Reaction products of propane-1,2-diamine with 2-[(2-methylphenoxy)methyl]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:

from 2018-01-25 to 2018-05-31

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

Remarks:

HPLC-MS

Details on sampling:

For determination of the test item concentrations, three parallel samples were taken from the test solutions and six parallel samples from the control. Formulation samples were diluted with mobile phase or directly measured and the control samples were directly injected.

Vehicle:

no

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: SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): 3 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.5 - 23.5 °C

pH:

8.07 - 9.15

Nominal and measured concentrations:

Nominal concentrations: 0, 0.05, 0.13, 0.3, 0.8 and 2 mg/L
Measured concentration: 0, 0.052, 0.126, 0.32, 0.77 and 2.06 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: glass, ~250 mL volume, 100 mL test medium
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 103.17 x 10^4 cells/mL
- No. of organisms per vessel: 0.1 mL algal biomass (10^7 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: 8053 lux

TEST CONCENTRATIONS
- Range finding study: yes, 3 range-finding tests were performed
- Test concentrations: Test 1: 0.01, 0.1, 1, 10, 50, 100 mg/L; Test 2: 0.005, 0.01, 0.1, 1, 5 mg/L; Test 3: 0.005, 0.01, 0.1, 1, 3 mg/L
- Results used to determine the conditions for the definitive study: At 3 mg/L in Test 3 a 100 % inhibition was observed. Therefore, the maximum test concentration for the definitive test was determined to be 2 mg/L.

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

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.237 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 0.120 - 0.350 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.732 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 0.522 - 1.101 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.077 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other: 95 % CI: 0.048 - 0.106 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.298 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other: 95 % CI: 0.236 - 0.379 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Results with reference substance (positive control):

The date of the last study with the reference item Potassium dichromate was: 12 – 15 September 2017. The following results were obtained:
The 72h ErC50: 0.82 mg/L, (95 % confidence limits: 0.57 – 1.25 mg/L)
The 72h EyC50: 0.51 mg/L, (95 % confidence limits: 0.45 – 0.57 mg/L)

Growth inhibition: The results of the statistical evaluation (Dunnett’s test (α = 0.05; 2-sided)) showed that the 0 - 72 h average specific growth rate and yield were statistically significantly different from the untreated control in the concentration range of 0.13 - 2 mg/L and not significantly different at the test concentration of 0.05 mg/L.

Shape of the algal cells: No morphological abnormalities were observed in the control and in the lowest concentration of 0.05 mg/L during the test. Some swollen cells were observed together with the normal cells in the concentrations of 0.13 and 0.3 mg/L. At the test concentration of 0.8 mg/L cells with pinches were noticed among normal and swollen cells, while at the highest test concentration of 2 mg/L only thin and swollen cells, furthermore, cells with pinches were found during the study.

Cell density in the control: The cell density in the control has increased from nominal N = 1E+4 cells/mL at the start of the test (0 hours) to N = 103.17E+4 cells/mL (mean value) after 72 hours in the in the control.

Table 1: Summary of Biological Endpoints

Endpoints (0-72 h)	Growth rate (µ) [mg/L]	Yield (y) [mg/L]
	Calculation based on measured concentrations
EC10	0.237	0.077
95 % conf. limits	0.120 – 0.350	0.048 – 0.106
EC20	0.349	0.122
95 % conf. limits	0.210 – 0.492	0.086 – 0.159
EC50	0.732	0.298
95 % conf. limits	0.522 – 1.101	0.236 – 0.379
NOEC	0.05	0.05
LOEC	0.13	0.13

 

Validity criteria fulfilled:

yes

Conclusions:

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata. The EC10 and EC50 based on growth rate were determined to be 0.237 mg/L and 0.732 mg/L, respectively. The NOEC was determined to be 0.05 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 under GLP. The study was conducted under static conditions with an initial cell density of 1E+4 cells/mL for a period of 72 hours. A concentration series of 0.05, 0.13, 0.3, 0.8 and 2 mg/L of the test item and control were tested. The measured concentrations of the test item at the start and at the end of the test were in the range of 92 - 106 % of the nominal concentration. The measured concentrations deviated not more than 20 % from the nominal concentration, therefore, the nominal concentration was used to determine exposure concentrations. All validity criteria were fulfilled, therefore, the test is considered to be valid. The EC50-values for inhibition of growth rate (ErC50) and for inhibition of yield (EyC50) were determined to be 0.732 mg/L and 0.298 mg/L, respectively. The ErC10 and EyC10 values were determined to be 0.237 mg/L and 0.077 mg/L, respectively. The NOEC was determined to be 0.05 mg/L. The obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata.

Description of key information

In a 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata. The EC10 and EC50 based on growth rate were determined to be 0.237 mg/L and 0.732 mg/L, respectively. The NOEC was determined to be 0.05 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.732 mg/L

EC10 or NOEC for freshwater algae:

0.237 mg/L

Additional information

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 under GLP. The study was conducted under static conditions with an initial cell density of 1E+4 cells/mL for a period of 72 hours. A concentration series of 0.05, 0.13, 0.3, 0.8 and 2 mg/L of the test item and control were tested. The measured concentrations of the test item at the start and at the end of the test were in the range of 92 - 106 % of the nominal concentration. The measured concentrations deviated not more than 20 % from the nominal concentration, therefore, the nominal concentration was used to determine exposure concentrations. All validity criteria were fulfilled, therefore, the test is considered to be valid. The EC50-values for inhibition of growth rate (ErC50) and for inhibition of yield (EyC50) were determined to be 0.732 mg/L and 0.298 mg/L, respectively. The ErC10 and EyC10 values were determined to be 0.237 mg/L and 0.077 mg/L, respectively. The NOEC was determined to be 0.05 mg/L. The obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata.