Benzenesulfonic acid, 2-[2-[5-(aminocarbonyl)-1-ethyl-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridinyl]diazenyl]-4-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-, sodium salt (1:2)

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

14 January 2002 to 17 January 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to EU & OECD test guidance in compliance with GLP. This study is read across to the structural analogue Reaktiv-Gelb F-97494, which is considered to be structurally equivalent to the substance to be registered. Details of structure are detailed below under "illustrations"

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

The study was conducted according to the Commission Directive 88/302/EEC; Official Journal of the EC L 133 Part C (1988). This test method is in most parts identical with OECD Guideline 209.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

See below

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: > 300 deg C
- Boiling point: Not measured
- Vapour pressure: < 10E-05 hPa
- Water solubility (under test conditions): 108 g/l
- Solubility in organic solvents: Not measured.
- log Pow: < -4.9
- Adsorption characteristics:
- ready biodegradation: 8% after 28 days

OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: 8% after 28-days in a manometric test
:

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

No sampling was conducted.
Test concentrations (mg/l): 100, 1000, 10000
* Test substance concentration in physico-chemical oxygen consumption control (mg/l): 10000
Type of application: direct weighing

Test solutions

Vehicle:

no

Details on test solutions:

Two controls without test substance are included in the test design, one at the start and the other at the end of the test series. Each batch of activated sludge is checked using a reference substance.

Concentration reference substance (3.5-DichIorophenol mg/l): 5, 10, 20

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Type: mixed population of aquatic microorganisms (activated sludge)
Origin: aeration tank of a waste water treatment plant treating predominantly domestic sewage (Wupper area water authority)
Pretreatment: aeration of the activated sludge; daily feed with synthetic medium
pH of the suspension before application: 7.3

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

No post exposure observation period

Test conditions

Hardness:

No data

Test temperature:

19.0 - 19.3 C

pH:

8.2 - 8.4

Dissolved oxygen:

5.3 - 6.3 mg/l - start
2.7 - 3.6 mg/l - end

Salinity:

Not applicable.

Nominal and measured concentrations:

Test concentrations (mg/l): 100, 1000, 10000
* Test substance concentration in physico-chemical oxygen consumption control (mg/l): 10000

Details on test conditions:

Test concentration of the activated sludge : 520 mg/l suspended solids
Test temperature: 20 ± 2° C
Incubation time: 3 hours with permanent aeration

The physico-chemical oxygen consumption control is carried out, since some substances can also consume oxygen by chemical reactivity. In order to be able to tell the difference between physico-chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test substance is tested without activated sludge.

Reference substance (positive control):

yes

Remarks:

3.5-DichIorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 10 000 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Because of strong respiration of the activated sludge only 0 .52 g/l ss were used.
The test substance has been added to about 130 ml deionised water and stirred overnight before testing (equilibration phase).
Test concentrations are given as nominal concentrations and have not been confirmed by analytical methods.

The physico-chemical oxygen consumption is determined at 10000 mg/l lest substance concentration. No physico-chemical oxygen consumption has been determined. Therefore lower concentrations of the test substance cause no physico-chemical oxygen consumption (deduced values).

Results with reference substance (positive control):

The reference substance concentration result from the quantities used in the preparations and have not been confirmed by analytical methods.
EC50 of the reference substance 3,5-dichlorophenol is in the range 5-30 mg/l

Reported statistics and error estimates:

All validity criteria of the test method were met: -
respiratory rate of the 2 controls differs less than 15 %
respiratory rate of the controls < 60 mg 02 /I . h

Any other information on results incl. tables

Measured values of test substance / control / reference substance

	Test concentration [mg/l]	O2start   [mg O2/l]	O2end   [mg O2/l]	Exposure time  [min.]	Temp.   [⁰C]	pH
Test substance	100	5.7	2.7	5	19.2	8.4
	1000	6.2	3.6	4	19.1	8.3
	10000	5.3	2.9	4	19.1	8.2
Control 1		6.3	3.1	5	19.0	8.4
Control 2		6.2	2.9	5	19.3	8.3
Physico-chemical oxygen consumption control	10000	8.8	8.8	9	19.2	7.3
Reference substance 3.5 -Dichlorophenol	5	6.4	2.7	7	19.1	8.4
	10	6.5	3.3	7	19.1	8.4
	20	7.8	6.4	8	19.2	8.4

 

Test substance: Reaktivgelb F97-494

 

Test concentration   [mg/l]	Respiratory rate test substance   [mg/l . h]	Phys-chem. O2consumption   [mg/l . h]	Respiratory rate - phys.-chem. O2consumption [mg/l . h]	Inhibition     [%]
100	36.0	0.0*	36.0	7.7
1000	39.0	0.0*	39.0	0.0
10000	36.0	0.0	36.0	7.7
Control, mean	39.0
Control 1	38.4
Control 2	39.6

* The physico-chemical oxygen consumption is determined at 10000mg/l test substance concentration. No physico-chemical oxygen consumption has been determined. Therefore lower concentrations of the test substance cause no physico-chemical oxygen consumption (deduced values).

 

Reference substance: 3.5-Dichlorophenol

 

Test concentration  [mg/l]	Respiratory rate  [mg/l . h]	Inhibition  [%]
5	31.7	18.7
10	27.4	29.7
20	10.5	73.1

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Reaktivgelb F97-494 showed 7.7 % respiration inhibition of activated sludge at a test substance concentration of 10000 mg/l.

EC50> 10000 mg/l

Executive summary:

This study is read across to the structural analogue Reaktiv-Gelb F97-494., which is considered to be structurally equivalent to the substance to be registered

A study was performed to assess the toxicity of Reaktivgelb F97-494 to bacteria.

The study was conducted in accordance with the Commission Directive 88/302/EEC; Official Journal of the EC L133 Part C (1988). Study performed in compliance with the Principles of Good Laboratory Practice (GLP).

 

The activated sludge was exposed to Reaktivgelb F97-494 at different concentrations. The respiration rate of each mixture was determined after aeration periods of 3 hours.

 

Test substance, by analogue of read across showed 7.7 % respiration inhibition of activated sludge at a test substance concentration of 10000 mg/l.

 

EC50>10000mg/l

 

The substance is not considered to be harmful to sludge micro-organisms.