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Ecotoxicological information

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 2021-03-22 to 2021-06-01 with the definitive exposure phase from 2021-05-05 to 2021-05-07
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)
Analytical monitoring:
yes
Remarks:
LC-MS/MS method
Details on sampling:
Sampling schedule
Each loading rate 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). The analytical method and the results of the method validation are given in the report.
The peak distribution of the WAF was analyzed in fresh prepared medium in the highest test item concentration, in a separate approach. The signal distribution was compared with an analytical standard prepared in solvent.

Sampling and pre-treatment At the start of the exposure and at the renewal (0 and 24 hours), samples were taken after preparation of each loading rate 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
Five 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 48 ± 1 hour and 24 ± 1 hour prior to the start of the exposure as well as 24 ± 1 hour prior to the renewal of the test solution.
For each loading rate, an appropriate amount of the test item was weighed out and transferred into a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature, as within this time interval attainment of equilibrium has been demonstrated. After a separation phase of 1 hour (at room temperature), the WAFs were removed by siphoning (from the approximate middle of the glass flask) and were centrifuged (17000 g for 20 minutes at 20 ± 1 °C) to separate undissolved test item. The WAFs 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 item loading
Five WAFs with the nominal loading rates of 4.27 – 9.39 – 20.7 – 45.5 - 100 mg/L of the test item were tested (separation factor 2.2).
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. The composition of the culture medium is presented in the report.

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
Limit test:
no
Total exposure duration:
48 h
Hardness:
262 and 219 [mgCaCO3/L]
Test temperature:
During the test period, the temperature in the incubator was 19 – 20 °C.
pH:
See "Any other information on materials and methods incl. tables" below.
Dissolved oxygen:
See "Any other information on materials and methods incl. tables" below.
Conductivity:
616 and 624 µS/cm
Nominal and measured concentrations:
Five WAFs with the nominal loading rates of 4.27 – 9.39 – 20.7 – 45.5 - 100 mg/L of the test item were tested (separation factor 2.2).
The measured concentrations of the test item 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) in the fresh media were 0% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of 13 to 60% of the initially measured test item concentrations. Adsorption to glass was found to be negligible.
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 loading rate 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 (see ‘Application’).

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.

Solubility Test (non-GLP)
WAFs with a nominal loading of 100 and 1000 µg/L was prepared as described in section 4.2. After 24, 48 and 72 hours of stirring samples were taken and analyzed via LC-MS.
A stirring phase of 24 hours was found to be suitable for the preparation of the WAFs. 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.

Range Finding Test (non-GLP)

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 1, 10 and 100 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 in section 4.2. 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). From the highest loading rate (100 mg/L) the adsorption of the test item to the test vessels was measured at the end of the exposure intervals (24 and 48 hours). Therefore, the test vessels were carefully rinsed with demineralized water. The measured concentrations were 0.766 mg/L (24 hours) and 0.0605 mg/L (48 hours).
The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle). In the range finding test, two replicates per loading rate and control, each with five daphnids, were tested.
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.
Key result
Duration:
48 h
Dose descriptor:
EL100
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
24 h
Dose descriptor:
EL100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
26.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Confidence intervals: 23.9 - 32.4 mg/L
Key result
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
EL10
Effect conc.:
17.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Confidence intervals: 14.7 - 20.3 mg/L
Key result
Duration:
24 h
Dose descriptor:
EL10
Effect conc.:
35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Confidence intervals: 20.7 - 73.4 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
0.259 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 0.259 mg/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.:
0.024 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Confidence intervals: 0.0191 - 0.0357 mg/L
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 0.259 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.011 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Confidence intervals: 0.00891 - 0.0141 mg/L
Key result
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
0.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Confidence intervals: 0.0145 - 0.157 mg/L
Details on results:
The tested WAFs and the control were colorless and visually clear throughout the exposure period.
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.
The test item concentrations of 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) were analytically verified via LC-MS/ in fresh media (0 hours and 24 hours) and old media (24 hours and 48 hours) of all tested concentration levels and the control via LC-MS/MS.

The measured concentrations of the test item 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) in the fresh media were 0% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of 13 to 60% of the initially measured test item concentrations. Adsorption to glass was found to be negligible.
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.
Reported statistics and error estimates:
Methods of evaluation
The EC/EL100 – values after 24 and 48 hours and the EC/EL50- value after 24 hours were estimated empirically from the observed immobilization rates.
All effect levels (EC/EL10 / 50 / 100) are given based on the nominal loading rates of the test item and the geometric mean measured test item concentrations.

ECx-values and statistical analyses
The EC values were calculated by linear regression (24 hours) and by sigmoidal dose response regression (48 hours). The respective 95% confidence limits were calculated from the standard error and the t distribution wherever possible. 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

Conditions of analysis

Gradient Table

Time [min] A [%] B [%]
0.00 80 20
0.30 80 20
1.30 5 95
2.00 5 95
2.10 80 20
2.50 80 20

 

Dilution Steps (fresh media)

test item concentration Factor* volume volume
[mg/L]   [mL] [mL]
100 200 0.1 10
45.5 100 0.1 5.0
20.7 50 0.2 5.0
9.39 20 0.1 1.0
4.27 10 0.2 1.0
Control 2 1.0 1.0

 

Dilution Steps (old media)

Nominal  Dilution Sample Final
test item concentration Factor* volume volume
[mg/L]   [mL] [mL]
100 20 0.1 1.0
45.5 10 0.2 1.0
20.7 5 0.4 1.0
9.39 2 1.0 1.0
4.27 2 1.0 1.0
Control 2 1.0 1.0

 

Dilution Steps (glass adsorption)

Nominal  Dilution Sample Final Sampling date
test item concentration Factor* volume volume
[mg/L]   [mL] [mL]
100 200 0.1 10 24 hours / 48 hours 45.5 100 0.1 5.0 24 hours / 48 hours
20.7 40 0.05 1.0 24 hours
20.7 2 1.0 1.0 48 hours
9.39 20 0.1 1.0 24 hours
9.39 2 1.0 1.0 24 hours / 48 hours
4.27 2 1.0 1.0 24 hours / 48 hours

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
Based on the nominal loading rates, the 48 hours-EL50 for Daphnia magna was 26.6 mg/L (confidence limits: 23.9 – 32.4 mg/L) for the test item 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2).
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) (batch number: 20190290) were determined at the test facility according to OECD 202 (2004) from 2021-03-22 to 2021-06-01 with the definitive exposure phase from 2021-05-05 to 2021-05-07 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 4.27 to 100 mg/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 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) 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 the control via LC MS/MS.
The measured concentrations of the test item 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) in the fresh media were 0% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of 13 to 60% of the initially measured test item concentrations. Adsorption to glass was found to be negligible.

Description of key information

There is one recently performed acute daphnia test available.

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) (batch number: 20190290) were determined at the test facility according to OECD 202 (2004) from 2021-03-22 to 2021-06-01 with the definitive exposure phase from 2021-05-05 to 2021-05-07 at the test facility.
The substance is poorly soluble in water and therefore 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 4.27 to 100 mg/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 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) 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 the control via LC MS/MS.
The measured concentrations of the test item 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) in the fresh media were 0% of the nominal values. At the end of exposure after 24 and 48 hours, the measured concentrations were in the range of 13 to 60% of the initially measured test item concentrations. Adsorption to glass was found to be negligible. EL50 = 26.6 mg/L (based on nominal loading) and the EC50 = 0.0237 mg/L based on geometric mean measured concentration.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Remarks:
EL50 based on nominal Loading
Effect concentration:
26.6 mg/L
Fresh water invertebrates
Dose descriptor:
EC50
Remarks:
Mean measured concentration
Effect concentration:
0.024 mg/L

Additional information

The substance is a poorly soluble multi-component substance. Such a substance cannot be dissolved into a homogeneous solution and the multiple components make monitoring impossible. Sorption to the test vessel, test organism, water solubility all present problems of interpretation, where each component of the mixture may behave differently. Consideration therefore needs to be given to using the data derived from the testing of water accommodated fractions (WAFs) for aquatic toxicity and the use of such data for classification and labeling (OECD 2001)

OECD (2001), OECD Series on Testing and Assessment No. 27: Guidance Document on Use of the Harmonized System for the Classification of Chemicals which are Hazardous for the Aquatic Environment, OECD, Paris..