Theobromine

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:

04 April 2016 - 07 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test method according to OECD 201. GLP 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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: M15015C
- Expiration date of the lot/batch: Retest date 01/2020
- Purity test date:12/01/2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)
- Stability under test conditions:The test item was found to be stable during the test period

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L
- Sampling method: Analytical measurements were performed from the control and at the applied test concentration level at the beginning and at the end of experiment.
- Sample storage conditions before analysis: No storage was necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a concentration of 100 mg/L was prepared with the test item and algal growth medium (OECD Medium). As a Limit Test was carried out, further dilution of stock solution was not be performed
- Controls: A concurrent untreated control with OECD medium was performed and a reference control (potassium dichromate) is performed at least twice a year to demonstrate satisfactory test conditions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. They were cultured under standardised conditions.
- Age of inoculum (at test initiation): Three days of incubation in exponential growth.
- Method of cultivation: Stock cultures were small algal colonies that are inoculated onto agar regularly. These were transferred to fresh agar medium at least once every two months and were maintained under standardised conditions according to the test guidelines.The pre-culture was intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days.

ACCLIMATION
- Acclimation period: Since the algae was supplied by the SAG they were cultured under standardized conditions.
- Culturing media and conditions: Same as test
- Any deformed or abnormal cells observed: When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

The OECD medium was in accordance with the OECD guideline 201.

Test temperature:

Temperature in the test media during test: 23.2-23.4 ºC

pH:

7.63 - 8.91

Salinity:

The OECD medium was in accordance with the OECD guideline 201.

Nominal and measured concentrations:

Nominal: 100 mg/LAnalytical: Start of the test= 91.5 ± 0.72End of the test = 91.2 ± 0.97 Control= Not detected.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): covered by air-permeable stoppers
- Material, size, headspace, fill volume: Volumes of 100 mL test solution per replicate in 250 mL sterile Erlenmeyer flasks.
- Initial cells density: 10000
- Control end cells density: mean of replicates = 733300
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized water

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuously iluminated.
- Light intensity and quality: 8145 lux equivalent to approximately 110 µE/m2/s which was ensured with fluorescent lamps (spectran range 400 - 700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: manual cell counting using a microscopic method with a counting chamber
- Other: No abnormal appearance of the algae was observed.

TEST CONCENTRATIONS
Range finding study:
- Test concentrations: 0.1, 1, 10 and 100 mg/L with two replicates per each test concentration.
- Results used to determine the conditions for the definitive study: Untreated control = 71.33 (average of cell number at 72 hours (x10000 cells(mL))0.1 mg/L = 71.00 (average of cell number at 72 hours (x10000 cells(mL))1 mg/L = 67.50 (average of cell number at 72 hours (x10000 cells(mL))100 mg/L = 67.50 (average of cell number at 72 hours (x10000 cells(mL))

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:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

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): No
- Unusual cell shape: No- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: The area under growth curve (0-72h) was 0.3 % higher at the end of the study than control group
- 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: As a limit test was performed at 100 mg/L the effects of concentrations exceeding solubility were not assessed.

Results with reference substance (positive control):

- Results with reference substance valid = YesThe 72h ErC 50: 0.84 mg/L, (95 % confidence limits: 0.77 – 0.92 mg/L)The 72h EbC 50: 0.59 mg/L, (95 % confidence limits: 0.54 – 0.65 mg/L)The 72h EyC 50: 0.55 mg/L, (95 % confidence limits: 0.50 – 0.60 mg/L). These values are within the range of laboratory ring test data ( ISO Guideline No. 8692).

Validity criteria fulfilled:

yes

Remarks:

The cell density in the control increased by a factor of 73.33 within the 72h-period. The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 10.80 %. The coefficient of variat

Conclusions:

The 72h-EC50 (based on biomass, growth rate or yield) of Thebromine in Pseudokirchneriella subcapitata was greater than 100 mg/L and the 72h-NOEC was 100 mg/L.

Executive summary:

A freshwater Alga growth inhibition test was performed with theobromine according to OECD Guideline 201 under GLP conditions. In accordance to a range-finding study a limit test was performed with Pseudokirchneriella subcapitata at 100 mg/L. 6 replicates of algal cultures were exposed to 100 mg/L theobromine. These cultures plus 6 replicates of untreated controls were incubated on an orbital shaker under continuous illumination for 72 h. Growth was monitored daily by measuring the number of cells in each culture. An analytical monitoring was performed and validated. The 72h-EC50 based on biomass, growth rate and yield was > 100 mg/L; the 72h-NOEC was 100 mg/L, based on nominal concentration.

Description of key information

Key study: OECD 201 and EU C.3. GLP study. The 72h-EC50 was greater than 100 mg/L in green algae (based on growht rate inhibition). The 48h-NOEC was 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Key study: A freshwater alga growth inhibition test was performed with theobromine according to OECD Guideline 201 under GLP conditions. In accordance to a range-finding study, a limit test was performed with Pseudokirchneriella subcapitata at 100 mg test item/L. Based on nominal concentration, the 72h-EC50 based on biomass, growth rate and yield was > 100 mg/L and the 72h-NOEC was 100 mg/L.