Reaction products of diazotised sodium 2-amino-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate coupled with sodium 2-(4-aminobenzene-1-sulfonyl)ethyl sulfate, subsequently coupled with diazotised sodium 2-(4-aminobenzene-1-sulfonyl)ethyl sulfate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Remarks:

Experimental data of read across substances

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline available

Principles of method if other than guideline:

WoE report is based on two toxicity to microorganisms studies as-
WoE 2 and WoE 3.

GLP compliance:

not specified

Analytical monitoring:

not specified

Details on sampling:

WoE 2 - Concentrations: 1, 3.2, 10, 32 and 100 mg/l

WoE 3- Concentrations: 0.01, 0.1, 1.00, 10 and 100 mg/l.

Vehicle:

no

Details on test solutions:

WoE 3 - PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The solution was prepared by dissolving 0.32mg ammonium sulphate in 970 ml deionised water.
- Test concentration separation factor: 10

Test organisms (species):

other: WoE 2- Activated sludge, WoE 3 - Nitrosomonas. sp

Details on inoculum:

WoE 2- sludge of a Husman apparatus

WoE 3 - Name and location of sewage treatment plant where inoculum was collected: Surface water samples (20ml) were collected in duplicate sets of 50ml sterile plastic containers from the upstream zone of Aba River located in Aba, Nigeria.
- Initial biomass concentration:. 3000,000 CFU
- Method of cultivation: ISOLATION OF NITROSOMONAS:
The isolation procedure was a modification of the method adapted from Colwell and Zambruskii (1972). The target microorganisms were first enriched by inoculating 10ml of water samples into 100ml of  broth contained in a triplicate set of 250 ml Erlenmeyer flasks. Incubation was  at 28±2°C for 4 days in the dark. The culture (10ml) was then transferred into fresh sterile 100ml broth in a triplicate set of 250ml Erlenmeyer flasks and incubated as above. Two   additional   enrichments   in   broth   as described  above  were  carried  out  after  which, 0.1 ml of  enriched culture was inoculated onto a replicate  set  of  agar  plates  by  spread -   plate method.  Plates were incubated at 28±2°C and observed for growth. Discrete rnucoid colonies which developed were picked, purified by repeated streaking and Gram stained. Isolates which were Gram-negative rods were picked and presumed to be Ntrosomonas. Isolates were further  identified by growth in nitrite-free broth. Cultures that were positive  for  presence of  nitrite  after  3  days  of incubation in the dark were tentatively identified as Nitrosomonas. Stock  cultures  were  prepared on Winogradsky agar slants and stored at 4°C. 

- Preparation of inoculum for exposure:
Cells were transferred from stock cultures into 20ml Winogradsky broth and incubated at 28±2°C with shaking for 48h for maximum biomass yield. The cells were washed in nitrite-free physiological saline using a vortex mixer and allowed to stand for  1 h.   The cell sediment was resuspended in fresh sterile physiological saline and washed.  The washing procedure was done repeatedly until nitrite-N was undetected thus ensuring no residual nitrite-N. Cell viability was determined by placing 1 ml inoculum into 20ml sterile ammonium sulphate solution (0.05mgL"1  ammonia-N) contained in a duplicate set of 150ml Erlenmeyer flasks and incubated as above for 3h. Samples (1ml) of the culture were tested periodically for the presence of nitrite-N. Cultures that were positive confirmed the viability of the cells and were used for bioassay. Controls consisted of autoclaved cultures. This was to show that the accumulation of nitrite-N in the experimental flasks was due to the metabolic activities of the cells and not abiotic factors.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

3 to 8 hrs

Test temperature:

WoE 2- 21.1°C
WoE 3- 28±2°C

Nominal and measured concentrations:

WoE 2: 1, 3.2, 10, 32 and 100 mg/l
WoE 3: 0.01, 0.1, 1.00, 10 and 100 mg/l.

Details on test conditions:

WoE 3: TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: 250 ml
- No. of vessels per concentration (replicates): Triplicate
- No. of vessels per control (replicates): Triplicate
-Photoperiod: Darkness

TEST MEDIUM / WATER PARAMETERS
- Medium: The Winogradsky medium phase II contained (gL·1): (NH4)2S04 2g; K2HP04 1.0; MgS04. 7H20 0.5g; NaCl 2g; FeS04. 7H20; 0.49; ZnCl2 trace amounts and deionised water 1,000ml. The pH of medium was 7.5. Sterilization was by membrane filtration (0.2µm pore size, Acrodisc). Solid medium was prepared by adding autoclaved agar No.1 (1.5%w/v) to the broth medium.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: 0.01, 0.1, 1.00, 10 and 100 mg/l.

- Incubation: In the dark at 28 ± 2°C with shaking

Reference substance (positive control):

yes

Remarks:

WoE 2- 3,5 Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 - < 332.208 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: Other details not known

Results with reference substance (positive control):

WoE 2- EC50 was within the range (10-25 mg O2/l)

Reported statistics and error estimates:

WoE 3- EC50 was estimated from the linear regression of the plot of ammonia oxidation rate against concentration of toxicant.

Validity criteria fulfilled:

not specified

Conclusions:

On the basis of the experimental studies of the structurally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, based of toxicity to microorganism study the 3 to 8 hr EC50 value of test chemical was determined to be > 100 to < 332.208 mg/l.

Executive summary:

Data available of the structurally similar read across chemicals has been reviewed to determine the effect of the test chemical on microorganisms. The studies are as mentioned below:

The first study of toxicity to microorganisms was carried out according to OECD 209 guideline. Activated sludge (sludge of a Husman apparatus) was taken as test organism. Duration of study was 3 hr. Concentrations of the test substance were 1, 3.2, 10, 32 and 100 mg/l. Temperature was 21.1°C. Oxygen-consumption was measured with an electrode system. 3,5-Dichlorophenol was used as reference substance and EC50 was within the required range (10-25 mg O2/l)On the basis of toxicity to microorganism study the 3 hr EC0 and EC50 value of test chemical was determined to be 100 and > 100 mg/l respectively.  

 

The second includes study of  potential inhibitory effect of test chemical on ammonia-N oxidation by nitrosomonas was investigated. Source of inoculum was surface water samples (20ml) were collected in duplicate sets of 50ml sterile plastic containers from the upstream zone of Aba River located in Aba, Nigeria. The isolation procedure was a modification of the method adapted from Colwell and Zambruskii (1972). The target microorganisms were first enriched by inoculating 10ml of water samples into 100ml of  broth contained in a triplicate set of 250 ml Erlenmeyer flasks. Incubation was  at 28±2°C for 4 days in the dark. The culture (10ml) was then transferred into fresh sterile 100ml broth in a triplicate set of 250ml Erlenmeyer flasks and incubated as above. Two   additional   enrichments   in   broth   as described  above  were  carried  out  after  which, 0.1 ml of  enriched culture was inoculated onto a replicate  set  of  agar  plates by spread-plate method.  Plates were incubated at 28±2°C and observed for growth. Discrete rnucoid colonies which developed were picked, purified by repeated streaking and Gram stained. Isolates which were Gram-negative rods were picked and presumed to be Ntrosomonas. Isolates were further  identified by growth in nitrite-free broth. Cultures that were positive  for  presence of  nitrite  after  3  days  of incubation in the dark were tentatively identified as Nitrosomonas. Stock  cultures  were  prepared on Winogradsky agar slants and stored at 4°C. Cells were transferred from stock cultures into 20ml Winogradsky broth and incubated at 28±2°C with shaking for 48h for maximum biomass yield. The cells were washed in nitrite-free physiological saline using a vortex mixer and allowed to stand for  1 h.   The cell sediment was resuspended in fresh sterile physiological saline and washed.  The washing procedure was done repeatedly until nitrite-N was undetected thus ensuring no residual nitrite-N. Cell viability was determined by placing 1 ml inoculum into 20ml sterile ammonium sulphate solution (0.05mgL"1  ammonia-N) contained in a duplicate set of 150ml Erlenmeyer flasks and incubated as above for 3h. Samples (1ml) of the culture were tested periodically for the presence of nitrite-N. Cultures that were positive confirmed the viability of the cells and were used for bioassay. Controls consisted of autoclaved cultures. This was to show that the accumulation of nitrite-N in the experimental flasks was due to the metabolic activities of the cells and not abiotic factors. Initial biomass concentration was 3000,000 CFU. Duration of test was 8 hr. Temperature was of 28±2°C. Test vessel was  Erlenmeyer flasks. Fill volume was of 250 ml.  Number of vessels per concentration (replicates)was in  triplicate. Number of vessels per control (replicates) was in triplicate.  The Winogradsky medium phase II contained (gL·1): (NH4)2S04 2g; K2HP04 1.0; MgS04. 7H20 0.5g; NaCl 2g; FeS04. 7H20; 0.49; ZnCl2 trace amounts and deionised water 1,000ml. The pH of medium was 7.5. Sterilization was by membrane filtration (0.2µm pore size, Acrodisc). Solid medium was prepared by adding autoclaved agar No.1 (1.5%w/v) to the broth medium. The solution was prepared by dissolving 0.32mg ammonium sulphate in 970 ml deionised water and dispensed in 97ml  amounts into triplicate 250ml Erlenmeyer flasks. Into each triplicate set of flasks was added the appropriate toxicant concentration of 0.01, 0.1, 1.00, 10 and 100 mg/l. EC50 was estimated from the linear regression of the plot of ammonia oxidation rate against the concentration of toxicant. The median effective concentration EC50 of test chemical was determined to be 332.208 mg/l in the duration of 8 hr. 

 

On the basis of the experimental studies of the structurally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, based of toxicity to microorganism study the 3 to 8 hr EC50 value of test chemical was determined to be > 100 to < 332.208 mg/l. As per the value of EC50 test chemical was consider as non-toxic to microorganisms.

Description of key information

On the basis of the experimental studies of the structurally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, based of toxicity to microorganism study the 3 to 8 hr EC50 value of test chemical was determined to be > 100 to < 332.208 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

Additional information

Data available of the structurally similar read across chemicals has been reviewed to determine the effect of the test chemical on microorganisms. The studies are as mentioned below:

The first study of toxicity to microorganisms was carried out according to OECD 209 guideline. Activated sludge (sludge of a Husman apparatus) was taken as test organism. Duration of study was 3 hr. Concentrations of the test substance were 1, 3.2, 10, 32 and 100 mg/l. Temperature was 21.1°C. Oxygen-consumption was measured with an electrode system. 3,5-Dichlorophenol was used as reference substance and EC50 was within the required range (10-25 mg O2/l)On the basis of toxicity to microorganism study the 3 hr EC0 and EC50 value of test chemical was determined to be 100 and > 100 mg/l respectively.  

 

The second includes study of  potential inhibitory effect of test chemical on ammonia-N oxidation by nitrosomonas was investigated. Source of inoculum was surface water samples (20ml) were collected in duplicate sets of 50ml sterile plastic containers from the upstream zone of Aba River located in Aba, Nigeria. The isolation procedure was a modification of the method adapted from Colwell and Zambruskii (1972). The target microorganisms were first enriched by inoculating 10ml of water samples into 100ml of  broth contained in a triplicate set of 250 ml Erlenmeyer flasks. Incubation was  at 28±2°C for 4 days in the dark. The culture (10ml) was then transferred into fresh sterile 100ml broth in a triplicate set of 250ml Erlenmeyer flasks and incubated as above. Two   additional   enrichments   in   broth   as described  above  were  carried  out  after  which, 0.1 ml of  enriched culture was inoculated onto a replicate  set  of  agar  plates by spread-plate method.  Plates were incubated at 28±2°C and observed for growth. Discrete rnucoid colonies which developed were picked, purified by repeated streaking and Gram stained. Isolates which were Gram-negative rods were picked and presumed to be Ntrosomonas. Isolates were further  identified by growth in nitrite-free broth. Cultures that were positive  for  presence of  nitrite  after  3  days  of incubation in the dark were tentatively identified as Nitrosomonas. Stock  cultures  were  prepared on Winogradsky agar slants and stored at 4°C. Cells were transferred from stock cultures into 20ml Winogradsky broth and incubated at 28±2°C with shaking for 48h for maximum biomass yield. The cells were washed in nitrite-free physiological saline using a vortex mixer and allowed to stand for  1 h.   The cell sediment was resuspended in fresh sterile physiological saline and washed.  The washing procedure was done repeatedly until nitrite-N was undetected thus ensuring no residual nitrite-N. Cell viability was determined by placing 1 ml inoculum into 20ml sterile ammonium sulphate solution (0.05mgL"1  ammonia-N) contained in a duplicate set of 150ml Erlenmeyer flasks and incubated as above for 3h. Samples (1ml) of the culture were tested periodically for the presence of nitrite-N. Cultures that were positive confirmed the viability of the cells and were used for bioassay. Controls consisted of autoclaved cultures. This was to show that the accumulation of nitrite-N in the experimental flasks was due to the metabolic activities of the cells and not abiotic factors. Initial biomass concentration was 3000,000 CFU. Duration of test was 8 hr. Temperature was of 28±2°C. Test vessel was  Erlenmeyer flasks. Fill volume was of 250 ml.  Number of vessels per concentration (replicates)was in  triplicate. Number of vessels per control (replicates) was in triplicate.  The Winogradsky medium phase II contained (gL·1): (NH4)2S04 2g; K2HP04 1.0; MgS04. 7H20 0.5g; NaCl 2g; FeS04. 7H20; 0.49; ZnCl2 trace amounts and deionised water 1,000ml. The pH of medium was 7.5. Sterilization was by membrane filtration (0.2µm pore size, Acrodisc). Solid medium was prepared by adding autoclaved agar No.1 (1.5%w/v) to the broth medium. The solution was prepared by dissolving 0.32mg ammonium sulphate in 970 ml deionised water and dispensed in 97ml  amounts into triplicate 250ml Erlenmeyer flasks. Into each triplicate set of flasks was added the appropriate toxicant concentration of 0.01, 0.1, 1.00, 10 and 100 mg/l. EC50 was estimated from the linear regression of the plot of ammonia oxidation rate against the concentration of toxicant. The median effective concentration EC50 of test chemical was determined to be 332.208 mg/l in the duration of 8 hr. 

 

On the basis of the experimental studies of the structurally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, based of toxicity to microorganism study the 3 to 8 hr EC50 value of test chemical was determined to be > 100 to < 332.208 mg/l. As per the value of EC50 test chemical was consider as non-toxic to microorganisms.