α,α,α-trifluoro-p-toluidine

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

7 January to June 2010

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Due to high mortality in the control, the test was terminated after 20 days, then 1 day before the normal duration of such a test. However until the 20th day all criteria were good enough to let think final results after 21 days would be identical.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

Test terminated after 20 days

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

Details on sampling:

Aliquots from newly prepared solutions from each test concentration and the control were taken at day -1, day 0 (test initiation), day 7 and 14 for the determination of the actual test concentrations. Samples were taken from a point approximatively midway between the surface, bottom and sides of the test vessels.
In addition recovery samples of nominal concentrations of 0.028 and 0.996 mg test item/L were prepared in dilution water and analyzed by TSP HPLC method to judge the precision and quality control of the analysis during the exposure period.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions with concentrations of 8.0 and 4.0 mg test item/mL were prepared prior to test initiation and about weekly thereafter by dissolving approximately 0.4 g of p-TFMA in 50 and 100 mL of acetone, respectively. A dilution series was prepared starting with the 4.0 mg test item/mL stock solution resulting in further stock solutions with concentrations of 2.0, 1.0 and 0.5 mg test item/mL. The stock solutions were observed to be clear and colorless with no visible undissolved test item.

- Controls: a dilution water control and a solvent control were selected for the definitive exposure.
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not known

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: from laboratory cultures maintained at Springborn Smithers Laboratories (Europe), originally obtained from a culture at Springborn Smithers Laboratories Wareham (MA), USA.
- Feeding during test: yes
- Food type and amount: Daphnids were fed 2.0 mL of a solution containing approximately 4 x 107 cells/mL of the unicellular green alga, Ankistrodesmus falcatus (ANK) and approximately four drops of a combination of yeast, cereal leaves and flaked fish food (YCT) daily prior to the definitive exposure.
- Frequency: During the weekends, the cultures are generally fed once. During the definitive test, the daphnids will be fed 3 times per day using 3 mL food suspension per feeding interval and replicate, i.e., 9 mL food suspension per day and replicate.

ACCLIMATION:
- Acclimation period: 8 days
- Acclimation conditions (same as test or not): Prior to testing, the daphnids were held in glass beakers under a photoperiod of 16 hours light and 8 hours darkness with a 30 minute transition period. The culture water consisted of natural filtered water of the Lake of Constance. Total hardness, total alkalinity, pH and specific conductivity of the dilution water were determined to verify that these parameters are within the acceptable ranges. Representative samples of the dilution water source used in the preparation of the culture medium were analyzed periodically for the presence of pesticides,
PCBs and toxic metals at Bachema, Schlieren, and Interlabor Belp AG, Belp, Switzerland.
None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed. Based on the results of these analyses and the ability of daphnids to survive and reproduce over multiple culture generations in the dilution water, it was confirmed that the dilution water used was of acceptable quality.
The daphnids used during the definitive exposure were maintained under the following conditions for eight days prior to testing:
Total Hardness: 140 - 144 mg/L CaCO3
Total Alkalinity: 114 - 116 mg/L CaCO3
Specific Conductivity: 235 - 238 μS/cm
pH: 8.03 – 8.17
Temperature: 19 – 21°C
- Type and amount of food: Daphnids were fed 2.0 mL of a solution containing approximately 4 x 107 cells/mL of the unicellular green alga, Ankistrodesmus falcatus (ANK) and approximately four drops of a combination of yeast, cereal leaves and flaked fish food (YCT) daily prior to the definitive
exposure.
- Feeding frequency: daily


QUARANTINE (wild caught)
- Duration:
- Health/mortality:

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

20 d

Remarks on exposure duration:

Due to high mortality in the control and solvent control on day 20, the test was terminated after 20 days and therefore, no body length measurements were performed.

Test conditions

Hardness:

Total hardness as CaCO3: 140 to 152 mg/L

Test temperature:

Continuously measured temperature: 19 to 21°C, apart from an outlier on day 4 where a temperature of 22°C was measured.
Single-point measured temperature: 20 to 21ºC.

pH:

pH: 7.79 to 8.42

Dissolved oxygen:

3.93 to 9.45 mg/L

Nominal and measured concentrations:

NOMINAL TEST CONCENTRATIONS: Control, solvent control (acetone), 0.05, 0.1, 0.2, 0.4 and 0.8 mg test item/L. The concentrations measured at days -1, 0, 7 and 14 ranged from 83.0 to 106% of the nominal concentrations. This demonstrated that the diluter was working accurately and nominal concentrations could be used to determined the effect concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: The test vessels are clear glass aquaria. Each glass aquarium contains approximately 1.4 litres of test solution. Each aquarium has two holes of approximately 2 cm diameter, drilled in the side of the glass walls. The holes are covered with approximately 300 micrometer-mesh screen for drainage and to prevent loss of daphnids. Each aquarium is located in a stainless steel vessel. This exposure system is entirely constructed of glass, stainless steel, and other inert materials. The system was chemically clean before the test is started. The exposure system has been labeled with study number, replicate number and test group.
- Type : open

- Type of flow-through (e.g. peristaltic or proportional diluter): A 200-mL proportional diluter (e.g., Mount and Brungs, 1967) has been employed to deliver five toxicant concentrations, a control, and a solvent control, to four replicate vessels. Each treatment level was at least 50% of the next higher concentration of the test item. The exposure system is entirely constructed of glass, silicone, polyethylene and nylon.

- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water was natural filtered water of the Lake of Constance. The dilution water was stored in 50, 100 or 4000-liter containers and aerated using an air pump and air stones to bring the pH and dissolved gases into equilibrium with the atmosphere.
The batches of natural filtered water used for the definitive test had a total hardness range from 140 to 152 mg/L as CaCO3
- Alkalinity: from 116 to 122 mg/L as CaCO3
- Conductivity: from 235 to 245 μS/cm.
- Intervals of water quality measurement: At test initiation and weekly thereafter, total hardness, alkalinity and specific conductivity were measured and recorded in one replicate vessel of the highest test concentration and the control. Temperature, dissolved oxygen concentration and pH were measured and recorded in each vessel at test initiation and weekly thereafter until test termination. In addition, the temperature, dissolved oxygen concentration and pH were measured in one vessel of each test concentration and the controls, daily. Measurements taken from one replicate were alternated among the four replicate test chambers. Temperature was monitored continuously in one test solution by using a minimum-maximum thermometer. Readings of temperature extremes were recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light, 8-hour dark photoperiod
- Light intensity: 214 to 345 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The number of immobilized adult daphnids and observations of abnormal behavior were recorded daily. Assessments of offspring released were determined on test days 8, 11, 13, 15, 18 and 20. Additionally, the first day of brood release was recorded. Since the test was terminated on day 20, the length of the daphnids was not determined.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
A non-GLP preliminary range-finding test was conducted at Springborn Smithers Laboratories (Europe) at nominal p-TFMA concentrations of 0.00010, 0.0010, 0.010, 0.10 and 1.0 mg test item/L, a dilution water control and a solvent control under flow-through conditions. Initially, ten daphnids were distributed to each of the two replicate vessels of all treatments used for the test. At test termination (21-day exposure), daphnid survival,
reproductive output and growth (total body length) was determined.
Based on these results and in consultation with the Study Sponsor, definitive test concentrations of 0.05, 0.1, 0.2, 0.4 and 0.8 mg test item/L were selected for the definitive exposure.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

20 d

Dose descriptor:

EC50

Effect conc.:

>= 0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

and behaviour

Remarks on result:

other: Survival of parental Daphnia magna

Effect concentrations 2

Duration:

20 d

Dose descriptor:

EC10

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

and behaviour

Remarks on result:

other: C.L.: 0.05-0.5; Survival of parental Daphnia magna

Effect concentrations 3

Duration:

20 d

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

and behaviour

Remarks on result:

other: Survival of parental Daphnia magna and Offspring per female Daphnia magna

Effect concentrations 4

Duration:

20 d

Dose descriptor:

LOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

and behaviour

Remarks on result:

other: Survival of parental Daphnia magna and Offspring per female Daphnia magna

Effect concentrations 5

Duration:

20 d

Dose descriptor:

EC50

Effect conc.:

0.61 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and behaviour

Remarks on result:

other: C.L.: 0.57-0.63; Offspring per female Daphnia magna

Effect concentrations 6

Duration:

20 d

Dose descriptor:

EC10

Effect conc.:

0.36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and behaviour

Remarks on result:

other: C.L.: 0.19-0.45; Offspring per female Daphnia magna

Effect concentrations 7

Duration:

20 d

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and behaviour

Remarks on result:

other: offspring per female Daphnia magna

Effect concentrations 8

Duration:

20 d

Dose descriptor:

LOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and behaviour

Remarks on result:

other: offspring per female Daphnia magna

Details on results:

Due to high mortality in the control and solvent control on day 20, the test was terminated after 20 days and therefore, no body length measurements were performed. A repeat study is therefore necessary.

Results with reference substance (positive control):

- Results with reference substance valid: yes
A 48-hour reference test with K2Cr2O7 under static conditions was conducted from 01 to 03 July 2009 at Springborn Smithers Laboratories (Europe) under study # 7000.141.110. The reference test was used to evaluate the reliability of the test conditions for daphnia testing.
The 48-hour EC50 value for Daphnia magna and K2Cr2O7 was 0.34 mg test item/L with a 95% confidence interval of 0.25 to 0.50 mg test item/L. The results are well comparable with historical data of 48-hour EC50 values ranging from 0.37 to 0.71 mg test item/L.
These results show that the test conditions used are appropriate for testing.

Any other information on results incl. tables

Nominal concentration(mg test item/l)	Meansurvival(%)	SD	mean # ofoffspring releasedper female	SD	First broodrelease (day)
Control	82.5	15.0	137	13.2	8
SolventControl	70.0	11.5	153	22.1	8
0.05	75.0	20.8	191	12.6	7 - 8
0.1	67.5	22.2	184	20.7	7 - 8
0.2	62.5	19.1	168	21.8	7 - 8
0.4	77.5	20.6	152	23.3	7 - 8
0.8	35.0	5.8	25	7.4	8 - 11

Applicant's summary and conclusion

Validity criteria fulfilled:

no

Remarks:

high mortality in the control and solvent control on day 20

Conclusions:

The toxicity of p-TFMA to the reproduction of daphnids was determined to give EC10-20d = 0.4 mg/L. However, the test was terminated after 20 days and therefore due to high mortality in the control and solvent control. The study has been repeated then third time and each time it has to be terminated before the 21 days for different reasons. Another definitive study is still on-going.

Executive summary:

The toxicity of p-TFMA to the reproduction of daphnids was studied under flow-through conditions. The test was performed under GLP and according to the OECD 211 guideline for Daphnia magna reproduction test (2008). The test concentrations were measured and were found to be within 80% of nominal concentrations. Therefore nominal concentrations were used to determine the effect concentrations at the end of the test. The test had to be terminated after 20 days due to high mortality of the adults in control and solvent control. Until the 19th day all parameters were still good and in line with the results in the range-finding test (performed during 21 days). Therefore even if the test cannot be considered as valid, the results give a good indication of the toxicity of the substance to the reproduction of the daphnids. An EC10-20d was determined at 0.36 mg/L (0.19 - 0.45 mg/L 95% confidence limit) based on the number of offspring per female. Other definitive tests have been conducted and had to be terminated again for different reasons. Then a new test is still on-going to confirm these first results.