Reaction mass of sulphuric acid, hydrogen peroxide and peroxomonosulphuric acid

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:

2012-11-14 to 2012-11-30

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

Deviations:

yes

Remarks:

variation in dissolved oxygen concentrations not considered to adversely affect the study because oxygen consumption rates were determined over linear portion of traces.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 2012-07-10; Date of signature 2012-11-30

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test water for the range-finding and definitive tests was deionised reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

Test organisms (species):

activated sludge, domestic

Details on inoculum:

SOURCE
- A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which predominantly treats domestic sewage.
- Collections took place on 18 October 2012 for the range-finding test and on 23 October 2012 for the definitive test.

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 (8 L) was fed syntheitc sewage sludge (400 mL).
- The pH of the sample on the day of the test was 7.7 measured using a WTW pH/oxi 340I pH and dissolved oxygen meter.
- Suspended solids in 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 that had been rinsed three times with 20 mL of deionised reverse osmosis water prior to drying in an oven. The filtration process was carried out 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 was repeated until a constant weight was attained and the suspended solids concentration was found to equal 3.0 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C in a temperature controlled room under normal laboratory lighting

pH:

- pH of synthetic sewage stock used to feed the activated sludge for the range-finding test was 7.4
- pH of synthetic sewage stock used for the range-finding test was 7.0
- pH of synthetic sewage stock used to feed the activated sludge for the definitive test was 7.2
- pH of synthetic sewage stock used for the definitive test was 7.1

Nominal and measured concentrations:

RANGE-FINDING TEST
- 10, 100 and 1000 mg/L (nominal concentration)

DEFINITIVE TEST
- 100, 180 and 320 mg/L (nominal concentration)

Details on test conditions:

SYNTHETIC SEWAGE
- A synthetic sewage 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 (0.4 g); MgSO4.7H2O 0.2 g; K2HPO4 (2.8 g) dissolved in 1 L of water with the aid of ultrasonication).
- pH values were measured using a WTW pH/oxi 340I pH and dissolved oxygen meter.

RANGE FINDING TEST
- Test conditions to be used in the definitive test were determined by preliminary range-finding.
- Activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1,000 mg/L.
- An amount (2500 mg) of test material was introduced directly into water with the aid of ultrasonication for approximately 5 min and the volume adjusted to 1 L to give a 2500 mg/L stock solution, from which dilutions were made to give 250 mg/L and 25 mg/L stock solutions.
- An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to give a final volume of 500 mL and a final concentration of 10 mg/L.
- Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare test concentrations of 100 mg/L and 1000 mg/L.
- The 1000 mg/L test concentration was prepared in triplicate.
- Volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
- pH of test material stock solutions was measured using a WTW pH/oxi 340I pH and dissolved oxygen meter and adjusted to between pH 7.0 and pH 8.0.

CONTROLS
- The control group was maintained under identical conditions but not exposed to the test material.
- 3,5-dichlorophenol was included in the range-finding test as a reference item. Concentrations of 3.2, 10 and 32 mg/L were used 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 5.6 using a WTW pH/oxi 340I pH and dissolved oxygen meter and subsequently adjusted to 7.0 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 give 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.

DEFINITIVE TEST
- Based on the results of the range-finding test, and in order to obtain a No Observed Effect Concentration (NOEC), concentrations of 100, 180 and 320 mg/L were selected for the definitive test.
- Amounts of test material (250, 450 and 800 mg) of test material were each separately dissolved in water with the aid of ultrasonication for approximately 5 min and the volumes adjusted to 1 L to give 250, 450 and 800 mg/L stock solutions respectively.
- An aliquot (200 mL) of the 250 mg/L stock solution was dispersed with synthetic sewage (250 mL) and water to give a final volume of 500 mL and the required concentration of 100 mg/L.
- Similarly, aliquots (200 mL) of the 450 mg/L and 800 mg/L stock solutions were used to prepare test concentrations of 180 mg/L and 320 mg/L.
- Five replicates of each test concentration were prepared.
- Volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
- pH values were measured using a WTW pH/oxi 340I pH and dissolved oxygen meter.

CONTROLS
- The control group was maintained under identical conditions but not exposed to the test material.
- 3,5-dichlorophenol was included in the range-finding test as a reference item. Concentrations of 3.2, 10 and 32 mg/L were used 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 5.6 using a WTW pH/oxi 340I pH and dissolved oxygen meter and subsequently adjusted to 7.1 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 give 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.

PREPARATION OF THE TEST SYSTEM
- At time zero, 16 mL of synthetic sewage was diluted to 250 mL with water in a 500 mL conical flask and 250 mL of inoculum was added (first control).
- The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 to 1 L/min.
- Thereafter, at 15 min 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 additional control vessels were then prepared prior to preparation of the test material vessels.
- Two further control vessels were then prepared.
- 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 approximately 7.0 mg oxygen/L and 2.0 mg oxygen/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.
- Observations of the test material vessels were made at zero hours prior to addition of the activated sludge and further observations of the test preparations throughout the test period.
- pH of the control, reference item and test item preparations was measured at zero hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.
- The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

180 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
- No statistically significant toxic effects were shown test concentrations of 10 mg/L and 100 mg/L.
- Statistically significant toxic effects were shown at 1000 mg/L.
- The dissolved oxygen concentration in several vessles after 30 min contact time was < 60 % of the dissolved oxygen saturation level of 8.9 mg oxygen/L. This deviation was not considered to adversely affect the study because all oxygen consumption values were measured/calculated over the linear portion of the traces.

DEFINITIVE TEST
- No statistically significant toxic effects were shown at test concentrations of 100 mg/L and 180 mg/L.
- Statistically significant toxic effects were shown at 320 mg/L.
- The coefficient of variation of oxygen uptake in the control vessels was 7.6 %.
- Specific respiration rate of the controls was 27.45 mg oxygen per gram dry weight of sludge per hour.
- In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guideline (7.0 mg oxygen/L and 2.0 mg oxygen/L respectively). This deviation was not considered to adversely affect the study because oxygen consumption rate was determined over the linear portion of the trace.

Results with reference substance (positive control):

Percentage inhibition was plotted against concentration for the reference item and the results are shown in Figure 1 (attached).

Derived results were EC20 (3h) 3.7 mg/L; EC50 (3h) 10 mg/L with 95 % confidence limits of 8-13 mg/L; EC80 (3h) 28 mg/L.

The validation criterion for the reference item EC50 was satisfied.

Reported statistics and error estimates:

The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlft software package (IDBS) and the EC20, EC50 and EC80 values were determined from the equation for the fitted line. 95% confidence limits were calculated for the reference item EC50 values using the method of Litchfield and Wilcoxon.

One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the oxygen consumption data 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.

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

Table 1 - pH values of the test item stock solutions prior to addition of inoculum in the range finding test (attached).

Table 2 - Dissolved oxygen concentration of the test preparations after 30 minutes contact time in the range finding test (attached).

Table 3 - Oxygen consumption rates and percentage inhibition values after 3 h contact time in the range finding test (attached).

Table 4 - pH values of the test preparations at the start and end of the exposure period in the range finding test (attached).

Table 5 - Observations on the test preparations throughout the test period in the range finding test (attached).

Table 6 - pH values of the test item stock solutions prior to addition of inoculum in the definitive test attached).

Table 7 - dissolved oxygen concentrations of the test preparation after 30 min contact time in the definitive test (attached).

Table 8 - Oxygen consumption rates and percentage inhibition values after 3 h contact time in the definitive test (attached).

Table 9 - pH values of the test preparations at the start and end of the exposure period in the definitive test (attached).

Table 10 - Observations on the test preparations throughout the test period in the definitive test (attached).

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on respiration of activated sewage sludge micro-organisms gave a No Observed Effect Concentration (NOEC) of 180 mg/L after an exposure time of 3 hours.

Description of key information

No Observed Effect Concentration (3h) 180 mg/L (OECD 209)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

180 mg/L