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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 FEB 2011 to 10 FEB 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented guideline study performed compliant to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Carnauba waxes, type OP

IUPAC Name:

Carnauba waxes, type OP

Details on test material:

Name: C-Wax Type OP
Chemical Name: Fatty acids, carnauba wax, butyleneglycol, partly saponified
Purity: 99,5 - 100%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The WAF and the control were analytically verified by analysis of organic carbon (DOC, according to DIN EN 1484) in the fresh media (0 and 24 h) and old media (24 and 48 h).
For the old media, separate replicates without test organisms were prepared and incubated under test conditions until analysis. The samples were stored at 6 +/- 2 deg C until analysis. Prior to DOC analysis the samples were brought to room temperature and filtrated with a membrane filter (RC, 0.45 µm, MACHEREY-NAGEL).

Test solutions

Vehicle:

no

Details on test solutions:

Due to the very poor water solubility, water accommodated fractions (WAF) were tested.
The WAF is defined as 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 test conditions in the test medium, acc. to OECD Series, No. 23.
WAFs with a nominal loading of 100 mg test item/L were prepared with dilution water one day prior to application (at 0 h) and at water renewal (after 24 h). The WAF was shaken with 20 rpm for 24 h at room temperature. After a separation phase of at least 1 h the WAF was taken from the homogeneous liquid phase.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna STRAUS (Clone 5)
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
Breeder: DR.U.NOACK-LABORATORIEN, Kathe-Paulus-Str. 1, 31157 Sarstedt, Germany
Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +/- 2 deg C, in an incubator, 16 h illumination, illumination strength max. 20 µE x mE(-2) x sE(-1)
Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
Culture feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 10E(6) cells/mL

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

Total hardness of 160 to 180 mg CaCO3/L

Test temperature:

20.4 - 20.9 deg C

pH:

Control: 7.56 - 7.98
Treatment: 7.51 - 7.88

Dissolved oxygen:

Control [mg/L]: 9.00 - 9.36
Treatament [mg/L]: 8.88 - 9.16

Nominal and measured concentrations:

The original WAF was tested in a limit test (100 mg/L nominal test item concentration).
Measured concentrations (DOC-analysis) were not significantly different than DOC-concentrations found in the control medium, as the test item is insoluble in water. Therefore, all concentrations are nominal concentations.

Details on test conditions:

TEST METHOD: Semi-static (water renewal after 24 h)
Test duration: 48 h
Test vessels: Glass beakers (4 cm ID x 7 cm H), 50 mL capacity
Test volume: 20 mL
Dilution water: ISO Test water, according to OECD 202, Annex 3; pH-value: 7.8 +/- 0.2
Number of daphnids and replicates: 20 daphnids, divided into 4 replicates, each with 5 daphnids per limit loading and control
Age of daphnids at the beginning of the test (0 h): 2 to 24 h old daphnids were used for the study. They were obtained by removing the mother animals twice within 22 h.
Application: 20 g of the test solution each was weighed out into the test vessels. The daphnids were inserted with a small amount of dilution water by using a pipette.
Test temperature: 18 - 22 deg C, +/- 1 deg C constant
Illumination / Photoperiod: Diffuse light, illumination strength max. 20 µE x mE(-2) x sE(-1) / 16/8 h light/dark cycle
Feeding: The daphnids were not fed during the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a. (Sigma), Batch No. MKBC1858

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

There was no effect on Daphnia magna observed in the water accomodated fracton (WAF) with a nominal loading of 100 mg/L. Neither in the treatment nor in the control were any immobilized daphnids observed.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. The EC50-value with 95 % confidence interval (CI) was calculated by sigmoidal dose-response regression to be:
EC50: 1.71 mg/L (CI 1.57 - 1.87 mg/L)
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Reported statistics and error estimates:

The effects of the WAF were determined directly from the observation data without calculations.
EC50-value was calculated for the reference item by sigmoidal dose-response regression. The confidence interval for EC50 was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism.
All data were computer processed and rounded for presentation from the full derived data. Consequently if calculated manually based on the presented data minor variations may occur from these figures. Calculations were carried out using the following software:
- GraphPad Prism4, GRAPHPAD SOFTWARE, INC.
- Microsoft(R) Office Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

DOC - Analysis of Fresh (0 and 24 h) and Old Media (24 and 48 h)

	2011-02-08			2011-02-09			2011-02-09			2011-02-10
Sampling date	0 h			24 h			24 h			48 h
	(Fresh medium)			(Old medium)			(Fresh medium)			(Old medium)
Start of analysis	2011-02-10			2011-02-10			2011-02-10			2011-02-10
C-Wax Type OP
Nominal Loading of the Water	DOC
Accommodated	Meas.		Mean	Meas.		Mean	Meas.		Mean	Meas.		Mean
Fraction(WAF)	conc.		conc.	conc.		conc.	conc.		conc.	conc.		conc.
[mg/L]	[mg/L]		[mg/L]	[mg/L]		[mg/L]	[mg/L]		[mg/L]	[mg/L]		[mg/L]
100	2.6	2.6	2.6	2.8	2.8	2.8	2.2	2.2	2.2	2.3	2.3	2.3
Control	2.4	2.4	2.4	3.0	3.0	3.0	2.4	2.4	2.4	2.6	2.7	2.7

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

With the water accommodated fraction (WAF) of the test item C-Wax Type OP with a nominal loading of 100 mg/L, no effects were observed in an acute immobilization test on Daphnia magna according to OECD 202.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS) according to OECD guideline 202 (2004) and performed compliant to GLP (reliability category 1) the effects of the water accommodated fraction (WAF) with a nominal loading of 100 mg/L of the submission substance were determined in a limit test from 2011-02-07 to 2011-02-10, with the definitive exposure phase from 2011-02-08 to 2011-02-10, at DR. U . NOACK-LABORATORIEN, Kathe-Paulus-Str. 1, 31157 Sarstedt, Germany.

The limit test was conducted under semi-static conditions over a period of 48 h. Twenty daphnids were exposed to the WAF and the control.

The WAF with a nominal loading of 100 mg/L and the control were analysed via DOC analysis in the fresh media (0 and 24 h) and in the old media (24 and 48 h). All effect values are given based on the nominal loading of the test item C-Wax Type OP.

The water quality parameters (pH-value and dissolved oxygen concentration), measured at 0, 24 and 48 h, were determined to be within acceptable limits. The validity criteria of the test guideline were fulfilled.

The following effect concentrations were determined:

NOEC (24 and 48h, nominal) = 100 mg/L

EC50 (24 and 48 h, nominal) > 100 mg/L

Neither in the treatment nor in the contro any immobilized daphnids were observed.