Registration Dossier

Administrative data

bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February - June 2009
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study. Not according to GLP, but a quality management system was applied. Trianzine and not triazine, as part of the test substance name, was used throughout the report.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
(No post-exposure period, too few analytical determinations)
according to guideline
other: The Guidelines for Testing of Chemicals (HJ/T153-2004)
according to guideline
other: The Guidelines for Testing of Chemicals NO. 305B (China Environmental Science Press, 2004)
GLP compliance:
A quality management system was in operation.

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material: 2,2'-iminobis(4,6-diamino-1,3,5-trianzine). [Trianzine and not triazine was used, at least in the English translation.]
- Code: S-007-2009.
- Physical state: Light yellow powder
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Details on sampling:
- Sampling intervals/frequency for test organisms: exposure for 28 days, samples from both test solution and test organisms were taken daily from the 26th to the last day of test period.
- Sampling intervals/frequency for test medium samples: pH, dissolved oxygen concentration and temperature were recorded every second day.
- Sample storage conditions before analysis: No.
- Details on sampling and analysis of test organisms and test media samples: Seven fish were removed randomly from the exposure test solutions at each sampling time. The sampled fish were rinsed quickly with water, killed with a pin and weighed. The sampled fish were then treated by extracting their lipid component by Soxhlet Extraction Method. At the end of the test, fish in the control chamber were all collected and treated as described. The actual concentration of the test substance in the test medium and the extracted solutions was analytically determined.

Test solutions

Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to low water solubility of the test substance, a supersaturated dispersion of the test substance was continuously stirred over 4 hours prior to each test medium preparation. Then the dispersion was filtered with a 0.45 µm membrane filter. This filtrate was used as stock solution to prepare the lower test substance group.
- Controls: Dilution water only (dechlorinated drinking water, total hardness between 10 and 250 mg CaCO3/L, pH 6.0 - 8.5 is preferable).
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Common name: Zebra fish
- Strain: Brachydanio rerio
- Source: Amazonas Commercial Aquarium, Shanghai
- Age at study initiation (mean and range, SD): No data.
- Length at study initiation: 2.814 +/- 0.16 cm (SD)
- Weight at study initiation: 0.36 +/- 0.07 g (SD)
- Weight at termination: See attachment.
- Health status: During holding and acclimatization no fish died and all fish were healthy.
- Description of housing/holding area: No data.
- Feeding during test: yes.
- Food type: commercial fish diet
- Amount: No data.
- Frequency: No data.

- Acclimation period: 1 week
- Acclimation conditions: Same as test.
- Health during acclimation (any mortality observed): No mortality observed, all fish were healthy.

Study design

Route of exposure:
Test type:
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
28 d

Test conditions

10 to 250 mg CaCO3/L are preferable, no further data.
Test temperature:
23 +/- 1 °C
in treatment and control groups: 6.22 - 7.86
Dissolved oxygen:
>= 69.1 %
No data.
Not applicable.
Details on test conditions:
- Test vessel: No data.
- Aeration: No.
- Renewal rate of test solution: semi-static, renewal of test media every two days.
- No. of organisms per vessel: test groups: 30 organisms/vessel, control group: 10 organisms/vessel
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: No data.

- Source/preparation of dilution water: Dechlorinated drinking water
- Intervals of water quality measurement: every second day (pH, dissolved oxygen, temperature)
- Intervals of test medium replacement: every second day.

- Adjustment of pH: No.
- Photoperiod: 12 h light, 12 h dark

RANGE-FINDING / PRELIMINARY STUDY: No range-finding study was conducted.
- Results used to determine the conditions for the definitive study: Based on the LC50 value of the test substance on fish and the detection limit of chemical analysis, an exposure test with 100 and 10 mg/L was performed.
Nominal and measured concentrations:
Nominal: 10 and 100 mg/L
Measured: 0.69 and 6.90 mg/L
Reference substance (positive control):
d-666 standard solution (100 µg/mL)
Details on estimation of bioconcentration:
The Bioconcentration factor was calculated as follows:

BCF = Cf/Cw

Cf: concentration of the test substance in the fish (mg/kg)
Cw: concentration of the test substance in water (mg/kg)

Results and discussion

Bioaccumulation factoropen allclose all
< 0.18 dimensionless
whole body w.w.
Calculation basis:
other: After 28 d of exposure: BCF = conc. of test substance in fish / conc. of test substance in water.
Remarks on result:
other: environment / dose:Nominal 10 mg/L. Measured 0.69 mg/L.
< 0.02 dimensionless
whole body w.w.
Calculation basis:
other: After 28 d of exposure: BCF = conc. of test substance in fish / conc. of test substance in water.
Remarks on result:
other: environment / dose:Nominal 100 mg/L. Measured 6.9 mg/L.
Details on kinetic parameters:
No data.
Results with reference substance (positive control):
The BDF of d-666 standard solution was calculated as 2.1 x 10 000 which fulfilled the criteria for a valid test (BCF of d-666 standard solution should range in 1.5 x 10 000 to 3.0 x 10 000
Details on results:
- Mortality of test organisms: No mortality or other adverse effects were observed.
- Behavioural abnormalities: None.

Any other information on results incl. tables

Validity criteria:

Temperature was maintained at 23 +/- 1 °C (guideline: temperature variation less than +/- 2 °C).

Dissolved oxygen concentration was always higher than 60 % (guideline: at least 60 %).

The actual concentration of the test substance was maintained within +/- 20 % of mean of the measured values.

No mortality occurred in the control group (guideline: mortality < 10 %).

Applicant's summary and conclusion

Validity criteria fulfilled:
BCF at nominal 10 mg/L: <0.18
BCF at nominal 100 mg/L: <0.02
Executive summary:

The bioconcentration effect of the test substance to zebra fish was determined in a 28-day semi-static test with a medium renewal every two days. An exposure test at 10 and 100 mg/L was performed. Due to low water solubility of the test substance a supersaturated dispersion was stirred for 4 hours and afterwards filtrated, this solution was used as stock solution.

Samples from both test solutions and test organisms were taken daily from the 26th day to the last day during exposure period. The samples from test organisms were treated by extracting their lipid component.

The analytically measured test substance concentrations in the exposure test medium were 0.69 mg/L and 7.05 mg/L, 0.67 mg/L and 6.79 mg/L, 0.70 mg/L and 6.88 mg/L respectively from the first day to the third day during the sampling period.

In the controls and the treatment groups no fish death was determined during the 28 days test period. Based on the measured concentrations in the exposure medium and the test organism, the BCF were calculated as <0.18 and <0.02, respectively at the nominal exposure of 10 mg/L and 100 mg/L.

In conclusion, the test substance had no obvious bioconcentration effects on zebra fish under the present conditions of test.