2H-1,3-benzoxazine-2,4(3H)-dione

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:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,3-benzoxazine-2,4-dione

Cas Number:

2037-95-8

IUPAC Name:

1,3-benzoxazine-2,4-dione

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sampling method:
Frequency at t=0 h, t = 24 h and t=72 h
Volume 2.0 mL
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start and at the end of the test period.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of 3,4-dihydro-2,4-dioxo-1,3-2h-benzoxazine tested was a white powder with a purity of approximately 99% which was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with the highest concentration of 100 mg/L applying approximately one hour of magnetic stirring followed by a treatment period of ultrasonic waves (i.e. 1-3 minutes) to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

23°C

pH:

7.4-8.4

Nominal and measured concentrations:

1.0. 3.2, 10, 32 and 100 mg/L.
Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were at the level of nominal (i.e. 90-94% relative to nominal). At the end of the test, the concentrations were measured to be at 44-53% of nominal. It should be noted that a small response was measured in the control throughout the test, however, was considered negligible as the maximum contribution was 0.96%. The origin of this response is unknown.
Based on these results, the average exposure concentrations being the Time Weighted Average (TWA) were calculated and used to express effect parameters.

Nominal conc. (mg/L) Measured concentration (mg/L) TWA (mg/L)
t=0h t=24h t=72 h
1.0 0.929 0.733 0.459 0.66
3.2 2.97 2.36 1.46 2.1
10 9.16 7.16 4.57 6.5
101 9.351 7.511 4.391 6.61
32 28.8 23.1 15.0 21
100 91.1 76.2 53.4 70

The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae (see Table 4). Hence, it can be stated that the presence of the algae had no effect on the concentration of the test item in test medium.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Static
- aeration: continuous
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 204.4 x 10^4 cells/mL
- No. of organisms per vessel: 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 1 x 10^4 cells/mL
- No. of vessels per control (replicates): 1 x 10^4 cells/mL
- No. of vessels per vehicle control (replicates): 1 x 10^4 cells/mL

GROWTH MEDIUM
- Standard medium used: yes
M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

TEST MEDIUM / WATER PARAMETERS
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous (24h)
- Light intensity and quality: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
pH At the beginning and at the end of the test.
Temperature of medium Continuously in a temperature control vessel.
Appearance of the cells At the end of the final test, microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae.
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =20 mm for the combined limit/range-finding test; pathlength=10 mm for the final test). Algal medium was used as blank.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Six replicates of exponentially growing algae were exposed to a control and a concentration of 100 mg/L. Three replicates per concentration were exposed to 0.10, 1.0 and 10 mg/L in the combined range-finding test.
- Results used to determine the conditions for the definitive study: yes, 1.0, 3.2, 10, 32 and 100 mg/L in the final test

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 70 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.5 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

29 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

45 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 21 and 70 mg/L when compared to the control.

Results with reference substance (positive control):

The EC50 for growth rate inhibition (72h-ERC50) was 1.1 mg/L with a 95% confidence interval ranging from 1.1 to 1.1 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.36 mg/L with a 95% confidence interval ranging from 0.35 to 0.36 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

No significant growth rate inhibition was observed at the three lowest test concentrations, while significant growth rate inhibition of respectively 4.9 and 36% was recorded at 21 and 70 mg/L at the end of the test. However, growth rate inhibition at 21 mg/L was considered not biologically relevant since the recorded effect was <10%.
No significant yield inhibition was observed at the three lowest test concentrations, while significant yield inhibition of respectively 25 and 88% was recorded at 21 and 70 mg/L, at the end of the test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), 3,4-dihydro-2,4-dioxo-1,3-2h-benzoxazine inhibited growth rate and yield of this fresh water algae species significantly at Time Weighted Average (TWA) concentrations of 21 and 70 mg/L.
The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range of concentrations tested, i.e. exceeded a TWA concentration of 70 mg/L being considered as the maximum soluble concentration of test item in test medium.
The 72h-EC50 for yield inhibition (EYC50) was 33 mg/L with a 95% confidence interval ranging from 30 to 36 mg/L.
The 72h-NOEC for growth rate inhibition was 6.5 mg/L based on statistical significance and 21 mg/L based on biological relevance.
The 72h-NOEC for yield inhibition was 6.5 mg/L based on both statistical significance and biological relevance.

Executive summary:

The objectiveofthe study was to evaluate 3,4-dihydro-2,4-dioxo-1,3-2h-benzoxazine for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀, EC₂₀and EC₅₀for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

The batch of 3,4-dihydro-2,4-dioxo-1,3-2h-benzoxazine tested was a white powder with a purity of approximately 99% which was completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a preceding combined limit/range-finding test.Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominally 1.0, 3.2, 10, 32 and 100 mg/L. The initial algal cell density was 10⁴cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. The measured concentrationsat the start of the testwere at the level of nominal (i.e. 90-94%). At the end of the test, the concentrations decreased to 44-53% of nominal. Based on these results, the average exposure concentrations being the Time Weighted Average (TWA) were calculated to be 0.66, 2.1, 6.5, 21 and 70 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters (based on TWA concentrations) obtained in this study are summarized in the table below.

Parameter (mg/L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	6.5	21	29	45	>70
	lower 95%-cl			27	43
	upper 95%-cl			32	47
Yield	Value	6.5	6.5	13	18	33
	lower 95%-cl			11	16	30
	upper 95%-cl			15	20	36

cl – confidence limit, * - based on statistical significance,^#- based on biological relevance.