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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 February 2015 - 18 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Test item: SE7B
Chemical name: Estolide Dimer, Trimer and Tetramer
Origin: Biosynthetic Technologies
Lot No.: 0000140602
CAS-No.: 1365345-64-7
Purity: 98%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Chemical analysis of the test item was performed at the test site BiochemA GmbH. Samples
were taken in duplicate from the lowest, intermediate and highest test loading rates and the
control at 0 h, 24 h, 48 h and 72 h from separate test vessels without algae. After 72 h
additional samples were taken from test vessels with algae to determine the effect of the
algae on the test item concentration. With every sampling 2 x 4 mL were taken out of the
control and test vessels and filled into glass vials. Samples were kept in the refrigerator at 5
± 3°C until being sent to the analytical laboratory. Since the test item adsorbs to glass
surfaces, acetone was used to rinse-off the test item from the glass vial directly prior
analytical analysis. A retain sample was kept in the refrigerator until finalization of the study.
Proper departure and arrival of the samples was documented.

Test solutions

Vehicle:
not specified
Details on test solutions:
Solution A1
Ammonium chloride p.a. NH4Cl 1.5 g
Magnesium chloride p.a. MgCl2 * 6 H2O 1.2 g
Calcium chloride dihydrate e.p. CaCl2 * 2 H2O 1.8 g
Magnesium sulfate heptahydrate p.a. MgSO4 * 7 H2O 1.5 g
Potassium dihydrogen phosphate p.a. KH2PO4 0.16 g
dissolved in 1000 mL deionised water

Solution A2
Iron (III) chloride hexahydrate p.a. Fe(III)Cl3 * 6 H2O 0.0640 g
Sodium-EDTA dihydrate Na2EDTA * 2 H2O 0.0226 g
dissolved in 1000 mL deionised water

Solution A3
Boric acid p.a. H3BO3 0.185 g
Manganese chloride tetrahydrate p.a. Mn(Cl2) * 4 H2O 0.415 g
Sodium molybdate dihydrate p.a. Na2MoO4 * 2 H2O 0.007 g
Zinc chloride p.a. ZnCl2 0.003 g
Cobalt chloride hexahydrate p.a. CoCl2 * 6 H2O 0.0015 g
Copper chloride dihydrate p.a. CuCl2 * 2 H2O 0.00001 g
dissolved in 1000 mL deionised water (for ZnCl2, CoCl2 and CuCl2 more concentrated solutions were
prepared and the required volumes used for A3)

Solution A4
Sodium hydrogen carbonate p.a. NaHCO3 50 g
dissolved in 1000 mL deionised water.
Solutions A1 - A3 were autoclaved, Solution A4 was sterilely filtered (membrane pore diameter 0.22 µm).

10-x concentrated medium
100 mL from stock solution A1 and 10 mL from each stock solution A2 - A4 were filled up to
1000 mL with deionized water. After aerating the medium for 1 h the pH was 8.4.
1-x concentrated medium was prepared by diluting 150 mL of the 10-x concentrated medium
with 1350 mL deionized water.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Tests were carried out with the green alga Pseudokirchneriella subcapitata (Strain No. 61.81
SAG) from the Culture Collection of Algae at the University of Goettingen and cultivated in
suspension culture. The strain used for this study has been cultured in the laboratory of
Hydrotox GmbH since November 2014. Twice a week, the stock suspension is diluted into
fresh Holm-Hansen medium under axenic conditions to keep it in
exponential growth.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
After 0, 24, 48, and 72 hours, 200 µl samples were taken out of each test vessel and chlorophyll fluorescence was measured in a 96-well micro-plate.

Test conditions

Test temperature:
The temperature in the test was in the range of 23.1 to 23.3°C, which is within the
requirements of the guideline (21-24°C). The variation in temperature was 0.2 K, which
was within the required range of +/- 2 K
pH:
Measured pH-values at the beginning (0 h) and at the end of the test (72 h) were in teh range pH 7.3-7.3 for all samples.
The shift in pH was maximal 0.1
Nominal and measured concentrations:
The algae test was performed with five nominal loading rates between 62 mg/L and 5000 mg/L.

Measured test
concentrations of the dimer compound were very low with highest measured values of 6.20
µg/L in the lowest to 11.9 µg/L in the highest nominal loading rate. As the test item is a
mixture of not readily soluble substances, results are reported as nominal loading rates and
no conversion to effective concentration can be given.
Details on test conditions:
Tests were carried out with the green alga Pseudokirchneriella subcapitata (Strain No. 61.81
SAG) from the Culture Collection of Algae at the University of Goettingen and cultivated in
suspension culture. The strain used for this study has been cultured in the laboratory of
Hydrotox GmbH since November 2014. Twice a week, the stock suspension is diluted into
fresh Holm-Hansen medium under axenic conditions to keep it in
exponential growth.
Reference substance (positive control):
not specified

Results and discussion

Effect concentrations
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
556 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The algae test was performed with five nominal loading rates between 62 mg/L and 5000 mg/L. The following results were obtained:

Growth Rate:
ErLR50(72h, nominal): n.d.
ErLR20(72h, nominal): n.d.
ErLR10(72h, nominal): 3241 mg/L (95%-CI: n.d.)
LOErLR(72h, nominal) : 1667 mg/L
NOErLR(72h, nominal) : 556 mg/L

Yield:
EyLR50(72h, nominal): 5569 mg/L (95%-CI: 2190 – 1028157 mg/L)
EyLR20(72h, nominal): 613 mg/L (95%-CI: 6.9 – 1524 mg/L)
EyLR10(72h, nominal): 193 mg/L (95%-CI: 0.03 – 637 mg/L)
LOEyLR(72h, nominal) : 556 mg/L
NOEyLR(72h, nominal) : 185 mg/L
Reported statistics and error estimates:
The data presented were calculated using the statistical software ToxRat (ToxRat solutions, Alsdorf, Germany)

Any other information on results incl. tables

Results tables attached ( Results - Algal Growth Inh OECD 201 - SE7B.pd)f

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The algae test was performed with five nominal loading rates between 62 mg/L and 5000 mg/L. The following results were obtained:

Growth Rate:
ErLR50(72h, nominal): n.d.
ErLR20(72h, nominal): n.d.
ErLR10(72h, nominal): 3241 mg/L (95%-CI: n.d.)
LOErLR(72h, nominal) : 1667 mg/L
NOErLR(72h, nominal) : 556 mg/L

Yield:
EyLR50(72h, nominal): 5569 mg/L (95%-CI: 2190 – 1028157 mg/L)
EyLR20(72h, nominal): 613 mg/L (95%-CI: 6.9 – 1524 mg/L)
EyLR10(72h, nominal): 193 mg/L (95%-CI: 0.03 – 637 mg/L)
LOEyLR(72h, nominal) : 556 mg/L
NOEyLR(72h, nominal) : 185 mg/L