Superphosphates

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Remarks:

Characterization of the test and reference substances were not conducted under these principles of Good Laboratory Practice.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal: 0.0 (control), 6.25, 12.5, 25.0, 50.0, and 100 mg GSTP/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.
The control solution consisted of freshwater algal nutrient medium and was the same medium used to prepare the 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-μm 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:

22.0 - 25.4°C

pH:

6.03 - 7.59 at test initiation and 6.44 - 9.31 at test termination

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

- Nominal concentrations (mg/L): 0, 6.25, 12.5, 25.0, 50.0 and 100 mg/L GTSP
- Calculated concentrations mean (mg/L): 0, 4.83, 10.3, 21.6, 42.6 and 87.6mg/L (77-88% of nominal).
GTSP concentrations decreased slightly during the exposure period. The abiotic replicates measured 87% of the nominal concentration at test
termination, which was similar to the recoveries of the 0- and 72-hour test concentrations.
Only the calculated measured concentration at the lowest nominal concentration of 6.25 mg/L had a recovery that was slightly lower than 80% (77%). Since the recovery at all other concentrations was >80% and no inhibition of algal growth was observed, effect concentrations are expressed as nominal 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: 92 x10*4 cells/mL
- No. of organisms per vessel: 93 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 7793 to 8367 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 GSTP/L.
- Results used to determine the conditions for the definitive study:
After 72-hours of incubation, cell counts for the four lowest test concentrations ranged from 94.3 to 112x104 cells/mL.
The control cell count was 103x104 cells/mL. The cell count in the 1000 mg GTSP/L solution was 3.75x104 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. The pH of this solution was 3.99 and 3.76 at test initiation and termination, respectively. The low pH was likely to have contributed to
the inhibition observed in the cell numbers at termination.

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: Nominal concentration confirmed by chemical analysis (>80% recovery, except for lowest concentration with 77% 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: Nominal concentration confirmed by chemical analysis (>80% recovery, except for lowest concentration with 77% recovery).

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 concentration confirmed by chemical analysis (>80% recovery, except for lowest concentration with 77% recovery).

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

phosphate

Basis for effect:

biomass

Remarks on result:

other: Nominal concentration confirmed by chemical analysis (>80% recovery, except for lowest concentration with 77% recovery).

Details on results:

GTSP produced a stimulation of algal growth(biomass) at all concentrations tested that ranged from 6 to 24% compared to the control biomass.
Algal growth rate was inhibited or consistent with the growth rate of the control algae for the 6.25 and 25 mg GTSP/L concentrations.
All other test concentrations demonstrated slight stimulation (2 to 5%) in the algal growth rate as compared to the control growth rate.

Results with reference substance (positive control):

No positive control.

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: 88

Conclusions:

Only the calculated measured concentration at the lowest nominal concentration of 6.25 mg/L had a recovery that was slightly lower than 80% (77%). Since the recovery at all other concentrations was >80% and no inhibition of algal growth was observed, effect concentrations are expressed as nominal concentrations.
GTSP produced a stimulation of algal growth(biomass) at all concentrations tested that ranged from 6 to 24% compared to the control biomass.
Algal growth rate was inhibited or consistent with the growth rate of the control algae for the 6.25 and 25 mg GTSP/L concentrations.
All other test concentrations demonstrated slight stimulation (2 to 5%) in the algal growth rate as compared to the control growth rate.
Therefore, EbC50 and ErC50 values for inhibition of algal growth could not be calculated.
The nominal NOEC was 100 mg GTSP/L based on the lack of significant inhibition or stimulation of algal growth
(biomass and growth rate) as compared to the control.

Description of key information

No reliable study with single superphosphate is present. Based on a reliable study with triple superphosphate the nominal 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 single superphosphate is present. However, in a OECD 201 guideline study, Selenastrum capricornutum algae were exposed for 72 hrs to 6.25, 12.5, 25.0, 50.0, and 100 mg/L of triple superphosphate under static conditions. Measured concentrations (mean calculated) were 4.83, 10.3, 21.6, 42.6 and 87.6 mg/L (77 -88% of nominal).

Only the calculated measured concentration at the lowest nominal concentration of 6.25 mg/L had a recovery that was slightly lower than 80% (77%). Since the recovery at all other concentrations was >80% and no inhibition of algal growth was observed, effect concentrations are expressed as nominal concentrations.

No adverse inhibitory toxic effects were observed. The substance produced a stimulation of algal growth (biomass) at all concentrations tested that ranged from 6 to 24% compared to the control biomass. Algal growth rate was inhibited or consistent with the growth rate of the control algae for the 6.25 and 25 mg/L concentrations. All other test concentrations demonstrated slight stimulation (2 to 5%) in the algal growth rate as compared to the control growth rate.

Therefore, EbC50 and ErC50 values for inhibition of algal growth could not be calculated. The nominal NOEC was determined to be 100 mg/L based on the lack of significant inhibition or stimulation of algal growth (biomass and growth rate) as compared to the control.

Based on similar physico-chemical properties of substances from the phosphate category, data obtained from toxicity studies with Triple superphosphate (TSP) can be used to read across to Single superphosphate (SSP). The rationale is attached in the target record and can be found in the appendix of the CSR.