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 attentionjarosova@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