3-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:

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 201 (Alga, Growth Inhibition Test)

Version / remarks:

1992

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:

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 0.9 ppm. A stock solution with a nominal concentration of 1,200 mg/L was prepared by bringing 1.2231 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 22 hours. The pH of the stock solution was adjusted to 7.5. The test concentrations were formulated by the addition of appropriate amounts of the stock solution directly to dilution water. Nominal concentrations of 3-chlorophthalic anhydride were 0 mg/L (control), 75, 150, 300, 600, and 1,200 mg/L. A stability blank sample set up at 1,200 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 23.5 to 24.3°C and the light intensity ranged from 50 to 51 microEin/m2sec (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 five 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

Test conditions

Hardness:

Not reported

Test temperature:

23.9 to 24.3°C

pH:

7.5 - 7.6 initial; 8.3 - 9.8 final

Nominal and measured concentrations:

Nominal: 0 mg/L (control) 75, 150, 300, 600, and 1,200 mg 3-CLPA/L
Measured: 0 mg/L (control), 71, 150, 290, 560, and 1,100 mg 3-CLPA/L

Details on test conditions:

A range-finding test was conducted with a control and five concentrations of 3-CLPA: 0.10, 1.0, 10, 100, and 1,000 mg/L. At the conclusion of the test, the number of cells/mL in the test vessels was greater than 80% of the control growth at all five tested concentrations. 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 3-CLPA were 0 mg/L (control), 75, 150, 300, 600, and 1,200 mg/L. A stability blank sample set up at 1,200 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 380 footcandles (also measured as approximately 50 to 51 microEin/m2sec). 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):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: similar EC50 when based on the number of cells and AUC

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

560 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Similar NOEC when based on the number of cells

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

290 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

other: AUC

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1 100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

Insoluble material was not observed at any time during the definitive toxicity test. Nominal concentrations of 3-chlorophthalic anhydride were: 0 mg/L (control), 75, 150, 300, 600, and 1,200 mg/L. Mean measured concentrations were ND (none detected; control), 71, 150, 290, 560, and 1,100 mg/L. These mean measured concentrations were 92 to 100% of nominal concentrations and stable throughout the 72 hour exposure. The mean measured concentration in the stability blank (a 1,200 mg/L 3 chlorophthalic anhydride solution without algae that was incubated under test conditions for 72 hours) was 100% of the nominal concentration.

The algal population grew well, resulting in an average of 1,088,000 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 23.9 to 24.3°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, growth rates, and areas under the growth curve 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 Dunnett’s test. The NOECs were determined using the mean measured concentrations of 3-CLPA and the number of cells/mL, the average specific growth rate, and the area under the growth curve. Treatment data were compared to control data for the determination of the NOEC values.

Any other information on results incl. tables

Measured concentrations of 3-chlorophthalic anhydride during the toxicity test with the freshwater alga, Selenastrum capricornutum

Nominal Concentration of 3-CLPA (mg/L)	Measured Concentration of 3-CLPA (mg/L)
	0 Hour	72 Hour	Mean	% Recovery
Test Media Samples
Control	ND	---	ND	---
75	73	69	71	95
150	150	140	150	100
300	290	280	290	97
600	570	540	560	93
1,200	1,100	1,100	1,100	92
Stability Blank
1,200	1,200	1,200	1,200	100
Laboratory Control Sample
300	290	280	290	97
Matrix Spike Sample
300	--	280
	--	280	280	93
Blank
0	ND	---	ND	---
ND = none detected at or above the limit of quantitation of 0.033 mg/L.

Cell growth data from the toxicity test with 3-chlorophthalic anhydride and the freshwater alga, Selenastrum capricornutum

Measured Concentration of 3-CLPA (mg/L)	Replicate	Number of Cells per Milliliter
		Hour of Exposure
		0	24	48	72
Control	1	10,000	64,000	380,000	970,000
	2	10,000	60,000	230,000	1,228,000
	3	10,000	62,000	196,000	1,066,000
	mean	10,000	62,000	269,000	1,088,000
71.0	1	10,000	68,000	230,000	1,274,000
	2	10,000	62,000	214,000	1,198,000
	3	10,000	58,000	204,000	1,116,000
	mean	10,000	63,000	216,000	1,196,000
	% control	100	102	80	110
150	1	10,000	62,000	210,000	1,030,000
	2	10,000	66,000	200,000	1,124,000
	3	10,000	56,000	200,000	1,084,000
	mean	10,000	61,000	203,000	1,079,000
	% control	100	98	75	99
290	1	10,000	52,000	210,000	1,048,000
	2	10,000	66,000	190,000	1,078,000
	3	10,000	62,000	200,000	1,092,000
	mean	10,000	60,000	200,000	1,073,000
	% control	100	97	74	99
560	1	10,000	49,000	240,000	916,000
	2	10,000	48,000	150,000	944,000
	3	10,000	58,000	250,000	1,010,000
	mean	10,000	52,000	213,000	957,000
	% control	100	84	79	88
1,100	1	10,000	42,000	204,000	848,000
	2	10,000	43,000	160,000	756,000
	3	10,000	50,000	170,000	766,000
	mean	10,000	45,000	178,000	790,000
	% control	100	73	66	73

Average specific growth rate from the toxicity test with 3-chlorophthalic anhydride and the freshwater alga, Selenastrum capricornutum

Measured Concentration of 3-CLPA(mg/L)		Average Specific Growth Rate
		Hour of Exposure
		24	48	72
Control	mean	0.076	0.069	0.065
71	mean	0.077	0.064	0.066
	% control	101	93	102
150	mean	0.075	0.063	0.065
	% control	99	91	100
290	mean	0.075	0.062	0.065
	% control	99	90	100
560	mean	0.069	0.064	0.063
	% control	91	93	97
1,100	mean	0.063	0.060	0.061
	% control	83	87	94

Area under the growth curve from the toxicity test with 3-chlorophthalic anhydride and the freshwater alga, Selenastrum capricornutum

Measured Concentration of 3-CLPA (mg/L)		Area Under the Growth Curve
		Hour of Exposure
		24	48	72
Control	mean	624,000	4,356,000	20,400,000
71	mean	636,000	3,744,000	20,448,000
	% control	102	86	100
150	mean	612,000	3,540,000	18,684,000
	% control	98	81	92
290	mean	600,000	3,480,000	18,516,000
	% control	96	80	91
560	mean	504,000	3,444,000	17,244,000
	% control	81	79	85
1,100	mean	420,000	2,856,000	14,232,000
	% control	67	66	70

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EC50 value was determined to be >1200 mg/L and the 72-h NOEC value was 560 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, 75, 150, 300, 600 and 1200 mg/L for 72 hours under static conditions. The test was performed in 3 replicates per test concentration. 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 mean value of the cell concentration for each test concentration was plotted against time to produce growth curves from which the percentage inhibition of biomass (i.e.cell growth as calculated by comparison of Areas Under the Growth curves (AUG)) and the average specific growth rate for exponentially growing cultures was calculated. The 72-h EC50 value for growth rate (ErC50) was determined at >1100 mg/L. The 72 -h NOEC value was 560 mg/L.