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Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data is from peer reviewed journal
Justification for type of information:
Data is from peer reviewed journal
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Toxicity to micro-organisms study was conducted using anaerobic granular sludge or methanogens for 72 hrs.
GLP compliance:
not specified
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material (IUPAC name): 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride
- Common name: Red Astrazon 6B
- Molecular formula: C24H30ClN2Cl
- Molecular weight: 417.421 g/mol
- Smiles notation: C1(=[N+](c2ccccc2C1(C)C)C)\C=C/c1c(cc(N(CCCl)CC)cc1)C.[ClH-]
- InChl: 1S/C24H30ClN2.ClH/c1-6-27(16-15-25)20-13-11-19(18(2)17-20)12-14-23-24(3,4)21-9-7-8-10-22(21)26(23)5;/h7-14,17H,6,15-16H2,1-5H3;1H/q+1;/p-1
- Substance type: Organic
- Physical state: Solid
- Other: Test chemical was provided by Comodepur S. p. a., Como Italy.
Analytical monitoring:
not specified
Details on sampling:
- Concentrations: Test chemical concentration used for the study were 0, 300, 600, 1200 and 2400 mg/l, respectively.
Vehicle:
not specified
Test organisms (species):
other: Anaerobic granular sludge or methanogens were used as a test organism during the study.
Details on inoculum:
- Laboratory culture: Anaerobic granular sludge sampled from a UASB reactor fed with soft-drink wastewater was used as active biomass.
Test type:
not specified
Water media type:
not specified
Total exposure duration:
72 h
Nominal and measured concentrations:
0, 300, 600, 1200 and 2400 mg/l (nominal concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: Serum vials (120 ml total volume) with butyl rubber stoppers were used as a test vessel during the study.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Serum vials contains the total volume 120 ml.
- No. of vessels per concentration (replicates): 4 replicates/concentration

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The final biomass concentration in each vial was determined at the end of the test. Methane production were monitored in each vial every 24 h for 3 or 4 days.

TEST CONCENTRATIONS
- Test concentrations: Test chemical concentration used for the study were 0, 300, 600, 1200 and 2400 mg/l, respectively.

Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
IC100
Effect conc.:
300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks:
biomass
Details on results:
Test chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (Red Astrazon 6B) completely inhibited the methanogenic activity at every test concentration.

Table:Specific methane yields (ml CHWgVSS) and production rates (ml CH4/gVSS d) as functions of dye concentrations expressed in percentages of the control values without addition of dyes.

 

Dyes

concentration

(mg/l)

Dye (D5)

 

Yield (%)

Rate (%)

0

300

600

1200

2400

100

0

0

0

0

100

0

0

0

0

 

Initial dye concentration range: 300~-2400 mg/l; incubation time: 72 h.

Validity criteria fulfilled:
not specified
Conclusions:
Based on the inhibitory effect of the chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride on the biomass of test organism anaerobic granular sludge or methanogens, the inhibitory conc. (IC100) value was determined to be 300 mg/l, respectively.
Executive summary:

Toxicity to micro-organisms study was conducted using anaerobic granular sludge or methanogens for 72 hrs. Anaerobic granular sludge or methanogens were used as a test organism during the study. Anaerobic granular sludge sampled from a UASB reactor fed with soft-drink wastewater was used as active biomass.Test chemical concentration used for the study were 0, 300, 600, 1200 and 2400 mg/l, respectively. Serum vials (120 ml total volume) with butyl rubber stoppers were used as a test vessel during the study. The basal medium M9 was used with laboratory grade yeast extract (1 g/l), trypticase (1 g/l), sodium acetate, a microelements solution. All manipulations were carried out in an anaerobic chamber. The autoclaved medium was aliquoted in the vials (50 ml/each), to which was added: approximately 0.5 ml of whole granules of the sludge; concentrated dye solutions to obtain final test dye concentrations of 0, 300, 600, 1200 and 2400 mg/l, respectively. For test chemical, four replicate vials were prepared.Methane production were monitored in each vial every 24 h for 3 or 4 days. The final biomass concentration in each vial was determined at the end of the test.Test chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (Red Astrazon 6B) completely inhibited the methanogenic activity at every test concentration. Based on the inhibitory effect of the chemical 2 -[2 -[4 -[(2 -chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride on the biomass of test organism anaerobic granular sludge or methanogens, the inhibitory conc. (IC100) value was determined to be 300 mg/l, respectively.

Description of key information

Toxicity to micro-organisms study was conducted using anaerobic granular sludge or methanogens for 72 hrs (F. Malpei et. al; 1998). Anaerobic granular sludge or methanogens were used as a test organism during the study. Anaerobic granular sludge sampled from a UASB reactor fed with soft-drink wastewater was used as active biomass.Test chemical concentration used for the study were 0, 300, 600, 1200 and 2400 mg/l, respectively. Serum vials (120 ml total volume) with butyl rubber stoppers were used as a test vessel during the study. The basal medium M9 was used with laboratory grade yeast extract (1 g/l), trypticase (1 g/l), sodium acetate, a microelements solution. All manipulations were carried out in an anaerobic chamber. The autoclaved medium was aliquoted in the vials (50 ml/each), to which was added: approximately 0.5 ml of whole granules of the sludge; concentrated dye solutions to obtain final test dye concentrations of 0, 300, 600, 1200 and 2400 mg/l, respectively. For test chemical, four replicate vials were prepared.Methane production were monitored in each vial every 24 h for 3 or 4 days. The final biomass concentration in each vial was determined at the end of the test.Test chemical 2-[2-[4-[(2-chloroethyl)ethylamino] -o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (Red Astrazon 6B) completely inhibited the methanogenic activity at every test concentration. Based on the inhibitory effect of the chemical 2 -[2 -[4 -[(2 -chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride on the biomass of test organism anaerobic granular sludge or methanogens,the inhibitory conc. (IC100) value was determined to be 300 mg/l, respectively.

Key value for chemical safety assessment

Additional information

Experimental key and supporting study for the target chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride(CAS No. 6441-82-3)were reviewed for toxicity to microorganism endpoint to summarize the following information:

 

In an experiment key study from peer reviewed journal (F. Malpei et. al; 1998),Toxicity to micro-organisms study was conducted using anaerobic granular sludge or methanogens for 72 hrs. Anaerobic granular sludge or methanogens were used as a test organism during the study. Anaerobic granular sludge sampled from a UASB reactor fed with soft-drink wastewater was used as active biomass.Test chemical concentration used for the study were 0, 300, 600, 1200 and 2400 mg/l, respectively. Serum vials (120 ml total volume) with butyl rubber stoppers were used as a test vessel during the study. The basal medium M9 was used with laboratory grade yeast extract (1 g/l), trypticase (1 g/l), sodium acetate, a microelements solution. All manipulations were carried out in an anaerobic chamber. The autoclaved medium was aliquoted in the vials (50 ml/each), to which was added: approximately 0.5 ml of whole granules of the sludge; concentrated dye solutions to obtain final test dye concentrations of 0, 300, 600, 1200 and 2400 mg/l, respectively. For test chemical, four replicate vials were prepared.Methane production were monitored in each vial every 24 h for 3 or 4 days. The final biomass concentration in each vial was determined at the end of the test.Test chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (Red Astrazon 6B) completely inhibited the methanogenic activity at every test concentration. Based on the inhibitory effect of the chemical 2 -[2 -[4 -[(2 -chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride on the biomass of test organism anaerobic granular sludge or methanogens, the inhibitory conc. (IC100) value was determined to be 300 mg/l, respectively.

 

Another supporting toxicity to micro-organisms study was conducted on Bacillus subtilis, strain IFO 3022 (Toshihiko Ogawa et. al; 1988). Bacillus subtilis (IFO 3022), a bacterial population in activated sludge, was obtained from Institute for Fermentation, Osaka.Spizien medium was used for the asynchronous culture and consisted of 0.2% (NH4)2SO4, 1.4% K2HP04, 0.6% KH2PO4, 0.1% sodium citrate-2H20, 0.02% MgSO4.7H2O, 0.0002% FeCI3.6H2O, 0.0002% MnCI2.4H20 and 0.5% glucose. The medium composition of the synchronous culture was the same as that described above but contained 0.0072% glucose as well. The cell population was synchronized by the stationary phase method. These precultivate broths, 1 ml each, were inoculated in 1000 ml mediums, and the cultures were shaken at 37°C. The cell concentrations of the asynchronous cultures were determined by measurement of transmittance at 660 nm, and those of the synchronous cultures were read under a microscope using Thoma's hemacytometer, After harvesting by centrifugation at 8,000 rpm for 15 min from the cultivate broth, RNA and DNA were each fractionated by the method of Schmidt, Thannhauser and Schneider. Their concentrations were determined respectively by measurement of absorbance at 260 and 270 nm. Based on the inhibitory effect of the chemical 2 -[2 -[4 -[(2 -chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride on the growth of test organism Bacillus subtilis, the inhibitory conc. (IC) value was determined to be 0.0229 mg/l, respectively.

 

Thus, based on the overall reported results for target chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (from peer reviewed journals),it can be concluded that the IC and IC100 value for the test chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride was determined to be 0.0229 and 300 mg/l, respectively.