4-fluoroaniline

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 2010-11-29 to 2011-09-27

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 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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:

- Concentration:
Nominal concentration: 20, 3.2, 1.0, 0.32 and 0.10 mg test item/L and a control (the highest test concentration should have been 10 mg/L but due to a calculation error a concentration of 20 mg/L was prepared). These nominal concentrations are corresponding to geometric mean measured concentrations of: 11.98, 1.57, 0.487, 0.157 and 0.049 mg test item/L and a control.

- Sampling methods:
- Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from 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 concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period).
- Analyses: The concentrations of the test item were analysed in the duplicate test media samples from all test concentrations above the NOEC determined in this test and both sampling times (0 and 72 hours). From the control samples only one of the duplicate samples was analysed from each of both sampling times. All samples were centrifuged before analysis, in order to remove the algae.
- Sample storage: All samples were stored in a freezer ≤ -10 °C), protected from light until analysis was performed.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTIONS
- Standard Solutions used for the Quantification:
- Stock Solution: The test item was used to prepare a stock solution. 49.92 mg of the test item were dissolved in 50 mL pure water to obtain a stock solution of approximately 1 g test item/L.
- Standard Solutions: Appropriate amounts of the stock solution were diluted with test water to obtain standard solutions in the range from 0.5 to 12.5 mg test item/L.

Dosage of test item: A stock solution of 20 mg test item/L was prepared by dissolving 20.37 mg test item into 1018.5 mL test water by intense stirring for 15 minutes. The pH of this stock solution was adjusted with 1N NaOH from 7.9 to 8.1. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations. The test media were prepared just before introduction of the algae (= start of the test).

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISMS
- Common name: Pseudokirchneriella subcapitata
- Strain: Strain No. 61.81 SAG
- Source: The algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum: 4 days
- Method of cultivation: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

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

Test temperature:

22-23 °C

pH:

pH values: at test start 8.1, at the end of the test 8.0 - 9.4.

Nominal and measured concentrations:

- Nominal concentration: 20, 3.2, 1.0, 0.32 and 0.10 mg test item/L and a control (the highest test concentration should have been 10 mg/L but due to a calculation error a concentration of 20 mg/L was prepared).
- These nominal concentrations are corresponding to geometric mean measured concentrations of: 11.98, 1.57, 0.487, 0.157 and 0.049 mg test item/L and a control.

Details on test conditions:

TEST SYSTEM:
- Test vessel: Erlenmeyer flasks of 50 mL volume with 50 mL of test medium
- Aeration: no
- Initial cells density: Inoculation of a biomass of 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test start under the same conditions as in the test
- Replicates: The test was performed with three replicates per test concentration and six replicates in the control. Volumes of 50 mL of algal suspension per replicate were continuously stirred with magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass dishes and incubated in a water bath.
- Blanks: Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometrical measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by test item.

GROWTH MEDIUM
- Standard medium used

TEST MEDIUM/WATER PARAMETERS:
- Dilution water according to guideline
- Culture medium equal from test medium
- Intervals of water quality measurement: After 0, 24, 48, 72 h

OTHER TEST CONDITIONS:
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality:. The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media. Mean light intensity: 5725 lux (range: 5610 - 5930 lux).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentration: The cell densities in the samples were determined by spectrophotometrical measurement. Therefore defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced.
- The algal cell densities were calculated by subtracting the absmption of the blanks, from each of the measured absorption of the test media (with algae).
- The cell densities in a number of samples from one control replicate were counted by microscope after 72 hours of test duration. Out of this control five defined dilutions were prepared and measured spectrophotometrically. Based on the counted cell densities and the absorption of the control sample and the dilutions a linear regression was performed for the calculation of the cell densities in all other samples measured spectrophotometrically during the test.

TEST CONCENTRATION:
- Nominal concentration: 20, 3.2, 1.0, 0.32 and 0.10 mg test item/L and a control (the highest test concentration should have been 10 mg/L but due to a calculation error a concentration of 20 mg/L was prepared). These nominal concentrations are corresponding to geometric mean measured concentrations of 11.98, 1.57, 0.487, 0.157 and 0.049 mg test item/L and a control.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits (11.6-17.7)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. limits (1.55-2.31)

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits (0.937-2.04)

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.504 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf.limits (0.313-0.664)

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.487 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: n.d.

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.487 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: n.d.

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.57 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: n.d.

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.57 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: n.d.

Details on results:

At the start of the test 82 % of the nominal test concentrations was found (average of all test concentrations above the NOEC). After 72 hours test duration, 34 % of the nominal value was determined (average of all test concentrations above the NOEC). Since the test item concentrations were not stable during the test duration, all reported results refer to geometric mean measured concentrations.
The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose response test. The 72-hour ErC50 value was calculated to be 14.0 mg test item/L (extrapolated) and the 72-hour EyC50 was calculated to be 1.84 mg test item/L. The 72-hour NOErC and the 72-hour NOEyC were determined to be 0.487 mg test item/L, the associated 72- hour LOErC and LOEyC were 1.57 mg test item/L and the associated 72- hour ErC10 was determined to be 1.48 mg test item/L and the EyC10 was determined to be 0.504 mg test item/L.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50, the corresponding EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis. For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by the Williams t-test (growth rate, yield), respectively.
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat® Solutions GmbH. All validity criteria were met.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The influence of the test substance on the growth of the freshwater algae Pseudokirchneriella subcapitata was assessed in a static dose response test according to the OECD guideline No 201 (2006). The 72-hour ECr50 value was calculated to be 14.0 mg test item/L (extrapolated), the 72-hour NOErC was determined to be 0.487 mg test item/L and the associated 72- hour EC10 was determined to be 1.48 mg test item/L.

Executive summary:

The toxicity of the test substance to the freshwater alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 (2006) and the Commission Regulation (EC) No 761/2009, C.3 (2009). The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions. This study encompassed 6 treatment groups (5 dose rates of the test item, control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement. The samples of the test media sampled at the start and at the end of the test after 72 hours of exposure were analysed via HPLC-method. The nominal test concentrations of 20, 3.2, 1.0, 0.32 and 0.10 mg test item/L and a control are corresponding to geometric mean measured concentrations of: 11.98, 1.57, 0.487, 0.157 and 0.049 mg test item/L and a control. The 72-hour ErC50 value was calculated to be 14.0 mg test item/L (extrapolated) and the 72-hour EyC50 was calculated to be 1.84 mg test item/L. The 72-hour NOErC and the 72-hour NOEyC were determined to be 0.487 mg test item/L, the associated 72- hour LOErC and LOEyC were 1.57 mg test item/L and the associated 72- hour ErC10 was determined to be 1.48 mg test item/L and the EyC10 was determined to be 0.504 mg test item/L.