[No public or meaningful name is available]

Toxicity to microorganisms

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:

24 November 2016 to 20 January 2017

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 (Carbon and Ammonium Oxidation))

Deviations:

yes

Remarks:

no impact on results or integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

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 19 January 2017 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 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (9 L) was fed synthetic sewage (450 mL).
- The pH of the sample on the day of the test was 7.3 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 3 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 ºC 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.
- The suspended solids of the inoculum was checked on collection however, this was not re-checked on the day of used. As the control vessels passed all the validation criteria this is considered to have no significant adverse effect on the integrity of the study.

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 to 21 ºC

pH:

7.0 to 7.5 in test item preparations (see Table 4, attached)

Dissolved oxygen:

1.9 to 4.9 mg O2/L in test item preparations (see Table 3, attached)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentrations of10, 100 and 1000 mg/L test item

Details on test conditions:

TEST WATER
- Deionised reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC) was used for the test.

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.
- The pH of the synthetic sewage stock used to feed the activated sewage sludge and the pH of the synthetic sewage stock used for the range finder test were both found to be 7.3 when measured using a Hach HQ160 Flexi handheld meter.

INITIAL RANGE FINDER TEST – TEST ITEM PREPARATION
- An initial test was conducted however; the Sponsor requested that additional vessels to be included with the addition of a synthetic sewage as a respiratory substrate. Therefore, the test was repeated.
- 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 deionized 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 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 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) and adjusted to between pH 7.0 and 8.0 if necessary.
- The exposure conditions for each flask were as described for the range finder test.
- 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 was 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.

INITIAL RANGE FINDER 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 21 minutes. The pH of this stock solution was measured to be pH 5.6 and was adjusted to pH 7.1 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld.
- 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.
- The exposure conditions for each flask were as described for the range finder test.

RANGER FINDER TEST – TEST ITEM PREPARATION
- In the range-finding test, 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 deionized 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 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 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) and adjusted to between pH 7.0 and 8.0 if necessary.
- The exposure conditions for each flask were as described below.
- 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 was 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.

RANGE FINDER 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 21 minutes. The pH of this stock solution was measured to be pH 5.6 and was adjusted to pH 7.1 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld.
- 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.
- The exposure conditions for each flask were as described below.

RANGE FINDER TEST – MINERAL OIL PREPARATION
- At the Sponsors request additional vessels were prepared containing mineral oil at the same concentrations as in the test item preparations; 4.2, 42 and 422 mg/L. The test item was dispersed directly in water.
- Nominal amounts of test item (2.1, 21.1 and 211 mg) 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 of 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 4, 42 and 422 mg/L.
- The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter (see Table 1) and adjusted to between pH 7.0 and 8.0.

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/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. The test item vessels were prepared as described in Section 3.3.2.1. Two further control vessels were prepared, followed by the mineral oil controls as described above.
- The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 20 and 21 ºC.

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 2, 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.
- The 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 litre 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.

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 SYSTEMS
- Building management: Delta control system
- TOC measurement: Shimadzu TOC

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (Sigma Aldrich; Batch MKBV7274V; Purity 99.6 %; Expiry date 01 September 2017; Room temperature storage conditions)

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

Details on results:

RANGE-FINDING TEST
- An initial test was conducted showing no statistically significant toxic effects were shown at all of the test concentrations employed. However; the Sponsor requested that additional vessels to be included with the addition of a synthetic sewage as a respiratory substrate. Therefore, the test was repeated.
- In the second 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 item, reference item and mineral oil control 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% of the dissolved oxygen saturation level of 8.9 mg O2/L.
- No statistically significant toxic effects were shown at all 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.

VALIDATION CRITERIA
- The coefficient of variation of oxygen uptake in the control vessels was 1.2% and the specific respiration rate of the controls was 28.25 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

INHIBITION OF RESPIRATION RATE
- Results are summarised in the attached table.
- In some instances, the initial and final dissolved oxygen concentrations were outside those
recommended in the test guidelines (7 mg 02/L and 2 mg 0 2/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 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 effect of the mineral oil on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 422 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 422 mg/L.

Results with reference substance (positive control):

- Percentage inhibition was plotted against concentration for the reference item, 3,5-dichlorophenol (see Figure 1, attached).
- The reference item gave a 3-Hour EC50 value of 8.8 mg/L, 95% confidence limits 6.9 to 11 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).
- In order to determine the NOEC, 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 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 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 effect of the mineral oil on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 422 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 422 mg/L. The reference item gave a 3-Hour EC50 value of 8.8 mg/L, 95 % confidence limits 6.9 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

An initial test was conducted however; the Sponsor requested that additional vessels to be included with the addition of a synthetic sewage as a respiratory substrate. Therefore, the test was repeated. 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 test concentration) for a period of 3 hours at measured temperatures of between 20 and 21 ºC with the addition of a synthetic sewage as a respiratory substrate. The test item was a complex mixture containing 42% mineral oil. At the Sponsors request additional vessels were prepared containing mineral oil at the same concentrations as in the test item preparations; 4.2, 4.2 and 422 mg/L. 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 effect of the mineral oil on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 422 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 422 mg/L. The reference item gave a 3-Hour EC50 value of 8.8 mg/L, 95 % confidence limits 6.9 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 effect of the mineral oil on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 422 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 422 mg/L. The reference item gave a 3-Hour EC50 value of 8.8 mg/L, 95 % confidence limits 6.9 to 11 mg/L (OECD 209).

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

An initial test was conducted however; the Sponsor requested that additional vessels to be included with the addition of a synthetic sewage as a respiratory substrate. Therefore, the test was repeated. 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 test concentration) for a period of 3 hours at measured temperatures of between 20 and 21 ºC with the addition of a synthetic sewage as a respiratory substrate. The test item was a complex mixture containing 42% mineral oil. At the Sponsors request additional vessels were prepared containing mineral oil at the same concentrations as in the test item preparations; 4.2, 4.2 and 422 mg/L. 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 effect of the mineral oil on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 422 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 422 mg/L. The reference item gave a 3-Hour EC50 value of 8.8 mg/L, 95 % confidence limits 6.9 to 11 mg/L.