Diphosphoric acid, zinc salt, compd. with 1,3,5-triazine-2,4,6-triamine (1:1:2)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD guidelines and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

; with regards to temperature and stated as non-critical

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

; with regards to temperatue and stated as non-critical

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 1,3,5-triazine-2,4,6-triamine and zinc bis(dihydrogen phosphate)

EC Number:

690-512-6

Cas Number:

1271172-98-5

Molecular formula:

Zn(C3H7N6)2(P2O7)

IUPAC Name:

Reaction products of 1,3,5-triazine-2,4,6-triamine and zinc bis(dihydrogen phosphate)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 1, 3.2, 10, 32 and 100 mg/L.
- Sampling method: To all samples 1% KCl solution (10% in water) and 1% HCl conc. were added. On each day of measurements, calibrations were performed in the range 0.01 - 2 mg/L Zn.
- Frequency: The content of Zn in the test vessels was measured at the start, every 24 hours and at the end of test (72 hours).
- Sample storage conditions before analysis: Not detailed, but assumed to be analysed on the day of sampling.

Test solutions

Vehicle:

no

Details on test solutions:

Before the experiment, saturated solutions of test substance were prepared. This was performed by shaking a nominal loading rate with the appropriate amount of deionised water for 24 hours, followed by membrane filration. The remaining concentrations to be tested were prepared by dilution of this solution with deionised water. Since the solutions were diluted by algal pre-culture and nutrient medium, 1.25 times the nominal amount was used in preparation of the saturated solution. The technique essentially provided a water soluble fraction of nominal loading rates, further characterised by chemical analysis.

For each treatment, 240 mL of the respective test solution was prepared and mixed with 60 mL of the test culture. In this mixture, the pH-value was measured and samples for analytical determination were taken.

The test vessels were filled with ca. 40 mL of the mixture and then incubated open for 72 hours, shaken on an orbital shaker.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: Chodat
- Family: Chlorophyceae
- Order: Chlorococcales
- Source (laboratory, culture collection): The culture used on study was obtained in February 2011 by the MBM Science-bridge GmbH and were kept as a stock culture on solid agar at 8°C.
- Method of cultivation: From an aliquot of this stock on agar, the pre-culture was prepared.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

The temperature was taken as 27°C (consistent throughout the test).

pH:

The pH values in the control ranged from 7.7 to 8.6

Nominal and measured concentrations:

Nominal: Control (0), 1, 3.2, 10, 32, and 100 mg/L.
Measured (calculated from experimentally determined Zn value based on the known composition; geometric mean of 0, 24, 48 and 72 hour results): 0.26, 0.69, 2.13, 12.33 and 31.22 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks
- Material, size, headspace, fill volume: 50 mL glask flasks filled to ca. 40 mL test media.
- Aeration: No
- Initial cells density: 15148 cells/mL
- Control end cells density: 751088 cells/mL
- No. of vessels per concentration (replicates): 4 replicates for each treatment group
- No. of vessels per control (replicates): 7 replicates for the control group

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium prepared and used as per OECD 201 guidance

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous
- Light intensity and quality: 4600 - 4700 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer (photometric measurement of optical density (absorption).

Microscopic observation was also conducted at the end of the test to assess the appearance of the algal cells. The EC50 in relation to growth rate, AUC and yield was calculated along with the respective NOEC and LOEC values.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.25 fold

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

;calculated based on measure Zn content (Zn content in test material known)

Basis for effect:

growth rate

Remarks on result:

other: 10-17 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

;calculated based on measure Zn content (Zn content in test material known)

Basis for effect:

growth rate

Remarks on result:

other: not determinable

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

;calculated based on measure Zn content (Zn content in test material known)

Basis for effect:

growth rate

Remarks on result:

other: not determinable

Details on results:

- Exponential growth in the control (for algal test): yes (validity criteria met)
- Observation of abnormalities (for algal test): Conducted microscopically
- Other: Normal growth observed at 0 to 3.2 mg/L, fewer cells observed at 10 mg/L with no cells observed at 32 to 100 mg/L.
- Any stimulation of growth found in any treatment: Yes at 1 and 3.2 mg/L (non-significant).

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 1.0 mg/L (95% confidence limits 0.81-1.4 mg/L), all validity criteria met and within historical range.

Reported statistics and error estimates:

For the treatments with the nominal concentrations 3.2 and 10 mg/L, it was tested whether the differences between treatment and control were significant. In order to select a suitable test for significance, it was checked whether equality of variance was given. The calculated value is compared with an F-Test table. If teh calculated value is smaller than teh tabular value, equality of variance is given. If equality of variance is given, the t-test is used; else, the WEIR test is used.

With the t-Test, it was checked whether the differences are significant. Significance is given if the calculated t-value is bugger than the limit of significance.

Any other information on results incl. tables

Biological results on the test substance:

Parameter	Geometric mean of measured conc. (mg/L)	95% confidence-interval
NOEC (growth rate) 72 hr	0.69	Not determinable
NOEC (AUC) 72 hr	0.69	Not determinable
NOEC (Yield) 72 hr	0.69	Not determinable
LOEC (growth rate) 72 hr	2.1	Not determinable
LOEC (AUC) 72 hr	2.1	Not determinable
LOEC (Yield) 72 hr	2.1	Not determinable
72 hr ErC50	13	10 – 17 mg/L
72 hr EbC50	4.4	2.4 – 6.5 mg/L
72 hr EyC50	2.4	Not determinable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EC50 value for growth rate inhibition of the test substance (ErC50) was determined to be 13 mg/L with the associated NOEC determined to be 0.69 mg/L.