Fatty acids C18-(unsaturated) lithium salts

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:

1 March 2010 and 8 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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 items, 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, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

LiTOFA

IUPAC Name:

LiTOFA

Details on test material:

Sponsor's identification : LiTOFA
Description : off white solid
Batch number : SF 173
Date received : 21 January 2010
Expiry date : 21 January 2012
Storage conditions : room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-finding tests
Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).
The loading rates to be used in the definitive test were determined by preliminary range-finding tests. The initial range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 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 prepared for each control and test concentration.
Amounts of test item (20 and 200 mg) were each separately added to the surface of 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering three times and showed there to still be some micro-dispersions/particles of undissolved test item present.
An aliquot (250 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (9.1 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the initial 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 haemocytometer and light microscope.
It was not possible to monitor algal growth using a Coulter® Multisizer Particle Counter at 72 hours due to the dispersed test item present interfering with the Coulter counts.
The results of the initial range-finding test showed significant inhibition of growth occurred at both 10 and 100 mg/l loading rate WAF and so a second range-finding test was conducted by exposing Desmodesmus subspicatus cells to nominal loading rates of 1.0 and 10 mg/l for a period of 72 hours.
Amounts of test item (20 mg) were each separately added to the surface of 20 and 2 litres of culture medium to give the 1.0 and 10 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering three times and showed there to still be some micro-dispersions/particles of undissolved test item present.
An aliquot (250 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (3.7 ml) to give the required test concentrations of 1.0 and 10 mg/l loading rate WAF.
At the start of the second 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.
Exposure conditions in the second range-finding test were the same as those in the initial test.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Method:
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/l.
Experimental Preparation
Amounts of test item (20, 64, 20, 64 and 200 mg) were each separately added to the surface of 20, 20, 2, 2 and 2 litres of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering three times and showed there to still be micro-dispersions/particles of test item present.
An aliquot (500 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (6 ml) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAF.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a haemocytometer and light microscope.
It was not possible to monitor algal cells using a Coulter® Multisizer Particle Counter due to the dispersed test item interfering with the Coulter counts.

Chemical analysis of test loading rates
Samples were taken from the control (replicates R1 - R6 pooled) and each loading rate WAF test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at each occasion and stored at approximately 20ºC for further analysis if necessary.
The method of analysis, stability, recovery and test preparation analyses are described in Appendix 4 - see attached

Evidence of undissolved material :
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of mixing the 1.0 and 3.2 mg/l loading rate WAFs were observed to have formed clear colourless media columns with particles of test item floating at the surface and dispersed throughout. The 10 mg/l loading rate WAF was observed to have formed a cloudy dispersion with particles of test item suspended within the vortex and dispersed throughout whilst the 32 and 100 mg/l loading rate WAFs were observed to have formed cloudy white dispersions with particles of test item suspended within the vortex and dispersed throughout. After stirring and following a 1-Hour settlement period the 1.0 and 3.2 mg/l loading rate WAFs were observed to have formed clear colourless media columns with test item floating at the media surface, dispersed throughout and settled on the bottom of the mixing vessel. The 10 mg/l loading rate WAF was observed to have formed a slightly cloudy media column with particles of test item floating at the media surface, dispersed throughout and settled on the bottom of the mixing vessel whilst the 32 and 100 mg/l loading rate WAFs were observed to have formed cloudy media columns with particles of test item floating at the media surface, dispersed throughout and settled on the bottom of the mixing vessel. Microscopic examination of the WAFs prior to siphoning showed there to be particles/micro-dispersions of test item present. Following filtration through a glass wool plug three times no particles or micro-dispersions of test item were observed to be present in the 1.0 and 3.2 mg/l loading rate WAFs. However, in the 10, 32 and 100 mg/l loading rate WAFs particles/micro-dispersions of test item were still observed to be present. It was considered that further filtration would not have resulted in the removal of any further amounts of undissolved test item.
At the start of the test all control cultures were observed to be clear colourless solutions. The test cultures were observed to range from clear colourless solutions at 1.0 mg/l loading rate WAF through to cloudy white homogenous dispersions at 100 mg/l loading rate WAF. After the 72-Hour test period all control and 1.0 mg/l loading rate WAF test cultures were observed to be green dispersions. The 3.2 mg/l loading rate WAF test cultures were observed to be pale green dispersions, the 10 mg/l loading rate WAF test cultures were observed to be very pale green dispersions whilst the 32 and 100 mg/l loading rate WAF test cultures were observed to be cloudy white homogenous dispersions.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test Species
The test was carried out using Desmodesmus subspicatus strain CCAP 276/20. Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. 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 illumination at 21 ± 1ºC.
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 10E+03 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 ± 1ºC until the algal cell density was approximately 10E+04 – 10E+05 cells/ml.

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.
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 (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

Any deformed or abnormal cells observed:
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 1.0, 3.2 and 100 mg/l loading rate WAF, however some cell clumping was observed to be present in the test cultures at 10 and 32 mg/l loading rate WAF.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not applicable

Test temperature:

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

pH:

The pH values of each test and control flask are given in Table 3 - see section any other information on results. Temperature was maintained at 24 ± 1ºC throughout the test.
The pH values of the control cultures (see Table 3- in section any other information on results) were observed to increase frompH 7.3 at 0 hours to pH 7.7 – 7.8 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 applicable

Salinity:

freshwater used

Nominal and measured concentrations:

Whilst the results of the second range-finding test showed no effect on growth at the loading rates of 1.0 and 10 mg/l, during the initial range-finding test significant inhibition was observed to have occurred at both 10 and 100 mg/l loading rate WAF. It was considered that whilst every precaution was taken to maintain consistency between the range-finding tests the difference observed in the inhibition values obtained at 10 mg/l loading rate WAF was considered to be due to preferential dissolution of the test item with slight variances in mixing temperature and speed.
Based on this information loading rates of 1.0, 3.2, 10, 32 and 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.

Details on test conditions:

TEST SYSTEM
As in the range-finding tests 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and three flasks each containing 100 ml were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 3.15 x 10e5 cells per ml. Inoculation of 500 ml of test medium with 6 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.


Control end cells density: algal cell density was approximately 2.40 10E5 cells/ml

EFFECT PARAMETERS MEASURED :
Determination of cell densities:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a haemocytometer and light microscope

Determination of ECx values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic
axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). EL*x values were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EL*50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: Not applicable

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: Not applicable

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 11 - 16 mg/l

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 3.9 - 5.7 mg/l

Details on results:

Exponential growth in the control (for algal test): yes


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 1.0, 3.2 and 100 mg/l loading rate WAF, however some cell clumping was observed to be present in the test cultures at 10 and 32 mg/l loading rate WAF.


Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatusto the test material during the range-finding tests are given in Table 1 and Table 2.


Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 72 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 : 3.34 x 10E+03 cells per ml
Mean cell density of control at 72 hours : 2.40 x 10E+05 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 14% 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 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

From the data given in Tables 3 and 5, it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus(CCAP 276/20) were 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

ErL*10 (0 - 72 h) : 4.9 mg/l loading rate WAF
ErL*20 (0 - 72 h) : 7.0 mg/l loading rate WAF
ErL*50 (0 - 72 h) : 13 mg/l loading rate WAF; 95% confidence limits 11 - 16 mg/l loading rate WAF

where ErL*x is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and all loading rates 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 between the control and 1.0 mg/l loading rate WAF (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 1.0 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 3.2 mg/l loading rate WAF.



Inhibition of yield

EyL*10 (0 - 72 h) : 1.3 mg/l loading rate WAF
EyL*20 (0 - 72 h) : 2.1 mg/l loading rate WAF
EyL*50 (0 - 72 h) : 4.7 mg/l loading rate WAF; 95% confidence limits 3.9 – 5.7 mg/l loading rate WAF
where EyL*x is the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as for the growth rate. There were no statistically significant differences between the control and 1.0 mg/l loading rate WAF (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 1.0 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 3.2 mg/l loading rate WAF.



*EL = Effective Loading Rate

Results with reference substance (positive control):

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:
ErC50 (0 – 72 h) : 0.49 mg/l*
EyC50 (0 – 72 h) : 0.18 mg/l, 95% confidence limits 0.16 – 0.21 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.125 mg/l

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

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 and yield data after 72 hours for the control and all test loading rates 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

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

Nominal Loading Rate (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	4.34E+03	8.57E+05
	R2	4.46E+03	7.53E+05	-	-
	Mean	4.40E+03	8.05E+05
10	R1	4.29E+03	4.37E+05
	R2	4.66E+03	4.63E+05	11	44
	Mean	4.47E+03	4.50E+05
100	R1	4.15E+03	2.67E+04
	R2	4.13E+03	2.67E+04	64	97
	Mean	4.14E+03	2.67E+04

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

R₁and R₂= Replicates 1 and 2

Table 2       Cell Densities and Percentage Inhibition of Growth from the Second Range-finding Test

Nominal Loading Rate (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	4.77E+03	2.63E+05
	R2	4.14E+03	2.84E+05	-	-
	Mean	4.45E+03	2.74E+05
1.0	R1	4.32E+03	2.69E+05
	R2	4.02E+03	2.17E+05	2	11
	Mean	4.17E+03	2.43E+05
10	R1	4.71E+03	2.52E+05
	R2	4.74E+03	2.47E+05	4	9
	Mean	4.73E+03	2.50E+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.

R₁and R₂= Replicates 1 and 2

Table 3       Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.3	3.35E+03	1.67E+04	5.50E+04	2.83E+05	7.7
	R2	7.3	3.35E+03	1.67E+04	6.00E+04	2.00E+05	7.7
	R3	7.3	5.00E+03	1.34E+04	7.00E+04	2.27E+05	7.8
	R4	7.3	3.35E+03	1.50E+04	6.17E+04	2.63E+05	7.8
	R5	7.3	1.65E+03	1.67E+04	6.00E+04	2.27E+05	7.7
	R6	7.3	3.35E+03	2.17E+04	5.67E+04	2.37E+05	7.8
	Mean		3.34E+03	1.67E+04	6.06E+04	2.40E+05
1.0	R1	7.3	5.00E+03	2.17E+04	6.00E+04	2.60E+05	7.7
	R2	7.3	3.35E+03	2.34E+04	7.84E+04	2.23E+05	7.7
	R3	7.3	3.35E+03	1.84E+04	5.67E+04	2.33E+05	7.7
	Mean		3.90E+03	2.12E+04	6.50E+04	2.39E+05
3.2	R1	7.3	3.35E+03	1.67E+04	3.50E+04	1.37E+05	7.7
	R2	7.3	1.65E+03	1.67E+04	3.17E+04	1.67E+05	7.6
	R3	7.3	3.35E+03	1.84E+04	3.00E+04	1.47E+05	7.6
	Mean		2.78E+03	1.73E+04	3.22E+04	1.50E+05
10	R1	7.2	3.35E+03	5.00E+03	5.67E+04	5.00E+04	7.6
	R2	7.2	5.00E+03	8.35E+03	6.00E+04	7.17E+04	7.6
	R3	7.2	3.35E+03	6.65E+03	5.67E+04	6.67E+04	7.6
	Mean		3.90E+03	6.67E+03	5.78E+04	6.28E+04
32	R1	7.3	3.35E+03	5.00E+03	3.35E+03	8.35E+03	7.5
	R2	7.3	3.35E+03	6.65E+03	5.00E+03	6.65E+03	7.5
	R3	7.3	3.35E+03	6.65E+03	5.00E+03	6.65E+03	7.5
	Mean		3.35E+03	6.10E+03	4.45E+03	7.22E+03
100	R1	7.8	3.35E+03	3.35E+03	1.65E+03	1.65E+03	7.6
	R2	7.8	1.65E+03	6.65E+03	3.35E+03	1.65E+03	7.6
	R3	7.8	5.00E+03	3.35E+03	1.65E+03	1.65E+03	7.6
	Mean		3.33E+03	4.45E+03	2.22E+03	1.65E+03

*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.

R₁- R₆= Replicates 1 to 6

Table 4       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	0.060	0.050	0.068
	R2	0.060	0.053	0.050
	R3	0.050	0.069	0.049
	R4	0.055	0.059	0.060
	R5	0.060	0.053	0.055
	R6	0.070	0.040	0.060
	Mean	0.059	0.054	0.057

R₁- R₆= Replicates 1 to 6

Table 5       Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.059		2.80E+05
	R2	0.054		1.97E+05
	R3	0.056		2.22E+05
	R4	0.058	-	2.60E+05	-
	R5	0.056		2.25E+05
	R6	0.057		2.34E+05
	Mean	0.057		2.36E+05
	SD	0.002		2.94E+04
1.0	R1	0.058	[2]	2.55E+05
	R2	0.056	2	2.20E+05
	R3	0.056	2	2.30E+05
	Mean	0.057	1	2.35E+05	1
	SD	0.001		1.82E+04
3.2	R1	0.049	14	1.34E+05
	R2	0.052	9	1.65E+05
	R3	0.050	12	1.44E+05
	Mean	0.050	12	1.48E+05	38
	SD	0.002		1.62E+04
10	R1	0.035	39	4.67E+04
	R2	0.040	30	6.67E+04
	R3	0.039	32	6.34E+04
	Mean	0.038	34	5.89E+04	75
	SD	0.003		1.07E+04
32	R1	0.010	82	5.00E+03
	R2	0.007	88	3.30E+03
	R3	0.007	88	3.30E+03
	Mean	0.008	86	3.87E+03	98
	SD	0.002		9.81E+02
100	R1	-0.012	121	-1.70E+03
	R2	-0.012	121	0.00E+00
	R3	-0.012	121	-3.35E+03
	Mean	-0.012	121	-1.68E+03	101
	SD	0.000		1.68E+03

[ ]

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

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

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

ErL*50 (0 – 72 h) : 13 mg/l loading rate WAF; 95% confidence limits 11 - 16 mg/loading rate WAF
EyL*50 (0 – 72 h) : 4.7 mg/l loading rate WAF, 95% confidence limits 3.9 – 5.7 mg/l loading rate WAF

No Observed Effect Loading Rate (NOEL) based on growth rate: 1.0 mg/l loading rate WAF
No Observed Effect Loading Rate (NOEL) based on yield: 1.0 mg/l loadign rate WAF
Lowest Observed Effect Loading Rate (LOEL) based on growth rate: 3.2 mg/l loading rate WAF
Lowest Observed Effect Loading Rate (LOEL) based on yield: 3.2 mg/l loading rate WAF


* EL = Effective Loading Rate

Executive summary:

A study was performed to assess the effect of the test item 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, "Fresh water Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 440/2008.

Following preliminary range-finding tests, Desmodesmus subspicatus was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 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 haemocytometer and light microscope.

Results. Exposure of Desmodesmus subspicatus to the test item gave the following results:

Response Variable	EL*50 (mg/l Loading Rate WAF)	95% Confidence Limits (mg/l Loading Rate WAF)			No Observed Effect Loading Rate (NOEL) (mg/l)	Lowest Observed Effect Loading Rate (LOEL) (mg/l)
Growth Rate	13	11	-	16	1.0	3.2
Yield	4.7	3.9	-	5.7	1.0	3.2

Analysis of the test samples at 0 hours showed measured test concentrations to range from 1.2 mg/l at 1.0 mg/l loading rate WAF through to 52 mg/l at 100 mg/l loading rate WAF. Analysis of the test preparations at 72 hours showed a decline in measured test concentrations in the range of less than the limit of quantitation (LOQ) of the analytical method employed (which was determined to be 0.016 mg/l) to 4 mg/l. This decline was inline with the preliminary stability analyses conducted which indicated that the test item was unstable in culture medium particularly at the lower test concentrations employed.

However, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.