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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental test result performed using standard OECD test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control) and 100 mg/L
- Sample storage conditions before analysis: Test samples were analysed immediately after sampling
Vehicle:
no
Details on test solutions:
The test solution was prepared by dissolving 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. The final solubility value obtained after analytical detection is 428.8 mg/L. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Length: 8 – 14 μm
- Weight: 2 - 3 μm
- Source: Sterile, unicellular, suspension cultures of algae were obtained from Denmark Technical University, Denmark and is maintained at Unique Ecotox Research Laboratory, Nagpur.
- Method of cultivation: OECD medium

ACCLIMATION
- Culturing media and conditions (same as test or not): The medium to be used for the growth of algae was OECD medium.
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
21 to 24 ± 2°C
Nominal and measured concentrations:
Test chemical concentrations used for the study were 0 (control) and 100 mg/l, respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study.
- Initial cells density: 10000cells/ml
- No. of organisms per vessel: 10000cells/ml
- No. of vessels per concentration (replicates): Six replicates for each test concentration
- No. of vessels per control (replicates): Six replicates for Control

GROWTH MEDIUM
- Standard medium used: yes, OECD medium was used as a test medium in the study.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(6000-8000Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were measured using microscope.
- Chlorophyll measurement: No data
- Other: The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer.

TEST CONCENTRATIONS
- Test concentrations: test concentration were: 0 (control) and 100 mg/l.
- Results used to determine the conditions for the definitive study: Mortality of test organisms

Other:
Incubation :
1. The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 22 ° C±2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (1500Lux).
3. The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
4. The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7) was used as a reference substance.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, sickle shaped and green throughout the test duration in the control.
Results with reference substance (positive control):
- Results with reference substance valid?
- EC50: 0.868 mg/l
Reported statistics and error estimates:
To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) was determined.

Table: Assessment of test concentrations

Sr. No
Concentrations mg/L
Wavelength (nm)
Absorbance
Temperature (°C)
1
blank
198
0.00
25°C
2
10
198
0.13
25°C
3
20
198
0.25
25°C
4
30
198
0.37
25°C
5
40
198
0.50
25°C
6
50
198
0.63
25°C
7
60
198
0.76
25°C
8
70
198
0.88
25°C
9
80
198
0.99
25°C
10
90
198
1.12
25°C
11
100
198
1.26
25°C

 

The absorbance and concentrations were recorded at 198 nm.

Table: Concentration after analytical Determination

    0 Hours 0 Hours 72 Hours 72 Hours
SR. No concentrations (mg/L) Absorbance (mean) Analytical concentrations Absorbance (mean) Analytical concentrations
1 control 0.00 0.00 0.00 0.00
2 100 1.15 90.76 1.11 89.10

The test concentrations were measured and were maintained within 80 – 120 % of nominal. Hence, the EC50 (>100 mg/L) was expressed based on nominal concentrations. 

Table: pH and Temperature

Test Concentration(mg/L) Experimental Flasks pH Temperature °C
0 Hours 72 Hours 0 Hours 72 Hours
control R1 7.34 8.16 25 25
control R2 7.70 8.00 25 25
control R3 7.80 8.10 25 25
control R4 7.74 8.12 25 25
control R5 7.70 8.09 25 25
control R6 7.76 8.15 25 25
100 R1 8.15 7.90 25 25
100 R2 8.00 7.91 25 25
100 R3 8.05 7.95 25 25
100 R4 8.01 7.98 25 25
100 R5 8.00 7.90 25 25

 

The pH was measured at the beginning of the test and after 72 hr of exposure. The pH of the control medium did not increase by more than 1.5 units during the test.

Table: Cell count and percent inhibition

Experimental Flasks and Test Concentration(mg/L)
0 Hr Cell
Count
24 Hr
Cell
Count
48 Hr
Cell Count
72 Hr
Cell Count
Avg Specific Growth Rate (µ) Mean Avg Specific Growth Rate (µ) Percent Inhibition(%)
control 10000 35000 105000 280000 1.11 1.12 -
control 10000 30000 120000 290000 1.12
control 10000 35000 115000 295000 1.13
control 10000 30000 115000 290000 1.12
control 10000 35000 120000 300000 1.13
control 10000 30000 120000 295000 1.13
100 10000 25000 100000 235000 1.05 1.04 7.05
100 10000 20000 95000 230000 1.05
100 10000 25000 100000 230000 1.05
100 10000 35000 115000 215000 1.02
100 10000 30000 120000 240000 1.06
100 10000 20000 100000 230000 1.05
Validity criteria fulfilled:
yes
Remarks:
*The avg. increase in cell count in control group is >16 fold. *The coefficent of variation of avg. specific growth rates was <7 %(i.e., reported as 0.79%) *coefficent of variation for specific growth rates in the control <35%(i.e., reported as 8.24%).
Conclusions:
Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the 72 hr median effect concentration (ErC50) value was determined to be >100 mg/l (nominal conc.).
Executive summary:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0 and 100 mg/l was from the stock test concentrations. Thus, limit test was performed. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21 to 24 ± 2°C and with a continuous uniform illumination of 6000 -10000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. All control and test vessel conc. were performed in six replicates. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 0.79%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 8.24%), respectively thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (ErC50) value was determined to be >100 mg/l (nominal conc.).Thus, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Description of key information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0 and 100 mg/l was from the stock test concentrations. Thus, limit test was performed. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21 to 24 ± 2°C and with a continuous uniform illumination of 6000 -10000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. All control and test vessel conc. were performed in six replicates. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 0.79%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 8.24%), respectively thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (ErC50) value was determined to be >100 mg/l (nominal conc.).Thus, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0 and 100 mg/l was from the stock test concentrations. Thus, limit test was performed. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21 to 24 ± 2°C and with a continuous uniform illumination of 6000 -10000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. All control and test vessel conc. were performed in six replicates. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 0.79%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 8.24%), respectively thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (ErC50) value was determined to be >100 mg/l (nominal conc.).Thus, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.