4-fluoroaniline

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:

from 2010-12-08 to 2011-09-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to generally valid and/or internationally accepted testing guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-fluoroaniline

EC Number:

206-735-5

EC Name:

4-fluoroaniline

Cas Number:

371-40-4

Molecular formula:

C6H6FN

IUPAC Name:

4-fluoroaniline

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling method:
- Determination of the test item concentration after 0, 24 and 48 h
- Sampling: Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test and at day 1.
- Analyses: For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the aged test media of all test concentrations and the control were collected at the end of the test and at day 1 (after 24 hours of exposure) by pouring together the contents of the test beakers of each treatment.
The concentrations of the test item were analysed in the duplicate test media samples from all sampling times. From the control samples only one of the duplicate samples was analysed from each sampling times.
- Samples were diluted with acetonitrile
- Sampling storage: All samples were stored in a freezer (≤ - 10 °C), protected from light until analysis was performed.
- Determination of the water quality parameters in the fresh media (0 h) and in the old media (48 h)
- Concentrations (fortified samples): Approximately 10 and 50 mg test item were diluted with 10 and 50 mL pure water to obtain a stock solution of 1 g test item/L. Appropriate amounts of the stock solution were diluted with test water to obtain fortified samples at concentrations of 15, 30, 75 and 450 µg test item/L.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTIONS

Dosage of test item: A stock solution of 20 mg/L was prepared by dissolving 15 .6 and 13.1 mg test item into 780 and 655 mL test water, respectively, by intense stirring for 5 minutes. Adequate volumes of these stock solutions were diluted with test water to prepare the test media of the desired test concentrations.The test media were prepared just before introduction of Daphnia (= start of the test) and at test medium renewal on Day 1.

Standard solutions used for the Quantification:
Stock solution: The test item was used to prepare the stock solutions. 50.54 and 10.68 mg of the test item were diluted with 50 and 10 mL pure water to obtain stock solutions of approx. 1 g test item/L.

Standard solutions: Appropriate amounts of the stock solutions were diluted with a mixture of 50% acetonitrile / 50% test water (v/v) to obtain standard solutions in the range from 5 to 500 µg test item/L.
- Control: Reconstituted water according to guideline. In the control, test water was used without addition of the test item.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, clone 5
- Source: laboratories of IBACON
- Age at study initiation: 3.75 to 19.5 hours old.
- Feeding during the test:
Food type: green algae (Desmodesmus subspicatus) freshly grown in the laboratories of IBACON
Frequency: The Daphnia in the stock culture were fed at least on all working days

ACCLIMATION
Acclimatation was not necessary, since the test was performed in the same medium as the culturing.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

2.5 mmol/L (~ 250 mg/L) as CaCO3

Test temperature:

20 °C in the freshly prepared media and 20 °C in the aged test media

pH:

7.9 to 8.0 in the freshly prepared media; 7.9 in the aged test media; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

8.5 to 9.1 mg/L in the freshly prepared media; 8.2 to 9 .0 mg/L in the aged test media

Salinity:

0.9 mmol/L

Nominal and measured concentrations:

Nominal concentrations: 0.45, 0.20, 0.093, 0.042 and 0.019 mg test item/L (nominal) and a control.
Measured concentrations: At the start of the test and at test medium renewal 96 % of the nominal test concentration was found (average of all test concentrations). After 24 and 48 hours test duration, 93 % of the nominal value was determined (average of all test concentrations). During the test the Daphnia were exposed to a mean of 95 % of nominal. Therefore, all reported results refer to nominal concentrations.

Details on test conditions:

TEST SYSTEM:
- Test vessel: Glass beakers of 100 mL volume containing approximately 60 mL of test medium.
- Type and size: The test vessels were covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
- Renewal rate or test solutions: A semi-static test procedure with a test medium renewal at Day 1 was perforrned to keep the test item concentrations in the test media as constant as possible.
- No. of organisms per vessel: 20 Daphnia per control and test concentration,
- No. of vessel per concentration (replicates): 4 groups of 5 animals, each group in 60 mL test medium
- No. vessels per control (replicates): 1

TEST MEDIUM/WATER PARAMETERS
- Test water: Reconstituted Water (Elendt "M4") according to guideline
- Water Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3
- Alkalinity: 0.9 mmol/L
- Culture medium different from test medium: No
- Intervals of water quality measurements: 24 h

OTHER TEST CONDITIONS:
- Adjustment of pH: No
- Photoperiod: 16 h light-8 h dark
- Light intensity: 650 - 830 lux

EFECT PARAMETERS MEASURED: The mobility of the Daphnia was determined by visual observation after 24 and 48 hours. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae).

TEST CONCENTRATIONS:
- Test concentrations:
Nominal concentrations: 0.45, 0.20, 0.093, 0.042 and 0.019 mg test item/L (nominal) and a control.
Measured concentrations: At the start of the test and at test medium renewal 96 % of the nominal test concentration was found (average of all test concentrations). After 24 and 48 hours test duration, 93 % of the nominal value was determined (average of all test concentrations). During the test the Daphnia were exposed to a mean of 95 % of nominal. Therefore, all reported results refer to nominal concentrations.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: n.d.

Effect concentrations 2

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

0.45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: n.d.

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.042 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: n.d.

Effect concentrations 4

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.093 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: n.d.

Effect concentrations 5

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.254 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: n.d.

Details on results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and the test item concentration up to and including nominal 0.042 mg/L.
At the nominal test concentration of 0.093 mg test item/L one Daphnia was immobile. Two immobile Daphnia were observed at the nominal test concentration of 0.20 mg test item/L. At the highest nominal test concentration of 0.45 mg test item/L, 20 animals were immobile.
The NOEC was determined to be 0.042 mg test item/L, the LOEC was determined to be 0.093 mg test item/L and the EC50 value was calculated to be 0.254 mg test item/L. All reported results refer to nominal concentration.
All validity criteria were met.

Results with reference substance (positive control):

The EC50 after 24 hours was determined to be 1.63 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 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 2.10.05, ToxRat® Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the semi-static immobilization test, young Daphnia magna STRAUSS (< 24 h old) were exposed to the test item for 48 hours according to OECD guideline No. 202 (2004). The NOEC was determined to be 0.042 mg test item/L, the LOEC was determined to be 0.093 mg test item/L and the EC50 value was calculated to be 0.254 mg test item/L. All reported results refer to the nominal concentrations.

Executive summary:

ln the acute immobilization test with Daphnia magna (STRAUS), the effects of the test substance with a nominal loading of 0.45, 0.20, 0.093, 0.042 and 0.019 mg test item/L and a control were determined according to OECD-Guideline for Testing of Chemicals No.202 (2004) and Council Regulation (EC) No. 440/2008 Method C.2 (2008). The study encompassed 6 treatment groups (5 dose rates of the test item, control) each containing 20 individuals. The mobility of the Daphnia was determined in a semi static 48-hour test by visual observation after 24 and 48 hours. The samples collected at start and after each sampling time were analyzed via HPLC-method. In the test with the reference item potassium dichromate performed in January 2011, the EC50 after 24 hours was determined to be 1.63 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), therefore all validity criteria were met. At the start of the test and at test medium renewal 96 % of the nominal test concentration was found (average of all test concentrations). After 24 and 48 hours test duration, 93 % of the nominal value was determined (average of all test concentrations). During the test the Daphnia were exposed to a mean of 95 % of nominal. Therefore, all reported results refer to nominal concentrations. The 48-hour NOEC was determined to be 0.042 mg test item/L, the 48-hour LOEC was determined to be 0.093 mg test item/L and the 48-hour EC50 value was calculated to be 0.254 mg test item/L.