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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A old study with GLP, but with poor description of the methods. No analytical determination of the concentrations used.

Qualifier:

according to guideline

Guideline:

other: PRO/FT Algae-AC090-6

Deviations:

not specified

Principles of method if other than guideline:

Algal growth inhibition test.

GLP compliance:

yes

Analytical monitoring:

not specified

Details on sampling:

no data

Vehicle:

not specified

Details on test solutions:

Special preparation/precautions: The sample was dissolved in the test medium by boiling, at a concentration of 1% (1g/100 ml). Great concentrations were not completely soluble. The boiled stock solution was then added to test flasks warm, but in small quantities so that the assay flasks showed no significant increase in temperature (ie. 1°C).

Sample preparation (technical note suggested by the QA auditor): Since the sample was heated to a boil in solution, the dilution test set-up was different from that described in the test procedure. Instead of adding stock solution 50:50 to algal solution, a more direct sample addition was done. Only up to 1 ml of warm stock solution was added to 99 ml medium in order to keep test temperature low.

Test organisms (species):

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

Details on test organisms:

Organism: Selenastrum capricornutum, Strain no. 22662
Source: American Type Culture Collection

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

no

Hardness:

Sterile OECD Algal Medium was used.

Test temperature:

Sterile OECD Algal Medium was used.

pH:

Sterile OECD Algal Medium was used.

Dissolved oxygen:

Sterile OECD Algal Medium was used.

Salinity:

Sterile OECD Algal Medium was used.

Nominal and measured concentrations:

Nominal Concentrations: Control, 1000ppm, 320ppm, 100ppm, 32ppm and 10 ppm.
No data about measured concentrations is given in the report.

Details on test conditions:

Project: Algal Growth Inhibition Test - OECD Method, Procedure: Testing was conducted in accordance with procedure PRO/FT Algae-AC 090-6 .
Test medium: sterile OECD Algal Medium (100 ml/flask).
Test vessels: 250 ml glass capped flasks.
Incubation: 23°C, continuous light, approximately 8000 Lux, shaking culture.
Test scheme: 3 replicates per concentration, 5 test material concentrations established by preliminary range-finding. Starting inoculum 1x10exp4 cells/ml.
Special preparation/precautions: The sample was dissolved in the test medium by boiling, at a concentration of 1% (1g/100 ml). Great concentrations were not completely soluble. The boiled stock solution was then added to test flasks warm, but in small quantities so that the assay flasks showed no significant increase in temperature (ie. 1°C).
Sample preparation: Since the sample was heated to a boil in solution, the dilution test set-up was different from that described in the test procedure. Instead of adding stock solution 50:50 to algal solution, a more direct sample addition was done. Only up to 1 ml of warm stock solution was added to 99 ml medium in order to keep test temperature low.

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

325 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: area under the growth curve

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Details on results:

Linear regression analysis, plotting percent growth versus log of concentration, yielded the following information:
Sample CT-338-87 48 hr 72 hr 96 hr
Median Algal Inhibition 325 ppm 196 ppm 325 ppm
EbC50
NOEC (no effect conc.) 97 ppm 31 ppm 98 ppm
Correlation coefficient .95 .94 .97
(r exp2)
Cell growth was insufficient at 24 hrs for establishing concentration-effect relationships for all concentrations and for the blank control.
The calculated correlation coefficients, above, help indicate that the 96 hour value may be the best estimate of the median algal inhibitory concentration. This was due to more developed cell growth with time and thus better enumeration and differentiation amount test concentrations.
The rate of cell growth was satisfactory (greater than 16 x inoculum level at 72 hrs) in controls for acceptable data transformation and test validity in accordance with OECD guidelines.

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

The correlation coefficient (r exp2) 96 hours is 0.97.

Validity criteria fulfilled:

not specified

Remarks:

except: The rate of cell growth was satisfactory (greater than 16 x inoculum level at 72 hrs) in controls for acceptable data transformation and test validity in accordance with OECD guidelines.

Conclusions:

The toxicity of melamine to algae, as measured by growth inhibition, is low.

Executive summary:

The test material produced the following median algal inhibition values based on the area under the growth curve:

EbC50 48h: 325 ppm, 72 h: 196 ppm, 96 h: 325 ppm.

The best estimate of effect, because of cell growth dynamics, is the 96 hour value.

Following NOEC are presented in the report but with questionable reliability as it is not stated how they were derived:

NOEC 48h: 97 ppm, 72 h: 31 ppm, 96 h: 98 ppm.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A old study without apparent GLP and with poor description of the methods. The methods are only referenced to the Dutch Standard, which is not available. No analytical determination of the concentrations used.

Qualifier:

according to guideline

Guideline:

other: Dutch draft Standard Method NEN 6506, 1979

Deviations:

not specified

Principles of method if other than guideline:

Algal growth inhibition test.

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
n.a.

Analytical monitoring:

not specified

Details on sampling:

No data.

Vehicle:

no

Details on test solutions:

A stock solution of the test substance was prepared by dissolving 2.0 g of Melamine in one l of micropore-filtered test medium, the composition of which is given in NEN 6506. The pH of the stock solution was adjusted to that of the test medium. The test solutions for the range-finding and the growth inhibition test were prepared by diluting the stock solution with micropore-filtered medium.

Test organisms (species):

Scenedesmus pannonicus

Details on test organisms:

The fresh-water green alga Scenedesmus pannonicus (CCAP 276/ 4a) was used as the test organism. A preculture of algae in the exponential growth phase was prepared according to the method described in NPR 6505. The tests were carried out with suspensions of algae initially containing about 1 x 10exp4 cells/ml. At the start and end of the growth inhibition test, the morphology of the algae was examined microscopically.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

90.5 h

Remarks on exposure duration:

Derived from the Tables

Post exposure observation period:

None.

Hardness:

No data are found in the report, possibly they are available in the Dutch Standard.

Test temperature:

No data are found in the report, possibly they are available in the Dutch Standard.

pH:

No data are found in the report, possibly they are available in the Dutch Standard.

Dissolved oxygen:

No data are found in the report, possibly they are available in the Dutch Standard.

Salinity:

N.a.

Nominal and measured concentrations:

The concentrations of Melamine tested were 0, 10, 32, 100, 320, 560, 1000 and 2000 mg/l.

Details on test conditions:

The growth inhibition test was conducted as detailed in the Dutch draft Standard Method NEN6506.
A suspension of algae containing about 2 x 10exp4 cells/ml was prepared from the preculture.
The required concentrations of test substance were prepared by dilution of a stock solution containing 2000 mg/l of Melamine with medium, the stock solution and medium both being sterilized by micropore filtration. The tests were carried out in duplicate with algae and completed with a single background control series of test substance without algae, in square 180-mI culture flasks. Fifty ml of the test substance solution was transferred to each flask, and 50 ml of the suspension of algae added. Only the highest concentration tested was prepared by adding the correct quantity of preculture of algae to 100 ml of stock solution of test substance. The concentrations of Melamine tested were 0, 10, 32, 100, 320, 560, 1000 and 2000 mg/l. All flasks were incubated. One sample was taken from each flask once a day on four consecutive days, and the number of algal cells per ml in the samples was determined.

Reference substance (positive control):

no

Duration:

90.5 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

90.5 h

Dose descriptor:

EC50

Effect conc.:

940 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL: 880-1000 mg/l

Details on results:

RANGE-FINDING TEST
The range finding test was carried out with test substance concentrations ranging from 10 to 1000 mg/l. The NOEC was found to be 320 mg/l. Even the highest concentrations tested (1000 mg/l) did not suppress growth completely.

GROWTH INHIBITION TEST
The EC50 with respect to an effect on the inoculum was found to be 940 mg/l, with a confidence interval of 880-1000 mg/l.
By comparison of the growth curves of algal suspensions exposed to the test substance with those of algal blanks, the no observed effect concentration (NOEC) of Melamine was estimated to be 320 mg/l.
Microscopical examination of the cells at the start and end of the incubation period revealed no morphological abnormalities. During the incubation period the pH of the media containing different concentrations of test substance remained constant.

Results with reference substance (positive control):

n.a.

Reported statistics and error estimates:

The confidence interval of the EC50 was 880-1000 mg/l.

Mean cell counts [particles/ml] 

time(h)	Concentration Melamine [mg/l]
	0.00	10.00	32.00	100.00	320.00	560.00	1000.00	2000.00
0.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
19.50	2.25	2.39	2.40	2.39	2.47	2.21	1.88	1.94
43.00	6.33	7.15	7.17	6.43	5.91	4.76	3.99	3.90
67.00	16.45	17.28	16.89	16.79	15.78	13.76	9.58	5.24
90.50	6.79	38.79	36.50	36.06	32.57	27.49	17.42	9.54

 

Validity criteria fulfilled:

not specified

Conclusions:

The toxicity of melamine to algae, as measured by growth inhibition, is low.

Executive summary:

The toxicity of the product Melamine to the fresh-water green alga Scenedesmus pannonicus was determined in a growth inhibition test.

Melamine was found to impair the growth of the alga at concentrations higher than 320 mg/l.

With respect to the number of active growing cells in the inoculum, the EC50 was found to be 940 mg/l with a 95 % confidence interval of 880 to 1000 mg/l.

The no observed effect concentration (NOEC) was found to be 320 mg/l.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, data originate in part from a secondary source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and 100 mg/L. Samples were taken after 0 and 72 hours.
The concentration oftest substance in the test solution was analyzed at the beginning (0 hr) and at the end (72 hrs) ofthe study. About 15 mL was taken from triplicate flask of each test concentration and pooled. 30 mL sample (10 mL x 3 replicate) was taken from pooled solution and put into 20 mL volume vial. 0.3 mL of nitric acid solution (61%) was added into the samples then the samples were sent to the chemical analysis laboratory at Korea Research Institute of Chemical Technology for analysis of calcium. Samples at 0 hour were not filtered additionally because the undissolved test substance in the test solutions were filtered using 0.45 um membrane filter (advantec, Inc., USA) when the test solutions were prepared. Samples at 72 hours were centrifuged slightly (5000 rpm, 10 min) to remove grown algae and preparation process was same as above.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: After 100 mg of the test substance (purity: 99.9%) was added in 1,000 mL beaker and dilution water (OECD nutrient medium) was added up to the mark of the beaker to produce the nominal concentration of 100 mg/L. For the control group, only the dilution water was used.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Strain No. ATCC 22662; UTEX 1648
- Source: The culture collection of algae, The University of Texas at Austin, USA (UTEX)- Strain No.: UTEX1648

ACCLIMATION
- Culturing media and conditions: An OECD culture medium was diluted to produce the biomass of 1×104 cells/mL and then sub-cultured continuously at intervals of 3 to 4 days. For pre-cultivation, the test algae were inoculated into the OECD culture medium at a biomass of 0.5-1.0×104 cells/mL 2 to 4 days prior to study initiation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.7 ± 0.5 ℃

pH:

Control: 9.06 (0 hour) - 8.36 (72 hours)
0.3 mg/L: 8.83 (0 hours) - 8.39 (72 hours)
1.0 mg/L: 8.84 (0 hours) - 8.37 (72 hours)
3.1 mg/L: 8.87 (0 hours) - 8.35 (72 hours)
9.8 mg/L: 8.89 (0 hours) - 8.30 (72 hours)
31.3 mg/L: 8.79 (0 hours) - 8.32 (72 hours)
100.0 mg/L: 8.57 (0 hours) - 8.44 (72 hours)

Nominal and measured concentrations:

Nominal test substance concentrations: control, 0.3, 1, 3.1, 9.8, 31.3, and 100 mg/L
Measured test concentrations at 0 h: 0.3, 0.4, 0.3, 0.8, 1.7, and 4.4 mg/L
Measured test concentrations at 72 h: n.d., n.d., n.d., 0.3, 1.6, and 4.7 mg/L.

Details on test conditions:

TEST SYSTEM
- Initial cells density: 0.5-1.0×104 cells/mL (2-4 days prior to test initiation)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD culture medium


OTHER TEST CONDITIONS
- Light intensity and quality: 4,440-8,880 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Morphological changes of algal cells were observed including expansion, aggregation, atrophy, and decoloration at 72 hours after exposure. Cell densities were determined by direct counts using a BECKMAN COULTER Multisizer 3 particles analyzer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: control, 0.1, 1, 10, and 100 mg/L.
- Results used to determine the conditions for the definitive study: tthe average specific growth rate was inhibited by 13.5% and by 51.4% for the area under growth curve method at the highest dose of 100 mg/L.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 4.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Algal cells were observed to maintain a similar shape to the control in all test solutions.

Table 1. Growth inhibition rates based on mean value during the test period

(1) Average growth rate

Nominal concentration (mg/L)	Mean measured concentration (mg/L)	Average growth rate	Relative inhibition (%)	Mean relative inhibition (%)
Control	ND	1.7038	-	-
0.3	0.3	1.7084	-0.3	0.0
		1.6861	1.0
		1.7180	-0.8
1	0.4	1.6385	3.8	4.2
		1.598	6.2
		1.6619	2.5
3.1	0.3	1.7464	-2.5	-3.4
		1.8030	-5.8
		1.7356	-1.9
9.8	0.8	1.8378	-7.9	-3.4
		1.7358	-1.9
		1.7090	-0.3
31.3	1.7	1.7674	-3.7	-2.2
		1.7312	-1.6
		1.7263	-1.3
100.0	4.4	1.7609	-3.4	-3.3
		1.7747	-4.2
		1.7453	-2.4

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to the analogue justification attached to section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

325 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: area under the growth curve

Remarks on result:

other: Source, WoE, RA-A, CAS 108-78-1, Drozdowski 1988

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Remarks on result:

other: Source, WoE, RA-A, CAS 108-78-1, Drozdowski 1988

Duration:

90.5 h

Dose descriptor:

EC50

Effect conc.:

940 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

CL: 880-1000 mg/l

Remarks on result:

other: Source, WoE, RA-A, CAS 108-78-1, Oldersma & Hanstveit 1982.

Duration:

90.5 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Source, WoE, RA-A, CAS 108-78-1, Oldersma & Hanstveit 1982.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Source, WoE, RA-A, CAS 7757-93-9, Kim, 2013

Description of key information

 No toxic effects on algae were recorded within the limit of water solubility of the substance.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

98 mg/L

Additional information

Data on the toxicity of the target substance diphosphoric acid, compound with 1,3,5-triamine (1:2) to aquatic algae are not available. The evaluation of the acute toxicity is based on a weight of evidence approach studies conducted with available data on the source substances melamine (CAS 108-78-7) and calcium hydrogenorthophosphate (CAS 7757-93-9). Whereby melamine represents the 1,3,5-triamine moiety of the target substance. The source calcium hydrogenorthophosphate was selected to assess the toxicity of the diphosphoric acid moiety. The read across approach is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. Further justification is given in the analogue justification in IUCLID section 13.

Two studies on the toxicity of melamine to algae are available. Both studies investigated a possible growth inhibition of green algae induced by melamine. Drozdowski et al. (1988) exposed Pseudokirchneriella subcapitata to test substance concentrations of 10, 32, 320, and 1000 mg/L for 96 hours. The determined EC50 (96 h) was 325 mg/L (NOEC (96 h): 98 mg/L). In the second available study Scenedesmus pannonicus was exposed to nominal substance concentrations of 0, 10, 32, 100, 320, 560, 1000 and 2000 mg/L for 90.5 hours (Oldersma & Hanstveit, 1982). The determined EC50 (90.5 h) was 940 mg/L (meas. initial). The NOEC (90.5 h) was 320 mg/L (nominal).

The acute toxicity of calcium hydrogenorthophosphate to aquatic algae was investigated in a study following OECD guideline 201 (Kim et al. 2013). Pseudokirchneriella subcapitata was exposed to nominal test substance concentrations of 0.3, 1, 3.1, 9.8, 31.3, and100 mg/L. The mean measured test concentrations at test start were 0.3, 0.4, 0.3, 0.8, 1.7, and 4.4 mg/L. Based on the algal growth rate an EC50 (72 h) >4.4 mg/L (measured) was determined (nominal: EC50 (72 h) >100 mg/L). Based on the results of the read across study detrimental effects of phosphate to aquatic algae are not expected.