How reliable monitoring tool can be the estrogenic in ... 2010... · Introduction - in vitro assay...
Transcript of How reliable monitoring tool can be the estrogenic in ... 2010... · Introduction - in vitro assay...
How reliable monitoring tool can be the estrogenic in vitro assay?
Barbora Jarošová, R. Loos, B. Gawlik, L. Bláha, K. Hilscherová
Introduction
WFDEQSs – limits for good status of watersNP = 0.3 μg/L (Annual Average) E2 = 0.4 ng/L (Annual Average) EE2 = 0.035 ng/L (Annual Average)
Introduction
Results in
[ng/L EEQ]
In vitro bioassay (e.g. MVLN cells)
Introduction - in vitro assay as risk assessment tool ?Po
tent
ial r
elat
ive
to E
2
0.00001
0.0001
0.001
0.01
0.1
1
10
E1 E2 E3 EE2 Nonylphenol
in vitro in vivo
91 EU WWTPs Effluents JRC EC Ispra 5 EU
laboratories 16 EU countries Western and Central EU Domestic / Industrial / Rain
waters Activated sludge treatment
Sampling sites
CY
160 polar organic chemicals20 inorganic trace elements
Methods
Estrogenity of 75 samples analyzed by MVLN reporter gene bioassay
Methods
MCF – 7 (MVLN bioassay)
Glass fibre filtration Extracts dilution (6 conc.)
(MeOH)
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
0
5
10
15
20
25
30
Median Average Max.
ng/ L
EEQ
EU This study (N 75)Switzerland (N 5)Sweden (N 20)France (N 4)Germany (N 16)Netherlands (N 10)Netherlands Ind. (N 3)Germany Ind. (N 2)
53
Aerni et al. 2004Anal. Bioanal. Chem. 378:688-696
Cargouet et al. 2004 Sci. Total. Environ. 324:55-66
Korner et al. 2001 Environ. Toxicol. Chem.20:2142-2151
Vethaak et al. 2005 Chemosphere 59: 511-524
Discussion – Comparison with chemical analyses
MVLN
?Spearman
Correl. Sweeters Pharm. Silox.Musks OPFRs PCPs Benzotriazoles Vet.Antib. PFSs Nitrophenols Pest. Anorg.
Sweeters
Pharm. 0.52Silox.,Musks 0.31 0.38OPFRs 0.12 0.33 0.34PCPs 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 0.42PFSs 0.06 -0.08 -0.18 0.11 -0.08 0.04 0.32Nitrophenols 0.12 0.06 -0.03 -0.14 0.07 -0.01 0.02 -0.31Pest. 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 -0.06
EEQ 0.11 0.03 -0.16 -0.17 -0.07 -0.19 -0.14 -0.08 0.14 -0.08 0.10
Discussion – Comparison with chemical analyses
MVLN
NO But
E1, E2, EE2 LOQ was 10 ng/L and no sample > LOQ
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)
Discussion
What caused in vitro estrogenity in other EU WWTP effluents?
> 90 %Desbrow et al. 1998 Environ. Sci. Technol. 34:1548–1558Korner et al. 2001 Environ. Toxicol. Chem. 20:2142-2151Thomas et al. 2002 Environ. Toxicol. Chem. 21:1456Houtman et al. 2004 Environ. Sci. Technol. 38(23):6415-23Aerni et al. 2004 Anal. Bioanal. Chem. 378:688-696 …Cargouet et al. 2004 Sci. Total. Environ. 324:55-66 …
All otherExceptions: e.g. Kanzaki River in Japan3
(Genistein)
1 UK WIR 2012 report, 2012/7/5 http://www.ukwir.org2 Johnson et al. 2005 Water Research 39, 47–583 Kawanishi et al. 2004 Environmental Science & Technology 23, 6424
Discussion
Is there any stable ratio of major steroidal estrogens in WWTPs effluents ?
Median of reviewed ratios
56%10%
17% 4%
E1 % E2 % E3 % EE2 %
0102030405060708090
100
%
E1 E2 E3 EE2
Comparison with:Mie`ge et al. 2009a Environ. Pollut. 57:1721–1726
Aerni et al. 2004 Anal. Bioanal. Chem. 378:688-696Avberšek et al. 2011 Sci. Total. Environ. 409(23):5069-75Furuichi et al. 2004. Water Res. 38(20):4491-501Gutendorf and Westendorf 2001 Toxicology 166(1-2):79-89Houtman et al. 2004 Environ. Sci. Technol. 38(23):6415-23Korner et al. 2001 Environ. Toxicol. Chem. 20:2142-2151Legler et al. 2002 Environ. Sci. Technol .36(20):4410-5Metcalfe et al. 2001 in Caldwell et al. 2012. Environ Toxicol Chem. 1396-406Pawlowski et al. 2004 Toxicol. In Vitro. 18(1):129-38Rutishauser et al. 2004 Environ. Toxicol. Chem. 23(4):857-64Routledge 1997 in Caldwell et al. 2012. Environ Toxicol Chem. Snyder et al. 2001 Environ. Sci. Technol. 35(18):3620-5Sonneveld et al. 2006 Toxicol. Sci. 89(1):173-87Svenson, Allard 2003 Water Res. 37(18):4433-43Van den Belt et al. 2004 Aquat. Toxicol. 66(2):183-95
Discussion
In vitro potentials
00.20.40.60.8
11.21.41.61.8
E1 E2 E3 EE2
YES (N 6)
E-screen (N 1)
ER-calux (N4)
MVLN (N4)
This study
0.0030.01
1 Caldwell et al. 2012 Environ. Toxicol. Chem. 31(6):1396-406 2 UK Environment Agency, Technical Report, Young et al. 20043 Holbech et al. 2006 Comp Biochem. Physiol. C Toxicol. Pharmacol. 144(1):57-66
Discussion
In vivo derived PNECs 1;2;3
PNECE1 6 ng/LPNECE2 2 ng/LPNECE3 60 ng/LPNECEE2 0.1 ng/L
…
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/LE1 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 exceededWhat 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/LE1 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/LPNECE1 6 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/LE2 relative potency to E2…………...…………..1
E2 conc. ………...……from…….. 0.01 ng/L…………….…to……..1.1 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/LE3 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
PNECE3 60 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/LEE2 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
PNECEE2 0.1 ng/L
DiscussionWorst case:
If steroidal estrogens (SE) responsible for measured EEQsandIf 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
WFDEQS – limit for good status of watersNP = 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 [email protected]
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