3,4-dihydro-2H-1,4-benzoxazin-6-ol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish embryo acute toxicity (FET)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-02-13 to 2020-08-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 236 (Fish embryo acute toxicity (FET) test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media of all treatment groups and the negative control were taken at test start and at every test medium renewal.
For the determination of the stability of the test item, respectively the maintenance of the test item concentrations under the test conditions, duplicate samples of the aged test media of all treatment groups were taken at every test medium renewal and at the end of the test.
All samples were diluted by a factor of 2 with acetonitrile and to each biological sample 0.5 mL of 1 g/L Cysteine Hydrochloride was added. Additional samples of the control blank and the dilution solvent acetonitrile were taken at each sampling without any sample treatment.
All samples were stored in a freezer (≤ -20 °C) immediately after sampling, and were kept stored until analysis.
The concentrations of the test item were analysed in the duplicate test media samples from all test concentrations, and in the duplicate control samples and the additional control and solvent samples, from all sampling times.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preconditioning of the Test Vessel:
11 and 7 days prior to the test all wells were preconditioned with the test water. Each well was filled with 2 mL freshly prepared test water with the respective concentration of the test item, reference item or negative control.
The test concentrations and media used for the preconditioning were discarded before start of the test. All test concentrations and controls were freshly prepared just before introduction of the embryos (= start of the test).

Dosage of Test Item:
For the pre-conditioning of the test vessels, the test medium of the highest test concentration of nominal 10 mg test item/L was prepared by dissolving 10.5 and 16.7 mg test item into 1050 and 1670 mL test water by intense stirring for 10 minutes. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations. The pre-conditioning media was prepared just before pre-conditioning.
For the main test, the test medium of the highest test concentration of nominal 10 mg test item/L was prepared by dissolving 9.0, 10.1, 9.3 and 9.7 mg test item into 900, 1010, 930 and 970 mL test water by intense stirring for 10 minutes. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations.
The test media were prepared just before introduction of the test fish embryos (= start of the test and test medium renewal on day 1, 2 and 3).

Appearance of the Test Item in Test Medium: There were no remarkable observations.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish (Danio rerio)
- Source: In-house breeding
- Age at study initiation (mean and range, SD): Embryos; The cell stage of the embryos introduced at test start: 2 to 4.
- Sex: Male and female
- Quality of the Eggs (Fertilisation rate): The fertilisation rate of the egg batch, determined from a subsample of 120 eggs, was 90 %.
- Spawning: Eggs were collected in spawning traps. Spawning traps were placed into the aquaria in the evening before the desired spawning day.
Brood Fish: Breeding stock of known holding conditions was used.
- Method of breeding: not specified

ACCLIMATION : Not appropriate

FEEDING DURING TEST : no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

142 to 178 mg CaCO3/L at test start
160 to 178 mg CaCO3/L at test end

Test temperature:

25.0 to 25.7 °C

pH:

7.4 to 7.8 in the freshly prepared test media
7.7 to 7.9 in the aged test media

Dissolved oxygen:

101 to 110 % of the air saturation value in the freshly prepared test media
91 to 101 % in the aged test media

Conductivity:

458 to 466 μS/cm at test start
426 to 482 μS/cm at test end

Nominal and measured concentrations:

nominal concentrations: 0.43, 0.94, 2.1, 4.5 and 10 mg test item/L
time weighted average values: 0.306*, 0.673, 1.68, 3.76 and 8.46 mg test item/L.
*complete level actually

Details on test conditions:

TEST SYSTEM
- Test vessel/Material, size, headspace, fill volume: Polystyrene 24 well plates with 2 mL test medium
- Type (delete if not applicable): open / closed : not specified
- Aeration: not specified
- Renewal rate of test solution (frequency/flow rate): renewal every day. For the test water renewals, the same well plate was used. Most of the old test media, leaving sufficient volume for the fish embryo to not be exposed to the air, was removed and freshly prepared test media (2 mL) was introduced to each well. With this procedure minimal disturbance or stress was exerted on the embryo.
- No. of organisms per vessel: 1 egg per well
- No. of vessels per concentration (replicates): 20 eggs on one plate for each test concentration
- No. of vessels per control (replicates): 20 eggs as positive control on one plate; 4 eggs in test water as internal plate control on each of the above plates; 24 eggs in test water as negative control on one plate.
Each egg was immersed in 2 mL test media.

Fish embryo handling: Eggs were immersed in the test media containing the 5 concentrations of the test item, the positive control, the negative control and the internal plate controls at cell stage 2 to 4. The eggs were inspected with stereo microscope in order to determine fertilisation rate and cell stages. The introduction of the eggs into the test media indicated the start of the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (ISO Medium):
In deionised water (conductivity ≤ 10 μScm-1) analytical grade salts were added to following nominal concentrations:
CaCl2 × 2H2O : 209.6 mg/L
MgSO4 × 7H2O : 87.7 mg/L
NaHCO3 : 46.4 mg/L
KCl : 4.13 mg/L
Water Hardness : 178.3 mg/L as CaCO3
Alkalinity : 0.55 mmol/L
Ratio of Ca : Mg = 4 : 1 (based on molarity)
Na : K = 10 : 1 (based on molarity)

- Culture medium different from test medium: no
- Intervals of water quality measurement: pH and dissolved oxygen was determined daily in the freshly prepared and aged test media of each treatment group whereas water hardness and conductivity was measured at test start and termination.
In the aged test media after each water exchange and at test end, parameters were measured in the pooled replicates to obtain sufficient volumes.
Additionally, temperature was recorded daily or continuously in the test chamber.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 14 h light : 10 h dark
- Light intensity: 760 to 820 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Apical Observations:
The embryos were observed at test start and approximately after 24, 48, 72 and 96 hours after start of the test for coagulated embryos, lack of somite formation and non-detachment of the tail. The lack of heartbeat was recorded after 48, 72 and 96 hours. All these test parameters were determined using a stereoscopic microscope.
Hatching:
Hatching was recorded in all test chambers after 48, 72, and 96 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study Yes
- Test concentrations: not specified

Reference substance (positive control):

yes

Remarks:

3,4-Dichloroaniline (4.0 mg/L)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

5.42 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CI: 5.01 - 5.86 mg test item/L

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.68 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC10

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

not determinable

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

3.76 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (based on the slight but statistically not significant mortality of 5 % and the observed apathetic behaviour at 3.76 mg test item/L)

Details on results:

In the control, 100 % survived until test end. In the test concentrations of 0.306 to 1.68 mg test item/L 100% survived until test end, at 3.76 mg test item/L 5 % mortality was observed. Additionally four fish of this treatment group were lying on the side and all fish were observed to be apathetic. At 8.46 mg test item/L all embryos were dead after 96 hours.
Hatching success in the control was 100%. In the test item concentrations of 0.43 to 10 mg test item/L, hatching success was 100 %.
In the internal plate control of the test concentrations, hatching success after 96 hours was 75 % in the two lowest concentrations and 100% at 2.1 to 10 mg test item/L.
In the positive control, containing 4.0 mg 3,4-Dichloroaniline/L, 90% of the embryos died and the remaining fish embryo did not hatch.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to time weighted average concentrations, since the test item concentrations were not within ± 20% of the nominal concentrations during the test.

Validity criteria:
Overall Fertilisation Rate: The overall fertilisation rate of all eggs collected was 90 % in the batch tested, thus this validity criterion was met.
Water Temperature: The water temperature was maintained at 25.0 to 25.7°C at the chamber at any time during the test, thus this validity criterion was met.
Survival of Embryos: The overall survival of embryos in the negative (test water) control was 100 % until test termination (96 hours), thus this validity criterion was met.
Hatching Rate: Hatching rate in the negative control was 100.0 % at test termination (96 hours), thus this validity criterion was met.
Dissolved Oxygen: At test termination (96 hours), the dissolved oxygen concentration in the negative control and the highest test concentration was 95 % and 91 % of saturation, respectively, thus this validity criterion was met.
Positive Control: The mortality in the positive control was 90 %, thus this validity criterion was met.

Reported statistics and error estimates:

Based on the embryo mortality, the 96-hour LC50 and where possible the 95 %-confidence limits was calculated by using the Trimmed Spearman Karber procedure. The 96-hour LC20 and LC10 could not be determined.
A statistical evaluation was performed by using the Step-down Cochran-Armitage test (one-sided greater, α = 0.05). Nevertheless, The NOEC and the LOEC were determined directly from the raw data. The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Validity criteria fulfilled:

yes

Remarks:

See above

Conclusions:

Based on the test results, the 96-hour LC50 of the test item for zebrafish embryos (Danio rerio) was determined to be 5.42 mg test item/L based on time weighted average concentrations. The NOEC was determined to be 1.68 mg test item/L also based on time weighted average concentrations.

Executive summary:

The purpose of this study was to evaluate the acute toxicity of the test item to fish embryos, according to OECD 236 guideline and following GLP. Zebrafish embryos (≤16 cell stage) were exposed in a Semi-Static test to various concentrations under defined conditions for 96 hours. The following endpoints were recorded as indicators of acute lethality in fish embryos: the coagulation of the embryo, lack of somite formation, the non-detachment of the tail-bud from the yolk sac, the lack of heartbeat. Additionally, the hatching rate was determined.

This study encompassed 7 treatment groups: 5 dose rates of the test item (nominal: 10, 4.5, 2.1, 0.94 and 0.43 mg test item/L, corresponding to 8.46, 3.76, 1.68, 0.673 and 0.306 mg test item/L (TWA), a positive control (4.0 mg/L 3,4-Dichloroaniline) each containing 20 individuals and a negative control containing 24 individuals. Additionally, an internal plate control of 4 individuals containing the test water without the test item was placed on the plates containing the 5 test item concentrations and the positive control. The acute toxicity to zebrafish embryos was determined in a semi-static.

Based on the test results, the 96-hour LC50 of the test item for zebrafish embryos (Danio rerio) was determined to be 5.42 mg test item/L based on time weighted average concentrations. The NOEC was determined to be 1.68 mg test item/L also based on time weighted average concentrations. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to time weighted average concentrations, since the test item concentrations were not within ± 20% of the nominal concentrations during the test.

All validity criteria were met.

Description of key information

Based on the test results, the 96-hour LC50 of the test item for zebrafish embryos (Danio rerio) was determined to be 5.42 mg test item/L based on time weighted average concentrations. The NOEC was determined to be 1.68 mg test item/L also based on time weighted average concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

5.42 mg/L