Behavioral and physiological changes in the passerine Agelaioides badius following the ingestion of coated seeds with imidacloprid in a 30-day experiment.

The wide use of neonicotinoid seed treatment represents a hazard for farmland birds that feed on treated seeds. This study aimed to characterize the long-term effects of the neonicotinoid imidacloprid (IMI) in the passerine grayish baywing (Agelaioides badius). The birds were fed ad libitum for 32 days only with seeds treated with 53.1 (Low, 11 % of LD50) and 514 (High, (112 % of LD50) mg IMI/kg seed; these concentrations representing respectively, 1.8 and 17.1 % of 3 g IMI/kg, an average application rate used to treat crop seeds in Argentina. The effects exerted by IMI on birds were evaluated at behavioral, physiological, hematological, genotoxic, and biochemical levels. No differences in food consumption were observed between Control and Low treatments birds, indicating a lack of aversion to treated seeds. High treatment birds only decreased their food consumption by 20 % in the first 3 days of exposure. Birds from High treatment experienced an early loss of body weight, reduction in their mobility, lack of response to threats (i.e., predator call and approaching person), and altered their use of the cage. On the contrary, birds from Low treatment experienced a delay in the onset of effects like reduction in mobility, lack of response to threats, and a tendency to reduce their body weight. At the end of exposure, glutathione S transferase activity in the plasma of treated birds decreased, and cholinesterase activity increased in the liver of treated birds. This study highlights that consumption equivalent to 1.8 % of the daily diet of baywings as IMI-treated seeds, is sufficient to generate behavioral and physiological alterations and death. In the wild, these effects may have ecological consequences, by impairing the survival of birds, representing a risk to farmland bird populations.

Behavioral and physiological response of the passerine bird Agelaioides badius to seeds coated with imidacloprid.

Neonicotinoids are globally used insecticides, and there are increasing evidence on their negative effects on birds. This study is aimed at characterizing the behavioral and physiological effects of the neonicotinoid imidacloprid (IMI) in a songbird. Adults of Agelaioides badius were exposed for 7 days to non-treated peeled millet and to peeled millet treated with nominal concentrations of 75 (IMI1) and 450 (IMI2) mg IMI/kg seed. On days 2 and 6 of the trial, the behavior of each bird was evaluated for 9 min by measuring the time spent on the floor, the perch, or the feeder. Daily millet consumption, initial and final body weight, and physiological, hematological, genotoxic, and biochemical parameters at the end of exposure were also measured. Activity was greatest on the floor, followed by the perch and the feeder. On the second day, birds exposed to IMI1and IMI2 remained mostly on the perch and the feeder, respectively. On the sixth day, a transition occurred to sectors of greater activity, consistent with the disappearance of the intoxication signs: birds from IMI1 and IMI2 increased their time on the floor and the perch, respectively. Control birds always remained most of the time on the floor. IMI2 birds significantly decreased their feed intake by 31% the first 3 days, compared to the other groups, and significantly decreased their body weight at the end of the exposure. From the set of hematological, genotoxic, and biochemical parameters, treated birds exhibited an alteration of glutathione-S-transferase activity (GST) in breast muscle; the minimal effects observed are probably related to the IMI administration regime. These results highlight that the consumption of less than 10% of the bird daily diet as IMI-treated seeds trigger effects at multiple levels that can impair bird survival.

Concentration of current-use pesticides in frogs from the Pampa region and correlation of a mixture toxicity index with biological effects.

Contamination with current-use pesticides is frequently mentioned as a key factor in global amphibian declines although a limited number of studies have examined the mixture of pesticides accumulated by free-living frogs. This study examined the presence of 46 different pesticide residues in the muscle and kidney tissues of two frog species living in close association with row crops in the Pampa region of Argentina: The terrestrial Leptodactylus latinasus and the semi-aquatic Leptodactylus latrans. A total of 20 different pesticides were identified in frog tissues; chlorpyrifos-methyl, pirimiphos-methyl and acetochlor being the most frequently detected molecules. Overall, one or more pesticide residues (up to 12 in a single frog) were detected in 40-57 % of L. latrans. L. latinasus was found to present more pesticide detections than L. latrans. Interestingly, frog sampled in a natural reserve where no pesticides are applied presented an equivalent frequency of detections as frogs living near a crop. In L. latrans, the calculation of a pesticide toxicity index (PTI) permitted to highlight the existence of a strong positive correlation between PTI and liver GSH contents of females whereas, in males, PTI was negatively correlated with the perimeter of testicular seminiferous tubules. Males also presented near significant negative correlations between PTI and both body condition and the scaled fat index. These results indicate that frogs inhabiting agricultural regions are exposed to a complex and diffuse contamination by pesticide mixtures which is likely responsible for a number of biological effects that may be relevant at the population level.

Tissue distribution and sublethal effects of imidacloprid in the South American grayish baywing (Agelaioides badius).

The neonicotinoids are globally used insecticides, which have been shown to cause negative impacts on birds. The current study aimed to evaluate the distribution of the neonicotinoid imidacloprid (IMI) in the tissues of a songbird and identify related physiological effects. Adults of the grayish baywing (Agelaioides baduis) were administered with a single dose of 35 mg IMI/kg, and the IMI concentration was evaluated in liver, kidney and plasma at 4, 12, 24, and 48 h after dosing. At the same time points, effects on hematological, genetic and enzymatic parameters were assessed. Results showed that IMI was absorbed before 4 h, and eliminated at 48 h, in every tissue, and the highest concentrations were detected in plasma. Baywings showed intoxication signs and reduced mobility within the first 5 min post-dosing. Hematological parameters: red blood cells, packed cell volume, hemoglobin, and their derived indices exhibited a transient elevation 24 h after dosing, which coincided with maximum concentrations of IMI in the tissues. No effects were observed on the genotoxicity parameters evaluated: micronuclei and comet assay. Treated birds exhibited an alteration of cholinesterases activity in the muscle and plasma, and of glutathione-S-transferase (GST) activity in the plasma, brain, liver, and muscle. Based on the results obtained, the combined detection of IMI and inhibition of GST activity in the plasma is suggested as a non-lethal biomarker of IMI exposure in wild birds. As efficient field monitoring depends on the availability of proven biomarkers, the current study provides valuable tools for bird conservation in agroecosystems.

Imidacloprid seed coating poses a risk of acute toxicity to small farmland birds: A weight-of-evidence analysis using data from the grayish baywing Agelaioides badius.

The aim of this study was to estimate the risk posed by imidacloprid (IMI) seed coating to passerine birds of the Pampa Region of Argentina using data specifically generated with the grayish baywing (Agelaioides badius). Median lethal dose (LD50) of the IMI-based formulation tested was 57.11 mg IMI/kg body weight (bw), with intoxication signs starting from 20.6 mg IMI/kg bw. The feed intake rate (FIR) was estimated experimentally as 4.895 g/day per bird, representing 12.43% of bw. It was calculated that the ingestion of 7-10% of the FIR as treated seeds would be enough to achieve the LD50 for sorghum, corn, sunflower, and alfalfa, whereas consumption of 31 and 54% of FIR was necessary for oat and wheat, respectively. Based on spill data values available in the literature, it was calculated that, for most crops, a baywing would have to forage an area of field corresponding to less than 60 m2 to obtain the number of seeds required to reach the LD50. It was also shown that this number of seeds is coherent with the amount of seeds ingested in a bout. In a pilot study, all grayish baywings fed with millet seeds treated with 3 g IMI/kg died within three to five days of exposure. In Tier I risk assessment, the trigger value was achieved for all crops except soybean and a weight-of-evidence risk assessment was performed. All lines of evidence examined are consistent with the view that grayish baywings, and probably other small farmland birds, are exposed to a risk of acute toxicity and mortality under both worst-case and mixed-ration exposure scenarios. The possible impacts on bird species calls for an urgent reconsideration of IMI seed coating practices currently approved in the Pampa Region of Argentina and the various parts of the world where this practice is still in use.

Biomarker and hematological fieldwork with amphibians: is it necessary to sample all night?

In the context of the global amphibian crisis, biomonitoring constitutes a valuable assessment tool to provide critical up to date information on the status and health of amphibians worldwide. The aim of the current study was to evaluate the possible confounding effects of sex, size, and time since capture on enzymatic biomarkers and hematologic parameters of the South American frog Leptodactylus latrans. Frogs were collected by hand between 9 pm and 12 am on two consecutive nights. On the first night, captured frogs were transported for 2 h by car to laboratory installations, maintained overnight in plastic containers, and blood and tissue sampled on the next morning. In contrast, frogs collected on the second night were blood and tissue sampled in the field, immediately after the capture period. Hematological parameters were analyzed, and enzymatic activities of catalase, cholinesterase (ChE), and glutathione S-transferase (GST) were determined in the plasma, liver, kidney, and muscle. A sex difference was observed only for total white blood cell counts (WBC), females exhibiting significantly greater values than males. The packed cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), WBC, and muscle ChE activity were significantly correlated with snout-vent length (SVL). The correlation was inversed in the case of MCHC, WBC, and muscle ChE, while the correlation was positive between PCV and SVL. Most examined parameters presented similar values when frogs were sampled at night following capture or the next morning. Total red blood cells (RBCs) count, and plasma enzymatic activities of ChE and GST were the only parameters that presented significantly increased values in morning samplings compared with night samplings. Overall, the current study indicates that it is best to sample the frogs as soon as possible after capture if hematologic or plasmatic biomarkers are examined. Nevertheless, it is possible to sample on the next morning if tissular biomarkers are employed.

Accumulation of current-use pesticides, cholinesterase inhibition and reduced body condition in juvenile one-sided livebearer fish (Jenynsia multidentata) from the agricultural Pampa region of Argentina.

The aim of this study was to characterize the level and nature of the pesticide contamination received by one-sided livebearer fish (Jenynsia multidentata) from a watercourse situated within the main agricultural region of Argentina, and to assess the effects of this contamination on fish health. Juvenile one-sided livebearer fish (Jenynsia multidentata) were collected in December 2011 and March 2012 from three sites along the Pergamino River. Pesticide contamination was characterized by extracting whole fish and analytically determining thirty different pesticide molecules. The biomarkers catalase, glutathione-S-transferase, and cholinesterases were assessed. Body condition was calculated as an estimate of the amount of energy reserves possessed by the fish. Seventeen different pesticides were detected in fish tissues with 81% of captured animals containing at least one pesticide molecule. The pyrethroid insecticides fenvalerate and bifenthrin were most frequently detected, being respectively found in 41.8 and 36.4% of samples tested. Highly toxic dichlorvos and pirimiphos-methyl were detected. Differential levels of contamination could not be established amongst sites but were observed within sites amongst the two sampling dates. The months when pesticide residues were most abundant from in Site A and B corresponded to the months when body condition was at its lowest in the two sites. The inhibition of Che activity in March when body condition was reduced also points to a role of insecticide contamination in the reduction of body condition. These findings provide strong new evidence that current-used agricultural pesticides can accumulate in wild fish and impact their health and energetics.

Toxicities of glyphosate- and cypermethrin-based pesticides are antagonic in the tenspotted livebearer fish (Cnesterodon decemmaculatus).

Although pesticide contamination of surface waters normally occurs in the form of mixtures, the toxicity and interactions displayed by such mixtures have been little characterized until now. The present study examined the interactions prevailing in equitoxic and non-equitoxic binary mixtures of formulations of glyphosate (Glifoglex(®)) and cypermethrin (Glextrin(®)) to the tenspotted livebearer (Cnesterodon decemmaculatus), a widely distributed South American fish. The following 96 h-LC50s were obtained when pesticide formulations were tested individually: Glifoglex(®) 41.4 and 53 mg ae glyphosate/L; Glextrin(®) 1.89 and 2.60 µg cypermethrin/L. Equitoxic and non-equitoxic mixtures were significantly antagonic in all combinations tested. The magnitude of the antagonism (factor by which toxicity differed from concentration addition) varied between 1.37 and 3.09 times in the different non-equitoxic mixtures tested. Antagonism was due to a strong inhibition of cypermethrin toxicity by the glyphosate formulation, the toxicity of the cypermethrin-based pesticide being almost completely overridden by the glyphosate formulation. Results obtained in the current study with fish are radically opposite to those previously observed in tadpoles where synergy was observed when Glifoglex(®) and Glextrin(®) were present in mixtures (Brodeur et al., 2014).

Synergy between glyphosate- and cypermethrin-based pesticides during acute exposures in tadpoles of the common South American toad Rhinella arenarum.

The herbicide glyphosate and the insecticide cypermethrin are key pesticides of modern management in soy and corn cultures. Although these pesticides are likely to co-occur in ephemeral ponds or aquatic systems supporting amphibian wildlife, the toxicological interactions prevailing in mixtures of these two pesticides have been little studied. The current study evaluated the toxicity of equitoxic and non-equitoxic binary mixtures of glyphosate- and cypermethrin-based pesticides to tadpoles of the common South American toad, Rhinella arenarum. Two different combinations of commercial products were tested: glyphosate Glifosato Atanor®+cypermethrin Xiper® and glyphosate Glifoglex®+cypermethrin Glextrin®. When tested individually, the formulations presented the following 96 h-LC50s: Glifosato Atanor® 19.4 mg ae L(-1) and Glifoglex 72.8 mg ae L(-1), Xiper® 6.8 mg L(-1) and Glextrin® 30.2 mg L(-1). Equitoxic and non-equitoxic mixtures were significantly synergic in both combinations of commercial products tested. The magnitude of the synergy (factor by which toxicity differed from concentration addition) was constant at around twofold for all tested proportions of the glyphosate Glifoglex®+cypermethrin Glextrin® mixture; whereas the magnitude of the synergy varied between 4 and 9 times in the glyphosate Glifosato Atanor®+cypermethrin Xiper® mixture. These results call for more research to be promptly undertaken in order to understand the mechanisms behind the synergy observed and to identify and quantify the extent of its environmental impacts.