Benzene, (1-methylethyl)-, oxidized, cumene residues, acetophenone fraction

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2001-10-03 - 2003-02-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Acetophenone

EC Number:

202-708-7

EC Name:

Acetophenone

Cas Number:

98-86-2

Molecular formula:

C8H8O

IUPAC Name:

1-phenylethanone

Details on test material:

- Name of test material (as cited in study report): acetophenone
- Substance type: aromatic ketone
- Physical state: liquid
- Analytical purity: 98.71% (test substance obtained from JLM Chemicals, Inc.)
- Impurities (identity and concentrations): phenol (0.068%), alpha methylstyrene (0.204%), water (0.13%)
- Purity test date: 22.05.2001
- Lot/batch No.: R104-044
- Expiration date of the lot/batch: unknown
- Stability under test conditions: not reported
- Storage condition of test material: Test substance was stored at room temperature in the dark.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal acetophenone concentrations of 0, 12.5, 25, 50, 100, and 200 mg/l were tested.
- Sampling method: At test initiation (test day 0), one replicate of each treatment was sacrificed for analytical verification of the acetophenone concentration. At test termination (72 h), the remaining three replicates of each treatment were also sampled for concentration verification. Each replicate was first subsampled for algal cell density and/or absorbance determination. To collect each analytical chemistry sample, approximately 100 mL of test solution was collected using a syringe, filtered through 0.45 µm polyethersulfone filters, transferred into one approximately labeled 100 mL glass bottle, and the bottles were sealed.
- Sample storage conditions before analysis: no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Each test concentration was prepared in a batch and was covered and mixed on a stir plate for approximately 10 min
to allow complete dissolution of the test substance. The test solutions were then gently poured into the replicate test containers
so as to avoid volatilization of acetophenone.
- Controls: performed (test substance omitted in the test medium)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Source (laboratory, culture collection): Raphidocelis subcapitata was obtained from the Fort Collins Environmental Toxicology Laboratory (FCETL) in-house culture (FCETL stock culture number 121301)
- Age of inoculum (at test initiation): 5 d
- Method of cultivation: R. subcapitata was typically cultured at FCETL using MSMBL medium. The algae used for this test were taken from a subculture grown in fortified US EPA algal assay procedure (AAP) growth medium specified by US EPA 1978 and ASTM 1996. This subculture was established five days prior to test initiation in order to obtain a log-phase culture for inoculum preparation. The subculture was grown at 24 +/- 2°C under continuous broad-spectrum lighting and was continuously agitated on a rotary shaker.

ACCLIMATION
- Acclimation period: 5 d
- Culturing media and conditions (same as test or not): R. subcapitata was typically cultured using MSMBL medium. The medium used in the test was fortified AAP according to US EPA 1978 and ASTM 1996.
- Any deformed or abnormal cells observed: not reported

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 +/- 2°C (the test temperature reached 27°C in the test chambers for the nominal test concentration of 25 mg/L at 24 and 48 h of testing, and in nominal concentration 100 mg/L at 24 h of testing)

pH:

0 h: 8.3-8.4 (in all test runs)
96 h: 8.3-9.4 (in all test runs)

Nominal and measured concentrations:

Nominal and measured acetophenone concentrations

Nominal acetophenone Measured acetophenone concentration (mg/L)
concentration (mg/L) 0 h 72 h
A B C Overall average
0 (control) 0.23* 0.23 0.23 0.23 0.23
12.5 11.9 12.4 12.5 12.0 12.2
25 23.8 25.0 25.2 25.1 24.8
50 46.5 49.9 49.9 48.9 48.8
100 94.7 102 102 100 99.7
200 192 198 197 199 196
*control acetophenone measurements which were < method reporting limit (MRL) are reported as half the MRL, or 0.23 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300-mL glass BOD bottles
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass/300 mL/50 mL/250 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): static test conditions
- Initial cells density: 1.4 x 10E4 cells/mL in the test chambers (mean of 3 measurements)
- Control end cells density: 9.4 x 10E5 (increase after 72 h: 67.1 determined by direct microscopic cell count//82.5x increase of absorbance at 72 h; )
- No. of organisms per vessel: 1.03 x 10E6 cells/mL
- No. of vessels per concentration (replicates): 7
- No. of vessels per control (replicates): 7
- No. of vessels per vehicle control (replicates): no vehicle used

GROWTH MEDIUM
- Standard medium used: yes (fortified algal assay procedure growth medium according to US EPA 1978 and ASTM 1996)
- Detailed composition if non-standard medium was used: see above

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dilution water was prepared according to US EPA using sterile, deionized (i.e., Milli-Q) water
- Conductivity: 622-630 µS/cm (initial value in the definite test runs)
- Culture medium different from test medium: yes
- Other: The test was conducted in BOD bottles stoppered with ground-glass stoppers and the necks were wrapped with Parafilm. To reduce the negative effect of the limited gas exchange on algal growth, the algal assay medium was fortified with 500 mg/L additional sodium bicarbonate.

OTHER TEST CONDITIONS
- Sterile test conditions: yes(medium was sterilized using a vacuum filtration apparatus)
- Adjustment of pH: the pH of the medium was adjusted to 7.5 +/- 0.1; the pH during the definite test was 8.3-8.4 (at the beginning of the test; 0 h) and 8.3-9.2 after 96 h
- Photoperiod: continuous illumination
- Light intensity and quality: 743 +/- 148 ft-c (8000 +/- 1600 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth rates were determined daily
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: growth rates were calculated from absorbance data measured by spectrophotometer at 750 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 0, 20, 60, 200, 600, and 2000 mg/L nominal
- Results used to determine the conditions for the definitive study: 72 h EC50=63.9 mg/L

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

40 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% C.L.: 22.9-57.1 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

86.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L.:74.6-96.2 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

24.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not reported
- Unusual cell shape: not reported
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

Calculations for individual growth rates, areas under the curve, and per cent inhibition values followed equations from OECD TG 201. The concentration estimated to cause 50% decrease in algal growth rate (relative to the control) after 72 h of exposure was calculated using a linear interpolation (ICp) method and expressed as ECr50 (0-72 h). Replicate growth rates were calculated from absorbance data at test initiation vs test termination (72 h) and overall average measured acetophenone concentrations were entered into the ICp program, which uses linear interpolation to generate the IC50/EC50 and 95% confidence interval (Norberg-King 1993). The concentration estimated to cause a 50% decrease in algal growth rate expressed as 'area under the curve' (relative to the control) throughout the 72 h exposure was calculated using nonlinear regression (SPSS 1997) and expressed as ECb50 (0-72 h). The NOEC was identified using Dunnett's Test after verifying test data normality and homogeneity using Shapiro-Wilk's and Bartlett's Tests, respectively (West, Inc. 1996).

Any other information on results incl. tables

The toxicity of acetophenone to Raphidocelis subcapitata was investigated in a study conducted according to OECD guideline 201. The following results were obtained:

1. Calculated area under the growth curve (calculated acc. To guideline)

Measured acetophenone concentration (mg/L)	AUC (area under growth curve)	% inhibition of AUC
Control	0.210	0
12.2	0.171	18.6
24.8	0.157	25.4
48.8	0.099	52.9
99.7	0.022	89.3
196	0.007	96.8

2. Final calculated algal specific growth rate (72 h)

Measured acetophenone concentration (mg/L)	Specific growth rate
	A	B	C	Average
Control	1.470	1.564	1.343	1.459
12.2	1.320	1.330	1.234	1.295
24.8	1.242	1.378	1.391	1.337
48.8	1.145	1.217	1.092	1.151
99.7	0.519	0.611	0.611	0.580
196	0.135	0.187	0.305	0.209

The NOEC was the measured concentration of 24.8 mg/L. The 72 h ECb50, based on area under the growth curve and average measured acetophenone concentrations, was 40 mg/L (95% C.I.: 22.9-57.1 mg/L). The 72 h-ECr50, based on growth rate and average measured acetophenone concentrations, was 86.4 mg/L (95% C.I.: 74.6-96.2 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a toxicity study with Raphidocelis subcapitata conducted according to OECD guideline 201, the NOEC, 72 h ECb50, and 72 h-ECr50 were determined to be 24.8 mg/L, 40 mg/L (95% C.I.: 22.9-57.1 mg/L), and 86.4 mg/L (95% C.I.: 74.6-96.2 mg/L; effective), respectively.