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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study was preformed according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
For treatment 0.1 / 0.32 / 1.0 / 3.2 and 10 mg/L 100 ml unfiltered sample will be acidified with HCL conc. and evaporated at 105°C. After addition of 10 µl HCl, conc. and 5 ml de-mineralised water the dry residue is dissolved under heating. The solution is transferred in a 10 mL volumetric flask.
For treatments 0.01 mg/L and 0.032 mg/L 200 ml unfiltered sample will be acidified with HCL conc. and evaporated at 105°C. After addition of 10 µl HCl, conc. and 2.5 ml demin-eralised water the dry residue is dissolved under heating. The consolidated solutions are transferred in a 10 mL volumetric flask.
After addition of 100 µl HCl conc. and 100 µL KCl 10% the flask is filled up to 10 ml with demineralised water. Then the solution is transferred in an AAS vial.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Specification
Unicellular freshwater green alga:
Species Desmodesmus subspicatus
Strain CHODAT
Family Chlorophyceae
Order Chlorococcales

Origin and Culture
The culture of Desmodesmus subspicatus is obtained from MBM Sciencebridge GmbH.. The alga are kept as stock culture on solid agar at 2 – 7 °C. From an aliquot of this stock culture, the pre-culture will be prepared.

Study design

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

Test conditions

Test temperature:
21-24 °C
pH:
ca. 8
Nominal and measured concentrations:
Treatments 0.01 / 0.032 / 0.1 / 0.32 / 1.0 / 3.2 / 10 mg/L

Details on test conditions:
Test Culture
Three or four days before the start of each experiment, an aliquot of the stock culture con-taining a few cells is brought into 50 mL algal medium. The mixture is incubated for 72 - 96 hours at 21 –24°C and 4440 - 8880 Lux. The resulting pre-culture is growing exponen-tially.
Before usage, the pre-culture will be checked for the absence of cell aggregates. After the adjustment to a cell concentration of about 6*104/mL through photometric measurement and addition of algal medium, the pre-culture can be used for the test. The adjusted pre-culture will be mixed with the same amount of nutrient solution, the resulting mixture is designated as the test culture.

Positive Control
The EC50 of potassium dichromate is determined twice a year. A stock solution is used to prepare seven concentrations (0.04 / 0.063 / 0.1 / 0.16 / 0.25 / 0.4 / 0.63 / 1.0 / 1.6 mg/L).

Performance of the Study
Preparations
On the day before the start of the test, a sufficient number of vessels are provided. Two hours before the start of the test, the light incubator is turned on for warming up.
Description of the Study Performance
For each treatment, 400 mL of the respective test item solution (concentration = concen-tration in the treatment * 1.25) is prepared and mixed with 100 mL of the test culture. In this test solution, the pH-value is measured.
For the control, deionised water is used instead of test item solution.
The test vessels are filled with 100 ± 5 mL of the respective test solution and incubated open for 72 hours, shaken on an orbital shaker. Before the start of incubation and every 24 hours, the values of the absorption of the cuvettes are recorded. After the test, the pH value of the treatments and the control is measured again.
At the end of the test, treatments and control are inspected microscopically in order to ver-ify a normal and healthy appearance of the algae.
At the beginning and at the end of the test, the content of the test item in the test solution is analytically determined. Samples must not be filtrated.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7 (Potassium dichromate)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 2.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 1.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
At the start and at the end of the test, the content of Pb as main component of the test item
(80.3 %) was measured via AAS. The measured Pb concentrations were unexpectedly low
and implausible. Because of similar measured concentrations in a daphnia toxicity test, an
additional experiment for the determination of solubility in deionised water and in daphnia
test medium was performed within the scope of the Daphnia study.
Because of reactions with the anions in the test medium, the measured Pb concentration
in daphnia test medium was only 0.5 % of the Pb concentration in deionised water.
Furthermore, in the study for the determination of solubility in water, it was verified that
due to the oxidation of the test item during the test in all flasks and high deviation
of the measurements in all flasks, no defined solubility of Blei-cyanamid can be stated.
That means determination of repeatable measured test item concentrations under test
conditions was not possible. Therefore, the determination of the biological results was
based on the nominal concentrations.
Results with reference substance (positive control):
- NOEC: 0.4 mg/L
- LOEC: 0.63 mg/L
- EC50: 0.35 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The presented data are reliable and adequate.
Executive summary:

The study was performed using seven concentrations ranging from 0.01 to 10 mg/L. The

treatments were used to incubate the unicellular freshwater green alga Desmodesmus

subspicatus for a period of 72 hours. the cell concentration of each replicate was determined

by measuring the absorption of the solution at 44ß nm every 24 hours with a spectral

photometer. With these measured values, the number of cells was calculated (linear

correlation between cell concentration and absorption given). then the growth rate µ, the

area under tehe growth curve (AUC) and the Yield were determined.

At the start and at the end of the test, the content of Pb as main component of the test item

(80.3 %) was measured via AAS. The measured Pb concentrations were unexpectedly low

and implausible. Because of similar measured concentrations in a daphnia toxicity test, an

additional experiment for the determination of solubility in deionised water and in daphnia

test medium was performed within the scope of the Daphnia study.

Because of reactions with the anions in the test medium, the measured Pb concentration

in daphnia test medium was only 0.5 % of the Pb concentration in deionised water.

Furthermore, in the study for the determination of solubility in water, it was verified that

due to the oxidation of the test item during the test in all flasks and high deviation

of the measurements in all flasks, no defined solubility of Blei-cyanamid can be stated.

That means determination of repeatable measured test item concentrations under test

conditions was not possible. Therefore, the determination of the biological results was

based on the nominal concentrations.

The following results for the test item Cyanamide, lead(2+) salt (1:1) were determined: 

Endpoint

NOEC

LOEC

EC50

Growth Rate

0.32 mg/L

1.0 mg/L

2.9 mg/L

AUC

0.32 mg/L

1.0 mg/L

1.1 mg/L

Yield

0.32 mg/L

1.0 mg/L

1.2 mg/L

AUC (Area Under Curve according to EU Method C.3) means the integral of the biomass.

Yield (according to OECD Guideline 201) is defined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period.