Disodium 4-nitrophenyl 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:

23 - 25 August 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:

Disodium 4-nitrophenyl phosphate

EC Number:

224-246-5

EC Name:

Disodium 4-nitrophenyl phosphate

Cas Number:

4264-83-9

Molecular formula:

C6H4NO6P.2Na

IUPAC Name:

disodium 4-nitrophenyl 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

Details on test material:

Batch Nr: 11674900
Storage: 2-8°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 (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 dissolving 100 mg/l test item in Daphnia medium. The resulting solution was used as the 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 dissolving 100 mg/l 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:

6.5 to 7.0

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:

712 to 769 µS cm-1

Nominal and measured concentrations:

The concentration of 4-Nitrophenyl phosphate disodium salt in the test medium was determined by HPLC analysis at the beginning and after 24 as well as after 48 h of exposure. These analyses revealed that the concentration of the test item was stable over the course of the 48 h exposure period and was within the 80-120% of the nominal concentration. The measured concentration of the test item at the beginning of the test was 96.5 mg/l and 93.2 mg/l (i.e. 97% of the initial value) after 48 hours of exposure, respectively.

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 1.5 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 4-Nitrophenyl phosphate disodium salt was performed. The HPLC determinations indicate that the test item concentrations are stable in the highest (100 mg/l) concentration. Static conditions were therefore applied in the definitive test, since a limittest at 100 mg/l 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
For the chemical analysis, the samples were prepared by diluting them into the calibration range with ultra-pure water containing 0.1% conc. phosphoric acid before HPLC analysis and not as mentioned in the study plan with ultra-pure water followed by an acidification step. Since the preparation of the standards and the samples was identical, this method is preferred and has got no impact on the outcome of the test.
The molecular formula indicated in the report was wrong (C6H6NO6P.2Na), the correct one is C6H4NO6P.2Na. This deviation has no impact on the outcome of the test.

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.:

> 100 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.:

100 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.:

> 100 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.:

100 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 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 4-Nitrophenyl phosphate disodium salt (CAS no. 4264-83-9) on Daphnia magna were estimated to be >100 mg/l nominal concentration after 24 as well after 48 h of exposure, respectively.
The NOEC values were determined to be 100 mg/l nominal concentration after 24 as well after 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 concentration ECx was assessed based on the nominal 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 4-Nitrophenyl phosphate disodium salt (CAS no. 4264-83-9) to Daphnia magna was determined in a 48 hour static test according to OECD guideline 202. The median effect concentrations (EC50) of 4-Nitrophenyl phosphate disodium salt on Daphnia magna were estimated to be >100 mg/l nominal concentration after 24 as well as after 48 h of exposure, respectively. The NOEC values were determined to be 100 mg/l nominal concentration after 24 as well as after 48 h of exposure, respectively. The results of the test can be considered reliable without restriction.

Executive summary:

The acute toxicity of 4-Nitrophenyl phosphate disodium salt (CAS no. 4264-83-9) to Daphnia magna was investigated according to test guideline OECD 202 under static exposure conditions over a period of 48 h.

The test item 4-Nitrophenyl phosphate disodium salt is solid, 71.5% pure and has a high solubility (=294 g/l at pH 8 and at 22 °C). Therefore, the test solution was prepared by dissolving 100 mg/l test item in Daphnia medium. The single concentration tested was 100 mg/l nominal.

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

The concentration of 4-Nitrophenyl phosphate disodium salt in the test medium was determined by HPLC analysis at the beginning and after 24 as well after 48 h of exposure. These analyses revealed that the concentration of the test item was stable over the course of the 48 h exposure period and was within 80-120% of the nominal concentration. Therefore, the effective concentrations ECx were assessed based on the nominal concentration.

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 concentration (EC50) of 4-Nitrophenyl phosphate disodium salt (CAS no. 4264-83-9) on Daphnia magna after 24 h as well as after 48 h of exposure was estimated to be >100 mg/l nominal.

The NOEC values after 24 h and after 48 h of exposure were both 100 mg/l nominal.

All validity criteria were fulfilled.