Chitobiase activity as an indicator of altered survival, growth and reproduction in Daphnia pulex and Daphnia magna (Crustacea: Cladocera) exposed to spinosad and diflubenzuron

Ecotoxicology and Environmental Safety 74 (2011) 800–810

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Chitobiase activity as an indicator of altered survival, growth and reproduction in Daphnia pulex and Daphnia magna (Crustacea: Cladocera) exposed to spinosad and diflubenzuron Claire Duchet a,b, Marı´lia Mitie Inafuku b,1, Thierry Caquet b, Michel Larroque c, Evelyne Franquet d, Christophe Lagneau a, Laurent Lagadic b,n a

´moustication du Littoral Me´diterrane ´en, 165 avenue Paul-Rimbaud, F-34184 Montpellier, France Entente Interde´partementale de De ´ cologie et Sante´ des E ´ cosyste mes, E ´quipe E ´cotoxicologie et Qualite´ des Milieux Aquatiques, Agrocampus Ouest, 65 rue de Saint Brieuc, F-35042 Rennes, France INRA, UMR985 E Laboratoire de Chimie Analytique, UMR Qualisud, Faculte´ de Pharmacie, Universite´ Montpellier I, 15 Avenue Charles-Flahault, BP14491, F-34093 Montpellier Cedex 5, France d ´ cologie et de Pale´oe´cologie, Faculte´ des Sciences et Techniques Saint Je ´en d’E ´rˆ Universite´ Paul Ce´zanne, Institut Me´diterrane ome, C31, F-13397 Marseille, France b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 July 2010 Received in revised form 5 November 2010 Accepted 8 November 2010 Available online 15 April 2011

Chitobiase is involved in exoskeleton degradation and recycling during the moulting process in arthropods. In aquatic species, the moulting fluid is released into the aqueous environment, and chitobiase activity present therein can be used to follow the dynamics of arthropod populations. Here, chitobiase activity was used for monitoring the impact of mosquito candidate larvicides on Daphnia pulex and Daphnia magna under laboratory conditions. Both species were exposed to spinosad (2, 4, 8 mg L  1) and diflubenzuron (0.2, 0.4, 0.8 mg L  1) for 14 days. Bacillus thuringiensis var. israelensis (Bti; 0.25, 0.5, 1 mL L  1) was used as the reference larvicide. Chitobiase activity, adult survival, individual growth and fecundity, expressed as the number of neonates produced, were measured every 2 days. Average Exposure Concentrations of spinosad were ten-fold lower than the nominal concentrations, whereas only a slight deviation was observed for diflubenzuron. In contrast to Bti, spinosad and diflubenzuron significantly affected both species in terms of adult survival, and production of neonates. As compared to D. pulex, D. magna was more severely affected by diflubenzuron, at low and medium concentrations, with reduced adult growth and much lower chitobiase activity. Chitobiase activity was positively correlated with the individual body length, number of neonates produced between two consecutive observation dates, and number of females and neonates. In addition, the significant positive correlations between chitobiase activity measured on the last sampling date before the first emission of neonates and the cumulative number of neonates produced during the whole observation period strongly support the potential of the activity of this chitinolytic enzyme as a proxy for assessing the dynamics of arthropod populations exposed to larvicides used for mosquito control. & 2010 Elsevier Inc. All rights reserved.

Keywords: Daphnia sp. Neurotoxicant Insect growth regulator Bti Chitobiase Population dynamics Bioindicator Environmental risk assessment

1. Introduction Chitobiase is one of the two chitinolytic enzymes involved in exoskeleton degradation and recycling during ecdysis in arthropods. A first enzyme, chitinase, hydrolyses chitin, a polymer of b-(1–4)-linked N-acetyl-b-glucosamine (NAG), to oligomers and trimers of NAG, while chitobiase subsequently hydrolyses oligomers and trimers of NAG to NAG monomers (Muzzarelli, 1977; Merzendorfer and Zimoch, 2003). Chitobiase activity is located in both the epidermis and hepatopancreas of arthropods (Spindler-

n

Corresponding author. Fax: + 33 223 485 440. E-mail address: [email protected] (L. Lagadic). 1 Present address: Laborato´rio de Ecotoxicologia, Centro de Energia Nuclear na Agricultura, Universidade de Sa~ o Paulo, av Centena´rio 303, Cx. 96, 13400-970 Piracicaba, Sa~ o Paulo, Brazil. 0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2010.11.001

Barth et al., 1990; Zou and Fingerman, 1999a). During ecdysis, the moulting fluid, containing chitobiase, is released into the aqueous environment. Thus, chitobiase theoretically offers the possibility to perform non-destructive measurements of moulting activity of arthropods under field or laboratory conditions. Indeed, analysis of chitobiase activity in water showed promising results as an assessment tool for estimating arthropod biomass, population dynamics and secondary production both in laboratory (Espie and Roff, 1995; Oosterhuis et al., 2000; Sastri and Roff, 2000; Vrba et al., 2004; Sastri and Dower, 2006) and field studies (Hanson and Lagadic, 2005; Sastri and Dower, 2006; Conley et al., 2009). Exposure to toxicants can influence moulting of arthropods with measurable changes in chitobiase activity. Such changes have been observed in crabs exposed to endocrine disruptors (Zou and Fingerman, 1999b, c; Zou, 2005), in crabs and shrimps exposed to metals (Zhang et al., 2006; Xie et al., 2009), and in Daphnia magna

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exposed to pharmaceuticals (Richards et al., 2008). For the vast majority of the chemicals that have been tested, inhibition of chitobiase activity was recorded, except with pharmaceuticals for which a more complex pattern of induction and inhibition has been observed (Richards et al., 2008). It is worth noting that pesticides have rarely been studied with respect to their action on chitinolytic enzyme activity and the moulting process in arthropods, the only reported data concerning the inhibition of chitobiase by endosulfan in Uca pugilator (Zou and Fingerman, 1999b). For mosquito control programmes, insecticides are mostly applied as larvicides. The compounds are directly introduced into aquatic systems inhabited by mosquito larvae (e.g., marshes, ponds and sanitation devices). Cladocerans and other zooplankton groups are water column-dwelling organisms that share the habitat and, at least in part, the food resources of mosquito larvae (Blaustein and Chase, 2007). They may therefore be exposed to larvicides in treated areas, and there is a need for methods that can be used for monitoring the impacts of mosquito control programmes on these non-target organisms. The present study was undertaken to assess the potential of chitobiase activity for monitoring the impact of the candidate larvicides spinosad and diflubenzuron on survival, growth and fecundity of Daphnia pulex and D. magna (Crustacea: Cladocera) under laboratory conditions. These two Daphnia species were chosen because they are frequently found in biotopes where mosquito larvae develop (Metge, 1986). The larvicides were chosen based upon their different modes of action, and possible use for mosquito control. Currently, Bacillus thuringiensis var. israelensis (Bti) is the only larvicide used in Europe as the result of the implementation of the EU Biocidal Products Directive 98/8/EC. It is also widely used for mosquito control all over the world (Boisvert and Lacoursie re, 2004). Therefore, the risk of resistance to Bti in target species should not be neglected, and there is an urgent need to develop new larvicides that could be used alternately. In the present study, Bti was considered as a reference compound since it has the most favourable ecotoxicological profile among all mosquito larvicides. Lebrun and Vlayen (1981) estimated the LC5024 h for D. magna at 2700 mL L  1, and assigned the mortality of daphnids to gill clogging, instead of toxic effects peculiar to Bti at this concentration. Spinosad (DowElanco, Indianapolis, IN, USA) is a new biological insecticide that seems promising for mosquito control (DowElanco, 1996). It is a mixture of spinosyns A and D known as fermentation products of a soil actinomycete (Saccharopolyspora spinosa; Crouse et al., 2001). Spinosad acts as a contact and stomach poison (DowElanco, 1996; Salgado, 1998), and persistently stimulates the insect central nervous system by interacting with nicotinic acetylcholine receptors (Watson, 2001). It is considered as a selective insecticide for insect pest species (Miles and Dutton, 2000), but it may also be toxic to non-target species (Nasreen et al., 2000; Tillman and Mulrooney, 2000; Consoli et al., 2001). In particular, spinosad reduced survival and altered life history traits of D. pulex at 10 mg L  1 in laboratory experiments (Stark and Vargas, 2003). Diflubenzuron is a halogenated benzoylphenylurea. It is an effective stomach and contact insecticide acting by inhibition of chitin synthesis and so interfering with the formation of arthropod cuticle (WHO, 1996; Zhang and Zhu, 2006), leading to death. In crab larvae, diflubenzuron markedly affected the incorporation of glucose and NAG in the premoult stage during secretion of exocuticle (Christiansen et al., 1984). Aquatic arthropods, especially cladocerans (e.g., LC5048 h for D. magna ¼3.7 mg L  1; EPA, 1997), are considered as highly susceptible to diflubenzuron (Pest Management Regulatory Agency, 2001). In this study, biological parameters related to survival, growth and fecundity of groups of control and larvicide-exposed daphnids were measured in laboratory conditions at various dates following

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the beginning of the exposure. Chitobiase activity in the exposure medium was measured in parallel. Correlations between chitobiase activity and survival, growth and fecundity were analysed. Special attention was given to the correlation between chitobiase activity measured on the last sampling date before the first emission of neonates and the cumulative number of neonates produced during the whole experiment, in order to assess the capacity of chitobiase activity to predict the reproduction dynamics of daphnid population. Results are discussed with regard to the possible use of chitobiase activity as an endpoint to be included into the framework of ecological risk assessment of substances used for mosquito control. 2. Materials and methods 2.1. Chemicals B. thuringiensis var. israelensis (Bti) was applied as VectoBacs 12AS (1.2% AI, i.e., 1200 ITU mg  1), a flowable formulation produced by Valent Biosciences (Libertyville, IL, USA). Spinosad was applied as Conserves 120SC (11.6% AI; DowElanco, Indianapolis, IN, USA). Diflubenzuron was applied as wettable powder (Dimilins WP 25, 25% AI, Uniroyal Chemical SARL, Switzerland). Substrate, standard, and buffer components for chitobiase activity measurements were obtained from Sigma–Aldrich Co., Lyon, France. All the products and solvents used for residue analysis were of analytical grade and were purchased from Carlo Erba Re´actifs (Val de Reuil, France). 2.2. Test organisms Experiments were carried out using the 4–6th brood offspring ( o 24 h old) of D. pulex and D. magna reared at the INRA Experimental Unit of Aquatic Ecology and Ecotoxicology (Rennes, France). Daphnid strains were originally from the field and maintained under laboratory conditions for 1 year prior to testing. They were reared in 20 L glass aquaria filled with dechlorinated, charcoal-filtered tap water at 207 1 1C in a light–dark regimen of 16:8 with light intensity of  15 mE m  2 s  1 (OECD, 1998). They were fed three times a week with a suspension of green microalgae (Desmodesmus subspicatus for D. pulex and Chlorella vulgaris for D. magna), batch-cultured according to AFNOR T90-304 (AFNOR, 1993). 2.3. Exposure conditions Tests were performed in 125 mL polystyrene beakers containing 100 mL of exposure medium (dechlorinated, charcoal-filtered tap water and green microalgae suspension). Three nominal concentrations were tested for each compound: 2, 4 and 8 mg L  1 for spinosad, 0.2, 0.4 and 0.8 mg L  1 for diflubenzuron, and 0.25, 0.5 and 1 mL L  1 for Bti. Bti concentrations encompassed the maximum registered rates for aerial treatments (0.50 mL L  1; ACTA, 2008), whereas 8 mg L  1 spinosad was the lowest concentration that affected D. pulex and D. magna populations in field microcosms (Duchet et al., 2008, 2010a), and 0.8 mg L  1 diflubenzuron corresponds to the minimum EC5048 h measured under static laboratory conditions on D. magna neonates (Majori et al., 1984). Concentrations were hence chosen to obtain sufficient survival in the beakers to measure chitobiase activities. Test solutions were obtained by diluting the commercial preparations of the three insecticides in dechlorinated water. Exposure to each concentration was performed in 5 replicates, and 15 beakers remained as untreated controls. Neonates ( o 24 h) of D. pulex (20 per beaker) or D. magna (15 per beaker) were introduced into each beaker at the beginning of the test (Sanchez et al., 2000). Duration of the test was 14 days for both species (time needed to observe at least 3 broods in our conditions). The exposure medium was renewed on each observation day using stock solutions prepared at the beginning of the experiment in order to avoid a sudden and unrealistic change in chemical composition and larvicide concentration of exposure water. 2.4. Spinosad and diflubenzuron residue analysis To determine spinosad and diflubenzuron concentrations in the treated beakers, exposure medium was collected on renewal days d2, d4, d7 and d10. Samples were stored at  20 1C in 125 mL amber glass bottles until analysis. For spinosad analysis, 100 mL of each sample were acidified with 6 mL HCl (pH 2). Spinosad was extracted 3 times with 50 mL dichloromethane. The pooled extracts were evaporated to dryness at 30 1C under a nitrogen flow, and residues were resuspended in 1 mL acetonitrile. Volumes of 50 mL were injected into an HPLC device (Thermoquest P4000) equipped with a UV detector set at 243 nm, and an Eclipse XDB C8 column (150  4.6 mm, 5 mm, Agilent Technologies, Santa Clara CA, USA). The mobile phase consisting of acetonitrile and ammonium formate buffer at

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150 mg L  1 (90:10, v/v) was delivered at 0.8 mL min  1 flow rate. Calibration curves were established using stock solutions of spinosyns A and D (150 and 165 mg L  1, respectively) by successive dilutions in acetonitrile in order to obtain concentrations ranging from 75 to 1500 ng mL  1 and from 82.5 to 1650 ng mL  1 for spinosyns A and D, respectively. The response was linear in the range of concentrations tested (spinosyn A: y ¼28.837x, n¼7, r2 ¼0.998; spinosyn D: y¼ 34.191x, n ¼7, r2 ¼ 0.996). Under these conditions, the limit of quantification was 0.2 mg L  1 for both compounds. Diflubenzuron (50 mL) was extracted 3 times with 40 mL dichloromethane. The pooled extracts were evaporated to dryness at 30 1C under a nitrogen flow, and residues were resuspended in 2.5 mL of an acetonitrile/water mixture (60/40, v/v). Volumes of 100 mL were injected into an HPLC device (Thermoquest P4000) equipped with a UV detector set at 260 nm, and a Nucleosil 100-5 C18 column ¨ (125 mm  3 mm; Macherey-Nagel GmbH and Co. KG, Duren, Germany). The mobile phase consisting of acetonitrile and water (60/40, v/v) was delivered at 0.3 mL min  1 flow rate. Calibration curves were established using stock solutions of diflubenzuron by successive dilutions in acetonitrile in order to obtain concentrations ranging from 20 to 2 mg L  1. The response was linear in the range of concentrations tested (y¼ 1052.6x, n ¼6, r2 ¼ 0.999). Under these conditions, the limit of quantification was 1 mg L  1. As a consequence, samples in triplicate were pooled before extraction and analysis. Average Exposure Concentrations (AEC) for the whole duration of the experiment (14 days) were calculated for spinosad and diflubenzuron, according to Van Wijngaarden et al. (1996): Pn c Dt AEC ¼ Pi n¼ 1 i i i ¼ 1 Dti where i is the sampling date, ci the concentration measured at ti and ci ¼ ðci þ ci1 Þ=2, Dti ¼ ti  ti  1. 2.5. Individual endpoint measurements Every 2 days immobile adults and newborns were counted and removed. The number of mobile adults observed per treated and control beaker on each observation date was used to assess the impacts of exposure on daphnid survival. Newborns were enumerated on each observation date, and the fecundity of daphnids was expressed as the cumulative number of newborns in each beaker on each observation date. For adult body length measurements, surviving individuals were collected separately with a pipette and transferred into a polystyrene cup where they were briefly deposited into a drop of exposure medium and photographed using a digital camera (S40 PowerShot, Canon Inc., Tokyo, Japan) fitted to a binocular dissecting microscope. Surviving adults were then reintroduced into the beakers and fed with green microalgae (equivalent of  0.1 mg carbon Daphnia  1 day  1; OECD, 1998). Body length was measured on the pictures from the eye to base of the tail spine (Boronat and Miracle, 1997) using an image analysis software (Ellixs software, Microvision Instruments, Evry, France). Mean growth was calculated from body length measurements as the difference between mean length values for two successive observation dates. 2.6. Chitobiase activity measurement The activity of chitobiase in water was determined immediately before renewing the exposure medium, every 2 days. Measurements were performed according to the method of Sastri and Roff (2000), as modified by Hanson and Lagadic (2005). This assay uses MUF-NAG as the substrate, which is cleaved by chitobiase into NAG and the fluorescent MUF (4-methylumbelliferone). A 5 mL aliquot of 100 mL of test medium stored at 4 1C was gently filtered through a sterile 0.2 mm polycarbonate filter (Millipore Corp., Molsheim, France) using a sterile syringe to remove bacteria and algal remains. A 150 mL aliquot of the filtered sample was then incubated for 1 h at 25 1C in 96 wells polystyrene microplates with 50 mL of 0.31 mM MUF-NAG in 0.15 M pH 5.5 citrate phosphate buffer. The MUF-NAG stock solution was made by dissolving MUF-NAG in methyl cellosolve to a concentration of 5 mM. The reaction was stopped by addition of 50 mL 0.25 M NaOH, and fluorescence was measured at 360 nm excitation/450 nm emission using a SpectraMax GeminiXS spectrofluorometer microplate reader (Molecular Devices, Saint Gre´goire, France). Fluorescence values were converted into MUF concentrations using a seven-point MUF calibration curve. Blanks were prepared using filtered samples in which MUF-NAG and NaOH were simultaneously added. Each sample was measured in triplicate, and the average of the three measures was used in all subsequent analyses. Chitobiase activity was expressed as nmol MUF liberated per hour (Sastri and Roff, 2000). 2.7. Data analysis Survival, fecundity and chitobiase activity data failed normality test (Kolmogorov–Smirnov), and log and square-root transformations of the data did not improve normality. As a consequence, data were analysed using non-parametric Kruskal–Wallis (KW) test followed by least significance difference (LSD) post-hoc test (Sprent and Ley, 1992) with a Bonferroni correction. One-way Repeated

Measures-ANOVA (RM-ANOVA) was used to analyse log-transformed body length data. When RM-ANOVA indicated a significant treatment effect, data were further analysed for each sampling date using one-way ANOVA followed by a Duncan post hoc test to detect when differences between treated and control groups occurred. The relation between chitobiase activity and the individual endpoints was analysed using Spearman’s correlation coefficients between chitobiase activity measured on each sampling date and the corresponding values for the number of surviving individuals, production of neonates, total number of individuals (females and neonates), body length and body length increment between two successive observation dates. Correlation between the chitobiase activity measured on the last observation date before the first emission of neonates and the cumulative number of neonates produced for the whole test duration was also analysed. All statistical tests were performed using Statisticas for Windows Version 6.0 (Statsoft, Tulsa, OK, USA). For all tests, significance was accepted at a ¼0.05.

3. Results 3.1. Spinosad and diflubenzuron exposure concentrations Spinosad AEC values of 0.23, 0.50 and 0.62 mg L  1 were computed for nominal concentrations of 2, 4 and 8 mg L  1, respectively. Diflubenzuron AEC values of 0.21, 0.26 and 0.44 mg L  1 were computed for nominal concentrations of 0.2, 0.4 and 0.8 mg L  1, respectively. Owing to the discrepancies between nominal and actual exposure concentrations, only AEC will be used thereafter. Decrease of the water concentration of both compounds was fast. Only 24.8%, 28.4% and 20.5% of the applied nominal spinosad concentrations (2, 4 and 8 mg L  1, respectively) were found in the beakers after two days of exposure. For diflubenzuron, the remaining concentrations after 2 days of exposure were 30%, 45% and 32.5% of the applied nominal concentrations (0.2, 0.4 and 0.8 mg L  1, respectively). For diflubenzuron residue analysis, the three replicates were pooled. Therefore, only one value per sampling date was available, preventing to estimate any error value and precluding from statistical comparison. For spinosad, comparison of concentration values (one-way ANOVA) indicated that there was a significant difference only between the values measured at d2 (p ¼0.008). Tukey post-hoc test indicated that there was a significant difference between the lowest concentration and the medium and highest concentrations (p ¼0.0185 and p ¼0.0115, respectively). The difference between the medium and highest concentrations was not significant (p ¼0.2). Considering that the first measurements were made only two days after the beginning of the experiment and that a significant difference was observed at this date, it can be considered that the levels of exposure at the beginning of the exposure (i.e., d0) were different. 3.2. Number of surviving adults The number of living adults progressively decreased with time in control beakers (Fig. 1). At the end of the experiment, the mean number (7SE) of surviving control individuals was 1670.7 for D. pulex and 1370.1 for D. magna. Exposure to Bti did not have any significant effect on the number of surviving adults at the different dates for either species (Fig. 2). Exposure to spinosad caused statistically significant differences in the number of living adults, as compared to the controls, from d6 and d8 to the end of the experiment for D. pulex exposed to 0.50 and 0.23 mg L  1, respectively (Fig. 3a, b), and from d2 and d8 to the end of the experiment for D. magna exposed to 0.50 and 0.62 mg L  1, respectively (Fig. 3e, f). A significant difference (KW and LSD post hoc tests; po0.05) was also observed at d10 for D. pulex exposed to the highest spinosad concentration (Fig. 3c). A different pattern was observed for adult daphnids exposed to diflubenzuron, with statistically significant differences in the number of living individuals, as compared to the controls, in groups exposed to 0.26 and 0.44 mg L  1 from d6 and d10,

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Fig. 1. Time-change in the mean (+ SE; n¼ 15) numbers of adults and neonates and mean (7 SE; n¼ 15) chitobiase activity in control Daphnia pulex and Daphnia magna during the observation period.

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Fig. 2. Time-change in the mean (+ SE) numbers of adults (black bars) and neonates (hatched bars) and mean ( 7 SE) chitobiase activity () in Daphnia pulex and Daphnia magna exposed to Bti (a, d: 0.25 mL L  1; b, e: 0.5 mL L  1; c, f: 1 mL L  1) for 14 days. Significant difference as compared to the controls (Fig. 1; KW and LSD post-hoc tests, with a Bonferroni correction): a: 0.01o p o0.05, b: 0.001o p o0.01, c: p o 0.001, for chitobiase activity. To facilitate comparisons, Y-axis has been maintained identical as in the controls (Fig. 1).

C. Duchet et al. / Ecotoxicology and Environmental Safety 74 (2011) 800–810

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Fig. 3. Time-change in the mean (+ SE) numbers of adults (black bars) and neonates (hatched bars) and mean ( 7SE) chitobiase activity () in Daphnia pulex and Daphnia magna exposed to spinosad (Spd) (a, d: 2 mg L  1; b, e: 4 mg L  1; c, f: 8 mg L  1; nominal concentrations; the corresponding AEC values were 0.23, 0.50 and 0.62 mg L  1) for 14 days. Significant difference as compared to the controls (Fig. 1; KW and LSD post-hoc tests, with a Bonferroni correction): *0.01 o po 0.05, **0.001o p o0.01, ***p o 0.001, for adult and neonate numbers. To facilitate comparisons, Y-axis has been maintained identical as in the controls (Fig. 1).

respectively, to the end of the experiment for D. pulex (Fig. 4b, c) and in groups exposed to 0.21 and 0.26 mg L  1 from d2 to the end of the experiment for D. magna (Fig. 4d, e).

3.3. Body length For both species, mean body length significantly increased during the experiment (Tables 1 and 2; RM-ANOVA; po0.001). The dynamics of body length increase in D. pulex was affected by spinosad at 0.62 mg L  1 at d6 and d8, and by diflubenzuron at 0.44 mg L  1 between d6 and d12 (Table 1; one-way ANOVA and Duncan post hoc test; po0.05). Mean body length of D. pulex was significantly lower, as compared to controls, for individuals exposed to spinosad at 0.23 and 0.50 mg L  1 at d8, and for individuals exposed to diflubenzuron at 0.21 mg L  1 at d10 (Table 1; one-way ANOVA and Duncan post hoc test; po0.05). Bti and spinosad did not affect the growth of D. magna

(Table 2). In contrast, D. magna individuals were significantly smaller from d4 to d6 and from d2 to d6 for the groups exposed to the lowest and medium diflubenzuron concentrations, respectively (Table 2; one-way ANOVA and Duncan post hoc test; po0.05). This effect was only transient since from d8 to the end of the experiment, no significant differences in the mean body length were shown between control and diflubenzuron-exposed groups. The mean body length of daphnids exposed to the lowest diflubenzuron concentration was even significantly higher than that of control organisms on d10 (oneway ANOVA and Duncan post hoc test; po0.05).

3.4. Production of neonates The first D. pulex neonates appeared 6 days after the beginning of the experiment in treated and control beakers (Figs. 1–3), except in those treated with diflubenzuron at 0.26 and 0.44 mg L  1 where

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**

c

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*** 6

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-1

Dfb 0.4 µg L

270

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160

-1

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80

Chitobiase activity (nmol MUF/hour)

Daphnia magna

Daphnia pulex 80

805

14

Duration of exposure (days)

Fig. 4. Time-change in the mean (+ SE) numbers of adults (black bars) and neonates (hatched bars) and mean ( 7 SE) chitobiase activity () in Daphnia pulex and Daphnia magna exposed to diflubenzuron (Dfb) (a, d: 0.2 mg L  1; b, e: 0.4 mg L  1; c, f: 0.8 mg L  1; nominal concentrations; the corresponding AEC values were 0.21, 0.26 and 0.44 mg L  1) for 14 days. Significant difference as compared to the controls (Fig. 1; KW and LSD post-hoc tests, with a Bonferroni correction): *0.01o p o 0.05, **0.001o po 0.01, ***po 0.001, for adult and neonate numbers, a: 0.01o p o 0.05, b: 0.001o po 0.01, c: p o 0.001, for chitobiase activity. To facilitate comparisons, Y-axis has been maintained identical as in the controls (Fig. 1).

they were first recorded on d8 (Fig. 4e, f). With only a few exceptions on some observation days, exposure of adult D. pulex to the three concentrations of spinosad and diflubenzuron had a significant negative effect on neonate production (Figs. 3 and 4). This was also the case for the cumulative number of neonates produced by the adults for the whole exposure period (Table 3). On average, reduction in the cumulative number of neonates ranged from 35% to 46% and from 34% to 52% of the controls for spinosad and diflubenzuron, respectively. D. magna neonates were first observed 8 days after the beginning of the experiment in treated and control beakers (Figs. 1–3), except in those treated with diflubenzuron at 0.21 and 0.26 mg L  1 where they were first recorded on d10 (Fig. 4d and e). From d8 to d14, exposure to the three concentrations of spinosad and diflubenzuron caused a significant reduction of the cumulative number of neonates, as compared to the controls, except for the adults exposed to 0.50 mg L  1 spinosad and 0.44 mg L  1 diflubenzuron

(Figs. 3e and 4f, respectively). Similar results were observed when considering the cumulative number of neonates produced for the whole exposure period (Table 3). On average, reduction in the cumulative number of neonates ranged from 16% to 39% and from 22% to 82% of the controls for spinosad and diflubenzuron, respectively.

3.5. Chitobiase activity Chitobiase activity did not exhibit a clear temporal pattern for control groups of D. pulex and D. magna (Fig. 1). Nevertheless, a first peak of activity occurred just before the production of neonates. A second peak was observed 8 and 10 days later, in D. pulex and D. magna, respectively, which could correspond to the first moult of neonates.

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Table 1 Mean ( 7SE) body length (in mm) of Daphnia pulex for control groups and groups exposed to Bti, spinosad (Spd) or diflubenzuron (Dfb). Significant difference from control (RM-ANOVA followed by one-way ANOVA per sampling date and Duncan post-hoc test): *0.01o p o 0.05; **0.001o po 0.01; ***p o0.001. Day (d) following the application of the larvicide Treatment (nominal concentrationa) d2 Control Bti 0.25 mL L  1 Bti 0.50 mL L  1 Bti 1.00 mL L  1 Spd 2 mg L  1 Spd 4 mg L  1 Spd 8 mg L  1 Dfb 0.2 mg L  1 Dfb 0.4 mg L  1 Dfb 0.8 mg L  1 a

d4

0.98 0.94 0.89** 0.93 0.93 0.93 0.94 0.98 0.95 0.97

(70.01) (70.03) (70.02) (70.02) (70.02) (70.02) (70.02) (70.02) (70.02) (70.02)

1.24 1.27 1.23 1.27 1.17 1.23 1.26 1.34 1.32 1.28

d6 (7 0.02) (7 0.03) (7 0.03) (7 0.02) (7 0.02) (7 0.03) (7 0.03) (7 0.03) (7 0.02) (7 0.03)

d8

1.53 1.58 1.46 1.54 1.46 1.49 1.41* 1.50 1.51 1.43*

(7 0.02) (7 0.03) (7 0.03) (7 0.03) (7 0.03) (7 0.03) (7 0.02) (7 0.03) (7 0.04) (7 0.03)

1.75 1.76 1.75 1.76 1.55*** 1.55*** 1.61** 1.69 1.70 1.59***

d10 (7 0.01) (7 0.02) (7 0.03) (7 0.02) (7 0.04) (7 0.04) (7 0.03) (7 0.03) (7 0.04) (7 0.03)

1.82 1.84 1.79 1.82 1.85 1.85 1.92** 1.71* 1.78 1.73

d12 (7 0.01) (7 0.03) (7 0.03) (7 0.02) (7 0.03) (7 0.02) (7 0.02) (7 0.04) (7 0.04) (7 0.03)

1.91 1.95 1.92 1.89 1.96 1.90 1.88 1.88 1.87 1.80**

(7 0.01) (7 0.02) (7 0.03) (7 0.03) (7 0.03) (7 0.03) (7 0.04) (7 0.03) (7 0.04) (7 0.04)

The corresponding Average Exposure Concentration (AEC) values were 0.23, 0.50 and 0.62 mg L  1 for spinosad, and 0.21, 0.26 and 0.44 mg L  1 for diflubenzuron.

Table 2 Mean ( 7SE) body length (in mm) of Daphnia magna for control groups and groups exposed to Bti, spinosad (Spd) or diflubenzuron (Dfb). Significant difference from control (RM-ANOVA followed by one-way ANOVA per sampling date and Duncan post-hoc test): *: 0.01 o po 0.05; ***: p o 0.001. Treatment (nominal concentrationa)

Day (d) following the application of the larvicide d2

Control Bti 0.25 mL L  1 Bti 0.50 mL L  1 Bti 1.00 mL L  1 Spd 2 mg L  1 Spd 4 mg L  1 Spd 8 mg L  1 Dfb 0.2 mg L  1 Dfb 0.4 mg L  1 Dfb 0.8 mg L  1 a

1.22 1.16 1.10 1.21 1.17 1.24 1.23 1.12 1.07* 1.19

d4 (7 0.01) (7 0.03) (7 0.02) (7 0.03) (7 0.03) (7 0.02) (7 0.02) (7 0.02) (7 0.02) (7 0.02)

d6

1.81 1.80 1.85 1.84 1.81 1.97 1.95 1.32*** 1.45*** 1.90

(7 0.02) (7 0.03) (7 0.04) (7 0.03) (7 0.04) (7 0.03) (7 0.03) (7 0.03) (7 0.05) (7 0.04)

d8

2.23 2.28 2.33 2.31 2.19 2.29 2.29 1.87*** 1.77*** 2.33

(70.02) (70.04) (70.03) (70.04) (70.05) (70.05) (70.04) (70.07) (70.09) (70.04)

2.31 2.38 2.38 2.35 2.30 2.45 2.49 2.47 2.36 2.44

d10 (7 0.02) (7 0.04) (7 0.04) (7 0.03) (7 0.05) (7 0.04) (7 0.03) (7 0.13) (7 0.12) (7 0.04)

2.47 2.46 2.49 2.52 2.50 2.46 2.60 2.75* 2.51 2.53

d12 (7 0.02) (7 0.04) (7 0.04) (7 0.03) (7 0.04) (7 0.05) (7 0.04) (7 0.10) (7 0.13) (7 0.04)

2.52 2.56 2.54 2.59 2.44 2.68 2.67 2.74 2.44 2.63

d14 (70.02) (70.04) (70.04) (70.04) (70.09) (70.04) (70.04) (7 0.31) (7 0.28) (70.05)

2.58 2.52 2.57 2.53 2.61 2.62 2.61 2.72 2.80 2.74

(7 0.02) (7 0.04) (7 0.03) (7 0.04) (7 0.04) (7 0.05) (7 0.06) (7 0.40) (70.17) (7 0.05)

The corresponding Average Exposure Concentration (AEC) values were 0.23, 0.50 and 0.62 mg L  1 for spinosad, and 0.21, 0.26 and 0.44 mg L  1 for diflubenzuron.

Table 3 Cumulative fecundity of Daphnia pulex and Daphnia magna expressed as the mean 7 SE cumulative numbers of neonates produced by the adults in control beakers and in beakers treated with Bti, spinosad or diflubenzuron at the end of the exposure period (d14). Significant difference from control (KW and LSD post-hoc tests, with a Bonferroni correction): *: 0.01 op o 0.05, **: 0.001o po 0.01, ***: p o 0.001. Treatment (nominal concentrationa)

Daphnia pulex

Daphnia magna

Control Bti 0.25 mL L  1 Bti 0.50 mL L  1 Bti 1.00 mL L  1 Spd 2 mg L  1 Spd 4 mg L  1 Spd 8 mg L  1 Dfb 0.2 mg L  1 Dfb 0.4 mg L  1 Dfb 0.8 mg L  1

2447 7 2687 19 2467 7 2367 11 1587 5** 1317 12*** 1567 9** 1497 14*** 1187 13*** 1617 8**

184 7 9 190 7 12 162 7 5 162 7 6 112 7 30* 154 7 4 122 7 12* 32 7 16*** 43 7 12*** 143 7 12

No significant difference in chitobiase activities between control and Bti-exposed D. magna groups were observed (Fig. 2d, e). Similarly, spinosad did not affect chitobiase activity in D. magna. In contrast, exposure to diflubenzuron resulted in a dramatic reduction in chitobiase activity, the more potent effects being observed with the two lowest concentrations (Fig. 4d and e). Spearman’s correlation coefficients (r) between chitobiase activity and the individual endpoints measured on daphnids are shown in Table 4. Correlation coefficients were positive and significant (p o0.05) for all the endpoints except for the body length increment between two dates for both species, and for the number of surviving adults for D. pulex. In particular, there was a significant positive correlation between chitobiase activity measured on the last observation date before the first emission of neonates and the cumulative number of neonates produced for the whole test duration (Table 3; r ¼0.45 and 0.60 for D. pulex and D. magna, respectively).

a The corresponding Average Exposure Concentration (AEC) values were 0.23, 0.50 and 0.62 mg L  1 for spinosad, and 0.21, 0.26 and 0.44 mg L  1 for diflubenzuron.

4. Discussion An increase in chitobiase activity was observed for D. pulex groups exposed to Bti, which was only significant for the highest concentration (Fig. 2c). No clear temporal pattern and no significant differences were observed for chitobiase activity measured for groups of D. pulex exposed to the two other larvicides, except for diflubenzuron where significant differences were occasionally observed for the two lowest concentrations (Fig. 4a and b).

In this study, exposure of D. pulex and D. magna to Bti, spinosad and diflubenzuron took into account the dissipation of the compounds from the water during the 14 days of the observation period. To a certain extent, such an experimental design mimicked possible field exposure patterns. The results of larvicide residue analysis showed that, over the whole experimental period, AEC values for spinosad and diflubenzuron were lower than the

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Table 4 Spearman’s correlation coefficient (r) between chitobiase activity values and values of survival, number of neonates produced between two consecutive observation dates, total number of individuals (females + neonates), body length, length increment between two consecutive observation dates, and cumulative fecundity (Table 3). Statistically significant coefficient values: *: p o 0.05 (n¼ 70). Correlation between chitobiase activity measured

(1) On each sampling date and The number of surviving adults The number of neonates produced between two consecutive observation dates The number of adult females+ neonates The mean body length The increment in mean adult body length between two consecutive observation dates

Daphnia pulex

Daphnia magna

0.17 0.42*

0.34* 0.49*

0.46* 0.42* 0.09

0.45* 0.62*  0.23

(2) On the last observation date before the first emission 0.45* of neonates and the cumulative number of neonates produced for the whole test duration (Table 3)

0.60*

targeted nominal concentrations. Spinosad AECs were about tenfold lower than the nominal concentrations, whereas only a slight deviation (1.5-fold) was observed for diflubenzuron. Several studies have already shown a rapid dissipation of spinosad from water in experimental exposure devices or aquatic ecosystems. For example, in in situ enclosures, Duchet et al. (2008) showed that spinosad concentrations dramatically decreased to 45.8%, 24.1% and 12.9% of the initial exposure levels, for nominal concentrations of 8, 17 and 33 mg L  1, respectively, 2 days after treatment. Cleveland et al. (2002) determined half-lives of 1–2 days for the sum of spinosyns A and D in outdoor microcosms. Rapid removal of spinosad from water mainly resulted from both photolysis and adsorption onto the sediments (Saunders and Bret, 1997; Cleveland et al., 2002). Adsorption may have also occurred onto the walls of the microcosms. Diflubenzuron is also known to adsorb rapidly onto the sediments and walls of vessels and pipes, and it may also partition into the surface film because of its low water solubility and high Kow (WHO, 2006). Adsorption processes of spinosad and diflubenzuron onto the wall of the beakers may also account for the low exposure concentrations recorded in the present experiment. Therefore, for both compounds, the effects that are described below resulted from low dose exposure, which is highly relevant for ecological risk assessment. Bti had no effect on the number of surviving individuals. It did not affect the production of neonates, and had no influence on the increase in adult body length of D. pulex and D. magna. This is in accordance with previous field studies on the same Daphnia species (Duchet et al., 2008, 2010a). This also confirms that Bti is poorly toxic for Cladocera (Boisvert and Boisvert, 2000; Boisvert and Lacoursie re, 2004). Whatever the exposure concentration, spinosad had negative effects on survival and reproduction of both Daphnia species, whereas their growth, expressed as individual body length timechange, was not affected. These results are consistent with the laboratory data published by Stark and Vargas (2003) who showed that exposure of D. pulex to 10 mg L  1 spinosad led to a decline in survival, birth rate, net reproductive rate, and intrinsic rate of population increase. In experiments performed in in situ enclosures, Duchet et al. (2008, 2010a) showed that both survival and size structure of natural populations of D. pulex and D. magna were affected by spinosad treatments at nominal concentrations Z8 mg L  1. Population recovery was observed for the lowest nominal concentration (8 mg L  1) after the first week of exposure in D. pulex (Duchet et al., 2008) but not in D. magna (Duchet et al., 2010a). Actual exposure concentrations of the D. pulex population were

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between 1.65 and 4.78 mg L  1 2 days after application (Duchet et al., 2008). The results obtained in the present study suggest that even lower spinosad concentrations are able to exert a significant negative impact on D. pulex and D. magna under laboratory conditions. The absence of a clear relationship between spinosad exposure concentration and either adult mortality or cumulative number of neonates does not allow to conclude whether the reduction in the cumulative number of neonates is only the direct consequence of a reduction in the number of adults in the test systems or if sublethal effects on reproduction also occurred. For both Daphnia species, exposure to the three diflubenzuron concentrations resulted in a decrease in the number of surviving individuals, and also negatively affected the production of neonates. In addition, in D. magna, a significant negative effect on body length was observed at the beginning of the experiment (up to d6), and maturity was slightly delayed for the individuals exposed to the lowest and medium diflubenzuron concentrations. In cladocerans, clutch size depends on size at maturity and brood size depends on body length, with a usually linear relationship between ¨ maximal brood size and body size (Hulsmann, 2001). The maximum brood size of a given size class (under non-limiting food conditions and without predation, which corresponds to our test conditions) may be influenced by morphologic variations (length– width relation) of the daphnids and by the egg size, both traits affecting the available space in the brood pouch. In the case of low food availability, daphnids produce few eggs regardless of the body size. In addition, they may require a longer time to produce their first brood, and each adult instar may not be able to produce eggs (McCauley et al., 1990). Our results suggest that, at least at the beginning of the exposure period, diflubenzuron-exposed daphnids may have dedicated more energy to maintenance and detoxication at the expense of growth and consequently of reproduction, as predicted by physiological models when energy becomes limiting (Kooijman, 1986; McCauley et al., 1990; Bradley et al., 1991). Chitobiase activities measured in the beakers where D. pulex groups were exposed to 1 mL L  1 Bti were sometimes higher than in the controls, while no significant differences were observed between these two groups for the various individual endpoints. In arthropods, chitobiase activity occurs in both the epidermis and gut where a specific isoform has been identified (Peters et al., 1998, 1999; Zou and Fingerman, 1999d). In Euphasia superba, this chitobiase isoform is located in the cytosol of gut epithelial cells (Peters et al., 1998, 1999), which are the target of Bti endotoxins (Van Frankenhuyzen, 1993; Knowles, 1994; Whalon and Wingerd, 2003). Histopathological observations revealed that, in Cladocera, midgut epithelial cells can be affected by Bti treatment, the clear cells being more vulnerable than the dark cells, without any consequence on adult survival (Rey et al., 1998). Harmlessness of Bti to Cladocera was interpreted as a consequence of the relative scarcity of clear cells and of their patchy distribution along the whole midgut together with important cellular renewal capacity of the epithelium (Rey et al., 1998). Such an impact of Bti on gut epithelial cells of D. pulex, with a release of chitobiase in water as a consequence, may have occurred in the present experiment. This could explain the higher enzyme activity in Bti-exposed D. pulex, as compared to the controls. Although exposure to spinosad significantly altered individual endpoints in both daphnid species, its effects on chitobiase activity were not so obvious. No clear tendencies and no significant differences were shown for both D. pulex and D. magna. As observed with spinosad, no clear pattern emerged from the chitobiase activity measurements in the beakers where D. pulex were exposed to diflubenzuron. Nevertheless, on occasion, chitobiase activities for the groups exposed to the two lowest larvicide concentrations were inferior to the values obtained for the controls. This is in

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accordance with the significant reduction in the number of individuals and in the production of neonates observed at these concentrations. For D. magna, large differences in chitobiase activity, as compared to the controls, were observed for groups exposed to 0.21 and 0.26 mg L  1 diflubenzuron, those groups exhibiting a decreased production of neonates and a reduced number of individuals, respectively. Changes in chitobiase activity in daphnids exposed to diflubenzuron reflected variations in the number of individuals, either adults or neonates, present in the beakers. This confirms the results published by Oosterhuis et al. (2000) who showed that the amount of chitobiase released in water in a given area is positively correlated to the number of aquatic organisms living in the same area. In D. magna exposed to low concentrations of diflubenzuron, the decreased chitobiase activity mirrors the reduction in the number of individuals, both adults and neonates (Fig. 4). This is consistent with the mode of action of this insecticide, which is known as a growth regulator in insects (Grosscurt and Jongsma, 1987). As shown by the significant positive correlation between chitobiase activity and body length in both Daphnia species (r¼ 0.42 and 0.62 for D. pulex and D. magna, respectively), measurements of the enzyme activity also mirrored the development of individuals. This is consistent with the results obtained by Sastri and Roff (2000) who showed that chitobiase activity globally increased with body length. In crustaceans, moulting is regulated by a multi-hormone system, but is under immediate control of ecdysteroids (Chang et al., 1993). Because the adverse impacts on crustacean moulting cannot be readily seen in the wild, disruption of moulting represents an invisible form of endocrine disruption (Zou, 2005). In theory, any event (moult inhibiting hormone synthesis and release, ecdysteroidogenesis, ecdysteroid receptor binding, etc.) in the endocrine cascades of moulting regulation could be the target of environmental chemicals. Chitobiase has been proven to be a product of a gene regulated by the moulting hormones (e.g., 20-hydroxyecdysone) in crustaceans (Zou, 2005; Meng and Zou, 2009). Diflubenzuron acts as a chitin-synthesis inhibitor by interfering with the endocrine-regulated process of moulting (McKennedy et al., 2004). Christiansen et al. (1984) showed that diflubenzuron inhibits both formation of exocuticle and endocuticle in larvae of the crab Rhithropanopeus harrisii. Moreover, chitobiase activity was found to correlate well with the profiles of ecdysteroids during the moulting cycle of U. pugilator (Zou, 2005). Zou and Fingerman (1999b) investigated changes in chitobiase activity following exposure to various moult-inhibiting agents. A 7-day exposure of U. pugilator to Aroclor 1242, PCB29, diethylstilbene, endosulfan and diethylphthalate resulted in decreased chitobiase activity in the epidermis. Inhibition of epidermal chitobiase activity can be partly responsible for the slowing of moulting caused by these compounds because this enzyme is necessary for complete digestion of exoskeletonal chitin (Zou, 2005). Overall, chitobiase activity can be considered as indicative of effects of xenobiotics on crustacean development, and our results support the idea that diflubenzuron, and to a lesser extent spinosad, can influence chitobiase activity and moulting process in daphnids. Interestingly, low concentrations of diflubenzuron are able to affect planktonic crustaceans since, as in the present study, effects on survival and development were reported for concentrations lower than 1 mg L  1 in the copepod Eurytemora affinis (Savitz et al., 1994). Measurement of chitobiase activity alone would probably not allow identifying the effects of larvicides as accurately as survival or production of neonates would. However, the existence of significant relationships between chitobiase activity and the individual endpoints measured in both Daphnia species, especially between the activity measured on the last observation date before the first emission of neonates and the cumulative number of

neonates produced for the whole test duration, supports the potential for chitobiase activity to be used as an indicator of population dynamics in terms of abundance and reproduction. All other things being equal, systems with the lowest chitobiase activity should contain the less abundant daphnid populations and/or populations with the lowest fecundity. Indeed, in this study, the lowest values for chitobiase activity and cumulative production of neonates were observed for exposure to diflubenzuron, whereas the highest values for both parameters were observed for Bti-exposed and control groups. Richards et al. (2008) observed a similar pattern in relation to the impact of pharmaceuticals on D. magna in laboratory. Therefore, chitobiase activity not only reflects crustacean biomass production, as shown by Oosterhuis et al. (2000), it also mirrors daphnid population dynamics. Santojanni et al. (1998) showed that D. magna body length change between 7 and 15 day-old individuals was linked to their cumulative fecundity under experimental conditions. This relationship between growth and fecundity held true for toxicity tests with pyridine, cadmium and chromium. Growth, development, and reproduction are ideal parameters to observe because they incorporate effects on the organism’s feeding, behaviour and mobility, for example. Therefore, chitobiase activity would be affected before the manifestation of these higher-level effects and may help to predict future changes in growth, development, and reproduction as a result of exposure to a toxicant, indicating its potential as a biomarker of effect (Newman and Unger, 2003).

5. Conclusions The present study shows that survival, growth and fecundity of D. pulex and D. magna can be negatively affected by mosquito larvicides exhibiting different modes of action (i.e., neurotoxicant and growth inhibitor), and that these effects can be detected using chitobiase activity measurements. As compared to D. pulex, D. magna showed a higher sensitivity to diflubenzuron. This confirms previous results obtained with the same Daphnia strains (Duchet et al., 2010b). The similar sensitivity of D. pulex and D. magna, that was deduced from laboratory tests with a wide range of synthetic chemicals (immobilisation test, 30 reference chemicals, Canton and Adema, 1978; LC50 and reproduction test, 15 compounds, Lilius et al., 1995), is therefore questionable, and requires further investigations. Using Bti as the reference innocuous larvicide in Daphnia sp., comparison of the results obtained with spinosad (neurotoxicant) and diflubenzuron (insect growth regulator) strongly supports the potential of chitobiase activity as a proxy for assessing the dynamics of daphnid populations when exposed to chemicals that affect growth and/or reproduction. Of course, like most of the biomonitoring tools, chitobiase activity alone cannot be used to identify the causative agents of an observed impact. It may only provide evidence that an effect has occurred (or may occur), and should therefore only be considered an indiscriminative bioindicator (McCarthy et al., 2002). However, chitobiase activity measurement, which is rapid to perform, inexpensive and nondestructive, has shown great potential for field monitoring of the quality of freshwater ecosystems (Hanson and Lagadic, 2005; Conley et al., 2009). Extensive baseline studies and calibration programmes are still needed in order to evaluate and validate this parameter for aquatic ecosystem health assessment. They should include field studies in ecosystems where chitobiase may be used for biomonitoring of the impact of mosquito control (e.g., coastal wetlands). Chitobiase activity should be monitored in combination with measurements of arthropod biomass for demonstrating effects beyond natural variation of arthropod population dynamics and for strengthening the potential of chitobiase activity as a biomonitoring tool.

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Acknowledgments The authors are grateful to Dow AgroSciences for the generous gift of Conserves 120SC, and to Alphonse Quemeneur (INRA Experimental Unit of Aquatic Ecology and Ecotoxicology, Rennes, France) for rearing the two Daphnia species that have been used in the present study. The authors are also indebted to Dr. Mark L. Hanson (University of Manitoba, Winnipeg, Canada) for his helpful comments which greatly improved the quality of this paper.

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