Tall oil, reaction products with ethanolamine

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:

05 January 2015 to 21 January 2015

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:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

TEST ITEM PREPARATION

RANGE-FINDING TEST
- 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 22 °C and 23 °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 if necessary.
- Exposure conditions for each flask were as described for the definitive test.
- As it was not a requirement of the test guidelines, no analysis was conducted to determine the homogenicity, concentration or stability of the test item formulation. This is an exception with regard to GLP and was reflected in the compliance statement.
- The control group was maintained under identical conditions but not exposed to the test item.

DEFINITIVE TEST
- Nominal amounts of test item (50, 160 and 500 mg) were separately dispersed directly into 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 21 °C and 22 °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 100, 320 and 1000 mg/L (5 replicates).
- The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter (see Table 6, attached) and adjusted to between pH 7.0 and pH 8.0 if necessary.
- The control group was maintained under identical conditions but not exposed to the test item.

PREPARATION OF THE TEST SYSTEM
- 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 additional control vessels were then prepared before test item vessel preparation.
- Two further control vessels were then prepared.
- The test was conducted under normal laboratory lighting in a temperature controlled room at 20 °C to 21 °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 06 January 2015 for the range-finding test and on 20 January 2015 for the definitive test.
- Microorganisms were obtained from the aeration stage of the Severn Trent Water plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

SYNTHETIC SEWAGE
- A synthetic sewage composed of 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) was prepared by dissolving in 1L of water with the aid of ultrasonication and then added to each test vessel to act as a respiratory substitute.
- In the range-finding test, the pH of the synthetic sewage was 7.0 adjusted to 7.1 using 1.0 NaOH.
- In the definitive test, the pH of the synthetic sewage stock used to feed the activated sewage sludge was 7.0 whilst the pH of the synthetic sewage stok was 7.2.
- The pH values were 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 overnight prior to use in the test.
- On the day of collection, the activated sewage sludge (10 L) was fed synthetic sewage (500 mL).
- The pH of the sample on the day of the test was 7.6 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 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:

None

Hardness:

Not reported

Test temperature:

20 °C to 21 °C

pH:

- Test item: pH 7.2 to 7.8 (see Table 9, attached)
- Control: pH 6.9 to 7.8 (see Table 9, attached)

Dissolved oxygen:

66 to 76 % of dissolved oxygen saturation level of 8.9 mg O2/L (see Table 2, attached)

Salinity:

Not applicable

Nominal and measured concentrations:

RANGE FINDING TEST
- Nominal concentrations of 10, 100 and 1000 mg/L

DEFINITIVE TEST
- Nominal concentrations of 100, 320 and 1000 mg/L

Details on test conditions:

TEST WATER

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

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

680 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

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

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 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
- 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 test preparations throughout the definitive test are shown 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 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, it was considered necessary to perform a definitive test in order to obtain a NOEC value for the test item.

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 items are shown 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 test preparations throughout the definitive test are shown in Table 10 (attached).
- Pecentage inhibition is plotted against concentration for the reference item 3,5-dichlorphenol and test item (see Figure 1 and Figure 2, attached).

VALIDATION CRITERIA
- The coefficient of variation of oxygen uptake in the control vessels was 2.2 % and the specific respiration rate of the controls was 31.39 mg oxygen per gram dry weight of sludge per hour.
- Validation criteria were satisfied.

Results with reference substance (positive control):

- The effect of the reference item on respiration rate of activated sewage sludge microorganisms gave a 3-hour EC50 value of 9.7 mg/L (see table attached).
- The validation criteria for the reference item EC50 value was satisfied.

Reported statistics and error estimates:

ECx AND NOEC
- Percentage inhibition values were plotted against concentration, a line fitted using the XIfit software package (IDBS) abd the EC10, EC20, EC50 and EC80 values were determined for the fitted line.
- The EC20, EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data.
- 95 % confidence limits were calculated for the reference item EC50 value 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 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).

INHIBITION OF RESIRATION RATE

- No statistically significant toxic effects were shown at the test concentration of 100 mg/L. However, statistically significant toxic effects (P < 0.05) were shown at test concentrations of 320 mg/L and 1000 mg/L.

- Based on these results, the No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L (see table 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). However, this was considered to have 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.

- 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.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge was assessed in accordance with OECD Guideline 209 and gave a 3 -hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration after 3 hours exposure was 100 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

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item t concentrations of 100, 320 and 1000 mg/L (5 replicates) for a period of 3 hours at a temperature of 20 to 21 °C with the addition of a synthetic sewage as a respiratory substitute.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and 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 after 3 hours exposure was 100 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 EC value of 9.7 mg/L with 95 % confidence limits of 8.0 to 12 mg/L.

CONCLUSION

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 after 3 hours exposure was 100 mg/L.

Description of key information

The effect of the test item on the respiration of activated sewage sludge was tested using OECD Guideline 209 and gave a 3 -hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration after 3 hours exposure was 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 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

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item t concentrations of 100, 320 and 1000 mg/L (5 replicates) for a period of 3 hours at a temperature of 20 to 21 °C with the addition of a synthetic sewage as a respiratory substitute.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and 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 after 3 hours exposure was 100 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 EC value of 9.7 mg/L with 95 % confidence limits of 8.0 to 12 mg/L.

CONCLUSION

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 after 3 hours exposure was 100 mg/L.