Phosphoric acid, mono- and di-C12-14-alkyl esters

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2019 to October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study to GLP certification with no deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Constituent 2

Reference substance name:

C12-C14 alcohol

IUPAC Name:

C12-C14 alcohol

Constituent 3

Reference substance name:

Mono (C12-C14 alkyl) phosphate ester

IUPAC Name:

Mono (C12-C14 alkyl) phosphate ester

Constituent 4

Reference substance name:

Di (C12-C14 alkyl) diphosphate ester

IUPAC Name:

Di (C12-C14 alkyl) diphosphate ester

Constituent 5

Reference substance name:

Tri (C12-C14 alkyl) triphosphate ester

IUPAC Name:

Tri (C12-C14 alkyl) triphosphate ester

Constituent 6

Reference substance name:

C12-C14 alkyl pyrophosphate esters

IUPAC Name:

C12-C14 alkyl pyrophosphate esters

Constituent 7

Reference substance name:

Ortho-phosphoric acid

IUPAC Name:

Ortho-phosphoric acid

Constituent 8

Reference substance name:

unknown

Molecular formula:

unknown

IUPAC Name:

unknown

Test material form:

solid: bulk

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 1023S17201
- Expiration date of the lot/batch: 09 July 2020
- Purity test date: 17 April 2019
- Purity: 100%
- Physical state: white solid
- Storage: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Nominal concentrations were 10, 18, 32, 56 and 100% v/v saturated solution
Geometric mean measured concentrations (worst case) were 0.0050, 0.0082, 0.017, 0.032 and 0.074 mg/L.

- Sampling method: concentration and stability of the test item was verified by chemical analysis at 0, 24 and 48 hours.
- Sample storage conditions before analysis: Samples were taken from the control and each of the test groups from the freshly prepared test preparations at 0 and 24 hours and from the old/expired pooled replicates at 24 and 48 hours for quantitative analysis. The 0 and 48-Hour samples were taken for immediate analysis. The 24-Hour samples were taken and stored frozen for analysis at a later date. Duplicate sets of samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary. Test samples were thawed in a water bath at ambient temperature prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the test substance is poorly soluble in water. Following ECETOC (1996) and OECD (2019) guidance on testing substances of high purity, yet poorly soluble in water, a slow-stir saturated solution was prepared by stirring an excess (50 mg/L) of test in test media for a period of 23 hours with a 1-hour settlement period prior to removing any undissolved test item present by filtration, (first 500 mL discarded) to give a 100% v/v saturated solution of the test item.

A nominal amount of test item (250 mg) was dispersed in 5 liters of test water and with the aid of magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and allowed to stand for 1-hour. Visual observations made on the slow-stir saturated solution following settlement indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the slow-stir saturated solution by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down , to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the slow-stir saturated solution removed by mid-depth siphoning (the first 500 mL discarded) to give a 100% v/v saturated solution. Microscopic observations of the slow-stir saturated solution were preformed after filtering and showed no micro-dispersions of test item to be present. A series of dilutions was made from this
saturated solution to give the remaining test concentrations of 10, 18, 32 and 56% v/v saturated solution.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity for the preparation at 0 hours. The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 and 48 hours.


- Eluate: 100% v/v
- Test concentrations: 10, 18, 32, 56 and 100% v/v saturated solution.
- Controls: Negative and positive (potassium dichromate) controls used
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Initially yes, but not following filtration.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

- The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
- Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 medium in a temperature controlled room.
- Temperature: 18 to 22 °C.
- Lighting cycle: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Feeding: Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata formerly known as Pseudokirchneriella subcapitata) and GEMMA Micro 300 fish food suspension.
Culture maintenance: conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

An initial range-finding test was performed to a series of nominal test concentrations of 0.1, 1.0, 10 and 100% v/v saturated solution for 48 hours. Based on results the definitive test was performed with 10, 18, 32, 56 and 100% v/v saturated solution.

Post exposure observation period:

None

Test conditions

Hardness:

Reconsistuted water (Elendt M4 medium) was used with hardness of 2.5 mmol/L (= 250 mg/l as CaCO3)

Test temperature:

21 deg C

pH:

pH ranged from 7.4 to 8.4 across the test concentrations, replicates and test duration.

Dissolved oxygen:

Dissolved oxygen ranged from 8.7 to 9.2 mg O2/l across the test concentrations, replicates and test duration.

Salinity:

not applicable

Conductivity:

no data

Nominal and measured concentrations:

Nominal test concentrations (% v/v saturated solution): 0, 10, 18, 32, 56, 100
Geometric mean measured test concentrations (mg/l): 0, 0.005, 0.0082, 0.017, 0.032, 0.074

Details on test conditions:

TEST SYSTEM
- Test vessel: 120 mL glass vessels containing approximately 100 ml of test preparation
- Type (delete if not applicable): covered to reduce evaporation
- Volume of solution: 100 ml
- Renewal rate of test solution (frequency): daily in semi-static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Source/preparation of dilution water: Reconstituted water
- Adjustment of pH: None
- Photoperiod: 16 hr light: 8 hr dark
- Light intensity: 801 to 987 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: An initial range-finding test was performed to a series of nominal test concentrations of 0.1, 1.0, 10 and 100% v/v saturated solution for 48 hours.
- Results used to determine the conditions for the definitive study: Based on results the definitive test was performed with 10, 18, 32, 56 and 100% v/v saturated solution.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.074 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.074 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

There was no immobilization in 20 daphnids exposed to geometric mean measured test concentrations of 0.0082, 0.017, 0.032 and 0.074 mg/L for a period of 48 hours. One daphnid was observed to be immobilized at 48 hours in the test concentration of 0.0050 mg/L. Given that this equivalated to only 5% immobilization in this test group his was considered not to be significant. The EC50 was >0.074 mg/l. The No Observed Effect Concentration (NOEC) after 24 and 48 hours exposure was 0.074 mg/L. Due to the nature of the data it was not possible to calculate the slope and error of the response curve at 24 or 48 hours.

Results with reference substance (positive control):

After 24 hours the EC50 was 1.2 mg/l (95% CI of 1.1 - 1.8 mg/l) and after 48 hours the EC50 was 0.71 mg/l (0.61 - 0.81 mg/l). The results were in the normal range for this reference item.

Reported statistics and error estimates:

Analysis of the immobilization data was carried out using the Weibull analysis using linear maximum-likelihood regression at 24 and 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item is poorly water soluble and prepared as a dilution series from 100% v/v saturated solution. Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.0054 to 0.15 mg/L. Analysis of the corresponding old/expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between less than the limit of quantification (LOQ), which was determined to be 0.0042 mg/L to 0.099 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

The geometric mean measured test concentrations were determined to be 0.0050, 0.0082, 0.017, 0.032 and 0.074 mg/L. Exposure of Daphnia magna to the test item gave EC50 values based on the geometric mean measured test concentrations of greater than 0.074 mg/L. The No Observed Effect Concentration (NOEC) was 0.074 mg/L.

This study showed that there were no toxic effects at saturation.

Executive summary:

The acute toxicity of the test item to Daphnia magna was performed using the OECD 202 Guideline Daphnia sp., Acute Immobilisation Test.

Preliminary solubility work indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation. Dissolved test item concentration of approximately 0.063 mg/L was obtained from a slow-stir saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. Following a preliminary range-finding test, 20 daphnids were exposed to aqueous solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of 21 °C under semi-static test conditions.

Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.0054 to 0.15 mg/L. Analysis of the corresponding old/expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between less than the limit of quantification (LOQ), which was determined to be 0.0042 mg/L to 0.099 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. 

The geometric mean measured test concentrations were determined to be 0.0050, 0.0082, 0.017, 0.032 and 0.074 mg/L. Exposure of Daphnia magna to the test item gave EC50 values based on the geometric mean measured test concentrations of greater than 0.074 mg/L. The No Observed Effect Concentration (NOEC) was 0.074 mg/L.

This study showed that there were no toxic effects at saturation.