Fate and toxicity of 2,4-D and fipronil in mesocosm systems.

2,4-D and fipronil are among Brazil's most used pesticides. The presence of these substances in surface waters is a concern for the aquatic ecosystem health. Thus, understanding the behavior of these substances under environmentally relevant conditions is essential for an effective risk assessment. This study aimed to determine the degradation profiles of 2,4-D and fipronil after controlled application in aquatic mesocosm systems under influencing factors such as environmental aspects and vinasse application, evaluate pesticide dissipation at the water-sediment interface, and perform an environmental risk assessment in water and sediment compartments. Mesocosm systems were divided into six different treatments, namely: control (C), vinasse application (V), 2,4-D application (D), fipronil application (F), mixture of 2,4-D and fipronil application (M), and mixture of 2,4-D and fipronil with vinasse application (MV). Pesticide application was performed according to typical Brazilian sugarcane management procedures, and the experimental systems were monitored for 150 days. Pesticide dissipation kinetics was modeled using first-order reaction models. The estimated half-life times of 2,4-D were 18.2 days for individual application, 50.2 days for combined application, and 9.6 days for combined application with vinasse. For fipronil, the respective half-life times were 11.7, 13.8, and 24.5 days. The dynamics of pesticides in surface waters resulted in the deposition of these compounds in the sediment. Also, fipronil transformation products fipronil-sulfide and fipronil-sulfone were quantified in water 21 days after pesticide application. Finally, performed risk assessments showed significant potential risk to environmental health, with RQ values for 2,4-D up to 1359 in freshwater and 98 in sediment, and RQ values for fipronil up to 22,078 in freshwater and 2582 in sediment.

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.

Environmental analysis of the eutrophication and spread of aquatic macrophytes in a tropical reservoir: a case study in Brazil.

Land use changes threaten the maintenance of water quality and challenge the management of tropical reservoirs. In particular, eutrophication alters several ecosystem functions and services, compromising multiple uses of water. For example, in the Lajeado Reservoir (Araraquara, São Paulo, Brazil), aquatic macrophytes rapidly spread and occupied more than 90% of the system's surface area (from 2016 to 2019). In such a scenario, this research aimed to evaluate the eutrophication causes and impacts to provide technical and scientific support to public agencies and propose remediation alternatives. First, a diagnosis of the study area was performed, using available data on land use, water quality, and climate (between 2010 and 2018). Second, water and sediment samples were collected for physical, chemical, and ecotoxicological analyses. The Ecological Risk Assessment consisted of a triad of Lines of Evidence, including physical-chemical, chemical, and ecotoxicological results. The expansion of agricultural activities (e.g., sugarcane cultivation) and diffuse pollution were highlighted among possible causes, and water quality was affected by increases in the concentration of nutrients (e.g., nitrogen and phosphorus), metals (e.g., iron), and emerging contaminants (e.g., pesticides and caffeine). As a result, the Lajeado Reservoir presented a high ecological risk, and alternatives for macrophyte removal are needed. This research involved several stakeholders (i.e., community, government agencies, and universities) who participated more effectively in environmental monitoring and recovery.

Irrigation with Water Contaminated by Sugarcane Pesticides and Vinasse Can Inhibit Seed Germination and Crops Initial Growth.

Sugarcane crops are dependent on chemicals for maintaining plantations. Therefore, environmental consequences concern adjacent areas that can be affected by contaminants in common use, including pesticides and vinasse (i.e., a by-product from the ethanol industry). This study aimed to evaluate phytotoxicity through two plant bioassays with water from mesocosms contaminated with the herbicide 2,4-D (447.0 µg L-1), the insecticide fipronil (63.5 µg L-1), and sugarcane vinasse (1.3%). First, the germination test (4 d) with Eruca sativa L. assessed water samples collected three times after the contamination (2 h, 14 d, and 30 d), considering germination, shoot, and root growth. The results from this bioassay indicated higher phytotoxicity for 2,4-D as it fully inhibited the shoot and root growth even in low concentrations (0.2 µg L-1). However, no significant effect was reported for fipronil and vinasse. Also, the 2,4-D effects drastically decreased due to an expressive concentration reduction (99.4% after 30 d in mixture with vinasse). Second, the irrigation test with Phaseolus vulgaris L. and Zea mays L. considered shoot and root growth and biomass under 21 days after plants emergence. The herbicide 2,4-D inhibited the initial growth of tested species, especially the roots (up to 45% inhibition). Furthermore, sugarcane vinasse caused harmful effects on plant growth (up to 31% inhibition). Therefore, our data showed that these contaminants could inhibit plant germination and initial growth under our tested conditions. These evaluations can endorse risk assessments and water management in sugarcane crops surrounding areas.

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.

Bioconcentration and toxicological impacts of fipronil and 2,4-D commercial formulations (single and in mixture) in the tropical fish, Danio rerio.

The insecticide fipronil and the herbicide 2,4-D are the most applied pesticides in sugarcane crops leading to aquatic contamination. The whole-body bioconcentration of fipronil and 2,4-D, single and in mixture, was evaluated in Danio rerio after 96-h exposure. The activities of catalase (CAT) and glutathione S-transferase(GST) in whole body and in the gills and the acetylcholinesterase (AChE) in muscle were determined. The gill histopathology and the morphology of the pavement (PVC) and the mitochondria-rich(MRC) cells at gill surface were analyzed. Bioconcentration occurred after exposure to fipronil (2.69 L kg-1) and 2,4-D (1.73 L kg-1) single and in mixture of fipronil (3.10 L kg-1) and 2,4-D (1.27 L kg-1). Whole-body CAT activity was unchanged, and its activity decreased in the gills after exposure to fipronil and increased after exposure to 2,4-D and mixture. GST and AChE increased after single exposure to each pesticide and mixture of both. Fish exposed to mixture increased the MRC fractional area (MRCFA) which suggested possible ionic regulation disturbance and reduced the microridge of the PVC surface. Synergistic interactions occurred in the CAT activity and MRCFA after exposure to mixture of pesticides. The results indicate that the recommended application dose of fipronil and 2,4-D, single or in mixture, for sugarcane crops affects this fish species altering its homeostasis.

Whole-body bioconcentration and biochemical and morphological responses of gills of the neotropical fish Prochilodus lineatus exposed to 2,4-dichlorophenoxyacetic acid or fipronil individually or in a mixture.

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and the insecticide fipronil have been used widely in agriculture and detected in aquatic ecosystems, where they threaten wildlife. This study evaluated the whole-body bioconcentration and the biochemical and morphological changes in the gills of the neotropical fish Prochilodus lineatus exposed for 96 h to 2,4-D or fipronil as single compounds or as a mixture (2,4-D + fipronil). Fish exposed to either compound alone bioconcentrated 2,4-D (77 ± 23 ng g - 1 fish dry mass) and fipronil (789 ± 178 ng g - 1 fish dry mass). Fish exposed to 2,4-D + fipronil bioconcentrated fipronil (683 ± 73 ng g - 1 fish dry mass) but not 2,4-D. In the gills, catalase (CAT) and glutathione-S-transferase (GST) activities and the lipid peroxidation (LPO) level increased after exposure to 2,4-D. GST activity increased after exposure to fipronil. Conversely, no changes occurred in CAT and GST activities and LPO upon exposure to 2,4-D + fipronil. Histopathological changes such as hyperplasia, cellular hypertrophy, epithelial lifting, and vascular congestion were frequent in the gills of fish exposed to 2,4-D or fipronil individually or 2,4-D + fipronil. The mitochondria-rich cell (MRC) density increased on gill surface in fish exposed to fipronil or 2,4-D + fipronil. Only exposure to 2,4-D alone induced oxidative stress in the gills. Most morphological changes showed defense responses against the pesticides; however, hypertrophy and the change in MRC indicated compensatory responses to maintain the gill osmoregulatory function. The 2,4-D + fipronil mixture showed antagonistic interaction, except for the MRC fractional area at gill surface, which showed synergistic interaction. This is the first report showing antagonistic interaction of 2,4-D and fipronil in the gills after exposing fish to the mixture of both pesticides. The biochemical and morphological changes in gills endanger the gill functions, a phenomenon that implies an energy cost for fish.

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.

Life-History Traits Response to Effects of Fish Predation (Kairomones), Fipronil and 2,4-D on Neotropical Cladoceran Ceriodaphnia silvestrii.

Aquatic environments are constantly exposed to a cocktail of contaminants mainly due to human activities. As polluted ecosystems may simultaneously present other multiple natural stressors, the objective of the present study was to evaluate joint effect of stressors (natural and anthropogenic) on life history traits of the Neotropical cladoceran, Ceriodaphnia silvestrii. For this purpose, the effects of water conditioned with predator kairomones (fish) and environmental concentrations (sublethal) of two pesticides widely used in sugarcane monoculture in Brazil, the insecticide Regent® 800 WG (active ingredient-a.i. fipronil) and the herbicide DMA® 806 BR (a.i. 2,4-D) were evaluated using chronic toxicity testing, isolated and in mixture, for this cladoceran species. The environmental risks of pesticides for tropical freshwater biota were also estimated from the risk quotient MEC/PNEC. Among the characteristics of the life history of C. silvestrii evaluated after 8 days of exposure, compared with the mean value of control, the age of primiparous females was not affected by any evaluated treatment. However, species average survival decreased in the treatment of kairomones mixed with fipronil (FK) and in the treatment with a mixture of fipronil, 2,4-D, and kairomones (MFKD). The body length of maternal females was shorter than in the control after exposure in treatments with only kairomones (K) and FK. Fecundity of this cladoceran was reduced when exposed to FK and MFKD treatments, and the intrinsic rate of population increase significantly decreased for organisms exposed to treatment with fipronil (F) and to mixtures of fipronil and 2,4-D (MFD), MFDK, and FK. The results indicated that the combination of anthropogenic and natural stressors causes changes in C. silvestrii life history traits, which can contribute to the decline in populations, and our preliminary risk assessment results are a matter of concern regarding biota conservation.

Exposure to environmental concentrations of fipronil and 2,4-D mixtures causes physiological, morphological and biochemical changes in Raphidocelis subcapitata.

The occurrence of pesticides and their mixtures in the environment can alter the ecological relationships between aquatic food chains. Since fipronil and 2,4-dichlorophenoxyacetic acid (2,4-D) are commonly found together in Brazilian water bodies, the present study aimed to investigate through an integrative approach the toxicity mechanisms of environmentally relevant concentrations of pesticides Regent® 800 WG (active ingredient - a.i. fipronil), DMA® 806 BR (a.i. 2,4-D) isolated and in mixtures on the green alga Raphidocelis subcapitata using multiple parameters: physiological (growth rate and chlorophyll a fluorescence), morphological (cell complexity and size), biochemical (composition of lipid classes) and related to the photosynthetic activity (variable fluorescence, the maximum quantum yield of the photosystem II - PSII - and the efficiency of the oxygen evolving complex - OEC - of PSII). The results indicated that fipronil significantly inhibited algal population growth, increased the chlorophyll a content (observed by fluorescence), cell size and lipid class content of triacylglycerol (TAG), free fatty acid (FFA) and acetone mobile polar lipid (AMPL) and, on the other hand, decreased variable fluorescence of algae. The tested concentrations of 2,4-D increased the chlorophyll a fluorescence, the cell size and the lipid classes TAG and FFA. The pesticide mixtures have had more effects on algae than isolated compounds, causing alterations in all parameters analyzed, including photosynthetic activity (maximum quantum yield and efficiency of the oxygen evolving complex of the PSII), in which no alterations were observed for the toxicity of the single pesticides. The results suggest that these analyses are important to evaluate pesticide toxicity mechanisms in ecological risk assessments of tropical regions. Thus, here we demonstrate the importance of using multiple parameters in ecotoxicological studies to obtain a better understanding of the toxicity of these compounds for phytoplankton.