Ammonium 2-ethylhexyl sulphate

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:

2019-09-24 to 2019-09-27

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)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
- Sampling method: For determination of the test item concentrations three replicate samples (5 mL per replicate) were taken from each test concentration and six replicates (5 mL per replicate) from the control group at the start and at the end of the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test item solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent. A stock solution was first prepared by dissolving an amount of 0.0808 g test item in 808 mL dilution water (OECD Medium, see section 5.4) resulting a nominal concentration of 100 mg/L. Test solutions of subsequent lower concentrations were prepared by appropriate dilution of this stock solution. After the formulation procedure the algal cells were immediately introduced into the test solution (start of experiment).

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: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany
- Age of inoculum: 4 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.6 - 23.4 °C (in the flask);
21.9 - 23.8 °C (in climate chamber)

pH:

7.63-9-16

Nominal and measured concentrations:

Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
Measured concentrations: within ± 20 % of the nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed, covered with air-permeable stoppers
- Material, size, fill volume: glass, ~250 mL volume, 100 mL test medium
- Initial cells density: 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: Continuous illumunation
- Light intensity and quality: 4684 lux, fluorescent lamps (spectral range of 400-700 nm)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Other: The cell morphology was examined in parallel.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 1st Preliminary Range-Finding Test: 0.1, 1, 10 and 100 mg/L; 2nd Preliminary Range-Finding Test: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: See results in the section "Any other information on results incl. tables".

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

57.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 34.6 - 137.6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. limits: 0.8 - 12.9

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

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: 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

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

16.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Results with reference substance (positive control):

- Results with reference substance valid? Yes

The date of the last study with the reference item Potassium dichromate was: 23 – 26 September 2019. The following results were obtained:
The 72h ErC50: 0.80 mg/L, (95 % confidence limits: 0.49 – 1.49 mg/L)
The 72h EyC50: 0.50 mg/L, (95 % confidence limits: 0.39 – 0.63 mg/L)

Reported statistics and error estimates:

Mean values and standard deviations were calculated for each concentration at each observation period using Microsoft Excel for Windows software.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values using analysis of variance (ANOVA) and Dunnett-t test (α= 0.05, 2-sided) by SPSS software.
For determination of the ECx values Probit analysis by SPSS software was performed.

Table 1: Results of the 1st preliminary range-finding test

Nominal concentrations [mg/L]	Untreated control	0.1	1	10	100
Average cell number at 72 hours (x 10^4cells/mL)	247.0	242.5	257.0	208.5	185.0
Growth Rates(µ) [0-72h]	0.0764	0.0760	0.0771	0.0741	0.0725
% Inhibition ofµ [0-72h]	–	0.5	-0.9	3.0	5.2
Yield(Y) [0-72h]	246.0	241.5	256.0	207.5	184.0
% Inhibition ofY [0-72h]	–	1.8	-4.1	15.7	25.2

Table 2: Results of the 2nd preliminary range-finding test

Nominal concentrations [mg/L]	Untreated control	1	10	100
Average cell number at 72 hours (x 10^4cells/mL)	71.0	85.5	81.0	30.0
Growth Rates(µ) [0-72h]	0.0592	0.0617	0.0610	0.0472
% Inhibition ofµ [0-72h]	–	-4.2	-3.1	20.2
Yield(Y) [0-72h]	70.0	84.5	80.0	29.0
% Inhibition ofY [0-72h]	–	-20.7	-14.3	58.6

Main test

Validity of the Test
- The cell density in the control cultures increased by a factor of 149.17 within 72 hours. This corresponds to a specific growth rate of 1.67 day-1.
- The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %
    - CV for section-by-section growth rate day 0-1: 8.03 %
    - CV for section-by-section growth rate day 1-2: 15.03 %
    - CV for section-by-section growth rate day 2-3: 9.79 %
The mean coefficient of variation for section-by-section specific growth rates: 10.95 %.
- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.58 %.

All validity criteria were met, therefore the study can be considered as valid.

Analytical results:

Five different concentrations arranged in a geometric series with a spacing factor of 2 and a concurrent control were tested in the main experiment. The nominal test item concentrations were6.25, 12.5, 25, 50 and 100 mg/L. The measured concentrations of the test item varied between 84 and 99 % of the nominal concentrations (i.e. within ± 20 %) during the experiment, therefore, the biological results are based on the nominal concentrations. In the untreated control group the test item was below the quantitation limit.

Table 2: Test Itemconcentration measured at the start (24 September 2019)

Nominal concentration mg/L	Mean of the measured concentrations mg/L	Measured concentration in % of the nominal
6.25	5.81 (RSD: 1.9 %)	93
12.5	11.7 (RSD: 0.9 %)	94
25	23.7 (RSD: 0.3 %)	95
50	48.9 (RSD: 0.5 %)	98
100	96.3 (RSD: 0.4 %)	96
Control	below the quantitation limit	-

Table 3: Test Itemconcentration measured at the end (27 September 2019)

Nominal concentration mg/L	Mean of the measured concentrations mg/L	Measured concentration in % of the nominal
6.25	5.25 (RSD: 0.2 %)	84
12.5	11.5 (RSD: 1.1 %)	92
25	24.5 (RSD: 1.0 %)	98
50	49.7 (RSD: 1.4 %)	99
100	98.0 (RSD: 1.5 %)	98
Control	below the quantitation limit	-

Average Specific Growth Rate
The results of the statistical evaluation showed that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value in the concentrations of 25, 50 and 100 mg/L and not significantly different in the concentrations of 6.25 and 12.5 mg/L.
Accordingly, the 72-h NOEC based on growth rate was determined to be 12.5 mg/L and the LOEC was determined to be 25 mg/L.
The 72-h EC10, EC20, EC50 values for growth rate were determined to be 57.1, > 100 and > 100 mg/L, respectively.

Yield
The results of the statistical evaluation showed that the 0-72 h yield was statistically significantly different from the untreated control value in the concentrations of 25, 50 and 100 mg/L and not significantly different in the concentrations of 6.25 and 12.5 mg/L.
Accordingly the 72-h NOEC based on yield was determined to be 12.5 mg/L and the LOEC was determined to be 25 mg/L.
The 72-h EC10, EC20, EC50 for yield were determined to be 6.4, 16.8 and > 100 mg/L, respectively.

Morphological Changes of Algal Cells
No abnormal shape of algal cells was noticed in the control and in the lowest concentration of 6.25 mg/L during the test.
In the concentrations of 12.5, 25, 50 and 100 mg/L swollen cells were observed together with normal cells during the test.

Validity criteria fulfilled:

yes

Conclusions:

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item Ammonium 2-ethylhexyl sulfate had inhibitory effects on the growth of Raphidocelis subcapitata. The 72-h EC10, EC20, EC50 for growth rate were determined to be 57.1, > 100 and > 100 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 6.4, 16.8 and > 100 mg/L, respectively. The 72-h NOEC value based on growth rate and yield was determined to be 12.5 mg/L. All validity criteria were met. The results are based on nominal test item concentrations.

Executive summary:

The effect of the test item on the growth of a unicellular green algal species, Raphidocelis subcapitata, was determined in a Growth Inhibition Test according to OECD guideline 201. Exponentially growing cultures of Raphidocelis subcapitata were exposed to nominal test item concentrations of 6.25, 12.5, 25, 50 and 100 mg/L under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. For determination of the test item concentrations, samples were taken from each concentration level and control at the start and at the end of the test. Concentrations were determined using HPLC method with UV detection. The analytically measured test item concentrations remained within ± 20 % of the nominal over the test period of 72 hours, therefore, the biological results are based on the nominal concentrations.
No abnormal shape of algal cells was noticed in the control and in the lowest concentration of 6.25 mg/L during the test. In the concentrations of 12.5, 25, 50 and 100 mg/L swollen cells were observed together with normal cells during the test. The results of the statistical evaluation showed that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value in the concentrations of 25, 50 and 100 mg/L and not significantly different in the concentrations of 6.25 and 12.5 mg/L.
In this 72-h algal growth inhibition test , the obtained results showed that the test item Ammonium 2-ethylhexyl sulfate had inhibitory effects on the growth of Raphidocelis subcapitata. The 72-h EC10, EC20, EC50 for growth rate were determined to be 57.1, > 100 and > 100 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 6.4, 16.8 and > 100 mg/L, respectively. The 72-h NOEC value based on growth rate and yield was determined to be 12.5 mg/L. All validity criteria were met. The results are based on nominal test item concentrations.

Description of key information

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item Ammonium 2-ethylhexyl sulfate had inhibitory effects on the growth of Raphidocelis subcapitata. The 72-h EC10, EC20, EC50 for growth rate were determined to be 57.1, > 100 and > 100 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 6.4, 16.8 and > 100 mg/L, respectively. The 72-h NOEC value based on growth rate and yield was determined to be 12.5 mg/L. All validity criteria were met. The results are based on nominal test item concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

57.1 mg/L

Additional information

The effect of the test item on the growth of a unicellular green algal species, Raphidocelis subcapitata, was determined in a Growth Inhibition Test according to OECD guideline 201. Exponentially growing cultures of Raphidocelis subcapitata were exposed to nominal test item concentrations of 6.25, 12.5, 25, 50 and 100 mg/L under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. For determination of the test item concentrations, samples were taken from each concentration level and control at the start and at the end of the test. Concentrations were determined using HPLC method with UV detection. The analytically measured test item concentrations remained within ± 20 % of the nominal over the test period of 72 hours, therefore, the biological results are based on the nominal concentrations.
No abnormal shape of algal cells was noticed in the control and in the lowest concentration of 6.25 mg/L during the test. In the concentrations of 12.5, 25, 50 and 100 mg/L swollen cells were observed together with normal cells during the test. The results of the statistical evaluation showed that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value in the concentrations of 25, 50 and 100 mg/L and not significantly different in the concentrations of 6.25 and 12.5 mg/L.
In this 72-h algal growth inhibition test , the obtained results showed that the test item Ammonium 2-ethylhexyl sulfate had inhibitory effects on the growth of Raphidocelis subcapitata. The 72-h EC10, EC20, EC50 for growth rate were determined to be 57.1, > 100 and > 100 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 6.4, 16.8 and > 100 mg/L, respectively. The 72-h NOEC value based on growth rate and yield was determined to be 12.5 mg/L. All validity criteria were met. The results are based on nominal test item concentrations.