Adenosine

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 - 11 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Adenosine

EC Number:

200-389-9

EC Name:

Adenosine

Cas Number:

58-61-7

Molecular formula:

C10H13N5O4

IUPAC Name:

adenosine

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

solid: particulate/powder

Details on test material:

Batch No.: 10004539
Storage at room temperature approx. 20°C

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following test vessels were set up:
- Test solution (Tn); containing Daphnia medium with test item (four replicates with five Daphnia each)
- Blank control (Bn); containing pure Daphnia medium (four replicates with five Daphnia each)
- Since the test item is soluble, the test solutions were prepared by directly dissolving the test item in Daphnia medium. The resulting solution was used as test solutions in the toxicity test. Pure Daphnia medium served as blank controls.
- O2 concentration and pH were measured in the test medium for each concentration including the blank control. If the O2 concentration was too low (>5.0 mg/l required) the media was aerated by stirring. Afterwards, the media were filled into the test vessels (50 ml per vessel).
- Daphnia, aged less than 24 h and already acclimatized to the Daphnia medium, were then introduced into the test media, and the vessels were covered with a glass plate. The Daphnia were not fed during the test and the test vessels were not aerated.
semi static conditions: After 24 h, the Daphnia were transferred to the fresh test media.
- Evidence of undissolved material: Not relevant. Since the test item is soluble, the test solutions were prepared by directly dissolving the test item in Daphnia medium

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISMS
- Test organism: Daphnia magna (Straus, 1820), derived from a healthy stock and not first brood progeny
- Breeding: Parental and young Daphnia held in 4 l glass aquaria (3 l medium) at 20±2°C
- Illumination: 16 h per day
- Medium: Continuously aerated Elendt M4 medium prepared with ultra-pure water (conductivity <1.5 µS/cm)
- Feed: Suspension of Desmodesmus subspicatus in Elendt M4 medium with an optical density OD680 of about 15 units

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

not reported

Test temperature:

18–22°C, controlled at ±1 °C

pH:

6.2 - 7.8

Dissolved oxygen:

The minimum dissolved oxygen concentration in the controls and the test vessels at the end of each 24 h-period was >=6.5 mg O2/l (required >=3 mg O2/l).

Salinity:

not reported

Conductivity:

661 to 684 µS cm-1

Nominal and measured concentrations:

The concentrations of Adenosine in the test medium were determined by HPLC analysis (LOQ 0.052 mg/l) at the beginning, after 24 h in the old and the fresh media, and after 48 h of exposure. These analyses confirmed that the test item was dissolved and the concentration was stable over each of the two 24 h test periods and remained within 80-120% of the nominal concentration. The measured concentration of the test item at the beginning of the test was 119 mg/l and 121 mg/l after 24 h in the old medium (99 and 101%, respectively, of the nominal concentration). After renewal of the test medium at 24 h, the measured concentration was 121 mg/l in the fresh medium as well as after 48 h in the old medium (100 and 101%, respectively, of the nominal concentration).

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml beakers, all-glass, with 50 ml of test medium,covered with a glass plate to avoid evaporation and contamination of the test solutions with dust
- Test medium: Elendt M4 medium; prepared with ultra-pure water (conductivity <1.5 µS/cm)
- Number of Daphnia: 20 individuals per test concentration, and for the blank, divided in 4 groups of 5 individuals
- Age: Less than 24 h
- Light: 16 h photoperiod a day, supplied by overhead white fluorescent tubes
- Temperature: 18–22 °C, controlled at ±1 °C
- pH: 6 to 9. The pH should normally not vary by more than 1.5units in one test.
- Feed: The Daphnia are not fed during the test
- Aeration: The test vessels were not aerated during the test.
- Test type: Semi-static exposure conditions; renewal of test medium after 24 h of exposure
- Test duration: 48 h

RANGE-FINDING STUDY
Prior to the definitive test a non-GLP range finding test, under static conditions, with nominal concentrations of 10 and 100 mg/l of Adenosine was performed.

EFFECT PARAMETERS MEASURED
Observed immobility (inability to swim) of the daphnids: Observations of immobile Daphnia were made after 24 and 48 h of exposure. Any abnormal behaviour or appearance was reported.

Reference substance (positive control):

yes

Remarks:

Acute reference test with potassium dichromate conducted twice a year

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No significant effects (<=10% immobilization) were observed at 120 mg/l nominal concentration or in the blank controls, neither after 24 h of exposure nor after 48 h. Therefore, the median effect concentrations (EC50) of Adenosine (CAS no. 58-61-7) on Daphnia magna after 24 h as well as after 48 h of exposure were estimated to be >120 mg/l nominal concentration
The NOEC values after 24 h and after 48 h of exposure were both 120 mg/l nominal concentration.

Results with reference substance (positive control):

Acute ref. test with K2Cr2O7 conducted twice a year. The EC50 value for the control of sensitivity for 24 h of exposure with K2Cr2O7 was estimated to be 0.89 mg/l (29.6.2016), which lies within the recomm. range of 0.6–2.1 mg/l acc. to OECD Guideline 202.

Reported statistics and error estimates:

The effective concentrations ECx were assessed based on the nominal concentration of the test item.
No statistical analysis was performed.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria i.e. immobilization in the controls <=10% and O2 concentrations at the end of the test >=3 mg/L were fulfilled.

Conclusions:

The acute toxicity of Adenosine (CAS no. 58-61-7) to Daphnia magna was determined in a 48 hour semi-static test according to OECD guideline 202. The median effect concentrations (EC50) of Adenosine on Daphnia magna after 24 h as well as after 48 h of exposure were estimated to be >120 mg/l nominal concentration. The NOEC values after 24 h and after 48 h of exposure were both 120 mg/l nominal concentration. The results of the test can be considered reliable without restriction.

Executive summary:

The acute toxicity of Adenosine (CAS no. 58-61-7) to Daphnia magna was investigated according to test guideline OECD 202, under semi-static exposure conditions over a period of 48 h.

The test item Adenosine is solid, has a high solubility (4.13 g/l at a pH of 7.0 to 7.6 and ca. 20 °C) and is 100% pure.Consequently, the test solution was prepared by directly dissolving the test item in Daphnia medium.

The test was performed as a limit test. Since preliminary tests indicated that the test concentrations were not stable in Daphnia medium over 48 h, the nominal concentration was set to 120 mg/l (so as to evaluate the toxicity of the test item at a concentration which would be surely >=100 mg/l).

20 individuals, divided in 4 groups of 5 individuals, were used for the test concentration and for the blank.

The concentration of Adenosine in the test medium was determined by HPLC analysis at the beginning of the test, as well as after 24 (in the fresh and old media) and 48 h of exposure. These analyses revealed that the concentration of the test item was stable over each of the two 24 h periods, and remained within 80-120% of the nominal concentration. Therefore, the effective concentrations ECx were assessed based on the nominal concentration of the test item.

No significant effects (<=10% immobilization) were observed at 120 mg/l nominal concentration or in the blank controls, neither after 24 h of exposure nor after 48 h.

Therefore, the median effect concentrations (EC50) of Adenosine (CAS no. 58-61-7) on Daphnia magna after 24 h as well as after 48 h of exposure were estimated to be >120 mg/l nominal concentration.

The NOEC values after 24 h and after 48 h of exposure were both 120 mg/l nominal concentration.

All validity criteria were fulfilled.