Tetrasodium 2,2''-diamino-8,8''-dihydroxy-4',4'''-{4-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,6-diyldiimino}bis(naphthalene-1-azobenzene-2',6-disulfonate)

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 22nd to 24th, 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Red LF 6339

IUPAC Name:

Red LF 6339

Sampling and analysis

Analytical monitoring:

no

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: ARA Ergolz II, Fullinsdorf, Switzerland
- Pretreatment: the sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenised aliquot of the final sludge suspension was weighed thereafter dried and the ratio of wet to dry weight was calculated. Based on ths ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding of two days prior to use, the sludge was fed daily with 50 ml synthetic wastewater per liter and was kept under continuous aeration until use at RT. Immediately 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 7.9 to 7.1 with a diluted sulfuric acid solution.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

22 °C (start)
19 °C (end)

Dissolved oxygen:

did not drop below 2.5 mg/l during the incubation period
just before the measurement of the respiration rates the dissolved oxugen was at least 8.3 mg/l

Nominal and measured concentrations:

10, 32, 100, 320 and 1000 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 ml glass flasks
- Material, size, headspace, fill volume: 200 ml activate sludge inoculum was added. the sludge was added in the intervals of 15 minutes first to a control, then to test solution of the reference item then to the test solutions of the test item and finally to a second control.
- Aeration: continuously aerated with compressed air at a flow appr. one liter per minute.
- No. of vessels per concentration (replicates): one
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 2.8 g/l dry weight corresponding to 1.1 g dry material per litre test medium
- Nitrification inhibitor used (delete if not applicable): none / N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:synthetic water as per the test guidelines
- Intervals of water quality measuremets: the pH values and the dissolved oxygen were determined at the start and at the end of the incubation The water temperature was measured at the start and at the end of the incubation in one control, and was continuously monitored in all test media and the controls during measurement of the respiration rates. Before the addition of the activated sludge, the appearance of the test media was recorded.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : a well-mixed sample of each test medium was poured into a BOD-flask after exactly 3 hours incubation time, and was not further aerated. The dissolved oxygen concentration was measured with an oxygen electrode and was continuouslly recorded for about ten to fifteen minutes. During measurement, the samples were stirred on a magnetic stirrer. The oxugen consumption rate was determined from the linear part of the respiration curve in the range of 6.2-2.5 mg/l oxygen/l.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:3.2
- Reference substance: 5, 16 and 50 mg/l

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:

growth inhibition

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:

growth inhibition

Details on results:

The test item had no significant inhibitory effect (< 15 %) on the respiration rate.
The oxygen consumption rates of the two controls differend only by 0.2 %

Results with reference substance (positive control):

EC50 (3h) = 11.5 mg/l (95 % CL: 10.7-12.4 mg/l)

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

EC50 (3h) > 1000 mg/l (nominal)
EC20 (3h) > 1000 mg/l (nominal)
NOEC (3h) = 1000 mg/l (nominal)

Executive summary:

The inhibitory effect of the test item 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 were tested: 10, 32, 100, 320 and 1000 mg/l. Concentrations exceeding 1000 mg/l were not tested. In 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 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 the test item 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.