fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2022-04-04 to 2022-04-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2016)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide

Batch number LAB AA101.20 Grobgut

Purity (certified) 100% (UVCB)

Chemical composition Disodium 2- (dodecanoylamino)pentanedioate (main component)

Appearance Colorless powder (solid)

Water Solubility 7.8 g/L

Stability under test
conditions Not specified

Expiry date 2023-08-23

Recommended storage Room temperature

Analytical monitoring:

yes

Remarks:

via LC-MS/MS

Details on sampling:

Sampling schedule
All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Sampling and pre-treatment
At the start, samples were taken from one additional replicate of each test item concentration and the control and at the end samples were taken from pooled replicates.

Vehicle:

no

Details on test solutions:

Stock solution, media preparation
100 mg test item/L was freshly prepared with dilution water. The dispersion was agitated until the solution was visually clear. The Tyndall effect was negative.

Preparation of the test item solutions
Out of the stock solution 6 concentrations were tested in a geometric series with a dilution factor of √10: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg test item/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP).

Test organisms (species):

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

Details on test organisms:

Test organism Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)

Synonyms
Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)

Reason for the selection of the test organism
Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.

Origin
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

Cultivation at test facility
Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.

Culture medium Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Remarks:

Dilution water (OECD TG 201 medium)

Limit test:

no

Total exposure duration:

72 h

Hardness:

see section "Any other information on results incl. tables" below.

Test temperature:

see section "Any other information on results incl. tables" below.

pH:

see section "Any other information on results incl. tables" below.

Nominal and measured concentrations:

Out of the stock solution 6 concentrations were tested in a geometric series with a dilution factor of √10: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg test item/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP).

The measured concentrations of N-Lauroyl-L-glutamic acid in the fresh media were in the range of 86 to 96% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of 109 to 120% of the nominal test item concentrations.

Details on test conditions:

Control
Six replicates were exposed under the same conditions as the test concentrations.

Reference Item
Potassium dichromate was tested as a reference item (for results of the most recent study, please refer to "Any other information on results incl. tables" ).

Test method Static procedure

Duration of the test 72 hours

Replicates Six replicates for the control, three replicates per concentration level

Test container Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.

Test volume 100 mL

Ultrapure water Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 µS/cm).

Preculture
A three days old preculture, prepared in dilution water, was used as inoculum.

Initial cell density
Nominal: approximately 5 x 103 - 104 cells/mL
Actual: 6864 cells/mL

Application
Application was carried out by adding an appropriate amount of algae inoculum to the test solutions.

Incubation The flasks were positioned randomly and repositioned daily.

Temperature
Nominal range: 21 - 24 °C, controlled at ± 2 °C

Agitation
Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

Light intensity (target) Approximately 4440 to 8880 lux

Light regime 24 hours/day light

Light homogeneity Within ± 15% over incubation area
 
Type and Frequency of Measurements
Biological Parameters

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 observed in the preliminary range finding test at the concentration of 100 mg/L.

Microscopic evaluation
The algae cells were evaluated microscopically at the start and the end of the incubation period. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

Physico-chemical Properties

The pH-value at the start of the exposure was measured in one additional replicate of each test item concentration and the control. At the end of the exposure, it was measured in a pooled sample of the test item concentrations and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (SIGMA ALDRICH, batch number BCCC1619, purity 100.0%, CAS RN 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

80.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: Confidence Intervals 61.2 - > 100 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

16.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

51.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Biological Data

Microscopic evaluation of the cells at the start and the end of exposure revealed no morphological abnormalities for all test item concentration levels and control. All effect values given are based on the nominal test item concentration of fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide.

Physico-chemical Data

The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test media were clear by eye throughout exposure period (possible turbidity related to algae growth not taken into account).

Measured Exposure Concentrations during the Definitive Test

The concentrations of N-Lauroyl-L-glutamic acid, component of the test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide, and the control were determined in fresh media (0 hours) and old media (72 hours) of all tested concentration levels and the control via LC-MS/MS.

The measured concentrations of N-Lauroyl-L-glutamic acid in the fresh media were in the range of 86 to 96% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of 109 to 120% of the nominal test item concentrations.

Results with reference substance (positive control):

Test of the Reference Item

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number BCCC1619, purity 100.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2021-11-01 to 2021-11-04. The reference item toxicity is in the valid range which was established by calculation of the average of the historic reference data since 2006, and the limits were set using the threefold standard deviation of these values.
For details see "Any other information on results incl. tables" below.

Reported statistics and error estimates:

EC-values and statistical analyses
EC10-,EC20- and EC50- values with confidence intervals of growth rate inhibition and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression with the GraphPad Prism Software.

NOEC, LOEC and analyses
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield.
The following statistical tests were conducted:

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

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

Monotonicity of Concentration/Response was tested by trend analysis by contrast with a significance level of 0.05 for growth rate.

Step-down Jonckheere-Terpstra Test Procedure was performed with a significance level of 0.05 for growth rate.

Dunnett’s multiple -t-test was performed with a significance level of 0.05 for yield.

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 the following software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.
• ToxRat Version 3.3.0, ToxRat Solutions GmbH

Cell Densities

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
100	1	6864	16516	79223	593052
	2	6864	15079	85835	432091
	3	6864	15493	89748	749312
	Mean	6864	15696	84935	591485
31.6	1	6864	18513	127298	920979
	2	6864	20603	127639	1022229
	3	6864	19870	123654	1048182
	Mean	6864	19662	126197	997130
10.0	1	6864	23819	157110	1129776
	2	6864	24458	156454	1146823
	3	6864	22948	160142	1256350
	Mean	6864	23742	157902	1177650
3.16	1	6864	20839	161500	1190814
	2	6864	21696	151317	1123793
	3	6864	24991	153224	1279559
	Mean	6864	22509	155347	1198055
1.00	1	6864	28391	200498	1372893
	2	6864	27863	205842	1363042
	3	6864	21855	163048	1471264
	Mean	6864	26036	189796	1402400
0.316	1	6864	27321	155005	1291299
	2	6864	22998	157371	1205207
	3	6864	25301	163929	1375547
	Mean	6864	25207	158768	1290684
Control	1	6864	29932	189793	1389041
	2	6864	28225	192096	1333940
	3	6864	24635	171090	1391874
	4	6864	28184	185862	1322470
	5	6864	28301	202549	1390750
	6	6864	22170	178305	1343701
	Mean	6864	26908	186616	1361963

 

Evaluation after 72 hours
Statistically significant differences of growth rates and yield compared to
control values are marked (s), not significant differences are marked (ns).

Nominal test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.49	16		586188	57
	2		1.38	22		425227	69
	3		1.56	11		742448	45
	Mean	(s)	1.48	16	(s)	584621	57
31.6	1		1.63	7		914115	33
	2		1.67	5		1015365	25
	3		1.68	5		1041318	23
	Mean	(s)	1.66	6	(s)	990266	27
10.0	1		1.70	4		1122912	17
	2		1.71	3		1139959	16
	3		1.74	2		1249486	8
	Mean	(s)*	1.71	3	(s)	1170786	14
3.16	1		1.72	3		1183950	13
	2		1.70	4		1116929	18
	3		1.74	1		1272695	6
	Mean	(s)*	1.72	2	(s)	1191191	12
1.00	1		1.77	0		1366029	-1
	2		1.76	0		1356178	0
	3		1.79	-1		1464400	-8
	Mean	(ns)	1.77	-1	(ns)	1395536	-3
0.316	1		1.75	1		1284435	5
	2		1.72	2		1198343	12
	3		1.77	0		1368683	-1
	Mean	(ns)	1.75	1	(ns)	1283820	5
Control	1		1.77			1382177
	2		1.76			1327076
	3		1.77			1385010
	4		1.75			1315606
	5		1.77			1383886
	6		1.76			1336837
	Mean		1.76			1355099

* = statistical significant, but biologically not relevant (Inhibition < 5%)

 

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

	Replicate No.	Specific Growth Rate [d^-1]			Mean	SD	CV	Mean CV [%]
		section-by-section			(0 - 72 hours)	±	[%]
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.47	1.85	1.99	1.77	0.267	15.1	20.0
	2	1.41	1.92	1.94	1.76	0.297	16.9
	3	1.28	1.94	2.10	1.77	0.434	24.5
	4	1.41	1.89	1.96	1.75	0.298	17.0
	5	1.42	1.97	1.93	1.77	0.307	17.3
	6	1.17	2.08	2.02	1.76	0.509	28.9
				Mean	1.76
				SD ±	0.01
				CV [%]	0.45

SD = Standard deviation CV = Coefficient of variation

 

Environmental Conditions

Nominal test item concentration				pH-values
[mg/L]				Start; 0 hours	End; 72 hours
100				7.67	8.74
31.6				7.61	9.00
10.0				7.55	9.24
3.16				7.54	9.28
1.00				7.58	9.26
0.316				7.58	9.29
Control				7.85	9.22
Room temperature [°C]:		min.: 22.0		max.: 23.0		mean value: 22.5
	n = 19		mean value: 5853				CV [%]: 8.09
Light intensity			range of the measured values: 5090 - 6586
[lux]			equalling -13.0 to 12.5

CV = Coefficient of variation n = number of measuring points

 

Water parameters of the Dilution Water

Parameters of the dilution water (measured on 2022-03-22)
Conductivity	Total hardness	Acidity	Alkalinity	Total organic carbon
[µS/cm]	[mg CaCO3/L]	[mmol/L]	[mmol/L]	[mg C/L]
137	34	0.1	0.6	< LOQ*

*Limit of quantification = 2.00 mg C/L

 

Measured Concentrations of N-Lauroyl-L-glutamic acid during the Definitive Test

Sampling date		Fresh media,		Old media,
		0 hours		72 hours
Nominal		N-Lauroyl-L-glutamic acid
Concentration of
the test item	the active substance	Meas. conc.	[%]	Meas. conc.	[%]
[mg/L]	[mg a.s./L]	[mg/L]		[mg/L]
100	58.1	50.4	87	68.1	117
31.6	18.4	16.4	89	21.5	117
10.0	5.81	5.01	86	6.93	119
3.16	1.84	1.72	93	2.00	109
1.00	0.581	0.527	91	0.654	112
0.316	0.184	0.177	96	0.221	120
Control		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percentage of the nominal concentration of the test item
LOQ = limit of quantification (0.100 mg test item/L, corresponding to 0.0581 mg a.s./L)

 

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)

	Current Study	Valid Range (average ± 3 x SD)
	Growth Rate inhibition
ErC50	1.09	0.787 ± 0.566
95% confidence interval	0.944 – 1.25
	Yield inhibition
EyC50	0.643	0.429 ± 0.348
95% confidence interval	0.581 – 0.712

SD = Standard deviation

Validity Criteria

Validity Criterion	Required	This study
Increase of the cell growth in the control cultures	Exponentially, ≥ 16-fold corresponding to a specific growth rate of 0.92 day^-1	198-fold
		(specific growth rate 1.76 day^-1)
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures	≤ 35%	20.0%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤ 7%	0.45%

 

Gradient Table

Time [min]	A [%]	B [%]
0.00	70	30
0.30	70	30
0.80	5	95
2.00	5	95
2.10	70	30
2.50	70	30

 

Dilution Steps

Nominal	Dilution	Sample	Final
test item concentration	Factor	volume	volume
[mg/L]		[mL]	[mL]
100	20000	0.05^1)	5.0^1)
		0.05^2)	5.0^2)
31.6	5000	0.05^1)	5.0^1)
		0.2^2)	5.0^2)
10.0	2000	0.05^1)	5.0^1)
		0.1^2)	1.0^2)
3.16	500	0.05^1)	5.0^1)
		0.4^2)	1.0^2)
1.00	200	0.05^1)	5.0^1)
0.316	50	0.2^1)	5.0^1)
Control	20	0.1^1)	1.0^1)

* including factor 2
1) first dilution step 2) second dilution step

 

Parameter, Acceptance Criteria and Results of the Method Validation

Parameter	Acceptance criteria	Result
Calibration	≥ 5 standard concentrations,	1.0 to 10 µg standard/L (n = 6),	ü
	r ≥ 0.99 (R^2≥ 0.9801 for 2^nd order regression)	r ≥ 0.99
	Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation
Matrix effects	Should be ≤ 20%	Matrix effects ≤ 20%	ü
Lowest calibration level (LCL)	S/N ≥ 9 for quantifier ion trace	S/N for 1.0 µg standard/L	ü
	S/N ≥ 3 for qualifier ion trace	2262 (Quantifier), 208 (Qualifier)
Limit of Quantification (LOQ)	Should be at or below lowest nominal test concentration	0.100 mg test item/L (1 x LOQ)	ü
		120 mg test item/L (1200 x LOQ)
Accuracy (Fortified samples)	Mean recovery rate of 70-120%	1 x LOQ: 99% (n = 5)	ü
	per fortification level (2 levels)	1200 x LOQ: 94% (n = 5)
Precision	Relative standard deviation ≤ 20% per fortification level	1 x LOQ: 4.6%	ü
		1200 x LOQ: 9.4%
Stability	Samples: Stable if 70 – 120% of nominal concentration	See section 18.1	ü
	Calibration standards: Stable if ±10% of freshly prepared standard
Specificity: LC-MS/(MS)	Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity).	quantifier [m/z]: 330.13 > 148.00	ü
		qualifier [m/z]: 330.13 > 129.98
	Blank values < 30% of the LOQ	Blank values < 30% of LOQ	ü
Procedural recovery	Procedural recoveries with experimental samples. The recovery has to be 70-120% of the nominal value.	See section 18.2	ü

 

Preparation of Fortified Samples

LOQ Level	Control	1	1200
Stock solution	-	6000	6000
[mg test item/L]		(Dilution medium 1)	(Dilution medium 1)
(Medium)
Spiking solution	-	10	Stock solution
[mg test item/L]		(Algae dilution water)
(Medium)
Replicates	2	5	5
Concentration of the LOQ	-	0.100	120
[mg test item/L]
Medium for preparation	Algae dilution water
Volume of spiking solution [mL]	-	0.05	0.1
Volume of medium [mL]	5.0	4.95	4.90
Dilution factor	20	20	20000
Dilution medium	Acetonitrile containing 2% formic acid^1)
	Dilution medium 2^2)3)
Sample volume [mL]	5^1)	5^1)	5^1)
	0.1^2)	0.1^2)	0.1^2)
			0.1^3)
Finale volume [mL]	10^1)	10^1)	10^1)
	1.0^2)	1.0^2)	10^2)
			10^3)
Final expected concentrations for analyses [µg/L]	-	5.0	6.0

Dilution medium 1: Acetonitrile : algae dilution water (50 : 50) containing 1% formic acid
Dilution medium 2: Acetonitrile : ultra-pure water (50 : 50) containing 1% formic acid

1) First dilution step
2) Second dilution step
3) Third dilution step

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of N-Lauroyl-L-glutamic acid, component of the test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide
Fortified concentrations*: 0.101 mg test item/L (1 x LOQ) and 121 mg test item/L (1200 x LOQ), corresponding to 0.0584 mg a.s./L (1 x LOQ) and 70.1 mg a.s./L (1200 x LOQ).

Replicate	N-Lauroyl-L-glutamic acid
	1 x LOQ		1200 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[mg/L]		[mg/L]
1	0.0568	97	75.4	108
2	0.0585	100	63.9	91
3	0.0620	106	68.5	98
4	0.0547	94	59.5	85
5	0.0586	100	62.6	89
Mean	0.0581	99	66.0	94
SD ±	0.003		6.2
CV [%]	4.6		9.4

Meas. conc. = Measured concentration of the test item, dilution factor taken into account
% = Percent concentration of the fortified sample
* = Weighing factor taken into account
a.s. = active substance
SD = Standard deviation
CV = Coefficient of Variation

 

Stability

The stability of the fortified samples was checked after 7 days and was 116% of the nominal concentration. Samples were found to be stable over this period.

The stability of the stock solution (100 mg standard/L in methanol) was checked after 33 days and was 102 and 106% of the nominal concentration. The stock solution was found to be stable over this period.

The responses of a 3 days old calibration standard (6 µg test item/L) was compared with the responses of a freshly prepared calibration standard with the same concentration. The deviation was < 10% and was found to be stable over this period.

 

 

Procedural Recovery

A procedural recovery (Quality Control) was freshly prepared on each day of analysis. It was treated in parallel to the test samples. For results, see Table 16.

Measured Concentrations and Percent of Nominal Concentration of the Quality Control during the Definitive Test

Sampling	0 hours		72 hours		72 hours^1)
date
Quality Control	N-Lauroyl-L-glutamic acid
Test item	Meas.	%	Meas.	%	Meas.	%
[mg/L]	conc.		conc.		conc.
	[mg a.s./L]		[mg a.s./L]		[mg a.s./L]
1.00	0.0512	88	0.0629	108	0.0579	99

Meas. conc. = measured concentration of the test item, dilution and weighing factors taken into account
% = percent of the nominal concentration
a.s. = active substance
1) = reanalyzed samples

 

Preliminary Test (non-GLP)

A non-GLP preliminary range finding test under static conditions over a period of 72 hours was conducted at the test facility. A stock solution with a concentration of 100 mg test item/L was freshly prepared with dilution water and agitated until the solution was visually clear. Out of this solution two concentration levels were prepared (by dilution) with the concentrations 1.00 and 10.0 mg/L.
In the range finding test, two replicates per concentration and four for the control were tested.

Results of the Range Finding Test (0 - 72 hours)

Nominal test item concentration	Growth Rate Inhibition	Yield Inhibition
[mg/L]	[%]	[%]
100	19	62
10.0	2	11
1.00	2	9

Measured Exposure Concentrations during the non-GLP Preliminary Range Finding Test
Determination of the test item via LC-MS

Sampling		0 hours		72 hours
		Start of the exposure		End of the exposure
		(with algae)		(with algae)
Nominal concentration of the		fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide
test item [mg/L]	active substance	Meas. conc.	%	Meas. conc.	%
	[mg a.s./L]	[mg a.s./L]		[mg a.s./L]
100	65.9	62.0	94	77.1	117
10.0	6.59	6.00	91	7.84	119
1.00	0.659	0.630	96	0.758	115
Control		< LCL		< LCL

Meas. Conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal test item concentration
a.s. = active substance (N-Lauroyl-L-glutamate, disodium salt)
LCL = lowest calibration level = 0.5 µg test item/L corresponding to 0.330 µg a.s./L

 

 

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

In this study, fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide 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 concentrations):
The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg/L and 80.7 (61.2 – > 100) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 10.0 and 1.00 mg/L, respectively.

Executive summary:

Aquatic effects of fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide (Batch no.: LAB AA101.20 Grobgut) to the unicellular freshwater green alga Pseudokirchneriella subcapitata were determined at the test facility according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2022-04-04 to 2024-04-07. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.
The study was conducted under static conditions with an initial cell density of 6864 cells/mL. A stock solution with a nominal concentration of 100 mg test item/L was prepared with dilution water (see Table 2). The dispersion was agitated until the solution was clear by eye. The Tyndall effect was negative. Six concentrations were tested in a geometrical series with a dilution factor of √10: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg test item/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.
The concentrations of the test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide and the control were determined in fresh media (0 hours) and old media (72 hours) of all tested concentration levels and the control via LC-MS. Details of the analytical method are presented in section 14.
The measured concentrations of N-Lauroyl-L-glutamate in the fresh media were in the range of 86 to 96% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of 109 to 120% of the nominal test item concentrations.  Therefore, all effect values are given are based on nominal test item concentrations.

NOEC, LOEC and ECx-values with 95% Confidence Intervals of fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide (0-72 hours)
based on the nominal test item concentrations[mg/L]

	Inhibition of Growth Rate
NOEC	10.0
LOEC	31.6
ErC10	51.2 (36.6 – 67.6)
ErC20	> 100
ErC50	> 100
	Inhibition of Yield
NOEC	1.00
LOEC	3.16
EyC10	6.41 (0.785 – 13.1)
EyC20	16.9 (9.68 – 27.5)
EyC50	80.7 (61.2 - > 100)

 

Description of key information

EC50 (72h) > 100 mg/L, EC10 (72h) = 51.2 mg/L (nominal) (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

51.2 mg/L