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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:
24-01-2012 to 23-02-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met with acceptable deviations.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Single replicate performed due to technical error.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
Single replicate performed due to technical error.
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: February 2000; signature: April 2000
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Preliminary range finding test (2nd): 0 (control), 0.0040, 0.040, 0.40 and 4.0 mg/L (nominal ; based on the results of the pre-study media preparation trial). Definitive Test: 0 (control), 1.9 mg/L (mean measured test item concentration, initial concentration at 0 hours was 2.27 mg/L)
Samples from control replicates and test group replicates of equal nominal concentration were pooled for analysis. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
- Sampling method: Samples were taken at 0 h and 72 h for quantitative analysis. Duplicate samples were taken at 0 hours and stored frozen (approximately -20°C) for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours. At 72 hours one of the replicate samples was lost due to spillage during analysis. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours and therefore only a single measured concentration at 72 hours was determined. This result showed that there had been a decline in concentration over the test period with a measured concentration of 1.55 mg/L being obtained. The results were treated using worst case methodology for effect level estimation.
- Sample storage conditions before analysis: See above.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct dissolution of test item in culture medium to prepare stock; serial dilution of stock to prepare test media. During validation: recovery analyses were prepared by separately dispersing amounts of test material (1.0 g) in culture medium (300 mL). Recoveries were in the range of 0.744 mg/L and 1.267 mg/L. During subsequent stability testing the test item was found to have evidence of instability in culture medium in light and dark conditions with 51% to 64% being found after 72 hours respectively. With 72% using an open vessel in dark conditions. A second media preparation trial was performed based on difficulties of preparing test item solutions. Media preparation trial 2 and 3 was utilised in the definitive test. Which indicated a consistent media preparation. The second trial was as follows: Duplicate test item dispersions at concentrations of 100 mg/L were prepared by dispersing known amounts of test item in known volumes of ASTM algal culture medium, reconstituted water or dechlorinated tap water and stirring each dispersion at a temperature of 25°C for 48 hours using a propeller stirrer at approximately 2000 rpm. After the 48-Hour stirring period the dispersions were cooled to 21°C or 14°C for 24 hours and the undissolved test item removed by filtration (0.2 µm) prior to samples being taken for analysis. In order to ensure that adsorption of the test item to the filter matrix was reduced to an insignificant level, the filter used was preconditioned by passing a suitable volume (1 litre) of the dispersion through the filter prior to collecting the saturated solution for testing. Analysis performed on the pretest trial preparation showed measured test concentrations of 2.51 and 2.77 mg/L for each of the two prepared saturated solutions. Given the consistency of the results it was considered that the method was suitable for use in the definitive test.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No precipitate reported using the second and third media preparation trials.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Scenedesmus subspicatus (or Desmodesmus subspicatus)
- Strain: CCAP 276/20.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium under constant aeration and illumination (light intensity: 7000 lux) at 21 ± 1 ºC temperature.

ACCLIMATION
- Acclimation period: No. However, prior to the start of the test exposure sufficient master culture was added to approximately 100 ml culture medium in flasks at 10^4 cells/mL and stirring at 100 – 150 rpm under constant conditions until algal density 10^4 – 10^5 cells/mL.
- Culturing media and conditions (same as test or not): Yes.
Culture medium: (mg/L) - ASTM medium, according to OECD 201, Annex 3
NaNO3 25.5, MgCI2.6H2O 12.164, CaCI2.2H2O 4.41, MgSO4.7H20 14.7, K2HPO4 1.044, NaHC03 15.0, H3BO3 0.1855, MnCI2.4H2O 0.415, ZnCI2 0.00327, CoCI2.6H2O 0.00143, CuCI2.2H2O 0.000012, NaMoO4.2H2O 0.00726, FeCl3.6H2O 0.159, Na2-EDTA 0.3, . pH 7.5 +/- 0.1.
- Any deformed or abnormal cells observed: None reported.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ± 1ºC
pH:
0-hours pH: 7.0
72-hours pH: 9.0 - 9.1.
This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the gaseous CO2 transfer rate. This effect was considered exaggerated by the requirement to conduct the test in conical flasks sealed with ground glass stoppers due to the possible volatile nature of the test item, this further reduced gaseous exchange between the test vessels and the atmosphere. This increase in the control cultures was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion (increased by factor 75).
Nominal and measured concentrations:
Preliminary range finding test (2nd): 0 (control), 0.0040, 0.040, 0.40 and 4.0 mg/L (nominal ; based on the results of the pre-study media preparation trial).
Definitive Test: 0 (control), 1.9 mg/L (mean measured test item concentration, initial concentration at 0 hours was 2.27 mg/L)
Samples from control replicates and test group replicates of equal nominal concentration were pooled for analysis. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
At 72 hours one of the replicate samples was lost due to spillage during analysis. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours and therefore only a single measured concentration at 72 hours was determined. This result showed that there had been a decline in concentration over the test period with a measured concentration of 1.55 mg/L being obtained. The results were treated using worst case methodology for effect level estimation.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): Closed – Static. Vessels glass stoppered.
- Headspace, fill volume: 100 mL fill volume, approximately 150 mL headspace
- Aeration: Vessel shaken continuously.
- Renewal rate of test solution (frequency/flow rate): Not applicable.
- Initial cells density: nominal: ca. 1x10^4 cells/mL
- Control end cells density: Definitive test: mean: 9.88 x10^5 cells/mL
- No. of vessels per concentration (replicates): 6 replicates of each test concentration
- No. of vessels per control (replicates): 3 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. ASTM medium, according to OECD 201, Annex 3
NaNO3 25.5, MgCI2.6H2O 12.164, CaCI2.2H2O 4.41, MgSO4.7H20 14.7, K2HPO4 1.044, NaHC03 15.0, H3BO3 0.1855, MnCI2.4H2O 0.415, ZnCI2 0.00327, CoCI2.6H2O 0.00143, CuCI2.2H2O 0.000012, NaMoO4.2H2O 0.00726, FeCl3.6H2O 0.159, Na2-EDTA 0.3, . pH 7.5 +/- 0.1.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Start and end of test.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: Intensity approximately 7000 lux
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Multisizer particle counter
- Other: Microscopic evaluation of the cells was carried out at the end of the exposure for abnormalities.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable. Limit test in definitive test justified from the results of the range finding study.
- Range finding study: Yes.
- Test concentrations: Two replicates per concentration were exposed to 0 (control), 0.0040, 0.040, 0.40 and 4.0 mg/L (nominal ; based on the results of the pre-study media preparation trial)
- Results used to determine the conditions for the definitive study: Yes. The results showed no effect on growth at all concentrations tested.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
mean measured concentration
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: - mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
mean measured concentration
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
mean measured concentration
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits: - mg/L
Details on results:
- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the end of the exposure period revealed no abnormalities in the test item concentrations or controls.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: Not reported. It was speculated in that adsorption to algal cells may also have been a significant factor in the decrease in measured test concentrations observed over the test period
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Low dose stimulation was observed in the range finder and in the definitive limit test.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Differences in nominal versus measured between with and without algae speculated to be due to test item adsorption to algal cells.
- Effect concentrations exceeding solubility of substance in test medium: No.
Reported statistics and error estimates:
Students t-test was carried out on the area under the growth curve data at 72 hours for the control and the test material cultures to determine any statistically significant differences between the test and control groups.
There were no statistically significant differences (P ≥ 0.05), between the control and test material group and therefore the "No Observed Effect Concentration"
(NOEC) based on the mean measured test concentration was 1.9 mg/L.

Table 2: Inhibition of growth rate and yield in the definitive test

Mean measured concentration (mg/L)

Area under curve at 72 hours

% inhibition

Growth Rate (0-72h)

% inhibition

0 (control)

1.49 x10^7

-

0.060

-

1.91

1.42x10^7

5

0.062

[3]

 

 

 

 

 

 

 

 

 

 

Where: [ ] = increase in growth relative to the controls.

Validity criteria fulfilled:
yes
Conclusions:
The test item 48h-EC50 was > 1.9 mg/L (C.I: – mg/L) based on mean measured concentrations. The corresponding NOEC was 1.9 mg/L.
Executive summary:

The algal growth inhibition to Scenedesmus subspicatus was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following preliminary range-finding tests (conducted over a range of nominal test concentrations of 0.0040, 0.040, 0.40 and 4.0 mg/L), algae was exposed to an aqueous solution of the test item at a mean measured test concentration of 1.9 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1°C under static conditions. In the range finding test two replicates were used for each test item concentration and control. In the definitive test, six replicates were tested for each test item concentration and three replicates for the control. The test solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer prior to removal of the aqueous phase by filtration (0.2µm). This "saturated" solution was then used to produce the required test concentration. Temperature was acceptably maintained during the test. The pH values of the control cultures were observed to increase from pH 7.0 at 0 hours to pH 9.0-9.1 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the gaseous CO2 transfer rate. This effect was considered exaggerated by the requirement to conduct the test in conical flasks sealed with ground glass stoppers due to the possible volatile nature of the test item, this further reduced gaseous exchange between the test vessels and the atmosphere. This increase in the control cultures was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion (increased by factor 75). The validity criteria of the test guideline were considered fulfilled. analysis of the test preparations at 0 hours showed the measured concentrations to be in the range of 2.26 mg/L to 2.27 mg/L. These results were in line with a media preparation trial conducted using an identical method of preparation to that employed in the definitive test. At 72 hours one of the replicate samples was lost due to spillage during analysis and therefore only a single measured concentration at 72 hours was determined. This result showed that there had been a decline in concentration over the test period with a measured concentration of 1.55 mg/L being obtained. This decline was in line with the stability analyses conducted which showed that the test item was unstable in culture medium over the test period. Adsorption to algal cells may also have been a significant factor in the decrease in measured test concentrations observed over the test period. On this basis effect levels were based on mean measured concentrations to give a worst case analysis of the data. The EC50 for growth rate reduction (72h-ErC50) was > 1.9 mg/L and the corresponding NOEC was 1.9 mg/L based on mean measured concentrations.

Description of key information

ErC50 (algae; growth rate) = > 1.9 (C.I. - ) mg/L based on mean measured concentrations, 72hour-freshwater, OECD TG 201, 2002

NOEC (algae) = 1.9 mg/L based on mean measured concentrations, 72hour-freshwater, OECD TG 201, 2002

Key value for chemical safety assessment

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

Additional information

Key study : OECD TG 201, 2002: The algal growth inhibition to Scenedesmus subspicatus was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following preliminary range-finding tests (conducted over a range of nominal test concentrations of 0.0040, 0.040, 0.40 and 4.0 mg/L), algae was exposed to an aqueous solution of the test item at a mean measured test concentration of 1.9 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1°C under static conditions. In the range finding test two replicates were used for each test item concentration and control. In the definitive test, six replicates were tested for each test item concentration and three replicates for the control. The test solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer prior to removal of the aqueous phase by filtration (0.2µm). This "saturated" solution was then used to produce the required test concentration. Temperature was acceptably maintained during the test. The pH values of the control cultures were observed to increase from pH 7.0 at 0 hours to pH 9.0-9.1 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the gaseous CO2 transfer rate. This effect was considered exaggerated by the requirement to conduct the test in conical flasks sealed with ground glass stoppers due to the possible volatile nature of the test item, this further reduced gaseous exchange between the test vessels and the atmosphere. This increase in the control cultures was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion (increased by factor 75). The validity criteria of the test guideline were considered fulfilled. analysis of the test preparations at 0 hours showed the measured concentrations to be in the range of 2.26 mg/L to 2.27 mg/L. These results were in line with a media preparation trial conducted using an identical method of preparation to that employed in the definitive test. At 72 hours one of the replicate samples was lost due to spillage during analysis and therefore only a single measured concentration at 72 hours was determined. This result showed that there had been a decline in concentration over the test period with a measured concentration of 1.55 mg/L being obtained. This decline was in line with the stability analyses conducted which showed that the test item was unstable in culture medium over the test period. Adsorption to algal cells may also have been a significant factor in the decrease in measured test concentrations observed over the test period. On this basis effect levels were based on mean measured concentrations to give a worst case analysis of the data. The EC50 for growth rate reduction (72h-ErC50) was > 1.9 mg/L and the corresponding NOEC was 1.9 mg/L based on mean measured concentrations.