Methyl 2-benzoylbenzoate

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:

November 2014 - January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: RCX 14-672
Chemical name: methyl-2-benzoylbenzoate
Batch no.: N14003
CAS no.: 606-28-0
EC no.: 210-112-3
Molecular formula: C15H12O3
Molecular mass: 240.3 g/mol
Description: white to light yellowish powder
Purity: >99% (gas chromatography)
Water solubility: 117.7 mg/l
Test item storage: at room temperature, protected from light
Stability: stable under storage conditions
Expiry date: 30 November 2015

Analytical monitoring:

yes

Details on sampling:

Analyses (by HPLC) of the test item concentrations were conducted in the definitive test at the beginning and after 24, 48 and 72 h of exposure.

Vehicle:

yes

Details on test solutions:

Based on the results of the range finding test, the definitive test with RCX 14-672 was performed at the following loading rates: 100, 50.0, 25.0, 12.5, 6.25 and 3.13 mg/l. The loadings of the test item were prepared by directly adding the respective amounts of test item to empty glass vessels. The algal medium was then added, moderately stirred for 24 h, followed by filtration (0.45 µm cellulose acetate membrane sterile syringe filter, VWR International). The resulting saturated solutions were used in the definitive test, and the actual concentrations of RCX 14-672 were measured by HPLC.

The pH of the OECD medium was adjusted before filtration (pH 8.0±0.2).

For each test series, the following test flasks were set up:
• Test suspension (Tn); containing test medium, test item and algae (three replicate samples)
• Blank control (Bn); containing test medium and algae (six replicate samples)

Test flasks were placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test organism: Desmodesmus subspicatus 86.81 SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany)

Culture: 250 ml flask containing 100 ml of sterile OECD medium inoculated with cell material from axenic slope cultures
Illumination: Continuous (2000–3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland)
Temperature: 22 ± 2 °C, thermo-controlled room

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

22±2 °C

pH:

8.0 ± 0.2.

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

100, 50.0, 25.0, 12.5, 6.25 and 3.13 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml flasks, all-glass, with 100 ml of test medium, shaken (about 130 rpm)
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: 0.50–0.85 μg/ml with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, recommended OECD medium

OTHER TEST CONDITIONS
- Adjustment of pH: the pH of the control medium should not increase by more than 1.5 units during the test
- Photoperiod: the light intensity is maintained within ±15% from the average light intensity over the incubation area
- Light intensity and quality: the light intensity amounts to about 3000 lux which corresponds to about 40 μE•m-2•s-2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: every 24 h using a spectrophotometer (680 nm wavelength). Aliquots of 5 ml were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the control vessels

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 10-30 and 100 mg/l
- Results used to determine the conditions for the definitive study: 74% inhibtion at 100 mg/l

Reference substance (positive control):

not required

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 9.10-15.2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

17.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 13.7-19.9

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

29.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 26.5-33.1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 7.8-11.5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

12.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 10.3-12.9

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 15.1-17.3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Determination of growth rate inhibition:
The ErCx and NOErC values of RCX 14-672 were assessed based on the measured concentrations (geometric mean, GM0–72h).
The median effect concentration with respect to growth rate (ErC50/0–3 days) of RCX 14-672 to Desmodesmus subspicatus was calculated to be 29.7 mg/l (95% confidence intervals: 26.5–33.1 mg/l). The low effect concentrations with respect to growth rate, ErC10 and ErC20, were calculated to be 12.4 mg/l (95% confidence intervals: 9.10–15.2 mg/l) and 17.1 mg/l (95% confidence intervals: 13.7–19.9 mg/l). The no observed effect concentration (NOErC) with respect to growth rate was 8.49 mg/l, as determined by Dunnett’s test.

Determination of algal yield (biomass production) inhibition:
The EyCx and NOEyC values of RCX 14-672 were assessed based on the geometric mean (GM0–72h) concentrations). The median effect concentration with respect to yield (EyC50/0–3 days) of RCX 14-672 to Desmodesmus subspicatus was calculated to be 15.8 mg/l (95% confidence intervals: 15.1–17.3 mg/l). The low effect concentrations with respect to yield, EyC10 and EyC20, were calculated to be 10.3 mg/l (95% confidence intervals: 7.84–11.5 mg/l) and 12.1 mg/l (95% confidence intervals: 10.3–12.9 mg/l). The no observed effect concentration (NOEyC) with respect to yield was 8.49 mg/l, as determined by Dunnett’s test.

Reported statistics and error estimates:

The statistical analysis with respect to ErCx/EyCx NOErC/NOEyC was run by excluding the lowest loading rate, 3.13 mg/l.

The inhibition at 3.13 mg/l loading was higher than at 6.25 and 12.5 mg/l loadings, respectively. This result cannot be explained. Moreover, given that the replicates of the 3.13 mg/l loading were quite variable, and that both 6.25 and 12.5 mg/l loadings showed no inhibition (as confirmed by Dunnett’s test), the results of the 3.13 mg/l loading can be regarded as outliers.

Validity criteria fulfilled:

yes

Executive summary:

The median growth inhibiting concentration (ErC50/EyC50), the low-effect concentrations (ErC10/EyC10,ErC20/EyC20) and the no-effect concentrations (NOErC/NOEyC) of RCX 14-672 to the green alga Desmodesmus subspicatus were investigated over a period of 72 h. This investigation was based on the average specific growth rate and the yield (algal biomass production) within 3 days.

The loading rates were 100, 50.0, 25.0, 12.5, 6.25 and 3.13 mg/l. Three parallel test vessels were used for each loading rate of the test item. Since RCX 14-672 has a limited water solubility (117,7 mg/l in water), the test concentrations were prepared by direct addition of the test item, stirring for 24 h followed by sterile filtration.

HPLC analyses were performed at the beginning and after 24, 48 h and 72 h of exposure. These analyses revealed that only part of the test item was dissolved; and that these concentrations declined over the whole test period, especially at low concentrations. The geometric mean of the measured concentrations (GM0-72h) corresponded to 81, 78, 57, 34, 8 and 36% of the initial values after 72 h at 100, 50.0, 25.0, 12.5, 6.25 and 3.13 mg/l loading rates, respectively. Therefore, the effective concentrations ECx and the no-observed-effect concentrations NOEC of RCX 14-672 were assessed based on the geometric mean of the measured concentrations (GM0-72 h).

The results of the growth rate (r) and yield (y) inhibition of RCX 14-672 to the green alga were reported (see "Results and Discussion") as EC values (Ec10, EC20 and EC50) with their 95% confidence limits based on the geometric mean of the measured concentrations (GM0–72h).

Description of key information

The EC50 for freshwater algae is 29.7 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

29.7 mg/L

EC10 or NOEC for freshwater algae:

12.4 mg/L