4-tert-Butoxystyrene

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:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

March 23, 2006

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control.
- Frequency: 0 h - 24 h - 72 h
- Sampling method:
Sampling vessels were pre-incubated with the respective test solutions before sampling. In addition, the glass wool used for preparation of the WSF was retained for possible analysis. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: samples were stored in a freezer until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: butoxystyrene was not completely soluble in test medium at the loading rate prepared. Preparation of test solutions started with a loading rate of 100 mg/l applying 24 hours of magnetic stirring in order to obtain maximum solubility of the test substance. Afterwards, the mixture was poured over glass wool to discard any undissolved material. The clear and colourless Water Soluble Fraction (WSF) was used for testing, together with several dilutions in OECD 201 (M2)-medium.
- Controls: test medium without test substance or other additives.
- Evidence of undissolved material: the mixture was poured over glass wool to discard any undissolved material.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source: in-house laboratory culture.
- Method of cultivation: stock cultures were started by inoculating growth (M1) medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C and light intensity of 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Acclimation period: 3 to 4 days before the start of the test, cells from the algal stock culture were inoculated in OECD 201 (M2)-medium at a cell density of 10000 cells/ml. The pre-culture was maintained under the same conditions as used in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/l as CaCO3

Test temperature:

21-22 °C

pH:

8.0-8.4

Nominal and measured concentrations:

The average exposure concentrations were calculated to be 0.087, 0.70, 1.2, 1.6 and 4.8 mg/l at 4.6, 10, 22, 46 and 100% of the WSF prepared at a loading rate of 100 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Initial cells density: 10000 cells/ml
- Control end cells density: 186 x 10^4 cells/ml
- No. of vessels per concentration (replicates): 3 of each test concentration + 1 extra for all test concentrations for sampling purposes.
- No. of vessels per control (replicates): 6 + 1 extra for sampling purposes.
In addition, 2 replicates of the highest test concentration without algae.

GROWTH MEDIUM
- Growth (M1) medium, according to NPR 6505, was formulated using Milli-RO water with the following composition:
NaNO3 500 mg/l
K2HPO4 40 mg/l
MgSO4.7H2O 76 mg/l
Na2CO3.10H2O 54 mg/l
C6H8O7.H2O 6 mg/l
NH4NO3 330 mg/l
CaCl2.H2O 36 mg/l
C6H5FeO7.xH2O 6 mg/l
H3BO3 2.9 mg/l
MnCl2.4H2O 1.81 mg/l
ZnCl2 0.11 mg/l
CuSO4.5H2O 0.08 mg/l
(NH4)6Mo7O24.4H2O 0.018 mg/l

TEST MEDIUM / WATER PARAMETERS
- Culture (M2) medium according to OECD 201, formulated using Milli-Q water.
- Culture medium different from test medium: no.

OTHER TEST CONDITIONS
- Photoperiod: continuously
- Light intensity and quality: TLD-lamps "cool-white" of 30 Watt with a light intensity within the range of 78 to 82 µE/m^-2.s^-1.
- Incubation: vessels were distributed at random in the incubator and the algal cells were kept in suspension by continuous shaking

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations:
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (path length = 20 mm). Algal medium was used as blank.

TEST CONCENTRATIONS
- Test concentrations: 0.087, 0.70, 1.2, 1.6, 4.8 mg/l
- Range finding study: Three replicates per concentration were exposed to 0.1, 1.0 and 10% of the WSF prepared at a loading rate of 100 mg/l. Inadvertently, no algae were added to the test group containing 10% of the WSF. Therefore, this concentration was omitted from further discussion.
- Results used to determine the conditions for the definitive study: The expected EC50 for growth rate reduction was above 1.5 mg/l. The expected EC50 for yield inhibition was between 0.15-0.48 and 1.5 mg/l.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Remarks on result:

other: 95 % CI ranging from 1.1 to 4.0 mg/l

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 4.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- ErC50 0-72h: 1.8 mg/l with a 95% CI ranging from 1.3-2.6 mg/l
- EyC50 0-72h: 0.79 mg/l with a 95% CI ranging from 0.51-1.2 mg/l

Validity criteria fulfilled:

yes

Executive summary:

A fresh water algal growth inhibition test was performed with butoxystyrene. Based on the results of a preceding combined limit/range-finding test a final test was performed. Preparation of test solutions started with a loading rate of 100 mg/l applying 24 hours of magnetic stirring in order to obtain maximum solubility of the test substance. Afterwards, the mixture was poured over glass wool to discard any undissolved material. The clear and colourless Water Soluble Fraction (WSF) was used for testing, together with several dilutions in M2-medium. One hour before the test was started, a part of each test solution was used for pre-incubation of the respective test vessels.

Exponentially grown algal cultures were exposed to a control group and to 4.6, 10, 22, 46 and 100% of the WSF prepared at a loading rate of 100 mg/l. The initial cell density was 10⁴ cells/ml and the total test period was 72 hours. Samples for analytical confirmation of actual test concentrations were taken at the start, after 24 hours of exposure and at the end of the test. The average exposure concentrations were calculated to be 0.087, 0.70, 1.2, 1.6, 4.8 mg/l.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Butoxystyrene reduced growth rate and inhibited the yield of this fresh water algae species significantly at average exposure concentrations of 1.6 and 1.2 mg/l, respectively.

The EC₅₀ for growth rate reduction (E_RC₅₀: 0-72h) was beyond 4.8 mg/l. The EC₅₀ for yield inhibition (E_YC₅₀: 0-72h) was 2.1 mg/l. The NOEC for growth rate reduction was 1.2 mg/l and the NOEC for yield inhibition was 0.70 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.8 mg/L

EC10 or NOEC for freshwater algae:

1.2 mg/L

Additional information

A GLP-compliant fresh water algal growth inhibition study was performed with butoxystyrene according to OECD201.

Exponentially grown algal cultures of Pseudokirchneriella subcapitata were exposed to a control group and to 4.6, 10, 22, 46 and 100% of Water Soluble Fraction prepared at a loading rate of 100 mg/l. The initial cell density was 10⁴ cells/ml and the total test period was 72 hours. Samples for analytical confirmation of actual test concentrations were taken at the start, after 24 hours of exposure and at the end of the test. The average exposure concentrations were calculated to be 0.087, 0.70, 1.2, 1.6 and 4.8 mg/l.

The 72h E_yC₅₀ was 2.1 mg/l and the NOEC for yield inhibition was 0.70 mg/l. The 72h E_rC₅₀ was beyond 4.8 mg/l and the NOEC for growth rate reduction was 1.2 mg/l.

The preferred observational endpoint in this study is algal growth inhibition because it is not dependent on test design, whereas yield depends both on growth rate of the test species as well as test duration and other elements of test design.