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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:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study is similar to the OECD Guideline 201 (Alga, Growth Inhibition Test) test, but no data was reported regarding GLP procedures. The authors do however provide detailed information concerning materials and methods. Thus we consider this a Klimisch 2e study as the study is well documented, meets generally accepted scientific principles and is acceptable for assessment

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Grenzwerte der Schadwirkung wassergefährdender Stoffe gegen Blaualgen (Microcystis aeruginosa) und Grünalgen (Scenedesmus quadricauda) im Zellvermehrungshemmtest
Author:
Bringmann G. and Kühn R.
Year:
1978
Bibliographic source:
Vom Wasser, 50: 45-60
Reference Type:
publication
Title:
Testing of substances for their toxicity threshold: model organisms Microcystis (Diplocystis) aeruginosa and Scenedesmus quadricauda
Author:
Bringmann G. and Kühn R.
Year:
1978
Bibliographic source:
Mitt. Internat. Verein. Limnol., 21: 275-284
Reference Type:
publication
Title:
Comparison of the toxicity thresholds of water pollutants to bacteria, algae, and protozoa in the cell multiplication inhibition test
Author:
Bringmann G. and Kühn R.
Year:
1980
Bibliographic source:
Water Research, 14: 231-241
Reference Type:
review article or handbook
Title:
alpha, alpha, alpha-trichlorotoluene (trichloromethylbenzene)
Author:
UNEP
Year:
2004
Bibliographic source:
OECD SIDS

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
at least test duration, and oxygen content and pH not reported
Principles of method if other than guideline:
The authors performed a cell multiplication inhibition test. Using this method the onset of the inhibition of cell multiplication under the influence of the test substance is determined.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): benzotrichlorid; alpha, alpha, alpha-trichlorotoluol (benzotrichloride)

No more data available

Sampling and analysis

Analytical monitoring:
not specified
Details on sampling:
no data

Test solutions

Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the test substance is dissolved in bidistilled water and prior to preparation of the test cultures the stock of the test solution with known concentration that will be used in the test is neutralized.
- Controls:
Negative controls were included which were not exposed to the test substance, had a fixed standardized offer of nutrients and were kept under identical conditions as the test cultures.
A second control was included which was exposed to the dilution series of the test substance. It had a fixed standardized offer of nutrients, was kept under identical conditions as the test cultures containing benzyl chloride but was not inoculated. This additional control was included so that this non-inoculated control could be used as a photometric blank for turbidimetry of the inoculated dilution series if, after termination of the test period in the dilution series, a colouration or turbity occured for chemical/physical reasons.

No more data available

Test organisms

Test organisms (species):
other: Microcystis aeruginosa and Scenedesmus quadricauda
Details on test organisms:
TEST ORGANISMS
- Both test organisms are ubiquitous species frequently found in Central Europe, proved to be physiologically constant over decades of culturing, grow in cultures as single cells and are particularly suitable for turbidimetric measurements.


No more data available

Study design

Test type:
static
Water media type:
not specified
Limit test:
no
Total exposure duration:
8 d
Post exposure observation period:
No data

Test conditions

Hardness:
No data
Test temperature:
27°C
pH:
7
No pH adjustement was done if the effect of the pH of the studied test solution was a part of the test.
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
No data
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: fill volume is 10 mL, culture tubes are stoppered with cotton-lined metal caps
- No. of organisms per vessel: the quantity of the cell material used for inoculation is determined turbimetrically (baffled against stray light) and is standardized. The extinction value of the monochromatic radiation at 578 nm for a 10 mm layer of the algal suspension of test cultures will correspond to a turbidity value of the Formazin standard suspension TE/F/578 nm = 20 after inoculation
- No. of vessels per concentration (replicates): 4 of which 3 are inoculated and one is not. The latter one is used as photometric blank for turbidimetry if, after termination of the test period in the dilution series, a colouration or turbity occured for chemical/physical reasons.
- No. of vessels per control (replicates): 12


GROWTH MEDIUM
- Standard medium used: nor the OECD nor the AAP medium were used.
- Detailed composition if non-standard medium was used: Composition of the growth and test media are given in table 1.


TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Culture medium and conditions of culturing are standardized for stock and pre-cultures. This medium however is not the same as the test medium (composition of the two media are given in table 1). Further details on the media preparation for the stock, pre- and test cultures as well as for the trace element stock solution (composition given in table 2) are described below.


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No pH adjustement was done if the effect of the pH of the studied test solution was a part of the test.
- Photoperiod: constant lighting
- Light intensity and quality: test and control cultures were kept under standardized conditions, namely on a white surface protected against daylight and exposed to constant lighting in the central field between two lateral luminescent tubes at 60 cm distance from each other (Osram L 40/30; 2800 lm; 0.70 cd/cm²)
- Relative humidity: 50%


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:
after termination of the test periode (i.e. 8 days) the culture tubes are shaken vigorously to obtain a uniform cell suspension immediately prior to the measurements. The algal suspension concentration is measured turbidimetrically and is expressed as the extinction of the primary light of the monochromatic radiation at 578 nm for a 10 mm layer. If after the termination of the test period colouration or turbidity occurs for chemical-physical reasons, the analogous steps of dilution of the non-inoculated series are used as photometric blank values for turbodimetry of the inoculated dilution series. An Eppendorf digital photometer with integrated computer for multiplication and calculus of differences and on-line digital printout was used. This measuring technology enables an elimination of colouration or secondary turbidity of the medium by balancing.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: a dilution serie with a factor 2 of the test substance stock solution was made with double-distelled water and prepared under sterile conditions.

No more data available
Reference substance (positive control):
not specified

Results and discussion

Effect concentrationsopen allclose all
Duration:
8 d
Dose descriptor:
other: Toxicity threshold (similar to EC3)
Effect conc.:
34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
turbidity of the test medium
Remarks on result:
other: Microcystis aeruginosa
Duration:
8 d
Dose descriptor:
other: Toxicity threshold (similar to EC3)
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
turbidity of the test medium
Remarks on result:
other: Scenedesmus quadriccauda
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
> 34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
turbidity of the test medium
Remarks on result:
other: Microcystis aeruginosa
Details on results:
no data
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
no data

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Conclusions:
Under the test conditions, the authors established the toxicity threshold, which is similar to the EC3, to 34 mg/L for Microcystis aeruginosa and >100 mg/L for Scenedesmus quadricauda. Thus, at this level of information, benzotrichloride should not be classified according to CLP-Regulation (EC) No 1272/2008 as the exposure duration is greater then the requested 72 hours. Microcystis aeruginosa however appears to be more sensitive to benzotrichloride than Scenedesmus quadricauda.
Executive summary:

The authors performed a cell multiplication inhibition test determining the onset of the inhibition of cell multiplication under the influence of benzotrichloride using Microcystis aeruginosa and Scenedesmus quadricauda. This principle of the test is similar to the OECD Guideline 201 (Alga, Growth Inhibition Test). The test organisms were exposed to benzotrichloride under static conditions for 8 days at 27°C and relative humidity of 50%. Furthermore the test and control cultures were kept on a white surface protected against daylight and exposed to constant lighting in the central field between two lateral luminescent tubes at 60 cm distance from each other (Osram L 40/30; 2800 lm; 0.70 cd/cm²).

The toxicity threshold (similar to EC3) was determined based on the extinction value of the monochromatic radiation at 578 nm for a 10 mm layer of the algal suspension using photoelectric measurement.

Hnece, in the test conditions, the toxicity threshold was established at 34 mg/L for Microcystis aeruginosa and >100 mg/L for Scenedesmus quadricauda. Any other effect was described by the authors. Therefore, at this level of information, benzotrichloride should not be classified according to CLP-Regulation (EC) No 1272/2008 as the exposure duration is greater then the requested 72 hours.

No data on GLP procedure is reported and limited data was provided to confirm the fulfillment of the validity criteria of the OECD guideline. However the authors provided very detailed information on the materials and methods and on the principle of the experiment. Therefore, at this level of details and as it is based on generally well accepted scientific principles, this study should be considered as reliable with restrictions, a Klimisch 2.e study.