Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-04-04 to 2017-01-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2011)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
No. 761/2009/method C.3 (2009)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: A saturated solution of the test item with a nominal loading of 1 mL/L was tested in a limit test. The limit concentration is based on the results of a preliminary range finding test.
- Sampling method: Analytical evaluation of the test concentration of the test substance and the control was carried out via LC-MS/MS from freshly prepared media after 0 hours (with algae) and old test media after 72 hours (with algae). The limit concentration and the control were diluted (factor 2) with acetonitrile containing 2% formic acid directly after sampling.
- Sample storage conditions before analysis: All samples were stored at room temperature until sample preparation and in an autosampler until start of analysis, if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution (The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium. acc. to OECO Series, No. 23 (2000)) with a nominal loading of 1 mL test substance/L was prepared once with demineralized water 95.5 h prior to the start of the exposure. The test item was placed onto the surface of the demineralized water with a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 72.5 h with a magnetic stirrer at 30 ± 1 °C followed by slow stirring for 23 hours at 22 ± 1 °C. After completion of stirring, the test item solution was allowed to stand for 1 hour for separation of the phases. Thereafter, the saturated solution was removed by siphon from the approximate bottom of the glass flask. Before application, the components of the dilution water were added to the saturated solution. The saturated solution was used as a limit concentration in the test.
- Controls: six replicates
- Evidence of undissolved material: The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). No Tyndall effect was observed.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata, green algae
- Strain: Pseudokirchneriella subcapitata HINDAK, SAG 61.81
- Source: Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen
- Age of inoculum (at test initiation): A four day old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar.

ACCLIMATION
- Acclimation period: A four day old preculture, prepared in dilution water, was used as inoculum.
- Culturing media and conditions (same as test or not): Nutrient medium Z
- Any deformed or abnormal cells observed: The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol Ca+Mg/L
Test temperature:
Nominal range: 22 °C, mean value 22 °C
pH:
nominal 21-24°C +/-2°C, current: 8.08 to 8.13 at 0 hours, 9.38 to 9.42 at 72 h
Nominal and measured concentrations:
A saturated solution of the test item with a nominal loading of 1 mL/L was tested in a limit test.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: sealed with cotton wool plugs
- Material, size, headspace, fill volume: volume: 250 mL, test volume 100 mL
- Initial cells density: nominal 5*10^3-10^4, current: 6305 cells/mL
- Control end cells density: 2.0*10^6
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: dilution water according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Alkalinity: pH-value 8.1 +/- 0.2
- Ca/mg ratio: 0.24 mmol Ca+Mg/L

OTHER TEST CONDITIONS
- Photoperiod: 24 h/day light
- Light intensity and quality: approx. 4440 to 8880 lux/ 60 to 120 µE*m^(-2)*s^(-1), current study: mean value 5435 lux (range 4814 to 5832 lux)
- Agitation:Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.
- Other: Microscopic evaluation of the cells at the start and end of the exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.
- The pH-value at the start of the exposure was measured in one additional replicate of the limit concentration and the control. At the end of the exposure it was measured in a pooled sample of the limit test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of the exposure.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: A saturated solution of the test substance (The maximum dissolved concentration of the test substance that can be achieved under the test conditions in the test medium, acc. to OECO Series, No. 23 (2000)) with a nominal loading of 1 mL/L was tested in a limit test. The limit concentration is based on the results of a preliminary range finding test.

Range finding study
- Results used to determine the conditions for the definitive study: A range finding study has been performed with a 100%, 10% and 1 % saturated solution. Growht rate inhibition of 2% with the 100% saturated solution. Yield inhibition 12-22%. Measured Test item concentrations during the range finding test were below system quantification limit (5 µg test substance/L). The limit concentration in the main test is based on the results of a preliminary range finding test. A saturated solution of the test item with a nominal loading of 1 mL/L was tested in a limit test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 other: mL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
based on the nominal loading of the saturated solution [mL/L].
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 other: mL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
based on the nominal loading of the saturated solution [mL/L].
Key result
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
> 1 other: mL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
based on the nominal loading of the saturated solution [mL/L].
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 1 other: mL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
based on the nominal loading of the saturated solution [mL/L].
Key result
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
> 1 other: mL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
based on the nominal loading of the saturated solution [mL/L].
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 other: mL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other:
Remarks:
based on the nominal loading of the saturated solution [mL/L].
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
> 1 other: mL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other:
Remarks:
based on the nominal loading of the saturated solution [mL/L].
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 other: mL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other:
Remarks:
based on the nominal loading of the saturated solution [mL/L].
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
> 1 other: mL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other:
Remarks:
based on the nominal loading of the saturated solution [mL/L].
Details on results:
Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities. All effect values are given based on nominal limit test item loadings. The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The saturated solution was clear and homogenous. The measured concentrations of the test item were < LOQ (6 µg/L of the test item) at the start {0 hours) and at the end of the exposure (72 hours).
Results with reference substance (positive control):
- Results with reference substance valid: The toxicity of potassium dichromate (CAS No.7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours (with headspace). The reference substance toxicity is in the valid range following test facility SOPs.
- EC50: 0.460 mg/L growth rate (95% Conf. intervall 0.443-0.481 mg/L), 72 hour. Valid range 0.780+/- 0.473.
Reported statistics and error estimates:
Stistical Significance (NOEC/LOEC) - growth rate and yield: The program (t-test) states no significant difference for saturated solution compared to the control.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The study meets the validity criteria of the guideline (327-fold increased cell growth (required <16-fold). 18.0% mean coef. of variation section-by-section spec. growth rates (req. <35%). 0.74% variation coef. of average spec. growth rates (req. <7%).).
Conclusions:
In this study no inhibition of growth on the freshwater green alga Pseudokirchneriel/a subcapitata was found after 72 hours exposure to the test substance. The nominal EC50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were >1.00 mL/L, respectively. The nominal NOEL-values for inhibition of growth rate and yield after 72 hours were 1.00 mL/L, respectively. All effect values given are based on nominal loading of the saturated solution of the test substance.

In the saturated solution with a nominal loading of 1.00 mL/L of the test substance, no effects on Pseudokirchneriel/a subcapitata were observed.