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]

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition 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

Details on test solutions:

The culture medium has been prepared by mixing the 4 stock solutions in order to obtain final concentrations specified in the following table:
SOLUTION N. NUTRIENT QUANTITY TO BE DISSOLVEDIN 500 ML OF DEIONIZED WATER
1 NH4Cl 0.75g
CaCl2 2H2O 0.9g
MgCl2 6H2O 0.6g
MgS0O4 7H2O 0.75g
KH2PO4 0.08 g
2 FeCl3 6H2O 0.032 g
Na2EDTA 2H2O 0.05 g
3 H3BO3 0.0925 g
MnCl2 4H2O 0.207 g
ZnCl2 1 mL of the following solution: 150 mg in 100 mL of deionized water
CoCl2 6H2O 1 mL ofthe following solution: 75 mg in 100 mL of deionized water
CuCl2 2H2O 1 mL ofthe following solution: 25 mg in 500 mL of deionized water, then diluting mL of it in10mL
Na2MoO4 2H2O 1 mL of the following solution: 35 mg in 10 mL of deionized water
4 NaHCO3 25g

Solutions 1, 2, 3 have been sterilised by autoclaving (121°C, 15 min). Solution 4 has been sterilised via membrane filtration (0.22 um). 10 mL of solution 1 and 1 mL of solutions 2, 3, 4 have been made up to 1000 mL with deionised water.
For higher volumes the medium has been prepared according to the same proportion.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata SAG 61.81
Test organism: Green alga
Genus: Pseudokirchneriella
Species: subcapitata
Family: Chlorophyceae
Order: Chlorococcalles

Test type:

static

Water media type:

other: The culture medium has been usedasdilution water.

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

72 ± 2 h

Test temperature:

23 ± 2 ℃

pH:

7.7-8.8

Nominal and measured concentrations:

0.3mg/L, 0.15mg/L, 0.075mg/L, 0.038mg/L, 0.019mg/L and 0 mg/L(nominal concentration)

Details on test conditions:

TEST SYSTEM
- Initial cells density: 10000 cell/mL
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates):

GROWTH MEDIUM
- Standard medium used: yes


OTHER TEST CONDITIONS
- Light intensity and quality: Continuous — with fluorescent lamp (7000- 8000lux)


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- 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:
After the results of the range finding test, the test item has been prepared at 20 mg/Lin culture medium. The solution has been extracted for 72 h, at 10 rpm, at room temperature. The extract from stock solution (20 mg/L) has been diluted at 0.50 mg/L, 0.42 mg/L, 0.35 mg/L, 0.29 mg/L and 0.24 mg/L solutions under stirring. However, test solutions were not well solubilized and then the analytical chemical results (EXC LV - 20AA6306)confirmed the heterogeneity.
A new stocksolution (20 mg/L) has been prepared using an acidified water (phosphoric acid 0.1%). The test has been performed on the new diluted nominal solutions: 5.00 mg/L, 2.50 mg/L, 1.25 mg/L,
0.63 mg/L and 0.31 mg/L. The test solutions appeared to be solubilized in the acidified medium but algae growth resulted inhibited at all tested concentrations.
So, a newstocksolution (20 mg/L) has been prepared using an acidified water (phosphoric acid 0.1%). Then 5 dilutions have been performed with culture medium. Definitive test has been performedonthe diluted nominal solutions: 0.3 mg/L, 0.15 mg/L, 0.075 mg/L, 0.038 mg/L, 0.019 mg/L and 0 mg/L

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.081 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The EC50 of the test item is 0.081 mg/L (measured concentration).

Validity criteria fulfilled:

yes

Executive summary:

On 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]" the growth inhibition test on a culture of algae Pseudokirchneriella subcapitata was performed on a culture in exponential growth phase. This approach, according to OECD 201, allowed to calculate the growth inhibition (effect) concentration to define a limit not toxic threshold.

During the range finding test the algae have been exposedto 5 different dilutions of the test item for 72 hours. After the results of the range finding test an EC50 test has been performed. The algae have been exposedto 5 dilutions of the test item for 72 hours. Algae have been measured using a Burker chamber.

At the end of the test, microscopic observation was performed to verify a normal and healthy appearanceof the inoculum culture and to observe any abnormal appearance of the algae.

At the beginning and at the end of the test (72 hours) pH has been measuredin all the vessels. Temperature of the ambient has been recorded every day.

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, can be summarised as follow:

EC50 (72 h) of the testitem “N4,N4’-HEXANE-1,6 -DIYLBIS[N-BUTYL-6-CHLORO-N,N’-BIS(2,2,6,6-T ETRAMETHYLPIPERIDIN-4-

YL)- 1,3,5-TRIAZINE-2,4-DIAMINE]” (measured concentration): 0.081 mg/L

Description of key information

During the range finding test the algae have been exposed to 5 different dilutions of the test item for 72 hours. After the results of the range finding test an EC50 test has been performed. The algae have been exposed to 5 dilutions of the test item for 72 hours. Algae have been measured using a Burker chamber.

At the end of the test, microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae.

At the beginning and at the end of the test (72 hours) pH has been measured in all the vessels. Temperature of the ambient has been recorded every day.

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, can be summarised as follow:

EC50 (72 h) of the testitem “N4,N4’-HEXANE-1,6 -DIYLBIS[N-BUTYL-6-CHLORO-N,N’-BIS(2,2,6,6-T ETRAMETHYLPIPERIDIN-4-

YL)- 1,3,5-TRIAZINE-2,4-DIAMINE]” (measured concentration): 0.081 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

0.081 mg/L