2-Thiophenecarboxylic acid, 5-chloro-

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:

5 Feb - 26 Feb 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

(1984)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Remarks:

The test item and reference substance concentrations are not confirmed by analytical methods as they are directly weighed into the test vessels.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct weighing into test flasks
- Controls: Yes, blank control
- Test concentration separation factor: 1.8

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Laboratory culture: mixed population of aquatic microorganisms (activated sludge)
- Name and location of sewage treatment plant where inoculum was collected: aeration tank of a domestic sewage treatment plant (Municipal STP Cologne-Stammheim)
- Method of cultivation: Stirring period of test item before start of incubation time: 16 h.
- Pretreatment: aeration of the activated sludge, daily fed with synthetic medium.
- Initial biomass concentration: 960 mg/L suspended solids

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

with permanent aeration

Test temperature:

19.0 to 19.4 °C

pH:

6.2 to 7.7

Dissolved oxygen:

4.7 to 7.9 mg O2/L at the start
4.9 to 7.9 mg O2/ L at the end

Nominal and measured concentrations:

560, 1000, 1800, 3200, 5600 mg/L (nominal concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel: Flask
- Aeration: Permanent aeration
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2 (blank control), 1 (positive control)
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 960 mg/L suspended solids
- Nutrients provided for bacteria: Daily feed with synthetic medium

EFFECT PARAMETERS MEASURED
- respiration inhibition at 3 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Reference substance (positive control):

yes

Remarks:

3.5-Dichlorophenol (Purity; 97%; Batch number: 15809KI-392) 2.5, 5, 10, 20, 40 mg/L (nominal concentration)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

737.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Blank controls oxygen uptake rate: 32.5 mg/L*h
- Abiotic control: no physico-chemical oxygen consumption was observed at that test substance concentration of 10000 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50=9.3 mg/L (confidence limits were 7.9 mg/L and 11.1 mg/L).

Oxygen content, temperature, and pH values during exposure phase

	Test concentration	O2 start	O2 end	Time (start-end)	Temp.	pH
	[mg/L]	[mg O2/L]	[mg O2/L]	[min.]	[°C]
Test item	560	5.6	4.9	2	19.2	7.4
	1000	6.3	5.8	3	19.2	7.0
	1800	6.3	5.9	3	19.2	7.2
	3200	7.2	7.0	4	19.3	7.0
	5600	7.4	7.2	6	19.4	7.0
control 1		5.0	2.8	4	19.0	7.7
Control 2		4.7	3.1	3	19.4	7.7
Physico-chemical oxygen consumption control	10000	7.9	7.9	7	19.2	6.2

Validity criteria for the measurement of the bacterial toxicity

Target condition according to guideline:	Actual condition according to the study:	Validity criteria met:
The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. If the rate is lower, the test should be repeated with washed activated sludge or with the sludge from another source. The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	The oxygen uptake rate of the controls were 32.0 and 33.0 mg/L*h (960 mg/L suspended solids).	Yes
Reference substances In a 2004 international ring test organized by ISO using activated sludge derived from domestic sewage, the EC50 of 3,5-dichlorphenol was found to lie in the range 2 mg/L to 25 mg/L for total respiration, 5 mg/L to 40 mg/L for heterotrophic respiration and 0.1 mg/L to 10 mg/L for nitrification respiration. If the EC50 of 3,5-DCP does not lie in the expected range, the test should be repeated with activated sludge from another source. The EC50 of copper (II) sulphate pentahydrate should lie in the range of 53-155 mg/L for the total respiration.	The EC50 value for the reference substance (3,5-DCP; total respiration) was 9.3 mg/L (confidence limits were 7.9 mg/L and 11.1 mg/L).	Yes
The test should be performed at a temperature within the range 20±2°C.	During the test the temperature in the test vessels was maintained in the range of 19.0 C° to 19.4 C°.	Yes

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on results incl. tables'.

Conclusions:

The test substance showed 93.8% respiration inhibition of activated sludge at a test item concentration of 5600 mg/L; EC50=737.2 mg/L (calculated concentration).

Executive summary:

The study was performed to assess the toxicity of the test substance to bacteria. The study was conducted in accordance with the Commission Directive 88/302/EEC Part C, Method 11 (1988). The activated sludge was exposed to the test substance at different concentration (560, 1000, 1800, 3200, 5600 mg/L; nominal concentration). The respiration rate of each mixture was determined after aeration period of 3 hours. The test substance showed 93.8% respiration inhibition of activated sludge at a test item concentration of 5600 mg/L. The following value was determined EC50=737.2 mg/L (calculated concentration). 

Description of key information

A study was performed to assess the toxicity of the test substance to bacteria. the study was conducted in accordance with the Commission Directive 88/302/EEC Part C, Method 11 (1988). The activated sludge was exposed to the test substance at different concentration (560, 1000, 1800, 3200, 5600 mg/L; nominal concentration). The respiration rate of each mixture was determined after aeration period of 3 hours. The test substance showed 93.8% respiration inhibition of activated sludge at a test item concentration of 5600 mg/L. The following value was determined EC50=737.2 mg/L (calculated concentration). 

Key value for chemical safety assessment

EC50 for microorganisms:

737.2 mg/L