Decyl dihydrogen phosphate, 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:

Experimental start date 23 June 2017 Experimental completion date 10 March 2018

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)

Specific details on test material used for the study:

Identification: Decyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7)
Trade Name: BEROL 522
CAS-No.: 68427-32-7
Batch: 1526886
Purity: 56%
Physical state/Appearance: Pale yellow liquid
Expiry Date: 31 March 2018
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Range-Finding Test
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

Definitive Test
Samples were taken from the control and each test group from the bulk test preparation at 0 hours, from additional samples incubated alongside at 24 and 48 hours, and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
The method of analysis,

Vehicle:

no

Details on test solutions:

Range-Finding Test
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 60 minutes and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (4.4 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
Nominal amounts of test item (89.5 and 100 mg) were each separately dissolved in culture medium with the aid of ultrasonication for approximately 30 minutes and the volumes adjusted to 500 mL and 1 liter to give the 179 and 100 mg/L stock solutions respectively. A series of dilutions was made from the 100 mg/L stock solution to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (4.1 mL) to give the required test concentrations of 1.0, 3.2, 10, 32, 100 and 179 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 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 10^4 to 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ±1 ºC throughout the test.
The temperature within the incubator was recorded daily.

pH:

The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter.
The pH value of the control cultures was observed to increase from pH 7.6 at 0 hours to pH 8.4 at 72 hours.
The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
The pH value of the definitive test was observed to increase from pH 7.5-8.2 at 0 hours to pH 8.3-9.0 at 72 hours.

Nominal and measured concentrations:

Range finding test
0.10, 1.0, 10 and 100 mg/L
Definitve test
Nominal: 1.0, 3.2, 10, 32, 100 and 179 mg/L
Measured: 0.59, 2.2, 8.3, 28, 90, 162 mg/L.

Details on test conditions:

Range-Finding Test
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Definitive Test
Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 3 flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.08 x 10^5 cells per mL. Inoculation of 500 mL of test medium with 4.1 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Assessments
Test Organism Observations
Samples were taken at 19, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
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.

Water Quality Criteria
The appearance of the test media was recorded daily.

Culture Medium
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 162 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

8.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

140 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

8.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 91 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Details on results:

Range-finding Test
The results showed no effect on growth rate at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.
Based on this information test concentrations of 1.0, 3.2, 10, 32, 100 and 179 mg/L, equivalent to 0.56, 1.8, 5.6, 18, 56 and 100 mg active ingredient (ai)/L) were selected for the definitive test.
Chemical analysis of the 1.0, 10 and 100 mg/L test preparations at 0 hours showed measured test concentrations to range from 99% to 105% of nominal. A decline in measured test concentration was observed at 72 hours in the range of 2% to 89% of nominal indicating that the test item was unstable under the conditions of the test.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours (see Annex 4) showed measured test concentrations to range from 0.90 to 162 mg/L. A concentration dependent decline in measured test concentrations was observed after each 24-Hour period in the range of 0.87 to 162 mg/L at 24 hours, 0.56 to 163 mg/L at 48 hours and from 0.055 to 164 mg/L at 72 hours.
Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
Accordingly the following results were determined from the data based on the geometric mean measured test concentrations:
Inhibition of Growth Rate
ErC10 (0 to 72 hour) : 66 mg/L (37 mg ai/L)
ErC20 (0 to 72 hour) : >162 mg/L (>91 mg ai/L)
ErC50 (0 to 72 hour) : >162 mg/L (>91 mg ai/L)
where ErCx is the test concentration that reduced growth rate by x%.
It was not possible to calculate ErC20 or ErC50 values as no more than 18% inhibition of growth rate occurred at the maximum test concentration employed.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis 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, 0.59 and 2.2 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 2.2 mg/L (1.2 mg ai/L). Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 8.3 mg/L (4.6 mg ai/L).
Inhibition of Yield
EyC10 (0 to 72 hour) : 1.2 mg/L (0.65 mg ai/L)
EyC20 (0 to 72 hour) : 6.8 mg/L (3.8 mg ai/L)
EyC50 (0 to 72 hour) : 140 mg/L (78 mg ai/L)
Where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 6.2.3. There were no statistically significant differences (P≥0.05), between the control, 0.59 and 2.2 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 2.2 mg/L (1.2 mg ai/L). Correspondingly the LOEC based on yield was 8.3 mg/L (4.6 mg ai/L).

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 310 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 1.55 x 10^6 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 11% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.59, 2.2 and 8.3 mg/L, however cell clumping was observed in the 28, 90 and 162 mg/L test cultures.

Observations on Test Item Solubility
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, 0.59, 2.2, 8.3 and 28 mg/L test cultures were observed to be green dispersions. The 90 mg/L test cultures were observed to be pale green dispersions whilst the 162 mg/L test cultures were observed to be slightly pale green dispersions.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

The geometric mean measured test concentrations were determined to be

Nominal Test Concentration		Geometric Mean Measured Test Concentration		Expressed as a Percentage of the Nominal Test Concentration (%)
mg/L	mg ai/L	mg/L	mg ai/L
1.0	0.56	0.59	0.33	59
3.2	1.8	2.2	1.2	69
10	5.6	8.3	4.6	83
32	18	28	16	88
100	56	90	50	90
179	100	162	91	91

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:
EC50 (mg/L):>162*
No Observed Effect Concentration (NOEC) (mg/L):2.2
Lowest Observed Effect Concentration (LOEC) (mg/L): 8.3
Yield
EC50 (mg/L): 140
No Observed Effect Concentration (NOEC) (mg/L): 2.2
Lowest Observed Effect Concentration (LOEC) (mg/L):8.3

Based on a test item active ingredient content of 56%, exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:
EC50 (mg/L):>91*
No Observed Effect Concentration (NOEC) (mg/L):1.2
Lowest Observed Effect Concentration (LOEC) (mg/L): 4.6
Yield
EC50 (mg/L): 78
No Observed Effect Concentration (NOEC) (mg/L): 1.2
Lowest Observed Effect Concentration (LOEC) (mg/L): 4.6

* It was not possible to calculate ErC50 values as no more than 18% inhibition of growth rate occurred at the maximum test concentration employed.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32, 100 and 179 mg/L, equivalent to 0.56, 1.8, 5.6, 18, 56 and 100 mg active ingredient (ai)/L) (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.90 to 162 mg/L. A concentration dependent decline in measured test concentrations was observed after each 24-Hour period in the range of 0.87 to 162 mg/L at 24 hours, 0.56 to 163 mg/L at 48 hours and from 0.055 to 164 mg/L at 72 hours.

Given this decline in measured test concentrations it was considered justifiable to base 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 0.59, 2.2, 8.3, 28, 90 and 162 mg/L (equivalent to 0.33, 1.2, 4.6, 16, 50 and 91 mg ai/L).

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC10 (mg/L)	EC20 (mg/L)	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	66	>162*	>162^*	2.2	8.3
Yield	1.2	6.8	140	2.2	8.3

Based on a test item active ingredient content of 56%, exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC10 (mg ai/L)	EC20 (mg ai/L)	EC50 (mg ai/L)	No Observed Effect Concentration (NOEC) (mg ai/L)	Lowest Observed Effect Concentration (LOEC) (mg ai/L)
Growth Rate	37	>91*	>91^*	1.2	4.6
Yield	0.65	3.8	78	1.2	4.6

*It was not possible to calculate E_rC₂₀or E_rC₅₀values as no more than 18% inhibition of growth rate occurred at the maximum test concentration employed

Description of key information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Based on a test item active ingredient content exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

EC50 >91 mg a.i./L)

No Observed Effect Concentration (NOEC) 1.2 mg a.i/L

It was not possible to calculate ErC50 values as no more than 18% inhibition of growth rate occurred at the maximum test concentration employed.

Key value for chemical safety assessment

EC50 for freshwater algae:

91 mg/L

EC10 or NOEC for freshwater algae:

1.2 mg/L