N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD 222 guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Principles of method if other than guideline:

In all treatments tested the soil moisture was more than 10% higher at the end of the test than the initial moisture (12.7% to 32.9%) as recommended by the guideline (100 ± 10% of the original moisture). These deviations are considered to have had no impact on the outcome and validity of this study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

EC Number:

401-990-0

EC Name:

N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Cas Number:

106990-43-6

Molecular formula:

C132 H250 N32

IUPAC Name:

N2-[2-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}[3-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}amino)propyl]amino)ethyl]-N2-[3-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}amino)propyl]-N4,N6-dibutyl-N4,N6-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine

Sampling and analysis

Analytical monitoring:

no

Test substrate

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used):
The test item was poorly soluble in water; therefore, mixtures of finely ground quartz sand and the test item were produced for the test item concentrations.
The test item was mixed in an amount of quartz sand (2% of the quartz sand from the total amount of the artificial soil had been separated) to prepare each test item concentration, respectively. The test item was incorporated into the remainder of the artificial soil by thoroughly mixing in the respective test item/quartz sand mixtures for 5 minutes. The treated artificial soil was filled into the test vessels. Per test vessel an amount corresponding to 500 g dw (dry weight) was used.
- Controls: water treated

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Source: ECT Oekotoxikologie GmbH, since 1994 (originally delivered by Co. Landenberger)
- Age at test initiation (mean and range, SD): >= 2 months - <= 1year, (with clitellum)
- Weight at test initiation (mean and range, SD): 250mg - 600 mg (differnce between individuals not more than 4 weeks)

ACCLIMATION
- Acclimation period: >= 24h
- Acclimation conditions (same as test or not): artificial soil under test conditions

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test conditions

Test temperature:

19-21.2°C

pH:

5.7-6.5

Moisture:

soil moisture(day 0): 49.3 – 51.9% (rec. 40 – 60% of the WHCmax),
soil moisture (day 56): 57.5 – 65.5% (rec. deviation ≤ 10% from day 0)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Bellaplast containers (Polarcup GmbH) of inert/non-toxic plastic (polystyrene), 170cm2 x 6 cm, covered with transparent lid
- Amount of soil or substrate: 500g dw
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate):
The composition of this artificial soil was based on OECD Guideline No. 222 (2004) (the percentages refer to the dry weight):
- 10% Sphagnum peat (no visible plant remains; finely ground and air-dried)
- 20% Kaolin-Clay (kaolinite content > 30%)
- 69.6% Quartz sand (fine sand content with particles between 50 and 200 μm higher than 50%)
- 0.4% Calcium carbonate (CaCO3)
The air dried peat was shredded in a chaff-cutter. Afterwards, all parts of the artificial soil were mixed homogeneously. The pH value was adjusted to 6 ± 0.5 using calcium carbonate. The moisture content was adjusted to 40-60% of WHCmax using deionised water. The water content of the test substrate was checked weekly by reweighing the test vessels and losses of water will be compensated.

OTHER TEST CONDITIONS
- Photoperiod/ Light intensity: light cycle: 16 : 8 h with a constant light intensity at the substrate of 442 - 583 lx (recommended 400 – 800 lx),

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- day 28 mortality and biomass
The adult worms were sampled out of the test containers after the feeding activity has been recorded for each test container. During this process morphological and behavioural changes and the number of surviving adult worms in each test container were recorded. Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end. The combined weight of the surviving adult worms from each replicate was determined.

- day 56 reproduction
The adult worms were sampled out of the test containers after the feeding activity has been recorded for each test container. During this process morphological and behavioural changes and the number of surviving adult worms in each test container were recorded. Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end. The combined weight of the surviving adult worms from each replicate was determined.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
62.5 mg test item/kg soil (dw)
125 mg test item /kg soil (dw)
250 mg test item /kg soil (dw)
500 mg test item /kg soil (dw)
1000 mg test item /kg soil (dw)

Nominal and measured concentrations:

62.5, 125, 250, 500, 1000 mg test item/kg artificial soil (dw);

Reference substance (positive control):

yes

Remarks:

Carbendazim

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 3

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass

Effect concentrations 4

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass

Effect concentrations 5

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 6

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Almost the whole food was consumed by the worms in all test vessels after 7, 14, 21 and 28 days of the application of the test item.
No other effects on behavior or morphology of the adult earthworms were observed.

Validity criteria:
mortality of the adults in the control = 0% (required: ≤ 10%);
mean number of juveniles in the control = 464.8 (required: ≥ 30)
CV for the number of juveniles in the control = 14.9 (required: ≤ 30%)

Results with reference substance (positive control):

Carbendazim as the active ingredient of the fungicide Derosal was tested as the toxic reference item from August 14 – October 09, 2013 (ECT Study No.: IRR1308). It was used in concentrations of 0.32, 1 and 3.2 mg a.s./kg.
The NOECReproduction for Carbendazim tested as a reference item was determined as 0.32 mg/kg soil (dw) and accordingly the LOECReproduction was determined as 1.0 mg/kg soil (dw). These observed effects are within the range expected from the guideline (1-5 mg Carbendazim/kg soil (dw)) and hence acceptable sensitivity of the test system is assured.

Reported statistics and error estimates:

(Dunnett’s t-test (2-sided, p ≤ 0.05), Dunnett’s t-test (1-sided, p ≤ 0.05);

Applicant's summary and conclusion

Validity criteria fulfilled:

yes