Isobutylbenzene

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

A biological safety cabinet was used to maintain test sterility from January 30 – Feb 02, 2018 as the flow hood was not available. There was no impact on the study as both instruments are able to maintain sterility.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Isobutylbenzene

EC Number:

208-706-2

EC Name:

Isobutylbenzene

Cas Number:

538-93-2

Molecular formula:

C10H14

IUPAC Name:

(2-methylpropyl)benzene

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Confirmatory analysis of the test solutions was performed on initial test solutions prepared for test initiation, 24-h, 48-h solutions, and on final solutions collected at test completion. The initial, 24 hour, 48 hour, and final test solutions were collected from each control and from each nominal test item loading rate. Aliquots of the initial test solutions were collected from the vessels used to prepare the bulk test solutions. The 24-h and 48-h solutions were collected from the additional non-inoculated vessels prepared at test initiation. At test completion, the test solutions from the inoculated vessels were pooled and centrifuged to remove the algal cell prior to collection. Approximately 40 mL of each test solution and each control were collected in glass containers of appropriate size and stored refrigerated (2°C to 8°C) until analysis. Approximately 1L of negative control water was also collected at test initiation. Sub-sample containers were filled completely to reduce headspace as much as possible.

Test solutions

Vehicle:

no

Details on test solutions:

The test item is a mono-constituent organic liquid; with a water solubility of 12 mg/L ± 1 mg/L at T = 20.0°C ± 0.5 °C. The test solutions were prepared as water accommodated fractions (WAFs) using the methodology described below. The test item was also found to be volatile in aqueous solutions; therefore extra effort was taken to reduce headspace and exposure to the laboratory environment.
Water accommodated fractions (WAFs) were prepared at 3.125, 6.25, 12.5, 25, 50, and 100 mg/L nominal loading rates (Table 6-3) and tested alongside a negative control and a WAF (method) control.
To prepare the WAFs, the test item was weighed and added directly to approximately 1L of sterile nutrient media in a 2L glass aspirator bottle. The solutions were then brought up to the 2L mark using dilution water. A WAF control (0 mg/L) was prepared alongside the test item WAFs; however, no test item was added, and a total of 2L was prepared. Immediately following preparation, WAFs were sealed with Parafilm®.
Each WAF solution was stirred using a magnetic stir bar and stirrer using a slow stir method. Stirring occurred for approximately 44 hours at which time the solution were allowed to settle undisturbed for approximately 2.5 hours. WAF solutions were prepared following aseptic techniques.
WAF solutions were removed from the mixing vessel and collected in a glass holding vessel (600mL glass beaker) by draining from the bottom third of the aspirator bottle, discarding the first ~50 mL, into an appropriate container and immediately covered with Parafilm®. WAF solutions were drained from low loading rate to high loading rate to prevent cross contamination.
The WAF solutions were prepared in a laminar flow hood on January 28th, 2018, according to the Safe Work Procedure for Laminar Flow Hoods and Biological Safety Cabinets (MEHSSWP-00031). On January 30th, 2018, the flow hood was out of service and a biological safety cabinet was used to maintain sterility for the remainder of the test. The biological safety cabinet was operated as per the Maxxam Work Instruction for Microbiology Lab-Equipment Maintenance and Calibration Program (COR1 WI-00012) and the Maxxam Work Instruction for Biosafety and Biosecurity in the Microbiology Laboratory (COR1 WI-00039) (deviation #1). As the test item was found to be volatile in aqueous solutions, extra effort was taken to reduce headspace and exposure to the laboratory environment.
The test item was handled according to the Work Instruction for Management of Test and Reference Items for GXP Studies (BBY1 WI-00008). All chemicals were weighed using analytical balances, which were operated according to the procedures outlined in the Work Instruction for Corporate Procedure for Balance Calibrations and Verifications (COR WI-00015). All containers and apparatus used were new or thoroughly cleaned according to the Work Instruction for Cleaning of Laboratory Ware (BBY WI-00001).
All equipment and glassware was sterilized according to the Work Instruction for the Operation and Maintenance of Sterilizers (BBY2 WI-00019) or by using a 70% ethanol solution.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): In-house culture; original culture was obtained from Canadian Phycological Culture Center (Waterloo, ON)
- Age of inoculum (at test initiation): 3 days


ACCLIMATION
- Acclimation period: not applicable
- Culturing media and conditions (same as test or not): yes

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23 °C

pH:

7.4 - 9.6

Nominal and measured concentrations:

Nominal: 0 (negative control); 0 (WAF control), 3.125, 6.25, 12.5, 25, 50, 100 mg/L
mean measured: BQL, BQL, 0.26, 0.63, 0.73, 2.27, 2.60, 5.25 mg/L
BQL - below limit of quantification (< 0.005 mg/L)

All biological results were refered to mean mesured test concentrations, as the analytical values deviates by more that 20 % from the nominal values.

Details on test conditions:

TEST SYSTEM
- Test vessel: ca. 42 mL sterile glass vials with Teflon lined caps, mixed on a horizontal shaker at approximately 50-65 rpm
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: ca. 42 mL
- Aeration: no
- Initial cells density: 0.99 x 10E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per WAF control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (Nutrient growth medium)
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: continuous
- Light intensity and quality: 5970 - 6420 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell Yield and Specific Growth Rate (SGR) after 24, 48 and 72 h
- Determination of cell concentrations: Replicate cell counts were performed on aliquots from each flask using the Cellometer X4HM Auto counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Reference substance (positive control):

yes

Remarks:

zinc sulphate heptahydrate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.42 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.26 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

Zink sulphate hexahydrate 72 h EyC50: 0.079 mg/L

Reported statistics and error estimates:

The 72 hour ELR50, ELR20, ELR10 along with their 95% confidence limits (based on both nominal loading rates and mean measured test item concentrations) and the NOELR and LOELR values for growth inhibition were determined using a computerised statistical package, CETIS™ (Version 1.9.2.4), a Comprehensive Environmental Toxicity Information System, recommended by Environment Canada. All reported values were rounded according to the Work Instruction for Significant Figures in Data Reporting (COR WI-00114).