Theobromine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 April 2016 - 07 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD 202. GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and 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: Stock solution was prepared with a nominal concentration of 100 mg/L and ISO medium.
- Eluate: ISO medium
- Controls: ISO medium without addition of the test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary (definitive test).
- Age at study initiation: Less than 24 years old at the beggining of the test.
- Method of breeding: The previous generation was reared with conditions similarly to the test conditions.
- Feeding during test: No
- Food type: Algal suspension of Pseudokirchneriella subcapitata.
- Frequency: At least three times per week

ACCLIMATION
- Acclimation period: To avoid the necessity of adaptation prior to the test, the water used in the test was similar to the culture conditions (water temperature, background colour etc.).
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Algal suspension of Pseudokirchneriella subcapitata.
- Feeding frequency: At least three times per week.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The reconstituted ISO medium had a total hardness of 249 mg/L as CaCO3

Test temperature:

20.1 -20.3 ºC in the test vessels and 19.6 - 20.5º C in the climate chamber.

pH:

7.82 - 7.94

Dissolved oxygen:

8.4 - 8.9 mg/L

Nominal and measured concentrations:

Nominal =100 mg/L
Measured concentration at the start of the test = 95.0 ± 1.06 mg/L
Measured concentration at the end of the test= 96.8 ± 4.23 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker
- Material, size, headspace, fill volume: 50 mL.
- Renewal rate of test solution: Static conditions
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized water was used to prepare the reconstitued water (ISO medium) according to the guideline OECD 202 .
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light/ 8 hours dark cycle

EFFECT PARAMETERS MEASURED: Immobilisation

TEST CONCENTRATIONS
Range finding study:
- Test concentration: A range-finding study was conducted with concentration of 0.1, 1, 10 and 100 mg/L in two replicates per concentration with 5 daphnids per replicate.
- Results used to determine the conditions for the definitive study: 1 daphnid was immobilised at the untreated control and 1 daphnid was immobilised at the 100 mg/L level.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

based on immobilisation

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

based on immobilisation

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

based on immobilisation

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

based on immobilisation

Details on results:

- Behavioural abnormalities: No abnormal behaviour or appearance of test organisms was observed
- Mortality of control: 1/20

Results with reference substance (positive control):

- Results with reference substance valid: Yes- 24h-EC50: 1.16 mg/L
- Other: Reference substance is tested at least twice a year.

Table 1:Immobilisation of the test animals

Test group	Number of animals	Number of immobilised animals
		24 h	48 h
Control	5	0	1
	5	0	0
	5	0	0
	5	0	0
100 mg/L (nominal)	5	0	0
	5	0	0
	5	0	0
	5	0	0

 

 Table 2:Temperature measured in the test vessels (°C)

Test group	Replicate	Measuring
		0 h	24 h	48 h
Control	1	20.3	20.1	20.1
	2	20.3	20.1	20.1
	3	20.3	20.1	20.1
	4	20.3	20.1	20.1
100 mg/L (nominal)	1	20.3	20.1	20.1
	2	20.3	20.1	20.1
	3	20.3	20.1	20.1
	4	20.3	20.1	20.2

Table 3:Oxygen concentrationmeasured in the test vessels (mg/L)

Test group	Replicate	Measuring
		0 h	48 h
Control	1	8.4	8.9
	2	8.4	8.9
	3	8.4	8.9
	4	8.4	8.9
100 mg/L (nominal)	1	8.2	8.8
	2	8.2	8.8
	3	8.2	8.8
	4	8.2	8.8

Table 4: pH measured in the test vessels

Test group	Replicate	Measuring
		0 h	48 h
Control	1	7.82	7.86
	2	7.82	7.84
	3	7.82	7.82
	4	7.82	7.84
100 mg/L (nominal)	1	7.94	7.88
	2	7.94	7.90
	3	7.94	7.90
	4	7.94	7.91

Validity criteria fulfilled:

yes

Remarks:

There were only one immobilised animal in the control group and the dissolved oxygen at the end of the test in control and test vessels was more than 3mg/L.

Conclusions:

The 48h EC50 and NOEC of theobromine in Daphnia magna was greater to and equal to 100 mg/L, respectively.

Executive summary:

A short-term toxicity test on aquatic invertebrates was performed with theobromine according to OECD Guideline 202 under GLP conditions. In accordance to a range-finding preliminary study, a limit test was performed with Daphnia magna at 100mg/L of test item. Twenty Daphnia magna (<24 h old, 5 per replicate) were exposed to either a control medium or 100 mg theobromine /L for 48 hours under static conditions. Validity criteria were fulfilled and an analytical monitoring was performed with a validated method. One daphnid was immobilised in the control medium and there was no immobilisation in vessels with theobromine, therefore the 48h-NOEC for theobromine was 100 mg/L based on nominal concentration.

Description of key information

Key study: OECD 202 and EU C.2. GLP study. The 48h-EC50 was greater than 100 mg/L in Daphnia Magna (based on mobility). The 48h-NOEC was 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

Key study: A short-term toxicity test on aquatic invertebrates was performed with theobromine according to OECD Guideline 202 under GLP conditions. In accordance with a range-finding preliminary study, a limit test was performed with Daphnia magna at 100 mg/L of test item. The 48h-EC50 and NOEC for theobromine in Daphnai magna was determined to be greater than 100 mg/L and equial to 100 mg/L based on nominal concentration (basis for effects: mobility).