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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-02-12 to 2020-08-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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 test concentrations and the control were taken at test start and the test medium renewal after 24 hours.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the renewal periods, at least duplicate samples were taken at the test medium renewal and at the end of the test from all aged test media and the control of the actual test by pooling the contents of the beakers of all replicates for each treatment.
All samples were diluted by a factor of 2 with acetonitrile. Additional samples of the control and the dilution solvent were taken at each sampling without any sample treatment.
All samples were stored in a freezer (≤ - 20 °C), protected from light, until analysis was performed.
The concentrations of the test item were analysed in the duplicate test media samples from all test concentrations and in the duplicate control samples from all sampling times. Additional samples of the control and the dilution solvent were not analysed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test medium of the highest test concentration of nominal 10 mg test item/L was prepared by dissolving 10.0 and 10.6 mg test item into 1000 and 1060 mL test water by intense stirring for 15 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
The test media were prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): There were no remarkable observations at the two lowest concentrations. At the concentration of nominal 2.1 mg test item/L a slight colouration was observed in the aged test medium on day two. At the concentration of nominal 4.5 and 10 mg test item/L a slight colouration was observed in the aged test medium on both days.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: clone 5
- Age at study initiation: From 2.50 to 23.00 hours old
- Method of breeding: The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
- Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.
- Feeding during test : no

ACCLIMATION : Was not necessary, since the test was performed in the same medium as the culturing

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Reconstituted Water (Elendt "M4"): 2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

18.4 to 19.3 °C in the freshly prepared media; 18.9 to 19.2°C in the aged test media

pH:

7.6 to 7.8 in the freshly prepared media; 7.6 to 7.9 in the aged test media; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

8.9 to 9.0 mg/L in the freshly prepared media; 8.6 to 8.8 mg/L in the aged test media

Details on test conditions:

TEST SYSTEM
- Test vessel/Material, size, headspace, fill volume, open / closed : Glass beakers of 100 mL volume containing approximately 60 mL of test medium. The test vessels were covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
- Aeration: not specified
- Renewal rate of test solution (frequency/flow rate): renewal at day 1
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 5 daphnia in 60 mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (Elendt "M4"):
Analytical grade salts and additives were added as described in the guideline.
- Culture medium different from test medium: no
- Intervals of water quality measurement: The water temperature, pH-values and the dissolved oxygen concentrations were determined in all freshly prepared and aged test media of each treatment group.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark
- Light intensity: The light intensity was 540 to 860 lux (measured once during the test).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those animals that are not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae).

VEHICLE CONTROL PERFORMED: none required

RANGE-FINDING STUDY : yes
- Test concentrations: not specified

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.57 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CI: 2.25 - 2.94

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1.79 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CI: 1.52 - 2.10

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.68 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

4.11 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the time weighted average test item concentration of 1.68 mg test item/L. At the time weighted average concentrations of 4.11 and 9.20 mg test item/L all animals were immobile.

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:
Control Immobilisation Rate: Was 0 % and furthermore no daphnid showed signs of disease or stress; thus the validity criterion was met.
Dissolved Oxygen Concentration: Was ≥ 8.8 mg O2/L in in all treatment groups at the end of the test; thus validity criterion was met.

Results with reference substance (positive control):

EC50-24H: 1.163 mg/L
The results indicate that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).

Reported statistics and error estimates:

The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by probit analysis.
The NOEC and LOEC after 24 and 48 hours were calculated by Step-down Cochran Armitage test procedure.
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:

The 48-hour NOEC was determined to be 1.68 mg test item/L. The 48-hour LOEC was determined to be 4.11 mg test item/L and the 48-hour EC50 value was calculated to be 2.57 mg test item/L.

Executive summary:

The purpose of this study was to determine the influence of the test item on the mobility of Daphnia magna, acording to OECD 202 guideline and following GLP.

Young daphnids (< 24 hours old) were exposed in a semi-static test to various concentrations. The study encompassed 6 treatment groups (10, 4.5, 2.1, 0.94 and 0.43 mg test item/L (spacing factor 2.2) corresponding to 9.20, 4.11, 1.68, 0.735 and 0.410 mg test item/L (measured TWA) and a control) each containing 20 individuals. The mobility of the daphnids was determined by visual observation after 24 and 48 hours. After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the time weighted average test item concentration of 1.68 mg test item/L. At the time weighted average concentrations of 4.11 and 9.20 mg test item/L all animals were immobile.

The 48-hour NOEC was determined to be 1.68 mg test item/L. The 48-hour LOEC was determined to be 4.11 mg test item/L and the 48-hour EC50 value was calculated to be 2.57 mg test item/L.

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

The 48-hour NOEC was determined to be 1.68 mg test item/L. The 48-hour LOEC was determined to be 4.11 mg test item/L and the 48-hour EC50 value was calculated to be 2.57 mg test item/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

2.57 mg/L