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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
Study period:
01 Feb - 12 Jul 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Remarks:
Federal Office for the Environment (FOEN), Bern, Switzerland
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 0.32, 1.0, 3.2, 10, 32, 100 mg/L
- Sampling method: Samples were taken at the beginning of exposure, 24 h (aged), 48 h (aged) and 72 h (aged). Immediately after sampling. Immediately after sampling 10 mL methanol per 10 mL sample volume were added in order to stabilize the sample. Pre-experiments showed stability of the samples under deep-frozen conditions.
- Sample storage conditions before analysis: stored deep-frozen (approx. - 20 °C).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 230.04 mg of the test item completely in 2300 mL of test water using intense stirring for 30 min at room temperature in the dark in a (glass-stoppered) Erlenmeyer flask. The Erlenmeyer flask was nearly completely filled, minimizing the air-space in the flasks and avoiding potential losses of test item during the 30 minutes stirring period. After the stirring period, the test medium of the highest test concentration was diluted with test water to prepare the test media for the lower test concentrations. The test media were prepared just before the start of the test.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Germany)
- Method of cultivation: Cultured according to test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
15 mg CaCO3/L
Test temperature:
22 - 23 °C
pH:
start: 7.6 - 7.7
end: 7.5 - 7.8
Nominal and measured concentrations:
nominal: control, 0.32, 1.0, 3.2, 10, 32, 100 mg/L
measured: < LOQ, 0.18, 0.24, 0.46, 1.1, 2.0, 93 mg/L (geometric mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (pro-conditioned with test medium concentration to avoid adsorption of the test item to the glass ware)
- Type (delete if not applicable): closed (glass-stoppered)
- Headspace, fill volume: fill volume was about 60 mL in order to avoid any headsapce due to volatility of the test item
- Aeration: incubated on an orbital shaker
- Initial cells density: 5000 cells/mL
- Control end cells density: 561333 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water according to guideline
- Culture medium different from test medium: no, same as test
- Intervals of water quality measurement: The pH was measured at test start and end. Temperature was measured continuously. Appearance of test medium was visually controlled daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no (sterile purified water was used)
- Adjustment of pH: to keep the pH as constant as possible 6 mmol/L HEPES buffer was added to the test medium
- Photoperiod: continuous illumination (LED light)
- Light intensity and quality: 62 µE s-1m-2 (range: 61 - 61 µE s-1m-2); measured at nine places of experimental area

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement (duplicate, excitation: 440 nm, emission: 680 nm)
- Other: The shape and size of the algal cells were visually inspected at the end of the study.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: control, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: Inhibition was -0.8% at 1 mg/L, 30% at 10 mg/L and 83% at 100 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.86 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.81 - 0.91 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 1.5 - 1.6 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.38 - 0.43 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.83 - 0.88 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.46 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination did not reveal any difference of exposed algal cells compared to the control algal cells.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No; undissolved particles were not recorded throughout the test.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: EC50 (72 h): 1 mg/L based on growth rate
- Other: The EC50 was in the recommended range of the guideline (EC50 (72 h): 0.92 - 1.46 mg/L)
Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression. For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by the Williams t-test or Welch t-test where appropriate. Statistical analysis was performed using ToxRat Professional®.

Table 1: Average growth rate and inhibition of growth

Nominal test concentration [mg/L]

Measured test concentration [mg/L]

Average growth rate µ [day-1] and inhibition of µ [Ir]

0-24 h

0-48 h

0 - 72 h

µ

Ir [%]

µ

Ir [%]

µ

Ir [%]

Control

-

1.587

0.0

1.683

0.0

1.574

0.0

0.32

0.18

1.468

7.4

1.690

-0.4

1.572

0.1

1.0

0.24

1.516

4.5

1.641

2.5

1.555

1.2

3.2

0.46

1.373

13.5

1.618

3.9

1.522

3.3

10

1.1

0.612

61.4

1.072

36.3

1.231

21.8

32

2.0

0.069

95.7

0.198

88.2

0.485

69.2

100

93

-0.386

124.3

-0.411

124.4

-0.276

117.6

Table 2: Analytical results

Sampling day

[day]

Nominal concentration [mg/L]

Determined concentration [mg/L]

% of Nominal concentration [%]

0 (fresh)

Control

< LOQ

n.a.

 

0.32

0.322

100

 

1.0

0.976

98

 

3.2

3.09

96

 

10

9.9

99

 

32

31.5

99

 

100

97.3

97

24 (aged)

Control

< LOQ

n.a.

 

0.32

< LOQ

n.a.

 

1.0

< LOQ

n.a.

 

3.2

0.659

21

 

10

6.60

66

 

32

25.0

78

 

100

94.2

94

48 (aged)

Control

< LOQ

n.a.

 

0.32

< LOQ

n.a.

 

1.0

< LOQ

n.a.

 

3.2

< LOQ

n.a.

 

10

< LOQ

n.a.

 

32

< LOQ

n.a.

 

100

90.2

90

72 (aged)

Control

< LOQ

n.a.

 

0.32

< LOQ

n.a.

 

1.0

< LOQ

n.a.

 

3.2

< LOQ

n.a.

 

10

< LOQ

n.a.

 

32

< LOQ

n.a.

 

100

91.4

91

LOQ: 0.298 mg test item/L

n.a.: not applicable

Validity criteria fulfilled:
yes
Conclusions:
This study was performed according to OECD 201 (GLP). Inhibition of growth of Pseudokirchneriella subcapitata was recorded resulting in an ErC50 (72 h) of 1.6 mg/L and an ErC10 (72 h) of 0.86 mg/L based on the geometric mean measured concentrations.

Description of key information

ErC50: 1.6 mg/L (geometric mean measured concentration, Pseudokirchneriella subcapitata, OECD 201)
ErC10: 0.86 mg/L (geometric mean measured concentration, Pseudokirchneriella subcapitata, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
1.6 mg/L
EC10 or NOEC for freshwater algae:
0.86 mg/L

Additional information

One study is available, in which the toxicity to freshwater algae was investigated according to GLP and OECD guideline 201. In a static test system, the test organism Pseudokirchneriella subcapitata was exposed to six nominal concentrations of 0.32, 1.0, 3.2, 10, 32, 100 mg/L corresponding to geometric mean measured concentrations of 0.18, 0.24, 0.46, 1.1, 2.0, 93 mg/L. The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC/UV measurements.

As the test item is a volatile substance, the test was performed using glass stoppered Erlenmeyer flasks (closed system without headspace), minimizing the airspace in the flasks and avoiding potential losses of test item by evaporation. At the highest test concentration of 100 mg/L the test item was sufficiently stable during the test period. However, at the lower test concentrations a significant decrease of test item concentration was observed, depending on the concentration of the test item in the respective test medium. This effect was attributed to specific properties of the test item and its specific behavior in test water under the conditions of the test. Due to the decrease of the test item concentrations, the biological results were related to the mean measured concentrations (calculated as the geometric mean of the concentrations measured at all sampling dates (0, 24, 48, and 72 hours).

The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 h were 1.6 mg/L (95% CI: 1.5 - 1.6 mg/L) and 0.85 mg/L (95% CI: 0.83 - 0.88 mg/L), respectively. The EC10 values were 0.86 mg/L (95% CI: 0.81 - 0.91 mg/L) for growth rate and 0.41 mg/L (95% CI: 0.38 - 0.43 mg/L) for yield.