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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:
1984-01-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Principles of method if other than guideline:
The purpose of this test method was to provide a rapid screening method whereby substances which may adversely affect aerobic microbial treatment plants can be identified, and to indicate suitable non-inhibitory concentrations of test items to be used in biodegradability tests.
Two controls without test item were included in the test design, one at the start and the other at the end of the test series. Each batch of activated sludge was checked using 3.5-Dichlorophenol as a reference compound.
The chemical oxygen consumption control was carried out, since some substances may consume oxygen by chemical reactivity. In order to be able to differentiate between chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test item was additionally tested without activated sludge.
To measure the oxygen consumption, 250 mL of sludge with test item (or control or reference compound) was incubated for 3 h in 300 mL closed Erlenmeyer flasks (with air inlet and outlet) and aerated through a glass tube at 50-100 L/h with clean oil-free air. For measurement, the content of the Erlenmeyer flasks was completely transferred to 250 mL BOD bottles and 02-content was measured with an 02-meter (redox electrode) with writer.
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
Test item concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
Test item concentration in chemical oxygen consumption control
Concentration of reference compound (3,5-Dichlorophenol): 1 and 100 mg/L
Vehicle:
no
Details on test solutions:
Before use the pH of the activated sludge was measured and adjusted to pH 6-8. 8 mL of the synthetic medium and 25 mL of activated sludge were added to the dissolved test item. The mixture was filled up to 250 mL with deionised water and aerated at 20 °C +/-2 °C.
The exposure medium with the reference substance was prepared by adding 8 mL of the sythetic medium, 25 mL of activated sludge and a defined amount of the stock solution to achieve the test concentrations and was filled up to 250 mL with deionised water.
Test organisms (species):
activated sludge of a predominantly industrial sewage
Details on inoculum:
Type : mixed population of aquatic microorganisms (activated sludge)
Origin: laboratory scale sewage treatment unit (3L), treating prdominantly industrial sewage. (Location: Bürrig / Germany)
Microbial inoculum:
- First, the sludge was settled and the supernatent was decanted.
- After centrifuging (20 min at 4000 rpm and 20 °C) the supernatent was decanted.
- Approx. 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 6 g/L (dry weight) suspended solids.
- The calculated amount of sludge was first dissolved in synthetic medium and than filled up to a defined end volume with deionised water.
Storage of sludge: Aeration of the activated sludge at 20 °C +/- 2°C daily fed with synthetic medium.
Synthetic medium: A synthetic waste water feed was made by dissolving the following amounts of substance in 1 litre of water:
16.0 g peptone
11.0 g meat extract
3.0 g urea
0.7 g NaCl
0.4 g CaCl2 x 2 H2O
0.2 g MgSO4 x 7 H2O
2.8 g K2HPO4
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
with permanent aeration
Post exposure observation period:
not reported
Hardness:
not reported
Test temperature:
20 °C +/- 2 °C
pH:
7.1 - 7.6
Dissolved oxygen:
not reported
Salinity:
not reported
Nominal and measured concentrations:
Nominal concentrations of test item: 6.25, 12.5, 25, 50, and 100 mg/L
Nominal concentration in chemical oxygen consumption control: 100 mg/L
Nominal concentration of reference compound (3,5-Dichlorophenol): 1 and 100 mg/L
Details on test conditions:
Test vessels: 300 mL glass Erlenmeyer flasks
Test concentration of the activated sludge: 0.38 g/L dry weight
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The substance showed no significant respiration inhibition of activated sludge at the highest test item concentration of 100 mg/L.
Results with reference substance (positive control):
The EC50 of 3,5-Dichlorophenol is in the range 5-30 mg/L.
Reported statistics and error estimates:
not reported

In the following table the respirationrate, the percentage of inhibition as well as the pH values at the different test concentrations were presented:

 

Test item concentration [mg/L]

Respirationrate

 

[mg/(L*min)]

Inhibition

 

%

pH

Test item

6.25

0.33

-

7.5

12.5

0.32

-

7.5

25

0.30

-

7.5

50

0.29

-

7.6

100

0.26

10.34

7.6

Control 1

 

0.25

 

7.5

Control 2

 

0.33

 

7.6

Chemical oxygen consumption control

100

0.07

 

7.1

Validity criteria fulfilled:
yes
Remarks:
(- respiratory rates of the 2 controls differ less than 15 % form each other; - EC50 of 3,5-Dichlorophenol is in the range 5-30 mg/L)
Conclusions:
The substance showed no significant respiration inhibition of activated sludge at the highest test item concentration of 100 mg/L.
Executive summary:

The toxicity of the substance to microorganisms was assessed in a study according to ISO 8192 "Test for Inhibition of Oxygen Consumption of Activated Sludge". This test method is in accordance with the OECD Guideline 209 (1983). The respiration rate of each mixture was determined after aeration periods of 3 hours. The substance showed no significant respiration inhibition of activated sludge at the highest concentration tested, and hence: IC50 > 100 mg/L (based on nominal concentrations).

Description of key information

The substance showed no significant respiration inhibition of activated sludge at the highest concentration tested, and hence the IC50 is > 100 mg/L (based on nominal concentrations). 

Key value for chemical safety assessment

Additional information

The toxicity of the substance to bacteria was assessed in a study according to ISO 8192 "Test for Inhibition of Oxygen Consumption of Activated Sludge". The substance showed no significant respiration inhibition of activated sludge at the highest concentration tested, and hence the IC50 is > 100 mg/L (based on nominal concentrations).

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