4-hydroxybenzophenone

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:

21 Nov 2018 to 04 Jan 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))

Version / remarks:

Adopted 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: White powder
Purity/Composition: 99.72%
Test item storage: At room temperature desiccated

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Test Item – Preparation and Test Concentrations
The batch of 4-Hydroxy-benzophenone tested was a white powder with a purity of 99.72% (HPLC). 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 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 (see Table 1, Table 2 and Table 3). Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
Any residual volumes were discarded.

Reference Item – Preparation and Test Concentrations
The batch of activated sludge was checked for sensitivity by testing the reference item 3,5-dichlorophenol. 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 in both full tests. 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:

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 8.0 and 7.3 on the day of testing for the first and second full test, respectively. 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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Approximately 10 minutes

Hardness:

0.4 g CaCl2.2H2O

Test temperature:

20 ± 2°C

pH:

7.5 ± 0.5.

Dissolved oxygen:

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.

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

10, 100 and 1000 mg/L

Details on test 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.

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 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.

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.

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

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

7.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

First full test: EC50 = 6.1 mg/L
Second full test: EC50 = 2.3 mg/L

Reported statistics and error estimates:

First full test:
lower 95%-cl 2.4
upper 95%-cl 9.8


Second full test:
lower 95%-cl 1.9
upper 95%-cl 2.8

Combined Limit/Range-Finding Test

Detailed study results are presented in Table 9 of Appendix 1.

The combined limit/range-finding showed 11%, 59% and 96% 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 10 and 100 mg/L.

There was no oxygen uptake from abiotic processes.

The temperature continuously measured in the temperature control vessels ranged between 19.3 and 23.5°C during the test, and was slightly outside the range prescribed by the study plan (20 ± 2°C, see also Appendix 5).

All test conditions, except the temperature, and acceptability criteria prescribed by the study plan were met.

First Full Test

Inhibition of the Respiration Rate

Detailed study results are presented in Table 10 of Appendix 1.

A summarized overview of the study results is presented in Table 6.

No significant inhibition of the respiration rate of the sludge was recorded at 3.8 mg 4-Hydroxy-benzophenone per liter. At higher concentrations the inhibitory effect of 4-Hydroxy-benzophenone on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 17% inhibition at 12 mg/L to 92% at 400 mg/L.

The effects observed were not in agreement with what was expected based on the results of the combined limit/range-finding test. A second full test was performed to verify the results of the first full test.

Table 6       

First Full Test – Overview of Results

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)¹
Control		35.27	23.52
T1	3.8	31.92	21.28	9.52
T2	12	29.23	19.49	17.13
T3	39	26.24	17.49	25.61
T4	125	25.36	16.91	28.11
T5	400	2.95	1.97	91.63

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

Experimental Conditions - First Full Test

The pH values in the individual vessels are presented in Table 10 of Appendix 1.

The pH in all test vessels, before addition of sludge, was between 7.1 and 7.3. After the 3 hour exposure period the pH was between 8.4 and 8.5.

The temperature continuously measured in the temperature control vessels ranged between 21.1 and 23.6°C during the test, and was slightly outside the range prescribed by the study plan (20 ± 2°C, see also Appendix 7).

Second Full Test

Inhibition of the Respiration Rate

Detailed study results are presented in Table 11 of Appendix 1.

A summarized overview of the study results is presented in Table 7.

No statistically significant inhibition of the respiration rate of the sludge was recorded at 12 mg 4-Hydroxy-benzophenone per liter (for more information on statistics see Appendix 6).  At higher concentrations the inhibitory effect of 4-Hydroxy-benzophenone on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 35% inhibition at 39 mg/L to 89% at 400 mg/L.

The results of the second full test were in agreement with was expected from the combined limit/range-finding test. The results of this test allowed for reliable determination of a NOEC and EC_x values.

Table 7       

Second Full Test – Overview of Results

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)¹
Control		36.98	24.65
T1	3.8	31.17	20.78	15.7
T2	12	33.57	22.38	9.22
T3	39	24.01	16.01	35.08*
T4	125	9.48	6.32	74.37*
T5	400	3.92	2.61	89.40*

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

Experimental Conditions – Second Full Test

The pH values in the individual vessels are presented in Table 11 of Appendix 1.

The pH in all test vessels, before addition of sludge, was between 7.3 and 7.4. After the 3 hour exposure period the pH was between 7.3 and 8.0.

The temperature continuously measured in the temperature control vessels ranged between 20.3 and 22.6°C during the test, and was slightly outside the range prescribed by the study plan (20 ± 2°C, see also Appendix 7).

Determination of Effect Parameters

Table 8 shows the effect parameters based on 4-Hydroxy-benzophenone concentrations; see also Appendix 5 and Appendix 6.

Table 8       

Effect Parameters

Parameter	4-Hydroxy-benzophenone Concentration (mg/L)
NOEC	12
EC10	7.7 (4.3 - 14)
EC20	15 (10 - 24)
EC50	57 (43 - 75)

(   ) Between brackets the 95% confidence intervals are given.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present test 4-Hydroxy-benzophenone was not toxic to waste water bacteria (activated sludge) at or below a concentration of 12 mg/L (NOEC).
The EC10 was 7.7 mg/L (95% confidence interval: 4.3 - 14 mg/L).
The EC20 was 15 mg/L (95% confidence interval: 10 - 24 mg/L).
The EC50 was 57 mg/L (95% confidence interval: 43 - 75 mg/L).

Executive summary:

The objective of the study was to evaluate 4-Hydroxy-benzophenone 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 4-Hydroxy-benzophenone 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 4-Hydroxy-benzophenone tested was a white powder with a purity of 99.72% (HPLC). 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.

Both full tests were performed based on the results of a preceding combined limit/range-finding test. Five concentrations were tested, ranging from 3.8 to 400 mg/L, and increasing with a factor 3.2. Five replicates per concentration and six replicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at 12 mg 4-Hydroxy-benzophenone per liter (for more information on statistics see Appendix 6). At higher concentrations the inhibitory effect of 4-Hydroxy-benzophenone on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 35% inhibition at 39 mg/L to 89% at 400 mg/L.

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.

4-Hydroxy-benzophenone was not toxic to waste water (activated sludge) bacteria at a concentration of 12 mg/L (NOEC).

The effect parameters for the inhibition of the respiration rate are presented below.

Parameter	4-hydroxy-benzophenone Concentration (mg/L)
NOEC	12
EC10	7.7 (4.3 - 14)
EC20	15 (10 - 24)
EC50	57 (43 - 75)

( ) Between brackets the 95% confidence intervals are given.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC50 for microorganisms:

57 mg/L

EC10 or NOEC for microorganisms:

12 mg/L