Butanedioic acid, 2-hydroxy-, 1,4-bis[5,7,7-trimethyl-2-(1,3,3-trimethylbutyl)octyl] ester

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-08-06 to 2015-10-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to international methods and GLP standards.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MAFF (12-Nousan-No.8147,2-7-7-1, 24 November, 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] 2-hydroxybutanedioate

EC Number:

807-422-1

Cas Number:

66918-01-2

Molecular formula:

C40H78O5

IUPAC Name:

1,4-bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] 2-hydroxybutanedioate

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamberThe cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber

Test solutions

Vehicle:

yes

Details on test solutions:

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for the test item and in both the range finding and definitive tests.

Test organisms

Test organisms (species):

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

Details on test organisms:

Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662).
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Test conditions

Hardness:

No information

Test temperature:

Temperature range in flasks : 22.8 – 22.9 °C
Temperature range within the climate chamber : 22.5 - 23.3 °C

pH:

7.71-8.64

Dissolved oxygen:

No information

Salinity:

No information

Nominal and measured concentrations:

Because the test items is very poorly soluble in water, a test solution was prepared according to OECD method No. 23.
Nominal concentration: 100 mg/L
The non-dissolved test material was removed to obtained the 100% staturated solution.

Details on test conditions:

Test performance
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.
The test was performed with six replicates per test concentration and six replicates in the control group as well. Volumes of 100 mL test solution per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.

The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 8141 lux (equivalent to ~110 µE/m2/s)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

EbC50

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

ErC50

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield (EyC50)

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Because significantly toxic response was not observed during the preliminary concentration Range-Finding Test, only one test concentration at the solubility level of the Test Item in the test medium (100 % saturated solution) and one control group was tested in a Limit-Test.

The ErC50, EbC50 and EyC50 values of the Test Item were determined from the raw data.
Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test ( = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by 2 Sample t-Test ( = 0.05) by TOXSTAT software.

Any other information on results incl. tables

Average Specific Growth Rates (µ)

The results of the statistical evaluation show that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100 % saturated solution.

Growth rate (µ) and Percentage inhibition of µ during the Test Period

	Growth rate µ and % inhibition of µ
	0 - 24 h		0 - 48 h		0 -72 h
Test Group	µ	%	µ	%	µ	%
Control	0.0589	0.0	0.0592	0.0	0.0586	0.0
100 % saturated solution	0.0573	2.6	0.0584	1.4	0.0583	0.5

Areas under the Growth curves

The results of the statistical evaluation show that the 0-72 h areas were not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100 % saturated solution.

Area under the growth curves (A) and Percentage inhibition of A during the Test Period

	Areas under the Growth curves (A) and % inhibition of A
	0 - 24 h		0 - 48 h		0 -72 h
Test Group	A	%	A	%	A	%
Control	38.0	0.0	270.0	0.0	1266.0	0.0
100 % saturated solution	36.0	5.3	258.0	4.4	1230.0	2.8

Yield

The results of the statistical evaluation show that the 0-72 h yield was not statistically significantly different from the untreated control value; accordingly the No Observed Effect Concentration (NOEC) was determined as 100 % saturated solution.

Yield (Y) and Percentage Inhibition of Y during the Test Period

Test Group		Yield (Y) and % inhibition of Y (0 -72 h)
		Y	%
Control		66.8	0.0
100% saturated solution		65.5	2.0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

according to the test guidelines

Conclusions:

The effect of Bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] malate Test Item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.
The 72h EbC50 value: > 100 % saturated solution (nominal)
The 72h ErC50 value: > 100 % saturated solution (nominal)
The 72h EyC50 value: > 100 % saturated solution (nominal)
The 72h NOEC: 100 % saturated solution (nominal)
The 72h LOEC: > 100 % saturated solution (nominal)
Based on the results of this study, the Bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] malate had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium.

Executive summary:

The effect of Bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] malate was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours. Because toxic response was not observed during the preliminary concentration range-finding test, a Limit Test was carried out using only one test concentrationat the limit of solubility of the Test Item in the test medium (100 % saturated solution) and one control group.The experiment was carried out without analytical support based on agreement with the Sponsor. The test design included six replicates at test concentration and six replicates for the untreated control. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a= 0.05) by TOXSTAT software. The E_rC_50, E_bC_{50 a}nd E_yC₅₀ values of the Test Item were determined directly from the raw data. 

The 72h E_bC₅₀ value:     >100 % saturated solution (nominal)

The 72h E_rC₅₀ value:      >100 % saturated solution (nominal)

The 72h E_yC₅₀ value:      >100 % saturated solution (nominal)

The72h NOEC:                  100 % saturated solution (nominal)

The72h LOEC :               >100 % saturated solution (nominal)

Based on the results of this study, the Bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] malate had no toxic effect at saturation; the EC₅₀results and the LOEC are higher than the solubility level of the Test Item in the test medium.