α,α,α-trifluoro-p-toluidine

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:

from 07 aug 2009 to 16 nov 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

α,α,α-trifluoro-p-toluidine

EC Number:

207-236-5

EC Name:

α,α,α-trifluoro-p-toluidine

Cas Number:

455-14-1

Molecular formula:

C7H6F3N

IUPAC Name:

4-(trifluoromethyl)aniline

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

A 100 mg test item/L stock solution was prepared prior to test initiation by dissolving 0.1014 g test item in 1000 mL algal medium. The stock solution was observed to be clear and colorless. Nominal test concentrations were prepared as follows:
1 2 3 4
100 NA NA 100
100 160 500 32
100 50 500 10
32 50 500 3.2
10 50 500 1.0
3.2 50 500 0.32
1.0 50 500 0.10

With: 1: Stock Solution Concentration (mg test item/L)
2: Volume of Stock Used (mL)
3: Diluted to Volume (mL) with Dilution Water
4: Nominal Concentration (mg test item/L)

All resulting test solutions were shaken by hand for 30 seconds and observed to be clear and colorless, with no visible undissolved test item.
Six dilution water control vessels containing only algal medium were established and maintained under the same conditions as the treatment level solutions.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): The alga was originally obtained from Albrecht-v.-Haller-Institut, Göttingen, Germany.
- Method of cultivation: was maintained in stock culture at Springborn Smithers Laboratories (Europe).

ACCLIMATION
- Acclimation period: Four days prior to test start, the culture was maintained within the following conditions: a shaking rate of 120 rpm, a temperature of 23ºC and continuous illumination of 6148 to 7590 lux. Lighting was supplied by fluorescent bulbs. Culture flasks were shaken continuously on an orbital shaker. Temperature was controlled using an environmental chamber. The inoculum used to initiate the toxicity test was taken from a stock culture that had been transferred to fresh medium four days before testing.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 2ºC

pH:

8.07 to 8.24

Nominal and measured concentrations:

Nominal: 0, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 250 mL Erlenmeyer flasks
- Aeration: no
- Initial cells density: 1*10E4 cells/mL.
- Control end cells density: yes, every 24h
- 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: The algal medium used to prepare the exposure solutions was the same as the culture medium. The medium were prepared using deionized water and were equilibrated to test temperature. The pH of the medium was 8.07.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light intensity
- Light intensity and quality: 4400 to 8800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At each subsequent 24-hour interval, cell counts were conducted on each replicate vessel of the treatment levels and the control using a hemocytometer (Neubauer Improved) and a Leica DMLS microscope. One sample was removed from each flask for counting. One or more hemocytometer field(s), each 0.1 cm x 0.1 cm in size and 0.01 cm deep, containing 0.0001 mL of culture, were examined for each sample until at least 400 cells or a total of four fields were counted. Observations of the health of the algal cells were also made at each 24-hour interval.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0.010, 0.10, 1.0, 10 and 100 mg test item/L and a control.
- Results used to determine the conditions for the definitive study: Following 72 hours of exposure, cells exposed to all treatment levels and the control were observed to be normal.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL: 8.23 - 8.69

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL: 2.14 - 2.41

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL: 3.42 - 3.65

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.61 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.96 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.67 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95%CL: 3.10 - 4.20

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95%CL: 0.88 - 2.41

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95%CL: 1.33 - 2.85

Details on results:

- Exponential growth in the control (for algal test): yes
At experimental completion (72 hours), cells exposed to all the treatment levels tested were observed to be normal. No change in the characteristics of the test solutions was observed throughout the duration of the test, apart from the highest test concentration, where at test termination a slight precipitation was observed on the edge of the test vessels.
Springborn Smithers Laboratories Study # 1141.000.430 Page 19
The 72-hour cell densities in the control and the 0.061, 0.19, 0.61, 1.96, 5.81, 19.0 and 56.1 mg test item/L treatment levels averaged 141, 139, 141, 144, 98.7, 21.2, 0.9 and 1.1 x 104 cells/mL, respectively.

The 0 - 72-hour growth rate in the control and the 0.061, 0.19, 0.61, 1.96, 5.81, 19.0 and 56.1 mg test item/L treatment levels averaged 1.64, 1.63, 1.64, 1.64, 1.52, 1.01, 0.02 and 0.10 day-1, respectively. Statistical analysis (Mann-Whitney Test, p < 0.05) demonstrated a significant reduction in growth rate among algae exposed to the 1.96, 5.81, 19.0 and 56.1 mg test item/L treatment levels when compared to the control. Therefore, the 72-hour NOEC and LOEC values for growth rate were determined to be 0.61 and 1.96 mg test item/L, respectively. The 72-hour EC10, EC20 and EC50 values for growth rate were determined to be 2.28 mg test item/L (95% confidence interval: 2.14 – 2.41 mg test item/L), 3.53 mg test item/L (95% confidence interval: 3.42 – 3.65 mg test item/L) and 8.48 mg test item/L (95% confidence interval: 8.23 – 8.69 mg test item/L), respectively.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- During this study, the 72-hour cell growth in the control was 141 x 104 cells/mL, which fulfills the above criterion of a 16 fold increase within 72 hours. The mean daily growth rate CV through 72 hours and the CV for the 0 to 72 hour average growth rate for the control were 18.1 and 0.84%, respectively, which also fulfill the criteria given for green algae.

Reported statistics and error estimates:

Mann-Whitney Test, p < 0.05

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Toxique to algae.

Executive summary:

In a 72 -hour toxicity study (Springbornsmithers, 2009), the effects of p-TFMA on the growth of algae Pseudohirchneriella subcapitata were evaluated according to OECD 201 guideline.

The cultures of algae were exposed to p-TFMA (purity 98.2%) at concentrations of 0, 0.1, 0.32, 1, 3.2, 10, 32 and 100 mg test item/L, under static conditions.

At experimental completion (72 hours), cells exposed to all the treatment levels tested were observed to be normal. No change in the characteristics of the test solutions was observed throughout the duration of the test, apart from the highest test concentration, where at test termination a slight precipitation was observed on the edge of the test vessels.

The EC50 and EC10 based on growth rate after 72 hours were 8.48 and 2.28 mg/L respectively.

The NOEC based on growth rate and biomass after 72 hours was 0.61 mg/L.

Based on these results, p-TFMA could be considered as toxic for algae.