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 fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

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 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: For determination of the test item concentrations, five replicate samples (5 mL/replicate) were taken from each test concentration and from the control.
- Sampling method: The samples were were taken from each test concentration and from the control at the start and at the end of each water renewal period. Five parallel samples were taken from the test solutions and from the control. The 50.0 and 31.3 mg/L nominal concentrations were analysed only at the start and end of the first renewal period as all fish died during the first two days of the test. 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. After the formulation procedure the test animals were immediately introduced into the test solution. For preparation of test solutions (at each renewal period) a stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.25 g test item in 2500 mL ISO medium using approx. 1 hour shaking to obtain clear solution. The test solutions of chosen test concentrations were prepared by appropriate dilution of this stock solution
- Controls:Untretated blank control was performed with the test solution without the test item.
- Evidence of undissolved material: No

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Akvárium magazin Kft. (Pasaréti Gyula) 1222 Budapest, Dévény u. 36, Hungary
- Age at study initiation: Juveniles were used
- Sex: Both female and male (were not separated).
- Length at study initiation: The body length of each fish used was within the range of 2 ± 1 cm.
- Weight at study initiation: 0.69 - 0.94 g

ACCLIMATION
- Acclimation period: Fish were held for at least 12 days before test initiation in the fish laboratory of TOXI-COOP ZRT.
- Acclimation conditions: Same as the test. Fish were held under the same conditions as used during the exposure period.
- Type and amount of food during acclimation: During holding, fish were fed with appropriate, commercial diet for fish.
- Feeding frequency during acclimation: At least three times per week until one day before the test start.
- Health during acclimation (any mortality observed): The health of the breeding was continuously monitored and any mortality or abnormal behaviour recorded. No significant mortality (less than 5 % of population) occurred in seven days before the start of the experiment, therefore the batch was considered to be acceptable for testing.

FEEDING DURING TEST
The fish were not fed during the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

None

Post exposure observation period:

Not performed

Hardness:

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

Test temperature:

The measured temperatures were within a range of 21.4 – 22.3 °C in the test aquariums and were constant (the maximum deviation did not exceed ± 2 °C).

pH:

The test was carried out without adjustment of pH. The measured pH values were in the range of 7.25 – 7.80 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 61.7 – 99.1 % of the air saturation value at the temperature used. Test solutions were not aerated during the test.

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

- Nominal concentration: 0 (control), 7.6, 12.2, 19.5, 31.3 and 50.0 mg/L
- Measured concentration: 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 77 – 111 % of the nominal at the start and 52 – 120 % at the end of the water renewal periods. The measured concentrations deviated more than 20 % from the nominal during the experiment therefore the geometric mean of the concentrations measured at the start and end of the water renewal periods were calculated to determine exposure concentrations. The corresponding calculated geometric mean test item concentrations were: 6.0, 11.6, 19.7, 32.0 and 53.2 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquariums
- Type : open
- Fill volume: 2 litre
- Aeration: Yes
- Renewal rate of test solution (frequency/flow rate): semi-static 48 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: The loading of the test aquariums was also calculated and were in the range of 0.35 – 0.47 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: For preparation of test water, separate stock solutions of individual substances are first prepared in deionised water. The ISO medium is prepared by adding 25 mL from each of four stock solutions to one litre deionised water.
- Culture medium different from test medium: No. Fish were held prior to the study in reconstituted water (ISO medium, prepared according to recommendation of Annex 2 of the OECD 203 guideline). The test was performed using the same water.
- Intervals of water quality measurement: The water temperature, pH value and dissolved oxygen concentration were determined at the start of the test (i.e. introduction of the fish) and on the subsequent days (once daily) in each test group and the control. Test conditions were determined from fresh and old solution at water renewal.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light: 8 h dark

EFFECT PARAMETERS MEASURED: The purpose of this study was to evaluate the acute toxicity of the test item on Zebrafish (Danio rerio). The recorded effects were mortality and symptoms of intoxication after 24, 48 and 96 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.6
- Range finding study
In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item.
- Test concentrations: 50, 10 and 1 mg/L
- Results used to determine the conditions for the definitive study: After 96 hours the EC100 was determined to be 50 mg/L and the NOEC was determined to be 10 mg/L.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

23.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95 % conf. limit: 17.9 – 32.6 mg/L)

Details on results:

- Behavioural abnormalities: No signs of abnormalities were observed in the control group and in the two lowest concentrations (6.0 and 11.6 mg/L measured) during the whole test period.
- Observations on body length and weight: None
- Other biological observations: None
- Mortality of control: There was no mortality observed in the control group and in the two lowest concentrations (6.0 and 11.6 mg/L measured) during the 96-h test period.
- Mortality: A single fish died in the concentration of 19.7 mg/L (measured) during the test (observed as dead at the 72-h observation period). All fish were observed as dead during the first two days at the two highest concentrations (32.0 and 53.2 mg/L measured).
- Other adverse effects control: None
- Abnormal responses: Not determined
- 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 77 – 111 % of the nominal at the start and 52 – 120 % at the end of the water renewal periods (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
- Toxic effects: The test item had significant toxic effects on fish. The 96-h LC50 value was determined to be 23.2 mg/L. The highest test concentration causing no mortality within the test period (96-h LC0) was determined to be 11.6 mg/L and the lowest test concentration causing 100% mortality within the test period (96-h LC100) was determined to be 32.0 mg/L.

Results with reference substance (positive control):

No determined.

Reported statistics and error estimates:

The LC50 value was calculated by Probit analysis using SPSS software. Other endpoints were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test substance to Zebrafish (Danio rerio) was determined according to OECD 203 under GLP conditions. The 96 hour LC50 was determined to be 23.2 mg/L.

Executive summary:

The acute toxicity of the test substance to Zebrafish (Danio rerio) was determined according to OECD 203 (1992) and EU Council Regulation (EC) No 440/2008/C1 (2008) under GLP conditions. Young fish were exposed in an acute toxicity test to aqueous test media containing the test item for 96 hours at a range of concentration in a semi-static test system (48-h renewal periods). The fish were observed at least on each day of the test for signs of intoxication and mortality. Body weights of fish were determined prior to test initiation; body length measurement was performed at the end of the test (or when any mortality occurred). Measurements of environmental conditions were performed daily (at the start and end of each water renewal period). The dilution water (ISO medium, without addition of the test item) was used as a control solution. The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent. The test solutions of chosen test concentrations were prepared by appropriate dilution of this stock solution. Based on the results of the preliminary experiment, nominal concentrations of 7.6, 12.2, 19.5, 31.3 and 50.0 mg/L were investigated in the main study. The quantification of the test item was performed by a previously validated analytical method. 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 from the control at the start and at the end of each water renewal period and analysed by HPLC- MS method. Measured concentrations of these components were in the range of 77 – 111 % of the nominal at the start and 52 – 120 % at the end of the water renewal periods. The corresponding measured test item concentrations (calculated as the geometric mean of the measured start and end values of each renewal periods) were: 6.0, 11.6, 19.7, 32.0 and 53.2 mg/L. All biological results are based on the measured geometric mean concentrations. In addition, no mortality was observed in the control during the test, the dissolved oxygen concentration in the test solutions did not fall below 60 % of air saturation value during the study and no significant change (more than one unit) in the pH value was observed during the test. All validity criteria were within acceptable limits and therefore the study was considered as valid. The 96-h LC50 value was determined to be 23.2 mg/L.

Description of key information

The acute toxicity of the test substance to Zebrafish (Danio rerio) was determined according to OECD 203 (1992) under GLP conditions. The 96 hour LC50 was determined to be 23.2 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

23.2 mg/L

Additional information

The acute toxicity of the test substance to Zebrafish (Danio rerio) was determined according to OECD 203 (1992) and EU Council Regulation (EC) No 440/2008/C1 (2008) under GLP conditions. Young fish were exposed in an acute toxicity test to aqueous test media containing the test item for 96 hours at a range of concentration in a semi-static test system (48-h renewal periods). The fish were observed at least on each day of the test for signs of intoxication and mortality. Body weights of fish were determined prior to test initiation; body length measurement was performed at the end of the test (or when any mortality occurred). Measurements of environmental conditions were performed daily (at the start and end of each water renewal period). The dilution water (ISO medium, without addition of the test item) was used as a control solution. The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent. The test solutions of chosen test concentrations were prepared by appropriate dilution of this stock solution. Based on the results of the preliminary experiment, nominal concentrations of 7.6, 12.2, 19.5, 31.3 and 50.0 mg/L were investigated in the main study. The quantification of the test item was performed by a previously validated analytical method. 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 from the control at the start and at the end of each water renewal period and analysed by HPLC- MS method. Measured concentrations of these components were in the range of 77 – 111 % of the nominal at the start and 52 – 120 % at the end of the water renewal periods. The corresponding measured test item concentrations (calculated as the geometric mean of the measured start and end values of each renewal periods) were: 6.0, 11.6, 19.7, 32.0 and 53.2 mg/L. All biological results are based on the measured geometric mean concentrations. In addition, no mortality was observed in the control during the test, the dissolved oxygen concentration in the test solutions did not fall below 60 % of air saturation value during the study and no significant change (more than one unit) in the pH value was observed during the test. All validity criteria were within acceptable limits and therefore the study was considered as valid. The 96-h LC50 value was determined to be 23.2 mg/L.