Adenosine 5'-(tetrahydrogen triphosphate), disodium salt

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 November - 24 November 2016

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 5'-(tetrahydrogen triphosphate), disodium salt

EC Number:

213-579-1

EC Name:

Adenosine 5'-(tetrahydrogen triphosphate), disodium salt

Cas Number:

987-65-5

Molecular formula:

C10H14N5O13P3.2Na

IUPAC Name:

disodium hydrogen [({[5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl phosphonato)oxy]phosphonate

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

impurity 2

Reference substance name:

Unknown impurities.

Molecular formula:

Not available as unknown impurities.

IUPAC Name:

Unknown impurities.

Test material form:

solid: crystalline

Details on test material:

Storage conditions: 2-8°C
Batch No: 11678500

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

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)
- Due to high solubility (= 573 g/l at pH 3 and at 22 °C, as determined in the GLP test OECD 105, ACH no. A16-01712), the test solutions were prepared by dilution of a stock solution: A 100 mg/l stock solution was prepared by adding the test item to sterile-filtered Algal medium (pH adjusted at 8.0 ± 0.2) in sterile glassware and stirred for 10 minutes. 100 ml of this stock 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. Due to high solubility (= 573 g/l at pH 3 and at 22 °C), the test solutions were prepared by dilution of a stock solution.

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: None – the culture is re-purchased annually

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: 21.7; Max: 23.8; Mean: 22.2

pH:

6.7 - 7.8

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

The concentrations of ATP, Di-Na 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 concentrations were stable over the 72h test period (98% of the initially measured concentration) and were within 80-120% of the nominal concentration. The initially measured concentration was 97.2 mg/l and the measured concentration after 72 h of exposure was 95.1 mg/l.
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 ATP, Di-Na 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. Based on the results of this non-GLP range finding test, the definitive test was performed as a limit test at the nominal concentration of 100 mg/l.

EFFECT PARAMETERS MEASURED
- Cell density: Cell concentrations were determined every 24 h using a spectrophotometer at 680nm 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.

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 effects were observed as compared to the blank controls.
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 ATP, Di-Na to Desmodesmus subspicatus was estimated to be >100 mg/l.
The low effect concentration with respect to growth rate ErC10 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 effects were observed as compared to the blank controls.
Based on these effects and the nominal concentrations of the test item, the median effect concentration with respect to yield (EyC50/0–3 days) of ATP, Di-Na to Desmodesmus subspicatus was estimated to be >100 mg/l.
The low effect concentration with respect to yield EyC10 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:

Statistical analysis was performed with respect to the no observed effect concentrations. The statistical analysis was conducted with the software ToxRat® Standard Version 3.2.1.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

In the control cultures: average specific growth rate of 1.38 (>0.92)/ coefficient of variation of average specific growth rates: 1.5% (=7%)/ mean coefficient of variation for section-by-section specific growth rates 23.4% (=35%).

Conclusions:

The acute toxicity of ATP, Di-Na (CAS no. 987-65-5) 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 ATP, Di-Na 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 ATP, Di-Na (CAS no. 987-65-5) 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.

The test item ATP, Di-Na is solid, has a high solubility (= 573 g/l at pH 3 and at 22 °C) and is 90.3% pure. Consequently, the test solutions were prepared by dilution of a stock solution. The single investigated nominal concentration was 100 mg/l. The test, therefore, represents a limit test.

Six parallel test vessels were used for the test item concentration and six vessels for the blank controls. The concentrations of ATP, Di-Na 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 concentrations were stable over the 72h test period (98% of the initially measured concentration) and were within 80-120% of the nominal concentration. Therefore, the effective concentrations ErCx/EyCx were assessed based on nominal concentration of the test item.

With respect to the endpoint growth rate and yield (biomass production), no significant effects were observed at the nominal concentration of 100 mg/l as compared to the blank controls. 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) and yield (EyC50/0–3 days) of ATP, Di-Na to Desmodesmus subspicatus was estimated to be >100 mg/l.

The no observed effect concentration with respect to growth rate (NOErC) and yield (NOEyC) was estimated to be 100 mg/l nominal concentration.

All validity criteria were fulfilled.