4-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:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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:

4-chlorophthalic anhydride

EC Number:

204-251-9

EC Name:

4-chlorophthalic anhydride

Cas Number:

118-45-6

Molecular formula:

C8-H3-Cl-O3

IUPAC Name:

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

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution was prepared at a nominal concentration of 500 mg/L by bringing 0.5330 grams of the test substance, 4-CLPA, to a total volume of 1,000 mL with deionized water in a 1,000 mL class A volumetric flask. The pH of the stock solution was adjusted from an initial pH of 3.7 to 7.7 using 1N sodium hydroxide and allowed to mix overnight on a magnetic stir plate. Nominal concentrations [0 (control), 3.7, 11, 33, 100, and 280 mg/L] were formulated by the addition of appropriate volumes of the stock solution to 500 mL of test solution without the use of a solvent.

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 6.3 g/L. The sludge was incubated at room temperature and aerated prior to use. It was adjusted to a suspended solids content of 4.3 g/L on the day of use for the definitive test. 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 adjusted to 7.7 with 1N sodium hydroxide at the time of use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

19.1 - 20.3°C

pH:

7.7

Dissolved oxygen:

Initial: 8.2 - 9.1 mg/L; Final: 2.4 - 3.8 mg/L

Nominal and measured concentrations:

0 (control), 3.7, 11, 33, 100, and 280 mg/L

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 less than 1 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 an appropriate amount of the 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 (Nominal concentrations of 5.1, 12, and 30 mg/L)

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 19.1 to 20.3°C.

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 4-chlorophthalic anhydride was greater than 280 mg/L, the highest tested concentration. Insoluble material was not observed at any tested concentration during the test.

Results with reference substance (positive control):

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 9.4 mg/L and within the acceptable range of 5 to 30 mg/L.

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 probit method.

Any other information on results incl. tables

Activated sludge respiration data from the test with 4-CLP

Nominal Concentration of 4-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	9.1	3.2	4.5	--	35	--
	2	8.6	2.8	3.0	--	35	--
3.7	1	8.6	2.9	4.0	--	34	3
	2	8.9	3.7	4.0	--	31	11
11	1	8.7	3.8	4.5	--	29	17
	2	8.6	3.6	4.0	--	30	14
33	1	8.6	2.6	3.5	--	36	-3
	2	8.7	3.2	4.0	---	33	6
100	1	8.2	2.4	3.0	10.0	35	0
	2	8.7	3.3	4.0	--	32	9
280	1	8.6	3.2	4.0	--	32	9
	2	8.5	2.8	3.5	--	34	3

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), 3.7, 11, 33, 100, 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. The 3-h EC50 was determined to be >280 mg/L.