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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2022-04-06 to 2022-09-29 with the definitive exposure phase from 2022-09-27 to 2022-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

(2004)

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
100 % (UVCB)
Chemical composition
Disodium 2- (dodecanoylamino)pentanedioate (main component)
Water solubility
7.8 g/L
pH
9.9 at concentration of: 7800 [mg/L]
Appearance
colorless powder (solid)
Expiry date
2023-08-23
Recommended storage
Room temperature

Analytical monitoring:

yes

Remarks:

via LC-MS/MS method

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data. The method was validated according to SANTE/2020/12830 , Rev.1 criteria provided in Annex I.

Sampling schedule
All concentration levels and the control were analytically verified via LC-MS/MS method in the fresh media at the start of exposure and at the renewal of the test solutions (0 and 24 hours) as well as in the 24-hours old media at the renewal and at the end of the exposure (24 and 48 hours).

Sampling and pre-treatment
At the start of the exposure and at the renewal (0 and 24 hours), samples were taken after preparation of the concentration levels and analyzed. At the renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken from the test vessels.

Vehicle:

no

Details on test solutions:

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

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS, obtained from continuous laboratory cultures.

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, 16 hours illumination, light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), is used. The composition of the culture medium is presented in Table 2.

Feeding of the culture stocks
The daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae were cultured at the test facility.

Test type:

semi-static

Water media type:

freshwater

Remarks:

ASTM soft test water was used as the test item precipitates in water with a higher hardness. (ASTM Soft Test Water (according to ASTM E729 - 96))

Limit test:

no

Total exposure duration:

48 h

Hardness:

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

Test temperature:

During the test period, the temperature in the incubator was 19 – 20 °C.
For details, see section "Any other information on results incl. tables" below

pH:

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

Dissolved oxygen:

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

Conductivity:

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

Nominal and measured concentrations:

Five concentrations were tested in a geometric series with a dilution factor of 2: 6.25 – 12.5 – 25.0 – 50.0 – 100 mg/L, corresponding to the geometric mean measured test item concentrations 4.16 – 9.26 – 22.4 – 46.4 – 90.9 mg/L.

Details on test conditions:

Control
Dilution water without test item incubated under the same conditions as the test groups.

Reference Test
A reference test with potassium dichromate was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility.

Test method
The study was performed under semi-static conditions with a renewal of the test solutions after 24 hours.

Test duration 48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume 20 mL

Dilution water
ASTM soft test water was used as the test item precipitates in water with a higher hardness

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each concentration level and the control.

Age of the daphnids at the start of the exposure
Less than 22 hours old daphnids from a healthy stock were used for the study. To achieve this, juvenile daphnids were removed from the culture vessels at the latest 22 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.

Acclimatization At least 2 hours in the dilution water (see Table 3)

Application
20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette.

Renewal of the test solutions
The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).

Test temperature (target) 18 - 22 °C, constant within ± 1 °C

Illumination (target) Diffuse light, light intensity of max. 1500 lx

Photoperiod (target) 16/8 hours light/dark cycle

Feeding The daphnids were not fed during the study.


Type and Frequency of Measurements
Biological Parameters

Immobilization and other observations
Immobilization was determined in all groups after 24 and 48 hours. A daphnid was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Observation of other adverse, sub-lethal effects (e.g. discoloration, abnormal behavior etc.) was done, but none occurred during the course of the study.

Water Quality Parameters

Dilution water
Prior to the start of the exposure and at the renewal of the test solutions (0 and 24 hours), the water quality parameters (i.e. pH-value, dissolved oxygen concentration, total hardness, conductivity and temperature) of the dilution water (control) were measured.

Test media
At the start of the exposure and at the renewal (0 and 24 hours), the water quality parameters of the fresh media (i.e. pH-value, dissolved oxygen concentration) were measured in one additional replicate (without daphnids) of each concentration level and the control.
At the renewal and at the end of the exposure (24 and 48 hours), the water quality parameters of the old media were measured in one appropriate replicate (containing daphnids) of each concentration level and the control. The replicate with the highest immobilization rate per each concentration level and the control were measured.

Temperature
The incubator temperature (measurement in air with a thermo-hygrograph) was recorded throughout the period of the test.

Criteria for the water quality parameters (target)
• The dissolved oxygen concentration in the 24-hours old media should be ≥ 3 mg/L in all concentration levels and in the control.
• The pH should be in the range of 6 – 9.
• The deviation of the final pH-values (old media) from the initial
values (fresh media) should not exceed 1.5 units.

Reference substance (positive control):

yes

Remarks:

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number BCCC1619, purity 100.0%, CAS RN 7778-50-9) was determined after 24 hours from 2022-07-26 to 2022-07-27.

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

90.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 90.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

32.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

90.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

13.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

16.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

27.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

30.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

The effect levels (EC10 / 50 / 100) are based on the nominal concentrations of the test item and the calculated geometric mean measured if the test item.

Additional Observations during the Definitive Test

The concentration levels and the control were clear and colorless by eye throughout the exposure period.
 
Water Quality Parameters

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits during the study. During the test period, the temperature in the incubator was 19 – 20 °C.

Measured Exposure Concentrations during the Definitive Test

The concentrations of N-Lauroyl-L-glutamic acid, main 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 24 hours) and old media (24 hours and 48 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 93 to 104% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of 36 to 90% of the nominal test item concentrations.

Results with reference substance (positive control):

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

Reported statistics and error estimates:

Methods of evaluation
The EC100 – value after 24 and 48 hours was estimated empirically from the observed immobilization rates.
All effect levels (EC10 / 50 / 100) are given based on the nominal concentrations of the test item and on the calculated geometric mean measured of the test item.

ECx-values and statistical analyses
The EC10/50-values after 24 and 48 hours of exposure were calculated by sigmoidal dose response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t distribution wherever possible. In the case that the confidence limits could not be calculated by the software, the respective confidence limits were empirically derived from the observation data. All calculations were carried out from the best-fit values with the software GraphPad Prism.
The concentration-effect relationships for immobilization after 48 hours are shown graphically.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Immobilization Rates after 24 and 48 hours of Exposure

(n = 20, divided into 4 replicates with 5 daphnids each)

Nominal concentration of the test item [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	60	40	60	40	50	100	100	100	100	100
50.0	60	20	40	20	35	80	60	60	80	70
25.0	0	0	0	20	5	20	40	40	20	30
12.5	0	0	0	0	0	0	0	0	0	0
6.25	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 Absolute Numbers of immobile Daphnids after 24 and 48 hours of Exposure

 (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal concentration of the test item [mg/L]	NUMBER OF IMMOBILE DAPHNIDS / TOTAL NUMBER OF DAPHNIDS
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	Sum	1	2	3	4	Sum
100	3 / 5	2 / 5	3 / 5	2 / 5	10 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
50.0	3 / 5	1 / 5	2 / 5	1 / 5	7 / 20	4 / 5	3 / 5	3 / 5	4 / 5	14 / 20
25.0	0 / 5	0 / 5	0 / 5	1 / 5	1 / 20	1 / 5	2 / 5	2 / 5	1 / 5	6 / 20
12.5	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
6.25	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

 

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

Sampling date	Fresh media,  0 hours		Old media,  24 hours		Fresh media,  24 hours		Old media,  48 hours
Nominal Concentration of the test item [mg/L]	N-Lauroyl-L-glutamic acid
	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]
100	98.2	98	87.4	87	99.7	100	79.7	80
50.0	48.6	97	44.9	90	51.7	103	41.0	82
25.0	24.5	98	21.1	85	23.3	93	20.8	83
12.5	12.5	100	8.08	65	12.7	102	5.73	46
6.25	6.15	98	2.28	36	6.51	104	3.29	53
Control	< LOQ		< LOQ		< LOQ		< LOQ
Nominal Concentration of the test item [mg/L]	Geometric mean measured concentration [mg/L]
100	90.9
50.0	46.4
25.0	22.4
12.5	9.26
6.25	4.16

Meas. conc. = measured concentration of the test item, dilution and weighing factors taken into account

%                   = percentage of the nominal concentration of the test item

LOQ              = limit of quantification (1.00 mg test item/L, corresponding to 0.581 mg a.s./L)

a.s.                = active substance

 

Test of the Reference Item

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number BCCC1619, purity 100.0%, CAS RN 7778-50-9) was determined after 24 hours from 2022-07-26 to 2022-07-27.

EC₅₀-Value (with Confidence Limits) of the Reference Item Potassium dichromate

 based on nominal concentrations [mg/L], (0 - 24 hours)

	Current Study	Valid Range
EC50	1.67 mg/L	0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000)
Confidence limits	1.00 - 4.00 mg/L

 

Water Quality Parameters in the fresh Media at the Start of the Exposure and at the Renewal
(0 and 24 hours)

(measured in one additional replicate (without daphnids) per concentration level and control)

Nominal concentration of the test item [mg/L]	0 hours		24 hours
	pH-value	Dissolved O2 concentration [mg/L]	pH-value	Dissolved O2 concentration [mg/L]
100	8.30	8.14	7.95	8.29
50.0	8.34	8.19	7.96	7.70
25.0	8.45	8.15	8.00	8.36
12.5	8.49	7.90	8.01	8.33
6.25	8.47	7.18	8.04	8.27
Control	8.64	7.65	8.60	8.02

Water Quality Parameters in the 24-hours old Media at the Renewal and at the End of the Exposure (24 and 48 hours)

  (measured in one replicate (containing daphnids) per concentration level and control)

Nominal concentration of the test item [mg/L]	24 hours			48 hours
	pH-value	Dissolved O2 concentration [mg/L]	Replicate number	pH-value	Dissolved O2 concentration [mg/L]	Replicate number
100	8.13	8.06	1	8.20	7.80	2
50.0	8.18	7.95	1	8.33	7.99	1
25.0	8.29	7.85	4	8.51	8.40	2
12.5	8.34	7.72	4	8.56	8.32	2
6.25	8.45	7.98	4	8.68	8.34	2
Control	8.67	7.42	4	8.51	8.20	2

 

 Water Quality Parameters of the Dilution Water at the Start of the Exposure and at the Renewal (0 and 24 hours)

Dilution water	pH-Value	Dissolved O2 concentration [mg/L]	Temperature   [°C]	Conductivity   [µS/cm]	Total hardness   [mg CaCO3/L]
2022-09-27   0 hours	8.64	7.65	21.0	138.6	50
2022-09-28 24 hours	8.60	8.02	21.4	141.2	51

 

 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 (fresh media)

Nominal test item concentration [mg/L]	Dilution Factor*	Sample volume [mL]	Final volume [mL]
100	20000	0.05^1) 0.1^2)	10^1) 5.0^2)
50.0	5000	0.05^1) 0.08^2)	10^1) 1.0^2)
25.0	5000	0.05^1) 0.08^2)	10^1) 1.0^2)
12.5	1000	0.1^1) 0.2^2)	10^1) 1.0^2)
6.25	1000	0.1^1) 0.2^2)	10^1) 1.0^2)
Control	200	0.1^1)	10^1)

* including factor 2

¹⁾ first dilution step                 ²⁾ second dilution step

 

 Dilution Steps (old media)

Nominal test item concentration [mg/L]	Dilution Factor*	Sample volume [mL]	Final volume [mL]
100	10000	0.1^1) 0.1^2)	10^1) 5.0^2)
50.0	5000	0.05^1) 0.08^2)	10^1) 1.0^2)
25.0	2500	0.1^1) 0.2^2)	5.0^1) 5.0^2)
12.5	1000	0.1^1) 0.2^2)	10^1) 1.0^2)
6.25	500	0.04^1)	10^1)
Control	200	0.1^1)	10^1)

* 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,  r ≥ 0.99 (R^2≥ 0.9801 for 2^nd order regression) Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation	1.0 to 10 µg standard/L (n = 6), r ≥ 0.99	ü
Matrix effects	Should be ≤ 20%	Matrix effects ≤ 20%	ü
Lowest calibration level (LCL)	S/N ≥ 9 for quantifier ion trace S/N ≥ 3 for qualifier ion trace	S/N for 1.0 µg standard/L 8329 (Quantifier), 8562 (Qualifier)	ü
Limit of Quantification (LOQ)	Should be at or below lowest nominal test concentration	1.0 mg test item/L (1 x LOQ) 120 mg test item/L (120 x LOQ)	ü
Accuracy (Fortified samples)	Mean recovery rate of 70-120%  per fortification level (2 levels)	1 x LOQ: 102% (n = 5) 120 x LOQ: 104% (n = 5)	ü
Precision	Relative standard deviation ≤ 20% per fortification level	1 x LOQ: 5.6% 120 x LOQ: 1.8%	ü
Stability	Samples: Stable if 70 – 120% of nominal concentration Calibration standards: Stable if ±10% of freshly prepared standard	See section 17.1	ü
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 qualifier [m/z]: 330.13 > 129.98	ü
	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 17.2	ü

 

Preparation of Fortified Samples

LOQ Level	Control	1	120
Stock solution [mg test item/L] (Medium)	-	6000 (Dilution medium 1)	6000 (Dilution medium 1)
Spiking solution [mg test item/L] (Medium)	-	100 (Daphnia dilution water)	Stock solution
Replicates	2	5	5
Concentration of the LOQ  [mg test item/L]	-	1.0	120
Medium for preparation	Daphnia dilution water
Volume of spiking solution [mL]	-	0.05	0.1
Volume of medium [mL]	5.0	4.95	4.90
Dilution factor	200	200	20000
Dilution medium	Acetonitrile containing 2% formic acid^1) Dilution medium 2^2)3)
Sample volume [mL]	5^1) 0.1^2)	5^1) 0.1^2)	5^1) 0.1^2) 0.1^3)
Finale volume [mL]	10^1) 10^2)	10^1) 10^2)	10^1) 10^2) 10^3)
Final expected concentrations for analyses [µg/L]	-	5.0	6.0

Dilution medium 1: Acetonitrile : daphnia 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

 

 

 

 

 

Validity Criteria

 

The study was performed according to OECD Guideline 202 (2004). The validity criteria were fulfilled:

 

Validity criteria (target)	Result	Valid
≤ 10% of the daphnids in the control immobile or showing signs of disease or stress, e.g. discoloration or unusual behavior such as trapping on the surface of the water, during the 48-hour test period	No daphnid	ü
≥ 3 mg O2 /L in the 24-hours old media at the renewal of the test solutions and at the end of the exposure in all concentration levels and in the control	≥ 7.42 mg/L	ü

 

 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*: 1.01 mg test item/L (1 x LOQ) and 121 mg test item/L (120 x LOQ), corresponding to 0.586 mg a.s./L (1 x LOQ) and 70.3 mg a.s./L (120 x LOQ).

Replicate	N-Lauroyl-L-glutamic acid
	1 x LOQ		120 x LOQ
	Meas. conc. [mg a.s./L]	%	Meas. conc. [mg a.s./L]	%
1	0.560	96	70.8	101
2	0.621	106	72.9	104
3	0.627	107	72.9	104
4	0.626	107	72.8	104
5	0.568	97	74.5	106
Mean	0.600	102	73	104
SD ±	0.03		1
CV [%]	5.6		1.8

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 (test item) was checked after 4 days and was 108% 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 7 days old calibration standard (6 µg standard/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

 

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

Sampling date	0 hours		24 hours		48 hours
Quality Control	N-Lauroyl-L-glutamic acid
Test item [mg a.s./L]	Meas. conc. [mg a.s./L]	%	Meas. conc. [mg a.s./L]	%	Meas. conc. [mg a.s./L]	%
0.581	0.614	106	0.612	105	0.605	104

Meas. conc. = measured concentration, dilution and weighing factors taken into account

%                   = percent of the nominal concentration

a.s.                = active substance

 

Preliminary Range Finding Tests (non-GLP)

 

First Preliminary Range Finding Test with Elendt M4 Medium

 

A non-GLP preliminary range finding test was conducted at the test facility under semi-static conditions over a period of 48 hours with three nominal test item concentrations of 1.00, 10.0 and 100 mg/L prepared with Elendt M4 medium.

A stock solution of 100 mg/L was freshly prepared with dilution water as described in section 4.2. The stock solution was further diluted to the concentration levels 1.00 and 10.0 mg/L with dilution water. At the start of the exposure (0 hours) and on the renewal of the test solutions (24 hours) the loading rate 100 mg/L was colorless and visually clear. In the old media (24 and 48 hours) white depositions of the test item on the bottom of test vessel and in the water phase were observed.

The loading rates 1.00, 10.0 mg/L and the control were colorless and visually clearly throughout the exposure period. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle).

In the range finding test, two replicates per concentration level and control, each with five daphnids, were tested.

 

Results of the First Range Finding Test (0 - 48 hours)

Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours			48 hours
	Replicates			Replicates
	1	2	MV	1	2	MV
100	80	40	60	100	100	100
10.0	0	0	0	0	0	0
1.00	0	0	0	0	0	0
Control	0	0	0	0	0	0

 

 

Measured Exposure Concentrations during the First non-GLP Preliminary Range Finding Test

Determination of the test item via LC-MS

Sampling date	Fresh media, 0 hours		Old media, 24 hours		Fresh media, 24 hours		Old media, 48 hours
Nominal test item loading rates	N-Lauroyl-L-glutamic acid
[mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
100	97.6	98	< LCL		100	100	67.5	68
10.0	10.2	102	< LCL		9.83	98	< LCL
1.00	1.10	110	< LCL		0.952	95	< LCL
Control	< LCL		< LCL		< LCL		< LCL

%                   = Percent of the nominal test item concentration

LCL               = lowest calibration standard = 0.5 µg/L

 

 Second Preliminary Range Finding Test with ASTM Soft Test Water

 

A non-GLP preliminary range finding test was conducted at the test facility under semi-static conditions over a period of 48 hours with three nominal test item concentrations of 1.00, 10.0 and 100 mg/L prepared with ASTM Soft Water.

A stock solution of 100 mg/L was freshly prepared with dilution water as described. The stock solution was further diluted to the concentration levels 1.00 and 10.0 mg/L with dilution water. All tested concentration levels were visually clear and colorless throughout the exposure period. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle).

In the range finding test, two replicates per concentration level and control, each with five daphnids, were tested.

 

Results of the Second Range Finding Test (0 - 48 hours)

Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours			48 hours
	Replicates			Replicates
	1	2	MV	1	2	MV
100	40	0	20	40	0	20
10.0	0	0	0	0	0	0
1.00	0	0	0	0	0	0
Control	0	0	0	0	0	0

 

 

Measured Exposure Concentrations during the Second non-GLP Preliminary Range Finding Test

Determination of the test item via LC-MS

Sampling date	Fresh media, 0 hours		Old media, 24 hours		Fresh media, 24 hours		Old media, 48 hours
Nominal test item loading rates	N-Lauroyl-L-glutamic acid
[mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
100	111	111	84.2	84	99.4	99	67.9	68
10.0	11.2	112	3.75	38	9.91	99	5.37	54
1.00	1.20	120	< LCL		1.20	120	< LCL
Control	< LCL		< LCL		< LCL		< LCL

%                   = Percent of the nominal test item concentration

LCL               = lowest calibration standard = 1 µg/L

 

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item, the 48 hours-EC50 for Daphnia magna was 36.0 mg/L (confidence limits: 32.4 – 40.2 mg/L) for the test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide.
Based on the geometric mean measured concentrations, the 48 hours-EC50 for Daphnia magna was 32.9 mg/L (confidence limits: 29.4 – 36.9 mg/L) for the test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide.

Executive summary:

Summary

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide (batch number: LAB AA101.20 Grobgut) were determined at the test facility according to OECD 202 (2004) from 2022-04-06 to 2022-09-29 with the definitive exposure phase from 2022-09-27 to 2022-09-29.

The study was conducted under semi-static conditions over a period of 48 hours with five concentrations. The test item is a UVBC substance with a solubility of 7.8 g/L. Therefore, test concentrations could be prepared. The test item concentration levels were prepared in the range of 6.25 to 100 mg/L set up in a geometric series with a factor of 2. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration and the control.

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 24 hours) and old media (24 hours and 48 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 93 to 104% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of 36 to 90% of the nominal test item concentrations.

The geometric mean measured test item concentrations were calculated to be: 4.16 – 9.26 – 22.4 – 46.4 – 90.9 mg/L.

The ECx-values are based on the nominal concentrations of the test item and on the calculated geometric mean measured of the test item.

The validity criteria of the test guideline were fulfilled.

 

 EC₁₀-, EC₅₀- (with Confidence Limits) and EC₁₀₀-Values

Effect values	Test duration [hours]	Nominal concentrations of the test item [mg/L]	Geometric mean measured test item concentrations [mg/L]
EC10 (with confidence limits)	24	30.1 (Cl: 20.8 – 40.5)	27.3 (Cl: 18.9 – 36.7)
	48	16.2 (Cl: 12.9 – 19.9)	13.5 (Cl: 10.3 – 17.2)
EC50 (with confidence limits)	24	100 (Cl: 55.0 – > 100)	90.9 (Cl: 52.0 – > 90.9)
	48	36.0 (Cl: 32.4 – 40.2)	32.9 (Cl: 29.4 – 36.9)
EC100	24	> 100	> 90.9
	48	100	90.9

Description of key information

Based on the nominal concentrations of the test item, the 48 hours-EC50 for Daphnia magna was 36.0 mg/L (confidence limits: 32.4 – 40.2 mg/L) for the test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide.
Based on the geometric mean measured concentrations, the 48 hours-EC50 for Daphnia magna was 32.9 mg/L (confidence limits: 29.4 – 36.9 mg/L) for the test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

Based on the geometric mean measured concentrations

Effect concentration:

32.9 mg/L