Imidacloprid-treated seed ingestion has lethal effect on adult partridges and reduces both breeding investment and offspring immunity☆
Introduction
In the last decades, farmland birds in Europe and North America have been suffering population declines at higher rates than birds from other habitats (EBCC, 2014). Farmland is being profoundly altered through agricultural intensification, posing a major challenge for biodiversity conservation today in many countries (Krebs et al., 1999). Recent studies have pointed out that a major cause of bird population declines is the use of pesticides, either because of indirect effects on habitat and food supply (Hallmann et al., 2014, Goulson, 2014) or because of direct toxic effects on the health of birds (Mineau and Whiteside, 2013). A greater probability of lethality in birds occurs when the ratio between the LD50 and the estimated field exposure dose is low (EFSA, 2009). Pesticides with higher LD50 or lower risk of exposure can produce a range of sub-lethal effects such as loss of physical condition, immunosuppression, neurological impairments or endocrine disruption (Fry, 1995). All these effects may ultimately affect survival or reproduction, and therefore impact on population dynamics.
Imidacloprid is a systemic insecticide belonging to the family of neonicotinoids and it is currently the first insecticide and the second agrochemical most used in the world (Jeschke et al., 2011, Goulson, 2013). This pesticide acts by binding to specific nicotinic acetylcholine receptors, thus interfering with the transmission of nerve impulses. Starting in December 2013, the European Union declared a moratorium on the use of three neonicotinoid insecticides (i.e. imidacloprid, thiamethoxam and clothianidin) for seed coating, soil treatment and foliar treatment due to its toxicity on pollinators, but their use for seed treatment of winter cereals, as well as after crop flowering and in crops harvested before flowering, continues to be approved (Regulation 485/2013). Imidacloprid oral acute LD50 for birds vary from 31 mg/kg in the Japanese quail (Coturnix japonica) to 152 mg/kg in the bobwhite quail (Colinus virginianus) (Tomlin, 2004/05). In the field, there are some documented cases of wild bird mortalities due to the ingestion of seeds treated with imidacloprid (Berny et al., 1999, Bro et al., 2010, Ibáñez et al., 2011, Mineau and Palmer, 2013).
Imidacloprid is predominantly used as seed coating in a large variety of crops (Jeschke et al., 2011, Goulson, 2013). In the 20th century seed coating was responsible for up to 50% of incidents on wildlife caused by approved pesticides in Europe (De Snoo et al., 1999). Since that, the niche market for insecticidal treated seed has tripled and neonicotinoids monopolized 77% of this market in 2005 (Elbert et al., 2008). Farmland birds are at risk of exposure to pesticide-treated seeds because sown seeds are not always properly buried in the field. The US Environmental Protection Agency estimated that about 1% of the drilled seeds remain accessible for granivorous vertebrates. In addition, occasional spillages during sowing activities (especially at field corners and extremes of sowing lines) may also be attractive for foraging birds and increase the risk of exposure to pesticides. Under such circumstances, treated seed ingestion can result in the intake of a high amount of a toxic pesticide over a short foraging time period. It is estimated that a farmland bird could get a lethal dose with the ingestion of less than five imidacloprid treated seeds (Goulson, 2013, Mineau and Palmer, 2013).
In a previous 10 day-exposure study performed in spring with adult red-legged partridges (Alectoris rufa), animals were exposed to seeds treated with the recommended application rate for cereal seed coating and with twice this rate (to assess the effects of potential abuses in pesticide application). The highest imidacloprid dose used in that study (twice the recommended application rate) was shown to reduce exposed partridge survival, whereas both doses produced physiological and biochemical changes, affected fertility and decreased offspring survival (Lopez-Antia et al., 2013). Similar adverse effects on biochemical, oxidative stress and immune system parameters have been reported in poultry (Siddiqui et al., 2007, Balani et al., 2011, Kammon et al., 2012, Gibbons et al., 2014). In the current work, we wanted to test if these effects still occur under a more realistic scenario, in which coated seeds represent only part (20%) of an adult partridge's diet. We also considered two exposure periods that correspond to the sowing seasons of autumn (long-cycle winter cereal) and late winter (short-cycle winter cereal) in Spanish farmlands. Moreover, our main interest was to study the impact of the insecticide on reproduction, as the published literature in this regard is limited to only one preliminary study (Lopez-Antia et al., 2013); in that experiment the reproductive results were obtained from a small number of breeding pairs and thus should be confirmed. In the current study, based on a larger sample size, we also studied new reproductive performance parameters (egg quality and content; offspring immunity), in order to obtain a better understanding of the indirect effects of imidacloprid on reproduction.
We hypothesized that exposure to field doses of imidacloprid would produce oxidative system imbalance and would impact on important health parameters, such as the immune system, or on the reproductive system (Banerjee et al., 2001, Sheweita et al., 2005, Agarwal et al., 2012). Carotenoid-based ornamentation has been proposed as a good indicator of oxidative damage and general health status (Pérez-Rodriguez and Viñuela, 2008) and is known to influence reproductive investment (Alonso-Alvarez et al., 2012). The simultaneous investigation of effects on all these variables should help us to better understand the range of effects and toxicity mechanism of imidacloprid.
Section snippets
Experimental design
The experiment was conducted in the Dehesa de Galiana experimental facilities (Ciudad Real, Spain). All experimental protocols were approved by the Committee on Ethics and Animal Experimentation of the University of Castilla-La Mancha. We used 96 (51 females and 45 males) captive-born, one year-old red-legged partridges from the experimental farm of the University of Castilla-La Mancha. The sex of individuals was determined genetically following Fridolfsson and Ellegren (1999). Partridges were
Effects on adult partridges
Imidacloprid treatment at the high dose killed all partridges in 21 days, with lethality occurring earlier in females than in males (χ2=7.74, p=0.005) (Fig. 1), the mean survival time for the high dose group was 6.7±1.1 days for females and 12.7±1.8 for males. The first deaths occurred on the third day of autumn treatment (10 partridges died this day, eight females and two males). This mortality rate in the high dose group (100%) was higher than in the low dose group (18.7%; χ2=44.75, p<0.001)
Discussion
The target dose of imidacloprid (the recommended application rate for cereal seed coating) killed all partridges, with mortality occurring more rapidly in females than in males. A sublethal dose of 20% of the recommended application rate reduced levels of some plasma biochemical parameters (glucose, Mg and LDH), increase SOD activity, produced changes in carotenoid-based integumental coloration, reduced the clutch size, delayed the first egg lay date, increased egg yolk vitamins and carotenoids
Conclusions
This study revealed that imidacloprid exposure not only have lethal effects, but also numerous deleterious sub-lethal effects on redox balance, secondary sexual traits and reproduction in a bird at sublethal levels equivalent to 20% of treated seed in diet during sowing periods. We also found a reduced cell-mediated immune response in the offspring not directly exposed to the toxic. In addition to seed treatment, imidacloprid is applied through the irrigation water and as foliar spray, after
Acknowledgments
This study financed by FEDENCA (Real Federación Española de Caza) and Oficina Nacional de Caza (Project number: UCTR100161) with the partnership of Fundación Biodiversidad. Funding sources had no involvement in study design or execution. We thank to Jordi Feliu, Pablo Camarero, Xurxo Piñeiro, Esther Garcia-de Blas, Jaime Rodriguez-Estival, Ines Sanchez Barbudo and Monica Martinez-Haro for their help.
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As acknowledged in the manuscript, all experimental protocols were approved by the Committee on the Ethics and Animal Expectation of the university of Castilla-La Mancha. Animal welfare was guaranteed in the terms established in the Directive 63/2010 of the European Council concering the protection of animals used for scientific purposes.The funding sources are all acknowledged in the manuscript.