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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 03, 2021 to July 23, 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:

yes

Remarks:

Interference of 35% was determined for the analyte 2,2’- (tetradecylimino)bisethanol at the LOQ level of 0.250 μg test item/L Calibration solutions extended over a range of the analytes in relevant analytical solutions plus 316% and minus 60%.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted March 23, 2006, corrected July 28, 2011

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Aggregate State at Room Temperature: Liquid
Colour: Slightly yellowish
pKa (predicted): 2,2´- (dodecylimino)bisethanol: 14.41 according to Chemical Book
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:

One sample of the freshly prepared stock solution and duplicate samples from the freshly prepared test media (without algae) of all test concentrations and from the control were taken at the start of the test. 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 test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment. Furthermore, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken after 24 and 48 hours test duration by pooling adequate volumes from the test vessels per treatment group. All samples were diluted by a factor of two with acetonitrile containing 0.5% formic acid. Additional samples of the untreated control and of the dilution solvent acetonitrile containing 0.5% formic acid were taken at each sampling without any sample treatment.
Storage: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen up to the date of the final report.
Storage of Standard Solutions and Final extracts:
All samples were analysed within 24 hours after sample preparation without further storage.

Vehicle:

no

Details on test solutions:

A stock solution of 5 mg test item/L was prepared by dissolving 4.8 mg test item into 960 mL test water by intense stirring for 35 minutes and ultrasonic treatment for 10 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 algae (= start of the test).

Test organisms (species):

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

Details on test organisms:

Origin: The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
Reference Item: For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least
twice a year to demonstrate satisfactory test conditions.

Test type:

static

Water media type:

other: Reconstituted Water (OECD Medium): Analytical grade salts were added to deionised water

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

none

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L)
as CaCO3.

Test temperature:

The temperature was measured daily in an Erlenmeyer flask filled
with water and incubated under the same conditions as the test
flasks. Water temperature was: 22.1 to 23.3 °C

pH:

The pH was measured in all test item concentrations and the control
at the start and the end of the test.
pH value in the control at test start: 8.1,
pH value in the control at test end: 8.4
pH values in the test item treatments at test start: 8.1,
pH values in the test item treatments at test end: 8.1 to 8.3

Nominal and measured concentrations:

100, 32.26, 10.41, 3.36 and 1.08 μg test item/L (spacing factor 3.10)
and a control,
corresponding to following geometric mean measured concentrations of the test item: 6.68, 4.72, 3.92, 2.13 and 1.57 μg test item/L,
and to the following initial mean measured concentrations of the test
item: 51.7, 16.3, 5.47, 1.97 and 0.911 μg test item/L.

Details on test conditions:

Test vessel: Erlenmeyer flasks of 50 mL volume with nominal 50 mL of test medium loosely covered with watch glasses.

Initial cells density: The test was started (0 hours) by inoculation of a biomass of nominal 5000 algal cells per mL test medium.
These cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test start under the same conditions as in the test.

Replicates: The test was performed with three replicates per test concentration and six replicates in the control.
Test Procedure: The test units were continuously stirred by magnetic stirrers. The flasks were incubated in a water bath, were placed in a random order
and were repositioned each day to minimize differences in test conditions.
Blanks: Additionally, one replicate of each test concentration and of the
control was prepared without algae to provide a "blank" for the spectrophotometric measurements. The additional replicates were
incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by the test item (see also "Determination of the Cell Density").

GROWTH MEDIUM
Standard medium used: yes (reconsituted water)

OTHER TEST CONDITIONS
Sterile test conditions: no
Light Regime: Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 5173 lux (range: 4970 to 5380 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae). Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

TEST CONCENTRATIONS
100, 32.26, 10.41, 3.36 and 1.08 μg test item/L (spacing factor 3.10) and a control,

Pre-experiments/Range finding study
To follow the recommendations of OECD 201 (2011) and 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 of the test vessels was performed.
- Recovery values determined at test start in some pre-experiments were below or strongly above the nominal range of 80 – 120%, likely caused by the adsorptive properties of the test substance that complicates homogenous application. However, pre-test results have shown that application via an organic solvent did not improve the initial recoveries. Therefore, aqueous application was performed in the main test of this study since actual present test item concentrations were determined via analytical dose verification. Thereby, special care was taken to use a stock solution concentration that is below the CMC and to vigorously saturate all pipette tips used for the dilution steps to avoid inhomogeneous test substance transfer.
- Since recoveries of the main constituents of the test substance decreased during the duration of the pre-tests, additional samples were taken after 24 and 48 hours test duration to be able to calculate reasonable geometric mean values to evaluate the biological results. A semi-static or flow-through test design is not applicable for the algae test.
- Enhanced recoveries of both main constituents were determined in samples taken after 24 or 48 hours test duration of test concentrations at which no or no strong algae growth inhibition occurred in case samples were directly diluted with an organic dilution solvent (e.g. acetonitrile) after sampling. This effect is likely caused by adsorption of the test substance to the algae cells and the adsorbed substance is washed from the cells after addition of the dilution solvent. To avoid this effect, it was investigated to remove the algae cells by centrifugation prior to the addition of a dilution solvent. Thereby, only less than 50% of nominal recoveries could be measured in the water phase. It is likely that the missing amount of substance is adsorbed to algae cells. To reflect the recommendations of OECD Guideline 23 as near as possible, it was decided to use the procedure of direct addition of a dilution solvent after sampling to ensure correct recoveries for samples without or with low amounts of algae cells. To reduce adsorption as much as possible, 0.5% formic acid was added to the dilution solvent as counter ion for potential adsorption of the positively charged test substance to negatively charged surfaces.
- 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 was not feasible.
- Since the test substance has a water solubility/critical micellar concentration (CMC) that is higher than the highest test concentration (see section 2.1), the use of a Water accommodated fraction (WAF) for test solution preparation is not necessary. An aqueous stock solution of 5 mg test item/L was prepared to be below the CMC of 7.5 mg/L.
- The use of toxicity mitigation for this cationic substance with increased dissolved organic carbon content (DOC) is not applicable since already performed studies with river-water including an increased DOC content were already performed (information according to the sponsor). The non-GLP pre-experiments were not performed in compliance with the GLP-Regulations

Reference substance (positive control):

yes

Remarks:

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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.51 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.62 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.25 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.92 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.48 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.59 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.92 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.02 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.36 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.59 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.97 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.47 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

37.2 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

5.75 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.17 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.97 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.47 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Growth Inhibition:
The 72-hour EyC50 was calculated to be 3.51 μg test item/L (initial mean: 5.02 μg test item/L) and the ErC50 6.13 μg test item/L (initial mean: 37.2 μg test item/L). The 72-hour EyC10 was calculated to be 2.25 μg test item/L (initial mean: 1.59 μg test item/L) and the ErC10 2.59 μg test item/L (initial mean: 2.17 μg test item/L). The 72-hour NOEyC was determined to be 2.13 μg test item/L (initial mean: 1.97 μg test item/L) and the associated 72-hour LOEyC of 3.92 μg test item/L (initial mean: 5.47 μg test item/L). The 72-hour NOErC was determined to be 2.13 μg test item/L (initial mean: 1.97 μg test item/L) and the associated 72-hour LOErC is 3.92 μg test item/L (initial mean: 5.47 μg test item/L).

Signs of Phytotoxicity evaluated by Microscopic Observations:
The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to the geometric mean measured test concentration of 6.68 μg test item/L (initial mean: 51.7 μg test item/L) and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Analytical 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, 69 % of the nominal test concentrations were found (average of all test concentrations). In the 24 hours aged, 48 hours aged and 72 hours
(test end) aged test media, 149 %, 77 % and 13 % of the nominal value was determined, respectively (each value is average of all test concentrations). 2,2’-(tetradecylimino)bisethanol In the freshly prepared test media at the start of the test, 36 % of the nominal test concentrations were found (average of all test concentrations). In the 24 hours aged, 48 hours aged and 72 hours (test end) aged test media, 45 %, 18 % and 3 % of the nominal value
was determined, respectively (each value is average of all test concentrations). Generally, a recovery decrease was determinable for both measured ingredients from test start to test end. The elevated
recoveries determined after 24 and 48 hours test duration in comparison to the recoveries determined at test start are mainly caused by high recovery values measured in the three lower test concentrations at which algae growth was not or not strongly reduced (see biological results). It is assumed that this phenomenon is caused by adsorption of the ingredients to the algae cells which are later washed from the cells by the applied sample preparation. This is supported by a performed pre-test without dilution solvent. In this pre-test, the centrifugation of samples led to a significant reduction of substance in the water phase while it can be assumed that the missing amount of substance is
adsorbed to the algae cells. This is substantiated by the fact that at the higher test concentrations of nominal 100 and 32.26 μg test item/L without or with significantly reduced algae growth, no increased recovery values were determinable after 24 and 48 hours test duration. Since the measured recoveries of the intermediate samples taken after 24 and 48 hours test duration skew the calculable geometric mean concentrations of the lower test concentrations, initial mean measured concentrations are additionally presented since they reflect actual dosing of the test item without alteration by cell
adsorption effects in the water phase and additionally reflect the amount of substance which is adsorbed to algae cells. The biological results of the study give rise that the adsorbed substance is still bioavailable to the algae.

Results with reference substance (positive control):

In the previous reference test, performed in February 2021 with Potassium Dichromate, the EC50 for yield was 0.393 mg test item/L and the EC50 for growth rate was 0.895 mg test item/L. The data show that the sensitivity of the test organism did not change significantly over time, thus confirming correct sensitivity of the tested algal species.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by probit. For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield and growth rate). The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Validity criteria fulfilled:

yes

Remarks:

Cell Density Increase in Control Cultures: 84.4-fold increase within 72 hours Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 9.9% Coefficient of Variation of Average Growth between Control Replicates: 2.0 %

Conclusions:

The influence of 2,2’-(C12-14 evennumbered alkylimino)diethanol on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test.
The 72-hour EyC50 was calculated to be 3.51 μg test item/L (initial mean: 5.02 μg test item/L) and the 72-hour ErC50 value was calculated to be 6.13 μg test item/L (initial mean: 37.2 μg test item/L). The 72-hour NOEyC was determined to be 2.13 μg test item/L (initial mean: 1.97 μg test item/L) and the associated 72-hour LOEyC was 3.92 μg test item/L (initial mean: 5.47 μg test item/L). The 72-hour NOErC was determined to be 2.13 μg test item/L (initial mean: 1.97 μg test item/L) and the associated 72-hour LOErC was 3.92 μg test item/L (initial mean: 5.47 μg test item/L). The initial concentrations and the maintenance of the exposure concentrations during the test were assessed in the analytical part. All reported results refer to geometric mean measured concentrations of the test item (weighted sum of geometric mean 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. In addition, all results were evaluated based on initial mean measured concentrations to compensate varying intermediate recoveries that were determined and to reflect actual dosage of the test item.

Executive summary:

Title:  2,2’-(C12-14 evennumbered alkylimino) diethanol: Toxicity to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test

Purpose: The purpose of this test was to determine the inhibitory effect of the test item 2,2’-(C12-14 evennumbered alkylimino) diethanol on the
growth of the freshwater green algae Pseudokirchneriella subcapitata. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The test method and the test species Pseudokirchneriella subcapitata 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: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by
spectrophotometric measurement.

Endpoints: Yield and growth rate of the algae

Test Concentrations: 100, 32.26, 10.41, 3.36 and 1.08 μg test item/L (spacing factor 3.10) and a control, corresponding to following geometric mean measured concentrations of the test item: 6.68, 4.72, 3.92, 2.13 and 1.57 μg test item/L, and to the following initial mean measured concentrations of the test item: 51.7, 16.3, 5.47, 1.97 and 0.911 μg test item/L.

Test Conditions: Water temperature: 22.1 to 23.3 °C; pH value in the control at test start: 8.1, pH value in the control at test end: 8.4; pH values in the test item treatments at test start: 8.1, pH values in the test item treatments at test end: 8.1 to 8.3; continuous illumination; mean light intensity: 5173 lux (4970 to 5380 lux).

Biological Results:

Validity Criteria: 417.5-fold increase of cell density within 72 hours; CV of sectional (daily) growth rate of control: 18.5 %; CV of average growth of control replicates: 0.9 %; and thus, the validity criteria were met.

Parameter	Yield	Growth rate
	geometric mean test item concentration	geometric mean test item concentration
	[µg test item/L]	[µg test item/L]
72-hour EC50	3.51	6.13
72-hour EC20	2.62	3.48
72-hour EC10	2.25	2.59
72-hour NOEC	2.13	2.13
72-hour LOEC	3.92	3.92
	initial mean measured test item concentration	initial mean measured test item concentration
	[µg test item/L]	[µg test item/L]
72-hour EC50	5.02	37.2
72-hour EC20	2.36	5.75
72-hour EC10	1.59	2.17
72-hour NOEC	1.97	1.97
72-hour LOEC	5.47	5.47

Analytical 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, 69 % of the nominal test concentrations were found (average of all test concentrations). In the 24 hours aged, 48 hours aged and 72 hours (test end) aged test media, 149 %, 77 % and 13 % of the nominal value was determined, respectively (each value is average of all test concentrations). 2,2’-(tetradecylimino)bisethanol In the freshly prepared test media at the start of the test, 36 % of the nominal test concentrations were found (average of all test concentrations). In the 24 hours aged, 48 hours aged and 72 hours (test end) aged test media, 45 %, 18 % and 3 % of the nominal value was determined, respectively (each value is average of all test concentrations). Generally, a recovery decrease was determinable for both measured ingredients from test start to test end. The elevated recoveries determined after 24 and 48 hours test duration in comparison to the recoveries determined at test start are mainly caused by high recovery values measured in the three lower test concentrations at which algae growth was not or not strongly reduced. It is assumed that this phenomenon is caused by adsorption of the ingredients to the algae cells which are later washed from the cells by the applied sample preparation. This is supported by a performed pre-test without dilution solvent. In this pre-test, the centrifugation of samples led to a significant reduction of substance in the water phase while it can be assumed that the missing amount of substance is adsorbed to the algae cells. This is substantiated by the fact that at the higher test concentrations of nominal 100 and 32.26 μg test item/L without or with significantly reduced algae growth, no increased recovery values were determinable after 24 and 48 hours test duration. Since the measured recoveries of the intermediate samples taken after 24 and 48 hours test duration skew the calculable geometric mean concentrations of the lower test concentrations, initial mean measured concentrations are additionally presented since they reflect actual dosing of the test item without alteration by cell adsorption effects in the water phase and additionally reflect the amount of substance which is adsorbed to algae cells. The biological results of the study give rise that the adsorbed substance is still bioavailable to the algae.

 

Description of key information

The influence of 2,2’-(C12-14 evennumbered alkylimino)diethanol on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 3.51 μg test item/L (initial mean: 5.02 μg test item/L) and the 72-hour ErC50 value was calculated to be 6.13 μg test item/L (initial mean: 37.2 μg test item/L). The 72-hour NOEyC was determined to be 2.13 μg test item/L (initial mean: 1.97 μg test item/L) and the associated 72-hour LOEyC was 3.92 μg test item/L (initial mean: 5.47 μg test item/L). The 72-hour NOErC was determined to be 2.13 μg test item/L (initial mean: 1.97 μg test item/L) and the associated 72-hour LOErC was 3.92 μg test item/L (initial mean: 5.47 μg test item/L).

The initial concentrations and the maintenance of the exposure concentrations during the test were assessed in the analytical part. All reported results refer to geometric mean measured concentrations of the test item (weighted sum of geometric mean 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. 

Generally, a recovery decrease was determinable for both measured ingredients from test start to test end. The elevated recoveries determined after 24 and 48 hours test duration in comparison to the recoveries determined at test start are mainly caused by high recovery values measured in the three lower test concentrations at which algae growth was not or not strongly reduced. It is assumed that this phenomenon is caused by adsorption of the ingredients to the algae cells which are later washed from the cells by the applied sample preparation.
This is supported by a performed pre-test without dilution solvent. In this pre-test, the centrifugation of samples led to a significant reduction of substance in the water phase while it can be assumed that the missing amount of substance is adsorbed to the algae cells. This is substantiated by the fact that at the higher test concentrations of nominal 100 and 32.26 μg test item/L without or with significantly reduced algae growth, no increased recovery values were determinable after 24 and 48 hours test duration.
Since the measured recoveries of the intermediate samples taken after 24 and 48 hours test duration skew the calculable geometric mean concentrations of the lower test concentrations, initial mean measured concentrations are additionally presented since they reflect actual dosing of the test item without alteration by cell adsorption effects in the water phase and additionally reflect the amount of substance which is adsorbed to algae cells. The biological results of the study give rise that the adsorbed substance is still bioavailable to the algae. Therefore, the effect concentrations based on inital mean measured concentrations are more realistic to derive the toxicity of the registration substance.

Key value for chemical safety assessment

EC50 for freshwater algae:

37.2 µg/L

EC50 for marine water algae:

2.17 µg/L