2-hydroxy-N-((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)octadecanamide

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:

2017-10-16 to 2017-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ceramide III

IUPAC Name:

Ceramide III

Test material form:

solid

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Remarks:

No analytical monitoring was carried out due to the low solubility of the test item.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
In view of the difficulties associated with the evaluation of aquatic
toxicity of poorly water soluble and multi-component test items, a
modification of the standard method for the preparation of aqueous
media was performed. An approach endorsed by several important
regulatory authorities in the EU and elsewhere (ECETOC 1996 and
OECD 2000) is to expose the organisms to a Water Accommodated
Fraction (WAF) of the test item in cases where the test item is a
complex mixture and is poorly soluble in water and in the permitted
solvents. Using this approach, aqueous media were prepared by
mixing the test item with water for a prolonged period sufficient to
ensure equilibration between the test item and the water phase. After
completion of mixing and following a settlement period, the test item
phase was separated by siphon and the test organisms were exposed
to the aqueous phase, the WAF (which may contain dissolved and/or
suspended and/or emulsified fractions of the test item mixture).
Exposure are expressed in terms of the original concentration of the
test item in water at the start of the mixing period (loading rate)
irrespective of the actual concentration of the test item in the WAF.

Preparation of the water
accommodated fraction
One water accommodated fraction (WAF) was prepared with a
nominal loading of the test item of 100 mg/L. The loading level are
based on the results of a preliminary range finding test (for results,
please refer to Annex I).
An appropriate amount of the test item was weighed out and
transferred into a measuring flask with an appropriate amount of the
test media (see Table 1 and Table 2). This dispersion was shaken for
at least 24 hours with 20 rpm at room temperature. After a separation
phase of 1 hour at room temperature, the aqueous phase or WAF was
removed by siphoning (from the approximate centre of the glass flask).
The resulting water accommodated fraction (WAF) was used in a limit
test.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Age at study initiation (mean and range, SD): 2 to 24 hours old daphnids
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2°C, in an incubator, 16 h illumination, light intensity of max. 20 µE m-2 s-1
- Feeding during test: no

ACCLIMATION
- Acclimation period: At least 2 hours in river water
- Acclimation conditions (same as test or not): same
- Type and amount of food: culture daphnids are 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 > 1E06 cells/mL. The algae are cultured at the test facility

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

river water: 80.1 mg CO3/L;
culture medium: 160 -180 mg CaCO3 /L

Test temperature:

19 - 20 °C

pH:

start: 7.92 (test 100 mg/l) 8.09 (control); end: 7.65 (test 100 mg/l) 7.60 (control)

Dissolved oxygen:

start: 8.74 mg/l (test 100 mg/l) 9.85 mg/l (control); end: 8.12 mg/l (test 100 mg/l) 8.43 mg/l (control)

Conductivity:

220 µS/cm

Nominal and measured concentrations:

100 mg/l water accomodated fraction (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm) with a capacity of 50 mL, loosely covered with watch glasses
- Renewal rate of test solution (frequency/flow rate): static
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
river water as specified in section "any other information on materials and methods"

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Diffuse light; light intensity of max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Immobilisation was determined in all groups after 24 and 48 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
Range finding study
- Test concentrations: 100 mg/l loading, nominal, prepared in natural river water
2 replicates with 10 daphnids each
- Results used to determine the conditions for the definitive study: no immobilisation

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal loading ot test item

Basis for effect:

mobility

Details on results:

- immobilisation of test item: 0 %
- immobilisation of control: 0 %

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.92 mg/L (95% CI 1.11 - 2.00 mg/L)

Reported statistics and error estimates:

EC-values and statistical analyses for reference item:
An EC50-value was calculated for the reference item by sigmoidal dose-response regression. The 95 % confidence limits for the EC50 were calculated from the best-fit values, the standard error and the t-distribution with the validated software GraphPad Prism.

Any other information on results incl. tables

The percentage of immobility, determined in the WAF and in the control after 24 and 48 hours, is given in Table 4. The absolute numbers of immobile daphnids are presented inTable 5. Other adverse effects did not appear.

The study was performed under static conditions in natural river water.

Table 4:     Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test

                (n = 20, divided into 4 replicates with 5 daphnids each)

CERAMIDEIII Nominal loading of the test item in the WAF [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Table5:     Absolute Numbers of immobile Daphnids after 24 and 48 hours of Exposure in the Definitive Test

                (n = 20, divided into 4 replicates with 5 daphnids each)

CERAMIDEIII Nominal loading of the test item in the WAF [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

 

Additional Observations during the Definitive Test

The WAF was visually clear with finely distributed test item particles, which precipitated at the surface of the test solution and at the bottom of the test vessel after 24 hours of the exposure.

Water Quality Parameters

The measured water quality parameters(i.e. pH-value, dissolved oxygen concentration, water temperature, conductivity and total hardness) were within the acceptable limits.

During the test period, the temperature in the incubator was 19 – 20 °C.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EL10 as well as EL50 to Daphnia magna (effect immobilisation) was > 100mg/L water accomodated fraction nominal.

Executive summary:

The 48-h-acute toxicity of Ceramide III to Daphnia magna was studied under static conditions according to OECD Guideline 202 (2004) in a limit test.  Daphnids were exposed to control and test chemical at a nominal concentration 100 mg/L water accommodated fraction in natural river water for 48 h.

Mortality/immobilization was observed daily. 

The 48-hour EL10 as well as EL50 was > 100mg/L water accomodated fraction nominal.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

 

Results Synopsis

Test Organism Age: max. 24 h old daphnids

Test Type: Static limit test

Water accommodated fraction

 

48-hour EL10: > 100mg/L

Endpoint(s) Effected:  mobility