Aluminium, 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex

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:

Data is from experimental study report.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

Short term toxicity study was carried out according to the OECD guideline.

GLP compliance:

no

Analytical monitoring:

no

Details on sampling:

No data available

Vehicle:

not specified

Details on test solutions:

The test solution was prepared in aseptic condition. The test chemical 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 and filter it 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 10E4cells/ml. Care was taken to have a homogeneous solution for the experiment.

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

The fresh water green alga Chlorella vulgaris, was used as the test organism. Sterile, unicellular, liquid cultures of algae. The culture was examined under the microscope to confirm that it was unicellular, healthy and not contaminated. 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.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

22 °C ±2°C.

pH:

6.7 to 7.4

Nominal and measured concentrations:

6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L, 200mg/L. All the six concentration were in geometric series spaced by a factor of 2.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Type (delete if not applicable): open / closed: No data
- Material, size, headspace, fill volume: 100 ml of conical flasks filled with 60ml of test solution
- Aeration: no data
- Initial cells density: 38.787 x104 cells/mL)
- Control end cells density: No data
- No. of organisms per vessel: No data
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: No data


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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer


TEST CONCENTRATIONS
- Spacing factor for test concentrations: No data
- Justification for using less concentrations than requested by guideline: No data
- Range finding study: No data
- Test concentrations: 6.25mg/l,12.5mg/l,25mg/l,50mg/l,100mg/l,200mg/l
- Results used to determine the conditions for the definitive study: Cell growth inhibition

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

136.57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: calculated from equation through probit analysis

Details on results:

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 0.1 units.

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	3.72 × 10^4	5.32 × 10^4	6.92 × 104
Replicate 2	4.08 × 10^4	5.68 × 10^4	7.28 × 10^4
Replicate 3	3.96 × 10^4	5.56 × 10^4	7.96 × 10^4
CAS No. 68653 -95 -9
6.25mg/l
Replicate 1	27600	30000	34400
Replicate 2	29200	27600	34000
12.5mg/l
Replicate 1	26800	26800	33200
Replicate 2	25200	26000	31600
25mg/l
Replicate 1	24400	25200	30800
Replicate 2	23600	25200	30000
50mg/l
Replicate 1	24400	25200	30000
Replicate 2	23200	24400	30000
100mg/l
Replicate 1	22800	22800	27600
Replicate 2	22000	21200	28000
200mg/l
Replicate 1	19600	20800	27200
Replicate 2	18800	19600	25200

 

 

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	0.645	R1	0.412	R1	0.400	R1	0.375	R1	0.366	R1	0.338	R1	0.334
	R2	0.662	R2	0.408	R2	0.384	R2	0.366	R2	0.366	R2	0.343	R2	0.308
	R3	0.691
Mean of Avg. Specific growth rate	0.666		0.410		0.392		0.371		0.366		0.341		0.321
Percentage Inhibition (%I)	_		38.457		41.178		44.356		45.014		48.827		51.829

 

 

 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	6.15	6.30
Replicate 2	6.25	6.34
Replicate 3	6.35	6.40
Average	6.25	6.35
CAS No. 68583 -95 -9
6.25mg/l
Replicate 1	6.35	6.31
Replicate 2	6.40	6.29
12.5mg/l
Replicate 1	6.50	6.50
Replicate 2	6.45	6.52
25mg/l
Replicate 1	6.49	6.53
Replicate 2	6.47	6.52
50mg/l
Replicate 1	6.48	6.55
Replicate 2	6.50	6.53
100mg/l
Replicate 1	6.42	6.50
Replicate 2	6.45	6.52
200mg/l
Replicate 1	6.49	6.56
Replicate 2	6.50	6.57

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to test chemical to various nominal test concentration, EC50 calculated from equation through probit analysis was determine to be 136.57 mg/L.

Executive summary:

The effect of test chemical 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 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L, 200mg/L. The test solution was prepared in aseptic condition. The test chemical 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 and filter it 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.

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 0.1 units.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 136.57 mg/L.

Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as toxic as per the CLP classification criteria.

Description of key information

After 72 hours of exposure to test chemical to various nominal test concentration, EC50 calculated from equation through probit analysis was determine to be 136.57 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

136.57 mg/L

Additional information

The effect of test chemical 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 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L, 200mg/L. The test solution was prepared in aseptic condition. The test chemical 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 and filter it 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.

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 0.1 units.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 136.57 mg/L.

Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as toxic as per the CLP classification criteria.