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Ecotoxicological information

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:
Dec 13 2012 - Dec 17 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: The day 0 samples are taken directly from the vials of the stock solutions. To the other sampling times (day 1, day 2 and day 3) sampling is performed as follows: Sampling is performed using glass pipettes directly from the test vials. The pipette and all used vials are preconditioned two times with about 2 mL each of the respective test solutions. Then about 1.5 mL of the test sample is taken.
The vials are centrifuged for 10 min at 13.000 rpm (Thermo Scientific Heraeus Fresco 17) to separate the algae. Then 1000 µL of the aqueous sample (or aliquots; or aliquots adjusted to 1000 µL with pure water) and 100 µL of methanol are pipetted into a HPLC vial with screw cap. The vial is closed, shaken shortly by hand and measured by LC-MS/MS. If the concentrations are too high, new diluted samples are prepared and measured. Each centrifuged algae sample is extracted with 500 µL of methanol to determine the residual amount of the test item (respective homologues) sticking to the algae. After short mixing (IKA Vortex-Schüttler VF2) the samples are centrifuged again. Then 100 µL of the methanolic sample and 1000 µL pure water are pipetted into an HPLC vial with screw cap. The vial is closed, shaken shortly by hand and the sample measured by LC-MS/MS.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: Chlorophycea, Chlorophyta
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D37073
Göttingen, Catalog No 61.81 SAG
- Age of inoculum (at test initiation):
- Method of cultivation: The stock cultures are maintained fulfilling the criteria of the OECD 201 guideline. Prior to testing a pre-culture will be established in standard OECD growth medium to obtain exponentially-growing algae for the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not stated
Test temperature:
22°C
pH:
7.2-7.9
Dissolved oxygen:
not stated
Salinity:
not stated
Nominal and measured concentrations:
A non-GLP range-finder test was performed with 3 nominal concentrations and chemical analysis: Control, 0.1, 1.0, and 10.0 mg test item/L.
Nominal concentrations for the definitive test: 0.625, 1.25, 2.50, 5.0 and 10.0 mg test item/L.
At test start, the measured test concentrations, based on the sum of the item fractions, were 0.610, 1.18, 2.24, 4.55 and 9.00 mg/L (i.e. 89.6– 97.6 % of nominal). By test end (72 hours), the measured test concentrations were 0.343, 0.723, 1.49, 3.34 and 7.31 mg/L (i.e. 54.9 – 73.1 % of nominal).
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile 250 mL conical glass flasks covered with air-permeable silicone-sponge caps.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume:
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density:
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: /

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod:
- Light intensity and quality: The culture vessels were continuously illuminated with a light intensity (day light: OSRAM “cool white”) adjusted to 4440 - 8880 lux close to the surface of the liquid (equivalent to 60-120 µE m-² s-²).
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (CASY 1 Model TT, Roche Diagnostics GmbH, Germany).
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: The test concentrations were: control, 0.036, 0.358 and 3.58 mg a.s./L, corresponding to 0.1, 1.0, and 10.0 mg test item/L.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.97 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 1.8 - 2.2
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.705 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 0.63 - 0.79
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.303 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: There was a concentration-dependant inhibition on the growth rate and the cell number increase (yield) of P. subcapitata. The inhibition of growth rate was -3.0, 7.7, 51.1, 70.7 and 97.3 % after 72 hours exposure at the test concentrations of 0.405, 0.845, 1.66, 3.64 and 7.95 mg/L (geometrical mean measured concentrations), respectively
Results with reference substance (positive control):
- EC50: The nominal ErC50 value of 2.91 mg/L (95 % confidence limits 2.35 - 3.63 mg/L) is in good agreement with the results of an international ring test with ErC50 of 3.38 ± 1.30 mg/L.
Reported statistics and error estimates:
Yield data showing inhibitions around 50% was statistically analysed to determine an EC50, EC20 and EC10 values together with 95% confidence intervals using Probit-analysis assuming log-normal distribution of the values. Individual replicate responses were used for the regression analysis.
The NOEC and LOEC values for growth rate and yield were determined using the Williams t-test. The computer program ToxRat Professional was used for all statistical evaluations.
Validity criteria fulfilled:
yes
Conclusions:
The mean measured EC50 for growth rate and yield was determined to be 0.705 mg a.i./L and 0.376 mg a.i./L, respectively. A mean measured NOEC of 0.303 mg a.i./L was statistically estimated for growth rate.

Description of key information

A full study was performed under GLP conditions and according to OECD TG 201 [(Wenzel (2013)]. The 72-h EC50 based on growth rate was 0.705 mg a.i./L with 95% confidence limits of 0.6 -0.8 mg a.i./L. The No Observed Effect Concentration was 0.303 mg a.i./L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.705 mg/L
EC10 or NOEC for freshwater algae:
0.303 mg/L

Additional information

Four ecotoxicity studies with algae are available on the test substance. The study considered as the key study for the risk assessment is a GLP study conducted according to the OECD Guideline 201 and EU Guideline C.3 [(Wenzel (2013)]. The 72-h EC50 based on growth rate was 0.705 mg a.i./L with 95% confidence limits of 0.6 -0.8 mg a.i./L. The No Observed Effect Concentration was 0.303 mg a.i./L.

Another study is available which gives a 72h NOEC of 0.19 mg a.i./L [Webley (2008)]. However this study was not considered reliable as no analytical verification of the tested concentrations was done. Therefore the study will not be used for the risk assessment. Two other algae studies [(Mead (2000) and Kamp (1997)] were considered valid studies but both resulted in 72h NOEC or 72h ErC10 values (1.25 mg a.i./L and 18.8 mg a.i./L respectively) that were higher than the NOEC value obtained in the study from Wenzel (2013).