Sodium 6-[(4-aminophenyl)azo]-5-hydroxynaphthalene-1-sulphonate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Study period:

2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Real Decreto 363/1995 (BOE núm. 133 de Junio de 1995).

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

7-amino-4-hydroxy-3-[[4-[(4-sulphophenyl)azo]phenyl]azo]naphthalene-2-sulphonic acid, compound with 2,2',2''-nitrilotriethanol (1:2)

EC Number:

265-016-4

EC Name:

7-amino-4-hydroxy-3-[[4-[(4-sulphophenyl)azo]phenyl]azo]naphthalene-2-sulphonic acid, compound with 2,2',2''-nitrilotriethanol (1:2)

Cas Number:

64683-40-5

Molecular formula:

C22H17N5O7S2.2C6H15NO3

IUPAC Name:

7-amino-4-hydroxy-3-({4-[(4-sulfophenyl)diazenyl]phenyl}diazenyl)naphthalene-2-sulfonic acid - 2,2',2''-nitrilotriethanol (1:2)

Test solutions

Vehicle:

not specified

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection Algae and Protozoo (Cumbria, United Kingdom)
- Method of cultivation:
Temperature: 22 ± 2 ° C
Intensity and quality of illumination: 2500 Lux (The surface where the study is done receives uniform and continuous fluorescent lighting (white-cold type)
Continuous orbital agitation
The reference substance used to verify the response of test organisms is potassium dichromate (K2Cr2O7). The result of periodic verification of the response of the test organisms is IC50 72h = 1.1 mg / L.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22 ± 2ºC

Nominal and measured concentrations:

Theoretical tested concentrations: 10 mg/L, 32 mg/L, 100 mg/L, 320 mg/L, 1000 mg/L, 3200 mg/L, 10000 mg/L
Actual concentration of the mother solution tested (active material): 2866,04 mg/L (10.460 mg/L of test material).
Actual tested concentrations (active material): 2,87 mg/L, 9,17 mg/L, 28,66 mg/L, 91,71 mg/L, 286,6 mg/L, 917,1 mg/L, 2866 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Test tubes (40 mL)
- Volume of culture used: 10 mL
- Type: closed with a material permeable to air
- Type of flow-through (e.g. peristaltic or proportional diluter): continuous agitation
- Initial cells density: 1,09 10E4 cells/mL
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

GROWTH MEDIUM
- Standard medium used: yes, culture medium defined in AFNOR T90-304, which has been validated by the laboratory since it gives quantitative results comparable to those described in the guideline
- Detailed composition if non-standard medium was used:

Solution 1: Ca(NO3)2·4H2O: 0.4 g / 100 ml H2O
Solution 2: KNO3: 10 g / 100 ml H2O
Solution 3: MgSO4·7H2O: 3 g / 100 ml H2O
Solution 4: K2HPO4: 4 g / 100 ml H2O
Solution 5:
CuSO4· 5H2O: 30 mg / l
(NH4 6Mo7O24·4H2O: 60 mg / l
ZnSO4·7H2O: 60 mg / l
CoCl2 6H2O: 60 mg / l
Mn (NO3)2·4H2O: 60 mg / l
C6H8O·7H2O: 60 mg / l
H3BO3: 60 mg / l
Solution 6:
C6H5FeO7 5H2O: 1.62 g / l
FeSO4·7H2O: 0.62 g / l
FeCl 3·6H2O: 0.62 g / l

Add to 1000 mL of deionized water
Solution 1: 1 mL
Solution 2:1 mL
Solution 3: 1 mL
Solution 4: 1 mL
Solution 5 : 0.5 mL
Solution 6: 0.5 mL

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: not specified
- Growth apparatus: Incubation chamber with orbital agitation (Ivymen system)
- Light intensity and quality: 2500 Lux (The surface where the study is done receives uniform and continuous fluorescent lighting (white-cold type)

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: 7+control
- Preparation of stock and test solutions: A stock solution has been prepared with a concentration of 10 g / L of the test material. Therefore, 10 g of the material have been dissolved in 100 ml of growth medium by shaking in a volumetric flask, Class A of 100 mL, so that a stock solution of 10000 mg / L has been obtained. Because the study material contains 27.4% of active material, the stock solution has a concentration 2740 mg / L of active material. The different solutions used in the test have been prepared from this stock solution of 1000 mg / L of active substance.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Key result

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

115 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Results with reference substance (positive control):

- Results with reference substance valid
- IC50: 1.1 mg/L


The validation criterion considered for this test is:
- the cell density of the control solutions must have been multiplied by a factor greater than 16 in 72h
- the pH of the control solutions must not have varied more than 1.5 units during the test
- The IC50 value 72h of potassium dichromate is in the range of 0.9 - 1.5 mg / l

Any other information on results incl. tables

The results of the cell concentration are shown in the table 1.
Table 1: Calculated cell concentration

replicates nº of cells/ml	control		concentration (mg/L)
	0h	72h	2.87	9.17	28.66	91.71	286.6	917.7	2866
1	1.09E4	2.46E5	2.56E5	2.21E5	1.96E5	1.41E5	9.34E4	7.01E4	5.93E4
2	1.09E4	2.63E5	2.43E5	2.23E5	1.93E5	1.38E5	9.59E4	6.84E4	6.13E4
average	1.09E4	2.55E5	2.50E5	2.22E5	1.95E5	1.40E5	9.47E4	6.97E4	6.03E4
sd	0	5303	8839	1768	1768	1768	1768	1768	74
CV	0	2	4	1	1	1	2	3	1

Inhibition of algal growth during the test: In the table 2, the percentage of growth inhibition is shown, defined as the biomass at the end of the exposure period (72h) minus the biomass at the beginning of the exposure period (0h).

Table 2: % inhibition algal growth

Conc. (mg/L)	% inhibition algal growth
control	0
2.12.87	2.1
9.17	13.5
28.66	24.9
91.71	47.2
286.6	65.8
917.1	76.7
2866	100

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The result obtained in this study indicate that the substance has an IC50 value (72h) equal to 115 mg / L active substance

Executive summary:

For the determination of inhibition of algal growth, the used algae has been the unicellular freshwater Pseudokirchneriella subcapitata (formerly Selenastrum Capricornutum). The guideline followed was the OECD 201 (1984) together with the Spanish Royal Decree 363/1995 (BOE No. 133 of June 1995).
The fresh water algae Selenastrum capricornutum has been exposed to the test substance at test concentrations: 10 mg / L, 32 mg / L, 100 mg / L, 320 mg / L, 1000 mg / L, 3200 mg / L and 10000 mg / L. The total duration of the trial has been 72 hours.
The result obtained in this test indicates that the test substance has an IC50 value (72h) of 115 mg /L based on active material.