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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 2019 - 14 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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: 394901004
- 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:

Samples were taken at 22, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Nominal amounts of test item (100 and 32 mg) were separately dissolved in culture medium and the volumes adjusted to 1 liter to give 100 and 32 mg/L stock solutions, respectively from which a series of dilutions was made to give further stock solutions of 10, 3.2 and 1.0 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (4.2 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

Study design

Test type:

static

Water media type:

other: Reverse osmosis purified deionized water

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

N/A

Post exposure observation period:

N/A

Test conditions

Hardness:

Not specified

Test temperature:

24 ±1 ºC

pH:

7.8 - 8.9

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Range finding (nominal) (mg/L): 0, 0.1, 1.0, 10.0 and 100.0.
Definitive (nominal) (mg/L): 0, 1.0, 3.2, 10.0, 32.0 and 100.0.
Definitive (measured) (mg/L): 0, 1.1, 3.6, 10.0, 28.0 and 97.0.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks each containing 100 mL of test preparation
- Type (delete if not applicable): closed - plugged with polyurethane foam bungs
- Type of flow-through (e.g. peristaltic or proportional diluter): static, constantly shaken at approximately 150 rpm
- Renewal rate of test solution:no
- Initial nominal cells density: 5.00 x 10^3 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
NaNO3: 25.5 mg/L
MgCl2.6H2O: 12.16 mg/L
CaCl2.2H2O: 4.41 mg/L
MgSO4.7H2O: 14.6 mg/L
K2HPO4: 1.044 mg/L
NaHCO3: 15.0 mg/L
H3BO3: 0.186 mg/L
MnCl2.4H2O: 0.415 mg/L
ZnCl2: 0.00327 mg/L
FeCl3.6H2O: 0.160 mg/L
CoCl2.6H2O: 0.00143 mg/L
Na2MoO4.2H2O: 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O: 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod: Continuous illumination
- Light intensity and quality: intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Range finding (nominal) (mg/L): 0, 0.1, 1.0, 10.0 and 100.0.
Definitive (nominal) (mg/L): 0, 1.0, 3.2, 10.0, 32.0 and 100.0.
Definitive (measured) (mg/L): 0, 1.1, 3.6, 10.0, 28.0 and 97.0.

Reference substance (positive control):

no

Remarks:

Potassium dichromate (0.25, 0.50, 1.0, 2.0 and 4.0 mg/L)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 3.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 10 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 3.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 10 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.1, 3.6 and 10 mg/L. Burst cells were observed to be present in the test cultures at 28 mg/L, and no cells were observed to be present in the 97 mg/L test cultures.
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.1, 3.6 and 10 mg/L test cultures were observed to be green dispersions. The 28 mg/L test cultures were observed to be light orange dispersions whilst the 97 mg/L test cultures were observed to be orange dispersions.

Results with reference substance (positive control):

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour): 1.5 mg/L; 95% confidence limits 1.3 to 1.7 mg/L
EyC50 (0 to 72 hour): 0.76 mg/L; 95% confidence limits 0.69 to 0.85 mg/L
No Observed Effect Concentration based on growth rate: 0.50 mg/L
No Observed Effect Concentration based on yield: 0.50 mg/L
Lowest Observed Effect Concentration based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration based on yield: 1.0 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item

Reported statistics and error estimates:

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 growth rate and yield data after 72 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

 Inhibitionof Growth Rate

E_rC₁₀(0 to 72 hour) : 8.7 mg/L

E_rC₂₀(0 to 72 hour): 14 mg/L

E_rC₅₀(0 to 72 hour): 29 mg/L; 95% confidence limits 23 to 38 mg/L

where E_rC_xis the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using 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). There were no statistically significant differences (P³0.05), between the control, 1.1 and 3.6 mg/L test concentrations; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 3.6 mg/L. Correspondingly the LOEC based on growth rate was 10 mg/L.

 Inhibition of Yield

E_yC₁₀(0 to 72 hour): 7.3 mg/L

E_yC₂₀(0 to 72 hour): 9.1 mg/L

E_yC₅₀(0 to 72 hour): 13 mg/L; 95% confidence limits 12 to 15 mg/L

Where E_yC_xis the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as inSection 6.2.3. There were no statistically significant differences (P³0.05), between the control, 1.1 and 3.6 mg/L test concentrations; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 3.6 mg/L. Correspondingly the LOEC based on yield was 10 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:
Growth Rate (95% Confidence Limits (mg/L): 23 - 38): EC50 (mg/L): 29; NOEC (mg/L): 3.6; LOEC (mg/L): 10
Yield (95% Confidence Limits (mg/L): 12 - 15): EC50 (mg/L): 13; NOEC (mg/L): 3.6; LOEC (mg/L): 10

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriellasubcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to aqueous solutions of the test item at concentrations of 1.0, 3.2, 10, 32 and100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

 Results.

Analysis of the test preparations at 0 and 24 hours showed measured test concentrations to range from 104% to 114% of nominal. A slight decline in measured test concentrations was observed at 48 and 72 hours in the range of 73% to 112% of nominal at 48 hours and from 71% to 108% of nominal at 72 hours. Given that some measured test concentrations fell below the acceptable level of 80% of nominal, it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be 1.1, 3.6, 10, 28 and 97 mg/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	29	23	-	38	3.6	10
Yield	13	12	-	15	3.6	10