Chlorothalonil

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:

18 Sep 2002 to 19 Sep 2002

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

Version / remarks:

1993

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

- Test substance: Test solutions were prepared by the direct addition of a known weight of the test substance to a total volume of 500 ml aerated dechlorinated water, synthetic sewage and activated sludge. During the test. 0.07302 g of the test substance were used to prepare the test solutions.
- Reference substance: A nominal 500 mg/L stock solution of the reference substance, 3,5-dichlorophenol, was prepared in deionised water. This stock solution was adjusted as necessary to be within the range pH 6 to 8 and was observed to be clear and colourless. The final pH of this stock on the day of use was measured as 6.52 and no subsequent adjustment was required.

Test organisms (species):

activated sludge

Details on inoculum:

- Collecting date and location: Activated sludge was obtained from Buckland Sewage Treatment Works, Newton Abbot, Devon, UK, 1 day prior to the study. This works treats sewage of predominantly domestic origin.
- Method of preparation: On return to the laboratory, the activated sludge was settled and the concentrated sludge washed with aerated dechlorinated water. The washed settled sludge was fed with 50 mL of OECD synthetic sewage feed per litre of sludge per day and aerated at room temperature, until it was used in the test.
- Synthetic sewage: A synthetic sewage mixture described by the OECD guideline was prepared containing the following constituents per litre of deionised water: 15.2 g of peptone, 10.5 g of meat extract, 2.9 g of urea, 0.7 g of NaCl, 0.4 g of CaCl2.2H2O, 0.2 g of MgSO4.7H2O and 2.8 g of K2HPO4.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2°C

pH:

- Start of the test: 6.98 - 7.04
- End of the test: 7.45 - 7.78

Nominal and measured concentrations:

Nominal concentration (both test substance and reference substance): 1.0, 3.2, 10, 32 and 100 mg/L. See Table 1 in 'Any other information on materials and methods incl. tables'

Details on test conditions:

TEST SYSTEM
- Aeration: Yes
- Replication: One vessel for each of the test substance concentration and positive control concentration and 2 vessles for the blank control.
- Final solids concentration: 1600 mg/L


TEST MEDIUM / WATER PARAMETERS
- pH: The pH of each flask was measured at the start and end of the test.
- Temperature: Flasks were set up at 10 minute intervals and aerated at 20 ± 2°C for 3 hours. The temperatures of the flask contents were measured at the end of the 3 hours aeration using a mercury-in-glass thermometer.

EFFECT PARAMETERS MEASURED:
- The respiration rate of each culture was measured after 3 hours and compared with the mean respiration rate of the two control cultures. The rate of oxygen uptake was measured in a confined perspex cell into which a polarographic oxygen electrode was inserted. The electrode was connected to a meter whose output was recorded on a potentiometric chart recorder. The dissolved oxygen concentration after the 3 hour
aeration period was at least 6.5 mg O2/L
- The respiration rate was calculated between approximately 7.5 and 4.0 mg O2/L, or over approximately a 6 minute period where respiration was low. The rates of oxygen uptake were expressed as mg O2/L/h.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

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

Details on results:

The respiration rate of the two controls were 26.0 and 27.0 mg O2/L/h respectively. In test flasks dosed with the test substance, 22% inhibition observed at the highest test concentration (100 mg/L), 11% inhibition at 3.2 mg/L and < 10% inhibition was observed in the remaining concentrations. An overview of the results is provided in Table 2 in 'Any other information on results incl. tables'.

Results with reference substance (positive control):

The reference substance, 3,5-dichlorophenol, caused substantial inhibition of the respiration rate of the activated sludge. From the results obtained the 3 hour EC50 value was estimated to be 6.4 mg/L. This is within the expected normal range of 2 to 25 mg/L indicating the sludge was responding normally and confirming the viability of the sludge organisms. The respiration rates in the two control flasks were within 15% of each other. Therefore, the mean control respiration rate was used in calculation of the percentage inhibition. An overview of the results is provided in Table 2 in 'Any other information on results incl. tables'.

Table 2. Inhibition results

Test substance	Nominal concentration (mg/L)	pH		Respiration rate (mg O2/L/h)	% mean control respiration rate^a	% inhibition
		Start	End
Control	Control	7.00	7.57	26.0	-	-
3,5-DCP	100	6.98	7.45	1.5	5.6	94
3,5-DCP	32	7.01	7.63	3.5	13.0	87
3,5-DCP	10	7.00	7.62	8.5	31.5	68
3,5-DCP	3.2	7.01	7.78	21.0	77.8	22
3,5-DCP	1.0	7.01	7.68	26.5	98.1	< 10
The test substance	100	7.01	7.51	21.0	77.8	22
The test substance	32	7.04	7.57	24.0	88.9	11
The test substance	10	7.00	7.59	26.0	96.3	< 10
The test substance	3.2	7.01	7.58	25.0	92.6	< 10
The test substance	1.0	7.00	7.60	26.0	96.3	< 10
Control	Control	7.01	7.59	28.0	-	-

a Mean control respiration rate = 27 mg O2/L/h

b 3,5-dichlorophenol

Validity criteria fulfilled:

not specified

Conclusions:

After the incubation period of three hours, the test substance had ≤ 22% inhibition on the respiration rate of activated sludge at concentrations up to and including 100 mg/L, the highest concentration tested. Based on the findings, the 3-hour EC50 was determined to be ≥100 mg/L and NOEC was determined to be 10 mg/L.

Executive summary:

The inhibitory effects of the test substance on the respiration of activated sludge micro-organisms were studied according to OECD TG No. 209 (1993) at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The study was conducted in compliance with GLP criteria. There were two separate controls and 3,5-dichlorophenol as a reference substance at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. Each flask contained an excess of the synthetic sewage, sufficient activated sludge to give final solids concentrations of 1600 mg/L, an appropriate quantity of either the test substance or 3,5-dichlorophenol stock solution and aerated dechlorinated water to give a final flask contents volume of 500 mL. The test was carried out at a temperature of 20 ± 2°C. After the incubation period of three hours, The respiration rate of the two controls were 26.0 and 27.0 mg O2/L/h respectively. In test flasks dosed with the test substance 22% inhibition observed at the highest test concentration (100 mg/L), 11% inhibition at 3.2 mg/L and <10% inhibition was observed in the remaining concentrations. Based on the findings, the 3-hour NOEC of the test substance was determined to be 10 mg/L and the EC50 was ≥ 100 mg/L. The EC50 of the reference substance was determined to be 6.4 mg/L. 

Description of key information

3-h NOEC ≥ 10 mg/L, Activated sludge, inhibition of total respiration, OECD TG 209, Wallace 2002

3-h EC50 > 100 mg/L, Activated sludge, inhibition of total respiration, OECD TG 209, Wallace 2002

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

10 mg/L

Additional information

The inhibitory effects of the test substance on the respiration of activated sludge micro-organisms were studied according to OECD TG No. 209 (1993) at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The study was conducted in compliance with GLP criteria. There were two separate controls and 3,5-dichlorophenol as a reference substance at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. Each flask contained an excess of the synthetic sewage, sufficient activated sludge to give final solids concentrations of 1600 mg/L, an appropriate quantity of either the test substance or 3,5-dichlorophenol stock solution and aerated dechlorinated water to give a final flask contents volume of 500 mL. The test was carried out at a temperature of 20 ± 2°C. After the incubation period of three hours, The respiration rate of the two controls were 26.0 and 27.0 mg O2/L/h respectively. In test flasks dosed with the test substance 22% inhibition observed at the highest test concentration (100 mg/L), 11% inhibition at 3.2 mg/L and <10% inhibition was observed in the remaining concentrations. Based on the findings, the 3-hour NOEC of the test substance was determined to be 10 mg/L and the EC50 was ≥ 100 mg/L. The EC50 of the reference substance was determined to be 6.4 mg/L.