Barium, 1,4-bis (C13-rich C11-14-isoalkyl) 2-sulfobutanedioate phosphate complex

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 14 May 2008 and 5 June 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies,which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/l) of test material in culture medium for a period of 48 hours prior to removing any undissolved test material present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test material.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21/08/2007 Date of Signature: 15/10/2007

Test material

Test material information

Constituent 1

Reference substance name:

dibarium(2+) bis(1,5-dioxo-1,5-bis(tridecyloxy)pentane-3-sulfonate) hydrogen phosphate

EC Number:

931-037-9

Molecular formula:

Molecular formula for each combination of chain length. C13,13: (C30H57O7S)2 Ba @ BaHOP4 C11,12: (C27H51O7S)2 Ba @ BaHOP4 C12,12: (C28H53O7S)2 Ba @ BaHOP4 C12,13: (C29H55O7S)2 Ba @ BaHOP4 C13,14: (C31H59O7S)2 Ba @ BaHOP4 C14,14: (C32H61O7S)2 Ba @ BaHOP4

IUPAC Name:

dibarium(2+) bis(1,5-dioxo-1,5-bis(tridecyloxy)pentane-3-sulfonate) hydrogen phosphate

Details on test material:

Sponsor's identification: Barium di(bistridecylsulfosuccinate) in mixture with Barium hydrogen phosphate
Description : pale yellow solid
Batch number : Y-T-1
Storage conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
A range of standard solutions covering 0.21 to 51 mg/l (exceeding the range of the working sample concentrations) was analysed.

- Sampling method:

The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of a single peak.
The method was developed by the Department of Analytical Services, Safepharm Laboratories Limited.


- Sample storage conditions before analysis:

Storage conditions : room temperature in the dark

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:

An amount of test material (1100 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
An aliquot (1 litre) of this saturated solution was inoculated with algal suspension (27 ml) to give the required test concentration of 100% v/v saturated solution.
The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0 and 72 hours (see Appendix 2 - attached background material).


- Eluate:
Not applicable

- Controls:

A positive control (Safepharm Laboratories Project Number: 0039/0994) used potassium dichromate as the reference material. An amount of reference material (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/l stock solution from which a series of dilutions were made to give further stock solutions of 10, 2.0, 1.0, 0.50, 0.25 and 0.125 mg/l. An aliquot (250 ml) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/l stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
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. Six replicate flasks were prepared for the control and three replicate flasks prepared for each test concentration.
The flasks were incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


- Chemical name of vehicle :
culture medium.


- Concentration of vehicle in test medium:
An aliquot (250 ml) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/l stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

- Evidence of undissolved material :
None recorded

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
Green Alga


- Strain:
Strain CCAP 276/20

- Source:
Obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Age of inoculum :
Not recorded

- Method of cultivation:
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 10E3 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 deg C until the algal cell density was approximately 10E4 - 10E5 cells/ml.

ACCLIMATION

- Acclimation period:
Not recorded.

- Culturing media and conditions:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 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
Na2SeO3.5H2O 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

* Elga Optima 15+ or Elga Purelab Option R-15 BP

- Any deformed or abnormal cells observed:
None recorded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

Observations on test material solubility
At the start of the test all control and test cultures were observed to be clear colourless solutions. There was a slight foam observed at the media surface in the test cultures. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Test conditions

Hardness:

Not recorded.

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

The pH values of the control cultures (see Table 2 - see results section) were observed to increase from pH 7.4 – 7.5 at 0 hours to pH 7.8 – 7.9 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.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

Range-finding test: nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
Definitive test: 100% v/v saturated solution.
Based on the result of the range-finding test a "limit test" was conducted. The test material solution for the definitive test was prepared by stirring an excess (100 mg/l) of the test material in culture medium for a period of time and then removing any undissolved test material by filtration to give a saturated solution.

Details on test conditions:

TEST SYSTEM
- Test vessel:
250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 250ml,
- Aeration: No

- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable

- Renewal rate of test solution (frequency/flow rate): Not applicable

- Initial cells density: 4 x 10E3 cells/ml

- Control end cells density: 3.60

- No. of organisms per vessel:
At initiation of the test the culture contained a nominal cell density of 4 x 10E3 cells per ml.

- No. of vessels per concentration (replicates):
six, each containing 100 ml of test preparation.

- No. of vessels per control (replicates):
Six, each containing 100 ml of test preparation.


GROWTH MEDIUM
- Standard medium used: yes
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.


TEST MEDIUM

Culture Medium
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 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
Na2SeO3.5H2O 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.




OTHER TEST CONDITIONS
- Sterile test conditions: yes

- Adjustment of pH:
The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Photoperiod:
Not recorded

- Light intensity and quality:
warm white lighting (380 – 730 nm)

EXPOSURE CONDITIONS :

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.47 x 10E5 cells per ml. Inoculation of 1 litre of test medium with 27 ml of this algal suspension gave an initial nominal cell density of 4 x 10E3 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 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

- Chlorophyll measurement:
Not recorded

TEST CONCENTRATIONS


Samples were taken from the control (replicates R1 - R6 pooled) and the 100% v/v saturated solution test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20ºC for further analysis if necessary.
The method of analysis, stability, recovery and test solution analyses are described in Appendix 2 (see attached background material).



- Range finding study:

Information provided by the Sponsor indicated that the water solubility of the test material was very low. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the material using traditional methods of preparation e.g. ultrasonication and high shear mixing. Therefore, as the test material is a preparation, it was considered appropriate to prepare the test material using a saturated solution method of preparation.

The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.

An amount of test material (1100 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (250 ml) of each of the stock solutions was separately inoculated with algal suspension (1.5 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

The test was conducted in 250 ml glass control 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 control group was maintained under identical conditions but not exposed to the test material.

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 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Cl not stated

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL not stated

Details on results:

RESULTS:
Range-finding Test:
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in Table 1.
The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
Based on this information a “Limit test” was conducted for the definitive test. The test material solution for the definitive test was prepared by stirring an excess (100 mg/l) of test material in culture medium for a period of time and then removing any undissolved test material by filtration to give a saturated solution.

Definitive Test:
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rates, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.
The mean cell densities versus time for the definitive test are presented in Figure 1 (see attached background material).

Validation criteria:
The following data show that the cell concentration of the control cultures increased by a factor of 94 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.
Mean cell density of control at 0 hours : 4.34 x 10E3 cells per ml
Mean cell density of control at 72 hours : 4.07 x 10E5 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 23% 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 – 72 h) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Growth data:
From the data given in Tables 2 and 4, it is clear that the growth rate (r), yield (y) and biomass (b) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test material over the 72-Hour exposure period.
Accordingly the following results were determined from the data:

Inhibition of growth rate:
ErC10 (0 - 72 h) : > 100% v/v saturated solution
ErC20 (0 - 72 h) : > 100% v/v saturated solution
ErC50 (0 - 72 h) : > 100% v/v saturated solution

where ErCx is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100% v/v saturated solution test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P≥0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100% v/v saturated solution.

Inhibition of yield:
EyC10 (0 - 72 h) : > 100% v/v saturated solution
EyC20 (0 - 72 h) : > 100% v/v saturated solution
EyC50 (0 - 72 h) : > 100% v/v saturated solution

where EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as for inhibition of growth rate. There were no statistically significant differences (P≥0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100% v/v saturated solution.

Inhibition of biomass integral:
EbC10 (0 - 72 h) : > 100% v/v saturated solution
EbC20 (0 - 72 h) : > 100% v/v saturated solution
EbC50 (0 - 72 h) : > 100% v/v saturated solution

where EbCx is the test concentration that reduced biomass integral (area under the growth curve) by x%.

Statistical analysis of the biomass integral data was carried out as for inhibition of growth rate. There were no statistically significant differences (P≥0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 100% v/v saturated solution.

Observations on cultures:
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

Observations on test material solubility:
At the start of the test all control and test cultures were observed to be clear colourless solutions. There was a slight foam observed at the media surface in the test cultures. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Physico-chemical measurements:
The pH values of each test and control flask are given in Table 2. Temperature was maintained at 24 ± 1ºC throughout the test.
The pH values of the control cultures (see Table 2) were observed to increase from pH 7.4 – 7.5 at 0 hours to pH 7.8 – 7.9 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

Verification of test concentrations:
Analysis of the test preparations at 0 and 72 hours (see attached background material - Appendix 2) showed measured test concentrations to be less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.21 mg/l.
This does not infer that no test material was in solution but that the dissolved concentration (i.e. bioavailable to the test organisms) was below the limit of quantitation. As such the results are based on nominal test concentrations only.

Results with reference substance (positive control):

The cell densities from exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material during the positive control (Safepharm Laboratories Project No: 0039/0994) are given in Table 5 and Figure 2. Daily specific growth rates for the control cultures are given in Table 6 whilst growth rates, yield and biomass integral values are given in Table 7 (see attached background material - attachment 3). Percentage inhibition values are plotted against test concentration in Figure 3, Figure 4 and Figure 5 (see attached background material)
Accordingly the following results were determined from the data:
ErC50 (0 - 72 h) : 0.57 mg/l; 95% confidence limits 0.48 - 0.66 mg/l
EyC50 (0 - 72 h) : 0.32 mg/l; 95% confidence limits 0.29 - 0.35 mg/l
EbC50 (0 - 72 h) : 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l
No Observed Effect Concentration (NOEC) based on growth rate : 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on biomass integral : 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.125 mg/l Lowest Observed Effect Concentration (LOEC) based on biomass integral : 0.125 mg/l

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

Reported statistics and error estimates:

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) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Table1               Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (% v/v saturated solution)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield/Biomass Integral
Control	R1	3.79E+03	2.34E+05
	R2	3.47E+03	1.29E+05	-	-
	Mean	3.63E+03	1.82E+05
0.10	R1	3.96E+03	1.97E+05
	R2	3.61E+03	2.04E+05	[2]	[10]
	Mean	3.79E+03	2.00E+05
1.0	R1	3.93E+03	2.37E+05
	R2	3.71E+03	1.71E+05	[2]	[12]
	Mean	3.82E+03	2.04E+05
10	R1	3.53E+03	1.81E+05
	R2	3.57E+03	1.28E+05	4	15
	Mean	3.55E+03	1.54E+05
100	R1	3.66E+03	2.19E+05
	R2	3.56E+03	1.55E+05	[2]	[3]
	Mean	3.61E+03	1.87E+05

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 fields of view for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2 [Increase in growth compared to controls].

Table 2               Cell Densities and pH Values in the Definitive Test

Nominal Concentration (% v/v saturated solution)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.5	4.80E+03	3.78E+04	1.05E+05	3.99E+05	7.9
	R2	7.4	4.19E+03	2.53E+04	8.74E+04	4.00E+05	7.9
	R3	7.4	4.44E+03	2.06E+04	6.77E+04	4.01E+05	7.9
	R4	7.4	4.02E+03	1.99E+04	5.84E+04	3.60E+05	7.8
	R5	7.4	4.22E+03	2.13E+04	8.07E+04	4.43E+05	7.9
	R6	7.4	4.40E+03	2.38E+04	8.05E+04	4.41E+05	7.9
	Mean		4.34E+03	2.48E+04	7.99E+04	4.07E+05
100	R1	7.0	4.46E+03	2.58E+04	1.34E+05	4.95E+05	8.0
	R2	6.9	4.74E+03	2.35E+04	1.17E+05	4.67E+05	8.0
	R3	6.9	4.70E+03	2.11E+04	1.16E+05	3.92E+05	8.0
	R4	7.0	3.39E+03	2.35E+04	1.23E+05	5.02E+05	8.1
	R5	7.0	4.87E+03	2.12E+04	1.10E+05	4.13E+05	8.1
	R6	6.9	4.43E+03	2.03E+04	1.12E+05	3.74E+05	8.1
	Mean		4.43E+03	2.26E+04	1.18E+05	4.40E+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.

R₁- R₆= Replicates 1 to 6

Table 3               Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/ml/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.094	0.042	0.056
	R2	0.077	0.052	0.063
	R3	0.068	0.049	0.074
	R4	0.067	0.045	0.076
	R5	0.070	0.056	0.071
	R6	0.074	0.051	0.071
	Mean	0.075	0.049	0.069

R₁- R₆= Replicates 1 to 6

Table 4               Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test

Nominal Concentration (% v/v saturated solution)		Growth Rate (cells/ml/hour)		Yield (cells/ml)		Biomass Integral
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*	0 – 72 h	% Inhibition
Control	R1	0.064		3.94E+05		7.97E+06
	R2	0.064		3.96E+05		7.27E+06
	R3	0.064		3.97E+05		6.69E+06
	R4	0.062	-	3.56E+05	-	5.96E+06	-
	R5	0.065		4.39E+05		7.53E+06
	R6	0.065		4.36E+05		7.55E+06
	Mean	0.064		4.03E+05		7.16E+06
	SD	0.001		3.10E+04		7.25E+05
100	R1	0.067	[5]	4.90E+05		9.52E+06	[33]
	R2	0.066	[3]	4.62E+05		8.73E+06	[22]
	R3	0.064	0	3.87E+05		7.75E+06	[8]
	R4	0.067	[5]	4.98E+05		9.30E+06	[30]
	R5	0.064	0	4.08E+05		7.86E+06	[10]
	R6	0.063	2	3.70E+05		7.42E+06	[4]
	Mean	0.065	[2]	4.36E+05	[8]	8.43E+06	[18]
	SD	0.002		5.47E+04		8.77E+05

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

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

Table 5               Cell Densities and pH Values in the Positive Control

Nominal Concentration (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.7	4.03E+03	1.92E+04	6.48E+04	4.29E+05	7.8
	R2	7.7	4.03E+03	2.09E+04	6.48E+04	3.62E+05	7.8
	R3	7.6	3.99E+03	2.41E+04	6.66E+04	4.00E+05	7.9
	R4	7.6	4.23E+03	1.90E+04	6.18E+04	3.29E+05	7.8
	R5	7.6	3.95E+03	2.07E+04	6.15E+04	4.35E+05	7.8
	R6	7.5	4.03E+03	1.89E+04	6.75E+04	4.30E+05	7.8
	Mean		4.04E+03	2.05E+04	6.45E+04	3.98E+05
0.0625	R1	7.5	3.92E+03	2.71E+04	6.70E+04	4.44E+05	7.7
	R2	7.5	4.17E+03	2.06E+04	7.91E+04	3.19E+05	7.8
	R3	7.5	4.16E+03	2.24E+04	5.72E+04	3.24E+05	7.8
	Mean		4.08E+03	2.33E+04	6.78E+04	3.62E+05
0.125	R1	7.5	4.00E+03	1.81E+04	5.20E+04	3.16E+05	7.7
	R2	7.4	4.16E+03	1.93E+04	6.09E+04	2.79E+05	7.7
	R3	7.4	4.31E+03	1.67E+04	5.48E+04	3.43E+05	7.7
	Mean		4.16E+03	1.80E+04	5.59E+04	3.13E+05
0.25	R1	7.4	4.24E+03	1.81E+04	3.39E+04	2.52E+05	7.7
	R2	7.4	4.30E+03	1.52E+04	4.32E+04	3.37E+05	7.7
	R3	7.4	3.99E+03	1.66E+04	4.46E+04	3.27E+05	7.7
	Mean		4.18E+03	1.67E+04	4.06E+04	3.05E+05
0.50	R1	7.4	3.88E+03	8.34E+03	2.03E+04	4.89E+04	7.6
	R2	7.4	4.02E+03	8.77E+03	1.76E+04	6.31E+04	7.5
	R3	7.4	4.13E+03	9.23E+03	1.64E+04	5.61E+04	7.5
	Mean		4.01E+03	8.78E+03	1.81E+04	5.60E+04
1.0	R1	7.4	4.09E+03	7.92E+03	7.80E+03	9.56E+03	7.4
	R2	7.4	4.10E+03	6.76E+03	6.40E+03	7.90E+03	7.4
	R3	7.3	4.16E+03	7.02E+03	8.71E+03	9.76E+03	7.4
	Mean		4.12E+03	7.23E+03	7.64E+03	9.07E+03

*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.

R₁- R₆= Replicates 1 to 6

Table 6               Daily Specific Growth Rates for the Control Cultures in the Positive Control

		Daily Specific Growth Rate (cells/ml/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.065	0.051	0.079
	R2	0.069	0.047	0.072
	R3	0.075	0.042	0.075
	R4	0.065	0.049	0.070
	R5	0.068	0.045	0.082
	R6	0.065	0.053	0.077
	Mean	0.068	0.048	0.076

R₁- R₆= Replicates 1 to 6

Table 7               Inhibition of Growth Rate, Yield and Biomass Integral in the Positive Control

Please see attached background material.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 100% v/v saturated solution. Correspondingly the No Observed Effect Concentration was 100% v/v saturated solution.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods. 

Information provided by the Sponsor indicated that the water solubility of the test material was very low. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the material using traditional methods of preparation e.g. ultrasonication and high shear mixing. As the test material is a preparation it was considered appropriate to prepare the test material using a saturated solution method of preparation.

Following a preliminary range-finding test, Desmodesmus subspicatuswas exposed to a solution of the test material at a test concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solution was prepared by stirring an excess (100 mg/l) of test material in culture medium using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 21°C for 48 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a saturated solution of the test material.

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

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatu swas exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 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. 

Exposure ofDesmodesmus subspicatusto the test material gave EC₅₀values of greater than 100% v/v saturated solution and correspondingly the No Observed Effect Concentration was 100% v/v saturated solution.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.21 mg/l.

This does not infer that no test material was in solution but that the dissolved concentration (i.e. bioavailable to the test organisms) was below the limit of quantitation. As such the results are based on nominal test concentrations only.

This study showed that there were no toxic effects at saturation.

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an E_rC₅₀(0 - 72 h) of 0.57 mg/l; 95% confidence limits 0.48 – 0.66 mg/l, an E_yC₅₀(0 - 72 h) of 0.32 mg/l; 95% confidence limits 0.29 ‑ 0.35 mg/l, and an E_bC₅₀(0 - 72 h) of 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/l respectively and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/l respectively.