Oxacycloheptadecan-2-one

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:

06 December 2016 - 16 December 2016

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:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Version / remarks:

2007

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Appearance: Colourless to very pale yellowish solid
- Test item storage: In refrigerator (2-8°C)
- Solubility in water: Insoluble
- Stability in water: Stable

Analytical monitoring:

no

Details on test solutions:

- Test substance: The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, 1-Litre test bottles were filled with 200 mL of test item mixtures in Milli- RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added to obtain loading rates of 10, 100 and 1000 mg/L.
- Reference substance: A 3,5-dichlorophenol solution with a final concentration of 1 g/L in Milli-RO water was prepared. The pH as used for the test was 7.7. 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. Four concentrations were tested: 1.0, 3.2, 10 and 32 mg/L.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: Micro-organisms in activated sludge form municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of sludge: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105 °C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.7 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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

17 - 22 °C

pH:

Start: 7.3 - 7.4
End: 7.2 - 8.1

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 10, 100 and 1000 mg/L. In addition, an abiotic control was tested at 1000 mg/L.
- Measured concentrations: no analysis performed (not required).

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass, open bottles/vessels.
- Aeration: yes, continuous with clean, oil-free air. 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.
- No. of vessels per concentration: 1 replicate at test concentrations of 10 and 100 mg/L, 3 replicates at test concentration of 1000 mg/L
- No. of vessels per blank control: 6
- No. of vessels per abiotic control: 1
- No. of vessels per reference control: 1
- Nutrients provided for bacteria: Synthetic medium (= sewage feed) containing 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2·2H2O, 0.2 g MgSO4·7H2O dissolved in Milli-RO water, made up to 1 litre and filtered.
- 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. Throughout the test, optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Sludge concentration: 3.0 g/L (weight of dry solids in sludge).
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: 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.

OTHER TEST CONDITIONS
- Temperature: The medium temperature was recorded continuously in a temperature control vessel(s). The temperature control vessel(s) 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.
- pH: The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls.
- Adjustment of pH: no
- Photoperiod: not relevant (3-h test)

EFFECT PARAMETERS MEASURED:
After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: Combined limit-range-finding test
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: not applicable (no effects up to 1000 mg/L)

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

other: NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: ELR50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

In the combined limit/range-finding no inhibition of the respiration rate was observed at any of the tested loading rates (for 1000 mg/L the mean value was considered). Therefore, the NOELR was considered to be the highest test loading rate. The ELR50 was above the highest loading rate tested (1000 mg/L). There was no significant oxygen uptake from abiotic processes.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Dose-response test: yes (test concentrations: 1.0, 3.2, 10 and 32 mg/L)
- EC50 = 5.9 mg/L

Reported statistics and error estimates:

- Determination of ELR50: For the reference item calculation of the EC50 value was based on a 3-parameter cumulative distribution definition (CDF) using a non-linear regression analyses with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item .
- Determination of NOELR: An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided, smaller).

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions®GmbH, Germany).

Table: Respiration rate/inhibition – Test substance

Loading rate (mg/L)	Replicate	Respiration rate		% inhibition of respiration rate (mean value)
		mg O2/L·h	mg O2/g·h
0	1	39.28	26.19	-
	2	39.53	26.35	-
	3	30.59	20.39	-
	4	44.65	29.77	-
	5	41.53	27.69	-
	6	47.35	31.57	-
	mean	40.49	26.99 (RC)	-
	SD	5.79	3.84	-
	CV (%)	14	14	-
10	1	40.83	27.22	-0.84
100	1	46.22	30.81	-14.16
1000	1	50.41	33.61	-24.51
	2	34.59	23.06	14.57
	3	38.36	25.57	5.26
	mean	41.12	27.41 (RT)	-1.56 (IT)

RC: Total respiration control

RT: Total respiration with test substance

IT: % inhibition of total respiration relative to Rc

 

Table: Respiration rate/inhibition – Reference control (R) and abiotic control (TA)

Loading rate (mg/L)	Replicate	Respiration rate		% inhibition of respiration rate (mean value)
		mg O2/L·h	mg O2/g·h
Reference control
1.0	1	35.15	23.43	13.18
3.2	1	22.59	15.06	44.21
10	1	17.46	11.64	56.88
32	1	8.76	5.84	78.36
Abiotic control
1000	1	0.26	0.17	99.36

 

Validity criteria fulfilled:

yes

Remarks:

see 'Overall remarks'

Conclusions:

Under the conditions of the test, the substance was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR). The ELR50 was >1000 mg/L.

Executive summary:

The influence of Oxacycloheptadecan-2 -one on the respiration rate of activated sludge was investigated in a study according to OECD TG 209 and in compliance with GLP criteria. In a combined limit/range-finding test, activated sludge of a predominantly domestic sewage was exposed to nominal loading rates of 0 (control), 10, 100 and 1000 mg/L for 3 hours. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, a reference control (at 1, 3.2, 10 and 32 mg/L) and abiotic control (at 1000 mg/L) were included in the test. As the test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water, Milli-RO water mixtures were magnetically stirred for a period of approximately 24 hours before being added to the synthetic medium, sludge and Milli-RO water. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during exposure. Thereafter, oxygen consumption was recorded for approximately 10 minutes. The reference control showed that the batch of activated sludge had normal sensitivity. No abiotic uptake was observed. The study met the acceptability criteria and was considered valid.

No statistically significant inhibition of the respiration rate of the sludge was recorded at any of the tested loading rates. Therefore, the substance is consluded to be not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR). The ELR50 was above 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L