N-(2-hydroxypropyl)oleamide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2014

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Incubation temperature was in the range 20.6°C – 21.1°C, (recommended range: 21°C - 24°C). Growth rate of algae, in control conditions met the validity criteria, it can be assumed that this deviation didn’t impact the results of this study.

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

Constituent 2

Reference substance name:

N-(2-Hydroxypropyl) Oleamide

IUPAC Name:

N-(2-Hydroxypropyl) Oleamide

Test material form:

other: White waxy solid ( at ambiant temprature)

Details on test material:

- Name of test material (as cited in study report): N-(2-Hydroxypropyl) Oleamide
- Molecular formula (if other than submission substance): C12H41NO2
- Molecular weight (if other than submission substance): 339.56 g/mol
- Solubility in water : poorly soluble ( in the range of 0.02 to 0.750 mg/L)
- Melting point : 35.9°C to 41.7°C
- Relative density: 0.883 to 0.891 g.cm-3
- Vapour pressure : 2.42.10-3mPa
- Analytical purity: 100% dry matter
- Lot/batch No.: T22221 without solvent
- Expiration date of the lot/batch: 29 may 2014
- Storage condition of test material: room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test item concentration has been measured in stock solutions, prepared in ethanol and in all test concentration daily sampled throughout the definitive test, according to a validated method provided by the sponsor (SOP-M 1735, 2011) .

- Sample treatment before analysis: Samples of test concentrations have been diluted in acetonitrile immediately after sampling, and, if necessary a second dilution step have been realized before analysis, to reach concentrations included in the linearity range of the analysis method.
dilution in acetonitrile
- Sample storage conditions before analysis: when needed, samples were stored at 4°C after dilution in acetonitrile

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 stock solutions.
According to the guidance document on aquatic toxicity testing of difficult substances and mixtures (OECD series on testing and assessment n° 23), the use of solvent, when needed, is accepted as long as the final concentration, in test concentrations, doesn’t exceed 0.01%
Therefore, a stock solution has been prepared in ethanol and diluted in ethanol (serial dilution) in order to obtain concentrated solutions.
Each concentrated solution has then been added to growth medium (10 µL in 100 mL), to obtain the selected test concentrations.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: CCAP 278/4
- Origin : Liquid culture obtained from the Culture Center for Algae and Protozoa (CCAP)
Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria, U.K.
- Inoculum from a preculture in exponential growth

Study design

Test type:

static

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not monitered

Test temperature:

20.6°C – 21.1°C

pH:

See table 2 for pH measurement at 0 and 72h.

Dissolved oxygen:

Not monitored

Salinity:

Not applicable

Nominal and measured concentrations:

Defintive test:
Nominal concentrations: 0.800 - 0.400 - 0.200 - 0.100 - 0.05 mg/L
Measured concentrations: 0.800 - 0.286 - 0.102 - 0.031 - 0.011 mg/L ( geometric mean of measured concentration)

Measured concentrations: see table 2 in "other informations"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks with air permeable stoppers filled with 100 mL of test concentration
- Initial cells density: 1x 10E4 cell/mL
- No. of vessels per concentration (replicates): 3 with algae, 1 without algae ( blank control), 1 for analysis
- No. of vessels per control (replicates): 6 without solvent
- No. of vessels per vehicle control (replicates): 6 with solvent ( 0.01%)


GROWTH MEDIUM
- Standard medium used: yes, Growth medium according to OECD 201


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity: 6 100 -7 860 Lux


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study : yes
- Test concentrations: in initial nominal concentration : 2 - 1 - 0.5 - 0.25 - 0.125 - 0.0625 - 0.03125 mg/L
- Results used to determine the conditions for the definitive study:
Absence of algae in concentrations between 0.25 and 2 mg/L, absence of growth inhibition between 0.03125 and 0.125 mg/L.
The definitive test has been performed at concentrations between 0.05 mg/L and 0.8 mg/L, according to the result of the preliminary test and in order to cover the highest value of the solubility range determined during the hydrosolubility test (0.750 mg/L).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits : 0.08 - 0.15 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.031 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No

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), when controls and solvent controls are tested, results have to be compared in order to determine if the two sets of controls are significantly different.
The comparison of control data (Wilcoxon test at 0.05% (Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming) showed that the two controls were significantly different.
Therefore, the set of data measured for solvent controls has been used for growth rate inhibition calculation.

Cells density measurements are presented in table 2

Average specific growth rate in control conditions are presented in table 3

Growth rates calculations and inhibitions are presented in table 4

Test concentration meausrements are presented in table 5

As test concentrations were not maintained in the range 80% - 120% of the initial concentration along the test, exposure concentrations have been calculated using the geometric mean of the measured values. For the nominal concentration at 0.05 mg/L, the test item has been detected at 72h but the value was under the limit of quantification. Therefore the exposure concentration has been estimated by calculating the geometric mean of measured values and using the half of the LOQ (0.001 mg/L) as value at 72h.

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- EC50: 0.91 mg/L
- Other: value included in the warning limits of the laboratory (historic geometric mean +/- 2 Standard deviation

Reported statistics and error estimates:

NOEC have been determined with a William’s test (Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming)
ErC 10 and ErC 50 (0 – 72 h) values have been calculated using log-logistic regression with bootstrap estimation of 95% confidence limits.

Any other information on results incl. tables

Table 2: Cell density determined with a particle counter

Test solutions (initial nominal concentrations) in mg/L	Cell Density x 10^4cell/mL (particle counter)								pH
	T0	Mean	T24h	Mean	T48h	Mean	T72h	Mean	T0	T72h
Control	1.00 1.00 1.00 1.00 1.00 1.00	1.00	4.80 5.65 4.21 4.82 3.64 3.35	4.41	26.64 23.71 16.39 19.22 14.21 22.79	20.49	205.44 129.49 75.32 153.04 110.38 161.87	139.26	8.3	9.0 8.2 7.9 8.7 8.3 8.6
Control with solvent (ethanol 0.01%)	1.00 1.00 1.00 1.00 1.00 1.00	1.00	4.70 2.80 5.41 4.27 3.56 4.26	4.17	19.52 19.33 22.12 15.06 12.38 20.13	18.09	67.00 78.39 129.15 79.78 50.56 76.72	80.27	8.0	7.7 7.9 8.0 7.9 7.8 7.9
0.05	1.00 1.00 1.00	1.00	4.52 3.19 4.01	3.91	21.25 21.17 17.36	19.93	118.52 100.52 65.19	94.74	8.1	8.1
0.1	1.00 1.00 1.00	1.00	5.76 3.50 3.53	4.26	18.86 19.67 21.91	20.15	75.62 72.75 95.99	81.45	8.1	7.8
0.2	1.00 1.00 1.00	1.00	2.20 1.94 1.95	2.03	2.51 2.82 2.55	2.63	6.79 6.08 6.19	6.35	8.2	7.6
0.4	1.00 1.00 1.00	1.00	1.76 1.69 1.61	1.69	2.02 1.85 1.94	1.94	3.00 2.43 2.86	2.76	8.2	7.6
0.8	1.00 1.00 1.00	1.00	1.55 1.50 1.60	1.55	1.83 1.83 1.72	1.79	2.74 2.22 2.37	2.44	8.1	7.6

Measurement done on blank control, performed for all concentrations, demonstrate the lack of interference for measurements with particles counter.

Table 3: Average specific growth rate in controls conditions (controls and solvent controls)

Test solutions (initial nominal concentrations)	Average specific growth rate (days^–1)
	0-24h	Mean	24-48h	Mean	48-72h	Mean	0-72h	Mean	sd
Control	1.569 1.732 1.437 1.573 1.292 1.209	1.469	1.714 1.434 1.359 1.383 1.362 1.917	1.528	2.043 1.698 1.525 2.075 2.050 1.960	1.892	1.775 1.621 1.441 1.677 1.568 1.696	1.630	0.116
Control with solvent (ethanol 0.01%)	1.548 1.030 1.688 1.452 1.270 1.449	1.406	1.424 1.932 1.408 1.260 1.246 1.533	1.471	1.233 1.400 1.764 1.667 1.407 1.338	1.468	1.402 1.454 1.620 1.460 1.308 1.447	1.449	0.101

Table 4 : Growth rates calculations and inhibition

Test solutions initial nominal concentrations) in mg/L	Average specific growth rate (days^–1) 0-72h	Mean	sd	Growth rate inhibition (%)
Control with solvent	1.402 1.454 1.620 1.460 1.308 1.447	1.448	0.101	/
0.050	1.592 1.537 1.392	1.507	0.103	-4
0.100	1.442 1.429 1.521	1.464	0.05	-1.1
0.200	0.638 0.602 0.608	0.616	0.02	57.5
0.400	0.366 0.296 0.350	0.337	0.037	76.7
0.800	0.336 0.266 0.288	0.296	0.036	79.5

Table 5 : Measured concentration of test item during the test

Nominal concentrations	Measured concentrations (mg/L)
Concentrated solutions prepared in ethanol analyzed at T 0h
0.50 g/L	0.51
1.00 g/L	1.03
2.00 g/L	2.00
4.00 g/L	4.34
8.00 g/L	8.83
Test concentrations
	T 0h	T 24h	T 48h	T 72h*
Control with solvent	< LOQ	< LOQ	< LOQ	< LOQ
0.050 mg/L	0.031	0.026	0.016	< LOQ
0.100 mg/L	0.066	0.063	0.045	0.005
0.200 mg/L	0.139	0.123	0.083	0.077
0.400 mg/L	0.316	0.316	0.264	0.253
0.800 mg/L	0.990	0.809	0.690	0.741

* Measured after storage, in acetonitrile, at 4°C for 48°C

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

-The biomass of controls with solvent increased by a factor of more than 16, corresponding to a specific growth rate of 1.45 day-1(mean cell density at T0h: 1 10E4 cell/.mL ; mean cell density at T72h: 80.27 10E4 cell/mL).
-The mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 and 2-3), for control with solvent, did not exceed 35% (mean coefficient of variation: 2.5%).
-The coefficient of variation of average specific growth rates, for controls with solvent, during the whole test period in replicate reached the maximum accepted value of 7%.
According to the validity criteria, the test is considered valid.

Executive summary:

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

Exponentially growing cells ofunicellular green algaePseudokirchneriella subcapitatahave been exposed to the test item at a range of concentration between 0.05 and 0.8 mg/L for a period of 72 hours, under continuous illumination. Algal cell concentrations have been determined every 24 hours and inhibition has been measured as a reduction in growth rate relative to control cultures grown under identical conditions.

Test item concentrtaion have been measured evry day during the test, and exposure concentrations have been calculated using the geometric mean of the measured values.

In these test conditions, inhibition of algal growth have been measured and an the following ErC50and a NOEC, based on measured concentrations, have been determined for the test item, after 72h :

                                     ErC50= 0.11 mg/L

                                     NOEC = 0.031 mg/L