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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 January 2019 to 23 May 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))

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: 2-(4-Phenylbenzoyl)benzoic acid
Batch/Lot Number: Sarex #7650
CAS number: 42797-18-2
EC number: 700-862-4
Description: white powder
Manufacture Date: 27 September 2016
Expiry Date: 31 December 2019
Purity: 99.6 %
Molecular weight: 302.32 g/mol
Molecular formula: C20H14O3
Storage Conditions: Controlled room temperature (15-25°C, below 70 RH%),
protected from light and humidity
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety. Causes skin irritation and serious eye damage

Analytical monitoring:

no

Remarks:

See below statement from the study director

Details on sampling:

No chemical analysis of the dose formulation as performed as part of this study. Traceability (equipment used, quantities of the test substance weighed, etc) of dosing form preparations was checked and revealed no abnormalities of consequences. Furthermore, for this study, the formulations were prepared just before the treatment. Consequently, the absence of dose formulation analysis data is considered not to prejudice the overall GLP status of the study and the sceintific reliability of the study conclusions.

Vehicle:

yes

Details on test solutions:

FORMULATION
The test substance was added by directly weighing the test substance into the bottles (and using ultrasonic bath for 10 minutes). The test formulation was freshly prepared at the beginning of the experiment, in the testing laboratory.
The pH of the solutions was checked but not adjusted due to the direct application of the test substance.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Species: Activated sludge, microorganisms from a domestic waste water treatment plant.

Source: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary

Conditioning: The activated sludge was supplied by the sewage plant for domestic sewage two days before the start of the experiment. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage per litre and kept aerated at 20 ± 2°C until use. The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in chlorine-free tap water and again centrifuged.

An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined.

Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.05. The activated sludge was used directly after conditioning.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

19.2 – 21.8 °C

pH:

7.25 - 8.11

Nominal and measured concentrations:

Preliminary test: 10, 100 and 1000 mg/L (nominal)
Definitive test: 89.41,143.05, 228.88, 366.21, 585.94, 937.50, 1500 mg/L (nominal)

Details on test conditions:

CONTROLS
Abiotic Control (A)
The abiotic control did not need to be tested as there was no oxygen uptake in the preliminary test.

Untreated Control (B)
Eight replicates (B1 – B8) of the untreated control group (deionised water, synthetic sewage and inoculum, without addition of the test item) were tested in parallel with all of the test series.

Reference Control (REF)
In parallel with the test item, the reference item 3,5-Dichlorophenol (REF1, REF2, REF3, REF4) was tested (at nominal test concentrations of 1.0; 3.2; 10 and 32 mg/L) under identical test conditions.
A stock solution of 3,5-Dichlorophenol was prepared according to the OECD Guideline No. 209 (1.0 g of powdered 3,5-Dichlorophenol dissolved in 1000 mL of water). Warm deionised water and ultrasonic bath was used to help the dissolution of the reference substance and the volume of the solution was made up to 1.0 L with deionised water and then cooled to room temperature. The pH of the solution was checked and adjusted, with NaOH solution (2 mol/L) to pH 7.45.

TEST CONDITIONS
Test units
-Type and size: Appropriate glass beakers for 500 mL volume and BOD bottles with 300 mL volume.
-Identification: Each test flask was uniquely identified with study code, treatment and replicate codes.

Test conditions
-Surrounding type: Temperature controlled laboratory
-Temperature: 19.2 – 21.8 °C
-Aeration: With compressed air (about 1 L/min)
-Recording: Test conditions were measured with suitable instruments and documented in the raw data.

PERFORMANCE OF THE TEST
Preliminary
Preparations of the test flasks
- One test solution with a final volume of 400 mL (ratio of composition of each test mixture referring to 500 mL according to the guideline) was prepared per treatment in a glass flask.
- A volume of 12.8 mL synthetic sewage and an adequate amount of the test substance were placed into the test flask by direct addition and filled up with deionised water to 200 mL before the start of the incubation. At the start of the test, 200 mL activated sludge inoculum with a sludge concentration of 3 g per litre of suspended solids was added to all flasks, with the exception of the abiotic control flasks.
- Abiotic controls in 5 parallels (A1, A2, A3, A4 and A5), and one untreated blank (B1) control was started as the first step of the test. Then at appropriate time intervals of approximately 30 minutes the further test groups were started. The second series were 4 different concentrations (without replicates) of the reference substance (REF1, REF2, REF3, REF4) and to the second blank control (B2). The third series was the 5 replicates of the test substance concentration of 10 mg/L nominal concentration (TI11, TI12, TI13, TI14 and TI15) and the third blank control (B3). The fourth series was the five replicates of test substance concentration 100 mg/L nominal concentration (TI21, TI22, TI23, TI24 and TI25) and the fourth blank control (B4). The final series was the five replicates of test substance concentration 1000 mg/L nominal concentration (TI31, TI32, TI33, TI34 and TI35) and the fifth blank control (B5).

In summary, 3 test substance concentration series and one abiotic control series were prepared in 5 parallels with one blank control series by series. In parallel with the above test mixtures four reference substance concentrations without replicates were also prepared with one blank control.
Since significant inhibition effect (on the total respiration rate of the microorganisms of the activated sludge) was detected in the preliminary test further testing was required. Influence of test item on oxygen consumption of activated sludge in the preliminary test are summarised in the table: Please check in the "Any other information on results incl. tables" section.

Definitive test
Preparations of the test flasks
- One test solution with a final volume of 400 mL (ratio of composition of each test mixture referring to 500 mL according to the guideline) was prepared per treatment in a glass flask.
- A volume of 12.8 mL synthetic sewage and an adequate amount of the test substance given into the flask by direct addition was filled up with deionised water to 200 mL before the start of the incubation. At the start of the test 200 mL activated sludge inoculum with a sludge concentration of 3 g per litre of suspended solids was added.
- Four reference controls (REF1–REF4) with different concentrations and one untreated blank (B1) control were started as the first step of the test. Then at appropriate time intervals of approximately 30 minutes the further test groups were started. The second series were the five replicates of the lowest test substance concentration of 89.41 mg/L (TI11, TI12, TI13, TI14, TI15) and the second blank control (B2).
- The third series was the five replicates of the next test substance concentration of 143.05 mg/L (TI21, TI22, TI23, TI24, TI25) and the third blank control (B3) and accordingly, to the highest test substance concentration of 1500 mg/L (TI71, TI72, TI73, TI74 and TI75) and the last blank control (B8).The abiotic control did not need to be tested as there was no oxygen uptake in the preliminary test.

In summary seven concentration series were prepared with 5 replicates and with one blank control series by series. In parallel with the above test mixtures four reference substance concentrations without replicates, were also prepared with one blank control.
The purpose of the test was only to assess the effects on total oxygen uptake and inhibition effect in respect of nitrification process was not determined in agreement with the Sponsor.
Influence of test substance on oxygen consumption of activated sludge in the definitive test are summarised in the table: Please check in the "Any other information on results incl. tables" section.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 810.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

(calculated based on the oxygen uptake rate and the percentage of inhibition of the respiration rate)

Remarks on result:

other: with 95 % confidence limits of 706.69 – 930.50 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

ca. 322.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

(calculated based on the oxygen uptake rate and the percentage of inhibition of the respiration rate)

Remarks on result:

other: with 95% confidence limits of 278.76 - 372.53 mg/L

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

ca. 2 040.54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

(calculated based on the oxygen uptake rate and the percentage of inhibition of the respiration rate)

Remarks on result:

other: with 95% confidence limits of 1604.67 – 2594.81 mg/L)

Details on results:

INHIBITION OF RESPIRATION RATE:

- In comparison to the inoculum controls the inhibition of the total respiration rate in the case of the activated sludge was between -5.27 % and 60.06 % in the examined concentration range of 89.41 – 1500 mg/L of test substance
- The average specific total respiration rate (RTS) of the untreated (Blank) controls (B1-B7) was 26.89 mg O2/L/h/g solid, so the validity criteria were met.
- The variation coefficient of oxygen uptake rate in above untreated replicates was 7.24 % and thus, the validity criteria were met.
- With respect to the inhibitory effect of the test substance on the total respiration rate, the 3 h EC50 value of test substance was determined to be in the range of 100 mg/L to 1000 mg/L (810.91 mg/L was calculated, with 95 % confidence limits of 706.69 – 930.50 mg/L).

EC20: 322.25 mg/L (278.76 – 372.53 mg/L)
EC50: 810.91 mg/L (706.69 - 930.50 mg/L)
EC80: 2040.54 mg/L (1604.67 – 2594.81 mg/L)

Results with reference substance (positive control):

INHIBITION OF RESPIRATION RATE:

- The following nominal concentrations of the reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as in the case of the test substance: 1.0, 3.2, 10.0 and 32.0 mg/L.
- In comparison to the controls the inhibition of the total respiration rate of the activated sludge was 76.02 % at the highest nominal concentration of 32.0 mg/L.
- At the nominal concentrations of 1.0, 3.2 and 10.0 mg/L 8.01 %, 24.24 % and 34.55 % inhibition of the respiration rate was calculated respectively.
- The 3 h EC50 of 3,5-Dichlorophenol was calculated to be 12.55 mg/L with 95% confidence limits of 9.67 – 16.30 mg/L.
- The EC50 values of the positive control substance 3,5-Dichlorophenol (3,5-DCP) were in the recommended range for total respiration using activated sludge derived from domestic sewage as well.

Reported statistics and error estimates:

The 3 h EC20, EC50 and EC80 values of the test substance and the 3 h EC50 values of the reference substance with their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.

Details on results:

Influence of test substance on oxygen consumption of activated sludge in the Preliminary test:

 

Test group		Conc. of test substance in test mix. (mg/L)	Total oxygen consumption rate (RT)(mg O2/L/h)	Specific respiration rate (RS) (mg O2/L/h/g solid)	Inhib. (%)	pH values^#		Temperature (C°) (measured in the Laboratory) *	Variation coefficient of O2 consumption rate (%)
ID	Name					start	end
A1	Abiotic	1000	-0.21	–	–	7.24	7.68	20.6/21.9	–
A2		1000	-0.79	–	–	7.24	7.72		–
A3		1000	-0.95	–	–	7.10	7.68		–
A4		1000	-0.46	–	–	7.05	7.65		–
A5		1000	-0.21	–	–	7.04	7.63		–
B1	Blank	0.00	32.22	21.48	–	7.09	7.15	20.6/21.9	6.49
B2^1		0.00	29.10	19.40	–	7.36	7.73
B3^1		0.00	28.80	19.20	–	7.49	8.00
B4		0.00	31.70	21.13	–	7.54	8.07
B5		0.00	33.42	22.28	–	7.52	8.05
REF1	Ref. item 3,5- DCP	1.00	25.60	17.07	15.86	7.35	7.67	20.6/21.9	–
REF2		3.20	20.54	13.70	32.15	7.36	7.68		–
REF3		10.00	16.18	10.78	46.22	7.36	7.71		–
REF4		32.00	5.78	3.85	79.70	7.36	7.73
TI1	Test item	10	31.53	21.02	-3.23	7.49	7.99	20.6/21.9	–
TI2		100	31.06	20.71	-1.72	7.55	8.08		–
TI3		1000	15.16	10.11	49.48	7.52	8.08		–

#: Individual pH values of the BOD flasks, except TI1-3, where the average values are reported

*:   Minimum / Maximum temperature (°C) measured in the Laboratory, during the experiment

Influence of test substance on the total oxygen consumption of activated sludge during the definitive test:

Test group		Conc. of test substance in test mix. (mg/L)	Total oxygen consumption rate (RT) (mg O2/L/h)	Specific respiration rate (RTS) (mg O2/L/h/g solid)	Inhib. (%)	pH values^#		Temperature (C°) (measured in the Laboratory)*	Variation coefficient of O2 consumption rate (%)
ID	Name					start	end
B1	Blank	0.00	43.20	28.80	–	7.31	7.68	19.2/21.8	7.24
B2		0.00	44.23	29.49	–	7.25	7.72
B3		0.00	42.60	28.40	–	7.41	7.56
B4		0.00	41.64	27.76	–	7.46	7.93
B5		0.00	38.80	25.87	–	7.49	8.04
B6		0.00	37.08	24.72	–	7.49	8.05
B7		0.00	36.76	24.51	–	7.52	8.08
B8		0.00	38.40	25.60	–	7.77	8.03
REF1	Ref. item	1.00	37.11	24.74	8.01	7.31	7.67	19.2/21.8	–
REF2		3.20	30.56	20.37	24.24	7.32	7.66		–
REF3		10.00	26.40	17.60	34.55	7.28	7.69		–
REF4		32.00	9.68	6.45	76.02	7.30	7.67		–
TI1	Test item	89.41	42.46	28.31	-5.27	7.25	7.72	19.2/21.8	–
TI2		143.05	39.79	26.52	1.37	7.42	7.54		–
TI3		228.88	35.23	23.49	12.67	7.46	7.95		–
TI4		366.21	27.46	18.30	31.94	7.49	8.04		–
TI5		585.94	20.84	13.89	48.35	7.50	8.09		–
TI6		937.5	17.16	11.44	57.47	7.56	8.11		–
TI7		1500	16.11	10.74	60.06	7.83	8.05		–

#: Individual pH values of the BOD flasks, except TI1-3, where the average values are reported

*:   Minimum / Maximum temperature (°C) measured in the Laboratory, during the experiment

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 3 h EC50 value of the of the study substance was determined to be 810.91 mg/L.

Executive summary:

A study was conducted to determine the effects of the of test substance, PBBA on the respiration rate of activated sludge microorganisms according to OECD Guideline 209, in compliance with GLP. Initially a preliminary test was conducted, where a significant inhibition effect on the total respiration rate of the microorganisms of the activated sludge was detected. Then a definitive test was conducted where the activated sludge was exposed to the test substance at nominal concentrations of 89.41, 143.05, 228.88, 366.21, 585.94, 937.50 and 1500 mg/L for 3 h. The abiotic control was not tested again as there was no oxygen uptake in the preliminary test. The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate. Based the inhibitory effect of the test substance on the total respiration rate, the 3 h EC50 value of test substance was determined to be 810.91 mg/L nominal (calculated, with 95 % confidence limits of 706.69 – 930.50 mg/L) (Sipos, 2019).

Description of key information

The 3 h EC50 value of the test substance for inhibition of respiration rate of the activated sludge was determined to be 810.91 mg/L (nominal).

Key value for chemical safety assessment

EC50 for microorganisms:

810.91 mg/L

Additional information

A study was conducted to determine the effects of the of test substance, PBBA on the respiration rate of activated sludge microorganisms according to OECD Guideline 209, in compliance with GLP. Initially a preliminary test was conducted, where a significant inhibition effect on the total respiration rate of the microorganisms of the activated sludge was detected. Then a definitive test was conducted where the activated sludge was exposed to the test substance at nominal concentrations of 89.41, 143.05, 228.88, 366.21, 585.94, 937.50 and 1500 mg/L for 3 h. The abiotic control was not tested again as there was no oxygen uptake in the preliminary test. The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate. Based the inhibitory effect of the test substance on the total respiration rate, the 3 h EC50 value of test substance was determined to be 810.91 mg/L nominal (calculated, with 95 % confidence limits of 706.69 – 930.50 mg/L) (Sipos, 2019).