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Ecotoxicological information

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

Constituent 1
Chemical structure
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 concentrationsopen allclose all
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
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
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
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
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.