Sublethal Endpoints Research Articles

Mixed toxicity of chlorpyrifos and dichlorvos show antagonistic effects in the endangered fish species golden mahseer (Tor putitora)

Golden mahseer (Tor putitora) is an economically important but endangered fish species in many countries. Increasing pesticide application can possess a threat to this species but their sensitivity to pesticides, typically chlorpyrifos and dichlorvos, is unknown. We determined 96 h-LC50 of chlorpyrifos and dichlorvos to be 0.753 mg/L and 12.964 mg/L, respectively, indicating higher toxicity of chlorpyrifos than dichlorvos. Despite the same mode of action, their joint effect was antagonistic, with an additive index value of - 0.58 at 96 h-LC50. Moreover, to get insights in the temporal sub-lethal effects, fish were exposed to 10% and 50% of the 96 h-LC50 values of the respective pesticides. Aerobic metabolism, opercular movements, and feeding behavior were examined for sub-lethal end-points following 24 h, 48 h, 72 h and 96 h exposure. Both chlorpyrifos and dichlorvos in single exposures induced a significant drop in oxygen consumption rate; while it was significantly elevated in the mixed pesticide exposure. Accelerated opercular movements were observed in all pesticide treatment groups but were more persistent in chlorpyrifos treatments. Reduced feeding attempts were more pronounced in chlorpyrifos and mixture treatments wherein feeding attempts dropped to zero. Overall, the acute toxicity data reported in the present study can be used to assess the maximum tolerance level of golden mahseer to chlorpyrifos and dichlorvos, and their mixture. Furthermore, the sub-lethal end point responses can be applied in monitoring the environmental risk posed by these waterborne pesticides either individually or in combination to the aquatic life.

Ecotoxicity Assessment of Graphene Oxide by Daphnia magna through a Multimarker Approach from the Molecular to the Physiological Level including Behavioral Changes.

The extensive use of engineered nanomaterials, such as graphene oxide (GO), is stimulating research about its potential environmental impacts on the aquatic ecosystem. This study is aimed to comprehensively assess the acute toxicity of a well-characterized GO suspension to Daphnia magna. Conventional ecotoxicological endpoints (lethality, immobilization) and more sensitive, sublethal endpoints (heartbeat rate, feeding activity, and reactive oxygen species (ROS)) production were used. The possible normalization of the heartbeat rate and feeding activity in clean test medium was also investigated. The fate, time-dependent, and concentration-dependent aggregation behaviour of GO was followed by dynamic light scattering, UV-Vis spectroscopy, and zeta potential measurement methods. The EC20 value for immobilization was 50 mg/L, while, for physiological and behavioural endpoints, it ranged from 8.1 mg/L (feeding activity) to 14.8 mg/L (immobilization). The most sensitive endpoint was the ROS production with EC20 = 4.78 mg/L. 24-h recovery experiments revealed that feeding activity was restored only up to a certain level at higher concentrations, indicating that the potential environmental health effects of GO cannot be neglected. Alterations of normal physiology (heart rate) and feeding activity may be associated with increased risk of predation and reproductive decline, highlighting that GO may have impacts on population and food web dynamics in aquatic ecosystems.

Comparison of imidacloprid, propiconazole, and nanopropiconazole effects on the development, behavior, and gene expression biomarkers of the Pacific oyster (Magallana gigas)

Coastal areas are final recipients of various contaminants including pesticides. The effects of pesticides on non-target organisms are often unclear, especially at environmentally relevant concentrations. This study investigated the impacts of insecticide imidacloprid (IMI) and fungicide propiconazole (PRO), some of the most detected pesticides in the Arcachon Bay in France. This work also included the research of propiconazole nanoformulation (nanoPRO). The effects were assessed studying the development of the early life stages of the Pacific oyster (Magallana gigas). Oyster embryos were exposed for 24, 30, and 42 h (depending on the endpoint) at 24 °C to environmentally relevant concentrations of the two pesticides as well as to nanoPRO. The research focused on sublethal endpoints such as the presence of developmental malformations, alterations of locomotion patterns, or changes in the gene expression levels. No developmental abnormalities were observed after exposure to environmental concentrations detected in the Arcachon Bay in recent years (maximal detected concentration of IMI and PRO were 174 ng/L and 29 ng/L, respectively). EC50 of PRO and nanoPRO were comparable, 2.93 ± 1.35 and 2.26 ± 1.36 mg/L, while EC50 of IMI exceeded 200 mg/L. IMI did not affect larval behavior. PRO affected larval movement trajectory and decreased average larvae swimming speed (2 μg/L), while nanoPRO increased the maximal larvae swimming speed (0.02 μg/L). PRO upregulated especially genes linked to reactive oxygen species (ROS) production and detoxification. NanoPRO effects on gene expression were less pronounced - half of the genes were altered in comparison with PRO. IMI induced a strong dose-response impact on the genes linked to the detoxification, ROS production, cell cycle, and apoptosis regulation. In conclusion, our results suggest that current pesticide concentrations detected in the Arcachon Bay are safe for the Pacific oyster early development, but they might have a small direct effect via altered gene expressions, whose longer-term impacts cannot be ruled out.

Environmental concentrations of antifouling paint particles are toxic to sediment-dwelling invertebrates.

Antifouling paint particles (APPs) and associated metals have been identified in sediments around boatyards and marinas globally, but the effects of APPs on benthic organisms are largely unknown. Sub-lethal endpoints were measured following laboratory exposures of the harbour ragworm (Hediste diversicolor) and the common cockle (Cerastoderma edule) to environmentally relevant concentrations of biocidal ('modern' and 'historic') and biocide-free ('silicone') APPs added to clean estuarine sediment. Further, the 5-day median lethal concentrations (LC50) and effects concentrations (EC50) for modern biocidal APPs were calculated. For ragworms, significant decreases in weight (15.7%; p<0.01) and feeding rate (10.2%; p<0.05) were observed in the modern biocidal treatment; burrowing behaviour was also reduced by 29% in this treatment, but was not significant. For cockles, the modern biocidal treatment led to 100% mortality of all replicates before endpoints were measured. In cockles, there was elevated levels of metallothionein-like protein (MTLP) in response to both modern and historic biocidal treatments. Ragworms had a higher tolerance to modern APPs (5-day LC50:19.9 APP g L-1; EC50: 14.6gL-1) compared to cockles (5-day LC50: 2.3gL-1 and EC50: 1.4gL-1). The results of this study indicate that modern biocidal APPs, containing high Cu concentrations, have the potential to adversely affect the health of benthic organisms at environmentally relevant concentrations. The findings highlight the need for stricter regulations on the disposal of APP waste originating from boatyards, marinas and abandoned boats.

The influence of organic carbon on the toxicity of sediment-associated dinonylnaphthalene sulfonic acids to the benthic invertebrates Tubifex tubifex and Hyalella azteca

Naphthalene sulfonic acids (NSAs) are used extensively in industrial applications as dispersants in dyes, rubbers, and pesticides, and as anti-corrosive agents in coatings, gels, and sealants. This study examined the toxicity of three NSA congeners, barium dinonylnaphthalene sulfonate (BaDNS), calcium dinonylnaphthalene sulfonate (CaDNS), and dinonylnaphthalene disulfonic acid (DNDS), to two benthic species, Tubifex tubifex and Hyalella azteca. Two substrates with different levels of organic carbon (sediment [2%] and sand [0%]) were used in toxicity tests. Juvenile production was the most sensitive endpoint for T. tubifex: the 28-d EC50s were <18.2, 22.2, and 64.0 μg/g dw in sand and 281.3, 361.6, and 218.9 μg/g dw in sediment for BaDNS, CaDNS, and DNDS, respectively. The 28-d LC50s for H. azteca were similar among compounds: 115.3, 82.1, and 49.0 μg/g dry weight (dw) in sand, and 627.3, 757.9, and >188.5 μg/g dw in sediment, for BaDNS, CaDNS, and DNDS, respectively. However, when LC50s were estimated based on concentrations of NSAs measured in overlying water (which can be an important route of exposure for H. azteca), BaDNS and CaDNS were 3–4 orders of magnitude more toxic than DNDS. The NSAs examined were >3-fold more toxic when present in substrates with no organic carbon (e.g., sand) for all H. azteca endpoints where LC/EC50s could be calculated and for sublethal endpoints for T. tubifex. The organic carbon content of the sediment appears to have acted as a sink and reduced NSA toxicity by decreasing bioavailability. Environmental sediment samples were collected from 12 river sites across southern Ontario. The maximum concentration of CaDNS observed in sediment collected from this region was 2.8 μg/g dw in sediment with 2% organic carbon; 100-fold lower than the lowest EC10 in the current study.

Nanoplastics affect moulting and faecal pellet sinking in Antarctic krill (Euphausia superba) juveniles

Plastic debris has been identified as a potential threat to Antarctic marine ecosystems, however, the impact of nanoplastics (<1 μm) is currently unexplored. Antarctic krill (Euphausia superba) is a keystone species of Southern Ocean pelagic ecosystems, which plays a central role in the Antarctic food webs and carbon (C) cycle. Krill has been shown to rapidly fragment microplastic beads through the digestive system, releasing nanoplastics with unknown toxicological effects. Here we exposed krill juveniles to carboxylic (COOH, anionic) and amino- (NH2, cationic) polystyrene nanoparticles (PS NPs) and we investigated lethal and sub-lethal endpoints after 48 h. The analysis of PS NP suspensions in Antarctic sea water (SW) media showed that PS-COOH formed large agglomerates (1043 ± 121 nm), while PS-NH2 kept their nominal size (56.8 ± 3 nm) during the exposure time. After 48 h, no mortality was found but increase in exuviae production (12.6 ± 1.3%) and reduced swimming activity were observed in juveniles exposed to PS-NH2. The microbial community composition in SW supports the release of krill moults upon PS NP exposure and stimulates further research on the pivotal role of krill in shaping Southern Ocean bacterial assemblages. The presence of fluorescent signal in krill faecal pellets (FPs) confirmed the waterborne ingestion and egestion of PS-COOH at 48 h of exposure. Changes in FP structure and properties were also associated to the incorporation of PS NPs regardless of their surface charge. The effects of PS NPs on krill FP properties were compared to Control 0 h as a reference for full FPs (plastic vs food) and Control 48 h as a reference for more empty-like FPs (plastic vs lack of food). Exposure to PS NPs led to a FP sinking rate comparable to Control 48 h, but significantly lower than Control 0 h (58.40 ± 23.60 m/d and 51.23 ± 28.60 m/d for PS-COOH and PS-NH2; 168.80 ± 74.58 m/d for Control 0 h). Considering the important role played by krill in the food web and C export in the Southern Ocean, the present study provides cues about the potential impact of nanoplastics on Antarctic pelagic ecosystems and their biogeochemical cycles.

Combined effects of exposure to engineered silver nanoparticles and the water-soluble fraction of crude oil in the marine copepod Calanus finmarchicus

While it is likely that ENPs may occur together with other contaminants in nature, the combined effects of exposure to both ENPs and environmental contaminants are not studied sufficiently. In this study, we investigated the acute and sublethal toxicity of PVP coated silver nanoparticles (AgNP) and ionic silver (Ag+; administered as AgNO3) to the marine copepod Calanus finmarchicus. We further studied effects of single exposures to AgNPs (nominal concentrations: low 15 μg L−1 NPL, high 150 μg L−1 NPH) or Ag+ (60 μg L−1), and effects of co-exposure to AgNPs, Ag+ and the water-soluble fraction (WSF; 100 μg L−1) of a crude oil (AgNP + WSF; Ag++WSF). The gene expression and the activity of antioxidant defense enzymes SOD, CAT and GST, as well as the gene expression of HSP90 and CYP330A1 were determined as sublethal endpoints. Results show that Ag+ was more acutely toxic compared to AgNPs, with 96 h LC50 concentrations of 403 μg L−1 for AgNPs, and 147 μg L−1 for Ag+. Organismal uptake of Ag following exposure was similar for AgNP and Ag+, and was not significantly different when co-exposed to WSF. Exposure to AgNPs alone caused increases in gene expressions of GST and SOD, whereas WSF exposure caused an induction in SOD. Responses in enzyme activities were generally low, with significant effects observed only on SOD activity in NPL and WSF exposures and on GST activity in NPL and NPH exposures. Combined AgNP and WSF exposures caused slightly altered responses in expression of SOD, GST and CYP330A1 genes compared to the single exposures of either AgNPs or WSF. However, there was no clear pattern of cumulative effects caused by co-exposures of AgNPs and WSF. The present study indicates that the exposure to AgNPs, Ag+, and to a lesser degree WSF cause an oxidative stress response in C. finmarchicus, which was slightly, but mostly not significantly altered in combined exposures. This indicated that the combined effects between Ag and WSF are relatively limited, at least with regard to oxidative stress.

Phytochemical analysis of ionic liquid-Graviola (Annona muricata) fruit extract and its acute toxicity on zebrafish early-life stages

Annona muricata, commonly known as soursop and Graviola, is a member of the Annonaceae family. Some of its phytochemicals were reported to have a neurotoxicity effect causing neurodegenerative diseases. However, different parts of this tree have been used for ages in traditional medicine due to their biological activities, such as anti-inflammatory, antimicrobial, antioxidant and anticancer effects. This study aimed to qualitatively screen the crude ionic liquid-Graviola fruit extract (IL-GFE) phytochemical composition, assess its acute toxicity and determine the lethal concentrations using zebrafish (Danio rerio) embryos. IL-GFE contains acetogenins, alkaloids, phenols, terpenoids, tannins and flavonoids. Acute toxicity effects of IL-GFE on zebrafish embryos were observed from 24 to 120 hours of post fertilisation (hpf). The survival rate, LC50, sublethal endpoints and effect of IL-GFE on the heart rate of zebrafish larvae were assessed. Results showed that the lethal concentration (LC50) of the crude IL-GFE was 173.45 μg/mL. Interestingly, no significant changes on the morphology of the treated zebrafish were observed at a concentration of 125 μg/mL. However, the heart rate of zebrafish larvae at 96 hpf was significantly decreased by 33.76% after treated with crude IL-GFE at 125 µg/mL (119.00 ± 4.72 beats/min) as compared to the untreated group (179.67 ± 4.66 beats/min). This preliminary finding showed that crude ionic liquid-Graviola fruit extract and its phytoconstituents might have the potential to be developed as a food supplement or herbal product. However, further tests need to be conducted to evaluate its medicinal properties and adverse effects on organisms of higher orders.

Sublethal Endpoints in Non-target Organism Testing for Insect-Active GE Crops.

Historically, genetically engineered (GE) plants that have incorporated genes conferring insect protection have primarily used Cry proteins derived from Bacillus thuringiensis (Bt) to achieve their insecticidal phenotype. As a result, regulators have developed a level of familiarity and confidence in reviewing plants incorporating these insecticidal proteins. However, new technologies have been developed that produce GE plants that incorporate pest protection by triggering an RNA interference (RNAi) response or proteins other than Bt Cry proteins. These technologies have new modes of action. Although the overall assessment paradigm for GE plants is robust, there are ongoing discussions about the appropriate tests and measurement endpoints needed to inform non-target arthropod assessment for technologies that have a different mode of action than the Bt Cry proteins. As a result, increasing attention is being paid to the use of sublethal endpoints and their value for environmental risk assessment (ERA). This review focuses on the current status and history of sublethal endpoint use in insect-active GE crops, and evaluates the future use of sublethal endpoints for new and emerging technologies. It builds upon presentations made at the Workshop on Sublethal Endpoints for Non-target Organism Testing for Non-Bt GE Crops (Washington DC, USA, 4–5 March 2019), and the discussions of government, academic and industry scientists convened for the purpose of reviewing the progress and status of sublethal endpoint testing in non-target organisms.

Developmental toxicity and transcriptome analysis of 4-epianhydrotetracycline to zebrafish (Danio rerio) embryos

As a primary degradation by-product of tetracycline (TC), 4-Epianhydrotetracycline (4-EATC) has been detected frequently in the aquatic environment, which may pose a potential environmental risk to aquatic organisms. Up to now, however, the toxicology study on 4-EATC to aquatic organisms is limited. In the present study, in order to better understand the toxic mechanism of 4-EATC, developmental toxicity including lethal and sublethal effects of 4-EATC and TC were investigated. The results showed that the developmental toxicity of 4-EATC to zebrafish embryos was stronger than that of TC. The 96 h LC50 value of 4-EATC to zebrafish embryos was 29.13 mg/L. Malformations seemed to be the most sensitive sublethal endpoint of 4-EATC exposure, and the 96 h EC50 value was 8.57 mg/L. Transcriptome response of 4-EATC to zebrafish embryos was determined. The results showed that 430 different expression genes (DEGs) caused by 4-EATC, and most enriched in tryptophan (TRP) metabolism pathway. Annotation of DEGs in the TRP metabolism demonstrated that expression of 4 gene products in tryptophan metabolized along the kynurenine (KYN) pathway were changed. Disorder of TRP catabolism in KYN pathway was a potential mechanism of 4-EATC toxicity to zebrafish embryos.

Assessing the toxicity of the benzamide fungicide zoxamide in zebrafish (Danio rerio): Towards an adverse outcome pathway for beta-tubulin inhibitors

Commercial benzamide fungicides are applied to crops to control damage caused by oomycete fungi and are used as veterinary pharmaceuticals in aquaculture. The mechanism of action of these fungicides is to induce mitotic arrest via binding to beta-tubulin, thus inhibiting tubulin polymerization. However, there are little toxicity data available for benzimidazole fungicides in fish. To address this knowledge gap, we conducted zebrafish embryo toxicity tests to assess deformities, survival, and sub-lethal responses following exposure to zoxamide (0, 0.5, 1.0, 2.5, 5.0 and 10 μM zoxamide). We hypothesized that skeletal deformities would be prevalent in zebrafish due to its mechanism of inhibiting beta-tubulin polymerization. Zoxamide was relatively toxic to zebrafish embryos and larvae, and survival was reduced ∼50 % at 2 days post fertilization (dpf) with 10 μM exposure and over time at 6 dpf, 2.5 μM exposure reduced survival by ∼20 %. Frequency of hatch was also reduced/delayed in zebrafish at 3 dpf with >2.5 μM zoxamide. Pericardial edema, body length shortening, and spine curvature were observed in larvae exposed to >5 μM. Mitochondrial bioenergetics were assessed in ∼30 hpf embryos (24-hour exposure) using an XFe24 Flux Analyzer and regression analysis revealed a negative relationship between basal respiration and zoxamide concentration. Superoxide dismutase 1 and caspase 3 mRNA levels were both decreased in 6 dpf larvae exposed to 2.5 μM zoxamide, but were not changed in expression at 0.5 nor 1 μM zoxamide. Continuous 6-day exposure to zoxamide reduced larval activity at 2.5 μM; conversely a 24-hour exposure (at 5–6 dpf) induced hyperactivity at 5 μM suggesting dose and time dependent effects on fish behavior. Based on sub-lethal endpoints, we conceptualize an adverse outcome pathway for chemicals that inhibit tubulin polymerization.

Using aerobic exercise to evaluate sub-lethal tolerance of acute warming in fishes

ABSTRACTWe investigated whether fatigue from sustained aerobic swimming provides a sub-lethal endpoint to define tolerance of acute warming in fishes, as an alternative to loss of equilibrium (LOE) during a critical thermal maximum (CTmax) protocol. Two species were studied, Nile tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus). Each fish underwent an incremental swim test to determine gait transition speed (UGT), where it first engaged the unsteady anaerobic swimming mode that preceded fatigue. After suitable recovery, each fish was exercised at 85% of their own UGT and warmed 1°C every 30 min, to identify the temperature at which they fatigued, denoted as CTswim. Fish were also submitted to a standard CTmax, warming at the same rate as CTswim, under static conditions until LOE. All individuals fatigued in CTswim, at a mean temperature approximately 2°C lower than their CTmax. Therefore, if exposed to acute warming in the wild, the ability to perform aerobic metabolic work would be constrained at temperatures significantly below those that directly threatened survival. The collapse in performance at CTswim was preceded by a gait transition qualitatively indistinguishable from that during the incremental swim test. This suggests that fatigue in CTswim was linked to an inability to meet the tissue oxygen demands of exercise plus warming. This is consistent with the oxygen and capacity limited thermal tolerance (OCLTT) hypothesis, regarding the mechanism underlying tolerance of warming in fishes. Overall, fatigue at CTswim provides an ecologically relevant sub-lethal threshold that is more sensitive to extreme events than LOE at CTmax.

Effect of Bt toxin Cry1Ab on two freshwater caddisfly shredders \u2013 an attempt to establish dose-effect relationships through food-spiking

Genetically modified organisms (GMOs), which produce Bacillus thuringiensis (Bt) toxins, are widely used in agriculture in some parts of the world. Despite this, ecotoxicological methods, tailored to GMOs, are lacking to assess effects on aquatic environments. With the objective to investigate a food-related exposure pathway for aquatic shredders, we used a new food-spiking method while caddisfly larvae (Chaetopteryx spec., Sericostoma spec.) served as test species. Pure Cry1Ab toxins were spiked on black alder leaf discs and subsequently used in a feeding experiment. The toxin did not influence larval mortality compared to the control. The results, however, showed significant effects on larval lipid content (Chaetopteryx spec.) and development (Sericostoma spec.) at concentrations of 17.2 and 132.4 ng Cry1Ab/mg leaf, respectively. These changes are indicative for impacts on the fitness of the specimen and thus relevant in a risk assessment context. Ultimately, the food-spiking method allowed applying different Bt toxin concentrations leading to the establishment of dose-response relationships for various response variables. The use of long test durations and sublethal endpoints (consumption, lipid content, growth, larval instars) is, moreover, advisable when testing GMO effects.

Oxygen consumption rate of Caenorhabditis elegans as a high-throughput endpoint of toxicity testing using the Seahorse XFe96 Extracellular Flux Analyzer

Caenorhabditis elegans presents functioning, biologically relevant phenotypes and is frequently used as a bioindicator of toxicity. However, most C. elegans in vivo effect-assessment methods are laborious and time consuming. Therefore, we developed a novel method to measure the oxygen consumption rate of C. elegans as a sublethal endpoint of toxicity. This protocol was tested by exposing 50 larval stage one C. elegans individuals for 48 h (at 20 °C) to different concentrations of two toxicants i.e. benzylcetyldimethylammonium chloride (BAC-C16) and cadmium (Cd). Following exposures, the oxygen consumption rate of the C. elegans individuals were measured using the high-throughput functionality of the Seahorse XFe96 Extracellular Flux Analyzer. Dose-response curves for BAC-C16 (R2 = 0.93; P = 0.001) and Cd (R2 = 0.98; P = 0.001) were created. Furthermore, a strong, positive correlation was evidenced between C. elegans oxygen consumption rate and a commonly used, ecologically relevant endpoint of toxicity (growth inhibition) for BAC-C16 (R2 = 0.93; P = 0.0001) and Cd (R2 = 0.91; P = 0.0001). The data presented in this study show that C. elegans oxygen consumption rate can be used as a promising functional measurement of toxicity.

Embryotoxicity of atrazine and its degradation products to early life stages of zebrafish (Danio rerio)

Triazine herbicide atrazine is considered to be moderately toxic to various aquatic animals. The aim of our study was to evaluate the acute embryotoxicity of atrazine and its two degradation products, desisopropylatrazine and desethylatrazine, and their mixture to the early life stages of zebrafish (Danio rerio) by means of a modified method of the Fish Embryo Acute Toxicity (FET) Test – OECD guideline 236. Toxic effects were studied by the evaluation of lethal endpoints and development of disorders. Furthermore, sublethal endpoints such as hatching rate, formation of somites, development of eyes, spontaneous movement, heartbeat, blood circulation, pigmentation and occurrence of edema at 24, 48, 72 and 96 h post fertilization were assessed. Newly fertilized eggs were exposed to various concentrations of atrazine, desisopropylatrazine and desethylatrazine, and their combination 0.3, 30, 100, 300, 1000, 3,000 and 10,000 μg/l, which represent environmentally relevant levels of these pollutants in surface waters and multiples of these concentrations to find out if the toxic effect depends on dose. Single substances and their combination were not associated with a negative effect on mortality. Rare malformations were observed during these embryonal toxicity tests. Only pericardial edema was recorded during the monitored observation. A significant increase in the occurrence of pericardial edema between the control 0% and the experimental group 17.6 %) was found only in the group exposed to the highest concentration of a triazine herbicide combination (10,000 μg/l) at 72 and 96 h post fertilization. Obtained results indicate that especially higher not environmentally relevant concentrations of atrazine, its metabolites or their combination present a potencial risk of embryotoxicity for zebrafish.

Environmentally relevant mixture of S-metolachlor and its two metabolites affects thyroid metabolism in zebrafish embryos

Herbicides and their metabolites are often detected in water bodies where they may cause adverse effects to non-target organisms. Their effects at environmentally relevant concentrations are often unclear, especially concerning mixtures of pesticides. This study thus investigated the impacts of one of the most used herbicides: S-metolachlor and its two metabolites, metolachlor oxanilic acid (MOA) and metolachlor ethanesulfonic acid (MESA) on the development of zebrafish embryos (Danio rerio). Embryos were exposed to the individual substances and their environmentally relevant mixture until 120 hpf (hours post-fertilization). The focus was set on sublethal endpoints such as malformations, hatching success, length of fish larvae, spontaneous movements, heart rate and locomotion. Moreover, expression levels of eight genes linked to the thyroid system disruption, oxidative stress defense, mitochondrial metabolism, regulation of cell cycle and retinoic acid (RA) signaling pathway were analyzed. Exposure to S-metolachlor (1 μg/L) and the pesticide mixture (1 μg/L of each substance) significantly reduced spontaneous tail movements of 21 hpf embryos. Few rare developmental malformations were observed, but only in larvae exposed to more than 100 μg/L of individual substances (craniofacial deformation, non-inflated gas bladder, yolk sac malabsorption) and to 30 μg/L of each substance in the pesticide mixture (spine deformation). No effect on hatching success, length of larvae, heart rate or larvae locomotion were found. Strong responses were detected at the molecular level including induction of p53 gene regulating the cell cycle (the pesticide mixture - 1 μg/L of each substance; MESA 30 μg/L; and MOA 100 μg/L), as induction of cyp26a1 gene encoding cytochrome P450 (pesticide mixture - 1 μg/L of each substance). Genes implicated in the thyroid system regulation (dio2, thra, thrb) were all overexpressed by the environmentally relevant concentrations of the pesticide mixture (1 μg/L of each substance) and MESA metabolite (1 μg/L). Zebrafish thyroid system disruption was revealed by the overexpressed genes, as well as by some related developmental malformations (mainly gas bladder and yolk sac abnormalities), and reduced spontaneous tail movements. Thus, the thyroid system disruption represents a likely hypothesis behind the effects caused by the low environmental concentrations of S-metolachlor, its two metabolites and their mixture.

Effects of pH and nitrites on the toxicity of a cypermethrin-based pesticide to zebrafish embryos

Physical and chemical water parameters have an important role on the toxicity of environmental contaminants by affecting their degradation, transformation and bioavailability. The objective of this study was to evaluate the effects of pH and nitrite variation on the toxicity of the cypermethrin based pesticide Barrage®. Zebrafish embryos (Danio rerio) were exposed to a combination of pH or nitrite and cypermethrin through the Barrage® formulation for 96 h and lethal and developmental endpoints assessed. Concerning mortality, pH seemed to increase Barrage® toxicity, while nitrite concentrations tested did not seem to change Barrage® lethality. At sublethal level, either for combinations involving pH or nitrites, results suggested some interactive effects of environmental and chemical factors, highlighting the need of further research. This work suggests that sublethal endpoints should be included in the assessment of combined effects of compounds given that they can provide important information not obtained through lethality.

Effects of two little-studied environmental pollutants on early development in anurans

Despite intensive ecotoxicological research, we still know relatively little about the ecological impacts of many environmental contaminants. Filling these knowledge gaps is particularly important regarding amphibians, because they play significant roles in freshwater and terrestrial ecosystems, and their populations are declining worldwide. In this study, we investigated two pollutants that have been poorly studied in ecotoxicology despite their widespread occurrence in surface waters: the herbicide terbuthylazine and the pharmaceutical drug carbamazepine. We exposed two anuran species throughout their larval development to each of two environmentally relevant concentrations of each pollutant, and recorded mortality and 17 sub-lethal endpoints up to several months after exposure. Mortality was low and unrelated to treatment. In agile frogs (Rana dalmatina), we found that treatment with 0.3 μg/L terbuthylazine decreased tadpole activity and reduced fat bodies in juveniles, whereas treatment with 50 μg/L carbamazepine decreased spleen size and increased spleen pigmentation. In common toads (Bufo bufo), treatment with 0.003 μg/L terbuthylazine increased body mass at metamorphosis, treatment with 0.3 μg/L terbuthylazine increased the size of optic tecta, and treatment with 0.5 μg/L carbamazepine decreased hypothalamus size. Treatment with 50 μg/L carbamazepine reduced the feeding activity of toad tadpoles, decreased their production of anti-predatory bufadienolide toxins, and increased their body mass at metamorphosis; juvenile toads in this treatment group had reduced spleen pigmentation. Neither treatments affected the time to metamorphosis, post-metamorphic body mass, or sex ratios significantly. These results show that environmental levels of both terbuthylazine and carbamazepine can have several sub-lethal effects on anurans, which may be detrimental to individual fitness and population persistence in natural conditions. Our findings further highlight that toxic effects cannot be generalized between chemicals of similar structure, because the terbuthylazine effects we found do not conform with previously reported effects of atrazine, a related and extensively studied herbicide.

Modulation of PAH toxicity on the freshwater organism G. roeseli by microparticles

Polycyclic aromatic hydrocarbons are widespread and environmentally persistent chemicals that readily bind to particles in air, soil and sediment. Plastic particles, which are also an ubiquitous global contamination problem, may thus modulate their environmental fate and ecotoxicity. First, the acute aqueous toxicity of phenanthrene in adult Gammarus roeseli was determined with a LC50 of 471 μg/L after 24 h and 441 μg/L after 48 h. Second, considering lethal and sublethal endpoints, effects of phenanthrene concentration on G. roeseli were assessed in relation to the presence of anthropogenic and natural particles. The exposure of gammarids in presence of either particle type with phenanthrene resulted after 24 and 48 h in reduced effect size. Particle exposure alone did not result in any effects. The observed reduction of phenanthrene toxicity by polyamide contradicts the discussion of microplastics acting as a vector or synergistically. Especially, no difference in modulation by plastic particles and naturally occurring sediment particles was measured. These findings can most likely be explained by the similar adsorption of phenanthrene to both particle types resulting in reduced bioavailability.

Lethal and sub-lethal effects of nanosized titanium dioxide particles on Hydropsyche exocellata Dufour, 1841

Nanosized titanium dioxide particles (nTiO2) are one of the most widely used nanoscale engineered materials. Evidence of the presence of nTiO2 in freshwater systems has been reported by recent ecotoxicological studies, which indicated the potential toxic effects of nTiO2 to aquatic organisms. This study aimed at exploring lethal and sub-lethal endpoints of aqueous exposure of the aquatic insect Hydropsyche exocellata Dufour, 1841 (Trichoptera, Hydropsychidae) to nTiO2 at different concentrations. Abdominal contractions significantly increased with nTiO2 exposure. Levels of lipid peroxidation (LPO) increased with nTiO2 dose, whereas the opposite was recorded for the antioxidant enzyme catalase activity (CAT). Moreover, the highest exposure (50.0 mg/L) resulted in the highest mortality (56% of mortality two weeks after pulse exposure to nTiO2). Overall, our results show that nTiO2 has potential toxic effects on H. exocellata at concentrations of 50.0 mg/L.