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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
5 September 1995 - 13 October 1995
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other:
Remarks:
Validity criteria were not met as no actual concentrations could be measured, except for a measurement of Al in the stock solution. Even though the test solutions were prepared by stirring in DMSO followed by filtration, the filtrate which was used to prepare the test concentration range was a turbid suspension. A dilution up to 18% of this filtrate did not indicate precipitation whereas in the second test a heavy precipiation was recorded at the higher concentrations. Based on the results of the study, it cannot be determined on whether 18% of the filtrate can be considered the maximum solubility of the test item in medium. A good interpretation of this study is not possible.
Qualifier:
according to guideline
Guideline:
other: EEC-Directive 92/69 C.3
Version / remarks:
1992
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Version / remarks:
1989
GLP compliance:
yes
Specific details on test material used for the study:
Stability under storage conditions: stable
Stability in water: not indicated
Solubility in water: <2.4 mg/L at 20 °C
Analytical monitoring:
yes
Details on sampling:
No analyitical method was available or could be developed for this compound. Therefore, quantative analyes were performed on Al and Mg. Consequently, no stability of the test substance could be tested. The analyses were performed by: TNO Institute of Environmental Sciences in Delft, the Netherlands

- Concentrations: in the first test: 1.0, 3.2 and 18% of the 5 µm filtrate
- Sampling method first test: according to schedule below
Frequency: at t=0 and at t=72 h
Volume: 25 mL
- Storage before sampling: samples were stored in a refrigerator until shipment to the TNO laboratories at Delft, the Netherlands. All samples were filtered through a 0.45 µm filter acidified before storage and subsequent shipment.
Also a sample was taken from the stock of 100 mg/L at t=0.

In the second test, samples of 10, 32 and 100% of the 5 µm filtrate were only taken at t=0.
Vehicle:
yes
Remarks:
Dimethylsulphoxide (DMSO)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 201.3 mg of test substance was dissolved in DMSO and mixed with 200 mL of ISO-medium providing a nominal concentration of 1000 mg/L. After 90 hours of stirring, the solution was a turbid suspension and was diluted up to 2000 mL with ISO medium. This suspension was stirred for another 2 hours. Subsequently, this suspension was filtered through a rough paper filter (Schleider and Schuell 604) to remove the larger particles. The filtrate, a turbid suspension, was used to prepare the test concentration range. The test solutions were prepared in ISO-medium containing 0.1 mL DMSO/L. Subsequently, a volume of 0.19 mL of the algal suspension was added. The final volume of the test solutions was 50 mL per vessel. At the start of the test all test solutions were clear and colorless. The higher concentrations tested in the second test were turbid, and precipitation occurred during the first 48 hours of exposure.
- Controls: Test medium without test substance or other additives and one control containing test medium with the additive used in the treatment of the stock solutions.
- Chemical name of vehicle: Dimethylsulphoxide, organic solvent
- Concentration of vehicle in test medium: 0.1 mL/L
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): not indicated
- Age of inoculum (at test initiation): not indicated
- Incubation: during incubation the algal cells were kept in suspension by continuous shaking.

ACCLIMATION: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
Between 22.5 and 23.5 °C throughout the test
pH:
Between 8.1 and 8.5 throughout the test
Nominal and measured concentrations:
Nominal concentrations in the first test: 1, 1.8, 3.2, 5.6, 10 and 18% of a 5 µm filtrate prepared at a nominal concentration of 100 mg/L.
Nominal concentration in the second test: 10, 32 and 100% of a 5 µm filtrate prepared at a nominal concentration of 100 mg/L.
Measured concentration in all sampled concentrations: < 1.53 mg/L. The concentration of test substance was based on the amount of Al calculated as follows: concentration Al * (588/54)

Measured concentration in the stock solution of 100 mg/L was 1.63 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Aeration: not indicated
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: blank control: 48.8 x 10^4 cells/mL, control with vehicle: 51.7 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6
- No. of vessels per highest concentration without algae: 1

GROWTH MEDIUM
- Standard medium used: yes, ISO medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water (tap water purified by reverse osmosis and then passed over activated carbon and ion-exchange cartridges (Millipore Corp., Bedford, Mass., USA)
- Culture medium different from test medium: not indicated

OTHER TEST CONDITIONS
- Sterile test conditions: not indicated
- Adjustment of pH: no
- Light intensity and quality: continuously using LTD-lamps of 18 Watt (Philips, Spain) yielding 7000-8000 lux.

EFFECT PARAMETERS MEASURED: Biomass and growth rate over a period of 72 hours. Additionally, pH was measured at the beginning and at the end of the test and temperature of the medium was measured everyday in a temperature control vessel.
- Determination of cell concentrations: at the beginning of the test cells were counted by microscope, using a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Lambda Spectrophotometer (Perkin-Elmer, Illinois, USA) with a cuvette of 5 cm path-length. Algal medium was used as a blank.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study concentrations: 0.1, 1, 10 and 100% of a 5 µm filtrate prepared at a nominal concentration of 100 mg/L.
- Results used to determine the conditions for the definitive study: yes, in the 100% filtrate, cell densities were found to have increased to an average of 5 x 10^4 cells/mL, whereas the cell density in the controls had increased to an average of 32 x 10^4 cells/mL. In the 10% filtrate, the cell density had increased to an average of 11 x 10^4 cells/mL after 72 hours. No inhibition of cell growth was observed at a nominal concentration of the 1% filtrate.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (July 1995)
Duration:
72 h
Dose descriptor:
EC50
Remarks on result:
other:
Remarks:
<10% effects were observed at a test concentration of 18% of a 5µm filtrate prepared at a nominal concentration of 100 mg/L.
Duration:
72 h
Dose descriptor:
NOEC
Remarks on result:
other:
Remarks:
< 10% effects were observed at a test concentration of 18% of a 5µm filtrate prepared at a nominal concentration of 100 mg/L.
Details on results:
- Analysis of the samples showed that the detected concentration of Al and Mg were not higher than those measured in samples taken from the controls. Hence, although a turbid suspension was attained dispersing the test substance with DMSO, no detectable concentration of Al or Mg could be found in samples containing the water soluble fraction after filtration through a 0.45 µm filter. Only a small amount of Al was detected in the sample taken from the stock solution prepared for the first full test. Based on this amount, a concentration of 1.63 mg/L of test substance in the stock solution prepared at 100 mg/L was determined.
- Exponential growth in the control (for algal test): yes
- No biological, behavioural or other abnormalities were observed.
- Any stimulation of growth found in any treatment: in the 24-72 hour interval, a stimulation of cell growth was indicated for the highest test concentrations.
- Effect concentrations exceeding solubility of substance in test medium: unknown, EC50 was higher than the highest tested concentration (18% of a 5 µm filtrate prepared at a loading rate of 100 mg/L), but actual concentrations could not be measured. As only at the higher test concentrations in the second test, precipitation occurred, it is unsure whether this value is considered to be the maximum solubility in medium.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50 for growth inhibition, 0-72h: 0.98 mg/L
- EC50 for growth rate reduction, 24-72h: 1.1 mg/L
- Other: these results were in line with historical data.

For more detailed results concerning the mean cell densities, percentage of cell growth and percentage reduction of growth rate, refer to the attached document. Also, for details on measured concentrations, refer to the attached document. (CRL 137813 - Tables.docx)

Validity criteria fulfilled:
no
Remarks:
Actual concentrations could not be measured.
Conclusions:
The 72h-EC50 for growth inhibition was > 18% of a 5µm filtrate prepared at a nominal concentration of 100 mg/L. The NOEC for growth inhibition was equal to 18% of a 5µm filtrate prepared at a nominal concentration of 100 mg/L. Because it cannot be concluded that this is the maximum solubility of the test substance in medium, a good interpretation of this study was not possible.
Executive summary:

A study was performed to assess the effect of the test substance on the growth rate of fresh water algae (Pseudokirchneriella subcapitata) after 72 hours of exposure. The study was conducted in accordance with OECD 201 and GLP and performed under static conditions.

Results of a range-finding test showed that no inhibition of growth took place after 72 hours at a concentration of 1% of a filtrate prepared at a nominal concentration of 100 mg/L. Inhibition of growth was observed in the 100 mg/L filtrate itself and in the 10% filtrate. Results of the range-finding test were used to determine the test concentrations for the final test. A full test with a duration of 72 hours was performed exposing three replicates of exponentially growing algal cultures to nominal test concentrations of 1.0, 1.8, 3.2, 5.6, 10 and 18% of a 5 µm filtrate prepared at a loading rate of 100 mg/L. In a second test concentrations of 10, 32 and 100% of a 5µm filtrate prepared at a loading rate of 100 mg/L were used. However, this test was not completed due to precipitation of test substance in the treated solutions. Dimethylsulphoxide was used as a solvent. Six replicates of exponentially growing algal cultures were used as a blank control and vehicle control.

Samples were taken from 1.0, 3.2 and 18% of the 5µm filtrate and from the solvent-control. In addition, a sample was taken from the stock of 100 mg/L at the start of the test. Analysis was based on the concentrations of Al and Mg in the test solutions. Analysis of the samples showed that the detected concentration of Al and Mg were not higher than those measured in samples taken from the controls. Hence, although a turbid suspension was attained dispersing the test substance with DMSO, no detectable concentration of Al or Mg could be found in samples containing the water soluble fraction after filtration through a 0.45 µm filter. Only a small amount of Al was detected in the sample taken from the stock solution prepared for the first full test. Based on this amount, a concentration of 1.63 mg/L of test substance in the stock solution prepared at 100 mg/L was determined.

Validity criteria were not met as no actual concentrations could be measured.

< 10% effects were observed at a test concentration of 18% of a 5 µm filtrate prepared at a nominal concentration of 100 mg/L. Testing at higher concentrations in the second test did not provide effect concentrations due to precipitation of the test substance in the solutions. EC50 and NOEC for growth inhibition were determined to be > and equal to 18% of a 5µm filtrate prepared at a nominal concentration of 100 mg/L, respectively. Because it cannot be concluded that this is the maximum solubility of the test substance in medium, a good interpretation of this study was not possible.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:

Description of key information

The substance is currently considered to be highly insoluble in water and this indicates that aquatic toxicity is unlikely to occur. A transformation/dissolution study is being performed which should give conclusive evidence on whether this consideration is correct.

Key value for chemical safety assessment

Additional information

A study was performed to assess the effect of the test substance on the growth rate of fresh water algae (Pseudokirchneriella subcapitata) after 72 hours of exposure. The study was conducted in accordance with OECD 201 and GLP and performed under static conditions.

Results of a range-finding test showed that no inhibition of growth took place after 72 hours at a concentration of 1% of a filtrate prepared at a nominal concentration of 100 mg/L. Inhibition of growth was observed in the 100 mg/L filtrate itself and in the 10% filtrate. Results of the range-finding test were used to determine the test concentrations for the final test. A full test with a duration of 72 hours was performed exposing three replicates of exponentially growing algal cultures to nominal test concentrations of 1.0, 1.8, 3.2, 5.6, 10 and 18% of a 5µm filtrate prepared at a loading rate of 100 mg/L. In a second test concentrations of 10, 32 and 100% of a 5µm filtrate prepared at a loading rate of 100 mg/L were used. However, this test was not completed due to precipitation of test substance in the treated solutions. Dimethylsulphoxide was used as a solvent. Six replicates of exponentially growing algal cultures were used as a blank control and vehicle control.

Samples were taken from 1.0, 3.2 and 18% of the 5µm filtrate and from the solvent-control. In addition, a sample was taken from the stock of 100 mg/L at the start of the test. Analysis was based on the concentrations of Al and Mg in the test solutions. Analysis of the samples showed that the detected concentration of Al and Mg were not higher than those measured in samples taken from the controls. Hence, although a turbid suspension was attained dispersing the test substance with DMSO, no detectable concentration of Al or Mg could be found in samples containing the water soluble fraction after filtration through a 0.45 µm filter. Only a small amount of Al was detected in the sample taken from the stock solution prepared for the first full test. Based on this amount, a concentration of 1.63 mg/L of test substance in the stock solution prepared at 100 mg/L was determined.

Validity criteria were not met as no actual concentrations could be measured, except for a small measurement of Al in the stock solution. Even though the test solutions were prepared by stirring in DMSO followed by filtration, the filtrate which was used to prepare the test concentration range was a turbid suspension. A dilution up to 18% of this filtrate did not indicate precipitation whereas in the second test a heavy precipiation was recorded at the higher concentrations. < 10% effects were observed at a test concentration of 18% of a 5µm filtrate prepared at a nominal concentration of 100 mg/L.

Based on the results of the tests, it cannot be determined on whether 18% of the filtrate can be considered the maximum solubility of the test item in medium. An good interpretation of this study is not possible.