Reaction mass of (3aR,5aS,9aS,9bR)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan and (3aS,5aR,9aR,9bS)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

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 17 October 2008 to 10 December 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 201 (2006) and the EC Method C.3 with GLP statement. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. This report was well documented and all validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

No. 440/2008

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

2008-11-12

Specific details on test material used for the study:

- Storage condition of test material: in a refrigerator at about 4°C.
- Water solubility: 1.88 mg/L

Analytical monitoring:

yes

Details on sampling:

- Sampling method: For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For the 72-hour stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 15 mL) was too small to perform the analyses. A volume of 200-500 mL per sample was necessary for analytical purposes. All samples were stored deep-frozen (at about -20 °C) immediately after sampling. Based on a pre-experiment the test item was shown to be stable under the storage conditions. The concentrations of the test item were determined in the duplicate test medium samples from the undiluted filtrate. The samples from the dilutions of 1:22, 1:10, 1.4.6 and 1:2.2 were not analyzed, since these concentrations were below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.
- Sample storage conditions before analysis: The samples were stored deep-frozen and protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared by dispersing 140 mg of the test item in 1400 mL of test water using ultrasonic treatment for 15 minutes. Then, the dispersion was stirred by a magnetic stirrer at room temperature in the dark over 96 hours to dissolve a maximum amount of the test item in the dispersion. The long stirring period of 96 hours was selected according to the results of a pre-experiment (non-GLP) which showed that the maximum concentration of test item was reached after the
stirring period of 96 hours. Furthermore it was shown within this pre-experiment that the test item was stable in water during a period of 96 hours. After the 96-hour stirring period, the dispersion was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm). The undiluted filtrate was used as the highest concentrated test medium and as stock solution for the preparation of the test media of lower test concentrations. For the preparation of the test media of the lower test concentrations, the filtrate was diluted with test water. The test media were prepared just before the start of the test (= addition of algae).
- Eluate: test water
- Differential loading: 1:22, 1:10, 1:4.6, 1:2.2 and the undiluted filtrate
- Controls: yes (test water without test item)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): yes in the loading rate of 100 mg/L only.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen/Germany).
- Age of inoculum (at test initiation): no data
- Method of cultivation: The algae were cultivated at Harlan Laboratories under standardized conditions according the the test guidelines.

ACCLIMATION
no data

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

The water harness (calculated) of the test water was 0.24 mmol/L ( = 24 mg/L as CaCO3).

Test temperature:

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22-23°C.

pH:

See table 2 in "Any other information on results incl. tables"

Dissolved oxygen:

No data

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 1:22, 1:10, 1:4.6, 1:2.2 and the undiluted filtrate (loading rate of 100 mg/L)
Measured concentrations: At the start of the test, the analytically determined concentration of the test item in the undiluted filtrate was 1.7 mg/L. At the end of the test, 1.2 mg/L were found. The mean measured concentration (calculated as geometric mean) was of 1.4 mg/L in the undiluted filtrate.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): covered with a glass dish
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks
- Aeration: During the test, the test solutions were continuously stirred by magnetic stirrers.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cells density: 10000 cells/mL, corresponding to 0.897 x 10^3 relative fluorescence units.
- Control end cells density: 57.4 x 10^3 relative fluorescence units.
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water was prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water. See table 1 in "Any other information on materials and methods incl. tables".
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no data
- Photoperiod: no data
- Light intensity and quality: The test flasks were illuminated by fluorescent tubes (Philips TLD 36 W/840), installed above the test flasks. The mean measured light intensity at the level of the test solutions was approximately 7400 Lux (range: 6200 to 8400 Lux, measured at nine places in the experimental area).
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Coulter Counter, Model ZM).
- Chlorophyll measurement: no
- Other: A small volume of the algal suspension was daily withdrawn from each test flask for the measurement of the biomass, and was not replaced. The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800). The measurements were performed in duplicate. At the end of the test, a sample was taken from the control and from the test medium with the loading rate of 100 mg/L. The shape and size of the algal cells were visually inspected.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: dilution factors (1:22, 1:10, 1:4.6, 1:2.2 and the undiluted filtrate)
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes (non-GLP)
- Results used to determine the conditions for the definitive study: no data

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Details on results:

The 72-hour EC10 and EC50 values of the test item could not be determined because of the absence of a significant inhibitory effect of the test item on the algal growth at the tested concentrations. For the determination of the NOEC, average growth rate and yield at the test concentrations were compared to the control values by Dunnett’s tests.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities were observed.
- Unusual cell shape: no data
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the test medium with a loading rate of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
- Any stimulation of growth found in any treatment: no stimulation of growth
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: The result of the latest positive control test performed in 2008 showed that the sensitivity of the test system was within the internal historical range (72-h EC50 for the growth rate: 1.20 mg/L (Study B83755), range of the 72-h EC50 for the growth rate from 2000 to 2008: 0.71-1.74 mg/L).
- Other: none

Reported statistics and error estimates:

None

Table 6.1.5/2: pH values in the treatments

Treatment/ Dilution	pH values
	Start	End
Control 1 :22 1 :10 1 :4.6 1 :2.2 Undiluted Filtrate	8.0 8.2 8.2 8.1 8.1 8.0	8.6 8.4 8.4 8.5 8.4 8.2

Table 6.1.5/3: Biomass of Algae

Treatment / Dilution	Rep. No.	Biomass of algae* (relative fluorescence units)
		24 h	48 h	72 h
Control	1 2 3 4 5 6	3.3 4.9 4.3 4.6 4.7 4.9	20.6 25.5 17.9 21.7 25.3 22.9	54.3 62.9 58.9 58.7 54.9 54.5
	Mean SD	4.4 0.6	22.3 2.9	57.4 3.5
1:22	1 2 3	4.7 4.9 5.0	23.5 22.2 19.2	94.3 78.9 76.6
	Mean SD	4.9 0.1	21.6 2.2	83.3 9.6
1:10	1 2 3	3.8 4.6 4.2	19.5 19.4 17.2	82.7 79.1 94.7
	Mean SD	4.2 0.4	18.7 1.3	85.5 8.2
1:4.6	1 2 3	3.7 5.0 4.6	18.2 20.8 19.3	79.5 101.0 97.4
	Mean SD	4.4 0.7	19.4 1.3	92.6 11.5
1:2.2	1 2 3	4.1 4.3 3.9	20.9 22.0 16.3	97.1 101.1 86.5
	Mean SD	4.1 0.2	19.7 3.0	94.9 7.6
Undiluted filtrate (loading rate: 100 mg/L)	1 2 3	4.4 3.9 4.5	24.9 23.4 24.2	68.3 54.1 67.3
	Mean SD	4.2 0.3	24.1 0.8	63.3 7.9

SD: Standard Deviation

*: The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 10^3). At the start of the test, the initial cell density was 10000 algal cells/mL, corresponding to 0.897 x 10^3 relative fluorescence units.

Table 6.1.5/4: Section-by-Section Growth Rates

Treatment / Dilution	Section by section growth rates (day^-1) and inhibition of the growth rates (Ir)
	0-24 h		24-48 h		48-72 h
	µ	Ir(%)	µ	Ir(%)	µ	Ir(%)
Control 1:22 1:10 1:4.6 1:2.2 Undiluted Filtrate	1.59 1.69 1.54 1.59 1.53 1.55	0.0 -6.3 3.2 0.2 4.2 2.4	1.62 1.49 1.50 1.49 1.56 1.74	0.0 7.8 7.4 8.0 3.6 -7.7	0.95 1.35 1.52 1.56 1.58 0.96	0.0 -41.7 -59.8 -64.0 -66.0 -0.9

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were fulfilled.

Conclusions:

The test item had no significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours up to and including the loading rate of 100 mg/L (corresponding on mean measured concentration of 1.4 mg/L). The 72-h NOEC was >= 1.4 mg/L and the 72-h EC50 was > 1.4 mg/L.

Executive summary:

This study, according to OECD Guideline 201 (2006) and the Commission Regulation (EC) No 440/2008 C.3 with GLP statement, was investigated to evaluate the influence of the test item on the growth of the freshwater green algae species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) in a 72 -hour static test.

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered through a 0.45 µm membrane filter. The undiluted filtrate of the dispersion was used as highest test concentration and as stock solution for the preparation of the lower test concentrations. The following treatments were tested: Dilution 1:22, 1:10, 1:4.6, 1:2.2 and the undiluted filtrate in parallel to a control. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Only the highest test concentration was analytically measured since it was determined as the NOEC in this test. At the start of the test, the analytically determined concentration of the test item in the undiluted filtrate was 1.7 mg/L. At the end of the test, 1.2 mg/L were found. The mean measured concentration (calculated as geometric mean) was of 1.4 mg/L. The biological effects are related to the loading of 100 mg/L and to the mean measured concentration of 1.4 mg/L.

The test item had no significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours up to and including the loading rate of 100 mg/L. The loading rate of 100 mg/L (mean measured concentration of 1.4 mg/L) was therefore determined to be the 72 -hour NOEC. This value might even be higher, but loading rates of the test item exceeding 100 mg/L were not tested, in accordance with the test guidelines. The 72 -hour EC50 was higher than the loading rate of 100 mg/L (mean measured concentration of 1.4 mg/L).

Description of key information

EU Method C.3, OECD Guideline 201, GLP, key study, validity 1:

The test item had no significant inhibitory effect on the growth of the algae, up to the maximum concentration testable and close to the limit of solubility.

Based on geometric mean measured concentrations, The 72h- EC50 (P. subcapitata) was > 1.4 mg/L and the 72-h NOEC (P. subcapitata) was ≥ 1.4 mg/L.

Additional information

One key study is available, according to OECD Guideline 201 (2006) and the Commission Regulation (EC) No 440/2008 C.3 with GLP statement, to evaluate the influence of the substance on the growth of the freshwater green algae species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) in a 72 -hour static test.

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered through a 0.45 µm membrane filter. The undiluted filtrate of the dispersion was used as highest test concentration and as stock solution for the preparation of the lower test concentrations. The following treatments were tested: Dilution 1:22, 1:10, 1:4.6, 1:2.2 and the undiluted filtrate in parallel to a control. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. Only the highest test concentration was analytically measured since it was determined as the NOEC in this test. At the start of the test, the analytically determined concentration of the test item in the undiluted filtrate was 1.7 mg/L. At the end of the test, 1.2 mg/L were found. The mean measured concentration (calculated as geometric mean) was of 1.4 mg/L. The biological effects are related to the loading of 100 mg/L and to the mean measured concentration of 1.4 mg/L.

The test item had no significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours up to and including the loading rate of 100 mg/L. The loading rate of 100 mg/L (mean measured concentration of 1.4 mg/L) was therefore determined to be the 72 -hour NOEC. The 72 -hour EC50 was higher than the loading rate of 100 mg/L (mean measured concentration of 1.4 mg/L, close to the solubility limt of the substance and the highest concentration it is possible to test under the conditions of the study).