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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:
05 Feb - 01 Mar 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)
Version / remarks:
2011
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Remarks:
Federal Office for the Environment (FOEN)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 0.32, 1.0, 3.2, 10 mg/L
- Sampling method: Duplicate samples were taken at the beginning (containing no algae) and at the end of exposure (containing algae).
- Sample storage conditions before analysis: Samples were deep-frozen at -20 °C immediately after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of nominal 100 mg/L was prepared by dissolving 109.91 mg of the test item in 1100 mL of test water. The solution was stirred intensively for 15 min at room temperature. This stock solution was diluted with test water to preapre the test media for the solution of 10 mg/L. The lower test concentration were prepared by further dilution of the 10 mg/L solution.
- 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, Göttingen, Germany)
- Method of cultivation: Cultivation was done under standardized condtions.

ACCLIMATION
- Acclimation period: An inoculum culture was set up four days before the start of exposure.
- Culturing media and conditions (same as test or not): same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.15 mmol/L (= 15 mg/L as CaCO3)
Test temperature:
23 °C
pH:
7.5 - 7.9
Nominal and measured concentrations:
nominal: control, 0.032, 0.10, 0.32, 1.0, 3.2, 10 mg/L
measured (0 h): < LOQ, 0.321, 0.988, 2.82, 8.55 mg/L; only the nominal concentrations >= 0.32 mg/L were analysed
measured (72 h): < LOQ, 0.256, 0.811, 28.1, 8.78 mg/L; only the nominal concentrations >= 0.32 mg/L were analysed
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks completely filled with 60 mL medium
- Type: closed due to volatility of test item
- Aeration: incubated on an orbital shaker
- Initial cells density: 5000 cells/mL
- Control end cells density: 231.1 E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted test water (AAP Medium)
- Culture medium different from test medium: no, same as test
- Intervals of water quality measurement: The pH was measured at the test start and at test end. The temperature was monitored continuously.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: To keep the pH of the test media as constant as possible 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to the test water.
- Photoperiod: continuous illumination
- Light intensity and quality: approx. 71 μE s-1 m-2

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: algal biomass measured by fluorescence measurement (excitation 440 nm, emission 680 nm)
- Observation of cells: Microscopic examination 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: 8.7% inhibition of growth rate at 1 mg/L, 101.8% inhibition of growth rate at 10 mg/L, 168.3% inhibition of growth rate at 100 mg/L; EC50 (72 h): 3.4 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 3.0 - 3.1 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 1.8 - 1.9 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 1.4 - 1.5 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.76 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.70 - 0.81 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Unusual cell shape: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 1.0 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: EC50 (72 h): 0.74 mg/L
Reported statistics and error estimates:
The 72-hour EC10 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 Welch t-test.

The test substance was confirmed to be sufficiently stable over the test (86 - 97% of nominal at test start; 80 - 88% of nominal after 72 h).

Table 1: Average specific growth rate

Nominal test item concentration [mg/L]

Average specific 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

1.845

0.0

1.877

0.0

1.749

0.0

0.032

1.964

-6.4

1.851

1.4

1.732

0.9

0.10

1.626*

11.9

1.851*

1.4

1.753

-0.3

0.32

1.857

-0.6

1.841*

1.9

1.740

0.5

1.0

1.714

7.1

1.736*

7.5

1.661*

5.0

3.2

1.216*

34.1

1.072*

42.9

0.831*

52.5

10

0.217*

88.3

-0.238*

112.7

-0.156*

108.9

 *: mean value statistically significantly lower than in the control

Validity criteria fulfilled:
yes
Conclusions:
The study resulted in an ErC50 (72 h) of 3.1 mg/L and an ErC10 (72 h) of 1.4 mg/L based on growth inhibition of Pseudokirchneriella subcapitata (OECD 201).
Executive summary:

The impact of the test substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to OECD 201 (GLP).

The nominal concentrations of 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L were tested in parallel with a control (test water without test item). As the test item is volatile, a closed system was applied.

The measured concentrations in the test media of the test concentrations of 0.32 to 10 mg/L were between 86 and 99% of the nominal values at the start of the test and between 80 and 88% at the end of the test.

Thus, the test substance was stable in the test media over the test period of 72 hours and the resulted effect values were related to the nominal concentrations.

The test substance had toxic effects on the algal growth; the 72-hour ErC50 was calculated to be 3.1 mg/L and the ErC10 (72 h) was 1.4 mg/L based on growth inhibition.

Description of key information

ErC50 (72 h): 3.1 mg/L based on growth inhibition of Pseudokirchneriella subcapitata (OECD 201)

ErC10 (72 h): 1.4 mg/L based on growth inhibition of Pseudokirchneriella subcapitata (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
3.1 mg/L
EC10 or NOEC for freshwater algae:
1.4 mg/L

Additional information

One experimental study is available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to OECD 201 (GLP) under static conditions using the freshwater algae Pseudokirchneriella subcapitata. Test concentrations of 0.032, 0.10, 0.32, 1.0, 3.2, 10 mg/L were chosen based on the results from a preliminary range-finding test. It was ensured by an appropriate test design that the substance does not volatilize from the test vessels. The suitability of the test system was confirmed by HPLC/UV analysis of the test concentrations. The measured concentrations were ≥80% of the nominal values at the end of the test. Thus, the effect concentrations were based on the nominal values. Inhibition of growth was recorded resulting in an ErC50 (72 h) of 3.1 mg/L and an ErC10 (72 h) of 1.4 mg/L.