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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:
13 July 2015 to 15 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
RANGE FINDING TEST
- Test item was dispersed directly in water.
- Nominal amounts of test item (5, 50 and 500 mg) were separately dispersed in approximately 200 mL of deionised reverse osmosis water in triplicate.
- Ultrasonication was applied for approximately 15 minutes followed by magnetic stirring for 24 hours at temperatures between 20 °C and 21 °C to maximise the concentration of the dissolved test item.
- All test vessels were shielded from light during mixing.
- Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
- The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter (see Table 1, attached) and adjusted to between pH 7.0 and pH 8.0.
- As it was not a requirement of the test guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and was reflected in the GLP compliance statement.
- The control group was maintained under identical conditions but not exposed to the test item.
- At time zero, synthetic sewage (16 mL) was diluted to 250 mL with water and 250 mL of inoculum was added to a 500 mL conical flask (first control).
- The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 L/min.
- Thereafter, at 15 minute intervals, the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added.
- Two further control vessels were then prepared.
- The test was conducted under normal laboratory lighting in a temperature controlled room at approximately 20 °C.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
TEST SYSTEM
- A mixed population of activated sewage sludge microorganisms was obtained on 30 July 2015 from the aeration stage of the Severn Trent Water plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

SYNTHETIC SEWAGE
- Synthetic sewage of the following composition was added to each test vessel to act as a respiratory substrate: peptone (16 g), meat extract (11 g), urea (3 g), NaCl (0.7 g), CaCl2.2H2O (0.4 g), MgSO4.7H2O (0.2 g) and K2HPO4 (2.8 g) dissolved in 1L of water with the aid of ultrasonication.
- The pH of the synthetic sewage stock was 7.1.
- The pH was measured using a Hach HQ40d Flexi handheld meter.

PREPARATION OF INOCULUM
- The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and was used on the day of collection.
- The pH of the sample was 7.2 measured using a Hach HQ40d Flexi handheld meter.
- Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (50 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper that had been rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven.
- Filtration was performed using a Buchner funnel which was then rinsed three times with 10 mL of deionised reverse osmosis water and filtration was continued for 3 minutes.
- The filter paper was then dried in an oven at approximately 105 °C for at least one hour and allowed to cool before weighing.
- The process of drying and weighing was repeated until a constant weight was obtained.
- The suspended solids concentration was equal to 3.0 g/L prior to use.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Not applicable
Hardness:
Not reported
Test temperature:
20 °C
pH:
Test item preparations after stirring and prior to addition of inoculum in the range-finding test were pH 7.1 to 7.9 after adjustment (see Table 1, attached)
Dissolved oxygen:
Dissolved oxygen concentration of the test item preparations after 30 minutes contact time in the range-finding test were 66 to 73 % of the dissolved oxygen saturation level of 8.9 mg O2/L (see Table 2, attached).
Salinity:
Not applicable
Nominal and measured concentrations:
10, 100 and 1000 mg/L nominal concentration
Details on test conditions:
TEST WATER
- Deionised reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC) was used for the range-finding and definitive tests.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 1 000 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:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 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:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
RANGE-FINDING TEST
- The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 2 (attached).
- Oxygen consumption rates and percentage inhibition values for the control, test and reference item after 3 hours contact time are given in Table 3 (attached).
- The pH values of the test preparations at the start and end of the exposure period are given in Table 4 (attached).
- Observations made on the test preparations throughout the range-finding test are given in Table 5 (attached).
- Dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70 % of the dissolved oxygen saturation level of 8.9 mg O2/L.
- No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
- It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
- Percentage inhibition was plotted against concentration for the reference item (see Figure 1, attached).
- In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

VALIDATION CRITERIA
- The coefficient of variation of oxygen uptake in the control vessels was 3.46 % and the specific respiration rate of the controls was 21.97 mg oxygen per gram dry weight of sludge per hour.
- Validation criteria were satisfied.
Results with reference substance (positive control):
INHIBITION OF RESPIRATION RATE BY 3,5-DICHLOROPHENOL
- EC10 (3 h) 1.7 mg/L
- EC20 (3 h) 2.4 mg/L
- EC50 (3 h) 7.3 mg/L (95 % confidence limits 5.8-9.3 mg/L)
- EC80 (3 h) 22 mg/L
- The validation criterion for the reference item EC50 was also satisfied.
Reported statistics and error estimates:
ECx AND NOEC
- Percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line.
- The EC10, EC20, EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data.
- Calculation of 95 % confidence limits for the reference item EC50 value was performed using the method of Litchfield and Wilcoxon (1949).
- One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data for the range-finding and definitive test after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups.
- All statistical analyses were performed using the SAS computer software package (SAS, 1999 -2001)
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge microorganisms gave a 3-hour EC50 value of > 1000 mg/L. The Observed No Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
Executive summary:

GUIDELINE

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method was designed to be compatible with OECD Guidelines for Testing of Chemicals (2010) No 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation).

METHODS

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L concentration) for a period of 3 hours at a temperature of approximately 20 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item (3,5 -dichlorophenol).

RESULTS

The effect of the test item on the respiration of activated sewage sludge gave a 3 -hour EC50 value of > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference item gave a 3 -hour EC50 value of 7.3 mg/L (95 % confidence limits 5.8 to 9.3 mg/L).

Description of key information

The effect of the test item on the respiration of activated sewage sludge microorganisms gave a 3-hour EC50 value of > 1000 mg/L. The Observed No Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L (OECD 209).

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

GUIDELINE

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method was designed to be compatible with OECD Guidelines for Testing of Chemicals (2010) No 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation).

METHODS

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L concentration) for a period of 3 hours at a temperature of approximately 20°C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item (3,5 -dichlorophenol).

RESULTS

The effect of the test item on the respiration of activated sewage sludge gave a 3 -hour EC50 value of > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference item gave a 3 -hour EC50 value of 7.3 mg/L (95 % confidence limits 5.8 to 9.3 mg/L).