Phosphoric acid, mixed esters with alcohols, C7-9-iso-, C8-rich and alcohols, C11-14-iso-, C13-rich, ethoxylated, potassium salt

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:

September 2017 - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006, corrected 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical samples were taken and analysed from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions.

Vehicle:

yes

Remarks:

test medium

Details on test solutions:

The medium used for the test was AAP-Medium (according to Annex 3 of OECD 201). The pH was adjusted to 7.5 ± 0.1 with NaOH or HCl, if necessary.

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata Hindák (Sphaeropleales: Selenastraceae), strain: SAG 61.81 purchased from MBM Sciencebridge GmbH, Germany.

The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from the commercial supplier.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

N/A

Post exposure observation period:

N/A

Hardness:

N/A

Test temperature:

22.6 – 23.3 °C

pH:

7.30 – 8.55

Dissolved oxygen:

N/A

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

Concentrations tested:
Nominal (per active ingredient): 100, 32, 10, 3.2, 1.0, 0.32, 0.10, 0.032, 0.010 mg/L and control
Nominal (per test material): 111.1, 35.1, 11.1, 3.51, 1.11, 0.351, 0.111, 0.0351, 0.0111 mg/L and control

Concentrations measured:
Per test material: 111.1 (actual: 112 [0h]; 117 [72h]), 35.1 (actual: 35.6 [0h]; 36.7 [72h]), 11.1 (actual: 10.9 [0h, 72h]), 3.51 (3.31 [0h], 3.67 [72h]) mg/L and control

The measured initial concentrations of test material ranged from 94 % to 101 % of nominal. In the aged samples the measured content was between 98 % and 105 % of nominal. Therefore all toxicological endpoints were evaluated using the nominal concentrations of the test item and presented in the values per active ingredient.

Details on test conditions:

The test organism was exposed to different concentrations of the test item under defined conditions in a synthetic growth medium for a time period of 72 h. Initial target cell density was 0.5 × 104 cells/mL. Three replicates were employed for each of the test item concentrations and six replicates for the control. By comparing the cell division under test conditions with and without the influence of the test item, an inhibition of the cell multiplication can be calculated. This inhibition is a value for toxicity.

At defined dates (24, 48 and 72 hours), the number of cells in each replicate was determined in duplicate. The determination was performed by fluorescence measurement. The fluorescence measurements were performed with a fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0).

By the means of a calibration curve, where fluorescence signals (y axis) were plotted versus cell numbers (x axis), the cell numbers were derived from the fluorescence signals. To establish a calibration curve, the cell numbers were counted with a Neubauer chamber after preparation of a dilution series of a logarithmic growing Pseudokirchneriella subcapitata culture.

The number of cells in each replicate was used to calculate effect concentrations for the test item which resulted in 10, 20 and 50 % inhibition of the cell growth rate (ErC10, 20, 50) and yield (EyC10, 20, 50). The test item concentration which did not cause any significant inhibition (NOEC) and the lowest observed effect concentration (LOEC) were also determined where possible.

Additionally, the morphological appearance of the algae cells was observed microscopically at the end of the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested in a separate study twice a year. The EC50 values calculated in this reference test were considered to be within an acceptable range therefore it can be considered that the test organism was sensitive to the test item.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

17.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

31.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.07 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

9.28 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

Validity criteria:
Biomass: Cell numbers, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 97.12, which exceeds the threshold of 16. It corresponds to a growth rate of 1.52536/day.
Coefficient of Variation (section by section): The mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 15 % and did not exceed 35 %.
Coefficient of Variation (average growth rate): The coefficient of variation of average growth rate in replicate control cultures was 2.2 % and did not exceed 7 % for the whole test period.

Analytical results:
The measured initial concentrations of test item ranged from 94% to 101% of nominal. In the aged samples the measured content was between 98% and 105% of nominal. Therefore toxicological endpoints were evaluated using the nominal concentrations of the test item.

Biological results:
After 72 h at termination of the test no statistically significant inhibition of growth rate or yield was observed for test item concentrations up to and including 3.2 mg/L. At higher test item concentrations the inhibition of growth rate peaked in 96.7 % and the inhibition of yield peaked in 99.8 % at a nominal test item concentration of 100 mg/L.

The morphology of the algae cells was observed microscopically at test end. The cells were considered normal for the control and up to and including a nominal test item concentration of 10 mg/L. Fewer cells were observed 32 mg/L test item concentration. No cells were observed at 100 mg/L nominal test item concentration.

Reported statistics and error estimates:

The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-ttest, left sided, for yield, Bonferroni-Holms corrected Welch test, left sided, for growth rate). The
EC10, 20, 50-values for growth rate and yield were determined by probit analysis following the normal and logistic distribution, respectively. Due to statistical reasons inhibition-values below 0 % were set to zero. Only concentrations within a clear concentration response relation were used for calculations.

Toxicological endpoints

	Test item[mg/L]
	nominal	95 % confidence limits
ErC10(Growth rate)^1)	12.8	10.1 - 15.3
ErC20^1)	17.3	14.4 - 20.1
ErC50^1)	31.1	27.3 - 35.5
EyC10(Yield)^2)	7.07	5.70 - 8.30
EyC20^2)	9.28	7.84 - 10.6
EyC50^2)	14.8	13.0 - 16.8
NOEC^3)	3.2
LOEC^3)	10

¹⁾Probit analysis following normal distribution

²⁾Probit analysis following logistic distribution

³⁾Following Bonferroni-Holms corrected Welch test (left-sided, p<0.05) for growth rate and Dunnetts-t-test (left-sided, p<0.05) for yield

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 10 mg/L and above. The overall LOEC was therefore determined to be 10 mg/L, the corresponding NOEC was set at 3.2 mg/L.

The EC10-value for growth rate (ErC10) was determined to be 12.8 mg/L. The EC10-value for yield (EyC10) was 7.07 mg/L.
The EC20-value for growth rate (ErC20) was determined to be 17.3 mg/L. The EC20-value for yield (EyC20) was 9.28 mg/L.
The EC50-value for growth rate (ErC50) was determined to be 31.1 mg/L. The EC50-value for yield (EyC50) was 14.8 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

31.1 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L

Additional information

NOEC = 3.2 mg/L

EC10 = 12.8 mg/L

All values for growth rate.