1.
ENDOCRINE DISRUPTING CHEMICALS and ENDOCRINE POTENCY of WASTEWATER measured by IN VIVO and IN VITRO ASSAYS
K. Ole Kusk1, Hanne Frederiksen2, Eva C. Bonefeld-Jørgensen4,
Anna-Maria Andersson2, Anne E. Lykkesfeldt5, Christine Nellemann3,
Kamilla M.S. Hansen1, Tanja Krüger4, Manhai Long4,
Camilla Taxvig3, Henrik R. Andersen1
1Dept. Environmental Engineering, Technical University of Denmark
2Dept. Growth and Reproduction, Copenhagen University Hospital, Denmark
3National Food Institute, Dept. of Toxicology and Risk Assessment, Technical University of Denmark
4Cellular & Molecular Toxicology, Centre of Arctic Environmental Medicine, Institute of Public Health, Aarhus University, Denmark
5Dept. Tumor Endocrinology, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark
Corresponding author:
We have a lot of authors since each is only measuring with a few methods and everybody has a boss who need to be on the paper too.
I am making this presentation on behaf of Ole who has been principial investigator but had to stay home and take care of the teaching while the group is enjoying the sun here with you.
DanEd

2.
Aim of the project
The question: Does wastewater cause other hormonal disrupting effects we need to consider in treatment evaluation besides estrogen agonists (– probably)
Evaluate presence of different type of hormonal disrupting effects
- 2x Influent to STPs
- 2x Effluent from STPs
Wastewater
inlet
Wastewater
outlet
We participate in a project about endocrine disruptors. Our task is to

3. Content Sampling and pre-treatment Chemical analyzes
Content
Sampling and pre-treatment
Chemical analyzes
Ecotoxicological test
Bioassays
Conclusions

4. 1- Sampling and pre-treatment
Influent and effluents from two sewage treatment plants (STPs) were sampled
Lynetten STP
( PE)
Usserød STP
( PE)
Source
Domestic, small industries
Domestic, hospital
Recipient
Sea
Small stream
Total Nitrogen
<15 ppm
<8 ppm
COD
25-75 ppm
10-30 ppm

5. 1- Sampling and pre-treatment
1- Sampling and pre-treatment
Sample preparation
Grab samples
Influent Effluent
pH → 3
pH → 3
Filter
500 ml subsamples
1000 ml subsamples
SPE
Dry. Freeze -18 °C

6. Treatment of extracts 0.5 or 1.0 L Chemical analysis Bioassay
1- Sampling and pre-treatment
SPE are distributed to laboratories
Organic solvent
Organic solvent
0.5 or 1.0 L
Bioassay
Chemical analysis µL
(concentrated
samples)

7. Chemical analyses (Agilent triplequad) (Varian GC Iontrap)
2- Chemical analyses
Chemical analyses
LC-MS/MS GC MS-MS
(Agilent triplequad) (Varian GC Iontrap)
13 phthalate metabolites 3 industrial phenols
5 parabens sunscreen chemicals
3 steroid estrogens

8.
2- Chemical analyses
Selected estrogenic compounds in effluent from two Danish sewage treatment plants, Usserød and Lynetten, quantified by GC-MS-MS
Concentrations in ng/L
LOQ*
Usserød
effluent
Lynetten
Industrial phenols
Octylphenol
2.5 16 50
isoNonylphenol 14 27
Bisphenol A 60
172
Sunscreen chemicals
Homosalate 25
N.D**
Benzophenone-3
158 81
Benzophenone-7 4 2
3-(4-Methylbenzyliden)camphor
7.5
N.D.
Octyl methoxycinnamate 23 12
Octyl dimethylaminobenzoate
Steroid estrogens
Estrone 1
< 1
17-β-Estradiol
Ethinyl estradiol
*LOQ Limit of quantification; ** N.D. not detected;

9.
2- Chemical analyses
Phthalate metabolites and parabens in influent and effluent from two Danish sewage treatment plants, Usserød and Lynetten, quantified by LC-MS/MS
LOD*
Usserød STP
Lynetten STP
Concentrations in ng/L
influent
effluent
Mono-ethyl phthalate
1.6
751
2.5
1814
< LOD
Mono-butyl phthalate
8.4
1970 46
2996
129
Mono-3-carboxypropyl phthalate
3.7
134
7.5
110 63
Mono-benzyl phthalate
2.3
1101
404
Mono(2-ethylhexyl) phthalate
3.0
383 37
891 52
Mono(2-ethyl-5-hydroxyhexyl) phthalate 83
245
Mono(2-ethyl-5-oxyhexyl) phthalate
1.5 53
126
Mono(2-ethyl-5-carboxyhexyl) phthalate
1.0 94
251
Mono-octyl phthalate
3.4
9.1
Mono-iso-nonyl phthalate
4.0 28
Mono-iso-nonyl phthalate (one hydroxy group) 30 58
Mono-iso-nonyl phthalate (one keto group) 16 34
Mono-iso-nonyl phthalate (one carboxy group)
2.4 57
119
Methyl paraben
2.8
17462
16322
262
Ethyl paraben
1.9
9193
10519
290
Propyl paraben
12105 50
18631
231
Butyl paraben
1.2
4483
4.6
5641
550
Benzyl paraben
0.9 14 12

10. Bioassays Steroid synthesis (H295R assay ) ER activation (YES assay)
3- Bioassays
Bioassays
Steroid synthesis (H295R assay )
ER activation (YES assay)
Cell proliferation (MCF-7 cell assay)
Expression of the estrogen inducible progesterone receptor (MCF-7 cell assay)
Transactivation of the ER (MCF-7 stably transfected MVLN cells)
Androgenic or anti-androgenic (AR) transactivation assay (Chinese Hamster Ovary CHO cells)
Thyroid hormone (TH) cell proliferation (GH3 cell proliferation (T-screen)
Dioxin-like compounds assayed with transfected mouse heptoma cells (Hepa1.12cR carrying the AhR-luciferase reporter gene)

11. H295R Assay Steroid synthesis
3- Bioassays
H295R Assay Steroid synthesis
Testosterone
500
1000
1500
2000
2500
3000
3500
Usserød
Lynetten
ng hormone/L SC
10 000
20 000
mL wastewater/L
Influent
Effluent *
SC: Solvent Control

12. H295R Assay Steroid synthesis
3- Bioassays
H295R Assay Steroid synthesis
Estradiol
300 *
Usserød
Lynetten *
250
200
ng hormone/L
150
100 * 50 SC
10 000
20 000
mL wastewater/L
Influent
Effluent
SC: Solvent Control

13. YES assay ER activation
3- Bioassays
YES assay ER activation

14. Wastewater concentration (ml sample/L)
MCF-7 cell assay (increased growth up to 100/2000 mL/L then cytotoxicity)
3- Bioassays
Tox
Wastewater concentration (ml sample/L)

15. GH3 cell proliferation Thyroid hormone effects
3- Bioassays
GH3 cell proliferation Thyroid hormone effects T -
Screen
Lynetten
Usserød
Lynetten
Usserød
Influent
Influent
Effluent
Effluent
6 000 * *
5 000 *# #
4 000
GH3 cell proliferation/L # # * *
3 000
2 000
1 000 SC
625
1 250
2 500
5 000
10 000 SC
625
1 250
2 500
5 000
10 000 SC
1 250
2 500
5 000
10 000
20 000 SC
1 250
2 500
5 000
10 000
20 000 SC SC SC SC
SC=Solvent Control Concentrations in: mL wastewater/L
Values represent the mean ± SD of at least two independent assays performed in triple.
* indicates a significant difference compared to the solvent control.
# indicates cytotoxicity.

16. AhR-transactivity Dioxine like effects
3- Bioassays
AhR-transactivity Dioxine like effects SC
260
640
1 600
4 000
10 000
130
320
800
2 000
5 000
SC=Solvent Control Concentrations in: mL wastewater/L
Effluent
Usserød
Lynetten
Influent

17. Bioequivalents of hormones in assays
3- Bioassays
Bioequivalents of hormones in assays
Assay
Lynetten
influent
effluent
Usserød
Influent
Bio-EEQ
(ng E2/L water)
YES  29
3.0
90% 26
2.7
ER transactivation
32 ± 0.3
2.5 ± 0.3
92%
31 ± 2.8
1.4 ± 0.2
96%
Bio-TEQ
(ng TCDD/L water)
AhR transactivation
7.6± 1.6
3.0± 0.6
61%
7.0± 1.4
1.7± 0.4
76%
Bio-T3EQ
(ng T3/L water)
GH3 proliferation
284
5.1
98%
5.8

18.
4- Ecotoxicity test
Ecotoxicity test
Early life-stage development test on a marine copepod Acartia tonsa

19. Early life-stage test with copepod
4- Ecotoxicity test
Early life-stage test with copepod
Lynetten influent
Lynetten effluent
Usserød influent
Usserød effluent
EC10
110
(89-140)
770
( )
320
( )
> 2000
EC50
230
( )
> 1000
510
( )
Effect concentrations (EC) (mL wastewater/L)
causing 10 and 50 % inhibition of the development of early life stages of Acartia tonsa

20. CONCLUSIONS Effects of wastewater extracts
5- Conclusions
CONCLUSIONS Effects of wastewater extracts
All EDC assays responded to in- and effluents from both STPs
Steroid synthesis and metabolism (Dose-response not good)
E2 receptor activation
E2 sensitive growth stimulation
Androgen receptor activation
AhR transactivation stimulated (presence of dioxine-like substances)
Thyroid hormone like activity was increased during treatment (Agonist-antagonist mixture)
Concentrations and effects of EDCs reduced but not eliminated in effluents.
The ecological relevance of these findings is unknown – but active substances (EDCs) are discharged and potentially they may affect wildlife.
Weak ecotoxic effect was observed in the effluent from one STP.

21. CONCLUSIONS Most relevant parameters for WW treatment
Chemical analysis
? Industrial phenols (Bisphenol A, Nonylphenol).
? Steroid estrogen.
+ Sunscreen (BP-3, OMC).
-/+ Paraben (butylparaben).
- Phatalate ester metabolites.
Cell based bioassays
? Testosterone & estradiol biosynthesis. (+ with fractionation).
+ Estrogen assays. YES or E-screen.
+ AhR (dioxin). Low treatment effectiveness.
-/+ Thyroid hormone assay. Not so relevant with well treated WW.
Ecotoxicity
-- Copepod development. Low sensitivity. No effect in effluents. €.