Supporting Information. Effects of activated carbon on PCB bioaccumulation and biological responses of Chironomus. riparius in full life cycle test

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1 Supporting Information Effects of activated carbon on PCB bioaccumulation and biological responses of Chironomus riparius in full life cycle test Inna Nybom *1, Sebastian Abel 1, Greta Waissi 1, Kristiina Väänänen 1, Kimmo Mäenpää 1, Matti T. Leppänen 2, Jussi V.K. Kukkonen 3, Jarkko Akkanen 1 1 University of Eastern Finland (UEF), Department of Environmental and Biological Sciences, P.O. Box 111, FI Joensuu, Finland 2 Finnish Environment Institute (SYKE), Ecotoxicology and risk assessment group, P.O. Box 35, FI University of Jyväskylä, Finland. 3 University of Jyväskylä, Department of Biological and Environmental Science, P.O. Box 35, FI University of Jyväskylä, Finland. Number of pages: 10 Number of Tables: 3 Number of Figures: 7 S1

2 Table S1. The endpoints and durations of the experiments. Table S2. Sediment dry weight (dw %), TOC (g/kg dw), BC (g/kg dw) and PCB (20 congeners) extracted from the sediments (µg/g sediment dw), average values ± standard deviation in KJ and TJ sediments. dw (%) TOC (g/kg dw) BC (g/kg dw) PCB ( 20) (µg/g sediment dw) Kernaalanjärvi (KJ) ± ± 1.14 a 1.53 ± 0.35 a 3.38 ±0.53 a b µm 2.52 ± ± µm ± ± µm ± ± µm 5.27 ± ± µm 2.69 ± ± <20 µm 13.91± ± Tervajoki (TJ) ± ± ± ± 0.19 b µm ± ± µm ± ± µm ± ± µm 8.08 ± ± µm 8.44 ± ± <20 µm ± ± a TOC, BC and PCB concentration of KJ sediment according to Nybom et al b Sediment grain size distribution (n=2): dw expressed as %, TOC g/kg dw in each fraction followed by the proportion (%) from the total TOC content S2

3 Table S3. Water and sediment quality parameters, average values ± standard deviation. The measurements between similar experimental sets were comparable, thus only values from representative experiments are presented. Emergence (1 st generation, P) and bioaccumulation (adult midges) experiments differed from the experimental design and therefore measurements from both are shown. The sediment ph value in bioaccumulation experiment was not monitored due to the insufficient sediment layer for the measurements. Kernaalanjärvi (KJ) Tervajoki (TJ) Water ph Sediment ph O2 (asv) a C Ammonium mg/l a air saturation value 0 % 0.5% 2.5% 0 % 0.5% 2.5% Emergence (P) 5.75 ± ± ± ± ± ± 0.65 Bioaccumulation 6.22 ± ± ± ± ± ± 0.57 Emergence (P) 5.54 ± ± ± ± ± ± 0.16 Emergence (P) ± ± ± ± ± ± 6.58 Bioaccumulation ± ± ± ± ± ± 7.37 Emergence (P) ± ± ± ± ± ± 0.44 Bioaccumulation ± ± ± ± ± ± 0.83 Emergence (P) 1.71 ± ± ± ± ± ± 0.18 Bioaccumulation 0.72 ± ± ± ± ± ± 0.15 S3

4 Figure S1. Chironomus riparius emergence times and the times of appearance of egg rods in P and F1 generations. Larval stages exposed to unamended and AC-amended (0.5% and 2.5%) PCB-contaminated sediments (KJ and TJ). S4

5 Figure S2. Female and male emergence during bioaccumulation experiment in PCB contaminated sediments (TJ, KJ). On top box plot for percentiles and median, the whiskers indicate minimum and maximum values mean value is indicated with +. Below cumulative emergence (%) of the midges during 28 days. *Significantly different compared to the unamended sediment p<0.05. Figure S3. Transmission electron micrographs (TEM) of C. riparius larvae microvilli layer exposed to unamended and AC amended sediments (KJ and TJ), 10-day experiment. Magnification 6000, scale bar 2000nm. S5

6 P C B µ g /g s e d im e n t (d w ) 8 6 H e p ta H exa P e n ta T e tra T ri K J T J Figure S4. PCB (20 congeners) extracted from the sediments KJ and TJ, (n=3). The KJ sediment is the same as used by Nybom et al. 2015, 1 and the PCB concentration and profile was determined therein. Figure S5. PCB (16 congeners) freely dissolved concentration (C free ) determined by PDMS passive samplers in KJ and TJ sediments as a function of sedimentary AC percentage. Note the difference in the scales of the graphs. S6

7 Discussion of Weight normalized concentrations and exuviae concentrations Weight-normalized PCB ( 20) concentrations extracted from C. riparius larvae were approximately half of the concentrations extracted from adult midges (Figure S6). During the metamorphosis the weight of the C. riparius decreased by 58%. The ww of the larvae on day 14 was 8.68 ± 1.25 mg, whereas the average weight of adult midges was 3.61 ± 0.30 mg. However, the lipids from the larvae, and thus also the PCBs bound in the lipids, were mostly transferred from the larvae to the midges. In previous studies, organochlorines (e.g. PCBs) have been shown mainly to be retained within the insect body during metamorphosis, which leads to increased concentrations in adults, which have a smaller mass than larvae. 2 More than 50% higher body residues in adult midges compared to fourth instar larvae have also been reported by Hwang et al. (2001) in an experiment where C. riparius were exposed to 12 C- and 14 C-labeled 2,2,4,4,5,5 - hexachlorobiphenyl. 3 In the present study the reduction in weight of the male midges was higher (2.12 ± 0.23 mg) compared to that found in the female midges (4.85 ± 0.23 mg), and the bioaccumulation of PCBs may also differ, depending on the sex of the midges. 3, 4 Within this study, the samples were pooled in order to gain a sufficient quantity of samples for extractions. The congener composition of the exuviae was similar to that found in of midges (Figure S6). Interestingly, the AC amendment did not affect the PCB concentration of the exuviae, although clear effects were observed in larvae and in midges. The exuviae of Chironomidae are primarily chitin-protein complexes with a waxy coating. 5, 6 The reason behind the inefficiency of AC as regards PCBs in exuviae may be due to the different environment in which the PCBs are bound (i.e. the AC amendment would affect mainly the lipid-bound fraction of PCBs present in larvae and midges). The lipid analysis made from the exuviae confirmed that the proportion of lipids was negligible. The determined lipid content of the exuviae was 0.12 % (±0.69) of the ww. S7

8 Figure S6. PCB (20 congeners, μg/g ww) extracted from Chironomus riparius larvae (n=3), adult midges (KJ n=2, TJ n=3) and exuviae (pooled sample, n=1) exposed to KJ and TJ sediments as a function of sedimentary AC percentage. Exposure period 14 days (larvae) and days (midges and exuviae). *Significantly different ( PCB) compared to the corresponding unamended sediment p<0.05. Note the difference in the scales of the graphs. S8

9 Figure S7. Comparison of lipid-normalized tissue concentrations (C Bio,Lip ) of PCBs in C. riparius larvae and midges and equilibrium partitioning concentrations of PCBs in lipids (C Lip Sed ) in KJ and TJ sediment as a function of sedimentary AC percentage. C Lip Sed were determined as described in Mäenpää et al. (2011) 7 and Mäenpää et al. (2015) 8 for the equilibrium concentrations in the passive sampler polymer equilibrated with sediment. The data is fitted to a linear regression and equation and coefficient of determination (R 2 ) are shown. Note the different scales of X- and Y-axes in the graphs. S9

10 References 1. Nybom, I.; Waissi-Leinonen, G.; Mäenpää, K.; Leppänen, M.T.; Kukkonen, J.V.K.; Werner, D.; Akkanen, J. Effects of activated carbon ageing in three PCB contaminated sediments: Sorption efficiency and secondary effects on Lumbriculus variegatus. Water Res. 2015, 85, ; /j.watres Kraus, J.M.; Walters, D.M.; Wesner, J.S.; Stricker, C.A.; Schmidt, T.S.; Zuellig, R.E. Metamorphosis Alters Contaminants and Chemical Tracers in Insects: Implications for Food Webs. Environ. Sci. Technol. 2014, 48 (18), ; /es502970b. 3. Hwang, H.; Fisher, S.; Landrum, P. Identifying body residues of HCBP associated with 10-d mortality and partial life cycle effects in the midge, Chironomus riparius. Aquatic Toxicology 2001, 52 (3-4), ; /S X(00) Maul, J.D.; Belden, J.B.; Schwab, B.A.; Whiles, M.R.; Spears, B.; Farris, J.L.; Lydy, M.J. Bioaccumulation and trophic transfer of polychlorinated biphenyls by aquatic and terrestrial insects to tree swallows (Tachycineta bicolor). Environmental Toxicology and Chemistry 2006, 25 (4), ; /05-309R Nation, J.L. Insect physiology and biochemistry. CRC Press: Boca Raton, Kavanaugh, R.; Egan, A.; Ferrington, J.L. Factors affecting decomposition rates of chironomid (Diptera) pupal exuviae. CHIRONOMUS Journal of Chironomidae Research 2014, (27), 16-24; /cjcr.v0i Mäenpää, K.; Leppänen, M.T.; Reichenberg, F.; Figueiredo, K.; Mayer, P. Equilibrium Sampling of Persistent and Bioaccumulative Compounds in Soil and Sediment: Comparison of Two Approaches To Determine Equilibrium Partitioning Concentrations in Lipids. Environ. Sci. Technol. 2011, 45 (3), ; /es Mäenpää, K.; Leppänen, M.; Figueiredo, K.; Mayer, P.; Gilbert, D.; Jahnke, A.; Gil-Allué, C.; Akkanen, J.; Nybom, I.; Herve, S. Fate of polychlorinated biphenyls in a contaminated lake ecosystem: Combining equilibrium passive sampling of sediment and water with total concentration measurements of biota. Environmental Toxicology and Chemistry 2015, 34 (11), ; /etc S10