Ammonium 2-ethylhexyl sulphate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-09-24 to 2019-10-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA Guideline 712-C-16-013: OCSPP 850.1010, “Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids”

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 19.8, 29.6, 44.4, 66.7 and 100 mg/L
- Sampling method: For determination of the test item concentrations, samples were taken from each test concentration and the control at the start and at the end of the tests. Four replicate samples were analysed (5 mL per replicate in Experiment I and 3 mL per replicate in Experiment II) in each case.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test solutions used in the test were prepared by mechanical dispersion without using any solubilising agent.

Experiment I
An amount of 0.0302 g test item was dissolved in 302 mL dilution water (ISO Medium) resulting a nominal concentration of 100 mg/L. After the formulation procedure test animals were immediately introduced into the test solution or control (start of Experiment I).

Experiment II
A stock solution was first prepared by dissolving an amount of 0.0804 g test item in 804 mL dilution water (ISO Medium) resulting a nominal concentration of 100 mg/L. Test solutions of subsequent lower concentrations were prepared by appropriate dilution of this stock solution. (see Table 4). After the formulation procedure test animals were immediately introduced into the test solutions or control (start of Experiment II).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Straus
- Age at study initiation: Less than 24 h old at the beginning of the test.
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Feeding during test : No
- Food type: The Daphnia were fed with concentrated algal suspension of Raphidocelis subcapitata during the holding.

ACCLIMATION
Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

249 mg/L (as CaCO3)

Test temperature:

21.1 – 21.4 °C (in the test vessels)
20.3 – 21.8 °C (in the climate chamber)

pH:

7.78 – 7.96

Dissolved oxygen:

7.96 – 8.26 mg/L

Nominal and measured concentrations:

Nominal concentrations: 19.8, 29.6, 44.4, 66.7 and 100 mg/L
Measured cncentrations: within ± 20 % of nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: Beaker
- Material, size, fill volume: glass, volume app. 50 mL, containing app. 40 mL of test solution
- Volume of solution: Each test vessel contained app. 40 mL of test solution
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to OECD guideline 202

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light (artificial illumination) and 8 hours darkness.
- Light intensity: Light intensity was determined at the start of the experiments as 748 lux (at Exp. I) and 712 lux (at Exp. II).

EFFECT PARAMETERS MEASURED:
The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile. In addition to immobility any abnormal behaviour or appearance is reported.

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No toxic effects were observed during the preliminary test, therefore, a single concentration of 100 mg/L and a concurrent control were tested in a limit test (Experiment I). As toxicity (30 % immobilization) was observed in the limit test at 100 mg/L, a complementary experiment (Experiment II) was carried out in a full test using a range of concentration. The following nominal concentrations were tested in Experiment II: 19.8, 29.6, 44.4, 66.7 and 100 mg/L; concurrent control ran.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested at least twice a year

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 108 calculated; 95% conf. limits: 72.6-404.1 mg/L

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

15.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. limits: 1.4-27.8 mg/L

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

30.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf- limits: 8.3-44.8 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

29.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

44.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: None
- Immobilisation of control: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes

The date of the last study with reference item Potassium dichromate was: 24 – 25 September 2019.
The 24-h EC50: 1.34 mg/L, (95 % confidence limits: 1.16 – 1.65 mg/L).

Reported statistics and error estimates:

For the determination of the LOEC and NOEC, ANOVA and Dunnett-t test (α = 0.05, 2-sided), for determination of the ECx values Probit analysis was used using SPSS software.

Analytical Results
The measured concentrations of the test item were in the range of 98 – 101 % of the nominal at the start and 100 – 113 % at the end of the tests (i.e. within ± 20 % during the experiment) therefore the biological results are based on the nominal concentrations.

Table 1: Test item concentration measured at the start of the experiments

Nominal concentration mg/L	Mean of the measured concentrations mg/L	Measured concentration in % of the nominal
100 (Experiment I)	98.1 (RSD: 0.6 %)	98
Control (Experiment I)	below the quantitation limit	-
19.8	20.0 (RSD: 2.4 %)	101
29.6	29.6 (RSD: 0.2 %)	100
44.4	44.1 (RSD: 0.7 %)	99
66.7	67.5 (RSD: 1.3 %)	101
100 (Experiment II)	98.8 (RSD: 0.4 %)	99
Control (Experiment II)	not detected	-

Table 2: Test item concentration measured at the end of the experiments

Nominal concentration mg/L	Mean of the measured concentrations mg/L	Measured concentration in % of the nominal
100 (Experiment I)	100.4 (RSD: 2.4 %)	100
Control (Experiment I)	below the quantitation limit	-
19.8	19.8 (RSD: 3.0 %)	100
29.6	31.6 (RSD: 2.8 %)	107
44.4	45.5 (RSD: 3.2 %)	102
66.7	75.5 (RSD: 1.4 %)	113
100 (Experiment II)	109.8 (RSD: 6.0 %)	110
Control (Experiment II)	below the quantitation limit	-

Biological Results
The results of the statistical evaluation showed that the 48-h immobilisation was statistically significantly different from the untreated control value in the test concentrations of 44.4, 66.7 and 100 mg/L and not significantly different in the concentrations of 19.8 and 29.6 mg/L. Accordingly the 48-h NOEC was determined to be 29.6 mg/L.
After 48-h exposure the observed immobility in the highest concentration of 100 mg/L (35 % including the results of both Experiment I and II; 30 % and 40 % in Exp. I and II respectively) was less than in the lower concentrations of 44.4 and 66.7 mg/L (40 % and 65 % immobility respectively). This high variability of immobilisation observed in the three highest concentrations (40 %, 65 % and 35 % immobility) which showed no concentration response pattern can be considered to be due to the flat concentration response curve of the test item and the low factor of 1.5 between test concentrations.
The 48-h-EC50 was determined to be > 100 mg/L (108.0 calculated) (95 % conf. limits: 72.6 – 404.1 mg/L).

Validity of the Study
No immobilisation was observed in the control group (≤ 10 %) and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L in all cases. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Validity criteria fulfilled:

yes

Conclusions:

In this 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item Ammonium 2-ethylhexyl sulfate had toxic effects on the mobility of Daphnia.
The 48-h EC50 value was determined to be > 100 mg/L (108.0 calculated) (95 % conf. limits: 72.6 – 404.1).
The 48-h NOEC was determined to be 29.6 mg/L.
All validity criteria were met. The results are based on nominal test item concentrations.

Executive summary:

The influence of the test item on the mobility, respectively survival of Daphnia magna was evaluated in an Immobilisation Test according to OECD guideline 202.

Young Daphnia were exposed for 48 hours to the limit concentration of 100 mg/L (Experiment I). As toxicity was observed in the limit test at 100 mg/L, a complementary experiment (Experiment II) was carried out in a full test. In the full test (Experiment II) daphnids were exposed to aqueous test item concentrations of 19.8, 29.6, 44.4, 66.7 and 100 mg/L for 48 hours. A control containing test medium (ISO medium) without test item was also included. The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure.
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 tests. Concentrations were determined using HPLC-UV method. The measured concentrations of the test item were in the range of 98 – 101 % of the nominal at the start and 100 – 113 % at the end of the tests (i.e. within ± 20 % during the experiment) therefore the biological results are based on the nominal concentrations. 

The results of the statistical evaluation showed that the 48-h immobilisation was statistically significantly different from the untreated control value in the test concentrations of 44.4, 66.7 and 100 mg/L and not significantly different in the concentrations of 19.8 and 29.6 mg/L. Accordingly the 48-h NOEC was determined to be 29.6 mg/L. After 48-h exposure the observed immobility in the highest concentration of 100 mg/L (35 % including the results of both Experiment I and II; 30 % and 40 % in Exp. I and II respectively) was less than in the lower concentrations of 44.4 and 66.7 mg/L (40 % and 65 % immobility respectively). This high variability of immobilisation observed in the three highest concentrations (40 %, 65 % and 35 % immobility) which showed no concentration response pattern can be considered to be due to the flat concentration response curve of the test item and the low factor of 1.5 between test concentrations. The 48-h-EC50 was determined to be >100 mg/L (108.0 calculated) (95 % conf. limits: 72.6 – 404.1 mg/L). All validity criteria were met.

Description of key information

In this 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item Ammonium 2-ethylhexyl sulfate had toxic effects on the mobility of Daphnia.

The 48-h EC50 value was determined to be > 100 mg/L (108.0 calculated) (95 % conf. limits: 72.6 – 404.1).

The 48-h NOEC was determined to be 29.6 mg/L.

All validity criteria were met. The results are based on nominal test item concentrations.

Additional information

The influence of the test item on the mobility, respectively survival of Daphnia magna was evaluated in an Immobilisation Test according to OECD guideline 202.

Young Daphnia were exposed for 48 hours to the limit concentration of 100 mg/L (Experiment I). As toxicity was observed in the limit test at 100 mg/L, a complementary experiment (Experiment II) was carried out in a full test. In the full test (Experiment II) daphnids were exposed to aqueous test item concentrations of 19.8, 29.6, 44.4, 66.7 and 100 mg/L for 48 hours. A control containing test medium (ISO medium) without test item was also included. The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure.
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 tests. Concentrations were determined using HPLC-UV method. The measured concentrations of the test item were in the range of 98 – 101 % of the nominal at the start and 100 – 113 % at the end of the tests (i.e. within ± 20 % during the experiment) therefore the biological results are based on the nominal concentrations. 

The results of the statistical evaluation showed that the 48-h immobilisation was statistically significantly different from the untreated control value in the test concentrations of 44.4, 66.7 and 100 mg/L and not significantly different in the concentrations of 19.8 and 29.6 mg/L. Accordingly the 48-h NOEC was determined to be 29.6 mg/L. After 48-h exposure the observed immobility in the highest concentration of 100 mg/L (35 % including the results of both Experiment I and II; 30 % and 40 % in Exp. I and II respectively) was less than in the lower concentrations of 44.4 and 66.7 mg/L (40 % and 65 % immobility respectively). This high variability of immobilisation observed in the three highest concentrations (40 %, 65 % and 35 % immobility) which showed no concentration response pattern can be considered to be due to the flat concentration response curve of the test item and the low factor of 1.5 between test concentrations. The 48-h-EC50 was determined to be >100 mg/L (108.0 calculated) (95 % conf. limits: 72.6 – 404.1 mg/L). All validity criteria were met.