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Organism size and growth curves are important biological characteristics. Current methods to measure organism size, and in particular growth curves, are often resource intensive because they involve many manual steps. Here we demonstrate a method for automated, high-throughput measurements of size and growth in individual aquatic invertebrates kept in microtiter well-plates. We use a spheroid counter (Cell 3 iMager, cc-5000) to automatically measure size of seven different freshwater invertebrate species. Further, we generated calibration curves (linear regressions, all p < 0.0001, r 2 >=0.9 for Ceriodaphnoa dubia , Asellus aquaticus , Daphnia magna , Daphnia pulex; r 2 >=0.8 for Hyalella azteca , Chironomus spec . larvae and Culex spec . larvae) to convert size measured on the spheroid counter to traditional, microscope based, length measurements, which follow the longest orientation of the body. Finally, we demonstrate semi-automated measurement of growth curves of individual daphnids ( C . dubia and D . magna ) over time and find that the quality of individual growth curves varies, partly due to methodological reasons. Nevertheless, this novel method could be adopted to other species and represents a step change in experimental throughput for measuring organisms’ shape, size and growth curves. It is also a significant qualitative improvement by enabling high-throughput assessment of inter-individual variation of growth.

Effect concentrations in the toxicity assessment of chemicals with fish and fish cells are generally based on external exposure concentrations. External concentrations as dose metrics, may, however, hamper interpretation and extrapolation of toxicological effects because it is the internal concentration that gives rise to the biological effective dose. Thus, we need to understand the relationship between the external and internal concentrations of chemicals. The objectives of this study were to: (i) elucidate the time-course of the concentration of chemicals with a wide range of physicochemical properties in the compartments of an in vitro test system, (ii) derive a predictive model for toxicokinetics in the in vitro test system, (iii) test the hypothesis that internal effect concentrations in fish (in vivo) and fish cell lines (in vitro) correlate, and (iv) develop a quantitative in vitro to in vivo toxicity extrapolation method for fish acute toxicity. To achieve these goals, time-dependent amounts of organic chemicals were measured in medium, cells (RTgill-W1) and the plastic of exposure wells. Then, the relation between uptake, elimination rate constants, and log KOW was investigated for cells in order to develop a toxicokinetic model. This model was used to predict internal effect concentrations in cells, which were compared with internal effect concentrations in fish gills predicted by a Physiologically Based Toxicokinetic model. Our model could predict concentrations of non-volatile organic chemicals with log KOW between 0.5 and 7 in cells. The correlation of the log ratio of internal effect concentrations in fish gills and the fish gill cell line with the log KOW was significant (r>0.85, p = 0.0008, F-test). This ratio can be predicted from the log KOW of the chemical (77% of variance explained), comprising a promising model to predict lethal effects on fish based on in vitro data.

If an organism does not feed, it dies of starvation. Even though some insecticides which are used to control pests in agriculture can interfere with feeding behavior of insects and other invertebrates, the link from chemical exposure via affected feeding activity to impaired life history traits, such as survival, has not received much attention in ecotoxicology. One of these insecticides is the neonicotinoid imidacloprid, a neurotoxic substance acting specifically on the insect nervous system. We show that imidacloprid has the potential to indirectly cause lethality in aquatic invertebrate populations at low, sublethal concentrations by impairing movements and thus feeding. We investigated feeding activity, lipid content, immobility, and survival of the aquatic arthropod Gammarus pulex under exposure to imidacloprid. We performed experiments with 14 and 21 days duration, both including two treatments with two high, one day pulses of imidacloprid and one treatment with a low, constant concentration. Feeding of G. pulex as well as lipid content were significantly reduced under exposure to the low, constant imidacloprid concentration (15 µg/L). Organisms were not able to move and feed--and this caused high mortality after 14 days of constant exposure. In contrast, feeding and lipid content were not affected by repeated imidacloprid pulses. In these treatments, animals were mostly immobilized during the chemical pulses but did recover relatively fast after transfer to clean water. We also performed a starvation experiment without exposure to imidacloprid which showed that starvation alone does not explain the mortality in the constant imidacloprid exposure. Using a multiple stressor toxicokinetic-toxicodynamic modeling approach, we showed that both starvation and other toxic effects of imidacloprid play a role for determining mortality in constant exposure to the insecticide.

Thanks to growing interest and research in the field, toxicokinetic–toxicodynamic (TKTD) models are close to realising their potential in environmental risk assessment (ERA) of chemicals such as plant protection products. A fundamental application is to find a multiplicative scale factor which—when applied to an exposure profile—results in some specified effect relative to a control. The approach is similar to applying assessment factors to experimental results, common in regulatory frameworks. It also relies on the same core assumption: that increasing the scaling always produces more extreme effects. Unlike experimental approaches, TKTD models offer an opportunity to interrogate this assumption in a mathematically rigorous manner. For four well-known TKTD models we seek to prove that the approach guarantees a unique scale factor for any percentage effect. Somewhat surprisingly, certain model configurations may have multiple scale factors which result in the same percentage effect. These cases require a more cautious regulatory approach and generate open biological and mathematical questions. We provide examples of the violations and suggest how to deal with them. Mathematical proofs provide the strongest possible backing for TKTD modelling approaches in ERA, since the applicability of the models can be determined exactly.

A critical understanding of how pests interact with active ingredients is essential for the development of new insect control solutions to maintain crop quality and quantity by reducing insect damage. Absorption of insecticides into insect bodies of targeted pest species is the first critical step that confounds the efficacy of insecticides. This study investigated how different feeding behaviour of two pests, Myzus persicae and Spodoptera littoralis , affects the absorption, metabolism, and excretion (AME) of seven insecticidally inactive test compounds. A feeding contact assay for the chewing pest (Lepidopteran larvae) and an oral ingestion assay for the sucking pest (aphids) was used to investigate the AME of test compounds with agrochemical-like structural motifs. The standardized assays comprised of an exposure period with treated diet and a subsequent depuration period with untreated diet. The results showed that S. littoralis larvae differed from M. persicae in their compound quantities absorbed into the insect body and in their excretion products at the end of the exposure or depuration periods. We suggest that this is caused by their different ingestion types and rates resulting in different absorption and excretion quantities. Further, we found differences in the metabolism (timing and biotransformation pathways) of compounds between both species. Notably, certain compounds remained detectable in both pests after the depuration period, suggesting compound and species-specific metabolism and excretion. Our results highlight the complex interplay between feeding biology of insects, in particular the critical role of excretion products, and the exposure to different compounds that lead to species-specific AME.

Current environmental risk assessments (ERA) do not account explicitly for ecological factors (e.g. species composition, temperature or food availability) and multiple stressors. Assessing mixtures of chemical and ecological stressors is needed as well as accounting for variability in environmental conditions and uncertainty of data and models. Here we propose a novel probabilistic ERA framework to overcome these limitations, which focusses on visualising assessment outcomes by construct-ing and interpreting prevalence plots as a quantitative prediction of risk. Key components include environmental scenarios that integrate exposure and ecology, and ecological modelling of relevant endpoints to assess the effect of a combination of stressors. Our illustrative results demonstrate the importance of regional differences in environmental conditions and the confounding interactions of stressors. Using this framework and prevalence plots provides a risk-based approach that combines risk assessment and risk management in a meaningful way and presents a truly mechanistic alternative to the threshold approach. Even whilst research continues to improve the underlying models and data, regulators and decision makers can already use the framework and prevalence plots. The integration of multiple stressors, environmental conditions and variability makes ERA more relevant and realistic.

The General Unified Threshold model of Survival (GUTS) provides a consistent mathematical framework for survival analysis. However, the calibration of GUTS models is computationally challenging. We present a novel algorithm and its fast implementation in our R package, GUTS, that help to overcome these challenges. We show a step-by-step application example consisting of model calibration and uncertainty estimation as well as making probabilistic predictions and validating the model with new data. Using self-defined wrapper functions, we show how to produce informative text printouts and plots without effort, for the inexperienced as well as the advanced user. The complete ready-to-run script is available as supplemental material. We expect that our software facilitates novel re-analysis of existing survival data as well as asking new research questions in a wide range of sciences. In particular the ability to quickly quantify stressor thresholds in conjunction with dynamic compensating processes, and their uncertainty, is an improvement that complements current survival analysis methods.

Toxicokinetic-toxicodynamic (TKTD) models quantify the time-course of internal concentration, which is defined by uptake, elimination and biotransformation (TK), and the processes which lead to the toxic effects (TD). TKTD models show potential in predicting pesticide effects in fluctuating concentrations, but the data requirements and validity of underlying model assumptions are not known. We calibrated TKTD models to predict survival of Gammarus pulex in propiconazole exposure and investigated the data requirements. In order to assess the need of TK in survival models, we included or excluded simulated internal concentrations based on pre-calibrated TK. Adding TK did not improve goodness of fits. Moreover, different types of calibration data could be used to model survival, which might affect model parameterization. We used two types of data for calibration: acute toxicity (standard LC50, 4 d) or pulsed toxicity data (total length 10 d). The calibration data set influenced how well the survival in the other exposure scenario was predicted (acute to pulsed scenario or vice versa). We also tested two contrasting assumptions in ecotoxicology: stochastic death and individual tolerance distribution. Neither assumption fitted to data better than the other. We observed in 10-d toxicity experiments that pulsed treatments killed more organisms than treatments with constant concentration. All treatments received the same dose, i.e. the time-weighted average concentration was equal. We studied mode of toxic action of propiconazole and it likely acts as a baseline toxicant in G. pulex during 10-days of exposure for the endpoint survival.

Insecticides prevent or reduce insect crop damage, maintaining crop quality and quantity. Physiological traits, such as an insect's feeding behavior, influence the way insecticides are absorbed and processed in the body (toxicokinetics), which can be exploited to improve species selectivity. To fully understand the uptake of insecticides, it is essential to study their total uptake and toxicokinetics independent of their toxic effects on insects. We studied the toxicokinetics (TK) of insecticidally inactive test compounds incorporating agro-like structural motifs in larvae of the Egyptian cotton leafworm (Spodoptera littoralis, Lepidoptera), and their distribution across all biological matrices, using laboratory experiments and modeling. We measured Spodoptera larval behavior and temporal changes of whole-body concentrations of test compounds during feeding on treated soybean leaf disks and throughout a subsequent depuration period. Differences in the distribution of the total quantities of compounds were found between the biological matrices leaf, larva, and feces. Rate constants for uptake and elimination of test compounds were derived by calibrating a toxicokinetic model to the whole-body concentrations. Uptake and elimination rate constants depended on the physicochemical properties of the test compounds. Increasing hydrophobicity increased the bioaccumulation potential of test compounds. Incomplete quantities in larval matrices indicated that some compounds may undergo biotransformation. As fecal excretion was a major elimination pathway, the variable time of release and number of feces pellets led to a high variability in the body burden. We provide quantitative models to predict the toxicokinetics and bioaccumulation potential of inactive insecticide analogs (parent compounds) in Spodoptera. Key words: agrochemistry, exposure, depuration, absorption, excretion

Aquatic mesocosms are complex test systems used within regulatory risk assessment of plant protection products. These model ecosystems allow researchers to capture interactions of multiple species under realistic environmental conditions. They enable assessment of direct and indirect effects of stressors at all trophic levels (i.e., from primary producers to secondary consumers) and impacts on ecosystem functions. Due to the limited ability to test the multitude of potential exposure scenarios, cross-linking aquatic mesocosm studies with virtual mesocosms, i.e., aquatic system models (ASMs), can serve to meet the demand for more environmental realism and ecological relevance in risk assessment. In this study, full control data sets from seven aquatic mesocosm studies conducted at a single test facility under GLP were analysed graphically and using descriptive statistics. Thereby, not only a comprehensive data base but also an insight into the species present, their dynamics over time, and variability in unchallenged mesocosms was observed. While consistency in dynamics could be discerned for physical and chemical parameters, variability was evident for several biological endpoints. This variability points to amplification of small differences over time as well as to stochastic processes. The outline of existing gaps and uncertainties in data leads to the estimation of what can be expected to be captured and predicted by ASMs.