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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:
2009-03-20 to 2009-04-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other:
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 2006-03-23
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-05-01
Analytical monitoring:
yes
Details on sampling:
At the start of the definitive test, four samples (10 mL) were taken from the freshly-prepared control and test media. After 72 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. Additional samples were also taken from a flask containing triethylmethylammonium tetrafluoroborate at 100 mg/L but with no algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells.
On each occasion, two of the samples was analysed and the others were stored in a freezer in case further analysis was required.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The method of preparation used during the definitive test was based on the results of a range finding test. The test medium was prepared by adding the test substance (100 mg) directly to algal culture medium (1000 mL).
An aliquot (9.65 mL) of the secondary algal inoculum was added to a portion (100 mL) of the test medium at each concentration, to give an initial cell density of 1 x 10^4 cells/mL. An aliquot (100 mL) of the appropriate 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
- Strain: Strain No. CCAP 278/4
- Source (laboratory, culture collection): Axenic, uni-cellular, liquid slope cultures of algae were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland.

PRE-CULTURE:
The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium (OECD TG 201 medium see "Salinity" below) 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 0.829 x 106 cells/mL.
No further information on the test organisms was stated.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
No data
Test temperature:
Control flasks: 22.4 °C (at 0h) and 23.0°C (at 72h)
Test flasks: 22.8°C (at 0h) and 23.0°C (at 72h)
pH:
Control flasks: 7.54 (at 0h) and 9.88 (at 72h)
Test flasks: 7.57 (at 0h) and 9.69 (at 72h)
Dissolved oxygen:
Not appropriate
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Nominal concentration: 0 (control) and 100 mg/L triethylmethylammonium tetrafluoroborate
Measured concentration: 0 (control) and 103 mg/L triethylmethylammonium tetrafluoroborate
Details on test conditions:
RANGEFINDING TEST:
The range finding test employed nominal test concentrations of 0(control), 1, 10 and 100 mg/L. After 72 hours, algal growth was not significantly inhibited at any test concentration. Based on these results the definitive (limit) test employed a single nominal concentration of 100 mg/L.

DEFINITIVE TEST:
A range finding test was followed by a definitive (limit) test with one test concentration, plus an algal nutrient medium control group.
Six flasks were established for the control group and test group, plus an additional flask at a nominal concentration of 100 mg/L, which contained test medium but no algal cells. All of the control and test flasks were incubated. The media remaining in the preparation flasks were used for water quality measurements at the start.
Before the start of the test, the required number of empty test vessels (250 mL conical flasks), were loosely stoppered with foam bungs, covered with aluminium foil that was secured by autoclave tape and sterilised by autoclaving (121°C for at least 15 minutes). After the addition of the inoculated test medium (100 mL), each flask was then loosely plugged with a foam bung.
The control cultures were prepared as for the test medium except that no test substance was added.

ENVIRONMENTAL CONDITIONS:
Conical flasks (250 mL) each containing control or test culture (100 mL) were placed in an illuminated orbital incubator according to a random number sequence. The cultures were incubated, without renewal of medium for 72 hours under continuous illumination of approximately 6258 to 6448 lux provided by 6 x 30 W “cool white” 1 metre fluorescent tubes.
Temperature and pH of control and test flasks at the start and end of the test were recorded.
Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at a nominal 150 cycles per minute. The minimum and maximum temperature and light intensity in five positions within the test area were determined each day. To minimise the impact of
differences in light intensity across the test area on algal growth, control and test flasks were re-positioned in the test area each day during the definitive test.

MEASUREMENT OF GROWTH:
Samples were taken from control and test flasks at 24, 48 and 72 hours and the cell densities measured 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 of each test level.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 103 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
triethylmethylammonium tetrafluoroborate
Basis for effect:
growth rate
Remarks:
EbC10
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 103 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
triethylmethylammonium tetrafluoroborate
Basis for effect:
growth rate
Remarks:
EbC50
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
103 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
triethylmethylammonium tetrafluoroborate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 103 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
triethylmethylammonium tetrafluoroborate
Basis for effect:
growth rate
Remarks:
ErC10
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 103 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
triethylmethylammonium tetrafluoroborate
Basis for effect:
growth rate
Remarks:
ErC50
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No microscopic abnormalities of the cells were detected.
- Other: After 72 hours, analysis of a sample of medium containingtriethylmethylammonium tetrafluoroborate at 100 mg/L, which had been incubated without algal cells gave similar results to test media incubated in the presence of algal cells. These results indicate that the presence of algal cells did not affect the stability of the test substance.
- At the start of the test the test media were colourless.

The pH of the control medium and the test concentration increase more than 1.5 units during the test. This was thought not to affect the test results.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The data were compiled in an Excel spreadsheet and analysed using SAS 8.2 (SAS INSTITUTE (1999) SAS OnlineDoc Version Eight. SAS Institute Inc., Cary, NC, USA.).
For the area under the curve and growth rate, Williams’ test (1971, 1972) was used to compare the treated group with control unless there was evidence of a non-monotonic dose-response relationship, in which case Dunnett’s test (1955, 1964) was used.
The test results have been expressed in terms of nominal concentrations of triethylmethylammonium tetrafluoroborate.

Validity criteria:

For the test to be valid, the cell concentration in control cultures should have increased by a factor of at least 16 within 72 hours; the mean coefficient of variation for daily growth rates (Days 0-1, 1-2 and 2-3) in the control cultures must not exceed 35% and the coefficient of variation of average specific growth rates during the test in replicate control cultures must not exceed 7%. All of these validity criteria were met and therefore the study is valid.

Validity criteria fulfilled:
yes
Conclusions:
The effects of test item triethylmethylammonium tetrafluoroborate on greenalgae Pseudokirchneriella subcapitata were investigated in an acute algae growth inhibition test according to OECD 201 (2006). The test was conducted as static limit test, meaning that the algae were exposed to a control and a single concentration of 100 mg/L triethylmethylammonium tetrafluoroborate dissolved in OECD 201 medium for 72 h in a static system. The measured concentrations of triethylmethylammonium tetrafluoroborate ranged from 99 and 108% of nominal concentration during the test. The 72 h-EC50 (growth rate) and the 72 h-NOEC were determined to be > 106 mg/L and 106 mg/L triethylmethylammonium tetrafluoroborate (mean measured), respectively. These results are considered reliable since all validity criteria of the study were met.

Description of key information

In a reliable GLP- and guideline- conform key study of triethylmethylammonium tetrafluoroborate according to OECD 201, the 72-h ErC50 and NOErC values for the inhibition of growth rate of Pseudokirchneriella subcapitata were estimated with > 103 mg/L and ≥ 103 mg/L (mean measured), respectively (Codling, 2009). Thus, triethylmethylammonium tetrafluoroborate appears to have a low potential for toxicity to freshwater algae.

Key value for chemical safety assessment

Additional information