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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-27 to 2009-11-12

Reliability:

1 (reliable without restriction)

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 (6.4 and 200 µg/L) and the control were analytically verified at the beginning and end of the test (prepared with algae).
- Sampling method: Separate replicates for the test item analysis after 0 h and 72 h (6.4, 200 µg/L and control) were prepared without algae for analysis.
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-values (the second highest test concentration of 200 µg/L) before analysis
- Sample storage condition: All samples were stored at room temperature, 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. After agitation, an equilibration phase for 1 h before application was carried out.
- Eluate: Natural river water
- Differential loading: 2.00 - 6.40 - 20.0 - 64.0 - 200 - 640 µg/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 – 0.5 mL aliquots of the test medium (i.e. samples of exposed algal cells) from test concentrations 20.0 – 640 µg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 3 – 12 days under test conditions. The test item effect was observed to be reversible at 20 – 640 µg/L. Therefore, there is potential for recovery following exposure up to 640 µg/L (highest test concentration).
For exact data please refer to information on materials and methods.

Hardness:

Not measured

Test temperature:

Mean: 23 °C, Min: 22 °C, Max: 24 °C

pH:

Nominal test item
concentration pH-value
[µg/L] Start; 0 h End; 72 h
640 7.97 7.97
200 7.97 8.05
64 7.98 8.10
20 7.95 8.12
6.4 7.97 8.28
2.0 7.97 8.32
Control 7.96 8.28

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 1703663 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 Innerste was used. This river is located in
Sarstedt / Ruthe. 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
Characterisation Data of Natural River Water
River Hüpeder Bach
Location D-30982 Pattensen, Am Pfingstanger
Sampling Date 2009-10-07
Weather on Day of Sampling dry, few clouds, ca. 22 °C
Colour Yellowish, clear
pH 7.79
Conductivity [µS/cm] 531
DOC [mg C/L] 3.8
TOC [mg C/L] 3.7
Ammonium-N [mg N/L] < 0.015
Nitrate-N [mg N/L] 3.06
o-Phosphate-P [mg P/L] 0.116
Total Phosphate [mg P/L] 0.140
Suspended Matter [mg/L] 17.6
Total Hardness [mg CO3/L] 170

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)

Nominal
Test Item Concentration Specific Growth
[mg/L] Rate Inhibition [%] Yield Inhibition [%]
10.0 100 100
1.00 100 100
0.10 61 96
0.01 4 19

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

107 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (94.6 - 123)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.16 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (7.17- 11.9)

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 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
Bis (2-hydroxyethyl) cocoalkylamine(CAS No. 61791-31-9) (0-72 h)

                                based on nominal test item concentrations [µg/L]

                               

	Rate-related inhibition
ErC10	9.16 (7.17 – 11.9)
ErC20	20.9 (16.8 – 25.7)
ErC50	107 (94.6 – 123)
	Inhibition of yield
EyC10	4.60 (3.97 – 5.31)
EyC20	6.82 (6.12 – 7.66)
EyC50	15.1 (13.8 – 16.3)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 h		24 h	48 h	72 h
640		1		9787		3381	368	2131
		2		9787		6394	570	3555
		3		9787		5491	2920	3574
		Mean		9787		5089	1286	3087
200		1		9787		14487	27732	71103
		2		9787		14144	29508	65557
		3		9787		13603	28761	62333
		Mean		9787		14078	28667	66331
64.0		1		9787		17401	25344	175406
		2		9787		18806	29861	171538
		3		9787		16898	36173	219995
		Mean		9787		17702	30459	188980
20.0		1		9787		22975	88089	669827
		2		9787		17871	81974	616801
		3		9787		17110	85419	668779
		Mean		9787		19319	85161	651802
6.40		1		9787		29316	180585	1342467
		2		9787		31247	172980	1434747
		3		9787		23431	203230	1446977
		Mean		9787		27998	185598	1408064
2.00		1		9787		29142	214181	1695668
		2		9787		26308	209608	1629553
		3		9787		32869	223045	1801709
		Mean		9787		29440	215611	1708977
Control		1		9787		26157	221353	1653979
		2		9787		29151	221193	1752256
		3		9787		31459	226015	1833932
		4		9787		24973	227355	1692641
		5		9787		38053	208071	1621882
		6		9787		29555	216150	1667289
		Mean		9787		29891	220023	1703663

Evaluation after 72 h

             

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[µg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
640	1		-0.508	100		-7656	100
	2		-0.338	100		-6232	100
	3		-0.336	100		-6213	100
	Mean		-0.394	100		-6700	100
200	1		0.661	61.6		61316	96.4
	2		0.634	63.1		55770	96.7
	3		0.617	64.1		52546	96.9
	Mean		0.637	62.9		56544	96.7
64.0	1		0.962	44.1		165619	90.2
	2		0.955	44.5		161751	90.5
	3		1.04	39.7		210208	87.6
	Mean		0.985	42.7		179193	89.4
20.0	1		1.41	18.1		660040	61.0
	2		1.38	19.7		607014	64.2
	3		1.41	18.1		658992	61.1
	Mean		1.40	18.6		642015	62.1
6.40	1		1.64	4.60		1332680	21.3
	2		1.66	3.31		1424960	15.9
	3		1.67	3.15		1437190	15.2
	Mean		1.66	3.69		1398277	17.5
2.00	1		1.72	0.07		1685881	0.47
	2		1.71	0.85		1619766	4.38
	3		1.74	-1.10		1791922	-5.79
	Mean		1.72	-0.06		1699190	-0.31
Control	1		1.71			1644192
	2		1.73			1742469
	3		1.74			1824145
	4		1.72			1682854
	5		1.70			1612095
	6		1.71			1657502
	Mean		1.72			1693876

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	0.983	2.14	2.01	1.71	0.633	37.0	31.5
	2	1.09	2.03	2.07	1.73	0.553	32.0
	3	1.17	1.97	2.09	1.74	0.503	28.8
	4	0.94	2.21	2.01	1.72	0.684	39.8
	5	1.36	1.70	2.05	1.70	0.348	20.4
	6	1.11	1.99	2.04	1.71	0.527	30.8
				Mean	1.72
				SD ±	0.01
				VC %	0.87

Validity criteria fulfilled:

yes

Conclusions:

Bis (2-hydroxyethyl) cocoalkylamine (CAS No. 61791-31-9) 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 9.16 (7.17 – 11.9) and 4.60 (3.97 – 5.31) µg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 107 (94.6 – 123) and 15.1 (13.8 – 16.3) µg/L, respectively.
The test item effect was observed to be reversible at 20 – 640 µg/L. Therefore, there is potential for recovery following exposure up to 640 µg/L (highest test concentration).
The concentrations of Bis (2-hydroxyethyl) cocoalkylamine were analysed at the concentration levels 6.4 and 200 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 83 – 87 % of the nominal values. At the end of the test Bis (2-hydroxyethyl) cocoalkylamine was analysed at concentration levels 6.4 and 200 mg/L (prepared without algae) and gave recoveries of 0 - 11 % 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 was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. No adsorption (< LOQ) of the test item to glassware was observed. Therefore all effect values are given based on nominal concentrations of the test item.

Executive summary:

The toxicity of Bis (2-hydroxyethyl) cocoalkylamine(CAS No. 61791-31-9) (Batch no.: S001016)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 October 27 to November12, 2009. 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.

Bis (2-hydroxyethyl) cocoalkylamine (CAS no. 61791-31-9) is poorly soluble in water and also 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 amines 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 amines 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 ethoxylated primary fatty amines 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. Six concentrations were tested in a geometrical series with a dilution factor of √10, nominal: 2.00 - 6.40 - 20.0 - 64.0 - 200 - 640 µg/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 Bis (2-hydroxyethyl) cocoalkylamine were analysed at the concentration levels 6.4 and 200 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 83 – 87 % of the nominal values. At the end of the test Bis (2-hydroxyethyl) cocoalkylamine was analysed at concentration levels 6.4 and 200 mg/L (prepared without algae) and gave recoveries of 0 - 11 % 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 was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. No adsorption ( LOQ) of the test item to glassware was observed. Therefore all effect values are given based on nominal concentrations of the test item. For details of the analytical results please refer to part 9.

Bis (2-hydroxyethyl) cocoalkylamine (CAS No. 61791-31-9) 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 9.16 (7.17 – 11.9) and 4.60 (3.97 – 5.31) µg/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 107 (94.6 – 123) and 15.1 (13.8 – 16.3) µg/L, respectively.

After 72 h algae were transferred from the nominal concentration of 20 – 640 µg/L and the control to fresh untreated medium and allowed to grow for further 3 – 12 d under test conditions. The test item effect was observed to be reversible at 20 – 640 µg/L. Therefore, there is potential for recovery following exposure up to 640 µg/L (highest test concentration).

Reference 2

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-12-05 to 2021-12-13, with the definitive exposure phase from 2021-12-07 to 2021-12-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 clear 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:

via LC-MS/MS

Details on sampling:

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

Sampling schedule
All loading rates and the control were analytically verified via LC-MS/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 8.00 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 concentration levels at the end of the exposure.

 
Sampling and pre-treatment
At the start of the exposure, after 24 and after 48 hours, samples were taken from one additional replicate of each test item loading rate and the control. At the end of the exposure, samples were taken from pooled replicates.
For the adsorption to glass the vessels were rinsed carefully with deionized 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.

Vehicle:

no

Details on test solutions:

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

Preparation of the water accommodated fraction
Seven water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 0.00800 to 8.00 µg/L set up in a geometric series with a factor of √10: 0.00800 – 0.0253 – 0.0800 – 0.253 – 0.800 – 2.53 – 8.00 µg/L.
The procedure according to ASTM D6081 (2019) as described below was 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 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 . This procedure was found to be optimal according to the results of a solubility test carried out in advance. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour at room temperature, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The first 25 mL were discarded.
The WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative for all WAFs. The resulting water accommodated fractions (WAF) were used in the test.

Test organisms (species):

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

Details on test organisms:

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

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

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

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

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

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

Test type:

static

Water media type:

freshwater

Remarks:

OECD TG 201 medium

Limit test:

no

Total exposure duration:

72 h

Hardness:

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

Test temperature:

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

pH:

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

Conductivity:

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

Nominal and measured concentrations:

Seven water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 0.00800 to 8.00 µg/L set up in a geometric series with a factor of √10: 0.00800 – 0.0253 – 0.0800 – 0.253 – 0.800 – 2.53 – 8.00 µg/L.

Details on test conditions:

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

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

Test method
Static procedure

Duration of the test
72 hours

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

Test container and Pre-Treatment
Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs were used. A coating phase (saturation of the test container) was carried out. The test containers were pre-treated with the appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure, the test container was emptied and refilled with freshly prepared test solution. Preparation of the test solutions for coating was documented in the raw data and in the final report.

Test volume 100 mL

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

Dilution water According to the guidelines

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

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

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

Incubation
The flasks were positioned randomly and repositioned daily.

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

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

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

Light regime
24 hours/day light

Light homogeneity
Within ± 15% over incubation area


Type and Frequency of Measurements

Biological Parameters

Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was observed in the preliminary range finding test at the loading rate of 1.00 mg/L. A calibration curve with a satisfactory correlation was used to calculate the cell density from fluorescence and given in the report.

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

Physico-chemical Properties

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

Reference substance (positive control):

yes

Remarks:

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

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.4 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks:

Inhibition

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

0.705 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks:

Inhibition

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.503 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks:

Inhibition

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

0.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks:

Inhibition

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.253 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks:

Inhibition

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.06 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Inhibition

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

1.95 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Inhibition

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.19 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Inhibition

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

0.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Inhibition

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.253 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Inhibition

Details on results:

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

Physico-chemical Data

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

Measured Exposure Concentrations during the Definitive Test

The test item concentrations of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) were analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) with algae. Additionally, the loading rate 8.00 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 concentration levels at the end of the exposure. Samples after 24, 48, 72 hours of exposure was centrifuged to separate the algae from the water phase. Both was analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae.
As the test item is a UVCB substance, all evaluations are based on the nominal loading rates of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2).

Results with reference substance (positive control):

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

Reported statistics and error estimates:

EL-values and statistical analyses
EL10-,EL20- and EL50- 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 and LOEL values
NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. The Dunnett’s method was used for the statistical significance calculations. A Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normal distribution of data. The BROWN-FORSYTHE test was done for analysis of variance homogeneity. P-values for both Normality and Equal Variance tests are 0.01. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

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.

Cell Densities

Nominal test item loading rates	Replicate	Cell density [cells/mL]
[µg/L]	No.	0 hours	24 hours	48 hours	72 hours
8.00	1	6864	10253	10318	4902
	2	6864	11533	10766	6144
	3	6864	9670	12041	4601
	Mean	6864	10485	11042	5216
2.53	1	6864	16622	44321	214858
	2	6864	17818	47744	270622
	3	6864	19337	58091	290076
	Mean	6864	17926	50052	258519
0.800	1	6864	19876	113381	838607
	2	6864	21586	115594	732904
	3	6864	23900	126254	842902
	Mean	6864	21787	118410	804804
0.253	1	6864	26423	177369	1183806
	2	6864	26171	160637	1003593
	3	6864	21302	154807	1025656
	Mean	6864	24632	164271	1071018
0.0800	1	6864	25416	162094	1128390
	2	6864	27608	165773	1131651
	3	6864	27617	162598	1092092
	Mean	6864	26880	163488	1117378
0.0253	1	6864	29156	191250	1138511
	2	6864	26970	173177	1231867
	3	6864	25483	166871	1152432
	Mean	6864	27203	177099	1174270
0.00800	1	6864	27012	178377	1158325
	2	6864	22867	150651	1067488
	3	6864	27593	149851	1053431
	Mean	6864	25824	159626	1093081
Control	1	6864	26275	141646	1092227
	2	6864	24698	156391	961312
	3	6864	22771	142006	1026398
	4	6864	28391	145047	1022193
	5	6864	25472	186986	1051497
	6	6864	27845	171891	1234993
	Mean	6864	25909	157328	1064770

 

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]		[%]
8.00	1		-0.11	100		-1962	100
	2		-0.04	100		-720	100
	3		-0.13	100		-2263	100
	Mean	(s)	-0.09	100	(s)	-1648	100
2.53	1		1.15	31.7		207994	80.3
	2		1.22	27.1		263758	75.1
	3		1.25	25.7		283212	73.2
	Mean	(s)	1.21	28.2	(s)	251655	76.2
0.800	1		1.60	4.68		831743	21.4
	2		1.56	7.35		726040	31.4
	3		1.60	4.57		836038	21.0
	Mean	(s)	1.59	5.53	(s)	797940	24.6
0.253	1		1.72	-2.16		1176942	-11.3
	2		1.66	1.11		996729	5.78
	3		1.67	0.68		1018792	3.70
	Mean	(ns)	1.68	-0.12	(ns)	1064154	-0.59
0.0800	1		1.70	-1.21		1121526	-6.01
	2		1.70	-1.27		1124787	-6.32
	3		1.69	-0.56		1085228	-2.58
	Mean	(ns)	1.70	-1.01	(ns)	1110514	-4.97
0.0253	1		1.70	-1.39		1131647	-6.97
	2		1.73	-2.95		1225003	-15.8
	3		1.71	-1.63		1145568	-8.29
	Mean	(ns)	1.71	-1.99	(ns)	1167406	-10.4
0.00800	1		1.71	-1.73		1151461	-8.84
	2		1.68	-0.11		1060624	-0.26
	3		1.68	0.15		1046567	1.07
	Mean	(ns)	1.69	-0.56	(ns)	1086217	-2.68
Control	1		1.69			1085363
	2		1.65			954448
	3		1.67			1019534
	4		1.67			1015329
	5		1.68			1044633
	6		1.73			1228129
	Mean		1.68			1057906

 

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	1.34	1.69	2.04	1.69	0.350	19.8
	2	2	1.28	1.85	1.82	1.65	0.318
	3	3	1.20	1.83	1.98	1.67	0.414
	4	4	1.42	1.63	1.95	1.67	0.268
	5	5	1.31	1.99	1.73	1.68	0.344
	6	6	1.40	1.82	1.97	1.73	0.296
				Mean	1.68
				SD ±	0.03
				CV [%]	1.69

SD = Standard deviation CV = Coefficient of variation

 

Environmental Conditions

Nominal test item loading rates				pH-values
[µg/L]				Start; 0 hours	End; 72 hours
8.00				7.13	7.66
2.53				7.20	7.78
0.800				7.21	8.18
0.253				7.30	8.50
0.0800				7.32	8.54
0.0253				7.40	8.71
0.00800				7.58	8.85
Control				7.77	8.92
Room temperature [°C]:		min.: 22.0		max.: 23.0		mean value: 22.5
	n = 23		mean value: 5264				CV [%]: 7.49
Light intensity			range of the measured values: 4561 - 5835
[lux]			equalling -13.3 to 10.9%

CV = Coefficient of variation n = number of measuring points

 

Water parameters of the Dilution Water

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

*Limit of quantification = 2.00 mg C/L

 

Measured Concentrations of the compound C10 Amine + 2EO of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) during the Definitive Test
(0, 24, 48, 72 hours)

Sampling date		0 hours		24 hours		48 hours		72 hours
Nominal		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
loading rate of the		C10 Amine + 2EO
test item	active substance	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
8.00^1)	0.582^1)	0.631	109	0.352	61	0.564	97	0.619	106
8.00	0.582	0.535	92	0.347	60	0.454	78	0.602	103
2.53	0.184	0.154	84	< LOQ		0.168	92	0.139	75
0.800	0.0582	< LOQ		< LOQ		< LOQ		< LOQ
0.0800	0.00582	< LOQ		< LOQ		< LOQ		< LOQ
0.0253	0.00184	< LOQ		< LOQ		< LOQ		< LOQ
0.00800	0.000582	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C10 Amine + 2EO of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LOQ = limit of quantification (2.00 µg/L of the test item, corresponding to 0.145 µg a.s./L)
1) = test item concentration without algae

 

Measured Concentrations of the compound C10 Amine + 2 EO of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) (Algae and glass adsorption)
(0, 24, 48, 72 hours)

Sampling date		24 hours		48 hours		72 hours		72 hours
Nominal		Algae adsorption						Glass Adsorption
loading rate of the		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
		C10 Amine + 2 EO
test item	active substance	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
8.00	0.582	0.0426	7	0.0435	7	< LOQ		< LOQ
2.53	0.184	< LOQ		< LOQ		< LOQ
0.800	0.0582	< LOQ		< LOQ		< LOQ		< LOQ
0.253	0.0184	< LOQ		< LOQ		< LOQ		< LOQ
0.0800	0.00582	< LOQ		< LOQ		< LOQ		< LOQ
0.0253	0.00184	< LOQ		< LOQ		< LOQ		< LOQ
0.00800	0.000582	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		-

Meas. conc. = measured concentration of the compound C10 Amine + 2 EO of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LCL = lowest calibration level (0.500 µg/L of the test item corresponding to 0.0364 µg a.s./L)
- = not determined

 

Measured Concentrations of the compound C12 Amine + 2EO of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) during the Definitive Test
(0, 24, 48, 72 hours)

Sampling date		0 hours		24 hours		48 hours		72 hours
Nominal		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
loading rate of the		C12 Amine + 2EO
test item	active substance	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
8.00^1)	4.21^1)	3.19	76	2.11	50	2.08	49	3.21	76
8.00	4.21	2.08	49	1.46	35	2.66	63	2.42	58
2.53	1.33	< LOQ		< LOQ		1.06	80	< LOQ
0.800	0.421	< LOQ		< LOQ		< LOQ		< LOQ
0.253	0.133	< LOQ		< LOQ		< LOQ		< LOQ
0.0800	0.0421	< LOQ		< LOQ		< LOQ		< LOQ
0.0253	0.0133	< LOQ		< LOQ		< LOQ		< LOQ
0.00800	0.00421	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C12 Amine + 2EO of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LOQ = limit of quantification (2.00 µg/L of the test item, corresponding to 1.05 µg a.s./L)
1) = test item concentration without algae

Measured Concentrations of the compound C12 Amine + 2 EO of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) (Algae and glass adsorption)
(0, 24, 48, 72 hours)

Sampling date		24 hours		48 hours		72 hours		72 hours
Nominal		Algae adsorption						Glass Adsorption
loading rate of the		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
		C12 Amine + 2 EO
test item	active substance	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
8.00	4.21	1.75	42	1.29	31	0.907	22	< LOQ
2.53	1.33	0.506	38	0.455	34	< LOQ		< LOQ
0.800	0.421	< LOQ		< LOQ		< LOQ		< LOQ
0.253	0.133	< LOQ		< LOQ		< LOQ		< LOQ
0.0800	0.0421	< LOQ		< LOQ		< LOQ		< LOQ
0.0253	0.0133	< LOQ		< LOQ		< LOQ		< LOQ
0.00800	0.00421	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		-

Meas. conc. = measured concentration of the compound C12 Amine + 2 EO of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LCL = lowest calibration level (0.500 µg/L of the test item corresponding to 0.263 µg a.s./L)
- = not determined

 

Measured Concentrations of the compound C14 Amine + 2EO of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) during the Definitive Test
(0, 24, 48, 72 hours)

Sampling date		0 hours		24 hours		48 hours		72 hours
Nominal		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
loading rate of the		C14 Amine + 2EO
test item	active substance	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
8.00^1)	1.49^1)	0.692	46	0.616	41	< LOQ		0.710	48
8.00	1.49	< LOQ		< LOQ		0.767	51	< LOQ
2.53	0.472	< LOQ		< LOQ		< LOQ		< LOQ
0.800	0.149	< LOQ		< LOQ		< LOQ		< LOQ
0.253	0.0472	< LOQ		< LOQ		< LOQ		< LOQ
0.0800	0.0149	< LOQ		< LOQ		< LOQ		< LOQ
0.0253	0.00472	< LOQ		< LOQ		< LOQ		< LOQ
0.00800	0.00149	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the compound C14 Amine + 2EO of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LOQ = limit of quantification (2.00 µg/L of the test item, corresponding to 0.373 µg a.s./L)
1) = test item concentration without algae

 

Measured Concentrations of the compound C14 Amine + 2 EO of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) (Algae and glass adsorption)
(0, 24, 48, 72 hours)

Sampling date		24 hours		48 hours		72 hours		72 hours
Nominal		Algae adsorption						Glass Adsorption
loading rate of the		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
		C14 Amine + 2 EO
test item	active substance	Meas.	%	Meas.	%	Meas.	%	Meas.	%
[µg/L]	[µg a.s./L]	conc.		conc.		conc.		conc.
		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]		[µg a.s./L]
8.00	1.49	0.896	60	0.477	30	0.389	26	0.162	11
2.53	0.472	0.190	40	0.161	34	< LCL		0.0944	20
0.800	0.149	< LOQ		< LOQ		< LOQ		< LOQ
0.253	0.0472	< LOQ		< LOQ		< LOQ		< LOQ
0.0800	0.0149	< LOQ		< LOQ		< LOQ		< LOQ
0.0253	0.00472	< LOQ		< LOQ		< LOQ		< LOQ
0.00800	0.00149	< LOQ		< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		-

Meas. conc. = measured concentration of the compound C14 Amine + 2 EO of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LCL = lowest calibration level (0.500 µg/L of the test item corresponding to 0.0932 µg a.s./L)
- = not determined

 

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	1.09	0.787 ± 0.566
95% confidence interval	0.944 – 1.25
	Yield inhibition
EyC50	0.643	0.429 ± 0.348
95% confidence interval	0.581 – 0.712

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	155-fold
		(specific growth rate 1.68 day^-1)
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures	≤ 35%	19.8%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤ 7%	1.69%

 

Percentage of the Relevant Amine

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

 

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

 

Parameter, Acceptance Criteria and Results of the Method Validation

Parameter	Acceptance criteria	Result
Linearity	≥ 5 standard concentrations,	0.5 to 20 µg test item/L (n = 7),	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
Lowest calibration level (LCL)	S/N ≥ 9 for quantifier ion trace	S/N for 0.5 µg test item/L	ü
	S/N ≥ 3 for qualifier ion trace	C10 Amine + 2 EO
		1430 (Quantifier), 432 (Qualifier)
		C12 Amine + 2 EO
		4847 (Quantifier), 8439 (Qualifier)
		C14 Amine + 2 EO
		792 (Quantifier), 2555 (Qualifier)
Matrix effects	Should be ≤ 20%	Matrix effects: < 20%	ü
Limit of Quantification (LOQ)	Should be at or below lowest test concentration	2.0 µg test item/L (1 x LOQ)*	ü
		10 µg test item/L (5 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: 98% (n = 5)
		5 x LOQ: 100% (n = 5)
		C12 Amine + 2 EO
		1 x LOQ: 98% (n = 5)
		5 x LOQ: 101% (n = 5)
		C14 Amine + 2 EO
		1 x LOQ: 103% (n = 5)
		5 x LOQ: 101% (n = 5)

* 2µg test item/L is above the lowest test concentration and represents the technical possible LOQ.

Precision^1)	Relative standard deviation ≤ 20% per fortification level	C10 Amine + 2 EO	ü
		1 x LOQ: 2.0%
		5 x LOQ: 2.1%
		C12 Amine + 2 EO
		1 x LOQ: 3.2%
		5 x LOQ: 3.1%
		C14 Amine + 2 EO
		1 x LOQ: 2.9%
		5 x LOQ: 3.2%
Stability	Samples: Stable if 70 – 120% of nominal concentration	See section 18.1	ü
	Calibration standards: Stable if ±10% of freshly prepared standard
Specificity^1):	Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity).	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
	Blank values < 30% of the LOQ	Blank values < 30% of LOQ	ü
Procedural recovery	Procedural recoveries with experimental samples. The recovery has to be 70-120% of the nominal value.	See section 18.2	ü

 = criterion fulfilled
- = not determined

 

Preparation of Fortified Samples of the Test Item

LOQ Level	Control	1	5
Stock solution	-	100 mg test item/L in Acetonitrile
Spiking solution	-	200	1000
[µg test item/L]		(dilution medium)	(dilution medium)
(Medium)
Replicates	2	5	5
Concentration of the LOQ	-	2.0	10
[µg test item/L]
Medium for preparation	Algae dilution medium
Volume of spiking solution [mL]	-	0.05	0.05
Volume of medium [mL]	5.0	34790	34790
Dilution factor	2	2	2
Dilution medium	Acetonitrile including 2% formic acid
Sample volume [mL]	5.0	5.0	5.0
Finale volume [mL]	10	10	10
Final expected concentrations for analyses [µg/L]	-	1 µg test item/L, corresponding to	5 µg test item/L, corresponding to
		0.0727 C10 Amine + 2EO	0.364 C10 Amine + 2EO
		0.526 C12 Amine + 2EO	2.63 C12 Amine + 2EO
		0.186 C14 Amine + 2EO	0.932 C14 Amine + 2EO

Dilution medium: Acetonitrile : ultra-pure water (50 : 50) containing 1% formic acid

 

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*: 2.07 µg test item/L (1 x LOQ) and 10.3 µg test item/L (5 x LOQ).

Active substance	1 x LOQ	5 x LOQ
	[µg a.s./L]	[µg a.s./L]
C10 Amine + 2 EO	0.150	0.751
C12 Amine + 2 EO	1.09	5.44
C14 Amine + 2 EO	0.385	1.93

* = 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 substance: C10 Amine + 2 EO

Replicate	Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
	C10 Amine + 2 EO
	1 x LOQ		5 x LOQ
	Meas.	%	Meas.	%
	conc.		conc.
	[µg a.s./L]		[µg a.s./L]
1	0.152	101	0.774	103
2	0.145	97	0.766	102
3	0.145	97	0.752	100
4	0.148	99	0.737	98
5	0.148	98	0.743	99
Mean	0.148	98	0.755	100
SD ±	0.003		0.02
CV [%]	2.0		2.1

Meas. conc. = measured concentration C10 Amine + 2 EO in the test item, dilution factor taken into account
a.s. = active substance
% = percent concentration of the fortified sample
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 substance: C12 Amine + 2 EO

Replicate	Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
	C12 Amine + 2 EO
	1 x LOQ		5 x LOQ
	Meas.	%	Meas.	%
	conc.		conc.
	[µg a.s./L]		[µg a.s./L]
1	1.11	102	5.66	104
2	1.02	94	5.57	102
3	1.08	99	5.53	102
4	1.05	96	5.48	101
5	1.07	99	5.21	96
Mean	1.07	98	5.49	101
SD ±	0.03		0.2
CV [%]	3.2		3.1

 

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 substance: C14 Amine + 2 EO

Replicate	Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
	C14 Amine + 2 EO
	1 x LOQ		5 x LOQ
	Meas.	%	Meas.	%
	conc.		conc.
	[µg a.s./L]		[µg a.s./L]
1	0.413	107	2.05	107
2	0.383	99	1.93	100
3	0.396	103	1.90	99
4	0.389	101	1.91	99
5	0.394	102	1.92	100
Mean	0.395	103	1.94	101
SD ±	0.01		0.1
CV [%]	2.9		3.2

Meas. conc. = measured concentration C12 Amine + 2 EO and C14 Amine + 2 EO, respectively, in the test item,
dilution factor taken into account
a.s. = active substance
% = percent concentration of the fortified sample
SD = standard deviation

CV = coefficient of variation

 

Stability

The stability of the standard stock solution (1000 mg test item/L in acetonitrile) was checked after 15 days and was 90 to 104% 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% for all measured components and was found to be stable over this period.

The stability of the fortified samples at the 1xLOQ was checked after 3 and after 7 days. Samples were found to be stable over this period.

 

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

	3 days		7 days
Component	Meas.	%	Meas.	%
	conc.		conc.
	[µg a.s.L]		[µg a.s.L]
C10 Amine + 2 EO	0.122	81	0.115	76
C12 Amine + 2 EO	0.858	79	0.812	75
C14 Amine + 2 EO	0.318	83	0.300	78

 

Procedural Recovery

A procedural recovery (Quality Control) on 1x LOQ Level was freshly prepared on each day of analysis. It was treated in parallel to the test samples.

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

Sampling	0 hours		24 hours		48 hours		72 hours
date
Component	Meas.	%	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	0.133	92	0.129	89	0.108	75	0.131	91
C12 Amine + 2 EO	0.960	92	0.937	90	1.06	102	0.915	88
C14 Amine + 2 EO	0.348	94	0.310	84	0.391	106	0.333	90
Sampling	72 hours^1)
date
Component	Meas.	%
	conc.
	[µg a.s.L]
C10 Amine + 2 EO	0.134	93
C12 Amine + 2 EO	1.06	102
C14 Amine + 2 EO	0.391	106

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
a.s. = active substance
Quality Control = 2.00 µg test item/L, weighing factor taken into account
1) = sample set for glass adsorption samples

 

Range Finding Test (non-GLP)

Solubility Test

Two WAFs were prepared as described in section 4.2. After 24, 48 and 72 hours samples were taken as shown behind and analyzed via LC-MS.

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	61.2	68.2	81.7
	C16 Component + 2EO	44.0	52.8	54.5
	C18 Component + 2EO	20.9	14.3	13.4
		35.5	40.2	39.9
Nominal test item loading rate 1.00 mg/L	C8 Component + 2EO	76.8	-
	C10 Component + 2EO	91.3
	C12 Component + 2EO	92.3
	C14 Component + 2EO	84.9
	C16 Component + 2EO	57.1
	C18 Component + 2EO	33.0
	C18:1 Component + 2EO	51.3

LCL = lowest calibration standard = 2 µg/L
Measured concentrations are relative concentrations and not corrected by the content.

Preliminary Range Finding Test

Two non-GLP preliminary range finding tests under static conditions over a period of 72 hours were conducted at the test facility. Water accommodated fractions (WAF) with loading rates of 10 – 100 – 1000 µg test item/L and 0.01 – 0.1 – 10 µg test item/L were freshly prepared as described. The WAFs were prepared on day -2 and on day -1. All loading rates were tested and were clear and colorless throughout the exposure period. The Tyndall effect was negative.
For the preparation of the WAFs a stirring phase of 24 hours is chosen for the definitive test. In the first preliminary range finding test with nominal loading rates of 10 – 100 – 1000 µg test item/L inhibition of growth rate and yield were 100% in all loading rates. Therefore, this test is not reported. For results of the second preliminary test encompassing the loading range of the definitive test, see the tables below.

 

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

Nominal test item loading rates	Growth Rate Inhibition	Yield Inhibition
[µg/L]	[%]	[%]
10	100	100
0.1	8	34
0.01	1	6

 

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 test item loading rates		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
		(C12 Component + 1EO)
[µg/L]	[µg a.s./L]	Meas. conc.	%	Meas. conc.	%
		[µg a.s./L]		[µg a.s./L]
10	5.26	6.44	122	11.1	211
0.1	0.0526	< LCL		< LCL
0.01	0.00526	< LCL		not plausible
Control		< LCL		< LCL

% = Percent of the nominal test item concentration
a.s. = active substance
LCL = lowest calibration standard = 2 µg/L (1.05 µg a.s./L)
Meas. conc. = measured concentration of the test item, enrichment and dilution factors taken into account

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

Sampling		0 hours		72 hours
		Start of the exposure		End of the exposure
		(with algae)		(with algae)
Nominal test item loading rates		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
		(C14 Component + 2EO)
[µg/L]	[µg a.s./L]	Meas. conc.	%	Meas. conc.	%
		[µg a.s./L]		[µg a.s./L]
10	1.86	1.38	74	3.73	200
0.1	0.0186	< LCL		< LCL
0.01	0.00186	< LCL		< LCL
Control		< LCL		< LCL

% = Percent of the nominal test item concentration
a.s. = active substance
LCL = lowest calibration standard = 2 µg/L (0.373 µg a.s./L)
Meas. conc. = measured concentration of the test item, enrichment and dilution factors taken into account

 

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

Sampling		0 hours		72 hours
		Start of the exposure		End of the exposure
		(with algae)		(with algae)
Nominal test item loading rates		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
		(C16 Component + 2EO)
[µg/L]	[µg a.s./L]	Meas. conc.	%	Meas. conc.	%
		[µg a.s./L]		[µg a.s./L]
10	0.846	< LCL		1.08	127
0.100	0.00846	< LCL		< LCL
0.0100	0.0000846	< LCL		< LCL
Control		< LCL		< LCL

% = Percent of the nominal test item concentration
a.s. = active substance
LCL = lowest calibration standard = 2 µg/L (0.169 µg a.s./L)
Meas. conc. = measured concentration of the test item, enrichment and dilution factors taken into account

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

Sampling		0 hours		72 hours
		Start of the exposure		End of the exposure
		(with algae)		(with algae)
Nominal test item loading rates		Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
		(C18:1 Component + 2EO)
[µg/L]	[µg a.s./L]	Meas. conc.	%	Meas. conc.	%
		[µg a.s./L]		[µg a.s./L]
10	0.480	< LCL		not plausible
0.100	0.00480	< LCL		not plausible
0.0100	0.000480	< LCL		not plausible
Control		< LCL		not plausible

% = Percent of the nominal test item concentration
a.s. = active substance
LCL = lowest calibration standard = 2 µg/L (0.0960 µg a.s./L)
Meas. conc. = measured concentration of the test item, enrichment and dilution factors taken into account

 

Fingerprint – MS spectrum

The peak distribution of the WAF was analyzed in fresh prepared medium in the highest test item concentration 8.00 µg/L and the control. An analytical standard of the test item was prepared in acetonitrile and diluted to 1.00 mg/L with dilution medium. The highest test item concentration and the control was diluted factor 2 with acetonitrile containing 2% formic acid to avoid an inhomogeneous sample. The standard dilution and the test item dilution were analytical verified via MS and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared. In general, the concentrations and the solubility of the test item is too low for significant MS spectra, therefore no conclusions can be drawn.

Analytical Results

The test item concentrations of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) were analytically verified via LC-MS/MS at the start (0 hours) and after 24, 48 and 72 hours with and without algae. Samples were centrifuged to separate the algae from the water phase. The supernatant was analyzed to determine the truly dissolved test item fraction. Details of the analytical method are presented in section 2, the analytical results are presented in Table 1 to Table 3.

The test item is poorly soluble in water and low concentrations at the start of the exposure could be observed.

 

Findings:

• The tendency for adsorption to the wall of the centrifuge tubes increases with the chain length of the analytes.


• In the higher nominal loading rates the results of the truly dissolved fraction partly fell below the limit of quantification in higher chain length.


• The results with and without algae are comparable at the start of the test (T0). At the end of the test (T72) there is a clear decline of the test substance when compared to the test without algae. The decline is larger for longer alkylchain constituents.


• In the lower nominal loading rates (0.800 to 0.008 µg/L) the nominal concentration of each component was below the limit of quantification and therefore not quantified in additional experiments.

 

 

Conclusion:

The determination of the truly dissolved fraction is not possible with Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2).

 

 

Table 1:       Measured Concentrations of the compound C10 Amine + 2EO of the test item

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

Nominal loading rate of the	test item [µg/L]	8.00			2.53
	active substance [µg a.s./L]	0.582			0.184
Sampling date		Samples with organic solvent
		Meas. conc. [µg a.s./L]	%^1)	%^2)	Meas. conc. [µg a.s./L]	%^1)	%^2)
0 hours	With algae	0.837	144^3)	-	0.202	110	-
	Without algae	0.778	134^3)	-	0.164	89	-
24 hours	With algae	0.731	126^3)	87	0.197	107	98
	Without algae	0.684	118	88	0.147	80	90
48 hours	With algae	0.698	120	83	< LOQ
	Without algae	0.729	125	94	< LOQ
72 hours	With algae	0.226	39	27	< LOQ
	Without algae	0.680	117	87	< LOQ
Sampling date		Truly dissolved test item (centrifuged samples)
		Meas. conc. [µg a.s./L]	%^1)	%^2)	Meas. conc. [µg a.s./L]	%^1)	%^2)
0 hours	Without algae	0.792	136^3)	-	0.197	107	-
24 hours	With algae	0.732	126	87	0.159	86	79
	Without algae	0.722	124	93	0.153	83	93
48 hours	With algae	0.657	113	79	0.146	79	72
	Without algae	0.726	125	93	0.157	86	96
72 hours	With algae	0.232	40	28	< LOQ
	Without algae	0.685	118	88	< LOQ

Meas. conc.    = measured concentration of the compound C10 Amine + 2EO of the test item,

 dilution factors taken into account

a.s.                   = active substance

%¹⁾                    = percent of the nominal concentration of the active substance

%²⁾                              = percent of the initially measured concentrations (with organic solvent)

LOQ                 = limit of quantification (2.00 µg/L of the test item, corresponding to 0.145 µg a.s./L)

³⁾                       = reanalyzed and confirmed

 

Table 2:       Measured Concentrations of the compound C12 Amine + 2EO of the test item

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

Nominal loading rate of the	test item [µg/L]	8.00			2.53
	active substance [µg a.s./L]	4.21			1.33
Sampling date		Samples with organic solvent
		Meas. conc. [µg a.s./L]	%^1)	%^2)	Meas. conc. [µg a.s./L]	%^1)	%^2)
0 hours	With algae	5.97	142	-	1.47	110	-
	Without algae	5.78	137	-	1.33	100	-
24 hours	With algae	5.28	126	89	1.43	108	97
	Without algae	5.06	120	88	< LOQ
48 hours	With algae	4.34	103	73	< LOQ
	Without algae	4.96	118	86	< LOQ
72 hours	With algae	< LOQ			< LOQ
	Without algae	4.64	110	80	< LOQ
Sampling date		Truly dissolved test item (centrifuged samples)
		Meas. conc. [µg a.s./L]	%^1)	%^2)	Meas. conc. [µg a.s./L]	%^1)	%^2)
0 hours	Without algae	3.79	90	-	0.801	60	-
24 hours	With algae	3.29	78	55	< LOQ
	Without algae	3.03	72	52	< LOQ
48 hours	With algae	2.13	51	36	< LOQ
	Without algae	2.75	65	48	< LOQ
72 hours	With algae	< LOQ			< LOQ
	Without algae	2.69	64	47	< LOQ

Meas. conc.    = measured concentration of the compound C12 Amine + 2EO of the test item,

 dilution factors taken into account

a.s.                   = active substance

%¹⁾                    = percent of the nominal concentration of the active substance

%²⁾                              = percent of the initially measured concentrations (with organic solvent)

LOQ                 = limit of quantification (2.00 µg/L of the test item, corresponding to 1.05 µg a.s./L)

 

Table 3:       Measured Concentrations of the compound C14 Amine + 2EO of the test item

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

Nominal loading rate of the	test item [µg/L]	8.00			2.53
	active substance [µg a.s./L]	1.49			0.472
Sampling date		Samples with organic solvent
		Meas. conc. [µg a.s./L]	%^1)	%^2)	Meas. conc. [µg a.s./L]	%^1)	%^2)
0 hours	With algae	1.70	114	-	0.457	97	-
	Without algae	1.88	126	-	0.534	113	-
24 hours	With algae	0.883	59	52	< LOQ
	Without algae	1.08	73	58	< LOQ
48 hours	With algae	0.483	32	28	< LOQ
	Without algae	0.840	56	45	< LOQ
72 hours	With algae	< LOQ			< LOQ
	Without algae	0.694	47	37	< LOQ
Sampling date		Truly dissolved test item (centrifuged samples)
		Meas. conc. [µg a.s./L]	%^1)	%^2)	Meas. conc. [µg a.s./L]	%^1)	%^2)
0 hours	Without algae	< LOQ			< LOQ
24 hours	With algae	< LOQ			< LOQ
	Without algae	< LOQ			< LOQ
48 hours	With algae	< LOQ			< LOQ
	Without algae	< LOQ			< LOQ
72 hours	With algae	< LOQ			< LOQ
	Without algae	< LOQ			< LOQ

Meas. conc.    = measured concentration of the compound C14 Amine + 2EO of the test item,

 dilution factors taken into account

a.s.                   = active substance

%¹⁾                    = percent of the nominal concentration of the active substance

%²⁾                              = percent of the initially measured concentrations (with organic solvent)

LOQ                 = limit of quantification (2.00 µg/L of the test item, corresponding to 0.373 µg a.s./L)

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

In this study, Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal test item loadings):
The EL50-value for inhibition of growth rate (ErL50) after 72 hours was 4.06 (3.81 – 4.36) µg/L. The NOEL-value for inhibition of growth rate after 72 hours was 0.253 µg/L.
The EL50-value for inhibition of yield rate (EyL50) after 72 hours was 1.40 (1.24 – 1.60) µg/L. The NOEL-value for inhibition of yield after 72 hours was 0.253 µg/L.
Daily analysis was carried out. The measured concentrations were to a large extent below the limit of quantification. Therefore, the calculation of mean time weighted average measured concentrations was not possible and no endpoints could be calculated based on measured concentrations.

Executive summary:

The toxicity of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) (batch no.: 20201158) 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 2021-12-05 to 2021-12-13, with the definitive exposure phase from 2021-12-07 to 2021-12-10 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6864 cells/mL. The test item is a UVCB substance with constituents of different water solubility and therefore seven 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 0.00800 to 8.00 µg/L set up in a geometric series with a factor of √10: 0.00800 – 0.0253 – 0.0800 – 0.253 – 0.800 – 2.53 – 8.00 µg/L. For preparation of the loading rates the test item was dissolved i n methanol (800, 80.0 and 0.800 mg/L). The test item with methanol was applied onto a curved glass slide and the methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. The resulting water accommodated fractions (WAF) were used in the test. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions during the test were within the acceptable limits.

The test media were clear by eye at the beginning of the exposure period .

The test item concentrations of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) were analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) with algae. Additionally, the loading rate 8.00 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 concentration levels at the end of the exposure. Samples after 24, 48, 72 hours of exposure was centrifuged to separate the algae from the water phase. Both was analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae.
As the test item is a UVCB substance, all evaluations are based on the nominal loading rates of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2). Time weighted average measured concentrations could not be calculated as to a large extent the concentrations were below the LOQ. However, with regard to the findings in the groups with higher nominal test concentrations it can be assumed that the test item concentrations were stable during exposure.

NOEL, LOEL and ELx-values and 95% Confidence Intervals of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) (0 - 72 hours)
based on the nominal test item loading rates [µg/L]

	Inhibition of Growth Rate
NOEL	0.253
LOEL	0.800
ErL10	1.19 (1.04 – 1.33)
ErL20	1.95 (1.83 – 2.06)
ErL50	4.06 (3.81 – 4.36)
	Inhibition of Yield
NOEL	0.253
LOEL	0.800
EyL10	0.503 (0.400 – 0.620)
EyL20	0.705 (0.591 – 0.826)
EyL50	1.40 (1.24 – 1.60)

 

Description of key information

Two algae studies are available for Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs. (PFAEO-C, New CAS no.: 71786-60-2; old name: Bis (2-hydroxyethyl) cocoalkylamine, Old CAS No.: 61791-31-9).

• one study is performed according to the Water Accommodate Fraction (WAF) approach and


• one under is performed according to the Bulk-approach.

The test item is a UVCB substance with constituents of different water solubility. This means that only a fraction of the total mass of the UVCB added to the test solution may be dissolved in the WAF. The term "loading rate" in place of "nominal concentration" has therefore been advocated for expressing exposures of UVCBs that neither wholly dissolve nor completely form a stable dispersion or emulsion over the required test range (Girling et al., 1992). The loading rate is the mass to volume ratio of the UVCB to test medium used in the preparation of a WAF. Results should be calculated from the loading rates of the entire UVCB as either a median lethal loading (LL50), median effective loading (EL50) or 10 percentile effective loading (EL10) value. These loading levels from the WAF are used directly for derivation of the PNEC and for classification and labelling (see also Annex I.4.5 of the CLP guidance document). 

The study according to the WAF approach resulted in 72h ErL10 and ErL50-values of 1.19 and 4.06 µg/L for inhibition of specific growth rate.

The study according to the Bulk approach resulted in 72h ErC10 and ErC50-values of 9.16 and 107 μg/L for inhibition of specific growth rate.

 

 

Key value for chemical safety assessment

EC50 for freshwater algae:

4.06 µg/L

EC10 or NOEC for freshwater algae:

1.19 µg/L

Additional information

Two algae studies are available for Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs. (PFAEO-C, New CAS no.: 71786-60-2; old name: Bis (2-hydroxyethyl) cocoalkylamine, Old CAS No.: 61791-31-9).

• one study is performed according to the Water Accommodate Fraction (WAF) approach and


• one under is performed according to the Bulk-approach.

 

PFAEO-C is a multicomponent mixture (UVCB) of cationic surface-active constituents with different water solubilities. The fate of cationic surfactants in general deviates from standard chemicals. These substances are therefore considered as difficult substances for which the results of standard guideline studies are very difficult to interpret when considering them in a standard way. The reasons are the intrinsic properties like the relatively low water solubility and strong sorption to equipment and organisms which have an impact on both the effect and exposure assessment.

Effect assessment: Classical ecotoxicity testing with these substances using reconstituted water often leads to test results which are poorly reproducible and are associated with high uncertainty.

Exposure assessment: Because of the complex sorption mechanisms (van der Waals and Ionic mechanisms) the actual dissolved exposure concentration cannot reliably be estimated with the equilibrium partitioning method.

 

The two available freshwater algae tests where therefore performed following two different approaches. Both studies are considered to be of equal quality but are performed for different purposes.

One test which is focused on determining the intrinsic toxicity of PFAEO-C (for C&L purposes) is performed according to the Water Accommodate Fraction (WAF) approach as described in “OECD guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (No. 23 Feb. 2019). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration.

 

The other test which is suited to derive a realistic risk ratio for the aquatic compartment is performed according to the PECaquatic bulk/PNECaquatic bulk approach as described in ECETOC Technical Report “Environmental Risk Assessment of difficult substances” (TR 88, 2003). The so called “Bulk approach” is used in the environmental risk assessment to cope with the earlier mentioned lack of realistic PEC estimation and problems observed with the reproducibility of the specific chemical analyses.

Instead of using the dissolved PECwater, the Bulk concentration (dissolved + sorbed) in water is used. This bulk approach requires a PNECwater, bulk that means that testing has to use river water which contains dissolved organic carbon and suspended organic and inorganic matter, instead of reconstituted lab water.

Tests according to the bulk approach were thus performed because the partitioning of cationic surfactants to soil, sediment or suspended matter is rather complex which explains why there is no alternative  equilibrium Partitioning Method (EPM, di Toro, 2008) formula for these substances available yet. The use of the Bulk approach however elegantly bypasses this deficiency as it eliminates the EPM on the exposure and effect side. (See figure 1 below; more details on the Bulk approach are attached to the dossier).

 

Main difference between the WAF and Bulk approach lies in the preparation of the test solutions and how the results should be interpreted. 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. There is however a clear difference in the evaluation of a standard aquatic ecotoxicity test and an ecotoxicity test performed using the Bulk approach. In order to class a 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 of the dissolved concentration.

For ecotoxicity tests performed using the bulk approach, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the dose-response relationship should thus be based on the sum of adsorbed and dissolved substance. Results from bulk-approach tests are therefore easier to interpret because nominal concentrations corrected for sorption to glassware can be used to quantify the dose. Because of the use of natural river water a bulk approach test is more environmentally realistic than the standard method and due to that considered to be a higher tier study. During the test, similar as in the environment, the DOC and suspended matter present will serve as passive dosing reservoirs maintaining a more constant exposure.

 

Study 1:  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 seven WAFs were prepared 24 ± 1 hours prior to the start of exposure with nominal loading rates of the test item in the range of 0.00800 to 8.00 μg/L set up in a geometric series with a factor of √10: 0.00800 – 0.0253 – 0.0800 – 0.253 – 0.800 – 2.53 – 8.00 μg/L. For preparation of the loading rates the test item was dissolved in methanol (800, 80.0 and 0.800 mg/L). The test item with methanol was applied onto a curved glass slide and the methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The WAFs were checked via laser beam  (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. The resulting water accommodated fractions (WAF) were used in the test. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions during the test were within the acceptable limits.

The test item concentrations of PFAEO-C (Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs, CAS no.: 71786-60-2) were analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) with algae. Additionally, the loading rate 8.00 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 concentration levels at the end of the exposure. 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 dissolved test item fraction and the fraction adsorbed to algae.

As the test item is a UVCB substance, all evaluations are presented based on the nominal loading rates of PFAEO-C.

The exposure concentrations were quantified by analyzing the 3 main constituents (C10, C12 and C14 which together represent 78.8% of the total active ingredient). The time weighted average measured concentrations in water could however not be calculated as to a large extent the concentrations were below the LOQ. Only for the highest test concentration of 8 µg/L, where there was no significant algae growth, the recovery ranged from <LOQ (C14) to 103% (C10).

The calculated ErL10 and ErL50 values with 95 % confidence intervals for inhibition of specific growth rate were 1.19 (1.04 – 1.33) and 4.06 (3.81 – 4.36) μg/L, respectively.

 

Because it is known that cationic surfactants sorb strongly to algae (van Wijk et al., 2009) also the sorption to algae and glassware was quantified for all samples but also these concentrations were in general <LOQ only at the highest test concentration there was parallel to the increase of the chain length an increase of the amount of test item sorbed to algae ranging from LOQ-7% of nominal for the C10, 22-42% of nominal for the C12 and 35 – 63% of nominal for the C14. Finally, a control standard with a loading rate of 8.00 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. The recoveries of these samples were very similar to that of the highest test concentration with algae.

When considering the results for the highest test concentration and also knowing that the test item is chemically stable, it can be assumed that the test item concentrations were stable during exposure.

 

Study 2: A study was conducted to determine the fresh water algal growth inhibition of the test substance, PFAEO-C (99.9% active), according to the OECD Guideline 201, in compliance with GLP. 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 2.00 - 6.40 - 20.0 - 64.0 - 200 – 640 µg/L. The test was carried out according to the bulk approach using enriched natural surface water with a low Dissolved Organic Carbon (DOC; 3.8 mg/L) and Total Suspended Solids (TSS; ± 17.6 mg/L) content, 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 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(≈NPOC). It should be noted that this composition is in perfect alignment with the risk assessment method developed 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.

Figure 1: Risk assessment scheme according to the Bulk approach.

 

 

The concentrations of PFAEO-C in the test were quantified at the concentration levels 6.4 and 200 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 83 – 87 % of the nominal values. At the end of the test Bis (2-hydroxyethyl) cocoalkylamine was analysed at concentration levels 6.4 and 200 mg/L (prepared without algae) and gave recoveries of 0 - 11 % 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 was attributed to additional sorption to suspended matter and DOC due to thermo-dynamically driven redistribution of the sorbed fraction. No adsorption (< LOQ) of the test item to glassware was observed. Therefore, all effect values are given based on nominal concentrations of the test item.

Bis (2-hydroxyethyl) cocoalkylamine (Old CAS No.: 61791-31-9) 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 9.16 (7.17 – 11.9) and 4.60 (3.97 – 5.31) μg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 107 (94.6 – 123) and 15.1 (13.8 – 16.3) μg/L, respectively.

As indicated, the test item is a UVCB substance with constituents of different water solubility. This means that only a fraction of the total mass of the UVCB added to the test solution may be dissolved in the WAF. The term "loading rate" in place of "nominal concentration" has therefore been advocated for expressing exposures of UVCBs that neither wholly dissolve nor completely form a stable dispersion or emulsion over the required test range (Girling et al., 1992). The loading rate is the mass to volume ratio of the UVCB to test medium used in the preparation of a WAF. Results should be calculated from the loading rates of the entire UVCB as either a median lethal loading (LL50), median effective loading (EL50) or 10 percentile effective loading (EL10) value. These loading levels from the WAF are used directly in the classification criteria (see also Annex I.4.5 of the CLP guidance document). 

The mitigation factor of the river water constituents in the bulk approach test can be calculated by taking the ratio between the bulk approach effect concentrations and the WAF approach effect concentrations based on loading:  ErC50 bulk/ErL50 WAF = 9.16/1.19 = 7.7  

 

 

• Droge, S.T.J. and Goss, K.W. 2013. Development and Evaluation of a New Sorption Model for Organic Cations in Soil: Contributions from Organic Matter and Clay Minerals. Environmental Science and Technology, 47:14233-14241.


• Di Toro, D 2008 Bioavailability of chemicals in Sediments and soils: toxicological and chemical interactions. SERDP/ESTCP Bioavailability workshop


• van Wijk, D., Gyimesi-van den Bos, M., Garttener-Arends, I., Geurts, M., Kamstra, J., Thomas, P., 2009. Bioavailability and detoxification of cationics, I. Algal toxicity of trimethylammonium salts in the presence of suspended matter and humic acid. Chemosphere 75 (3), 303–309.