1,3,5-triazine-2,4,6(1H,3H,5H)-trithione

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

Study period:

2006-12-25 to 2006-12-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "Method concerning tests relating to new chemical substances"

Version / remarks:

No.1121002 by the Pharmaceutical and Food Safety Bureau, 2003; No.2 by the Manufacturing Industries Bureau, 2003; No. 031121002 Environmental Health Department, Policy Division

Principles of method if other than guideline:

method similar to OECD201 performed under GLP; with analytical verification of test substance

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,3,5-triazine-2,4,6(1H,3H,5H)-trithione

EC Number:

211-322-8

EC Name:

1,3,5-triazine-2,4,6(1H,3H,5H)-trithione

Cas Number:

638-16-4

Molecular formula:

C3H3N3S3

IUPAC Name:

1,3,5-triazine-2,4,6(1H,3H,5H)-trithione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Solubility in water: 1420 mg/L (25 °C) (calculated value), less than or equal to 200, less than 500 mg/L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations were sampled at test start and test end.
- Sampling method: Samples were taken using micropipettes.
- Sample storage conditions before analysis: Samples were kept at room temperature and analysed the same day.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material (100 mg) was weighed in a 30 mL beaker, approx. 5 mL of dilution water was added and the total amount was transferred to a 1 L graduated flask using a Pasteur pipette. 5 mL dilution water was again placed in the 30 mL beaker and then transferred to the flask. This procedure was repeated 4 times before the beaker was washed with a small amount of water using an ultrasonic bath to dissolve any test material clinging to the wall of the beaker. The graduated flask was filled to volume and placed in an ultrasonic bath for 10 min (24 °C). The concentration levels were prepared by dilution with dilution water except for the highest test concentration. The test solutions were filter sterilized (pore size 0.22 µm) and filled in Erlenmeyer flasks.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection , received on 07 Sep 2002
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Aseptical subcultures using slant medium of Gorham's medium

ACCLIMATION
- Acclimation period: For the test, algae were transplanted from the slant medium to ATCC liquid medium and stationary culture was performed for 7 days.
- Culturing media and conditions (same as test or not): other than test
- Pre-culture: Culture period with exponential growth for 3 days after acclimation. Same environmental conditions as test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.7 °C - 22.9 °C

pH:

6.3 - 7.7 at test start and 6.0 - 8.6 at test end

Nominal and measured concentrations:

Nominal concentrations: control mg/L, 3.0 mg/L, 7.3 mg/L, 17.4 mg/L, 41.7 mg/L, 100.0 mg/L
Measured concentrations / geometric mean: control, 2.0 mg/L, 5.7 mg/L, 15.2 mg/L, 38.1 mg/L, 94.9 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): vessels closed with gas permeable silicon
- Material, size, fill volume: Glass, 300 mL, 100 mL
- Aeration: None; shaking at 100 rpm
- Initial cells density: 1E04 cells/mL
- Control end cells density: approx. 130E04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD guideline medium was used.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD guideline test medium
- Culture medium different from test medium: yes
- Intervals of water quality measurement: The pH of the test media was measured at test start and test end.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous lighting
- Light intensity and quality: 60 - 90 mmol/m*s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were determined using a coulter counter after 24, 48 and 72 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.4
- Range finding study
- Test concentrations: 4.0, 20.0 and 100 mg/L (nominal), 3.0, 17.4 and 98.9 mg/L (measured)
- Results used to determine the conditions for the definitive study: ErC50 (72h): > 98.9 mg/L; EbC50 (72h): 21.9 mg/L; NOEC: 3.0 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 94.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 130.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

1,3,5-triazine-2,4,6(1H,3H,5H)-trithione, trisodium salt (TMT)

Basis for effect:

growth rate

Remarks on result:

other: recalculated value

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

1,3,5-triazine-2,4,6(1H,3H,5H)-trithione, trisodium salt (TMT)

Basis for effect:

growth rate

Remarks on result:

other: recalculated value

Details on results:

- Exponential growth in the control (for algal test): yes, replication by a factor of 131.5
- Effect concentration based on biomass: EbC50 (72h) 43.2 mg/L (95% CL 37.2 - 51.1 mg/L), NOEbC (72 h) 5.7 mg/L.
- Inhibition of growth rate: The maximum inhibition was 24.3% at the highest test concentration
- Analytics: The measured concentrations were in the range from 90 to 102% of nominal values at test start and from 51 to 90% of nominal values at test end. The effect levels have been calculated based on the geometric mean of the measured values.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50 (72h): 1.01 mg/L (95% CL: 0.94 - 1.10 mg/L); EbC50 (72h): 0.54 mg/L (95% CL: 0.50 - 0.58 mg/L)
- Date of experiment: 23 Oct - 26 Oct 2006

Reported statistics and error estimates:

The NOEC values were calculated using EcoTox-Statistics (version 2.6, Oita University). Test of variance bonds was performed using the Bartlett method, a one-way analysis of variance was performed in the case of variance, a multiple comparison test was performed when there was significance among the test groups. A Kruskal-Wallis rank sum test was performed when there was no variance and a multiple comparison was performed by the non-parametric Dunnett method if there was no significance among the test groups.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to the test substance at measured concentrations (geom. mean) of 2.0 mg/L, 5.7 mg/L, 15.2 mg/L, 38.1 mg/L, 94.9 mg/L under static conditions. The NOEC and the EC50 values based on growth rate were 5.7 mg/L and > 94.9 mg/L, respectively.