Ammonium dihydrogenorthophosphate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 August 2000 - 13 December 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP-compliant, according to OECD201 in unicellular green algae Pseudokirchneriella subcapitata. However, purity test substance unknown.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The purity, expiration date, and lot number of the test substance were not given.

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material: No data

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal: 0.0 (control), 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg DAP/L
- Sampling method: At test initiation (0 hrs), 15-mL samples were collected from each parent solution. At test termination (72 hrs), 4-mL samples were removed from each individual replicate and composited by treatment into a 12-mL total sample.
- Sample storage conditions before analysis:no data.

Vehicle:

no

Details on test solutions:

STOCK AND TEST SOLUTION AND THEIR PREPARATION
- Vehicle, solvent: no
- Concentration of vehicle/ solvent: 0 mL/L
- Other procedures: 100 and 50 mg/L were prepared by direct addition to freshwater algal nutrient medium (FWAM) followed by sonication and 50% serial dilutions were prepared from the 50 mg/L solution in FWAM to prepare subsequent test solutions.

DILUTION WATER
- Source: freshwater algal nutrient medium by addition of nutrients to ABC reagent water. After preparation, the algal medium
was pH-adjusted to 7.5 ± 0.1 (using 0.1 N HCl) and filtered through a 0.45-μm Millipore filter.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: Pseudokirchnerella subcapitata
- Source (laboratory, culture collection): Department of Botany, Culture Collection of Algae, University of Texas at Austin, Austin, Texas
- Age of inoculum (at test initiation): 6 days old culture
- Method of cultivation: In 250-mL Erlenmeyer flasks fitted with foam stoppers. Positioned on a rotary shaker set at 100 rpm and incubated for 72 hours at 24 ± 2°C under continuous cool-white fluorescent lighting. Light intensity was maintained at approximately 8600 ± 860 lux.

ACCLIMATION
- Acclimation period: No data.
- Culturing media and conditions (same as test or not): Freshwater algal nutrient medium (FWAM), same as test. The medium was prepared by the
addition of appropriate reagent grade nutrients to ABC reagent water. After preparation, the medium was pH-adjusted to 7.5 ± 0.1 (using 0.1 N HCl) and filtered through a 0.45-:m Millipore filter.
- Any deformed or abnormal cells observed: No data

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Cell counts were performed every 24 ± 1 hour after inoculation

Post exposure observation period:

no data

Hardness:

No data

Test temperature:

22.5-25.7 °C

pH:

ranged from 7.39 to 7.42 at test initiation and from 7.56 to 9.35 at test termination

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

- Nominal concentrations (mg/L): 0, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg/L DAP
- Calculated concentrations at initiation: 0, 1.65, 3.57, 6.41, 12.0, 24.1, 47.4 and 97.1 mg/L (95-114% of nominal).
DAP concentrations decreased during the exposure period (43-86% of nominal) which was likely due to incorporation of the phosphate into the algal biomass rather than instability of the test substance. Therefore, effect values were determined based on initial concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL Erlenmeyer flask containing 100 mL solution, fitted with foam stoppers.
- Initial cells density: 1.05 x10*4 cells/mL
- Control end cells density: 103 x10*4 cells/mL
- No. of organisms per vessel: 103 x10*6 cells/vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):3
- No. of vessels per vehicle control (replicates): n/a (no vehicle)

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
Light intensity in the environmental chamber ranged from 8034 to 8788 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were performed every 24 ± 1 hour after inoculation on all replicates of the control and
each test concentration. The algal cell counts were accomplished utilizing a hemacytometer and an optical microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2-fold
- Range finding study
- Test concentrations: nominal test concentrations of 0.10, 1.0, 10, 100, and 1000 mg DAP/L.
- Results used to determine the conditions for the definitive study:
Cell counts for the four lowest test concentrations ranged from 95.0 to 140x10*4 cells/mL. The control cell count was 103x10*4 cells/mL. The cell count in the 1000 mg DAP/L solution was 13.3x10*4 cells/mL. This test solution was cloudy and contained particulate matter throughout the test, indicating that the test substance was not soluble at this concentration.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

phosphate

Basis for effect:

growth rate

Remarks on result:

other: No inhibition of algal growth observed in study. Nominal initial concentration confirmed by chemical analysis (>80% recovery).

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

phosphate

Basis for effect:

biomass

Remarks on result:

other: No inhibition of algal growth observed. Nominal initial concentration confirmed by chemical analysis (>80% recovery).

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

phosphate

Basis for effect:

growth rate

Remarks on result:

other: Nominal initial concentration confirmed by chemical analysis (>80% recovery)

Details on results:

There was some growth stimulation at 6.25 mg/L and above. This was consistent with the known stimulatory effect of phosphate compounds. The reported NOEC reflects this stimulatory response and is not considered to be an adverse effect.

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

Statistical analysis to determine EbC50 and ErC50 values was not performed due to the lack of inhibition of growth during the definitive test.
The NOEC was calculated using Dunnett’s test as performed by SAS.

.

Validity criteria fulfilled:

yes

Remarks:

Growth factor control: 98

Conclusions:

The nominal 72h-ErC50 is > 100 mg/L
The nominal NOErC = 100 mg/L (no inhibition of algal growth observed in study).
Nominal initial concentrations were confirmed by chemical analysis (>80% recovery)
No adverse inhibitory effects were observed. There was some growth stimulation at 6.25 mg/L and above. This was consistent with the known stimulatory effect of phosphate compounds. The reported NOEC reflects this stimulatory response and is not considered to be an adverse effect.

Description of key information

No reliable study with ammonium dihydrogenorthophosphate is available for algae. Based on a reliable study with diammonium hydrogenorthophosphate the EC50 for freshwater algae is >100 mg/L and the NOEC is 100 mg/L.  The read-across rationale can be found in the category approach document attached in Section 13 of IUCLID and is fully incorporated in the CSR. 

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

No reliable study with ammonium dihydrogenorthophosphate is available for algae. However, in a OECD 201 guideline study, Selenastrum capricornutum algae were exposed for 72 hrs to 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg/L of diammonium hydrogenorthophosphate (DAP) under static conditions.

Measured DAP concentrations were 95-114% of nominal at the beginning of the test period. The measured concentrations decreased during the exposure period (43-86% of nominal) which was likely due to incorporation of the phosphate into the algal biomass rather than instability of the test substance. Therefore, effect values were determined based on initial concentrations. Results are expressed as nominal initial concentrations as the recovery of these concentrations was >80%.

No adverse inhibitory toxic effects were observed. There was some growth stimulation at 6.25 mg/L and above. This was consistent with the known stimulatory effect of phosphate compounds.