Porcellio scaber is one of the most frequently used species in toxicity testing (van Gestel et al. 2018). The parent population of animals used in this study originated from an unpolluted environment near the city of Kamnik (46°12'01.8"N, 14°35'31.7"E) in Slovenia (Europe). Animals were bred for several years in large glass containers with a mixture of limestone sand and soil at the bottom, at room temperature (20–23 °C), high relative air humidity (~ 95%) and a natural diurnal light regime. They were left to feed ad libitum on maple (Acer sp.) leaf litter, with regular additions of carrots and potatoes.

For the experiments, laboratory raised individuals were used. On the day of the recording, adult male specimens in the intermoult phase (Zidar et al. 1998) were selected from the breeding containers. Body size (measured from the anterior edge of the head to the base of the uropods) of the selected isopods ranged between 10 and 12 mm.

Isolated individuals or individuals accompanied by a conspecific were monitored in free-choice experiments where animals could select between uncontaminated and pyrethrin-contaminated soils. For this purpose, circular transparent polypropylene (PP) pots meant for food packaging (diameter 9.5 cm, height 6.0 cm) were used as test arenas. Arenas were divided into two equally sized chambers with a 3.5 cm high PP barrier (Fig. 1). In the middle of the barrier was a passage (diameter of 1.2 cm), which was large enough for the isopods to easily pass through. At the bottom of the arena there was a 10 mm thick plaster of Paris darkened with charcoal (Sørensen et al. 1997).

Approximately 20 min prior recording a 3.0 g of Lufa 2.2 soil (Speyer, Germany) was added on top of the plaster that was previously saturated with tap water. Soil was previously dried, grinded, and sifted through a 0.5 mm sieve. Homogeneously granulated substrate prevented additional tactile stimuli that can affect animals’ activity (Anselme 2013). In control groups uncontaminated soil was added to both test arena chambers simultaneously. In treatment groups uncontaminated and contaminated soils were added separately to each chamber in the following manner. First, the passage between the chambers was closed with an adhesive tape. Next, uncontaminated soil was added to one of the chambers (chamber A), previously marked with a number (Fig. 1). Afterwards, contaminated soil was added to the adjacent chamber (chamber B) and, finally, the adhesive tape blocking the passage was removed.

For each new recording a new substrate was prepared, both plaster and soil, but pots were re-used after thorough washing with tap water. This prevented any other chemical stimuli besides insecticides to affect animals’ activity. For example, it is known that pheromones in woodlice faeces promote aggregation behaviour (Takeda 1980).

Figure 1. 

The two-chambered test arena for video tracking experiments with Porcellio scaber. Animals could select between uncontaminated (chamber A) and pyrethrin-contaminated soils (chamber B).

Pyrethrins were used as a soil contaminant in all experiments in this study. Pyrethrins are chemicals of natural origin with insecticidal action that have been used to control pests indoor and outdoor since the early 1800’s (Casida 1980; Tod et al. 2003). They prolong the openness of sodium channels in nerve cells, which paralyses the animal at higher doses. Therefore, longer exposure to an insecticide reduces animal’s chances of survival.

Soil was contaminated for 1 h prior the recording with the insecticide product Flora Kenyatox Verde Plus (Unichem, Slovenia) which contained 0.2% of pyrethrin. The insecticide product was well shaken and 50 µL, 100 µL, 150 µL, and 200 µL was added to 20 g of dry soil and mixed well with a spatula. The obtained concentrations were 2.5, 5.0, 7.5, and 10.0 mL of insecticide formulation per kg dry soil which roughly correspond to 5.2, 10.4, 15.7, and 20.8 mg of pyrethrin per kg dry soil.

One-hour prior video recording animals were marked dorsally across several tergites with partly dried enamel white paint to increase the visual contrast between woodlice and the substrate. In the case of two animals per arena only one animal was marked. It was reported previously that some external markers (e.g., nail polish) may affect diurnal activity and food consumption in some isopod species (Drahokoupilová and Tuf 2012; Kenne et al. 2019). However, our previous experiments have shown that the marking we used is less persistent and has no effect on animal activity. Until recordings, animals were kept on moist plaster to prevent desiccation.

At the start of recordings one or two animals were placed in test arenas, always in chamber A containing uncontaminated soil. The animals were then continuously recorded for 3 h. In similar studies, animals’ activity was tracked for 2 or 4 hours (Bayley et al. 1997; Sørensen et al. 1997; Engenheiro et al. 2005). Our preliminary observations have shown that P. scaber is highly active up to 3 h after introducing into the arena but then its activity decreases due to habituation (Anselme 2013), therefore recording longer than 3 h is unnecessary.

To provide a recording environment isolated from the outside light and other unwanted perturbances, a specially designed recording box was used. The box measured 55, 100, and 100 cm in depth, width, and height, respectively. Animals were recorded only under infrared light (850 nm) to avoid any light-induced behaviour (Devigne et al. 2011; Broly et al. 2015). Videos were captured with two webcams (Logitech C920) simultaneously. Cameras have been modified to improve video quality for recordings in infrared light. Each camera recorded two arenas. A 140-led IR illuminator was used to ensure adequate illumination of test arenas.

Videos were captured in VirtualDub 1.10.4 at 5 frames per second and a Full HD resolution (1920 × 1080 pixels). One pixel corresponded to 0.13 mm.

Videos were first analysed via video-tracking in Bonsai 2.4.0 (Lopes et al. 2015). The area of each test arena chamber was isolated by cropping. The white spot at the back of the marked animal was extracted from the background by thresholding and the spot’s centroid coordinates inside each chamber were determined for each video frame. In the case of paired animals only the marked animal was analysed. Altogether 44 isolated and 44 paired animals were analysed (n = 12 for 0 mg of pyrethrin per kg dry soil; n = 8 for 2.5, 5.0, 7.5, and 10.0 mg of pyrethrin per kg dry soil).

Next, videos were examined also manually. In this way a sequence of several hundred frames when animals were at rest with antennae close to the body and no detectable movements was selected. Based on allocations of the centroid between two consecutive frames in this sequence the upper limit for noise was determined (0.2 pixels or 0.022 mm per frame). The upper limit for non-locomotor activity (0.8 pixels or 0.088 mm per frame) was determined based on a sequence of several hundred frames when animals were feeding or digging. All larger allocations of the centroid between two consecutive frames (> 0.8 pixels) were considered as locomotor activity. Additionally, the number of visits to contaminated soil (chamber B) was counted.

Finally, raw trajectories were imported to MS Excel and used to calculate behavioural variables: the proportion of time that isopods spent on the uncontaminated soil (chamber A), the overall duration of locomotor and non-locomotor activity. Change in animal’s position during locomotor activity was calculated as path length. Average speed was calculated as the total path length divided by the total time of locomotor activity.

All statistical analyses were performed in R 4.1.1. (R Core Team), except for the probit regression and Pearson’s correlation which were performed in SPSS 27.0. All plots were drawn using the latter software as well.

Sample sizes were relatively small, data were often non-normally distributed, and between-group variance was often heteroscedastic, therefore robust statistical methods implemented in the R package WRS2 were employed (Mair and Wilcox 2020). Instead of mean or median, a robust measure of central tendency was used, i.e., either a modified one-step M-estimator based on Huber’s Psi (est parameter set to “mom”) or a 20% trimmed mean (see below for details).

The avoidance response was determined in four different ways. In the first approach, we assumed that avoidance of contaminated soil was successful if animals spent more than half of the recording time on uncontaminated soil. Therefore, the percentage of time on uncontaminated soil was tested against a fixed value of 50% for each concentration and the control. To do so a robust one-sample test was applied using the function onesampb(), the M-estimator of central tendency, and 10,000 bootstraps (to estimate the 95% confidence intervals).

In the second approach, data on the time spent on uncontaminated soil were used to estimate the median effective concentration of pyrethrin (EC₅₀) for avoidance response. Data were first transformed by a formula adapted from ISO (2008): AR = ((t_i-t_c)/t)*100 (t_i – time on contaminated soil; t_c – time on uncontaminated soil; t – total time of observation), and then the probit regression was applied. Negative values were considered as 0% of avoidance.

In the third approach, the avoidance response was assessed by the number of visits to contaminated soil. A robust two-way ANOVA was applied using the number of visits to contaminated soil as a dependent variable, while concentration treatment (0, 2.5, 5.0, 7.5, 10.0) and number of animals (isolated, paired) were used as independent categorical variables whose interaction was tested as well. For two-way ANOVA the function pbad2way(), the M-estimator of central tendency, and 5,000 bootstraps were used. Next, post-hoc comparisons were performed to find at which concentrations the response differed from the control in isolated (4 tests) and paired (4 tests) animals, as well as to find at which concentration treatments the response of isolated and paired animals was mutually different (5 tests). For this, robust independent two-sample tests were applied using the function pb2gen(), the M-estimator of central tendency, and 10,000 bootstraps (to estimate the 95% confidence intervals). P-values were adjusted via the method of Benjamini and Hochberg (1995).

In the fourth approach to estimate avoidance response, the location of animals at the end of the 3-h recordings was used. The percentage of animals on the uncontaminated soil was calculated.

Behavioural variables on isopod activity (duration, path length, average speed) were analysed as described for the third approach to avoidance response estimation. First, a robust two-way ANOVA was performed with the specific behavioural variable as a dependent variable, while concentration treatment and number of animals were used as independent categorical variables whose interaction was also tested. Upon significant effects, the same post-hoc comparisons procedure as stated above was applied. Additionally, Pearson’s correlation between the duration of locomotor activity and path length was calculated.

Data on average speed at contaminated (chamber B) and uncontaminated (chamber A) soil involved two measurements per individual. To account for repeated measurements, a robust two-way between-within subjects ANOVA was applied. Separate models were fitted for isolated and paired animals. In both, average speed was used as a dependent variable, while concentration treatment and arena chamber (A – uncontaminated, B – contaminated) were used as independent categorical variables whose interaction was also tested. In this case the function bwtrim() and the 20% trimmed mean as an estimate of central tendency were used. Next, post-hoc comparisons were performed to find at which concentration treatments average speed differed between the two arena chambers in isolated (5 tests) and paired (5 tests) animals. For this, robust dependent two-sample tests were applied using the function yuend() and the 20% trimmed mean as an estimate of central tendency. P-values were adjusted according to Benjamini and Hochberg (1995). Additionally, effect sizes, i.e., magnitude of between group mean differences, were calculated as proposed by Wilcox and Tian (2011).

Although all animals started the experiment in chamber A, the control animals showed that the initial position of animals in the arena does not affect the time the control animals spent in each chamber of the arena (A or B, both containing uncontaminated soil). In published avoidance behaviour test protocols, animals were introduced into the test arenas differently: in the middle between control and test soils (Loureiro et al. 2005), randomly on both soils (Zidar et al. 2012; Škarkova et al. 2016) or on the uncontaminated soil (Zidar et al. 2019; the present study). Our result confirmed previous reports by Anselme (2013, 2015), that P. scaber shows high exploratory activity in a new environment and can explore both chambers of the arena in a short period of time.

During 3 h of exposure isolated isopods clearly avoided soil with pyrethrin formulation already at a concentration of 5.0 mL of formulation per kg dry soil, which roughly corresponds to 10.4 mg of pyrethrin per kg dry soil. This concentration is the lowest observed effective concentration (LOEC) in this study. According to our previous study (Zidar et al. 2012), this concentration can be considered as an upper sublethal concentration for P. scaber. In paired animals, the LOEC was twice as high, i.e., 20.8 mg/kg dry soil, at the concentration that is considered lethal to P. scaber (Zidar et al. 2012). Such weaker or delayed avoidance response in paired animals effectively means longer exposer to pyrethrin. Potentially, locomotor dysfunctions that the insecticide may induce makes the isopods even harder to retreat from the contaminated soil. Such positively reinforced feedback loop due to insecticide poisoning was reported previously for springtails exposed to dimethoate (Pereira et al. 2013).

The EC₅₀ value obtained in this study for isolated animals (2.8 mL of formulation or 5.9 mg of pyrethrin per kg dry soil) was almost twice lower than the EC₅₀ value obtained in the 48-h avoidance test with animals in groups (9.7 mg of pyrethrin per kg dry soil; Zidar et al. 2012). This was so even though in the latter study pyrethrin formulation contained piperonyl butoxide that enhances the effects of pyrethrin (Kakko et al. 2000). In paired animals the obtained EC₅₀ (7.9 mL of formulation or 16.5 mg of pyrethrin per kg dry soil) was almost 3× higher compared to isolated animals and also considerably higher than in our previous study (Zidar et al. 2012).

The frequency at which isolated animals visited contaminated soil decreased with the increased concentration of pyrethrin in soil and was in concordance with the time that animals spent on the contaminated soil. In paired animals the frequency of visits to contaminated soil was generally lower than that of isolated animals but their retention time on contaminated soil was much higher. As reported by Zidar et al. (2003), the number of visits does not necessarily reflect the exposure time.

The location of animals (on uncontaminated vs. contaminated soil) at a given time also does not necessarily reflect the avoidance response as has been reported previously (Odendaal and Reinecke 1997; Pereira et al. 2013). In our study, animals have often passed between chambers and after 3 h many animals were observed on contaminated soil, although they spent significantly more time on uncontaminated soil. However, the discrepancy between the animals’ location at a given time and avoidance success can be avoided by using average time spent on uncontaminated soil as a measure of avoidance (this study), or to record the location of animals during the exposure more frequently (see Madžarić et al. 2018).

Our results show that the accepted standard toxicity tests relying on avoidance behaviour of a group of individuals as an endpoint should probably be reconsidered when performed with gregarious animals like isopods that exhibit strong aggregation behaviour. These tests tend to underestimate the effect of the toxicant. The reason for this is twofold and originates from the dynamic hierarchy of the two independent stimuli of an opposite sign provided by the toxicant (negative) and the presence of conspecifics (positive) against the background environment (neutral). When isopods are introduced to a novel environment such as the test arena, they first explore it in approximately random movement. Note that the location of the negative stimuli is fixed while the positive stimuli move(s) randomly within the arena. When both stimuli appear on different halves of the arena the choice is clear. The dilemma arises when both negative and positive stimuli occur at the same arena half. In such scenarios, the animals’ response will depend on the relative strength of both stimuli. When toxicant concentration is high, it will prevail over conspecific attraction and animals will eventually aggregate at the optimal arena half. However, when toxicant concentration is low, conspecific attraction prevails over the negative effects of toxicant and animals will aggregate at the non-optimal arena half. The observed result is a lack of avoidance response interpreted as no effect of toxicant. However, when individual animals, and not groups, are tested at the same low toxicant concentration, they show clear avoidance signalling harmful effects. Thus, aggregation can mask the real effect of the toxicant and tests with groups of animals will tend to overestimate the effective toxicant concentration.

The real concern is that in natural populations animals will practically always be in a group. Groups of aggregating animals avoid high toxicant concentrations but are much less effective at avoiding low and moderate concentrations although harmful. Consequently, in a heterogeneously polluted environment these concentrations might eventually cause more damage as animals will be exposed to them longer and accumulate their negative effects, while they will retreat from higher concentrations. The standard toxicity tests with avoidance behaviour will however fail to reveal this. Thus, for gregarious animals we should rather estimate the effective concentration for both individual animals and those in a group. Although counterintuitive at first sight, the range of concentrations between these two values may be effectively most harmful to the natural populations.

Finally, group size and social composition of its members are additional factors that for sure add to the variation of toxicity tests results and should be considered in model species for which this is relevant and possible. Further investigation is needed in this direction. From the broadest perspective, aggregation of gregarious animals will likely affect the outcome and interpretation of any kind of choice tests with any kind of aversive (e.g., light, predator pheromones) or favourable (e.g., humidity, thigmotactic shelters) stimuli.

Locomotor behaviour of terrestrial isopods was recognised as a sensitive biomarker of exposure to different pollutants (Bayley et al. 1995, 1997; Sørensen et al. 1997; Engenheiro et al. 2005). Bayley et al. (1995) reported increased locomotory time, speed, and path length of P. scaber when exposed to sublethal concentrations of insecticide dimethoate. Later, studies showed that increased locomotor activity strongly correlate with the assimilated dimethoate and consequently acetylcholinesterase inhibition (Bayley and Beatrupe 1996; Jensen et al. 1997). On the other hand, Engenheiro et al. (2005) reported a decreased locomotory activity and path length of Porcellionides pruinosus exposed to higher concentrations of dimethoate. Decreased locomotor activity was recorded also in Oniscus asellus collected at polluted sites (Bayley et al. 1997; Sørensen et al. 1997). In our study, in contrast to the above examples, the choice for uncontaminated soil was offered, the locomotor and non-locomotor response and path length of isolated animals to pyrethrin contaminated soil was reduced. The activity of paired animals was generally low, in control and exposed animals, therefore exposed animals did not differ from the control animals. The total activity in control groups, non-locomotor and locomotor activity together, ranged 16–54 min/h in isolated and 7–27 min/h in paired animals. The activity of control isolated animals was comparable to control animals from the study of Bayley et al. (1995), while the activity of our paired animals was lower. The next obvious difference between isolated and paired isopods was the speed of locomotion on contaminated soil. Isolated animals were moving on contaminated soil much faster than on uncontaminated soil, but this was less obvious in paired animals. Faster locomotion in an environment with adverse stimuli is expected as it enhances the chance of finding a more suitable space, while slowing down can indicate the onset of favorable conditions (Fraenkel and Gunn 1961; Breed and Moore 2021). Although the rate of locomotion is often proportional to the intensity of the adverse stimulus, we did not observe such a response. On the other hand, different speed of locomotion on contaminated and uncontaminated soil also indicated that lower activity of exposed animals has not been caused by pyrethrin poisoning, as reported for dimethoate (Bayley and Beatrupe 1996).