2,2'-(C12-14 evennumbered alkyl imino) diethanol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 03, 2021 to April 27, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: SANTE/2020/12830 Rev.1: Guidance Document on Pesticide Analytical Methods for Risk Assessment and Post-approval Control and Monitoring Purposes

Version / remarks:

24. February 2021

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals",

Version / remarks:

2nd Ed., February 08, 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted April 13, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Colour: Slightly yellowish
pKa (predicted): 2,2´- (dodecylimino)bisethanol: 14.41 according to Chemical Book (see references)
Critical Micellar Concentration (CMC): 7.5 mg/L according to information provided by the sponsor
Expiry Date: October 01, 2022
Storage Conditions at the Test Facility: At 20 ± 5 °C, in the dark

Analytical monitoring:

yes

Details on sampling:

Sampling: Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test and at day 1.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the aged test media of all test concentrations and the control were collected at day 1 (after 24 hours of exposure) and at the end of the test by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of 2 with acetonitrile.
Furthermore, samples of the untreated control and the sample dilution solvent acetonitrile were taken at each sampling point. These samples remained undiluted.
Storage of Test Samples: All samples were stored in a freezer (≤ - 20 °C), protected from light, until analysis was performed. Afterwards the samples were again stored deep frozen and will be kept stored up to the date of the final report.

Vehicle:

no

Details on test solutions:

A stock solution of 10 mg test item/L was prepared by dissolving 12.5 and 13.3 mg test item into 1250 and 1330 mL test water by intense stirring for 25 and 15 minutes and ultrasonic treatment for 5 and 0 minutes. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations.
The test media were prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 1.75 to 20.25 hours old
Origin: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.
Breeding Conditions: The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Acclimatisation: Was not necessary, since the test was performed in the same medium as the culturing.

Test type:

semi-static

Water media type:

other: Reconstituted Water (Elendt "M4"): Analytical grade salts and additives were added at the following nominal concentrations in deionised water

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

Water Temperature:
18.7 to 19.5 °C in the freshly prepared media; 19.6 to 19.8°C in the aged test media

pH:

7.9 in the freshly prepared media;
7.9 in the aged test media;
and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

9.0 to 9.3 mg/L in the freshly prepared media
8.6 to 8.9 mg/L in the aged test media

Nominal and measured concentrations:

3.0, 1.4, 0.62, 0.28 and 0.13 mg test item/L (spacing factor 2.2),
corresponding to following time-weighted average concentrations of the test item:
2342, 1064, 449, 182 and 81.9 μg test item/L, and a control.

Details on test conditions:

Test vessel: Glass beakers of 100 mL volume containing approximately 60 mL of test medium. The test vessels were covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.

A semi-static test system with a test medium renewal on Day 1 was
chosen to keep the concentrations of test item as constant as possible in the test media.

20 daphnids per control and test concentration, divided into 4 groups of 5 animals, each group in one test vessel.
The test was performed with four replicates per treatment group.

TEST MEDIUM
Reconstituted water (Elendt "M4") prepared according to SOP.

Photoperiod:
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 480 to 510 lux (measured once during the test).

EFFECT PARAMETERS MEASURED:
The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those animals that are not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae). Any signs of disease or stress or unusual behaviour were documented in the raw data.

RANGE-FINDING STUDY
Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
To follow the recommendations of OECD 23 (2019) as close as possible, the test design was chosen based on the obtained pre-test results as follows:
Although the test substance is known to be highly adsorptive, pre-tests have shown that pre-saturation of the test vessels did not lead to increased solubility or stability. Therefore, no pre-saturation was performed.
Since recoveries of both main constituents of the test substance decreased in the range-finder, the test was carried out in a semi-static design to maintain the test concentrations as stable as possible throughout the test.
The main constituent has a pKa of 14.41 and is therefore present in fully ionized form at the relevant pH range of this study. Therefore, adjusting the pH to get the constituents into a non-ionised form is not feasible.
Since the test substance has a water solubility/critical micellar concentration (CMC) that is higher as the highest test concentration (see section 6.1), the use of a Water accommodated fraction (WAF) for test solution preparation was not necessary.
The use of toxicity mitigation for this cationic substance with increased dissolved organic carbon content (DOC) is not applicable since studies with river-water including an increased DOC content were already performed (information according to the sponsor).

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

910 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

588 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

449 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

1 064 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

2 023 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

1 826 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

1 064 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

2 342 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the time-weighted average test item concentration of 449 μg test item/L. At the time-weighted average concentration of 1064 μg test item/L 15 daphnids were immobile and 20 daphnids were immobile at the time-weighted average concentration of 2342 μg test item/L.
Daphnia were moving slower compared to the control at the time-weighted average test concentrations of 449 μg/L (9 daphnids) and 1064 μg/L (5 daphnids), respectively.

Analytical results:
Summary of Analytical Results for 2,2’-(dodecylimino)bisethanol
Time Weighted Average concentration:
Control: not pplicable
130 µg test item/L: 90.9 µg test item/L or 68.9 µg a.i./L
280 µg test item/L: 200 µg test item/L or 152 µg a.i./L
620 µg test item/L: 491 µg test item/L or 372 µg a.i./L
1400 µg test item/L: 1139 µg test item/L or 863 µg a.i./L
3000 µg test item/L: 2510 µg test item/L or 1903 µg a.i./L

Summary of Analytical Results for 2,2’-(dodecylimino)bisethanol
Time Weighted Average concentration:
Control: not pplicable
130 µg test item/L: 63.8 µg test item/L or 12.9 µg a.i./L
280 µg test item/L: 147 µg test item/L or 29.8 µg a.i./L
620 µg test item/L: 381 µg test item/L or 77.4 µg a.i./L
1400 µg test item/L: 990 µg test item/L or 201 µg a.i./L
3000 µg test item/L: 2164 µg test item/L or 439 µg a.i./L


Summary of Analytical Results for the test item
Time Weighted Average concentration:
Control: not pplicable
130 µg test item/L: 81.9µg test item/L
280 µg test item/L: 182 µg test item/L
620 µg test item/L: 449 µg test item/L
1400 µg test item/L: 1064 µg test item/L
3000 µg test item/L: 2342 µg test item/L

Results with reference substance (positive control):

Results of the most recent Test with the Reference Item Potassium dichromate
EC50 24 h: 0.918 mg test item/L
EC50 48 h: 0.685 mg test item/L
The results indicate that the sensitivity of the daphnidswas consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L)

Reported statistics and error estimates:

The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by weibull analysis.
The NOEC and LOEC after 24 and 48 hours were calculated by Step-down Cochran Armitage test procedure.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Validity criteria fulfilled:

yes

Remarks:

Control Immobilisation Rate: Was 0 % and furthermore no daphnid showed signs of disease or stress Dissolved Oxygen Concentration: Was ≥ 8.6 mg O2/L in in all treatment groups at the end of the test

Conclusions:

The toxic effect of the test item 2,2’-(C12-14 evennumbered alkylimino) diethanol to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was calculated to be 449 μg test item/L. The 48-hour LOEC was calculated to be 1064 μg test item/L and the 48-hour EC50 value was calculated to be 910 μg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to time-weighted average concentrations of the test item (weighted sum of time-weighted average values of both measured main ingredients) since the concentrations of the main ingredients were not within ± 20% of the nominal concentrations during the test for all test concentrations and different recovery decrease was determinable for each ingredient.

Executive summary:

Title: 2,2’-(C12-14 evennumbered alkylimino) diethanol: Acute Toxicity to Daphnia magna in a Semi-Static 48-hour Immobilisation Test
Purpose: The purpose of this study was to determine the influence of the test item 2,2’-(C12-14 evennumbered alkylimino) diethanol on the mobility of Daphnia magna. For this purpose, young daphnids (< 24 hours old) were exposed in a semi-static test to various concentrations under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours.
The test method and the test species Daphnia magna are recommended by the test guidelines. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Test Species: Daphnia magna, clone 5; 1.75 to 20.25 hours old.
Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) each containing 20 individuals. The mobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours.
Endpoints: Number of immobile organisms after 24 and 48 hours

Test Concentrations: 3.0, 1.4, 0.62, 0.28 and 0.13 mg test item/L (spacing factor 2.2), corresponding to following time-weighted average concentrations of the test item: 2342, 1064, 449, 182 and 81.9 μg test item/L, and a control.

Test Conditions: Water temperature: 18.7 to 19.8 °C; pH value: 7.9; dissolved oxygen concentration: 8.6 to 9.3 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 480 to 510 lux; and thus were within the ranges requested by guideline OECD 202

Validity Criteria: Control immobilisation rate: 0 %, dissolved oxygen concentration at test end: ≥ 8.6 mg O2/L; and thus, the validity criteria were met.

Biological Test Results: After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the time-weighted average test item concentration of 449 μg test item/L. At the time-weighted average concentration of 1064 μg test item/L 15 daphnids were immobile and 20 daphnids were immobile at the time-weighted average concentration of 2342 μg test item/L.
Daphnia were moving slower compared to the control at the time-weighted average test concentrations of 449 μg/L (9 daphnids) and 1064 μg/L (5 daphnids), respectively.

EC50 - 24 h: 2023 μg test item/L, EC50 - 48 h: 910 μg test item/L
EC20 - 24 h: 1902 μg test item/L, EC20 - 48 h: 700 μg test item/L
EC10 - 24 h: 1826 μg test item/L, EC10 - 48 h: 588 μg test item/L
NOEC - 24 h: 1064 μg test item/L, NOEC - 48 h: 449 μg test item/L
LOEC - 24 h: 2342 μg test item/L, LOEC - 48 h: 1064 μg test item/L

Analytical Test Results: The quantification of the main ingredients 2,2’-(dodecylimino)bisethanol and 2,2’-(tetradecylimino)bisethanol of the test item 2,2’-(C12-14 evennumbered alkylimino) diethanol in the test samples was performed using liquid chromatography with MS/MS detection.
2,2’-(dodecylimino)bisethanol:
In the freshly prepared test media at the start of the test and at the renewal of the test media at day 1, 82 % of the nominal test concentrations were found (average of all test concentrations). In the 24 hours aged test media after 24 and 48 hours test duration, 72 % of the nominal value was determined (average of all test concentrations). During the test the test organism were exposed to a mean of 77 % of nominal.
2,2’-(tetradecylimino)bisethanol
In the freshly prepared test media at the start of the test and at the renewal of the test media at day 1, 73 % of the nominal test concentrations were found (average of all test concentrations). In the 24 hours aged test media after 24 and 48 hours test duration, 51 % of the nominal value was determined (average of all test concentrations).During the test the test organism were exposed to a mean of 62 % of nominal.

Conclusion:
The toxic effect of the test item 2,2’-(C12-14 evennumbered alkylimino) diethanol to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was calculated to be 449 μg test item/L. The 48-hour LOEC was calculated to be 1064 μg test item/L and the 48-hour EC50 value was calculated to be 910 μg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to time-weighted average concentrations of the test item (weighted sum of time-weighted average values of both measured main ingredients) since the concentrations of the main ingredients were not within ± 20% of the nominal concentrations during the test for all test concentrations and different recovery decrease was determinable for each ingredient.

Description of key information

The toxic effect of the test item 2,2’-(C12-14 evennumbered alkylimino) diethanol to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was calculated to be 449 μg test item/L. The 48-hour LOEC was calculated to be 1064 μg test item/L and the 48-hour EC50 value was calculated to be 910 μg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to time-weighted average concentrations of the test item (weighted sum of time-weighted average values of both measured main ingredients) since the concentrations of the main ingredients were not within ± 20% of the nominal concentrations during the test for all test concentrations and different recovery decrease was determinable for each ingredient.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

910 µg/L