Reaction mass of (R*,R*)-7-methoxy-3,7-dimethyl-2-octanol and (R*,S*)-7-methoxy-3,7-dimethyl-2-octanol

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:

2017-02-20 to 2017-XX-XX

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.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

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. QA statement)

Remarks:

signed on 10 January 2017

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

ADMINISTRATION OF THE TEST AND REFERENCE SUBSTANCE
The test vessels were 1 L cylindrical glass bottles closed with PTFE screw caps. A magnetic stirring bar was placed in each test vessel. Blank controls were prepared with 16 mL synthetic sewage feed and 234 mL test water. Test mixtures were prepared by addition of the required amounts of test item in pre-filled test vessels (with synthetic sewage feed and test water). The 3,5-DCP stock solution was diluted with test water as necessary to obtain the required test concentrations into reference mixtures after filling with synthetic sewage and water. Then, 250 mL sludge suspension were sequentially added in all test vessels. After addition of the inoculum, the test vessels were immediately closed and the mixtures were stirred under aeration for 3 hours.
The test was carried out without adjustment of the pH.

TEST AND REFERENCE CONCENTRATIONS
- Test mixtures: Based on the results of the range-finding test, test solutions were prepared to obtain the following nominal test concentrations: 100, 320 and 1000 mg test item.L-1 in order to determine a NOEC and to demonstrate that EC50 was higher than 1000 mg.L-1.
- Controls: Test water without test item but treated in the same way as the test mixtures.
- Reference mixtures: The 3,5-DCP stock solution was diluted to obtain the following nominal concentrations (spaced by a factor of approximately 1.68): 5.0, 8.4, 14.1, 23.7 and 40.0 mg 3,5-DCP.L-1.

SYNTHETIC SEWAGE FEED
The medium was prepared by adding each of the following constituents at the stated amounts to each litre of ultrapure water:
Peptone: 16 g; Meat extract: 11 g; Urea: 3 g; NaCl: 0.7 g; CaCl2.2H2O: 0.4 g; MgSO4.7H2O: 0.2 g; K2HPO4: 2.8 g
The pH of this solution was 7.5 ± 0.5. This nutrient solution was refrigerated and stored in the dark before use.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Origin: Activated sludge was collected (20/02/2017) from a wastewater treatment plant (Aureilhan, France) receiving predominantly domestic sewage.
- Pre-treatment: Upon arrival in the test facility, the sludge was sieved (using a 1 mm2 mesh sieve), washed 4 times with tap water and re-suspended in tap water. The activated sludge was aerated and fed with the synthetic sewage feed (50 mL per L of sludge suspension) until the next day (final test). The dry weight of the sludge suspension was determined and was adjusted to approximately 3.0 g.L-1.

Test type:

not specified

Water media type:

other: Deionised water, containing less than 1 mg.L-1 DOC (Dissolved Organic Carbon).

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None

Hardness:

None

Test temperature:

between 18.01 and 21.51°C throughout the test (average value: 20.59°C). Comply with the requirements (20°C ± 2°).

pH:

The pH of the reaction mixtures at the end of the test period ranged from 7.788 to 8.167

Dissolved oxygen:

None

Salinity:

None

Conductivity:

No data

Nominal and measured concentrations:

Nominal test item concentration: 100, 320 and 1000 mg test item.L-1

Details on test conditions:

TEST PROCEDURE AND CONDITIONS
- Test duration: 3 hours
- Test vessels: All-glass bottles of approximately 1 L capacity closed with PTFE screw caps, containing 500 mL of reaction mixture. Each test vessel was uniquely identified with study code, replicate number, start time of the test and type of series. At the end of incubation, samples were withdrawn to measure the rate of decrease of dissolved O2 in completely filled all-glass BOD (Biological Oxygen Demand) bottles of approximately 300 mL capacity.
- Test water: Deionised water, containing less than 1 mg.L-1 DOC (Dissolved Organic Carbon).
- Inoculum: The activated sludge concentration in all test, reference and blank mixtures was approx. 1.5 g.L-1 of suspended solids.

STUDY DESIGN
- 6 replicates bottles containing synthetic sewage feed, test water and inoculum (blank controls)
- 5 replicates bottles per test concentration containing synthetic sewage feed, test water, test item and inoculum (test mixtures)
- 1 replicate per reference concentration containing synthetic sewage feed, test water, reference substance and inoculum (reference mixtures)
The test item was not known to have strong reducing properties, therefore it was judged not necessary to perform abiotic controls.
(The content of the test vessels is given in "additional information on materials and methods" section).
- Test environment: Controlled environment room (20°C ± 2°C)
- Aeration: All suspensions were aerated and stirred during the exposure period.

MEASUREMENT AND RECORDING
- Dissolved [02] (DO): After the exposure period, samples of each mixture were poured into BOD bottles to continuously measure the oxygen concentration using an oxygen electrode and determine the rate of decrease of dissolved O2. The measurement of total oxygen uptake inhibition was only determined (carbon and ammonium oxidation not separated).
- pH: At the end of the test from all test, reference and control mixtures.
- Temperature of medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the study period.

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:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

After 3 hours of exposure, a slight effect on the respiration rate of activated sludge was observed at 320 and 1000 mg.L-1, confirming the observations of the range-finding test. Based on these results, the highest nominal test concentration without observed effects (= NOEC) was 100 mg.L-1; the 3-hour EC50 was estimated to be ≥ 1000 mg.L-1.

Results with reference substance (positive control):

The EC50 of 3,5-DCP was 12.58 mg.L-1 for total respiration and thus remained within the prescribed range of 2 to 25 mg.L-1.

Reported statistics and error estimates:

None

Table 6.1.7/2: Test results (oxygen uptake rate / inhibition, pH at the end of the test).

Content	Concentration (mg.L^-1)	Oxygen uptake rate (mg O2.g^-1.h^-1)	Inhibition (%)	pH
Control (1)	0	33.869	-	7.921
Control (2)	0	35.839	-	7.928
Ref (1)	5.01	28.847	12.178	8.078
Ref (2)	8.41	25.966	20.948	7.888
Ref (3)	14.22	12.213	62.819	8.167
Ref (4)	23.83	5.845	82.205	8.097
Ref (5)	40.06	3.648	88.895	8.087
Control (3)	0	31.019	-	7.910
Control (4)	0	32.931	-	7.897
Test item	100.82	n.d.	n.d.	7.895
Test item	100.72	30.211	8.025	7.921
Test item	100.44	n.d.	n.d.	7.884
Test item	100.44	32.828	0.059	7.918
Test item	100.44	33.125	-0.846	7.901
Test item	320.68	n.d.	n.d.	7.863
Test item	321.90	28.241	14.022	7.976
Test item	321.76	29.407	10.472	7.818
Test item	323.02	28.333	13.744	7.945
Test item	320.86	28.780	12.382	7.916
Test item	1001.40	27.540	16.155	7.903
Test item	1001.28	27.083	17.547	7.905
Test item	1000.90	27.682	15.725	7.980
Test item	1001.10	26.989	17.833	7.941
Test item	1004.98	27.098	17.502	7.898
Control (5)	0	30.842	-	7.797
Control (6)	0	32.586	-	7.788

n.d.: not determined sinceafter 3 hours of exposure, the oxygen uptake observed at 1000 mg.L^-1was so fast that it was not possible to plot a graph of oxygen concentration versus time (8 mg O₂.L^-1to < 2 mg O₂.L^-1were consumed in less than 30 s).

Environmental conditions throughout the test

The pH of the reaction mixtures at the end of the test period ranged from 7.788 to 8.167.

Temperatures were situated between 18.01 and 21.51°C throughout the test (average value: 20.59°C), and complied with the requirements (20°C ± 2°).

Validity of the Test

Blank controls:                      

- Mean oxygen uptake rate in the blank controls was 32.847 mg O₂.g^-1dry weight activated sludge.h^-1, and therefore was well above the (minimum) required value of 20 mg O₂.g^-1.h^-1.

- The coefficient of variation of oxygen uptake rate in control replicates was 5.7% at the end of the definitive test (below the recommended upper limit of 30%).

Reference substance:

The EC50 of 3,5-DCP was 12.58 mg.L-1 for total respiration and thus remained within the prescribed range of 2 to 25 mg.L-1.

The validity criteria for OECD 209 were successful.

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal concentrations, the 3-hour EC50 value was higher than 1000 mg test item.L-1. The estimated NOEC value was 100 mg test item.L-1.

Executive summary:

The inhibitory effect of test item 7-METHOXY-3,7-DIMETHYLOCTAN-2-OL MULTICONSTITUENT on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3-hour respiration inhibition test according to Guideline OECD 209 and in compliance with GLP.

A definitive test was performed following the results of a range-finding test. Microorganisms from activated sludge were exposed to the test item, over a range of nominal concentrations of 100, 320 and 1000 mg test item/L (five replicates per test concentration), and to six blank controls in order to determine a NOEC, and to demonstrate that EC₅₀was higher than 1000 mg/L. Furthermore, an exposure of the test organism to chemical 3,5-dichlorophenol(3,5-DCP) was performed in order to check the sensitivity of the batch of activated sludge used on the day of exposure. All the mixtures contained an activated sludge concentration of about 1.5 g/L of suspended solids. After 3 hours of exposure under aeration and stirring, samples of each mixture were poured into BOD bottles to continuously measure the oxygen concentration using an oxygen electrode and to determine the rate of decrease of dissolved O₂. The measurement of total oxygen uptake inhibition was only determined (carbon and ammonium oxidation not separated).

The validity of the test was demonstrated by an oxygen uptake rate in the blank controls higher than 20 mg O2/g/h^{, a coefficient of variation of oxygen uptake rate in control replicates below 30% and an EC}₅₀ of 12.58 mg/L for 3,5-DCP (within the prescribed range of 2 to 25 mg/L for total respiration). After 3 hours of exposure, a slight effect on the respiration rate of activated sludge was observed at 320 and 1000 mg/L (but not exceeding 20% inhibition), confirming the observations of the range-finding test. No effect on the respiration rate was observed at 100 mg/L.

Therefore, based on nominal concentrations, the 3-hour EC50 value was higher than 1000 mg test item/L. The estimated NOEC value was 100 mg test item/L.

Description of key information

Based on nominal concentrations, the 3-hour EC50 value was higher than 1000 mg test item/L. The estimated NOEC value was 100 mg test item/L.

The substance is therefore considered as non-harmful for microorganisms.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

A reliable experimental study was available in order to assess the inhibitory effect of the test item on the respiration rate of aerobic wastewater micro-organisms of activated sludge. This study was investigated in a 3-hour respiration inhibition test according to Guideline OECD 209 in compliance with GLP.

Microorganisms from activated sludge were exposed to the test item over a range of nominal concentrations of 100, 320 and 1000 mg test item.L^-1(five replicates per test concentration) and to six blank controls. As a positive control, reference item 3,5-dichlorophenol was tested.

This study complied with the requirements of the guideline because all the validity criteria were respected. The test item is classified as not harmful and the study was considered acceptable for that endpoint.