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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
The study was performed on the primary hydrolysis product of BPA-DA, 4,4-Bisphenol A Tetra-Acid (BPA-TA; CAS 38103-05-8).
Reason / purpose:
other: Read across target
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
extended time period
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Concentrations of DAH were measured in the controls and all test solutions at test initiation and termination. The stock solution concentration was also measured at test initiation.
Details on test solutions:
The nominal DAH concentrations used in this test were 770, 1540, 3079, 6415, and 12830 mg/L. The dilution factor between the concentrations was 2.0, except between 2880 and 6000 mg/L, where the dilution factor was 2.1. These concentrations bracketed the approximate 72 h EC50 value estimated from the Range-Finding Test.
The stock solution was prepared with BPA-DA on the day prior to test initiation to ensure complete hydrolysis to DAH occurred. After preparation, the stock solution was stored in the dark at 4 ± 2 °C. Dilution water with a concentration of 0.1 mL/L ACN was prepared a day prior to initiation for use in the test solution preparation.
On the day of test initiation, a total of 500 mL of each test concentration was prepared by adding an aliquot of the appropriate DAH stock solution to the dilution water containing 0.1 mL/L ACN. The test vessels were pre-conditioned with each test solution. Each test solution was thoroughly mixed before dispensing a 50 mL aliquot into each of the test vessels.
Two sets of negative controls, one with only dilution and one with 0.1 mL/L ACN and dilution water, were prepared. The controls were treated the same as the test solutions, as outlined above.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
S. capricornutum, strain UTCC 37, was obtained from the Department of Botany Culture Collection, the University of Toronto, Ontario, Canada as a sterile liquid starter culture and a sterile algal slant (Organism Lot #UT040225). These starter cultures were used to initiate new cultures of Selenastrum. The algae was cultured in 250-mL glass Erlenmeyer flasks, using sterile nutrient media (SNM) and according to the procedures in the Vizon SOPs “Selenastrum capricornutum 72-h Growth Inhibition Test” and “OECD Alga, Growth Inhibition Test.”
Test type:
static
Water media type:
freshwater
Total exposure duration:
96 h
Test temperature:
23 ± 2 °C
pH:
6.9 - 7.7
Nominal and measured concentrations:
Nominal: 770, 1540, 3079, 6415 and 12830 mg DAH/L
Adjusted: 767, 1533, 3066, 6388 and 12777 mg DAH/L
Details on test conditions:
The temperature of the test chamber was monitored daily using a min/max thermometer. The pH of the test solutions was determined at test initiation and test termination and conductivity was measured at test initiation.
At 24-, 48-, 72- and 96-h, a minimum of four cell counts were performed on aliquots (10 µL) from each flask, using a Bright-Line haemocytometer (Hausser Scientific, Horsham, PA). An average of four counts (10 µL volume) was multiplied by 10^4 to estimate the cell concentration in each replicate (cells/mL). Additional counts were made in some of the test concentrations when the four counts varied excessively. On the haemocytometer, 25 squares were counted. In all counts, cells touching the outer top or left border were not counted, whereas, cells touching the outer bottom or right border were counted. The average of these counts was used as the cell yield for that flask.
The mean cell concentration was plotted against time for each test concentration, including the controls to examine the effect of DAH on the growth curves. The measured number of cells/mL at time was calculated by taking the average of the counts from each replicate flask during each counting period. The area under the growth curves (AUGC) for exponentially growing cultures was determined. The concentration-response relationship was examined by plotting the AUGC and mean AUGC values vs. mean measured concentration of DAH. This plot was used to select an appropriate model for non-linear regression analysis to fit the experimental data and determine the 96-h EC50. This plot was also used to estimate initial values for model parameters.
The non-linear regression function was used to estimate the 96-h EC50 and 95% confidence limits. The no-observed-adverse-effect-concentration, NOAEC, and the lowest-observed-adverse-effect concentration, LOAEC, were determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test performed using SAS JMP 4.0.2 (SAS Institute 2000). The mean measured DAH concentrations were used to calculate the test endpoints, as the measured concentrations were not within 80 to 120% of the adjusted nominal concentrations.
For the results of a growth inhibition test to be acceptable and the test to be considered valid, the following conditions must be satisfied: the number of alga cells in the standard controls must have increased by a factor of at least 16 in 72 h; and the disappearance of the test substance from the water into the algal biomass does not necessarily invalidate the test.
A test with a concurrent reference substance test was not performed, however, a reference substance test, with zinc sulphate heptahydrate, was conducted as a separate study to assess the relative sensitivity of the test organisms and the precision of data produced by the laboratory.
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
1 713 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: hydrolysis product
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
other: NOAEC
Effect conc.:
< 877 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: hydrolysis product
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
other: LOAEC
Effect conc.:
877 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: hydrolysis product
Basis for effect:
growth rate
Details on results:
The 1,533 and 6,388 mg DAH/L test solutions were analysed in duplicate and the relative difference between the two analyses were 0.5 and 2%, respectively. The average observed/nominal DAH concentration ratio in the matrix spiked samples was 101 ± 0 %. Measured DAH concentrations were 97 to 103% of adjusted nominal DAH concentrations at test initiation and 109 to 131% of nominal DAH concentrations at test termination. The measured concentrations were higher at test termination than at test initiation. Mean measured DAH concentrations were 105 to 115% of nominal DAH concentrations.
The cell counts in each 24-h period during the test and the mean AUGC data are summarised in the following table. The concentration-response data was examined by plotting the AUGC versus concentration based on mean measured DAH concentrations. The mean measured DAH concentrations were used for the test endpoint calculations because the measured concentrations were not within 80 to 120% of nominal concentrations.
The toxicity test met the test validity, as the number of algae cells in the dilution water controls increased by a factor of 151 in 72 h. The pH values were within 1 pH unit at test initiation and termination and were within of the tolerance limits (6.5 to 8.5) of the test organisms. There was a slight hormetic effect (stimulation) on algal growth observed in the solvent control, as compared to the dilution water control. The 96-h EC50 and 95% confidence limits, based on mean measured DAH concentrations, were 1713 mg DAH/L (1420 – 2086). The NOAEC was <877 mg DAH/L and the LOAEC was 877 mg DAH/L.
Results with reference substance (positive control):
The separate study conducted with zinc sulphate heptahydrate indicated that the test organisms responded normally to the reference substance.

Measured DAH Concentrations in Test Solutions (mg/L)

Adjusted Nominal DAH Concentration

Test Initiationa

Test Terminationa

Meana

Control

Less than DLb

Less than DLb

0

Solvent Control

Less than DLb

Less than DLb

0

767

752 (98%)

1001 (131%)

877 (114%)

1533

1546 (101%)

1665 (109%)c

1606 (105%)

3066

3155 (103%)

3421 (112%)

3288 (107%)

6388

6367 (100%)

8310 (130%)d

7339 (115%)

12777

12,846 (101%)

14808 (116%)

13827 (108%)

25553 (Stock)

24,700 (97%)

N/A

N/A

a Measured concentrations as a percentage of the adjusted nominal concentrations are listed in parentheses.

b The detection limit was 3 mg/L.

c Mean of duplicate analysis: 1673 and 1657 mg/L.

d Mean of duplicate analysis: 8452 and 8168 mg/L.

Adjusted Nominal DAH Concentration (mg/L)

Mean Measured DAH Concentration (mg/L)

Average Cell Counts (x104/mL)

Mean Area Under the Growth Curve

0-h

24-h

48-h

72-h

96-h

Control

0

0.97

3.5

18.0

149.0

179.8

6168

Solvent Control

0

0.97

3.9

18.8

154.8

193.1

6492

767

877

0.97

3.2

15.4

123.4

205.3

5790

1533

1606

0.97

3.1

15.3

85.9

108.9

3729

3066

3288

0.97

1.7

5.8

17.5

31.8

897

6388

7339

0.97

0.8

1.9

0.8

1.6

21

12777

13827

0.97

1.2

0.5

0.7

0.8

-17

Validity criteria fulfilled:
yes
Conclusions:
The 96-h EC50 and 95 % confidence limits, based on mean measured DAH concentrations, were 1713 mg DAH/L (1420 – 2086). The NOAEC was < 877 mg DAH/L and the LOAEC was 877 mg DAH/L.
Executive summary:

The acute toxicity of the read-across test material to algae was investigated in a study conducted using methodology equivalent to OECD 201 under GLP conditions (Vizon SciTec Inc., 2003).

Selenastrum capricornutum were exposed to adjusted nominal concentrations of 767, 1533, 3066, 6388 and 12777 mg/L under static conditions for 96 hours.

The 96 h EC50, based on mean measured concentrations, was 1713 mg/L (95 % confidence limits 1420 – 2086 mg/L). The NOAEC was < 877 mg/L and the LOAEC was 877 mg/L.

Description of key information

The 96 h EC50 and NOAEC in Selenastrum capricornutum were 1713 and < 877 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
1 713 mg/L
EC10 or NOEC for freshwater algae:
877 mg/L

Additional information

The acute toxicity of the read-across test material to algae was investigated in a study conducted using methodology equivalent to OECD 201 under GLP conditions (Vizon SciTec Inc., 2003). The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Selenastrum capricornutum were exposed to adjusted nominal concentrations of 767, 1533, 3066, 6388 and 12777 mg/L under static conditions for 96 hours.

The 96 h EC50, based on mean measured concentrations, was 1713 mg/L (95 % confidence limits 1420 – 2086 mg/L). The NOAEC was <877 mg/L and the LOAEC was 877 mg/L.