Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

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:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD 201 (2011)
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material: tert-butylbenzene
- IUPAC name: tert-Butylbenzene
- Molecular formula: C10H14
- Molecular weight: 134.22 g/mol
- Substance type: Organic
- Physical state: Liquid
Analytical monitoring:
not specified
Vehicle:
no
Details on test solutions:
The primary stock solution used in the range-finding test was prepared by dissolving 0.0250 g of the test substance in 250 mL with OECD medium, resulting in a concentration of 100 mg.L-. The stock solution of 10 mg.L-1 was prepared from dilution of the primary stock solution.

The primary stock solution used in the definitive test was prepared by dissolving 0.320 g of the test substance in 1000 mL with OECD medium, resulting in a concentration of 320 mg.L-1. The stock solution of 10 mg.L-1 was prepared from dilution of the primary stock solution.

From these initial stock solutions, a series of dilutions was prepared in order to obtain the desired tested concentrations

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The unicellular green alga species Pseudokirchneriella subcapitata used is part of the Chloroficeae group, Chlorococcales order.

- Test System Selection Justification
The alga species used as test system is recommended in the Guideline OECD 201 (2011).

- Origin of the Test System
The test system was obtained from Culture Collection of Algae at Goettingen University - SAG 61.81 on October, 08th, 2013 and is recommended by the Guideline OECD 201 (2011).

- Stock Culture Maintenance
The stock culture was prepared and maintained under lab-controlled conditions in solid medium with protease peptone. Its composition is described in below.
Product Final concentration (g.L-1)
Agar 10
KNO3 0.2
K2HPO4 0.02
MgSO4.7H2O 0.02
Protease peptone 1


The inoculated solid medium was kept inside a culture room, with controlled temperature of 21 to 24°C and luminosity of 2100  10% lux. After algae cultures reached an ideal growth (about one week), they were maintained at temperature of 2 to 6°C and luminosity of 100 to 200 lux in order to slow down the algae growth.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22.1 to 22.2 °C
pH:
8.10 to 8.11
Nominal and measured concentrations:
Nominal concentrations: 0 (control); 1; 3.2; 10; 32; 100 and 320 mg.L-1
Measured concentrations: < LQ (lower than the practical limit of quantification (0.20 µg.mL-1)); 0.9737; 3.0846; 10.2708; 29.5118; 102.7077; 295.1180 mg.L-1
Details on test conditions:
TEST SYSTEM
- Glassware Preparation: All glassware used in the test was washed with detergent, commercial acetone, 5% nitric acid and rinsed thoroughly with tap water and followed with deionized water.
- Test vessel: 250 mL Erlenmeyer flasks containing 100 mL of test solution.
- Initial algae cell concentration: 5e+3 cells.mL-1
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

- Light intensity: 5991 lux (average)
- Photoperiod: continuous light

- Determination of cell concentrations: A Neubawer chamber and a microscope were used to determine the cell number (cells.mL-1).
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 22.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 290.44 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 3.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The 72-hours EyC50 value of test substance, obtained in this study with Pseudokirchneriella subcapitata, was 22.40 mg.L-1 (95% confidence limits = 17.69 to 28.37 mg.L-1) and the 72-hours ErC50 value was 290.44 mg.L-1 (95% confidence limits = 215.95 to 390.61 mg.L-1). The highest concentration of the test substance in which was not observed any statistically significant adverse effect on algal growth (NOEC) over the test period (72 hours) was 1 mg.L-1 and the lowest test substance concentration at which was observed significant reducing effect on growth (LOEC) was 3.2 mg.L-1.

The 72-hours EyC50 value of the reference substance potassium dichromate was 0.65 mg.L-1 (confidence limits at 95% = 0.59 to 0.71 mg.L-1), demonstrating that the toxic sensitivity of the algae was within the accepted range (ISO, 1989), which report toxicity ranged between 0.60 and 1.03 mg.L-1.

The growth curve of the test system (Pseudokirchneriella subcapitata) at different concentrations of test substance during 72 hours of exposure in the definitive test is shown.

In order to check the validity of the test, the biomass in the control cultures should increase exponentially by a factor of at least 16 within 72 hours. The mean coefficient of variation (CV) for section-by-section specific growth rates in the control cultures must not exceed 35% and the CV of average specific growth rate during the entire test period in replicate control cultures must not exceed 7%. In fact, in the present study, the biomass in the control cultures increased 577 times at end of the test . The mean coefficient of variation (CV) of the growth rates in the control group was 32.5% and the CV of average specific growth rate during the entire test period in replicate control cultures was 0.3% , showing therefore, the validity of this study.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.65 mg/L (based on yield)
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 72 hour EC50 (growth rate) of the test material was determined to be 290.44 mg/l.
Executive summary:

In this guideline (OECD 201) study conducted with GLP certification, the objective of this study was to evaluate the toxicity of tert-butylbenzene on alga Pseudokirchneriella subcapitata. A range-finding test was conducted to find the concentrations that cause algae growth inhibition. Based on the results of the range-finding test, a definitive test was carried out with the following concentrations of test substance: 0 (control); 1; 3.2; 10; 32; 100 and 320 mg/L. The test was incubated during 72 hours inside chambers with controlled conditions of luminosity, temperature, and agitation. The 72-hours EyC50 value was 22.40 mg/L and the 72-hours ErC50 value was 290.44 mg/L. The highest test substance concentration which no observed significant adverse effect (NOEC) on algal growth was 1 mg.L-1 and the lowest test substance concentration at which was observed significant reducing effect on growth (LOEC) was 3.2 mg/L.

Description of key information

Study carried out according to guideline and with GLP compliance.

Key value for chemical safety assessment

EC50 for freshwater algae:
290.44 mg/L
EC10 or NOEC for freshwater algae:
1 mg/L

Additional information

The key study for the determination of the inhibition of algal growth was conducted according to the Study Plan and procedures described in the Guideline 201 of the Organisation for Economic Co-operation and Development (OECD, 2011). The 72-hours EyC50 value of test substance, obtained in this study with Pseudokirchneriella subcapitata, was 22.40 mg/L (95% confidence limits = 17.69 to 28.37 mg/L) and the 72-hours ErC50 value was 290.44 mg/L (95% confidence limits = 215.95 to 390.61 mg/L). The highest concentration of the test substance in which was not observed any statistically significant adverse effect on algal growth (NOEC) over the test period (72 hours) was 1 mg/L and the lowest test substance concentration at which was observed significant reducing effect on growth (LOEC) was 3.2 mg/L.