Andreia C.M. Rodrigues

Life history and biochemical effects of chlorantraniliprole on Chironomus riparius

The need to overcome pesticide resistance has led to the development of novel insecticides such as chlorantraniliprole (CAP), an anthranilic diamide. CAP disrupts calcium homeostasis in nerve and muscle cells and is used in several agricultural crops due to its potency and selectivity. However, chronic toxicity data for aquatic invertebrates are limited. Our objective was to evaluate the toxicity of CAP at different levels of biological organization using Chironomus riparius. Organismal endpoints (survival, larval growth and emergence), and 5 biomarkers associated with important physiological functions (acetylcholinesterase - AChE; catalase - CAT; glutathione-S-transferase - GST; total glutathione - TG; and lipid peroxidation - LPO) were investigated. Effects of CAP on cellular energy allocation (CEA) were also assessed. Acute tests revealed a 48 h LC50 for C. riparius of 77.5 μg/L and life-cycle tests revealed a chronic LOEC of 3.1 μg/L based on effects on C. riparius larval growth and emergence. C. riparius females exposed as larvae to low concentrations of CAP emerged at a smaller size which might also translate into effects on reproduction. Chironomid larvae were not under oxidative stress, since short exposures to CAP did not affect LPO levels, despite the significant inhibition of GST (0.6-9.6 μg/L) and CAT (9.6 μg/L). It seems that detoxification of reactive intermediates and ROS is still achieved due to glutathione consumption, since TG levels were significantly decreased in organisms exposed to CAP (0.6-9.6 μg/L). Moreover, it was observed that CEA was disturbed due to increased activity of the electron transport system (ETS), suggesting extra energy expenditure in larvae. These results show that environmental concentrations of CAP can impair the fitness of C. riparius natural populations and at the same time that chironomids, as for most insecticides, are suitable test organisms to evaluate the organismal and biochemical effects of anthranilic diamides.

Behavioural responses of freshwater planarians after short-term exposure to the insecticide chlorantraniliprole.

Recent advances in video tracking technologies provide the tools for a sensitive and reproducible analysis of invertebrate activity under stressful conditions nurturing the field of behavioural ecotoxicology. This study aimed to evaluate behavioural responses of the freshwater planarian Dugesia subtentaculata exposed to a model compound, chlorantraniliprole (CAP). This compound is an anthranilic diamide insecticide and due to its neurotoxic action can, at low concentrations, impair behaviour of exposed organisms. Behavioural endpoints measured included feeding and locomotor activities. Feeding responses were based on planarian predatory behaviour using Chironomus riparius larvae as prey. Locomotion was measured by the traditional planarian locomotor velocity (pLMV) assay and additionally using an automated video tracking system using a Zebrabox(®) (Viewpoint, France) device. While feeding and pLMV were significantly impaired at 131.7μg/L CAP, the video tracking system showed that total distance covered by planarians was significantly reduced at concentrations as low as 26.2μg/L CAP. Our results show that more advanced automated video recording systems can be used in the development of sensitive bioassays allowing a reliable, time- and cost-effective quantification of behaviour in aquatic invertebrates. Due to their ecological relevance, behavioural responses should not be disregarded in risk assessment strategies and we advocate the suitability of planarians as suitable organisms for behavioural ecotoxicological studies.

Assessment of thiamethoxam toxicity to Chironomus riparius.

The insecticide thiamethoxam (TMX) is a systemic neonicotinoid widely used for pest control in several agricultural crops. TMX mimics the action of acetylcholine causing uncontrolled muscular contraction eventually leading to insect death. TMX is being found in freshwater ecosystems at concentrations of up to 225µg/L. Still, chronic toxicity data for freshwater invertebrates is limited. Therefore, the aim of this study was to evaluate the acute and chronic effects (at organismal and biochemical levels) of TMX on the freshwater insect Chironomus riparius. C. riparius life history responses were significantly affected by TMX exposure, namely with a decrease in growth and delay in emergence. Concerning the biochemical responses, after a short exposure (48h) to TMX, our results showed that low concentrations of TMX significantly reduced CAT activity and LPO levels of C. riparius. No effects were observed in AChE, GST and ETS activities. Effects in terms of survival, development rates and biochemical responses of C. riparius exposed to low concentrations of TMX observed in this study suggest potential deleterious effects of this neonicotinoid on aquatic insects inhabiting freshwaters environments near agricultural areas.

Sensitivity of the sea snail Gibbula umbilicalis to mercury exposure--linking endpoints from different biological organization levels.

Mercury contamination is a common phenomenon in the marine environment and for this reason it is important to develop cost-effective and relevant tools to assess its toxic effects on a number of different species. To evaluate the possible effects of Hg in the sea snail Gibbula umbilicalis, animals were exposed to increasing concentrations of the contaminant in the ionic form for 96 h. After this exposure period, mortality, feeding and flipping behavior, the activity of the biomarkers glutathione S-transferase, superoxide dismutase, catalase, lactate dehydrogenase and cholinesterase, the levels of lipid peroxidation and cellular energy allocation were measured. After 96 h of exposure to the highest Hg concentration (≈LC20), there was a significant inhibition of the cholinesterase activity as well as impairment in the flipping behavior and post-exposure feeding of the snails. Cholinesterase inhibition was correlated with the impairment of behavioral responses also caused by exposure to Hg. These endpoints, including the novel flipping test, revealed sensitivity to Hg and might be used as relevant early warning indicators of prospective effects at higher biological organization levels, making these parameters potential tools for environmental risk assessment. The proposed test species showed sensitivity to Hg and proved to be a suitable and resourceful species to be used in ecotoxicological testing to assess effects of other contaminants in marine ecosystems.

Exposure to chlorantraniliprole affects the energy metabolism of the caddisfly Sericostoma vittatum

Caddisflies have been included in ecotoxicological studies because of their sensitivity and ecological relevance. The present study aimed to assess the sublethal effects of an anthranilic diamide insecticide, chlorantraniliprole (CAP), to Sericostoma vittatum. Used worldwide, CAP is a persistent compound that has been found in surface waters at concentrations from 0.1 μg/L to 9.7 μg/L. It targets the ryanodine receptors, and the present ecotoxicological assessment focused on biomarkers related to neurotransmission, biotransformation, oxidative stress damage, and endpoints related to energy processing (feeding, energy reserves, and cellular metabolism). Six days of exposure trials revealed that feeding activity was significantly decreased in S. vittatum larvae exposed to 0.9 μg/L CAP. Concomitantly, a reduction in cellular metabolism and a significant decrease in protein content were also observed in caddisfly larvae exposed to CAP, suggesting metabolic depression. The results show that sublethal concentrations of CAP can cause detrimental sublethal effects on S. vittatum total glutathione content at concentrations as low as 0.2 μg/L. Bioenergetics can be used to assess physiological effects of contaminants, and the present results show that exposure to low, environmentally relevant, concentrations of CAP alter energy acquisition and metabolism in nontarget aquatic insects with potential population level effects. Environ Toxicol Chem 2017;36:1584-1591.

Combined effects of predation risk and food quality on freshwater detritivore insects

Because both predation risk and resource quality have a pivotal role in ecological communities, their combined effects were assessed in stream detritivores. Bioassays were conducted using a simplified trophic chain, coupling effects of predation risk and basal resources with different nutritious qualities, namely, a predator planarian Dugesia subtentaculata, a caddisfly shredder Sericostoma vittatum, a dipteran collector Chironomus riparius, native alder Alnus glutinosa and invasive eucalypt Eucalyptus globulus. We investigated whether individual performance of C. riparius larvae is affected under predation risk and whether the response can be mediated by resource quality. We also assessed whether shredder–collector interactions are altered under these conditions. Predation risk and food quality influenced leaf decomposition and C. riparius growth independently. Chironomus riparius fed preferentially on alders, resulting in increased growth rate. Litter processing decreased under planarian presence, in both leaf types, impairing the growth of C. riparius larvae, this effect being exacerbated with eucalypts. Chironomus riparius growth was also reduced in the presence of the caddisfly, suggesting competition between these species, irrespective of planarian presence, but dependent on leaf type. The present study highlighted the importance of assessing predator influence along detritus-based processing chains, because predation risk may result in sublethal costs, with potential cascading effects.

Invasive Species Mediate Insecticide Effects on Community and Ecosystem Functioning

Anthropogenic activities increase pesticide contamination and biological invasions in freshwater ecosystems. Understanding their combined effects on community structure and on ecosystem functioning presents challenges for an improved ecological risk assessment. This study focuses on an artificial stream mesocosms experiment testing for direct and indirect effects of insecticide (chlorantraniliprole - CAP) exposure on the structure of a benthic macroinvertebrate freshwater community and on ecosystem functioning (leaf decomposition, primary production). To understand how predator identity and resource quality alter the community responses to chemical stress, the mediating effects of an invasive predator species (crayfish Procambarus clarkii) and detritus quality (tested by using leaves of the invasive Eucalyptus globulus) on insecticide toxicity were also investigated. Low concentrations of CAP reduced the abundance of shredders and grazers, decreasing leaf decomposition and increasing primary production. Replacement of autochthonous predators and leaf litter by invasive species decreased macroinvertebrate survival, reduced leaf decomposition, and enhanced primary production. Structural equation modeling (SEM) highlighted that CAP toxicity to macroinvertebrates was mediated by the presence of crayfish or eucalypt leaf litter which are now common in many Mediterranean freshwaters. In summary, our results demonstrate that the presence of these two invasive species alters the effects of insecticide exposure on benthic freshwater communities. The approach used here also allowed for a mechanistic evaluation of indirect effects of these stressors and of their interaction on ecosystem functional endpoint, emphasizing the value of incorporating biotic stressors in ecotoxicological experiments.