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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 23, 1998 - June 24, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Test material (Fosfomycin PEA salt): 100 ml of an aqueous Fosfomycin PEA salt stock solution at 500 mg/l were prepared and transferred to the suitable 500 ml beaker and kept gently stirred unter the start of the contact time. The concentration of use was 100 mg/l.
Reference substance (3,5-dichlorophenol): A stock solution at 500 mg/l was prepared. 0.05 g of standard were dissolved in 1 ml 1N NaOH, diluted to about 3 ml with bidistilled water. Excess NaOH was neutralized with 1N H2SO4 to the point of incipient precipitation. Thereafter the mixture was made up to 0.1 litres.
- Controls: Yes.
Test organisms (species):
other: Activated sludge from the RBM laboratory water treatment plant.
Details on inoculum:
- Preparation of inoculum for exposure: 2 L of wet sludge were suspended in water in order to obtain an activated sludge with a liquor/suspended solids level of 4 g/l was calculated. 2 L of wet sludge were suspended in water in order to obtain an activated sludge with a liquor/suspended solids level of 4 g/l. To that mixture 50 ml/l of synthetic sewage feed were added.
- Initial biomass concentration: 4 g/l (dwt)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
After 3 hours, sample's oxygen measurement at 15 minute intervals.
Test temperature:
20 ºC
pH:
6.82-7.00
Nominal and measured concentrations:
Nominal concentrations: 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: Beakers
- Material, size, headspace, fill volume: Glass, 500 ml
- Aeration: Air-flow using compressed air.
- No. of organisms per vessel: 4 g/l of activated sludge
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: See the table below.
Synthetic sewage feed (1 litre of water):
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2·2H2O
0.2 g MgSO4·7H2O
2.8 g K2HPO4
- Intervals of water quality measurement: Starting pH of each sample.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen consumption over a period of 10 minutes

TEST CONCENTRATIONS
- Test concentrations: Nominal concentrations: 100 mg/l
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (1, 3.2, 10, 32 and 50 mg/l)
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
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 was 10.8 mg/L (95% CL: 10.7-10.9)
Reported statistics and error estimates:
The EC50 was calculated by the Probit method modified by the Flemish Institue for Technological Research (VITO).

Table 2. Summary of results.

Beaker

Chemical

Concentration

Oxygen consumption

(mgO2/L min)

Inhibition

(%)

Starting pH

7

Fosfomycin PEA salt

100

0.45

18.9

6.82

1

Control

-

0.58

-

7.0

8

Control

-

0.53

-

7.0

2

3,5-dichlorophenol

50

0.03

95

6.96

3

3,5-dichlorophenol

32

0.10

82

6.96

4

3,5-dichlorophenol

10

0.44

20.7

6.98

5

3,5-dichlorophenol

3.2

0.45

18.9

6.97

6

3,5-dichlorophenol

1.0

0.46

17.1

6.99

Validity criteria fulfilled:
yes
Remarks:
Variation between the respiration rates of the 2 control samples < 15%. Inhibitory effect of 3,5-dichlorophenol was within the range of the EC50 value of 5 to 30 mg/l
Conclusions:
EC50 value (based on respiration inhibition) on activated sludge at 3 hours exposure period was > 100 mg/l.
Executive summary:

The assessment of the effect of the test article Fosfomycin PEA salt on microorganisms by measuring the respiration rate followed the OECD Guideline 209. 4 g/l of activated sludge from RMB laboratory sewage plant were exposed for 3 hours of aeration at 20 ºC to test material at the nominal concentration of 100 mg/l. The reference substance, 3,5 -dichlorophenol was tested at 1, 3.2, 10, 32 and 50 mg/l and two control tests were carried out.

At 100 mg/l the test article showed 18.9 % inhibition on the respiration rate. The EC50 can be defined as >100 mg/l. The EC50 of the reference compound under the same conditions was 10.8 mg/l, which is in the accepted guideline range (5 -30 mg/l).

Description of key information

Key study: Test method according to OECD Guideline 209. GLP study.

The EC50 value (based on respiration inhibition) on activated sludge at 3 hours exposure period was > 100 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

Key study: Test method according to OECD Guideline 209. GLP study. The EC50 value (based on respiration inhibition) on activated sludge at 3 hours exposure period was > 100 mg/l.