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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:
20 Sep 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
April 1984
Deviations:
yes
Remarks:
Settled sludge was used
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- 500.0 mg test substance were mixed and made up to 1000 mL with water (stock solution for test concentration 100 mg/L).
- 160 mL of the stock solution were mixed and made up to 500 mL with water (dilution 160 mg/L for test concentration 32 mg/L).
- 50 mL of the stock solution were mixed and made up to 500 mL with water (dilution 50 mg/L for test concentration 10 mg/L).
- 50 mL of dilution 160 mg/L were mixed and made up to 500 mL with water (dilution 16 mg/L for test concentration 3.2 mg/L).
- 50 mL of dilution 50 mg/L were mixed and made up to 500 mL with water (dilution 5 mg/L for test concentration 1 mg/L).
The test solution was freshly prepared at the start of the test.
Test organisms (species):
activated sludge
Details on inoculum:
- Source: The sludge was collected from a sewage treatment plant
- pH: The pH after collection was 8.0. The pH of the sludge before use was 8.4.
- Preparation of inoculum for exposure: The preparation was carried out according to the method described in the guideline. The sludge was separated from the aqueous layer by settling instead of centrifugation.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
18 - 22 °C
pH:
8.4 - 8.6 (after 3h)
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 1.0, 3.2, 10.0, 32.0 and 100.0 mg/L
- Measured concentrations: no analysis performed (or required)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL BOD flasks with gas inlet
- Aeration: Yes
- No. of vessels per concentration: 1
- No. of vessels per control: 2
- No. of vessels per positive control: 1
- Sludge concentration (weight of dry solids per volume): 1.46 g/L
- Nutrients provided for bacteria: Yes, see 'Nutrient solution' below
- Biomass loading rate: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Dechlorinated drinking water
- Nutrient solution: 16 g Peptone, 11 g Meat extract, 3.0 g Urea, 0.7 g NaCl, 0.4 g CaCl.2H2O, 0.2 g MgSO4.7H2O and 2.8 g K2HPO4 were dissolved and the volume nade up to one liter with dechlorinated drinking water.

OTHER TEST CONDITIONS
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED: Oxygen consumption/h
Oxygen consumption per hour in mg/liter was measured with an ORION - Electrode Type 97-08,ORION Microprocessor Ionalizer 901 and plotted on a recorder. The inhibitory effect of the test substance is expressed as a percentage of the mean respiration rates of two controls.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: not reported, but determined by the assessor.
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Whilst no significant inhibition is observed in the 1, 3.2, 10 and 100 mg/L test vessels, inhibitory effects are observed in the 50 mg/L test vessel. However, as this does not follow a dose-response relationship, this result is not considered relevant. An overview of the results is provided in 'Any other information on results incl. tables'.
- The negative inhibition observed in the 10 and 100 mg/L vessels are a consequence of the comparison of oxygen consumption values obtained in the blank, and the higher oxygen consumption values obtained in the presence of the test substance, which are due to biological variations between the various test samples.
- Although no NOEC was determined in the study report, based on the complete abscence of inhibition of respiration at the highest concentration, it is determined at ≥100 mg/L (by the assessor).
Results with reference substance (positive control):
The 3-h EC50 of the reference substance was determined to be 15.8 mg/L.
Reported statistics and error estimates:
The inhibitory values were calculated on the basis of the measured time depend oxygen consumption of a Blank and Test solution. The Results (ECxx Values) were determined after calculation of the linear regression.

Table: Consumption rate and inhibitory effects of the test substance and reference

Sample

Conc. [mg/L]

Consumption rate [mg/L/h]

Inhibition [%]

pH

Blank 1

0.0

70.6

-1

8.4

Blank 2

0.0

69.2

1

8.6

Reference 1

32.0

15.6

78

8.5

Reference 2

10.0

53.3

24

8.5

Reference 3

3.2

64.9

7

8.6

Test conc. 1

100.0

77.8

-11

8.5

Test conc. 2

32.0

50.0

28

8.6

Test conc. 3

10.0

72.7

-4

8.6

Test conc. 4

3.2

64.9

7

8.6

Test conc. 5

1.0

69.2

1

8.6

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'.
Conclusions:
The 3-h EC50 and EC20 values are >100 mg/L for microorganisms present in activated sludge from a sewage treatment plant. The NOEC is determined to be ≥100 mg/L.
Executive summary:

The toxicity of the substance to microorganisms was investigated in an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria. In this study non-adapted activated sludge from a sewage was exposed to nominal test substance concentrations of 0 (control), 1.0, 3.2, 10.0, 32.0, and 100 mg/L for 3 hours. Nominal test concentrations were not analytically verified. The effects on microorganisms was determined by measuring total respiration rates and comparing these to an unexposed control. Whilst no significant inhibition is observed in the 1, 3.2, 10 and 100 mg/L test vessels (-11 to 7 % inhibition), inhibitory effects are observed in the 50 mg/L test vessel (28% inhibition). The negative inhibition is a consequence of the comparison of oxygen consumption values obtained in the blank, and the higher oxygen consumption values obtained in the presence of the test substance, which are due to biological variations between the various test samples. Based on these findings,3-h EC50 and 3-h EC20 values of >100 mg/L were derived. Although no NOEC was derived in the study report, the absence of inhibition of respiration in the highest tested concentration of 100 mg/L justifies the NOEC to be determined at ≥100 mg/L.

Description of key information

No quantitative acute effect value (EC50) can be determined as no ≥50% effect was reached.

The NOEC is ≥100 mg/L in microorganisms in STP sludge, OECD 209, Grade 1996

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

Table: Overview of available data on the toxicity to microorganisms:

Species

Guideline / GLP

Endpoint

Effect value

Comment

Reference

Activated sludge, domestic

OECD 209 / GLP

3-h EC50

3-h EC20

3-h NOEC

> 100 mg/L

> 100 mg/L

≥ 100 mg/L*

Negative inhibition observed. No test substance analysis performed (or required)

Grade, 1996

* No NOEC is given in the study report. It is determined based on the complete absence of inhibition in to the highest tested concentration.