Adenosine phosphate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 February - 2 March 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006, Annex 5 corrected 28 July 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

20 May 2008, amended 7 December 2015, for the purpose of its adaptation to technical progress, by Commission Regulation (EU) 2016/266

Deviations:

no

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

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
- Test solution (Tn); containing test medium, test item and algae (six replicates)
- Blank control (Bn); containing test medium and algae (six replicates)
- Since the test item is soluble, the test solution was prepared by adding the test item to sterile filtered Algal medium (pH adjusted at 8.0 ± 0.2) in sterile glassware and stirring it until dissolution. 100 ml of each test solution was added to the sterile test flasks and inoculated with an exponentially growing preculture of algae.
Pure Algal medium, also sterile filtered into sterile test flasks and inoculated with the exponentially growing preculture of algae mentioned above, served as blank controls. For the incubation, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.
- Evidence of undissolved material: Not relevant. Since the test item is soluble, the test solution was prepared by adding the test item to sterile filtered Algal medium (pH adjusted at 8.0 ± 0.2).

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Test organism: Axenic slope culture of Desmodesmus subspicatus 86.81SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany
- Culture: 250 ml flask containing 100 ml of sterile OECD medium inoculated with cell material from an axenic slope culture
- Illumination: Continuous (2000–3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland)
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- Control of sensitivity: Twice per year, with potassium dichromate

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

not reported

Test temperature:

Min: 22.4; Max: 22.9; Mean: 22.7

pH:

6.4 - 8.0

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

The concentrations of Adenosine-5’-monophosphate (AMP) in the test media were measured by HPLC analysis at the beginning and after 24, 48 h and 72 h of exposure. These analyses revealed that the test item was fully dissolved and the test concentration was satisfactorily maintained over the 72 h test period (84% of the initially measured concentration).
Therefore, the effective concentrations ErCx/EyCx were assessed based on nominal concentration of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml flasks, all-glass, with 100 ml of test medium, shaken (130 rpm)
- Test medium: Recommended OECD medium
- Preculture: Exponentially growing culture of Desmodesmus subspicatus
- Illumination: Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland)
- Light intensity: the light intensity amounts to about 3000 lux which corresponds to about 40 µE•m-2•s-2
- Homogeneity: the light intensity is maintained within ±15% from the average light intensity over the incubation area. The test vessels are repositioned every day within the incubation area to minimize variation.
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- pH: Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test
- Test type: Static exposure conditions over a period of 72 h
- Starting cell density: 0.5–0.85 µg/ml with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units)

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. The results indicated that the test item is well soluble and that the test solutions can be prepared by respective dilutions of a stock solution. It also showed, that at a concentration of 100 mg/l the test item is rather stable in algal medium over 72 h. Given the very limited and biologically non-significant effects observed based on the growth rate in this non-GLP range finding test the definitive test run as a limit-test at 100 mg/l.

EFFECT PARAMETERS MEASURED
- Cell density: Cell concentrations were determined every 24 h using a spectrophotometer at 680 nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). Aliquots of 5 ml were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the blank controls.

DEVIATIONS FROM GUIDELINES
The HPLC stock solution for the standards (test item dissolved in ultra-pure water) was diluted with OECD algal medium and not as described in the study with a 0.01 M phosphate buffer pH 4.0. Therefore, the method was not validated in the course of the OECD 105 test (A17-00028) but in a separate validation. These deviations had no influence on the test since the two tests are independent and the described test is based on the new validation.

Reference substance (positive control):

not required

Remarks:

Control of sensitivity: None – the culture is re-purchased annually

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

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

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

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
- Any stimulation of growth found in any treatment: yes (growth rate and yield)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

With respect to the endpoint growth rate, no significant toxic effects were observed at the nominal concentration of 100 mg/l as compared to the blank controls. Rather, a slight activation of the cell growth was observed.
Based on these effects and the nominal concentrations of the test item, the median effect concentration with respect to growth rate (ErC50/0–3 days) of Adenosine-5’-monophosphate (AMP) to Desmodesmus subspicatus was estimated to be >100 mg/l.
The no observed effect concentration (NOErC) with respect to growth rate was determined to be 100 mg/l.

With respect to the endpoint yield (biomass production), no significant toxic effects were observed at the nominal concentration of 100 mg/l as compared to the blank controls. Rather, a slight activation of the cell growth was observed.
Based on these effects and the nominal concentration of the test item, the median effect concentration with respect to yield (EyC50/0–3 days) of Adenosine-5’-monophosphate (AMP) to Desmodesmus subspicatus was estimated to be >100 mg/l.
The no observed effect concentration (NOEyC) with respect to yield was determined to be 100 mg/l.

Reported statistics and error estimates:

The statistical significance of differences between effects observed for control and test item treatments was determined by Dunnett’s test or Student-t test using the ToxRat® Standard Version 2.10.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

In the control cultures: average specific growth rate of 1.31 (>0.92)/ coefficient of variation of average specific growth rates: 1% (<=7%)/ mean coefficient of variation for section-by-section specific growth rates 21.8% (<=35%).

Conclusions:

The acute toxicity of Adenosine-5’-monophosphate (AMP) (CAS no. 61-19-8) to Desmodesmus subspicatus was determined in a 72 hour static test according to OECD guideline 201. The median effect concentration with respect to growth rate (ErC50/0–3 days) of Adenosine-5’-monophosphate (AMP) to Desmodesmus subspicatus was estimated to be >100 mg/l. The no observed effect concentration (NOErC) with respect to growth rate was determined to be 100 mg/l. The results of the test can be considered reliable without restriction.

Executive summary:

The effective concentrations (ErC10, ErC50 and EyC10, EyC50) as well as the no observed effect concentrations (NOErC and NOEyC), of Adenosine-5’-monophosphate (AMP) (CAS no. 61-19-8) to

the green alga Desmodesmus subspicatus based on the average specific growth rate and the yield (algal biomass production) were investigated over a period of 72 h, following guideline OECD 201.

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 sterile-filtered Algal medium and stirring it until dissolution.

The only nominal concentration was 100 mg/l. Six parallel test vessels were used for the test item and six for the blank controls.

The concentrations of Adenosine-5’-monophosphate (AMP) in the test media were measured by HPLC analysis at the beginning and after 24, 48 and 72 h of exposure. These analyses revealed that the test item was fully dissolved and that the concentration was stable over the 72 h test period (84% of the initially measured concentration). Therefore, the effective concentrations ErCx/EyCx were assessed based on the nominal concentration of the test item.

With respect to the endpoint growth rate, no significant effects were observed at 100 mg/l nominal concentration, as compared to the blank controls.

With respect to the endpoint yield (algal biomass), no significant effects were observed at 100 mg/l nominal concentration, as compared to the blank controls.

Therefore, the 72h ErC50 and EyC50 values of Adenosine-5’-monophosphate (AMP) (CAS no. 61-19-8) on the green alga Desmodesmus subspicatus were both estimated to be >100 mg/l nominal concentration.

The NOErC and NOEyC values were both 100 mg/l nominal concentration.

All validity criteria were fulfilled.