p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

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:

16-23 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2021

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

impurity 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

impurity 2

Reference substance name:

Acetone

EC Number:

200-662-2

EC Name:

Acetone

Cas Number:

67-64-1

Molecular formula:

C3H6O

IUPAC Name:

acetone

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Constituent 1

Reference substance name:

p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

EC Number:

269-198-6

EC Name:

p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Cas Number:

68189-42-4

Molecular formula:

C6H6NO6P.2C4H11NO3

IUPAC Name:

4-nitrophenyl dihydrogen phosphate - 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Test material form:

solid

Details on test material:

Batch / Lot No. 46587100

Specific details on test material used for the study:

Batch no.: 46587100
Test item storage: 2–8 °C, protected from light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test media including the control at the start of the test (without algae) and after 24, 48 and 72 hours of exposure (containing algae). For sampling at the start of the test the samples were taken from the freshly prepared single test solution as well as from the pure test water serving as control. For sampling at 24, 48 and 72 hours, the replicates per treatment were pooled prior to sampling. These samples were centrifuged at 4500 g for 10 minutes to separate the algae.
All samples were diluted with acetonitrile containing conc. 1% phosphoric acid to obtain a composition of sample:acetonitrile (9:1; v:v) containing 0.1% conc. phosphoric acid. Subsequently, the samples were stored deep-frozen (at about -20 °C) until analysis. In non-GLP preliminary experiments, the test item proved to be stable under these storage conditions.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
• Test solution: containing test water, test item and algae (six replicates)
• Control: containing test water and algae (six replicates)
Since the test item is soluble, the single test solution was prepared by adding 100 mg/l of test item to test water in sterile glassware and stirring it until dissolution. 100 ml of the resulting test media were added to the sterile test flasks and inoculated with an exponentially growing preculture of algae.
For the incubation, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min. The test flasks were repositioned daily during the exposure.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Test organism: Raphidocelis subcapitata (SAG Nr. 61.81), supplied by the SAG Culture Collection of Algae at Göttingen University, 37073 Göttingen, Germany.
- Test vessel: 250 ml flasks, all-glass, with 100 ml of test medium, shaken (130 rpm), capped with air permeable stoppers.
- Preculture: Exponentially growing liquid culture of Raphidocelis subcapitata kept under the same environmental conditions as in the test.
- Illumination: Continuous, 4440 – 8880 lux ± max. 15% variation,
source: Osram Fluora L 18W/77 and LEDVANCE Cool light, 10 W, 4000 K, 1000 lm
- Temperature: 21–24 °C maintained at ±2 °C in a thermo-controlled room.
- Control of sensitivity: For evaluation of the algal quality and the test procedure, potassium dichromate is tested as a positive control twice a year.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

The calculated water hardness of the test water is 24.2 mg/l as CaCO3.

Test temperature:

21–24 °C maintained at ±2 °C in a thermo-controlled room.

pH:

The pH was 8.0±0.2. The pH of the control should not increase by more than 1.5 units during the test.

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

NOMINAL CONCENTRATIONS
Since no relevant inhibitory effects were observed in the non-GLP range-finding test, the definitive test was performed as a limit test at a nominal concentration of 100 mg/l.

MEASURED CONCENTRATIONS
The concentrations of 4-NPP, di-TRIS in the test media were determined by HPLC with UV detection in the samples from the test start as well as in the samples after 24, 48 and 72 hours of exposure.
These analyses showed that the single test concentration was correctly dosed and remained within 80–120% of the nominal concentration over the duration of the test.
Therefore, the effective concentrations ECx were assessed based on the 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), capped with air permeable stoppers.
- Test water: Reconstituted water (OECD medium) prepared according to the test guidelines was used for algal cultivation and testing.
- Preculture: Exponentially growing liquid culture of Raphidocelis subcapitata kept under the same environmental conditions as in the test.
- Illumination: Continuous, 4440 – 8880 lux ± max. 15% variation,
source: Osram Fluora L 18W/77 and LEDVANCE Cool light, 10 W, 4000 K, 1000 lm
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- pH: Initially adjusted to 8.0±0.2; the pH of the control medium should not increase by more than 1.5 units during the test
- Test design: Static exposure conditions over a period of 72 h
- Initial cell density: Nominal density of 5,000 cells/ml in all test vessels.

RANGE FINDING STUDY
Prior to the definitive test a non-GLP range finding test (three replicates for the control and two replicates per test concentration) with nominal concentrations of 1, 10 and 100 mg/l test item was performed. No relevant inhibitory effects were observed in the non-GLP range-finding test.

EFFECT PARAMETERS MEASURED
- Biomass: Algal biomass is determined at 24, 48 and 72 hours of exposure using a spectrophotometer at 680 nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH 4153 Reinach). Aliquots of 5 ml are sampled from each test flask. At test start (0 hours) the nominal value is applied.

Reference substance (positive control):

yes

Remarks:

Control of sensitivity: Potassium dichromate is tested as a positive control twice a year.

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:

AVERAGE SPECIFIC GROWTH RATE
With respect to the endpoint average specific growth rate no significant effects (Student t-test, one-sided smaller, ¿ = 0.05) were observed at a nominal concentration of 100 mg/l 4-NPP, di-TRIS compared to the control.
The 72-hour EC50 based on average specific growth rate of Raphidocelis subcapitata was therefore estimated to be >100 mg/l, while the NOEC was determined to be 100 mg/l.
YIELD
With respect to the endpoint yield no significant effects (Student t-test, one-sided smaller, ¿ = 0.05) were observed at a nominal concentration of 100 mg/l 4-NPP, di-TRIS compared to the control.
The 72-hour EC50 based on yield of Raphidocelis subcapitata was therefore estimated to be >100 mg/l, while the NOEC was determined to be 100 mg/l.

OBSERVATIONS AND ENVIRONMENTAL CONDITIONS
The microscopic examination of the algal cells at the end of the test showed no abnormal appearance of the algae growing at the single test concentration of nominal 100 mg/l. The appearance of the algal cells was not affected by the test item up to at least this concentration.
No remarkable observations were made concerning the appearance of the test media.The pH value in the control replicates changed by 0.7 units. The light intensity was maintained at 5306 ± 6%. The temperature remained at 23 °C over the duration of the test. All values were within the guideline ranges

Results with reference substance (positive control):

The result of the positive control test performed in July 2020 showed that the sensitivity of the test system based on average specific growth rate (72 h EC50 = 1.25 mg/l) was in line with a ring test yielding 1.19 mg/l, SD = 0.27 (ISO 8692:2012: Water quality - Fresh water algal growth inhibition test with unicellular green algae).

Reported statistics and error estimates:

The lowest observed effect concentration (LOEC) is the lowest concentration tested at which the substance is observed to have a statistically significant reducing effect on the respective endpoint (p < 0.05) when compared with the control within a stated exposure period. The LOEC values were determined using the software ToxRat® Pro. In case of no significant effects, the LOEC is not reported. The no observed effect concentration (NOEC) is the test concentration immediately below the LOEC.

Any other information on results incl. tables

Validity of the test:

 

Validity criterion (applies to control cultures)	Required value	Achieved value
Average specific growth rate [d^-1]	>=0.92	1.9
CV of average specific growth rate over 3 days [%]	<=7	0.8
CV of section-by-section specific growth rates [%]	<=35	9.9

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See table in section "any other information on results incl. tables".

Conclusions:

4-NPP, di-TRIS had no significant inhibitory effects on the green algae Raphidocelis subcapitata up to the nominal concentration of 100 mg/l under the conditions of the test.

Executive summary:

The growth inhibitory effects of 4-NPP, di-TRIS (CAS no. 68189-42-4) to the green alga Raphidocelis subcapitata were investigated according to the OECD guideline 201 over a period of 72 hours.
The test item 4-NPP, di-TRIS is solid, 99.03% (w/w) pure and soluble in test water up to at least 100 mg/l. The single nominal concentration tested was 100 mg/l.
The concentrations of 4-NPP, di-TRIS in the test media were determined by HPLC with UV detection in the samples from the test start as well as in the samples after 24, 48 and 72 hours of exposure. These analyses showed that the single test concentration was correctly dosed and remained within 80–120% of the nominal concentration over the duration of the test. Therefore, the effective concentrations ECx were assessed based on the nominal concentration of the test item.
With respect to the endpoint average specific growth rate no significant effects were observed at a nominal concentration of 100 mg/l compared to the control.
With respect to the endpoint yield no significant effects were observed at a nominal concentration of 100 mg/l compared to the control.
The results of the average specific growth rate and yield inhibition of 4-NPP, di-TRIS to the green alga Raphidocelis subcapitata are summarised in the following table based on nominal concentration:

 

Parameter (0-72 h)	Growth rate [mg/l]	Yield [mg/l]
EC50	>100	>100
NOEC	100	100

In conclusion, 4-NPP, di-TRIS had no significant inhibitory effects on the green algae Raphidocelis subcapitata up to the nominal concentration of 100 mg/l under the conditions of the test.
All validity criteria were fulfilled.