Interactive%20Effects%20of%20Two%20Environmental%20Stressors,

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Interactive Effects of Two Environmental
Stressors, PAH Contamination and Hypoxia, in the
Brown Shrimp, Penaeus aztecus Enmin
Zou Department of Biological Sciences Nicholls
State University Thibodaux, LA 70310
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Hypoxic Zone in Northern Gulf of Mexico
Nutrients input
Algal Blooms
Degradation of Organic Materials
Depletion of Oxygen
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Hypoxia is an environmental stress for aquatic
organisms, such as penaeid shrimps, inhabiting
these waters.
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Contamination of polycylcic aromatic
hydrocarbons (PAHs) from gas and petroleum
production on the northern continental slope of
the Gulf of Mexico
Total PAH in Gulf of Mexico sediments lt 0.005
36.7 ppm (Wade et al., 1988)
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Aquatic animals in the northern Gulf of Mexico
are subject to dual stresses of hypoxia and PAH
contamination
PAH Contamination
Hypoxia
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• The brown shrimp, Penaeus aztecus, is an
• important commercial shrimp, whose life
• history overlaps with the hypoxic zone in the
• northern Gulf of Mexico.
• Penaeus aztecus is faced with two stressors
• Hypoxia
• PAH contamination

Hypoxia
PAHs
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Objectives 1. To investigate whether acute
exposure to naphthalene, a representative
petroleum PAH, can make shrimps more vulnerable
to hypoxia. 2. To investigate whether hypoxia
can promote naphthalene bioaccumulation.
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Impacts of hypoxia on oxyregulation of the brown
shrimp, Penaeus aztecus
OCR C/(a bC)
Oxygen Consumption Rate (OCR)
Oxyregulation
Oxyconformation
0
Cc
O2 Concentration (C)
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S ?Ca/b(OCR-A)dC ?CsC (OCR-B)dC minimum

Or when S 0
Cc (aCs/b)1/2 Oxyregulating Capacity a/b
OCR b a/C (Zou et al., 1993)
Oxygen Consumption Rate (OCR)
0
a/b
O2 Concentration (C)
Cc
Cs
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a/b, an index for oxyregulation, reflects an
animals susceptibility to hypoxia. The greater
a/b, the smaller the oxyregulating capacity, i.e.
the sooner onset of hypoxic stress.
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• Four groups of brown shrimps were respectively
• exposed to
• Clean seawater
• Seawater with 0.05 v/v acetone
• Seawater with 0.5 mg/l naphthalene
• Seawater with 2.0 mg/l naphthalene

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Cc 2.53 mg/l
Clean Seawater (wt 9.00 g)
0.05 v/v Acetone (wt 7.02 g)
0.5 mg/l Naphthalene (wt 8.17 g)
2.0 mg/l naphthalene (wt 7.53 g)
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Effects of acute exposure to naphthalene on
oxyregulating capacity of Penaeus aztecus
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Changes in critical oxygen concentration (Cc)
due to naphthalene
Clean Seawater 2.53 mgO2/l
Seawater with 0.05v/v acetone 2.89 mg O2 /l
0.5 mg/l Naphthalene 3.76 mg O2 /l
2.0 mg/l Naphthalene 4.22 mgO2/l
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Conclusion Acute exposure to naphthalene
reduces oxyregulating capacity of the brown
shrimp, Penaeus aztecus, subjected to
progressive hypoxia, thereby making shrimps more
suceptible to hypoxia.
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Impacts of hypoxia on naphthalene
bioaccumulation in the brown shrimp, Penaeus
aztecus
Working Hypothesis
Hypoxia
Increased Ventilation
Increased Water-flow over Gills
Enhanced Bioaccumulation of Naphthalene
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Schematic diagram of hypoxia experimental system
Degassing Tank
Pump
N2 Tank
Shrimps
Exposure Tank
Exposure Tank O2 Concentration 1.32 2.61 mg/l
Oxygenation Tank
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Hypoxia system 1 Exposure tank 2
Oxygenation tank 3 Nitrogen saturated tank
3
1
2
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Ten groups of 25 shrimps each were respectively
exposed to

• Clean Seawater
• 0.005 v/v Acetone Normoxia
• 0.005 v/v Acetone Hypoxia
• 10 ?g/l Naphthalene Normoxia
• 10 ?g/l Naphthalene Hypoxia
• Clean Seawater
• 0.01 v/v Acetone Normoxia
• 0.01 v/v Acetone Hypoxia
• 250 ?g/l Naphthalene Normoxia
• 250 ?g/l Naphthalene Hypoxia

At days 3 and 7 shrimps were sacrificed for
branchial and Hepatopancreatic tissues.
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Naphthalene bioaccumulation in Penaeus
aztecus under hypoxia
10 ?g/L, day3
10 ?g/L, day 7
9.8 (8)
7.62 (8)
9.59 (8)
3.35 (6)
0.51 (8)
1.37 (8)
0.43 (8)
0.67 (8)
250 ?g/L, day 3
250 ?g/L, day 7
8.16 (7)
26.33 (6)
18.98 (7)
4.06 (7)
1.21 (7)
1.00(7)
0.66 (7)
0.80 (6)
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• Conclusions
• Naphthalene was found to mainly accumulate in
  the
• hepatopancreas---Naphthalene burden in the
• hepatopancreas up to 60 times that in the
  gills.
• Hypoxia did not significantly alter naphthalene
• bioaccumuluation in either organs.

Uptake
Naphthalene Bioaccumulation
Elimination
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Naphthalene Metabolism
Naphthalene
CYP 1A (Ethoxyresorufin O-deethylase, or EROD)
Naphthalene epoxide
Glutathione S-transferase (GST)
Glutathione conjugates
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EROD activities in the hepatopancreas of Penaeus
aztecus subjected to various treatments
(pM/min/mg protein)

10 ?g/L, day 7
10 ?g/L, day 3
250 ?g/L, day 7

250 ?g/L, day 3

Ace, acetone Nor, normoxia Hyp, hypoxia Nap,
naphthalene (A) p lt 0.05, versus Nap Nor
(B) p lt 0.05, versus Nap Nor (C) plt 0.05,
plt 0.01, versus Clean Nor (D) p lt 0.01,
versus Ace Nor
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Summary of results for EROD activities in the
hepatopancreas

• Naphthalene significantly induced
  hepatopancreatic
• EROD activity under normoxia.
• Hypoxia had no significant effects on
  hepatopancreatic
• EROD activity in the presence of naphthalene.
• Acetone significantly inhibited EROD activity
• in the hepatopancreas.

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EROD activities in the gills of Penaeus aztecus
subjected to various treatments (pM/min/mg
protein)
10 ?g/L, day 3
10 ?g/L, day 7


250 ?g/L, day 3

250 ?g/L, day 7

Ace, acetone Nor, normoxia Hyp, hypoxia Nap,
naphthalene (A) p lt 0.01, versus Nap Nor
(C) p lt 0.01, versus Clean Nor (D) p lt
0.05, versus Clean Nor, p lt 0.01, versus Ace
Nor and Nap Nor, p lt 0.05, versus Ace
Hyp
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Summary of results for EROD activities in the
gills

• Hypoxia significantly inhibited branchial EROD
• activities when naphthalene was present.
• Acetone significantly suppressed branchial EROD
• activities under normoxia
• Under hypoxia, naphthalene at 250 ?g/l inhibited
  EROD
• activities in the gills.

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GST activities in the hepatopancreas of Penaeus
aztecus subjected to various treatments
(nM/min/mg protein)
A

10 ?g/L, day 3
10 ?g/L, day 7
250 ?g/L, day 3
250 ?g/L, day 7



Ace, acetone Nor, normoxia Hyp, hypoxia Nap,
naphthalene (B) p lt0.05, p lt 0.01, versus
Nap Nor (C) p lt 0.05, versus Clean Nor,
p lt 0.01, versus Nap Nor (D) p lt 0.01,
versus Nap Hyp
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Summary of results for hepatopancreatic GST
activities

• Naphthalene significantly induced GST activities
• in the hepatopancreas under both normoxia and
• hypoxia.
• Hypoxia significantly elevated GST activities in
  the
• hepatopancreas when naphthalene was present.

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GST activities in the gills of Penaeus aztecus
subjected to various treatments (nM/min/mg
protein)
10 ?g/L, day 3
10 ?g/L, day 7
B

250 ?g/L, day 3
250 ?g/L, day 7

Ace, acetone Nor, normoxia Hyp, hypoxia Nap,
naphthalene (A) p lt 0.05, p lt 0.01, versus
Nap Nor (B) p lt 0.01, versus Nap Nor (C)
p lt 0.05, p lt 0.01, versus Ace Nor
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Summary of results for branchial GST activities

• Hypoxia significantly decreased branchial GST
• activities in the presence of naphthalene.
• Under normoxia, naphthalene at 250 ?g/l
  significantly
• suppressed branchial GST activities.

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Effects of hypoxia on activities of EROD and
GST in the presence of naphthalene
EROD GST
Hepatopancreas No effect
Gill
Uptake
Naphthalene Bioaccumulation
Elimination

• The significant increase in GST activity may
  explain the absence
• of increased naphthalene bioaccumulation in
  the hepatopancreas
• when Penaeus aztecus is subjected to hypoxia.
• Alterations in activities of EROD and GST have
  no bearings on
• naphthalene bioaccumulation in the gills
  because gills are not an
• ideal organ for bioaccumulation of chemicals.

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• Take-home messages
• Acute exposure to PAH compound naphthalene
  reduces oxyregulating capacity of the brown
  shrimp.
• Hypoxia does not promote bioaccumulation of the
  PAH compound naphthalene in the brown shrimp. The
  absence of such a significant effect is
  attributed to increased naphthalene metabolism in
  the hepatopancreas when the shrimp is subjected
  to hypoxia

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Acknowledgements
Appreciation to the funding agencies, NOAA and
USGS, and my students Rongzhong Ye and Ben
Stueben.
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Questions?