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Ecotoxicological information

Toxicity to microorganisms

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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 May 2006 to 24 July 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD Guideline 209

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Copper (II) sulphate pentahydrate instead of the prescribed 3,5- dichlorophenol as a reference substance. The usage of dichlorophenol is unsuitable for routine lab use.
GLP compliance:
yes (incl. certificate)
Remarks:
GLP according to German "Chemikaliengesetz", EC and OECD Guideline principles

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was weighed out directly into an Erlenmeyer flask and filled up with 250 mL autoclaved tap water.
- Dispersion treatment: stirring for 24 h
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Before stirring the test item was insoluble, floated on the surface and stuck to the side of the flask. After stirring the test item was partly dispersed and floated on the surface and stuck to the side of the flask.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Pretreatment: The sludge was washed twice with autoclaved tap water and adjusted corresponding to a dry weight of 4.0 g/l ± 10 %.
- Dry sludge concentration 4.17 g/L

Non adapted activated sludge from the sewage plant at Sarstedt, Germany, is well suited as it comprises mostly municipal sewage and very
little industrial waste water.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Test temperature:
21.2 °C
pH:
6.79 (pH of the activated sludge)
Nominal and measured concentrations:
1000 mg/l nominal, no analytical measurements
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks, DIN 12380
- Fill volume: 500 mL
- Aeration: Permanent
- No. of vessels per concentration (replicates): one
- No. of vessels per control (replicates): two
- Biomass loading rate: 200 ml inoculum/500 ml

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 284 mL autoclaved tap water and 16 mL synthetic waste water according to OECD 209 per 500 mL flask

OTHER TEST CONDITIONS
The pH-value of the activated sludge was measured prior to testing and the temperature in the measuring cell once during the measurement.
The room temperature during the test was recorded continuously by a hygrothermograph.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen depletion measured after 3 h incubation and recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1 nominal limit concentration
Reference substance (positive control):
yes
Remarks:
copper (II) sulphate pentahydrate, p.a.

Results and discussion

Effect concentrations
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:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Before stirring the test item was insoluble, floated on the surface and stuck to the side of the flask. After stirring the test item was partly dispersed and floated on the surface and stuck to the side of the flask.
- Effect concentrations exceeding solubility of substance in test medium: see above

Results with controls: see Table 1 below
Results with reference substance (positive control):
- Results with reference substance valid? Yes (see below)
- Relevant effect levels: EC50 = 125 mg/l , i.e. within the recommended confidence range of 52-157 mg/l
Reported statistics and error estimates:
The EC50-value of the reference item was calculated with probit analysis using software SIGMAPLOT (Windows) rel. 8.02, 2002, SPSS CORPORATION

Any other information on results incl. tables

Table 1: Respiration rates and inhibition of respiration of test and reference item concentrations

 

Concentration

Respiration rate

Inhibition

 

[mg/L]

[mg 02/L x h]

[%]

 Test item

1000

40.0

-16

 Control 1

-

36.4

-

 Control 2

-

32.8

-

 Reference item

180

10.8

69

 

100

21.2

39

 

58

30.0

13

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The difference of the two control respiration rates in the study was 10 % and the EC50 of the reference substance was in the accepted range (see above).
Conclusions:
The test item is not toxic at concentrations ¿ 1000 mg/L to activated sludge of a municipal sewage treatment plant.
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was performed with the test item (C.I. Pigment Red 122) on 2006-06-09 at DR.U.NOACK-LABORATORIEN, D-31157 Sarstedt. Test system was activated sludge of the municipal treatment plant of D-31157 Sarstedt. The test was performed under static conditions with the nominal limit concentration of 1000 mg/L. The respiration rates of control, reference and test item concentrations were measured after a contact time of three hours, and the inhibitory effects of the test and reference concentrations were determined in comparison to the control respiration rates. No inhibition of the test item was observed at the tested concentration.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper-(II)-sulphate-pentahydrate as reference item and the reference toxicity was determined. The EC50- value for the reference item was 125 mg/L.

Validity criteria of the test guideline were fulfilled.

The test item is not toxic at concentrations ¿ 1000 mg/L to activated sludge of a municipal sewage treatment plant.