The Impact of Pesticides - Academy Publish

Study area and SamplingThis study was carried out in the northwest of the Region of Murcia which is locatedin southeastern Spain (Figure 1). The study area covers about 250,000 ha andelevation ranges from 550 to 1,521 m. The climate is Mediterranean with a meanannual rainfall of 400 mm. The landscape is characterized by mountain slopescovered by Aleppo pine forests (Pinus halepensis) interspersed with farming crops(cereals, vineyards, olive and woody crops) and far from any urban, industrial ormining zones. The nearest industrial area, urban area (population >350,000 h), andhighway are situated 70, 60, and 70 km away, respectively. The study area has oneof the highest densities of Booted eagle and Short-toed eagle (Circaetus gallicus) inEurope (Martínez, 2002) with one pair per 3-4 km 2 (Martínez et al., 2006). TheBooted eagle population in the area was estimated at 50 breeding pairs between1999 and 2004 (Martínez et al., 2006; unpublished results).From the 1999-2003 breeding seasons, the reproductive behaviour of the Bootedeagle was observed from the beginning of courtship (March-April) until nestlingsleft their nests (June-July). Close to the predicted hatching date, nests were visitedtwice weekly. The same nest was visited each year. A total of 62 nestlings werestudied and blood samples (2.0 ml) were taken by puncturing the radial vein using ahypodermic needle and syringe and taken immediately to the laboratory inrefrigerated conditions and frozen at –40ºC until processing. Nestlings weresampled at between 35 and 45 days old in order to obtain sufficient blood sampleswithout damage the nestlings’ health. Careful steps were taken in order to avoidstressing the nestlings. The head of the nestlings was covered before the animal wasput into a bag and brought down to the ground, where a blood sample was taken bythe veterinarian from the “Santa Faz” Wildlife Recovery Centre (Alicante, Spain),who also evaluated the health status of the nestlings. All individuals were markedwith a numbered steel band. Finally, nestlings were returned to the nest. These nestswere monitored until the chicks commenced flying. No chicks were injured duringthe sampling and all were able to fly satisfactorily.Organochlorine AnalysisBlood samples were analyzed for dichlorodiphenyltrichloroethane (p,p’-DDT),dichlorodiphenyldichloroethane (p,p’-DDD), and dichlorodiphenyldichloroethylene(p,p’-DDE).Reagents and StandardsAll reagents used for the analysis were of a trace analysis grade. Hexane, acetone,petroleum ether, diethyl ether were supplied by Lab-scan Analytical Sciences andanhydrous sodium sulphate by Merck Co. (Darmstadt). SepPak Florisil columnswere supplied by Waters. Pesticide standard (Pesticide Mix 4-8858 dissolved inmethyl alcohol-methylene chloride 98:2) was procured from Supelco (USA). Priorto analytical procedures, all glassware was rinsed several times with acetone andhexane.AcademyPublish.org - The Impact of Pesticides324