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This is to certify that the

thesis entitled

mRPHOLOGICAL EFFECTS OF BISPHENOL A ON THE
EARLY LIFE STAGES OF MEDAKA (Oryzias latipes)

presented by

STEPHANIE D . PASTVA

has been accepted towards fulfillment
of the requirements for

14.3. degree in FISHERIES AND WILDLIFE

   

Major pr essor

JBHn P. Giesy

 
   

Date May 2000

 

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

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11m mm.w5p.14

MORPHOLOGICAL EFFECTS OF BISPHENOL-A ON THE EARLY LIFE
STAGES OF MEDAKA (ORYZIAS LATIPES)
By

Stephanie D. Pastva

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Fisheries and Wildlife

May 2000

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ABSTRACT
MORPHOLOGICAL EFFECTS OF BISPHENOL-A ON THE EARLY LIFE
STAGES OF MEDAKA (ORYZIAS LATIPES)
3)!

Stephanie D. Pastva

Bisphenol-A (BPA). a widely used polycarbonate plasticizer, has been of concern
because it has been shown to leach out of plastics and other epoxy products.
Primary sources of environmental releases are expected to be from BPA and
epoxy manufacturing facilities. Although environmental concentrations may be
limited, little is known about the effects of this compound on fish. particularly
. during their most sensitive early life stages. A 96-hr, 200pg BPA/L, lethality
exposure was conducted with newly hatched larvae, but no differences in
mortality between treatments were observed. In addition, medaka embryos were
exposed beginning 5-hr post-fertilization, for 9-d at 25°C, to concentrations of 20
pg BPA/L or 200 pg BPA/L (24-h static renewal). Embryos were monitored daily
for stage of development and gross abnormalities. Embryos exposed to 200 pg
BPA/L did not exhibit abnormalities until after day 4, between days 4-8 the
severity index score of embryos was significantly greater than that for embryos
exposed to lesser concentrations. By day 9, severity index scores were no
longer statistically different among treatments. BPA caused transient embryonic
deformities in medaka embryos at environmentally relevant concentrations, but

these deformities healed prior to hatching.

DEDICATION

This thesis is dedicated to the Pastva family...

I would like to thank my parents, Dawn and Robert who have always encouraged
me to do my best and also to figure things out for myself. They even made me
look things up in the dictionary when I asked how to spell a word! I know I didn't
appreciate it then, but I'm thankful for the way it made me ask questions and

pursue the answers.

I would also like to thank my grandparents, Betty and Elmer Pastva. They have
done so many things that have helped me get where I am today that I couldn’t
possibly mention them all. They have taught me what the word family really

means.

ACKNOWLEDGMENTS

A big thanks to Dr. Giesy for taking a chance and taking me on as a grad
student— even though I didn’t have a toxicology background. The opportunities I
have been presented with are enormous. I hope I have met and continue to both
meet and exceed his expectations.

During this time, K. Kannan was instrumental in teaching me about the
chemistry lab. Some of the lessons were hard and quite frustrating, however, I
now have a much better understanding of and appreciation for chemistry.

Although his name is listed last, it is by no means an indication of how I
feel about him. Alex Villalobos was there for me the entire time during my MS.
research. Although his job was only as a post-doc, he was much more. He was
my mentor, someone that I looked to for help and advice (at work and with
personal problems). He taught me all I know about medaka and their
development. Alex was (and still is) also a good friend, he’s always been there

for the good and bad in my life cheering me on and with good advice.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................ vi
LIST OF FIGURES ............................................................................................. vii
INTRODUCTION ................................................................................................... 1
METHODOLOGY .................................................................................................. 5
Culture of Broodstock and Egg Collection: ........................................................ 5
Embryo Exposure: ............................................................................................. 5
Severity Index: ................................................................................................... 7
96-hour larval mortality experiment .................................................................. 11
Statistical Analysis ........................................................................................... 11
RESULTS ........................................................................................................... 13
Embryo Exposure ............................................................................................ 13
Larval exposure ............................................................................................... 13
DISCUSSION ...................................................................................................... 17
Larval exposure ............................................................................................... 17
Advantages and disadvantages of scoring system .......................................... 17
Embryo exposure ............................................................................................. 20
REFERENCES .................................................................................................... 22

LIST OF TABLES

Table 1. Physical and chemical properties of bisphenol-A . ................................. 4
Table 2. Salts needed to make Embryo Rearing Medium ................................... 6
Table 3. Scoring guide for the assessment of a Severity Index. ........................ 10
Table 4. Number of embryos with deformities for each concentration ................ 16

Table 5. Comparison of results of freshwater fish species exposed to

BPA. ......................................................................................................... 19

vi

LIST OF FIGURES

Figure 1. Hemorrhage ........................................................................................ 14
Figure 2. Pericardial Edema ............................................................................... 14
Figure 3. Average Severity Index scores. .......................................................... 15

vii

INTRODUCTION

The embryonic and/or larval periods have consistently been shown to be
the most sensitive life stage of a variety of fish species to chemical stressors
(McKim, 1985). These early life stages are thought to be especially sensitive due
to the numerous critical events that occur in a short time period (McKim, 1985).
In vivo assays, instead of in vitro assays, maintain biological complexity and
observations about effects can be directly observed instead of inferred. Thus,
partial exposures are relevant for determining the potential effects of chemicals
on fish.

The medaka (Oryzias Iatipes) serves as an excellent fish model for early
life-stage tests for the following reasons: 1.) adults are small (3-4 cm long); 2.) It
is easily cultured in the laboratory; 3.) under proper conditions females produce
eggs daily (Hyodo-Taguchi and Egami, 1989), and 4.) adults do not need be
killed to obtain eggs. In addition, the chorion, is clear allowing observation,
without damage, of the developing embryo under a light microscope. Finally, the
embryological and larval stages of medaka development have already been well-
documented (lwamatsu, 1994). Observing the developing embryo provides an
abundance of information including survival, growth, and developmental
abnormalities.

Bisphenol-A (4,4' isopropylidenediphenol or 2,2'-Bis(p-hydroxyphenyl
propane)), BPA, is an industrially important compound, widely used as a

monomer in the production of polycarbonates, epoxy resins, and coatings. BPA

is very soluble in water and has a relatively short environmental half-life (Table
1). In 1993, 109 tons of BPA were estimated to have been released into the air,
surface water, or wastewater treatment plants from the United States alone
(Staples, et al., 1998). BPA has been described as "slightly to moderately toxic"
to select aquatic organisms (Staples, et al., 1998). BPA has also been confirmed
to be an endocrine disrupter by interacting with the estrogen receptor (Brotons, et
al., 1995, Krishnan, et al., 1993, Perez, et al., 1998, Steinmetz, et al., 1997). It is
for this reason that BPA has attracted so much attention.

BPA occurs at relatively small concentrations in streams and rivers. In the
1970's, BPA from various rivers in the Tokyo area were reported to range from
0.06 ug/L - 1.9 pg/L (Matsumoto, 1982). According to a 1996 study, in five US.
receiving streams of facilities that manufacture or use BPA in the manufacturing
process, BPA was < 1.0 pg/L (Markham, et al., 1998). In 1997, BPA
concentrations in these same 5 streams ranged from < 1.0 ug/L - 8 ug/L
(Staples, et al., 2000).

Previous studies have examined acute toxicity of BPA to adults of various
fish (Alexander, et al., 1988, Staples, et al., 1998). Although BPA is classified as
slightly to moderately toxic (Staples, et al., 1998), information about the sublethal
effects of BPA to fish, especially during their early life stages is lacking. The
overall objective of this research was to determine if waterborne BPA
concentrations affected medaka early life stages. To accomplish this objective,
medaka embryos were exposed to BPA to quantify morphological effects on

embryological stages and acute lethality to newly hatched larvae. This study

only looked at embryological developmental deformities as an endpoint. Further
research should be designed to determine if BPA at environmentally relevant
concentrations causes abnormalities related to the endocrine system in fish

species.

Table 1. Physical and chemical properties of bisphenol-A (Staples, et al., 1998).

 

i“:
HO O 13 O OH

CH3

CAS No. 80-05-7

Formula C15H1602

Molecular Weight 228 g/mol

pKa 9.6 - 11.3

Water Solubility 120 mg/l - 300 mg/l

Log Kow 2.2 - 3.82

Half-life 2.5 - 4 days

 

 

 

METHODOLOGY

Culture of Broodstock and Egg Collection:

Adults were housed in glass aquaria at a constant temperature of 25°C (i
2°C), with a photoperiod of 16:8 (lightzdark) hours. Adults were fed brine shrimp
and 1:1 TetraMin® (Tetra; Blacksburg, VA) flake foodzground trout chow pellets
were fed ad lib daily.

Eggs were collected less than 5-hr after fertilization, from individually
netted females. Clusters of eggs were carefully removed from the female’s
abdomen using fingertips. Filaments attaching adjacent eggs together were
removed by gently rolling the clusters between moistened fingertips, and using a
sharp pair of forceps to pull filaments from the egg. The cleaned eggs were then
observed using a dissecting microscope. Eggs that were greater than stage 10
(lwamatsu, 1994), unfertilized, or abnormal in appearance were discarded.
Eggs were then disinfected by placing them in a 0.9% solution of hydrogen
peroxide for 10 min, after which eggs were placed in embryo rearing medium
(ERM) until initiation of exposure. ERM (Table 2) is osmotically balanced with

the medaka embryo.

Embryo Exposure:

Embryos were exposed in 10 ml scintillation vials (Research Products
International; Mount Prospect, IL). Vials were washed beforehand using Luqui-

nox detergent (VWR Scientific Products; South Plainfield, NJ) followed by

Table 2. Salts needed to make Embryo Rearing Medium

 

 

Salt Amount needed
(ggmslliter)
NaCl 1.0
KCl 0.03
CaCl2'2H20 -OR- 0.04 -OR-
CaCl2 anhydrous 0.03
MgSO4 0.08

aerate and adjust pH (7+/- 0.2) with

0.14M NaHC03(0.259/20ml H20)

 

acetone (Burdick and Jackson; Muskegon, MI) and finally hexane (Burdick and
Jackson). Powder BPA was obtained from Sigma Chemical Company (St. Louis,
MO). Dilutions were made from a 20 mg/L BPA standard dissolved in nanopure
water, no organic carrier solvent was used. ERM was used for the controls and
to make the 200 pg/L and 20 ug/L dilutions.

Individual eggs were randomly placed in vials until there were 5 embryos
in 5 ml of test solution. There were 5 replicates per concentration tested, for a
total N=25 embryos per concentration. Individual vials were labeled and capped
with a double layer of teflon tape. The vials were placed in a water bath, and
maintained at 25° C. The exposure was a 24-hr static renewal. Due to the short
half-life of BPA, a new 20 mg BPA/L standard was made daily, diluted, and

replaced in the vials.

Severity Index:

Observations of individual embryos were made daily. A published atlas of
normal medaka development (lwamatsu, 1994) was used to determine if
embryos were developing normally. Recorded abnormalities were given a daily
severity index (SI) score as described in Villalobos et al (Villalobos, et al., 2000).
Briefly, abnormalities in individual embryos were recorded each. day and a
severity index score was calculated (Equation 1) on a scale ranging from 0 = no
abnormalities observed, to a maximum of 11.5 = death (Table 3). Those
unfamiliar with embryological lesions are encouraged to read Sharp (1990) as it
provides pictures and definitions of embryological lesions. However, the results

section of this paper provides pictures of cardiovascular lesions (Figures 1 and

2). When more than one lesion was observed in an embryo, SI scores for each
lesion were summed. Observations were recorded for a period of 9-d, at which

time surviving embryos were euthanized with MS—222 (Sigma Chemical Co).

Equation 1. Severity Index computation, from Villalobos et al ( 2000)

n n n n
SI: 2 (CRin)+ Z (CVxEj)+ Z (SKxEk)+ Z (OAxEl) +embryosperdose
i=1 i=1 k=1 [:1

Where E,= no. embryos with craniofacial (CR) value, E,= no. embryos with
cardiovascular (CV) value, E,= no. embryos with myoskeletal (SK) value, and

E,= no. embryos with other anomaly (OA) value.

 

Table 3. Scoring guide for the assessment of a Severity Index according to gross abnormalities observed in the medaka early life (embryonic and/or larval)

stage assay (Villalobos et al., 2000).

 

 

Cranio- Observed effect Cardio- Observed effect Myo— Observed effect Other Observed effect
facial vascular skeletal anomaly
value - value - value - value-
CR CV SK OA
0 No observable lesion 0 No observable lesion 0 No observable lesion 0 No observable lesion
1 Minor, transient defect in structure 1 Bradycardia, 1 Minor spinal curvature, 1 Minor, transient yolk sac edema
OI’SIZG ("6‘ less expansron Of Arrhythmia, Minor, delayed development (yse)
brain, less head Width, less eye ll bod 'dth /size t arl Ch . ll bl dd
pigmentation) Enlarged heart, (sma, er y W a e y anges m ga a er
stages) coloration
Mild pericardial edema
2 Moderate anomaly in structure or 2 Moderate edema (i.e. pericardial, 2 Marked (one or multiple) spinal 2 Delayed and/or prolonged hatch
size, not compromising survival peritoneal), sometimes reversible curvatures (i.e. lordosis, kyphosis, (i.e. > 24 h),
(l'e‘ deformed jaw) Hemostasis, SCOI'OSIS) Abnormal (Le. head first) hatch
Synophthalmia, Exophthalmia Some hemorrhaging Moderate yse
3 Severe anomaly in structure or 3 Severe edema, in most cases 3 Growth reduction within proportion 3 Diminished yolk resorption,
srze, irreversible and often irreverSIbIe of body srze or length (stunting), No swim bladder inflation
compromisrng survrval Sube idermal (include e e) that may be compensated at a
(microphthalmia, microcephalia) p edema y later time Larval weakness
Extended hemorrhaging
4 Anencephaly 4 Tubular heart (extreme edema), 4 Complete stunted development 4 Larval failure to thrive (loss of
. . (no proportion in body size/length) equilibrium, lack of mobility,
Anophthalmla Acardia . permanent recumbency)
. . . Body opaCIty
Cyclopia Brain hemorrhaging

11.5 Mortality (embryo / larval)

 

 

96-hour larval mortality experiment

The major differences between the larval exposure and the embryo
exposure were the use of a solvent to make stock solutions, duration of
exposure, and the only endpoint assessed was mortality. 250-ml l-Chem jars
(Nalge Nunc International Corp; Rochester, NY) were used to hold the newly
hatched (<24-hr) medaka larvae for this static daily renewal test. 1 ml of ethanol
was used to rapidly dissolve the BPA into 1 liter of ERM. The stock solution was
used to make the dilution to 200 ug/L, resulting in medaka being exposed to only
0.1% ethanol. A control, solvent control, and 200 ug/L BPA were tested. There
were 3 replicates of each test solution. Each jar contained 150 ml of test solution
and ten larvae, for a total of 30 individuals tested per solution. This was a 24-hr
static renewal exposure, with new stock solution and dilutions made daily. Jars
were kept in a 25°C water bath. Larvae were also checked daily for mortalities

and were fed ad lib with TetraMin baby food.

Statistical Analysis

Differences in SI values among treatments were examined in SAS (SAS,
1996) by the repeated measures function (proc mixed) using an autoregressive
correlation pattern, with each vial serving as the replicate. The rationale for this
statistical analysis was that: the same endpoint was being measured daily
(severity of deformity) and daily measurements were not independent of one
another (i.e. if an embryo exhibited hemostasis on day 6, on day 7 the embryo

would probably still exhibit hemostasis). Effects of treatments on the number of

11

embryos expressing deformities at day 9 follows the binomial distribution which
was used to determine if there were differences in deformity rates between
controls and the greatest concentration (200 ug/L). Day 9 was chosen for the
analysis to provide the most biologically meaningful differences. Differences in
larval mortality rates per replicate between after 96-hr of exposure data of the

larvae was tested for statistical differences in mortality with a t-test (SAS, 1996).

12

RESULTS

Embryo Exposure

No deformities were observed until after day 3 (Fig. 3). Statistical analysis
revealed that on days 5-8 the SI of embryos exposed to 200 pg/L BPA were
significantly greater than controls (p < 0.05). However, by day 9, embryos
exposed to 200 ug/L BPA were no longer significantly different from those
exposed to 0 pg/L BPA. There were no significant differences in SI between
embryos exposed to BPA over all 9 days (p = 0.13). A regression of SI, on day
9, as a function of BPA concentration, was not significant (r2 = 0.04, p > 0.05).

Most embryos exhibited no deformities (Table 4). Most deformities
observed were cardiovascular in nature (Figures 1 and 2). Although by day 9
embryos exposed to 200 ug/L BPA had a 24% rate of deformity (Table 4), there

was not a significant difference between this group and the controls (p = 0.0994).

Larval exposure

Only one larva died during the 4-d exposure period. This individual was
from the solvent control group. There were no differences in larval mortality
among treatments (p > 0.05). Long-term effects were not assessed since larvae

were not grown out to adulthood.

13

 

Figure 1. Hemorrhage
Embryo on the right was exposed to 200 ug BPA/L and has a large hemorrhage

(arrow) at the left duct of Cuvier region. The hemorrhage appears as a large
diffuse red coloring below the eye. The embryo on the left is a normally

developing control individual of the same age.
Figure 2. Pericardial Edema

This embryo was exposed to 200

ug BPA/L and has a severe
pericardial edema (arrow). A
pericardial edema is a sac of fluid
surrounding the heart region. It
appears as a clear wedge

between the head and yolk sac.

 

 

 

 

11.5 I T
Conc (pg BPA/L
9.2 '_ . 0 ‘—
* 20
A 200
9 6.9 — —
o
0
(I)
<7) 4.6 - _.
2.3 — —
0.0 4 - - ' I

 

0 3 6 9
Days After Fertilization

Figure 3. Average Severity Index scores with error bars over time.

15

Table 4. Number of embryos with deformities for
each concentration tested. Sample size is 25

individuals per concentration.

 

 

 

 

Concentration #deformed % deformed
(pg/L)
0
20
200 6 24

 

 

 

 

 

16

DISCUSSION

Larval exposure
Survival of newly hatched medaka larvae was not affected by exposure to

200 ug/L of BPA after 96-hr. This result is consistent with previous acute toxicity
testing with freshwater fish, which reported LCsos in the mg/L range (Table 5).
Environmentally relevant concentrations of BPA do not appear to be acutely toxic

to medaka or fathead minnows (Pimephelas promelas) (Table 5).

Advantages and disadvantages of scoring system

Computing 3 severity index is a very time intensive process. It involves
daily monitoring of each embryo. In addition, the scorer must be experienced at
determining abnormalities and then assigning them to a mild, moderate, or
severe category. Depending on the chemical and dose tested, there may be
numerous zeros (embryos with no abnormalities) at the end of the exposure. If
this occurs, the data will not be normally distributed. However, non-parametric
versions of the repeated measures test (based on rank scores) produces similar
results. Even though the repeated measures procedure has some difficulties,
there are several advantages to using the daily severity index. Using this index
allows for quantification of differences between embryos and treatments instead
of just anecdotal observations. In addition, changes over time (when toxicity
begins to occur and potential healing) can be determined. However, if this
amount of discrimination is not needed, then assessing the embryos on the final

day of exposure would be more efficient and easier to analyze statistically. In

17

addition, the severity index data on day 9 yielded very similar p-values to the

binomial analysis of the day 9 data.

18

 

 

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19

Embryo exposure

Embryos exposed to 200 ug/L BPA did not exhibit abnormalities until after
day 4, and between days 4-8, the severity index score of embryos was
significantly greater than for lesser concentrations (Figure 3). The lower 200
pg/L Sl scores on day 9 were a result of individual embryos healing. There was
a lessening in the severity of observed hemorrhages, the resulting lower SI
scores were responsible for a non-significant value on day 9. Results of other
exposure studies, in which medaka embryos have been exposed to various
toxicants, indicate stage-specificity of abnormalities. Medaka embryos exposed
to TCDD (Wisk and Cooper, 1990) and a PCN mixture (Halowax 1014)
(Villalobos, et al., 2000) did not exhibit lesions until day 4 or 5. It is interesting to
note that day 4 coincides with formation of the liver, and consequently it has
been hypothesized that the mechanism of action of resulting deformities is
metabolism by cytochrome P-450 enzymes.

Since all organs are developed by day 9, the medaka embryo may be
metabolizing and/or excreting BPA more quickly than it is being exposed.
Although the pathways of BPA metabolism by bacteria have been well
documented (Lobos, et al., 1992, Spivack, et al., 1994), little has been done to
investigate how BPA is metabolized in fish species. This study did not attempt to
determine if the embryos were metabolizing the BPA or which enzyme system
may be responsible for the metabolism of BPA. Studies that investigate possible
metabolism may give additional insight into the mechanism of action for the

embryological deformities that were observed.

20

Since the route of exposure was waterborne instead of injection, it can be
argued that the chorion protected the developing embryo by decreasing
exposure. However, the chorion is semi-permeable. Previous work with TCDD
indicated that medaka egg waterborne exposure and egg nanoinjection yielded
similar LC/LDsos (Wright, et al., 1997). Recently, zebrafish (Danio rerio) embryos
were used to study the uptake and clearance of C“ labeled atrazine. They found
that in a waterborne exposure protocol, atrazine penetrated the chorion quickly
followed by a slowing to steady state (Wiegand, et al., 2000). Within 3-hr after
the medium was changed to atrazine-free water, 75% of the absorbed atrazine
was released from the embryo to the medium (Wiegand, et al., 2000). As
atrazine has a similar Log Kow (2.75) and water solubility (30 mg/L) as BPA
(Wiegand, et al., 2000), waterborne exposures to hydrophilic compounds (such
as BPA) may be more meaningful than a nanoinjection protocol.

This study indicates that BPA does result in embryonic deformities at
environmental concentrations, but that the medaka embryo is capable of healing
these deformities prior to hatching. Furthermore, environmentally relevant
concentrations of BPA do not cause acute lethality to medaka larvae. Therefore,
fishes in the wild, with similar or lesser sensitivity to BPA, may be affected

similarly by BPA.

21

 

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Alexander H.C., Dill D.C., Smith L.W., Guiney P.D., Dorn PB. 1988. Bisphenol-
A: Acute aquatic toxicity. Environ. Toxicol. 7:19-26.

Brotons J.A., Olea-Serrano M.F., Villalobos M., Pedraza V., Olea N. 1995.
Xenoestrogens released from lacquer coatings in food cans. Environ.
Health Perspect. 103(6):608-612.

Hyodo-Taguchi Y., Egami N. 1989. Use of small fish in biomedical research, with
special reference to inbred strains of medaka. In A. Woodhead, ed.,
Nonmammalian animal models for biomedical research. CRC Press, Inc,
Boca Raton, FL, USA. p. 185-214.

lwamatsu T. 1994. Stages of normal development in the Medaka (Oryzias
latipes). Zool. Sci. 11:825-839.

Krishnan A.V., Stathis P., Permuth S.F., Tokes L., Feldman D. 1993. Bisphenol-
A: an estrogenic substance is released from polycarbonate flasks during
autoclaving. Endocrinology. 132:2279-2286.

Lobos J.H., Leib T.K., Su T.-M. 1992. Bidegradation of Bisphenol-A and other
bisphenols by a gram-negative aerobic bacterium. Appl. Environ.
Microbiology. 58(6):1 823-1831 .

Markham D.A., McNett D.A., Birk J.H., Klecka G.M., Bartels M.J., Staples CA.
1998. Quantitative determination of bisphenol-a in river water by cool on-
column injection-gas chromatography-mass spectrometry. Int. J. Environ.
Anal. Chem. 69(1):83-98.

Matsumoto G. 1982. Comparative study on organic constituents in polluted and
unpolluted inland aquatic environments-Ill. Phenols and aromatic acids in
polluted and unpolluted waters. Water Research. 16:551-557.

McKim J.M. 1985. Early life stage toxicity tests. In G. M. Rand and S. R.
Petrocelli, ed., Fundamentals of aquatic toxicology: Methods and
applications. Hemisphere Publishing Corporation, New York, New York,
USA. p. 58-95.

Perez P., Pulgar R., Olea-Serrano F., Villalobos M., Rivas A., Metzler M.,
Pedraza V., Olea N. 1998. The estrogenicity of bisphenol A related
diphenylalkanes with various substituents at the central carbon and the
hydroxy groups. Environ. Health Perspect. 106(3):167—174.

SAS. 1996. SAS, 6.12 ed. The SAS Institute, Cary, NC.

22

Sharp JR 1990. The influence of toxicants on the teratogenic response of
fishes: A guide to the literature, a glossary of terms and a pictorial atlas.
Trace Substances in Environ. Health. 24:63-80.

Spivack J., Leib T.K., Lobos J.H. 1994. Novel pathway for bacterial metabolism
of Bisphenol A, rearrangements and stilbene cleavage in Bisphenol A
metabolism. J. Biol. Chem. 269(10):7323-7329.

Staples C.A., Dorn P.B., Klecka G.M., O'Block S.T., Branson D.R., Harris LR.
2000. Bisphenol A concentrations in receiving waters near US
manufacturing and processing facilities. Chemosphere. 40:521-525.

Staples C.A., Dorn P.B., Klecka G.M., O'Block S.T., Harris LR. 1998. A review of
the environmental fate, effects, and exposures of Bisphenol-A.
Chemosphere. 36(10):2149-2173.

Steinmetz R., Brown N.G., Allen D.L., Bigsby R.M., Ben Jonathan N. 1997. The
environmental estrogen bisphenol A stimulates prolactin release in vitro
and in vivo [see comments]. Endocrinology. 138(5):1780-6.

Villalobos S.A., Papoulias D.M., Meadows J., Blankenship A.L., Pastva S.D.,
Kannan K., Tillitt D.E., Giesy JP. 2000. Toxic responses of medaka (d-rR)
strain to polychlorinated naphthalene mixtures after embryonic exposure
by in ovo nanoinjection: a partial life cycle assessment. Environ. Toxicol.
Chem. 19(2):432-440.

Wiegand C., Pflugmacher S., Giise M., Frank H., Steinberg C. 2000. Uptake,
toxicity, and effects on detoxification enzymes of atrazine and
trifluoroacetate in embryos of zebrafish. Ecotoxicology and Environ.
Safety. 45: 1 22-1 31 .

Wisk J.D., Cooper KR. 1990. The stage specific toxicity of 2,3,7,8-
tetrachlorodibenzo-p-dioxin in embryos of the Japanese medaka (Oryzias
latipes). Environ. Toxicol. Chem. 921159-1169.

Wright P.J., Papoulias D.M., Tillitt DE. 1997. Presented at the Society of
Toxicology and Chemistry, San Francisco, CA, USA.

23

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