Dibutyl peroxydicarbonate

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:

28 September - 5 October 2017

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

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: 1233236-01
- Expiration date of the lot/batch: 15-12-2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: storage in original container, keep away from heat and direct sun irradiation
- Stability under test conditions: instable

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant Staufener Bucht was used as a test system. The treatment plant has a capacity of 140,000 inhabitant equivalents and clarifies predominantly domestic wastewater. The sampling date of the activated sludge was 4 October 2017. The activated sludge was kept aerated until use at test temperature. The activated sludge was washed twice by settling sludge, decanting the supernatant and re-suspending the sludge in chlorine-free tap water. The dry solid content of the activated sludge was determines as 4.5 mg/L by weight measurements before and after drying at 105 °C (mean of triplicate measurements). Before using, the activated sludge was diluted to 3 g/L dry solids with clorine-free tap water to obtain a final concentration of 1.5 g/L solids in the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20.4-20.7 °C

pH:

7.3

Dissolved oxygen:

4.99-7.63 mg/l

Nominal and measured concentrations:

100mg/L test material nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: Breakers à 1000 ml, Schott, Mainz, Germany
- Type (delete if not applicable): Open
- Fill volume: 500 mL for set-up
- Test materials:
- Membrane pump for aeration connected with glass pipes with PolyEthylene (PE)-tubes
- Graduated cylinder 1000 ml, Schott, Mainz, Germany
- Graduated cylinder 250 ml, Schott, Mainz, Germany
- Graduated cylinder 50 ml, Schott, Mainz, Germany
- Graduated pipette 20, Brand, Wertheim, Germany
- Adjustable pipettes, 1000 µl and 5 mL, Eppendrof, Wesseling, Berzdorf, Germany
- Oxygen electrode and meter Multi 9430, WTW, Weilhem, Germany
- 250 - 300 ml BOD bottles with adapter for oxygen electrode, Germany
- pH electrode and pH meter pH 330, WTW, Weilheim, Germany
- Scale BP 221 S, Sartorius, Göttingen, Germany
- Min/Max.-thermometer

- Sludge concentration (weight of dry solids per volume): 1.5 g/L

TEST MEDIUM / WATER PARAMETERS
Chlorine-free tap water was used.

SYNTHETIC SEWAGE FEED
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2 * 2 H2O
0.2 g MgSo4 * 7 H2O
2.8 g K2HPO4
Dissolved in demineralised water and made up to 1 litre.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen consumption/ Respiration rate

TEST CONCENTRATIONS
Limit test

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 9.24 mg/L

Reported statistics and error estimates:

The EC50 calculation was performed with the ToxRat software by probit analysis using linear max. likelihood regression (ToxRat Standard Version 2.09, © ToxRat Solutions GmbH). The EC50 was calculated from the slope of the curve.

Table 1: Respiration rate of activated sludge after application of the test item

No.	Vessel	O2 concentration [mg/L]		consumption rate [mg/(l*h)]	consumption rate [mg/(g sludge*h)]	inhibition [%]
		first measurement	last measurement
1	Blank 1	7.04	5.19	44.4	29.6
2	Blank 2	7.43	5.64	43	28.6
3	Blank 3	7.86	6.85	24.2	16.2
17	Blank 4	7.39	5.89	36	24
18	Blank 5	7.43	5.93	36	24
19	Blank 6	8.17	6.63	37	24.6
12	test item 1 (100 mg/L)	6.35	4.47	45.1	30.1	-23
13	test item 2 (100 mg/L)	7.63	6.3	31.9	21.3	13
14	test item 3 (100 mg/L)	4.99	2.82	52.1	34.7	-42
15	test item 4 (100 mg/L)	5.34	3.62	41.3	27.5	-12
16	test item 5 (100 mg/L)	7.62	6.47	27.6	18.4	25
	coefficient of variation of blanks:		0.18
	mean consumption rate blanks:		24.5
	mean consumption rate test vessels:		26.4

Validity criteria fulfilled:

yes

Conclusions:

The inhibition observed in the test vessels with 100 mg/L test concentration was between -42 and 25 % but the mean respiration rates of the test vessels were not significantly lower than the respiration rate of the controls (Student's t-test).
EC50 > 100 mg/L
NOEC ≥ 100 mg/L
PEROXAN NBC-50 had no toxic effect on the total respiration rate of activated sludge micro-organism at the concentration of 100 mg/L after the exposure period of 3 hours.

Executive summary:

The study procedure was based on OECD Guideline No. 209 (July 22nd, 2010) and performed under GLP.

A limit test with the concentration of 100 mg/L was conducted. The mean respiration rate in the treated test vessels was 26.4 mg oxygen consumption per gram sludge per hour. The mean respiration rate in the blank control was 24.5 mg/g*h. Hence, the mean respiration rate in the treated test vessels was even slightly higher than in the blank controls. This shows that the test item had no toxic effect on the total respiration rate of activated sludge microorganism at the concentration of 100 mg/L after the exposure period of 3 hours. Additionally the result of the student-t test showed no significant difference between the treated vessels abd the blank control.

Since all validity criteria of the test were met, this study is considered to be valid.

EC₅₀(3 h, nominal) > 100 mg/L

LOEC                     > 100 mg/L

NOEC (3 h, nominal) ≥ 100 mg/L

Description of key information

An activated sludge respiration inhibition test conducted according to OECD guideline 209 and under GLP showed no toxic effect on microorganisms up to 100 mg/L.

As the test substance has peroxide content of 50.6 %, the NOEC for the peroxide itself is 50.6 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

50.6 mg/L

Additional information

To investigate potential harmful effects of the test item on microorganisms, an activated sludge respiration inhibition test was performed with domestic sewage sludge according to the OECD guideline 209 and under GLP. The inhibition observed in the test vessels with 100 mg/L test concentration was -42 and 25 % but the mean respiration rates of the test vessels were not significantly lower than the respiration rate of the controls (Student's t-test). Therefore, an EC50 of > 100 mg/L and a NOEC of ≥ 100 mg/L were determined.