DOI: 10.5061/dryad.xgxd254n7
title: EPDM microplastic and earthworm effects on plants

description: Soil microplastic pollution can have negative effects on organisms,
 including plants, but the underlying mechanisms are not fully understood.
 We tested whether structural or chemical properties of a microplastic
 cause its effects on plant above- and belowground growth and whether these
 effects can be influenced by earthworms. We conducted a factorial
 experiment in a greenhouse with seven common Central European grassland
 species. Microplastic granules of the synthetic rubber ethylene propylene
 diene monomer (EPDM), a frequently used infill material of artificial
 turfs, and cork granules with a comparable size and shape to the EPDM
 granules were used to test for structural effects of granules in general.
 To test for chemical effects, EPDM-infused fertilizer was used, which
 should have contained any leached water-soluble chemical components of
 EPDM. Two Lumbricus terrestris individuals were added to half of the pots,
 to test whether these earthworms modify effects of EPDM on plant growth.
 As response parameters aboveground, belowground, and total biomass, as
 well as several root traits were measured, and further parameters derived
 from the measured values. EPDM granules had a clear negative effect on
 plant growth, but since cork granules had a negative effect of similar
 magnitude, with an average decrease in biomass of 37 % in presence of
 granules, this is likely due to the structural properties of granules
 (i.e., size and shape). For some belowground plant traits, EPDM had a
 stronger effect than cork, which shows that there must be other factors
 playing into the effects of EPDM on plant growth. The EPDM-infused
 fertilizer did not have any significant effect on plant growth by itself,
 but it had in interaction with other treatments. Earthworms had an overall
 positive effect on plant growth and mitigated most of the negative effects
 of EPDM. These results show that EPDM microplastic can have negative
 effects on plant growth, and that these might be more related to its
 structural than to its chemical properties.
description: <em>1. Material and
 methods</em><br><em>2.1  Study species and
 pre-cultivation</em> The study was conducted on
 seven grassland species (<em>Alopecurus pratensis</em> L.,
 <em>Daucus carota</em> L., <em>Festuca
 guestfalica</em> Boenn. Ex Rchb., <em>Galium album</em>
 Mill., <em>Leucanthemum ircutianum</em> Turcz. ex DC.,
 <em>Lolium perenne</em> L., <em>Prunella
 vulgaris</em> L.) from five families (Table S1). These seven species
 were chosen because they are very common in central European grasslands,
 represent different guilds (grasses and forbs), and because a previous
 study had shown that their growth was reduced by the presence of EPDM
 microplastics in the soil (van Kleunen <em>et al</em>. 2020).
 Using multiple species from different guilds increases the generality of
 the results (van Kleunen <em>et al.</em> 2014), and therefore
 the results should be more representative for Central European grassland
 communities in general. Seeds were sown in cultivation pots (Kulturschalen
 TK1214; Pöppelmann GmbH & Co. KG TEKU ®, Lohne, Germany) filled
 with peat soil (Einheitserde ® CL P; Einheitserdewerke Patzer Gebr. Patzer
 GmbH & Co. KG, Sinntal-Altengronau, Germany). As the species were
 known to differ in the time required for germination, they were sown on
 different dates (Table S1). The germination took place in a greenhouse
 with a temperature of 18°C during the night and 20°C (±2°C) during the
 day. Additional lighting was used to extend the daily light period to 14
 hours. <em>2.2  Experimental
 set-up</em> We used three main treatments
 according to a full factorial design: granules treatment (none, cork,
 ethylene propylene diene monomer [EPDM]), EPDM-infusion (yes, no) and
 earthworm presence (yes, no). We used four replicates of every treatment
 combination for each of the seven species, resulting in a total of 336
 pots. The pots were randomly allocated to positions on three benches in
 the greenhouse. For the experiment, we used 3-L pots
 (SMV pots; Soparco, Chaingy, France). In each pot, we put a circular piece
 of chiffon fabric (Chiffon Stoff weiß; Modern Textiles GmbH, Berlin,
 Germany) at the bottom to avoid the substrate leaking out from the bottom
 of the pots. We filled all pots with 1.2 L of a 1:1 mix of sand
 (Quarzsand; Emil Steidle GmbH & Co.KG, Sigmaringen, Germany) and
 vermiculite (Isola Vermiculite GmbH, Sprockhövel, Germany). The
 sand-vermiculite mixture had a grain size of 0.3 to 0.8 mm. As substrate
 for the earthworms, we added a layer of 600 ml of organic substrate to
 each pot on top of the sand-vermiculite mix. The organic substrate was a
 1:1 mix of soil (Oberbodengemisch; Corthum Nordschwarzwald GmbH, Marxzell,
 Germany) and special earthworm cultivation soil on basis of peat-mix,
 containing food, proteins, vitamins and minerals for the earthworms
 (Regenwurmzuchterde; Superwurm e.K., Düren, Germany). For the treatments
 with cork and EPDM, this organic top layer consisted of 30 ml of granules
 mixed into 570 ml of the organic substrate. The black EPDM granules (Gummi
 Appel GmbH + Co.KG, Kahl am Main, Germany; Figure S1), which are sold for
 use as infill material for artificial turfs (e.g., sports fields) had a
 size of 1.0 mm to 2.5 mm (approximate size distribution according to the
 producer: 1% 1.0 – 1.2 mm; 3% 1.2 – 1.4 mm; 15% 1.4 – 1.6 mm; 33% 1.6 –
 1.8 mm; 48% 1.8 – 2.5 mm). The 5 % volume concentration of EPDM in the
 organic substrate was chosen, because it was shown to have negative
 effects on plant growth without resulting in high plant mortality (van
 Kleunen et al. 2020). Since this concentration is equal to a concentration
 of ca. 53.4 g microplastic per kg substrate, it is comparable to the 67.5
 g microplastics per kg found in soils of an industrial area (Fuller
 & Gautam 2016). The cork granules were used to determine whether
 the effects of EPDM on plant growth result from the physical presence of
 granules, irrespective of the material. The cork granules (cork-shop.com,
 Friedbert Bleile Handel und Dienstleistungen, Umkrich, Germany; Figure S1)
 consisted of natural cork without any additives and ranged from 0.5 mm to
 2.0 mm in size and are also sold for use as infill material for artificial
 turfs.    On the 6th of May 2019, we transplanted the
 seedlings into the pots. Within the first week, 14 seedlings that had died
 were replaced. The temperature in the greenhouse was set to 16°C/18°C
 (night/day) with ventilation starting at temperatures above 20°C/22°C
 (night/day), and no artificial lighting was used. The plants were watered
 regularly until saturation of the substrate with approximately equal
 amounts of tap water using a hose throughout the whole experiment to keep
 the soil permanently moist and avoid drought stress. To
 test the effect of the presence of earthworms, we added two
 <em>Lumbricus terrestris</em> individuals to half of the pots
 (Wurmwelten.de, Stadtoldendorf, Germany). This number is representative of
 the number of earthworms typically found in Central European grasslands
 (Jänsch et al. 2013). As the first batch of earthworms we had ordered did
 not include enough individuals to have two worms per pot, we first added
 only one worm per pot four days after transplanting the seedlings. Three
 days later, after we had received a second batch of earthworms, we added
 the second worm to each pot. Before adding the earthworms to the pots, we
 washed and weighed each one of them separately. The total earthworm mass
 per earthworm-treatment pot was between 6.13 g and 9.77 g, with a mean ±
 SE of 7.96 ± 0.05 g. To reduce earthworm escape from pots, we put a sleeve
 of white fleece (Thermovlies M85; Hermann Meyer KG, Rellingen, Germany)
 with an approximate height of 6 to 9 cm around each pot (i.e., also around
 the pots without earthworms, Figure S2). To test
 whether effects of EPDM on plant growth could result from  water-soluble
 chemical components of the EPDM granules (including any additives added
 during production or residual reactants), we added EPDM-infused fertilizer
 to half of the pots. We soaked 5 % volume concentration of EPDM in 1 ‰
 Universol ® Blue fertilizer solution (ICL Deutschland Vertriebs GmbH,
 Nordhorn, Germany; main macronutrients contained in the fertilizer: 18 %
 nitrogen, 11 % phosphate, 18 % potassium oxide) for one week. Afterwards,
 it was stored at 15°C in a dark room. As control, we used 1 ‰ Universol ®
 Blue fertilizer solution, stored in the same room. Before applying 50 ml
 of EPDM-infused fertilizer to each plant in the infusion treatment, the
 EPDM granules were removed from the fertilizer solution with a 200 μm
 sieve. We applied the infusion treatment once a week, starting from the
 second week, resulting in five applications in total. No significant
 difference in pH was observed between the infused fertilizer and control
 fertilizer, as measured before each application with a pH-meter.
 <em>2.3  Measurements</em><br><em>2.3.1
 Initial measurements</em> Two and three days
 after transplanting, we took initial size measurements of the plants. For
 this, we measured on each plant the length and width of the biggest
 cotyledon, the length and width of the biggest true leaf (if present) and
 counted the numbers of cotyledons and true leaves. These measurements were
 used to calculate a proxy of total initial leaf area of each plant as
 number of cotyledons × length of cotyledon × width of cotyledon + number
 of true leaves × length of true leaf × width of true leaf.
 <em>2.3.2 Harvest</em> The plants
 were harvested from the 17th to the 21st of June 2019, after a growth
 period of at least 6 weeks. As two plants had died, the total number of
 harvested plants was 334. The aboveground and belowground parts were
 harvested separately. The aboveground parts were cut off, dried in a
 drying oven at 70°C for at least three days and weighed. The belowground
 parts were washed free from substrate and stored at 6°C in 60 ml plastic
 cups filled with demineralized water. Since the top third of the substrate
 was organic soil, it was not possible to get the roots completely clean.
 To measure root morphological traits, we scanned a subsample of distal
 fine roots, which are most likely the absorptive roots, from the lower 1/3
 of the root system of each plant, using an EPSON ® Scanner (Expression
 12000 XL & 11000 XL). This was done within four days after
 harvesting the roots. We analysed the scanned root subsamples with
 WinRHIZOTM (Pro Version 2017a; Regent Instruments Canada Inc.), which
 calculated the root length, root volume and average diameter for each root
 subsample. After scanning, the subsample and remaining root system were
 dried separately in a drying oven at 70°C for at least two days and
 weighed. From the WinRHIZOTM data and the root biomass, we calculated the
 total root length, specific root length (SRL; root length/root dry weight)
 and root tissue density (RTD: root dry weight/root volume).  Seventy-four
 of the initial 336 earthworms were missing (9 pots with no earthworms, 56
 pots with one earthworm instead of two), indicating that they had died or
 escaped. At the time of harvest the total earthworm mass per
 earthworm-treatment pot ranged from 0 g, when both earthworms had died or
 escaped, to 10.66 g, with a mean ± SE of 5.84 ± 0.18 g (7.36 ± 0.11 g when
 considering only the pots that still had two earthworms).
 <em>2.4 Statistical analysis</em>
 All analyses were conducted with R 4.2.2 (R Core Team 2022). To
 test the effects of the different treatments and their interactions on
 aboveground biomass, belowground biomass, total biomass, root weight ratio
 (RWR), total root length, SRL, RTD and average root diameter, we fitted a
 linear mixed effects model for each of these response variables with the
 lme function of the R package <em>nlme</em> (Pinheiro et al.
 2018). As fixed factors, we used the three treatments and all possible
 two- and three-way interactions. To account for differences in initial
 size and in the number of days until harvest, we added total initial leaf
 area and growth duration (i.e., number of days between initial size
 measurements and harvest) as scaled and centred covariates. As we did not
 have specific expectations for differences among the seven study species,
 and we considered them merely as representatives of common grassland
 species in Central Europe, we included species identity as well as its
 interactions with the three treatments as random terms. From the analyses,
 we excluded nine pots in which both earthworms had disappeared, two pots
 in which the plants had died, and one pot of the non-earthworm treatment
 in which we found an earthworm. As preliminary analyses did not reveal any
 difference between pots with one or two surviving earthworms, we did not
 include the number of alive earthworms as covariate.
 Since none of the models met the assumptions of normality, the
 response variables were transformed (square-root transformation for
 biomass parameters, RWR, total root length and SRL; log-transformation for
 RTD and average root diameter). Since most of the models with did not meet
 the assumption of homoscedasticity due to differences in variance among
 the species, we modelled different variances per species with the varIdent
 function of the R package <em>nlme </em>(Pinheiro et al.
 2018). The latter, however, was not required for SRL. To estimate the
 standard deviations of the random terms, the restricted maximum likelihood
 method (REML) of the linear mixed effects model was used. As the granules
 treatment had three levels, we used a priori orthogonal contrasts to
 compare the presence of granules (EPDM + cork) with the absence of
 granules (C<sub>Present</sub>), and the presence of EPDM
 granules with the presence of cork granules
 (C<sub>EPDM</sub>). To test for significance of each fixed
 term, the maximum likelihood method (ML) of the linear mixed effects model
 was used. We then used the anova function to compare the log-likelihood of
 models including and excluding the factor of interest. The test statistic
 used to compare the models was the log-likelihood ratio, which is
 approximately chi-squared distributed. In addition to
 the orthogonal contrasts among the granule treatments, we also added to
 the figures letters to indicate the significant differences for all
 pairwise combinations of treatments based on the Sidak multiple comparison
 method. It should be noted, however, that these pairwise multiple
 comparisons have a lower statistical power than the planned contrasts in
 the mixed effect models. Therefore, the result and discussion sections
 focus on the outcome of the latter approach.   In
 addition to the R packages that were already mentioned, we used
 <em>RVAideMemoire</em> (Maxime Hervé 2019) for visual
 inspection of models’ assumptions, <em>plyr </em>(Wickham
 2011) and <em>dpylr</em> (Wickham <em>et al.</em>
 2019) for data wrangling, and <em>plotrix</em> (Lemon 2006),
 <em>ggplot2</em> (Wickham 2016) and
 <em>ggpattern</em> (FC <em>et al</em>. 2022) for
 data visualization, and <em>emmeans </em>(Lenth 2021) and
 <em>multcompView</em> (Graves <em>et al</em>.
 2019) for pairwise comparisons.
description: The data file is a txt file which can be opened in Microsoft
 Excel, R and most other statistical softwares.
description: \# Negative effects of EPDM microplastic and cork granules on plant growth
 are mitigated by earthworms and likely caused by their structural
 properties Science of the Total Environment Elina Rittelmann-Woods, Tom
 Lachaise and Mark van Kleunen Soil microplastic pollution can have
 negative effects on organisms, including plants, but the underlying
 mechanisms are not fully understood. We tested whether structural or
 chemical properties of a microplastic cause its effects on plant above-
 and belowground growth and whether these effects can be influenced by
 earthworms. We conducted a factorial experiment in a greenhouse with seven
 common Central European grassland species. Microplastic granules of the
 synthetic rubber ethylene propylene diene monomer (EPDM), a frequently
 used infill material of artificial turfs, and cork granules with a
 comparable size and shape to the EPDM granules were used to test for
 structural effects of granules in general. To test for chemical effects,
 EPDM-infused fertilizer was used, which should have contained any leached
 water-soluble chemical components of EPDM. Two Lumbricus terrestris
 individuals were added to half of the pots, to test whether these
 earthworms modify effects of EPDM on plant growth. As response parameters
 aboveground, belowground, and total biomass, as well as several root
 traits were measured, and further parameters derived from the measured
 values. EPDM granules had a clear negative effect on plant growth, but
 since cork granules had a negative effect of similar magnitude, with an
 average decrease in biomass of 37 % in presence of granules, this is
 likely due to the structural properties of granules (i.e., size and
 shape). For some belowground plant traits, EPDM had a stronger effect than
 cork, which shows that there must be other factors playing into the
 effects of EPDM on plant growth. The EPDM-infused fertilizer did not have
 any significant effect on plant growth by itself, but it had in
 interaction with other treatments. Earthworms had an overall positive
 effect on plant growth and mitigated most of the negative effects of EPDM.
 These results show that EPDM microplastic can have negative effects on
 plant growth, and that these might be more related to its structural than
 to its chemical properties. \## Description of the data and file structure
 Column Explanation Rand Random number, for randomization in greenhouse No
 Plant ID number Species Lol_per = Lolium perenne ; Alo_pec = Alopecurus
 pratensis ; Leu_irc = Leucanthemum ircutianum ; Prun_vul = Prunella
 vulgaris; Dau_car = Daucus carota ; Fes_gue = Festuca guestfalica ;
 Gal_alb = Galium album Guild Forb or grass Treatment Control = no
 granules, no EPDM-Infusion, no earthworms; EPDM = EPDM granules; Cork =
 cork granules; Infused = EPDM-Infusion; EW = 2 individuals of Lumbricus
 terrestris added; several treatments seperated by / = combination of
 treatments Granulate shows if and which granule was present in pot; GranC
 = No granules EPDM 0 = no EPDM present ; 1 = EPDM present Cork 0 = no Cork
 present ; 1 = Cork present Infused 0 = no EPDM-Infustion treatment ; 1 =
 EPDM-Infusion treatment EW 0 = no earthworms present ; 1 = earthworms
 present Control 0 = no Control ; 1 = Control (non of the treatments were
 applied) Number_C initial number of cotyledons Length_Cotyledon initital
 length of (biggest) cotyledon in mm; just blade was measured
 Width_Cotyledon initital width of (biggest) cotyledon in mm; measured at
 broadest spot Number_TL initial number of true leaves Length_Trueleaf
 initital length of biggest true leaf in mm; just blade was measured
 Width_Trueleaf initial width of biggest true leaf in mm; measured at
 broadest spot Total_L_area calculated from initial size measurements in
 mm^2 Day_in_measure days between transplanting and initial size
 measurements Day_harvest days between transplanting and harvest
 Growth_duration days between initial size measurements and harvest
 Plant_dead 0 = plant alive ; 1 = plant dead at time of harvest Above_mass
 aboveground biomass in g (dry weight) Main_root_mass biomass of main root
 system in g (dry weight) Sub_root_mass biomass of subsample of root
 system, which was used for root scanning, in g (dry weight) Below_mass
 total belowground biomass in g(dry weight) Total_mass Total biomass in g
 (dry weight) RWR root weight ratio = Below_mass/Total_mass Root_length
 root lengh of scanned subsample in cm, calculated with WinRhizo
 Total_root_length root length of whole root system in cm = Root_lenght *
 Below_mass / Sub_root_mass SRL specific root length in m/g =
 (Total_root_length / Below_mass) / 100 Root_volume root volume of scanned
 subsample in cm^3, calculated with WinRhizo Total_root_volume root volume
 of whole root system in cm^3 = Root_volume * Below_mass / Sub_root_mass
 RTD root tissue density in g/cm^3 = Below_mass / Total_root_volume
 Avg_root_diameter average root diameter of scanned subsampled in mm,
 calculated with WinRhizo EW_in initial weight of earthworms per pot in g
 (both earthworms together) EW_replaced weight of earthworms that were put
 in pot later to replace dead EW in g EW_end final weight of earthworms per
 pot in g EW_alive alive number of earthworms found in pot at harvest; pots
 in EW treatment with EW_alive = 0 were taken out of analysis EW_dead
 number of missing earthworms and earthworms that were found dead at
 harvest EW_difference difference between initial and final earthworm
 weight per pot in g Soil_loose 0 = soil not loose ; 1 = soil loose ; soil
 was visually inspected 3 days before harvest; indication for earthworm
 activity Cells with missing data are indicated with NA (= not available).
 \## Sharing/Access information Links to other publicly accessible
 locations of the data: NA Data was derived from the following sources:
 data was collected by the authors conducting a greenhouse experiment at
 the University of Konstanz