Agnieszka J. Bednarska

Interactions between toxic chemicals and natural environmental factors — A meta-analysis and case studies

The paper addresses problems arising from effects of natural environmental factors on toxicity of pollutants to organisms. Most studies on interactions between toxicants and natural factors, including those completed in the EU project NoMiracle (Novel Methods for Integrated Risk Assessment of Cumulative Stressors in Europe) described herein, showed that effects of toxic chemicals on organisms can differ vastly depending purely on external conditions. We compiled data from 61 studies on effects of temperature, moisture and dissolved oxygen on toxicity of a range of chemicals representing pesticides, polycyclic aromatic hydrocarbons, plant protection products of bacterial origin and trace metals. In 62.3% cases significant interactions (p< or =0.05 or less) between natural factors and chemicals were found, reaching 100% for the effect of dissolved oxygen on toxicity of waterborne chemicals. The meta-analysis of the 61 studies showed that the null hypothesis assuming no interactions between toxic chemicals and natural environmental factors should be rejected at p=2.7 x 10(-82) (truncated product method probability). In a few cases of more complex experimental designs, also second-order interactions were found, indicating that natural factors can modify interactions among chemicals. Such data emphasize the necessity of including information on natural factors and their variation in time and across geographic regions in ecological risk assessment. This can be done only if appropriate ecotoxicological test designs are used, in which test organisms are exposed to toxicants at a range of environmental conditions. We advocate designing such tests for the second-tier ecological risk assessment procedures.

Environmental conditions enhance toxicant effects in larvae of the ground beetle Pterostichus oblongopunctatus (Coleoptera: Carabidae)

The wide geographical distribution of ground beetles Pterostichus oblongopunctatus makes them very likely to be exposed to several environmental stressors at the same time. These could include both climatic stress and exposure to chemicals. Our previous studies demonstrated that the combined effect of nickel (Ni) and chlorpyrifos (CHP) was temperature (T)-dependent in adult P. oblongopunctatus. Frequently the different developmental stages of an organism are differently sensitive to single stressors, and for a number of reasons, such as differences in exposure routes, their interactions may also take different forms. Because of this, we studied the effects of the same factors on the beetle larvae. The results showed that all factors, as well as their interactions, influenced larvae survival. The synergistic effect of Ni and CPF was temperature-dependent and the effect of Ni x T interaction on the proportion of emerged imagines indicated stronger toxicity of Ni at 25 degrees C than at 10 degrees C.

More ecological ERA: incorporating natural environmental factors and animal behavior.

We discuss the importance of selected natural abiotic and biotic factors in ecological risk assessment based on simplistic laboratory bioassays. Although it is impossible to include all possible natural factors in standard lower-tier ecotoxicological testing, neglecting them is not an option. Therefore, we try to identify the most important factors and advocate redesigning standard testing procedures to include theoretically most potent interactions. We also point out a few potentially important factors that have not been studied enough so far. The available data allowed us to identify temperature and O2 depletion as the most critical factors that should be included in ecotoxicity testing as soon as possible. Temporal limitations and fluctuations in food availability also appear important, but at this point more fundamental research in this area is necessary before making decisions on their inclusion in risk assessment procedures. We propose using specific experimental designs, such as Box-Behnken or Central Composite, which allow for simultaneous testing of 3 or more factors for their individual and interactive effects with greater precision and without increasing the effort and costs of tests dramatically. Factorial design can lead to more powerful tests and help to extend the validity of conclusions. Finally, ecological risk assessment procedures should include information on animal behavior, especially feeding patterns. This requires more basic studies, but already at this point adequate mechanistic effect models can be developed for some species.

Combined effect of environmental pollutants (nickel, chlorpyrifos) and temperature on the ground beetle, Pterostichus oblongopunctatus (Coleoptera: Carabidae)

Terrestrial organisms in the field often are exposed to a combination of stress factors of various origins, but little is known about interactions between different types of stressors. In the present study, we demonstrate the results of a study on interactions between Ni, chlorpyrifos (CPF), and temperature in the ground beetle, Pterostichus oblongopunctatus. The results revealed that all factors, and their interactions, influenced life-cycle parameters of the beetles (survival and reproduction). Significant three-factor interactions were found for effects on beetle survival, indicating that the combined negative effect of Ni and CPF was temperature dependent. In addition, significant effects of body mass were found: The survival of beetles treated with CPF and the reproduction of beetles exposed to Ni were positively correlated with body mass. All studied endpoints were affected by temperature. The results indicate that understanding interactions between temperature and toxicants, as well as among chemicals themselves, is essential for proper ecological risk assessment.

Two-Phase Uptake of Nickel in the Ground Beetle Pterostichus oblongopunctatus (Coleoptera: Carabidae): Implications for Invertebrate Metal Kinetics

We studied nickel (Ni) kinetics in the ground beetle Pterostichus oblongopunctatus exposed to different, potentially stressful, temperatures. We found unexpected Ni kinetics in metal-exposed adult and larval beetles. Instead of the pattern observed commonly for other metals, i.e., an increase in metal concentration followed by stabilisation in the uptake phase and a decrease after transfer to uncontaminated food, the Ni-fed beetles apparently switched to decontamination soon after the start of Ni exposure while they were still being fed Ni-spiked food. In addition, internal body Ni concentrations showed high variance. The traditional first-order, one-compartment model with the switch to decontamination set to the last day of the uptake phase appeared inadequate and in most cases was nonsignificant. Instead, the model with a regression-estimated point of switching to decontamination fit the data better, explaining 57.2–91.5% of the temporal variability of mean Ni body concentrations (weighted regression) in adult beetles and 44.1–62.3% in larvae. Temperature did not affect Ni toxicokinetics in adults, but in larvae there were some temperature-dependent differences in kinetic parameters.

Toxicokinetics of Metals in Terrestrial Invertebrates: Making Things Straight with the One-Compartment Principle

In this analysis, we first performed a critical review of one-compartment models used to describe metal toxicokinetics in invertebrates and found mathematical or conceptual errors in almost all published studies. In some publications, the models used do not represent the exact solution of the underlying one-compartment differential equations; others use unrealistic assumptions about constant background metal concentration and/or zero metal concentration in uncontaminated medium. Herein we present exact solutions of two differential-equation models, one describing simple two-stage toxicokinetics (metal toxicokinetic follows the experimental phases: the uptake phase and the decontamination phase) and another that can be applied for more complex three-stage patterns (toxicokinetic pattern does not follow two phases determined by an experimenter). Using two case studies for carabids exposed via food, based on previously published data, we discuss and compare our models to those originally used to analyze the data. Our conclusion is that when metal toxicokinetic follows a one-compartment model, the exact solution of a set of differential equations should be used. The proposed models allow assimilation and elimination rates to change between toxicokinetic stages, and the three-stage model is flexible enough to fit patterns that are more complex than the classic two-stage model can handle.

The toxicokinetics cell demography model to explain metal kinetics in terrestrial invertebrates

Metal toxicokinetics in invertebrates are usually described by one-compartment first-order kinetic model. Although the model gives an adequate description of the toxicokinetics in certain cases, it has been shown to fail in some situations. It also does not seem acceptable on purely theoretical grounds as accumulation and excretion rates may change depending on instantaneous toxicant concentration in the gut. We postulate that the mechanism behind such changes is connected with the toxic effect of metals on gut epithelial cells. Based on published data, we have constructed a mechanistic model assuming a dynamic rate of replacement of epithelial cells with increasing contamination. We use a population-type modeling, with a population of gut epithelial cells characterized by specific death and birth rates, which may change depending on the metal concentration in food. The model shows that the equilibrium concentration of a toxicant in an organism is the net result of gut cell death and replacement rates. At low constant toxicant concentrations in food, the model predicts that toxicant-driven cell mortality is moderate and the total amount of toxicant in the intestine increases slowly up to the level resulting from the gradual increase of the cell replacement rate. At high constant concentration, total toxicant amount in the gut increases very fast, what is accompanied by massive cell death. The increased cell death rate results in reduced toxicant absorption, which in turn brings its body load down. The resulting pattern of toxicokinetic trajectory for high metal concentration closely resemble that found in empirical studies, indicating that the model probably describes the actual phenomenon.

Regulation of body metal concentrations: Toxicokinetics of cadmium and zinc in crickets

Previous studies indicated that essential and xenobiotic metals differ substantially in terms of their toxicokinetics. Whether these differences are due to different assimilation rates, different elimination rates, or both, and whether all metals are regulated in a similar manner but with different efficiency remains unclear. To compare the mechanisms responsible for the regulation of different metals, parameters for toxicokinetic models have to be tested under exposures to the identical molar concentration of those metals. In this study, the cricket Gryllus assimilis was exposed to Zn or Cd at 2.5, 10, and 40mMkg(-1) dry food. The body concentrations of the metals were not perfectly regulated by the crickets. For Zn, a clear increase in the body concentration was found only at the highest treatment; whereas at the lowest treatment, the internal concentration remained unchanged throughout the experiment. At the lowest Zn concentration, the assimilation (kA) [day(-1)] and elimination (kE) [day(-1)] rate constants were balanced (kA=0.024, kE=0.024). When increasing the Zn exposure, kA decreased to 0.018 at 10mMkg(-1) and 0.01 at 40mMkg(-1), and kE increased to 0.05 and 0.07, respectively. Therefore, the body concentration of Zn was regulated by simultaneously changing the assimilation and elimination rate. By contrast, even at the lowest treatment, a significant increase in Cd concentration was observed in the crickets. The equilibrium Cd concentration resulted almost exclusively from increasing kE from 0.17, through 0.28 to 0.61 at 2.5, 10 and 40mMkg(-1). The kA for Cd did not reveal any clear trend. Zn was more efficiently regulated by crickets than was Cd: a 16-fold increase in exposure concentration (from 2.5 to 40mM Znkg(-1)) resulted only in a twofold increase of internal concentration, whereas the identical increase in Cd exposure concentration resulted in almost a sevenfold increase in internal concentration of this metal.

Subcellular partitioning of cadmium and zinc in mealworm beetle (Tenebrio molitor) larvae exposed to metal-contaminated flour.

By studying the internal compartmentalization of metals in different subcellular fractions we are able to better understand the mechanisms of metal accumulation in organisms and the transfer of metals through trophic chains. We investigated the internal compartmentalization of cadmium (Cd) and zinc (Zn) in mealworm beetle (Tenebrio molitor) larvae by breeding them in flour contaminated with either Cd at 100, 300 and 600mgkg(-1), or Zn at 1000 and 2000mgkg(-1). We separated the cellular components of the larvae into 3 fractions: the S1 or cytosolic fraction containing organelles, heat-sensitive and heat-stable proteins, the S2 or cellular debris fraction and the G or metal-rich granule fraction. The concentration of Cd and Zn in each fraction was measured at 0, 7, 14 and 21 days of being fed the flour. The concentration of Cd in the flour affected the concentration of Cd measured in each larval subcellular fraction (p≤0.0001), while the concentration of Zn in the flour only affected the Zn concentration in the S2 and G fractions (p≤0.02). Both Cd and Zn concentrations in mealworms remained relatively constant during the exposure (days 7, 14 and 21) in all three fractions, but the Cd concentrations were much higher than those found in larvae before the exposure (day 0). The concentration of Cd in the flour, however, did not affect the percentage of Cd in the S1 fraction. The contribution of Cd in the G fraction to the total Cd amount was similar (30-40%) in all Cd treatments. The percentage of Zn in all three fractions was not affected by the concentration of Zn in the flour and the relative contributions of each subcellular fraction to the total burden of Zn remained generally constant for both control and treated larvae. In general, larvae sequestered approximately 30% of Cd and Zn in the S1 fraction, which is important for the transport 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.