Reaction Products of C4 alcohols and C4 alkenes obtained as by-products from the manufacturing of butan-2-ol by sulfuric acid esterification and hydrolysis of butene

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 January 2010 and 5 February 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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Products of C4 alcohols and C4 alkenes obtained as by-products from the manufacturing of butan-2-ol by sulfuric acid esterification and hydrolysis of butene

EC Number:

701-242-6

Molecular formula:

A complex and variable combination of C4 hydrocarbons, 2,2'-oxybisbutane and butan-2-ol.

IUPAC Name:

Reaction Products of C4 alcohols and C4 alkenes obtained as by-products from the manufacturing of butan-2-ol by sulfuric acid esterification and hydrolysis of butene

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Chemical analysis of test loading rates
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. Duplicate samples were taken at each occasion and stored at approximately 20ºC for further analysis if necessary.

Given the volatile nature of the test item, an additional control and test replicate were prepared and incubated alongside the test. These remained unopened until the end of the test. A sample from each of these additional test replicates was taken for chemical analysis at 72 hours.
The method of analysis, stability, recovery and test preparation analyses are described in attached Appendix 4.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding test
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 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 completely filled with test preparation and sealed with a ground glass stopper to reduce evaporation and minimise losses due to the test items volatile nature. Two replicate flasks were used for each control and test concentration.
Amounts of test item (24 and 240 mg) were each separately added to the surface of 2.4 litres of culture medium in completely filled and sealed vessels 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. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. An aliquot (1 litre) of each of the WAFs was separately inoculated with algal suspension (18.4 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 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.

Definitive test
Based on the results 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.

Experimental Preparation
An amount of test item (550 mg) was added to the surface of 5.5 litres of culture medium in a completely filled and sealed vessel to give the 100 mg/l loading rate. 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 mixture allowed to stand for 1 hour. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
An aliquot (3 litres) of the WAF was inoculated with algal suspension (25.5 ml) to give the required test concentration of 100 mg/l loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours (see Appendix 4 in attached section.

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.

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

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 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 ± 1ºC until the algal cell density was approximately 104 – 105 cells/ml.
A positive control (Harlan Laboratories Ltd Project Number: 0039/1127) used potassium dichromate as the reference item.

Details of the positive control are given in Appendix 1. The positive control was conducted between 12 January 2010 and 15 January 2010.

- 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).

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.
For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/l) was added to the prepared culture medium prior to use.

- Any deformed or abnormal cells observed:
None recorded.

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 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 each test and control flask are given in Table 2 see in any other information on materials and methods section. Temperature was maintained at 24 ± 1ºC throughout the test.
The pH values of the control cultures (see Table 2 in any other information on materials and methods section) were observed to increase from pH 7.6 – 7.7 at 0 hours to pH 8.9 – 9.0 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.

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 320pH meter.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l
Based on the results 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:

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: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 3.65 x 10E5 cells per ml. Inoculation of 1 litre of test medium with 11 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.

- Control end cells density: algal cell density was approximately 6.24 10E5 cells/ml

- No. of organisms per vessel:
initial cell density of approximately 4 x 10E3 cells/ml.

- No. of vessels per concentration (replicates):
Three replicate flasks per concentration.

- No. of vessels per control (replicates):
Six replicate flasks.


GROWTH MEDIUM
- Standard medium used: yes
For the purpose of the definitive test, the test material was dissolved directly in 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 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).


TEST MEDIUM

Amounts of test material (23, 46, 92, 184 and 368 mg) were each separately added to the surface of 2.3 litres of culture medium in a sealed vessel with minimal headspace to give the 10, 20, 40, 80 and 160 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 dimple was formed at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10, 20, 40, 80 and 160 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.
An aliquot (1 litre) of each of the loading rate WAFs was separately inoculated with algal suspension (11 ml) to give the required test concentrations of 10, 20, 40, 80 and 160 mg/l loading rate WAF.
Total Organic Carbon (TOC) analysis was performed on the uninoculated test preparations at 0 and 72 hours.



OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Not applicable

- 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 completely filled with 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 item.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 4.70 x 105 cells per ml. Inoculation of 3 litres of test medium with 25.5 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 sealed with ground glass stoppers and incubated (INFORS Multitron¿ Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
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). ECxvalues were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949). 


- Chlorophyll measurement:
Not recorded

TEST CONCENTRATIONS
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 20, 40, 80 and 160 mg/l.

- Range finding study:
The loading rates to be used in the definitive test were 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 completely filled 250 ml glass conical flasks sealed with ground glass stoppers to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. The test material was prepared as a Water Accommodated Fraction (WAF).
Amounts of test material (24 and 230 mg) were each separately added to the surface of 2.4 and 2.3 litres of culture medium respectively in sealed vessels with minimal headspace 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. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.
An aliquot (1 litre) of each of the loading rate WAFs was separately inoculated with algal suspension (15.7 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.

POSITIVE CONTROL:
A positive control (Safepharm Laboratories Project Number: 0039/0994) used potassium dichromate as the reference material. An amount of reference material (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 10, 2.0, 1.0, 0.50, 0.25 and 0.125 mg/l. An aliquot (250 ml) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/l stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Six replicate flasks were prepared for the control and three replicate flasks prepared for each test concentration.
The flasks were incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
-The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
-The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
-The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL not stated

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL nit stated

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL not stated

Details on results:

Validation of Mixing Period
Pre-study work (see Appendix 3 in any other information on materials and methods section) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours. As such, for the purposes of testing, the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour standing period.

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test item during the range-finding test are given in Table 1 see in any other information in results section.
The results showed no effect on growth at 10 and 100 mg/l loading rate WAF.
Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.

Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 see in any other information on results section. Daily specific growth rates for the control cultures are given in Table 3 see in any other information on results section. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 see in any other information on results section.
The mean cell densities versus time for the definitive test are presented in Figure 1 see attached section.

Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 17 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 : 4.33 x 103 cells per ml
Mean cell density of control at 72 hours : 7.17 x 104 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 31% 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 2 and 4, see in any other information on results section. 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 item 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. 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.

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 item solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At both the start and end of the mixing period and following the 1-Hour standing period the WAF was observed to have formed a clear colourless media column with small oily globules of test item floating at the media surface. Microscopic examination of the WAF after stirring showed there to be no globules or micro-dispersions of test item 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 and test cultures were observed to be pale green dispersions.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface (see Table 5 see in any other information in results section).
Chemical analysis of test loading rates
Analysis of the test preparations at 0 hours (see Appendix 4 see attached section) showed measured test concentrations to range from 63 to 64 mg/l. A slight decline in measured test concentrations to 53 mg/l (84% of the mean 0-Hour measured test concentration) was observed at 72 hours.
Due to the volatile nature of the test item, an additional test replicate was prepared at 0 hours and incubated alongside the test to provide a sample for unopened vessel analysis at 72 hours. Analysis of this preparation showed a measured test concentration of 60 mg/l was obtained indicating that no losses due to volatility occurred.
The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole the results were based on nominal loading rates only.

Results with reference substance (positive control):

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 0039/1127) used potassium dichromate as the reference item 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 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:

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

Table 1              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	5.43E+03	1.77E+05
	R2	4.98E+03	1.79E+05	-	-
	Mean	5.20E+03	1.78E+05
10	R1	4.82E+03	1.89E+05
	R2	5.10E+03	2.05E+05	[4]	[11]
	Mean	4.96E+03	1.97E+05
100	R1	4.62E+03	1.87E+05
	R2	5.18E+03	2.11E+05	[4]	[12]
	Mean	4.90E+03	1.99E+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 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	24 h	48 h	72 h	72 h
Control	R1	7.6	5.17E+03	9.02E+03	1.99E+04	7.65E+04	8.9
	R2	7.7	4.09E+03	8.40E+03	1.93E+04	6.73E+04	9.0
	R3	7.6	4.41E+03	8.03E+03	1.69E+04	6.38E+04	8.9
	R4	7.6	4.54E+03	8.63E+03	1.91E+04	9.19E+04	9.0
	R5	7.6	3.94E+03	8.83E+03	2.01E+04	6.32E+04	9.0
	R6	7.6	3.86E+03	8.46E+03	2.11E+04	6.77E+04	9.0
	Mean		4.33E+03	8.56E+03	1.94E+04	7.17E+04
100	R1	7.8	4.58E+03	6.78E+03	1.99E+04	7.63E+04	9.1
	R2	7.8	3.68E+03	7.78E+03	2.13E+04	1.03E+05	9.3
	R3	7.8	4.10E+03	6.72E+03	1.90E+04	1.02E+05	9.0
	R4	7.9	4.03E+03	7.62E+03	1.96E+04	7.77E+04	9.1
	R5	7.9	4.87E+03	7.55E+03	1.87E+04	6.75E+04	9.1
	R6	7.9	4.30E+03	8.96E+03	1.95E+04	6.48E+04	9.1
	Mean		4.26E+03	7.57E+03	1.97E+04	8.18E+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.

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	0.034	0.033	0.056
	R2	0.031	0.035	0.052
	R3	0.029	0.031	0.055
	R4	0.032	0.033	0.065
	R5	0.033	0.034	0.048
	R6	0.031	0.038	0.049
	Mean	0.032	0.034	0.054

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	0.041		7.13E+04
	R2	0.039		6.32E+04
	R3	0.038		5.93E+04
	R4	0.044	-	8.74E+04	-
	R5	0.038		5.93E+04
	R6	0.039		6.39E+04
	Mean	0.040		6.74E+04
	SD	0.002		1.07E+04
100	R1	0.041	[3]	7.17E+04
	R2	0.045	[13]	9.90E+04
	R3	0.045	[13]	9.77E+04
	R4	0.041	[3]	7.37E+04
	R5	0.039	3	6.26E+04
	R6	0.039	3	6.05E+04
	Mean	0.042	[4]	7.75E+04	[15]
	SD	0.003		1.69E+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

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)	17.5	17.5	34	34
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 item 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.

Executive summary:

Introduction.

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, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 440/2008.

Methods. 

Following a preliminary range-finding test,Desmodesmus subspicatus was exposed to a Water Accommodated Fraction (WAF) of the test item, 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.

The test item was known to be volatile and hence testing was conducted in completely filled, stoppered test vessels in order to minimise possible losses due to volatilisation. Following the recommendations of published data (Hermanet al1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

Results.

Exposure of Desmodesmus subspicatusto the test item 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.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 63 to 64 mg/l. A slight decline in measured test concentrations to 53 mg/l (84% of the mean 0-Hour measured test concentration) was observed at 72 hours.

Due to the volatile nature of the test item, an additional test replicate was prepared at 0 hours and incubated alongside the test to provide a sample for unopened vessel analysis at 72 hours. Analysis of this preparation showed a measured test concentration of 60 mg/l was obtained indicating that no losses due to volatility occurred.

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.

*EL = Effective Loading Rate

Conclusion.

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

*EL = Effective Loading Rate