Effects of climate conditions on the avoidance behavior of Folsomia candida and Enchytraeus crypticus towards metal(loid)- contaminated soils

Global climate changes are predicted for the 21 st century. Alterations in soil contaminants' availability and soil invertebrates’ behavior are expected, which may interfere with the avoidance capacity that invertebrates may have towards contaminated soils and, therefore, compromise their role in soil functioning. This study aimed to assess the individual effects of air temperature, ultraviolet (UV) radiation and atmospheric CO 2 concentration on the avoidance behavior of the arthropod Folsomia candida and the soft-bodied oligochaete Enchytraeus crypticus towards metal(loid)- contaminated soils. Avoidance behavior was evaluated under distinct climate treatments (simulating increases in air temperature, UV radiation exposure or atmospheric CO 2 concentration) and compared to the response obtained at the standard conditions recommended by ISO guidelines. Both soil invertebrate species behave differently under standard conditions, with F. candida not avoiding the contaminated soils while E. crypticus did. Increases in air temperature and exposure to UV radiation did not change F. candida behaviour towards contaminated soils. However, high atmospheric CO 2 concentration modified this pattern and induced avoidance towards contaminated soils. As for E. crypticus , contaminated soils were also avoided under the different climate treatments simulated. Thus, our study shows that, depending on the species and the climate factor, changes in climate conditions may alter soil invertebrates’ behavioral patterns towards meta(loid)-contaminated soils.


Introduction
Soil invertebrates have the ability to avoid soil contamination.They possess sensory organs (chemical, mechanical) that allow them to detect and escape from unfavorable and/or harmful soil conditions (Slifer and Sekhon, 1978;Moment and Johnson, 1979;Edwards and Bohlen, 1992;Curry and Schmidt, 2007).Avoidance behavior varies among invertebrate species and may depend on multiple factors: i) organism development stage (e.g., juveniles or adults); ii) organism mode of life traits (e.g., physiological, morphological and behavioural traits); iii) soil characteristics (e.g., texture, pH, salinity, organic matter, nutrients availability); iv) contaminants presence in the system and their availability; v) climate conditions (e.g., different temperature, moisture level) (Boyd et al., 2002;Loureiro et al., 2005;Lukkari and Haimi, 2005;Natal-da-Luz et al., 2008;Chelinho et al., 2014;Gainer et al., 2019;González-Alcaraz et al., 2019).The vast majority of studies have been focused on the characteristics of invertebrates and/or the soil environment.Still, there is a lack of literature on the influence of climate conditions.
Climate conditions may modify the availability of soil contaminants, due to changes in key soil parameters (e.g., pH, salinity, organic matter), but also the performance of soil invertebrates and/or their susceptibility to contaminants (Peijnenburg and Jager, 2003;Maraldo et al., 2009;Duval et al., 2011;Karmakar et al., 2016).This may have significant implications for the risk assessment of contaminants and contaminated soils in the context of climate change, especially considering the IPCC predictions for the end of the century (IPCC, 2013(IPCC, , 2014a)).In fact, some authors have already pointed out that soil ecotoxicity risks may be altered when changing the climate conditions (e.g., Holmstrup et al., 2010; González-Alcaraz and van Gestel, 2015;Lima et al., 2015;Jegede et al., 2017).These studies have mostly looked at traditional endpoints (i.e., Journal Pre-proof J o u r n a l P r e -p r o o f 4 invertebrates' survival and reproduction) and found synergistic interactions in most case studies, while behavioral endpoints have received less attention.
To our knowledge, so far, only two studies have evaluated the possible effects of climate conditions on the avoidance behavior of soil invertebrates (Lobe et al., 2018;González-Alcaraz et al., 2019).Lobe et al. (2018) found that heat stress may influence the avoidance behavior of Enchytraeus albidus towards silver nitrate and silver nanoparticles.As for González-Alcaraz et al. (2019), the authors showed that the capacity of Folsomia candida and Enchytraeus crypticus to avoid metal(loid)contaminated soils might be compromised under water stress situations (high and low soil water availability due to intense rainfalls/floods and dry spells, respectively) and that this behavioral shift may be related to changes in the composition of the soil pore water.Both studies were developed modifying the climate conditions (air temperature and soil moisture content, respectively) recommended by the existing standardized ISO guidelines to evaluate invertebrate avoidance behavior (ISO, 2008(ISO, , 2011)).Despite this first step, more research is needed on the effects of climate factors, including others different to temperature and moisture, on soil invertebrate avoidance behavior.
Considering all this, the present study aimed to assess the individual effects of three climate factors (air temperature, ultraviolet -UVradiation and atmospheric CO 2 concentration) on the avoidance behavior of different soil invertebrate species towards metal(loid)-contaminated soils.The model invertebrate species F. candida (phylum Uniramia, class Collembola, family Isotomidae) and E. crypticus (phylum Annelida, class Oligochaeta, family Enchytraeidae) were selected due to their key role in soil functioning processes (e.g., organic matter/nutrient cycling, soil bioturbation, soil structure improvement) and their use as bioindicators of stress conditions (Didden and Römbke, 2001;Fountain and Hopkin, 2005).Avoidance behavior tests were performed Journal Pre-proof J o u r n a l P r e -p r o o f 5 under different climate treatments, simulated by individually changing specific climate factors, and comparing to the response under the standardized climate conditions.We initally hypothesized that changes in air temperature, UV radiation and atmospheric CO 2 concentration would affect the avoidance behavior of F. candida and E. crypticus towards metal(loid)-contaminated soils.

Culture conditions of soil invertebrate species
Soil invertebrates were cultured under controlled laboratory conditions.Folsomia candida was cultured in plastic boxes containing moist plaster of Paris mixed with activated charcoal (9:1 w:w) and kept at 20 °C with a 16:8 h light:dark photoperiod (ISO, 1999).Once or twice a week, granulated dry baker's yeast (Sacchromyces cerevisae) was added as food source and distilled water was replenished to maintain the medium moist, as needed.Age-synchronized collembolans (10-12 d old) were used for the performance of the avoidance behavior tests (ISO, 2011).Enchytraeus crypticus was cultured in plastic boxes containing agar medium dissolved in aqueous soil extracts and kept at 20 °C under complete darkness.The culture was fed once or twice a week with a mixture of oatmeal, dry yeast, yolk powder and fish oil, as needed.Enchytraeids with clearly visible clitellum and similar size (~1 cm length) were used for the performance of the avoidance behavior tests.In both cases, only organisms showing no visible problems were used.

Metal(loid)-contaminated test soils
Journal Pre-proof J o u r n a l P r e -p r o o f 6 Two soils contaminated with metal(loid)s from different types of anthropogenic sources were selected in central-northern Portugal: a soil from the abandoned mining district of Braçal (Sever do Vouga, Northeast of Aveiro; hereafter mining soil) (Anjos et al., 2012;Vidal et al., 2012), and a soil from a former agricultural area near the Estarreja chemical complex (Estarreja, Northeast of Aveiro; hereafter agricultural soil) (Inácio et al., 2008;Rodrigues et al., 2012).Both test soils showed loamy sand texture (~ 77-86% sand, ~ 8-16% silt, and ~ 6-7% clay; data not shown), acidic pH (~ 4.7-5.9 in 0.01M CaCl 2 ), low salinity (electrical conductivity -EC -≤ 0.3 dS m -1 ) and low values of cation exchange capacity (CEC ≤ 10 cmol c kg -1 ) (Table 1).Both test soils showed different contents of total organic carbon (TOC ~ 46 mg kg -1 in the mining soil and ~ 26 mg kg -1 in the agricultural soil), but similar contents of total nitrogen (TN ~ 1.7-1.8mg kg -1 ) (Table 1).Despite this, the agricultural soil showed greater concentrations of dissolved organic carbon (DOC ~ 42 mg kg -1 in the mining soil and ~ 125 mg kg -1 in the agricultural soil) and total dissolved nitrogen (TDN ~ 13 mg kg -1 in the mining soil and ~ 22 mg kg -1 in the agricultural soil) (Table 1).The concentrations of total and exchangeable (extracted with 0.01M CaCl 2 ) metal(loid)s (Al, As, Cd, Cu, Mn, Pb and Zn) were high in both test soils (Table S1, Supplementary material).A complete description of the characterization of both test soils is provided by González-Alcaraz et al. (2019).
Soil samples were taken from the top 20 cm, air-dried, sieved (2 mm mesh), thoroughly homogenized and stored at 4 °C prior to the performance of the avoidance behavior tests.

Avoidance behavior evaluation
Journal Pre-proof J o u r n a l P r e -p r o o f Invertebrates' avoidance behavior was evaluated following the instructions of the standardized ISO guidelines 17512 (ISO, 2008(ISO, , 2011)), by giving F. candida and E. crypticus the option to choose between a clean soil (hereafter control soil) and one of the metal(loid)-contaminated test soils.The natural standard soil Lufa 2.2 (Speyer, Germany) was used as control soil (sandy loam texture; pH in 0.01M CaCl 2 ~ 5.3; EC ~ 0.1 dS m -1 ; WHC ~ 45.3%).Before starting the avoidance behavior tests, both the control soil and the metal(loid)-contaminated test soils were moistened at 50% of their maximum WHC (i.e., soil moisture content recommended by the ISO guidelines).
Avoidance behavior tests were performed in two-section containers (cylindrical plastic containers of ~ 8 cm diameter and ~ 6 cm height), where one section contained the control soil and the other the metal(loid)-contaminated soil to be tested (n = 5).Dual control tests (both sections filled with the control soil) were also performed to prove the validity of the avoidance behavior tests according to the ISO guidelines (n = 5).
Organisms were placed in the soil midline between both sections (20 for F. candida and 10 for E. crypticus).Test containers were covered with a perforated plastic lid and kept for 48 h in acclimatized chambers/rooms with a 16:8 h light:dark photoperiod and under the different climate treatments simulated (see Section 2.3.2).After 48 h, a removable plastic split (~ 1 mm thickness) was placed in the soil midline between both sections and the number of surviving organisms in each test container section was recorded: F. candida (simultaneous addition of water to both container sections, gentle soil agitation and count of organisms by eye); E. crypticus (soil transfer to 250 μm sieves, water addition to removing soil particles and count of organisms by eye).For more details on the procedure see González-Alcaraz et al. (2019).
The avoidance behavior of F. candida and E. crypticus towards each metal(loid)contaminated test soil was calculated by applying Equation 1 (in %): C (number of Journal Pre-proof organisms in control soil); T (number of organisms in metal(loid)-contaminated test soil); N (total number of surviving organisms).Positive values indicated that soil invertebrates avoided the metal(loid)-contaminated test soil (i.e., avoidance response) and negative values that invertebrates preferred the metal(loid)-contaminated test soil (i.e., preference response or non-avoidance response).When avoidance behavior equals zero it means that invertebrates were equally distributed between the control soil and the metal(loid)-contaminated test soil (i.e., neutral response).
(Equation 1) Additionally, during the performance of the avoidance behavior tests, extra containers without soil invertebrates were prepared for each of the metal(loid)contaminated test soils and the control soil to check for the loss of soil moisture content under the different climate treatments simulated (n = 3).

Climate treatments simulated
The standardized ISO guidelines 17512 (ISO, 2008(ISO, , 2011) ) recommend evaluating the avoidance behavior of soil invertebrates in acclimatized chambers/rooms with a constant air temperature of ~ 20 °C, without controlling both the UV radiation exposure and the atmospheric CO 2 concentration (hereafter standard conditions) (Table S2, Supplementary material).To evaluate the individual effects of air temperature, UV radiation and atmospheric CO 2 concentration, a set of climate treatments were established based on the IPCC projections for southern Europe for the year 2100 (IPCC, 2013(IPCC, , 2014a(IPCC, , 2014b) and compared to the response under the standard conditions (Figure 1; Table S2, Supplementary material).S2, Supplementary material).
For the climate factor air temperature, three treatments were established considering the air temperature rise predictions (Figure 1a; Table S2, Supplementary material).They From this time on the air temperature gradually decreased until 4:00 when the minimum air temperature was reached again and continued until 8:00 to start a new air temperature regime cycle.Avoidance behavior tests with air temperature treatments were performed in a KBWF 720 Binder (Germany) acclimatized chamber.
For the climate factor UV radiation, one treatment was established (Figure 1b; Table S2, Supplementary material).It consisted of a repetitive UV radiation regime of 24 h duration (6 h with UV radiation emission and 18 h without UV radiation).S3, Supplementary material).Avoidance behavior tests with UV radiation emission were performed in an acclimatized room.UV radiation was provided by two UV lamps (Spectroline XX15F/B, Spectronics Corporation, USA) with peak emissions at 365 nm for UVA (~ 320-400 nm) and 312 nm for UVB (~ 280-320 nm).The UV lamps were covered by cellulose acetate sheets, previously burned under the lamps for 12 h (Figure S1, Supplementary material).This allowed the stabilization of the UV radiation intensity passing through and prevented the exposure of the avoidance behavior test containers to UVC, which is not typically found in nature due to its strong adsorption to    = ( 2) For the climate factor atmospheric CO 2 concentration, three treatments were established considering the atmospheric CO 2 rise predictions (Figure 1c; Table S2, Supplementary material).The treatments consisted of setting the atmospheric CO 2 concentration at three different constant levels, with a difference of 200 ppm of CO 2 : i) 600 ppm (low exposure scenario for atmospheric CO 2 concentration); ii) 800 ppm (intermediate exposure scenario for atmospheric CO 2 concentration); iii) 1000 ppm (worst-case scenario for atmospheric CO 2 concentration).The atmospheric CO 2 concentration was controlled by a IRGAs system coupled to the acclimatized chamber, where the avoidance behavior tests were performed (FitoclimaD1200 Aralab, Portugal).
Avoidance behavior tests under standard conditions were performed separately for each of the climate factors evaluated.

Statistical analyses
Statistical analyses were performed with GraphPad Prism 6.0 and IBM SPSS Statistics 22 softwares; differences were considered significant at p < 0.05.Student's t-test was used to check for differences in the survival of organisms and the loss of soil moisture content under the different climate treatments simulated, by comparing each climate treatment with the standard conditions.

Validity of avoidance behavior tests with soil invertebrates
Avoidance behavior tests need to fulfill two assumptions to be considered valid (ISO 2008(ISO , 2011)): i) the number of surviving organisms > 80% for F. candida and > 90% for  2 and 3; Tables S4 and S5, Supplementary material).
For F. candida, survival decreased significantly (t-test, p < 0.05) at 25 -35 ºC in the dual control tests and in the test performed with the agricultural soil, compared to the standard conditions (~ 71% and 74% of surviving organisms, respectively), and no homogeneous distribution of organisms was observed in the dual control tests (77% to 23%; significant differences, Fisher's test, p < 0.05) (Table 2; Table S4, Supplementary material).For E. crypticus, survival decreased at 25 -35 ºC compared to the standard conditions in the test performed with the mining soil (~ 84% of surviving organisms) and organisms were not homogeneously distributed in the dual control tests (74% to 27%; significant differences, Fisher's tests, p < 0.05) (Table 3; Table S5, Supplementary material).

Avoidance behavior of soil invertebrates under standard conditions
Both soil invertebrate species showed a different behavior towards the metal(loid)contaminated test soils under the standard conditions and their response was consistent across the different tests performed (Figure 2).The collembolan F. candida did not avoid the metal(loid)-contaminated test soils (Figure 2).Organisms were equally distributed between the control soil and the metal(loid)-contaminated soil tested or showed a slight preference for the contaminated section of the containers, especially in the presence of the agricultural soil (~ 14-16% of preference).Contrariwise, E.
Journal Pre-proof J o u r n a l P r e -p r o o f 14

Air temperature
In general, when exposed to the different air temperature treatments, F. candida did not avoid both metal(loid)-contaminated test soils and a higher number of collembolans were found in the contaminated section of the containers (Figure 2a).This was significant (Fisher's test, p < 0.05) for the mining soil at 25 -35 ºC (~ 30% of preference) and the agricultural soil both at 15 -25 ºC and 25 -35 ºC (~ 82% and ~ 66% of preference, respectively).The two-way ANOVA showed that F. candida behavior was significantly (p < 0.05) affected by the soil type, the climate conditions and their interaction (Table S6, Supplementary material) due to the greater preference response for the agricultural soil with increasing air temperature.In the case of E. crypticus, avoidance response was observed towards both metal(loid)-contaminated test soils when exposed to most of the air temperature treatments simulated (Figure 2a).
Enchytraeids avoided significantly (Fisher's test, p < 0.05) the mining soil at 20 -30 ºC (~ 64% of avoidance) and the agricultural soil, under all air temperature treatments (~ 31-76% of avoidance).The two-way ANOVA showed that E. crypticus behavior was significantly (p < 0.05) affected only by the factor soil type (Table S6, Supplementary material), due to the higher avoidance response towards the agricultural soil.

UV radiation
When exposed to UV radiation, F. candida showed a neutral response in the case of the mining soil (collembolans were equally distributed between both container sections), while it significantly (Fisher's test, p < 0.05) preferred the agricultural soil (~ 36% of preference) (Figure 2b).On the contrary, E. crypticus avoided significantly (Fisher's test, p < 0.05) both metal(loid)-contaminated test soils with UV radiation Journal Pre-proof J o u r n a l P r e -p r o o f 15 exposure (~ 40% and ~ 52% of avoidance towards the mining and the agricultural soils, respectively) (Figure 2b).For both soil invertebrate species, the two-way ANOVA did not show effects for any of the factors analyzed (soil type and climate conditions) or their interaction (Table S7, Supplementary material).

Atmospheric CO 2 concentration
When exposed to rising atmospheric CO 2 concentration, F. candida tended to avoid both metal(loid)-contaminated test soils (Figure 2c).This was more evident as atmospheric CO 2 concentration increased, with collembolans avoiding significantly (Fisher's test, p < 0.05) the mining soil at 800 and 1000 ppm of atmospheric CO 2 (~ 29-48% of avoidance) and the agricultural soil at 1000 ppm of atmospheric CO 2 (~ 56% of avoidance).The two-way ANOVA showed a significant (p < 0.05) effect of the factor climate conditions (Table S8, Supplementary material), with the highest avoidance behavior towards metal(loid)-contaminated soils occurring at 1000 ppm of atmospheric CO 2 .As for E. crypticus, enchytraeids avoided significantly (Fisher's test, p < 0.05) both metal(loid)-contaminated test soils regardless of the atmospheric CO 2 concentration tested (~ 38-100% and ~ 34-72% of avoidance towards the mining and agricultural soils, respectively) (Figure 2c).The two-way ANOVA showed that E.
crypticus behavior was significantly (p < 0.05) affected by the interaction soil type x climate conditions (Figure S8, Supplementary material).

Soil moisture content loss during the performance of avoidance behavior tests
There was a general decrease in soil moisture content after 48 h of exposure to the  S2c, Supplementary material).These decreases were significant (t-test, p < 0.05) only for the agricultural soil at 600 and 1000 ppm of atmospheric CO 2 .

Effects of climate factors on the performance of soil invertebrates without metal(loid) soil contamination
The experimental set-up with dual control tests helped relate the performance of soil invertebrates directly to the climate conditions to which they were exposed for 48 h, without the influence of metal(loid) contamination.This set-up, essential to understanding the main effects of climate factors, also facilitated the evaluation of invertebrates response when confronted with the metal(loid)-contaminated test soils.
Air temperature was the climate factor inducing greater effects in the absence of metal(loid)s.In the case of F. candida, its survival decreased at 25 -35 ºC (~ 28% lower compared to the standard conditions) (Table 2).This negative effect could have been related to the exposure of F. candida to temperatures above its thermal optimum Journal Pre-proof J o u r n a l P r e -p r o o f 17 (~ 10-26 ºC) (Snider and Butcher, 1973;Fountain and Hopkin, 2005;Jänsch et al., 2005a), but also to the high loss of soil moisture content at these elevated temperatures (~ 44% decrease) (Figure S2a, Supplementary material).Without any possible shelter besides soil particles, the only option would have been to burrow into the soil to reduce heat stress.However, collembolans were most of the time on the first centimeters of the topsoil (authors' visual observation).This agrees with the findings of Boiteau and MacKinley (2013) that showed no relocation of F. candida to deeper soil layers to avoid the stress conditions induced by temperature changes.
Unlike F. candida, the survival of E. crypticus was not affected by any of the air temperature regimes tested (Table 3).This difference could have been related to the greater resistance of E. crypticus to high temperatures (thermal tolerance limit ~ 15-30 ºC) (Jänsch et al., 2005a).Moreover, the burying behavior of enchytraeid species could have guaranteed some desiccation protection (Didden, 1993;Jänsch et al., 2005b) 2 and 3).This could have been related to the fact that the soil does not dry evenly when exposed to high temperatures, and invertebrates could have remained in specific spots with higher soil moisture content and/or grouped to minimize their body dehydration.This possible seek for areas with greater soil moisture content was already reported by González-Alcaraz et al. ( 2019) who found a clear avoidance response of both invertebrate species towards soil dryness when having the option to choose between different soil moisture levels.
Different from air temperature, the exposure to both UV radiation and atmospheric CO 2 concentration did not induce any effect on invertebrates' performance in duals Journal Pre-proof J o u r n a l P r e -p r o o f 18 control tests (Tables 2 and 3).In the case of F. candida, Cardoso et al. (2014) also showed no effects of UV radiation on collembolan survival (UV doses from 1121 to 3795 J m -2 ).As for the effects of UV radiation on E. crypticus or atmospheric CO 2 on both soil invertebrate species, the absence of previous studies to our knowledge does not allow results comparison and emphasizes the need for considering climate factors.

Effects of climate factors on the avoidance behavior of soil invertebrates towards metal(loid) soil contamination
Under standard conditions, both soil invertebrate species showed an opposite response towards metal(loid) soil contamination (Figure 2).The collembolan F. candida was homogeneously distributed between the test container sections or showed a slight preference for the contaminated section, especially for the agricultural soil (~ 14-16% of preference).The latter could have been favored by the high availability of DOC and TDN of the agricultural soil (Table 1) (Natal-da-Luz et al., 2008).On the contrary, E.
González-Alcaraz et al. ( 2019) also found the same behavior in both invertebrate species under varying soil moisture conditions.The authors pointed out that this behavioral response could be related to the different sensitivity/resistance of both invertebrate species to metal(loid) soil contamination.Collembolans are generally more tolerant than enchytraeids to metal(loid)s due to the different main routes of exposure (Fountain and Hopkin, 2005;Römbke, 2003).In addition, F. candida is able to minimize the internal concentration of metal(loid)s through the exfoliation of the midgut epithelium as part of the detoxification system (Fountain andHopkin, 2001, 2005).
Journal Pre-proof J o u r n a l P r e -p r o o f 19 When changing the climate factors air temperature and UV radiation F. candida did not modify its response towards the metal(loid)-contaminated test soils (Figures 2a and   2b).Similar to the standard conditions, collembolans showed a homogeneous distribution in most of the climate treatments simulated or, in the case of the agricultural soil, a clear preference for the contaminated section of the test containers (≥ 36% of preference).No major differences were observed among soils in terms of loss of moisture content after 48 h exposure to the different air temperature and UV radiation treatments simulated (Figures S2a and S2b, Supplementary material).Hence, once again, the high DOC and TDN concentrations of the agricultural soil (Table 1) could have been the major factor that led F. candida to remain in this soil, even taking into consideration the stress related to air temperature and UV radiation treatments (Figures 2a and 2b).
Unlike air temperature and UV radiation, when avoidance behavior tests were performed with atmospheric CO 2 , F. candida completely changed its behavioral pattern towards the metal(loid)-contaminated test soils (Figure 2c).As atmospheric CO 2 concentration increased, collembolans started avoiding the contaminated section of the test containers and tended to stay in the control soil, especially when exposed to 1000 ppm of atmospheric CO 2 (≥ 48% of avoidance).Soil invertebrates are assumed to be relatively tolerant to elevated CO 2 pressure since they live in a medium with higher CO 2 concentration than in the atmosphere (Glinski and Stepniewski, 1985;Zinkler and Platthaus, 1996).Despite this, exposure to high atmospheric CO 2 concentration could have interfered with the ability of F. candida to compensate for high metal(loid) concentrations through internal regulation.If so, with increasing atmospheric CO 2 , collembolans may have felt threatened by the metal(loid)-contaminated test soils and, to avoid this pressure, they may have needed to escape to a more favorable place.Rising Journal Pre-proof J o u r n a l P r e -p r o o f 20 atmospheric CO 2 concentration could have also led to some modifications in key soil parameters, indirectly affecting the response of F. candida.Although we did not evaluate soil physico-chemical characteristics after avoidance behavior tests, we hypothesize that greater CO 2 diffusion to the soil could have lowered the pH due to the formation of carbonic acid and the dissociation of H + ions.This could have been favored by the low buffer capacity of the metal(loid)-contaminated test soils (Table 1).between the test container sections (Figure 2a).The latter could have been related to the exposure of E. crypticus to temperatures above its upper thermal tolerance limit (> 30 ºC), which could result in a higher vulnerability of enchytraeids to metal(loid)s.In fact, González-Alcaraz and van Gestel (2015) and González-Alcaraz et al. (2015) found greater toxicity of soils affected by metal(loid) mine wastes to E. crypticus with increasing air temperature.In addition, the high vulnerability of enchytraeid species to desiccation could have led E. crypticus to lose, to some extent, their capacity to avoid metal(loid) soil contamination at these elevated temperatures (Lindberg et al., 2002;Maraldo et al., 2008Maraldo et al., , 2009)).Indeed, González-Alcaraz et al. (2019) demonstrated that E. crypticus might lose its capacity to avoid metal(loid)-contaminated soils under dry soil conditions.The consistent avoidance response of E. crypticus towards the metal(loid)-contaminated soils tested, regardless of the climate factors evaluated, highlights the importance of considering these organisms for the risk assessment of natural contaminated sites.If metal(loid) contamination jeopardizes soil invertebrates, namely enchytraeids in this case, all soil functions and services provided by them may be impaired.

Conclusions
The soil invertebrate species F. candida and E. crypticus presented different  (600, 800 and 1000 ppm).In the UV radiation treatment "on" indicates that the UV lamps were emitting UV radiation (in total 6 h per day) and "off" that there was no UV radiation emission (in total 18 h per day).For each climate factor modulated the remaining factors were kept at the levels recommended by the standardized ISO guidelines (20 °C and no control of UV radiation and atmospheric CO 2 concentration).
Soil moisture content was established at 50% of the soil maximum water holding capacity for all the climate treatments simulated.Journal Pre-proof  (Chapman, 1965).Ammonium concentration determination by spectrophotometry at λ = 670 nm (Lambda 25 UV/Vis spectrometer Perkin Helmer) (NEIKER, 2005).f Soil saturation with water for 3 h followed by 2 h of water excess removal (ISO, 1998).g Bouyouco's densimeter method (Gee and Bauder, 1986).
Journal Pre-proof Journal Pre-proof Data are average ± SD (n = 5).For organism distribution between sections asterisks (*) indicate significant differences between observed and expected values (Fisher's test, p < 0.05).

Climate
The different climate treatments established were simulated by varying one individual climate factor (air temperature, UV radiation or atmospheric CO 2 concentration) while keeping the remaining factors at Journal Pre-proof J o u r n a l P r e -p r o o f the levels recommended by the standardized ISO guidelines (Table consisted of repetitive air temperature regimes of 24 h duration and thermal amplitude of 10 °C mimicking increase and decrease in daily temperature (minimummaximum air temperature reached): i) 15 -25 °C (characteristic air temperature regime for central-northern Portugal during the summer period); ii) 20 -30 °C (intermediate exposure scenario for air temperature); iii) 25 -35 °C (worst-case scenario for air temperature).Each regime started from the minimum air temperature (15, 20 and 25 °C, respectively) at 8:00 and gradually increased until 12:00 when the maximum air temperature was reached (25, 30 and 35 °C, respectively) and maintained until 16:00.
For creating the regime we considered the UV radiation data existing for Lisbon (Portugal) since it is the closest city to the area where the metal(loid)-contaminated test soils were collected with a detailed UV radiation record (Tropospheric Emission Monitoring Internet Service, TEMIS; http://www.temis.nl/index.php).The regime was based on the UV index data of the summer period (higher UV exposure) between 2002 and 2018, selecting the average minimum (~ 7) and maximum (~ 10) UV indexes.The regime Journal Pre-proof J o u r n a l P r e -p r o o f 10 started without UV radiation emission from 8:00 to 10:00.At this time, UV radiation corresponding to a UV index of ~ 7 started emitting until 12:00 when it was intensified up to ~ 10.The UV index of ~ 10 was maintained until 14:00 when it decreased again to ~ 7 until 16:00.From this time on, the UV radiation emission ceased until 8:00 to start a new cycle of the UV radiation regime.This regime resulted in a daily UV dose of ~ 4400 J m -2 (regular daily UV dose in Lisbon during the summer period) (Table the stratosphere ozone layer.The different UV indexes selected were obtained by keeping the UV lamps in permanent, static position respect to fixed platforms where the test containers were located (FigureS1, Supplementary material).One lamp was set at ~ 75 cm distance from the test containers to reach the UV index of ~ 7 and the other lamp at ~ 65 cm distance to reach the UV index of ~ 10.The test containers were moved between platforms to be exposed to the corresponding UV indexes and the perforated plastic lids were removed during the periods of UV radiation emission.To ensure that the selected UV indexes were reached, a spectroradiometer connected to a monochromator (Bentham Instruments, UK) was used to measure the UV irradiance reaching the test containers (FigureS1, Supplementary material); the spectroradiometer was placed in between the avoidance behavior test containers.The UV irradiance Journal Pre-proof measurements were corrected according to a weighing factor concerning the erythema reference action spectrum established by the International Commission on Illumination (CIE) to obtain the biologically effective UV irradiance.Then, the daily UV dose (total amount of biologically effective UV irradiance reaching the avoidance behavior test containers per day) was calculated by applying Equation 2 (in J m -2 ): I i (biologically effective UV irradiance in mW m -2 ); t i (exposure time in s); i (UV regime phase: 1 and 3 refers to the UV radiation exposure period to the UV index of ~ 7, and 2 to the UV exposure period to the UV index of ~ 10).
Fisher's Journal Pre-proof J o u r n a l P r e -p r o o f 12 exact test was used to check for differences in the avoidance behavior of soil invertebrates towards each metal(loid)-contaminated soil when varying individual climate factors, by comparing the observed distribution of organisms between both test container sections with an expected distribution where no avoidance response occurred (Natal-da-Luz et al., 2008): i) one-tailed tests were applied to analyze the results from the avoidance behavior tests (control soil vs. metal(loid)-contaminated test soil); ii) twotailed tests were applied to analyze the results from the dual controls (control soil vs.control soil).To deepen into the effects of the climate factors evaluated, the total number of organisms in the control section of the test containers was compared by a two-way ANOVA followed by Tukey's post-hoc test(Natal-da-Luz et al., 2008).The factors included in the analysis were the soil type (mining and agricultural) and the climate conditions (climate treatments simulated within each climate factor).Data were log-transformed when they failed to pass the Leven's test for the homogeneity of variance.

E
. crypticus per combination tested, and ii) homogeneous distribution of organisms in dual control tests (40% to 60% in each test container section).Taking this into account, Journal Pre-proof J o u r n a l P r e -p r o o f 13 the validity criteria were met for both soil invertebrate species in all the cases (standard conditions and climate treatments simulated) except at the highest air temperature regime tested (25 -35 ºC) (Tables different climate treatments simulated, including on the standard conditions (Figure S2, Supplementary material).Exposure to the standard conditions led to decreases in the soil moisture content of ~ 15-17% in all the soils (control, mining and agricultural Journal Pre-proof J o u r n a l P r e -p r o o f 16 soils).The simulation of different air temperature treatments led to significant (t-test, p < 0.05) losses of soil moisture content, especially at the highest temperatures (Figure S2a, Supplementary material): control soil (~ 30-44% decrease; only significant at 15 -25 ºC); mining soil (~ 27% decrease at 15 -25 °C and ~ 51-52% decrease at 20 -30 °C and 25 -35 °C); agricultural soil (~ 34% decrease at 15 -25 °C and ~ 56-61% decrease at 20 -30 °C and 25 -35 °C).Exposure to UV radiation led to significant (t-test, p < 0.05) decreases of soil moisture content in all the soils (~ 39-44%) (Figure S2b, Supplementary material).Exposure to atmospheric CO 2 led to losses of soil moisture content in all the soils (control soil ~ 23-31%, mining soil ~ 36-42% and agricultural soil ~ 25-47%) (Figure and, thus, diminished the effects of these elevated temperatures.Besides invertebrates' survival, at 25 -35 ºC, both F. candida and E. crypticus tended to stay in one of the container sections of the dual control tests instead of showing a homogeneous distribution as in the other temperature treatments (Tables Caramanna et al. (2013) reported a decrease in soil pH after one hour of CO 2 injection into soil columns.He et al. (2016) also found soil acidification after short periods of CO 2 injection into plant germination boxes.Soil acidification could have promoted greater availability of some of the metals present in the contaminated test soils and turned the medium less favorable for F. candida.Unfortunately, the lack of studies on the effects of atmospheric CO 2 concentration on the avoidance behavior of soil invertebrates towards soil contamination does not allow comparing the results obtained.Moreover, the information gathered highlights how climate change might compromise and impact F. candida in anthropogenic-contaminated areas.The migration (avoidance or escape) of collembolans may lead to changes in the structure of soil communities and/or loss of the beneficial functions they perform, if no other organisms can assume their role, impairing ecosystems functionality and sustainability.Differently to F. candida, the avoidance behavior that E. crypticus showed towards metal(loid) soil contamination under standard conditions was not modified by the different climate factors evaluated (Figure2).Enchytraeids avoided both metal(loid)contaminated test soils when exposed to the distinct treatments of air temperature, UV radiation and atmospheric CO 2 concentration simulated, which highlights how unfavorable the test soils were for these organisms.The only difference observed was the high data variability obtained at the highest air temperature regime tested(25 -35    Journal Pre-proof J o u r n a l P r e -p r o o f 21 ºC), especially for the mining soil in which enchytraeids were equally distributed behavioral strategies towards metal(loid) soil contamination.Folsomia candida did not avoid the metal(loid)-contaminated soils tested, neither when exposed to the standard conditions recommended by the ISO guidelines nor when increasing air temperature or when exposed to UV radiation.However, under rising atmospheric CO 2 concentration, Journal Pre-proof J o u r n a l P r e -p r o o f 22 F. candida changed its behavioral pattern and avoided the metal(loid)-contaminated soils.On the contrary, E. crypticus always avoided the metal(loid)-contaminated soils tested, regardless of the exposure climate conditions.Therefore, the present study shows that changes in climate conditions may alter the behavioral patterns of soil invertebrates towards meta(loid)-contaminated soils.However, this depends on the invertebrate species tested and the climate factor modulated.Moreover, the information gathered highlights how climate change might compromise and impact soil-dwelling organisms, preventing them from performing vital processes that may potentially impair terrestrial ecosystems functionality and sustainability.

Figure captions Figure 1 .
Figure captions

Figure 2 .
Figure 2. Avoidance behavior of Folsomia candida and Enchytraeus crypticus in tests

Table 2 .
Survival and organism distribution between sections of Folsomia candida in the dual control tests (both test container sections filled with Lufa 2.2 soil) for the standard conditions (climate conditions recommended by the standardized ISO guidelines) and the climate treatments simulated by individually modulating the climate factors air temperature, ultraviolet (UV) radiation and atmospheric CO 2 concentration.Data are average ± SD (n = 5).For survival asterisks (*) indicate significant differences between climate treatments and standard conditions (Student t-test; p < 0.05).For organism distribution between sections asterisks (*) indicate significant differences between observed and expected values (Fisher's test, p<0.05).

Table 3 .
Survival and organism distribution between sections of Enchytraeus crypticus in the dual control tests (both test container sections filled with Lufa 2.2 soil) for the standard conditions (climate conditions recommended by the standardized ISO guidelines) and the climate treatments simulated by individually modulating the climate factors air temperature, ultraviolet (UV) radiation and atmospheric CO 2 concentration.