Reaction mass of Fatty acids, montan-wax and Montan wax

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:

2008-11-19 - 2009-01-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to relevant guidelines and compliant to GLP. Results are plauslible and well reportred.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German Chemikaliengesetz and Directive 88/320/EEC

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Fatty acids, montan-wax and Montan wax

EC Number:

914-468-7

Molecular formula:

R-CH2-COOH mainly

IUPAC Name:

Reaction mass of Fatty acids, montan-wax and Montan wax

Details on test material:

TEST ITEM: Licowax S

Chemical characterisation: Fatty acids, montan wax
Appearance: Pale yellow powder
Solubility in water: Not specified
Recommended storage: Cool and dry
Storage at test facility: 6 ± 2 °C, protected from moisture and light
Retention of test item: At least 1 g has been retained
Identification parameter at test facility: Name, batch number, state, consistency and colour

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of TOC-concentration at the start of the test. However, due to the very poor solubility of the test item in water the concentration was below the detection limit.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution / Dispersion treatment:
A stock solution of 100 mg/L was prepared with dilution water and agitated at 20 rpm for 48 h at room temperature. After a separation phase of 1 h the water accomodated fraction* was taken from the homogenous liquid phase.

Loading level of the water accommodated fraction*:
One loading level of 100 mg/L was tested in a limit test. Per definition of the water accomodated fraction, all terms related to concentration levels will be given as loading levels as partly dissolved compounds and mixtures cannot be related to concentrations.

*) Water accommodated fractions: The aqueous medium containing only the fraction of a multi-component test item that is dissolved and / or present as a stable dispersion/emulsion under the test conditions in the test medium, acc. to OECD Series, No. 23.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test organism: Desmodesmus subspicatus CHODAT SAG 86.81 (formerly Scenedesmus subspicatus CHODAT)
Reason for the selection of the test system: According to the guideline Desmodesmus subspicatus CHODAT SAG 86.81 is suitable for this kind of study.
Origin: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
Cultivation at test facility: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE * mE(-2)*sE(-1) for 24 h per day.
Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

0.24 mmol/L (Ca2+ and Mg2+)

Test temperature:

minimum 21°C, mean 22.5°C maximum 24°C

pH:

treatment:
Start: 8.30 End: 8.54

Control:
Start: 7.83 End 8.53

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Nominal and measured concentrations:

As the solubility of the test item is very poor, a water accomodated fraction of a nominal loading of 100 mg/L had been used for a limit test.

Details on test conditions:

CONTROL: Six replicates (without test item) were tested under the same test conditions as the test replicates.
TREATMENT: Six replicates
SELF FLUORESCENCE: No self fluorescence has been found up to the water accommodated fraction of 100 mg/L.

REFERENCE ITEM Potassium dichromate p.a. (Sigma), CAS No. 7778 - 50 - 9
Purity: >= 99.5 %
Batch No.: 06719MH
Test concentrations: 0.28 - 0.51 - 0.90 - 1.6 mg/L
Experimental date: September 22 to 25th, 2008

TEST METHOD:
Static procedure
Exposure duration: 72 h
Replicates: Six replicates per limit concentration and control
Test container: Sterile Erlenmeyer flasks, volume: 250 mL
Test volume: 100 mL
Dilution water: OECD-medium according to the guideline
Preculture: A three day old preculture was used as inoculum. Incubation was performed in 500 mL Erlenmeyer flasks with dilution water. For the start of the test the preculture was directly pipetted to the test and control replicates to receive an initial cell concentration of approximately 2 ¿ 5 x 10E(3) cells/mL in the replicates.
Application: Application was carried out by adding appropriate volumes of the water accommodated fraction to the test replicates.
Temperature: Nominal range: 21 ¿ 24 °C, controlled at ± 2 °C
Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
Light intensity: 60 - 120 µE * mE(-2) * sE(-1)
Light regime: 24 h/d light

TYPE AND FREQUENCY OF MEASUREMENT
Cell density was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. The pH-value at the beginning of the test was measured for one additional replicate of the water accommodated fraction and the control. At the end it was measured from one pool of all replicates of the water accommodated fraction and the control. The room temperature was measured continuously by a hygro-thermograph. Light intensity was measured prior to test start. Microscopic evaluation of the cells at the start and end of the incubation were carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of alga cells.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Due to poor solubility of the test item, a water accomodated frachtion at nominal 100 mg/L had been used for a limit test

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Due to poor solubility of the test item, a water accomodated frachtion at nominal 100 mg/L had been used for a limit test

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Due to poor solubility of the test item, a water accomodated frachtion at nominal 100 mg/L had been used for a limit test

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to poor solubility of the test item, a water accomodated frachtion at nominal 100 mg/L had been used for a limit test

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to poor solubility of the test item, a water accomodated frachtion at nominal 100 mg/L had been used for a limit test

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to poor solubility of the test item, a water accomodated frachtion at nominal 100 mg/L had been used for a limit test

Details on results:

Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities. Environmental conditions, pH-value (measured at 0 and 72 h) and room temperature (measured continuously) were determined to be within the acceptable limits.

Results with reference substance (positive control):

The toxicity of potassium dichromate to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 h from September 22 to 25, 2008. The reference item toxicity is in the valid range:
EC50 (growth rate, 72h) = 0.69 mg/L (95% CL 0.64-0.74)
EC50 (biomass, 72h) = 0.31 mg/L (95% CL 0.30-0.32)

Reported statistics and error estimates:

Not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a 72h growth inhibition study on the unicellular alga Desmodesmus subspicatus (reliability category 1, compliant to GLP), the test item showed no inhibitory effect at the nominal test concentration of 100 mg/L (water accomodated fraction, limit test): EC50 (72h, growth rate and biomass) > 100 mg/L nominal concentration.
The results with the tested reference substance potassium dichromate proove the sensitivity of the applied test system.

Executive summary:

The toxicity of Licowax S to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at DR.U.NOACK-LABORATORIEN in 31157 Sarstedt, Germany from January 12 to 15, 2009. The aim of the study was to assess the effects on growth rate and biomass over a period of 72 h in a limit test. The study was conducted under static conditions with an initial cell density of approximately 2 - 5 x 10E(3) cells/mL. A water accommodated fraction* of 100 mg/L was prepared. Per definition of the water accommodated fraction all terms related to concentration levels will be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. Six replicates were tested for the water accommodated fraction and the control. Environmental conditions were determined to be within the acceptable limits. The concentration of Licowax S was analysed at test start via TOC-analysis. Due to the poor solubility of the test item the actual concentration was below the detection limit. All effect levels are given based on the water accomodated fraction of the test item:

EC10 (72h, growth rate and biomass) > 100 mg/L

EC20 (72h, growth rate and biomass) > 100 mg/L

EC50 (72h, growth rate and biomass) > 100 mg/L

*) Water accommodated fractions: The aqueous medium containing only the fraction of a multi-component test item that is dissolved and / or present as a stable dispersion/emulsion under the test conditions in the test medium, acc. to OECD Series, No. 23.