How reliable monitoring tool can be
the estrogenic in vitro assay?
Barbora Jarošová, R. Loos, B. Gawlik, L. Bláha, K. Hilscherová
Introduction
WFD
EQSs – limits for good status of waters
NP = 0.3 μg/L (Annual Average)
E2 = 0.4 ng/L (Annual Average)
EE2 = 0.035 ng/L (Annual Average)
Introduction
In vitro bioassay (e.g. MVLN cells)
Results in
[ng/L EEQ]
Potential relative to E2
Introduction - in vitro assay as risk assessment tool ?
in vitro
10
in vivo
1
0.1
0.01
0.001
0.0001
0.00001
E1
E2
E3
EE2
Nonylphenol
Sampling sites
 91 EU WWTPs Effluents
 JRC EC Ispra  5 EU
laboratories
 16 EU countries
 Western and Central EU
 Domestic / Industrial / Rain
waters
 Activated sludge treatment
CY
Methods
 160 polar organic chemicals
20 inorganic trace elements
Methods
 Estrogenity of 75 samples analyzed by MVLN reporter
gene bioassay
Glass fibre filtration
Extracts dilution (6 conc.)
(MeOH)
MCF – 7
(MVLN bioassay)
Results – estrogenity - MUNICIPAL WWTPs
 16 out of 48 MUNICIPAL WWTPs showed EEQ > LOD 0.5 ng/L.
Results – INDUSTRIAL and unknown WWTPs
 5 out of 12 INDUSTRIAL WWTPs showed EEQ > LOD 0.5 ng/L.
Cytotoxicity
Discussion
 EEQs ̴ well comparable to other EU studies
Aerni et al. 2004
Anal. Bioanal. Chem.
378:688-696
53
30
EU This study (N 75)
Cargouet et al. 2004
Sci. Total. Environ.
324:55-66
Switzerland (N 5)
25
Sweden (N 20)
ng / L EEQ
France (N 4)
20
Korner et al. 2001
Environ. Toxicol.
Chem.
20:2142-2151
Germany (N 16)
Netherlands (N 10)
15
Netherlands Ind. (N 3)
Germany Ind. (N 2)
Vethaak et al. 2005
Chemosphere
59: 511-524
10
5
0
Median
Average
Max.
Discussion – Comparison with chemical analyses
?
MVLN
Spearman
Correl.
Sweeters
Pharm. Silox.Musks OPFRs
PCPs
Benzotriazoles Vet.Antib.
PFSs
Nitrophenols
Pest. Anorg.
Sweeters
Pharm.
0.52
Silox.,Musks
0.31
0.38
OPFRs
0.12
0.33
0.34
PCPs
0.17
0.31
0.24
0.22
Benzotriazoles
0.05
0.24
0.01
0.05
0.21
Vet.Antib.
0.14
-0.22
-0.03
0.13
-0.21
0.42
PFSs
0.06
-0.08
-0.18
0.11
-0.08
0.04
0.32
Nitrophenols
0.12
0.06
-0.03
-0.14
0.07
-0.01
0.02
-0.31
Pest.
0.08
0.11
0.06
0.09
0.05
0.22
0.21
0.10
-0.18
Anorg.
-0.26
-0.39
-0.35
-0.15
-0.16
-0.16
-0.22
-0.19
0.02
EEQ
0.11
0.03
-0.16
-0.19
-0.14
-0.08
0.14
-0.17 -0.07
-0.06
-0.08 0.10
Discussion – Comparison with chemical analyses
MVLN
NO 
But
E1, E2, EE2 LOQ was 10 ng/L and no sample > LOQ
Discussion
 What caused in vitro estrogenity in other EU WWTP effluents?
> 90 %
Desbrow et al. 1998 Environ. Sci.
Technol. 34:1548–1558
Korner et al. 2001 Environ. Toxicol.
Chem. 20:2142-2151
Thomas et al. 2002 Environ. Toxicol.
Chem. 21:1456
Houtman et al. 2004 Environ. Sci.
Technol. 38(23):6415-23
Aerni et al. 2004 Anal. Bioanal. Chem.
378:688-696 …
Cargouet et al. 2004
Sci. Total. Environ. 324:55-66 …
1
Nonylphenol (NP)
> 100 µg/L ~ main cause
< 1 µg/L ~ less than 1%
UK 95%ile: 0.49 µg/L 1
17 EU WWTPs: 0.05 -1.31 µg/L2
e.g. Sole et al. 2000 Environ. Sci. Technol.
34:5076-5083 (289 µg/L)
UK WIR 2012 report, 2012/7/5 http://www.ukwir.org
2 Johnson et al. 2005 Water Research 39, 47–58
3 Kawanishi et al. 2004 Environmental Science & Technology 23, 6424
All other
Exceptions:
e.g. Kanzaki
River in
Japan3
(Genistein)
Discussion
 Is there any stable ratio of major steroidal estrogens
in WWTPs effluents ?
Median of reviewed ratios
100
90
4%
17%
80
70
56%
%
60
10%
50
40
30
E1 %
E2 %
E3 %
EE2 %
20
10
0
E1
E2
E3
EE2
Comparison with:
Mie`ge et al. 2009a Environ. Pollut.
57:1721–1726
Discussion
In vitro potentials
1.8
YES (N 6)
1.6
E-screen (N 1)
1.4
ER-calux (N4)
1.2
MVLN (N4)
1
This study
0.8
0.6
0.4
0.01
0.003
0.2
0
E1
E2
E3
EE2
Aerni et al. 2004 Anal. Bioanal.
Chem. 378:688-696
Avberšek et al. 2011 Sci. Total.
Environ. 409(23):5069-75
Furuichi et al. 2004. Water Res.
38(20):4491-501
Gutendorf and Westendorf
2001 Toxicology 166(1-2):79-89
Houtman et al. 2004 Environ.
Sci. Technol. 38(23):6415-23
Korner et al. 2001 Environ.
Toxicol. Chem. 20:2142-2151
Legler et al. 2002 Environ. Sci.
Technol .36(20):4410-5
Metcalfe et al. 2001 in Caldwell
et al. 2012. Environ Toxicol
Chem. 1396-406
Pawlowski et al. 2004 Toxicol.
In Vitro. 18(1):129-38
Rutishauser et al. 2004 Environ.
Toxicol. Chem. 23(4):857-64
Routledge 1997 in Caldwell et
al. 2012. Environ Toxicol Chem.
Snyder et al. 2001 Environ. Sci.
Technol. 35(18):3620-5
Sonneveld et al. 2006 Toxicol.
Sci. 89(1):173-87
Svenson, Allard 2003 Water
Res. 37(18):4433-43
Van den Belt et al. 2004 Aquat.
Toxicol. 66(2):183-95
Discussion
In vivo derived PNECs 1;2;3
PNECE1
6 ng/L
PNECE2
2 ng/L
PNECE3 60 ng/L
PNECEE2 0.1 ng/L
…
1
Caldwell et al. 2012 Environ. Toxicol. Chem. 31(6):1396-406
2
UK Environment Agency, Technical Report, Young et al. 2004
3
Holbech et al. 2006 Comp Biochem. Physiol. C Toxicol. Pharmacol. 144(1):57-66
Discussion
 Estimation
of probable conc. of E1, E2, E3 and EE2 :
Example: Total EEQ (E2 equiv.)…………….……….....……..2 ng/L
E1 ratio…………………………………………….. 19 - 99 %
 E1 (E2 equiv) .........from.......0.20 x 2 = 0.38 ng/L
...………to…....0.99 x 2 = 1.98 ng/L
E1 relative potency to E2…………...………….…..0.13
 E1 conc. …………from... 0.38 / 0.13 = 2.9 ng/L
……………to……..1.8 / 0.13 = 15.2 ng/L
PNECE1 6 ng/L might have been exceeded
What if steroidal estrogens responsible only for 50 % ?
Discussion
 Estimation
of probable conc. of E1, E2, E3 and EE2 :
Example: Total EEQ (E2 equiv.)…………….………………..2 ng/L
E1 ratio……………………………………….. 19 - 99 %
 E1 (E2 equiv) .....from......0.20 x 2 = 0.38 ng/L
...…to…....0.99 x 2 = 1.98 ng/L
E1 relative potency to E2…………...………….…..0.13
 E1 conc. ……from…. 0.38 / 0.13 = 2.9 ng/L
……to…….1.8 / 0.13 = 15.2 ng/L
E2 ratio……………………………………...0.5 - 55 %
 E2 (E2 equiv) ...from......0.005 x 2 = 0.01 ng/L
……to…....0.55 x 2 = 1.11 ng/L
E2 relative potency to E2…………...…………..1
 E2 conc. ………...……from…….. 0.01 ng/L
…………….…to……..1.1 ng/L
PNECE1 6 ng/L
PNECE2 2 ng/L
E3 ratio………………………………………...0.5 - 72 %
 E3 (E2 equiv) .....from.....0.005 x 2 = 0.01 ng/L
………to…....0.72 x 2 = 1.44 ng/L
E3 relative potency to E2…………...……………...0.11
 E3 conc. …….from…0.01 / 0.11 = 0.9 ng/L
...……to…..1.44 / 0.11 13.6 ng/L
EE2 ratio……………………………………...0.2 - 19 %
 EE2 (E2 equiv) ..from. 0.002 x 2 = 0.004 ng/L
………to…....0.19 x 2 = 0.38 ng/L
EE2 relative potency to E2…………...…………..1.09
 EE2 con. . from…0.004 / 1.09 = 0.004 ng/L
….…to…….. 0.38 / 1.09 = 0.35 ng/L
PNECE3 60 ng/L
PNECEE2 0.1 ng/L
Discussion
 Worst case:
If steroidal estrogens (SE) responsible for measured EEQs
and
If actual ratio of SE was similar to any of the reported values
(27 WWTPs)
than:
max. 25 out of 75 EU WWTPs effluents exceeded any of PNEC(E1, E2, E3, EE2)
(max. 33% of tested samples, EEQ > 0.6 ng/L)
 Is the worst case scenario realistic ? (municipal vs. industrial WWTPs)
 Most frequently exceeded PNECEE2 and PNECE1
 Dilution in river!
Conclusions – in vitro assay as risk assessment tool ?
 In
vitro EEQ ≠ In vivo EEQ
 In vitro assays  estimation of concentrations
of responsible compounds (steroidal estrogens)
 if well determined in vitro potencies
 range of ratios of responsible compounds
 Estimated concentrations – can be compared
to PNECs (in vivo based)
Conclusions
WFD
EQS – limit for good status of waters
NP = 0.3 μg/L (Annual Average)
E2 = 0.4 ng/L (Annual Average)
EE2 = 0.035 ng/L (Annual Average)
 Estimated concentrations – can be compared to EQSs
Conclusions
 Determination of negative samples by in vitro assay
 Comparison of calculated SE concentration
with SE modeled concentrations
Acknowledgment
• Project CETOCOEN (CZ.1.05/2.1.00/01.0001) from the
European Regional Development Fund
• JRC EC Ispra, Italy
• John P. Giesy, Department of Biomedical Veterinary
Sciences and Toxicology Centre, University of
Saskatchewan, Saskatoon, Saskatchewan, Canada
• RECETOX, Masaryk University, Brno, Czech Republic
Thank you for your attention
jarosova@recetox.muni.cz
References (not previously cited in presentation)
Baronti et al. 2000 Environ. Sci. Technol. 34:5059–5066 107
Björkblom et al. 2008 Chemosphere 73:1064–1070
Caldwell et al. 2012 Environ. Toxicol. Chem.. 1396-406
Claraa et al. 2005 Water Res. 39:97–106
Labadie, Budzinski 2005 Environ. Sci. Technol. 39(14):5113-20
Legler et al. 2002. Environ. Sci. Technol. 36(20):4410-5
Muller et al. 2008 Environ. Toxicol. Chem. 27(8):1649-58
Pothitou, Voutsa 2008 Chemosphere 73(11):1716-23
Thomas et al. 2007 J. Environ. Monit. 9(12):1410-8