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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
equivalent or similar to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Test solutions concentrations were not measured pre or post incubation.
GLP compliance:
Published study not conducted to GLP
Specific details on test material used for the study:
Not reported
Analytical monitoring:
Details on sampling:
- Concentrations: 15 mg/L stock solution measured, only.
- Sampling method: Subsample of stock taken for HPLC analysis
- Sample storage conditions before analysis: Not reported
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 15 mg/L stock solution prepared by dissolving and stirring bisphenol AF in ultra-pure water on a magnetic stirrer for 24 h.
- Eluate: ultra-pure water
- Differential loading: not reported
- Controls: dilution water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna, water flea
- Strain/clone: Straus 1820 (Clone A)
- Justification for species other than prescribed by test guideline: n/a
- Source: ECT Oekotoxikologie, Flörsheim, Germany.
- Age of parental stock (mean and range, SD): not reported
- Feeding during test: not reported
- Food type: not reported
- Amount: not reported
- Frequency: not reported

- Acclimation period: not reported
- Acclimation conditions (same as test or not): not reported
- Type and amount of food: not reported
- Feeding frequency: not reported
- Health during acclimation (any mortality observed): not reported

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


Neonates, < 24 h old used. Collection procedures not reported.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
not reported
Test temperature:
21 ± 1 ºC
not reported
Dissolved oxygen:
not reported
not reported
not reported
Nominal and measured concentrations:
nominal: 0.90, 1.0, 2.0, 3.0, 4.0, 5.0, 8.0, 12.0 mg/L
Details on test conditions:
- Test vessel: not reported
- Type (delete if not applicable): not reported
- Material, size, headspace, fill volume: not reported
- Aeration: not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): n/a
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): n/a
- Biomass loading rate: n/a

- Source/preparation of dilution water: not reported
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: not reported
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Conductivity: not reported
- Salinity: not reported
- Culture medium different from test medium: not reported
- Intervals of water quality measurement: not reported

- Adjustment of pH: not reported
- Photoperiod: not reported
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility measured at 24 and 48 h.


- Test concentrations: n/a
- Results used to determine the conditions for the definitive study: n/a
Reference substance (positive control):
Key result
48 h
Dose descriptor:
Effect conc.:
2.7 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Behavioural abnormalities: not reported
- Observations on body length and weight: not reported
- Other biological observations: not reported
- Mortality of control: not reported
- Other adverse effects control: not reported
- Abnormal responses: not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium: not reported
Results with reference substance (positive control):
not reported
Reported statistics and error estimates:
Values were calculated by non-linear regression model using GraphPad Prism (version 6.0).

24/48 h EC20: 2.3 / 1.6 mg/L
24 h EC50 (with 95 % C.L.): 3.4 (2.8 - 4.0) mg/L
48 h EC50 (with 95 % C.L.): 2.7 (1.7 - 4.3) mg/L
24/48 h EC80: 4.9 / 4.6 mg/L
Validity criteria fulfilled:
not specified
Immobility in the controls was not reported and DO concentration was not reported.
The 48 h EC50 was determined to be 2.7 (95 % C.L.: 1.7 - 4.3) mg/L.
Executive summary:

The 48–hr-acute toxicity of bisphenol AF to Daphnia magna was studied under static conditions.  Daphnids were exposed to control, 0.90, 1.0, 2.0, 3.0, 4.0, 5.0, 8.0 and 12.0 mg/L for 48 hr.  Mortality/immobilisation and sublethal effects were observed at 24 and 48 hr.  The 48– hour EC50 was 2.7 mg/L.

Based on the results of this study, bisphenol AF would be classified as moderately toxic to Daphnia magna in accordance with the classification system of the U.S. EPA.

This study is classified as [enter acceptability classification e.g. acceptable/unacceptable/ supplementary) and {satisfies/does not satisfy} the guideline requirements for an acute toxicity study with freshwater invertebrates.

Results Synopsis;

Test Organism Age: < 24 h

Test Type: Static

Endpoint: 48 h EC50 = 2.7 (95 % C.L.: 1.7 - 4.3) mg/L

Description of key information

EC50 = 2.7 mg/L, ISO 6341 (2012), Tišler et al., 2016

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2.7 mg/L

Additional information

In a single key study by Tisler et al., 2016, a suite of aquatic ecotoxicity tests were conducted to identify hazards to the aquatic environment and conduct a risk assessment using measured concentration of BPAF from the environment to determine a risk characterisation ratio (i.e. predicted exposure concentration (PEC)/predicted no effect concentration (PNEC). Firstly, a suite of low tier aquatic toxicity tests were conducted on V. fischeri (bacteria), Desmodesmus subspicatus (green algae), Daphnia magna (invertebrate) and Danio rerio (fish; common name zebrafish).  

Specifically for the Daphnia magna test the authors quote the use of the ISO 6341 (2012) guideline being followed (Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity test).  However, the test is also equivalent or similar to the OECD 202 (2004) and OPPTS850.1010 (1996) guidelines. For the husbandry water fleas were kept in 3-L aquariums covered with glass plates (20 daphnids in 2.5 L of Elendt M4 medium) at 21 ± 1 °C in a 16 h light/8 h dark photoperiod. Water fleas were fed daily as follows: four times with green algae D. subspicatus (0.13 mg of C/daphnid), twice per week with Tetramin (20 mg blended in deionized water/aquarium) and once a week with dry yeast (5 mg blended in deionized water/aquarium).  Two replicate vessels (30 mL medium/ medium-test-item) containing 10 daphnids (Straus 1820 (Clone A)) each were used at each concentration and the dilution water control.  The concentrations tested were 0.90, 1.0, 2.0, 3.0, 4.0, 5.0, 8.0, 12.0 mg/L. Immobilisation of water fleas was determined by 15 seconds gentle agitation of the test vessel, those water fleas which were not able to swim during this time were considered as immobile.  Immobility was checked at 24 and 48 hours. Although there was no chemical analytics conducted during the test the authors also ran a chronic test.  During the test they assessed the stability of BPAF in the Daphnia medium (Elendt M4 medium) and showed it to be stable over 3 days, therefore the use of nominal concentrations can be used in line with the guideline.  After dosing, BPAF stayed at 90 % of nominal concentrations.  This showed that dosing technique was appropriate as well as the stability of the test item in the test system.  Water quality parameters were also tested in the chronic test and deemed acceptable.  Furthermore, DuPont (2002), showed that there was minimal impact of BPAF on pH and dissolved oxygen up to 1000 mg/L of BPAF.  It can be assumed that the water quality parameters were met.  From the sigmoidal plot of the data it can be seen that the control mortality was <= 10 %. The validity criteria was likely met during testing.

Concentration-response curves for each tested species exposed to BPAF were plotted using Origin (version 8.1) data analysis and graphing software (OriginLab). The EC50 with 95% confidence limits, values were calculated by non-linear regression model using GraphPad Prism (version 6.0, GraphPad Software Inc., San Diego).

The EC50 of BPAF was determined to be 2.7 mg/L (95 % confidence limits = 1.7-4.3 mg/L).  The value will be used in the chemical safety assessment.