Fate of estrogens in sewage treatment process andeffect of treated sewage on fish
Koya Komori *, Yuji Okayasu*, Kiyoshi Miyajima*, Tadashi Higashitani*, Norihide Nakada*
and Yutaka Suzuki ** Water Environment Research Group, Public Works Research Institute, 1-6, Minamihara, Tsukuba City, Ibaraki, 305-8516,
Japan( komori@pwri.go.jp ; okayasu@pwri.go.jp ; miyajima@pwri.go.jp ; higashi44@pwri.go.jp ; nakada55@pwri.go.jp ;ysuzuki@pwri.go.jp )Abstract: Estrogens in treated sewage are suspected to pose an endocrine disrupting effect on fish, but it is becoming clear that mostof the estrogens can be removed in a sewage treatment process. However, the knowledge about the relation between operation
conditions and removal efficiency and the accurate fate in a sewage treatment process are restricted. Moreover, it cannot be said thatsufficient information has been obtained about the effect of treated sewage on fish. This paper reports the results of a review of therelationship between MLDO concentration and estrogen removal in aeration tanks of activated sludge process, and the biologicalimpact of treated sewage. The batch experiment where MLDO is controlled was conducted. Although no major change in E1 wasobserved when MLDO was still at a low level, it was confirmed that E1 concentration suddenly dropped when MLDO reached 3
mg/L. The rate of E1 reduction is speculated to depend closely on the concentration of dissolved oxygen. The exposure of medaka totreated sewage lead to the clarification of the relationship between estrogenic activity and the vitellogenin concentration in the sample
water. It was also confirmed that the higher estrogenic activity, the higher the rise seen in vitellogenin concentration.
Keywords: estrogens, estrone, 17ȕ-estradiol, feminization of fish, medaka,

Introduction³Our Stolen Future,´ authored by Dr. Theo Colborn et al., was translated into Japanese and published in September 1997. This bookbrought the problem of endocrine disruptors (EDs), popularly called environmental hormones, to public notice in Japan. Since then, avariety of investigations and surveys have been conducted in the field of sewage treatment. Since environmental hormones are known
to be able to affect life at even extremely low concentrations, they have drawn a veryhigh level of public concern. The Ministry ofLand, Infrastructure andTransport (MLIT), as well as local governments and research institutes, have engaged in active research fromthe viewpoint of preventing any impact on human health and the ecosystem.
Following an investigation of 47 sewage treatment plants conducted jointly by MLIT and local governments, a report was issued
noting that levels of environmental hormones in sewage influent were greatly decreased during the sewage treatment process, andmany substances decreased by over 90%,. Estrogens originating in humans and animals (17ȕ-estradiol or E2 and estrone or E1)
showed a steep drop in levels in discharged water. Most of those substances were in the form of estrones [MLIT, 2001]. However,there is a conspicuous lack knowledge of the details of the behavior of estrogens during the sewage treatment process and therelationship between the operating conditions of the plant and the removal efficiency. To help sewage treatment plants adopt specificstrategies to minimize outflow of these trace chemicals in the future, it is necessary to accumulate the basic information required tohelp predict and evaluate the impact of the operational conditions of the sewage treatment plant on the discharge of these chemicals tothe environment.
The numerous reports have been issued in Europe and North America on anomalies in reproductive organs of wildlife or theirabnormal reproductive conduct have propelled the environmental hormone issue to the top of the environmental agenda [e. g., Jobling
et al., 1996; Routledge et al., 1998; Gray et al., 1999; Metcalfe et al., 2001; Jones et al., 2003]. Some of the phenomena reported
include the feminization of male river fish living near the discharge ports of sewage treatment plants, with hermaphrodite fish observed

1- 381 -

to have eggs inside their testes [Routledge et al., 1998]. The causes of these phenomena are probably disturbance of the fish endocrine
system by chemicals emitted as a result of human activity. The chemicals that predominantly cause feminization are reported to be
estrogens, including E2 and E1, and a synthetic estrogen, 17D-ethynylestradiol or, EE2. However, there are fewer reports in Japan on
the biological impact of treated sewage and sufficient findings have not been obtained. In urban areas, the ratio of treated sewage in theflow of rivers to which sewage treatment plants discharge water is increasing, making it urgentlynecessary to clarify the biologicalimpact of treated sewage.
This paper reports the results of a review of the relationship between MLDO concentration and estrogen removal in aeration tanks ofactivated sludge process, and the biological impact of treated sewage.

Estrogen-like Activity in Sewage Influent, Treated Sewage and River water

Non--exceeding probability (%)

The EDs that most noticeably cause feminization are called estrogen-like substances, since they cause conditions similar to thosegenerallyresulting from the presence of estrogen inside the body. They can be measured as estrogenic activity (17ȕ-estradiol or E2
activity equivalent) using DNArecombinant yeasts with estrogen receptor genes. In our experiments, estrogenic activity was measured
using a DNArecombinant yeast stock provided by Prof. John Sumpter of Brunel University, UK. The DNArecombinant yeast isdesigned to generate ȕ-galactosidase when estrogens or estrogen-like substances bond with estrogen receptors (ERs). For themeasurement of estrogenic activity using the DNArecombinant
100%
yeast method, we followed the method developed byYakou et al.
90%
80%[Yakou, 1999], itself an improved version of the method ofTreated sewage
70%
n=77
Routledge et al.
60%
Figure 1 shows the distribution of estrogenic activity concentrationin sewage treatment plants and rivers in Japan. About 80% of
estrogenic activity was removed in the treatment processes ofsewage treatment plants on average; however, the treated sewage
still carries a concentration of 16 ng/l-E2 activity equivalent(medium) on discharge to the environment. This concentration is ahundred times higher than the median concentration in rivers,
which predicts a rise in estrogenic activity if the ratio of treatedsewage to the entire flow of a river increases.

50%
40%
30%
20%
10%
0%
0.01

River water
n=167

0.1

Sewage influent
n=87

1

10

100

1000

10000

Estrogenic activity as E2 equivalent (ng/L)

Figure 1:䇭Concentration distribution of estrogenic activity
in sewage influent, treated sewage and river water

Estrogenic activity䇭䋨ng/L-E2䋩

Theoretical activity was calculated from the concentrations of E1, E2 and nonylphenol (NP) obtained from chemical analysis of the
sewage influent and treated sewage of 25 sewage treatment plants. We studied to what extent these substances contributed to estrogenic
activity as obtained using the DNA recombinant yeast method. The estrogenic activity at each treatment plant and the corresponding
theoretical values of E1, E2 and NP are shown in Figure 2.
Although there are numerous samples that can be explained by
Theoretical values
YESNP
the presence of these three substances, many still defy such
100
E2
Sewage influent
E180
explanation, with the ratio between them varying according to60
treatment plant. It was also found that more than half of the 25
40
20
samples taken from both the sewage influent and treated0㪐 㪈 㪏 㪎 㪌 㪊 㪈㪏 㪉㪋 㪈㪐 㪉㪈 㪉㪌 㪉 㪈㪊 㪍 㪈㪇 㪉㪇 㪈㪎 㪉㪉 㪈㪈 㪈㪋 㪉㪊 㪋 㪈㪍 㪈㪌
sewage had a high content of estrogen-like active substancesthat cannot be traced to these three substances alone. To
100
Treated sewage
80
ascertain the contribution of each such substance, although60
there are some samples in the sewage influent that contain E1,40
20
E2 and NP each at certain levels, we took numerous samples0㪐 㪈㪈 㪈㪎 㪏 㪉㪇 㪉㪈 㪋 㪈㪇 㪉㪊 㪈㪏 㪉㪌 㪈㪐 㪊 㪉 㪍 㪉㪋 㪎 㪉㪉 㪈㪋 㪈㪌 㪈 㪈㪍 㪌 㪈㪉 㪈㪊from treated sewage that had a high E1 content. It was found
WWTP
that E1 is an important contributory substance for the
Figure 2: Contribution of each substance to estrogenic activity
estrogenic activity seen in treated sewage.

2- 382 -

Investigation of Behavior of Estrogen in Sewage Treatment Plants
Investigation method
We investigated a sewage treatment plant that serves 30,500 people with a conventional activated sludge process with a daily capacityof 20,400 m3 in January and July 2004. For both investigations, samples were taken from each treatment process every 2 hours over 24hours, and composite samples over 24 hours for flow proportion were prepared. The samples were analyzed for E2, E1, nonylphenol,
estrogenic activityas well as the items listed in environmental quality standards related to the preservation of the living environment,such as SS and BOD.

Results and discussion
The samples from both dates show a good removal efficiency (over 90%) for SS, BOD and COD, but the removal ratio of T-N turned
out to be low for the winter samples (16% in winter as opposed to 41% in summer). The samples from both seasons had a highremoval efficiency for E2 (70% in winter and 87% in summer), whereas the concentration of E1 increased as treatment proceeded(740% up in winter and 50% up in summer) with this trend particularly prominent in winter. For estrogenic activity (Fig. 3), theactivity level for the winter investigation rose as the treatment proceeded (removal efficiency of ±97% in winter, but 39% in summer).
in Winter survey

Recycle flow

44

1895
1355

1140 1450
500 mg/day
E2 equivalent

A

B

Drained sludge

CD

976 mg/day
E2 equivalent

Return sludge

36

1048

in Summer survey
Recycle flow

1036
739 mg/day
E2 equivalent

A

886

440

459
C

B

D

Drained sludge
Return sludge

1462

339 mg/day
E2 equivalent

Dissolved
Suspended

Figure 3: Mass balance of estrogenic activity (a, in mg/day, E2equivalent)through physicochemical treatment (A and C), biological treatment (B), and chlorination
process (D) during winter and summer in a municipal sewage treatment plant in Japan.
A: Primary settling tank; B: Aeration tank; C: Final settling tank D: Chlorination tankAccording to E1, E2 and NP concentration quantified by LC/MS/MS or HPLC analysis, the majority of estrogenic activity values
detected in samples of each sewage treatment process were attributable to E1. In the summer, the plant was operated with a level of
SRT and DO concentrations that promote nitrification, but in winter, the plant was operated at a reduced DO concentration in theaeration tank to reduce nitrification. These operation conditions mayhave affected the treatment of estrogen.

Relationship between MLDO concentration in the aeration tank and estrogen removal
As mentioned earlier, it is possible to speculate that removal of estrogen is affected by its relationship with the operational conditions of
the sewage treatment plant (DO control).A batch experiment was therefore conducted to clarify the relationship between the MLDO
concentration in the aeration tank and estrogen removal to find appropriate operation conditions that can stably reduce estrogen in the
sewage during treatment.

MethodAnalysis of the substances under study
Eight estrogens were to be investigated, including E2, E1, estriol (E3), and EE2 as well as sulfate conjugates of E2, E1 and E3, or
estrone-3-sulfate (E1-S), ȕ-estradiol-3-sulfate (E2-S), estriol-3-sulfate (E3-S), and estradiol 3, 17-disulfate (E2-diS). These substances

3- 383 -

were measured using the solid-phase extraction-LC/MS/MS method. The details of the analysis method are shown in the literature[Komori et al., 2004].

Investigation method
An aerobic batch experiment to simulate the treatment process in the plug flow type aeration tank (experiment duration: 8 hours) was
conducted using the effluent from the primary settling tank and returned sludge to investigate changes in the levels of estrogen-related
substances in the aeration tank of sewage treatment plant. The effluent from the primary settling tank and returned sludge were obtained
from the conventional activated sludge process pilot plant (aeration tank 2 m3 in capacity) using the sewage influent of the plant. The
experimental equipment used is a 150-L stainless steel container fitted with an agitator, into which 55 L of the effluent from the primary
settling tank and 25 L of returned sludge were fed and agitated. The water was aerated by air pumps through the air stones, and theaeration blow was adjusted to give an oxygen level of 3.0 mg/L by controlling the blow synchronously with the readings of theadditionally fitted dissolved oxygen meter.

Results and discussion

5.0
4.0
3.0
2.0
1.0
0.0

0

2

4

6

8

Elapse of time [hour]

E2

120

E1

E3

Concentration [ng/L]

DO

Concentration [ng/L]

pH

8.5
8.0
7.5
7.0
6.5
6.0
5.5

DO [mg/L]

pH [䋭]

Figure 4 shows temporal changes in pH, MLDO and estrogen in the aeration tank solution. When the experiment began, the dissolved
oxygen supplied by aeration was initiallyrapidly consumed, leading to pseudo-anaerobic conditions. After one hour had elapsed,
MLDO began to appear. It reached the set value of 3.0 mg/L in 7 hours. Levels of E2, which is a free estrogen, declined to below thedetection limit under these pseudo-anaerobic conditions in 2 hours. There were no major changes in E1 until 6 hours after the start ofthe experiment. However, its concentration suddenly dropped when MLDO began to rise. As explained above, it is assumed that the
reduction rate of E1 closely depends on the dissolved oxygen concentration. No EE2 was detected in any of the samples (detection
limit: 0.5 ng/L). For the estrogen sulfate conjugates, E1-S and E3-S were lower than the level of free estrogen, while E2-S is almost
equal to the free estrogen. E2-diS was detected at high concentrations. E1-S, E2-S and E3-S declined in the initial stages of theexperiment, while E2-diS showed almost no change.

100
80
60
40
20
0
0

2

4

6

Elapse of time [hour]

8

1000

E1-3S

E2-S

E3-S

E2-diS

100
10
1
0

2

4

6

8

Elapse of time [hour]

Figure 4: Temporal changes in pH, MLDO and estrogens

Effect of Treated Sewage on Fish
Effect of estrogen on Japanese medaka (Oryzias latipes)
An exposure experiment using adult male medaka in the laboratorywas conducted with E2 and E1 as the substances under study toobtain basic knowledge on the effects of exposure to medaka of the treated sewage. Vitellogenin concentration in the livers of thesample medaka was measured to identify the minimum effective concentration and maximum ineffective concentration of E2 and E1
and the relationship between the exposure time and the concentration of vitellogenin.

Methods
Five test concentrations of E2 and E1 were used: 5.0, 12.6, 31.6, 79.5 and 200 ng/L. The sample medaka were about 5 months old,
mature male medaka hatched from eggs taken from a group continuously raised in captivity for over three generations. The sample
medaka were selected from a group of medaka that had been raised up to four months, to the maturing stage, as above, and werescreened by sex based on their secondary sexual characteristic (shape of anal fin). Male fish alone were selected from the group andraised together until the experiment.
Sixty sample fish were used for each test section. They were exposed to the test water in flowing-water exposure equipment placed in aconstant-temperature chamber lined with a stainless steel interior finish. The test water tank was made of hard glass with an effective
water capacity of 10 L. The exposure time was 14 days.

4- 384 -



Results and discussion
100000Figures 5 and 6 plot changes in vitellogenin concentration in theliver during the exposure time for E2 and E1. For vitellogenin in
the control section, every individual was subjected to aconcentration of lower than 1 ng/mg throughout the exposure
period.
For E2, vitellogenin in the liver tends to rise in concentration in
proportion to E2 concentration for each exposure period. On the
2nd and 7th day of exposure, no significant difference wasobserved between the measurement in the 5.0 ng/L section andthat in the control section, but a significant difference appeared(p < 0.01) for the measurements on the 14th day of exposure.
For E1, no significant difference was observed at a standard of
p < 0.05, in comparison with the control section in eachexposure period for the below 31.6 ng/L concentration sections.

Control
31.6 ng/L

5.0 ng/L
79.5 ng/L

12.6 ng/L
200 ng/L

Vitellogenin conc. (ng/mg-liver)

10000

1000

100

10

1

0.1
0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Exposure days

Figure 5: Temporal changes in vitellogenin concentration in
the liver as result of exposure to E2

Vitellogenin conc. (ng/mg-liver)

For the 79.5 ng/L and 200 ng/L sections, a significant
100000Control
5 ng/Ldifference (p < 0.01) was observed from the measurement in
12.6 ng/L
31.6 ng/L
the control section on the 3rd, 7th and 14th day of exposure.
79.5 ng/L
200 ng/L
10000
Of the concentrations of vitellogenin in each exposure period,
1000
the lowest concentration (set concentration) that showed asignificant difference (p < 0.05) from the control section is100
defined as the minimum effective concentration, while the10highest concentration showing no significant difference is1defined as the maximum ineffective concentration. The
minimum effective concentration of E2 was 12.6 ng/L for the
0.1
2 day and 7 day exposure period and 5.0 ng/L for the 14 day
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14
period. The maximum ineffective concentration was 5.0 ng/L
Exposure days
Figure 6: Temporal changes in vitellogenin concentration inand <5.0 ng/L, respectively. The minimum effective
the liver as a result of exposure E1
concentration of E1 was 79.5 ng/L for the 3-day, 7-day and
14-day exposure periods, while the maximum ineffective concentration was 31.6 ng/L for all. Effective concentrations of 17ȕ-estradiol
and estrone are put together in a list as shown in Table 1.

Table 1: Effective concentration of 17Ǫ-estradiol and estrone
Substances under study

Exposure time(day)

17E-estradiol

EstroneMaximum ineffective concentration Minimum effective concentration(ng/L)(ng/L)

2

5.0

12.6

7

5.0

12.6

14

< 5.0

5.0

3

31.6

79.5

7

31.6

79.5

14

31.6

79.5

Impact on medaka of treated sewage
Methods
The influence of treated sewage on medaka was analyzed using the fish exposure test equipment placed in the sewage treatment plant.
The equipment consists of three water tanks: the first tank removes sand and mud, the second tank maintains the water temperature at
25 ÛC, after which the water is led to the third tank (30 L) for exposure of medaka to the treated sewage.
The exposure test was conducted on adult medaka fish in treated sewage and sand-filtered water (Test A). Sixty male medaka and 10
female medaka were put in both the treated sewage section and the sand-filtered water section and exposed to the water for 6 weeks.

5- 385 -



The other exposure test was conducted in treated sewage and sand-filtered water, on juvenile and freshly hatched medaka (Test B).
Twenty freshly hatched fish were put in both the treated sewage section and the sand-filtered water section and exposed for 8 weeks.
Estrogenic activity in the treated water and vitellogenin concentration in the medaka fish were measured during the test periods of both
Test Aand B.

Results and discussion
VTG concentrationࡆ࠹ࡠࠫࠚ࠾ࡦỚᐲ

Estrogen-like
Estrogenic
activity
activityࠛࠬ࠻ࡠࠥࡦ᭽ᵴᕈ
ࠛࠬ࠻ࡠࠥࡦᵴᕈ
40

8Effluent fromsecondary settling tank
ੑᰴಣℂ᳓

6

20

4

Test A

8th week

6th week

4th week

2nd week

Test start

6th week

4th week

2nd week0

Test start

Vitellogenin concentration (ng/mg-liver)20

Test B40

8Effluent from sand filtration⍾䉐ㆊ᳓

6

Estrogenic activity (ng/L-E2)

Changes in the level of estrogenic activity and the concentrationof vitellogenin in the test are shown in Figure 7. In Test A,
vitellogenin was found to have formed in the male medaka in
the treated sewage section and the sand-filtered section. The
level of estrogenic activity in both test sections was above 20
ng/L-E2 on the start of the test, but it subsequentlynever rose
above 10 ng/L-E2. This fact suggests that the enhanced level of
estrogen action seen at the start of the test was responsible forformation of vitellogenin in the medaka. The authors speculatethat vitellogenin, once formed, will stay inside the body until
metabolized. For Test B, male medaka produced vitellogen in
the 8th week in the treated sewage section, where a rise inestrogenic activity beyond 20 ng/L-E2 in the 6th week was
observed. As explained earlier, the concentration of estrogens,attributable to the formation of vitellogen in male medaka was
12.6 ng/L for the minimum effective concentration of E2 (2ndand 7th day of exposure), and the maximum ineffective
concentrations were 5.0 ng/L (2nd and 7th day of exposure).
Judging from this observation, it is possible that vitellogenin wasformed as early as on the 2nd day of exposure when exposed to
E2 at above 10 ng/L. This appears to be in agreement with theresults of TestsAand B obtained from the experiment at the
sewage treatment plant.

20

4

Test A

8th week

6th week

4th week

2nd week

Test start

6th week

4th week

Test start0

2nd week

20Test BVTG concentration ng/mg-liver)

Figure 7: Changes in estrogenic activity and vitellogenin
concentration in the experiment

The relationship between estrogenic activity and vitellogenin
concentration in the sample water when medaka were exposedto the treated sewage is shown in Figure 8. Although there is alarge variation, there is a tendency to show a higher estrogenic
activity the more the vitellogenin concentration rises.

10000
1000
100
10
1
0
0

1
10
Estrogenic activity㧔ng/L-E2㧕

100

Figure 8: Relationship between estrogenic activity in thesamples and VTG concentration in the livers of the mal
medaka individuals in the exposure test

Conclusions
1) The concentration levels of estrogenic activity in the sewage influent, treated sewage and river water show that the level of estrogenic
activity in the sewage decreases by about 80% during the treatment process at the sewage treatment plant, but the treated sewage
containing a concentration of 16 ng/L-E2 activity equivalent (median) is discharged to the environmental water. This level is two digits
higher than the concentration in the river water (median). As the ratio of treated sewage to the flow of a river rises, the level of estrogenic
activity is predicted to rise.
2) Estrogenic activity in the sewage indicates that substances not traced back to E1, E2 and NP are contained in water samples. For thecontribution of each substance, E1, E2 and NP are respectively responsible for raising the level of estrogenic activity in the sewage
influent. It is thus confirmed that E1 has a high contributoryrole in the treated sewage and that E1 plays an important role as a major
substance that enhances estrogenic activity in the treated sewage.

6- 386 -

3) Behavior of estrogens in the sewage treatment plant was investigated in winter and summer. Although the removal ratio for E2 wasfound to be high in both seasons (70% for winter and 87% for summer), the concentration of E1 rises with the progress of treatment(740% up in winter and 50% up in summer), and this tendency is particularly conspicuous in winter.
4) The batch experiment where MLDO is controlled was conducted. Although no major change in E1 was observed when MLDO was
still at a low level, it was confirmed that E1 concentration suddenly dropped when MLDO reached 3 mg/L. The rate of E1 reduction is
speculated to depend closely on the concentration of dissolved oxygen.
5) The influence of estrogen on the formation of vitellogenin in male medaka was investigated. The maximum ineffective andminimum effective concentration of E2 in the 14-day exposure test were <5.0 ng/L and 5.0 ng/L, respectively. The maximum
ineffective and minimum effective concentrations of E1 were 31.6 ng/L and 79.5 ng/L, respectively.
6) Male medaka fish were exposed to treated sewage and sand-filtered water. The results confirmed that vitellogenin had formed in the
medaka in the treated sewage in which the estrogenic activityhad risen above 20 ng/L-E2. The exposure of medaka to treated sewage
lead to the clarification of the relationship between estrogenic activity and the vitellogenin concentration in the sample water. It wasalso confirmed that the higher estrogenic activity, the higher the rise seen in vitellogenin concentration.

Acknowledgment
The authors thank the local governments for kindly providing wastewater samples. We also thank the laboratory staff who prepared thesamples for the estrogen-like activity and the LC/MS/MS analyses.

References
Gray, M. A.; Thather, K. L. and Metcalfe, C. D. (1999): Reproductive success and behavior of Japanese medaka (oryzias latipes)
exposed to 4-tert-octylphenol, Environmental Toxicology and Chemistry (18) 11, 2587-2594
Jobling, S.; Sheahan, D.; Osborne, J. A.; Matthiessen, P. and Sumpter, J. P. (1996): Inhibition of testicular growth in rainbow trout(oncorhynchs mykiss) exposed to estrogenic alkylphenolic chemicals, Environmental Toxicology and Chemistry (15) 2,
194-202
Jones, E. T.; Thorpe, K.; Harrison, N.; Thomas, G.; Morris, C.; Hutchinson, T,; Woodhead, S. and Tyler, C. (2003): Dynamics of
estrogen biomarker responses in rainbow trout exposed to 17D-ethnylestrdiol, Environmental Toxicology and Chemistry(22) 12, 3001-3008
Komori, K.; Tanaka, H.; Okayasu, Y.; Yasojima, M. and Sato, C. (2004): Analysis and Occurrence of Estrogen in Wastewater in Japan,
Water Science & Technology (50) 5, 93-100
Metcalfe, C. D.; Metcalfe, T. L.; Kiparissis, Y.; Koenig, B. G.; Khan, C.; Hughes, R. J.; Croley, T. R.; March, R. E. and Potter, T.
(2001): Estrogenic potency of chemicals detected in sewage treatment plant effluents as determind by in vivo assays withJapanese medaka (oryzias latipes), Environmental Toxicology and Chemistry (20) 2, 297-308
Ministry of Land, Infrastructure andTransport, MLIT, (2001): FY2000 Results of a Fact-finding Study of Endocrine Disruptors in
Water Environments, Ministry of Land, Infrastructure and Transport (in Japanese).
Routledge, E.J.; Sheahan, D.; Desbrow, C.; Brighty, G. C.; Waldock, M. and Sumpter, J. P. (1998): Identification of estrogenic
chemicals in STW effluent. 2. in vivo responses in Trout and Roach, Environmental Science & Technology, 32, 1559-1565
Yakou, Y.; Takahashi, A.; Higashitani, T. and Tanaka, H. (1999): Measurement of Estrogen-like Activity in Wastewater Using
Recombinant Yeast, Environmental Engineering Research, 36, 199-208, Japan Society of Civil Engineering, (in Japanese).

7- 387 -



- 388 -9 Introduction and Objectives
9 Estrogn-like activity in sewage influent,treated sewage and river water
9 Investigation of behavior of estrogen in
sewage treatment plants
9 Relationship between MLDO concentrationin the aeration tank and estrogen removal
9 Effect of treated sewage on fish
9 Conclusion

Outline of the presentation

Water Environment Research Group
Public Works Research Institute (PWRI)

Koya Komori, Yuji Okayasu, Kiyoshi Miyajima,
Tadashi Higashitani, Norihide Nakada and Yutaka Suzuki

Fate of estrogens in sewage
treatment process andeffect of treated sewage on fish

10th Japanese German Workshop on Water Technology
Hotel Courtyard Marriott, Berlin
09.10-10.10.2006

3

1“Our Stolen Future” wastranslated into Japanese andpublished in September 1997

“Our Stolen Future”

translated by
Tsutomu Nagasaka

byTheo Colborn,
Dianne Dumanoski
andJohn Peterson Myers

9 Introduction and Objectives
9 Estrogn-like activity in sewage influent,treated sewage and river water
9 Investigation of behavior of estrogen in
sewage treatment plants
9 Relationship between MLDO concentrationin the aeration tank and estrogen removal
9 Effect of treated sewage on fish
9 Conclusion

Outline of the presentation

42

- 389 - The biological impact of treated sewage(Effect of treated sewage on fish) A review of the relationship between
MLDO concentration and estrogen removal inaeration tanks of activated sludge process

Objectives

1) MLIT, (2001): FY2000 Results of a Fact-finding Study of Endocrine Disruptors in
Water Environments, Ministry of Land, Infrastructure and Transport (in Japanese)

Substances

7

5

RemovalMedian concentration (μg/L)
Sewage influent Treated sewage efficiency
Nonylphenol (NP)
4.4
Trace (0.2)
95 %Bisphenol-A
0.53
Trace (0.02)
96 %2,4-dichlorophenol
0.07
N.D.
99 %
Diethyl phtalate
3.1
N.D.
99 %
Di-n-butyl phthalate
2.6
N.D.
99 %
Di (2-ethylhexyl) phthalate
12
Trace (0.4)
97 %Di (2-ethylhexyl) adipate
0.09
N.D.
99 %
Benzophenone
0.17
0.05
71 %
ELISA
0.042
0.01
76 %
17b-estradiol (E2)
LC/MS/MS
0.0081
N.D.
99 %Estrone
LC/MS/MS
0.043
0.006485 %Concentration of environmental hormones insewage influent and treated sewage 1)
Place

Influences

Probablecausative
substances
organotin
components
nonylphenol*not confirmed
nonylphenol*not confirmed
Researchers

9 Introduction and Objectives
9 Estrogn-like activity in sewage influent,treated sewage and river water
9 Investigation of behavior of estrogen in
sewage treatment plants
9 Relationship between MLDO concentrationin the aeration tank and estrogen removal
9 Effect of treated sewage on fish
9 Conclusion

Outline of the presentation

seashores invirilescence,
Horiguchi et al.
shellfish
reishiaJapan
population(1994)
rivers in thevirilescence,
Sumpter et al.
rainbow trout
United Kingdom
population(1985)
Purdom et al.
roachrivers in thehermaphroditism
fish(1994)(a kind of carp) United Kingdom
thyroid
Great Lakes inLeatherland
hyperplasia,
unknown
salmonthe United(1992)
population
Statesorganochlorine
male penis
agriculturalatrophy,lakes in Florida
chemicals such Guillette et al
decrease of
reptilescrocodile
State in the(1994)
as DDT
hatchability ofUnited Statesdischarged into
ovum,
lakes
population
Cf. Quoted works are based on "the interim reports by the workshop concerning exogenous endocrinedisrupting chemicals issues."2) http://www.env.go.jp/en/chemi/ed/speed98/sp98t2.html

Wildlife

The report concerning the influences on wildlife 2)86

- 390 -

1

10

100

N=87

1000

MedianSewage Influent

10000(E2 : 17β-estrdiol)

Estrogenic activity，ng/L,E2-equivalent (ng/l)

0.1Median 16

N=167

N=77

Treated sewage

MedianRiver water

Outline of the presentation

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
0.01

9 Introduction and Objectives
9 Estrogn-like activity in sewage influent,treated sewage and river water
9 Investigation of behavior of estrogen in
sewage treatment plants
9 Relationship between MLDO concentrationin the aeration tank and estrogen removal
9 Effect of treated sewage on fish
9 Conclusion

Non-exceeding probability

Concentration distribution of estrogenic activity insewage influent, treated sewage and river water

11

9

4

8

2 19 17 9 18 3 12 10 23 14 25 21 1 20 15 24 4 16 5

6 13 11 8 22 7

1 14 24 21 3 25 20 7 15 10 18 17 16 19 5 23 12 11 6Treated Sewage

2 13 9Sewage Influent

YES
E2
E1

Drained sludge

A

1036

Recycle flow

36

886

1140 1450

Drained sludge

A

44

Recycle flow

Capacity: 20,400m3/day

739 mg/day
E2 equivalent

in Summer survey

500 mg/day
E2 equivalent

in Winter survey

Return sludge

BB

459

Return sludge

BB

1895

C

C

1462

440

1048

1355

D

D

12

Dissolved
Suspended(mg/day, E2equivalent)

339 mg/day
E2 equivalent

A: Primary settling tank
B: Aeration tank
C: Final settling tank
D: Chlorination tank

976 mg/day
E2 equivalent

10

Nonyl phenol

Mass balance of estrogenic activity in a municipal
sewage treatment plant

0

20

40

60

80

100

0

20

40

60

80

100

Contribution of Estrogens and Nonylphenol in Estrogenic
Activity of Sewage Influent and Treated Sewage

Estrogenic Activity, ng/L-E2 equivalent

0

20

40

60

80

100

120

0

4

E1

6

Elapse of time [hour]

2

E2

E3

8

0

0

2

6

8

1

10

100

1000

4

6

E2-diS

E3-S

Elapse of time [hour]

2

E2-S

E1-3S

8

0.0

5.5

Elapse of time [hour]

1.0

6.0
4

2.0

6.5

7.0

3.0

5.0
4.0

DO

7.5

pH

8.0

8.5

9 Introduction and Objectives
9 Estrogn-like activity in sewage influent,treated sewage and river water
9 Investigation of behavior of estrogen in
sewage treatment plants
9 Relationship between MLDO concentrationin the aeration tank and estrogen removal
9 Effect of treated sewage on fish
9 Conclusion

Outline of the presentation

Temporal changes inpH, MLDO andestrogens

Concentration [ng/L]

pH [－]

Concentration [ng/L]- 391 DO [mg/L]

15

13

Air pump

DO
controlunit

9 Introduction and Objectives
9 Estrogn-like activity in sewage influent,treated sewage and river water
9 Investigation of behavior of estrogen in
sewage treatment plants
9 Relationship between MLDO concentrationin the aeration tank and estrogen removal
9 Effect of treated sewage on fish
9 Conclusion

Outline of the presentation

E2, E1,E3, EE2,
E1-S, E2-S, E3-S,
E2-diS

16

14

Analytical estrogens

Experimental apparatus

Experiment duration: 8 hours

Collecting Sample

Mixer

DO meter

10 L

10 L

Overflow

HRT; 12 h

42 ml/min

10 L

P

Overflow

HRT; 12 h

42 ml/minP

10 L

Stock solution

Overflow

HRT; 12 h

42 ml/minP

10 L

Stock solution

0.1

1

10

100

1000

10000

100000

0

1

2

3

4

S.Control
31.6ng/L

5

7

8

Exposure days

6

5ng/L
79.5ng/L

910

12.6ng/L
200ng/L

11

12

13

14

Temporal changes in vitellogenin (VTG)
concentration in the liver as a result of exposure E1

Vitellogenin concentration in
the livers of the sample fishB
C
D
E
(12.6 ng/l) (31.6 ng/l) (79.5 ng/l) (200 ng/l)

Overflow

HRT; 12 h

P

Stock solutionz Test water: E2 & E1z Sample fish: 60 for each tankz Exposure time: 14 daysA
(5.0 ng/l)

Overflow

HRT; 12 h

42 ml/min

42 ml/min

P

Stock solution

Stock solution

19

17

0

1

2

3

4

Control
31.6ng/L

5

7

8
Exposure days

6

5.0ng/L
79.5ng/L

9

10

11

12.6ng/L
200ng/L

12

13

14

E1

E2

Substances
under study

Maximum
ineffective
concentration(ng/L)
5.0
5.0
< 5.0
31.6
31.6
31.6

Exposure time
(day)
2
7
14
3
7
14

12.6
12.6
5.0
79.5
79.5
79.5

Minimum
effective
concentration(ng/L)

Effective concentration of E2 and E1

0.1

1

10

100

1000

10000

100000Temporal changes in vitellogenin (VTG)
concentration in the liver as result of exposure to E2

Vitellogenin conc. (ng/mg-liver)

Effect of estrogen on Japanese medaka

Vitellogenin conc. (ng/mg-liver)- 392 20

18

Test B; juvenile and freshly
hatched medaka

Test A; adult medaka fish

Test Waterz Treated sewage
z Sand filtered water

0

1

10

100

1000

10000

0

1
10
Estrogenic activity（ng/L-E2）

100

Relationship between estrogenic activity in thesamples and VTG concentration in the livers of themal medaka individuals in the exposure test

30 L

The third tank(fish exposure tank)

23

21

Test A

Test A

Test B

Effluent from sand filtration
砂ろ過水

Test B

0

20

40

0

20

40

Vitellogenin concentration (ng/mg-liver)

9 Introduction and Objectives
9 Estrogn-like activity in sewage influent,treated sewage and river water
9 Investigation of behavior of estrogen in
sewage treatment plants
9 Relationship between MLDO concentrationin the aeration tank and estrogen removal
9 Effect of treated sewage on fish
9 Conclusion

Outline of the presentation

0

2

4

6

8

0
4th week
4th week

2
6th week
6th week

4

Test start
Test start

The second tank(maintains the water temperature)

Effluent fromsecondary settling tank
二次処理水

Estrogen-like
Estrogenic
activity
activity
エストロゲン様活性
エストロゲン活性

2nd week
2nd week

4th week
4th week

6

Test start
Test start

VTG concentration
ビテロジェニン濃度

6th week
6th week

8

8th week
8th week

The first tank(removes sand & mud)2nd week
2nd week

Changes in estrogenic activity andvitellogenin concentration in the experiment

Estrogenic activity (ng/L-E2)

Impact on medaka of treated sewage

VTG concentration ng/mg-liver)- 393 24

22

- 394 -z The estrogenic activity had risen above 20
ng/L-E2z The higher estrogenic activity, the higher
the rise seen in VTG concentration

3) Male medaka fish were exposed to treatedsewage and sand-filtered water

Conclusions (3/3)

27

We also thank the laboratory staff who
prepared the samples for the estrogen-like
activity and the LC/MS/MS analyses

The authors thank the local governmentsfor kindly providing wastewater samples

Acknowledgment

28

26

E2
E1
z Max. ineffective conc.; <5.0 ng/L 31.6 ng/L
z Min. effective conc. ; 5.0 ng/L 79.5 ng/L

Although no major change in E1 wasobserved when MLDO was still at a low level, it
was confirmed that E1 concentration suddenly
dropped when MLDO reached 3 mg/L
252) The influence of estrogen on the formationof VTG in male medaka was investigated

Conclusions (2/3)

1) The batch experiment where MLDO iscontrolled was conducted

Conclusions (1/3)

- 395 -its

Collaboration with Murata Keisokuki Service Co., Ltd.
& Kyoto University
29

z Development of simultaneous analysis of pharmaceuticals inaqueous samples using LC-MS/MS

Collaboration with Teijin Eco-Science Ltd .

z Analytical method of clarithromycin，erythromycin andmetabolite in waste waters

Collaboration with Towa Kagaku Co., Ltd.

z Development of analytical method for LVFX, CAM and AZM
in sewage samples
z Occurrence of LVFX, CAM and AZM in sewage treatment
plants in JapanThe latest research

30

Thank you for your attention

10th Japanese German Workshop on Water Technology
Hotel Courtyard Marriott, Berlin
09.10-10.10.2006