Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2022-05-14 to 2022-06-03 with the definitive exposure periods from 2022-05-16 to 2022-05-20 (first definitive study) and 2022-05-30 to 2022-06-03 (second definitive study)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

(2019)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)

Batch number 20201158

CAS No. 71786-60-2

Purity (certified) 100% UVCB

Chemical name Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs

Density 910 kg/m3 at 20 °C

Appearance Light yellow liquid

Water Solubility Dispersible / insoluble

Stability under
test conditions Not specified

Expiry date 2023-02-10

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

Analytical monitoring:

yes

Remarks:

Samples were analyzed with a LC-MS/MS method.

Details on sampling:

The test loadings and the controls were analytically verified daily in the fresh media (after 0, 24, 48, 72 hours) and in the 24-hours old media (after 24, 48, 72 and 96 hours).
Peak distribution (Finger print)
The peak distribution (fingerprint) of the WAF was analyzed in freshly prepared medium in the highest loading rate of the test item once during the test. The signal distribution was compared with an analytical standard prepared in solvent and a control.

Vehicle:

no

Details on test solutions:

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

Test loadings
10.0 – 22.0 – 48.4 – 106 – 234 – 515 – 1134 µg/L (factor 2.2), corresponding to geometric mean measured concentrations of 5.37 – 11.3 – 32.3 – 72.1 – 191 – 651 - 1498 µg/L.
The concentrations are chosen due to the results of a preliminary range finding test (see Annex II) and an Early-Life Stage Toxicity Test (Noack Study Code SO21177/FSZ19203).
In the first definitive study no EC50-values could be determined since only partial mortality (<50%) was observed A second definitive study was carried out with two additional test loadings (control, 515 & 1134 µg/L), which prolonged the concentration series of the first definitive study.

Preparation of the water accommodated fractions
Seven water accommodated fractions (WAF) were prepared separately with the nominal loading rates mentioned above.
The procedure according to ASTM D6081 (2019) as described below will be carried out. 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 slides with the test item were inserted in glass flasks with an appropriate amount of dilution water.
The test item is a poorly soluble UVCB, therefore a prolonged slow stirring procedure was applied for 24 ± 1 hours at room temperature. The stirring time was chosen based on the preliminary investigations of a daphnia reproduction test. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour at room temperature, the aqueous phase of the WAF was removed by siphoning (from the approximate middle (positioned in the middle third) of the glass flask). The first 25 mL were discarded.
The WAFs were checked using a laser beam (Tyndall effect) for undissolved test item (formation of emulsion). No presence of undissolved test item during the test was observed. The Tyndall effect was negative. The resulting water accommodated fractions (WAF) were used in the test.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Test organism
Danio rerio (zebrafish)
Vertebrata, Gnathostomata, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae

Reason for the selection of the test system
According to the guideline, Danio rerio is suitable for this kind of study.

Origin
All fish used in the test were gained at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)

Holding
Holding was performed at the test facility at 23 ± 2 °C and diffuse light (540 – 1000 Lux, photoperiod of 12 to 16 h light / 12 to 8 h dark). The water was changed at least once per week. The dissolved oxygen concentration was more than 80% of the air saturation value.
Zebrafish with at least 9 days of acclimatization and mortality < 5% within these days prior to the start of the exposure were used in the test.
No mortality was observed during this time. No disease treatments were administered throughout holding and testing.

Water
Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 40 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Oxygen saturation: ≥ 80 % of air saturation value
Acidity: 0.3 mmol/L (measurement 2022-03-22)
0.3 mmol/L (recent measurement 2022-05-30)
Alkalinity: 0.8 mmol/L (measurement 2022-03-22)
0.8 mmol/L (recent measurement 2022-05-30)
Conductivity: 172 µS/cm (measurement 2022-03-22)
168 µS/cm (recent measurement 2022-05-30)

Acclimatization
A sufficient number of fish were acclimatized to dilution water and environmental conditions equivalent to the test conditions for at least 9 days. The environmental conditions were equivalent to the test conditions.

Feeding
Food was provided 3 times per week. Food was given to satiation (4% of the fish body weight) per feeding day. The test fish were not fed 24 h before test start.

Food Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany

Test type:

semi-static

Water media type:

freshwater

Remarks:

Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.

Limit test:

no

Total exposure duration:

96 h

Hardness:

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

Test temperature:

The temperature during test solution preparation in both studies ranged from 22.0 to 23.5 °C.
For details see section "Any other information on results incl. tables" below.

pH:

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

Dissolved oxygen:

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

Nominal and measured concentrations:

10.0 – 22.0 – 48.4 – 106 – 234 – 515 – 1134 µg/L (factor 2.2), corresponding to geometric mean measured concentrations of 5.37 – 11.3 – 32.3 – 72.1 – 191 – 651 - 1498 µg/L.

Details on test conditions:

Controls
7 fish in dilution water (without test item but treated in the same way as the test solution) were tested under the same test conditions as the test replicates.

Reference item
No reference item is recommended for this test according to the guidelines.

Duration of the test 96 hours

Test vessel and Pre-Treatment
Glass aquaria of 3 L were used (dimensions: 11.5/13/20 cm, depth of water: approx. 17 cm) and covered with glass plates. A coating phase (saturation of the test vessels) was carried out. The test vessels were pre-treated with the appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure, the test vessels were emptied and refilled with freshly prepared test solution. The test solutions for coating were prepared as described above.

Test volume About 2.5 L per vessel

Aeration No aeration was provided.

Dilution water Same as used for holding

Acclimatization
Acclimatization was not necessary, because the quality of the dilution water and the environmental conditions were equivalent to the water used for holding.

Number of fish
7 zebrafish were used for the test loadings and the controls. For the whole study 69 fish were used.

Loading, length of fish
First definitive study:
Fish density in the tanks was 0.104 g fish per liter test solution.
Average body length at the test end: 1.89 cm
Average body weight at the test end: 0.037 g
Second definitive study:
Fish density in the tanks was 0.115 g fish per liter test solution.
Average body length at the test end: 1.94 cm
Average body weight at the test end: 0.041 g

Age of fish Juvenile

Replicates One replicate each for the limit loading and the control

Introduction of fish The fish were introduced randomly to individual replicates.

Feeding of test fish The fish were not fed during the exposure.

Water temperature (target) 21 - 25 °C, controlled at ± 1 °C

Dissolved oxygen Concentration (target)
Not less than 60% of air saturation value.

Light intensity (target) 540 – 1000 Lux

Photoperiod
A photoperiod (light / dark cycle: 16 / 8 h) was maintained during exposure.


Type and Frequency of Measurements
Biological Parameters

Observation intervals
Observations were made after 2 ± 0.5 h and 5 ± 1 h after the start of the exposure. On days 1-4 of the test, all vessels with living fish were inspected twice per day.

Criteria of effects
Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behavior, respiratory function, pigmentation, etc.).

Measurement of fish size and weight
For measurements of size and weight all control fish were used at the end of the exposure.


Physico-chemical Properties

Water quality and light intensity measurements
The pH-value, temperature and oxygen saturation were measured daily in all test vessels. Chlorine and nitrate were measured from the dilution water used in the test.
Total hardness and TOC was determined at the start of the exposure in the dilution water. During the test, the water temperature was recorded continuously (once per hour) with a data logger. The light intensity was measured at the start of the exposure at one central spot on the surface per the test aquaria.
The solution appearance and behavior were observed daily before and after renewal.

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

72.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 100% Active ingredient

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

106 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

651 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL100

Effect conc.:

515 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

199 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 100% Active ingredient

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

241 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 100% Active ingredient

Key result

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

208 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 100% Active ingredient

Key result

Duration:

72 h

Dose descriptor:

LL50

Effect conc.:

248 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 100% Active ingredient

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

356 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 100% Active ingredient

Key result

Duration:

48 h

Dose descriptor:

LL50

Effect conc.:

348 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

988 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

24 h

Dose descriptor:

LL50

Effect conc.:

764 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Biological Data

All effect levels are given based on the nominal test item loading concentration. The LL0 after 96 hours was the nominal test item loading rate of 106 µg/L corresponding to an overall arithmetic mean measured concentration of 72.1 µg/L.

Physicochemical Data

The environmental conditions (pH-value, temperature and oxygen-saturation) were determined to be within the acceptable limits. Light intensity was measured at the start of the exposure and ranged from 680 to 840 (mean value 776) Lux in the first definitive test and from 680 to 790 (mean value 761) Lux in the second definitive test.

Measured Exposure Concentrations during the Definitive Test

The concentrations of the four compounds C10 Amine + 2EO, C12 Amine + 2EO, C14 Amine + 2EO and C16 Amine + 2EO of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) were analytically verified daily via LC MS/MS in the fresh media (after 0, 24, 48, 72 hours) and in the 24-hours old media (after 24, 48, 72 and 96 hours) in all test item concentrations and the control.

The measured concentrations of all compounds of the test item in the fresh media at the start of an exposure-renewal interval (0, 24, 48 and 72 hours) were between < LOQ to 169% of the nominal values.
The measured concentration of the test item in the old media at the end of an exposure-renewal interval (24, 48, 72 and 96 hours) were between < LOQ and 156% of the nominal values. All test solutions were clear and showed no turbidity during sampling because of an negative Tyndall effect.

The peak distribution (fingerprint) of the WAF was analyzed in freshly prepared medium in the limit loading of the test item after 0 hours. The signal distribution was compared with an analytical standard prepared in solvent and a control.

Reported statistics and error estimates:

Evaluation 2. Definitive test
LL50-values and corresponding confidence intervals were calculated by standard procedures (sigmoidal dose-response regression), as revised in OECD Series on Testing and Assessment No. 54 (2006).
After 2 ± 0.5 h and 5 ± 1 h no mortality occurred.
After 24 hours only 0 and 100% mortality occurred. The LL50 was calculated as a geometric mean of the corresponding concentrations. These concentrations were chosen as lower and upper confidence limits.

After 48 h the LL50-values and the confidence limits were calculated by sigmoidal regression.
After 72 to 96 hours one partial response of mortality occurred. Therefore, the LL50-values were calculated by sigmoidal regression and the corresponding concentrations were chosen as lower and upper confidence limits.
The test loadings leading to 0 and 100% mortality (LL0 and LL100) after 96 h will be determined directly from the test results.

Geometric mean measured concentrations
The geometric mean measured concentrations were determined by the sumproduct function of excel, referring to the recoveries and purities of the four compounds C10 ethoxylated alkylamine C12 ethoxylated alkylamine, C14 ethoxylated alkylamine and C16 ethoxylated alkylamine.

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

Sublethal observations / clinical signs:

Preparation of the WAFs in the First Definitive Study

Stock solution [mg/L] in MeOH	1000
Preparation volume of the stock solution [mL]	20
Application volume of test item [µL/20 mL] for the stock solution (density of the test item taken into account)	21.98
Preparation volume per WAF [L]	2
Application volume of the stock solution placed	20.0	44.0	96.8	212	468
on a curved glass slide [µL/2L]
Final test item Loading rate [µg/L]	10.0	22.0	48.4	106	234

 

Preparation of the WAFs in the Second Definitive Study

Stock solution [mg/L] in MeOH	5000
Preparation volume of the stock solution [mL]	20
Application volume of test item [µL/20 mL] for the stock solution (density of the test item taken into account)	109.9
Preparation volume per WAF [L]	2
Application volume of the stock solution placed	206	454
on a curved glass slide [µL/2L]
Final test item Loading rate [µg/L]	515	1134

 

Observations of Sub Lethal Effects and Normal Behavior in the Test Vessels in the First
Definitive Study
(n = 7, number of fish)

Nominal Test Item loading	Effect *	Number of fish affected at observation time [hours]
[µg/L]		day 0		day 1		day 2		day 3		day 4
		1.5	5	18.5	25.5	42.5	49.5	67	73.5	90.5	96
234	-1	7	7	7	7	3	1	1	0	0	0
	(2.1)	0	0	0	0	0	0	0	1	0	0
	(2.2)	0	0	0	0	1	1	0	0	0	0
	(2.4)	0	0	0	0	0	3	4	1	3	3
	(2.5)	0	0	0	0	0	2	1	3	2	2
	(2.7)	0	0	0	0	0	3	4	4	3	3
	(2.9)	0	0	0	0	4	0	0	0	0	0
	(2.10)	0	0	0	0	0	5	0	1	1	1
	(3.5)	0	0	0	0	0	5	5	6	5	5
	(4.1)	0	0	0	0	0	0	1	0	0	0
	(5.4)	0	0	0	0	0	1	1	3	3	3
	(E)	0	0	0	0	0	1	1	1	2	2
106	-1	7	7	7	7	7	7	7	7	7	7
48.4	-1	7	7	7	7	7	7	7	7	7	7
22.0	-1	7	7	7	7	7	7	7	7	7	7
10.0	-1	7	7	7	7	7	7	7	7	7	7
Control	-1	7	7	7	7	7	7	7	7	7	7

*) The numbers in brackets correspond to the following observations:
(1) = Normal behavior (2.1) = Lethargy/Immobility (2.2) = Lateral position
(2.4) = Slow escape reflex (2.5) = Missing escape reflex (2.7) = Hypoactivity
(2.9) = Arresting on the ground (2.10) = Arresting below the surface (3.5) = Clamped fins
(4.1) = Hyperventilation (5.4) = Descent of the fish (E) = Exitus lethalis

 

Observations of Sub Lethal Effects and Normal Behavior in the Test Vessels in the
Second Definitive Study
(n = 7, number of fish)

Nominal Test Item loading	Effect *	Number of fish affected at observation time [hours]
[µg/L]		day 0		day 1		day 2		day 3		day 4
		2	4	21	28	45	52	69	76	93	96
1134	-1	7	4	0	100 % mortality
	(2.4)	0	3	0
	(E)	0	0	7
515	-1	7	7	6	0	0	0	0	100 % mortality
	(2.4)	0	0	1	7	0	0	0
	(2.5)	0	0	0	0	2	1	0
	(3.5)	0	0	0	7	2	1	0
	(5.4)	0	0	0	0	2	1	0
	(E)	0	0	0	0	5	6	7
Control	-1	7	7	7	7	7	7	7	7	7	7

*) The numbers in brackets correspond to the following observations:
(1) = Normal behavior (2.4) = Slow escape reflex (2.5) = Missing escape reflex
(3.5) = Clamped fins (5.4) = Abnormal vertical orientation (E) = Exitus lethalis

 

Cumulative Mortality % in the Test Vessels in the First Definitive Study

Nominal Test Item loading	Cumulative mortality [%] at observation time [hours]
[µg/L]	day 0		day 1		day 2		day 3		day 4
	1.5	5	18.5	25.5	42.5	49.5	67	73.5	90.5	96
234	0	0	0	0	0	14	14	14	29	29
106	0	0	0	0	0	0	0	0	0	0
48.4	0	0	0	0	0	0	0	0	0	0
22.0	0	0	0	0	0	0	0	0	0	0
10.0	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Cumulative Mortality % in the Test Vessels in the Second Definitive Study

Nominal Test Item loading	Cumulative mortality [%] at observation time [hours]
[µg/L]	day 0		day 1		day 2		day 3		day 4
	2	4	21	28	45	52	69	76	93	96
1134	0	0	100	100	100	100	100	100	100	100
515	0	0	0	0	71	86	100	100	100	100
Control	0	0	0	0	0	0	0	0	0	0

 

Environmental Conditions in the First Definitive Study

	Nominal Test item loading	Oxygen Saturation		pH Value		Temperature
	[µg/L]	[%]
						[°C]
		new	old	new	old	new	old
Day 0	234	93		7.34		23.8
	106	94		7.12		23.8
	48.4	92		7.16		23.8
	22.0	92		7.36		23.8
	10.0	95		7.08		23.7
	Control	99		7.60		23.6
Day 1	234	96	86	7.50	7.49	24.0	24.2
	106	97	83	7.38	7.52	24.1	23.8
	48.4	92	80	7.48	7.43	24.1	23.8
	22.0	97	76	7.46	7.41	24.6	23.8
	10.0	96	85	7.46	7.41	23.8	23.8
	Control	98	72	7.44	7.18	23.2	23.8
Day 2	234	97	86	7.44	7.42	24.0	23.8
	106	96	81	7.44	7.38	24.0	23.9
	48.4	98	81	7.43	7.40	24.0	23.9
	22.0	97	81	7.41	7.36	24.0	24.0
	10.0	97	81	7.33	7.35	23.8	23.9
	Control	98	73	7.18	7.17	23.3	23.7
Day 3	234	97	80	7.50	7.33	24.0	24.0
	106	97	88	7.48	7.40	24.0	24.0
	48.4	98	95	7.48	7.44	24.0	24.0
	22.0	97	79	7.45	7.38	24.1	24.0
	10.0	98	85	7.46	7.41	23.8	23.9
	Control	100	74	7.37	7.28	23.7	23.8
Day 4	234		81		7.37		23.8
	106		86		7.37		24.0
	48.4		87		7.37		24.1
	22.0		83		7.29		24.1
	10.0		77		7.19		24.0
	Control		87		7.29		23.9

 

Environmental Conditions in the Second Definitive Study

	Nominal Test item loading	Oxygen Saturation		pH Value		Temperature
	[µg/L]	[%]
						[°C]
		new	old	new	old	new	old
Day 0	1134	97		7.38		23.6
	515	97		7.39		23.4
	Control	100		7.54		23.4
Day 1	1134	-	83	-	7.39	-	23.4
	515	98	84	7.47	7.42	23.5	23.2
	Control	100	84	7.55	7.24	23.7	23.4
Day 2	1134	-	-	-	-	-	-
	515	97	81	7.66	7.40	23.4	22.9
	Control	99	83	7.51	7.48	23.3	23.9
Day 3	1134	-	-	-	-	-	-
	515		84		7.46		22.9
	Control	99	84	7.44	7.24	22.9	23.0
Day 4	1134		-		-		-
	515
	Control		84		7.41		23.2

 

Water Temperature in the Control (Continuous Measuring) in the First and Second Definitive Test

Period of Measurements	2022-05-16 to 2022-05-20	2022-05-30 to 2022-06-03
Minimum Temperature [°C]	22.9	22.5
Maximum Temperature [°C]	24.2	23.5
Mean Temperature [°C]	23.7	23.0

 

Residual Chlorine from the Dilution Water in the First and Second Definitive Test

Batch	For Study Days	[mg/L]
		First Definitive Test	Second Definitive Test
Day 0	0 to 1	< 0.01	< 0.01
Day 1	1 to 2	< 0.01	< 0.01
Day 2	2 to 3	< 0.01	< 0.01
Day 3	3 to 4	< 0.01	< 0.01

Nitrate from the Dilution Water in the First and Second Definitive Test

Batch	For Study Days	[mg/L]
		First Definitive Test	Second Definitive Test
Day 0	0 to 1	2.14	1.31
Day 1	1 to 2	1.86	1.42
Day 2	2 to 3	2.08	1.86
Day 3	3 to 4	1.55	1.58

TOC and Total Hardness from the Dilution Water

Parameter	Study Days	[mg/L]
		First Definitive Test	Second Definitive Test
TOC	0 to 4	< 2	< 2
Total Hardness	0 to 4	62	70

 

Measured Concentrations of the Test Item during the Definitive Test (C10 Amine + 2 EO)

Sampling date		Fresh media			Old media		Fresh media		Old media
		0 hours			24 hours		24 hours		48 hours
Nominal		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
concentration of the		C10 Amine + 2 EO
Test item	a.s.	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]
1134^1)	82.4	128	155		128	156	-	-	-	-
515^1)	37.4	56.5	151		55.4	148	52.2	139	51.9	139
234^2)	17.0	18.3	107		17.5	103	17.8	105	17.3	101
106	7.73	8.34	108		7.68	100	7.83	102	7.89	102
48.4	3.53	3.80	108		3.69	105	3.42	97	3.40	97
22.0	1.60	1.68	105		1.27	79	1.74	109	1.62	101
10.0	0.729	0.727		100	0.688	95	0.781	107	0.753	104
Control^1)		< LOQ			< LOQ		< LOQ		< LOQ
Control		< LOQ			< LOQ		< LOQ		< LOQ
Sampling date		Fresh media			Old media		Fresh media		Old media
		48 hours			72 hours		72 hours		96 hours
Nominal		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
concentration of the		C10 Amine + 2 EO
Test item	a.s.	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]
1134^1)	82.7	-	-		-	-	-	-	-	-
515^1)	37.5	56.5	151		52.2	139	-	-	-	-
234	17.0	18.3	107		12.5	73	17.2	101	15.2	90
106	7.73	7.98	104		5.46	71	7.24	94	5.76	75
48.4	3.53	3.86	110		2.35	67	3.42	97	2.78	79
22.0	1.60	1.62	101		0.982	61	1.36	85	0.962	60
10.0	0.729	0.765		105	0.591	81	0.624	86	0.246	34
Control^1)		< LOQ			< LOQ		< LOQ		-
Control		< LOQ			< LOQ		< LOQ		< LOQ

Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 0.145 µg a.s./L)
a.s. = Active substance
1) = 2nd definitive study
2.) = 1st definite study
- = not determined

 

Measured Concentrations of the Test Item during the Definitive Test (C12 Amine + 2 EO)

Sampling date		Fresh media			Old media		Fresh media		Old media
		0 hours			24 hours		24 hours		48 hours
Nominal		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
concentration of the		C12 Amine + 2 EO
Test item	a.s.	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]
1134^1)	597	661	111		852	143	-	-	-	-
515^1)	271	234	86		359	132	387	143	358	132
234	123	131	107		108	88	129	105	109	88
106	55.8	59.7	107		41.4	74	58.6	105	48.8	87
48.4	25.5	28.0	110		19.9	78	24.4	96	19.6	77
22.0	11.6	12.1	105		< LOQ		12.6	109	6.17	53
10.0	5.26	5.32		101	2.77	53	5.08	97	3.57	68
Control^1)		< LOQ			< LOQ		< LOQ		< LOQ
Control		< LOQ			< LOQ		< LOQ		< LOQ
Sampling date		Fresh media			Old media		Fresh media		Old media
		48 hours			72 hours		72 hours		96 hours
Nominal		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
concentration of the		C12 Amine + 2 EO
Test item	a.s.	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]
1134^1)	597	-	-		-	-	-	-	-	-
515^1)	271	419	155		346	128	-	-	-	-
234	123	127	103		63.2	51	123	100	84.6	69
106	55.8	54.7	98		21.1	38	46.6	84	13.3	24
48.4	25.5	27.7	109		5.17	20	22.3	87	9.62	38
22.0	11.6	10.7	93		1.59	14	7.46	64	< LOQ
10.0	5.26	4.84		92	2.44	46	2.09	40	< LOQ
Control^1)		< LOQ			< LOQ		< LOQ		-
Control		< LOQ			< LOQ		< LOQ		< LOQ

Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 1.05 µg a.s./L)
a.s. = Active substance
1) = 2nd main study
- = not determined

 

Measured Concentrations of the Test Item during the Definitive Test (C14 Amine + 2 EO)

Sampling date		Fresh media			Old media		Fresh media		Old media
		0 hours			24 hours		24 hours		48 hours
Nominal		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
concentration of the		C14 Amine + 2 EO
Test item	a.s.	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]
1134^1)	211	357	169		256	121	-	-	-	-
515^1)	96	155	162		95.1	99	136	142	94.9	99
234	43.6	45.9	105		27.5	63	48.3	111	28.1	64
106	19.8	18.8	95		3.88	20	23.2	117	10.5	53
48.4	9.02	9.57	106		1.61	18	7.30	81	3.86	43
22.0	4.10	3.69	90		< LOQ		4.23	103	< LOQ
10.0	1.86	1.41		76	< LOQ		1.17	63	< LOQ
Control		< LOQ			< LOQ		< LOQ		< LOQ
Sampling date		Fresh media			Old media		Fresh media		Old media
		48 hours			72 hours		72 hours		96 hours
Nominal		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
concentration of the		C14 Amine + 2 EO
Test item	a.s.	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]
1134^1)	211	-	-		-	-	-	-	-	-
515^1)	96	147	153		98.2	102	-	-	-	-
234	43.6	40.1	92		4.57	10	40.4	93	12.3	28
106	19.8	14.2	72		0.630	3	8.61	44	< LOQ
48.4	9.02	6.17	68		< LOQ		4.78	53	< LOQ
22.0	4.10	2.30	56		< LOQ		1.49	36	< LOQ
10.0	1.86	0.813		44	< LOQ		< LOQ		< LOQ
Control		< LOQ			< LOQ		< LOQ		< LOQ

Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 0.373 µg a.s./L)
a.s. = Active substance
1) = 2nd main study
- = not determined

 

Measured Concentrations of the Test Item during the Definitive Test (C16 Amine + 2 EO)

Sampling date		Fresh media			Old media		Fresh media		Old media
		0 hours			24 hours		24 hours		48 hours
Nominal		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
concentration of the		C16 Amine + 2 EO
Test item	a.s.	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]
1134^1)	96	133	139		87.6	91	-	-	-	-
515^1)	44	61.7	142		12.5	29	59.1	136	30.7	71
234	19.8	16.6	84		6.45	33	17.5	88	5.74	29
106	8.97	5.85	65		0.803	9	6.65	74	1.72	19
48.4	4.09	2.88	70		< LOQ		2.05	50	0.327	8
22.0	1.86	0.813	44		< LOQ		0.964	52	< LOQ
10.0	0.846	0.307		36	< LOQ		0.243	29	< LOQ
Control		< LOQ			< LOQ		< LOQ		< LOQ
Sampling date		Fresh media			Old media		Fresh media		Old media
		48 hours			72 hours		72 hours		96 hours
Nominal		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
concentration of the		C16 Amine + 2 EO
Test item	a.s.	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]
1134^1)	96	-	-		-	-	-	-	-	-
515^1)	44	59.8	137		19.3	44	-	-	-	-
234	19.8	12.1	61		0.272	1	13.8	70	0.955	5
106	8.97	2.75	31		< LOQ		1.10	12	< LOQ
48.4	4.09	0.846	21		< LOQ		1.27	31	< LOQ
22.0	1.86	0.555	30		< LOQ		0.349	19	< LOQ
10.0	0.846	0.172		20	< LOQ		< LOQ		< LOQ
Control		< LOQ			< LOQ		< LOQ		< LOQ

Meas. Conc. = Measured concentration (dilution factor taken into account)
% = Percent of nominal concentration of the test item
LOQ = Limit of quantification (2.0 µg/L of the test item, corresponding to 0.169 µg a.s./L)
a.s. = Active substance
1) = 2nd main study
- = not determined

 

Calculated Overall Mean Measured Test Item Concentrations of the study

Sampling day	Fresh media	Old media	Fresh media	Old media
	0 hours	24 hours	24 hours	48 hours
Nominal	Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs
concentration of the test item	(CAS: 71786-60-2)
[µg/L]	Calculated mean measured concentration per study day [µg/L]
1134^1)	1472	1523	-	-
515^1)	583	599	730	616
234	244	184	245	183
106	106	61.8	111	78.8
48.4	50.9	29.0	42.8	31.3
22.0	21.1	2.36	22.5	9.22
10.0	8.92	4.31	8.39	5.29
Sampling day	Fresh media	Old media	Fresh media	Old media
	48 hours	72 hours	72 hours	96 hours
Nominal	Tallow amine propoxylate (CAS: 1309955-79-0)				Overall mean measured concentration of the test item [µg/L]
concentration of the test item
[µg/L]	Calculated mean measured concentration per study day [µg/L]
1134^1)	-	-	-	-	1498
515^1)	785	593	-	-	651
234	227	91.7	224	130	191
106	91.6	31.4	73.4	22.4	72.1
48.4	44.4	8.87	36.4	14.6	32.3
22.0	17.5	3.29	12.2	2.01	11.3
10.0	7.58	3.77	3.45	1.20	5.37

1) = 2nd definitive study , all other results are from the first study
- = not determined

Fingerprint of the Test Item (non-GLP)

The peak distribution of the test concentration was analyzed in freshly prepared medium in the nominal test item concentration 1134 µg/L. A solution of the analytical standard of the test item was prepared in acetonitrile and diluted to 1.00 mg/L with dilution medium. A sample of the nominal test item concentration 1134 µg/L and a control were diluted factor 2 with acetonitrile containing 2% formic acid to avoid an inhomogeneous sample. The solutions were analytically verified via high resolution MS and evaluated by the software. The detected signals of the analytical standard and the test item solution were compared. In test item and standard solutions 6 masses were observed and could be assigned to the test item. Compared to the standard in dilution medium the test item in fresh media showed a relative increase in the amounts of the compounds with 274 Da (C12), 302 Da (C14) and 358 Da (C18), whereas the relative amounts of compounds < 274 Da (C10 and C8) are decreased. In general, however, the concentrations and solubility of the test item is too low for significant MS spectra.

 

Percentage of the Relevant Amine

	[%]
C10 Amine + 2EO	7.27
C12 Amine + 2EO	52.62
C14 Amine + 2EO	18.64
C16 Amine + 2EO	8.46

 

Gradient Table

Time [min]	A [%]	B [%]
0.00	75	25
0.30	75	25
1.30	10	90
2.00	10	90
2.10	75	25
2.50	75	25

 

Dilution steps

Nominal	Dilution	Sample	Final
test item	Factor*	volume	volume
concentration		[mL]	[mL]
[µg/L]
1134	200	1	10
		0.1	1.0
1134	80	1	10
		0.25	1.0
515^1)	100	1	10
		0.2	1.0
515^1)	40	1	10
		0.5	1.0
515^2)	100	0.2	10
515^2)	40	0.05	1.0
234	20	0.1	1.0
234	2	1.0	1.0
106	10	0.2	1.0
106	2	1.0	1.0
48.4	10	0.2	1.0
48.4	2	1.0	1.0
22.0	2	1.0	1.0
10.0	2	1.0	1.0
Control	2	1.0	1.0

* including factor 2
1) samples 48h and later
2) samples 0h and 24h

 

Parameter, Acceptance Criteria and Results of the Method Validation

Parameter	Acceptance criteria	Result
Linearity	≥ 5 standard concentrations,	0.2 to 20 µg test item/L (n = 8),	ü
	r ≥ 0.99 (R^2≥ 0.9801 for 2^nd order regression)	r ≥ 0.99
	Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation
Matrix effects	Should be ≤ 20%	Matrix effects: < 20%	ü
Lowest calibration level (LCL)	S/N ≥ 9 for quantifier ion trace	S/N for 0.2 µg test item/L	ü
	S/N ≥ 3 for qualifier ion trace	C10 Amine + 2 EO
		187 (Quantifier), 110 (Qualifier)
		C12 Amine + 2 EO
		937 (Quantifier), 460 (Qualifier)
		C14 Amine + 2 EO
		493 (Quantifier), 354 (Qualifier)
		C16 Amine + 2 EO
		185 (Quantifier), 176 (Qualifier)
Limit of Quantification (LOQ)	Should be at or below lowest test concentration	2.0 µg test item/L (1 x LOQ)	ü
		1400 µg test item/L (700 x LOQ)
Accuracy^1)	Mean recovery rate of 70-120%	C10 Amine + 2 EO	ü
(Fortified samples)	per fortification level (2 levels)	1 x LOQ: 88% (n = 5)
		700 x LOQ: 105% (n = 5)
		C12 Amine + 2 EO
		1 x LOQ: 92% (n = 5)
		700 x LOQ: 106% (n = 5)
		C14 Amine + 2 EO
		1 x LOQ: 91% (n = 5)
		700 x LOQ: 105% (n = 5)
		C16 Amine + 2 EO
		1 x LOQ: 89% (n = 5)
		700 x LOQ: 107% (n = 5)
Precision^1)	Relative standard deviation ≤ 20% per fortification level	C10 Amine + 2 EO	ü
		1 x LOQ: 7.9%
		700 x LOQ: 2.3%
		C12 Amine + 2 EO
		1 x LOQ: 7.8%
		700 x LOQ: 2.1%
		C14 Amine + 2 EO
		1 x LOQ: 8.2%
		700 x LOQ: 2.5%
		C16 Amine + 2 EO
		1 x LOQ: 14%
		700 x LOQ: 2.2%

Stability	Samples: Stable if 70 – 120% of nominal concentration	See section 15.1	ü
	Calibration standards: Stable if ±10% of freshly prepared standard
Specificity:	Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity).	C10 Amine + 2 EO	ü
LC-MS/(MS)		quantifier [m/z]: 246.20 > 88.13
		qualifier [m/z]: 246.20 > 70.17
		C12 Amine + 2 EO
		quantifier [m/z]: 274.30 > 88.13
		qualifier [m/z]: 274.30 > 70.23
		C14 Amine + 2 EO
		quantifier [m/z]: 302.36 > 88.12
		qualifier [m/z]: 302.36 > 70.29
		C16 Amine + 2 EO
		quantifier [m/z]: 330.36 > 88.12
		qualifier [m/z]: 330.36 > 70.29
	Blank values < 30% of 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 15.2	ü

 = criterion fulfilled
- = not determined

Preparation of the fortified samples

The method was validated at the 1 x LOQ (2.0 µg test item/L) and at the 700 x LOQ 1400 µg test item/L).

 

Preparation of Fortified Samples

LOQ Level	Control	1	700
Stock solution	-	100 mg test item/L in Acetonitrile
Spiking solution	-	0.2	Stock solution
[mg test item/L]		(Dilution medium)
(Medium)
Replicates	2	5	5
Concentration of the LOQ	-	2.0	1400
[µg test item/L]
Medium for preparation	Fish dilution medium
Volume of spiking solution [mL]	-	0.05	0.07
Volume of medium [mL]	5.0	4.95	4.93
Dilution factor	-	2	100
Dilution medium	Acetonitrile containing 2% formic acid ^1)
	Dilution medium ^2)
Sample volume [mL]	5.0^1)	5.0^1)	5.0^1)
			0.1^2)
Finale volume [mL]	10^1)	10^1)	10^1)
			5.0^2)
Test item concentration in the final solution [µg/L]	-	1.0	14.0

Dilution medium Acetonitrile : ultra-pure water (50 : 50 v/v) containing 1 % formic acid
1) First dilution step
2) Second dilution step

 

Nominal Concentrations of the Fortified Samples of the active ingredients of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Fortified concentrations*: 2.02 µg test item/L (1 x LOQ) and 1419 µg test item/L (700 x LOQ).

Active ingredient	1 x LOQ*	700 x LOQ*
	[µg a.s./L]	[µg a.s./L]
C10 Amine + 2 EO	0.147	103
C12 Amine + 2 EO	1.06	747
C14 Amine + 2 EO	0.376	265
C16 Amine + 2 EO	0.171	120

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

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Active ingredient: C10 Amine + 2 EO and C12 Amine + 2 EO

Replicate	Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
	C10 Amine + 2 EO
	1 x LOQ		700 x LOQ
	Meas.conc.	%	Meas.conc.	%
	[µg a.s./L]		[µg a.s./L]
1	0.147	101	108	105
2	0.125	85	112	108
3	0.123	84	109	106
4	0.127	87	106	103
5	0.125	85	106	102
Mean	0.13	88	108	105
SD ±	0.01		2
CV [%]	7.9		2.3
Replicate	Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
	C12 Amine + 2 EO
	1 x LOQ		700 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]
1	1.11	104	814	109
2	0.956	90	797	107
3	0.941	89	782	105
4	0.922	87	776	104
5	0.948	89	773	104
Mean	0.98	92	789	106
SD ±	0.08		17
CV [%]	7.8		2.1

Meas. conc. = measured concentration of each component of the test item, dilution factor taken into account
% = percent concentration of the fortified sample
* = weighing factor taken into account
SD = standard deviation CV = coefficient of variation

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
Active ingredient: C14 Amine + 2 EO and C16 Amine + 2 EO

Replicate	Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
	C14 Amine + 2 EO
	1 x LOQ		700 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]
1	0.389	103	284	107
2	0.346	92	284	107
3	0.318	85	279	106
4	0.327	87	270	102
5	0.329	87	270	102
Mean	0.34	91	277	105
SD ±	0.03		7
CV [%]	8.2		2.5
Replicate	Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
	C16 Amine + 2 EO
	1 x LOQ		700 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[µg a.s./L]		[µg a.s./L]
1	0.187	109	131	109
2	0.149	87	131	109
3	0.135	79	130	108
4	0.138	81	126	105
5	0.151	89	126	105
Mean	0.15	89	129	107
SD ±	0.02		3
CV [%]	14		2.2

Meas. conc. = measured concentration of each component of the test item, dilution factor taken into account
% = percent concentration of the fortified sample
* = weighing factor taken into account
SD = standard deviation
CV = coefficient of variation

Stability

The stability of the fortified samples at the 1 x LOQ was checked after 3 days and was for C10 Amine + 2 EO, C12 Amine + 2 EO, C14 Amine + 2 EO and C16 Amine + 2 EO between 105 and 109% of the nominal concentration for each component.

The stability of the standard stock solution (1000 mg test item/L in acetonitrile) was checked after 7 days and was 103 to 106% of the nominal concentration and was found to be stable over this period.

The responses of a 3 days old calibration standard (8 µg test item/L) was compared with the responses of a freshly prepared calibration standard with the same concentration. The deviation was < 10% and was found to be stable over this period. 

Procedural Recovery

A procedural recovery (Quality Control) 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	Fresh media		Old media		Fresh media		Old media
date	0 hours		24 hours		24 hours		48 hours
Component	Meas.	%	Meas.	%	Meas.	%	Meas.	%
	conc.		conc.		conc.		conc.
	[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
C10 Amine + 2 EO	0.144	96	0.147	98	0.143	96	0.130	88
C12 Amine + 2 EO	1.08	100	1.09	101	1.09	101	1.02	95
C14 Amine + 2 EO	0.380	99	0.389	102	0.381	100	0.358	94
C16 Amine + 2 EO	0.186	107	0.176	101	0.170	98	0.166	95
Sampling	Fresh media		Old media		Fresh media		Old media
date	48 hours		72 hours		72 hours		96 hours
Component	Meas.	%	Meas.	%	Meas.	%	Meas.	%
	conc.		conc.		conc.		conc.
	[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
C10 Amine + 2 EO	0.136	91	0.104	70	0.157	105	0.147	99
C12 Amine + 2 EO	1.04	96	0.852	79	1.12	104	1.05	97
C14 Amine + 2 EO	0.361	94	0.299	78	0.362	95	0.364	95
C16 Amine + 2 EO	0.170	98	0.132	76	0.172	99	0.161	93

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
Quality Control = 2.00 µg test item/L, weighing factor taken into account

 

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

Sampling	Fresh media		Old media		Fresh media		Old media
date	0 hours		24 hours		24 hours		48 hours
Component	Meas.	%	Meas.	%	Meas.	%	Meas.	%
	conc.		conc.		conc.		conc.
	[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
C10 Amine + 2 EO	0.117	79	0.113	76	0.113	76	0.140	95
	0.139	94	0.139	94	0.139	94
C12 Amine + 2 EO	1.02	96	1.04	98	1.04	98	1.11	104
	1.08	101	1.08	101	1.08	101
C14 Amine + 2 EO	0.358	95	0.350	93	0.350	93	0.369	98
	0.366	97	0.366	97	0.366	97
C16 Amine + 2 EO	0.137	80	0.147	86	0.147	86	0.126	73
	0.130	76	0.130	76	0.130	76
Sampling	Fresh media		Old media		Fresh media		Old media
date	48 hours		72 hours		72 hours		96 hours
Component	Meas.	%	Meas.	%	Meas.	%	Meas.	%
	conc.		conc.		conc.		conc.
	[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
C10 Amine + 2 EO	0.140	95	0.103	70	0.103	70	-	-
C12 Amine + 2 EO	1.11	104	0.890	83	0.890	83	-	-
C14 Amine + 2 EO	0.369	98	0.317	84	0.317	84	-	-
C16 Amine + 2 EO	0.126	73	0.163	95	0.163	95	-	-

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
Quality Control = 2.00 µg test item/L, weighing factor taken into account

- = No measurements due to 100 % mortality

 

Preliminary (non-GLP)

Solubility Test

A solubility test was performed within the framework of a study on Daphnia chronic reproduction. Two water accommodated fractions were prepared. 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 Elendt M4 medium according to OECD 211, Annex 2. A slow stirring procedure was applied for 72 hours at room temperature. The WAFs were checked via laser beam (Tyndall effect) for undissolved test item. No presence of undissolved test item during the test was observed. For the preparation of the water accommodated fractions a stirring phase of 24 hours was found to be suitable.

Measured Concentrations of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) during the Stability Test (non-GLP)
Analytical system: LC-MS

	Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
	Components	Measured concentration [µg/L]
Sampling date		24 hours	48 hours	72 hours
Nominal test item loading rate 100 µg/L	C14 Component + 2EO	66.9	68.2	81.7
	C16 Component + 2EO	52.1	52.8	54.5
	C18 Component + 2EO	27.7	14.3	13.4
	C18:1 Component + 2EO	38.7	40.2	39.9
Nominal test item loading rate 1000 µg/L	C8 Component + 2EO	768	-
	C10 Component + 2EO	913
	C12 Component + 2EO	923
	C14 Component + 2EO	849
	C16 Component + 2EO	571
	C18 Component + 2EO	330
	C18:1 Component + 2EO	513

LCL = lowest calibration standard = 2 µg/L

 

Range Finding Test
A non-GLP preliminary range finding test was conducted at the test facility under semi-static conditions over a period of 96 hours.
The range finding test was carried out with the test loading of 10 µg/L. The water accommodated fraction was prepared as described above. A coating phase (saturation of the test vessels) was carried out as described above. The Tyndall effect was negative.

Observations of Sub Lethal Effects and Normal Behavior in the Test Vessels
(n = 3 , number of fish)

Nominal	Effect *	Number of fish affected at observation time
Test Item Loading
[µg/L]		day 0	day 1	day 2	day 3	day 4
10	-1	3	3	3	3	3
Control	-1	3	3	3	3	3

*) The numbers in brackets correspond to the following observations:
(1) = Normal behavior

 Cumulative Mortality % in the Test Vessels

Nominal	Cumulative mortality [%] at observation time
Test Item Loading
[µg/L]	day 0	day 1	day 2	day 3	day 4
10	0	0	0	0	0
Control	0	0	0	0	0

 

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

Sampling date		Fresh media,		Old media,		Fresh media,		Old media,
		Day 0		Day 1		Day 3		Day 4
Nominal test item loading rates		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
		(C14 Component + 2 EO)
[µg/L]	[µg a.s./L]	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
10.0	1.86	1.72	92	0.460	25	1.24	67	< LCL
Control		< LCL		< LCL		< LCL		< LCL
Sampling date		Fresh media,		Old media,		Fresh media,		Old media,
		Day 0		Day 1		Day 3		Day 4
Nominal test item loading rates		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
		(C16 Component + 2 EO)
[µg/L]	[µg a.s./L]	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%	Meas. conc.	%
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
10.0	0.846	0.462	55	< LCL		0.272	32	< LCL
Control		< LCL		< LCL		< LCL		< LCL

% = Percent of the nominal test item concentration
a.s. = active substance
LCL = 0.5 µg test item/L (0.0932 µg C14 Component + 2 EO/L; 0.0423 µg C16 Component + 2EO))
Meas. Conc. = measured concentration of the test item, dilution factors taken into account

 

Fingerprint – MS spectrum

The peak distribution of the test concentration was analyzed in freshly prepared medium in the nominal test item concentration 1134 µg/L. A solution of the analytical standard of the test item was prepared in acetonitrile and diluted to 1.00 mg/L with dilution medium. A sample of the nominal test item concentration 1134 µg/L and a control were diluted factor 2 with acetonitrile containing 2% formic acid to avoid an inhomogeneous sample. The solutions were analytically verified via high resolution MS and evaluated by the software. The detected signals of the analytical standard and the test item solution were compared. In test item and standard solutions 6 masses were observed and could be assigned to the test item. Compared to the standard in dilution medium the test item in fresh media showed a relative increase in the amounts of the compounds with 274 Da (C12), 302 Da (C14) and 358 Da (C18), whereas the relative amounts of compounds < 274 Da (C10 and C8) are decreased. In general, however, the concentrations and solubility of the test item is too low for significant MS spectra.

Validity criteria fulfilled:

yes

Conclusions:

The effect levels are given based on the nominal loading rates and the overall arithmetic mean measured concentrations of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2). In this study Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) was found to cause lethal effects to rainbow trout after 96 hours at the nominal test item loadings 234 – 515 – 1134 µg/L, corresponding to overall arithmetic mean measured concentrations of 191 – 651 – 1498 µg/L and higher. The LL50 after 96 hours was 241 µg test item/L (nominal loading rate), corresponding to an overall arithmetic mean measured concentration of 199 µg/L. The LL0 and LL100 after 96 hours were 106 and 515 µg test item/L (nominal loading rate), respectively, corresponding to overall arithmetic mean measured concentrations of 72.1 and 651 µg/L.

Executive summary:

The acute toxicity of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) (batch no. 20201158) to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (2019) in two definitive studies from 2022-05-14 to 2022-06-03 with the definitive exposure periods from 2022-05-16 to 2022-05-20 (first definitive study) and 2022-05-30 to 2022-06-03 (second definitive study) at the test facility.

The test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) is a light yellow liquid, poorly soluble but dispersible in water. It is a UVCB substance with constituents of different water solubility. Water accommodated fractions (WAFs) were prepared 24 ± 1 hour prior to the start of exposure with the nominal loading rates of the test item of 10.0 – 22.0 – 48.4 – 106 – 234 – 515 – 1134 µg/L, corresponding to the overall arithmetic mean measured concentrations of 5.37 – 11.3 – 32.2 – 72.1 – 191 – 651 - 1498 µg/L. For the preparation of the loading rate the test item was dissolved in methanol applied onto curved glass slides. The methanol was evaporated. The glass slides with the test item were inserted in glass flasks 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, 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. The test vessels were pre-treated with the appropriate test solution for at least 12 hours under test conditions.
The selection of the test loadings are based on the results of preliminary range finding tests (acute, see Annex II) and Early-Life Stage Toxicity Test (Noack Study Code SO21177/FSZ19203).
In the first definitive study no EC50-values could be determined. A second definitive study was carried out with only two additional test loadings (control, 515 & 1134 µg/L), which prolonged the concentration series of the first definitive study.

The studies were performed over a period of 96 hours under semi-static conditions with daily renewal of the test media. Seven test organisms were exposed to the test loadings and the controls. No significant mortality but sub-lethal effects were observed in the first definitive test. 100% mortality was observed in the test loadings 515 and 1134 µg/L. . Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.

The measured concentrations of all compounds of the test item in the fresh media at the start of an exposure-renewal interval (0, 24, 48 and 72 hours) were between < LOQ to 169% of the nominal values.
The measured concentration of the test item in the old media at the end of an exposure-renewal interval (24, 48, 72 and 96 hours) were between < LOQ and 156% of the nominal values.

All effect values given were based on the nominal loading of the test item and the overall mean measured concentration of the test item.

All validity criteria of the OECD 203 (2019) test guideline were met. The effect levels (LL0 / LL100) given in the table below are based on the nominal concentrations and the overall arithmetic mean measured concentrations of the test item.

LL-Values (96 hours) of the Test item
based on the nominal test item loading rates and the overall arithmetic mean measured concentration [µg/L]

 

	Based on nominal test item loading rates [µg/L]		Based on overall arithmetic
			mean measured test item concentrations [µg/L]
Test duration [h]	LL50	Confidence interval	LC50	Confidence interval
24	764	(515 – 1134)	988	(651 – 1498)
48	348	(344 – 351)	356	(345 – 368)
72	248	(234 – 515)	208	(191 – 651)
96	241	(234 – 515)	199	(191 – 651)
LL100 =	515		651
Lowest test item loading with
100 % mortality after 96 h
LL0 =	106		72.1
Highest test item loading with
0 % mortality after 96 h

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-12-2009 - 24-4-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, well conducted. No major restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Storage of samples: samples were analysed immediately after sampling without storage.
Samples treatment: samples were diluted in methanol.

Vehicle:

no

Details on test solutions:

A stock solution 5 mg/L CECAJEL 210 at was prepared on the day when the test was started and then on each day the solutions were renewed, Ie. after 24, 48 and 72 hours. Solutions were prepared by mixing 10 mg of the test item in 2 litres of dilution water and diluted in order to achieve the
desired range of concentrations.

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, colourless \
over the period of the test. No precipitation was observed at the end of the test

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The organisms used for the test were Dania rerio (Teleostei, Cyprinidae). Batch n° 09/Br/02/1 was supplied by Elevage de la Grande Riviere (69490 Saint-Forgeux). The fish were placed under the test conditions (medium, identical or lower biological loading, temperature, lighting, etc.) 20 days before the beginning of the test. The fish selected for the test were all from the same batch and were of the same age (date of birth stated by the supplier: 20/08/08). Their size (determined by measuring 10 randomly sampled fish) was 3.2 ± 0.18 cm. The mortality in the aquarium containing
the batch of fish used for the tests was less than 5 % (observed mortality: 0 %) over the 7 days preceding the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

22.4 ± 0.5 °C

pH:

7.43-8.05

Dissolved oxygen:

Did not fall below 77 % saturation.

Nominal and measured concentrations:

0.500, 0.370, 0.274, 0.203 and 0.151 mg/L (Nominal)

Details on test conditions:

The study was carried out in a thermostatically controlled room. The temperature, which was measured on a continuous basis in a test flask, was 22.4 ± 0.5 °C. The room was equipped with a programmed lighting system which produced a controlled lighting sequence: intensity between 400 and 800 lux at the surface of the aquaria; photo-period of 12 hours of darkness per 24 h. Physico-chemical parameters were measured using a METTLER TOLEDO 345 pH-meter for measurement of pH and with a WTW OXI 538 oxymeter for dissolved oxygen measurement. The study was performed using aquariums with a nominal capacity of 5 Iitres, covered with Altuglas®cover plates drilled with holes to allow the test solutions to be aerated. Volume of test medium per aquarium was 2.5 L.

At the beginning of the test, appropriate volumes of the stock solution of CECAJEL 210 (nominal concentration 5 mg/L) were poured directly into the test aquarium, which were topped up with dilution water to obtain the above mentioned range of nominal concentrations (without pH-adjustment).

Number of fish :10 for each test concentration and for the control (2 replicates). The definitive test was carried out under semi-static conditions.
Five fish were added in each test aquarium which were covered and incubated. Two aquaria were prepared for each concentration. The fish were examined after 1 hour and at intervals not exceeding 24 hours. They were considered to be dead if no reaction was obtained when their caudal peduncle was stimulated and if no respiratory movement was observed. The visible anomalies were noted,
as were any sub-lethal effects such as loss of balance, pigmentation, changes in swimming behaviour, respiratory distress, etc.

Solutions were renewed after 24, 48 and 72 hours and physical parameters were measured daily.

Reference substance (positive control):

yes

Remarks:

Conducted on every fish batch

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 0.29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 %(0.24-0.28)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% (0.11-0.18)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: ND

Details on results:

The test chemical displays significant toxicity to fish.

Results with reference substance (positive control):

The sensitivity of the test organisms was checked at least once for each new batch of fish, by determining the LC50-24h of the potassium dichromate. The results of the last test, carried out on 12/03/09, indicated a LC50-24h of 250 mglL. For reference, the former AFNOR standard NF T 90-303 states that the LC5Q-24h of thepotassium dichromate with respect to Dania rerio must be between 200 and 400 mglL. The fish batch was considered acceptable for testing.

Reported statistics and error estimates:

The mortality data are analyzed using an Excel sheet program. It was designed to calculate the LC50 value and the 95% confidence interval. Probit
analysis is generally used to calculate the 24-, 48-, 72 and 96-hour LC50 values.

Sublethal observations / clinical signs:

Percentage Mortality

Nominal mg/L	Mean Measured mg/L	24	48	72	96
0.50	0.29	0	70	100	100
0.37	0.22	0	30	90	100
0.27	0.14	0	0	30	60
0.20	0.09	0	0	10	20
0.15	0.05	0	0	0	10
Control	-	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

Test well conducted with in general with validated analytical method ,GLP accreditation and analytical certificate. CAS number is contradictory will need clarifying. May be a toxicity overestmation for risk assesment purposes. Suitable for classification and labeling. Some minor discrepancies. Can be considered reliable without significant restrictions.
This study is considered to be reliable without restriction now the identity of the test substance has been clarified.

Executive summary:

The acute toxicity (assessed by lethality) of the test item CECAJEL 210 to Danio reria (freshwater fish) for a duration of 96 hours under semi-static conditions was assessed according to the OECO Guideline 203.
Fish were exposed to a range of concentrations of CECAJEL 210 dissolved in dilution water.
The effective concentrations killing 50% of fish at 24h, 48h, 72h, and 96h (LC50-24h, 48h, 72h and 96h) were calculated by regression analysis using the Probit/log model. The results are presented in the following table:

Duration of the exposure period	LC50 value, mg/L (95% CI)
24h	>29
48h	0.25 (0.24 - 0.28
72h	0.14 (0.11 - 0.18
96h	0.10 (ND)

CI: confidence Interval; ND: not determined
The

Description of key information

There are many acute toxicity to fish studies available for the primary fatty amine ethoxylates (2EO) from which most are considered supporting studies mainly because they lack quantification of the exposure concentrations or sufficient identification of the test substance used. The available short-term toxicity test results indicate that fish are generally less sensitive to the primary fatty amine ethoxylates when compared to algae and short-term daphnia. The available acute fish data for the primary fatty amine ethoxylates which covers an alkyl chain length range from C12 to C18 reveals only a slight increase of the toxicity in relation to an increasing alkyl chain length.

 

Two studies however are considered adequate to address the acute fish test endpoint. Both were performed semi-statically with daily renewal of the test solutions and with quantification of the exposure concentrations. One study where the test solutions were prepared via dilution of a stock solution is performed with PFAEO-O (CAS no.: 25307-17-9) and is due to the availability of a test with the test substance, Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs. (CAS no.: 71786-60-2; Old name: Bis(2-hydroxyethyl) coco amine; abbreviation: PFAEO-C) considered supporting to the key study performed with the test substance.  

 

The acute toxicity of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (PFAEO-C; CAS no.: 71786-60-2) to fish (zebrafish) was determined according to OECD TG 203 (2019) under GLP conditions.

The test item PFAEO-C is poorly soluble but dispersible in water. It is a UVCB substance with constituents of different water solubility. Water accommodated fractions (WAFs) were therefore prepared 24 ± 1 hour prior to the start of exposure with the nominal loading rates of the test item of 10.0 – 22.0 – 48.4 – 106 – 234 – 515 – 1134 μg/L, corresponding to the overall arithmetic mean measured concentrations of 5.37 – 11.3 – 32.2 – 72.1 – 191 – 651 - 1498 μg/L. For the preparation of the loading rate the test item was dissolved in methanol applied onto curved glass slides. The methanol was evaporated. The glass slides with the test item were inserted in glass flasks 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, 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. The test vessels were pre-treated with the appropriate test solution for at least 12 hours under test conditions.

The selection of the test loadings was based on the results of preliminary range finding tests and Early-Life Stage Toxicity Test (Noack Study Code SO21177/FSZ19203).

In the first definitive study no EC50-values could be determined. A second definitive study was carried out with only two additional test loadings (control, 515 & 1134 μg/L), which prolonged the concentration series of the first definitive study.

The studies were performed over a period of 96 hours under semi-static conditions with daily renewal of the test media. Seven test organisms were exposed to the test loadings and the controls. No significant mortality but sub-lethal effects were observed in the first definitive test.

100% mortality was observed in the test loadings 515 and 1134 μg/L. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.

The measured concentrations of all compounds of the test item in the fresh media at the start of an exposure-renewal interval (0, 24, 48 and 72 hours) were between < LOQ to 169% of the nominal values.

The measured concentration of the test item in the old media at the end of an exposure-renewal interval (24, 48, 72 and 96 hours) were between < LOQ and 156% of the nominal values.

on the nominal loading of the test item and the overall mean measured concentration of the test item.

All validity criteria of the OECD 203 (2019) test guideline were met. The effect levels (LL0 / LL100) given in Table 1 are based on the nominal concentrations and the overall arithmetic mean measured concentrations of the test item. 

All effect values given are based on the nominal test item loading rates (LLxx) and the overall arithmetic mean measured concentration (LCxx).

The 96h LL50/LC50 values calculated from the effects observed are: 241 and 199 µg/L

 

Test duration [h]	LL50	Confidence interval	LC50	Confidence interval
24	764	(515 – 1134)	988	(651 – 1498)
48	348	(344 – 351)	356	(345 – 368)
72	248	(234 – 515)	208	(191 – 651)
96	241	(234 – 515)	199	(191 – 651)
LL100 =Lowest test item loading with 100 % mortality after 96 h	515		651
LL0 =Highest test item loading with 0 % mortality after 96 h	106		72.1

 

 

 

 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Remarks:

Test performed according to the WAF approach. LL50 = 0.241 mg/L

Effect concentration:

0.241 mg/L

Additional information

Most of the short-term toxicity fish tests were conducted in a period when no reliable specific method of analyses was available. The concentrations were therefore not analytically verified, the reliability of the results is limited because of the poor solubility of the test substances and partial sorption onto the walls of test vessels. Hence, most of the tests were considered to be valid with restrictions. The available data reveal a comparable toxicity independent of the chain length.

Two studies are however considered adequate to address the acute fish test endpoint. Both were performed semi-statically with daily renewal of the test solutions and with quantification of the exposure concentrations. One study where the test solutions were prepared via dilution of a stock solution is performed with PFAEO-O (CAS no.: 25307-17-9) and is due to the availability of a test with the test substance, Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs. (CAS no.: 71786-60-2; PFAEO-C) considered supporting to the key study which was performed with the test substance.  All effect values for this study are given based on the nominal test item loading rates (LLxx) and the overall arithmetic mean measured concentration (LCxx). The 96h LL50/LC50 values calculated from the effects observed are: 241 and 199 µg/L.

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.

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


• 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.


• OECD (2019): Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals. OECD series on testing and assessment no. 23 (second edition), ENV/JM/MONO(2000)6/REV1