Amines, N'-[3-[(3-aminopropyl)amino]propyl]-N,N-di-C16-18-alkyltrimethylenedi-

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:

2016-04-26 to 2016-10-27

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Food was provided on day 0 instead of day 1 due to good experience with this procedure. One worm had an individual weight of 0.62 g at start and thus above recommended 0.60 g. Due to technical reasons the temperature increased to about 23 °C for 14 h.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

Application
On day -1, for homogenization, the test item was heated up to 40 °C in an incubator for about one hour. Afterwards the respective test item amount for preparation of a stock solution of 8000 mg/L was weighed out in a Schott bottle with a stirring rod (total volume 1000 mL). Approximately 500 mL pre-warmed demineralised water was added. The solution was acidified to pH 3.3 with HCL (2 M) under stirring conditions (1100 rpm). When the emulsion was homogenous, the solution was neutralized to pH 5.5. Finally the bottle was filled up with demineralised water to 1000.02 g. This stock solution was stirred overnight at room temperature and was used for preparation of the respective test item solutions on day 0.
On day 0 the spiking solutions were added to the artificial soil. Additional demineralised water was added to the artificial soil to adjust the humidity to a moisture of 54 % of the maximum water holding capacity.
Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 600 g soil dry weight was filled into test vessels.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Test system Eisenia fetida (Annelida, Lumbricidae)

Reason for the selection
Eisenia 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 - 23 °C
The room temperature increased to 23 °C for about 14 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 6.81 6.57
62.5 6.90 6.51
125 6.92 6.50
250 6.88 6.49
500 6.88 6.48
1000 6.82 6.44

Moisture:

Table 20: pH-Values, Moisture and WHCmax of the Test Media
Application rate
[mg/kg SDW] Moisture [%] of dry weight WHCmax of artificial soil
[g/100 g DW]
day 0 day 56
Control 1.3 26.1* 38.7
62.5 21.2 26.2*
125 21.3 26.0*
250 21.1 25.9*
500 21.1 25.7*
1000 22.9 27.3*
WHC = Water Holding Capacity
* = The soil moisture deviated by more than 10 % from the initial value.

Details on test conditions:

Experimental Procedure

Test item
TETRAMEEN 2HT

Application rates
62.5 – 125 – 250 – 500 – 1000 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 (see “Synchronisation”).

Body weight (actual)
0.32 to 0.62 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 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)
- 0.27 % 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 (30000 g) was pre-moistened with 2010 g demineralised water to achieve a moisture content corresponding to 20 % 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
On day -1, for homogenization, the test item was heated up to 40 °C in an incubator for about one hour. Afterwards the respective test item amount for preparation of a stock solution of 8000 mg/L was weighed out in a Schott bottle with a stirring rod (total volume 1000 mL). Approximately 500 mL pre-warmed demineralised water was added. The solution was acidified to pH 3.3 with HCL (2 M) under stirring conditions (1100 rpm). When the emulsion was homogenous, the solution was neutralized to pH 5.5. Finally the bottle was filled up with demineralised water to 1000.02 g. This stock solution was stirred overnight at room temperature and was used for preparation of the respective test item solutions on day 0.
On day 0 the spiking solutions were added to the artificial soil. Additional demineralised water was added to the artificial soil to adjust the humidity to a moisture of 54 % of the maximum water holding capacity.
Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 600 g soil dry weight was filled into 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 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Carbendazim

Duration:

8 wk

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

8 wk

Dose descriptor:

EC10

Effect conc.:

617 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 significant mortality of adult earthworms was observed in the controls or all treatment rates after 28 days of exposure to the test item.
- Total mass of adults at beginning of test:
At test start the earthworms had individual weights in the recommended range of 0.32 - 0.62 g.
- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
There were no statistically significant differences in earthworm body weights in all test item concentrations compared to the control.
- No. of offspring produced:
The reproduction rate (average number of juveniles produced) was 131 in the control and ranged from 61 to 112 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 to 500 mg/kg soil dry weight compared to the control. However, at the test item concentrations 1000 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 500 mg/kg SDW. The EC50-value for reproduction was determined to be > 1000 mg TETRAMEEN 2HT/kg SDW

Results with reference substance (positive control):

Reference Item - Reproduction Rate (Number of Juveniles after 8 Weeks)
Application rate
[mg a.i./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.i. = 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 -value (acceptable probability of incorrectly concluding that there is a difference) was  = 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 were calculated. For details, please refer to section 13.

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

  Adult mortality

No earthworm mortality 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		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

 

 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

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 D

10/10 -

9/10 1/10

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 obvious pathological symptoms      D = spontaneous segmentation and separation

 

 Live Weight

At test start, the earthworms had individual weights of 0.32 - 0.62 g. There were no statistically significant differences in earthworm body weights in all treatment rates compared to the control.

 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	0.48	0.53	0.05	10.4	0.10± 0.03	21.1 ± 5.86	–
	2	0.47	0.59	0.12	25.5
	3	0.48	0.57	0.09	18.8
	4	0.47	0.60	0.13	27.7
	5	0.47	0.56	0.09	19.1
	6	0.48	0.56	0.08	16.7
	7	0.47	0.59	0.12	25.5
	8	0.48	0.60	0.12	25.0
62.5	1	0.47	0.63	0.16	34.0	0.13 ± 0.03	27.4 ± 6.21	No
	2	0.48	0.62	0.14	29.2
	3	0.47	0.56	0.09	19.1
	4	0.48	0.61	0.13	27.1
125	1	0.48	0.57	0.09	18.8	0.10 ± 0.02	20.0 ± 4.41	No
	2	0.47	0.54	0.07	14.9
	3	0.48	0.58	0.10	20.8
	4	0.47	0.59	0.12	25.5
250	1	0.47	0.56	0.09	19.1	0.10 ± 0.01	21.1 ± 2.44	No
	2	0.48	0.59	0.11	22.9
	3	0.47	0.58	0.11	23.4
	4	0.48	0.57	0.09	18.8
500	1	0.48	0.62	0.14	29.2	0.13 ± 0.02	26.9 ± 3.14	No
	2	0.47	0.61	0.14	29.8
	3	0.48	0.60	0.12	25.0
	4	0.47	0.58	0.11	23.4
1000	1	0.47	0.62	0.15	31.9	0.15 ± 0.01	30.5 ± 2.54	No
	2	0.48	0.62	0.14	29.2
	3	0.47	0.60	0.13	27.7
	4	0.48	0.64	0.16	33.3

SD = Standard deviation                                                                       

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

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate  [mg/kg dry soil weight]	Replicate	Number of juveniles	Mean±SD	CV              [%]	[%] of control	Sig.
Control	1	126	131 ± 17.4	13.3	-	-
	2	117
	3	165
	4	120
	5	111
	6	132
	7	130
	8	146
62.5	1	80	100 ± 31.2	31.2	76.3	No
	2	71
	3	109
	4	140
125	1	101	107 ± 13.3	12.5	81.7	No
	2	127
	3	98
	4	103
250	1	131	112 ± 26.4	23.6	85.5	No
	2	90
	3	138
	4	88
500	1	73	108 ± 37.8	35.0	82.4	No
	2	111
	3	87
	4	159
1000	1	61	61 ± 4.20	6.89	46.6	Yes
	2	66
	3	56
	4	59

SD = Standard deviation        CV = Coefficient of variation      

Sig. = Statistical Significance (Williams Test,α= 0.05) compared to the control

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, TETRAMEEN 2HT did not induce evident earthworm mortality nor significant pathological symptoms or changes in the behaviour of adult earthworms in the control and all test item concentrations. There were no statistically significant differences in earthworm body weights in all test item concentrations compared to the control. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 to 500 mg/kg soil dry weight compared to the control. However, at the test item concentrations 1000 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 500 mg/kg SDW. The EC50-value for reproduction was determined to be > 1000 mg TETRAMEEN 2HT/kg SDW.

Executive summary:

Effects of TETRAMEEN 2HT on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2004) from April 26^thto October 27^th, 2016 with the definitive exposure phase from August 31^thto October 25/26^th, 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 – 1000mg Tetrameen 2 HT /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 0.32 and 0.62 g at experimental starting.

 

After 28 days of exposure in soil, neither earthworm mortalities 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 all test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 131 in the control and ranged from 61 to 112 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 to 500 mg/kg soil dry weight compared to the control. However, at the test item concentrations 1000 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 500 mg/kg SDW. The EC₅₀-value for reproduction was determined to be > 1000 mg Tetrameen 2 HT/kg SDW.

 

All validity criteria recommended by the test guidelines were fulfilled.

Summary of All Observed Effects in the Application rates

Effects	TETRAMEEN 2HT
Application rates [mg/kg SDW]	62.5	125	250	500	1000
Mortality of adult earthworms	No	No	No	No	No
Body Weight Gain	No	No	No	No	No
Reduced reproduction rate (Number of juveniles)	No	No	No	No	Yes
Summary of all effects	No	No	No	No	Yes

 

NOEC, LOEC and EC-values

Endpoint	[mg/kg SDW]
LOECmortality, biomass	≥1000
NOECmortality, biomass	1000
LOECreproduction	1000
NOECreproduction	500
EC-valuesreproduction (95 % Confidence Interval)	EC10: 617 (not determined) EC50: > 1000

Description of key information

There is one long term terrestrial toxicity test available for Tetramine diC16 -18. The long earthworm test resulted in a NOEC of 500 mg/kg dw. Because the applicability of the equilibrium partitioning method is questioned for this use of cationic surfactants the reliability of the long term earthworm test is considered higher than the results of the EPM. The observed NOEC of 500 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:

500 mg/kg soil dw

Additional information

There is one long term terrestrial toxicity test available for Di-C16 -18 Tetramine. The long earthworm test resulted in a NOEC of 500 mg/kg dw. Because there applicability of the equilibrium partitioning method is questioned for the use of cationic surfactants, the reliability of the long term earthworm test is considered higher than the results of the EPM. The observed NOEC of 500 mg/kg dw is therefore used for the derivation of the PNECsoil.