Reaction mass of orthophosphoric acid and potassium dihexadecyl phosphate and dipotassium hexadecyl phosphate

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:

weight of evidence

Study period:

From December 18, 2017 to December 21, 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:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

To demonstrate that test vessels were dosed with nominal exposure concentrations of test substance, samples were analysed using the gas chromatography mass spectrometry method.

Details on sampling:

Samples for analysis at test start were taken from excess test solutions and at test end from a single test replicate solution from the control and each test concentration.

Vehicle:

yes

Details on test solutions:

Preparation of test solutions
The study was run with a culture medium control and nominal exposure concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L. Two primary stock concentrates of test substance, with nominal concentrations of 5 and 10 mg/L, were prepared. The 5 mg/L stock was prepared by adding a nominal 0.010 g of test substance (actual weight of 0.01011 g) and making up to 2000 mL volume with culture medium. The 10 mg/L stock was prepared by adding a nominal 0.010 g of test substance (actual weight of 0.01001 g) and making up to 1000 mL volume with culture medium. Both stocks were ultrasonicated for 100 minutes and assessed as homogenous dispersions with very fine particles visible. The 5 and 10 mg/L stocks were used directly as the 5 and 10 mg/L test solutions. The 5 mg/L stock was also used to prepare the remaining test solutions by direct addition of the appropriate amount to culture medium. The control consisted of culture medium only. In all cases the final solutions contained nutrients. The control, 0.625 and 1.25 mg/L test solutions were observed to be clear and colourless. The 2.5, 5 and 10 mg/L were observed to be homogenous dispersions with very fine particles visible. 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. The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test was AAP-medium.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 ± 2ºC

pH:

7.20 to 7.71

Nominal and measured concentrations:

A 10 mg/L test concentration was selected as the highest concentration based on the dispersibility of the test substance demonstrated in a non-GLP solubility trial and the results of a non GLP range-finding study.
0, 0.625, 1.25, 2.5, 5.0 and 10 mg/L (nominal)
0, 0, 0, 0, 0 and 0.78 mg/L (mean measured)

Details on test conditions:

Test appratus
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. 0.78 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.:

> 0.78 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.:

> 0.78 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.:

> 0.78 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 (LOQ) of test substance in this study was 0.8 mg/L for all test solutions. The instrument LOQ was 0.4 mg/L but during analysis samples were diluted ×2, doubling the LOQ. Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R² value of 0.995. The maximum percentage difference from nominal concentration for standards at the LOQ is less than 20% and less than 15% at levels greater tha the LOQ. All analytical values are quoted to two significant figures and percentages to the nearest integer. 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. The algal cell particle densities measured at each time period are given in Table 1.

Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	Replicate	Algal cell particle density (×10^4cells/mL)
			24 h	48 h	72 h
Culture medium control	0	A	1.67	6.34	33.30
		B	1.47	5.59	31.20
		C	1.51	7.42	34.80
		D	1.63	6.41	37.20
		E	1.63	7.02	33.00
		F	1.49	6.96	31.80
		Mean	1.57	6.62	33.55
0.625	0	A	1.55	7.21	33.70
		B	1.67	6.90	31.30
		C	1.70	7.26	31.40
		Mean	1.64	7.12	32.13
1.25	0	A	1.69	7.43	31.50
		B	1.59	7.46	35.30
		C	1.59	6.61	31.60
		Mean	1.62	7.17	32.80
2.5	0	A	1.77	6.63	31.90
		B	1.68	6.29	27.40
		C	1.72	8.14	33.60
		Mean	1.72	7.02	30.97
5.0	0	A	1.77	6.34	27.50
		B	1.94	6.20	31.00
		C	2.19	7.03	32.60
		Mean	1.97	6.52	30.37
10	0.78	A	2.15	7.18	29.80
		B	2.29	5.53	29.20
		C	2.71	5.81	29.50
		Mean	2.38	6.17	29.50

Table 1: Algal cell particle density

 

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 t-tests to identify significant differences (p <0.05) from the control. The EC₅₀, EC₂₀ and EC₁₀ values with their associated confidence intervals were not calculated in this case due to the lack of biological response and analytical data.

The mean growth rates are given in Table 2, together with the rates expressed as percentages of the control.

Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	Mean growth rate / day (0-72 h)	Mean growth rate / day 95% Cl	Percentage of control (%)
Culture medium control	0	1.386	1.363 – 1.408	-
0.625	0	1.372	1.337 – 1.407	99
1.25	0	1.378	1.324 – 1.432	99
2.5	0	1.358	1.271 – 1.446	98
5.0	0	1.352	1.280 – 1.424	98
10	0.78	1.343	1.335 – 1.352	97

Table 2: mean growth rate over the test period

All values are quoted to 3 decimal places.

The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows:

	Test substance (mg/L)	95% confidence limits
NOEC	0.78	-
LOEC	>0.78	-
ErC50	>0.78	n/a

 

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 t-tests to identify significant differences (p <0.05) from the control. The EC₅₀, EC₂₀ and EC₁₀ values with their associated confidence intervals were not calculated in this case due to the lack of biological response and analytical data.

The yield mean values are shown in Table 3, together with the yields expressed as percentages of the control.

Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	Mean yield (0-72 h) (×104 cells/mL)	Percentage of control (%)
Culture medium control	0	33.0	-
0.625	0	31.6	96
1.25	0	32.3	98
2.5	0	30.4	92
5.0	0	29.8	90
10	0.78	29.0	88

Table 3: Mean yields over the test period

The results obtained from these statistical analysis, were as follows:

	Test substance (mg/L)	95% confidence limits
NOEC	0.78	-
LOEC	>0.78	-
EyC50	>0.78	n/a

 

Additional biological data

The microscopic observations, made at test end, showed that for the control and all test concentrations, the cells appeared normal.

Validity criteria

The validity criteria specified in the OECD 201 guideline are;

- To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72 h test period. In this test, cell particle density increase (measured as a surrogate for biomass) was 64 times over the 72 h.

- The mean coefficients of variation for control replicates sectional (daily) specific growth rates must not exceed 35% and in this test, was determined to be 20.6% for the control.

- 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 1.5% for the control.

The study has met all validity criteria.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, 72 h ErC50, EyC50, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.78, >0.78, 0.78 and >0.78 mg/L, respectively.

Executive summary:

A study was conducted to determine the acute toxicity potential of the test substance, mono- and di- C16 PSE, H3PO4 (purity: 100%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. Each replicate test vessel was inoculated with 0.594 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.524 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.625, 1.25, 2.5, 5.0 and 10 mg/L in AAP medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored at start (0 h) and end of the test (72 h), using a GC/MS method of analysis. Except for the measured values of the top test concentration at 0 h (i.e., determined to be 1.6 mg/L), the remaining measurements at 0 and 72 h were found to be below the limit of quantification (LOQ). Therefore, the test concentrations were presented as mean measured concentrations i.e., 0, 0, 0, 0, 0 and 0.78 mg/L. 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 determined to be >0.78 and >0.78 mg/L respectively. The NOEC and the LOEC values were found to be 0.78 mg/L and >0.78 mg/L, respectively, for both growth rate and cell particle densities. All validity criteria were met during this study. Under the study conditions, 72 h ErC50, EyC50, NOEC and LOEC values were determined to be >0.78, >0.78, 0.78 and >0.78 mg/L, respectively (Scymaris, 2018).

Description of key information

Based on the study results, 72 h ErC50, EyC50, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.78, >0.78, 0.78 and >0.78 mg/L (measured), respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.78 mg/L

EC10 or NOEC for freshwater algae:

0.78 mg/L

Additional information

A study was conducted to determine the acute toxicity potential of the test substance, mono- and di- C16 PSE, H3PO4 (purity: 100%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. Each replicate test vessel was inoculated with 0.594 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.524 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.625, 1.25, 2.5, 5.0 and 10 mg/L in AAP medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored at start (0 h) and end of the test (72 h), using a GC/MS method of analysis. Except for the measured values of the top test concentration at 0 h (i.e., determined to be 1.6 mg/L), the remaining measurements at 0 and 72 h were found to be below the limit of quantification (LOQ). Therefore, the test concentrations were presented as mean measured concentrations i.e., 0, 0, 0, 0, 0 and 0.78 mg/L. 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 determined to be >0.78 and >0.78 mg/L respectively. The NOEC and the LOEC values were found to be 0.78 mg/L and >0.78 mg/L, respectively, for both growth rate and cell particle densities. All validity criteria were met during this study. Under the study conditions, 72 h ErC50, EyC50, NOEC and LOEC values were determined to be >0.78, >0.78, 0.78 and >0.78 mg/L, respectively (Scymaris, 2018).