3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride

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:

key study

Study period:

02.12.2016 – 05.12.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

To assess the presence or absence of toxic effects of test chemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride (CAS No. 68391-31-1) on growth of algae.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: No data
- Expiration date of the lot/batch: No data
- Purity test date: No data

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: No data
- Specific activity: No data
- Locations of the label: No data
- Expiration date of radiochemical substance: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: No data
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No data
- Preliminary purification step (if any): No data
- Final dilution of a dissolved solid, stock liquid or gel: No data
- Final preparation of a solid: No data

FORM AS APPLIED IN THE TEST (if different from that of starting material) No data

OTHER SPECIFICS: No

Analytical monitoring:

no

Details on sampling:

No data

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The stock solution 100 mg/L was prepared by dissolving orange powder in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

- Controls: OECD medium as specified in OECD 201, used as a control and for dilution of the stock solution.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Institute of botany of the ASCR
- Age of inoculum (at test initiation): 5x103 cells/ml
- Method of cultivation: No data

ACCLIMATION
- Acclimation period: No data
- Culturing media and conditions (same as test or not): No data
- Any deformed or abnormal cells observed: No data

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

±1 hour

Post exposure observation period:

No data

Hardness:

No data

Test temperature:

23°C ± 2°C

pH:

The sample at concentration 85.0 mg/L : pH = 7.4 changed to pH = 7.7 during the test,
Control : pH = 8.0 changed to pH = 7.7 during the test

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

0, 10, 17, 29, 50 and 85 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50ml glass vessel
- Type : closed (with air-permeable stopper)
- Material, size, headspace, fill volume: 7.5 ml
- Aeration: No data
- Type of flow-through (e.g. peristaltic or proportional diluter): No data
- Renewal rate of test solution (frequency/flow rate): No data
- Initial cells density: 5x103 cells/mL
- Control end cells density: No data
- No. of organisms per vessel: No data
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): No data
- No. of vessels per vehicle control (replicates): No data

GROWTH MEDIUM
- Standard medium used: Yes

OTHER TEST CONDITIONS
- Adjustment of pH: Without adjustment
- Photoperiod: Continuous
- Light intensity and quality: 6000 lx - 8000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell counting: Microscope with counting chamber Cyrus I or electronic particle counter.
- Chlorophyll measurement: No
- 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

Remarks:

ErC50 = EC50

Effect conc.:

67.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 61.7 – 74.7 mg/L

Details on results:

No data

Results with reference substance (positive control):

Results with reference substance valid
- ErC50: 0.69 mg/L K2Cr2O7

Reported statistics and error estimates:

No data

Control Solution: OECD growth medium without the sample, the coefficient of variation of average growth rate in replicates during the whole test period was 0.6 %.

Validity criteria fulfilled:

yes

Conclusions:

The median effective concentration (ErC50) for the test substance, 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride, in a freshwater algae Desmodesmus subspicatus was determined to be 67.9 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 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H- pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride according to OECD Guideline 201.

The test substance was dissolved in OECD growth medium and tested at the concentrations 0, 10, 17, 29, 50 and 85 mg/L. Effects on the growth rate of the organism were studied.

The median effective concentration (ErC50) for the test substance, 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride, in Desmodesmus subspicatus was determined to be 67.9 mg/L and also the substance was found to be not rapidly degradable. Thus considering the CLP criteria for aquatic classification of the substance, it is concluded that 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride exhibits toxicity to aquatic algae (Desmodesmus subspicatus) in the chronic 3 classification category.

Description of key information

On the basis of median effective concentration (ErC50) for the test substance, 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride observed in Desmodesmus subspicatus, it is concluded that the test chemical exhibits toxicity to aquatic algae (Desmodesmus subspicatus) in the chronic 3 classification category as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

67.9 mg/L

Additional information

Various short term studies available for the test chemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride (CAS No. 68391-31-1)were reviewed to determine the toxic nature of test chemical on the growth of aquatic invertebrates. The studies are as mentioned below;

 

The Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H- pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride according to OECD Guideline 201. The test substance was dissolved in OECD growth medium and tested at the concentrations 0, 10, 17, 29, 50 and 85 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride, in Desmodesmus subspicatus was determined to be 67.9 mg/L and also the substance was observed to be not rapidly degradable. Thus considering the CLP criteria for aquatic classification of the substance, it is concluded that 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride exhibits toxicity to aquatic algae (Desmodesmus subspicatus) in the chronic 3 classification category.

 

The next study was designed to assess the toxic effects of the test compound 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride (CAS No. 68391-31-1) on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test solution was prepared in aseptic condition. The test item 3-[(4,5-dihydro-3-methyl-5-oxo- 1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 24 hours to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. 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 he 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 0.602 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 10.506 %. 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 2.363 %. Hence, the test is considered valid as per OECD guideline, 201.

 

After 72 hours of exposure to test item 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride (CAS No. 68391-31-1) to various nominal test concentrations, EC50 was determine to be 109.09 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride (CAS No. 68391-31-1)can be considered as non-toxic to algae and thus can be consider as not classified as per the CLP classification criteria.

 

On the basis of median effective concentration (ErC50) for the test substance, 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride observed in Desmodesmus subspicatus, it is concluded that the test chemical exhibits toxicity to aquatic algae (Desmodesmus subspicatus) in the chronic 3 classification category as per the CLP classification criteria.