Effects Of Pesticide Mixtures Research Articles

Synergistic toxic effects and mechanistic insights of beta-cypermethrin and pyraclostrobin exposure on hook snout carp (Opsariichthys bidens): A biochemical, transcriptional, and molecular approach

The extensive utilization of pesticides results in their frequent detection in aquatic environments, often as complex mixtures, posing risks to aquatic organisms. The hook snout carp (Opsariichthys bidens) serves as a valuable bioindicator for evaluating the impacts of environmental pollutants in aquatic ecosystems. However, few studies examined the toxic effects of pesticides on O.bidens, let alone the characterization of the combined effects resulting from their mixtures. This study aims to elucidate the toxic effects of beta-cypermethrin and pyraclostrobin on O.bidens, individually and in combination, focusing on biochemical, transcriptional, and molecular responses. By organizing and analyzing the toxicogenomic databases, both pesticides were identified as a contributor to processes such as apoptosis, oxidative stress, and inflammatory responses. The acute toxicity test revealed comparable acute toxicity of beta-cypermethrin and pyraclostrobin on O.bidens, with LC50 being 0.019 and 0.027 mg/L, respectively, whereas the LC50 decreased to 0.0057 and 0.0079 mg/L under the combined exposure, indicating potential synergistic effects. The activities of enzymes involved in oxidative stress and detoxification were significantly altered after exposure, with superoxide dismutase (SOD) and catalase (CAT) increasing, while malondialdehyde (MDA) levels decreased. The activity of CYP450s was significantly changed. Likewise, the expression levels of genes (mn-sod, p53, esr, il-8) associated with oxidative stress, apoptosis, endocrine and immune systems were significantly increased. Combined exposure to the pesticides significantly exacerbated the aforementioned biological processes in O.bidens. Furthermore, both pesticides can modify protein activity by binding to the surface of SOD molecules and altering protein conformation, contributing to the elevated enzyme activity. Through the investigation of the synergistic toxic effects of pesticides and molecular mechanisms in O.bidens, our findings highlight the importance of assessing the combined effects of pesticide mixtures in aquatic environments.

Assessing the impact of imidacloprid, glyphosate, and their mixtures on multiple biomarkers in Corbicula largillierti

Pesticide mixtures are frequently utilized in agriculture, yet their cumulative effects on aquatic organisms remain poorly understood. Aquatic animals can be effective bioindicators and invasive bivalves, owing to their widespread distribution, provide an opportunity to assess these impacts. Glyphosate and imidacloprid, among the most prevalent pesticides globally, are frequently detected in freshwater systems in South America. This study aims to understand the cumulative effects of pesticide mixtures on aquatic organisms, using invasive Corbicula largillierti clams from a natural stream in northwestern Argentina. We conducted 48-hour exposure experiments using two concentrations of imidacloprid (20 and 200 μg L−1 a.i), two concentrations of glyphosate (0.3 and 3 mg L−1 a.i), and two combinations of these pesticides (both at low and high concentrations, respectively), simulating the direct contamination of both pesticides based on their agronomic recipe and observed values in Argentine aquatic environments. Clam metabolism was assessed through the examination of multiple oxidative stress parameters and measuring oxygen consumption rate as a proxy for standard metabolic rate (SMR). Our findings revealed that imidacloprid has a more pronounced effect compared to glyphosate. Imidacloprid significantly decreased clam SMR and cellular levels of reduced glutathione (GSH). However, when both pesticides were present, also cellular glycogen and thiobarbituric acid-reactive substances (TBARS) were affected. Proteins and glutathione S-Transferase (GST) activity were unaffected by either pesticide or their mixture at the assayed concentrations, highlighting the need to test several stress parameters to detect toxicological impacts. Our results indicated additive effects of imidacloprid and glyphosate across all measured parameters. The combination of multiple physiological and cytological biomarkers in invasive bivalves offers significant potential to enhance biomonitoring sensitivity and obtain insights into the origins and cellular mechanisms of chemical impacts. These studies can improve pollution regulatory policies and pesticide management.

Joint toxic mechanism of clothianidin and prochloraz in the earthworm (Eisenia fetida)

Pesticides are typically present as combinations within soil ecosystems and have detrimental effects on untamed surroundings. However, the collective impacts and fundamental mechanisms of pesticides on soil living beings are currently inadequately assessed. In our current work, we evaluated the interactive consequences of clothianidin (CLO) and prochloraz (PRO) on earthworms (Eisenia fetida) using several toxicological tests, such as acute adverse effects, biocatalytic activity, and alterations in transcriptional activity. The findings revealed that CLO (with a 14-day LC50 value of 6.08 mg kg−1) exhibited greater toxicity compared to PRO (with a 14-day LC50 value of 79.41 mg kg−1). Moreover, the combinations of CLO and PRO had synergistic acute effects on E. fetida. Additionally, the activities of POD, CAT, and GST were significantly varied in most instances of single and mixed treatments when compared to the control. Surprisingly, the transcriptional levels of four genes (gst, sod, crt, and ann), related to oxidative load, metabolic detoxification systems, endoplasmic reticulum, and oxytocin neuropeptide, respectively, were also altered in response to single and mixture exposures, as compared to the control. Alterations in enzyme activity and gene transcriptional level could serve as early indicators for detecting co-exposure to pesticides. The findings of this research offered valuable holistic understanding regarding the toxicity of pesticide combinations on earthworms. Further research should be conducted to investigate the persistent effects of pesticide mixtures on terrestrial invertebrates in order to draw definitive conclusions about the associated risks.

An adverse outcome pathway-based approach to assess the neurotoxicity by combined exposure to current-use pesticides

Exposure to multiple pesticides in daily life has become an important public health concern. However, the combined effects of pesticide mixtures have not been fully elucidated by the conventional toxicological testing used for individual chemicals. Grouping of chemicals by mode of action using common key events (KEs) in the adverse outcome pathway (AOP) as endpoints could be applied for efficient risk assessment of combined exposure to multiple chemicals. The purpose of this study was to investigate whether exposure to multiple pesticides has synergistic neurotoxic effects on mammalian nervous systems. According to the AOP-based approach, we evaluated the effects of 10 current-use pesticides (4 neonicotinoids, 4 pyrethroids and 2 phenylpyrazoles) on the common KEs in AOPs for neurotoxicity, such as KEs involving mitochondrial and proteolytic functions, in a mammalian neuronal cell model. Our data showed that several pyrethroids and phenylpyrazoles partly shared the effects on several common KEs, including decreases in mitochondrial membrane potential and proteasome activity and increases in autophagy activity. Furthermore, we also found that combined exposure to a type-I pyrethroid permethrin or a type-II pyrethroid deltamethrin and the phenylpyrazole fipronil decreased the cell viability and the benchmark doses much more than either single exposure, indicating that the pair exhibited synergistic effects, since the combination indexes were less than 1. These findings revealed that novel pairs of different classes of pesticides with similar effects on common KEs exhibited synergistic neurotoxicity and provide new insights into the risk assessment of combined exposure to multiple chemicals.

Synergistic effects on oxidative stress, apoptosis and necrosis resulting from combined toxicity of three commonly used pesticides on HepG2 cells

The widespread use of pesticides performs a vital role in safeguarding crop yields and quality, providing the opportunity for multiple pesticides to co-exist, which poses a significant potential risk to human health. To assess the toxic effects caused by exposures to individual pesticides (chlorpyrifos, carbofuran and acetamiprid), binary combinations and ternary combinations, individual and combined exposure models were developed using HepG2 cells and the types of combined effects of pesticide mixtures were assessed using concentration addition (CA), independent action (IA) and combination index (CI) models, respectively, and the expression of biomarkers related to oxidative stress, apoptosis and cell necrosis was further examined. Our results showed that both individual pesticides and mixtures exerted toxic effects on HepG2 cells. The CI model indicated that the toxic effects of pesticide mixtures exhibited synergistic effects. The results of the lactate dehydrogenase (LDH) release and apoptosis assay revealed that the pesticide mixture increased the release of LDH and apoptosis levels. Moreover, our results also showed that individual pesticides and mixtures disrupted redox homeostasis and that pesticide mixtures produced more intense oxidative stress effects. In conclusion, we have illustrated the enhanced combined toxicity of pesticide mixtures by in-vitro experiments, which provides a theoretical basis and scientific basis for further toxicological studies.

Evaluating the Impact of Individual and Combined Toxicity of Imidacloprid, Cycloxaprid, and Tebuconazole on Daphnia magna.

The risks posed by chemicals in the environment are typically assessed on a substance-by-substance basis, often neglecting the effects of mixtures. This may lead to an underestimation of the actual risk. In our study, we investigated the effects of three commonly used pesticides-imidacloprid (IMI), cycloxaprid (CYC), and tebuconazole (TBZ)-both individually and in combination, using various biomarkers to assess their impact on daphnia. Our findings indicated that the order of toxicity, from highest to lowest, was TBZ, IMI, and CYC, as determined by acute toxicity as well as reproduction. The effects of the ITmix (IMI and TBZ) and CTmix (CYC and TBZ) combinations on immobilization and reproduction were evaluated by MIXTOX, revealing a higher risk of immobilization at low concentrations for ITmix. The effect on reproduction differed depending on the ration of pesticides in the mixture, with synergism observed, which may be caused mainly by IMI. However, CTmix showed antagonism for acute toxicity, with the effect on reproduction depending upon the composition of the mixture. The response surface also exhibited a switch between antagonism and synergism. Additionally, the pesticides extended the body length and inhibited the development period. The activities of superoxide dismutase (SOD) and catalase (CAT) content was also significantly induced at different dosage points in both the single and combination groups, indicating changes in the metabolic capabilities of detoxifying enzymes and target site sensitivity. These findings highlight the need for more attention to be focused on the effects of pesticide mixtures.

Mixture effects of thiamethoxam and seven pesticides with different modes of action on honey bees (Aplis mellifera)

Even though honey bees in the field are routinely exposed to a complex mixture of many different agrochemicals, few studies have surveyed toxic effects of pesticide mixtures on bees. To elucidate the interactive actions of pesticides on crop pollinators, we determined the individual and joint toxicities of thiamethoxam (THI) and other seven pesticides [dimethoate (DIM), methomyl (MET), zeta-cypermethrin (ZCY), cyfluthrin (CYF), permethrin (PER), esfenvalerate (ESF) and tetraconazole (TET)] to honey bees (Aplis mellifera) with feeding toxicity test. Results from the 7-days toxicity test implied that THI elicited the highest toxicity with a LC50 data of 0.25 (0.20–0.29) μg mL−1, followed by MET and DIM with LC50 data of 4.19 (3.58–4.88) and 5.30 (4.65–6.03) μg mL−1, respectively. By comparison, pyrethroids and TET possessed relatively low toxicities with their LC50 data from the range of 33.78 (29.12–38.39) to 1125 (922.4–1,442) μg mL−1. Among 98 evaluated THI-containing binary to octonary mixtures, 29.59% of combinations exhibited synergistic effects. In contrast, 18.37% of combinations exhibited antagonistic effects on A. mellifera. Moreover, 54.8% pesticide combinations incorporating THI and TET displayed synergistic toxicities to the insects. Our findings emphasized that the coexistence of several pesticides might induce enhanced toxicity to honey bees. Overall, our results afforded worthful toxicological information on the combined actions of neonicotinoids and current-use pesticides on honey bees, which could accelerate farther comprehend on the possible detriments of other pesticide mixtures in agro-environment.

Effect of temperature and dietary pesticide exposure on neuroendocrine and olfactory responses in juvenile Chinook salmon (Oncorhynchus tshawytscha)

Projected water temperature increases based on predicted climate change scenarios and concomitant pesticide exposure raises concern about the responses of aquatic organisms. To better understand the effect of pesticide mixtures and influence of water temperature to fish, juvenile Chinook salmon (Oncorhynchus tshawytscha) were dietarily exposed to a mixture of legacy and current use pesticides (p,p’-DDE, bifenthrin, chlorpyrifos, esfenvalerate, and fipronil) at concentrations detected from field-collected prey items in the Sacramento-San Joaquin Delta, California (Delta) and exposed under current and predicted future water temperature scenarios, 11, 14, or 17 °C, for 14 days. The expression of a subset of genes (deiodinase 2-dio2, gonadotropin releasing hormone 2-gnrh2, and catechol-o-methyltransferase-comt) involved in neuroendocrine, dopaminergic, and olfactory function previously shown to be altered by individual pesticide exposures germane to this study were determined and olfactory function assessed using a Y-maze behavioral assay. When total body burdens of pesticides were measured, a significant decrease in dio2 expression was observed in Chinook salmon exposed at 14 °C compared to fish kept at 11 °C. Increases in gnrh2 expression were also observed in fish exposed to 14 °C. Similarly, increases in comt expression was noted at 14 and 17 °C. Additionally, altered expression of all transcripts was observed, showing interactions between temperature and individual pesticide concentrations. Chinook salmon spent significantly more time actively avoiding the odorant arm at baseline conditions of 11 °C in the Y-maze. At higher temperatures, Chinook spent significantly more time not making a choice between the odorant or clean arm following exposure to the low pesticide mixture, relative to 11 °C. These results suggest that dietary exposure to pesticide mixtures can potentially induce neuroendocrine effects and behavior. Impaired olfactory responses exhibited by Chinook salmon could have implications for predator avoidance in the wild under increased temperature scenarios and impact populations in the future.

Biomarker development for neonicotinoid exposure in soil under interaction with the synergist piperonyl butoxide in Folsomia candida

Pesticide toxicity is typically assessed by exposing model organisms to individual compounds and measuring effects on survival and reproduction. These tests are time-consuming, labor-intensive, and do not accurately capture the effect of pesticide mixtures. Moreover, it is unfeasible to screen the nearly infinite combinations of mixtures for synergistic effects on model organisms. Therefore, reliable molecular indicators of pesticide exposure have to be identified, i.e., biomarkers. These biomarkers can form the basis of rapid and economical screening procedures to assess the toxicity of pesticides even under synergistic interaction with other pollutants. In this study, we screened the expression patterns of eight genes for suitability as a biomarker for neonicotinoid exposure in the soil ecotoxicological model Folsomia candida (springtails). Springtails were exposed to the neonicotinoids imidacloprid and thiacloprid either alone or with various levels of piperonyl butoxide (PBO), which inhibits cytochrome P450 enzymes (CYPs): a common point of synergistic interaction between neonicotinoid and other pesticides. First, we confirmed PBO as a potency enhancer for neonicotinoid toxicity to springtail fecundity, and then used it as a tool to confirm biomarker robustness. We identified two genes that are reliably indicative for neonicotinoid exposure even under metabolic inhibition of CYPs by PBO, nicotinic acetylcholine receptor–subunit alpha 1 (nAchR) and sodium-coupled monocarboxylate transporter (SMCT). These results can form the basis for developing high-throughput screening procedures for neonicotinoid exposure in varying mixture compositions.

The acute toxicity of pesticide mixtures to honeybees.

Honeybees (Apis mellifera) frequently live in complex environments where exposure to mixtures of pesticides is possible. Although several studies have expressed concern regarding the combined effects of pesticide mixtures, other studies did not find increased toxicity. Thus, the primary objective of this study was to identify peer-reviewed literature measuring the toxicity of pesticide mixtures to honeybees and determine how frequently synergistic interactions occur. Many experiments (258) were identified that met the criteria for inclusion. When considering all experiments, 34% of experiments had model deviation ratios (MDR; expected toxicity/observed toxicity) greater than 2, suggesting greater-than-additive toxicity. Twelve percent of experiments had MDR values greater than 5, with several studies exceeding 100. However, most experiments that had higher MDRs included azole fungicides or acaricides as a component of the mixture. After removal of these groups, only 8% of experiments exceeded an MDR of 2, and no experiments exceeded 5. Moreover, the influence of the azole fungicides was dose dependent. If only experiments that used azole exposure at environmentally relevant concentrations were considered, azole fungicides had limited impact on neonicotinoid insecticides. However, pyrethroid insecticides still had greater than expected toxicity with 80% of experiments having MDR values greater than 2. Acaricides also had greater than expected incidence of synergy with approximately 30% of studies reporting MDR values greater than 2. It should be noted that even the azole studies considered environmentally relevant frequently used maximum exposure rates and worst-case exposure scenarios. The primary finding is that synergy is uncommon except for a few cases where known synergists (azole fungicides) and pesticides with variable metabolism potential, such as some pyrethroids, are in combination. Future work is still needed to refine the relevance of azole fungicides at commonly occurring environmental concentrations. Integr Environ Assess Manag 2022;18:1694-1704. © 2022 SETAC.

Гігієнічне регламентування безпечного застосування комбінованих пестицидів в системі захисту яблуневих насаджень від шкідників, хвороб і бур’янів

Background: The quality and safety of agricultural product, grown with the application of chemical plant protection products, is an urgent problem of preventive medicine. The occurrence of multiple pesticide residues in the foodstuffs is of concern to the general public and is engaging scientists in the search for approaches to the assessment and control of multi-residues. Objective: We analyzed a range of the active substances of combined pesticides used in Ukraine in modern apple orchard protection systems, characterized a hazard and assessed the combined exposure to several pesticides at their joint ingress with apples into the human body. Materials and methods: In the work, we applied the methods of analytical research of the range of active substances of pesticides, registered and used in Ukraine in modern systems of apple orchard protection, by functional purpose (herbicides, insecticides, acaricides, fungicides); used the normative documents of national legislation on hygienic regulation of pesticides and methodological approaches to the assessment of the combined effects of pesticide mixtures in the foodstuffs. Results: According to the results of the analysis of the residues of active substances of pesticides in apples, the conclusion about the low level of short-term (acute) risk for health consumers has been made. However, it is impossible to ignore a presence of many pesticides with a similar mechanism of biological action in food, and hence, a possibility of their additive or synergistic effect on health. Conclusions: Further research should be aimed at assessing the exposure and identifying the health hazards of the consumers from the combined effect of pesticides, applied for vegetables and cereals, by established groups of substances with a general effect mode / mechanism.

A targeted transcriptomics approach for the determination of mixture effects of pesticides

While real-life exposure occurs to complex chemical mixtures, toxicological risk assessment mostly focuses on individual compounds. There is an increasing demand for in vitro tools and strategies for mixture toxicity analysis. Based on a previously established set of hepatotoxicity marker genes, we analyzed mixture effects of non-cytotoxic concentrations of different pesticides in exposure-relevant binary mixtures in human HepaRG hepatocarcinoma cells using targeted transcriptomics. An approach for mixture analysis at the level of a complex endpoint such as a transcript pattern is presented, including mixture design based on relative transcriptomic potencies and similarities. From a mechanistic point of view, goal of the study was to evaluate combinations of chemicals with varying degrees of similarity in order to determine whether differences in mechanisms of action lead to different mixtures effects. Using a model deviation ratio-based approach for assessing mixture effects, it was revealed that most data points are consistent with the assumption of dose addition. A tendency for synergistic effects was only observed at high concentrations of some combinations of the test compounds azoxystrobin, cyproconazole, difenoconazole, propiconazole and thiacloprid, which may not be representative of human real-life exposure. In summary, the findings of our study suggest that, for the pesticide mixtures investigated, risk assessment based on the general assumption of dose addition can be considered sufficiently protective for consumers. The way of data analysis presented in this paper can pave the way for a more comprehensive use of multi-gene expression data in experimental studies related to mixture toxicity.

Pesticide mixture effects on liver protein abundance in HepaRG cells

Toxicological effects of chemicals are mostly tested individually. However, consumers encounter exposure to complex mixtures, for example multiple pesticide residues, by consuming food such as crops, fruits or vegetables. Currently, more than 450 active substances are approved in the European Union, and there is little data on effects after combined exposure to several pesticides. Toxicological animal studies would increase enormously, if pesticide combinations had to be analyzed in vivo. Therefore, in vitro methods addressing this issue are needed. We have developed 32 immunoaffinity-based mass spectrometry assays to investigate the impact of hepatotoxic active substances on liver proteins in human HepaRG cells. Five compounds were selected based on their (dis)similar capability to modulate protein levels, and on their combined use in commercially available formulations. Four binary mixtures were prepared from these five substances and tested in different concentrations over three time points. We applied a novel statistical method to describe deviations from additivity and to detect antagonistic and synergistic effects. The results regarding the abundance of hepatotoxicity-related proteins showed additive behavior for 1323 out of 1427 endpoints tested, while 104 combinatorial effects deviating from additivity, such as antagonism or synergism were observed.

Effects of pesticide mixtures on xenobiotic efflux transporter P‐glycoprotein in the European Honeybee

About 35% of the world's food crops depend on insect pollinators like the European Honeybee, Apis mellifera, to reproduce. The sudden loss of a colony's worker bee population, often referred to as colony collapse disorder (CCD), has been linked to certain crop pesticide exposures. Multidrug resistance (MDR) transporters of the ATP-binding Cassette (ABC) family, such as P-glycoprotein, are the first line of defense in biological barriers, including the intestine and brain, and a key determinants of xenobiotic uptake in all organisms. Insect homologs of P-glycoprotein (P-gp) have a major function in developing resistance against broad-spectrum insecticides. However, the effects of pesticide mixtures on the protective efflux function of insect P-glycoprotein are not known, particularly mixtures of agricultural and in-hive pesticides. In this study, we cloned two variants of honeybee P-glycoprotein and quantified relative P-gp expression in head, thorax and abdomen using the C219 antibody. Honeybee P-gp was primarily detected in head and thorax that contain the brain and ganglion, indicating a key function in protecting the bee brain towards neurotoxic pesticides. To further explore the effects of pesticide exposure on bee P-gp function, we will express both P-gp variants in insect cells and determine their molecular interactions with selected pesticides using an ATPase activity assay. Purified bee P-gp will be used to determine the interactions of 15 current-use agricultural pesticides (see table 1) for the top ten bee-pollinated crops in California, including sunflowers, almonds, and avocados. We will categorize pesticide interactions into P-gp substrates (ATPase stimulation), inhibitors (ATPase inhibition), and non-interactors that do not interfere with ATPase activity. We hypothesize that binary combinations of inhibitory pesticides or pesticide substrates with competing binding sites can promote synergistic P-gp inhibition and ultimately lead to pesticide accumulation in the honeybee nervous system and subsequent neurotoxic effects. Pesticide combinations that show synergistic or antagonistic effects in the in vitro bee P-gpATPase activity assay will be subjected to an in vivo behavioral (navigation, coordination) and mortality study using live honeybees. The findings of this project will provide critical baseline data for future development of pollinator-safe pesticides and inform good agricultural practices.

Synergy Assessment for Plant Growth by Independent Joint Action Theory

Calculating the predicted biological efficacy of a mixture and determining the significance of the difference between the predicted efficacy and the measured efficacy of that mixture are fundamental when assessing the synergy of mixtures. The Independent Joint Action theory and Bliss’s formula are well-known and widely accepted for predicting pesticide mixture effects that are expressed in terms of percent mortality. Bliss’s formula, however, is not applicable to growth-affecting components, such as plant growth regulators. Therefore, there is an unmet need of critical importance: an appropriate method for assessing synergy of growth-affecting mixtures needs to be identified within the scientific community. The formula, G(1 + 2..n) = G1G2…Gn/(GCTL)n−1, which was derived from the Independent Joint Action theory, is presented for calculating the predicted efficacy for mixtures of growth-affecting components that either promote or inhibit growth. Its application is demonstrated by analyzing data from a greenhouse assay in which a mixture of S-abscisic acid and gibberellic acid was used to promote the growth of corn seedlings.

How to use statistics to claim antagonism and synergism from binary mixture experiments.

We review statistical approaches applicable for the analysis of data from binary mixture experiments, which are commonly used in pesticide science for evaluating antagonistic or synergistic effects. Specifically, two different situations are reviewed, one where every pesticide is only available at a single dose level and a mixture simply combines these doses, and one where the pesticides and their mixture are used at increasing doses. The former corresponds to using factorial designs whereas the latter corresponds to fixed-ratio designs. We consider dose addition and independent action as references for lack of antagonistic and synergistic effects. Data from factorial designs should be analyzed using two-way analysis of variance models whereas data from fixed-ratio designs should be analyzed using non-linear dose-response analysis. In most cases, independent action seems the more natural choice for factorial designs. In contrast, dose addition is more appropriate for fixed-ratio designs although dose addition is not equally compatible with all types of dose-response data. Fixed-ratio designs should be preferred as they allow validation of the assumed dose-response relationship and, consequently, provide much stronger claims about antagonistic and synergistic effects than factorial designs. Finally, it should be noted that, in any case, simple ways of summarizing pesticide mixture effects may come at the price of more or less restrictive modeling assumptions. © 2021 Society of Chemical Industry.

Synergy between glyphosate and cypermethrin formulations on zooplankton: evidences from a single-specie test and a community mesocosm experiment.

Agrochemicals can reach freshwater bodies by drift, leaching, or runoff, where they constitute complex mixtures. Given that glyphosate and cypermethrin are within the most worldwide used pesticides, they are likely to co-occur in freshwater bodies. The aim of this study was to analyze the interaction between glyphosate and cypermethrin formulations on the cladoceran Ceriodaphnia dubia (Richard 1894)through an acute toxicity test and on a zooplankton assemblage through a mesocosm (30 L) experiment. The 24-h LC50 of both isolated pesticides and their equitoxic mixture was obtained for C. dubia. The mesocosm was performed by exposing a zooplankton assemblage to both pesticides isolated and in combination. The acute toxicity of the equitoxic mixture in C. dubia was 3 and 4 times higher than the isolated toxicity of glyphosate and cypermethrin, respectively. The total toxic units of the mixture were 0.53, indicating a synergistic interaction. In the mesocosm experiment, both pesticides also interacted causing a synergistic negative effect in Cladocera and Copepoda abundances. No interactions between pesticides were found for Rotifera; therefore, the mixture effect was considered additive. It is suggested to continue analyzing pesticide mixture effects on the basis of complementary scales of analysis to reach more environmentally relevant information.

Neurological effects of long-term exposure to low doses of pesticides mixtures in male rats: Biochemical, histological, and neurobehavioral evaluations

Humans are usually exposed to multiple pesticides in real life, but little is known as yet about the safety of low-dose pesticides mixtures. This study was conducted to evaluate the effects of long-term exposure to very low doses of pesticide mixtures on biochemical, histological, and neurobehavioral alterations in the rat model. For 90 days, four groups of male Wistar rats were given a mixture of five pesticides (in drinking water) in doses of 0, 0.25, 1 and 5 times the legally permitted levels (mg/kg body weight/day). After three-month exposure, the neurobehavioral effects of pesticide mixtures were evaluated by the Morris water maze, elevated plus maze and the open field tests. Then the biochemical and histopathological alterations in the hippocampus of studied animals were evaluated. Results showed that long-term exposure to a combination of five pesticides affected the nervous system in dose-dependent manner. As expected, nearly all of the parameters determined in this study were adversely changed in the high dose group. Exposure to medium dose (permitted level of pesticides mixture) was also able to induce oxidative stress and impaired memory and learning ability, although not all parameters were significantly changed in this group. It means that pesticides may behave differently when mixed. Interestingly, the administration of low doses of these chemicals induced an adaptive response by stimulating the redox system. In conclusion, it seems that the prolonged exposure to pesticide mixtures may cause adverse neurobehavioral effects, even at permitted levels.

Synergistic effect of fenpropathrin and paclobutrazol on early life stages of zebrafish (Danio rerio)

Aquatic organisms are usually exposed to various co-existing pollutants. However, toxic effects of pesticide mixtures on aquatic organisms and its potential underlying mechanism still remain unclear. The joint effects of fenpropathrin (FEN) and paclobutrazol (PAC) on zebrafish (Danio rerio) using diverse toxicological endpoints were investigated in the current work. Our data exhibited that the 96-h LC50 values of FEN to zebrafish at multiple life phases ranged from 0.0029 (0.0013–0.0042) to 0.16 (0.082–0.23) mg a.i. L−1, which were lower by comparison to PAC ranging from 13.16 (8.564–21.03) to 23.43 (17.94–29.91) mg a.i. L−1. Combination of FEN and PAC displayed synergistic effect on embryonic zebrafish. Activities of T-SOD, Cu/Zn-SOD and CYP450 were remarkably changed in the majority of single and mixture treatments by comparison to the untreated group. The mRNA levels of 17 genes related to oxidative stress, cellular apoptosis, immune system and endocrine system were assessed, and the data suggested that embryonic zebrafish were affected by both single pesticides and their mixtures. Five genes (P53, tsh, ERα, crh and cxcl-clc) showed greater alterations when exposed to pesticide mixtures by comparison to their individual chemicals. Therefore, it is urgently necessary to conduct more studies on mixture toxicities of different pesticides to explore the chemical mixtures with synergistic interactions.

Pesticide Mixtures Cause Short‐Term, Reversible Effects on the Function of Autotrophic Periphyton Assemblages

In a laboratory experiment we investigated the effects of pesticide mixtures on the structure and function of freshwater biofilms, with focus on their photoautotrophic component. We identified 6 herbicides and 1 fungicide commonly found in Swedish streams at relatively high concentrations and created 3 ternary mixtures that were tested in concentration series ranging from observed environmental concentrations to up to 100 times higher. Biofilms were exposed to these pesticide mixtures for 8 d and then allowed to recover for another 12 d. Our results show a rapid and consistent inhibition of photosynthesis after just 24-h exposure to the highest test concentration of pesticides, as well as in some treatments with lower concentrations (i.e., 10 times the environmental level), on exposure. Interestingly, the observed effects were reversible because biofilm photosynthesis recovered rapidly and completely in clean media in all but one treatment. In contrast to the functional response, no effects were observed on the algal assemblage structure, as assessed by diagnostic pigments. We conclude that the pesticide mixtures induce a rapid but reversible inhibition of photosynthesis, without short-term effects on biofilm structure. Environ Toxicol Chem 2020;39:1367-1374. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.