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Toxicity to microorganisms

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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 27 APR 2005 to 28 APR 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guideline and compliant to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Reference substance copper (II) sulphate pentahydrate instead of the prescribed 3,5-dichlorophenol. Test performed as limit test (one treatment replicate)
GLP compliance:
yes (incl. certificate)
Remarks:
GLP according to German Chemikaliengesetzt and Directive 88/320/EEC

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
Test concentrations 1000 mg/L
Pretreatment The test item was weighed out directly into an Erlenmeyer flask, filled with 250 mL autoclaved tap water and stirred for 24 h.
Dispersion treatment Agitation
CONTROL 2 control samples without test item were included, one at the beginning and one at the end of the test.

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
Non adapted activated sludge from the municipal sewage treatment plant of D-31157 Sarstedt
The sludge was washed twice with autoclaved tap water and diluted corresponding to a dry weight of 4.0 g/L ± 10 %.
Date of receipt: 2005-04-27
Dry sludge concentration: 4.12 g/L

Study design

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

Test conditions

Test temperature:
21 °C
pH:
6.95 for the activated sludge
Nominal and measured concentrations:
Treatment: 1000 mg/L nominal concentration
Control (1 and 2): 0 mg/L
Details on test conditions:
REFERENCE ITEM Copper (II) sulphate pentahydrate p.a. MERCK
CAS RN 7758-99-8
Lot A265990
Purity 99.9 %
Expiry date 2005-11-30
Test concentrations 58 - 100 - 180 mg/L
Range of validity (EC50) 53 - 155 mg/L

TEST METHOD Static
Test duration 3 h
Replicates One per concentration and two per control
Test vessels 500 mL Erlenmeyer flasks, DIN 12380
Volume of the study medium 500 mL

Nutrient solution Synthetic waste water according to OECD 209

Application:
200 mL inoculum
16 mL synthetic waste water
284 mL autoclaved tap water
Test item concentration (directly weighing)

Ventilation Permanent

The pH-value of the activated sludge was measured prior to testing and the temperature in the measuring cell once during the measurement.
The oxygen depletion of the control, reference and test item was measured after 3 h incubation by means of oximeter and recorded for at least 3 minutes on a flat bed recorder.
Room temperature: continuously recorded by a hgrothermograph

A range-finding test (NON-GLP-state) was carried out with three concentrations: 10 - 100 - 1000 mg/L.
Based on the results of the preliminary test the study was carried out with a limit test concentration of 1000 mg/L.
2 control replicates without test item were included.
A fresh sample of activated sludge taken from the sewage plant Sarstedt was used for the test procedure. It was washed twice and diluted with autoclaved tap water to a dry sludge concentration of 4.0 g/L +/- 10 %.
The reference item was tested in three concentrations within the concentration range 58 - 180 mg/L with a dilution factor of 1.8. The respiration rates of all samples were determined subsequently to an incubation period of 3 h.
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate, CAS RN 7758-99-8

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
Remarks:
respiration rate
Details on results:
After stirring for 24 h the test item was mostly dispersed. Only part of the test item stuck to the side of the Erlenmeyer flask.
For details see "Any other information on results incl. tables"!
Results with reference substance (positive control):
In the reference test the EC50 -value for copper(II)-sulphate-pentahydrate was determined by probit analysis with:
EC50 = 107 mg/L
This is in the recommended confidence range of 53 - 155 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. Probit values were taken from WEBER (1986). Confidence intervals were calculated according to a standard procedure (BREITIG & TÜMPLING 1982).

Any other information on results incl. tables

Table 1: Respiration Rates and Inhibition of Respiration of Test and Reference Item Concentrations:

 

Concentration
[mg/L]

Respiration rate
[mgO
2/L x h]

Inhibition
[%]

Test item

1000

30.8

5

Control 1

-

33.2

-

Control 2

-

31.6

-

Reference item

180

8.4

74

 

100

16.8

48

 

58

25.6

21

Difference of the control respiration rates: 5 %

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a respiration inhibition test with acitvated municipial sludge (OECD 209) the following effect concentration was determined for the submission substance:
EC50 (3h) > 1000 mg/L (nominal concentration)
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was performed with the test item. 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 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 was observed at the tested limit 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 107 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.