Benzoic acid, 2-([1,1'-biphenyl]-4-ylcarbonyl)-

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:

From May 30, 2017 to June 02, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Because the test substance is poorly soluble in water, a saturated test solution was prepared according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. A saturated solution (stock solution with nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of the test substance into the test medium (ISO Medium) two days before the start of the study. This solution was shaken for about 24 h at approximately 30°C and then equilibrated for about 24 h at approximately 20°C. The non-dissolved test substance was removed by filtration through a fine (0.22 µm) filter to give the 100% saturated solution. The test solutions were prepared by dilutions of this stock solution in a series corresponding to: 100; 50; 25; 12,5; 6,25 [% saturated solution].

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, Germany. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
Justification of species: Pseudokirchneriella subcapitata is a fast-growing species, convenient for culturing and testing and is a recommended species by relevant guidelines.
Culture conditions: stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not specified

Test temperature:

The temperature was in the range of 22.7–22.8°C measured in the flask and between 21.9 and 23.5°C measured within the climate chamber. Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber.

pH:

The range of the pH was 7.21 – 8.81 during the experiment. The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test.

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Range-finding test:
Nominal: 0.1; 1; 10; 100 [% saturated solution]

Definitive test:
Nominal: 100; 50; 25; 12,5; 6,25 [% saturated solution]:
Measured concentrations - geometric mean: 4.75, 9.45, 18.90, 38.15 and 76.15 mg/L

Details on test conditions:

Light intensity:

The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 8373 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15% and therefore provided equal conditions for each test vessel.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 76.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: WAF

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

38.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 76.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: WAF

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: WAF

Basis for effect:

biomass

Details on results:

Although statistically significant differences were noted at the tested concentration range of 9.45 - 76.15 mg/L (measured) in case of growth rates, biomass and yield as well, the observed differences in case of growth rates (inhibition: µ = 3.5, 4.3 and 8.1 %) at 9.45, 18.90 and 38.15 mg/L, respectively and in case of biomass (inhibition: A = 9.6 %) at 9.45 mg/L are not biologically significant, but are considered to be due to the biological variability of the test system.
Therefore the NOEC was determined as 38.15 mg/L (measured) and LOEC was determined as 76.15 mg/L (measured) in case of growth rates, while the NOEC was determined as 9.45 mg/L (measured) and the LOEC was determined as 18.90 mg/L (measured) in case of biomass.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study with the reference substance Potassium dichromate is: 07 – 10 March 2017.

The 72h ErC 50: 0.88 mg/L, (95% confidence limits: 0.81 – 0.96 mg/L)
The 72h EyC 50: 0.54 mg/L, (95% confidence limits: 0.50 – 0.59 mg/L)
The 72h EbC 50: 0.64 mg/L, (95% confidence limits: 0.59 – 0.70 mg/L)

These values are within the range of laboratory ring test data.

Reported statistics and error estimates:

The specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (Days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50 and EbC50 values of the test item could not be calculated (due to the slight effect observed) and therefore determined directly from the raw data.
The EyC50 values of the test item was calculated using Probit analysis by TOXSTAT software.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h EC50 and NOEC of the test substance to aquatic algae were determined to be >76.15 mg/L and 38.15 mg/L (measured geometric mean), respectively.

Executive summary:

A study was conducted to determine the acute toxicity of the test substance, PBBA to the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum) according to OECD 201 Guideline, EU Method C.3 and EPA Guideline OPPTS 850.5400, in compliance with GLP. A water accommodated fraction (WAF) at a nominal loading rate of 100 mg/L was prepared for preliminary and main test. Inhibition was observed at the highest examined concentration level during the preliminary range-finding test. These results served as basis to select five test concentrations in a geometric series (factor 2.0) for the main experiment. Controls were added to demonstrate the adequacy of the procedure. Subsequent dilutions were made from the WAF stock solution in a geometric series corresponding to 6.25, 12.5, 25, 50 and 100% . Test concentrations were analytically determined at the start and end of the experiment. The test design included 3 replicates at each test concentration and 6 replicates for the untreated controls. The biological results are based on the measured test substance concentrations, which were found to be: 4.75, 9.45, 18.90, 38.15 and 76.15 mg/L (geometric mean). Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. All results were assessed to be significantly different from that of the control groups. Under the study conditions, the 72 h EC50 and NOEC of the test substance to aquatic algae, based on growth rate, were determined to be >76.15 and 38.15 mg/L (measured geometric mean), respectively (Sipos, 2017).

Description of key information

The 72 h EC50 and NOEC of the test substance to aquatic algae, based on growth rate, were determined to be >76.15 and 38.15 mg/L (measured geometric mean), respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

76.15 mg/L

EC10 or NOEC for freshwater algae:

38.15 mg/L

Additional information

A study was conducted to determine the acute toxicity of the test substance, PBBA to the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum) according to OECD 201 Guideline, EU Method C.3 and EPA Guideline OPPTS 850.5400, in compliance with GLP. A water accommodated fraction (WAF) at a nominal loading rate of 100 mg/L was prepared for preliminary and main test. Inhibition was observed at the highest examined concentration level during the preliminary range-finding test. These results served as basis to select five test concentrations in a geometric series (factor 2.0) for the main experiment. Controls were added to demonstrate the adequacy of the procedure. Subsequent dilutions were made from the WAF stock solution in a geometric series corresponding to 6.25, 12.5, 25, 50 and 100% . Test concentrations were analytically determined at the start and end of the experiment. The test design included 3 replicates at each test concentration and 6 replicates for the untreated controls. The biological results are based on the measured test substance concentrations, which were found to be: 4.75, 9.45, 18.90, 38.15 and 76.15 mg/L (geometric mean). Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. All results were assessed to be significantly different from that of the control groups. Under the study conditions, the 72 h EC50 and NOEC of the test substance to aquatic algae, based on growth rate, were determined to be >76.15 and 38.15 mg/L (measured geometric mean), respectively (Sipos, 2017).