Interactive Effects of Two Environmental Stressors, PAH Contamination and Hypoxia, in the

Interactive Effects of Two Environmental Stressors, PAH Contamination and Hypoxia, in the

Brown Shrimp, Penaeus aztecus

Enmin Zou

Department of Biological SciencesNicholls State University

Thibodaux, LA 70310

Dissolved OxygenContours (mgl)

Hypoxic Zone in NorthernGulf of Mexico

Algal Blooms

Depletion of Oxygen

Degradation of Organic Materials

Nutrients input

Hypoxia is an environmental stress for aquatic organisms, such as penaeid shrimps, inhabiting thesewaters.

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:

< 0.005 – 36.7 ppm< 0.005 – 36.7 ppm (Wade et al., 1988)

Aquatic animals in the northern Gulf of Mexico are subject to dual stresses of hypoxia and PAH contamination

Hypoxia PAH Contamination

The brown shrimp, Penaeus aztecus, is animportant 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

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 promotenaphthalene bioaccumulation.

Oxy

gen

Con

sum

ptio

n R

ate

(OC

R)

0O2 Concentration (C)

Oxyregulation

Oxy

conf

orm

atio

n

Cc

OCR = C/(a + bC)

Impacts of hypoxia on oxyregulation of the brown shrimp,Penaeus aztecus

O2 Concentration (C)

Oxy

gen

Con

sum

ptio

n R

ate

(OC

R)

0Cc Cs

Cc = (aCs/b)1/2

Oxyregulating Capacity = a/b

OCR = b – a/C (Zou et al., 1993)

S = ∫Ca/ba/b(OCR-A)dC + (OCR-A)dC + ∫Cs

C (OCR-B)dC(OCR-B)dC = minimum

Oxy

conf

orm

atio

nA

Oxyregulation

B

Or when S = 0•

a/b

a/b, an index for oxyregulation, reflects an animal’s susceptibility to hypoxia.

The greater a/b, the smaller the oxyregulatingcapacity, i.e. the sooner onset of hypoxic stress.

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

0.00

1.00

2.00

3.00

4.00

5.00

6.00

0.00 2.00 4.00 6.00 8.00

Oxygen Concentration (mg/l)

OC

R (

gO2

• g

-1 •

min

-1)

OCR = 3.64 - 1.54/C ( p < 0.05)A

0.00

1.00

2.00

3.00

4.00

5.00

6.00

0.00 2.00 4.00 6.00 8.00


OC

R (

gO2

• g

-1 •

min

-1)

OCR = 4.81 - 2.18/C (p < 0.001)

B

0.001.002.003.004.005.006.00

0.00 2.00 4.00 6.00 8.00


OC

R (

gO2

• g

-1 •

min

-1)

OCR = 4.99 - 4.32/C (p < 0.0005)C

0.001.002.003.004.005.006.00

0.00 2.00 4.00 6.00 8.00


OC

R (

gO2

• g

-1 •

min

-1)

OCR = 7.21 - 14.55/C (p < 0.0005)

D

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)

Cc = 2.53 mg/l

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50a/

b

(6)(20)

(12)

(18)

Control A Control B 0.5 mg/lNaphthalene

2.0 mg/lNaphthalene

#***

Effects of acute exposure to naphthalene on oxyregulating capacity of Penaeus aztecus

Changes in critical oxygen concentration (Cc) due to naphthalene

Clean Seawater 2.53 mgO2/l

Seawater with 0.05%v/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

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.

Impacts of hypoxia on naphthalene bioaccumulationin the brown shrimp, Penaeus aztecus

Working Hypothesis

Hypoxia

Increased Ventilation

Increased Water-flow over Gills

Enhanced Bioaccumulationof Naphthalene

N2 Tank

Degassing Tank

Exposure Tank

Oxygenation Tank

Pump

Schematic diagram of hypoxia experimental system

Shrimps

Exposure Tank O2 Concentration: 1.32 – 2.61 mg/l

Hypoxia system 1 Exposure tank

2 Oxygenation tank

3 Nitrogen saturated tank

3

2

1

• 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

Ten groups of 25 shrimps each were respectively exposed to:

At days 3 and 7 shrimps were sacrificed for branchial andHepatopancreatic tissues.

0

5

10

15

20

25

Normoxia Hypoxia Normoxia Hypoxia

A

Hepatopancreas Gill

0

5

10

15

20

25


B

Hepatopancreas Gill

0

5

10

15

20


C

Hepatopancreas Gill

01020304050607080


D

Hepotapancreas Gill

10 g/L, day3 10 g/L, day 7

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

7.62 (8) 9.59 (8)

0.51 (8)1.37 (8)

3.35 (6)

9.8 (8)

0.43 (8) 0.67 (8)

8.16 (7)

4.06 (7)

1.21 (7) 1.00(7)

18.98 (7)

26.33 (6)

0.66 (7)0.80 (6)

Naphthalene bioaccumulation in Penaeus aztecusunder hypoxia

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.

NaphthaleneBioaccumulation

Uptake

Elimination

Naphthalene

Naphthalene epoxide

Glutathione conjugates

Naphthalene Metabolism

CYP 1A (Ethoxyresorufin O-deethylase, or EROD)

Glutathione S-transferase(GST)

EROD activities in the hepatopancreas of Penaeus aztecus subjected to various treatments (pM/min/mg protein)

Ace, acetone; Nor, normoxia; Hyp, hypoxia; Nap, naphthalene; (A) * p < 0.05, versus Nap + Nor; (B) * p < 0.05, versus Nap + Nor; (C) * p< 0.05, ** p< 0.01, versus Clean + Nor; (D) ** p < 0.01, versus Ace + Nor ;

0

20

40

60

80

100

120

Ace+Nor Ace+Hyp Nap+Nor Nap+Hyp Clean+Nor

A

01020304050607080


C

*

0

20

40

60

80

100


D

01020304050607080


B10 g/L, day 3 10 g/L, day 7

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

*

** **

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.

0

20

40

60

80

100


B

EROD activities in the gills of Penaeus aztecus subjected to various treatments (pM/min/mg protein)

Ace, acetone; Nor, normoxia; Hyp, hypoxia; Nap, naphthalene; (A) ** p < 0.01, versus Nap + Nor; (C) ** p < 0.01, versus Clean + Nor; (D) * p < 0.05, versus Clean + Nor, ** p < 0.01, versus Ace + Nor and Nap + Nor, # p < 0.05, versus Ace + Hyp;

0

50

100

150

200

250

300


A

**

0

10

20

30

40

50

60

70


C**

0

20

40

60

80

100

120


D*

#

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

250 g/L, day 3250 g/L, day 7

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.

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

Ace, acetone; Nor, normoxia; Hyp, hypoxia; Nap, naphthalene; (B) * p <0.05, ** p < 0.01, versus Nap + Nor; (C) * p < 0.05, versus Clean + Nor, ** p < 0.01, versus Nap + Nor; (D) ** p < 0.01, versus Nap + Hyp;

0

5

10

15

20

Ace+Nor Ace+Hyp Nap+Nor Nap+Hyp Clean+Nor0

5

10

15

20


B*

0

5

10

15

20

25

30


C

0

5

10

15

20

25


D

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

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

A

** ****

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.

0

5

10

15

20

25

30


D

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

Ace, acetone; Nor, normoxia; Hyp, hypoxia; Nap, naphthalene; (A) * p < 0.05, ** p < 0.01, versus Nap + Nor; (B) ** p < 0.01, versus Nap + Nor; (C) * p < 0.05, ** p < 0.01, versus Ace + Nor;

B

0

5

10

15

20

25


A

02468

10121416


02468

10121416


C

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

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

*

**

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.

EROD GST

Hepatopancreas No effect +

Gill – –

Effects of hypoxia on activities of EROD and GSTin the presence of naphthalene

NaphthaleneBioaccumulation

Uptake

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.

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

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

Acknowledgements

Questions?

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Interactive Effects of Two Environmental Stressors, PAH Contamination and Hypoxia, in the