Reaction mass of fatty acids, montan-wax, 1-methyl-1,3- propanediyl esters and fatty acids, montan-wax, ethylene esters and fatty

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2008-11-19 - 2009-05-14

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

Principles of method if other than guideline:

Not applicable

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:

Montan waxes, Type E

IUPAC Name:

Montan waxes, Type E

Details on test material:

TEST ITEM: Licowax E

Components:
1. Ethylenglycol ester of montanic acids
2. Montanic acid ester (genuin)
3. Montanic acids

CAS No:
1. 73138-45-1
2. 8002-53-7
3. 68476-03-9


Chemical characterisation: Fatty acids, montan wax, ethylene esters
Appearance: Pale yellow shelter, solid
Solubility in water: Approx. 24 mg/L (room temperature)
pH value: Slightly acidic
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

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

TOC analysis was carried out at test start and end of the test from the control and the loading levels 32.0 ¿ 320 mg/L. Separate replicates were prepared without algae.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution / Dispersion treatment:
Water accommodated fractions* (WAF) of 10, 32, 100 and 320 mg/L were prepared with dilution water. The WAFs were shaken with 20 rpm for 48 h at room temperature. After a separation phase of 1 h the WAF was taken from the homogenous liquid phase.

Loading level of the water accommodated fraction*:
Per definition of the WAF all terms related to concentration levels were given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. Five loading levels were tested: 3.20 - 10.0 - 32.0 - 100 - 320 mg/L (factor 3.16). The loading levels of 10.0 - 320 mg/L were prepared from the WAF. The lowest loading level of 3.20 mg/L was prepared by dilution of the WAF of 10 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.

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:

no

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°C maximum 23°C

pH:

treatment:
Start: 7.86-7.92 End: 9.43-9.74

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, water accomodated fractions of a nominal loading had been prepared:
3.20 - 10.0 - 32.0 - 100 - 320 mg/L (factor 3.16). The lowest loading level of 3.20 mg/L was prepared by dilution of the WAF of 10 mg/L.
In spite of the poor solubility, analytical monitoring by TOC-measurement at the start and the end of the experiment had been tried. For results see "Any other information on materials and methods inc. tables".

Details on test conditions:

CONTROL: Six replicates (without test item) were tested under the same test conditions as the test replicates.
TREATMENTS: Three replicates
SELF FLUORESCENCE: No self fluorescence has been found up to the water accommodated fraction of 320 mg/L of the test item.

REFERENCE ITEM Potassium dichromate p.a. (Sigma), CAS No. 7778 - 50 - 9
Purity: >= 99.5 %
Batch No.: 06719MH
Test concentrations: 0.25 - 0.50 - 1.0 mg/L
Experimental date: March 09 to 12, 2009

TEST METHOD:
Static procedure
Exposure duration: 72 h
Replicates: Three replicates for each dilution level, six for the control
Test container: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.
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 each loading level.
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 out of one additional replicate from each loading level and the control. At the end it was measured from one pooled replicates of each loading level 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:

EC20

Effect conc.:

> 320 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, water accomodated fractions had been prepared

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 320 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, water accomodated fractions had been prepared

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 320 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, water accomodated fractions had been prepared

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 320 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, water accomodated fractions had been prepared

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 320 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, water accomodated fractions had been prepared

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 - < 320 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, water accomodated fractions had been prepared

Details on results:

Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities. Environmental conditions ( pH-value, room temperature, light intensity) 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 March 09 to 12, 2009. The reference item toxicity is in the valid range:
EC50 (biomass, 72h) = 0.320 mg/L (95% CL 0.294-0.358)
EC50 (growth rate, 72h) = 0.695 mg/L (95% CL 0.657-0.729)

Reported statistics and error estimates:

EC-values and statistical analyses:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response
regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.

NOEL and LOEL values:
The NOEL and LOEL were determined by calculation of statistical significance of biomass integrals and growth rates.
One Way Analysis of Variance and DUNNETT'S test were carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value for ANOVA was a=0.05.

Any other information on results incl. tables

Evaluation after 72h:

 Statistically significant differences of growth rates and yield compared tocontrol values are marked (+), not significant differences are marked (-).

 

Nominal loading level of the test item [mg/L]	Replicate No.	Growth rate [d^-1]	Rate-related inhibition [%]	Yield [cells/mL]	Inhibition of yield [%]
	1	1.711	4.42	821374	21.60
320	2	1.797	-0.43	1067006	-1.84
	3	1.727	3.51	862530	17.67
	Mean	(-) 1.745	2.50	(-) 916970	12.48
	1	1.815	-1.39	1123788	-7.26
100	2	1.799	-0.5	1070815	-2.21
	3	1.823	-1.87	1152975	-10.05
	Mean	(-) 1.812	-1.25	(-) 1115859	-6.51
	1	1.719	3.93	843095	19.53
32.0	2	1.768	1.23	975680	6.87
	3	1.785	0.25	1028456	1.84
	Mean	(-) 1.757	1.80	(-) 949077	9.41
	1	1.805	-0.85	1091482	-4.18
10.0	2	1.720	3.9	844558	19.39
	3	1.725	3.64	856432	18.26
	Mean	(-) 1.750	2.23	(-) 930824	11.16
	1	1.734	3.13	880324	15.98
3.20	2	1.790	-0.03	1044197	0.33
	3	1.822	-1.79	1147972	-9.57
	Mean	(-) 1.782	0.44	(-) 1024164	2.25
	1	1.787		1032683
	2	1.823		1152364
	3	1.776		999696
Control	4	1.725		858064
	5	1.806		1096208
	6	1.822		1147190
	Mean	1.790		1047701

 negative inhibitions = increase of growth

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 statistically significant inhibitory effect up to the nominal test concentration of 320 mg/L (water accomodated fractions):
EC50 (72h, growth rate and biomass) > 320 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 E 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 May 08 to 14, 2009. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately 2 - 5 x 10E(3) cells/mL. Water accommodated frations* (WAF) were prepared. Per definition of the WAF all terms related to concentration levels were given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. Five loading levels (prepared from the WAF) were tested: 3.20 - 10.0 - 32.0 - 100 - 320 mg/L (factor 3.16). Three replicates were tested for each loading level and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

TOC analysis was carried out at test start and end of the test from loading levels 32.0 ¿ 320 mg/L and the control. All effect values are given based on nominal loading levels of the test item.

EC10 (72h, growth rate) > 320 mg/L

EC20 (72h, growth rate) > 320 mg/L

EC50 (72h, growth rate) > 320 mg/L

100 mg/L < EC10 (72h, biomass) > 320 mg/L

EC20 (72h, biomass) > 320 mg/L

EC50 (72h, biomass) > 320 mg/L

Licowax E caused no significant inhibiting effects on algal growth when tested with the water accommodated fractions.

*) 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