Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Nov 2012 - 12 Feb 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the solvent control and the 100% v/v solution (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 12 and 19 and of the old expired test preparations on Days 3, 7, 14 and 21. Samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test the test item was prepared using a preliminary solution in dimethylformamide. An amount of test item (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (200 µL) of the 100mg/10 mL solvent stock solution was dispersed in a final volume of 2 liters of Elendt M7 media with the aid of a magnetic stirrer for 10 minutes to give a 1.0 mg/L stock solution. After stirring, any undissolved test item was removed from the media by filtration using a 0.2 µm Gelman Acrocap filter to give the 100% v/v solution test concentration. Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 5, 12 and 19 (fresh media) and on days 3, 7, 14 and 21 (old media).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: in-house laboratory culture
- Age: 1st instar, less than 24 hours old
- Feeding during test: yes
- Food type: algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Amount: 2 to 7 µL algal suspension, 10 to 32 µL flake food suspension; Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
- Frequency: daily

ACCLIMATION
Culture conditions:
Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

210 - 288 mg/L as CaCO3

Test temperature:

20 - 21 °C

pH:

7.2 - 8.1

Dissolved oxygen:

5.9 - 9.2 mg O2/L

Nominal and measured concentrations:

100% v/v

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with plastic lids
- Material, size, headspace, fill volume: 150 mL glass flasks, filled with 100 mL test solution
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 3 times per week on Days 0, 3, 5, 7, 10, 12, 14, 17 and 19
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M7 medium) was used for the range-finding and definitive test and was the same as that used to maintain the stock animals.
Reconstituted water (Elendt M7 medium):

Solution Concentration of stock solution (mg/L)
(I) H3BO3 57190
(II) MnCl2 * 4H2O 7210
(III) LiCl 6120
(IV) RbCl 1420
(V) SrCl2 * 6H2O 3040
(VI) NaBr 320
(VII) Na2Mo04 * 2H2O 1260
(VIII) CuCl2 * 2H2O 335
(IX) ZnCl2 260
(X) CoCl2 * 6H2O 200
(XI) KI 65
(XII) Na2SeO3 43.8
(XIII) NH4VO3 11.5
(XIV) Na2EDTA * 2H2O 5000
FeS04 * 7H2O 1991


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 829 to 958 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): live and dead parental and filial Daphnia, and discarded unhatched eggs were counted daily

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: A range of concentrations showed no toxicity at the limit of solubility.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: parental generation

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: parental generation

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: length

Remarks on result:

other: parental generation

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 100 other: % v/v solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: parental generation

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 100 other: % v/v solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: parental generation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: % v/v solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: parental generation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: % v/v solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: parental generation

Reported statistics and error estimates:

For the estimation of the "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) the numbers of live young produced per adult over the duration of the test for the control, solvent control and 100% v/v solution test group were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and the Williams test for differences between treatment means when several dose levels are compared with a zero dose control (Williams 1971). Results from the control, solvent control and 100% v/v solution test group Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Effects on the Filial Generation (Fi):

Information on the effects of the test item on the Fi generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by the 100% v/v solution test group were in the same general condition as the young produced by the solvent controls over the duration of the test. Young were first produced in the solvent control test group on Day 9 of the test. There were no unhatched eggs or dead young observed in the test.

Verification of Test Concentrations:

Analysis of the freshly prepared 100% v/v solution on days 0, 5, 12 and 19, and of the old or expired media on days 3, 7, 14 and 21 showed measured concentrations of less than the limit of quantitation calculated to be 0.0039 mg/L. This does not infer that there was no test item in solution, just that the test item which was in solution, was at a concentration of less than 0.0039 mg/L. It was therefore considered justifiable to base the results on nominal concentrations.

Table: Summary of findings following the exposure of Daphnia magna for 21 days

Nominal Concentration (% v/v solution)	% Survival of P1	Number of Live Young		Number of Dead Young		Number of Unhatched Eggs
		Total	Per Female (cumulative)	Total	Per Female (cumulative)	Total	Per Female (cumulative)
Control	90	1298	144	0	0	0	0
Solvent Control	90	1366	152	0	0	0	0
100	80	1122	140	0	0	0	0

Description of key information

No effects in the range of water solubility

Additional information

One study investigating the chronic toxicity of propane-1,2,3-triyl 3,5,5-trimethylhexanoate (CAS 56554-53-1) is available (Parr, 2013). The long-term toxicity to Daphnia magna was investigated in a limit test set up according to OECD guideline 211 under semi-static conditions. The exposure period was 21 days. Due to the low water solubility of the test substance (< 0.05 mg/L), the test solution were prepared by dissolving the substance in dimethylformamide. The stock solution was dispersed in Elendt M4 media and undissolved test substance was removed by filtration. The test substance concentration was analytically verified (GC). No effect on parent survival or reproduction rate was observed. At test termination a NOEC (21 d): 100% v/v (nominal) was determined (EC50 (21 d) > 100% v/v (nominal)). Thus, chronic effects of propane-1,2,3-triyl 3,5,5-trimethylhexanoate (CAS 56554-53-1) on aquatic invertebrates are not expected up to the limit of water solubility of the substance.