Essential oil of Ylang Ylang III obtained from the flowers of Cananga odorata (Annonaceae) by steam distillation

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:

07 Dec 2017 - 05 Jan 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 July 2010

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Ylang Ylang III
Appearance: Pale yellow clear liquid
Batch: 17106567
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2019 (expiry date)

Additional information
Irritant or corrosive: Yes
Specific gravity / density: 0.914

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The batch of Ylang Ylang III tested was a pale yellow clear liquid (UVCB). 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. The test item was pipetted directly into the Milli-RO water and stirred briefly to mix. 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.

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
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The pH was 7.7 on the day of testing.
- Pretreatment: The batch of sludge was used one day after collection. Therefore, 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids, i.e. 3.0 g/L of sludge. The amount of suspended solids in the final test mixture was 1.5 g/L.

Test type:

not specified

Water media type:

other: water Tap water purified by reverse osmosis (Millipore Corp.,Bedford, Mass., USA)

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3-hour exposure period.

Test temperature:

20- 22°C

pH:

T 0hr control: 7.3 -7.6
T 3hr control: 7.9 - 8.1

T 0hr test item: 7.3
T 3hr test item: 7.8 - 8.2

T 0hr 3,5 DCP: 7.3 - 7.4
T 3hr 3,5 DCP: 8.1 - 8.3

Dissolved oxygen:

>60-70% of air saturation at test start

Nominal and measured concentrations:

Test substance
Nominal: 3.2, 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: all glass open bottles/vessels
- Total fill volume: 500 mL (250 mL test solution (incl. synthetic medium) + 250 mL activated sludge in test medium)
- Aeration: yes (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 (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per positive control (replicates): 4
- Sludge concentration (weight of dry solids per volume): 1.5 g/L sludge
- Nutrients provided for bacteria: synthetic medium acc. to OECD 209
- Nitrification inhibitor used : no

TEST MEDIUM / WATER PARAMETERS
- Source/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
- Adjustment of pH: no
- Details on termination of incubation: 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.
- 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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen uptake

TEST CONCENTRATIONS
- Spacing factor for test concentrations:factor 3.2
- Range finding study
In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/L were tested. The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. In addition, a control (6 replicates) and an abiotic control (1 replicate) were tested.
Test procedure and conditions were similar to those applied in the final test with the following exceptions:
•The pH of the sludge was 7.5 on the day of testing.
- Results used to determine the conditions for the definitive study: The combined limit/range-finding test showed 11% inhibition at 10 mg/L, no inhibition at 100 mg/L and 20% inhibition of the respiration rate at a concentration of 1000 mg Ylang Ylang III per liter.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

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:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Inhibition of the Respiration Rate:
The effects observed were in agreement with what was expected based on the results of the combined limit/range-finding test. The results of this test allowed for reliable determination of a NOEC and ECx values. No statistically significant inhibition of the respiration rate of the sludge was recorded at 320 mg Ylang Ylang III per L. At the highest concentration tested the inhibitory effect of Ylang Ylang III on aerobic waste water (activated sludge) bacteria was 17%.

Acceptability of the test:
1. The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (24 mg oxygen per one gram of activated sludge).
The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test (12 %).
2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (10.7 mg/L).

Results with reference substance (positive control):

The batch of activated sludge was tested for sensitivity with the reference item 3,5- dichlorophenol, and showed normal sensitivity.
EC50 = 10.7 mg/L (95% Cl: 8.0 - 13.5) (falls within the OECD reported range for total respiration, i.e. 2-25 mg/L)

Reported statistics and error estimates:

ECx:
For the reference item, calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For Ylang Ylang III, no EC50 value could be calculated because effects were below 50% (EC50 > 1000 mg/L).

NOEC determination:
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 (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Final Test – Overview of the 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		36.62	24.41
T1	3.2	33.41	22.28	8.74
T2	10	38.66	25.77	-5.59
T3	32	32.76	21.84	10.54
T4	100	38.88	25.92	-6.19
T5	320	36.90	24.60	-0.77
T6	1000	30.47	20.31*	16.79

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

* Statistically significantly different compared to control.

Validity criteria fulfilled:

yes

Remarks:

see details on results

Conclusions:

Ylang Ylang III was not toxic to waste water bacteria (activated sludge) at or below a concentration of 320 mg/L (NOEC). The EC50 was above 1000 mg/L.

Executive summary:

This study was performed to assess the effect of Ylang Ylang III on the respiration of activated sewage sludge according to OECD 209 and GLP guidelines. A final test was performed based on the result of a preceding combined limit/range-finding test. Microorganisms were exposed to six concentrations ranging from 3.2 to 1000 mg/L (5 replicates) and an untreated control group (6 replicates) for 3 hours. Optimal contact between the test item and test medium was ensured by applying continuous stirring and aeration during that period. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes (no aeration but continuous stirring).The study met the acceptability criteria prescribed by the study plan and was considered valid. No statistically significant inhibition of the respiration rate of the sludge was recorded at 320 mg Ylang Ylang III per L. At the highest concentration tested the inhibitory effect of Ylang Ylang III on aerobic waste water (activated sludge) bacteria was 17%.The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.To conclude, Ylang Ylang III was not toxic to waste water bacteria (activated sludge) at or below a concentration of 320 mg/L (NOEC). The EC50 was determined to be above 1000 mg/L.

Description of key information

This study was performed to assess the effect of Ylang Ylang III on the respiration of activated sewage sludge according to OECD 209 and GLP guidelines. A final test was performed based on the result of a preceding combined limit/range-finding test. Microorganisms were exposed to six concentrations ranging from 3.2 to 1000 mg/L (5 replicates) and an untreated control group (6 replicates) for 3 hours. Optimal contact between the test item and test medium was ensured by applying continuous stirring and aeration during that period. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes (no aeration but continuous stirring).The study met the acceptability criteria prescribed by the study plan and was considered valid. No statistically significant inhibition of the respiration rate of the sludge was recorded at 320 mg Ylang Ylang III per L. At the highest concentration tested the inhibitory effect of Ylang Ylang III on aerobic waste water (activated sludge) bacteria was 17%.The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.To conclude, Ylang Ylang III was not toxic to waste water bacteria (activated sludge) at or below a concentration of 320 mg/L (NOEC). The EC50 was determined to be above 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

320 mg/L

Additional information

It should be noted that the EC50 was determined to be above 1000 mg/L