Myrac Aldehyde

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:

2 - 20 July 2020

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 15 November 2018

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: 10, 100 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 (02/07/2020) 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 3 times with chlorine-free tap water and re-suspended in tap water. The activated sludge was aerated and fed with the synthetic sewage feed (30 mL per L of sludge suspension) until the next day (final test). The dry weight of the sludge suspension was determined, then the sludge was diluted in chlorine-free tap water to obtain the required sludge solids concentration of 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 20.1 and 22.7°C throughout the test (average value: 21.8°C) which was slightly higher than the requirements laid down in the study plan (20 ± 2 °C). This minor deviation was considered not to affect the results of the test as no impact was observed on the blank controls (all validity criteria of the test have been fulfilled; .

pH:

The pH of the reaction mixtures at the end of the test period ranged from 7.388 to 7.736.

Dissolved oxygen:

None

Salinity:

None

Conductivity:

No data

Nominal and measured concentrations:

Nominal test item concentration: 10, 100 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.:

10 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, an effect on the respiration rate of activated sludge was observed at 100 and 1000 mg.L-1 (average inhibition of 29.0 and 42.1%, respectively). Nevertheless, based on these results, the 3-hour EC50 was estimated to be ≥ 1000 mg.L-1. The highest nominal test concentration without observed effects (= NOEC) was 10 mg.L-1.

Results with reference substance (positive control):

The EC50 of 3,5-DCP was 6.8 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	23.140	-	7.736
Control (2)	0	25.174	-	7.705
Ref (1)	5.00	15.533	39.919	7.651
Ref (2)	8.40	11.773	54.461	7.582
Ref (3)	14.10	6.077	76.494	7.601
Ref (4)	23.70	2.965	88.531	7.577
Ref (5)	40.00	2.084	91.940	7.601
Control (3)	0	25.013	-	7.633
Control (4)	0	24.177	-	7.560
Test item	10.10	24.738	4.312	7.492
Test item	9.94	24.462	5.379	7.601
Test item	10.08	24.430	5.503	7.620
Test item	9.96	25.351	1.942	7.641
Test item	10.74	25.342	1.975	7.603
Test item	99.80	17.578	32.006	7.512
Test item	100.56	17.730	31.419	7.388
Test item	100.08	19.192	25.766	7.478
Test item	99.88	17.886	30.816	7.467
Test item	100.90	19.377	25.050	7.479
Test item	1001.08	15.211	41.162	7.480
Test item	1003.00	15.374	40.534	7.512
Test item	1001.74	15.093	40.219	7.475
Test item	1001.18	15.455	17.833	7.941
Test item	999.84	13.655	47.184	7.453
Control (5)	0	29.259	-	7.576
Control (6)	0	28.853	-	7.554

Environmental conditions throughout the test

The pH of the reaction mixtures at the end of the test period ranged from 7.388 to 7.736.

Temperatures recorded continuously were situated between 20.4 and 22.7°C throughout the test (average value: 21.8°C), which was slightly higher than the requirements laid down in the study plan (20 ± 2 °C). This minor deviation was considered not to affect the results of the test as no impact was observed on the blank controls (all validity criteria of the test have been fulfilled)

Validity of the Test

Blank controls:                      

- Mean oxygen uptake rate in the blank controls was 25.9 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 9.3% at the end of the definitive test (below the recommended upper limit of 30%).

Reference substance:

The EC50 of 3,5-DCP was 6.8 mg.L-1 for total respiration and thus remained within the prescribed range of 2 to 25 mg.L-1.

Thus all validity criteria of the test have been fulfilled in the present study, which can be considered valid.

Validity criteria fulfilled:

yes

Conclusions:

The effect of test item 3-cyclohexene-1-carboxaldehyde multiconstituent on microorganisms from activated sludge was investigated in a respiration inhibition test. Under the experimental conditions, and based upon nominal concentrations, the 3-hour EC50 value was estimated to be higher than 1000 mg test item.L-1. The NOEC value was 10 mg test item.L-1, and the LOEC 100 mg.L-1.

Executive summary:

A study was performed to assess the effects of test item 3-cyclohexene-1-carboxaldehyde multiconstituent on microorganisms from activated sludge by performing a respiration inhibition test. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 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 at nominalconcentrations of 10, 100 and 1000 mg test item.L^-1(five replicates for each concentration) and tosix blank controlsin order to determine a concentration with no significant effect and todemonstrate that EC₅₀was higher than 1000 mg.L^-1. Furthermore, an exposure of the test organism to chemical3,5-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 anactivated sludge concentration of about.L^-1of suspended solids.

After 3 hours of exposure under aeration and stirring,samples of each mixturewere poured into BOD bottles to continuously measure the oxygen concentrationusing an oxygen electrodeand determine the rate of decrease of dissolved O₂.The measurement of total oxygen uptake inhibition was determined only (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 an EC}₅₀of 6.8 mg.L^-1 for 3,5-DCP (within the prescribed range of 2 to 25mg.L^-1 for total respiration). At the end of the test, the 3-hour EC₅₀was estimated to be ≥ 1000 mg.L^-1, although an effect on the respiration rate of activated sludge was observed at 100 and 1000 mg.L^-1(average inhibition of 29.0 and 42.1%, respectively). No effect on the respiration rate was observed at 10 mg.L^-1.

The effect of test item 3-cyclohexene-1-carboxaldehyde multiconstituent on microorganisms from activated sludge was investigated in a respiration inhibition test. Under the experimental conditions, and based upon nominal concentrations, the 3-hour EC₅₀ value was estimated to be higher than 1000 mg test item.L^-1. The NOEC value was 10 mg test item.L^-1, and the LOEC 100 mg.L^-1.

Description of key information

The effect of test item 3-cyclohexene-1-carboxaldehyde multiconstituent on microorganisms from activated sludge was investigated in a respiration inhibition test. Under the experimental conditions, and based upon nominal concentrations, the 3-hour EC50 value was estimated to be higher than 1000 mg test item.L-1. The NOEC value was 10 mg test item.L-1, and the LOEC 100 mg.L-1.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

10 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 00, 100 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.