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

1. 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

2. Dissolved OxygenContours (mgl) Hypoxic Zone in Northern Gulf of Mexico Nutrients input Algal Blooms Degradation of Organic Materials Depletion of Oxygen

3. Hypoxia is an environmental stress for aquatic organisms, such as penaeid shrimps, inhabiting these waters.

4. 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 (Wade et al., 1988)

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

6. 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

7. 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.

8. 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)

9. B Oxyregulation A Oxyconformation 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 Cc O2 Concentration (C) Cs

10. a/b, an index for oxyregulation, reflects an animal’s susceptibility to hypoxia. The greater a/b, the smaller the oxyregulating capacity, i.e. the sooner onset of hypoxic stress.

11. 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

12. 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)

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

14. Changes in critical oxygen concentration (Cc) due to naphthalene

15. 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.

16. 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

17. 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

18. Hypoxia system 1 Exposure tank 2 Oxygenation tank 3 Nitrogen saturated tank 3 1 2

19. 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.

20. 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) 8.16 (7) 250 g/L, day 3 26.33 (6) 250 g/L, day 7 18.98 (7) 4.06 (7) 1.21 (7) 1.00(7) 0.66 (7) 0.80 (6)

21. 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

22. Naphthalene Metabolism Naphthalene CYP 1A (Ethoxyresorufin O-deethylase, or EROD) Naphthalene epoxide Glutathione S-transferase (GST) Glutathione conjugates

23. B * 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 < 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 ;

24. 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.

25. 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 < 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;

26. 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.

27. 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 <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;

28. 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.

29. 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 < 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;

30. 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.

31. Effects of hypoxia on activities of EROD and GST in the presence of naphthalene 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.

32. 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

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

34. Questions?