cyclic C12/14-Glucamide;cyclic N-methyl-N-dodecanoyl/tetradecanoyl-glucamine;anhydro-N-dodecanoyl/tetradecanoyl-N-methylglucamine;Amides, C12-C14, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated

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 2020-02-09 to 2021-02-17 with the definitive exposure phase from 2021-02-09 to 2021-02-11

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 cyclic Glucamide C12-14
Batch number S148866
Purity 100 % (UVCB)
Sum formula
main compounds : C19H37NO5 (C12-acyl-derivative);
C21H41NO5 (C14-acyl-derivative)
Molecular weight main compounds :
359.5 g/mol (C12-derivative); 387.5 g/mol (C14-derivative)
Density 1.07 g/cm3
Water solubility soluble/miscible
Appearance slightly turbid brown solid mass
Expiry date 2022-09-30
Recommended storage Room temperature, dry

Analytical monitoring:

yes

Remarks:

via LC-MS/MS method

Details on sampling:

Sampling schedule
All loading rates 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 loading rates 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:

Water Accommodated Fraction
Water accommodated fractions (WAF) were prepared because the test item is an UVCB substance with constituents of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2018).
Using this approach, aqueous media was prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

Preparation of the water accommodated fractions
Five water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 0.854 to 20.0 mg/L set up in a geometric series with a factor of 2.2: 0.854 – 1.88 – 4.14 – 9.09 – 20.0 mg/L.
For each loading rate, an appropriate amount of the test item were weighed out and transferred into a measuring flask with an appropriate amount of dilution water. The WAF’s were treated with ultrasound for 5 minutes at room temperature.

Test loading
Per definition of the WAF all terms related to concentration level were given as nominal loading rates because partly dissolved compounds and mixtures cannot be related to concentrations. The loading rates are based on the results of a preliminary range finding test (non-GLP, open system with headspace).

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:

Composition of the Culture Medium Elendt M4 according to OECD 202, Annex 3 (2004)

Limit test:

no

Total exposure duration:

48 h

Hardness:

See section "Any other information on results incl. tables"

Test temperature:

See section "Any other information on results incl. tables"

pH:

See section "Any other information on results incl. tables"

Dissolved oxygen:

See section "Any other information on results incl. tables"

Conductivity:

See section "Any other information on results incl. tables"

Nominal and measured concentrations:

Five water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 0.854 to 20.0 mg/L set up in a geometric series with a factor of 2.2: 0.854 – 1.88 – 4.14 – 9.09 – 20.0 mg/L.
The measured concentrations of the test item cyclic Glucamide C12-14 in the fresh media were in the range of 108 to 154% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of 75 to 131% of the nominal test item concentrations.

Details on test conditions:

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 Same composition as the culture medium (see Table 2)

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 24 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 24 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
Acclimatization is not necessary, because the composition of the dilution water is equivalent to the culture medium.

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 loading rate 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 loading rate and the control. The replicate with the highest immobilization rate per loading 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.

Reference substance (positive control):

yes

Remarks:

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number BCCC1619, purity 100%, CAS RN 7778-50-9) was determined after 24 hours from 2021-02-02 to 2021-02-03.

Key result

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

9.09 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EL100

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

3.68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

9.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

1.37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EL10

Effect conc.:

4.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Results of the Definitive Test

Biological Data

The percentage of immobility, determined in all tested loading rates and in the control after 24 and 48 hours and the absolute numbers of immobile daphnids are presented section "Any other information on results incl. tables" below.

Additional Observations during the Definitive Test

The tested WAFs were colorless and visually clear 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 20 – 21 °C.

Measured Exposure Concentrations during the Definitive Test

The concentrations of the test item cyclic Glucamide C12-14 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 the test item cyclic Glucamide C12-14 in the fresh media were in the range of 108 to 154% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of 75 to 131% of the nominal test item concentrations.

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number BCCC1619, purity 100%, CAS RN 7778-50-9) was determined after 24 hours from 2021-02-02 to 2021-02-03. For results of the most recent of the monthly performed reference tests, see section "Any other information on materials and methods incl. tables" above.

Reported statistics and error estimates:

Methods of evaluation
The EL100 – values after 24 and 48 hours were estimated empirically from the observed immobilization rates.
All effect levels (EL10 / 50 / 100) are given based on the nominal loading rates of the test item, since the test item is an UVCB substance. Per definition of the WAF, all terms related to the concentration were given as loading rate because partly dissolved compounds and mixtures cannot be related to concentrations.

ECx-values and statistical analyses
The EL10- values after 24 and 48 hours and the EL50- values after 48 hours were calculated by sigmoidal dose-response regression. The confidence limits were calculated from the standard error and the t distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism. The concentration-effect relationships of the test item 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 loading rate	IMMOBILIZATION [%]
of the test item	24 hours					48 hours
[mg/L]	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
20.0	100	100	100	100	100	100	100	100	100	100
9.09	40	40	60	40	45	100	100	100	100	100
4.14	0	20	20	0	10	60	40	60	40	50
1.88	0	0	0	0	0	20	20	40	20	25
0.854	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 loading rate	Number of immobile Daphnids / Total number of Daphnids
of the test item	24 hours					48 hours
[mg/L]	Replicates					Replicates
	1	2	3	4	Sum	1	2	3	4	Sum
20.0	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
9.09	2 / 5	2 / 5	3 / 5	2 / 5	9 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
4.14	0 / 5	1 / 5	1 / 5	0 / 5	2 / 20	3 / 5	2 / 5	3 / 5	2 / 5	10 / 20
1.88	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	1 / 5	1 / 5	2 / 5	1 / 5	5 / 20
0.854	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

The concentrations of the test item cyclic Glucamide C12-14 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 the test item cyclic Glucamide C12-14 in the fresh media were in the range of 108 to 154% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of 75 to 131% of the nominal test item concentrations.

 

Measured Concentrations of the Test Item cyclic Glucamide C12-14 during the Definitive Test

Sampling date		Fresh media,		Old media,		Fresh media,		Old media,
		0 hours		24 hours		24 hours		48 hours
Nominal Concentration of the		C12-acyl-derivative
Test item	active substance	Meas.	[%]	Meas.	[%]	Meas.	[%]	Meas.	[%]
[mg/L]	[mg a.s./L]	conc.		conc.		conc.		conc.
		[mg a.s./L]		[mg a.s./L]		[mg a.s./L]		[mg a.s./L]
20.0	13.2	14.9	113	12.6	95
9.09	6.01	7.16	119	6.33	105	6.58	109	5.94	99
4.14	2.74	3.04	111	2.70	99	3.16	116	3.07	112
1.88	1.24	1.911)	154^1)	1.62	131	1.491)	1201)	1.25	101
0.854	0.564	0.6831)	121^1)	0.581	103	0.622	110	0.532	94
Control		< LOQ		< LOQ		< LOQ		< LOQ
Nominal Concentration of the		Geometric mean Test item concentration
Test item	active substance	Meas. conc.
[mg/L]	[mg a.s./L]	[mg a.s./L]
20.0	13.2	13.7
9.09	6.01	6.49
4.14	2.74	2.99
1.88	1.24	1.55
0.854	0.564	0.602

Meas. conc. = measured concentration of the active substance, dilution factors taken into account
% = percentage of the nominal concentration of the test item
a.s. = active substance
LOQ = limit of quantification (0.100 mg test item/L corresponding to 0.0661 mg a.s./L)
1) = reanalyzed and confirmed

 

Measured Concentrations of the Test Item cyclic Glucamide C12-14 during the Definitive Test

Sampling date		Fresh media,		Old media,		Fresh media,		Old media,
		0 hours		24 hours		24 hours		48 hours
Nominal Concentration of the		C14-acyl-derivative
Test item	active substance	Meas.	[%]	Meas.	[%]	Meas.	[%]	Meas.	[%]
[mg/L]	[mg a.s./L]	conc.		conc.		conc.		conc.
		[mg a.s./L]		[mg a.s./L]		[mg a.s./L]		[mg a.s./L]
20.0	3.96	4.48	113	3.51	89
9.09	1.80	2.12	118	1.81	101	2.00	111	1.64	91
4.14	0.820	0.925	113	0.706	86	0.932	114	0.790	96
1.88	0.372	0.5611)	151^1)	0.438	118	0.448^1)	120^1)	0.287	77
0.854	0.169	0.1971)	117^1)	0.151	89	0.183	108	0.126	75
Control		< LOQ		< LOQ		< LOQ		< LOQ
Nominal Concentration of the		Geometric mean Test item concentration
Test item	active substance	Meas. conc.
[mg/L]	[mg a.s./L]	[mg a.s./L]
20.0	3.96	3.97
9.09	1.80	1.88
4.14	0.820	0.833
1.88	0.372	0.422
0.854	0.169	0.162

Meas. conc. = measured concentration of the active substance dilution factors taken into account
% = percentage of the nominal concentration of the test item
a.s. = active substance
LOQ = limit of quantification (0.100 mg test item/L corresponding to 0.0198 mg a.s./L)
1) = reanalyzed and confirmed

 

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	≥ 5.77 mg/L	ü

 

Physicochemical Data

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 loading level and control)

Nominal loading rate	0 hours		24 hours
of the test item
[mg/L]	pH-value	Dissolved	pH-value	Dissolved
		O2 concentration		O2 concentration [mg/L]
		[mg/L]
20.0	7.36	8.62	Not determined, due to 100% mortality after 24 hours
9.09	7.49	8.68	7.28	9.31
4.14	7.55	8.65	7.40	9.29
1.88	7.54	8.45	7.54	9.29
0.854	7.60	8.62	7.60	9.27
Control	7.60	8.64	7.63	9.10

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 per loading level and control)

Nominal loading rate	24 hours			48 hours
of the test item
[mg/L]	pH-value	Dissolved	Replicate number	pH-value	Dissolved	Replicate number
		O2 concentration			O2 concentration
		[mg/L]			[mg/L]
20.0	6.78	8.26	3	Not determined, due to 100% mortality after 24 hours
9.09	6.80	8.31	3	6.96	6.27	3
4.14	6.82	8.47	3	7.05	5.78	3
1.88	6.94	8.49	3	7.06	5.77	3
0.854	6.95	8.67	3	7.17	6.92	3
Control	6.92	8.88	3	7.20	8.41	3

 

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	Temperature	Conductivity	Total hardness
		O2 concentration
		[mg/L]	[°C]	[µS/cm]	[mg CaCO3/L]
0 hours:	7.71	8.93	20.0	591	262
24 hours:	7.62	8.69	19.8	615	245

 

Gradient Table

Time [min]	A [%]	B [%]
0.00	90.0	10.0
0.50	90.0	10.0
1.20	5.0	95.0
2.50	5.0	95.0
2.70	90.0	10.0
3.20	90.0	10.0

 

Dilution Steps

Nominal	Dilution	Sample	Final
test item concentration	Factor*	volume	volume
[mg/L]		[mL]	[mL]
20.0	4000	0.05^1)	10^1)
		0.1^2)	1.0^2)
9.09	1250	0.16^1)	10^1)
		0.1^2)	1.0^2)
4.14	800	0.1^1)	10^1)
		0.25^2)	1.0^2)
1.88	400	0.05	10
0.854	125	0.16	10
Control	20	0.1	1.0

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

 

Parameter, Acceptance Criteria and Results of the Method Validation

Parameter	Acceptance criteria	Result
Linearity	5 standard concentrations,	1 to 10 µg test item/L (n = 6),	ü
	r ≥ 0.99	r ≥ 0.99
Lowest calibration standard	S/N ≥ 9 for quantifier ion trace	1 µg test item/L	ü
	S/N ≥ 3 for qualifier ion trace	C12-acyl derivative
		S/N: 6218 (quantifier), 282 (quantifier)
		C14-acyl derivative
		S/N: 3679 (quantifier), 86 (quantifier)
Limit of Detection (LOD)	Determination only necessary when S/N LCL ≤ 30.	Not necessary	-
	S/N of 3-5 for the signal which is used for the identification. If significant blank values are observable, LOD has to be 3-5 times higher as the mean value plus standard deviation of 5 to 10 blank measurements.
Limit of Quantification (LOQ)	At least 20% above lowest calibration standard after sample preparation	0.100 mg test item/L (1 x LOQ)	ü
		24.0 mg test item/L (240 x LOQ)
Accuracy^1)	Mean recovery rate of 70-110% (ideally 80-100%) per	C12-acyl derivative	ü
(Fortified samples)	fortification level (2 levels)	1 x LOQ: 97% (n = 5)
		240 x LOQ: 108% (n = 5)
		C14-acyl derivative
		1 x LOQ: 97% (n = 5)
		240 x LOQ: 103% (n = 5)

Precision^1)	Relative standard deviation ≤ 20% per fortification level	C12-acyl derivative	ü
		1 x LOQ: 1.7 %
		240 x LOQ: 4.1%
		C14-acyl derivative
		1 x LOQ: 1.9%
		240 x LOQ: 4.2%
Specificity	Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity) per compound.	C12-acyl derivative	ü
(LC-MS/MS)		360.36 > 178.20 (Quantifier)
		360.36 > 160.13 (Qualifier)
		C14-acyl derivative
		388.39 > 178.20 (Quantifier)
		388.39 > 160.13 (Qualifier)
	Blank values < 30 % of LOQ	Blank values < 30% of LOQ
Procedural recovery	At least one procedural recovery on 1x LOQ Level is freshly prepared on each day of analysis. It will be treated in parallel to the test samples. The recovery should be above 90% in comparison to the mean recovery of the fortified samples (in the range of the mean recovery +/- 2x Relative standard deviation, if the recovery is < 90 %).	For details, see section 17.1.	ü

 

Preparation of Fortified Samples

LOQ Level	Control	1	240
Stock solution	-	1000	100
[mg test item/L]		(Methanol)	(Daphnia dilution water)
(Medium)
Spiking solution	-	10.0	Stock solution
[mg test item/L]		(Methanol)
(Medium)
Replicates	2	5	5
Concentration of the LOQ	-	0.100	24.0
[mg test item/L]
Medium for preparation	Daphnia dilution water
Volume of spiking solution [mL]	-	0.05	1.2
Volume of medium [mL]	5.0	4.95	3.8
Dilution factor	2	20	4000
Dilution medium	Acetonitrile containing 2% formic acid^1)
	Dilution medium^2)3)
Sample volume [mL]	5.0^1)	5.0^1)	5.0^1)
		0.1^2)	0.25^2)
			0.1^3)
Finale volume [mL]	10^1)	10^1)	10^1)
		1.0^2)	5.0^2)
			10^3)

Dilution medium: ultra-pure water : acetonitrile (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 cyclic Glucamide C12-14
Fortified concentrations*: 0.101 mg test item/L, corresponding to 0.0666 mg C12 acyl derivative/L and 0.0200 mg C14 acyl derivative/L (1 x LOQ) and 23.5 mg test item/L corresponding 15.5 mg C12 acyl derivative/L and 4.66 mg C14 acyl derivative/L (240 x LOQ).

Replicate	C12-acyl-derivative
	1 x LOQ		240 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[mg a.s./L]		[mg a.s./L]
1	63.2	95	16.1	104
2	65.8	99	16.1	103
3	64.5	97	17.7	114
4	63.5	95	16.9	109
5	65.1	98	16.9	109
Mean	64	97	16.8	108
SD ±	1		0.7
CV [%]	1.7		4.1
Replicate	C14-acyl-derivative
	1 x LOQ		240 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[mg a.s./L]		[mg a.s./L]
1	19.1	95	4.65	100
2	19.7	99	4.59	98
3	19.1	96	5.09	109
4	19.0	95	4.80	103
5	19.8	99	4.87	105
Mean	19.3	97	4.8	103
SD ±	0.4		0.2
CV [%]	1.9		4.2

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

The stability of the fortified samples was checked after 34 day and was 106% (C12-acyl-derivative) and 101% (C14-acyl-derivative) of the initially measured concentration and were 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	Fresh media,		Old media,		Fresh media,		Old media,
date	0 hours		24 hours		24 hours		48 hours
Quality Control	C12-acyl-derivative
Test item	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[mg a.s./L]	conc.		conc.		conc.		conc.
	[mg a.s./L]		[mg a.s./L]		[mg a.s./L]		[mg a.s./L]
0.0685	0.146^1)	107^1)	0.0633	92	0.0694	101	0.0691	101
Quality Control	C14-acyl-derivative
Test item	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[mg a.s./L]	conc.		conc.		conc.		conc.
	[mg a.s./L]		[mg a.s./L]		[mg a.s./L]		[mg a.s./L]
0.0205	0.0413^1)	101^1)	0.0200	98	0.0223	109	0.0209	102

Meas. conc. = measured concentration of the active substance, dilution and weighing factors taken into account
% = percent of the nominal concentration
1) = erroneously QC was spiked twice (concentration of 2 x LOQ)

 

Preliminary Range Finding Tests (non-GLP)

A non-GLP preliminary range finding test under semi-static conditions over a period of 48 hours was conducted at the test facility with four WAFs at nominal loading rates of the test item of 0.10, 1.00, 10.0 and 100 mg/L prepared with dilution water.
The WAFs were prepared prior to the start of the exposure (0 hours) and prior to the renewal of the test solutions (24 hours) as specified in section 4.2 for the definitive test.
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.

Immobilization Rates in the non-GLP Preliminary Range Finding Test
(n = 10, divided into 2 replicates with 5 daphnids each)

Nominal loading rate of the test item in the WAF	IMMOBILIZATION [%]
[mg/L]	24 hours			48 hours
	Replicates			Replicates
	1	2	MV	1	2	MV
100	100	100	100	100	100	100
10.0	60	40	50	100	100	100
1.00	0	0	0	0	0	0
0.10	0	0	0	0	0	0
Control	0	0	0	0	0	0

 

Measured Exposure Concentrations of the C12 Compound during the non-GLP Preliminary Range Finding Test
Determination of the test item via LC-MS/MS

Sampling date	Fresh media,		Old media,		Fresh media,		Old media,
	0 hours		24 hours		24 hours		48 hours
Nominal loading rate of the test item in the WAF	cyclic Glucamide C12-14
[mg/L]	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%
	[mg/L]		[mg/L]		[mg/L]		[mg/L]
100	111	111	100	100	not determined
10.0	10.3	103	10.5	105	8.78	88	10.0	100
1.00	1.33	133	1.30	130	0.940	94	0.854	85
0.100	Results not plausible				0.0938	94	0.0770	77
Control	< LCL		< LCL		< LCL		< LCL

Meas. conc. = measured concentration of the test item, enrichment and dilution factors taken into account
% = percent of the nominal concentration of the test item
< LCL = < lowest calibration level (0.1 µg/L of the test item)

 

Measured Exposure Concentrations of the C14 Compound during the non-GLP Preliminary Range Finding Test
Determination of the test item via LC-MS/MS

Sampling date	Fresh media,		Old media,		Fresh media,		Old media,
	0 hours		24 hours		24 hours		48 hours
Nominal loading rate of the test item in the WAF	cyclic Glucamide C12-14
[mg/L]	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%
	[mg/L]		[mg/L]		[mg/L]		[mg/L]
100	120	120	95.1	95	not determined
10.0	9.95	100	9.52	95	8.80	88	8.66	87
1.00	1.31	131	1.11	111	0.904	90	0.697	70
0.100	Results not plausible				0.0922	92	0.0602	60
Control	< LCL		< LCL		< LCL		< LCL

Meas. conc. = measured concentration of the test item, enrichment and dilution factors taken into account
% = percent of the nominal concentration of the test item
< LCL = < lowest calibration level (0.1 µg/L of the test item)

 

 

 

 

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal loading rates, the 48 hours-EL50 for Daphnia magna was 3.68 mg/L (95% confidence limits: 3.11 – 4.29 mg/L) for the test item cyclic Glucamide C12-14.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item cyclic Glucamide C12-14 (batch number: S148866) were determined at the test facility according to OECD 202 (2004) from 2020-02-09 to 2021-02-17 with the definitive exposure phase from 2021-02-09 to 2021-02-11 at the test facility.
The study was conducted under semi-static conditions over a period of 48 hours with five water accommodated fractions (WAF). The WAFs were prepared separately with nominal loading rates of the test item in the range of 0.854 to 20.0 mg/L set up in a geometric series with a factor of 2.2. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each loading rate and the control.
The concentrations of the test item and the control were determined in fresh media (0 hours and 24 hours) and old media (24 hours and 48 hours) of all tested WAFs and the control via LC-MS/MS.
The measured concentrations of the test item cyclic Glucamide C12-14 in the fresh media were in the range of 108 to 154% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of 75 to 131% of the nominal test item concentrations.
The EL10 / 50 / 100-values based on the nominal loading rates of the test item and are given in the table below.
The validity criteria of the test guideline were fulfilled.

EL10-, EL50- (with Confidence Limits) and EL100-Values
(based on the nominal loading rates of the test item)

Effect values	Test	Nominal loading rates
	duration	of the test item
	[hours]	[mg/L]
EL10	24	4.20 (Cl: 3.12 – 5.42)
(with confidence limits)	48	1.37 (Cl: 0.85 – 1.91)
EL50	24	9.75 (Cl: 8.85 – 11.1)
(with confidence limits)	48	3.68 (Cl: 3.11 – 4.29)
EL100	24	20.0
	48	9.09

 

Description of key information

Based on the nominal loading rates, the 48 hours-EL50 for Daphnia magna was 3.68 mg/L (95% confidence limits: 3.11 – 4.29 mg/L) for the test item cyclic Glucamide C12-14.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

EL50

Effect concentration:

3.68 mg/L