3-{3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propoxy}-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate

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:

experimental study

Adequacy of study:

key study

Study period:

18 June 2013 - 11 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 or 100 mg/L nominal loading rate
- Sampling method:
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test item (1000 mg) was added to the surface of 10 L of reconstituted water to give the 100 mg/L loading rate. After the addition of the test item, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 ours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescoseal. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 100 mg/L loading rate WAF.
- Eluate: Reconstituted water
- Differential loading: N/A
- Controls: Reconstituted water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea (Daphnia)
- Strain: Daphnia magna
- Source: In house laboratory cultures
- Age at study initiation (mean and range, SD): 1st instar
- Weight at study initiation (mean and range, SD): NDA
- Length at study initiation (length definition, mean, range and SD): NDA
- Method of breeding: parthenogenises
- Feeding during test: The daphnids received no food during exposure

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

not applicable

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

The temperature was maintained at 22-25°C throughout the duration of the test.

pH:

Control: 7.8-8.2
Test item: 7.9-8.1

Dissolved oxygen:

The dissolved oxygen concentration was approximately air-saturation value

Salinity:

NDA

Nominal and measured concentrations:

nominal concentrations: 0 or 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL covered jar
- Material: glass
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- 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): N/A
- Biomass loading rate:NDA


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Total organic carbon: NDA
- Particulate matter: NDA
- Metals:NDA
- Pesticides:NDA
- Chlorine:NDA
- Alkalinity:NDA
- Ca/mg ratio:NDA
- Conductivity:NDA
- Culture medium different from test medium: No difference
- Intervals of water quality measurement: N/A


OTHER TEST CONDITIONS
- Adjustment of pH: Using NaOH or HCl were necessary
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods
- Light intensity: NDA


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (non-concurrent)

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: none
- Observations on body length and weight: N/A
- Other biological observations: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: NDA
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: NDA
- Effect concentrations exceeding solubility of substance in test medium: N/A

Results with reference substance (positive control):

- Results with reference substance valid? Yes
The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.71 mg/L.

The No Observed Effect Concentration after 48 hours was 0.32 mg/L. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

Time (h) 24
EC50 (mg/L) = 1.0
95% Confidence limits (mg/L) = 0.91-1.2

Time (h) 48
EC50 (mg/L) = 0.71
95% Confidence limits (mg/L) = 0.65-0.76

Validity criteria fulfilled:

yes

Conclusions:

The 48-h EL50 based on nominal loading rates was > 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, 'Daphnia sp., Acute Immobilisation Test' referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at single nominal loading rate of 100 mg/L for 48 hours at a temperature of 22 °C to 25 °C under static test conditions. The number of immobilized Daphnia and any adverse reaction to exposure were recorded after 24 and 48 hours.

Results

Exposure of Daphnia magna to the test item gave EL*50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Analysis of the 100 mg/L loading rate WAF at 0 and 48 hours showed that measured concentrations of 0.0213 and 0.00463 mg/L were obtained respectively. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

*EL = Effective Loading Rate

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04 to 06 October 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accomodated Fraction (WAF) method: A nominal 1000 mg/l stock solution was produced by WAF method. The stock solution was
prepared by stirring the test material in dilution water for 20 hours, followed by settling for 4 hours. The aqueous layer was then removed for testing. The test solutions were prepared by adding the 1000 mg/l stock solution to dilution water in appropriate quantities to obtain the desired
concentrations for the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: Reading University
- Age at study initiation (mean and range, SD): Less than 24 hours

BREEDING
- Type and amount of food: A suspension of Chlorella vulgaris, at a rate of 1 mg organic carbon per litre of culture water
- Feeding frequency: Each working day

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

156 mg/L CaCO3

Test temperature:

0 hours, each concentration: 21°C
48 hours, each concentration: 21°C

pH:

0 hours, each concentration: 7.5
48 hours, each concentration: 7.3

Dissolved oxygen:

0 hours, each concentration: 84 % ASV
48 hours, each concentration: 90-93% ASV

Nominal and measured concentrations:

Nominal concentrations: 0, 100, 220, 450 and 1000 mg/L (see "Remarks on results")

Details on test conditions:

TEST SYSTEM
- Test vessel: 50ml crystallising dishes
- Type (delete if not applicable): Closed with a transparent perspex sheet, it was placed over the tops of the crystallising dishes.
- Material, size, headspace, fill volume: 25ml of the test sample concentration
- 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: Dilution water of typical hardness 156 mg/l CaCO3, adjusted to 20 ± 1°C, aerated until the dissolved oxygen
concentration had reached a minimum of 60% air saturation and the pH stabilised between 7.0 and 8.0.
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH value, dissolved oxygen and temperature were measured immediately prior to initiating the test for
the dilution water, and at the end of the test for the control and test concentrations.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The number of immobilised Daphnia were recorded after 24 and 48 hour
exposure periods.

TEST CONCENTRATIONS
- Range finding study
- Results used to determine the conditions for the definitive study: A preliminary study had identified EC50 (48h) > 1000 mg/l.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: At 24 hours some daphnids were held at the meniscus at all test concentrations, but continued to show normal
swimming behaviour. At 48 hours those daphnids that were held at the meniscus showed slower swimming behaviour.

At a WAF loading rate of 1000 mg/l an immobilisation of 45 % could be observed. The EL50 (48h) was > 1000 mg/l. This indicate, that the EC50 is > water solubility of the test substance.

Because of the serial dilution from the highest concentration (1000 mg/L), only the 1000 mg/L test concentration was valid and could be used for evaluation of toxicity of the test substance. Without chemical analytics of the diluted concentrations, no information about the exact test concentration could be stated.

After 24 h some daphnids were held at the miniscus at all test concentrations. As these daphnids showed no immobilisation after 48 h, no further effect on daphnids would be expected.

Table 1: Cumulative percent immobilisation of Daphnia magna after 24 and 48 hours

Concentration [mg/l]	Number immobilised		% immobilisation
	24 h	48 h	24 h	48 h
0	0	0	0	0
100	0	1	0	5
220	0	0	0	0
450	0	0	0	0
1000	0	9	0	45

 

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1990

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Original report not available and documentation insufficient for assessment

Principles of method if other than guideline:

48h-Daphnia magna test, no guideline stated.

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Analytical monitoring:

not specified

Vehicle:

not specified

Test organisms (species):

Daphnia magna

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

15.86 mg/L

Basis for effect:

mobility

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: daphnids were agglutinated to test material swimming on the surface of dilution water.

As only raw data are available, the documentation is insufficient for assessment.

The EC50 (48h) was stated as 15.86 mg/L. Due to the low water solubility of the test substance, the EC50 is > water solubility of the substance (< 0.3 mg/L).

Table 1: Number of immobilised Daphnia magnaafter 48 hours.

Concentration mg/l	Number of immobile daphnids					% immobilisation
	A	B	C	D	Total/20
0	0	0	0	0	0	0
4.8	0	0	0	0	0	0
6.2	0	0	2	1	3	15
8	0	1	0	0	1	5
10	3	3	3	2	11	55
13	3	2	1	2	8	40
17	3	3	3	2	11	55
22	3	3	2	3	11	55
29	4	5	4	3	16	80
37	5	4	4	4	17	85

 

 

 

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 January to 19 January 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fraction (WAF): This WAF was prepared by moderately stirring 100 mg test substance per liter of medium overnight
followed by filtration (MILLIPORE AP15 glass fiber filter).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: Straus
- Length at study initiation (length definition, mean, range and SD): < 2mm
- Method of breeding: Parental and young Daphnia were held in 40L glass aquaria.

BREEDING
- Breeding conditions (same as test or not): Reconstituted water (ISO 6341) was the same as test, prepared with distilled water. The medium in the
breeding system was continuously aerated in comparison to the test.
- Type and amount of food: 15 g sera micropan (sera GmbH, Heinsberg) homogenized in 1L deionized water
- Feeding frequency: Once a day, except weekends

ACCLIMATION
- Acclimation period: Daphnia, which had been selected in size, were acclimatized to the reconstituted water for 4 hours before introduction into
the test media.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

20.0 ± 0.5 °C

pH:

control: 7.3 at 0h, 7.7 at 48h
100 mg/l: 7.5 at 0h, 7.7 at 48h

Dissolved oxygen:

control: 6.2 mg/l at 0h, 6.0 mg/l at 48h
100 mg/l: 6.0 mg/l at 0h, 5.9 mg/l at 48h

Nominal and measured concentrations:

nominal: 100 mg/l (WAF)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 100 mL flasks, all-glass, with 50 mL of test medium
- Aeration: no
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO 6341), prepared with distilled water. The ratio of Ca:Mg ions is 4:1 and of Na:K ions is 10:1.
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1
- Culture medium different from test medium: No, but test medium was not aerated as the culture medium.
- Intervals of water quality measurement: Temperature determined in a control flask at the start and at the end of the test. Oxygen and pH were
determined in all vessels prior to the addition of Daphnia to the test medium and at the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 h a day, supplied by overhead white fluorescent tubes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of immobile Daphnia were made after 24 and 48 h of
exposure

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

The test substance show no acute toxicity for daphnids under test conditions.

Table 1: Immobilisation of Daphnia magna after 24 and 48 hours

Nominal concentration (mg/l)	Code	Number of individuals per test vessel	Immobile Daphnia (No./vessel) 24h	Immobile Daphnia (No./vessel) 48h	% immobile Daphnia after 24h of exposure	% immobile Daphnia after 48h of exposure
Control 0	A B C	20 20 20	0 1 0	0 1 0	0 5 0	0 5 0
100	A B	20 20	0 1	1 1	0 5	5 5

 

Reference 5

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

06 April - 09 April 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fraction (WAF): The weighted sample of 10000 mg/l was mixed up to the equilibrium concentration. A beaker was
appointed with a glass tube, by what the aqueous phase could be take off witout contamination of the unsolved film of the test substance. Before the
take off of the aqueous phase the preparation was left for 1 hour for complete phase separation.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Waterflea
- Strain: Klon 5
- Source: Umweltbundesamt

HOUSING
- Acclimation conditions (same as test or not): not, in a non conditioned room, light/ dark = 14h/10h, light: GRO-Lux, Aqua-Star
- Type and amount of food: Scenedesmus subspicatus
- Feeding frequency: 2 - 3 times daily

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

2.5 mmol/L

Test temperature:

19.2 - 20.5 °C

pH:

6.8 - 7.1

Dissolved oxygen:

≥ 96%

Nominal and measured concentrations:

nominal: 10000 mg/L (WAF)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 50 ml beaker filled with 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
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4 : 1


OTHER TEST CONDITIONS
- Photoperiod: 14 h light, 10 h dark (Gro-Lux)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobile daphnids; daphnids, which are not immobile, but attached on the
meniscus; daphnids which are arranged on the bottom and which are not immobile; all parameters were observed once to thrice daily, at least after
0h, 24h and 48h

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: No effects within the range of water solubility of the test substance were observed.

Details on results:

- Behavioural abnormalities: Some daphnids at 10000 mg/l were attached at the meniscus after 24 respectively 48 h. The activity of the daphnids at
10000 mg/l was in comparison to the control reduced at the end of the test. Immobilisation could not be observed. The amount of ecdysis was 100%
in comparison to the control.

No immobilisation of the daphnids could be observed in the control and the test concentration (10000 mg/l WAF) at 24h and 48h under conditions tested.

Some daphnids at 10000 mg/l were attached at the meniscus after 24 respectively 48 h. The daphnids were hit back in the water with a pipette. The activity of the daphnids at 10000 mg/l was in comparison to the control reduced at the end of the test. Immobilisation could not be observed. The amount of ecdysis was 100% in comparison to the control.

Overall no effect within the range of water solubility of the test substance was observed under conditions tested.

Reference 6

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accommodated fraction (WAF) were prepared by shaking the product in the dilution water in a closed bottle for 20 - 24 hours in the
dark at 20 °C. After standing for 1 - 2 hours the WAF was drawn from the centre of the water column. Separate WAFs were prepared for each
concentration.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: A
- Source: Sheffield University

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 +/- 0.5 °C

pH:

control: 7.9-7.9 at the start, 8.0-7.9 at the end
highest concentration (1000 mg/l): 7.9-7.8 at the start, 7.9 at the end

Dissolved oxygen:

End control: 8.4 ppm
End highest concentration: 7.8-8.2 ppm

Nominal and measured concentrations:

nominal: 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- No. of organisms per vessel: 5 - 10
- No. of vessels per concentration (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7
- Culture medium different from test medium: yes, stock cultured in 5 µm filtered natural lake water with addition of Elendt M7 (Elendt 1990).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

590 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: EC50 calculation not correct, depending on results: EC50 > 1000 mg/L

Results with reference substance (positive control):

EC50 (24h) = 1.53 mg/l

The Water Accommodated Fraction (WAF) loading rates up to 100 mg/L showed no toxicity to Daphnia magna. 100 mg/L demonstrated a limit value, as this is the highest value used for classification of a substance. Therefore can be stated, that no toxicity in the range of water solubility would be expected.

The immobilisation observed at 10 mg/L has been disregarded since less effect was observed at two higher concentrations.

Table 1: Number of immobilised Daphnia magna after 24 and 48 hours incubation.

Concentration	Number of animals	Immobilised 24h	Immobilised 48h
Control	40	1	1
10 mg/l	40	9	26
32 mg/l	20	0	1
100 mg/l	40	1	5
320 mg/l	19	2	10
1000 mg/l	40	21	32

 

Reference 7

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2003/01/13 - 2003/01/16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: OECD 202 "Daphnia sp, Acute Immobilisation Test and Reproduction Test" (1984)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, date of inspection 28 February 2000

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water samples were taken from the control (replicates R1-R2 pooled) and the 1000 mg/L loading rate WAF test group (replicates R1-R2 and R3-R4 pooled) at 0 and 48 h.
- Sample storage conditions before analysis: Duplicate samples of the 1000 mg/L loading rate WAF test groups at 0 h were taken and stored frozen (approx -20°C) for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of test material (10 g) was added to the surface of 10 L of reconstituted water to give a 1000 mg/L loading rate. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. This was stirred for 23 h. After stirring the mixture was allowed to stand for 1 h. The aqueous phase was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1000 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test material to be present.
- Controls: Reconstituted water
- Evidence of undissolved material: no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: 1st instar of an in-house laboratory culture
- Age at study initiation (mean and range, SD): < 24 h
- Method of breeding: Adult Daphnia were maintained in polypropylene vessels containing approx. 2 L of reconstituted water in a temperature controlled room at 21 °C. The lighting cycle was 16 h light and 8 h dark. Each culture was fed daily with a suspension of algae (Chlorella sp.). Reproduction was by parthenogenesis.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

21.8 °C (0 h), 20.6 °C (24 h), 20.8 °C (48 h)

pH:

7.6-7.9

Dissolved oxygen:

90-98%

Nominal and measured concentrations:

Nominal concentrations: control - 1000 mg/L
Measured concentrations: Less than the limit of quantification (0.032 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars containing approx. 200 mL of test preparation
- Type: covered to reduce evaporation
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water


OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark with 20 min dawn and dusk transition periods


EFFECT PARAMETERS MEASURED: immobilisation at 24 and 48 h


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10 - 100 - 1000 mg/L (WAFs)
- Results used to determine the conditions for the definitive study: No immobilisation was observed

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: no mortality was observed in the controls
- Effect concentrations exceeding solubility of substance in test medium: The nominal concentration of 1000 mg/L exceeded the water solubility of the test item.

No effects were observed in the range of water solubility at a nominal concentration of 1000 mg/L (WAF). Analysis of the test preparations at 0 and 48 h showed the measured test concentrations to be less than the limit of quantification of the analytical method (< 0.032 mg/L) (Table 1).

Table 1: Chemical analysis of the test loading rates

Samples	Nominal loading rate [mg/L]	Concentration found [mg/L]
0 h	control	< LOQ
	1000 R1-R2	< LOQ
	1000 R3-R4	< LOQ
48 h	control	< LOQ
	1000 R1-R2	< LOQ
	1000 R3-R4	< LOQ

Reference 8

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: none
- Observations on body length and weight: N/A
- Other biological observations: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: NDA
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: NDA
- Effect concentrations exceeding solubility of substance in test medium: N/A

Results with reference substance (positive control):

- Results with reference substance valid? Yes
The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.71 mg/L.

The No Observed Effect Concentration after 48 hours was 0.32 mg/L. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

Time (h) 24
EC50 (mg/L) = 1.0
95% Confidence limits (mg/L) = 0.91-1.2

Time (h) 48
EC50 (mg/L) = 0.71
95% Confidence limits (mg/L) = 0.65-0.76

Validity criteria fulfilled:

yes

Conclusions:

The 48-h EL50 based on nominal loading rates was > 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, 'Daphnia sp., Acute Immobilisation Test' referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at single nominal loading rate of 100 mg/L for 48 hours at a temperature of 22 °C to 25 °C under static test conditions. The number of immobilized Daphnia and any adverse reaction to exposure were recorded after 24 and 48 hours.

Results

Exposure of Daphnia magna to the test item gave EL*50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Analysis of the 100 mg/L loading rate WAF at 0 and 48 hours showed that measured concentrations of 0.0213 and 0.00463 mg/L were obtained respectively. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

*EL = Effective Loading Rate

Reference 9

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: At 24 hours some daphnids were held at the meniscus at all test concentrations, but continued to show normal
swimming behaviour. At 48 hours those daphnids that were held at the meniscus showed slower swimming behaviour.

At a WAF loading rate of 1000 mg/l an immobilisation of 45 % could be observed. The EL50 (48h) was > 1000 mg/l. This indicate, that the EC50 is > water solubility of the test substance.

Because of the serial dilution from the highest concentration (1000 mg/L), only the 1000 mg/L test concentration was valid and could be used for evaluation of toxicity of the test substance. Without chemical analytics of the diluted concentrations, no information about the exact test concentration could be stated.

After 24 h some daphnids were held at the miniscus at all test concentrations. As these daphnids showed no immobilisation after 48 h, no further effect on daphnids would be expected.

Table 1: Cumulative percent immobilisation of Daphnia magna after 24 and 48 hours

Concentration [mg/l]	Number immobilised		% immobilisation
	24 h	48 h	24 h	48 h
0	0	0	0	0
100	0	1	0	5
220	0	0	0	0
450	0	0	0	0
1000	0	9	0	45

 

Reference 10

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

15.86 mg/L

Basis for effect:

mobility

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: daphnids were agglutinated to test material swimming on the surface of dilution water.

As only raw data are available, the documentation is insufficient for assessment.

The EC50 (48h) was stated as 15.86 mg/L. Due to the low water solubility of the test substance, the EC50 is > water solubility of the substance (< 0.3 mg/L).

Table 1: Number of immobilised Daphnia magnaafter 48 hours.

Concentration mg/l	Number of immobile daphnids					% immobilisation
	A	B	C	D	Total/20
0	0	0	0	0	0	0
4.8	0	0	0	0	0	0
6.2	0	0	2	1	3	15
8	0	1	0	0	1	5
10	3	3	3	2	11	55
13	3	2	1	2	8	40
17	3	3	3	2	11	55
22	3	3	2	3	11	55
29	4	5	4	3	16	80
37	5	4	4	4	17	85

 

 

 

Reference 11

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

The test substance show no acute toxicity for daphnids under test conditions.

Table 1: Immobilisation of Daphnia magna after 24 and 48 hours

Nominal concentration (mg/l)	Code	Number of individuals per test vessel	Immobile Daphnia (No./vessel) 24h	Immobile Daphnia (No./vessel) 48h	% immobile Daphnia after 24h of exposure	% immobile Daphnia after 48h of exposure
Control 0	A B C	20 20 20	0 1 0	0 1 0	0 5 0	0 5 0
100	A B	20 20	0 1	1 1	0 5	5 5

 

Reference 12

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: No effects within the range of water solubility of the test substance were observed.

Details on results:

- Behavioural abnormalities: Some daphnids at 10000 mg/l were attached at the meniscus after 24 respectively 48 h. The activity of the daphnids at
10000 mg/l was in comparison to the control reduced at the end of the test. Immobilisation could not be observed. The amount of ecdysis was 100%
in comparison to the control.

No immobilisation of the daphnids could be observed in the control and the test concentration (10000 mg/l WAF) at 24h and 48h under conditions tested.

Some daphnids at 10000 mg/l were attached at the meniscus after 24 respectively 48 h. The daphnids were hit back in the water with a pipette. The activity of the daphnids at 10000 mg/l was in comparison to the control reduced at the end of the test. Immobilisation could not be observed. The amount of ecdysis was 100% in comparison to the control.

Overall no effect within the range of water solubility of the test substance was observed under conditions tested.

Reference 13

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

read-across source

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

590 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: EC50 calculation not correct, depending on results: EC50 > 1000 mg/L

Results with reference substance (positive control):

EC50 (24h) = 1.53 mg/l

The Water Accommodated Fraction (WAF) loading rates up to 100 mg/L showed no toxicity to Daphnia magna. 100 mg/L demonstrated a limit value, as this is the highest value used for classification of a substance. Therefore can be stated, that no toxicity in the range of water solubility would be expected.

The immobilisation observed at 10 mg/L has been disregarded since less effect was observed at two higher concentrations.

Table 1: Number of immobilised Daphnia magna after 24 and 48 hours incubation.

Concentration	Number of animals	Immobilised 24h	Immobilised 48h
Control	40	1	1
10 mg/l	40	9	26
32 mg/l	20	0	1
100 mg/l	40	1	5
320 mg/l	19	2	10
1000 mg/l	40	21	32

 

Reference 14

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: no mortality was observed in the controls
- Effect concentrations exceeding solubility of substance in test medium: The nominal concentration of 1000 mg/L exceeded the water solubility of the test item.

No effects were observed in the range of water solubility at a nominal concentration of 1000 mg/L (WAF). Analysis of the test preparations at 0 and 48 h showed the measured test concentrations to be less than the limit of quantification of the analytical method (< 0.032 mg/L) (Table 1).

Table 1: Chemical analysis of the test loading rates

Samples	Nominal loading rate [mg/L]	Concentration found [mg/L]
0 h	control	< LOQ
	1000 R1-R2	< LOQ
	1000 R3-R4	< LOQ
48 h	control	< LOQ
	1000 R1-R2	< LOQ
	1000 R3-R4	< LOQ

Description of key information

One reliable study is available on an analogue of the registered substance. In this study Daphnia magna were exposed to a Water Accommodated Fraction of the substance at a nominal loading rate of 100 mg/L under static conditions for 48 hours. The EL50(48 h) value was > 100 mg/L loading rate WAF. The substance is not acutely harmful to aquatic invertebrates.

Additional information

One reliable study is available on an analogue of the registered substance. In this study (Harris, 2013) performed under GLP according to OECD TG 202 and EC Method C.2, Daphnia magna were exposed to the substance under static conditions for 48 hours at a temperature of 22 °C to 25 °C. Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, twenty daphnids ( 4 replicates of 5 animals) were exposed to a WAF of the test material at a nominal loading rate of 100 mg/L. The number of immobilized Daphnia and any adverse reaction to exposure were recorded after 24 and 48 hours. The EL50 (48 h) value was > 100 mg/L loading rate WAF.

The results of this study are supported by data available for read-across substances fatty acid polyols (Fatty acids, C5-9, esters with pentaerythritol (EC 270-290-3, CAS 68424-30-6) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)). The substance with the CAS No. 131459-39-7 is a structural analogue of one of the read-across substances (CAS No. 68424-30-6) and can be used for read-across also.

The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Four studies, investigating the short-term toxicity to aquatic invertebrates of Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS-No. 11138-60-6), were available. These studies were conducted with Daphnia magna and according to OECD 202. All measured results from 48 -hour toxicity studies indicate the test substance is of low toxicity to Daphnia magna.

Häner (2007) determined EL 50 (48h) > 100 mg/L (WAF loading rate) and Kuttler (1998) observed no immobilisation at 10000 mg/L (EL50 > 10000 mg/L, WAF loading rate). Both other studies (Källquist 1995 and Craig 2005) calculated EL50 > 1000 mg/L (WAF loading rate) with an immobilisation under 50% at 1000 mg/L (Källquist: 32%; Craig: 45%). In addition, a 15-day reproduction test (Low, 1996, entered in 6.1.4) showed no immobile daphnia during the whole test duration at a WAF loading rate of 2570 mg/L. One other available test result (EC50 = 15.86 mg/L) should be evaluated with caution because the original report was not available and therefore the documentation was insufficient for assessment (Draguez, 1991). Nonetheless, all data show toxicities of Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS-No. 11138-60-6) far in excess of natural occurring concentrations (water solubility < 0.3 mg/L). Therefore, the substance is not harmful to aquatic invertebrates.

In an addition study on 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS-No. 131459-39-7). In this GLP-study withDaphnia magnaaccording to OECD 202 (1984) no effect on immobilisation was observed at the tested WAF loading rates (Sewell and McKenzie, 2003). The observed EL50 (48h) > 1000 mg/L with a NOELR ≥ 1000 mg/L. Chemical analysis of the WAF loading rates at 0 and 48 h showed measured test concentrations less than or at the limit of quantification of the analytical method (< 0.032 mg/L).