2,4,6-trimethylbenzene-1,3-diamine

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

Study period:

2016-10-21 to 2017-04-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Vehicle:

no

Details on test solutions:

Stock solutio: 400 mg test item/L, freshly prepared with dilution water. The stock solution was agitated until the test item is completely dissolved. Five dilution levels out of the stock solution were tested in a geometrical series with a dilution factor of 2: 25.0 - 50.0 - 100 - 200 - 400 mg/L. The concentrations are based on the result of a preliminary range finding test

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.

Origin Sammlung von Algenkulturen (SAG)
Pflanzenphysiologisches Institut der Universität Göttingen
Nikolausberger Weg 18, D-37073 Göttingen

Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE·m-2·s-1 for 24 hours per day.

A four days old preculture, prepared in dilution water, was used as inoculum.
Initial cell density Nominal: approximately 5 x 103 - 104 cells/mL
Current: 6305 cells/mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2 °C
Room temperature [°C]: min.: 21.0 max.: 22.0 mean value: 21.5

pH:

The pH-value at the start of exposure was measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-value was measured from pooled samples of each test item concentration and the control.

Geometric mean measured
test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
400 7.80 8.10
200 7.85 8.26
100 7.81 8.58
50 7.84 8.92
25 7.82 9.31
Control 7.92 9.39

Nominal and measured concentrations:

Nominal: 25, 50, 100, 200 and 400 mg/L
Measured (72 h): 27.9, 54.1, 107.0, 208.0 and 418.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x E3 - E4 cells/mL, Actual: mean 6305 cells/mL
- Control end cells density: Mean 2231485 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:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH 8.1  0.2

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the definitive test at the saturated solution.

TEST CONCENTRATIONS

Results of the Range Finding Test (0-72 hours)
Nominal Test Item Concentration

[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 6 26
10 -1 -3
1 1 5
0.1 2 11
Negative values = growth stimulation

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

333 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

NOEC, LOEC, ECx-values and 95 % Confidence Intervals of the test item (0 - 72 hours) based on nominal test item concentrations [mg/L]

Growth Rate Inhibition
Nominal test item concentration [mg/L]
NOEC 50.0
LOEC 100
ErC10 119 (77.1 – 156)
ErC20 191 (162 – 241)
ErC50 333 (280 – 369)

Yield Inhibition
Nominal test item concentration [mg/L]
NOEC 50.0
LOEC 100
EyC10 30.9 (25.4 – 36.3)
EyC20 41.8 (35.4 – 48.1)
EyC50 103 (92.2 – 116)

Results with reference substance (positive control):

based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.435 0.763 ± 0.514
95% confidence interval 0.414 – 0.458

Yield inhibition
EyC50 0.230 0.402 ± 0.293
95% confidence interval 0.179 – 0.322

SD = Standard deviation

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 333 (280 – 369) mg/L and 103 (92.2 – 116) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 50.0 mg/L. All effect level are based on the nominal test item concentrations.

Executive summary:

The toxicity of the test item to the unicellular freshwater green algae Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3.

The study was conducted under static conditions with an initial cell density of 6305 cells/mL. A stocksolution of 400 mg/L was prepared by direct addition of the test item to the dilution water and shaken until the test item was completely dissolved. Five concentrations were tested in a geometrical series with a dilution factor of 2 (nominal): 25.0 - 50.0 - 100 - 200 - 400mg/L

Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.The test media were clear throughout the test period.

 

The concentrations of thetest item were analytically verified by HPLC-DAD at the start and the end of the exposure of all test concentrations and the control. The test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC₅₀-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC₅₀) after 72 hours were 333 (280 – 369) mg/L and 103 (92.2 – 116) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 50.0 mg/L.All effect values are based on the geometric mean measured test item concentrations of the test item.

Description of key information

The toxicity of the test itemto the unicellular freshwater green algae Pseudokirchneriella subcapitatawas determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 761/2009/Method C.3.Five concentrations were tested in a geometrical series with a dilution factor of 2 (nominal):25.0 - 50.0 - 100 - 200 - 400mg/L

Three replicates were tested for each test item concentration and six replicates for the control.The test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC₅₀-values with 95% confidence intervals for inhibition of growth rate (ErC₅₀) and yield (EyC₅₀) after 72 hours were 333 (280 – 369) mg/L and 103 (92.2 – 116) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 50.0 mg/L. All effect values are based on the geometric mean measured test item concentrations of the test item.

Key value for chemical safety assessment

EC50 for freshwater algae:

333 mg/L

EC10 or NOEC for freshwater algae:

50 mg/L