Polar modified Rice Bran Wax

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-09-16 to 2015-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Determination of the TOC
All concentration levels and the control were verified via analysis
of the total organic carbon (TOC, according to DIN EN 1484), at the start (0 hours) and at the end of the exposure (48 hours).

Sampling for the analytical monitoring
At the start of the exposure (0 hours), sampling was carried out after preparation of the concentration levels.
At the end of the exposure (48 hours), samples were taken from additional replicates with test media, but without daphnids. These replicates were incubated
under test conditions until sampling

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
A saturated solution with a nominal concentration of 100 mg/L of the
test item was prepared with dilution water (see Table 2) one day prior to the start of the exposure (at -24 hours). The test item was mixed with the dilution
water by constant stirring with a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, undissolved test item particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption
during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow for adsorption and saturation of the
filter material with the dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated
solution, was used as the highest concentration in the test and as a stock solution for the preparation of further concentration levels. During filtration, the
filter was always kept covered.

Test concentrations
5 concentration levels of the test item in a geometric series with a separation factor of 2, prepared by diluting the saturated solution with dilution water, were tested as follows:
6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution
The test concentrations were selected based on the results of a non-GLP preliminary range finding test as specified

Control
Dilution water without test item incubated under the same conditions as the test groups

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5).

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the of test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu),
14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination, light intensity of max. 20 µEm-2  s-1

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used. The composition of the culture medium is presented in Table 2.

Culture feeding
The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany



Composition of the Culture Medium Elendt M4
according to OECD 202, Annex 3 (2004)
Component Concentration [mg/L]
CaCl2 x 2 H2O 176*
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 4.30
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Na2EDTA x 2 H2O 5.00
FeSO4 x 7 H2O 1.99
H3BO3 0.29
MnCl2 x 4 H2O 0.36
LiCl 0.30
SrCl2 x 6 H2O 0.15
RbCl 0.071
NaBr 0.016
Na2MoO4 x 2 H2O 0.063
CuCl x 2 H2O 0.017
ZnCl2 0.013
CoCl2 x 6 H2O 0.010
KJ 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2  0.8
* = original recipe: 293.8 mg/L, modified to achieve a total water hardness of
160 to 180 mg CaCO3/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Dilution water dated:

0 hours: 2016-04-12 Total hardness [mg CaCO3/L]: 178

Test temperature:

18 - 22°C, constant within ± 1°C
2016-04-12 20.4 °C

pH:

Water Quality Parameters at the Start of the Exposure (0 hours) (measured in one additional replicate per dilution level and control)
Dilution level of the
saturated solution

[%] pH-value Dissolved
O2 concentration [mg/L]
100* 7.85 7.25
50.0 8.22 7.50
25.0 8.21 8.28
12.5 8.09 8.56
6.25 7.85 8.60
Control 8.82 9.87
* = saturated solution


Water Quality Parameters at the End of the Exposure (48 hours) (measured in all replicates per dilution level and control)
Dilution level of the
saturated solution
[%] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
100* 7.91 7.89 7.89 7.88 7.82 7.66 7.86 7.84
50.0 7.89 7.88 7.89 7.91 7.96 7.90 8.00 7.80
25.0 7.85 7.84 7.85 7.86 7.83 7.80 7.95 7.96
12.5 7.82 7.83 7.83 7.82 7.94 7.89 7.73 7.89
6.25 7.64 7.71 7.76 7.78 7.67 7.79 7.68 7.63
Control 7.72 7.74 7.76 7.80 7.78 7.84 7.86 7.90
* = saturated solution

Dissolved oxygen:

Water Quality Parameters at the Start of the Exposure (0 hours) (measured in one additional replicate per dilution level and control)
Dilution level of the
saturated solution

[%] pH-value Dissolved
O2 concentration [mg/L]
100* 7.85 7.25
50.0 8.22 7.50
25.0 8.21 8.28
12.5 8.09 8.56
6.25 7.85 8.60
Control 8.82 9.87
* = saturated solution


Water Quality Parameters at the End of the Exposure (48 hours) (measured in all replicates per dilution level and control)
Dilution level of the
saturated solution
[%] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
100* 7.91 7.89 7.89 7.88 7.82 7.66 7.86 7.84
50.0 7.89 7.88 7.89 7.91 7.96 7.90 8.00 7.80
25.0 7.85 7.84 7.85 7.86 7.83 7.80 7.95 7.96
12.5 7.82 7.83 7.83 7.82 7.94 7.89 7.73 7.89
6.25 7.64 7.71 7.76 7.78 7.67 7.79 7.68 7.63
Control 7.72 7.74 7.76 7.80 7.78 7.84 7.86 7.90
* = saturated solution

Nominal and measured concentrations:

All concentration levels of the test item LICOCARE RBW 106 TP and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure (0 hours) and at the end of the exposure (48 hours).
The measured TOC concentrations at the start of the exposure (0 hours) were in the range of 3.24 - 4.62 mg C/L for the dilution levels of 6.25 to 100 % of the saturated solution and the control. At the end of the exposure (after 48 hours), the measured TOC concentrations were in the range of 3.08 - 4.56 mg C/L. The results are presented

Measured TOC Concentrations during the Definitive Test



Sampling date 2016-04-12 2016-04-14
Start of the exposure, 0 hours End of the exposure, 48 hours
Start of analysis 2016-04-12 2016-04-14

Dilution level of the
saturated solution
of the test item [%]

Total Organic Carbon (TOC)
Measured concentration1) Measured concentration1)
[mg C/L] [mg C/L]
100* 4.62 4.56
50.0 4.21 3.75
25.0 4.22 3.85
12.5 4.47 4.13
6.25 4.41 3.95
Control 3.24 3.08
1) = measured TOC concentration, mean value of 2 injections, dilution factor taken into account
* = saturated solution of the test item

The limit of quantification of the analytical method is set to 2.00 mg/L.

Details on test conditions:

Test method
Static procedure

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Dilution water
Same as culture medium (see Table 2)

Number of daphnids
20 daphnids, divided into 4 replicates, each with 5 daphnids, were
and replicates used for each concentration level and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for
the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded.
The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny will be used for the test.
 
Acclimatisation
Acclimatisation of the daphnids was not necessary, because the dilution water was equivalent to the culture medium.

Application 2
0 g test solution per replicate was weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small
amount of dilution water by a pipette.

Test temperature (target)
18 - 22 °C, constant within ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 20 µEm-2  s-1 (max. 1340 lx)

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

> 50 - < 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 50 - < 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Results with reference substance (positive control):

A reference test was conducted as an acute immobilisation test (acc. to AQS P 9/2 and OECD 202) in ISO test water (acc. to OECD 202 (2004), Annex 3) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented in section 7.2.3.

Reference item Potassium dichromate p.a. (SIGMA)

Purity 99.0%

Batch No. MKBV0900V

Storage stability 2016-12-16

Test concentrations 1.00 – 2.00 – 4.00 mg/L

Ranges of validity EC50 (24 hours): 0.6 mg/L - 2.4 mg/L, according to AQS P 9/2 (clone 5)
EC50 (24 hours): 0.6 mg/L - 2.1 mg/L, according to OECD 202 (clone A)

Test duration 2016-04-05 to 2016-04-06

Reported statistics and error estimates:

Methods of evaluation
The EC10- , EC50- and the EC100-values (after 24 and 48 hours) were deduced directly from the observation data. Only the biological relevant concentration
levels (i.e. the two highest concentration levels) were taken into account. All other concentration levels were biologically not relevant ( partial effect (no immobilisation rates ≥ 50%) was observed, the calculation of the EC10- and EC50-values (after 24 and 48 hours) was not possible and,
therefore, estimated directly from the observation data. The concentration-effect relationships after 24 and 48 hours are shown graphically.
All effect levels (EC10 / 50 / 100) were based on the nominal concentrations the test item LICOCARE RBW 106 TP, since no specific analysis was carried out.

EC-values and statistical analyses for reference item
An EC50-value was calculated only for the reference item by sigmoidal
dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried
out from the best-fit values with the software GraphPad Prism5.

SoftwareAll data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual
calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item LICOCARE RBW 106 TP, the 48 hour EC50 for Daphnia magna was > 100 mg/L.

Executive summary:

1        Summary

 

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the test item LICOCARE RBW106 TP were determined according to OECD 202 (2004) from 2016-04-11 to 2016-04-14, with the definitive exposure phase from 2016-04-12 to 2016-04-14,at the test facility.

The study was conducted under static conditions over a period of 48 hours with a saturated solution (prepared with a nominal concentration of 100 mg/Lof the test itemLICOCARE RBW106 TP) and a further four dilution levels of the saturated solution in a geometric series with a separation factor of2(nominal concentrations of6.25 to 50.0%= 6.25 to 50 mg/L).

The tested saturated solution and the following dilution levels of the saturated solution were visually clear throughout the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration leveland the control.

All concentration levels of the test item LICOCARE RBW 106 TP and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure (0 hours) and at the end of the exposure (48 hours).

The measured TOC concentrations at the start of the exposure (0 hours) were in the range of 3.24 - 4.62 mg C/L for the dilution levels 6.25 to 100 % of the saturated solution and the control. At the end of the exposure (after 48 hours), the measured TOC concentrations were in the range of 3.08 - 4.56 mg C/L. The results are presented.

Since no specific analysis was carried out, the effect values given inTable1are based on the nominal concentrations of the test itemLICOCARE RBW106 TP.

The validity criteria of the test guidelines were fulfilled.

 

Table1:   EC₁₀-, EC₅₀- and EC₁₀₀-Values

(based on the nominal concentrations of the test item)

LICOCARERBW106 TP
Effect levels	Test duration   [hours]	Toxicity endpoint based on the nominal concentrations the test item [mg/L]
EC10 (with 95% confidence limits)	24	50.0 < EC10< 100
	48	50.0 < EC10< 100
EC50 (with 95% confidence limits)	24	> 100
	48	> 100
EC100	24	> 100
	48	> 100

 

Description of key information

Based on the nominal concentrations of the test item LICOCARE RBW 106 TP, the 48 hour EC50 for Daphnia magna was > 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 100 mg/L