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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:
2017-06-06 to 2017-09-22
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:
adopted 28 July, 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 6.25, 12.5, 25, 50, 100.50 mg/L nominal
- Sampling method: Samples (at least 2 x about 5 mL) were taken from all five test solutions and controls at the beginning of the test prior to addition of the algae (2 x 6 samples, analysis and retain samples). At the end of the growth test, samples were taken directly from pooled replicates per test concentration and controls (2 x 6 samples). For stabilisation of the samples, all samples were given into 5 mL of methanole.
- Sample storage conditions before analysis:If samples could not be measured on the day of sampling, they were immediately stored at ≤ -18 °C until analysis. All samples will be discarded after finalisation of the study.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To prepare the highest test concentration, an amount of 100.50 mg of the test item (considering a purity of the test item of 99.48%) was transferred into a graduated sterile 1 L glass flask, filled up with sterile growth medium. An acetone washed, sterilised stirring bar was added to the bottle and the test item solution was stirred for 2 hour at room temperature (about 20°C) to achieve maximum solubility of the test item. This test solution was serially diluted with sterile growth medium to obtain the other test concentrations.
- Controls: yes, test medium only
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Raphidocelis subcapitata, Selenastraceae, Chlorophycea, Chlorophyta (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum)
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG
- Method of cultivation: The stock cultures are maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980).

ACCLIMATION
- Acclimation period: Prior to testing a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test. The culture duration of the pre-cultures was 3 days.
- Culturing media and conditions (same as test or not): same as test conditions
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21-24°C ± 2°C
pH:
from 7.46 at test start to 8.13 at test end
Nominal and measured concentrations:
Nominal: 0, 6.25, 12.5, 25, 50, 100 mg/L
Arithmetic mean measured concentration: 7.77, 14.0, 29.4, 55.1, 125.3 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: conical glass flasks
- Type (delete if not applicable): closed with air permeable silicone sponge caps.
- Material, size, headspace, fill volume: Glass flasks with 250 mL nominal volume, fill-volume: 100mL
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 10000 cells/mL
- Control end cells density: 152.6E04 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified water processed using an ELGA „PURELAB Ultra“
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The pH of the medium is a result of the equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air
- Light intensity and quality:The light intensity (OSRAM Standard “cool white”) adjusted to 60 - 120 µE m-2 s-1 (4440 - 8880 lux) close to the surface of the liquid.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 1.0, 10.0, 100.0 mg/L
- Results used to determine the conditions for the definitive study: Since there was no significant inhibition of growth rate and yield, the test susbtance was applied up to the maximum concentration tested in the range finder with a separation factor of 2 between each concentrations.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
> 125.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
> 125.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs in a non-GLP test twice a year. The latest ErC50 value of 2.29 mg/L (nominal; 95% confidence limits: 2.12 – 2.48 mg/L) for inhibition of growth rate is in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L.
Reported statistics and error estimates:
The computer program ToxRat® was used for statistical evaluations.

Table1: Growth rate (G) and its inhibition relative to control (%I) as computed from the raw data for test intervals selected.

Arithmetic mean measured conc.
[mg/L]

0-24 h

0-48 h

0-72 h

G

%I

G

%I

G

%I

Control

1.727

0.00

1.684

0.00

1.674

0.00

7.77

1.699

1.61

1.661

1.36

1.679

-0.29

14.0

1.656

4.12

1.643

2.43

1.660

0.86

29.4

1.754

-1.54

1.681

0.17

1.661

0.81

55.1

1.713

0.82

1.659

1.48

1.656

1.09

125.3

1.702

1.44

1.644

2.33

1.676

-0.08

Table2: Yield (Y) and its inhibition relative to control (%I) as computed from the raw data for test intervals selected.

Arithmetic mean measured conc.
[mg/L]

0-24 h

0-48 h

0-72 h

Y

%I

Y

%I

Y

%I

Control

4.636

0.00

28.125

0.00

151.550

0.00

7.77

4.479

3.37

26.955

4.16

156.500

-3.27

14.0

4.248

8.35

25.765

8.39

144.775

4.47

29.4

4.784

-3.21

27.970

0.55

145.725

3.84

55.1

4.558

1.68

27.170

3.40

143.250

5.48

125.3

4.497

2.98

25.852

8.08

151.825

-0.18

Validity criteria fulfilled:
yes
Remarks:
Validity crieteria as outlined under "any other information on material and methods" were fulfiled.
Conclusions:
The NOEC of the alpha methyl glucoside to the green alga Raphidocelis subcapitata based on growth rate was > 125.3 mg/L a.s., this value is based on the geometric mean exposure concentration. Since no EC50 value could be obtained due to the absence of any adverse effect, the determined NOAEL is also considered the EC0 value.
Executive summary:

In a 72 hour acute toxicity study, the cultures of green alga, Raphidocelis subcapitata were exposed to alpha methyl glucoside at nominal concentrations of 0, 6.25, 12.5, 25.0, 50.0 and 100.0 mg a.i./L} under static conditions in accordance with the test guideline OECD 201.  The NOEC value based on growth rate and yield were >125 and ≥ 125 mg a.i./L, respectively.  The % growth inhibition in the treated algal culture as compared to the control ranged from -0.29 to 1.09 and were not significant compared to the controls. No abnormalities were noted. Thus, due to the absence of any adverse effect the NOAEL value is also considered to be the EC value.

Test Organism: green alga Raphidocelis subcapitata

Test Type : Static

72 hr EC0: > 125 mg a.i./L

72 hr EC50: > 125 mg a.i./L

72 hr NOEC: ≥ 125mg a.i./L

Values were determined for growth rate and yield.

Endpoint(s) Effected: None

Description of key information

- OECD guideline study (201), GLP, RL1, NOEC > 125.3 mg/L a.s.

Key value for chemical safety assessment

EC50 for freshwater algae:
125 mg/L
EC10 or NOEC for freshwater algae:
125 mg/L

Additional information

In a 72 hour acute toxicity study, the cultures of green alga, Raphidocelis subcapitata were exposed to methyl-a-D-glucopyranoside at nominal concentrations of 0, 6.25, 12.5, 25.0, 50.0 and 100.0 mg a.i./L} under static conditions in accordance with the test guideline OECD 201.  The NOEC value based on growth rate and yield were >125 and ≥ 125 mg a.i./L, respectively.  The % growth inhibition in the treated algal culture as compared to the control ranged from -0.29 to 1.09 and were not significant compared to the controls.No abnormalities were noted. 

Test Organism: green alga Raphidocelis subcapitata

Test Type : Static

 

72 hr EC50: > 125 mg a.i./L

72 hr NOEC: ≥ 125mg a.i./L

Endpoint(s) Effected: None