6-methyl-3,4-dihydro-2H-1,4-benzoxazine

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2019 - 01 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6-methyl-3,4-dihydro-2H-1,4-benzoxazine

EC Number:

837-106-9

Cas Number:

71472-57-6

Molecular formula:

C9H11NO

IUPAC Name:

6-methyl-3,4-dihydro-2H-1,4-benzoxazine

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identity: 6-methyl-3,4-dihydro-2H-1,4-benzoxazine
- CAS Number: 71472-57-6
- Batch: 1394901004
- Purity: 99.8% [a/a]
- Physical State/Appearance: Light yellow liquid
- Expiry Date: 01 September 2019
- Storage Conditions: Approximately 4 °C in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The pH of the test item stock solutions were measured using a Hach HQ40d Flexi handheld meter and dissolved oxygen meter

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item was dissolved in water with the aid of ultrasonication and vigorous shaking for approximately 15 minutes and the volume adjusted to 1 liter to give required stock solution from which dilutions were made. An aliquots of the stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge and water, to a final required volume and concentration. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity.
- Controls: Yes

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Composition of synthetic sewage:
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

Study design

Test type:

not specified

Water media type:

other: deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC)

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

Not specified

Test temperature:

20 - 21 °C

pH:

7.3 - 7.4

Dissolved oxygen:

Dissolved oxygen concentrations of the test preparations after 30 minutes contact time in the Definitive Test (mg O2/L):
Control: 5.88 - 7.83
Test item 3.2 mg/L: 6.81 - 7.82
Test item 10 mg/L: 6.47 - 6.72
Test item 32 mg/L: 5.96 - 6.59
Test item 100 mg/L: 6.11 - 7.01
Test item 320 mg/L: 5.97 - 7.33
Reference: 7.61 - 8.95

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Range finding test (mg/L): 10, 100 and 1000
Definitive test (mg/L): 3.2, 10, 32, 100 and 320

Details on test conditions:

TEST SYSTEM
-Fill volume: 500 mL
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per reference (replicates): 1
- Suspended solids concentration: 3.0 g/L

OTHER TEST CONDITIONS
- Adjustment of pH: yes with 1.0 M NaOH
- Photoperiod: normal laboratory lighting:

TEST CONCENTRATIONS
- Range finding study : Yes

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

ca. 64 mg/L

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 32 mg/L

Details on results:

It was not possible to determine an EC80 value for the test item as no concentration tested resulted in greater than 80% inhibition.

No statistically significant toxic effects were shown at the test concentrations of 3.2, 10 and 32 mg/L; however statistically significant toxic effects (P < 0.05) were shown at the test concentrations of 100 and 320 mg/L.

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.

Results with reference substance (positive control):

The reference item, 3,5 dichlorophenol, gave a 3 Hour EC50 value of 5.8 mg/L, 95% confidence limits 4.3 to 7.8 mg/L confirming the acceptability of the inoculum used in the test system.

Reported statistics and error estimates:

The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values for the reference item determined from the equation for the fitted line.
The 95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
In order to determine the NOEC, one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours to determine any statistically significant differences between the test and control groups.
All statistical analyses were performed using the SAS computer software package (SAS, 1999 2001).

Any other information on results incl. tables

	6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine		3,5-Dichlorophenol
	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	64	-	1.0	-
EC20	81	-	1.6	-
EC50	150	*	5.8	4.3–7.8
EC80	>320	-	21	-
NOEC	32	-	-	-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro organisms gave a 3 Hour EC10 value of 64 mg/L. The NOEC after 3 hours exposure was 32 mg/L.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods

Following a preliminary range‑finding test and at the request of the Sponsor, a test to determine the No Observed Effect Concentration (NOEC) and the 3‑Hour EC₁₀value for the test item was performed. Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of3.2,10,32,100and320 mg/L (five replicates) for a period of 3 hours at a measured temperature of between 20 °C and 21 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5‑dichlorophenol.

Results

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3‑Hour EC₁₀value of 64 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 32 mg/L.

The reference item, 3,5‑dichlorophenol, gave a 3‑Hour EC₅₀value of 5.8 mg/L, 95% confidence limits 4.3 to 7.8 mg/L confirming the acceptability of the inoculum used in the test system.