C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

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:

2012-12-16 to 2013-01-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

EC Number:

701-479-5

Molecular formula:

C42H82O7

IUPAC Name:

C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

Details on test material:

- Name of test material (as cited in study report): Diglycerol diisostearate
- Physical state: Viscous liquid, light yellow
- Analytical purity: 100 % (w/w) UVCB
- Purity test date: 2012-02-29
- Lot/batch No.: ESD0012304
- Expiration date of the lot/batch: 2013-06-09
- Stability under test conditions: Stable
- Storage condition of test material: Keep container tightly closed and dry.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

- Concentrations: 6.25 - 12.5 - 25 - 50 - 100 mg/L (WAF)
- Sampling method: All tested loading levels of the test item and the control were analytically verified by analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure (0 h) and at the end of the exposure (48 h).
For the old media, an additional replicate of each loading level and the control was prepared, with test media, but without daphnids, and incubated under test conditions until sampling.
- Sample storage conditions before analysis: The samples were stored at 6 ± 2 °C until analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Five water accommodated fractions (WAF) were prepared with nominal loadings of the test item in the range of 6.25 to 100 mg/L, set up in a geometric series with a separation factor of 2.
For each loading level, an appropriate amount of the test item was weighed out and transferred into a measuring flask with an appropriate amount of dilution water. For details, see the table below.
These dispersions were stirred for 72 h with 1100 rpm at room temperature. After a separation phase of 1 h, the WAFs were taken from the homogeneous liquid phase (the approximate centre of the glass flask) and used for testing.
The test loadings are based on the results of a non GLP preliminary range finding test under static conditions.
- Eluate: Dilution water
- Differential loading: 6.25 - 12.5 - 25 - 50 - 100 mg/L
- Controls: Dilution water without test item, tested under the same conditions as the test goups

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µExm-2 x s-1
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

DILUTION WATER:
Dilution water ISO test water, according to OECD 202, Annex 3:
Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2  2 H2O 294
MgSO4  7 H2O 123
pH-value: 7.8  0.2

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water at test start: 246 (mg CaCO3/L)

Test temperature:

21.5 °C (Water)

pH:

Water Quality Parameters at the Start of the Exposure (0 h)
(measured in one additional replicate per concentration level and control)
Diglycerol diisostearate
Nominal Loading Rate
of the Water Accommodated
Fraction (WAF)
[mg/L] pH-Value




100 7.64
50.0 7.68
25.0 8.02
12.5 8.03
6.25 7.99
Control 7.87

Water Quality Parameters at the End of the Exposure (48 h)
(measured in all replicates)
Diglycerol diisostearate
Nominal Loading Rate
of the Water Accommodated
Fraction (WAF)
[mg/L] pH-Values
Replicates
1 2 3 4
100 7.69 7.68 7.67 7.67
50.0 7.70 7.68 7.68 7.66
25.0 7.71 7.71 7.71 7.71
12.5 7.72 7.73 7.72 7.72
6.25 7.74 7.73 7.74 7.72
Control 7.81 7.76 7.75 7.76

Dissolved oxygen:

Water Quality Parameters at the Start of the Exposure (0 h)
(measured in one additional replicate per concentration level and control)
Diglycerol diisostearate
Nominal Loading Rate
of the Water Accommodated
Fraction (WAF)
[mg/L] Dissolved
O2-Concentration


[mg/L]
100 8.48
50.0 8.57
25.0 8.44
12.5 8.55
6.25 8.68
Control 8.52

Water Quality Parameters at the End of the Exposure (48 h)
(measured in all replicates)
Diglycerol diisostearate
Nominal Loading Rate
of the Water Accommodated
Fraction (WAF)
[mg/L] Dissolved O2 –Concentration [mg/L]
Replicates
1 2 3 4
100 8.46 8.43 8.41 8.39
50.0 8.28 8.32 8.36 8.37
25.0 8.35 8.31 8.35 8.43
12.5 8.43 8.45 8.39 8.36
6.25 8.39 8.45 8.34 8.40
Control 8.67 8.33 8.42 8.44

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: see above
- Conductivity: Dilution water at test start:632 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


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

TEST CONCENTRATIONS
- Range finding study:
Immobilization Rates in the non GLP Preliminary Range Finding Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Diglycerol diisostearate
Nominal Loading Rate
of the Water Accommodated
Fraction (WAF)
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
100 60 70 65 100 100 100
10 0 0 0 0 0 0
1 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Loading rate is > water solubility

Details on results:

- Mortality of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.60 (CI 1.40 - 1.80) mg/L

Any other information on results incl. tables

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

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

Diglycerol diisostearate Nominal Loading Rate of the Water Accommodated Fraction (WAF) [mg/L]	IMMOBILIZATION [%]
	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
100	60	40	20	60	45	80	100	80	80	85
50.0	0	20	20	0	10	20	40	80	40	45
25.0	0	0	0	0	0	20	20	20	40	25
12.5	0	0	0	0	0	0	0	0	0	0
6.25	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal loading rates of the test item Diglycerol diisostearate, the NOELR after 48 h was 12.5 mg/L.. This loading rate is above water solubility based on TOC measurements (see EP Summary Aquatic toxicity). This no hazards were identified up to the water solubility. Effects observed at higher loading rates are most likely effects related inhibition of filtration by the micro droplet of the test emulsion. This assumption is backed by the fact that no toxicity is observed in acute tests of fish and algae up to loading rates of 100 mg/L respectively 250 mg/L.