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

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:

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Adopted April 13, 2004

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: At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sampling method:
Frequency at t=0 h and t=48 h
Volume 1.6 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Test solutions

Vehicle:

yes

Remarks:

water

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.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: In-house laboratory culture with a known history.
- Justification for species other than prescribed by test guideline: This system has been selected as an internationally accepted invertebrate species.
- Source: In-house laboratory culture with a known history.
- Age of parental stock (mean and range, SD): <4 weeks
- Feeding during test: no
- Food type: /
- Amount: /
- Frequency: /

ACCLIMATION
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Daily, a suspension of fresh water algae

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Test temperature:

20-21°C

pH:

7.0-8.0

Dissolved oxygen:

7.1-9.3 mg/L

Nominal and measured concentrations:

10, 18, 32, 56 and 100% of the saturated solution (SS).

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Type (delete if not applicable): static
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: 5 per vessel containing 50 mL of test solution
- No. of vessels per concentration (replicates): 4 for the control and the highest test concentration.
2 per intermediate test concentration.
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Composition of medium M7:
The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
The hardness of test medium expressed as CaCO3: 180 mg/L with a pH between 6 and 9.

The following salts and vitamins were added to freshly prepared test medium (see section 4.8.2.2) to reach the following concentrations:

Salts: H3BO3 0.71 mg/L
FeSO4.7H2O 0.25 mg/L
MnCl2.4H2O 0.090 mg/L
LiCl 0.076 mg/L
RbCl 0.018 mg/L
SrCl2.6H2O 0.038 mg/L
Na2MoO4.2H2O 0.015 mg/L
NaBr 0.0040 mg/L
CuCl2.2H2O 0.0042 mg/L
ZnCl2 0.013 mg/L
CoCl2.6H2O 0.010 mg/L
KI 0.0032 mg/L
Na2SeO3 0.0022 mg/L
NH4VO3 0.00057 mg/L
Na2EDTA.2H2O 0.62 mg/L
Na2SiO3.5H2O 7.5 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L
Vitamins: Thiamine hydrochloride 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A daily photoperiod of 16 hours.
- Light intensity: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality) At 24 hours and at 48 hours.
pH and Dissolved oxygen At the beginning and at the end of the test, for the highest concentration and the control.
Temperature of medium Continuously in a temperature control vessel, beginning at the start of the test.


VEHICLE CONTROL PERFORMED: yes (medium)

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.00, 10 and 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:

24 h

Dose descriptor:

EC50

Effect conc.:

27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 22 - 33 mg/L

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% C.I.: 15 - 19 mg/L

Details on results:

No immobility was observed in the control and at the lowest test concentration throughout the exposure period. At the end of the test, a dose-related increase of immobility was observed at 18% of the SS prepared at a loading rate of 100 mg/L and higher, reaching ≥90% immobility at the three highest test concentrations.
The responses recorded in this test allowed for reliable determination of an EC50 and were in agreement with what was expected based on the results of the combined limit/range-finding test.

Results with reference substance (positive control):

The 24h-EC50 in this reference test with K2Cr2O7 was within the expected range of 0.6 and 2.1 mg/L as specified in ISO International Standard 6341, October 2012. The 48h-EC50 was within the expected range of 0.28 and 0.75 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.
The 24h-EC50 was 0.92 mg/L with a 95% confidence interval between 0.78 and 1.1 mg/L.
The 48h-EC50 was 0.55 mg/L with a 95% confidence interval between 0.47 and 0.64 mg/L.

Reported statistics and error estimates:

See table below for statistics

Any other information on results incl. tables

Parameter	CH02672 Average exposure conc. (mg/L)	95%-confidence interval (mg/L)
24h-EC50	27	22 - 33
48h-EC50	17	15 - 19

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to CH02672 was 17 mg/L based on average exposure concentrations (95% confidence interval between 22 and 33 mg/L).

Executive summary:

The objective of the study was to evaluate CH02672 for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC₅₀at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004. 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 combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 7.6, 15, 26, 45 and 86 mg/L, respectively. During the exposure period, the concentrations decreased to 74 - 81% of initial at the end of the test. Based on these results, the average exposure concentrations were calculated and used to express effect parameters.

No immobility was observed in the control and at the lowest test concentration throughout the exposure period. At the end of the test, a dose-related increase of immobility was observed at 18% of the SS prepared at a loading rate of 100 mg/L and higher, reaching ≥90% immobility at the three highest test concentrations.

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

In conclusion, the 48h-EC₅₀for Daphnia magna exposed to CH02672 was 17 mg/L based on average exposure concentrations (95% confidence interval between 15 and 19 mg/L).