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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 July 2019 - 09 July 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 203 (Fish, Acute Toxicity Test)

Version / remarks:

The test was conducted according to the new threshold approach recommended by ECHA

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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
- Source and lot/batch No.of test material: 1394901004
- Expiration date of the lot/batch: 01 September 2019
- Purity: >99% (by HPLC area % 225 nm bandwidth 50 nm)
- Physical State/Appearance: Light yellow liquid
- Storage Conditions: Approximately 4 °C in the dark, will not be exposed to the atmosphere

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and 3.9 mg/L test group, from fresh and old media throughout the 96 Hour test period, for quantitative analysis. The samples at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) were taken for analysis immediately. All other samples were taken and stored frozen prior to analysis if necessary.
Duplicate sets of samples at 0 and 72 hour (fresh media), 24 and 96 hour (old media) were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

The concentration of the test item in the sample solutions was determined by HPLC.
Limit of Quantification (LOQ) was determined as the lowest usable standard concentration, which was 0.005 mg/L.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout (Oncorhynchus mykiss)
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK, maintained in house since 02 April 2019.
- Age at study initiation: juvenile
- Length at study initiation: 5.3 cm (standard deviation = 0.36)
- Weight at study initiation: 1.29 g (standard deviation = 0.20)
- Maintenance of the brood fish: Glass fiber tank with a "single pass" water renewal system

ACCLIMATION
- Acclimation period: 24 June 2019 to 01 July 2019
- Type and amount of food during acclimation: commercial trout pellets which was discontinued approximately 24½ hours prior to the start of the definitive test
- Health during acclimation: mortality observed

FEEDING DURING TEST - No

TEST WATER
Laboratory tap water was dechlorinated by passage through an activated carbon filter and partly softened giving water with a total hardness of approximately 140 mg/L as CaCO3.

Study design

Test type:

semi-static

Water media type:

other: Laboratory tap water was dechlorinated by passage through an activated carbon filter and partly softened

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

N/A

Post exposure observation period:

N/A

Test conditions

Hardness:

140 mg/L as CaCO3.

Test temperature:

13 °C to 14 °C

pH:

7.7 - 8.3

Dissolved oxygen:

≥ 9.7 mg O2/L

Salinity:

Not specified

Conductivity:

497 μS/cm at 20 °C

Nominal and measured concentrations:

3.9 mg/L
Chemical analysis of the freshly prepared test solutions at 0 and 72 hours, and of the corresponding old or expired test solutions at 24 and 96 hours showed measured test concentrations to range from 82% to 100% of nominal and as such the results are based on the nominal test concentration only.

Details on test conditions:

TEST SYSTEM
- Test vessel: 25 to 30 L glass vessels with 20 L of media
- Type: semi-static, a daily renewal
- Aeration: via narrow bore glass tubes
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
Aluminium: <6 mg Al/L
Chloride: 23.85 mg/L
Coliform bacteria: 0 no./100mL
Colour: <0.65 mg/l Pt/Co
Conductivity: 497 μS/cm at 20 °C
E.Coli bacteria: 0 no./100mL
Fluoride: 0.36 mg F/L
Iron: <27 μg Fe/L
Manganese: 1.3 μg Mn/L
Nitrate: 12.35 mg NO3/L
Odour: 0 Dilution number
Pesticides: <0.003 μg/L
pH: 7.61 pH Value
Sodium: 38.5 mg Na/L
Copper: 0.0653 mg Cu/L
Lead: <0.9 μg Pb/L
Nickel: 4.8 μg Ni/L

OTHER TEST CONDITIONS
- Temperature: 13 °C to 14 °C
- Photoperiod: 16 hours light and 8 hours darkness and 20 minute dawn and dusk transition periods

TEST PREPARATION
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give a 100 mg/L stock solution. An aliquot (780 mL) of this stock solution was dispersed in a final volume of 20 liters of test water to give the required test concentration of 3.9 mg/L.
The stock solution was inverted several times to ensure adequate mixing and homogeneity. The 3.9 mg/L test preparation was stirred with a flat bladed mixer for approximately 1 minute to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 72 and 96 hours.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

>= 3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

There were no mortality and no sub lethal effects of exposure observed in the test.
Sub-Lethal Effects: There were no sub lethal effects of exposure observed in the test.
Validation Criteria: The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was equal to or greater than 60% of ASV in the control and test vessels.
An estimate of the LC50 values was given by inspection of the mortality data.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Cumulative Mortality Data in the Definitive Test

Nominal Concentration (mg/L)	Cumulative Mortality (Initial Population = 7)							Mortality (%)
	1 Hour	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0	0	0	0	0
3.9	0	0	0	0	0	0	0	0

Results for Test Samples

Time Point	Nominal Concentration (mg/L)	Measured Concentration (mg/L)	Percentage of Nominal Concentration (%)
0	Control 3.9	< LOQ 3.90	- 100
24 (old)	Control 3.9	< LOQ 3.79	- 97
72 (fresh)	Control 3.9	< LOQ 3.18	- 82
96 (old)	Control 3.24	<LOQ 3.24	- 83

LOQ = Limit of quantification (0.005 mg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on the nominal test concentration gave a 96 Hour LC50 of greater than 3.9 mg/L. The No Observed Effect Concentration (NOEC) was 3.9 mg/L.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

In accordance with the recommendations of REACh, the test was conducted according to the new threshold approach recommended by ECHA, rather than the traditional OECD 203 approach of using a range of test concentrations. When using this new threshold approach, the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as a provisional threshold concentration, and a sensitivity “Limit test” is then conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 value is greater than the provisional threshold concentration. If, however, one or more fish from the test group die, the threshold test is terminated and a repeat test is conducted. The repeat test then follows the traditional OECD 203 approach of using a range of test concentrations to determine an EC50 value. The EC50 value obtained from the Acute Toxicity to Daphnia magna test (Covance Study Number YC81RJ) was the lowest of these two EC50 values, and hence this Acute Toxicity to Fish test was conducted at a single nominal concentration of 3.9 mg/L.

Seven fish were exposed to an aqueous solution of the test item, at a single nominal concentration of 3.9 mg/L for a period of 96 hours at a temperature of 13 ºC to 14 °C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

Chemical analysis of the freshly prepared test solutions at 0 and 72 hours, and of the corresponding old or expired test solutions at 24 and 96 hours showed measured test concentrations to range from 82% to 100% of nominal and as such the results are based on the nominal test concentration only.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave an LC50 value based on the nominal test concentration of greater than 3.9 mg/L. The No Observed Effect Concentration (NOEC) was 3.9 mg/L.