3-nitrobenzoic acid

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22.09.2017-25.09.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experiment test result performed using standard test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

Short term toxicity of test material to aquatic algae was performed according to the 201 OECD guideline in a static system.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The stock solution 100 mg/l was prepared by dissolving white powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Institute of botany of the ASCR
- Initial biomass concentration: 5x10(3) cells /ml
- Method of cultivation: No data available

ACCLIMATION - No data available

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

±1 hour

Test temperature:

23±2°C

pH:

At concentration 10 mg/l: 7.7 (changed to 7.9 during test)
At concentration 18 mg/l: 7.6 (changed to 7.7 during test)
At concentration 32 mg/l: 7.4 (changed to 7.3 during test)
At concentration 58 mg/l: 7.1 (changed to 6.9 during test)
At concentration 100 mg/l: 6.4 (changed to 5.2 during test)
Control: 8.1 (changed to 8.3 during test)

Nominal and measured concentrations:

0, 10, 18, 32, 58, 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml Glass vessel
- Type (delete if not applicable): closed (with air permeable stopper)
- Sample volume: 15 ml
- Initial cells density: 5x10(3) cells/ml
- No. of vessels per concentration (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 6000-8000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microscope with counting chamber Cyrus I or electronic particle counter.
- Other: ErC50 was calculated using non-linear regression by the software Prism 4.0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

106.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 98.6-114.9

Results with reference substance (positive control):

Results with reference substance valid
- EC50: 0. 77 mg/L

Reported statistics and error estimates:

ErC50 is calculated from the inhibition of growth rate by non-linear regression using software Prism 4.0, GraphPad.

Validity criteria fulfilled:

yes

Conclusions:

The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 106.4 mg/L on the basis of effects on growth rate in a 72 hour study.

Executive summary:

Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance for total exposure period of 72 hrs. Test conducted according to OECD Guideline 201. The stock solution 100 mg/l was prepared by dissolving white powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture and tested at the concentrations 0, 10, 18, 32, 58, 100  mg/L. Effects on the growth rate of the organism were studied by using positive reference substance Potassium dichromate (K2Cr2O7).

The median effective concentration (EC50) for Potassium dichromate in Desmodesmus subspicatus was determined to be 0.77 mg/L. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 106.4 mg/L with 95% CI was 98.6 - 114.9 mg/l.

This value indicates that the substance test substance is likely to be non-hazardous to aquatic algae and cannot be classified as toxic and not classified as per the CLP criteria.

Description of key information

Toxicity to aquatic algae and cyanobacteria:

Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance for total exposure period of 72 hrs. Test conducted according to OECD Guideline 201. The stock solution 100 mg/l was prepared by dissolving white powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture and tested at the concentrations 0, 10, 18, 32, 58, 100  mg/L. Effects on the growth rate of the organism were studied by using positive reference substance Potassium dichromate (K2Cr2O7).

The median effective concentration (EC50) for Potassium dichromate in Desmodesmus subspicatus was determined to be 0.77 mg/L. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 106.4 mg/L with 95% CI was 98.6 - 114.9 mg/l.

This value indicates that the substance test substance is likely to be non-hazardous to aquatic algae and cannot be classified as toxic and not classified as per the CLP criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

106.4 mg/L

Additional information

Toxicity to aquatic algae and cyanobacteria:

The effect of test material was evaluated for aquatic algae and cyanobacteria based on results of two experimental report as described below:

The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized.The test solution was prepared in aseptic condition. The test item  was prepared by adding 50mg of test item in 250ml of BBM to get the final concentration of 200mg/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.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. 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) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations 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. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

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 0.358 per day. Secondly 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 33.42%. Thirdly 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 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 335.805 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

The above report was supported by another eperimental study which suggests , that the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized.The test solution was prepared in aseptic condition. The test item  was prepared by adding 50mg of test item in 250ml of BBM to get the final concentration of 200mg/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.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. 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) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations 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. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

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 0.358 per day. Secondly 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 33.42%. Thirdly 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 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item to various nominal test concentrations, EC10 value was determined to be 9.05±5.93 graphically and through probit analysis.