Sublethal Effects Research Articles

Sublethal and transgenerational effects of insecticides used in whitefly control on biological traits of the parasitoid Eretmocerus hayati

Sublethal and transgenerational effects of insecticides used in whitefly control on biological traits of the parasitoid Eretmocerus hayati

Sublethal Effects of Lambda-Cyhalothrin, Imidacloprid, and Indoxacarb on Some of the Behavioral and Physiological Patterns of German Cockroach (Dictyoptera: Blattellidae)

German cockroach, Blattella germanica (L.), is a common household pets and a serious global public health risk. This study aimed to investigate the sublethal effects of lambda-cyhalothrin, imidacloprid, and indoxacarb on aggregation behavior, sexual communication, feeding efficiency, bodyweight, and nutritional indices of B. germanica. However, the study showed that the LD10 and LD25 doses of these insecticides impaired aggregation behavior and sexual communication of B. germanica; In addition, the data analyses of Excess Proportion Index (EPI), indicated that the aggregation response and sexual communication of adult cockroaches were decreased due to its exposure to LD10 and LD25 compared to the control treatment (LD0). Although the cockroaches’ food intake increased, their bodyweight decreased after being exposed to LD10 and LD25 compared with the control treatment. Moreover, a negative impact of LD10 and LD25 was observed on some nutritional indices e.g. approximate digestibility (AD), relative growth rate (RGR), and efficiency of conversion of digested food (ECD). On the other hand, the LD10 and LD25 have positively affected on other nutritional indices, including efficient conversion of ingested food (ECI) and relative consumption rate (RCR) when compared with control group. This study showed that the sublethal effects of lambda-cyhalothrin, imidacloprid, and indoxacarb employed to control insect pests widely, have considerable effects on the insect’s behavioral and physiological patterns.

Spirotetramat- and thiamethoxam-induced sublethal effects increase spread of tomato chlorosis virus by its vector Bemisia tabaci

Spirotetramat- and thiamethoxam-induced sublethal effects increase spread of tomato chlorosis virus by its vector Bemisia tabaci

Cadmium as an Endocrine Disruptor That Hinders the Reproductive and Developmental Pathways in Freshwater Fish: A Review

Cadmium (Cd) is a non-essential element with sub-lethal effects even at low concentrations. The persistent nature of Cd and its tendency to bioaccumulate eventually create harmful effects on water biota, including fish. Cd affects various aspects of hormonal action in fish since it bioaccumulates in the endocrine system and hinders the synthesis, secretion, and metabolic activity of hormones, causing severe damage along the hypothalamus–pituitary–gonadal axis. Linking reproductive and developmental impairments in fish with ecologically relevant concentrations of individual metals can be challenging due to the complexity of aquatic ecosystems. This review deliberated the significant and novel trends of toxicological difficulties and approaches, including elucidating environmental sources’ bioavailability and Cd-induced toxic effects in freshwater fish. Both acute and chronic exposure to Cd can cause a range of adverse effects, such as growth inhibition, impaired reproductive capacity, endocrine disruption, and developmental abnormalities in freshwater fish, as evidenced by the present review. These investigations support the concept of Cd as a naturally available pollutant that causes irreversible damage in fish. These findings will help to understand the etiology of environmental circumstances that pose substantial dangers to fish health and are also crucial for preventing and treating exposure-related reproductive disturbances in freshwater fish due to environmental pollution.

The Lethal and Sublethal Effects of Lambda-Cyhalothrin and Emamectin Benzoate on the Soybean Pest Riptortus pedestris (Fabricius).

Riptortus pedestris (Fabricius, 1775) (Hemiptera: Alydidae) is a major soybean pest in East Asia that can cause soybean staygreen syndrome. To date, no insecticides have been registered for the control of R. pedestris in China, and these insects are primarily controlled in the field through the application of broad-spectrum insecticides including lambda-cyhalothrin (LCT) and emamectin benzoate (EMB). Here, the lethal and sublethal effects of LCT and EMB on R. pedestris were comprehensively evaluated. LCT and EMB were both found to exhibit high levels of toxicity and concentration-dependent repellent effects for R. pedestris. The exposure of third instar nymphs from the F0 generation to LC30 concentrations of LCT and EMB resulted in a significant increase in the duration of nymph development and adult pre-oviposition period (APOP), together with reductions in fifth instar nymph and adult body weight, longevity, oviposition days, fecundity, vitellarium length, lateral oviduct diameter, and vitellogenin (Vg) gene expression as compared to control treatment. Strikingly, these suppressive effects were transmitted to the F1 generation, which similarly experienced the prolongation of preadult development and the preoviposition period (TPOP). Relative to control-treated populations, the F1 generation for these insecticide-treated groups also exhibited significant decreases in population parameter values. Overall, these data offer new insight into the impact that LCT and EMB treatment can have on R. pedestris, providing a valuable foundation for the application of these pesticides in the context of integrated pest management strategies aimed at soybean crop preservation.

Lethal and sublethal effects of chronic exposure to insecticide residues on reared Alphitobius diaperinus

Abstract Edible insects such as lesser mealworm (Alphitobius diaperinus) are a promising new protein source for food and feed. The feed substrate on which these insects are reared may be contaminated with residues of insecticides originating from agricultural products that may impact insect performance. In this study, two generations of A. diaperinus were chronically exposed to spinosad (2.0 and 0.2 mg/kg) and imidacloprid (0.1 and 0.01 mg/kg) in the substrate. The aim was to determine sublethal effects on performance measures (total biomass (yield), mean individual weight, number of alive individuals) of larvae, pupae, and adult beetles, as well as pupation and eclosion. Exposure to spinosad at 2.0 mg/kg resulted in significant adverse effects on most performance measures of larvae, of both generations. Imidacloprid caused a reduction in yield and mean individual weight of the larvae as compared to the control at 0.1 mg/kg, while an increase in those measures was observed at 0.01 mg/kg. Significant adverse effects on adult beetles were only observed for imidacloprid at 0.1 mg/kg, and no significant effects of this insecticide on pupation and eclosion were observed. The concentrations of tested substances in larval samples were negligible for both generations, however, transfer from substrate to larval biomass was higher in the offspring generation relative to the parent generation. More research is needed to fully assess the hazard of insecticide residues to cause sublethal effects on A. diaperinus, for which method development for more cost-efficient designs is required.

Sublethal effects of imidacloprid on the fitness of two species of wheat aphids, Schizaphis graminum (R.) and Rhopalosiphum padi (L.).

Imidacloprid is a neonicotinoid insecticide that efficiently controls piercing-sucking mouthparts pests. However, the impact of low lethal concentration of imidacloprid on key demographic parameters of wheat aphids, Schizaphis graminum (R.) and Rhopalosiphum padi (L.) has been scarcely studied. In this study, we used the age stage, two-sex life table approach to investigate the sublethal effects of imidacloprid on the biological traits of S. graminum and R. padi. Bioassays showed that imidacloprid possesses high toxicity to adult S. graminum and R. padi, with LC50 of 3.59 and 13.78 mg L-1 following 24 h exposure. A low lethal concentration of imidacloprid (LC25) significantly decreased adult longevity and total longevity of progeny generation aphids (F1) of S. graminum. Nevertheless, imidacloprid (LC25) had no significant effects on the fecundity and longevity of directly exposed parental parental S. graminum and R. padi (F0). Our results showed that the low lethal concentration of imidacloprid affected the demographic parameters that ultimately impact on the population of S. graminum. This study provides detailed information about the overall effects of imidacloprid on S. graminum and R. padi that might help to manage these two key pests.

High temperatures are associated with reduced cognitive performance in wild southern pied babblers.

Global temperatures are increasing rapidly. While considerable research is accumulating regarding the lethal and sublethal effects of heat on wildlife, its potential impact on animal cognition has received limited attention. Here, we tested wild southern pied babblers (Turdoides bicolor) on three cognitive tasks (associative learning, reversal learning and inhibitory control) under naturally occurring heat stress and non-heat stress conditions. We determined whether cognitive performance was explained by temperature, heat dissipation behaviours, individual and social attributes, or proxies of motivation. We found that temperature, but not heat dissipation behaviours, predicted variation in associative learning performance. Individuals required on average twice as many trials to learn an association when the maximum temperature during testing exceeded 38°C compared with moderate temperatures. Higher temperatures during testing were also associated with reduced inhibitory control performance, but only in females. By contrast, we found no temperature-related decline in performance in the reversal learning task, albeit individuals reached learning criterion in only 14 reversal learning tests. Our findings provide novel evidence of temperature-mediated cognitive impairment in a wild animal and indicate that its occurrence depends on the cognitive trait examined and individual sex.

Using dietary exposure to determine sub-lethal effects from imidacloprid in two springtail (Collembola) species

Standard toxicity tests expose springtails (Collembola) through soil, while dietary exposure tests with animals visible on a surface are less commonly applied. We refined a method for dietary chemical exposure for two widely distributed and abundant Collembola species: Folsomia quadrioculata and Hypogastrura viatica as existing methods were sub-optimal. Newly hatched Collembola were offered bark with a natural layer of Cyanobacteria that was either moistened with a solution of the neonicotinoid insecticide imidacloprid using a micropipette or soaked in the solution overnight. The first method was superior in producing a measured concentration close to the nominal (0.21 and 0.13 mg/kg dry bark, respectively), and resulting in sub-lethal effects as expected. The adult body size was reduced by 8% for both species, but egg production only in H. viatica. Contrastingly, soaked bark resulted in a measured concentration of 8 mg/kg dry bark, causing high mortality and no egg production in either species. Next, we identified the sub-lethal concentration-range by moistening the bark to expose H. viatica to 0, 0.01, 0.04, 0.13, 0.43 and 1.2 mg imidacloprid/kg dry bark. Only the highest concentration affected survival, causing a mortality of 77%. Imidacloprid reduced moulting rate and the body size at first reproduction. The age at first reproduction appeared delayed as some replicates did not reproduce within the experiment duration. The method of moistened bark for dietary exposure proved optimal to continuously study life history traits, such as growth and reproductive outcomes, which are important to understand effects on key events crucial for population viability and growth.

Application of 3D-printed pollen traps as a useful tool for exposure and risk assessment of pesticide residues on bumblebees

The study was designed to test the feasibility of using 3D-printed pollen traps for long-term monitoring of Bombus terrestris colonies' exposure to pesticide residues in pollen loads collected by them, along with an assessment of the resulting risks to the bumblebee's adults, larvae, and queens. Bumblebee colonies were placed in the vicinity of flowering orchards, winter oilseed rape, allotments, or home gardens for 6 weeks of the experiment. Pollen traps printed in 3D technology were installed in the hive inlets. The weight of bumblebee pollen loads obtained using pollen traps was in the range of 0.036–5.83 g. Pollen load samples were analyzed for residues of up to 261 pesticides and their metabolites by liquid and gas chromatography techniques coupled to tandem mass spectrometry (LC-MS/MS and GC-MS/MS). Residues of 18 fungicides, 12 herbicides, 6 insecticides, and an acaricide were detected. Herbicide – pendimethalin, fungicide – thiophanate-methyl, and insecticide – chlorpyrifos-ethyl were the most commonly detected pesticides. Chlorpyrifos and thiacloprid residues were detected in pollen load samples in the next year after their ban from use as plant protection products in the European Union. The risk of acute or chronic effects was assessed as negligible or low, although the chronic risk of bumblebee queens to insecticide chlorpyrifos and the acute risk of larvae exposed to acaricide fenpyroximate could be interpreted as moderate. The risk of sublethal effects related to chronic exposure of adult bumblebees and queens to pollen loads contaminated by chlorpyrifos-ethyl and cypermethrin cannot be excluded. The risk of chronic toxicity or sublethal effects may be particularly relevant for bumblebee queens, especially during their foraging in the initial period of establishing a new colony.

Exposure to closed-loop scrubber washwater alters biodiversity, reproduction, and grazing of marine zooplankton

Shipping is a large industry responsible for atmospheric emissions of hazardous substances including SOX, NOX, and particulate matter. Many ships have installed exhaust gas cleaning systems (scrubbers) to remove primarily SOX from the exhaust, but the hazardous substances are instead transferred to the water used in the scrubbing process. Ships with closed-loop scrubbers recirculate the water but can still discharge around 126-150 m3 directly to the surrounding marine environment every day. The discharged water contains metals and organic substances, such as polycyclic aromatic hydrocarbons, that are known to be toxic to marine zooplankton. Here we show that closed-loop scrubber washwater is toxic to communities of marine mesozooplankton at our lowest tested dilution, 1.5% (v/v), and affects survival, reproduction, diversity, and ability to predate on microzooplankton. The cumulative toxic unit of the undiluted closed-loop scrubber washwater was estimated to 17, which indicates that the water could be toxic at levels below what was tested in this study. Among all detected substances, vanadium, copper, benzo[ghi]perylene, nickel, and zinc were identified as toxicity-driving substances in the order listed. Closed-loop scrubber washwater has been shown to affect development and survival in single species of copepods, but here we find evidence of toxicity at the community level, irrespective of seasonal community structure, and that the exposure has potential to disrupt the interactions between trophic levels in the pelagic food web. We show that the closed-loop scrubber washwater cause both lethal and sublethal effects in marine zooplankton, due to contaminants, some of which are persistent in the marine environment.

Sublethal concentrations of thiamethoxam induce transgenerational hormesis in cotton aphid, Aphis gossypii Glover

In agroecosystems, insects have to compete with chemical insecticides, which are frequently present at sublethal concentrations. The exposure of insects to these modest stresses is now well-established to generate hormesis effects, which has implications for controlling insect pests. In this study, we assessed the sublethal effects of thiamethoxam on the biological parameters of Aphis gossypii Glover (Hemiptera: Aphididae), adults (F0) and subsequent transgenerational impacts, i.e., on the progeny (F1 generation), using an age stage, two-sex life table analysis. Results showed that thiamethoxam exhibited high toxicity against adult A. gossypii with the LC50 of 0.313 mg L−1 after 48 h exposure. The LC5 and LC10 of thiamethoxam considerably reduced the adult cotton aphids (F0) longevity and fecundity, while the reproductive days were reduced only at LC10. The pre-adult stage was decreased, while the adult longevity, total longevity, and fecundity were significantly extended in F1 aphids after exposure of F0 aphids to the sublethal concentrations of thiamethoxam. Moreover, the key demographic parameters such as intrinsic rate of increase, finite rate of increase and reproductive days were significantly increased, while mean generation time and total prereproductive were significantly reduced in the progeny. No significant effects were observed on the net reproductive rate. Taken together, these results showed that the sublethal concentrations of thiamethoxam affect the directly exposed aphids (F0) while causing transgenerational hormetic effects on the F1 generation of A. gossypii. In conclusion, our research showed that thiamethoxam has both sublethal and transgenerational effects on cotton aphids; it could be effective in IPM programs targeting this key pest.

Beauveria bassiana biogenic nanoparticles for the control of Noctuidae pests.

Biogenic metallic and oxide metal nanoparticles have potential as alternatives for several current problems in agriculture, such as the control of caterpillars which cause huge losses in the production of important crops. In the present study, capped and uncapped silver, iron oxide and titanium dioxide nanoparticles were synthesized from the filtrate of Beauveria bassiana and evaluated in regard to physico-chemical characteristics, capping composition, cytotoxicity, genotoxicity and biological activity on Helicoverpa armigera and Spodoptera frugiperda caterpillars. A difference in the physico-chemical parameters of the capped and uncapped nanoparticles was observed, with larger aggregation and lower stability of the uncapped. In regard to the study of the capping, the presence of functional groups of biomolecules as well as the activity of B. bassiana hydrolytic enzymes were observed. Cytotoxic effects on the tested cell lines were not observed and DNA damage levels increased with more intense effects of the uncapped nanoparticles. In regard to the biological activity against Noctuidae pests, the uncapped and capped iron oxide, and uncapped titanium dioxide nanoparticles occasioned higher mortality (76%, 60% and 51%, respectively) but no differences in LC50 were recorded. Moreover, sublethal effects were reported on Helicoverpa armigera whereas Spodoptera frugiperda showed low susceptibility to the nanoparticles. The results demonstrate that biogenic metallic and oxide metal nanoparticles might show promising effects for the control of caterpillars which cause damage on important agricultural crops. Further investigations are necessary to understand the mechanisms of action and optimize the biological activity of these new nanomaterials. © 2023 Society of Chemical Industry.

Testing the heat treatment dose for Agrilus planipennis (Coleoptera: Buprestidae) prepupae using the Humble water bath.

The lethal heat treatment dose (time and temperature) for the prepupal life stage of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), emerald ash borer (EAB), was determined through an in vitro application using a carefully calibrated heat treatment apparatus. The lethal and sublethal effects of heat on A. planipennis prepupae were assessed through a ramped heat delivery application, simulating industrial kilns and conventional heat chamber operations, for treatments combining target temperatures of 54 °C, 55 °C, and 56 °C, and exposure durations of 0 min (i.e., kiln temperature ramp only), 15 min, or 30 min. Prepupal EAB larvae did not survive exposure to 56 °C for 15 min or longer, or to 55 °C for 30 min. Sublethal effects were observed for all other treatments. Sublethal effects included delayed development and failure to complete the pupal and adult life stages.

Sublethal effect of chlorpyrifos on predatory behavior and physiology of Eocanthecona furcellata (Hemiptera: Pentatomidae).

Insecticides have been known to reduce the predation efficacy of natural enemies. However, the mechanism of the sublethal effect of insecticides on the functional response of predators remains unclear. This study investigated the sublethal effects of the broad-spectrum insecticide chlorpyrifos on the predatory bug Eocanthecona furcellata (Wolff), which is a potential biological control agent against pests in integrated pest management (IPM) programs. After exposure to a sublethal concentration of chlorpyrifos, the predation capacity and the maximum predatory number of E. furcellata increased by 11.27 and 15.26%, respectively, with prey handling time decreased by 15.07%, and the searching efficiency increased by 5.88-12.61%. Additionally, the intraspecific interference effect was enhanced. Glutathione S-transferase (GST) activity was significantly decreased after 12- to 60-h treatment. At 12 h after treatment, the expression levels of GST gene (GST3), acetylcholinesterase gene (AChE), and cytochrome P450 monooxygenasegene (cyp6B1) were significantly up-regulated by 1.47-, 1.48-, and 2.05-fold, respectively, while GST gene (GST1) was significantly down-regulated by 16.67-fold. These results indicated that a sublethal chlorpyrifos concentration inhibited the GST activity and stimulated the predatory behavior of E. furcellata. The results will advance our understanding of the toxicological mechanism of predatory stink bug responses to insecticides and predict chlorpyrifos' effects on predators in an IPM program.

Metabolomic response of striped marsh frog (Limnodynastes peronii) tadpoles exposed to the fire retardant Phos-Chek LC95W

Climate change and other factors have contributed to an increased frequency and intensity of global wildfires in recent years. Ammonium-based fire retardants are widely used to suppress or delay the spread of fire and have generally been regarded as presenting a low risk of acute toxicity to fauna. However, studies have raised concerns about their potential to cause indirect or sub-lethal effects, and toxicity information regarding the potential for such impacts in aquatic species is limited. To address these knowledge gaps, we used an untargeted metabolomics approach to evaluate the sub-lethal physiological and metabolic responses of striped marsh frog (Limnodynastes peronii) tadpoles exposed to a concentration gradient of the ammonium polyphosphate (APP)-based fire retardant Phos-Chek LC95W (PC). Acute exposure (96 h) to PC significantly altered the relative abundance of 14 metabolites in whole tadpoles. The overall metabolic response pattern was consistent with effects reported for ammonia toxicity and also suggestive of energy dysregulation and osmotic stress associated with alterations to physicochemical water quality parameters in the PC treatments. Results suggest that run-off or accidental application of this formulation into waterways can have significant sub-lethal consequences on the biochemical profiles (i.e., the metabolome) of aquatic organisms and may be a concern for frog species that breed and develop in small, often ephemeral, waterbodies. Our study highlights the benefits of integrating untargeted metabolomics with other ecological and toxicological endpoints to provide a more holistic characterisation of the sub-lethal impacts associated with bushfire-fighting chemicals and with environmental contaminants more broadly.

Fish microplastic ingestion may induce tipping points of aquatic ecosystems.

Microplastics can be ingested by a wide range of aquatic animals. Extensive studies have demonstrated that microplastic ingestion-albeit often not lethal-can affect a range of species life-history traits. However, it remains unclear how the sublethal effects of microplastics on individual levels scale up to influence ecosystem-level dynamics through cascading trophic interactions. Here we employ a well-studied, empirically fed three-species trophic chain model, which was parameterized to mimic a common type of aquatic ecosystems to examine how microplastic ingestion by fish on an intermediate trophic level can produce cascading effects on the species at both upper and lower trophic levels. We show that gradually increasing microplastics in the ingested substances of planktivorous fish may cause population structure effects such as skewed size distributions (i.e. reduced average body length vs. increased maximal body size), and induce abrupt declines in fish biomass and reproduction. Our model analysis demonstrates that these abrupt changes correspond to an ecosystem-level tipping point, crossing which difficult-to-reverse ecosystem degradation can happen. Importantly, microplastic pollution may interact with other anthropogenic stressors to reduce safe operating space of aquatic ecosystems. Our work contributes to better understanding complex effects of microplastic pollution and anticipating tipping points of aquatic ecosystems in a changing world. It also calls attention to an emerging threat that novel microplastic contaminants may lead to unexpected and abrupt degradation of aquatic ecosystems, and invites systematic studies on the ecosystem-level consequences of microplastic exposure.

Field-realistic exposure to neonicotinoid and sulfoximine insecticides impairs visual and olfactory learning and memory in Polistes paper wasps.

Exposure to insecticides may contribute to global insect declines due to sublethal insecticide effects on non-target species. Thus far, much research on non-target insecticide effects has focused on neonicotinoids in a few bee species. Much less is known about effects on other insect taxa or newer insecticides, such as sulfoxaflor. Here, we studied the effects of an acute insecticide exposure on both olfactory and visual learning in free-moving Polistes fuscatus paper wasps. Wasps were exposed to a single, field-realistic oral dose of low-dose imidacloprid, high-dose imidacloprid or sulfoxaflor. Then, visual and olfactory learning and short-term memory were assessed. We found that acute insecticide exposure influenced performance, as sulfoxaflor- and high-dose imidacloprid-exposed wasps made fewer correct choices than control wasps. Notably, both visual and olfactory performance were similarly impaired. Wasps treated with high-dose imidacloprid were also less likely to complete the learning assay than wasps from the other treatment groups. Instead, wasps remained stationary and unmoving in the testing area, consistent with imidacloprid interfering with motor control. Finally, wasps treated with sulfoxaflor were more likely to die in the week after treatment than wasps in the other treatment groups. Our findings demonstrate that sublethal, field-realistic dosages of both neonicotinoid- and sulfoximine-based insecticides impair wasp learning and short-term memory, which may have additional effects on survival and motor functioning. Insecticides have broadly detrimental effects on diverse non-target insects that may influence foraging effectiveness, pollination services and ecosystem function.

Ecotoxicity effect factors for plastic additives on the aquatic environment: New approach for life cycle impact assessment

All plastic contains additives. Once in the environment, these will start to leach out and will expose and harm aquatic biota, causing potentially lethal and sub-lethal toxic effects. Even though life cycle assessment covers the toxic impacts of several thousands of chemicals, models to assess the toxic impacts of plastic additives are only emerging. We gathered 461 data points from the literature (266 for freshwater and 195 for marine ecosystems) for 75 species belonging to 9 different phyla. The endpoints effective concentration and lethal concentration, no observed effects concentrations and lowest observed effect concentration tested in acute and chronic exposure, were harmonized into chronic values by applying extrapolation factors. The collected data points covered 75 main plastic additives. This allowed us to calculate 25 Effect factors, 19 for single chemicals and four for overarching categories (alkylphenols, benzophenones, brominated flame retardants and phosphates. In addition, we calculated an aggregated effect factor for chemicals that did not fit in any of the previous groups, as well as a Generic effect factor including 404 gathered data points. The estimated potentially affected fraction (PAF) for the single additives varied between 20.69 PAF·m3·kg−1 for diethyl phthalate and 11081.85 PAF·m3·kg−1 for 4-Nonylphenol. The factors can in future be combined with fate and exposure factors to derive a characterization factor for toxicity caused by additives in aquatic species. This is an important advancement for the assessment of the impacts of plastic debris on aquatic species, thus providing information for decision-makers, as well as guiding policies for the use of additives, ultimately aiming to make the plastic value chain more sustainable.

Chronic toxicity testing including transcriptomics-based molecular profiling in Cloeon dipterum

The so-called EPT taxa have been shown to be highly sensitive to various environmental pollutants. However, there are only few published studies on toxicity testing with EPT representatives and there is a particular lack of protocols for chronic toxicity testing, e.g., for integration into species sensitivity distribution (SSD) approaches. To address this gap, we performed a long-term 38-day semi-static toxicity test with the European mayfly species Cloeon dipterum using the insecticide fipronil as model substance. The functionality of the test system was confirmed by the high emergence rate of 85% in the control condition. We found a high sensitivity with regard to larval development with an EC50 of 180 ng/L and a NOEC of 38.0 ng/L after 7 days exposure. After 38 days, an LC50 value of 185 ng/L and an EC50 value of 160 ng/L for emergence (both: NOEC = 38.0 ng/L) were calculated. In a short-term 7-day toxicity test, we found a similar effect on larval development. In addition to the physiological endpoints, we examined fipronil-induced gene expression changes at the transcriptome level in this test. Our results revealed a concentration-dependent increase in the number of differentially expressed genes, as well as observed effects on larval development. Notably, we identified marker gene candidates involved in nervous system development, mirroring the known mode-of-action of fipronil in C. dipterum. The affected genes primarily play crucial roles in neurological processes. Concluding, within this two-step approach we were able to identify fipronil effects on the sublethal physiological endpoint larval development and to complement these effects at the molecular level by gene expression changes in the transcriptome. Thus, this assay proved to be suitable to assess sublethal effects as well as the mode-of-action of substances in the non-standard organism C. dipterum already after a short-term exposure of 7 days. However, further testing is required to validate the procedure.