1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

fish adult: (sub)lethal effects

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19 Dec 2011 to 09 Jan 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD TG 229 (Fish Short Term Reproduction Assay)

Version / remarks:

2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS Guideline 890.1350

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

EC Number:

262-104-4

EC Name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Cas Number:

60207-90-1

Molecular formula:

C15H17Cl2N3O2

IUPAC Name:

1-{[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl}-1H-1,2,4-triazole

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Test organisms

Aquatic vertebrate type:

fish

Test organisms (species):

Pimephales promelas

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Reference substance (positive control):

no

Results and discussion

Any other information on results incl. tables

- Biological observation: No abnormal observations in behavior or appearance, including secondary sex characteristics were noted during this study.

- Behavioral Appearance and Observations: During the in-life exposure, no notable observations occurred with regards to behaviour, coloration/banding, changes in ovipositor appearance or size of dorsal nape pad.

- Fecundity: The mean number of eggs per female per reproductive day for the control was 12. The mean number of eggs per female per reproductive day for the 0.010, 0.12 and 1.0 mg/L treatment levels was 16, 17 and 3.9, respectively. Statistical analysis (Dunnett’s Multiple
Comparison Test) determined a significant difference (increase and decrease, respectively) in mean number of eggs per female per day among fish exposed to the 0.12 and 1.0 mg/L treatment levels compared to the control. The increase at 0.12 mg/L is not deemed biologically significant.

- Fertilization Success: The percentage of viable eggs in the control was 98%. The percentage of viable eggs in the 0.010, 0.12 and 1.0 mg/L treatment levels was 97, 96 and 76%, respectively. Statistical analysis (Jonckheere-Terpstra Step-Down Test) determined a significant decrease in percentage of viable eggs in the 1.0 mg/L treatment level compared to the control.

- Survival:Mean survival among males in the control was 100%. Mean survival among male fish exposed to the 0.010, 0.12 and 1.0 mg/L treatment levels was 75, 100 and 100%, respectively. One male mortality occurred in the 1.0 mg/L treatment. This mortality was inadvertent and caused by technician error; therefore, this mortality was not considered a toxicant-related mortality and was not included in the mean survival results. Statistical analysis (Cochran-Armitage Trend Step-Down Test) determined no significant difference in mean survival among male fish in any of the treatment levels tested compared to the control. Mean survival among females in the control was 100%. Mean survival among female fish exposed to the 0.010, 0.12 and 1.0 mg/L treatment levels was 94, 100 and 81%, respectively. Statistical analysis (Cochran-Armitage Trend Step-Down Test) determined a significant decrease in mean survival among female fish exposed to the 1.0 mg/L treatment level compared to the control.

- Tubercle Score: No tubercles were observed on any female fish, therefore they were not scored. For male fish, the mean tubercle score in control was 31. Mean male tubercle scores in the 0.010, 0.12 and 1.0 mg/L treatment levels were 31, 32 and 19, respectively. Statistical analysis using the Jonckheere-Terpstra Step-Down Test in conjunction with Monte Carlo Trials determined no significant difference in mean
tubercle scores in any of treatment levels tested compared to the control.

- Gondal Somatic Indices: The mean male GSI in the control was 1.2%. The mean male GSI among fish exposed to the 0.010, 0.12 and 1.0 mg/L treatment levels was 1.1, 1.3 and 2.0%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined a significant increase in mean male GSI among fish exposed to the 1.0 mg/L treatment level compared to the control.
The mean female GSI in the control was 14%. The mean female GSI in the 0.010, 0.12 and 1.0 mg/L treatment levels was 14, 15 and 19%, respectively. Statistical analysis (Jonckheere-Terpstra Step-Down Test) determined a significant increase in mean female GSI in fish exposed to the 1.0 mg/L treatment level compared to the control.

- Length: The mean male length in the control was 52.0 mm. The mean male length in the 0.010, 0.12 and 1.0 mg/L treatment levels was 49.1, 50.3 and 53.4 mm, respectively. The mean female length in the control was 39.7 mm. The mean female length in the 0.010, 0.12 and 1.0 mg/L treatment levels was 39.5, 40.3 and 41.4 mm, respectively.

- Weight: The mean male weight in control was 3.18 g. The mean male weight in the 0.010, 0.12 and 1.0 mg/L treatment levels was 2.68, 2.89 and 2.88 g, respectively. Statistical analysis (Dunnett's Multiple Comparison Test) determined no significant difference in mean
weight among fish exposed to any of the treatment levels tested compared to the control. The mean female weight in control was 1.31 g. The mean female weight in the 0.010, 0.12 and 1.0 mg/L treatment levels was 1.29, 1.33 and 1.48 g, respectively. Statistical analysis (Dunnett's Multiple Comparison Test) determined no significant difference in mean weight among female fish exposed to any of the treatment levels tested compared to the control.

- Blood Plasma Vitellogenin Concentration: Mean male vitellogenin concentration in control was 200 ng/mL. Mean male vitellogenin
concentration in the 0.010, 0.12 and 1.0 mg/L treatment levels was 360, 210 and 480 ng/mL, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in male vitellogenin concentration in any treatment levels tested compared to the control. Two fish were considered outliers (replicates B and C of the 0.010 mg/L treatment level). Data for male VTG were analyzed, both including the outliers and excluding the outliers. No statistical differences were detected in either of the data sets for this endpoint. The mean female vitellogenin concentration in control was 1.1E+06 ng/mL. Mean female vitellogenin concentration in the 0.010, 0.12 and 1.0 mg/L treatment levels was 1.2E+06, 1.1E+06 and 2.0E+05 ng/mL, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined a significant decrease in mean female vitellogenin concentration in fish exposed to the 1.0 mg/L treatment level compared to the control.

- Histopathology: Tissue-sections of the ovary and testes were examined from control and test substance-exposed fathead minnows. Based on histopathology evaluation, increased oocyte atresia was observed at the 1.0 mg/L treatment level. No additional exposure-related findings in male or female fish were observed.

Deviations from the protocol

The protocol states that sampling will be done at test termination. During this study, the final sampling was done on test day 18, three days prior to test termination. All analytical recoveries to that point were near nominal; there were no diluter malfunctions during the last three days of exposure nor changes in water quality parameters which would be indicative of problems with the diluter. Therefore, this deviation did not have a negative impact on the results or interpretation of the study.
The protocol states that total dissolved oxygen levels will be maintained at > 60% of air saturation. On test day 6, the dissolved oxygen levels were recorded to be 59% of saturation in replicate B of the control. The dissolved oxygen level was rechecked later in the day and measured at 64% saturation. Since replicate B of the control met all control criteria, this deviation did not have a negative impact on the results or interpretation of the study.

Performance Criteria
Following 21 days of exposure, the mean percent survival in control was 100%, meeting the guideline performance criteria of ≥ 90%. Mean control fecundity was 12 eggs/female/reproductive day, with successful spawning occurring at least every four days. This meets the guideline performance criteria for evidence of active spawning. The percentage of viable eggs in control was 98%, meeting the guideline performance criteria of > 95% fertility. Therefore, the study is considered valid. 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on results incl. tables'

Conclusions:

In a fish short-term reproduction assay performed following EPA 890.1350 and OECD TG 229 guidelines, female survival, fecundity, fertilization success, female vitellogenin were decreased in 1.0 mg/L tretment. GSI (males and females) was increased in 1.0 mg/L treatment. Female mortalities (one female in three of four replicates) were observed at the high treatment level. Based on histopathology evaluation, increased oocyte atresia was observed at the 1.0 mg/L treatment level. No additional exposure-related findings in male or female fish were observed.

Executive summary:

This study was performed to determine the potential of the test substance to interact with the endocrine system in fathead minnow (Pimephales promelas) exposed under flow-through conditions following OPPTS Guideline 890.1350 and OECD Guideline 229. The study complied with CLP criteria. The reproduction assay ran for 21 days and the endpoints evaluated were fecundity (number of eggs per female per reproductive day), fertilization success, nuptial tubercle score, blood plasma vitellogenin (VTG) concentration, gonadosomatic index (GSI), weight, survival, histological examination of gonadal tissues, as well as behaviour and appearance, including secondary sexual characteristics. Four replicate vessels were established for each treatment level (i.e., 0.012, 0.12 and 1.2 mg/L nominal, 0.010, 0.12 and 1.0 mg/L mean measured), and a control. Test vessels were maintained at  25 ± 1 ºC, photoperiod of 16 hours of light at an intensity range of 540 to 1100 lux (50 to 100 footcandles) and 8 hours of darkness. Samples were removed from two replicates (A and C) of each treatment level and the control at day 0 (exposure initiation) and day 10 and two replicates (B and D) on days 3 and 18.

Statistical analyses were performed for male and female fish separately, where appropriate. The analysis involved the comparison of the mean or median responses per replicate tank between the concentration and control groups. No abnormal behaviour or notable changes in secondary sex characteristics were observed throughout the 21-day study. Decreased female survival, decreased fecundity, decreased fertilization success, decreased female vitellogenin and increased GSI (males and females) were observed at 1.0 mg/L. Female mortalities (one female in three of four replicates) were observed at the high treatment level. Based on histopathology evaluation, increased oocyte atresia was observed at the 1.0 mg/L treatment level. No additional exposure-related findings in male or female fish were observed.