1-ethylazepan-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:

08 Mar 2018 - 13 Apr 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 (Carbon and Ammonium Oxidation))

Version / remarks:

22 Jul 2010

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was pipetted directly into the amber test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis). 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.
- Differential loading: Yes
- Controls: Test medium without test item, treated in the same way as the test item solutions (6 replicates)

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: No
- Inoculum source: Municipal sewage treatment plant 'Watershap Aa en Maas', 's-Hertogenbosch, The Netherlands
- Preparation of sludge: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added.
- Pretreatment: The batch of sludge was used 1 d after collection. Therefore, 50 mL of synthetic medium ( = sewage feed) was added per Liter of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Sludge concentration (suspended solids): 3.0 g/L, as used for the test
- Final concentration of sludge at test start: 1.5 g/L (250 mL sludge in a final volume of 500 mL test solution)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 - 21 °C

pH:

Start: 7.4 - 7.5
End: 8.1 - 8.5

Nominal and measured concentrations:

Control, 100, 180, 320, 560, and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles
- Aeration: Yes, continuous aeration with clean, oil-free air throughout the test. The aeration was adjusted such that the dissolved oxygen concentration at the start was > 60-70% saturation (60% air saturation is > 5.0 mg/L at 20 °C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1 (only in combined limit/range-finding test)
- Sludge concentration (weight of dry solids per volume): 3.0 g/L
- Nutrients provided for bacteria: Synthetic feed
- Performance of the test: The synthetic medium (= 16 mL sewage feed) and an appropriate amount of test item stock were mixed and made up to 250 mL with Milli-RO water (tap water purified by reverse osmosis). Then, 250 mL activated sludge was added and this was the start of the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO-medium was prepared with RO water (tap water purified by reverse osmosis)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Other: The test vessels were constantly aerated and stirred during exposure.

EFFECT PARAMETERS MEASURED:
- Oxygen consumption: After 3 h contact time, the oxygen consumption was recorded for a period of approximately 10 min. During the measurement, the sample was not aerated but continuously sitrred on a magnetic stirrer.
- Oxygen recording: Oxygen measurements were performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multi-channel measuring and controlling system.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study : Yes, a combined limit/range-finding test was performed, in which the highest concentration level was tested in triplicate and lower concentration levels only in 1 replicate.
- Test concentrations: 10, 100, and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes. No inhibition, 4% and 32% inhibition of the respiration rate was observed at 10, 100, and 1000 mg/L treatment levels, respectively. The EC50 was above the highest concentration tested (1000 mg/L).

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

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:

EC10

Effect conc.:

446 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% confidence interval: 263 - 561 mg/L

Details on results:

- Adsorption: None
- Blank controls oxygen uptake rate: The mean control oxygen uptake rate was 31 mg oxygen per one gram of activated sludge in the final test and thus was higher than the required 20 mg oxygen/g activated sludge (dw of suspended solids).
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation of oxygen uptake in control replicates was 8% and thus did not exceed 30%.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 7.1 mg/L, 95% confidence interval: 3.9 - 10.3 mg/L

Reported statistics and error estimates:

Data analysis was performed with ToxRat Professional v3.2.1 (ToxRat Solutions GmbH, Germany).
EC10 and EC20 values were based on Probit analysis using linear maximum likelihood regression, with the percentage of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.
However, no EC50 values could be calculated for the test item because the effects were < 50% (EC50 > 1000 mg/L).
The NOEC was determined with a Williams Multiple Sequential t-test Procedure (alpha = 0.05, one-sided, smaller). An effect was considered significant if the statistical analysis of the data obtained for the test concentrations showed significant inhibition of the respiration rate compared to the control.

VALIDITY CRITERIA

The study fulfilled the validity criteria of the guideline (Table 1) and is thus valid.

 

Table 1: Validity criteria for OECD 209.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.	The mean oxygen uptake of the control was 31 mg oxygen per gram activated sludge.	Yes
The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	The coefficient of variation of oxygen uptake in control replicates was 8%.	Yes

 

RESULTS

 

No statistically significant inhibition of the respiration rate of the sludge was recorded at a test item concentration of 320 mg/L. At higher concentrations, the inhibitory effect of the test item on aerobic waste water (activated sludge) bacteria increased with increasing concentration, resulting in 14% inhibition at 560 mg/L and 31% at 1000 mg/L (Table 2).

However, the inhibitory effect on aerobic waste water bacteria observed at 1000 mg/L was below 50%. Therefore, the NOEC was considered to be the highest concentration tested where no statistically significant effects were observed. The effect values are summarized in Table 3.

 

Table 2. Results of the final test.

Treatment	Concentration [mg/L]	Mean respiration rate		mean % inhibition of the respiration rate
		[mg O2/L h]	[mg O2/g h]^1
Control	0	46.87	31.25
1	100	44.84	29.89	4.35
2	180	46.06	30.70	1.74
3	320	46.13	30.76	1.58
4	560	40.51	27.01*	13.58
5	1000	32.40	21.60*	30.87

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

* Statistically significantly different compared to control.

 

Table 3. Effect parameters of final test.

Parameter	test item concentration [mg/L]
NOEC	320
EC10	446 (263 – 561 mg/L)*
EC20	708 (563 – 846 mg/L)*
EC50	> 1000

*The 95% confidence intervals are given in brackets.

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

No inhibition of STP microorganisms expected. EC50 (3 h) > 1000 mg/L.

Additional information

One experimental study is available testing the toxicity of the substance to aquatic microorganisms. The study was conducted using activated sludge of a STP treating predominantly domestic sewage. The study procedure followed test guideline OECD 209 (GLP). The respiration of activated sludge in a control without test substance was compared to the oxygen consumed by the inoculum at test concentrations of 100, 180, 320, 560, and 1000 mg/L (nominal). The experimental duration was 3 hours. The determined EC50 (3 h) was > 1000 mg/L. Based on the results N-ethylcaprolactam is not expected to affect the degradation process in STPs.