Phosphoric acid, hexadecyl phosphate esters, potassium salts with cetostearyl alcohol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 23, 2017 to October 27, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

To demonstrate that nominal exposure concentrations were being achieved, the concentrations of test substance in the test solutions were measured using the high-performance liquid chromatography / mass spectrometry method.

Details on sampling:

At the start of the test, samples were taken from excess solutions and at the end of the test, replicate vessels of the dilution water control, solvent control and each test concentration were taken for whole sample analysis.

Vehicle:

yes

Remarks:

Tetrahydrofuran (THF)

Details on test solutions:

Preparation of test solutions
The study was run with a culture medium control, solvent control and nominal exposure concentrations of 0.125, 0.25, 0.5, 1.0 and 2.0 mg/L. The test substance was ground to a fine white powder in a pestle and mortar and a primary stock concentrate of test substance, with a nominal concentration of 20 g/L, was prepared by weighing a nominal 0.2 g of test substance into a 10 mL volumetric flask and making up to volume with tetrahydrofuran (THF). The resultant stock was observed to be a cloudy white solution. The stock solution was used to prepare the test solutions. This was achieved by the direct addition of the appropriate amount of concentrate to dilution water via a microliter syringe into a stirring solution in a volumetric flask. The solvent control was prepared in the same way using solvent only. The control consisted of culture medium only. In all cases the final solutions contained nutrients. A solvent was used in this study to assist in dosing the test compound due to its apparent low solubility in test media, the concentration of solvent used in all exposure solutions with the exception of the control was 100 µL/L. The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.

Test organisms (species):

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

Details on test organisms:

The test species was the unicellular green alga Pseudokirchneriella subcapitata strain CCAP 278/4 from laboratory cultures maintained under axenic conditions. A 3-d old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium, and under the environmental conditions, described for the test.

Test type:

not specified

Water media type:

other: AAP-medium

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 ± 2ºC

pH:

7.06 to 8.13

Nominal and measured concentrations:

Control, solvent control and nominal concentrations of 0.125, 0.25, 0.5, 1.0 and 2.0 mg/L
Control, solvent control and mean measured concentrations of 0.063, 0.13, 0.27, 0.46 and 0.80 mg/L
Due to low solubility of the test substance, 2.0 mg/L was selected as the highest concentration based on a non-GLP solubility trial and range-finding study. This concentration was dosed to the media via a solvent carrier (tetrahydrofuran (THF)) to assist in dosing the test compound. The concentration of solvent used in all exposure solutions with the exception of the control was 100 µL/L.

Details on test conditions:

Test apparatus
The test vessels were glass conical flasks of 250 mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 22 ± 2°C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking at 160 rpm.

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.46 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: growth rate and biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 0.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: growth rate and biomass

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 0.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

> 0.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Results

Analytical data

The limit of quantification of test substance in this study was 0.05 mg/L. All analytical values are quoted to two significant figures and percentages to the nearest integer. The measured concentrations at the start of the study were 95 -106% of nominal and at the end were 0 -20% of nominal. On the basis of the analytical data the mean measured concentrations were used for the calculation and reporting of results.

Biological data

Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass.

Growth rates

The growth rate (0 to 72 h) was calculated for each replicate culture. The growth rates were examined by one-way analysis of variance and a two‑sample t test to identify significant differences (p<0.05) between the control and solvent control. There was no significant effect (p=0.2584) between the control and solvent control. Therefore, the solvent control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t test to identify significant differences (p<0.05). The results obtained from growth rate analyses, based on mean measured test concentrations, were as follows:

 

	Test substance (mg/L)
NOEC	0.46
LOEC	0.80
ErC50	>0.80
ErC20	>0.80
ErC10	>0.80

Yield

This response is defined as the biomass at the end of the test minus the starting biomass. For the purposes of calculation, the cell particle density count (cell particles per unit volume) is an acceptable surrogate for biomass. These cell particle densities were examined by one-way analysis of variance and two-sample t test to identify significant differences (p<0.05) from the control and solvent control. There was no significant effect (p=0.2172) between the control and solvent control. Therefore, the solvent control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t test to identify significant differences (p<0.05). The EC₅₀, EC₂₀and EC₁₀values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method. The results obtained from statistical analysis, based on mean measured test concentrations, were as follows:

	Test substance (mg/L)
NOEC	0.46
LOEC	0.80
EyC50	>0.80
EyC20	0.719
EyC10	0.546

Additional biological data

The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the solvent control, 0.063, 0.13, 0.27, 0.46 and 0.80 mg/L test concentrations appeared normal.

Validity criteria

The validity criteria specified in the OECD 201 guideline are;

1) To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72 h test period. In this testcell particle density increase (measured as a surrogate for biomass) was 88.5 and 82.6 over the 72 h for the control and solvent control respectively.

2) The mean coefficients of variation for control replicate sectional (daily) specific growth rates must not exceed 35% and in this test, was determined to be 12% for the control and 10% for the solvent control.

3) The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7% and was calculated as 2.7% for the control and 1.0% for the solvent control

Validity criteria fulfilled:

yes

Conclusions:

Under study conditions, 72 h ErC50, EyC50, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.80, >0.80, 0.46 and 0.80 mg/L, respectively.

Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, test substance, 'mono- and di- C16 PSE, K+ and C16-18-OH', to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and solvent control, whereas triplicates of each concentration of the test substance were employed. Three replicate algal cultures, with a nominal cell density of approximately 0.5 x 10⁴cells/mL, were exposed to test substance at nominal concentrations of 0, 0.125, 0.25, 0.5, 1 and 2 mg/L in AAP medium for 72 h. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.514 × 10⁴cells/mL and was used for growth calculations. The exposure levels of test substance in aqueous samples of test media were monitored using a HPLC method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0.063, 0.13, 0.27, 0.46 and 0.80 mg/L. The test results were expressed in terms measured concentration of test substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. ErC50 value (for growth rate) and EyC50 value (Cell particle density) were found to be > 0.80 mg/L. The NOEC and LOEC values were found to be 0.46 mg/L and 0.80 mg/L, respectively, for both growth rate and cell particle densities. The study results were considered to have fulfilled the all the validity criteria. Under study conditions, 72 h ErC50, EyC50, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.80, >0.80, 0.46 and 0.80 mg/L, respectively (Scymaris, 2017).

Description of key information

The 72 h ErC50, EyC50, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.80, >0.80, 0.46 and 0.80 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.8 mg/L

EC10 or NOEC for freshwater algae:

0.46 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, test substance, 'mono- and di- C16 PSE, K+ and C16-18-OH', to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and solvent control, whereas triplicates of each concentration of the test substance were employed. Three replicate algal cultures, with a nominal cell density of approximately 0.5 x 10⁴cells/mL, were exposed to test substance at nominal concentrations of 0, 0.125, 0.25, 0.5, 1 and 2 mg/L in AAP medium for 72 h. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.514 × 10⁴cells/mL and was used for growth calculations. The exposure levels of test substance in aqueous samples of test media were monitored using a HPLC method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0.063, 0.13, 0.27, 0.46 and 0.80 mg/L. The test results were expressed in terms measured concentration of test substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. ErC50 value (for growth rate) and EyC50 value (Cell particle density) were found to be > 0.80 mg/L. The NOEC and LOEC values were found to be 0.46 mg/L and 0.80 mg/L, respectively, for both growth rate and cell particle densities.The study results were considered to have fulfilled the all the validity criteria.Under study conditions, 72 h ErC50, EyC50, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.80, >0.80, 0.46 and 0.80 mg/L, respectively (Scymaris, 2017).

Based on the available information, signficant toxicity was not observed up to the highest soluble test concentration, therefore the EC50 value of the test substance, 'mono- and di- C16 PSE, K+ and C16-18-OH' was established as >0.8 mg/L.