Reaction mass of 1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane and cineole

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:

2016-12-06 to 2017-08-03

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

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

Specific details on test material used for the study:

Not applicable

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, a limit test was performed at 1000 mg test item.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 (05/12/2016) 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:

yes

Total exposure duration:

3 h

Post exposure observation period:

None

Hardness:

None

Test temperature:

between 17.8 and 19.5°C throughout the test.
This was slightly lower than the requirements as laid down in the study plan (20°C ± 2 °C). This minor deviation was considered not to affect the results of the test as no impact was observed on the controls respiration at the end of the test.

pH:

The pH of the reaction mixtures at the end of the test period ranged from 7.882 to 8.184

Dissolved oxygen:

None

Salinity:

None

Conductivity:

No data

Nominal and measured concentrations:

Nominal test item concentration: 1000 mg/L (limit test)

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 1.593 g.L-1 of suspended solids.

STUDY DESIGN
- 6 replicates bottles containing synthetic sewage feed, test water and inoculum (blank controls)
- 3 replicates bottles for the limit test concentration containing synthetic sewage feed, test water, test item and inoculum (test mixtures)
- one replicate per reference concentration containing synthetic sewage feed, test water, reference substance and inoculum (reference mixtures)
(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.:

>= 1 000 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, no effect on the respiration rate of activated sludge was observed at the limit test concentration of 1000 mg.L-1, confirming the observations of the range-finding test where results showed no significant effect at any of the tested concentrations.

Results with reference substance (positive control):

The EC50 of 3,5-DCP was 13.681 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.
Statistical analyses were performed by the software ToxRat® Professional

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	29.844	-	8.107
Control (2)	0	28.869	-	8.105
Ref (1)	5.0	27.292	13.854	8.070
Ref (2)	8.4	26.965	14.884	8.023
Ref (3)	14.1	13.680	56.821	8.070
Ref (4)	23.7	5.862	81.496	8.184
Ref (5)	40.0	4.399	86.115	8.166
Control (3)	0	35.031	-	7.937
Control (4)	0	32.634	-	8.009
Test item	1002.2	31.695	-0.045	7.882
Test item	1001.3	32.041	-1.136	7.920
Test item	1000.9	31.515	0.524	7.987
Control (5)	0	32.764	-	7.984
Control (6)	0	30.944	-	8.008

Environmental conditions throughout the test

The pH of the reaction mixtures at the end of the test period ranged from 7.882 to 8.184

Temperatures recorded continuously were situated between 17.8 and 19.5°C throughout the test. This was slightly lower than the requirements as laid down in the study plan (20°C ± 2 °C). This minor deviation was considered not to affect the results of the test as no impact was observed on the controls respiration at the end of the test.

Validity of the Test

Blank controls:                      

- Mean oxygen uptake rate in the blank controls was 31.681 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 7.1% at the end of the definitive test (below the recommended upper limit of 30%).

Reference substance: The EC50 of 3,5-DCP was 13.681 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 and the highest nominal concentration without observed effects values were estimated to be higher than 1000 mg test item.L-1. Thus, the NOEC value estimated was ≥ 1000 mg test item.L-1.

Executive summary:

The inhibitory effect of test item REACTION MASS OF 1,4-CINEOLE AND 1,8-CINEOLE on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3-hour respiration inhibition test according to OECD Guideline 209 in compliance with GLP.

A limit test was performed following the results of a range-finding test. Microorganisms from activated sludge were exposed to the test item at a nominal concentration of 1000 mg test item.L^-1(3 replicates) and tosix blank controls. 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 1.593 g.L^-1of 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 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 anoxygen uptake rate in the blank controls higher than 20 mg O₂.g^-1.h^{-1, a coefficient of variation of oxygen uptake rate in control replicates below 30% and a 3,5-DCP-EC}₅₀ of 13.681 mg.L^-1 for total respiration (within the prescribed range of 2 to 25 mg.L^-1). After 3 hours of exposure, no effect on the respiration rate of activated sludge was observed at the limit test concentration of 1000 mg.L^-1, confirming the observations of the range-finding test where results showed no significant effect at any of the tested concentrations.

Therefore, based on nominal concentrations, the 3-hour EC50 and the highest nominal concentration without observed effects values were estimated to be higher than 1000 mg test item.L^-1. Thus, the NOEC value estimated was ≥ 1000 mg test item.L^-1.

Description of key information

Based on nominal concentrations, the 3-hour EC50 and the highest nominal concentration without observed effects values were estimated to be higher than 1000 mg test item.L^-1.

Thus, the NOEC value estimated was ≥ 1000 mg test item.L-1.

Therefore, the substance is considered as not harmful to microorganisms.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

A reliable experimental study was available in order to assess the inhibitory effect of test item REACTION MASS OF 1,4-CINEOLE AND 1,8-CINEOLE 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 OECD Guideline 209 and in compliance with GLP.

A limit test was performed following the results of a range-finding test. As a positive control, reference item 3,5-dichlorophenol was tested.

The validity of the test was demonstrated by an oxygen uptake rate in the blank controls higher than 20 mg O₂.g^-1.h^{-1, a coefficient of variation of oxygen uptake rate in control replicates below 30% and a 3,5-DCP-EC}₅₀ of 13.681 mg.L^-1 for total respiration (within the prescribed range of 2 to 25 mg.L^-1). After 3 hours of exposure, no effect on the respiration rate of activated sludge was observed at the limit test concentration of 1000 mg.L^-1, confirming the observations of the range-finding test where results showed no significant effect at any of the tested concentrations.

The test item is classified as not harmful and the study was considered acceptable for that endpoint.