Acute Toxicity, Mutagenicity, and Estrogenicity
of Biodegradation Products of Bisphenol-A
Michihiko Ike,1 Min-Yu Chen,1 Chang-Suk Jin,2 Masanori Fujita1
1Department of Environmental Engineering, Graduate School of Engineering, Osaka
University, 2-1, Yamada-Oka, Suita, Osaka 565-0871 Japan
2Kyonggi Environmental Technology Center, Myongji University, San 38-2 Nam-dong,
Yongin-city, Kyonggi-do 449-728 Korea
Received 20 December 2001; revised 5 April 2002; accepted 1 June 2002
ABSTRACT: Biodegradation of bisphenol-A (BPA), which is known as an estrogenic chemical, proceeds
via complicated metabolic routes and leads to formation of several kinds of biodegradation products.
Through the major route BPA can be completely mineralized; however, p-hydroxyacetophenone (p-HAP),
p-hydroxybenzaldehyde (p-HBAL), and p-hydroxybenzoic acid (p-HBA) are transiently accumulated at
relatively high concentrations. On the other hand, degradation of BPA through the minor route tends to
cause the accumulation of 2,3-bis(4-hydroxyphenyl)-1,2-propanediol and p-hydroxyphenacyl alcohol as
the dead-end products. To fully assess the impact of BPA discharge into the environment, the consid-
erable BPA degradation products p-HAP, p-HBAL, and p-HBA and the mixture of the dead-end products
were examined for their acute toxicity, mutagenicity, and estrogenicity using the Daphtoxkit (Creasel Ltd.),
umu test system, and yeast two-hybrid system, respectively. BPA was moderately toxic to Daphnia
magna (48-h EC50 was 10 mg/L) and weakly estrogenic, with activity that was 5 orders of magnitude lower
than that of 17
-estradiol in the yeast screen, though no mutagenicity was observed. All the tested BPA
biodegradation products showed very low acute toxicity compared with BPA, and none was mutagenic.
A slight estrogenic activity was detected only for p-HAP among the tested degradation products. It was
concluded that biodegradation can remarkably reduce the toxic effects of BPA. © 2002 Wiley Periodicals,
Inc. Environ Toxicol 17: 457–461, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.
10079
Keywords: bisphenol-A; biodegradation products; acute toxicity; mutagenicity; estrogenicity
INTRODUCTION
Bisphenol-A [BPA; 2,2-bis(4-hydroxyphenyl)propane] is an
industrially important chemical that is abundantly used as a
primary raw material for the production of plastics and resins.
The annual production of BPA in the world exceeded 930
million pounds in the beginning of 1990s (Lobos et al., 1992),
and the amount is increasing. According to EPA standard
evaluation procedures, BPA was determined to be moderately
to slightly toxic to fish and invertebrates (Alexander et al.,
1988). BPA is not recognized as a mutagen (Schweikl et al.,
1998); however, it has been recently identified as a weakly
estrogenic chemical that modifies natural endocrine functions
by binding to the estrogen receptor, and it causes adverse
effects on human health and wildlife (Colborn et al., 1996).
Because BPA is commonly found in all kinds of aquatic and
terrestrial environments because of its widespread use (Dorn et
al., 1987; Alexander et al., 1988), it has become very important
to fully elucidate the environmental behavior of BPA, espe-
cially its ecotoxicological aspects.
BPA is considered readily biodegradable in the aquatic
environment (Staples et al., 1998). Studies using pure cul-Correspondence to: Michihiko Ike; e-mail ike@env.eng.osaka-u.ac.jp
© 2002 Wiley Periodicals, Inc.
457

tures of BPA-degrading bacteria clarified that biodegrada-
tion of BPA proceeds via complicated metabolic routes and
leads to formation of several kinds of biodegradation prod-
ucts (BDPs). Although known BPA-degrading bacteria
could grow on BPA as a sole carbon source and about
70%–85% of the total organic carbon (TOC) was mineral-
ized into CO2 and/or cell materials, the remaining 15%–
30% fraction was accumulated in the culture medium as
soluble BDPs (Lobos et al., 1992; Ike et al., 2000). Accord-
ing to the metabolic pathway of BPA (Fig. 1) proposed by
Spivack et al. (1994), the major route (
80%) is its cleav-
age to produce p-hydroxyacetophenone (p-HAP) and p-
hydroxybenzaldehyde (p-HBAL), followed by further deg-
radation of both BDPs via p-hydroxybenzoic acid (p-HBA).
In addition, about 20% of BPA is converted to 2,3-bis-
(4-hydroxyphenyl)-1,2-propanediol (Tetraol-IV) via bis(4-
hydroxyphenyl)-1-propanol in the minor route. Though
Tetraol-IV can be slowly degraded to generate p-hydroxy-
phenacyl alcohol (p-HPOH), these minor-route BDPs were
accumulated longer in the medium. BPA degradation by
mixed microbial consortia like activated sludge and river
water microcosms also led in most cases to the accumula-
tion of minor-route BDPs, and Tetraol-IV and p-HPOH
were generally identified as the dead-end BDPs (Ike et al.,
2000). Thus, it is likely that the discharge of BPA into the
environment will cause secondary pollution by the above-
mentioned BDPs.
As mentioned above, several researchers have focused
on the toxic effects of BPA, especially its estrogenicity, and
much data are available. However, much less attention has
been paid to the toxicity of its BDPs, and little is known (Jin
et al., 1998, acute toxicity of p-HAP and p-HBAL; Ku¨hn et
al., 1998, acute toxicity of p-HBA; Lemini et al., 1997;
Pedersen et al., 2000; estrogenicity of p-HBA), although
research on their possible ecological impact is urgently
needed. In this study an investigation was done of the acute
toxicity, mutagenicity, and estrogenicity of some of the
BPA BDPs including dead-end ones compared with the
parent chemical, BPA.
MATERIALS AND METHODS
Biodegradation Products of Bisphenol-A
Because p-HAP, p-HBAL, and p-HBA are major BDPs,
they were used for the toxicity assays. These three chemi-
cals were purchased from Tokyo Chemical Industry Co.
Ltd. (Tokyo, Japan), Wako Pure Chemical Industries Ltd.
(Osaka, Japan), and Kishida Chemical Co. Ltd. (Osaka,
Japan), respectively, as commercial products.
Because BDPs occurring in the minor route were not
commercially available, the dead-end BDPs were extracted
from the culture broth resulting from BPA degradation tests
by Pseudomonas paucimobilis FJ-4 (Ike et al., 1995). The
BPA-degrading bacterium FJ-4 was grown at 30°C for
about 1 day in a minimal salt medium containing 100 mg/L
of BPA as the sole carbon source, and dead-end BDPs
accumulated in the culture broth were extracted from the
cell-free supernatant with ethyl acetate after acidification to
pH 3.0 with hydrochloric acid. The ethyl acetate was evap-
orated, and the residual BDPs were dissolved in ethanol or
dimethyl sulfoxide (DMSO) to make the 100-fold concen-
trated solution, which was used for the toxicity assays. The
BDPs of BPA were monitored with high-pressure liquid
chromatography (HPLC) and identified with gas chroma-
tography–mass spectrometry (GC-MS). Details of the anal-
yses of BDPs were described previously (Jin et al., 1996).
Acute Toxicity Assay
Daphtoxkit F magna (Creasel BVBA; Deinze, Belgium),
which makes use of Daphnia magna as a test species, were
applied to the acute toxicity assay. The acute immobiliza-
tion tests were performed using this commercial test kit
according to the operational procedure manual given by the
supplier, which fully adheres to OECD Guideline 202. The
24- and 48-h 50% effective concentrations (24-h EC50 and
48-h EC50) for the test chemicals were determined based on
the immobilization of D. magna.
Mutagenicity Assay
For the evaluation of the mutagenicity, umu tests were
carried out according to Oda et al. (1985). Salmonella
typhimurium TA1535 harboring plasmid pSK1002 carrying
the umuC-lacZ fusion gene was used as a tester strain. A
DNA-damaging chemical would induce umuC expression;
Fig. 1. Proposed biodegradation pathway of BPA.
458 IKE ET AL.