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Ecotoxicological information

Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2008 - June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: no melting point/range (Decomposition at temperatures > 200 °C)
- Boiling point: no boiling point (decomposition before boiling)
- Vapour pressure: <1 * 10-12 hPa at 25°C (calculation)
- Water solubility: 125.76 g/L at 20°C
- log Pow: <-2
- pKa: = 7,00+/-0,04
Analytical monitoring:
no
Details on sampling:
No analytical monitoring
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: a stock solution of an appropriate concentration in deionised water was prepared. The stock solution then was used to prepare the treatments.

First Test: Limit test, 1000 mg/L (nominal concentration)
Main Study: 5 concentrations ranging from 2000 to 125 mg/L

Controls: two replicates before and two replicates after measuring reference and test item
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
-PRE-TREATMENT:
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treamtent plant. It was collected on the same day as
the respective experiment was performed and aereated during transport. Upon arrival in the test facility, sludge was washed with tap water three
times, then centrifuged n re-suspended in tap water.
The nutrition was freshly prepared. The inocolum was taken from its source, wahsed, aerated and the dry matter was determined.

-CHEMICALS AND NUTRITIENT SOLUTION:
all chemicals used in the test were "analytical grade" or "for use in microbiology":
Peptone (8.0 g)
Meat Extract (5.5g)
Urea (1.5g)
NaCl (0.35g)
CaCl2 * 2 H20 (0.2 g)
MgSO4*7 H2o (0.1 g)
K2HPO4 (1.4g)
Deionised water (ad 500mL)


- INITIAL BIOMASS CONCENTRATION:
dry matter of sludge (mg suspended solids/litre), on February 13, 2008 : 4.3
dry matter in the test (mg suspended solids/litre): on February 13, 2008: 1.72

dry matter of sludge (mg suspended solids/litre), on February 15, 2008 : 3.8
dry matter in the test (mg suspended solids/litre): on February 15, 2008: 1.52
Test type:
other: aerobic
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
no data given in the test report
Hardness:
deionised water was used
Test temperature:
20 +/- 2°C
pH:
7.1 -7.6
Dissolved oxygen:
no data given in the test report
Salinity:
no data given in the test report
Nominal and measured concentrations:
First Test (limit test): 1000 mg/L nominal conc.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Aeration: purified air, using pasteur pipettes, flow approx. 0.75 L/min
- Feeding: nutrient solution, 16 mL/vessel

Reference substance (positive control):
yes
Remarks:
3,5-Dichlorphenole (CAS 591-35-5)
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
< 125 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
ca. 270 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 2 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Any observations: The test item showed inhibition values which were not correlated to its concentration. At Concentrations 2000 and 1000 mg/L, approx. the same inhibition was found. It is believed that the test system, consisting of a wide variety of microorganisms, is not uniformly affected by the test item.

Results with reference substance (positive control):
results considered valid
Reported statistics and error estimates:
none

In both studies, the inhibition which was caused by the test item did not rise above 30 %, which were observed in the treatments 2000 and 1000 mg/L. At the lowest concentration of 125 mg/L, 15.6 % inhibition were observed.

It might be possible that the test item shows inhibitory action only to part of the microorganisms which are present in the activated sludge, so that respiration of these microorganisms is nearly completely inhibited by approx. 500 mg/L test item concentration. The micro-organisms which are not susceptible to toxic effects of the test item are not inhibited at all up to the highest concentrations tested. Therefore, no exact 3h EC50 value and no exact NOEC value could be determined. The 3h EC50 value is given as > 2000 mg/L and the NOEC is stated as 125 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The following results for the test item were determined:
3h NOEC = below 125 mg/L
3h EC20 = 270 mg/L (95% confidence interval: 110 - 430 mg/L)
Executive summary:

Two valid experiments were performed:

First, a limit-test with two replicates using the nominal concentration 1000 mg/L was performed.

The treatment 1000 mg/L showed a mean inhibition of the respiration rate of the activated sludge of 31%.

Then, a main study was performed using five concentrations ranging from 2000 to 125 mg/L.

The following inhibitions of the respiration rate of the activated sludge were detected: At 2000 mg/L 29.5 %, at 1000 mg/L 30.0 %, at 500 mg/L 24.5 %, at 250 mg/L 19.5 % and at 125 mg/L 15.8% inhibition were detected.

The test item showed inhibition values which were not correlated to its concentration. At Concentrations 2000 and 1000 mg/L, approx. the same inhibition was found. It is believed that the test system, consisting of a wide variety of micro-organisms, is not uniformly affected by the test item.

The following results for the test itemwere determined:

3h NOEC = below 125 mg/L

3h EC20 = 270 mg/L (95% confidence interval: 110 - 430 mg/L)

3h EC50 > 2000 mg/L

Description of key information

Not acute toxic to bacteria.
3h NOEC < 125 mg/L
3h EC20 = 270 mg/L
3h EC50 > 2000 mg/L
EC10(16 h) = 2600 mg/L
EC50(16 h) = 7500 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
2 000 mg/L

Additional information

Two studies were determined.

In the key study two valid experiments were performed:

First, a limit-test with two replicates using the nominal concentration 1,000 mg/L

The treatment 1,000 mg/L showed a mean inhibition of the respiration rate of the activated sludge of 31%.

Then, a main study was performed using five concentrations ranging from 2,000 to 125 mg/L.

The following inhibitions of the respiration rate of the activated sludge were detected: At 2000 mg/L 29.5 %, at 1000 mg/L 30.0 %, at 500 mg/L 24.5 %, at 250 mg/L 19.5 % and at 125 mg/L 15.8% inhibition were detected.

The following results for the test item were determined:

3h NOEC = below 125 mg/L

3h EC20 = 270 mg/L (95% confidence interval: 110 - 430 mg/L)

3h EC50 > 2000 mg/L

In the supporting study the following values were determined:

EC10(16 h) = 2600 mg/L

EC50(16 h) = 7500 mg/L

The substance is not acute toxic for bacteria.