TOMILAC 214

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:

7 July 2009 - 20 July 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recently conducted GLP compliant study using the most recent test methods

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

and OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): JKY-214
- Substance type: Organic
- Physical state: white solid
- Analytical purity: 97.5%
- Lot/batch No.: Y002E
- Storage condition of test material: Room temperature in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Range Finding study: Nominal test concentrations of 0.0022, 0.022 and 0.22 mg/l
Definitive study: 0.74 mg/l (see below)

- Sampling method: By direct extraction

- Sample storage conditions before analysis: Not applicable; prepared and dosed.

Test solutions

Vehicle:

yes

Details on test solutions:

Pre-study media preparation trial

Information provided by the Sponsor indicated that the test material was insoluble in water. Pre-study solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 2.0 mg/l (by visual inspection) was obtained using a preliminary solution in dimethylformamide.
Based on this information the test material was categorised as being a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

Saturated solution preparation

An amount of test material (1125 mg) was dispersed, in duplicate in 22.5 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21 °C for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
1) Centrifugation at 10000 g for 30 minutes
2) Centrifugation at 40000 g for 30 minutes
3) Filtration through a 0.2 urn Sartorius Sartopore filter (approximately 1 litre discarded in order to pre-condition the filter)
4) Filtration through a 0.2 urn Sartorius Sartopore filter (approximately 2 litres discarded in order to pre-condition the filter)

Solvent spike preparation

An amount of test material (200 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (200 pi) of this 200 mg/10 ml solvent stock solution was dispersed, in triplicate, in 2 litres of reconstituted water with the aid of magnetic stirring for approximately 10 minutes, 24 hours or 48 hours prior to taking samples for chemical analysis after the following pre-treatments:
1) Untreated
2) Centrifugation at 10000 g for 30 minutes
3) Centrifugation at 40000 g for 30 minutes
4) Filtration through a 0.2 urn Gelman Acrocap filter (approximately 100 ml discarded in order to pre-condition the filter)
5) Filtration through a 0.2 urn Gelman Acrocap filter (approximately 500 ml discarded in order to pre-condition the filter)

Range-finding test

Based on the results obtained from the pre-study media preparation trial the test material was prepared using a solvent spike method of preparation prior to removal of any undissolved test material by centrifugation at 40000 g for 30 minutes to give a nominal test concentration of 0.22 mg/l.

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 0.0022, 0.022 and 0.22 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 material was prepared using a preliminary solution in dimethylformamide.

An amount of test material (200 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (50 pi) of this solvent stock solution was dispersed in 500 ml of culture medium with the aid of magnetic stirring for approximately 10 minutes prior to removing any undissolved test material by centrifugation at 40000 g for 30 minutes to give a 0.22 mg/l stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 0.022 and 0.0022 mg/l. An aliquot (200 ml) of each of the stock solutions was separately inoculated with algal suspension (1.2 ml) to give the required test concentrations of 0.0022, 0.022 and 0.22 mg/l.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 pl/l of dimethylformamide.

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°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.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Definitive test

Based on the result of the range-finding test a "limit test" was conducted at a nominal concentration of 0.22 mg/l to confirm that at the highest attainable concentration no effect on algal growth was observed.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations in the range of 0.73 to 0.75 mg/l were obtained. The differences observed between the concentrations obtained during the pre-study media preparation trial and definitive test were considered to be due the different media types.

Experimental Preparation

For the purpose of the definitive test, the test material was prepared using a preliminary solution in dimethylformamide.

An amount of test material (200 mg) was dissolved in dimethylformamide with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (200 pi) of this solvent stock solution was dispersed in 2 litres of culture medium with the aid of magnetic stirring for approximately 10 minutes prior to removing any undissolved test material by centrifugation at 40000 g for 30 minutes to give a stock solution with a 0-Hour mean measured test concentration of 0.74 mg/l. An aliquot (1 litre) of the 0.74 mg/l stock solution was inoculated with algal suspension (10.5 ml) to give the test concentration of 0.74 mg/l.

The stock solutions and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: (CCAP 276/20)
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Approximately 72 hours prior to innoculation
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C. The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.


ACCLIMATION
- Acclimation period: As above
- Culturing media and conditions (same as test or not): As above
- Any deformed or abnormal cells observed: Not specified.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

Not specified.

Test temperature:

24 ± 1°C

pH:

7.5 ± 0.1

Dissolved oxygen:

Not specified.

Salinity:

Not applicable - freshwater.

Nominal and measured concentrations:

Range Finding study: Nominal test concentrations of 0.0022, 0.022 and 0.22 mg/l
Definitive study: 0.74 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flask
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: 100 ml volume per flask
- Aeration: No; Sealed with polyurethane foam bungs to reduce evaporation
- Initial cells density: 4x 103 cells/ml
- Control end cells density: 4x 103 cells/ml
- No. of vessels per concentration (replicates): 6 replicate vessels
- No. of vessels per control (replicates): 6 replicate vessels
- No. of vessels per vehicle control (replicates): 6 replicate vessels

GROWTH MEDIUM
- Standard medium used: Yes, ASTM

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water

-Algal culture details:

NaNO3 25.5 mg/l
MgCI2.6H2O 12.164 mg/l
CaCI2.2H2O 4.41mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCI2.4H2O 0.415 mg/l
ZnCI2 0.00327 mg/l
FeCI3.6H2O 0.159 mg/l
CoCI2.6H2O 0.00143 mg/l
Na2Mo04.2H2O 0.00726 mg/l
CuCI2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2Se03.5H2O 0.000010 mg/l

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCl. The pH of each control, solvent 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 temperature within the incubator was recorded daily.

- Photoperiod: Constant illumination .
- Light intensity and quality: approximately 7000 lux
- Salinity (for marine algae): Not applicable.
- Control / Solvent groups: The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 µ/l of dimethylformamide.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Electronic particle counter; Coulter
- Chlorophyll measurement: Not measured

TEST CONCENTRATIONS
Range Finding study: Nominal test concentrations of 0.0022, 0.022 and 0.22 mg/l
Definitive study: 0.74 mg/l

Verification of test concentrations

Samples were taken from the solvent control (replicates - R6 pooled) and the 0.74 mg/l test group (replicates R1 – R3 and R4 – R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 72 hours and stored at approximately -20°C for further analysis if necessary.

Evaluation of data

Comparison of growth rates

The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass by equation:
The average specific growth rate over the test duration was calculated for each replicate control and test material vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test material vessel was calculated by equation:


Comparison of Yield

Yield is calculated as the increase in biomass over the exposure period by equation:

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following

Determination ofECx values

ECX values were determined by inspection of the growth rate and yield data after 72 hours.

Statistical analysis

A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the solvent control and the 0.74 mg/l test concentration to determine any statistically significant differences between the test and solvent control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Geometric mean measured test concentrations

The geometric mean measured test concentrations of the samples were calculated using the measured test concentrations of replicates R1 - R3 and replicates R4- R6 pooled

Validation Criteria

The results of the test are considered valid if the following performance criteria are met:

The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.

The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.

The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.65 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:

EC50

Effect conc.:

> 0.65 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.65 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.65 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Pre-Study Media Preparation Trial

The results of the pre-study media preparation trial indicated that for both the solvent spike and saturated solution methods of preparation filtration was not appropriate as significant losses were observed suggesting that the test material was adsorbing to the filter matrices. The results from the saturated solution samples following centrifugation were variable indicating that centrifugation was not appropriate for the removal of the greater excess of test material present using this method of preparation. The results from the solvent spike method of preparation indicated that centrifugation at 10000 g was not sufficient to ensure all undissolved test material was removed. No increase in the dissolved test material concentration obtained from the solvent spike method of preparation was observed with increased preparation time.

It was therefore considered that the most appropriate method of preparation for the test material was as a solvent spike with any undissolved test material being removed by centrifugation at 40000 g for 30 minutes to give a nominal test concentration of approximately 0.22 mg/l.

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 below.

The results showed no effect on growth at the nominal test concentrations of 0.0022, 0.022 and 0.22 mg/l.

Based on this information a single nominal test concentration of six replicates, of 0.22 mg/l was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations in the range of 0.73 to 0.75 mg/l were obtained. The differences observed between the concentrations obtained during the pre-study media preparation trial and definitive test were considered to be due the different media types used.

Definitive Test

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 below. Daily specific growth rates for the control cultures are given in Table 3 below. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 below.

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 39 after 72 hours and the cell concentration of the solvent control cultures increased by a factor of 55 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 : 4.05 x 103 cells per ml
Mean cell density of control at 72 hours: 1.59 x 105 cells per ml
Mean cell density of solvent control at 0 hours : 4.06 x 103 cells per ml
Mean cell density of solvent control at 72 hours: 2.24 x 105 cells per ml

The mean coefficients of variation for section by section specific growth rate for the control and solvent cultures were 24% and 25% respectively and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficients of variation for average specific growth rate for the control and solvent control cultures over the test period (0 - 72 h) were 2% and 4% respectively 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) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test material at a 0-Hour mean measured test concentration of 0.74 mg/l over the 72-Hour exposure period.
The test concentration of 0.74 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline.

Accordingly the following results were determined from the data based on the 0-Hour mean measured test concentration:

5.3.2.1 Inhibition of growth rate

ErC10 (0 - 72 h) : > 0.74 mg/l
ErC20 (0 - 72 h) : > 0.74 mg/l
ErC50 (0 - 72 h) : > 0.74 mg/l

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

Statistical analysis of the growth rate data was carried out for the solvent control and 0.74 mg/l 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 decreases in growth rate (P>0.05), between the solvent control and 0.74 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.74 mg/l.

Inhibition of yield

EyC10 (0 - 72 h) : > 0.74 mg/l
EyC20 (0 - 72 h) : > 0.74 mg/l
EyC50 (0 - 72 h) : > 0.74 mg/l

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

Statistical analysis of the yield data was also carried out. There were no statistically significant decreases in yield (P>0.05), between the solvent control and 0.74 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.74 mg/l.

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.

Observations on test material solubility

At the start of the test all control, solvent control and 0.74 mg/l test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, solvent control and 0.74 mg/l test cultures were observed to be pale green dispersions.

Physico-chemical measurements

The pH values of each test and control flask are given in Table 2. Temperature was maintained at 24 ± 1°C throughout the test.

The pH values of the control cultures were observed to increase from pH 7.6 - 7.7 at 0 hours to pH 7.7 - 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 hours showed measured test concentrations to range from 0.73 to 0.75 mg/l. A slight decline in measured test concentrations was observed at 72 hours in the range of 0.54 to 0.58 mg/l. This decline was in line with the preliminary stability analyses conducted which indicated that the test material was unstable.

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 following results were determined from the data based on the geometric mean measured test concentrations:

Growth rate
ErC10 (0 - 72 h) : > 0.65 mg/l
EryC20 (0 - 72 h) : > 0.65 mg/l
ErC50 (0 - 72 h) : > 0.65 mg/l
No Observed Effect Concentration (NOEC) = 0.65 mg/l

Yield

EyC10 (0 - 72 h) : > 0.65 mg/l
EyC20 (0 - 72 h) : > 0.65 mg/l
EyC50 (0 - 72 h) : > 0.65 mg/l
No Observed Effect Concentration (NOEC) = 0.65 mg/l

The use of the geometric mean measured test concentrations in the calculation of the EC50 and NOEC values had no significant effect on the outcome of the study.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project No: 0039/1088) used potassium dichromate as the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following results:

ErC50 (0 - 72 h) 0.79 mg/l
EyC50 (0 - 72 h) 0.30 mg/l, 95% confidence limits 0.27 - 0.34 mg/l

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l

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

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the solvent control and 0.74 mg/l 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 decreases in growth rate (P>0.05), between the solvent control and 0.74 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.74 mg/l.
Statistical analysis of the yield data was also carried out. There were no statistically significant decreases in yield (P>0.05), between the solvent control and 0.74 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.74 mg/l.

Any other information on results incl. tables

Table 1 Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

 

Nominal Concentration (mg/l)	Cell Densities[1](cells per ml)		Inhibition Values (%)
	0 Hours	72 Hours	Growth Rate	Yield
Control R1 R2 Mean	4.00E+03 4.34E+03 4.17E+03	2.66E+05 2.20E+05 2.43E+05	-	-
Solvent Control R1 R2 Mean	4.02E+03 4.22E+03 4.12E+03	3.19E+05 2.51 E+05 2.85E+05	-	-
0.0022 R1 R2 Mean	4.39E+03 4.02E+03 4.21 E+03	3.36E+05 3.24E+05 3.30E+05	[3]	[16]
0.022 R1 R2 Mean	4.38E+03 4.62E+03 4.50E+03	3.87E+05 2.59E+05 3.23E+05	0	[13]
0.22 R1 R2 Mean	4.37E+03 4.54E+03 4.46E+03	3.04E+05 2.81 E+05 2.93E+05	2	[3]

 

 

Table 2 - Cell Densities and pH Values in the Definitive Test

 

0-Hour Mean Measured Test Concentration (mg/l)	pH	Cell Densities* (cells per ml)				pH
	0h	0h	24 h	48 h	72 h	72 h
Control R1	7.6	4.06E+03	1.78E+04	4.85E+04	1.59E+05	7.9
R2	7.7	4.10E+03	1.95E+04	5.06E+04	1.65E+05	7.8
R3	7.6	4.02E+03	1.83E+04	3.98E+04	1.34E+05	7.8
R4	7.6	4.02E+03	1.58E+04	4.25E+04	1.66E+05	7.8
R5	7.6	4.07E+03	1.60E+04	3.91 E+04	1.73E+05	7.7
R6	7.6	4.04E+03	1.72E+04	4.13E+04	1.54E+05	7.7
Mean		4.05E+03	1.74E+04	4.36E+04	1.59E+05
Solvent Control         R1	7.5	4.06E+03	2.08E+04	5.65E+04	2.60E+05	7.7
R2	7.5	4.12E+03	1.78E+04	4.09E+04	1.89E+05	7.6
R3	7.5	4.02E+03	1.90E+04	5.04E+04	2.05E+05	7.6
R4	7.4	4.02E+03	2.05E+04	5.99E+04	2.18E+05	7.6
R5	7.4	4.06E+03	1.60E+04	4.45E+04	2.67E+05	7.6
R6	7.4	4.11E+03	1.57E+04	4.12E+04	2.04E+05	7.6
Mean		4.06E+03	1.83E+04	4.89E+04	2.24E+05
0.74                 R1	7.4	4.14E+03	2.60E+04	5.86E+04	3.47E+05	7.7
R2	7.4	4.18E+03	2.17E+04	7.17E+04	3.87E+05	7.7
R3	7.3	4.13E+03	2.29E+04	4.78E+04	2.96E+05	7.7
R4	7.4	4.02E+03	2.03E+04	4.26E+04	3.01 E+05	7.7
R5	7.4	4.09E+03	2.45E+04	5.57E+04	2.82E+05	7.7
R6	7.3	4.07E+03	2.12E+04	4.83E+04	2.61 E+05	7.7
Mean		4.11E+03	2.28E+04	5.41 E+04	3.12E+05

 

 

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	0.062	0.042	0.049
	R2	0.066	0.040	0.049
	R3	0.063	0.032	0.050
	R4	0.057	0.041	0.057
	R5	0.058	0.037	0.062
	R6	0.061	0.037	0.055
	Mean	0.061	0.038	0.054
Solvent Control	R1	0.069	0.042	0.064
	R2	0.062	0.035	0.064
	R3	0.065	0.041	0.058
	R4	0.068	0.045	0.054
	R5	0.058	0.043	0.075
	R6	0.057	0.040	0.067
	Mean	0.063	0.041	0.064

 

 

Table 4 - Inhibition of Growth Rate and Yield in the Definitive Test

 

0-Hour Mean Measured Test Concentration (mg/l)	Growth Rate		Yield
	(cells/ml/hour)		(cells/ml)
	0-72h	% Inhibition	0-72h	% inhibition
Control
R1	0.051		1.55E+05
R2	0.052		1.61 E+05
R3	0.049		1.30E+05
R4	0.052	-	1.62E+05	-
R5	0.052		1.69E+05
R5	0.051		1.50E+05
Mean	0.051		1.55E+05
SD	0.001		1.38E+04
Solvent Control      R1	0.058		2.56E+05
R2	0.054		1.85E+05
R3	0.055		2.01 E+05
R4	0.056	-	2.14E+05	-
R5	0.058		2.63E+05
R6	0.055		2.00E+05
Mean	0.056		2.20E+05
SD	0.002		3.19E+04
0.74             R1	0.062	[11]	3.42E+05
R2	0.064	[14]	3.83E+05
R3	0.060	[7]	2.92E+05
R4	0.060	[7]	2.97E+05
R5	0.059	[5]	2.78E+05
R6	0.058	[4]	2.57E+05
Mean	0.061	[8]	3.08E+05	[40]
SD	0.002		4.63E+04

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 based on the 0-Hour mean measured test concentration gave EC50 values of greater than 0.74 mg/l. Correspondingly the No Observed Effect Concentration was 0.74 mg/l.

Based on the geometric mean measured test concentrations the EC50 values were estimated to be greater than 0.65 mg/l. Correspondingly the No Observed Effect Concentration was 0.65 mg/l.

Executive summary:

The substance was not inhibitory to the growth of algae at the limit of achievable concentration within the test. As no mortality or clinical signs were observed at the highest attainable test concentration the substance is considered not acutely toxic to algae.

No classification or labelling is applicable.