N-(2-hydroxypropyl)oleamide

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - February 2014

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

At the beginning of the test, room temperature was in the range 16°C – 18°C, Afterward, temperature has been stabilized between 18 and 20°C. As validity criteria have been fulfilled for controls conditions, this deviation didn't invalidate the results.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate is includedin the report (date of inspection : 22-24 January 2013)

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

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test item concentration has been measured in concentrated solutions, and in all test concentrations throughout the reproduction test before and after renewal, according to a validated method provided by the sponsor (SOP-M 1735, 2011)
To determine test item concentration in test solutions before and after renewal, samples have been diluted in acetonitrile as follows :

Test concentrations before renewal (Old):
40 mL collected from one vessel af each test condition
Addition of sample in 40 mL of acetonitrile (first dilution 1/2)
At day 2 only, dilution of 2 mL of the first dilution in 8 mL of acetonitrile (second dilution 1/5)

Test concentrations after renewal (New):
Preparation of a specific flask per concentration, containing 50 mL of test concentration
Addition of 50 mL of acetonitrile per the flask, and stirring (first dilution 1/2)
Only for the two highest concentrations, dilution of 2 mL of the first solution in 8 mL of acetonitrile (second dilution 1/5)

Concentrated solutions : dilution in acetonitrile in two successive steps

For test solutions not analysed on the sampling day, samples were stored at 4°C until analysis

Test solutions

Vehicle:

yes

Details on test solutions:

The test item “N-(2-hydroxypropyl) Oleamide” was poorly soluble in water, with a solubility estimated in the range between 0.02 and 0.75 mg/L. Considering its complete solubility in ethanol, this solvent has been used to prepare concentrated solutions.
Concentrated solutions have been prepared in ethanol (one per test concentration).
Each concentrated solution has then been added to test medium as follows:
*Each individual glass bottle has been filled with 25 mL of test medium (Elendt M4)
*5 µL of the corresponding concentrated solution prepared in ethanol have been added in the middle of the bottle
*The bottles have been completed with 25 mL of test medium (Elendt M4)
Solvent controls have been prepared by adding 5 µL of ethanol in test medium, with the same procedure
Concentrated solutions have been prepared once, the first day of the reproduction test, stored at 4°C and used for all renewals

In order to reduce potential adsorption on test vessels, two series of flasks have been used for renewals and have been coated with test concentrations

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain/clone: A
- Feeding during test :yes
- Food type: Chlorella vulgaris (algae)
- Amount: 26 µL , corresponding to 0.15 mg of carbon per daphnia
- Frequency: at day 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

none

Test conditions

Hardness:

260 mg/L expressed as CaCO3

Test temperature:

day 0-9: 16 – 18°C
day 10 – 21: 18 – 20°C

pH:

See Table 1

Dissolved oxygen:

See Table 1

Salinity:

Not applicable

Nominal and measured concentrations:

Initial nominal concentrations: 0.029 – 0.088 – 0.226 – 0.677 – 1.930 mg/L
Initial measured concentrations: 0.019 – 0.052 – 0.147 – 0.511 – 1.566 mg/L (average of concentrations measured at each renewal),
Measured test concentrations after and before renewal are presented in Table 3

Details on test conditions:

The conditions selected for the definitive test have been established on the basis of the results of the preliminary test combined with the information gathered during the first days of the ready biodegradability test (CO2 evolution test : 0.8% biodegradation at 24h and 32.0 % of biodegradation at 96h). Consequently, the following conditions have been applied:
- Preparation of one concentrated solution in ethanol per test concentrations and preparation of each flask independently (Cf.protocol description: Chapter IV - 4 - page 15)
- Use of two series of flasks, coated with test concentrations, during the test to reduce the potential adsorption of the test item on the glass
- Reduction of the maximal renewal period from 72h to 48h
- Analysis of each test concentrations before and after renewal
- Dilution of the test concentrations in acetonitrile before analysis, according to the protocol described in Chapter IV - 5 - page 16.
- Storage of the test concentrations diluted in acetonitrile (50%) at 4°C when analysis could not be performed extemporaneously.
TEST SYSTEM
- Test vessel: disposable glass bottles of 100 mL capacity filled with 50 mL of test solution
- Type : open
- Aeration: none within the test period
- Renewal of test solution : at day 2, 4, 5, 7, 9, 11, 13, 15, 17, 19
- No. of organisms per vessel: one
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- No. of vessels for analysis : 1 bottle per condition for analysis of renewal solution

TEST MEDIUM / WATER PARAMETERS
- Dilution water: M4 elendt medium (see composition in table 2)
- pH: 8.2 – 8.4
- Conductivity: 670 - 698 µS/cm
- Dissolved oxygen concentration : 8.9 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 hours light / 8 hours night
- Light intensity: 1018 – 1480 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observation at days : 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 21
Mortality among the parent
Offspring (living and death) produced by each parent animal


VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.052 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.147 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 3

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.147 - < 1.566 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 4

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.147 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Population growth (based on parents living at the start of the test)

Effect concentrations 5

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.511 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Population growth (based on parents living at the start of the test)

Effect concentrations 6

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.147 - < 1.566 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Population growth (based on parents living at the start of the test)

Effect concentrations 7

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.147 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Reproduction (based on parents alive at the end of the test)

Effect concentrations 8

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.511 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Reproduction (based on parents alive at the end of the test)

Details on results:

Measured concentrations of test item in freshly prepared solutions (New) and in exposed solutions ( Old) : see Table 3

Test concentrations (in initial nominal concentrations) prepared for this reproduction test covered the solubility range estimated during the hydrosolubility test (0.020 – 0.750 mg/L).
Comparison of these initial nominal concentrations and the initial measured concentrations showed that, for the two highest test concentrations, initial measured concentrations at each renewal, corresponded to 68 to 90% of the nominal concentration.
The variability was more important for lower test concentrations: 55 - 87% for 0.226 mg/L, 44 - 81% for 0.088 mg/L and 31 - 134% for 0.029 mg/L. This may reflect a difference of test item behavior depending on concentration in the test medium.
Despite the coating of the flask, no test item was measured in “old” test concentrations (except at 1.930 mg/L). Considering the nature and the behavior of the test item some assumptions can be made:
- A reaction between the test item and the test medium, leading to a modification of the molecular mass and to a lack of detection by the analysis method. Indeed, the analytical system (LC MS/MS) used in this study is a very accurate detection system based on the molecular weight of the test item analysed.
- An adsorption of the test item on daphnia and/or algae


Number of living offspring per test flask and number of surviving parents : see Table 4

Date of the first brood : see Table 5


Determination of effect on the population growth and on the reproduction

According to OECD document: “Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application” (OECD series on testing and assessment n°54), the two controls set (without and with solvent) have been compared using a Wilcoxon Rank Sum Tests at 0.05% (Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming).
This comparison showed that the output reproductive in the two controls were significantly different.
Therefore, the results of solvent controls have been used for the following calculations (NOEC – EC50...).

According to OECD 211, the effect of the test item in reproduction can be calculated in two ways:
*based on the total number living offspring produced at the end of the test per parent at the start of the test excluding accidental and/or inadvertent mortality (population growth inhibition)
*based on the living offspring produced per surviving parental animal (reproduction inhibition)

In this study, the mortality of one parent in the solvent controls can be considered as an inadvertent mortality.

Percentages of effect, for each concentration, have been calculated for the two endpoints (reproduction and population) : See Table 6

Results with reference substance (positive control):

Sensitivity of the test organism was checked using potassium dichromate.
The test carried out from January 9th to Januay 10th 2014 (in ISO 6341) gave the following result:

EC50 24h: 1.26 mg/L (1.17 – 1.32 mg/L).

The EC50 24 hours was calculated using a logistic regression analysis with a bootstrap estimation of the 95% confidence limits.
The EC50 24h is in the recommended range: 0.6 – 2.1 mg/L. This result confirms the appropriate sensitivity of the strain of Daphnia magna.

Reported statistics and error estimates:

NOEC and LOEC Determination based on the total number living offspring produced at the end of the test per parent living at the start of the test

The normality and the homogeneity of variance have not been verified for this set of data (Chi square and Levene’s test). Therefore, a Wilcoxon’s Rank Sum test at 0.05% (non parametric test corresponding to a Mann Witney test) has been performed (Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming).

NOEC and LOEC Determination based on the living offspring produced per surviving parental animal

The normality and the homogeneity of variance has been verified for this set of data (Chi square and Levene’s test) and a Willam’s test has been performed (Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming)

Any other information on results incl. tables

Table 3 : Measured concentration of test item in freshly prepared solutions (New) and in exposed solutions (Old)

Initial nominal concentration (mg/L)

Measured concentrations (mg/L)

Controls

New

/

<LOQ

0.005

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

/

Old

/

<LOQ

<LOQ

0.005

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

Solvent controls

New

/

<LOQ

<LOQ

<LOQ

<LOQ

0.006

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

/

Old

/

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

0.029

New

<LOQ

<LOQ

0.025

0.012

0.009

0.039

0.018

0.018

0.019

0.016

0.015

/

Old

/

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

0.088

New

0.058

0.058

0.052

0.048

0.039

0.061

0.057

0.071

0.050

0.048

0.044

/

Old

/

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

0.226

New

0.197

0.172

0.144

0.147

0.133

0.167

0.149

0.189

0.131

0.140

0.124

/

Old

/

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

0.677

New

0.505

0.495

0.466

0.521

0.591

0.534

0.458

0.540

0.471

0.501

0.514

/

Old

/

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

0.017

0.008

<LOQ

0.013

<LOQ

<LOQ

1.93

New

1.734

1.626

1.421

1.662

1.460

1.665

1.580

1.710

1.373

1.631

1.592

/

Old

/

0.065

<LOQ

0.054

0.010

<LOQ

0.024

0.008

0.009

0.011

<LOQ

<LOQ

Table 4 : Number of living offspring per test flask and number of surviving parents

Test flasks	Controls	Solvent controls	N-(2-hydroxypropyl) Oleamide (initial measured concentration mg/L)
			0.019	0.052	0.147	0.511	1.566
1	115	8	82	85	12	42	0
2	126	94	83	101	29	85	0
3	94	83	100	90	69	64	0
4	119	93	86	66	142	52	0
5	93	108	111	86	70	0	49
6	108	70	96	84	64	0	0
7	101	95	93	105	118	0	0
8	104	93	91	91	20	63	0
9	115	96	106	86	1	64	0
10	113	74	107	104	0	99	76
Total of offspring	1088	814	955	898	525	469	125
Total of surviving parent	10	9	10	10	5	7	2

In blue cells, offspring born from a parent dead at the end of the test

Table 5: Date of the first brood

	Controls	Solvent controls	N-(2-hydroxypropyl) Oleamide (initial measured concentration mg/L)
			0.019	0.052	0.147	0.511	1.566
Exposure time (days)	10	10	10	9	9	10	13

Table 6 : Inhibition of population growth and reproduction of Daphnia magna after 21 days

	Initial measured concentration (mg/L)
	Solvent controls	0.019	0.052	0.147	0.511	1.566
Number of parents at the beginning of the test	9*	10	10	10	10	10
Number of parents alive at the end of the test	9	10	10	5	7	2
Mean of offspring per total parent	89.6	95.5	89.8	52.5	46.9	12.5
SD	11.84	10.32	11.60	49.07	35.99	27.11
Coefficient of variation	0.13	0.11	0.13	0.93	0.77	2.17
% of inhibition	/	-6.6	-0.3	41.4	47.6	86.0
Mean of offspring per alive parent at the end of the test	89.6	95.5	89.8	92.6	67.0	62.5
SD	30.44	10.32	11.60	54.17	35.99	27.11
Coefficient of variation	0.34	0.11	0.13	0.58	0.54	0.43
% of inhibition		-6.6	-0.3	-3.4	25.2	30.2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

No mortality observed in the controls and 10% in the solvent controls
The mean number of living offspring produced per parent animal surviving at the end of the test was higher than 60.
The test was therefore considered as valid.

Effect concentration are expressed in initial measured concentrations

The 21 day EC50 based on mortality of adults was in the range 0.147 - 1.566 mg/L and the NOEC has been determined at 0.052 mg/L.
Reproduction NOEC and LOEC values were equal to 0.147 mg/L and 0.511 mg/L, based either on parents living at the start of the test or on parents alive at the end of the test

Executive summary:

The 21-day-chronic toxicity of N-(2-Hydroxypropyl) Oleamide to Daphnia magna was studied under semi - static conditions (renewal at least every 48h) in a GLP-study.

The test item N-(2-Hydroxypropyl) Oleamide was poorly soluble in water and soluble in ethanol. Therefore, concentrated test item solutions have been prepared in ethanol, and the daphnia magna reproduction test has been performed with tests concentrations containing 0.01% of ethanol.

Test item concentration have been measured in concentrated solution (prepared in 100 % ethanol) and in all test concentrations throughout the reproduction test before and after renewal.

Despite a preparation protocol designed to optimise exposure concentrations, and a renewal of test solutions every 48h, it was not possible to measure test item concentrations in solutions before renewal except for the highest test concentration.

Therefore, results have been calculated using initial measured concentration (initial measured concentrations).

Daphnids were exposed test item at initial measured concentrations of 0.019, 0.052, 0.147, 0.511, 1.566 mg./L. Controls and solvent controls (containing 0.01 % of ethanol) have been performed . Reproduction inhibition rates have been calculated using solvent controls values The 21 day EC₅₀based on mortality of adults was in the range 0.147 - 1.566 mg/L and the NOEC has been determined at 0.052 mg/L Reproduction NOEC and LOEC values were equal to 0.147 mg/L and 0.511 mg/L, based either on parents living at the start of the test or on parents alive at the end of the test