3-chlorophthalic anhydride

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-chlorophthalic anhydride

EC Number:

204-179-8

EC Name:

3-chlorophthalic anhydride

Cas Number:

117-21-5

Molecular formula:

C8H3ClO3

IUPAC Name:

4-chloro-1,3-dihydro-2-benzofuran-1,3-dione

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

The test substance was used to prepare a 500 mg/L stock solution in deionized water and the pH was adjusted from 3.2 to 7.0 using 1.0 N sodium hydroxide. The stock solution was mixed overnight and the pH was adjusted from 3.7 to 7.4. This solution was then diluted with deionized water to prepare test solutions at nominal concentrations of 3.3, 10, 31, 93 and 280 mg/L.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge used for the test was obtained from the municipal wastewater treatment plant at Merrimac, Massachusetts. This treatment plant treats predominantly domestic waste. As collected, the suspended solids concentration of the sludge used in the definitive test was 4.8 g/L. The sludge was incubated at room temperature and aerated prior to use. It was adjusted to a suspended solids content of approximately 3.8 g/L. Because the sludge was not used the day it was collected, synthetic sewage, consisting of 16 g/L peptone, 11 g/L meat extract, 3 g/L urea, 0.7 g/L NaCl, 0.34 g/L CaCl2, 0.2 g/L MgSO4 . 7H2O, and 2.8 g/L K2HPO4 was combined with the sludge at the rate of approximately 50 mL/L. The pH of the sludge was 8.1 on the day of collection and was adjusted to 7.1 on the day of the activated sludge respiration inhibition test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

20.0 - 20.9 °C

pH:

7.1

Nominal and measured concentrations:

0 (control), 3.3, 10, 31, 93 and 280 mg/L (nominal)

Details on test conditions:

Water used for dilution of the sludge and for all toxicity testing was deionized water collected at Marblehead, Massachusetts with a hardness of 4 mg/L as CaCO3. The test was conducted under static conditions at 20 +/- 2°C. At time 0 for each test vessel, 16 mL of synthetic sewage and the appropriate amount of the 500 mg/L test substance stock solution were brought up to 300 mL in dilution water in a 1.5 liter glass beaker. A 200 mL aliquot of microbial inoculum was added to this mixture within 5 minutes of its preparation. Test vessels were established at 15 minute intervals and vessels were aerated with 0.5 to 1.0 liters of air per minute for 3 hours. The first and last vessels established were controls. After the 3 hour incubation period, a portion of the contents of each test vessel were transferred to a BOD bottle equipped with a magnetic stirrer and the dissolved oxygen concentration in this sealed bottle was monitored for 10 minutes. Test vessels were maintained under ambient laboratory lighting during the test. Dissolved oxygen was measured.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 280 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The control respiration rates were within 15% of each other and the temperature during the test was 20.0 to 20.9°C. The EC50 value determined during a reference toxicant test with the batch of activated sludge used for the definitive test and 3,5 dichlorophenol was 11 mg/L and within the acceptable range of 5 to 30 mg/L.

The test substance did not inhibit respiration of the activated sludge in the tested range of concentrations. The 3-hour EC50 for activated sludge exposed to 3-chlorophthalic anhydride was greater than 280 mg/L. Insoluble material was not observed during the test.

Reported statistics and error estimates:

The respiration rate was calculated as the mg oxygen/liter/hour consumed between 6.5 (or the initial dissolved oxygen concentration if it was less than 6.5 mg/L) and 2.5 mg/L or over a 10 minute period if the respiration rate was low. The percent inhibition, which was used to determine the EC50 values, was calculated as (1 minus [2 times the respiration rate of the test substance concentration] divided by the sum of the 2 control respiration rates) times 100.

The 3-hour EC50 value for the definitive activated sludge respiration inhibition test could not be calculated for the test substance by standard statistical techniques because there was less than 50% inhibition at all tested concentrations. The 3-hour EC50 value for the reference substance was calculated by the binomial method.

Any other information on results incl. tables

Activated sludge respiration data from the test with 3-Chlorophthalic Anhydride

Nominal Concentration of 3-CLPA (mg/L)	Rep.	Dissolved Oxygen Concentration (mg/L)		Time to 6.5 mg/L Oxygen (mins)	Time to 2.5 mg/L Oxygen (mins)	Respiration Rate (mg/L/hr)	Inhibition (%)
		Initial	Final
0 (control)	1	7.4	0.2	1.0	6.0	48	--
	2	7.7	0.2	2.0	6.5	53	--
3.3	1	7.6	0.2	1.5	6.0	53	-5
	2	7.5	0.2	1.0	5.5	53	-5
10	1	7.6	0.2	1.5	6.0	53	-5
	2	7.9	0.2	1.5	6.5	48	5
31	1	8.6	0.2	2.5	7.0	53	-5
	2	8.3	0.2	2.0	7.0	48	5
93	1	8.2	0.2	2.0	7.0	48	5
	2	7.8	0.2	1.5	6.5	48	5
280	1	8.0	0.2	1.5	6.0	53	-5
	2	7.9	0.2	1.5	6.5	48	5

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 3-h EC50 value for microorganisms in activated sludge is >280 mg/L.

Executive summary:

The toxicity of the substance to microorganisms was investigated in an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria. In this study non-adapted activated sludge from a predominantly domestic sewage was exposed to nominal test substance concentrations of 0 (control), 1.0, 3.3, 10, 31, 93 and 280 mg/L for 3 hours. Test concentrations were not analytically verified. The effects on microorganisms was determined by measuring total respiration rates and comparing these to an unexposed control. No inhibition in respiration of the activated sludge was observed at any of the tested concentrations. Also, no insolubility of the test material was observed. The 3-h EC50 was determined to be >280 mg/L.