[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:

13 June 2016 to 31 August 2016

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:

lower than expected dissolved oxygen concentrations in control vessels after 30 minutes contact time with no adverse effect on study (see below)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

TEST WATER
- The test water used for the range-finding test, initial experiment and definitive test was deionised reverse osmosis water containing < 1 mg/L Dissolved Organic Carbon (DOC).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

SOURCE
- Mixed populations of activated sewage sludge microorganisms were obtained on three dates for the range-finding test (13 June 2016), the initial experiment (04 July 2016) and the definitive test (31 August 2016).
- Activated sewage sludge microorganisms were obtained on each occasion 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 and was used on the day of collection.
- The pH of the sample was measured as 7.8 using a Hach HQ40d Flexi handheld meter.
- Determination of the suspended solids level of the activated sewage sludge was carried out using a Buchner funnel by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper. The filter paper had previously been rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven.
- The filter paper used in the Buchner funnel was 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 was repeated until a constant weight was obtained.
- The suspended solids concentration was equal to 3.0 g/L.

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 to 21 °C in the definitive test

pH:

7.5 to 7.9 in the definitive test (see Table 9, attached)

Dissolved oxygen:

1.5 to 6.0 mg O2/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
- Initial experiment: nominal concentrations of 32, 56 and 100 mg/L
- Definitive test: nominal concentrations of 320, 560 and 1000 mg/L

Details on test conditions:

SYNTHETIC SEWAGE
- A 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 1 L of water with the aid of ultrasonication.
- In the range-finding test, the pH of the synthetic sewage stock used to feed the activated sewage sludge was pH 7.1 and the pH of the synthetic sewage stock used for the definitive test was 7.2. The pH values were measured using a Hach HQ40d Flexi handheld meter.

TEST ITEM PREPARATION - RANGE-FINDING TEST
- The test concentrations to be used in the definitive test were determined by a preliminary range-finding test and an initial experiment.
- In the range-finding test, activated sewage sludge microorganisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water.
- To maximise the dissolved test item concentration, 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 between 20 °C and 21 °C.
- 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 8.0 if necessary.
- The control group was maintained under identical conditions but not exposed to the test item.

REFERENCE ITEM PREPARATION - RANGE-FINDING TEST
- 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 15 minutes.
- The pH of the stock solution was measured as 6.2 and adjusted to pH 7.2 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d 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 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.

INITIAL EXPERIMENT
- An initial experiment was performed using the test concentrations of 32, 56 and 100 mg/L
- This experiment showed that the NOEC value was ≥ 100 mg/L and a definitive test was performed to confirm whether the NOEC value was > 100 mg/L.

TEST ITEM PREPARATION - DEFINITIVE TEST
- Based on the results of the range-finding test and initial experiment, concentrations of 320, 560 and 1000 mg/L were assigned to the definitive test.
- The test item was dispersed directly in water.
- To maximise the dissolved test item concentration, nominal amounts of test item (160, 280 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 20 °C to 22 °C.
- 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 320, 560 and 1000 mg/L (5 replicates of each).
- The pH of the test item dispersions were measured after stirring using a Hach HQ40d handheld meter (see Table 6, attached) and adjusted to between pH 7.0 and 8.0 if necessary.
- 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.

REFERENCE ITEM PREPARATION - DEFINITIVE TEST
- The reference item 3,5-dichlorophenol was used.
- 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 15 minutes.
- The pH of the stock solution was measured as 5.8 and adjusted to pH 7.0 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d 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 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.

PREPARATION OF DEFINITIVE TEST SYSTEM
- At time zero, synthetic sewage (16 mL) was diluted to 250 mL with water and inoculum (250 mL) was 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 into which appropriate amounts of the reference item were added.
- Two additional control vessels were then prepared prior to preparation of the test item vessels.
- Finally, two further control vessels were prepared.
- The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of 20 °C to 21 °C.

OBSERVATIONS
- Observations were made on the test preparations throughout the test period.
- Observations of 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 start of the test (after addition of activated sludge) and at the end of the 3-hour incubation period.
- Measurements were made 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 RATES
- As each vessel reach 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 was measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe.
- The contents of the measuring vessel were constantly stirred 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 rapid oxygen consumption, measurements may have been made 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 a period of approximately 10 minutes.

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 portion 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 phases (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)

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
- The results of the study were considered valid if the following conditions were met:
(i) The EC50 (3-hour contact time) for 3,5-dichlorophenol was within the range 2 to 25 mg/L for total respiration.
(ii) The pecific respiration rate of the blank controls was 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 was not more than 30 % at the end of the test.

MAJOR COMPUTERISED SYSTEMS
- Building management: Delta control system

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (Sigma Aldrich; batch MKBV7274V, 97 % purity, expiry date 22 January 2017)

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

560 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: oxygen consumption

Details on results:

RANGE-FINDING TEST
- Dissolved oxyen 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 tiem 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 concentration of 10 and 100 mg/L. However, statistically significant toxic effects were shown at the test concentration of 1000 mg/L.

INITIAL EXPERIMENT
- The NOEC value was determined to be ≥ 100 mg/L and a definitive test was performed to confirm whether the value was > 100 mg/L.
- Based on the results of the initial experiment, concentrations of 320, 560 and 1000 mg/L were selected for the definitive test.

DEFINITIVE TEST
- 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 item 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).

VALIDATION CRITERIA
- The coefficient of variation of oxygen uptake in the control vessels was 5.0 % and the specific respiration rate of the controls was 24.88 mg oxygen per gram dry weight of sludge per hour. The validation criteria were therefore satisfied.
- Validation criteria for the reference item were also satisfied (see table below).

INHIBITION OF RESPIRATION RATE
- Results obtained with the reference substance are shown in the table below.
- No statistically signifcant toxic effects were seen at test item concentrations of 320 and 560 mg/K. However, statistically significant toxic effects (p < 0.05) were shown at test item concentration of 1000 mg/L.
- Based on these results, the No Observed Effect Concentration (NOEC) after 3 hours exposure to the test item was 560 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 was determined over the linear portion of the oxygen consumption trace.

Results with reference substance (positive control):

- Percentage inhibition plotted against concentration for the reference item is shown in Figure 1 (attached).
- The EC10, EC20, EC50 and EC80 values calculated for the reference substance are shown in the table below.

Reported statistics and error estimates:

ECx AND NOEC
- For the reference item only, percentage inhibition values were plotted against concentration, a line was fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values were determined from the equation for the fitted line. Calculation of 95 % confidence limits for the reference item EC50 value used 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 after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups and identify the NOEC.
- All statistical analyses were performed using the SAS computer software package (SAS, 1999-2000).

RESULTS WITH REFERENCE ITEM (3,5-DICHLOROPHENOL)

	ECx (3 h) mg/L	95 % confidence limits (mg/L)
EC10	1.6	-
EC20	2.4	-
EC50	7.5	5.9 - 9.6
EC80	23	-

Validity criteria fulfilled:

yes

Conclusions:

The No Observed Effect Concentration (NOEC) after 3 hours exposure to the test item was 560 mg/L. The reference item gave a 3-hour EC50 value of 7.5 mg/L (95 % confidence limits 5.9 to 9.6 mg/L).

Executive summary:

GUIDELINE

A study was performed to assess the effect of the test item on 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 and an initial experiment, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 320, 560 and 1000 mg/L for a period of 3 hours at measured temperatures of 20 to 21 °C with the addition of 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 No Observed Effect Concentration (NOEC) after 3 hours exposure to the test item was 560 mg/L. The reference item gave a 3-hour EC50 value of 7.5 mg/L (95 % confidence limits 5.9 to 9.6 mg/L).

Description of key information

The No Observed Effect Concentration (NOEC) after 3 hours exposure to the test item was 560 mg/L (OECD 209).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

560 mg/L

Additional information

GUIDELINE

A study was performed to assess the effect of the test item on 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 and an initial experiment, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 320, 560 and 1000 mg/L for a period of 3 hours at measured temperatures of 20 to 21 °C with the addition of 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 No Observed Effect Concentration (NOEC) after 3 hours exposure to the test item was 560 mg/L. The reference item gave a 3-hour EC50 value of 7.5 mg/L (95 % confidence limits 5.9 to 9.6 mg/L).