Influence of salinity and fish species on PAH uptake from dispersed crude oil

1. Influence of salinity and fish species on PAH uptake from dispersed crude oil 在不同鹽度下魚種攝取被分散原油PAH之影響 Shahunthala D. Ramachandran , Michael J. Sweezey , Peter V. Hodson , Monica Boudreau , Simon C. Courtenay , Kenneth Lee , Thomas King , Jennifer A. Dixon Marine Pollution Bulletin 2006 ; 52:1182-1189 Reporter : Bei-Chan Liu

2. Introduction

3. Oil spills

4. The coastal areas Salinity Temperature • The solubility of toxic hydrocarbons from the crude oil. • The effectiveness of chemical dispersants. • The binding characteristics of residual oil fractions onto • suspended particles. The accumulation of hydrocarbons by aquatic organisms could also be affected by their osmoregulatory adaptations.

5. Crude oil PAH(多環碳氫化合物) More soluble than alkanes that comprise an equal number of carbon atom. Rank among the most toxic component of crude oil.

6. PAH Solubility The solubility of toluenein different salinity (McAuliffe 1987) The mean reductionin solubility for 12 aromatic hydrocarbons (Sutton and Calder 1975) Fresh water The low salinity coastal water or estuaries and would have a greater adverse impact on aquatic organisms. Seawater 68±4.4%

7. Salinity may modify the performance of chemical dispersants • Much of the work on dispersant effectiveness has tested marine conditions (32–34 salinity), with few freshwater tests. • Dispersants would not be used in shallow waters where dispersion would be limited. • Most dispersants are formulated to work within a narrow range of water salinities, close to that of seawater.

8. Fish can accumulate soluble petroleum hydrocabons very rapidly. (Collier et al. 1995) Gills are primary route of hydrocarbon uptake and excretion, usually by diffusion. (Thomas and Rice 1982) The lighter PAHs Volatilize and solubilize easier The heavier and more toxic PAHs Less soluble If use dispersants ,the hydrophobic nature of the more toxic fractions enables them to partition directly from crude oil to lipid-rich tissues coming into contact with oil droplets.

9. Objective • This research was to measure changes in exposure of fish to PAH when MESA crude oil was dispersed at a range of salinities. • Exposure was estimated by measuring the induction of hepatic cytochrome P450 (CYP1A) activity, an indicator of PAH uptake.

10. Materials and methods

11. Crude oil and dispersant Crude oil :MESA sour crude Dispersant : Corexit 9500 MESA sour crude is insoluble in water and non-volatile when dispersed. Corexit 9500 is meant to be used on higher viscosity oils and emulsions.

12. Test fishes • Juvenile (8–10 weeks) rainbow trout: was chosen to enable comparisons with freshwater data (0–15‰). • Mummichogs were chosen for • exposure bioassays at 15‰ and • 30‰ salinity.

13. Preparation of WAF The crude oil was weathered by sparging with air for 130h. A 1:9 mixture of oil and water 0 ‰ 15 ‰ 30 ‰ Mixture for 18h and settled for 1h The WAF layer was separated from surface oil to use as exposure solution

14. Preparation of CEWAF A 1:9 mixture of oil and water 0 ‰ 15 ‰ 30 ‰ Mixture for 18h of stirring Corexit at a ratio of 1:20 of the oil was added with a further 1 h of stirring and then settled for 1 h. The CEWAF emulsion layer was separated from surface oil to use as exposure solution.

15. Exposure tests five fish A series of WAF concentrations A series of CEWAF concentrations After 48h fish were anaesthetized with 100 mg/L of MS-222. fish were killed by severing the spinal cord. Their livers were removed, weighed.

16. livers microcentrifuge tubes Centrifuges The supernatant (S9 fraction) wasremoved frozen in liquid nitrogen ,and stored at -80 ℃. A model CYP1A inducer, b-naphthoflavone (BNF,10 μg/L), served as a positive control. 0 ‰ 15 ‰ 30 ‰

17. EROD assay In liver CYP1A (EROD) activity was expressed as pmol of resorufin produced per min per mg protein in the S9 fraction.

18. 300 ml PAH analysis Gas chromatography 20 ml dichloromethane Dried by filtration through sodium sulfate concentrated to 1.0 mL GC TPH PAH

19. 20 ml Spectrofluorometry 2 ml hexane Shaken for 20 min and left for10 min The hexane layer Total PAH Spectrofluorometry

20. Statistical analysis • Analyses of variance (ANOVA) were calculated from EROD activity values which had been log transformed to achieve normal distribution. • A one-way ANOVA with treatment as a factor was applied to detect differences among treatments (control, WAF and CEWAF). • Median effect concentrations (EC50) for the WAF and CEWAF exposures of each oil were calculated from induction curves using Graph Pad–Prism fitting a linear regression.

21. Results

22. 0.01 0.1 1 10 EROD activity potency decreased by 20- to 50-fold EROD induction for BNF was similar Mummichog EROD activity CEWAF concentrations similar to trout EROD activity. 0.01 0.1 1 10

23. Comparisons among EC50s The ratio dropped by about 35-fold

24. PAH concentrations in bioassay treatments 0.1 v/v CEWAF 0ppt > 15ppt

25. Total PAH

26. Discussion

27. Decreased exposure with increasing salinity • These experiments corroborate earlier work on • increased exposure to PAH with chemical dispersion • of crude oil (Ramachandran et al., 2004). salinity EROD activity dispersant effectiveness PAH solubility binding capacity onto suspendedparticulate matter osmoregulation in test fish

28. Dispersant effectiveness Dispersant effect did not seem to change between salinities of 0‰ and 15‰. The dispersant was less effective at high salinities. Binding capacity onto suspendedparticulate matter Interactions between PAH and particulates might also be affected by salinity. Fish were not fed for 48 h prior to testing.

29. PAH solubility Salinity effects on PAH uptake by fish is that salinity controls PAH solubility and bioavailability. Two (naphthalenes) and three (phenanthrene) ring compounds. PAH molecule weight The low molecular weight (LMW) The higher molecular weight (HMW) Four (fluorene) and five (pyrene , chrysene) ringed compounds. Solubility : LMW ＞HMW

30. LMW HMW

31. Osmoregulation in test fish Hypo-osmotic environment Fish are subjected to diffusion of water from the surrounding medium into the gill, as is the case with freshwater fish. Hypo-osmotic environment iso-osmotic conditions This process slows until iso-osmotic conditions. In this study, responses of fish to PAH did not change between 0‰ and 15‰ salinity.

32. 15‰ 30‰ Reduced PAH uptake from CEWAF by mummichogs. EROD activity in mummichog exposed to BNF was reduced by one half The reduction in PAH uptake at higher salinities might be due to water and PAH efflux in response to osmotic gradients.

33. Conclusion

34. Oil spills PAH will be up to 60-fold Full salinity Low salinity Use of dispersants 10 times 250 times • The increased solubility of PAH at lower salinities, especially • the lower molecular weight two- and three-ringed homologs. • This solubility effect is enhanced by the apparent increased • effectiveness of chemical dispersion at low salinities. • The potential risks to aquatic life of PAH toxicity following oil • spills are enhanced in lower salinity waters such as estuaries • and near coastalzones.

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