Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: data from peer- reviewed journals

Qualifier:

equivalent or similar to guideline

Guideline:

other: Microtox Vibrio Fischeri assay

Principles of method if other than guideline:

The Microtox acute toxicity assay was performed by using a modified strain of Vibrio fischeri

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
no data

Analytical monitoring:

not specified

Details on sampling:

Sample storage conditions before analysis: Frozen samples were brought to room temperature, and centrifuged.

Vehicle:

yes

Details on test solutions:

no data

Test organisms (species):

Vibrio fisheri

Details on inoculum:

Details on test organisms
Laboratory culture: From eight serial dilutions, the percent concentration to decrease 20% of the luminescence of amodified strain of Vibrio fischeri
(EC20) after 5 min incubation was calculated with the Microtox data analysis program
Method of cultivation: No data
Preparation of inoculum for exposure: Frozen samples were brought to room temperature, and centrifuged. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70 μl 1 M NaOH. Colour correction was done at 490 nm.
Pretreatment: No data
Initial biomass concentration: No data

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

5 min

pH:

The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70 μl 1 M NaOH

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

measured concentrations

Details on test conditions:

TEST SYSTEM
Test vessel: Microtox 500 Analyzer
No. of vessels per concentration (replicates): triplicate analysis.

OTHER TEST CONDITIONS
Adjustment of pH: The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70 μl 1 M NaOH
Photoperiod:
Light intensity: Colour correction was done at 490 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The percent concentration to decrease 20% of the luminescence of amodified strain of Vibrio fischeri (EC20) after 5 min incubation was calculated with the Microtox data analysis program [Microtox Omni Software
(1999) Azur Environmental, Newark, Del.].

Reference substance (positive control):

yes

Remarks:

A solution of 1 g/l ZnSO4.7H2O was used as the positive control

Duration:

5 min

Dose descriptor:

other: EC20

Effect conc.:

44.6 other: % dilution

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: EC20 is the percent dilution of the sample (v/v) to cause 20% decrease of bioluminescence in the Microtox assay

Results with reference substance (positive control):

Result with reference substance (positive control)
Results with reference substance valid?: Yes
Other: Glucose was used as negative control and it was non- toxic

Table 1 : Toxicity of the components in the Microtox assay

 

Sample	EC20 (% DILUTION)
Positive control [ZnSO4.7H20]	0.72±0.1
Distilled water	>100
100mg/l Amaranth	44.6±11.6
1g/l Glucose	>100

 

EC20 is the % dilution of the sample (v/v) to cause 20% decrease of bioluminescence in the Microtox assay

>100% indicates no toxic effect

Validity criteria fulfilled:

yes

Remarks:

glucose was used as a negative control

Conclusions:

Microtox assay was conducted using a modified strain of Vibrio fischeri.
The toxicity of 100mg/l of Amaranth determined in terms of EC20 (% dilution) was 44.6 ± 11.6.
According to the ranking scheme for Microtox assay using EC20 values, Amaranth can be categorized under moderately toxic category.

Executive summary:

The Microtox acute toxicity assay was performed by using a modified strain of Vibrio fischeri

Frozen samples were brought to room temperature, and centrifuged. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70μl 1 M NaOH. Colour correction was done at 490 nm. The Microtox acute toxicity assay was performed in a Microtox 500 Analyzer on samples before and after decoloration according to the test protocols defined by the manufacturer From eight serial dilutions, the percent concentration to decrease 20% of the luminescence of amodified strain ofVibrio fischeri(EC20)after 5 min incubation was calculated with the Microtox data analysis program [Microtox Omni Software(1999) Azur Environmental, Newark, Del.]. A solution of 1 g/l ZnSO4·7H2O was used as the positive control and 1 g/l glucose as the negative control. Each EC20 reported is the average of triplicate analysis.

The concentration to decrease 50% of the bacterial luminescence in the Microtox acute assay (EC50) is normally reported. However, in most of these studies, the EC50 before or after decoloration was greater than 100% indicating that there was no toxicity or toxicity change. To better evaluate whether the decoloration process affected toxicity, the dilution required to decrease 20% of the bacterial luminescence relative to the control (EC20)was reported instead. The following rating was adapted from Coleman & Qureshi (1985) –

EC20:>100%=nontoxic;

>75–100%=slightly non-toxic;

 >50–75%=toxic;

>25–50%=moderately toxic;

<25% very toxic.

The toxicity of 100mg/l of Amaranth determined in terms of EC20 (% dilution) was 44.6 ± 11.6.

According to the ranking scheme for Microtox assay using EC20 values, Amaranth can be categorized under moderately toxic category.

Description of key information

The Microtox acute toxicity assay was performed by using a modified strain of Vibrio fischeri . Frozen samples were brought to room temperature, and centrifuged. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70μl 1 M NaOH. Colour correction was done at 490 nm. The Microtox acute toxicity assay was performed in a Microtox 500 Analyzer on samples before and after decoloration according to the test protocols defined by the manufacturer From eight serial dilutions, the percent concentration to decrease 20% of the luminescence of amodified strain ofVibrio fischeri(EC20)after 5 min incubation was calculated with the Microtox data analysis program [Microtox Omni Software(1999) Azur Environmental, Newark, Del.]. A solution of 1 g/l ZnSO4·7H2O was used as the positive control and 1 g/l glucose as the negative control. Each EC20 reported is the average of triplicate analysis. The concentration to decrease 50% of the bacterial luminescence in the Microtox acute assay (EC50) is normally reported. However, in most of these studies, the EC50 before or after decoloration was greater than 100% indicating that there was no toxicity or toxicity change. To better evaluate whether the decoloration process affected toxicity, the dilution required to decrease 20% of the bacterial luminescence relative to the control (EC20)was reported instead. The following rating was adapted from Coleman & Qureshi (1985) –

EC20:>100%=nontoxic;

>75–100%=slightly non-toxic;

 >50–75%=toxic;

>25–50%=moderately toxic;

<25% very toxic. The toxicity of 100mg/l of Amaranth determined in terms of EC20 (% dilution) was 44.6 ± 11.6.

Additional information

Based on the various experimental data for the target chemical study have been reviewed to determine the toxic nature of target chemical aluminium, 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex (915 -67 -3). The studies are as mentioned below:

In the first key study for target chemical (915-67-3) Biotechnology Letters, 2002. The Microtox acute toxicity assay was performed by using a modified strain of Vibrio fischeri . Frozen samples were brought to room temperature, and centrifuged. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70μl 1 M NaOH. Colour correction was done at 490 nm. The Microtox acute toxicity assay was performed in a Microtox 500 Analyzer on samples before and after decoloration according to the test protocols defined by the manufacturer From eight serial dilutions, the percent concentration to decrease 20% of the luminescence of amodified strain ofVibrio fischeri(EC20)after 5 min incubation was calculated with the Microtox data analysis program [Microtox Omni Software(1999) Azur Environmental, Newark, Del.]. A solution of 1 g/l ZnSO4·7H2O was used as the positive control and 1 g/l glucose as the negative control. Each EC20 reported is the average of triplicate analysis. The concentration to decrease 50% of the bacterial luminescence in the Microtox acute assay (EC50) is normally reported. However, in most of these studies, the EC50 before or after decoloration was greater than 100% indicating that there was no toxicity or toxicity change. To better evaluate whether the decoloration process affected toxicity, the dilution required to decrease 20% of the bacterial luminescence relative to the control (EC20)was reported instead. The following rating was adapted from Coleman & Qureshi (1985) –

EC20:>100%=nontoxic;

>75–100%=slightly non-toxic;

 >50–75%=toxic;

>25–50%=moderately toxic;

<25% very toxic. The toxicity of 100mg/l of Amaranth determined in terms of EC20 (% dilution) was 44.6 ± 11.6.

 

Similarly in the second supporting study from (Indian J Microbiol 2011). This investigation was aimed at identifying the effects of the Amaranth dye and its degradation products on microbial growth. Amaranth dye was purchased from Hi-media Laboratories Pvt. Ltd., Mumbai, India. Aspergillus ochraceus NCIM 1146 was obtained from National Chemical Laboratory, Pune, India. E. coli MTCC 452, B. subtilis MTCC 6910 and Penicillium ochrochloron MTCC 517 were obtained from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh, India. It was regularly maintained and preserved at 4°C on nutrient agar slants contained in (g/l); bacteriological peptone 10.0, beef extract 10.0 and NaCl 5.0 Microbial toxicity of control dye amaranth and metabolites obtained after its decolorization (final concentration 1,000 ppm) was carried out in relation to E. coli, Bacillus substilis, Aspergillus ochraceus and Penicillium ochrochloron MTCC 517 and zone of inhibition (diameter in mm) was recorded. The diameter of the discs used was 10mm. Amaranth and its degradation products were not toxic to Aspergillus ochraceus and Penicillium ochrochloron MTCC 517 at 1,000 ppm concentration. Amaranth inhibited growth of E. coli and Bacillus substilis.

 

 

The third study for the chemical (915-67-3) was used (from, TOXICOLOGY AND APPLIED PHARMACOLOGY, 1977). The death of Paramecium caudatum (PC), a unicellular animal, can be observed more readily and in far less time than that of small animals. Hence a bioassay was conducted to study the toxic effect of Amaranth. Paramecium Caudatum was maintained at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter aerogenes. Amaranth was tested in 0.1% and 1% concentration. The test concentrations were put in a hollow slide glass, and an equal volume of 0.04 M phosphate buffer, pH 7.0, was added. After 5 to 10 test organisms were added, their survival times were measured microscopically. Thirty to forty test organisms for each concentration were tested by the same method, and the mean survival time and the death rate were calculated. The survival time was defined as the time required until death was observed for each concentration. Death was assumed to have occurred when there was no movement. The death rate was defined as the percentage of deaths observed during 20 minutes. The mean survival time (in sec) of test organism Paramecium caudatum was determined to be 695 seconds.  The death rate of the test organism at 10000mg/l was 77.4%. Therefore the Effective concentration causing more than 50% death of Paramecium caudatum was reported as 10000 mg/l.