N-(2-hydroxypropyl)oleamide

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Remarks:

Reproduction and Growth of the Earthworm

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 18 August 2017 and 22 October 217

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxypropyl)oleamide

EC Number:

203-828-2

EC Name:

N-(2-hydroxypropyl)oleamide

Cas Number:

111-05-7

Molecular formula:

C21H41NO2

IUPAC Name:

N-(2-hydroxypropyl)oleamide

Test material form:

other: waxy solid

Specific details on test material used for the study:

Name: N-(2-hydroxypropyl) Oleamide
CAS Number: 111-05-7
Purity: 100%
Batch number: 160602013073 w/o solvent
Expiration date: 30 May 2018
Appearance: Beige waxy solid
Storage conditions: Room temperature (15 -25°C)

Sampling and analysis

Analytical monitoring:

no

Test substrate

Vehicle:

no

Details on preparation and application of test substrate:

Prior to treatment the maximum water holding capacity (MWHC) of the soil was determined as 56.71% and the 55% capacity calculated as 0.312 g water/g dry artificial soil which provided a suitable soil structure for earthworm development. No pre-moistening of the soil was required as the water content was already >27.5% of the MWHC.

The test substance was weighed separately for each rate of application, placed into glass vials and melted in a water bath at 40 -50°C,. The melted test substance was poured over 50 g dry sand and allowed to solidify before being added to a 2150 g dry weight equivalent of moist soil. Reverse osmosis (RO) water (239.42 mL) was then added to bring the water content to 55% of MWHC. The sand and water were mixed into a 2150 g dry weight equivalent of moist soil using a hand held electric mixer.

For the water control 457.07 mL RO water was mixed into a 4104.55 g soil dry weight equivalent of moist soil to which 95.45 g dry sand had been added. The mixing procedure was as for the N-(2-hydroxypropyl) Oleamide treatments.

For the toxic reference treatment 42µL of Ringer (500 g/L carbendazim) was added to a final volume of 100 mL RO water to give a solution containing 0.21 mg a.i./mL. This was mixed into a 4104.55 g dry weight equivalent of moist soil to which 95.45 g of untreated dry sand had been added. A further 357.07 mL of RO water was added to bring the soil to 55% of the maximum water holding capacity.

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

nematods

Details on test organisms:

Earthworms (Eisenia fetida) and dried rabbit manure were purchased from a reputable supplier of laboratory livestock. Prior to study start the worms were maintained in animal manure and fed regularly. Details of suppliers are recorded only in the raw data to maintain supplier confidentiality.
Selection of adult worms for the study was based primarily on individual weights falling within the specified weight range of 300 – 600 mg at the start of the treatment period. Prior to treatment, the batch of worms was inspected for reproductive maturity, i.e. production of egg cocoons or presence of a clitellum, the presence of which confirmed suitability for use.
Approximately 24 hours prior to the study start adult worms were transferred to untreated artificial soil.

Medium (Test Soil)
An artificial soil (OECD 207) of the following composition was used as the test medium:

Industrial quartz sand: 70 % w/w
Kaolin clay: 20 % w/w
Peat: 10 % w/w

pH was adjusted to 6.0 ± 0.5 using calcium carbonate
(Untreated soil prior to treatment pH 6.5)
Soil batch: WS1704.

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

Adult worms were removed from the soil four weeks after treatment and the juvenile worms reared for a further four weeks.

Test conditions

Test temperature:

19.6 - 21.9 ºC

pH:

6.6 - 6.9

Moisture:

At termination, group replicate mean moisture content values were found to be approximately 29.45 to 31.77%, equivalent to 51.94 – 56.03% MWHC.

Details on test conditions:

Experimental Design and Study Conduct
Prior to application selected worms were rinsed in reverse osmosis water, blotted dry and individually weighed to ensure that all individual weights fell within the specified range (300 - 600 mg) before being reweighed in replicates of ten.
Tests were conducted in plastic containers measuring approximately 11 x 17 x 5 cm, each covered with a tight fitting lid. After application, a weight equivalent to 500 g dry soil at 55% of the maximum water holding capacity was transferred into each of eight replicate containers for the water control, four replicate containers for each treatment rate and eight for the positive control. Containers were labeled with the name of the study director, study number, test substance and rate of application, replicate number and date of application. The replicates of ten worms were then allocated randomly to each treatment using a bodyweight stratification procedure with the aid of a random number table.
Dried rabbit manure was used as food. One day after application of the test item, 5 g of dried manure was uniformly distributed onto the surface of the soil in each test container and wetted with 5 mL of reverse osmosis water. Worms at each treatment rate were fed weekly in a similar way if two or more replicates per rate were assessed as having consumed 50% or more of the food. The quantity of water given was based on the amount of water loss from the moisture control box each week.
Any worms visible on the soil surface were observed daily during the adult exposure phase for behavioural and pathological signs.
On Day 28 of the study the soil was removed from the containers and the numbers and replicate weights of live adults were recorded. The soil was returned to the containers for the juvenile development phase. The juvenile worms were fed by carefully mixing 5 g of food (dried rabbit manure) into the substrate of each container. Juvenile worms were not fed further during the four-week rearing period.

Weights
Surviving adult worms were rinsed in reverse osmosis water, blotted dry and weighed in replicate groups at the end of the four week exposure period. They were then humanely euthanized by freezing before being discarded as clinical waste.

Juvenile Worms
The number of surviving juvenile worms in each replicate was determined on Day 56. Test containers were placed in a water bath set at 60°C to encourage juvenile worms to come to the soil surface. Worms visible on the soil were removed with tweezers and counted. Test containers were then removed from the water bath and emptied onto a tray. As soon as possible after removal from the water bath, a sample of soil was taken from each test container, checked to ensure it contained no worms and weighed accurately, before being dried and used to determine the moisture content at termination. The remaining soil from each container was searched carefully by hand and any remaining worms removed with tweezers and counted.

Test Conditions
The worms were maintained in a room which was designed to provide a satisfactory range of environmental conditions for the species. Minimum and maximum ambient temperatures and light intensity were recorded daily. The lights were controlled by a time switch set to provide a 16-hour light: 8-hour dark photoperiod.
The temperature ranged from 19.6 – 21.9ºC. The light intensity range was 433 – 794 lux.


Test concentrations - Definitive study
Ten treatments were employed in the study. These consisted of:
i) Water control.
ii) N-(2-hydroxypropyl) Oleamide applied at 16.3 mg a.i./kg dry soil.
iii) N-(2-hydroxypropyl) Oleamide applied at 29.4 mg a.i./kg dry soil.
iv) N-(2-hydroxypropyl) Oleamide applied at 52.9 mg a.i./kg dry soil.
v) N-(2-hydroxypropyl) Oleamide applied at 95.3 mg a.i./kg dry soil.
vi) N-(2-hydroxypropyl) Oleamide applied at 171.5 mg a.i./kg dry soil.
vii) N-(2-hydroxypropyl) Oleamide applied at 308.6 mg a.i./kg dry soil.
viii) N-(2-hydroxypropyl) Oleamide applied at 555.6 mg a.i./kg dry soil.
ix) N-(2-hydroxypropyl) Oleamide applied at 1000 mg a.i./kg dry soil.
x) Carbendazim applied at 5 mg a.i./kg dry soil.

Termination
On Day 56 at termination of the test, the number of juvenile worms per container was determined. All test worms were then humanely euthanized by freezing prior to being discarded as clinical waste.

Reference substance (positive control):

yes

Remarks:

Carbendazim

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

28 d

Dose descriptor:

LC50

Basis for effect:

mortality

Remarks on result:

not determinable

Remarks:

No dose-response relationship

Effect concentrations 2

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Effect concentrations 3

Duration:

28 d

Dose descriptor:

EC50

Basis for effect:

other: Adult bodyweight

Remarks on result:

not determinable

Remarks:

No dose-response relationship

Effect concentrations 4

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult bodyweight

Effect concentrations 5

Duration:

56 d

Dose descriptor:

EC50

Basis for effect:

reproduction

Remarks:

number of juveniles

Remarks on result:

not determinable

Remarks:

No dose-response relationship

Effect concentrations 6

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

number of juveniles

Details on results:

Health and mortality
Adult mortality of 0, 0, 2.5, 7.5, 5.0, 0, 0 and 0% was recorded at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil respectively. No mortality was recorded in the water control.
The 95.3 mg a.i./kg dry soil N-(2-hydroxypropyl) Oleamide treated group had significantly higher mortality (p<0.05*) than the water control group. However as no mortality was observed at the three highest rates of 308.6, 555.6 and 1000 mg a.i./kg of dry soil this is not considered to be biologically significant.
The LC50 for the mortality on Day 28 could not be estimated because there was no evidence of a dose-response relationship.
The NOEC for mortality on Day 28 was 1000 mg a.i./kg dry soil.

Bodyweights
There were no statistically significant differences between the groups for mean body weight of the adult earthworms.
The EC50 for the mean bodyweight of the adult earthworm at Day 28 could not be estimated because there was no evidence of a dose-response relationship.
The NOEC for the mean bodyweight at Day 28 was 1000 mg a.i./kg dry soil.

Juvenile Worms
Water control group productivity was acceptable (mean of 195 juveniles per replicate). The coefficient of variation of the number of juveniles in the water control group was 5.12%.
The mean number of juveniles produced at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil was 156, 175, 168, 170, 190, 171, 178 and 185 respectively compared to 195 in the water control. A mean of 1 juvenile per replicate was produced in the carbendazim treatment.
The 16.3 mg a.i./kg dry soil N-(2-hydroxypropyl) Oleamide treated group had significantly fewer juveniles (p<0.01**) than the water control group, this is not considered to be biologically significant as no significant reduction in juveniles was observed at any higher treatment rate.
The EC50 for the number of juveniles on Day 56 could not be estimated because there was not a dose-response relationship.
The NOEC for the number of juvenile worms on Day 56 was 1000 mg a.i./kg dry soil.

Results with reference substance (positive control):

Health and mortality
No mortality was recorded in the 5% in the toxic reference group.

Bodyweights
There were no statistically significant differences between the groups for mean body weight of the adult earthworms.

Juvenile Worms
There was a statistically significant reduction (p<0.001***) in the mean number of juveniles in the carbendazim group at 5 mg a.i./kg dry soil.

Any other information on results incl. tables

% Adult Mortality and Treatment Mean Bodyweights (mg) 

Treatment	Rate (mg a.i./kg dry soil)	Adult mortality		Adult Body weight
		% Mortality	pvalue	Day of study		Change (%)	pvalue
				Time 0 (mg)	Day 28^(a)(mg)
Water control	0	0	-	428	509	+18.9	-
N-(2-hydroxypropyl) Oleamide	16.3	0	>0.999	429	518	+20.7	0.998
	29.4	0	>0.999	434	465	+7.1	0.097
	52.9	2.5	0.333	428	513	+19.9	1.000
	95.3	7.5	0.035*	429	524	+22.1	0.966
	171.5	5.0	0.109	429	544	+26.8	0.268
	308.6	0	>0.999	428	512	+19.6	1.000
	555.6	0	>0.999	426	526	+23.5	0.912
	1000	0	>0.999	427	503	+17.8	1.000
Carbendazim	5	5.0	0.060	429	301	-29.8	<0.001***

 

p values for adult mortality are in comparison with the water control using Fisher’s exact test

p values for bodyweight are in comparison with the water control using Dunnett’s test

^(a) Mean adjusted bodyweight

- Not applicable

*p<0.05, ***p<0.001

% Adult Mortality and Treatment Mean Bodyweights (mg) 

Treatment	Rate (mg a.i./kg dry soil)	Adult mortality		Adult Body weight
		% Mortality	pvalue	Day of study		Change (%)	pvalue
				Time 0 (mg)	Day 28^(a)(mg)
Water control	0	0	-	428	509	+18.9	-
N-(2-hydroxypropyl) Oleamide	16.3	0	>0.999	429	518	+20.7	0.998
	29.4	0	>0.999	434	465	+7.1	0.097
	52.9	2.5	0.333	428	513	+19.9	1.000
	95.3	7.5	0.035*	429	524	+22.1	0.966
	171.5	5.0	0.109	429	544	+26.8	0.268
	308.6	0	>0.999	428	512	+19.6	1.000
	555.6	0	>0.999	426	526	+23.5	0.912
	1000	0	>0.999	427	503	+17.8	1.000
Carbendazim	5	5.0	0.060	429	301	-29.8	<0.001***

 

p values for adult mortality are in comparison with the water control using Fisher’s exact test

p values for bodyweight are in comparison with the water control using Dunnett’s test

^(a) Mean adjusted bodyweight

- Not applicable

*p<0.05, ***p<0.001

Mean Number of Juvenile Worms per Treatment Replicate 

Treatment	Rate (mg a.i./kg dry soil)	n	Mean	sd	pvalue
Water control	0	8	195	10.0	-
N-(2-hydroxypropyl) Oleamide	16.3	4	156	22.6	0.001**
	29.4	4	175	25.3	0.238
	52.9	4	168	14.0	0.051
	95.3	4	170	7.7	0.095
	171.5	4	190	8.7	0.999
	308.6	4	171	10.1	0.119
	555.6	4	178	11.1	0.452
	1000	4	185	23.0	0.912
Ringer (carbendazim)	5	8	1	0.5	<0.001***

 n      Number of replicates
sd    Standard deviation

p values for mean number of juveniles are in comparison with the water control using Dunnett’s test

CV (%) = 5.12% (coefficient of variation for the water control group)

**p<0.01,***p<0.001

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Adult mortality of 0, 0, 2.5, 7.5, 5.0, 0, 0 and 0% was recorded at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil respectively. No mortality was recorded in the water control and 5% in the toxic reference group.
The 95.3 mg a.i./kg dry soil N-(2-hydroxypropyl) Oleamide treated group had significantly higher mortality (p<0.05*) than the water control group. However as no mortality was observed at the highest three rates of 308.6, 555.6 and 1000 mg a.i./kg of dry soil this is not considered to be biologically significant.
The LC50 for the mortality on Day 28 could not be estimated because there was no evidence of a dose-response relationship.
The NOEC for mortality on Day 28 was 1000 mg a.i./kg dry soil.
There were no statistically significant differences between the groups for mean body weight of the adult earthworms.
The EC50 for the mean bodyweight of the adult earthworm at Day 28 could not be estimated because there was no evidence of a dose-response relationship.
The NOEC for the mean bodyweight at Day 28 was 1000 mg a.i./kg dry soil.
A mean of 195 juveniles per replicate was produced in the water control with a coefficient of variation of 5.12%. A mean of 1 juvenile per replicate was produced in the carbendazim treatment.
The 16.3 mg a.i./kg dry soil N-(2-hydroxypropyl) Oleamide treated group had significantly fewer juveniles (p<0.001**) than the water control group, this is not considered to be biologically significant as no significant reduction in juveniles was observed at any higher treatment rate. The 5 mg a.i./kg dry soil carbendazim treated group had significantly fewer juveniles than the water control group (p≤0.001, t- test).
The EC50 for the number of juveniles on Day 56 could not be estimated because there was no evidence of a dose-response relationship.
The NOEC for the number of juvenile worms on Day 56 was 1000 mg a.i./kg dry soil.
The study was considered valid as there was ≤ 10% adult mortality at four weeks and ≥30 juveniles had been produced in each water control replicate by the end of the test with the coefficient of variation of reproduction ≤ 30%. In addition application of the toxic reference carbendazim at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.

Executive summary:

Summary

A study was performed to determine the effects of N-(2-hydroxypropyl) Oleamide on the reproduction and growth of the earthworm, Eisenia fetida. The method followed was that described in OECD 222 Guideline for the Testing of Chemicals, Earthworm Reproduction Test (Eiseniafetida/Eisenia andrei) 2004.

A definitive study was conducted at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil. Ten groups of worms were allocated to the study. Eight groups of 40 worms were treated with the test item at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil. A water control group of 80 worms was maintained in untreated soil to act as negative control. A positive control group, 80 worms, was treated with Ringer (active ingredient carbendazim) at 5 mg a.i./kg dry soil. Adult worms were removed from the soil four weeks after treatment and the juvenile worms reared for a further four weeks.

Findings

Adult mortality of 0, 0, 2.5, 7.5, 5.0, 0, 0 and 0% was recorded at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil respectively. No mortality was recorded in the water control and 5% in the toxic reference group.

The 95.3 mg a.i./kg dry soil N-(2-hydroxypropyl) Oleamide treated group had significantly higher mortality than the water control group, where no mortality occurred. However as no mortality was observed at the three highest rates of 308.6, 555.6 and 1000 mg a.i./kg dry soil this is not considered to be biologically significant.

The LC₅₀for the mortality on Day 28 could not be estimated because there was no evidence of a dose-response relationship. 

The NOEC for mortality on Day 28 was 1000 mg a.i./kg dry soil. 

There were no statistically significant differences between the groups for mean bodyweight of the adult earthworms.

The EC₅₀for the mean bodyweight of the adult earthworm at Day 28 could not be estimated because there was no evidence of a dose-response relationship.

The NOEC for the mean bodyweight at Day 28 was 1000 mg a.i./kg dry soil.

Juvenile worms

The coefficient of variation of the number of juveniles in the water control group was 5.12%.

The 16.3 mg a.i./kg dry soil N-(2-hydroxypropyl) Oleamide treated group had significantly fewer juveniles than the water control group, this is not considered to be biologically significant as no significant reduction in juveniles was observed at any higher treatment rate. The 5 mg a.i./kg dry soil carbendazim treated group had significantly fewer juveniles than the water control group. 

The EC₅₀for the number of juveniles on Day 56 could not be estimated because there was not a dose-response relationship.

The NOEC for the number of juvenile worms on Day 56 was 1000 mg a.i./kg dry soil.

The study was considered valid as there was≤10% adult mortality at four weeks and ≥30 juveniles had been produced in each water control replicate by the end of the test with the coefficient of variation of reproduction ≤ 30%. In addition, application of the toxic reference carbendazim at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.