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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:
not given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: National Guideline study with acceptable restrictions.

Data source

Reference
Reference Type:
publication
Title:
Testing toxicity of volatile substances with algae.
Author:
Galassi S and Vighi M
Year:
1981
Bibliographic source:
Chemosphere, 10(19), 1123-1126.

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: U.S. EPA, Algal Assay Procedure - Bottle Test (AAPBT)
Deviations:
yes
Remarks:
test temperature: 20 ± 1°C
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,2,4-trichlorobenzene
- Analytical purity: not given
- Impurities (identity and concentrations): not given

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- gaschromatographic analysis of toxicant concentration after 24, 48 and 96 hours
- aqueous solutions were injected directly into a gaschromatograph

The testing flasks were closed by screw caps. The caps were pierced by stainless steel needle dipping in to the culture medium. Sampling for measurement of algal growth and toxicant concentration was made through the needle by means of a syringe.

Capped flasks were shaken for 24 hours at 20°C to let vapour and liquid phases equilibrate.

After a 24 hours equilibration period, concentrations in culture media remained almost constant. Differences in the measurements carried out at different times (at the end of the equilibration period and after 48 and 96 hours of treatment) lie in the range of acceptable analytical variability.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Stock solution was prepared as follows: an amount of the chemical ten times higher than the saturation solubility was added to distilled water in a closed vessel, stirred for 48 hours and decanted for 24 hours. The supernatant was filtered through paper-filters and the concentration was measured.

Final solutions were made by: adding 10 ml of stock culture medium (10 times standard concentration) to different amounts of toxicant stock solution. Solutions were diluted to 100 ml with distilled water and quickly transferred into the culture flask.

Capped flasks were shaken for 24 hours at 20°C to let vapour and liquid phases equilibrate.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The algal inoculum was added, after reaching the equilibrium concentration of the test substance (24 hours) at a starting cell concentration of 5 x 10 Exp+6 cells/l.

Algal growth was measured at 24, 48 and 96 hours by in vivo fluorescence. Results were expressed as a percentage of the growth in the control culture and graphically elaborated for the evaluation of the EC50. Only the 96 hours data are reported in the publication.

Study design

Test type:
static
Water media type:
freshwater
Total exposure duration:
96 h

Test conditions

Hardness:
not given
Test temperature:
in exception to AAPBT the temperature was 20 ± 1°C
pH:
not given
Dissolved oxygen:
not given
Salinity:
not given
Nominal and measured concentrations:
Initial concentrations could not be measured, due to high volatility of 1,2,4-TCB. Therefore the nominal concentrations calculated from the dilution ofthe titrated stock solutions were assumed as Ci.

Ci Ceq Ci/Ceq
1.66 0.40 4.15
3.32 0.62 5.32
6.64 1.52 4.37
9.96 2.29 4.35
13.28 2.39 3.92
16.60 2.79 5.95
23.24 4.68 4.97
4.72 (± 0.67)

Ci: initial concentration
Ceq: concentration at equilibrium calculated as a mean of the measurements after the equilibration period and during treatment
Details on test conditions:
- culture medium and test conditions were similar to the "Algal Assay Procedure-Bottle Test" (AAPBT)
=> with exception that the temperature was 20 ± 1°C


TEST SYSTEM
- Test vessel:
- Type: closed
=> by screw cap with both silicone rubber (4 mm thick) and teflon gaskets
=> The caps were pierced by stainless steel needle dipping in to the culture medium. The outer end of the needle was closed with Parafilm.

- Material, size, headspace, fill volume:
=> 2 litre spherical flasks
=> the volume of the culture was 100 ml
=> the measured medium to flask volume ratio (0.047) was low enough to avoid notable carbon dioxide deficiency

- Initial cells density: 5 x 10 EXP+6 cells/ml


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: Algal growth was measured at 24, 48 and 96 hours by in vivo fluorescence.


TEST CONCENTRATIONS

A
Final solutions were made by adding 10 ml of stock culture medium (10 times standard concentration) to different amounts of toxicant stock solution. Solutions were diluted to 100 ml with distilled water and quickly transferred into the culture flask.

B
Initial concentrations could not be measured, due to high volatility of 1,2,4-TCB. Therefore the nominal concentrations calculated from the dilution ofthe titrated stock solutions were assumed as Ci.

Ci Ceq Ci/Ceq
1.66 0.40 4.15
3.32 0.62 5.32
6.64 1.52 4.37
9.96 2.29 4.35
13.28 2.39 3.92
16.60 2.79 5.95
23.24 4.68 4.97
4.72 (± 0.67)
Ci: initial concentration
Ceq: concentration at equilibrium calculated as a mean of the measurements after the equilibration period and during treatment
(The algal inoculum was added, after reaching the equilibrium concentration of the test substance (24 hours) at a starting cell concentration of 5 x 10 Exp+6 cells/l.)


RANGE FINDING STUDY
- no data
Reference substance (positive control):
not required

Results and discussion

Effect concentrations
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): no data
- Observation of abnormalities (for algal test): no data
Results with reference substance (positive control):
- reference substance not required
Reported statistics and error estimates:
no data

Any other information on results incl. tables

max tested conc. of 0% eff. mg/l: 0.37

min tested conc. of 100% eff. mg/l: 4.9

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Executive summary:

Galassi S and Vighi M (1981)

A modificated Algal Assay Procedure-Bottle Test (US EPA) for testing of volatile substances was performed. The procedure permits maintenance of a constant concentration of toxicant in the culture. The test algalSelenastrum capricornutumwas added to the test solution after reaching the equilibrium of 1,2,4-TCB between vapour and liquid phase (24 h after preparation of test solution). Both concentration of 1,2,4-TCB and algal growth rate were measured at 24, 48 and 96 hours. After 96 hours duration an EC50 of 1.4 mg/l was found. Effect concentrations at 24 and 48 hours are not stated in the publication.

 

Beside a test performed by Calamari et al. (1983) the study by Galassi and Vighi (1981) provides the only valid result on toxicity to algal according to EU Risk Assessment Report (2003). Most other studies didnt consider the volatility of 1,2,4-TCB.