Black HM 2482

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 10th to November 12th, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

1984

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EU Commission Directive 88/302/EEC, 1988

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

400-920-6

EC Name:

-

Cas Number:

89857-06-7

Molecular formula:

C50 H53 N11 O14 S

IUPAC Name:

5'-[2-(7-{2-[4-(2-{1'-[3-(dimethylazaniumyl)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-8-hydroxy-6-sulfonatonaphthalen-2-yl)diazen-1-yl]-6'-hydroxy-3,4'-dimethyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium bis(2-hydroxypropanoate)

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

For measurement of the respiration rate, a well-mixed sample of each test medium was poured into a BOD-flask after exactly three hours incubation time, and was not further aerated.

Test solutions

Vehicle:

no

Details on test solutions:

The test item is soluble at concentrations above 5 g/L. Therefore, a stock solution of test item with
a nominal concentration of 5.0 g/L was prepared by dissolving 1.00 g of test item completely in 200 mL of tap water by stirring for 10 minutes at room temperature. Adequate volumes of the stock solution were added to tap water to prepare the test media with the different test item concentrations.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: aerobic activated sludge from a wastewater treatment plant (ARA Ergolz ll, Füllinsdorf, Switzerland) treating predominantly domestic wastewater
- Preparation of inoculum for exposure: 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. 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. 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 6.8.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

20 °C at the start
19 °C at the end of the incubation period

Dissolved oxygen:

2.5 mg/l during the incubation period
7.9 mg/l just before the measurements of the respiration rates

Nominal and measured concentrations:

Test substance: 10, 32, 100, 320, and 1000 mg/l (nominal)
Reference substance: 5, 6 and 50 mg/l (nominal)

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 with compressed air at a flow of approximately one liter per minute.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): The sludge concentration of the inoculum was 3.2 g dry weight per liter (corresponding to about 1.3 g dry material per liter test medium in the test flasks).
- Nutrients provided for bacteria: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 x 2H2O, 0.2 g MgSO4 x 7H2O, 2.8 g K2HPO4.
- Biomass loading rate:


EFFECT PARAMETERS MEASURED
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. The water temperature was measured in one control at the start and end of the incubation period, and was continuously monitored in all test media and the controls during measurement of the respiration rate. Before the addition of activated sludge and synthetic wastewater, the appearance of the test media was recorded.


EVALUATIONS
The dissolved oxygen concentration was measured with an oxygen electrode (WTW TriOxmatic 300 and an oxygen meter WTW Oxi 539, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim/Germany), and was continuously recorded for about ten to fifteen minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2 L-1 minute-1) was determined from the linear part of the respiration curve in the range of 6.5 - 2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption, the rate was determined over a period of at least 10 minutes.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol 5, 16 and 50 mg/l (nominal)

Results and discussion

Effect concentrations

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

Details on results:

The oxygen consumption rates of the two controls (run at the start or at the end of the test) differed only by 0.3% (guideline-recommended maximum variation: 15%).

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 11 mg/L (the 95%-confidence limits were not calculable). The 3-hour EC50 is within the guideline recommended range of 5-30 mg/L, confirming the suitability of the activated sludge used.

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

EC50 (3h) > 1000 mg/l

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 88/3O2/EEC, Part C.11, and the OECD Guideline, No. 209.
The following nominal concentrations of test item were tested: 10, 32, 100, 320, and 1000 mg/L. Concentrations above 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 the suitability of the activated sludge and the method used.
Up to and including the 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 (EC15) of test item to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher but concentrations above 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.