Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate

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)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

The test solution was prepared in aseptic condition. The test item disodium 4,4’-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate was prepared by adding 140 mg of test item in 350 ml of BBM to get the final concentration of 400 mg/L. 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 10(4)cells/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

Hardness:

No data

Test temperature:

24 °C ±2°C.

pH:

6.7 to 7.4

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l, 200 mg/l and 400mg/l. All the six concentration were in geometric series spaced by a factor of 2.

Details on test conditions:

Light Quality White Fluoroscent Light
Light Intensity 1500Lux
Photoperiod 16 Hour Light Period : 8 Hour Dark Period
RPM Speed 120 Revolutions per minute
Water Deionized Waer
Test vessel size 100ml
Test solution volume 60ml
Dilution water Bold’s Basal Medium
Test concentration Six test concentration were:
Replicates per concentration
Three replicates for Control
Two replicates for each test concentration

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

379.668 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: calculated graphically through probit analysis.

Details on results:

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 100mg/l, 200mg/l and 400mg/l. EC50 was found to be 379.668 mg/l graphically through probit analysis

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

Test vessels and test concentration	24 Hours	48 Hours	72 Hours
Control
Replicate 1	41.5 x 10^4	70 x 10^4	89 x 10^4
Replicate 2	49 x 10^4	65 x 10^4	86 x 10^4
Replicate 3	44.5 x 10^4	71.5 x 10^4	91.5x 10^4
CAS No. 4553-89-3
12.5mg/l
Replicate 1	36.5 x 10^4	43.5 x 10^4	55.5 x 10^4
Replicate 2	41.5 x 10^4	46 x 10^4	58.5 x 10^4
25mg/l
Replicate 1	35 x 10^4	51.5 x 10^4	32 x 10^4
Replicate 2	34 x 10^4	35.5 x 10^4	35 x 10^4
50mg/l
Replicate 1	39 x 10^4	43.5x 10^4	29.5 x 10^4
Replicate 2	35.5 x 10^4	41 x 10^4	30 x 10^4
100mg/l
Replicate 1	41 x 10^4	43.5 x 10^4	28.5 x 10^4
Replicate 2	42.5 x 10^4	40 x 10^4	22 x 10^4
200mg/l
Replicate 1	26 x 10^4	20.5 x 10^4	16.5 x 10^4
Replicate 2	27 x 10^4	23 x 10^4	14.5 x 10^4
400mg/l
Replicate 1	16 x 10^4	12 x 10^4	7 x 10^4
Replicate 2	12 x 10^4	9.5 x 10^4	9 x 10^4

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

	CONTROL		12.5mg/l		25mg/l		50mg/l		100mg/l		200mg/l		400mg/l
Average Specific Growth rate (µ )	R1	1.496	R1	1.339	R1	1.155	R1	1.128	R1	1.117	R1	0.934	R1	0.649
	R2	1.485	R2	1.356	R2	1.185	R2	1.134	R2	1.030	R2	0.891	R2	0.732
	R3	1.505
Mean of Avg. Specific growth rate	1.495		1.348		1.170		1.131		1.073		0.913		0.691
Percentage Inhibition (%I)	_		9.891		21.752		24.377		28.218		38.955		53.826

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	6.5	7.1
Replicate 2	6.6	7.0
Replicate 3	6.6	7.0
CAS No. 4553-89-3
12.5 mg/l
Replicate 1	6.5	6.9
Replicate 2	6.5	6.8
25 mg/l
Replicate 1	6.5	6.8
Replicate 2	6.5	6.7
50 mg/l
Replicate 1	6.5	6.7
Replicate 2	6.5	6.7
100 mg/l
Replicate 1	6.5	6.7
Replicate 2	6.5	6.6
200 mg/l
Replicate 1	6.5	6.6
Replicate 2	6.5	6.6
400 mg/l
Replicate 1	6.5	6.6
Replicate 2	6.5	6.6

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to various nominal test concentrations of disodium 4,4’-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate (CAS No. 4553-89-3) to freshwater green alga Chlorella vulgaris, EC50 was determined to be 379.668 mg/l graphically through probit analysis.

Executive summary:

The effect of test item disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate, CAS No. 4553-89-3 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 concentrations chosen for the study were 12.5mg/l, 25mg/l, 50 mg/l, 100mg/l, 200 mg/l and 400mg/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 379.668 mg/l.

Based on the above LC50 value it can be concluded that the target substance 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate is non toxic to green algae and thus can not be classified under Aquatic toxic category.

Description of key information

After 72 hours of exposure to various nominal test concentrations of disodium 4,4’-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate (CAS No. 4553-89-3) to freshwater green alga Chlorella vulgaris, EC50 was determined to be 379.668 mg/l graphically through probit analysis.

Key value for chemical safety assessment

EC50 for freshwater algae:

379.668 mg/L

Additional information

Available Experimental and predicted data of the target substance Disodium 4,4’-(2,4-dihydroxy-5- hydroxymethyl-1,3-phenylene bis-azo) di- (naphthalene-1-sulfonate) for the Short term toxicity to aquatic invertebrate are summarized as follows:

For key study, the effect of test item disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate, CAS No. 4553-89-3 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 concentrations chosen for the study were 12.5mg/l, 25mg/l, 50 mg/l, 100mg/l, 200 mg/l and 400mg/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 379.668 mg/l.

Supporting above experimental study, toxicity to aquatic algae and cyanobacteria of Disodium 4,4’-(2,4-dihydroxy-5- hydroxymethyl-1,3-phenylene bis-azo) di- (naphthalene-1-sulfonate) was estimated using QSAR Toolbox version 3.3. The Effective concentration (LC50) of the substance Disodium 4,4’-(2,4-dihydroxy-5- hydroxymethyl-1,3-phenylene bis-azo) di- (naphthalene-1-sulfonate) in algae Pseudokirchneriella subcapitata in a 72 hour study is estimated to be 155.21 mg/L. based on the effects on population.

Based on the above LC50 values it can be concluded that the target substance 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate is not likely to be toxic to algae and thus cannot be classified under Aquatic toxic category as per CLP criteria.