2-Propenenitrile, reaction products with 2,2,4(or 2,4,4)-trimethyl-1,6-hexanediamine

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:

from 2018-05-23 to 2018-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentrations, three replicate samples (1 mL per replicate) were taken from each test solution at the start and at the end of the test. From the control one replicate (1 mL per replicate) was analysed at the start and at the end of the test.

Vehicle:

no

Details on test solutions:

The test solutions were prepared by mechanical dispersion without using any solubilising agents. A stock solution was first prepared by dissolving an amount of 0.01 g test item in 1000 mL dilution water (OECD Medium) to obtain the nominal concentration of 10 mg/L. The test solutions of subsequent lower test concentrations were prepared by appropriate diluting of this stock solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): 4 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

in the flasks: 21.5 - 21.9 °C
in the climate chamber: 21.3 - 22.2 °C

pH:

7.88 - 8.87

Nominal and measured concentrations:

Nominal concentrations: 0.5, 1.1, 2.3, 4.8 and 10.0 mg/L
Measured concentrations: 0.7, 1.4, 2.9, 5.4 and 10.8 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: covered with air-permeable stoppers
- Material, size, headspace, fill volume: glass, ~250 mL volume, 100 mL test medium
- Initial cells density: 1E+4 cells/mL
- No. of organisms per vessel: 0.1 mL algal biomass (1E+7 algal cells per mL)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD Medium prepared according to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: 7901 lux, fluorescent lamps (range 400 - 700 nm)

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: 0.5 % inhibition at 1 mg/L and 100 % inhibition at 10 mg/L. Therefore, the EC50 lies between 1 and 10 mg/L.

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: microscope with counting chamber at 24, 48 and 72 hours

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.8 mg/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.:

3.4 mg/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.:

1.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Results with reference substance (positive control):

The date of the last study with the reference item Potassium dichromate was: 17 - 20 September 2018. The following results were obtained:
The 72h ErC50: 0.69 mg/L, (95 % confidence limits: 0.59 – 0.81 mg/L)
The 72h EyC50: 0.41 mg/L, (95 % confidence limits: 0.36 – 0.46 mg/L)

Preliminary test

After 72 hours of exposure a significant effects on the growth of the algae was observed. At concentration level 10 mg/L, 100 % inhibition was observed, while at 1 mg/L only 0.5 % of inhibition was observed. The concentrations for the main test were based on these results.

Table 1. Results from the preliminary test

Nominal concentrations [mg/L]	Control	0.1	1	10	100
Average cell number at 72 hours (x 10^4 cells/mL)	199.7	197.0	194.5	1.0	1.0
Growth Rates (µ) [0-72h]	0.0736	0.0734	0.0732	0.0000	0.0000
% Inhibition of µ [0-72h]	–	0.3	0.5	100.0	100.0
Yield (Y) [0-72h]	198.7	196.0	193.5	0.0	0.0
% Inhibition of Y [0-72h]	–	1.3	2.6	100.0	100.0

 

Main Test

Analytical results - The measured concentrations were in the range of 110 - 145 % of the nominal at the start and 106 - 145 % at the end of the exposure period. As the measured concentrations deviated more than 20 % from the nominal concentrations, the exposure concentrations were calculated as the geometric mean of the measured start and and concentrations.

Growth inhibition - Biological results are based on the measured geometric mean concentrations. The results of the statistical evaluation showed that the average specific growth rate and yield were statistically significantly different from the untreated control at the concentrations of 2.9, 5.4 and 10.8 mg/L and not significantly different at 0.7 and 1.4 mg/L.

Shape of the algal cells - No morphological abnormalities were observed in the control and in the two lowest concentrations of 0.7 and 1.4 mg/L during the test. In the concentration of 2.9 mg/L mostly thin cells and some swollen cells were observed. Determination of the shape of algal cells was not completely possible in the two highest concentrations of 5.4 and 10.8 mg/L as no or very few cells were found during cell counting.

Table 2. Biological results from the main test

Endpoints (0-72 h)	Growth rate (µ) [mg/L]	Yield (y) [mg/L]
	Calculation based on measured concentrations
EC10	2.5	1.8
95 % conf. limits	2.4 – 2.6	1.7 – 1.9
EC20	2.8	2.1
95 % conf. limits	2.7 – 2.9	2.0 – 2.2
EC50	3.4	2.6
95 % conf. limits	3.3 – 3.5	2.5 – 2.7
NOEC	1.4	1.4
LOEC	2.9	2.9

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EC10 and EC50 based on growth rate were determined to be 2.5 and 3.4 mg/L, respectively. The NOEC based on growth rate and yield was determined to be 1.4 mg/L.

Executive summary:

The effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata was determined according to OECD 201, Regulation (EC) No. 226/2016 Method C.3 and EPA OPPTS 850.5400 and under GLP. The study was conducted under static conditions with an initial cell density of 1E4 cells/mL for a period of 72 hours. A concentration series of 0.5, 1.1, 2.3, 4.8 and 10.0 mg/L and control was performed. The measured concentrations of the test item at the start and at the end of the test were in the range of 106 - 145 % of the nominal concentration. The measured concentrations deviated more than 20 % from the nominal concentrations, therefore, the measured geometric mean concentrations were determined used for the exposure concentrations - 0.7, 1.4, 2.9, 5.4 and 10.8 mg/L. The EC50-values for inhibition of growth rate (ErC50) and for inhibition of yield (EyC50) were determined to be 3.4 and 2.6 mg/L, respectively. The ErC10 and EyC10 values were determined to be 2.5 and 1.8 mg/L, respectively. The NOEC was determined to be 1.4 mg/L. The test item showed a significant toxic effect on the green unicellular algae.

Description of key information

The 72-h EC10 and EC50 based on growth rate were determined to be  2.5 and 3.4 mg/L, respectively. The NOEC based on growth rate and yield was determined to be 1.4 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.4 mg/L

EC10 or NOEC for freshwater algae:

2.5 mg/L

Additional information

The effect of the test item on the growth of an unicellular green algal species Raphidocelis subcapitata was determined according to OECD 201, Regulation (EC) No. 226/2016 Method C.3 and EPA OPPTS 850.5400 and under GLP. The study was conducted under static conditions with an initial cell density of 1E4 cells/mL for a period of 72 hours. A concentration series of 0.5, 1.1, 2.3, 4.8 and 10.0 mg/L and control was performed. The measured concentrations of the test item at the start and at the end of the test were in the range of 106 - 145 % of the nominal concentration. The measured concentrations deviated more than 20 % from the nominal concentrations, therefore, the measured geometric mean concentrations were determined used for the exposure concentrations - 0.7, 1.4, 2.9, 5.4 and 10.8 mg/L. The EC50-values for inhibition of growth rate (ErC50) and for inhibition of yield (EyC50) were determined to be 3.4 and 2.6 mg/L, respectively. The ErC10 and EyC10 values were determined to be 2.5 and 1.8 mg/L, respectively. The NOEC was determined to be 1.4 mg/L. The test item showed a significant toxic effect on the green unicellular algae.