Impact of a perfluorinated organic compound PFOS on ... - Springer Link

Ecotoxicology (2011) 20:447–456 DOI 10.1007/s10646-011-0596-2

Impact of a perfluorinated organic compound PFOS on the terrestrial pollinator Bombus terrestris (Insecta, Hymenoptera) Veerle Mommaerts • An Hagenaars • Johan Meyer • Wim De Coen • Luc Swevers Hadi Mosallanejad • Guy Smagghe

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Accepted: 10 January 2011 / Published online: 21 January 2011 Ó Springer Science+Business Media, LLC 2011

Abstract Perfluorinated organic chemicals like perfluorooctane sulfonic acid (PFOS) are persistent environmental pollutants that have been measured in a great diversity of wildlife worldwide, especially in the aquatic compartment. However, little information is available on the presence and effects of PFOS in the terrestrial compartment. Therefore, we investigated in this project the risks for effects, bioaccumulation and potential mechanisms of activity of PFOS in the bumblebee Bombus terrestris L. (Hymenoptera: Apidae) that is an important worldwide pollinator in the terrestrial compartment of wildflowers and cultivated crops. The exposure to PFOS occurred orally via the drinking of treated sugar water in a wide range from 1 lg/l up to 10 mg/l, containing environmentally relevant as well as high concentrations, and this was done with use of Veerle Mommaerts and An Hagenaars are equally contributed to this study. V. Mommaerts G. Smagghe Laboratory of Cell Genetics, Department of Biology, Faculty of Sciences, Free University of Brussels, Brussels, Belgium V. Mommaerts H. Mosallanejad G. Smagghe (&) Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium e-mail: guy.smagghe@ugent.be A. Hagenaars J. Meyer W. De Coen Laboratory of Ecophysiology, Biochemistry and Toxicology, Department of Biology, University of Antwerp, 2020 Antwerp, Belgium L. Swevers Insect Molecular Genetics and Biotechnology, Institute of Biology, National Centre for Scientific Research ‘‘Demokritos’’, Aghia Paraskevi Attikis, Athens, Greece

microcolonies of B. terrestris in the laboratory. A chronic toxicity assay demonstrated high bumblebee worker mortality (up to 100%) with an LC50 of 1.01 mg/l (R2 = 0.98). In addition, PFOS posed strong detrimental reproductive effects, and these concerted with a dramatic reduction in ovarian size. HPLC–MS demonstrated a bioaccumulation factor of 27.9 for PFOS in bumblebee workers fed with sugar water containing 100 lg/l PFOS during 5 weeks (2184 ± 365 ng/g BW). Finally, potential mechanisms of activity were investigated to explain the significant impact of PFOS on survival and reproduction capacity of B. terrestris. Exposure of bumblebee workers to PFOS resulted in a significant decrease in mitochondrial electron transport activity (p = 0.035) and lipid amounts (p = 0.019), while the respective p-values were 0.58 and 0.12 for protein and glucose amounts. Hence, addition of PFOS to ecdysteroid responsive Drosophila melanogaster S2 cells resulted in a strong antagonistic action on the EcR-b.act.luc reporter construct, demonstrating that PFOS may exert its effects partially through an endocrine disrupting action via the insect molting hormone or ecdysteroid receptor. Keywords PFOS Bumblebee Survival Reproduction Electron transport Energy content Antagonistic activity

Introduction Perfluorooctane sulfonate acid (PFOS) is a chemical used in pharmaceuticals, cosmetics, paper coatings, fire retardants and insecticides (Giesy and Kannan 2001; Austin et al. 2003; Hoff et al. 2005). Biomonitoring studies showed that many perfluorinated-based consumer products like the insecticide sulfuramid can have PFOS as degradation product (Manning et al. 1991; Grossman et al. 1992;

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Renner 2001; Houde et al. 2006). Hence, the persistence and the easy bioaccumulation of perfluorochemicals in the environment have contributed to the worldwide detection of PFOS in different trophic levels of both the aquatic and terrestrial compartment like drinking waters, fishes, invertebrates, birds, mammals and humans (Giesy and Kannan 2001; Kannan et al. 2001; Van de Vijver et al. 2003; Hoff et al. 2004, 2005; Martin et al. 2004; Tomy et al. 2004; Harada et al. 2006; Skutlarek et al. 2006; So et al. 2006; Dauwe et al. 2007; Ehresman et al. 2007). Multiple studies were performed during the last decade to evaluate sideeffects of PFOS exposure, but mainly on vertebrates and on aquatic invertebrates. However, following the recovery of high PFOS concentrations in wood mice, Hoff et al. (2004) argued bioavailability of PFOS also in the terrestrial ecosystem. Lethal as well as sublethal effects of PFOS have been reported, but nearly all studies on focused on vertebrates (see for review Lau et al. 2007). In rats, PFOS exposure affected the general condition of the organism and reproduction via interference of the estrous cycle (Austin et al. 2003). This endocrine disruptive potential of PFOS was also reported by Shi et al. (2008) for zebrafish embryos and was accompanied by developmental toxicity and mortality. More recently, the impact of PFOS on aquatic invertebrates was investigated. In water fleas (Daphnia magna and Moina macrocopa), a multi-generation study with PFOS showed reproductive changes and elevated mortality in the progeny (Ji et al. 2008). Mortality and reproductive effects were also reported in damesfly (Enallagma cyathigerum) larvae after exposure to PFOS, but in addition behavioral changes were observed (Van Gossum et al. 2009; Bots et al. 2010). Within the terrestrial compartment, bumblebees or Bombus terrestris fulfill an important ecological role as pollinator of most wild flowers (Goulson 2010). Therefore side-effects of anthropogenic compounds on their biology should be avoided. Considering the effects of PFOS so far, only short-term toxicity was assessed on pollinating honeybees or Apis mellifera (Beach et al. 2006). Nonetheless, based on the sublethal effects on reproduction and on the impairments with the foraging behavior, as reported for aquatic invertebrates, long-term exposure to PFOS could result in colony loss. In addition, the presence of pollutants and the increased use of chemicals might also negatively affect biodiversity. In this context several authors reported that bumblebee microcolonies can be used as unique and reliable setup for assessing the side-effects of pesticides and other environmental contaminants on worker mortality, reproduction and behavior (Tasei et al. 2000; Mommaerts et al. 2006, 2010; Gradish et al. 2010). The aims of this project were to evaluate the accumulation, effects and potential mechanisms of PFOS in the

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pollinator B. terrestris. Hereto, microcolonies of bumblebee workers were orally exposed via the drinking of treated sugar water in the laboratory to environmentally relevant as well as to higher concentrations of PFOS. The lethal and sublethal effects on survival, nest growth, reproduction and foraging behavior were followed. The PFOS accumulation in the total body of intoxicated workers was evaluated by use of liquid chromatography coupled to high-resolution mass spectrometry (HPLC–MS). To better understand the mechanism and potential endocrine disrupting effects that are behind the strong negative effects of PFOS, analyses of the electron transport system (ETS) and the energy content in the form of lipid, protein and glucose amounts were performed and the antagonistic/agonistic capacity of PFOS with the insect molting hormone or ecdysteroid receptor (EcR) was tested.

Materials and methods Products The experiments were performed with PFOS (tetraethylammonium salt, 98%) that was purchased with Sigma– Aldrich (Bornem, Belgium), CAS number 56773423. Insects All experiments were performed with bumblebee workers obtained from a continuous mass rearing program (Biobest NV, Westerlo, Belgium) and conducted under standardized laboratory conditions of 28–30°C, 60–65% relative humidity and continuous darkness. The insects were provided ad libitum with commercial sugar water and pollen (Apihurdes, Spain) as energy and protein source, respectively (Mommaerts et al. 2006). Assessment of lethal and sublethal effects of PFOS with use of microcolonies In the laboratory, microcolonies were made by placing five newly emerged bumblebee workers, which were collected from the bumblebee colony, in an artificial plastic nest box (15 9 15 9 10 cm) as described by Mommaerts et al. (2006). The bumblebee workers were exposed orally via the drinking of sugar water treated with PFOS at high concentrations: 1, 2, 5 and 10 mg/l, and low concentrations: 1, 10 and 100 lg/l. For the negative control, bumblebee workers were exposed ad libitum to plain sugar water which resulted in worker mortality \10% after 11 weeks, while in the positive controls bumblebee workers were fed on sugar water treated with 200 ppm imidacloprid (i.e.,

Impact of PFOS on the terrestrial pollinator Bombus terrestris

neonicotinoid insecticide) that resulted in all cases in 100% mortality. In the artificial nest boxes, the survival of the bumblebee workers was measured on a weekly basis over a period of 11 weeks. In parallel, the adverse sublethal effects on reproduction were weekly monitored by scoring the numbers of drones produced per nest. Per treatment four artificial nests were exposed to assess lethal and sublethal effects and the whole experiment was repeated twice. Data were statistically analyzed in SPSS v16.0 (SPSS Inc., Chicago, IL) with use of the Kolmogorov–Smirnov test to confirm normal distribution of the data (p = 0.05), and then by one-way analysis of variance (ANOVA). Means ± SEM were separated with a post-hoc Tukey– Kramer test (p = 0.05). In addition, medium response concentrations (LC50s) and corresponding 95% confidence intervals were calculated in GraphPad Prism v4 (GraphPad Software, La Jolla, CA); the accuracy of data fitting to the curve model was evaluated on R2 values. Effect of PFOS on ovary growth in bumblebee workers To investigate the severe detrimental effects of PFOS on reproduction seen when bumblebee workers were exposed to 1 mg/l eight microcolonies consisting each of five workers were started up. During a period of 11 weeks four nests were orally exposed to PFOS at 1 mg/l via the drinking of treated sugar water, while the other nests were fed with untreated sugar water and served as negative control. Then, in each nest the dominant worker was killed by freezing and the ovaries were dissected out in phosphate buffer (pH 7.6). The length of the ovaries of the dominant workers was measured and compared with the controls. In addition, the number of eggs present per ovary was determined under the binocular for both groups. Accumulation of PFOS in bumblebee workers To assess the accumulation of PFOS in the bumblebee worker body, individual workers were placed in plastic boxes and provided with sugar water treated with PFOS at 100 lg/l (the highest concentration which was shown safe for bumblebee workers) as described above. The 10 individual female workers were allowed to consume the treated sugar water for 5 weeks ad libitum and then killed by freezing at -20°C. The PFOS concentration was measured in whole body bumblebee worker extracts using HPLC–MS. PFOS was extracted according to the solvent extraction method of Powley et al. (2005). HPLC was carried out on a CapLC system (Waters, Milford, MA) connected to a Quattro II triple quadrupole MS (Micromass, Manchester, UK). Aliquots of 5 ll were loaded on an Optiguard C18 pre-column

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(10 9 1 mm2 inner diameter, Alltech, Deerfield, IL). The analysis was performed on a Fluophase column (50 9 1 mm2 inner diameter, Sercolab, Antwerp, Belgium) at a flow rate of 40 ll/min. The mobile phase was 4 mM NH4OAc/CH3OH and an elution gradient was used starting at 45% CH3OH and going to 90% CH3OH in 3 min. After 5 min, the initial conditions were resumed. PFOS was measured under negative electrospray ionization using single reactant monitoring (m/z 499-99) and the internal standard (1H,1H,2H,2H-perfluorooctane sulfonic acid) was measured under the same conditions (m/z 427-81). The dwell time was 0.1 s. The electrospray-capillary voltage was set at -3.5 kV and the cone voltage was 24 V. The source temperature was 80°C and the pressure in the collision cell was 3.3 10-5 mm Hg(Ar). Effect of PFOS on mitochondrial electron transport system (ETS) activity in bumblebee workers As described above individual bumblebee workers were exposed to PFOS at 1 mg/l, a concentration responsible for severe detrimental effects on bumblebee reproduction, via the drinking of treated sugar water during a period of 5 weeks. In the control group, the workers were kept on untreated sugar water. Mitochondrial ETS activity was quantified in triplicate for ten individual treated and ten individual control bumblebee workers according to De Coen and Janssen (1997). In brief, bumblebee workers were individually homogenized, the homogenate centrifuged and the supernatant used to quantify ETS activity. NADH/NADPH solution (NADH, NADPH, Tris HCl, Triton X-100, pH 8.5) was added and the reaction was started by adding INT solution (2-(p-iodophenyl)3(p-nitrophenyl)-5-phenyl tetrazolium chloride, 8 mM). The absorbance was measured kinetically at 490 nm for 15 min at room temperature. The amount of formazan formed was determined using the formula of Lambert–Beer (e = 15900/M.cm). The ETS activity was calculated based on the theoretical stoichiometrical relationship that 1 lmol of O2 was consumed in the ETS for each 2 lmol of formazan formed. Normal distribution was confirmed for all data with the Kolmogorov–Smirnov test (p = 0.05), and then means were separated with a two-tailed unpaired t-test (p = 0.05). Effect of PFOS on energy content in bumblebee workers To evaluate the impact of PFOS on the energy content, bumblebee workers were individually exposed to PFOS at 1 mg/l, a concentration responsible for severe detrimental effects on bumblebee reproduction, in sugar water during 5 weeks as described above. In the control group,

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bumblebee workers were exposed to plain sugar water. Thereafter whole body homogenates of 20 individual control and 19 individual treated bumblebee workers were analyzed for total protein, sugar (sugar and their methyl derivatives, oligosaccharides and polysaccharides) and lipid amounts according to De Coen and Janssen (2003). All measurements were performed in triplicate. In brief, proteins were precipitated using trichloroacetic acid (TCA). After centrifugation, supernatant fractions were kept for sugar determination and the remaining pellet for total protein content. The pellet was resuspended in NaOH, incubated for 30 min at 60°C and neutralized with HCl. Total protein content was determined in accord to the Bradford assay. The absorbance was quantified in an Ultramicroplate reader ELx808 (Bio-Tek instruments, Winooski, VT) at 600 nm. Protein concentrations were calculated by means of a standard curve with BSA (United States Biochemical Corp., Cleveland, OH). The total sugar content of the supernatant fraction was determined using 5% phenol and concentrated H2SO4. The absorbance was measured at 490 nm and glucose was used as standard. To extract the total lipids, homogenates were mixed with chloroform, methanol and purified water. After centrifugation, the bottom chloroform phase was incubated with H2SO4 for 15 min at 200°C. The absorbance was measured at 340 nm. Lipid concentrations were calculated by means of a standard curve of tripalmitine (Sigma– Aldrich, Bornem, Belgium). Data were analyzed for normal distribution with the Kolmogorov–Smirnov test (p = 0.05), and then means were separated with a two-tailed unpaired t-test (p = 0.05). Reporter assay to determine EcR interaction of PFOS with ecdysteroid-responsive insect S2 cells PFOS was assayed for its ability to activate and inhibit transcription of an ecdysteroid-inducible luciferase reporter gene using an ecdysteroid-responsive insect cell line. S2 cells (S2-Mt-Dl), derived from fruit fly D. melanogaster embryo (Schneider 1972), were cultured in HyQ SFXInsectTM medium (Perbio Science, Erembodegem, Belgium) (Soin et al. 2008). S2 cells were transiently transfected with the reporter TM construct using Lipofectin (Invitrogen) and subsequently assayed for the activation of the transcription of the ecdysteroid-inducible luciferase reporter gene. The ‘EcREb.act.luc’ reporter construct is composed of seven copies of EcRE (Riddihough and Pelham 1987), a basal actin promoter from Bombyx (b.act), the luciferase reporter gene (luc), and a termination signal (Soin et al. 2008). In brief, each well of a 6-well plate was filled with 3 9 106 S2 cells. The transfection medium was prepared by pre-incubating

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lipofectin for 45 min at room temperature in culture medium without serum before incubating it for 15 min in the presence of 1.5 lg of the reporter construct. The cells were washed twice with serum-free medium and subsequently incubated for 5 h with the transfection medium. In order to test EcR/USP agonistic activity, transfected cells at a density of 2–3 9 104 cells in 100 ll medium were incubated for 24 h with 1 ll of different concentrations of PFOS. Control cells were treated with 1 ll ethanol per 100 ll medium. The Steady-GloÒ luciferase assay system kit (Promega, Leiden, the Netherlands) was used to obtain luciferase reporter genedependent luminescence, which was measured with a Tecan M200 luminometer (Tecan, Mechelen, Belgium). In these experiments, data were compared to the response of 1 lM 20-hydroxyecdysone that was set as maximum ecdysteroid agonistic response (Soin et al. 2008). The results are expressed as total RLUs measured (means ± SEM) and based on three measurements per treatment and the experiment was two times repeated. For testing antagonistic activity, transfected S2 cells at a density of 2–3 9 104 cells in 100 ll were incubated for 24 h with serial dilution of PFOS in the presence of 1 lM of 20E (a concentration that elicits maximum response; data not shown). Data were expressed and analyzed as described above. Next to reporter activation, S2 cells exposed to PFOS were also investigated for cell viability effects as described before (Soin et al. 2008). Hereto, 2 9 105 S2 cells in 100 ll were incubated for 5 days with PFOS at 1, 10 and 100 lM, and the control cells to 1 ll ethanol in 100 ll cell suspension. For each concentration four replicates were done, and each experiment was repeated two times. After incubation, the cell numbers were counted with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) technique (Decombel et al. 2004). Briefly, 100 ll of cell solution was transferred to a microtube (Eppendorf) and 100 ll of 1 mg/ml MTT solution was added. After 3 h incubation at 27°C, the formazan crystals were collected by centrifugation for 7 min at 20000 g at 4°C. Then, the formazan crystals were dissolved in isopropanol, and after centrifugation the absorbance at 560 nm as measure of cell viability was determined in a microtiter plate reader (PowerWare X340, Bio-Tek Instruments Inc.). Then means (±SEM) were analyzed with a two-tailed unpaired t-test (p = 0.05).

Results Assessment of lethal effects of PFOS on bumblebee workers with use of microcolonies The highest PFOS concentrations tested, 5 and 10 mg/l, resulted in 100% worker mortality after 14 days. Also at a


lower concentration, 2 mg/l, 100% worker mortality was observed but the effect was delayed: there was 11 ± 4, 53 ± 4, 90 ± 2 and 100 ± 0% worker mortality after 1, 2, 3 and 4 weeks, respectively. Exposure to lower concentrations via the drinking of treated sugar water resulted in lower or no toxicity. With PFOS at 1 mg/l, the first dead workers were seen only after 6 weeks and after 11 weeks mortality had reached 46 ± 3%. At lower concentrations of 100, 10 and 1 lg/l, there was no significant mortality over the control groups: the respective percentages were 10 ± 4, 4 ± 2 and 14 ± 1% after 11 weeks. The LC50 value for PFOS after 11 weeks was 1.01 mg/l (95% confidence interval 0.6–1.8 mg/l; R2 = 0.98). For the positive controls that were treated with imidacloprid, all these showed 100% worker mortality. Assessment of sublethal effects of PFOS on reproduction with use of microcolonies Next to lethal effects also sublethal effects on reproduction were scored of both high and low PFOS concentrations. For the high concentrations of PFOS at 2, 5 and 10 mg/l, a total loss of reproduction was seen as a consequence of the severe worker mortality (100%) that occurred at these concentrations (see above). As depicted in Table 1, a lower concentration of 1 mg/l resulted also in a significant effect as no males were produced after 11 weeks. In these nests no eggs were laid by the dominant workers. However, it should be noted here that the bumblebee workers were not apathic as sugar pots were made and filled by foraging workers. In contrast, when bumblebees were exposed to PFOS at 1, 10 and 100 lg/l the nest reproduction was not significantly different (p [ 0.05) from that in the control groups. The dominant workers started to lay eggs after 8–9 days and hatching of the first drones was observed in week 5. Effect of PFOS on ovary growth in bumblebee workers To document the reproductive effects of PFOS at 1 mg/l as described above, the ovaries of the dominant workers were investigated. After dissection, it was apparent that the dominant worker ovaries were reduced in length by more than 50% (7 ± 1 mm) as compared to those of dominant workers from control nests (17 ± 1 mm). Despite the reduction, the ovaries consisted each of four ovarioles as in the controls. In addition, the number of eggs present in the ovaries of the PFOS-treated workers could not be counted under the binocular as they were totally degenerated. In the control nests, 27 ± 3 eggs per ovary were counted.

451 Table 1 Sublethal effects of different concentrations of PFOS on the reproduction of B. terrestris after oral exposure via the drinking of treated sugar water over a period of 11 weeks Treatment

Mean number of drones per nest ± SEM

10 mg/l PFOS

0±0c

5 mg/l PFOS

0±0c

2 mg/l PFOS 1 mg/l PFOS

0±0c 0±0c

100 lg/l PFOS

31.4 ± 0.6 a

10 lg/l PFOS

36.6 ± 1.4 b

1 lg/l PFOS

32.3 ± 0.3 ab

Control

33.8 ± 0.8 ab

The data are expressed as mean numbers of drones per nest ± SEM, based on four artificial nests per treatment and five workers per nest, and with the experiment being repeated twice. ANOVA resulted in three groups (F = 261.720, df = 63, p \ 0.001). Values that are followed by a different letter (a–c) are significantly different after a post-hoc Tukey–Kramer test with p = 0.05

Accumulation of PFOS in bumblebee workers To determine the accumulation of PFOS in the terrestrial insect upon oral exposure, bumblebee workers were fed with sugar water supplemented with PFOS at 100 lg/l during 5 weeks. The bumblebee workers survived this treatment and were active as in the controls. As determined by HPLC–MS, the treated bumblebee workers had accumulated 2184 ± 365 ng/g wet body weight resulting in a bioaccumulation factor (BAF) of 27.9 for PFOS in bumblebees. Effect of PFOS on mitochondrial ETS activity in bumblebee workers In the untreated control bumblebee workers, the ETS activity yielded 1.84 nmol O2 per worker and per min (Table 2). Chronic exposure to PFOS at 1 mg/l via the drinking of treated sugar water resulted in a significant decrease in ETS activity of 26% (t = 2.285; df = 18; p = 0.03). Effect of PFOS on energy content in bumblebee workers In bumblebee workers exposed to PFOS at 1 mg/l, the total energy content was not significantly different (p [ 0.05) when compared to the controls and this was also the case for the protein content (t = 0.5606; df = 37; p = 0.58) and glucose content (t = 1.450; df = 37; p = 0.13) (Table 2). In contrast, the lipid content was significantly lower compared to the control group (t = 2.456; df = 37; p = 0.019).

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Table 2 Effects of PFOS at 1 mg/l on the electron transport activity (ETS; oxygen consumption), energy reserves with lipid, protein and sugar amounts, and body weight after oral exposure via the drinking of treated sugar water over a period of 5 weeks Energy reserves (mean mg/g wet weight ± SEM) Treatment ETS (mean nmol O2/organism min ± SEM) Lipid content Protein content

Sugar content

Control

1.84 ± 0.12 a

52.1 ± 6.1 a

PFOS

1.36 ± 0.17 b (p = 0.035) 18.1 ± 1.6 b (p = 0.019) 303 ± 27 a (p = 0.58) 44.0 ± 4.5 a (p = 0.12) 275 ± 11 a (p = 0.06)

24.6 ± 2.1 a

322 ± 22 a

Total body weight (mean mg ± SEM) 308 – 12 a

Data are expressed as mean ± SEM. Significant differences between control of PFOS treatment are denoted with different letters (a–b) after an unpaired t-test (p = 0.05)

Reporter assay to determine EcR interaction of PFOS with ecdysteroid-responsive insect S2 cells

150000

Discussion Over the past decade, different publications have described the toxicity of PFOS in the aquatic and terrestrial ecosystem, however, this is the first study that evaluated the effects of a long-term oral exposure of PFOS in an important insect of the terrestrial compartment, namely workers of the pollinating bumblebee B. terrestris. A chronic exposure to different PFOS concentrations by use of microcolonies in the laboratory resulted in worker mortality (lethal effect) and decreased reproduction (sublethal effect), and these effects occurred in a concentrationdependent manner. At high PFOS concentrations of C2 mg/l, severe lethal effects with 100% mortality of the

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RLU

100000

As depicted in Fig. 1, PFOS performed no agonistic activity on the EcR as incubation of transfected cells, containing the EcRE-b.act.luc reporter construct, during 24 h with PFOS resulted in a mean luminescence response not higher than the blank control series. The mean measured luminescence was 55 ± 6, 203 ± 27, 158 ± 5 and 204 ± 16 RLU for PFOS at 100, 10 and 1 lM and the blank, respectively. In contrast, 20E at 1 lM showed a strong agonistic activity with 130634 ± 4566 RLU that is considered as maximum, based on previous concentration– response experiments (Soin et al. 2008). Incubation of S2 cells with PFOS and 1 lM of 20E demonstrated a strong antagonistic activity. The mean luminescence response was 61522 ± 4118 , 53132 ± 7053 and 154 ± 13 RLU with addition of PFOS at 1, 10 and 100 lM, respectively, which corresponds to a respective reduction of 53, 59 and 100% as compared to 1 lM 20E without PFOS (Fig. 1). With the MTT test no significant effects (p [ 0.05) were seen on the cell viability of the S2 cells after exposure for 5 days to the different PFOS concentrations of 1, 10 and 100 lM as compared to control cells (data not shown).

50000

0 blank

1 µM 20E

1 µM

10 µM

100 µM

PFOS agonistic activity

1 µM

10 µM

100 µM

PFOS antagonistic activity

Treatment

Fig. 1 Comparison of the agonistic and antagonistic activity of PFOS in ecdysteroid-responsive Schneider (S2) cells of Drosophila melanogaster. The reporter construct was first transiently introduced into the cells (see ‘‘Reporter assay to determine EcR interaction of PFOS with ecdysteroid-responsive insect S2 cells’’ section for details) and harvested. For agonistic activity, the cells were then treated with PFOS at different concentrations (1, 10 and 100 lM) and finally assayed for luciferase expression activity. For antagonistic activity, the cells were exposed to 1 lM of 20-hydroxyecdysone (20E) together with serial concentrations of PFOS and then assayed for luciferase expression activity. The control cells (blank) were treated with ethanol alone. The results are expressed as total RLUs measured (means ± SEM)

exposed bumblebee workers were scored, while mortality was moderate with 46 ± 3% at 1 mg/l. As reported before (Beach et al. 2006), PFOS can pose strong lethal effects, but the mechanism(s) behind is still unknown. Austin et al. (2003) and Bilbao et al. (2010) reported a lowered food intake and narcotic effects that concerted with a fitness reduction by PFOS, but none of these symptoms were seen in the treated bumblebee workers in this study. However, we could demonstrate that the mitochondrial ETS activity was lowered by 26% in the treated bumblebee workers. Previously, Li et al. (2003) and Muller et al. (2004) reported that a lower ETS activity led to a decreased ATP production and oxygen consumption by the mitochondria and to the formation of reactive oxygen species (ROS). Moreover, Hu et al. (2003) demonstrated that PFOS increases the non-selective permeability of membranes and decreases the mitochondrial membrane potential. This increased permeability and decreased membrane potential


can induce the release of proapoptotic molecules and triggers cell death (Crompton 1999). Therefore, we believe that PFOS in the bumblebee workers caused an inhibition of the respiratory chain, and this in turn has contributed to the high levels of acute mortality observed in the current experiments. However, before making final conclusions on risks in terrestrial insects, PFOS and related perfluorochemicals should be investigated in more realistic field-related situations for the assessment of potentially deleterious lethal and sublethal effects on insect survival, reproduction and foraging behavior. For bumblebees this should be done with queenright colonies of B. terrestris, and where bumblebee workers need to forage/fly for food that is placed at a distance (i.e., 3 m) from their hives. Herewith, effects towards electron transport and the respiratory chain should be confirmed at molecular level. Hence, it is clear that these experiments should be done with environmentally relevant concentrations. Second, based on the current HPLC–MS analysis it was clear that PFOS were strongly bioaccumulated to a high extent in bumblebee workers. When exposed to PFOS at 100 lg/l via the drinking of treated sugar water for 5 weeks, the bumblebee workers demonstrated 2184 ± 365 ng/g PFOS accumulation in their body which corresponds to 655 ± 109 ng PFOS per bumblebee worker. Interestingly, this high bioaccumulation is close to a calculated value of 970 ng PFOS per bumblebee worker when a bumblebee worker drinks 277 ll of sugar water per day (Mommaerts et al. 2010). Although this high bioaccumulation of PFOS in the body of bumblebee workers agrees with previous reports in other organisms (Giesy and Kannan 2001; Houde et al. 2006), it remains to be investigated how much of PFOS is accumulated in specific critical organs as the reproductive system. We believe that this information can be helpful to document detrimental effects of PFOS on biodiversity by accumulation in specific species after direct exposure and also in higher trophic level biota. In this context, Ankley et al. (2005) reported a high specific accumulation of PFOS in the ovaries of fathead minnows Pimphales promelas (64.6 lg/g ovary) after 21 days of oral exposure to 30 lg/l PFOS, and interestingly this agreed with histopathological changes in the ovaries. Because the oral exposure in the latter experiment was about 3.3 times lower in concentration (30 lg/l versus 100 lg/l) and 1.7 times shorter in period (21 days versus 35 days) as compared to the exposure with bumblebee workers, it can be expected that also in the treated bumblebee workers PFOS was highly accumulated in their ovaries. Although exposure to PFOS at 100, 10 and 1 lg/l during 11 weeks showed no effects, here it is of interest to note that PFOS at 1 mg/l (and higher) was highly detrimental for the bumblebee workers and their ovaries were totally affected. Based on Van Gossum et al. (2009) the

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highest environmental concentration reported (so far) is 8.6 lg/l PFOS in fresh water, which is a concentration far below 1 mg/l. However, we believe that it should not be underestimated that higher doses of PFOS can be accumulated over longer periods of exposure and in specific sensitive organs like the ovaries, particularly because PFOS is not readily biodegradable (Houde et al. 2006). Besides, Moody et al. (2002) reported about accidental release of fire fighting foam, leading to high PFOS concentrations of 2.2 mg/l in the environment. Although the latter is accidental, such concentrations have been reported in specific cases and the current data demonstrated that exposure to such doses is highly toxic for bumblebee workers. In addition, PFOS can be accumulated also at higher trophic levels. Recently, Stahl et al. (2009) reported on carryover of PFOS from soil to crops, and so PFOS can be transported throughout the plant and into nectar and the pollen. Given that nectar and pollen are the two main food sources of pollinators, it is likely that these beneficial insects are exposed to the risks of being poisoned by PFOS during their foraging. In conclusion, we feel that all data so far are indicative of the risks for strong effects by PFOS due to a high bioaccumulation, which was also confirmed in bumblebee workers of B. terrestris in this study. Another interesting effect of PFOS is that treatment caused a strong reduction of reproduction in treated bumblebee workers. Typically, the ovaries in workers treated with 1 mg/l were highly affected and any normal egg (follicle) in the ovary was included in this, leading to a total loss of drone production. In the past, negative effects by PFOS have been observed in rat and mice with reduced uterus implantation and fetal death, and in zebrafish with embryonic and larval malformations (Luebker et al. 2005; Lau et al. 2004, 2006, 2007; Shi et al. 2008). Besides, Kannan et al. (2005) demonstrated that PFOS accumulation in the embryo occurred via oviparous transfer. Here, to explain the reproductive effects as seen in treated bumblebee workers, we postulate two potential mechanisms: namely reduction of the lipid reserves and an endocrine disrupting effect. Indeed PFOS caused in bumblebee workers a significant effect (p = 0.019) on the lipid reserves of the energy budget which indicates a reduction of the energy stores vital for the organism. This agrees with the metabolic cost hypothesis of Rowe et al. (2001) on the suppression of non-vital processes as growth and reproduction. Indeed, the inability to allocate lipids to the reserves was shown to be important for the maintenance of reproduction in D. magna and D. melanogaster (De Coen and Janssen 2003; Folk and Bradley 2005). Also for bumblebees, lipid reserves are important energy sources as they supply energy for flight and reproduction (Cvacˇka et al. 2006). However, to date there are insufficient data available to identify whether PFOS may cause an inhibition

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of the lipid synthesis and/or increase in the lipid breakdown. This lack of information was also discussed by Hagenaars et al. (2008) for the glycogen reserves in the liver of carp fish after exposure to PFOS at 1 mg/l. Next to metabolic costs, there was a clear antagonistic EcR activity in the presence of 20E by all PFOS concentrations tested and this without loss of viability of the reporter S2 cells, providing strong evidence of interference with the hormonal regulation of insect development and reproduction. In the past, multiple studies have reported endocrine disrupting properties for PFOS, and these authors specifically referred to alterations in hormone biosynthesis and hormone responsive genes (Biegel et al. 1995; Austin et al. 2003; Lau et al. 2003; Ankley et al. 2005; Hu et al. 2005; Oakes et al. 2005; Shi et al. 2007; Hagenaars et al. 2008; Jensen and Leffers 2008; Wei et al. 2008; Bilbao et al. 2010). Most notable, the current experiments with ecdysteroid-responsive S2 cells demonstrated for the first time that PFOS can mediate endocrine effects via interference with the function of the ecdysteroid hormone receptor EcR. It is generally accepted that the EcR plays a crucial role in insect reproduction; for instance in oogenesis to control the production of yolk proteins (Raikhel et al. 2002; Terashima and Bownes 2004), in follicular epithelium morphogenesis (Romani et al. 2009) and in eggshell formation (Cherbas et al. 2003; Bernardi et al. 2009). In the bumblebee B. terrestris, oocyte growth in the ovary can be correlated with increasing titers of ecdysteroids that likely are involved in the regulation of ovarian follicle development and/or extra-ovarian reproductive functions as in other insects (Bloch et al. 2000; Geva et al. 2005; Swevers and Iatrou 2009). Any impairment of normal EcR functioning will lead to aberration of normal ovarian, oocyte and egg growth as was also seen in the treated bumblebees. Before, Liu et al. (2007) suggested with use of tilapia fish an interaction of perfluorochemicals with the estrogenic activities however, no experimental prove was given. Thus, the current data provide for the first time strong conceptual evidence that PFOS has antagonistic activity towards the EcR as tested with EcR-reporter S2 insect cells. Furthermore, substantial interference is observed at concentrations as low as 1 lM, corresponding to *500 lg/l, indicating that normal functioning of the ecdysteroid signalling pathway during bumblebee reproduction can be affected at low (sublethal) concentrations. Whether PFOS affects EcR directly requires additional biochemical tests as suggested for other potential antagonists (Soin et al. 2010). Besides, it is possible that PFOS cause indirectly impairment of EcR function because of high sensitivity to small disturbances in general cell metabolism, but without loss of cell viability. Therefore, we believe that the strong antagonistic interference of PFOS in the ecdysteroid signaling may have (at least in part) contributed to the detrimental effects on

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growth and reproduction as seen in treated bumblebee workers. However, before making final conclusions on the endocrine disrupting activity via the EcR for future risk assessment, PFOS and/or perfluorochemicals should be evaluated in more realistic conditions with whole organisms and at physiological and molecular level. Hence, such tests should also include a good knowledge of environmentally relevant concentrations. Acknowledgments A. Hagenaars is recipient of a doctoral grant by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). The authors acknowledge Biobest (Westerlo, Belgium) for the kind gift of bumblebees. This work is also supported in part by the Research Council of VUB (Brussels, Belgium), the Fond National de la Recherche Luxembourg (FNR) (Luxembourg) and the Fund for Scientific Research (FWO)Flanders (Brussels, Belgium). Research on ecdysteroid agonists at the Insect Molecular Genetics and Biotechnology group at the Institute of Biology, National Center for Scientific Research ‘‘Demokritos’’ was supported by two bilateral scientific and technological cooperation grants (Greece-Japan & Greece-Spain) from the General Secretariat for Research and Technology, Ministry of Development, in Greece.

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