CONTINUING PROPOSAL: Effects of dispersed oil on growth and development of juvenile
sheepshead minnow (Cyprinodon variegatus).
PI:
Robert J. Griffitt
Cluster:
Understand
Approximate Budget: $110,000
PROPOSAL SUMMARY:
The research objective of this proposal is to identify the
effects of simultaneous exposure to dispersed crude oil and chronic hypoxia, on estuarine
organisms. The hypothesis to be tested is that exposure to dispersed oil under hypoxic
conditions will impair the ability to eliminate these xenobiotics (through interruption of the
CYP1A pathway), and that this will result in increased PAH tissue burdens, reduced larval
survival, decreased reproductive output, and increased hepatic cellular proliferation in marine
and estuarine species.
INTRODUCTION: Previous research has indicated that dispersants such as Corexit may have
serious adverse effects to marine organisms [1, 2], with effects including reduced fertilization
capability [3] and developmental abnormalities [4] in larval fish exposed to oil:dispersant
mixtures. Importantly, co-exposure of crude oil and dispersant has been shown to increase the
uptake rate of oil components from the Water-Accommodated Fraction (WAF) [5, 6]. Therefore,
the high levels of dispersant that were applied in the Deepwater Horizon incident will
significantly increase the fractions of crude oil that are dispersed in the Water Accommodated
Fraction (WAF), increasing bioavailability to pelagic organisms. The impact of the oil release is
exacerbated by the fact that the northern Gulf of Mexico is subject to periodic episode of hypoxia
throughout much of the same area impacted by the oil release [7]. Oxygen depletion has been
documented in 32 of 38 GOM estuaries surveyed in 1994-1995 (USEPA, 1999), while Engle et
al. [8] estimated that between 5.2 and 29.3% of the total estuarine area in the northern GOM was
affected by hypoxia.
Given the size and extent of the release of oil into the northern Gulf of Mexico marine and/or
estuarine organisms are likely to be simultaneously exposed to high levels of dispersed oil and
hypoxia. This has serious implication for the ability of organisms to cope with the
exposure, as the two stressors share a common cellular defense pathway. Key proteins in
each cellular defense pathway (ARNT and HIF1 β) have been shown to be the same protein [9],
implying that simultaneous exposure to hydrocarbons and hypoxia may result in synergistic
toxicity via competition for ARNT/HIF1β dimerization. Exposure to acute hypoxia in the
presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased expression of CYP1A in
zebrafish [10], also indicating crosstalk between the two pathways. Further, modulation of both
CYP1A [11] and HIF1 [12, 13] pathways are associated with impaired reproduction, implying
that simultaneous exposure to chronic hypoxia and dispersed oil may have serious implications
1
for reproductive success in estuarine organisms.
No data exists on the ability of chronic hypoxia
to modulate the CYP1A pathway in estuarine
species following exposure to complex,
dispersed hydrocarbon mixture. The effects of
an interrupted CYP1A pathway on
reproduction and larval development are
similarly unclear.
RESULTS TO DATE:
Embryonic SHM (1
dpf) were exposed to 0, 0.1, or 1.0 % Water
Accommodated Fraction (WAF) for 3 weeks.
Endpoints assessed were hatching success, larval
length, and CYP1A1 induction. WAF was
produced according to established protocols.
Samples of CEWAF were analyzed for PAH
content by MicroMethods (Table 1). Our results
indicate that 1.0% WAF had a significant impact
on hatching success (Fig 1A), as hatch success
was significantly reduced beginning at 120 hours
post fertilization (hpf), and continuing through to
216 hpf. After hatching, juvenile SHM were
Figure 1. Effects of exposure to WAF on Sheepshead
minnow. A. Hatch success. B. Larval growth. Stars
exposed to 0, 0.1, or 1.0% WAF for another 14
indicate significant differences (p<0.05).
days, resulting in a significant (approximately
10%) reduction in length in both WAF treatments compared to controls (Figure 1B). Our results
indicate that relatively low concentrations of dispersed oil offer the possibility of altering growth
and reproduction dynamics in an ecologically sensitive member of the MS estuarine community.
OBJECTIVES:
We hypothesize that high levels of Corexit 9500 result in higher
concentrations of PAHs present in the WAF, leading to enhanced bioavailability and uptake, that
the ability to detoxify these xenobiotics is be impaired by chronic hypoxia, and that the
combination of the two stressors will result in reduced larval survival, decreased reproductive
output, and hepatotoxicity. To test these hypotheses, we will expose juvenile and adult
sheepshead minnow (Cyprinodon variegatus, SHM) to dispersed crude oil under hypoxic or
normoxic conditions. Sheepshead minnow will be used, as they are indigenous to the region and
are ecologically relevant. Additionally, we currently have a large SHM colony present in culture
at GCRL that have been used to examine interactive effects of PAH and hypoxic stress [14, 15].
APPROACH:
Experiment 1:
Chronic hypoxia will impair the ability of juvenile SHM to eliminate
dispersed oil through interruption of the CYP1A pathway.
2
Juvenile (<1dph) SHM will be exposed for 28 days to Normoxia, Hypoxia, WAF+Normoxia,
and WAF+Hypoxia treatments. We will use Quantitative PCR (QPCR) to examine induction of
4 genes involved in hydrocarbon or hypoxia defense pathways. CYP1A activity will be assessed
in juvenile SHM using the ethyoxyresorufin-o-dethylase (EROD) assay [16, 17], modified for
use with juvenile fish [18, 19]. Exposed SHM will be assessed for tissue burdens of TPH and
dispersant.
Table 1. Concentration of oil components in WAF.
Component
1,2,4- Trimethylbenzene
2-Butanone
Acetone
Acrylonitrile
Benzene
Ethylbenzene
m,p-Xylene
o-Xylene
Toluene
Phenanthrene
Naphthalene
2-Methylnaphthalene
Fluorene
Chrysene
Isopropylbenzene
Naphthalene
n-Butylbenzene
n-Propyl Benzene
sec-Butyl Benzene
Concentration (ug/L)
462
214
340
300
11500
534
2410
1070
8680
40.0
211
229
16.4
13.5
48.0
199
21.8
64.8
11.7
Experiment 2:
Synergistic effects between
hypoxia and dispersed oil will lead to reduced larval
survival, altered growth and maturation dynamics,
and reduced fecundity in SHM.
Previous research has shown a direct relationship
between increased CYP1A induction and decreased
vitellogenin synthesis in rainbow trout liver cells [20],
and indicated a link between CYP1A expression and
genes associated with reproduction in largemouth
bass [11]. As exposure to crude and dispersed oil
would normally result in induction of CYP1A, there
is a clear need to investigate the effects of dispersed
oil/hypoxia exposure on reproductive dynamics in
organisms exposed to dispersed oil.
Experiment 3:
Coexposure of WAF and hypoxia will increase liver cellular proliferation
in SHM.:
The reduction in biotransformation ability in SHM co-exposed to WAF and
hypoxia may lead to increased liver tissue burdens and hepatotoxicity. A common marker of this
is increased hepatic cell proliferation, which can be evidenced by PCNA staining. Cell
proliferation is considered an initial response important in development of neoplasia when it is
sustained over time rather than being brief and transient [21].
EXPECTED RESULTS. We will gain critical knowledge about the interactive effects of
simultaneous exposure to dispersed oil and chronic hypoxia on an environmentally relevant
species of estuarine fish. In particular, we will identify 1) the effect of chronic hypoxia on
uptake of PAHs from complex dispersed oil in estuarine fish, 2) to what extent chronic hypoxia
alters crosstalk between the CYP1A and HIF1 pathways in estuarine fish exposed to complex,
dispersed oil, and 3) what the consequences of the dispersed oil:hypoxia induced modulation of
molecular pathways are on reproductive success and larval development.
3
BUDGET
CATEGORIES
A. Personnel
P.I.
Tox Facility Manager
Research Scientist
Lab Technician
Undergraduate student
TOTAL SALARIES
B. Fringe Benefirts
P.I.
Tox Facility Manager
Research Scientist
Project Manager
Undergraduate student
TOTAL FRINGE
TOTAL PERSONNEL
29.62%
32.60%
32.45%
32.60%
0.70%
Name
Robert Griffitt
TBD
Nancy B Peterson
Idrissa Boube
Jeremy Lindsey
Monthly salary
Months
$6,444
1
$3,500
2
$3,548
1
$3,500
6
$823
6
Robert Griffitt
TBD
Nancy B Peterson
Idrissa Boube
Jeremy Lindsey
$1,909
$1,141
$1,151
$1,141
$6
C. Travel
$6,444
$7,000
$3,548
$21,000
$4,940
$42,932
$1,909
$2,282
$1,151
$6,846
$35
$12,223
$55,155
$1,500
D. Supplies
Organisms
Consumables
$2,500
$10,000
shipping
$500
F. Contractual services
histology
hydrocarbon
$1,000
$4,000
TOTAL DIRECT
Indirect Costs (46.5%)
TOTAL
$74,655
$34,714
$109,369
4
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