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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-09-27 - 2019-10-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD Guideline for Testing of Chemicals, Section 2, No. 202, “Daphnia sp., Acute Immobilisation Test”, adopted 13th April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Council Regulation (EC) No 440/2008, Annex Part C, C.2: „Daphnia sp. Acute Immobilisation Test“. Dated May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

EPA Guideline 712-C-16-013: OCSPP 850.1010, “Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids” October, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, No. 23 (Second Edition)

Version / remarks:

Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, No. 23 (Second Edition), ENV/JM/MONO(2000)6/REV1, 8 February 2019

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L nominal, control
- Sampling method: 2 x 2 mL were taken from the test item treated group at the start and at the end of each renewal period, 1 x 2 mLwere taken from the control group at the start of each renewal period
- Sample storage conditions before analysis: not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The preparation of test solution was performed by direct weighing and stirring for 24 h with subsequent filtration. Therefore, 0.0504 g of the test item was were suspended in the 504 mL dilution water (ISO Medium) in order to give the loading rate of 100 mg test item/L in the first renewal period and 0.0508 g of the test item was suspended in the 508 mL dilution water (ISO Medium) in order to give the loading rate of 100 mg test item/L in the second renewal period. The solutions were handled by ultrasonic bath for approx. 15 minutes andthereafter stirred for a period of approximately 24 hours thereafter to achieve an equilibrated concentration. Then, the solutions were filtrated through a membrane filter (0.45 µm) to separate any non-dissolved test material.
- Controls: The dilution water (ISO Medium) without test item added was used as untreated control solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna (Straus)
- Age at study initiation (mean and range, SD): Less than 24 h old at the beginning of the test.
- Stage and instar at study initiation: neonate
- Source:
Origin: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection), 2100 Gödöllő, Kotlán S. u. 3. Hungary
Breeding: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT.
- Feeding during test : The Daphnia were fed with concentrated algal suspension of Raphidocelis subcapitata during the holding. Test animals were not fed during the exposure.

ACCLIMATION
- Other: Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore, additional acclimatisation before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The reconstituted water (ISO Medium) has an approximate theoretical total hardness of 249 mg/L (as CaCO3).

Test temperature:

The test temperature was in the range of 21.1 – 21.3 °C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 20.8 – 21.8 °C.

pH:

The pH of the test solutions was not adjusted and did not vary by more than 1.5 units in any test. The pH was in the range of 7.85 – 7.99 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 7.41 –8.34 mg/L during the test.

Nominal and measured concentrations:

nominal: 100 mg/L nominal loading rate
geometric mean measured: 0.88 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, volume app. 50 mL
- Volume of solution: approximately 40 mL
- Aeration: Test solutions were not aerated.
- Renewal rate of test solution (frequency/flow rate): 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Other: The test vessels were kept in a climate chamber under controlled environmental conditions during the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO Medium, according to OECD 202) was used as dilution water in the experiment. Separate stock solutions of individual substances were first prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE ELIX 3 water purification system). The ISO Medium was prepared by adding 25 mL from each of four stock solutions to one litre deionised water proportionally. For further information on the detailed composition of the ISO Medium please see "Any other information on materials and methods incl. tables".
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: Light-Dark Cycle: 16 h light : 8 h dark
- Light intensity: The measured light intensity was 703 lux in the climate chamber at the start of the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile. In addition to immobility any abnormal behaviour or appearance is reported. The water temperature, dissolved oxygen concentration and pH were measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the test. Additionally, the ambient temperature was measured continuously using a min/max thermometer in the climate chamber.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: No immobilisation of the daphnids was observed in a static and a semi-static range-finding pre-test. Based on the results of the non-GLP preliminary range-finding tests, the main test was performed as limit test.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study was: 2019-03-12 to 13.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.88 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 0.88 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.88 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.88 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No abnormal behaviour of the test animals was observed in the control group and the test concentration of nominal 100 mg/L, corresponding to a geometric mean measured concentration of 0.88 µg/L, after 48 hours of exposure.
- Mortality of control: No mortality was observed in the control group.
- Immobilisation of control: No immobilisation was observed in the control group.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- ECx: The 24-h EC50: 1.58 mg/L (95 % conf. limits: 1.34 – 1.85 mg/L).

Reported statistics and error estimates:

A limit test was performed and no toxic effects were observed, therefore, statistical analysis was not necessary. The EC50, the NOEC and LOEC values of the test item were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD Guideline 202 and EU Method C.2 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 daphnids.
No immobilisation or any abnormal behaviour was observed in the control group and the limit concentration of nominal 100 mg/L after 48h of exposure. Accordingly, the 48-hour EC50 and the 48-hour EC10 were determined to be > 100 mg test item/L, corresponding to a geometric mean measured concentration of > 0.88 µg test item/L and representing the saturation concentration or the limit of solubility. The 48-hour NOEC was determined to be ≥ 100 mg test item/L and the associated 48-hour LOEC is > 100 mg/L based on the nominal concentration. Based on the geometric mean measured concentration the 48-hour NOEC was determined to be ≥ 0.88 µg test item/L and the associated 48-hour LOEC is > 0.88 µg test item/L, respectively.

Executive summary:

The test substance was assessed using Daphnia magna in a 48-hour acute immobilisation test according to OECD 202 under GLP. In the semi-static test, young daphnia were exposed to the test item in aqueous test media for 48 hours under defined conditions. Based on the results of the non-GLP preliminary tests the main test was performed as a limit test. The test concentration of nominal 100 mg/L and a control were tested in order to demonstrate that the test item has no influence on the mobility of Daphnia magna up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration).

Due to the poor water solubility of the test substance the test concentration of nominal 100 mg/L was stirred for 24h and filtrated through a membrane filter to remove any non-dissolved test material. As the test item was previously shown not to be stable in the ISO Medium for 48 hours a semi-static test was performed. The analytically measured test item concentration did not remain within ± 20 % of the nominal concentration during the test period of 48 hours.

Healthy, young female daphnids with known origin, history and acclimatisation conditions were used. Twenty daphnia (divided into 4 replicates) were tested in each test group, exposed to the saturation concentration (equivalent to 100 mg/L nominal concentration) test item or run as control. Each test vessel contained approximately 40 mL test solution. Fully characterised (content, physico-chemical characteristics) ISO Medium was used as test medium.

The immobilisation of the test animals was recorded 24 and 48 hours after treatment. Environmental conditions were recorded. All measured values remained within acceptable ranges.

No immobilisation or any abnormal behaviour of the test animals was observed in the control group and no immobilisation or any abnormal behavior of the test animals was observed at the test concentration of nominal 100 mg/Lafter 48 hours of exposure.

Accordingly, the 48-hour EC₅₀ and the 48 -hour EC₁₀ were determined to be > 100 mg test item/L, corresponding to a geometric mean measured concentration of > 0.88 µg test item/L and representing the saturation concentration or the limit of solubility. The 48-hour NOEC was determined to be ≥ 100 mg test item/L and the associated 48-hour LOEC is > 100 mg/L based on the nominal concentration. Based on the geometric mean measured concentration the 48-hour NOEC was determined to be ≥ 0.88 µg test item/L and the associated 48-hour LOEC is > 0.88 µg test item/L, respectively.

The results were determined directly from the raw data.

All validity criteria were met and, therefore, the study can be considered as valid.

Description of key information

Key, short-term toxicity to aquatic invertebrates: EC₅₀ (48h) > 100 mg/L nominal, corresponding to > 0.88 µg/L based on geometric mean measured concentrations of the test item for Daphnia magna (OECD 202, semi-static, limit-test, GLP)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The study was conducted under GLP according to OECD Guideline 202 and EU Method C.2 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.

The toxic effect of the test item to Daphnia magna was assessed 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 stirred for 24h and filtrated through a membrane filter to remove any non-dissolved test material. No immobilisation or any abnormal behaviour was observed in the control group and the limit concentration of nominal 100 mg/L after 48h of exposure. Accordingly, the 48-hour EC₅₀ and the 48 -hour EC₁₀ were determined to be > 100 mg test item/L, corresponding to a geometric mean measured concentration of > 0.88 µg test item/L and representing the saturation concentration or the limit of solubility. The 48-hour NOEC was determined to be ≥ 100 mg test item/L and the associated 48-hour LOEC is > 100 mg/L based on the nominal concentration. Based on the geometric mean measured concentration the 48-hour NOEC was determined to be ≥ 0.88 µg test item/L and the associated 48-hour LOEC is > 0.88 µg test item/L, respectively.