Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in accordance with an internationally recognised method

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: a preliminary study had identified the 72 hour EC50 as being approximately 39mg/l by Biomass integral and therefore the definitive test concentrations were prepared as Control (0) 10. 18. 32, 56 and 100mg
- Sampling method: the samples are transferred to 8m1 polypropylene vials and analysed by Ion Chromatography.
- Sample storage conditions before analysis: test substance specific analysis of each test concentration was carried out as soon as possible after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: a 100 mg/1 solution of the sample was prepared in culture medium. A clear colourless solution was obtained. The test concentrations prepared by addition of the appropriate volume of this solution to the algal culture medium. The definitive test concentrations
were prepared as Control (0) I 0, 18, 32, 56 and 100 mg/1.

Note: after 72 hours incubation the test solutions were observed to be faintly green and cloudy at 100 mg/1 due to the low cell density. At lower concentrations the exposure solutions had a strong green colouration and turbidity due to the high algal cell density. There was no sedimented
material apparent.

- Controls: as for test solution but without addition of test substance
-
Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable, none used

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 mg/L stock.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): there was no sedimented material apparent.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source: culture collection
- Method of cultivation: from a pre-culture growing in exponential phase. Inoculum level adjusted to give an initial cell density of 1 x 10E4
cells/ml. Culture conditions same as test conditions (ca. 6000 to 10000 lux continuous white light, temperature 23 ± 2°C, shaking at 200 rpm).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

23 ± 2 °C

pH:

0 mg/l 7.8 (start) 8.4 (end)
10 mg/l 7.4 (start) 8.4 (end)
18 mg/l 7.1 (start) 7.5 (end)
32 mg/l 7.0 (start) 6.9 (end)
56 mg/l 6.4 (start) 6.5 (end)
100 mg/l 5.9 (start) 5.9 (end)

Dissolved oxygen:

not reported

Salinity:

not applicable - freshwater study

Nominal and measured concentrations:

Nominal: 0, 10, 18, 32, 56 and 100mg/I.
Measured: -, 14%, 42%, 53%, 83% and 94%

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml conical flask
- Fill volume: 100 ml
- Aeration: no
- Initial cells density: 1 x 10E4 cells/ml
- Control end cells density: 202 x 10E4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: de-ionised, filtered and sterilised by autoclaving at 120°C for 30 minutes.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: adjusted to 8.0 ± 0.2 to obtain the culture medium.
- Photoperiod: continuous
- Light intensity and quality: 6000 - 10000 lux white light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: the cell density was determined at 24, 48 and 72 hours on a small volume removed from each replicate flask. The cell counts were made using a haemocytometer and microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: yes
- Test concentrations: Control (0), 10, 18, 32, 56 and 100 mg/1.

- Results used to determine the conditions for the definitive study: A preliminary study identified the 72 hour EC50 as being approximately 39mg/l by Biomass integral.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

The water quality measurements of the test solutions (pH and temperature) were within acceptable limits.

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50:
- Other:

Reported statistics and error estimates:

NOEC estimated as <10 mg/1 as determined by the Bonferroni T test, (P = 0.05)

Percent inhibition by biomass integral and growth rate

Nominal concentration (mg/l)	Percent inhibition by biomass integral		Percent inhibition by growth rate
	0-48hours	0-72hours	0-48hours	0-72hours
10	8	21	6	6
18	15	18	7	4
32	IS	27	4	8
56	26	66	14	32
100	56	83	30	50

The 72 hour EC50 of MPP to Pseudokirclmeriella subcapitata was estimated as 44mg/l using the biomass integral and 98mg/l by growth rate calculation, nominal concentrations. The 48 hour EC50 was 93mg/l by biomass integral and > 1OOmg/1 by growth rate, nominal concentrations. The 72 hour NOEC was estimated as < 10 mg/1 as determined by the Bonferroni T test.

The growth curves showed that at test concentrations 10 to 32 mg/l, the growth of the algae was good with a low level of inhibition at 48 hours, increasing a little at 72 hours. At higher concentrations of 56 to l00 mg/1 a clearer dose response was recorded with very little increase in cell density between 48 and 72 hours.

Validity criteria fulfilled:

yes

Remarks:

pH and temperature of test medium within acceptable limits. Cell density of controls increases by a factor of at least 16 within 72hrs.

Conclusions:

The 72 hour EC50 by growth rate was determined to be 98 mg/l.

Description of key information

The 72 hour EC50 by growth rate was determined to be 98 mg/l.  

Key value for chemical safety assessment

EC50 for freshwater algae:

98 mg/L

Additional information

In a test for algal growth inhibition conducted over 72 hours, the growth curves showed that at test concentrations 10 to 32 mg/l, the growth of the algae was good with a low level of inhibition at 48 hours, increasing a little at 72 hours. At higher concentrations of 56 to l00 mg/1 a clearer dose response was recorded with very little increase in cell density between 48 and 72 hours.