Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 June 2013-15 January 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test item (250 mg) was added to the surface of 2.5 lites of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium 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 hours and the mixture was 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 Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
An aliquot (1 litre) of te 100 mg/L loading rate WAF was inoculated with algal suspension (6.2 mL) to give the required test concentration of 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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in house
- Age of inoculum (at test initiation): NDA
- Method of cultivation: In-house cultures were maintained in the laboratory by the periodic replenishment of culture medium


ACCLIMATION
- Acclimation period: until the algal cell density was approximately 10E04 – 10E05 cells/mL
- Culturing media and conditions (same as test or not):Same as test
- Any deformed or abnormal cells observed: NDA

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

N/A

Hardness:

NDA

Test temperature:

23-25 °C

pH:

pH of culture medium = 7.6 at 0 h; 8.2 at 72 h

Dissolved oxygen:

NDA

Salinity:

NDA

Nominal and measured concentrations:

nominal concentrations: 0 or 100 mg/L

Details on test conditions:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water Elga Optima 15+ or Elga Purelab Option R-15 BP
- 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: Same medium used
- Intervals of water quality measurement: NDA


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24h
- Light intensity and quality: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)
- Salinity (for marine algae):N/A


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter ® Multisizer Particle Counter
- Chlorophyll measurement: None


TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A
- Justification for using less concentrations than requested by guideline: Limit test based on results of range finding study
- Range finding study
- Test concentrations: Nominal loading rates: 0, 10 or 100 mg/L

The results showed no significant effect on growth rate at 10 or 100 mg/L nominal loading rate.

Based on this information a concentration of 100 mg/L nominal loading rate WAF was selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (non concurrent)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: ErC50 (0-72h) = 1.2 mg/L; 95% confidence limits 1.0-1.3 mg/L

Reported statistics and error estimates:

Statistical analysis of the growth rate data and the yield data was carried out for the control and 100 mg/L loading rate WAF test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There was no statistically significant differences (p>=0.05) between the control and the 100 mg/L loading rate WAF test group and therefore the NOEL based on either growth rate or yield was 100 mg/L loading rate WAF.

See attached document for tables and figures.

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EL50 was > 100 mg/L loading rate WAF. The NOELR was 100 mg/L loading rate WAF.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, 'Freshwater Alga and Cyanobacteria, Growth Inhibition Test' referenced as Method C.3 of Commission Regulation (EC) 761/2009.

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, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 +/- 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group using a Coulter Multisizer Particle Counter.

Results

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Chemical analysis of the 100 mg/L loading rate WAF at 0 and 72 hours showed measured test concentrations of 0.00068 and 0.0038 mg/L respectively. Analysis of the control preparation at 72 hours showed a peak was present, the profile of which was consistent with that of the test item. Given that this was not observed at 0 hours it was considered that possible contamination had occurred during the test. As such it was considered appropriate to perform a further confirmatory test. Analysis of the control preparations from this confirmatory test also showed a peak profile similar to that of the test item or a possible trace contaminant which had a characteristic profile similar to that of the test item. Given that no adverse effects were observed in the control group, this was considered to have had no impact on the outcome of the test.

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.

Description of key information

One reliable study is available. In this study Pseudokirchneriella subcapitata were exposed to a Water Accommodated Fraction of the substance at a nominal loading rate of 100 mg/L under static conditions for 72 hours. The ErL50(72 h) value was > 100 mg/L loading rate WAF. The NOELR was 100 mg/L loading rate WAF. The substance is not  harmful to algae.

Additional information

One reliable study is available. In this study (Vryenhoef, 2014) performed under GLP according to OECD TG 201 and EC Method C.3, green alga Pseudokirchneriella subcapitata were exposed to the substance under static conditions for 72 hours at 24 °C. 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, Pseudokirchneriella subcapitata (6 replicates) was exposed to a WAF of the test material at a nominal loading rate of 100 mg/L. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group using a Coulter Multisizer Particle Counter. ErL50 (72 h) value was > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

The results of this study are supported by data available for the 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)) and their analogues is applicable based on the similarity in structure and physico-chemical properties. 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 therefore be used for read-across.

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

Two studies, investigating the toxicity to freshwater algae of Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234 -392 -1, CAS-No. 11138-60-6), were available.

During both static Algal Growth Inhibition tests no inhibiting effects on growth rate were observed within 72 hour test period at the tested concentrations. The first study (Häner, 2007) according to OECD 201 with Desmodesmus subspicatus determined a no-observed-effect concentration for growth rate (ErL0) of ≥ 100 mg/L and an ErL50 > 100 mg/L (WAF loading rate). In the second study (Craig, 2005) similar to OECD 201 with Pseudokirchnerella subcapitata an ErL50 and ErL10 > 1000 mg/L (WAF loading rate) was estimated. In addition, another algae toxicity test (Kuttler, 1998) with Desmodesmus subspicatus showed no toxicity of the test substance after 72 h at a WAF loading rate of 10000 mg/L. All results show no toxicities of Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS-No.11138-60-6) in the range of water solubility (< 0.3 mg/L). Therefore, the test substance is not harmful to algae.

In a static GLP study on 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS-No. 131459-39-7), (Mead, 1999) with Scenedesmus subspicatus according to OECD 201, no inhibition on growth rate and biomass was observed at the tested WAF loading rates (EL50 (72h) > 100 mg/L, NOELR (72h) ≥ 100 mg/L). An increase in growth rate and biomass (growth rate: 2 - 4%, biomass: 2 -20%) in comparison to the controls was determined. Chemical analysis showed measured test concentrations of 2 -18 mg/L. No toxic effects were observed within the water solubility under the conditions tested.