Producto P0310

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

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

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the analysis of the actual test item concentrations the following samples were taken:
Just before the start of the test:
- duplicate samples from each test medium (without algae)
- duplicate samples from the control (without algae)
After 72 hours:
- duplicate samples from each test medium (with algae) (stability samples)
- duplicate samples from the control (with algae)
For the 72-hour stability samples, the contents of the test flasks of each treatment were poured together. All samples were deep-frozen (at about -20 ºC) immediately after sampling. Based on pre-experiments for investigation of the storage stability, the test item is sufficiently stable in the test water under these storage conditions. The concentration of the test item was analyzed in the duplicate test medium samples from the highest test concentration of nominal 100 mg/L from both sampling times (0 and 72 hours). The samples from the lower test concentrations were not analyzed, since these concentrations were below the determined 72-hour NOEC. From the control samples, only one of the duplicate samples was analyzed from the corresponding sampling times.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal test item concentration of 100 mg/L was prepared by dissolving 50.1 mg of the test item completely in 500 mL of test water using ultrasonic treatment (15 minutes) and intense stirring (15 minutes at room temperature in the dark). Adequate volumes of this intensively mixed test medium were diluted with test water to prepare the test media with the lower test item concentrations. The test media were prepared just before the start of the test (= addition of algae).

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany).
- Method of cultivation: The algae are cultivated in laboratories under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L as CaCO3).

Test temperature:

They were incubated in a temperature controlled water bath at a temperature of 22–23 ºC.

pH:

At the start of the test, the pH value in the test media and the control was 8.3. At the end of the test, pH values of 8.6 to 8.9 were measured

Salinity:

Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:
Macro-nutrients:
NaHCO3 50.0 mg/L CaCl2 × 2 H2O 18.0 mg/L NH4Cl 15.0 mg/L MgSO4 × 7 H2O 15.0 mg/L MgCl2 × 6 H2O 12.0 mg/L KH2PO4 1.6 mg/L
Trace elements:
Na2EDTA × 2 H2O 100.0 μg/L FeCl3 × 6 H2O 80.0 μg/L MnCl2 × 4 H2O 415.0 μg/L H3BO3 185.0 μg/L Na2MoO4 × 2 H2O 7.0 μg/L ZnCl2 3.0 μg/L CoCl2 × 6 H2O 1.5 μg/L CuCl2 × 2 H2O 0.01 μg/L

Nominal and measured concentrations:

The following nominal concentrations were tested: 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control was tested in parallel (test water without test item).
The mean measured test concentration (calculated as the geometric mean over all measurements of the test concentration) was 26 mg/L (i.e., 26% of the nominal value).

Details on test conditions:

TEST SYSTEM
- Test vessel: 50-mL Erlenmeyer flasks
- Type (delete if not applicable): The flasks were covered with glass dishes.
- Initial cells density: The test was started by inoculation of 10,000 algal cells per mL of test medium. These cells were taken from an exponentially growing pre-culture, which was set up four days prior to the test under the same conditions as in the test. One day before the start of the test, the pre-culture was diluted threefold to keep the algae in exponential growth.
- 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
- Intervals of water quality measurement: The pH was measured and recorded in each 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.

OTHER TEST CONDITIONS
- Photoperiod: continuously illuminated
- Light intensity and quality: at a measured light intensity of about 5850 Lux (mean value), range: 5500 to 6500 Lux (minimum and maximum value of measurements at nine places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed about 35 cm above the test flasks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:
Small volumes of the test media and the control (1.0 mL) were taken from all test flasks after 24, 48 and 72 hours of 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 of exposure, a sample was taken from the control and from the highest test concentration of (nominal 100 mg/L). The shape and size of the algal cells were examined microscopically in these samples.

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: The test concentrations were based on the results of a range-finding test and the results of the pre-experiment to determine the solubility of the test item in test water. Concentrations above nominal 100 mg/L have not been tested in compliance with EU Commission Directive 92/69/EEC. An enlarged spacing factor of 3.2 between the test concentrations was chosen because, according to the results of the range-finding test, the concentration-effect relationship was rather flat and thus, a large concentration range had to be tested.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

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.:

> 26 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The test item had no statistically significant inhibitory effect on the growth (yield and growth rate) of Pseudokirchneriella subcapitata during the test period of 72 hours up to and including the highest nominal test concentration of 100 mg/L (mean measured concentration of 26 mg/L).

Results with reference substance (positive control):

For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test. The latest result of the positive control test in 2006 (72-h EC50 for the growth rate: 1.70 mg/L) showed that the toxic performance was valid and within the historical range (from 2000 to 2006: 72-h EC50: 0.71–1.74 mg/L).

Validity criteria fulfilled:

yes

Remarks:

(increase of cell density by at least a factor of 16 within three days; the mean coefficient of variation of the daily growth rates was lower than 35%; the coefficient of variation of the average specific growth rates in the controls was below 7%)

Conclusions:

The test concentration of 100 mg/L (mean measured concentration of 26 mg/L) was, therefore, determined to be the 72-hour NOEC.

Executive summary:

The influence of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, C.3 (1992), and the OECD Guideline 201 (2006). The nominal test concentrations were 1.0, 3.2, 10, 32 and 100 mg/L in parallel with a control. At the start of the test, the analytically determined test item concentration in the analyzed test medium (nominal of 100 mg/L) was 91% of the nominal value, proving a correct dosage of the test item. During the test period of 72 hours, a considerable decrease of the test item concentration in the test medium was determined. At the end of the test, 7.5% of the nominal value was found. This low value is probably the result of a photolytic degradation (intense irradiation of the test flasks). The mean measured test concentration was 26 mg/L. The reported biological results are related both to the nominal and to the mean measured test item concentrations. The test item had no statistically significant inhibitory effect on the growth (yield and growth rate) of Pseudokirchneriella subcapitata during the test period of 72 hours up to and including the highest nominal test concentration of 100 mg/L (mean measured concentration of 26 mg/L).

The test concentration of 100 mg/L (mean measured concentration of 26 mg/L) was, therefore, determined to be the 72-hour NOEC (highest concentration tested without toxic effects after the test period of 72 hours). This value might even be higher, but nominal concentrations of the test item above 100 mg/L were not tested (according to the guidelines). The 72-hour LOEC (lowest concentration tested with toxic effects after the test period of 72 hours) and the 72-hour EC10 and EC50 for the growth rate and yield could not be quantified due to the absence of a toxic effect of the test item at the tested concentrations. Accordingly, these parameters were clearly higher than nominal concentration of 100 mg/L (mean measured concentration of 26 mg/L).

Description of key information

Key study: OECD guideline 201 and EU method C.3. GLP study.

The test concentration of 100 mg/L (mean measured concentration of 26 mg/L) was determined to be the 72-hour NOEC.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Key study: OECD guideline 201 and EU method C.3. GLP study.

The test concentration of 100 mg/L (mean measured concentration of 26 mg/L) was determined to be the 72-hour NOEC.