[8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard OECD test guidelines

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

This study was designed to assess the effect of the test item (8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride (CAS No. 68391-32-2) on the growth of green alga Chlorella vulgaris. The study was conducted in accordance with “OECD guideline for testing of chemicals No. 201 – Alga, growth inhibition test”.

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): (8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride
- Molecular formula (if other than submission substance): C19H20N5O3.Cl
- Molecular weight (if other than submission substance): 401.852 g/mol
- Smiles notation (if other than submission substance):[N+](c1cc(\N=N\c2c3cc([N+](C)(C)C)ccc3ccc2O)ccc1N)([O])=O.[ClH]
- InChl (if other than submission substance): 1S/C19H19N5O3.ClH/c1-24(2,3)14-7-4-12-5-9-18(25)19(15(12)11-14)22-21-13-6-8-16(20)17(10-13)23(26)27;/h4-11H,1-3H3,(H2-,20,21,22,25);1H
- Structural formula attached as image file (if other than submission substance): No data available
- Substance type: Organic
- Physical state: Solid

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The test solution was prepared in aseptic condition. The test item [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium 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 2 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 of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name:green alga
- Strain:Chlorella vulgaris
- Source (laboratory, culture collection): 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.
- Method of cultivation: Bold’s Basal Medium(BBM)

ACCLIMATION
- Culturing media and conditions (same as test or not):The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM.
- Any deformed or abnormal cells observed:no

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

22 °C ±2°C

pH:

6.8 ±0.3

Nominal and measured concentrations:

Six test concentration were: 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l,100mg/l, and 200mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Type (delete if not applicable): No data available
- Material, size, headspace, fill volume: Conical flasks of 100 ml size was used for the study.
- Aeration: No data available
- Type of flow-through (e.g. peristaltic or proportional diluter): No data available
- Renewal rate of test solution (frequency/flow rate): No data available
- Initial cells density: 21.39x104 cells/mL
- Control end cells density: No data available
- No. of organisms per vessel: 10000 cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control
- No. of vessels per vehicle control (replicates): No data available

GROWTH MEDIUM
- Standard medium used: No data available
- Detailed composition if non-standard medium was used: The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No data available
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(1500Lux)
- Salinity (for marine algae): No data available

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 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.
- Chlorophyll measurement: No data available
- Other: 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 2.
- Justification for using less concentrations than requested by guideline: No data available
- Range finding study: No data available
- Test concentrations: Six test concentration were: 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l,100mg/l, and 200mg/l, respectively (Nominal concentrations)
- Results used to determine the conditions for the definitive study: No data available

6.1. Test solution: The test solution was prepared in aseptic condition. The test item [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium 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 2 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 of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.

6.2. Test vessels: All the tests were carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60ml so that a sufficient amount of head space was left.

6.3. Replicates: For the assessment of algal growth, the study was conducted in replicates. The control vessel was maintained in triplicates as recommended in the OECD guideline and the test concentrations were selected in geometric series which were maintained in duplicates.

6.4. Incubation:
i) 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.
ii) The test vessels were incubated with a continuous, uniform fluorescent illumination(1500Lux).
iii) 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.
iv) 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.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

204.52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Control vessels: The microscopic observations were noted down in each of the control vessel. All the cells appeared healthy, round and green throughout the study duration in the control. Also, the drift in pH in the control vessels did not increase by >1.5 units when observed on 72 hours as compared to 0 hours. The average pH drift observed in the control vessels was 0.15 units.

Test vessels: The effect of the test item on the green algae Chlorella vulgaris culture was observed at nominal test concentration of >6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/L. All the six concentrations were in geometric series spaced by a factor of 2. The microscopic observations were also noted in each of the test vessel. All the cells appeared healthy, round and green throughout the study duration and no significant changes were observed up to the concentration of 50 mg/l whereas, black dotted rings were observed inside the algal cells in concentrations of 50 mg/l, 100 mg/l and 200 mg/l. EC50 was found to be 204.52 mg/l graphically through probit analysis.

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 1: Showing the average cell count using Haemocytometer of the test vessels at an equal interval of 24hrs, 48hrs and 72hrs

Test vessels and test concentration	24 Hours	48 Hours	72 Hours
Control
Replicate 1	21600	36800	50800
Replicate 2	20800	36000	50400
Replicate 3	22800	38000	50800
CAS No. 68391 -32 -2
6.25 mg/l
Replicate 1	18800	28400	35600
Replicate 2	18000	28400	35200
12.5 mg/l
Replicate 1	18000	26000	33200
Replicate 2	20400	25200	32800
25 mg/l
Replicate 1	20400	23600	32800
Replicate 2	18000	22000	30800
50 mg/l
Replicate 1	18000	20400	28800
Replicate 2	18000	18400	26800
100 mg/l
Replicate 1	15600	15200	25200
Replicate 2	13200	14400	23600
200 mg/l
Replicate 1	15600	14400	23200
Replicate 2	14000	13200	21600

 

 

Table 2: Showing the values of average specific growth rate and percentage inhibition after an interval of 72 hours

	CONTROL		6.25mg/l		12.5mg/l		25mg/l		50mg/l		100mg/l		200mg/l
Average Specific Growth rate (µ )	R1	0.542	R1	0.423	R1	0.400	R1	0.396	R1	0.353	R1	0.308	R1	0.281
	R2	0.539	R2	0.419	R2	0.396	R2	0.375	R2	0.329	R2	0.286	R2	0.257
	R3	0.542
Mean of Avg. Specific growth rate	0.541		0.421		0.398		0.385		0.341		0.297		0.269
Percentage Inhibition (%I)	_		22.097		26.424		28.736		37.030		45.062		50.339

 

 

 Table 3: Depicting pH values at 0 Hours and after 72 Hours of test item exposure to algae

Test vessels and test concentration	0 Hours	72 Hours
CONTROL
Replicate 1	7.30	7.37
Replicate 2	7.28	7.43
Replicate 3	7.26	7.50
Average	7.28	7.43
CAS No. 68391 -32 -2
6.25 mg/l
Replicate 1	7.29	7.31
Replicate 2	7.27	7.23
12.5 mg/l
Replicate 1	7.26	7.27
Replicate 2	7.24	7.24
25 mg/l
Replicate 1	7.20	7.20
Replicate 2	7.21	7.26
50 mg/l
Replicate 1	7.24	7.26
Replicate 2	7.25	7.23
100 mg/l
Replicate 1	7.23	7.24
Replicate 2	7.24	7.32
200 mg/l
Replicate 1	7.25	7.33
Replicate 2	7.26	7.33

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to test item [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-
naphthyl]trimethylammonium chloride (CAS No. 68391-32-2) to various nominal test concentrations, EC50 was found to be 204.52 mg/l graphically and through probit analysis.

Executive summary:

The effect of test item [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride, CAS No. 68391-32-2 was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/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 204.52 mg/L. Thus based on this value, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

Description of key information

The effect of test item [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride, CAS No. 68391-32-2 was studied on the growth of fresh water green alga Chlorella vulgaris (UERL Study report, Sustainability Support Services (Europe) AB, 2016). The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/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 204.52 mg/L. Thus based on this value, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

204.52 mg/L

Additional information

The effect of test item [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride, CAS No. 68391-32-2 was studied on the growth of fresh water green alga Chlorella vulgaris (UERL Study report, Sustainability Support Services (Europe) AB, 2016). The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/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 204.52 mg/L. Thus based on this value, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.