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Size-dependent effects of polystyrene plastic particles on the nematode Caenorhabditis elegans as related to soil physicochemical properties
Journal Pre-proof Size-dependent effects of polystyrene plastic particles on the nematode Caenorhabditis elegans as related to soil physicochemical properties Shin Woong Kim, Dasom Kim, Seung-Woo Jeong, Youn-Joo An PII:
S0269-7491(19)34034-5
DOI:
https://doi.org/10.1016/j.envpol.2019.113740
Reference:
ENPO 113740
To appear in:
Environmental Pollution
Received Date: 22 July 2019 Revised Date:
5 December 2019
Accepted Date: 6 December 2019
Please cite this article as: Kim, S.W., Kim, D., Jeong, S.-W., An, Y.-J., Size-dependent effects of polystyrene plastic particles on the nematode Caenorhabditis elegans as related to soil physicochemical properties, Environmental Pollution (2020), doi: https://doi.org/10.1016/j.envpol.2019.113740. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
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Size-dependent effects of polystyrene plastic particles on the nematode Caenorhabditis elegans as related to soil physicochemical properties
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Shin Woong Kim a1, Dasom Kima1, Seung-Woo Jeongb, and Youn-Joo Ana*
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Department of Environmental Health Science, Konkuk University, 120 Neungdong-ro, Gwangjingu, Seoul 05029, Korea
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b
Department of Environmental Engineering, Kunsan National University, Kunsan 573-701, Korea
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These authors contributed equally to this work.
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*Corresponding author.
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Tel.: +82 2 2049 6090
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Fax: +82 2 2201 6295
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E-mail: [email protected] (Y.-J. An)
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ABSTRACT
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Plastic polymers are widely used in various applications and are thus prevalent in the environment.
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Over time, these polymers are slowly degraded into nano- and micro-scale particles. In this study,
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the free-living nematode, Caenorhabditis elegans, was exposed to polystyrene particles of two
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different sizes (42 and 530 nm) in both liquid and soil media. The number of offspring significantly
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(p < 0.05) decreased at polystyrene concentrations of 100 mg/L and 10 mg/kg in liquid and soil
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media, respectively. In soil media, but not liquid media, C. elegans was more sensitive to the larger
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particles (530 nm) than the smaller particles (42 nm), and the median effective concentration (EC50)
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values of the 42 and 530 nm-sized particles were found to be >100 and 14.23 (8.91–22.72) mg/kg,
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respectively. We performed the same toxicity bioassay on five different field-soil samples with
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different physicochemical properties and found that the size-dependent effects were intensified in
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clay-rich soil samples. A principal component analysis showed that the bulk density, cation
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exchange capacity, clay content, and sand content were the dominant factors influencing the
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toxicity of the 530 nm-sized polystyrene particles. Therefore, we conclude that the soil composition
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has a significant effect on the toxicity induced by these 530 nm-sized polystyrene particles.
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Capsule: Soil texture composition is strongly linked to the toxicity of polystyrene plastic particles
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in the soil nematode Caenorhabditis elegans.
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KEYWORDS: Caenorhabditis elegans; Microplastic; Nanoplastic; Soil texture; Toxicity values
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1. Introduction
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Owing to their versatility and durability, plastic polymers are one of the most widely used
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materials. They are often released into the natural aquatic and terrestrial environments, thus
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continuously exposing the inhabitants to their hazardous constituents (Browne, 2010; Rillig, 2012).
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Plastic polymer fragments slowly convert into nano- (< 100 nm) and micro- (< 5 mm) particles
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(Lambert and Wagner, 2018; PlasticEurope, 2015). Numerous previous studies have reported that
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these plastic particles have adverse physical and biological effects on marine and freshwater
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organisms and induce complex toxic effects in combination with other pollutants (Antunes et al.,
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2013; Guzzetti et al., 2018; Prokić et al., 2019; Rist and Hartmann, 2018; Wright et al., 2013).
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Numerous problems associated with plastic particles in soil and terrestrial ecosystems have been
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highlighted previously (Rillig, 2012) and have started to be addressed recently (Chae and An,
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2018). The plastic particles originate from various plastic sources, including domestic sewage
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(Talvitie et al., 2017; Ziajahromi et al., 2017), fertilizers (Horton et al., 2017; Nizzetto et al., 2016),
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and vinyl mulch used in agriculture (Farmer et al., 2017; Sintim and Flury, 2017). Monitoring of
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microplastic particles has shown that they can be found at a concentration of 300–67,500 mg/kg in
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industrial soils (Fuller and Gautam, 2016) and at 55.5 mg/kg in floodplains at a depth of 0–5 cm
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(Scheurer and Bigalke, 2018). They can be transported to deeper soil layers and might as well be
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ingested by soil organisms such as earthworms and insects.
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Previous studies have simulated the exposure of collembolans (Maaß et al., 2017; Zhu et al.,
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2018a,b), various earthworm species (Gaylor et al., 2013; Hodson et al., 2017; Huerta Lwanga et
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al., 2016, 2017; Rodriguez-Seijo et al., 2017), isopods (Kokalj et al., 2018), mites (Zhu et al.,
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2018a), and nematodes to plastic particles (Lei et al., 2018; Zhao et al., 2017). It has been reported
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that earthworms can ingest microplastic particles and transport them through their burrowing and
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excretion activities (Huerta Lwanga et al., 2016; Rillig, 2012). Furthermore, several studies have
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reported that microplastic particles can induce sub-lethal effects on earthworm species at the
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individual and immune levels (Hodson et al., 2017; Huerta Lwanga et al., 2016, 2017; Rodriguez-
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Seijo et al., 2017). Although numerous reliable studies have supported the assertion that nano- and
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micro-plastic exposures can induce adverse biological effects, these studies do not represent real
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soil environmental conditions because several of them were conducted in non-soil media such as
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liquids or mixtures of plaster and activated charcoal (Kokalj et al., 2018; Lei et al., 2018; Maaß et
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al., 2017; Zhao et al., 2017; Zhu et al., 2018a). In general, these studies have reported that
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nanoplastics can cause a decrease in the growth and reproduction of various nematode species (Lei
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et al., 2018; Zhao et al., 2017). For example, Zhu et al. (2018c) found that an exposure to nano-
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sized polystyrene changes intestinal microbial diversity in soil oligochaetes. Although these studies
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improve our understanding of the toxicity mechanisms of plastic particles, the exposure media used,
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including the essential nutrients for each species, were liquid.
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It is well known that the adverse effects of general toxicants (metals and organic pollutants) are
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strongly linked to soil properties. For example, soil organic carbon and matter can alter toxicant
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bioavailability in soil receptors, including plants, soil invertebrates, and microbes (Marschner and
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Kalbitz, 2003; Pinto et al., 2004). In addition, the partitioning between solid and solution phases
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depends on specific soil factors, such as pH and presence of clay minerals (Lamb et al., 2009; Sauvé
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et al., 2000). In microplastic research, however, the interactions between plastic toxicity and soil
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properties have not been fully addressed. In the present study, we conducted bioassays in both
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liquid and soil media to evaluate the media-dependent effects of nano- and micro-plastic particles,
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using a variety of field soils. Based on our previous finding, the free-living nematode,
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Caenorhabditis elegans, was chosen as the test species, and the number of offspring was selected as
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the primary indicator of the effects of these particles (Kim et al., 2014; 2018). The plastic particles
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of two different sizes and five soil samples were used to assess whether the effects of these particles
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were dependent on particle size and soil physicochemical properties.
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2. Materials and methods
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2.1. Materials and target organism
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Polystyrene (PS) beads of two different sizes, 42 nm (42PS; product number FS02F-12719) and
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530 nm (530PS; product number FS03F-9360), were obtained as dispersions from Bangs
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Laboratories, Inc. (Fishers, IN, USA). These beads exhibited strong fluorescent properties with <
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480 nm excitation and > 520 nm emission wavelengths. To confirm the PS particle type, a Fourier
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transform infrared (FTIR, 4100typeA, JASCO, Japan) spectral analysis was carried out using dried
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samples at a wavenumber range of 4000–650 cm-1 (Fig. S1). The morphologies and average
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diameters of 42PS and 530PS particles were measured using a field emission transmission electron
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microscope (FE-TEM, JEM-2100F, JEOL, Japan). The hydrodynamic sizes and zeta potential (mV)
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of each PS particle type in deionized water (DW) and K-medium (0.032 M KCl, 0.051 M NaCl)
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(Brenner, 1974) were determined under specified test conditions, using a dynamic light-scattering
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analyzer (Zetasizer Nano ZS, Malvern Instruments, UK). As shown in Fig. S2, the 42PS and 530PS
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particles were moderately spherical with average diameters (n = 100) of 68 ± 8 and 496 ± 14 nm,
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respectively. The hydrodynamic diameters of the 42PS particles in DW and K-medium were 62 and
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59 nm and those of the 530PS particles were 559 and 527 nm, respectively. The zeta potentials of
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the 42PS particles in DW and K-medium were 45.1 ± 0.6 and −28.7 ± 0.6 mV and those of the
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530PS particles were 49.5 ± 0.5 and −12.8 ± 0.5 mV, respectively.
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The target organism, C. elegans (wild type, Bristol strain N2), was obtained from the Animal
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Genomics Laboratory, Konkuk University (Seoul, South Korea). The cultures were maintained on
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the nematode growth medium (NGM; NaCl 3 g/L, peptone 2.5 g/L, agar 17 g/L, 1 M potassium
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phosphate 25 mL/L, 1 M CaCl2․2H2O 1 mL/L, 1 M MgSO4·7H2O 1 mL/L, cholesterol 1 mL/L),
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with Escherichia coli strain OP50 supplied as its food source (Brenner, 1974). All cultures were
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maintained at 20 ± 2°C in the dark. To synchronize worm age, the C. elegans strains that were at
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least 3 d old were treated with a Clorox solution for 10 min, after which the suspension was
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centrifuged at 400 × g for 2 min to collect the pellets containing embryos. Subsequently, the pellets
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were washed thrice with K-medium, and the embryos were placed on new NGM agar plates with E.
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coli strain OP50. The plates were incubated until the nematodes reached the early-adult stage (54–
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58 h synchronization after the Clorox solution treatment). All media components were obtained
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from BD (Sparks, MD, USA), except for sodium chloride and potassium chloride, which were
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obtained from Duksan (Asan, Korea).
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2.2. Liquid and soil media tests
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Liquid media tests were performed according to previous studies with some modifications
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(Williams and Dusenbery, 1990). K-medium (1 mL) with different nominal concentrations of 42PS
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and 530PS particles (0.01, 0.1, 1, 10, and 100 mg/L) was placed into each well of a 24-well plate,
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followed by the inoculation of 10 age-synchronized worms (54–58 h) into each well. Each PS
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particle type was assessed using eight replicates and a negative control (plastic-free condition). The
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plates were maintained at 20 ± 2°C for 24 h in the dark. Subsequently, the number of offspring was
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counted under a microscope, and the data were expressed as a percentage (%) of the mean of the
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control group.
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Soil media testing was performed using the nematode offspring counting assay, as described
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in our previous studies (Kim et al., 2014; 2018). The nematodes were exposed to the test soil using
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a standardized toxicity method (ASTM, 2001; ISO, 2010); the offspring in the test soil were
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attracted by a microbial food source on an agar plate. Landwirtschaftliche Untersuchungs und
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Forschungsanstalt (LUFA 2.2, LUFA Speyer, Germany) was selected as the test soil, and air-dried
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for 7 days after sieving (< 2 mm). The stock solutions of both PS particle types (42PS and 530PS)
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were prepared in K-medium to adjust their final soil concentrations, which were 0 (plastic-free
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condition), 0.01, 0.1, 1, 10, and 100 mg/kg (n = 8). K-medium (0.2 mL) and 10 age-synchronized
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worms were added to each well of the 24-well plate containing 0.3 g of the test soil. After 24 h, the
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soil containing worms was placed on soil-agar isolation plates (Kim et al., 2014, 2018). To prepare
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these plates, E. coli strain OP50 was cultured in Luria-Bertani medium (25 g/L) at 37°C for 16 h,
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and 150 µL of the cell suspension (optical density 1.1–1.2, 8.0–9.6 × 108 cells/mL) was smeared
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onto each side of the NGM agar plate. The test soils were arranged linearly in the central area of the
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soil-agar isolation plates, and the number of offspring being attracted from the soil to each side of
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the NGM agar plate was counted and expressed as a percentage (%) of the average value of the
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control group.
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2.3. Physiochemistry-dependent toxicity of PS beads in soil
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Five field soils (S1–S5) were collected from metalliferous sites in South Korea. These sites
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were located on the perimeter of an arsenic smelter and had been remediated using a soil washing
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process. The soil conditions were unpredictable owing to large variations in soil physicochemical
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properties, especially pH and texture (Kim et al., 2017). Soils were hand sorted, air-dried, and
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sieved to a size of < 2 mm. Aggregated stability (AS) was calculated by weighing the soil after
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slowly wetting on a 0.25-mm sieve according to the soil quality test kit guide (USDA, 1999). Bulk
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density (BD) was measured as described previously (Friedman et al., 2001; Tan, 2005). The cation-
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exchange capacity (CEC) was determined by extracting the cations with ammonium acetate and
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potassium chloride solutions. Electrical conductivity (EC) was calculated by an instrumental
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analysis after 1:1 (soil:water) extraction (EC meter, Multi Meter CP-500L; Istek, Seoul, Korea).
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Organic matter (OM) was determined via the loss on ignition test (700–800°C, 4 h). The soil pH
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was measured after DW extraction (1:5), and the soil texture (percentage of clay, sand, and silt) was
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studied using a Soil Texture Test Kit (model 1067; LaMotte, Chestertown, MD, USA). Water
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holding capacity (WHC) was assessed by weighing the soil after performing the wet-dry process for
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3 h (Table S1) (Harding and Ross, 1964; USDA, 1999, 2001). The total concentrations of metals,
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including arsenic (As), cadmium (Cd), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn), were
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analyzed using an inductively coupled plasma atomic emission spectrophotometer (ICP-AES; JY
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138, Ultrace, Jobin Yvon, France) after performing an acid digestion process (Table S1). Dry
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samples of each soil (0.2 g) were transferred into a Teflon vessel and treated with a mixture of nitric
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acid (HNO3), perchloric acid (HClO4), and hydrofluoric acid at 200°C. Thereafter, 20 mL of DW
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was poured into the vessel, after which a metal analysis was performed using ICP-AES. The
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detection limits of ICP-AES were 0.5 (As), 0.05 (Cd), 0.05 (Cu), 0.05 (Ni), 0.1 (Pb), and 0.05 (Zn)
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µg/L.
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A nematode offspring counting assay was used to assess the toxicity of PS particles in
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different field soils. Soil samples S1–S5 (0.3 g) were added to each well of a 24-well plate and
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moistened with K-medium. The stock solutions of both 42PS and 530PS particles were prepared in
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K-medium to adjust their soil concentrations; the nominal concentrations of PS in soil were
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determined to be 0 (plastic-free condition), 0.01, 0.1, 1, 10, and 100 mg/kg (n = 4). The water
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content of each soil sample was adjusted to 140–145% of WHC (i.e., the same water content as the
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LUFA test). Ten age-synchronized worms were exposed to the soil samples for 24 h, after which
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the nematode offspring counting assay was performed as described above.
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2.4. Data analysis
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To determine the factors that are primarily responsible for the toxicity of PS particles, we
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used a principal component analysis (PCA) based on minimum data set (MDS) selection (Andrews
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et al., 2002; Govaerts et al., 2006). Offspring number measurements for each concentration (100,
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10, 1, and 0.1 mg/kg) of the 42PS and 530PS particles were used as the test data set; the data for ten
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physicochemical properties and concentrations of six metals were also considered. Only the
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principal components (PC) with an eigenvalue >1 were considered for selection, and the
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physicochemical properties with an eigenvector within 20% of the measurement data for each
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concentration of the 42PS and 530PS particles were determined as the dominant factors influencing
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PS toxicity (Andrews et al., 2002; Wander and Bollero, 1999).
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The Dunnett’s Program (ver. 1.5) was used to calculate the 10% effective concentration
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(EC10) and significant differences (p < 0.05) between the mean values of the control and treatment
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groups (USEPA, 1999). The median effective concentration (EC50) values were calculated using the
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Trimmed Spearman-Karber method (Hamilton et al., 1977).
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3. Results
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3.1. Size-dependent toxicity of PS in different test media
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As shown in Fig. 1A, 100 mg/L of the 42PS and 530PS particles in liquid media caused a
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significant decrease in offspring number (p < 0.05). There was no evidence of a size-dependent
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effect since both 42PS and 530PS particles produced similar effects. In the soil media test, the
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number of offspring started to decrease significantly (p < 0.05) at 10 mg/kg of the 530PS particles;
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however, for the 42PS particles, significant decreases (p < 0.05) were observed only at 100 mg/kg
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(Fig. 1B). The EC10 and EC50 values for the 42PS and 530PS particles in liquid and soil (LUFA)
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media are shown in Table S2. Some toxicity values could not be calculated because of the large chi-
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square value or high level of trimming (>70%) in the statistical program.
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3.2. Physiochemistry-dependent toxicity of PS beads in soil
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Next, we investigated whether the different physicochemical properties of each soil sample
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(S1–S5) had a significant effect on the number of offspring in the absence of PS treatment. The S2,
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S4, and S5 soil samples had significant effects on the nematode population (p < 0.05) (Fig. S3).
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Each data point for the 42PS and 530PS particles was expressed as a percentage (%) of the average
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value of each control group. The metal concentrations in the target field soils were < 43.9, N.D., <
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57.9, < 18.1, < 96.3, < 124.0 mg/kg for As, Cd, Cu, Ni, Pb, and Zn, respectively (Table S1). Our
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previous study found that metal concentrations < 200 mg/kg produced no significant toxic effects in
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the nematode bioassay (Kim et al., 2018). In soil sample S1, the 530PS particles significantly
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decreased the number of offspring (p < 0.05) at all tested concentrations (0.01–100 mg/kg), while a
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significant decrease in offspring number (p < 0.05) could only be observed at 100 mg/kg of the
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42PS particles (Fig. 2A). For soil sample S2, significant reductions in offspring number (p < 0.05)
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began at 0.1 mg/kg of the 530PS particles and at 10 mg/kg of the 42PS particles (Fig. 2B). The S3
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and S4 soil samples exhibited trends similar to the LUFA soil (Fig. 2C and D), while in soil sample
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S5, the toxicity of the 530PS particles was significantly higher than that of the 42PS particles (Fig.
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2E). The EC10 and EC50 values for each soil sample are shown in Table S2.
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3.3. Determination of factors strongly related to PS toxicity
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Different concentrations of the 42PS particles exhibited different correlations with various
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soil factors, while the 530PS particles had constant correlations with BD, CEC, clay content, Pb
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(T), and sand content even at different concentrations. The first (PC1), second (PC2), third (PC3),
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and fourth (PC4) principal components were classified as useful data loading sets (eigenvalue >1)
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for each concentration of the 42PS and 530PS particles (Table S3–S6) and are shown as the PC1
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and PC2 loading plots in Fig. 3. The PCA data are generally used to explain the positive or negative
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directions between each data set. The clay content in all figures was located opposite the sand
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content and in the same direction as WHC and CEC (Fig. 3A–D). As shown in the supplementary
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tables, the eigenvectors were used to determine the soil factors related to PS toxicity. For example,
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at the concentration of 100 mg/kg, the total variations were calculated as 40%, 28%, 20%, and 8%
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for PC1, PC2, PC3, and PC4, respectively (Table S3). The eigenvector for the 42PS particles (100
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mg/kg) was 0.082 in PC1, which showed a positive correlation with Ni (T). The eigenvector for the
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530PS particles (100 mg/kg) was −0.335 in PC1, which exhibited positive correlations with both
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BD and Pb (T) and negative correlations with CEC, clay content, and WHC (Fig. 3A and Table S3).
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In summary, the dominant soil factors at each 42PS concentration were determined to be AS, Cu
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(T), EC, Ni (T), OM, Pb (T), and pH, with no certain direction in each data set. In the data analysis
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for the 530PS particles, the soil factors included BD, CEC, clay content, sand content, and WHC.
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4. Discussion
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Previous studies have reported the toxicity of plastic particles towards C. elegans using
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liquid media tests. Lei et al. (2018) have reported that an exposure (2 days) to 5.0 mg/m2 of
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microplastics can significantly impact the survival, growth, and reproduction of C. elegans. Zhao et
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al. (2017) found that a chronic exposure (4.5 days) to nano-polystyrene induces alterations in the
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behavior, survival, and reproduction of C. elegans at concentrations higher than 10 µg/L, and these
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effects could be transferred to the next generation. In the present study, we found that the number of
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offspring significantly (p < 0.05) decreased at 100 mg/L in liquid media. The liquid media test
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showed no evidence of size-dependent effects, whereas the soil media test exhibited size-dependent
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effects beginning at 10 mg/kg. This phenomenon indicates two important facts; a) the toxicity of PS
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particles can increase in soil media, and b) the 530PS particles were more toxic than the 42PS
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particles in soil media. Because there are considerable differences in the units used and the actual
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exposed concentration, it could be argued that this media-dependent comparison might not be
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reasonable. Despite this, our results showed a clear difference in offspring number (approximately
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40%; Fig. 1) obtained by using similar concentrations of the 530PS particles, i.e., 10 mg/L and 10
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mg/kg in the liquid and soil media, respectively. In previous studies, different media tests have been
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used to explain the interaction between solid and liquid phases in soils (Eom et al., 2007; Juvonen et
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al., 2000; Pandard et al., 2006). Test organisms, such as C. elegans, generally show a higher
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sensitivity to PS particles, even at similar concentrations, in liquid media than that in in soil media
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(Donkin and Dusenbery, 1994).
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Previous studies have also reported size-dependent effects of plastic polymers in live
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organisms. Jeong et al. (2016) found that the smaller polystyrene particles (0.05 µm) have a greater
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toxic effect than that of the larger particles (0.5 and 6 µm) on the fecundity and life span of rotifer
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species. They also found that the smaller polyethylene particles (1–4 µm) had significant
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immobilization effects on Daphnia, unlike the larger ones (90–106 µm) (Reche et al., 2016). These
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findings might be linked to the feeding behavior of organisms because the smaller particles are
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easier to accumulate (Wang et al., 2009), while the larger particles are more effectively egested
266
(Jeong et al., 2016). Ziajahromi et al. (2018) reported that the sediment-dwelling invertebrate
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Chironomus tepperi was affected by polyethylene particles, and the effect was size-dependent. They
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found that the smaller polyethylene particles (10-27 µm) induce significant adverse effect on the
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growth and survival of C. tepperi than the larger ones (100-126 µm). However, very limited studies
270
have been conducted on the size-dependent effects of plastic particles in soil systems. Jiang et al.
271
(2019) found that nano-sized polystyrene (100 nm) has a higher genotoxic and oxidative damage
272
than micro-sized one (5 µm) on higher plant Vicia faba in culture solution. Lei et al. (2018) reported
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that C. elegans showed a more sensitive response to the smaller (0.1–1.0 µm) PS particles compared
274
with that of the larger particles (5.0 µm). However, their experiments were performed in filter paper
275
and liquid media. In previous studies of other nanomaterials, such as metallic nanoparticles, C.
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elegans showed a more sensitive response to smaller Al2O3 (Wang et al., 2009), CeO2 (Roh et al.,
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2010), Fe oxide (Höss et al., 2015), TiO2 (Roh et al., 2010; Wang et al., 2009), and ZnO (Wang et
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al., 2009) nanoparticles. These studies posited that the aggregate size and surface area of
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nanoparticles are highly interactive with the size-dependent effects on C. elegans; however, these
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studies also used liquid media.
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The S1, S2, and S5 samples showed significant decreases in offspring number upon exposure to
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the 530PS particles at very low concentrations (0.01–0.1 mg/kg). Although some reports have
283
estimated that 43,000–63,000 tons of microplastic are added into the agricultural system annually
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(Nizzetto et al., 2016), the relevant concentrations of nanoplastic particles in the soil environment
285
have not yet been reported. Hence, our test concentrations cannot be directly compared with actual
286
environmental concentrations. The S3 and S4 soil samples showed a trend similar to that seen in the
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LUFA soil, in that the only significant decrease occurred at a concentration of 100 mg/kg for both
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42PS and 530PS particles. This difference from the other soil samples (S1, S2, and S5) might be
289
strongly related to the soil physicochemical properties. As shown in Table S1, the S1, S2, and S5
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groups had high levels of AS, EC, and clay, whereas the LUFA, S3, and S4 groups showed high
291
levels of sand. Clay generally consists of particles with sizes < 2 µm and can form residual (< 0.2
292
µm) and storage (0.2–30 µm) pores (Greenland, 1977). Thus, particles in the size range 0.2–2 µm,
293
which includes the size range of 530PS, can be classified as clay particles. The composition of the
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test soils containing 530PS particles might differ from the control soil, and this can affect the
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nematode population because it has been shown to be negatively correlated with fine-textured soil
296
(Elliot et al., 1980; Ward, 1975; Yeates, 1981). These effects are not as pronounced in coarse-
297
textured soils, such as in the LUFA, S3, and S4 groups, because these soils have very low levels of
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clay (Table S1). Our test results with the field soil samples enabled us to conclude that soil texture
299
fractions strongly influence PS toxicity in soil environments.
300
PCA is commonly used to evaluate linear and monotonic relationships between specific variables
301
and can provide insights into how several parameters significantly influence each other (Andrews et
302
al., 2002). Using this approach, we found that the toxicity of PS particles is conditioned by the soil
303
physical characteristics including texture fractions. We found that the correlations for the 42PS
304
particles were inconclusive because no trends were noted between the data loadings. In the 530PS
305
data set, correlations were observed among BD, CEC, clay content, and sand content (Fig. 3; Table
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S3–6). Because CEC is generally positively related to the clay content in soil systems (Lambooy
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1984), we concluded that the soil textures (clay, sand, and silt) are strongly related to PS toxicity.
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The data obtained with different concentrations (100, 10, 1, and 0.1 mg/kg) of both 42PS and
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530PS particles were reconstituted according to the sand content (%), and a dot graph was
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constructed as shown in Fig. 4. At the concentration of 100 mg/kg of both 42PS and 530PS particles
311
(Fig. 4A and B), no relationships were observed between each data set at different sand contents. A
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weak relationship was observed for the 42PS particles (Fig. 4C, E, and G), whereas the 530PS
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particles showed increasing trends with high R2 values of 0.7347, 0.6268, and 0.6646 for
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concentrations 10, 1, and 0.1 mg/kg, respectively (Fig. 4D, F, and H). A clay content (%)-dependent
315
dot graph was also constructed, as shown in Fig. 5. The data for 100 mg/kg 42PS and 530PS
316
particles showed trends similar to those of the sand content-dependent dot graph (Fig. 5A and B).
317
For the concentrations of 10, 1, and 0.1 mg/kg of the 530PS particles, the high R2 values were
318
found to be 0.7987, 0.7844, and 0.7019, respectively (Fig. 5D, F, and H), and a decreasing trend
319
was observed for each concentration. According to these data, the increase in the sand content
320
appeared to be strongly related to the reduction of PS toxicity in the soil system.
321
We confirmed the media-dependent changes in PS toxicity, showing that C. elegans exhibited a
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more sensitive response in soil media than that in liquid media. The PS size-dependent effects
323
occurred in soil media, with texture fractions strongly related to PS toxicity. However, our results
324
have some limitations; the threshold PS concentration causing adverse effects in the nematode
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species was observed at a very low plastic concentration (0.01 mg/kg) in soil media, and it might be
326
caused by other factors such as contaminants or organic matter. Although these limitations should
327
be addressed in future studies, our results add to the existing knowledge on the occurrence and
328
effects of plastic particles in soil systems.
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Acknowledgement
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This paper was supported by Konkuk University in 2019. We thank the Korean Basic Science
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Institute (KBSI) for their assistance with our dynamic light scattering and TEM analyses.
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Figure legends
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Fig. 1. The number of Caenorhabditis elegans offspring following exposure to the 42PS and 530PS particles in (A) liquid (mg/L) and (B) soil (mg/kg) media. All data were normalized to each control group. Error bars indicate standard deviations, and asterisks (*) indicate significant (p < 0.05) differences compared with the control.
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Fig. 2. The number of Caenorhabditis elegans offspring following exposure to the 42PS and 530PS particles in five field-soil samples; (A) S1, (B) S2, (C) S3, (D) S4, and (E) S5. All data were normalized to each control group. Error bars indicate standard deviations, and asterisks (*) indicate significant (p < 0.05) differences compared with the control.
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Fig. 3. Loading plots of the 42PS and 530PS data and physicochemical properties for (A) 100 mg/kg, (B) 10 mg/kg, (C) 1 mg/kg, and (D) 0.1 mg/kg of soil. Loading plots were generated by a principal component analysis (PCA). The x- and y-axes indicate the first (PC1) and second (PC2) principal components, respectively. The blue lines represent the loading per data, and the 42PS and 530PS data are shown in green. AS = aggregate stability, BD = bulk density, CEC = cation exchange capacity, Clay = clay content (%), EC = electrical conductivity, OM = organic matter, Sand = sand content (%), Silt = silt content (%), WHC = water holding capacity, Metals (T) = total concentration of each metal (As, Cd, Cu, Ni, Pb, and Zn).
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Fig. 4. Reconstituted dot plot according to sand content; (A) 42PS 100 mg/kg, (B) 530 PS 100 mg/kg, (C) 42PS 10 mg/kg, (D) 530PS 10 mg/kg, (E) 42PS 1 mg/kg, (F) 530PS 1 mg/kg, (G) 42PS 0.1 mg/kg, and (H) 530PS 0.1 mg/kg. The x- and y-axes indicate the sand content (%) and offspring number (% control), respectively. All data were from LUFA (Landwirtschaftliche Untersuchungs und Forschungsanstalt Germany) and five field-soil samples (S1–S5). Error bars indicate standard deviations, and asterisks (*) indicate significant (p < 0.05) differences compared with measurements of the lowest sand content.
525 526 527 528 529 530 531
Fig. 5. Reconstituted dot plot according to clay content; (A) 42PS 100 mg/kg, (B) 530 PS 100 mg/kg, (C) 42PS 10 mg/kg, (D) 530PS 10 mg/kg, (E) 42PS 1 mg/kg, (F) 530PS 1 mg/kg, (G) 42PS 0.1 mg/kg, and (H) 530PS 0.1 mg/kg. The x- and y-axes indicate clay content (%) and offspring number (% control), respectively. All data were from LUFA (Landwirtschaftliche Untersuchungs und Forschungsanstalt Germany) and five field-soil samples (S1–S5). Error bars indicate standard deviations, and asterisks (*) indicate significant (p < 0.05) differences compared with measurements of the lowest clay content.
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Fig. 1.
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Fig. 2.
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Fig. 3
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Fig. 4
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Fig. 5
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HIGHLIGHTS We assessed the nano- and micro- plastic toxicity using nematode species in soil. C. elegans showed more sensitive response in soil than in liquid. The nematodes showed high sensitivity to larger particles than smaller ones. The size-dependent effects were intensified in clay-rich soil samples. We concluded that the plastic toxicity is highly linked with soil properties.
Author statement Shin Woong Kim: Conceptualization, Methodology. Writing - Original Draft Dasom Kim: Investigation, Writing - Original Draft Seung-Woo Jeong: Writing - Review & Editing Youn-Joo An: Supervision, Writing - Review & Editing, Funding acquisition
Conflict of interest The authors declare no conflicts of interest.
S0269-7491(19)34034-5
DOI:
https://doi.org/10.1016/j.envpol.2019.113740
Reference:
ENPO 113740
To appear in:
Environmental Pollution
Received Date: 22 July 2019 Revised Date:
5 December 2019
Accepted Date: 6 December 2019
Please cite this article as: Kim, S.W., Kim, D., Jeong, S.-W., An, Y.-J., Size-dependent effects of polystyrene plastic particles on the nematode Caenorhabditis elegans as related to soil physicochemical properties, Environmental Pollution (2020), doi: https://doi.org/10.1016/j.envpol.2019.113740. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
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Size-dependent effects of polystyrene plastic particles on the nematode Caenorhabditis elegans as related to soil physicochemical properties
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Shin Woong Kim a1, Dasom Kima1, Seung-Woo Jeongb, and Youn-Joo Ana*
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a
Department of Environmental Health Science, Konkuk University, 120 Neungdong-ro, Gwangjingu, Seoul 05029, Korea
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b
Department of Environmental Engineering, Kunsan National University, Kunsan 573-701, Korea
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1
These authors contributed equally to this work.
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*Corresponding author.
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Tel.: +82 2 2049 6090
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Fax: +82 2 2201 6295
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E-mail: [email protected] (Y.-J. An)
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ABSTRACT
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Plastic polymers are widely used in various applications and are thus prevalent in the environment.
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Over time, these polymers are slowly degraded into nano- and micro-scale particles. In this study,
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the free-living nematode, Caenorhabditis elegans, was exposed to polystyrene particles of two
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different sizes (42 and 530 nm) in both liquid and soil media. The number of offspring significantly
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(p < 0.05) decreased at polystyrene concentrations of 100 mg/L and 10 mg/kg in liquid and soil
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media, respectively. In soil media, but not liquid media, C. elegans was more sensitive to the larger
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particles (530 nm) than the smaller particles (42 nm), and the median effective concentration (EC50)
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values of the 42 and 530 nm-sized particles were found to be >100 and 14.23 (8.91–22.72) mg/kg,
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respectively. We performed the same toxicity bioassay on five different field-soil samples with
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different physicochemical properties and found that the size-dependent effects were intensified in
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clay-rich soil samples. A principal component analysis showed that the bulk density, cation
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exchange capacity, clay content, and sand content were the dominant factors influencing the
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toxicity of the 530 nm-sized polystyrene particles. Therefore, we conclude that the soil composition
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has a significant effect on the toxicity induced by these 530 nm-sized polystyrene particles.
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Capsule: Soil texture composition is strongly linked to the toxicity of polystyrene plastic particles
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in the soil nematode Caenorhabditis elegans.
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KEYWORDS: Caenorhabditis elegans; Microplastic; Nanoplastic; Soil texture; Toxicity values
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1. Introduction
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Owing to their versatility and durability, plastic polymers are one of the most widely used
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materials. They are often released into the natural aquatic and terrestrial environments, thus
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continuously exposing the inhabitants to their hazardous constituents (Browne, 2010; Rillig, 2012).
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Plastic polymer fragments slowly convert into nano- (< 100 nm) and micro- (< 5 mm) particles
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(Lambert and Wagner, 2018; PlasticEurope, 2015). Numerous previous studies have reported that
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these plastic particles have adverse physical and biological effects on marine and freshwater
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organisms and induce complex toxic effects in combination with other pollutants (Antunes et al.,
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2013; Guzzetti et al., 2018; Prokić et al., 2019; Rist and Hartmann, 2018; Wright et al., 2013).
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Numerous problems associated with plastic particles in soil and terrestrial ecosystems have been
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highlighted previously (Rillig, 2012) and have started to be addressed recently (Chae and An,
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2018). The plastic particles originate from various plastic sources, including domestic sewage
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(Talvitie et al., 2017; Ziajahromi et al., 2017), fertilizers (Horton et al., 2017; Nizzetto et al., 2016),
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and vinyl mulch used in agriculture (Farmer et al., 2017; Sintim and Flury, 2017). Monitoring of
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microplastic particles has shown that they can be found at a concentration of 300–67,500 mg/kg in
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industrial soils (Fuller and Gautam, 2016) and at 55.5 mg/kg in floodplains at a depth of 0–5 cm
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(Scheurer and Bigalke, 2018). They can be transported to deeper soil layers and might as well be
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ingested by soil organisms such as earthworms and insects.
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Previous studies have simulated the exposure of collembolans (Maaß et al., 2017; Zhu et al.,
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2018a,b), various earthworm species (Gaylor et al., 2013; Hodson et al., 2017; Huerta Lwanga et
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al., 2016, 2017; Rodriguez-Seijo et al., 2017), isopods (Kokalj et al., 2018), mites (Zhu et al.,
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2018a), and nematodes to plastic particles (Lei et al., 2018; Zhao et al., 2017). It has been reported
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that earthworms can ingest microplastic particles and transport them through their burrowing and
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excretion activities (Huerta Lwanga et al., 2016; Rillig, 2012). Furthermore, several studies have
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reported that microplastic particles can induce sub-lethal effects on earthworm species at the
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individual and immune levels (Hodson et al., 2017; Huerta Lwanga et al., 2016, 2017; Rodriguez-
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Seijo et al., 2017). Although numerous reliable studies have supported the assertion that nano- and
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micro-plastic exposures can induce adverse biological effects, these studies do not represent real
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soil environmental conditions because several of them were conducted in non-soil media such as
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liquids or mixtures of plaster and activated charcoal (Kokalj et al., 2018; Lei et al., 2018; Maaß et
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al., 2017; Zhao et al., 2017; Zhu et al., 2018a). In general, these studies have reported that
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nanoplastics can cause a decrease in the growth and reproduction of various nematode species (Lei
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et al., 2018; Zhao et al., 2017). For example, Zhu et al. (2018c) found that an exposure to nano-
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sized polystyrene changes intestinal microbial diversity in soil oligochaetes. Although these studies
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improve our understanding of the toxicity mechanisms of plastic particles, the exposure media used,
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including the essential nutrients for each species, were liquid.
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It is well known that the adverse effects of general toxicants (metals and organic pollutants) are
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strongly linked to soil properties. For example, soil organic carbon and matter can alter toxicant
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bioavailability in soil receptors, including plants, soil invertebrates, and microbes (Marschner and
75
Kalbitz, 2003; Pinto et al., 2004). In addition, the partitioning between solid and solution phases
76
depends on specific soil factors, such as pH and presence of clay minerals (Lamb et al., 2009; Sauvé
77
et al., 2000). In microplastic research, however, the interactions between plastic toxicity and soil
78
properties have not been fully addressed. In the present study, we conducted bioassays in both
79
liquid and soil media to evaluate the media-dependent effects of nano- and micro-plastic particles,
80
using a variety of field soils. Based on our previous finding, the free-living nematode,
81
Caenorhabditis elegans, was chosen as the test species, and the number of offspring was selected as
82
the primary indicator of the effects of these particles (Kim et al., 2014; 2018). The plastic particles
83
of two different sizes and five soil samples were used to assess whether the effects of these particles
84
were dependent on particle size and soil physicochemical properties.
85
5
86
2. Materials and methods
87
2.1. Materials and target organism
88
Polystyrene (PS) beads of two different sizes, 42 nm (42PS; product number FS02F-12719) and
89
530 nm (530PS; product number FS03F-9360), were obtained as dispersions from Bangs
90
Laboratories, Inc. (Fishers, IN, USA). These beads exhibited strong fluorescent properties with <
91
480 nm excitation and > 520 nm emission wavelengths. To confirm the PS particle type, a Fourier
92
transform infrared (FTIR, 4100typeA, JASCO, Japan) spectral analysis was carried out using dried
93
samples at a wavenumber range of 4000–650 cm-1 (Fig. S1). The morphologies and average
94
diameters of 42PS and 530PS particles were measured using a field emission transmission electron
95
microscope (FE-TEM, JEM-2100F, JEOL, Japan). The hydrodynamic sizes and zeta potential (mV)
96
of each PS particle type in deionized water (DW) and K-medium (0.032 M KCl, 0.051 M NaCl)
97
(Brenner, 1974) were determined under specified test conditions, using a dynamic light-scattering
98
analyzer (Zetasizer Nano ZS, Malvern Instruments, UK). As shown in Fig. S2, the 42PS and 530PS
99
particles were moderately spherical with average diameters (n = 100) of 68 ± 8 and 496 ± 14 nm,
100
respectively. The hydrodynamic diameters of the 42PS particles in DW and K-medium were 62 and
101
59 nm and those of the 530PS particles were 559 and 527 nm, respectively. The zeta potentials of
102
the 42PS particles in DW and K-medium were 45.1 ± 0.6 and −28.7 ± 0.6 mV and those of the
103
530PS particles were 49.5 ± 0.5 and −12.8 ± 0.5 mV, respectively.
104
The target organism, C. elegans (wild type, Bristol strain N2), was obtained from the Animal
105
Genomics Laboratory, Konkuk University (Seoul, South Korea). The cultures were maintained on
106
the nematode growth medium (NGM; NaCl 3 g/L, peptone 2.5 g/L, agar 17 g/L, 1 M potassium
107
phosphate 25 mL/L, 1 M CaCl2․2H2O 1 mL/L, 1 M MgSO4·7H2O 1 mL/L, cholesterol 1 mL/L),
108
with Escherichia coli strain OP50 supplied as its food source (Brenner, 1974). All cultures were
109
maintained at 20 ± 2°C in the dark. To synchronize worm age, the C. elegans strains that were at
6
110
least 3 d old were treated with a Clorox solution for 10 min, after which the suspension was
111
centrifuged at 400 × g for 2 min to collect the pellets containing embryos. Subsequently, the pellets
112
were washed thrice with K-medium, and the embryos were placed on new NGM agar plates with E.
113
coli strain OP50. The plates were incubated until the nematodes reached the early-adult stage (54–
114
58 h synchronization after the Clorox solution treatment). All media components were obtained
115
from BD (Sparks, MD, USA), except for sodium chloride and potassium chloride, which were
116
obtained from Duksan (Asan, Korea).
117 118
2.2. Liquid and soil media tests
119
Liquid media tests were performed according to previous studies with some modifications
120
(Williams and Dusenbery, 1990). K-medium (1 mL) with different nominal concentrations of 42PS
121
and 530PS particles (0.01, 0.1, 1, 10, and 100 mg/L) was placed into each well of a 24-well plate,
122
followed by the inoculation of 10 age-synchronized worms (54–58 h) into each well. Each PS
123
particle type was assessed using eight replicates and a negative control (plastic-free condition). The
124
plates were maintained at 20 ± 2°C for 24 h in the dark. Subsequently, the number of offspring was
125
counted under a microscope, and the data were expressed as a percentage (%) of the mean of the
126
control group.
127
Soil media testing was performed using the nematode offspring counting assay, as described
128
in our previous studies (Kim et al., 2014; 2018). The nematodes were exposed to the test soil using
129
a standardized toxicity method (ASTM, 2001; ISO, 2010); the offspring in the test soil were
130
attracted by a microbial food source on an agar plate. Landwirtschaftliche Untersuchungs und
131
Forschungsanstalt (LUFA 2.2, LUFA Speyer, Germany) was selected as the test soil, and air-dried
132
for 7 days after sieving (< 2 mm). The stock solutions of both PS particle types (42PS and 530PS)
133
were prepared in K-medium to adjust their final soil concentrations, which were 0 (plastic-free
134
condition), 0.01, 0.1, 1, 10, and 100 mg/kg (n = 8). K-medium (0.2 mL) and 10 age-synchronized
7
135
worms were added to each well of the 24-well plate containing 0.3 g of the test soil. After 24 h, the
136
soil containing worms was placed on soil-agar isolation plates (Kim et al., 2014, 2018). To prepare
137
these plates, E. coli strain OP50 was cultured in Luria-Bertani medium (25 g/L) at 37°C for 16 h,
138
and 150 µL of the cell suspension (optical density 1.1–1.2, 8.0–9.6 × 108 cells/mL) was smeared
139
onto each side of the NGM agar plate. The test soils were arranged linearly in the central area of the
140
soil-agar isolation plates, and the number of offspring being attracted from the soil to each side of
141
the NGM agar plate was counted and expressed as a percentage (%) of the average value of the
142
control group.
143 144
2.3. Physiochemistry-dependent toxicity of PS beads in soil
145
Five field soils (S1–S5) were collected from metalliferous sites in South Korea. These sites
146
were located on the perimeter of an arsenic smelter and had been remediated using a soil washing
147
process. The soil conditions were unpredictable owing to large variations in soil physicochemical
148
properties, especially pH and texture (Kim et al., 2017). Soils were hand sorted, air-dried, and
149
sieved to a size of < 2 mm. Aggregated stability (AS) was calculated by weighing the soil after
150
slowly wetting on a 0.25-mm sieve according to the soil quality test kit guide (USDA, 1999). Bulk
151
density (BD) was measured as described previously (Friedman et al., 2001; Tan, 2005). The cation-
152
exchange capacity (CEC) was determined by extracting the cations with ammonium acetate and
153
potassium chloride solutions. Electrical conductivity (EC) was calculated by an instrumental
154
analysis after 1:1 (soil:water) extraction (EC meter, Multi Meter CP-500L; Istek, Seoul, Korea).
155
Organic matter (OM) was determined via the loss on ignition test (700–800°C, 4 h). The soil pH
156
was measured after DW extraction (1:5), and the soil texture (percentage of clay, sand, and silt) was
157
studied using a Soil Texture Test Kit (model 1067; LaMotte, Chestertown, MD, USA). Water
158
holding capacity (WHC) was assessed by weighing the soil after performing the wet-dry process for
159
3 h (Table S1) (Harding and Ross, 1964; USDA, 1999, 2001). The total concentrations of metals,
8
160
including arsenic (As), cadmium (Cd), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn), were
161
analyzed using an inductively coupled plasma atomic emission spectrophotometer (ICP-AES; JY
162
138, Ultrace, Jobin Yvon, France) after performing an acid digestion process (Table S1). Dry
163
samples of each soil (0.2 g) were transferred into a Teflon vessel and treated with a mixture of nitric
164
acid (HNO3), perchloric acid (HClO4), and hydrofluoric acid at 200°C. Thereafter, 20 mL of DW
165
was poured into the vessel, after which a metal analysis was performed using ICP-AES. The
166
detection limits of ICP-AES were 0.5 (As), 0.05 (Cd), 0.05 (Cu), 0.05 (Ni), 0.1 (Pb), and 0.05 (Zn)
167
µg/L.
168
A nematode offspring counting assay was used to assess the toxicity of PS particles in
169
different field soils. Soil samples S1–S5 (0.3 g) were added to each well of a 24-well plate and
170
moistened with K-medium. The stock solutions of both 42PS and 530PS particles were prepared in
171
K-medium to adjust their soil concentrations; the nominal concentrations of PS in soil were
172
determined to be 0 (plastic-free condition), 0.01, 0.1, 1, 10, and 100 mg/kg (n = 4). The water
173
content of each soil sample was adjusted to 140–145% of WHC (i.e., the same water content as the
174
LUFA test). Ten age-synchronized worms were exposed to the soil samples for 24 h, after which
175
the nematode offspring counting assay was performed as described above.
176 177
2.4. Data analysis
178
To determine the factors that are primarily responsible for the toxicity of PS particles, we
179
used a principal component analysis (PCA) based on minimum data set (MDS) selection (Andrews
180
et al., 2002; Govaerts et al., 2006). Offspring number measurements for each concentration (100,
181
10, 1, and 0.1 mg/kg) of the 42PS and 530PS particles were used as the test data set; the data for ten
182
physicochemical properties and concentrations of six metals were also considered. Only the
183
principal components (PC) with an eigenvalue >1 were considered for selection, and the
184
physicochemical properties with an eigenvector within 20% of the measurement data for each
9
185
concentration of the 42PS and 530PS particles were determined as the dominant factors influencing
186
PS toxicity (Andrews et al., 2002; Wander and Bollero, 1999).
187
The Dunnett’s Program (ver. 1.5) was used to calculate the 10% effective concentration
188
(EC10) and significant differences (p < 0.05) between the mean values of the control and treatment
189
groups (USEPA, 1999). The median effective concentration (EC50) values were calculated using the
190
Trimmed Spearman-Karber method (Hamilton et al., 1977).
191 192
3. Results
193
3.1. Size-dependent toxicity of PS in different test media
194
As shown in Fig. 1A, 100 mg/L of the 42PS and 530PS particles in liquid media caused a
195
significant decrease in offspring number (p < 0.05). There was no evidence of a size-dependent
196
effect since both 42PS and 530PS particles produced similar effects. In the soil media test, the
197
number of offspring started to decrease significantly (p < 0.05) at 10 mg/kg of the 530PS particles;
198
however, for the 42PS particles, significant decreases (p < 0.05) were observed only at 100 mg/kg
199
(Fig. 1B). The EC10 and EC50 values for the 42PS and 530PS particles in liquid and soil (LUFA)
200
media are shown in Table S2. Some toxicity values could not be calculated because of the large chi-
201
square value or high level of trimming (>70%) in the statistical program.
202 203
3.2. Physiochemistry-dependent toxicity of PS beads in soil
204
Next, we investigated whether the different physicochemical properties of each soil sample
205
(S1–S5) had a significant effect on the number of offspring in the absence of PS treatment. The S2,
206
S4, and S5 soil samples had significant effects on the nematode population (p < 0.05) (Fig. S3).
207
Each data point for the 42PS and 530PS particles was expressed as a percentage (%) of the average
208
value of each control group. The metal concentrations in the target field soils were < 43.9, N.D., <
209
57.9, < 18.1, < 96.3, < 124.0 mg/kg for As, Cd, Cu, Ni, Pb, and Zn, respectively (Table S1). Our
10
210
previous study found that metal concentrations < 200 mg/kg produced no significant toxic effects in
211
the nematode bioassay (Kim et al., 2018). In soil sample S1, the 530PS particles significantly
212
decreased the number of offspring (p < 0.05) at all tested concentrations (0.01–100 mg/kg), while a
213
significant decrease in offspring number (p < 0.05) could only be observed at 100 mg/kg of the
214
42PS particles (Fig. 2A). For soil sample S2, significant reductions in offspring number (p < 0.05)
215
began at 0.1 mg/kg of the 530PS particles and at 10 mg/kg of the 42PS particles (Fig. 2B). The S3
216
and S4 soil samples exhibited trends similar to the LUFA soil (Fig. 2C and D), while in soil sample
217
S5, the toxicity of the 530PS particles was significantly higher than that of the 42PS particles (Fig.
218
2E). The EC10 and EC50 values for each soil sample are shown in Table S2.
219 220
3.3. Determination of factors strongly related to PS toxicity
221
Different concentrations of the 42PS particles exhibited different correlations with various
222
soil factors, while the 530PS particles had constant correlations with BD, CEC, clay content, Pb
223
(T), and sand content even at different concentrations. The first (PC1), second (PC2), third (PC3),
224
and fourth (PC4) principal components were classified as useful data loading sets (eigenvalue >1)
225
for each concentration of the 42PS and 530PS particles (Table S3–S6) and are shown as the PC1
226
and PC2 loading plots in Fig. 3. The PCA data are generally used to explain the positive or negative
227
directions between each data set. The clay content in all figures was located opposite the sand
228
content and in the same direction as WHC and CEC (Fig. 3A–D). As shown in the supplementary
229
tables, the eigenvectors were used to determine the soil factors related to PS toxicity. For example,
230
at the concentration of 100 mg/kg, the total variations were calculated as 40%, 28%, 20%, and 8%
231
for PC1, PC2, PC3, and PC4, respectively (Table S3). The eigenvector for the 42PS particles (100
232
mg/kg) was 0.082 in PC1, which showed a positive correlation with Ni (T). The eigenvector for the
233
530PS particles (100 mg/kg) was −0.335 in PC1, which exhibited positive correlations with both
234
BD and Pb (T) and negative correlations with CEC, clay content, and WHC (Fig. 3A and Table S3).
11
235
In summary, the dominant soil factors at each 42PS concentration were determined to be AS, Cu
236
(T), EC, Ni (T), OM, Pb (T), and pH, with no certain direction in each data set. In the data analysis
237
for the 530PS particles, the soil factors included BD, CEC, clay content, sand content, and WHC.
238 239
4. Discussion
240
Previous studies have reported the toxicity of plastic particles towards C. elegans using
241
liquid media tests. Lei et al. (2018) have reported that an exposure (2 days) to 5.0 mg/m2 of
242
microplastics can significantly impact the survival, growth, and reproduction of C. elegans. Zhao et
243
al. (2017) found that a chronic exposure (4.5 days) to nano-polystyrene induces alterations in the
244
behavior, survival, and reproduction of C. elegans at concentrations higher than 10 µg/L, and these
245
effects could be transferred to the next generation. In the present study, we found that the number of
246
offspring significantly (p < 0.05) decreased at 100 mg/L in liquid media. The liquid media test
247
showed no evidence of size-dependent effects, whereas the soil media test exhibited size-dependent
248
effects beginning at 10 mg/kg. This phenomenon indicates two important facts; a) the toxicity of PS
249
particles can increase in soil media, and b) the 530PS particles were more toxic than the 42PS
250
particles in soil media. Because there are considerable differences in the units used and the actual
251
exposed concentration, it could be argued that this media-dependent comparison might not be
252
reasonable. Despite this, our results showed a clear difference in offspring number (approximately
253
40%; Fig. 1) obtained by using similar concentrations of the 530PS particles, i.e., 10 mg/L and 10
254
mg/kg in the liquid and soil media, respectively. In previous studies, different media tests have been
255
used to explain the interaction between solid and liquid phases in soils (Eom et al., 2007; Juvonen et
256
al., 2000; Pandard et al., 2006). Test organisms, such as C. elegans, generally show a higher
257
sensitivity to PS particles, even at similar concentrations, in liquid media than that in in soil media
258
(Donkin and Dusenbery, 1994).
12
259
Previous studies have also reported size-dependent effects of plastic polymers in live
260
organisms. Jeong et al. (2016) found that the smaller polystyrene particles (0.05 µm) have a greater
261
toxic effect than that of the larger particles (0.5 and 6 µm) on the fecundity and life span of rotifer
262
species. They also found that the smaller polyethylene particles (1–4 µm) had significant
263
immobilization effects on Daphnia, unlike the larger ones (90–106 µm) (Reche et al., 2016). These
264
findings might be linked to the feeding behavior of organisms because the smaller particles are
265
easier to accumulate (Wang et al., 2009), while the larger particles are more effectively egested
266
(Jeong et al., 2016). Ziajahromi et al. (2018) reported that the sediment-dwelling invertebrate
267
Chironomus tepperi was affected by polyethylene particles, and the effect was size-dependent. They
268
found that the smaller polyethylene particles (10-27 µm) induce significant adverse effect on the
269
growth and survival of C. tepperi than the larger ones (100-126 µm). However, very limited studies
270
have been conducted on the size-dependent effects of plastic particles in soil systems. Jiang et al.
271
(2019) found that nano-sized polystyrene (100 nm) has a higher genotoxic and oxidative damage
272
than micro-sized one (5 µm) on higher plant Vicia faba in culture solution. Lei et al. (2018) reported
273
that C. elegans showed a more sensitive response to the smaller (0.1–1.0 µm) PS particles compared
274
with that of the larger particles (5.0 µm). However, their experiments were performed in filter paper
275
and liquid media. In previous studies of other nanomaterials, such as metallic nanoparticles, C.
276
elegans showed a more sensitive response to smaller Al2O3 (Wang et al., 2009), CeO2 (Roh et al.,
277
2010), Fe oxide (Höss et al., 2015), TiO2 (Roh et al., 2010; Wang et al., 2009), and ZnO (Wang et
278
al., 2009) nanoparticles. These studies posited that the aggregate size and surface area of
279
nanoparticles are highly interactive with the size-dependent effects on C. elegans; however, these
280
studies also used liquid media.
281
The S1, S2, and S5 samples showed significant decreases in offspring number upon exposure to
282
the 530PS particles at very low concentrations (0.01–0.1 mg/kg). Although some reports have
283
estimated that 43,000–63,000 tons of microplastic are added into the agricultural system annually
13
284
(Nizzetto et al., 2016), the relevant concentrations of nanoplastic particles in the soil environment
285
have not yet been reported. Hence, our test concentrations cannot be directly compared with actual
286
environmental concentrations. The S3 and S4 soil samples showed a trend similar to that seen in the
287
LUFA soil, in that the only significant decrease occurred at a concentration of 100 mg/kg for both
288
42PS and 530PS particles. This difference from the other soil samples (S1, S2, and S5) might be
289
strongly related to the soil physicochemical properties. As shown in Table S1, the S1, S2, and S5
290
groups had high levels of AS, EC, and clay, whereas the LUFA, S3, and S4 groups showed high
291
levels of sand. Clay generally consists of particles with sizes < 2 µm and can form residual (< 0.2
292
µm) and storage (0.2–30 µm) pores (Greenland, 1977). Thus, particles in the size range 0.2–2 µm,
293
which includes the size range of 530PS, can be classified as clay particles. The composition of the
294
test soils containing 530PS particles might differ from the control soil, and this can affect the
295
nematode population because it has been shown to be negatively correlated with fine-textured soil
296
(Elliot et al., 1980; Ward, 1975; Yeates, 1981). These effects are not as pronounced in coarse-
297
textured soils, such as in the LUFA, S3, and S4 groups, because these soils have very low levels of
298
clay (Table S1). Our test results with the field soil samples enabled us to conclude that soil texture
299
fractions strongly influence PS toxicity in soil environments.
300
PCA is commonly used to evaluate linear and monotonic relationships between specific variables
301
and can provide insights into how several parameters significantly influence each other (Andrews et
302
al., 2002). Using this approach, we found that the toxicity of PS particles is conditioned by the soil
303
physical characteristics including texture fractions. We found that the correlations for the 42PS
304
particles were inconclusive because no trends were noted between the data loadings. In the 530PS
305
data set, correlations were observed among BD, CEC, clay content, and sand content (Fig. 3; Table
306
S3–6). Because CEC is generally positively related to the clay content in soil systems (Lambooy
307
1984), we concluded that the soil textures (clay, sand, and silt) are strongly related to PS toxicity.
308
The data obtained with different concentrations (100, 10, 1, and 0.1 mg/kg) of both 42PS and
14
309
530PS particles were reconstituted according to the sand content (%), and a dot graph was
310
constructed as shown in Fig. 4. At the concentration of 100 mg/kg of both 42PS and 530PS particles
311
(Fig. 4A and B), no relationships were observed between each data set at different sand contents. A
312
weak relationship was observed for the 42PS particles (Fig. 4C, E, and G), whereas the 530PS
313
particles showed increasing trends with high R2 values of 0.7347, 0.6268, and 0.6646 for
314
concentrations 10, 1, and 0.1 mg/kg, respectively (Fig. 4D, F, and H). A clay content (%)-dependent
315
dot graph was also constructed, as shown in Fig. 5. The data for 100 mg/kg 42PS and 530PS
316
particles showed trends similar to those of the sand content-dependent dot graph (Fig. 5A and B).
317
For the concentrations of 10, 1, and 0.1 mg/kg of the 530PS particles, the high R2 values were
318
found to be 0.7987, 0.7844, and 0.7019, respectively (Fig. 5D, F, and H), and a decreasing trend
319
was observed for each concentration. According to these data, the increase in the sand content
320
appeared to be strongly related to the reduction of PS toxicity in the soil system.
321
We confirmed the media-dependent changes in PS toxicity, showing that C. elegans exhibited a
322
more sensitive response in soil media than that in liquid media. The PS size-dependent effects
323
occurred in soil media, with texture fractions strongly related to PS toxicity. However, our results
324
have some limitations; the threshold PS concentration causing adverse effects in the nematode
325
species was observed at a very low plastic concentration (0.01 mg/kg) in soil media, and it might be
326
caused by other factors such as contaminants or organic matter. Although these limitations should
327
be addressed in future studies, our results add to the existing knowledge on the occurrence and
328
effects of plastic particles in soil systems.
329 330
Acknowledgement
331
This paper was supported by Konkuk University in 2019. We thank the Korean Basic Science
332
Institute (KBSI) for their assistance with our dynamic light scattering and TEM analyses.
333
15
334
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Fig. 1. The number of Caenorhabditis elegans offspring following exposure to the 42PS and 530PS particles in (A) liquid (mg/L) and (B) soil (mg/kg) media. All data were normalized to each control group. Error bars indicate standard deviations, and asterisks (*) indicate significant (p < 0.05) differences compared with the control.
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Fig. 2. The number of Caenorhabditis elegans offspring following exposure to the 42PS and 530PS particles in five field-soil samples; (A) S1, (B) S2, (C) S3, (D) S4, and (E) S5. All data were normalized to each control group. Error bars indicate standard deviations, and asterisks (*) indicate significant (p < 0.05) differences compared with the control.
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Fig. 3. Loading plots of the 42PS and 530PS data and physicochemical properties for (A) 100 mg/kg, (B) 10 mg/kg, (C) 1 mg/kg, and (D) 0.1 mg/kg of soil. Loading plots were generated by a principal component analysis (PCA). The x- and y-axes indicate the first (PC1) and second (PC2) principal components, respectively. The blue lines represent the loading per data, and the 42PS and 530PS data are shown in green. AS = aggregate stability, BD = bulk density, CEC = cation exchange capacity, Clay = clay content (%), EC = electrical conductivity, OM = organic matter, Sand = sand content (%), Silt = silt content (%), WHC = water holding capacity, Metals (T) = total concentration of each metal (As, Cd, Cu, Ni, Pb, and Zn).
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Fig. 4. Reconstituted dot plot according to sand content; (A) 42PS 100 mg/kg, (B) 530 PS 100 mg/kg, (C) 42PS 10 mg/kg, (D) 530PS 10 mg/kg, (E) 42PS 1 mg/kg, (F) 530PS 1 mg/kg, (G) 42PS 0.1 mg/kg, and (H) 530PS 0.1 mg/kg. The x- and y-axes indicate the sand content (%) and offspring number (% control), respectively. All data were from LUFA (Landwirtschaftliche Untersuchungs und Forschungsanstalt Germany) and five field-soil samples (S1–S5). Error bars indicate standard deviations, and asterisks (*) indicate significant (p < 0.05) differences compared with measurements of the lowest sand content.
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Fig. 5. Reconstituted dot plot according to clay content; (A) 42PS 100 mg/kg, (B) 530 PS 100 mg/kg, (C) 42PS 10 mg/kg, (D) 530PS 10 mg/kg, (E) 42PS 1 mg/kg, (F) 530PS 1 mg/kg, (G) 42PS 0.1 mg/kg, and (H) 530PS 0.1 mg/kg. The x- and y-axes indicate clay content (%) and offspring number (% control), respectively. All data were from LUFA (Landwirtschaftliche Untersuchungs und Forschungsanstalt Germany) and five field-soil samples (S1–S5). Error bars indicate standard deviations, and asterisks (*) indicate significant (p < 0.05) differences compared with measurements of the lowest clay content.
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HIGHLIGHTS We assessed the nano- and micro- plastic toxicity using nematode species in soil. C. elegans showed more sensitive response in soil than in liquid. The nematodes showed high sensitivity to larger particles than smaller ones. The size-dependent effects were intensified in clay-rich soil samples. We concluded that the plastic toxicity is highly linked with soil properties.
Author statement Shin Woong Kim: Conceptualization, Methodology. Writing - Original Draft Dasom Kim: Investigation, Writing - Original Draft Seung-Woo Jeong: Writing - Review & Editing Youn-Joo An: Supervision, Writing - Review & Editing, Funding acquisition
Conflict of interest The authors declare no conflicts of interest.