Chlorpyrifos

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

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

Qualifier:

according to guideline

Guideline:

other: EC Directive 88/302, Biodegradation - Activated Sludge Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch Number: RMM 2236
Purity: 97.8%

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was insufficiently soluble in water to allow the preparation of an aqueous stock solution. The test substance was also considered to be volatile, so, a concentrated stock solution (0.5 g/mL) was prepared in acetone and then diluted to establish two further stock solutions (0.15 g/mL, 0.05 g/mL). Aliquots (0.1 mL) of the respective stock solutions were then added using a micro syringe to conical flasks containing dechlorinated tap water, activated sludge and synthetic sewage.
- Controls: Control and reference cultures were established with acetone (0.1 ml) and a further control culture was established without acetone in order to assess the effect of acetone on its respiration rate.
- Chemical name of vehicle: Organic solvent (acetone)

Test organisms (species):

other: activated sludge and synthetic sewage

Details on inoculum:

- Laboratory culture: In the laboratory, the sample was maintained under aerobic conditions until required.
- Name and location of sewage treatment plant where inoculum was collected: Oakley Sewage Treatment Works, Suffolk
- Preparation of inoculum for exposure: On the day of collection, aliquots (25 mL) of the activated sludge were filtered through dried and pre-weighed Whatman's GFC filter papers which were then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated and adjusted to 4 g/L by the removal of supernatant. Synthetic sewage (50 mL/L) was added and the mixture aerated overnight. On the day of the test, the MLSS content of the sludge was determined and adjusted to 4 g/L by the addition of dechlorinated tap water. The pH of the sludge was also measured.

Test type:

not specified

Water media type:

other: dechlorinated tap water

Limit test:

no

Total exposure duration:

3.5 h

Remarks on exposure duration:

Two sets of mixtures were prepared; the first was used to determine respiration rates after thirty minutes of aeration and the second to determine respiration rates after three hours.

Test temperature:

20 ± 2°C

pH:

7.5-8.1

Nominal and measured concentrations:

0, 10, 30, 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Aeration: Aeration was achieved by placing the cultures in conical flasks, onto an orbital shaking table, operated at a shaking speed of 200 rpm.
- No. of vessels per concentration (replicates): 10 and 30 mg/L single mixture; 100 mg/L triplicate mixture
- No. of vessels per blank control (replicates): 1
- No. of vessels per vehicle control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used to prepare synthetic sewage was tap water that had been softened and treated by reverse osmosis (Elga Ltd, Prima 4 reverse osmosis unit) and then purified (Elga Ltd, UHP) to give a resistivity of 18 Megohm/cm.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (3,5-DCP)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: highest dose tested

Key result

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: highest dose tested

Duration:

3 h

Dose descriptor:

other: EC20 and EC80

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: highest dose tested

Duration:

30 min

Dose descriptor:

other: EC20 and EC80

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: highest dose tested

Details on results:

- Blank controls oxygen uptake rate: The specific respiration rate of the control cultures established at the end of the thirty-minute and three-hour aeration periods (30.0 and 33.3 mgO2/g s.s./h, respectively) were 114 and 102% of those established at the start (26.3 and 32.6 mgO2/g s.s./h, respectively). These results show that the test was valid and that the sample of activated sludge employed was sensitive to inhibition.
- Vehicle control: The respiration rates of the cultures that were not treated with acetone (28.3 and 34.9 mgO2/g/h) were 100 and 106% of the mean control rates of mixtures that were treated with acetone (28.2 and 33.0 mgO2/g/h) after thirty minutes and three hours of exposure, respectively. These results confirmed that acetone did not adversely affect the respiration rate of the sludge.

Results with reference substance (positive control):

Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The thirty-minute and three-hour 50% effect concentrations (EC50) for 3,5-DCP were calculated by the Moving Average method to be 12.5 mg/L (95% confidence limits 9.6-16.9 mg/L) and 20.0 mg/L (95% confidence limits 16.8-24.1 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

EC20, EC50, EC80 (respiration rate activated sludge): >100 mg/L (highest dose tested)

Executive summary:

The effect of the test substance on the respiration rate of activated sludge was assessed using methods based on EC Directive 88/302 and OECD guideline 209.

Samples of activated sludge (suspended solids, 1.6 g s.s/L), fed with synthetic sewage, were exposed to the test substance at nominal concentrations of 10,30 and 100 m/L for 30 minutes and 3 hours. Single mixtures were prepared at 10 and 30 mg/L and the highest level was prepared in triplicate. Two sets of control and test mixtures were established in two litre conical flasks. Test mixtures were established by the addition of appropriate volumes of stock solutions prepared in acetone. Acetone was added to controls and reference mixtures and omitted from an additional mixture, which was established to assess its effect on the respiration rate of the sludge. Each mixture was aerated for 30 minutes or 3 hours by continuous agitation on an orbital shaker. Their rates of oxygen consumption were then determined using an oxygen electrode and compared with those of the initial and final controls.

The reference inhibitor 3,5-dichlorophenol was employed in each test series at 3.0, 10.0 and 32.0 mg/L, as a positive control.

The specific respiration rates of the control cultures established at the end of the 30-minute and 3-hour aeration periods (30.0 and 33.3 mgO2g s.s./h, respectively) were 114 and 102% of the rates of those established at the start (26.3 and 32.6 mgO2g s.s./h). The 30-minute and 3-hour 50% effect concentration (EC50) for 3,5-DCP were calculated to be 12.5 mg/L (95% confidence limits 9.6-16.9 mg/l) and 20.0 mg/L (95% confidence limits 16.8-24.1 mg/L), respectively. These results show that both tests were valid and that the samples of activated sludge employed were sensitive to inhibition.

The respiration rates of the cultures that were not treated with acetone (28.3 and 34.9 mgO2g/h) were 100 and 106% of the mean control rates of those that were treated with acetone (28.2 and 33.0 mgO2/g/h) after 30 minutes and 3 hours of exposure, respectively. These results confirmed that acetone did not adversely affect the respiration rate of the sludge.

The test substance had no biologically significant inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test, after exposure times of 30 minutes and 3 hours. Respiration rates were decreased, at most, by 7% of the mean control value (28.2 mgO2g/h) at nominal concentrations of 10, 30 and in one replicate at 100 mg/L after 30 minutes of exposure and by 3% of the mean control value (33.0 mgO2/g/h) at 10 mg/L after 3 hours. The EC20, EC50 and EC80 could not, therefore, be calculated in either test series but these must be greater than 100 mg/L, the highest level tested.

Description of key information

EC20, EC50, EC80 (respiration rate activated sludge): >100 mg/L (highest dose tested); OECD 209, EC Directive 88/302: Reliability = 1

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L