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Diss Factsheets

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 10, 2002- October 7, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
1988
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
-
EC Number:
444-050-5
EC Name:
-
IUPAC Name:
dodecasodium 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate
Test material form:
solid
Details on test material:
Identity: Red Rwa 4565
Appearance: Solid, red powder
Storage: at room temperature at about 20°C

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
No data.

Test solutions

Vehicle:
no
Details on test solutions:
Please refer to "Details on test conditions" and "Any other information on materials and methods".

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf, Switzerland) treating predominantly domestic wastewater.
- Preparation of inoculum for exposure:
During the holding of two days prior to use, the sludge was fed daily with 50 mL synthetic wastewater* per liter and was kept at room temperature under continuous aeration until use. lmmediately before use, the dry weight of the activated sludge was measured again in the inoculum used in the test. The pH of the activated sludge inoculum was adjusted from 8.1 to 7.1 with a diluted sulfuric acid solution.
- Pretreatment:
The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter.

*Synthetic wastewater: [16 g peptone; 11 g meat extract; 3 g urea; 0.7 g NaCl; 0.4 g CaCl2 · 2H20; 0.2 g MgS04 · 7H20; 2.8 g K2HP04] filled up to a final volume of 1 liter with deionized water.

Study design

Test type:
static
Water media type:
other: Tap water and synthetic wastewater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
None
Post exposure observation period:
None

Test conditions

Hardness:
Not specified.
Test temperature:
The water temperature was measured in one control at the start and at the end of the incubation period, and was continuously monitored in all test media and the controls during measurement of the respiration rate.
pH:
The pH values and the dissolved oxygen concentrations were determined in all test media and the controls at the start and at the end of the 3-hour incubation period.
Dissolved oxygen:
During the incubation period of exactly 3 hours all test media and the controls were continuously aerated by intense stirring on magnetic stirrers. The concentration of dissolved oxygen did not drop below 2.5 mglL during the incubation period, and just before measurement of the respiration rates the dissolved oxygen concentrations were at least 6.9 mg/L.
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal concentrations: 10, 32, 100, 320 and 1000 mg/L.Concentrations exceeding 1000 mg/L were not tested.

According to the results of a pre-test (non-GLP), no stock solution could be prepared due to the insufficient water solubility of Red Rwa 4565.
Therefore, test item amounts of 5.0, 16.1, 51.0, 160.2 and 500.2 mg were directly weighed (by means of an analytical balance) into the test flasks. Then 284 mL of tap water was added to each flask. The test item was mixed into the tap water by ultrasonic treatment for ten minutes and intense stirring Íor 24 hours at roomtemperature in the dark to dissolve a maximum amount of the test item ancl/or disperse it as homogeneously as possible. Then,16 mL of synthetic wastewater and 200 mL of the activated sludge inoculum were added.

Additionally, two controls (tap water, synthetic wastewater and activated sludge inoculum, without test item) and the reference item 3,S-dichlorophenol (positive control) at nominal concentrations of 5, 16 and 50 mg/L were tested in parallel under otherwise identical test conditions.
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL glass flasks
- Aeration: During the incubation period of exactly 3 hours all test media and the controls were continuously aerated by intense stirring on magnetic stirrers. The concentration of dissolved oxygen did not drop below 2.5 mglL during the incubation period, and just before measurement of the respiration rates the dissolved oxygen concentrations were at least 6.9 mg/L
- Sludge concentration (weight of dry solids per volume): At the start of the test 200 ml activated sludge inoculum with a sludge concentration of 2.3 g dry weight/l (corresponding to about 0.9 g dry material per liter test medium) was added. The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, then to the test solutions of the reference item, then to the test solutions of the test item, and finally to the second control.

TEST CONCENTRATIONS
- Test concentrations: 10, 32, 100, 320 & 1000 mg/L

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total 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 total respiration
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The test item Red Rwa 4565 had no significant inhibitory etfect (<15%) on the respiration rate of activated sludge after the incubation period of three hours up to and including the highest test item concentration of 1000 mg/L. Thus, the 3-hour NOEC of Red Rwa 4565 to activated sludge microorganisms was at least 1000 mg/L. This value might be even higher, but concentrations exceeding 1000 mg/L were not tested. The 3-hour 8C20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/L.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,S-dichlorophenol (positive control) was calculated to be 13mg/L (the 95% confidence limits were not calculable). The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/L, confirming suitability of the activated sludge used.
Reported statistics and error estimates:
No statistical analysis was performed.

Any other information on results incl. tables

Influence of 3.5 -dichlorophenol and Red Rwa 4565 on oxygen consumption of activated sludge

 

Flask

Test chemical

Nominal concentration of test chemical (mg/L)

Oxygen consumption rate

(mg O2/L min-1)

Inhibition

(%)

pH values

Oxygen concentration (mg O2/L)

 

Start*

End*

Start*

End*

 

1

control

0

1.187

 

7.8

8.1

8.3

8.6

 

10

control

0

1.187

 

7.7

7.8

8.5

6.9

 

Mean deviation (%)

1.237

8.4

 

 

 

 

 

2

3,5-dichlorophenol

5

1.113

10.0

7.8

8.1

8.5

8.7

 

3

3,5-dichlorophenol

16

0.388

68.6

7.8

8.2

8.6

8.6

 

4

3,5-dichlorophenol

50

0.113

90.8

7.7

8.3

8.6

8.6

 

5

Red Rwa 4565

10

1.254

-1.4

7.8

8.1

8.5

8.3

 

6

Red Rwa 4565

32

1.357

-9.7

7.8

8.0

8.6

8.2

 

7

Red Rwa 4565

100

1.249

-1.0

7.8

7.9

8.4

8.2

 

8

Red Rwa 4565

320

1.264

-2.2

7.8

7.9

8.6

8.4

 

9

Red Rwa 4565

1000

1.089

12.0

7.8

7.9

8.5

7.5

 

*Start and end of the 3-hour incubation period

- % inhibition: increased oxygen consumption rate relative to that of control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
When Red Rwa 4565 was assessed for its toxicity to waste water bacteria, the following result was obtained:
- the 3-hour NOEC of Red Rwa 4565 to activated sludge microorganisms was at least 1000 mg/L;
- the 3-hour EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/L.
Executive summary:

The inhibitory effect of the test item Red Rwa 4565 on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 87/302/EEC, and the OECD Guideline for Testing of Chemicals, No. 209.

 

The following nominal concentrations of Red Rwa 4565 were tested: 10, 32, 100, 320 and 1000 mg/L. Concentrations exceeding 1000 mg/L were not tested.

 

ln addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16 and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

 

Up to and including the highest concentration of 1000 mg/L the test item Red Rwa 4565 had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. Thus, the 3-hour NOEC of Red Rwa 4565 to activated sludge microorganisms was at least 1000 mg/L. This value might be even higher, but concentrations exceeding 1000 mg/L were not tested. The 3-hour EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/L.