[1,1'-Biphenyl]-2,2'-diol, 3,3'-bis(1,1-dimethylethyl)-5,5'-dimethoxy-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 12 February 2009 and 27 March 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, 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. Pre-study work showed that a preparation period of 24 hours was 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)

Remarks:

Date of Inspection: 19/08/08 Date of signature: 04/03/09

Test material

Test material information

Constituent 1

Reference substance name:

L17_Precursor

IUPAC Name:

L17_Precursor

Details on test material:

Sponsor's identification : L17_Precursor
Description : pale yellow powder
Batch number : DALA046312
Date received : 11 November 2008
Storage conditions : room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
100 mg/l loading rate WAF test group
Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.
A volume of test sample had 30% (w/v) sodium chloride added and was then extracted with dichloromethane (3 x 50 ml). The extracts were filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in dichloromethane to give a final theoretical concentration of 10 mg/l.

- Sampling method:
Samples were taken from the control (replicates R1 - R6 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis.


- Sample storage conditions before analysis:
Samples used immediately upon sampling. Duplicate samples were taken and stored frozen (approximately -20 degC) for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
An amount of test material (250 mg) was added to the surface of 2.5 litres of culture medium to give the 100 mg/l loading rate. 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 mixture allowed to stand for 1 hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAF by filtering three times 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 100 mg/l loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed there to be no micro-dispersions of test material present.
An aliquot (2 litres) of the WAF was separately inoculated with algal suspension (16.4 ml) to give the required test concentration of 100 mg/l loading rate WAF.

- Eluate:
Not applicable

- Controls:
A positive control (Harlan Laboratories Ltd Project No: 0039/1066) 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.

- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of stirring the WAF was observed to have formed a clear colourless media column with test material floating at the media surface and within the dimple. After both the end of stirring and the 1-Hour settlement period the test material was observed to be floating at the media surface and within the media column.
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 and test cultures were observed to be pale green dispersions.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
Algae

- Strain:
Strain CCAP 276/20

- Source:
Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Age of inoculum :
Not recorded

- Method of cultivation:
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 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.

ACCLIMATION
- Acclimation period:
Not recorded.

- Culturing media and conditions:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/l of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).

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:
None recorded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not recorded.

Test temperature:

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

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.
The pH values of the control cultures (see Table 2) were observed to increase from pH 7.3 at 0 hours to pH 7.5 – 7.6 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:

The loading rate 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 loading rates of 10 and 100 mg/l for a period of 72 hours.
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

Details on test conditions:

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

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

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

- Initial cells density:
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.

- Control end cells density:
algal cell density was approximately 1.53 x 105 cells per ml

- No. of organisms per vessel:
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 4.87 x 105 cells per ml. Inoculation of 2 litres of test medium with 16.4 ml of this algal suspension gave an initial nominal cell density of 4 x 103 cells per ml and had no significant dilution effect on the final test concentration.

- No. of vessels per concentration (replicates):
6

- No. of vessels per control (replicates):
6


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


TEST MEDIUM
- Source/preparation of dilution water:
Reverse osmosis purified deionised water (Elga Optima 15+)

- Total organic carbon:
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic materials, in the WAF. A WAF of a nominal loading rate of 100 mg/l was prepared in duplicate in deionised reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis.
The results are summarised as follows:

Nominal
Loading Rate Time (Hours)
(mg/l) 24 96
mg C/l mg C/l Corrected for Control mg C/l mg C/l Corrected for Control
Control 100 1.47 1.47 1.56 1.56

It is evident from this work that increasing the stirring period did not significantly increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.

- Particulate matter:
None recorded

- Culture medium different from test medium:
Same as test medium

- Intervals of water quality measurement:
Not recorded


OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Continuous lighting.

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

EXPOSURE CONDITIONS :

As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l loading rate WAF 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 4.87 x 105 cells per ml. Inoculation of 2 litres of test medium with 16.4 ml of this algal suspension gave an initial nominal cell density of 4 x 103 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.

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples were taken at 0, 23, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

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

- Chlorophyll measurement:
Not recorded

- Other:
Evaluation of data
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

µ = InNn - InN1 / tn - t1

where:
μ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement
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 using the following equation:

Ir = µc - µt / µc x 100

where:
Ir = percentage inhibition of average specific growth rate
μc = mean average specific growth rate for the control cultures
μt = average specific growth rate for the test culture

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

Y = Nn - N0

where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test
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 equation:

Iy = (Yc - Yt) / Yc x 100

where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group


TEST CONCENTRATIONS

- Range finding study:
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 rate 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 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 100 mg/l loading rate WAF 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 three times and showed there to be no micro-dispersions of test material present.

An aliquot (250 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (2.6 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.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

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

- Results used to determine the conditions for the definitive study:
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

VALIDATION:
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:

EL50

Effect conc.:

>= 100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

growth rate, yield and biomass integral

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

growth rate, yield and biomass integral

Details on results:

- Exponential growth in the control (for algal test): yes
Please see Table 3

- Observation of abnormalities (for algal test):
No abnormalities observed

- Any stimulation of growth found in any treatment:
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 over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Accordingly the following results were determined from the data:

Inhibition of growth rate
ErL*10 (0 - 72 h) : > 100 mg/l loading rate WAF
ErL*20 (0 - 72 h) : > 100 mg/l loading rate WAF
ErL*50 (0 - 72 h) : > 100 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 100 mg/l loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P>=0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/l loading rate WAF.

Inhibition of yield
EyL*10 (0 - 72 h) : > 100 mg/l loading rate WAF
EyL*20 (0 - 72 h) : > 100 mg/l loading rate WAF
EyL*50 (0 - 72 h) : > 100 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 in Section 5.3.2.1. There were no statistically significant differences between the control and 100 mg/l loading rate WAF (P0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/l loading rate WAF.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
Chemical analysis of the test preparations at 0 hours (see Appendix 5) showed measured test concentrations to range from 1.4 to 1.5 mg/l. A decline in measured concentrations was observed at 72 hours in the range of 0.59 to 0.63 mg/l. Given that the preliminary stability analyses conducted indicated that the test material was stable over the test period this decline was considered to be due to adsorption of the test material to the algal cells present. This was inline with preliminary recovery analyses conducted in the presence of algal cells which indicated that adsorption of the test material was occurring.

However, 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.

- Effect concentrations exceeding solubility of substance in test medium:
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of stirring the WAF was observed to have formed a clear colourless media column with test material floating at the media surface and within the dimple. After both the end of stirring and the 1-Hour settlement period the test material was observed to be floating at the media surface and within the media column.

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 and test cultures were observed to be pale green dispersions.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project No: 0039/1066) 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) : 0.52 mg/l, 95% confidence limits 0.43 – 0.62 mg/l
EyC50 (0 – 72 h) : 0.29 mg/l, 95% confidence limits 0.25 – 0.33 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.125 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.25 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:

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 control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

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

Nominal Loading Rate (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	4.52E+03	4.81E+05
	R2	4.30E+03	4.85E+05	-	-
	Mean	4.41E+03	4.83E+05
10	R1	4.55E+03	6.36E+05
	R2	4.22E+03	3.85E+05	[2]	[6]
	Mean	4.39E+03	5.10E+05
100	R1	4.50E+03	5.60E+05
	R2	4.52E+03	5.29E+05	[3]	[13]
	Mean	4.51E+03	5.44E+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

[Increase in growth compared to controls]

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	23 h	48 h	72 h	72 h
Control	R1	7.3	3.82E+03	1.15E+04	3.07E+04	1.62E+05	7.6
	R2	7.3	3.78E+03	1.10E+04	3.17E+04	1.71E+05	7.5
	R3	7.3	4.00E+03	1.25E+04	3.79E+04	1.74E+05	7.5
	R4	7.3	4.21E+03	1.08E+04	3.97E+04	1.68E+05	7.6
	R5	7.3	4.57E+03	1.06E+04	2.94E+04	1.13E+05	7.5
	R6	7.3	4.38E+03	1.19E+04	3.76E+04	1.33E+05	7.6
	Mean		4.13E+03	1.14E+04	3.45E+04	1.53E+05
100	R1	7.2	4.06E+03	1.04E+04	3.38E+04	1.84E+05	7.5
	R2	7.2	4.17E+03	1.07E+04	3.35E+04	1.76E+05	7.5
	R3	7.2	4.06E+03	1.08E+04	3.50E+04	1.98E+05	7.5
	R4	7.1	4.02E+03	1.14E+04	3.14E+04	1.33E+05	7.5
	R5	7.2	4.74E+03	1.07E+04	3.34E+04	1.52E+05	7.5
	R6	7.2	4.30E+03	1.05E+04	3.40E+04	2.19E+05	7.5
	Mean		4.23E+03	1.08E+04	3.35E+04	1.77E+05

 

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

R₁- R₆= Replicates 1 to 6

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

		Daily Specific Growth Rate (cells/ml/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.046	0.039	0.069
	R2	0.044	0.042	0.070
	R3	0.050	0.044	0.063
	R4	0.043	0.052	0.060
	R5	0.042	0.041	0.056
	R6	0.047	0.046	0.053
	Mean	0.045	0.044	0.062

_R1- R₆= Replicates 1 to 6

Table 4              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	0.051		1.58E+05
	R2	0.052		1.67E+05
	R3	0.052		1.70E+05
	R4	0.052	-	1.64E+05	-
	R5	0.046		1.08E+05
	R6	0.049		1.28E+05
	Mean	0.050		1.49E+05
	SD	0.002		2.50E+04
100	R1	0.053	[6]	1.80E+05
	R2	0.053	[6]	1.72E+05
	R3	0.054	[8]	1.94E+05
	R4	0.049	2	1.29E+05
	R5	0.051	[2]	1.48E+05
	R6	0.056	[12]	2.15E+05
	Mean	0.053	[5]	1.73E+05	[16]
	SD	0.002		3.12E+04

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

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Table 5              Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		100
	*	+	*	+
Height of Media Column (cm)	15	15	15	15
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

*= Start of mixing period

+= End of mixing period

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

Given that no toxicity was observed at the maximum test concentration employed of 100 mg/l loading rate WAF in accordance with Council Directive 67/548/EEC the test material will not be classified as being harmful to aquatic organisms.

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) No 440/2008.

Methods. 

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/l (six replicate flasks) 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 o fDesmodesmus subspicatus to the test material gave EL*₅₀values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.4 to 1.5 mg/l. A decline in measured concentrations was observed at 72 hours in the range of 0.59 to 0.63 mg/l. Given that the preliminary stability analyses conducted indicated that the test material was stable over the test period this decline was considered to be due to adsorption of the test material to the algal cells present. This was inline with preliminary recovery analyses conducted in the presence of algal cells which indicated that adsorption of the test material was occurring. 

However, 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.

Given that no toxicity was observed at the maximum test concentration employed of 100 mg/l loading rate WAF in accordance with Council Directive 67/548/EEC the test material will not be classified as being harmful to aquatic organisms.

*EL = Effective Loading Rate