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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:
04 July 2012 - 24 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006, corrected 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
EC 761/2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
June 2012
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method: 10 mL of sample taken, acetonitrile (10 mL) was added and the sample was further diluted by a factor of 10 with acetonitrile. Further dilutions where required were performed with acetonitrile.
- Sample storage conditions before analysis: On each occasion, two of the samples were analysed and the other two were stored in a freezer in case further analysis was required.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance (7.64 mg, corrected for purity) was stirred in dilution medium (20L) for approximately 30 minutes and then used as the test medium at a nominal concentration of 360 μg/L. Test glassware was pre-exposed overnight to the test medium before use in the test to minimise loss of BMI by adsorption. An aliquot (4.0 mL) of the secondary algal inoculum was added to a portion (700 mL) of the
test medium, to give an initial cell density of 1 x 10^4 cells/mL. An aliquot (100 mL) of the inoculated test medium was added to each of the test vessels.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): 3 days
- Method of cultivation:
The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterised by a cell density of 1.74 x 10^6 cells/mL.

Culture medium: algal nutrient medium according to OECD 201.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22.5 - 23.4 °C
pH:
7.76 - 9.22 (Control)
7.82 - 9.14 (test concentration)
Nominal and measured concentrations:
Nominal concentration: 360 μg/L
Measured concentration: 141-153 ug/L (t=0h), 33-36 ug/L (t=72h). Overall mean 135 μg/L (geometric mean).
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks, glass, 250 mL, loosely plugged with a foam bung. 100mL medium.
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: 1 x10E4 cells/mL
- Control end cells density: 1747667 cells/mL (mean of 6)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 4440 to 8880 lux. provided by 6 x 30 W “cool white” 1 metre fluorescent tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities measured at 24, 48 and 72 hours using a Coulter Z Series Particle Count and Size Analyser. The estimate of cell numbers in each sample was based on the mean of three consecutive counts, corrected for background counts of uninoculated dilution media. The presence of any abnormal cells was also noted during screening for each group.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: NA
- Justification for using less concentrations than requested by guideline: Limit test done at limit of solubility of test substance in water
- Range finding study: done at 360 μg/L
- Test concentrations: 360 μg/L
- Results used to determine the conditions for the definitive study: no growth inhibition observed after 72 hrs, though may have been affected by the use of THF to aid dissolution of the test substance
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 135 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
135 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Individual cell densities for each culture and the mean values are given in Table 1 in 'Overall remarks'. The calculated average specific growth rate and yield values are given in Table 2 and are expressed in terms of percentage inhibition by comparing the mean test group value with that of the control. No significant inhibition was observed.
The mean coefficient of variation (CoV) for section by section daily growth rates in the control cultures was 8.5% during the definitive test and the CoV for the average specific growth rates of the control culture was 0.493 during the 72 hour exposure period.
No microscopic abnormalities of the cells were detected.
At the start of the test, the measured concentrations of BMI in samples of the test medium ranged between 39 and 43% of the nominal value. After 72 hours, measured levels ranged between 33 and 36% of the nominal value (85% of its initial mean measured value). Based on a geometric mean, the overall mean measured level of BMI was 135 μg/L, which was used to determine the study end points. After 72 hours, analysis of the samples of medium containing BMI at 360 μg/L, which had been incubated without algal cells gave similar results (24 and 26% of the nominal value) to test medium incubated in the presence of algal cells, indicating that the presence of algal cells did not affect the stability of the test substance.
Reported statistics and error estimates:
none.
Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure to BMI, the EC50 value in terms of growth rate and yield could not be calculated because insufficient inhibition occurred but was considered to be >135 μg/L.
The “no observed effect concentration” (NOEC) for growth rate and yield was determined to be 135 μg/L, and the lowest observed effect concentration must be >135 μg/L.
Executive summary:

An algal growth inhibition study was undertaken to determine the effect of BMI on algae (algae used: Pseudokirchnerella subcapitata). The study was conducted in accordance with OECD guideline 201 and EC method C3. Six algal cultures, with an initial cell density of ca. 1 x 10E4 cells/mL, were exposed to BMI dispersed in algal nutrient medium at a single nominal concentration of 360 μg/L (the stated limit of aqueous solubility). The test medium was prepared by the direct addition of the test substance to OECD medium; to aid dispersion, stirring was employed. To minimise loss of the test substance by sorption, all test glassware was pre-exposed to BMI at the test concentration overnight before use. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 22.5 to 23.4C for 72 hours.

At the start of the test, the measured concentrations of BMI in samples of the test medium ranged between 39 and 43% of the nominal value. After 72 hours, measured levels ranged between 33 and 36% of the nominal value (85% of its initial mean measured value). Based on a geometric mean, the overall mean measured level of BMI was 135 μg/L, which was used to determine the study end points.

Cell numbers were counted daily to monitor growth. Test results are expressed in terms of the growth rate and yield. No significant inhibition was observed compared to the control; therefore, the EC50 value was considered to be >135 μg/L. The no observed effect concentration (NOEC) was 135 μg/L, and the lowest observed effect concentration was >135 μg/L.

Description of key information

NOEC for freshwater algae was 135 μg/L; EC50 >135 μg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
135 µg/L
EC10 or NOEC for freshwater algae:
135 µg/L

Additional information

An algal growth inhibition study was undertaken to determine the effect of BMI on algae (algae used: Pseudokirchnerella subcapitata). The study was conducted in accordance with OECD guideline 201 and EC method C3. Six algal cultures, with an initial cell density of ca. 1 x 10E4 cells/mL, were exposed to BMI dispersed in algal nutrient medium at a single nominal concentration of 360 μg/L (the stated limit of aqueous solubility). The test medium was prepared by the direct addition of the test substance to OECD medium; to aid dispersion, stirring was employed. To minimise loss of the test substance by sorption, all test glassware was pre-exposed to BMI at the test concentration overnight before use. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 22.5 to 23.4C for 72 hours.

At the start of the test, the measured concentrations of BMI in samples of the test medium ranged between 39 and 43% of the nominal value. After 72 hours, measured levels ranged between 33 and 36% of the nominal value (85% of its initial mean measured value). Based on a geometric mean, the overall mean measured level of BMI was 135 μg/L, which was used to determine the study end points.

Cell numbers were counted daily to monitor growth. Test results are expressed in terms of the growth rate and yield. No significant inhibition was observed compared to the control; therefore, the EC50 value was considered to be >135 μg/L. The no observed effect concentration (NOEC) was 135 μg/L, and the lowest observed effect concentration was >135 μg/L.