4-chlorophthalic anhydride

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Version / remarks:

1992

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:

yes

Details on sampling:

Start (from stock solutions prior to distribution to test chambers) and end of the test (from pooled test replicates)

Test solutions

Vehicle:

no

Details on test solutions:

Water used for acclimation of test organisms and for all toxicity testing was sterile freshwater AAP medium adjusted to pH of 7.5 +/- 0.1. Dilution water collected at the start of the definitive toxicity test had a measured total organic carbon concentration of 4.6 ppm. A stock solution with a nominal concentration of 1,000 mg/L was prepared by bringing 1.0661 g of test substance to a total volume of 1,000 mL with dilution water in a glass volumetric flask. The solution was mixed for approximately 27 hours. The pH of the stock solution was adjusted to 7.5 using 1 N sodium hydroxide. The test concentrations were formulated by the addition of appropriate amounts of the stock solution directly to dilution water. Nominal concentrations of 4 CLPA were 0 mg/L (control), 65, 130, 250, 500, and 1,000 mg/L. A stability blank sample set up at 1,000 mg/L was placed among the test vessels.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Algae used for the test (Selenastrum capricornutum, UTEX 1648) were from a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin and delivered to T.R. Wilbury Laboratories on July 17, 2001. The culture was transferred to sterile enriched media identical to media used for this test and maintained at test conditions for at least 14 days before the definitive test. During the 14 day acclimation period prior to the start of the test, the temperature ranged from 23.8 to 24.0°C and the light intensity ranged from 50 to 51 microEin/m^2sec (24 hours light/0 hours dark photoperiod). During the acclimation period, the culture was actively growing in at least two subcultures prior to the start of the toxicity test. The subsample of algae used to inoculate media at the start of the definitive test came from a four day old culture. Identification of the culture organisms, which are also referred to as Raphidocelis subcapitata, was verified using an appropriate taxonomic key.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Test temperature:

24.0 - 24.5°C

pH:

7.4 - 7.5 initial; 7.8 - 9.5 final

Nominal and measured concentrations:

Nominal: 0 mg/L (control) 65, 130, 250, 500, and 1,000 mg 4-CLPA/L
Mean measured: ND (not detected at or above the LOQ; control), 59.7, 124, 239, 478 and 933 mg 4 CLPA/L

Details on test conditions:

A range-finding test was conducted with a control, solvent control (0.1 ml/L dimethylformamide), and four concentrations of 4 CLPA: 0.10, 1.0, 10, and 20 mg/L. At the conclusion of the test, the number of cells/mL in the test vessels qualled the following percent of the number of cells/mL in the control vessels: solvent control = 100%, 0.10 mg/L = 98%, 1.0 mg/L = 106%, 10 mg/L = 82%, and 20 mg/L = 103%. Fine white particles were observed on the media surface in test vessels with a nominal concentration of 20 mg/L at the start of the test. No other insoluble material was observed during the range-finding test.

A second range-finding test was conducted with a control and two concentrations of 4 CLPA: 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 to 7.5 using 0.1N sodium hydroxide. At the conclusion of the test, the number of cells/mL in the test vessels equaled the following percent of the number of cells/mL in the control vessels: 100 mg/L = 106% and 1,000 mg/L = 104%. No insoluble material was observed during the range-finding test.

The definitive toxicity test was conducted at 24 +/- 2°C with five concentrations of test substance and a control. Nominal concentrations of 4-CLPA were 0 mg/L (control), 65, 130, 250, 500, and 1,000 mg/L. A stability blank sample set up at 1,000 mg/L was placed among the test vessels. No algae was inoculated into the stability blank. Water quality measurements were made and each solution was subdivided into three clear glass 250 mL Erlenmeyer flasks. Flasks were inoculated with approximately 10,000 algal cells/mL and capped with inverted glass beakers. Test vessels were randomly arranged on a rotary shaker adjusted to approximately 100 rpm in an incubator during the test (a random numbers table was used to select the location for each vessel) and vessels were repositioned daily. A 24 hour light and 0 hour dark photoperiod was automatically maintained with cool white fluorescent lights that provided a light intensity of approximately 400 footcandles (also measured as approximately 52 to 55 microEin/m^2sec). The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a hemocytometer at 24, 48, and 72 hours. Temperature of the incubator was measured and recorded daily and the temperature in a representative vessel of water incubated with the test vessels was continuously recorded. The pH of test solutions was measured and recorded in each test solution prior to distribution to the test vessels at the start of the test and in each test vessel at the end of the test.

Reference substance (positive control):

not required

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 933 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Number of cells/mL

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

478 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Number of cells/mL

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 933 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Number of cells/mL

Effect concentrations 4

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 933 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Number of cells/mL

Details on results:

Insoluble material was not observed at any time during the definitive toxicity test. Nominal concentrations of 4-CLPA were: 0 mg/L (control), 65, 130, 250, 500, and 1,000 mg/L. Mean measured concentrations were ND (none detected; control), 59.7, 124, 239, 478, and 933 mg/L. These mean measured concentrations were 92 to 96% of nominal concentrations. The mean measured concentration in the stability blank (a 1,000 mg/L 4 CLPAsolution without algae that was incubated under test conditions for 72 hours) was 95% of the nominal concentration.

The algal population grew well, resulting in an average of 1,230,000 cells/mL in the control after 72 hours. No effects (relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells) were noted during the test. Water quality throughout the test was within acceptable limits. The incubator temperature ranged from 24.0 to 24.5°C

Reported statistics and error estimates:

The average specific growth rate was calculated as the natural log of the number of cells/mL at time t1 minus the natural log of the number of cells/mL at time t0 divided by the exposure period. The 24, 48, and 72 hour EC50 values could not be calculated using standard statistical procedures because cell growth and growth rates at all tested concentrations were greater than 50% of the control. The no observed effect concentration (NOEC) was determined using a one-way analysis of variance (ANOVA) and William’s test. The NOECs were determined using the mean measured concentrations of 4-CLPA and the number of cells/mL and average specific growth rate. Treatment data were compared to control data for the determination of the NOEC values.

Any other information on results incl. tables

Measured concentrations of 4-CLPA during the toxicity test with the freshwater alga, Selenastrum capricornutum

Nominal Concentration of 4-CLPA (mg/L)	Measured Concentration of 4-CLPA (mg/L)
	0 Hour	72 Hour	Mean	% Recovery
Test Media Samples
Control	ND	---	ND	---
65	59.3	60	59.7	92
130	123	124	124	95
250	234	244	239	96
500	461	495	478	96
1,000	907	959	933	93
Stability Blank
1,000	925	982	954	95
Laboratory Control Sample
250	196	228	212	85
Blank
0	ND	---	ND	---
ND = none detected at or above the limit of quantitation of 0.0251 mg/L.

Cell growth data from the toxicity test with 4-CLPA and the freshwater alga, Selenastrum capricornutum

Measured Concentration of 4-CLPA (mg/L)	Replicate	Number of Cells per Milliliter
		Hour of Exposure
		0	24	48	72
Control	1	10,000	58,000	132,000	1,180,000
	2	10,000	52,000	180,000	1,270,000
	3	10,000	50,000	186,000	1,240,000
	mean	10,000	53,000	166,000	1,230,000
59.7	1	10,000	64,000	238,000	1,260,000
	2	10,000	54,000	258,000	1,270,000
	3	10,000	56,000	222,000	1,230,000
	mean	10,000	58,000	239,000	1,253,000
	% control	100	109	144	102
124	1	10,000	48,000	248,000	1,300,000
	2	10,000	46,000	200,000	1,150,000
	3	10,000	54,000	214,000	1,100,000
	mean	10,000	49,000	221,000	1,183,000
	% control	100	92	133	96
239	1	10,000	50,000	226,000	970,000
	2	10,000	58,000	258,000	1,160,000
	3	10,000	54,000	228,000	1,570,000
	mean	10,000	54,000	237,000	1,233,000
	% control	100	102	143	100
478	1	10,000	48,000	224,000	1,160,000
	2	10,000	58,000	298,000	1,140,000
	3	10,000	48,000	248,000	1,170,000
	mean	10,000	51,000	257,000	1,157,000
	% control	100	96	155	94
933	1	10,000	42,000	182,000	860,000
	2	10,000	52,000	216,000	1,030,000
	3	10,000	56,000	224,000	840,000
	mean	10,000	50,000	207,000	910,000
	% control	100	94	125	74

Average specific growth rate from the toxicity test with 4-chlorophthalic anhydride and the freshwater alga, Selenastrum capricornutum

Measured Concentration of 4-CLPA(mg/L)		Average Specific Growth Rate
		Hour of Exposure
		24	48	72
Control	mean	0.069	0.059	0.067
59.7	mean	0.073	0.066	0.067
	% control	106	112	100
124	mean	0.066	0.064	0.066
	% control	96	108	99
239	mean	0.070	0.066	0.067
	% control	101	112	100
478	mean	0.068	0.068	0.066
	% control	99	115	99
933	mean	0.067	0.063	0.063
	% control	97	107	94

Applicant's summary and conclusion

Conclusions:

The 72-h EC50 value was determined to be >1000 mg/L and the 72-h NOEC value was 500 mg/L.

Executive summary:

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, exponentially growing freshwater alga (Pseudokirchneriella subcapitata) were exposed to nominal test substance concentrations of 0, 65, 130, 250, 500, and 1,000 mg/L for 72 hours under static conditions. Test concentrations were analytically verified at the start and the end of exposure and measured concentrations remained within ±20% of nominal. The cell concentration in each flask was determined at the start of the test and after 24, 48, and 72 hours exposure. The 72-h ErC50 value for growth rate (ErC50) and biomass (cells/mL) was determined at >1000 mg/L. The 72 hour NOEC is 500 mg/L 4-chlorophthalic anhydride when determined using the number of cells/mL or the average specific growth rate when treatment data are compared to control data.