2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

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: Guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

The WHCmax, the moisture content and the pH of the artificial soil were determined. Two days before the test start, the artificial soil (20 kg) was pre-moistened by adding 2120 g demineralised water to achieve a moisture content corresponding to 27 % of the maximum water capacity of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.

- Method of mixing into soil (if used):
The respective test item amount for each concentration was weighed out, dissolved in demineralised water and added to the artificial soil. Additional demineralised water was added to adjust the humidity of the artificial soil to a moisture of 40 – 60 % of the WHCmax. Subsequently, the test medium was mixed thoroughly to ensure a homogenous distribution, and a soil amount corresponding to about 600 g DW was filled into each test vessel.

- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution):
- Evaporation of vehicle before use:

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida (Annelida, Lumbricidae)
- Source: breeding stock culture maintained at the test facility.
- Age at test initiation: 2-12 months
- Weight at test initiation (mean and range, SD):between 0.31 and 0.57 g at the start of exposure
- Acclimation period: two days prior to the test start.

Breeding:
- Organisms of the species Eisenia fetida are bred at the test facility in covered plastic vessels containing potting compost.

Synchronisation of the earthworm population:
Earthworms of homogeneous age and body weight were used in the definitive test. Synchronisation of the population was achieved by placing adult earthworms into breeding boxes and removing the adults after 4 weeks. Offspring from the remaining cocoons reached the adult age after 2 months at the earliest.

Feeding:
During breeding, the earthworms were fed with a litter of dried stinging nettle leaves and porridge oats. A sufficient amount of the food was provided depending on the feeding rate and the density of the earthworm population in the vessels.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

20 +/- 2 °C

pH:

6.0 +/- 0.5

Moisture:

27 % of the maximum water capacity of the artificial soil

Details on test conditions:

TEST SYSTEM
- Test container (material, size): round plastic boxes with an inner diameter of 15.0 cm, corresponding to a bottom surface area of 177 cm2, and a height of 14 cm. Transparent and perforated lids enabled sufficient gas exchange, light input and prevented the test substrate from drying
- Amount of soil or substrate:
- No. of organisms per container (treatment): 10 adult earthworms (with clitellum) aged 2-12 months were used per replicate.
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
The artificial soil consisted of the following components:
- 10 % peat, air-dried and finely ground
- 20 % kaolin, kaolinite content > 30 %
- 69 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
- 0.40 % calcium carbonate (CaCO3) to achieve a pH of 6.0 +/- 0.5

OTHER TEST CONDITIONS
- Photoperiod: 16 h
- Light intensity: 400 - 800 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): After the first four weeks, the adult earthworms were removed.
Observation parameters:
A) no obvious pathological symptoms
B) no reaction to touching
C) no negative phototactical reaction
D) spontaneous segmentation and separation
E) spasmodic winding
F) yellow secretion from the oral aperture
G) ulceration or bleeding of the skin
H) dead earthworms

Day 56: determination of number of offspring

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Nominal and measured concentrations:

Nominal: 62.5, 125, 250, 500 and 1000 mg/kg dw

Reference substance (positive control):

yes

Remarks:

Carbendazim

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

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

After 28 days of exposure in soil, no earthworm mortalities, pathological symptoms or changes in the behaviour of adult earthworms were observed in any treatment. No statistically significant differences in growth (earthworm body weight gain) were observed at any test item concentration.
After a further four weeks, the reproduction rate (mean number of juveniles produced per replicate) was 118 in the control and ranged from 118 to 142 in the test item treatment replicates. Compared to the control, no statistically significant reduction of reproduction was observed in any test item concentration. Consequently, the NOEC of the test item concerning growth and reproduction was determined to be ≥ 1000 mg/kg SDW.

Reproduction Rate
The mean number of juveniles per replicate in the control was 118. The average number of juveniles per replicate in the treated groups ranged from 118 to 142 (Table 7, Figure 1). No statistically significant reduction of reproduction was observed in any treatment compared to the control. The EC50-value was not determined since no reduction of reproduction ≥ 50 % occurred.
The coefficient of variation calculated for the reproduction of the control was 13.9 % and thus lower than 30 %.

Results with reference substance (positive control):

Test concentrations: 0.5 – 1 – 2 – 4 mg a.s./kg dw
The test is conducted once within twelve months. The most recent test was carried out from March 4th to May 4th, 2015. According to the OECD 222, significant effects of Carbendazim should be observed between 1 and 5 mg a.s./kg SDW. This criterium was fulfilled.

Mortality [%] of Adult Earthworms after 28 Days of Exposure

Replicate

[mg/kg SDW]	1	2	3	4	5	6	7	8	Mean
Control	0	0	0	0	0	0	0	0	0
62.5	0	0	0	0	–				0
125	0	0	0	0					0
250	0	0	0	0					0
500	0	0	0	0					0
1000	0	0	0	0					0

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Replicate

[mg/kg soil dry weight]	Observation parameter	1	2	3	4	5	6	7	8
Control	A	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
62.5	A	10/10	10/10	10/10	10/10	–
125	A	10/10	10/10	10/10	10/10
250	A	10/10	10/10	10/10	10/10
500	A	10/10	10/10	10/10	10/10
1000	A	10/10	10/10	10/10	10/10

A) No visible symptoms or behavioural changes    

Body Weight Changes of the Adult Earthworms

Test item concentration [mg/kg SDW]	Replicate	Mean body weights per replicate		Mean body weight gain of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sig.
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.42	0.51	0.09	21.4	0.11 ± 0.02	26.5 ± 3.83	–
	2	0.42	0.53	0.11	26.2
	3	0.42	0.54	0.12	28.6
	4	0.42	0.55	0.13	31.0
	5	0.41	0.51	0.10	24.4
	6	0.41	0.52	0.11	26.8
	7	0.41	0.50	0.09	22.0
	8	0.41	0.54	0.13	31.7
62.5	1	0.41	0.56	0.15	36.6	0.12 ± 0.03	29.5 ± 6.78	No
	2	0.42	0.56	0.14	33.3
	3	0.42	0.51	0.09	21.4
	4	0.41	0.52	0.11	26.8
125	1	0.42	0.51	0.09	21.4	0.11 ± 0.02	25.3 ± 5.79	No
	2	0.42	0.54	0.12	28.6
	3	0.41	0.49	0.08	19.5
	4	0.41	0.54	0.13	31.7
250	1	0.41	0.55	0.14	34.1	0.09 ± 0.03	22.4 ± 8.21	No
	2	0.41	0.50	0.09	22.0
	3	0.42	0.49	0.07	16.7
	4	0.42	0.49	0.07	16.7
500	1	0.41	0.52	0.11	26.8	0.11 ± 0.03	25.4 ± 6.27	No
	2	0.41	0.54	0.13	31.7
	3	0.42	0.53	0.11	26.2
	4	0.42	0.49	0.07	16.7
1000	1	0.41	0.54	0.13	31.7	0.13 ± 0.01	31.3 ± 3.56	No
	2	0.42	0.53	0.11	26.2
	3	0.41	0.55	0.14	34.1
	4	0.42	0.56	0.14	33.3

Reproduction Rate (Number of Juveniles after eight weeks)           

Test item concentration [mg/kg SDW]	Replicate	Number of juveniles	Mean±SD	CV              [%]	[%] Reduction relative to the control	Sig.
Control	1	105	118 ± 16.4	13.9	-	-
	2	102
	3	112
	4	120
	5	150
	6	102
	7	125
	8	127
62.5	1	132	131 ± 3.30	2.52	-11	No
	2	127
	3	135
	4	131
125	1	129	142 ± 15.8	11.1	-20	No
	2	127
	3	152
	4	158
250	1	123	119 ± 49.8	41.9	-1	No
	2	151
	3	47
	4	154
500	1	176	129 ± 34.8	27.0	-9	No
	2	133
	3	95
	4	113
1000	1	121	118 ± 9.47	8.03	0	No
	2	126
	3	119
	4	104

SD = Standard deviation        CV = Coefficient of variation      Sig. = Statistical Significance (Dunnett’s Multiple t-test Procedure) compared to the control

Validity criteria fulfilled:

yes

Conclusions:

Compared to the control, no statistically significant reduction of reproduction was observed in any test item concentration. Consequently, the NOEC of the test item concerning growth and reproduction was determined to be ≥ 1000 mg/kg SDW.

Executive summary:

Effects or the test item on mortality, growth and the reproductive potential of the earthworm species Eisenia fetida were determined according to OECD Guideline 222 (2004) from May 15th to July 11th, 2015 at Dr. U.Noack-Laboratorien in Sarstedt, Germany. The study was conducted over 8 weeks with test item concentrations of 62.5, 125, 250, 500 and 1000 mg test item/kg dw, which were mixed into artificial soil. A control containing untreated artificial soil was also set up. Sexually mature earthworms were inserted into 8 control replicates and into 4 replicates per test item concentration (10 earthworms per replicate).

After 28 days of exposure in soil, no earthworm mortalities, pathological symptoms or changes in the behaviour of adult earthworms were observed in any treatment. No statistically significant differences in growth (earthworm body weight gain) were observed at any test item concentration.

After a further four weeks, the reproduction rate (mean number of juveniles produced per replicate) was 118 in the control and ranged from 118 to 142 in the test item treatment replicates. Compared to the control, no statistically significant reduction of reproduction was observed in any test item concentration. Consequently, the NOEC of the test item concerning growth and reproduction was determined to be ≥ 1000 mg/kg dw.

Description of key information

Effects or the test item on mortality, growth and the reproductive potential of the earthworm species Eisenia fetida were determined according to OECD Guideline 222 (2004) from May 15th to July 11th, 2015 at Dr. U.Noack-Laboratorien in Sarstedt, Germany. The study was conducted over 8 weeks with test item concentrations of 62.5, 125, 250, 500 and 1000 mg test item/kg dw, which were mixed into artificial soil. A control containing untreated artificial soil was also set up. Sexually mature earthworms were inserted into 8 control replicates and into 4 replicates per test item concentration (10 earthworms per replicate).
After 28 days of exposure in soil, no earthworm mortalities, pathological symptoms or changes in the behaviour of adult earthworms were observed in any treatment. No statistically significant differences in growth (earthworm body weight gain) were observed at any test item concentration.
After a further four weeks, the reproduction rate (mean number of juveniles produced per replicate) was 118 in the control and ranged from 118 to 142 in the test item treatment replicates. Compared to the control, no statistically significant reduction of reproduction was observed in any test item concentration. Consequently, the NOEC of the test item concerning growth and reproduction was determined to be ≥ 1000 mg/kg dw.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:

1 000 mg/kg soil dw