(4aR*, 8aR*)-1-bromo-4a,5,9,10,11,12-hexahydro-3-methoxy-6H-benzofuro[3a,3,2-ef] [2]benzazepin-6-one hydrochloride (1:1)

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:

2001-10-08 to 2002-03-11

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

Remarks:

: There were no amendments to the study plan.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Remarks:

: There were no amendments to the study plan

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: no data
- Sample storage conditions before analysis: deep frozen at -20 deg C immediately after sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest test substance concentration of nominal 70 mg/L was prepared by dissolving 43 mg of the test substance completely in 6000 mL test water by ultrasonic treatment for 10 min and intense stirring for 10 min at room temperature in the dark. Adequate volumes of this intensively mixed test substance were diluted with test water to prepare the test media with the lower test substance concentrations. The test media were prepared just before the addition of algae (= start of the test).
- Eluate: water
- Differential loading: dilutions
- Controls: Yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): clear test medium solutions

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen Göttingen (SAG, Experimentelle Phykologie und Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universitat Göttingen, D-37073 Göttingen, Germany).
- Age of inoculum (at test initiation): exponentially growing pre-culture, which was set-up three days prior to the test at the same conditions as in the test.
- Method of cultivation: algae were cultivated in synthetic water according to guideline

ACCLIMATION
- Acclimation period: three days
- Culturing media and conditions (same as test or not): synthetic water same as the test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22-23 deg C

pH:

7.7 - 8.1 start of the test (T = 0)
7.8 - 8.7 end of the test (T = 72h)

Nominal and measured concentrations:

Nominal concentrations: 1.4, 3.2, 7.0, 14, 32 and 70 mg/L
Measured concentrations: not analyzed, 1.7 (54% of nominal), 4.1 (59% of nominal), 8.8 (63% of nominal), 21 (66% of nominal), 49 (70% of nominal) mg/L

During the test period of 72 hours a decrease of the test item concentrations in the test media was determined. At the end of the test 25 to 46% of the nominal values were found. The mean measured test concentrations (calculated as the average over all measurements per test concentration) varied in the range of 54 to 70% of the nominal values. Therefore the reported biological results are based on the mean measured test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): covered with glass dishes
- Material, size, headspace, fill volume: 50 mL erlenmeyer flask filled up to a volume of 15 mL with the alge suspension
- Initial cells density: 10000 algal cells per mL medium
- Control end cells density: 10000 algal cells per mL medium
- No. of vessels per concentration (replicates): three replicates
- No. of vessels per control (replicates): six replicates

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic test water
- Alkalinity: calculated hardness of the test water was 24 mg/L as CaCO3
- Intervals of water quality measurement: ge

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no data
- Photoperiod: continuously
- Light intensity and quality: Continuously illuminated at a measured light intensity of about 8300 Lux (mean value), range: 7800 to 8900 Lux (minimum and maximum value of measurements at 9 places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks.
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 24, 48 and 72h exposure
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement: Yes
- Other: Growth rate: Yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric series as requested in guideline
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes (Not GLP certified)
- Test concentrations: no data
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

24 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: could not be determined

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0 -8.2 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities
- Unusual cell shape: not applicable
- Colour differences: not applicable
- Flocculation: not applicable
- Adherence to test vessels: not applicable
- Aggregation of algal cells: not applicable
- Other: not applicable
- Any stimulation of growth found in any treatment: not applicable
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: the decrease of test substance concentration in the test medium might have been related to the instability of the test substance in the test medium.
- Effect concentrations exceeding solubility of substance in test medium: not applicable
- 72 h LOEC based on growth rate = 8.8 mg/L
- 72 h EC90 based on growth rate > 49 mg/L

Reported statistics and error estimates:

The calculations of the EC values are based on the mean measured test item concentrations. The biological results of the lowest test concentration of nominal 1.4 mg/L were not taken into account at the Probit Analysis because this test concentration was below the 72-hour NOEC, and thus not analyzed. For the determination of the LOEC and NOEC, the calculated mean biomass and the mean growth rate at the test concentrations were tested for significant differences compared to the control values by a Dunnett-test.

Table 1. Algal cell densities during the test period of 24h, 48h and 72h.

Nominal test item concentration (mg/L)	m (mean value) s (standard deviation) n (number of flasks)	24 h	48 h	72 h
Control	msn	5.370.846	20.071.796	93.627.926
1.4(n.a.)	msn	4.950.363	19.331.753	90.355.833
3.2(1.7)*	msn	4.430.083	19.001.093	85.274.033
7.0(4.1)*	msn	3.770.433	14.750.513	65.325.053
14(8.8)*	msn	2.380.063	4.420.083	15.181.133
32(21)*	msn	1.630.283	2.980.393	7.874.323
70(49)*	msn	1.620.063	2.070.183	8.022.353

At the start 10000 algal cells/mL were incubated; * mean measured test concentration; n.a. - not analyzed

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Scenedesmus subspicatus resulted in a 72-h NOEC and EC50 (based on growth rate) of 4.1 and 24 mg/L, respectively. The results of the test can be considered reliable without restriction.

Description of key information

In the guideline study (Piether, 2002), a 72 -h growth inhibition test with the unicellular green alga Scenedesmus subspicatus was performed with T002102. In this test, 72-h NOEC and EC50 (based on growth rate) of 4.1 and 24 mg/L, respectively were obtained. The results of the test can be considered reliable without restriction.

Key value for chemical safety assessment

EC50 for freshwater algae:

24 mg/L

EC10 or NOEC for freshwater algae:

4.1 mg/L