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Toxicity to microorganisms

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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Started on 27th April 1993 and completed on 27th April 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: The aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, treating predominantly domestic sewage.
- Preparation of inoculum for exposure: The sample was maintained at 21ºC with continuous aeration and used on the day of collection. The pH was equal to 6.7 and the suspended solids equal to 3.7 g/l prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
Approximately 50 mg/L as CaCO3
Test temperature:
21 ºC
Nominal and measured concentrations:
Nominal concentration: 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 ml conical flasks
- Aeration: Compressed air via narrow bore glass tubes.
- No. of vessels per concentration (replicates): Three replicates
- No. of vessels per control (replicates): Two replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water, dechlorinated by sodium thiosulphate and aged by recirculation through a gravel filter.

OTHER TEST CONDITIONS
- Photoperiod: Normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : As each vessel reached 30 minutes contact time the contents were poured into the measuring vessel and the rate of respiration measured. The procedure was repeated after a 3-hour contact period.

Reference substance (positive control):
yes
Remarks:
(3,5-dichlorophenol at 3.2, 10 and 32 mg/L)
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
Variation in respiration rates of controls 1 and 2: ±2%
Results with reference substance (positive control):
- Results with reference substance valid
- Relevant effect levels:
EC50 (30 minutes) = 16 mg/L
EC50 (3 hours) = 9 mg/L

Table 1: Oxygen consumption rates and percentage inhibition for 3 hour contact time

 

Concentration

(mg/L)

O2 consumption rates

(mg O2/L/min)

% inhibition

Control R1

0.67

-

Control R2

0.65

-

Test substance 1000 mg/L R1

0.63

5

Test substance 1000 mg/L R2

0.61

8

Test substance 1000 mg/L R3

0.61

8

Reference substance 3.2 mg/L

0.57

14

Reference substance 10 mg/L

0.28

58

Reference substance 32 mg/L

0.11

83

 

Validity criteria fulfilled:
yes
Remarks:
(the two control respiration rates are within 15% of each other; the results for the referene substance are in the accepted range).
Conclusions:
The EC50 (3-h) was greater than 1000 mg/L.
Executive summary:

This study describes the methods used and results obtained during a study to assess the inhibitory effect of the test substance on the respiration of activated sewage sludge. The method followed the OECD Guideline 209 and EU method C.11. At time "0" 16 ml of synthetic sewage was made up to 300 ml with water in a 500 ml conical flask. 200 ml inoculum were added and the mixture aerated at approximately 0.5-1 litre per rninute. Thereafter, at 15 minute intervals the procedure was repeated wit h appropriate amounts of the test or reference material being added. As each vessel reached 30 minutes contact time the contents were poured into the measuring vessel and the rate of respiration rneasured. The procedure was repeated after a 3-hour contact period. The EC50 (3-h) for the test material was greater than 1000 mg/L.

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 16th April 1996 to 17th July 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Standard national method. GLP study
Qualifier:
according to
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Source: German collection of micro-organisms (D-38124 Braunschweigh, FRG)
- Laboratory culture:
- Method of cultivation:
- Preparation of inoculum for exposure:
- Pretreatment:
- Initial biomass concentration:
Test type:
static
Total exposure duration:
16 h
Test temperature:
21 ± 1 ºC
Nominal and measured concentrations:
Nominal concentrations:
Experiment 1: 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0 and 8.0 g/L
Experiment 2: 4.2, 8.4, 16.9, 33.8, 67.5, 135.0, 270.0, 540.0 and 864.0 g/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 ml Erlenmeyer
- No. of vessels per concentration (replicates): Three replicates

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell density: The absorption of the bacterial suspension was determined at 436 nm.
Reference substance (positive control):
no
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
50.6 g/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Conclusions:
An inhibition of 50 % (EC 50) was reached at a concentration of 50.6 g/L. In conclusion it can be stated that the test article did not inhibit bacterial proliferation under the conditions described and within the limits of the test system used up to a concentration of 10 g/L. This concentration is recommended as the upper limit when testing potentially water hazardous materials. At higher concentrations a dose dependent inhibition of bacterial proliferation occurred.
Executive summary:

This study was performed to assess the potential of the test article to inhibit the reproduction of Pseudomonas putida. Two experiments were performed since no inhibition of bacterial growth was detected in the concentration range of the first experiment.

Each concentration was tested in triplicate and the controls fivefold. The test article was tested at the following concentrations:

Experiment 1: 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0 and 8.0 g/L

Experiment 2: 4.2, 8.4, 16.9, 33.8, 67.5, 135.0, 270.0, 540.0 and 864.0 g/L

Growth reduction of the bacteria was observed at concentrations above 10 g/L.

An inhibition of 50 % (EC 50) was reached at a concentration of 50.6 g/L.

An inhibition of 10 % (EC 10) was reached at a concentration of 17.0 g/L.

In conclusion it can be stated that the test article did not inhibit bacterial proliferation under the conditions described and within the limits of the test system used up to a concentration of 10 g/L. This concentration is recommended as the upper limit when testing potentially water hazardous materials. At higher concentrations a dose dependent inhibition of bacterial proliferation occurred.

Description of key information

Key study: Experimental results: OECD guideline 209 and EU method C.11. GLP study.
The EC50 (3-h) was greater than 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

Key study: Experimental results: OECD guideline 209 and EU method C.11. GLP study.

The EC50 (3-h) was greater than 1000 mg/L.