Assessment of thiamethoxam toxicity to Chironomus riparius

Ecotoxicology and Environmental Safety 137 (2017) 240–246

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Assessment of thiamethoxam toxicity to Chironomus riparius a,b

a,b

b

b

Althiéris S. Saraiva , Renato A. Sarmento , Andreia C.M. Rodrigues , Diana Campos , ⁎ Ganna Fedorovac, Vladimír Žlábekc, Carlos Gravatob, João L.T. Pestanaa,b, , Amadeu a,b M.V.M. Soares

MARK

a Departmento de Produção Vegetal, Universidade Federal do Tocantins, Campus Universitário de Gurupi, Rua Badejós, Lote 7, Chácaras 69/72, Zona Rural, PO box 66, CEP: 77402-970 Gurupi, Tocantins, Brazil b Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal c University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Hydrocenoses, Zatisi 728/II, 389 25 Vodnany, Czechia

A R T I C L E I N F O

A B S T R A C T

Keywords: Neonicotinoids Freshwater insects Life-history traits Biochemical responses

The insecticide thiamethoxam (TMX) is a systemic neonicotinoid widely used for pest control in several agricultural crops. TMX mimics the action of acetylcholine causing uncontrolled muscular contraction eventually leading to insect death. TMX is being found in freshwater ecosystems at concentrations of up to 225 µg/L. Still, chronic toxicity data for freshwater invertebrates is limited. Therefore, the aim of this study was to evaluate the acute and chronic effects (at organismal and biochemical levels) of TMX on the freshwater insect Chironomus riparius. C. riparius life history responses were significantly affected by TMX exposure, namely with a decrease in growth and delay in emergence. Concerning the biochemical responses, after a short exposure (48 h) to TMX, our results showed that low concentrations of TMX significantly reduced CAT activity and LPO levels of C. riparius. No effects were observed in AChE, GST and ETS activities. Effects in terms of survival, development rates and biochemical responses of C. riparius exposed to low concentrations of TMX observed in this study suggest potential deleterious effects of this neonicotinoid on aquatic insects inhabiting freshwaters environments near agricultural areas.

1. Introduction Neonicotinoids represent one of the most widely used class of insecticides nowadays (Morrissey et al., 2015). The insecticide thiamethoxam (TMX), introduced in 1998 under the trademark Actara® (foliar application and irrigation, soil treatment) and Cruiser® (seed treatment), belongs to the 2nd generation of neonicotinoids, characterized by the replacement of a cloropiridinil group (present in 1st generation neonicotinoids), by a clorotiazolidil group (Maienfisch et al., 2001). The insecticide TMX is used in a wide variety of agricultural crops and forestry (EFSA, 2013; FAO, 2014) to control insects of the orders Hemiptera, Thysanoptera, Coleoptera, Lepidoptera and Diptera (Uneme, 2011). As all neonicotinoids, TMX mimics the action of acetylcholine and is not degraded by acetylcholinesterases (Nauen et al., 2003). The continuous activation of acetylcholine receptors causes hyperexcitability of the central nervous system, inducing uncontrolled muscular contraction and eventually leading to death of insects (Rancan et al., 2006).

⁎

TMX is a highly leachable compound in certain types of soil, contaminating ground waters (Sánchez-Bayo et al., 2013) and is frequently detected on surface waters near agricultural areas (Samson-Robert et al., 2014; Anderson et al., 2015; Main et al., 2015; Schaafsma et al., 2015). In addition, average residue levels of TMX in water systems have increased over the past 15 years reflecting the worldwide trend in usage of this compound (Sánchez-Bayo et al., 2016). In some freshwater ecosystems located in areas of intensive agriculture, such as crop and grassland playas, concentrations of TMX can reach 20.1 and 225 µg/L, respectively (Anderson et al., 2013). As such this insecticide conveys a potential risk for freshwater invertebrates, and for insects in particular (Uğurlu et al., 2015). In fact, some deleterious effects of TMX on non-target aquatic organisms have been already reported (Anderson et al., 2015; Smit et al., 2015). Among the freshwater model organisms in aquatic toxicology, Chironomus riparius Meigen (Diptera: Chironomidae), has been one of the species widely used in standard laboratory tests to evaluate the effects of pesticides in freshwater ecosystems (Pérez et al., 2013; Morrissey et al., 2015). C. riparius has a short life-cycle, their larval

Corresponding author at: Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal. E-mail address: [email protected] (J.L.T. Pestana).

http://dx.doi.org/10.1016/j.ecoenv.2016.12.009 Received 27 July 2016; Received in revised form 5 December 2016; Accepted 7 December 2016 0147-6513/ © 2016 Elsevier Inc. All rights reserved.

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viously burned sand at 500 °C; < 1 mm), and 100 mL of test solutions prepared by diluting the stock solution in ASTM hard water medium. Based on preliminary tests and data available in the literature for TMX and other neonicotinoids, we exposed six day old C. riparius larvae to a gradient of TMX concentrations (0, 32, 51.2, 81.92, 131.07, 209.71, and 335.54 µg/L), plus a control treatment (ASTM hard water only) during 48 h. These survival tests were performed with three replicates per treatment, each containing 10 larvae. Exposure of organisms were performed at 20 ± 1 °C and 16:8 h light: dark photoperiod. During exposure, organisms were deprived of food. At the end of the exposure, the sediment was sieved and mortality was assessed with any immobile larva considered dead.

stages live in close contact with sediments and can be easily maintained in the laboratory (Learner, 1966; Faria et al., 2007; Muñoz et al., 2014). In addition, this midge species is widely distributed in the northern hemisphere at temperate latitudes being found in lentic and lotic environments (Péry and Garric, 2006). The evaluation of xenobiotic toxicity to C. riparius is usually assessed at the organismal level by measuring larvae survival and life-history responses, such as growth and emergence (OECD, 2004). In addition, biochemical responses have been used as complementary analysis tools to predict the toxicity at sub-individual level (Rodrigues et al., 2015a, 2015b; Campos et al., 2016). The aim of this study was thus to investigate the lethal and sublethal effects (at individual and sub-individual levels) of TMX on C. riparius. The effects of TMX exposure were evaluated at the organismal level by assessing C. riparius larvae survival and life-history responses (growth and emergence) in response to TMX acute and chronic exposures. Also, several different biochemical responses were evaluated. Acetylcholinesterase (AChE) activity which has been used as a neurotoxicity biomarker of exposure to organophosphorus and carbamate pesticides (Amiard-Triquet, 2009). TMX does not target AChE directly but, this enzyme has been used as biomarker of exposure to neonicotinoids, such as imidacloprid (Azevedo-Pereira et al., 2011b). In addition we evaluated effects in terms of oxidative stress defense mechanism focusing on an enzymatic antioxidant defense (Catalase – CAT activity), a phase II biotransformation enzyme (Glutathione-Stransferase – GST activity) and oxidative damage (measured by lipid peroxidation – LPO levels). CAT is an enzyme that act as ROS scavenger (Felton and Summers, 1995), and the measurement of CAT activity has been used as indicators of pesticide induced-stress (Rodrigues et al., 2015a, 2015b). GST activity has been reported as important cellular defense mechanism against chemical-induced stress (Rodrigues et al., 2015b; Campos et al., 2016). LPO levels reflect thus the oxidative damage caused by the free radical production and the impairment in antioxidant defense (Kehrer, 1993Khan et al., 2005; Kapoor et al., 2009). Finally, we evaluated the effects of TMX exposure on the energy consumption of C. riparius larvae, assessed by measuring the activity of the electron transport system (ETS). This endpoint allows for an insight of the metabolic activity of the organism and can reflect energetic costs of exposure and detoxification mechanisms (Smolders et al., 2004; Rodrigues et al., 2016).

2.2.2. Partial life-cycle test The chronic exposure of C. riparius followed OECD guideline no. 219 (OECD, 2004). Chironomids were exposed to 4, 6.5, 10.5 and 18 µg/L TMX for a period of 28 days. These concentrations were prepared from a stock solution of 1 mg/L of TMX. 1st instar larvae (2-days old) were introduced into 200 mL glass vials containing a 1.5 cm layer of inorganic sediment ( < 1 mm), previously burnt at 500 °C for 5 h, and 150 mL of aerated experimental solutions. For each treatment, sixteen replicates with five organisms each were used. After 10 days, chironomid larvae were collected from eight replicates per treatment and stored in ethanol (70%) for posterior measurement of body length (mm) in a stereoscopic microscope. The remaining replicates were left until the 28th day and C. riparius imagoes were collected daily for the assessment of percentage of emergence (derived by dividing the number of adult midges by the number of introduced larvae) and female/male ratios. Throughout the exposure period, food (TetraMin®) was provided every two days (0.5 mg/larva/day) and physico-chemical parameters (pH, temperature, conductivity and dissolved oxygen) were monitored (OECD, 2004). 2.3. Short exposure for biochemical experiments C. riparius 12-days old larvae (4th instar larvae) were exposed to three concentrations of TMX (4.5, 9 and 18 µg/L) plus control treatment (ASTM hard water only) during 48 h. The exposure was performed in 500 mL crystallizing dishes containing 200 mL of experimental solutions. Fifteen larvae per replicate were used, with a total of seven replicates per treatment, without sediment or food. After TMX exposure, C. riparius was quickly dried on filter paper, weighed, frozen in liquid nitrogen, and stored at −80 °C until further analysis. Homogenization of the samples was performed on ice, 1600 µL of ultrapure water were added to the samples and them homogenized by sonication (Brason Sonifier 250). From each replicate, an aliquot of 300 µL was taken for ETS, 200 µL for determination of LPO, whereas 500 µL homogenate was diluted with 500 µL of 0.2 M K-phosphate buffer, pH 7.4, centrifuged for 20 min at 10,000g (4 °C) and the postmitochondrial supernatant (PMS) divided into 3 microtubes. Those PMS samples were kept at- −80 °C until further analyses of biomarkers.

2. Material and methods 2.1. Thiamethoxam Thiamethoxam (EZ)-3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl1,3,5-oxadiazinan-4-ylidene (nitro) amine, (purity of 99.6%) was purchased from Sigma-Aldrich (UK). TMX is water-soluble (up to 4.1 g/L) with a log Kow (octanol/water partition coefficient)=−0.13 (FAO, 2014). The stock solution was prepared in ultrapure water, kept at 4 °C and protected from light to avoid degradation of TMX. 2.2. Chironomus riparius culture conditions

2.3.1. Biochemical responses assessment The protein concentration of each sample was determined in 10 µL of PMS following the Bradford method (Bradford, 1976), adapted from BioRad's Bradford microassay setup in a 96 well plate, using bovine γglobuline as a standard. The absorbance was read at 590 nm. AChE activity was measured on 50 µL of PMS following the method described by Ellman et al. (1961), adapted to microplate by Guilhermino et al. (1996). The absorbance was read at 414 nm. The molar extinction coefficient (ε)=13.6×103 M−1 cm−1 was used to determine the enzymatic activity, expressed in nmol/min/mg protein. CAT activity was determined in 10 µL of PMS by measuring the decomposition of the substrate hydrogen peroxide (H2O2) (Clairborne,

Chironomid culture was maintained in ASTM hard water medium (ASTM, 1980), with controlled aeration in plastic containers, containing 1–2 cm of inorganic fine sediment ( < 1 mm), previously burnt at 500 °C for 5 h. C. riparius were kept at 20 ± 2 °C, with a photoperiod of 16:8 h light: dark. Organisms were fed three times a week with macerated fish food (TetraMin®, Tetrawerke, Melle, Germany). 2.2.1. Acute test The acute tests were performed in glass crystallizing dishes (Ø=7.5 cm) containing 1.5 cm layer of inorganic fine sediment (pre241

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1985). The results were expressed as µmol/min/mg protein using a ε=40 M−1 cm−1. GST activity was measured based on the method describe by Habig et al. (1974) on 50 µL of PMS, reading the conjugation of GSH with 1chloro-2,4-dinitrobenzene at 340 nm. The enzymatic activity was expressed in nmol/min/mg protein using a ε=9.6×103 M−1 cm−1. LPO was determined on 200 µL of homogenate, treated with 4 µL 4% BHT. LPO levels were measured as thiobarbituric acid-reactive substances (TBARS) in accordance with Ohkawa et al. (1979) and Bird and Draper (1984) The samples were maintained in dark and absorbance was read at 535 nm. The results were expressed as nmol TBARS/ g weight, using ε=1.56×105 M−1 cm−1. ETS activity was measured on 300 µL of homogenate following a protocol adapted from De Coen and Janssen (1997) with slight modifications (Rodrigues et al., 2015a). To homogenate samples, 150 µL of homogenization buffer were added and centrifuged (1000g, 10 min, 4 °C). Fifty microliters of supernatant and 150 µL of buffered solution; and 100 µL of INT solution (p-iodonitrotetrazolium) were added to a microplate. The absorbance was measured kinetically over a 3 min period at 490 nm. The cellular oxygen consumption rate conversion was performed based on the stoichiometric relationship, whereby for every 2 µmol of formazan formed, 1 µmol of oxygen is consumed. The formula of Beer-Lambert was then applied to quantify the oxygen consumed: A=ε×l×c (A=absorbance; ε for INT-formazan=15,900 M−1 cm−1; l=0.9 cm; and c=oxygen consumed).

Table 1 TMX concentrations (µg/L, mean ± SD) measured in overlying water after ten days in chronic exposure and after 48 h in short exposure for biochemical analyses.

Chronic exposure (10 days)

Short exposure (48 h)a

Nominal concentrations (µg/L)

Measured concentrations (µg/L)

4 6.5 10.5 18 4.5 9 18

3.8 ± 0.56 5.9 ± 0.42 10.5 ± 0.70 18 ± 0 4.5 9.4 17

a In short exposure experiments only one composite sample per treatment was analyzed.

3. Results 3.1. TMX concentrations in water The measured TMX concentration of the stock solution (7.4 mg/L) was higher than nominal concentration (6 mg/L). Consequently, TMX nominal concentration used for the acute test was adjusted. Nominal and measured concentrations of chronic and short exposure, did not differ more than 13.8% and 5.6%, respectively. The values of nominal and measured concentrations for the chronic test (10 days) and short exposure (48 h) for biochemical analyses are presented in Table 1.

2.4. Chemical analysis

3.2. Acute test

TMX in water samples was analyzed by LC/MS/MS using a triple stage quadrupole TSQ Quantum Ultra Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled to an Accela 1250 LC pump (Thermo Fisher Scientific) and an HTS XT-CTC autosampler (CTC Analytics AG, Zwingen, Switzerland). A Hypersil GOLD column (50 mm×2.1 mmID×3 µm particles; Thermo Fisher Scientific, San Jose, CA, USA) was used for the separation of target analytes. Heated electrospray (HESI-II) in positive ion mode was used for ionization of the target compound. Two SRM transitions were monitored for each analyte according to the European Commission Decision 2002/657/EC of 17 August 2002 concerning the performance of analytical methods and interpretation of results. Quantification of TMX was performed by internal standard method using labeled metolachlor. Good linearity and limits of quantification (LOQ) were observed: R-squared values was 0.99; LOQs was 1.5 ng/mL. Quality control was confirmed by analysis of blank samples to assure that target analytes were not introduced from sampling or laboratory procedures and sample handling.

The estimated TMX 48-h LC50 (95% CI) was 86.41 µg/L (74.35– 100.04 µg/L; R2=0.91; four-parameter logistic curve: Y=5.91+(98.27−5.91)/(1+10^((1.93−x)*1.17)). At the end of the exposure 100% of living organisms were found in the control treatment and a monotonic decreased in survival was observed through the range of concentrations tested. 3.3. Chronic assay After 10 days of exposure to TMX, C. riparius survival was above 80% in the control treatment, 4, 6.5, and 10.5 µg/L, but survival at 18 µg/L, decreased to 35.5%. A significant reduction in C. riparius larvae growth after 10 days of exposure to 18 µg/L TMX was observed (F4,34=20.02, p < 0.001, Fig. 1), in comparison with the control treatment. No observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) values were established at 10.5 and 18 µg/L TMX, respectively.

2.5. Statistical analysis The 48 h LC50 of TMX in C. riparius was estimated by dose-response analysis using a four parameter logistic curve: Y=Bottom+(TopBottom)/(1+10^((LogLC50-x)*HillSlope)). Normality of data was assessed using the Kolmogorov-Smirnov test and homogeneity of variance was tested by the Bartlett's test. Growth and biochemical data of C. riparius, were analyzed by analysis of variance (ANOVA), followed by Dunnett's post-hoc tests. For all statistical tests the significance level was set at p < 0.05. Non-parametric test (Kruskal-Wallis test, by Dunn posthoc tests) was conducted for percentage emergence of C. riparius. Concerning biochemical endpoints, CAT and GST data were Log transformed. Statistical analyses were performed using the software GraphPad Prism version 6.0 for Windows (GraphPad Software, La Jolla, California, USA). The sex ratio of C. riparius was analyzed using contingency tables and Chi-square test, using SigmaPlot version 13.0 statistical software (Systat Software, Inc., San Jose California, USA).

Fig. 1. Effects of TMX exposure on body length of C. riparius (mm; mean ± SE); endpoint assessed after 10 days of exposure. *denote significant (p < 0.05) differences compared to the control treatment (Dunnett's test).

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spite no significant differences found by post hoc tests in comparison with the control treatment. In contrast, ETS activity in C. riparius larvae was not significantly altered by exposure to TMX (F3,24=0.81 p=0.5), compared to the control treatment. 4. Discussion Knowledge on the potential ecotoxicological effects of TMX is still limited concerning freshwater insects (Directive 98/8/EC, 2008; FAO, 2014). Our study shows that TMX is highly toxic to C. riparius, since chronic exposure to low concentrations caused significant decrease on growth and emergence rate. The acute toxicity of TMX to C. riparius (estimated LC50 of 86.41 µg/ L) suggests that chironomids can be at risk of acute exposure in some freshwater systems near agricultural areas (TMX measured concentrations up to 225 µg/L) (Anderson et al., 2013). In addition, the toxicity data obtained in this study is similar to toxicity observed for other neonicotinoids towards C. riparius, as shown in Table 3. Our results suggest that C. riparius is more sensitive to TMX exposure than the caddisfly Sericostoma vittatum (estimated 48-h LC50 of 203.87 µg/L – nominal concentrations, unpublished data) and the freshwater planarian Dugesia tigrina (LC50 96 h > 60.000 µg/L) (Saraiva et al., 2016). Nevertheless, TMX is slightly less toxic than imidacloprid to C. riparius (LC50 values of 12.9–19.9 µg/L) (Azevedo-Pereira et al., 2011a; Pestana et al., 2009a). TMX presented also less toxicity to Chironomus dilutus compared to others neonicotinoids (14 days LC50 values of 1.52, 2.41 and 23.6 µg/L for imidacloprid, clothianidin and TMX, respectively) (Cavallaro et al., 2016). Moreover our study shows that C. riparius NOEC values are several orders of magnitude lower that values observed for Daphnia magna (NOEC=100 mg/L) (FAO, 2014). In addition, TMX is reported as nontoxic to crustaceans (Gammarus sp. Thamnocephalus platyurus), molluscs (Lymnea stagnalis and Radix peregra) and fishs (Oncorhynchus mykiss and Lepomis macrochirus). However, TMX is highly toxic to crustaceans of the Ostracoda class (EC50=180 µg/L), and to other insects such as the ephemeropteran Cloeon sp. (EC50=14 µg/L) (Directive 98/8/EC, 2008) and the dipteran Chironomus dilutus (EC50=4.13 µg/L) with similar sensitivity to C. riparius (Cavallaro et al., 2016). Brock and Wijngaarden (2012) point out that for insecticides with very specific mode of action, like neonicotinoids, data on toxicity on Daphnia cannot always be used to protect sensitive invertebrates, such as freshwater insects (Beketov and Liess, 2008; Anderson et al., 2015) and thus research on the potential ecological effects of TMX in freshwaters should be focused on non-target aquatic insects. Furthermore, the present study also showed effects of TMX on the sex ratio of C. riparius. This indication of sex-related developmental toxicity, with females being more sensitive than males, is in accordance with previous studies where higher toxicity of neonicotinoids towards female chironomids have been reported (Kunce et al., 2015). Similar effects have been also observed on chironomids exposed to other insecticides such as pyrethroids (Agra and Soares, 2009; Rodrigues et al., 2015a). Differences in susceptibility to toxicants of female and male midges might be related to their differences in energy homeostasis since females need higher levels of energetic reserves allocated into egg masses production (Goedkoop and Spann, 2010; Rodrigues et al., 2015b). As such deleterious effects of neonicotinoids in terms of fertility and fecundity of aquatic insects are to be expected. Concerning biochemical responses, our results show that short-term exposure to TMX do not impair AChE activity which is in agreement with other studies showing that neonicotinoid-induced inhibition of AChE activity in C. riparius was only observed after 96 h of exposure (Azevedo-Pereira et al., 2011b). Thus the short exposure time (48 h) may not have been sufficient to reveal TMX indirect effects on AChE activity, which obviously does not exclude its neurotoxicity since these compounds act as mimics of acetylcholine impairing neurofunction. Secondly, it is known that cholinesterase activity in C. riparius is mainly

Fig. 2. Effects of TMX exposure on cumulative percentage of C. riparius emergence. Percentage emergence was derived by dividing the number of adult midges by the number of introduced larvae. Endpoint assessed after 28 days of exposure. *denote significant (p < 0.05) differences compared to the control treatment (Dunn test).

At the end of the 28 days of exposure, emergence rate in the control treatment reached 77.5%. Exposure to 10.5 and 18 µg/L TMX caused a significant reduction in C. riparius emergence rate (H=19.42, df=3, p < 0.001, Fig. 2), when compared with the control treatment. NOEC and LOEC values were set at 6.5 and 10.5 µg/L, respectively. In the 10.5 µg/ L TMX treatment, 45% of larvae reached the pupal state, but failed to emerge until the 28th day of the test, so the emergence percentage did not exceed 12.5%. However, at the highest concentration tested no chironomids reached the adult stageduring the 28days of experiment (100% mortality). A significant imbalance in the ratio of females to males (χ2=19.82, df=3, p < 0.001) was observed. The female: male ratiovaried from 0.8 in the control treatment to 1.2 and 0.4, in 4 and 6.5 µg/L TMX treatments, respectively. Only males emerged in the 10.5 µg/L TMX treatment, and in the 18 µg/L TMX treatment no adults emerged until the 28th day, thus these treatments were not included in this analysis.

3.4. Biochemical responses TMX exposure caused a significant reduction in CAT activity (F3,24=3.92, p < 0.01) and LPO (F3,23=6.66, p < 0.01), compared to the control treatment (Table 2), with LOEC of 18 μg/L TMX. TMX exposure also caused significant effects on AChE activity (F3,24=5.18, p < 0.01) and GST activity (F3,24=8.37, p < 0.01), deTable 2 Effects of short-term (48 h) exposure to sub-lethal concentrations of TMX on the biochemical endpoints in C. riparius (4th instar larvae). All values are presented as mean ± SD. Biochemical endpoints

AChE activity (nmol/min/ mg protein) CAT Activity (µmol/min/ mg protein) GST Activity (nmol/min/ mg protein) LPO TBARS (nmol/g wet weight) ETS Activity (mJ/mg organism/h)

TMX concentration (µg/L) Control

4.5

9

18

11.9 ± 2.26

14.1 ± 3.21

9.4 ± 1.65

11.9 ± 1.51

6.6 ± 2.51

6.2 ± 2.18

5.6 ± 1.44

3.5 ± 0.58*

16.3 ± 2.25

18.9 ± 3.11

14.1 ± 0.9

14.3 ± 1.06

81.5 ± 3.49

84.5 ± 6.63

71.3 ± 8.92

66.6 ± 12.09*

113.9 ± 11.72

116.6 ± 11.0

109.2 ± 8.20

109.3 ± 11.49

* Denotes a significant difference compared to the control treatment (p < 0.05, Dunnett's test).

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Table 3 Toxicity of neonicotinoids to Chironomus riparius. Pesticide

Endpoint

Test duration

Mensuration

Toxicity (μg/L)

Reference

Thiamethoxam

Survival Growth rate Emergence LPO levels CAT Activity Survival Emergence Survival Emergence Survival Emergence Survival Emergence Survival AChE Activity

48 h 10 d 10 d 48 h 48 h 48 h 30 d 48 h 28 d 48 h 28 d 96 h 28 d 48 h 4d 6d 24 h 28 d 10 d after oviposition 17 d after oviposition 34 d after oviposition 28 d

LC50 NOEC/LOEC NOEC/LOEC NOEC/LOEC NOEC/LOEC LC50 NOEC/LOEC LC50 NOEC EC50 EC50 EC50 NOEC/LOEC LC50 LOEC LOEC LC50 EC50 LC50 LC50 LOEC NOEC

86.41 10.5/18 6.5/10.5 9/18 9/18 35 10/11.4 19.6 5 22 1 12.94 0.4/1.2 19.9 1.2 0.3 55.2 3.11 5.18 1.57 0.5 0.5

Our study

Acetamiprid Clothianidin Imidacloprid

Thiacloprid

Survival Emergence Survival Survival Emergence Emergence

Directive 98/8/EC (2008) Nisso Chemical Europe GmbH European Regulatory Affaires (2015) EC Review report (2004) EPA (2003) Morrissey et al., 2015 Pestana et al. (2009a) Pestana et al. (2009a) Azevedo-Pereira et al. (2011a) Azevedo-Pereira et al. (2011b) Azevedo-Pereira et al. (2011b) EFSA Scientific report (2008) EFSA Scientific report (2008) Langer-Jaesrich et al. (2010) Langer-Jaesrich et al. (2010) Langer-Jaesrich et al. (2010) ECHA (2015)

h: hours; d: days.

et al., 2015; Démares et al., 2016). However, in our study, short exposures to TMX did not cause significant changes in the ETS activity of C. riparius larvae, which might be related to compensatory physiological and behavioural mechanisms such as alteration in feeding, assimilation or locomotion (Agostini et al., 2013; Rodrigues et al., 2016). Nevertheless, a deeper investigation on effects of longer exposures on bioenergetics parameters would be interesting to address the sensitivity of these endpoints and their relation with life-history responses concerning TMX exposures. In summary, the present study highlight the high toxicity of TMX towards C. riparius. Further research on the potential deleterious ecological effects of TMX ecotoxicity should focus on multigenerational effects in order to verify if the responses here evaluated in terms of emergence rate and sex ratio will translate into population dynamics. Previous studies have reported that other neonicotinoids such as imidacloprid, may weaken the ability of aquatic insect detritivores including chironomids to detect predators, forage for food (Pestana et al., 2009a; Azevedo-Pereira et al., 2011a) and consequently affect organic matter processing (Pestana et al., 2009b). Multispecies tests are thus necessary to understand the direct and indirect effects of TMX on freshwater invertebrate communities and ecosystem functions (Pestana et al., 2009a).

due to AChE functioning (Pérez et al., 2013) associated with both isoforms previously identified in Chironomus (Huchard et al., 2006). In fact, it was shown that these two AChE isoenzymes, AChE-1 and AChE2, might confer insect resistance to organophosphate and carbamate insecticides (Weill et al., 2002; Russell et al., 2004; Baek et al., 2005; Yu, 2006; Brown et al., 2009; Li et al., 2010), but there are no clear evidences of such differential sensitivity of AChE1 and AChE-2 towards neonicotinoids. On the other hand, our study shows that short-term exposures to low concentrations of TMX affect antioxidant defences reflected by a decrease of CAT activity in C. riparius larvae. It is interesting to note that neonicotinoids do not necessarily elicit the same type of responses in other aquatic organisms. In mussels, for example, imidacloprid exposure caused a decrease in AChE activity while thiacloprid induced an opposite effect with mixture of neonicotinoids increasing the activity of AChE (Dondero et al., 2010). In addition, the neonicotinoid guadipyr increased the activities of AChE and GST but had no effects on CAT activity in D. magna (Qi et al., 2013). In zebrafish livers, short exposures to sub-lethal concentrations of neonicotinoids have been shown to affect the protective capability of GST against DNA damage and decrease of GST activity, while increasing CAT activity (Ge et al., 2015). The impairment of antioxidant defences, however, did not translate into higher levels of oxidative tissue damage. Actually, the LPO levels do not depend only on generation of oxygen free radicals and antioxidants activity, but are also dependent on presence of lipid substrates (Miller et al., 1998). Thus, we hypothesize that short exposure to TMX caused a reduction in the lipid content of C. riparius larvae and consequently a decrease in LPO level measured in the highest concentration tested. In fact, it has been shown that exposure to neonicotinoids such as thiamethoxam and imidacloprid, promote a decrease in the total lipids contents in aphids (Aphis gossypii), since lipids may be allocated for energy production (Gerami, 2013). The stress adaptation and tolerance to xenobiotics, as well as organism's survival is also dependent of energy metabolism (Sokolova et al., 2012). Concerning energy consumption, Rodrigues et al. (2015a) suggested that ETS activity, used as a proxy for cellular metabolism, appears to be a good indicator of stress and also correlated with effects at higher levels of biological organization. In addition, the consumption of energy reserves may increase to overcome the energetic requirements for detoxification processes, with consequent effects in terms of energy available for growth and development (Choi et al., 2001; Rand

Acknowledgement This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES, Brazil (Edital 71/2013 – Programa Ciência Sem Fronteiras – Modalidade Pesquisador Visitante EspecialPVE – Projeto: A058_2013) and The National Council for Scientific and Tecnhological Development of the Ministry of Science, Technology and Innovation (CNPq/MCTI) - Young Talent Fellowship (314907/2014-9). We thank to Fundação para Ciência e Tecnologia (FCT) - Portugal for the doctoral grants of Diana Campos (SFRH/BD/87370/2012) and Andreia C.M. Rodrigues (SFRH/BD/79424/2011) and the post-doctoral fellowship of João Pestana (SFRH/BPD/103897/2014). We also thank FCT and POPH/FSE (Programa Operacional Potencial Humano/Fundo Social Europeu) for the contract of Carlos Gravato (IF/1401/2014). Support was also given to G. Fedorova and V. Žlábek by the Ministry of Education, Youth and Sports of the Czech Republic - projects “CENAKVA“ (No. CZ.1.05/2.1.00/01.0024) and “CENAKVA II“ (No. LO1205 under the NPU I program) and by the Czech Science 244

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