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Toxicity to aquatic algae and cyanobacteria

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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)
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
- The test substance was stirred in demineralized water for about 20 minutes at approximately 20 +/- °C.
- The nominal concentration of the stock solution was 125 mg/L.
- The pH-value of 9.9 of the stock solution was adjusted to 7.6.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
- Pre-culture techniques: a seed culture was incubated for 7 days at 23 °C +/- 2°C final cell density 384 x 10E04cells/ml
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 °C
pH:
7.9 - 8.1
Nominal and measured concentrations:
- Nominal test concentrations (mg/L): 0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100
- The analytical verifications of the test substance were investigated at different concentrations in OECD-medium. The analytical results yielded 80 % or higher recoveries; they varied between 101 and 110 % of the nominal concentrations at test initiation and between 103 to 108 % at test termination. Therefore, all biological results are related to the nominal concentrations of the test item.
Details on test conditions:
- Test vessels: Erlenmeyer flasks (nominal volume 250 mL) plugged with gas permeable siliconsponge caps
- Test volume: 100 mL
- Number of replicates/conc.: 3
- Number of replicates/conc.: 3
- Illumination: artificial light, type universal white (e.g. Osram L 25), permanent illumination
- Light intensity: about 60 - 120 µE/(m2xs) at a wave length of 400 - 700 nm
- Measurement of fluorescence: after 0, 24, 48 and 72 h
- Cell counting: after 72 h in a counting chamber (Neubauer improved)
- Measurement of temperature: continuously during the whole test period in the climate chamber
- Measurement of pH-values: after 0 h and 72 h in an additional uninoculated replicate and after 72 h in the inoculated replicate No. 1 of each concentration
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.66 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC90
Effect conc.:
10.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Inhibition related to biomass
EbC10 = 2.13 mg/L
EbC50 = 4.34 mg/L
EbC90 = 7.98 mg/L
Validity criteria fulfilled:
yes
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-10-07 to 2006-01-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The purity of the test substance is not indicated, no analytical monitoring was performed.
Qualifier:
according to
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA (1985) Methods for measuring the acute toxicity of effluents to freshwater and marine organisms. EPA/600/4-85/013.
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO 5667-16 (1998) Water quality sampling -guidance on biotesting on samples.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was prepared as a water accomodated fractions (WAFs). WAFs were prepared by the direct addition of the required nominal weights or volumes to seawater followed by gentle stirring for approximately 20 hours followed by a settling period of approximately one hour. After this settling period, the middle phase of the preparation is siphoned, avoiding incorporation of undissolved particles, if present.
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Common name: Skeletonema costatum
- Method of cultivation: The cultures were cultivated by inoculating nutrient medium (ISO culture medium) to a cell density of approximately 2 x 10^3 to 10^4 cells per millilitre.
The pre-cultures were incubated at approximately 20 +/- 2 °C under constant illumination for 3d +/- 1d and were used as the inoculum source for subsequent toxicity tests.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
no data
Test temperature:
20.4 - 21.6 °C
pH:
8.18 - 8-68
Dissolved oxygen:
not measured
Salinity:
no data
Conductivity:
51.2 - 52.1 mS/cm
Nominal and measured concentrations:
Nominal concentrations: 1.0, 3.2, 10, 32, 100 mg/L
No measured concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml borosilicate glass conical flasks,
- Material, size, headspace, fill volume: 80 ml of test medium seawater was added.
- Renewal rate of test solution: No renewal
- Initial cells density: 146.5 x 10 ^4 cells mL
- No. of organisms per vessel: Approximately 10^4 cells per millilitre.
- No. of vessels per concentration: 3
- No. of vessels per control: 4

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium is prepared from natural seawater supplied by pump from Scapa Flow, Orkney. All seawater was UV sterilised and filtered to 0.2 µm. The filtered treated seawater was then enriched with nutrients and vitamins in accordance with ISO guidelines.
- Conductivity: 51.2 - 52.1 mS/cm

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Constant illumination for 3 d +/- 1 d
- Light intensity and quality: 7460 (0h), 7690 (24 h), 7080 (48h), 7430 (72h) lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Counts of algal cell numbers were carried out daily by microscope using either haemocytometer or Sedgewick-Rafter chamber, depending upon cell numbers present or by Fluorometer measurements. Three readings were performed on each test vessel.

TEST CONCENTRATIONS
- Range finding study: Rangefinding tests were conducted over 72h to determine the approximate concentrations at which effects were observed.
- Test concentrations: 1.0, 3.2, 10, 32, 100 mg/L
Reference substance (positive control):
yes
Remarks:
3.5 Dichlorophenol
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
21 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % Confidence limits: 18.85 - 21 mg/L
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
Duration:
72 h
Dose descriptor:
EC90
Effect conc.:
29.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass integral
Details on results:
- Other: There was a difference in results between the rangefinding and definitive tests, possibly due to flask contamination or insufficient cells added.

In the first definitive test there was growth at 10 mg/L^-1, the highest test concentration.
The second definitive test range was therefore derived from the initial definitive test and was 1.0, 3.2, 10, 32 and 100 mg/L^-1, the 72h EC50 was 18.71 mg/L^-1.
A third definitive test was requested by the client with three replicates per test concentration and the 72h EC50 was 21.00 mg/L^-1 (water accommodated fractions) to the marine phytoplankton Skeletonema costatum in the aqueous phase.

Results with reference substance (positive control):
- Results with reference substance valid: Yes
- EC50: The 72 h EC50 was determined to be 3.82 mg/L
Validity criteria fulfilled:
yes

Description of key information

Freshwater: The test substance was tested in an OECD guideline 201 study with Desmodesmus subspicatus. The EC50 was determined to be 6.66 mg/L and EC10 was determined to be 3.4 mg/L.

 

Saltwater: The test substance was tested in a guideline study with Skeletonema costatum. The test substance exhibited a 72 h EC50 of 21 mg/L and a NOEC value of 10 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
6.6 mg/L
EC50 for marine water algae:
21 mg/L
EC10 or NOEC for freshwater algae:
3.4 mg/L
EC10 or NOEC for marine water algae:
10 mg/L

Additional information

Freshwater

Key

The test substance was tested in an OECD guideline 201 study with Desmodesmus subspicatus. The nominal concentrations tested were 0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 mg/L. The analytical verifications of the test substance were investigated at different concentrations in OECD-medium. The analytical results yielded 80 % or higher recoveries; they varied between 101 and 110 % of the nominal concentrations at test initiation and between 103 to 108 % at test termination. Therefore, all biological results are related to the nominal concentrations of the test item. The test was conducted under static conditions. The EC50 was determined to be 6.66 mg/L and EC10 was determined to be 3.4 mg/L.

Supporting

The test substance was tested in an OECD guideline study with Desmodesmus subspicatus. The EC50 was determined to be 6.4 mg/L the EC10 was determined to be 5 mg/L.

Conclusion: Two OECD guideline studies were conducted. The results of both studies lay in the same range. As a worst case the EC50 value of 6.66 mg/l and the EC10 value of 3.4 mg/L was chosen as key values.

Salt water

The test substance was tested in a guideline study with Skeletonema costatum. The test substance was added to the test system via water accommodated fractions. 5 nominal concentrations were tested: 1, 3.2, 10, 32 and 1000 mg/L. Pre-cultures in the exponential growth phase were prepared from stock laboratory cultures by inoculating nutrient medium (ISO culture medium) to a cell density of approximately 2 x 10^3 to 10^4 cells per millilitre.The test substance exhibited a 72 h EC50 of 21 mg/L and a NOEC value of 10 mg/L. All relevant validity criteria were met.