N-{3-[C12-14-(even numbered)-alkyloxy]propyl}propane-1,3-diamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2022-02-01 to 2022-02-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

via LC-MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) with algae

Details on sampling:

Determination of the test item
Samples were analyzed with a LC-MS method, implemented under non-GLP and documented finally in the GLP raw data. The method was validated according to Annex I. Additionally, the peak distribution of the WAF was analyzed in freshly prepared medium in the highest test item concentration once during the test. The signal distribution was compared with an analytical standard prepared in solvent.

Sampling schedule
All loading rates and the control were analytically verified via LC-MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) with algae.

Peak distribution (Finger print)
The peak distribu tion (fingerprint) of the WAF was analyzed in highest loading of the test item (50 µg/L) after 0 hours. The signal distribution was compared with an analytical standard prepared in solvent. Details of the method and results are given in section 20.

 
Sampling and pre-treatment At the start of the exposure, after 24 and after 48 hours, samples were taken from one additional replicate of each test item loading rate and the control. At the end of the exposure, samples were taken from pooled replicates.
Samples after 24, 48, 72 hours of exposure were centrifuged to separate the algae from the water phase. Both were analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae. The centrifuge tubes were rinsed with the appropriate test solutions to minimize adsorption to the walls of the centrifuge tubes.

Vehicle:

no

Details on test solutions:

Water Accommodated Fraction
Water accommodated fractions (WAFs) were prepared because the test item is a UVCB substance with compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2019) and ASTM D6081 (2019). Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. 

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 1.28 to 50.0 µg/L set up in a geometric series with a factor of 2.5: 1.28 – 3.20 – 8.00 – 20.0 – 50.0 µg/L, corresponding to the time weighted average concentrations of 0.499 – 0.632 – 2.01 – 6.79 – 14.8 µg/L.
The procedure according to ASTM D6081 (2019) as described below was carried out. The test item was melted at 80 °C before preparation of the WAFs. 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. 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 at room temperature, 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 WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative for all WAFs. The resulting water accommodated fractions (WAF) were used in the test.

Test loading
Per definition of the WAF all terms related to concentration level are given as 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):

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

Details on test organisms:

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

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

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

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

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

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

Test type:

static

Water media type:

freshwater

Remarks:

OECD TG 201 medium

Limit test:

no

Total exposure duration:

72 h

Hardness:

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

Test temperature:

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

pH:

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

Conductivity:

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 1.28 to 50.0 µg/L set up in a geometric series with a factor of 2.5: 1.28 – 3.20 – 8.00 – 20.0 – 50.0 µg/L, corresponding to the time weighted average concentrations of 0.499 – 0.632 – 2.01 – 6.79 – 14.8 µg/L.

Details on test conditions:

Control
The control solution was prepared without test item following the same method as specified for the WAFs. Six replicates were exposed under the same conditions as the test concentrations. 

Reference item
Potassium dichromate is tested twice per year as a reference item. Results of the most recent test was provided in the report.

Test method Static procedure

Duration of the test 72 hours

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

Test container and Pre-Treatment
Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs were used. 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, the test container was emptied and refilled with freshly prepared test solution. Preparation of the test solutions for coating was documented in the raw data and in the final report.

Test volume 100 mL

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

Dilution water According to the guidelines

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

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

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

Incubation
The flasks were positioned randomly and repositioned daily.

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

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

Light intensity (target)
Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

Light regime 24 hours/day light

Light homogeneity Within ± 15% over incubation area


Type and Frequency of Measurements
Biological Parameters

Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was observed in the preliminary range finding test at the loading rate of 500 µg/L. A calibration curve with a satisfactory correlation was used to calculate the cell density from fluorescence and given in the report.

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

Physico-chemical Properties

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

Reference substance (positive control):

yes

Remarks:

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number BCCC1619, purity 100.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2021-11-01 to 2021-11-04.

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

8.88 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

25.05 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

4.55 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

14.67 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.91 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

3.23 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

2.49 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

6.79 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

2.01 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

8.9 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

5.93 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

4.56 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

6.79 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

2.01 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

15.6 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

11.4 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

9.38 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

20 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

25.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

18 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

14.7 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

20 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Definitive Test
Biological Data
Microscopic evaluation of the cells at the start and the end of exposure revealed no morphological abnormalities. All effect values given are based on the nominal test item loading rates and the time weighted average concentrations of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2).

Physico-chemical Data

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

Measured Exposure Concentrations during the Definitive Test

The test item concentrations of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) were analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) with algae. Samples after 24, 48, 72 hours of exposure were centrifuged to separate the algae from the water phase. Both was analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae.
As the test item is a UVCB substance, all evaluations are based on the nominal loading rates of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2).
The peak distribution (fingerprint) of the WAF was analyzed in freshly prepared medium in the highest loading of the test item (50 µg/L) after 0 hours. The signal distribution was compared with an analytical standard prepared in solvent.

Results with reference substance (positive control):

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

Reported statistics and error estimates:

EL-values and statistical EL10-,EL20- and EL50- values with confidence intervals of growth
analyses rate inhibition and for yield inhibition after 72 hours were calculated by sigmoidal dose-response regression with the GraphPad Prism Software.

NOEL and LOEL values NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. The Dunnett’s method was used for the statistical significance calculations. A Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normal distribution of data. The BROWN-FORSYTHE test was done for analysis of variance homogeneity. P-values for both Normality and Equal Variance tests are 0.01. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. Normality test and equal variance test were failed for growth rate, therefore the data were transformed (Y=Log(Y)).

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

Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Preparation of the WAFs in the Definitive Test

	Stock solution 1		Stock solution 2*
Stock solution	1000		100
[mg/L in MeOH]
Weighing out [mg]	10		1 mL of Stock solution 1
Volume of the stock solution [mL]	10		10
Volume of the stock solution placed on a curved glass slide [µL/L]	50.0	20.0	80.0	32.0	12.8
Final test item	50.0	20.0	8.00	3.20	1.28
Loading rate [µg/L]

* = These stock solution will be prepared out of stock solution 1

 

Cell Densities

Nominal test item loading rates	Replicate	Cell density [cells/mL]
[µg/L]	No.	0 hours	24 hours	48 hours	72 hours
50.0	1	6864	<LOQcell density	<LOQcell density	<LOQcell density
	2	6864	<LOQcell density	<LOQcell density	<LOQcell density
	3	6864	<LOQcell density	<LOQcell density	<LOQcell density
	Mean	6864	n.a.	n.a.	n.a.
20.0	1	6864	4318	4615	206903
	2	6864	6317	23702	268211
	3	6864	6427	28148	342698
	Mean	6864	5687	18822	272604
8.00	1	6864	17663	122300	1004039
	2	6864	19996	161748	1044093
	3	6864	23909	203723	1071554
	Mean	6864	20523	162590	1039895
Mrz 20	1	6864	26632	208806	1102657
	2	6864	26738	232267	1019017
	3	6864	25308	187962	1102657
	Mean	6864	26226	209678	1074777
Jan 28	1	6864	36872	272506	1057407
	2	6864	30678	238322	1099261
	3	6864	24772	218827	1140868
	Mean	6864	30774	243218	1099179
Control	1	6864	32073	259849	1083991
	2	6864	35111	256305	1197228
	3	6864	37952	289014	1120604
	4	6864	30199	256799	1082641
	5	6864	29187	226861	1083901
	6	6864	35788	261792	1046905
	Mean	6864	33385	258437	1102545

LOQcell density = Limit of quantification of the cell density (2280 cells/mL). n.a. = not applicable

 

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

Nominal test item loading rates	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[µg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
50.0	1		n.d.	100		n.d.	100
	2		n.d.	100		n.d.	100
	3		n.d.	100		n.d.	100
	Mean	(s)	n.d.	100	(s)	n.d.	100
20.0	1		1.14	33		200039	82
	2		1.22	28		261347	76
	3		1.30	23		335834	69
	Mean	(s)	1.22	28	(s)	265740	76
8.00	1		1.66	2		997175	9
	2		1.68	1		1037229	5
	3		1.68	1		1064690	3
	Mean	(ns)	1.67	1	(ns)	1033031	6
3.20	1		1.69	0		1095793	0
	2		1.67	2		1012153	8
	3		1.69	0		1095793	0
	Mean	(ns)	1.68	1	(ns)	1067913	3
1.28	1		1.68	1		1050543	4
	2		1.69	0		1092397	0
	3		1.70	-1		1134004	-4
	Mean	(ns)	1.69	0	(ns)	1092315	0
Control	1		1.69			1077127
	2		1.72			1190364
	3		1.70			1113740
	4		1.69			1075777
	5		1.69			1077037
	6		1.68			1040041
	Mean		1.69			1095681

n.d. = not determinable

 

NOEL, LOEL and ELx-values and 95% Confidence Intervals of
C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) (0 - 72 hours)
based on the nominal test item loading rates [µg/L]

	Inhibition of Growth Rate
NOEL	8.00
LOEL	20.0
ErL10	14.7 (10.9 – 17.5)
ErL20	18.0 (14.5 – 19.1)
ErL50	25.1 (22.5 – 34.1)
	Inhibition of Yield
NOEL	8.00
LOEL	20.0
EyL10	9.38 (7.82 – 11.9)
EyL20	11.4 (9.67 – 13.3)
EyL50	15.6 (13.8 – 16.8)

 

NOEL, LOEL and ELx-values and 95% Confidence Intervals of
C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) (0 - 72 hours)
based on the time weighted average concentration of the test item [µg/L]

	Inhibition of Growth Rate
NOEL	2.01
LOEL	6.79
ErL10	4.56 (3.05 – 5.66)
ErL20	5.93 (4.54 – 6.38)
ErL50	8.90 (7.91 – 11.4)
	Inhibition of Yield
NOEL	2.01
LOEL	6.79
EyL10	2.49 (1.95 – 3.39)
EyL20	3.23 (2.60 – 3.98)
EyL50	4.91 (4.21 – 5.40)

 

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

	Replicate No.	Specific Growth Rate [d^-1]			Mean	SD	CV	Mean CV [%]
		section-by-section			(0 - 72 hours)	±	[%]
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.54	2.09	1.43	1.69	0.355	21.0	19.2
	2	1.63	1.99	1.54	1.72	0.236	13.7
	3	1.71	2.03	1.36	1.70	0.338	19.9
	4	1.48	2.14	1.44	1.69	0.393	23.3
	5	1.45	2.05	1.56	1.69	0.320	19.0
	6	1.65	1.99	1.39	1.68	0.303	18.1
				Mean	1.69
				SD ±	0.02
				CV [%]	0.91

SD = Standard deviation CV = Coefficient of variation

 

Environmental Conditions

Nominal test item loading rates				pH-values
[µg/L]				Start; 0 hours	End; 72 hours
50.0				8.04	8.14
20.0				8.06	9.69
8.00				8.08	9.55
3.20				8.11	9.63
1.28				8.12	9.72
Control				8.23	9.68
Room temperature [°C]:		min.: 23.5		max.: 24.0		mean value: 23.8
	n = 19		mean value: 5462				CV [%]: 6.99%
Light intensity			range of the measured values: 4842 to 6063 equalling -11.4 to 11.0%
[lux]

CV = Coefficient of variation n = number of measuring points

Water parameters of the Dilution Water

Parameters of the dilution water (measured on 2021-11-09)
Conductivity	Total hardness	Acidity	Alkalinity	Total organic carbon
[µS/cm]	[mg CaCO3/L]	[mmol/L]	[mmol/L]	[mg C/L]
136	40	0.2	0.6	< LOQ*

*Limit of quantification = 2.00 mg C/L

 

Measured Exposure Concentrations during the Definitive Test

The test item concentrations of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) were analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) with algae. Samples after 24, 48, 72 hours of exposure were centrifuged to separate the algae from the water phase. Both was analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae.
As the test item is a UVCB substance, all evaluations are based on the nominal loading rates of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2).
The peak distribution (fingerprint) of the WAF was analyzed in freshly prepared medium in the highest loading of the test item (50 µg/L) after 0 hours. The signal distribution was compared with an analytical standard prepared in solvent.

Measured Concentrations of the compound C12 Diamine of the test item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) during the Definitive Test
(0, 24, 48, 72 hours)

Sampling date		Fresh medium,		Old medium,		Old medium,		Old medium,
		0 hours		24 hours		48 hours		72 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C12 ether diamine
test item	active substance	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
50.0	31.5	29.9	95	14.3	45	8.04	26	3.56	11
20.0	12.6	11.5	91	5.90	47	3.58	28	2.52	20
8.00	5.04	3.64	72	1.47	29	1.09	22	0.782	16
3.20	2.02	1.27	63	< LOQ		< LOQ		< LOQ
1.28	0.806	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C12 Diamine of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LOQ = limit of quantification (1.00 µg/L of the test item, corresponding to 0.630 µg a.s./L)

 

Measured Concentrations of the compound C12 Diamine of the test item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) (Algae adsorption and the water phase)
(24, 48, 72 hours)

Sampling date		Algae adsorption,		Algae adsorption		Algae adsorption
		24 hours		48 hours		72 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C12 ether diamine
test item	active substance	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
50.0	31.5	9.27	29	6.31	20	3.47	11
20.0	12.6	2.83	22	1.87	15	2.25	18
8.00	5.04	1.25	25	1.04	21	0.792	16
3.20	2.02	< LOQ		< LOQ		< LOQ
1.28	0.806	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Sampling date		Water phase,		Water phase,		Water phase,
		24 hours		48 hours		72 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C12 ethder diamine
test item	active substance	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
50.0	31.5	2.21	7	2.81	9	< LOQ
20.0	12.6	1.30	10	2.78	22	< LOQ
8.00	5.04	< LOQ		< LOQ		< LOQ
3.20	2.02	< LOQ		< LOQ		< LOQ
1.28	0.806	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C12 Diamine of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LOQ = limit of quantification (1.00 µg/L of the test item, corresponding to 0.630 µg a.s./L)

 

Measured Concentrations of the compound C14 Diamine of the test item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) during the Definitive Test
(0, 24, 48, 72 hours)

Sampling date		Fresh medium,		Old medium,		Old medium,		Old medium,
		0 hours		24 hours		48 hours		72 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C14 ether diamine
test item	active substance	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
50.0	10.0	5.71	57	1.89	19	1.45	14	0.607	6
20.0	4.00	2.67	67	0.805	20	0.619	15	0.451	11
8.00	1.60	0.831	52	0.257	16	0.238	15	< LOQ
3.20	0.640	0.231	36	< LOQ		< LOQ		< LOQ
1.28	0.256	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C14 Diamine of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LOQ = limit of quantification (1.00 µg/L of the test item, corresponding to 0.200 µg a.s./L)

 

Measured Concentrations of the compound C14 Diamine of the test item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) (Algae adsorption and the water phase)
(24, 48, 72 hours)

Sampling date		Algae adsorption,		Algae adsorption		Algae adsorption
		24 hours		48 hours		72 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C14 ether diamine
test item	active substance	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
50.0	10.0	0.816	8	1.05	11	0.528	5
20.0	4.00	0.468	12	0.347	9	0.407	10
8.00	1.60	0.212	13	0.233	15	< LOQ
3.20	0.640	< LOQ		< LOQ		< LOQ
1.28	0.256	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ
Sampling date		Water phase,		Water phase,		Water phase,
		24 hours		48 hours		72 hours
Nominal		C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
loading rate of the		C14 ether diamine
test item	active substance	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
50.0	10.0	0.835	8	0.553	6	< LOQ
20.0	4.00	0.284	7	0.439	11	< LOQ
8.00	1.60	< LOQ		< LOQ		< LOQ
3.20	0.640	< LOQ		< LOQ		< LOQ
1.28	0.256	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C14 Diamine of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LOQ = limit of quantification (1.00 µg/L of the test item, corresponding to 0.200 µg a.s./L)

 

Validity Criteria

The study meets the validity criteria of the guideline:

Validity Criteria

Validity Criterion	Required	This study
Increase of the cell growth in the control cultures	Exponentially, ≥ 16-fold corresponding to a specific growth rate of 0.92 day^-1	161-fold
		(specific growth rate 1.69 day^-1)
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures	≤ 35%	19.2%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤ 7%	0.91%

 

Gradient Table

Time [min]	A [%]	B [%]
0.00	85	15
0.50	85	15
1.50	5	95
2.50	5	95
2.60	85	15
3.00	85	15

 

Dilution steps of the samples

Nominal concentration of test item	Dilution factor*	Sample Volume	Final volume	Sampling date
[mg/L]		[mL]	[mL]
50.0	20	0.1	1.0	0 hours, 24 hours
50.0	10	0.2	1.0	48 hours
50.0	4	0.5	1.0	72 hours
20.0	10	0.2	1.0	0 hours, 24 hours
20.0	2	1.0	1.0	48 hours, 72 hours
8.00	2	1.0	1.0	each sampling point
3.20	2	1.0	1.0	each sampling point
1.28	2	1.0	1.0	each sampling point
Control	2	1.0	1.0	each sampling point

*including dilution factor 2

 

Further dilution steps of the algae samples

Nominal concentration of test item	Dilution factor*	Sample Volume	Final volume	Sampling date
[mg/L]		[mL]	[mL]
50.0	10	0.1	1.0	24 hours, 48 hours
50.0	4	0.25	1.0	72 hours
20.0	4	0.25	1.0	48 hours

*including enrichment factor 1

Further dilution steps of the water phase

Nominal concentration of test item	Dilution factor*	Sample Volume	Final volume	Sampling date
[mg/L]		[mL]	[mL]
50.0	20	0.1	1.0	48 hours
20.0	10	0.2	1.0	48 hours

*including dilution factor 2

 

Parameter, Acceptance Criteria and Results of the Method Validation

Parameter	Acceptance criteria	Result
Calibration	≥ 5 standard concentrations,	0.1 to 5.0 µg test item/L (n = 7),	ü
	r ≥ 0.99 (R^2≥ 0.9801 for 2^nd order regression)	R^2≥ 0.9801
	Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation
Matrix effects	Matrix effects are considered significant if they exceed ±20%.	Matrix effects: 15 to 28%	ü
		(matrix-matched calibration‑standards were used)
Lowest calibration level (LCL)	S/N ≥ 9 for quantifier ion trace	S/N for 0.1 µg standard/L	ü
	S/N ≥ 3 for qualifier ion trace	C12 Diamine
		5002 (Quantifier), 3382 (Qualifier)
		C14 Diamine
		460 (Quantifier), 481 (Qualifier)
Limit of Quantification (LOQ)	Should be at or below lowest nominal test concentration	1.0 µg test item/L (1 x LOQ)	ü
		60 µg test item/L (60 x LOQ)
Accuracy (Fortified samples)	Mean recovery rate of 70-120%	C12 Diamine	ü
	per fortification level (2 levels)	1 x LOQ: 106% (n = 5)
		60 x LOQ: 117% (n = 5)
		C14 Diamine
		1 x LOQ: 110% (n = 5)
		60 x LOQ: 117% (n = 5)
Precision	Relative standard deviation ≤ 20% per fortification level	C12 Diamine	ü
		1 x LOQ: 8.0%
		60 x LOQ: 8.6%
		C14 Diamine
		1 x LOQ: 14%
		60 x LOQ: 10%

Stability	Samples: Stable if 70 – 120% of nominal concentration	See section 17.1	ü
	Calibration standards: Stable if ±10% of freshly prepared standard
Specificity: LC-MS/(MS)	Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity).	C12 Diamine	ü
		quantifier [m/z]: 301.35 > 98.11
		qualifier [m/z]: 301.35 > 70.16
		C14 Diamine
		quantifier [m/z]: 329.38 > 98.17
		qualifier [m/z]: 329.38 > 312.45
	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	60
Stock solution	-	1000 mg test item/L in acetonitrile
Spiking solution	-	100	2000
[µg test item/L]		(acetonitrile)	(acetonitrile)
(Medium)
Replicates	2	5	5
Concentration of the LOQ	-	1.0	60
[µg test item/L]
Medium for preparation	Algae dilution water
Volume of spiking solution [mL]	-	0.05	0.15
Volume of medium [mL]	5.0	4.95	4.85
Dilution factor	2	2	100
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)
			0.2^3)
Finale volume [mL]	10^1)	10^1)	10^1)
			1.0^2)
			1.0^3)
Final expected concentrations for analyses	-	0.5 µg test item/L	0.6 µg test item/L
		corresponding to	corresponding to
		0.315 µg C12 Diamine/L,	0.378 µg C12 Diamine/L,
		0.100 µg C14 Diamine/L	0.120 µg C14 Diamine/L

Dilution medium: Algae dilution water : acetonitrile (50 : 50) containing 1% formic acid

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
Fortified concentrations*: 1.03 µg test item/L (1 x LOQ) and 62.0 µg test item/L (60 x LOQ), corresponding to 0.651 µg C12 Diamine/L and 0.207 µg C14 Diamine/L (1 x LOQ) and 39.1 µg C12 Diamine/L and 12.4 µg C14 Diamine/L (60 x LOQ)

Replicate	C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
	C12 Diamine
	1 x LOQ		60 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]
1	0.594	91	42.5	109
2	0.714	110	42.4	109
3	0.736	113	47.0	120
4	0.697	107	51.9	133
5	0.709	109	44.7	114
Mean	0.69	106	46	117
SD ±	0.06		4
CV [%]	8.0		8.6
Replicate	C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
	C14 Diamine
	1 x LOQ		60 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]
1	0.235	113	13.0	105
2	0.243	117	15.2	122
3	0.175	85	13.2	106
4	0.227	110	16.6	134
5	0.255	123	14.8	119
Mean	0.23	110	15	117
SD ±	0.03		2
CV [%]	14		10

Meas. conc. = measured concentration of the test item, dilution factor taken into
Account
a.s. = active substance
% = percent concentration of the fortified sample
* = weighing factor taken into account
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 28 days and was 107 to 110% of the nominal concentration and was found to be stable over this period.

The responses of a 2 days old calibration standard (1 µg test item/L) was compared with the responses of a freshly prepared calibration standard with the same concentration. The deviation was > 10% for C14 Diamine and was not found to be stable over this period. Therefore, the calibration standards were freshly prepared of each day of analysis.

The stability of the fortified samples at the 1xLOQ was checked after 2 and after 7 days. Samples after 2 days were found to be stable over this period.

Measured Concentrations and Percent of Nominal Concentration of the stored fortified samples

	2 days		7 days
Component	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]
C12 Diamine	0.651	100	0.420	64
C14 Diamine	0.224	108	0.116	56

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
a.s. = active substance

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

Sampling	0 hours		24 hours		48 hours		72 hours
date
Component	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
C12 Diamine	0.607	93	0.473	73	0.631	97	0.537	83
C14 Diamine	0.185	89	0.160	78	0.217	105	0.171	83

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
a.s. = active substance
Quality Control = 1.00 µg test item/L, weighing factor taken into account

 

Range Finding Test (non-GLP)

Solubility Test

Two WAFs were prepared as described. After 24 and 48 hours of slow stirring, samples were taken as shown behind and analyzed via LC-MS. Two components were measured.

Measured Concentrations of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) during the Solubility Test (non-GLP)
Analytical system: LC-MS

	C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
	Components	24 hours		48 hours
Sampling date		Meas. conc.	%	Meas. conc.	%
		[µg/L]		[µg/L]
Nominal test item loading rate 500 µg/L	C12 Component	400	80	406	81
	C14 Component	239	48	233	47

% = Percent of the nominal test item concentration
LCL = lowest calibration level = 0.5 µg/L
Meas. conc. = measured concentration of the test item, dilution factors taken into account

A slow stirring phase of 24 hours was found to be sufficient.

 Preliminary Range Finding Test

A non-GLP preliminary range finding tests under static conditions over a period of 72 hours were conducted at the test facility. Water accommodated fractions (WAF) with loading rates of 5 – 50 – 500 µg test item/L were freshly prepared as described. The WAFs were prepared on day -2 and on day -1. All loading rates were tested and were clear and colorless throughout the exposure period. The Tyndall effect was negative.
For the preparation of the WAFs a stirring phase of 24 hours is chosen for the definitive test.

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

Nominal test item loading rates	Growth Rate Inhibition	Yield Inhibition
[µg/L]	[%]	[%]
500	100	100	50	100	100
5	2	10

 

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

Sampling	0 hours		72 hours		72 hours
	Start of the exposure		End of the exposure		End of the exposure
	(with algae)		(with algae)		(glass adsorption)
Nominal test item loading rates	C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
[µg/L]	(C12 Component)
	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
500	420	84	334	67	< LCL
50.0	47.5	95	5.51	11	6.10	12
5.00	3.02	60	< LCL		< LCL
Control	< LCL		< LCL		< LCL

% = Percent of the nominal test item concentration
LCL = lowest calibration level = 0.5 µg/L
Meas. conc. = measured concentration of the test item, dilution factors taken into account

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

Sampling	0 hours		72 hours		72 hours
	Start of the exposure		End of the exposure		End of the exposure
	(with algae)		(with algae)		(glass adsorption)
Nominal test item loading rates	C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2)
[µg/L]	(C14 Component)
	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
500	263	53	79.3	16	< LCL
50.0	24.0	48	< LCL		2.98	6
5.00	1.16	23	< LCL		< LCL
Control	< LCL		< LCL		< LCL

% = Percent of the nominal test item concentration
LCL = lowest calibration level = 0.5 µg/L
Meas. conc. = measured concentration of the test item, dilution factors taken into account

 

Fingerprint – MS spectrum

The peak distribution of the test item was analyzed in fresh prepared medium in the highest test item concentration 50.0 µg/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 acetonitrile. The highest test item concentration and the control was diluted factor 2 with acetonitrile containing 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 mass of C12 Ether Diamine (301.35 Da ± 0.5 Da) were found in both spectra. The concentration of C14 Ether Diamine in the highest test item concentration is too low for a significant signal. The mass of C14 Ether Diamine (329.38 Da± 0.5 Da) were found in the spectrum of the standard.
For analyses the C12 and C14 ether diamine 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.

Validity criteria fulfilled:

yes

Conclusions:

In this study, C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal test item loadings and time weighted average concentration of the test item):
Based on nominal loadings:
The EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 25.1 (22.5 – 34.1) µg/L and 15.6 (13.8 – 16.8) µg/L, respectively.

Based on time weighted average concentrations:
The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 8.90 (7.91 – 11.4) µg/L and 4.91 (4.21 – 5.40) µg/L, respectively.

Executive summary:

The toxicity of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) (batch no.: S20210420) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2022-01-30 to 2022-02-04, with the definitive exposure phase from 2022-02-01 to 2022-02-04 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6864 cells/mL. The test item is a UVCB substance with constituents of different water solubility and therefore five water accommodated fractions (WAFs) were prepared 24 ± 1 hour prior to the start of exposure with nominal loading rates of the test item in the range of 1.28 to 50.0 µg/L set up in a geometric series with a factor of 2.5: 1.28 – 3.20 – 8.00 – 20.0 – 50.0 µg/L, corresponding to the time weighted average concentrations of 0.499 – 0.632 – 2.01 – 6.79 – 14.8 µg/L. For preparation of the loading rates the test item was suspended in methanol (1000 and 100 mg/L). The test item with methanol was applied onto a curved glass slide and the methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water (see Table 3). A slow stirring procedure was applied for 24 ± 1 hour at room temperature. 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 WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. The resulting water accommodated fractions (WAF) were used in the test. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions during the test were within the acceptable limits. The test media were clear by eye during the exposure period of 72 hours.

The test item concentrations of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) were analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) with algae. Samples after 24, 48, 72 hours of exposure were centrifuged to separate the algae from the water phase. Both was analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae.

The test item is a UVCB substance with constituents of different water solubility which means that per definition of the WAF approach, the dose-response can only be based to loading rates because partly dissolved compounds and mixtures cannot be related to concentrations.

NOEL, LOEL and ELx-values and 95% Confidence Intervals of
C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) (0 - 72 hours)
based on the nominal test item loading rates [µg/L]

	Inhibition of Growth Rate
NOEL	8.00
LOEL	20.0
ErL10	14.7 (10.9 – 17.5)
ErL20	18.0 (14.5 – 19.1)
ErL50	25.1 (22.5 – 34.1)
	Inhibition of Yield
NOEL	8.00
LOEL	20.0
EyL10	9.38 (7.82 – 11.9)
EyL20	11.4 (9.67 – 13.3)
EyL50	15.6 (13.8 – 16.8)

 

NOEL, LOEL and ELx-values and 95% Confidence Intervals of
C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) (0 - 72 hours)
based on the time weighted average concentration of the test item [µg/L]

	Inhibition of Growth Rate
NOEL	2.01
LOEL	6.79
ErL10	4.56 (3.05 – 5.66)
ErL20	5.93 (4.54 – 6.38)
ErL50	8.90 (7.91 – 11.4)
	Inhibition of Yield
NOEL	2.01
LOEL	6.79
EyL10	2.49 (1.95 – 3.39)
EyL20	3.23 (2.60 – 3.98)
EyL50	4.91 (4.21 – 5.40)

 

 

 

Description of key information

The algae test has been performed by using the WAF approach. The EL50 for inhibition of specific growth rate was determined to be 25.05 µg/L (99.8% active) and the EL10 for inhibition of specific growt rate was determined to 14.67 µg/L (99.8% active). 

Key value for chemical safety assessment

EC50 for freshwater algae:

25.05 µg/L

EC10 or NOEC for freshwater algae:

14.67 µg/L

Additional information

One algae study for etherdiamine C12-14 is available. The study was performed under static conditionbs according to OECD TG 201 with specific chemical analysis (LC-MS/MS) under GLP conditions.

 

Etherdiamine C12-14 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 intrinsic properties like the relatively low water solubility and strong sorption to equipment and organisms. Classical ecotoxicity testing with this type of substance 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 algae test was performed according to 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) under static conditions. The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration.

 

The test solutions for the WAF approach were prepared by gentle mixing the test item with test medium for a prolonged period (24 hours) sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period (1 hour), the WAF was separated by siphoning. Five WAFs were prepared and tested at nominal loading rates 1.28 - 3.20 – 8.00 – 20.0 – 50.0 µg/L (separation factor 2.5), corresponding to the time weighted mean measured test item concentrations were 0.499 – 0.632 – 2.0 – 6.79 – 14.8 µg/L. No undissolved or emulsified material was observed in the WAF solutions based on the Tyndall effect check. Adsorptive losses to glass vessels was kept as low as possible by pre-conditioning the test vessels already with appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure, the test containers were emptied and refilled with freshly prepared test solutions.

The results are presented based on nominal test loadings and on time weighted average (TWA) measured concentrations. The EC₅₀-values for inhibition of growth rate (E_rC₅₀) and yield (E_yC₅₀) after 72 hours were 8.90 (7.91 – 11.4) µg/L and 4.91 (4.21 – 5.40) µg/L, respectively. The EC₁₀-values for inhibition of growth rate (E_rC₁₀) and yield (E_yC₁₀) after 72 hours were 4.56 (3.05 – 5.66) µg/L and 2.49 (1.95 – 3.39) µg/L, respectively. The TWA results are given despite the fact that per definition of the WAF, all terms related to concentration level should be given as loading rates (mass-to-volume ratio of the substance to the medium) because partly dissolved compounds and mixtures cannot be related to concentrations. Analytical verifications of selected components can be helpful and deliver supporting information, but they do not represent the whole test substance and therefore, toxicity results will be evaluated based on WAF loading rate (Wheeler, Lyon et al. 2020). Several guidance documents suggest to use the WAF loading rate for the environmental hazard classification of chemical substances e.g. the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (OECD 2002, OECD 2019) as well as OECD guidance documents on the classification of chemicals which are Hazardous for the Aquatic Environment

 

 

 

References:

OECD (2002). Guidance Document on the Use of the Harmonised System for the Classification of Chemicals which are Hazardous for the Aquatic Environment.

 

OECD (2019). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.

 

Wheeler, J. R., D. Lyon, C. Di Paolo, A. Grosso and M. Crane (2020). "Challenges in the regulatory use of water-accommodated fractions for assessing complex substances." Environmental Sciences Europe 32(1): 1-10.