Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers, ethoxylated

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:

2017-07-31 to 2017-08-03

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers, ethoxylated

IUPAC Name:

Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers, ethoxylated

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The WAF and the control were analytically verified via analysis of the total organic carbon (TOC, according to DIN EN 1484) in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours).
Analytical results are presented

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of the WAFs and analyzed.
At the end of the exposure (48 hours), samples of the old media were taken from additional replicates, which were prepared with test media but without daphnids. These replicates were incubated under test conditions until sampling.

Test solutions

Vehicle:

no

Details on test solutions:

Loading levels
Five water accommodated fractions (WAF) were prepared with nominal loading levels of the test item of:
6.25 – 12.5 – 25.0 – 50.0 – 100 mg/L (separation factor 2).
Per definition of the WAF, all terms related to concentration level will be given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.
The WAF is based on the results of a non-GLP preliminary range finding test conducted at the test facility under static conditions.

Water Accommodated Fraction (WAF)
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, 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.

Preparation of test solutions
For the water accommodated fractions (WAF) an appropriate amount of the test item was weighed out and transferred into a brown glass flask with dilution water one day prior to application, respectively. The WAFs were shaken with 20 rpm for 24 hours at room temperature. After a separation phase of 1 hour the WAFs were taken from the homogeneous liquid phase from the middle of the water body. The WAFs were checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion).

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

Test organisms

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 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 ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, was 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 according to ELENDT (1990)
Component Concentration [mg/L]
CaCl2 x 2 H2O 147
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
Cyanocobalamine 0.0010
Biotin 0.00075
pH 8.2  0.8

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water dated:

0 hours: 2017-08-01 Total hardness [mg CaCO3/L]: 168

Test temperature:

18 - 22°C, constant within ± 1°C
2017-08-01 20.8 °C

pH:

Water Quality Parameters in the fresh Media at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) WAF and control)
Nominal loading level of
the test item in the water
accommodated fraction
[mg/L] pH-value
Dissolved
O2 concentration

[mg/L]
100 8.45 8.37
50 8.55 8.34
25 8.52 8.37
12.5 8.52 8.40
6.25 8.31 8.50
Control 8.60 8.39


Water Quality Parameters in the old Media at the End of the Exposure (48 hours)
(measured in all replicates containing daphnids)
Nominal loading level of
the test item in the water
accommodated fraction
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
100 7.71 - - - 8.35 - - -
50 7.72 - - - 8.35 - - -
25 7.73 - - - 8.53 - - -
12.5 7.72 - - - 8.48 - - -
6.25 7.74 - - - 8.29 - - -
Control 7.80 - - - 8.68 - - -

Dissolved oxygen:

Water Quality Parameters in the fresh Media at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) WAF and control)
Nominal loading level of
the test item in the water
accommodated fraction
[mg/L] pH-value
Dissolved
O2 concentration

[mg/L]
100 8.45 8.37
50 8.55 8.34
25 8.52 8.37
12.5 8.52 8.40
6.25 8.31 8.50
Control 8.60 8.39


Table 10: Water Quality Parameters in the old Media at the End of the Exposure (48 hours)
(measured in all replicates containing daphnids)
Nominal loading level of
the test item in the water
accommodated fraction
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
100 7.71 - - - 8.35 - - -
50 7.72 - - - 8.35 - - -
25 7.73 - - - 8.53 - - -
12.5 7.72 - - - 8.48 - - -
6.25 7.74 - - - 8.29 - - -
Control 7.80 - - - 8.68 - - -

Conductivity:

Water Quality Parameters of the Dilution Water at the Start of the Exposure (0 hours)
Dilution water
dated: pH-Value




pH-Value undissolved O2 concentration [mg/L] Temperature [°C] Conductivity [µS/cm] Total hardness [mg CaCO3/L]
2017-08-01 8.60 8.39 20.8 435 168
 

Nominal and measured concentrations:

All concentration levels of the test item 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). At the end of the exposure (48 hours), samples of the old media were taken for analysis from additional replicates, which were prepared with test media, but without daphnids and incubated under test conditions until sampling.
The measured TOC concentrations of the loading levels in fresh media (0 hours) were in the range of 3.01 to 8.36 mg C/L and < LOQ at the control.
In the old media (48 hours), the measured TOC concentrations of the loading levels were in the range of 2.82 to 7.42 mg C/L and < LOQ at the control. The results are presented in Table 7.

Measured TOC Concentrations during the Definitive Test
Sampling date 2017-08-01
Start of the exposure interval,
0 hours 2017-08-03
End of the exposure interval,
48 hours
Start of analysis 2017-08-01 2017-08-03

Nominal loading level
of the test item in the water accommodated fraction
[mg/L] Total Organic Carbon (TOC)
Measured
concentration1)
[mg/L] Measured
concentration1)
[mg/L]
100 8.36 7.42
50 5.43 4.83
25 3.86 3.53
12.5 3.01 2.82
6.25 3.29 2.99
Control < LOQ < LOQ
1) = measured TOC concentration, mean value of 2 injections

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

Details on test conditions:

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids were
used for each loading 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 was used for the test.

Acclimatization
Acclimatization was not necessary, because the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate were 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 pipette.

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

Illumination (target)
Diffuse light, light intensity of max. 1500 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

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

other: EL 10,50,100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

other: EL 10,50,100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

other: NOEL

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium (see Table 2) 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 7.3.

Purity 99.0%

Batch No. MKBV0900V

Expiry date 2021-11-25

Test concentrations 1.00 - 2.00 – 4.00 mg/L

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

Exposure phase 2017-08-03 to 2017-08-04

Reported statistics and error estimates:

Methods of evaluation
Because no effects were observed, no calculations were made.
All effect levels (EL10 / 50 / 100) were given based on the nominal loading of the test item.

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

Software
All 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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the WAFs with a nominal loading in the range of 6.25 to 100 mg/L of the test item, no effects on Daphnia magna were observed.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004), from 2017-07-31 to 2017-08-03, with the definitive exposure phase from 2017-08-01 to 2017-08-03.

The study was conducted under static conditions over a period of 48 hours with five Water Accommodated Fractions (WAF). Each WAF was prepared separately in the range of 6.25 to 100 mg/L.

The test itemis a yellowish wax (solid) and insoluble in water. The WAFs of the test item were prepared in Elendt M4 medium.

All WAFs and the control were visually clear throughout the exposure period, but showed a positive Tyndall effect. For the control the Tyndall effect was negative.

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

The loading levels of the test item and the controlwereanalytically 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 in fresh media (0 hours) were in the range of 3.01 to 8.36 mg C/L for the loading levels and < LOQ for the control.
In the old media (48 hours), the measured TOC concentrations were in the range of 2.82 to 7.42 mg C/L for the loading levels and < LOQ for the control.

Per definition of the WAF, all terms related to concentration levels have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to conentrations. Therefore, the EL_{10 / 50 / 100}-values were based on the nominal loading level.

The validity criteria of the test guideline were fulfilled.

 

Table1:   EL₁₀-, EL₅₀- and EL₁₀₀-Values

                  (based on the nominal loading level)

Effect levels		Test duration [hours]		Nominal test item loading level [mg/L]
EL10, 50, 100		24		> 100
		48		> 100