Aquatic Toxicology 150 (2014) 83–92
Contents lists available at ScienceDirect
Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox
In situ effects of urban river pollution on the mudsnail Potamopyrgus
antipodarum as part of an integrated assessment
Radka Zounkova a , Veronika Jalova a , Martina Janisova a , Tomas Ocelka b , Jana Jurcikova b ,
Jarmila Halirova c , John P. Giesy d,e,f,g,h , Klara Hilscherova a,∗
a
Masaryk University, Faculty of Science, RECETOX, Kamenice 753/5, 62500 Brno, Czech Republic
Institute of Public Health, Partyzánské nám. 7, 70200 Ostrava, Czech Republic
c
Czech Hydrometeorological Institute, Kroftova 2578/43, 61600 Brno, Czech Republic
d
Department Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK S7N 5B4 Saskatchewan,
Canada
e
Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA
f
Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong Special
Administrative Region
g
School of Biological Sciences, University of Hong Kong, Hong Kong Special Administrative Region
h
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China
b
a r t i c l e
i n f o
Article history:
Received 13 November 2013
Received in revised form 7 February 2014
Accepted 27 February 2014
Available online 11 March 2014
Keywords:
Mortality
Reproduction
Passive sampling
Gastropoda
Sediment, In vitro
a b s t r a c t
The freshwater mudsnail (Potamopyrgus antipodarum) is sensitive to toxicity of both sediment and water
and also to the endocrine disrupting compounds (EDC) at environmentally relevant concentrations. This
study determined effects of in situ exposure of P. antipodarum as a part of a complex assessment of
the impact of a city metropolitan area with large waste water treatment plant (WWTP) for 0.5 million
population equivalents on two urban rivers. The study combined the in situ biotest with detailed chemical
analyses and a battery of in vitro bioassays of both sediment and water. Passive sampling of river water
was conducted during the course of exposure of the mudsnail. P. antipodarum was exposed for 8 weeks
in cages permeable to sediment and water at localities up- and down-stream of the city of Brno, Czech
Republic and downstream of the WWTP in two rivers. Greater mortality and significantly decreased
embryo 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 greater
mortality, while numbers of embryos produced by surviving individuals were comparable or slightly
greater 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 and
concentrations of metals. The results of this study confirm the applicability of this novel field biotest
with P. antipodarum for the evaluation of the effects of river pollution on metazoans, especially as suitable
in situ part of integrative contamination assessment.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Invertebrates are essential elements of aquatic ecosystems.
Among key species in aquatic ecosystems are insects, molluscs and
crustaceans. Sub-lethal effects of chemicals on different physiological processes, such as reproduction, growth, ecdysis, behavior,
or morphological changes have been observed in all these groups
∗ Corresponding author. Tel.: +420 549 493 256; fax: +420 549 492 840.
E-mail address: hilscherova@recetox.muni.cz (K. Hilscherova).
http://dx.doi.org/10.1016/j.aquatox.2014.02.021
0166-445X/© 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 affect
populations, and consequently survival of species and structures
or functions of ecosystems (Oehlmann et al., 2007; Oetken et al.,
2004). Therefore, surrogate and sentinel species are needed for the
assessment of the effect of contaminated water and sediment on
these organisms.
Some organisms in the class Gastropoda have been found to be
sensitive to toxicity associated with sediments and surface waters,
and specifically effects of (xeno-)hormones (Schulte-Oehlmann
et al., 2000; Tillmann et al., 2001). The freshwater mudsnail
84
R. Zounkova et al. / Aquatic Toxicology 150 (2014) 83–92
(Potamopyrgus antipodarum) is a cosmopolitan organism with
some advantages for use as an indicator organism, including
continuous fertility of parthenogenetic females, few maintenance
requirements and sensitivity to environmentally-relevant compounds that might affect reproduction (Duft et al., 2003a, b; Jobling
et al., 2004; Mazurová et al., 2008; Stange et al., 2012). Because this
mudsnail can live under various environmental conditions, which
is documented by its worldwide distribution, it is a suitable model
organism for use in biotests with water or sediment of different
physical and chemical characteristics.
Effects of waste water treatment plant (WWTP) effluents and
estrogenic compounds have been investigated using several species
of molluscs, including freshwater gastropods Viviparus viviparus
or Planorbarius corneus (Ramshorn snail) (Benstead et al., 2011).
The ramshorn snail exhibited significant concentration-dependent
increase in fecundity and in overall duration of the reproductive
cycle in adult snails exposed to WWTP effluent (Clarke et al., 2009).
P. antipodarum has been shown to be affected by chemicals
with endocrine disruptive potential such as bisphenol A, organotins, estradiols, alkylphenols, UV screens or fadrozole. Greater
reproduction of this mudsnail was observed after exposure to artificial sediment spiked with these chemicals (Duft et al., 2003a,
b; Gust et al., 2010b; Jobling et al., 2004; Oehlmann et al., 2000;
Schmitt et al., 2008). P. antipodarum was also successfully used as
a test organism in contact tests with whole sediment (Galluba and
Oehlmann, 2012; Mazurová et al., 2008; Schmitt et al., 2010a, 2011),
in laboratory tests with waste water (Jobling et al., 2003, 2004) or in
on-site flow-through tests with whole WWTP effluent (Magdeburg
et al., 2012; Stalter et al., 2010). The 28–56 day sediment contact
test with reproduction of P. antipodarum as the measurement endpoint was selected for standardization within the OECD framework
(Duft et al., 2007; Oehlmann et al., 2007).
While instrumental analyses of water or sediment provide information on presence and concentration of known target substances,
there can be also a pool of unknown substances contributing to
observed effects in these matrices. In addition, all effects of known
substances and especially their interactions are not known. For this
reason it is appropriate to use biotests and chemical analyses simultaneously, especially in cases of complex pollution. Both in vitro and
in vivo tests can be conducted in the laboratory with water and/or
sediment collected from the field. However, some characteristics
of the sampled materials representing the environmental matrices
can change after their removal from the environment. It is therefore
difficult to simulate the real environmental situation and varying conditions in laboratory exposure, especially for dynamic river
ecosystems. Thus, the most relevant way of exposure is direct field
exposure of model species (Burton and Nordstrom, 2004). There is
little information on suitable model species of molluscs to be used
for studies directly in field. Only two studies have been published
on in situ exposure of P. antipodarum, indicating its potential applicability in assessment of contamination in field (Gust et al., 2010a;
Schmitt et al., 2010b).
The overall objective of the research, results of which are presented here, was characterization of effects of a city with large
municipal WWTP on urban rivers pollution. A major focus of this
study was characterization of the influence of in situ exposure
to river sediments and water on survival and reproduction of P.
antipodarum and examination of the sensitivity of this species and
its suitability for direct exposure in urban rivers. Another important aim was to investigate the relationship between results of
the in situ contact biotest with data from chemical analyses and
in vitro biotests of water and sediment. In addition to collection of
grab samples also passive sampling of water was conducted to get
more representative estimate of time-weighted concentrations of
contaminants. In this study, two types of passive samplers were
used. These included Polar Organic Chemical Integrative Samplers
(POCIS), which sequester waterborne hydrophilic contaminants,
and semipermeable membrane devices (SPMD) for monitoring
waterborne hydrophobic pollutants. Detailed characterization of
contamination included in vitro biotests on cytotoxicity, dioxin-like
toxicity, (anti)estrogenicity and (anti)androgenicity, and chemical
analysis of several classes of pollutants, including hydrophobic organic pollutants, pharmaceuticals, pesticides, perfluorinated
organic compounds (PFOCs) and alkylphenols, some of which are
known as endocrine disrupting chemicals (EDCs) (Groshart and
Okkerman, 2000).
2. Materials and methods
2.1. Localities and sampling design
Selection of locations was based on a larger project concerned
with a long-term assessment of impact of the metropolitan region
of Brno (Czech Republic) on fluvial environment in two urban
rivers Svratka and Svitava (Grabic et al., 2010). Brno, with 404,000
inhabitants, is the second-largest city in the Czech Republic with
traditional sources of urban water pollution as sewage, industrial wastewater and surface runoff from construction sites and
urban roads. A large WWTP with a capacity of 513,000 population equivalent is located downstream of the city and is processing
waste waters from Brno and surrounding settlements. Waste water
is subjected to primary (mechanical) treatment followed by biological stage of activation with pre-denitrification and anaerobic
phosphorus removal (system of circulatory activation with change
of anaerobic, anoxic and aerated zones). Excess activated sludge
is then anaerobically stabilized (Brněnské vodárny a kanalizace,
2010; Ministry of the Environment of the Czech Republic, 2010).
Samples of sediments and grab and passive samples of water
were taken from six locations. The study locations were chosen to
examine the contamination in the rivers, its changes along the flow
of the rivers through and downstream of the city of Brno and effects
on biota. Two sampling locations (upstream and downstream the
city) were chosen at each river to observe the influence of the city,
and two sampling locations were located downstream the confluence of both rivers and the WWTP effluent discharge to observe the
impact of the WWTP. Thus, this field study included the following
locations (Fig. 1): Kníničky (location 1a) – Svratka upstream of the
city of Brno, downstream of the dam of Brno reservoir; Přízřenice 1
(location 1b) – Svratka downstream of Brno, upstream of the confluence with the Svitava River; Bílovice nad Svitavou (location 2a)
– a small town on the Svitava River upstream of Brno; Přízřenice 2
Fig. 1. Map of metropolitan region of Brno and sampling locations. 1a – Kníničky
– Svratka upstream of Brno; 1b – Přízřenice 1 – Svratka downstream of Brno, 2a –
Bílovice nad Svitavou – Svitava upstream of Brno; 2b – Přízřenice 2 – Svitava downstream of Brno; 3 – Modřice – Svratka downstream of confluence with Svitava,
downstream of WWTP; 4 – Rajhradice – 3 km downstream of the confluence of the
rivers Svratka and Svitava, downstream of the regional WWTP.
R. Zounkova et al. / Aquatic Toxicology 150 (2014) 83–92
(location 2b) – Svitava downstream of Brno, upstream of the confluence with Svratka River; Modřice (location 3) – Svratka downstream
of the confluence with the Svitava River, downstream of the WWTP
effluent discharge; Rajhradice (location 4) – a small town 3 km
downstream of the confluence of the Svratka and Svitava Rivers,
downstream of the WWTP effluent discharge. Only sediment was
sampled at location 3. Additionally, samples of effluent water were
taken and passive samplers were installed into the effluent of the
WWTP.
Passive samplers were exposed from the beginning of May
2008 until the beginning of June 2008 (4 weeks). Standard sampling arrangement described in Grabic et al. (2010) and Jalova
et al. (2013) with a combination of POCIS (Polar Organic Chemical Integrative Sampler) and several SPMDs (Semipermeable
Membrane Device) was used. SPMD and POCIS were obtained
from Exposmeter AB, Tavelsjo, Sweden. One POCIS was used
for both chemical analysis and bioassay testing. Two SPMDs
were used in duplicates for chemical analysis, one SPMD was
used for toxicity assessment. SPMDs for chemical analysis contained performance reference compounds (PRC) used as onsite
SPMDs calibration. Four deuterated PAHs ([2 H10 ]acenaphthene,
[2 H10 ]fluorene, [2 H10 ]phenanthrene, and [2 H12 ]chrysene) and four
13 C -labeled PCBs (PCB 3, 8, 37, and 54) were used as PRCs. Passive
12
samplers were placed in special racks in protective shroud places.
Samplers were installed and deployed in 0.5–1 m water depth and
exposed for a 4-week period. Temperature was recorded in detail
(every hour) during the 4-week deployment of passive samplers
by temperature loggers placed on the sampling racks. The temperatures and the overall ranges of their fluctuations (day–night,
min–max) were very similar across studied river sites (see Table S1
in Supplementary Materials). Composite bottom sediment samples
and grab samples of water were collected on May 12, 2008. Samples
of sediment were taken from the upper layer of fresh sediments
by use of a method designed by the Czech Hydrometeorological
Institute, which was developed in accordance with ISO 5667-12
standard (ISO, 1995). Representative composite sediment samples
were prepared by thorough mixing and homogenization of surface sediments collected from several spots (six to eight individual
grabs) within each sampling locality (10 m2 area). Samples of water
were taken by use of a telescopic sampling device. Passive sampling
was conducted according to validated protocols and general rules
for passive sampling (Huckins et al., 1993, 1999) and EN ISO/IEC
17025 standard (ISO, 2005). All samples were refrigerated after collection (4 ◦ C) and transported to laboratory, where they were stored
at −18 ◦ C until the analysis, which was started within one month
of the sampling.
2.2. Identification and quantification of residues
Samples of water, sediment, and organic extracts of SPMD and
POCIS samplers were analyzed for wide range of organic compounds. Extraction and cleanup of passive samplers, as well as
the methodology for chemical analysis of studied compounds,
have been described previously (Grabic et al., 2010; Jalova et al.,
2013). Briefly, SPMDs were dialyzed with hexane, POCIS eluted
with methanol:toluene:dichloromethane (1:1:8, v/v/v; Jalova et al.,
2013). Sediment samples were homogenized, lyophilized and
sieved prior to analysis. Metals (Al, As, Ba, Cd, Co, Cr, Cu, Mo,
Ni, Pb, Se, Ti, Zn) were extracted from sediment (fraction < 2 mm)
by nitric acid and measured by ICP-MS method (Elan 6100 with
autosampler AS-90; Perkin-Elmer Sciex, USA). Determination of
the total mercury content in sediments was performed by means
of atomic absorption spectrophotometric (AAS) method using a
single-purpose cold vapor Advanced Mercury Analyzer AMA-254
(ALTEC Ltd., Czech Republic). Organic pollutants were extracted
from sediments (fraction < 1 mm) by microwave extraction with
85
hexane:acetone mixture and cleaned on a silica gel column. A
portion of each organic extract of POCIS, SPMD and sediments was
transferred into DMSO for testing in bioassays.
After removal of particulate matter and addition of internal
standards water samples were directly injected on analytical HPLC
column Phenomenex Aqua 5 ␮ C18 125 A (50 mm × 2 mm), where
individual analytes were separated and further detected by MS/MS
system. All methods were validated in accordance with EN ISO/IEC
17025 standard (ISO, 2005).
Extracts of SPMD and sediments were analyzed for polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls
(PCBs), organochlorine pesticides, hexachlorobenzene (HCB),
␣-, ␤-, ␥-stereoisomers of hexachlorocyclohexane (HCH),
dichlorodiphenyltrichloroethane (DDT) and its degradation products dichlorodiphenyldichloroethylene (DDE) and
dichlorodiphenyldichloroethane (DDD), triclosan and its environmental transformation product methyl triclosan (Me-triclosan)
and polybrominated diphenyl ethers (PBDEs), expressed as the
sum of congeners. Eluates from POCIS were analyzed for polar pesticides, pharmaceuticals and perfluorinated organic compounds
(PFOCs). Samples of water were analyzed for polar pesticides,
pharmaceuticals and alkylphenols. A complete list of individual
pollutants analyzed is attached in footnotes to Table 1b. Concentrations of HCB, HCHs, PCBs, PBDEs, DDT and its degradation products
as well as triclosan and Me-triclosan after derivatization were
determined by GC/MS–MS using isotope dilution. GC/MS was used
for quantification of PAHs. PAHs with more rings were analyzed
by HPLC using a FLD detector with deuterated internal standards.
Polar pesticides, pharmaceuticals, PFOCs and alkylphenols were
measured by standard method direct injection HPLC/MS–MS.
Set of carbon 13 C12 -labeled internal standards were included
in the analyses as described in Jalova et al. (2013). The native
standards were purchased from Dr. Ehrenstorfer, AccuStandards,
and Absolute Standards via Labicom.
2.3. In vitro bioassays
Four transactivation reporter gene bioassays were used to measure receptor-mediated potencies of organic extracts of sediments
and passive samplers by procedures described in Jalova et al.
(2013). AhR-mediated (dioxin-like) potency was determined by
use of the H4IIE-luc bioassay, a rat hepatoma cell line, which contains a luciferase reporter gene under control of dioxin-responsive
enhancers (DRE) (Sanderson et al., 1996; Hilscherova et al., 2001;
Villeneuve et al., 2002). Estrogen receptor (ER)-mediated potency
was evaluated by use of the MVLN bioassay, a human breast carcinoma cell line which has been transfected with a luciferase gene
under 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-kb2
cells, a human breast carcinoma cell line stably transfected with
luciferase reporter gene under control of functional endogenous
androgen receptor (AR) and glucocorticoid receptor (GR) (Wilson
et al., 2002).
Cells were cultured in dark in incubator at 37 ◦ C and
assays conducted on 96-well microplates. Approximately 24 h
after plating, cells were exposed to samples, calibration reference or solvent control. Standard calibration was performed
with 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 MVLN
and dihydrotestosterone (DHT; Sigma–Aldrich, Czech Republic;
1 pM–10 ␮M) for MDA-kb2. Effects of extracts on MVLN and MDAkb2 cells were assessed either singly or in combination with
competing endogenous ligand. Antiestrogenicity was determined
by simultaneous exposure of sample extract and 17␤-estradiol
86
R. Zounkova et al. / Aquatic Toxicology 150 (2014) 83–92
Table 1a
Concentration of pollutants in sediment (per g dry mass of sediment) from the six study sites around Brno.
Sediment
1a
2a
1b
2b
3
4
Triclosan
Me-triclosan
Sum of PBDEs
Sum of PCBs
Sum of HCHs
HCB
p,p -DDT
Sum of DDT and metabolites
ng/g
0.6
0.1
0.07–1.15
5.9
1.0–1.9
0.85
0.38
1.47–1.86
ng/g
2.4
0.73
0–3
137
0–1.85
1.1
1.3
5.2–6.0
ng/g
13
2.7
1.06–2.84
123
0–0.98
2.1
680
757
ng/g
5.5
1.4
0.48–1.84
20
0–0.96
0.74
5.7
12.0–12.2
ng/g
34
10
1.08–3.45
38.3
0–1.68
2.0
5.4
20.5–20.8
ng/g
57
7.2
1.75–3.67
59.5
0–0.79
1.7
5.4
29.7
Sum of PAHs
Al
As
Ba
Cd
Co
Cr
Cu
Hg
Mo
Ni
Pb
Se
Ti
Zn
␮g/g
2.3
1630
0.35
43
0.12
4.28
5.72
5.86
0.01
0.015
7.84
16.1
0.37
20.4
44.8
␮g/g
15.2
3910
1.47
100
1.72
5.35
25.7
23.5
0.12
0.037
15.6
36.6
0.49
31.8
178
␮g/g
14.6
6070
1.82
179
2.51
8.03
30.1
79.6
0.89
0.089
24.4
68.9
0.56
50.6
329
␮g/g
19.2
3530
1.44
96
0.46
3.09
16.1
24.5
0.68
0.051
11.7
22.9
0.49
32.3
155
␮g/g
12.3
5030
1.89
124
1.32
5.49
24.1
51.7
0.96
0.087
15.5
50.6
0.54
39.6
214
␮g/g
26
5490
0.91
130
1.12
6.19
25.8
59.2
0.96
0.098
18.3
48.7
0.63
47.7
259
Ranges: the sum of detected compounds – the sum of detected compounds plus limits of detection of non-detected compounds.
Table 1b
Concentrations of pollutants in water and passive samplers from study sites and WWTP effluent.
1a
2a
1b
2b
WWTP effluent
4
Water
Sum of pesticides
Sum of sulfonamides
Sum of other antibiotics
Sum of other pharmaceuticals
Sum of alkylphenols + BPA
ng/l
30–1000
11–58
71–179
38–48
44–63
ng/l
885–1909
24–79
102–192
103–115
n.a.
ng/l
194–1151
0–85
69–181
39–68
5–45
ng/l
1050–1949
65–108
80–181
90–101
23–63
ng/l
961–1889
3290–3330
501–563
2150–2160
278–279
ng/l
534–1365
340–384
82–174
280–292
42–73
POCIS
Sum of pesticides
Sum of sulfonamides
Sum of other antibiotics
Sum of other pharmaceuticals
Sum of PFOCs
ng/POCIS
523–627
44–61
29–87
115–120
2–7
–
–
–
–
–
–
–
–
–
–
ng/POCIS
3100–3190
165–173
14–23
397–401
24–27
ng/POCIS
10,370–11,720
3990–4140
534–682
12,550–12,610
140–175
ng/POCIS
1310–1420
287–297
30–36
722–726
14–17
SPMD
Triclosan
Me-triclosan
Sum of PBDEs
Sum of PCBs
Sum of HCHs
HCB
p,p -DDT
Sum of DDT and metabolites
pg/l
127
210
14.8–28.2
597
186–199
103
27.3
260
–
–
–
–
–
–
–
–
pg/l
182
257
18.9–31.4
390
157–164
153
50.9
534
pg/l
659
446
24.9–31.2
2127
145–159
189
208
715
pg/l
34,005
13,991
173–175
2077
646–654
354
62.4
480
pg/l
3374
1779
32.6–39.1
1041
183–193
142
103
587
ng/l
14.4
–
ng/l
26.4
ng/l
60.0
ng/l
41.9
ng/l
36.2
Sum of PAHs
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 of
PAHs: 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, propiconazole, 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, sulfachloropyridazin, 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.
R. Zounkova et al. / Aquatic Toxicology 150 (2014) 83–92
(33 pM) and antiandrogenicity was tested in combination with
dihydrotestosterone (1 nM) – given concentrations are near their
EC50 value. Several dilutions of extracts were tested in triplicate to
provide a concentration–response curve for each sample. After 24 h
of exposure, medium was removed, and cells were washed with
phosphate-buffered saline (PBS) and lysed. Intensity of luciferase
luminescence corresponding to the respective receptor activation
was measured by use of Promega Steady Glo Kit (Promega, USA)
in case of assays with H4IIE-luc and MVLN cells and with prepared
luciferase reagent (Wilson et al., 2002) in MDA-kb2 assay. Noncytotoxic sample concentrations to be used in bioassays with cell lines
were determined by use of the neutral red uptake assay (Freyberger
and Schmuck, 2005). At the end of the incubation period, neutral
red solution (0.5 mg/ml of media) was added and cells incubated for
1 h at 37 ◦ C. Medium was removed, cells washed with PBS and lysed
with 1% acetic acid in 50% ethanol. Absorbance was measured in a
microplate spectrophotometer at 570 nm. Yeast strain of recombinant Saccharomyces cerevisiae constitutively expressing luciferase
was used for detailed cytotoxicity assessment (Leskinen et al., 2005;
Michelini et al., 2005).
Statistical evaluation of in vitro bioassays was performed by
nonlinear logarithmic regression of concentration–response curves
(Graph Pad Prism, GraphPad® Software, San Diego, CA, USA). Relative potencies expressed as TCDD equivalents/E2 equivalents/DHT
equivalents were calculated by relating the EC50 value of standard
calibration with the concentration of the tested sample inducing
the same response (Villeneuve et al., 2000). Cytotoxicity, antiestrogenicity and antiandrogenicity corresponded to the decrease in
detected luminescence/absorbance signal given by solvent control
in case of cytotoxicity and specified amount of competing standard
ligand for the other effects. The IC50 values for antiestrogenicity and
antiandrogenicity were calculated from concentration–response
curves expressed in percentage of signal of competitive concentration of added natural ligand (33 pM E2, 1 nM DHT). For better clarity
of the trends the values are expressed as an index of antiestrogenicity (AE) or antiandrogenicity (AA), which corresponds to reciprocal
value of IC50 . Similarly, the index of cytotoxicity was derived as the
reciprocal value of IC50 (or IC20 values in case the 50% response was
not reached) for the cytotoxic response.
Concentrations of analyzed compounds as well as the biological potencies determined in bioassays for SPMD extracts
were recalculated to the concentrations in water to take into
account the differences in sampling rates among SPMDs from different locations. Performance reference compounds (PRC) were
used for in situ calibration of sampling rates. Details of the
calculation are described in Jalova et al. (2013). Results of
POCIS samples were compared on the basis of concentrations
and toxic equivalents in sampler extracts (ng/POCIS). No correction of POCIS sampling rates was made because the water
flow in sampling localities did not vary significantly. Also,
only minor influence of water flow rate on the accumulation of pollutants into POCIS has been demonstrated (Li et al.,
2010).
2.4. In vivo biotest
P. antipodarum (Gray 1843) (Mollusca, Gastropoda, Caenogastropoda) is a parthenogenetic and ovoviviparous freshwater
mudsnail, which is indigenous to New Zealand, but is currently
widely distributed in aquatic environments around the world.
These snails inhabit upper layers of aquatic sediments and feed on
plants and detritus. A population from clean reference unpolluted
location in sand-pond Stratov (district Nymburk, Czech Republic)
was used in the test. The specimens were collected one month
before the test. Adult individuals 3.6–3.9 mm length (Duft et al.,
2003a) were used for the in situ test.
87
The field contact biotest with P. antipodarum was conducted at
the 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 one
meter from the river bank and fixed ashore. Six cages were placed
at every locality one week before start of the test. About 30 g of
sediment was added into each cage and they were kept on river
bottom to microfilm overgrow. Eight adult mudsnails were placed
into each cage one week later. Thus, a total of 48 mudsnails were
exposed at each location. Microfilm and sediment particles served
as food and exogenous food was not provided. Cages were covered with nylon net and exposed in the river environment for
eight weeks. After the exposure, the cages were transported to
the laboratory, adult mudsnails were euthanized in 10% MgCl2
solution, dissected and reproduction success and mortality was
evaluated. Reproduction was evaluated by counting the number
of embryos in the brood pouch of 20 randomly selected maternal snails per site or of all surviving adults in case they were less
than 20.
Normality was checked by the Kolmogorov–Smirnov test and
homogeneity of variance was confirmed by use of Levene’s test.
The statistical significance of differences between groups was
evaluated using non-parametric Mann–Whitney U test. Calculations 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 2008
both by chemical analysis and in vitro bioassays revealed Svratka
upstream of the city (1a) as generally the least polluted location
(Tables 1a, b and 2). A detailed report of concentrations of individual compounds is included in Supplementary Materials (Tables
S2–S5). Unfortunately, POCIS samplers from locations 2a and 1b
and SPMD sampler from 2a were damaged or stolen during the
exposure period, so it was not possible to conduct all comparisons.
Sediments contained greater concentrations of several groups of
non-polar pollutants (PCBs, DDTs, Me/triclosan, PAHs) and metals
at location 1b compared to 1a, demonstrating impact of the sources
in the city on contamination of the River Svratka. The analysis
of SPMDs also confirmed greater concentrations of most of these
pollutants directly downstream of the city. The Svitava River was
more contaminated upstream of the city (location 2a), such that the
effect of sources in the city was not obvious. Results from water
grab sample and POCIS also document greater concentrations of
pesticides and pharmaceuticals in the Svitava than in the Svratka
River. Concentrations of triclosan and its metabolite, sulfonamides
and some other pharmaceuticals were greater downstream of the
WWTP. Also, concentrations of PAHs in sediments were greatest
in 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 concentrations in sediments from site 1b directly downstream of the
Brno city and from the two most downstream sites under WWTP
(3, 4, Table 1a). The differences were most pronounced for Cu, Pb
and Zn.
Results of the in vitro bioassays documented the presence of
androgenic and estrogenic compounds in the polar fraction of
WWTP effluent. Estrogenicity was also detected in POCIS from the
Svitava River downstream of Brno (2b) and at the most downstream
location (4; Table 2). At the same time, the greatest antiandrogenic potency was observed in sediments from these two locations.
88
R. Zounkova et al. / Aquatic Toxicology 150 (2014) 83–92
Table 2
Results of in vitro assessment of extracts of samples from localities where cages with mudsnails were placed.
1a
2a
1b
2b
3
4
Sediment
Dioxin-like toxicity [ng TCDD eq./g sed.]
Estrogenicity [ng E2 eq./g sed.]
Androgenicity [ng DHT eq./g sed.]
Index of cytotoxicitya
Index of antiestrogenicitya
Intex of antiandrogenicitya
9.08
n.d.
n.d.
22.4
1301
762
13.8
n.d.
n.d.
99.1
2649
655
11.7
n.d.
n.d.
166
1473
246
8.15
n.d.
n.d.
66.8
325
1332
10.5
n.d.
n.d.
160
2270
722
19.4
n.d.
n.d.
290
1353
1602
POCIS
Dioxin-like toxicity [ng TCDD eq./POCIS]
Estrogenicity [ng E2 eq./POCIS]
Androgenicity [ng DHT eq./POCIS]
Index of cytotoxicitya
Index of antiestrogenicitya
Index of antiandrogenicitya
n.d.
n.d.
n.d.
146
220
270
–
–
–
–
–
–
–
–
–
–
–
–
n.d.
0.47
n.d.
323
620
324
WWTP effluent
1.77
2.77
27.7
1238
727
n.d.
n.d.
0.56
n.d.
156
423
375
SPMD
Dioxin-like toxicity [pg TCDD eq./l]
Estrogenicity [ng E2 eq./l]
Androgenicity [ng DHT eq./l]
Index of cytotoxicitya
Index of antiestrogenicitya
Index of antiandrogenicitya
n.d.
n.d.
n.d.
2.58
3.21
n.d.
–
–
–
–
–
–
23.0
n.d.
n.d.
13.1
14.3
8.02
10.6
n.d.
n.d.
1.46
0.74
6.51
7.80
n.d.
n.d.
11.6
5.83
6.17
7.58
n.d.
n.d.
1.31
2.99
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.
a
Index of cytotoxicity, antiestrogenicity, antiadrogenicity = reciprocal value of IC50 (IC20 in case of cytotoxicity of SPMD extract). Units are [1/(g/ml)] for sediment extracts,
[1/(POCIS/ml)] for POCIS extracts or [1/(l/ml)] for SPMD extracts, respectively.
Unfortunately, the passive samples from location 2a in this sampling (spring 2008) were lost, but POCIS exposed at this location in
the same period in 2007 elicited estrogenicity (0.18 ng E2/POCIS). In
general, extracts of sediments and SPMDs exhibited antiestrogenic
and antiandrogenic potencies. Dioxin-like activity was detected in
hydrophobic fraction of water (SPMD) downstream of Brno and in
all sediments, but only in WWTP effluent in the case of POCIS. Alternatively, in vitro cytotoxicity was detected in all types of samples
from all locations. In sediments, it was greatest downstream of the
city on the Svratka River and at the localities downstream of the
WWTP. Both dioxin-like toxicity and cytotoxicity of sediments and
SPMD from the Svratka River were greater directly downstream of
the city (location 1b) compared to upstream (1a).
The in situ contact test with P. antipodarum after eight weeks
exposure to river sediment and water led to various magnitudes of
mortality of adult mudsnails and number of embryos at the study
locations (Fig. 2). Mortality was proportional to the general magnitudes of pollution at locations. The least mortality was observed
at the most upstream location on the River Svratka (1a), where
the least contamination was determined by chemical analysis and
toxicity in vitro biotests. Alternatively, there was about three-fold
greater mortality at the location on the Svratka River directly downstream of Brno (1b), which exhibited greater contamination of
sediments (Tables 1a and b). The greatest mortalities were observed
at locations 3 and 4 downstream of the city and of the WWTP, where
greater concentrations of triclosan and Me-triclosan, polycyclic
aromatic hydrocarbons, sulfonamides and other pharmaceuticals
were detected.
Reproduction of mudsnails varied among locations (Fig. 3).
There were, on average, approximately two-times lesser numbers
of embryos in brood pouches of adults exposed at locations directly
downstream of Brno (1b, 2b) compared to the locations upstream of
Brno on the same rivers (1a, 2a). In the case of the Svitava River, this
difference was statistically significant. The number of embryos was
greatest in mudsnails held in the Svitava River upstream of Brno,
and was almost twice as great as for mudsnails held in the Svratka
River upstream of Brno, which is considered to be the least polluted location. Numbers of embryos of surviving mudsnails from
localities downstream of WWTP (3, 4), where the greatest mortality was observed, were, on average, slightly greater than those
number of embryos
20
*
*
*
*
*
15
10
5
0
1a
Fig. 2. Mortality of adults of P. antipodarum after 8 weeks in situ exposure. The insert
shows the location of sampling sites on the rivers. 48 individuals were exposed at
each site (six cages of eight adult mudsnails).
2a
1b
2b
3
4
Fig. 3. Average number of embryos in the brood pouch of adults of P. antipodarum
after 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.
R. Zounkova et al. / Aquatic Toxicology 150 (2014) 83–92
at the least polluted location 1a (by 40 and 20%, respectively).
However, this difference was not statistically significant, similarly
to the comparison with the most downstream locations on the
Svitava River before the confluence (2b). Alternatively, numbers
of embryos in mudsnails at locations 3 and 4 were significantly
greater (more than twice as great) when compared to mudsnails exposed in the Svratka River, directly downstream of Brno
(1b, Fig. 3).
4. Discussion
Effects of chemical pollution, especially of compounds classified
as endocrine disruptors, were documented in previous studies with
P. antipodarum. Also correlations between effects of some known
estrogenic chemicals (17␣-ethinylestradiol, EE2, bisphenol-A, and
4-tert octylphenol) and of mixture of pollutants in WWTP effluent
water on reproduction of P. antipodarum and estrogenic effect on
fish, such as greater production of vitellogenin, have been observed
(Jobling et al., 2003). Effects on reproduction of P. antipodarum
after laboratory exposure to sediments containing compounds
with dioxin-like, estrogenic and anti-androgenic activity were
described also in our previous study with sediment from Lake
Pilnok, which has been used as a dumping site for powdered waste
coal (Mazurová et al., 2008). However, studies conducted in the
laboratory cannot accurately simulate natural conditions and their
changes to which organisms are exposed in field, as well as the
complex mixture of compounds contained in WWTP effluents and
in surface waters.
Results of the present study revealed effects on reproduction
and mortality of mudsnails exposed for 8 weeks at different localities in the Brno metropolitan area. Two studies testing in situ exposure of P. antipodarum have been published previously, but both of
them used only 4 week exposure (Gust et al., 2010a; Schmitt et al.,
2010b). During this shorter duration of exposure those authors
observed as much as 25% mortality, which corresponds to the more
upstream locations in the study, the results of which are presented
here. The greater mortality at downstream locations observed in
this study can be affected not only by the longer duration of exposure, but also by the magnitude and composition of pollution in the
studied rivers. Only one of the two previous in situ studies presented
data on concentrations of contaminants (Schmitt et al., 2010b),
and the comparison to data presented here shows that there were
greater concentrations of PAHs in our study, namely at the downstream locations. In this study exposure was characterized by use
of both chemical analysis and in vitro bioassays of sediment and
water. Since the results from grab water samples represent only
the instantaneous conditions, a four week long passive sampling
of water for both hydrophobic and hydrophilic pollutants was conducted during the course of exposure of the mudsnails in river to
get representative estimate of longer-term exposure. A battery of
in vitro tests was employed to provide complementary information
to quantification of individual residues, which can only account
for the known compounds and do not take into consideration the
possible interactions within mixture. Quantification of individual
residues showed that concentrations of some pollutants in the
Svratka River increased during its course through Brno. The fact that
greater concentrations of DDT, PCBs, metals, triclosan, and PAHs,
were found in sediments from the Svratka River directly downstream of Brno than in sediment from upstream location, suggests
that there are sources of these pollutants in the city such as urban
runoff. Concentrations of some pollutants, including triclosan, Metriclosan and some pharmaceuticals increased downstream of the
WWTP, which indicates that the WWTP despite its effectiveness and up-to-date methods of treatment could still contribute
contaminants to the river. Similar observations were reported
89
during a recent study conducted in the same area in 2007, which
also documented efficient treatment of the WWTP for cytotoxic
compounds, xenoestrogens and xenoandrogens (Jalova et al.,
2013).
Results of two previous studies showed different trends in
numbers of embryos in P. antipodarum after in situ exposure. Significantly more embryos in the brood pouch at more polluted
locations were observed by Schmitt et al. (2010a,b), whereas Gust
et al. (2010a) reported fewer embryos downstream of WWTPs.
These contradictory results can be explained by different composition and concentrations of mix of compounds contained in
water and sediments (e.g. estrogenic compounds may cause inhibition of reproduction at high concentrations (Jobling et al., 2003)).
The differences are explained by the results of another study by
Schmitt et al. (2011), which tried to identify compounds responsible for these effects. Effect-directed analysis showed that two
out of six fractions stimulated reproduction of P. potamopyrgus,
while two other fractions inhibited reproduction. Fractions which
stimulated reproduction also exhibited greater estrogenic potency
in the ER-LUC assay using reporter cell line BG-1. Results of the
study demonstrate that some WWTP effluents and thus surface
waters can contain compounds both stimulating and inhibiting
reproduction. The resulting effect might depend on quantity and
ratio of these compounds. These results correspond well with those
of this study, where lesser numbers of embryos were observed
in snails from both rivers directly downstream of Brno compared
to upstream locations. In the case of the River Svratka there was
more than two-fold greater mortality and two-fold lesser numbers of embryos at the location directly downstream of Brno (1b),
which corresponds with the greatest magnitude of pollution by
PCBs, DDTs and metals. This observation also corresponds with
the greater in vitro cytotoxic potency of extracts from both sediments and SPMDs from this location (Table 2). In the case of
the Svitava River, the situation was different, since the pollution
of this river is already greater upstream of Brno. Relatively great
pollution with PCBs, PAHs, polar pesticides, some pharmaceuticals and metals was observed upstream of Brno on the Svitava
River. That pollution could be linked to recently increasing habitation density due to moving from the center of the city to suburbs
upstream of location 2a. There is a small WWTP for this area
with insufficient capacity for the new settlements which could
contribute to the river pollution. Some of these pollutants could
be related to greater numbers of embryos in mudsnails at location 2a. There was no strong influence of the city sources on the
pollution in this river. Pollution of the Svitava River was mostly
comparable or lower at the location downstream of Brno and mortality and number of embryos of P. antipodarum at this location
were comparable to those observed at the least polluted location
(1a).
Greatest mortalities were observed on locations 3 and 4
downstream of Brno, the confluence of the two rivers and of
the spot where WWTP effluent enters the river. Mortality was
probably affected by cytotoxic compounds quantified by the
in vitro assay and triclosan and Me-triclosan in sediments at both
these locations. Moreover, the greatest concentrations of sulfonamides and other pharmaceuticals in water, and of polycyclic
aromatic hydrocarbons and dioxin-like potency in sediments
were found at the most downstream location 4. Despite greater
mortality, number of embryos of surviving mudsnails was greater
than in the Svratka River directly downstream of Brno (1b), but
not significantly different (20–40% greater) than at the cleanest
location. Individuals at these locations (3, 4) are exposed to multiple stressors, including cytotoxic compounds at the same time
with hormonally active compounds. This is demonstrated by the
estrogenic and androgenic potency in POCIS from WWTP effluent
and estrogenic activity also at location 4, but also by ubiquitous
90
R. Zounkova et al. / Aquatic Toxicology 150 (2014) 83–92
80
mortality (%)
600
mortality
hazardous
elements
400
60
40
200
sum of hazardous
elements (mg/kg)
A 100
was also observed between mortality of exposed snails and cytotoxicity of organic extracts of sediments detected in vitro (Fig. 4B),
which indicates relation to other chemicals than metals. Mudsnails are exposed to the whole mixture of both analyzed and
unknown pollutants and it is not surprising that the total cytotoxicity corresponds better to the mortality than any individual group
of compounds. These two correlations indicate that both inorganic
and organic pollutants affect their survival.
20
5. Conclusions
0
0
1a
2a
1b
2b
3
4
B 100
400
mortality (%)
80
cytotoxicity
300
60
200
40
100
20
0
index of cytotoxicity
mortality
0
1a
2a
1b
2b
3
4
Fig. 4. Mortality of adults of P. antipodarum after 8 weeks in situ exposure and sum of
concentrations of metals classified as hazardous elements (As, Cd, Co, Cr, Cu, Hg, Ni,
Pb, Zn; MoA, 2009) in sediments (A), or index of cytotoxicity of extracts of sediments
from study sites, respectively (B). Number of specimen as in Fig. 2.
antiestrogenic and antiandrogenic potencies. The relatively good
reproduction at these locations where greater mortality was
observed could be affected by the presence of endocrine disruptive
compounds.
The hormonal system of molluscs is insufficiently known. The
estrogen receptor (ER) has been identified in P. antipodarum (Stange
et al., 2012) and some hormones were detected in molluscs
(Lafont and Mathieu, 2007), but there is a lack of information
about their function. There is little information about effects
of non-endocrine disruptive compounds on reproduction of P.
antipodarum. It has been shown that nitrates, as well as fluorides and copper (Cu) nanoparticles reduce reproduction of P.
antipodarum (Alonso and Camargo, 2011, 2013; Pang et al., 2012).
Changes of reproduction and mortality of the freshwater mudsnail
P. antipodarum can thus be the consequence of effects on different magnitudes of general stress, endocrine disruption through
receptors, changes of metabolism of hormones or enzymes and
others.
Sediment and water samples from localities downstream of
Brno and the regional WWTP contained relatively high concentrations of organic pollutants and metals known for their negative
effects on biota. Some of these compounds, such as organochlorine
pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, pharmaceuticals, belong to the group of EDCs (Depledge
and Billinghurst, 1999; Groshart and Okkerman, 2000). However,
no correlation between concentrations of any single chemical or
group of chemicals and numbers of embryos in mudsnails was
observed. Alternatively, mortality was generally in a good accord
with concentrations of hazardous metals (Fig. 4A). Negative effects
of metals on survival of pulmonate snails have been documented in
previous studies (Gupta et al., 1981; Laskowski and Hopkin, 1996;
Allah et al., 1997). However, greater mortality at localities 3 and
4 might be caused also by contribution of other pollutants, which
have not been analyzed in the sediments. Good correspondence
Results of the present study proved the suitability of freshwater
mudsnail P. antipodarum as a model organism for in situ assessment
of effects of urban rivers contamination on biota. It demonstrated
effects of various sources of pollution in the studied area. The in situ
assays with P. antipodarum document the presence of toxic compounds in the complex contaminant mixture in sediments as well
as effects on reproduction in mudsnails. This is the first study that
brings together this in situ test with simultaneous passive sampling for the determination of time-weighted exposure and detailed
characterization of exposure through both chemical analysis and
in vitro bioassays of both sediment and water. A battery of in vitro
tests provided complementary information to chemical analysis
taking into account also unanalyzed compounds and interactions
within mixture. This approach enabled to indicate groups of compounds contributing to the observed effects.
Chemical pollution resulting from runoff and waste waters from
the Brno metropolitan area had negative effect on survival of the
freshwater mudsnail. Greater mortality was observed to be consistent with concentrations of metals and in vitro cytotoxicity.
Number of embryos was also affected by pollution from the Brno
metropolitan area as well as by suburb sources with a small WWTP
of insufficient capacity. The early development of embryos in the
brood pouch reflects effects of those toxicants that immediately
affect 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 Chair
program, a Visiting Distinguished Professorship in the Department of Biology and Chemistry and State Key Laboratory in Marine
Pollution, City University of Hong Kong, the 2012 “High Level Foreign Experts” (#GDW20123200120) program, funded by the State
Administration of Foreign Experts Affairs, the P.R. China to Nanjing University and the Einstein Professor Program of the Chinese
Academy of Sciences.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.aquatox.
2014.02.021.
References
Allah, A.T.A., Wanas, M.Q.S., Thompson, S.N., 1997. Effects of heavy metals on survival and growth of Biomphalaria glabrata Say (Gastropoda: Pulmonata) and
interaction with Schistosome infection. J. Mollus. Stud. 63 (1), 79–86.
Alonso, A., Camargo, J.A., 2011. Toxic effects of fluoride ion on survival, reproduction and behaviour of the aquatic snail Potamopyrgus antipodarum (Hydrobiidae
Mollusca). Water Air Soil Pollut. 219 (1–4), 81–90.
Alonso, A., Camargo, J.A., 2013. Nitrate causes deleterious effects on the behaviour
and reproduction of the aquatic snail Potamopyrgus antipodarum (Hydrobiidae
Mollusca). Environ. Sci. Pollut. R. 20 (8), 5388–5396.
R. Zounkova et al. / Aquatic Toxicology 150 (2014) 83–92
Benstead, R.S., Baynes, A., Casey, D., Routledge, E.J., Jobling, S., 2011. 17betaOestradiol may prolong reproduction in seasonally breeding freshwater
gastropod molluscs. Aquat. Toxicol. 101 (2), 326–334.
Brněnské vodárny a kanalizace, 2010. Odvádění a čištění odpadních vod/ČOV
Brno–Modřice (Sewage water treatment at WWTP Brno), Available on-line on
http://www.bvk.cz/o-spolecnosti/odvadeni-a-cisteniodpadnich-vod/cov-brnomodrice/ (in Czech).
Burton, G.A., Nordstrom, J.E., 2004. An in situ toxicity identification evaluation
method part II: field validation. Environ. Toxicol. Chem. 23 (12), 2851–2855.
Clarke, N., Routledge, E.J., Garner, A., Casey, D., Benstead, R., Walker, D., Watermann,
B., Gnass, K., Thomsen, A., Jobling, S., 2009. Exposure to treated sewage effluent
disrupts reproduction and development in the seasonally breeding Ramshorn
snail (Subclass: Pulmonata, Planorbarius corneus). Environ. Sci. Technol. 43 (6),
2092–2098.
Demirpence, E., Duchesne, M.J., Badia, E., Gagne, D., Pons, M., 1993. MVLN cells – a
bioluminescent MCF-7-derived cell-line to study the modulation of estrogenic
activity. J Steroid Biochem. 46 (3), 355–364.
Depledge, M.H., Billinghurst, Z., 1999. Ecological significance of endocrine disruption
in marine invertebrates. Mar. Pollut. Bull. 39 (1–12), 32–38.
Duft, M., Schulte-Oehlmann, U., Tillmann, M., Markert, B., Oehlmann, J., 2003a.
Toxicity of triphenyltin and tributyltin to the freshwater mudsnail Potamopyrgus antipodarum in a new sediment biotest. Environ. Toxicol. Chem. 22,
145–152.
Duft, M., Schulte-Oehlmann, U., Weltje, L., Tillmann, M., Oehlmann, J., 2003b. Stimulated embryo production as a parameter of estrogenic exposure via sediments
in the freshwater mudsnail Potamopyrgus antipodarum. Aquat. Toxicol. 64 (4),
437–449.
Duft, M., Schmitt, C., Bachmann, J., Brandelik, C., Schulte-Oehlmann, U., Oehlmann, J.,
et al., 2007. Prosobranch snails as test organisms for the assessment of endocrine
active chemicals – an overview and a guideline proposal for a reproduction test
with the freshwater mudsnail Potamopyrgus antipodarum. Ecotoxicology 16 (1),
169–182.
Freyberger, A., Schmuck, G., 2005. Screening for estrogenicity and anti-estrogenicity:
a critical evaluation of an MVLN cell-based transactivation assay. Toxicol. Lett.
155 (1), 1–13.
Galluba, S., Oehlmann, J., 2012. Widespread endocrine activity in river sediments in
Hesse, Germany, assessed by a combination of in vitro and in vivo bioassays. J.
Soils Sediments 12 (2), 252–264.
Grabic, R., Jurcikova, J., Tomsejova, S., Ocelka, T., Halirova, J., Hypr, D., Kodes, V.,
2010. Passive sampling methods for monitoring endocrine disruptors in the
Svratka and Svitava Rivers in the Czech Republic. Environ. Toxicol. Chem. 29
(3), 550–555.
Groshart, C., Okkerman, P.C., 2000. Towards the Establishment of a Priority List of
Substances for Further Evaluation of Their Role in Endocrine Disruption. European commission DG ENV, Delft, Zeist.
Gupta, P., Khangarot, B., Durve, V., 1981. The temperature dependence of the acute
toxicity of copper to a freshwater pond snail, Viviparus bengalensis L. Hydrobiologia 83 (3), 461–464.
Gust, M., Buronfosse, T., Geffard, O., Mons, R., Queau, H., Mouthon, J., Garric, J.,
2010a. In situ biomonitoring of freshwater quality using the New Zealand mudsnail Potamopyrgus antipodarum (Gray) exposed to waste water treatment plant
(WWTP) effluent discharges. Water Res. 44 (15), 4517–4528.
Gust, M., Garric, J., Giamberini, L., Mons, R., Abbaci, K., Garnier, F., Buronfosse, T.,
2010b. Sensitivity of New Zealand mudsnail Potamopyrgus antipodarum (Gray)
to a specific aromatase inhibitor. Chemosphere 79 (1), 47–53.
Hilscherova, K., Kannan, K., Kang, Y.S., Holoubek, I., Machala, M., Masunaga,
S., Nakanishi, J., Giesy, J.P., 2001. Characterization of dioxin-like activity
of sediments from a Czech river basin. Environ. Toxicol. Chem. 20 (12),
2768–2777.
Hilscherova, K., Kannan, K., Holoubek, I., Giesy, J.P., 2002. Characterization of estrogenic activity of riverine sediments from the Czech Republic. Arch. Environ.
Contam. Toxicol. 43 (2), 175–185.
Huckins, J.N., Manuweera, G.K., Petty, J.D., Mackay, D., Lebo, J.A., 1993.
Lipid-containing semipermeable membrane devices for monitoring organic
contaminants in water. Environ. Sci. Technol. 27 (12), 2489–2496.
Huckins, J.N., Petty, J.D., Orazio, C.E., Lebo, J.A., Clark, R.C., Gibson, V.L., Gala,
W.R., Echols, K.R., 1999. Determination of uptake kinetics (sampling rates)
by lipid-containing semipermeable membrane devices (SPMDs) for polycyclic aromatic hydrocarbons (PAHs) in water. Environ. Sci. Technol. 33 (21),
3918–3923.
ISO, 1995. ISO 5667-12:1995 Water Quality – Sampling – Part 12: Guidance on Sampling of Bottom Sediments. International Standard. International Organization for
Standardization, Geneva, Switzerland.
ISO, 2005. ISO/IEC 17025:2005 General Requirements for the Competence of Testing
and Calibration Laboratories. International Standard. International Organization
for Standardization, Geneva, Switzerland.
Jalova, V., Jarosova, B., Blaha, L., Giesy, J.P., Ocelka, T., Grabic, R., Jurcikova, J., Vrana,
B., Hilscherova, K., 2013. Estrogen-, androgen- and aryl hydrocarbon receptor
mediated activities in passive and composite samples from municipal waste
and surface waters. Environ. Int. 59, 372–383.
Jobling, S., Casey, D., Rodgers-Gray, T., Oehlmann, J., Schulte-Oehlmann, U.,
Pawlowski, S., Baunbeck, T., Turner, A.P., Tyler, C.R., 2003. Comparative responses
of molluscs and fish to environmental estrogens and an estrogenic effluent.
Aquat. Toxicol. 65 (2), 205–220.
Jobling, S., Casey, D., Rodgers-Gray, T., Oehlmann, J., Schulte-Oehlmann, U.,
Pawlowski, S., Baunbeck, T., Turner, A.P., Tyler, C.R., 2004. Erratum to:
91
“Comparative responses of molluscs and fish to environmental estrogens and
an estrogenic effluent” (vol. 65, pg 205, 2003). Aquat. Toxicol. 66 (2), 207–222.
Lafont, R., Mathieu, M., 2007. Steroids in aquatic invertebrates. Ecotoxicology 16 (1),
109–130.
Laskowski, R., Hopkin, S.P., 1996. Effect of Zn, Cu, Pb, and Cd on fitness in snails (Helix
aspersa). Ecotoxicol. Environ. Saf. 34 (1), 59–69.
Lenihan, H.S., Kiest, K.A., Conlan, K.E., Slattery, P.N., Konar, B.H., Oliver, J.S., 1995.
Patterns of survival and behavior in Antarctic benthic invertebrates exposed to
contaminated sediments: field and laboratory bioassay experiments. J. Exp. Mar.
Biol. Ecol. 192 (2), 233–255.
Leskinen, P., Michelini, E., Picard, D., Karp, M., Virta, M., 2005. Bioluminescent yeast
assays for detecting estrogenic and androgenic activity in different matrices.
Chemosphere 61, 259–266.
Li, H., Vermeirssen, E.L., Helm, P.A., Metcalfe, C.D., 2010. Controlled field evaluation of water flow rate effects on sampling polar organic compounds
using polar organic chemical integrative samplers. Environ. Toxicol. Chem. 29,
2461–2469.
Magdeburg, A., Stalter, D., Oehlmann, J., 2012. Whole effluent toxicity assessment at
a wastewater treatment plant upgraded with a full-scale post-ozonation using
aquatic key species. Chemosphere 88 (8), 1008–1014.
Michelini, E., Leskinen, P., Virta, M., Karp, M., Roda, A., 2005. A new recombinant cellbased bioluminescent assay for sensitive androgen-like compound detection.
Biosens. Bioelectron. 20, 2261–2267.
Mazurová, E., Hilscherová, K., Jálová, V., Köhler, H., Triebskorn, R., Giesy, J.P., Bláha, L.,
2008. Endocrine effects of contaminated sediments on the freshwater snail Potamopyrgus antipodarum in vivo and in the cell bioassays in vitro. Aquat. Toxicol.
89 (3), 172–179.
Ministry of the Environment of the Czech Republic, Czech Environmental
Inspectorate, 2010. Report on the reconstruction and modernization of
the WWTP in Brno 2010; Available online on http://www.cizp.cz/(b1obdbr
(in
4uyzcvq454q1mucvb)/default.aspx?id=511&ido=362&sh=-711127208.
Czech).
MoA, 2009. Direction No. 257/2009 Coll. for sediment use on agricultural soils. Ministry of Agriculture and Ministry of Environment of Czech Republic, Prague. (in
Czech).
Mothershead II, R.F., Hale, R.C., 1992. Influence of Ecdysis on the accumulation
of polycyclic aromatic hydrocarbons in field exposed blue crabs (Callinectes
sapidus). Mar. Environ. Res. 33 (2), 145–156.
Oehlmann, J., Schulte-Oehlmann, U., Tillmann, M., Markert, B., 2000. Effects of
endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part I: Bisphenol A and octylphenol as xeno-estrogens. Ecotoxicology 9
(6), 383–397.
Oehlmann, J., Di Benedetto, P., Tillmann, M., Duft, M., Oetken, M., Schulte-Oehlmann,
U., 2007. Endocrine disruption in prosobranch molluscs: evidence and ecological
relevance. Ecotoxicology 16 (1), 29–43.
Oetken, M., Bachmann, J., Schulte-Oehlmann, U., Oehlmann, J., 2004. Evidence for
endocrine disruption in invertebrates. Int. Rev. Cytol. 236, 1–44.
Pang, C.F., Selck, H., Misra, S.K., Berhanu, D., Dybowska, A., Valsami-Jones, E., Forbes,
V.E., 2012. Effects of sediment-associated copper to the deposit-feeding snail,
Potamopyrgus antipodarum: a comparison of Cu added in aqueous form or as
nano- and micro-CuO particles. Aquat. Toxicol. 106, 114–122.
Péry, A.R.R., Gust, M., Vollat, B., Mons, R., Ramil, M., Fink, G., Ternes, T., Garric, J.,
2008. Fluoxetine effects assessment on the life cycle of aquatic invertebrates.
Chemosphere 73 (3), 300–304.
Sanderson, J.T., Aarts, J., Brouwer, A., Froese, K.L., Denison, M.S., Giesy, J.P., 1996. Comparison of Ah receptor-mediated luciferase and ethoxyresorufin-O-deethylase
induction in H4IIE cells: implications for their use as bioanalytical tools for the
detection of polyhalogenated aromatic hydrocarbons. Toxicol. Appl. Pharm. 137
(2), 316–325.
Schmitt, C., Oetken, M., Dittberner, O., Wagner, M., Oehlmann, J., 2008. Endocrine
modulation and toxic effects of two commonly used UV screens on the aquatic
invertebrates Potamopyrgus antipodarum and Lumbriculus variegatus. Environ.
Pollut. 152 (2), 322–329.
Schmitt, C., Balaam, J., Leonards, P., Brix, R., Streck, G., Tuikka, A., Bervoets, L., Brack,
W., van Hattum, B., Meire, P., de Deckere, E., 2010a. Characterizing field sediments from three European river basins with special emphasis on endocrine
effects – a recommendation for Potamopyrgus antipodarum as test organism.
Chemosphere 80 (1), 13–19.
Schmitt, C., Vogt, C., Van Ballaer, B., Brix, R., Suetens, A., Schmitt-Jansen, M., de
Deckere, E., 2010b. In situ cage experiments with Potamopyrgus antipodarum
– a novel tool for real life exposure assessment in freshwater ecosystems. Ecotoxicol. Environ. Saf. 73 (7), 1574–1579.
Schmitt, C., Streck, G., Lamoree, M., Leonards, P., Brack, W., de Deckere, E., 2011.
Effect directed analysis of riverine sediments – the usefulness of Potamopyrgus antipodarum for in vivo effect confirmation of endocrine disruption. Aquat.
Toxicol. 101 (1), 237–243.
Schulte-Oehlmann, U., Tillmann, M., Markert, B., Oehlmann, J., 2000. Effects of
endocrine disruptors on prosobranch snails (Mollusca; Gastropoda) in the laboratory. Part II: Triphenyltin as a xeno-androgen. Ecotoxicology 9 (6), 399–412.
Stalter, D., Magdeburg, A., Oehlmann, J., 2010. Comparative toxicity assessment of
ozone and activated carbon treated sewage effluents using an in vivo test battery.
Water Res. 44 (8), 2610–2620.
Stange, D., Sieratowicz, A., Horres, R., Oehlmann, J., 2012. Freshwater mudsnail
(Potamopyrgus antipodarum) estrogen receptor: identification and expression
analysis under exposure to (xeno-)hormones. Ecotoxicol. Environ. Saf. 75,
94–101.
92
R. Zounkova et al. / Aquatic Toxicology 150 (2014) 83–92
Tillmann, M., Schulte-Oehlmann, U., Duft, M., Markert, B., Oehlmann, J., 2001. Effects
of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the
laboratory. Part III: Cyproterone acetate and vinclozolin as antiandrogens. Ecotoxicology 10 (6), 373–388.
Villeneuve, D.L., Blankenship, A.L., Giesy, J.P., 2000. Derivation and application of
relative potency estimates based on in vitro bioassay results. Environ. Toxicol.
Chem. 19 (11), 2835–2843.
Villeneuve, D.L., Khim, J.S., Kannan, K., Giesy, J.P., 2002. Relative potencies of individual polycyclic aromatic hydrocarbons to induce dioxinlike and estrogenic
responses in three cell lines. Environ. Toxicol. 17 (2), 128–137.
Wilson, V.S., Bobseine, K., Lambright, C.R., Gray, L.E., 2002. A novel cell line MDA-kb2,
that stably expresses an androgen- and glucocorticoid-responsive reporter for
the detection of hormone receptor agonists and antagonists. Toxicol. Sci. 66 (1),
69–81.
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
7.5.2008 14:00
13.62
14.09
12.78
13.34
14
14.47
12.75
12.75
9.5.2008 11:00
9.5.2008 12:00
13.25
13.34
13.06
13.15
13.43
13.53
14.23
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
7.5.2008 17:00
14.84
14.93
13.9
14.09
15.12
14.84
13.83
13.89
9.5.2008 14:00
9.5.2008 15:00
13.81
14.09
13.72
14.28
14.75
15.21
13.48
13.67
7.5.2008 18:00
7.5.2008 19:00
13.34
12.78
14.18
14.37
14.56
14.18
13.73
13.61
9.5.2008 16:00
9.5.2008 17:00
14.47
14.47
14.65
14.84
15.5
15.4
14.11
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
7.5.2008 22:00
12.87
12.78
14.47
14.47
13.43
12.87
13.76
13.89
9.5.2008 19:00
9.5.2008 20:00
12.78
12.59
15.03
15.12
15.12
14.84
14.23
14.11
7.5.2008 23:00
8.5.2008 0:00
12.78
12.68
14.37
13.25
12.59
12.4
14.26
14.48
9.5.2008 21:00
9.5.2008 22:00
12.59
12.59
15.12
15.21
14.56
14.09
14.23
14.23
8.5.2008 1:00
8.5.2008 2:00
12.59
12.59
12.68
12.49
12.12
11.93
14.01
13.61
9.5.2008 23:00
10.5.2008 0:00
12.59
12.49
15.12
14.84
13.72
13.25
14.33
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
8.5.2008 5:00
12.49
12.31
12.4
12.4
11.55
11.46
13.51
13.32
10.5.2008 2:00
10.5.2008 3:00
12.4
12.31
14
13.72
12.68
12.49
14.51
14.39
8.5.2008 6:00
8.5.2008 7:00
12.31
12.49
12.31
12.31
11.36
11.27
13.1
12.94
10.5.2008 4:00
10.5.2008 5:00
12.31
12.21
13.53
13.43
12.4
12.21
14.23
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
8.5.2008 10:00
12.59
12.68
12.4
12.49
11.55
12.12
12.79
12.88
10.5.2008 7:00
10.5.2008 8:00
12.49
12.68
13.15
13.06
12.12
12.21
14.05
14.01
8.5.2008 11:00
8.5.2008 12:00
12.96
13.34
12.59
12.96
12.78
13.62
13.23
13.36
10.5.2008 9:00
10.5.2008 10:00
12.78
12.78
12.96
13.06
12.49
12.96
14.01
14.08
8.5.2008 13:00
8.5.2008 14:00
13.72
14.18
13.53
13.9
14.18
14.47
12.72
12.85
10.5.2008 11:00
10.5.2008 12:00
13.25
13.62
13.15
13.25
13.62
14.47
14.23
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
8.5.2008 17:00
14.84
14.93
14.28
14.47
14.65
14.65
13.86
13.92
10.5.2008 14:00
10.5.2008 15:00
14.37
14.75
14.37
14.75
16.06
16.62
13.76
14.23
8.5.2008 18:00
8.5.2008 19:00
14.65
13.15
14.56
14.65
14.65
14.47
13.86
13.89
10.5.2008 16:00
10.5.2008 17:00
15.03
15.12
14.93
15.12
16.34
15.96
14.79
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
8.5.2008 22:00
12.96
12.96
14.65
14.65
13.9
13.53
14.01
14.23
10.5.2008 19:00
10.5.2008 20:00
12.78
12.78
15.31
15.31
15.12
14.65
14.73
14.67
8.5.2008 23:00
9.5.2008 0:00
12.96
12.87
14.84
14.18
13.25
12.87
14.39
14.6
10.5.2008 21:00
10.5.2008 22:00
12.96
12.78
15.4
15.4
14.28
14
14.73
14.94
9.5.2008 1:00
9.5.2008 2:00
12.87
12.87
13.81
13.62
12.68
12.59
14.48
14.26
10.5.2008 23:00
11.5.2008 0:00
12.78
12.78
14.84
14
13.72
13.34
15.28
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
9.5.2008 5:00
12.78
12.68
13.34
13.25
12.4
12.31
13.92
13.92
11.5.2008 2:00
11.5.2008 3:00
12.68
12.59
13.34
13.15
12.78
12.59
14.64
14.39
9.5.2008 6:00
9.5.2008 7:00
12.59
12.49
13.15
12.96
12.31
12.21
13.92
13.89
11.5.2008 4:00
11.5.2008 5:00
12.49
12.4
13.15
12.96
12.49
12.4
14.36
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
9.5.2008 10:00
12.87
13.06
12.96
13.06
12.59
13.06
13.83
13.92
11.5.2008 7:00
11.5.2008 8:00
12.59
12.87
12.96
12.87
12.31
12.4
14.08
13.98
1b
2b
4
11.5.2008 9:00
1a
12.96
1b
12.87
2b
12.68
4
13.89
14.5.2008 0:00
1a
13.53
16.34
14.93
16.46
11.5.2008 10:00
11.5.2008 11:00
13.15
13.43
12.96
13.06
13.25
13.81
13.92
13.86
14.5.2008 1:00
14.5.2008 2:00
13.43
13.43
15.59
15.12
14.56
14.37
16.37
16.1
11.5.2008 12:00
11.5.2008 13:00
13.81
14.18
13.25
13.43
14.65
15.59
14.48
14.23
14.5.2008 3:00
14.5.2008 4:00
13.34
13.34
14.84
14.65
14.18
14.09
15.89
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
11.5.2008 16:00
14.84
15.21
14.18
14.65
16.81
16.99
14.14
14.26
14.5.2008 6:00
14.5.2008 7:00
13.25
12.96
14.37
14.28
13.9
14
15.53
15.62
11.5.2008 17:00
11.5.2008 18:00
15.4
15.5
15.31
15.5
16.9
16.81
14.64
14.91
14.5.2008 8:00
14.5.2008 9:00
13.53
13.53
14.18
14.28
14.09
14.37
15.62
15.62
11.5.2008 19:00
11.5.2008 20:00
13.15
13.06
15.59
15.68
16.24
15.78
15.1
15.22
14.5.2008 10:00
14.5.2008 11:00
13.72
14.09
14.28
14.28
14.93
15.59
15.71
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
11.5.2008 23:00
13.06
12.96
15.96
16.24
14.84
14.47
15.25
15.46
14.5.2008 13:00
14.5.2008 14:00
14.93
15.31
14.75
15.21
17.28
18.03
15.56
15.46
12.5.2008 0:00
12.5.2008 1:00
12.96
12.96
16.15
15.78
14.18
13.81
15.71
15.71
14.5.2008 15:00
14.5.2008 16:00
15.59
15.87
15.68
15.96
18.5
18.5
15.56
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
12.5.2008 4:00
12.87
12.78
14.93
14.47
13.25
13.06
15.22
15.07
14.5.2008 18:00
14.5.2008 19:00
15.68
14.09
16.15
16.24
17.46
16.53
16.43
16.22
12.5.2008 5:00
12.5.2008 6:00
12.68
12.59
14
13.72
12.96
12.78
14.91
14.82
14.5.2008 20:00
14.5.2008 21:00
14.28
14
16.34
16.53
16.34
15.87
16.22
16.37
12.5.2008 7:00
12.5.2008 8:00
12.68
13.06
13.53
13.43
12.78
12.96
14.76
14.85
14.5.2008 22:00
14.5.2008 23:00
14.09
14
16.71
16.9
15.4
15.12
16.67
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
12.5.2008 11:00
13.34
13.53
13.43
13.53
13.72
14.37
15.1
15.22
15.5.2008 1:00
15.5.2008 2:00
14
14
15.87
15.31
14.47
14.37
16.49
16.25
12.5.2008 12:00
12.5.2008 13:00
13.81
14.09
13.62
13.9
15.21
16.06
15.22
14.85
15.5.2008 3:00
15.5.2008 4:00
13.9
13.9
15.03
14.75
14.37
14.28
15.95
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
12.5.2008 16:00
14.75
15.31
14.75
15.21
16.99
17.09
14.94
15.34
15.5.2008 6:00
15.5.2008 7:00
13.72
13.53
14.47
14.37
13.9
13.81
15.71
15.62
12.5.2008 17:00
12.5.2008 18:00
15.5
15.59
15.21
15.31
17.18
16.99
15.62
15.71
15.5.2008 8:00
15.5.2008 9:00
13.72
13.81
14.28
14.28
13.81
14
15.53
15.49
12.5.2008 19:00
12.5.2008 20:00
13.25
13.34
15.5
15.68
16.24
15.96
15.49
15.37
15.5.2008 10:00
15.5.2008 11:00
13.9
14.28
14.37
14.37
14.37
14.75
15.49
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
12.5.2008 23:00
13.25
13.25
15.87
16.24
15.31
14.93
15.46
15.62
15.5.2008 13:00
15.5.2008 14:00
15.03
15.4
14.75
15.12
16.24
16.9
15.37
15.31
13.5.2008 0:00
13.5.2008 1:00
13.25
13.25
15.96
15.5
14.65
14.37
15.98
15.98
15.5.2008 15:00
15.5.2008 16:00
15.78
15.78
15.5
15.78
17.28
17.09
15.56
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
13.5.2008 4:00
13.15
12.96
14.65
14.37
13.81
13.62
15.43
15.22
15.5.2008 18:00
15.5.2008 19:00
15.96
14.09
16.24
16.34
16.99
16.81
16.19
16.19
13.5.2008 5:00
13.5.2008 6:00
12.96
12.87
14.18
14
13.43
13.25
15.04
15.01
15.5.2008 20:00
15.5.2008 21:00
14.09
14.09
16.53
16.53
16.71
16.24
16.07
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
13.5.2008 9:00
13.25
13.34
13.9
13.9
13.25
13.62
15.22
15.22
15.5.2008 23:00
16.5.2008 0:00
14.09
14.09
16.9
16.71
15.5
15.12
16.4
16.46
13.5.2008 10:00
13.5.2008 11:00
13.62
13.9
13.9
13.9
14.09
14.84
15.37
15.34
16.5.2008 1:00
16.5.2008 2:00
14
14
16.24
15.87
14.93
14.65
16.49
16.25
13.5.2008 12:00
13.5.2008 13:00
14.18
14.65
14.09
14.28
15.59
16.53
15.34
15.43
16.5.2008 3:00
16.5.2008 4:00
14
14
15.5
15.31
14.56
14.56
16.1
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
13.5.2008 16:00
15.5
15.87
15.31
15.68
17.93
18.12
15.31
15.74
16.5.2008 6:00
16.5.2008 7:00
13.9
13.72
15.12
15.03
14.56
14.65
15.86
15.92
13.5.2008 17:00
13.5.2008 18:00
16.06
16.24
15.96
16.06
17.93
17.65
16.1
16.19
16.5.2008 8:00
16.5.2008 9:00
13.9
13.9
15.03
15.03
14.65
14.84
15.89
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
13.5.2008 21:00
13.53
13.72
16.24
16.34
16.62
16.15
16.04
16.04
16.5.2008 11:00
16.5.2008 12:00
14.28
14.37
15.03
15.03
15.59
16.15
15.86
15.86
13.5.2008 22:00
13.5.2008 23:00
13.53
13.53
16.43
16.81
15.87
15.4
16.07
16.19
16.5.2008 13:00
16.5.2008 14:00
14.56
14.93
15.31
15.59
16.34
16.62
15.59
15.59
1b
2b
4
16.5.2008 15:00
1a
15.12
1b
15.78
2b
16.9
4
15.95
19.5.2008 6:00
1a
14.37
15.03
15.68
16.19
16.5.2008 16:00
16.5.2008 17:00
15.31
15.5
15.96
16.15
17.18
17.46
16.31
16.28
19.5.2008 7:00
19.5.2008 8:00
14.28
14.47
14.93
14.93
15.5
15.31
16.04
15.89
16.5.2008 18:00
16.5.2008 19:00
15.5
14.47
16.34
16.34
17.18
16.9
16.19
16.04
19.5.2008 9:00
19.5.2008 10:00
14.56
14.65
14.93
14.93
14.84
14.84
15.77
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
16.5.2008 22:00
14.09
14.28
16.43
16.53
16.24
16.15
16.07
16.19
19.5.2008 12:00
19.5.2008 13:00
14.65
14.65
14.65
14.65
14.93
14.84
15.25
15.07
16.5.2008 23:00
17.5.2008 0:00
14.28
14.18
16.71
16.24
15.96
15.59
16.43
16.73
19.5.2008 14:00
19.5.2008 15:00
14.65
14.65
14.65
14.75
14.84
14.75
15.01
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
17.5.2008 3:00
14.18
14.09
15.4
15.21
15.21
15.12
16.19
15.98
19.5.2008 17:00
19.5.2008 18:00
14.75
14.47
14.84
14.84
14.75
14.75
14.76
14.79
17.5.2008 4:00
17.5.2008 5:00
14.09
14.09
15.03
14.93
15.03
14.84
16.01
16.07
19.5.2008 19:00
19.5.2008 20:00
14.47
14.47
14.84
14.84
14.65
14.37
14.82
14.82
17.5.2008 6:00
17.5.2008 7:00
14.09
13.9
14.84
14.75
14.65
14.65
16.04
16.01
19.5.2008 21:00
19.5.2008 22:00
14.47
14.47
14.84
14.75
14.28
14.09
14.79
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
17.5.2008 10:00
14.28
14.56
14.75
14.84
14.84
15.21
15.86
16.19
20.5.2008 0:00
20.5.2008 1:00
14.37
14.37
14.28
14.18
13.81
13.72
14.51
14.23
17.5.2008 11:00
17.5.2008 12:00
14.75
15.12
14.84
15.03
15.78
16.62
16.07
16.07
20.5.2008 2:00
20.5.2008 3:00
14.28
14.28
14.09
14
13.53
13.34
14.11
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
17.5.2008 15:00
15.68
16.15
15.87
16.24
17.56
18.12
15.71
16.1
20.5.2008 5:00
20.5.2008 6:00
14.09
14
13.9
13.81
13.06
12.96
13.8
13.73
17.5.2008 16:00
17.5.2008 17:00
16.62
16.71
16.53
16.71
18.4
18.5
16.52
16.73
20.5.2008 7:00
20.5.2008 8:00
14.18
14.37
13.81
13.81
12.78
12.78
13.61
13.48
17.5.2008 18:00
17.5.2008 19:00
16.71
14.18
16.81
16.9
18.31
18.03
16.67
16.58
20.5.2008 9:00
20.5.2008 10:00
14.47
14.47
13.72
13.72
12.78
12.78
13.39
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
17.5.2008 22:00
14.37
14.28
16.99
17.09
17.46
17.09
16.67
16.82
20.5.2008 12:00
20.5.2008 13:00
14.47
14.47
13.43
13.72
12.78
12.78
13.04
12.98
17.5.2008 23:00
18.5.2008 0:00
14.37
14.37
17.37
17.09
16.71
17.28
17
17.39
20.5.2008 14:00
20.5.2008 15:00
14.37
14.37
13.15
13.9
12.96
12.96
13.1
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
18.5.2008 3:00
14.28
14.28
16.06
16.24
16.71
16.62
16.91
16.91
20.5.2008 17:00
20.5.2008 18:00
14.47
14.37
13.9
13.9
12.78
12.59
13.48
13.51
18.5.2008 4:00
18.5.2008 5:00
14.28
14.28
16.43
16.53
16.34
16.24
16.82
16.79
20.5.2008 19:00
20.5.2008 20:00
14.37
14.37
13.9
13.72
12.4
12.21
13.55
13.55
18.5.2008 6:00
18.5.2008 7:00
14.28
14.09
16.53
16.43
16.06
15.96
16.73
16.73
20.5.2008 21:00
20.5.2008 22:00
14.37
14.37
13.81
14
12.12
12.02
13.58
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
18.5.2008 10:00
14.28
14.37
16.34
16.24
16.34
16.62
16.79
16.94
21.5.2008 0:00
21.5.2008 1:00
14.28
14.18
14.09
14.09
11.84
11.65
13.23
13.26
18.5.2008 11:00
18.5.2008 12:00
14.56
14.65
16.24
16.15
17.09
17.37
17.15
17.15
21.5.2008 2:00
21.5.2008 3:00
14.09
14.09
14
14
11.55
11.46
13.32
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
18.5.2008 15:00
15.21
15.31
16.43
16.43
18.03
17.74
16.88
17
21.5.2008 5:00
21.5.2008 6:00
14.09
14
13.9
13.9
11.36
11.27
13.26
13.23
18.5.2008 16:00
18.5.2008 17:00
15.31
15.4
16.53
16.71
18.12
18.59
17.18
16.82
21.5.2008 7:00
21.5.2008 8:00
14.09
14.28
13.72
13.72
11.27
11.27
13.04
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
18.5.2008 20:00
14.75
14.65
17.18
17.18
17.74
17.37
16.67
16.85
21.5.2008 10:00
21.5.2008 11:00
14.28
14.28
13.53
13.53
11.18
11.18
12.56
12.4
18.5.2008 21:00
18.5.2008 22:00
14.47
14.56
17.28
17.28
16.99
16.99
17
17.15
21.5.2008 12:00
21.5.2008 13:00
14.18
14.18
13.34
13.43
11.36
11.46
12.24
12.21
18.5.2008 23:00
19.5.2008 0:00
14.47
14.47
16.62
15.87
16.71
16.34
17.15
16.91
21.5.2008 14:00
21.5.2008 15:00
14.28
14.28
13.62
13.81
11.55
11.65
12.34
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
19.5.2008 3:00
14.47
14.47
15.4
15.31
16.06
15.96
16.67
16.52
21.5.2008 17:00
21.5.2008 18:00
14.37
14.09
13.9
14
11.74
11.74
12.98
13.01
19.5.2008 4:00
19.5.2008 5:00
14.47
14.47
15.21
15.12
15.87
15.78
16.4
16.28
21.5.2008 19:00
21.5.2008 20:00
14.28
14.28
14
14
11.74
11.65
13.23
13.23
1b
2b
4
21.5.2008 21:00
1a
14.18
1b
13.9
2b
11.46
4
13.23
24.5.2008 12:00
1a
14
14.18
13.34
13.23
21.5.2008 22:00
21.5.2008 23:00
14.09
14.18
13.9
13.9
11.36
11.18
13.23
13.04
24.5.2008 13:00
24.5.2008 14:00
14.18
14.37
15.21
15.21
13.53
13.62
13.23
13.92
22.5.2008 0:00
22.5.2008 1:00
14.09
14.09
13.72
13.62
11.08
10.99
12.79
12.5
24.5.2008 15:00
24.5.2008 16:00
14.47
14.75
15.21
15.21
13.34
13.43
14.23
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
22.5.2008 4:00
14
13.9
13.53
13.53
10.89
10.89
12.72
12.72
24.5.2008 18:00
24.5.2008 19:00
14
13.81
15.21
15.31
13.81
13.72
14.79
14.85
22.5.2008 5:00
22.5.2008 6:00
13.9
13.81
13.43
13.43
10.8
10.8
12.59
12.47
24.5.2008 20:00
24.5.2008 21:00
13.81
13.81
15.31
15.31
13.53
13.34
14.82
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
22.5.2008 9:00
14.09
14.09
13.34
13.25
10.89
10.99
12.21
12.08
24.5.2008 23:00
25.5.2008 0:00
13.72
13.72
15.21
14.47
12.87
12.78
14.76
14.51
22.5.2008 10:00
22.5.2008 11:00
14
14
13.25
13.25
11.18
11.46
11.98
11.95
25.5.2008 1:00
25.5.2008 2:00
13.72
13.72
14.18
14.18
12.59
12.49
14.14
14.26
22.5.2008 12:00
22.5.2008 13:00
14
14
13.34
13.9
11.65
11.65
11.98
12.08
25.5.2008 3:00
25.5.2008 4:00
13.72
13.62
14.09
14.09
12.4
12.31
14.33
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
22.5.2008 16:00
14.18
14.09
14.18
14.28
11.65
11.65
12.75
13.01
25.5.2008 6:00
25.5.2008 7:00
13.43
13.43
13.9
14
12.21
12.21
13.8
13.73
22.5.2008 17:00
22.5.2008 18:00
14.09
14.09
14.37
14.18
11.65
11.65
13.26
13.32
25.5.2008 8:00
25.5.2008 9:00
13.81
13.9
14.09
14.37
12.31
12.59
13.64
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
22.5.2008 21:00
14.09
13.9
14.09
14.09
11.46
11.27
13.32
13.23
25.5.2008 11:00
25.5.2008 12:00
13.9
14.18
14.47
14.47
13.72
14.28
13.83
13.95
22.5.2008 22:00
22.5.2008 23:00
14
14
14.18
14.18
11.27
11.27
12.85
13.04
25.5.2008 13:00
25.5.2008 14:00
14.47
14.75
15.5
15.59
14.75
15.4
13.86
14.51
23.5.2008 0:00
23.5.2008 1:00
13.9
13.9
14.09
13.9
11.27
11.27
13.23
13.23
25.5.2008 15:00
25.5.2008 16:00
15.03
15.5
15.59
15.87
15.5
15.78
15.07
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
23.5.2008 4:00
13.9
13.9
13.9
13.9
11.27
11.27
13.23
13.23
25.5.2008 18:00
25.5.2008 19:00
14.65
13.62
15.68
15.5
15.4
15.03
15.59
15.8
23.5.2008 5:00
23.5.2008 6:00
13.81
13.72
13.9
13.9
11.27
11.27
13.1
13.01
25.5.2008 20:00
25.5.2008 21:00
13.53
13.53
15.21
15.12
14.65
14.28
15.92
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
23.5.2008 9:00
13.9
13.81
13.81
13.81
11.46
11.55
12.82
12.72
25.5.2008 23:00
26.5.2008 0:00
13.53
13.53
15.68
14.56
13.72
13.53
15.95
15.56
23.5.2008 10:00
23.5.2008 11:00
13.53
13.9
13.81
14.65
11.84
12.12
12.63
12.66
26.5.2008 1:00
26.5.2008 2:00
13.53
13.53
14.28
14
13.43
13.34
14.97
14.79
23.5.2008 12:00
23.5.2008 13:00
14.09
14.28
14
14.56
12.4
12.68
12.72
12.82
26.5.2008 3:00
26.5.2008 4:00
13.43
13.43
14.09
14.09
13.25
13.15
14.85
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
23.5.2008 16:00
15.03
14.84
14.84
14.93
12.78
12.68
13.48
13.8
26.5.2008 6:00
26.5.2008 7:00
13.25
13.34
14.09
14.18
12.87
12.87
14.33
14.23
23.5.2008 17:00
23.5.2008 18:00
14.84
14.93
14.93
14.84
12.78
12.78
13.98
14.11
26.5.2008 8:00
26.5.2008 9:00
13.62
13.72
14.56
14.28
12.96
13.15
14.14
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
23.5.2008 21:00
13.9
13.81
14.65
14.56
12.49
12.4
14.23
14.23
26.5.2008 11:00
26.5.2008 12:00
13.62
13.9
14.37
14.47
14
14.37
14.23
14.14
23.5.2008 22:00
23.5.2008 23:00
13.81
13.81
14.56
14.65
12.21
12.21
14.05
13.92
26.5.2008 13:00
26.5.2008 14:00
14.09
14.28
15.31
15.5
14.93
15.4
14.08
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
24.5.2008 2:00
13.81
13.81
14
13.81
12.02
11.93
13.55
13.29
26.5.2008 16:00
26.5.2008 17:00
14.93
15.21
15.5
15.4
16.15
16.34
15.22
15.22
24.5.2008 3:00
24.5.2008 4:00
13.72
13.72
13.72
13.62
11.74
11.74
13.32
13.51
26.5.2008 18:00
26.5.2008 19:00
14
13.62
15.31
15.31
16.15
15.78
15.37
15.56
24.5.2008 5:00
24.5.2008 6:00
13.62
13.62
13.62
13.72
11.65
11.65
13.58
13.48
26.5.2008 20:00
26.5.2008 21:00
13.9
14
15.31
15.31
15.5
15.21
15.71
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
24.5.2008 9:00
13.72
13.72
13.9
14.09
11.74
12.02
13.29
13.29
26.5.2008 23:00
27.5.2008 0:00
14
14
15.5
14.56
14.65
14.37
16.19
16.07
24.5.2008 10:00
24.5.2008 11:00
13.81
13.81
15.21
16.62
12.31
12.87
13.23
13.26
27.5.2008 1:00
27.5.2008 2:00
14
13.9
14.28
14.37
14.18
14.09
15.53
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
27.5.2008 5:00
13.9
13.81
14.37
14.47
13.72
13.72
14.97
14.85
29.5.2008 19:00
29.5.2008 20:00
15.4
14.84
17.93
18.12
19.62
19.25
18.12
18.09
27.5.2008 6:00
27.5.2008 7:00
13.81
13.72
14.37
14.37
13.62
13.72
14.73
14.57
29.5.2008 21:00
29.5.2008 22:00
14.65
14.84
18.31
18.5
18.87
18.4
18.12
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
27.5.2008 10:00
13.9
14.18
14.47
14.56
14.09
14.47
14.6
14.76
30.5.2008 0:00
30.5.2008 1:00
14.75
14.75
18.78
18.59
17.56
17.28
18.45
18.42
27.5.2008 11:00
27.5.2008 12:00
14.37
14.75
14.65
14.93
15.12
15.96
14.91
14.97
30.5.2008 2:00
30.5.2008 3:00
14.75
14.65
18.31
18.03
17.09
16.81
18.21
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
27.5.2008 15:00
15.5
16.06
16.15
16.53
17.74
18.21
15.46
16.1
30.5.2008 5:00
30.5.2008 6:00
14.65
14.65
17.37
16.99
16.62
16.53
17.74
17.62
27.5.2008 16:00
27.5.2008 17:00
16.53
17.37
16.81
16.9
18.59
18.87
16.55
16.67
30.5.2008 7:00
30.5.2008 8:00
14.28
14.75
16.62
16.43
16.53
16.71
17.54
17.54
27.5.2008 18:00
27.5.2008 19:00
14.93
14.09
17.18
17.37
18.68
18.5
16.58
16.58
30.5.2008 9:00
30.5.2008 10:00
14.75
15.21
16.43
16.43
16.99
17.46
17.65
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
27.5.2008 22:00
14.09
14.18
17.56
17.74
17.74
17.28
16.97
17.27
30.5.2008 12:00
30.5.2008 13:00
15.87
16.34
16.62
16.9
18.87
19.72
17.8
18.09
27.5.2008 23:00
28.5.2008 0:00
14.09
14.09
18.03
17.09
16.81
16.53
17.65
17.92
30.5.2008 14:00
30.5.2008 15:00
17.09
17.65
17.18
17.65
20.47
21.04
18.56
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
28.5.2008 3:00
14.18
14.18
15.78
15.59
15.96
15.78
17.42
17.18
30.5.2008 17:00
30.5.2008 18:00
18.68
18.78
18.4
18.68
21.33
21.14
19.03
18.88
28.5.2008 4:00
28.5.2008 5:00
14.18
14.18
15.59
15.5
15.78
15.68
17.15
17.15
30.5.2008 19:00
30.5.2008 20:00
18.68
15.59
18.78
18.87
20.85
20.57
19.03
19.03
28.5.2008 6:00
28.5.2008 7:00
14.09
13.72
15.5
15.5
15.68
15.68
17.15
17
30.5.2008 21:00
30.5.2008 22:00
14.93
15.03
18.97
19.06
20.1
19.72
19.03
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
28.5.2008 10:00
14.37
14.37
15.68
15.78
16.15
16.62
16.91
16.82
31.5.2008 0:00
31.5.2008 1:00
15.03
15.03
19.34
19.44
18.87
18.5
19.38
19.49
28.5.2008 11:00
28.5.2008 12:00
14.65
14.84
15.87
16.24
17.18
17.84
16.73
16.61
31.5.2008 2:00
31.5.2008 3:00
15.03
15.03
19.34
19.15
18.21
17.93
19.4
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
28.5.2008 15:00
15.68
16.62
17.18
17.37
19.25
19.62
16.73
17.39
31.5.2008 5:00
31.5.2008 6:00
15.03
15.03
18.31
18.03
17.56
17.46
18.82
18.71
28.5.2008 16:00
28.5.2008 17:00
17.18
17.37
17.56
17.74
19.91
20
17.86
17.86
31.5.2008 7:00
31.5.2008 8:00
14.47
15.03
17.65
17.37
17.56
17.74
18.59
18.56
28.5.2008 18:00
28.5.2008 19:00
17.56
14.75
17.84
17.93
19.72
19.44
17.71
17.65
31.5.2008 9:00
31.5.2008 10:00
15.21
15.4
17.28
17.28
18.12
18.59
18.62
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
28.5.2008 22:00
14.84
14.75
18.12
18.21
18.59
18.21
17.71
17.89
31.5.2008 12:00
31.5.2008 13:00
16.24
16.62
17.56
17.84
20
20.76
19.03
19.14
28.5.2008 23:00
29.5.2008 0:00
14.84
14.84
18.5
17.74
17.84
17.37
18.09
18.33
31.5.2008 14:00
31.5.2008 15:00
17.18
17.84
18.12
18.5
21.52
22.09
19.14
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
29.5.2008 3:00
14.84
14.84
16.34
15.96
16.9
16.81
17.89
17.68
31.5.2008 17:00
31.5.2008 18:00
18.97
19.15
19.15
19.34
22.28
21.99
19.78
19.98
29.5.2008 4:00
29.5.2008 5:00
14.75
14.65
15.78
15.68
16.71
16.62
17.62
17.51
31.5.2008 19:00
31.5.2008 20:00
15.5
15.68
19.44
19.53
21.71
21.33
19.95
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
29.5.2008 8:00
14.37
14.65
15.59
15.59
16.53
16.62
17.45
17.39
31.5.2008 22:00
31.5.2008 23:00
15.68
15.78
19.62
19.81
20.47
20.1
19.86
19.98
29.5.2008 9:00
29.5.2008 10:00
14.47
14.84
15.68
15.68
16.9
17.28
17.36
17.54
1.6.2008 0:00
1.6.2008 1:00
15.68
15.68
20.1
20
19.62
19.34
20.18
20.24
29.5.2008 11:00
29.5.2008 12:00
15.21
15.78
15.78
15.96
17.65
18.4
17.56
17.48
1.6.2008 2:00
1.6.2008 3:00
15.68
15.78
19.81
19.53
18.97
18.78
20.12
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
29.5.2008 15:00
16.43
17.28
16.43
16.9
19.72
20.19
17.21
17.24
1.6.2008 5:00
1.6.2008 6:00
15.68
15.68
18.68
18.21
18.4
18.21
19.55
19.35
29.5.2008 16:00
29.5.2008 17:00
17.56
17.84
17.28
17.56
20.38
20.29
17.68
17.95
1.6.2008 7:00
1.6.2008 8:00
15.31
15.5
17.93
17.74
18.31
18.5
19.2
19.14
1a
1b
2b
4
1.6.2008 9:00
15.78
17.74
18.87
19.26
1.6.2008 10:00
1.6.2008 11:00
15.87
15.96
17.74
17.84
19.44
19.91
19.49
19.69
1.6.2008 12:00
1.6.2008 13:00
16.24
16.53
17.84
18.12
20.38
21.23
19.55
19.72
1.6.2008 14:00
16.9
18.4
21.9
20.01
1.6.2008 15:00
1.6.2008 16:00
17.28
17.65
18.59
18.78
21.61
21.52
19.72
19.58
1.6.2008 17:00
1.6.2008 18:00
18.12
18.03
19.06
19.15
21.23
20.85
19.72
19.84
1.6.2008 19:00
1.6.2008 20:00
15.68
16.43
19.15
19.06
20.66
20.38
19.98
19.95
1.6.2008 21:00
16.24
19.06
19.91
19.86
1.6.2008 22:00
1.6.2008 23:00
16.24
16.24
19.06
19.06
19.72
19.34
19.81
19.78
2.6.2008 0:00
2.6.2008 1:00
16.24
16.24
18.97
18.78
18.97
18.68
19.69
19.52
2.6.2008 2:00
16.24
18.59
18.5
19.32
2.6.2008 3:00
2.6.2008 4:00
16.24
16.15
18.4
18.21
18.4
18.21
19.11
19.03
2.6.2008 5:00
2.6.2008 6:00
16.06
15.96
17.93
17.74
18.03
17.93
18.85
18.71
2.6.2008 7:00
2.6.2008 8:00
15.59
15.87
17.56
17.46
17.84
17.93
18.62
18.56
2.6.2008 9:00
15.96
17.46
18.12
18.62
2.6.2008 10:00
2.6.2008 11:00
15.78
15.78
17.46
17.37
18.68
19.34
18.56
18.59
2.6.2008 12:00
2.6.2008 13:00
16.15
16.62
17.65
18.03
20.1
20.85
18.85
19.03
2.6.2008 14:00
17.18
18.4
21.61
18.88
2.6.2008 15:00
2.6.2008 16:00
17.65
18.21
18.4
18.5
22.18
22.47
19.4
19.78
2.6.2008 17:00
2.6.2008 18:00
18.59
18.31
18.68
18.87
22.38
22.09
19.72
19.4
2.6.2008 19:00
2.6.2008 20:00
15.87
15.78
18.97
19.06
21.61
21.14
19.2
19.17
2.6.2008 21:00
15.78
19.15
20.66
19.26
2.6.2008 22:00
2.6.2008 23:00
15.87
15.87
19.25
19.62
20.19
19.81
19.4
19.61
3.6.2008 0:00
3.6.2008 1:00
15.87
15.87
19.06
18.21
19.34
18.97
19.86
19.69
3.6.2008 2:00
15.87
17.84
18.59
19.35
3.6.2008 3:00
3.6.2008 4:00
15.87
15.78
17.56
17.46
18.21
18.03
19.11
18.88
3.6.2008 5:00
3.6.2008 6:00
15.68
15.68
17.46
17.37
17.84
17.65
18.8
18.71
3.6.2008 7:00
15.12
17.37
17.65
18.71
3.6.2008 8:00
3.6.2008 9:00
15.96
15.96
17.37
17.37
17.84
18.21
18.68
18.65
3.6.2008 10:00
3.6.2008 11:00
16.06
15.96
17.46
17.46
18.68
19.44
18.62
18.56
3.6.2008 12:00
3.6.2008 13:00
16.53
16.99
17.65
18.31
20.19
21.14
18.59
18.56
3.6.2008 14:00
17.18
18.78
21.9
18.56
3.6.2008 15:00
3.6.2008 16:00
17.74
18.03
18.97
19.15
22.57
22.47
19.29
19.32
Mean
Std.dev.
14.45
1.24
15.49
1.76
15.40
2.83
15.71
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
0.6
0.1
2.4
0.73
13
2.7
5.5
1.4
34
10
57
7.2
PBDEs (ng/g)
PBDE 28
PBDE 47
PBDE 99
PBDE 100
PBDE 153
PBDE 154
PBDE 183
<0.061
0.069
<0.074
<0.074
<0.29
<0.19
<0.39
<0.17
<0.14
<0.2
<0.2
<0.78
<0.5
<1
<0.061
0.53
0.53
<0.12
<0.5
<0.35
<0.75
<0.073
0.32
0.16
<0.1
<0.37
<0.25
<0.57
<0.12
0.54
0.54
<0.18
<0.68
<0.43
<0.96
<0.057
0.75
1.00
<0.1
<0.43
<0.34
<0.99
PCBs (ng/g)
PCB 28+31
PCB 52
PCB 101
PCB 118
PCB 138
PCB 153+168
PCB 170
PCB 180
0.35
0.11
0.46
0.26
1.2
2.2
0.40
0.92
1.3
1.4
11.9
2.9
29.7
45.8
12.7
31.2
8.8
3.2
6.6
3.0
25
33.9
12.5
30.2
1.0
0.47
1.4
0.69
3.9
6.5
1.7
3.8
2.0
0.62
1.7
0.9
7.2
10.8
4.1
11
6.1
0.43
3.15
1.3
12.3
16.9
5.4
13.9
HCHs (ng/g)
alfa-HCH
beta-HCH
delta-HCH
gama-HCH
<0.23
<0.31
<0.36
1.0
<0.36
<0.49
<0.56
<0.44
<0.19
<0.26
<0.30
<0.23
<0.19
<0.25
<0.29
<0.23
<0.33
<0.44
<0.51
<0.40
<0.15
<0.21
<0.24
<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
p,p-DDD
o,p-DDE
p,p-DDE
o,p-DDT
p,p-DDT
<0.1
0.41
<0.13
0.68
<0.16
0.38
0.5
1.7
<0.36
1.7
<0.45
1.3
7.3
47.3
0.58
17.9
4.1
680
0.61
2.4
<0.15
2.6
0.72
5.7
1.1
3.7
<0.27
6.1
4.2
5.4
3.5
10.5
0.3
9.0
1.0
5.4
PAHs (µg/g)
phenantrene
anthracene
fluoranthene
pyrene
benzo(a)anthracene
chrysene
benzo(b)fluoranthene
benzo(k)fluoranthene
benzo(a)pyrene
benzo(ghi)perylene
dibenzo(ah)anthracene
indeno(1,2,3-cd)pyrene
0.11
0.029
0.42
0.33
0.35
0.18
0.17
0.12
0.27
0.14
0.009
0.2
2.0
0.47
3.0
2.1
1.9
1.1
0.95
0.6
1.3
0.65
0.075
1.1
1.0
0.15
2.6
1.9
1.8
1.1
1.2
0.76
1.6
0.92
0.086
1.5
2.1
0.44
4.0
2.8
2.4
1.4
1.2
0.77
1.7
0.86
0.083
1.4
0.77
0.1
2.1
1.6
1.5
0.87
1.0
0.62
1.4
0.82
0.083
1.4
2.7
0.56
4.9
3.4
3.3
1.9
1.9
1.2
2.5
1.3
0.14
2.2
Metals (µg/g)
Hg
Al
Cd
Cu
Pb
0.011
1630
0.12
5.86
16.1
0.12
3910
1.72
23.5
36.6
0.89
6070
2.51
79.6
68.9
0.68
3530
0.46
24.5
22.9
0.96
5030
1.32
51.7
50.6
0.96
5490
1.12
59.2
48.7
Triclosan (ng/g)
Me-triclosan (ng/g)
Zn
As
Ba
Co
Cr
Mo
Ni
Se
Ti
44.8
0.35
43
4.28
5.72
0.015
7.84
0.37
20.4
178
1.47
100
5.35
25.7
0.037
15.6
0.49
31.8
329
1.82
179
8.03
30.1
0.089
24.4
0.56
50.6
155
1.44
96
3.9
16.1
0.051
11.7
0.49
32.3
214
1.89
124
5.49
24.1
0.087
15.5
0.54
39.6
259
0.91
130
6.19
25.8
0.098
18.3
0.63
47.7
Table S3. Concentrations of studied pollutants in water (µg/l) from the study sites around Brno.
PESTICIDES
acetochlor
alachlor
atrazin
bromacil
carbofuran
cyanazin
desmetryn
diazinon
dichlobenil
diuron
chlorbromuron
chlorotoluron
isoproturon
linuron
hexazinon
metalaxyl
metobromuron
metolachlor
metoxuron
metribuzin
monolinuron
prometryn
simazin
terbuthylazine
terbutryn
2,4,5-T
2,4-D
MCPP
MCPA
dimethoat
desethylatrazin
methamidophos
methidathion
phorate
phosphamidon
tri-allate
methabenzthiazuron
bentazone
metamitron
nicosulfuron
rimsulfuron
tebuconazole
desisopropylatrazin
imazethapyr
thifensulfuron-methyl
thiophanate-methyl
ethofumesat
azoxystrobin
propyzamide
fenhexamid
fenarimol
fipronil
kresoxim-methyl
propiconazole
phosalone
fluazifop-p-butyl
pyridate
desethyldesisopropylatrazin
1a
2a
1b
2b
WWTP effluent
4
<0.019
<0.005
<0.009
<0.017
<0.017
<0.013
<0.002
<0.003
<0.013
<0.015
<0.041
<0.02
<0.015
<0.025
0.010
<0.005
<0.018
<0.01
<0.01
<0.015
<0.026
<0.005
<0.006
0.006
<0.003
<0.019
<0.017
<0.018
0.018
<0.005
<0.015
<0.019
<0.019
<0.028
<0.019
<0.036
<0.007
<0.016
<0.011
<0.004
<0.027
<0.009
<0.01
<0.006
<0.011
<0.014
<0.033
<0.01
<0.021
<0.017
<0.014
<0.044
<0.039
<0.013
<0.019
<0.028
<0.041
<0.012
0.230
0.029
0.013
<0.019
<0.019
<0.015
<0.002
<0.004
<0.015
0.080
<0.045
0.330
<0.017
<0.028
<0.008
<0.006
<0.02
<0.011
<0.012
<0.017
<0.029
<0.005
<0.007
0.190
<0.004
<0.021
<0.02
<0.021
<0.021
<0.005
<0.017
<0.022
<0.022
<0.031
<0.021
<0.04
<0.008
<0.018
<0.012
<0.004
<0.031
<0.01
<0.011
<0.007
<0.012
<0.016
<0.036
<0.012
<0.024
<0.019
<0.016
<0.049
<0.044
<0.014
<0.021
<0.032
<0.046
<0.013
0.054
0.027
<0.009
<0.018
<0.017
<0.013
<0.002
<0.004
<0.014
<0.016
<0.042
0.045
<0.016
<0.026
<0.008
<0.005
<0.018
<0.01
<0.011
<0.016
<0.027
<0.005
<0.006
0.017
<0.003
<0.021
<0.019
<0.02
<0.022
<0.005
<0.016
0.038
<0.02
<0.028
<0.019
<0.037
<0.007
<0.018
<0.011
<0.004
<0.028
<0.01
<0.01
<0.006
<0.011
<0.015
<0.034
<0.011
<0.022
<0.018
<0.015
<0.045
<0.04
<0.013
<0.019
<0.029
<0.043
<0.012
0.310
0.029
0.011
<0.018
<0.017
<0.014
<0.002
<0.004
<0.014
0.089
<0.042
0.350
<0.016
<0.026
<0.008
<0.005
<0.018
<0.01
<0.011
<0.016
<0.027
<0.005
<0.006
0.160
0.004
<0.019
<0.018
<0.018
0.046
<0.005
0.018
<0.02
<0.02
<0.029
<0.02
<0.038
<0.008
<0.016
<0.011
<0.004
<0.029
<0.01
<0.01
<0.006
<0.011
<0.015
<0.034
<0.011
<0.022
<0.018
<0.015
<0.046
<0.041
<0.013
<0.019
<0.029
<0.043
0.014
<0.021
0.033
0.032
<0.019
<0.019
<0.015
<0.002
0.015
<0.015
0.150
<0.045
<0.022
<0.017
<0.028
<0.008
<0.006
<0.02
<0.011
<0.012
<0.017
<0.029
<0.005
<0.007
0.200
0.037
<0.019
<0.017
<0.018
0.048
<0.005
0.043
0.078
<0.021
<0.031
<0.021
<0.04
<0.008
0.200
<0.012
<0.004
<0.031
0.011
0.013
<0.007
<0.012
0.031
<0.036
<0.012
<0.024
<0.019
<0.016
<0.049
<0.044
<0.014
<0.021
<0.032
<0.046
0.070
0.130
0.036
0.013
<0.017
<0.016
<0.013
<0.002
<0.003
<0.013
0.056
<0.039
0.087
<0.015
<0.024
<0.007
<0.005
<0.017
<0.009
<0.01
<0.015
<0.025
<0.004
<0.006
0.080
0.007
<0.02
<0.019
<0.02
0.046
<0.005
0.018
<0.019
<0.019
<0.027
<0.018
<0.035
<0.007
0.029
<0.01
<0.003
<0.027
<0.009
<0.01
<0.006
<0.01
<0.014
<0.032
<0.01
<0.021
<0.017
<0.014
<0.042
<0.038
<0.012
<0.018
<0.027
<0.04
0.012
2-hydroxyatrazin
bromoxynil
dichlorprop
0.014
<0.021
<0.018
0.013
<0.024
<0.021
0.013
<0.023
<0.02
0.022
<0.021
<0.018
<0.012
<0.021
<0.018
0.020
<0.022
<0.02
SULFONAMIDES
sulfomethoxazol
sulfapyridin
sulfamethazin
sulfamethoxypyridazin
sulfachloropyridazin
0.011
<0.009
<0.009
<0.011
<0.018
0.024
<0.011
<0.011
<0.012
<0.021
<0.017
<0.013
<0.014
<0.015
<0.026
0.022
0.043
<0.011
<0.012
<0.02
0.69
2.6
<0.01
<0.011
<0.019
0.080
0.26
<0.011
<0.012
<0.021
OTHER ANTIBIOTICS
trimetoprim
metronidazol
cefalexin
ofloxacin
norfloxacin
ciprofloxacin
enrofloxacin
erythromycin
doxycyclin
<0.009
<0.015
<0.016
<0.017
0.071
<0.014
<0.018
<0.002
<0.017
0.015
<0.014
<0.015
<0.016
0.082
<0.013
<0.016
0.005
<0.016
<0.013
<0.015
<0.016
<0.017
0.069
<0.014
<0.018
<0.002
<0.017
0.013
<0.016
<0.017
<0.018
0.063
<0.014
<0.018
0.004
<0.018
0.14
0.11
<0.015
<0.016
0.14
0.047
<0.016
0.064
<0.015
0.027
<0.015
<0.015
<0.016
0.045
<0.013
<0.017
0.010
<0.016
OTHER PHARMACEUTICALS
diclofenac
carbamazepine
diaverdin
0.010
0.028
<0.01
0.039
0.064
<0.012
<0.014
0.039
<0.015
0.023
0.067
<0.011
1.2
0.95
<0.01
0.13
0.15
<0.012
ALKYLPHENOLS
t-octylphenol
n-octylphenol
4-n-nonylphenol
p-nonylphenol
monoNPE
diNPE
Bisphenol A
0.013
<0.004
<0.005
0.028
0.003
<0.004
<0.006
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
0.005
<0.005
<0.007
<0.012
<0.003
<0.005
<0.008
<0.001
<0.007
<0.01
0.023
<0.004
<0.007
<0.011
0.014
<0.001
0.003
0.22
0.036
0.005
0.003
0.004
<0.006
<0.009
0.032
0.006
<0.006
<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.
1a
PESTICIDES
acetochlor
alachlor
atrazin
bromacil
carbofuran
cyanazin
desmetryn
diazinon
dichlobenil
diuron
chlorbromuron
chlorotoluron
isoproturon
linuron
hexazinon
metalaxyl
metobromuron
metolachlor
metoxuron
metribuzin
monolinuron
prometryn
simazin
terbuthylazine
terbutryn
2,4,5-T
2,4-DP
2,4-D
MCPP (mecoprop)
MCPA
dimethoat
desethylatrazin
methamidophos
methidathion
phorate
phosphamidon
tri-allate
methabenzthiazuron
bentazone
metamitron
nicosulfuron
rimsulfuron
tebuconazole
imazethapyr
thifensulfuron-methyl
thiophanate-methyl
ethofumesat
azoxystrobin
propyzamide
fenhexamid
fenarimol
fipronil
kresoxim-methyl
propiconazole
phosalone
fluazifop-p-butyl
31.3
4.1
21.4
<2.9
<2.2
<2.5
<0.36
<0.63
<2.5
10
<6.4
48.5
34.1
<4.1
14.4
1.5
<2.7
3.9
<0.88
4.1
<4.3
1.1
1.5
48.2
3.8
<1.6
1.1
11.3
34
35.7
<0.79
18.6
3.7
<2.9
<4.5
<3.2
<6
<1.3
5.3
<2
<0.57
<3.4
4.1
<1.2
<1.7
<3.4
<7.1
<1.8
<3.6
4.1
<1.7
<8.5
<3.9
4.9
<2.9
<3.9
2a
-
1b
-
2b
792
19.4
123
<2.9
2.1
<2.4
<0.36
3.1
<2.4
339
<6.3
474
45.3
26.1
14
2.2
<2.6
51.2
<0.87
7.5
<4.2
4.3
15.2
811
21
2.3
4.3
31.7
20
97.9
<0.78
78.7
<3.4
<2.8
<4.5
<3.2
<6
<1.3
5.7
<1.9
1.6
<3.4
23
1.4
<1.6
10.1
<7.1
3.2
<3.6
<1.4
<1.7
<8.4
<3.9
28.2
<2.9
<3.9
WWTP effluent
4
(duplicates)
(duplicates)
439
<6.7
893
<29
<22
<25
<3.7
56
<25
9212
<65
<34
272
<41
15.5
16.8
<27
<15
<9
<29
<44
31.1
58.9
2082
324
<17
<9.1
<41.4
69
139
8.3
279
<35
<29
<46
<33
<62
<13
69.7
<20
<5.8
<35
74.9
<12
<17
<17
<73
37.2
<37
104
<18
<87
<40
139
<30
<40
265
13.4
388
<24
<18
<20
<3
27
<20
4034
<53
<27
95
<33
<12
<7.7
<22
<12
<7.3
26.4
<35
15
26
921
150
<16
<8.6
<39
28
113
<6.5
113
<29
<24
<37
<27
<50
<11
92.9
<16
<4.7
<28
33.8
<9.7
<14
<14
<59
15.8
<30
<12
<14
<70
<33
51.4
<24
<32
130
5.1
63.6
<2.7
<2
<2.3
<0.34
3.6
<2.3
442
<6
100
27.9
8.8
10
2.3
<2.5
8.1
<0.83
10.1
<4
2.9
7.3
251
20.8
<1.6
<0.84
7.9
13
24.7
<0.74
34.8
<3.3
<2.7
<4.3
<3.1
<5.7
<1.2
10.1
<1.8
<0.54
<3.2
10.7
<1.1
<1.6
<3.2
<6.7
2.4
<3.4
<1.3
<1.6
<8
<3.7
13.8
<2.8
<3.7
180
3.7
59.2
<2.8
<2.1
<2.4
<0.35
3.5
<2.4
386
<6.1
92.4
31.1
5.6
8.2
1.2
<2.6
10.2
<0.85
9.5
<4.1
3
6.6
266
20.5
<1.8
1.8
15
20
46.3
<0.76
31.3
3.8
<2.8
<4.4
<3.1
<5.8
<1.3
11.8
<1.9
0.56
<3.3
10.9
<1.1
<1.6
<3.3
<6.9
2.3
<3.5
5.2
<1.7
<8.2
<3.8
<13.8
<2.8
<3.8
pyridate
clopyralid
bromoxynil
<3.5
174
<3.8
-
-
<3.4
40.3
<3.9
<36
<297
<40.8
<29
<280
<38.4
<3.3
91.4
<3.8
<3.4
90.6
<4.3
SULFONAMIDES
sulfomethoxazol
sulfapyridin
sulfamethazin
sulfamethoxypyridazin
sulfachloropyridazin
30
14
<5.7
<5.9
<5.5
-
-
87
71
6.7
<4.4
<4.1
3514
2223
46
<48
<44
1303
888
<73
<76
<69
160
99
5.5
<5.3
<4.9
202
102
4.9
<5
<4.6
OTHER ANTIBIOTICS
trimetoprim
metronidazol
cefalexin
ofloxacin
norfloxacin
ciprofloxacin
enrofloxacin
erythromycin
12
<11
<15
<11
15
<11
<10
1.9
-
-
13
<1.4
<1.9
<1.4
<1.3
<1.4
<1.3
0.9
503
18
<15
<11
13
<11
<10
32
212
113
<51
89
<68
<63
<67
87
26
1.8
<1.8
<1.3
<1.2
1.6
<1.2
1.4
26
1.9
<1.6
<1.1
<1.1
<1.1
<1.1
1.5
OTHER PHARMACEUTICALS
diclofenac
carbamazepine
diaverdin
30
85
<5.4
-
-
148
249
<4.1
11958
6536
<44
4185
2412
<75
358
356
<4.8
388
341
<4.6
PFOCs
PFHxS
FHUEA
FOSA
N-methyl FOSA
PFOA
PFOS
PFNA
<1.1
<1.2
<0.49
<0.53
1.7
<1.3
<0.47
-
-
<0.93
<1.1
<0.42
<0.45
9.1
9.5
5.7
<7.7
<8.8
<3.4
<3.7
184
18
6.7
<10
<10
<4.5
<3.9
72
<13
<4.3
<0.89
<1
<0.4
<0.43
9.9
3.9
0.82
<1.1
<1.3
<0.49
<0.53
9.6
3.2
<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).
1a
2a
1b
2b
4
WWTP effluent
(duplicates)
(duplicates)
Triclosan
Me-triclosan
127
210
-
182
257
659
446
37499
16319
30512
11664
3281
1598
3467
1961
PBDEs
PBDE28
PBDE47
PBDE100
PBDE99
PBDE154
PBDE153
PBDE183
<2.5
10.2
4.6
<2.8
<2.5
<1.6
<4.0
-
<1.9
12.9
6.0
<2.6
<2.4
<1.4
<4.2
<1.2
14.3
7.0
2.4
<1.8
1.2
<3.3
6.3
130
53.0
7.6
2.0
1.8
<1.6
4.4
90.3
33.6
10.1
2.7
3.3
<3.5
<1.8
20.0
9.1
<2.2
2.5
<1.3
<3.5
1.6
22.2
9.8
<1.0
<0.9
<0.6
<1.6
PCBs
PCB 28+31
PCB 52
PCB 101
PCB 118
PCB 138
PCB 153+168
PCB 170
PCB 180
18.1
28.8
86.8
130
136
158
16.5
23.5
-
191
46.1
33.3
12.0
29.1
55.1
7.3
16.2
1179
251
202
47.3
115
259
21.6
51.1
731
334
428
115
266
531
30.0
73.8
490
200
305
63.1
166
342
22.9
56.3
353
120
126
31.3
88.7
182
19.9
48.1
406
126
139
36.3
107
219
25.0
55.6
HCHs
alfa-HCH
beta-HCH
delta-HCH
gama-HCH
24.3
<6
161
<7
-
17.0
10.0
130
<7
14.3
<7
131
<7
29.7
24.9
598
<7
29.4
7.9
602
<8
13.1
<7
163
<7
14.9
7.5
168
<7
HCB
103
-
153
189
409
299
144
140
DDT and metabolites
o,p-DDE
p,p-DDE
o,p-DDD
p,p-DDD
o,p-DDT
p,p-DDT
2.3
106
24.7
77.1
22.6
27.3
-
6.3
157
69.5
192
58.3
50.9
8.1
146
74.2
190
88.6
208
12.2
192
81.7
134
66.2
70.9
9.3
145
63.4
93.5
38.1
53.9
6.9
147
70.3
191
54.6
94.2
6.6
155
73.5
209
54.0
112
PAHs
phenantrene
anthracene
fluoranthene
pyrene
benzo(a)anthracene
chrysene
benzo(b)fluoranthene
benzo(k)fluoranthene
benzo(a)pyrene
benzo(ghi)perylene
dibenzo(ah)anthracene
indeno(1,2,3-cd)pyrene
4354
239
5911
3045
313
48
151
79
72
80
<20
67
-
3219
333
10410
7332
2839
851
531
288
263
170
27
134
8884
1754
19328
16559
8672
2209
953
552
517
296
37
244
1523
556
13510
14529
13205
1629
788
473
740
293
47
174
1372
536
10549
11072
9524
1287
600
365
539
213
38
139
6231
1032
12804
9947
3445
1272
620
360
389
279
37
179
5312
959
12407
10138
4141
1084
611
362
391
276
32
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).