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]

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

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 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

no

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

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, adapted

Details on inoculum:

For the preparation of the inoculum a sample of aerobic sludge has been selected by the mixed treatmentplant of urban (about 66%) and industrial (about 34%) liquid sewagesituated at San Rocco-Monza (MB), Italia. The plant of treatment is managed by “BRIANZACQUE SRL (Monza)”.
In the laboratory, the sampled muds have been mixed and let settle, keeping them in aerobic conditions for 2 days.
The mud samples, before their use, have been analysed to check its ability to form colony units. Then it has been centrifuged, washed and analyzed to quantify the suspended solids concentration for the inoculum preparation. The inoculum was prepared in order to have a concentration of suspended solid of about 4 mg/L.

Duration of test (contact time):

28 d

Initial conc.:

23.57 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

1. Medium preparation:
Solution A Formula Concentration (g/L)
Anhydrous potassium dihydrogenphosphate KH2PO4 8.50
Anhydrous dipotassium hydrogenphosphate K2HPO4 21.75
Disodium hydrogenphosphate dihydrate Na2HPO4*H2O 33.40
Ammonium chloride NH4CI 0.50
pH = 7.42
Solution B Formula Concentration (g/L)
Calcium chloride dihydrate CaCl2*2H2O 36.40
Solution C Formula Concentration (g/L)
Magnesium sulfate heptahydrate MgSO4*7H2O 22.50
Solution D Formula Concentration (g/L)
Iron chloride (III) hexahydrate FeCl2 6H2O 0.25
All solutions have been made with deionized water(MilliQ Millipore).
Then for eachlitre of medium 10 mL of Solution A have been transferred into a 1000 mL volumetric flask and diluted with 800 mL of deionised water; 1 mL of Solution B, 1 mL of Solution C and 1 mL of Solution D have been added and the solution has been diluted to volume with deionised water. For higher volumes the medium has been prepared according to the same proportion. The preparationof the solutions has been reported in an internal logbook.
2. Bottles preparation
Bottles have been used as follows:
Bottles type N. of total Replica prepared N. oftotal Replica used N. Replica/ Check points (days)
Blank 23 17 3 replica / once a week 5 replica at 28th day
Test Samlpe 23 17 3 replica / once a week 5 replica at 28th day
Reference substance 23 17 3 replica / once a week 5 replica at 28th day
Test Sample + Reference 8 8 3 replica / t=1st day 5 replica at 28th day
Abiotic check 8 8 3 replica / t=1st day 5 replica at 28th day
to which the inoculum of the muds has been added.
All dilutions have been done using deionised water.

3. For assay execution the following preparations have been done using deionised water. Bottles not used have been eliminated at the end of the test.

4. Test sample
The elemental analysis of the test item has been determined by REDOX SnC, (see results inattachment N. 2). Because of the slight solubility of the test item, the sample was prepared by ultrasonic dispersion. About 10 g of the test sample was added to 400 mL of deionised water. Then the sample was stirred for 40 minutes at 40°C. Finally the sample was letsettle (1 hour) and the surplus was removed. The sample was analyzed at TOC analyzer and the value obtained was considered asthe initial measured concentration.
The sample has been used with the measuredinitial concentration of 23.57 mg/L of organic carbon (TOC).

5. Reference substance
Considering 1-Octanol as reference substance using its molecular formula, a starting measured concentration of 16.30 mg/l of organic carbon (TOC) has been used.
Blank
Blank has been used which contained water, culture medium and the inoculum of the mud.
Test sample + Reference substance
Test sample and reference substance have been put together in order to have a final concentration given by the sum of organic carbon (TOC) of the 2 substances (see attachment N. 3 for results).
Abiotic check
Test item has been put together with 50 mg/L HgCl, in order to check a possible abiotic degradation.

6. Conditions of assay
The inoculum has been addedin all bottles (see bottle preparation), closed and incubation continued understirring in darkness at 20 + 1 °C for 28 days. Data recording is performed continuously by a validated informatic monitoring system (Labguard Evisense). At least 1 hour before every determination of inorganic carbon (TIC) the reaction in one bottle of blank, one of reference substance and one ofthe test item has been stopped with the addition of 6 mL of sodium hydroxide (1 M). Triplicate bottles have been analyzed to detect inorganic carbon (IC) at each check point, weekly, exceptat the end of the test wherefive bottles have been analyzed.

7. Determination of total organic carbon (TOC) and total inorganic carbon(TIC)
Determination of total inorganic carbon has been done using an automatic TOC analyser. It is a high sensitivity instrument based on the combustion catalytic oxidation method (680°C). The carbon dioxide generated by oxidation has been detected using an infrared gas analyzer (NDIR). Samples, properly diluted with deionised water with low TOC contents, have been analysed by means of an integrated sampling system directly from vials containing 40 mL volume.

Reference substance:

other: 1-Octanol

Parameter:

% degradation (inorg. C analysis)

Value:

3

Sampling time:

1 d

Parameter:

% degradation (inorg. C analysis)

Value:

5

Sampling time:

7 d

Parameter:

% degradation (inorg. C analysis)

Value:

5

Sampling time:

17 d

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

21 d

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

2

Sampling time:

28 d

Results with reference substance:

The degradation of reference substance 1-Octanol after 28 days is 89%. Details see blow:
Check Point(Days) % of Biodegradation
0 0
1 46
7 80
17 83
21 81
28 89

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

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 analysis of inorganic carbon for the evaluation in an aqueous medium of the aerobic biodegradability has been performed following screening method described in OECD 310: 2014.

For this purpose the amount of developed inorganic carbon has been measured and reported in comparison to the blank.

The test has been done using the test item with a fixed concentration of organic carbon (see Par. Assay Execution). Samples have been kept at the temperature of 20 + 1 °C for the whole period ofthe test (28 days).

On the basis of results obtained, interpreted in accordance to OECD 310:2014, 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]’ is considered not biodegradable in aerobic conditions.

Description of key information

On the basis of results obtained, interpreted in accordance to OECD 310:2014, 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]’ is considered not biodegradable in aerobic conditions.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater