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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 2019 - 04 June 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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% (by HPLC area % 225 nm bandwidth 50 nm)
- Physical State/Appearance: Light yellow liquid
- Expiry Date: 01 September 2019
- Storage Conditions: Approximately 4 °C in the dark, will not be exposed to the atmosphere

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sample storage conditions before analysis: frozen

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
A nominal amount of test item (20 mg) was dissolved in test water and the volume adjusted to 2 liters to give the 10 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 5.6, 3.2, 1.8 and 1.0 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Name: Daphnia magna
- Age at study initiation: less than 24 hours old
- Source: In house laboratory
- Food type: mixture of algal suspension (Pseudokirchneriella subcapitata) and GEMMA Micro 300 fish food suspension
- Frequency: daily

Study design

Test type:

static

Water media type:

other: Reconstituted water (Elendt M4 medium)

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

N/A

Post exposure observation period:

Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure.

Test conditions

Hardness:

2.5 mmol/L (= 250 mg/L as CaCO3)

Test temperature:

21 °C to 23 °C

pH:

7.9 ±0.3

Dissolved oxygen:

8.1 - 8.4

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal Concentrations:
Range finding Test (mg/L): 0, 0.1, 1.0, 10.0, 100.0.
Definitive Test (mg/L): 0, 1.0, 1.8, 3.2, 5.6 and 10.0

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 89% to 96% of nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass vessels containing 100 mL Elendt M4 medium
- Type: static, no renewal of test media
- Aeration: No
- Type (delete if not applicable): closed
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- CaCl2.2H2O: 293.8
- MgSO4.7H2O: 123.3
- KCl: 5.8
- NaHCO3: 64.8
- Na2SiO3.9H2O: 10.0
- NaNO3: 0.274
- KH2PO4: 0.143
- K2HPO4: 0.184
- H3BO3: 500
- FeSO4.7H2O: 200
- MnCl2.4H2O: 100
- LiCl: 50
- RbCl: 50
- SrCl2.6H2O: 50
- NaBr: 12.4
- Na2MoO4.2H2O: 25.2
- CuCl2.2H2O: 6.4
- ZnCl2: 6.3
- CoCl2.6H2O: 2.5
- KI: 2.5
- Na2SeO3: 1.0
- NH4VO3: 0.3
- Na2EDTA.2H2O: 2500
- Thiamine HCl: 75
- Cyanocobalamine (B12): 1.0
- Biotin (B6): 0.75

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods
- Light intensity: between 200 and 1200 Lux
- Temperature: 18 to 22 °C
- Feeding during test: No

RANGE-FINDING STUDY
- Test concentrations (mg/L): 0.10, 1.0, 10 and 100

Reference substance (positive control):

yes

Remarks:

Potassium dichromate at concentrations: 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Effect concentrations 3

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

ca. 3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

No sub lethal effects of exposure were observed throughout the test

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Test concentrations (mg/L): 0.32, 0.56, 1.0, 1.8 and 3.2
- Limit test: No
- Resutls:
24 hour: EC50 (mg/L) - 1.1; 95% Confidence Limits (mg/L) - 0.95 - 1.3; NOEC (mg/L) - 0.56; LOEC (mg/L) - 1.0
48 hour: EC50 (mg/L) - 0.8; 95% Confidence Limits (mg/L) - 0.12 - 1.5; NOEC (mg/L) - 0.56; LOEC (mg/L) - 1.0

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum Likelihood regression.
The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) at 24 and 48 hours were calculated using the Step-down Cochran-Armitage test.
All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Any other information on results incl. tables

Cumulative Immobilization Data and Observations in the Range‑finding Test

Nominal Concentration (mg/L)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative ImmobilizedDaphnia		Observations		Cumulative ImmobilizedDaphnia		Observations
	R1	R2	R1	R2	R1	R2	R1	R2
Control	0	0	5N	5 N	0	0	5 N	5 N
0.10	0	0	5 N	5 N	0	0	5 N	5 N
1.0	0	0	5 N	5 N	0	0	5 N	5 N
10	2	2	3 N	3 P & T	5	5	AI	AI
100	5	5	AI	AI	5	5	AI	AI

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (mg/L)	24 Hours
	Cumulative ImmobilizedDaphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	5 N	5 N	5 N
1.0	0	0	0	0	0	0	5 N	5 N	5 N	5 N
1.8	0	0	0	0	0	0	5 N	5 N	5 N	5 N
3.2	0	2	1	1	4	20	5 N	3 N	4 N	4 N
5.6	2	4	2	2	10	50	3 N	1 N	3 N	3 N
10	2	1	3	4	10	50	3 N	4 N	2 N	1 N

Nominal Concentration (mg/L)	48 Hours
	Cumulative ImmobilizedDaphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
1.0	0	1	0	0	1	5	5 N	4 N	5 N	5 N
1.8	0	0	0	0	0	0	5 N	5 N	5 N	5 N
3.2	0	2	3	1	6	30	5 N	3 N	2 N	4 N
5.6	5	5	4	4	18	90	AI	AI	1 N	1 N
10	4	4	5	5	18	90	1 N	1 N	AI	AI

R          =Replicate

N      = Normal

P      = Pale

T      = Trapped at surface

AI        = AllDaphniaimmobilized

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item gave the following results at 48-hour:
EC50 (mg/L): 3.9
95% Confidence Limits (mg/L) 2.2 - 6.8
NOEC (mg/L): 1.8
LOEC (mg/L): 3.2

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 Methods

Following a preliminary range‑finding test, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous solutions of the test item at nominal concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L for 48 hours at a temperature of 21°C to 23 °C under static test conditions. The number of immobilized Daphnia was recorded after 24 and 48 hours.

Results

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 89% to 96% of nominal and so the results are based on nominal test concentrations.

Exposure of Daphnia magna to the test item gave the following results:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
48	3.9	2.2	-	6.8	1.8	3.2