Salts of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthenium chloride and 4-{[1-hydroxy-6-({[5-hydroxy-6-(phenyldiazo)-7-sulfonaphthalen-2-yl]carbamoyl}amino)-3-sulfonaphthalen-2-yl]diazo}benzoic acid (2:1)

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-11-19

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

not applicable

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:

not required

Details on sampling:

not applicable

Test solutions

Vehicle:

no

Details on test solutions:

Before the start of the test defined amounts of the test item were directly weighed into the test flasks and mechanically dispersed using short ultrasonic bath treatment for 10 min. The test concentrations (10, 31, 100, 313 and 1000 mg/L) were chosen to permit the determination of the EC50. Concentrations in excess of nominal 1000 mg test item/L were not tested. Two controls (deionised water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and a ratio of wet sludge to its dry weight determined. Based on this ratio, calculated amounts of wet sludge were suspended in isotonic saline solution to yield a concentration equivalent to 4 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.14. The activated sludge was used directly after conditioning. The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

not applicable

Test conditions

Hardness:

not applicable

Test temperature:

19.7 – 20.6 C (during the incubation)
18.6 – 20.6 C (during oxygen measurement)

pH:

The pH of the activated sludge inoculum was determined to be pH 7.14

Dissolved oxygen:

The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 7.5 mg O2/L.

Salinity:

not applicable

Nominal and measured concentrations:

nominal: 10, 31, 100, 313 and 1000 mg/L
no concentrations measured

Details on test conditions:

aerobic incubation

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

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

Remarks:

respiration rate

Effect concentrations 2

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

Remarks:

respiration rate

Details on results:

The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were not inhibited and influenced dose dependently in the whole concentration range, and the observed slight inhibition (13.0 %) at the highest examined concentration level of 1000 mg/L was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.470 (at 1000 mg/L) was in the historical control data range (0.513 ± 0.079), and the deviation from the control was within ± 15 %, can be considered as a biological variability of the test system.

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-Dichlorophenol for the used activated sludge batch was determined to be 8.94 mg/L with 95 % confidence limits of 7.17 to 11.14 mg/L and thus the EC50 was in the acceptable the range of 5 to 30 mg/L.

Reported statistics and error estimates:

not applicable

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

EC50 (3h) > 1000 mg/l

Executive summary:

In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited in the concentration range of 10 to 1000 mg F 213 Red /L. The respiration rate was inhibited between 1.9 % (10 mg F 213 Red/ L) and 13.0 % (1000 mg F 213 Red/ L) in the examined nominal test concentration range. Concentrations exceeding 1000 mg/L nominal were not tested.

NOEC (3h) and EC50 (3h) > 1000 mg/l