Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

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:

from 2017-12-11 to 2018-02-14

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from each test concentrations and from the control.
- Sampling method: Samples were taken at the start (0 hours) and at the end of exposure (48 hours) and analysed using a HPLC- MS method. Four parallel samples were taken from the test solutions and from the control. Formulation samples were diluted with mobile phase A and analysed by an HPLC method with MS detection. Control samples were directly injected.
- Sample storage conditions before analysis: The samples were kept in containers tightly closed in a dry, room temperature and well-ventilated place until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent. For preparation of test solutions a stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.05 g test item in 500 mL ISO medium using approx. 1 hour shaking to obtain clear solution. The test solutions of subsequent concentrations were prepared by appropriate dilution of this stock solution.
- Controls: Untreated control was run parallel in the test. A positive control with the reference substance potassium dichromate was also performed.
- Evidence of undissolved materia: No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea.
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Age at the test start: < 24 hours old
- Feeding during test: No. The test animals were not fed during the exposure.
- Breeding conditions: Daphnia were bred in the Laboratory of TOXI-COOP ZRT. under similar temperature and light conditions as in the test, and in reconstituted water of a similar quality regarding to pH, components of the main ions and total hardness as the test water used in the test. The animals were fed with centrifuged green alga suspension.

ACCLIMATION
Test animals were bred under similar conditions as that used during the exposure period, therefore additional acclimatisation before the test was not necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

Not performed

Hardness:

The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).

Test temperature:

The test temperature was in the range of 20.4 – 20.8 °C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 20.0 – 20.9 °C.

pH:

The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was in the range of 7.74 – 8.11 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 6.98 – 8.20 mg/L during the test.

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

- Nominal concentration: The following nominal concentrations were tested: 6.25, 12.5, 25, 50 and 100 mg/L.
- Measured concentrations: Measured concentrations of the components of the test substance ((Phosphoric acid mono(2-ethylhexyl) ester, Pyrophosphoric acid di(2-ethylhexyl) ester and Phosphoric acid bis(2-ethylhexyl) ester)) were in the range of 82 – 103 % of the nominal at the start and 60 – 121 % at the end of the test. The corresponding calculated geometric mean test item concentrations were: 5.3, 12.0, 25.5, 49.5 and 94.8 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers
- Fill volume: 40 mL
- Aeration: No
- 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: ISO medium (according to OECD 202) was used as dilution water in the experiment.
- Culture medium different from test medium: No
- Intervals of water quality measurement: During the test, the test vessels were kept in a climate chamber under controlled conditions. The water temperature, dissolved oxygen concentration and pH were measured in each test group at the start (before test solutions distributed into test vessels) and in each test vessel (i.e. each replicate) at the end of the test. Additionally, the ambient temperature was measured continuously using a min/max thermometer in the climate chamber.

OTHER TEST CONDITIONS
- Adjustment of pH: No. The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test.
- Photoperiod: The light-dark cycle during the test was 16 hours light (artificial illumination) and 8 hours darkness.
- Light intensity: Light intensity was measured at the start of the experiment and determined as 621 lux.

EFFECT PARAMETERS MEASURED: The purpose of this study was to evaluate the influence of the test item on the mobility respectively survival of the test organisms after 24 and 48 hours.

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY
- Test concentrations: A non-GLP preliminary test was performed at the nominal concentations of 100, 10, 1 and 0.1 mg/L
- Results used to determine the conditions for the definitive study: The EC50 in the preliminary test was determined to be between 10 and 100 mg/L.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % conf. limits: 6.5 – 9.4 mg/L

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: Not observed
- Other biological observations: Not perfomed
- Mortality of control: There was no mortality of the test organisms in the control.
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations that might cause a difference between measured and nominal values: Yes. Measured concentrations of the components of the test substance ((Phosphoric acid mono(2-ethylhexyl) ester, Pyrophosphoric acid di(2-ethylhexyl) ester and Phosphoric acid bis(2-ethylhexyl) ester)) were in the range of 82 – 103 % of the nominal at the start and 60 – 121 % at the end of the test (i.e. more than ± 20 % deviation) therefore the exposure concentrations were calculated as the geometric mean of the concentrations (based on the mean value of the three components) measured at the start and end of the test.
- Effect concentrations exceeding solubility of substance in test medium: No
- Toxicity results: The 48-h EC50 value was determined to be 7.7 mg/L, the 48-h NOEC was determined to be 5.3 mg/L, the 48-h EC10 was determined to be 5.6 mg/L and the 48-h LOEC was determined to be 12.0 mg/L.

Results with reference substance (positive control):

The 24h EC50 of the reference substance potassium dichromate was determined to be 1.53 mg/L, (95 % confidence limits: 1.30 – 1.78 mg/L)

Reported statistics and error estimates:

- The 48-h ECx values were calculated by Probit analysis using SPSS software.
- The NOEC, LOEC values were determined directly from the raw data.
- Biological results are based on the mean measured test item concentrations.

Validity criteria fulfilled:

yes

Conclusions:

In a static immobilization GLP test Daphnia magna (< 24 h old) were exposed to the test item for 48 hours according to the OECD 202 (2004). The 48h-EC50 was determined to be 7.7 mg/L.

Executive summary:

In a static immobilization GLP test Daphnia magna (< 24 h old) were exposed to the test item for 48 hours according to Comission Regulation (EC) No 440/2008 Part C, Method 2 and the OECD 202 (2004). The Daphnia were exposed to a nominally range of concentrations of 0 (Control), 6.25, 12.5, 25.0, 50.0 and 100 mg/L. The test solutions used in the test were prepared by mechanical dispersion. An appropriate amount of test item was dissolved in the dilution water (ISO medium) in order to obtain the concentration of 100 mg/L. The further test solutions were prepared by appropriate dilution of this stock solution. The components of the test substance (Phosphoric acid mono(2-ethylhexyl) ester, Pyrophosphoric acid di(2-ethylhexyl) ester and Phosphoric acid bis(2-ethylhexyl) ester) were used for the quantitation of the Test Item. Samples were taken from the test concentrations and the control at the start and at the end of the experiment and analyzed by HPLC method with MS detection. The corresponding measured geometric mean test item concentrations were: 5.3, 12.0, 25.5, 49.5 and 94.8 mg/L. Measured concentrations of these components were in the range of 82 – 103 % of the nominal at the start and 60 – 121 % at the end of the test. As the analytically measured concentrations remained not within ± 20 % of the nominal concentration, biological results and endpoints are based on the of the measured geometric mean test item concentrations. An untreated control (ISO-Medium without addition of the test item) and a toxic reference control with potassium dichromate were performed. The 24h-EC50 of potassium dichromate was determined to be 1.53 mg/L. The validity criteria were fulfilled. The 48h-EC50 of the test substance was determined to be 7.7 mg/L.

Description of key information

In a static immobilization GLP test Daphnia magna (< 24 h old) were exposed to the test item for 48 hours according to the OECD 202 (2004). The 48h-EC50 was determined to be 7.7 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

7.7 mg/L

Additional information

In a static immobilization GLP test Daphnia magna (< 24 h old) were exposed to the test item for 48 hours according to Comission Regulation (EC) No 440/2008 Part C, Method 2 and the OECD 202 (2004). The Daphnia were exposed to a nominally range of concentrations of 0 (Control), 6.25, 12.5, 25.0, 50.0 and 100 mg/L. The test solutions used in the test were prepared by mechanical dispersion. An appropriate amount of test item was dissolved in the dilution water (ISO medium) in order to obtain the concentration of 100 mg/L. The further test solutions were prepared by appropriate dilution of this stock solution. The components of the test substance (Phosphoric acid mono(2-ethylhexyl) ester, Pyrophosphoric acid di(2-ethylhexyl) ester and Phosphoric acid bis(2-ethylhexyl) ester) were used for the quantitation of the Test Item. Samples were taken from the test concentrations and the control at the start and at the end of the experiment and analyzed by HPLC method with MS detection. The corresponding measured geometric mean test item concentrations were: 5.3, 12.0, 25.5, 49.5 and 94.8 mg/L. Measured concentrations of these components were in the range of 82 – 103 % of the nominal at the start and 60 – 121 % at the end of the test. As the analytically measured concentrations remained not within ± 20 % of the nominal concentration, biological results and endpoints are based on the of the measured geometric mean test item concentrations. An untreated control (ISO-Medium without addition of the test item) and a toxic reference control with potassium dichromate were performed. The 24h-EC50 of potassium dichromate was determined to be 1.53 mg/L. The validity criteria were fulfilled. The 48h-EC50 of the test substance was determined to be 7.7 mg/L.