(2,4-dioxo-1,3-benzoxazin-7-yl) ethyl carbonate

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:

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 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:

(2,4-dioxo-1,3-benzoxazin-7-yl) ethyl carbonate

EC Number:

605-404-6

Cas Number:

16535-55-0

Molecular formula:

C11 H9 N O6

IUPAC Name:

(2,4-dioxo-1,3-benzoxazin-7-yl) ethyl carbonate

Test material form:

solid: particulate/powder

Details on test material:

Off-white crystalline powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. 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 and t=72 h
Volume 1.6 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 10% of the SS but without algae and samples for analysis were taken at the start and at the end of the test period.
Additionally, reserve samples of 1.6 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 CH02672 tested was an off-white crystalline powder with a purity of >99.9% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a 10-minute period of ultrasonic waves. During the combined limit/range-finding test this was followed by a three-day period of magnetic stirring while during the final test a one-day period of magnetic stirring was applied, to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS 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 10^4 cells/mL.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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:

between 21 and 23°C

pH:

7.3 - 8.2

Nominal and measured concentrations:

Solutions containing 1.0, 3.2, 10, 32 and 100% of the SS, prepared at a loading rate of 100 mg/L

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: 115.5 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: Continuously using TLD-lamps with a light intensity within the range of 79 to 81 µE.m-2.s-1.

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 (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.


TEST CONCENTRATIONS
- Range finding study (0.10, 1.0, 10 and 100 mg/L)
- Test concentrations: Six replicates of exponentially growing algae were exposed to a control and a concentration of 100 mg/L.
- Results used to determine the conditions for the definitive study: A final test was performed based on the results of a preceding combined limit/range-finding test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.90, 3.0, 8.8, 28 and 92 mg/L, respectively. During the exposure period, the concentrations decreased to 41 – 66% of initial at the end of the test.
Based on these results, the average exposure concentrations were calculated and used to express effect parameters .

The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae. Hence, it can be stated that the presence of the algae did not affect the concentration of the test item in test medium throughout the test.

Inhibition of growth rates and yield increased with increasing concentration of CH02672 from 2.1 mg/L upwards resulting in 100% inhibition at the highest concentration tested. Statistically significant inhibition of growth rates and yield was found at test concentrations of 2.1 mg/L and higher. However, growth rate inhibition was considered to be biologically not relevant at 2.1 mg/L, where the observed inhibition was below 10%. The NOEC based on biological relevance was thus set at 2.1 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 2.1 mg/L when compared to the control.

Results with reference substance (positive control):

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 1.11 mg/L with a 95% confidence interval ranging from 1.10 to 1.13 mg/L.
The 72h-ERC50 was within the expected range of 0.92 and 1.46 mg/L as specified in ISO International Standard 8692, February 2012, and the historical range of 0.86 and 2.3 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.
In conclusion, the sensitivity of this culture of Raphidocelis subcapitata was in agreement with ISO International Standard 8692, February 2012 and the historical data collected at the Test Facility.

Reported statistics and error estimates:

See table below

Any other information on results incl. tables

Parameter (mg/L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	0.58	2.1	2.4	3.4	6.8
	lower 95%-cl			2.3	3.3	6.6
	upper 95%-cl			2.5	3.6	7.0
Yield	Value	0.58	0.58	1.4	1.8	3.0
	lower 95%-cl			1.2	1.7	2.8
	upper 95%-cl			1.5	1.9	3.1

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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, CH02672 inhibited growth rate and yield of this freshwater algae species significantly at TWA concentrations of 2.1 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 6.8 mg/L with a 95% confidence interval ranging from 6.6 to 7.0 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was 3.0 mg/L with a 95% confidence interval ranging from 2.8 to 3.1 mg/L.
The 72h-NOEC for growth rate inhibition was 0.58 mg/L based on statistical significance and 2.1 mg/L based on biological relevance.
The 72h-NOEC for yield inhibition was 0.58 mg/L based on statistical significance and biological relevance.

Executive summary:

The objectiveofthe study was to evaluate CH02672 for its ability to generate toxic effects in Raphidocelis 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. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23,2019.

The batch of CH02672 tested was an off-white crystalline powder with a purity of >99.9% and not completely soluble in test medium at the loading rate initially prepared.

A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

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 solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 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 0.90, 3.0, 8.8, 28 and 92 mg/L, respectively. During the exposure period, the concentrations decreased to 41 – 66% of initial at the end of the test.Based on these results, the average exposure concentrations were calculated and used to express effect parameters.

Inhibition of growth rates and yield increased with increasing concentration of CH02672 from 2.1 mg/L upwards resulting in 100% inhibition at the highest concentration tested (74 mg/L)). Statistically significant inhibition of growth rates and yield was found at test concentrations of 2.1 mg/L and higher. However, growth rate inhibition was considered to be biologically not relevant at 2.1 mg/L because the observed inhibition was below 10%. Biologically relevant inhibition of growth rate was observed at concentrations of 6.7 mg/L and higher.

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

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	0.58	2.1	2.4	3.4	6.8
	lower 95%-cl			2.3	3.3	6.6
	upper 95%-cl			2.5	3.6	7.0
Yield	Value	0.58	0.58	1.4	1.8	3.0
	lower 95%-cl			1.2	1.7	2.8
	upper 95%-cl			1.5	1.9	3.1

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