bis(2,2,6,6-tetramethyl-1-(phenylthio)piperidin-4-yl)carbonate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-08-16 - 2019-10-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 203 “Fish, Acute Toxicity Test” (Adopted: 18 June 2019)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MEP, P.R. China, the Guidelines for the Testing of Chemicals, Effects on Biotic Systems (the Second Edition), No. 203 “Fish Acute Toxicity Test”. China Environmental Press, 2013.8

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment No. 23 (Second Edition) “Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals. (8 February 2019)

Deviations:

not applicable

Qualifier:

according to guideline

Guideline:

other: Guidance document on analytical quality control and validation procedures for pesticide residues analysis in food and feed, SANCO/12571/2013, 19 November 2013 rev.0, Implemented by 01/01/2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L nominal, control
- Sampling method: Samples were taken in 0-hour and 72-hour fresh solution and 24-hour and 96-hour old solution of the only test item concentration and the control. About 20 mL sample was taken from each test solution and control solution.
- Sample storage conditions before analysis: The test solutions were stored in refrigerator (2-8 °C) for up to 15 days before analysis by the subsequently adapted and amended analytical method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1.000 g (1.0003 g - 1.0007 g) of test item were weighed and added directly into 10 L test water. The solution was mixed by magnetic stirrer for approximately 48 hours and filtered with a 0.45 μm membrane filter. The 5 L filtered solution was used for exposure.
An additional test solution was prepared as above to validate the initial concentration of the test medium. 1.0004 g of test item was weighed to prepare this solution.
- Controls: dilution water (without test item)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish (Danio rerio)
- Strain: Batch no.: Zebra fish 20190531
- Source: The fish were reproduced in the test facility. Their parental generation was introduced from Hangzhou Hunter Biotechnology Inc.
- Length at study initiation (length definition, mean, range and SD): 1.8 ± 0.14 cm
- Weight at study initiation (mean and range, SD): 0.0562 ± 0.0129 g

ACCLIMATION
- Acclimation conditions (same as test or not): The fish are held in water of the quality and under the illumination conditions identical with those used in the test during the acclimation period.
Light condition: 12 hours light: 12 hours darkness
Temperature: 23 ± 2 °C
Oxygen concentration: At least 80% of air saturation value (ASV).
- Type and amount of food during acclimation: not specified.
- Feeding frequency during acclimation: Twice on weekday and one time on weekend until 24 hours before the test was started.
- Health during acclimation (any mortality observed): From hatching, there were not any signs of disease or significant numbers of mortality.

FEEDING DURING TEST
- Frequency: Fish were not fed during the period of the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

The water hardness was 180 mg/L (as CaCO3).

Test temperature:

23.7 - 24.1 °C

pH:

8.08 - 8.30

Dissolved oxygen:

84.1 - 99.7 % ASV

Nominal and measured concentrations:

nominal: 100 mg/L nominal loading rate
mean measured concentration: < LOQ (0.5 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: Rectangle glass tanks (length: 30 cm, width: 20 cm, height: 30 cm)
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Test solution volume: 5 L
- Aeration: not specified
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: < 0.8 g wet weight fish/L water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water treated with Aquapro Pure Water System.
- Total organic carbon: < 2 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: The periodic measurements of TOC in the laboratory were performed every three months and lower than 2 mg/L. For further information on the periodic characteristics of test water quality by authorized facility please see "Any other information on materials and methods incl. tables".
- Other:
Water hardness: 40 to 250 mg/L (as CaCO3)
pH: 6.0 to 8.5 pH units
Aeration: Before usage, the water was aerated for more than 24 hours to attain dissolved oxygen saturation.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 hours light: 12 hours darkness (cool white fluorescent lighting)
- Light intensity: 540 - 1000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Biological observation: Observation intervals: at 2 ± 0.5 h, 5 ± 1 h and 24 ± 2 h after the start of the exposure (day 0-1). On days 2-4 of the test, all vessels with living fish were inspected twice per day (preferably early morning and late afternoon to best cover the 24-hour periods).
- Method: Mortalities and visible abnormalities were recorded. Fish are considered dead if there is no visible movement (e.g. gill movements) and if touching of the caudal peduncle produces no reaction. Dead fish should be removed as soon as they are observed. Visible abnormalities may include abnormalities in regard to equilibrium (e.g. loss of balance, head up or down, floating at surface or sinking), appearance (weak or dark pigmentation, exophthalmia), ventilatory behaviour (e.g. hyper, hypo or irregular ventilation, coughing) and swimming behaviour (hyper or hypo activity, immobility, convulsions, near surface or bottom, dense or loss of schooling).
- Water quality and physicochemical measurements:
Water total hardness: at the beginning of the test.
Dissolved oxygen and pH: in fresh and old media for both control and test groups, measured at the beginning and daily during the 96-hour exposure period.
Temperature: monitored continuously by automatic temperature monitoring system.
Light intensity: at the beginning of the test.

TEST CONCENTRATIONS
- Range finding study : In a non-GLP range-finding test, a saturated solution was prepared by preparing a solution at a nominal concentration of 100 mg/L and stirring for approximately 48 hours and filtering with 0.45 μm membrane. The filtered solution was used for exposure. The test fish was Rare Minnow and 5 fish were used for exposure. The solutions were renewed every 48 hours. During the period of 96 hours, neither death nor any visible abnormalities were observed in the test group. Based on the results of the non-GLP preliminary range-finding tests, the main test was performed as limit test.
The concentration of the test substance in the test medium sample was assessed by HPLC. The measured concentration of the test item in the test medium sample was below the limit of quantification of instrument (0.5 mg/L).

Reference substance (positive control):

yes

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: No visible abnormalities were observed in both the control group and the test group.
- Observations on body length and weight: no
- Other biological observations: no
- Mortality of control: No mortality was observed in the control group.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- LC50: 24-hour LC50 = 257.10 mg/L (95% confidence limits: 232.50 - 284.10 mg/L)

Reported statistics and error estimates:

The test was a limit test. No statistical method was adopted.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD Guideline 203 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the registered substance towards fish.
During the 96-hour exposure period, no death or visible abnormalities were observed in both the control group and the test group. Accordingly, the 96-hour LC50 was determined to be > 100 mg test item/L (nominal concentration), representing the saturation concentration or the limit of solubility. The analytical concentration was

Executive summary:

A fish acute toxicity study of the test item was conducted with zebra fish (Danio rerio) over a 96-hour exposure period according to OECD 203 under GLP.

The toxic effect of the test item to zebra fish (Danio rerio) was assessed over a 96-hour exposure period in a semi-static limit test at the saturation concentration. Based on the results of the preliminary test, the definitive test was carried out with a limit test at a nominal concentration of 100 mg/L (equivalent to 100 mg/L nominal loading rate). Due to poor water solubility 1.00 g of the test item were weighed and added directly into 10 L test water. The solution was mixed by magnetic stirrer for approximately 48 hours and filtered through a 0.45 μm membrane filter to remove any non-dissolved test material. The 5L filtered solution was used for exposure. A blank control was performed concurrently. Seven fish were used for each test group and the control group, respectively.

The test was performed with a semi-static system, and test solutions were renewed every 24 hours to maintain the test concentration as constant as possible. Dissolved oxygen and pH were measured daily during the 96-hour exposure period in the control and test solutions. Temperature was monitored continuously by automatic temperature monitoring system.

During the 96-hour exposure period, no death or visible abnormalities were observed in both the control group and the test group.

The concentration of the test item was determined in samples of the test media by HPLC, including 0-hour and 72-hour fresh solution and 24-hour and 96-hour old solution (i.e. the solution at renewal after 24 hours exposure). As the substance could not be detected by use of the initial developed and validated HPLC method anymore, the analytical method was adapted and amended correspondingly.The samples of the test solutions stored in the refrigerator (2-8 °C) for 15 days (the longest storage period, 0-hour fresh solution) were used for re-analysis and a fresh test solution was prepared in addition to validate the initial concentration of the test medium. The results showed that the determined concentrations in fresh and old media were <LOQ (0.5 mg/L).

According to the results of the test, the 96-hour LC₅₀ of the test item to zebra fish (Danio rerio) was greater than 100 mg/L (nominal concentration), representing the saturation concentration or the limit of solubility. The analytical concentration was <LOQ (0.5 mg/L), which was the saturated solution, and no death and visible abnormalities were observed under this condition.

Description of key information

Key, short-term toxicity to fish: LC₅₀ (96h) > 100 mg/L nominal, analytical concentration <LOQ (0.5 mg/L) for zebra fish (Danio rerio) (OECD 203, semi-static, limit-test, GLP)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

The study was conducted under GLP according to OECD Guideline 203 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test item towards fish.

The toxic effect of the test item to zebra fish (Danio rerio) was assessed over a 96-hour exposure period in a semi-static limit test at the saturation concentration (equivalent to 100 mg/L nominal loading rate). Due to the poor water solubility of the test substance the test concentration of nominal 100 mg/L was weighed directly, stirred for 24h and filtrated through a membrane filter to remove any non-dissolved test material. During the 96-hour exposure period, no death or visible abnormalities were observed in both the control group and the test group. Accordingly, the 96-hour LC₅₀ was determined to be > 100 mg test item/L (nominal concentration), representing the saturation concentration or the limit of solubility. The analytical concentration was <LOQ (0.5 mg/L), which was the saturated solution, and no death and visible abnormalities were observed under this condition.