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The Effects of Bisphenol-A on the Immune System of Wild
Yellow Perch, Perca flavescens
James A. Rogers &Reehan S. Mirza
Received: 14 October 2012 /Accepted: 16 August 2013 / Published online: 5 September 2013
#Springer Science+Business Media Dordrecht 2013
Abstract Bisphenol-A (BPA) is an environmental con-
taminant used in the manufacturing of polycarbonate
plastics and epoxy resins, which has been discovered in
freshwater systems worldwide as a result ofeffluent from
manufacturing. This bioactive molecule is an estrogen
mimic and has become a concern for exposure, especial-
ly during development, resulting in its removal from
baby bottles and other consumer products. BPA is an
endocrine disruptor in a variety of species and has been
classified as a toxic substance in multiple countries. In
this study, we examined the effect of BPA exposure on
leukocyte counts in wild yellow perch, Perca flavescens.
Yellow perch were exposed to either 2, 4, and 8 ppb
BPA; Saprolegnia; or a blank control for a period of
7 days. Leukocyte blood counts were significantly
higher in Saprolegnia, 4 ppb BPA, and 8 ppb BPA
treatments compared to control. To test compound ef-
fects of BPA and Saprolegnia on leukocyte counts over a
7-day period, perch were exposed to either 4 ppb BPA,
4 ppb BPA+Saprolegnia, or control. Leukocyte counts
were significantly higher in the 4 ppb BPA treatment
relative to control. The 4 ppb BPA+Saprolegnia treat-
ment was numerically elevated from the control,
exhibiting a 153 % increase relative to control. BPA
represents a contaminant with immunomodulatory
properties that remain to be determined.
Keywords Bisphenol-A .Leukocyte .Immune system .
Yellow perch .Saprolegnia .Endocrine disruptor
1 Introduction
Bisphenol-A (BPA) is a potent endocrine disrupting mol-
ecule which has been seen to have carcinogenic proper-
ties (Cavalieri and Rogan 2010). It is an estrogen mimic,
or xenoestrogen, most commonly examined in water-
ways where concentrations are highest and most persis-
tent worldwide (Klečka et al. 2009). This molecule is
used in manufacturing processes and can be found in
relatively high levels in runoff from industries that man-
ufacture polycarbonate plastics and epoxy resins (Staples
et al. 1998). Reported production for 2008 was
5,108,500 t with an expected 7 % growth in demand
peryear(Burridge2008). Bisphenol-A has been found
to leach out of these plastics under high heat conditions
and also as a result of photo leaching (reviewed in Staples
et al. 1998). As a result of runoff into river systems, BPA
has been identified in many water bodies worldwide in
varying concentrations as high as 12 ppb in North
America and 43 ppb in Europe (Klečka et al. 2009).
Bisphenol-A has been seen to accumulate in the tis-
sues of fishes (Belfroid et al. 2002). As well as being a
potent endocrine disruptor, BPA has been shown to in-
crease lymphocyte proliferation at low levels, 0.005–
50 mg/L in aquacultured goldfish, Carassius auratus,
but actually inhibits macrophage proliferation at high
concentrations, 500–1,000 mg/L (Yin et al. 2007).
Much of the research looking at effects of BPA in aquatic
Water Air Soil Pollut (2013) 224:1728
DOI 10.1007/s11270-013-1728-5
J. A. Rogers :R. S. Mirza (*)
Department of Biology and Chemistry, Nipissing University,
100 College Dr., North Bay, ON P1B 8L7, Canada
e-mail: reehanm@nipissingu.ca
species has been conducted at concentrations of parts per
million which are orders of magnitude above concentra-
tionsfoundinaquatic ecosystems (Klečka et al. 2009).
However, relatively little research has been conducted at
ecological levels of BPA in aquatic environments in
aquatic ecosystems on wild fish populations.
Fishes can serve as bioindicators for contaminants
such as BPA (reviewed in Milla et al. 2011). Fishes are
easy to collect in contaminated areas and their immune
systems have been documented to be altered by pollut-
ants (Hoeger et al. 2004). White blood cells count is an
indicator of immune parameter change in fishes and has
been used as a marker of immunological dysfunction
(Mekkawy et al. 2011). The majority of previous studies
using fish to examine BPA have used laboratory-reared
fish (Yin et al. 2007; Minghong et al. 2011). These
fishes can be used as a model but do not necessarily
illustrate the effects of BPA in a natural population.
Yellow perch (Perca flavescens)areawidelydistributed
species throughout North America and they are relative-
ly abundant (Scott and Crossman, 1973). Due to their
prevalence in lakes and rivers throughout North
America, yellow perch could serve as an ecologically
relevant bioindicator (Daoust et al. 1989).
In this study, we examine the effects of BPA at
environmentally relevant concentrations on the immune
response of wild yellow perch after a 7-day exposure.
As a measure of immune response, we conducted a
leukocyte count which is a well-established measure of
immune function (Forson and Storfer 2006). To deter-
mine whether the response from BPA exposure repre-
sented a true immune response, we also exposed fish to
Saprolegnia spp. Saprolegnia is a ubiquitous water
mold and an opportunistic pathogen (Van West, 2006).
It can cause infection if fish are experiencing immune
deficiency or their immune system is stressed by another
biological factor (Muzzarelli et al. 2001). To further test
immune deficiency, perch were exposed to a combina-
tion of BPA and Saprolegnia. A combined exposure of
pathogen and BPA has the potential to elicit an even
stronger immune response than BPA alone.
2 Methods
2.1 Collection and Maintenance of Fish
Wild yellow perch, 7.5–18 cm SL, were caught by
angling from Lake Nipissing, North Bay, ON, Canada
and held at 20 °C under a 16:8 h (light/day) photoperiod
in 37 L glass aquaria (50×30×20 cm) with a small corner
box filter containing filter floss and ammo chips. No
activated carbon was used as it may remove some BPA
from water (Kazner et al. 2008). Fish were acclimated to
the laboratory setting and fed after the first 48 h. Perch
were fed daily on worms (Lumbricus terrestris)
obtained from a local bait shop. Uneaten food was
removed every 24 h.
2.2 Saprolegnia Culture
Saprolegnia spp. was cultured from dead minnows ac-
quired from a local bait shop. After 24 h in water,
Saprolegnia was observed on fish tissue and was cul-
turedoncornmealagarplatesobtainedfromPhyto
Technologies Laboratories (Shawnee Mission, KS,
USA). The Saprolegnia culturing procedure was modi-
fied from Neish (1975) using 4×10
3
μg/ml of penicillin
G and 1×10
3
μg/ml of streptomycin sulfate as opposed
to carbenicillin. Purified segments of Saprolegnia were
transferred in a sterile environment to new plates of
cornmeal agar.
Whole hemp seeds were obtained from Chi Hemp
Industries Inc (Victoria, BC, Canada). Seeds were cracked
open and autoclaved for 30 min. Hemp seeds were added
to cornmeal agar plates and Saprolegnia was allowed to
grow over seeds while held in an incubator at 21 °C.
Covered seeds were used for Saprolegnia exposures.
2.3 Treatments
Bisphenol-A treatments were administered from a 1 g/L
stock solution of BPA prepared in acetonitrile. In exper-
iment1,fishreceived2,4,or8ppb(60,120,or240μl)
BPA, Saprolegnia or no treatment (control) for a period
of 7 days (n=4). Saprolegnia treatments each received
one hemp seed covered in Saprolegnia.Thisseedwas
not replaced throughout the exposure periods and was
allowed to continuously develop. We conducted 50 %
water changes every 72 h to maintain solution concen-
tration and water quality. After each 50 % water change,
a half treatment was re-administered to each tank to
maintain consistent levels of BPA.
In experiment 2, control tanks received acetonitrile
alone as a vehicle control. Based on the first experiment,
we used 4 ppb BPA; therefore, an equal volume of aceto-
nitrile (160 μl) was used for the control treatment. Fish
were exposed to acetonitrile, 4 ppb BPA or 4 ppb BPA+
1728, Page 2 of 6 Water Air Soil Pollut (2013) 224:1728
Saprolegnia (n=4 per treatment) for 7 days. Again, 50 %
treatment was re-administered after each water change.
2.4 Blood Sampling and Leukocyte Counting
At intervals of both 48 h and 7 days, individuals were
removed from their tanks and placed in a 185 mg/L
solution of MS-222 (Tricaine methanesulfonate) for
anesthesia. A caudal vein extraction method was used
to extract blood from each individual and blood samples
(at least 20 μl) were placed on a glass slide and smeared
as per traditional methods; all smears were fixed with
high-performance liquid chromatography grade metha-
nol (Howen 2000). After extraction of blood samples,
fishes were placed in a recovery tank of dechlorinated
water with an aerator until they were observed to be
swimming normally and then returned to their study
tanks. For final blood samples, fishes were euthanized
with a 1 g/L solution of MS-222 prior to extraction of
blood. Wright's stain was prepared and staining proce-
dure was followed as outlined by Howen (2000).
The leukocyte counting method was modified from
that of Forson and Storfer (2006). A 10×10 grid eyepiece
was used to keep density of counting areas relatively
consistent. All areas were held to standards that each grid
segment of the eyepieces must contain at least one type of
blood cell; however, there could be no clumping of red
blood cells. For each slide, 10 random areas were counted
at ×400 magnification within the entire grid area and
means were obtained per slide (Fig. 1).
2.5 Statistical Analysis
Due to small sample sizes, we conducted pairwise com-
parisons of leukocyte counts of each treatment to the
control in experiment 1 and among all treatments in
experiment 2 using Mann–Whitney Utests. To correct
for inflation of the family-wise type I error rate, we used a
modified Bonferroni correction (Keppel, 1982). In
experiment 1, the corrected pvalue was 0.05 and for
experiment 2, it was p=0.033). Based on the work of
Yin et al. (2007)whereBPAcausedanincreaseincell
counts, we conducted one-tailed tests. All statistics were
performed using SPSS 18 (SPSS Inc., Chicago, IL, USA).
3 Results
For the first group of exposures, no significant differ-
ence in mean leukocyte count was observed after 48 h of
exposure to 2, 4, and 8 ppb BPA or Saprolegnia com-
pared to the blank control (all pvalues are >0.05; Fig. 2).
After 7 day of exposure, an increase of 195–230 % was
observed in mean leukocyte count of yellow perch
exposed to 4 and 8 ppb BPA and Saprolegnia exposures
compared to the blank control (p=0.042, 0.017, and
0.017, respectively; Fig. 2).
In the second group of exposures, no significant
difference in mean leukocyte count of yellow perch
was observed after 48 h of exposure to 4 ppb BPA or
4 ppb BPA+Saprolegnia when compared to the aceto-
nitrile control (all pvalues>0.033; Fig. 3). However,
after 7 days of exposure, a significant increase of
148 % in mean leukocyte count was seen in the 4 ppb
BPA exposure compared to the control (p=0.025;
Fig. 3). An increase is seen in the mean leukocyte
count of 4 ppb BPA+Saprolegnia treatment by 153 %
but is not statistically significant compared to control
(p=0.0385, Fig. 3). Between 4 ppb BPA and 4 ppb
BPA+Saprolegnia, there is no observed statistical
difference (pvalues>0.033; Fig. 3).
4 Discussion
Overall, we found that BPA exposure at environmentally
relevant concentrations affected the short-term immune
response of yellow perch by elevating leukocyte counts.
Fig. 1 Blood smears stained
with Wright's stain. Repre-
sentative of a control smear
(left) and a 4 ppb BPA (right)
after 7 days. Arrow denotes
area of high leukocyte con-
centration. ×400
magnification
Water Air Soil Pollut (2013) 224:1728 Page 3 of 6, 1728
After 7 days of exposure, leukocyte counts increased by
asmuchas230%whenexposedto4ppbBPAcompared
to the blank control. We saw similar increases in leuko-
cytes when we exposed yellow perch to Saprolegnia and
Fig. 2 Mean±SE leukocyte
count in yellow perch after
exposure to either:
Saprolegnia, 2 ppb BPA,
4 ppb BPA, or 8 ppb BPA or
a blank control for 48 h (top)
and 7 days (bottom). Letters
denote significant difference
at p≤0.05 from the control
(see text for details)
Fig. 3 Mean±SE leukocyte
count in yellow perch after
exposure to either: acetoni-
trile control, 4 ppb BPA, or
4 ppb BPA+Saprolegnia af-
ter 48 h (top) and 7 days
(bottom). Letters denote a
significant difference at
p≤0.033 (see text for details)
1728, Page 4 of 6 Water Air Soil Pollut (2013) 224:1728
to a combination of BPA+Saprolegnia. Bisphenol-A typ-
ically biodegrades rapidly in aquatic systems (Staples
et al. 1998), but if there was continual introduction into
the environment, then BPA levels could be maintained
keeping any aquatic organisms at risk. Therefore, short-
term exposure to BPA is sufficient to affect immune
response and could lead to larger implications.
In experiment 1, environmentally relevant levels of 4
and8ppbBPAinitiatedanimmuneresponseinyellow
perch after 7 days of exposure. Moreover, exposure to
Saprolegnia led to a similar leukocyte increase indicating
that the immune response to BPA is comparable to path-
ogen exposure. In the second experiment, the initial
leukocyte count of the control was higher than seen in
the first experiment, but then dropped to similar counts by
day 7. Increase in leukocytes could be due to the presence
of acetonitrile in the control tank which may have initiat-
ed an immune response, but then was dealt with by the
end of day 7. The 4 ppb BPA treatment was statistically
different from the control demonstrating a 148 % increase
in leukocyte counts, although this was not as great an
increase as found in the first experiment. Compound
exposure was not significantly different from control,
though a biological effect was observed. Leukocyte
counts in the compound exposure were similar to 4 ppb
BPA; however, significance was not reached due to
higher variation among individuals within the group.
Fish exposed to environmental contaminants that stress
the immune system show increased susceptibility to path-
ogens (Rice et al. 1996); due to this, we hypothesized an
increase susceptibility to Saprolegnia. However, we saw
no increase in leukocyte response or signs of infection
due to concurrent BPA and Saprolegnia exposure.
Tetrapods diverged from fishes over 450 Ma ago;
however, physiologically the two groups still share
many similarities and fish may represent significant
bioindicators due to conservation of immune functions
(Davis et al. 2008; Ohta and Flajnik 2006; Gerwick et al.
2007). Tumor necrosis factor-like genes have been iden-
tified in fish; which are homologues of those identified in
humans (Plouffe et al. 2005). Immune interactions with
the endocrine system are well documented in vertebrates
(O'Halloran et al. 1998). In mammals, BPA upregulates
estrogen receptor expression and has been found to in-
crease the production of autoantibodies representing im-
mune dysfunction (Yurino et al. 2004). BPA and various
analogues were found to act with agonistic effects on a
mammalian model of breast cancer increasing prolifera-
tion of cancer cells through natural sex hormone-
controlled pathways (Rivas et al. 2002). Weng et al.
(2010) found that exposure to 4 nM BPA altered gene
expression and DNA methylation in human breast epi-
thelial cells suggesting developmental exposures may
represent increase risks of immunological dysfunction
and exposures to BPA at environmentally relevant levels
represents a serious threat. Milla et al. (2011)arguethat
fishes provide an ideal model organism to use for study-
ing the effects of BPA and other endocrine disrupting
compounds making them beneficial for the study of
affected waterways and toxicity in aquatic populations.
5 Conclusion
We found that BPA exposure at environmentally relevant
concentrations over a 7-day period caused an increase in
leukocyte counts. Kalair et al. (1993) has previously
stated that an increase in the number of leukocytes due
to treatment represents an impairment of the immune
system and Singh and Srivastava (2010) suggest that
hematological parameters are important bioindicators in
teleosts. It remains to be determined if, over a short-term
exposure, the leukocyte increase is detrimental or bene-
ficial. However, a short term immunostimulatory re-
sponse may be beneficial in that it offsets the negative
effects of costs associated with detoxification. Using wild
fish populations as bioindicators for studying the effects
of BPA in the short- and long-term could provide
new breakthroughs in understanding how EDCs
affect immune function. Further work is needed
to understand the environmental effects of BPA
in aquatic systems.
Acknowledgments We would like to thank Todd Bowerman
and Doug McIntyre for help in collecting yellow perch. Ashley
Ryan assisted with all analytical work and photography. Thank
you to Rebecca Mulligan for helping culture Saprolegnia. This
study was funded by Nipissing University and all work was
conducted under Nipissing University Animal Care Committee
protocol #PR2010-04-04-20.
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