Saponification and oxidation product of carnauba wax with acidic sodium dichromate solution esterified with 1-methyl-1,3-propanediol and subsequent saponification with calcium dihydroxide

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

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

Adequacy of study:

key study

Study period:

2009-07-27 to 2009-08-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study. For justification of read-across within the category please see chapter 1 of the Chemical Safety Report.

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 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

and ISO 11268-1 (1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Test material

Test material information

Constituent 1

Reference substance name:

Montan waxes, Type OP

IUPAC Name:

Montan waxes, Type OP

Constituent 2

Reference substance name:

Reaction mass of Fatty acids, montan-wax and Fatty acids, montan-wax, 1-methyl-1,3-propanediyl esters and Fatty acids, montan-wax, calcium salts and Montan wax

EC Number:

914-460-3

IUPAC Name:

Reaction mass of Fatty acids, montan-wax and Fatty acids, montan-wax, 1-methyl-1,3-propanediyl esters and Fatty acids, montan-wax, calcium salts and Montan wax

Details on test material:

- Name of test material (as cited in study report): Licowax OP
- Chemical characterisation: Fatty acids, montan wax, 1-methyI-1,3-propanediylesters
- Physical state: Pale yellow flakes
- Analytical purity: 100 % (w/w) certified
- Purity test date: 2008-03-01

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

No analytical monitoring

Test substrate

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The test item was weighed out and thoroughly mixed with quartz sand, which was mixed into the artificial soil. Demineralised water was added to adjust the humidity of artificial soil to a moisture of 54 % of the maximum water holding capacity.
- Controls: Artificial soil moistended with demineralised water without test or reference item was used as control medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Quartz sand
- Concentration of vehicle in test medium (stock solution and final test solution): 10 g per replicate
- Evaporation of vehicle before use: Not necessary

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm Eisenia fetida Savigny 1826
- Source: Own breeding
- Weight at test initiation (mean and range, SD): 0.4 +/- 0.04


ACCLIMATION
- Acclimation period: 3 days
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): No mortality observed

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Post exposure observation period:

No

Test conditions

Test temperature:

18 - 22 °C

pH:

See "Any other information on materials and methods incl. tables", Table 1.

Moisture:

See "Any other information on materials and methods incl. tables", Table 1.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Glass dishes, volume 1.5 L, covered with perforated plastic film
- Amount of soil or substrate: 555 g (dry weight)
- No. of organisms per container (treatment): 10 per replicate
- No. of replicates per treatment group: 4
- No. of replicates per control: 4



SOURCE AND PROPERTIES OF SUBSTRATE (if soil)

- Composition (if artificial substrate):
- Organic carbon (%): 10 % peat
- Maximum water holding capacity (in % dry weigth): 63
- Storage (condition, duration): under test conditions for 13 days



OTHER TEST CONDITIONS
- Photoperiod: 24 h
- Light intensity: 531 +/- 81 lx


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

The live weight of the earthworms was measured individually at the day of application (day 0) and at day 14.
Mortality, behaviour and morphological changes of the earthworms were recorded 7 and 14 days after application.


VEHICLE CONTROL PERFORMED: no


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test
- Range finding study
- Test concentrations: 10 - 100 - 1000 mg/kg soil DW
- Results used to determine the conditions for the definitive study:
Range Finding Test (14 d): Mortality and Change of Live Weight [%]
See "Any other information on materials and methods incl. tables", Table 2 for results of range finding study!

Nominal and measured concentrations:

Nominal: Limit test concentration of 1000 mg/kg soil DW

Reference substance (positive control):

yes

Remarks:

2-Chloroacetamide

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

loss of body weight, other symptoms

Effect concentrations 2

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, loss of body weight, other symptoms

Details on results:

- Mortality at end of exposure period: 0 %
- Total mass of adults at beginning of test: Not determined
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: see table
- Morphological abnormalities: None
- Behavioural abnormalities: None
- Other biological observations: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:
NOEC and LC-Values after 14 d for 2-Chloroacetamide: See table!

Reported statistics and error estimates:

NOEC values of biomass A t-test was carried out for the determination of statistically
significant differences compared to the control. A Normality Test and an Equal Variance Test were conducted prior to the
t-test. P-values for both Normality and Equal Variance Test are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is no difference) is alpha=0.05.

NOEC values of symptoms and mortality: No statistics had to be performed since no mortality and no symptoms were observed.

Any other information on results incl. tables

Earthworm Mortality in [%] after 7 and 14 Days of Exposure

Mortality [%]
Nominal test item concentration
	Day 7					Day 14
[mg/kg soil dry weight]	1	2	3	4	MV	1	2	3	4	MV
Control	0	0	0	0	0	0	0	0	0	0
1000	0	0	0	0	0	0	0	0	0	0

No earthworm showed any symptoms or behavioural changes in the control as well as in the limit test item concentration of 1000 mg/kg soil dry weight.

Earthworm Behaviour and Pathological Symptoms after 7 and 14 Days of Exposure

Nominal test item concentration	Effect	Day 7                                Replicate				Day 14                    Replicate
[mg/kg soil dry weight]		1	2	3	4	1	2	3	4
Control	a)	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
1000	a)	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10

a) no obvious pathological symptoms or behavioural changes

During the test the biomass loss of the earthworms was below 20 % in the controls. After 14 days of exposure the earthworm biomass was not statistically significant different at the limit test item concentration of 1000 mg/kg soil dry weight compared to the control

Mean Biomass and Mean Loss of Body Weight

	Mean live weight			Comparison versus Control
Nominal test item concentration	Test start MV ± SD	Test end MV ± SD	Test end CV	Loss	Significance
[mg/kg soil dry weight]	[g/worm]	[g/worm]	[%]	[%]
Control	0.399 ± 0.04	0.372 ± 0.05	13.4	7	-
1000	0.399 ± 0.03	0.362 ± 0.04	11.1	9	No

Results with the reference item: The acute toxicity of 2-Chloroacetamide on the earthwormEisenia fetida fetida(SAvIGNY) wasdetermined over aperiod of two weeks from 2009-02-12 to 2009-02-26. The LC₅₀-value ranged between 20 and 80 mg/kg soil dry weight as recommended by the guideline.

NOEC and LC-Values after 14 d for 2-Chloroacetamide

LC-Values/ NOEC	Concentration [mglkg soil dry weight]	95%- Confidence Interval [mg/kg soll dry weight]
LC50	50.1	45.8 - 55.5
NOEC	30.0	-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

No mortality as well as no pathological symptoms or changes in the earthworm behaviour were observed at the limit test item concentration of 1000 mg/kg soil dry weight after 14 days of exposure to Licowax OP. Compared to the control the biomass of earthworms exposed to Licowax OP at the limit test item concentration of 1000 mg/kg soil dry weight was not statistically significantly reduced at test end.
Consequently, the No Observed Effect Concentration (NOEC) of Licowax OP with respect to mortality, loss of body weight and other symptoms was determined to be at 1000 mg/kg soil dry weight.

Executive summary:

The acute effects of Licowax OP on the earthworm Eisenia fetida were determined according to the OECD Guideline 207 and DIN ISO 11268-1 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from July 27^th to August 11^th, 2009. The study was conducted with a limit test item concentration of 1000 mg/kg soil dry weight throughout 14 days. Artificial soil without test item was used as control. 40 test organisms with an individual weight between 0.3 - 0.6 g, divided into four replicates, were tested at the limit test item concentration and control in artificial soil containing 10 % peat.

No mortality as well as no pathological symptoms or changes in the earthworm behaviour were observed at the limit test item concentration of 1000 mg/kg soil dry weight after 14 days of exposure (Table 1). Compared to the control, the biomass of the earthworms at the limit test item concentration of 1000 mg/kg soil dry weight was not statistically significantly reduced at test end.

Consequently, the no observed effect concentration (NOEC) with respect to mortality, loss of biomass and other symptoms was determined to be

at 1000 mg/kg soil dry weight.

All validity criteria of the test guideline were fulfilled.

Summary of all Effects

Effects	Licowax OP [mg/kg soil dry weight]
LOEC Mortality	> 1000
LOEC Loss of body weight	> 1000
LOEC Behaviour and pathological symptoms	> 1000
NOEC Mortality, loss of body weight, other symptoms	1000