics were added to 1000 g of dried soil and thoroughly mixed.
Immediately after germination one seedling per clover cultivar (Aber Ace, Aber Hera, Aber Swan) was transplanted individually into each of ten 70 mm x 70 mm x 85 mm (deep) pots containing approximately 276 g of soil with PE or PLA/PBAT blend at 0.1% or 0.2% concentration, with a further ten seedlings per cultivar planted into unamended soil (the Control). These pots were randomly arranged on a wire-mesh bench (to prevent cross-contamination of plastics) in an unheated greenhouse with natural light for three months (May - July 2024). The mean daily minimum and maximum temperatures during this period were 12.6 °C (± 0.7) and 24.3 °C (± 0.3), respectively. Pots were watered to capacity and their positions re-randomised weekly. The Fv/Fm samples were collected from all plants the day prior to the end of the experiment. All plants (i.e., ten replicates per plastic treatment per cultivar) were harvested at termination for sampling.

Collection/Generation/Transformation methods
We assessed Fv/Fm ratios as a measure of leaf photosynthetic efficiency by dark adapting one leaf on each individual plant with clips for thirty minutes prior to taking Fv/Fm readings using a Hansatech PEA chlorophyll fluorimeter. Upon termination of the experiment, the plants were cleaned of any adhering compost before roots and shoots were separated and oven-dried at 50 °C for 48 h before weighing to determine plant root, shoot and total dry weight biomass. Immediately before drying, we quantified the number of stolons produced by each plant and also selected the longest root branch to quantify the number of rhizobia nodules per unit root length. Data were noted on paper and then transcribed to Excel spreadsheets before conversion to csv format for data storage. Root:Shoot and Fv/Fm ratios were logit-transformed prior to analysis.