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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
Study period:
2002-04-26 - 2002-05-13 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
OECD Guideline for Testing of Chemicals, No. 201: Alga, Growth Inhibition Test, 1984.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
EU Commission Directive 92/69/EEC, C.3: Algal Inhibition Test, 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature at about 20 °C, away from direct sunlight
- Stability under test conditions: Stability in water: Max. 90 days

OTHER SPECIFICS:
Solubility in water: 5.5 g/L
pH in aqueous solution: 7
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the only test concentration of nominal 100 mg/l was prepared by dissolving 31 mg test item completely in 300 ml test water by ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature.
- Controls: A control was tested in parallel (test water without test item).
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG
- Source (laboratory, culture collection): Supplied by the Sammlung von Algenkulturen Göttingen (SAG, Experimentelle Phykologie und Sammlung von Algenkulturen, Albrecht-von-Haller-lnstitut für Pflanzenwissenschaften, Universitat Göttingen, D-37073 Göttingen, Germany). The algae had been grown in the laboratories under standardized conditions according to the test guidelines.
- Age of inoculum (at test initiation): The test was started (0 hours) by inoculation of 10,000 algal cells per ml test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test under the same conditions as in the test.
- Method of cultivation:
The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:
Macro-nutrients:
NaHCO3 50.0 mg/L
CaCl2 x 2 H2O 18.0 mg/L
NH4CI 15.0 mg/L
MgSO4 x 7 H2O 15.0 mg/L
MgCI2 x 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L

Trace elements:
Na2EDTA x 2 H2O 100.0 µg/L
FeCI3 x 6 H2O 80.0 µg/L
MnCl2 x 4 H2O 415.0 µg/L
H3BO3 185.0 µg/L
Na2MoO4 x 2 H2O 7.0 µg/L
ZnCI2 3.0 µg/L
CoCl2 x 6 H2O 1.5 µg/L
CuCI2 x 2 H2O 0.01 µg/L
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
Calculated water hardness of the test water: 0.24 mmol/l (=24 mg/l) as CaCO3
Test temperature:
Algae were incubated in a temperature controlled water bath at a temperature between 22 and 23 °C
pH:
At the start of the test, the pH value in the test medium and the control was 8.0. At the end of the test, pH value of 9.0 was measured.
Nominal and measured concentrations:
0, 100 mg/l (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks
- Type (delete if not applicable): The flasks were covered with glass dishes.
- Material, size, headspace, fill volume: Volumes of 15 ml algal suspension for each replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks,
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 10,000 algal cells per ml test medium
- Control end cells density: 76.18 x 10,000 cells/ml (72h)
- No. of vessels per concentration (replicates): three replicates per test concentration
- No. of vessels per control (replicates): six replicates of the control

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in the single test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test medium was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: not stated
- Photoperiod: continuously illuminated
- Light intensity and quality: measured light intensity of about 8700 Lux (mean value), range: 8000 to 9200 Lux (minimum and maximum value of measurements at 9 places distributed over the experimental area at the surface of the test medium). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Small volumes of the test medium and the control (1.0 ml) were taken out of all test flasks after 24, 48 and 72 hours exposure, and were not replaced. The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter®, Model ZM), with at least two measurements per sample.
In addition, after 72 hours exposure, a sample was taken from the control and from the single test concentration (nominal 100 mg/L). The shape of the algal cells was microscopically examined in these samples.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: The test concentration was based on the results of a range-finding test (without GLP).
- Test concentrations: 0, 100 mg/l
Reference substance (positive control):
yes
Remarks:
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control at least once per year to demonstrate satisfactory test conditions.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
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 abnormalities stated
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: none
Results with reference substance (positive control):
not stated
Validity criteria fulfilled:
yes
Conclusions:
The test was performed scientifically reasonable according to OECD 201 under GLP, the report is well documented and the validity criteria are met. Hence, the results can be considered as sufficiently reliable to assess the toxicity of 1,3-Dimethyl-4-aminouracil to algae.
The mean biomass and growth rate of the algae grown at the test concentration of 100 mg/l were inhibited by only 10 and 3.9% respectively. This low inhibition was not considered to be of a biological relevance. This is also confirmed by the 72-hour results of the range-finder test, where the mean algal cell density in the test medium of 100 mg/l was even slightly higher compared to the control value. The 72-hour LOEC (lowest concentration tested with toxic effects), and the 72-hour EC50 for the algal biomass b and growth rate r could not be quantified due to the absence of a toxic effect of 1,3-DimethyI-4-aminouracil at the tested concentration. However, these parameters were clearly higher than 100 mg/L.
With an ErC50 > 100 mg/l, 1,3-Dimethyl-4-aminouracil does not need to be classified as hazardous to the environment acc. Regulation 1272/2008, neither acute nor chronic.
Executive summary:

The influence of the test item 1,3-Dimethyl-4-aminouracil on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, C.3, 1992, and the OECD Guideline No. 201, 1984, under GLP.

A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on Scenedesmus subspicatus up to and including the concentration of nominal 100 mg/l. Thus the only concentration tested was nominal 100 mg/l and a control.

The analytically determined test item concentration in the analyzed test medium was 104 and 103% of the nominal value at the start and the end of the test. Consequently, the test item was stable during the test period of 72 hours under the test conditions, and the reported biological results are based on the nominal concentration of the test item.

The test item 1,3-Dimethyl-4-aminouracil had no significant inhibitory effect on the growth of Scenedesmus subspicatus during the exposure period of 72 hours at the test concentration of 100 mg/l. The 72-hour NOEC (highest concentration tested without toxic effects after the exposure period of 72 hours) of 1,3-Dimethyl-4-aminouracil to Scenedesmus subspicatus was determined to be at least 100 mg/l. Concentrations in excess of 100 mg/l have not been tested, according to EU Commission Directive 92/69/EEC.

The 72-hour EC50 and the 72-hour EC100 were clearly higher than 100 mg/L. These values could not be quantified due to the absence of toxicity of 1,3-Dimeihyl-4-aminouracil up to the tested concentration.

1,3-Dimethyl-4-aminouracil does not need to be classified as hazardous to the environment acc. Regulation 1272/2008, neither acute nor chronic.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, Guideline Study. Scientifically acceptable.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on test solutions:
A stock solution (125 mg/l, nominal) was prepared using deionized water and was then treated ultrasonically for 3 minutes. Following this, the stock solution was stirred for about 10 min at approximately 20 +/- 2 °C. Serial dilutions of this stock solution were subsequently made in order to obtain the desired test concentrations.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: strain SAG 86.81
- Source (laboratory, culture collection): University of Goettingen
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 +/- 2 °C
Nominal and measured concentrations:
Nominal test concentrations: 1.56. 3.13, 6.25, 12.5, 25.0, 50.0, 100.0 mg/l Additionally, a culture medium control was tested in parallel.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (nominal volume 250 ml) plugged with gas permeable siliconsponge caps
- Material, size, headspace, fill volume:
- Initial cells density: 10000
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes/no
- Detailed composition if non-standard medium was used:


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD guideline 201.



OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH:
- Photoperiod: 16:8 h day-night regime
- Light intensity and quality: artificial light, type universal white, permanent illumination, about 60 - 120 microE/(m2*s) at a wave length of 400 - 700 nm
- Salinity (for marine algae):


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: in vivo chlorophyll-a-fluorescence (pulsed excitation with light flashes having a wavelength of 435 nm), after 72 h in a counting chamber (Neubauer improved) in replicate No. 2 of the inoculated control


Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 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.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Reported statistics and error estimates:
The EC50 was determined directly from the raw data.
Effect on the development of biomass:
EbC10 (72 h) = 91.1 mg/l
EbC50 (72 h) > 100 mg/l

No observed effect concentration (95 % significance level)
NOEbC (72 h) = 50 mg/l
NOErC (72 h) = 6.25 mg/l

Lowest observed effect concentration (95 % significance level)
LOEbC (72 h) = 100 mg/l
LOErC (72 h) = 12.5 mg/l

Chemical analysis:
Analytical recovery rates (control analyses of 1.56, 12.5 and 100 mg/l):
after 0 h = 92.9 - 97.6 %
after 72 h = 100 - 107.2 %

Description of key information

Toxicity to aquatic algae

- ErC50 > 100 mg/l, ErC0 ≥ 100 mg/l for Desmodesmus subspicatus over 72h (OECD 201, static, GLP)

- ErC50 > 100 mg/l, ErC0 ≥ 100 mg/l, NOEC = 100 mg/l for Desmodesmus subspicatus over 72h (OECD 201, static, GLP)

Key value for chemical safety assessment

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

Additional information

According to Regulation 1272/2008, Table 4.1.0, Classification categories for hazardous to the aquatic environment, a substance must be classified as Acute (short-term) aquatic hazard, Acute Category 1, if i.a. the 72 or 96 hr ErC50 (for algae or other aquatic plants) is ≤ 1 mg/l. It must be classified as Chronic Category 3 if the 72 or 96 hr ErC50 (for algae or other aquatic plants) is > 10 to ≤ 100 mg/l, and the substance is not rapidly degradable and/or the experimentally determined BCF ≥ 500 (or, if absent, the log Kow ≥ 4).

In both available studies, the following values were determined: ErC50 > 100 mg/l, EC0/NOEC ≥ 100 mg/l, for Desmodesmus subspicatus. Hence, the substance does not need to be classified as hazardous to the aquatic environment, neither acute nor chronic.