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Endpoint:
additional ecotoxicological information
Remarks:
Fish Sexual Development Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-02-2018 to 26-04-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 234 Fish Sexual Development Test
Version / remarks:
July 2011
GLP compliance:
yes (incl. certificate)
Type of study / information:
Early life stage toxicity and sexual development in fish

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RÜTGERS Germany GmbH, Varziner Strasse 49, 47138 Duisburg (Germany)
- Lot/Batch n°: Lab Sample 6.9.2017
- Expiration date of the lot/batch: 31-12-2018
- Purity test date: 06-09-2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions of test material: room temperature, dry
- Storage stability of the test item in frozen samples: Spiked samples frozen on 15-01-2018 were thawed 26-04-2018 and analysed alongside the t63 samples.
The analysis of the thawed samples confirmed the storage stability.

Results and discussion

Any other information on results incl. tables

A. Sex ratio

Table 1 Biological effects at test termination (63 dpf) (excerpt: endocrine effects only, from Report, Table 11, p. 50)

(see also Diagram in Attached Document)

 

Nominal concentration 4-(1-phenylethyl)-phenol [μg/L]

Control

2.0

6.3

20.0

63.0

200.0

Mean measured concentration 4-(1-phenylethyl)phenol [μg/L]

Control

2.1

6.4

19.7

61.8

187.9

No of replicates

4

4

4

4

4

4

 

 

 

 

 

 

 

 

Sex ratio [% females]*

Mean

51.3

53.6

49.5

40.8

50.2

34.9

SD

6.0

13.7

7.3

10.2

11.6

10.7

RSD

11.6

25.6

14.8

25.1

23.1

30.8

Sex ratio [% females/ stage 0]*

Mean

8.9

13.6

20.5**

33.0**

29.1**

52.5**

SD

8.6

13.4

4.8

8.5

12.4

17.7

RSD

96.5

98.1

23.6

25.6

42.7

33.8

Sex ratio [% males]*

Mean

26.0

27.2

30.0

22.4

18.6

0.0***

SD

16.6

8.7

3.2

15.8

6.8

0.0

RSD

63.8

32.1

10.5

70.8

36.8

-

dpf = days post-fertilization

SD = Standard deviation; RSD = Relative standard deviation

* Only mature fish with clearly identified sex (histopathology) were considered for evaluation. Fish which could not be determined histopathologically
 (unidentified) or immature fish (female/stage 0; transition phase) were excluded from calculation.

** Williams t-test, p<0.05; one-sided greater

*** Jonckheere-Terpstra test, p<0.05; one-sided smaller

The sex ratio was determined based on histopathological examinations of the gonads. The examined fish were assigned to five different categories: female, male, female/stage 0, transition phase or unidentified. Female/stage 0 described individuals, where the gonads showed only undeveloped ovaries with oogonia to perinucleolar oocytes. The gonads of these animals were most probably arrested in the development into testis due to the treatment with the test item, and afterwards developedtowards female. Animals in transition phase described animals with gonads consisting of undeveloped germ cells to oogonia exclusively. For these animals, it was not possible to confirm the sex. The

category ‘unidentified animals’ described fish where no gonads were visible on the histological slide, even after recutting.

For determination of statistically significant differences, the percentage of mature males was considered. Furthermore, statistical differences with respect to the percentage of females/stage 0 werecalculated.

In controls and in the four lower test concentration, the mean value of %males ranged between 18.6% [61.6 μg/L (mean measured concentration)] and 30.0% [6.4 μg /L (mean measured concentration)]. No mature males were found in any of the replicates of the highest test concentration [187.9 μg/L (mean measured concentration)]. A statistically significant difference (p<0.05) in the sex ratio in terms of %males was thus observed for the highest test concentration.

There was a concentration-related, statistically significant increase (p<0.05) in the mean value of %females/stage 0 from the concentration of 6.4 μg/L.

The NOEC for the endpoint sex ratio (%males) was thus defined as 61.8 μg/L (mean measured concentration). Taking into account the dose-related increase in the number of females/stage 0, the NOEC was defined as 2.1 μg/L (mean measured concentration).

--------------------------------------------------------------------------------------

B. Vitellogenin (VTG) content in blood plasma

VTG content in females and males(63 dpf) (excerpt: endocrine effects only, from Report, Table 12, p. 51)

 

Nominal concentration 4-(1-phenylethyl)-phenol [μg/L]

Control

2.0

6.3

20.0

63.0

200.

Mean measured concentration 4-(1-phenylethyl)phenol [μg/L]

Control

2.1

6.4

19.7

61.8

187.9

No of replicates

4

4

4

4

4

4

 

 

 

 

 

 

 

 

VTG / total protein [ng/μg]; females*

Mean

480.8

407.8

500.3

404.5

451.9

729.7**

SD

204.0

82.2

65.5

163.4

181.2

307.4

RSD

42.4

20.2

13.1

40.4

40.1

42.1

VTG / total protein [ng/μg]; males*#

Mean

0.07

0.39

0.77

0.04

0.07

-

SD

0.02

0.61

1.04

0.02

0.08

-

RSD

27.9

158.2

134.6

52.9

108.4

-

dpf = days post-fertilization

SD = Standard deviation; RSD = Relative standard deviation

* Only mature fish with clearly identified sex (histopathology) were considered for evaluation. Fish which could not be determined histopathologically
 (unidentified) or immature fish (female/stage 0; transition phase) were excluded from calculation.

** Williams t-test, p<0.05; one-sided greater

*** Jonckheere-Terpstra test, p<0.05; one-sided smaller

# No values could be obtained for the highest concentration as no mature male fish could be identified.

The VTG content in the blood plasma of all individuals was determined. However, only the values of mature males and females were considered for analysis. Fish which could not be determined histopathologically (unidentified) or immature fish (female/stage 0; transition phase) were excluded from calculation.

A statistically significant increase in mean VTG values was reported in females, only at the highest test concentration (187.9 μg/L). There were no significant differences in VTG contents for males at any concentration level. Of note, VTG content could not be determined at the highest concentration, as no mature males were present. Based on the results for mature females, the NOEC for the biomarker VTG was determined at 61.8 μg/L (mean measured concentration).

Applicant's summary and conclusion

Conclusions:
Interpretation of the obtained results (an increase in the VTG content in females as well as the absence of males in the highest test concentration) suggests a strong estrogenic mode of action for the test substance. This interpretation follows the OECD TG 234. Based on these endocrine-related endpoints, the following NOEC was determined (based on the measure %males, corresponding to an increase in the measured VTG concentration in female blood plasma): NOEC of 61.8 μg/L 4-(1-phenylethyl)phenol (mean measured concentration).
Executive summary:

A fish sexual development test (FSDT) was performed with zebrafish (Danio rerio). The objective of this study was the assessment of effects of continuous exposure to 4-(1-phenylethyl)phenol (4-MSP) on the early life stages and sexual differentiation of zebrafish (Danio rerio), following the OECD test guideline 234. (see also Toxicological Section: Early Life Stages)

The study was conducted with nominal concentrations of 2.0, 6.3, 20.0, 63.2 and 200 μg/L in four replicates each under flow-through conditions. An untreated control was run in parallel. Exposure was started with 30 fertilised eggs per test vessel and replicate. Biological effects were based on mean measured concentrations (2.1 μg/L; 6.4 μg/L; 19.7 μg/L; 61.8 μg/L;187.9 μg/L).

Endpoints which were not indicative of endocrine-mediated effects included hatching success and rates, and mortalities during the early life stage and the juvenile growth. At day 35 post-fertilisation (pf) and when groups were terminated (day 63 pf), fish were digitally photographed. Fish lengths were determined by evaluating photographs using electronically supported analysis. Single wet weights (blotted dry) were determined on day 63 pf (test end).

Endpoints which are indicative of endocrine-mediated effects and which could be determined during the study were the sex ratio and the vitellogenin (VTG) concentration in blood plasma. Sex ratios were determined macroscopically by inspection of the gonads and by histopathological verification in the fish gonads.

Endocrine related endpoints and biomarkers: Sex ratio (based on histopathology)

When compared to controls, a statistically significant test-item-related effect on sex ratio in terms of %males was reported at 187.9 μg/L. No mature males were found in any of the replicates of the highest test concentration, whereas the mean value of %males ranged between 18.6% and 30.0% in controls and the four lower test concentrations. Hence, the NOEC for the endpoint sex ratio (%males) was defined as 61.8μg/L (mean measured concentration).

The mean value of %females/stage 0 (individuals where the gonads showed only undeveloped ovaries with oogonia to perinucleolar oocytes) ranged between 8.9% (controls) and 52.5% (187.9 μg/L), with a monotonous concentration- response relationship. A statistically significant difference was observed for concentrations ≥ 6.4 μg/L. Regarding the number of females/stage 0, the NOEC was defined as 2.1 μg/L (mean measured concentration).

Endocrine-related endpoints and biomarkers: Vitellogenin content in blood plasma

A statistically significant test item-related increase in vitellogenin level was reported at 187.9μg/L in females only (mean VTG value of 729.7 ng/μg protein vs. 480.8 ng/μg protein in controls).

VTG contents for males could be determined only for controls and for the four lower test concentrations, as no mature males were present at the highest test level. No significant differences in VTG content were observed.

Based on the results for mature females, the NOEC for the biomarker VTG was determined at 61.8 μg/L (mean measured concentration).

Conclusion

Based on these endocrine-related endpoints, the following NOEC was determined (based on the measure %males, corresponding to an increase of the measured VTG concentration in female blood plasma):  NOEC of 61.8 μg/L 4-MSP (mean measured concentration), corresponding to NOEC of 63.0 μg/L 4-MSP (nominal concentration).