Amines, N-(C16-18 and C18-unsatd. alkyl)-N,N',N'-trimethyltrimethylenedi-

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

Study period:

September 27th to November 24th, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Food was provided on day 0 due to good experience with this procedure. Due to technical reasons the temperature increased up to 24 °C for 26 hours in total. At test end, the soil moisture deviated by > 10 % from the initial value in all treatments.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All stock solutions and the control were analytically verified via LC-MS/MS. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Vehicle:

no

Details on preparation and application of test substrate:

One day before worm insertion (Day -1), the respective test item amount for each concentration was weighed out, dispersed 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 54 % of the WHCmax.
Subsequently, the test medium was mixed thoroughly to ensure a homogenous distribution, was covered to prevent any evaporation and stored in the dark under test conditions. On day 0, after further mixing, a soil amount corresponding to about 600 g SDW was filled into the respective test vessels.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Test system
Eisenia fetida (Annelida, Lumbricidae)
Reason for the selectionEisenia fetida is suitable for this kind of study and is one of the recommended species according to the guideline.

Source
Breeding stock culture maintained at the test facility.

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 are 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:

Nominal: 20 +/- 2 °C
Actual: 18 - 24 °C
The room temperature increased to 24 °C for about 26 hours. This deviation is considered to have no impact on quality and integrity of the study.

pH:

Application rate
[mg/kg SDW] pH-value
day 0 day 56
Control 6.27 5.95
62.5 6.24 6.13
125 5.98 6.00
250 6.07 6.06
500 5.94 6.21
1000 6.02 6.11
2000 5.91 6.43

Moisture:

Application rate
[mg/kg SDW] pH-value Moisture [%] of dry weight
day 0 day 56
Control 19.5 25.2
62.5 19.4 24.0
125 19.3 23.6
250 19.0 24.5
500 19.4 24.9
1000 19.1 23.9
2000 18.9 24.1
The soil moisture deviated by more than 10 % from the initial value.

Details on test conditions:

Test item
N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine
Application rates
62.5 – 125 – 250 – 500 – 1000 - 2000 mg/kg soil dry weight (Factor 2)
Control
Artificial soil, moistened with demineralised water, without test or reference item was used as control medium
Test duration
8 weeks
Number of replicates
8 replicates per control and 4 replicates per test item concentration
Number and age of the earthworms
10 adult earthworms (with clitellum) aged 2-12 months were used per replicate. The difference of age did not deviate by more than 1 month.
Body weight (actual)
0.36 to 0.59 g
Adaptation
Earthworms were adapted to the artificial soil to be used for the test two days prior to test start.
Homogeneity of groups
The worms were washed with demineralised water and dried gently on a paper towel. The homogeneity of the population was checked by weighing the earthworms. After confirmation of homogeneity, groups of 10 earthworms (chosen by a randomised procedure) were weighed and introduced into each test vessel.
Test medium
Artificial soil consisted of the following dry components:
- 5 % peat, air-dried and finely ground
- 20 % kaolin, kaolinite content > 30 %
- 74 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
- 0.20 % calcium carbonate (CaCO3) to achieve a pH of 6.0 ± 0.5
The WHCmax, the moisture content and the pH-value of the artificial soil were determined and adjusted. Two days before test start the artificial soil (22000 g) was pre-moistened with 1892 g demineralised water to achieve a moisture content corresponding to 27 % of the WHCmax of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.
Test container
Test units containing about 600 g soil dry weight were 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 will enable sufficient gas exchange, light input and prevent the test substrate from drying.
Application
One day before worm insertion (Day -1), the respective test item amount for each concentration was weighed out, dispersed 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 54 % of the WHCmax.
Subsequently, the test medium was mixed thoroughly to ensure a homogenous distribution, was covered to prevent any evaporation and stored in the dark under test conditions. On day 0, after further mixing, a soil amount corresponding to about 600 g SDW was filled into the respective test vessels.
Temperature
20 +/- 2 °C
Photoperiod
16 h
Light intensity
400 - 800 lx
Feeding
The earthworms were fed with air-dried cattle manure weekly during the first 4 weeks as presented in Table 3. At test start and at day 28 the food was mixed carefully into the soil, on the other days the food was placed onto the soil surface.
The cattle manure was delivered by LEHR- UND FORSCHUNGSGUT RUTHE, Schäferberg 1, 31157 Sarstedt, Germany.
Moistening
Throughout the test the replicates were weighed weekly. Evaporated water was replenished by adding demineralised water to maintain the soil moisture which should not deviate by more than 10% of the initial value at the end of the test. Additional moistening was performed in case the soil appeared too dry.

Feeding and Additional Moistening During the Test
Day Amount of food [g]
Cattle manure Amount of demineralised water
[mL]
0 5 –
7, 14, 21, 28 5 10
35, 42, 49 – –

Nominal and measured concentrations:

62.5 – 125 – 250 – 500 – 1000 - 2000 mg/kg soil dry weight (Factor 2)

Reference substance (positive control):

yes

Remarks:

CARBENDAZIM: 0.5 - 1 - 2 - 4 mg a.s./kg SDW The study was carried out from January 21th to March 18th, 2016 (Study no. RBR1601), by application into the artificial soil by mixing.

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

2 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

8 wk

Dose descriptor:

EC10

Effect conc.:

1 165 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

8 wk

Dose descriptor:

other: EC20

Effect conc.:

1 530 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

8 wk

Dose descriptor:

EC10

Effect conc.:

192 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

8 wk

Dose descriptor:

other: EC20

Effect conc.:

275 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

8 wk

Dose descriptor:

EC50

Effect conc.:

547 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality at end of exposure period: No evident earthworm mortality (≤ 10 %) was observed in the controls and all test item concentrations
- Total mass of adults at beginning of test: At test start, the earthworms had individual weights of 0.36 - 0.59 g.
- Changes in body weight of live adults (% of initial weight) at end of exposure period: There were no statistically significant differences in earthworm body weights in the treatment rates 62.5 to 1000 mg test item/kg SDW compared to the control. However, at the test item concentration of 2000 mg/kg SDW the earthworm body weight statistically significantly decreased.
- No. of offspring produced: The reproduction rate (average number of juveniles produced) was 128 in the control and ranged from 0 to 115 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 to 250 mg/kg soil dry weight compared to the control. However, at the test item concentrations 500 - 2000 mg/kg the earthworm reproduction was statistically significantly reduced (Table 7). The EC50-value for reproduction was determined to be 547 mg N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine/kg SDW.

Results with reference substance (positive control):

Reference Item - Reproduction Rate (Number of Juveniles after 8 Weeks)
Application rate
[mg a.s./kg DW] Rep. Number of juveniles Mean ± SD CV % of control Significance*
Control 1 35 36 ± 9.33 25.9 _ _
2 37
3 19
4 39
5 32
6 34
7 37
8 53
0.5 1 46 46 ± 2.45 5.33 128 No
2 43
3 46
4 49
1 1 28 44 ± 14.1 32.1 122 No
2 61
3 38
4 48
2 1 14 9 ± 5.32 59.1 25.0 Yes
2 3
3 12
4 5
4 1 0 0 ± 0.0 - 0.0 Yes
2 0
3 0
4 0

Rep. = Replicate a.s. = active ingredient SD = Standard deviation CV = Coefficient of variation
Sig. = Statistical Significance (ANOVA. α = 0.05) compared to control

According to OECD 222 significant effects of Carbendazim should be observed between 1 and 5 mg a.s./kg soil DW.

Reported statistics and error estimates:

Adult Mortality
The arithmetic mean and NOEC were determined. Since no mortality occurred the LCx – values were not determined.
Biomass, Reproduction
Significant differences in biomass and reproduction were determined in comparison with the control group by probit analysis.
When running the test, a Shapiro-Wilk Normality Test and a Levene’s Equal Variance Test were done first. The α-value (acceptable probability of incorrectly concluding that there is a difference) was α = 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated. The EC10, 20, 50 - values for reproduction and the EC10, 20 - values for biomass were calculated.
Software
The data presented in the tables of the report were computer-generated and have been rounded for presentation. Thus, manual
re-calculation of the data based on the results presented in this report may result in minor deviations from these figures.
Calculations were carried out using software
- Excel, MICROSOFT
- ToxRat Professional, TOXRAT SOLUTIONS GMBH

No evident earthworm mortality (≤ 10 %) was observed in the controls and all test item concentrations. <

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

Application rate		Replicate
[mg/kg SDW]		1		2		3		4		5		6		7		8		Mean
Control		0		10		0		0		0		10		0		0		2.5
62.5		10		10		0		0		–					5
125		0		0		0		0						0
250		0		0		0		0						0
500		0		0		0		10						2.5
1000		0		0		0		0						0
2000		20		0		20		0						10

 

Pathological Symptoms and Changes in Behaviour

No significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and all test item concentrations.

 Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate

Replicate

[mg/kg SDW]

Observation parameter

1

2

3

4

5

6

7

8

Control

A H

10/10 -

9/10 1/10

10/10 -

10/10 -

10/10 -

9/10 1/10

10/10 -

10/10 -

62.5

A H

9/10 1/10

9/10 1/10

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 H

10/10 -

10/10 -

10/10 -

9/10 1/10

1000

A

10/10

10/10

10/10

10/10

2000

A H

8/10 2/10

10/10 -

8/10 2/10

10/10 -

A = no obvious pathological symptoms      H = Earthworm dead

Live Weight

At test start, the earthworms had individual weights of 0.36 - 0.59 g. There were no statistically significant differences in earthworm body weights in the treatment rates 62.5 to 1000 mg test item/kg SDW compared to the control. However, at the test item concentration of 2000 mg/kg SDW the earthworm body weight statistically significantly decreased.

 

Body Weight Changes of the Adult Earthworms        

Application rate [mg/kg SDW]	Replicate	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sig.
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.47	0.62	0.15	31.9	0.15± 0.03	30.6 ± 6.65	–
	2	0.47	0.67	0.20	42.6
	3	0.48	0.62	0.14	29.2
	4	0.48	0.63	0.15	31.3
	5	0.48	0.57	0.09	18.8
	6	0.48	0.62	0.14	29.2
	7	0.47	0.60	0.13	27.7
	8	0.47	0.63	0.16	34.0
62.5	1	0.48	0.71	0.23	47.9	0.18 ± 0.04	37.8 ± 7.82	No
	2	0.48	0.67	0.19	39.6
	3	0.47	0.61	0.14	29.8
	4	0.47	0.63	0.16	34.0
125	1	0.48	0.57	0.09	18.8	0.15 ± 0.04	30.6 ± 9.21	No
	2	0.48	0.66	0.18	37.5
	3	0.47	0.65	0.18	38.3
	4	0.47	0.60	0.13	27.7
250	1	0.47	0.62	0.15	31.9	0.15 ± 0.04	31.5 ± 7.59	No
	2	0.47	0.57	0.10	21.3
	3	0.48	0.67	0.19	39.6
	4	0.48	0.64	0.16	33.3
500	1	0.47	0.61	0.14	29.8	0.16 ± 0.02	33.7 ± 4.45	No
	2	0.48	0.66	0.18	37.5
	3	0.47	0.61	0.14	29.8
	4	0.48	0.66	0.18	37.5
1000	1	0.48	0.58	0.10	20.8	0.10 ± 0.05	21.7 ± 9.60	No
	2	0.47	0.51	0.04	8.5
	3	0.47	0.60	0.13	27.7
	4	0.47	0.61	0.14	29.8
2000	1	0.47	0.47	0.00	0.0	-0.02 ± 0.02	-4.23 ± 5.18	Yes
	2	0.48	0.45	-0.03	-6.3
	3	0.48	0.48	0.00	0.0
	4	0.47	0.42	-0.05	-10.6

SD = Standard deviation                                                                       

Sig. = Statistical Significance (Williams Multiple Sequential t-test Procedure,α= 0.05) compared to control

Reproduction Rate

The reproduction rate (average number of juveniles produced) was 128 in the control and ranged from 0 to 115 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 to 250 mg/kg soil dry weight compared to the control. However, at the test item concentrations 500 - 2000 mg/kg the earthworm reproduction was statistically significantly reduced . The EC₅₀-value for reproduction was determined to be 547 mgN-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine/kg SDW.

The coefficient of variation calculated for the reproduction of the control was 24 % and thus lower than 30 %.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine did not induce evident earthworm mortalities (≤ 10 %) or pathological symptoms or changes in the behaviour of adult earthworms. There were no statistically significant differences in earthworm body weights in the test item concentrations 62.5 to 1000 mg test item/kg soil dry weight compared to the control. However, at the test item concentration of 2000 mg/kg soil dry weight the earthworm body weight statistically significantly decreased. The EC10 – value for biomass was determined to be 1165 mg test item/kg SDW. After a further four weeks, the reproduction rate (average number of juveniles produced) was 128 in the control and ranged from 0 to 115 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 to 250 mg test item/kg soil dry weight compared to the control. However, at the test item concentrations 500 to 2000 mg/kg the earthworm reproduction was statistically significantly reduced. Overall, the NOEC of the test item concerning mortality, biomass and reproduction was determined to be 250 mg/kg SDW. The EC50-value for reproduction was determined to be 547 mg N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine/kg SDW.

Executive summary:

Effects of N-C16 -18 -alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3 -diamine on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2004) from September 27^th to November 24^th, 2016 with the definitive exposure phase from September 28^th to November 22^th/23^rd, 2016 at the test facility in 31157 Sarstedt, Germany.

 

The study was conducted under static conditions over 8 weeks with the test item concentrations 62.5 – 125 – 250 – 500 – 1000 - 2000 mg N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine/kg SDW. Each application rate was mixed into artificial soil containing 5 % peat. A control using untreated artificial soil was tested under the same conditions as the test item treatments. 80 test organisms were inserted into 8 control replicates and 40 test organisms were divided into 4 replicates for each treatment (10 earthworms per replicate). They had an individual body weight between 0.36 and 0.59 g at experimental starting.

After 28 days of exposure in soil, neither evident earthworm mortalities (≤ 10 %) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls or all test item concentrations. There were no statistically significant differences in earthworm body weights in the test item concentrations 62.5 to 1000 mg test item/kg soil dry weight compared to the control. However, at the test item concentration of 2000 mg/kg soil dry weight the earthworm body weight statistically significantly decreased. The EC₁₀– value for biomass was determined to be 1165 mg test item/kg SDW. After a further four weeks, the reproduction rate (average number of juveniles produced) was 128 in the control and ranged from 0 to 115 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 to 250 mg test item/kg soil dry weight compared to the control. However, at the test item concentrations 500 to 2000 mg/kg the earthworm reproduction was statistically significantly reduced. Overall, the NOEC of the test item concerning mortality, biomass and reproduction was determined to be 250 mg/kg SDW. The EC₅₀-value for reproduction was determined to be 547 mgN-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine/kg SDW.

 

Each concentration level and control were analysed via LC-MS analysis after preparation on day -1.

The measured concentrations of the test item stock solutions of all active ingredients were within the range of 90 to 115% of the nominal values, demonstrating the right preparation of the tested concentrations as well as a homogenous application. The method was validated according to SANCO/3029/99 rev.4 (2000) prior to the start of exposure.

Summary of All Observed Effects in the Application rates

Effects	N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine
Application rates [mg/kg SDW]	62.5	125	250	500	1000	2000
Mortality of adult earthworms	No	No	No	No	No	No
Body Weight Gain	No	No	No	No	No	Yes
Reduced reproduction rate (Number of juveniles)	No	No	No	Yes	Yes	Yes
Summary of all effects	No	No	No	Yes	Yes	Yes

NOEC, LOEC and EC-values

Endpoint	[mg/kg SDW]
LOECmortality	>2000
NOECmortality	≥ 2000
LOECbiomass	2000
NOECbiomass	1000
LOECreproduction	500
NOECreproduction	250
EC-valuesbiomass (95 % Confidence Interval)	EC10: 1165 (925 – 1467) EC20: 1530 (1199 – 1968)
EC-valuesreproduction (95 % Confidence Interval)	EC10: 192 (104 – 355) EC20: 275 (152 – 498) EC50: 547 (266 – 1114)

Description of key information

There is one long term terrestrial toxicity test available for N-C16-18-alkyl-(even numbered, C18 unsaturated) trimethylpropane-1,3-diamine. The long earthworm test resulted in a NOEC of 250 mg/kg dw for reproduction. The reliability of the long term earthworm test is considered higher than the results of the EPM. The observed NOEC of 250 mg/kg dw is therefore used for the derivation of the PNECsoil.

Key value for chemical safety assessment

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

250 mg/kg soil dw