**Semi Aquatic Top-Predators as Sentinels of Diversity and Dynamics of Pesticides in Aquatic Food Webs: The Case of Eurasian Otter (***Lutra lutra***) and Osprey (***Pandion haliaetus***) in Loire River Catchment, France**

Charles Lemarchand1, René Rosoux2 and Philippe Berny1 *1Toxicology laboratory, VetAgro Sup, Marcy l'Etoile, 2Muséum des Sciences Naturelles d'Orléans, France* 

### **1. Introduction**

288 Pesticides in the Modern World - Risks and Benefits

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The Eurasian otter (*Lutra lutra, Lutrinae*, figure 1) and the osprey (*Pandion haliaetus, Pandionidae*, figure 2), formerly widespread in Europe and in France, have strongly declined during the 20th Century, following direct persecutions, habitat alteration and pollution, and consecutively decline of main prey. Direct persecutions were perpetrated because both species were considered as active competitors for fishing activity: otters were massively trapped for fur, and osprey populations were dismantled by direct shot or egg destruction in nests. Otter and osprey are both semi-aquatic top-predators species: diet is highly dominated by fish, which constitute at least 80 % and almost 98-100 % of the averaged prey biomass consumed by otter and osprey, respectively. However, diet studies of otter and osprey never showed any strong predation impact on fish diversity or biomass in rivers (reviews in Poole, 1989; Thibault et al. 2001; Clavero et al. 2003; Britton et al. 2005; Kruuk, 2006; Dennis, 2008). This diet specificity influence otter physiological characteristics: by comparison with other mammalian carnivores, a specific diversity and accumulation pattern of essential fatty acids of aquatic origin from food to otter tissues was recently shown (Koussoroplis et al. 2008). Another diet characteristic of both species is the diversity of prey and their opportunistic hunting behaviour: almost all fish species available in otter or osprey local habitat are able to be consumed, depending on hunting conditions, with important diet variations between seasons, during life cycle or between populations. This is particularly true concerning osprey, a migrating species present in northern and western Europe during reproductive season, and wintering from southwestern Europe to sub-Saharan Africa, but was also observed concerning sedentary otter. Because of their high trophic level, habitat requirements and main ecological characteristics, otter and osprey can be considered as good sentinels and indicator species of global contamination and biomagnification of toxic contaminants in aquatic food webs of large rivers, estuaries, reservoirs and lakes.

Semi Aquatic Top-Predators as Sentinels of Diversity and Dynamics of Pesticides in Aquatic

et al. 2000; Rattner et al. 2004; Lemarchand et al. 2010).

Fig. 2. Osprey (*Pandion haliaetus*, adult female; photo C. Lemarchand)

Related to their plant-specific metabolic action, water solubility and poorly lipophilic characters, herbicides are documented as less toxic pesticides to vertebrates than insecticides

concerning top predators species.

Food Webs: The Case of Eurasian Otter (*Lutra lutra*) and Osprey (*Pandion haliaetus*) in Loire … 291

aquatic systems or wild environment of these countries was recently underlined (UNEP, 2008). Due to environmental stability and persistence of DDT and metabolites, and probably following post-ban use of old stocks, these compounds are still present in environment and were recently detected in otters or ospreys (Kannan et al. 2004; Lemarchand et al. 2007, 2010; Henny et al. 2008). For this latter species, continuing of DDT use in some developing countries is an additional threat during the wintering period. Lindane, and to a lesser extent, Aldrin, Endosulfan and Methoxychlor were the main other OC pesticides quantified in otter or osprey during previous studies. However, most of studies reported low concentrations of these compounds when compared to DDT residues, and therefore a limited contribution to total OC contamination (Wiemeyer et al. 1988; Mason and Macdonald, 1993a,b, 1994; Elliott

Cholinesterase inhibitors as organophosphate (OP) and carbamate (CA) pesticides were widely used worldwide as insecticides for the protection of cultivated plants or livestock. Direct and indirect toxicity of cholinesterase inhibitors (*e.g.* Carbofuran, Mevinphos) were underlined on insect-consumer birds, birds of prey and scavengers like white-tailed sea eagle (*Haliaetus albicilla*) or red kite (*Milvus milvus*) (Hart et al. 1993; Elliot et al. 1996; Berny and Gaillet, 2008). These insecticides are highly toxic to birds of prey, nevertheless intoxication cases following OP and CA pesticides contamination remained rare when compared to total reported deaths (Fleischli et al. 2004). Pyrethroids insecticides were recently preferred to OP and CA pesticides uses. Indeed, pyrethroids pesticides are considered as a safer method of pest control because of their lower direct toxicity on mammals and birds (Martin et al. 1998; Chu et al. 2005). Nevertheless, data on pyrethroids insecticides diversity, persistence or toxicity in wild fauna are very poor in literature

Fig. 1. Eurasian Otter (*Lutra lutra*, adult male, photo R. Rosoux).

Indeed, after direct destructions, contamination by persistent pollutants (*e.g.* pesticides, polychlorinated biphenyls (PCBs) or heavy metals) is blamed to be the causative agent of the decline of otter and osprey populations, throughout Europe as elsewhere in the world. On seldom occasions, *acute poisoning* after direct exposure or secondary poisoning by ingestion of highly contaminated prey were observed on otters in France, especially after oil spills, other industrial accidents or following heavy treatments with insecticides, avicides or rodenticides (Fournier-Chambrillon et al. 2004; Lafontaine et al. 2005; Berny, 2007; Lemarchand et al. 2010). Elsewhere in the world, acute poisoning of many marine otters (*Enhydra lutris*) were reported after Exxon Valdez oil spill in 1989 in Alaska (Garshelis and Jonhson, 2001). On the other side cases of acute poisoning were very rarely observed on ospreys. Potential long-term effects of trophic originating toxic compounds (*i.e. chronic poisoning*) on otters and ospreys were often investigated through reports of contamination or intoxication cases on monitored populations. Pesticides, and particularly organochlorine (OC) pesticides were the most commonly analyzed elements (but also heavy metals and PCBs, these latter generally associated with OC pesticides to assess the total OC contamination). Pesticides uses in the European Union are clearly specified by Directive EC 91/414, but illegal poisoning of wild, game and domestic animals still occurs, and often results from pesticides abuse or illegal use (Berny, 2007; Berny and Gaillet, 2008). Among OC pesticides, dichorodiphenyltrichloroethane (DDT) and its main metabolites, particularly dichlorodiphenyldichloroethylene (DDE), were shown to accumulate in otter and osprey, causing body condition alteration, direct reproductive failure (like eggshell thinning observed on osprey) and consecutively population decline (Spitzer et al. 1978; Wiemeyer et al. 1988; Mason and Macdonald 1993a, b; Ewins et al. 1999; Elliott et al. 2000; Ruiz-Olmo et al. 2000; Henny et al. 2008). As these deleterious effects were documented on a very large spectrum of wild and domestic species after insect pests control, DDT uses were severely controlled or banned from the 1970's in developed countries (ban from 1973 in France). Nevertheless, DDT is still used in developing countries, particularly in India or Africa by indoor spraying during anti-malaria campaigns. The risk of DDT flow into agricultural and

Indeed, after direct destructions, contamination by persistent pollutants (*e.g.* pesticides, polychlorinated biphenyls (PCBs) or heavy metals) is blamed to be the causative agent of the decline of otter and osprey populations, throughout Europe as elsewhere in the world. On seldom occasions, *acute poisoning* after direct exposure or secondary poisoning by ingestion of highly contaminated prey were observed on otters in France, especially after oil spills, other industrial accidents or following heavy treatments with insecticides, avicides or rodenticides (Fournier-Chambrillon et al. 2004; Lafontaine et al. 2005; Berny, 2007; Lemarchand et al. 2010). Elsewhere in the world, acute poisoning of many marine otters (*Enhydra lutris*) were reported after Exxon Valdez oil spill in 1989 in Alaska (Garshelis and Jonhson, 2001). On the other side cases of acute poisoning were very rarely observed on ospreys. Potential long-term effects of trophic originating toxic compounds (*i.e. chronic poisoning*) on otters and ospreys were often investigated through reports of contamination or intoxication cases on monitored populations. Pesticides, and particularly organochlorine (OC) pesticides were the most commonly analyzed elements (but also heavy metals and PCBs, these latter generally associated with OC pesticides to assess the total OC contamination). Pesticides uses in the European Union are clearly specified by Directive EC 91/414, but illegal poisoning of wild, game and domestic animals still occurs, and often results from pesticides abuse or illegal use (Berny, 2007; Berny and Gaillet, 2008). Among OC pesticides, dichorodiphenyltrichloroethane (DDT) and its main metabolites, particularly dichlorodiphenyldichloroethylene (DDE), were shown to accumulate in otter and osprey, causing body condition alteration, direct reproductive failure (like eggshell thinning observed on osprey) and consecutively population decline (Spitzer et al. 1978; Wiemeyer et al. 1988; Mason and Macdonald 1993a, b; Ewins et al. 1999; Elliott et al. 2000; Ruiz-Olmo et al. 2000; Henny et al. 2008). As these deleterious effects were documented on a very large spectrum of wild and domestic species after insect pests control, DDT uses were severely controlled or banned from the 1970's in developed countries (ban from 1973 in France). Nevertheless, DDT is still used in developing countries, particularly in India or Africa by indoor spraying during anti-malaria campaigns. The risk of DDT flow into agricultural and

Fig. 1. Eurasian Otter (*Lutra lutra*, adult male, photo R. Rosoux).

aquatic systems or wild environment of these countries was recently underlined (UNEP, 2008). Due to environmental stability and persistence of DDT and metabolites, and probably following post-ban use of old stocks, these compounds are still present in environment and were recently detected in otters or ospreys (Kannan et al. 2004; Lemarchand et al. 2007, 2010; Henny et al. 2008). For this latter species, continuing of DDT use in some developing countries is an additional threat during the wintering period. Lindane, and to a lesser extent, Aldrin, Endosulfan and Methoxychlor were the main other OC pesticides quantified in otter or osprey during previous studies. However, most of studies reported low concentrations of these compounds when compared to DDT residues, and therefore a limited contribution to total OC contamination (Wiemeyer et al. 1988; Mason and Macdonald, 1993a,b, 1994; Elliott et al. 2000; Rattner et al. 2004; Lemarchand et al. 2010).

Cholinesterase inhibitors as organophosphate (OP) and carbamate (CA) pesticides were widely used worldwide as insecticides for the protection of cultivated plants or livestock. Direct and indirect toxicity of cholinesterase inhibitors (*e.g.* Carbofuran, Mevinphos) were underlined on insect-consumer birds, birds of prey and scavengers like white-tailed sea eagle (*Haliaetus albicilla*) or red kite (*Milvus milvus*) (Hart et al. 1993; Elliot et al. 1996; Berny and Gaillet, 2008). These insecticides are highly toxic to birds of prey, nevertheless intoxication cases following OP and CA pesticides contamination remained rare when compared to total reported deaths (Fleischli et al. 2004). Pyrethroids insecticides were recently preferred to OP and CA pesticides uses. Indeed, pyrethroids pesticides are considered as a safer method of pest control because of their lower direct toxicity on mammals and birds (Martin et al. 1998; Chu et al. 2005). Nevertheless, data on pyrethroids insecticides diversity, persistence or toxicity in wild fauna are very poor in literature concerning top predators species.

Fig. 2. Osprey (*Pandion haliaetus*, adult female; photo C. Lemarchand)

Related to their plant-specific metabolic action, water solubility and poorly lipophilic characters, herbicides are documented as less toxic pesticides to vertebrates than insecticides

Semi Aquatic Top-Predators as Sentinels of Diversity and Dynamics of Pesticides in Aquatic

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

Environnement") and regional naturalists were contributors for this study.

**2.2 Animals monitoring and sampling** 

**2. Materials and methods** 

**2.1 Study area** 

economic activity.

Food Webs: The Case of Eurasian Otter (*Lutra lutra*) and Osprey (*Pandion haliaetus*) in Loire … 293

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

Concerning such rare species, it is particularly difficult to obtain sufficient sample material from enough individuals to support analysis and statistics. For ethical reasons it was not imaginable to trap or kill otters or ospreys for analyses. To avoid any vital risk related to handling, capture and bleed of animals were not considered. Furthermore, otter and osprey are fully protected by national and international laws, and listed as species of interest by the European Community (Habitats Directive 92/43/EC, Birds Directive 79/409/CEE). All operations were therefore entirely conducted under appropriate authorizations by a noninvasive approach. A large network, constituted by people in charge of otter and osprey studying and monitoring in mainland France was built to organize and enhance sampling under the coordination of the Muséum d'Orléans. The national agency for game and wildlife (ONCFS), hunting federations (FDC), the national agency for water and aquatic environments (ONEMA), health centres of the national union (UFCS) and of the birds protection league (LPO – French representative of Bird Life International), the national research centre on birds population biology (CRBPO, associated with the French national museum of natural history MNHN and Mr Rolf Wahl, in charge of osprey ringing program in France), the French Ministry of Environment (MEEDDM and DREAL Centre), the national agency for forests (ONF), private land owners and companies, museums, associations ("Loiret Nature

Concerning otters, only road-traffic killed individuals and those found dead in the wild in study area were collected. Based on visual observation, carcasses found more than 24h (during summer) or 48h (during winter) after road collision were considered too degraded

(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).
