6H-Furo[2',3':4,5]oxazolo[3,2-a]pyrimidin-6-one, 2,3,3a,9a-tetrahydro-3-hydroxy-2-(hydroxymethyl)-7-methyl-, (2S,3S,3aR,9aS)-

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:

30 June - 03 July 2008

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.10, 1.0, 10 and 100 mg/l.
- Sampling method: During the combined limit/range-finding test singular samples for possible analysis were taken from all test concentrations and the control.
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 3 ml

- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The batch of Anhydrothymidine tested was a white powder with a purity of 99.7% and completely soluble in test medium at the concentrations tested.
Preparation of test solutions started with the highest test solution of 100 mg/l. No special treatment was necessary to completely dissolve the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name:
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): not indicated
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.


ACCLIMATION
- Acclimation period: 3 days before the start of the test
- Culturing media and conditions (same as test or not): The pre-culture was maintained under the same conditions as used in the test.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

22.8 - 23.4°C

pH:

8.0-8.1

Nominal and measured concentrations:

See the table: "Concentrations of the test substance in test medium" below

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Type: open
- Initial cells density: 1 x 10*4 cells/ml.
- No. of organisms per vessel:
6 replicates of 100 mg/l and the control,
3 replicates of 0.10, 1.0 and 10 mg/l,
1 replicate of the highest concentration without algae,
1 replicate of each test concentration without algae,
1 extra replicate of each test group for sampling purposes.



GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges
- Culture medium different from test medium: no


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: 8.1 ± 0.2
- Photoperiod: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 82 to 93 E.m-2.s-1.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
pH
At the beginning and at the end of the test.
The pH of the solutions should preferably not deviate by more than 1.5 units during the test.

Temperature of medium
Continuously in a temperature control vessel.

Appearance of the cells
At the end of the final test microscopic observations were performed to verify a normal and healthy appearance of the inoculum culture and to observe for any abnormal appearance of the algae.



TEST CONCENTRATIONS
- Test concentrations: 0.10, 1.0, 10 and 100 mg/l.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none

Results with reference substance (positive control):

- Results with reference substance valid? yes

- EC50:
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.8 mg/l with a 95% confidence interval ranging from 1.3 to 2.6 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the present batch corresponds with this range.
The EC50 for yield inhibition (EYC50: 0-72h) was 0.79 mg/l with a 95% confidence interval ranging from 0.51 to 1.2 mg/l. Historical ranges are not yet available.

Concentrations of the test substance in test medium

Time of sampling [hours]	Concentration
	Nominal [mg/l]	Analysed [mg/l]	Relative to nominal [%]	Relative to initial [%]
0	0	^3	n.a.
	100	102	102
	100 ^2	105	105
24	0	^3	n.a.	n.a.
	100	103	103	101
	100 ^2	109	109	104
72	0	^3	n.a.	n.a.
	100	104	104	103
	100 ^2	107	107	102

¹           Samples were stored in the freezer until the day of analysis.

²           Without algae.

³           A small response at the retention time of the test substance was observed. It was not considered to derive from the test samples since it was also observed in the analytical blanks.

n.a.      Not applicable.

Percentage reduction of growth rate (total test period) and percentage inhibition of yield

Concentration	Mean growth rate		Yield (0-72 h)
Anhydrothymidine
(mg/l)	µ (0-72 h)	Reduction (%)	x10^4 cells/ml	Inhibition (%)
Control	0.08603		489.82
0.10	0.08574	0.3	479.11	2.2
1.0	0.08659	-0.7	509.45	-4.0
10	0.08609	-0.1	491.92	-0.4
100	0.08573	0.3	479.39	2.1

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no reduction of growth rate or inhibition of yield was recorded at any of the concentrations of the tested substance.

The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) exceeded 100 mg/l, the regulatory limit concentration.

The NOEC for growth rate reduction and yield inhibition was 100 mg/l.

Description of key information

Under the conditions of the present study with Pseudokirchneriella subcapitata, no reduction of growth rate or inhibition of yield was recorded at any of the concentrations of the tested substance.

The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) exceeded 100 mg/l, the regulatory limit concentration.

The NOEC for growth rate reduction and yield inhibition was 100 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L