Registration Dossier

Ecotoxicological information

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:
fish: other
Type of information:
experimental study
Remarks:
Preliminary results are reported. After finalizing of the study report, the endpoint study record will be updated and the report will be attached asap.
Adequacy of study:
key study
Study period:
biological test Jan 29-Apr 11, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
ECHA decision on substance evaluation: Request of Fish Sexual Development Test, OECD 234

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2109

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD TG 234 (Fish Sexual Development Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder
Details on test material:
CAS number: 94-13-3
Specific details on test material used for the study:
CAS number: 94-13-3
Molecular mass: 180.2 g/mol
Purity (according to CoA): 99.7 %
Date of analysis: April 05, 2018
Physical state: Powder
Colour: White
Aqueous solubility: 500 mg/L (20 °C)
Log Pow (25°C): 3.04

Sampling and analysis

Analytical monitoring:
yes

Test organisms

Aquatic vertebrate type:
fish
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
70 d

Test conditions

Hardness:
5.5 to 6.0 mmol/L
Test temperature:
The mean water temperature was in the range of 26.8 °C to 27.5 °C in all test vessels
pH:
Mean 7.52- 7.66 Min 7.2 Max 7.83
Dissolved oxygen:
Mean 89 98 %, Min 78%
Conductivity:
229-254 µS/cm
Nominal and measured concentrations:
The concentrations of propyl 4-hydroxybenzoate were assessed by chemical analysis using LC-MS/MS. The LOQ was set to 0.75 µg propyl 4-hydroxybenzoate/L. At test start, after 7 days and after 14 days, samples were taken from all test vessels in order to confirm correct dosing. During the remaining course of the study, samples were taken on a weekly basis, alternating between the vessels that were served by the same dosage pump. At test end, samples of all vessels were analysed.
If samples were found outside the desired concentration range of 80 – 120 % of nominal concentrations, retain samples were analysed alongside two QCs to confirm the correct concentration in the tank. For calculations of the mean, the mean of the original and the retain sample was calculated. This mean value was used for calculation of the vessel and concentration mean.
Mean concentrations per treatment of the propyl 4-hydroxybenzoate during the course of the study were between 80.5 % (nominal concentration of 640.0 µg propyl 4-hydroxybenzoate/L) and 127.4 % (nominal concentration of 6.4 µg propyl 4-hydroxybenzoate/L) of the nominal concentration of the test item.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
70 d
Dose descriptor:
NOEC
Effect conc.:
640 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Post hatch survival at 70dpf
Key result
Duration:
70 d
Dose descriptor:
LOEC
Effect conc.:
640 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Post hatch survival at 70 dpf da
Key result
Duration:
70 d
Dose descriptor:
NOEC
Effect conc.:
640 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
70 d
Dose descriptor:
LOEC
Effect conc.:
640 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
70 d
Dose descriptor:
NOEC
Effect conc.:
640 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
70 d
Dose descriptor:
LOEC
Effect conc.:
640 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight

Any other information on results incl. tables

Biological effects at test termination

 

Nominal concentration propyl 4-hydroxybenzoate [µg/L]

Control

6.4

20.2

64.0

202.4

640.0

 

Mean measured concentration propyl 4-hydroxybenzoate [µg/L]

Control

7.66

25.45

59.93

164.73

517.58

No of replicates

4

4

4

4

4

4

Survival,

70 dpf [%]

Mean

82.5

82.5

80.8

84.2

83.3

87.5

SD

5.7

3.2

1.7

10.3

11.2

4.2

RSD

6.9

3.9

2.1

12.3

13.5

4.8

Length, all individuals;

70 dpf [cm]

Mean

3.3

3.3

3.4

3.4

3.5

3.6

SD

0.1

0.0

0.1

0.1

0.1

0.3

RSD

3.7

1.2

1.9

2.9

3.1

7.2

Weight, all individuals;

70 dpf [g]

Mean

0.381

0.379

0.422

0.400

0.464

0.503

SD

0.036

0.019

0.031

0.040

0.054

0.146

RSD

9.4

5.0

7.3

10.0

11.6

29.0

Length males;

70 dpf [cm]

Mean

-to be included after histopathology -

SD

RSD

Length females;

70 dpf [cm]

Mean

SD

RSD

Weight males;

70 dpf [mg]

Mean

SD

RSD

Weight females;

70 dpf [mg]

Mean

SD

RSD

Sex ratio
[% females]

Mean

SD

RSD

Sex ratio
[% males]

Mean

SD

RSD

Sex ratio
[% undifferentiated]

Mean

SD

RSD

SD = Standard deviation

RSD = Relative standard deviation


 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Preliminary Conclusions
Statistical evaluation revealed no effect of the test item propyl 4-hydroxybenzoate on any apical endpoint (hatch, post-hatch survival, growth).
Based on these endpoints, the following NOEC was determined:
NOEC of ≥ 517.58 μg propyl 4-hydroxybenzoate/L (mean measured concentration),
corresponding to
NOEC of ≥ 640.0 μg propyl 4-hydroxybenzoate/L (nominal concentration).


Sex ratios will be determined macroscopically by inspection of the gonads and by histopathological verification. Blood samples of all fish will be taken and measured for the vitellogenin (VTG) concentration. Furthermore, a histopathological examination of the fish gonads will be performed.

Executive summary:

A fish sexual development test (FSDT) with zebrafish (Danio rerio), sponsored by Clariant, was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME). The objective of this study was the assessment of effects of continuous exposure to the test item on the early life stages and sexual differentiation of zebrafish (Danio rerio), following the OECD test guideline 234[5]. The study was conducted with nominal concentrations of 6.4, 20.2, 64.0, 202.4, 640.0 µg propyl 4-hydroxybenzoate/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. Endpoints that were determined included hatching success and rates, and mortalities during the early life stage and the juvenile growth. At day 35 pf and when groups were terminated (day 70 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 70 pf (test end).

Sex ratios will be determined macroscopically by inspection of the gonads and by histopathological verification. Blood samples of all fish will be taken and measured for the vitellogenin (VTG) concentration. Furthermore, a histopathological examination of the fish gonads will be performed.

Early life stage

Hatching rate

First larvae started to hatch at 3 dpf across all treatment levels. Hatch was completed at 5 dpf in all replicates, with no difference between treatments. Complete hatch was observed in all replicates.

 

Post-hatch survival at 35 dpf

At 21 dpf, the post-hatch survival rate in controls reached a mean value of 84.2 %. At 35 dpf, post-hatch survival in control vessels was also at 83.3 % and thus met the validity criterion for survival in controls of ≥ 70 %. Survival rates under treatment conditions at 35 dpf varied between 82.5 % (mean measured concentration of 7.66 µg propyl 4-hydroxybenzoate/L) and 89.2 % (mean measured concentration of 517.58 µg propyl 4-hydroxybenzoate/L). Statistical analyses of post-hatch survival at 35 dpf revealed no significant differences between control and treatments (Williams Test,α=0.05; one-sided smaller).

 

Total length at 35 dpf

In this study, mean total length of 2.17 cm was observed in controls. The mean length of larvae in the treatment conditions were found in the range of 2.20 cm (59.93 µg/L) and 2.35 cm (517.58 µg/L). The larvae were thus larger in terms of length under treatment conditions. As this was not considered as negative effect, statistical analysis was performed in direction of smaller size. Statistical analyses revealed no significant decrease in size compared to control values (Jonckheere-Terpstra Test,α=0.05; one-sided smaller).

 

Test termination

Post-hatch survival at 70 dpf

Only few fish were found dead during the juvenile growth phase. No other signs of disease were observed. Statistical analyses of post-hatch survival at 70 dpf revealed no significant differences between control and treatments (Williams Test,α=0.05; one-sided smaller). The NOEC for the endpoint post-hatch survival at test end was defined at ≥ 517.58 µg propyl 4-hydroxybenzoate/L (mean measured concentration).

 

Total length and wet weight at 70 dpf

At 70 dpf, growth in terms of length was determined. Juvenile fish in the control displayed a mean measured length of 3.3 cm. The mean length of fish under treatment conditions varied between 3.3 cm and 3.6 cm. The mean measured weight of fish in controls was measured at 0.381 g, the mean weight of fish under treatment conditions varied between 0.379 g and 0.503 g (Table 9).Thus, the size of the juvenile fish was increasing with increasing test item concentrations

Statistical analyses revealed no significant decrease in size in terms of length (Williams Test, α=0.05; one-sided smaller) and weight (Jonckheere-Terpstra Test, α=0.05; one-sided smaller) at test end compared to control values.

 

Values evaluated separately for males and females to be included after histopathology.

 

Sex ratio

- to be included after histopathology –

 

Vitellogenin content in blood plasma

- to be included after histopathology –