Phosphoric acid, 2-ethylhexyl ester, sodium salt

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 2019-05-13 to 2019-07-18

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)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

LC/MS/MS

Details on sampling:

For determination of the test item concentration six replicate samples (3 mL per replicate) from the test concentration and six replicate samples (3 mL per replicate) from the control group were taken at the start and at the end of the test.

Vehicle:

no

Details on test solutions:

The test solution was prepared by mechanical dispersion without using any solubilizing agent. An amount of 0.0806 g test item was dissolved in 806 mL dilution water (OECD Medium) in order to obtain the concentration of 100 mg/L. Thereafter, this solution was placed into an ultrasonic bath for approximately 10 minutes.

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 (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Method of cultivation: on agar

ACCLIMATION
- Acclimation period: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21.4 - 23.3 °C

pH:

7.33 - 8.87

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentration: within ± 20 % of the nominal concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Material, size, fill volume: glass, 250 mL, fill volume 100 mL
- Aeration: none
- Initial cells density: 1E4 cells/mL
- Control end cells density: 65.8E4 cells/mL
- No. of vessels per concentration (replicates): 6
- 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: none
- Photoperiod: continuously illuminated with fluorescent lamps
- Light intensity and quality: 7024 lux

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

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: After 72-h exposure only 1.4 % inhibition was observed in the test item group. Therefore, a limit test was performed as the main test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Results with reference substance (positive control):

For the evaluation of the reliability of the applied test system and the experimental conditions Potassium dichromate is tested at least twice a year. The date of the latest study with the reference item Potassium dichromate was: 11 – 14 March 2019.
The 72-h ErC50: 0.83 mg/L, (95 % confidence limits: could not be calculated)
The 72-h EyC50: 0.57 mg/L, (95 % confidence limits: 0.45 – 0.72 mg/L)

Preliminary Range-Finding Test

Table 1: Results of the preliminary range-finding test

Nominal concentrations [mg/L]	Untreated  control	100.0
Average cell number at 72 hours (x 1E4 cells/mL)	97.67	92.00
Growth Rates (µ) [0 - 72 h]	0.0636	0.0627
% Inhibition of µ [0 - 72 h]	–	1.42
Yield (Y) [0 - 72 h]	96.67	91.00
% Inhibition of Y [0 - 72 h]	–	5.86

No toxic effect was observed during the preliminary test, therefore, only one test concentration (100 mg/L nominal) and one control group were tested in a limit test.

Analytical Results

The concentration of the test item was analytically determined at the start and at the end of the test. The mean of the measured concentrations was 95 % of the nominal at the start and 102 % of the nominal at the end of the test. The measured concentrations were within ± 20 % during the experiment. Therefore, the biological results are based on the nominal concentration.

Biological Results

A limit test was performed and no toxic effects were observed, therefore, statistical analysis was not necessary. The LOEC, NOEC and the ECx values were given directly from the raw data.

Average Specific Growth Rate

The 72-h NOEC based on growth rate was determined to be 100 mg/L, while the 72-h LOEC based on growth rate was determined to be > 100 mg/ L. The 72-h EC10, EC20 and EC50 values were determined to be > 100 mg/ L.

Table 2: Growth Rates (µ) and Percentage Inhibition of µ during the Test Period

Nominal Concentration [mg/L]	Growth Rates (µ) and % Inhibition of µ
	0 – 24 h		0 – 48 h		0 – 72 h
	µ	%	µ	%	µ	%
Control	0.0297	-	0.0579	-	0.0582	-
100	0.0345	-16.2	0.0568	1.9	0.0577	0.9

Remark: negative inhibition means growth stimulation

Yield

The 72-h NOEC based on yield was determined to be 100 mg/L, while the 72-h LOEC based on yield was determined to be > 100 mg/ L. The 72-h EC10, EC20 and EC50 values were determined to be > 100 mg/ L.

Table 3: Yield (Y) and Percentage Inhibition of Y during the Test Period

Nominal Concentration [mg/L]	Yield (y) and % Inhibition of y
	0 – 72 h
	y	%
Control	65.8	-
100	64.2	2.5

Morphological Changes of Algal Cells

No abnormal shape of algal cells was noticed in the control and at the test item concentration (100 mg/L nominal) during the study.

Validity criteria fulfilled:

yes

Conclusions:

The 72-h NOEC based on growth rate and yield was determined to be 100 mg/L, while the 72-h LOEC based on growth rate and yield was determined to be > 100 mg/L. The 72-h EC10, EC20 and EC50 values were determined to be > 100 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 and Regulation (EC) No. 226/2016 Method C.3 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 limit test with one test concentration of 100 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 95 - 102 % of the nominal concentration. The measured concentrations deviated not more than ± 20 % from the nominal concentration, therefore, the nominal concentration was used to determine the biological results. The EC50-values for inhibition of growth rate (ErC50) and for inhibition of yield (EyC50) were >100 mg/L. The ErC10 and EyC10 values were determined to be >100 mg/L. The NOEC was determined to be 100 mg/L.

Description of key information

The 72-h NOEC based on growth rate and yield was determined to be 100 mg/L, while the 72-h LOEC based on growth rate and yield was determined to be > 100 mg/L. The 72-h EC10, EC20 and EC50 values were determined to be > 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 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 and Regulation (EC) No. 226/2016 Method C.3 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 limit test with one test concentration of 100 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 95 - 102 % of the nominal concentration. The measured concentrations deviated not more than ± 20 % from the nominal concentration, therefore, the nominal concentration was used to determine the biological results. The EC50-values for inhibition of growth rate (ErC50) and for inhibition of yield (EyC50) were >100 mg/L. The ErC10 and EyC10 values were determined to be >100 mg/L. The NOEC was determined to be 100 mg/L.