Methyl 4-hydroxybenzoate

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

fish: other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 05, 2018 - December 10, 2019

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:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD TG 234 (Fish Sexual Development Test)

GLP compliance:

yes (incl. QA statement)

Test material

Specific details on test material used for the study:

Test item name: Methyl 4-hydroxybenzoate
CAS number: 99-76-3
Molecular formula: C8H8O3
Molecular mass: 152.2 g/mol
Purity (according to CoA): 99.8 %
Physical state: Powder

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:

In a previous study, conducted at Fraunhofer IME (study number: CLA-002/4-48/A, see OECD 234 FSTD EXP.1 ), a concentration range of 0.1; 0.32; 1.0; 3.2; 10.0 mg methyl 4-hydroxybenzoate/L was applied, following a respective ECHA decision.
Mortality of the early life stages was observed in that study, which occurred in a concentration dependent manner.

Based on the most sensitive endpoint post-hatch survival, the following NOEC was determined in the study CLA-002-4/48/A:

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

In order to be able to assess endocrine mediated effects, test concentrations should be chosen in a range avoiding increased mortality. Thus, it was agreed to set a lower concentration range.

Based on the results obtained in the study CLA-002/4-48/A, the following range of test concentrations was set:

320; 100; 32; 10; 3.2 μg methyl 4-hydroxybenzoate /L.

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


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)

Details on test organisms:

Zebrafish (Danio rerio) was chosen according to OECD test guideline 234 where it is recommended as a test organism, representative of aquatic vertebrates.

Species: Zebrafish (Danio rerio, Teleostei, Cyprinidae)
Source: Test facility bred
Holding: Fertilized eggs for the test were obtained from individuals reared in the laboratory of the Fraunhofer Institute, Schmallenberg, Germany.
Origin of the used strain of zebrafish:
West Aquarium GmbH, 37431 Bad Lauterberg, Germany

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

70 d

Remarks on exposure duration:

A prolongation of the in-life phase by one week was agreed to ensure gonadal maturation by as much fish as possible.

Post exposure observation period:

No

Test conditions

Hardness:

Total hardness: 1.0-1.3 mmol/L
Calcium hardness: 0.9-1.1 mmol/L
Magnesium hardness: 0.1-0.2 mmol/L

Test temperature:

The mean water temperature was in the range between 26.1 °C and 26.9 °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 25.7 and 27.4°C.

pH:

The mean pH-values in the test vessels were between 7.65 and 7.82 throughout the test period.
Single pH values during the in life phase were measured to be between 7.36 and 8.07.

Dissolved oxygen:

The mean oxygen saturation in all test vessels was determined to be between 89 and 97 %, with single values between 76 % and 104 %.

Conductivity:

247.0- 295.0 µS/cm

Nominal and measured concentrations:

Based on the results obtained in the study CLA-002/4-48/A (see details on test solutions and OECD 234 FSTD EXP.1), the following range of test concentrations was set:

320; 100; 32; 10; 3.2 μg methyl 4-hydroxybenzoate /L.

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

Preliminary data!!!!! Preliminary data!!!!!

As reported for the Study CLA-002/4-48/A (see OECD 234 FSTD EXP.1), a prolonged equilibration time of several weeks was necessary to achieve acceptably stable concentrations of the test item in the test tanks. However, a high variation of measured values could finally not be prevented. It was found, that the test item is highly biodegradable under the test conditions applied. Moreover, bacterial matrix in terms of microbial grow-up was observed and increased in the upper treatment levels. Visible grow-up was found at nominal concentrations 100 and 320 µg methyl 4-hydroxybenzoate/L.

Mean concentrations per treatment of methyl 4-hydroxybenzoate during the course of the study were between 75.0% and 106% of the nominal concentration of the test item.
As single measurements were found to be outside a 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 3.39; 7.96; 24.0; 78.4 and 250 µg methyl 4-hydroxybenzoate/L.
Mean measured results are shown in Any other information on materials and methods.
Details on the analytical method can be found in Annex 2 of the attached report

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 70), all fish were sacrificed and the individual lengths and weights were measured.

A prolongation of the in-life phase by one week was agreed with the sponsor to ensure gonadal maturation by as much fish as possible.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

70 d

Dose descriptor:

NOEC

Effect conc.:

24 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Key result

Duration:

70 d

Dose descriptor:

LOEC

Effect conc.:

78.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

For details of the results please refer to the attached report and the executive summary.

Reported statistics and error estimates:

See Annex 7: Statistical Analysis in the report attached

Any other information on results incl. tables

Full report is attached

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

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.

A decrease of hatching success was detected at 250 µg methyl 4-hydroxybenzoate/L.
The survival of fish over the whole test period was found to be significantly reduced at ≥ 78.4 µg methyl 4-hydroxybenzoate/L.

Thus, the NOEC for overall survival was determined as follows:

NOEC = 24.0 µg methyl 4-hydroxybenzoate/L (mean measured), corresponding to
NOEC = 32.0 µg methyl 4-hydroxybenzoate/L (nominal concentration).

Enhanced growth of fish over time was observed and was assumed to be a secondary effect of a treatment related increase of microbial matrix in the test vessels (NOEC total weight, all fish = 24.0 µg methyl 4-hydroxybenzoate/L, corresponding to 32.0 µg methyl 4-hydroxybenzoate/L (nominal concentration)). This increase in grow-up was most prominent for female fish.

The evaluation of the endocrine related endpoints revealed no effect on the male to female ratio (NOEC ≥ 250 µg methyl 4-hydroxybenzoate/L (mean measured), corresponding to
NOEC = 320 µg methyl 4-hydroxybenzoate/L (nominal concentration).

A significant increase of vitellogenin (VTG) concentrations could be detected for the female fish. This increase was statistically significant at ≥ 24.0 µg methyl 4-hydroxybenzoate/L, corresponding to 32.0 µg methyl 4-hydroxybenzoate/L (nominal concentration).

As no increase of VTG concentrations in male fish was observed, it can be postulated that the increase in females was not induced by an interaction of test item and the estrogen receptor and thus was mainly the result of an improved maturation of the fish in the treatment groups.

As no adverse effect on a population relevant endpoint was observed in this study, a primary endocrine potential of the substance could not be confirmed.

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[6].

 

This study was a repeat of a first study run, reported under the study number CLA-002/4-48/A (see OECD 234 FSTD EXP.1). The reason for the repeat was that the first study showed enhanced and concentration dependent mortality of early life stages and secondly an insufficient maturation of the exposed fish over the test period of 63 days. It was agreed to repeat the study with an adapted concentration range. Lower test item concentrations were chosen to prevent mortality or other signs of systemic toxicity.

 

The study reported here was conducted with nominal concentrations of 3.20, 10.0, 32.0, 100 and 320 µg 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 70 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.

 

To ensure that sufficient maturation of the fish was achieved the study was prolonged to in total 70 days of in-life testing. After 70 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

Mean concentrations per treatment of methyl 4-hydroxybenzoate during the course of the study were between 75.0% and 106.0% of the nominal concentration of the test item.

As the mean values were found to be outside a range of 80 – 120% of nominal values, it was decided to base the evaluation of biological effects on the mean measured concentrations.

The mean measured concentrations were calculated to be 3.39; 7.96; 24.0; 78.4 and 250 µg methyl 4-hydroxybenzoate/L.

 

Biological effects

Early life stages

 

Hatching rate

First larvae hatched on day 2 pf (post fertilization) across all treatment levels.

On day 3 pf, hatch was slightly delayed in the treatment groups, but hatching rates fully matched with controls already on the next day. Thus, a substance impact was refused at this stage.

On day 4 pf, hatch was found to be >90% in all test vessels of control and treatments up to 78.4 µg methyl 4-hydroxybenzoate/L.

Hatch was completed after 5 days for control and treatments up to 78.4 µg methyl 4-hydroxybenzoate/L. Hatch in controls was ≥ 80% and thus in line with the guideline requirements specified for zebrafish.

However, hatch was clearly impacted in two test vessels at 250 µg methyl 4-hydroxybenzoate/L. Finally, the mean hatching success on day 5 pf was calculated to be 53.3%, and was significantly reduced compared to control.

Thus, the NOEC for hatching success was determined to be 78.4 µg methyl 4-hydroxy-benzoate/L.

 

Post-hatch survival at 35 dpf

Survival was recorded daily on a visual basis and 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.

After 21 dpf and after 35 dpf, the mean post-hatch survival rates in controls were calculated to be 80.0 and 79.2%, respectively. Thus, the validity criterion for post hatch survival in controls of ≥ 70 % was met.

Only few dead fish were found between day 21 and 35 pf. At 250 µg methyl 4-hydroxybenzoate/L, mortality mainly was related to the embryo development and the hatching period. Only few dead fish were found later.

Post hatch survival at day 35 pf was calculated to be 78.3, 88.3, 68.3, 61.7 and 88.9 % for 3.39, 7.96, 24.0, 78.4 and 250 µg methyl 4-hydroxybenzoate/L, respectively.

Due to a lack of a clear concentration-response relationship, no significant difference between control and treatments could be detected.

Thus, the NOEC for the endpoint post-hatch survival during the early life stage was determined at ≥ 250 µg methyl 4-hydroxybenzoate/L.

 

Length at 35 dpf

On day 35 pf, larval growth in terms of total length was measured. A mean length of 2.00 cm was calculated for controls. The mean total lengths of larvae under treatment conditions were found in the range of 2.05 cm (at 3.39 and 7.96 µg methyl 4-hydroxybenzoate/L) to 2.19 cm (at 250 µg methyl 4-hydroxybenzoate/L).

Statistical analyses revealed a significant increase in total length compared to control at 78.4 µg methyl 4-hydroxybenzoate/L. Thus, the NOEC based on the endpoint growth in terms of total length after the ELS phase was defined as 24.0 µg methyl 4-hydroxybenzoate/L. The enhanced growth was assumed to be the result of the microbial matrix in the test tanks inducing an additional carbon source and thus an improved nutrition to the fish larvae.

 

Test termination

Total survival at day 70 pf

There was no mortality between day 35 pf and test end in control and at 3.39 µg methyl 4-hydroxybenzoate/L. One, one, two and no fish were found dead at 7.96, 24.0, 78.4 and 250 µg methyl 4-hydroxybenzoate/L, respectively.

No clinical signs of disease were observed.

Thus, the NOEC for survival between day 35 and 70 pf was determined at ≥ 250 µg methyl 4-hydroxybenzoate/L (mean measured concentration).

The overall survival, e.g. the number of surviving fish related to the number of fertilized eggs introduced, was calculated to be 79.2% 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 ≥ 78.4 µg methyl 4-hydroxybenzoate/L (NOEC: 24.0 µg methyl 4-hydroxybenzoate/L), which was the result of mortality of embryos in the hatching period and the observed mortality of fish in the early life stage phase.

 

Length and weight at day 70 pf, all fish

On day 70 pf, growth in terms of total length and wet weight (blotted dry) was measured. Juvenile fish in the control displayed a mean total length of 3.13 cm. The mean length of fish under treatment conditions varied between 3.15 cm (7.96 µg methyl 4-hydroxybenzoate/L) and 3.33 cm (78.4 µg methyl 4-hydroxybenzoate/L).

The mean wet weight of fish in controls was measured to be 0.301 g, the mean wet weight of fish under treatment conditions varied between 0.305 g (at 7.96 µg methyl 4-hydroxybenzoate/L) and 0.388 g (at 250 µg methyl 4-hydroxybenzoate/L).

The growth parameter for control clearly fulfilled the criterion specified in the guideline. The requirements are to achieve at least 75 mg wet weight (blotted dry) and at least 14 mm of standard length.

A concentration related increase of both total length and wet weight was observed.

A significant difference to control was found for total length of all fish at ≥ 24.0 µg methyl 4-hydroxybenzoate/L. However, the overall effect size was <10% compared to control and thus considered to be of minor biological relevance.

A more prominent and finally statistical significant increase was detected for wet weights at ≥ 78.4 µg (NOEC: 24.0 µg methyl 4-hydroxybenzoate/L).

These findings are consistent with the length increase observed on day 35 pf. The treatment related growth increase was assumed to be the secondary effect of microbial grow-up induced by rapid biodegradation of the test item. This additional matrix finally resulted in an improved nutrition for the fish larvae and juvenile fish.

 

Length and weight at day 70 pf, after histological examination

Total length and wet weights were finally evaluated, after the results of histology were available. The data were separately assessed for males, females and undifferentiated fish.

The mean total length in controls was 3.1, 3.3 and 3.0 cm for males, females and undifferentiated fish, respectively.

The mean total lengths of male fish in the treatments were calculated to be between 3.2 and 3.3 cm. The mean female total lengths in the treatments were calculated to be between 3.3 and 3.5 cm. The mean total lengths of the undifferentiated fish were determined to be between 2.9 and 3.2 cm.

A slight treatment related length increase was detected for male fish, but this was not significant.

The mean wet weight of controls was measured to be 0.282, 0.369 and 0.269 g for males, females and undifferentiated fish, respectively. The mean male weights in the treatments were calculated to be between 0.293 g and 0.340 g. The mean female weights in the treatments were calculated to be between 0.363 g and 0.447 g. The mean weights of the undifferentiated fish were determined to be between 0.238 g and 0.306 g.

A statistical significant increase of wet weights was detected for female fish at 250 µg methyl 4-hydroxybenzoate/L (NOEC: 78.4 µg methyl 4-hydroxybenzoate/L).

There was no effect on wet weight for both males and undifferentiated fish (NOEC:≥250 µg methyl 4-hydroxybenzoate/L).

 

Endocrine related parameters

Male to female ratio

The data presented in this report were evaluated in two ways. First, only the three sex categories ‘male’, ‘female’, and ‘undifferentiated’ were considered. This approach followed the evaluation described in the OECD TG 234. Secondly, we provided a more detailed evaluation, showing all phases of development, in order to allow a more detailed interpretation of the data.

Initially, we determined the female to male ratio [%] in controls in order to determine if the validity criterion regarding sex ratio was fulfilled. For this purpose, only mature individuals were considered. For this evaluation, fish assigned as ‘undifferentiated’ were not counted in the total number of fish. We observed a ratio of 40.4 % males to 59.6 % females in controls, and thus met the validity criterion of 30 – 70 % for males and females. For the treatment levels, there was no difference in the distribution of male and female fish. Mean male ratios were calculated to be between 40.7 and 55.6%, mean female ratios were determined to be between 44.4 and 59.3%.

 

An enhanced maturation was observed for both males and females. Consequently, the number of undifferentiated fish decreased in a concentration dependent manner. The proportion of undifferentiated fish decreased from 51.7 % of all fish in controls to 11.6 % at the highest treatment level.

 

The more detailed examination of the fish revealed that most of the undifferentiated gonads were at a stage of a protogyne gonad. Only few fish were related to the stage of juvenile gonads, e.g. 4.1 % in controls. The numbers of undifferentiated fish decreased in the treatment levels, suggesting a faster maturation. This finding is consistent with reported increase of the growth parameters observed on day 35 and 70 pf. The treatment related growth increase was assumed to be the secondary effect of microbial grow-up induced by rapid biodegradation of the test item. This additional matrix finally resulted in an improved nutrition for the fish larvae and developing fish.

 

Histopathological lesions

Histopathological lesions in the ovaries were characterized by few findings of oocyte atresia, egg debris and granulomatous inflammation in females, and testis-ova in males.

 

Females

Only one female in control showed signs of oocyte atresia and granulomatomous inflammation. In total, 3 female fish at the highest concentration expressed oocyte atresia, egg debris and inflammation. All in all, there was no obvious pathology in the female gonads.

 

Males

Single immature oocytes were found in the testis tissue (testis-ova) of one individual in control and one animal in the test concentrations < 250 µg methyl 4-hydroxybenzoate/L. No concentration-related change in frequency of this effect could be identified. Pathological alterations of testicular morphology were not observed.

 

Biomarker VTG concentration

The vitellogenin (VTG) concentrations were measured from blood plasma samples of each fish. The results were evaluated for fish identified as male, female and undifferentiated.

Mean VTG concentrations in control males were determined to be 0.04 ng/µg. No impact on VTG concentrations of the treatment groups was observed. Mean VTG concentrations were between 0.05 and 0.13 ng/µg.

For females, the mean VTG concentration in controls was calculated to be 154.7 ng/µg.

A concentration related increase of VTG amount compared to control could be detected in the treatments. The maximum mean VTG concentration was calculated to be 1032.2 ng/µg at 78.4 µg methyl 4-hydroxybenzoate/L. This increase was statistically significant at ≥ 24.0 µg methyl 4-hydroxybenzoate/L.

For the class of undifferentiated fish a mean VTG concentration in controls was determined to be 0.05 ng/µg. The mean VTG concentrations in the treatments were also very low and were calculated to be between 0.06 and 0.18 ng/µg. No substance related effect was found for both male and undifferentiated fish.

It can be assumed, that the increased VTG levels in female fish are a consequence of an improved grow-up and enhanced maturation. Enhanced growth and maturation was already displayed in the grow parameters derived on day 35 and 70 pf. Furthermore, the histological evaluation revealed a higher number of mature fish in the treatment concentrations.

 

An interaction with test item and the estrogen receptor would also induce an increase of VTG concentrations in male fish, but this was not observed at any treatment level.

 

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.

A decrease of hatching success was detected at 250 µgmethyl 4-hydroxybenzoate/L.

The survival of fish over the whole test period was found to be significantly reduced at ≥78.4 µg methyl 4-hydroxybenzoate/L.

 

Thus, the NOEC for overall survival was determined as follows:

 

NOEC = 24.0 µg methyl 4-hydroxybenzoate/L (mean measured), corresponding to

NOEC = 32.0 µg methyl 4-hydroxybenzoate/L (nominal concentration).

 

Enhanced growth of fish over time was observed, and was assumed to be a secondary effect of a treatment related increase of microbial matrix in the test vessels (NOEC total weight, all fish = 24.0 µg methyl 4-hydroxybenzoate/L, corresponding to 32.0 µgmethyl 4-hydroxybenzoate/L (nominal concentration)). This increase in grow-up was most prominent for female fish.

 

The evaluation of the endocrine related endpoints revealed no effect on the male to female ratio (NOEC≥250 µg methyl 4-hydroxybenzoate/L (mean measured), corresponding to

NOEC = 320 µg methyl 4-hydroxybenzoate/L (nominal concentration).

 

A significant increase of vitellogenin (VTG) concentrations could be detected for the female fish. This increase was statistically significant at≥24.0 µg methyl 4-hydroxybenzoate/L, corresponding to 32.0 µg methyl 4-hydroxybenzoate/L (nominal concentration).

 

As no increase of VTG concentrations in male fish was observed, it can be postulated that the increase in females was not induced by an interaction of test item and the estrogen receptor and thus was mainly the result of an improved maturation of the fish in the treatment groups.

 

As no adverse effect on a population relevant endpoint was observed in this study, a primary endocrine potential of the substance could not be confirmed.

 

Validity

Validity criteria according to the OECD Test Guideline 234 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 histological evaluation revealed a male to female ratio in controls of 40.4% male to 59.6% female fish and thus was in line with the guideline requirements.

It has to be stated, that a high number of undifferentiated fish was detected in controls and the treatment groups.The proportion of undifferentiated fish decreased from 51.7 % of all fish in controls to 11.6 % at the highest treatment level.

 

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	100 / 320**	78.4 / 250**
Post-hatch survival at 35 dpf	≥ 320 / > 320	≥ 250 / > 250
Total length at 35 dpf	32 / 100*^2)	24.0 / 78.4*^2)
Survival between day 35,  and day 70 pf	≥ 320 / > 320	≥ 250 / > 250
Total survival at 70 dpf	32 / 100**	24.0 / 78.4**
Total length at 70 dpf (all fish)	≥ 320 / > 320^1)	≥ 250 / > 250^1)
Wet weight at 70 dpf (all fish)	32 / 100^2)	24.0 / 78.4^2)
Length at 70 dpf (males)	≥ 320 / > 320	≥ 250 / > 250
Length at 70 dpf (females)	≥ 320 / > 320	≥ 250 / > 250
Weight at 70 dpf (males)	≥ 320 / > 320	≥ 250 / > 250
Weight at 70 dpf (females)	100 / 320*^3)	78.4 / 250*^3)
Sex ratio (male to female ratio)	≥ 320 / > 320	≥ 250 / > 250
Sex determination [% males]	≥ 320 / > 320^4)	≥ 250 / > 250^4)
Sex determination [% females]	≥ 320 / > 320^4)	≥ 250 / > 250^4)
Sex determination [% undifferentiated]	≥ 320 / > 320^4)	≥ 250 / > 250^4)
Vitellogenin concentration in females	10.0 / 32.0**	7.96 / 24.0**
Vitellogenin concentration in males	≥ 320 / > 320	≥ 250 / > 250
Vitellogenin concentration in undifferentiated fish	≥ 320 / > 320	≥ 250 / > 250

¹) A significant increase compared to control was found for total length of fish at ≥ 24 µg methyl 4-hydroxybenzoate/L. However, the overall effect size was <10% compared to control and thus considered to be of minor biological relevance.

²) A concentration related increase of growth parameters was observed at day 35 pf, and at day 70 pf (test end). A statistical significant increase was detected at ≥ 78.4 µg (NOEC: 24.0 µg methyl 4-hydroxybenzoate/L).

³) A concentration related increase of growth parameters was observed at day 70 pf (test end). A statistical significant increase was detected at 250 µg (NOEC: 78.4 µg methyl 4-hydroxybenzoate/L).

⁴) An enhanced maturation was observed for both males and females. Consequently, the number of undifferentiated fish decreased in a concentration dependent manner. As presented for the parameter “male to female ratio”, no shift in sex ratio was observed.

 

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

** Statistical evaluations were performed with the Jonckheere-Terpstra Test (α=0.05)