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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:
from January 1991 to 16 October 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform GLP study without deviations
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0 (DMSO control), 32, 65, 130, 250 and 500 µg/L (with and without algae)
- Sampling method: not reported
- Sample storage conditions before analysis: stored cool and dark
- Sampling time points: day 0 and day 4
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 50 mg 2-ethylanthraquinone was dissolved in 10 mL DMSO to obtain a concentration of 5000 mg/L. This stock solution was diluted with DMSO to obtain stock solutions of 2500, 1300,650 and 320 mg/L. To erlenmeyers of 1 litre, wrapped in aluminium foil and containing 400 mL algae medium, 40 µL of these stock solutions was added. These solutions were stirred for 2 hours to obtain homogeneous nominal test concentrations of 500,250,130,65 and 32 µg/L .
- Eluate: not applicable
- Differential loading: not reported
- Controls: Algae medium with DMSO (0.1 mL/L, DMSO control) and algae medium only (control)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMSO
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Algae
- Strain: not reported
- Source (laboratory, culture collection): Culture is in house since September 1986 and originates from TNO, the Netherlands.
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: The algae are cultured in 100-mL Erlenmeyers with 50 mL algae medium. Each week an inoculum (1 mL) of this culture is transferred into fresh algae medium. The culture chamber is illuminated 24 hours a day with fluorescent lamps.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
4 d
Hardness:
not reported
Test temperature:
23.0 - 25.4
pH:
7.3 - 7.9
Dissolved oxygen:
not reported
Nominal and measured concentrations:
Nominal: 0 (control), 0 (DMSO control), 32, 65, 130, 250 and 500 µg/L
Mean measured concentration: 0 (control), 0 (DMSO control), 20, 38, 66, 149, 281 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel fill volume: 250 mL
- Aeration: not reported
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 2.5 · 10^4 cells/mL
- Control end cells density: 120 · 10^4 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not reported
- Algae medium:
Per litre distilled water:
NaNO3: 25.5 mg
MgCl2 · 6H2O: 12.2. mg
CaCl2 · 2H2O: 4.4 mg
MgSO4 · 7H2O: 14.7 mg
K2HPO4: 1.0 mg
NaHCO3: 15 mg
Micronutrient stock solution: 1 mL

The micronutrient stock solution contains per liter distilled water:
H3BO3: 185.5 mg
MnCl2 · 4H2O: 415.6 mg
ZnCl2: 3.3 mg
CoCl2 · 6H2O: 1.4 mg
CuCl2 · 2H2O: 0.012 mg
Na2MoO4 · 2H2O: 7.3 mg
FeCl3 · 6H2O: 160.0 mg

This medium is filtered on a 0.20 µm sterile Pall filter (Millipore). After filtration, the pH of the medium is 7.5.

- Culture medium different from test medium: no
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: illuminated 24 hours a day with fluorescent
- Light intensity and quality: All wavelengths below 600 nm were filtered by a red sheet below the lamps (checked by spectrophotometer). In this way the light intensity was reduced from 8000 to 2000 lux. This measure was necessary because 2-ethylanthraquinone is unstable in water under normal light conditions.
- Temperature in incubator: 24 ± 1 °C
- Shaking speed in the incubator: 100 rotations per minute

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer
- Chlorophyll measurement: not done

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 2
- Range finding study: Various range-finding tests were performed.
- Test concentrations: not reported
- Results used to determine the conditions for the definitive study: The toxicity of the test substance was approximately determined in the range-finding tests.
Reference substance (positive control):
yes
Remarks:
Once a year, a toxicity test with Selenastrum capricornutum and the reference substance potassium bichromate is conducted.
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 280 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
150 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
280 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
66 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: not reported
- Colour differences: not reported
- Flocculation: No
- Adherence to test vessels: Not reported
- Aggregation of algal cells: Not reported
- Any stimulation of growth found in any treatment: Not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The concentration of the test substance was not maintained constant during the test, because the test substance is not stable in light in aqueous solution. Furthermore adsorption to or
degradation by the algae occurred as well.
- Effect concentrations exceeding solubility of substance in test medium: ECr50 above solubility limit, NOErC below solubility limit, ECb50 slightly above solubility limit, NOEbC below solubility limit
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.1 mg/L

Exposure concentration

Since the concentration was only measured at the start and at termination of the study the real exposure is not well known, but can be estimated by calculating the mean of the measured values. This figure is not very accurate and the final results were therefore rounded to two significant figures.

Mean measured test concentration

Nominal test concentration

(µg/L)

Mean measured test concentration (µg/L)

with algae

without algae

day0

day4

mean day0-4

day0

day4

0(control)

<27

<27

<27

<27

<27

0(DMSO-control)

<27

<27

<27

<27

<27

32

34

<27

<30

34

<27

65

58

<27

38

58

54

130

110

<27

66

110

105

250

217

81

149

217

206

500

414

147

281

414

329

Mean absorption versus mean measured concentrations

Nominal test concentration

(µg/L)

Mean adsorption

day0

day1

day 2

day3

day4

0 (control)

0.016

0.027

0.071

0.178

0.344

0 (DMSO-control)

0.016

0.028

0.074

0.188

0.382

32

0.016

0.027

0.066

0.177

0.339

65

0.016

0.026

0.061

0.150

0.292

130

0.016

0.027

0.067

0.164

0.333

250

0.016

0.025

0.055

0.125

0.252

500

0.016

0.024

0.044

0.091

0.180

Mean number of algae cells per mL

Mean measured concentration (µg/L)

Number 10^4 cells/mL

Day 0

Day 1

Day 2

Day 3

Day 4

0 (control)

2.5

6.0

20

60

120

0 (DMSO-control)

2.5

6.0

20

60

140

30

2.5

6.0

18

60

120

38

2.5

6.0

16

47

110

66

2.5

6.0

18

52

120

149

2.5

5.4

14

37

86

281

2.5

5.4

11

26

60

At day 0, the number of algae cells per mL were based on the number determined with the haemocytometer.

Validity criteria fulfilled:
yes
Conclusions:
For growth rate the EC50 is > 280 µg/L and the NOEC is equal to 150 µg/L.
Executive summary:

The toxicity of 2-ethylanthraquinone to the alga Selenastrum capricornutum (Chlorophyceae) was tested according to OECD Guideline 201 (OECD, 1984) at nominal test concentrations ranging from 32 to 500 µg per litre algal medium. As 2-ethylanthraquinone is unstable in water under normal light conditions, these tests were conducted in red light with wavelengths above 600 nm. Samples of the test concentrations were taken at day 0 and at day 4 and analysed by HPLC. The measured concentrations of the test substance in solution ranged from 34 to 414 µg/L at the start, but decreased to below the limit of detection and 147 µg/L after 4 days, respectively. The results based on the mean of the concentrations measured at the start and the end of the test are ECr50>280 µg/L, NOErC=150 µg/L, Eb50=280 µ/L and NOEbC=66 µg/L. The biomass is more affected by 2-ethylanthraquinone than the growth rate. For biomass the EC50 of this chemical is equal to 280 µg/L which is at or slightly above the solubility limit and the NOEC is 66 µg/L. For growth rate the EC50 is > 280 µg/L and the NOEC is equal to 150 µg/L.

Description of key information

In a short-term toxicity study with algae, the ECr50 was >280 µg/L, NOErC was 150 µg/L,  Eb50 was 280 µ/L and NOEbC was 66 µg/L.

Key value for chemical safety assessment

Additional information

The toxicity of 2-ethylanthraquinone to the alga Selenastrum capricornutum (Chlorophyceae) was tested according to OECD Guideline 201 (OECD, 1984) at nominal test concentrations ranging from 32 to 500 µg per litre algal medium. The test substance was dissolved in DMSO prior to the preparation of test solutions due to its low water solubility. As 2-ethylanthraquinone is unstable in water under normal light conditions, these tests were conducted in red light with wavelengths above 600 nm. Samples of the test concentrations were taken at day 0 and at day 4 and analysed by HPLC. The measured concentrations of the test substance in solution ranged from 34 to 414 µg/L at the start, but decreased to below the limit of detection and 147 µg/L after 4 days, respectively. The results based on the mean of the concentrations measured at the start and the end of the test were ECr50>280 µg/L, NOErC=150 µg/L, Eb50=280 µ/L and NOEbC=66 µg/L. Effects on biomass are more sensitive compared to effects on growth rate, however only the latter should be used for classification purposes. In this respect the most significant result is the NOErC=150 µg/L which eventually leads to a CLP classification as aquatic chronic toxic category 3, H412 (for details see endpoint summary 6.1 Aquatic toxicity).