Avoidance behaviour of isopods (Porcellio scaber) exposed to food or soil contaminated with Ag- and CeO2- nanoparticles

Applied Soil Ecology 141 (2019) 69–78

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Avoidance behaviour of isopods (Porcellio scaber) exposed to food or soil contaminated with Ag- and CeO2- nanoparticles

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Primož Zidara, , Monika Kosa, Eva Iliča, Gregor Maroltb, Damjana Drobnea, Anita Jemec Kokalja a b

Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia

A R T I C LE I N FO

A B S T R A C T

Keywords: Ecotoxicology Engineered nanomaterials Avoidance response Metal bioaccumulation Glutathione S-transferase Biomarkers

The main goal of the study was to evaluate the avoidance behaviour of isopods (Porcellio scaber) to food or soil contaminated with different nanoparticles (NPs): Ag-NPs, sulfidized Ag-NPs (Ag2S-NPs) or CeO2-NPs. These NPs are characterized by different physiochemical properties, and antimicrobial and toxic potentials. Isopods were exposed in three scenarios: (i) 14 d feeding test where animals were fed solely on contaminated leaves; (ii) 14 d food selection test where leaves spiked with NPs were offered simultaneously with uncontaminated leaves; and (iii) 48 h soil avoidance test where animals could choose between uncontaminated and soil spiked with NPs. The isopods decreased feeding on Ag-NP contaminated food and clearly avoided contact with Ag-NP contaminated soil. Substantial accumulation of Ag in the body of isopods was found both in feeding and food selection test. This means that food selection might mitigate but could not prevent Ag accumulation in woodlice. In contrast, no such behavioural response of isopods was found for CeO2-NP. These data suggest that the presence of metal ions in the case of Ag-NPs may be the main reason for differential behaviour. Interestingly, moderate avoidance behaviour was observed also towards Ag2S-NPs although no Ag+ was detected in stock suspensions. This avoidance was not statistically significant and needs to be further explored. Behavioural test with isopods showed again their value in estimation of potential adverse effects of pollutants comparable to collembolans and earthworms.

1. Introduction Terrestrial isopods (woodlice) are an important group of macrodecomposers. They are involved in nutrient cycling by fragmentation of dead plant material that enhances further bacterial decomposition (Kautz and Topp, 2000). Inhabiting the upper layer of soil they are frequently exposed to various pollutants (e.g. pesticides or heavy metals) via soil and food (Hopkin, 1989; Zimmer, 2002; Vijver et al., 2006). In the last decade a great deal of attention is being paid to engineered nanoparticles (NPs) as their production and diversity of applications are expanding. Therefore, it is expected that concentration of NPs in the terrestrial environment will increase, mainly through waste disposal and sewage sludge discharges. Sewage sludge is often applied to agricultural fields as fertilizer which provides a considerable input of NPs to soils, posing a potential risk to terrestrial organisms (Keller et al., 2013; Kampe et al., 2018). Silver nanoparticles (Ag-NPs) have found a way into many consumer products primarily due to its antimicrobial activity (reviewed by Nowack et al., 2011; Vance et al., 2015). There is a high potential for

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Ag-NPs to be used in agriculture as a fungicide (Kim et al., 2012). For Ag-NPs, global productions were reported ranging from 5.5 to 500 tons annually (reviewed by Sun et al., 2014) from which the majority of Ag reach the terrestrial environment mainly through sewage sludge discharges (Keller et al., 2013). The mobility, reactivity, and toxicity of Ag-NPs are closely related to its transformation in environmental and biological media: aggregation, surface oxidation, release of Ag+ and interaction with (predominantly) sulfides, chlorides and organic compounds (reviewed in Levard et al., 2012; Reidy et al., 2013; McShan et al., 2014). During wastewater transport and treatment metallic AgNPs are transformed into sulfidized Ag-NPs, also referred to as Ag sulfides (Ag2S) (Kaegi et al., 2011; Levard et al., 2012). However, despite the fact that Ag2S-NPs are sparingly soluble there are studies indicating that sulfidation of Ag-NPs under realistic conditions results also in the formation of amorphous Ag2S-NPs. This form may exhibit higher dissolution as compared to crystalline Ag2S-NPs resulting in higher rate of Ag+ release (Kraas et al., 2017). In line with this, Ag+ was assimilated in the digestive glands of terrestrial isopods exposed to Ag2S-NPs (Kampe et al., 2018). It was concluded that some forms of

Corresponding author. E-mail address: [email protected] (P. Zidar).

https://doi.org/10.1016/j.apsoil.2019.05.011 Received 5 December 2018; Received in revised form 13 May 2019; Accepted 15 May 2019 Available online 22 May 2019 0929-1393/ © 2019 Elsevier B.V. All rights reserved.

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dibasic and monobasic potassium phosphate, 1‑chloro‑2,4‑dinitrobenzene (CDNB), L‑glutathione (GSH) and sodium hydrogen carbonate. BCA Protein Assay Reagents A and B, cadmium chloride, and potassium dichromate were purchased from Pierce (USA). Silver nanoparticles (Ag-NPs) were provided by Colorobbia S.p.A. (Firenze, Italy). The same batch of Ag-NPs has been characterized and described previously (Zou et al., 2015; Böhme et al., 2015; Jemec et al., 2016; Kos et al., 2016; Romih et al., 2016). Particles were supplied as aqueous dispersion with surfactant polyvinylpyrrolidone (PVP) in ultrapure (MQ) water with a nominal particle concentration of 40 g L−1 (mean total Ag concentration 41.14 g L−1 was measured by flame atomic absorption spectrometry as reported in Jemec et al., 2016). The nominal size as measured by transmission electron microscope (TEM) was 20.4 ± 6.8 nm (Jemec et al., 2016), mean hydrodynamic diameter (z-average value) was 123.8 ± 12.2 nm and the share of Ag+-species in the supplied dispersion was 46% (Jemec et al., 2016). Ag2S-NPs were obtained from Applied Nanoparticles SL (Barcelona, Spain; batch number DR-027). Mean size measured with TEM was 21.5 ± 6.1 nm. Stock concentration of Ag2S-NPs was 1.38 g L−1. Particles were stabilised with 1 g L−1 PVP. Cerium (IV) oxide nanopowder (CeO2-NPs) (batch number 544841, < 25 nm particle size, Brunauer, Emmett and Teller technique) was purchased from Sigma-Aldrich Co. (Darmstadt, Germany) and was product of supplier Engi-Mat Co. (Lexington, USA). No additional surfactant was used to stabilize CeO2-NPs dispersion.

Ag2S-NPs are bioavailable to organisms and may exert toxic effects (Wang et al., 2015; Kraas et al., 2017). When silver enters the tissues it might cause cell damages (protein dysfunction, DNA and membrane damage) as well as depletion of glutathione and changes in the activity of various antioxidant enzymes like glutathione S-transferase (GST). In the significant part of toxicological studies with Ag-NPs the observed effects to biota have been attributed mostly to free ions dissolved from NPs or inherently present in the test suspension due to incomplete removal after synthesis (Ivask et al., 2014; Bondarenko et al., 2016; Jemec et al., 2016). In contrast to Ag-NPs, cerium (IV) oxide nanoparticles (CeO2-NPs) are almost insoluble in aqueous media (Cornelis et al., 2011; Dahle and Arai, 2015). The majority of CeO2-NPs are released into the terrestrial environment through atmospheric deposition of diesel automobile exhausts and through the application of sewage sludge to landfills and farmlands (Park et al., 2008; Piccinno et al., 2012; Keller et al., 2013). The CeO2-NPs are estimated to persist in soil due to its structural properties and association with soil particles (Pang et al., 2002; Cassee et al., 2011). Once in the soil, CeO2-NPs might be assimilated and translocated to edible tissues of various plants (Hawthorne et al., 2014; Majumdar et al., 2016) and potentially also to terrestrial invertebrates (Collin et al., 2014; Lahive et al., 2014). The biological activity of CeO2NPs is contradictory (Dahle and Arai, 2015). Namely, the reduction of Ce4+ to Ce3+ could cause oxidative stress due to reactive oxygen species (ROS) generation and possibly initiate metal unloading from metallothioneins. However, there are reports suggesting CeO2-NPs may be beneficial, capable of scavenging ROS and acting as antioxidants (reviewed in Dahle and Arai, 2015). The observed biological effects of CeO2-NPs could be attributed predominately to particulate fraction assuming that no Ce2+ contamination occurs (Kos et al., 2017). Many studies have shown that woodlice are capable of distinguishing between food or soil spiked with different concentrations of pollutants including metals (Zidar et al., 2003, 2005; Tourinho et al., 2015a), pesticides (Loureiro et al., 2005, 2009; Santos et al., 2010; Zidar et al., 2012), pharmaceuticals (Žižek and Zidar, 2013), and chars (Madžarić et al., 2018). Avoidance behaviour to suboptimal conditions enables woodlice to escape or mitigate the adverse effects of pollutants. They might avoid already very low concentrations of pollutants where no effect on other parameters such as growth are evidenced (Žižek and Zidar, 2013; Silva et al., 2014; Tourinho et al., 2015a). Avoidance behaviour of organisms is considered as environmentally relevant endpoint, because their habitat function is decreased when they avoid contaminated substrate (ISO 17512-1, 2008). In this study the avoidance behaviour of isopods was chosen as the focus end-point trying to elucidate whether soil or food contaminated with Ag-NPs, sulfidized Ag-NPs (hereafter termed Ag2S-NPs) and CeO2NPs is perceived by isopods as an unpleasant environment. The novelty of the study is that the avoidance behaviour was investigated using three experimental set-ups applying different exposure substrates (soil/ food) and durations of exposure (14 days and 48 h). Also, this is the first study to compare pristine and sulfidized Ag-NPs (Ag2S-NPs). In addition to avoidance behaviour, we also assessed metal assimilation to evaluate the bioavailability of metals and induction of detoxification processes by measuring GST activity. This was to elucidate whether isopods successfully cope with tested exposure concentrations of NPs.

2.2. Preparation and characterisation of the NP dispersions in ultrapure water The characterisation was performed on freshly prepared Ag-NP and CeO2-NP stock dispersions in ultrapure (MQ) water at nominal concentration 2 g L−1. NP dispersions of Ag-NPs (2 g L−1), CeO2-NPs (2 g L−1) and Ag2S-NPs (1 g L−1) were prepared just before application to soil and leaves. The Ag-NPs and Ag2S-NPs dispersions were mixed by 5 min vigorous vortexing (at 3000 rpm, IKA Vortex 4 basic, IKA, Germany) with no additional sonication. The CeO2-NPs were dispersed in MQ by 5 min vigorous vortexing, followed by ultrasonication at 20 kHz for 5 min in an ice-cooled bath using 130 W sonicator probe at 31% of its maximum power (Sonics, VibraCell, VCX 130 PB). The morphology and chemical composition of CeO2-NPs were investigated with field emission scanning electron microscope and energy dispersive X-ray spectroscopy (SEM-EDX; at 15 kV; SEM: Jeol JSM-7600F, EDX: INCA Wave 500, Oxford Instruments Analytical, Ltd., UK). Dynamic light scattering (DLS) was used to determine the hydrodynamic particle size (given as z-average) and polydispersity of both NPs stock dispersions (ZetaPALS instrument, Brookhaven Instruments Corporation, USA). To determine the dissolution of Ag-NP, Ag2S-NP and CeO2-NP stock dispersions in MQ, the presence of free Ag+ and Ce3+/4+-species was determined. The ultracentrifugation of stock dispersion, digestion of samples and measurements of metals were done as described in detail by Kos et al. (2017) using FAAS instrument for Ag (Perkin Elmer AAnalyst 100, Massachusetts, USA) and ICP-MS instrument for Ce (Agilent 7500ce, Agilent Technologies, Palo Alto, CA, USA). We prepared the following aqueous concentrations of Ag-NPs and CeO2-NPs: 0.1, 0.5, 1, and 2 g L−1 to prepare 100, 500, 1000 and 2000 mg NPs kg−1 soil/food. In the case of Ag2S-NPs we tested only one concentration (nominal 100 mg Ag2S-NPs kg−1) because stock concentration of Ag2S-NPs was not high enough to conduct experiments with higher exposure concentrations.

2. Materials and methods 2.1. Chemicals The following chemicals were used: trace analytical-grade 65% nitric acid (HNO3) and 30% hydrogen peroxide (H2O2) (Carlo Erba Milano, Italy), single element Ag and Ce standard solutions for flame atomic absorption spectrometry (FAAS) and inductively coupled plasma mass spectrometry (ICP-MS) (Perkin Elmer, Waltham, USA). The following chemicals were purchased from Sigma (Germany):

2.3. Test organisms Terrestrial isopods, Porcellio scaber (Latreille, 1804), originated from the laboratory culture at the Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia. Animals were derived from an unpolluted site near Kamnik, Slovenia. They were bred for several 70

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isopods were given a choice between NPs treated and control food (Fig. 1A). This resulted in 5 combinations (experimental groups) for each NP: “0–0”, “0–100”, “0–500”, “0–1000”, and “0–2000” mg NP kg−1 dry food. Each experimental group in the food selection test included 20 animals. Five identical setups containing only leaves with no isopods were used as an additional control. Both tests were performed in a climate chamber at 22 ± 1 °C, 80% relative humidity and a 16/8 h light/dark period. Animals were monitored on a daily basis and filter papers were moistened with MQ if necessary. All dead animals were removed and excluded from further analyses. After a week faecal pellets were removed and food was replaced. After 14 days animals were weighed and then their guts were removed to get rid of the contents of the gut. Animals were stored in plastic tubes and snap frozen in liquid nitrogen for metal quantification and GST activity analysis. Food remnants were air dried for 3 days and weighed to determine food consumption. The calculated amount of food consumed does not take into account potential coprophagy.

months before use in glass containers with moist loamy soil and peat at the bottom in a climate chamber at 22 ± 1 °C with a 16/8 h light/dark period. They were regularly fed with fallen leaves of various trees, with periodical additions of fresh vegetables and apples. For the test only adult animals (40 ± 15 mg fresh body mass) with undamaged antennae were used. Individuals prior moulting and gravid females were excluded. 2.4. Experimental setup 2.4.1. Food preparation Animals were fed with partially decomposed hazel leaves (Corylus avellana L.) derived from unpolluted environment. The dispersion were prepared as described in Section 2.2 and instantly applied to the air dried leaves and spread over the surface by paintbrush. The amount of dispersion was adjusted according to leaves dry mass. Our past studies have shown that such a method of applying a pollutant ensures that the nominal concentration does not deviate from the measured by > 20% (Romih et al., 2016). For food selection test leaves were halved, one half was spiked with Ag-NPs or CeO2-NPs (treated food) and the other one with MQ only (control food). After application, leaves were allowed to dry for 3 days in the dark at room temperature and re-weighed prior to use. The nominal concentrations applied were 100, 500, 1000 and 2000 mg NP kg−1 dry food. These concentrations were chosen based on previous experiments with isopods (Pipan-Tkalec et al., 2011; Tourinho et al., 2015a, 2015b; Malev et al., 2017).

2.4.4. 48 h soil avoidance behaviour test In the 48 h soil avoidance behaviour test isopods were given a choice between untreated soil (control) and soil treated with Ag-NPs, Ag2S-NPs or CeO2-NPs (treated soil). For Ag-NPs and CeO2-NPs a variety of concentrations: 0, 100, 500, 1000, and 2000 mg kg−1 dry soil and control soil were tested. This resulted in 5 combinations per NP: “0–0”, “0–100”, “0–500”, “0–1000”, and “0–2000” mg NP kg−1 dry soil. In the case of Ag2S-NPs only combination “0–100” was tested, due to a very low concentration of NPs in a stock solution. To check whether isopods might avoid PVP, animals were exposed to soil spiked with supernatant left from ultracentrifugation of Ag2S-NPs dispersion. Five replicates per combination with 10 animals per replicate were tested in the case of Ag-NPs, CeO2-NPs and supernatant; 10 replicates were tested in the case of Ag2S-NPs. The experimental setup was similar as described in Škarková et al. (2016). Two pots (0.2 L, 6 cm diameter) with perforated lids were filled with 30 g of moist Lufa 2.2 soil and bridged by a transparent tube (Fig. 1B). One pot contained control soil and the other treated soil. Isopods were introduced individually, five on each side of the test container. The test took place in a climate chamber at 22 ± 1 °C, with < 60% relative humidity and continuous darkness. No food was offered to animals during the experiment. During the 48hour exposure period, test pots were inspected after 12 and 48 h and the location of individual animal was recorded (control vs. treated soil).

2.4.2. Soil preparation We used natural Lufa 2.2 soil characterized as loamy soil with 1.59 ± 0.13% organic matter, 7.7 ± 1.7% clay, pH 5.4 ± 0.2, cation exchange capacity 9.7 ± 0.4 meq/100 g, and water holding capacity (WHC) 43.5 ± 2.8 g water per 100 g dry soil. Individual NP dispersions, Ag-NPs, Ag2S-NPs or CeO2-NPs, were prepared in a volume of MQ necessary to reach a final moisture content equivalent with 40% of the soil WHC. Dispersions were mixed thoroughly with the necessary amount of soil and then kept in closed containers for the next 24 h at room temperature in the dark. Control soil was moistened with MQ only. The pH and conductivity of the test soil were measured just prior the soil selection tests (Supplementary data, Table S1) according to ISO 10390 (1994) and ISO 11265 (1994) using a pH/conductivity meter (Thermo Scientific Orion Star A215 Benchtop pH/Conductivity Meter, Thermo Fisher Scientific, Waltham, Massachusetts, USA).

2.5. Metal concentration analyses

2.4.3. 14 days feeding test and 14 days food selection test The 14 d feeding test and the 14 d food selection test were designed due to the protocol described by Zidar et al. (2004) and carried out concurrently. Isopods were separated, weighed, and kept individually in a plastic Petri dish (d = 9 cm) with moist filter paper at the bottom. Food was offered at libitum in a small plastic dish (d = 2 cm, height of rim 3 mm) to prevent contact with the filter paper. Each of the five experimental groups in the feeding test (0, 100, 500, 1000, and 2000 mg NPs kg−1 dry food) included 15 animals. In food selection test

Freeze dried animal samples were acid digested in the microwave lab station as described previously in Kos et al. (2017). Ag concentration in the individual animal was measured by FAAS (AAnalyst 100, Perkin Elmer, USA), while Ce concentration was measured by ICP-MS (Agilent 7500ce, Agilent Technologies, USA). To verify the analytical procedure, the lobster hepatopancreas reference material for trace metals (TORT-2, National Research Council of Canada) was used. The numbers of analysed animals are presented in Fig. 5.

Fig. 1. Scheme of the food selection (A) and soil selection (B) test set up. 71

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2.6. Analysis of GST activity Whole animals were homogenised and the GST activities were measured as described by Kos et al. (2017) following the method by Habig et al. (1974) (Anthos Zenyth 3000, Great Britain). Final concentrations of chemicals in the well were: 1 mM CDNB and 1 mM GSH in potassium phosphate buffer (100 mM, pH 6.5). The reaction was followed spectrophotometrically at 340 nm at 25 °C for 15 min. The GST activity was expressed in nmoles of conjugated GSH min−1 mg−1 of the proteins (ε340 = 9600 M−1 cm−1). All samples were measured in triplicates. The numbers of analysed animals are presented in Fig. 4. 2.7. Data analysis The recovery of measured concentrations of stock NPs dispersions (before ultracentrifugation) was calculated as the quotient of measured elemental concentration and nominal elemental concentration, and expressed in %. The share of the free Ag+ or Ce3+/4+-species in the supernatant is quotient of measured elemental concentration in the supernatant after ultracentrifugation and measured elemental concentration before ultracentrifugation, and expressed in %. Mortality was calculated for 14 d feeding and food selection tests. The experiments were considered valid if the mortality of controls did not exceed 20% (Hornung et al., 1998). In food selection tests the amount of consumed food in 14 d was calculated as the sum of both leaves offered (control and treated with NPs). Food selection was calculated as the quotient of food consumption of a control/NPs treated leaf and of total food consumption, and expressed in %. The numbers of analysed animals are presented in Figs. 2–3. In the soil avoidance behaviour test a percentage of animals distributed on clean or Ag-NP/Ag2S-NP/CeO2-NP treated soil was calculated. Tests were considered valid if animals in control group were on average distributed into one or the other side within the ratio 60:40 (ISO 17512-1, 2008). The soil selection of P. scaber was further expressed as the avoidance response (AR) after 48 h calculated according to the formula: AR = ((nc − ni) / N) × 100 (nc – number on clean side; ni - number of animals on contaminated side; N – total number of animals) (ISO 17512-1, 2008). Negative values were considered as 0% of avoidance; > 80% avoidance response was considered as the criterion for limited habitat function (ISO 17512-1, 2008). In feeding tests we used a non-parametric Mann-Whitney U test to compare data (food consumption, GST activity and elemental body concentration) between the Ag-NP or CeO2-NP treated animals and controls. In the case of food selection tests and soil avoidance behaviour test the calculated percentages were tested with the one sample Student's t-test to the hypothetic 50% as it was presumed that animals can eat either food offered or distribute on either soil with equal probability. Calculations were done with the computer programmes OriginPro 8.0 software (OriginLab, Northampton, Massachusetts, USA) and Microsoft's Excel Office.

2 g L−1 stock was 47.63 ± 1.90% of the measured total Ag concentrations (1.900 ± 0.008 g L−1) for Ag-NPs (Table 1). This is in agreement with the data reported by Jemec et al. (2016). The share of Ag+ in the case of Ag2S-NPs was below the limit of detection (1.5 μg L−1). The share of free Ce3+/4+-species was only 0.33 ± 0.10% of the measured total Ce concentrations (1.902 ± 0.031 g L−1) (Table 1).

3. Results

3.2. 14 d feeding and food selection tests

3.1. Characteristics of the NPs dispersions

3.2.1. Mortality Mortality of control animals did not exceed 10% in any of the tests. No significant increase in mortality was recorded in isopods exposed to CeO2-NP treated food. However, mortality in Ag-NP exposed groups was in general slightly higher compared to CeO2-NP exposed groups. The highest mortality (25%) was observed in the treatment “0–500” mg Ag-NPs kg−1 dry food in food selection test (Table 2).

Fig. 2. Food consumption of isopods Porcellio scaber that were exposed to AgNP (A) or CeO2-NP (B) treated food for 14 days in the feeding or food selection test. Symbols on the box plot: outlier (°), arithmetic mean (▪); asterisks indicate statistically significant differences between the test groups and the respective controls (Mann-Whitney U test: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***)); n = number of data.

SEM micrographs of the CeO2-NPs show predominantly rounded particles which formed bigger aggregates (Supplementary data, Fig. S1). The hydrodynamic size (z-average) of Ag-NP and CeO2-NP stock dispersions (2 g L−1) were 120.3 ± 3.77 nm and 203.6 ± 4.03 nm, respectively. The polydispersity index of Ag-NP and CeO2-NP stock dispersions (2 g L−1) were 0.231 ± 0.017 and 0.283 ± 0.013, respectively, meaning both dispersions were moderately polydispersed. All measured total Ag and Ce concentrations were within ± 7% of the nominal concentrations (Table 1), therefore we refer to nominal Ag-NP, Ag2S-NP and CeO2-NP concentrations throughout the manuscript (as recommended in OECD 202, 2004). The share of free Ag+-species in

3.2.2. Food consumption Food consumption in the feeding test with Ag-NPs significantly (Mann-Whitney U test, p < 0.05) decreased at 500, 1000 and 2000 mg Ag-NPs kg−1 dry food (Fig. 2A), while no concentration dependent alterations in food consumption in comparison to group “0–0” 72

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3.2.3. Food selection In the food selection test, where a control food was offered concurently with Ag-NP treated food, isopods preferred control food at all tested concentrations (One sample Student's t-test; Fig. 3A). In the food selection test with CeO2-NPs (Fig. 3B) the rejection of CeO2-NP treated food was less pronounced. In this case, i sopods preferred control food (One sample Student's t-test, p < 0.01) only at the lowest treatment concentration (“0–100”). 3.2.4. GST activity In the feeding test with Ag-NPs the GST activity in woodlice increased only in the treatment of 100 mg kg−1 of dry food (MannWhitney U test, p < 0.05) (Fig. 4A). Contrary, in the feeding test with CeO2-NPs the GST activity in woodlice decreased at the lowest exposure concentration (Fig. 4B). In the food selection test no changes in GST activity due to Ag-NP exposure were noticed. In the food selection test with CeO2-NPs GST activity decreased in the group of “0–500” mg kg−1 of dry food (Mann-Whitney U test, p < 0.01). 3.2.5. Concentration of Ag and Ce in isopods after 14 d exposure In the feeding test with Ag-NP treated food (Fig. 5A) the concentration of Ag in animal was significantly higher in all exposures to NPs in comparison to control exposure and no clear dose-related accumulation was found. The highest concentration was found in a group fed with 500 mg Ag kg−1 dry food. In contrast, in the food selection test where a control food was offered simultaneously with Ag-NP treated food, body concentration of Ag evidently increased with the increasing concentration of Ag-NPs in a treated food (Mann-Whitney U test, p < 0.05). In the feeding test and food selection test with CeO2-NPs (Fig. 5B) body concentration of Ce was very low but still measurable in animals that were fed with 500 mg kg−1 dry food or higher concentration of CeO2-NPs (Mann-Whitney U test, p < 0.05). The trend was similar as in Ag-NP treated animals. 3.3. 48 h soil avoidance behaviour tests No mortality was recorded in either of the tests. In the 48 h avoidance behaviour test isopods evidently avoided Ag-NP treated soil at all treatment concentrations (One sample Student's t-test, p < 0.001). The recorded AR values were 96% and 100% in the two lowest and the two highest treatments, respectively (Fig. 6A–B). In the case of CeO2-NPs isopods did not avoid nor prefer CeO2-NP treated soil either. The only exception was “0–1000” CeO2-NPs group of isopods that preferred control soil to treated ones (One sample Student's t-test, p < 0.05; AR = 68%) (Fig. 6C–D). The calculated 48 h AR values for Ag2S-NPs and supernatant were 48% and 20%, respectively (Fig. 6E–F). No significant differences (One sample Student's t-test, p < 0.05) between percentage of animals on Ag2S-NPs treated or control soil were recorded.

Fig. 3. Food selection of isopods Porcellio scaber that had been exposed to control and Ag-NP (A) or control and CeO2-NP (B) treated food in the food selection tests for 14 days. The bars show average portion ( ± SE) of consumed control (white bars) and Ag-NP (A) or CeO2-NP (B) treated food (grey bars) in 14 days. Asterisks indicate statistically significant differences between food consumption of control and treated food (One sample Student's t-test: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***)); horizontal dotted line indicates hypothetical value of 50%; n = number of data.

was noted in the food selection test. On average, isopods consumed more food when they were simultaneously offered control and Ag-NP treated food in the food selection test in comparison to the Ag-NPs feeding test. Food consumption in the first and in the second week was comparable as well as trend of food consumption and faecal production (data not shown). In the feeding test with CeO2-NPs a significant (Mann-Whitney U test, p < 0.05) decrease in food consumption was observed at the two highest exposure concentrations, 1000 and 2000 mg CeO2-NPs kg−1 dry food (Mann-Whitney U test, p < 0.001) (Fig. 2B). In the food selection test with a group of animals exposed to CeO2-NPs, no clear-dose response was observed. Animals consumed less food in the case of “0–500” group and more in the case of ‘0–2000’ group compared to control (“0–0”). At the two highest exposure concentrations isopods consumed significantly more food when they were simultaneously offered control and CeO2-NP treated food in comparison to the feeding test offering only treated food (Mann-Whitney U test, p < 0.05).

4. Discussion Isopods are important decomposers of organic material in the terrestrial environment. Their positive attitude towards the decomposing substrate is therefore essential for their habitat function. This comprehensive avoiding behaviour study revealed that isopods show evidently repulsion towards Ag-NPs contaminated substrate, because they significantly: (i) decrease the feeding on Ag-NPs contaminated food if they have no choice of uncontaminated food, (ii) choose uncontaminated food over Ag-NPs contaminated food when offered simultaneously and (iii) this choice occurs already in acute exposure period when exposed to soil. Another isopod species Porcellionides pruinosus (Brandt, 1833) also decreased their feeding when offered Ag-NPs dosed food (29 mg Ag kg−1 dry food) and avoided Ag-NPs dosed soil after 48 h (36 mg Ag kg−1 dry soil) in spite of different experimental design 73

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Table 1 Characterisation of Ag-NP, Ag2S-NPs and CeO2-NP dispersions in ultrapure water. The table comprises the measured Ag/Ce concentration before and after ultracentrifugation (UC) in acid-digested samples, calculated recovery (%), calculated share of the free Ag+/Ce3+/4+-species (%), hydrodynamic size (given as Zaverage), and polydispersity index (PDI). The results are given as a mean of three independent measurements ± standard deviation (SD). NP nominal conc. (g L−1) Ag-NPs 0 0.1 0.5 1 2 CeO2-NPs 0 0.1 0.5 1 2 Ag2S-NPs 1#

Measured Ag or CeO2 conc. (before UC) ± SD (g L−1)

Recovery ± SD (%)

Measured Ag or CeO2 conc. (after UC) ± SD (g L−1)

Free Ag+/Ce3+/4+ -species ± SD (%)

Z-average (nm) ± SD

PDI ± SD

< LOD 0.093 ± 0.474 ± 0.940 ± 1.900 ±

/ 93.32 94.81 94.00 95.02

± ± ± ±

1.61 0.39 0.76 0.41

/ / / / 0.905 ± 0.040

/ / / / 47.63 ± 1.90

/ / / / 120.3 ± 3.77

/ / / / 0.231 ± 0.017

0.00001a 0.105 ± 0.002a 0.499 ± 0.003a 0.967 ± 0.010a 1.902 ± 0.031a

/ 105.3 99.87 96.67 94.39

± ± ± ±

1.69a 0.49a 0.98a 2.05a

/ / / / 0.006 ± 0.002

/ / / / 0.33 ± 0.10

/ / / / 203.6 ± 4.03

/ / / / 0.283 ± 0.013

0.958 ± 0.056b

102.6 ± 0.06

< LOD

< LOD

/

/

0.002 0.002 0.008 0.008

−1

LOD – limit of detection (1.5 μg L ); / - not measured. a Results are given for CeO2. b Note that nominal concentration is given for Ag2S, but measured concentration before ultracentrifugation is given for Ag concentration. Table 2 Mortality of isopods Porcellio scaber during 14 days of exposure to food spiked with Ag-NPs or CeO2-NPs in two types of experiments: feeding tests and food selection tests. (n = number). Ag-NPs Treatment (mg kg−1 dry food) Feeding test 0 100 500 1000 2000 Food selection test 0–0 0–100 0–500 0–1000 0–2000

n dead animals

0 0 1 1 3 1 3 5 1 2

CeO2-NPs Mortality (%) (n = 15) 0 0 6.7 6.7 20 (n = 20) 5 15 25 5 10

n dead animals

0 1 0 1 1 2 1 2 1 0

Mortality (%) (n = 15) 0 6.7 0 6.7 6.7 (n = 20) 10 5 10 5 0

(Tourinho et al., 2015a). Contrary to our expectations, isopods showed a moderate avoidance response also to Ag2S-NP contaminated soil, however, it was not statistically significant. No such data are yet available for Ag2S-NPs exposed to other isopod species. In the case of CeO2-NPs, isopods showed different behaviour. Avoidance to CeO2-NP treated soil was observed only in the “0–1000” group. This is most probably not a response to contamination because in the “0–2000” group no avoidance was noticed. In this latter group almost all animals in five replicates were found on treated soil after 24 and 48 h. This choice could not be just due to aggregation of animals as reported before (Loureiro et al., 2005; Zidar et al., 2012) although social interactions could outweigh an individual's preferences in the collective decision-making by leading the group towards suboptimal choices (Devigne et al., 2011). It probably just means that soil was not perceived as unpleasant environment. In 14 days of food exposure the isopods decreased their food consumption at 1000 mg CeO2 kg−1 dry food and higher concentrations. However, as in Ag-NP dosed food, isopods might have consumed also some faeces as coprophagy was not prevented. The dry weight of faeces in general followed the dry weight of leaves consumed, both in Ag and Ce exposure, which most probably means that NPs did not accelerate coprophagy. In similar experiment of Malev et al. (2017) the feeding rate of isopods increased at 1000 mg CeO2-NPs kg−1 dry food and decreased at and above

Fig. 4. Glutathione S-transferase (GST) activity in Porcellio scaber that had been exposed for 14 days to Ag-NP (A) or CeO2-NP (B) treated food in the feeding or food selection test. Symbols on the box plot: outlier (°), arithmetic mean (▪); asterisks indicate statistically significant differences between the test groups and the respective controls (Mann-Whitney U test: p < 0.05 (*)); n = number of data. 74

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dispersion may dissolve under physiological conditions in the gut which has been implied by evident bioavailability of Ag in isopods exposed to Ag2S-NPs (Kraas et al., 2017; Kampe et al., 2018). In the literature, three explanations of what may trigger the avoidance behaviour in terrestrial isopods are most often mentioned. The first option is that changes in microbiota result in altered palatability of the isopods food (Hassall and Rushton, 1982; Zimmer et al., 1996; Weißenburg and Zimmer, 2003). Ag-NPs and Ag+ have documented antiseptic action and alter microbial community structure (Kumar et al., 2014), while this was not shown for CeO2-NPs (Li et al., 2012) which could explain the observed difference in the isopods behaviour towards Ag-NPs and CeO2-NPs contaminated food. However, some observations in our study do not speak in favour of this. For example, in our study the effect is not dose-dependent which would be expected due to dose-dependent antimicrobial action of Ag-NPs. In addition, in food selection test isopods consumed a considerable amount of Ag-NP dosed food even at higher exposure concentration (up to 45% of the total consumed) although they also had a choice of uncontaminated food. Therefore we suggest that altered microbial community structure was not the prime reason for food discrimination in our case. In addition, an altered microbial structure does not explain a rapid response to soil Ag-NP contamination as evidenced after just 24 h or a moderate avoidance response to Ag2S-NP contaminated soil. Despite numerous reports on Ag-NPs antimicrobial action, Shoults-Wilson et al. (2011) found no statistically significant effects of Ag-NP treatment on microbial community in soil and similarly concluded that the antimicrobial activities of Ag+ and Ag-NPs and thus changed microbial activity cannot explain the avoidance behaviour of Eisenia fetida (Savigny, 1826). The second explanation of food selection that was previously suggested in the literature is that isopods might detect metal contamination by chemoreception (Weißenburg and Zimmer, 2003). For isopods different gustatory and olfactory chemoreceptors located on both pairs of antennas have been described (reviewed in Schmalfuss, 1998). Therefore, isopods might sense some chemicals directly or some through other parameters in the environment related to contamination. Namely, isopods are able to distinguish between all different sorts of chemicals, as has been found in numerous studies, for example among: different pesticides (Loureiro et al., 2005, 2009; Santos et al., 2010; Zidar et al., 2012), veterinary drugs (Žižek and Zidar, 2013) or chars (Madžarić et al., 2018). In our study both Ag-NPs and Ag2S-NPs were stabilised with PVP which could potentially affect animal behaviour. However, isopods did not respond to supernatant left from ultracentrifugation of Ag2S-NP dispersion. Considering previous studies with Ag-NO3 and Ag-NPs without PVP (Tourinho et al., 2015a) where a clear avoidance of isopods to Ag+ was reported we conclude that in our study the effect was mainly on the account of Ag+ and not PVP. The third possible explanation is that the avoidance response arises from the adverse effect caused by the pollutant that enters the body. It has been presumed that by feeding on fungi a large influx of metals might cause an aversion to that diet in isopods (Hopkin, 1993). This argument sufficiently explains the avoidance response to Ag-NPs and CeO2-NPs in the case of feeding test; however, it is less likely expected after 24 h soil exposure to Ag-NPs as evidenced in our study because isopods do not use soil as a predominant food source. However, moderate avoidance response to Ag2S-NPs might be explained solely by that. Our study also showed that the avoidance to NPs dosed food is not sufficient to prevent metal assimilation in isopods. In the case where the isopods consumed only contaminated food (both Ag- and CeO2-NPs) the assimilated metal did not increase along with exposure concentrations because the isopods reduced their feeding rate and metal intake at higher concentrations. However, in the food selection test, the Ag concentration in woodlice increased with the increased Ag concentration in treated food, even though they preferred control food. This was because the isopods in the choice test consumed the same or even more food in total than animals fed just treated food across all tested concentrations and on average around 40% or more of this total consumed

Fig. 5. Elemental body concentration (Ag or Ce) in isopods Porcellio scaber that had been exposed for 14 days to Ag-NPs (A) or CeO2-NPs (B) treated food in the feeding or food selection test. Please note that the scales of Y axis in the figures are much different. Symbols on the box plot: outlier (°), arithmetic mean (▪); asterisks indicate statistically significant differences between the test groups and the respective controls (Mann-Whitney U test: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***)); n = number of data.

2000 mg CeO2-NPs kg−1 dry food. However, Malev et al. (2017) used in-house synthesized NPs while we used commercial ones. This means that food type and different applications of NPs may influence the results. Differences in avoidance response of isopods to Ag-NPs, Ag2S-NPs and CeO2-NPs contaminated food or soil was most probably due to different physico-chemical properties of NPs. These NPs differ in a number of properties, such as size, shape and zeta potential, but the most notable differences were the presence of surfactant PVP in the case of Ag-NPs and Ag2S-NPs and the share of metal ions in the test dispersions. The dispersion of Ag-NPs had a high proportion of Ag+ ions (47.63 ± 1.90% in MQ) (Böhme et al., 2015; Zou et al., 2015; Jemec et al., 2016), the dispersion of Ag2S-NPs contained no Ag+ ions (below limit of detection - LOD), while the share of Ce ions in the CeO2-NP dispersion was very low (0.33 ± 0.10%). Besides, it is common knowledge that Ag-NPs are dissoluble in aqueous media under normal (physiological) conditions due to the oxidation of the surface, resulting in the release of silver ions (Ag+) (McShan et al., 2014; Romih et al., 2016), while CeO2-NPs are poorly dissoluble (Brunner et al., 2006; Batley et al., 2012; Ivask et al., 2014). On the other hand Ag2S-NPs 75

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Fig. 6. The avoidance behavioural response of Porcellio scaber to Ag-NPs (A, B), CeO2-NPs (C, D) or Ag2S-NPs (E, F) treated Lufa 2.2 soil after 24 and 48 h. The dotted line represent the criterion for limited habitat function (> 80% avoidance response). For Ag-NPs, CeO2-NPs and supernatant n = 5, for Ag2S-NPs n = 10. Supernatant was derived from ultracentrifugation of Ag2S-NPs dispersion. Circle - replicate result; bold horizontal line – average value.

assimilation in woodlice. In absolute terms, the assimilation of Ag in the isopods was significantly higher than those of Ce. This was because of high Ag ionic content available to the isopods and potential additional dissolution of Ag-NPs inside the isopods alimentary system (Romih et al., 2016). In addition, Ag has very low elimination rate constant as

food was dosed with NPs. Similar results were published by Zidar et al. (2004, 2005, 2012) in the case of Cd, Cu and pyrethrins dosed food where woodlice preferred control leaves but did also consume a considerable amount of leaves treated with chemicals. This means that food selection might mitigate but could not prevent the pollutant intake and 76

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doi.org/10.1016/j.apsoil.2019.05.011.

shown for P. pruinosus and P. scaber (Tourinho et al., 2016; Kampe et al., 2018). In line with expectations we detected very low concentrations of Ce since very low concentration of Ce ions was present in the food and the dissolution of CeO2-NPs under physiological conditions in the alimentary system of invertebrates is not documented yet but highly unlikely (Djinović et al., 2009). Most probably, the measured cerium content is due to adsorption of NPs on animals' body surface as NPs have high affinity to attach or adsorb to body surfaces (Novak et al., 2018). It also could not be attributed to gut content as gut was excluded from analysis. It seems that woodlice successfully cope with the assimilated Ag+ without the induction of GST. GST has several physiological roles however there is a clearly established link with cellular detoxification processes as its activity might increase in the case of metal exposure (Elumalai et al., 2007). On the other side it is known that Ag-NPs might cause the depletion of glutathione (McShan et al., 2014) which may result in decreased GST activity. In our study we recorded no relation between Ag-NP or CeO2-NP food contamination and GST activity. The only instance where GST was elevated was in the group exposed to 100 mg Ag-NPs kg−1 dry food in the feeding test. This was also the only group where no feeding reduction was recorded. Ag+ are most probably neutralised into sulfur-rich granules in small cells (Pipan-Tkalec et al., 2011; Tourinho et al., 2016) while a feeding reduction of treated food prevents an excessive influx of metals. Successful cellular detoxification processes probably resulted also in fairly low mortality of test animals. In the case of Ag-NPs the mortality slightly increased with the increased concentration of Ag-NPs and reached 20% at the highest concentration (2000 mg Ag-NPs kg−1 dry food). However, 20% effect is still considered within normal biological variability. Hornung et al. (1998) proposed 20% of mortality among control animals as a limit value for the validity of the test. In similar tests with P. pruinosus (Tourinho et al., 2015a) 20% mortality was recorded at 114 mg Ag kg−1 dry food. No relation between contamination and mortality was noticed in the case of CeO2-NPs as was expected due to published data (Tourinho et al., 2015b; Malev et al., 2017).

References Batley, G.E., Kirby, J.K., McLaughlin, M.J., 2012. Fate and risks of nanomaterials in aquatic and terrestrial environments. Acc. Chem. Res. 46 (3), 854–862. Böhme, S., Stärk, H.J., Reemtsma, T., Kühnel, D., 2015. Effect propagation after silver nanoparticle exposure in zebrafish (Danio rerio) embryos: a correlation to internal concentration and distribution patterns. Environ. Sci. Nano 2, 603–614. Bondarenko, O.M., Heinlaan, M., Sihtmäe, M., Ivask, A., Kurvet, I., Joonas, E., Jemec, A., Mannerström, M., Heinonen, T., Rekulapelly, R., Singh, S., 2016. Multilaboratory evaluation of 15 bioassays for (eco) toxicity screening and hazard ranking of engineered nanomaterials: FP7 project NANOVALID. Nanotoxicology 10, 1229–1242. Brunner, T.J., Wick, P., Manser, P., Spohn, P., Grass, R.N., Limbach, L.K., Bruinink, A., Stark, W.J., 2006. In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility. Environ. Sci. Technol. 40 (14), 4374–4381. Cassee, F.R., van Balen, E.C., Singh, C., Green, D., Muijser, H., Weinstein, J., Dreher, K., 2011. Exposure, health and ecological effects review of engineered nanoscale cerium and cerium oxide associated with its use as a fuel additive. Crit. Rev. Toxicol. 41 (3), 213–229. Collin, B., Oostveen, E., Tsyusko, O.V., Unrine, J.M., 2014. Influence of natural organic matter and surface charge on the toxicity and bioaccumulation of functionalized ceria nanoparticles in Caenorhabditis elegans. Environ. Sci. Technol. 48, 1280–1289. Cornelis, G., Ryan, B., McLaughlin, M.J., Kirby, J.K., Beak, D., Chittleborough, D., 2011. Solubility and batch retention of CeO2 nanoparticles in soils. Environ. Sci. Technol. 45 (7), 2777–2782. Dahle, J., Arai, Y., 2015. Environmental geochemistry of cerium: applications and toxicology of cerium oxide nanoparticles. Int. J. Environ. Res. Public Health 12 (2), 1253–1278. Devigne, C., Broly, P., Deneubourg, J.L., 2011. Individual preferences and social interactions determine the aggregation of woodlice. PLoS One 6 (2), e17389. Djinović, P., Batista, J., Levec, J., Pintar, A., 2009. Influence of morphological, redox and surface acidity properties on WGS activity of CuO–CeO2 catalysts. J. Chem. Eng. Jpn 42, 3–9. Elumalai, M., Antunes, C., Guilhermino, L., 2007. Enzymatic biomarkers in the crab Carcinus maenas from the Minho River estuary (NW Portugal) exposed to zinc and mercury. Chemosphere 66 (7), 1249–1255. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases the first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Hassall, M., Rushton, S.P., 1982. The role of coprophagy in the feeding strategies of terrestrial isopod Porcellio scaber. Oecologia 53, 374–381. Hawthorne, J., De la Torre Roche, R., Xing, B., Newman, L.A., Ma, X., Majumdar, S., Gardea-Torresdey, J., White, J.C., 2014. Particle-size dependent accumulation and trophic transfer of cerium oxide through a terrestrial food chain. Environ. Sci. Technol. 48 (22), 13102–13109. Hopkin, S.P., 1989. Ecophysiology of Metals in Terrestrial Invertebrates. Elsevier Applied Science Publishers, London. Hopkin, S.P., 1993. Deficiency and excess of copper in terrestrial isopods. In: Dallinger, R., Rainbow, P.S. (Eds.), Ecotoxicology of Metals in Invertebrates. SETAC Special Publication Series. Lewis Publishers, Boca Raton, pp. 359–382. Hornung, E., Farkas, S., Fischer, E., 1998. Tests on the isopod Porcellio scaber. In: Løkke, H., van Gestel, C.A.M. (Eds.), Handbook of Soil Invertebrate Toxicity Tests. Ecological and Environmental Toxicology Series. Wiley, Chichester, pp. 207–226. ISO 10390, 1994. Soil Quality — Determination of pH. ISO 11265, 1994. Soil Quality – Determination of the Specific Electrical Conductivity. ISO 17512-1, 2008. Soil Quality - Avoidance Test for Determining the Quality of Soils and Effects of Chemicals on Behaviour - Part 1: Test with Earthworms (Eisenia fetida and Eisenia andrei). Ivask, A., Juganson, K., Bondarenko, O., Mortimer, M., Aruoja, V., Kasemets, K., Blinova, I., Heinlaan, M., Slaveykova, V., Kahru, A., 2014. Mechanisms of toxic action of Ag, ZnO and CuO nanoparticles to selected ecotoxicological test organisms and mammalian cells in vitro: a comparative review. Nanotoxicology 8 (S1), 57–71. Jemec, A., Kahru, A., Potthoff, A., Drobne, D., Heinlaan, M., Böhme, S., Geppert, M., Novak, S., Schirmer, K., Rekulapally, R., Singh, S., 2016. An interlaboratory comparison of nanosilver characterisation and hazard identification: harmonising techniques for high quality data. Environ. Int. 87, 20–32. Kaegi, R., Voegelin, A., Sinnet, B., Zuleeg, S., Hagendorfer, H., Burkhardt, M., Siegrist, H., 2011. Behavior of metallic silver nanoparticles in a pilot wastewater treatment plant. Environ. Sci. Technol. 45 (9), 3902–3908. Kampe, S., Kaegi, R., Schlich, K., Wasmuth, C., Hollert, H., Schlechtriem, C., 2018. Silver nanoparticles in sewage sludge: bioavailability of sulfidized silver to the terrestrial isopod Porcellio scaber. Environ. Tox. and Chem. 37, 1606–1613. Kautz, G., Topp, W., 2000. Acquisition of microbial communities and enhanced availability of soil nutrients by the isopod Porcellio scaber (Latr.)(Isopoda: Oniscidea). Biol. Fertil. Soils 31 (2), 102–107. Keller, A.A., McFerran, S., Lazareva, A., Suh, S., 2013. Global life cycle releases of engineered nanomaterials. J. Nanopart. Res. 15, 1692. Kim, S.W., Jung, J.H., Lamsal, K., Kim, Y.S., Min, J.S., Lee, Y.S., 2012. Antifungal effects of silver nanoparticles (AgNPs) against various plant pathogenic fungi. Mycobiology 40 (1), 53–58. Kos, M., Kahru, A., Drobne, D., Singh, S., Kalčíková, G., Kühnel, D., Rohit, R., Žgajnar Gotvajn, A., Jemec, A., 2016. A case study to optimise and validate the brine shrimp Artemia franciscana immobilisation assay with silver nanoparticles: the role of harmonisation. Environ. Pollut. 213, 173–183.

5. Conclusions This study revealed that terrestrial isopod species Porcellio scaber behaviourally respond only to nanoparticles that release free metal ions like pristine Ag-NPs. Interestingly, this study implies that isopods might behaviourally respond also to Ag2S-NPs that usually occur in the environment after transformation of pristine Ag-NPs, but this remains to be further explored. Avoidance behavioural test with isopods showed again their value in estimation of potential adverse effects of bioavailable pollutants. Acknowledgements This work was financed by Slovenian Research Agency (ARRS), through Research programs “Integrative zoology and speleobiology P10184” and H2020 NanoFASE (grant No. 646002). Work of PhD student Monika Kos was supported by ARRS within the framework of young researchers. Dr. Gregor Marolt acknowledges support from the ARRS (Grant No. P1-0153). We thank Eva Kranjc from the Jožef Štefan Institute (Ljubljana, Slovenia) for the characterisation of CeO2-NPs, who acknowledges support of ISO-FOOD Project "ERA Chair for Isotope Techniques in Food Quality, Safety and Traceability" (grant agreement No. 621329). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Mention of trade names of commercial products and companies does not constitute endorsement or recommendation for use. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// 77

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P. Zidar, et al.

Santos, M.J., Soares, A.M., Loureiro, S., 2010. Joint effects of three plant protection products to the terrestrial isopod Porcellionides pruinosus and the collembolan Folsomia candida. Chemosphere 80 (9), 1021–1030. Schmalfuss, H., 1998. Evolutionary strategies of the antennae in terrestrial isopods. J. Crustacean Biol. 18 (1), 10–24. Shoults-Wilson, W.A., Zhurbich, O.I., McNear, D.H., Tsyusko, O.V., Bertsch, P.M., Unrine, J.M., 2011. Evidence for avoidance of Ag nanoparticles by earthworms (Eisenia fetida). Ecotoxicology 20 (2), 385–396. Silva, P.V., Silva, A.R.R., Mendo, S., Loureiro, S., 2014. Toxicity of tributyltin (TBT) to terrestrial organisms and its species sensitivity distribution. Sci.Total Environ. 466, 1037–1046. Sun, T.Y., Gottschalk, F., Hungerbühler, K., Nowack, B., 2014. Comprehensive probabilistic modelling of environmental emissions of engineered nanomaterials. Environ. Pollut. 185, 69–76. Škarková, P., Kos, M., Drobne, D., Vávrová, M., Jemec, A., 2016. Effects of food salinization on terrestrial crustaceans Porcellio scaber. Appl. Soil Ecol. 100, 1–7. Tourinho, P.S., van Gestel, C.A.M., Jurkschat, K., Soares, A.M., Loureiro, S., 2015a. Effects of soil and dietary exposures to Ag nanoparticles and AgNO3 in the terrestrial isopod Porcellionides pruinosus. Environ. Pollut. 205, 170–177. Tourinho, P.S., Waalewijn-Kool, P.L., Zantkuijl, I., Jurkschat, K., Svendsen, C., Soares, A.M., Loureiro, S., van Gestel, C.A.M., 2015b. CeO2 nanoparticles induce no changes in phenanthrene toxicity to the soil organisms Porcellionides pruinosus and Folsomia candida. Ecotox. Environ. Safe. 113, 201–206. Tourinho, P.S., van Gestel, C.A.M., Morgan, A.J., Kille, P., Svendsen, C., Jurkschat, K., Mosselmans, F.W.J., Soares, A.M., Loureiro, S., 2016. Toxicokinetics of Ag in the terrestrial isopod Porcellionides pruinosus exposed to Ag NPs and AgNO 3 via soil and food. Ecotoxicology 25 (2), 267–278. Vance, M.E., Kuiken, T., Vejerano, E.P., McGinnis, S.P., Hochella Jr., M.F., Rejeski, D., Hull, M.S., 2015. Nanotechnology in the real world: redeveloping the nanomaterial consumer products inventory. Beilstein J. Nanotechnol. 6, 1769. Vijver, M.G., Vink, J.P., Jager, T., Van Straalen, N.M., Wolterbeek, H.T., Van Gestel, C.A.M., 2006. Kinetics of Zn and Cd accumulation in the isopod Porcellio scaber exposed to contaminated soil and/or food. Soil Biol. Biochem. 38 (7), 1554–1563. Wang, P., Menzies, N.W., Lombi, E., Sekine, R., Blamey, F.P.C., Hernandez-Soriano, M.C., Cheng, M., Kappen, P., Peijnenburg, W.J., Tang, C., Kopittke, P.M., 2015. Silver sulfide nanoparticles (Ag2S-NPs) are taken up by plants and are phytotoxic. Nanotoxicology 9 (8), 1041–1049. Weißenburg, M., Zimmer, M., 2003. Balancing nutritional requirements for copper in the common woodlouse, Porcellio scaber (Isopoda: Oniscidea). Appl. Soil Ecol. 23 (1), 1–11. Zidar, P., Kaschl, U.I., Drobne, D., Božič, J., Štrus, J., 2003. Behavioural response in paired food choice experiments with Oniscus asellus (Crustacea, Isopoda) as an indicator of different food quality. Arh. Hig. Rada Toksikol. 54 (3), 177–182. Zidar, P., Drobne, D., Štrus, J., Van Gestel, C.A.M., Donker, M., 2004. Food selection as a means of Cu intake reduction in the terrestrial isopod Porcellio scaber (Crustacea, Isopoda). Appl. Soil Ecol. 25 (3), 257–265. Zidar, P., Božič, J., Štrus, J., 2005. Behavioral response in the terrestrial isopod Porcellio scaber (Crustacea) offered a choice of uncontaminated and cadmium-contaminated food. Ecotoxicology 14 (5), 493–502. Zidar, P., Hribar, M., Žižek, S., Štrus, J., 2012. Behavioural response of terrestrial isopods (Crustacea: Isopoda) to pyrethrins in soil or food. Eur. J. Soil Biol. 51, 51–55. Zimmer, M., 2002. Nutrition in terrestrial isopods (Isopoda: Oniscidea): an evolutionaryecological approach. Biol. Rev. 77 (4), 455–493. Zimmer, M., Kautz, G., Topp, W., 1996. Olfaction in terrestrial isopods (Crustacea: Oniscidea): responses of Porcellio scaber to the odour of litter. Eur. J. Soil Biol. 32 (3), 141–147. Žižek, S., Zidar, P., 2013. Toxicity of the ionophore antibiotic lasalocid to soil-dwelling invertebrates: avoidance tests in comparison to classic sublethal tests. Chemosphere 92 (5), 570–575. Zou, J., Hannula, M., Misra, S., Feng, H., Labrador, R.H., Aula, A.S., Hyttinen, J., Pyykkö, I., 2015. Micro CT visualization of silver nanoparticles in the middle and inner ear of rat and transportation pathway after transtympanic injection. J. Nanobiotechnol. 13 (1), 5.

Kos, M., Kokalj, A.J., Glavan, G., Marolt, G., Zidar, P., Božič, J., Novak, S., Drobne, D., 2017. Cerium (IV) oxide nanoparticles induce sublethal changes in honeybees after chronic exposure. Environ. Sci.: Nano 4, 2297–2310. Kraas, M., Schlich, K., Knopf, B., Wege, F., Kägi, R., Terytze, K., Hund-Rinke, K., 2017. Long-term effects of sulfidized silver nanoparticles in sewage sludge on soil microflora. Environ. Toxicol. Chem. 36 (12), 3305–3313. Kumar, S., Singh, M., Halder, D., Mitra, A., 2014. Mechanistic study of antibacterial activity of biologically synthesized silver nanocolloids. Colloid. Surface. 449, 82–86. Lahive, E., Jurkschat, K., Shaw, B.J., Handy, R.D., Spurgeon, D.J., Svendsen, C., 2014. Toxicity of cerium oxide nanoparticles to the earthworm Eisenia fetida: subtle effects. Environ. Chem. 11, 268–278. Levard, C., Hotze, E.M., Lowry, G.V., Brown Jr, G.E., 2012. Environmental transformations of silver nanoparticles: impact on stability and toxicity. Environ. Sci. Technol. 46 (13), 6900–6914. Li, Y., Zhang, W., Niu, J., Chen, Y., 2012. Mechanism of photogenerated reactive oxygen species and correlation with the antibacterial properties of engineered metal-oxide nanoparticles. ACS Nano 6 (6), 5164–5173. Loureiro, S., Soares, A.M., Nogueira, A.J., 2005. Terrestrial avoidance behaviour tests as screening tool to assess soil contamination. Environ. Pollut. 138 (1), 121–131. Loureiro, S., Amorim, M.J., Campos, B., Rodrigues, S.M., Soares, A.M., 2009. Assessing joint toxicity of chemicals in Enchytraeus albidus (Enchytraeidae) and Porcellionides pruinosus (Isopoda) using avoidance behaviour as an endpoint. Environ. Pollut. 157 (2), 625–636. Madžarić, S., Kos, M., Drobne, D., Hočevar, M., Jemec Kokalj, A., 2018. Integration of behavioral tests and biochemical biomarkers of terrestrial isopod Porcellio scaber (Isopoda, Crustacea) is a promising methodology for testing environmental safety of chars. Environ. Pollut. 234, 804–811. Majumdar, S., Trujillo-Reyes, J., Hernandez-Viezcas, J.A., White, J.C., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2016. Cerium biomagnification in a terrestrial food chain: influence of particle size and growth stage. Environ. Sci. Technol. 50 (13), 6782–6792. Malev, O., Trebše, P., Piecha, M., Novak, S., Budič, B., Dramićanin, M.D., Drobne, D., 2017. Effects of CeO 2 nanoparticles on terrestrial isopod Porcellio scaber: comparison of CeO 2 biological potential with other nanoparticles. Arch. Environ. Contam. Toxicol. 72, 303–311. McShan, D., Ray, P.C., Yu, H., 2014. Molecular toxicity mechanism of nanosilver. J. Food Drug Anal. 22 (1), 116–127. Novak, S., Jemec Kokalj, A., Hočevar, M., Godec, M., Drobne, D., 2018. The significance of nanomaterial post-exposure responses in Daphnia magna standard acute immobilisation assay: example with testing TiO2 nanoparticles. Ecotox. Environ. Safe. 152, 61–66. Nowack, B., Krug, H.F., Height, M., 2011. 120 years of nanosilver history: implications for policy makers. Environ. Sci. Technol. 45 (4), 1177–1183. OECD 202, 2004. Daphnia Sp. Acute Immobilisation Test. Pang, X., Li, D., Peng, A., 2002. Application of rare-earth elements in the agriculture of China and its environmental behavior in soil. Environ. Sci. Pollut. Res. 9, 143–148. Park, B., Donaldson, K., Duffin, R., Tran, L., Kelly, F., Mudway, I., Morin, J.-P., Guest, R., Jenkinson, P., Samaras, Z., Giannouli, M., Kouridis, H., Martin, P., 2008. Hazard and risk assessment of a nanoparticulate cerium oxide-based diesel fuel additive - a case study. Inhal. Toxicol. 20 (6), 547–566. Piccinno, F., Gottschalk, F., Seeger, S., Nowack, B., 2012. Industrial production quantities and uses of ten engineered nanomaterials in Europe and the world. J. Nanopart. Res. 14, 1109. Pipan-Tkalec, Ž., Drobne, D., Vogel-Mikuš, K., Pongrac, P., Regvar, M., Štrus, J., Pelicon, P., Vavpetič, P., Grlj, N., Remškar, M., 2011. Micro-PIXE study of Ag in digestive glands of a nano-Ag fed arthropod (Porcellio scaber, Isopoda, Crustacea). Nucl. Instrum. Meth. 269, 2286–2291. Reidy, B., Haase, A., Luch, A., Dawson, K., Lynch, I., 2013. Mechanisms of silver nanoparticle release, transformation and toxicity: a critical review of current knowledge and recommendations for future studies and applications. Materials 6 (6), 2295–2350. Romih, T., Jemec, A., Kos, M., Hočevar, S.B., Kralj, S., Makovec, D., Drobne, D., 2016. The role of PVP in the bioavailability of Ag from the PVP-stabilized Ag nanoparticle dispersion. Environ. Pollut. 218, 957–964.

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