Reaction products of 2-[[2-(dimethylamino)ethyl]methylamino]ethanol di(hexanoate) and ethyl oxirane

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:

2015-10-20 to 2015-10-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP principles

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via LC-MS/MS after 0 hours (start of exposure) and 72 hours (end of exposure). Separate replicates for the test item analysis at the beginning of exposure were prepared with algae. For the test item analysis after 72 hours, samples were taken out of the test replicates.

Vehicle:

no

Details on test solutions:

A stock solution of 500 mg/L was freshly prepared with dilution water. The stock solution was treated with ultrasound for 5 min at room temperature. Thereafter the solution was stirred for 30 min (1100 rpm at room temperature). 5 concentrations were tested in a geometrical series with a
dilution factor of 2.5: 12.8 - 32.0 - 80.0 - 200 - 500 mg/L. The concentrations were based on the results of a preliminary range finding test (non-GLP).

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline. Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE·m-2·s-1 for 24 hours per day. The algae are kept in Nutrient medium Z according to LÜTTGE et al. (1994). A four days old preculture, prepared in dilution water, was used as inoculum.
Initial cell density: Nominal: approximately 5 x 103 - 104 cells/mL
Current: 6153 cells/mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2 °C

Nominal and measured concentrations:

nominal: 12.8 - 32.0 - 80.0 - 200 - 500 mg/L

Measured Concentrations and Percentage of the Nominal Concentration of the test item in Fresh Medium (0 hours) and Old Medium (72 hours)

Sampling 2015-10-20 2015-10-23
date 0 h, fresh medium 72 h, old medium

Nominal test item
concentration
[mg/L] Meas. conc. [%] Meas. conc. [%]
[mg/L] [mg/L]
500 518 104 494 99
200 198 99 195 98
80.0 77.0 96 77.7 97
32.0 32.8 103 32.6 102
12.8 12.2 95 12.7 99
Control < LOQ < LOQ

Meas. Conc = Measured concentration of the test item (dilution factor taken into account, two injections each)
% = Percent of the nominal concentration of the test item
LOQ = Limit of Quantification (1 mg/L test item)

Details on test conditions:

Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels. The flasks were positioned randomly and repositioned daily. Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm. Light intensity (target) was 60 - 120 µE · m-2 · s-1 with a light regime of 24 hours/day light.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

96.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (81.8 – 111)

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

153 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (141 – 163)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

293 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (273 – 317)

Results with reference substance (positive control):

Results of the latest test with reference substance:
ErC50 0.564 mg/L
95 % confidence interval 0.546 - 0.582
Valid Range (average ± 3 x SD): 0.797 ± 0.428

Reported statistics and error estimates:

EC10-, EC20- and EC,50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha =0.05.

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 293 (273 – 317) mg/L and 103 (89.2 – 120) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 32.0 mg/L. All effect level are based on nominal concentration which had been analytically vertified.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3 The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6153 cells/mL. A stock solution of 500 mg/L was prepared by direct addition of the test item to the dilution water. Five concentrations were tested in a geometrical series with a dilution factor of 2.5 (nominal): 12.8 - 32.0 - 80.0 - 200 - 500 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The concentrations of the test item were determined in the fresh media (0 h) and old media (72 h) of all tested concentration levels and the control via LC-MS/MS. The measured concentrations in the fresh media (0 h) were in the range of 95 to 104 % of the nominal values of all concentrations. The measured concentrations in the old media (72 h) were in the range of 97 to 102 % of the nominal values of all concentrations. Therefore, all effect values given are based on the nominal test item concentrations.

The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 293 (273 – 317) mg/L and 103 (89.2 – 120) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 32.0 mg/L. All effect level are based on nominal concentration which had been analytically vertified.

Description of key information

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3 The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of growth rate and yield over a period of 72 hours.
The study was conducted under static conditions with an initial cell density of 6153 cells/mL. A stock solution of 500 mg/L was prepared by direct addition of the test item to the dilution water. Five concentrations were tested in a geometrical series with a dilution factor of 2.5 (nominal): 12.8 - 32.0 - 80.0 - 200 - 500 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.
The concentrations of the test item were determined in the fresh media (0 h) and old media (72 h) of all tested concentration levels and the control via LC-MS/MS. The measured concentrations in the fresh media (0 h) were in the range of 95 to 104 % of the nominal values of all concentrations. The measured concentrations in the old media (72 h) were in the range of 97 to 102 % of the nominal values of all concentrations. Therefore, all effect values given are based on the nominal test item concentrations.

The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 293 (273 – 317) mg/L and 103 (89.2 – 120) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 32.0 mg/L. All effect level are based on nominal concentration which had been analytically vertified.

Key value for chemical safety assessment

EC50 for freshwater algae:

293 mg/L

EC10 or NOEC for freshwater algae:

32 mg/L

Additional information

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 293 (273 – 317) mg/L and 103 (89.2 – 120) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 32.0 mg/L.

All effect level are based on nominal concentration which had been analytically vertified.