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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 - 07 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Study conducted in accordance with international guidelines and in accorance with GLP. All guideline validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not reported
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: not reported
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: n/a
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): stock and treatment solutions prepared in AAP nutrient medium.

OTHER SPECIFICS:
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 62.5, 125, 250, 500 and 1000 µg/L and blank control
- Sampling method: Aliquots from treatment solution submitted on Day 0. Test solution replicates pooled on Day 3 and an aliquot of the pool submitted.
- Sample storage conditions before analysis: Refrigerated
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions prepared from a 50 mg/L stock solution.
- Eluate: AAP nutrient medium
- Differential loading: 10000 cells/mL
- Controls: AAP medium and algae only
- 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.): visually inspected with no visible particulate.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: P. subcapitata
- Source (laboratory, culture collection): Cultured on-site. Original culture sourced from Dpeartment of Botany, University of Texas, USA.
- Age of inoculum (at test initiation): n/a
- Method of cultivation: Cultured based on published literature (see reference 1 and 2 of attached study report).

ACCLIMATION
- Acclimation period: Not reported
- Culturing media and conditions (same as test or not): As per main test conditions
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
23.8 ºC
pH:
0 h: 7.85 - 8.47
72 h: 7.65 - 8.29
Dissolved oxygen:
Not reported
Salinity:
n/a
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 62.5, 124, 250, 500 and 1000 µg/L
0 h measured: 53.6, 121, 206, 411, 849 µg/L
72 h measured: 50.8, 117, 195, 385 and 768 µg/L
Mean measured: 52.2, 119, 200, 398 and 808 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flask
- Type (delete if not applicable): closed / gas permeable
- Material, size, headspace, fill volume: Sterile glass flask containing 50 mL test media. Cotton wool plug permitted gas exchange.
- Aeration: Cotton wool plug allowed gas exchange- no forced aeration.
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): n/a
- Initial cells density: 10000 cells/mL
- Control end cells density: ca. 2.1 x 10E6
- No. of organisms per vessel: n/a
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n/a

GROWTH MEDIUM
- Standard medium used: yes (AAP)
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: To prepare 1 L of AAP nutrient medium, 1 mL of each of the 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution are added to approximately 800 ml of Mili-Q water with mixing after each addition. The final volume was brought to 1 L with Milli-Q water. The medium was adjusted to pH 7.57 with 0.1 N hydrochloric acid and filter sterilised through 0.22 µm cellulose acetate filters sterile containers.
- 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
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: Continuous light
- Light intensity and quality: 6690 - 7430 lux
- Salinity (for marine algae): n/a

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell count, area under the growth curve, growth rate
- Determination of cell concentrations: hemacytometer and a compound microscope
- Chlorophyll measurement: not reported
- Other: n/a

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 62.5, 125, 250, 500 and 1000 µg/L based on the results of a preliminary range-finding test.
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: not reported
- Results used to determine the conditions for the definitive study: yes, although results not reported.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 808 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
52.2 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not reported
- Unusual cell shape: not reported
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported
- Other: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
not conducted
Reported statistics and error estimates:
The data for healthy cell count, area under the growth curve, and growth rate based on healthy cell count were determined to be non-normally distributed (Sharpiro-Wilk test). Therefore, a non-parametric analysis was performed (Kruskal-Wilk test) and the Jonckheere-Terpstra test was used to determine the LOEC and NOE values. No outlier were found in the data for healthy cell count, area under the growth curve, and growth rate based on healthy cell count. The EbC50, EyC50 and ErC50 (and 95 % confidence intervals) for healthy cell count, are under the growth curve, and growth rate based on healthy cell count were obtained by the Bruce-Versteeg regression model.

Table 1:       Nominal and measured concentrations of technical end product (TEP) and active ingredient (a.i.)

Nominal Test

Material

Concentration

Measured Concentration

(µg/L)

Mean Measured Concentration

(µg/L)

% Recovery Mean Measured

(%)

TEP

(mg/L)

a.i.

(mg/L)

Day 0

Day 3

Dilution control

nd

nd

nd

-

62.5

61.8

53.6

50.8

 

84

125

124

121

117

119

96

250

247

206

195

200

81

500

495

411

385

398

80

1000

989

849

768

808

82

Abiotic control

(1000)

989

849e

792

820

83

a defined in terms of bisphenol AF/L nutrient medium

b nominal bisphenol AF concentration corrected for 98/93 % purity

c calculated as (Day 0 measured concentration + Day 3 measured concentration)/2

d based on corrected bisphenol AF concentrations

e no separate analysis was conducted since the 1000 µg/L test solution and the Day 0 abiotic control solution were the same solution. Therefore, the measured concentration obtained for the test concentration solution can be used for the abiotic control.

nd not detected (limit of detection = 3.75 µg/L)

Table 2:       Effect of bisphenol AF on biomass (area under the growth curve) for P. subcapitata

Treatment  concentration

(mg/l)

Cell density

% Inhibition of biomass

Nominal

Measured*

Rep

24 h

48 h

72 h

Mean 72 h

c.o.v.

(%)

Dilution  control

1

70000

550000

2115000

1871667

8.0

-

2

10000

435000

1785000

3

35000

365000

1765000

4

10000

370000

1825000

5

30000

405000

1745000

6

75000

530000

1995000

62.5

52.2

1

10000

480000

2060000

2068333

1.6

-11

2

20000

555000

2040000

3

30000

570000

2105000

125

119

1

15000

145000

680000

771667

25.2

59

2

65000

305000

995000

3

0

100000

640000

250

200

1

35000

145000

745000

785000

4.8

58

2

0

70000

820000

3

20000

110000

790000

500

398

1

5000

35000

510000

511667

19.1

73

2

15000

95000

610000

3

0

0

415000

1000

808

1

10000

30000

315000

230000

34.2

88

2

0

0

160000

3

0

0

215000

Table 3:       Growth Rate Data Summary

Treatment concentration

(mg/L)

Growth rate

Cells/mL

Nominal

Measured*

Rep

Day 0-1

Day 1-2

Day 2-3

Mean

c.o.v.

(%)

Mean

Day 0-3

Dilution control

1

2.71

1.71

1.03

1.82

46.54

1.82

2

1.10

3.33

0.81

1.75

79.17

1.75

3

2.08

2.01

1.31

1.80

23.75

1.80

4

1.10

3.16

1.14

1.80

65.56

1.80

5

1.95

2.30

1.05

1.77

49.44

1.77

6

2.77

1.57

1.04

1.79

49.44

1.79

Mean

1.95

2.35

1.06

-

50.17

1.79

c.o.v. (%)

37.80

31.61

15.43

1.46

-

1.46

% inhibition

-

-

-

-

-

-

62.5

52.2

1

1.10

3.43

0.88

1.81

78.35

1.81

2

1.61

3.04

0.63

1.76

68.84

1.76

3

1.95

2.69

0.69

1.78

56.80

1.78

Mean

1.55

3.05

0.74

-

68.00

1.78

c.o.v. (%)

27.50

12.22

18.06

-

-

1.35

 

-

-

-

-

-

0

125

119

1

1.39

1.79

1.26

1.48

18.64

1.48

2

2.64

0.94

1.06

1.55

61.14

1.55

3

0.00

3.04

1.44

1.50

101.79

1.50

Mean

1.34

1.93

1.26

-

60.52

1.51

c.o.v. (%)

98.38

54.83

15.26

-

-

2.34

% inhibition

-

-

-

-

-

16

250

200

1

2.08

0.69

1.89

1.55

48.37

1.55

2

0.00

n/a

2.14

1.07

141.42

1.64

3

1.61

1.10

2.10

1.60

31.34

1.60

Mean

1.23

0.90

2.05

-

73.71

1.60

c.o.v. (%)

88.69

32.01

6.59

-

-

2.62

% inhibition

-

-

-

-

-

11

500

398

1

0.69

1.10

2.72

1.50

71.30

1.50

2

1.39

1.25

1.87

1.50

21.66

1.50

3

0.00

n/a

3.73

1.86

141.42

1.47

Mean

0.69

0.11

0.93

-

78.13

1.49

c.o.v. (%)

100.00

9.27

33.48

-

-

1.19

% inhibition

-

-

-

-

-

16

1000

808

1

1.10

0.00

2.93

1.34

110.18

1.34

2

0.00

n/a

3.50

1.75

141.42

1.17

3

0.00

0.00

n/a

0.00

n/a

1.27

Mean

0.37

0.00

3.21

-

125.80

1.26

c.o.v. (%)

173.21

n/a

12.55

-

-

7.04

% inhibition

-

-

-

-

-

30

Validity criteria fulfilled:
yes
Conclusions:
The 72 h ErC50 and NOEC (for growth rate) were estimated to be > 808.0 µg/L and 52.2 µg/L, respectively.
The 72 h EyC50 and NOEC (for yield) were estimated to be 156 µg/L and 119 µg/L, respectively.

Study satisfies guideline validity criteria.
Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriealla subcapitata were exposed to Bisphenol AF at mean measured concentrations of 52.2, 119, 200, 398 and 808 µg/L under static conditions in accordance with the OECD 201 Guideline.  The NOEC (for yield) and EyC50 values based on cell density were 119 and 156 (c.l.: 107 - 228) µg/L, respectively.  The NOEC (for growth rate) and ErC50 values based on cell density were 52.2 and >808 µg/L, respectively. The % growth inhibition in the treated algal culture as compared to the control ranged from 0 - 90 %.

This toxicity study is classified as acceptable and satisfies the guideline requirements for freshwater alga toxicity study.

Results Synopsis

Test Organism: Pseudokirchneriella subcapitata

Test Type: Static

72 h ErC50: > 808 µg/L

72 h EyC50: 156 (107 - 228) µg/L

72 h NOEC for growth rate: 52.2 µg/L

72 h NOEC for yield: 119 µg/L

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test:

Determine the effects of Bisphenol AF to freshwater green alga. Test organism was exposed to the test item over a range of concentrations for 72 h. Growth growth inhibition were quantified from measurements of the algal biomass over time.

- Short description of test conditions:

8 concnetrations tested in triplicate plus a control.
Test conducted under continuous illumination for 72 h at 7000 lux.
Alge growth was quantified by fluometric method using a fluoresence spectrometer at 450 and 680 nm.

- Parameters analysed / observed:

Cell growth used to determine EC20, EC50 and EC80.
GLP compliance:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not reported
- Stability under test conditions: not reported
- Solubility and stability of the test substance in the solvent/vehicle: not reported
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Stock solution prepared at 15 mg/L in ultrapure water by mixing for 24 h.
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: stock solution = 15 mg/L
- Final preparation of a solid: no

FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid

OTHER SPECIFICS: no
Analytical monitoring:
yes
Remarks:
Stock solution only
Details on sampling:
- Concentrations: 0.25, 0.50, 1.0, 2.0, 4.0, 5.0, 10.0 and 15.0 mg/L.
- Sampling method: Stock analysed prior to preparation of treatment solutions.
- Sample storage conditions before analysis: not reported
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 15 mg/L stock solution used to prepare treatment solutions at 0.25, 0.50, 1.0, 2.0, 4.0, 5.0, 10.0 and 15.0 mg/L. Treatment solutions prepared in algae growth medium.
- Eluate: Algae growth medium
- Differential loading: 10E4
- Controls: Yes, algae growth medium.
- 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):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subpicatus
- Strain: Chodat 1926
- Source (laboratory, culture collection): SAG 86.81; Collection of Algal Cultures, University of Göttingen, Germany
- Age of inoculum (at test initiation): Not reported
- Method of cultivation: In a temperature controlled room (21 ± 1 ºC) on an orbital shaker at 150 rpm, continuous light (4000 lux). Algae nutrient solution prepared according to Jaworski (Thompson et al., 1988).

ACCLIMATION
- Acclimation period: not reported
- Culturing media and conditions (same as test or not): not reported
- Any deformed or abnormal cells observed: not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
not reported
pH:
not reported
Dissolved oxygen:
not reported
Salinity:
not reported
Conductivity:
not reported
Nominal and measured concentrations:
Nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask (100 mL)
- Type (delete if not applicable): not reported
- Material, size, headspace, fill volume: 40 mL fill volume
- 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
- Initial cells density: 10E4
- Control end cells density: 10E4
- No. of organisms per vessel: n/a
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): n/a

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: Nutrient solution prepared according to Thompson et al., 1988).

TEST MEDIUM / WATER PARAMETERS
- 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
- Culture medium different from test medium: not reported
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: not reported
- Photoperiod: Continuous illumination
- Light intensity and quality: 7000 lux
- Salinity (for marine algae): n/a

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell growth/ density at 72 h
- Determination of cell concentrations:fluorecence spectrometer
- Chlorophyll measurement: no
- Other: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: none
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study: no
- Test concentrations: n/a
- Results used to determine the conditions for the definitive study: none
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
IC50
Effect conc.:
3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: IC20
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: IC80
Effect conc.:
6.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): not reported
- Observation of abnormalities (for algal test): not reported
- Unusual cell shape: not reported
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported
- Other: not reported
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: n/a
- Effect concentrations exceeding solubility of substance in test medium:
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
IC20 IC50 and IC 80 values calculated by non-linear regression model using GraphPad Prism (version 6.0, USA).

Table 1:       72 h Endpoints for Freshwater Algae, D. subspicatus after Treatment with Bisphenol AF

Statistical parameter

Endpoint

(mg/L)

95 % Confidence Intervals

IC20

1.3

-

IC50

3.0

1.9 – 4.6

IC80

6.5

-

Validity criteria fulfilled:
not specified
Conclusions:
The IC20, IC 50 and IC80 were estimated to be 1.3, 3.0 and 6.5 mg/L, respectively.

Executive summary:

The effect of Bisphenol AF on the freshwater green algae Desmodesmus subspicatus was tested at 8 nominal concentrations; 0.25, 0.50, 1.0, 2.0, 4.0, 5.0, 10.0 and 15.0 mg/L, in algae growth media. Treatement solutions were prepared from an initial 15 mg/L stock solution and innoculated with 10E4 cells/mL. Treatment vessels were continuosly agitated and illuminated for 72 h, after which time the growth of algae was quantified by fluorometric method using a fluorescence spectrometer. The growth rates of algae and percent inhibition for each treatment concentration were calculated.

The 72 h IC 20, IC50 and IC80 values for Bisphenol AF were 1.3, 3.0 (c.l.: 1.9 - 4.6) and 6.5 mg/L.

Description of key information

ErC50 = 3.0 mg/L, ISO 8692 (2012), Tišler et al., 2016

NOEC for growth rate = 52.2 µg/L, OECD 201 (2006), DuPont 2007

Key value for chemical safety assessment

EC50 for freshwater algae:
3 mg/L
EC10 or NOEC for freshwater algae:
0.052 mg/L

Additional information

There are 2 key studies on the growth inhibition of algae exposed to BPAF.  The studies were equivalent or similar to the Freshwater Alga and Cyanobacteria, Growth Inhibition Test (OECD 201, 2006), Algal Toxicity Test (OPPTS850.5400, 1996) and Fresh Water Algal Growth Inhibition Test With Unicellular Green Algae (ISO 8692, 2012).

In the study by Tisler et al., 2016, they sought to run a suite of aquatic ecotoxicity tests 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 onV. fischeri (bacteria), Desmodesmus subspicatus(green algae), Daphnia magna (invertebrate) and Danio rerio (fish; common name zebrafish).  

Specifically for the algae test the authors quote the use of the ISO 8692 (2012) guideline being followed (Fresh Water Algal Growth Inhibition Test With Unicellular Green Algae).  However, the test is also equivalent or similar to the OECD 201 (2006) and OPPTS850.5400 (1996) guidelines. For the husbandry Desmodesmus subspicatus Chodat 1926 (SAG 86.81; Collection of Algal Cultures, University of Gottingen, Germany) were maintained in a nutrient solution of Jaworski medium in temperature-controlled room (21 ± 1 °C) on an orbital shaker at 150 rpm, alternating 15 min agitation and 15 min resting under continuous fluorescent illumination (4000 lux).  Three replicate vessels (40 mL medium/ medium-test-item) containing 10^4 algae cells/mL each were used at each concentration and the dilution water control.  The concentrations tested were 0.25, 0.50, 1.0, 2.0, 4.0, 5.0, 10.0, 15.0 mg/L. After 72 h, the growth of algae was quantified by fluorometric method. Fluorescence of algae was measured in 10 mm cuvette with continuous mixing at excitation and emission wavelengths of 450 and 680 nm, respectively. The growth rates of algae and the percentages of inhibition in comparison to the control were calculated for each tested concentration.

Although there was no chemical analytics conducted during the test the authors also ran a chronic Daphnia magna 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.  After dosing, BPAF stayed at 90 % of nominal concentrations, therefore the use of nominal concentrations can be used in line with the guideline.  Both the algae and Daphnia medium are similar in complexity and it is expected that the concentrations in the algae test remained at 80-120 % of nominal. Furthermore, the analytical results showed that dosing technique was appropriate as well as the stability of the test item in the test system.  However, whether the study met the validity criteria of the guidelines is not documented.

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 ErC50 of BPAF was determined to be 3.0 mg/L (95 % confidence limits = 1.9-4.6 mg/L). This value is used for chemical safety assessment.

In 2002 a GLP guideline compliant study was conducted at the E.I. du Pont de Nemours and Company, Haskell Laboratory for Health and Environmental Sciences.  The study followed the OECD Freshwater Alga and Cyanobacteria, Growth Inhibition Test (OECD 201, 2006) guideline.    

Pseudokirchneriella subcapitata cultures were maintained under photoperiod, shaking speed, and temperature conditions similar to those used in the study. Illumination was maintained at 5005 ± 805 lux. The organisms were cultured in flasks containing approximately 50 mL of filter-sterilized AAP nutrient medium and were aseptically transferred to fresh medium every 3 to 7 days. The flasks were fitted with sterilized foam stoppers to permit gas exchange. The P. subcapitata culture used to inoculate test vessels was aseptically transferred to fresh medium 4 days prior to use.

The methodology of the test strictly followed the OECD 201 guidance.  The concentrations tested were 52.2, 119, 200, 398, 808, and 820 μg/L. A non-parametric analysis of the data was performed (Kruskal-Wallis test) and the Jonckheere-Terpstra test was used to determine the LOEC and NOEC values. It was not possible to determine the ErC50.

The water quality parameters for the duration of the test were as follows: temperature was 23.8 °C and the pH ranged from 7.65 to 8.47.   All water quality parameters were within acceptable limits during the exposure.  Light intensity remained between 6000 to 8000 lux. The test substance is well defined but no expiration date is noted.  The test organism used was a recommended organism as per all guidelines (i.e. Pseudokirchnerialla subcapitata).  All validity criteria were met as per the guideline. Analytics were conducted to confirm the concentration of the test material in the test system during the study period, and showed that BPAF remained above or equal 80 % of nominal. Therefore, test item was deemed stable and effects were determined based on nominal dosing.  

Although an ErC50 was not determinable, a NOEC for growth rate was.  The NOEC was determined to be 52.2 µg/L. This value is used for chemical safety assessment.