2-[[3-(dimethylamino)propyl]methylamino]ethanol

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:

01 DEC 2014 - 04 DEC 2014 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted March 2006, corrected July 28, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analysis of the Test Item Concentrations

Sampling:
Duplicate samples from the freshly prepared test media (containing 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.
All samples were diluted by a factor of two with acetonitrile directly after sampling.
One additional sample of the blank control and the solvent control was taken at test start and test end without any sample treatment.

Analyses:
The concentrations of the test item were analysed in the duplicate test media samples from all test concentrations and in the duplicate control samples each from both sampling times (0 and 72 hours). All samples were centrifuged before analysis, in order to remove the algae. The blank control and solvent control from both sampling times were analysed without prior centrifugation.

Storage of Samples:
All samples were stored in a freezer (≤ - 10 °C), protected from light until analysis was performed.

Fortified Samples:
Two stock solutions were prepared. Approximately 50 mg of the test item were dissolved in 50 mL acetonitrile to obtain a stock solution of approximately 1 g test item/L. Appropriate amounts of these stock solutions were diluted with test water to obtain fortified samples at a level of 0.4 and 60 mg test item/L. Exact values were documented in the raw data.


Sample Preparation:
The samples were shaken, then and ultrasonicated for 1 minute after thawing to obtain homogenous samples. The samples were further diluted with acetonitrile / test water (1/1, v/v) to match the calibration range, if necessary. The samples were centrifuged (13000 rpm, 10 minutes) before analysis.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

- Species: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)

- Origin: The algae were supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", Göttingen, Germany.

- Breeding Conditions: The algae were cultivated under standardised conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

0.24 mmol/l, corresponding to 24 mg/l, as CaCO3

Test temperature:

21 to 22 °C

pH:

8.0 to 8.3 at test start
8.3 to 9.1 at test end

Salinity:

Reconstituted water was used for the test: Analytical grade salts were added to deionised water.

Nominal and measured concentrations:

- Nominal 50, 15.8, 5.01, 1.58, 0.50 mg test item/L.
- At the start of the test 108 % of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 102 % of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 105 % of nominal. Therefore, all reported results refer to nominal concentrations.

Details on test conditions:

- 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.

- Type and Size: Erlenmeyer flasks of 50 mL volume with approximately 50 mL of test
medium

- Identification: Each test unit was uniquely identified with the serial number of the study, treatment and replicate number.

- 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: 5703 lux (range: 5400 to 6000 lux)

- Recording: Test conditions were recorded

- Introduction of Algae: The test was started (0 hours) by inoculation of a biomass of approx. 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre‐culture, which was set up 3 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. Volumes of approx. 50 mL of algal suspension per replicate were continuously stirred with magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass dishes and incubated in a water bath. The flasks 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 spectrophotometrical 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.

- Determination of the Cell Density: The cell density on each observation time was determined by spectrophotometrical 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.

- Exposure Time: 72 hours.

Reference substance (positive control):

yes

Remarks:

The reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23.1 mg/L

95% CI:

17.3 - 32

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: not determinable

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

8.18 mg/L

95% CI:

4.27 - 11.7

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

37.7 mg/L

95% CI:

33.1 - 43

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.75 mg/L

95% CI:

1.87 - 759

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

17.6 mg/L

95% CI:

13.5 - 21.2

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield and growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

15.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield and growth rate

Details on results:

- Growth Inhibition: The 72-hour EyC50 was calculated to be 23.1 mg test item/L and the ErC50 > 50 mg test item/L. The 72-hour EyC10 was calculated to be 4.75 mg test item/L and the ErC10 17.6 mg test item/L. The 72-hour NOEyC was determined to be 5.01 mg test item/L and the associated 72-hour LOEyC of 15.8 mg test item/L. The 72-hour NOErC was determined to be 5.01 mg test item/L and the associated 72-hour LOErC is 15.8 mg test item/L.

- Microscopic Examination: 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 a nominal test concentration of 15.8 mg 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. At a concentration of 50 mg test item/L the cells were deformed.

Results with reference substance (positive control):

The reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
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 Bonferroni-Welch t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat Solutions GmbH.

Validity criteria fulfilled:

yes

Conclusions:

- 72-hour EyC50: 23.1 mg test item/L (nominal);
- 72-hour ErC50: >50 mg test item/L (nominal);
- 72-hour NOEyC and NOErC: 5.01 mg test item/L (nominal);
- 72-hour LOEyC and LOErC: 15.8 mg test item/L (nominal).

Executive summary:

A static dose-response test was performed to assess the effect of the test material 2-[[3-(dimethylamino)propyl]methylamino]ethanol on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2011) No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

Following a preliminary range-finding test and initial experiment, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 50, 15.8, 5.01, 1.58 and 0.50 mg test item/L (3 replicate flasks per concentration) for 72 hours, under constant illumination and a temperature of 21 to 22°C. 

Samples of the algal populations were removed daily and the cell density determined for each control and treatment group, using spectrophotometrical measurement.

The validity criteria for the analytical method were met. At the start of the test 108 % of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 102 % of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 105 % of nominal. Therefore, all reported results refer to nominal concentrations.

The 72-hour E_yC50 was calculated to be 23.1 mg test item/L and the 72-hour E_rC50 value was calculated to be >50 mg test item/L. The 72-hour NOE_yC was determined to be 5.01 mg test item/L and the associated 72-hour LOE_yC was 15.8 mg test item/L. The 72-hour NOE_rC was determined to be 5.01 mg test item/L and the associated 72-hour LOE_rC was 15.8 mg test item/L.

Description of key information

toxicity of substance 2-[[3-(dimethylamino)propyl]methylamino]ethanol to freshwater algae Pseudokirchneriella subcapitata (OECD 201):

- 72h-E_yC₅₀: 23.1 mg test item/L (nominal, based on yield);
- 72h-E_rC₅₀: >50 mg test item/L (nominal, based on growth rate)
- 72h-NOE_yC and NOE_rC: 5.01 mg test item/L (nominal, based on yield and growth rate);
- 72h-LOE_yC and LOE_rC: 15.8 mg test item/L (nominal, based on yield and growth rate).

Key value for chemical safety assessment

EC50 for freshwater algae:

50 mg/L

EC10 or NOEC for freshwater algae:

5.01 mg/L