N4,N4'-hexane-1,6-diylbis[N-butyl-6-chloro-N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine]

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 190701
- Expiration date of the lot/batch: 24 July 2021
- Purity: >90%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature
- Stability under storage conditions: Stable

Analytical monitoring:

yes

Remarks:

HPLC method

Vehicle:

yes

Remarks:

Because of the poorly solubility in water of the test item and relating results on other organisms (crustacean and algae) a stock solution (1 g/L) has been prepared using an acidified water (phosphoric acid 0.1%).

Details on test solutions:

Range finding test:
Because of the poorly solubility in water of the test item an extraction has been performed to extract the soluble components of the test item in water, according to OECD Guidance N. 23. The test item has been prepared at 100 mg/L and 10 mg/L in tap water. The solutions have been extracted for 72 h, at 100 rpm, at room temperature. From extract at 10 mg/L the solutions at 1 mg/L, 0.1 mg/L and 0.01 mg/L have been prepared understirring. 5 different dilutions with ten-fold dilutions (100 mg/L, 10 mg/L, 1 mg/L, 0.1 mg/L and 0.01 mg/L) have been tested. The test has been performed on nominal concentrations.

Definitive test 2nd experimentation:
After results of 1st experimentation, a new experimentation has been performed.
Because of the poorly solubility in water of the test item has been prepared using an acidified water (phosphoric acid 0.1%). Then 5 dilutions have been performed with tap water. To animal welfare only
acidified control has been prepared andtested.
The test has been performed on the diluted nominal solutions: 0.0125 mg/L, 0.025 mg/L, 0.05 mg/L, 0. 1 mg/L and 0.2 mg/L(nominal concentrations)

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Danio rerio
Family: Teleostei
Length: 1-2 cm
Biomass-density: < 0.8 g wet weight fish/L
Supplier: Universita’ Milano Bicocca

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

250 mg CaCO3/L

Test temperature:

24°C + 1°C

pH:

8.21-8.45

Dissolved oxygen:

76-80 % of saturation of dissolved oxygen

Nominal and measured concentrations:

0.0125 mg/L, 0.025 mg/L, 0.05 mg/L, 0. 1 mg/L and 0.2 mg/L(nominal concentrations)

Details on test conditions:

- Test vessel: Tanks
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- Aeration: continuous with porous stones

- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: lower than 0.8 g/L
To animal welfare only acidified control has been prepared andtested.

OTHER TEST CONDITIONS
- Photoperiod: Tanks have beenlighted with white light lamps with 12 hours photoperiod daily.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: factor 2
- Range finding study
- Test concentrations: 100 mg/L, 10 mg/L, 1 mg/L, 0.1 mg/L and 0.01 mg/L
- Results used to determine the conditions for the definitive study: After results of 15 experimentationa new experimentation has been performed. The test concentrations are 0.0125 mg/L, 0.025 mg/L, 0.05 mg/L, 0. 1 mg/L and 0.2 mg/L(nominal concentration).

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.053 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

LC50 of the test item at 96 hours is 0.053 mg/L (measured concentration).
The sample is not considered stable because the measured concentration at the end and at the beginning of the test has not been maintained within + 20 %. Only the highest tested concentration has been evaluated.
Therefore, the statistical analysis (LCso) has been performed using the geometric mean concentration of the highest tested concentration and for the others a protective factor has been used. The percentage recovery (74.5%) related to analytical chemical results of the higher tested concentration has been used for the others.

Validity criteria fulfilled:

yes

Executive summary:

The acute toxicity of the test item, N4,N4’-HEXANE-1,6-DIYLBIS[N-BUTYL-6-CHLORO-N,N’- BIS(2,2,6,6-TETRAMETHYLPIPERIDIN-4-YL)- 1,3,5-TRIAZINE-2,4-DIAMINE] has been evaluated according to OECD guideline N. 203.

In particular, the assay of acute toxicity with fish Danio rerio has been performed by means of a static assay in which the solution has not been changed for the whole test period.

After a range finding test, two definitive test have been performed. For each test 42 fish have been used, 35 of them treated with the test item at different concentrations and 7 fish used as control, kept in the same assayconditions without adding the test item.

Fish have been observed for a period of 96 hours and mortality and/or possible anomalous behavioursif observed, both in control and in treated groups, have been reported. Temperature, pH and percentage of saturation of oxygen in the water of each tank have been measured at the beginning of the treatment and with intervals of 24 hours.

Stability of the test item in the definitive test has been performed using HPLC technique equipped with UV detector.

The test item has been measured at the beginning and at the end of the test in the control and in the treated samples.

The obtained results, in compliance with test acceptability criteria, for the extract of the test item “N4,N4’- HEXANE-1,6-DIYLBIS[N-BUTYL-6-CHLORO-N,N'-BIS(2,2,6,6-TETRAMETHYLPIPERIDIN-4-YL)- 1,3,5 - TRIAZINE-2,4-DIAMINE]” can be summarised as follow:

LC50 of the test item at T= 96 h (measured concentration): 0.053 mg/L

Description of key information

The obtained results, in compliance with test acceptability criteria, for the extract of the test item “N4,N4’- HEXANE-1,6-DIYLBIS[N-BUTYL-6-CHLORO-N,N'-BIS(2,2,6,6-TETRAMETHYLPIPERIDIN-4-YL)- 1,3,5 - TRIAZINE-2,4-DIAMINE]” can be summarised as follow:

LC50 of the test item at T= 96 h (measured concentration): 0.053 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.053 mg/L