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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Nov 2017 - 08 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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 : Cedarwood Oil Virginia
Appearance: Pale yellow to yellow liquid
Batch: 1002960562
Purity/Composition: 100.0% (UVCB)
Test item storage: At room temperature
Stable under storage conditions until: 28 February 2018 (expiry date)

Additional information
Test facility test item number: 207801/A
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name:Essential oil of Junipers Virginiana L. (Cupressaceae) obtained from the wood by steam distillation
pH (1% in water, indicative range): 4.57– 4.85 (determined by Charles River Den Bosch)
Specific gravity/density: 0.945-0.965 (d 20/20)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The batch of Cedarwood Oil Virginia tested was a pale yellow to yellow 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 7.5 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: 7.6
T 3hr control: 7.6-7.9

T 0hr test item: 7.5 - 7.6
T 3hr test item: 7.6-7.9

T 0hr 3,5 DCP: 7.6
T 3hr 3,5 DCP: 7.9-8.1
Dissolved oxygen:
>60-70% of air saturation at test start
Nominal and measured concentrations:
Test substance
Nominal: 20, 60, 180, 540 and 1620 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 3.0
- Range finding study
- Test concentrations: loading rates of 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes; The combined limit/range-finding showed no inhibition at a concentration of 10 mg/L, and 14% and 32% inhibition of the respiration rate at a concentration of 100 and 1000 mg/L, respectively. Therefore, the expected EC50 was at a concentration above 1000 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 620 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.:
301 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% CL: 69-685 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
60 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 generally as expected, but slightly lower than 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 60 mg Cedarwood Oil Virginia per L. Cedarwood Oil Virginia showed a statistically significant inhibitory effect of 12%, 10% and 17% at a concentration of 180, 540 and 1620 mg/L, respectively.

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 (7%).
2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (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 = 6.94 mg/L (95% Cl: 3.36 - 10.62) (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 Cedarwood Oil Virginia, no EC50-value could be calculated because effects were below 50% (EC50 > 1620 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

 

36.46

24.30

 

T1

20

35.43

23.62

2.82

T2

60

35.75

23.83

1.94

T3

180

32.20

21.47 *

11.67

T4

540

32.88

21.92 *

9.80

T5

1620

30.23

20.15 *

17.09

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:
Cedarwood Oil Virginia was not toxic to waste water bacteria (activated sludge) at or below a concentration of 60 mg/L (NOEC). The EC10 and EC50 were 301 mg/L (95% confidence interval: 69 - 685 mg/L) and >1620 mg/L respectively.
Executive summary:

This study was performed to assess the effect of Cedarwood Virginia oil 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 five concentrations ranging from 20 to 1620 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 60 mg Cedarwood Oil Virginia per L. Cedarwood Oil Virginia showed a statistically significant inhibitory effect of 12%, 10% and 17% at concentrations of 180, 540 and 1620 mg/L, respectively.The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.The EC10 and EC50 were determined as 301 mg/L (95% confidence interval: 69 - 685 mg/L) and >1620 mg/L respectively.

Description of key information

This study was performed to assess the effect of Cedarwood Virginia oil 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 five concentrations ranging from 20 to 1620 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 60 mg Cedarwood Oil Virginia per L. Cedarwood Oil Virginia showed a statistically significant inhibitory effect of 12%, 10% and 17% at concentrations of 180, 540 and 1620 mg/L, respectively.The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.The EC10 and EC50 were determined as 301 mg/L (95% confidence interval: 69 - 685 mg/L) and >1620 mg/L respectively.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
301 mg/L

Additional information

EC50 > 1620 mg/L