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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Exprimental data using OECD recommended test guidelines

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:

Test material

Test material form:
solid: crystalline
Details on test material:
CAS No: 104-94-9
Chemical Name: 4-methoxyaniline (p-anisidine)
Nature of chemical: Organic

Sampling and analysis

Analytical monitoring:
not specified

Test solutions

Details on test solutions:
The test solution was prepared in aseptic condition. The test item p-anisidine was prepared by adding 10mg of test item in 400ml of BBM to get the final concentration of 25mg/L. This stock solution was kept for stirring for 30 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.
• Three replicates for Control
• Two replicates for each test concentration

Test organisms

Test organisms (species):
Chlorella vulgaris
Details on test organisms:
The fresh water green alga Chlorella vulgaris, was used as the test system (organism). Sterile, unicellular, suspension cultures of algae were obtained from National Environmental Engineering Research Institute (NEERI), Nagpur and maintained at Unique Ecotox Research Laboratory, Nagpur. The culture was examined under the microscope to confirm that it was unicellular, healthy and not contaminated.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

No Data
Test temperature:
24 °C ±2°C.
6.8 to 7.7
Dissolved oxygen:
No Data
No Data
Nominal and measured concentrations:
Test concentration: Six test concentration were: 0.08 mg/L, 0.2 mg/L, 0.5 mg/L, 1.25mg/L, 3.125 mg/L and 7.813mg/L
Details on test conditions:
The Test Conditions are summarized in a table below
S. No Parameter Conditions
1. Study Type Static
2. Exposure Duration 72 Hours
3. Temperature 24 °C ±2°C.
4. Light Quality White Fluorescent Light
5. Light Intensity 1500Lux
6. Photoperiod 16 Hour Light Period : 8 Hour Dark Period
7. RPM Speed 120 Revolutions per minute
8. Water Deionized Water
9. Test vessel size 100ml
10. Test volume 60ml
11. Dilution water Bold’s Basal Medium
12. Test concentration Six test concentration were: 0.08 mg/l, 0.2 mg/l, 0.5 mg/l, 1.25mg/l, 3.125 mg/l and 7.813mg/l
13. Replicates per concentration Three replicates for Control; Two replicates for each test concentration
Reference substance (positive control):

Results and discussion

Effect concentrations
72 h
Dose descriptor:
Effect conc.:
0.937 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: EC50 value was obtained graphically and through probit analysis.

Any other information on results incl. tables


According to the test validity criteria the following criteria are met in this test: 

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 1.511 per day.

The mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 10.544%.

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 0.379%.

Hence, the test is considered valid as per OECD guideline, 201.

Applicant's summary and conclusion

Validity criteria fulfilled:
After 72 hours of exposure to test item p-anisidine (CAS No. 104-94-9) to various nominal test concentrations, EC50 was found to be 0.937mg/l graphically and through probit analysis.
Executive summary:

The effect of test item p-anisidine, CAS No. 104-94-9 was studied on the growth of fresh water green algaChlorella vulgaris.The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 0.08mg/l, 0.2mg/l, 0.5mg/l, 1.25mg/l, 3.125mg/l and 7.813mg/l. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 0.937mg/L.