Reaction products resulting from the esterification of C18 unsaturated fatty acid with glycerol and glycerol oligomers

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:

2015-05-10 to 2015-05-13

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 (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The WAF and the control were analytically verified via analysis of the total organic carbon (TOC, according to DIN EN 1484), measured as NPOC
(Non Purgeable Organic Carbon), at the start of the exposure (0 hours) and at the end of the exposure (48 hours).
At the start of the exposure, sampling was carried out after preparation of the WAF.
At the end of the exposure (48 hours), samples were taken from an additionally prepared replicate of the WAF and the control.
The additional replicates were prepared with test media but without daphnids and incubated under test conditions.
The samples were acidified with 150 µL of 2 M HCl (ROTH) per 30 mL sample volume.

Recovery of the measured TOC in the Water Accommodated Fraction (WAF) should be within ± 20 % of the initially measured concentration.
Exposures have to be expressed in terms of the original concentration of the test item in water at the start of the mixing period (limit loading rate)
irrespective of the actual concentration of the test item in the WAF.

Vehicle:

no

Details on test solutions:

Water Accommodated In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items,
Fraction (WAF) 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, an aqueous medium was
prepared by mixing the test item with ISO test 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 organisms exposed to the aqueous phase, the WAF (which may
contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposure is 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 The water accommodated fraction (WAF) was prepared with al imit loading level of 100 mg/L as specified below.
accommodated fraction 100 mg/L of the test item were weighed out and transferred into a brown glass flask with an appropriate amount of
dilution water. This dispersion was shaken for 24 hours with 20 rpm at room temperature. After a separation phase of
1 hour, the aqueous phase or WAF was removed by siphoning from the approximate centre of the glass flask to give the
100 mg/L loading level WAF.

Limit loading level A WAF with a limit loading level of 100 mg/L was tested. Per definition of the WAF, all terms related to concentrations
have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to
concentrations. The WAF mentioned above is based on the results of a non GLP preliminary range finding test
conducted at the test facility under static conditions.

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

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Clone 5
- Source:
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Age at study initiation (mean and range, SD): 2 to 24 hours old daphnids from a healthy stock were used for the study
- Method of breeding:
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 x m-2 x s-1
Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
Cultur feeding: 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
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: At least 2 h in dilution water.
- Acclimation conditions (same as test or not): Same as test conditions.
- Health during acclimation (any mortality observed): No mortality observed.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

267 mgCaCO3/L

Test temperature:

18 - 22°C, constant within ± 1°C
At start of exposure: 19.1 °C

pH:

pH at the Start of the Exposure (0 hours)
(measured in one additional replicate of the WAF and the control)

Nominal test item
concentration
[mg/L] pH-value

100 6.10
Control 7.61


pH at the End of the Exposure (48 hours)
(measured in all replicates)

Nominal test item
concentration
[mg/L] pH-values
Replicates
1 2 3 4
100 7.43 7.36 7.34 7.34
Control 7.65 7.65 7.66 7.65

Dissolved oxygen:

O2-concentration at the Start of the Exposure (0 hours)
(measured in one additional replicate of the WAF and the control)

Nominal test item
concentration
[mg/L]
Dissolved
O2-concentration
[mg/L]
100 8.34
Control 8.70



O2-concentration at the End of the Exposure (48 hours)
(measured in all replicates)

Nominal test item
concentration
[mg/L] Dissolved O2 -concentration [mg/L]
Replicates
1 2 3 4
100 8.18 8.10 8.22 7.89
Control 7.99 7.68 7.78 7.75

Nominal and measured concentrations:

A WAF with a nominal loading level of 100 mg/L was tested in a limit test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
- Test volume: 20 mL
- Aeration: None
- 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
Component Concentration [mg/L]
KCl 5.75
NaHCO3 64.8
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123

- Water Quality Parameters of the Dilution Water prior to the Start of the Exposure (0 hours):
pH-value Dissolved O2 concentration [mg/L] Temperature [°C] Conductivity [µS/cm] Total hardness [mg CaCO3/L]
8.00 9.47 19.1 680 267

- Culture medium different from test medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: test start (0 hours) and test end (48 hours)

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 20 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilisation, 24 hours interval

TEST CONCENTRATIONS
A WAF with a nominal loading level of 100 mg/L was tested in a limit test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

other: EL10,50,100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Duration:

48 h

Dose descriptor:

other: EL10,50,100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

other: NOEL

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

other: LOEL

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 hours. The EC50-value with 95 % confidence limits (CI) was calculated by
sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.92 mg/L (CI 1.71 - 2.29 mg/L)
The EC50-value of the reference item potassium dichromate after 24 hours is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality
criteria according to AQS P 9/2 (02/2000) 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.

Biological Data

The percentage immobility, determined in the WAF and the control after 24 and 48 hours under static conditions, is given in the table below.

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

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

Nominal loading of the water accommodated fraction (WAF)   [mg/L]	IMMOBILISATION [%]
	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

 

The WAF was slightly turbid throughout the exposure period.

 

Measured Exposure Concentrations during the Definitive Test

 

The WAF of the test item Reaction mass of C18 (unsaturated) esters with glycerine oligomers and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484), measured as NPOC (Non Purgeable Organic Carbon) at the start of the exposure

(0 hours) and at the end of the exposure (48 hours).
The measured TOC concentration was 68.6 mg/L at the start of the exposure (0 hours) and 68.5 mg/L at the end of the exposure (48 hours).

The analytical results are presented in the table below.

The measured TOC concentration remained stable within ± 20 % of the initially measured value. This indicates that the test item was successfully maintained for the duration of the test.

Table:      Measured TOC Concentrationsand Percent of initially measured TOCin the Definitive Test

Sampling date	2015-05-11 0 hours Start of the exposure	2015-05-13 48 hours End of the exposure
Start of analysis	2015-05-11	2015-05-13
Nominal loading of the water accommodated fraction (WAF) [mg/L]	Total Organic Carbon (TOC)^1)
	Measured concentration^2) [mg/L]	Measured concentration^2) [mg/L]	%
100	68.6	68.5	100
Control	< 2.00	< 2.00	—

¹⁾          = measured as NPOC

²⁾          = measured NPOC concentration, mean value of 2 injections, dilution factor taken into account

%        = percent of the initially measured NPOC

 

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

Validity criteria fulfilled:

yes

Conclusions:

At the Water Accommodated Fraction with a nominal loading of 100 mg/L of the test item Reaction mass of C18 (unsaturated) esters with glycerine
oligomers, no effects were observed on Daphnia magna.

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of a Water Accommodated Fraction (WAF) of the test item Reaction mass of C18 (unsaturated) esters with glycerine oligomers(batch number: ESD0017954) with a nominal loading of 100 mg/L was determined according to OECD 202 (2004). The limit test was conducted under static conditions over a period of 48 hours from 2015-05-10 to 2015-05-13, with the definitve exposure phase from 2015-05-11 to 2015-05-13, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

The test item is an UVCB substance and a yellow liquid. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble 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, the aqueous medium was prepared by shaking the test item with dilution water at 20 rpmfor 24 hours at room temperature to ensure equilibration between the test item and the water phase. 100.1 mg of the test item were weighed out and transferred with 1000 mL of dilution water (ISO test water, acc. to OECD202, Annex 3) into a brown glass flask. After completion of shaking and following a 1-hour settlement period, the test item phase was separated by siphon from the approximate center of the brown glass flask. The WAF was slightly turbid throughout the exposure period.

Twenty daphnids were exposed to the WAF and the control.

The WAF of the test item Reaction mass of C18 (unsaturated) esters with glycerine oligomers and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484), measured as NPOC (Non Purgeable Organic Carbon) at the start of the exposure

(0 hours) and at the end of the exposure (48 hours).
The measured TOC concentration was 68.6 mg/L at the start of the exposure (0 hours) and 68.5 mg/L at the end of the exposure (48 hours).

The measured TOC concentration remained stable within ±20 % of the initially measured value. This indicates that the test item was successfully maintained for the duration of the test.

Per definition of the WAF, all terms related to concentrations have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. Therefore, the effect values given in the table below were based on the nominal loading level of 100 mg/L of the test item Reaction mass of C18 (unsaturated) esters with glycerine oligomers.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 hours) and at the end of the exposure

(48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

 

 

At the Water Accommodated Fraction with a nominal loading of 100 mg/Lof the test item

Reaction mass of C18 (unsaturated) esters with glycerine oligomers,

no effects were observed onDaphnia magna.

Table:   EL-Values, NOEL and LOEL

(based on the nominal loading of the test item)

Effect levels	Test duration [h]	Reaction mass ofC18 (unsaturated) esters with glycerine oligomers
		Nominal test item concentration [mg/L]
EL10 /50 / 100	24	> 100
	48	> 100
NOEL	48	100
LOEL	48	> 100

 

Description of key information

In the daphnia limit test (OECD202) no effects were observed up to the nominal loading rate of 100 mg/l corresponding to a TOC measured concentration on 68.5 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 100 mg/L