Identification of pesticide exposure-induced metabolic changes in mosquito larvae

Science of the Total Environment 643 (2018) 1533–1541

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Identification of pesticide exposure-induced metabolic changes in mosquito larvae Renato Russo a,b, Sven-Bastiaan Haange c,d, Ulrike Rolle-Kampczyk c, Martin von Bergen c, Jeremias Martin Becker a,b, Matthias Liess a,b,⁎ a

UFZ, Helmholtz Centre for Environmental Research, Department of System-Ecotoxicology, Permoserstraße 15, 04318 Leipzig, Germany RWTH Aachen University, Institute for Environmental Research (Biology V), Worringerweg 1, 52074 Aachen, Germany UFZ, Helmholtz Centre for Environmental Research, Department of Molecular System Biology, Permoserstraße 15, 04318 Leipzig, Germany d University of Leipzig, Institute of Biochemistry, Leipzig, Germany b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Pesticides affect ecosystems at concentrations below regulatory thresholds. • We observed metabolic changes at concentrations considered safe in risk assessment. • The insecticide clothianidin induced energy-related metabolic changes in mosquitoes. • Effects were weaker but lasted longer after exposure to very low concentrations. • Low concentrations may induce environmental effects through increased energy demand.

a r t i c l e

i n f o

Article history: Received 5 April 2018 Received in revised form 22 June 2018 Accepted 22 June 2018 Available online xxxx Editor: Yolanda Picó Keywords: Metabolic change Low-concentration exposure Freshwater macroinvertebrates Neonicotinoids

a b s t r a c t The European regulatory framework for pesticides generally applies an assessment factor of up to 100 below the acute median lethal concentration (LC50) in laboratory tests to predict the regulatory acceptable concentrations (RACs). However, long-term detrimental effects of pesticides in the environment occur far below the RACs. Here, we explored the metabolic changes induced by exposure to the neonicotinoid insecticide clothianidin in larvae of the mosquito Culex pipiens. We exposed the test organisms to the insecticide for 24 h and then measured the levels of 184 metabolites immediately and 48 h after the pulse contamination. We established a link between the exposure to clothianidin and changes in the level of three specific classes of metabolites involved in energy metabolism, namely, glycerophospholipids, acylcarnitines and biogenic amines. Remarkably, exposure to concentrations considered to be safe according to the regulatory framework (2–4 orders of magnitude lower than the acute LC50), induced longer-term effects than exposure to the highest concentration. These results suggest that a specific detoxification mechanism was only triggered by the highest concentration. We conclude that even very low insecticide concentrations increase the energy demands of exposed organisms, which potentially translates into a decline in sensitive species in the field. © 2018 Elsevier B.V. All rights reserved.

Abbreviations: LC50, Median lethal concentration; RACs, Regulatory acceptable concentrations; PFOS, Perfluorooctanesulfonic acid; LC, Liquid chromatography; FIA, Flow injection analysis; MS, Mass spectrometry; MRM, Multi-reaction monitoring; ESI, Electrospray ionization; CCD, Colony collapse disorder; ATP, Adenosine triphosphate; RNA, Ribonucleic acid; LD50, Median lethal dose; HBCD, Hexabromocyclododecane. ⁎ Corresponding author at: UFZ, Helmholtz Centre for Environmental Research, Department of System-Ecotoxicology, Permoserstraße 15, 04318 Leipzig, Germany. E-mail address: [email protected] (M. Liess).

https://doi.org/10.1016/j.scitotenv.2018.06.282 0048-9697/© 2018 Elsevier B.V. All rights reserved.

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1. Introduction Within the European risk assessment framework, an assessment factor of up to 100 below the acute LC50 (the concentration that is lethal to 50% of test organisms in laboratory standard tests) is commonly applied to predict the regulatory acceptable concentrations (RACs) of pesticides in the natural environment (EFSA, 2013). However, the current framework is clearly not protective enough as several studies on non-target freshwater macroinvertebrates have detected the detrimental effects of pesticides at concentrations considered to be safe (Beketov et al., 2013; Liess et al., 2013; Liess and Von Der Ohe, 2005; Münze et al., 2017; Nyman et al., 2013; Schäfer et al., 2012). Low pesticide concentrations, such as RACs, might not have short-term lethal effects. Nevertheless, such concentrations may result in delayed lethal effects (Beketov and Liess, 2008), interact synergistically with other stressors in the field (Janssens et al., 2017; Liess et al., 2016; Beketov and Liess, 2005; Coors and De Meester, 2008; Relyea and Mills, 2001) and show longterm cumulative effects in cases of repeated or chronic exposure (Alkassab and Kirchner, 2016; Liess et al., 2013; Russo et al., 2018). In 2013, EU Commission Regulation 284 (EU, 2013a) indicated that resolving the uncertainties about the effects of pesticides at low concentrations is crucial for refining the risk assessment framework for pesticides. In this respect, understanding the biochemical processes that underlie pesticide toxicity, particularly at low concentrations, may increase the predictive power of toxicity tests. In fact, the identification of responses in key metabolites for use as regulatory endpoints may allow a prompt detection of detrimental effects even at sub-lethal concentrations. The increased knowledge of toxicity mechanisms can thus contribute to the design of cost- and time-effective analyses and, ultimately, to more realistic extrapolations of the laboratory data to the field. In recent years, due to the advancement in metabolomic techniques, new high-throughput and multi-endpoint methods for analysing metabolic changes have become available (Aardema and MacGregor, 2002). These techniques have been increasingly applied to characterize the effects of toxicants on organisms (Mckelvie et al., 2009; Marx-Stoelting et al., 2015; Tralau et al., 2015) and to identify potential biomarkers of exposure to specific compounds (Lin et al., 2006; Robertson, 2005). The link between the changes in the levels of key natural metabolites and the exposure to a specific toxicant is relatively well established for humans. An increase in mercapturic acid, for instance, has been linked to exposure to atrazine (Lucas et al., 1993), while an increase in 1hydroxypyrene has been linked to exposure to polycyclic aromatic hydrocarbons (Jongeneelen, 2001). Recently, Zhang et al. (2014) proposed using increases in serine and testosterone as biomarkers of arsenic exposure. In rats, the fluctuations of various metabolites have been linked to exposure to hydrazine (Nicholls et al., 2001) and γ radiation (Lanz et al., 2009), and Wu et al. (2013) observed decreased levels of amino acids and increased levels of betaine and fumarate in clams exposed to arsenic. Similarly, various potential biomarkers of toxicant exposure have been developed for earthworms in the past decades: an increase in histidine, for instance, has been proposed as a biomarker of exposure to copper (Gibb et al., 1997). More recently, fluctuations in the levels of various metabolites have been identified as potential indicators of PFOS exposure (Lankadurai et al., 2013). Mckelvie et al. (2009) explored in the use of increasing concentrations of alanine as a potential biomarker of organochlorine insecticide exposure. However, few studies to date have investigated the metabolic responses of freshwater invertebrates after exposure to various substances (Jones et al., 2012; Nagato et al., 2013) despite the relevance of this taxonomic group for the risk assessment of pesticides. Recent evidence has suggested that alanine and lysine play key roles in the toxicities of copper, arsenic and lithium in Daphnia (Nagato et al., 2013), and vitellogenin has been identified as a biomarker of exposure to oestrogenic compounds in aquatic environments (Matozzo et al.,

2008). Additionally, Martin-Park et al. (2017) explored the metabolic profiles of contaminated mosquitoes for the first time and proposed that an increase in acylcarnitines is a potential tool for diagnosing pyrethroid insecticide exposure. The higher sensitivity of metabolomic techniques compared to traditional toxicity tests enables the detection of effects at low and environmentally relevant concentrations (Tufi et al., 2015). Furthermore, the identification of key metabolites as biomarkers in freshwater invertebrates may contribute to the design of more effective toxicological tests without the use of either mammal systems, which are too expensive and undesirable for ethical reasons (EU, 2016), or cell culture systems, which do not present the required functional complexity (Jones et al., 2012). Accordingly, in the present study, we explored the link between insecticide exposure and metabolic changes in the larvae of a common freshwater macroinvertebrate and relevant target of pest control, the mosquito Culex pipiens. In the laboratory, we aimed to identify key metabolic profiles that reveal sub-lethal effects at low concentrations that potentially lead to delayed/synergistic effects in the field. The metabolic changes in C. pipiens were investigated at high (10,000 ng/L, the same order of magnitude as LC50), medium (100 ng/L) and very low (1 ng/L) concentrations of clothianidin, a neonicotinoid insecticide commonly applied in agriculture. The high concentration is of great regulatory relevance, being at the same order of magnitude as the LC50 while the medium concentration resembled environmentally realistic conditions (Knillmann et al., 2018). Finally, we applied the very low concentration to test the experimental hypothesis that metabolic changes can be detected even below the RACs. We investigated changes at two different time points in order to explore the transient nature of metabolic changes: the first, directly after the exposure, to investigate the changes induced by the toxicity of clothianidin; the second, approximately at half of the larval stage (Gerberg et al., 1969), to explore the combination of the effects of toxicity with the biological response of the larvae. It is pivotal to understand the transient nature of such metabolic changes in order to ensure their detection and, eventually, apply them as regulatory endpoint in the future. 2. Materials and methods 2.1. Study design We exposed newborn larvae of the mosquito Culex pipiens pipiens to three different concentrations of clothianidin (1 ng/L, 100 ng/L and 10,000 ng/L) for 24 h. The metabolite contents were quantified directly after the 24 h of exposure and 48 h after the end of the pulse exposure by LC-MS-MS. 2.2. Study species The mosquito Culex pipiens (Linnaeus, 1758 - Insecta: Diptera) is a potential vector of diseases such as West Nile fever, Rift Valley fever and Elephantiasis (Chandre et al., 1998; Kumar and Pillai, 2011; Paul et al., 2005; Rodriguez et al., 1993; Scott et al., 2015). The larvae of this species are common in ephemeral ponds and are therefore frequently exposed to various insecticides either through vector control measures or through run-off from agricultural fields. We used the biotype molestus (subsequently referred to as simply C. pipiens), which has adapted to subterranean habitats, because it is relatively easy to culture. Unlike other mosquitoes, C. pipiens can lay its first egg raft without a blood meal (Clements, 1999) and mates in small cages without swarming, which makes this species suitable for laboratory culture. The mosquito culture was obtained from the Federal Environment Agency, UBA, Berlin, Germany, and was grown in a rearing chamber at 25 °C with 60% humidity and a 16:8 light:dark regime. The egg rafts were collected in plastic trays containing tap water. The hatched larvae were cultured in tap water in white plastic trays that were constantly aerated using an aquarium pump. The larvae were fed with a 1:1

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(weight ratio) mixture of ground dog biscuits (Hd-H biscuits, ssniff Spezialdiäten GmbH, Soest, Germany) and stinging nettle powder (Folia urticae, Caesar & Loretz GmbH, Hilden, Germany) until pupation (approximately 10 days). The pupae were placed into glass trays, transferred into the cages and covered with a mesh to prevent adult mosquito oviposition until emergence (24–72 h). The emerged mosquitoes were released to mate inside the cages and fed with sponges soaked in glucose-, fructose- and honey-saturated solutions. Female mosquitoes placed their egg rafts into open trays filled with water, which were subsequently transferred into plastic trays outside the cages to restart the cycle. 2.3. Insecticide The test organisms were exposed to the neonicotinoid insecticide clothianidin. Neonicotinoid insecticides were commercialized in 1991 and have dominated the global agricultural insecticide market since 2000 (Simon-Delso et al., 2015). Clothianidin [(E)-1-(2-chloro-1,3thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine] is one of the latest members of neonicotinoid insecticides. This compound was invented and developed by Sumitomo Chemical Takeda Agro Company, Ltd. and has been effectively used against Diptera, Coleoptera and Lepidoptera pests. The chemical structure of clothianidin is characterized by the presence of a thiazolyl ring. Clothianidin and other neonicotinoids are of increasing concern (Commission Regulation (EU) No. 485, EU, 2013b) because evidence suggests that these compounds impact nontargeted, beneficial insects (Mullin et al., 2005; Pecenka and Lundgren, 2015), especially honeybees (Brandt et al., 2016; Di Prisco et al., 2013; Jeschke and Nauen, 2008; Laurino et al., 2011). Due to its chemical and physical properties, clothianidin has the potential to reach surface waters (APVMA, 2007) and has often been detected in agricultural streams (Whiting et al., 2014; Münze et al., 2017) at concentrations of up to 1000 ng/L (Knillmann et al., 2018). Clothianidin was obtained as granulated powder (weight ratio 1:1) from DANTOP® (Spiess-Urania Chemical GmbH, Germany) and prepared by diluting 0.125 g of the powder in 0.5 L of deionized water (to a final concentration of 0.125 g clothianidin/L) and then stirring the solution for 12 h. During this time, the solution was protected against photodegradation by covering it with aluminium foil. The three test solutions were obtained from serial dilutions prepared by mixing the stock solution with appropriate quantities of deionized water (for more details on the preparation of test solutions, see Table S7). Since the lowest concentration of clothianidin applied in our study was out of the detection range of most standard methods for this substance, we performed analytical measurements only for (i) the last dilution step before obtaining the test concentrations and (ii) the medium test concentration. On average, the measured concentrations deviated from the nominal concentrations by ±3.5% (Table S8). Therefore, all results reported in the subsequent sections refer to the nominal concentrations. In preliminary tests, the LC50 for C. pipiens was 14.85 μg/L (Fig. S1), which is comparable with data in the literature on the toxicity of neonicotinoid to aquatic macroinvertebrates (PPDB website; Beketov and Liess, 2008; Mo et al., 2002). 2.4. Acute toxicity tests On day 1, up to 24-h-old C. pipiens larvae were placed in 2-L glass vessels filled with 1 L of M4 Elendt medium (OECD guideline No. 211, 1998, annex 2) for the exposure to clothianidin for 24 h. Test temperature was 18 ± 1 °C. Each vessel contained 20 mosquito larvae. We applied four contamination levels: control group (non-exposed), 1 ng/L (4 orders of magnitude below the LC50), 100 ng/L (2 orders of magnitude below the LC50) and 10,000 ng/L (the same order of magnitude as the LC50). Five glass vessels (up to a total of 100 larvae) were prepared for each concentration level.

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On day 2 (directly after the 24 h of exposure), the mortality was always below 5% at each concentration level (Fig. S2). While such low mortality was expected for the lowest and the medium concentrations, this seemed unexpected for the highest concentration. However, the LC50 calculation referred to mortality measured after 48 h of exposure, while in the present study we exposed the mosquito larvae to clothianidin only for 24 h. In fact, when comparing our results with the LC50 calculation (Table S9), the mortality after 24 h of exposure at 10,000 ng/L appeared comparable. Sixty surviving larvae from each of the four different contamination levels were frozen in 1.5-mL Eppendorf tubes at −80 °C for the metabolic analysis. The amount of larvae needed to achieve enough biomass to allow metabolomics measurements were estimated based on calculations of average dry weight of larvae at different developmental stages (Table S10). The remaining larvae were placed in 2-L glass vessels filled with 1 L of fresh (non-contaminated) medium and fed with 1.2 mg/larvae of a mixture of ground dog biscuits (Hd-H biscuits, ssniff Spezialdiäten GmbH, Soest, Germany) and stinging nettle powder (Folia urticae, Caesar & Loretz GmbH, Hilden, Germany) at a final weight ratio of 1:1 and a final concentration of 12 mg/mL. On day 4 (48 h after the insecticide exposure), 20 larvae per condition were frozen in 1.5-mL Eppendorf tubes at −80 °C for the metabolic analysis. We repeated the experiment nine times over a span of five months; however, due to measurement issues, we could only consider seven of the nine replicates for statistical analysis. All the analytical measurements were performed together at the end of the last experimental replication. 2.5. Sample preparation for LC-MS-MS measurement The sample tubes were centrifuged for 15 min (15,000 rpm at 5 °C), and up to 500 μL of medium was removed. Subsequently, the samples were (i) dried in a vacuum centrifuge (VAQ at 45 °C for 4 h), (ii) diluted in 1 mL of a mix of methanol/acetonitrile (1:1), (iii) treated with ultrasound under cooling with ice for 1 h, and (iv) centrifuged again (15,000 rpm at 5 °C). Prior to the analysis, the supernatant was dried again in a nitrogen evaporator (1 h at 4 bar) and then placed in 100 μL of methanol. 2.6. Metabolomic measurement by MS-MS With metabolomics having only been recently applied to aquatic invertebrates, information on the links between pesticide exposure and related changes in metabolite profiles is sparse. As such, a sound and reproducible extraction method has not been developed. Therefore, we used a commonly applied metabolomics kit (Absolute IDQ p180, Biocrates LIFE Science AG, Innsbruck, Austria). The kit has been designed to cover metabolic pathways that are known to be of central biological relevance – e.g., acylcarnitines are involved in mitochondrial function (Imam et al., 2018; Kenéz et al., 2016; Moser et al., 2015), phosphatidylcholines influence lipoprotein structure (Jackson et al., 1976), and biogenic amines are involved in cell cycle controls (Heby, 1981) and detoxification (Frieling et al., 2012). The kit identifies and quantifies 184 metabolites from 6 compound classes (Table 1) and provides highly robust, quantitative metabolomics data with excellent accuracy and precision. Its outstanding inter-instrument, inter-laboratory, as well as long-term reproducibility has been proven by an independent, international ring trial (Siskos et al., 2017). The analyses were carried out as previously described (Kenéz et al., 2016; Oberbach et al., 2011). In brief, a flow injection tandem mass spectrometry method (FIA-MS/MS) was employed for the unipolar metabolites, and an LC-MS/MS method was employed for the polar metabolites. The assay required only very small amounts of substrate (10 μL). The quantification of analytes utilized stable isotope-labelled or chemically homologous internal standards. Quality controls (QCs) were included for three different concentration levels. For calibration, the kit

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contained a calibrator mix with seven different concentrations. The measurements were carried out with electrospray ionization (ESI) in multi-reaction monitoring (MRM) mode to ensure high specificity and sensitivity. For the FIA measurements, two injections were needed. In total, 158 MRM pairs were measured in positive ion mode (including 13 internal standards (ISs)), and two MRM pairs were measured in negative ionization mode (including one IS). In the LC-MS/MS method, 65 MRM pairs were measured only in the positive mode (including 25 Iss). Therefore, only one injection was necessary for the LC part. The kit needed the following additional chemicals: water, Millipore; ITC, Fluka (derivatisation of amino acids); pyridine, Fluka (p.a.); methanol, Merck; LiChrosolv for LC/MS; Acetonitril, Merck; LiChrosolv for LC/MS, Fluka; and formic acid, Fluka. The kit has been used for several biofluids and tissue samples in various species, ranging from human to yeast (Ferrario et al., 2016; Papathanassiu et al., 2017; Rußmayer et al., 2015), but to our knowledge, using this kit for mosquito larvae is new. 2.7. Data analysis Statistical analyses were conducted with RStudio for Mac (version 1.0.153). The dataset used consisted of 5152 (184 × 4 × 7 × 2) observations resulting from 184 metabolites that were measured at four different contamination levels over seven experimental replications on day 2 and day 4 (Tables S1–S6). To investigate which pesticide concentrations affected the (i) metabolite content as a whole and (ii) the contents of each metabolite class on day 2 and day 4, we constructed linear mixed-effects models with the lme4 (Bates et al., 2014; version 1.1–7) and lmerTest packages (Kuznetsova et al., 2017). To explore the effects of the pesticide on the metabolite content, we included only the pesticide concentrations as a fixed factor; the effect of individual metabolites, nested within the classes, and the effect of experimental replications were included as random intercepts. When investigating the effects of the pesticide on the average metabolite content within each metabolite class, we applied similar models as above, with the only difference being that the metabolite classes were not included in the random intercept because a separate model was constructed for each class. To compare the effects of different pesticide concentrations in a post hoc analysis, we specified custom contrasts between the pesticide concentrations that matched our specific hypotheses using the phia package (De Rosario-Martinez, 2015; version 0.2–0). Finally, to explore the effects of the pesticide on each individual metabolite, we compared the concentrations of each metabolite in pesticide-treated larvae and in the control group using paired t-tests with the seven experimental replications included as the grouping factor. To avoid type I error inflation by multiple comparisons, all p values were adjusted to the number of classes, the number of metabolites within a given class, or the number of custom contrasts used in a post hoc test by applying the fdr correction. The metabolite concentrations were log transformed prior to the analyses to improve homoscedasticity and to normalize the distribution of the residuals, as is required by

Table 1 List of compounds measured by mass spectrometry. The analysis was carried out by a flow injection tandem mass spectrometry method (FIA-MS/MS) for the unipolar metabolites, and an LC-MS/MS method was used for the polar metabolites. Compound class

Subclasses

Acylcarnitines Amino acids Proteinogenic, citrulline, ornithine Glycerophospholipids Phosphatidylcholines, lysophosphatidylcholines Sphingolipids Sphingomyelines Biogenic amines Hexoses

No. of compounds 40 19 76, 14 15 19 1

parametric tests. In the plots, we report the effects of pesticide exposure as the metabolite concentration of the exposure group relative to that of the non-exposed control group. 3. Results 3.1. Average metabolite content We found that all the applied clothianidin concentrations had significant effects on the average content across all metabolites (Fig. 1). On day 2, directly after the 24-h contamination period (Fig. 1a), exposure to 10,000 ng/L resulted in a 27% reduction in the overall metabolite content (t = −9.4; df = 2392 following Satterthwaite approximation; p b 0.001), while exposure to 100 ng/L resulted in only a 13% reduction (t = −4.2; df = 2392; p b 0.001), and exposure to 1 ng/L resulted in a 17% reduction (t = −5.6; df = 2393; p b 0.001). Thus, the highest pesticide concentration resulted in stronger effects than the medium concentration (χ2 = 28.1; df = 1; p b 0.001) or the lowest concentration (χ2 = 14.8; df = 1; p b 0.001). In contrast, on day 4 (48 h after exposure, Fig. 1b), the highest pesticide concentration did not result in significant changes in the overall metabolite content, while the medium and lowest concentrations resulted in reductions of 16% (t = −4.6; df = 2411; p b 0.001) and 12% (t = −2.9; df = 2402; p = 0.005), respectively. 3.2. Metabolite classes Directly after pesticide exposure (day 2), we observed significant changes in the average metabolite contents within three (out of six) metabolic classes, namely, biogenic amines, glycerophospholipids and acylcarnitines (Fig. 2a). In particular, on day 2, exposure to 10,000 ng/L reduced the contents of biogenic amines (t = −3.0; df = 240; p = 0.006), glycerophospholipids (t = −14.0; df = 1145; p b 0.001), and acylcarnitines (t = −6.9; df = 513; p b 0.001). Also exposure to 100 ng/L marginally reduced the biogenic amine content (t = −2.0; df = 239; p = 0.084) and significantly reduced the glycerophospholipid content (t = −6.5; df = 1146; p b 0.001) but increased the acylcarnitine content (t = 3.3; df = 513; p = 0.003). Similarly, exposure to 1 ng/L significantly reduced the biogenic amine content (t = −2.5; df = 240; p = 0.038) and glycerophospholipid content (t = −7.0; df = 1145; p b 0.001) but marginally increased the acylcarnitine content (t = 2.0; df = 513; p = 0.089). The highest pesticide concentration decreased the glycerophospholipid content more strongly than the medium (χ2 = 54.3; df = 1; p b 0.001) and the lowest concentrations (χ2 = 23.1; df = 1; p b 0.001). However, the decrease in the biogenic amine content did not vary significantly between the different pesticide concentrations. The decrease in acylcarnitines at the highest pesticide concentration differed significantly from the increase in acylcarnitines at the lowest (χ2 = 62.51; df = 1; p b 0.001) and medium (χ2 = 85.31, df = 1, p b 0.001) concentrations; however, the changes in the acylcarnitine content were generally small (b10%) compared to those of the other metabolite classes. However, two days after pesticide exposure (day 4), only the glycerophospholipid and acylcarnitine contents were affected, and this result was observed only after exposure to the low and medium pesticide concentrations (Fig. 2b). Decreased glycerophospholipid contents were observed after exposure to both 100 ng/L (t = −9.4; df = 1163; p b 0.001) and 1 ng/L (t = −5.3; df = 1163; p b 0.001) of clothianidin, and the effect was stronger at 100 ng/L (χ2 = 11.55; df = 1, p b 0.001). Notably, the glycerophospholipid content tended to increase (even though not significantly) after the exposure to 10,000 ng/L. Similarly, the content of acylcarnitines was reduced after exposure to both 100 ng/L (t = −4.9; df = 513; p b 0.001) and 1 ng/L (t = −6.6; df = 513; p b 0.001) clothianidin, while tended to increase after the exposure to 10,000 ng/L. The reduction was marginally stronger at 1 ng/L (χ2 = 3.17; df = 1, p = 0.075). A detailed list of all measured factors of change

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Fig. 1. Percent change of average metabolites levels at different clothianidin concentrations. The relative overall metabolite content (percent of the metabolite content of the non-exposed control group) after exposure to 1 ng/L, 100 ng/L and 10,000 ng/L clothianidin for 24 h measured (a) directly after exposure and (b) 48 h hours later. The analysis is based on a linear mixedeffects model with 5152 observations. The relative changes are shown with their associated 95% confidence intervals. The asterisks below the bars report significant deviations from the control group, while significant differences between doses of exposure are displayed on the top, where “⁎⁎” indicates p b 0.01 and “⁎⁎⁎” indicates p b 0.001.

Fig. 2. Percent change of metabolite classes level at different clothianidin concentrations. The relative changes in the contents of the different metabolite classes (percent of the metabolite content of the non-exposed control group) after exposure to 1 ng/L, 100 ng/L and 10,000 ng/L clothianidin for 24 h measured (a) directly after exposure and (b) 48 h later. The analysis is based on linear mixed-effects models with 5152 observations. Only classes with significant changes are reported. The relative changes are shown with their associated 95% confidence intervals. The asterisks below the bars report significant deviations from the control group, while significant differences between the exposure doses are displayed on the top, where “.” denotes 0.05 b p ≤ 0.1; “⁎” denotes p ≤ 0.05; and “⁎⁎” denotes p b 0.01 and “⁎⁎⁎” p b 0.001.

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for all metabolite classes at each of the three pesticide concentrations on day 2 and day 4 is provided in the supplementary material (Table S11). 3.3. Single metabolites After adjusting the p values for the numerous comparisons performed, we revealed significant changes in only three of the 184 metabolites measured. These three metabolites belonged to the same classes identified above, namely, glycerophospholipids, acylcarnitines, and biogenic amines. On day 2 (directly after the 24-h exposure to clothianidin), we observed a 26% decrease (t = −8.2; df = 6; p = 0.002) of the glycerophospholipid phosphatidylcholine diacyl C26:0 (PC aa C26:0) after exposure to clothianidin at 1 ng/L (Fig. 3a). In contrast, on day 4, we observed a marginally significant increase of the two biogenic amines histamine (8%; t = 3.8; df = 6; p = 0.086) and taurine (16%; t = 4.0; df = 6; p = 0.086) after exposure to clothianidin at 10,000 ng/L (Fig. 3b). A detailed list of all measured metabolite contents at each of the three pesticide concentrations on day 2 and day 4 is provided in the supplementary information (Table S1–S6). 4. Discussion 4.1. Effects of clothianidin exposure on the overall metabolite content High pesticide doses exhibited strong but short-term effects on general metabolite contents, while much lower doses resulted in weaker but longer-term effects. Pulse exposure to low concentrations of pesticides is already known to have sub-lethal but long-term effects on freshwater (Liess et al., 2013; Beketov et al., 2008; Wieczorek et al., 2018)

and terrestrial invertebrates (Maute et al., 2017). Remarkably, we could detect metabolic changes even at a concentration 4 orders of magnitude lower than the acute LC50, which did not cause additional mortality in exposed larvae as compared to the control group (Fig. S2). This result highlights the urgency of improving the understanding of the mechanisms that underlie the toxicity of contaminants at very low doses that are considered safe according to governmental risk assessments (EFSA, 2013). 4.2. Effects on different metabolite classes Three different classes of metabolites, namely, glycerophospholipids, acylcarnitines and biogenic amines, responded significantly to insecticide exposure. Glycerophospholipids decreased immediately after exposure. Both down- and up-regulation of glycerophospholipids in rats have been previously linked to exposure to high doses (the same order of magnitude as the acute LD50 – PPDB website) of the phenylpyrazole insecticide fipronil (Moser et al., 2015). In mosquitoes, glycerophospholipids are known to be a major component of cellular membranes (Ecker and Liebisch, 2014). Glycerophospholipids also act as modulators of energy metabolism, the mitochondrial electron transport system and of signalling pathways (Atella and Shahabuddin, 2002; Soares et al., 2015) involved in growth, survival and proliferation (Kerr and Colucci, 2011). Both fipronil and clothianidin target the central nervous system of insects through the hyperexcitation of nerves and muscles (Simon-Delso et al., 2015). Therefore, the observed reduction in glycerophospholipids in mosquito larvae may result from an insecticide-induced disturbance of neural cell membranes. However, we observed that the highest

Fig. 3. Percent change of single metabolites level at different clothianidin concentrations. The relative changes in the contents of the individual metabolites (percent of the metabolite content of the non-exposed control group) after exposure to 1 ng/L, 100 ng/L and 10,000 ng/L clothianidin for 24 h measured (a) directly after exposure and (b) 48 h hours later. This analysis is based on paired t-tests that have been adjusted for multiple comparisons with 5152 observations. Only the contents of those metabolites showing significant changes were reported for the three different concentration treatments. The relative changes are shown with their associated 95% confidence intervals. The asterisks below the bars report significant deviations from the control group, where “.” denotes 0.05 b p ≤ 0.1 and “⁎” denotes p ≤ 0.05. The baseline concentrations in the non-exposed control group were as follows: 1.77 μM (phosphatidylcholine diacyl C26:0 on day 2), 0.62 μM (histamine on day 4), and 2.37 μM (taurine on day 4).

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pesticide concentration only reduced the glycerophospholipid content immediately after exposure, while the smaller decrease caused by lower pesticide concentrations lasted for at least 48 h. We have interpreted our outcomes as follows: given that the observed changes in metabolite levels stemmed from a combination of both (i) the toxicity mechanisms of clothianidin and (ii) the biological response of the larvae after exposure, we can assume that different doses of the insecticide activated different defence mechanisms, resulting in different levels of metabolites. For example, Shi et al. (2018) observed different dosedependent responses of earthworms exposed to various concentrations of HBCD, a brominated flame retardant. The response to low doses in earthworms mainly involved the breakdown of proteins for energy production; in fact, greatly increased amino acid and adenosine triphosphate [ATP] levels were detected. In contrast, exposure to high doses also enhanced the expression of genes for detoxifying enzymes; in addition to a mild increase in amino acid and ATP levels, a large increase in RNA coding for detoxifying enzymes was detected. Similarly, in our study, we can hypothesize that after the Culex pipiens larvae were exposed to low insecticide concentrations (≤100 ng/L), only basic compensatory mechanisms (increased energy production) were activated, which resulted in a long-lasting decrease in the overall metabolite content and particularly in glycerophospholipid levels. However, more specific defence mechanisms – including detoxification and the restoration of membranes through the modulation of gene expression and the upregulation (even though not significant) of the glycerophospholipid production - may have been involved in the response to the high insecticide concentration, resulting in shorter-term effects. The acylcarnitine levels initially increased after insecticide exposure at the lowest and the medium concentrations but decreased at the highest insecticide concentration. In contrast, 48 h later, the acylcarnitine levels decreased following exposure to the lowest and the medium concentration. Recently, Martin-Park et al. (2017) associated exposure to a pyrethroid insecticide with a decrease in acylcarnitine levels and indicated that this result was due to the increased energy demand for the production of detoxifying enzymes. Acylcarnitines represent a transport stage of fatty acids before betaoxidation for energy production in mitochondria; therefore, acylcarnitines are well-established biomarkers for the early diagnosis of metabolic disorders in mammals (Rodrıguez-Sanchez et al., 2015; Roschinger et al., 2000). However, recent investigations of mammalian brains have also suggested that acylcarnitines play a role in modulating neurotransmission through the cerebral synthesis of acetylcholine (Jones et al., 2010; Lemhonwah et al., 2008) and in altering and stabilizing the membrane composition (Jones et al., 2010). These additional functions may explain the short-term increase in acylcarnitines after exposure to low levels of neonicotinoid insecticides, which target the nicotinic acetylcholine receptors located in the cell membranes of insect nervous systems (Sparks and Nauen, 2015). At a later stage, or after exposure to higher insecticide concentrations that may induce specific detoxification and restoration pathways, the decrease in acylcarnitines suggests an increased energy demand. Toxicant-induced changes in the contents of biogenic amines have never been investigated in insects. However, it is known that biogenic amines act as neurotransmitters, neuromodulators and neurohormones (Evans, 1980). Additionally, these compounds are involved in various functions such as reproduction (Gruntenko and Rauschenbach, 2008), responsiveness to olfactory stimuli (Mercer and Menzel, 1982) and orientation (Casagrand and Ritzmann, 1992). Clothianidin has raised many concerns due to its potential effects on colony collapse disorder (CCD Di Prisco et al., 2013; Brandt et al., 2016; Laurino et al., 2011) of honeybees. One of the primary symptoms of CCD is the failure of the bees to return to the hive. Thus, the neonicotinoid-induced reduction of biogenic amines, as measured in the present study, may play a relevant role in the impairment of olfactory functions and the orientation of exposed organisms. The reduction of biogenic amines should be investigated further as a potential indicator of exposure to cholinergic

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pesticides (Williamson and Wright, 2013) such as neonicotinoids. In particular, more insights should be gained to explore the transient nature of changes measured in the present investigation. 4.3. Effects on individual metabolites Finally, we explored the possibility of identifying individual metabolites as potential indicators of neonicotinoid exposure. Our outcomes suggest that a few individual metabolites responded more strongly to the pesticide exposure than most other metabolites from the same classes. We identified a significant reduction in the glycerophospholipid phosphatidylcholine diacyl C26:0 directly after exposure as well as a marginally significant increase in the two biogenic amines histamine and taurine 48 h later. The increase in histamine and taurine two days after insecticide exposure was unexpected since biogenic amines did not show significant changes at the same tested time when analysed as whole class. However, over the 441 levels (21 biogenic amines ∗ 7 experimental repetitions ∗ 3 clothianidin concentrations) measured two days after insecticide exposure, 57% were above, and 43% below, the control level. Therefore, it is likely that the two single metabolites alone showed significant increase that was masked when the whole group of 21 biogenic amines was analysed. Such increase may result from the roles of these metabolites as signal modulators in the activation of detoxification mechanisms. However, information in the literature on the biological roles of these metabolites in aquatic invertebrates is scarce. Therefore, we can suggest that phosphatidylcholine diacyl C26:0, taurine and histamine are only putative biomarkers of neonicotinoid exposure. Further studies should investigate the consistency of changes in metabolic profiles across different contaminants, doses, species, and time intervals in order to (i) clarify the role of individual metabolites in the toxicity of neonicotinoids and the subsequent biological response and to (ii) ensure that the identified biomarkers can be successfully applied. 5. Conclusions Taken together, our outcomes showed that very low concentrations of the neonicotinoid insecticide clothianidin, which did not induce mortality in the mosquito Culex pipiens, showed metabolic effects that indicated an increased energy demand. Even though no impact is expected at such low concentrations according to the European regulatory risk assessment, this increased energy demand can potentially translate into reduced fitness and may explain changes in the freshwater macroinvertebrate community under field conditions. We suggest acylcarnitines, glyerophospholipids and biogenic amines as potential regulatory endpoints to detect effects even at low sublethal concentrations. However, most metabolic changes lasted for 48 h only after exposure to low insecticide concentrations. We conclude that the transient changes in the metabolic profile reflect dose-dependent biological responses. Very low insecticide concentrations may disturb metabolism in a non-specific way that increases energy demand for an extended time, while higher concentrations may trigger a specific detoxification response that results in stronger but short-lived metabolic changes. Author contributions ML, RR and MvB conceived the research question. URK and RR developed the design of the investigation. RR performed the laboratory tests. RR, JMB, ML and SBH analysed and interpreted the results. The manuscript was written by RR, with contributions of ML and JMB. Funding sources Helmholtz long-range strategic research funding (POF III). The funding source was not involved in the study design, in the collection,

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analysis and interpretation of data, in the writing of the article; nor in the decision to submit the article for publication. Acknowledgment We thank the Helmholtz long-range strategic research funding (POF III) for the financial support. Appendix A. Supplementary data Two files of supplementary material are provided: the file named “Data” includes information on the level measured in each sample for every metabolite, divided into classes (Tables S1 – S6); the file name “Supplementary info” include data on mortality during the toxicity tests, LC50 calculations, preparation of test solutions, analytical measurements of clothianidin and details on dry weight of mosquitoes larvae and significant metabolic changes measured. Supplementary data to this article can be found online at https://doi.org/10.1016/j. scitotenv.2018.06.282. References Aardema, M.J., MacGregor, J.T., 2002. Toxicology and genetic toxicology in the new era of “toxicogenomics”: imoact of “-omics” technologies. Mutat. 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