The Importance of Including Variable Exposure Concentrations When Assessing Toxicity of Sediment-Associated Pharmaceuticals to an Amphipod.

Pharmaceuticals have been classified as an environmental concern due to their increasing consumption globally and potential environmental impact. We examined the toxicity of sediment-associated diclofenac and citalopram administered as both single compounds and in a mixture to the sediment-living amphipod Corophium volutator. This laboratory-based study addressed the following research questions: (1) What is the toxicity of sediment-associated diclofenac and citalopram to C. volutator? (2) Can the mixture effect be described with either of the two mixture models: concentration addition (CA) or independent action (IA)? (3) What is the importance of the choice of (i) exposure measure (start concentration, time-weighted average [TWA], full exposure profile) and (ii) effect model (concentration-response vs. the toxicokinetic-toxicodynamic model general unified threshold model for survival in its reduced form [GUTS-RED]) for the derived effect concentration values? Diclofenac was more toxic than citalopram to C. volutator as a single compound (10-day exposure). Diclofenac exposure to C. volutator provided median lethal concentrations (LC50s) within the same range (11 µg g-1 dry wt sediment) using concentration-response based on TWA and both GUTS-RED models. However, concentration-response based on measured start concentrations provided an approximately 90% higher LC50 (21.6 ± 2.0 µg g-1 dry wt sediment). For citalopram, concentration-response parameters were similar regardless of model or concentration used (LC50 85-97 µg g-1 dry wt sediment), however, GUTS-RED with the assumption of individual tolerance resulted in a lower LC50 (64.9 [55.3-74.8] µg g-1 dry wt sediment). The mixture of diclofenac and citalopram followed the CA quite closely, whereas the result was synergistic when using the IA prediction. In summary, concentration-response based on TWA and GUTS-RED provided similar and reasonably good fits compared to the data set. The implications are that GUTS-RED will provide a more flexible model, which, in principle, can extend beyond the experimental period and make predictions based on variable exposure profiles (toxicity at different time frames and at different variable exposure scenarios) compared to concentration-response, which provides contaminant toxicity at one point in time. Environ Toxicol Chem 2024;00:1-11. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Quantifying synergistic interactions: a meta-analysis of joint effects of chemical and parasitic stressors.

The global biodiversity crisis emphasizes our need to understand how different stressors (climatic, chemical, parasitic, etc.) interact and affect biological communities. We provide a comprehensive meta-analysis investigating joint effects of chemical and parasitic stressors for 1064 chemical-parasitic combinations using the Multiplicative model on mortality of arthropods. We tested both features of the experimental setup (control mortality, stressor effect level) and the chemical mode of action, host and parasite phylogeny, and parasite-host interaction traits as explanatory factors for deviations from the reference model. Synergistic interactions, defined as higher mortality than predicted, were significantly more frequent than no interactions or antagony. Experimental setup significantly affected the results, with studies reporting high (> 10%) control mortality or using low stressor effects (< 20%) being more synergistic. Chemical mode of action played a significant role for synergy, but there was no effects of host and parasite phylogeny, or parasite-host interaction traits. The finding that experimental design played a greater role in finding synergy than biological factors, emphasize the need to standardize the design of mixed stressor studies across scientific disciplines. In addition, combinations testing more biological traits e.g. avoidance, coping, and repair processes are needed to test biology-based hypotheses for synergistic interactions.

Single and mixture toxicity of selected pharmaceuticals to the aquatic macrophyte Lemna minor.

Plants represent uncommon targets to evaluate pharmaceuticals toxicity. In this work, Lemna minor was employed as a plant model to determine the toxicity of selected pharmaceuticals, and to assay if such toxicity could be predicted by QSAR models based on green algae. Among eight compounds, measurable toxicity was determined for ketoprofen (EC50 = 11.8 ± 1.9 mg/L), fluoxetine (EC50 = 27.0 ± 8.7 mg/L) and clindamycin 2-phosphate (EC50 = 57.7 ± 1.7 mg/L). Even though a correlation of r2 = 0.87 was observed between experimental toxicity towards algae and L. minor, QSAR estimations based on algae data poorly predicted the toxicity of pharmaceuticals on the plant. More experimental data for L. minor are necessary to determine the applicability of these predictions; nonetheless, these results remark the importance of measuring experimental ecotoxicological parameters for individual taxa. The toxicity of pharmaceutical binary mixtures (ketoprofen, fluoxetine and clindamycin) revealed in some cases deviations from the concentration addition model; nonetheless these deviations were small, thus the interactions are unlikely to be of severe biological significance. Moreover, the EC50 concentrations determined for these pharmaceuticals are significantly higher than those detected in the environment, suggesting that acute effects on L. minor would not take place at ecosystem level.

Using TKTD Models in Combination with In Vivo Enzyme Inhibition Assays to Investigate the Mechanisms behind Synergistic Interactions across Two Species.

The aim of this study is to compare the azole synergy across an insect, Chironomus riparius, and a crustacean species, Daphnia magna. We use a combination of in vivo measurements of cytochrome P450 monooxygenase (CYP) biotransformation potential and toxicokinetic (TK) and toxicodynamic (TD) modeling to understand the mechanism behind the synergy of two azole fungicides: the imidazole prochloraz and the triazole propiconazole on the pyrethroid insecticide α-cypermethrin. For both species, the synergistic effect of prochloraz was well-described by its effect on in vivo CYP activity, which corresponded to the biotransformation rate of the TK model parameterized on the survival data of the mixture experiment. For propiconazole, however, there were 100-fold and 50-fold differences between the 50% effect concentration of in vivo CYP activity and the modeled biotransformation rate for C. riparius and D. magna, respectively. Propiconazole, therefore, seems to induce synergy through a mechanism that cannot be quantified solely by the CYP activity assay used in this study in either of the two species. We discuss the differences between prochloraz and propiconazole as synergists across the two species in the light of the type and time dynamics of affected biotransformation processes.

Environmental monitoring and risk assessment in a tropical Costa Rican catchment under the influence of melon and watermelon crop pesticides.

A monitoring network was established in streams within a catchment near the Costa Rican Pacific coast (2008-2011) to estimate the impact of pesticides in surface water (84 samples) and sediments (84 samples) in areas under the influence of melon and watermelon production. A total of 66 (water) and 47 (sediment) pesticides were analyzed, and an environmental risk assessment (ERA) was performed for four taxa (algae, Daphnia magna, fish and Chironomus riparius). One fungicide and seven insecticides were detected in water and/or sediment; the fungicide azoxystrobin (water) and the insecticide cypermethrin (sediments) were the most frequently detected pesticides. The insecticides endosulfan (5.76 µg/L) and cypermethrin (301 µg/kg) presented the highest concentrations in water and sediment, respectively. The ERA revealed acute risk in half of the sampling points of the melon-influenced area and in every sampling point from the watermelon-influenced area. Safety levels were exceeded within and around the crop fields, suggesting that agrochemical contamination was distributed along the catchment, with potential influence of nearby crops. Acute risk was caused by the insecticides chlorpyrifos, cypermethrin and endosulfan to D. magna, fish and C. riparius; the latter was the organism with the overall highest/continuous risk. High chronic risk was determined in all but one sampling point, and revealed a higher number of pesticides of concern. Cypermethrin was the only pesticide to pose chronic risk for all benchmark organisms. The results provide new information on the risk that tropical crops pose to aquatic ecosystems, and highlight the importance of including the analysis of sediment concentrations and chronic exposure in ERA.

Grandmother's pesticide exposure revealed bi-generational effects in Daphnia magna.

Man-made chemicals are a significant contributor to the ongoing deterioration of numerous ecosystems. Currently, risk assessment of these chemicals is based on observations in a single generation of animals, despite potential adverse intergenerational effects. Here, we investigate the effect of the fungicide prochloraz across three generations of Daphnia magna. We studied both the effects of continuous exposure over all generations and the effects of first-generation (F0) exposure on two subsequent generations. Effects at different levels of biological organization from genome-wide gene expression, whole organism metabolite levels, CYP enzyme activity and key phenotypic effects, such as reproduction, were monitored. Acclimation to prochloraz was found after continuous exposure. Following F0-exposure, embryonically exposed F1-offspring showed no significant effects. However, in the potentially germline exposed F2 animals, several parameters differed significantly from controls. A direct association between these F2 effects and the toxic mode of action of prochloraz was found, showing that chemicals can be harmful not only to the directly exposed generation, but also to prenatally exposed generations and in that way effects may even appear to skip a generation. This implies that current risk assessment practices are neglecting an important aspect of toxicity, such as delayed effects across generations due to a time gap between chemical exposure and emergence of effects.

A Nonmechanistic Parametric Modeling Approach for Benchmark Dose Estimation of Event-Time Data.

We propose benchmark dose estimation for event-time data, using a two-step approach. This approach avoids estimation of complex models and has been previously shown to give robust results for summarizing relevant parameters for risk assessment. In the first step, the probability of the event of interest to occur (in a certain time interval) is described as a function of time, resulting in an event-time model; such a model is fitted allowing an individual curve for each dose, and relevant estimates are extracted. In the second step, a dose-response model is fitted to the estimates of t50 obtained from the event-time model in the first step. Given a predefined benchmark response, the benchmark dose is then estimated from the resulting model. This novel approach is demonstrated in two examples. Our application of the time-to-event model showed a gain in power compared to the traditional analysis of end-of-study summary data.

Can Organophosphates and Carbamates Cause Synergisms by Inhibiting Esterases Responsible for Biotransformation of Pyrethroids?

Hydrolysis catalyzed by general esterases (GEs) is the most efficient route for hydrolyzation of pyrethroid insecticides. Organophosphate (OP) and carbamate (CB) insecticides are known to inhibit GEs in addition to acetylcholinesterase (AChE), which is their main target. We hypothesize that synergies can be induced by OPs and CBs when mixed with pyrethroids, due to their inhibition of GE-dependent detoxification of pyrethroids. To test this hypothesis, we conducted mixture toxicity experiments with Daphnia magna using α-cypermethrin (α-cyp) in combination with the noninsecticidal OP tetraisopropyl pyrophosphoramide (iso-OMPA) and five AChE inhibitors diazinon, chlorpyrifos, chlorfenviphos, parathion, and aldicarb. In addition, the in vivo GE activity inhibition was measured for all compounds. Up to 10-fold synergy was found between α-cyp and iso-OMPA, and the degree of synergy correlated linearly with the inhibition of the GE activity. No synergy, however, was found in any of the insecticide mixtures nor was the GE activity inhibited within the nonlethal concentration range tested. It was concluded that the effect of the insecticides on AChE occurred at lower concentrations than their effect on GEs, making the daphnids become immobilized before any synergistic effects on mortality could be observed. The implications of the findings are discussed from a risk assessment perspective.

Application of General Unified Threshold Models of Survival Models for Regulatory Aquatic Pesticide Risk Assessment Illustrated with an Example for the Insecticide Chlorpyrifos.

Mathematical models within the General Unified Threshold models of Survival (GUTS) framework translate time-variable chemical exposure information into expected survival of animals. The GUTS models are species and compound specific and explicitly describe the internal exposure dynamics in an organism (toxicokinetics) and the related damage and effect dynamics (toxicodynamics), thereby connecting the external exposure concentration dynamics with the simulated mortality or immobility over time. In a recent scientific opinion on toxicokinetic-toxicodynamic (TKTD) models published by the European Food Safety Authority (EFSA), the GUTS modeling framework was considered ready for use in the aquatic risk assessment for pesticides and aquatic fauna. The GUTS models are suggested for use in risk assessment, if they are sufficiently validated for a specific substance-species combination. This paper aims to illustrate how they can be used in the regulatory environmental risk assessment for pesticides for a specific type of refinement, that is, when risks are triggered by lower tiers in acute as well as in chronic risk assessment and mortality or immobility is the critical endpoint. This approach involves the evaluation of time-variable exposure regimes in a so-called "Tier-2C" assessment. The insecticide chlorpyrifos was selected as an example compound because a large data set was available. The GUTS models for 13 different freshwater arthropods and 8 different theoretical aquatic exposure profiles were used to calculate a series of GUTS-based risk estimates, including exposure profile-specific multiplication factors leading to 50% mortality or immobility at the end of the tested profile (LP50/EP50) as "margins of safety." To put the use of GUTS models within the tiered aquatic risk assessment into perspective, GUTS models for the 13 aquatic arthropods were also used to predict the environmental risks of a measured chlorpyrifos exposure profile from an experimental ditch study (Tier-3 approach), and the results are discussed in the context of calibration of the tiered approach. Integr Environ Assess Manag 2021;17:243-258. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

bmd: an R package for benchmark dose estimation.

The benchmark dose (BMD) methodology is used to derive a hazard characterization measure for risk assessment in toxicology or ecotoxicology. The present paper's objective is to introduce the R extension package bmd, which facilitates the estimation of BMD and the benchmark dose lower limit for a wide range of dose-response models via the popular package drc. It allows using the most current statistical methods for BMD estimation, including model averaging. The package bmd can be used for BMD estimation for binomial, continuous, and count data in a simple set up or from complex hierarchical designs and is introduced using four examples. While there are other stand-alone software solutions available to estimate BMDs, the package bmd facilitates easy estimation within the established and flexible statistical environment R. It allows the rapid implementation of available, novel, and future statistical methods and the integration of other statistical analyses.

Linking Morphology, Toxicokinetic, and Toxicodynamic Traits of Aquatic Invertebrates to Pyrethroid Sensitivity.

Pyrethroid insecticides are known to be highly toxic to most aquatic nontarget organisms, but little is known about the mechanisms causing some species to be highly sensitive while others are hardly affected by the pyrethroids. The aim of the present study was to measure the sensitivity (EC50-values) of 10 aquatic invertebrates toward a 24 h pulse of the pyrethroid cypermethrin and subsequently test if the difference in sensitivity could be explained by measured morphological and physiological traits and modeled toxicokinetic (TK) and toxicodynamic (TD) parameters. Large differences were observed for the measured uptake and elimination kinetics, with bioconcentration factors (BCFs) ranging from 53 to 2337 at the end of the exposure. Similarly, large differences were observed for the TDs, and EC50-values after 168 h varied 120-fold. Modeling the whole organism cypermethrin concentrations indicated compartmentation into a sorbed fraction and two internal fractions: a bioavailable and non-bioavailable internal fraction. Strong correlations between surface/volume area and the TK parameters (sorption and uptake rate constants and the resulting BCF) were found, but none of the TK parameters correlated with sensitivity. The only parameter consistently correlating with sensitivity across all species was the killing rate constant of the GUTS-RED-SD model (the reduced general unified threshold models of survival assuming stochastic death), indicating that sensitivity toward cypermethrin is more related to the TD parameters than to TK parameters.

Timing of sub-lethal insecticide exposure determines parasite establishment success in an insect-helminth model.

Environmental toxicants are pervasive in nature, but sub-lethal effects on non-target organisms and their parasites are often overlooked. Particularly, studies on terrestrial hosts and their parasites exposed to agricultural toxicants are lacking. Here, we studied the effect of sequence and timing of sub-lethal exposures of the pyrethroid insecticide alpha-cypermethrin on parasite establishment using the tapeworm Hymenolepis diminuta and its intermediate insect host Tenebrio molitor as a model system. We exposed T. molitor to alpha-cypermethrin (LD20) before and after experimental H. diminuta infection and measured the establishment success of larval tapeworms. Also, we conducted in vitro studies quantifying the direct effect of the insecticide on parasite viability. Our results showed that there was no direct lethal effect of alpha-cypermethrin on H. diminuta cysticercoids at relevant concentrations (LD10 to LD90 of the intermediate host). However, we observed a significantly increased establishment of H. diminuta in beetles exposed to alpha-cypermethrin (LD20) after parasite infection. In contrast, parasite establishment was significantly lower in beetles exposed to the insecticide before parasite infection. Thus, our results indicate that environmental toxicants potentially impact host-parasite interactions in terrestrial systems, but that the outcome is context-dependent by enhancing or reducing parasite establishment depending on timing and sequence of exposure.

Differences in life stage sensitivity of the beetle Tenebrio molitor towards a pyrethroid insecticide explained by stage-specific variations in uptake, elimination and activity of detoxifying enzymes.

It is widely accepted that sensitivity towards pesticides varies significantly between species. Much less is known about the potential differences in pesticide sensitivity and its biological mechanism throughout the lifecycle of a single species. In the present study we used three life-stages (larvae, pupae and adult) of the holometabolous insect Tenebrio molitor to investigate: i) Life-stage specific differences in sensitivity towards the pyrethroid insecticide α-cypermethrin after topical exposure, and ii) whether these differences can be explained by the degree of uptake and/or excretion. Finally, we investigated if an efficient excretion coincided with higher activities of the detoxifying enzymes cytochrome P450 (P450), esterases (EST) and glutathione-S-transferease (GST). We found that mobility of adults of T. molitor was more affected by α-cypermethrin treatment than larvae and pupae. Mortality was relatively low for all life stages and did not vary significantly with dose within the duration of the experiment, which indicated that death was (at least partly) due to starvation (indirect effect of paralysis) rather that direct effects of the insecticide. Insecticide treatment during the pupal stage further impaired normal development from pupa to adult. Toxicokinetic measurements showed that cuticle penetration of α-cypermethrin differed significantly between life-stages. Approximately 50% of the applied insecticide had penetrated the adult cuticle after 1 h, whereas a maximum of 30% and 16% had penetrated the waxier cuticle of larvae and pupae. Further, the pupal stage lacked the ability to excrete compounds, and hence internal insecticide concentrations in pupae increased or stagnated until emergence of the adult. Finally, quantification of detoxification enzymes showed a markedly higher activity of P450 in adults and larvae compared to pupae. These findings suggest that assessing toxicity and/or risk of pesticides collectively for a species may not be adequate without taking into account the potential sensitivity differences between life stages.

A comparative study of acetylcholinesterase and general-esterase activity assays using different substrates, in vitro and in vivo exposures and model organisms.

Acetylcholinesterase (AChE) and general-esterase (GE) activities are important to understand detoxification processes of xenobiotics. The assays to quantify them have employed different substrates, inhibitors, types of experiments (in vitro and in vivo) and model organisms. The aim of this work was to give a systematic overview of the effect of the above factors on the outcome of AChE and GE activity measurements. We showed that AChE activity could be measured with the substrate acetylthiocholine iodide (AChI) but not with acetylcholine bromide (AChB) and only in in vitro assays. For GE activity, Michaelis-Menten kinetics differed between the substrates 4-methylumbellifery butyrate (4-MUB) and 1-naphtyl acetate (1-NA) in the measurements of in vitro activity, but their inhibition curves and IC50 values for the general inhibitor tetraisopropyl pyrophosphoramide (iso-OMPA) were similar, confirming that both substrates targeted the same group of enzymes. The GE substrate 4-MUB was applicable both in vitro and in vivo, while 1-NA was only applicable in vitro due to its high acute toxicity. When comparing the zooplankton crustacean Daphnia magna and the sediment dwelling Chironomus riparius, the latter had a four-fold higher maximal AChE activity (Vmax) and a higher susceptibility to the AChE inhibitor BW284c51 (four-fold lower 50% inhibitory concentration, IC50), but a lower maximal GE activity and lower susceptibility to iso-OMPA (higher IC50), indicating significant species differences between in C. riparius and D. magna. We conclude that both choice of substrate and exposure method matters for the outcome of esterase assays and that esterase compositions between species may vary significantly.

Stability of saponin biopesticides: hydrolysis in aqueous solutions and lake waters.

Saponins form a group of plant-produced glycosides with potential as biopesticide ingredients. The environmental fate of saponins has never been fully investigated. In the present study, we use QS-18, a specific saponin from Quillaja saponaria as an example, to quantify hydrolysis under different conditions of pH, temperature and water chemical composition. Saponin hydrolysis in buffer solutions was base-catalyzed and followed first-order kinetics. Thus, hydrolysis was slow at pH 5.1 with a half-life of 330 ± 220 d (26 °C), which increases to 0.06 ± 0.01 d at pH 10.0. Hydrolysis rates were highly sensitive to temperature with an activation energy of 56.9 ± 14.2 kJ mol-1 at pH 7.2. In strong contrast, hydrolysis in lake waters (pH 6.4-8.2) produced different patterns with a fast initial dissipation of 25 to 60% of the added saponin within the first five hours, followed by an extremely slow reaction with 25 to 75% unreacted saponin left after reaction times longer than 120 h. The fast dissipation followed by slow hydrolysis in lake water was hypothesized to be attributed to sorption and/or flocculation of saponins by inorganic nanoparticles and/or solutes in the lake water followed by inactivation of hydrolysis due to the sorption/flocculation. The present study demonstrates that saponins may hydrolyze slowly under acidic and cold conditions. In addition, it demonstrates that dissipation kinetics in natural waters may deviate substantially from the kinetics predicted based on laboratory experiments with "clean" buffered solutions. This emphasizes the need for a deeper understanding of the processes affecting the dissipation kinetics of potential toxins under natural conditions, as fate models based on laboratory derived kinetic data may be seriously flawed.

Enantioselective mixture toxicity of the azole fungicide imazalil with the insecticide α-cypermethrin in Chironomus riparius: Investigating the importance of toxicokinetics and enzyme interactions.

The fungicide imazalil is a chiral compound with one R- and one S-enantiomer. Enantiomers, while having the same chemical properties, can differ in their biological activity expressed as efficacy/toxicity as well as in their degradation kinetics and pathways. Azoles such as imazalil have been shown to synergize the effect of pyrethroid insecticides like α-cypermethrin through inhibition of cytochrome P450 monooxygenase responsible for pyrethroid detoxification. The aim of this study was to investigate, if the enantiomers of imazalil are selective in their synergistic potential in a mixture with a pyrethroid insecticide tested in Chironomus riparius. Potential enantioselectivity was studied on the level of uptake and elimination, inhibition of cytochrome P450 activity measured in vitro and in vivo and on synergistic potential of α-cypermethrin induced immobilization. Synergy was measured as an increase in α-cypermethrin toxicity after 144h applying a constant non-lethal imazalil concentration of 0.65 µmol/L. The R- and S-imazalil enantiomers increased α-cypermethrin toxicity from an EC50 of 1580 ±â€¯980 pmol/L to an EC50 of 83 ±â€¯10 pmol/L and 53 ±â€¯8 pmol/L, respectively. The relatively small potency difference between imazalil enantiomers could not be explained by the in vitro cytochrome P450 inhibition, as the IC50 values were similar (0.11 ±â€¯0.01 and 0.09 ±â€¯0.01 µmol/L for R- and S-imazalil). Measuring in vivo P450 inhibition and the toxicokinetic of imazalil did not show a clear trend of selectivity towards one or the other enantiomer. The study therefore suggests that cytochrome P450 enzymes involved in detoxification in C. riparius are not enantioselective for imazalil.

Guidance on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals.

This Guidance document describes harmonised risk assessment methodologies for combined exposure to multiple chemicals for all relevant areas within EFSA's remit, i.e. human health, animal health and ecological areas. First, a short review of the key terms, scientific basis for combined exposure risk assessment and approaches to assessing (eco)toxicology is given, including existing frameworks for these risk assessments. This background was evaluated, resulting in a harmonised framework for risk assessment of combined exposure to multiple chemicals. The framework is based on the risk assessment steps (problem formulation, exposure assessment, hazard identification and characterisation, and risk characterisation including uncertainty analysis), with tiered and stepwise approaches for both whole mixture approaches and component-based approaches. Specific considerations are given to component-based approaches including the grouping of chemicals into common assessment groups, the use of dose addition as a default assumption, approaches to integrate evidence of interactions and the refinement of assessment groups. Case studies are annexed in this guidance document to explore the feasibility and spectrum of applications of the proposed methods and approaches for human and animal health and ecological risk assessment. The Scientific Committee considers that this Guidance is fit for purpose for risk assessments of combined exposure to multiple chemicals and should be applied in all relevant areas of EFSA's work. Future work and research are recommended.

The synergistic potential of azole fungicides does not directly correlate to the inhibition of cytochrome P450 activity in aquatic invertebrates.

The ability of azole fungicides to inhibit cytochrome P450 dependent metabolism is proposed to be the main mechanism for their synergizing effect on pyrethroid insecticide toxicity in aquatic invertebrates. This study investigates the correlation between inhibition strength and synergistic potential of azole fungicides in the crustacean Daphnia magna and the insect larvae Chironomus riparius. Inhibition strength was measured in vivo toward the cytochrome P450 catalysed conversion of 7-ethoxycoumarin to 7-hydroxycoumarin (ECOD). Synergistic potentials were determined as the ratio between predicted and observed toxicity of mixtures based on the model of concentration addition (CA) and independent action (IA). Azoles (n = 9-11) enhanced the toxicity of α-cypermethrin in D. magna (Synergy ratios CA: 0.8 - 16; IA: 1.1 - 22) and inhibited cytochrome P450 activity by different degrees (IC50: 0.0023 - 36 µM for D. magna and 0.08 - 24 µM for C. riparius). Inhibition strengths were strongly correlated in the two organisms (r: 0.937 p: 0.019 for triazoles and r: 0.903 p: 0.097 for imidazoles). Lipophilicity governed the inhibition strength of triazoles in both species (r > 0.9, p < 0.05). No correlation was observed between inhibition strengths and synergistic potentials. Several reasons for the apparent lack of correlation were discussed.

Similar recovery time of microbial functions from fungicide stress across biogeographical regions.

Determining whether the structural and functional stress responses of communities are similar across space and time is paramount for forecasting and extrapolating the consequences of anthropogenic pressures on ecosystems and their services. Stream ecosystems are under high anthropogenic pressure; however, studies have only examined the response of stream communities across large scales over multiple generations. We studied the responses of leaf-associated microbial communities in streams within three European biogeographical regions to chemical stress in a microcosm experiment with multiple cycles of fungicide pollution and resource colonisation. Fungal community composition and the ecosystem function leaf decomposition were measured as response variables. Microbial leaf decomposition showed similar recovery times under environmental levels of fungicide exposure across regions. Initially, the decomposition declined (between 19 and 53%) under fungicide stress and recovered to control levels during the third cycle of pollution and colonisation. Although community composition and its stress response varied between regions, this suggests similar functional community adaptation towards fungicide stress over time. Genetic, epigenetic and physiological adaptations, as well as species turnover, may have contributed to community adaptation but further studies are required to determine if and to which extent these mechanisms are operating. Overall, our findings provide the first evidence of a similar functional response of microbial leaf decomposition to chemical stress across space and time.

Combined effects of antifouling biocides on the growth of three marine microalgal species.

The toxicity of the antifouling compounds diuron, irgarol, zinc pyrithione (ZnPT), copper pyrithione (CuPT) and copper was tested on the three marine microalgae Tisochrysis lutea, Skeletonema marinoi and Tetraselmis suecica. Toxicity tests based on the inhibition of growth rate after 96-h exposure were run using microplates. Chemical analyses were performed to validate the exposure concentrations and the stability of the compounds under test conditions. Single chemicals exhibited varying toxicity depending on the species, irgarol being the most toxic chemical and Cu the least toxic. Selected binary mixtures were tested and the resulting interactions were analyzed using two distinct concentration-response surface models: one using the concentration addition (CA) model as reference and two deviating isobole models implemented in R software; the other implementing concentration-response surface models in Excel®, using both CA and independent action (IA) models as reference and three deviating models. Most mixtures of chemicals sharing the same mode of action (MoA) were correctly predicted by the CA model. For mixtures of dissimilarly acting chemicals, neither of the reference models provided better predictions than the other. Mixture of ZnPT together with Cu induced a strong synergistic effect on T. suecica while strong antagonism was observed on the two other species. The synergy was due to the transchelation of ZnPT into CuPT in the presence of Cu, CuPT being 14-fold more toxic than ZnPT for this species. The two modelling approaches are compared and the differences observed among the interaction patterns resulting from the mixtures are discussed.