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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was conducted with the analogue substance CAS no. 40027-38-1, which is primarily composed of C18:1 diamine and oleic acid (> 80% of the composition when accounting for those two main constituents) as well as other fatty acids, mono- and diamines with carbon chains of similar length, at significantly lower concentrations. This composition is very similar to that of the registered substance (see section 1.2, boundary composition). Therefore, the test item is considered representative of the boundary composition of the registered substance and the experimental toxicity to algae study is considered reliable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: the nominal test item loadings are 0.0800 -0.253 – 0.800 - 2.53 - 8.00 mg/L.
- Sampling method: At the start of the exposure (0 hours) and at the end (72 hours), samples of the fresh and old media respectively were taken from the tests vessels for analyses.
- Sample storage conditions before analysis:All original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

Water Accommodated Fraction (WAF)
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble and multi component test items, a modification of the standard method for the preparation of aqueous media was performed.

An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. After completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms were exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fraction
Five water accommodated fractions (WAF) were prepared with a nominal loading of the test item of 0.0800 – 0.253 – 0.800 – 2.53 – 8.00 mg/L (spacing factor v10). The loading levels are based on the results of a preliminary range finding test (non-GLP, open system).

For the loading levels of 8.00 and 2.53 mg/L an appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a brown glass flask with an appropriate amount of demineralized water. These dispersions were shaken for at least 24 hours with 20 rpm at room temperature. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, Macherey-Nagel). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following water soluble fraction (WSFs) were used in the test. During filtration, the filter was always be kept covered. The solutions were checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. The components of the dilution water were added to the WSF. The loading levels 0.0800, 0.253 and 0.800 were prepared by diluting the WAF with a loading of 2.53 mg/L with dilution water.

Test loading
Per definition of the WAF, all terms related to concentration level were given as loading level.

Control Six replicates (without test item) were tested under the same test conditions as the test item replicates.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK CCAP 278/4
- Strain: CCAP 278/4
- Source (laboratory, culture collection):
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2590 – 5180 lux corresponding to 35 - 70 µE · m-2 · s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

min.: 22.0 max.: 24.0 mean value: 23.0

pH:

Please see below the pH values in the table on materials and methods part.

Salinity:

Not applicable (freshwater conditions)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 6442 cells/mL
- Control end cells density: Mean 1759810 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Composition of Dilution Water according to the guideline OECD 201 (see below the details of the component concentrations in mg/L)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE · m-2 · s-1, mean value: 6031 lux,measured: 5053 - 6234 w,ith a mean of 5560 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the definitive test in the saturated solution.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.232 mg/L

95% CI:

>= 0.099 - <= 0.638

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.276 mg/L

95% CI:

>= 0.116 - <= 0.669

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.1 mg/L

95% CI:

0.08 - <= 0.236

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.197 mg/L

95% CI:

0.091 - <= 0.247

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.005 mg/L

95% CI:

0.003 - 0.016

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.006 mg/L

95% CI:

>= 0.003 - <= 0.017

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.003 mg/L

95% CI:

>= 0.003 - <= 0.006

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.005 mg/L

95% CI:

>= 0.003 - <= 0.006

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test media were clear throughout the test period.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid : Yes
The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2018-09-25 to 2018-09-28. The reference item toxicity is in the valid range following test facility SOPS.
- EC50: Values of the Reference Item based on nominal concentrations, (0-72 hours) :
Growth Rate Inhibition : ErC50 = 0,799 mg/L (95% CI : 0.707 – 1.30); Valid Range : 0,753 mg/L +/- 0,544
Yield Inhibition : EyC50 = 0,607 mg/L (95% CI : 0,404 - 0694); Valid Range : 0,407 mg/L +/- 0,313

Reported statistics and error estimates:

EL10-, EL20- and EL50-values (based on nominal loadings) and EC10-, EC20- and EC50-values (based on geometric mean measured test item concentration) with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEL, LOEL and statistical analyses
The NOEL and LOEL were determined by calculation of statistically significant differences of growth rates and yield.
The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was calculated with a significance level of 0.01.

Levene’s test on variance homogeneity was calculated with a significance level of 0.01.

Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm was done for growth rate with a significance level of 0.05.

Monotonicity of Concentration/Response was calculated by trend analysis by contrasts (significance level 0.05) for yield.

Williams Multiple Sequential t-test Procedure was performed for yield with a significance level of 0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.


Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.
• ToxRat Version 3.2.1, ToxRat Solutions Gmb

Cell Densities

Replicate   No.	Cell density [cells/mL]
	0 hours	24 hours	48 hours	72 hours
1	6442	<LOQCell density	<LOQCell density	<LOQCell density
2	6442	<LOQCell density	<LOQCell density	<LOQCell density
3	6442	<LOQCell density	<LOQCell density	<LOQCell density
Mean	6442	n.a.	n.a.	n.a.
1	6442	<LOQCell density	<LOQCell density	<LOQCell density
2	6442	<LOQCell density	<LOQCell density	<LOQCell density
3	6442	<LOQCell density	<LOQCell density	<LOQCell density
Mean	6442	n.a.	n.a.	n.a.
1	6442	<LOQCell density	<LOQCell density	<LOQCell density
2	6442	<LOQCell density	<LOQCell density	<LOQCell density
3	6442	<LOQCell density	<LOQCell density	<LOQCell density
Mean	6442	n.a.	n.a.	n.a.
1	6442	6703	30197	253967
2	6442	8671	75180	569021
3	6442	8912	53473	566961
Mean	6442	8095	52950	463316
1	6442	17968	223234	1464633
2	6442	23797	245556	1511453
3	6442	24244	122146	1907411
Mean	6442	22003	196979	1627832
1	6442	23378	299903	1875933
2	6442	26888	304858	2041367
3	6442	27481	249806	2054673
4	6442	24969	255576	1205609
5	6442	26262	290996	1607327
6	6442	28062	260332	1773952
Mean	6442	26173	276912	1759810

LOQ_celldensity= 2653 N/mL                    n.a. = not available

Evaluation after 72 hours   

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item loading (mg/L)	No.	[d-1]		[%]	[cells/mL]		[%]
	1	n.a.		100	n.a.		100
8	2	n.a.		100	n.a.		100
	3	n.a.		100	n.a.		100
	Mean	(+)	n.a.	100	(+)	n.a.	100
	1	n.a.		100	n.a.		100
2.53	2	n.a.		100	n.a.		100
	3	n.a.		100	n.a.		100
	Mean	(+)	n.a.	100	(+)	n.a.	100
	1	n.a.		100	n.a.		100
0.8	2	n.a.		100	n.a.		100
	3	n.a.		100	n.a.		100
	Mean	(+)	n.a.	100	(+)	n.a.	100
	1	1.23		34	247525		86
0.253	2	1.49		20	562579		68
	3	1.49		20	560519		68
	Mean	(+)	1.40	25	(+)	456874	74
	1	1.81		3	1458191		17
0.08	2	1.82		2	1505011		14
	3	1.90		-2	1900969		-8
	Mean	(-)	1.84	1	(-)	1621390	8
	1	1.89			1869491
	2	1.92			2034925
	3	1.92			2048231
Control	4	1.74			1199167
	5	1.84			1600885
	6	1.87			1767510
	Mean	1.87			1753368

 

Section-by-Section and Average Specific Growth Rates of the Control Group (0 – 72 hours)

	No.	0 - 24 hours	24 - 48 hours	48 – 72 hours	Mean (0-72 hours)	SD ±	CV [%]	Mean CV (%)
Control	1	1.29	2.55	1.83	1.89	0.633	33.5	26.4
	2	1.43	2.43	1.90	1.92	0.500	26.0
	3	1.45	2.21	2.11	1.92	0.411	21.4
	4	1.36	2.33	1.55	1.74	0.513	29.4
	5	1.41	2.41	1.71	1.84	0.513	27.9
	6	1.47	2.23	1.92	1.87	0.380	20.3
				Mean	1.87
				SD ±	0.07
				CV [%]	3.57

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values based on nominal test item loadings and the geometric mean measured test item concentrations:
The EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 0.276 (0.116 – 0.669) mg/L and 0.197 (0.0908 – 0.247) mg/L, respectively. The EL10-values for inhibition of growth rate (ErL10) and yield (EyL10) after 72 hours were 0.232 (0.0993 – 0.638) mg/L and 0.0995 (< 0.0800 – 0.236) mg/L, respectively. The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.00644 (0.00268 – 0.0166) mg/L and 0.00473 (0.00258 – 0.00583) mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.00539 (0.00254 – 0.0158) mg/L and 0.00278 (< 0.00250 – 0.00571) mg/L, respectively.

Executive summary:

The toxicity of the test item to freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 C.3. The aim of the study was the determination of NOEL, LOEL, EL10, EL20- and EL50-values and NOEC, LOEC, EC10, EC20- and EC50-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6442 cells/mL. As the test item is poorly water soluble substance, five water accommodated fractions (WAF) with the nominal test item loadings 0.0800 -0.253 – 0.800 - 2.53 - 8.00 mg/L (factor√10) were prepared as described in section 4.2.Three replicates were tested for each test item loading and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were clear throughout the test period. The concentrations of the test item in all test concentration levels and the control were analytically verified by LC-MS/MS at the start and at the end of exposure.

The measured concentrations of the test item were between 1 and 6% of the nominal value at the start of the exposure (0 hours) and between 1 and 2% of the nominal value at the end of the exposure (72 hours).

Therefore, all effect values given are based on the nominal test item loadings.

 

NOEL, LOEL, ELx-values and 95% Confidence Intervals (0 - 72 hours)

based on nominal test item loadings [mg/L]

	Growth Rate Inhibition Nominal test item loading [mg/L]
NOEL	0.0800
LOEL	0.253
ErL10	0.232 (0.0993 – 0.638)
ErL20	0.248 (0.107 – 0.644)
ErL50	0.276 (0.116 – 0.669)
	Yield Inhibition Nominal test item loading [mg/L]
NOEL	0.0800
LOEL	0.253
ErL10	0.0995 (< 0.0800 – 0.236)
ErL20	0.136 (0.0823 – 0.239)
ErL50	0.197 (0.0908 – 0.247)

NOEC, LOEC, ECx-values and 95% Confidence Intervals (0 - 72 hours)

based on geometric mean measured test item concentration [mg/L

 

	Growth Rate Inhibition Geometric mean measured test item concentration [mg/L]
NOEC	0.00250
LOEC	0.00590
ErC10	0.00539 (0.00254 – 0.0158)
ErC20	0.00577 (0.00258 – 0.0160)
ErC50	0.00644 (0.00268 – 0.0166)

	Yield Inhibition
	Geometric mean measured test item concentration [mg/L]
NOEC	0.00250
LOEC	0.00590
ErC10	0.00278 (< 0.00250 – 0.00571)
ErC20	0.00346 (0.00252 – 0.00574)
ErC50	0.00473 (0.00258 – 0.00583)

 

 

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-07-16 to 2012-07-23

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The study was conducted with river water.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Nominal concentrations: 0.0100 - 0.0316 - 0.100 - 0.316 - 1.00 mg/L
- Sampling method: Each test item concentration and the control were analytically verified at the beginning and the end of the test via LC-MS/MS analysis. At test start and end samples were taken out of additional replicates provided without algae. Sorption of the test item to the glassware of the test vessel was determined in one replicate of a concentration level close to the 72h-ErC10.
- Sample storage conditions before analysis: All original samples were stored at room temperature until sample preparation, if necessary, prepared samples were stored at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1.00 g/L of test item was freshly prepared with distilled water with a pH of 2. The stock solution was stirred at 400 rpm for 24 h at 50 °C. Thereafter the stock solution was mixed with an ultraturrax (1 min, 17000 rpm). The stock solution was a homogenous and turbid dispersion.
- Eluate: Distilled water
- Differential loading: 0.0100 - 0.0316 - 0.100 - 0.316 - 1.00 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDAK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

After 72 hours algae were transferred from the highest dosage level (nominal test item concentration 1.00 mg/L) and the control into fresh untreated medium. 1.0 and 0.02 mL algae suspension from each test replicate and from two control replicates were filled up with dilution water to 10 mL and allowed to grow for further 3 days to determine whether the effect of the test item was reversible, respectively.

Hardness:

Not measured

Test temperature:

Mean: 21.8 °C, Min: 21.5 °C, Max: 22.0 °C

pH:

Nominal concentration pH-value
[mg test item/L] Start; 0 hours End; 72 hours
1.00 8.01 8.02
0.316 8.01 8.21
0.100 8.03 8.22
0.0316 8.02 8.22
0.0100 8.02 8.23
Control 7.98 8.23

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information on materials and methods

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 8446 cells/mL
- Control end cells density: Mean 990926 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS

Composition of Dilution water (OECD)
Component Concentration [mg/L]
NH4Cl 15
MgCl2 ¿ 6 H2O 12
CaCl2 ¿ 2 H2O 18
MgSO4 ¿ 7 H2O 15
KH2PO4 1.6
FeCl3 ¿ 6 H2O 0.064
Na2EDTA ¿ 2 H2O 0.1
H3BO3 0.185
MnCl2 ¿ 4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4 ¿ 2 H2O 7 x 10-3
CoCl2 ¿ 6 H2O 1.5 x 10-3
CuCl2 ¿ 2 H2O 1 x 10-5
NaHCO3 50
pH-value 8.1 ¿ 0.2

This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.

Test medium: Water of the river Innerste was used. This river is located near D-31157 Ruthe-Sarstedt, Germany (Latitude 52.25°, Longitude 9.83°). Water parameters are given. Additionally 50 % of the components concentrations of the dilution water (total application volume 6.5 mL/L) acc. to the guideline was added to enable a sufficient growth of algae.
Water Parameters of the River Water

River Innerste
Location Amtshof K514, km 26,
31157 Ruthe-Sarstedt, Germany
Latitude:52.25°, Longitude: 9.83°
Sampling Date 2012-03-20
Weather on Day of Sampling Sunny, slightly clouded; approx. 15°C
Colour Yellowish to brownish, clear
pH 7.93
Conductivity [µS/cm] 856
Dissolved Oxygen [mg O2/L] 11.2
DOC [mg C/L] 2.80*
TOC [mg C/L] 3.66*
Ammonium-N [mg N/L] 0.07
Nitrate-N [mg N/L] 3.40
Total Nitrogen [mg N/L] 3.80
o-Phosphate-p [mg P/L] < 0.20
Total Phosphate [mg P/L] < 0.20
Suspended Matter [mg/L] 14.0
Total Hardness [mg CO3/L] 271
Storage conditions 15 ± 2°C


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.

TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0 – 72 hours)

Nominal
Test Item Concentration
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
10.0 100 100
1.00 100 100
0.1 3 13
0.01 -2 -10
0.001 -1 -6

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.323 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

99.32% active ingredient

Basis for effect:

growth rate

Remarks on result:

other: (0.213 - 0.663)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

99.32% active ingredient

Basis for effect:

growth rate

Remarks on result:

other: (0.328 - 0.715)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.718 (CI 0.699 - 0.737) mg/L
Yield Inhibition: 0.422 (CI 0.386 - 0.466) mg/L

Reported statistics and error estimates:

NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEL/LOEL calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The ¿-value (acceptable probability of incorrectly concluding that there is a difference) is ¿=0.05.
Detection of statistically significant differences was considered negligible when inhibition values are = 10 %. These findings were marked “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant.

NOEC, LOEC, EC-Values and 95 % Confidence Intervals of

                   N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate](0-72 hours)

                   based on nominal test item concentrations [mg/L]

	Inhibition of Growth Rate
NOEC	0.316
LOEC	1.00
ErC10	0.323 (0.213 - 0.663)
ErC20	0.353 (0.320 - 0.673)
ErC50	0.410 (0.328 - 0.715)
	Inhibition of Yield
NOEC	0.0100
LOEC	0.0316
EyC10	0.0624 ( 0.0100 - 0.113)
EyC20	0.139 (0.0889 - 0.213)
EyC50	0.423 (0.326 - 0.521)

Cell Densities

Nominal concentration	Replicate	Cell density [cells/mL]
[mg test item/L]	No.	0 hours	24 hours	48 hours	72 hours
1.00	1	5724	2854	LOQ	3420*
	2	5724	LOQ	LOQ	4560*
	3	5724	2962	LOQ	4940*
	Mean	5724	n.a.	n.a.	4307*
0.316	1	5724	16265	122100	654281
	2	5724	16868	154911	672919
	3	5724	21920	185981	706955
	Mean	5724	18351	154331	678052
0.100	1	5724	30816	329104	998896
	2	5724	23856	245805	760231
	3	5724	31562	281447	899614
	Mean	5724	28745	285452	886247
0.0316	1	5724	26720	300491	865482
	2	5724	33350	309883	886818
	3	5724	25761	238007	901011
	Mean	5724	28610	282794	884437
0.0100	1	5724	30638	335568	1130246
	2	5724	28917	330742	1176537
	3	5724	29949	307374	1095670
	Mean	5724	29835	324561	1134151
Control	1	5724	27549	252364	1041314
	2	5724	25822	288890	1134278
	3	5724	33940	350021	1212573
	4	5724	27549	269909	947239
	5	5724	33308	306181	934443
	6	5724	27365	250009	1052109
	Mean	5724	29256	286229	1053659

Evaluation after 72 hours

              Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-)

 

Nominal concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg test item/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		-0.172	100		-2304	100
	2		-0.076	100		-1164	100
	3		-0.049	100		-784	100
	Mean	(+)	-0.099	100	(+)	-1417	100
0.316	1		1.58	9.06		648557	38.1
	2		1.59	8.52		667195	36.3
	3		1.61	7.58		701231	33.1
	Mean	(+)*	1.59	8.39	(+)	672328	35.8
0.100	1		1.72	0.94		993172	5.23
	2		1.63	6.18		754507	28.0
	3		1.69	2.95		893890	14.7
	Mean	(+)*	1.68	3.36	(+)	880523	16.0
0.0316	1		1.67	3.69		859758	18.0
	2		1.68	3.23		881094	15.9
	3		1.69	2.92		895287	14.6
	Mean	(+)*	1.68	3.28	(+)	878713	16.2
0.0100	1		1.76	-1.43		1124522	-7.31
	2		1.78	-2.20		1170813	-11.7
	3		1.75	-0.83		1089946	-4.01
	Mean	(-)	1.76	-1.49	(-)	1128427	-7.68
Control	1		1.74			1035590
	2		1.76			1128554
	3		1.79			1206849
	4		1.70			941515
	5		1.70			928719
	6		1.74			1046385
	Mean		1.74			1047935

Section-by-Section and Average Specific Growth Rates of the Control Group

                   (0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.57	2.22	1.42	1.74	0.423	24.4	29.1
	2	1.51	2.42	1.37	1.76	0.569	32.3
	3	1.78	2.33	1.24	1.79	0.546	30.6
	4	1.57	2.28	1.26	1.70	0.526	30.9
	5	1.76	2.22	1.12	1.70	0.554	32.6
	6	1.57	2.21	1.44	1.74	0.416	23.9
				Mean	1.74
				SD ±	0.03
				CV [%]	1.94

Validity criteria fulfilled:

yes

Conclusions:

In this study N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (nominal concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.410 (0.328 – 0.715) mg test item/L and 0.423 (0.326 – 0.521) mg test item/L, respectively. The EC10-values after 72 hours for inhibition of growth rate and inhibition of yield were 0.323 (0.213 – 0.663) mg test item/L and 0.0624 (< 0.0100 – 0.113) mg test item/L, respectively. All effect values are given based on the nominal concentrations of N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate].

Executive summary:

The toxicity of N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] (Batch no.: 30411) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The test period was between2012-07-16 and with the definitive exposure phase from to at Dr.U.Noack-Laboratorienin 31157 , . The aim of the study was the determination of NOEC, LOEC, EC10-, EC20 - and EC50 - values of growth rate and yield in natural river water over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5724 cells/mL. Five concentration levels were tested in a geometrical series with a dilution factor of (nominal): 0.0100 - 0.0316 - 0.100 - 0.316 - 1.00 mg/L. Three replicates were tested for each concentration level and six replicates for the control. Environmental conditions (pH and room temperature) were determined to be within the acceptable limits.

The tested concentration levels of the test item N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminiumdi[(9Z)-octadec-9-enoate] (CAS No. 34140-91-5) and the control were analytically verified by LC-MS/MS of samples on days 0 (fresh media, 0 hours) and day 3 (old media, 72 hours).

In the fresh media (0 h), the measured concentrations of the test item N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] (CAS No. 34140-91-5) were in the range of 74 – 91 % of the nominal values. The recoveries in the old media were in the range of M- 18 % (72 hours) of the nominal values.
Additionally, the adsorption to the glassware in a test vessel which contained algae at the nominal test concentration level of 0.316 was determined to be 2 %.

The inhibition values are given in Table 1. All effect values are given based on the nominal concentrations of N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminiumdi[(9Z)-octadec-9-enoate] (CAS No. 34140-91-5).

 

 

NOEC, LOEC, EC-Values and 95 % Confidence Intervals of

                   N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate](0-72 hours)

                   based on nominal test item concentrations [mg/L]

 

	Inhibition of Growth Rate
NOEC	0.316
LOEC	1.00
ErC10	0.323 (0.213 - 0.663)
ErC20	0.353 (0.320 - 0.673)
ErC50	0.410 (0.328 - 0.715)
	Inhibition of Yield
NOEC	0.0100
LOEC	0.0316
EyC10	0.0624 ( 0.0100 - 0.113)
EyC20	0.139 (0.0889 - 0.213)
EyC50	0.423 (0.326 - 0.521)

Description of key information

The 72h-EC10 and 72h-EC50 for growth rate are 0.323 and 0.41 mg/L respectvely in an OECD guideline 201 test in natural river water with Pseudokirchneriella subcapitata under GLP (reliability 1).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.41 mg/L

EC10 or NOEC for freshwater algae:

0.323 mg/L

Additional information

The toxicity of N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The test duration was 72 hours, test was performed in natural river water. Nominal test concentrations were: 0.0100 - 0.0316 - 0.100 - 0.316 - 1.00 mg/L. The measured concentrations of the test item were in the range of 74 – 91 % of the nominal values. Based on nominal concentrations the 72h-EC50 and 72h-EC10 for growth rate were found to be 0.41 and 0.323 mg/L respectively.

 

Additionnaly, a GLP study according to the TG OECD 201 with a similar substance is available to assess the inhibition of the growth of the freshwater green alga Pseudokirchneriella subcapitata in a standard medium. Due to the complex structure of the test item, and the very low solubility the WSF approach was used for ecotoxicity testing. The following effect values based on nominal test item loadings were derived: the EL50 -values for inhibition of the growth rate (ErL50) and yield (EyL50) after 72 hours were 0.276 (0.116 – 0.669) mg/L and 0.197 (0.0908 – 0.247) mg/L, respectively. The EL10 -values for inhibition of growth rate (ErL10) and yield (EyL10) after 72 hours were 0.232 (0.0993 – 0.638) mg/L and 0.0995 (< 0.0800 – 0.236) mg/L, respectively.

 

According REACH Annex IX, both testing proposals have been submited for chronic aquatic toxicity on fish and daphnia (TG OECD 210 and 211). Those test will be performed under a standard medium on the registered substance. When the full dataset in standard medium will be available, the Algae key value for chemical safety assessment will be re-assessed.