2,2'-(C16-18 (evennumbered) alkyl imino) diethanol

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 2021-05-31 to 2022-03-23 with the definitive exposure phase from 2022-03-16 to 2022-03-18

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 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4)

Batch number 20192048

CAS No. 1218787-30-4

Purity (certified) 100% UVCB
Water content: 0.07 w/w%

Appearance Yellowish to light brown, solid

Water Solubility Slightly soluble

Stability under test conditions
Not specified

Expiry date 2022-10-25

Recommended storage
Store container tightly closed in a dry, well-ventilated place. Avoid elevated temperatures.

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 compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2019).
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 6.25 to 100 mg/L set up in a geometric series with a factor of 2:
6.25 – 12.5 – 25.0 – 50.0 – 100 mg/L, corresponding to the geometric mean measured test item concentrations of 12.5 – 23.5 – 52.0 – 70.3 – 103 µg/L.
For each loading rate an appropriate amount of stock solution containing the test item with methanol was placed on a curved glass slide. The methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. For the preparation of the water accommodated fractions a stirring phase of 24 hours was found to be suitable. Longer stirring phases led to a decrease of the loading in the WAFs (< LCL). Therefore, a stirring phase of 24 hours is chosen for the definitive test. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The first 25 mL were discarded. The WAF were checked via laser beam (Tyndall effect) for undissolved test item. No presence of undissolved test item during the test was observed. The resulting water accommodated fractions (WAF) were used in the test.

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.

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:

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

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

A coating phase (saturation of the test container) was carried out. The test containers were pre-treated with the appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure and the renewal.

Hardness:

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

Test temperature:

During the test period, the temperature in the incubator was 19.5 – 20 °C.

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

pH:

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

Dissolved oxygen:

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

Conductivity:

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

Nominal and measured concentrations:

Five water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 6.25 to 100 mg/L set up in a geometric series with a factor of 2:
6.25 – 12.5 – 25.0 – 50.0 – 100 mg/L, corresponding to the geometric mean measured test item concentrations of 12.5 – 23.5 – 52.0 – 70.3 – 103 µg/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 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 coated set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette.

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.

Pre-treatment
A coating phase (saturation of the test container) was carried out. The test containers were pre-treated with the appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure and the renewal, the test containers were emptied and refilled with freshly prepared test solution.


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 2022-01-19 to 2022-01-20.

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 103 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 103 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

70.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

51.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 103 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

68.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

47.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

103 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

EL10

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

The test item contains 0.07% water.
Additional Observations during the Definitive Test:

The tested WAF’s and the control were colorless and visually clear throughout the exposure period.

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 19.5 – 20 °C.

The concentrations of the test item 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4) 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 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4) at the end of exposure after 24 and 48 hours were in the range of 14 to 82% of the initially measured 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 2022-01-19 to 2022-01-20.

Reported statistics and error estimates:

Methods of evaluation
The EC/EL100 – values after 24 and 48 hours and the EC/EL50- value after 24 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.
The effect levels (EC10 / 50 / 100) given are based on the geometric mean measured concentrations of the test item.

ECx-values and statistical analyses
The EC/EL10- values after 24 and 48 hours and the EC/EL50- values after 48 hours 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. 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.

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

Geometric mean measured concentrations
The geometric mean measured concentrations were determined by the sumproduct function of excel, referring to the recoveries and purities of the two lead components of the samples. This procedure takes into account the mean measured concentration of main constituent as well as their content in the product.

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

Component	Concentration [mg/L]
CaCl2 x 2 H2O	294
MgSO4 x 7 H2O	123
KCl	5.80
NaHCO3	64.8
Na2SiO3 x 5 H2O	7.47
NaNO3	0.274
KH2PO4	0.143
K2HPO4	0.184
Na2EDTA x 2 H2O	2.50
FeSO4 x 7 H2O	0.996
H3BO3	2.86
MnCl2 x 4 H2O	0.361
LiCl	0.306
SrCl2 x 6 H2O	0.152
RbCl	0.0710
NaBr	0.0160
Na2MoO4 x 2 H2O	0.0615
CuCl x 2 H2O	0.0168
ZnCl2	0.0130
CoCl2 x 6 H2O	0.0100
KI	0.00325
Na2SeO3	0.00219
NH4VO3	0.000575
Thiaminhydrochloride	0.075
Cyanocobalamin	0.0010
Biotin	0.00075
pH	8.2 ± 0.8

 

Preparation of the WAFs in the Definitive Test

	SL 1
Stock solution	200
[g/L in MeOH]
Weighing out [g]	2
Volume of the stock solution [mL]	10
Volume of the stock solution placed on a curved glass slide [µL/L]	500	250	125	62.5	31.25
Final test item	100	50.0	25.0	12.5	6.25
Loading rate [mg/L]

 

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
100	0	20	20	0	10	100	100	80	100	95
50.0	0	0	0	0	0	60	40	20	20	35
25.0	0	0	0	0	0	0	0	0	0	0
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 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
100	0 / 5	1 / 5	1 / 5	0 / 5	2 / 20	5 / 5	5 / 5	4 / 5	5 / 5	19 / 20
50.0	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	3 / 5	2 / 5	1 / 5	1 / 5	7 / 20
25.0	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 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 the compound C16 Amine + 2EO of the test item 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4) during the Definitive Test

Sampling date		Fresh medium,	Old medium,		Fresh medium,	Old medium,
		0 hours	24 hours		24 hours	48 hours
Nominal		2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4)
loading rate of the		C16 Amine + 2EO
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]
100	32.8	0.113	0.0783	69	0.0850	0.0432	51
50.0	16.4	0.0625	0.0383	61	0.0704	0.0363	52
25.0	8.20	0.0682	0.0241	35	0.0579	0.0217	37
12.5	4.10	0.0231	0.0191	82	0.0196	0.0139	71
6.25	2.05	0.0179	0.00345	19	0.0203	0.00864	43
Control		< LOQ	< LOQ		< LOQ	< LOQ

Meas. conc. = measured concentration of the compound C16 Amine + 2EO of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the initially measured concentration of the active substance
LOQ = limit of quantification (0.200 µg/L of the test item, corresponding to 0.0656 µg a.s./L)

 

Measured Concentrations of the compound C18 Amine + 2EO of the test item 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4) during the Definitive Test

Sampling date		Fresh medium,	Old medium,		Fresh medium,	Old medium,
		0 hours	24 hours		24 hours	48 hours
Nominal		2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4)
loading rate of the		C18 Amine + 2EO
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]
100	60.8	0.0374	0.0233	62	0.0273	0.00831	30
50.0	30.4	0.0337	0.0103	30	0.0371	0.00518	14
25.0	15.2	0.0194	0.0102	53	0.00979	0.00682	70
12.5	7.60	0.00852	0.00123	14	0.00447	0.00277	62
6.25	3.80	0.00223	0.000657	30	0.00279	0.00119	43
Control		< LOQ	< LOQ		< LOQ	< LOQ

Meas. conc. = measured concentration of the compound C18 Amine + 2EO of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the initially measured concentration of the active substance
LOQ = limit of quantification (0.200 µg/L of the test item, corresponding to 0.122 µg a.s./L)

 

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 (= yes)
≥ 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	≥ 7.34 mg/L	ü

 

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]
100	7.38	8.11	7.72	7.37
50.0	7.37	8.40	7.74	7.42
25.0	7.33	8.42	7.74	7.34
12.5	7.29	8.41	7.77	8.27
6.25	7.26	8.37	7.77	8.35
Control	7.20	8.37	7.93	8.25

 

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]
100	7.75	7.98	2	7.98	7.54	1
50.0	7.82	8.07	2	8.03	8.41	1
25.0	7.82	8.00	2	8.06	8.13	1
12.5	7.83	8.21	2	8.07	8.09	1
6.25	7.81	8.18	2	8.10	8.13	1
Control	7.83	8.12	2	8.12	8.24	1

 

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	7.20	8.30	21.5	612	249
24 hours	7.93	8.25	21.9	608	256

 

Gradient Table

Time [min]	A [%]	B [%]
0.00	80	20
0.30	80	20
1.30	5	95
2.00	5	95
2.10	80	20
2.50	80	20

 

Dilution steps

loading rate		[mL]	[mL]
[mg/L]
100	800	0.1^1)	10^1)
		0.25^2)	1.0^2)
100	200	0.1	10
100	100	0.1	5.0
50.0	800	0.1^1)	10^1)
		0.25^2)	1.0^2)
50.0	400	0.1^1)	10^1)
		0.5^2)	1.0^2)
50.0	200	0.1	10
50.0	100	0.1	5.0
50.0	50	0.2	5.0
25.0	400	0.1^1)	10^1)
		0.5^2)	1.0^2)
25.0	250	0.2^1)	5.0^1)
		0.2^2)	1.0^2)
25.0	100	0.1	5.0
25.0	50	0.2	5.0
12.5	250	0.2^1)	5.0^1)
		0.2^2)	1.0^2)
12.5	100	0.1	5.0
12.5	10	0.2	1.0
6.25	100	0.1	5.0
6.25	50	0.2	5.0
6.25	20	0.1	1.0
6.25	10	0.2	1.0

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

 

Requirements of the According to SANCO 3029/99 rev.4 (2000) and SANTE2020/12830
method validation Rev. 1 (24/02/2021) using the following criteria:

Parameter, Acceptance Criteria and Results of the Method Validation

Parameter	Acceptance criteria	Result
Linearity	≥ 5 standard concentrations,	0.05 to 0.8 µg test item/L (n = 7),	ü (=yes)
	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
Lowest calibration level (LCL)	S/N ≥ 9 for quantifier ion trace	S/N for 0.05 µg test item/L	ü
	S/N ≥ 3 for qualifier ion trace	C16 Amine + 2 EO
		159 (Quantifier), 109 (Qualifier)
		C18 Amine + 2 EO
		406 (Quantifier), 259 (Qualifier)
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)	Should be at or below lowest test concentration	0.200 µg test item/L (1 x LOQ)	ü
		200 µg test item/L (1000 x LOQ)
Accuracy^1)	Mean recovery rate of 70-120%	C16 Amine + 2 EO	ü
(Fortified samples)	per fortification level (2 levels)	1 x LOQ: 107% (n = 5)
		1000 x LOQ: 89% (n = 5)
		C18 Amine + 2 EO
		1 x LOQ: 106% (n = 5)
		1000 x LOQ: 88% (n = 5)

Precision^1)	Relative standard deviation ≤ 20% per fortification level	C16 Amine + 2 EO	ü
		1 x LOQ: 8.4%
		1000 x LOQ: 6.8%
		C18 Amine + 2 EO
		1 x LOQ: 6.1%
		1000 x LOQ: 6.1%
Specificity^1):	Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity).	C16 Amine + 2 EO	ü
LC-MS/(MS)	(Compounds: C16 Component + 2EO; C18 Component + 2EO)	quantifier [m/z]: 330.30 > 88.06
		qualifier [m/z]: 330.30 > 70.22
		C18 Amine + 2 EO
		quantifier [m/z]: 358.33 > 88.12
		qualifier [m/z]: 358.33 > 70.16
	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

 

 = criterion fulfilled
- = not determined

 

Preparation of Fortified Samples of the Test Item

LOQ Level	Control	1	1000
Stock solution	-	1000 mg test item/L in acetonitrile
Spiking solution	-	20.0	20 000
[µg test item/L]		(Dilution medium)	(Daphnia dilution medium)
(Medium)
Replicates	2	5	5
Concentration of the LOQ	-	0.200	200
[µg test item/L]
Medium for preparation	Daphnia dilution medium
Volume of spiking solution [mL]	-	0.05	0.05
Volume of medium [mL]	5.0	4.95	4.95
Dilution factor	2	2	400
Dilution medium	Acetonitrile containing 0.2% fomic acid^1)
	Dilution medium^2)
Sample volume [mL]	5.0^1)	5.0^1)	5.0^1)
			0.05^2)
Finale volume [mL]	10^1)	10^1)	10^1)
			10^2)

1) First dilution step
2) Second dilution step
Dilution medium = acetonitrile : daphnia dilution medium (50 : 50 v/v) containing 0.1% formic acid

 

Nominal Concentrations of the Fortified Samples of the active ingredients of 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4)
Fortified concentrations*: 0.202 µg test item/L (1 x LOQ) and 200 µg test item/L (1000 x LOQ).

Active ingredient	1 x LOQ	1000 x LOQ
	[µg a.s./L]	[µg a.s./L]
C16 Amine + 2 EO	0.0663	65.6
C18 Amine + 2 EO	0.123	122

* = weighing factor taken into account
a.s. = active substance

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4)

Replicate	2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4)
	C16 Amine + 2 EO				C18 Amine + 2 EO
	1 x LOQ		1000 x LOQ		1 x LOQ		1000 x LOQ
	Meas.	%	Meas.	%	Meas.	%	Meas.	%
	conc.		conc.		conc.		conc.
	[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
1	0.0709	107	52.6	80	0.131	107	105	86
2	0.0802	121	56.2	86	0.142	116	109	90
3	0.0729	110	63.2	96	0.126	103	114	94
4	0.0676	102	59.7	91	0.126	103	108	89
5	0.0643	97	58.8	90	0.122	99	97.0	80
Mean	0.071	107	58	89	0.130	106	107	88
SD ±	0.006		4		0.008		6
CV [%]	8.4		6.8		6.1		6.1

Meas. conc. = measured concentration of the active substance, dilution factor taken into account
a.s. = active substance
% = percent concentration of the fortified sample
SD = standard deviation
CV = coefficient of variation

 

Stability

The stability of the standard stock solution (1000 mg test item/L in acetonitrile) was checked after 15 days and was between 90 and 92% of the nominal concentration for each component.

The stability of the fortified samples at the 1xLOQ was checked after 2 days. The recovery to the nominal concentrations was 92% (C16 Amine + 2 EO) and 100% (C18 Amine + 2 EO).

Procedural Recovery

A procedural recovery (Quality Control) on 1x LOQ Level 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

Component	C16 Amine + 2 EO		C18 Amine + 2 EO
Analysis date	Meas. conc.	%	Meas. conc.	%
	[µg a.s.L]		[µg a.s.L]
16.03.2023	0.0115	172^1)	0.126	102
17.03.2023	0.0525	79	-	-
	0.0596	89	0.128	104
	0.0559	84	0.128	104
18.03.2023	0.0532	80	-	-
	0.0542	81	0.108	87
23.03.2023	0.0716	107	0.133	107

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
a.s. = active substance
Quality Control = 0.200 µg test item/L, weighing factor taken into account
1) = reanalyzed on 2023-03-17

 

Preliminary Range Finding Tests (non-GLP)
Solubility Test (non GLP)
WAFs with a nominal loading of 100 and 1000 µg/L were prepared as described. After 24, 48 and 72 hours of stirring samples were taken and analyzed via LC-MS.
A stirring phase of 24 hours was found to be suitable for the preparation of the WAFs. Longer stirring phases led to a decrease of the loading in the WAFs. Therefore, a stirring phase of 24 hours is chosen for the definitive test.

Measured Concentrations of of C16 Component of the Test Item during the Stability Test (non-GLP)
Analytical system: LC-MS

	2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4)
	(C16 Component + 2EO)
	Measured concentration [µg a.s./L]
Nominal test item loading	24 hours	48 hours	72 hours
[µg/L]
100	20.4	16.6	17.2
1000	164	Not determined

 

Measured Concentrations of of C18 Component of the Test Item during the Stability Test (non-GLP)
Analytical system: LC-MS

	2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4)
	(C18 Component + 2EO)
	Measured concentration [µg a.s./L]
Nominal test item loading	24 hours	48 hours	72 hours
[µg/L]
100	2.17	1.58	2.29
1000	13.9	Not determined

 

Range Finding Test (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 three WAFs at nominal loading rates of the test item of 1.00, 10.0 and 100 mg/L. The test vessels were pre-treated with the appropriate test solution for at least 12 hours under test conditions. For each loading rate an appropriate amount of stock solution containing the test item with methanol was placed on a curved glass slide. The methanol was evaporated. The glass slide with the test item will be inserted in a glass flask with an appropriate amount of dilution water. All WAFs and the control were visually clear and colorless throughout the exposure. The test item was analytically verified via LC-MS/MS in the WAFs and in the control in the fresh media at the start of the exposure (0 hours) and on the renewal of the test solutions (24 hours) and in the old media (24 and 48 hours).
In the range finding test, two replicates per loading rate and control, each with five daphnids, were tested.

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

Nominal loading of the	IMMOBILIZATION [%]
test item	24 hours			48 hours
[mg/L]	Replicates			Replicates
	1	2	MV	1	2	MV
100	100	100	100	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 of C16 Component of the Test Item during the non-GLP Preliminary Range Finding Test
Determination of the test item via LC-MS

Sampling date		Fresh media,	Old media,		Fresh media,	Old media,
		0 hours	24 hours		24 hours	48 hours
Nominal test item loading rates		2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4)
		(C16 Component + 2EO)
[mg/L]	[mg a.s./L]	Meas. conc.	Meas. conc.	%	Meas. conc.	Meas. conc.	%
		[µg a.s./L]	[µg a.s./L]		[µg a.s./L]	[µg a.s./L]
100	32.8	43.8	3.25	7	not determined
10.0	3.28	1.84	0.151	8	1.79	2.17	121
1.00	0.328	1.29	< LCL		0.0532	0.399	749
Control		< LCL	< LCL		< LCL	< LCL

% = Percent of the initially measured concentrations
a.s. = active substance
LCL = lowest calibration standard = 0.05 µg/L (0.0164 µg a.s./L)
Meas. Conc. = measured concentration of the test item, dilution factors taken into account

 

Measured Exposure Concentrations of C18 Component of the Test Item during the non-GLP Preliminary Range Finding Test
Determination of the test item via LC-MS

Sampling date		Fresh media,	Old media,		Fresh media,	Old media,
		0 hours	24 hours		24 hours	48 hours
Nominal test item loading rates		2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4)
		(C18 Component + 2EO)
[mg/L]	[mg a.s./L]	Meas. conc.	Meas. conc.	%	Meas. conc.	Meas. conc.	%
		[mg a.s./L]	[mg a.s./L]		[mg a.s./L]	[mg a.s./L]
100	60.8	0.0266	0.000436	2	not determined
10.0	6.08	0.0448	< LCL		0.00104	0.000515	80
1.00	0.608	< LCL	< LCL		< LCL	0.000362	-
Control		< LCL	< LCL		< LCL	< LCL

% = Percent of the initially measured concentrations
a.s. = active substance
LCL = lowest calibration standard = 0.05 µg/L (0.0304 µg a.s./L)
- = no intial measured concentration
Meas. Conc. = measured concentration of the test item, dilution factors taken into account

 

Fingerprint

The peak distribution of the test item was analyzed in fresh prepared medium in the highest test item concentration 100 mg/L (WAF approach) from the main study and the control. An analytical standard of the test item was prepared in acetonitrile and diluted to 10.0 mg/L with dilution medium. The highest test item concentration and the control were diluted factor 2 with acetonitrile containing 0.2% formic acid to avoid an inhomogeneous sample. The standard dilution and the test item dilution were analytical verified via MS and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared. The concentrations of the analytes C16 Amine + 2EO (330.30 Da ± 0.5 Da) and C18 Amine + 2EO (358.33 Da ± 0.5 Da) in the highest test item concentration are too low for significant signals. Both masses were found in the spectrum of the standard.
For analyses, the C16 and C18 amine + 2EO were chosen, because they were both found in the standard solution. During analysis of the test item solutions of the study, another detection mode (MRM) was chosen, because this is more specific and sensitive for concentration control analysis. This modus was suitable to detect both analytes. However, the MRM mode is not suitable for the scan of masses in the fingerprint.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

Based on the nominal loading rates, the 48 hours-EL50 for Daphnia magna was 51.1 mg/L (confidence limits: 26.3 – 97.3 mg/L) for the test item 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4).
Based on the geometric mean measured concentrations, the 48 hours-EC50 for Daphnia magna was 70.9 µg/L (confidence limits: 53.1 – 94.8 µg/L) for the test item 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4) (batch number: 20192048) were determined at the test facility according to OECD 202 (2004) from 2021-05-31 to 2022-03-23 with the definitive exposure phase from 2022-03-16 to 2022-03-18 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 6.25 to 100 mg/L set up in a geometric series with a factor of 2. The test containers were pre-treated with the appropriate test solution for at least 12 hours under test conditions. 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 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4) was quantified by measurement of the C16 and C18 amine + 2EO constituents. The concentrations of the test item 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 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4) at the end of exposure after 24 and 48 hours were in the range of 14 to 82% of the initially measured test item concentrations.
The geometric mean measured test item concentrations were calculated to be: 12.5 – 23.5 – 52.0 – 70.3 – 103 µg/L.

The EL10/50/100- values given in the table below are based on the nominal loading rates of the test item. Additionally, EC10/50/100-values based on the calculated geometric mean measured test item concentrations are given in the table below.
The validity criteria of the test guideline were fulfilled.

EC / EL10-, EC / EL50- and EC / EL100-Values

Effect values	Test	Nominal loading rates	Geometric mean measured test item concentrations
	duration	of the test item	[µg/L]
	[hours]	[mg/L]
EC / EL10	24	100 (Cl: 25.8 – > 100)	103 (Cl: 51.7 – > 103)
(with confidence limits)	48	47.5 (Cl: 25.0 – 95.9)	68.8 (Cl: 52.0 – 93.9)
EC / EL50	24	> 100	> 103
(with confidence limits)	48	51.1 (Cl: 26.3 – 97.3)	70.9 (Cl: 53.1 – 94.8)
EC / EL100	24	> 100	> 103
	48	> 100	> 103

 

 

Description of key information

There is one short term toxicity test available performed with the substance. 

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4) were determined according to OECD 202 (2004) under GLP conditions.

2,2’-(C16-18(even numbered) alkyl imino) diethanol (PFAEO-HT) is a multicomponent mixture (UVCB) of cationic surface-active constituents with different water solubilities. The fate of cationic surfactants in general deviates from standard chemicals. These substances are therefore considered as difficult substances for which the results of standard guideline studies are very difficult to interpret when considering them in a standard way. The reasons are the intrinsic properties like the relatively low water solubility and strong sorption to equipment and organisms. Classical ecotoxicity testing with these substances using reconstituted water often leads to test results which are poorly reproducible and are associated with high uncertainty. Because of the complex sorption mechanisms (van der Waals and Ionic mechanisms) the actual dissolved exposure concentration cannot reliably be estimated.

The acute daphnia test was therefore performed following the Water Accommodated Fraction (WAF) approach as described in “OECD guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (No. 23 Feb. 2019) with a daily refreshment of the test solutions.

The test solutions for the WAF approach were prepared separately by gentle mixing the test item with test medium for a prolonged period sufficient to ensure equilibration between the test item and the water phase with nominal loading rates of the test item in the range of 6.25 to 100 mg/L set up in a geometric series with a factor of 2. At the completion of mixing and following a settlement period, the WAF was separated by siphoning. This procedure was followed for each renewal of the test solutions.

Five WAFs were prepared and tested at nominal loading rates 6.25 – 12.5 – 25.0 – 50.0 – 100 mg/L (separation factor 2). No undissolved or emulsified material was observed in the WAF solutions based on the Tyndall effect check. The test vessels were pre-saturated with the appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure and the renewal, the test containers were emptied and refilled with freshly prepared test solution.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each loading rate and the control. The concentration of PFAEO-HT was quantified by measurement of the C16 and C18 amine + 2EO constituents (representing 96% of the actives). The concentration of the test item was 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 PFAEO-HT at the end of exposure after 24 and 48 hours were in the range of 14 to 82% of the initially measured test item concentrations. The geometric mean measured test item concentrations were calculated to be: 12.5 – 23.5 – 52.0 – 70.3 – 103 µg/L.

It should be noted that the geometric mean concentrations are given here even though per definition of the WAF, all terms related to concentration level should be given as “loading rates” because partly dissolved compounds and mixtures cannot be related to concentrations and considering the stability of the test substance and the daily renewal of the test solutions, it is considered justified to use the results based on nominal loading rates for Classification and Labeling purposes.

In Table 1 the results are presented based on nominal Test Loadings and on geometric mean measured concentrations based on quantification of the concentration of 2 main constituents representing 96% of the active ingredient. The observed EL50 and EC50 for mobility after 48 hours are respectively 70.9 µg/L and 51.1 mg/L.

 

 

 

 

Table 1:      EC / EL₁₀-, EC / EL₅₀- and EC / EL₁₀₀-Values

Effect values	Test duration [hours]	Nominal loading rates of the test item [mg/L]	Geometric mean measured test item concentrations [µg/L]
EC / EL10 (with confidence limits)	24	100 (Cl: 25.8 – > 100)	103 (Cl: 51.7 – > 103)
	48	47.5 (Cl: 25.0 – 95.9)	68.8 (Cl: 52.0 – 93.9)
EC / EL50 (with confidence limits)	24	> 100	> 103
	48	51.1 (Cl: 26.3 – 97.3)	70.9 (Cl: 53.1 – 94.8)
EC / EL100	24	> 100	> 103
	48	> 100	> 103

 

 

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

The effect concentration is based on nominal loading rate and not based on geometric mean measured concentration.

Effect concentration:

51.1 µg/L

Additional information

There is one short term toxicity test with the substance available. This study was conducted reliably according to the OECD 202 test guideline. Chemical analysis was conducted and a detailed Certificate of Analysis is included. Critical validity criteria were met. Some small but non critical discrepancies. Toxicity may be considered an overestimation for risk assesment purposes and sufficient for classification and labelling.