phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

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:

The study was conducted between 11 December 2018 and 08 March 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

EC Number:

828-215-2

Cas Number:

172796-84-8

Molecular formula:

C8H12NO7P2Na

IUPAC Name:

phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

impurity 1

Reference substance name:

4-aminophenylacetic acid

EC Number:

214-828-7

EC Name:

4-aminophenylacetic acid

Cas Number:

1197-55-3

Molecular formula:

C8H9NO2

IUPAC Name:

4-aminophenylacetic acid

impurity 2

Reference substance name:

EBP dimer

Molecular formula:

C16H22N2O12P4

IUPAC Name:

EBP dimer

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-,monosodium salt (“EBP”)
CAS Number: 172796-84-8
Batch: CHPC071917EBP
Purity: >98% pure
Physical State/Appearance: Beige colored powder
Expiry Date: 01 July 2019
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Remarks:

high performance liquid chromatography with tandem quadrapole mass spectrometry (HPLC-MS/MS)

Details on sampling:

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. All samples were taken for immediate analysis. A set of duplicate samples was taken at 0 and 72 hours and stored frozen for further analysis if necessary. Separate samples of the control and all test groups were taken from the inoculated preparations and incubated alongside the test vessels to provide samples for 24 and 48 hours, which were stored frozen for analysis if required.

Test solutions

Vehicle:

no

Details on test solutions:

Range-Finding Tests
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for 20 minutes and shaking by hand before the volume was adjusted to 500 mL to give a 100 mg/L stock solution. A series of dilutions was made to give further stock solutions of 0.10, 1.0 and 10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.1 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
The results of the first range-finding test showed significant inhibition of growth occurred at all test concentrations employed and a NOEC was not achieved. Therefore a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal test concentrations of 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for 20 minutes and shaking by hand before the volume was adjusted to 500 mL to give a 100 mg/L stock solution. A series of dilutions was made to give further stock solutions of 0.0010, 0.010, 0.10, 1.0 and 10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (1.5 mL) to give the required test concentrations of 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/L.

Initial Experiment
Based on the results of the second range-finding test the following test concentrations were assigned to an initial test (originally planned as the definitive test): 1.0, 3.2, 10, 32 and 100 mg/L.
A nominal amount of test item (200 mg) was dissolved in culture medium, and the volume adjusted to 2000 mL to give a 100 mg/L stock solution. This stock solution was sonicated for 10 minutes and shaken by hand, from which a series of dilutions was made to give further stock solutions of 1.0, 3.2, 10 and 32 mg/L. One liter of each of the stock solutions was separately inoculated with algal suspension (8.6 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The test was conducted in 250 mL glass conical flasks. Six flasks each containing 100 mL of solution were used for the control and three flasks each containing 100 mL were used for each treatment group.

Definitive Test
Based on the results of the range-finding tests and initial experiment the following test concentrations were assigned to the definitive test: 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.

Experimental Preparation
A nominal amount of test item (200 mg) was dissolved in culture medium, and the volume adjusted to 2000 mL to give a 100 mg/L stock solution. This stock solution was sonicated for 15 minutes and shaken by hand. A series of dilutions was made from the 100 mg/L stock solution to give further stock solutions of 1.0, 3.2, 10 and 32 mg/L; these were made up to 1 liter in volume except for the 1.0 mg/L preparation which was made to 2 liters as this preparation was used as a stock to prepare the lower test concentrations of 0.032, 0.10 and 0.32 mg/L, each made to a total volume of 1 liter. An aliquot (1 liter) of each of the stock solutions was separately inoculated with algal suspension (7.2 mL) to give the required test concentrations of 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solutions and each of the prepared concentrations 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 and 72 hours.

Test organisms

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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ±1 °C

pH:

The pH value of the control cultures was observed to be pH 7.8 at 0 and 72 hours. There was no pH deviation in the control cultures after 72 hours and therefore was within the limits (less than 1.5 pH units) given in the Test Guidelines.
The test item solutions were tested without pH adjustment. At 95 mg/L the pH was 5.7 and 5.8 at 0 and 72 hours respectively; as significant levels of inhibition were also observed at 8.6 and 31 mg/L, where pH values were within the range not expected to have an impact on algal growth, it was considered that the effects exhibited were primarily attributable to the test item.

Nominal and measured concentrations:

Range-Finding Tests:
nominal: 0.10, 1.0, 10 and 100 mg/L

Second Range-Finding Test:
Nominal: 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/L

Initial Experiment:
Nominal: 1.0, 3.2, 10, 32 and 100 mg/L

Definitive Test:
Nominal: 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L

Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.032 to 98 mg/L; between 92% and 103% of the nominal concentrations. At 72 hours test concentrations ranged from 0.015 to 93 mg/L (between 48% and 93% of the nominal concentrations) with the greatest losses occurring at the lower concentrations of 0.032, 0.10 and 0.32 mg/L, to between 49% and 69% of their starting concentrations. At 1.0, 3.2, 32 and 100 mg/L, concentrations were maintained between 86% and 97% of their starting concentrations and at 10 mg/L to the slightly lower level of 76% of the starting concentration. The 24 and 48 hour samples were not analyzed because a decline in measured test concentration was not seen in all the test concentrations. 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.
The geometric mean measured test concentrations were determined to be:
Nominal Test Concentration (mg/L) Geometric Mean Measured Test Concentration (mg/L) Expressed as a Percentage of the Nominal Test Concentration (%)
0.032 0.0220 69
0.10 0.0826 83
0.32 0.254 79
1.0 0.957 96
3.2 2.90 91
10 8.62 86
32 30.5 95
100 95.4 95

Details on test conditions:

Culture Medium
The culture medium used for the range-finding tests, initial experiment and definitive test was the same as that used to maintain the stock culture.
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 ±0.1 with 0.1N NaOH or HCl.

Range-Finding Tests:
The test concentrations to be used in the initial experiment were determined by two preliminary range-finding tests. The first range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.
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.
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for 20 minutes and shaking by hand before the volume was adjusted to 500 mL to give a 100 mg/L stock solution. A series of dilutions was made to give further stock solutions of 0.10, 1.0 and 10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.1 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
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. A sample of each test concentration was taken for immediate chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. Duplicate samples were taken on each occasion and stored for further analysis if required.
The results of the first range-finding test showed significant inhibition of growth occurred at all test concentrations employed and a NOEC was not achieved. Therefore a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal test concentrations of 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for 20 minutes and shaking by hand before the volume was adjusted to 500 mL to give a 100 mg/L stock solution. A series of dilutions was made to give further stock solutions of 0.0010, 0.010, 0.10, 1.0 and 10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (1.5 mL) to give the required test concentrations of 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/L.
The control group was maintained under identical conditions but not exposed to the test item.
Exposure conditions in the second range-finding test were the same as those in the first range-finding test. No analytical samples were taken for the second range-finding test since stability of the test item had already been established during the first range-finding test.

Initial Experiment
Based on the results of the second range-finding test the following test concentrations were assigned to an initial test (originally planned as the definitive test): 1.0, 3.2, 10, 32 and 100 mg/L.
A nominal amount of test item (200 mg) was dissolved in culture medium, and the volume adjusted to 2000 mL to give a 100 mg/L stock solution. This stock solution was sonicated for 10 minutes and shaken by hand, from which a series of dilutions was made to give further stock solutions of 1.0, 3.2, 10 and 32 mg/L. One liter of each of the stock solutions was separately inoculated with algal suspension (8.6 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The test was conducted in 250 mL glass conical flasks. Six flasks each containing 100 mL of solution were used for the control and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained concurrently under identical conditions but was not exposed to the test item.
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.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
A NOEC was not achieved from the initial experiment therefore the definitive test was conducted with lower test concentrations and the findings of the second range-finding test were considered to have been anomalous.

Definitive Test
Based on the results of the range-finding tests and initial experiment the following test concentrations were assigned to the definitive test: 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.

Experimental Preparation
A nominal amount of test item (200 mg) was dissolved in culture medium, and the volume adjusted to 2000 mL to give a 100 mg/L stock solution. This stock solution was sonicated for 15 minutes and shaken by hand. A series of dilutions was made from the 100 mg/L stock solution to give further stock solutions of 1.0, 3.2, 10 and 32 mg/L; these were made up to 1 liter in volume except for the 1.0 mg/L preparation which was made to 2 liters as this preparation was used as a stock to prepare the lower test concentrations of 0.032, 0.10 and 0.32 mg/L, each made to a total volume of 1 liter. An aliquot (1 liter) of each of the stock solutions was separately inoculated with algal suspension (7.2 mL) to give the required test concentrations of 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solutions and each of the prepared concentrations 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 and 72 hours.

Exposure Conditions
As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of culture media were used for the control and three flasks each containing 100 mL of test preparation 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 6.95 x 10^5 cells per mL. Inoculation of 1 liter of test medium with 7.2 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.96 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Tests
For the first range-finding test the results showed inhibition of growth at all the test concentrations. Therefore, as a NOEC was not achieved the second range-finding test was conducted using an extended range of 0.0010, 0.010, 0.10, 1.0, 10, 100 mg/L.
Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near to nominal indicating that the test item was stable under test conditions.
For the second range-finding test the results showed no significant inhibition at 0.0010, 0.010 and 1.0 mg/L. For 0.10 mg/L the yield was reduced by 13% , however as yield was only inhibited by 5% at the higher concentration of 1.0 mg/L the inhibition at 0.10 mg/L was considered not to be related to test item effects. No analytical samples were taken as stability had already been determined in the first range-finding test.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the initial experiment.

Initial Experiment
In this experiment the results showed inhibition of growth at all the test concentrations. Therefore as a NOEC was not achieved the definitive test was conducted using the extended range of 0.032, 0.1, 0.32, 1.0, 3.2, 10, 32, 100 mg/L. The results of the second range-finding test are believed to have been anomalous and as there was no analysis of samples of the test media, the measured test item concentrations for the second range-finding test cannot be confirmed.
Chemical analysis of the test preparations at 0 hours (not reported) showed the measured test item concentrations to be near to nominal, confirming the test system had been correctly dosed. No samples were taken for analysis at 72 hours since further testing would be required.

Growth 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) : 1.2 mg/L
ErC20 (0 to 72 hour) : 4.1 mg/L
ErC50 (0 to 72 hour) : 34 mg/L*
where ErCx is the test concentration that reduced growth rate by x%.
* 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

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 and 0.022 to 0.96 mg/L test concentrations; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.96 mg/L. Correspondingly the LOEC based on growth rate was 2.9 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour) : 0.17 mg/L
EyC20 (0 to 72 hour) : 0.45 mg/L
EyC50 (0 to 72 hour) : 2.5 mg/L; 95% confidence limits 1.8 to 3.5 mg/L
where EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences (P≥0.05) between the control, 0.022 and 0.083 mg/L test concentrations; however, all other test concentrations were significantly different (P<0.05), therefore the NOEC based on yield was 0.083 mg/L. Correspondingly the LOEC based on yield was 0.25 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 193 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 : 9.63 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 6% 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 0.022, 0.083, 0.25 and 0.96 mg/L. Some misshapen cells were observed to be present in the test cultures at 2.9 and 8.6 mg/L whilst lots of cell debris and no intact cells visible, was observed in the test cultures at 31 and 95 mg/L.

Water Quality Criteria
Temperature was maintained at 24 ±1 ºC throughout the test.
The pH value of the control cultures was observed to be pH 7.8 at 0 and 72 hours. There was no pH deviation in the control cultures after 72 hours and therefore was within the limits (less than 1.5 pH units) given in the Test Guidelines.
The test item solutions were tested without pH adjustment. At 95 mg/L the pH was 5.7 and 5.8 at 0 and 72 hours respectively; as significant levels of inhibition were also observed at 8.6 and 31 mg/L, where pH values were within the range not expected to have an impact on algal growth, it was considered that the effects exhibited were primarily attributable to the test item.

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, 0.022 and 0.083 mg/L test cultures were observed to be green dispersions, whilst the 0.25, 0.96 and 2.9 mg/L test cultures were very pale green or pale green dispersions and the 8.6 mg/L test cultures were an extremely pale green dispersions. The 31 mg/L test cultures were observed to be clear colorless solutions and the 95 mg/L test cultures were observed to be light peach colored solutions.

Results with reference substance (positive control):

A positive control (Envigo study number YJ31TQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control was conducted between 12 November 2018 and 03 December 2018.
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) : 1.5 mg/L; 95% confidence limits 1.3 to 1.7 mg/L
EyC50 (0 to 72 hour) : 0.76 mg/L; 95% confidence limits 0.69 to 0.85 mg/L
No Observed Effect Concentration based on growth rate: 0.50 mg/L
No Observed Effect Concentration based on yield: 0.50 mg/L
Lowest Observed Effect Concentration based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration based on yield: 1.0 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item*.

Any other information on results incl. tables

Cell Densities and Percentage Inhibition of Growth from the First Range-finding Test

Nominal Concentration (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	6.10E+03	1.51E+06	-	-
	R2	5.78E+03	1.05E+06
	Mean	5.94E+03	1.28E+06
0.10	R1	5.46E+03	8.65E+05	11	42
	R2	6.48E+03	6.33E+05
	Mean	5.97E+03	7.49E+05
1.0	R1	7.60E+03	4.27E+05	24	64
	R2	8.27E+03	5.16E+05
	Mean	7.93E+03	4.71E+05
10	R1	7.57E+03	1.39E+05	47	91
	R2	6.60E+03	1.14E+05
	Mean	7.08E+03	1.27E+05
100	R1	9.59E+03	2.14E+04	83	99
	R2	6.86E+03	1.98E+04
	Mean	8.23E+03	2.06E+04

*       Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from              3 counts for each of the replicate flasks

R      = Replicate

-       =Not applicable

Cell Densities and Percentage Inhibition of Growth from the Second Range-finding Test

Nominal Concentration (mg/L)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	6.63E+03	3.02E+05	-	-
	R2	4.81E+03	1.02E+06
	Mean	5.72E+03	6.61E+05
0.0010	R1	4.81E+03	8.06E+05	[11]	[38]
	R2	4.99E+03	1.02E+06
	Mean	4.90E+03	9.12E+05
0.010	R1	6.89E+03	1.10E+06	[8]	[69]
	R2	6.37E+03	1.13E+06
	Mean	6.63E+03	1.12E+06
0.10	R1	6.78E+03	8.37E+05	[3]	[35]
	R2	6.10E+03	9.46E+05
	Mean	6.44E+03	8.92E+05
1.0	R1	6.75E+03	1.08E+06	[5]	[46]
	R2	6.86E+03	8.55E+05
	Mean	6.81E+03	9.66E+05
10	R1	5.51E+03	2.96E+05	24	71
	R2	5.31E+03	9.27E+04
	Mean	5.41E+03	1.95E+05
100	R1	7.27E+03	4.08E+04	64	95
	R2	5.87E+03	3.15E+04
	Mean	6.57E+03	3.61E+04

Note: Control replicate 1 data was excluded from calculations, however is shown for completeness.

*        Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R      = Replicate

-       = Not applicable

[ ]    = Increase in growth compared to controls

Inhibition of Growth Rate and Yield in the Initial Experiment

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.069	-	7.14E+05	-
	R2	0.068		6.49E+05
	R3	0.069		7.15E+05
	R4	0.069		7.15E+05
	R5	0.070		7.48E+05
	R6	0.069		6.92E+05
	Mean	0.069		7.06E+05
	SD	0.001		3.32E+04
1.0	R1	0.060	13	3.79E+05
	R2	0.063	9	4.57E+05
	R3	0.056	19	2.85E+05
	Mean	0.060	14	3.74E+05	47
	SD	0.004		8.60E+04
3.2	R1	0.057	17	3.09E+05
	R2	0.059	14	3.57E+05
	R3	0.043	38	1.08E+05
	Mean	0.053	23	2.58E+05	63
	SD	0.009		1.32E+05
10	R1	0.042	39	1.00E+05
	R2	0.042	39	9.99E+04
	R3	0.040	42	8.17E+04
	Mean	0.041	40	9.39E+04	87
	SD	0.001		1.06E+04
32	R1	0.040	42	8.41E+04
	R2	0.040	42	8.39E+04
	R3	0.042	39	9.69E+04
	Mean	0.041	41	8.83E+04	87
	SD	0.001		7.48E+03
100	R1	0.031	55	4.19E+04
	R2	0.029	58	3.41E+04
	R3	0.032	54	4.69E+04
	Mean	0.031	56	4.10E+04	94
	SD	0.002		6.41E+03

*      In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R      = Replicate

SD   = Standard Deviation

-       = Not applicable

Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Concentration (mg/L)		pH	Cell Densities*(cells per mL)			pH
		0 Hours	24 Hours	48 Hours	72 Hours	72 Hours
Control	R1	7.8	2.95E+04	1.69E+05	1.12E+06	7.8
	R2		2.63E+04	1.59E+05	9.30E+05
	R3		2.31E+04	1.54E+05	8.61E+05
	R4		3.09E+04	1.60E+05	9.11E+05
	R5		3.10E+04	1.66E+05	1.03E+06
	R6		2.57E+04	1.63E+05	9.45E+05
	Mean		2.78E+04	1.62E+05	9.65E+05
0.022	R1	7.6	2.53E+04	1.50E+05	8.97E+05	7.9
	R2		3.14E+04	1.50E+05	9.05E+05
	R3		2.71E+04	1.28E+05	7.00E+05
	Mean		2.80E+04	1.43E+05	8.34E+05
0.083	R1	7.4	2.78E+04	1.67E+05	1.11E+06	7.9
	R2		2.82E+04	1.36E+05	7.23E+05
	R3		2.94E+04	1.62E+05	8.14E+05
	Mean		2.85E+04	1.55E+05	8.82E+05
0.25	R1	7.4	2.81E+04	1.75E+05	7.64E+05	7.8
	R2		2.50E+04	1.66E+05	7.81E+05
	R3		2.59E+04	1.40E+05	6.79E+05
	Mean		2.64E+04	1.61E+05	7.41E+05
0.96	R1	7.3	2.59E+04	1.59E+05	7.52E+05	7.7
	R2		2.92E+04	1.53E+05	7.64E+05
	R3		2.64E+04	1.52E+05	5.26E+05
	Mean		2.72E+04	1.55E+05	6.81E+05

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R   = Replicate

Geometric mean Measured Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	24 Hours	48 Hours	72 Hours	72 Hours
2.9	R1	7.2	2.73E+04	1.73E+05	6.57E+05	7.7
	R2		3.12E+04	1.54E+05	6.50E+05
	R3		2.89E+04	1.25E+05	4.80E+05
	Mean		2.91E+04	1.51E+05	5.96E+05
8.6	R1	7.0	3.01E+04	1.13E+05	2.87E+05	7.5
	R2		2.23E+04	5.03E+04	1.19E+05
	R3		2.10E+04	4.33E+04	9.32E+04
	Mean		2.44E+04	6.90E+04	1.66E+05
31	R1	6.5	1.86E+04	3.37E+04	6.98E+04	7.2
	R2		1.92E+04	3.53E+04	6.49E+04
	R3		2.19E+04	3.18E+04	6.48E+04
	Mean		1.99E+04	3.36E+04	6.65E+04
95	R1	5.7	1.20E+04	1.69E+04	2.85E+04	5.8
	R2		1.19E+04	2.02E+04	3.91E+04
	R3		1.04E+04	2.21E+04	4.12E+04
	Mean		1.14E+04	1.97E+04	3.63E+04

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R   = Replicate

Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Treatment		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.074	0.073	0.078
	R2	0.069	0.075	0.073
	R3	0.064	0.079	0.072
	R4	0.076	0.068	0.073
	R5	0.076	0.070	0.076
	R6	0.068	0.077	0.073
	Mean	0.071	0.074	0.074

R   = Replicate

Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.075	-	1.11E+06	-
	R2	0.073		9.25E+05
	R3	0.072		8.56E+05
	R4	0.072		9.06E+05
	R5	0.074		1.02E+06
	R6	0.073		9.40E+05
	Mean	0.073		9.60E+05
	SD	0.001		9.18E+04
0.022	R1	0.072	1	8.92E+05
	R2	0.072	1	9.00E+05
	R3	0.069	5	6.95E+05
	Mean	0.071	2	8.29E+05	14
	SD	0.002		1.16E+05
0.083	R1	0.075	[3]	1.11E+06
	R2	0.069	5	7.18E+05
	R3	0.071	3	8.09E+05
	Mean	0.072	2	8.77E+05	9
	SD	0.003		2.03E+05
0.25	R1	0.070	4	7.59E+05
	R2	0.070	4	7.76E+05
	R3	0.068	7	6.74E+05
	Mean	0.069	5	7.36E+05	23
	SD	0.001		5.45E+04
0.96	R1	0.070	4	7.47E+05
	R2	0.070	4	7.59E+05
	R3	0.065	11	5.21E+05
	Mean	0.068	6	6.76E+05	29
	SD	0.003		1.34E+05

*        In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R      =Replicate

SD   = Standard Deviation

[ ]    = Increase in growth compared to controls

-       = Not applicable

Geometric Mean Measured Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
2.9	R1	0.068	7	6.52E+05
	R2	0.068	7	6.45E+05
	R3	0.063	14	4.75E+05
	Mean	0.066	9	5.91E+05	38
	SD	0.003		1.00E+05
8.6	R1	0.056	23	2.82E+05
	R2	0.044	40	1.14E+05
	R3	0.041	44	8.82E+04
	Mean	0.047	36	1.61E+05	83
	SD	0.008		1.05E+05
31	R1	0.037	49	6.48E+04
	R2	0.036	51	5.99E+04
	R3	0.036	51	5.98E+04
	Mean	0.036	50	6.15E+04	94
	SD	0.001		2.87E+03
95	R1	0.024	67	2.35E+04
	R2	0.029	60	3.41E+04
	R3	0.029	60	3.62E+04
	Mean	0.027	62	3.13E+04	97
	SD	0.003		6.82E+03

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R   = Replicate

Applicant's summary and conclusion

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:
Response Variable EC50 (mg/L) 95% Confidence Limits (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate 34 Not determined* 0.96 2.9
Yield 2.5 1.8 - 3.5 0.083 0.25
* 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

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" and Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Following preliminary range-finding tests and an initial experiment, Pseudokirchneriella subcapitata was exposed to aqueous solutions of the test item at concentrations of 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/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 0.032 to 98 mg/L (between 92% and 103% of the nominal concentrations). At 72 hours test concentrations ranged from 0.015 to 93 mg/L (between 48% and 93% of the nominal values), with the greatest losses occurring at 0.032, 0.10 and 0.32 mg/L (between 49% and 69% of their starting 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. The geometric mean measured concentrations were 0.022, 0.083, 0.25, 0.96, 2.9, 8.6, 31 and 95 mg/L.

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

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	34	Not determined*			0.96	2.9
Yield	2.5	1.8	-	3.5	0.083	0.25

*It was not possible to calculate 95% confidence limits for the E_rC₅₀value as the data generated did not fit the models available for the calculation of confidence limits