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Low-density polyethylene microplastics as a source and carriers of
agrochemicals to soil and earthworms.
Cite this article as:
Rodríguez-Seijo Andrés, Santos Bruna, Ferreira da Silva Eduardo, Cachada
Anabela, Pereira Ruth (2019) Low-density polyethylene microplastics as a source
and carriers of agrochemicals to soil and earthworms. Environmental Chemistry
16, 8-17 https://doi.org/10.1071/EN18162
The full version can be found at CSIRO https://doi.org/10.1071/EN18162
1
Low-density polyethylene microplastics as a source and carriers of agrochemicals to soil and
1
earthworms
2
3
Andrés Rodríguez-Seijo1*; Bruna Santos1, Eduardo Ferreira da Silva2, Anabela Cachada1,3,4, Ruth Pereira1
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1GreenUPorto and Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal.
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2Department of Geosciences, Geobiotec Research Centre, University of Aveiro, Aveiro, Portugal.
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3Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto,
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Terminal de Cruzeiros do Porto de Leixões, Matosinhos, Portugal.
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4CESAM, University of Aveiro, Campus de Santiago, Aveiro, Portugal.
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*Corresponding author:
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Andrés Rodríguez Seijo
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GreenUPorto and Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal.
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andresrodriguezseijo@hotmail.com
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Abstract
17
Microplastics (MPs) are an environmental issue on marine ecosystems due to the remarkable evidences of
18
their presence and the adverse effects on organisms, but studies addressing the problem on soils and its
19
biota are scarce. Several questions can arise related with this major environmental problem, and its
20
impacts on terrestrial ecosystems, mainly whether MPs could transport contaminants (e.g. pesticides) to
21
the soil matrix and if they could be a carrier of pesticides to soil biota through ingestion. In order to
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contribute to the understanding of these issues, earthworms (Eisenia fetida) were exposed for 14 days to
23
soil containing MPs with two different sizes (5 mm and 0.25 µm-1 mm), previously sprayed or not with
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chlorpyriphos (CPF). Acetylcholinesterase activity and thiobarbituric acid reactive substances were
25
measured to track the exposure of earthworms to the MPs alone or combined with CPF. The behaviour of
26
earthworms in the test containers was also assessed, as well as the movement of MPs on soil. The
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concentration of CPF in the soil at the end of the experiment differed between treatments with MPs of
28
different sizes (17.9 ng g-1 and 2442 ng g-1 for large and small MPs, respectively), probably due to the
29
2
highest specific surface area of small MPs. Despite the ability of the MPs to release the pesticide CPF to
30
the soil, the earthworms have firmly avoided the contaminated MPs when the concentrations in soil were
31
higher. No evidences of MPs uptake were recorded. In summary, MPs may contribute with pesticides to
32
the soil, and may have impacts on soil biota with important ecosystem functions.
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Keywords: Acetylcholine; earthworms; ecotoxicology, low-density polyethylene, microplastics,
34
oxidative stress; terrestrial ecosystems; thiobarbituric acid reactive substances.
35
Abbreviations
36
AChE - Acetylcholinesterase; CPF – chlorpyrifos; CTL – control soil; DTNB - Ellman's Reagent; LDPE -
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Low-density polyethylene; MPs - Microplastics; PE – Polyethylene; TBARS - Thiobarbituric acid
38
reactive substances.
39
3
Introduction
40
Microplastics (MPs), small plastic pieces with less than 5 mm, are a well-known threat to aquatic
41
ecosystems (rivers, lakes, oceans, seas) and hundreds of studies have assessed the risks to biota,
42
especially in what regards marine environments (Derraik et al. 2002; Rillig 2012; Horton et al. 2017;
43
Bläsing and Amelung 2018). Microplastics in soil are a growing concern, which only started to receive
44
the attention of the scientific community a few years ago (Rillig 2012). Indeed, their distribution in soils
45
has not been thoroughly characterized, although it is known that terrestrial ecosystems are the primary
46
source of MPs to freshwater and marine ecosystems (Nizzetto et al. 2016; Bläsing and Amelung 2018;
47
Chae and An 2018; Ng et al. 2018; Rochman 2018). This might be occurring given the difficulties
48
associated with extracting and quantifying MPs in a complex matrix like the soil (Rodríguez-Seijo et al.
49
2017; Bläsing and Amelung 2018; Hurley et al. 2018), as well as, to the more evident impacts caused by
50
the effects on marine biota, which made public opinion and authorities more concerned about problems at
51
sea.
52
Plastic wastes are continuously entering the soils through four main pathways: i) inputs from
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agricultural practices; ii) the use of sewage sludge for soils fertilization; iii) inappropriate deposition of
54
plastics and, iv) water and wind erosion (Nizzetto et al. 2016; Chae and An 2018; Ng et al. 2018;
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Rochman 2018). Geyer at al. (2017) studied the production, use and fate of all plastics ever made and
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concluded that 79 % of plastic waste generated (4900 of 6300 million tons) were accumulated in landfills
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or across terrestrial and aquatic ecosystems. Plastic litter, once in the environment, can go through a
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process of both degradation and disintegration, as a result of physical and chemical weathering,
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generating MPs (Barnes et al. 2009). This is considered a secondary form of MPs, whereas the primary
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forms can be found in cosmetic and industrial products (Rillig 2012).
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Since the middle of the last century, plastic polymers such as polyethylene or polypropylene have
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been widely used in agriculture, allowing farmers to increase crop production due to their use for
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mulching and greenhouses (Kyrikou and Briassoulis 2007; Kasirajan and Ngouajio 2012). Furthermore,
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Nizzetto et al. (2016) estimated that 125-850 tons of MPs million-1 inhabitants were added annually to
65
soils in Europe, through the application of sewage sludge both directly and as biosolids on agricultural
66
soils, for fertilisation purposes. As a result of this final-end of sewage sludge, recommended by European
67
policies like the sludge directive (European Union 1986), Zubris and Richards (2005) showed that
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synthetic fibres can be easily detected in agricultural soils 15 years after sludge application. Weithmann et
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4
al. (2018), also showed that organic fertilisers from recycled biowastes, depending on how they were
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composted/partially digested, still contain MPs and agrochemicals.
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In this context, and for decades, plastics and agrochemicals have been deliberately applied to soils
72
(e.g., Nerin et al. 1996; Gonçalves et al. 2005; Ramos et al. 2015; Steinmetz et al. 2016). Ramos et al.
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(2015) showed how plastics and agrochemicals are highly related by the adsorption of pesticides on the
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surface of agricultural plastics, as well as the role of the resulting MPs as carriers of these pollutants
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(Nerin et al. 1996; Nerin and Battle 1999; Steinmetz et al. 2016). Ramos et al. (2015) concluded that
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pesticides accumulated in a plastic film could migrate to matrixes like soil and the atmosphere. Also,
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when present in agricultural soils, they can act as a sink and source of pesticides (584-2284 µg pesticide
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g-1 plastic in mulch film; 24-32 µg pesticide g-1 soil). However, this will depend on the chemical
79
composition of the substance. As Kleinteich et al. (2018) showed, MPs could act as carriers of
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hydrophobic pollutants (like polycyclic aromatic hydrocarbons), but their bioavailability was reduced due
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to the sorption of these organic compounds to the MPs. However, and once again, although the role of
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MPs as carriers of inorganic and organic contaminants has been studied for aquatic ecosystems
83
(Koelmans et al. 2015; Horton et al. 2017), research on soils is poor and recent (Horton et al. 2017; Yang
84
et al. 2018).
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The main goal of this study was to assess MPs influence in pesticides transport to soil and soil biota,
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using earthworms as an experimental organism. As a starting point, it was intended to answer two main
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questions: i) Can MPs be a source of pesticides to the soil? ii) Can MPs be vectors of the same
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contaminants to the biota that ingest them?
89
Chlorpyrifos (CPF) was the chosen active ingredient because it is a broad-spectrum
90
organophosphorothionate pesticide with several agricultural applications to a variety of food and feed
91
crops It is widely used all over the world, and its effect on earthworms have been intensively studied
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(Venkateswara Rao et al. 2003; Zhou et al. 2007; Pelosi et al. 2013; Muangphra et al. 2016).
93
Organophosphates inhibit acetylcholinesterase activity and cause an accumulation of acetylcholine in the
94
synaptic clefts (Dai et al. 2001), a neurotransmitter responsible by sending nervous signals to muscle
95
cells, at neuromuscular junctions and nerve synapses. As a result, acetylcholine can be accumulated and
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make an over-stimulation of the muscles and the central nervous system, causing death by asphyxiation.
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To answer the above described questions, a preliminary trial and two assays with MPs were performed.
98
5
The preliminary trial was done to determine the lowest sub-lethal concentration of CPF at which a
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response of earthworms can be detected. The concentrations were chosen based on the data from other
100
works. Later, a commercial formulation of CPF was diluted to the dose recommended by the supplier and
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used to contaminate MPs with 5 mm of diameter, to infer whether they could transport the pesticide to the
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soil matrix. As it was known from previous studies that MPs <3mm could potentially be ingested by
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earthworms (Shumway and Koide 1994; Zaller and Saxler 2007; Clause et al. 2011), MPs with a diameter
104
of less than 1mm were used to test if smaller MPs can also act as carriers of CPF to earthworms.
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Acetylcholinesterase (AChE) was the enzyme used as a biomarker in this study to track the exposure
106
of earthworms both to CPF desorbed from MPs to the soils, as well as to the pesticide hypothetically
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transported to the gut of earthworms by the MPs. This enzyme catalyses the hydrolysis of acetylcholine at
108
cholinergic synapses and neuromuscular junctions. It has been widely used to assess effects of
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organophosphate pesticides to organisms, as it is their primary target, along with other cholinesterase’s
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(Čolović et al. 2013; Pelosi et al. 2013; Sanchez-Hernandez et al. 2014). Thus, it is a specific biomarker
111
for these class of pesticides.
112
Organic contaminants, such as chlorpyrifos, and MPs have also been reported to cause elevated levels
113
of oxidative stress in earthworms (Wang et al. 2012; Muangphra et al. 2015; Rodríguez-Seijo et al.
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unpubl. data), specifically from peroxidation of lipid tails in the membrane structure (Markad et al. 2012;
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Zhang et al. 2013). Lipid peroxidation produces several complex products that can react with protein and
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DNA and cause damages (Abd El-Hakim et al. 2018). Thiobarbituric acid reactive substances (TBARS),
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produced as a result of lipid peroxidation, are used to quantify oxidative stress, and can be detected using
118
thiobarbituric acid as a reagent (Oakes et al. 2003). In that sense, TBARS acts as an effect biomarker, and
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it was selected to pursue the potential uptake of MPs and/or adsorbed CPF and the subsequent oxidative
120
stress effects.
121
122
Experimental
123
Test soil and earthworms
124
In this study, the OECD artificial soil was used as test substrate. It was prepared to be composed by 70 %
125
air-dried quartz sand (< 2mm), 20% kaolin clay and 10% peat (pH 6.5 ± 0.5; adjusted using calcium
126
6
carbonate). The water-holding capacity (WHC) of the soil was adjusted to 40% of its WHCmax (OECD
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1984; 2004).
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Earthworms of the species E. fetida (Savigny, 1826) were selected for this study and obtained from a
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laboratorial culture, kept under environmentally controlled conditions (photoperiod 16 hL:8 hD;
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temperature 20 ± 2 ºC) as described by Gavina et al. (2016). The organisms from the cultures were fed
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with defaunated horse manure or oatmeal once a week and grown in a medium composed of sphagnum
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peat, horse manure and deionised water. Defaunated horse manure is obtained through three freeze-
133
thawing cycles (48 h at -20 °C followed by 48 h at 25 °C) to eliminate the original soil fauna (Alves et al.
134
2015). The culture medium is moistened with deionised water. For each assay, adult individuals (with
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clitellum) weighing between 300 and 600 mg were used (OECD 2004). Before the tests, the earthworms
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were acclimatised 24 h in test containers with the OECD artificial soil, under the same conditions as the
137
ones described for culture maintenance.
138
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Microplastics and pesticide
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Low-density polyethylene (LDPE) pellets (C2H4)n from Sigma-Aldrich (CAS Number 9002 88 4, density
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0.918 g ml-1 at 25 ºC), with 5mm diameter (MPs5mm) and a diameter between 250 µm and 1mm
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(MPs1mm), were used. As it was known from previous studies that small MPs (<1 mm) could be ingested
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by earthworms (Huerta Lwanga et al. 2016; Rodríguez-Seijo et al. 2017), MPs were cut in a mill, and the
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resulting fragments were sieved on a calibrated vibratory sieve shaker Retsch AS200 (250 µm to 1 mm)
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following the methodology previously described by Rodríguez-Seijo et al. (2017). To remove any
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contamination or potential particles attached to the MPs was followed the procedure described by Karami
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et al. (2017). MPs were rinsed twice with deionised water and once with ethanol (70 %) and were dried
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overnight on aluminium foil (40 °C). The shape of smaller MPs was described in a previous study
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(Rodríguez-Seijo et al. 2017), which the same methodology was used.
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An aqueous solution of chlorpyrifos (CPF), prepared by the dilution of a commercial formulation
151
(Ciclone® 48EC, 43 % w/w chlorpyrifos, SAPEC AGRO, S.A., Setúbal, Portugal), was used in the
152
experiment. CPF has a typical half-degradation time DT50 of 386 days in laboratory conditions and 27.6
153
days at field conditions (PPDB 2018).
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7
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Experimental setup
156
Test 1: AChE as a biomarker of CPF exposure: preliminary test for the determination of the lowest sub-
157
lethal CPF concentration causing a response in E. fetida.
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For the preliminary assay, five concentrations were tested, ranging from 9.9 to 50 mg kg-1 soil dw (9.9;
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14.8; 22.2; 33.3 and 50), as well as a control (CTL) of non-contaminated soil (0 mg kg-1 soil dw). For each
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concentration, three replicates of 500 g of OECD soil were prepared. The soils were contaminated with
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Ciclone® 48 EC by using a stock solution of 5000 mg of CPF L-1. Through a series of dilutions in the
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deionized water volume needed to adjust the WHC of the soil, the pesticide was thoroughly mixed with
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the soil in the above-described concentrations, and the replicates were left 10 days at controlled
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conditions for stabilisation. After that, ten earthworms were added to the plastic containers (with
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perforated plastic lids), containing the contaminated and the CTL soil. Based on the results of previous
166
studies (Tomlin 2006; De Silva et al. 2010; Cang et al. 2017), no mortality was expected from any of the
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containers. The trial went on for 14 days, and no food was provided during the assay. The soil moisture of
168
each replicate was monitored and adjusted with deionised water whenever necessary. The test was carried
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out in the same conditions previously described for the maintenance of the culture of earthworms.
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Tests 2 and 3: MPs as carriers of pesticides to soil and biota: earthworms’ exposure to MPs5mm and
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MPs1mm contaminated with CPF
172
The first test, with large MPs (5mm), was performed including three different treatments with four
173
replicates each, namely: soil with MPs5mm + CPF, soil with MPs5mm, and the CTL (without MPs and
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CPF). For the first treatment (MPs5mm+CPF) MPs were sprayed with the pesticide before being added to
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the soil. A stock solution of the commercial formulation of the pesticide was prepared to spray a plastic
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film (area: 1 m2), where 40 MPs5mm were spread on the surface, of which 32 were randomly selected
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and used for the test. A figure of MPs exposure to the pesticide was included in the supplementary
178
material (Fig. S1). The stock solution of CPF has prepared in order to follow the dose recommended by
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the supplier (4L ha-1). The MPs were left to dry on the surface of the plastic film at greenhouse conditions
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for seven days. After this period, contaminated as well as non-contaminated MPs (MPs5mm), were added
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to the soil. For each replicate, eight MPs5mm or MPs5mm+CPF were placed on 500 g of OECD soil
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(moistened to attain 40% of its WHCmax) at 2 cm below the top of the surface (the location marked with
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8
a line in the test container, for better identification). The MPs were left on the soil for 10 days, then ten
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earthworms weighing between 300 mg to 600 mg were picked and added to the plastic containers on the
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soil surface, and left to burrow. They were kept in the same conditions as stated before: 14 days without
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feeding and with the periodical addition of deionised water to kept soil moisture constant and at the same
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light and temperature conditions described for the cultures.
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After that, a final trial with small MPs (< 1 mm) was carried out. A control soil (CTL) was done once
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again, as well as treatments with both MPs without pesticide (MPs1mm) and MPs sprayed with pesticide
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(MPs1mm+CPF), four replicates of each. For each treatment, between 180-200 MPs particles were added
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to achieve the same mass than 8 MPs (± 0.21 g). Microplastics less than 1mm were sprayed with an
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aqueous solution of the commercial formulation of CPF as described before. For easier identification, all
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the MPs were randomly placed 2 cm below the surface of the soil once again, and its location marked on
194
the the container. Microplastics stayed in the soil for 10 days, after which ten earthworms from 300 mg to
195
600 mg were added, as described for the previous trial, and stayed in the soil for 14 days.
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Finally, the movement of MPs was evaluated when removing the soil from the test containers. It was
197
slowly and carefully done with a metal spoon, followed by direct visual observation of each portion of
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soil. It was paid attention to the line on the 2 cm, outlining their original placement outside the
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experimental vessel. It was recorded if MPs were at top two centimetres, at the two centimetres or were
200
moved along by the test vessel up to the bottom (8 cm far away to the MPs line). For the MPs1mm, MPs
201
movement was recorded following visual identification under a binocular stereo microscope (Woodall et
202
al. 2014; Martin et al. 2017; Klein et al. 2018) and the methodology proposed by Rillig et al. (2017a) with
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soil cores as experimental units to extrapolate the results.
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Earthworms’ movement was verified, by visual identification of their position when carefully
205
removing the soil and MPS. It was recorded if earthworms were at the first two centimetres, at the two
206
centimetres or were at lower depths the experimental unit.
207
Determination of chlorpyrifos (CPF) concentrations in soils
208
A composite soil sample of each treatment was obtained at the end of each assay, and soils were frozen,
209
freeze-dried and analysed for it CPF content. Ten grams of soil were extracted with a mixture of
210
hexane:acetone (1:1), followed by a clean-up of extracts with Florisil© and elution with hexane:acetone
211
(9:1) (Caetano et al. 2012; Patinha et al. 2018). Chlorpyrifos was quantified using gas chromatography
212
9
with mass spectrometry detection (Agilent GC 7890B/ 5977A MSD), using helium as a carrier gas (1.2
213
ml min-1), and a Guardian ZB5-MS fused silica capillary column (30 m x 0.25 mm x 0.25 μm,
214
Phenomenex). The detection was achieved using electron ionization in the single ion monitoring mode
215
(SIM). The injection volume was 1 µL, and spitless injection mode was used. The injector temperature
216
was 275 °C, and the MS transfer line temperature was 250 °C. The oven temperature was programmed as
217
follows: 70 °C for 1.5 minutes, increase at a rate of 10 °C min-1 until 200 °C, followed by 7 °C min-1 until
218
320 °C and kept for 3 minutes. An internal standard calibration was used, ranging from 50 to 500 ppb (5
219
points, r2 > 0.9999), prepared using a Chlorpyrifos standard solution (100 mg · ml-1, CPAChem). The
220
internal standard (Chlorpyrifos D10, 100 mg · ml-1, Dr. Ehrenstorfer) was added to the samples before
221
analysis. The precision of the method was lower than 20 %, over the working range. Accuracy was
222
assessed by recoveries of fortified samples, and it was within an acceptable range (86-104 %). Method
223
blanks were used in every tenth sample to detect possible interferences/contamination from the reagents,
224
glassware and other processing hardware, and results were always below the instrumental detection limit
225
(5 ng · g-1). Relative Standard Deviation in intermediate precision conditions ranged between 9 and 20 %.
226
Molecular biomarkers: AChE and lipid peroxidation
227
After each assay, a pool of five earthworms, randomly selected per each replicate, were frozen in liquid
228
nitrogen, and stored at -80 ºC in a freezer before homogenization. After that, the tissues were
229
homogenised in an ice-cold phosphate buffer (50 mM, pH=7.0 with 0.1% Triton X-100) using a tissue
230
homogeniser (T10 basic ULTRA-TURRAX®), at 4 °C. Homogenized samples were centrifuged (3000
231
rpm for 10min at 4 °C), and the supernatants were divided in aliquots and frozen in Eppendorf tubes at -
232
20 ºC, according to the methodology described by Correia et al. (2017).
233
Acetylcholinesterase activity (AChE) was determined according to Ellman et al. (1961). The
234
procedure was done with a reaction solution composed of phosphate buffer (0.1 M; pH = 7.2),
235
acetylcholine 0.075 M and DTNB 10 mM. The product of AChE activity was measured at a wavelength
236
of 414 nm and presented in nmol min-1 mg-1 protein.
237
Lipid peroxidation was measured through the quantification of thiobarbituric acid reactive substances
238
(TBARS), according to Buege and Aust (1978). The method is based on how malondialdehyde (MDA), a
239
by-product of lipid peroxidation, reacts with 2-thiobarbituric acid (TBA), after precipitation of protein
240
10
with trichloroacetic acid (Correia et al. 2017). The absorbance was then measured at a wavelength of 535
241
nm and the MDA content estimated and expressed in nmol MDA equivalents by mg-1 of protein.
242
In both cases, the protein concentration of the samples was determined according to Bradford’s
243
protocol (Bradford 1976), adapted to a microplate and measured spectrophotometrically at 595 nm. All
244
spectrophotometric readings (AChE, TBARS and protein) were measured using a spectrophotometer
245
equipped with a microplate reader (Thermo Scientific™ Multiskan™ GO UV/Vis microplate
246
spectrophotometer).
247
248
Statistical analysis
249
To assess significant differences between treatments for the measured variables (AChE, TBARS), and
250
after checking the homogeneity of variances (Levene’s test), a one-way analyses of variance (ANOVA)
251
were carried out, with a level of significance of 0.05, and followed by the LSD and Dunnett’s post-hoc
252
tests to determine significant differences from the control (non-contaminated OECD soil without MPs).
253
Earthworms distribution in the test containers were analysed with a Chi-square Test (level of significance
254
of 0.05) to test for significant differences between the CTL and the treatments with MPs. Regarding the
255
distribution of MPs, a similar approach was followed to compare only the treatments with MPs (with and
256
without CPF). Statistical analyses were carried out using Prism 6 for Windows (GraphPad Software Inc,
257
USA).
258
Results
259
AChE as a biomarker of exposure to CPF (Test 1)
260
After 14 days of exposure, no mortality was recorded in all the replicates of the different treatments
261
tested. There were significant differences between treatments and the control soil in earthworms’ weight
262
(ANOVA: F = 7.930; df: 5, 12; p = 0.001). Chlorpyrifos had a significant inhibitory effect on AChE
263
activity in the earthworms exposed to all the concentrations tested even at the lowest one (ANOVA: F:
264
23.95; df: 5, 48; p = 0.000) (Fig. 1). Thus, the lowest observed effect concentration of CPF for the
265
inhibition of AChE in E. fetida was LOEC ≤ 9mg kg-1.
266
267
11
Molecular biomarkers: AChE and lipid peroxidation
268
For the first experiment (MPs with 5mm), no mortality was recorded after 14 days of exposure, except in
269
one experimental replicate (MPs5mm+CPF), where one earthworm was missing/died at the end of the
270
experiment, which was assumed has occurred by chance. Furthermore, no significant differences between
271
treatments and control soil were observed for the weight of earthworms (ANOVA: F = 0.8474; df: 2, 7; p
272
= 0.468). Significant differences were observed in AChE activity between treatments and the control
273
(ANOVA: F = 9.014; df: 2, 34; p = 0.0007) (Fig. 2a). The AChE activity was significantly inhibited in
274
earthworms exposed to soils with MPs5mm sprayed with CPF. A significant reduction in TBARS, when
275
compared with the control, was recorded especially for the treatment MPs5mm+CPF (ANOVA: F =
276
8.308; df: 2, 33; p = 0.001) (Fig. 2b).
277
Regarding the experiment conducted with smaller MPs (0.25-1 mm), no mortality was recorded.
278
There were detected significant differences between treatments and control soil in earthworm’s weight
279
(ANOVA: F = 15.29; df: 2, 7; p = 0.0028) for MPs1mm+CPF, where the percentage of weight loss was
280
lower (Loss weight percentage: CTL= 9.49 ± 1.05 %; MPs1mm= 5.95 ± 2.12 %; MP1mm+CPF=2.62 ±
281
1.56 %). No significant differences were recorded in the activity of AChE between the treatments
282
(ANOVA: F=0.188; df: 2,17; p = 0.8629) (Fig. 2c). Significant differences were recorded between the
283
treatment with MPs1mm+CPF and the control in terms of TBARS (ANOVA: F=22.38; df: 2, 21; p =
284
0.0001) (Fig. 2d).
285
286
Movement of earthworms and MPs in the experimental units
287
Earthworms showed a different behaviour in the different treatments (Fig. 3a and 3b). In the control soils,
288
they were more homogeneously distributed in the test containers, and no earthworms were found in the
289
bottom of the pots. In the treatment with MPs (without CPF), this homogeneous distribution was
290
maintained, but a great percentage of earthworms was found between the 2 cm and the bottom for both
291
microplastics sizes. Indeed, the chi-squared analyses showed significant differences for MPs5mm (X2 =
292
5.87, df = 3, p = 0.035) and MPs1mm (X2 = 7.38, df = 3, p = 0.02) treatments compared to the control.
293
However, in the treatment with contaminated MPs (with CPF), earthworms were significantly more
294
concentrated in the bottom of the test containers and were detected significant differences in the
295
12
distribution in comparison with control soil, MP5mm+CPF (X2 = 53.66, df = 3, p = 0.00001) and
296
MP1mm+CPF treatment (X2 = 50.03, df = 3, p = 0.00001).
297
The movement/transport of microplastics by earthworms was also registered for experiments with
298
different MPs size and are graphically represented together (Fig. 4). There was a slight displacement of
299
MPs but only in the top 2 cm of the test containers. They were not displaced for greater depths of soil, at
300
least during the time of exposure. No significant differences were observed for MP5mm (X2 = 1.109, df =
301
2, p = 0.574) and for MP1mm (X2 = 3.45, df = 2, p = 0.063) treatment.
302
303
Chlorpyrifos (CPF) content in soils
304
Chlorpyrifos was detected only in the samples with MPs5mm+CPF and MPs1mm+CPF with a
305
remarkable difference between both treatments. For MPs5mm+CPF treatment it was detected 17.9 ± 4.5
306
ng CPF g-1 soil dw, while for MPs1mm+CPF treatment it was recorded a concentration of 2442 ± 238 ng
307
CPF g-1 soil dw, around 135 times more regarding MPs5mm+CPF treatment (Table 1).
308
309
Discussion
310
AChE as a biomarker of exposure to CPF
311
Chlorpyrifos decreased acetylcholinesterase’s activity as expected, as it belongs to a group of
312
organophosphorus pesticides designed to act as an inhibitor of cholinesterase’s (PPDB 2018), and this
313
was precisely the reason why it was selected as a biomarker of exposure to CPF. In our study, this effect
314
was recorded for this species even at the lowest concentration tested, but we were not able to obtain a
315
precise value for LOEC since lower concentrations should have been tested. However, Muangphra et al.
316
(2016), in a study with the earthworm Pheretima peguana (Rosa, 1890) exposed the organisms to 0.1, 1,
317
10, 100 mg kg-1 of chlorpyrifos for 14 days and reported inhibition of the AChE activity at the lowest
318
concentration of 0.1 mg kg-1. In the same sense, Sanchez-Hernandez et al. (2014), reported for the
319
earthworm Aporrectodea caliginosa (Savigny1826), a significant inhibition in AChE activity, in a 21-day
320
exposure to chlorpyrifos-spiked soils (0.51 and 10 mg kg−1 soil dw). Reinecke and Reinecke (2007) tested
321
concentrations of 0.5, 1, 2.5, 8 µg kg-1 of chlorpyrifos and reported a significant inhibition of AChE
322
13
activity on A. caliginosa for all concentrations in comparison to the control soil one day after the first
323
pesticide application. Therefore, and although available data is for other species and other exposure
324
conditions (e.g. different test substrates), a positive response of E. fetida to CPF is also expected at
325
concentrations lower than those recorded in this study.
326
Microplastics as a source of some pesticides to the soil
327
Regarding our first question, we found a positive answer at least for LDPE MPs used in this study and for
328
organophosphorus pesticides with a chemical structure similar to CPF. In both experiments, CPF was
329
recorded on soil samples from the treatments with MPs that were previously sprayed with the pesticide.
330
This agrees with the findings of previous authors that showed that, in a general way, the sorption of
331
different types of pesticides to LDPE plastic easily occurs after different contact times and requires low
332
activation energy (Nerin et al. 1996; Teuten et al. 2007; Rochman et al. 2013; Allen et al. 2018). Our
333
study also demonstrated that desorption is possible especially for more soluble formulations of pesticides
334
like the commercial formulation of CPF. Nevertheless, this phenomenon must also be analysed under
335
environmental conditions, since the sorption rates of pesticides to MPs will vary according to plastic
336
ageing, contaminant degradation conditions, contaminant mixtures and formulations and biofouling
337
(Nerin et al. 1996).
338
A remarkable difference was recorded, with the highest concentration of CPF being displayed in the
339
soil with the small MPs1mm. In our treatment with MPs1mm, between 180-200 MP particles with sizes
340
among 250 µm-1mm were added, thus a great difference in the specific surface area of adsorption (SSA)
341
existed in the treatments with MPs of different sizes. According to Teuten et al. (2007, 2009), an increase
342
in SSA will increase the capacity of MPs for sorption and/or transport of organic compounds (e.g.
343
chlorpyrifos). In this sense, several authors (Teuten et al. 2007, 2009; Bakir et al. 2014; Karapanagioti
344
and Werner 2018) indicated that polyethylene has a high sorption and desorption capacity compared to
345
other types of plastic polymers. In fact, it was already suggested that plastic fragmentation increases SSA,
346
leading higher sorption of pollutants over time (Rochman et al. 2013). This concern was also highlighted
347
by Hodson et al. (2017) for commercial plastic bags and their role as Zn carrier since a higher SSA to
348
mass ratio could change the role of MPs as vectors of metals as well. Nevertheless, and since earthworms
349
had a clear avoidance behaviour of several pesticides, including organophosphorus pesticides (Pereira et
350
al. 2010), at least for this species, its ability to contribute for the biotransport, to great depths in the soil,
351
14
of contaminated MPs seems to be limited. This was shown in our study since in the treatments with MPs
352
of both sizes sprayed with CPF, the greatest majority of earthworms were found in the bottom of the test
353
containers displaying a clear avoidance behaviour, when compared with their behaviour in the CTL or in
354
the test container with MPs only. In fact, and according to our data, only some transportation in the first
355
2cm was recorded, likely caused by the bioturbation of soil during earthworm’s movement. However, we
356
assume that the distribution of small MPs in the soil containers, may be biased by our ability to detect
357
MPs lower than 1mm in size. Our results are not in accordance to papers published within this topic,
358
where earthworms and/or collembolans played a key role in MPs movement in the soil profile through
359
both horizontal and vertical movement (Maaß et al. 2017; Rillig et al. 2017a, 2017b). Rillig et al. (2017a)
360
indicated that earthworms could transport MPs particles up to a depth of 10 cm, and a distribution
361
between soil depths is dependent to particle size (smallest particles at depth layers) and suggested that this
362
movement could be through earthworm’s activities (uptake/egestion, burrowing, adherence to organism’s
363
exterior or by casts). Previously, Huerta-Lwanga et al. (2016) showed that earthworms could incorporate
364
microplastics through their burrowing activities from the surface. In the case of collembolans, Maaβ et al.
365
(2017) showed how Folsomia candida (Willem, 1902) and Proisotoma minuta (Tullberg, 1871) can
366
translocate and mobilise MPs particles (urea-formaldehyde particles) through horizontal movements.
367
Later, Zhu et al. (2018) also reported that F. candida and Hypoaspis aculeifer (Canestrini, 1883) can
368
transport polyvinyl chloride (PVC) particles (diameter between 80-250 μm) up to a depth of 9 cm, due to
369
a small body size that can favour incorporation of MPs into soil pores. In any case, more analyses are
370
needed to confirm small MPs movement, for example by using soil extractions according to the recent
371
papers on this topic (Rodríguez-Seijo and Pereira 2017; Hurley et al. 2018; Zhang and Liu 2018; Zhang et
372
al. 2018). However, it is important to take into account that the contamination of MPs with substances
373
that induce the avoidance of earthworms may change the pattern observed in these previous studies.
374
Microplastics as carriers of pesticides to biota
375
Our data was not able to provide a precise answer to our second question. In fact, a significant inhibition
376
of AChE activity was detected in the MPs5mm+CPF treatment (Fig. 2a), suggesting that the earthworms
377
were exposed to CPF and that the concentration released from the MPs to the soil was enough to cause a
378
neurological response. Nevertheless, and given the size of these MPs, this response was induced by
379
exposure to the pesticide in the soil and via dermal absorption or through soil uptake, as earthworms were
380
not able to ingest MPs of 5 mm of diameter. The detection of CPF by chemosensory organs present in
381
15
their cuticle also lead earthworms to avoid the MPs and the most contaminated soil, probably located in
382
the upper 2cm of soil. In fact, the soil samples analysed in this study, were composite soil samples
383
obtained after the homogenization of soil of the three replicates of each treatment. Nevertheless, it was
384
likely that a gradient of CPF existed in the test containers, with the highest concentration close to the 2
385
cm of soil, where the MPs were placed. However, in the second experiment and in the treatment with
386
MPs1mm+CPF, no response was recorded in terms of AChE activity, contradicting what was expectable
387
based on the previous mentioned treatment, as a higher concentration of CPF was found in soil with
388
smaller MPs. However, we hypothesize that it was precisely this highest concentration that likely lead
389
earthworms to firmly and immediately avoid the upper soil and to refuge in the bottom of the test
390
containers, where they stayed immobile very soon after the exposure start. A more rapid detection of CPF
391
by chemoreceptors and a more pronounced response has likely prevented earthworms to eat soil being
392
exposed to levels able to induce a neurological response, and for this reason, a no significant inhibition
393
was recorded in the AChE activity on earthworms exposed to MPs1mm+CPF. In parallel, a more difficult
394
detection and the slowest avoidance of MPs with CPF in the treatment with MP5mm, where a low
395
concentration of the pesticide was recorded, has contributed for the activation of the antioxidant defence
396
system of the earthworms and subsequently for the significant reduction of TBARS content in these
397
earthworms. The opposite was recorded in earthworms exposed to MP1mm, which displayed a significant
398
increase in TBARS content in their body. De Bono et al. (2002) studying the nematode Caenorhabditis
399
elegans observed that repulsive and environmental stress conditions are important inducers of rapid
400
locomotion and aggregation, behaviours that are regulated by two different sensory pathways. Although
401
few studied, the same aggregation behaviour has been reported for earthworms, as a strategy to reduce
402
their collective surface-to-volume ratio and reduce their vulnerability to different stresses (Zirbes et al.
403
2012). The rapid induction of movement by a high CPF concentration in soil and the persistence of this
404
aggregation behaviour may have led to anxiety on earthworms and subsequently to the oxidative stress
405
expressed as an increase in TBARS content. In fact, as stated by Kar and Choudhury (2016), oxidative
406
stress can be the result as well as the cause of stress and anxiety. When an individual is under stress or
407
experiences anxiety, its metabolism is affected, and the generation of oxidative free radicals is promoted,
408
which in turn, through a cascade of biochemical events lead to the generation and persistence of
409
anxiety/stress.
410
16
Although the small size of MPs in the MPs1mm and MPs1mm+CPF treatment allowed them to be
411
ingested by the earthworms, it is unlikely that such uptake has occurred being responsible by the
412
oxidative stress recorded, for three reasons: i) a high content of TBARS was recorded only on earthworms
413
exposed to the MPs1mm+CPF, but not in the MP1mm treatment as well; ii) the strong avoidance by
414
earthworms of the layer of the soil where MPs were placed, while the MPs persist in the upper 2cm of
415
soil; iii) the absence of a response in AChE activity denoting that earthworm were not exposed to CPF,
416
both through dermal absorption or to ingestion of MPs, at least at concentrations able to inhibit this
417
enzyme.
418
419
Conclusions
420
In summary, and regarding our first question, this study shows that LDPE MPs may contribute to the
421
input of pesticides to the soil matrix. Nevertheless, earthworms seemed to have few impacts in the
422
transportation of contaminated MPs to greater depths in the soil, as they avoided these materials.
423
However, such transport may occur with soil management practices, such as tillage. Furthermore, this
424
behavioural response of earthworms with so many important functions on soils, need to be analysed in
425
depth as it may indirectly compromise several soils functions and services (Bloiun et al. 2013). E. fetida
426
is an epigeic species, inhabiting the litter layer, however the presence of MPs with pesticides in the soil
427
surface seemed to lead these earthworms to escape for deeper layers in the soil. Additionally, the
428
contribution of MPs for the persistence of pesticides in soils needs to be addressed as well in future
429
studies. Our data also suggested that MPs may have few relevance as carriers of some organic substances
430
to biota like earthworms, especially of those that can easily desorb from MPs surface and/or of those that
431
can induce a clear avoidance response of these organisms. Nevertheless, more studies are needed to
432
provide a more consistent answer to our second question.
433
434
Supplementary material
435
The supplementary figure contains microplastics exposure to the pesticide before and after pesticide
436
application (Fig. S1).
437
Conflicts of Interest
438
17
The authors declare no conflicts of interest.
439
440
Acknowledgements
441
This work has been supported by the LABEX DRIIHM, French programme "Investissements d'Avenir"
442
(ANR-11-LABX-0010) which is managed by the ANR (Agence Nationale de la Recherche) within the
443
Observatoire Hommes-Milieux Estarreja (OHM-E/2018/Proj.4), and by the Strategic Funding
444
UID/Multi/04423/2013 (CIIMAR), UID/GEO/04035/2013 (GEOBIOTEC), and UID/AMB/50017/2013
445
(CESAM RU), through national funds provided by FCT (Foundation for Science and Technology) and
446
European Regional Development Fund (ERDF), in the framework of the PT2020 Partnership Agreement.
447
FCT, also supported the work through an individual research grant attributed to A. Cachada
448
(SFRH/BPD/100429/2014). We would like to thank Ana Cláudia Dias (technician from University of
449
Aveiro) for her assistance and her support with the pesticide analyses.
450
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Figure captions
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Fig. 1. Acetylcholinesterase activity (nmol · min-1 · mg-1 protein) after 14 days of exposure to soil
678
contaminated with chlorpyrifos at different concentrations (from 9.9 to 50 mg kg-1 soil dw). Error bars
679
represent the standard deviation. Asterisks indicate a significant difference with the control (p<0.05).
680
Fig. 2. Acetylcholinesterase activity (Figs. 2a and 2c) and Thiobarbituric acid reactive substances content
681
(Figs. 2b and 2d) after exposure for 14 days to soils with MPs (MPs5mm and MPs1mm, respectively) and
682
to soil with MPs and pesticide (MPs5mm+CPF and MPs1mm+CPF, respectively). For all cases, error
683
bars represent the standard deviation and asterisks indicate a significant difference with the control (p <
684
0.05).
685
Fig. 3a (left) and 3b (right). Mean relative distribution of earthworms recovered from the test soil above
686
2cm, at 2cm, below 2cm of depth and at the bottom of the test container at the end of 14 days of exposure.
687
Letters above bars indicate a significant difference between treatments (a for treatment without pesticide,
688
b for treatment with pesticide) and the control using chi-square test (p < 0.05).
689
Fig. 4. Mean relative distribution of MPs recovered from the test soil above 2cm, at 2cm, below 2cm of
690
depth and at the bottom test vessel at the end of 14 days of exposure.
691
Tables
Table 1. Concentrations (ng g−1 soil dw) of chlorpyrifos in each treatment.
OECD5mm
OECD1mm
MPs5mm
MPs1mm
MPs5mm+CPF
MPs1mm+CPF
BDL
BDL
BDL
BDL
17.9 ± 4.5
2442 ± 238
BDL=Below detection limit (5 ng g-1).
SUPPLEMENTARY MATERIAL
Low-density polyethylene microplastics as a source and carriers of agrochemicals to soil and
earthworms
Andrés Rodríguez-Seijo1*; Bruna Santos1, Eduardo Ferreira da Silva2, Anabela Cachada1,3,4, Ruth Pereira1
1GreenUPorto and Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal
2Department of Geosciences, Geobiotec Research Centre, University of Aveiro, Aveiro, Portugal
3Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto,
Terminal de Cruzeiros do Porto de Leixões, Matosinhos, Portugal
4CESAM, University of Aveiro, Campus de Santiago, Aveiro, Portugal
*Corresponding author:
Andrés Rodríguez Seijo
GreenUPorto and Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal.
andresrodriguezseijo@hotmail.com
Figures: 1.
Figure S1. Application of pesticide to the microplastics in the experimental unit (1m2).
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