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Endocrine disrupter testing in aquatic vertebrates – in vivo

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Administrative data

Endpoint:
fish: other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 25, 2017- December 28, 2017 (Biological test and chemical analysis)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Echa decision on a substance evaluation. OECD 234 was requested

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

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

Test material

Specific details on test material used for the study:
CAS number: 99-76-3
Molecular formula: C8H8O3
Molecular mass: 152.2 g/mol
Purity (according to CoA): 99.9 %

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Prior to the proposed test start, the flow through system was equilibrated and samples from all test vessels were taken for chemical analysis. This was performed concomitantly to ensure the right test concentrations and the correct adjustment of the dosing pump system. Samples were immediately transferred to the chemical analysis laboratory and measured.
If the test concentrations could not be confirmed, the dosing system was re-adjusted and after a further equilibration phase with at least one total exchange of test media, again samples from all test vessels were taken and the procedure was repeated.
The analytical verification of the test item in the pre-exposure period was performed under non-GLP conditions.
From each sample, an aliquot was taken and used for analytical measurement. The remaining amount of the samples was immediately be stored at ≤ -18 °C to enable additional analyses on request of the study director, the chemical investigator or the study monitor. Additional samples were taken and stored at ≤ -18 °C, if technical irregularities, e.g. malfunction of the dosing system, occurred. All samples are going to be discarded after finalization of the study.
When the right test concentrations were confirmed, the experimental phase was started by introducing the fertilized eggs. In the following, samples from all test vessels including controls were taken once weekly. When the analytical results showed stable concentrations in both replicate vessels during the equilibration phase and at test start, only one vessel per replicate pair was sampled weekly thereafter. The two vessels of one vessel pair were sampled alternately.

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of stock solutions
For preparation of the stock solution, an appropriate amount of methyl 4-hydroxybenzoate was weighted out and was transferred to a clean glass bottle. This was filled with dilution water until its final volume of 10 L. This solution was finally acidified with 2500 µL hydrochloric acid (37 %) and was stirred for approx. 24 h. These solutions served as application solutions in the flow through device.
To achieve the final concentrations in the test vessels, the application solutions were mixed with dilution water in adequate volumes via the dosing pumps.

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:
63 d

Test conditions

Hardness:
Total hardness [mmol/L] 1.1 - 1.2
Calcium hardness [mmol/L] 0.8 – 0.9
Magnesium hardness [mmol/L] 0.2 - 0.4
Test temperature:
The mean water temperature was in the range between 26.1 °C and 27.0 °C in all test vessels throughout the in-life phase, and thus in line with the guideline requirements. Single values of water temperatures during the in life phase were measured to be between 25.0 and 27.8°C.
pH:
pH 8.04 – 8.10
Dissolved oxygen:
Dissolved oxygen content
94.4 - 109.7[%]
8.64 - 10.14 [mg/L]
Conductivity:
[µS/cm] 261.0 – 277.0
Nominal and measured concentrations:
The concentration range was defined in a respective ECHA conclusion. In detail, it was based on available data from fish studies with the same test substance on acute toxicity, i.e. the acute LC50 value. This was determined in an acute fish toxicity test according to the OECD TG 203, for the test species medaka (Oryzias latipes). The LC50 was determined to be 59.6 mg Methyl 4-hydroxybenzoate/L (data as specified in the respective Material safety data sheet provided by the sponsor).

Based on the respective ECHA conclusion, the following range of test concentrations was set:

0.1; 0.32; 1.00; 3.2; 10.0 mg methyl 4-hydroxybenzoate/L.

A spacing factor of √10 (i.e. 3.16) was applied in order to cover a preferably wide concentration range.

The concentrations of methyl 4-hydroxybenzoate were assessed by chemical analysis using an HPLC method. The LOQ was set to 0.030 mg methyl 4-hydroxybenzoate/L. Mean concentrations per treatment of methyl 4-hydroxybenzoate during the course of the study were between 87% and 111% of the nominal concentration. As single samples differed from the range of 80 – 120% of nominal values, it was decided to base the evaluation of biological effects to the mean measured concentrations. The mean measured concentrations were calculated to be 0.11, 0.36, 1.07, 2.78 and 9.64 mg methyl 4-hydroxybenzoate/L.


For more data see any other information on materials and methods
Details on test conditions:
Oxygen concentration and pH were measured in each test vessel directly before adding the eggs and afterwards at least twice per week. The water temperature was measured each working day in all test vessels and in the control. Additionally, the water temperature was measured continuously in at least two control vessels. Uneaten food and faeces were removed from the fry cages and the test aquaria at regular intervals.
At test start, 2 x 15 fertilized and randomized eggs were placed in the stainless steel chambers serving as fry cages. To allow better observation of eggs and larvae, the chambers were placed in the mid part of each test vessel. Each aquarium was equipped with two fry cages. 120 eggs (4 x 30) were used for each test concentration. From day 7 on (dpf = days post fertilization), larvae were fed once daily with ground breeding food (TetraMin Baby, Tetra Werke, Melle, Germany). From the same date on, brine shrimp nauplii (Artemia salina) were added twice daily and. From day 14 (dpf) on, ground TetraMin flakes were added once daily to the fish feed. The larvae were retained in the fry chambers until 14 dpf before the release in the main test vessel.
After 21 and 35 days, survival was determined by photographic counting. After 35 days, length of each individual fish was measured by digital photography. At test end (day 63), all fish were sacrificed and the individual lengths and weights were measured.
At termination of the fish groups, the fish were prepared as follows: the fish were anaesthetized using chloro-butanol. A blood sample was taken from each fish afterwards via heart puncture. After determination of length and weight, the fish were killed with a dorsal cut. All fish were transferred to an appropriate fixative to allow a histopathological analysis of fish tissue.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
0.11 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Post-hatch survival at 35 dpf
Key result
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
0.36 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Post-hatch survival at 35 dpf
Key result
Duration:
63 d
Dose descriptor:
NOEC
Effect conc.:
0.11 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Total survival at 63 dpf
Key result
Duration:
63 d
Dose descriptor:
LOEC
Effect conc.:
0.36 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Total survival at 63 dpf
Details on results:
see: Executive summary

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Remarks:
Most validity criteria are fulfilled but most relevant endpoints could not be evaluated since fish did not reach the needed developmental stages.
Conclusions:
A fish sexual development test (FSDT) was performed to assess the 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 [6].
Evaluation of all endpoints revealed a clear dose dependent effect of the test item methyl 4-hydroxybenzoate on post-hatch survival of early life stages after 21 days post fertilization.
Based on the most sensitive endpoint post-hatch survival, the following NOEC was determined:

NOEC = 0.11 mg methyl 4-hydroxybenzoate/L (mean measured)

Total length of the early life stages was significantly reduced at 9.64 mg methyl 4-hydroxybenzoate/L (NOEC: 2.78 mg methyl 4-hydroxybenzoate/L).
Between day 35 and 63 pf no increased mortality of fish could be observed. As a result of the lethal effects of the early life stages, the overall survival was significantly reduced at ≥ 0.36 mg methyl 4-Hydroxybenzoate/L (NOEC: 0.11 mg methyl 4-hydroxybenzoate/L).
The examination of gonads at test end revealed that most of the fish in controls and under exposure conditions did not reach sexual maturity during the study. Thus, it was not possible to derive information on possible impacts of the test item on sexual development.
Growth of fish at test end was significantly affected at 9.64 mg methyl 4-hydroxybenzoate/L, in terms of reduced wet weight (NOEC: 2.78 mg methyl 4-hydroxybenzoate /L).

Since the relevant parameters for sexual development could not be measured the test was repeated.
Executive summary:

A fish sexual development test (FSDT) with zebrafish (Danio rerio) 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 methyl 4-hydroxybenzoate on the early life stages and sexual differentiation of zebrafish (Danio rerio), following the OECD test guideline 234[7].

The study was conducted with nominal concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg test item/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, e.g. 120 fertilised eggs in control and each treatment concentration. Endpoints that were determined included hatching success, survival and growth during the early life and juvenile stage. At test end after 63 days, fish were measured for total length and wet weight (blotted dry). At day 21 and 35 post fertilization (pf), fish were digitally photographed. Total fish lengths were determined on day 35 pf by evaluating photographs using electronically supported analysis.

 

After 63 days, the in-life phase was finished and all fish were individually measured for total length and wet weight (blotted dry). A blood sample was taken from each fish for measurement of vitellogenin concentration. Sex ratios were determined by macroscopically inspection of the gonads. To confirm the results, a histological examination of the fish gonads was performed.

 

Chemical analysis

The concentrations of methyl 4-hydroxybenzoate were assessed by chemical analysis using an HPLC method. The LOQ was set to 0.030 mg methyl 4-hydroxybenzoate/L. Mean concentrations per treatment of methyl 4-hydroxybenzoate during the course of the study were between 87% and 111% of the nominal concentration. As single samples differed from the range of 80 – 120% of nominal values, it was decided to base the evaluation of biological effects to the mean measured concentrations. The mean measured concentrations were calculated to be 0.11, 0.36, 1.07, 2.78 and 9.64 mg methyl 4-hydroxybenzoate/L.

 

Biological effects

Early life stages

Hatching rate

First larvae hatched on day 3 pf (post fertilization) across all treatment levels. A slight delay of hatch compared to control could be observed at two dosage levels ≥ 2.78 mg methyl 4-hydroxybenzoate/L on day 3 and 4. The delay of hatch originates most likely from a difference in water temperatures between controls and top concentration levels. The difference was limited to the initial phase of the study. The lower temperature values were within the limits as specified in the guideline, but however, led to delay in development of the sensitive embryonic stage.

 

On day 6 pf, hatch was found to be > 80% in all test vessels. Hatch in controls was ≥ 80% and thus in line with the guideline requirements specified for zebrafish.

Hatch was completed after 9 days in all test vessels. Finally, no dose related impact on hatch could be observed (NOEC≥9.64 mg methyl 4-hydroxybenzoate/L).

 

Post-hatch survival at 35 dpf

Survival was recorded daily by visual inspection and the fish numbers were confirmed on day 21 and 35 pf by photographic counting of larvae and juveniles. For calculation of post hatch survival rates, the numbers of the photographic counting were used.

Between day 9 pf and day 21 pf, increased larval mortality was observed across the test levels ≥ 0.36 mg methyl 4-hydroxybenzoate/L.

On day 21 pf and on day 35 dpf, the mean post-hatch survival rate in controls was calculated to be 96.7%, thus met the validity criterion for survival in controls of ≥ 70 %.

Only few dead fish were found afterwards between day 21 and 35 pf.

Mortality of larvae thus occurred mainly in the initial phase of the study, i.e. during the phase of transition from yolk sac feeding to external feeding. Surviving larvae did not display any signs of disease. Statistical analyses of post-hatch survival on day 35 pf revealed significant differences between control and treatments at concentrations ≥ 0.36 mg methyl 4-hydroxybenzoate/L. Thus, the NOEC for the endpoint post-hatch survival during the early life stage was determined at 0.11 mg methyl 4-hydroxybenzoate/L.

 

Length, day 35 pf

On day 35 pf, larval growth in terms of total length was determined. A mean total length of 1.52 cm was calculated for controls. The mean lengths of larvae under treatment conditions were found in the range of 1.36 cm (9.64 mg methyl 4-hydroxybenzoate/L) to 1.76 cm (0.36 mg methyl 4-hydroxybenzoate/L).

Statistical analyses revealed a significant decrease in total length compared to control at 9.64 mg methyl 4-hydroxybenzoate/L. Thus, the NOEC based on the endpoint size in terms of total length after the ELS phase was defined as 2.78 mg methyl 4-hydroxybenzoate/L.


 

Test termination

Survival on day 63 pf

Three fish at 0.36 mg methyl 4-hydroxybenzoate/L, one fish at 1.07 mg methyl 4-hydroxybenzoate/L and three fish at 9.64 mg methyl 4-hydroxybenzoate/L were found dead during the juvenile growth phase between day 35 and day 63 (test end). No signs of disease were observed.

Statistical analyses of survival between day 35 and 63 pf revealed no significant differences between control and treatment concentrations. Thus, the NOEC for survival between day 35 and 63 pf was determined at ≥ 9.64 mg methyl 4-hydroxybenzoate/L (mean measured concentration).

The total survival was calculated to be 96.7% for controls and was thus in line with quality criteria for this parameter, i.e. at least 70%. A treatment related and significant decrease compared to control was observed at ≥ 0.36 mg methyl 4-hydroxybenzoate/L (NOEC: 0.11 mg methyl 4-hydroxybenzoate/L), which was the result of mortality of the early life stages as reported above.

 

Length and weight on day 63 pf

On day 63 pf, growth performance was measured in terms of total length and wet weight (blotted dry). Juvenile fish in the control displayed a mean total length of 2.6 cm. The mean length of fish under treatment conditions varied between 2.4 cm and 2.9 cm. The fish exposed to the highest test concentration were smaller than the fish in the controls and other treatments. However, no significant change compared to control was detected (NOEC≥9.64 mg methyl 4-Hydroxybenzoate/L).

The mean wet weight of fish in controls was measured to be 0.156 g, the mean wet weight of fish under treatment conditions varied between 0.112 g and 0.236 g. The weight of fish exposed to the highest test concentration was found to be reduced compared to the control fish. Statistical analyses revealed a significant decrease at this concentration step, i.e. the NOEC was determined to be 2.78 mg methyl 4-hydroxybenzoate/L.

 

Histopathology

The sex ratio of all fish groups of controls and treatment levels was determined. Sex determination was performed macroscopically by inspection of the gonads and furthermore histologically in order to confirm the results of the macroscopic inspection.

 

The inspection of gonads at test termination revealed that most of the fish did not reach sexual maturity during the test period. The fish samples were send to the histopathological lab to perform a more detailed analysis.

 

The examination revealed, that a high number of fish were still in a transition phase from a protogyne gonad towards a mature gonad which could be attributed to be male or female.

The maturation stages found were thus classified as follows:

-          Female fish

-          Male fish

-          Male fish in transition phase

-          Protogynic gonad

-          juvenile.

 

The stages of maturation can be sorted as follows, starting with the lowest maturity stage:

 

Juvenile < protogynic gonad < female, male.

 

In this study, 80.3 % of all control fish expressed characteristics of a protogynic gonad. Only < 5% of the fish could be clearly identified to be male or female.

As a consequence, the final sex ratio (%males/%females) of the controls and exposed fish groups could not be determined.

The failure of sex ratio determination also prevented a more detailed, i.e. sex-specific, evaluation of growth parameters. The examination revealed, that a high number of fish were still in a transition phase from a protogyne gonad towards a mature gonad which could be attributed to be male or female.

 

Vitellogenin in blood plasma

An initial measurement of vitellogenin (VTG) concentrations revealed that most control fish expressed only very low concentrations in blood plasma, mostly below the limit of quantification of the detection method (ELISA). Due to the immature gonadal stage, the expression of significant VTG levels could not be expected. Thus, the VTG analysis was skipped from this study.

 


 

Conclusion

A fish sexual development test (FSDT) was performed to assess the 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 [6].

Evaluation of all endpoints revealed a clear dose dependent effect of the test item methyl 4-hydroxybenzoate on post-hatch survival of early life stages after 35 days post fertilization.

Based on the most sensitive endpoint post-hatch survival, the following NOEC was determined:

 

NOEC = 0.11 mgmethyl 4-hydroxybenzoate/L (mean measured), corresponding to

NOEC = 0.10 mg methyl 4-hydroxybenzoate/L (nominal).

 

Total length of the early life stages was significantly reduced at 9.64 mg methyl 4-hydroxybenzoate/L (NOEC: 2.78 mg methyl 4-hydroxybenzoate/L).

Between day 35 and 63 pf no increased mortality of fish could be observed. As a result of the lethal effects of the early life stages, the overall survival was significantly reduced at ≥ 0.36 mg methyl 4-Hydroxybenzoate/L (NOEC: 0.11 mg methyl 4-hydroxybenzoate/L).

 

The examination of gonads at test end revealed that most of the fish in controls and under exposure conditions did not reach sexual maturity during the study. Thus, it was not possible to derive information on possible impacts of the test item on sexual development.

 

Growth of fish at test end was significantly affected at 9.64 mg methyl 4-hydroxybenzoate/L, in terms of reduced wet weight (NOEC: 2.78 mg methyl 4-hydroxybenzoate /L).

 

Validity

Validity criteria according to the OECD Test Guideline 234 [6] were adhered to. These included:

•          The dissolved oxygen concentration was at least 60 % of the air saturation value throughout the test.

•          The water temperature did not differ by more than ± 1.5 °C between successive days at any time during the test and was within 27.0 ± 2.0 °C.

•          Hatching success of eggs in controls was greater than 80 %.

•          Post-hatch survival of fish larvae, fry and juveniles in the controls was greater than 70%.

•          In controls, fish reached a mean length of > 14 mm (total length) and a mean weight (wet weight, blotted dry) of > 75 mg.

•          Sex ratio (% males or % females) in the controls:

The examination of gonads at test end revealed that most of the fish in controls and under exposure conditions did not reach sexual maturity during the study. Thus, it was not possible to derive information on possible impacts of the test item on sexual development.

The number of fish clearly determined as mature male or female was below 5% of all fish examined in controls.

Since the relevant parameters for sexual development could not be measured the test was repeated (see OECD 234 FSTD EXP.2, 2019).

 

Table1:         Fish SDT with methyl 4-hydroxybenzoate: Summary of NOEC/LOEC determination during the course of the study

Parameter

NOEC / LOEC

Nominal concentration

methyl 4-hydroxybenzoate [mg/L]

NOEC / LOEC

Mean measured concentration methyl 4-hydroxybenzoate [mg/L]

Hatching success

≥ 10.0 / > 10.0

≥ 9.64 / > 9.64

Post-hatch survival at day 35 pf

0.10 / 0.32*

0.11 / 0.36*

Total length at day 35 pf

3.2 / 10.0*

2.78 / 9.64*

Total survival at day 63 pf

0.10 / 0.32*

0.11 / 0.36*

Total length at day 63 pf (all fish)

≥ 10.0 / > 10.0**

≥ 9.64 / > 9.64**

Wet weight at day 63 pf (all fish)

3.2 / 10.0**

2.78 / 9.64**

Sex ratio1)

n.d.

n.d.

Vitellogenin concentration2)

n.d.

n.d.

1) The examination of gonads at test end revealed that most of the fish in controls and under exposure conditions did not reach sexual maturity during the study. Thus, it was not possible to derive information on possible impacts of the test item on sexual development (n.d. = not determined).

2) Initial measurements of VTG revealed very low concentrations below the LOQ already for controls. As many immature fish were found also for the treatment levels, it was agreed to skip the biomarker measurements in this study.

* Statistical evaluations were performed with the Williams T-Test (α=0.05)

** Statistical evaluations were performed with the Dunnett’s T-Test (α=0.05)