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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:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted under GLP
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Test system: municipal Activated sludge
Age: 1 day
Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany
Collection of the test system: 13 Oct 2014
Arrival in the test facility: 13 Oct 2014
After arrival of the activated sludge suspension in the test facility the suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L Dw.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19.5 - 20.0 °C
Dissolved oxygen:
oxygen concentration during aeration: > 2 mg/L
oxygen concentration immediately before measurement: > 7 mg/L
Salinity:
n/a
Nominal and measured concentrations:
15.6, 7.8, 3.9, 2.0 and 1.0 mg/L (nominal)
Details on test conditions:
Test concentrations:
- 15.6, 7.8, 3.9, 2.0 and 1.0 mg/L (nominal) without correction of the purity and blank controls.
- 100, 10, 1 mg/L as nominal concentration based on reference substance.

Test replicates:
- 6 replicates for the control
- 3 replicates for each test substance concentration
- 2 replicates for each reference substance concentration

Application of the test substance: Direct addition by initial weight into the test flasks. The test substance was weighed in the required amounts for the test concentrations directly to the testvessels.

Reference substance preparation:
1000.0 mg of the reference substance was added to about 800 mL of deionized water and stirred at room temperature until the reference substance was completely dissolved. The pH value of the stock solution was measured and adjusted to 7.1 with 1 M sodium hydroxide solution. Following this the stock solution was made up to 1L with deionized water. The stock solution appeared colorless-clear and no undissolved reference substance was visible.

Test conditions
Incubation time: 3 hours
Test temperature: 19.5 - 20.0 °C
Test vessels: Glas-beakers (nominal volume 1L)
Test volume: 500 mL
Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium
Oxygen concentration during aeration: > 2 mg/L
Oxygen concentration immediately before measurement: > 7 mg/L
Duration of the measurement of oxygen consumption: about 8 - 10 min
Sludge concentration in the test vessel: 1.5 g/L Dw

The test substance was added in the required amounts according to the test concentrations directly to the test vessels with about 234 mL deionized water. Aliquots of the reference substance stock solution were dosed to the test vessels and made up with deionized water to a volume of 234 mL. 16 mL synthetic medium were dosed to each test vessel with test substance and reference substance afterwards. To prepare the blank control assays 234 mL of deionized water and 16 mL synthetic medium were mixed. The pH-values in all test vessels were checked. An adjustment was not necessary.

After addition of 250 mL of inoculum suspension (3 g/L Dw) the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test- or reference substance.

After 3 hours incubation at 20 ± 2°C the mixtures in the test vessels were placed subsequently for oxygen measuring. The content of oxygen at the start of the measurements was > 7 mg/L.

The total oxygen consumption was measured in the sequence BC1-3 and RS1, RS2-5, RS6 and TS1-3, TS4-7, TS8-11 and TS12-15. The oxygen consumption of the blank control BC4- 6 were measured at last. No abiotic control was tested.

The oxygen uptake was measured for a period of about 8 to 10 minutes or until the oxygen concentration fell below 2 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
3.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 2.6/4.9
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 4.0/6.3
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
9.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 8.3/11.2
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 15.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: n/a
Details on results:
The results in this study are consistent with all validity criteria and the test is valid according to the guidelines used for this study.
No deviations from the test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.
Results with reference substance (positive control):
In order to verify that the microorganisms are responding normally to toxic stress, tests with a reference substance are conducted according to OECD 209 guideline. The results from the reference substance test are compared to EC50 values published in OECD 209 guideline, which represent the typical response range.

According to the test guideline OECD 209 the EC50 values of the reference substance 3,5-dichlorophenol should be in the range of 2-25 mg/L after 3 hours of incubation.

The EC50 of the reference substance 3,5-dichlorophenol was in the range of 2-25 mg/L in 3 hours.

These results indicate that the microorganisms are responding normally to toxicant stress.
Validity criteria fulfilled:
yes

Description of key information

Depending on local conditions and existing concentrations, disturbances in the biodegradation process of activated sludge are possible. 

Key value for chemical safety assessment

Additional information

The toxic potential of the compound towards activated sludge was investigated in an OECD 209 guideline study under consideration of GLP. The incubation time of the activated sludge was 3 hours, the tested concentration were nominal 1.0, 2.0, 3.9, 7.8 and 15.6 mg/L. The EC10, EC20, EC50 and EC80 were 3.6, 5.0, 9.6 and > 15.6 mg/L, respectively. Depending on local conditions and existing concentrations, disturbances in the biodegradation process of activated sludge are possible.