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:
2008-12-01 to 2008-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
, 2006
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2006-06-01

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Naringin Dihydrochalcone (DHC)
- Physical state: solid, whitish powder
- Storage condition of test material: room temperature, dark and dry
No further details are given.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No other test substance was used.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentration of the test item was analytically determined from all test concentrations and the control after 0 (test start), 24, 48 and 72 hours (test end).
- Concentrations: 0, 70.7, 98.9, 138, 194, 271, 380 mg/L
- Sampling method: Separate samples for the test start, 24, 48 and 72 hours were provided.
- Sample storage conditions before analysis: All samples were stored at 6 °C until start of analysis.
- Preparation of samples: The samples were dilzted with mobile phase prior to analysis. The control was analysed directly.

Furthermore, separate samples of the highest and lowest test concentration were prepared, incubated in the dark and analysed at the end of the test..

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 380 mg test item/L was prepared with dilution water. Solution was treated with ultrasound at 40°C for 30 minutes and undissolved particles were removed by filtration.
Dilution factor: 1.4

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen
- Age of inoculum (at test initiation):
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35-70 µE x m-2 x s-1 for 24 hours per day.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: same as test

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
nominal hardness of 0.24 mmol Ca+Mg/L
Test temperature:
nominal range 21-24°C,
controlled at +/- 2°C,
mean value: 22.5°C
pH:
test item (test start): pH 7.10 - pH 7.88,
test item (test end): pH 7.13 - pH 7.97,
control (test start): pH 8.05,
control (test end): pH 8.43
see also attached table "environmental conditions"
Dissolved oxygen:
not required
Salinity:
Composition of Dilution water :
NH4Cl: 15 mg/L
MgCl2 x 6 H2O: 12 mg/L
CaCl2 x 2 H2O: 18 mg/L
MgSO4 x 7 H2O: 15 mg/L
KH2PO4: 1.6 mg/L
FeCl3 x 6 H2O: 0.064 mg/L
Na2EDTA x 2 H2O: 0.1 mg/L
H3BO3: 0.185 mg/L
MnCl2 x 4 H2O: 0.415 mg/L
ZnCl2: 3 x 10-3 mg/L
Na2MoO4 x 2 H2O: 7 x 10-3 mg/L
CoCl2 x 6 H2O: 1.5 x 10-3 mg/L
CuCl2 x 2 H2O: 1 x 10-5 mg/L
NaHCO3: 50 mg/L
pH-value: 8.1 +/- 0.2
This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.
Nominal and measured concentrations:
Test concentrations (nominal): 70.7, 98.9, 138, 194, 271 and 380 mg/L (factor 1.4);
Test concentrations (geometric mean concentration): 54.8, 77.1, 117, 177, 254 and 354 mg/L
see also attached table
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile Erlenmeyer flasks, volume 250 mL
- Type (delete if not applicable): closed with cotton wool plugs
- Material, test volume: glass flasks, 25 mL
- Control end cells density: 2-5 x 10^3 cell/mL
- No. of vessels per concentration (replicates): three replicates for each concentration level
- No. of vessels per control (replicates): six replicates
- No. of vessels per vehicle control (replicates): six replicates (without test item) were tested under the same conditions as the test replicates

GROWTH MEDIUM
- Standard medium used: yes, nutrient medium Z according to Lüttge et al. (1994)

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

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24 hours/day
- Light intensity and quality: 60-120 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: Cell density was measured daily via chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm.
- Other: Self fluorescence occurred after 48 hours in the test concentrations 117 -351 mg/L and after 72 hours in each concentration level. The self fluorescense was measured daily in one separate replicate of each concentration without alga. It was tested under the same conditions as the test group.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.4
Reference substance (positive control):
yes
Remarks:
Potassium dichromate p.a. (Sigma), >99.5% pure

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
177 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
growth rate
Remarks on result:
other: rate-related inhibition
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
254 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
growth rate
Remarks on result:
other: rate-related inhibition
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
196 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
growth rate
Remarks on result:
other: rate-related inhibition, 175-215 mg/L 95% confidence interval
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
251 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
growth rate
Remarks on result:
other: rate-related inhibition, 232-266 mg/l confidence interval
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 354 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
growth rate
Remarks on result:
other: rate-related inhibition, above water solubility
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
117 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
biomass
Remarks on result:
other: inhibition of yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
177 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
biomass
Remarks on result:
other: inhibition of yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
167 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
biomass
Remarks on result:
other: inhibition of yield, 157-176 mg/l 95% confidence interval
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
177 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
biomass
Remarks on result:
other: inhibition of yield, 168-186 mg/l 95% confidence interval
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
211 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
biomass
Remarks on result:
other: inhibition of yield, 200-224 mg/l 95% confidence interval
Details on results:
The concentrations of Naringin Dihydrochalcone (DHC) were analysed at all concentration levels after 0, 24, 48 and 72 h (same algae density as test replicates) via HPLC-DAD analysis. The measured concentrations at test start were in the range of 96 - 102 % of the nominal values. At the end of the test the test item gave recoveries in the range of 45 – 89 %. Separate replicates of the highest and lowest test concentration were incubated in the dark and analysed at test end. They gave recoveries of 92 % for 380 mg/L and 100 % for 70.7 mg/L (see attached tables).
Results with reference substance (positive control):
- Results with reference substance are valid
- EC50 rate-related inhibition: 0.69 mg/L (CI = 0.64-0.74 mg/L)
- EC50 yield inhibition: 0.31 mg/L (CI = 0.3-0.32 mg/L)
Reported statistics and error estimates:
The NOEC and LOEC were determined by calculation of statistical significance of growth rates and yield.
One Way Analysis of variance was carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of variance a normality test and an equal variance test were done first. P-values for both normality and equal variance tests were 0.05. The a-value for ANOVA was a=0.05. The equal variance tests of growth rate data failed. The data were transformed with Graph Pad Prism function Y = Y squared. Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant.
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see above "overall remarks"
Conclusions:
In this study Naringin Dihydrochalcone (DHC) was found to inhibit the growth of the freshwater green alga Desmosdesmus subspicatus after 72 hours with the following effect values: the EC50 for specific growth rate = >354 mg/L, and the EC50 for yield = 211 (200-224) mg/L. All effect levels are given based on geometric mean exposure concentrations of the test item.