Reaction mass of [2,2,4-trimethyl-1-(2-methylpropanoyloxy)pentan-3-yl] benzoate and [2,2,4-trimethyl-3-(2-methylpropanoyloxy)pentyl] 2-methylpropanoate and (3-benzoyloxy-2,2,4-trimethylpentyl) benzoate

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:

14 January 2013 - 26 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: ASTM E1218-04

GLP compliance:

yes

Specific details on test material used for the study:

Description: clear colorless liquid
Batch: V046212201
Expiry / Retest date: 30 April 2014
Storage conditions: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a Coulter Multisizer Particle Counter.

Vehicle:

not specified

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of the test organisms were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban; Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and illumination at 21+1 1 C.
prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flsak to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100-150 rpm) and constant illumination at 24+-1 C until the algal cell density was approximately 10^4-10^5 cells/mL.
A positive control (Harlan study number: 41200591) used zinc chloride as the refernce item.
Details on the positive control are given in Appendix 2. The psoitive control was conducted between 13 Feb 2012 and 17 Feb 2012.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

not specified

Test temperature:

24+-1 C

pH:

7.1-7.7

Dissolved oxygen:

not specified

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

not applicable

Reference substance (positive control):

yes

Remarks:

zinc chloride

Key result

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Validation of Mixing Period
Pre-study work indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours.
Range finding test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the renage-finding test are given in Table 1.
The results showed no effect on growth at 10 and 100 mg/L loading rate WAF.
Based on this informayion a single lodading rate of six replicates of 100 mg/L, using a stirring period of 23 hours followed by a 1-hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confim that no effect on gowth was observed.
Chemical analysis of the 100 mg/L loading rate WAF at 0 hours showed a measured test concentration of 1.2 mg/L was obtained. A significant decline in measured test concentration was observed at 96 hours to 0.045 mg/L. This decline was considered to be due to the unstable nature of the test item.
Definitive test
Cell density values determined at each sampling time and pH values at 0 and 96 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate, yield inhibition and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4. Growth rate, yield and biomass integral values for the control and test cultures after 96 hours and percentage inhibition values are given in Table 5.
The mean cell densities versus time for the definitive test are presented in Figure 1.
Validation criteria
The following data shows that the cell concentration of the control cultures increased by a factor of 122 after 72 hours and by a factor of 206 after 96 hours. This increase was in line with the ASTM Guideline that states the enhancement must be at least by a factor of 16 after 72 hours and the OPTTS Guidelines that states the enhancement must be at least by a fator of 100 after 96 hours.
Mean cell density of control at 0 hours : 1.10 x 10^4 cells per mL
Mean cell density of control at 72 hours : 1.35 x 10^6 cells per mL
Mean cell density of control at 96 hours : 2.27 x 10^6 cells per mL
The coefficient of variation in replicate control cultures after 72 hours was 1% for average specific growth rate and 4% for yield and hence satisfied the validation criterion which states that these must not exceed 7% and 20% respectively.
The coefficient of variation of the cell density values in replicate control cultures was 4 % after 72 hours and 6% after 96 hours and hence satisfied the validation criterion which sates that this must not exceed 20%.
Growth data
From the data given in Tables 2, 4 and 5, it is clear that the growth rate (r), yield (y) and biomass integral (b) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the prsence of the test item over the 96-hour exposure period.
It was considered unneccessary and unrealistic to test at loading rates in excess of 100 mg/L.
Observation on cultures
All test and control cultures were inspected microscopically at 96 hours. There were no abnormalities detected in any of the control or test cultures.
Observation on test item solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of mixing the 100 mg/L loading rate WAF was observed to have formed a clear colorless media column with oily globules of test item floating on the surface and a few oily globules dispersed throughout. Microscopic examination of the WAF showed there to be no micro-dispersions or globules of test item present.
At the start of the test all control and 100 mg/L loading rate WAF test cultures were observed to be clear colorless solutions. After the 96-hour test period all control and test cultures were observed to be dark green dispersions.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test itme on the growth of Pseudokirchneriella subcapitata has been investigated and 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.

Description of key information

The effect of the test itme on the growth of Pseudokirchneriella subcapitata has been investigated and 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.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L