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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:
2017-05-23 - 2017-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Version adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011
Deviations:
yes
Remarks:
Deviations from the Study Plan, see "Any other information on material and methods incl. tables", but none from the guideline
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Version adopted 07. December 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Pyridine-2-carbaldehyde oxime
EC Number:
212-849-6
EC Name:
Pyridine-2-carbaldehyde oxime
Cas Number:
873-69-8
Molecular formula:
C6H6N2O
IUPAC Name:
pyridine-2-carbaldehyde oxime
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in a tightly closed vessel at room temperature (20 ± 5 °C).

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: not specified. Samples for the analytical determination were taken at test start and test end.
- Sample storage conditions before analysis: not specified

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing the load of 100.0 mg/L in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments were prepared by dilution of this stock solution with nutrient medium.
- Controls: medium without any test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus (unicellular freshwater green alga)
- Strain: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen [SAG], Strain-No. 86.81
- Source (laboratory, culture collection): The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): An aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours. The resulting culture grew exponentially.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.
- Any deformed or abnormal cells observed: no

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
22.0 - 23.1 °C
pH:
7.7 at test start
7.6 - 7.8 at test end
Nominal and measured concentrations:
Nominal: 0, 1.0, 3.2, 10, 32 and 100 mg/L
Found (t0): Found (t72h): LOQ (Limit of quantification) = 0.2 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): open (covered with perforated plastic foil)
- Material, size, headspace, fill volume: glass flasks with a total volume of 65 mL, filled with 45 ± 1 mL of the respective test solution
- Aeration: no
- Initial cells density: 3.9 *103 cells/mL
- Control end cells density: 149640 cells/mL (mean) +/- 11906 cells/mL (standard deviation)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

Stock Solution I:
NH4Cl 1500 mg
MgCl2*6H2O 1200 mg
CaCl2*2H2O 1800 mg
MgSO4*7H2O 1500 mg
KH2PO4 160 mg
H2O deionised ad 1000 mL

Stock Solution II:
FeCl3*6H2O 64 mg
Na2EDTA*2H2O 100 mg
H2O deionised ad 1000 mL

Stock Solution III:
H3BO3 185 mg
MnCl2*4H2O 415 mg
ZnCl2 3 mg
CoCl2*6H2O 1.5 mg
CuCl2*2H2O 0.01 mg
Na2MoO4*2H2O 7 mg
H2O deionised ad 1000 mL

Stock Solution IV:
NaHCO3 50 g
H2O deionised ad 1000 mL

Algal Medium:
Stock Solution I 10.0 mL
Stock Solution II 1.0 mL
Stock Solution III: 1.0 mL
Stock Solution IV 1.0 mL
H2O deionised ad 1000 mL

Stock solutions I and III and the used demineralised water were sterilized using an autoclave. Stock solutions II and IV were sterilized using filtration. The algal medium corresponds to the OECD TG 201 medium.
Deviations from the nominal weighted loads were less than 5%.

- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous
- Light intensity and quality: 7200 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell number was determined with an electronic particle counter before the start of incubation and every 24 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125
- Test concentrations: 0, 1.0, 3.2, 10, 32 and 100 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
8.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): normal and healthy appearance of the algae at the end of the test
- Unusual cell shape: no
- Colour differences: no
- Flocculation: not stated
- Adherence to test vessels: not stated
- Aggregation of algal cells: not stated
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
The 72h-EC50s of potassium dichromate were determined in a separate reference test. The values were within the range of the laboratory. The results of the last study with the positive control K2Cr2O7 were:
72h ErC50 = 0.94 mg/L (0.90 - 0.98 mg/L)
72h EyC50 = 0.42 mg/L (0.39 - 0.45 mg/L)
The study was performed under GLP conditions in February 2017.
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1. Biological results are stated using two significant digits.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study was performed according to OECD TG 201 with no relevant deviations (only minor deviation from the study plan with no influence on the integrity of the study, see "Any other information on materials and methods incl. tables"), the results were so obtained via a scientifically reasonable method. The validity criteria for the study as given by the OECD Guideline 201 were met and the study is therefore considered to be valid. Hence, there is no doubt that the obtained results are not reliable: The toxic effect was investigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 hours. The maximum concentration investigated was 100 mg/L (nominal).
At the start and at the end of the test, the content of the test item in the test solutions with and without algae was determined using HPLC.
The measured concentrations lay between 89 % and 99 % of the nominal concentrations at the beginning of the test and between 98 % and 111 % of the nominal concentrations at the end of the test. As the measured concentrations were within ± 20 % of the nominal concentrations, the determination of the results was based on nominal concentrations.
The following EC-values (72h; growth rate; nominal) were determined accordingly:
LOEC (growth rate and yield) = 3.2 mg/L
NOEC (growth rate and yield) = 1.0 mg/L
EC50 (growth rate) = 48 mg/L
Based on these results, the test item does not need to be classified as acute toxic to the aquatic environment. With regard to chronic toxicity, taking into account the facts that the test item is soluble in water, i.e. above the determined EC50 value, and not readily biodegradable, the test item should be classified as aquatic chronic Cat. 3 according to the Regulation (EC) No. 1272/2008.
Executive summary:

The study was performed according to OECD TG 201 under GLP using 5 concentrations ranging from 1 to 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the following concentrations: 3.2 / 10 / 32 / 100 mg/L nominal concentration

At the start and at the end of the test, the content of the test item in the test solutions with and without algae was determined using HPLC. The measured concentrations lay between 89 % and 99 % of the nominal concentrations at the beginning of the test and between 98 % and 111 % of the nominal concentrations at the end of the test. As the measured concentrations were within ± 20 % of the nominal concentrations, the determination of the results was based on nominal concentrations.

The 72h-EC50s of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 - 0.66 mg/L).

The following results for the test item pyridine-2-carbaldehyde oxime were determined:

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

1.0 mg/L

3.2 mg/L

12 mg/L

48 mg/L

Yield

1.0 mg/L

3.2 mg/L

8.2 mg/L

20 mg/L