Assessing the Risk of Fipronil-Treated Seed Ingestion and Associated

Article pubs.acs.org/est

Assessing the Risk of Fipronil-Treated Seed Ingestion and Associated Adverse Effects in the Red-Legged Partridge Ana Lopez-Antia,*,† Manuel E. Ortiz-Santaliestra,†,‡ Pablo R. Camarero,† François Mougeot,† and Rafael Mateo† †

Institute of Research in Game Resources (Instituto de Investigación en Recursos Cinegéticos, IREC), CSIC-UCLM-JCCM, Ciudad Real, Spain ‡ Institute for Environmental Sciences, University of Koblenz-Landau, Landau, Germany S Supporting Information *

ABSTRACT: Fipronil is an insecticide commonly used in agriculture, but there are growing concerns over its environmental impacts (e.g., harmful effects on pollinators). Fipronil-treated seed ingestion might threaten granivorous farmland birds, in particular, Gallinaceous birds that are particularly sensitive to this insecticide. We report here on exposure risk and effects in a game bird of high socioeconomic importance, the red-legged partridge (Alectoris rufa). We fed captive birds with untreated maize (controls) or with a mixture of untreated−treated maize (ratio 80:20; exposed birds) during 10 days at the beginning of the breeding period (n = 12 pairs in each group). We first show that exposed partridges did not reject treated seeds but reduced food intake and lost body condition. We further studied the effects of treated seed ingestion on adult survival, oxidative balance, plasma biochemistry, carotenoid-based coloration, cellular immune response, steroid hormone levels, and reproduction. Fipronil exposure altered blood biochemistry and sexual hormone levels and reduced cellular immune response, antioxidant levels, and carotenoid-based coloration. Exposed pairs also had reduced egg fecundation rate and produced eggs with fewer antioxidants and offspring that had reduced cellular immune response. These negative effects on adult partridges, their reproductive performance, and offspring quality highlight that fipronil-treated seed ingestion is a significant threat to wild birds. sensitive to fipronil; studying its effects on species of this Order is therefore vitally important. In March 2014, the European Union declared a 2-year moratorium on the use of fipronil treated seeds (maize and sunflower) due to the high risk of acute toxicity to bees associated with the exposure to dust or to residues present in pollen and nectar. The potential threat of fipronil-treated seeds to granivorous birds was also recognized as requiring urgent attention.17 The last report published by the European Food Safety Authority18 also highlighted that the consumption of fipronil-treated maize could occur and cause lethal effects on wild granivorous birds. This report also concluded that the risk assessments for fipronil-treated seeds should focus on the acute and short-term effects, as there is no indication that fipronil affects reproduction. However, Kitulagodage et al.19,20 found a reduction of hatching success and maternal transfer of fipronil residues to egg yolk and chicks in female zebra finch (Taeniopygia guatatta) exposed to a single dose of 1 mg/kg of body weight (b.w.) and a reduction of feeding activity and adverse neurological effects in bobwhite quail dosed with 11.3 mg/kg b.w. In rats, fipronil administration was also shown to

1. INTRODUCTION Changes in agricultural practices that occurred since the middle of the 20th century have led to population declines in many bird species associated with agroecosystems.1,2 Pesticide use is a key factor explaining farmland bird declines, because of indirect effects on habitats and food supply3−6 or direct toxic effects compromising bird health at lethal or sublethal levels.7−9 The latter, depending on targets and the degree of adverse effects, can ultimately impair survival or reproduction and therefore affect population dynamics. Fipronil is a phenyl pyrazole insecticide discovered and produced by the French company Rhône-Poulenc Agro in 1985−1987 and in commercial use since 1993.10,11 Fipronil binds to the gamma-aminobutyric acid (GABA) receptors producing an uncontrolled activity of the central nervous system and subsequent death in insects. It also binds to vertebrate GABA receptors, although with less affinity than to those of insects.10−12 Fipronil has been mainly used as seed treatment because of its systemic effect.11 The LD50s reported for fipronil greatly varied among studied birds: from >2500 mg/kg for the mallard duck (Anas platyrhynchos)13 to 11.3 mg/kg for the bobwhite quail (Colinus virginianus),14 31 mg/kg for the ring-necked pheasant (Phasianus colchicus),15 or 34 mg/kg for the red-legged partridge (Alectoris rufa).16 Gallinaceous birds seem particularly © 2015 American Chemical Society

Received: Revised: Accepted: Published: 13649

August 7, 2015 October 7, 2015 October 8, 2015 October 8, 2015 DOI: 10.1021/acs.est.5b03822 Environ. Sci. Technol. 2015, 49, 13649−13657

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Environmental Science & Technology

hematocrit was measured in a 75 μL aliquot, and the remaining was centrifuged at 10 000 × g for 10 min at 4 °C to separate plasma from the cellular fraction (pellet), which were stored separately at −80 °C for later analysis. Partridge pairs were kept in their cages from April through to June in order to monitor their reproduction. 2.2. Food and Treated Seed Consumption. Treated seeds used in this experiment were commercial Regent TStreated seeds (BASF, Barcelona, Spain) and red colored. The package information indicated that seeds were treated with the insecticide fipronil and with the fungicides fludioxonil and metalaxyl, although the exact concentration of each pesticide was not indicated. According to the manufacturer, the application rate for maize seeds was 250 cc of Regent TS (fipronil 50% w/v) per 100 kg of seeds. The half-life of fipronil when exposed to sunlight was estimated at 3.6 h in water and 34 days in loamy soil.30 The half-life on vegetation is 3−7 months, and some studies showed that one of its metabolites, MB 46513, can bioaccumulate in fatty tissues.31 To measure the food consumed by each pair of partridges (that shared the same cage), we weighed the amount of food put in the feeders at the start of the experiment and that left in the feeders at the end of the experiment. We placed plastic bags under the cages to collect the food spillages, which were taken into account when quantifying the food remaining. In the case of exposed pairs, we weighed separately the treated (red colored) and untreated maize (uncolored) seeds left. 2.3. Survival and Body Condition of Adult Partridges. During experiment, we checked partridges’ survivorship daily. We initially measured tarsus length and weighed each partridge before and after the exposure period to calculate body condition (scaled mass index32). 2.4. Immune Response of Adult Partridges. We used the phytohemagglutinin (PHA) skin test to estimate cellmediated immune response. We performed the test on April 24, 4 days after the end of the exposure period. Details on the methodology are described in Mougeot et al.33 2.5. Reproduction. The first egg laying occurred on April 9, i.e., 1 day before exposure started. Once the experiment had begun, we checked all cages daily and collected eggs, which were measured (maximum length and width) and kept at 15 °C to temporarily prevent development. Egg checking and collection continued beyond the end of the experiment until the end of the laying season (average clutch size is 9). Every 15 days, we transferred all eggs collected thus far to an automatic incubation chamber (Masalles Valltrade, Sant Cugat del Valles, Barcelona, Spain), where they were incubated for 21 days (37.7 °C, 45% humidity, and constant movement). Throughout the experiment, a total of five consecutive incubation sets (or batches) were performed. Before transferring eggs to the incubation chamber, those laid in 4th, 8th, and 12th position of the laying sequence of each pair were separated and kept frozen at −80 °C for analyzing levels of egg yolk vitamins, carotenoids, and fipronil/fipronil sulfone. On the 21st day of incubation, eggs were placed in individual cages and moved to a hatching chamber where they were incubated at 37.7 °C with constant humidity but without movement. We checked the chamber daily to monitor hatching events. We noted hatching date, tarsus length, and body mass of each hatchling, which were individually marked and housed in closed rooms with a heat source, water, and food (partridge growth fodder, NantaNutreco, Tres Cantos, Spain). We remeasured tarsus length, reweighed all chicks at 8, 16, 24, and 32 days of age, and

impair reproduction,21 alter behavior,22 and cause thyroid hormone disruption.23 The red-legged partridge is a Gallinaceous game bird of high socioeconomic value in Spain, whose populations are currently in decline.24,25 The species is granivorous and, in most of its range, inhabits agricultural areas where maize and sunflower are cultivated. Analysis of crop contents has shown that red-legged partridges usually consume seeds treated with pesticides,26 so they are at a high risk of fipronil exposure if these ingested seeds were treated with this insecticide. In Spain, maize is sown in April, coinciding with the red-legged partridge reproductive season (and that of many other farmland birds). As mentioned above, Gallinaceous birds are known to be especially sensitive to fipronil. This, together with the fact that red-legged partridge breeds well in captivity, makes this bird an ideal model species to experimentally study the sublethal effects of fipronil.27 In this experiment, we fed captive partridges with a mixture of untreated and treated maize seeds during 10 days in April (when most birds start laying), mirroring the potential exposure during the sowing season. We studied food consumption (distinguishing between treated and untreated seeds) and investigated the possible avoidance of treated seeds. We also studied the effects of fipronil-treated seed ingestion on adult birds (body condition, plasmatic biochemistry, oxidative stress, cellular immune response, ornamental carotenoid-based coloration, and sex steroid hormones levels), on their reproductive performance (clutch size, egg size, egg shell thickness, antioxidant content, and hatching success), and on their offspring (chick development, survival, and cellular immune response). Finally, we measured maternal transfer of the insecticide to the eggs. Detecting sublethal effects in these variables should help to better understand fipronil toxicity mechanisms and risks to exposed birds.

2. MATERIAL AND METHODS 2.1. Experimental Design. The experiment was conducted in the Dehesa de Galiana experimental facilities (Ciudad Real, Spain) with the approval of the Universidad de Castilla-La Mancha’s Committee on Ethics and Animal Experimentation. For this experiment, we used 53 (24 pairs and 5 extra males) captive-born, 1-year-old red-legged partridges. Each individual was sexed genetically, following Fridolfsson and Ellegren.28 Partridges were housed in pairs or individually in the case of the five extra males in outdoor cages (95 × 40 × 42 cm) and acclimatized to the facility during 4 months before starting the experiment. Birds were provided ad libitum with tap water and a commercial partridge feed (Partridge maintenance fodder, Nanta-Nutreco, Tres Cantos, Spain) mixed with wheat. Each pair was randomly assigned to one of two treatments (control vs exposed; 12 pairs in each group, with two and three extra males in the control and exposed groups, respectively). The exposure to treated seeds was designed to coincide with the maize sowing time in Spain, began on April 10 after egg laying was confirmed, and finished on April 20, 2012. During the exposure period, partridges were fed exclusively with a mixture of untreated−treated maize seeds (ratio 80:20) provided ad libitum (exposed group) or with untreated maize seeds, also provided ad libitum (control group). This estimation of 20% of treated seed occurrence in the diet is based on published data29 about seed consumption by redlegged partridges in the wild. On April 20, we took 1 mL of blood (drawn by jugular venipuncture) from each partridge. Blood samples were kept refrigerated in heparinized tubes, the 13650

DOI: 10.1021/acs.est.5b03822 Environ. Sci. Technol. 2015, 49, 13649−13657

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the homogenates using the Bradford method.36 Enzyme activities were calculated relative to protein levels (per mg). We measured malondialdehyde (MDA) levels in the RBC homogenates (lipid peroxidation levels).37 For this we used an Agilent 1100 Series HPLC system (Agilent Technologies, Waldbronn, Germany) coupled with a fluorescence detector (FLD) and a 5 μm ODS-2 C-18 (4.0 × 250 mm × 5 μm) column. We determined plasma levels of retinol (vitamin A), αtocopherol (vitamin E), and carotenoids (zeaxanthin and lutein) using HPLC coupled to photodiode (DAD) and fluorescence (FLD) detectors.38 We extracted antioxidants from the egg yolk39 and used the same analytical method as for plasma antioxidants. We also used the automatic spectrophotometer analyzer A25 to determine plasma biochemistry levels, specifically, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LHD), creatine phosphokinase (CPK), albumin, total protein, glucose, cholesterol, triglycerides, calcium, magnesium, phosphorus, creatinine, urea, and uric acid. We used the reaction kits available for each parameter (BioSystems, Barcelona, Spain). We quantified testosterone and estradiol levels in plasma samples by enzyme-linked immunosorbent assays using commercial kits (DRG instruments GmbH, Marburg, Germany). We obtained calibration curves using the kit standards and adjusted the obtained values to a four-parameter logistic model using the software XLSTAT (Addinsoft SATL, Paris, France). 2.8. Color Measurements of Adult Partridges. We measured the red coloration of the beak and eye rings of partridges, which are pigmented by carotenoids and are indicators of individual health33 and investment in reproduction.40 We quantified the percentage of eye-ring area pigmented by carotenoids33 and measured the beak and eye-ring color using a portable spectrophotometer (Minolta CM-2600 d; Tokyo, Japan39). Eye-ring pigmentation area, beak color, and eye-ring color were measured for each bird on February 14 (before experiment) and on April 20 (end of the exposure period). 2.9. Statistical Analyses. Survival of adults and chicks was analyzed using a Kaplan−Meier survival analysis. We used a t test for independent samples to test for differences in food consumption between control and exposed partridges and a one sample t test to compare the ratio of treated seeds ingested with the ratio of treated seeds offered (20%). We used a General Linear Model (GLM) of repeated measures to analyze differences between experimental groups in body condition change throughout the experiment. We used Generalized Linear Models (GLMz) with the experimental group and the sex as fixed factors in order to test for fipronil effects on all adult partridge traits, being the sex × experimental group interaction removed from the models when nonsignificant. In the analysis of treatment effects on carotenoid-based coloration, we included the color measurement prior to experiment as a covariate. We tested relationships between variables with Pearson’s correlations, and when a variable was significantly correlated (p < 0.05) with body condition, we added body condition as a covariate in the GLMz. To test for fipronil effects on egg and chick parameters, we performed GLMz with the experimental group as fixed factor, including also the breeding pair as a nested factor within the experimental group for egg parameters, chick condition, and

calculated their body condition as for adults. Some chicks from the third and fourth incubation rounds were selected for the PHA test, excluding the first chick of each pair. The PHA test was performed as described for adults. Chick sex was determined genetically following Fridolfsson and Ellegren.28 Unhatched eggs were opened and examined to determine fertility (based on the presence of an embryo or germinal disk). Eggshell thickness was also measured (see Lopez-Antia et al.34 for details on the method). 2.6. Fipronil and Fipronil Sulfone Levels in Seeds, Dead Partridges, and Egg Yolk Samples. The concentrations of fipronil and fipronil sulfone, the primary biological metabolite of fipronil, were measured by LC-MS in different tissues, crop contents, and seeds using different extraction methods depending on the matrix. For tissues (liver, brain, and egg content), 1 g (wet weight) of sample was homogenized with 10 g of sulfate sodium anhydride in a mortar, extracted with 20 mL of dichloromethane (BHD Prolabo) by 10 min of horizontal shaking, 5 min of sonication, and then filtered. The extract was evaporated in a rotary evaporator, dissolved in 1 mL of mobile phase (ethyl acetate:cyclohexane 1:1), and passed through a gel permeation column of Bio-Beads S-X3 (Bio-Rad Laboratories; Madrid, Spain). The first 45 mL of elute were discarded, and the next 15 mL were collected, evaporated, and dissolved again in 500 μL of acetonitrile. For seeds, crops, and gizzard contents we followed the method described in LopezAntia et al.34 Pesticide analysis was done using an Agilent 1100 series highperformance liquid chromatography (HPLC) system coupled with an Agilent 6110 Quadrupole LC/MS multimode (Agilent Technologies, Waldbronn, Germany). The column used was a Zorbax Eclipse XDB-C18 (4.6 × 150 mm, ×5 μm) with a precolumn Zorbax Eclipse XBD-C18 (4.6 × 12.5 mm, 5 μm). The injection volume was 20 μL, and the mobile phase had a flow rate of 0.8 mL/min. The chromatographic conditions consisted of a gradient mode using two phases (A, formic acid 0.1% in water; B, formic acid 0.1% in acetonitrile); the initial conditions were 95% of A and 5% of B during 5 min, then to 50% of each phase in 7.5 min, and to 10% of A and 90% of B in 16.5 min. These conditions were maintained for 3 min and then returned to the starting conditions over a 3 min period. The analyzed compounds were detected using negative ionization mode with the following MM-ES+APCI conditions: temperature of gas and vaporization were 250 and 200 °C, respectively, flow rate was 8 L/min and 35 psi of pressure, the capillary voltage was 3000 V, and charge 1000 V with amperage of 4.0 μA. The fragmentation voltage and retention time are shown in Table S1. The recoveries of the two compounds in seeds, liver, and egg content are shown in Table S2. Concentrations in samples were corrected by recovery rates when these were 0.62, N = 27, p ≤ 0.001). When body condition was included as a covariate in the models, the treatment effect became nonsignificant for lutein (p = 0.13) but remained significant for zeaxanthin (p = 0.03). Regarding carotenoid-based coloration, we found that exposed partridges had a lower percentage of eye-ring pigmentation (85.8 ± 1.5%) than controls (96.5 ± 0.9%) after exposure (p < 0.001; Figure S2). The percentage of eye13652


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Table 1. Reproductive Parameters (N, %, or mean ± SE) of Partridge Pairs from the Control and Fipronil-Exposed Groups

ring pigmentation was positively associated with body condition (R = 0.61, N = 51, p < 0.001), and the treatment effect remained significant when body condition was introduced as a covariate in the model (p < 0.001). We did not find differences between control and exposed partridges for the eyering hue or chroma or for the beak chroma, and a marginally significant treatment effect on beak hue was found (p = 0.08): exposed partridges displayed slightly redder beaks than controls after exposure. Regarding steroid hormone levels, we found that testosterone levels were lower in exposed than in control males (p = 0.04; Figure 1) and unaffected by treatment in females. In males, testosterone levels tended to increase with body condition (R = 0.37, N = 27, p = 0.06) and the treatment effect became nonsignificant when body condition was introduced in the model. For estradiol levels, we did not find significant differences between control and exposed partridges. All plasma biochemical parameters are summarized in Table S3. Compared with controls, fipronil-exposed partridges presented reduced plasma levels of albumin (p = 0.042), magnesium (p = 0.015), total proteins (p = 0.006), triglycerides (p = 0.04), and calcium, the latter being significantly reduced in females only (p = 0.03). Uric acid levels were higher in exposed partridges than in controls (p < 0.001). All these parameters correlated positively with body condition after the experiment in the treated group (all R > 0.54, N = 20, all p < 0.03), and all these treatment effects became nonsignificant when body condition was introduced as a covariate in the models. In the case of uric acid, plasma values negatively correlated with body condition in a marginally significant manner (R = −0.41, N = 20, p = 0.069). 3.2. Effects on Reproduction and Indirect Effects on Offspring. Reproductive parameters for control and exposed partridges are summarized in Table 1. Clutch size did not differ between exposed and control partridges, but egg fertility was lower for fipronil-exposed pairs than for controls (p = 0.036). There were no differences in hatching rate between treatment groups. Eggs laid by exposed partridges were of similar size and had similar shell thickness as those laid by controls but were significantly heavier (p = 0.026; Table 1, Figure S3). Eggs laid by treated partridges had lower levels of tocopherol (p < 0.001) and zeaxanthin (p < 0.049) in their yolk than those laid by control partridges (Table 1, Figure S4). There were no significant treatment effects for retinol and lutein levels in egg yolk. Fipronil and fipronil sulfone residues were detected in all eggs laid by exposed females that were analyzed (n = 9), with mean (±SE) levels of 0.69 ± 0.40 and 347 ± 112 ng/g for fipronil and fipronil−sulfone, respectively. Fipronil and fipronil−sulfone levels within eggs were positively correlated (R = 0.81, N = 9, p < 0.01). Analyzed eggs were laid between 20 and 66 days after the end of the treatment. Fipronil−sulfone levels decreased with the time elapsed since the end of the treatment (R = −0.82, N = 8, p = 0.01), while fipronil levels were not correlated with the laying date. Chicks from parents exposed to fipronil were heavier at hatching than those from controls (p = 0.012; Table 1). Chick body weight at hatching positively correlated with egg weight (R = 0.844, N = 59, p = 0.012), and the treatment effect became nonsignificant when egg weight was introduced as a covariate in the model. We did not find differences between treatment groups in the body condition of chicks at hatching, chick sex ratio, chick growth (up to 32 days), or chick survival (Table 1).

parameter

control

exposed

no. of pairs no. of laying females total no. of eggs clutch size per laying female egg length (mm)a egg width (mm)a egg mass (g)a elongation indexa shell thickness (μm)a retinol in yolk (nmol/g)a tocoferol in yolk (nmol/g)a lutein in yolk (nmol/g)a zeaxanthin in yolk (nmol/g)a fertile eggs (%)b1 hatching rate of fertile eggs (%)b2 hatching rate of total eggs (%)b3 total number of chicks brood size chick body weight at birth chick body condition at birth chick mortality (%)b4 female ratio in the offspringc chick mean survival time after hatching (days) chick cellular immune response (wing web swelling (μm))

10 10 89 8.9 ± 1.8 38.9 ± 0.1 28.9 ± 0.1 17.4 ± 0.1 1.34 ± 0.01 223 ± 4.0 500 ± 17.1 1495 ± 37 64.2 ± 6.2 46.1 ± 3.0 85 ± 4 67 ± 6 56 ± 6 40 4 ± 1.0 11.77 ± 0.16 12.01 ± 0.18 30 ± 7 58 ± 8 25.0 ± 1.9

11 9 52 6.0 ± 1.3 39.1 ± 0.2 29.1 ± 0.1 18.0 ± 0.1*d 1.34 ± 0.01 222 ± 3.9 493 ± 24 1178 ± 51**d 47.7 ± 8.7 35.7 ± 4.2*d 67 ± 7*d 69 ± 9 47 ± 8 20 2.3 ± 0.8 12.11 ± 0.22*d 11.76 ± 0.25 32 ± 11 65 ± 11 26.0 ± 2.5

103 ± 20

75 ± 20*d

a

Marginal means for the nested models. bMarginal means of weighted models 1fertile eggs/total eggs, 2hatched eggs/fertile eggs, 3hatched eggs/total eggs, and 4died chicks/total chicks. cNumber of female chicks/total number of chicks d(*,**) Significantly different from controls at the p ≤ 0.05 and p ≤ 0.01 levels, respectively.

However, chicks from exposed partridges showed a reduced inflammatory response to PHA compared with those from control partridges (p = 0.021; Table 1). PHA response was negatively correlated with chick condition at hatching (p = 0.002).

4. DISCUSSION Our results show that birds can be exposed to toxic doses of fipronil through the ingestion of coated seeds and that the consequences can be observed to impact on several functions important for survival and reproduction. The treated seeds contained a mixture of pesticides, which means that we cannot directly attribute the observed toxic effects to the sole action of fipronil. However, fipronil residues measured in the treated seeds were more than five times above the oral LD50 for rats, whereas residues of the different fungicides were at least 78 times below the corresponding oral LD50 value. We may thus suppose that fipronil was the main source of toxicity of treated seeds on exposed partridges. 4.1. Food Consumption, Exposure Levels, and Effects on Adult Body Condition. Partridges exposed to a fipronil dose averaging 8.73 ± 0.18 mg/kg b.w. reduced their food consumption and consumed 63.5−75.5% less maize than controls. This reduction could be due to a rejection of the mixture, because of the treated seed characteristics (color, odor, or taste), or could be due to sickness produced by a sublethal intoxication.27,41,42 Other studies have also reported a reduction 13653


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Environmental Science & Technology in “clean” food consumption by birds subjected to a forced fipronil exposure (e.g., northern bobwhite quail,20 red legged partridge,16 other gallinaceous birds14,15). In our experiment, loss of body mass and condition in the exposed group were not explained by total food consumption or treated seed consumption. This suggests that, besides the effect of fipronil exposure on appetite, other toxic effects also influenced the loss of body weight. Contrary to these studies with galliforms, Avery et al.43 did not register reductions in food consumption and body weight in three passerine bird species fed with fiproniltreated rice seeds (500 mg/kg). These species-specific differences in feeding behavior after exposure to fipronil may be important to explain the greater sensitivity of gallinaceous birds to this insecticide. High tier risk assessment requires considering the avoidance of treated food as a modulator of the real exposure risk in the field. In our study, partridges did not distinguish between treated and untreated maize seeds, despite the fact that treated seeds were conspicuously colored in red. In a similar way, the EFSA scientific report for fipronil18 documented mortality cases of grey partridges during an avoidance study, indicating that this species can consume enough treated seeds to reach a lethal dose. On the other hand, we cannot fully extrapolate our results to a field situation, where treated seeds and clean food could be more easily differentiated and thus treated seed avoidance could occur.44 Risk of exposure of farmland birds to pesticide-treated seeds can also be mitigated by dehusking seeds before ingesting,45 significantly reducing the chances of pesticide uptake. However, maize seeds are difficult to dehusk, and some studies have shown that red-legged partridge do not dehusk seeds,46 which agrees with our observations during the present experiment. Therefore, dehusking behavior would not be a relevant way to reduce exposure of this species to fiproniltreated seeds. 4.2. Physiological Effects on Adult Partridges. Partridges exposed to fipronil-treated seeds exhibited a range of physiological alterations. Some of these appeared explained by the loss of body condition, while others can be explained by a more direct effect of the insecticide. All these effects could have consequences on partridges’ survival or reproduction. All of the detected effects of fipronil exposure on plasma biochemical parameters (Table S3) were symptomatic of food restriction or subfeeding. Moreover, the increase in uric acid levels indicated that exposed partridges had reached the third phase of fasting. The recovery after this phase becomes a difficult process, and some internal organs could be damaged.47 Treatment effects on uric acid levels in our experiment were not completely explained by body condition, and this may be in part explained by the antioxidant role of uric acid (see below). We found that exposed females reduced circulating calcium levels more than exposed males. This difference is probably due to the extra calcium required for eggshell formation and oviduct contraction during egg laying. We also found that exposed partridges had a lower cellular immune response (reduced wing web swelling) than controls, a response known to be highly correlated with body condition.33,48 The reduced cellular immune response in fipronil-exposed birds was mediated by body condition loss, suggesting that fipronil does not have a direct immunotoxic effect, as demonstrated in other insecticides.49 Although the reduced immune response is unlikely to be a direct immunotoxic effect of the insecticide, it still could have important implications for partridge survival.

The phase I detoxification process of pesticides produces free radicals50 that the body antioxidant system should neutralize to avoid tissue damage. Contrary to what we expected, MDA levels measured in red blood cells were lower for exposed partridges than for controls. This parameter measures the phospholipid peroxidation in cellular membranes and should therefore be higher in animals exposed to a toxicant. Moreover, birds in worst body condition should have a worsened antioxidant status.51 We have several possible explanations for this phenomenon. The first could be a hormetic effect of the insecticide that increased antioxidant defenses in excess of the damage produced. We registered marginally greater levels of SOD activity in exposed partridges (only in males) than in controls. Uric acid is also an important antioxidant molecule in birds,52 so the higher levels produced during the exposure to the insecticide could have improved the oxidative status of partridges. Moreover, lower lipid levels in blood, in particular triglycerides, may reduce the possibility of MDA formation by lipid peroxidation chain reaction.53,54 Another nonexclusive explanation could be a worsened oxidative status in control partridges due to their greater reproductive effort, known to cause oxidative stress.55 Control partridges in our experiment invested more in reproduction (redder ornamental coloration, larger clutches) and did so more successfully than exposed partridges, which may have come with an increased “oxidative cost”. We found treatment effects on plasma carotenoid levels (Figure 1; only in males) and on carotenoid-based coloration, specifically on the amount of pigmentation of the eye ring, which was significantly reduced in exposed partridges. These two variables are positively associated in red-legged partridge33 as carotenoids are the pigments that color the red sexual ornaments displayed by partridges.56 Moreover, both variables correlated positively with body condition, as found in previous studies.33,57 Here, treatment effects on lutein plasma levels were explained by variations in body condition, but effects on zeaxanthin plasma levels and eye-ring pigmentation were only partially explained by changes in body condition. This suggests a direct effect of fipronil on zeaxanthin and coloration. Carotenoids are very susceptible to oxidative stress,58 and oxidative stress may have reduced zeaxanthin levels more than other antioxidant (retinol or tocopherol) levels. The decrease in carotenoid plasma levels (lutein and zeaxanthin) induced by fipronil exposure was also sex dependent and only detected in males (Figure 1). This could happen because males use carotenoids for self-maintenance and for ornamental coloration only, while females also need to allocate them to egg yolks. The lack of fipronil effect on carotenoid plasma levels in females could be offset by a lower investment in reproduction. Finally, we detected reduced levels of testosterone in exposed males. This reduction was also explained by body condition losses due to the effect of fipronil, as found in a previous study. Body condition and testosterone levels could also be independently affected by fipronil and spuriously correlated. An alternative explanation could be that fipronil affects the central nervous system, causing an imbalance in the hormonal regulation. This neuroendocrine effect of fipronil was proposed by Ohi et al.21 after detecting that 280 mg/kg of fipronil topically administered to Wistar rats significantly altered plasma progesterone and estradiol levels 96 h after the exposure. Sex steroid hormone levels, body condition, and immune response are parameters extensively interrelated and associated with the expression of ornamental traits and courtship.33,59−62 13654


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Environmental Science & Technology 4.3. Effects on Reproduction and on the Offspring. Overall, treated partridges produced fewer chicks than controls, although this difference was not statistically significant (Table 1). Eggs laid by exposed partridges had lower antioxidant levels than those laid by control partridges, these differences being significant for tocopherol and zeaxanthin levels. In female redlegged partridges, one of the greatest investments of carotenoid reserves is for egg yolks.63 As mentioned above, fipronil exposure affected plasma carotenoid levels both directly and indirectly (through the reduced food consumption). Our hypothesis is that this reduction led to a lower investment in reproduction (reduction of antioxidant levels in eggs and of eye-ring pigmentation in females; reduction of eye-ring pigmentation in males). In red-legged partridge, coloration is known to influence mate choice and reproductive investment: females mated with redder males invest more in reproduction.40 Another important finding is the reduced fertile egg ratio in exposed partridges (Table 1), which could be explained by the reduced levels of sex hormones observed in males. In males, testosterone is involved in spermatogenesis, ornamental coloration, and reproductive behaviors.64 Kitulagodage et al.19 also detected a reduced hatching success in zebra finches after the exposure of breeding females to a single dose of fipronil of 1 mg/kg of b.w. In rats, reproductive effects of fipronil exposure, such as reduced pregnancy index, have also been registered.10,21 Chicks hatched from eggs laid by exposed partridges were heavier at hatching than those from controls. This may be because of resource allocation constraints and a trade-off between producing fewer heavier eggs or larger clutches with lighter eggs, depending on female body condition. The differences in chicks’ weight at hatching between control and exposed partridges quickly disappeared: 8 days after hatching, we did not find differences between groups in body weight or body condition. Finally, we found that chicks from exposed parents had a reduced cellular immune response compared with those from control parents (Table 1). Carotenoid and vitamin levels in the egg yolk confer antioxidant protection during the embryonic development and in the early days immediately following hatching.65 Moreover, the fipronil and fipronil−sulfone residues detected in the egg yolk could generate extra oxidative stress when fipronil is metabolized by the embryo. Free radicals play an important role in immune system regulation,66 and alterations of the oxidative status may be responsible for this effect on immune response.67 Overall, our experimental results provide evidence that fipronil exposure has a range of adverse effects on reproduction that had been overlooked in previous assessments.18 The negative effects on adult partridges and on their reproductive performance reported here highlight that the use of fiproniltreated seed in agriculture can be a significant threat to wild granivorous birds.

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egg weight from both experimental groups along the different incubation sets, and vitamin and carotenoid levels in the yolk of eggs laid by control and exposed partridges; tables showing ESI+APCI-MS parameters of analyzed compounds, recovery rates of fipronil analysis, mortality rate, body condition, hematocrit, and wing web swelling in response to PHA and plasma biochemistry levels in control and fipronil exposed partridges after the exposure period, and levels of fipronil and fipronil− sulfone detected in the gizzard, liver, and brain of the three exposed partridges that died during experiment (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: +34 926295450; fax: +34 926295451; e-mail: ana. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The present study was financed by FEDENCA (Real Federación Española de Caza) and Oficina Nacional de Caza with the partnership of Fundación Biodiversidad. Funding sources had no involvement in study design or execution. We thank to D. González-Barrio, and J. Feliu for their help during laboratory and field work.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b03822. Additional graphical representation of body condition before and after exposure in controls and fipronil exposed partridges, proportion of the eye-ring area pigmented by carotenoids in males and females from the control and exposed groups after the exposure period, 13655


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Assessing the Risk of Fipronil-Treated Seed Ingestion and Associated

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