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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
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 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Concentrations of DAH were measured in the control and all test solutions at test initiation, before and after renewal on Day 2, and at test termination. The stock solution concentration was also measured at test initiation and before renewal on Day 2.
Details on test solutions:
The concentrations used in the test were chosen based on the results of a range-finding test conducted previously. The OECD Guideline states that the highest concentration in a definitive test should not exceed 1000 mg/L, however, the measured DAH concentration of the highest concentration tested in the range-finding test (nominal 1283 mg DAH/L) was 866 mg/L. Therefore, to compensate for lower than expected concentration of the hydrolysis product, a slightly higher (1283 mg DAH/L) concentration than the 1000 mg/L limit was tested to ensure that the concentration of hydrolysis product was within 20% of the 1000 mg/L limit.
Stock solutions were 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.
The pH of the lowest concentration (78 mg DAH/L) was 7.9 and was within the pH tolerance range (6.0 to 8.5) of the test organisms; therefore, it was not necessary to prepare any lower concentrations.
The test solutions were renewed on Day 2. Approximately 3 h prior to test solution renewal, 80 uL each of Chlorella, Selenastrum and YCT were added to each test vessel. The test solutions were prepared as outlined above for Day 0, using the previously prepared stock solution and the previously used glassware.
Test organisms (species):
Daphnia magna
Details on test organisms:
The Daphnia cultures were originally obtained from Pacific Environmental Science Centre, North Vancouver, Canada and then maintained in an in-house culture. Prior to the test, the Daphnia cultures were fed 2.8 mL each of green algae (Chlorella sp. and Selenastrum capricornutum) and yeast-cereal-grass fermented-trout-chow (YCT) three times per week. The neonates used in the test were fed 6 mL of concentrated Chlorella sp. and 6 mL of concentrated S. capricornutum on the day of test initiation. The algae cultures were obtained from the University of Toronto Culture Collection, Toronto, ON. The yeast, Fleischmann’s Traditional Dry Active Yeast, was obtained from Fleishmann’s Yeast, LaSalle, QC; the cereal grass, Ward’s Cereal Grass Media, was obtained from Ward’s Natural Science, St. Catherine’s, ON, Canada; and trout chow, Nutra-Plus 0 Crumble, was obtained from Skretting, Vancouver, BC, Canada.
Test type:
semi-static
Total exposure duration:
96 h
Hardness:
95 mg CaCO3/L (Test Initiation)
98 mg CaCO3/L (Renewal Day 2)
Test temperature:
19.3 - 20.9 °C
pH:
8.0 (Test Initiation)
7.8 (Renewal Day 2)
Dissolved oxygen:
8.6 to 9.2 mg/L (96 to 101% saturation)
Details on test conditions:
To reduce adsorption losses, all glassware used in this study was pre-conditioned with the appropriate test solution prior to use.
The concentrations used in the test were chosen based on the results of a range-finding test conducted previously.
Stock solutions were 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.
The pH of the lowest concentration (78 mg DAH/L) was 7.9 and was within the pH tolerance range (6.0 to 8.5) of the test organisms; therefore, it was not necessary to prepare any lower concentrations.
The test solutions were renewed on Day 2. Approximately 3 h prior to test solution renewal, 80 µL each of Chlorella, Selenastrum and YCT were added to each test vessel. The test solutions were prepared as outlined above for Day 0, using the previously prepared stock solution and the previously used glassware. The Daphnia were temporarily transferred from the test solutions into glass vials, the test solutions were replaced and the Daphnia were transferred into the freshly prepared test solutions using a glass, wide-bore pipette.
Conductivity was measured at test initiation and in the freshly prepared solutions on Day 2. The temperature, pH, and dissolved oxygen (DO) concentrations were measured in the test solutions at test initiation, before and after renewal on Day 2, and at test termination. Initial measurements were taken from a separate test vessel to avoid contamination of the test solutions. This test vessel was filled with the same solutions and treated as the other test vessels. Interim and final measurements were taken in each replicate.
General observations on behaviour, such as immobilisation, were made and recorded at test initiation and at 24-, 48-, 72- and 96-h of exposure. Immobilisation was defined as the inability to swim during a period of 15 sec following gentle agitation of the test solution. The test was terminated at 96-h.
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.:
> 1 248 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1 248 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 1 248 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The 312 and 1248 mg/L test solutions were analysed in duplicate and the relative difference between the two analyses were 1 and 0%, respectively. The average observed/nominal DAH concentration ratio in the matrix spiked samples was 101 ± 0 %. Measured DNA concentrations were 97 – 101%, 96 – 101%, 99 – 103% and 99 – 105% of the adjusted nominal DAH concentrations at test initiation, on Day 2 (before), on Day 2 (after) and at test termination, respectively. Mean measured DAH concentrations were 98 – 102% of adjusted nominal DAH concentrations. The 48-h LC50 and 95% confidence intervals for the reference substance test were within the acceptable range (± 2 SD) of previous tests conducted in this laboratory. These results indicate that the test organisms were responding normally to the reference substance. The toxicity test met the test validity criteria, because there were no immobilised organisms in the controls, and because the dissolved oxygen concentrations at the test termination were > 60% saturation. The water quality measurements (e.g. dissolved oxygen, pH) were also within the tolerance limits of the test organism. After the 96-h exposure, there was one Daphnia (5%) immobilised at 624 mg/L and 7 Daphnia (35%) immobilised at 1248 mg DAH/L.
Results with reference substance (positive control):
The 48-h LC50 and 95% confidence intervals for the reference substance test were within the acceptable range (± 2 SD) of previous tests conducted in this laboratory. These results indicate that the test organisms were responding normally to the reference substance.

Adjusted Nominal DAH Concentration

mg/L

Test Initiationa 

mg/L

Day 2 Beforea 

mg/L

Day 2 Aftera 

mg/L

Test Terminationa 

mg/L

Meana 

mg/L

Control

Less than DLb

Less than DLb

Less than DLb

Less than DLb

0

Solvent Control

Less than DLb

Less than DLb

Less than DLb

Less than DLb

0

78

75.3 (97%)

74.5 (96%)

78.4 (100%)

77.8 (100%)

76.5 (98%)

156

155 (99%)

153 (98%)

155 (99%)

154 (99%)

154 (99%)

312

316 (101%)c

314 (101%)

312 (100%)

320 (103%)

316 (101%)

624

606 (97%)

608 (97%)

624 (100%)

628 (101%)

617 (99%)

1248

1251 (100%)

1250 (100%)

1271 (102%)d

1309 (105%)

1270 (102%)

10,403 (Stock)

10,405 (100%)

N/A

10,739 (103%)

N/A

10,572 (102%)

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: 314 and 318 mg/L.

d Mean of duplicate analysis: 1271 and 1270 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 96-h EC50 estimate was >1248 mg/L, based on adjusted nominal concentrations. The NOEC and LOEC were 1248 and >1248 mg/L, respectively.
Executive summary:

The acute toxicity of the read-across test material to Daphnia magna was investigated in a study conducted using methodology equivalent to OECD 202 under GLP conditions (Vizon SciTec Inc., 2005).

Daphnids were exposed to adjusted nominal concentrations of 78, 156, 312, 624 and 1248 mg/L under semi-static conditions for 96 hours.

After the 96-h exposure, there was one Daphnia (5 %) immobilised at 624 mg/L and 7 Daphnia (35 %) immobilised at 1248 mg/L. Therefore, the 96-h EC50 estimate was >1248 mg/L, based on adjusted nominal concentrations. The NOEC and LOEC were 1248 and >1248 mg/L, respectively.

Description of key information

The 96 h EC50 and NOEC in Daphnia magna were >1248 and 1248 mg/L, respectively.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1 248 mg/L

Additional information

The acute toxicity of the read-across test material to Daphnia magna was investigated in a study conducted using methodology equivalent to OECD 202 under GLP conditions (Vizon SciTec Inc., 2005). The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Daphnids were exposed to adjusted nominal concentrations of 78, 156, 312, 624 and 1248 mg/L under semi-static conditions for 96 hours.

After the 96-h exposure, there was one Daphnia (5 %) immobilised at 624 mg/L and 7 Daphnia (35 %) immobilised at 1248 mg/L. Therefore, the 96-h EC50 estimate was >1248 mg/L, based on adjusted nominal concentrations. The NOEC and LOEC were 1248 and >1248 mg/L, respectively.