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Endocrine disrupter testing in aquatic vertebrates – in vivo

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Administrative data

Endpoint:
fish: other
Remarks:
Sexual Development Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 August 2019 - 16 April 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is performed in accordance with GLP and to the relevant test guideline, with no deviations impacting the integrity of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD TG 234 (Fish Sexual Development Test)
Version / remarks:
28 July 2011
Deviations:
yes
Remarks:
Light: sometimes <540 lux. Water temp. occasionally >1.5ºC between test chambers. Test solution conc. not maintained within ±20% of mean meas. values. Dissolved O2: sometimes <60% ASV. Stat. sig. difference between solvent control & control for survival
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctan-1-ol
EC Number:
211-477-1
EC Name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctan-1-ol
Cas Number:
647-42-7
Molecular formula:
C8H5F13O
IUPAC Name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctan-1-ol
Test material form:
liquid
Details on test material:
Molecular Formula: C8H5F13O
Appearance: Colourless / light-yellow liquid
Storage: Room temperature (15 - 30 ºC)
Expiry Date: 07 April 2021

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.030, 0.096, 0.30, 0.96 and 3.0 mg/L; control and solvent control
- Sampling method: Triplicate 25 mL samples of freshly prepared test media and the corresponding 24 h old media from the controls and each test concentration were taken for analysis weekly throughout the duration of the test. A single replicate was sampled on each occasion with alternative replicates sampled each time with the exception of Day 0 (pre-hatch) and the end of the test samples when each replicate was sampled.
- Sample storage conditions before analysis: At each sample timing, triplicate samples were taken. One for chemical analysis and two as ‘back-up’ samples, stored frozen, in case further analysis was required.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Initial solvent stock solutions were prepared in acetone. The solvent stock solutions were prepared as required during the test and stored for a maximum of 6 days at 2 - 8°C.

Weight or vol. used Final vol. (mL) Nominal conc. (mg/mL) Media code
0.88 mL of
test substance 5 300 SM1
1.6 mL of SM1 5 96 SM2
0.5 mL of SM1 5 30 SM3
0.5 mL of SM2 5 9.6 SM4
0.5 mL of SM3 5 3.0 SM5

The preparation of the test concentrations for each phase are detailed in the attached Appendix document. The dilution water was aerated for approximately 24 hours prior to use to saturate the oxygen concentration.

- Eluate: N/A
- Differential loading: N/A
- Controls: Yes, blank control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 10µL/L in test concentrations and solvent control
- Test concentration separation factor: 3.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported. The test preparations were observed to be colourless solutions throughout the duration of the test.
- Other relevant information: N/A

Test organisms

Aquatic vertebrate type:
fish
Test organisms (species):
Oryzias latipes
Details on test organisms:
TEST ORGANISM
- Common name: Japanese Medaka
- Strain: N/A
- Source: In-house laboratory breeding system.
- Life stage: Eggs, up to the stage of sexual maturity
- Age at study initiation (mean and range, SD):
Pre-hatch phase: eggs < 12 h old
Post-hatch phase: Started once all viable eggs were hatched (10 d following egg addition)
- Length at study initiation (length definition, mean, range and SD): N/A
- Weight at study initiation (mean and range, SD): N/A
- Method of breeding:
The Oryzias latipes eggs were obtained from in-house cultures (details maintained within the centrally held facility records). Viable fish eggs were obtained from breeding pairs of O. latipes. On collection, the eggs were gently rolled to remove filaments and the stage of egg development was established. The eggs were added to the test system, typically before the first cleavage of the blastodisc or within 12 hours of fertilisation.
The breeding pairs in the breeding system were held in a temperature-controlled room under artificial light (16 hour light:8-hour dark photo-period and ~30 minute dawn/dusk period) in holding tanks appropriate to their size, under continuous water renewal (flow-through) conditions.
The breeding pairs of O. latipes were fed 2 to 4 times daily using 24 to 48 hour old brine shrimp (Artemia salinis) nauplii and also with Tetramin® flake food, which was added to the holding tank in quantities dictated by the size of the fish. The feeding frequency used ensured the fish received adequate amounts of food and to prevent build-up of excess uneaten food which could affect water quality and hence the health of the fish. The food was not considered to contain contaminants likely to affect the outcome of the study. Uneaten food and debris were siphoned or cleaned from the tanks as required.
- Pre-exposure reproductive information: N/A

ACCLIMATION - of eggs
- Acclimation period: At least 1 hour prior to being added to the vessels.
- Acclimation conditions (same as test or not): Yes
- Type and amount of food during acclimation: N/A
- Feeding frequency during acclimation: N/A
- Health during acclimation (any mortality observed): Not reported

QUARANTINE (wild caught): N/A

FEEDING DURING TEST
- Food type: Following hatching, fish were fed Tetramin® flake food and/or live brine shrimp (Artemia) nauplii, (~48 hours old), depending on the size of the fish
- Amount: Dictated by the size of the fish
- Frequency: 1 - 3 times a day. The feeding regime was considered appropriate to ensure fish were supplied with an adequate amount of food whilst also ensuring that a build-up of uneaten food did not occur (which could affect the water quality when using a sealed system).

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): > 20 breeding pairs
- Method of collection of fertilised eggs: The fertilised eggs were collected from the breeding groups, gently rolled to remove filaments and acclimatised to laboratory conditions for at least 1 hour prior to being added to the vessels.
- Subsequent handling of eggs: Thirty fertilised eggs were added into glass scintillation vials containing treated mains water and randomly assigned to the test vessels (total 120 eggs per concentration). The eggs from each vial were transferred using a pipette into the corresponding vessel by releasing the eggs under the water surface. During the pre-hatch period, where possible, non viable or necrotic eggs were removed, avoiding disturbance of adjacent viable eggs.
The post-hatch phase started once all of the viable eggs were considered to have hatched. At the start of the post-hatch phase an initial estimate of hatching success was made.
- Subsequent handling of juvenile fish: To accommodate juvenile fish growth, test vessels were periodically increased in size.

POST-HATCH FEEDING
- Start date: Once hatched
- Type/source of feed: Tetramin® flake food and/or live brine shrimp (Artemia) nauplii
- Amount given: Dictated by the size of the fish
- Frequency of feeding: 1 - 3 times a day. The feeding regime was considered appropriate to ensure fish were supplied with an adequate amount of food whilst also ensuring that a build-up of uneaten food did not occur (which could affect the water quality when using a sealed system).
Excess food (and waste material) was siphoned out of the test vessels as required.

Study design

Test type:
semi-static
Water media type:
other: Laboratory treated mains supply - the water was pumped to the laboratory through an activated carbon filter
Limit test:
no
Total exposure duration:
122 d
Remarks on exposure duration:
112 d post-hatch (122 d total)
Post exposure observation period:
N/A

Test conditions

Hardness:
Range across test duration:
46 - 77 mg/L as CaCO3
See attached Appendix document for full output
Test temperature:
Range across test duration:
23.0 - 26.8 ºC
Range for continuously measured vessel:
23.8 - 26.8 ºC
See attached Appendix document for full output. All values were considered to be within acceptable limits with the exception that on three occasions, the water temperature varied by more than 1.5°C (Days 54, 67 and 82) over the duration of the test. This was considered not to affect the integrity of the test given that the temperatures remained within the 25 ± 2°C range specified in the protocol.
pH:
Range across test duration:
6.3 - 8.5
See attached Appendix document for full ouput
Dissolved oxygen:
Range across test duration:
40.9 - 107.6 % air saturation value
See attached Appendix document for full output.
Salinity:
N/A
Conductivity:
Range across test duration:
175 - 230 µS/cm
See attached Appendix document for full output.
Nominal and measured concentrations:
Nominal: 0.030, 0.096, 0.30, 0.96 and 3.0 mg/L; corresponding to geometric mean measured: 0.00933, 0.0137, 0.0231, 0.0537 and 0.0953 mg/L.
See attached Appendix document for full output.
Details on test conditions:
TEST SYSTEM
- Test vessel: Constructed glass aquaria
- Type (delete if not applicable): Closed (due to the volatile nature of the test substance)
- Material, size, headspace, fill volume: Glass, completely filled, sealed.
The size of the test vessel was increased periodically as the size of the fish increased. Initially the test vessels contained ~1 L of media per vessel; then increased to ~2 L per vessel at 6 days post-hatch, ~3.5 L per vessel at 48 days post-hatch and further increased to ca 7 L per vessel at 61 days post-hatch.
- Aeration: Yes. Initially aeration was not provided to the test vessels due to the volatile nature of the test substance. However, at 7 days post-hatch, the fish were observed to be swimming at the surface of the vessels and dissolved oxygen concentration was measured to be below 60% air saturation value (ASV). From this point, and for the remainder of the test, gentle aeration was provided to the test vessels to ensure dissolved oxygen concentration was maintained at or above 60% ASV and maintain fish health.
- Type of flow-through: The test substance was known to be highly volatile therefore extensive media preparation work was conducted to determine the most appropriate method of preparation for the test.
The preferred method for dosing this type of study is use of flow-through. However, despite using several designs and dosing both with and without the use of auxiliary solvents, neither nominal nor consistent concentrations could be achieved using a flow-through system. Therefore, a semi-static (daily renewal) test system was used, with a solvent spike.
- Renewal rate of test solution (frequency/flow rate): Daily. At each media renewal, ca 90 % of the old media was removed from the test vessel and replaced with freshly prepared media.
- No. of organisms per vessel: Nominally 30 fertilised eggs were added directly to test vessels.
- No. of organisms per mL or well: 1 egg per 33.3 mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Vehicle control performed: Yes
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: Not specified
- Stocking density: 120 eggs per concentration (30 per vessel)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Treated mains water that had been treated via activated carbon filters. The dilution water was aerated for approximately 24 h prior to use to saturate the oxygen concentration.
- Total organic carbon: Not specified
- Particulate matter: Turbidity: < 1.4 NTU
- Metals: See attached Appendix document for the typical Certificate of Analysis
- Pesticides: See attached Appendix document for the typical Certificate of Analysis
- Chlorine: 10.8 mg/L
- Alkalinity: as CaCO3: 14.2 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement:
pH, temperature and dissolved oxygen concentration of each test vessel was determined at the start and the end of the test and then weekly (for fresh media and corresponding old media) for the duration of the test.
Conductivity and total hardness were determined in each test vessel at the start and the end of the test.
Temperature was also monitored continuously in one of the control vessels.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour day:8-hour dark period with an approximate 30-minute dawn/dusk transition period
- Light intensity: The light intensity was measured in the test area to range from 526 to 584 lux at the start and from 510 to 591 lux at the end of the test. The lower end of the light intensity range was slightly below the 540 to 1080 lux range recommended in the Guideline. As a sealed vessel system with solvent was used, a lower light intensity was considered justifiable to try to minimise any potential bacterial build-up which could adversely affect the oxygen concentration within the test vessel. Given that no adverse effects were observed in the control and solvent control groups, this slightly lower light intensity was considered not to affect the integrity of the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Appearance, colour and behaviour of the test substance in the test media were recorded throughout the test.
- The eggs were observed daily during the pre-hatch period. The fish were observed daily throughout the exposure period for survival and general behaviour.
- Any mortality was recorded and, where possible, the sex of the fish determined by macroscopic examination of the gonads.
- Any abnormal behaviour (compared to the controls), for example, territorial aggressiveness, changes to body colour, colourations or shape, was also recorded.
- Hatching success
- Organism length and weight (female and male)
- Genetic sex ratio
- Phenotypic sex ratio (gonad histology)
- Vitellogenin (VTG) concentration (male and female)
- Secondary sex characteristics (anal fin papillae count)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: N/A

RANGE FINDING STUDY
- Test concentrations: N/A
- Results used to determine the conditions for the definitive study: The nominal concentrations used in this test were based on information supplied by the Sponsor from previous tests.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
122 d
Dose descriptor:
NOEC
Effect conc.:
0.014 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: overall NOEC (based on all endpoints)
Duration:
122 d
Dose descriptor:
NOEC
Effect conc.:
0.054 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Remarks:
body length and weight (male and female)
Duration:
122 d
Dose descriptor:
NOEC
Effect conc.:
0.023 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks:
(hatching success)
Duration:
122 d
Dose descriptor:
NOEC
Effect conc.:
0.054 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
(survival)
Duration:
122 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.095 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
genetic/phenotypic sex ratio
Remarks:
(genetic & phenotypic sex ratio, % males)
Duration:
122 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.095 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
vitellogenin level
Remarks:
(female)
Duration:
122 d
Dose descriptor:
NOEC
Effect conc.:
0.014 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
vitellogenin level
Remarks:
(male)
Duration:
122 d
Dose descriptor:
NOEC
Effect conc.:
0.054 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
secondary sexual characteristics
Remarks:
(male anal fin papillae count)
Details on results:
A statistically significant effect was observed on hatching success at nominal concentrations of 0.96 mg/L and above. In addition, statistically significant effects were observed on survival and growth (male and female body length and weight) at the highest nominal test concentration of 3.0 mg/L.
Sex ratio was not affected by the test substance at the concentrations employed, with no statistically significant differences observed between the test concentrations and solvent control in terms of either genetic or phenotypic sex ratio.
Whilst no statistically significant increase or decrease in VTG concentration in female fish was observed, statistically significant effects were observed for male secondary sex characteristics and male VTG concentration. A significant decrease in the number of anal fin papillae was observed at the 3.0 mg/L nominal test concentration whilst a significant increase in VTG concentration was observed at the 0.30 mg/L nominal test concentration and above.
The overall NOEC covering all endpoints tested was considered to be 0.096 mg/L based on nominal test concentrations (0.0137 mg/L based on geometric mean measured concentrations).

- Mortality/survival at embryo, larval, juvenile, and adult stages:
Hatching success: Mean survival in treatments: 89 % - 96 %
Overall survival of hatched larvae: Mean survival in treatments: 77 % - 91 %
- Numbers and time of dead and malformed organisms: Not specified
- Days to hatch: 10
- Number of healthy fish at end of test: Not specified
- Type of and number with morphological abnormalities: In general, no abnormal appearance was observed during the test.
- Type of and number with behavioural abnormalities: In general, no abnormal behaviour was observed during the test. At the highest nominal concentration, some of the fish were observed to be lethargic and at the bottom of the vessel during the test. However, these effects were only briefly observed and did not persist.
- Other biological observations: N/A
- Effect concentrations exceeding solubility of substance in test medium: No
- Observed effects at each concentration for each observation time: See 'any other details on results incl. tables' for tabulated data
- Cumulative mortality at each concentration and for each recommended observation time if possible: Not specified
- Mortality in the controls: Control: 8 %; solvent control: 16 %
- Incidents in the course of the test which might have influenced the results:
For survival and phenotypic sex ratio, a statistically significant difference was observed between the control and solvent control (see 'evaluation of potential effects of the solvent' below). Initially, aeration was not provided to the test vessels. However, at 7 dph, the fish were observed swimming at the vessel surface and dissolved oxygen concentration was < 60% ASV. From this point onwards, gentle aeration was provided to the test vessels to ensure dissolved oxygen concentration was maintained at <= 60% ASV and maintain fish health. On three occasions, the water temperature between vessels varied by more than 1.5°C (Days 54, 67 and 82) over the duration of the test. This was considered not to affect the integrity of the test given that the temperatures remained within the 25 ± 2°C range specified in the protocol.
The test duration was extended due to fish in the highest test concentration experiencing a delay in begining to reach sexual maturity until 83 dph (based on eggs being observed), compared to the control and solvent control groups. The control and solvent control groups began to reach sexual maturity at 75 dph. The test duration (112 dph) was longer than the Guideline-recommended length (60 dph).
- Fish weights (individual and mean values):
Please see the attached Appendix document for individual fish weights and lengths; and 'any other information on results incl. tables' for a summary table of the data (Table 3).

- Evaluation of potential effects of the solvent: For survival and phenotypic sex ratio, the analysis showed statistically significant differences between the control and solvent control group. However, the effect was only a 9 % reduction and therefore the biological relevance of this is questionable, based on allowances for control mortality in chronic aquatic toxicity testing. All statistical analyses of effects were therefore limited to the solvent control group, instead of pooled controls, and is not considered to negatively impact the validity of the study.

-The test preparations were observed to be colourless solutions throughout the duration of the test.
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
Statistical analysis was performed with CETIS v 1.8.6.8.
Each NOEC and LOEC analysis included Bartlett’s Equality of Variance test and Shapiro-Wilks Test to evaluate homogeneity and normality of the data, respectively. In addition, Grubbs Extreme Value test was also included to determine presence of outliers. Where data were non-monotonic, CETIS alerts the user so an appropriate method can be selected instead.
Data from control and solvent control groups were compared using a Fisher Exact Test (hatching success, survival and sex ratio), an Equal Variance Two-Sample t-Test (male length, female length and weight data and male anal fin papillae counts), an Unequal Variance Two-Sample t-Test (male weight data) or Wilcoxon Rank Sum Two-Sample Test for VTG concentration. With the exception of survival and phenotypic sex ratio, the analysis showed no statistically significant differences between control and solvent control groups. Given that significant effects were observed, results from test substance groups were analysed against the solvent control data.
With the exception of hatching success and final survival, all other endpoints were analysed separately for male and female fish. Any fish where genotypic and phenotypic sex did not match, or where one evaluation was missing, were excluded from statistical analysis. Data were analysed using one-way ANOVA to determine any negative effect of the test concentrations compared to the control (C>T) with the exception of the male VTG data where analysis was conducted using a one-way ANOVA to determine any positive effect of the test concentrations compared to the control (CThe hatching success and final survival was analysed using the Cochran-Armitage Trend Step-Down Test.
For the final length and weight of the male and female fish, genetic and phenotypic sex ratio, male secondary sex characteristics (papillary process counts) and VTG concentrations, the data were analysed using the Jonckheere-Terpstra Step-Down Test.

Any other information on results incl. tables

A summary of the NOEC and LOEC values for the various end points assessed are given below:

Response

Nominal concentration
(mg/L)

Geometric Mean Measured Concentration (mg/L)

NOEC

LOEC

NOEC

LOEC

Hatching success

0.30

0.96

0.0231

0.0537

Final survival

0.96

3.0

0.0537

0.0953

Female growth (lengths and weights)

0.96

3.0

0.0537

0.0953

Male growth (lengths and weights)

0.96

3.0

0.0537

0.0953

Genetic sex ratio (% males)

≥3.0

>3.0

≥0.0953

>0.0953

Phenotypic sex ratio (% males)

≥3.0

>3.0

≥0.0953

>0.0953

VTG concentration (female)

≥3.0

>3.0

≥0.0953

>0.0953

VTG concentration (male)

0.096

0.30

0.0137

0.0231

Secondary sex characteristics

0.96

3.0

0.0537

0.0953

Validity criteria

The following validity criteria were achieved and therefore the test is considered valid:

  • The dissolved oxygen concentration should be at least 60% air saturation value (ASV) for the duration of the test. The oxygen concentration was observed to be below 60% ASV in several instances. Following observations, the aeration was increased and the oxygen concentration returned to >60% ASV. This was considered not to affect the integrity of the test given that no adverse effects were observed at the time
  • The water temperature did differ by more than 1.5ºC between test chambers on occasion during the test, but was maintained at 25 ± 2ºC
  • A validated analytical method was available with a detection limit of 0.005 mg/L. Concentrations of the test solution were not maintained within ±20% of the mean measured values due to the highly volatile nature of the test substance
  • Hatching success in the control treatments was >80% (actual: control, 87%, solvent control, 94%)
  • Post-hatch survival of fish larvae in the control treatments was ≥70% (actual: control, 92%, solvent control, 84%)
  • The weight and length of the control fish was >150 mg and >20 mm, respectively, at the end of the test
  • Sex ratio (% males or females) in the control treatments (based on phenotypic evaluation) was 30 – 70%
  • A statistical significant reduction in survival was observed for the solvent control when compared to the control group. However, the effect was only a 9 % reduction and therefore the biological relevance of this is questionable, based on allowances for control mortality in chronic aquatic toxicity testing. All statistical analyses of effects were therefore limited to the solvent control group, instead of pooled controls, and is not considered to negatively impact the validity of the study

Endpoint results

For mean values per replicate vessel and individual results where relevant, please see the attached Appendix document.

General observations

In general, no abnormal behaviour or appearance was observed during the test. At the highest nominal concentration, some of the fish were observed to be lethargic and at the bottom of the vessel during the test. However, these effects were only briefly observed and did not persist.

Hatching success

The mean hatching success of embryos in the control and solvent control vessels was 87 % and 94 %, respectively. Mean hatching success in the treatments ranged between 89 % - 96 %. There were no statistically significant effects (p<0.05) on hatching success at nominal test concentrations of 0.030, 0.096 and 0.30 mg/L compared to the solvent control.  Statistically significant effects were observed at the nominal concentrations of 0.96 and 3.0 mg/L compared to the solvent control. The NOEC and LOEC values for hatching success were therefore considered to be 0.30 and 0.96 mg/L, respectively, based on nominal concentrations (0.0231 and 0.0537 mg/L, respectively, based on geometric mean measured concentrations). 

Table 1. Hatching success

Nominal concentration (mg/L)

Geometric mean measured concentration (mg/L)

Hatching success (%)

Mean

SD

Control

Control

87

3

Solvent control

Solvent control

94

5

0.030

0.00933

96

3

0.096

0.0137

92

8

0.30

0.0231

93

3

0.96

0.0537

89

9

3.0

0.0953

91

8

SD = standard deviation

Survival

The mean survival in the control and solvent control groups was 92 % and 84 % respectively.

Mean survival in the treatments ranged between 77 % - 91 %. There were no statistically significant effects (p<0.05) on survival at nominal test concentrations of 0.03 to 0.96 mg/L compared to the solvent control. However, a significant reduction in survival was observed at the nominal 3.0 mg/L test concentration compared to the solvent control. The NOEC and LOEC values for survival were therefore considered to be 0.96 and 3.0 mg/L, respectively, based on nominal concentrations (0.0537 and 0.0953 mg/L, respectively, based on geometric mean measured concentrations).

Table 2. Percentage post-hatch larval survival

Nominal concentration (mg/L)

Geometric mean measure concentrations (mg/L)

Survival at the end of the test (%)

Mean

SD

Control

Control

92

8

Solvent control

Solvent control

84

8

0.030

0.00933

88

3

0.096

0.0137

91

7

0.30

0.0231

81

7

0.96

0.0537

84

7

3.0

0.0953

77

7

SD = Standard deviation

Fish total lengths and wet weights

There were no statistically significant effects (p<0.05) on male or female fish weight and length at nominal test concentrations of 0.030 to 0.96 mg/L compared to the solvent control. Statistically significant reduction in length and weight in both male and female fish was observed at the highest nominal test concentration of 3.0 mg/L compared to the solvent control. Taking into account both length and wet weight, the NOEC and LOEC values for growth up to sexual maturity were therefore considered to be 0.96 and 3.0 mg/L, respectively, based on nominal concentrations (0.0537 and 0.0953 mg/L, respectively, based on geometric mean measured concentrations).

Table 3. Fish total lengths and wet weights

Nominal concentration (mg/L)

Geometric mean measured concentration (mg/L)

Male fish

Female fish

Total length (mm)

Wet weight (mg)

Total length (mm)

Wet weight (mg)

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Control

Control

27.5

0.9

180.8

14.1

26.8

0.9

173.5

12.1

Solvent control

Solvent control

27.9

0.4

194.5

0.4

27.1

0.4

179.5

9.1

0.030

0.00933

27.0

0.4

176.4

4.8

26.8

0.3

173.4

6.2

0.096

0.0137

27.1

0.5

179.7

11.4

26.4

0.7

163.2

12.8

0.30

0.0231

27.5

1.1

185.1

19.1

26.6

0.4

164.9

9.1

0.96

0.0537

27.6

1.3

185.2

30.4

27.0

1.1

179.2

20.4

3.0

0.0953

25.8

0.9

161.4

24.6

25.4

0.4

157.2

10.8

SD = Standard deviation

Sex ratio

Statistical analysis showed no significant difference (p<0.05) at any of the test concentrations compared to the solvent controls in terms of either genetic or phenotypic sex ratio. The NOEC and LOEC values for sex ratio were therefore considered to be ≥3.0 and >3.0 mg/L, respectively, based on nominal concentrations (≥0.0953 and >0.0953 mg/L, respectively, based on geometric mean measured concentrations).

Table 4. Genetic and phenotypic sex ratio

Nominal concentration (mg/L)

Geometric mean measured concentration (mg/L)

Genetic sex

Phenotypic sex

Proportion of total number (%)

Proportion of total number (%)

Female fish

Male fish

Female fish

Male fish

Control

Control

36

64

34

66

Solvent control

Solvent control

48

52

48

52

0.030

0.00933

47

53

48

52

0.096

0.0137

44

56

45

55

0.30

0.0231

45

55

47

53

0.96

0.0537

53

47

53

47

3.0

0.0953

41

59

49

51

Secondary Sex Characteristics

Statistically significant effects were observed at the 3.0 mg/L nominal test concentration compared to the solvent control in terms of the mean number of anal papillae per male fish. The NOEC and LOEC values for the secondary sex characteristics were considered to be 0.96 and 3.0 mg/L, respectively, based on nominal concentrations (0.0537 and 0.0953 mg/L, respectively, based on geometric mean measured concentrations). 

Table 5. Secondary sex characteristics - anal fin papillae count

Nominal concentration
(mg/L)

Geometric mean measured concentrations (mg/L)

Number of anal fin papillae

Mean

SD

Control

Control

67

8

Solvent control

Solvent control

58

9

0.030

0.00933

49

7

0.096

0.0137

61

5

0.30

0.0231

62

6

0.96

0.0537

33

10

3.0

0.0953

1

1

SD = standard deviation

Vitellogenin analysis

Statistical analysis showed no significant difference (p<0.05) at any of the test concentrations compared to the solvent control in terms of VTG concentration for female fish. The NOEC and LOEC values for VTG concentration in female were therefore considered to be ≥3.0 and >3.0 mg/L, respectively, based on nominal concentrations (≥0.0953 and >0.0953 mg/L, respectively, geometric based on mean measured concentrations).

Statistically significant increases in VTG concentrations in male fish were observed at the 0.30, 0.96 and 3.0 mg/L nominal test concentrations compared to the solvent control. The NOEC and LOEC values for VTG concentration in male were therefore considered to be 0.096 and 0.30 mg/L, respectively, based on nominal concentrations (0.0137 and 0.0231 mg/L, respectively, geometric based on mean measured concentrations).

Table 6. Vitellogenin analysis

Nominal concentration
(mg/L)

Geometric mean measured concentration (mg/L)

VTG concentration (ng/mL)

Female Fish

Male Fish

Mean

SD

Mean

SD

Control

Control

0.74 x 105

0.31 x 105

6.57

11.22

Solvent control

Solvent control

0.98 x 105

0.67 x 105

13.82

13.19

0.030

0.00993

0.74 x 105

0.12 x 105

394.72

496.25

0.096

0.0137

0.92 x 105

0.29 x 105

18.86

23.21

0.30

0.0231

0.70 x 105

0.11 x 105

2874.79

1557.83

0.96

0.0537

0.84 x 105

0.16 x 105

0.53 x 105

0.13 x 105

3.0

0.0953

0.84 x 105

0.30 x 105

0.78 x 105

0.24 x 105

SD = standard deviation

Histological evaluation

Phenotypic sex was determined to be male, female, or intersex for all fish in the study. Three fish were designated as intersex: two of these had primarily female gonads with at least two stages of spermatogenesis also present and one had a primarily male gonad with a stage of oogenesis also present. No relationship between intersex designation and test item exposure was present on the study.

Otherwise, at least two or more stages of spermatogenesis or oogenesis were observed in each of the gonads examined. However, fish exposed to nominal 0.96 or 3.0 mg/L often had smaller gonads in comparison to controls and lower concentration groups, as well as fewer stages of spermatogenesis/oogenesis present within the tissue. Comparison of genetic sex and phenotypic sex indicated some feminisation of male fish at the highest nominal test concentration of 3.0 mg/L.

Based on microscopic examination, the phenotypic sex ratios (male:female) were determined to be 2:1 for controls and 1:1 for solvent controls and all test item-exposed groups.

Table 7. Histological evaluation

Nominal concentration (mg/L)

Number of individuals

XX male

XY female

Intersex

Control

2

-

-

Solvent control

1

1

-

0.030

1

2

-

0.096

-

-

1

0.30

-

1

-

0.96

-

1

2

3.0

1

7

-

- = Not applicable

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Some validity criteria were not met but these were not deemed to have impacted the integrity of the study. See 'any other information on results incl. tables'.
Conclusions:
The overall NOEC covering all endpoints tested was considered to be 0.096 mg/L based on nominal test concentrations, or 0.0137 mg/L based on geometric mean measured concentrations.
Executive summary:

A GLP test in accordance with OECD 234 (2011), the in vivo Fish Sexual Development Test, has been performed in order to assess the endocrine-disrupting potential of the test item - in particular, the potential for an estrogenic mode of action was investigated.

Eggs were obtained from in-house breeding pairs of Oryzias latipes. The test was initiated by adding fertilised eggs to test vessels (120 eggs per concentration; < 12 h old), which were exposed to a range of concentrations of the test substance dissolved in water under semi-static test conditions (with daily renewal of the test media). Due to difficulties encountered with maintaining concentrations with a flow-through system, based on the results of media preparation trials, a solvent spike (acetone) using semi-static (daily) renewal was used as the dosing method for this study. The test was continued until fish were sexually differentiated at 112 days post-hatch (dph) (total exposure duration: 122 days).

The definitive test was conducted at nominal test concentrations of 0.030, 0.096, 0.30, 0.96 and 3.0 mg/L. Control and solvent control groups were also included. Four replicate vessels were used for the controls and each test concentration.

At the end of the exposure, once sexually dimorphic, the adult fish were assessed for growth (weight and length), secondary sex characteristics (anal fin papillae count), genetic sex (by PCR method) and vitellogenin (VTG; by ELISA method). In addition, gonad histology was performed to determine the phenotypic sex of the fish. Hatching success and final survival were also examined.

Chemical analysis was conducted weekly throughout the test on freshly prepared media and the corresponding 24 h old media. Nominal concentrations were generally not achieved and were not maintained over the 24 h semi-static exposure periods. This was due to the highly volatile nature of the test substance and the requirement to aerate the vessels to maintain fish health. Given the variability in measured concentrations throughout the test, the results have been calculated based on both nominal and mean measured concentrations. The geometric mean measured concentrations were calculated to be 0.00933, 0.0137, 0.0231, 0.0537 and 0.0953 mg/L.

Results

A statistically significant effect was observed on hatching success at nominal concentrations of 0.96 mg/L and above. In addition, statistically significant effects were observed on survival and growth (male and female length and weight) at the highest nominal test concentration of 3.0 mg/L.

Sex ratio was not affected by the test substance at the concentrations employed with no statistically significant differences observed between the test concentrations and solvent control in terms of either genetic or phenotypic sex ratio.

Whilst no statistically significant increase or decrease in VTG concentration in female fish was observed, statistically significant effects were observed for male secondary sex characteristics and male VTG concentration. A significant decrease in the number of anal fin papillae was observed at the 3.0 mg/L nominal test concentration whilst a significant increase in VTG concentration was observed at the 0.30 mg/L nominal test concentration and above.

The overall NOEC covering all endpoints tested was considered to be 0.096 mg/L based on nominal test concentrations, or 0.0137 mg/L based on geometric mean measured concentrations.

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