**2. Materials and methods**

#### **2.1 Study area**

292 Pesticides in the Modern World - Risks and Benefits

(Berny, 2007). Data on herbicides diversity and toxicity on vertebrates or predators, especially otters or ospreys, are particularly rare. Nevertheless, some studies demonstrated a direct effect of herbicides on herbivorous mammals or bird diversity or abundance during land use modifications (Santillo et al. 1989a,b). Bioaccumulation potential of herbicides to a top predator was recently confirmed by a study in Washington State (USA) on sediments, fish and ospreys (Chu et al. 2007). Furthermore, recent studies underlined a direct impact of herbicides, particularly triazines, on fish and amphibians' reproduction or survival (Langlois et al. 2009; Tillitt et al. 2010). Direct impact of herbicides on fish or amphibians' populations would indirectly affect otters and ospreys by a reduction in food resource. Therefore toxicity of some persistent herbicides on vertebrates could be underestimated by an insufficient risk evaluation. At the beginning of the 1980's in France, otters only survived in two distinct populations: in the Massif Central mountains (centre), and along Atlantic Ocean and western wetlands of the country (Bouchardy, 1986). At the same period, osprey had disappeared of continental France as a nesting species. Legal protection of the otter and the osprey was decided from 1976. First signs of species recovery or return were recorded soon after. From 1985 increase and expanding of otter populations were proved and monitored in the whole repartition area of the species in France (Bouchardy, 1986; Rosoux and Bouchardy, 2002). In spring 1984, one pair of osprey stopped its migration towards northern Europe and built a nest along Loire River. Species is nesting again in continental France since 1985 (Coll., 1996). As osprey is a semicolonial and philopatric species, other pairs quickly mated close to the first one, starting a new expanding population. European directives and national action plans allowed the protection and / or the restoration of both species habitat (Rosoux et al, 1999; Nadal and Tariel, 2008; Kuhn, 2009). The main characteristic of these species recoveries is their entirely natural process. Indeed, otter and osprey were never been reintroduced or reinforced in France, in order to establish habitats requirements, main natural and anthropogenic limits to populations, to locate colonization corridors and major sites for reproduction and breeding. After about three decades of protection, otter population in France is still increasing, formerly isolated populations met from the beginning of the 2000's and the repartition area of the species covers the whole Massif Central related to the western third of the country (Bouchardy et al. 2001; Kuhn, 2009; Lemarchand and Bouchardy 2011). 37 reproductive pairs of ospreys wee noted in 2010 in continental France, mainly distributed along the medium part of the Loire River, but a geographical expansion of the species towards other river systems was recently noted (Nadal and Tariel, 2008). Increase of otter and osprey populations particularly concerns Loire River catchment, a major dispersal corridor that should be decisive for species conservation and dispersion in the whole country. As many predators, otter and osprey suffered from a bad reputation, but are now associated with preserved habitats and food resource (Chanin, 2003; Whitfield et al. 2003; Grove et al. 2009). However, otter and osprey remain sparse in France and are listed on UICN National Red Lists as "Minor preoccupation

(LC)" and "Vulnerable (VU)", respectively (UICN France et al. 2008, 2009).

Objectives of this study were to evaluate the contamination of two flagship species (European otter and osprey) by a wide spectrum of pesticides, using a standard protocol of pesticides analyses in wild or domestic fauna and a non-invasive animal approach during a natural recolonization process in Loire River catchment. Since 2004 for the otter and 2007 for the osprey, a large toxicological program was launched during the "Plan Loire Grandeur Nature" program in France. 45 pesticides, including herbicides, organochlorine, organophosphate, carbamate and pyrethroids pesticides and a few main metabolites were systematically analyzed in otters and ospreys (but also in great cormorants, freshwater fish

and invertebrates) from Loire River catchment (Lemarchand et al. 2007, 2009, 2010).

The study area corresponded to the whole Loire River and main tributaries catchment in France (Fig. 3). Loire River catchment (117000 km2, total length of rivers and tributaries of about 40000 km) is characterized by an important diversity of habitats and species, and is considered as one of the most preserved large hydrosystems in Western Europe. A national and European action plan, "Plan Loire Grandeur Nature", is running since 1994 to study and conserve this diversity, but also to protect inhabitants from floods and to maintain economic activity.

Fig. 3. Map of the Loire River (bold) catchment in mainland France
