Earthworms, soil and microplastic

For these experiments we used the anecic earthworm Lumbricus terrestris L. (Oligochaeta, Lumbricidae; obtained from Wurmwelten, Dassel, Germany), a species native to Europe that is frequently used in lab and field trials (e.g. refs 12, 16 and 17). In the experiment we used four adult, healthy earthworms with a body weight between 3 to 5 g each per experimental unit; the individuals were carefully washed, dried on filter paper and weighed prior to their addition to the experimental pots.

As experimental soil we used material from the top horizon of an Albic Luvisol (73.6% sand, 18.8% silt and 7.6% clay; 6.9 mg/100 g P; 5.0 mg/100 g K; pH 7.3)18, which was field collected from a meadow in Berlin, sieved (5 cm) to remove stones or debris, and then steamed (90 °C, 2 h) to eliminate other soil biota that could move microplastic.

For this study we used clear, approximately spherical polyethylene (PE) microplastics (Cospheric, Santa Barbara, CA, USA), containing no additives or solvents (density 0.96 g cm−3). PE is among the major plastic types that are used worldwide3. We assumed, based on a previous study12, which also used PE, that earthworms might ingest and/or transport microparticles of different sizes to a different extent. For this reason, we used four commercially available different particle size ranges: 710–850 μm (PE-1), 1180–1400 μm (PE-2), 1700–2000 μm (PE-3) and 2360–2800 μm (PE-4). All particles were white in appearance, facilitating their retrieval from soil.

Experimental setup

We carried out a fully factorial experiment in which each added PE-microplastic size was combined with earthworm presence/absence (n = 5), for a total of 40 pots. Additionally, there was a control without microplastics, but with earthworms (n = 10) to ascertain any effects on earthworms. This 21-day experiment was carried out in an air-conditioned greenhouse with a temperature of 20 °C (±2 °C). As containers (experimental units) we used plant pots (volume: 3 L; height: 19.2 cm; diameter: 17.0 cm) for which the bottom was sealed with permeable black fleece in order to avoid standing water and to prevent the earthworms from escaping during the experiment. This setup was not designed to ascertain the maximum depth to which earthworms could move particles, as this earthworm species can produce burrows to >0.3 m depth14, but to clearly demonstrate the movement of particles. Preliminary trials had shown that earthworms can be reared in containers of this size. All containers were filled with 2.5 kg of soil, and all containers received 5.0 g of dried Populus spp. leaf litter (suitable material for these earthworms)12, added as chopped material to the soil surface, to provide a sufficient amount of organic matter for the earthworms. We did not monitor soil water content, but all pots were watered with 100 mL of water at the same time of day every 2 d (starting 10 d before earthworm addition), which was found suitable in preliminary trials. Pots could freely drain during the study. Microplastic particles were added to the soil surface by weight (750 mg of the differently sized PE-microplastic particles) at the beginning of the experiment. This amount of microplastic did not have overt toxic effects on earthworms in preliminary trials. For the different sizes this translated to 2625 particles (PE-1), 424 particles (PE-2), 203 particles (PE-3) and 75 particles (PE-4).

Harvest and measurements

At the end of the experiment (21 d), the remaining leaf litter and the earthworm casts (around the earthworm burrows) were collected from the soil surfaces. The visible microplastic particles at the soil surfaces were collected and counted, and any presence of microplastic in surface middens was noted. Earthworms were then extracted by hand: this occurred during carefully extracting cores for microplastics (see below), if earthworms were encountered, and all remaining earthworms were extracted after the coring for microplastics was completed. All earthworms were washed, carefully dried off on paper towels, weighed and placed into empty, moistened Petri dishes. The earthworms were kept for 36 h to ensure that the earthworms empty their guts completely, and to search for any microplastic particles in the casts, since the species L. terrestris needs around 20 h to fully empty their gut19. At the end of this period, any presence of microplastic particles in casts was noted.

We approached the analysis of microplastic in soil by using soil cores to sample the experimental units to three depths. In each pot we took two soil cores (diameter: 40 mm), taken to avoid spots with visible earthworm burrows but otherwise randomly. We sliced these cores into three equal portions of 3.5 cm (top: 0–3.5 cm, middle: 3.5–7.0 cm, and bottom: 7.0–10.5 cm), and dried the soils at 40 °C for 48 h before further processing. We then used an aqueous extraction/flotation method to extract the microplastic particles, exploiting their low density (0.96 g cm−3). Briefly, soil samples were suspended in 25 mL water, the suspension was then vortexed (10 s), centrifuged (Thermo Scientific Heraeus Multifuge, 2500 rpm, 5 min, 21 °C) and decanted through a series of sieves. Microplastic particles were collected on a sieve (250 μm) and then counted. We did not formally assess efficiency of this extraction method, since it was not necessary for our question, but re-extractions of soils did not yield any further microplastic particles. We expressed data as relative counts of microplastic particles. This was done either as a percentage of particles added for particles remaining on the surface (i.e. number of particles retrieved from the pot surface/number of particles added to the pot), or as a percentage of particles retrieved in the cores for the depth distribution (i.e. number of particles extracted from the respective layer in the pot/number of particles extracted from all layers in the pot) in order to compare among the different particle sizes.

Statistical analyses

We separated the downward movement of microplastic particles into two components, reflecting the measurements at harvest time: (a) the disappearance of material from the soil surface and (b) the vertical distribution of these transported particles in the soil profile. For the first component, we applied a linear model to test the effects of earthworm presence, microplastic particle size and their interaction on the relative proportion of particles recovered from the surface of the pots at the end of the experiment. For the latter component, we used a linear mixed effects model with layer, earthworm presence and microplastic particle size as fixed effects and pot id as random effect to account for the repeated analysis of the same pot in different layers.

Data on earthworm mortality and change in body mass during the experiment were analyzed by a generalized linear model with binomial error distribution and linear model, respectively in both cases with the type of microplastic as predictor variable.

Model assumptions for all models were validated using diagnostic plots of residuals. All analyses were conducted in R version 3.3.020 with the R packages nlme21 and sciplot22 and all data are provided in the Supplementary Information.