Sodium 5-oxo-1-palmitoyl-L-prolinate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 10 November 2008 and 16 October 2009.

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.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EXAMPLE

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

EXAMPLE

Deviations:

no

Principles of method if other than guideline:

LCE08010 is known to be poorly soluable in water. 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, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material 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 material with water for a prolonged period. Previous experience gained from studies conducted on poorly water soluble test materials has shown that a mixing period of 24 hours is sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Sponsor's identification: LCA08010
Description: greenish brown turbid viscous liquid
Batch number: 0729900003
Date received: 2 July 2008
Storage conditions: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.32, 1.0, 3.2, 10 and 32 mg/l loading rate WAF.

Samples were taken from the additional control and each loading rate WAF test group containing no algal cells at 0 and 72 hours for analysis.

Duplicate samples were taken and stored frozen (approximately -20 deg C) for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Procedure

Range-finding tests

Due to the low aqueous solubility and complex nature of the test material for the purposes of the test the test material 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 material (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 material, 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 material 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 and showed there to be no globules or micro-dispersions of test material present.
An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (4.2 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 material.
The results obtained indicated that significant inhibition of growth occurred at both 10 and 100 mg/l loading rate WAF. As such a second range-finding test was conducted at nominal loading rates of 1.0 and 10 mg/l.
Amounts of test material (20 and 20 mg) were each separately added to the surface of 20 and 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test material, 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 material 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 and showed there to be no micro-dispersions of test material present.
An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (1.2 ml) to give the required test concentrations of 1.0 and 10 mg/l loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test material.

At the start of the range-finding tests 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.

Definitive test
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.32, 1.0, 3.2, 10 and 32 mg/l loading rate WAF.

Experimental Preparation
Amounts of test material (6.4 and 20 mg) were each separately weighed onto a glass slide and suspended within 20 litres of culture medium to give 0.32 and 1.0 mg/l loading rate WAFs respectively, whilst the 3.2, 10 and 32 mg/l loading rate WAFs were prepared by separately dispensing 64, 200 and 640 mg of test material on to the surface of 20 litres of culture medium via a plastic disposable syringe. After the addition of the test material, 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 material 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 0.32, 1.0, 3.2, 10 and 32 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test material present. The weighing of test material onto a glass slide allowed for accurate weighing of small amounts of test material.
An aliquot (1 litre) of each of the loading rate WAFs was separately inoculated with algal suspension (16.8 ml) to give the required test concentrations of 0.32, 1.0, 3.2, 10 and 32 mg/l loading rate WAF.
An additional 150 ml of the control and each loading rate WAF test group containing no algal cells was run alongside the test to provide samples for Total Organic Carbon (TOC) analysis at 0 and 72 hours (see Appendix 4). It was considered appropriate to omit the presence of algal cells in the samples prepared for TOC analysis as their presence would artificially increase the TOC concentration obtained.

Physico-chemical measurements
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 temperature within the incubator was recorded daily.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period - see attached table
Evaluation of data
Comparison of growth rates and comparison of yield - please see in attached section

Determination of EL*x values
EL*x values were determined by inspection of the growth rate, yield and biomass intergral data after 72 hours.

Statistical analysis
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 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).

3.4 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%.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

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 (See below). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 deg C.

Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 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 104 – 105 cells/ml.

A positive control (Harlan Laboratories Ltd Project Number: 0039/1088) used potassium dichromate as the reference material.

Details of the positive control are given in section - results with reference substance (positive control).

The positive control was conducted between 26 May 2009 and 29 May 2009.

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

See below 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Not recorded.

Test temperature:

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

pH:

The pH values of the control cultures (see Table 3 in section any other information on results) were observed to increase from pH 7.2 – 7.3 at 0 hours to pH 7.6 – 7.7 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:

Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.32, 1.0, 3.2, 10 and 32 mg/l loading rate WAF.

Details on test conditions:

Exposure conditions
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 material.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.38 x 10e5 cells per ml. Inoculation of 1 litre of test medium with 16.8 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, 22, 47 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits not reported

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% Confidence Limits not reported

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% Confidence Limits not reported

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits not reported

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% Confidence Limits not reported

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% Confidence Limits not reported

Details on results:

RESULTS
Range-finding Tests
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding tests are given in Table 1 and Table 2 - in section any other information.
The results showed no significant effect on growth rate at 1.0 mg/l loading rate WAF. However, growth was observed to be reduced at the 10 and 100 mg/l loading rate WAF.
Based on this information loading rates of 0.32, 1.0, 3.2, 10 and 32 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.

Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 3 - in section any other information on results. Daily specific growth rates for the control cultures are given in Table 4 - in section any other information.

Growth rates, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 5 - in section ant other information on materials.

The mean cell densities versus time for the definitive test are presented in Figure 1 (see attached)

Percentage inhibition values are plotted against loading rate in Figure 2, Figure 3 and Figure 4 (see attached)

Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 84 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 : 4.17 x 10e3 cells per ml
Mean cell density of control at 72 hours : 3.50 x 10e5 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 33% 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 5% 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 3 (see section any other information on results) and 5 (see section any other information on materials), it is clear that the growth rate (r), yield (y) and biomass (b) 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) : 8.3 mg/l loading rate WAF
ErL*20 (0 - 72 h) : 12 mg/l loading rate WAF
ErL*50 (0 - 72 h) : 20 mg/l loading rate WAF; 95% confidence limits 17 -24 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, 0.32, 1.0 and 3.2 mg/l loading rate WAFs (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 3.2 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 10 mg/l loading rate WAF.
5.2.2.2 Inhibition of yield
EyL*10 (0 - 72 h) :9.1 mg/l loading rate WAF
EyL*20 (0 - 72 h) : 9.3 mg/l loading rate WAF
EyL*50 (0 - 72 h) : 10 mg/l loading rate WAF; 95% confidence limits 9.7 - 10 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. There were no statistically significant differences between the control, 0.32, 1.0, 3.2 and 10 mg/l loading rate WAFs (P <0.05), however the 32 mg/l loading rate WAF was significantly different (P<0.05). Inspection of the yield data showed that significant inhibition (52%) occurred at 10 mg/l loading rate WAF. The lack of a statistically significant response at this concentration was considered to be due to the significant negative inhibition observed at 0.32, 1.0 and 3.2 mg/l loading rate WAF. Therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was considered to be 3.2 mg/l loading rate WAF and correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was considered to be 10 mg/l loading rate WAF.

Inhibition of biomass integral
EbL*10 (0 - 72 h) : 4.3 mg/l loading rate WAF
EbL*20 (0 - 72 h) : 5.5 mg/l loading rate WAF
EbL*50 (0 - 72 h) : 10 mg/l loading rate WAF; 95% confidence limits 8.2 - 12 mg/l loading rate WAF
where EbL*x is the loading rate that reduced biomass integral by x%.
Statistical analysis of the biomass integral data was carried out. There were no statistically significant differences between the control, 0.32, 1.0, 3.2 and 10 mg/l loading rate WAFs (P<0.05), however the 32 mg/l loading rate WAF was significantly different (P<0.05). Inspection of the biomass integral data showed that significant inhibition (50%) occurred at 10 mg/l loading rate WAF. The lack of a statistically significant response at this concentration was considered to be due to the significant negative inhibition observed at 0.32, 1.0 and 3.2 mg/l loading rate WAF. Therefore the "No Observed Effect Loading Rate" (NOEL) based on biomass integral was considered to be 3.2 mg/l loading rate WAF and correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on biomass integral was considered to be 10 mg/l loading rate WAF.

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
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of mixing the 0.32 and 1.0 mg/l loading rate WAFs were observed to have formed clear colourless media columns with test material adhered to the glass slide suspended within the media column whilst test material was observed to be floating at the media surface and within the media column of the 3.2, 10 and 32 mg/l loading rate WAFs. After both the 23-Hour mixing period and 1-Hour settlement period the 0.32 and 1.0 mg/l loading rate WAFs were observed to have formed clear colourless media columns with test material adhered to the glass slide whilst test material was observed to be floating at the media surface and dispersed throughout the media column of the 3.2 mg/l loading rate WAF. The 10 mg/l loading rate WAF was observed to have formed a very slightly cloudy media column with particles of test material floating a the media surface and within the media column whilst the 32 mg/l loading rate WAF was observed to have formed a cloudy media column with particles of test material floating at the media surface and within the media column.
Microscopic examination of the WAFs indicated that micro-dispersions of test material were present and as such it was considered appropriate to remove the aqueous phase for testing by filtration through a glass wool plug. Following filtration microscopic examination of the WAFs showed there to be no micro-dispersions of test material present.
At the start of the test all control and test cultures were observed to be clear colourless solutions.

After the 72-Hour test period all control, 0.32, 1.0 and 3.2 mg/l loading rate WAFs were observed to have formed green dispersions, the 10 mg/l loading rate WAFs were observed to have formed pale green dispersions whilst the 32 mg/l loading rate WAFs were observed to have formed extremely pale green dispersions.

Results with reference substance (positive control):

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:

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 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).

Table1              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/Biomass Integral
Control	R1	4.84E+03	3.11E+05
	R2	5.72E+03	3.53E+05	-	-
	Mean	5.28E+03	3.32E+05
10	R1	4.75E+03	4.57E+04
	R2	5.00E+03	2.51E+04	52	91
	Mean	4.88E+03	3.54E+04
100	R1	4.74E+03	2.92E+04
	R2	4.89E+03	3.00E+04	57	92
	Mean	4.82E+03	2.96E+04

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

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/Biomass Integral
Control	R1	4.62E+03	2.43E+05
	R2	3.92E+03	2.81E+05	-	-
	Mean	4.27E+03	2.62E+05
1.0	R1	4.91E+03	1.92E+05
	R2	4.58E+03	2.12E+05	9	23
	Mean	4.75E+03	2.02E+05
10	R1	4.20E+03	1.75E+04
	R2	4.42E+03	1.85E+04	65	95
	Mean	4.31E+03	1.80E+04

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

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	22 h	47 h	72 h	72 h
Control	R1	7.3	4.08E+03	2.67E+04	5.86E+04	3.46E+05	7.7
	R2	7.3	4.37E+03	1.96E+04	5.59E+04	3.66E+05	7.6
	R3	7.3	3.64E+03	2.08E+04	4.55E+04	2.28E+05	7.6
	R4	7.2	4.74E+03	1.52E+04	5.02E+04	4.50E+05	7.6
	R5	7.2	4.14E+03	1.48E+04	5.22E+04	3.66E+05	7.6
	R6	7.2	4.08E+03	1.70E+04	4.86E+04	3.46E+05	7.6
	Mean		4.17E+03	1.90E+04	5.18E+04	3.50E+05
0.32	R1	7.1	5.46E+03	2.62E+04	5.67E+04	4.50E+05	7.7
	R2	7.2	4.14E+03	2.05E+04	5.92E+04	4.54E+05	7.6
	R3	7.2	3.98E+03	1.97E+04	5.41E+04	3.89E+05	7.6
	Mean		4.53E+03	2.21E+04	5.66E+04	4.31E+05
1.0	R1	7.1	4.79E+03	2.15E+04	7.58E+04	6.99E+05	7.6
	R2	7.2	3.75E+03	2.22E+04	4.81E+04	4.17E+05	7.6
	R3	7.1	4.16E+03	1.95E+04	6.52E+04	4.90E+05	7.6
	Mean		4.23E+03	2.11E+04	6.30E+04	5.35E+05
3.2	R1	7.1	3.98E+03	2.10E+04	9.21E+04	7.52E+05	7.6
	R2	7.1	3.11E+03	2.18E+04	5.06E+04	3.58E+05	7.7
	R3	7.2	3.51E+03	1.71E+04	4.85E+04	3.33E+05	7.7
	Mean		3.53E+03	1.99E+04	6.37E+04	4.81E+05
10	R1	7.1	4.66E+03	2.03E+04	3.44E+04	1.89E+05	7.6
	R2	7.1	4.56E+03	1.62E+04	2.56E+04	1.48E+05	7.6
	R3	7.1	4.44E+03	1.76E+04	1.84E+04	1.70E+05	7.5
	Mean		4.55E+03	1.80E+04	2.61E+04	1.69E+05
32	R1	7.0	4.35E+03	5.18E+03	1.08E+04	1.42E+04	7.4
	R2	7.0	4.35E+03	4.92E+03	8.34E+03	9.61E+03	7.4
	R3	7.0	4.02E+03	4.22E+03	9.90E+03	1.21E+04	7.4
	Mean		4.24E+03	4.78E+03	9.67E+03	1.20E+04

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

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	0.086	0.031	0.071
	R2	0.072	0.042	0.075
	R3	0.075	0.031	0.064
	R4	0.061	0.048	0.088
	R5	0.059	0.050	0.078
	R6	0.066	0.042	0.079
	Mean	0.070	0.041	0.076

R₁- R₆= Replicates 1 to 6

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results:
Response Variable EL*50 95% Confidence Limits No Observed Effect Loading Rate Lowest Observed Effect Loading Rate
(mg/l Loading Rate WAF) (mg/l Loading Rate WAF) (NOEL) (mg/l) (LOEL) (mg/l)
Growth Rate 20 17 - 24 3.2 10
Yield 10 9.7 - 10 3.2 10
Biomass 10 8.2 - 12 3.2 10


* EL = Effective Loading Rate

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 Regulation (EC) 440/2008.

Methods.

Information provided by the Sponsor indicated that the test material was a mixture of components. Given this and also the poor solubility of the test material in water it was considered that the most suitable method of preparation for this material was as a Water Accommodated Fraction (WAF).

Method development work conducted for the determination of dissolved test material concentrations using HPLC-MS analysis gave a good response in single ion mode at 354 and 396 m/z. However, as the test material is a mixture of components it was considered that chemical analysis was not suitable for detecting the total dissolved test material concentration present. As such it was considered appropriate to determine the dissolved test material concentration present in the test samples by Total Organic Carbon (TOC) analysis.

Following preliminary range-finding tests, Desmodesmus subspicatus was exposed to WAFs of the test material over a range of nominal loading rates of 0.32, 1.0, 3.2, 10 and 32 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 of Desmodesmus subspicatus to the test material 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	20	17	-	24	3.2	10
Yield	10	9.7	-	10	3.2	10
Biomass	10	8.2	-	12	3.2	10

Total Organic Carbon (TOC) analysis of the test preparations at 0 hours showed a concentration dependant increase in measured test concentrations in the range of 1.18 to 15.5 mg C/l whilst measured concentrations in the range of less than the limit of quantitation (LOQ) which was considered to be 1.0 mg C/l to 15.8 mg C/l were obtained at 72 hours. Overall there were no significant changes in the measured concentrations obtained between 0 and 72 hours.

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

*EL = Effective Loading Rate

Description of key information

Introduction.

A study was performed to assess the effect of the test material on the growth of the green algaDesmodesmus 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 Regulation (EC) 440/2008.

Methods.

Information provided by the Sponsor indicated that the test material was a mixture of components. Given this and also the poor solubility of the test material in water it was considered that the most suitable method of preparation for this material was as a Water Accommodated Fraction (WAF).

Method development work conducted for the determination of dissolved test material concentrations using HPLC-MS analysis gave a good response in single ion mode at 354 and 396 m/z. However, as the test material is a mixture of components it was considered that chemical analysis was not suitable for detecting the total dissolved test material concentration present. As such it was considered appropriate to determine the dissolved test material concentration present in the test samples by Total Organic Carbon (TOC) analysis.

Following preliminary range-finding tests,Desmodesmus subspicatuswas exposed to WAFs of the test material over a range of nominal loading rates of 0.32, 1.0, 3.2, 10 and 32 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 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	20	17	-	24	3.2	10
Yield	10	9.7	-	10	3.2	10
Biomass	10	8.2	-	12	3.2	10

Total Organic Carbon (TOC) analysis of the test preparations at 0 hours showed a concentration dependant increase in measured test concentrations in the range of 1.18 to 15.5 mg C/l whilst measured concentrations in the range of less than the limit of quantitation (LOQ) which was considered to be 1.0 mg C/l to 15.8 mg C/l were obtained at 72 hours. Overall there were no significant changes in the measured concentrations obtained between 0 and 72 hours.

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

*EL = Effective Loading Rate

Key value for chemical safety assessment

EC50 for freshwater algae:

20 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L