Branched and linear octadecanoic acid, esters with glycerol oligomers

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from similar mixture/product

Adequacy of study:

key study

Study period:

19 - 21 Dec 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test design with deficiencies: all loading levels above water solubility; physical effects

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all tested loading levels (6.25 - 100 mg/L) and the control at the start (0 h) and at the end of the exposure (48 h)
- Sampling method: For the old media, an additional replicate of each loading level and the control was prepared, with test media, but without daphnia, and incubated under test conditions until sampling.
- Sample storage conditions before analysis: The samples were stored at 6 ± 2 °C until analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Due to the poorly water soluble test item, the organisms were exposed to a Water Accommodated Fraction (WAF) of the test item. 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. At the completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organsisms exposed to the aqueous phase, the WAF. Five water accomoated fractions (WAF) were prepared with nominal loadings of the test item in the range of 6.25 - 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. These dispersion were stirred for 72 h with 1100 rpm at room temperature. After a separation phase of 1 h, the WAFs were taken from the homogenous liquid phase (the approximate centre of the glass flask) and used for testing. Exposures were expressed in terms of the original concentration of the test item in water at the start of the mixin period (loading rate), irrespective of the actual concentration of the test item in the WAF.
- Differential loading: Yes, 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.
- Controls: Yes, dilution water without test item, tested under the same conditions as the test groups.
- Evidence of undissolved material: The loading level of 100 mg/L was slightly turbid throughout the exposure. All other loading levels and the control were visually clear throughout the exposure.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Clone 5
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), in-house culture
- Age of daphnia at experimental starting: 2 to 24 h old
- Feeding during test: No.
- Culturing: 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.
- Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
- Culture feeding: Yes.
- Food type: A mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: > 1E+06 cells/mL
- Frequency: At least 5 times per week, ad libitum.

ACCLIMATION
- Acclimation period: At least 2 h
- Acclimation conditions: in dilution water

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Removal of the parental daphnia twice within 22 h.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

246 mg CaCO3/L (dilution water)

Test temperature:

19 - 20 °C (incubator)

pH:

0 h: 7.64 - 8.03
48 h: 7.66 - 7.81

Dissolved oxygen:

0 h: 8.44 - 8.68 mg O2/L
48 h: 8.28 - 8.67 mg O2/L

Conductivity:

623 µS/cm (dilution water)

Nominal and measured concentrations:

nominal: control, 6.25, 12.5, 25.0, 50.0 and 100 mg/L
measured: < 2.00/< 2.00, < 2.00/2.43, 2.63/2.78, 3.21/3.13, 3.31/3.17, and 8.08/8.16 mg C/L (0 h/48 h, means of 3)

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Material, size, headspace, fill volume: Material: glass; Size: 4 cm ID x 7 cm H, 50 mL; Test volume per vessel: 20 mL.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202, Annex 3
- Total organic carbon: < 2.00 mg/L
- Conductivity: 623 µS/cm
- Ca/mg ratio: 294 mg CaCl2 2H2O/L and 123 mg MgSO4 7H2O/L
- Culture medium different from test medium: Yes. culture medium different from test medium. Daphnia were cultured in Elent M4 culture medium and the test media were prepared in dilution water (ISO test water).
- Intervals of water quality measurement: Prior to the start of the exposure (0 h), the water quality parameters of the dilution water were measured, inlcuding pH values, dissolved oxygen concentration, temperature, conductivity and total hardness. The water quality paramaters (i.e. pH value and dissolved oxygen concentration) of the test groups and the control at the start of the exposure (0 h) were measured in one additional replicate per loading level and control. At the end of the exposure (48 h), the water quality parameters were measured in all replicates per loading level and control. For the old media, an additional replicate of each loading level and the control was prepared, with test media, but without daphnia, and incubated under test conditions until sampling.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light/ 8 h dark cycle
- Light intensity: Diffuse light, max. 20 µE*m^-2*s^-1

EFFECT PARAMETERS MEASURED:
Immobilization: In all groups after 24 h and 48 h.

RANGE-FINDING STUDY
- Test concentrations: Control, 1, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, the test loadings are based on the results of a non GLP preliminary range finding test under static conditions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a.

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

- Other biological observations: No
- Mortality of control: 0%
- Other adverse effects control: 0%

Results with reference substance (positive control):

- Results with reference substance valid? Yes (04 - 05 Dec 2012)
- Relevant effect levels: EC50 (24 h) = 1.60 mg/L (95% CI: 1.40 - 1.80 mg/L)
- Limit test: No, test concentrations: 0.625, 1.00, 1.60, and 2.56 mg/L (Potassium dichromate p.a.)

Reported statistics and error estimates:

The EL50 values after 24 h, the EL100 values after 24 and 48 h, the NOELR and the LOELR after 48 h were estimated directly from the observation data, without mathematical calculation.

ANALYTICAL RESULTS

The measured concentrations of total organic carbon at the start of the exposure (0 h) were in the range of < 2.00 to 8.08 mg C/L in the loading levels of 6.25 - 100 mg/L, and < 2.00 mg C/L in the control. At the end of the exposure (48 h), the measured concentrations of total organic carbon wree in the range of 2.43 - 8.16 mg C/L in the concentration levels of 6.25 to 100 mg/L, and < 2.00 mg C/L in the control.

All effect values were based on the nominal loading rates of the test item.

BIOLOGICAL RESULTS

After 48 h effects were observed at loading rates of ≥ 25.0 mg/L.

Table 1. Immobilization Rates in the Definitive Test after 24 and 48 h of Exposure (n = 20, divided into 4 replicates with 5 daphnia each).

Test substance 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	20	20	0	10	20	40	80	40	45
25	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

MV = mean value

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

- Other biological observations: No
- Mortality of control: 0%
- Other adverse effects control: 0%

Results with reference substance (positive control):

- Results with reference substance valid? yes (04 - 05 Dec 2012)
- Relevant effect levels: EC50 (24 h) = 1.60 mg/L (95% CI: 1.40 - 1.80 mg/L)
- Limit test: No, test concentrations: 0.625, 1.00, 1.60, and 2.56 mg/L (Potassium dichromate p.a.)

Reported statistics and error estimates:

The EL50 values after 24 h, the EL100 values after 24 and 48 h, the NOEL and the LOEL after 48 h were estimated directly from the observation data, without mathematical calculation. The EL10 values and the EL50 value after 48 h were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad Prism 5. The EC50 value for the reference item and its confidence limits were calculated accordingly.

ANALYTICAL RESULTS

The measured concentrations of total organic carbon at the start of the exposure (0 h) were in the range of < 2.00 to 8.08 mg C/L in the loading levels of 6.25 - 100 mg/L, and < 2.00 mg C/L in the control. At the end of the exposure (48 h), the measured concentrations of total organic carbon wree in the range of 2.43 - 8.16 mg C/L in the concentration levels of 6.25 to 100 mg/L, and < 2.00 mg C/L in the control.

All effect values were based on the nominal loading rates of the test item.

BIOLOGICAL RESULTS

After 48 h effects were observed at loading rates of ≥ 25.0 mg/L.

Table 1. Immobilization Rates in the Definitive Test after 24 and 48 h of Exposure (n = 20, divided into 4 replicates with 5 daphnia each).

Test substance 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	20	20	0	10	20	40	80	40	45
25	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

MV = mean value

Validity criteria fulfilled:

yes

Description of key information

EL50 (48 h) > 50 mg/L (nominal, OECD 202)

Additional information

There are two GLP studies available investigating the acute immobilization effects of the target substance (key study) and a structurally related source substance (supporting study) on aquatic invertebrates according to the OECD guideline 202. The source substance in the supporting study (Di(isooctadecanoic) acid, diester with oxydi(propanediol), CAS 67938-21-0) is one main constituent of the target substance. Therefore, its effect concentrations are expected to be in the same range as the target substance Polyaldo 2 -1 -IS (CAS 73296-86-3). In accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5, the results of the supporting study are therefore considered informative supplementary information for this endpoint (see read-across justification in IUCLID section 13).

In the key study with the target substance, Daphnia magna was exposed to five nominal water accommodated fractions (WAFs) ranging from 6.25 – 100 mg/L for 48 h in a static exposure design. The loading rates at the beginning and end of exposure were analytically determined by total organic carbon (TOC) analysis. TOC analysis confirmed the presence of the test substance in the test solution and indicated that the concentration of the target test item was stable throughout the test. 80% immobilization of D. magna was observed at a loading rate of 100 mg/L, whereas 35% immobilization was recorded at 50 mg/L, 10% at 25 mg/L and 0% at the lower loading rates. The substance is known to be surface active but no information on visual observations of the test solutions were included in the test report. However, in the available study investigating the water solubility (according to the OECD 105), there was clear evidence that the test item forms micelles in water at test concentrations of at least 100 mg/L. It was observed that mixtures of the test item in water at concentrations ranging from 100 to 1000 mg/L were visually milky, stable formulations forming micelles suspected to cause physical effects. Thus, it is justified to exclude at least the highest loading rate from the assessment. Consequently, an EL50 (48 h) of > 50 mg/L was derived.

In the supporting study with the source substance the findings of the key study were confirmed. In this study, D. magna was exposed to five WAFs of nominal loading rates ranging from 6.25 – 100 mg/L for 48 h in a static test design. All loading rates are above the water solubility limit of the source substance (3 mg/L). The tested loading levels were analytically verified by TOC analysis at the beginning and end of exposure. The analytical results indicated that the concentration of the test item in the aqueous phase was stable for the duration of the test. Moreover, the results indicate that it is very likely that undissolved particles were present at the loading rate of 100 mg/L since the TOC strongly increased at the highest loading rate. This was supported by visual observations. The highest loading rate was turbid and therefore excluded from assessment. 85% immobilization of D. magna was observed at a loading rate of 100 mg/L, whereas 45% immobilization was recorded at 50 mg/L, 25% at 25 mg/L and 0% at the lower loading rates. The derived EL50 (48 h) was set to > 50 mg/L (nominal). The NOELR (48 h) was 12.5 mg/L and the derived LOELR (48 h) was 25.0 mg/L (nominal), respectively.

In conclusion, the observed effects in both studies were recorded at loading rates greater than the water solubility and micelle formation is very likely and confirmed by investigations of the water solubility. Thus, it is likely that physical rather than intrinsic effects of the substance caused the immobilization observed at the highest loading rate which was consequently excluded from the assessment.