Ecotoxicology and Environmental Safety 52, 8}12 (2002) Environmental Research, Section B doi:10.1006/eesa.2002.2166, available online at http://www.idealibrary.com on Toxicity and Bioaccumulation of Fipronil in the Nontarget Arthropodan Fauna Associated with Subalpine Mosquito Breeding Sites P. F. Chaton, P. Ravanel, M. Tissut, and J. C. Meyran Laboratoire Ecosyste` mes et Changements Environnementaux, Centre de Biologie Alpine, UniversiteH Joseph Fourier, BP 53, F-38041 Grenoble Cedex 9, France Received March 13, 2001 species resistant to other insecticides. At present, "pronil In order to examine ecological impact of 5pronil use for larval appears to be one ofthe most e !ective synthetic insecticides culicine control in natural hydrosystems, toxicity and bioac- for mosquito larvae control (Chaton et al., 2001). Bioac- cumulation of this new insecticide were analyzed on aquatic cumulation studies have indicated a low direct bioavailabil- species representative of the nontarget arthropodan fauna (non- ity in the aquatic environment, in addition to a low-rate culicine larval Diptera:Chaoboridae, Chironomidae; planktonic integumental route for penetration into larvae. Moreover, Crustacea:Cladocera, Copepoda, Ostracoda) associated with importance ofthe digestive route forinsecticide accumula- target larval mosquito populations in the subalpine breeding tion into larvae has suggested that ingestion might be the sites. Standard toxicological bioassays using 5pronil aqueous best way of "pronil administration to mosquito larvae solutions from 1 to 2000 nM indicated di4erent sensitivity levels among species. Insecticide bioaccumulation analyses, using (Chaton et al., 2001). [14C]5pronil solutions in simpli5ed laboratory ecosystem, also However, before considering the operational use of "p- indicated large di4erences among species. These di4erences may ronil for reducing target populations of larval Culicidae, come from biological parameters characteristic of each species. some ecotoxicological information on this relatively new Taking into account these nontarget e4ects of 5pronil, a possible insecticide against the nontarget arthropodan fauna is re- strategy of use of this insecticide for integrated mosquito control quired. This investigation is currently being carried out on management was proposed, which is based upon selective dietary planktonic crustacean and nonculicine larval dipteran spe- absorption of the insecticide by larval Culicidae. cies. Planktonic Crustacea represent an important part of 2002 Elsevier Science (USA) the "ltering fauna in the subalpine mosquito breeding sites Key Words: 5pronil; biocidal activity; bioaccumulation; aqua- (TeH tard, 1974; Amoros, 1984). Among nonculicine larval tic environment; Diptera; Crustacea. dipteran species, Chaoboridae are predators (Durieu and Thomas, 1995), thus involved in a food chain including larval mosquitoes (Rey et al., 2000a). Chironomidae, which INTRODUCTION are sediment-dwelling organisms, are involved in recycling organic detritus (Ali, 1980) and are major items for numer- Among the new chemical insecticides likely to be widely ous aquatic predators (see references in Dickman, 2000). used in the environment, "pronil is a promising phenyl- Such a comparative analysis ofthe nontarget e !ects of pyrazole compound (Colliot et al., 1992) because ofits "pronil will allow understanding ofthe biological para- unique toxicity mechanism (Buckingam et al., 1994; Cole meters involved in the toxicity ofthis insecticide in aquatic et al., 1993). Due to the e!ect of "pronil being highly speci"c organisms, and adjustment ofa new possible selective insec- to the invertebrate -aminobutyric acid (GABA)-receptor ticide strategy usable in the aquatic medium. (Hainzl and Casida, 1996), it was used primarily in the terrestrial environment against insect pests (Gaulliard, 1996; Martin et al., 1996; Balania and De Visscher, 1997; Valles MATERIALS AND METHODS et al., 1997). In the aquatic environment, preliminary evalu- Test Organisms ations in laboratory (Ali et al., 1998) and in the "eld (Ali et al., 1999; Sulaiman et al., 1997) have suggested a possible Test species analyzed here originated from di!erent sub- use of "pronil against larval culicine and chironomid midge alpine mosquito breeding sites known for the abundance of their nontarget arthropodan populations (Pautou, 1981; To whom correspondence should be addressed. Fax: 33 4 76 51 44 63. Rey et al., 2000a). Among the Crustacea, three ecologically E-mail: [email protected]. di!erent subclasses were chosen and the most representative 8 0147-6513/02 $35.00 2002 Elsevier Science (USA) All rights reserved. NONTARGET EFFECT OF FIPRONIL IN MOSQUITO BREEDING SITES 9 species ofeach subclass were studied (Cladocera: Daphnia TABLE 1 pulex de Geer; Copepoda: Acanthocyclops robustus Jurine, Di4erential Sensitivity to Fipronil among Species of the Non- Diaptomus castor Jurine; Ostracoda: Eucypris virens Jurine). target Arthropodan Fauna:Log Probit Determination of LC 50 Among the larval nonculicine dipteran fauna, Chironomus (Median Lethal Concentrations) after 48 Hours of Treatment annularius Meigen (Chironomidae) and Chaoborus crystal- 48-h LC linus de Geer (Chaoboridae) were selected. Immediately Species n Slope$SE (nM) 95% CL after collecting, specimens were determined and then sub- jected to bioassay and bioaccumulation processing. Crustacea Cladocera Daphnia pulex 900 Insensitive Bioassay Protocol Copepoda Acanthocyclops robustus 900 4.8$0.1 194.2 143.9}262.1 Tolerance to "pronil was analyzed by standard bioassay Diaptomus castor 600 4.7$0.1 7.9 6.4}9.9 technique (WHO, 1981) adapted to planktonic Crustacea Osracoda following Rey et al. (2000b) and to the larval nonculicine Eucypris virens 900 Insensitive dipteran fauna following Rey et al. (1998). Bioassays were Diptera conducted in triplicate on samples of20 same-sized speci- Chaoboridae Chaoborus crystallinus 600 1.5$0.3 1478.5 1056.2}2096.3 mens placed in disposable plastic vials containing 20 ml Chironomidae solution without food. Controls were reared in de- Chironomus annularius 300 1.2$0.4 5.6 3.6}24.5 chlorinated tap water. Treated specimens were reared in aqueous solutions with di!erent concentrations (i.e., 1, 10, Note. n"total number ofspecimens assayed. 25, 50, 100, 250, 500, 1000, 1500, and 2000 nM) of "pronil (99.9% purity, Aventis Crop Science, La Dargoire, Lyon, France). These concentrations were obtained by serial dilu- tion in tap water ofa 0.1 M initial ethanol stock solution. water. Fipronil samples and animal homogenates were ana- Bioassay medium for chironomid larvae included 5 g of lyzsed by liquid scintillation counting, using a 1414 Win- sterilized sand (Prolabo, granulometry from 150 to 210 m) spectral (EG & G Wallac) and Ready Safe scintillation #uid in order to prevent larval cannibalism (Ali et al., 1998). The (Beckman). Each experiment was repeated three times. absence ofinsecticide loss to the sand was checked using [C]"pronil. RESULTS The mortality was recorded 48 h after the beginning of Diwerential Toxicity of Fipronil the treatment. After correction by Abbott's formula (Ab- bott, 1925), dose}mortality data were subjected to log- Table 1 reveals large di!erences in tolerance levels to probit analysis (Raymond, 1985). "pronil among species after 48 h of treatment. Among Crus- tacea, D. pulex and E. virens are insensitive, even to concen- trations close to the solubility limit of "pronil in water. Bioaccumulation Studies Strong di!erences in tolerance exist between species within Fipronil accumulation was studied on 800 specimens for the same subclass (e.g., within Copepoda: between A. robust- each species. Each assay was performed for 48 h on 20 us, less sensitive, and D. castor, more sensitive). specimens placed in plastic vials containing 20 ml ofwater Larval nonculicine dipteran species appear either sensi- solutions of[ C]"pronil at concentrations used for the tive, such as C. annularius, or strongly insensitive, such as toxicological bioassays. These radiolabeled "pronil solu- C. crystallinus, which shows a larval mortality ofmerely tions were obtained by serial dilution in tap water of 68.7% after 48 h of treatment for the maximum "pronil a 4.35 mM [C]"pronil ethanol solution (Aventis Agricul- concentration used. ture Ltd, Ongar, UK) (speci"c activity, 947.2 MBq mmol\; radiopurity, 97}99%). [C]Fipronil Accumulation After the 48 h treatments, accumulation of [C]"pronil within the treated organisms was measured and the accu- After the 48 h treatments, the AF varied according to the mulation factor (AF) was calculated. The AF corresponds to species (Fig. 1). Among Dipteran larvae, the AF was less the ratio obtained at the end ofthe experiment between the than 1 for C. crystallinus and more than 60 for C. annularius. [C] concentration per gram fresh weight of animal and Among Crustacea, the AF was close to 10 for E. virens, close the [C] concentration per milliliter ofmedium. A 1-ml to 30 for D. pulex and D. castor, and more than 40 for A. sample ofthe "pronil solution was taken and the whole robustus. No clearcut relationship was observed between treated animal sample was rinsed in 2 ml distilled water, AF and LC, except for C. crystallinus, where a very low weighed, ground, and then homogenized in 5 ml distilled AF might explain the high LC value. 10 CHATON ET AL. FIG. 1. Comparison of "pronil toxicity (expressed as 48-h LC, nM) and bioaccumulation (expressed as AF) among nontarget arthropodan species (Crustacea: D. pulex, A. robustus, D.