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Ecotoxicological information

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 - 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
Deviations:
no
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

Constituent 1
Chemical structure
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 9-{5-O-[hydroxy({[(hydroxyphosphinato)oxy]phosphinato}oxy)phosphoryl]pentofuranosyl}-9H-purin-6-amine
impurity 1
Chemical structure
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 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)
- Due to high solubility (= 573 g/l at pH 3 and at 22 °C, as determined in the GLP test OECD 105, test report ACH no. A16-01712), the test solution was prepared by dilution of a stock solution (test item dissolved in Daphnia medium) with aerated Daphnia medium. The resulting solutions were 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.
After 24 h, the Daphnia were transferred to the fresh test media.
- 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):
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:
6.5 to 7.5
Dissolved oxygen:
The minimum dissolved oxygen concentration in the controls and the test vessels at the end of each 24h-period was >=6.5 mg O2/l (required >=3 mg O2/l).
Salinity:
not reported
Conductivity:
693 to734 µS cm-1
Nominal and measured concentrations:
The concentrations of ATP, Di-Na in the test medium were measured by HPLC analysis at the beginning, after 24 h in the fresh and old media, and after 48 h of exposure. These analyses confirmed that the test item concentrations were unstable in Daphnia medium over the test period and did not remain within 80-120% of the nominal concentration. The measured concentrations of the test item in the fresh media at the beginning of the test and after 24 h of exposure were 94.3
and 95.3 mg/l, respectively. The measured concentrations of the old media after 24 and 48 h of exposure were 19.4 and 21.1 mg/l, respectively (i.e. respectively 19 and 21% of the initial value). The geometric mean concentration after 24 h was 42.8 mg/l and the arithmetic mean of the geometric mean of the measured concentrations after 48 h of exposure was 43.8 mg/l.
The effective concentrations ECx were assessed based on the arithmetic mean of 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 (Table 2 and Table 3); 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 1.5units 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 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 shows, that the test item is not stable in Daphnia medium and that its concentrations decreased over time. 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. Since the test concentrations decreased over time, the definitive test was run under semi-static exposure conditions (the test medium was renewed after 24 h of exposure).

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 concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 43.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
43.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 42.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
42.8 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. Therefore, the median effect concentrations (EC50) of ATP, Di-Na on Daphnia magna were estimated to be >42.8 mg/l and >43.8 mg/l measured concentration after 24 and 48 h of exposure, respectively.
The NOEC values were determined to be 42.8 mg/l and 43.8 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 arithmetic mean of 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 ATP, Di-Na (CAS no. 987-65-5) to Daphnia magna was determined in a 48 hour semi-static test according to OECD guideline 202. The median effect concentrations (EC50) of ATP, Di-Na on Daphnia magna were estimated to be >42.8 mg/l and >43.8 mg/l measured concentration after 24 and 48 h of exposure, respectively. The NOEC values were determined to be 42.8 mg/l and 43.8 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 ATP, Di-Na (CAS no. 987-65-5) to Daphnia magna were investigated under semi-static exposure conditions over a period of 48 h, following guideline OECD 202.

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 media were prepared by respective dilutions of a stock solution. Since the test item was shown to be unstable in Daphnia medium, the test run under semi-static conditions with renewal of the test solutions after 24 h of exposure.

The test run as limit test at the nominal concentration of 100 mg/l. 40 individual Daphnia divided into two test vessels were exposed to the test item, and 60 Daphnia divided into three test vessels were used as blank controls.

The concentrations of ATP, Di-Na in the test medium were measured by HPLC analysis at the beginning, after 24 h in the old and fresh media, and 48 h of exposure. These analyses confirmed that the test item concentrations were unstable in Daphnia medium over the test period and did not remain within 80-120% of the nominal concentration. Therefore, the effective concentrations ECx were assessed based on 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. Therefore, the median effect concentrations (EC50) of ATP, Di-Na on Daphnia magna were estimated to be >42.8 mg/l and >43.8 mg/l measured concentration after 24 and 48 h of exposure, respectively.

The NOEC values were determined to be 42.8 mg/l and 43.8 mg/l measured concentration after 24 and 48 h of exposure, respectively.

All validity criteria were fulfilled.