Benzene-1,3,5-tricarboxylic acid

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:

2017-03-20 to 2017-05-11

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 solution: 100 mg test item/L, freshly prepared with dilution water and treated with ultrasound at room temperature for 10 minutes. The stock solution and four dilution levels out of the stock solution (nominal) were tested in a geometrical series with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/LThe 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.: 22.0 max.: 24.0 mean value: 22.0

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
100 (pH control) 8.25 9.05
100 4.00 4.01
31.6 7.12 7.79
10.0 7.24 9.22
3.16 7.62 9.30
1.00 7.96 9.42
Control 8.03 9.19

Nominal and measured concentrations:

Nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L.
Measured (0 h): 1.08 - 3.42 - 10.9 - 33.5 - 100.5 mg/L
Measured (72 h): 1.08 - 3.45 - 11.0 - 34.3 - 106.0 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 5 x E3 - E4 cells/mL, Actual: mean 6839 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: Three replicates of the highest test concentration, adjusted with 1M NaOH and HCl to pH 8.1 ± 0.2, were tested under the same test conditions as the test vessels.
- 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.
- Microscopic evaluation: at the start and the end of exposure; The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS

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

Nominal test item concentration
[mg/L] Growth Rate Inhibition [%] Yield Inhibition[%]
100 100 100
10 2 11
1 -1 -6
100 (pH-control) 10 39

Negative values = growth stimulation

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

69.7 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.:

3.16 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 3.16
LOEC 10.0
ErC10 38.6 (32.6 – 96.9)
ErC20 49.4 (35.0 – 97.2)
ErC50 69.7 (39.6 – 98.3)


Yield Inhibition
Nominal test item concentration [mg/L]
NOEC 3.16
LOEC 10.0
EyC10 7.92 (< 1.00 – 19.4)
EyC20 21.0 (10.3 – 30.2)
EyC50 52.6 (39.0 – 78.5)

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 69.7 (39.6 – 98.3) mg/L and 52.6 (39.0 – 78.5) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 3.16 mg/L.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga 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 6839 cells/mL. A stock solution of 100 mg/L was prepared with dilution water and treated 10 minutes with ultrasound. Five concentrations were tested in a geometrical series with a dilution factor of 3.16 (nominal):1.00 - 3.16 - 10.0 - 31.6 - 100 mg/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 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 69.7 (39.6 – 98.3) mg/L and 52.6 (39.0 – 78.5) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 3.16 mg/L.

Description of key information

The toxicity of the test itemto the unicellular freshwater green algaPseudokirchneriella subcapitatawas determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3.

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 69.7 (39.6 – 98.3) mg/L and 52.6 (39.0 – 78.5) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 3.16 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

69.7 mg/L

EC10 or NOEC for freshwater algae:

3.16 mg/L