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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-02-13 to 2020-08-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition 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.
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 test period, samples of the test vessels of all treatment groups were taken in duplicate at the end of the test (after the 72 hours test period) by pooling the content of the test beakers of all replicates for each treatment.
Additionally, samples were taken after 24 and 48 hours by pooling the content of the test 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) 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 from sampling points, as requested by sponsor as follow:
in case of the test item concentration of 1.0 and 3.2 mg test item/L - from all times (0, 24, 48 and 72)
in case of the test item concentration of 10, 32 and 100 mg test item/L - from sampling times of 0 and 72 hours.
Additional samples of the controls 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 100 mg test item/L was prepared by dissolving 100.1 mg test item into 1001 mL test water by intense stirring for 20 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 algae (= start of the test).
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): There were no remarkable observations in the freshly prepared solutions. A slight brown colouration was observed at the concentration of 3.2 mg test item/L after 72 hours. After 24, 48 and 72 hours slight brown colouration was observed at 10 to 32 mg test item/L and brown colouration was observed at 100 mg test item/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (KORSHIKOV),
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): The test was started (0 hours) by inoculation of a biomass of nominal 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: not needed.
- Culturing media and conditions (same as test or not): yes

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 22.5 to 23.2 °C

pH:

The pH was measured in all test item concentrations and the control at the start and the end of the test.
pH value in the control at test start: 8.1,
pH value in the control at test end: 8.7;
pH values in the test item treatments at test start: 7.7 to 7.8,
pH values in the test item treatments at test end: 8.0 to 8.8

Nominal and measured concentrations:

Nominal: 100, 32, 10, 3.2 and 1.0 mg test item/L
Measured (TWA): 57.2, 16.3, 4.53, 1.66 and 0.479 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel/ Material, size, headspace, fill volume, open / closed: Erlenmeyer flasks of 50 mL volume with nominal 50 mL of test medium loosely covered with watch glass.
- Aeration: not specified
- Initial cells density: 5000 algal cells per mL test medium
- Control end cells density: 78420 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
The test units were continuously stirred by magnetic stirrers. The flasks were covered loosely with watch glass and incubated in a water bath. The flasks were placed in a random order and were repositioned each day to minimize differences in test conditions.
Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by the test item.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (OECD Medium):
Analytical grade salts were added as described in the guideline.
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3. The culture medium was prepared 3 days before test start to allow pH to stabilise.
- Culture medium different from test medium: no
- Intervals of water quality measurement:
The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The pH was measured in all test item concentrations and the control at the start and the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: not specified
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 4932 lux (range: 4710 to 5120 lux).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: yes
- Test concentrations: not specified

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.09 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 7.13-9.16

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.25 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 3.62-5.01

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.03 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 2.46-3.71

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.66 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.53 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.84 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI: 2.99-4.92

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.86 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI: 1.83-4.4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.45 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI: 1.32-4.51

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.66 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.53 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L (time weighted average concentration of 57.2 mg test item/L) and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested
- Colour differences: not specified
- Flocculation: not specified
- Adherence to test vessels: not specified
- Aggregation of algal cells: not specified
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not specified
- Effect concentrations exceeding solubility of substance in test medium: NA

In the test item treated groups a dose related reduction of algal growth was observed. However, the inhibition at the nominal concentration of 100 mg test item/L (time weighted average concentration of 57.2 mg test item/L) did not fit into this relation, since the reduction of growth was less, compared to the nominal concentration of 32 mg test item/L (time weighted average concentration of 16.3 mg test item/L). The test item solutions also showed a dose related colouration. The more pronounced colouration led to higher blank values, which may have a stronger impact on the calculation of the cell numbers if they are variable. It is assumed that the blank used for the nominal concentration of 100 mg test item/L was not representative for the measured samples of this concentration (the absorption of 0.325 is assumed to be too low). This assumption is supported by the value measured for the blank sample in the range finder, which was 0.505). Therefore, the nominal concentration of 100 mg test item/L was excluded from the statistical evaluation and the determination of the endpoints.

Validity Criteria of the Study:
Cell Density Increase in Control Cultures: 157.0-fold increase within 72 hours and thus, the validity criterion was met.
Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 9.2 % and thus, the validity criterion was met.
Coefficient of Variation of Average Growth between Control Replicates: 1.2 % and thus, the validity criterion was met.

Results with reference substance (positive control):

72-hour ErC50: 0.858 mg/L
72-hour ErC10: 0.464 mg/L

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hour ErC50 and the 72 hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by a three parametric normal concentration distribution function (CDF).
For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield) and Bonferroni-Welch t-test (growth rate).
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 72-hour EyC50 was calculated to be 3.84 mg test item/L and the ErC50 8.09 mg test item/L. The 72-hour EyC10 was calculated to be 2.45 mg test item/L and the ErC10 3.03 mg test item/L. The 72-hour NOEyC was determined to be 1.66 mg test item/L and the associated 72-hour LOEyC of 4.53 mg test item/L. The 72-hour NOErC was determined to be 1.66 mg test item/L and the associated 72-hour LOErC is 4.53 mg test item/L.

Executive summary:

The purpose of this test was to determine the inhibitory effect of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata, according to OECD 201 guideline and following GLP. Exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The study encompassed 6 treatment groups (5 dose rates of the test item (nominal: 100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16), corresponding to 57.2, 16.3, 4.53, 1.66 and 0.479 mg test item/L (TWA)) and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometric measurement.

The 72-hour EyC50 was calculated to be 3.84 mg test item/L and the ErC50 8.09 mg test item/L. The 72-hour EyC10 was calculated to be 2.45 mg test item/L and the ErC10 3.03 mg test item/L. The 72-hour NOEyC was determined to be 1.66 mg test item/L and the associated 72-hour LOEyC of 4.53 mg test item/L. The 72-hour NOErC was determined to be 1.66 mg test item/L and the associated 72-hour LOErC is 4.53 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 72-hour EyC50 was calculated to be 3.84 mg test item/L and the ErC50 8.09 mg test item/L. The 72-hour EyC10 was calculated to be 2.45 mg test item/L and the ErC10 3.03 mg test item/L. The 72-hour NOEyC was determined to be 1.66 mg test item/L and the associated 72-hour LOEyC of 4.53 mg test item/L. The 72-hour NOErC was determined to be 1.66 mg test item/L and the associated 72-hour LOErC is 4.53 mg test item/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

8.09 mg/L

EC10 or NOEC for freshwater algae:

3.03 mg/L