Combined effects of three insecticides with different modes of action on biochemical responses of the solitary bee Osmia bicornis.

Bees are simultaneously exposed to a variety of pesticides, which are often applied in mixtures and can cause lethal and sublethal effects. The combined effects of pesticides, however, are not measured in the current risk assessment schemes. Additionally, the sublethal effects of pesticides on a variety of physiological processes are poorly recognized in bees, especially in non-Apis solitary bees. In this study, we used a full-factorial design to examine the main and interactive effects of three insecticide formulations with different modes of action (Mospilan 20 SP, Sherpa 100 EC, and Dursban 480 EC) on bee biochemical processes. We measured acetylcholinesterase (AChE), glutathione S-transferase (GST) and esterase (EST) activities, as well as a nonenzymatic biomarker associated with energy metabolism, i.e., ATP level. All studied endpoints were affected by Sherpa 100 EC, and the activities of AChE and EST as well as ATP levels were affected by Dursban 480 EC. Moreover, complex interactions between all three insecticides affected ATP levels, showing outcomes that cannot be predicted when testing each insecticide separately. The results indicate that even if interactive effects are sometimes difficult to interpret, there is a need to study such interactions if laboratory-generated toxicity data are to be extrapolated to field conditions.

Mortality Pattern of Poecilus cupreus Beetles after Repeated Topical Exposure to Insecticide─Stochastic Death or Individual Tolerance?

The mortality of organisms exposed to toxicants has been attributed to either stochastic processes or individual tolerance (IT), leading to the stochastic death (SD) and IT models. While the IT model follows the principles of natural selection, the relevance of the SD model has been debated. To clarify why the idea of stochastic mortality has found its way into ecotoxicology, we investigated the mortality of Poecilus cupreus (Linnaeus, 1758) beetles from pesticide-treated oilseed rape (OSR) fields and unsprayed meadows, subjected to repeated insecticide treatments. We analyzed the mortality with the Kaplan-Meier estimator and general unified threshold model for survival (GUTS), which integrates SD and IT assumptions. The beetles were exposed three times, ca. monthly, to the same dose of Proteus 110 OD insecticide containing thiacloprid and deltamethrin, commonly used in the OSR fields. Kaplan-Meier analysis showed that the mortality of beetles from meadows was much higher after the first treatment than after the next two, indicating the IT model. Beetles from the OSR displayed approximately constant mortality after the first and second treatments, consistent with the SD model. GUTS analysis did not conclusively identify the better model, with the IT being marginally better for beetles from meadows and the SD better for beetles from OSR fields.

Effects of agricultural landscape structure and canola coverage on biochemical and physiological traits of the ground beetle Poecilus cupreus.

The intensifications in the agricultural landscape and the application of pesticides can cause adverse effects on the fitness of organisms in that landscape. Here, we investigated whether habitats with different agricultural pressures influenced acetylcholinesterase (AChE) activity - a biomarker for exposure to pesticides, respiration rate, and resistance to starvation in the ground beetle Poecilus cupreus. Two differently structured landscapes were selected for the study, one dominated by small (S) and another by large (L) fields. Within each landscape three habitat types were selected: in the S landscape, these were habitats with medium (M), small (S) and no canola (meadow, 0) coverage (i.e., SM, SS, S0), and in the L landscape habitats with large (L), medium (M) and no canola (meadow, 0) coverage (i.e., LL, LM, L0), representing different levels of agricultural pressure. The activity of AChE was the highest in beetles from canola-free habitats (S0 and L0), being significantly higher than in beetles from the SM and SS habitats. The mean respiration rate corrected for body mass was also the highest in S0 and L0 beetles, with significant differences between populations from L0 vs. SS and from S0 vs. SS. Only beetles from S0, SS, L0, and LM were numerous enough to assess the resistance to starvation. Individuals from the LM habitat showed better survival compared to the canola-free habitat in the same landscape (L0), whereas in S landscape the SS beetles survived worse than those from S0, suggesting that characteristics of L landscape may lead to developing mechanisms of starvation resistance of P. cupreus in response to agricultural pressure.

Floral resources,energetic value and pesticide residues in provisions collected by Osmia bicornis along a gradient of oilseed rape coverage.

Pollinators in agricultural landscapes are facing global decline and the main pressures include food scarcity and pesticide usage. Intensive agricultural landscapes may provide important food resources for wild pollinators via mass flowering crops. However, these are monofloral, short-term, and may contain pesticide residues. We explored how the landscape composition with a different proportion of oilseed rape (6-65%) around Osmia bicornis nests affects floral diversity, contamination with pesticides, and energetic value of provisions collected by this species of wild bees as food for their offspring. Altogether, the bees collected pollen from 28 plant taxa (6-15 per nest) and provisions were dominated by Brassica napus (6.0-54.2%, median 44.4%, 12 nests), Quercus sp. (1.2-19.4%, median 5.2%, 12 nests), Ranunculus sp. (0.4-42.7%, median 4.7%, 12 nests), Poaceae (1.2-59.9%, median 5.8%, 11 nests) and Acer sp. (0.6-42%, median 18.0%, 8 nests). Residues of 12 pesticides were found in provisions, with acetamiprid, azoxystrobin, boscalid, and dimethoate being the most frequently detected at concentrations up to 1.2, 198.4, 16.9 and 17.8 ng/g (median 0.3, 10.6, 11.3, 4.4 ng/g), respectively. Floral diversity and energetic value of provisions, but not the Pesticide Risk Index depended on landscape structure. Moreover, pollen diversity decreased, and energetic value increased with landscape diversity. Thus, even a structurally simple landscape may provide diverse food for O. bicornis if the nest is located close to a single but resource-diverse patch. Both B. napus and non-crop pollen were correlated with pesticide concentrations.

Homogeneity of agriculture landscape promotes insecticide resistance in the ground beetle Poecilus cupreus.

The intensification of agriculture leads to increased pesticide use and significant transformation from small fields towards large-scale monocultures. This may significantly affect populations of non-target arthropods (NTA). We aimed to assess whether the multigenerational exposure to plant protection products has resulted in the evolution of resistance to insecticides in the ground beetle Poecilus cupreus originating from different agricultural landscapes. Two contrasting landscapes were selected for the study, one dominated by small and another by large fields. Within each landscape the beetles were collected at nine sites representing range of canola coverage and a variety of habitat types. Part of the collected beetles, after acclimation to laboratory conditions, were tested for sensitivity to Proteus 110 OD-the most commonly used insecticide in the studied landscapes. The rest were bred in the laboratory for two consecutive generations, and part of the beetles from each generation were also tested for sensitivity to selected insecticide. We showed that the beetles inhabiting areas with medium and large share of canola located in the landscape dominated by large fields were less sensitive to the studied insecticide. The persistence of reduced sensitivity to Proteus 110 OD for two consecutive generations indicates that either the beetles have developed resistance to the insecticide or the chronic exposure to pesticides has led to the selection of more resistant individuals naturally present in the studied populations. No increased resistance was found in the beetles from more heterogeneous landscape dominated by small fields, in which spatio-temporal diversity of crops and abundance of small, linear off-crop landscape elements may provide shelter that allows NTAs to survive without developing any, presumably costly, resistance mechanisms.

Toxicokinetics of three insecticides in the female adult solitary bee Osmia bicornis.

The worldwide decline of pollinators is of growing concern and has been related to the use of insecticides. Solitary bees are potentially exposed to many insecticides through contaminated pollen and/or nectar. The kinetics of these compounds in solitary bees is, however, unknown, limiting the use of these important pollinators in pesticide regulations. Here, the toxicokinetics (TK) of chlorpyrifos (as Dursban 480 EC), cypermethrin (Sherpa 100 EC), and acetamiprid (Mospilan 20 SP) was studied for the first time in Osmia bicornis females at sublethal concentrations (near LC20s). The TK of the insecticides was analysed in bees continuously exposed to insecticide-contaminated food in the uptake phase followed by feeding with clean food in the decontamination phase. The TK models differed substantially between the insecticides. Acetamiprid followed the classic one-compartment model with gradual accumulation during the uptake phase followed by depuration during the decontamination phase. Cypermethrin accumulated rapidly in the first two days and then its concentration decreased slowly. Chlorpyrifos accumulated similarly rapidly but no substantial depuration was found until the end of the experiment. Our study demonstrates that some insecticides can harm solitary bees when exposed continuously even at trace concentrations in food because of their constant accumulation leading to time-reinforced toxicity.

Effects of agricultural landscape structure, insecticide residues, and pollen diversity on the life-history traits of the red mason bee Osmia bicornis.

Agricultural landscapes have changed substantially in recent decades, shifting from the dominance of small fields (S) with diverse cropping systems toward large-scale monoculture (L), where landscape heterogeneity disappears. In this study, artificial nests of the red mason bee, Osmia bicornis, were placed in S and L landscape types on the perimeter of oilseed rape fields representing different oilseed rape coverages (ORC, % land cover). The local landscape structure around each nest was characterised within a 100, 200, 500, and 1000 m radius using ORC and 14 landscape characteristics, which were then reduced by non-metric multidimensional scaling (nMDS) to two axes: nMDS1 characterised the dataset primarily according to land fragmentation and the main crop, whereas nMDS2 captured the prevalence of more natural areas in the landscape. Pollen diversity and insecticide risk levels in the pollen provisions collected by the bees were analysed, and their dependence on the landscape structure was tested. Thereafter, the effects of pollen diversity, insecticide risk, and landscape structure on the life-history traits of bees and their sensitivity to topically applied Dursban 480 EC were determined. Pollen taxa richness in a single nest ranged from 3 to 12, and 34 pesticides were detected in the pollen at concentrations of up to 320 ng/g for desmedipham. The O. bicornis foraging range was relatively large, indicating that the landscape structure within a radius of ~1000 m around the nest is important for this species. Pollen diversity in the studied areas was of minor importance for bee performance, but the ORC or landscape structure significantly affected the life-history traits of the bees. Contamination of pollen with insecticides affected the bees by decreasing the mass of newly emerged adults but their sensitivity to Dursban 480 EC was not related to environmental variables.

Physiological and biochemical response of the solitary bee Osmia bicornis exposed to three insecticide-based agrochemicals.

The physiological and biochemical stress induced by pesticides need to be addressed in economically and ecologically important non-Apis solitary bees, particularly at lower than field-applied concentrations. Thus, the aim of the present study was to analyse the physiological and biochemical changes in female adult Osmia bicornis bees upon continuous oral exposure to three insecticide-based agrochemicals - i.e. Dursban 480 EC (active ingredient - a.i. chlorpyrifos), Sherpa 100 EC (a.i. cypermethrin), and Mospilan 20 SP (a.i. acetamiprid), in a toxicokinetic manner (feeding with either insecticide-contaminated food or uncontaminated food (controls) for 8 d in the contamination phase followed by 8 d of decontamination (i.e. feeding with uncontaminated food)). All three tested agrochemicals altered the energetic budget of bees by the deprivation of energy derived from lipids and carbohydrates (but not proteins) and/or a decrease in respiration based metabolic rate (energy consumption) compared to the controls. The activities of acetylcholinesterase and glutathione-S-transferase enzymes were not altered by insecticides at tested concentrations. These results show that chronic exposure to at least some pesticides even at relatively low concentrations may cause severe physiological disruptions that could potentially be damaging for the solitary bees.

Different effects of Zn nanoparticles and ions on growth and cellular respiration in the earthworm Eisenia andrei after long-term exposure.

In this study, the effects of zinc nanoparticles (ZnO-NPs) and ions (ZnCl2) on the mortality, growth, maturation, and cellular respiration of the earthworm Eisenia andrei were assessed. Earthworms were individually exposed for 98 days, starting from the juvenile stage, to soils contaminated with either ZnO-NPs or ZnCl2 (125, 250, 500 and 1000 mg Zn kg-1 dry weight (dw)). Exposure to the highest-concentration ionic treatments (500 and 1000 mg kg-1) caused 100% mortality, while for other treatments, mortality did not exceed 15% at the end of exposure. Compared to the control treatment, both 125-1000 mg kg-1 ZnO-NPs and 125 or 250 mg kg-1 ZnCl2 stimulated earthworm growth, which might be due to a hormetic effect. ZnO-NPs and ZnCl2 caused different responses at medium Zn concentrations (250 and 500 mg kg-1): earthworms exposed to ionic treatment at 250 mg kg-1 were characterized by a significantly lower growth constant, lower cellular respiration rate, later inflection point, and higher final body weight than those exposed to ZnO-NPs treatments at the same (250 mg kg-1) or twice as high (500 mg kg-1) nominal Zn concentrations. However, differences were not observed in all examined parameters between the studied forms when the highest-concentration ZnO-NPs treatment was compared with the lowest-concentration ionic treatment, which was likely due to the same levels of available Zn concentrations in those treatments. Overall, different growth and maturation strategies accompanied by pronounced differences in cellular respiration were adopted by earthworms exposed to low and medium levels of either ZnO-NPs or ZnCl2.

Supporting non-target arthropods in agroecosystems: Modelling effects of insecticides and landscape structure on carabids in agricultural landscapes.

Intensification of agricultural practices is one of the most important drivers of the dramatic decline of arthropod species. We do not know, however, the relative contribution to decline of different anthropogenic stressors that are part of this process. We used high-resolution dynamic landscape models and advanced spatially-explicit population modelling to estimate the relative importance of insecticide use and landscape structure for population dynamics of a widespread carabid beetle Bembidion lampros. The effects of in-crop mitigation measures through the application of insecticides with reduced lethality, and off-crop mitigation measures by increasing abundance of grassy field margins, were evaluated for the beetle along the gradient of landscape heterogeneity. Reducing the insecticide-driven lethality (from 90 to 10%) had larger positive impacts on beetle density and occupancy than increasing the abundance of field margins in a landscape. The effects of increasing field margins depended on their width and overall abundance in the landscape, but only field margins 4 m wide, applied to at least 40% of fields, resulted in an increase in beetle population density comparable to the scenario with the smallest reduction of insecticide-driven lethality we considered. Our findings suggest the importance of field margins rather as a supporting not stand-alone mitigation measure, as they generally improved effects of reduction of insecticide-driven lethality. Therefore, adding sufficiently broad off-field habitats should help to maintain viable beetle populations in agricultural landscapes even with moderate use of insecticides. In general, the less persistent the insecticides are in the environment, the larger positive impacts of applied mitigation measures on beetle populations were found. We also showed that the effectiveness of applied mitigation measures strongly depends on landscape and farmland heterogeneity. Thus, to achieve the same management or mitigation target in different landscapes might require different strategies.

The development of the solitary bee Osmia bicornis is affected by some insecticide agrochemicals at environmentally relevant concentrations.

Solitary bees provide essential pollination services for many arable crops, but are prone to global decline. Agricultural intensification, which is connected with pesticide usage, is among major threats to bees and, thus, to the food security and ecosystem stability. As it may not be possible to cease pesticide usage currently because of the growing demand for food, it is crucial to understand the pesticide toxicities to bees for better protection of pollinator populations. The majority of studies have focused on social bees, and those on solitary bees studied effects of adult exposure, whereas these bees are also likely to be exposed as larvae via the consumption of contaminated pollen. Here, the effects of three commonly used insecticide-based plant protection products on the development of the solitary bee, Osmia bicornis (red mason bee), were studied by exposing larvae to insecticide-contaminated multifloral pollen. The tested insecticides were: Dursban480EC, containing the organophosphate chlorpyrifos (CHP), Sherpa100EC, containing the pyrethroid cypermethrin (CYP), and Mospilan20SP with the neonicotinoid acetamiprid (ACT). When compared to the control larvae fed with uncontaminated-pollen, both CHP and CYP significantly reduced the O. bicornis larval survival and their body mass at all tested concentrations. In contrast, ACT did not affect either larval survival or body mass, but the length of larval stage to cocoon formation was significantly shortened compared to controls. None of studied insecticides affected the mass of cocooned individuals. However, at least 80% of individuals exposed to any of the tested insecticides died before reaching the adult stage, whereas 43% of the controls emerged successfully after overwintering. Although no clear monotonic dose-response relationships were found, our study showed that at least some insecticide formulations affect the development of O. bicornis even at concentrations actually found in pollen in the field, indicating an urgent need for revising current pesticide usage recommendations.

Unravelling the ZnO-NPs mechanistic pathway: Cellular changes and altered morphology in the gastrointestinal tract of the earthworm Eisenia andrei.

A major uptake route of nanoparticles (NPs) occurs via the gastrointestinal (GI) tract. When GI tract cells are exposed, NPs cytotoxic effects are observed that subsequently adversely affect the GI tract morphology and have consequences for the whole organism. The aim of this study was to understand the mechanism of effects caused by ZnO-NPs compared to Zn ions on the earthworm Eisenia andrei. The following aspects of individually exposed earthworms were investigated: 1) qualitative structural alterations in the gut epithelium and chloragogen cells of the GI tract, 2) quantitative changes within chloragogen tissues after 48 h of exposure (using morphometric analysis), and 3) the ADP/ATP ratio in homogenized tissue of the whole organism after 21 days of exposure to contaminated soil (contamination phase) followed by 14 days of elimination in clean soil (decontamination phase) to identify possible recovery. Both ZnO-NPs and Zn ions adversely affect the gut epithelium and chloragogen tissue of earthworms after 48 h of exposure to contaminated soil. Morphometric measurements revealed that the proportions of debris vesicles in the chloragocytes were significantly lower in worms exposed to ZnO-NPs than in worms exposed to Zn ions. Moreover, numerous spherite granules were observed in the chloragocytes of ionic Zn-treated worms, but not the ZnO-NPs-treated worms, suggesting differential regulation of these Zn forms. The Zn cytotoxic effect was not reflected in ADP/ATP ratio measurements. Our study provides new insights into nano-specific effects that are distinctive from ion regulation inside the GI tract and furthers our understanding of the relationship between effects at the cellular and whole-body levels.

Effects of Cadmium Bioavailability in Food on Its Distribution in Different Tissues in the Ground Beetle Pterostichus oblongopunctatus.

In most laboratory studies with oral exposure of terrestrial invertebrates to metals an artificial food, which is easy to handle, is used. The bioavailability of metals from this artificial food may, however, be much higher than from more field relevant food sources. Such differences may affect toxicokinetic processes in different tissues. To test the effect of bioavailability of Cd in food on Cd toxicokinetics and internal distribution in terrestrial invertebrates, we performed the experiment using the ground beetle Pterostichus oblongopunctatus exposed to Cd via food differing in their soluble Cd pool. We showed that in carabids Cd accumulation and elimination pattern in different tissues is not governed by the metal availability in food.

Energy reserves and respiration rate in the earthworm Eisenia andrei after exposure to zinc in nanoparticle or ionic form.

The energy budget is an indicator of an organism's overall condition. Changes in energy reserves and/or energy consumption have been used as biomarkers of toxic stress. To understand the effects of different forms and concentrations of Zn and the costs of effective Zn regulation by the earthworm Eisenia andrei, we performed a toxicokinetic experiment in which individuals were sampled over time to determine the available energy reserves (total carbohydrate, protein, and lipid content), energy consumption (measured at the cellular level and as the whole-animal respiration rate), and internal Zn concentration. The earthworms were exposed to ZnCl2 or zinc nanoparticles (ZnO-NPs) in Lufa 2.2 soil for 21 days (contamination phase), followed by 14 days of elimination in clean soil (decontamination phase). Carbohydrates were the only energy reserves with significantly lower levels following ZnO-NP 1000 treatment than following other treatments (p ≤ 0.00001) in the contamination phase. The total available energy reserves and protein content did not differ among treatments, but a significant effect of exposure time was observed (p ≤ 0.0001). Exposure to Zn (both ions and NPs) increased energy consumption at the cellular level, reflecting the high energy demand of the stress response. The results indicated that E. andrei can regulate internal Zn concentrations efficiently, regardless of form or concentration, without considerable impact on energy reserves or respiration rate.

Ground beetle communities in a mountain river subjected to restoration: The Raba River, Polish Carpathians.

Effects of passive restoration of mountain rivers on the organisms inhabiting exposed riverine sediments are considerably less understood than those concerning aquatic biota. Thus, the effects of a recovery of the Raba River after abandonment of maintenance of its channelization scheme on ground beetle (Coleoptera: Carabidae) communities were investigated by comparing 6 unmanaged cross-sections and 6 cross-sections from adjacent channelized reaches. In each cross-section, ground beetles were collected from 12 sampling sites in spring, summer, and autumn, and 8 habitat parameters characterizing the cross-sections and sampling sites were determined. Within a few years after abandonment of the Raba River channelization scheme, the width of this gravel-bed river increased up to three times and its multi-thread pattern became re-established. Consequently, unmanaged river cross-sections had significantly larger channel width and more low-flow channels and eroding cutbanks than channelized cross-sections. Moreover, sampling sites in the unmanaged cross-sections were typified by significantly steeper average surface slope and larger average distance from low-flow channels than the sites in channelized cross-sections. In total, 3992 individuals from 78 taxa were collected during the study. The ground beetle assemblages were significantly more abundant and richer in species in the unmanaged than in the channelized cross-sections but no significant differences in carabid diversity indices between the two cross-section types were recorded. Redundancy Analysis indicated active river zone width as the only variable explaining differences in abundance and species richness among the cross-sections. Multiple regression analysis indicated species diversity to predominantly depend on the degree of plant cover and substrate grain size. The study showed that increased availability of exposed sediments in the widened river reaches allowed ground beetles to increase their abundance and species richness within a few years after the onset of river restoration, but more time may be needed for development of more diverse carabid communities.

Effect of cadmium bioavailability in food on its compartmentalisation in carabids.

Metals assimilated by organisms are sequestered in various compartments and some forms are more stable than others. Sequestration mechanisms used by invertebrates to detoxify metals and prevent interaction with important biomolecules include metal binding to proteins and other ligands, and storage in inorganic granules. The rate and extent at which metal concentrations in different compartments respond to metal concentrations in food and food characteristics has not received much attention, despite being of great relevance. We performed an experiment on the carabid beetle Pterostichus oblongopunctatus exposed to Cd via food made of ground mealworm (Tenebrio molitor) larvae, either reared on Cd contaminated medium or artificially spiked after grinding with CdCl2 solution. Thus, in both cases we used the same type of food, differing only in the soluble Cd pool available to the predators, represented by P. oblongopunctatus. Subcellular compartmentalisation of Cd into organelles, heat-sensitive and heat-stable proteins (the first supernatant, S1 fraction), cellular debris (the second supernatant, S2 fraction) and metal-rich granules (G fraction) was checked a few times during the contamination (90 d) and decontamination (24 d) phases in a toxicokinetic experiment by using different centrifugation steps. The results showed no effect of the type of food (naturally, Cd-N, vs. artificially contaminated with Cd, Cd-A) on Cd sequestration kinetics in P. oblongopunctatus, but the amount of Cd sequestered in the S1 and G fractions were in general higher in the Cd-A than the Cd-N treatment, indicating that Cd transfer in the food web depends on the speciation of the metal in the food. The proportional distribution of Cd over different fractions was, however, similar in beetles fed both food types. Most of the accumulated Cd in the beetles existed as fraction S1 (ca. 35%), which is important for the transfer of metals to higher trophic levels in a food web.

Toxicokinetics of zinc-oxide nanoparticles and zinc ions in the earthworm Eisenia andrei.

The toxicokinetics of zinc in the earthworm Eisenia andrei was investigated following exposure for 21 days to ionic zinc (ZnCl2) or zinc oxide nanoparticles (ZnO-NPs) in Lufa 2.2 soil, followed by 21 days elimination in clean soil. Two concentrations were tested for both ZnCl2 (250 and 500µg Zn g-1) and ZnO-NPs (500 and 1000µg Zn g-1), corresponding to EC25 and EC50 for effects on reproduction. Based on the measured internal Zn concentrations in the earthworms over time of exposure, the kinetics parameters ka - assimilation rate constant (gsoil g-1body weight day-1) and ke - elimination rate constant (day-1) were estimated using a one-compartment model for either total Zn concentrations in the soil or porewater Zn concentrations. In the ZnCl2 treatments, ka was higher for total Zn concentrations in soil, whereas in the ZnO-NP treatments, ka was higher for porewater Zn concentrations. The value of ke did not differ between the two Zn forms (ZnCl2 vs ZnO-NPs) for either EC50 or EC25 when related to total Zn concentrations in soil, but for EC50, ke related to porewater Zn concentrations was significantly higher for ZnCl2 than for ZnO-NPs. It is concluded that differences in kinetic parameters between treatments were connected with exposure concentrations rather than with the form of Zn. Zinc was efficiently regulated by the earthworms in all treatments: a 2-fold increase in exposure concentration resulted in a less than 2-fold increase in internal concentration, and after transfer to uncontaminated soil the internal Zn concentrations in the earthworms returned to ca 111µgg-1 dw in all treatments.

Combined effects of chlorpyriphos, copper and temperature on acetylcholinesterase activity and toxicokinetics of the chemicals in the earthworm Eisenia fetida.

In polluted environments organisms are commonly exposed to a combination of chemicals with different modes of action, and their effects can be additionally modified by natural abiotic conditions. One possible mechanism for interactions in mixtures is via toxicokinetics, as chemicals may alter the uptake, distribution, biotransformation and/or elimination of each other, and all these processes can be affected by temperature. In this study, the effect of temperature (T) on the toxicokinetics of copper (Cu) and chlorpyriphos (CHP), applied either singly or in binary mixtures, was studied in the earthworm Eisenia fetida. The experiments were conducted at 10 or 20 °C and the earthworms were exposed to environmentally realistic concentrations of Cu and/or CHP for 16 d, followed by a depuration period of 4 d in uncontaminated soil. The earthworms were sampled for body Cu and/or CHP concentrations and acetylcholinesterase (AChE) activity measurements. The CHP degradation rate in the soil was substantially higher at 20 °C and in soil treated with Cu. The significant (p < 0.05) inhibition of AChE activity in the earthworms exposed to CHP was found. The effect of Cu was significant only at p < 0.1. No synergistic effect of the parallel CHP and Cu exposure was found. Four days after transferring the earthworms to uncontaminated soil, the AChE activity recovered to the level observed in control animals. The temperature effect on the toxicokinetic parameters was more pronounced for CHP than for Cu. In the case of CHP, the assimilation rate constant (kA) was significantly higher at 20 °C than at 10 °C, both in CHP-only and CHP + Cu treatments. A similar trend was found for the elimination rate constant (kE), but the difference was statistically significant only for non-Cu treatments. In the case of Cu, the general trend of higher kA and kE at 20 °C and in the absence of CHP was observed.

Metal toxicokinetics and metal-driven damage to the gut of the ground beetle Pterostichus oblongopunctatus.

Toxicokinetics makes up the background for predicting concentrations of chemicals in organisms and, thus, ecological risk assessment. However, physiological and toxicological mechanisms behind toxicokinetics of particular chemicals are purely understood. The commonly used one-compartment model has been challenged recently, showing that in the case of metals it does not describe the pattern observed in terrestrial invertebrates exposed to highly contaminated food. We hypothesised that the main mechanism shaping toxicokinetics of metals in invertebrates at high exposure concentrations in food is the cellular damage to the gut epithelial cells. Gut damage should result in decreased metal assimilation rate, while shedding the dead cells - in increased elimination rate. We performed a typical toxicokinetic experiment, feeding the ground beetles Pterostichus oblongopunctatus food contaminated with Cd, Ni or Zn at 40 mM kg-1 for 28 days, followed by a depuration period of 14 days on uncontaminated food. The male beetles were sampled throughout the experiment for body metal concentrations and histopathological examinations of the midgut. All metals exhibited a complex pattern of internal concentrations over time, with an initial rapid increase followed by a decrease and fluctuating concentrations during further metal exposure. Histopathological studies showed massive damage to the midgut epithelium, with marked differences between the metals. Cd appeared the most toxic and caused immediate midgut cell degeneration. The effects of Ni were more gradual and pronounced after at least 1 week of exposure. Zn also caused extensive degeneration in the gut epithelium but its effects were the weakest among the studied metals.