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 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

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)

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:

using an acidified water (phosphoric acid 0.1%)

Details on test solutions:

As dilution water, reconstituted water has been used; it has been prepared dissolving thefollowing salts in 1 litre of deionised water:
N. SOLUTION REAGENTS FORMULA QUANTITY(g/L)
A Bihydrated calcium chloride CaCl2 2H2O 11.76
B Eptahydrated magnesium sulphate MgSO, 7H20 4.93
C Acid sodium bicarbonate NaHCO3 2.59
D Potassium chloride KCl 0.23

25 mL of solutions+ A, B, C and D have been taken to 1000 mL volume with deionised water.
Medium has been prepared according to the same proportion and reported on internal logbook.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organism: Daphnia magna straus, Crustacean, Cladocero
Age: 6-24 hours
Origin: Breederin Eurofins Biolab Test Facility
N. of animals: 20 for each treated — 20 for control — 20 for control acidified
N. of animals/beaker: 5
N. of replications: 4 for control and 4 for each treated tested concentration
N. of concentrations: 5 for range finding test and ECs, test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

between 140 and 250 mg CaCO3/L

Test temperature:

20°C + 2°C

pH:

Group of control acidiied: 7.75-7.89
Group of control: 8.04-8.09
Group of treated: 7.83-7.95

Dissolved oxygen:

> 3 mg/L

Nominal and measured concentrations:

nominal concentration:1.00mg/L, 0.45mg/L,0.21mg/L, 0.09mg/L, 0.04mg/L, 0.00mg/L, 0.00mg/L(acidified).

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Beaker (500 ml).
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4


OTHER TEST CONDITIONS
- Light intensity: None during the course of the test.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 1000 mg/L, 100 mg/L, 10 mg/L, 1 mg/L and 0.1 mg/L
- Results used to determine the conditions for the definitive study:
The test item in the solutions was not well solubilized and analytical chemical results (EXC LV - 20AA6307) confirmed. A new stock solution (20 mg/L) has been prepared using an acidified water (phosphoric acid 0.1%). Then 5 dilutions have been performed with culture medium. Two controls have been prepared: one with culture medium and onewith culture medium acidified.
The test has been performed on the diluted nominal solutions: 0.04, 0.09, 0.21, 0.45, 1.00 mg/L.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.042 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The sample is considered not stable because the measured concentration of the test item at the end of the test has not been maintained within + 20 % to the concentration at the measured concentrations at the
beginning of the test.
The test item resulted not stable during the test, therefore the statistical analysis (EC50) has been performed usingtheinitial measured test concentration as required by the guideline OECD 202.
For the two lower concentrations a protective factor has been used. The lower percentage recovery (55.6%) related to analytical chemical results has been used.
Validity criteria have been satisfied：
% of immobilized Daphnie in control: 0.

Validity criteria fulfilled:

yes

Executive summary:

The aim of the study is to determine the ecotoxicological effects 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]”on biotic systems: in particular aquatic organism such as Daphnia magna was used as

test system in order to calculate the EC50 value, according to OECD guideline N. 202.

After a range finding test, the organisms have been exposed to 5 differentdilutions of the test item for a total period of 48 hours.

Daphnia have been kept under observations for a period of 48 hours and the number of immobilised organisms and/or possible abnormal behaviours both in control and in treatment group vessel have been observed.

At the beginning and at the end of the test (48 hours) dissolved oxygen and pH have been measuredin the highest concentration and in control group. Temperature of the assay environment have been recorded at the beginning and at the end of the test.

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, EC50 of the test item at T= 48 h (measured concentration) = 0.042 mg/L

Description of key information

The obtained results of OECD 202, in compliance with test acceptability criteria, EC50 of the test item at T= 48 h (measured concentration) = 0.042 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.042 mg/L