Fatty acids, C18 unsat, reaction products with diethylenetriamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2021-02-07 to 2021-02-12, with the definitive exposure phase from 2021-02-09 to 2021-02-12

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item
Fatty acids, C18 unsat, reaction products with diethylenetriamine

Commercial name
ARMOHIB Cl-219

Batch number
1865159

CAS No.
1226892-43-8

Purity (certified)
100% UVCB

Appearance
Liquid, amber, green

Water Solubility
Critical micelle concentration (CMC): 99 mg/L (pH 7, 23 °C)

Stability under test conditions
Not specified

Expiry date
2022-07-08

Recommended storage
Keep container tightly closed in a dry and well ventilated place under nitrogen.

Analytical monitoring:

yes

Remarks:

via LC-MS/MS

Details on sampling:

Determination of the test item
Samples were analyzed with a LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data.

Sampling schedule
All loading rates and the control 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. Additionally, the loading rate 50.0 µg/L was 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) without algae. Additionally, adsorption to glass was analytically verified from all loading rates at the end of the exposure.
The peak distribution of the WAF was analyzed in fresh prepared medium in the highest loading rate, at the start of the exposure. Therefore, a high resolution MS (Q-ToF) was used.

Sampling and pre-treatment
At the start, after 24 and after 48 hours samples were taken from one additional replicate of each test item loading rate and the control and at the end samples were taken from pooled replicates.
For the adsorption to glass the vessels were rinsed carefully with demineralized water before analysis to remove adhering algae.
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.

Details on test solutions:

Water Soluble Fraction
Water accommodated fractions (WAFs) 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) and ASTM D6081 (2014). 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
Six water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 1.58 to 500 µg/L set up in a geometric series with a factor of √10: 1.58 – 5.00 – 15.8 – 50.0 – 158 – 500 µg/L, corresponding to the time-weighted average concentrations of 0.700 – 0.915 – 1.07 – 1.29 – 7.85 – 152 µg/L. The procedure according to ASTM D6081 (2019) as described below was carried out. For each loading rate an appropriate amount of stock solutions containing the test item in 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 center 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.

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:

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:

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

Limit test:

no

Total exposure duration:

72 h

Hardness:

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

Test temperature:

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

pH:

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

Nominal and measured concentrations:

Six water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 1.58 to 500 µg/L set up in a geometric series with a factor of √10: 1.58 – 5.00 – 15.8 – 50.0 – 158 – 500 µg/L, corresponding to the time-weighted average concentrations of 0.700 – 0.915 – 1.07 – 1.29 – 7.85 – 152 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 item loading rates.

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 loading rate.

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.

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: 5375 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 5000 µg/L. A calibration curve with a satisfactory correlation was used to calculate the cell density from fluorescence.

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 per test item loading rate and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested twice per year as a reference item. Results of the most recent test see section "Any other information on results incl. tables" below.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.12 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

21.2 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.04 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

13 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.931 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

6.37 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.915 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

5 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.7 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1.58 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.27 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.:

44.7 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.17 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

28 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.11 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.:

20.7 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.07 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

15.8 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.915 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

5 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

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 of Fatty acids, C18 unsat, REACTION PRODUCTS WITH DIETHYLENETRIAMINE. For details see section "Any other information on results incl. tables" below.

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

The test item concentrations of Fatty acids, C18 unsat, reaction products with diethylenetriamine 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 were 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 Fatty acids, C18 unsat, reaction products with diethylenetriamine.

Range Finding Test (non-GLP)

Solubility Test

Two WAFs was prepared as described. After 24, 48 and 72 hours samples were taken as shown behind and analyzed via LC-MS.

Preliminary Range Finding Test

A non-GLP preliminary range finding test under static conditions over a period of 72 hours was conducted at the test facility with three water accommodated fractions (WAFs) of the test item with nominal loadings of 50.0 – 500 - 5000 µg/L.
Each WAF was prepared as described in section 4.2 with a stock solution of 50 g/L in MeOH and application of 100 – 10 – 1 µL/L on a curved glass slide. The Tyndall effect was concentration related positive. In the range finding test, two replicates per concentration and four for the control were tested.
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. Therefore, a stirring phase of 24 hours is chosen for the definitive test.

MS-Fingerprints (non-GLP)

The peak distribution of the WAF was analyzed in fresh prepared medium in the highest test item concentration 500 µg/L (Figure 14). An analytical standard of the test item was prepared in acetonitrile and diluted to 160 µg/L with acetonitrile. The highest test item concentration was diluted factor 2 with acetonitrile containing 2% trifluoroacetic 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. In both solutions two main groups could be identified, around 349 m/z and 611 m/z. The ratio of the higher masses compared to the lower ones is higher in the standard compared to the WAF concentration of 500 µg/L. No additional groups could be identified.

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 2020-10-20 to 2020-10-23. 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/EC-values and statistical analyses
EL/EC10-,EL/EC 20- and EL/EC 50- values with confidence intervals of growth rate inhibition and for yield inhibition after 72 hours were calculated by sigmoidal dose-response regression with the GraphPad Prism Software.

NOEL/NOEC, LOEL/LOEC and analyses
The NOEL/NOEC and LOEL/LOEC was determined by calculation of statistical significance of growth rate and yield. The following statistical tests were conducted: Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.

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

Multiple Sequentially-rejective Welch-t-test after Bonferroni-Holm was done with a significance level of 0.05 for growth rate.

Williams Multiple Sequential t-test Procedure was performed with a significance level of 0.05 for yield.

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

Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.
• ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH

NOEL/NOEC, LOEL/LOEC, ELx/ECx-values of ARMOHIB Cl-219(0 - 72 hours)
based on nominal test item loadings and time-weighted average concentrations [µg/L]

	Growth Rate Inhibition
	Nominal test item loading [µg/L]	Time-weighted average
		concentration [µg/L]
NOEL/NOEC	5.00	0.915
LOEL/LOEC	15.8	1.07
ErL10/ErC10	20.7 (16.8 – 26.1)	1.11 (1.07 – 1.15)
ErL20/ErC20	28.0 (23.6 – 32.3)	1.17 (1.14 – 1.19)
ErL50/ErC50	44.7 (41.9 – 47.0)	1.27 (1.25 – 1.28)
	Yield Inhibition
	Nominal test item loading [µg/L]	Time-weighted average
		concentration [µg/L]
NOEL/NOEC	1.58	0.700
LOEL/LOEC	5.00	0.915
EyL10/EyC10	6.37 (< 1.58 – 10.6)	0.931 (< 0.700 – 1.00)
EyL20/EyC20	13.0 (10.9 – 14.8)	1.04 (1.01 – 1.06)
EyL50/EyC50	21.2 (19.0 – 25.3)	1.12 (1.10 – 1.15)

 

Cell Densities

Nominal test item loading rates	Replicate	Cell density [cells/mL]
[µg/L]	No.	0 hours	24 hours	48 hours	72 hours
500	1	5375	<LOQcell density	<LOQcell density	<LOQcell density
	2	5375	<LOQcell density	<LOQcell density	<LOQcell density
	3	5375	<LOQcell density	<LOQcell density	<LOQcell density
	Mean	5375	n.a.	n.a.	n.a.
158	1	5375	<LOQcell density	<LOQcell density	<LOQcell density
	2	5375	<LOQcell density	<LOQcell density	<LOQcell density
	3	5375	<LOQcell density	<LOQcell density	<LOQcell density
	Mean	5375	n.a.	n.a.	n.a.
50.0	1	5375	5092	7924	46045
	2	5375	2677	12943	76307
	3	5375	2313	4725	37896
	Mean	5375	3361	8531	53416
15.8	1	5375	15992	106033	790374
	2	5375	16456	108114	907369
	3	5375	12510	93717	829323
	Mean	5375	14986	102621	842355
5.00	1	5375	10829	90199	974027
	2	5375	11628	100569	987051
	3	5375	23078	127408	1107613
	Mean	5375	15178	106059	1022897
1.58	1	5375	22976	134856	1130631
	2	5375	20608	131005	1035848
	3	5375	24234	137173	1198634
	Mean	5375	22606	134345	1121704
Control	1	5375	22428	153300	1213515
	2	5375	22609	138160	1246722
	3	5375	23748	139440	1082812
	4	5375	19821	126558	1040857
	5	5375	25199	143946	1192598
	6	5375	23784	151433	1317119
	Mean	5375	22932	142140	1182271

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]		[%]
500	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
158	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
50.0	1		0.716	60		40670	97
	2		0.884	51		70932	94
	3		0.651	64		32521	97
	Mean	(s)	0.750	58	(s)	48041	96
15.8	1		1.66	7		784999	33
	2		1.71	5		901994	23
	3		1.68	7		823948	30
	Mean	(s)	1.68	6	(s)	836980	29
5.00	1		1.73	4		968652	18
	2		1.74	3		981676	17
	3		1.78	1		1102238	6
	Mean	(s)*	1.75	3	(s)	1017522	14
1.58	1		1.78	1		1125256	4
	2		1.75	2		1030473	12
	3		1.80	0		1193259	-1
	Mean	(ns)	1.78	1	(ns)	1116329	5
Control	1		1.81			1208140
	2		1.82			1241347
	3		1.77			1077437
	4		1.76			1035482
	5		1.80			1187223
	6		1.83			1311744
	Mean		1.80			1176896

* = statistically significant but biologically not relevant (inhibition < 5%)

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

	Replicate No.	Specific Growth Rate [d^-1]			Mean	SD	CV	Mean CV [%]
		section-by-section			(0 - 72 hours)	±	[%]
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.43	1.92	2.07	1.81	0.335	18.6	18.9
	2	1.44	1.81	2.20	1.82	0.382	21.0
	3	1.49	1.77	2.05	1.77	0.282	15.9
	4	1.31	1.85	2.11	1.76	0.410	23.4
	5	1.55	1.74	2.11	1.80	0.289	16.1
	6	1.49	1.85	2.16	1.83	0.338	18.4
				Mean	1.80
				SD ±	0.03
				CV [%]	1.64

SD = Standard deviation CV = Coefficient of variation

 

Environmental Conditions

Nominal test item loading rates				pH-values
[µg/L]				Start; 0 hours	End; 72 hours
500				8.40	7.91
158				8.37	7.95
50.0				8.42	8.14
15.8				8.43	8.40
5.00				8.22	8.49
1.58				8.28	8.66
Control				8.19	8.79
Room temperature [°C]:		min.: 22.0		max.: 23.0		mean value: 22.5
	n = 20		mean value: 5552				CV [%]: 6.29
Light intensity			range of the measured values: 5021 - 6121
[lux]			equalling -9.56 to 10.3

CV = Coefficient of variation n = number of measuring points

 

Water parameters of the Dilution Water

Parameters of the dilution water (measured on 2020-10-28)
Conductivity	Total hardness	Acidity	Alkalinity	Total organic carbon
[µS/cm]	[mg CaCO3/L]	[mmol/L]	[mmol/L]	[mg C/L]
142	37	0.2	0.6	< LOQ*

*Limit of quantification = 2.00 mg C/L

 

Measured Concentrations of the Test Item during the Definitive Test
(Algae and Glass adsorption)

Sampling date	Day 1		Day 2		Day 3		Day 3
	Old medium		Old medium		Old medium		Old medium
	24 hours		48 hours		72 hours		72 hours
							Glass Adsorption
Nominal	Fatty acids, C18 unsat, reaction products with diethylenetriamine
concentration
of the test item	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[µg/L]	conc.		conc.		conc.		conc.
	[µg/L]		[µg/L]		[µg/L]		[µg/L]
500	54.5	11	49.2	10	34.5	7	11.6	2
158	41.7	26	37.6	24	34.7	22	14.7	9
50.0	5.22	10	< LOQ		2.11	4	3.40	7
15.8	< LOQ		< LOQ		< LOQ		1.06*	7*
5.00	< LOQ		< LOQ		< LOQ		< LOQ
1.58	< LOQ		< LOQ		< LOQ		< LOQ
Control	< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration of the test item
LOQ = limit of quantification (1.40 µg/L of the test item)
* = < LOQ but > 70% of the LOQ

Measured Concentrations of the Test Item during the Definitive Test

Sampling date	Day 0			Day 1
	Fresh medium			Old medium
	0 hours			24 hours
Nominal	Fatty acids, C18 unsat, reaction products with diethylenetriamine
concentration
of the test item	Meas. conc.	%		Meas. conc.		%
[µg/L]	[µg/L]			[µg/L]
500	338	68		140		28
158	111	71		7.52		5
50.0	27.1	54		< LOQ
50.0^1)	32.1	64		4.70		9
15.8	8.82	56		< LOQ
5.00	3.49	70		< LOQ
1.58	< LOQ			< LOQ
Control	< LOQ			< LOQ
Sampling date	Day 2				Day 3				Time-weighted average concentrations
	Old medium				Old medium
	48 hours				72 hours
Nominal	Fatty acids, C18 unsat, reaction products with diethylenetriamine
concentration
of the test item	Meas. conc.		%		Meas. conc.		%		[µg/L]
[µg/L]	[µg/L]				[µg/L]
500	121		24		126		25		152
158	2.10		1		8.47		5		7.85
50.0	< LOQ				< LOQ				1.29
50.0^1)	5.91		12		10.1	20			-
15.8	< LOQ				< LOQ				1.07
5.00	< LOQ				< LOQ				0.915
1.58	< LOQ				< LOQ				0.700
Control	< LOQ				< LOQ				-

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration of the test item
LOQ = limit of quantification (1.40 µg/L of the test item)
1) = test item concentration without algae

 

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

	Current Study	Valid Range (average ± 3 x SD)
	Growth Rate inhibition
ErC50	0.903	0.775 ± 0.558
95% confidence interval	0.858 – 0.946
	Yield inhibition
EyC50	0.441	0.422 ± 0.339
95% confidence interval	0.421 – 0.462

SD = Standard deviation

 

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	220-fold
		(specific growth rate 1.80 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%	18.9%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤ 7%	1.63%

 

MS Parameter

Parent Ion (Da)	Daughter Ion (Da)	Cone voltage (V)	Collision energy (eV)
104.08	87.09	20	8
346.28	97.15	66	48
	303.25		28
348.30	97.90	60	48
	305.26		28
350.31	97.09	68	48
	307.28		30
364.29	97.40	52	48
	304.22		22
366.31	306.24	48	22
	348.39		16
368.32	308.25	48	24
	350.46		16
606.50	97.34	74	68
	303.42		40
608.51	97.34	72	72
	302.96		42
610.53	97.27	74	78
	305.44		42
612.54	97.27	80	78
	305.05		42
614.56	97.27	80	80
	307.58		42
624.57	97.40	72	76
	319.48		44
626.59	97.93	74	52
	321.50		44
628.54	98.00	72	54
	321.69		42
630.55	98.00	68	54
	306.02		40
632.57	307.97	70	44

 

Dilution Steps

Nominal	Dilution	Sample	Final
concentration	Factor*	volume	volume
[µg/L]		[mL]	[mL]
500 (day 0+1)	200	0.05	5.0
500 (day 2)	40	0.05	1.0
500 (day 3)	20	0.1	1.0
158 (day 0)	20	0.1	1.0
158 (day 1+2+3)	2	1.0	1.0
50.0 (day 0)	10	0.2	1.0
50.0 (day 1+2+3)	2	1.0	1.0
15. 8	2	1.0	1.0
5.00	2	1.0	1.0
1.58	2	1.0	1.0
Control	2	1.0	1.0

*including factor 2

 

Dilution Steps (Algae Samples)

Nominal	Enrichment factor*	Sample	Final
concentration		volume	volume
[µg/L]		[mL]	[mL]
500 (day 1+3)	10	0.1	1.0
500 (day 2)	25	0.2^1)	1.0^1)
		0.2^2	1.0^2)
500 (day 2)	5	0.2	1.0
158	5	0.2	1.0

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

 

Dilution Steps (Glass adsorption)

Nominal	Enrichment factor*	Sample	Final
concentration		volume	volume
[µg/L]		[mL]	[mL]
500	0.2	0.2	1.0
158	0.2	0.2	1.0

*including enrichment factor 1

 

Method Validation (non-GLP)
Requirement of the According to SANCO 3029/99 rev.4 (2000) using following
method validation criteria.

Parameter	Acceptance criteria	Result
Linearity	5 standard concentrations,	0.5 to 10 µg test item/L (n = 7),	ü
	r ≥ 0.99	r ≥ 0.99
Lowest calibration standard	S/N ≥ 9 for quantifier ion trace	0.5 µg test item/L,	ü
		S/N: 241
Limit of Detection (LOD)	Determination only necessary when S/N LCL ≤ 30.	No necessary, S/N LCL ≥ 30	-
	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 level after sample	1.40 µg test item/L (1 x LOQ)	ü
	preparation	601 µg test item/L (429 x LOQ)
Accuracy^1)	Mean recovery rate of 70-110% (ideally 80-100%)	1 x LOQ: 94 % (n = 5)	ü
(Fortified samples)	per fortification level (2 levels)	429 x LOQ: 108 % (n = 5)
Precision^1)	Relative standard deviation ≤ 20% per fortification level	1 x LOQ: 7.0 %	ü
		429 x LOQ: 3.9 %
Specificity	Total Ion Count (TIC) of 17 compounds of the test item with 32 different daughter ions.	For detailed ions please refer to	ü
(LC-MS/MS)		Table 13
	Blank values < 30 % of LOQ	Blank values < 30% of LOQ
Procedural recovery	Procedural recoveries with experimental samples. Ideally, the recovery should be in the range of the mean recovery +/- 2x relative standard deviation of the 1xLOQ in comparison to the method validation. If the criterion is not matched, the recovery has to be at least 70-110 % of the nominal value.	For details, see section 17.1	ü

 = criterion fulfilled
- = not determined

 

Preparation of Fortified Samples

LOQ Level	Control	1	429
Stock solution	-	1000
[mg test item/L]		(Acetonitrile)
(Medium)
Spiking solution	-	0.140	60.0
[mg test item/L]		(Dilution medium)	(Dilution medium)
(Medium)
Replicates	2	5	5
Concentration of the LOQ	-	1.40	600
[µg test item/L]
Medium for preparation	Dilution medium
Volume of spiking solution [mL]	-	0.05	0.05
Volume of medium [mL]	10	9.95	9.95
Dilution factor	2	2	100
Dilution medium	Dilution medium
Sample volume [mL]	10^1)	10^1)	10^1)
			0.1^2)
Finale volume [mL]	10^1)	10^1)	10^1)
			5^2)

Dilution medium = Acetonitrile : algae dilution medium (50 . 50) containing 1% trifluoro acetic acid
1) First dilution step (dilution factor 2 included)
2) Second dilution step

 

Recovery Rates of the Fortified Samples of the Test Item
Fortified concentrations*:
1.40 µg test item/L (1 x LOQ) and 601 µg test item/L (429 x LOQ)

Replicate	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	1 x LOQ		429 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[µg/L]		[µg/L]
1	1.23	88	635	104
2	1.33	95	660	108
3	1.34	96	644	105
4	1.46	104	700	114
5	1.25	89	678	111
Mean	1.32	94	663	108
SD ±	0.09		26
CV [%]	7.0		3.9

Meas. conc. = measured concentration, dilution factor taken into account
% = percent of the fortified concentration
* = weighing factor taken into account

SD = standard deviation
CV = coefficient of variation

 

Procedural Recovery

A procedural recovery (Quality Control) on 1x LOQ Level were freshly prepared on each day of analysis. They were 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
date
Quality Control	Fatty acids, C18 unsat, reaction products with diethylenetriamine
[µg/L]	Meas. conc.	%	Meas. conc.	%
	[µg/L]		[µg/L]
1.40	1.31	94	1.36	98
Sampling	48 hours		72 hours
date
Quality Control	Fatty acids, C18 unsat, reaction products with diethylenetriamine
[µg/L]	Meas. conc.	%	Meas. conc.	%
	[µg/L]		[µg/L]
1.40	1.41	101	1.52	109

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

 

Range Finding Test (non-GLP)

Solubility Test

Two WAFs was prepared as described. After 24, 48 and 72 hours samples were taken as shown behind and analyzed via LC-MS.

Measured Concentrations of ARMOHIB Cl-219 during the Stability Test (non-GLP)
Analytical system: LC-MS

	ARMOHIB Cl-219
	Measured concentration [µg/L]
Sampling date	24 hours	48 hours	72 hours
Nominal test item loading rate 1000 µg/L	354	300	238
Nominal test item loading rate 10 µg/L	< LCL	< LCL	< LCL

LCL = lowest calibration standard = 5 µg/L

 

Preliminary Range Finding Test

A non-GLP preliminary range finding test under static conditions over a period of 72 hours was conducted at the test facility with three water accommodated fractions (WAFs) of the test item with nominal loadings of 50.0 – 500 - 5000 µg/L.
Each WAF was prepared as described in section 4.2 with a stock solution of 50 g/L in MeOH and application of 100 – 10 – 1 µL/L on a curved glass slide. The Tyndall effect was concentration related positive. In the range finding test, two replicates per concentration and four for the control were tested.
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. Therefore, a stirring phase of 24 hours is chosen for the definitive test.

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

Nominal test item loadings	Growth Rate Inhibition	Yield Inhibition
[µg/L]	[%]	[%]
5000	100	100
500	100	100
50	10	43

 

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

Sampling	0 hours		72 hours
	Start of the exposure		End of the exposure
	(with algae)		(with algae)
Nominal loading level of the water accommodated fraction	Fatty acids, C18 unsat, reaction products with diethylenetriamine
[µg/L]	Meas. conc.	%	Meas. conc.	%
	[µg/L]		[µg/L]
5000	4194	84	2883	58
500	399	80	174	35
50	25.5	51	2.12	4
Control	< LCL		< LCL

% = Percent of the nominal test item concentration
LCL = lowest calibration standard = 1 µg/L
Meas. Conc. = measured concentration of the test item

 

 

 

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

In this study, Fatty acids, C18 unsat, REACTION PRODUCTS WITH DIETHYLENETRIAMINE 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 [ELx] and on time-weighted average concentrations [ECx]):
The EL10-values for inhibition of growth rate (ErL10) after 72 hours were 20.7 (16.8 – 26.1) µg/L.
The EL10-values for inhibition of yield (EyL10) after 72 hours were 6.37 (< 1.58 – 10.6) µg/L.
The EL50-values for inhibition of growth rate (ErL50) after 72 hours were 44.7 (41.9 – 47.0) µg/L.
The EL50-values for inhibition of yield (EyL50) after 72 hours were 21.2 (19.0 – 25.3) µg/L.

The EC10-values for inhibition of growth rate (ErC10) after 72 hours were 1.11 (1.07 – 1.150) µg/L.
The EC10-values for inhibition of yield (EyC10) after 72 hours were 0.931 (< 0.700 – 1.00) µg/L.
The EC50-values for inhibition of growth rate (ErC50) after 72 hours were 1.27 (1.25 – 1.28) µg/L.
The EC50-values for inhibition of yield (EyC50) after 72 hours were 1.12 (1.10 – 1.15) µg/L.

Executive summary:

The toxicity of Fatty acids, C18 unsat, REACTION PRODUCTS WITH DIETHYLENETRIAMINE (Batch no.: 1865159) 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 2021-02-07 to 2021-02-12, with the definitive exposure phase from 2021-02-09 to 2021-02-12 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 5375 cells/mL. Six 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 1.58 to 500 µg/L set up in a geometric series with a factor of √10: 1.58 - 5.00 - 15.8 - 50.0 - 158 - 500 µg/L, corresponding to the time-weighted average concentrations of 0.700 – 0.915 – 1.07 – 1.29 – 7.85 – 152 µg/L. For preparation of the loading rates the test item was dissolved in methanol (5 and 0.500 g/L). The test item in 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. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. After a separation phase of one hour the WAFs were removed and checked via laser beam 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 loadings and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were visually clear throughout the test period. The concentrations of the test item Fatty acids, C18 unsat, REACTION PRODUCTS WITH DIETHYLENETRIAMINE were determined at the start of the exposure (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) of all tested loading levels and the control via LC-MS/MS with algae. Additionally, the loading rate 50.0 µg/L was analytically verified without algae determined at the start of the exposure (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) via LC-MS/MS.  All effect values are given as nominal loading rates and time-weighted average concentrations.

 

NOEL/NOEC, LOEL/LOEC, ELx/ECx-values of ARMOHIB Cl-219(0 - 72 hours)
based on nominal test item loadings and time-weighted average concentrations [µg/L]

	Growth Rate Inhibition
	Nominal test item loading [µg/L]	Time-weighted average
		concentration [µg/L]
NOEL/NOEC	5.00	0.915
LOEL/LOEC	15.8	1.07
ErL10/ErC10	20.7 (16.8 – 26.1)	1.11 (1.07 – 1.15)
ErL20/ErC20	28.0 (23.6 – 32.3)	1.17 (1.14 – 1.19)
ErL50/ErC50	44.7 (41.9 – 47.0)	1.27 (1.25 – 1.28)
	Yield Inhibition
	Nominal test item loading [µg/L]	Time-weighted average
		concentration [µg/L]
NOEL/NOEC	1.58	0.700
LOEL/LOEC	5.00	0.915
EyL10/EyC10	6.37 (< 1.58 – 10.6)	0.931 (< 0.700 – 1.00)
EyL20/EyC20	13.0 (10.9 – 14.8)	1.04 (1.01 – 1.06)
EyL50/EyC50	21.2 (19.0 – 25.3)	1.12 (1.10 – 1.15)

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-02-16 to 2010-03-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, All validity criteria fulfilled, complete identification of test substance, including chemical analyses

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: From the series of concentrations the second lowest and the second highest test concentration (0.320 and 3.20 mg/L) and the control were analytically verified at the beginning of the test.
- Sampling method: Separate replicates for the test item analysis after 0 and 72 h were prepared without algae. Analysis after 72 h of these concentrations were carried out. Sorption to the walls of the glass container was checked out of one test replicate (prepared with algae) of a concentration level close to the EC50-value (the second lowest test concentration of 0.320 mg/L was analysed).
- Sample storage conditions before analysis: The samples were stored under room temperature until start of analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshly prepared. Dispersion treatment was ultrasound for 5 min at room temperature. An equilibration phase for 1 h before application was carried out.
- Eluate: Natural water
- Differential loading: 0.100 - 0.320 - 1.00 - 3.20 - 10.0 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDAK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

After 72 h of exposure, 0.2 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from test concentrations 1.00 – 3.20 mg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 3 – 10 days under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 3.20 mg/L (second highest test concentration).
For results please refer to "Any other information on materials and methods".

Hardness:

Not measured

Test temperature:

Mean: 22.5 °C, Min: 22 °C, Max: 23 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
10.0 8.23 7.87
3.20 8.10 7.81
1.00 8.08 7.71
0.320 8.07 8.19
0.100 8.06 8.56
Control 8.07 8.64

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

please refer to information on materials and methods

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 9787 cells/mL
- Control end cells density: Mean 2221965 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: Yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Water of the river Leine was used. This river is located near D-31171 Nordstemmen, Marienbergstraße, Germany (52° 10’ 14.98‘’, 9° 46’ 7.64’’). Water parameters are given in Table 1. Additionally 50 % of the components concentrations of the dilution water (total application volume 6.5 mL/L) acc. to the guideline were added to enable a sufficient growth of algae
River Leine
Location D-31171 Nordstemmen, Marienbergstrasse
Sampling Date 2009-12-15
Weather on Day of Sampling cloudy, ca. - 1 °C
Colour Yellowish, clear
pH 7.97
Conductivity [µS/cm] 386
DOC [mg C/L] 3.9
TOC [mg C/L] 3.9
Ammonium-N [mg N/L] 0.042
Nitrate-N [mg N/L] 2.62
o-Phosphate-P [mg P/L] 0.062
Total Phosphate [mg P/L] 0.053
Suspended Matter [mg/L] 16.2
Total Hardness [mg CO3/L] 154

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0 – 72 h)
Results of the Range Finding Test (0 – 72 h)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition[%] Yield Inhibition [%]
10.0 100 100
1.00 28 76
0.10 - 1 - 5
0.01 - 1 - 6
0.001 1 3

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.343 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

94.4% a.i.

Basis for effect:

growth rate

Remarks on result:

other: (0.325 - 0.371)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.505 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

94.4% a.i.

Basis for effect:

growth rate

Remarks on result:

other: (0.461-0.568)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.82 (CI 0.78 - 0.85) mg/L
Yield Inhibition: 0.37 (CI 0.36 - 0.40) mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals of EC10-, EC20- and EC50-values were carried out from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

EC₁₀-_, EC₂₀-and EC₅₀- values and 95 % Confidence Intervals of
Tall oil diethylenetriamine imidazoline (CAS No. 68442-97-7) (0-72 h)

                                based on nominal test item concentrations [mg/L]

                               

	Rate-related inhibition
ErC10	0.343 (0.325 – 0.371)
ErC20	0.395 (0.372 – 0.436)
ErC50	0.505 (0.461 – 0.568)
	Inhibition of yield
EyC10	0.265 (0.249 – 0.277)
EyC20	0.295 (0.286 – 0.305)
EyC50	0.356 (0.347 – 0.367)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
10.0		1		9787		-426	-1047	0
		2		9787		-1220	-2222	0
		3		9787		-233	-2264	0
		Mean		9787		-626	-1844	0
3.20		1		9787		-2052	-2137	1520
		2		9787		-1742	-1977	380
		3		9787		-1690	-1789	760
		Mean		9787		-1828	-1968	887
1.00		1		9787		-562	1031	5722
		2		9787		975	1745	5327
		3		9787		1623	2958	13622
		Mean		9787		679	1911	8224
0.320		1		9787		37860	278636	1622761
		2		9787		39750	304557	1657894
		3		9787		32695	198158	1317148
		Mean		9787		36768	260450	1532601
0.100		1		9787		48125	569717	2285269
		2		9787		61459	628674	2188829
		3		9787		57248	553027	2240600
		Mean		9787		55611	583806	2238233
Control		1		9787		54164	640687	2232516
		2		9787		58145	665385	2138901
		3		9787		63141	667914	2230335
		4		9787		56905	633096	2290857
		5		9787		56914	642755	2150971
		6		9787		63677	653038	2288211
		Mean		9787		58824	650479	2221965

Evaluation after 72 h

             

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
10.0	1		0.000	100		-9787	100
	2		0.000	100		-9787	100
	3		0.000	100		-9787	100
	Mean		0.000	100		-9787	100
3.20	1		-0.621	100		-8267	100
	2		-1.083	100		-9407	100
	3		-0.852	100		-9027	100
	Mean		-0.852	100		-8900	100
1.00	1		-0.179	100		-4065	100
	2		-0.203	100		-4460	100
	3		0.110	93.9		3835	99.8
	Mean		-0.090	98.0		-1563	99.9
0.320	1		1.70	5.79		1612974	27.1
	2		1.71	5.39		1648107	25.5
	3		1.63	9.63		1307361	40.9
	Mean		1.68	6.94		1522814	31.2
0.100	1		1.82	-0.52		2275482	-2.86
	2		1.80	0.27		2179042	1.50
	3		1.81	-0.16		2230813	-0.840
	Mean		1.81	-0.14		2228446	-0.730
Control	1		1.81			2222729
	2		1.80			2129114
	3		1.81			2220548
	4		1.82			2281070
	5		1.80			2141184
	6		1.82			2278424
	Mean		1.81			2212178

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

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	VC %	Mean VC %
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	1.71	2.47	1.25	1.81	0.617	34.1	32.7
	2	1.78	2.44	1.17	1.80	0.635	35.4
	3	1.86	2.36	1.21	1.81	0.579	32.0
	4	1.76	2.41	1.29	1.82	0.564	31.0
	5	1.76	2.42	1.21	1.80	0.609	33.9
	6	1.87	2.33	1.25	1.82	0.539	29.6
				Mean	1.81
				SD ±	0.01
				VC %	0.54

Validity criteria fulfilled:

yes

Conclusions:

Tall oil diethylenetriamine imidazoline (CAS No. 68442-97-7) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values (nominal concentrations): The EC10-values with 95 % confidence intervals for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 0.343 (0.325 – 0.371) and 0.265 (0.249 – 0.277) mg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 0.505 (0.461 – 0.568) and 0.356 (0.347 – 0.367) mg/L, respectively.

The concentrations of Tall oil diethylenetriamine imidazoline were analysed at the concentration levels 0.320 and 3.20 mg/L (prepared without algae) and the control at test start via LC-MS/MS analysis. The measured concentrations at test start were in the range of 79 – 89 % of the nominal values. At the end of the test Tall oil diethylenetriamine imidazoline was analysed at concentration levels 0.320 and 3.20 mg/L (prepared without algae) and gave recoveries of < LOQ - 22 % of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The decrease is attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. No test item could be recovered from the glassware (lower than 5 µg/L, which was the lowest analytical standard). This means that less than 1.6 % of the nominal concentration was observed sorbed to glassware. Therefore all effect values are given based on nominal concentrations of the test item.

The test item effect was observed to be reversible at 1.00 – 3.20 mg/L. Therefore, there is potential for recovery following exposure up to 3.20 mg/L (second highest test concentration).

Executive summary:

The toxicity of Tall oil diethylenetriamine imidazoline (CAS No. 68442-97-7) (Batch no.: S000922)to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from February 16 to March 01, 2010 with the definitive exposure phase from February 16 to 19, 2010. The aim of the study was the determination of EC₁₀- , EC₂₀ - and EC₅₀ - values of growth rate and yield over a period of 72 h.

Tall oil diethylenetriamine imidazoline (CAS no. 68442-97-7) is insoluble in water and has a strong tendency to adsorb to negatively charged surfaces such as suspended matter, algae and test vessels or organic material (including dissolved organic matter such as humic acids). Many cationic substances in general but long chain amido amines/imidazolines in particular rank among the most difficult substances to test in environmental toxicology. Standard guideline studies are inappropriate to test substances with such properties and the current REACH Guidance Documents do not provide sufficient guidance concerning bioavaibility and exposure assessment for cationic surface-active substances like the amido amines/imidazolines as these were written with normal hydrophobic chemicals in mind, failing to take into account the lack of bioavaibility that occurs in the environment with cationic surface-active substances.

The aquatic ecotoxicity tests with amido amines/imidazolines were therefore performed in river water to allow a PEC_aquatic,bulk/PNEC_aquatic,bulkapproach and is considered to be conservative but more environmentally realistic than the standard method. This approach is based on PEC estimations representing 'total aquatic concentrations'. To characterize the risk to the aquatic compartment the PEC_aquatic,bulkis compared with the PNEC_aquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2003).

         

In order to class standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method / conditions and test organism used – suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels.

         

For ecotoxicity tests performed using the bulk approach, however, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the concentration-effect relationship should be based on the sum of adsorbed and dissolved substance in the volume of the medium tested. One of the advantages of the bulk approach tests with these difficult substances is that in the presence of suspended matter, humic acids and/or algae, the residual sorption to glassware will be negligible. The results of these bulk approach tests are therefore much easier and more realistic, and if compared to PEC_bulk clearly provide a more appropriate assessment of risks for the environment.

An individual test design was applied. Natural river water was used as dilution medium

The study was conducted under static conditions with an initial cell density of approximately 1 x     10⁴ cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of √10, nominal: 0.100 - 0.320 - 1.00 - 3.20 - 10.0 mg/L. Three replicates were tested for the test item concentrations and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Tall oil diethylenetriamineimidazolinewere analysed at the concentration levels 0.320 and 3.20 mg/L (prepared without algae) and the control at test start via LC-MS/MS analysis. The measured concentrations at test start were in the range of 79 – 89 % of the nominal values. At the end of the test Tall oil diethylenetriamineimidazolinewas analysed at concentration levels 0.320 and 3.20 mg/L (prepared without algae) and gave recoveries of  LOQ - 22 % of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The decrease is attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. No test item could be recovered from the glassware (lower than 5 µg/L, which was the lowest analytical standard). This means that less than 1.6 % of the nominal concentration was observed sorbed to glassware. Therefore all effect values are given based on nominal concentrations of the test item.

Tall oil diethylenetriamine imidazoline (CAS No. 68442-97-7) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values (nominal concentrations): The EC₁₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rC₁₀) and yield (E_yC₁₀) after 72 h were 0.343 (0.325 – 0.371) and 0.265 (0.249 – 0.277) mg/L, respectively. The EC₅₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rC₅₀) and yield (E_yC₅₀) after 72 h were 0.505 (0.461 – 0.568) and 0.356 (0.347 – 0.367) mg/L, respectively.

After 72 h algae were transferred from the nominal concentrations of 1.00 – 3.20 mg/L and the control to fresh untreated medium and allowed to grow for further 3 – 10 d under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 3.20 mg/L (second highest test concentration).

Description of key information

The dose response results are calculated based on nominal Test Loadings. The E_rL₁₀/E_rC₁₀ for reproduction after 72 hours is 20.7/1.11 µg/L. The E_rL₅₀ /E_rC₅₀ = after 72 hours is 44.7/1.27 µg/L. 

 

Key value for chemical safety assessment

EC50 for freshwater algae:

44.7 µg/L

EC10 or NOEC for freshwater algae:

20.7 µg/L

Additional information

Three algae studies are available for AAI-DETA

• One supporting study performed to assess the algae toxicity of a range of comparable imidazoline products and this study is not further described in detail.


• Two key studies performed to evaluate the toxicity to algae according to two different approaches: the WAF and Bulk approach.

Main practical difference between the WAF and Bulk approach lies in the preparation of the test solutions and how the results should be interpreted.

For the Bulk approach, instead of the dissolved concentration of test substance in the test, the Bulk concentration (dissolved + sorbed) in water is used to derive a PNECwater, bulk.

This means that aquatic ecotoxicity testing has to use river water that contains dissolved organic carbon and suspended organic and inorganic matter, instead of reconstituted lab water to prepare the test solutions. The test substance is considered stable under test conditions which means that the test organisms were thus fully exposed to the Bulk concentration of the substance during the test. For risk assessment the PNECwater, bulk is then compared to the PECwater, bulk. This PECwater, bulk is calculated without using the EPM as no calculation of the dissolved concentration is needed.

Tests according to the bulk approach were thus performed because it elegantly bypasses the deficiencies of the EPM on the exposure and analytical problems on the effect side.

However due to the use of non-standard test medium (natural river water) the results of bulk approach test are considered inadequate by regulators involved in C&L because they do not fulfill to the narrow criteria set to quantify the intrinsic toxicity. To address this shortcoming, new tests according to the WAF approach (as described in “OECD guidance document no. 23) were performed.

Key study 1 (Scheerbaum, 2021, Study ID.: SPO19060):

As indicated the test item is a UVCB substance with constituents of different water solubility and therefore, in agreement with OECD guidance 23, Water Accommodated Fractions (WAF) were prepared. For this algae test six WAFs were prepared 24 ± 1 hours prior to the start of exposure with nominal loading rates of the test item in the range of 1.58 to 500 µg/L set up in a geometric series with a factor of √10: 1.58 - 5.00 - 15.8 - 50.0 - 158 - 500 µg/L. For preparation of the loading rates the test item was dissolved in methanol (5 and 0.500 g/L). The test item in 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. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. The time needed to reach equilibrium was preliminary to the final test, measured in a solubility test. After a separation phase of one hour the WAFs were removed and checked via laser beam 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 loadings and six replicates for the control. The test vessels were presaturated with the WAFs for at least 12 hours. Before the start, the test solutions were replaced with fresh test solutions. Three replicates were tested for each test substance concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The AAI-DETA main constituent concentrations were quantified at the start of the exposure (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) of all tested concentration levels and the control via LC-MS/MS with algae. Additionally, the loading rate 50 µg/L without algae was analytically verified at the start of the exposure (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours).

The dose response results are calculated based on nominal Test Loadings and on Time Weighted Average (TWA) measured concentrations. The E_rL₁₀/E_rC₁₀ for reproduction after 72 hours is 20.7/1.11 µg/L. The E_rL₅₀/E_rC₅₀ after 72 hours is 44.7/1.27 µg/L. The TWA results are presented here despite the fact that 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.

The analytical evaluation of the test item and the control were carried out via LC-MS/MS in isocratic mode. An electrospray tandem mass spectrometer operating in positive ion mode was used as detector. The test item is an UVCB substance. Therefore, all ionizable compounds were fragmented, detected and summed up in a TIC (total ion count) signal. The test item was used as external standard for calibration. The test concentrations based on loading corresponded with the following timeweighted average concentrations:  0.700 – 0.915 – 1.07 – 1.29 – 7.85 – 152 μg/L.

 

Key study 2: (Scheerbaum, 2010, Study ID.: SPO13565):

A study was conducted under static conditions to determine the fresh water algal growth inhibition of the test substance, AAI-DETA according to the OECD Guideline 201 under GLP conditions. The toxicity of the test substance to an exponentially growing culture of Pseudokirchnerella subcapitata was determined over an exposure period of 72 h with nominal concentrations of 0.1, 0.32, 1.00, 3.20 and 10.0 mg/L. The test was carried out according to the bulk approach using enriched natural surface water with a Dissolved Organic Carbon concentration (DOC) of 3.9 mg/L and Total Suspended Solids (TSS) content of 16.2 mg/L, allowing a more environmentally realistic determination of the effects of the test substance.

Droge & Goss (2013) have shown that sorption of cationic surfactants to soil and sediment is mainly driven by electrostatic interaction and to a lesser extent by hydrophobic interaction. This means that both the suspended matter and dissolved organic carbon in surface water are the key surface water properties determining the bioavailability of the test substance.

The natural surface water used was therefore characterized in detail and selected to contain a realistic worst-case suspended matter concentration of 15±3.5 mg/L and ± 3.5 mg/L DOC(≈Non Purgeble Organic Carbon). It should be noted that this composition is in perfect alignment with the risk assessment method applied by ECHA, as the concentration of suspended matter in surface water is considered to be 15 mg/L in CHESAR III for risk assessment (see ECHA’s guidance R.16, v3.0, Feb 2016, p. 88).

When applying the bulk approach, the truly dissolved concentration is eliminated from the PEC/PNEC equation (see figure 1), only confirmation that the initial exposure concentrations are within 20% of the nominal concentrations is needed and sorption to glassware needs to be taken into account.

The concentrations of AAI-DETA were quantified at the test concentration levels of 0.320 and 3.20 mg/L (prepared without algae) and the control at test start via LC-MS/MS analysis. The measured concentrations at test start were in the range of 79 – 89 % of the nominal values

During the test a decrease of the concentration is observed of about 70%. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The concentration decrease observed is attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. No test item could be recovered from the glassware (lower than 5 µg/L, which was the lowest analytical standard). This means that less than 1.6 % of the nominal concentration was observed to be sorbed to glassware. All effect values are therefore given based on nominal concentrations of the test item.

AAI-DETA was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values (nominal concentrations): The EC₁₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rC₁₀) and yield (E_yC₁₀) after 72 h were 0.343 (0.325 – 0.371) and 0.265 (0.249 – 0.277) mg/L, respectively. The EC₅₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rC₅₀) and yield (E_yC₅₀) after 72 h were 0.505 (0.461 – 0.568) and 0.356 (0.347 – 0.367) mg/L, respectively.

The effects observed in the definitive study are in good agreement with the results observed in the supporting study where an E_rC₁₀ was observed of 0.57 mg/L.

 

Mitigation of the toxicity by river water constituents:

The degree of mitigation of the toxicity to algae due to the use of natural river water can be calculated by taking the ratio of the results observed for the bulk approach test and the results observed for the WAF approach test.

 

The mitigation factor for the chronic effect (EC₁₀) to algae is 343/20.7 is 16.6. 

The mitigation factor for the acute effect (EC₅₀) to algae is 505/44.7 is 11.3

 

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