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Aquatic Toxicology 150 (2014) 83–92

Contents lists available at ScienceDirect

Aquatic Toxicology

j ourna l ho me pa ge: www.elsev ier .com/ locate /aquatox

n situ effects of urban river pollution on the mudsnail Potamopyrgusntipodarum as part of an integrated assessment

adka Zounkovaa, Veronika Jalovaa, Martina Janisovaa, Tomas Ocelkab, Jana Jurcikovab,armila Halirovac, John P. Giesyd,e,f,g,h, Klara Hilscherovaa,∗

Masaryk University, Faculty of Science, RECETOX, Kamenice 753/5, 62500 Brno, Czech RepublicInstitute of Public Health, Partyzánské nám. 7, 70200 Ostrava, Czech RepublicCzech Hydrometeorological Institute, Kroftova 2578/43, 61600 Brno, Czech RepublicDepartment Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK S7N 5B4 Saskatchewan,anadaDepartment of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USADepartment of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong Specialdministrative RegionSchool of Biological Sciences, University of Hong Kong, Hong Kong Special Administrative RegionState Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 13 November 2013eceived in revised form 7 February 2014ccepted 27 February 2014vailable online 11 March 2014

eywords:ortality

eproductionassive samplingastropodaediment, In vitro

a b s t r a c t

The freshwater mudsnail (Potamopyrgus antipodarum) is sensitive to toxicity of both sediment and waterand also to the endocrine disrupting compounds (EDC) at environmentally relevant concentrations. Thisstudy determined effects of in situ exposure of P. antipodarum as a part of a complex assessment ofthe impact of a city metropolitan area with large waste water treatment plant (WWTP) for 0.5 millionpopulation equivalents on two urban rivers. The study combined the in situ biotest with detailed chemicalanalyses and a battery of in vitro bioassays of both sediment and water. Passive sampling of river waterwas conducted during the course of exposure of the mudsnail. P. antipodarum was exposed for 8 weeksin cages permeable to sediment and water at localities up- and down-stream of the city of Brno, CzechRepublic and downstream of the WWTP in two rivers. Greater mortality and significantly decreasedembryo production of P. antipodarum were observed immediately downstream of the city of Brno. P.antipodarum exposed at locations downstream of the metropolitan area and WWTP exhibited greatermortality, while numbers of embryos produced by surviving individuals were comparable or slightlygreater than for individuals held at the least polluted location. Comparisons with results of chemical
analysis and in vitro assays indicate occurrence of groups of compounds contributing to observed effects.Differences in mortalities of mudsnails among sites corresponded well with in vitro cytotoxicity andconcentrations of metals. The results of this study confirm the applicability of this novel field biotestwith P. antipodarum for the evaluation of the effects of river pollution on metazoans, especially as suitablein situ part of integrative contamination assessment.
© 2014 Elsevier B.V. All rights reserved.

. Introduction

Invertebrates are essential elements of aquatic ecosystems.mong key species in aquatic ecosystems are insects, molluscs and
rustaceans. Sub-lethal effects of chemicals on different physio-ogical processes, such as reproduction, growth, ecdysis, behavior,r morphological changes have been observed in all these groups
∗ Corresponding author. Tel.: +420 549 493 256; fax: +420 549 492 840.E-mail address: [email protected] (K. Hilscherova).

ttp://dx.doi.org/10.1016/j.aquatox.2014.02.021166-445X/© 2014 Elsevier B.V. All rights reserved.

(Mothershead II and Hale, 1992; Lenihan et al., 1995; Oetken et al.,2004; Péry et al., 2008). Changes in these processes might affectpopulations, and consequently survival of species and structuresor functions of ecosystems (Oehlmann et al., 2007; Oetken et al.,2004). Therefore, surrogate and sentinel species are needed for theassessment of the effect of contaminated water and sediment onthese organisms.

Some organisms in the class Gastropoda have been found to besensitive to toxicity associated with sediments and surface waters,and specifically effects of (xeno-)hormones (Schulte-Oehlmannet al., 2000; Tillmann et al., 2001). The freshwater mudsnail

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city of Brno, downstream of the dam of Brno reservoir; Prízrenice 1(location 1b) – Svratka downstream of Brno, upstream of the con-fluence with the Svitava River; Bílovice nad Svitavou (location 2a)– a small town on the Svitava River upstream of Brno; Prízrenice 2

Fig. 1. Map of metropolitan region of Brno and sampling locations. 1a – Knínicky

4 R. Zounkova et al. / Aquat

Potamopyrgus antipodarum) is a cosmopolitan organism withome advantages for use as an indicator organism, includingontinuous fertility of parthenogenetic females, few maintenanceequirements and sensitivity to environmentally-relevant com-ounds that might affect reproduction (Duft et al., 2003a, b; Joblingt al., 2004; Mazurová et al., 2008; Stange et al., 2012). Because thisudsnail can live under various environmental conditions, which

s documented by its worldwide distribution, it is a suitable modelrganism for use in biotests with water or sediment of differenthysical and chemical characteristics.

Effects of waste water treatment plant (WWTP) effluents andstrogenic compounds have been investigated using several speciesf molluscs, including freshwater gastropods Viviparus viviparusr Planorbarius corneus (Ramshorn snail) (Benstead et al., 2011).he ramshorn snail exhibited significant concentration-dependentncrease in fecundity and in overall duration of the reproductiveycle in adult snails exposed to WWTP effluent (Clarke et al., 2009).

P. antipodarum has been shown to be affected by chemicalsith endocrine disruptive potential such as bisphenol A, organ-

tins, estradiols, alkylphenols, UV screens or fadrozole. Greatereproduction of this mudsnail was observed after exposure to arti-cial sediment spiked with these chemicals (Duft et al., 2003a,; Gust et al., 2010b; Jobling et al., 2004; Oehlmann et al., 2000;chmitt et al., 2008). P. antipodarum was also successfully used as

test organism in contact tests with whole sediment (Galluba andehlmann, 2012; Mazurová et al., 2008; Schmitt et al., 2010a, 2011),

n laboratory tests with waste water (Jobling et al., 2003, 2004) or inn-site flow-through tests with whole WWTP effluent (Magdeburgt al., 2012; Stalter et al., 2010). The 28–56 day sediment contactest with reproduction of P. antipodarum as the measurement end-oint was selected for standardization within the OECD frameworkDuft et al., 2007; Oehlmann et al., 2007).

While instrumental analyses of water or sediment provide infor-ation on presence and concentration of known target substances,

here can be also a pool of unknown substances contributing tobserved effects in these matrices. In addition, all effects of knownubstances and especially their interactions are not known. For thiseason it is appropriate to use biotests and chemical analyses simul-aneously, especially in cases of complex pollution. Both in vitro andn vivo tests can be conducted in the laboratory with water and/orediment collected from the field. However, some characteristicsf the sampled materials representing the environmental matricesan change after their removal from the environment. It is thereforeifficult to simulate the real environmental situation and vary-

ng conditions in laboratory exposure, especially for dynamic rivercosystems. Thus, the most relevant way of exposure is direct fieldxposure of model species (Burton and Nordstrom, 2004). There isittle information on suitable model species of molluscs to be usedor studies directly in field. Only two studies have been publishedn in situ exposure of P. antipodarum, indicating its potential appli-ability in assessment of contamination in field (Gust et al., 2010a;chmitt et al., 2010b).

The overall objective of the research, results of which are pre-ented here, was characterization of effects of a city with largeunicipal WWTP on urban rivers pollution. A major focus of this

tudy was characterization of the influence of in situ exposureo river sediments and water on survival and reproduction of P.ntipodarum and examination of the sensitivity of this species andts suitability for direct exposure in urban rivers. Another impor-ant aim was to investigate the relationship between results ofhe in situ contact biotest with data from chemical analyses andn vitro biotests of water and sediment. In addition to collection of
rab samples also passive sampling of water was conducted to getore representative estimate of time-weighted concentrations of
ontaminants. In this study, two types of passive samplers weresed. These included Polar Organic Chemical Integrative Samplers

cology 150 (2014) 83–92

(POCIS), which sequester waterborne hydrophilic contaminants,and semipermeable membrane devices (SPMD) for monitoringwaterborne hydrophobic pollutants. Detailed characterization ofcontamination included in vitro biotests on cytotoxicity, dioxin-liketoxicity, (anti)estrogenicity and (anti)androgenicity, and chemicalanalysis of several classes of pollutants, including hydropho-bic organic pollutants, pharmaceuticals, pesticides, perfluorinatedorganic compounds (PFOCs) and alkylphenols, some of which areknown as endocrine disrupting chemicals (EDCs) (Groshart andOkkerman, 2000).

2. Materials and methods

2.1. Localities and sampling design

Selection of locations was based on a larger project concernedwith a long-term assessment of impact of the metropolitan regionof Brno (Czech Republic) on fluvial environment in two urbanrivers Svratka and Svitava (Grabic et al., 2010). Brno, with 404,000inhabitants, is the second-largest city in the Czech Republic withtraditional sources of urban water pollution as sewage, indus-trial wastewater and surface runoff from construction sites andurban roads. A large WWTP with a capacity of 513,000 popula-tion equivalent is located downstream of the city and is processingwaste waters from Brno and surrounding settlements. Waste wateris subjected to primary (mechanical) treatment followed by bio-logical stage of activation with pre-denitrification and anaerobicphosphorus removal (system of circulatory activation with changeof anaerobic, anoxic and aerated zones). Excess activated sludgeis then anaerobically stabilized (Brnenské vodárny a kanalizace,2010; Ministry of the Environment of the Czech Republic, 2010).

Samples of sediments and grab and passive samples of waterwere taken from six locations. The study locations were chosen toexamine the contamination in the rivers, its changes along the flowof the rivers through and downstream of the city of Brno and effectson biota. Two sampling locations (upstream and downstream thecity) were chosen at each river to observe the influence of the city,and two sampling locations were located downstream the conflu-ence of both rivers and the WWTP effluent discharge to observe theimpact of the WWTP. Thus, this field study included the followinglocations (Fig. 1): Knínicky (location 1a) – Svratka upstream of the

– Svratka upstream of Brno; 1b – Prízrenice 1 – Svratka downstream of Brno, 2a –Bílovice nad Svitavou – Svitava upstream of Brno; 2b – Prízrenice 2 – Svitava down-stream of Brno; 3 – Modrice – Svratka downstream of confluence with Svitava,downstream of WWTP; 4 – Rajhradice – 3 km downstream of the confluence of therivers Svratka and Svitava, downstream of the regional WWTP.

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location 2b) – Svitava downstream of Brno, upstream of the conflu-nce with Svratka River; Modrice (location 3) – Svratka downstreamf the confluence with the Svitava River, downstream of the WWTPffluent discharge; Rajhradice (location 4) – a small town 3 kmownstream of the confluence of the Svratka and Svitava Rivers,ownstream of the WWTP effluent discharge. Only sediment wasampled at location 3. Additionally, samples of effluent water wereaken and passive samplers were installed into the effluent of the

WTP.Passive samplers were exposed from the beginning of May

008 until the beginning of June 2008 (4 weeks). Standard samp-ing arrangement described in Grabic et al. (2010) and Jalovat al. (2013) with a combination of POCIS (Polar Organic Chem-cal Integrative Sampler) and several SPMDs (Semipermeable

embrane Device) was used. SPMD and POCIS were obtainedrom Exposmeter AB, Tavelsjo, Sweden. One POCIS was usedor both chemical analysis and bioassay testing. Two SPMDsere used in duplicates for chemical analysis, one SPMD wassed for toxicity assessment. SPMDs for chemical analysis con-ained performance reference compounds (PRC) used as onsitePMDs calibration. Four deuterated PAHs ([2H10]acenaphthene,2H10]fluorene, [2H10]phenanthrene, and [2H12]chrysene) and four3C12-labeled PCBs (PCB 3, 8, 37, and 54) were used as PRCs. Passiveamplers were placed in special racks in protective shroud places.amplers were installed and deployed in 0.5–1 m water depth andxposed for a 4-week period. Temperature was recorded in detailevery hour) during the 4-week deployment of passive samplersy temperature loggers placed on the sampling racks. The tem-eratures and the overall ranges of their fluctuations (day–night,in–max) were very similar across studied river sites (see Table S1

n Supplementary Materials). Composite bottom sediment samplesnd grab samples of water were collected on May 12, 2008. Samplesf sediment were taken from the upper layer of fresh sedimentsy use of a method designed by the Czech Hydrometeorologicalnstitute, which was developed in accordance with ISO 5667-12tandard (ISO, 1995). Representative composite sediment samplesere prepared by thorough mixing and homogenization of sur-

ace sediments collected from several spots (six to eight individualrabs) within each sampling locality (10 m2 area). Samples of waterere taken by use of a telescopic sampling device. Passive samplingas conducted according to validated protocols and general rules

or passive sampling (Huckins et al., 1993, 1999) and EN ISO/IEC7025 standard (ISO, 2005). All samples were refrigerated after col-

ection (4 ◦C) and transported to laboratory, where they were storedt −18 ◦C until the analysis, which was started within one monthf the sampling.

.2. Identification and quantification of residues

Samples of water, sediment, and organic extracts of SPMD andOCIS samplers were analyzed for wide range of organic com-ounds. Extraction and cleanup of passive samplers, as well ashe methodology for chemical analysis of studied compounds,ave been described previously (Grabic et al., 2010; Jalova et al.,013). Briefly, SPMDs were dialyzed with hexane, POCIS elutedith methanol:toluene:dichloromethane (1:1:8, v/v/v; Jalova et al.,

013). Sediment samples were homogenized, lyophilized andieved prior to analysis. Metals (Al, As, Ba, Cd, Co, Cr, Cu, Mo,i, Pb, Se, Ti, Zn) were extracted from sediment (fraction < 2 mm)y nitric acid and measured by ICP-MS method (Elan 6100 withutosampler AS-90; Perkin-Elmer Sciex, USA). Determination ofhe total mercury content in sediments was performed by means
f atomic absorption spectrophotometric (AAS) method using aingle-purpose cold vapor Advanced Mercury Analyzer AMA-254ALTEC Ltd., Czech Republic). Organic pollutants were extractedrom sediments (fraction < 1 mm) by microwave extraction with
cology 150 (2014) 83–92 85

hexane:acetone mixture and cleaned on a silica gel column. Aportion of each organic extract of POCIS, SPMD and sediments wastransferred into DMSO for testing in bioassays.

After removal of particulate matter and addition of internalstandards water samples were directly injected on analytical HPLCcolumn Phenomenex Aqua 5 � C18 125 A (50 mm × 2 mm), whereindividual analytes were separated and further detected by MS/MSsystem. All methods were validated in accordance with EN ISO/IEC17025 standard (ISO, 2005).

Extracts of SPMD and sediments were analyzed for poly-cyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls(PCBs), organochlorine pesticides, hexachlorobenzene (HCB),�-, �-, �-stereoisomers of hexachlorocyclohexane (HCH),dichlorodiphenyltrichloroethane (DDT) and its degrada-tion products dichlorodiphenyldichloroethylene (DDE) anddichlorodiphenyldichloroethane (DDD), triclosan and its environ-mental transformation product methyl triclosan (Me-triclosan)and polybrominated diphenyl ethers (PBDEs), expressed as thesum of congeners. Eluates from POCIS were analyzed for polar pes-ticides, pharmaceuticals and perfluorinated organic compounds(PFOCs). Samples of water were analyzed for polar pesticides,pharmaceuticals and alkylphenols. A complete list of individualpollutants analyzed is attached in footnotes to Table 1b. Concentra-tions of HCB, HCHs, PCBs, PBDEs, DDT and its degradation productsas well as triclosan and Me-triclosan after derivatization weredetermined by GC/MS–MS using isotope dilution. GC/MS was usedfor quantification of PAHs. PAHs with more rings were analyzedby HPLC using a FLD detector with deuterated internal standards.Polar pesticides, pharmaceuticals, PFOCs and alkylphenols weremeasured by standard method direct injection HPLC/MS–MS.Set of carbon 13C12-labeled internal standards were includedin the analyses as described in Jalova et al. (2013). The nativestandards were purchased from Dr. Ehrenstorfer, AccuStandards,and Absolute Standards via Labicom.

2.3. In vitro bioassays

Four transactivation reporter gene bioassays were used to mea-sure receptor-mediated potencies of organic extracts of sedimentsand passive samplers by procedures described in Jalova et al.(2013). AhR-mediated (dioxin-like) potency was determined byuse of the H4IIE-luc bioassay, a rat hepatoma cell line, which con-tains a luciferase reporter gene under control of dioxin-responsiveenhancers (DRE) (Sanderson et al., 1996; Hilscherova et al., 2001;Villeneuve et al., 2002). Estrogen receptor (ER)-mediated potencywas evaluated by use of the MVLN bioassay, a human breast car-cinoma cell line which has been transfected with a luciferase geneunder control of estrogen receptor activation (Demirpence et al.,1993; Hilscherova et al., 2002; Freyberger and Schmuck, 2005).(Anti)androgenicity was assessed in a bioassay with MDA-kb2cells, a human breast carcinoma cell line stably transfected withluciferase reporter gene under control of functional endogenousandrogen receptor (AR) and glucocorticoid receptor (GR) (Wilsonet al., 2002).

Cells were cultured in dark in incubator at 37 ◦C andassays conducted on 96-well microplates. Approximately 24 hafter plating, cells were exposed to samples, calibration ref-erence or solvent control. Standard calibration was performedwith 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; Ultra Scientific,USA; dilution series 1–500 pM) in case of H4IIE-luc, 17�-estradiol (E2; Sigma–Aldrich, Czech Republic; 1–500 pM) for MVLNand dihydrotestosterone (DHT; Sigma–Aldrich, Czech Republic;
1 pM–10 �M) for MDA-kb2. Effects of extracts on MVLN and MDA-kb2 cells were assessed either singly or in combination withcompeting endogenous ligand. Antiestrogenicity was determinedby simultaneous exposure of sample extract and 17�-estradiol

86 R. Zounkova et al. / Aquatic Toxicology 150 (2014) 83–92

Table 1aConcentration of pollutants in sediment (per g dry mass of sediment) from the six study sites around Brno.

Sediment 1a 2a 1b 2b 3 4

ng/g ng/g ng/g ng/g ng/g ng/gTriclosan 0.6 2.4 13 5.5 34 57Me-triclosan 0.1 0.73 2.7 1.4 10 7.2Sum of PBDEs 0.07–1.15 0–3 1.06–2.84 0.48–1.84 1.08–3.45 1.75–3.67Sum of PCBs 5.9 137 123 20 38.3 59.5Sum of HCHs 1.0–1.9 0–1.85 0–0.98 0–0.96 0–1.68 0–0.79HCB 0.85 1.1 2.1 0.74 2.0 1.7p,p′-DDT 0.38 1.3 680 5.7 5.4 5.4Sum of DDT and metabolites 1.47–1.86 5.2–6.0 757 12.0–12.2 20.5–20.8 29.7

�g/g �g/g �g/g �g/g �g/g �g/gSum of PAHs 2.3 15.2 14.6 19.2 12.3 26Al 1630 3910 6070 3530 5030 5490As 0.35 1.47 1.82 1.44 1.89 0.91Ba 43 100 179 96 124 130Cd 0.12 1.72 2.51 0.46 1.32 1.12Co 4.28 5.35 8.03 3.09 5.49 6.19Cr 5.72 25.7 30.1 16.1 24.1 25.8Cu 5.86 23.5 79.6 24.5 51.7 59.2Hg 0.01 0.12 0.89 0.68 0.96 0.96Mo 0.015 0.037 0.089 0.051 0.087 0.098Ni 7.84 15.6 24.4 11.7 15.5 18.3Pb 16.1 36.6 68.9 22.9 50.6 48.7Se 0.37 0.49 0.56 0.49 0.54 0.63Ti 20.4 31.8 50.6 32.3 39.6 47.7Zn 44.8 178 329 155 214 259

Ranges: the sum of detected compounds – the sum of detected compounds plus limits of detection of non-detected compounds.

Table 1bConcentrations of pollutants in water and passive samplers from study sites and WWTP effluent.

1a 2a 1b 2b WWTP effluent 4

Water ng/l ng/l ng/l ng/l ng/l ng/lSum of pesticides 30–1000 885–1909 194–1151 1050–1949 961–1889 534–1365Sum of sulfonamides 11–58 24–79 0–85 65–108 3290–3330 340–384Sum of other antibiotics 71–179 102–192 69–181 80–181 501–563 82–174Sum of other pharmaceuticals 38–48 103–115 39–68 90–101 2150–2160 280–292Sum of alkylphenols + BPA 44–63 n.a. 5–45 23–63 278–279 42–73

POCIS ng/POCIS ng/POCIS ng/POCIS ng/POCISSum of pesticides 523–627 – – 3100–3190 10,370–11,720 1310–1420Sum of sulfonamides 44–61 – – 165–173 3990–4140 287–297Sum of other antibiotics 29–87 – – 14–23 534–682 30–36Sum of other pharmaceuticals 115–120 – – 397–401 12,550–12,610 722–726Sum of PFOCs 2–7 – – 24–27 140–175 14–17

SPMD pg/l pg/l pg/l pg/l pg/lTriclosan 127 – 182 659 34,005 3374Me-triclosan 210 – 257 446 13,991 1779Sum of PBDEs 14.8–28.2 – 18.9–31.4 24.9–31.2 173–175 32.6–39.1Sum of PCBs 597 – 390 2127 2077 1041Sum of HCHs 186–199 – 157–164 145–159 646–654 183–193HCB 103 – 153 189 354 142p,p′-DDT 27.3 – 50.9 208 62.4 103Sum of DDT and metabolites 260 – 534 715 480 587

ng/l ng/l ng/l ng/l ng/lSum of PAHs 14.4 – 26.4 60.0 41.9 36.2

Ranges: the sum of detected compounds – the sum of detected compounds plus limits of detection of non-detected compounds.Sum of PBDEs: PBDE 28, PBDE 47, PBDE 99, PBDE 100, PBDE 153, PBDE 154, PBDE 183; sum of PCBs: PCB 28 + 31, PCB 52, PCB 101, PCB 118, PCB 138, PCB 153 + 168,PCB 170, PCB 180; sum of HCHs: alfa-HCH, beta-HCH, delta-HCH, gama-HCH; sum of DDT and metabolites: op-DDE, pp-DDE, op-DDD, pp-DDD, op-DDT, pp-DDT; sum ofPAHs: Phenantrene, Anthracene, Fluoranthene, Pyrene, Benzo(a)anthracene, Chrysene, Benzo(b)fluoranthene, Benzo(k)fluoranthene, Benzo(a)pyrene, Benzo(g,h,i)perylene,Dibenzo(a,h)anthracene, Indeno(1,2,3-c,d)pyrene; sum of pesticides: 2,4,5-T, 2,4-D, 2,4,-DP (dichlorprop), acetochlor, alachlor, atrazin, azoxystrobin, bentazone, bromacil,bromoxynil, carbofuran, cyanazin, desethylatrazin, desmetryn, diazinon, dichlobenil, dimethoat, diuron, ethofumesat, fenarimol, fenhexamid, fipronil, fluazifop-p-butyl,hexazinon, chlorbromuron, chlorotoluron, imazethapyr, isoproturon, kresoxim-methyl, linuron, MCPA, MCPP (mecoprop), metalaxyl, metamitron, methabenzthiazuron,methamidophos, methidathion, metobromuron, metolachlor, metoxuron, metribuzin, monolinuron, nicosulfuron, phorate, phosalone, phosphamidon, prometryn, propi-conazole, propyzamide, pyridate, rimsulfuron, simazin, tebuconazole, terbuthylazine, terbutryn, thifensulfuron-methyl, thiophanate-methyl, tri-allate; +clopyralid in POCIS;+desisopropylatrazin, desethyldesisopropylatrazin, 2-hydroxyatrazin in water; sum of sulfonamides: sulfapyridin, sulfamethazin, sulfamethoxypyridazin, sulfachloropyri-dazin, sulfamethoxazol; sum of other antibiotics: metronidazol, cefalexin, ofloxacin, norfloxacin, ciprofloxacin, enrofloxacin, erythromycin, trimetoprim; +doxycyclin in water;sum of other pharmaceuticals: diaveridin, carbamazepin, diclofenac; sum of alkylphenols + BPA: t-octylphenol, n-octylphenol, 4-n-nonylphenol, p-nonylphenol, monoNPE,diNPE, bisphenol A; sum of PFOCs: PFHxS, FHUEA, FOSA, N-methyl FOSA, PFOA, PFOS, PFNA. n.a. – value not available.– Sample not available.

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33 pM) and antiandrogenicity was tested in combination withihydrotestosterone (1 nM) – given concentrations are near theirC50 value. Several dilutions of extracts were tested in triplicate torovide a concentration–response curve for each sample. After 24 hf exposure, medium was removed, and cells were washed withhosphate-buffered saline (PBS) and lysed. Intensity of luciferase

uminescence corresponding to the respective receptor activationas measured by use of Promega Steady Glo Kit (Promega, USA)

n case of assays with H4IIE-luc and MVLN cells and with prepareduciferase reagent (Wilson et al., 2002) in MDA-kb2 assay. Noncyto-oxic sample concentrations to be used in bioassays with cell linesere determined by use of the neutral red uptake assay (Freyberger

nd Schmuck, 2005). At the end of the incubation period, neutraled solution (0.5 mg/ml of media) was added and cells incubated for

h at 37 ◦C. Medium was removed, cells washed with PBS and lysedith 1% acetic acid in 50% ethanol. Absorbance was measured in aicroplate spectrophotometer at 570 nm. Yeast strain of recombi-

ant Saccharomyces cerevisiae constitutively expressing luciferaseas used for detailed cytotoxicity assessment (Leskinen et al., 2005;ichelini et al., 2005).Statistical evaluation of in vitro bioassays was performed by

onlinear logarithmic regression of concentration–response curvesGraph Pad Prism, GraphPad® Software, San Diego, CA, USA). Rela-ive potencies expressed as TCDD equivalents/E2 equivalents/DHTquivalents were calculated by relating the EC50 value of standardalibration with the concentration of the tested sample inducinghe same response (Villeneuve et al., 2000). Cytotoxicity, antie-trogenicity and antiandrogenicity corresponded to the decrease inetected luminescence/absorbance signal given by solvent control

n case of cytotoxicity and specified amount of competing standardigand for the other effects. The IC50 values for antiestrogenicity andntiandrogenicity were calculated from concentration–responseurves expressed in percentage of signal of competitive concentra-ion of added natural ligand (33 pM E2, 1 nM DHT). For better clarityf the trends the values are expressed as an index of antiestrogenic-ty (AE) or antiandrogenicity (AA), which corresponds to reciprocalalue of IC50. Similarly, the index of cytotoxicity was derived as theeciprocal value of IC50 (or IC20 values in case the 50% response wasot reached) for the cytotoxic response.

Concentrations of analyzed compounds as well as the bio-ogical potencies determined in bioassays for SPMD extracts

ere recalculated to the concentrations in water to take intoccount the differences in sampling rates among SPMDs from dif-erent locations. Performance reference compounds (PRC) weresed for in situ calibration of sampling rates. Details of thealculation are described in Jalova et al. (2013). Results ofOCIS samples were compared on the basis of concentrationsnd toxic equivalents in sampler extracts (ng/POCIS). No cor-ection of POCIS sampling rates was made because the waterow in sampling localities did not vary significantly. Also,nly minor influence of water flow rate on the accumula-ion of pollutants into POCIS has been demonstrated (Li et al.,010).

.4. In vivo biotest

P. antipodarum (Gray 1843) (Mollusca, Gastropoda, Caenogas-ropoda) is a parthenogenetic and ovoviviparous freshwater

udsnail, which is indigenous to New Zealand, but is currentlyidely distributed in aquatic environments around the world.

hese snails inhabit upper layers of aquatic sediments and feed onlants and detritus. A population from clean reference unpolluted

ocation in sand-pond Stratov (district Nymburk, Czech Republic)as used in the test. The specimens were collected one month

efore the test. Adult individuals 3.6–3.9 mm length (Duft et al.,003a) were used for the in situ test.

cology 150 (2014) 83–92 87

The field contact biotest with P. antipodarum was conducted atthe six locations (see section on locations and sampling design)around the metropolitan area of Brno from April to June 2008.Cages consisted of stainless tube filter ½ (Valvosanitaria Bugatti,Castegnato, Italy), iron cover 5/4 (Pumpa, Brno, Czech Republic)and nylon net. Cages were laid on the river bottom, about onemeter from the river bank and fixed ashore. Six cages were placedat every locality one week before start of the test. About 30 g ofsediment was added into each cage and they were kept on riverbottom to microfilm overgrow. Eight adult mudsnails were placedinto each cage one week later. Thus, a total of 48 mudsnails wereexposed at each location. Microfilm and sediment particles servedas food and exogenous food was not provided. Cages were cov-ered with nylon net and exposed in the river environment foreight weeks. After the exposure, the cages were transported tothe laboratory, adult mudsnails were euthanized in 10% MgCl2solution, dissected and reproduction success and mortality wasevaluated. Reproduction was evaluated by counting the numberof embryos in the brood pouch of 20 randomly selected mater-nal snails per site or of all surviving adults in case they were lessthan 20.

Normality was checked by the Kolmogorov–Smirnov test andhomogeneity of variance was confirmed by use of Levene’s test.The statistical significance of differences between groups wasevaluated using non-parametric Mann–Whitney U test. Calcula-tions were performed using Microsoft Excel® (Microsoft, Redmond,WA, USA) and Statistica® for Windows 6.0 (StatSoft, Tulsa, OK,USA).

3. Results

The analyses of all types of samples collected in spring 2008both by chemical analysis and in vitro bioassays revealed Svratkaupstream of the city (1a) as generally the least polluted location(Tables 1a, b and 2). A detailed report of concentrations of indi-vidual compounds is included in Supplementary Materials (TablesS2–S5). Unfortunately, POCIS samplers from locations 2a and 1band SPMD sampler from 2a were damaged or stolen during theexposure period, so it was not possible to conduct all comparisons.Sediments contained greater concentrations of several groups ofnon-polar pollutants (PCBs, DDTs, Me/triclosan, PAHs) and metalsat location 1b compared to 1a, demonstrating impact of the sourcesin the city on contamination of the River Svratka. The analysisof SPMDs also confirmed greater concentrations of most of thesepollutants directly downstream of the city. The Svitava River wasmore contaminated upstream of the city (location 2a), such that theeffect of sources in the city was not obvious. Results from watergrab sample and POCIS also document greater concentrations ofpesticides and pharmaceuticals in the Svitava than in the SvratkaRiver. Concentrations of triclosan and its metabolite, sulfonamidesand some other pharmaceuticals were greater downstream of theWWTP. Also, concentrations of PAHs in sediments were greatestin the Svratka River, 3 km downstream of the WWTP (location 4).Concentration of most metals including the hazardous elements(MoA, 2009) have shown similar spatial trends with greatest con-centrations in sediments from site 1b directly downstream of theBrno city and from the two most downstream sites under WWTP(3, 4, Table 1a). The differences were most pronounced for Cu, Pband Zn.

Results of the in vitro bioassays documented the presence ofandrogenic and estrogenic compounds in the polar fraction of
WWTP effluent. Estrogenicity was also detected in POCIS from theSvitava River downstream of Brno (2b) and at the most downstreamlocation (4; Table 2). At the same time, the greatest antiandro-genic potency was observed in sediments from these two locations.

88 R. Zounkova et al. / Aquatic Toxicology 150 (2014) 83–92

Table 2Results of in vitro assessment of extracts of samples from localities where cages with mudsnails were placed.

1a 2a 1b 2b 3 4

SedimentDioxin-like toxicity [ng TCDD eq./g sed.] 9.08 13.8 11.7 8.15 10.5 19.4Estrogenicity [ng E2 eq./g sed.] n.d. n.d. n.d. n.d. n.d. n.d.Androgenicity [ng DHT eq./g sed.] n.d. n.d. n.d. n.d. n.d. n.d.Index of cytotoxicitya 22.4 99.1 166 66.8 160 290Index of antiestrogenicitya 1301 2649 1473 325 2270 1353Intex of antiandrogenicitya 762 655 246 1332 722 1602

POCIS WWTP effluentDioxin-like toxicity [ng TCDD eq./POCIS] n.d. – – n.d. 1.77 n.d.Estrogenicity [ng E2 eq./POCIS] n.d. – – 0.47 2.77 0.56Androgenicity [ng DHT eq./POCIS] n.d. – – n.d. 27.7 n.d.Index of cytotoxicitya 146 – – 323 1238 156Index of antiestrogenicitya 220 – – 620 727 423Index of antiandrogenicitya 270 – – 324 n.d. 375

SPMDDioxin-like toxicity [pg TCDD eq./l] n.d. – 23.0 10.6 7.80 7.58Estrogenicity [ng E2 eq./l] n.d. – n.d. n.d. n.d. n.d.Androgenicity [ng DHT eq./l] n.d. – n.d. n.d. n.d. n.d.Index of cytotoxicitya 2.58 – 13.1 1.46 11.6 1.31Index of antiestrogenicitya 3.21 – 14.3 0.74 5.83 2.99Index of antiandrogenicitya n.d. – 8.02 6.51 6.17 3.61

Standard deviations around the determined values were up to 20% for dioxin-like activity, cytotoxicity and anti/androgenicity and up to 15% for anti/estrogenicity.– Sample not available.n.d. Not detected.

a0 (IC2

[

UltgahanfcWSt

emlnat

Fse

River upstream of Brno, which is considered to be the least pol-

Index of cytotoxicity, antiestrogenicity, antiadrogenicity = reciprocal value of IC5

1/(POCIS/ml)] for POCIS extracts or [1/(l/ml)] for SPMD extracts, respectively.

nfortunately, the passive samples from location 2a in this samp-ing (spring 2008) were lost, but POCIS exposed at this location inhe same period in 2007 elicited estrogenicity (0.18 ng E2/POCIS). Ineneral, extracts of sediments and SPMDs exhibited antiestrogenicnd antiandrogenic potencies. Dioxin-like activity was detected inydrophobic fraction of water (SPMD) downstream of Brno and inll sediments, but only in WWTP effluent in the case of POCIS. Alter-atively, in vitro cytotoxicity was detected in all types of samples

rom all locations. In sediments, it was greatest downstream of theity on the Svratka River and at the localities downstream of the

WTP. Both dioxin-like toxicity and cytotoxicity of sediments andPMD from the Svratka River were greater directly downstream ofhe city (location 1b) compared to upstream (1a).

The in situ contact test with P. antipodarum after eight weeksxposure to river sediment and water led to various magnitudes ofortality of adult mudsnails and number of embryos at the study

ocations (Fig. 2). Mortality was proportional to the general mag-
itudes of pollution at locations. The least mortality was observedt the most upstream location on the River Svratka (1a), wherehe least contamination was determined by chemical analysis and
ig. 2. Mortality of adults of P. antipodarum after 8 weeks in situ exposure. The inserthows the location of sampling sites on the rivers. 48 individuals were exposed atach site (six cages of eight adult mudsnails).

0 in case of cytotoxicity of SPMD extract). Units are [1/(g/ml)] for sediment extracts,

toxicity in vitro biotests. Alternatively, there was about three-foldgreater mortality at the location on the Svratka River directly down-stream of Brno (1b), which exhibited greater contamination ofsediments (Tables 1a and b). The greatest mortalities were observedat locations 3 and 4 downstream of the city and of the WWTP, wheregreater concentrations of triclosan and Me-triclosan, polycyclicaromatic hydrocarbons, sulfonamides and other pharmaceuticalswere detected.

Reproduction of mudsnails varied among locations (Fig. 3).There were, on average, approximately two-times lesser numbersof embryos in brood pouches of adults exposed at locations directlydownstream of Brno (1b, 2b) compared to the locations upstream ofBrno on the same rivers (1a, 2a). In the case of the Svitava River, thisdifference was statistically significant. The number of embryos wasgreatest in mudsnails held in the Svitava River upstream of Brno,and was almost twice as great as for mudsnails held in the Svratka

luted location. Numbers of embryos of surviving mudsnails fromlocalities downstream of WWTP (3, 4), where the greatest mor-tality was observed, were, on average, slightly greater than those

0

5

10

15

20

1a 2a 1b 2b 3 4

num

ber o

f em

bryo

s

**

** *

Fig. 3. Average number of embryos in the brood pouch of adults of P. antipodarumafter 8 weeks in situ exposure. Number of examined maternal snails was 20 for 1a,2a, 1b, 2b; 10 for site 3, and 5 for site 4. *Significant difference.

ic Toxi

aHtSogs(

4

aPe4wfi(awdPclcci

atst2ouhtssdagsowtodgitfprSgwstrtWnc


t the least polluted location 1a (by 40 and 20%, respectively).owever, this difference was not statistically significant, similarly

o the comparison with the most downstream locations on thevitava River before the confluence (2b). Alternatively, numbersf embryos in mudsnails at locations 3 and 4 were significantlyreater (more than twice as great) when compared to mud-nails exposed in the Svratka River, directly downstream of Brno1b, Fig. 3).

. Discussion

Effects of chemical pollution, especially of compounds classifieds endocrine disruptors, were documented in previous studies with. antipodarum. Also correlations between effects of some knownstrogenic chemicals (17�-ethinylestradiol, EE2, bisphenol-A, and-tert octylphenol) and of mixture of pollutants in WWTP effluentater on reproduction of P. antipodarum and estrogenic effect onsh, such as greater production of vitellogenin, have been observedJobling et al., 2003). Effects on reproduction of P. antipodarumfter laboratory exposure to sediments containing compoundsith dioxin-like, estrogenic and anti-androgenic activity wereescribed also in our previous study with sediment from Lakeilnok, which has been used as a dumping site for powdered wasteoal (Mazurová et al., 2008). However, studies conducted in theaboratory cannot accurately simulate natural conditions and theirhanges to which organisms are exposed in field, as well as theomplex mixture of compounds contained in WWTP effluents andn surface waters.

Results of the present study revealed effects on reproductionnd mortality of mudsnails exposed for 8 weeks at different locali-ies in the Brno metropolitan area. Two studies testing in situ expo-ure of P. antipodarum have been published previously, but both ofhem used only 4 week exposure (Gust et al., 2010a; Schmitt et al.,010b). During this shorter duration of exposure those authorsbserved as much as 25% mortality, which corresponds to the morepstream locations in the study, the results of which are presentedere. The greater mortality at downstream locations observed inhis study can be affected not only by the longer duration of expo-ure, but also by the magnitude and composition of pollution in thetudied rivers. Only one of the two previous in situ studies presentedata on concentrations of contaminants (Schmitt et al., 2010b),nd the comparison to data presented here shows that there werereater concentrations of PAHs in our study, namely at the down-tream locations. In this study exposure was characterized by usef both chemical analysis and in vitro bioassays of sediment andater. Since the results from grab water samples represent only

he instantaneous conditions, a four week long passive samplingf water for both hydrophobic and hydrophilic pollutants was con-ucted during the course of exposure of the mudsnails in river toet representative estimate of longer-term exposure. A battery ofn vitro tests was employed to provide complementary informationo quantification of individual residues, which can only accountor the known compounds and do not take into consideration theossible interactions within mixture. Quantification of individualesidues showed that concentrations of some pollutants in thevratka River increased during its course through Brno. The fact thatreater concentrations of DDT, PCBs, metals, triclosan, and PAHs,ere found in sediments from the Svratka River directly down-

tream of Brno than in sediment from upstream location, suggestshat there are sources of these pollutants in the city such as urbanunoff. Concentrations of some pollutants, including triclosan, Me-
riclosan and some pharmaceuticals increased downstream of the
WTP, which indicates that the WWTP despite its effective-ess and up-to-date methods of treatment could still contributeontaminants to the river. Similar observations were reported

cology 150 (2014) 83–92 89

during a recent study conducted in the same area in 2007, whichalso documented efficient treatment of the WWTP for cytotoxiccompounds, xenoestrogens and xenoandrogens (Jalova et al.,2013).

Results of two previous studies showed different trends innumbers of embryos in P. antipodarum after in situ exposure. Sig-nificantly more embryos in the brood pouch at more pollutedlocations were observed by Schmitt et al. (2010a,b), whereas Gustet al. (2010a) reported fewer embryos downstream of WWTPs.These contradictory results can be explained by different com-position and concentrations of mix of compounds contained inwater and sediments (e.g. estrogenic compounds may cause inhi-bition of reproduction at high concentrations (Jobling et al., 2003)).The differences are explained by the results of another study bySchmitt et al. (2011), which tried to identify compounds respon-sible for these effects. Effect-directed analysis showed that twoout of six fractions stimulated reproduction of P. potamopyrgus,while two other fractions inhibited reproduction. Fractions whichstimulated reproduction also exhibited greater estrogenic potencyin the ER-LUC assay using reporter cell line BG-1. Results of thestudy demonstrate that some WWTP effluents and thus surfacewaters can contain compounds both stimulating and inhibitingreproduction. The resulting effect might depend on quantity andratio of these compounds. These results correspond well with thoseof this study, where lesser numbers of embryos were observedin snails from both rivers directly downstream of Brno comparedto upstream locations. In the case of the River Svratka there wasmore than two-fold greater mortality and two-fold lesser num-bers of embryos at the location directly downstream of Brno (1b),which corresponds with the greatest magnitude of pollution byPCBs, DDTs and metals. This observation also corresponds withthe greater in vitro cytotoxic potency of extracts from both sed-iments and SPMDs from this location (Table 2). In the case ofthe Svitava River, the situation was different, since the pollutionof this river is already greater upstream of Brno. Relatively greatpollution with PCBs, PAHs, polar pesticides, some pharmaceuti-cals and metals was observed upstream of Brno on the SvitavaRiver. That pollution could be linked to recently increasing habi-tation density due to moving from the center of the city to suburbsupstream of location 2a. There is a small WWTP for this areawith insufficient capacity for the new settlements which couldcontribute to the river pollution. Some of these pollutants couldbe related to greater numbers of embryos in mudsnails at loca-tion 2a. There was no strong influence of the city sources on thepollution in this river. Pollution of the Svitava River was mostlycomparable or lower at the location downstream of Brno and mor-tality and number of embryos of P. antipodarum at this locationwere comparable to those observed at the least polluted location(1a).

Greatest mortalities were observed on locations 3 and 4downstream of Brno, the confluence of the two rivers and ofthe spot where WWTP effluent enters the river. Mortality wasprobably affected by cytotoxic compounds quantified by thein vitro assay and triclosan and Me-triclosan in sediments at boththese locations. Moreover, the greatest concentrations of sulfon-amides and other pharmaceuticals in water, and of polycyclicaromatic hydrocarbons and dioxin-like potency in sedimentswere found at the most downstream location 4. Despite greatermortality, number of embryos of surviving mudsnails was greaterthan in the Svratka River directly downstream of Brno (1b), butnot significantly different (20–40% greater) than at the cleanestlocation. Individuals at these locations (3, 4) are exposed to mul-
tiple stressors, including cytotoxic compounds at the same timewith hormonally active compounds. This is demonstrated by theestrogenic and androgenic potency in POCIS from WWTP effluentand estrogenic activity also at location 4, but also by ubiquitous

90 R. Zounkova et al. / Aquatic Toxi

0

200

400

600

0

20

40

60

80

100

1a 2a 1b 2b 3 4

sum

of h

azar

dous

el

emen

ts (m

g/kg

)

mor

talit

y (%

)

mortality

hazardous elements

0

100

200

300

400

0

20

40

60

80

100

1a 2a 1b 2b 3 4

inde

x of

cyt

otox

icity

mor

talit

y (%

)

mortali ty

cytotoxicity

A

B

Fig. 4. Mortality of adults of P. antipodarum after 8 weeks in situ exposure and sum ofcPf

aroc

ee(aoaraCPero

BtepcangowopA4h

oncentrations of metals classified as hazardous elements (As, Cd, Co, Cr, Cu, Hg, Ni,b, Zn; MoA, 2009) in sediments (A), or index of cytotoxicity of extracts of sedimentsrom study sites, respectively (B). Number of specimen as in Fig. 2.

ntiestrogenic and antiandrogenic potencies. The relatively goodeproduction at these locations where greater mortality wasbserved could be affected by the presence of endocrine disruptiveompounds.

The hormonal system of molluscs is insufficiently known. Thestrogen receptor (ER) has been identified in P. antipodarum (Stanget al., 2012) and some hormones were detected in molluscsLafont and Mathieu, 2007), but there is a lack of informationbout their function. There is little information about effectsf non-endocrine disruptive compounds on reproduction of P.ntipodarum. It has been shown that nitrates, as well as fluo-ides and copper (Cu) nanoparticles reduce reproduction of P.ntipodarum (Alonso and Camargo, 2011, 2013; Pang et al., 2012).hanges of reproduction and mortality of the freshwater mudsnail. antipodarum can thus be the consequence of effects on differ-nt magnitudes of general stress, endocrine disruption througheceptors, changes of metabolism of hormones or enzymes andthers.

Sediment and water samples from localities downstream ofrno and the regional WWTP contained relatively high concentra-ions of organic pollutants and metals known for their negativeffects on biota. Some of these compounds, such as organochlorineesticides, polychlorinated biphenyls, polycyclic aromatic hydro-arbons, pharmaceuticals, belong to the group of EDCs (Depledgend Billinghurst, 1999; Groshart and Okkerman, 2000). However,o correlation between concentrations of any single chemical orroup of chemicals and numbers of embryos in mudsnails wasbserved. Alternatively, mortality was generally in a good accordith concentrations of hazardous metals (Fig. 4A). Negative effects

f metals on survival of pulmonate snails have been documented in
revious studies (Gupta et al., 1981; Laskowski and Hopkin, 1996;llah et al., 1997). However, greater mortality at localities 3 and
might be caused also by contribution of other pollutants, whichave not been analyzed in the sediments. Good correspondence

cology 150 (2014) 83–92

was also observed between mortality of exposed snails and cyto-toxicity of organic extracts of sediments detected in vitro (Fig. 4B),which indicates relation to other chemicals than metals. Mud-snails are exposed to the whole mixture of both analyzed andunknown pollutants and it is not surprising that the total cytotox-icity corresponds better to the mortality than any individual groupof compounds. These two correlations indicate that both inorganicand organic pollutants affect their survival.

5. Conclusions

Results of the present study proved the suitability of freshwatermudsnail P. antipodarum as a model organism for in situ assessmentof effects of urban rivers contamination on biota. It demonstratedeffects of various sources of pollution in the studied area. The in situassays with P. antipodarum document the presence of toxic com-pounds in the complex contaminant mixture in sediments as wellas effects on reproduction in mudsnails. This is the first study thatbrings together this in situ test with simultaneous passive samp-ling for the determination of time-weighted exposure and detailedcharacterization of exposure through both chemical analysis andin vitro bioassays of both sediment and water. A battery of in vitrotests provided complementary information to chemical analysistaking into account also unanalyzed compounds and interactionswithin mixture. This approach enabled to indicate groups of com-pounds contributing to the observed effects.

Chemical pollution resulting from runoff and waste waters fromthe Brno metropolitan area had negative effect on survival of thefreshwater mudsnail. Greater mortality was observed to be con-sistent with concentrations of metals and in vitro cytotoxicity.Number of embryos was also affected by pollution from the Brnometropolitan area as well as by suburb sources with a small WWTPof insufficient capacity. The early development of embryos in thebrood pouch reflects effects of those toxicants that immediatelyaffect the general health condition and reproduction.

Acknowledgements

This research was supported by the Czech Ministry of Education(LO1214) and the European Union Seventh Framework Programme(FP7) under the Project SOLUTIONS with grant agreement No.603437. Prof. Giesy was supported by the Canada Research Chairprogram, a Visiting Distinguished Professorship in the Depart-ment of Biology and Chemistry and State Key Laboratory in MarinePollution, City University of Hong Kong, the 2012 “High Level For-eign Experts” (#GDW20123200120) program, funded by the StateAdministration of Foreign Experts Affairs, the P.R. China to Nan-jing University and the Einstein Professor Program of the ChineseAcademy of Sciences.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.aquatox.2014.02.021.

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Supplementary materials

to In situ effects of urban river pollution on the mudsnail Potamopyrgus antipodarum as part of an integrated assessment

by Radka Zounkova, Veronika Jalova, Martina Janisova, Tomas Ocelka, Jana Jurcikova, Jarmila Halirova, John P. Giesy, Klara Hilscherova

Table S1. Water temperatures (oC) during the deployment period of passive samplers at river sites where mudsnails were exposed

1a 1b 2b 4

1a 1b 2b 4 7.5.2008 13:00 13.62 12.78 14 12.75

9.5.2008 11:00 13.25 13.06 13.43 14.23

7.5.2008 14:00 14.09 13.34 14.47 12.75

9.5.2008 12:00 13.34 13.15 13.53 13.76 7.5.2008 15:00 14.37 13.72 15.03 13.36

9.5.2008 13:00 13.53 13.34 13.9 13.36

7.5.2008 16:00 14.84 13.9 15.12 13.83

9.5.2008 14:00 13.81 13.72 14.75 13.48 7.5.2008 17:00 14.93 14.09 14.84 13.89

9.5.2008 15:00 14.09 14.28 15.21 13.67

7.5.2008 18:00 13.34 14.18 14.56 13.73

9.5.2008 16:00 14.47 14.65 15.5 14.11 7.5.2008 19:00 12.78 14.37 14.18 13.61

9.5.2008 17:00 14.47 14.84 15.4 14.36

7.5.2008 20:00 12.87 14.47 13.9 13.64

9.5.2008 18:00 14.47 15.03 15.31 14.3 7.5.2008 21:00 12.87 14.47 13.43 13.76

9.5.2008 19:00 12.78 15.03 15.12 14.23

7.5.2008 22:00 12.78 14.47 12.87 13.89

9.5.2008 20:00 12.59 15.12 14.84 14.11 7.5.2008 23:00 12.78 14.37 12.59 14.26

9.5.2008 21:00 12.59 15.12 14.56 14.23

8.5.2008 0:00 12.68 13.25 12.4 14.48

9.5.2008 22:00 12.59 15.21 14.09 14.23 8.5.2008 1:00 12.59 12.68 12.12 14.01

9.5.2008 23:00 12.59 15.12 13.72 14.33

8.5.2008 2:00 12.59 12.49 11.93 13.61

10.5.2008 0:00 12.49 14.84 13.25 14.54 8.5.2008 3:00 12.49 12.49 11.84 13.58

10.5.2008 1:00 12.49 14.37 12.96 14.73

8.5.2008 4:00 12.49 12.4 11.55 13.51

10.5.2008 2:00 12.4 14 12.68 14.51 8.5.2008 5:00 12.31 12.4 11.46 13.32

10.5.2008 3:00 12.31 13.72 12.49 14.39

8.5.2008 6:00 12.31 12.31 11.36 13.1

10.5.2008 4:00 12.31 13.53 12.4 14.23 8.5.2008 7:00 12.49 12.31 11.27 12.94

10.5.2008 5:00 12.21 13.43 12.21 14.11

8.5.2008 8:00 12.59 12.4 11.27 12.82

10.5.2008 6:00 12.21 13.25 12.12 14.01 8.5.2008 9:00 12.59 12.4 11.55 12.79

10.5.2008 7:00 12.49 13.15 12.12 14.05

8.5.2008 10:00 12.68 12.49 12.12 12.88

10.5.2008 8:00 12.68 13.06 12.21 14.01 8.5.2008 11:00 12.96 12.59 12.78 13.23

10.5.2008 9:00 12.78 12.96 12.49 14.01

8.5.2008 12:00 13.34 12.96 13.62 13.36

10.5.2008 10:00 12.78 13.06 12.96 14.08 8.5.2008 13:00 13.72 13.53 14.18 12.72

10.5.2008 11:00 13.25 13.15 13.62 14.23

8.5.2008 14:00 14.18 13.9 14.47 12.85

10.5.2008 12:00 13.62 13.25 14.47 14.23 8.5.2008 15:00 14.84 14.18 14.65 13.48

10.5.2008 13:00 14 13.72 15.31 13.86

8.5.2008 16:00 14.84 14.28 14.65 13.86

10.5.2008 14:00 14.37 14.37 16.06 13.76 8.5.2008 17:00 14.93 14.47 14.65 13.92

10.5.2008 15:00 14.75 14.75 16.62 14.23

8.5.2008 18:00 14.65 14.56 14.65 13.86

10.5.2008 16:00 15.03 14.93 16.34 14.79 8.5.2008 19:00 13.15 14.65 14.47 13.89

10.5.2008 17:00 15.12 15.12 15.96 14.97

8.5.2008 20:00 12.96 14.65 14.28 13.92

10.5.2008 18:00 12.96 15.21 15.5 14.85 8.5.2008 21:00 12.96 14.65 13.9 14.01

10.5.2008 19:00 12.78 15.31 15.12 14.73

8.5.2008 22:00 12.96 14.65 13.53 14.23

10.5.2008 20:00 12.78 15.31 14.65 14.67 8.5.2008 23:00 12.96 14.84 13.25 14.39

10.5.2008 21:00 12.96 15.4 14.28 14.73

9.5.2008 0:00 12.87 14.18 12.87 14.6

10.5.2008 22:00 12.78 15.4 14 14.94 9.5.2008 1:00 12.87 13.81 12.68 14.48

10.5.2008 23:00 12.78 14.84 13.72 15.28

9.5.2008 2:00 12.87 13.62 12.59 14.26

11.5.2008 0:00 12.78 14 13.34 15.59 9.5.2008 3:00 12.78 13.43 12.4 13.98

11.5.2008 1:00 12.68 13.53 12.96 15.07

9.5.2008 4:00 12.78 13.34 12.4 13.92

11.5.2008 2:00 12.68 13.34 12.78 14.64 9.5.2008 5:00 12.68 13.25 12.31 13.92

11.5.2008 3:00 12.59 13.15 12.59 14.39

9.5.2008 6:00 12.59 13.15 12.31 13.92

11.5.2008 4:00 12.49 13.15 12.49 14.36 9.5.2008 7:00 12.49 12.96 12.21 13.89

11.5.2008 5:00 12.4 12.96 12.4 14.3

9.5.2008 8:00 12.78 12.96 12.21 13.83

11.5.2008 6:00 12.31 12.96 12.31 14.23 9.5.2008 9:00 12.87 12.96 12.59 13.83

11.5.2008 7:00 12.59 12.96 12.31 14.08

9.5.2008 10:00 13.06 13.06 13.06 13.92

11.5.2008 8:00 12.87 12.87 12.4 13.98

1a 1b 2b 4

1a 1b 2b 4 11.5.2008 9:00 12.96 12.87 12.68 13.89

14.5.2008 0:00 13.53 16.34 14.93 16.46

11.5.2008 10:00 13.15 12.96 13.25 13.92

14.5.2008 1:00 13.43 15.59 14.56 16.37 11.5.2008 11:00 13.43 13.06 13.81 13.86

14.5.2008 2:00 13.43 15.12 14.37 16.1

11.5.2008 12:00 13.81 13.25 14.65 14.48

14.5.2008 3:00 13.34 14.84 14.18 15.89 11.5.2008 13:00 14.18 13.43 15.59 14.23

14.5.2008 4:00 13.34 14.65 14.09 15.71

11.5.2008 14:00 14.65 13.72 16.34 14.08

14.5.2008 5:00 13.25 14.47 13.9 15.56 11.5.2008 15:00 14.84 14.18 16.81 14.14

14.5.2008 6:00 13.25 14.37 13.9 15.53

11.5.2008 16:00 15.21 14.65 16.99 14.26

14.5.2008 7:00 12.96 14.28 14 15.62 11.5.2008 17:00 15.4 15.31 16.9 14.64

14.5.2008 8:00 13.53 14.18 14.09 15.62

11.5.2008 18:00 15.5 15.5 16.81 14.91

14.5.2008 9:00 13.53 14.28 14.37 15.62 11.5.2008 19:00 13.15 15.59 16.24 15.1

14.5.2008 10:00 13.72 14.28 14.93 15.71

11.5.2008 20:00 13.06 15.68 15.78 15.22

14.5.2008 11:00 14.09 14.28 15.59 15.86 11.5.2008 21:00 13.06 15.87 15.31 15.22

14.5.2008 12:00 14.37 14.47 16.43 15.89

11.5.2008 22:00 13.06 15.96 14.84 15.25

14.5.2008 13:00 14.93 14.75 17.28 15.56 11.5.2008 23:00 12.96 16.24 14.47 15.46

14.5.2008 14:00 15.31 15.21 18.03 15.46

12.5.2008 0:00 12.96 16.15 14.18 15.71

14.5.2008 15:00 15.59 15.68 18.5 15.56 12.5.2008 1:00 12.96 15.78 13.81 15.71

14.5.2008 16:00 15.87 15.96 18.5 16.07

12.5.2008 2:00 12.87 15.4 13.53 15.46

14.5.2008 17:00 15.87 16.15 18.12 16.28 12.5.2008 3:00 12.87 14.93 13.25 15.22

14.5.2008 18:00 15.68 16.15 17.46 16.43

12.5.2008 4:00 12.78 14.47 13.06 15.07

14.5.2008 19:00 14.09 16.24 16.53 16.22 12.5.2008 5:00 12.68 14 12.96 14.91

14.5.2008 20:00 14.28 16.34 16.34 16.22

12.5.2008 6:00 12.59 13.72 12.78 14.82

14.5.2008 21:00 14 16.53 15.87 16.37 12.5.2008 7:00 12.68 13.53 12.78 14.76

14.5.2008 22:00 14.09 16.71 15.4 16.67

12.5.2008 8:00 13.06 13.43 12.96 14.85

14.5.2008 23:00 14 16.9 15.12 16.85 12.5.2008 9:00 13.06 13.34 13.25 14.97

15.5.2008 0:00 14 16.34 14.75 16.76

12.5.2008 10:00 13.34 13.43 13.72 15.1

15.5.2008 1:00 14 15.87 14.47 16.49 12.5.2008 11:00 13.53 13.53 14.37 15.22

15.5.2008 2:00 14 15.31 14.37 16.25

12.5.2008 12:00 13.81 13.62 15.21 15.22

15.5.2008 3:00 13.9 15.03 14.37 15.95 12.5.2008 13:00 14.09 13.9 16.06 14.85

15.5.2008 4:00 13.9 14.75 14.28 15.71

12.5.2008 14:00 14.47 14.28 16.43 14.73

15.5.2008 5:00 13.72 14.65 14.09 15.65 12.5.2008 15:00 14.75 14.75 16.99 14.94

15.5.2008 6:00 13.72 14.47 13.9 15.71

12.5.2008 16:00 15.31 15.21 17.09 15.34

15.5.2008 7:00 13.53 14.37 13.81 15.62 12.5.2008 17:00 15.5 15.21 17.18 15.62

15.5.2008 8:00 13.72 14.28 13.81 15.53

12.5.2008 18:00 15.59 15.31 16.99 15.71

15.5.2008 9:00 13.81 14.28 14 15.49 12.5.2008 19:00 13.25 15.5 16.24 15.49

15.5.2008 10:00 13.9 14.37 14.37 15.49

12.5.2008 20:00 13.34 15.68 15.96 15.37

15.5.2008 11:00 14.28 14.37 14.75 15.49 12.5.2008 21:00 13.34 15.68 15.68 15.37

15.5.2008 12:00 14.65 14.47 15.5 15.71

12.5.2008 22:00 13.25 15.87 15.31 15.46

15.5.2008 13:00 15.03 14.75 16.24 15.37 12.5.2008 23:00 13.25 16.24 14.93 15.62

15.5.2008 14:00 15.4 15.12 16.9 15.31

13.5.2008 0:00 13.25 15.96 14.65 15.98

15.5.2008 15:00 15.78 15.5 17.28 15.56 13.5.2008 1:00 13.25 15.5 14.37 15.98

15.5.2008 16:00 15.78 15.78 17.09 15.86

13.5.2008 2:00 13.15 15.03 14.09 15.74

15.5.2008 17:00 16.06 15.96 17.09 16.22 13.5.2008 3:00 13.15 14.65 13.81 15.43

15.5.2008 18:00 15.96 16.24 16.99 16.19

13.5.2008 4:00 12.96 14.37 13.62 15.22

15.5.2008 19:00 14.09 16.34 16.81 16.19 13.5.2008 5:00 12.96 14.18 13.43 15.04

15.5.2008 20:00 14.09 16.53 16.71 16.07

13.5.2008 6:00 12.87 14 13.25 15.01

15.5.2008 21:00 14.09 16.53 16.24 16.19 13.5.2008 7:00 12.87 14 13.25 15.07

15.5.2008 22:00 14.09 16.71 15.78 16.28

13.5.2008 8:00 13.25 13.9 13.25 15.22

15.5.2008 23:00 14.09 16.9 15.5 16.4 13.5.2008 9:00 13.34 13.9 13.62 15.22

16.5.2008 0:00 14.09 16.71 15.12 16.46

13.5.2008 10:00 13.62 13.9 14.09 15.37

16.5.2008 1:00 14 16.24 14.93 16.49 13.5.2008 11:00 13.9 13.9 14.84 15.34

16.5.2008 2:00 14 15.87 14.65 16.25

13.5.2008 12:00 14.18 14.09 15.59 15.34

16.5.2008 3:00 14 15.5 14.56 16.1 13.5.2008 13:00 14.65 14.28 16.53 15.43

16.5.2008 4:00 14 15.31 14.56 15.89

13.5.2008 14:00 15.21 14.65 17.37 15.25

16.5.2008 5:00 13.9 15.12 14.65 15.86 13.5.2008 15:00 15.5 15.31 17.93 15.31

16.5.2008 6:00 13.9 15.12 14.56 15.86

13.5.2008 16:00 15.87 15.68 18.12 15.74

16.5.2008 7:00 13.72 15.03 14.65 15.92 13.5.2008 17:00 16.06 15.96 17.93 16.1

16.5.2008 8:00 13.9 15.03 14.65 15.89

13.5.2008 18:00 16.24 16.06 17.65 16.19

16.5.2008 9:00 13.9 15.03 14.84 15.92 13.5.2008 19:00 13.43 16.15 17.18 16.19

16.5.2008 10:00 14 15.03 15.21 15.89

13.5.2008 20:00 13.53 16.24 16.62 16.04

16.5.2008 11:00 14.28 15.03 15.59 15.86 13.5.2008 21:00 13.72 16.34 16.15 16.04

16.5.2008 12:00 14.37 15.03 16.15 15.86

13.5.2008 22:00 13.53 16.43 15.87 16.07

16.5.2008 13:00 14.56 15.31 16.34 15.59 13.5.2008 23:00 13.53 16.81 15.4 16.19

16.5.2008 14:00 14.93 15.59 16.62 15.59

1a 1b 2b 4

1a 1b 2b 4

16.5.2008 15:00 15.12 15.78 16.9 15.95

19.5.2008 6:00 14.37 15.03 15.68 16.19 16.5.2008 16:00 15.31 15.96 17.18 16.31

19.5.2008 7:00 14.28 14.93 15.5 16.04

16.5.2008 17:00 15.5 16.15 17.46 16.28

19.5.2008 8:00 14.47 14.93 15.31 15.89 16.5.2008 18:00 15.5 16.34 17.18 16.19

19.5.2008 9:00 14.56 14.93 14.84 15.77

16.5.2008 19:00 14.47 16.34 16.9 16.04

19.5.2008 10:00 14.65 14.93 14.84 15.49 16.5.2008 20:00 14.18 16.34 16.62 16.01

19.5.2008 11:00 14.65 14.84 14.84 15.37

16.5.2008 21:00 14.09 16.43 16.24 16.07

19.5.2008 12:00 14.65 14.65 14.93 15.25 16.5.2008 22:00 14.28 16.53 16.15 16.19

19.5.2008 13:00 14.65 14.65 14.84 15.07

16.5.2008 23:00 14.28 16.71 15.96 16.43

19.5.2008 14:00 14.65 14.65 14.84 15.01 17.5.2008 0:00 14.18 16.24 15.59 16.73

19.5.2008 15:00 14.65 14.75 14.75 14.88

17.5.2008 1:00 14.18 15.68 15.4 16.49

19.5.2008 16:00 14.65 14.84 14.75 14.79 17.5.2008 2:00 14.18 15.4 15.21 16.19

19.5.2008 17:00 14.75 14.84 14.75 14.76

17.5.2008 3:00 14.09 15.21 15.12 15.98

19.5.2008 18:00 14.47 14.84 14.75 14.79 17.5.2008 4:00 14.09 15.03 15.03 16.01

19.5.2008 19:00 14.47 14.84 14.65 14.82

17.5.2008 5:00 14.09 14.93 14.84 16.07

19.5.2008 20:00 14.47 14.84 14.37 14.82 17.5.2008 6:00 14.09 14.84 14.65 16.04

19.5.2008 21:00 14.47 14.84 14.28 14.79

17.5.2008 7:00 13.9 14.75 14.65 16.01

19.5.2008 22:00 14.47 14.75 14.09 14.73 17.5.2008 8:00 14.28 14.75 14.65 15.92

19.5.2008 23:00 14.37 14.56 13.9 14.73

17.5.2008 9:00 14.28 14.75 14.84 15.86

20.5.2008 0:00 14.37 14.28 13.81 14.51 17.5.2008 10:00 14.56 14.84 15.21 16.19

20.5.2008 1:00 14.37 14.18 13.72 14.23

17.5.2008 11:00 14.75 14.84 15.78 16.07

20.5.2008 2:00 14.28 14.09 13.53 14.11 17.5.2008 12:00 15.12 15.03 16.62 16.07

20.5.2008 3:00 14.28 14 13.34 14.01

17.5.2008 13:00 15.31 15.31 16.99 15.74

20.5.2008 4:00 14.18 14 13.25 13.89 17.5.2008 14:00 15.68 15.87 17.56 15.71

20.5.2008 5:00 14.09 13.9 13.06 13.8

17.5.2008 15:00 16.15 16.24 18.12 16.1

20.5.2008 6:00 14 13.81 12.96 13.73 17.5.2008 16:00 16.62 16.53 18.4 16.52

20.5.2008 7:00 14.18 13.81 12.78 13.61

17.5.2008 17:00 16.71 16.71 18.5 16.73

20.5.2008 8:00 14.37 13.81 12.78 13.48 17.5.2008 18:00 16.71 16.81 18.31 16.67

20.5.2008 9:00 14.47 13.72 12.78 13.39

17.5.2008 19:00 14.18 16.9 18.03 16.58

20.5.2008 10:00 14.47 13.72 12.78 13.29 17.5.2008 20:00 14.18 16.99 17.74 16.58

20.5.2008 11:00 14.47 13.62 12.78 13.23

17.5.2008 21:00 14.37 16.99 17.46 16.67

20.5.2008 12:00 14.47 13.43 12.78 13.04 17.5.2008 22:00 14.28 17.09 17.09 16.82

20.5.2008 13:00 14.47 13.72 12.78 12.98

17.5.2008 23:00 14.37 17.37 16.71 17

20.5.2008 14:00 14.37 13.15 12.96 13.1 18.5.2008 0:00 14.37 17.09 17.28 17.39

20.5.2008 15:00 14.37 13.9 12.96 13.23

18.5.2008 1:00 14.37 16.24 16.99 17.27

20.5.2008 16:00 14.28 13.9 12.78 13.39 18.5.2008 2:00 14.28 16.06 16.71 16.91

20.5.2008 17:00 14.47 13.9 12.78 13.48

18.5.2008 3:00 14.28 16.24 16.62 16.91

20.5.2008 18:00 14.37 13.9 12.59 13.51 18.5.2008 4:00 14.28 16.43 16.34 16.82

20.5.2008 19:00 14.37 13.9 12.4 13.55

18.5.2008 5:00 14.28 16.53 16.24 16.79

20.5.2008 20:00 14.37 13.72 12.21 13.55 18.5.2008 6:00 14.28 16.53 16.06 16.73

20.5.2008 21:00 14.37 13.81 12.12 13.58

18.5.2008 7:00 14.09 16.43 15.96 16.73

20.5.2008 22:00 14.37 14 12.02 13.48 18.5.2008 8:00 14.28 16.43 16.06 16.76

20.5.2008 23:00 14.28 14.09 11.93 13.29

18.5.2008 9:00 14.28 16.34 16.34 16.79

21.5.2008 0:00 14.28 14.09 11.84 13.23 18.5.2008 10:00 14.37 16.24 16.62 16.94

21.5.2008 1:00 14.18 14.09 11.65 13.26

18.5.2008 11:00 14.56 16.24 17.09 17.15

21.5.2008 2:00 14.09 14 11.55 13.32 18.5.2008 12:00 14.65 16.15 17.37 17.15

21.5.2008 3:00 14.09 14 11.46 13.36

18.5.2008 13:00 14.93 16.34 17.65 16.85

21.5.2008 4:00 14.09 14 11.36 13.36 18.5.2008 14:00 15.21 16.43 18.03 16.88

21.5.2008 5:00 14.09 13.9 11.36 13.26

18.5.2008 15:00 15.31 16.43 17.74 17

21.5.2008 6:00 14 13.9 11.27 13.23 18.5.2008 16:00 15.31 16.53 18.12 17.18

21.5.2008 7:00 14.09 13.72 11.27 13.04

18.5.2008 17:00 15.4 16.71 18.59 16.82

21.5.2008 8:00 14.28 13.72 11.27 12.94 18.5.2008 18:00 14.75 16.99 18.21 16.55

21.5.2008 9:00 14.18 13.72 11.18 12.79

18.5.2008 19:00 14.75 17.18 17.74 16.67

21.5.2008 10:00 14.28 13.53 11.18 12.56 18.5.2008 20:00 14.65 17.18 17.37 16.85

21.5.2008 11:00 14.28 13.53 11.18 12.4

18.5.2008 21:00 14.47 17.28 16.99 17

21.5.2008 12:00 14.18 13.34 11.36 12.24 18.5.2008 22:00 14.56 17.28 16.99 17.15

21.5.2008 13:00 14.18 13.43 11.46 12.21

18.5.2008 23:00 14.47 16.62 16.71 17.15

21.5.2008 14:00 14.28 13.62 11.55 12.34 19.5.2008 0:00 14.47 15.87 16.34 16.91

21.5.2008 15:00 14.28 13.81 11.65 12.63

19.5.2008 1:00 14.56 15.59 16.24 16.73

21.5.2008 16:00 14.28 13.9 11.74 12.85 19.5.2008 2:00 14.47 15.4 16.06 16.67

21.5.2008 17:00 14.37 13.9 11.74 12.98

19.5.2008 3:00 14.47 15.31 15.96 16.52

21.5.2008 18:00 14.09 14 11.74 13.01 19.5.2008 4:00 14.47 15.21 15.87 16.4

21.5.2008 19:00 14.28 14 11.74 13.23

19.5.2008 5:00 14.47 15.12 15.78 16.28

21.5.2008 20:00 14.28 14 11.65 13.23

1a 1b 2b 4

1a 1b 2b 4 21.5.2008 21:00 14.18 13.9 11.46 13.23

24.5.2008 12:00 14 14.18 13.34 13.23

21.5.2008 22:00 14.09 13.9 11.36 13.23

24.5.2008 13:00 14.18 15.21 13.53 13.23 21.5.2008 23:00 14.18 13.9 11.18 13.04

24.5.2008 14:00 14.37 15.21 13.62 13.92

22.5.2008 0:00 14.09 13.72 11.08 12.79

24.5.2008 15:00 14.47 15.21 13.34 14.23 22.5.2008 1:00 14.09 13.62 10.99 12.5

24.5.2008 16:00 14.75 15.21 13.43 14.33

22.5.2008 2:00 14.09 13.62 10.89 12.53

24.5.2008 17:00 15.03 15.21 13.72 14.57 22.5.2008 3:00 14 13.53 10.89 12.72

24.5.2008 18:00 14 15.21 13.81 14.79

22.5.2008 4:00 13.9 13.53 10.89 12.72

24.5.2008 19:00 13.81 15.31 13.72 14.85 22.5.2008 5:00 13.9 13.43 10.8 12.59

24.5.2008 20:00 13.81 15.31 13.53 14.82

22.5.2008 6:00 13.81 13.43 10.8 12.47

24.5.2008 21:00 13.81 15.31 13.34 14.82 22.5.2008 7:00 14 13.34 10.89 12.34

24.5.2008 22:00 13.81 15.21 13.15 14.82

22.5.2008 8:00 14.09 13.34 10.89 12.21

24.5.2008 23:00 13.72 15.21 12.87 14.76 22.5.2008 9:00 14.09 13.25 10.99 12.08

25.5.2008 0:00 13.72 14.47 12.78 14.51

22.5.2008 10:00 14 13.25 11.18 11.98

25.5.2008 1:00 13.72 14.18 12.59 14.14 22.5.2008 11:00 14 13.25 11.46 11.95

25.5.2008 2:00 13.72 14.18 12.49 14.26

22.5.2008 12:00 14 13.34 11.65 11.98

25.5.2008 3:00 13.72 14.09 12.4 14.33 22.5.2008 13:00 14 13.9 11.65 12.08

25.5.2008 4:00 13.62 14.09 12.31 14.23

22.5.2008 14:00 14.09 14.18 11.65 12.37

25.5.2008 5:00 13.53 14 12.31 13.98 22.5.2008 15:00 14.18 14.18 11.65 12.75

25.5.2008 6:00 13.43 13.9 12.21 13.8

22.5.2008 16:00 14.09 14.28 11.65 13.01

25.5.2008 7:00 13.43 14 12.21 13.73 22.5.2008 17:00 14.09 14.37 11.65 13.26

25.5.2008 8:00 13.81 14.09 12.31 13.64

22.5.2008 18:00 14.09 14.18 11.65 13.32

25.5.2008 9:00 13.9 14.37 12.59 13.61 22.5.2008 19:00 14.09 14.09 11.46 13.36

25.5.2008 10:00 13.9 14.93 12.96 13.73

22.5.2008 20:00 14.09 14.09 11.46 13.32

25.5.2008 11:00 13.9 14.47 13.72 13.83 22.5.2008 21:00 13.9 14.09 11.27 13.23

25.5.2008 12:00 14.18 14.47 14.28 13.95

22.5.2008 22:00 14 14.18 11.27 12.85

25.5.2008 13:00 14.47 15.5 14.75 13.86 22.5.2008 23:00 14 14.18 11.27 13.04

25.5.2008 14:00 14.75 15.59 15.4 14.51

23.5.2008 0:00 13.9 14.09 11.27 13.23

25.5.2008 15:00 15.03 15.59 15.5 15.07 23.5.2008 1:00 13.9 13.9 11.27 13.23

25.5.2008 16:00 15.5 15.87 15.78 15.28

23.5.2008 2:00 13.9 13.72 11.18 13.23

25.5.2008 17:00 15.78 15.87 15.78 15.37 23.5.2008 3:00 13.9 13.9 11.27 13.23

25.5.2008 18:00 14.65 15.68 15.4 15.59

23.5.2008 4:00 13.9 13.9 11.27 13.23

25.5.2008 19:00 13.62 15.5 15.03 15.8 23.5.2008 5:00 13.81 13.9 11.27 13.1

25.5.2008 20:00 13.53 15.21 14.65 15.92

23.5.2008 6:00 13.72 13.9 11.27 13.01

25.5.2008 21:00 13.53 15.12 14.28 15.95 23.5.2008 7:00 13.72 13.81 11.36 12.91

25.5.2008 22:00 13.62 15.68 14 15.98

23.5.2008 8:00 13.9 13.81 11.46 12.82

25.5.2008 23:00 13.53 15.68 13.72 15.95 23.5.2008 9:00 13.81 13.81 11.55 12.72

26.5.2008 0:00 13.53 14.56 13.53 15.56

23.5.2008 10:00 13.53 13.81 11.84 12.63

26.5.2008 1:00 13.53 14.28 13.43 14.97 23.5.2008 11:00 13.9 14.65 12.12 12.66

26.5.2008 2:00 13.53 14 13.34 14.79

23.5.2008 12:00 14.09 14 12.4 12.72

26.5.2008 3:00 13.43 14.09 13.25 14.85 23.5.2008 13:00 14.28 14.56 12.68 12.82

26.5.2008 4:00 13.43 14.09 13.15 14.73

23.5.2008 14:00 14.65 14.65 12.87 13.23

26.5.2008 5:00 13.34 14.09 12.87 14.51 23.5.2008 15:00 15.03 14.84 12.78 13.48

26.5.2008 6:00 13.25 14.09 12.87 14.33

23.5.2008 16:00 14.84 14.93 12.68 13.8

26.5.2008 7:00 13.34 14.18 12.87 14.23 23.5.2008 17:00 14.84 14.93 12.78 13.98

26.5.2008 8:00 13.62 14.56 12.96 14.14

23.5.2008 18:00 14.93 14.84 12.78 14.11

26.5.2008 9:00 13.72 14.28 13.15 14.23 23.5.2008 19:00 13.72 14.84 12.59 14.23

26.5.2008 10:00 13.72 14.28 13.53 14.23

23.5.2008 20:00 13.9 14.65 12.49 14.23

26.5.2008 11:00 13.62 14.37 14 14.23 23.5.2008 21:00 13.81 14.56 12.4 14.23

26.5.2008 12:00 13.9 14.47 14.37 14.14

23.5.2008 22:00 13.81 14.56 12.21 14.05

26.5.2008 13:00 14.09 15.31 14.93 14.08 23.5.2008 23:00 13.81 14.65 12.21 13.92

26.5.2008 14:00 14.28 15.5 15.4 14.73

24.5.2008 0:00 13.72 14.28 12.12 13.73

26.5.2008 15:00 14.56 15.5 15.78 15.13 24.5.2008 1:00 13.81 14 12.02 13.55

26.5.2008 16:00 14.93 15.5 16.15 15.22

24.5.2008 2:00 13.81 13.81 11.93 13.29

26.5.2008 17:00 15.21 15.4 16.34 15.22 24.5.2008 3:00 13.72 13.72 11.74 13.32

26.5.2008 18:00 14 15.31 16.15 15.37

24.5.2008 4:00 13.72 13.62 11.74 13.51

26.5.2008 19:00 13.62 15.31 15.78 15.56 24.5.2008 5:00 13.62 13.62 11.65 13.58

26.5.2008 20:00 13.9 15.31 15.5 15.71

24.5.2008 6:00 13.62 13.72 11.65 13.48

26.5.2008 21:00 14 15.31 15.21 15.86 24.5.2008 7:00 13.53 13.81 11.65 13.39

26.5.2008 22:00 14.09 15.4 14.84 15.98

24.5.2008 8:00 13.72 13.9 11.74 13.29

26.5.2008 23:00 14 15.5 14.65 16.19 24.5.2008 9:00 13.72 14.09 12.02 13.29

27.5.2008 0:00 14 14.56 14.37 16.07

24.5.2008 10:00 13.81 15.21 12.31 13.23

27.5.2008 1:00 14 14.28 14.18 15.53 24.5.2008 11:00 13.81 16.62 12.87 13.26

27.5.2008 2:00 13.9 14.37 14.09 15.1

1a 1b 2b 4

1a 1b 2b 4 27.5.2008 3:00 13.9 14.37 13.9 15.07

29.5.2008 18:00 17.84 17.84 20 18.09

27.5.2008 4:00 13.9 14.37 13.72 14.97

29.5.2008 19:00 15.4 17.93 19.62 18.12 27.5.2008 5:00 13.81 14.47 13.72 14.85

29.5.2008 20:00 14.84 18.12 19.25 18.09

27.5.2008 6:00 13.81 14.37 13.62 14.73

29.5.2008 21:00 14.65 18.31 18.87 18.12 27.5.2008 7:00 13.72 14.37 13.72 14.57

29.5.2008 22:00 14.84 18.5 18.4 18.21

27.5.2008 8:00 13.81 14.37 13.72 14.51

29.5.2008 23:00 14.75 18.68 18.03 18.36 27.5.2008 9:00 13.9 14.47 14.09 14.6

30.5.2008 0:00 14.75 18.78 17.56 18.45

27.5.2008 10:00 14.18 14.56 14.47 14.76

30.5.2008 1:00 14.75 18.59 17.28 18.42 27.5.2008 11:00 14.37 14.65 15.12 14.91

30.5.2008 2:00 14.75 18.31 17.09 18.21

27.5.2008 12:00 14.75 14.93 15.96 14.97

30.5.2008 3:00 14.65 18.03 16.81 18.09 27.5.2008 13:00 15.21 15.5 16.99 14.97

30.5.2008 4:00 14.65 17.74 16.62 17.86

27.5.2008 14:00 15.5 16.15 17.74 15.46

30.5.2008 5:00 14.65 17.37 16.62 17.74 27.5.2008 15:00 16.06 16.53 18.21 16.1

30.5.2008 6:00 14.65 16.99 16.53 17.62

27.5.2008 16:00 16.53 16.81 18.59 16.55

30.5.2008 7:00 14.28 16.62 16.53 17.54 27.5.2008 17:00 17.37 16.9 18.87 16.67

30.5.2008 8:00 14.75 16.43 16.71 17.54

27.5.2008 18:00 14.93 17.18 18.68 16.58

30.5.2008 9:00 14.75 16.43 16.99 17.65 27.5.2008 19:00 14.09 17.37 18.5 16.58

30.5.2008 10:00 15.21 16.43 17.46 17.68

27.5.2008 20:00 14 17.46 18.12 16.73

30.5.2008 11:00 15.5 16.43 18.03 17.8 27.5.2008 21:00 14.09 17.56 17.74 16.97

30.5.2008 12:00 15.87 16.62 18.87 17.8

27.5.2008 22:00 14.18 17.74 17.28 17.27

30.5.2008 13:00 16.34 16.9 19.72 18.09 27.5.2008 23:00 14.09 18.03 16.81 17.65

30.5.2008 14:00 17.09 17.18 20.47 18.56

28.5.2008 0:00 14.09 17.09 16.53 17.92

30.5.2008 15:00 17.65 17.65 21.04 18.48 28.5.2008 1:00 14.09 16.24 16.24 17.71

30.5.2008 16:00 18.31 18.03 21.33 18.68

28.5.2008 2:00 14.18 15.78 15.96 17.42

30.5.2008 17:00 18.68 18.4 21.33 19.03 28.5.2008 3:00 14.18 15.59 15.78 17.18

30.5.2008 18:00 18.78 18.68 21.14 18.88

28.5.2008 4:00 14.18 15.59 15.78 17.15

30.5.2008 19:00 18.68 18.78 20.85 19.03 28.5.2008 5:00 14.18 15.5 15.68 17.15

30.5.2008 20:00 15.59 18.87 20.57 19.03

28.5.2008 6:00 14.09 15.5 15.68 17.15

30.5.2008 21:00 14.93 18.97 20.1 19.03 28.5.2008 7:00 13.72 15.5 15.68 17

30.5.2008 22:00 15.03 19.06 19.72 19.09

28.5.2008 8:00 14.18 15.59 15.87 16.94

30.5.2008 23:00 15.03 19.25 19.25 19.23 28.5.2008 9:00 14.37 15.68 16.15 16.91

31.5.2008 0:00 15.03 19.34 18.87 19.38

28.5.2008 10:00 14.37 15.78 16.62 16.82

31.5.2008 1:00 15.03 19.44 18.5 19.49 28.5.2008 11:00 14.65 15.87 17.18 16.73

31.5.2008 2:00 15.03 19.34 18.21 19.4

28.5.2008 12:00 14.84 16.24 17.84 16.61

31.5.2008 3:00 15.03 19.15 17.93 19.23 28.5.2008 13:00 15.31 16.62 18.59 16.46

31.5.2008 4:00 15.03 18.78 17.74 19.03

28.5.2008 14:00 15.68 17.18 19.25 16.73

31.5.2008 5:00 15.03 18.31 17.56 18.82 28.5.2008 15:00 16.62 17.37 19.62 17.39

31.5.2008 6:00 15.03 18.03 17.46 18.71

28.5.2008 16:00 17.18 17.56 19.91 17.86

31.5.2008 7:00 14.47 17.65 17.56 18.59 28.5.2008 17:00 17.37 17.74 20 17.86

31.5.2008 8:00 15.03 17.37 17.74 18.56

28.5.2008 18:00 17.56 17.84 19.72 17.71

31.5.2008 9:00 15.21 17.28 18.12 18.62 28.5.2008 19:00 14.75 17.93 19.44 17.65

31.5.2008 10:00 15.4 17.28 18.59 18.71

28.5.2008 20:00 14.93 18.12 19.06 17.65

31.5.2008 11:00 15.87 17.28 19.25 18.71 28.5.2008 21:00 14.84 18.12 18.59 17.71

31.5.2008 12:00 16.24 17.56 20 19.03

28.5.2008 22:00 14.75 18.21 18.21 17.89

31.5.2008 13:00 16.62 17.84 20.76 19.14 28.5.2008 23:00 14.84 18.5 17.84 18.09

31.5.2008 14:00 17.18 18.12 21.52 19.14

29.5.2008 0:00 14.84 17.74 17.37 18.33

31.5.2008 15:00 17.84 18.5 22.09 19.35 29.5.2008 1:00 14.75 16.9 17.09 18.15

31.5.2008 16:00 18.4 18.87 22.28 19.49

29.5.2008 2:00 14.84 16.34 16.9 17.89

31.5.2008 17:00 18.97 19.15 22.28 19.78 29.5.2008 3:00 14.84 15.96 16.81 17.68

31.5.2008 18:00 19.15 19.34 21.99 19.98

29.5.2008 4:00 14.75 15.78 16.71 17.62

31.5.2008 19:00 15.5 19.44 21.71 19.95 29.5.2008 5:00 14.65 15.68 16.62 17.51

31.5.2008 20:00 15.68 19.53 21.33 19.81

29.5.2008 6:00 14.65 15.59 16.53 17.51

31.5.2008 21:00 15.68 19.53 20.95 19.81 29.5.2008 7:00 14.37 15.59 16.53 17.45

31.5.2008 22:00 15.68 19.62 20.47 19.86

29.5.2008 8:00 14.65 15.59 16.62 17.39

31.5.2008 23:00 15.78 19.81 20.1 19.98 29.5.2008 9:00 14.47 15.68 16.9 17.36

1.6.2008 0:00 15.68 20.1 19.62 20.18

29.5.2008 10:00 14.84 15.68 17.28 17.54

1.6.2008 1:00 15.68 20 19.34 20.24 29.5.2008 11:00 15.21 15.78 17.65 17.56

1.6.2008 2:00 15.68 19.81 18.97 20.12

29.5.2008 12:00 15.78 15.96 18.4 17.48

1.6.2008 3:00 15.78 19.53 18.78 19.95 29.5.2008 13:00 15.96 16.15 19.15 17.3

1.6.2008 4:00 15.68 19.15 18.59 19.75

29.5.2008 14:00 16.43 16.43 19.72 17.21

1.6.2008 5:00 15.68 18.68 18.4 19.55 29.5.2008 15:00 17.28 16.9 20.19 17.24

1.6.2008 6:00 15.68 18.21 18.21 19.35

29.5.2008 16:00 17.56 17.28 20.38 17.68

1.6.2008 7:00 15.31 17.93 18.31 19.2 29.5.2008 17:00 17.84 17.56 20.29 17.95

1.6.2008 8:00 15.5 17.74 18.5 19.14

1a 1b 2b 4 1.6.2008 9:00 15.78 17.74 18.87 19.26

1.6.2008 10:00 15.87 17.74 19.44 19.49 1.6.2008 11:00 15.96 17.84 19.91 19.69 1.6.2008 12:00 16.24 17.84 20.38 19.55 1.6.2008 13:00 16.53 18.12 21.23 19.72 1.6.2008 14:00 16.9 18.4 21.9 20.01 1.6.2008 15:00 17.28 18.59 21.61 19.72 1.6.2008 16:00 17.65 18.78 21.52 19.58 1.6.2008 17:00 18.12 19.06 21.23 19.72 1.6.2008 18:00 18.03 19.15 20.85 19.84 1.6.2008 19:00 15.68 19.15 20.66 19.98 1.6.2008 20:00 16.43 19.06 20.38 19.95 1.6.2008 21:00 16.24 19.06 19.91 19.86 1.6.2008 22:00 16.24 19.06 19.72 19.81 1.6.2008 23:00 16.24 19.06 19.34 19.78 2.6.2008 0:00 16.24 18.97 18.97 19.69 2.6.2008 1:00 16.24 18.78 18.68 19.52 2.6.2008 2:00 16.24 18.59 18.5 19.32 2.6.2008 3:00 16.24 18.4 18.4 19.11 2.6.2008 4:00 16.15 18.21 18.21 19.03 2.6.2008 5:00 16.06 17.93 18.03 18.85 2.6.2008 6:00 15.96 17.74 17.93 18.71 2.6.2008 7:00 15.59 17.56 17.84 18.62 2.6.2008 8:00 15.87 17.46 17.93 18.56 2.6.2008 9:00 15.96 17.46 18.12 18.62

2.6.2008 10:00 15.78 17.46 18.68 18.56 2.6.2008 11:00 15.78 17.37 19.34 18.59 2.6.2008 12:00 16.15 17.65 20.1 18.85 2.6.2008 13:00 16.62 18.03 20.85 19.03 2.6.2008 14:00 17.18 18.4 21.61 18.88 2.6.2008 15:00 17.65 18.4 22.18 19.4 2.6.2008 16:00 18.21 18.5 22.47 19.78 2.6.2008 17:00 18.59 18.68 22.38 19.72 2.6.2008 18:00 18.31 18.87 22.09 19.4 2.6.2008 19:00 15.87 18.97 21.61 19.2 2.6.2008 20:00 15.78 19.06 21.14 19.17 2.6.2008 21:00 15.78 19.15 20.66 19.26 2.6.2008 22:00 15.87 19.25 20.19 19.4 2.6.2008 23:00 15.87 19.62 19.81 19.61 3.6.2008 0:00 15.87 19.06 19.34 19.86 3.6.2008 1:00 15.87 18.21 18.97 19.69 3.6.2008 2:00 15.87 17.84 18.59 19.35 3.6.2008 3:00 15.87 17.56 18.21 19.11 3.6.2008 4:00 15.78 17.46 18.03 18.88 3.6.2008 5:00 15.68 17.46 17.84 18.8 3.6.2008 6:00 15.68 17.37 17.65 18.71 3.6.2008 7:00 15.12 17.37 17.65 18.71 3.6.2008 8:00 15.96 17.37 17.84 18.68 3.6.2008 9:00 15.96 17.37 18.21 18.65

3.6.2008 10:00 16.06 17.46 18.68 18.62 3.6.2008 11:00 15.96 17.46 19.44 18.56 3.6.2008 12:00 16.53 17.65 20.19 18.59 3.6.2008 13:00 16.99 18.31 21.14 18.56 3.6.2008 14:00 17.18 18.78 21.9 18.56 3.6.2008 15:00 17.74 18.97 22.57 19.29 3.6.2008 16:00 18.03 19.15 22.47 19.32

Mean 14.45 15.49 15.40 15.71 Std.dev. 1.24 1.76 2.83 2.06 Min 12.21 12.31 10.8 11.95 Max 19.15 20.1 22.57 20.24

Table S2. Concentrations of studied pollutants in sediments (per g dry mass of sediment) from the study sites around Brno.

1a 2a 1b 2b 3 4

Triclosan (ng/g) 0.6 2.4 13 5.5 34 57 Me-triclosan (ng/g) 0.1 0.73 2.7 1.4 10 7.2

PBDEs (ng/g) PBDE 28 <0.061 <0.17 <0.061 <0.073 <0.12 <0.057

PBDE 47 0.069 <0.14 0.53 0.32 0.54 0.75 PBDE 99 <0.074 <0.2 0.53 0.16 0.54 1.00 PBDE 100 <0.074 <0.2 <0.12 <0.1 <0.18 <0.1 PBDE 153 <0.29 <0.78 <0.5 <0.37 <0.68 <0.43 PBDE 154 <0.19 <0.5 <0.35 <0.25 <0.43 <0.34 PBDE 183 <0.39 <1 <0.75 <0.57 <0.96 <0.99

PCBs (ng/g) PCB 28+31 0.35 1.3 8.8 1.0 2.0 6.1

PCB 52 0.11 1.4 3.2 0.47 0.62 0.43 PCB 101 0.46 11.9 6.6 1.4 1.7 3.15 PCB 118 0.26 2.9 3.0 0.69 0.9 1.3 PCB 138 1.2 29.7 25 3.9 7.2 12.3 PCB 153+168 2.2 45.8 33.9 6.5 10.8 16.9 PCB 170 0.40 12.7 12.5 1.7 4.1 5.4 PCB 180 0.92 31.2 30.2 3.8 11 13.9

HCHs (ng/g) alfa-HCH <0.23 <0.36 <0.19 <0.19 <0.33 <0.15

beta-HCH <0.31 <0.49 <0.26 <0.25 <0.44 <0.21 delta-HCH <0.36 <0.56 <0.30 <0.29 <0.51 <0.24 gama-HCH 1.0 <0.44 <0.23 <0.23 <0.40 <0.19

HCB (ng/g) 0.85 1.1 2.1 0.74 2.0 1.7

DDT and metabolites (ng/g) o,p-DDD <0.1 0.5 7.3 0.61 1.1 3.5

p,p-DDD 0.41 1.7 47.3 2.4 3.7 10.5 o,p-DDE <0.13 <0.36 0.58 <0.15 <0.27 0.3 p,p-DDE 0.68 1.7 17.9 2.6 6.1 9.0 o,p-DDT <0.16 <0.45 4.1 0.72 4.2 1.0 p,p-DDT 0.38 1.3 680 5.7 5.4 5.4

PAHs (µg/g) phenantrene 0.11 2.0 1.0 2.1 0.77 2.7

anthracene 0.029 0.47 0.15 0.44 0.1 0.56 fluoranthene 0.42 3.0 2.6 4.0 2.1 4.9 pyrene 0.33 2.1 1.9 2.8 1.6 3.4 benzo(a)anthracene 0.35 1.9 1.8 2.4 1.5 3.3 chrysene 0.18 1.1 1.1 1.4 0.87 1.9 benzo(b)fluoranthene 0.17 0.95 1.2 1.2 1.0 1.9 benzo(k)fluoranthene 0.12 0.6 0.76 0.77 0.62 1.2 benzo(a)pyrene 0.27 1.3 1.6 1.7 1.4 2.5 benzo(ghi)perylene 0.14 0.65 0.92 0.86 0.82 1.3 dibenzo(ah)anthracene 0.009 0.075 0.086 0.083 0.083 0.14 indeno(1,2,3-cd)pyrene 0.2 1.1 1.5 1.4 1.4 2.2

Metals (µg/g) Hg 0.011 0.12 0.89 0.68 0.96 0.96

Al 1630 3910 6070 3530 5030 5490 Cd 0.12 1.72 2.51 0.46 1.32 1.12 Cu 5.86 23.5 79.6 24.5 51.7 59.2 Pb 16.1 36.6 68.9 22.9 50.6 48.7

Zn 44.8 178 329 155 214 259 As 0.35 1.47 1.82 1.44 1.89 0.91 Ba 43 100 179 96 124 130 Co 4.28 5.35 8.03 3.9 5.49 6.19 Cr 5.72 25.7 30.1 16.1 24.1 25.8 Mo 0.015 0.037 0.089 0.051 0.087 0.098 Ni 7.84 15.6 24.4 11.7 15.5 18.3 Se 0.37 0.49 0.56 0.49 0.54 0.63 Ti 20.4 31.8 50.6 32.3 39.6 47.7

Table S3. Concentrations of studied pollutants in water (µg/l) from the study sites around Brno.


PESTICIDES acetochlor <0.019 0.230 0.054 0.310 <0.021 0.130

alachlor <0.005 0.029 0.027 0.029 0.033 0.036 atrazin <0.009 0.013 <0.009 0.011 0.032 0.013 bromacil <0.017 <0.019 <0.018 <0.018 <0.019 <0.017 carbofuran <0.017 <0.019 <0.017 <0.017 <0.019 <0.016 cyanazin <0.013 <0.015 <0.013 <0.014 <0.015 <0.013 desmetryn <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 diazinon <0.003 <0.004 <0.004 <0.004 0.015 <0.003 dichlobenil <0.013 <0.015 <0.014 <0.014 <0.015 <0.013 diuron <0.015 0.080 <0.016 0.089 0.150 0.056 chlorbromuron <0.041 <0.045 <0.042 <0.042 <0.045 <0.039 chlorotoluron <0.02 0.330 0.045 0.350 <0.022 0.087 isoproturon <0.015 <0.017 <0.016 <0.016 <0.017 <0.015 linuron <0.025 <0.028 <0.026 <0.026 <0.028 <0.024 hexazinon 0.010 <0.008 <0.008 <0.008 <0.008 <0.007 metalaxyl <0.005 <0.006 <0.005 <0.005 <0.006 <0.005 metobromuron <0.018 <0.02 <0.018 <0.018 <0.02 <0.017 metolachlor <0.01 <0.011 <0.01 <0.01 <0.011 <0.009 metoxuron <0.01 <0.012 <0.011 <0.011 <0.012 <0.01 metribuzin <0.015 <0.017 <0.016 <0.016 <0.017 <0.015 monolinuron <0.026 <0.029 <0.027 <0.027 <0.029 <0.025 prometryn <0.005 <0.005 <0.005 <0.005 <0.005 <0.004 simazin <0.006 <0.007 <0.006 <0.006 <0.007 <0.006 terbuthylazine 0.006 0.190 0.017 0.160 0.200 0.080 terbutryn <0.003 <0.004 <0.003 0.004 0.037 0.007 2,4,5-T <0.019 <0.021 <0.021 <0.019 <0.019 <0.02 2,4-D <0.017 <0.02 <0.019 <0.018 <0.017 <0.019 MCPP <0.018 <0.021 <0.02 <0.018 <0.018 <0.02 MCPA 0.018 <0.021 <0.022 0.046 0.048 0.046 dimethoat <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 desethylatrazin <0.015 <0.017 <0.016 0.018 0.043 0.018 methamidophos <0.019 <0.022 0.038 <0.02 0.078 <0.019 methidathion <0.019 <0.022 <0.02 <0.02 <0.021 <0.019 phorate <0.028 <0.031 <0.028 <0.029 <0.031 <0.027 phosphamidon <0.019 <0.021 <0.019 <0.02 <0.021 <0.018 tri-allate <0.036 <0.04 <0.037 <0.038 <0.04 <0.035 methabenzthiazuron <0.007 <0.008 <0.007 <0.008 <0.008 <0.007 bentazone <0.016 <0.018 <0.018 <0.016 0.200 0.029 metamitron <0.011 <0.012 <0.011 <0.011 <0.012 <0.01 nicosulfuron <0.004 <0.004 <0.004 <0.004 <0.004 <0.003 rimsulfuron <0.027 <0.031 <0.028 <0.029 <0.031 <0.027 tebuconazole <0.009 <0.01 <0.01 <0.01 0.011 <0.009 desisopropylatrazin <0.01 <0.011 <0.01 <0.01 0.013 <0.01 imazethapyr <0.006 <0.007 <0.006 <0.006 <0.007 <0.006 thifensulfuron-methyl <0.011 <0.012 <0.011 <0.011 <0.012 <0.01 thiophanate-methyl <0.014 <0.016 <0.015 <0.015 0.031 <0.014 ethofumesat <0.033 <0.036 <0.034 <0.034 <0.036 <0.032 azoxystrobin <0.01 <0.012 <0.011 <0.011 <0.012 <0.01 propyzamide <0.021 <0.024 <0.022 <0.022 <0.024 <0.021 fenhexamid <0.017 <0.019 <0.018 <0.018 <0.019 <0.017 fenarimol <0.014 <0.016 <0.015 <0.015 <0.016 <0.014 fipronil <0.044 <0.049 <0.045 <0.046 <0.049 <0.042 kresoxim-methyl <0.039 <0.044 <0.04 <0.041 <0.044 <0.038 propiconazole <0.013 <0.014 <0.013 <0.013 <0.014 <0.012 phosalone <0.019 <0.021 <0.019 <0.019 <0.021 <0.018 fluazifop-p-butyl <0.028 <0.032 <0.029 <0.029 <0.032 <0.027 pyridate <0.041 <0.046 <0.043 <0.043 <0.046 <0.04 desethyldesisopropylatrazin <0.012 <0.013 <0.012 0.014 0.070 0.012

2-hydroxyatrazin 0.014 0.013 0.013 0.022 <0.012 0.020 bromoxynil <0.021 <0.024 <0.023 <0.021 <0.021 <0.022 dichlorprop <0.018 <0.021 <0.02 <0.018 <0.018 <0.02

SULFONAMIDES sulfomethoxazol 0.011 0.024 <0.017 0.022 0.69 0.080

sulfapyridin <0.009 <0.011 <0.013 0.043 2.6 0.26 sulfamethazin <0.009 <0.011 <0.014 <0.011 <0.01 <0.011 sulfamethoxypyridazin <0.011 <0.012 <0.015 <0.012 <0.011 <0.012 sulfachloropyridazin <0.018 <0.021 <0.026 <0.02 <0.019 <0.021

OTHER ANTIBIOTICS trimetoprim <0.009 0.015 <0.013 0.013 0.14 0.027

metronidazol <0.015 <0.014 <0.015 <0.016 0.11 <0.015 cefalexin <0.016 <0.015 <0.016 <0.017 <0.015 <0.015 ofloxacin <0.017 <0.016 <0.017 <0.018 <0.016 <0.016 norfloxacin 0.071 0.082 0.069 0.063 0.14 0.045 ciprofloxacin <0.014 <0.013 <0.014 <0.014 0.047 <0.013 enrofloxacin <0.018 <0.016 <0.018 <0.018 <0.016 <0.017 erythromycin <0.002 0.005 <0.002 0.004 0.064 0.010 doxycyclin <0.017 <0.016 <0.017 <0.018 <0.015 <0.016

OTHER PHARMACEUTICALS diclofenac 0.010 0.039 <0.014 0.023 1.2 0.13

carbamazepine 0.028 0.064 0.039 0.067 0.95 0.15 diaverdin <0.01 <0.012 <0.015 <0.011 <0.01 <0.012

ALKYLPHENOLS t-octylphenol 0.013 n.a. 0.005 <0.001 0.014 0.004

n-octylphenol <0.004 n.a. <0.005 <0.007 <0.001 <0.006 4-n-nonylphenol <0.005 n.a. <0.007 <0.01 0.003 <0.009 p-nonylphenol 0.028 n.a. <0.012 0.023 0.22 0.032 monoNPE 0.003 n.a. <0.003 <0.004 0.036 0.006 diNPE <0.004 n.a. <0.005 <0.007 0.005 <0.006 Bisphenol A <0.006 n.a. <0.008 <0.011 0.003 <0.01

n.a. value not available

Table S4. Concentrations of studied pollutants in POCIS (ng/POCIS) exposed for 4 weeks at the study sites around Brno.


(duplicates) (duplicates)

PESTICIDES acetochlor 31.3 - - 792 439 265 130 180

alachlor 4.1 - - 19.4 <6.7 13.4 5.1 3.7 atrazin 21.4 - - 123 893 388 63.6 59.2 bromacil <2.9 - - <2.9 <29 <24 <2.7 <2.8 carbofuran <2.2 - - 2.1 <22 <18 <2 <2.1 cyanazin <2.5 - - <2.4 <25 <20 <2.3 <2.4 desmetryn <0.36 - - <0.36 <3.7 <3 <0.34 <0.35 diazinon <0.63 - - 3.1 56 27 3.6 3.5 dichlobenil <2.5 - - <2.4 <25 <20 <2.3 <2.4 diuron 10 - - 339 9212 4034 442 386 chlorbromuron <6.4 - - <6.3 <65 <53 <6 <6.1 chlorotoluron 48.5 - - 474 <34 <27 100 92.4 isoproturon 34.1 - - 45.3 272 95 27.9 31.1 linuron <4.1 - - 26.1 <41 <33 8.8 5.6 hexazinon 14.4 - - 14 15.5 <12 10 8.2 metalaxyl 1.5 - - 2.2 16.8 <7.7 2.3 1.2 metobromuron <2.7 - - <2.6 <27 <22 <2.5 <2.6 metolachlor 3.9 - - 51.2 <15 <12 8.1 10.2 metoxuron <0.88 - - <0.87 <9 <7.3 <0.83 <0.85 metribuzin 4.1 - - 7.5 <29 26.4 10.1 9.5 monolinuron <4.3 - - <4.2 <44 <35 <4 <4.1 prometryn 1.1 - - 4.3 31.1 15 2.9 3 simazin 1.5 - - 15.2 58.9 26 7.3 6.6 terbuthylazine 48.2 - - 811 2082 921 251 266 terbutryn 3.8 - - 21 324 150 20.8 20.5 2,4,5-T <1.6 - - 2.3 <17 <16 <1.6 <1.8 2,4-DP 1.1 - - 4.3 <9.1 <8.6 <0.84 1.8 2,4-D 11.3 - - 31.7 <41.4 <39 7.9 15 MCPP (mecoprop) 34 - - 20 69 28 13 20 MCPA 35.7 - - 97.9 139 113 24.7 46.3 dimethoat <0.79 - - <0.78 8.3 <6.5 <0.74 <0.76 desethylatrazin 18.6 - - 78.7 279 113 34.8 31.3 methamidophos 3.7 - - <3.4 <35 <29 <3.3 3.8 methidathion <2.9 - - <2.8 <29 <24 <2.7 <2.8 phorate <4.5 - - <4.5 <46 <37 <4.3 <4.4 phosphamidon <3.2 - - <3.2 <33 <27 <3.1 <3.1 tri-allate <6 - - <6 <62 <50 <5.7 <5.8 methabenzthiazuron <1.3 - - <1.3 <13 <11 <1.2 <1.3 bentazone 5.3 - - 5.7 69.7 92.9 10.1 11.8 metamitron <2 - - <1.9 <20 <16 <1.8 <1.9 nicosulfuron <0.57 - - 1.6 <5.8 <4.7 <0.54 0.56 rimsulfuron <3.4 - - <3.4 <35 <28 <3.2 <3.3 tebuconazole 4.1 - - 23 74.9 33.8 10.7 10.9 imazethapyr <1.2 - - 1.4 <12 <9.7 <1.1 <1.1 thifensulfuron-methyl <1.7 - - <1.6 <17 <14 <1.6 <1.6 thiophanate-methyl <3.4 - - 10.1 <17 <14 <3.2 <3.3 ethofumesat <7.1 - - <7.1 <73 <59 <6.7 <6.9 azoxystrobin <1.8 - - 3.2 37.2 15.8 2.4 2.3 propyzamide <3.6 - - <3.6 <37 <30 <3.4 <3.5 fenhexamid 4.1 - - <1.4 104 <12 <1.3 5.2 fenarimol <1.7 - - <1.7 <18 <14 <1.6 <1.7 fipronil <8.5 - - <8.4 <87 <70 <8 <8.2 kresoxim-methyl <3.9 - - <3.9 <40 <33 <3.7 <3.8 propiconazole 4.9 - - 28.2 139 51.4 13.8 <13.8 phosalone <2.9 - - <2.9 <30 <24 <2.8 <2.8 fluazifop-p-butyl <3.9 - - <3.9 <40 <32 <3.7 <3.8

pyridate <3.5 - - <3.4 <36 <29 <3.3 <3.4 clopyralid 174 - - 40.3 <297 <280 91.4 90.6 bromoxynil <3.8 - - <3.9 <40.8 <38.4 <3.8 <4.3

SULFONAMIDES sulfomethoxazol 30 - - 87 3514 1303 160 202

sulfapyridin 14 - - 71 2223 888 99 102 sulfamethazin <5.7 - - 6.7 46 <73 5.5 4.9 sulfamethoxypyridazin <5.9 - - <4.4 <48 <76 <5.3 <5 sulfachloropyridazin <5.5 - - <4.1 <44 <69 <4.9 <4.6

OTHER ANTIBIOTICS trimetoprim 12 - - 13 503 212 26 26

metronidazol <11 - - <1.4 18 113 1.8 1.9 cefalexin <15 - - <1.9 <15 <51 <1.8 <1.6 ofloxacin <11 - - <1.4 <11 89 <1.3 <1.1 norfloxacin 15 - - <1.3 13 <68 <1.2 <1.1 ciprofloxacin <11 - - <1.4 <11 <63 1.6 <1.1 enrofloxacin <10 - - <1.3 <10 <67 <1.2 <1.1 erythromycin 1.9 - - 0.9 32 87 1.4 1.5

OTHER PHARMACEUTICALS diclofenac 30 - - 148 11958 4185 358 388

carbamazepine 85 - - 249 6536 2412 356 341 diaverdin <5.4 - - <4.1 <44 <75 <4.8 <4.6

PFOCs PFHxS <1.1 - - <0.93 <7.7 <10 <0.89 <1.1

FHUEA <1.2 - - <1.1 <8.8 <10 <1 <1.3 FOSA <0.49 - - <0.42 <3.4 <4.5 <0.4 <0.49 N-methyl FOSA <0.53 - - <0.45 <3.7 <3.9 <0.43 <0.53 PFOA 1.7 - - 9.1 184 72 9.9 9.6 PFOS <1.3 - - 9.5 18 <13 3.9 3.2 PFNA <0.47 - - 5.7 6.7 <4.3 0.82 <0.48

- sample not available

Table S5. Concentrations of studied pollutants measured in SPMDs recalculated to the concentrations in water (pg/l) by use of performance reference compounds (PRC).


(duplicates) (duplicates)

Triclosan 127 - 182 659 37499 30512 3281 3467 Me-triclosan 210 - 257 446 16319 11664 1598 1961

PBDEs PBDE28 <2.5 - <1.9 <1.2 6.3 4.4 <1.8 1.6

PBDE47 10.2 - 12.9 14.3 130 90.3 20.0 22.2 PBDE100 4.6 - 6.0 7.0 53.0 33.6 9.1 9.8 PBDE99 <2.8 - <2.6 2.4 7.6 10.1 <2.2 <1.0 PBDE154 <2.5 - <2.4 <1.8 2.0 2.7 2.5 <0.9 PBDE153 <1.6 - <1.4 1.2 1.8 3.3 <1.3 <0.6 PBDE183 <4.0 - <4.2 <3.3 <1.6 <3.5 <3.5 <1.6

PCBs PCB 28+31 18.1 - 191 1179 731 490 353 406

PCB 52 28.8 - 46.1 251 334 200 120 126 PCB 101 86.8 - 33.3 202 428 305 126 139 PCB 118 130 - 12.0 47.3 115 63.1 31.3 36.3 PCB 138 136 - 29.1 115 266 166 88.7 107 PCB 153+168 158 - 55.1 259 531 342 182 219 PCB 170 16.5 - 7.3 21.6 30.0 22.9 19.9 25.0 PCB 180 23.5 - 16.2 51.1 73.8 56.3 48.1 55.6

HCHs alfa-HCH 24.3 - 17.0 14.3 29.7 29.4 13.1 14.9

beta-HCH <6 - 10.0 <7 24.9 7.9 <7 7.5 delta-HCH 161 - 130 131 598 602 163 168 gama-HCH <7 - <7 <7 <7 <8 <7 <7

HCB 103 - 153 189 409 299 144 140

DDT and metabolites o,p-DDE 2.3 - 6.3 8.1 12.2 9.3 6.9 6.6

p,p-DDE 106 - 157 146 192 145 147 155 o,p-DDD 24.7 - 69.5 74.2 81.7 63.4 70.3 73.5 p,p-DDD 77.1 - 192 190 134 93.5 191 209 o,p-DDT 22.6 - 58.3 88.6 66.2 38.1 54.6 54.0 p,p-DDT 27.3 - 50.9 208 70.9 53.9 94.2 112

PAHs phenantrene 4354 - 3219 8884 1523 1372 6231 5312

anthracene 239 - 333 1754 556 536 1032 959 fluoranthene 5911 - 10410 19328 13510 10549 12804 12407 pyrene 3045 - 7332 16559 14529 11072 9947 10138 benzo(a)anthracene 313 - 2839 8672 13205 9524 3445 4141 chrysene 48 - 851 2209 1629 1287 1272 1084 benzo(b)fluoranthene 151 - 531 953 788 600 620 611 benzo(k)fluoranthene 79 - 288 552 473 365 360 362 benzo(a)pyrene 72 - 263 517 740 539 389 391 benzo(ghi)perylene 80 - 170 296 293 213 279 276 dibenzo(ah)anthracene <20 - 27 37 47 38 37 32 indeno(1,2,3-cd)pyrene 67 - 134 244 174 139 179 181

- sample not available

Figure S1. Examples of the calibrations and dose-response curves of the samples for the effects studied by in vitro bioassays for POCIS (androgenicity and estrogenicity) and sediments (dioxin-like activity, cytotoxicity, antiestrogenicity and antiandrogenicity).

aquatic toxicology - university of saskatchewan · of zoology, and center for integrative...

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