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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 January 2019 to 26 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: 2-Benzoylbenzoic acid
Appearance: Off-white powder
Stable under storage conditions until: 11 March 2020 (expiry date)
Vehicle:
no
Details on test solutions:
Test Item:
The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the amber coloured test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The test item – Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added, resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring. Any residual volumes were discarded.

Reference Item:
The batch of activated sludge was checked for sensitivity by testing the reference item 3,5-dichlorophenol.
For the combined limit range finding, a 1.0 g/L stock solution was prepared by dissolving 250.5 mg 3,5-dichlorophenol in Milli RO water and making up to a total volume of 250 mL. The pH as used for the test was 7.8. For the two full tests and the final test, a 1.0 g/L stock solution was prepared by dissolving 249.1 mg 3,5-dichlorophenol in Milli RO water and making up to a total volume of 250 mL. The pH as used for the test was 7.9. The 3,5 dichlorophenol stock solution was stored in a freezer (≤-15°C) until use. The reference item solution was defrosted at room temperature and diluted to reach the test concentrations. Volumes of 0.5, 1.6, 5.0 and 16 mL from the stock solution were added to the test medium, resulting in the final test concentrations of 1.0, 3.2, 10 and 32 mg/L, respectively.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Test system: Micro-organisms in activated sludge.
- Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas','s -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of the sludge: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The concentration of the suspended solids was determined (3.0 g/L, as used for the test). The pH was 7.5 on the day of testing. The batch of sludge was used one day after collection. Therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Medium: Adjusted ISO-medium, formulated using RO water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L
- Rationale: Recognized by international guidelines as the recommended test system.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
The temperature continuously measured in the temperature control vessels ranged between 20 and 21°C during the test, and complied with the requirements as laid down in the Study Plan (20 ± 2°C).
pH:
The pH in all test vessels, before addition of sludge, was between 6.5 and 7.6. After the 3 hour exposure period the pH was between 5.4 and 8.2.
The pH in the neutralized pH vessels, before neutralisation, was between 5.7 and 7.3. The vessel with a pH below 7 (two vessels) were neutralized to a pH of 7.0. After the 3 hour exposure period the pH was between 5.7 and 7.1. The two neutralized pH vessels didn’t remain stable during the test period (pH 5.7 and 6.4).
Nominal and measured concentrations:
2-Benzoylbenzoic acid
Five concentrations: 3.2, 10, 32, 100, 320, 1000 mg/L and 1000 mg/L with neutralized pH at the start of the test.
Replicates: 5 replicates per test group and 6 replicates for the control.
Control: Test medium without test item, treated in the same way as the test item solutions.
Details on test conditions:
Final Test

Test Procedure and Conditions
- Contact time: 3 hours, during which aeration and stirring took place.
- Vessels: All glass open bottles/vessels.
- Milli-RO / Milli-RO water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
- Synthetic medium (=sewage feed):
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
- Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.
- Air supply: Clean, oil-free air.
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- Oxygen recording: Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

Performance of the test: The synthetic medium (16 mL) made up to 50 mL with Milli- RO and 200 mL test item solution were mixed (total volume 250 mL). The pH was determined. Thereafter, 250 mL activated sludge was added. This was the start of the test. After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels was/were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
not specified
Details on results:
Combined Limit/Range-Finding Test
The combined limit/range-finding showed 7%, 6% and 99% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. Therefore, the expected EC50 was at a concentration between 100 and 1000 mg/L.
There was an oxygen release of 9%, from abiotic processes. Since this value was considered to be unreliable, a second abiotic control was performed. The second abiotic control showed no significant oxygen uptake or release from abiotic processes.
The temperature continuously measured in the temperature control vessels ranged between 20 and 23°C during the test, and was slightly outside the range prescribed by the Study Plan (20 ± 2°C).
All test conditions, except for the temperature, and acceptability criteria prescribed by the Study Plan were met.

First Full Test
Inhibition of the Respiration Rate
The effects observed were lower with what was expected based on the results of the combined limit/range-finding test.
In the present test 2-Benzoylbenzoic acid was toxic to waste water (activated sludge) bacteria at all concentrations tested. Therefore, a NOEC could not be determined. Based on these results it was decided to perform a second full test with lower concentrations

Second Full Test
Inhibition of the Respiration Rate
The effects observed were slightly lower with what was expected based on the results of the combined limit/range-finding test.
In the second full test 2-Benzoylbenzoic acid was toxic to waste water (activated sludge) bacteria at all concentrations tested. Therefore, a NOEC could not be determined. The effect observed at 1000 mg/L was lower than the inhibition observed at the combined limit range-finding and the first full test. It should be noted that the effect of the test item on the pH at 1000 mg/L, is different in all the three tests performed. The higher pH at the end of the test could explain the lower inhibition of the respiration rate. Based on these results it was decided to perform a final test with lower concentrations and an addition of a 1000 mg/L with a neutralized pH at the start of the test.

Final Test
Inhibition of the Respiration Rate
The effects observed were comparable with what was expected based on the results of the combined limit/range-finding test.
No statistically significant inhibition of the respiration rate of the sludge was recorded at and below 100 mg 2-Benzoylbenzoic acid per litre. At higher concentrations the inhibitory effect of 2-Benzoylbenzoic acid on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 24% inhibition at 320 mg/L to 85% at 1000 mg/L. The results of the 1000 mg/L with neutralized pH at the start of the test showed an inhibition of the respiration rate ranging from no inhibition to 89% inhibition. The variance in the inhibition of the respiration rate might related to the pH at the end of the test. Since stabilisation of the pH above 6.0 is not possible, the EC50 cannot me determined for a neutralized pH.
The results of this test allowed for reliable determination of a NOEC and ECx values. It should be noted that the EC50 value is an worst case value, since the pH of the solution at the end of the test might influenced the inhibition of the respiration rate, which resulted in a lower EC50 value.
Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present test 2-Benzoylbenzoic acid was not toxic to waste water bacteria (activated sludge) at or below a concentration of 100 mg/L (NOEC).
The EC10 was 229 mg/L (95% confidence interval: 145 - 300 mg/L).
The EC20 was 312 mg/L (95% confidence interval: 221 - 388 mg/L).
The EC50 was 528 mg/L (95% confidence interval: 425 - 634 mg/L).
Executive summary:

The objective of the study was to evaluate 2-Benzoylbenzoic acid for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the no - observed effect concentration (NOEC).

The influence of 2-Benzoylbenzoic acid on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based on the OECD guideline No. 209, 2010.

The batch of 2-Benzoylbenzoic acid tested was an off-white powder with a purity of 98.8%.  No correction was made for the purity/composition of the test item.

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L.  Therefore, weighed amounts were added to the test bottles containing Milli-RO water.  The test item – Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, synthetic medium, sludge and Milli - RO water were added, resulting in the required concentrations.  Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period.  Thereafter, oxygen consumption was recorded for approximately 10 minutes.

The final test was performed based on the result of a preceding combined limit/range-finding test and in total three full tests were performed due to the different effect of the test item on the pH of the test solution. The final study was used to determine the NOEC and EC50 value.. In this final, six concentrations were tested, ranging from 3.2 to 1000 mg/L and increasing with a factor 3.2. In addition an extra concentration of 1000 mg/L was performed with a neutralized pH at the start of the test. Five replicates per concentration and six replicates of an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at 100 mg 2-Benzoylbenzoic acid per Litre. At higher concentrations the inhibitory effect of 2 Benzoylbenzoic acid on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 24% inhibition at 320 mg/L to 85% at 1000 mg/L.  The results of the 1000 mg/L with neutralized pH at the start of the test showed an inhibition of the respiration rate ranging from no inhibition to 89% inhibition. Due to the variation in the inhibition at 1000 mg/l with neutralized pH at the start of the test, the EC50 for neutralized pH could not be determined.

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.

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

2-Benzoylbenzoic acid was not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/L (NOEC).

Description of key information

Study performed under GLP requirements.

In conclusion, under the conditions of the present test 2-Benzoylbenzoic acid was not toxic to waste water bacteria (activated sludge) at or below a concentration of 100 mg/L (NOEC).

The EC10 was 229 mg/L (95% confidence interval: 145 - 300 mg/L).

The EC20 was 312 mg/L (95% confidence interval: 221 - 388 mg/L).

The EC50 was 528 mg/L (95% confidence interval: 425 - 634 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:
528 mg/L

Additional information