Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1)

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:

2012-05-31 to 2012-07-27

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:

no

Principles of method if other than guideline:

Food was provided on day 0 instead of day 1.
The soil moisture of the vessels of the test item concentrations 62.5, 125 and 500 mg/kg soil dw deviated by more than 10 % of the initial moisture at the end of the test since it was decided to add additional water during the study due to a dry soil surface.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The respective test item amount was weighed out on Petri dishes for each test item concentration. Quartz sand (10 g per replicate) was given to the test item and mixed thoroughly with it. Due to the test item consistence small globules appeared which were reduced to small pieces with a spatula. The spiked quartz sand was added to the natural soil. Additional 214 mL demineralised water was added to the soil to adjust the humidity to a moisture content of 52 % 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.
- Controls: Natural soil with the addition of quartz sand (10 g per replicate) without test or reference item was used as control medium.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida SAVIGNY 1826, subspecies Eisenia fetida fetida
- Source: Own breeding
- Age at test initiation (mean and range, SD): synchronized earthworm culture, not deviating more than 1 month
- Weight at test initiation (mean and range, SD): 0.42

ACCLIMATION
- Acclimation period: 2 days prior to test start
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): healthy

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

Removement of adults after 28 days

Test temperature:

18 - 22°C

pH:

Application rate
[mg/kg soil dry weight] pH-value
day 0 day 56
Control 5.67 6.96
62.5 5.74 7.00
125 5.72 6.80
250 5.73 6.95
500 5.74 6.91
1000 5.78 7.00

Moisture:

Application rate
[mg/kg soil dry weight] Moisture [%] of dry weight

day 0 day 56
Control 21.4 22.4
62.5 21.0 25.1*
125 21.2 24.0*
250 21.1 22.1
500 21.0 24.6*
1000 21.0 23.1

Details on test conditions:

TEST SYSTEM
- Test container (material, size): round plastic boxes, ID: 15 cm, bottom surface area: 177 cm3, height: 14 cm, transparent and perforated lids enabled sufficient gas exchange and light penetration and prevented the test substrate from drying
- Amount of soil or substrate: 600 g DW
- No. of organisms per container (treatment): 10 (40)
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition:
Parameter

*) data provided by LUFA Speyer
#) acc. to German DIN classification LUFA-soil 2.2
Batch-No. SP2.21712
pH-value* 5.5 ± 0.2
Soil moisture before application [%] 11.7
Maximum water capacity* [g/100 g soil dry weight] 41.8 ± 3.0
Particle size distribution acc. to DIN*
Sand:
0.63 – 2.0 mm ¿%¿ 0.8 ± 0.3
0.2 – 0.63 mm ¿%¿ 43.4 ± 2.0
0.063 – 0.2 mm ¿%¿ 33.3 ± 1.8
Silt:
0.02 – 0.063 mm ¿%¿ 7.3 ± 0.9
0.006 – 0.02 mm ¿%¿ 4.5 ± 1.0
0.002 – 0.006 mm ¿%¿ 3.5 ± 0.7
Clay:
< 0.002 mm ¿%¿ 7.3 ± 1.2
Organic carbon content ¿%¿* 1.77 ± 0.20
Cation exchange capacity [mVal/100 g]*
at pH 10.1 ± 0.5
Soil type loamy sand (IS)

- Pretreatment of soil: none
- Storage (condition, duration): 23 days at 6 +/- 2°C

OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 511 +/- 93 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Adult mortality, biomass, behaviour and pathological symptoms after 28 days
Reproduction (Number of juveniles) after 55/56 days

Nominal and measured concentrations:

Nominal application rate: 62.5 - 125 – 250 – 500 – 1000 mg test item/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

0.503 - 1.26 - 2.52 mg Carbendazim/ kg soil DW

Key result

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

Remarks:

mortality, body weight

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

993.2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

mortality, body weight

Details on results:

- Mortality at end of exposure period:
After 28 days of exposure to the test item no evident mortality (<10%) of adult earthworms occurred in the control as well as in all test item concentrations
- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
At test start the earthworms had an individual weight of 0.30 - 0.49 g. The body weights of the earthworms increased in the control and in all test item concentrations throughout the first 28 days. The biomass increase of the adult earthworms throughout the first 28 days was not statistically significant different compared to the control at all application rates
- No. of offspring produced:
The mean number of juveniles in the control was 212. The average number of juveniles in the test item concentrations ranged between 189 and 240. Compared to the control the reproduction of the earthworms did not statistically significant differ at all test item concentrations.
The coefficient of variation calculated for the reproduction of the control was lower than 30 %.
- Behavioural abnormalities:
No symptoms or changes in the behaviour of the earthworms were observed in the control as well as at all test item concentrations after 28 days of exposure to the test item

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 0.63 mg Carbendazim/kg soil DW

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA) and DUNNETT’s Method were carried out for the determination of statistically significant differences compared to the control. A Normality Test and an Equal Variance Test were conducted prior to running the ANOVA. P-values for both Normality and Equal Variance Test are 0.05. The a-value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.

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

Application rate [mg/kg soil dry weight]	Mortality [%]
	Replicate								Mean
	1	2	3	4	5	6	7	8
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	10	0					2.5
1000	0	0	0	0					0

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate [mg/kg soil dry weight]	Effect	Replicate
		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) H)	10/10 -	10/10 -	9/10 1/10	10/10 -
1000	A)	10/10	10/10	10/10	10/10

Body Weight Changes of the Adult Earthworms      

Application rate [mg/kg soil dry weight]	Rep.	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sign.*
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.41	0.59	0.18	43.9	0.17 ± 0.02	40.8 ± 5.70	–
	2	0.41	0.56	0.15	36.6
	3	0.41	0.61	0.20	48.8
	4	0.41	0.58	0.17	41.5
	5	0.43	0.61	0.18	41.9
	6	0.43	0.57	0.14	32.6
	7	0.43	0.58	0.15	34.9
	8	0.43	0.63	0.20	46.5
62.5	1	0.41	0.56	0.15	36.6	0.16 ± 0.01	38.7 ± 2.25	No
	2	0.41	0.58	0.17	41.5
	3	0.43	0.60	0.17	39.5
	4	0.43	0.59	0.16	37.2
125	1	0.41	0.62	0.21	51.2	0.19 ± 0.02	45.3 ± 4.56	No
	2	0.41	0.58	0.17	41.5
	3	0.43	0.63	0.20	46.5
	4	0.43	0.61	0.18	41.9
250	1	0.41	0.59	0.18	43.9	0.19 ± 0.02	44.6 ± 4.51	No
	2	0.41	0.58	0.17	41.5
	3	0.43	0.61	0.18	41.9
	4	0.43	0.65	0.22	51.2
500	1	0.41	0.59	0.18	43.9	0.21 ± 0.03	55.6 ± 9.74	Yes^1)
	2	0.37	0.62	0.25	67.6
	3	0.37	0.58	0.21	56.8
	4	0.37	0.57	0.20	54.1
1000	1	0.37	0.57	0.20	54.1	0.19 ± 0.01	51.4 ± 3.87	Yes^1)
	2	0.37	0.56	0.19	51.4
	3	0.37	0.57	0.20	54.1
	4	0.37	0.54	0.17	45.9

Rep. = Replicate                     SD = Standard deviation                    *) Statistical significance compared to control

1) These slight increases were not considered to be biologically significant.

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg/kg soil dry weight]	Rep.	Number of juveniles	Mean ± SD	CV               [%]	[%] of control	Significance compared to control
Control	1	204	212 ± 19.2	9	–	–
	2	202
	3	189
	4	212
	5	242
	6	193
	7	237
	8	213
62.5	1	218	202 ± 20.6	10	95	No
	2	194
	3	177
	4	220
125	1	263	240 ± 27.1	11	113	No
	2	251
	3	201
	4	245
250	1	184	214 ± 20.2	9	101	No
	2	226
	3	226
	4	220
500	1	269	214 ± 38.1	18	101	No
	2	182
	3	202
	4	201
1000	1	230	189 ± 31.4	17	89	No
	2	172
	3	158
	4	194

SD = Standard deviation       CV = Coefficient of variation       Rep. = Replicate     

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] (CAS no 34140-91-5) did not induce evident mortality or pathological symptoms of adult earthworms after an exposure to the test item concentrations of 62.5, 125, 250, 500 and 1000 mg test item/kg soil dry weight for 28 days. The earthworm biomass increased in the control and in all tested application rates over 28 days without any statistically significant differences. After further four weeks the earthworm reproduction rate was not statistically significantly reduced compared to the control in all test item concentrations.

Thus, the No Observed Effect Concentration (NOEC) of N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] (CAS no 34140-91-5) with respect to mortality, body weight and reproduction of Eisenia fetida after 8 weeks of exposure in natural soil was determined to be = 1000 mg test item/kg soil dry weight.

Executive summary:

Effects of N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] (CAS no 34140-91-5) (batch number 30411) on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Savigny) were determined according to the Guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994) from May 31^th, 2012 to July 27^th, 2012 with the definitive exposure phase from May 31^th, 2012 to July 26^th, 2012 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany. The study was conducted under static conditions over 8 weeks with five different application rates of 62.5 – 125 - 250 - 500 - 1000 mg test item/kg soil dry weight, which were mixed into natural soil. Eighty sexually mature earthworms were inserted into 8 control replicates (10 earthworms per replicate) and forty sexually mature earthworms were divided into 4 replicates for test item concentrations each. They had an individual body weight between 0.30 and 0.49 g.

After 28 days no evident mortality (10%) as well as no pathological symptoms or changes in the behaviour of the adult earthworms was observed in the control and all tested application rates. Slight increases of earthworm biomass were not considered to be biologically significant. After further four weeks the reproduction rate (average number of juveniles) was 212 in the control and ranged between 189 and 240 in the test item application rates. The earthworm reproduction rate was not statistically significant reduced compared to the control in all test item concentrations.

Overall, the NOEC of N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] (CAS no 34140-91-5) concerning mortality, biomass and reproduction of earthworms was determined to be = 1000 mg/kg soil dry weight.

All validity criteria recommended by the guidelines were fulfilled.

Summary of All Observed Effects in the Tested Doses

Effects	N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] (CAS no 34140-91-5)
mg test item/kg soil dry weight	62.5	125	250	500	1000
Mortality of adult earthworms	No	No	No	No	No
Reduction of body weight	No	No	No	No	No
Reduced reproduction rate   (Number of juveniles)	No	No	No	No	No
Summary of all effects	No	No	No	No	No

NOEC and LOEC for Reproduction, Mortality and Body Weight

N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] (CAS no 34140-91-5)
	Application rate
	[mg test item/kg soil dry weight]
NOECreproduction, mortality, body weight	= 1000
LOECreproduction, mortality, body weight	> 1000

Description of key information

The 56d-NOEC (reproduction, mortality and body weight) is = 993.2 mg/kg dry weight in a OECD guideline 222 test with Eisenia fetida under GLP (reliability 1). 

Key value for chemical safety assessment

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

993.2 mg/kg soil dw

Additional information

Effects of N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Savigny) were determined according to the Guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994). The study was conducted under static conditions over 8 weeks with five different application rates of 62.5 - 125 - 250 - 500 - 1000 mg test item/kg soil dry weight, which were mixed into natural soil. Overall, the NOEC of N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] concerning mortality, biomass and reproduction of earthworms was determined to be = 993.2 mg/kg soil dry weight (based on active ingredient).