Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1999-02-01 to 1999-02-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tri (hexyl, octyl, decyl) citrate
- Substance type: pure active substance
- Physical state: liquid
- Stability under test conditions: in water for at least 2 hours
- Storage condition of test material: in original container, at room temperature, in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: -29°C (freezing point: -35.5°C)
- Boiling point: test substance showed decomposition above 260°C
- Vapour pressure: 8.5 x 10E-3 p at 25°C
- Water solubility (under test conditions): < 1 mg/L
- Solubility in organic solvents: soluble
- log Pow: > 6
- pKa: not applicable
- Base or acid catalysis of test material: no data
- UV absorption: no data
- Stability of test material at room temperature: stable
- pH dependance on stability: no data


OTHER PROPERTIES (if relevant for this endpoint)
- Adsorption characteristics: no data

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
In a pre-experiment (without GLP) the dissolved organic carbon content (DOC) of the test substance preparation (prepared as in test) was determined. In the sample the same amount of DOC was determined as in the blank control. Thus, no dissolved test substance could be verified in test water.
Therefore, no analytical dose verification in the test media of the algal growth inhibition test could be done

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A supersaturated stock suspension of the test substance with a nominal concentration of 100 mg/L was prepared by weighing 50 mg of the test substance into 500 mL test water, following ultrasonic treatment for 10 minutes and intense stirring. The supersaturated stock suspension was stirred on a magnetic stirrer at room temperauter in the dark over 3 days . Thereafter the suspension was filtered through a cellulose-nitrate filter with a defined pore size of 0.2 µm just before the start of the test.
- Controls: test water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no, suspension was filtered

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name. green algae
- Strain: Scenedesmus subspicatus CHODAT Strain No. 86.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut, University of Göttingen, Germany
- Age of inoculum (at test initiation): 3 days old pre-culture (exponentially growing)
- Method of cultivation: in the testing laboratory under standardised conditions according to the test guidelines


ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not mentioned

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol/L as CaCO3 (calculated)
Test temperature:
23.0 - 23.3 °C
pH:
start of test: 7.6 - 7.8
end of test: 9.6 - 10.0 (The increase of the pH was obviously caused by the CO2-consumption of the algae due to their rapid growth respectively their high densities)
Dissolved oxygen:
not mentioned
Salinity:
In deionized water (conductivity < 5 µS/cm) analytical grade salts were added to following nominal concentrations:
Macro-nutrients: 50.0 mg/L NaHCO3; 18.0 mg/L CaCl2 x 2 H2O; 15.0 mg/L NH4Cl; 15.0 mg/L MgSO4 x 7 H2O; 12.0 mg/L MgCl2 x 6 H2O; 1.6 mg/L KH2PO4
Trace elements: 100 µg/L Na2EDTA x 2 H2O; 80 µg/L FeCl3 x 5 H2O; 415 µg/L MnCl2 x 4 H2O; 185 µg/L H3BO3; 7 µg/L Na2MoO4 x 2 H2O; 3 µg/L ZnCl2; 1.5 µg/L CoCl2 x 6 H2O; 0.01 µg/L CuCl2 x 2 H2O
Nominal and measured concentrations:
WAF, no analytical determination of concentration
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks with 50 mL test medium covered with glass dishes, continuously stirred by magnetic stirrers, incubated in a water bath
- Aeration: not aerated
- Initial cells density: 10000 algal cells/mL
- Control end cells density: 92.23 x 10E4 cells/mL
- 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: deionized water (conductivity < 5 µS/cm)
- Ca/mg ratio: not mentioned
- Culture medium different from test medium: no
- Intervals of water quality measurement: not mentioned


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 7910 Lux (mean value, range: 7830 to 8000 Lux), achieved by fluorescent tubes (Universl Weiss L36W/25-1), installed above the water bath



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometrical measurement of cell density after 26, 48 and 72 hours (The cell density in one control was counted by microscope after 72 hours. Based on the counted cell densities and based on the determined absorption of the control and five dilutions of the control a linear regression was performed for the calculation of the cell densities in all other samples measured spectrophotometrical during the test.)
- Chlorophyll measurement: no
- Other:
For the determintation of an influence of the test substance on the algal cells, from the filtate a sample was taken after 72 hours test duration. The shape of the treated algal cells compared to the control was microscopically examined.
The behaviour of the test substance in the filtrate was determined daily.
Measurement of pH-values in samples of the test media and the controls at the start and at the end of the test.
The test media temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Reference substance (positive control):
yes
Remarks:
potassium dichromate, tested at least twice a year

Results and discussion

Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): no abnormalities observed
- 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 remarkable observation made concerning the appearance of the test substance in the filtrate
- Effect concentrations exceeding solubility of substance in test medium: no concentrations exceeding solubility tested
Results with reference substance (positive control):
not mentioned
Reported statistics and error estimates:
The LOEC, the EbC50 and ErC50 and the corresponding EC10 and their 95%-confidence limits could not be calculated due to the absence of toxicity of the test substance up to the water solubility limit of the test substance.

Any other information on results incl. tables

Table: Algal cell densities during the test period of 72 hours

 Treatment     Density of algal cells (x 10E4/mL)*  after 
   24 h  48 h  72 h
 control 8.10 ± 0.41  22.20 ± 1.98  92.23. ± 0.79
test substance filtrate   11.57 ± 0.49  20.07 ± 0.99  91.47 ± 0.49

* mean values ± standard deviation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
cell density in control cultures increased by a factor of at least 16 within 72 hours
Conclusions:
Due to the low water solubility of the test substance a filtrate of a supersaturated stock suspension of nominal 100 mg/L was tested. Thus, no concentration above the solubility limit of the test substance in the used test water were tested. Also due to the low water solubility no analytical verification could be done in the present test. Therefore, the biological results could not be related to a specific concentration of the test substance but only to the water solubility limit in the test medium.
The 72-hour NOEC was determined to be at least up to the solubility limit of the test substance. The NOEC might even be higher than this concentration, but concentrations in excess of the solubility limit had not been tested. The 72-hour LOEC and the 72-hour EC50 were clearly higher than the solubility limit.
Executive summary:

Due to the low water solubility of the test substance a filtrate of a supersaturated stock suspension of nominal 100 mg/L was tested. Thus, no concentration above the solubility limit of the test substance in the used test water were tested. Also due to the low water solubility no analytical verification could be done in the present test. Therefore, the biological results could not be related to a specific concentration of the test substance but only to the water solubility limit in the test medium.

The 72-hour NOEC was determined to be at least up to the solubility limit of the test substance. The NOEC might even be higher than this concentration, but concentrations in excess of the solubility limit had not been tested. The 72-hour LOEC and the 72-hour EC50 were clearly higher than the solubility limit.