Impact of BBP and Acetone Exposure on F.

heteroclitus Health
and Behavior
Austin Carbone, Kira Diaz, Andrea LeBoeuf, Lisa A. E. Kaplan, Ph.D.
Department of Biological Sciences, 275 Mount Carmel Ave Hamden, CT
06518

Abstract

Results

The objective of this study was to examine the impact of

benzyl butyl phthalate (BBP) and acetone on Fundulus
heteroclitus (common mummichog) health and feeding
behavior. F. heteroclitus (N = 60) were assigned to one of
three groups (0.1 ppm BBP, 0.1 ppm acetone, and
unexposed). Over the course of a 28-day exposure
period, behavior and food consumption were observed
and recorded on a daily basis. When comparing data from
days 3, 4, 5 and 6 to days 25, 26, 27 and 28, it was clear
that behavior and somatic health indices changed
significantly. The time needed to feed increased in all
treatment groups, with acetone exhibiting the greatest
change (A > BBP > control). A change in the relative
speed of food consumption was also observed (from
acetone > BBP > control to BBP > control > acetone).
Agitation only increased among control fish. Body weight
decreased in all treatment groups with the largest
decrease among control fish and the smallest decrease
among BBP exposed fish. Somatic index decreased in
groups with the largest decline in controls and the
smallest in the BBP group. In summary, acetone and BBP
alter behavior and health as compared to control, but
captive housing conditions also appear to negatively
impact health.

Summary
Somatic Index
Decreased for all treatment groups (fig. 2)
Control had the most significant decline (fig. 2)
Feeding
The time needed to feed increased significantly for acetone treated fish (fig. 3)
By the end of the treatment period, both BBP and acetone-treated fish took significantly longer to feed than
control fish (fig. 3)
Agitation
Increased significantly among controls when comparing initial and final time periods (fig. 4)

Initial SI

Initial Tine to
Feed

Final SI

Introduction

Fish exhibit many behavioral patterns associated with

mating, feeding, and predator avoidance7. Behavioral
patterns are an essential endpoint in toxicology because
they are an important indicator of fitness and play a huge
role in survival not only for the individual, but also for the
entire social group. Studies indicate that these essential
behaviors can be altered by exposure to chemicals
including anti-depressants (Venlafaxine)1,2, Fluoxetine3,4,
and plasticizers5.

BBP is of particular interest because it is a plasticizer that

is primarily used to increase the flexibility of plastic
products, including toys and packaging materials. When
plasticizers such as this are used, they are not
permanently bound to the plastics and can migrate into
the surrounding environment. Since BBP is used in the
packaging of food, there is a good possibility that it is
ingested6. This property makes BBP an interesting
chemical of choice for this study.

F. heteroclitus is a fish species that makes for a good

animal model6 for behavioral toxicology. These fish live in
estuaries along the east coast of North America,
stretching from Newfoundland to Florida. They are easily
obtained in large quantities, have a defined home range
(which can be used to track episodic exposure events), a
defined growth rate (45mm per year) that makes age or
cohort determination fairly simple, and they are easily
maintained in a laboratory setting. Recently, these fish
have been used in behavioral studies to identify the
effects of antidepressants on predation1,2 and courtship
behaviors7 as well as the effects of benzyl butyl phthalate
(BBP) on the anti-predator behaviors, shoaling and
agitation8.

This study focused specifically on how the feeding

behaviors of F. heteroclitus changed over a 28-day period
of exposure to either acetone (the delivery vehicle) or
Chemicals: Acetone (CAS 85-68-7), BBP (CAS 85-86-7),
BBP.
Instant Ocean

Control

Acetone

BBP

Treatment Group

Figure 2. Alterations in Average Somatic Index Preand Post-exposure. Somatic Index declined over the
course of the treatment period in all groups
(0.0007P0.02), but a more precipitous decline was
observed among controls (P=0.05).
Initial

Figure 3. Change in Feeding Time Pre- and Postexposure. A significant increase in the time needed to
feed was observed only among acetone exposed
individuals (P=0.0017).

Final

Figure 4. Effect of Treatment on Agitation Scores.

Only controls exhibited a significant increase in agitation
over time (P = 0.001).

Figure 5. Effects of Treatment on f F.

heteroclitus Mortality. Both acetone and BBPexposed groups had a higher mortality rate (20%)
than the controls (0%).

Discussion
The results of this experiment suggest chemical exposure and captive housing negatively affect F. heteroclitus behavior
(time needed to feed and agitation) and overall health (somatic index). Our vehicle of choice, acetone, is a commonly
used solvent in plastic manufacturing. By the end of the 28-day acetone exposure, fish exposed to acetone consumed
food at a significantly slower rate (61%) as compared to the first few days of the experiment. BBP (dissolved in the
acetone vehicle) decreased feeding time by only 20%, which was comparable to the change observed in the unexposed
control group. This suggests a possible antagonistic relationship between BBP and acetone that minimizes the effect of
acetone.

Materials &
Methods

Somatic index is a good indicator of health (weight standardized to length). A decrease in this morphometric index was
observed among all three treatment groups indicating a decline in fish health over the course of the treatment period.
The largest decrease occurred in the control (31%) indicating that captive housing conditions were insufficient to
maintain the overall health of the fish. It appears that while all fish lost weight, those exposed to chemicals were able to
somehow minimize weight loss as compared to control. This is in line with behavioral observations supporting a more
frenetic feeding pattern among fish exposed to acetone or BBP.

Animal Collection: F. heteroclitus (N=60) were collected

from Long Island Sound (Milford, CT) using minnow traps
(Figure 1). They were transported to the laboratory, where
the following morphometric indices were recorded: sex,
weight,
and length. B
C
A

At the beginning of the experiment while fish were in the process of acclimating to captivity, general behavior was
assessed as lethargic (slow swim speed, long periods of immobility). Over the course of the experiment, control fish
appeared to increase activity (swim pattern, speed, and duration) to a more typical level. This was initially interpreted
as an increase in agitation. Fish exposed to BBP or acetone, however, remained in a lethargic state throughout the
entire treatment period. Initially, we thought fish were exhibiting normal behavior within three to four days of entering
the laboratory. When reviewing the behavioral changes, however, we concluded that initial fish behavior was atypical
(lethargic) rather than normal. Thus, the increased agitation for the control group by the end of the treatment period
(fig. 5) more accurately suggests that control acclimated and resumed more active (typical) behavior. BBP or acetone
exposure either prevented this acclimation or enforced lethargy by negatively impacting health of the individuals.

Figure 1. Baiting, casting, and setting minnow traps at Milford Point

Estuary, Long Island Sound, Milford, CT.

Citations

Future studies will focus on this potential chemical interaction between BBP and its vehicle acetone by employing
different vehicles for BBP exposure in an attempt to isolate the impact of BBP alone.
Animal Care: Fish were randomly assigned to one of six
30-gallon
aquaria
(N=10
per
aquarium),
and
supplemented with air and biological filtration. They were
maintained close to capture conditions (21ppth salinity
and 23C).
Exposure: After a four-day depuration period, acetone (0.1
ppm) and BBP (0.1 ppm) treatment groups were exposed
daily for 28 consecutive days. Control fish remained
unexposed for the same duration.
Feeding and Agitation Assessment: Fish were fed once a
day with 25mg of Tetramin Fish Flakes. The food was
released from one of several pre-prepared containers,
during which a stopwatch was activated and recorded the
time elapsed during consumption in seconds. The
agitation of the fish was recorded daily, marked 1
(lethargic), 2 (normal), or 3 (agitated).

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8
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1

Acknowledgments

We thank the College of Arts & Sciences and the Department of Biological Sciences at Quinnipiac University for supporting
this project. We also thank the staff at Milford Point Audubon Center for the use of their access point to Long Island Sound.
Finally, we thank Nadia Jarvis for her assistance in animal care and data collection.