Residual determination and acute toxicity of the neonicotinoid clothianidin in the neotropical stingless bee Tetragonisca angustula Latreille, 1811 (Apidae: Meliponini).

Bees play a crucial role as natural pollinators, ensuring the maintenance and stability of the world's biodiversity and agricultural crops. Native bees in neotropical regions belong to the Meliponini tribe, a larger group that differs significantly in behavior and biology from honeybees (e.g., Apis mellifera) and solitary bees (e.g., Osmia spp.). Hence, the exposure and effects of pesticides is also likely to vary among these different species. The aim of this study was to develop an analytical method to determine the presence of the neonicotinoid clothianidin in the Brazilian native stingless bee Tetragonisca angustula (local common name: Jataí). The method used for the chemical analysis involved a QuEChERS technique combined with UHPLC-MS/MS analysis. The developed method was subsequently used to analyze collected field samples. In addition, the acute toxicity of the pesticide to T. angustula was evaluated in a laboratory bioassay evaluating both lethal and sublethal endpoints. The analytical method was successfully developed with detection and quantification limits of 1.55 and 5 µg L-1, respectively, along with a linear range of 1-5 ng mL-1. Clothianidin was detected in environmental samples (9.2-32.9 ng g-1), and the exposure experiments demonstrated acute oral toxicity to adults of T. angustula, (24 h-LD50 of 0.16 ng a.i./bee), as well as no significative interference in acetylcholinesterase activity. Considering the obtained toxicity endpoints for T. angustula and those reported in the literature for other bee species, this study revealed that T. angustula is more (lethally) sensitive to clothianidin than other bee species, including those commonly used in environmental risk assessment studies. This thus also supports the call for using native test species in (regional) risk assessment evaluations.

Abamectin and difenoconazole monitoring in strawberry flowers and pollen sampled from Tetragonisca angustula (Latreille) (Hymenoptera: Apidae) hives located in crop vicinities.

The increase in agricultural productivity associated with the emergence and the extensive use of pesticides is undeniable. However, strong evidence indicates that this continuous demand is causing serious environmental impacts and bringing toxic effects to associated biota as pollinating insects. The present work aims the determination of the insecticide abamectin (ABA) and the fungicide difenoconazole (DIF) in strawberry flowers (Fragaria x ananassa DUCH.) and pollen sampled from beehives of the stingless bee Tetragonisca angustula Latreille (Hymenoptera: Apidae) located nearby strawberry fields. For analysis, QuEChERS method was optimized, and the analytical performance of those two pesticides was verified. Then, the method was applied to strawberry flowers and the pollen was sampled during three field campaigns. While abamectin was not detected, the systemic fungicide difenoconazole was determined in almost all flowers and pollen samples, demonstrating the major persistence of this pesticide in investigated matrices. The results were then discussed about the difenoconazole application rate and transport to colonies to estimate a preliminary environmental risk assessment for stingless native bees. All calculations were proceeded considering exposure rates and toxicity data from the literature, adapted from Apis mellifera studies. In this sense, the determination, application, and discussion about risk assessment figure out as an important tool to the knowledge about the preliminary risks of native bees exposed to pesticides.

Toxicity of Binary Metal Mixtures to the Tropical Ostracod Strandesia Trispinosa.

The ecotoxicity of metals is generally assessed individually, in part because current knowledge does not allow for the accurate prediction of the toxicity of metal mixtures to aquatic organisms. The objective of this study was to investigate the toxic effects of binary combinations of metal salts (copper sulphate-CuSO4, cadmium chloride-CdCl2, mercury chloride-HgCl2 and manganese sulphate-MnSO4) on the tropical ostracod Strandesia trispinosa through acute toxicity tests. To this end, ostracods were exposed to each individual metal salt as well as to their combinations by applying a full factorial design. The model that best explained the effects of the mixtures CuSO4 x CdCl2, CuSO4 x HgCl2 and CuSO4 x MnSO4 on the survival of S. trispinosa was Concentration Addition, whereas this was Independent Action for the CdCl2 x HgCl2 mixture. The observed synergistic interactions are likely to result in unacceptable risks to aquatic ecosystems under real field conditions. This is especially the case if CuSO4 predominates the metal mixture, as observed for its combination with mercury and manganese.

Responses of Chironomus sancticaroli to the simulation of environmental contamination by sugarcane management practices: Water and sediment toxicity.

Sugarcane management practices include the application of pesticides, including the herbicide 2,4-D and the insecticide fipronil. In addition, a by-product from the ethanol industry, called vinasse, is commonly applied to fertilize sugarcane areas. The potential risks of these practices to the edge-of-field aquatic ecosystems were assessed in the present study. This was done by contaminating mesocosms with (single and mixtures of) both pesticides and vinasse and evaluating the effects on the midge Chironomus sancticaroli through in-situ and laboratory bioassays. To this end, outdoor mesocosms were treated with fipronil (F), 2,4-D (D), and vinasse (V) alone and with the mixture of fipronil and 2,4-D (M), as well as with both pesticides and vinasse (MV). C. sancticaroli was deployed in mesocosms before contamination in cages, which were taken out 4- and 8-days-post-contamination. Water and sediment samples were also taken for laboratory bioassays on the first day of contamination, as well as 7-, 14-, 21-, 30-, 45-, and 75-days post-contamination. The responses assessed in subchronic assays (8-day) were survival, growth, head capsule width, development, and mentum deformities. Low survival occurred in the in-situ experiments of all treatments due to the low oxygen levels. In the laboratory tests, effects on survival occurred for F, V, and M over time after exposure to both water and sediment. All organisms died post-exposure to water samples from the MV treatment, even 75-days-post-contamination. Impairments in body length and head capsule width occurred for F, V, and M for water and F, V, M, and MV for sediment samples over time. All treatments increased mentum deformities in exposed larvae for any of the sampling periods. The negative effects observed were more significant in the mixture mesocosms (M and MV), thus indicating increased risks from management practices applying these compounds together or with a short time interval in crops.

Modified QuEChERS Method for Extracting Thiamethoxam and Imidacloprid from Stingless Bees: Development, Application, and Green Metrics.

In the present study, a method for the determination of residues of the neonicotinoid insecticides thiamethoxam and imidacloprid in the stingless bee Melipona scutellaris Latreille (1811) was optimized through a factorial design, tested using green metrics, and then applied to exposed bees. It combines the extraction with a modified quick, easy, cheap, effective, rugged, and safe method and the determination by liquid chromatography-tandem mass spectrometry analysis. Different parameters such as the mass of the sample, dispersive sorbents, and elution solvents were assessed. Method validation parameters were checked and include sensitivity, specificity, and linearity. The limit of quantification of 0.0025 µg g-1 was obtained for both insecticides, where accuracy was 94%-100% with satisfactory intraday and interday precisions (relative standard deviation <10%). The qualified method was applied to orally and topically exposed bee samples, and the results indicated that it is suitable for the determination and quantification of neonicotinoid pesticide residues in this species. Moreover, green analytical metrics like the National Environmental Methods Index, Eco Scale score, high-performance liquid chromatography with an environmental assessment tool (HPLC-EAT), waste generation, and amount of sample were compared with methods described in the literature involving neonicotinoid analysis in honeybees. As a result, the present study displayed the highest Eco Scale score and HPLC-EAT score and the second smallest amount of sample and waste generated. Thus, the method meets green analytical metrics more than other methods. In this sense, besides the application, the multicriteria decision analysis tool employed suggests that this is a good option as a green analytical method. Environ Toxicol Chem 2022;41:2365-2374. © 2022 SETAC.

Chronic environmentally relevant levels of pesticides disrupt energy reserves, feeding rates, and life-cycle responses in the amphipod Hyalella meinerti.

When pesticides reach the aquatic environment, they can distribute in water and sediment, increasing the risks to benthic organisms, such as amphipods that play a key role in the aquatic food webs. Thus, the present study assessed the consequences of exposure to the insecticide fipronil and herbicide 2,4-D (alone and in mixture) on biochemical markers, feeding rates and the partial life-cycle of Hyalella meinerti. Three concentrations of fipronil (0.1, 0.3, and 0.7 µg L-1) and 2,4-D (19, 124, and 654 µg L-1), and six mixture combinations were assessed. The first experiment was carried out with males and females separately assessing the feeding rates, total carbohydrate content, and lipid profile. The second (partial life-cycle) lasted 49 days, and the survival, growth, and reproductive endpoints were assessed. Both pesticides and their mixture caused decreases in feeding rates, mainly in females. Females also suffered a change in the total carbohydrate content. In addition, there were changes in the percentage of triacylglycerol and phospholipids in males and females. Furthermore, alterations occurred in the percentual of triacylglycerol and phospholipids to both sexes. In the second experiment, fipronil and the mixtures caused decreases in the survival of H. meinerti over time. Exposure to 2,4-D, fipronil, and their mixture impaired the 28-day growth leading to biomass loss ranging from 17-23%, 54-60%, and 22-49%, respectively. The insecticide and mixture caused increases in time to sexual maturation of up to 10 and 6 days, respectively, and reduced the number of formed couples. Furthermore, fipronil decreased reproduction up to 36 times and no juveniles were produced in some mixture combinations. In addition, the pesticides on isolation decreased the juvenile size. Finally, exposure to both pesticides, alone or in a mixture, decreased the intrinsic rate of population growth. The results were observed in concentrations already quantified in water bodies, with risks for ecosystems functioning due to the importance of amphipods in aquatic ecosystems.

Multi-generational exposure to fipronil, 2,4-D, and their mixtures in Chironomus sancticaroli: Biochemical, individual, and population endpoints.

Conventional farming delivers a range of pesticides to aquatic ecosystems leading to implications for the indigenous species. Due to the multiple applications and persistence of molecules, organisms may be exposed for a prolonged period over multiple generations. The present study outlines a full life-cycle design over three generations of Chironomus sancticaroli exposed to the insecticide fipronil, the herbicide 2,4-D, and their mixtures. The experiment started with newly hatched larvae from the parental generation and lasted with the emerged adults from the second generation. Five nominal concentrations of fipronil and 2,4-D were tested, as well as six combinations of both pesticides. As additional responses, the total carbohydrates and the lipid classes were evaluated in the parental generation. The first and second generations were more susceptible to the tested compounds compared with the parental ones. Survival of larvae and pupae was decreased by both pesticides and their mixtures along with the generations. Only fipronil impaired the survival of emerged adults. Both pesticides (isolated and in the mixture) altered the emergence and the fraction of males and females. Moreover, the number of eggs produced, and their hatchability decreased. Only one combination of the pesticides increased the content of carbohydrates. Fipronil, 2,4-D, and its mixture altered the profile of the lipid classes. All mixture treatments and the three highest concentrations of fipronil extinguished the population of C. sancticaroli at the end of the first generation. In the remaining treatments with the insecticide, the population did not survive the second generation. Only three concentrations of 2,4-D and the control persisted until the end of the experiment. The results indicate that a prolonged exposition to these pesticides may disrupt the natural populations of exposed organisms with consequences to ecosystems' functioning, considering the importance of chironomids to aquatic and terrestrial environments.

Toxicity of fipronil and 2,4-D formulations (alone and in a mixture) to the tropical amphipod Hyalella meinerti.

Conventional farming uses a large volume of pesticides that may reach aquatic ecosystems. This is also the case for the insecticide fipronil and the herbicide 2,4-D, which are widely used in many crops. This study aimed at evaluating the individual and mixture toxicity of these pesticides to the tropical amphipod Hyalella meinerti. To this end, acute toxicity tests (96 h) were conducted. Chronic bioassays (10 days) were also carried out, in which the body length and dry biomass were evaluated as endpoints. In addition, a complete factorial mixture chronic toxicity test was carried out. H. meinerti was sensitive to fipronil in the acute toxicity tests, with a LC50-96-h of 0.86 µg L-1 (95% CI 0.26-0.46), and no acute effects were observed after 2,4-D exposure even at the highest test concentration of 100 mg L-1. In the chronic toxicity tests, all tested concentrations of both pesticides decreased the growth of H. meinerti, in which losses on biomass reached 45% and 65% for 2,4-D and fipronil, respectively. The pesticide mixture indicated antagonism although it still significantly decreased the body growth. The results obtained indicate a high sensitivity of H. meinerti exposed to environmentally realistic concentrations, demonstrating that there are risks for the species in real field conditions.

Impact of 2,4-D and fipronil on the tropical midge Chironomus sancticaroli (Diptera: Chironomidae).

Increased use of pesticides in conventional agriculture implies potential risks to the environment. In aquatic ecosystems, benthic organisms may be exposed to pesticides via contaminated water and sediment, leading to several potential cascading effects on the food web. The aim of this study was to assess the functional implications of environmental realistic concentrations of the herbicide 2,4-D and the insecticide fipronil (alone and in combination) to the native tropical chironomid Chironomus sancticaroli. These two pesticides are widely applied to different crops and have frequently been detected (together) in surface water bodies in Brazil and elsewhere. Commercial products containing fipronil (Regent® 800WG) and 2,4-D (DMA® 806BR) were evaluated in 8-day toxicity tests for their effects on larval survival, growth (body length and biomass), head capsule width, development, and mentum deformities. Fipronil decreased the larval survival at the highest test concentration and the effective concentrations (EC) after eight days of exposure were: EC10 = 0.48 µg L-1 (0.395-0.565), EC20 = 1.06 µg L-1 (0.607-1.513), and EC50 = 3.70 µg L-1 (1.664-5.736). All sublethal test concentrations of fipronil decreased the larval growth, causing reductions in biomass up to 72%. The two highest test concentrations of fipronil decreased the head capsule width and after exposure to 3.7 µg fipronil L-1, only half of the larvae reached the fourth instar. The incidence of deformities was increased by fipronil in a concentration dependent manner with an increase ranging from 23% to 75%. The highest test concentration of 2.4-D (426 µg L-1) decreased the head capsule width, but larval development was unaffected at all concentrations evaluated. In the mixture tests, antagonism was observed at lower fipronil concentrations and synergism at higher fipronil concentrations for growth. The incidence of deformities rose with increasing fipronil concentrations. The results showed that environmental realistic concentrations of fipronil may have serious ecological implications for C. sancticaroli populations and that a mixture with the herbicide 2,4-D can have synergistic effects, potentiating the risks to the aquatic ecosystem.

Functional responses of Hyalella meinerti after exposure to environmentally realistic concentrations of 2,4-D, fipronil, and vinasse (individually and in mixture).

Sugarcane crops management in Brazil includes the use of pesticides, as well as alternative organic fertilizers such as vinasse obtained from waste of the ethanol industry. In order to assess the effects of the environmental contamination generated by such sugarcane practices, this study was aimed to investigate the effects of the pesticides 2,4-Dichlorophenoxyacetic acid (2,4-D) and fipronil, as well as vinasse, on the survival, behavior, and reproduction of the native epibenthic macroinvertebrate Hyalella meinerti through in situ and laboratory experiments. In situ assays were conducted in mesocosms with six treatments, i.e. untreated control, 2,4-D, fipronil, and vinasse, the mixture of the two pesticides, and both pesticides mixed with vinasse. Survival, swimming behavior, and reproduction were evaluated over time post contamination, from 0-96 h (T1) and 7-14 days (T2) through in situ experiments and 30-44 days (T3) and 75-89 days (T4) post contamination by laboratory bioassays with mesocosm water. In the T1 period, survival of H. meinerti was registered only in controls and mesocosms treated with 2,4-D. In the T2 period, treatments containing fipronil and vinasse (isolated or in both mixture treatments) still caused 100 % of mortality. Survival was recorded only in 2,4-D and control treatments, whereas reproduction only occurred in the control. In the T3 period, no survival occurred to fipronil and both mixture treatments. Vinasse and 2,4-D decreased total reproduction in comparison to control. In the T4 period, amphipods survival was detected when exposed to fipronil and its mixture with 2,4-D. However, these same treatments decreased the amplexus rates and total reproduction, with synergism denoted for the pesticide mixture. The swimming activity of males, females, and couples was decreased in surviving organisms exposed to 2,4-D, fipronil, vinasse, and the mixture of pesticides along all experimental periods. Our study showed that the application of fipronil, 2,4-D, and vinasse isolated or mixed at realistic concentrations of actual sugarcane management practices may negatively impact functional responses of indigenous amphipods in natural aquatic systems.

Effects of abamectin-based and difenoconazole-based formulations and their mixtures in Daphnia magna: a multiple endpoint approach.

This study evaluated the toxicity of pesticide formulations Kraft® 36 EC (active ingredient-a.i. abamectin) and Score® 250 EC (a.i. difenoconazole), and their mixtures in Daphnia magna at different biological levels of organization. Survival, reproduction and biochemical markers (cholinesterase (ChE), catalase (CAT) and lipid peroxidation (LPO)) were some of the endpoints evaluated. Total proteins and lipids were also studied together with energy consumption (Ec). D. magna neonates were exposed for 96 h to Kraft (2, 4, and 6 ng a.i./L) and Score (12.5, 25, and 50 µg a.i./L) for the biochemical experiments, and for 15 days to abamectin (1-5 ng a.i./L) and to difenoconazole (3.12-50 µg a.i./L) to assess possible changes in reproduction. Exposures of organisms to both single compounds did not cause effects to antioxidant and detoxifying enzymes, except for LPO occurring at the highest concentration of difenoconazole tested. For ChE and CAT there was enzymatic induction in mixture treatments organisms, occurring at minor pesticides concentrations for CAT and at the two highest concentrations for ChE. There were no significant differences for total protein in D. magna but lipids showed an increase at the highest concentrations of pesticide mixture combinations. There was a significant increase of Ec in individuals of all treatments tested. In the chronic test, increased fecundity occurred for D. magna under difenoconazole exposures and mixtures. This study demonstrated that mixtures of these pesticides caused greater toxicity to D. magna than when tested individually, except for Ec. Therefore, effects of mixtures are very hard to predict only based on information from single compounds, which most possibly is the result of biological complexity and redundancy in response pathways, which need further experimentation to become better known.

Dynamics of (total and methyl) mercury in sediment, fish, and crocodiles in an Amazonian Lake and risk assessment of fish consumption to the local population.

Increasing concerns have been raised about the toxicity of mercury (Hg) to humans, especially for those that consume a great amount of fish. High Hg concentrations have previously been measured in Amazonian waterbodies, both resulting from natural and anthropogenic sources. However, few studies have been conducted so far in Amazonian lakes that are fished by local populations. In addition, few of those studies included methylmercury (MeHg), the most toxic and bioaccumulative Hg form, and evaluated the influence of physico-chemical conditions and season on Hg dynamics. In the present study, total Hg (THg) and MeHg concentrations were measured in bottom sediment as well as in two fish and two crocodile species of the Amazonian Cuniã Lake. Bottom sediment MeHg concentrations were higher in the dry season than in the wet season, which is related to differences in physico-chemical (pH and electrical conductivity) conditions. Diet appeared to be related with animal tissue MeHg concentrations, with the herbivorous fish having lower MeHg levels than the predatory fish and crocodiles. Based on the measured tissue concentrations and published data on local person weight and fish consumption, MeHg risk to Cuniã Lake populations was estimated. Although the MeHg fish tissue concentrations did not exceed national and international standards, a significant risk to the local population is anticipated due to their high fish consumption rates. Graphical abstract.

Acute toxicity of four metals to three tropical aquatic invertebrates: The dragonfly Tramea cophysa and the ostracods Chlamydotheca sp. and Strandesia trispinosa.

The relatively low availability of toxicity data for indigenous tropical species has often been discussed. In addition, several taxonomic groups of invertebrates are understudied, such as dragonflies and ostracods. The aim of the present study was therefore to evaluate the acute toxicity of four metals (cadmium - Cd, copper - Cu, manganese - Mn, and mercury - Hg) to the tropical dragonfly nymphs of Tramea cophysa and two tropical ostracod species (Chlamydotheca sp. and Strandesia trispinosa). Toxicity data for other invertebrates were also mined to allow comparing the sensitivity of the three test species with that of other (temperate and tropical) invertebrates. The order of metal sensitivity was different for the three test species: T. cophysa: Cu > CdHg > Mn, Chlamydotheca sp.: Cd > Cu > Hg > Mn, and S. trispinosa: Cd > Hg > Cu > Mn. However, manganese was the least toxic metal tested for all three species, which is hypothesized to be due to a possible metal transfer to the cuticle of the moulting test species. The sensitivity ranking of the three test species to the metals was S. trispinosa > Chlamydotheca sp.>T. cophysa (except for Cu for which the ranking was Chlamydotheca sp.>T. cophysa > S. trispinosa). Overall, the test species are concluded to be suitable test organisms for tropical toxicity evaluations. Future studies should also evaluate the chronic toxicity and include other important metal exposure routes such as sediment and food.

Freshwater neotropical oligochaetes as native test species for the toxicity evaluation of cadmium, mercury and their mixtures.

The toxicity of metals, whether isolated or in mixtures, involves changes in biochemical processes as well as in cell membranes, which may lead to deleterious short- and long-term effects on the affected organisms. Among metals, cadmium and mercury stand out due to their abundance in nature, frequent use for industrial activities and biological accumulation, with high levels of residence in trophic chains. Benthic communities are particularly prone to metal pollution since metals usually accumulate in sediments. The aim of this study was to evaluate the acute toxicity of mercury and cadmium, single and in mixture, to two native species of epibenthic oligochaetes: Allonais inaequalis and Dero furcatus. In order to assess the potential of these species as bioindicators, we compared their sensitivity with those of other internationally used species by applying the species sensitivity distribution approach. The 96h-LC50 of cadmium chloride was 627 and 364 µg L-1 for A. inaequalis and D. furcatus, respectively, evidencing that the latter species is almost twice as sensitive to this metal than A. inaequalis. For mercury chloride, the 96h-LC50 was 129 µg L-1 for A. inaequalis and 92 µg L-1 for D. furcatus. The sensitivities of these oligochaetes were superior or similar to that of other frequently used oligochaete test species such as Tubifex tubifex and Lumbriculus variegatus. The metal mixtures had synergism in general (D. furcatus) or at high doses only (A. inaequalis), implying a potentiation of their toxic effects when both metals co-occur in the environment. By comparing the derived toxicity values with concentrations of cadmium and mercury measured in the field, it can be concluded that aquatic organisms are likely to be at risk when exposed to the environmental relevant concentrations of cadmium and mercury here tested, especially when they are both present.

Sensitivity of tropical cladocerans to chlorpyrifos and other insecticides as compared to their temperate counterparts.

The use of temperate toxicity data in tropical risk assessments has often been disputed. Previous sensitivity comparisons between temperate and tropical species, however, have not shown a consistent sensitivity difference between climatically-distinct species. Such comparisons were often limited by a small tropical toxicity dataset. In addition, differences in the taxonomic compositions of the temperate and tropical species assemblages used to construct species sensitivity distributions curves also hampered direct comparisons (e.g. type and ration of crustaceans and insects). The aim of the present study was to compare the sensitivity of temperate and tropical cladocerans to insecticides. Acute laboratory toxicity tests were conducted with five Neotropical cladocerans exposed to a concentration series of the insecticide chlorpyrifos. Subsequently, their EC50 values were compared with those reported in the literature for non-tropical cladocerans. An additional literature toxicity data search for insecticides other than chlorpyrifos was also conducted for both temperate and tropical cladocerans to enable a comparison for a wider range of insecticides and taxa. The order of sensitivity of the native cladocerans to chlorpyrifos was Ceriodaphnia silvestrii (0.039 µg L-1) > Diaphanosoma birgei (0.211 µg L-1) = Daphnia laevis (0.216 µg L-1) > Moina micrura (0.463 µg L-1) = Macrothrix flabelligera (0.619 µg L-1). A regulatory acceptable concentration based on temperate cladoceran toxicity data of both chlorpyrifos and other insecticides also appeared to be sufficiently protective for tropical cladoceran species. Implications for the use of temperate toxicity data in tropical risk assessments and indications for tropical cladoceran test species selection are discussed.

Lethal and sublethal toxicity of abamectin and difenoconazole (individually and in mixture) to early life stages of zebrafish.

In recent years, the need for the development of alternative test methods for the conventional acute fish toxicity test (AFT) with adult fish has often been discussed. In addition, concerns have been raised on the potential risks related with environmentally realistic pesticide mixtures since risk evaluations have traditionally been based on individual pesticides. The insecticide/acaricide abamectin and the fungicide difenoconazole are the main pesticides that are intensively used in Brazilian strawberry crop and are hence likely to occur simultaneously in edge-of-field waterbodies. The aim of the present study was therefore to evaluate the lethal and sublethal toxicity of single and mixture exposures of these pesticides to zebrafish early life stages (embryos and juveniles). By comparing the derived toxicity data of the individual compounds with that previously determined for zebrafish adults, the order of life stage sensitivity was juvenile > adult > embryo. The pesticide mixture revealed a dose-level dependent deviation of the independent action model, with antagonism at low dose levels and synergism at high dose levels. Sublethal parameters (especially those related with locomotion) were considerably more sensitive than lethality. Subsequently, the inclusion of sublethal parameters may greatly improve the sensitivity of FET tests and hence its suitability as a substitution of adult fish testing in risk assessment evaluations.

Sensitivities of three tropical indigenous freshwater invertebrates to single and mixture exposures of diuron and carbofuran and their commercial formulations.

As compared to their temperate counterparts, few toxicity tests have been conducted so far into the evaluation of the sensitivity of indigenous tropical species to pesticides. Especially mixture toxicity assessments appear to be scarce. To contribute to increase our knowledge in this arena, we evaluated the acute toxicity of diuron and carbofuran and their mixtures to the neotropical oligochaetes Allonais inaequalis and Dero furcatus, and the ostracod Strandesia trispinosa. Tests were performed with both the pure active ingredients, as well as their formulated products. The toxicity of the latter to the three test organisms was generally greater than that of the pure active ingredients, although absolute differences were rather small. The sensitivity of the indigenous species was slightly greater than temperate test species from the same taxonomic groups. The concentration addition conceptual model best described the results of the mixture toxicity data. Derived deviations of this model appeared to be dependent on the test organism and as to whether the pesticides were applied as active ingredients or their commercial products. Reported field concentrations of the two pesticides indicate risks to freshwater biota, especially if they are both present. The test species used in the present study are concluded to be suitable candidates as surrogate test organisms in local pesticide risk evaluations.

Toxicity of abamectin and difenoconazole mixtures to a Neotropical cladoceran after simulated run-off and spray drift exposure.

Aquatic risk assessments of pesticides in tropical countries have often been disputed for being largely based on risk evaluations conducted in temperate regions. Although pesticide sensitivity comparisons between temperate and tropical freshwater organisms have indeed not revealed consistent differences, risk assessments are currently still based on a relatively small tropical toxicity dataset. In addition, greater levels of runoff and spray drift may be expected in tropical than in temperate agroecosystems, indicating that aquatic life in edge-of-field water bodies is likely to be subjected to higher concentrations of pesticides and their mixtures. The aim of the present study was to evaluate the toxicity of Kraft® 36 EC (a.i. abamectin), Score® 250 EC (a.i. difenoconazole) and their mixture to the Neotropical cladoceran Macrothrix flabelligera. Laboratory toxicity tests with the individual formulated products indicated EC50-48h values of 3.1 and 659µg a.i./L given as nominal test concentrations, respectively. Mixtures of the two pesticides revealed a concentration-dependent deviation of the independent action model, with antagonism at low and synergism at high pesticide mixture concentrations. Laboratory toxicity tests were also conducted with microcosm water that was treated with the individual or mixtures through runoff or direct overspray. Microcosm tanks receiving runoff water from experimental soil plots applied with recommended doses of the individual pesticides did not show toxicity to the test organism. Microcosms that received runoff water containing the pesticide mixture, however, did cause a short-term effect on immobility. The microcosms that were treated by direct overspray of both pesticide formulations showed the most pronounced toxic effects. Study findings suggest a potential risk of these pesticides at environmentally relevant concentrations, especially when they are both present.

Impact of runoff water from an experimental agricultural field applied with Vertimec® 18EC (abamectin) on the survival, growth and gill morphology of zebrafish juveniles.

Edge-of-field waterbodies in tropical agroecosystems have been reported to be especially prone to pesticide contamination through runoff resulting from intensive irrigation practices and tropical rainfall. In the present study, the effects of runoff from an experimental agricultural field applied with Vertimec(®) 18EC (active ingredient: abamectin) on zebrafish were evaluated. To this end, the experimental field was applied with the Vertimec(®) 18EC dose recommended for strawberry crop in Brazil, whereas another field was treated with water only to serve as control. No effects of runoff water from either plot were recorded on survival. Water from the treated field led to increased growth and gill alterations. In general, these alterations were of the first and second degree, including proliferation of cells between the secondary lamellae, dilation at the lamellar apex, detachment of the respiratory epithelium and aneurism. These results confirm the high toxic potential of Vertimec(®) 18EC and provide evidence that environmental risks are likely to occur in areas subject to runoff containing this pesticide.