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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Water samples were collected at test initiation (0 h) and at test end (72 h), combining samples from the 3 replicates for each test concentration, with a total of 100 ml sample per test concentration used for analysis.
Vehicle:
no
Details on test solutions:
A stock solution was prepared by dissolving m-toluic acid in OECD TG 201 medium. This stock solution was diluted to prepare appropriate test solutions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: ATCC22622
- Source: American Type Culture Collection
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The stock cultures are maintained fulfilling the criteria of the OECD 201 guideline.

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions: same as test (OECD TG 201 medium; T: 23 ± 2 °C; continuous light, 4000-5000 lux)
- Any deformed or abnormal cells observed: If deformed or abnormal cells occurred, cultures were not used for testing
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23.9-24.0 °C
pH:
7.6-10.4
Nominal and measured concentrations:
Nominal: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46, 100 mg/L
Measured (at 0 h): < 0.05 (control), 1.13, 2.53, 5.33, 11.3, 25.2, 52.3, 114 mg/L
Measured (at 72 h): < 0.05 (control, 1.08, 2.41, 5.01, 10.8, 23.9, 47.8, 105 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 ml glass Erlenmeyer flask
- Type: closed
- Fill volume: 100 ml test medium
- Initial cells density (0 h): 10000 cells/ml
- Control end cells density (72 h): 685200 cells/ml (SD: 58000 cells/ml)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201 medium (Amount of FeCl3 * 6 H2O: 0.08 mg/L instead of 0.064 mg/L)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Shaking: 100 rpm
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 4000-5000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Coulter Z), every 24 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ≤ 2.2
- Range finding study: yes
- Test concentrations: 1.0, 10, 100 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL: 15-21 mg/L; Based on specific growth rate from 24 h to 72 h
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Based on specific growth rate from 24 h to 72 h
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CL: 8.4-12 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Growth in control vessels was exponential. Measured concentrations were at 104-116 % of the nominal values. A decrease in pH could be observed with increasing concentrations of the test substance. Further, an increase in pH over the test period of 72 h could be observed for all test concentrations as well as for the control (see table below). pH of the controls and the treatment groups with concentrations ranging from 1.0 to 4.6 mg/L at termination (72 h) were 1.5 pH units higher than the corresponding pH values at test start. Since all validity criteria of the test were met and no growth inhibition > 10% were observed for these treatment groups, this pH increase is considered not to have influenced the validity of the test.
Results with reference substance (positive control):
EC50 (72 h): 0.90 mg/L (November 1998)
EC50 (72 h): 0.82 mg/L (June 1999)
Reported statistics and error estimates:
EC50 values were calculated using linear regression analysis. NOEC values were calculated using Dunnett's multiple comparison method (p < 0.05).

Table 1: Nominal and measured concentrations of m-toluic acid at 0 and 72 h

Nominal conc. (mg/L)

Measured conc. (mg/L)

Time-weighted mean (mg/L)

Percent of nominal

0 h

72 h

0 h

72 h

Control

< 0.05

< 0.05

-

-

-

1.0

1.13

1.08

1.1

113

108

2.2

2.53

2.41

2.47

115

110

4.6

5.33

5.01

5.17

116

109

10

11.3

10.8

11.0

113

108

22

25.2

23.9

24.5

115

109

46

52.3

47.8

50.0

114

104

100

114

105

109

114

105

Table 2: pH value of test solutions at 0 and 72 h

Nominal conc. (mg/L)

pH

0 h

72 h

Control

7.9

9.9

1.0

8.0

10.4

2.2

7.9

10.3

4.6

7.9

9.8

10

7.8

9.3

22

7.6

7.8

46

7.3

7.5

100

6.4

6.7

Table 3: Temperature in the incubation chamber over the 72 h test period

Exposure period (h)

Temperature (°C)

0

23.9

24

23.9

48

23.9

72

24.0

Table 4: Percentage inhibition of cell growth over the 72 h exposure period

Nominal conc. (mg/L)

Area under growth curve (average) (0-72 h)

Inhibition (%)

Control

13,459,200

-

1.0

14,202,000

-5.52

2.2

12,980,800

3.55

4.6

11,004,000

18.24

10

8,309,600

38.26

22

861,600

93.60

46

278,000

97.93

100

32,000

99.76

Table 5: Percentage inhibition of the average specific growth rate

Nominal conc. (mg/L)

Specific growth rate µ (24-72 h)

Inhibition (%) (24-72 h)

0

0.062407

-

1

0.05682038

8.95

2.2

0.05927807

5.01

4.6

0.05818345

6.77

10

0.05817723

6.78

22

0.01456425

76.66

46

0.00165868

97.34

100

0.00345201

94.47

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of m-toluic acid to the growth of the freshwater green microalga Pseudokirchneriella subcapitata (previous: Selenastrum capricornutum) were determined in a 72-h growth inhibition test according to OECD TG 201. Alga were cultured in OECD TG 201 medium. An appropriate exponential growth of the control was observed. Inhibiting effects of m-toluic acid on the specific growth rate were observed; the 72-h EC50 amounts to 18 mg/L and the corresponding NOEC to 10 mg/L (all nominal).

Description of key information

In a GLP-conform OECD 201 growth inhibition test on the freshwater green microalgae Pseudokirchneriella subcapitata (previous: Selenastrum capricornutum), inhibiting effects of m-toluic acid on the specific growth rate were observed; the 72 h EC50 amounts to 18 mg/L and the corresponding NOEC to 10 mg/L (all nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:
18 mg/L
EC10 or NOEC for freshwater algae:
10 mg/L

Additional information