Adenosine phosphate

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:

14 - 28 February 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:

yes

Remarks:

When the test concentrations are not stable over the whole 48-h test period, it is advised to repeat the test under semi-static conditions. Although the test concentration at 48h was only 66% of the starting value, the test was not repeated.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

30 May 2008

Deviations:

yes

Remarks:

When the test concentrations are not stable over the whole 48-h test period, it is advised to repeat the test under semi-static conditions. Although the test concentration at 48h was only 66% of the starting value, the test was not repeated.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Adenosine phosphate

EC Number:

200-500-0

EC Name:

Adenosine phosphate

Cas Number:

61-19-8

Molecular formula:

C10H14N5O7P

IUPAC Name:

adenosine phosphate

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: crystalline

Remarks:

crystalline powder

Details on test material:

Batch No: 11679700
Storage: at room temperature, protected from light

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 (two replicates with 20 Daphnia each)
- Blank control (Bn); containing pure Daphnia medium (three replicates with 20 Daphnia each)
- Since the test item is soluble, the test solution was prepared by direct dissolution of the test item in Daphnia medium. The resulting solution was used as test solution 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.
- Static exposure conditions
- Evidence of undissolved material: Not relevant. Since the test item is soluble, the test solution was prepared by direct dissolution of 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:

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:

6.0 - 6.9

Dissolved oxygen:

The minimum dissolved oxygen concentration in the controls and the test vessels at the end of each 24h-period was >=7.4 mg O2/l (required >=3 mg O2/l).

Salinity:

not reported

Conductivity:

685 to 695 µS cm-1

Nominal and measured concentrations:

The concentrations of Adenosine-5’-monophosphate (AMP) in the test medium were measured by HPLC analysis at the beginning and after 24 and 48 h of exposure. These analyses revealed that the concentrations of test item decreased from the first over the second day. The measured concentrations at the beginning of the test, after 24h and at the end of the test were 94.2, 92.7 and 62.3 mg/l respectively (i.e. 94, 93 and 62%, respectively, of the nominal concentration).
Therefore, the effective concentrations ECx were assessed based on the geometric mean of the measured concentrations.

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: 40 individuals per test concentration, 20 per vessel
- 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 1.5 units in one test.
- Feed: The Daphnia are not fed during the test
- Aeration: The test vessels were not aerated during the test.
- Test type: Static exposure conditions
- Test duration: 48 h

RANGE-FINDING STUDY
Prior to the definitive test a non-GLP range finding test with nominal concentrations of 1, 10 and 100 mg/l of Adenosine-5’-monophosphate (AMP) 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.

DEVIATIONS FROM GUIDELINES
When the test concentrations are not stable over the whole 48-h test period, it is advised to repeat the test under semi-static conditions. Although the test concentration at 48h was only 66% of the starting value, the test was not repeated, in accordance with the study monitor.

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.:

> 81.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

81.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 93.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

93.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No significant effects (<=10% immobilization) were observed at 100 mg/l nominal concentration or in the blank controls, neither after 24 h of exposure nor after 48 h. Based on these data and the measured concentrations of the test item, the median effect concentrations (EC50) of Adenosine-5’-monophosphate (AMP) on Daphnia magna were estimated to be >93.4 mg/l and >81.6 mg/l measured concentration after 24 and 48 h of exposure, respectively.
The NOEC values were determined to be 93.4 mg/l and 81.6 mg/l measured concentration after 24 and 48 h of exposure, respectively.

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 geometric mean of the measured concentrations.

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-5’-monophosphate (AMP) (CAS no. 61-19-8) to Daphnia magna was determined in a 48 hour static test according to OECD guideline 202. The median effect concentrations (EC50) of Adenosine-5’-monophosphate (AMP) on Daphnia magna were estimated to be >93.4 mg/l and >81.6 mg/l measured concentration after 24 and 48 h of exposure, respectively. The NOEC values were determined to be 93.4 mg/l and 81.6 mg/l measured concentration after 24 and 48 h of exposure, respectively. The results of the test can be considered reliable without restriction.

Executive summary:

The median effect concentration (EC50) and the no-effect concentration (NOEC) of Adenosine-5’-monophosphate (AMP) (CAS no. 61-19-8) to Daphnia magna were investigated under static exposure conditions over a period of 48 h, following guideline OECD 202.

The test item Adenosine-5’-monophosphate (AMP) is solid, 94.9% pure, and has a high solubility (3.51 g/l at a pH of 3.1 and ca. 20 °C). The test solution was prepared by adding the test item to Daphnia medium and stirring it until dissolution.

The single investigated nominal concentration was 100 mg/l.

40 individual Daphnia divided into 2 test vessels were exposed to the test item, and 60 Daphnia divided into 3 test vessels were used as blank controls.

The concentrations of Adenosine-5’-monophosphate (AMP) in the test medium were determined by HPLC analysis at the beginning and after 24 and 48 h of exposure. These analyses revealed that the concentrations of test item decreased from the first to the second day. The measured concentrations at the beginning of the test, after 24h and at the end of the test were 94.2, 92.7 and 62.3 mg/l respectively (i.e. 94, 93 and 62%, respectively, of the nominal concentration). Therefore, the effective concentrations ECx were assessed based on the geometric mean of the measured concentrations.

No significant effects (<=10% immobilization) were observed at 100 mg/l nominal concentration or in the blank controls, neither after 24 h of exposure nor after 48 h.

Based on these data and the measured concentrations of the test item, the median effect concentration (EC50) of Adenosine-5’-monophosphate (AMP) (CAS no. 61-19-8) on Daphnia magna after 24 h of exposure was estimated to be >93.4 mg/l measured concentration; and the EC50 after 48h was estimated to be >81.6 mg/l measured concentration.

The NOEC value after 24 h of exposure was 93.4 mg/l measured concentration, and the NOEC after 48h was 81.6 mg/l measured concentration.

All validity criteria were fulfilled.