3-chloroperbenzoic acid

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:

11-Oct-2017 through 13-Feb-2018

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

Version / remarks:

According to OECD 209, with activated sludge dose response study, 5 concentrations, 5 replicates each, 6 control replicates, determination of total respiration including range-finder.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A0378021
- Expiration date of the lot/batch: 31 October 2019
- Purity: 72%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: Sparingly soluble
- Solubility and stability of the test substance in the solvent/vehicle: No vehicle was used
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No vehicle was used

REFERENCE ITEM DETAILS
- 3,5-Dichlorophenol
- CAS number: 591-35-5
- Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany
- Batch number: MKBZ0947V
- Expiration date of the lot/batch: 25 October 2022
- Purity: 100.0 %

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 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 dark brown 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 and subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Concentration: 3.2, 10, 32, 100, 320 mg/L


PREPARATION OF REFERENCE ITEM SOLUTION: 3,5-dichlorophenol
- Method: A 1.0 g/L stock solution was prepared by dissolving 250.8 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 in both the combined limit/range-finding and final test. The 3,5-dichlorophenol stock solution was stored in a freezer (≤-15℃) 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.
:- Concentration: 1.0, 3.2, 10, 32 mg/L

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas'
's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Pretreatment: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration:3.0 g/L of sludge

Test type:

static

Water media type:

other: 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

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

3 hours, during which aeration and stirring took place.

Hardness:

Synthetic medium 16 g peptone
(=sewage feed) 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.

Test temperature:

The temperature continuously measured in the temperature control vessels ranged between 19 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 7.5. After the 3 hour exposure period the pH was between 7.5 and 8.2.

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.

Nominal and measured concentrations:

- Nominal Concentration: 3.2, 10, 32, 100, 320 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles/vessels.
- Type: open
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1

Reference substance (positive control):

yes

Key result

Duration:

3 h

Dose descriptor:

other: NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: ELR10

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: ELR20

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: ELR50

Effect conc.:

64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Treatment	Loading rate (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)¹
Control		36.16	24.10
T1	3.2	32.84	21.89	9.17
T2	10	32.74	21.82	9.46
T3	32	28.64	19.09*	20.79
T4	100	11.36	7.57*	68.58
T5	320	0.33	0.22*	99.09

Conclusions:

Under the conditions of this present test 3- Chloroperoxybenzoic Acid was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 10 mg/L (NOELR).
The ELR10 was at a loading rate of 20 mg/L (95% confidence interval: 16 - 23 mg/L).
The ELR20 was at a loading rate of 29 mg/L (95% confidence interval: 25 - 33 mg/L).
The ELR50 was at a loading rate of 64 mg/L (95% confidence interval: 58 – 69 mg/L).

Executive summary:

The objective of the study was to evaluate 3- Chloroperoxybenzoic Acid for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the ELR₅₀and/or the no-observed effect loading rate (NOELR).

The influence of 3- Chloroperoxybenzoic 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 3- Chloroperoxybenzoic Acid tested had a purity of 72% (iodometric titration). 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 and subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. 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. Five loading rates were tested, ranging from 3.2 mg/L to 320 mg/L and increasing with a factor 3.2. Five replicates per loading rate and sixreplicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 10 mg 3- Chloroperoxybenzoic Acid per litre. At higher loading rates the inhibitory effect of 3- Chloroperoxybenzoic Acid on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 21% inhibition at 32 mg/L to 99% at 320 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.

3- Chloroperoxybenzoic Acid was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 10 mg/L (NOELR). The effect parameters for the inhibition of the respiration rate are presented below:

NOELR = 10 mg/L

ELR₁₀ = 20 mg/L (16 -23 mg/L)

ELR₂₀ = 29 mg/L (25 -33 mg/L)

ELR₅₀= 64 mg/L (58 -69 mg/L)

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

Description of key information

Under the conditions of this present test, 3- Chloroperoxybenzoic Acid was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 10 mg/L (NOELR).

The ELR10 was at a loading rate of 20 mg/L (95% confidence interval: 16 - 23 mg/L).

The ELR20 was at a loading rate of 29 mg/L (95% confidence interval: 25 - 33 mg/L).

The ELR50 was at a loading rate of 64 mg/L (95% confidence interval: 58 – 69 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

64 mg/L

EC10 or NOEC for microorganisms:

20 mg/L