4-chlorophthalic anhydride

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other:

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Remarks:

OECD GLP

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:

yes

Details on sampling:

Collected from each replicate test vessel at start and end of the test

Test solutions

Vehicle:

no

Details on test solutions:

Water used for the acclimation of test organisms and for all toxicity testing was carbon filtered, deionized water collected at T.R. Wilbury Laboratories in Marblehead, Massachusetts. Water was adjusted to a hardness of 160 to 180 mg/L as CaCO3 and stored in polyethylene tanks where it was aerated and recirculated through particle filters, activated carbon, and an ultraviolet sterilizer. Prior to use, the pH of the dilution water was adjusted to less than 8.0. A sample of dilution water collected at the start of the definitive test had a hardness of 160 mg/L as CaCO3. A stock solution with a nominal concentration of 1,000 mg/L was prepared by bringing 2.1322 g of test substance to a total volume of 2,000 mL with dilution water in a glass volumetric flask. The solution was mixed for approximately 23 hours. The pH of this solution was adjusted from 4.3 to 7.2 using 1N sodium hydroxide. Appropriate amounts of the stock solution were added directly to dilution water to formulate the test concentrations. Nominal concentrations of the active ingredient were 0 mg/L (control), 150, 250, 400, 600 and 1,000 mg/L.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Juvenile daphnids, Daphnia magna, less than 24 hours old, employed as test organisms were from a single source and were identified using an appropriate taxonomic key. They were produced by an in-house culture of adult daphnids that were maintained under test conditions at T.R. Wilbury Laboratories. The original culture was obtained from Aquatic Biosystems Inc., Fort Collins, Colorado, on November 6, 2001. At the conclusion of the test the control daphnids had an average wet weight of 0.32 mg (loading rate during the definitive toxicity test was ≤40 daphnids per liter). Prior to testing, the daphnid culture was maintained in 100% dilution water under static, renewal conditions, and the test organisms were not treated for disease. No mortality was observed during the 48 hours prior to the beginning of the test and at the beginning of the test organisms were apparently free of disease, injuries, and abnormalities. The culture used to produce neonates for the definitive toxicity test produced young by day 7 and produced an average of at least 3 young/female/day during the 7 days prior to the start of the test. During the 18 days prior to the beginning of the test, the temperature of the culture ranged from 19.0 to 20.6°C, the pH ranged from 7.1 to 7.3, and the dissolved oxygen concentration was always at least 8.4 mg/L. Test organisms were fed the freshwater algae, Selenastrum capricornutum, and a mixture of yeast and trout chow once each day before the test. Daphnids were not fed during the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Test conditions

Hardness:

160 mg/L as CaCO3 (at study start)

Test temperature:

19.7 - 20.7°C

pH:

7.0 - 7.2

Dissolved oxygen:

8.2 - 9.0 mg/L

Nominal and measured concentrations:

Nominal: 0 mg/L (control), 150, 250, 400, 600 and 1,000 mg 4-CLPA/L
Mean measured: ND (not detected at or above the LOQ; control), 148, 255, 401, 597 and 982 mg 4 CLPA/L

Details on test conditions:

A range-finding test with 4-CLPA was conducted under static conditions with a control, a solvent control (0.10 mL/L dimethylformamide), and four concentrations of test substance: 0.10, 1.0, 10, and 20 mg/L. After 48 hours there was 100% survival in the control and solvent control, and at all tested concentrations. No sublethal effects were observed at any tested concentration. A small amount of fine white particulates was observed in the test vessels with a nominal concentration of 20 mg/L at 0 and 48 hours. No other insoluble material was observed.

A second range-finding test with 4-CLPA was conducted under static conditions with a control and two concentrations of test substance: 100 and 1,000 mg/L. A stock solution with a nominal concentration of 1,000 mg/L was prepared without the use of a solvent and allowed to mix for approximately 24 hours. The pH of the stock solution was adjusted from 5.5 to 7.0 using 0.1N sodium hydroxide. After 48 hours there was 100% survival in the control and at both tested concentrations. No sublethal effects or insoluble material were observed at either tested concentration.

The definitive toxicity test was conducted under static conditions at 20 +/- 1°C with five concentrations of test substance and a dilution water control. A stock solution with a nominal concentration of 1,000 mg/L was prepared by bringing 2.1322 g of test substance to a total volume of 2,000 mL with dilution water in a glass volumetric flask. The solution was mixed for approximately 23 hours. The pH of the stock solution was adjusted from 4.3 to 7.2 using 1N sodium hydroxide. Appropriate amounts of the stock solution were added directly to dilution water to formulate the test concentrations. Nominal concentrations of the active ingredient were 0 mg/L (control), 150, 250, 400, 600, and 1,000 mg/L.

Twenty daphnids were randomly distributed among two replicates of each treatment. The test was performed in 300 mL glass beakers that contained 250 mL of test solution (water depth was approximately 9 cm). Test vessels were randomly arranged in a water bath during the 48-hour test (random numbers tables were used to add test organisms to test vessels, to select the location of each vessel, as well as the assignment of test concentrations). A 16 hour light and 8 hour dark photoperiod was automatically maintained with cool white fluorescent lights that provided a light intensity of approximately 37 footcandles. A 15 minute transition period was provided between dark and light. Aeration was not required to maintain dissolved oxygen concentrations above acceptable levels. The numbers of surviving organisms, the occurrence of immobility and sublethal effects, and the presence of insoluble material were determined visually and recorded after 0, 24, and 48 hours. Dead test organisms were removed when first observed. Dissolved oxygen, pH, conductivity, and temperature were measured and recorded daily in each test vessel. The temperature in a representative vessel was recorded continuously during the test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 982 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 982 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

982 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Effect concentrations 4

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 982 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 982 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

No insoluble material was observed during the definitive toxicity test. Nominal concentrations of 4-CLPA were: 0 mg/L (control), 150, 250, 400, 600 and 1,000 mg/L. Mean measured concentrations were: ND (not detected; control), 148, 255, 401, 597 and 982 mg/L. The mean measured concentrations were 98 to 102% of the nominal concentration. One hundred percent survival occurred in the control and at all tested concentrations, and no sublethal effects were observed.

Reported statistics and error estimates:

Results of the toxicity test could not be interpreted by standard statistical techniques because 100% of the organisms exposed to each tested concentration survived the toxicity test without sublethal effects. The no observed effect concentration is the highest tested concentration of test substance that did not cause toxicant-related mortality or sublethal effects.

Any other information on results incl. tables

Measured concentrations of 4-CLPA during the toxicity test with the daphnid, Daphnia magna.

Nominal Concentration of 4-CLPA (mg/L)	Measured Concentration of 4-CLPA (mg/L)
	0 Hour	48 Hours	Mean	% Recovery
Test Media Samples
Control	ND	ND	ND	--
150	150	150	148	99
	147	145
250	253	255	255	102
	255	258
400	389	401	401	100
	411	402
600	592	595	597	100
	602	600
1,000	967	986	982	98
	989	987
Laboratory Control Sample 1,200
400	376	372	380	95
	398	383
	393	355
Blank
0	ND	---	ND	---
ND = none detected at or above the limit of quantitation of 0.103 mg/L.

Survival and sublethal effect data from the toxicity test with 4-CLPA and the daphnid, Daphnia magna

Mean Measured Concentration of 4-CLPA  (mg/L)	R	Number Alive			Number Affected
		Hour of Exposure
		0	24	48	0	24	48
Control	1	10	10	10	0	0	0
	2	10	10	10	0	0	0
148	1	10	10	10	0	0	0
	2	10	10	10	0	0	0
255	1	10	10	10	0	0	0
	2	10	10	10	0	0	0
401	1	10	10	10	0	0	0
	2	10	10	10	0	0	0
597	1	10	10	10	0	0	0
	2	10	10	10	0	0	0
982	1	10	10	10	0	0	0
	2	10	10	10	0	0	0
R: Replicate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48-h EC50 was determined to be >1000 mg/L.

Executive summary:

The acute toxicity to aquatic invertebrates was determined in an OECD OECD 202 study and in compliance with GLP criteria. In this study daphnids, (D. magna, in total 20 per concentration) were exposed to nominal concentrations of 0, 150, 250, 400, 600 and 1,000 for 48 hours under static conditions. The nominal test concentration was analytically verified at the start and end of exposure and remained within ±20% of nominal. Therefore, the effect value is expressed as nominal. Immobilization was recorded after 24 and 48 hours exposure. No immobilization was observed in the test. Based on these findings, the 48-h EC50 value was determined to be >1000 mg/L.