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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:
19 March 2010
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
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Prior to weighing the test substance was ground using a mortar and pestle. Since Single super phosphate (SSP) was not soluble in water at 0.5 g/L under the condition of this test, test concentrations were prepared separately in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Two 1 litre test bottles were filled with 250 mL of test substance mixtures with initial loading rates of 200 mg/L. These mixtures were stirred in closed dark glass bottles for approximately 27 hours. Subsequently, 16 mL synthetic sewage feed, 200 ml sludge and Milli-RO water up to 500 mL were added resulting in a loading rate of 100 mg/L. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring during 3-hours exposure period.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (4.0 g/L of sludge, as used for the test). The pH was 7.9 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
Oxygen consumption was measured and recorded for approximately 10 minutes afther the 3 hours exposure period.
Test temperature:
between 18.3 and 19.2°C.
pH:
7.4 - 7.5
Dissolved oxygen:
Oxygen consumption:
Controls Single super phosphate (SSP):55-60 mg O2/L/h
Single super phosphate (SSP): 51-55 mg O2/L/h
The oxygen concentration at the start of measurements was at least 7.3 mg O2 /l
Nominal and measured concentrations:
Loading rate: 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: open
- Material, size, headspace, fill volume: All glass, approximately 300 mL oxygen bottles and 1 litre test bottles.
- Aeration: during exposure with clean, oil-free air
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates):2
- Biomass loading rate: 1.6 g/L suspended solids in final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO)



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The respiration rate from each vessel, in mg O2/L/hr, was calculated from the linear part of the respiration curve, which was generally between 2.5 and 6.5 mg O2/L.

TEST CONCENTRATIONS
limit test at loading rate of 100 mg/L
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
Remarks:
respiration rate
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:
respiration rate
Details on results:
11% inhibition of respiration rate of the sludge was recorded at a loading rate of 100 mg/L
Single super phosphate (SSP) per litre. In the duplicate measurement no significant inhibition of respiration rate of the sludge was recorded. Therefore, no further testing was needed. Hence, the EC50 of Single super phosphate (SSP) exceeded a loading rate of 100 mg/L and the NOEC was 100 mg/L..
Results with reference substance (positive control):
Results with reference substance valid? Yes
The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/L (6.0 mg/L)
Reported statistics and error estimates:
For the reference substance the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.
For Single super phosphate (SSP) no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > 100 mg/L).
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, the EC50 of Single super phosphate (SSP) exceeded a loading rate of 100 mg/L and the NOEC was 100 mg/L.

Description of key information

In a reliable study with single superphosphate the 3-hr EC-50 was determined to >100 mg/L, and the NOEC 100 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information

In a reliable OECD 209 and EC C.11 guideline study, the respiration rate of the predominantly domestic sludge was determined after exposure to 100 mg/L single superphosphate for three hours. No significant inhibition of respiration rate of the sludge was recorded at this dose level (11% inhibition in first experiment, in the duplicate measurement no significant inhibition of respiration rate of the sludge was recorded). The substance was not toxic to waste water (activated sludge) bacteria at 100 mg/L. Therefore, no further testing was needed. Hence, the EC50 of single superphosphate exceeded a loading rate of 100 mg/L.