Isodecyl 3,5,5-trimethylhexanoate

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:

08 August to 11 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

23 March 2006 (corrected 28 July 2011)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test Substance
Test Substance Name: Isodecyl 3,5,5-trimethylhexanoate
CAS Number: 59231-35-5
Purity: 100%
Receipt Date: 01 March 2017
Storage on Receipt: Room Temperature (15 – 30°C)
Disposal: None hazardous
Hazard: None hazardous

Test substance details were supplied by the Sponsor. A Certificate of Analysis was not available for the compound; therefore, the client confirmed that the substance was a UVCB, a mixture if isomers and therefore assumed to be 100%. Emails included in study data.

Analytical monitoring:

yes

Vehicle:

no

Remarks:

WAF

Details on test solutions:

- Preliminary Solubility Trial:
Initial solubility work was conducted under Smithers Viscient study number 3201861 (acute toxicity to daphnia magna) and indicated that a WAF (water accommodated fraction) preparation method would be most appropriate based on the nature of the compound and the low water solubility.

- Appearance of Test Media:
The appearance, colour and behaviour of the test substance in the test media were recorded at the start and end of the test.

- Media Preparation Work:
After the range-finder test was conducted without chemistry analysis a media trial was conducted to assess test substance stability in the test system. Media were prepared in uplicate, using a WAF preparation method. New media samples were taken at 0 hours, media were then stored under test conditions for ca 72 hours. Old media samples were then taken to assess stability.

- Range-finding Test:
The range-finding test was conducted with a control and a nominal test substance concentration of 100 mg/L LR WAF, only one concentration was used to confirm results from the initial experiment.

- Definitive Limit Test:
Based on the results of the range-finding test, the definitive limit test was conducted with a control and nominal test concentration of 100 mg/L LR WAF.
At the start of the test, an amount of test substance (99.98 mg) was added to 1000 mL of EC medium. The preparation was stirred for ca 24 hours (with a vortex no deeper than 1 cm), media were then allowed to settle for ca 1 hour The final media was then
syphoned from the mid-section (aqueous phase) of the vessel. A control treatment was prepared by adding EC medium only to the control vessels. The appearance, colour and behaviour of the test substance in the test media were recorded at the start and end of the test.

Test organisms (species):

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

Details on test organisms:

The test organism, Pseudokirchneriella subcapitata (Strain 278/4), was originally obtained from the Culture Collection of Algae and Protozoa (CCAP) and is a representative species of the freshwater aquatic phytoplankton.
Prior to testing, duplicate starter cultures were prepared and incubated under test conditions (as below) to obtain sufficient algal cells in exponential growth and to achieve a starting algae cell density of 1 × 104 cells/mL.

- Culturing of Pseudokirchneriella subcapitata and Water Quality:
- Introduction:
Semi-axenic cultures of Pseudokirchneriella subcapitata were maintained in liquid culture. These were used to inoculate starter liquid cultures, which in turn were used to inoculate test vessels containing control and test media at the start of each test.

- Source:
The axenic strain of Pseudokirchneriella subcapitata (CCAP 278/4) was obtained from a concentrated liquid slope culture from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Oban, UK. On receipt from the CCAP, the slope culture was stored in the fridge at 2-8°C. A typical shelf life for each slope was 8 months, after which the slopes were discarded.

- Preparation of Algal Medium:
To prepare the algal culture medium, stock solutions containing the various nutrients were prepared with reverse osmosis (RO) water. Aliquots of each of the stock solutions were then added to RO water. The algal culture medium was autoclaved. Once cooled, an aliquot of a stock solution of NaHCO3 was added to the algal culture medium through a sterile filter (0.2 µm filter pore size). The following tables present the nominal nutrient concentrations used in the preparation of the algal (EC) culture medium.

Nutrient Final concentration (mg/L) Nutrient Final concentration (mg/L)
NH4Cl 15 FeCl3.6H2O 0.08
MgCl2.6H2O 12 Na2EDTA.2H2O 0.1
CaCl2.2H2O 18 NaHCO3 50
MgSO4.7H2O 15 ZnCl2 0.003
KH2PO4 1.6 CoCl2.6H2O 0.0015
H3BO3 0.185 Na2MoO4.2H2O 0.007
MnCl2.4H2O 0.415 CuCl2.2H2O 1 × 10-5
* FeCl3.6H2O is quoted in the OECD guideline to be added to the stock solution at a rate of 0.064 mg/L however this is a contradiction to the elemental composition which states that the media should contain 0.017 mg/L of Fe. In order to achieve an elemental composition of 0.017 mg/L of Fe in the test media, 80 mg/L of FeCl3.6H2O must be added to the stock solution.

- Inoculation of Test Cultures:
Prior to each test, a sub-sample (ca 200 µL) of a current liquid slope culture was added to two starter cultures (conical flasks) containing 100 mL EC medium. Each culture was incubated (under the same conditions as used in the test) for at least 72 hours prior to the start of each test. Following incubation, the cell density of a single starter culture was established using a haemocytometer and microscope. An inoculum volume was then calculated and added to each test vessel, to achieve a starting alga cell density of 1 × 104 cells/mL. All flasks for a particular test were inoculated from a single starter culture.
Regular tests are conducted using a reference toxicant to ensure that cultures are of the highest quality and sensitivity. The results from the latest reference test are presented in the Annex.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not reported.

Test temperature:

22.0-23.1 °C

pH:

Without Algae: 7.71 - 7.83
With Algae: 9.30 - 9.61

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Range-finding test: 100 mg/l loading rate
Definitive test: 100 mg/l leading rate

Details on test conditions:

- Test Vessel Preparation:
The test vessels were sterile autoclaved glass 250 mL Erlenmeyer (conical) flasks, into which 100 mL of the appropriate control or test media was added. Sterile foam bungs were used to cover the top of the vessels.
Each test and control vessel was inoculated with sufficient Pseudokirchneriella subcapitata cells to achieve a starting algae cell concentration of 1 × 104 cells/mL. An additional inoculated vessel was prepared for the control and each test concentration for initial water quality analysis.

- Test Vessel Sampling:
At approximately 24-hour intervals after the start of the incubation period, pre-determined volumes of test media (1.0 mL at 24 hours and 0.5 mL at 48 and 72 hours) were removed from each incubated test vessel, and transferred to individually identified cell counting vials. The contents of each vial were diluted to a 10 mL final volume with an electrolyte solution. The cell density of the vial contents was then determined using a particle counter (Z2 Coulter Counter®).

- Environmental Conditions:
All flasks were loosely-capped and incubated in a temperature controlled laboratory under a light bank. The vessels were placed on an orbital shaker (ca 100 rpm) under conditions of constant light (4440 to 8880 Lux), using florescent tube lights, emitting
light across the visible portion of the spectrum (400 - 700 nm). The light intensity within the test area was monitored at the start and end of the test.
At the start of the test, the pH of freshly prepared test media was determined. The pH in each test vessel was also determined at the end of the test.
The laboratory temperature was set within the range 21 to 24°C and maintained within ± 2°C for the duration of the test. The temperature was recorded continuously using a digital thermometer.

- Data Interpretation:
The algal cell concentration data were evaluated using three approaches:

(a) Comparison of average specific growth rates (µ)
The average specific growth rate (µ) for test algae in each test vessel was calculated using the equation:

µ = (Loge Nm - Loge N0) / (tn - t1)

where:
N0 = initial measured cell concentration at time t0 (cells/mL)
N1 = measured cell concentration at time t1 (cells/mL)
Nn = measured cell concentration at time tn (cells/mL)
N2 = measured cell concentration at time t2 (cells/mL)
t1 = time of first measurement after the beginning of the test (h)
t2 = time of second measurement after the beginning of the test (h)
tn = time of nth measurement after the beginning of the test (h)



The percentage inhibition (IA%) of cell growth was calculated using the following
equation:

I (%) = ((Ac - As) / Ac) x 100

where:
Ac = mean values of A or µ determined for the control treatment
As = mean values for A or µ determined for each treatment concentration (As)
Section-by-section percentage inhibition in growth rate and section-by-section growth
rates for control vessels are also reported.

(b) Final Yield (Y)
Yield (in terms of biomass) was determined using the following formula:
Yield = final time point biomass (cells/mL) – initial (inoculum) biomass (cells/mL)
Yields were calculated for each test vessel and a mean yield per treatment was
determined.

- Statistical Analysis:
Statistical analysis was not performed as no toxicity was observed during the test.

- Chemical Analysis:
Analytical chemistry was performed during the definitive phase, however, the analytical batch run at 0 hours failed to meet acceptance criteria. Therefore, a decision was made alongside the client to not report the analytical data, as over the course of
the study analytical method criteria was not met, this was considered to be due to issues with the compound and method (discussed further in Test Substance Properties and Related Issues section of the report). As analytical work was not reliable results were based on nominal loading rate concentrations (mg/L LR WAF).

- Mathematical Rounding Statement:
For presentation purposes, some numerical data may have been rounded, therefore manual recalculation may result in slightly different values to those presented in the results section.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Range-finder test:
Key results from the range-finding test are summarised in table 1.
At the start of the test, the control and test concentration were observed to be colourless solutions. At 72 hours, the control and test concentration were observed to be green, homogenous hazy dispersions of algal cells.
Chemical analysis was not conducted during the range-finding test.
The results of the range-finding test suggested that the 72-hour EC50 values for yield, specific growth rate and area under the curve (biomass) were likely to be greater than 100 mg/L LR WAF, based on nominal loading rate test substance concentration.

- Definitive Test:
- Test Substance Properties and Related Issues:
Chemistry analysis results did not meet acceptance during the definitive test. The reasons for this, and the decision to not report analytical results are discussed below. The test substance Isodecyl 3,5,5-trimethylhexanoate, is a moderately volatile compound with limited water solubility. As such, it presents as a good candidate for analysis by gas chromatography/mass spectrometry (GC/MS). This technique relies on initial separation on a chromatography column, mainly by the mechanism of differences in boiling point/volatility. Further separation and selectivity is provided by mass analysis, which also provides quantitative information, via the peak response for the analyte. Normally, GC/MS is considered to be a highly selective technique. However, many of the ions produced by the test substance are quite generic and might reasonably be expected to be produced by endogenous materials in the samples (e.g. lipids, fatty alcohols etc.).

Although the test substance, Isodecyl 3,5,5-trimethylhexanoate is a single chemical species, the isodecyl moiety within the molecule consists of a series of isomeric homologues. As each isomeric homologue has a slightly different boiling point, when the test article is analysed by GC/MS, a series of partially separated peaks, eluting as a broad “hump” is observed. This transforms the total peak response into a low, wide peak, with less well defined start and end points. This is at odds with the normal expectation in chromatographic techniques of measuring a single, sharp peak.

This has several impacts. First, by reducing the effective peak height there is a worsening of the signal to noise ratio, which is used to establish the limit of quantification (LOQ) of the analytical method. Therefore, the absolute sensitivity of the method is reduced.

Second, the start and end points of the peak are less well defined, which reduces both the accuracy and the precision of the measurement of peak areas (and hence the concentration).

Third, since the peak elutes across a wider time window than a sharp, narrow peak, it is much more difficult to say with certainty that there are no interferences extracted from the matrix lying beneath the analyte peak. Interferences producing the same ions
as the test substance would not be excluded from the peak response, which will reduce the accuracy of the measurement.

Finally, Isodecyl 3,5,5-trimethylhexanoate was found to be more volatile than expected during concentration of the sample extract (performed to improve the LOQ). Although steps were taken to control and reduce these loses, it may have further contributed to the difficulties with the methodology.

Smithers Viscient Study Number 3201857 found the solubility of the compound to be less than the LOQ, the solubility was therefore considered to be < 0.01 mg/L.

An analytical method was developed and validated for use with the compound, however, batches ran throughout the duration of the study showed several issues. Achieved solubility were calculated as below the LOQ, meaning results could not be reported, other batches did not meet method criteria and therefore could also not be reported. The decision was made alongside the client to base results on nominal loading rate concentrations. It was considered that solubility of the compound was below the LOQ.

Media trial results are summarised below.

Results at 0 hours were much higher than expected, results at 72 hours showed no concentration; the analytical work was not conclusive. Therefore, the Definitive phase was run semi-static to maintain concentrations as much as possible.

- Environmental Conditions:
The results of the environmental conditions and pH determined during the test are presented in table 2.
Temperatures remained within 21 - 24ºC, and were maintained within ± 2ºC range established for the test, temperature range during the test was 22.0 – 23.1°C.
The light intensity was measured at the start and end of the test; 7890 Lux at the start and 6710 Lux at the end of the test (mean of two readings carried out at each time point).
The pH of the test solutions ranged from 7.81 to 7.83 at test initiation in solutions with algae. The pH tended to increase relative to increases in algal densities, which is typical for tests conducted with P. subcapitata. The pH in the control increased by a maximum of 1.77 units, which exceeds the OECD 201 guideline requirement of an increase, not greater than 1.5 units. This change in pH was not considered to be detrimental to this study since increases in pH of greater than 1.5 units are commonly observed and this change had no impact on the growth of the control, which met the established validity criteria for the test.

- Test Media Descriptions:
The freshly prepared test media at 0 hours appeared as colourless solutions. The appearance of the test media following the 72-hour exposure period are summarised in the table below.

- Growth Test:
Individual replicate data for cell density and mean treatment cell density are presented in table 3. Results of the growth test, as specific growth rate, area under curve and yield are presented in table 4 to table 7.
Growth curves of Pseudokirchneriella subcapitata exposure to Isodecyl 3,5,5-trimethylhexanoate are presented in Figure 1.
The 72-hour yield (EyLx), area under the growth curve (EbLx) and growth rate (ErLx) toxicity values, with corresponding NOEL values, are presented in the table below.
The results indicated that there was no toxicity at the limit of solubility of the test substance (estimated as <0.01 mg/L).
Based on nominal loading rate concentrations, the 72-hour EyL50, EbL50 and ErL50 values were calculated to be >100 mg/L LR WAF.
The corresponding NOEL values for yield, biomass and specific growth rate after 72 hours were 100 mg/L LR WAF.
All validity criteria were met therefore the test was considered valid.

- Validity Criteria:
The validity criteria specified in the OECD 201 Test Guideline (Reference 1) are;
i. To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72-hour test period. In this test, the cell density increase (as a surrogate for biomass) was approximately 96 fold over the 72 hours.
ii. The mean coefficient of variation for the control replicate sectional (daily) specific growth rates must not exceed 35%. This was determined to be 9.19% in this test.
iii. The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7%. This was 2.22% in this test.

Results with reference substance (positive control):

- Study Title & Number: 3201656 - Inhibition of Growth to the Alga Pseudokirchneriella subcapitata
- GLP: Yes
- Protocol signed: 05 September 2016
- Final report signed: 17 January 2017
- Amended final report signed: 23 February 2017
- Test Dates: 27 September 2016 – 30 November 2016
- Test Substance: Potassium dichromate, Batch No. 13D290030, Purity: 100%, expiry date: 31 Jan 2018,
- Test Medium: EC
- Test Guideline: The study was designed in accordance with OECD Chemicals Testing Guideline No.201 Alga, Growth Inhibition Test (adopted 23 March 2006, Annex 5 corrected 28 July 2011)
- Objective: The objective of the study is to determine the effects of the reference substance against algal growth by exposing the green alga, Pseudokirchneriella subcapitata, at the exponential growth phase to the reference substance during a 72-hour test period.
- Treatment Rates: Control, 0.1, 0.32, 1.0 and 3.2 mg/L.
- Preparation method: An amount of test substance (ca 10 mg) was dissolved in 1000 mL of EC Medium to give a 10 mg/L stock solution, the remaining test mediums were prepared by serial dilution from the stock solution down.
- Environmental Conditions: Between 21 and 24°C, 24 hours light photoperiod.
- Test Design: Static regime, with test media preparation at 0 hours only
- Test Species: Pseudokirchneriella subcapitata
- Origin of Test Species: PThe Pseudokirchneriella subcapitata, culture is derived from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Oban, UK
- Test Vessels: 250 mL glass conical flasks
- Observations: Cell counts performed at 24, 48 and 72 hours.
- Study Duration: 72 hours
- Test System: 1000 cells/mL at 0 hours
- Replication: 6 replicates at Control and 3 replicates at each exposure concentration
- Optimal algal growth pH: 6.5 – 9.5
- Statistical Analysis
- System: CETIS v1.8.6.8, CETIS – Comprehensive Environmetal Toxicity Information System. 2001-2012. Tidepool Scientific, LLC
- Statistical Analysis Test: Bonferoni Adj t Test for NOEC values and Linear interpolation for EC10, 20 and 50 values.
- Conclusion: The 72-hour EC50 value for Yield, Area Under the growth Curve and Growth Rate were determined to be 1.19, 1.21 and 1.93 mg/L, respectively. The 72-hour NOEC value for Yield, Area Under the growth Curve and Growth Rate were determined to be 0.32 mg/L, respectively. This result is within the expected range and all validity criteria were met, therefore, the test is considered valid.
- 95% Confidence Limits Lower confidence limit = LCL Upper confidence limit = UCL

Reference substance results:

Endpoint	Results
	Yield	LCL(mg/l) UCL (mg/l)	Area Under Curve	LCL(mg/l) UCL (mg/l)	Growth Rate	LCL(mg/l) UCL (mg/l)
EC10 (mg/l)	0.081	0.0118-0.773	0.0706	0.0192-0.650	0.791	0.349-1.43
EC20 (mg/l)	0.449	NA-1.03	0.416	NA-0.874	1.16	0.963-1.49
EC50 (mg/l)	1.19	0.514-2.01	1.21	0.504-1.93	1.93	1.69-2.44
NOEC	0.32	NA	0.32	NA	0.32	NA
LOEC	1	NA	1	NA	1	NA

Media trial test results:

Nominal concentration (mg/l LR WAF)	Measured Concentrations (mg/l)
	0 hours	72 hours
100 (a)	5.85*	**
100 (b)	2.01*	**

LOQ 0.01 mg/l

* Considered to be sampling error as achieved concs are much greater than solubility level

** Batch failed to meet acceptance criteria

Test media descriptions at the end of the definitive test

Nominal Concentrations (mg/l LR WAF)	Colour of inoculated test medium	State of test medium
Control	Green	Homogenous hazt dispersion of algal cells
100

Test substance toxicity:

Parameter		Toxicity Values (mg/l LR WAF)		Statistical Test
		72 hours	Confidence limits (LCL-UCL)
Yield	EyL10	>100	NC	Results derived empirically
	EyL20	>100	NC
	EyL50	>100	NC
	NOEL	100	NC
Area Under the Curve (Biomass)	EbL10	>100	NC
	EbL20	>100	NC
	EbL50	>100	NC
	NOEL	100	NC
Growth Rate	ErL10	>100	NC
	ErL20	>100	NC
	ErL50	>100	NC
	NOEL	100	NC

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal loading rate concentrations, the 72-hour EyL50, EbL50 and ErL50 values were calculated to be >100 mg/L LR WAF.
The corresponding NOEL values for yield, biomass and specific growth rate after 72 hours were 100 mg/L LR WAF.
All validity criteria were met therefore the test was considered valid.

Executive summary:

The objective of the study was to determine the effects of the test substance on thegrowth of the green alga, Pseudokirchneriella subcapitata, during a 72-hour growthinhibition test.

The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201.

Based on the results of a range-finding test, for which the key results only have been reported, a definitive limit test was conducted at a nominal test concentration of 100 mg/L LR WAF (loading rate water accommodated fraction). Six replicate vessels were prepared for the control and 100 mg/L LR WAF concentration.

Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with 1 × 104 algae cells/mL and incubated at 21 – 24°C (under 4440 – 8880 Lux light

intensity) for 72 hours with cell counts at 24-hour intervals.

Analytical chemistry was performed during the definitive phase, however, the analytical batch run at 0 hours failed to meet acceptance criteria. Therefore, a decision was made alongside the client to not report the analytical data, as over the course of the study analytical method criteria were not met, this was considered to be due to issues with the compound and method (discussed further in Test Substance Properties section of the report). As analytical work was not reliable, results were based on nominal loading rate concentrations (mg/L LR WAF). Smithers Viscient study number 3201857 (Determination of Physicochemical Properties) found the water solubility to be less than the limit of quantification (LOQ) of 0.01 mg/L, therefore, it was considered that the water solubility level was <0.01 mg/L.

The 72-hour yield (EyLx), area under the growth curve (EbLx) and growth rate (ErLx) toxicity values, with corresponding no observed effect loading rates (NOEL) are presented in the table on the next page.

The results indicated that there was no toxicity at the limit of solubility of the test substance (estimated to be <0.01 mg/L). .

Parameter		Toxicity Values (mg/l LR WAF)		Statistical Test
		72 hours	Confidence limits (LCL-UCL)
Yield	EyL10	>100	NC	Results derived empirically
	EyL20	>100	NC
	EyL50	>100	NC
	NOEL	100	NC
Area Under the Curve (Biomass)	EbL10	>100	NC
	EbL20	>100	NC
	EbL50	>100	NC
	NOEL	100	NC
Growth Rate	ErL10	>100	NC
	ErL20	>100	NC
	ErL50	>100	NC
	NOEL	100	NC

Based on nominal loading rate concentrations, the 72-hour EyL50, EbL50 and ErL50 values were calculated to be >100 mg/L LR WAF.

The corresponding NOEL values for yield, biomass and specific growth rate after 72 hours were 100 mg/L LR WAF.

All validity criteria were met therefore the test was considered valid.

Description of key information

The key study was conducted according to internationally recognised testing guidelines and with GLP certification.

Additional information

Based on nominal loading rate concentrations, the 72-hour EyL50, EbL50 and ErL50 values were calculated to be >100 mg/L LR WAF.

The corresponding NOEL values for yield, biomass and specific growth rate after 72 hours were 100 mg/L LR WAF.

All validity criteria were met therefore the test was considered valid.