1-[4-(4-chlorophenoxy)-2-(trifluoromethyl)phenyl]ethanone

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-25 to 2015-10-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13. April 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

30. May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Version / remarks:

15. Dezember 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Institute für biologische Analytik und Consulting IBACON GmbH

Test material

Test material information

Constituent 1

Reference substance name:

1-[4-(4-chlorophenoxy)-2-(trifluoromethyl)phenyl]ethanone

EC Number:

813-142-0

Cas Number:

1417782-28-5

Molecular formula:

C15 H10 Cl F3 O2

IUPAC Name:

1-[4-(4-chlorophenoxy)-2-(trifluoromethyl)phenyl]ethanone

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: L85-76
- Expiration date of the lot/batch: 01.10.2016
- Purity test date: 14.10.2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ≤25 °C, in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 3.0, 1.37, 0.62, 0.28, 0.13, 0.06 and 0.03 mg/L
- Sampling method: For the analysis of the test item concentration, triplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentration during the test period, triplicate test media samples were collected from the test concentration and the controls of the aged test media at test end. All samples were diluted by a factor of 2 with acetonitrile. Additional samples of the control and the dilution solvent were taken at test start and test end without any sample treatment. The concentrations of the test item were analysed in two of the triplicate test media samples from all test concentrations, and in two of the triplicate control samples, from both sampling times (0 and 48 hours).
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 10 °C), protected from light until analysis was performed

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Before test start 35.6 mg of the test item was dissolved in 35.6 mL acetone, resulting in an application solution containing 1 mg test item/mL. 10 mL of this solution were pipetted into a 1 L wide neck inert glass flask. The entire bottom and walls of this flask were then coated with a thin layer of this solution by slowly rotating the flask around. The solvent was then allowed to evaporate completely (approximately 45 minutes), leaving behind a thin layer of test item on the glass surface. Then 1 L of test water was added, which resulted in a loading rate of 10 mg test item/L. This dispersion was then shaken for approximately 48 hours in an overhead shaker to achieve equilibrium. A centrifugation step was performed after the shaking period (20 minutes at 4700 rpm) to ensure that any undissolved test item was removed. The centrifuged solution was used as the highest test concentration. All other concentrations were prepared by serial diluting the highest test concentration. Centrifuge tubes, mixing vessels and test vessels were pre-conditioned for approximately 30 minutes. The test media were prepared just before introduction of the Daphnia (= start of the test).
- Controls: Yes. Control containing reconstituted water and a solvent control containing reconstituted water from a vessel which was treated the same way with acetone as the vessel of the stock solution but without addition of the test item.
- Chemical name of vehicle: Acetone
- Evidence of undissolved material: After the shaking period and prior to centrifugation the stock solution was slightly turbid and test item was observed on the bottom of the flask and at the surface of the solution. After the centrifugation step all solutions were visually clear and there were no remarkable observations

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea (Daphnia magna)
- Strain/clone: Daphnia magna (Straus), clone 5
- Source: Ibacon's in-house laboratory culture
- Age at test start: From 1.75 to 18.25 hours old
- Feeding during test: No

ACCLIMATION
- Acclimation period: Was not necessary, since the test was performed in the same medium as the culturing
- Acclimation conditions: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium.
- Type of food: The Daphnia in the stock culture were fed with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of Ibacon.
- Feeding frequency: At least on all working days

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

1.068 mmol/L as carbon hardness

Test temperature:

20 to 21 °C at test start; 20 °C at test end

pH:

8.1 to 8.8 at test start; 7.9 to 8.2 at test end (pH-value did not vary by more than 1.5 units)

Dissolved oxygen:

8.8 to 9.1 mg/L at test start; 8.5 to 8.7 mg/L at test end

Conductivity:

686 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 3.0, 1.37, 0.62, 0.28, 0.13, 0.06 and 0.03 mmg/L
Mean measured concentrations: 3.78, 1.81, 0.856, 0.431, 0.161, 0.083 and 0.041 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel (material, size, fill volume): Glass beakers of 100 mL volume containing approximately 60 mL of test medium.
- Type: closed
- No. of organisms per vessel: 5
- 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: According to guideline (Elendt M4 medium)
- Alkalinity: 0.348 mmol
- Conductivity: 686 µS/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark
- Light intensity: 290 - 390 lux

EFFECT PARAMETERS MEASURED: The mobility of the test species was determined by visual observation after 24 and 28 hours.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations:
1st experiment:
In the first experiment 9.99 mg of the test item were dissolved in acetone. The bottom and walls of a suitable vessel (1 L wide neck inert glass flask) were coated with a thin layer of the test item while the solvent was allowed to evaporate completely. Then 999 mL of test water were added, which results in a loading rate of 10 mg test item/L. This solution was gently shaken for 48 hours to achieve equilibrium. The resulting test solution was visually clear. With the same procedure but without the test item, the solvent control was prepared. In the subsequent Daphnia exposure limit test over 48 h 100 % immobility was observed at the only test concentration while no animal was immobile in the control.
In this first experiment a concentration of approximately 10 mg test item/L was analytically confirmed.

2nd experiment:
The biological pre-experiment was repeated in the same way as described above but a centrifugation step was included after the shaking period to ensure that non-dissolved test item is removed from the stock solution. Additionally, several dilutions of the centrifugate (1:10, 1:100 and 1:1000) were tested. Like in the first experiment all test organisms were immobile at the highest test concentration. 40 % immobility was observed at the 1:10 dilution and no immobility occurred at the 1:100 and 1:1000 dilutions. The analytical measurements to this pre-experiment demonstrate that the centrifugation reduced the concentration from 9 mg test item/L prior to centrifugation to 3 mg test item/L after 20 min centrifugation at 4700 rpm (0.3 mg/L in 1:10 and 0.03 mg/L in 1:100 dilution).

3rd experiment:
A third preliminary experiment was run after a shaking period of 96 hours and a subsequent filtration step (0.45 μm). In the filtrate of nominal 10 mg test item/L complete immobility of the Daphnia occurred already after 24 hours. The analytical measurement demonstrated a concentration of the test item of 4 mg/L.

All analytical experiments (reported in more detail in the raw data file) demonstrate that the test item is stable under the conditions of the daphnia test. Therefore, it was decided to perform the definitive test with a static design. In view of the high content of the main component the decision was taken to prepare the test concentrations by dilution of a stock solution (whose concentration is determined analytically).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.237 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: n.d.

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: n.d.

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.161 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.431 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: None

Results with reference substance (positive control):

In the most recent test with the reference item potassium dichromate performed in January 2015 the EC50 after 24 hours was determined to be 1.85 mg test item/L indicating that the sensitivity of the Daphnia was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).

Reported statistics and error estimates:

The 24-hour and 48-hour EC50, EC20 and EC10 and the 95% confidence limits were calculated by probit analysis.
The NOEC and LOEC after 24 and 48 hours were determined directly from the raw data.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.0.0, ToxRat® Solutions GmbH.

Any other information on results incl. tables

After 48 hours of exposure no immobilisation of the test animals was observed in the control, in the solvent control and in the test item concentration of 0.03 (0.041) mg test item/L. At the concentration of 0.06 (0.083) mg test item/L, one animal was immobile. This is not considered to be substance related as no immobility occurred at the higher concentration of 0.13 (0.161) mg test item/L. All test organisms were immobile at the nominal concentrations of 0.280 (0.431), 0.620 (0.856), 1.37 (1.81) and 3.0 (3.78) mg test item/L. Values presented in the text above refer to nominal test concentrations. Values in parenthesis refer to mean measured concentrations.

Table 1: Summary of Biological Results

Test concentration [mg test item/L]	% of immobilized Daphnia after
	24 hours	48 h
Control	0	0
Solvent control	0	0
0.041	0	0
0.083	5	5
0.161	0	0
0.431	70	100
0.856	100	100
1.81	100	100
3.78	100	100

Values refer to mean measured concentrations or, in case of the lowest concentration, to a value extrapolated from mean recovery of all other measured concentrations.

Endpoint	24 hours	48 hours
EC50 [mg/L]	0.321	0.237
95 % CI [mg/L]	0.161 – 0.583	n.d.
EC20 [mg/L]	0.209	0.175
95 % CI [mg/L]	0.049 – 0.327	n.d.
EC10 [mg/L]	0.166	0.150
95 % CI [mg/L]	< 0.041 – 0.268	n.d.
NOEC [mg/L]	0.161	0.161
LOEC [mg/L]	0.431	0.431

Values refer to mean measured concentrations

CI: Confidence interval

n.d.: not detectable

NOEC and LOEC were determined directly from the raw data.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a static acute test with Daphnia magna a 48-h EC50 of 0.237 mg test item/L (mean measured concentration) was determined.

Executive summary:

The influence of the test substance on the mobility of Daphnia magna was investigated in a 48-h static immobilisation test according to OECD guideline 202 (April 2004). Young Daphnia (<24 hours) were exposed for 48 hours to nominal test item concentrations of 3.0, 1.37, 0.62, 0.28, 0.13, 0.06 and 0.03 mg/L as well as to a control and a solvent control. The nominal concentrations correspond to mean measured concentrations of 3.78, 1.81, 0.856, 0.431, 0.161, 0.083 and 0.041 mg/L. After 48 hours of exposure no immobilisation of the test animals was observed in the control and the solvent control. The 48-h LOEC and NOEC were determined to be 0.431 and 0.161 mg/L, respectively. The 48 -h EC50 value was calculated to be 0.237 mg/L. Values refer to mean measured concentrations.