Bisphenol A: Acute Aquatic Toxicity

Bisphenol A: Acute Aquatic Toxicity
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  Environmenlal Toxicology and Cheinisiry, \’ol I p. 19-26, 1988 Printed in the US4. ergamon Journals Ltd. 0730 7268 88 $3 00 + 00 Copkright C 1988 SET.\C Environmental Toxicology BISPHENOL A: ACUTE AQUATIC TOXICITY HOWARD . ALEXANDER* nd DENNIS . DILL The Dou Chemical Companq, Midland, Michigan 48674 LADD W. SMITH PATRICK . GUINEY PHILIP ORN General Electric Companq, Pittsfield, Massachusetts 01201 U S Steel Chemicals, Pittsburgh, Pennsqhania 15230 Shell Development Companq , Westhollow Research Center, Houston, Texas 7725 1 (Receited 10 March 1987, Accepted 7 July 1987) Abstract-The Society of the Plastics Industry, Inc., ad hoc Bisphenol A Task Group determined that freshwater and saltwater environmental effects testing on bisphenol A should be conducted. This decision was based upon the nation’s high production capacity for bisphenol A, which is man- ufactured at many sites, its potential for entering the environment in substantial quantities and the general lack of relevant ecological effects data. The freshnater test results were as follows: the 96-h EC50 algal toxicity to Selenustrum capricornutum was 2.7 mg/L based on cell count, and 3.1 mg/L based on cell volume; the 48-h EC50 to the invertebrate Daphnia magna \+as 10 (9.2-11) rng/L; and the 96-h LCjOs to the fathead minnow, Pimephalesprornelas, was 4.7 (4.0-5.5) mg/L in a static test and 4.6 (3.6 to 5.4) mg/L in a flow-through test. The saltwater test results were as follows: the 96-h EC50 algal toxicity to the diatom Skeletonemu costatum was 1 O mg/L based on cell count and relative fluorescence, and 1.8 mg/L based on chlorophyll a content; the 96-h LCSO to the mpsid Mysidopsis bahia was 1.1 (0.92 to 1.2) mg/L; and the 96-h LC50 to the Atlantic silverside, Menidia menidia, was 9.4 (8.3 to 11) mg/L. According to current U.S. Environmental Protection Agency standard evaluation procedures, bisphenol A was moderately to slightly toxic to the fish and invertebrates tested, with LC50 or EC50 values of from 1.1 to 10 mg/L. These data did not trigger freshwater or saltwater chronic tests. The acute toxicity data together with the fact that bisphenol A rapidly biodegrades in surface waters indicate a low potential for chronic exposure or toxicity. Keywords-Bisphenol A Acute toxicity INTRODUCTION Bisphenol A is used in many plastics applica- tions, for example, plastic pipe, epoxy resins and coatings. There exists a high production capacity for the compound, estimated at 420,000 metric tons (930 million pounds), and it is manufactured at many sites. Freshwater and saltwater environ- mental effects testing were conducted because the use of bisphenol A may result in its entering the environment directly, because there is a general lack of relevant bisphenol A ecological effects data and because existing aquatic toxicity tests have not been supported by analytically verified test concen- trations. Scientific representatives of producers of bisphenol A through the Society of the Plastics Industry, Inc. (SPI), formed an ad hoc Bisphenol *To whom correspondence may be addressed. 19 Toxic substances test rule A Task Group. The task group reviewed the chem- ical fate, health effects and ecological effects data available for bisphenol A with U.S. nvironmen- tal Protection Agency (EPA) personnel, and a study plan and protocols for two tiers of fresh- water and saltwater tests were developed. The first tier of tests would consist of a series of acute tests conducted using analyzed test expo- sure concentrations. The second tier would consist of a chronic toxicity test and would be triggered, according to the EPA, if (a) the 96-h acute LC50 value for any vertebrate or invertebrate test species was equal to or less than 1.0 ppm, or (b) there were indications of “chronicity” (i.e., the ratio of the 48-h LC50 to the 96-h LC50 was equal to or greater than 2) in the acute test. The chronic toxi- city test would be conducted with the most sensitive vertebrate or invertebrate species (i.e., the one with the lowest acute LC50 value, or, in the absence of  20 H. c ALEXANDER T AL. an LC50 lower than 1 ppm, the test species that showed the greatest tendency for chronicity). The following tests were conducted with bisphenol A: freshwater acute tests with the alga Selenastrum capricornutum Printz, the invertebrate Daphnia magna Straus and the fathead minnow Pimephales promelas Rafinesque; and saltwater acute tests with the diatom Skeletonema costatum (Greville) Clevel, the invertebrate Mysidopsis bahia Molenock, and the Atlantic silverside Menidia menidia (Linnaeus). MATERIALS AND METHODS Test material The test material used in all of the studies was polycarbonate-grade bisphenol A, obtained from The Dow Chemical Company, Texas Division, (Freeport, TX) and identified as Lot No. TB 84071221. Molar purity, determined by differential scanning calorimetry, was 99.93% (Dow Chemical Company, unpublished data). The compound’s solubility in water is less than 0.1 at 25°C and 0.34% at 83°C [l]. Stability: Analytical method The stability of bisphenol A in the test systems was determined by analysis of the test concentra- tions during each of the tests. A reversed-phase liquid chromatographic method was used to mea- sure bisphenol A in water. The samples were fil- tered through a 0.45-pm membrane filter before being injected into the liquid chromatographic sys- tem. Quantitation was by comparison of the peak areas of bisphenol A on the sample chromato- grams with the peak areas on the standard chro- matograms using a Hewlett-Packard integrator. Fresh water tests The algal toxicity test with S. capricornutum followed procedures recommended by the EPA [2]. The daphnid static acute, fish static acute and flow-through acute toxicity tests were based on the procedures recommended by the American Society for Testing and Materials (ASTM) [3]. The test specifications for the freshwater acute tests are given in Table 1. Saltwater tests The algal toxicity tests with S. costatum fol- lowed by the EPA procedures outlined in both the Ocean Disposal Permit Program and the Algal Acute Toxicity Test (EG-8) [4,5]. The flow-through acute test with the mysid and the Atlantic silver- side followed ASTM procedures [3]. The test spec- ifications for the saltwater acute test are given in Table 2. Statistical analysis The mean analyzed exposure concentrations were used to calculate all toxicity values. In the algal tests, percent inhibition values and the log,,, of the test concentrations were used to estimate the ECSO values using nonlinear interpolation. Dun- nett’s t test [6] was used to compare treatment means with the control means in the freshwater algal test; William’s test [7] was used in the salt- water algal test. Table 1. Specifications for freshwater acute toxicity tests with algae, daphnids and fish Alga Daphnid Fathead minnow Species Selenastrum capricornutum Daphnia mugnu Pnnephales promelas Length of test 96 h 48 h 96 h Source of organisms Lab culture Lab reared Lab reared Dilution water Algal assay medium Lake Huron water Lake Huron water (softened to -85 mg/L hardness as CaCO,, pH 7.9-8.1) Temperature 24 t 2°C 20 * 1°C 17 t 1°C Photoperiod 16 h light: 8 h dark Test vessel size 125-ml Erlenmeyer flasks 250 ml Pyrex beakers 12 liters for static; Test volume 50 ml 200 ml 10 liters for static; >5 volume Test chamber Environmental chamber Environmental chamber Constant-temperature Daily observations Cell count, cell volume Dissolved oxygen, pH, Dissolved oxygen, pH, Effect criteria Cell counts, total cell volume Mortality/sublethal effects Mortality/sublethal effects (-170 mg/L hardness as CaCO,, pH 8.1) 16 h light: 8 h dark 4 h light Illumination 4,300 k 430 lux 2,000 300 lux -400 IUX 7 liters for flow-through changes/d for flow-through waterbath temperature temperature  Acute toxicity of bisphenol A 21 Table 2. Suecifications for saltwater acute toxicity tests with algae, mysid, and fish Alga (diatom) Mysid Atlantic silverside Species Length of test Type of test Source of organisms Dilution water Temperature Photoperiod Illumination Salinity Test vessel size Test volume Test chambei DailS obserxations Effect criteria Skeletonerna costatum 96 h Static Lab culture Saltwater algal assay medium 20 i 2°C 14 h light: 10 h dark -4,500 lux -2O%l 125 ml 50 m Environmental chamber In iivo chlorophyll a fluorescence Gromth (cell count and chlorophyll a fluorescence at 96 h) Mysidopsis bahia 96 h Flow-through Lab culture Seawater (Cape Cod Canal; pH 7.5-8.1) 25 °C 16 h light: 8 h dark 300-1,000 UX - 0%m 19.5 liters 11.0 liters; >I4 volume changesld Constant-temperature waterbath Dissolved oxygen, pH, temperature Mortality/sublethal effects Menidia menidia 96 h Flow-through Commercial supplier Seawater (Cape Cod Canal; pH 7.9-8.2) 22 °C 16 h light: 8 h dark 300-1,000 lux - 0 0 19.5 liters 11 O liters; >6.7 volume changesld Constant-temperature uaterbath Dissolved oxygen, pH, temperature Mortality/sublethal effects The fish and invertebrate EC50 or LC50 values and their 95% confidence intervals were calculated using a computer program that analyzed the con- centration-effect data with three statistical meth- ods (C. Stephan, personal communication): the binomial method [8], Thompson’s moving average [9-111; and probit analysis [12]. RESULTS Fresh water tests The 96-h acute algal test results for S. cupricor- nutum are presented in Table 3. There was no sig- nificant inhibition of algal growth at 0.69 and 1.2 mg/L bisphenol A. At 2.0 mg/L and higher, bisphenol A significantly inhibited algal growth. The 96-4 EC50 for cell count and total cell volume were 2.7 and 3.1 mg/L, respectively. The D. magna 24-h and 48-h acute EC50 val- ues (and 95% confidence intervals) were 16 (14-17) and 10 (9.2-11) mg/L bisphenol A respectively (Table 4). The acute freshwater toxicity to the fathead minnow was determined with both a static and a flow-through acute test to demonstrate the appli- cability of static acute data in testing a material that is stable in the test system. The static acute Table 3. Test results for the alga Selenastrum capricornurum Printz exposed to bisphenol A for 96 h Bisphenol A concentration (mg/L) Analyzed Cell count Total cell Lolume Nominal (mean - SD) cells/mL (x 10‘) Percent inhibition” bm‘/mL (x 10‘) Percent inhibition” Control NDh 0.78 0.69 .02 1.30 1.17 i 0.10 2.16 1.99 i 0.16 3.60 3.41 + 0.13 6.00 5.86 i 0.11 10.00 9.74 0.06 EC50 (mg/L)d 287 0 131 0 302 5 147 - 12 268 7 128 2 220 23 107 18’ 89 69’ 56 57’ 17 7 94‘ 18 2 86‘ 2 34 99‘ 2 29 98’ 2.73 (cell count) 3 10 (cell bolume) “Decrease of cell count or total cell volume as compared aith controls after 96 h. bNot detected at 0.05 mg/L. ‘p > 0.05, one-tailed Dunnett t test on transformed data. ‘EC50 calculated b nonlinear Interpolation.  22 H. C. ALEXANDER T AL. Table 4. Static acute test results for daphnids exposed to bisphenol A for 48 h Bisphenol A concentration (mg/L) Percent immobile (number immobile) Analyzed Organisms Nominal (mean t D exposed 24 h 48 h 20.0 12.0 7.20 4.32 2.60 1.55 0.93 Control EC50 (mg/L)d 95 C.1. (mg/L) 19.34 i 0.04 11.66 t 0.08 6.97 t 0.10 4.1 1 t 0.03 2.49 i 0.04 1.46 t 0.01 0.90 t 0.04 ND' 30 30 30 30 30 30 30 30 "Two daphnids distressed on surface of test vessel. 'Not detected at 0.05 mg/L. dEC50 and 95 confidence interval determined by Thompson's moving average. daphnid distressed on surface of test vessel. LC50 value (and 95 confidence interval) of 4.7 (4.0-5.5) mg/L bisphenol A did not change after 24 h (Table 5). The final test concentrations, when compared with the initial test concentrations, aver- aged 103 and ranged from 78 t 119 , which indicates that bisphenol A was stable in the static acute test system. In the flow-through acute test, the LC50 (and 95 confidence interval) at 24 h was 4.7 (3.6-5.4) mg/L; the value was 4.6 (3.6- 5.4) mg/L after 48 h (Table 6) and did not change thereafter. Concentrations of bisphenol A were analyzed at 0 and 96 h in the static acute test and the mean values are given in Table 5 In the flow-through acute test, the exposure vessels were analyzed daily and the mean values are given in Table 6. Saltwater tests The 96-h acute algal test results with S. costa- rum are given in Table 7. After 96 h, little growth was detected (96 inhibition or greater) at concen- trations of 2.8 mg/L bisphenol A and greater. The Table 5. Static acute test results for fathead minnows exposed to bisphenol A for 96 h Bisphenol A concentration (mg/L) Percent dead (number dead) Analyzed Fish Nominal (mean SD) exposed 24 h 48 h 72 h 96 h 13.3 8.20 5.6 3.64 2.37 1.54 1 oo Control 14.54 t 1.80 8.54 t 0.64 5.51 rir 0.17 3.97 t 0.40 2.26 t 0.05 1.43 t 0.07 0.99 t 0.17 ND' LC50 (mg/L)d 95 C.I. (mg/L)' 10 10 10 10 10 10 10 1 4.7 (4.0-5.5) 100 (10) 100 (10) 100 (10) 0 (0) 0 (0) 0 0) 4.7 (4.0-5.5) 0 ~~~ "All fish were lethargic and melanized. bMost fish appeared recovered; one fish showed loss of equilibrium and was lethargic and melanized. 'Not detected at 0.05 mg/L. dLC50 obtained by nonlinear interpolation. '95 confidence interval obtained by the binomial method.  Acute toxicity of bisphenol A 23 Table 6. Flow-through acute test results for fathead minnows exposed to bisphenol A for 96 h ~~ ~~ ~ Bisphenol A concentration (mg/L) Analyzed Fish Nominal (mean * SD exposed Percent dead (number dead) 24 h 48 h 72 h 96 h 8.65 8.66 t 3.5 20 5.65 5.41 0.22 20 3.65 3.58 0.36 20 2.37 2.28 0.11 20 I .54 1.52 k 0.09 20 1 oo 0.79 k 0.08 20 Control ND' 20 LCSO (mg/L)d 4.7 4.6 4.6 4.6 95% C.I. (mg/L)' (3.6-5.4) (3.6-5.4) (3.6-5.4) (3.6-5.4) "Surviving fish at 24 h showed loss of equilibrium; one surviving fish appeared recovered at 96 h. bFish showed a loss of equilibrium: 9 fish at 24 h, 4 fish at 48 h, 2 fish at 72 h; all fish appeared normal at 96 h. 'Not detected at 0.05 mg/L. dLC50 obtained by nonlinear interpolation. '95% confidence interval obtained by the binomial method. Table 7. Test results for the alga Skeletonemu costarum exposed to bisphenol A for 96 h Bisphenol A concentration (mg/L) Cell count Chlorophyll a Analyzed Percent Fluorescence Percent Nominal (mean t SD) Cells/ml (x in hi bi t ion" unitsh inhibition" 15 9.0 5.4 3.2 1.9 1.1 0.72 Control EC5O (mg/L)' 15 i 1.0 8.8 i 0.6 5.2 k 0.5 2.8 0.3 1.6 i 0.1 1.1 i 0.0 0.66 t 0.04 NDd 1 O (cell count) 100 100 97' 96' 51' 54' 27' 0 (0) 0 0) 8 (4) 22 (7) 650 (10) 687 (30) 853 (160) 993 (63) 100' 100' 99' 98L 34' 31' 14' - 1.8 (chlorophyll a ~ ~ ~~ ~~ ~~ ~~~~~~~ ~~~~~~~~ ~~ ~~ ~~~ "Decrease of cells/ml or relative fluorescence as compared with control after 96 h. hStandard deviation in parentheses. 'Significantly reduced; Williams teFt. "Not detected at 0.03 mg/L. 'EC50 calculated by nonlinear interpolation. 96-h EC50 values based on cell count and chlo- rophyll u measurements were 1.0 and 1.8 mg/L, respectively. The 96-h flow-through acute LC50 to the mysid M. bahiu was 1.1 (0.92-1.2) mg/L. The no observ- able effect concentration was 0.51 mg/L, based on only one death during the test. There was also one death observed in the control concentration. Ta- ble 8 summarizes the 24- to 96-h acute toxicity test results. The 96-h flow-through acute LC50 to the At- lantic si verside, M. menidiu, was 9.4 (8.3-1 1) mg/L bisphenol A. The test results are summa- rized in Table 9. The no observable effect concen- tration was estimated to be 5.9 mg/L. DISCUSSION Freshwater tests In an earlier test with S. cupricornutum con- ducted using nominal concentrations, an EC50 of 2.5 mg/L bisphenol A was reported [13]. The tox-
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