Reaction products of 7-amino-4-hydroxynaphthalene-2-sulfonic acid reacted with 2-((1,3,5-triazin-2-yl)amino)ethan-1-ol, coupled with diazotised 3-amino-4-hydroxybenzenesulfonic acid, subsequently coupled with diazotised 2-amino-5-methoxybenzenesulfonic acid, chelated with copper, sodium salt

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 1991-07-23 to 1991-07-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

No concentrations of the test substance were measured.

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The aeration stage of the Severn Trent Pic sewage treatment plant at Belper, Derbyshire, treating predominantly domestic sewage.
- Type: A mixed population of activated sewage sludge micro-organisms.
- Data of Collection: 1991-07-23
- Preparation of inoculum for exposure: Direct dispersion in water. At time "0" 16 mL of synthetic sewage was made up to 300 mL with water in a 500 mL conical flask. 200 mL inoculum were added and the mixture aerated at approsimately 0.5 - 1 Litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropiate amounts of the test of reference material being added. As each vessel reached 30 minutes contact time the contents were poured into the measuring vessel and the rate of respiration measured. The procedure was repeated after 3-hour contact period.
- Pretreatment: No pre-treatment was performed.
- Initial biomass concentration: 3.9 g/L ss

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

Exposure Conditions Contact time: 30 minutes and 3 hours. Vessels: 500 mL conical flasks. Aeration: Compressed air via narrow bore glass tubes.

Hardness:

50 mg/L as CaCO3

Test temperature:

22 °C

pH:

The pH was equal to 7.2

Dissolved oxygen:

0.5 - 7.9 mgO2/L

Nominal and measured concentrations:

- Nominal concentrations:
Test material: 1000 mg/L, in triplicate.
Reference material: 3.2, 10 and 32 mg/L.
- Measured concentrations: No concentrations were measured.

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flasks.
- Measuring apparatus: 250 mL darkened glass BOO bottle fitted with a Yellow Springs BOD probe, (Model 54) aid a magnetic stirrer.
- Aeration: Compressed air via narrow bore glass tubes.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 3.9 g/L
- Synthetic Sewage:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g HgSO4.7H2O
2.8 g K2HPO4
- dissolved in 1 litre tap water.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water, dechlorinated by sodium
thiosulphate (hardness approximately 50 mg/L as CaCO3).

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Normal laboratory lighting

EFFECT PARAMETERS MEASURED: Inhibition of the respiration rate was measured for a 10 minutes period after 30 minutes and 3 hours contact time.

TEST CONCENTRATIONS
- Range finding study: No

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: The study was carried out as a "limit test" at a single test concentration of 1000 mg/L using 3 replicate test vessels. Values of between 31 - 33% Inhibition were obtained after 3 hours contact time.

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none
After 3 hours the EC50 was determined to be > 1000 mg/L

Results with reference substance (positive control):

- Results with reference substance valid? yes
The EC50 of the reference substance was determined to be 14 mg/L after 30 minutes and 9.4 mg/L after 3 hous.

R1-R3: Replicates 1-3

Method: OECD Guideline No.209 and EEC Commission Directive 87/302/EEC

 

Contact time: 30 min and 3 h

 

Temperature: 22 °C

 

Results:

EC₅₀(30 min) : >1000 mg/L

EC₅₀(3 h) : > 1000 mg/L

Results:

The rate of respiration for each culture was measured for a 10-minute period
(between approximately 7.9 mg O2/L and 0.5 mg O2/L) after 30 minutes and 3 hours contact time.

 

 

30 minutes contact:

Concentration (mg/L)	O2consumption rates (mg O2/L/min)	%Inhibition
Control R1	0.54	-
Control R2	0.52	-
Test 1000 R1	0.47	11
Test 1000 R2	0.46	14
Test 1000 R3	0.47	11
Reference 3.2	0.53	0 (?)
Reference 10	0.40	25
Reference 32	0.12	77

 

 

3 hours contact:

Concentration (mg/L)	O2consumption rates (mg O2/L/min)	%Inhibition
Control R1	0.49	-
Control R2	0.50	-
Test 1000 R1	0.33	33
Test 1000 R2	0.33	33
Test 1000 R3	0.34	31
Reference 3,2	0.42	15
Reference 10	0.22	56
Reference 32	0.08	84

 

R1-R3: Replicates 1-3

percentage inhibition values as calculated from the equation:

%Inhibition = %Inhibition =[1 -2RS/(RC1 +RC2]x100

Where RS = Oxygen consumption rate for test or reference samples.
Rc1 + Rc2 - Oxygen consumption rates for Controls 1 and 2.

 

Validity criteria fulfilled:

yes

Conclusions:

After 3 hours the EC50 was determined to be > 1000 mg/L.
The "limit test" at a single test concentration was carried out as opposed to a full EC50 determination over a range of test concentrations as the results of the preliminary range-finding studies indicates that testing at concentrations in excess of 1000 mg/L would not result in percentage inhibition values in excess of 50% and so enable determination of an EC50 value.
It is considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L and so, therefore it was considered justifiable to carry out a "limit test" at 1000 mg/L and to state the percentage inhibition, EC50, values being >1000 mg/L

Executive summary:

The "limit test" at a single test concentration was carried out as opposed to a full EC50 determination over a range of test concentrations as the results of the preliminary range-finding studies indicates that testing at concentrations in excess of 1000 mg/L would not result in percentage inhibition values in excess of 50% and so enable determination of an EC50 value.

It is considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L and so, therefore it was considered justifiable to carry out a "limit test" at 1000 mg/L and to state the percentage inhibition, EC50, values being >1000 mg/L

 

Temperature: 22 °C

Method of Preparation: Direct dispersion in water

 

Reference Material: 3,5-dichlorophenol

Inoculum:

Type:A nixed population of activated sewage sludge micro-organisms.

Source: The aeration stage of the Severn Trent Pic sewage treatment plant at Belper, Derbyshire,
treating predominantly domestic sewage.

Results Synopsis:

 

Percentage inhibition for the reference material, 3,5-dlchlorophenol, is plotted against concentration and the EC₅₀values derived by inspection for 30 minutes and 3 hours contact time.

 

Kayarus Supra Rubine BLN EC₅₀(30 minutes): >1000 mg/L
3,5 dlchlorophenolEC₅₀(30 minutes) : 14 mg/L

 

Variation in respiration rates of Controls 1 and 2: ± 2%

 

Kayarus Supra Rubine BLN EC₅₀(3 hours): >1000 mg/L
3,5 dichlorophenol EC₅₀(3 hours) : 9.4 mg/L

Variation In respiration rates of Controls 1 and 2: ± 1%

 

 

Description of key information

A test on toxicity to microorganisms was performed according to OECD 209. After 3 hours the EC50 was determined to be > 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L