Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 September 2016 to 14 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
TEST WATER
- Deionised reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC) was used for the test.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
TEST SYSTEM
- A mixed population of activated sewage sludge micro-organisms was obtained on 23 September 2016 for the range-finding test and on 13 October 2016 for the definitive test.
- Activated sewage sludge microorganisms were obtained on both occasions from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

PREPARATION OF INOCULUM
- The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21°C overnight prior to use in the test.
- On the day of collection, the activated sewage sludge (10 L) was fed synthetic sewage (495 mL).
- The pH of the sample on the day of the test was 7.1 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 (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper (rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven) using a Buchner funnel which was then rinsed three times with 10 mL of deionized reverse osmosis water and filtration 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.
- This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.

PREPARATION OF TEST SYSTEM
- At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in 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 per minute.
- 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. The test item vessels were prepared as described above. Finally, two further control vessels were prepared.
- The test was conducted under normal laboratory lighting in a temperature controlled room at a measured temperature of between 21 °C.

OBSERVATIONS
- Observations were made on the test preparations throughout the test period.
- Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.

MEASUREMENT OF pH
- The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.

OXYGEN CONCENTRATION
- oxygen concentrations in all vessels were measured after 30 minutes contact time (see Table 7, attached).

MEASUREMENT OF RESPIRATION RATE
- As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe.
- The contents of the measuring vessel were stirred constantly by magnetic stirrer.
- Rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L).
- In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve.
- In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

CALCULATION OF OXYGEN UPTAKE RATES
- The respiration rate, R, expressed in milligrams oxygen per liter per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph using the equation R = [(Q1 – Q2) / Δt] x 60 where Q1 = oxygen concentration at the beginning of the selected section of the linear phase (mg/L); Q2 = oxygen concentration at the end of the selected section of the linear phase (mg/L); Δt = time interval between the beginning and end of the selected section of the linear phase (min).
- The specific respiration rate, RS, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg O2/g/h) was deduced using the equation Rs = R / SS where SS = concentration of suspended solids in the test mixture (g dry weight/L).

CALCULATION OF PERCENTAGE INHIBITION
- Percentage inhibition was calculated using the equation % inhibition = [1 – (R/Rbc)] x 100 where Rbc = mean respiration rate of the blank controls.
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:
21 °C in the definitive test
pH:
6.6 to 7.5 in the definitive test (see Table 9, attached)
Dissolved oxygen:
1.5 to 6.7 mg/L in the definitive test (see Table 8, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
RANGE-FINDING TEST
- Nominal concentrations of 10, 100 and 1000 mg/L

DEFINITIVE TEST
- Nominal concentrations of 320, 560 and 1000 mg/L
Details on test conditions:
SYNTHETIC SEWAGE
- Synthetic sewage was added to each test vessel to act as a respiratory substrate.
- The synthetic sewage contained 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 a final volume of 1 L water with the aid of ultrasonication.
- In the range-finding test, the pH of the synthetic sewage stock was found to be 7.3 when measured using a Hach HQ40d Flexi handheld meter.
- In the definitive test, the pH of the synthetic sewage stock used to feed the activated sewage sludge and used for the definitive test was 7.3 when measured using a Hach HQ40d Flexi handheld meter.

RANGE-FINDING TEST – TEST ITEM PREPARATION
- Activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L.
- The test item was dispersed directly in water.
-- Nominal amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionised reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures of between 20 °C and 21 °C, in order to maximise the dissolved test item concentration.
- All test vessels were shielded from the 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 Hach HQ40d Flexi handheld meter (see Table 1, attached).
- The control group was maintained under identical conditions but not exposed to the test item.

RANGE-FINDING TEST – REFERENCE ITEM PREPARATION
- A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum.
- A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 10 minutes.
- The pH of this stock solution was measured as 5.6 using a Hach HQ40d Flexi handheld meter and was adjusted to pH 7.2 using 1.0 M NaOH.
- Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L.
- The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

DEFINITIVE TEST – TEST ITEM PREPARATION
- Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 320, 560 and 1000 mg/L.
- The test item was dispersed directly in water.
- Nominal amounts of test item (160, 280 and 500 mg (five replicates of each)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures between 20 °C and 21 °C, in order to maximize the dissolved test item concentration.
- All test vessels were shielded from the 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 320, 560 and 1000 mg/L (five replicates of each).
- The pH of the test item dispersions were measured after stirring using Hach HQ40d Flexi handheld meter (see Table 6, attached) and adjusted to between pH 7.0 to 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.

DEFINITIVE TEST – REFERENCE ITEM PREPARATION
- A stock solution of 0.5 g/L 3,5-dichlorophenol was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 10 minutes.
- The pH of this stock solution was measured as 6.0 and adjusted to pH 7.3 using 1.0 M NaOH.
- The pH values were measured using a Hach HQ40d Flexi handheld meter.
- Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to give the final concentrations of 3.2, 10 and 32 mg/L.
- The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

VALIDATION CRITERIA
- Results of the study are considered valid if:
(i) The EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 2 to 25 mg/L for total respiration.
(ii) The specific respiration rate of the blank controls is not less than 20 mg oxygen per gram dry weight of sludge per hour for total respiration.
(iii) The coefficient of variation of oxygen uptake rate in control replicates is not more than 30% at the end of the test.

MAJOR COMPUTERISED SYSTEM
- Building management: Delta control system
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (Sigma Aldrich; batch MKBV7274V; purity 99.6 %; expiry date 01 September 2017; room temperature storage)
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
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
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).
- The 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 the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L.
- Based on these results and after discussion with the sponsor, it was considered necessary to perform a definitive test in order to obtain a NOEC value for the test item. Based on this information test concentrations of 320, 560 and 1000 mg/L were selected for the definitive test.

DEFINITIVE TEST
- The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 7 (attached).
- Oxygen consumption rates and percentage inhibition values for the control, test and reference items after 3 hours contact time are given in Table 8 (attached).
- The pH values of the test preparations at the start and end of the exposure period are given in Table 9 (attached).
- Observations made on the test preparations throughout the definitive test are given in Table 10 (attached).
- Percentage inhibition was plotted against concentration for the reference item, 3,5-dichlorophenol (see Figure 1, attached).

VALIDATION CRITERIA
- The coefficient of variation of oxygen uptake in the control vessels was 12 % and the specific respiration rate of the controls was 21.49 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.
- The validation criterion for the reference item EC50 value was also satisfied.

Results with reference substance (positive control):
RESULTS WITH THE REFERENCE ITEM
- The EC10, EC20, EC50 and EC80 values calculated for the reference substance are shown in the table below.
- It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
- 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.
- The 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 significantly toxic effects were shown at the test concentrations of 560 and 1000 mg/L, however statistically significantly toxic effects (P < 0.05) were shown at the test concentration of 320 mg/L which could possibly be due to insoluble item coating the membrane of the oxygen probe.
- Based on the test concentrations at 560 and 1000 mg/L showing no significant inhibition of respiration, the No Observed Effect Concentration (NOEC) after 3 hours exposure was considered to be 1000 mg/L.
Reported statistics and error estimates:
ECx AND NOEC
- The 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.
- The 95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (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 after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups to determine the NOEC.
- All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

RESULTS WITH REFERENCE ITEM (3,5-DICHLOROPHENOL)

 

ECx (3h) mg/L

95 % confidence limits (mg/L)

EC10

2.1

-

EC20

3.0

-

EC50

8.6

6.8 to 11

EC80

25

-

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 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 8.6 mg/L, 95% confidence limits 6.8 to 11 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 followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

 

METHODS

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 320, 560 and 1000 mg/L (5 replicates) for a period of 3 hours at a measured temperature of 21 °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 greater than 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 8.6 mg/L, 95% confidence limits 6.8 to 11 mg/L.

Description of key information

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 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 8.6 mg/L, 95% confidence limits 6.8 to 11 mg/L (OECD 209).

Key value for chemical safety assessment

Additional information

GUIDELINE

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

 

METHODS

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 320, 560 and 1000 mg/L (5 replicates) for a period of 3 hours at a measured temperature of 21 °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 greater than 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 8.6 mg/L, 95% confidence limits 6.8 to 11 mg/L.