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Supporting Information Internal concentrations in gammarids reveal increased risk of organic micropollutants in wastewater-impacted streams Nicole A. Munz1,2, Qiuguo Fu1, Christian Stamm1, Juliane Hollender1,2* 1Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland 2Institute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, 8092 Zürich, Switzerland *Corresponding author. Present address: Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland. Tel: +41 58 765 54 93. E-mail: [email protected] Content: Number of pages: 43, number of figures:6, number of tables: 15, number of sections: 6 Figure S1: Sites where gammarids were collected S3 Table S1. Number and weight of gammarids collected in the field at the different sites and time points. S4 Section S1. Additional information on substance selection and wastewater sample for laboratory experiment S5 Table S2. Number and weight of exposed gammarids (G.pulex) during the laboratory exposure experiment. S5 Section S2. Details on experimental setup of flume experiments S5 Table S3. Number and weight of gammarids used for determination of internal concentrations in flume experiments. S6 Section S3. Details on online SPE LC-HRMS/MS S7 Table S4. Performance of the chemical analysis of the field gammarids (including flume experiment) and the laboratory experiment. S8 Table S5. List of target compounds screened in the 5 most polluted gammarid samples S10 Section S4. Determination of water and lipid content in gammarids S24 S1 Table S6. Water and lipid contents of gammarids at selected sites and time points S24 Section S5. Calculation of logDow S25 Table S7. The geometric mean of acute EC50 values of invertebrates S26 Table S8. Internal concentrations in field gammarids in ng/g w.w. S27 Table S9. Water concentrations in ng/L at the sampling site Herisau before (May 2015) and after (June 2016) the upgrade of the WWTP with powdered activated carbon and at the site Ellikon (October 2015). S29 Table S10. Internal concentrations in gammarids (ng/g w.w.) and determined apparent BAF (L/kg) in the 48 h - lab experiment with dilutions of wastewater (30%, 60%, 90% WW) and defined substance mixture (Spike). S30 Table S11. Exposure media concentrations in ng/L of 48 h - lab experiment with dilutions of wastewater (30%, 60%, 90% WW) and defined substance mixture (Spike). S31 Section S6. Details on performance of lab and flume experiments S33 Table S12. Dilutions of the waste water in flumes in experiment 1 S34 Figure S2. Correlation of dilutions in flumes of experiment 1 calculated with conductivity and nitrate concentration S34 Table S13. Internal concentrations in gammarids and determined BAF in the flume experiments 1 and 3. S35 Table S14. Water concentrations in the flume experiments 1 and 3 S36 Table S15. Comparison of experimental BAFs from this study (from the experiment with the defined spiked substance mixture) with values reported in other studies S37 Figure S3. Comparison of BAFs from laboratory and flume experiments. S38 Figure S4. Correlation of logDow (pH 7.9) with the apparent BAF from the exposure experiment with the defined spiked substance mixture S41 Figure S5. Comparison of the BAFs in the laboratory experiment with the defined substance mixture with the BAFs determined in the field S41 Figure S6. Correlation between the sumTU based on internal concentrations and the SPEAR index S42 S2 Figure S1. Sites (with abbreviations) where gammarids were collected. The gammarid collection was performed at the 12 “2014-sites” and the site Herisau described in Munz et al. 20171. At the sites Birmensdorf, Muri and Reinach (not shown on map) no or not enough gammarids could be collected for the analysis of internal concentrations. S3 Table S1. Number and weight of gammarids collected in the field at the different sites and time points. Sep-14 Jan-15 Oct-15 May-15 Jun-15 Weight # of Weight # of Weight # of Weight # of Weight # of Site Location Replicate Major species (mg) gammarids (mg) gammarids (mg) gammarids (mg) gammarids (mg) gammarids 1 507 59 532 37 US 2 493 88 529 50 G.fossarum 1 322 57 516 30 Aadorf DS 2 - - 520 31 1 513 54 518 27 US 2 518 56 515 30 G.fossarum Elgg 1 511 59 520 29 DS 2 519 54 529 30 1 420 63 518 27 520 55 US 2 - - 503 30 507 53 G.fossarum 1 500 57 543 24 506 37 Ellikon DS 2 - - 547 27 499 38 1 536 54 537 25 US 2 544 65 535 25 G.fossarum 1 530 52 534 27 Knonau DS 2 542 34 529 25 1 458 22 533 23 US 2 342 39 538 21 G.fossarum/ 1 508 31 535 15 Dikerogammarus DS Marthalen 2 522 65 519 17 1 501 60 536 41 US 2 525 74 537 43 G.fossarum dingen 1 497 54 534 24 DS Unterehren- 2 333 36 526 32 1 509 37 521 24 US 2 396 45 525 24 G.pulex 1 489 49 509 20 DS Val-de-Ruz 2 496 28 501 19 1 515 34 515 28 US 2 509 43 509 24 G.fossarum 1 499 30 534 35 Villeret DS 2 504 37 533 34 1 502 56 526 34 US 2 507 59 526 28 G.fossarum 1 501 47 525 25 Zullwil DS 2 292 34 523 26 1 498 38 549 43 US 2 502 44 554 49 not identified 1 541 55 502 40 Herisau DS 2 534 59 434 33 S4 Section S1. Additional information on substance selection and wastewater sample for laboratory experiment The list of substances was selected based on the study described in Munz et al. (2017)1 and also includes simvastatin, dexamethason, fenoxycarb and hydrocortisone. To avoid high toxicity during the exposure, some toxic insecticides (chlorpyrifos, chlorpyrifos-methyl, diazinon, dimethoate, fipronil, imidacloprid) were excluded and also the x-ray contrast agent iopromide and other substances were not included due to analytical issues (e.g. high limit of quantification/detection (LOQ/LOD); gabapentin, sitagliptin). See Table S4 for the list of spiked substances. Wastewater was collected as final effluent from the WWTP Bachwis (Fällanden), Switzerland as 24h composite sample on April 25, 2017. Table S2. Number and weight of exposed gammarids (G.pulex) during the laboratory exposure experiment. dead Exposure weight # of animals condition (mg) gammarids at t48 Spike_1 758 14 8 Spike_2 639 15 6 Spike_3 569 14 4 WW30_1 716 16 0 WW30_2 592 14 1 WW30_3 527 13 1 WW60_1 746 16 2 WW60_2 587 14 1 WW60_3 507 15 3 WW90_1 700 14 1 WW90_2 519 13 0 WW90_3 655 14 0 Section S2. Details on experimental setup of flume experiments The cages for the exposure of the gammarids were made of 50 mL falcon tubes which were cut at both sites and covered with fine 500 µm pore size nets (each gammarid in one cage; nine cages were attached together to one unit; 2 units per flume; each cage contained a leaf disc as food source). In experiment 1 (August 2014), the channels were run over five weeks with four different dilutions of treated wastewater (0 = river water, 10, 50, 90%). For experiment 3 (June 2015), the channels were again fed with upstream river water and an artificial mixture of micropollutants and nutrients. For acclimatization, the gammarids were placed in the flumes (running only with stream water) two weeks before the start of the 4-week- exposure. The four treatments consisted of a control (C; only river water), technical control (TC; river water with spiked methanol), spiked micropollutants (“MP”), and spiked micropollutants and nutrients (“MP.N”). Eighteen micropollutants were spiked to the flumes with constant, nominal concentrations (ng/L): amisulpride (106), atenolol (217), benzotriazole (1098), carbamazepine (283), citalopram (55), clarithromycin (63), diazinon (457), diclofenac (603), diuron (75), fexofenadine (322), tebuconazole (24), iopromide (1629), metformin (5014), sucralose (1175), triclosan (55), valsartan (677), β-estradiol (0.35), and Zn (6040). In all experiments, each of the four treatment combinations was present in each block. S5 Table S3. Number and weight of gammarids used for determination of internal concentrations in flume experiments. In experiment 1 G.pulex were collected in the Chriesbach (Dübendorf, Switzerland). In experiment 3 G.fossarum from a pristine stream (Bäntalbach, Kollbrunn, Switzerland) were used. Per channel 12 gammarids were exposed. Weight # of Pooled Final weight of Treatment Channel Collection Date (mg) gammarids channels replicates (mg) Baseline - 597.3 26 25/08/2014 - 597.3 2 264.6 6 09/09/2014 13 233.3 7 10/09/2014 2, 8, 13 706.1 0% 8 208.2 5 09/09/2014 10 525.2 11 10/09/2014 10 525.2 6 169.3 4 09/09/2014 6, 11 470 11 300.7 7 10/09/2014 10% 1 224.2 5 09/09/2014 1, 16 400.9 16 176.7 4 10/09/2014 4 309.5 6 09/09/2014 4, 7 662.5 Experiment 1 Experiment 7 353 8 09/09/2014 50% 9 437.2 10 10/09/2014 9, 15 476.3 15 39.1 1 10/09/2014 3 446.3 12 09/09/2014 3 446.3 5 562.1 11 09/09/2014 5 562.1 90% 12 366.4 8 10/09/2014 12 366.4 14 518.5 10 10/09/2014 14 518.5 Baseline - 325.1 32 21/10/2014 - 597.3 2 184.1 8 27/05/2015 8 160.1 10 27/05/2015 C 2, 8, 10, 13 637.8 10 151.6 10 27/05/2015 13 142 8 27/05/2015 4 277.8 13 27/05/2015 4,7 465.8 7 188 10 27/05/2015 TC 9 157.8 9 27/05/2015 9,15 344.9 15 187.1 10 27/05/2015 1 229.9 12 27/05/2015 Experiment 3 Experiment 6 230.6 14 27/05/2015 MP 1,6,11,16 761.6 11 146.6 9 27/05/2015 16 154.5 10 27/05/2015 3 153.7 8 27/05/2015 5 197.9 9 27/05/2015 MP.N 3,5,12,14 711.7 12 219.1 12 27/05/2015 14 141 8 27/05/2015 S6 Section S3.
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