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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2019 to February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study carried out under GLP conditions for the purposes of REACH Regulation.

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

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
- Batch number of test material: 1023S17201
- Expiration date of the lot/batch: 09 July 2020
- Purity: 100%
- Appearance: White solid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Substance of low water solubility. Presence of undissolved test item was observed floating on surface of water column.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: A series of dilutions were made of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (900 mL) of the 1.0, 3.2, 10 and 32% v/v stock solutions was separately inoculated with 4.4 mL of algal suspension and an aliquot (500 mL) of the 100% v/v stock solution was inoculated with 2.4 mL of algal suspension to give an initial nominal cell density of 5.00 x 103 cells/mL.
- Sampling method: Aliquot removed.
- Sample storage conditions before analysis: Frozen until analysis.

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Samples inoculated with algae were also taken at 24 and 48 hours from media run alongside the test. The 0 and 72 hour samples were analysed on the day of sampling, with the 24 and 48 hour samples being taken and stored frozen and then analysed with the 72 hour samples. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A nominal amount of test item (250 mg) was dispensed in 5 liters of culture medium with the aid of magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface and was observed to be a clear colorless water column with some test item floating at the media surface and some test item floating throughout the media column. 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) and one sheet of filter paper. 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 and also one sheet of coarse filter paper (the first 500 mL discarded) to give a 100% v/v saturated solution. Microscopic observations of the slow-stir saturated solution was performed after filtering and showed no micro-dispersions of test item to be present.

- Test concentrations: A series of dilutions were made from this stock solution to give futher stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (900 mL) of the 1.0, 3.2, 10 and 32% v/v stock solutions was separately inoculated with 4.4 mL of algal suspension and an aliquot (500 mL) of the 100% v/v stock solution was inoculated with 2.4 mL of algal suspension to give an initial nominal cell density of 5.00 x 103 cells/mL.

- Controls: 0% control and a positive control were performed. A positive control test using potassium dichromate as the reference item was performed twice
in a 12 month period to demonstrate satisfactory conditions of the test.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No evidence at the start of the test, but undissolved material was observed on the water surface at the end of the test.

- Other relevant information: The stock solutions and each prepared concentration were inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age and method of inoculum: Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 105 to 106 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None - observations concluded at 72 hours.

Test conditions

Hardness:

No data

Test temperature:

24 degrees C (+/- 1 deg C)

pH:

pH ranged from 6.8 to 7.7 at the start of the test and 8.2 to 9.1 at 72 hours across the test concentrations.

Dissolved oxygen:

No data

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

Test item was prepared at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution. The geometric mean measured test concentrations were determined to be 0.0016, 0.0056, 0.017, 0.066 and 0.19 mg/L.
Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.0032 mg/L to 0.39 mg/L.
Analysis at 24, 48, and 72 hours showed a concentration dependent decline in the measured test concentrations during the test to range from less than the LOQ to 0.23 mg/L at 24 hours, less than the LOQ to 0.13 mg/L at 48 hours and less than the LOQ to 0.11 mg/L at 72 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flask
- Type (delete if not applicable): closed
- Aeration: No
- Initial cells density: An aliquot (900 mL) of the 1.0, 3.2, 10 and 32% v/v stock solutions was separately inoculated with 4.4 mL of algal suspension and an
aliquot (500 mL) of the 100% v/v stock solution was inoculated with 2.4 mL of algal suspension to give an initial nominal cell density of 5000 cells/mL.
- Control end cells density: 1,100,000 cells/ml
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately
150 rpm for 72 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 25, 50 and 72 hours and the cell densities determined using a haemocytometer and light microscope. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density. It was not possible to monitor algal cells using a Coulter® Multisizer Particle Counter due to the presence of undissolved test item floating at the surface of the water column observed initially at 24 hours. To determine the potential effect of the test item on the appearance of algal cells, a sample
was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Range-finding test was performed initially.
- Range finding study: yes
- Test concentrations: The range-finding test was conducted to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.
- Results used to determine the conditions for the definitive study: Growth rate inhibition values (%) for the range-finding test were 0, 4, 0, 1, and 23% for the control, 0.10, 1.0, 10 and 100% v/v saturated solutions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate at 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l performed between 16 and 19 July 2019.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.017 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.017 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Details on results:

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.0016 and 0.017 mg/L test cultures were observed to be bright green dispersions. The 0.066 mg/L test cultures were observed to be green dispersions with test item particles present, whilst the 0.19 mg/L test cultures were observed to be pale green dispersions with test item particles observed at the surface and present through the media column. The 0.0056 mg/L test cultures were observed to remain clear colorless solutions.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.0032 mg/L to 0.39 mg/L. Analysis at 24, 48, and 72 hours showed a concentration dependent decline in the measured test concentrations during the test to range from less than the LOQ to 0.23 mg/L at 24 hours, less than the LOQ to 0.13 mg/L at 48 hours and less than the LOQ to 0.11 mg/L at 72 hours. Given this decline in measured test concentrations the laboratory based the results 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 0.0016, 0.0056, 0.017, 0.066 and 0.19 mg/L.

The EC50 and NOEC measured mean concentrations are below the limit of quantification and limit of solubility of the test substance.

Results with reference substance (positive control):

- Results with reference substance valid - yes
- EC50 for growth rate: 1.3 mg/l (confidence limits 1.2 to 1.4 mg/l)

Reported statistics and error estimates:

50% inhibition of growth was not achieved in the test. Instead the EC50 value was determined by extrapolation of the growth curve as no more than 39% inhibition of growth rate occurred at the maximum attainable dissolved test item concentration of 0.19mg/L. It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Any other information on results incl. tables

Tables of results are as follows:

Verification of test concentrations

l test concentration (%v/v saturated solution)	Geometric mean measured test concentration (mg/l)	Expressed as a % of the 0-hour measured test concentration
1.0	0.0016	not applicable
3.2	0.0056	50
10	0.017	41
32	0.066	54
100	0.19	48

Definitive test results

For growth rate, the ErC50 value was determined by extrapolation of the growth curve as no more than 39% inhibition of growth

rate occurred at the maximum attainable dissolved test item concentration of 0.19 mg/L. Statistical analysis of the growth rate was carried out for the control, 0.0016, 0.017, 0.066 and 0.19 mg/L test concentrations using one way anlaysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P≥0.05), between the control, 0.0016 and 0.017 mg/L test concentrations; however, all other test concentrations were significantly different (P≥0.05) and, therefore the No Observed Effect Concentration (NOEC) based on growth rate was 0.017 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 0.066 mg/L.

For yield, there were no statistically significant differences (P≥0.05), between the control, 0.0016 and 0.017 mg/L test concentrations; however, all other test concentrations were significantly different (P≥0.05) and, therefore the NOEC based on yield was 0.017 mg/L. Correspondlingly the LOEC based on yield was 0.066 mg/L.

Definitive test results

nse variable	Reponse value (mg/l)	95% confidence limits (mg/l)	Comments
Growth rate	ErC10 = 0.07	Not determined
Growth rate	ErC20 = 0.12	Not determined
Growth rate	ErC50 = 0.29	Not determined	The ErC50 value was determined by extrapolation of the growth curve as no more than 39% inhibition of growth rate occurred at the maximum attainable dissolved test item concentration of 0.19 mg/L.
Growth rate	NOEC = 0.017
Growth rate	LOEC = 0.066
Yield	EyC10 = 0.03
Yield	EyC20 = 0.045
Yield	EyC50 = 0.086	0.074 to 0.10
Yield	NOEC = 0.017
Yield	LOEC = 0.066

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance is of poor water solubility and analysis at 24, 48, and 72 hours showed a concentration dependent decline in the measured test concentrations during the test to range from less than the LOQ to 0.23 mg/L at 24 hours, less than the LOQ to 0.13 mg/L at 48 hours and less than the LOQ to 0.11 mg/L at 72 hours. The limit of quantification (LOQ) was determined to be 0.0032 mg/L to 0.39 mg/L.

Given this decline in measured test concentrations the laboratory based the results 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 0.0016, 0.0056, 0.017, 0.066 and 0.19 mg/L. The EC50 for growth rate was calculated to be 0.29 mg/l, but this was determined by extrapolation of the growth curve as no more than 39% inhibition of growth rate
occurred at the maximum attainable dissolved test item concentration of 0.19 mg/L. Therefore, it is concluded that the test item is not acutely toxic (EC50) at its limit of solubility. The NOEC for growth rate of 0.017 mg/l is also within the range for the LOQ of the test item. The laboratory calculated a statistical difference in growth inhibition between the test concentrations 0.017 mg/l (10% v/v saturation) and 0.066 mg/l (32% v/v saturation), however undissolved particles of test item were observed amongst the green algal dispersion at 0.066 mg/l indicating toxicity is unlikely at the limit of solubility of the test substance.

Executive summary:

The effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was assessed using the OECD 201 Guideline "Freshwater Alga and Cyanobacteria, Growth Inhibition Test". The test substance is poorly water soluble and following a preliminary range-finding test, the alga was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination.

Daily analysis showed the test substance limit of quantification (LOQ) was determined to be 0.0032 mg/L to 0.39 mg/L. The test item concentrations declined through the 72 hour test. The laboratory then based the results 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 0.0016, 0.0056, 0.017, 0.066 and 0.19 mg/L. The EC50 for growth rate was calculated to be 0.29 mg/l, but this was determined by extrapolation of the growth curve as no more than 39% inhibition of growth rate occurred at the maximum attainable dissolved test item concentration of 0.19 mg/L. Therefore, it is concluded that the test item is not acutely toxic (EC50) at its limit of solubility. The NOEC for growth rate of 0.017 mg/l is also within the range reported for the LOQ of the test item and may be uncertain. The laboratory calculated a statistical difference in growth inhibition between the test concentrations 0.017 mg/l (10% v/v saturation) and 0.066 mg/l (32% v/v saturation), however undissolved particles of test item were observed amongst the green algal dispersion at 0.066 mg/l indicating toxicity is unlikely at the limit of solubility of the test substance.