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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-07 to 2017-07-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 209 with GLP statement. All validity criteria were fulfilled.
Justification for type of information:
Read-across from an analogue of the test item. Lavender oil (source data) and lavandin oil (target) originate from the same plant family (labiatae) and the same genus (lavandula). They are both obtained from flowering tops by steam distillation and they have similar composition.
In addition, it has been shown than both substances have similar ecotoxic effects, lavender oil being even more toxic to aquatic organisms than lavandin oil. Therefore, a similar toxicity pattern to microorganisms is expected for lavandin oil and lavender oil. Thereby, results from the experimental OECD 209 study assessing the toxicity of lavender oil to microorganisms can be extrapolated to the registered substance lavandin oil.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
signed on 14 January 2016
Specific details on test material used for the study:
Not applicable
Analytical monitoring:
no
Vehicle:
yes
Remarks:
deionized water
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Based on the Sponsor’s information, the test item is not soluble in water and was consider overall respiration only; it was directly diluted in osmosed water. No analytical estimation was done during this study.

- Preparation:
In order to evaluate information about the range of concentrations used in the main test, a range-finding test, non GLP, was done. The test solutions of the range finding test were: 10, 100 and 1000 mg/L. This test had indicated that inhibiting effects could be expected around 1000 mg test item /L (nominal concentration).
According to these preliminary results, 5 concentrations were tested (geometric series): 305 – 488 – 781 – 1250 and 2000 mg/L
According to OECD 209, the abiotic control do not need to be repeated because there was no oxygen uptake in the preliminary test.

- Protocol:
The test was carried out into 1000 mL glass flasks containing 500 mL as below:
- 16.0 ml synthetic sewage feed
- Adequate weight of test item and osmosed water for different concentrations (final volume: 500 mL)
- 250 mL of sludge suspension
Replicates: Six replicates for controls (Control a to Control f)
Three replicates (a to c) per treatment (1 to 5) for the test item
One replicate per treatment for the reference item (3,5 DCP)

The concentrations for the 3,5 DCP were : 0,5 – 1,6 – 5,1 – 16,4 – 52,4 mg/L

At the start of the test, the first control (Control a) was prepared and covered with parafilm. Then, every 10 -15 minutes the others flasks with test item and concentrations with reference item were prepared in turn. Finally, the last control (Control f) was prepared.

All suspensions were aerated with purified air (0.5 to 1 L/min), and stirred during 3 hours at 20 ± 2°C. Then, each suspension was poured into Karlsruher flask (following the order preparation).

The Oxygen was measured every minute, during 10 minutes, with a Dissolved Oxygen Sensor (WTW StirrOx G) integrated Stirrer, inserted into the Karlsruher flask.

- Test medium:
The medium was prepared from concentrated stock solutions in ultrapure water as described in the guideline.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: The inoculum was a fresh sample of activated sludge and was collected from the aeration tank of a sewage treatment plant receiving predominantly domestic sewage: STEP de Lille, 33290 BLANQUEFORT - FRANCE.
- Justification: Activated sludge micro-organisms have been selected following the recommendations of OECD Guideline No 209.
- Preparation of inoculum for exposure: A filtration through a fine sieve (around 1 mm) was performed in order to remove coarse particles. After removal of any coarse particles, the sludge was washed by decantation in osmosed water and test medium 50/50 (v/v) to adjust concentration of dry extract around 3 g/L.
The activated sludge was continuously aerated until usage in the test and fed daily with 50 mL/L synthetic sewage feed for one or two additional days.

Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
None
Test temperature:
20 ± 2 °C
pH:
7.6 to 8.2
Dissolved oxygen:
7.8 mg/L
Salinity:
None
Conductivity:
No data
Nominal and measured concentrations:
Nominal test item concentrations: 305 – 488 – 781 – 1250 and 2000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Test was performed in 1000 mL glass flasks containing 500 mL of incubation mixture: 16.0 ml synthetic sewage feed; Adequate weight of test item and osmosed water for different concentrations (final volume: 500 mL); 250 mL of sludge suspension.
- Synthetic Sewage Feed (The synthetic sewage feed was prepared to contain the following constituents per liter: 16 g peptone, 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). The pH of this solution was measured to be pH 7.5 +/- 0.5.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): Six blank controls

TEST MEDIUM / WATER PARAMETERS
Na2HPO4, 2H2O: 33.4 mg/l; CaCL2, 2H2O: 36.4 mg/l; K2HPO4: 21.75 mg/l; MgSO4, 7H2O: 22.5 mg/l; NH4Cl: 0.5 mg/l; FeCl3, 6H2O: 0.25 mg/l; KH2PO4: 8,5 mg/l
This mineral medium was diluted 50/50 (v/v) with osmosed water for the preparation of the activated sludge

EFFECT PARAMETERS MEASURED
- The inhibition of respiration of an activated sludge by the test item was performed in this test. The respiration rates of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of three hours.

TEST CONCENTRATIONS
- Range finding study: Based on the test guideline, a range-finding test using the following three nominal test item concentrations of 10 mg/L, 100 mg/L and 1000 mg/L was performed.
- Results used to determine the conditions for the definitive study: The results of the preliminary test showed an inhibitory effect at 1000 mg/L (nominal test item concentration)
Based on the results of the range-finding test and in agreement with the Sponsor a definitive test with the following nominal test item concentrations was performed (geometric series): 305 – 488 – 781 – 1250 and 2000 mg/L to primarily permit the determination of the 3h-EC50.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 230 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% Cl: 1000 - 1580 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
488 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
781 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
EC50 value including the associated lower and upper 95% confidence limit was calculated with Minitab 17 (scientific software). To compute the EC50, the inhibitions means (y) versus Logarithm concentrations (x) were subjected to regression analysis.

The equation was: Y=-268.3+103.0 Log(x)
Y: % average inhibition
X: concentration

With EC50 = 1230 mg/L (1000 – 1580)

NOEC/LOEC was determined with Dunnett test (Minitab 17):
The concentration 488 mg/L was stated as NOEC.
The concentration 781 mg/L was stated as LOEC.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 9.4 mg/L. The 3-hour EC50 is within the guideline-recommended range of 2–25 mg/L, confirming suitability of the activated sludge used.
Reported statistics and error estimates:
None

O2 consumption and inhibition

Treatment

Name

O2consumption in

mg O2/(L*h)

% inhibition

pH (end of the test)

Inoculum Control

Control a

43.500

/

8.0

Control b

32.667

/

8.2

Control c

45.000

/

8.1

Control d

50.571

/

8.1

Control e

51.429

/

8.2

Control f

48.000

/

8.2

Reference Item

3,5 DCP Conc 1

43.5

3.75

8.2

3,5 DCP Conc 2

39.3

13.04

8.1

3,5 DCP Conc 3

40.7

9.94

8.0

3,5 DCP Conc 4

10.7

76.32

7.8

3,5 DCP Conc 5

6.0

86.72

7.7

Test Item

1a

51.00

-12.8

7.7

1b

45.60

-0.9

7.9

1c

51.00

-12.8

7.9

2a

35.00

22.6

7.8

2b

46.00

-1.8

7.8

2c

50.00

-10.6

7.8

3a

32.00

29.2

7.7

3b

32.00

29.2

7.7

3c

34.00

24.8

7.8

4a

22.00

51.3

7.6

4b

21.33

52.8

7.6

4c

16.67

63.1

7.7

5a

14.67

67.5

7.6

5b

13.33

70.5

7.6

5c

12.67

72.0

7.6

 

-      Inoculum Control (Control a to f)

-      Reference item (3,5 DCP C1 to C5)

-      Test item 305 mg/L (1a to 1c)

-      Test item 488 mg/L (2a to 2c)

-      Test item 781 mg/L(3a to 3c)

-      Test item 1250 mg/L(4a to 4ac)

-      Test item 2000 mg/L (5a to 5c)

Validity of the Test

Validity criteria

Conformity for OECD 209

Conformity for the test

Oxygen uptake in the control

≥ 20mg O2/g activated sludge
(dry weight of suspended solids)/ h

Yes
(29.1 mg O2/g sludge/h)

Coefficient of variation of O2uptake rate in control replicates at the end of the definitive test

≤ 30%

Yes
(6.2 to 15.9%)

EC50of 3,5 dichlorophenol for total respiration

Between 2 to 25 mg/L

Yes
(9.4 mg/L)

The validity criteria for OECD 209 were successful.

Validity criteria fulfilled:
yes
Conclusions:
The 3-hour EC50 of the test item LAVENDER OIL POPULATIONwas found to be 1230 mg/L with confidence interval 95% between 1000 and 1580 mg/L. The 3-hour NOEC was determined to be 488 mg/L and the 3h-LOEC was 781 mg/L.
Executive summary:

The inhibitory effect of the test item LAVENDER OIL POPULATION on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the OECD Guideline 209 in compliance with GLP.

The study was performed using five concentrations, ranging from 305 to 2000 mg/L nominal concentrations. The dry matter of the activated sludge was determined as 3.11 g suspended solids/L, giving a concentration of 1.555 g suspended solids/L in this test.

As a positive control, the reference item 3,5-dichlorophenol was tested.

The result of this study is presented as follows:

 

Validity criteria

Conformity for OECD 209

Conformity for the test

Oxygen uptake in the control

≥ 20mg O2/g activated sludge
(dry weight of suspended solids)/ h

Yes
(29.1 mg O2/g sludge/h)

Coefficient of variation of O2uptake rate in control replicates at the end of the definitive test

≤ 30%

Yes
(6.2 to 15.9%)

EC50of 3,5 dichlorophenol for total respiration

Between 2 to 25 mg/L

Yes
(9.4 mg/L)

 

The validity criteria for OECD 209 were successful.

 

 

CE50-3h “LAVENDER OIL POPULATION”

(confidence interval 95%)

1230 mg/L

( 1000 –1580)

NOEC

488 mg/L

LOEC

781 mg/L

The results are presented in nominal test item concentration.

Description of key information

Based on a read-across from an experimental ASRIT study performed on the substance Lavender oil according to the OECD 209 guideline and under GLP conditions, the following toxicity value has been extrapolated to the registered substance:

-       3-hour EC50 = 1230 mg/L with confidence interval 95% between 1000 and 1580 mg/L;

-       3-hour NOEC = 488 mg/L;

-       3h-LOEC = 781 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 230 mg/L
EC10 or NOEC for microorganisms:
488 mg/L

Additional information

For that endpoint, a study on the registered substance was not available. Therefore, in order to assess the toxicity of the registered substance on microorganisms, the results from an experimental ASRIT study performed on the analogue substance Lavender oil have been used.

In this study, the inhibitory effect of the test item LAVENDER OIL POPULATION on the respiration rate of aerobic wastewater micro-organisms of activated sludge was determined. This study was investigated in a 3-hour respiration inhibition test according to the OECD Guideline 209 and in compliance with GLP.

The study was performed using five concentrations, ranging from 305 to 2000 mg/L nominal concentrations. The dry matter of the activated sludge was determined as 3.11 g suspended solids/L, giving a concentration of 1.555 g suspended solids/L in this test. As a positive control, the reference item 3,5-dichlorophenol was tested.

The results of the study presented in the box above are expressed in nominal test item concentration.

All the validity criteria were successful and the study respected the requirements of the guideline. This study was therefore considered acceptable for that endpoint and the read-across justification is provided in the iuclid study record.