Tribenzylamine

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:

2020-12-19 to 2021-02-25

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)

Version / remarks:

adopted on March 23, 2006; Annex 5 corrected: July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

7 December 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Two replicates of each sample were directly taken after preparation (0 hour) and analysed. The samples were analyzed undiluted due to due to the results of the method implementation. The concentrations of the test item were determined using the calibration curve.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A preparation with a test item concentration of 100 mg/L was freshly prepared. For that purpose, the test item was weighed in a calibrated flask and reconstituted water was added. The preparation was stirred with a magnetic stirrer for 24 hours. Then the formulation was passed through a filter membrane (pore size: 0.2 µm). The filtrate was used for the study. The pH was not adjusted.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green algae
- Strain: 61.81 SAG
- Source: Pflanzenphysiologisches Institut der Universität Göttingen, Germany
- Method of cultivation: The permanent algae cultures, the pre-culture and the algae cultures of the study were cultivated in an air-conditioned room with a water temperature of 21.0 – 24.0 °C, controlled at ± 2 °C. For cultivation, 300 mL Erlenmeyer flasks filled with 100 mL algae suspension were covered with air permeable stoppers. The cultures were shaken continuously at about 120 rpm and lit between 4440 and 8880 Lux. Fluorescent tubes were installed above the flasks served for lighting.

ACCLIMATION
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

21.4 to 22.5 °C

pH:

7.99 to 9.25

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal conc.: 100 mg/L
Measured conc.: < LOD and thus could not be determined

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks 250 mL
- Type: open
- Material, size, headspace, fill volume: glass, 250 mL, 100 mL medium
- Initial cells density: 10 000 cells per mL
- No. of vessels per concentration: 6
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous lighting
- Light intensity and quality: 6422 Lux ± 2.9 % and 6481 Lux ± 2.7 % prior to and at the end of the exposure period, respectively

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The algal biomass in each flask was determined daily during the exposure period with an electronic particle counter.

TEST CONCENTRATIONS
- Test concentrations: 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > 0.545 mg/L (LOD = Limit of detection (0.545 mg/L))

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: > 0.545 mg/L (LOD = Limit of detection (0.545 mg/L))

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > 0.545 mg/L (LOD = Limit of detection (0.545 mg/L))

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: > 0.545 mg/L (LOD = Limit of detection (0.545 mg/L))

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: ≥ 0.545 mg/L (LOD = Limit of detection (0.545 mg/L))

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: ≥ 0.545 mg/L (LOD = Limit of detection (0.545 mg/L))

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > 0.545 mg/L (LOD = Limit of detection (0.545 mg/L))

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: > 0.545 mg/L (LOD = Limit of detection (0.545 mg/L))

Details on results:

The test media were clear preparations and stayed unchanged throughout the study.

No morphological changes of the treated algae in comparison to the control algae were observed.

Results with reference substance (positive control):

- Results with reference substance valid? YES
- EC50: The 72 h ErC50: 0.86 mg/L, (95 % confidence limits: 0.81 – 0.91 mg/L)
The 72 h EyC50: 0.39 mg/L, (95 % confidence limits: 0.38 – 0.41 mg/L)

Validity of the Test

In the control group, the biomass increased by a factor of 178.6 over 72 hours. Therefore, the validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control group (section-by-section growth rates) during 72 hours was 31.94 %. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35 %. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control group after 72 hours was 1.12 %. According to the OECD test guideline, the coefficient of variation must not be higher than 7 %. Thus, the validity criterion was fulfilled.

 

EC-, LOEC- and NOEC-Values

For the test item the following EC, LOEC and NOEC values were determined:

Parameter (0 – 72 h)	Growth rate	Yield
EC10 95 % confidence interval	> 0.545 mg/L (nominal > 100 mg/L) n.d.	> 0.545 mg/L (nominal > 100 mg/L) n.d.
EC20 95 % confidence interval	> 0.545 mg/L (nominal > 100 mg/L) n.d.	> 0.545 mg/L (nominal > 100 mg/L) n.d.
EC50 95 % confidence interval	> 0.545 mg/L (nominal > 100 mg/L) n.d.	> 0.545 mg/L (nominal > 100 mg/L) n.d.
LOEC	> 0.545 mg/L (nominal > 100 mg/L)	> 0.545 mg/L (nominal > 100 mg/L)
NOEC	> 0.545 mg/L (nominal > 100 mg/L)	> 0.545 mg/L (nominal > 100 mg/L)

 

Concentrations of the Test Item in Control and Test Medium

The results of the analyses of all samples for analyze time point 0 hours are summarized in the following table. Each value represents the mean concentration of three measurements. Nominal and corresponding analytical concentrations of samples are given in the following table:

Nominal test item concentration [mg/L]	Analytical results
	0 h
	Meas. Conc. [mg/L]	[%]
0	< LOD	-
100	< LOD	-

LOD = Limit of detection (0.545 mg/L)

The test item concentration directly after preparation was < LOD (LOD: 0.545 mg/L) and, thus, could not be determined. Therefore, no further analysis of the test item concentrations was performed during the study.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae. The 72 h EC50 for growth rate and yield were > 0.545 mg/L (nominal > 100 mg/L) and, thus, could not be determined in this study.

Executive summary:

The effect of the source test item on algal growth was assessed using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Raphidocelis subcapitata and Selenastrum capricornutum) in a study according to OECD TG 201 under GLP conditions. For this purpose, exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to one concentration of the test item (limit test) for a period of 72 hours. Growth of the algae was determined after 24, 48 and 72 hours exposure and compared with the growth of the control group. From the results, EC values for growth rate and yield were calculated. The study comprised of one control group and one test item group with six replicates each. A nominal test item concentration of 100 mg/L was prepared in reconstituted water. The initial algae cell density in each replicate was 10 000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group. Samples of the test and control media were taken directly after preparation and the test item concentration was analyzed via HPLC with UV detection. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The test item concentration directly after preparation was < LOD (LOD: 0.545 mg/L) and, thus, could not be determined. Therefore, no further analysis of the test item concentrations was performed during the study. No significant effect on growth rate and yield of the algae was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility.

Description of key information

The 72 h EC₅₀ for growth rate and yield were > 0.545 mg/L (nominal > 100 mg/L) and, thus, could not be determined in this study (reference 6.1.5-1).

Additional information

The effect of the source test item on algal growth was assessed using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Raphidocelis subcapitata and Selenastrum capricornutum) in a study according to OECD TG 201 under GLP conditions. For this purpose, exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to one concentration of the test item (limit test) for a period of 72 hours. Growth of the algae was determined after 24, 48 and 72 hours exposure and compared with the growth of the control group. From the results, EC values for growth rate and yield were calculated. The study comprised of one control group and one test item group with six replicates each. A nominal test item concentration of 100 mg/L was prepared in reconstituted water. The initial algae cell density in each replicate was 10 000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group. Samples of the test and control media were taken directly after preparation and the test item concentration was analyzed via HPLC with UV detection. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The test item concentration directly after preparation was < LOD (LOD: 0.545 mg/L) and, thus, could not be determined. Therefore, no further analysis of the test item concentrations was performed during the study. No significant effect on growth rate and yield of the algae was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility.