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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January - 12 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the solvent control and the 0.65 mg/L test group at 0, 24 and 72 hours for immediate quantitative analysis. Duplicate samples were taken at 0 hours and stored at approx. -20°C for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 24 and 72 hours.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Pre-study media preparation trial:
Pre-study solubility work showed that the highest attainable test concentration (by visual inspection) that could be prepared was 1.0 mg/L using a preliminary solution in tetrahydrofuran. At higher test concentrations precipitation of the test material was observed on addition of the test material solvent stock solution to water. Based on this information the test material fell into the category of a 'difficult substance' as defined by the OECD Guidance document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 23, 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

Saturated solution preparation:
An amount of test material (550 mg) was dispersed, in duplicate, in 11 litres of reconstituted water with the aid of propeller stirring at approx. 1500 rpm at a temperature of approx. 21°C for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
- Centrifugation at 10000 g for 30 min
- Centrifugation at 40000 g for 30 min
- Filtration through a 0.2 µm Sartorius Sartopore filter (initial approx. 500 mL discarded in order to pre-condition the filter)
- Filtration through a 0.2 µm Sartorius Sartopore filter (initial approx. 1 L discarded in order to pre-condition the filter)

Solvent spike preparation:
An amount of test material (100 mg) was dissolved in tetrahydrofuran and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (1000 µL) of this solvent stock solution was dispersed in 10 litres of reconstituted water with the aid of magnetic stirring for approx. 10 minutes to give a 1.0 mg/L test concentration. Samples were taken for chemical analysis after the following pre-treatments:
- Untretaed
- Centrifugation at 10000 g for 30 min
- Centrifugation at 40000 g for 30 min
- Filtration through a 0.2 µm Gelman AcroCap filter (initial approx. 100 mL discarded in order to pre-condition the filter)
- Filtration through a 0.2 µm Gelman AcroCap filter (initial approx. 500 mL discarded in order to pre-condition the filter)

The remainder of the 1.0 mg/L test concentration was returned to the magnetic stirrer and stirred for a further 48 hours with samples being taken for analysis after both 24 and 48 hours stirring.

Range-finding test:
The test concentration to be used in the difinitive test was determined by a preliminary range-finding test, where Desmodesmus subspicatus cells were exposed to a series of nominal test concentrations of 0.0085, 0.085, and 0.85 mg/L for a period of 72 hours. The test material was prepared using a preliminary solution in tetrahydrofuran. An amount of test material (100 mg) was dissolved in tetrahydrofuran and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (100 µL) of this solvent stock solution was dispersed in 1 litre of culture medium with the aid of magnetic stirring for approx. 10 minutes prior to removal of any undissolved test material by centrifugation at 40000 g for 30 min to give a 0.85 mg/L stock solution. A series of dilutions was made from the 0.85 mg/L stock solutions of 0.0085, 0.085 and 0.85 mg/L. An aliqout (200 mL) of each of the stock solutions was seperately inoculated with algal suspension (3.2 mL) to give the required test concentrations of 0.0085, 0.085 and 0.85 mg/L.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive test:
Based on the results of the range-fining test a "Limit test" was conducted at a nominal concentration of 0.85 mg/L to confirm that at the highest attainable test concentration of 0.85 mg/L, no effect on algal growth was observed.
For the purpose of the definitive test the test material was prepared using a preliminary solution in tetrahydrofuran. An amount of test material (100 mg) was dissolved in tetrahydrofuran and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (300 µL) of this solvent stock solution was dispersed in 3 litres of culture medium with the aid of magnetic stirring for approx. 10 minutes prior to removal of any undissolved test material by centrifugation at 40000 g for 30 min to give a stock solution with a 0-hour measured test concentration of 0.65 mg/L. An aliquot (2.5 litres) of the 0.65 mg/L stock solution was inoculated with algal suspension (16.8 mL) to give the required test concentration of 0.65 mg/L
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage MArine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation):
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium, constant aeration and illumination at 21 ± 1°C. Prior to the start of the test sufficient master culture was added to approx. 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approx. 1000 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°C until the algal cell density was approx. 10^4 - 10^5 cells/mL.


ACCLIMATION
- Culturing media and conditions (same as test or not): yes
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ± 1°C
pH:
7.2 - 7.8
Nominal and measured concentrations:
0.65 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: 250 mL glass conical flasks filled with 100 mL of test preparations and controls
- Initial cells density: 4x10^3 cels
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: continuous illumination
- Light intensity and quality: approx. 7000 lux, 380 -730 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.0085, 0.085, and 0.85 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.65 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.65 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
other: for each of growth rate, biomass, and yield
Details on results:
- Exponential growth in the control (for algal test): yes
- no abnormalities observed
- At the start of the test all control, solvent control and 0.65 mg/I test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, solvent control and 0.65 mg/I test cultures were observed to be pale green dispersions.

The following data show that the cell concentration of the control cultures increased by a factor of 50 after 72 hours and the cell concentration of the solvent control cultures increased by a factor of 59 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0 hours: 3.83E03 cells per ml
Mean cell density of control at 72 hours: 1.93E05 cells per ml
Mean cell density of solvent control at 0 hours: 3.50E03 cells per ml
Mean cell density of solvent control at 72 hours: 2.06E05 cells per ml
The mean coefficients of variation for section by section specific growth rate for the control and solvent control cultures were 15% and 24% respectively and hence satisfied the validation criterion given in the OEGD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control and solvent cultures over the test period (0 - 72 h) was 2% and hence satisfied the validation criterion given in the OEGD Guideline which states that this must not exceed 7%.
Results with reference substance (positive control):
- Results with reference substance valid? yes
ErC50 (0 - 72 h): 0.52 mgll, 95% confidence. limits 0.43 - 0.62 mg/I
EyC50 (0 - 72 h): 0.29 mgll, 95% confidence limits 0.25 - 0.33 mg/I
EbC50 (0 - 72 h): 0.30 mgll, 95% confidence limits 0.26 - 0.34 mg/I



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, yield and biomass integral data after 24 and 72 hours for the solvent control and the 0.65 mg/L test concentration to determine any statistically significant differences between the test and solvent control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Growth rate, yield and biomass integral of Desmodesmus subspicatus were not affected by the presence of the test material at 0-hour measured test concentration of 0.65 mg/L over the 72-hour exposure period. Accordingly the results were determined from the data based on the 0-hour measured test concentrations.

Analysis of the test preparations at 24 hours showed a decline in measured test material concentrations to 0.11 mg/L. At 72 hours measured concentrations observed over the test period was inline with the preliminary stability analyses conducted which indicated that the test material was unstable. Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.022 mg/L) was used to enable calculation of the geometric mean measured concentration.

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

Validity criteria fulfilled:
yes
Conclusions:
ErC50 >0.65 mg/L, nominal
NOEC=0.65 mg/L, nominal
Executive summary:

A study according to OECD TG 201 was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus.


Pre-study solubility work conducted indicated that the test material was insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. The highest dissolved test material that could be obtained (by visual inspection) was 1.0 mg/l using a preliminary solution in tetrahydrofuran. Based on this information the test material fell into the category of a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 23, 2000).
A pre-study media preparation trial indicated that the use of a solvent spike method of preparation followed by centrifugation to remove the undissolved test material was the most appropriate method of preparation for the test material giving a dissolved test material concentration of approximately 0.85 mg/l.
Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at a 0-Hour measured test concentration of
0.65 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 material was known to be unstable. Based on this and at the request of the Sponsor chemical analysis of the test preparations was conducted at 0, 24 and 72 hours.


Analysis of the test preparations at 0 hours showed measured test material concentrations to range from 0.57 to 0.73 mg/l.
Exposure of Desmodesmus subspicatus to the test material gave ECs» values based on the 0-Hour measured test concentrations of greater than 0.65 mg/l and correspondingly the No Observed Effect Concentration was 0.65 mg/I.
Analysis of the test preparations at 24 hours showed a decline in measured test material concentrations to 0.11 mg/l.
Exposure of Desmodesmus subspicatus to the test material gave ECs values based on the 0 — 24 Hour geometric mean measured test material concentrations of greater than 0.27 mg/l and correspondingly the No Observed Effect Concentration was 0.27 mg/l. At 72 hours measured concentrations of less than the LOQ were obtained. The decline in measured concenirations observed over the test period was inline with.the preliminary stability analyses conducted.


These values don't represent the real environmental conditions because the test substance is rapidly oxidized by the oxygen content in the aqueous phase. Therefore, nominal values will be used for hazard assessment.

Description of key information

ErC50 (72 h) > 0.65 mg/L (nominal) (Desmodesmus subspicatus)


ErC10 (72 h) > 0.65 mg/L (nominal) (Desmodesmus subspicatus)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.65 mg/L
EC10 or NOEC for freshwater algae:
0.65 mg/L

Additional information

A study according to OECD TG 201 was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus.


Pre-study solubility work conducted indicated that the test material was insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. The highest dissolved test material that could be obtained (by visual inspection) was 1.0 mg/l using a preliminary solution in tetrahydrofuran. Based on this information the test material fell into the category of a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 23, 2000).
A pre-study media preparation trial indicated that the use of a solvent spike method of preparation followed by centrifugation to remove the undissolved test material was the most appropriate method of preparation for the test material giving a dissolved test material concentration of approximately 0.85 mg/l.
Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at a 0-Hour measured test concentration of
0.65 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 material was known to be unstable. Based on this and at the request of the Sponsor chemical analysis of the test preparations was conducted at 0, 24 and 72 hours.


Analysis of the test preparations at 0 hours showed measured test material concentrations to range from 0.57 to 0.73 mg/l.
Exposure of Desmodesmus subspicatus to the test material gave ECs» values based on the 0-Hour measured test concentrations of greater than 0.65 mg/l and correspondingly the No Observed Effect Concentration was 0.65 mg/I.
Analysis of the test preparations at 24 hours showed a decline in measured test material concentrations to 0.11 mg/l.
Exposure of Desmodesmus subspicatus to the test material gave ECs values based on the 0 — 24 Hour geometric mean measured test material concentrations of greater than 0.27 mg/l and correspondingly the No Observed Effect Concentration was 0.27 mg/l. At 72 hours measured concentrations of less than the LOQ were obtained. The decline in measured concenirations observed over the test period was inline with.the preliminary stability analyses conducted.


These values don't represent the real environmental conditions because the test substance is rapidly oxidized by the oxygen content in the aqueous phase. Therefore, nominal values will be used for hazard assessment.

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