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Ecotoxicological information

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:
25 July 2008 and 30 October 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: An aliquot (250 ml) of each of the stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l.
- Sampling method: The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of a single peak. The test samples were analysed following filtration through glass wool to remove the algal cells.
- Sample storage conditions before analysis:
Sponsor's identification : NA-Na
Description : white crystalline solid
Batch number : 7001
Date received : 12 May 2008
Storage conditions : room temperature in the dark

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test, the test material was dissolved directly in culture medium.An amount of test material (100 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 200 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 20, 2.0 and 0.20 mg/l. An aliquot (100 ml) of each of the stock solutions was separately mixed with algal suspension (100 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
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.

- Eluate: Not Applicable
- Controls: The control group was maintained under identical conditions but not exposed to the test material.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.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc):None

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): Not available
- 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°C until the algal cell density was approximately 104 - 105 cells/ml.

ACCLIMATION
- Acclimation period: Not available
- Culturing media and conditions (same as test or not):The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
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* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

Study design

Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Post exposure observation period:
All test and control cultures were inspected microscopically at 72 hours.

Test conditions

Hardness:
Not available
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 2) were observed to increase from pH 7.5 – 7.6 at 0 hours to pH 7.9 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 320 pH meter.
Dissolved oxygen:
Not available
Salinity:
Not available
Nominal and measured concentrations:
The range-finding test :nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l
definitive test: 10, 20, 40, 80 and 160 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel:250 ml glass conical flasks
- Type (delete if not applicable): closed,plugged with polyurethane foam bungs to reduce evaporation.
- Material, size, headspace, fill volume: 250 ml glass conical flasks each containing 100 ml of test preparation
- Aeration: None
- 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: 4 x 10^3 cells per ml
- Control end cells density: 5.68 x 10^5 cells per ml
- No. of organisms per vessel: Not applicable
- No. of vessels per concentration (replicates):6
- No. of vessels per control (replicates):6
- No. of vessels per vehicle control (replicates): Not applicable


GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:Not applicable


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water.
- Total organic carbon:No Data


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:No
- Photoperiod:continuous illumination
- Light intensity and quality:intensity approximately 7000 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
- Chlorophyll measurement: Not applicable
- Other:
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 th e 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 CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline:Not applicable
- Range finding study
- Test concentrations: The test concentrations 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 test concentrations of 0.10, 1.0, 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 used for each control and test concentration. The test material was dissolved directly in culture medium.
An amount of test material (100 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 200 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 20, 2.0 and 0.20 mg/l. An aliquot (100 ml) of each of the stock solutions was separately mixed with algal suspension (100 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
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 results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 20, 40, 80 and 160 mg/l.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.
- Any stimulation of growth found in any treatment: No
From the data given in Tables 2 and 4, it is clear that the growth rate (r), yield (y) and biomass integral (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
ErC10 (0 - 72 h) : 17 mg/l
ErC20 (0 - 72 h) : 25 mg/l
ErC50 (0 - 72 h) : > 160 mg/l
where ErCx is the test concentration that reduced growth rate by x%.
It was not possible to calculate an ErC50 value no concentration tested resulted in greater than 50% inhibition of growth rate.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control and 10 mg/l test concentration (P0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 10 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 20 mg/l.

Inhibition of yield
EyC10 (0 - 72 h) : 10 mg/l
EyC20 (0 - 72 h) : 14 mg/l
EyC50 (0 - 72 h) : 22 mg/l
where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 5.2.2.1. There were no statistically significant differences between the control and 10 mg/l test concentration (P0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 10 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 20 mg/l.

Inhibition of biomass integral
EbC10 (0 - 72 h) : 12 mg/l
EbC20 (0 - 72 h) : 17 mg/l
EbC50 (0 - 72 h) : 35 mg/l
where EbCx is the test concentration that reduced biomass integral (area under the growth curve) by x%.
Statistical analysis of the biomass integral data was carried out as in Section 5.2.2.1. There were no statistically significant differences between the control and 10 mg/l test concentration (P0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 10 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on biomass integral was 20 mg/l.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:No Data
- Effect concentrations exceeding solubility of substance in test medium: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 10 mg/l test cultures were observed to be pale green dispersions. The 20 and 40 mg/l test cultures were observed to be very pale green dispersions, the 80 mg/l test cultures were observed to be very pale green/brown dispersions whilst the 160 mg/l test cultures were observed to be pale green/brown dispersions.
Results with reference substance (positive control):
- Results with reference substance valid?Yes
- EC50: The cell densities from exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material during the positive control (Safepharm Laboratories Project No: 0039/0994) are given in Table 5 and Figure 5. Daily specific growth rates for the control cultures are given in Table 6 whilst growth rate, yield and biomass integral values are given in Table 7. Percentage inhibition values are plotted against test concentration in Figure 6, Figure 7 and Figure 8.
Accordingly the following results were determined from the data:
ErC50 (0 - 72 h) : 0.57 mg/l; 95% confidence limits 0.48 - 0.66 mg/l
EyC50 (0 - 72 h) : 0.32 mg/l; 95% confidence limits 0.29 - 0.35 mg/l
EbC50 (0 - 72 h) : 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l
No Observed Effect Concentration (NOEC) based on growth rate : 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on biomass integral : 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on biomass integral : 0.125 mg/l
The results from the positive control with potassium dichromate were within the normal range for this reference material.

Reported statistics and error estimates:
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control and 10 mg/l test concentration (P0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 10 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 20 mg/l.

Any other information on results incl. tables

Tables and figures are attached.

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%.

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 over a 72-Hour period and gave an ErC50 (0 - 72 h) of greater than 160 mg/l*, an EyC50 (0 - 72 h) of 22 mg/l and an EbC50 (0 - 72 h) of 35 mg/l. The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 20 mg/l, and the No Observed Effect Concentration was 10 mg/l.
Executive summary:

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 Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods. Following a preliminary range-finding test,Desmodesmus subspicatuswas exposed to an aqueous solution of the test material at concentrations of 10, 20, 40, 80 and 160 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.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatuswas exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 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. In terms of growth rate, exposure ofDesmodesmus subspicatusto the test material gave an ErC50(0 - 72 h) value of greater than 160 mg/l*. The Lowest Observed Effect Concentration based on inhibition of growth rate was 20 mg/l and the No Observed Effect Concentration was 10 mg/l.

In terms of yield, exposure ofDesmodesmus subspicatusto the test material gave an EyC50(0 - 72 h) value of 22 mg/l. The Lowest Observed Effect Concentration based on yield was 20 mg/l and the No Observed Effect Concentration was 10 mg/l.

In terms of biomass integral (area under growth curve), exposure ofDesmodesmus subspicatusto the test material gave an EbC50(0 - 72 h) value of 35 mg/l. The Lowest Observed Effect Concentration based on inhibition of biomass integral was 20 mg/l and the No Observed Effect Concentration was 10 mg/l.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal with the exception of the 160 mg/l test concentration at 72 hours which showed a measured concentration of 78% of nominal. Given that over the test duration the algae were exposed to an average concentration in excess of 80% of nominal it was considered acceptable to calculate the results based on nominal test concentration alone.

Exposure ofDesmodesmus subspicatusto the reference material, potassium dichromate, gave an ErC50(0 - 72 h) of 0.57 mg/l; 95% confidence limits 0.48 – 0.66 mg/l, an EyC50(0 - 72 h) of 0.32 mg/l; 95% confidence limits 0.29 ‑ 0.35 mg/l, and an EbC50(0 - 72 h) of 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l. The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/l respectively and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/l respectively. 


*It was not possible to calculate an ErC50value as no concentration tested resulted in greater than 50% inhibition of growth rate.