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:
from 2008-11-04 to 2008-11-07
Reliability:
1 (reliable without restriction)
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
92/69/EEC
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Details on test material:
Batch no: 002FL08;
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
NA

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
At the start of the test (0h) one sample was taken from each concentration level and at 24h, at 48h and at the end of the test (72h) one sample was taken from each replicate test vessel. Three samples were taken from the control medium.
Storage of the Samples: The samples were analysed directly after sampling.

Test solutions

Vehicle:
no
Details on test solutions:
The test item solution was prepared according to the WAF method (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000).
A supersaturated test item solution was prepared by dispersing/dissolving the test item amount (nominal load of 100 mg/L, limit concentration) without the use of any organic solvent into the Test Medium (OECD Medium) two days before the start of the study (on day -2). This solution was shaken for 24 hours at app. 30 ºC, then was left settling for 24 hours at app. 20 ºC and thereafter filtrated through a paper filter.
The test solutions were prepared by appropriate dilution of the stock solution on day 0.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) (Printz-Starr).

Origin: The algae were supplied by the Georg-August-Universität Göttingen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften Experimentelle Phykologie und Sammlung von Algenkulturen (SAG), Nikolausberger Weg 18, 37073 Göttingen, Germany

Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The preculture was incubated for three days at this test.)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no post exposure observation period

Test conditions

Hardness:
NA
Test temperature:
The cultures were maintained at a temperature in the range of 21 – 25 C  2 C, which was checked and recorded at the beginning of the study and every 24 hours in a flask filled with water. In addition, the temperature was continuously measured (with a min/max thermometer) within the climate chamber.
measured temperature: 23.3 - 23.7 °C
pH:
The pH was checked at the beginning and at the end of the study in the controls and every concentration.
pH 7.46 to 7.57 at test start
pH 7.77 to 8.83 at test end
Dissolved oxygen:
NA
Salinity:
NA
Nominal and measured concentrations:
Based on the results of the preliminary tests five different concentrations arranged in a geometric series and an untreated control group was tested in the main study. The nominal test item concentrations were 0.16; 0.31; 0.63; 1.25 and 2.50 mg test item/L.

The highest nominal concentration was the Stock solution (2.50 mg/L measured). The nominal test item concentrations were S.S./16; S.S./8; S.S./4; S.S./2 and S.S./1 (where S.S.=Stock Solution).

The measured test item concentrations were out the ±20 % range of the nominal concentrations during the test.
The corresponding measured mean test item concentrations were:
0.05; 0.18; 0.31; 0.63; 0.53 and 0.85 mg/L.
Therefore, all reported biological results are related to the geometric mean of the measured test item concentrations during the test calculated by EXCEL Software Program.
Details on test conditions:
Test Units:
Type and Size: Erlenmeyer flasks of 250 mL volume with 100 mL test medium
Identification: Each test unit was uniquely identified with study code, test item concentration and replicate number.
Course of the test:
Introduction of Algae: The test was started (0 hours) by inoculation of a biomass of approximately 10.000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, set up three days prior to the test start using the same conditions as in the test.
Replicates: The test was performed with three replicates per test concentration and six replicates in the untreated control. On a laboratory orbital shaker, volumes of 100 mL algal suspensions per replicate were continuously shaken in 250 mL Erlenmeyer flasks, covered with air-permeable stoppers.

Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.73 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% conf. limits: 0.62-0.85
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.31 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.34 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: 0.99-1.81
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.05 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.61 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% conf. limits: 0.55-0.69
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.05 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
Microscopic Examinations:
Shape of the Algal Cells: The shape of the algal cells growing was obviously not affected.
Cell Density in Control: In the control the cell density has increased from nominal N= 1 x 104 cells/mL at the start of the test (0 hours) to
N= 67.33 x 104 cells/mL (mean value) after 72 hours by a factor of 67.33. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.
Results with reference substance (positive control):
The date of the last study (Study Code: 08/712-022AL) with reference item Potassium dichromate is: 20 – 23 June 2008.
The ErC 50 : 0.90 mg/L, (95 % confidence limits: 0.82 – 0.98 mg/L)
The EbC 50 : 0.60 mg/L, (95 % confidence limits: 0.54 – 0.66 mg/L)
The EyC 50 : 0.48 mg/L, (95 % confidence limits: 0.44 – 0.53 mg/L)
Reported statistics and error estimates:
Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 0 h, 24 h, 48 h and at the end of the test (72 hours after the start of the test) using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399)..
Percentage inhibition of area A, growth rate r and yield Y were calculated using EXCEL for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The EC50 values of the test item and their confidence limits were calculated using Probit analysis. The analysis was done using the statistical software program “TOXSTAT 3.5”.
For the determination of the LOEC and NOEC, the calculated mean biomass b (area under the growth curve), growth rates r and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test and Williams’test by TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance for Levene’s Test.

Any other information on results incl. tables

NA

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
72-h growth rate EC50 = 1.34 mg/L.
72-h growth rate NOEC = 0.05 mg/L.
Executive summary:

In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with AVE 5530 EVA-KET, the 72-h EC50 based on growth rate was determined as 1.34 mg/L. The growth rate NOEC was determined at 0.05 mg/L. Analysis showed that the deviation from the nominal concentrations was higher than ± 20 % during the study, therefore the mean of measured values were used for calculation.