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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2021-06-07 to 2021-06-25 with the definitive exposure phase from 2021-06-23 to 2021-06-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
(2004)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item 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
Water: 0.1%

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:
with a LC-MS/MS method
Details on sampling:
Sampling schedule
All loading rates and the control were analytically verified via LC-MS/MS method in the fresh media at the start of exposure and at the renewal of the test solutions (0 and 24 hours) as well as in the 24-hours old media at the renewal and at the end of the exposure (24 and 48 hours).

Sampling and pre-treatment
At the start of the exposure and at the renewal (0 and 24 hours), samples were taken after preparation of the loading rates and analyzed. At the renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken from the test vessels.

Determination of adsorption of the test item on glass
Adsorption to glass walls of the test vessels was analytically
verified from all loading rates at the end of the exposure intervals. After sampling for the test medium analysis (aqueous phase), the glassware was emptied and an appropriate amount of a suitable solvent was added. The concentration of the test item in this solution was measured and the adsorbed test item amount was calculated from this concentration. Before this procedure, the test vessels were carefully rinsed with demineralized water to remove the remaining test media.
Vehicle:
no
Details on test solutions:
Water Accommodated Fraction
Water accommodated fractions (WAF) were prepared because the test item is an UVCB substance with compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2019).
Using this approach, aqueous media was prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

Preparation of the water accommodated fractions
Five water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 42.8 to 1000 µg/L set up in a geometric series with a factor of 2.2: 42.8 – 94.1 – 207 – 455 – 1000 µg/L, corresponding to the geometric mean measured test item concentrations of 31.6 – 74.5 – 180 – 411 – 865 µg/L.
The procedure according to ASTM D6081 (2019) as described below was carried out. For each loading rate an appropriate amount of stock solutions containing the test item with methanol was placed on a curved glass slide. The methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The first 25 mL were discarded.
The WAF were checked via laser beam (Tyndall effect) for undissolved test item. No presence of undissolved test item during the test was observed. The resulting water accommodated fractions (WAF) were used in the test.

Test loading
Per definition of the WAF all terms related to concentration level are given as loading rates because partly dissolved compounds and mixtures cannot be related to concentrations.
The loading rates are based on the results of a preliminary range finding test.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test system
Daphnia magna STRAUS, obtained from continuous laboratory cultures.

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, 16 hours illumination, light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), is used.

Feeding of the culture stocks
The daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae were cultured at the test facility.
Test type:
semi-static
Water media type:
freshwater
Remarks:
Medium Elendt M4 according to OECD 202, Annex 3 (2004)
Limit test:
no
Total exposure duration:
48 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.
Dissolved oxygen:
see section "Any other information on results incl. tables" below.
Conductivity:
see section "Any other information on results incl. tables" below.
Nominal and measured concentrations:
Five water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 42.8 to 1000 µg/L set up in a geometric series with a factor of 2.2: 42.8 – 94.1 – 207 – 455 – 1000 µg/L, corresponding to the geometric mean measured test item concentrations of 31.6 – 74.5 – 180 – 411 – 865 µg/L.
Details on test conditions:
Control
Dilution water without test item incubated under the same conditions as the test groups.

Reference Test
A reference test with potassium dichromate was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility.

Test method
The study was performed under semi-static conditions with a renewal of the test solutions after 24 hours.

Test duration 48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume 20 mL

Dilution water Same composition as the culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each concentration level and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. To achieve this, juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization is not necessary, because the composition of the dilution water is equivalent to the culture medium.

Application
20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette.

Renewal of the test solutions
The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette.

Test temperature (target) 18 - 22 °C, constant within ± 1 °C

Illumination (target) Diffuse light, light intensity of max. 1500 lx

Photoperiod (target) 16/8 hours light/dark cycle

Feeding The daphnids were not fed during the study.

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

Type and Frequency of Measurements
Biological Parameters

Immobilization and other observations
Immobilization was determined in all groups after 24 and 48 hours. A daphnid was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Observation of other adverse, sub-lethal effects (e.g. discoloration, abnormal behavior etc.) was done, but none occurred during the course of the study.

Water Quality Parameters

Dilution water
Prior to the start of the exposure and at the renewal of the test solutions (0 and 24 hours), the water quality parameters (i.e. pH-value, dissolved oxygen concentration, total hardness, conductivity and temperature) of the dilution water (control) were measured.

Test media
At the start of the exposure and at the renewal (0 and 24 hours), the water quality parameters of the fresh media (i.e. pH-value, dissolved oxygen concentration) were measured in one additional replicate (without daphnids) of each loading rate and the control.
At the renewal and at the end of the exposure (24 and 48 hours), the water quality parameters of the old media were measured in one appropriate replicate (containing daphnids) of each loading rate and the control. The replicate with the highest immobilization rate per loading level and the control was measured.

Temperature
The incubator temperature (measurement in air with a thermo-hygrograph) was recorded throughout the period of the test.
Reference substance (positive control):
yes
Remarks:
The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number BCCC1619, purity 100%, CAS RN 7778-50-9) was determined after 24 hours from 2021-04-07 to 2021-04-08.
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 865 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 865 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
575 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 865 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
198 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
508 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
EL100
Effect conc.:
> 1 000 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
EL100
Effect conc.:
> 1 000 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
649 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
EL10
Effect conc.:
221 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
EL10
Effect conc.:
570 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Additional Observations during the Definitive Test

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

Water Quality Parameters

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits during the study. During the test period, the temperature in the incubator was 19 – 20 °C.

Measured Exposure Concentrations during the Definitive Test

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

The measured concentrations of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) in the fresh media were for all four components in the range of 44 to 114% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of < LOQ to 107% of the nominal test item concentrations. Further glass adsorption in the test vials at end of the exposure intervals was analyzed.
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.


Results with reference substance (positive control):
The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number BCCC1619, purity 100%, CAS RN 7778-50-9) was determined after 24 hours from 2021-04-07 to 2021-04-08. For results see section "Any other information on materials and methods incl. tables" above.
Reported statistics and error estimates:
Methods of evaluation
The EL/EC100 – values after 24 and 48 hours and the EL/EC50- value after 24 hours were estimated empirically from the observed immobilization rates.
All effect levels (EL10 / 50 / 100) are given based on the nominal loading rates of the test item, since the test item is an UVCB substance. Additionally, the EC10 / 50 / 100-values are given based on the calculated geometric mean measured test item concentrations.

ECx-values and statistical analyses
The EL/EC10- values after 24 were calculated by straight line regression. The EL/EC10- values and the EL/EC50- values after 48 hours were calculated by sigmoidal regression. The confidence limits were estimated empirically from the observed immobilization rates. All calculations were carried out from the best-fit values with the software GraphPad Prism. The concentration-effect relationships of the test item are shown graphically.

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

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

Immobilization Rates after 24 and 48 hours of Exposure
(n = 20, divided into 4 replicates with 5 daphnids each)














































































































Nominal loading rateIMMOBILIZATION [%]
of the test item24 hours48 hours
[µg/L]ReplicatesReplicates
 1234MV1234MV
100040204040356010010010090
455000004020402030
2070000020200010
94.10000000000
42.80000000000
Control0000000000

 


Absolute Numbers of immobile Daphnids after 24 and 48 hours of Exposure
(n = 20, divided into 4 replicates with 5 daphnids each)














































































































Nominal loading rateNumber of immobile Daphnids / Total number of Daphnids
of the test item24 hours48 hours
[µg/L]ReplicatesReplicates
 1234Sum1234Sum
10002 / 51 / 52 / 52 / 57 / 203 / 55 / 55 / 55 / 518 / 20
4550 / 50 / 50 / 50 / 50 / 202 / 51 / 52 / 51 / 56 / 20
2070 / 50 / 50 / 50 / 50 / 201 / 51 / 50 / 50 / 52 / 20
94.10 / 50 / 50 / 50 / 50 / 200 / 50 / 50 / 50 / 50 / 20
42.80 / 50 / 50 / 50 / 50 / 200 / 50 / 50 / 50 / 50 / 20
Control0 / 50 / 50 / 50 / 50 / 200 / 50 / 50 / 50 / 50 / 20

 


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

























































































































Sampling dateFresh medium,Old medium,Fresh medium,Old medium,
0 hours24 hours24 hours48 hours
NominalEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
loading rate of theC10 Amine + 2EO
test itemactive substanceMeas.%Meas.%Meas.%Meas.%
[mg/L][mg a.s./L]conc. conc. conc. conc. 
  [mg a.s./L][mg a.s./L][mg a.s./L][mg a.s./L]
1.000.07270.07361010.0719990.07471030.0753104
0.4550.03310.03431040.0328990.03491050.032498
0.2070.01500.0149990.0147980.01521010.0150100
0.09410.006840.007501100.006921010.006991020.0067599
0.04280.003110.003561140.003281050.003401090.00334107
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)


 


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 – Glass adsorption












































































Sampling dateOld medium,Old medium,
24 hours48 hours
NominalEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
loading rate of theC10 Amine + 2EO
test itemactive substanceMeas.%Meas.%
[mg/L][mg a.s./L]conc. conc. 
  [mg a.s./L][mg a.s./L]
1.000.07270.00029000.0004021
0.4550.0331< LOQ0.0001530
0.2070.01500.0001631< LOQ
0.09410.00684< LOQ< LOQ
0.04280.00311< 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)


 


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

























































































































Sampling dateFresh medium,Old medium,Fresh medium,Old medium,
0 hours24 hours24 hours48 hours
NominalEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
loading rate of theC12 Amine + 2EO
test itemactive substanceMeas.%Meas.%Meas.%Meas.%
[mg/L][mg a.s./L]conc. conc. conc. conc. 
  [mg a.s./L][mg a.s./L]][mg a.s./L][mg a.s./L]
1.000.5260.5361020.466890.5501050.529101
0.4550.2390.2511050.216900.2501040.21891
0.2070.1090.107980.0975900.1101010.098490
0.09410.04950.05331080.0376760.0485980.041283
0.04280.02250.02501110.0132590.02271010.017980
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)


 


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 – Glass adsorption
















































































Sampling dateOld medium,Old medium,
24 hours48 hours
NominalEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
loading rate of theC12 Amine + 2EO
test itemactive substanceMeas.%Meas.%
[mg/L][mg a.s./L]conc. conc. 
  [mg a.s./L][mg a.s./L]
1.000.5260.010820.005001
0.4550.2390.0015710.002281
0.2070.1090.0036430.001682
0.09410.04950.0011020.002675
0.04280.0225< 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)



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

























































































































Sampling dateFresh medium,Old medium,Fresh medium,Old medium,
0 hours24 hours24 hours48 hours
NominalEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
loading rate of theC14 Amine + 2EO
test itemactive substanceMeas.%Meas.%Meas.%Meas.%
[mg/L][mg a.s./L]conc. conc. conc. conc. 
  [mg a.s./L]][mg a.s./L][mg a.s./L][mg a.s./L]
1.000.1860.177950.112600.1881010.17192
0.4550.08480.08471000.0574680.0843990.062874
0.2070.03860.0373970.0248640.0366950.025867
0.09410.01750.01761010.00462260.0149850.0078445
0.04280.007980.00793990.00130160.00635800.0025732
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)


 


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 – Glass adsorption


















































































Sampling dateOld medium,Old medium,
24 hours48 hours
NominalEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
loading rate of theC14 Amine + 2EO
test itemactive substanceMeas.%Meas.%
[mg/L][mg a.s./L]conc. conc. 
  [mg a.s./L][mg a.s./L]
1.000.1860.0268140.009085
0.4550.08480.0041550.005106
0.2070.03860.00849220.003479
0.09410.01750.00191110.0066638
0.04280.007980.000821100.0016621

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)



Measured Concentrations of the compound C16 Amine + 2EO of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) during the Definitive Test
























































































































Sampling dateFresh medium,Old medium,Fresh medium,Old medium,
0 hours24 hours24 hours48 hours
NominalEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
loading rate of theC16 Amine + 2EO
test itemactive substanceMeas.%Meas.%Meas.%Meas.%
[mg/L][mg a.s./L]conc. conc. conc. conc. 
  [mg a.s./L][mg a.s./L][mg a.s./L][mg a.s./L]
1.000.08460.0650770.0126150.0754890.040047
0.4550.03850.0326850.00777200.0330860.0099226
0.2070.01750.0132750.00259150.0125710.0028216
0.09410.007960.00541680.00042350.00450570.0007259
0.04280.003620.0022061< LOQ0.00161440.0002908
Control< LOQ< LOQ< LOQ< LOQ

Meas. conc. = measured concentration of the compound C16 Amine + 2EO of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LOQ = limit of quantification (2.00 µg/L of the test item, corresponding to 0.169 µg a.s./L)


 


Measured Concentrations of the compound C16 Amine + 2EO of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) during the Definitive Test – Glass adsorption


















































































Sampling dateOld medium,Old medium,
24 hours48 hours
NominalEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
loading rate of theC16 Amine + 2EO
test itemactive substanceMeas.%Meas.%
[mg/L][mg a.s./L]conc. conc. 
  [mg a.s./L][mg a.s./L]
1.000.08460.0393460.022827
0.4550.03850.00622160.0057115
0.2070.01750.0134760.0063836
0.09410.007960.00138170.0043755
0.04280.003620.000526150.0011131

Meas. conc. = measured concentration of the compound C16 Amine + 2EO of the test item,
dilution factors taken into account
a.s. = active substance
% = percent of the nominal concentration of the active substance
LOQ = limit of quantification (2.00 µg/L of the test item, corresponding to 0.169 µg a.s./L)


 


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




















Validity criteria (target)ResultValid
≤ 10% of the daphnids in the control immobile or showing signs of disease or stress, e.g. discoloration or unusual behavior such as trapping on the surface of the water, during the 48-hour test periodNo daphnidü
≥ 3 mg O2 /L in the 24-hours old media at the renewal of the test solutions and at the end of the exposure in all concentration levels and in the control≥ 6.80 mg/Lü

 


Water Quality Parameters in the fresh Media at the Start of the Exposure and at the Renewal
(0 and 24 hours)
(measured in one additional replicate (without daphnids) per loading level and control)








































































Nominal loading rate0 hours24 hours
of the test item
[µg/L]pH-valueDissolvedpH-valueDissolved
 O2 concentrationO2 concentration [mg/L]
 [mg/L] 
10007.427.706.826.80
  4557.147.267.426.89
  2077.707.577.546.80
    94.17.277.787.327.07
    42.87.847.417.427.06
Control8.726.827.537.05

 


Water Quality Parameters in the 24-hours old Media at the Renewal and at the End of the Exposure (24 and 48 hours)
(measured in one replicate (containing daphnids) per per loading level and control)






















































































Nominal loading rate24 hours48 hours
of the test item
[µg/L]pH-valueDissolvedReplicate numberpH-valueDissolvedReplicate number 
 O2 concentrationO2 concentration
 [mg/L][mg/L]
10007.367.1517.328.423
  4556.907.3717.398.363
  2076.878.2817.598.341
    94.16.877.3817.378.353
    42.86.837.1417.428.283
Control6.666.8017.348.423

 


Water Quality Parameters of the Dilution Water at the Start of the Exposure and at the Renewal (0 and 24 hours)









































Dilution waterpH-ValueDissolvedTemperatureConductivityTotal hardness
O2 concentration   
[mg/L][°C][µS/cm][mg CaCO3/L]
0 hours8.726.8221.0623278
24 hours7.537.0521.0623289

 


Percentage of the Relevant Amine

























 [%]
C10 Amine + 2EO7.27
C12 Amine + 2EO52.62
C14 Amine + 2EO18.64
C16 Amine + 2EO8.46

 


Gradient Table








































Time [min]A [%]B [%]
0.007525
0.307525
1.301090
2.001090
2.107525
2.507525

 


Dilution steps









































































NominalDilutionSampleFinal
test itemFactor*volumevolume
loading rate [mL][mL]
[mg/L]   
1.001000.51)5.01)
0.22)1.02)
0.455500.51)5.01)
0.42)1.02)
0.207200.11.0
0.0941100.21.0
0.042840.51.0
Control21.01.0

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


 


Further dilution steps (glass adsorption)





















































NominalDilutionSampleFinal
test itemFactor*volumevolume
loading rate [mL][mL]
[mg/L]   
1.00400.255.0
0.455200.11.0
0.207100.21.0
0.094140.51.0

*including dilution step 2


 


Parameter, Acceptance Criteria and Results of the Method Validation















































































































































ParameterAcceptance criteriaResult
Linearity≥ 5 standard concentrations,0.2 to 20 µg test item/L (n = 7),ü
r ≥ 0.99 (R2 ≥ 0.9801 for 2nd 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 traceS/N for 0.2 µg test item/Lü
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)
Matrix effectsShould be ≤ 20%Matrix effects: < 20%ü
Limit of Quantification (LOQ)Should be at or below lowest test concentration2.0 µg test item/L (1 x LOQ)ü
1200 µg test item/L (600 x LOQ)
Accuracy1) Mean recovery rate of 70-120%C10 Amine + 2 EOü
(Fortified samples)per fortification level (2 levels)1 x LOQ: 115% (n = 5)
  600 x LOQ: 102% (n = 5)
  C12 Amine + 2 EO
  1 x LOQ: 104% (n = 5)
  600 x LOQ: 104% (n = 5)
  C14 Amine + 2 EO
  1 x LOQ: 104% (n = 5)
  600 x LOQ: 106% (n = 5)
  C16 Amine + 2 EO
  1 x LOQ: 92% (n = 5)
  600 x LOQ: 107% (n = 5)
   





















































































































Precision1)Relative standard deviation ≤ 20% per fortification levelC10 Amine + 2 EOü
1 x LOQ: 14%
600 x LOQ: 0.9%
C12 Amine + 2 EO
1 x LOQ: 1.9%
600 x LOQ: 1.6%
C14 Amine + 2 EO
1 x LOQ: 0.94%
600 x LOQ: 1.8% 
C16 Amine + 2 EO
1 x LOQ: 4.1%
600 x LOQ: 1.7% 
 
StabilitySamples: Stable if 70 – 120% of nominal concentrationSee section 17.1ü
Calibration standards: Stable if ±10% of freshly prepared standard
Specificity1)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 EOü
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 the LOQBlank values < 30% of LOQü
Procedural recoveryProcedural recoveries with experimental samples. The recovery has to be 70-120% of the nominal value.See section 17.2ü

 = criterion fulfilled
- = not determined


 


Preparation of Fortified Samples of the Test Item





























































































LOQ LevelControl1600
Stock solution-100 mg test item/L in Acetonitrile120 mg test item/L in Acetonitrile
Spiking solution-200Stock solution
[µg test item/L](Dilution medium)
(Medium) 
Replicates255
Concentration of the LOQ-2.01200
[µg test item/L]
Medium for preparationDaphnia dilution medium
Volume of spiking solution [mL]-0.050.05
Volume of medium [mL]5.04.954.95
Dilution factor22100
Dilution mediumAcetonitrile including 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)

1) First dilution step
2) Second dilution step


Dilution medium = Acetonitrile: ultrapure water (50 : 50 v/v), including 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*: 2.02 µg test item/L (1 x LOQ) and 1208 µg test item/L (600 x LOQ).


































Active substance1 x LOQ600 x LOQ
[µg a.s./L][µg a.s./L]
C10 Amine + 2 EO0.147  87.9
C12 Amine + 2 EO1.06636
C14 Amine + 2 EO0.376225
C16 Amine + 2 EO0.171102

* = 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






















































































ReplicateEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
C10 Amine + 2 EO
1 x LOQ600 x LOQ
Meas.%Meas.%
conc.conc.
[µg a.s./L][µg a.s./L]
10.1329091.0104
20.15810789.5102
30.18312589.5102
40.18112489.5102
50.18812888.8101
Mean0.1711589.7102
SD ±0.02 0.8 
CV [%]14 0.9 

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






















































































ReplicateEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
C12 Amine + 2 EO
1 x LOQ600 x LOQ
Meas.%Meas.%
conc.conc.
[µg a.s./L][µg a.s./L]
11.07101675106
21.12106645102
31.13106662104
41.11104659104
51.12105656103
Mean1.11104659104
SD ±0.02 11 
CV [%]1.9 1.6 

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






















































































ReplicateEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
C14 Amine + 2 EO
1 x LOQ600 x LOQ
Meas.%Meas.%
conc.conc.
[µg a.s./L][µg a.s./L]
10.396105245109
20.391104235104
30.393104238106
40.389104238106
50.386103234104
Mean0.391104238106
SD ±0.004 4 
CV [%]0.94 1.8 

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


 


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: C16 Amine + 2 EO






















































































ReplicateEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
C16 Amine + 2 EO
1 x LOQ600 x LOQ
Meas.%Meas.%
conc.conc.
[µg a.s./L][µg a.s./L]
10.15691112110
20.15490107105
30.16798109107
40.16295108105
50.15088110108
Mean0.15892109107
SD ±0.007 2 
CV [%]4.1 1.7 

Meas. conc. = measured concentration of C16 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


Stability


The stability of the fortified samples at the 1xLOQ was checked after 4 days and was for C12 Amine + 2 EO, C14 Amine + 2 EO and C16 Amine + 2 EO between 90 and 108% of the nominal concentration for each component. For C10 Amine + 2 EO, mean recovery was 148% due to interferences in the chromatography. As a minor component of the test item this has no impact on the integrity of the study.


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.


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



















































































































ComponentC10 Amine + 2 EOC12 Amine + 2 EOC14 Amine + 2 EOC16 Amine + 2 EOMean recovery of all components
Sampling dateMeas. conc.%Meas. conc.%Meas. conc.%Meas. conc.%%
[µg a.s./L][µg a.s./L][µg a.s./L][µg a.s./L]
Fresh medium0.1501041.161110.4071100.192114110
0 hours
Old medium0.1561081.141100.4051100.183109109
24 hours
Fresh medium0.1641141.131080.3821030.172102107
24 hours/
Glass adsorption
24 hours
Old medium0.1491041.071030.3771020.175104103
48 hours/
Glass adsorption
48 hours
Additional analysis0.1451001.091050.3891050.180107104

Meas. conc. = measured concentration of the test item, mean value of two replicates,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


 


Preliminary Tests (non-GLP)
Solubility Test


Two WAFs were prepared as described. After 24, 48 and 72 hours samples were taken as shown behind and analyzed via LC-MS. The equilibrium has been attained after 24 h of stirring.


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)
ComponentsMeasured concentration [µg/L]
Sampling date24 hours48 hours72 hours
Nominal test item loading rate 100 µg/LC14 Component + 2EO66.968.281.7
C16 Component + 2EO52.152.854.5
C18 Component + 2EO27.714.313.4
C18:1 Component + 2EO38.740.239.9
Nominal test item loading rate 1000 µg/LC8 Component + 2EO768-
C10 Component + 2EO913
C12 Component + 2EO923
C14 Component + 2EO849
C16 Component + 2EO571
C18 Component + 2EO330
C18:1 Component + 2EO513

LCL = lowest calibration standard = 2 µg/L



Range Finding Test


A non-GLP preliminary range finding test under semi-static conditions over a period of 48 hours was conducted at the test facility with three WAFs at nominal loading rates of the test item of 0.01, 0.10 and 1.00 mg/L. The test vessels were pre-treated with the appropriate test solution for at least 12 hours under test conditions. The WAFs were prepared on day -2, -1 and 0 as specified. All WAFs and the control were visually clear and colorless throughout the exposure.
The test item was analytically verified via LC-MS/MS in the WAFs and in the control in the fresh media at the start of the exposure (0 hours) and on the renewal of the test solutions (24 hours) and in the old media (24 and 48 hours).
In the range finding test, two replicates per loading rate and control, each with five daphnids, were tested.
For the preparation of the WAFs a stirring phase of 24 hours was found to be suitable. Longer stirring phases led to a decrease of the loading in the WAFs. Therefore, a stirring phase of 24 hours is chosen for the definitive test.


 


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






























































Nominal loading of theIMMOBILIZATION [%]
test item24 hours48 hours
[mg/L]ReplicatesReplicates
 12MV12MV
      1.00100100100100% mortality after 24 hours
      0.10404040604050
      0.01000000
Control000000

 


Measured Exposure Concentrations of C12 Component of the Test Item during the non-GLP Preliminary Range Finding Test
Determination of the test item via LC-MS




















































































Sampling dateFresh media,Old media,Fresh media,Old media,
0 hours24 hours24 hours48 hours
Nominal test item loading ratesEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
(C12 Component + 2EO)
[mg/L][mg a.s./L]Meas. conc.%Meas. conc.%Meas. conc.%Meas. conc.%
[µg a.s./L][µg a.s./L][µg a.s./L][µg a.s./L]
1.000.52857210851898--
0.1000.052861.511648.59262.911962.0117
0.01000.005285.661074.88925.451035.2499
Control< LCL< LCL< 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 of C14 Component of the Test Item during the non-GLP Preliminary Range Finding Test
Determination of the test item via LC-MS




















































































Sampling dateFresh media,Old media,Fresh media,Old media,
0 hours24 hours24 hours48 hours
Nominal test item loading ratesEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
(C14 Component + 2 EO)
[mg/L][mg a.s./L]Meas. conc.%Meas. conc.%Meas. conc.%Meas. conc.%
[µg a.s./L][µg a.s./L][µg a.s./L][µg a.s./L]
1.000.18714907961310702--
0.1000.0187154822116619167892135722
0.01000.0018713.67278.1843715.58298.23440
Control< LCL< LCL< 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 of C16 Component of the Test Item during the non-GLP Preliminary Range Finding Test
Determination of the test item via LC-MS




















































































Sampling dateFresh media,Old media,Fresh media,Old media,
0 hours24 hours24 hours48 hours
Nominal test item loading ratesEthanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2)
(C16 Component + 2 EO)
[mg/L][mg a.s./L]Meas. conc.%Meas. conc.%Meas. conc.%Meas. conc.%
[µg a.s./L][µg a.s./L][µg a.s./L][µg a.s./L]
1.000.0849738869348410--
0.1000.0084968.981116.41941060106030.6360
0.01000.0008490.0159191.1813969.36931.03121
Control< LCL< LCL< 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


 


Fingerprint – MS spectrum


The peak distribution of the WAF were analyzed in fresh prepared medium in the highest test item concentration 1.00 mg/L and the control. An analytical standard of the test item was prepared in acetonitrile and diluted to 500 µg/L with acetonitrile. The highest test item concentration and the control was diluted factor 2 with acetonitrile containing 0.2% formic acid to avoid an inhomogeneous sample. The standard dilution and the test item dilution were analytical verified via MS and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared and quite similar.


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 

Validity criteria fulfilled:
yes
Conclusions:
Based on the nominal loading rates, the 48 hours-EL50 for Daphnia magna was 649 µg/L (confidence limits: 507 – 839 µg/L) for the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2).
Based on the geometric mean measured concentrations, the 48 hours-EL50 for Daphnia magna was 575 µg/L (confidence limits: 455 – 731 µg/L) for the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2).
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) (batch number: 20201158) were determined at the test facility according to OECD 202 (2004) from 2021-06-07 to 2021-06-25 with the definitive exposure phase from 2021-06-23 to 2021-06-25 at the test facility.
The study was conducted under semi-static conditions over a period of 48 hours with five water accommodated fractions (WAF). The WAFs were prepared separately with nominal loading rates of the test item in the range of 42.8 to 1000 µg/L set up in a geometric series with a factor of 2.2. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each loading rate and the control.
The concentrations of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) and the control were determined in fresh media (0 hours and 24 hours) and old media (24 hours and 48 hours) of all tested concentration levels and for the four compounds C10 Amine + 2EO, C12 Amine + 2EO, C14 Amine + 2EO and C16 Amine + 2EO and the control via LC MS/MS.


The measured concentrations of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) in the fresh media were for all four components in the range of 44 to 114% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of < LOQ to 107% of the nominal test item concentrations. Further glass adsorption in the test vials at end of the exposure intervals was analyzed.
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.
The geometric mean measured test item concentrations were calculated to be: 31.6 – 74.5 – 180 – 411 – 865 µg/L.
The EL10 / 50 / 100- values given are based on the nominal loading rates of the test item. Additionally, EC10 / 50 / 100-values based on the calculated geometric mean measured test item concentrations are given.
The validity criteria of the test guideline were fulfilled.


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
























































Effect valuesTestNominal loading ratesGeometric mean measured test item concentrations
durationof the test item[µg/L]
[hours][µg/L] 
EC / EL1024570 (Cl: 474 – 654)508 (Cl: 427 – 579)
(with confidence limits)48221 (Cl: 107 – 340)198 (Cl: 90.3 – 308)
EC / EL5024> 1000> 865
(with confidence limits)48649 (Cl: 507 – 839)575 (Cl: 455 – 731)
EC / EL10024> 1000> 865
48> 1000> 865

 


 


 


 

Description of key information

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) were determined according to OECD 202 (2004) under GLP conditions.


The study was conducted under semi-static conditions over a period of 48 hours with five water accommodated fractions (WAF). The WAFs were prepared separately with nominal loading rates of the test item in the range of 42.8 to 1000 µg/L set up in a geometric series with a factor of 2.2. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each loading rate and the control.


The concentrations of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) and the control were determined in fresh media (0 hours and 24 hours) and old media (24 hours and 48 hours) of all tested concentration levels and for four constituents i.e. C10 Amine + 2EO, C12 Amine + 2EO, C14 Amine + 2EO and C16 Amine + 2EO and the control via LC‑MS/MS. 


The measured concentrations of the test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (CAS: 71786-60-2) in the fresh media were for all four components in the range of 44 to 114% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of < LOQ to 107% of the nominal test item concentrations. Further glass adsorption in the test vials at end of the exposure intervals was analyzed. 


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. The geometric mean measured test item concentrations were calculated to be: 31.6 – 74.5 – 180 – 411 – 865 µg/L.


The EL10 / 50 / 100- values given in Table 1 are based on the nominal loading rates of the test item. Additionally, EC10 / 50 / 100-values based on the calculated geometric mean measured test item concentrations are given in Table 1. 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.


The validity criteria of the test guideline were fulfilled.


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













































Effect values



Test


duration


[hours]



Nominal loading rates


of the test item


[µg/L]



Geometric mean measured test item concentrations


[µg/L]



EC / EL10


(with confidence limits)



24



570 (Cl: 474 – 654)



508 (Cl: 427 – 579)



48



221 (Cl: 107 – 340)



198 (Cl: 90.3 – 308)



EC / EL50


(with confidence limits)



24



> 1000



> 865



48



649 (Cl: 507 – 839)



575 (Cl: 455 – 731)



EC / EL100



24



> 1000



> 865



48



> 1000



> 865



 


The 48h EC50 observed 0.649 mg/L nominal and 0.575 mg/L based on geometric mean are in good agreement with 0.84 mg/L observed in the study from Mark (1994) with ethanol, 2,2'-iminobis-, N-coco alkyl derivs (CAS no.: 61791-31-9) and somewhere in the middle of the 48 h EC50 of the pure C12 and C14 alkyl chain (CAS no.: 1541-67-9 and 18924-66-8) primary fatty amine ethoxylate.


These studies are however considered as supporting as they lack the quantification of the exposure concentrations as at the time of performance, no reliable specific method of analyses was available. Hence, most of the tests were considered to be valid with restrictions and are because of the availability of better data just used to complete the ecotoxicity picture of this substance. Based on the limited reliability of the acute studies it was decided to perform a higher tier long-term (21 d) invertebrate study with 2,2'-(C12-18 evennumbered alkyl imino) diethanol. The 21d EC50 value from this study is used as a worst-case for the short term hazard assessment.



  • 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

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Remarks:
EL50. The substance is an UVCB with constituents of different water solubility. This means that the WAF approach was used. The term "loading rate" in place of "nominal concentration" has therefore been advocated for expressing exposures of UVCBs.
Effect concentration:
0.649 mg/L

Additional information

In addition to the key study from Scheerbaum (2022) which resulted in an EL50 0.649 mg/L, there is one supporting acute test result available for PFAEO-C (old CAS no.: 61791-31-9) and two supporting studies with the pure C12 (CAS no.: 1541-67-9) and pure C14 (CAS no.: 18924-66-8) primary fatty amine ethoxylate. These studies are considered as supporting as they lack the quantification of the exposure concentrations as at the time of performance, no reliable specific method of analyses was available. Hence, most of the tests were considered to be valid with restrictions and are because of the availability of better data just used to complete the ecotoxicity picture of this substance.