NATIONAL CONFERENCEATDR A D G Bendale Mahila MahaVidyalay , Jalgoan

12th & 13th Jan, 2012

Relative sensitivity of a marine clams to WSFs of crude oil

Prin. Dr. (Mrs.) Snehal S. Donde. MSc, PhD, PGDEM, MBA

HIND SEVA PARISADS PUBLIC NIGHT DEGREE COLLEGE(Affiliated to University of Mumbai ) Santacruz (E) Mumbai-400 055

Inevitable oil spills

Two Panamanian cargo ships - MSC Chitra and the MV Khalijia-III collided off the Mumbai coast, triggering an oil spill, on August 7, 2010-800 tonnes

Mumbai-Uran pipeline spill- January 21, 2011-55 tones

The oil slick as seen from space by NASAs Terra satellite on May24, 2010 Location Gulf of Mexico near

Disposal of Ballast water

Objectives of study

To study histopathological changes and changing profile of proteins in different tissues of clam Dosinia fibula exposed to 10% stock WSF dilution concentrations of BH crude oil

Crude oil pollution needs greater attention in view of its complicated interaction with biological communities as it is complex mixture of aliphatic, acyclic and aromatic hydrocarbons.

Aromatic hydrocarbons are tainting components and exhibit carcinogenic effect (Rice et.al.1977) and are potentially toxic because they are relatively soluble in water (Siron et.al.1987).

Comparative acute toxicity study of WSFs of BH and PG crude oil on commercially important bivalves by static bioassay method .

Investigate effect of WSFs of crude oil on species of Venerid clams namely, hard shelled Gafrarium divaricatum and soft shelled Dosinia fibula ( Reeve).

IntroductionAfter a spill, hydrocarbons are subjected to physicochemical processes such as evaporation or photochemical oxidation which produce changes in oil composition.

The heavy fraction, made of long-chain saturated and polyaromatic hydrocarbons and a considerable fraction of asphaltenes and resins, is generally non-degradable.

Intertidal organisms are vulnerable to chronic pollution through oil spills on beaches and coated habitat may retain oil for long periods

Mollusk as sentinel organism to monitor marine environment health (Krahn and Stein, 1998)

Many work reported on effect of specific hydrocarbons on various marine organisms but investigations on the effects of WSFs of crude oil on marine bivalves are scanty.

Materials and methods

Clams (length 30-35mm) acclimatized for 7days and were kept in filtered sea water during experimental period of 120 hrs.

A series of stock WSFs were prepared (10%, 20%, and 30 % concentrations by v/v oil-water mixing ) by proportional oil and sea water mixing at constant temperature (Neff and Anderson,1977).

All the samples were analysed five times and all values are averages of five extractions of clams (Rice et al., 1979, Lee et al.,1972).

Weights of hydrocarbon residues in the test solutions ranged from 5.8 ppb to 583.6 ppb. (The sub-lethal petroleum hydrocarbon concentrations ranged from 1750.8 ugl 1 (30%WSF stock ) to 175.80ug l-1 (10%WSF stock dilutions ).

Tissue PAH determined as per the method of Pierce et. al., 1986.

Material & Method Cont-----

WSFs measured by UV fluorescence analysis of hexane extracts using an excitation wavelength of 310 nm and an emission wavelength of 360 nm.

Computation of LC50 values by PROBIT analysis (Finney,1978) and values were then subjected to the test for parallelism and relative potency of WSFs of crude oil for 48, 72, 96 and 120hrs according to the nomographic method given by Litchfield and Wilcoxon (1949).

The LC50 values were analysed for testing the significance of season related variability using ANOVA technique (Single classification) and single tail-t-test. The temperature and LC50 data were further analysed for correlation between the two variables temperature as independent and LC50 value as dependent variable.

The protein from hepatopancreas, gill, adductor muscle and whole body tissue of clam were estimated by the method of Lowry et al., (1951).

General observationsAll clams in pure concentration of WSF stock were found dead by first week.

In 1%, 10%, and 25% dilution concentrations all clams survived till the end of exposure period

The clams generally showed high accumulation in higher concentrations exposure initially and there after the levels dropped by the end of experiment and gragual increase in lower concentrations.

Mainly the depuration was more rapid.

WSFs of PG crude oil was found more toxic than BH crude oil and the Potency Ratio of the two differed significantly in all experimental periods.

Shell thickness Gafrarium divaricatum - upper (0.250 + 0.11 cm), middle (0.272 + 0.09 cm) and lower (0.305 + 0.01 cm)Dosinia fibula upper (0.228 + 0.03), middle (0.161 +0.01cm) and lower (0.152 + 0.10 cm).

LC50 LC50 values of WSF (ppb) of PG crude oil48, 72, 96 and 120hrs readings recorded

Comparison of seasonal variation changes of LC 50 values. (Values are in g /l total aromatic hydrocarbons)

PH =7.5 + 1.57; Dissolved Oxygen = 5.6 + 1.22 ml/l; Salinity = 29.15 + 1.59 %0.

Season/ Temp. Exposure period (h)24487296WinterLC50437.7366.19325.28266.7195% CL439.11- 433.02364.55 - 373.91324.22 - 327.70267.87 - 269.08SF2.212.252.01.7MonsoonLC50875.40551.19400.37395.2095% CL873.21- 877.09550.48 - 552.79399.07 - 401.55322.33 - 327.08SF2.272.252.211.43SummerLC50981.90583.79437.71350.1995% CL980.07 - 982.33582.11 - 584.07436.11 - 438.05349.02 - 352.17SF2.212.202.01.5

Relative Potency and Parallelism between WSFs of BH and PG crude oil (1.5 times greater) during different exposure periods in Gafrarium divaricatm

( a ) SR < fSR - Hence curves are parallel within limits of experimental error ( b ) PR > fPR - Hence the WSFs of two crude oils compared differ significantly in their potency *Factors fSR and fPR read from Nomograph no: 4 ( Litchfield and Wilcoxon, 1949 ) .

Particulars48 hrs72 hrs96 hrs120 hrsBHPG BHPGBHPGBHPGLC50981.94629.4583.66530.3437.68412.7350.1350.0895% Confidence Limit980.48 983.39627.22 631.58581.81585.51529.3531.22431.44443.92412.14413.26347.533352.66348.28351.88Slop Function 2.2502.27052.25372.25022.02.24981.5831.4331Slop Function Ratio ( SR ) 1.00911 1.00155 1.1249 1.1045Factor of SR ( fSR ) 1.65 1.60 1.50 1.18Potency Ratio( PR ) 1.85505 1.39106 1.5462 1.1126Factor of PR (fPR ) 1.36 1.35 1.34 1.08

Relative potency and parallelism between Gafrarium divaricatum and Dodinia fibula toWSFs of PG crude oil. (Values are mean + SD of five determinations)

SR < fSR hence curves are parallel within experimental error. PR > fPR the two compared differ significantly in their potency

Particulars48 hrs72 hrs96 hrs120 hrsG.D.D. F.G.D.D.F.G.D.D.F.G.D.D.F.LC 50629.4489.25529.4358.80412.7243.0351.0127.995% Confidencelimit638.39485.48492.24523.02535.77357.61359.99408.51416.89239.39246.61346.86355.14122.66133.14Slop Function2.273.612.253.251.832.251.582.18Slop Function Ratio( SR )1.451.401.130.63Factor of SR ( f SR )1.51.451.281.1Potency Ratio (PR )1.6172.1563.2342.696Factor of PR ( f PR )1.51.461.321.28

Accumulation and depuration of PAH in two species of marine bivalve in 10% WSF stock ( g / g )(Values are mean + SD of five determinations )PH- 7.5+1.5 ; Dissolved Oxygen - 5.8+ 0.9 mg / l ; Temperature - 27 + 2 o C ;Salinity - 30.2+ 1.05 %o *P < 0.01 **P

*Accumulation and depuration of PAH (g / g) in G. divaricatum

The relation of oil accumulation to the wet wt and lipid content of Gafrarium divaricatum and Dosinia fibula during 96 hr of exposure

Experimental animal Size of Clam (mm)Wet weight of avg. 5 clams (g)WSF conc./g wet wt tissue (ug /g)Lipid Content/g wet weight tissue (mg/g)PAH accumulated in 5 clams (ug/g)PAH /g lipid (mg/g)G. divaricatum22.07 - 28.032.36212.61528.41129.777.465D. fibula21.88 - 27.222.71323.8332.19651.7110.034

Protein content in D fibula during exposure to WSF dilutions of 96 hrs. (Mean + SD of 5 determination)

*P< 0.001; **P

Protein content of hepatopancreas (mg / g wet wt) in Dosinia fibula exposed to 10% WSF (Values are mean of 5 determinations)

Period (days) Concentration of WSF dilutions (%)Exposure0510255075100486.3090.0091.0792.12102.09119.01120.20790.8195.0090.1285.0679.1565.0669.151490.0078.5661.7956.2855.1948.1142.912889.8761.5955.0040.12Recovery186.9970.3965.1645.18785.9275.3271.0759.71

Protein content of Gill (mg/g wet wt) in D. Fibula exposed to 10% WSF dilutions of BH crude oil.

(Values are mean of 5 determinations)

Period (days) Concentration of WSF dilutions (%)Exposure05102550751004105.11111.03115.0109.76115.10111.0112.037101.05112.11111.198.1296.1982.1375.4414105.3399.0783.0487.0075.2966.5539.1828105.5577.5569.0569.1350.11Recovery1106.0980.2169.3952.137111.3283.1978.1564.00

Protein content of adductor muscle (mg /g wet wt) of D. fibula exposed to 10% WSF dilutions of BH crude oil (Values are mean of 5 determinations)

Period (days)Concentration of WSF dilutions (%)Exposure0510255075100494.5593.2698.19103.78111.29110.51112.00798.1199.5999.7889.3671.1163.6754.601499.3989.6381.3773.1268.8259.8842.732896.4380.1279.4463.2256.19Recovery195.1882.8383.6165.17784.3391.7189.9981.11

Concentration of PAH in the various tissues of D fibula

Chart1

0.030.030.020.03

11.193.031.980.65

26.845.9222.751.35

37.410.556.853.17

0.020.020.020.02

6.883.051.220.65

11.025.092.080.93

18.928.453.611.01

0.030.0310.030.031

20.3214.736.453.66

39.0124.6312.095.88

52.8931.0219.167.07

0.010.010.010.011

3.542.091.110.78

6.513.831.971.09

10.115.832.661.15

28 th day accumulation

3rd day depuration

7th day depuration

14 th day depuration

Concentration of WSF dilutions (%)

Conc. of PAH ( ug /g wet wt tissue)

Fig 6.1B : Concentration of PAH in the various tissues of Dosinis fibula

Sheet1

Period051025051025051025051025

28 th day accumulation0.0311.1926.8437.40.026.8811.0218.920.0320.3239.0152.890.013.546.5110.11

3rd day depuration0.033.035.92210.550.023.055.098.450.03114.7324.6331.020.012.093.835.83

7th day depuration0.021.982.756.850.021.222.083.610.036.4512.0919.160.011.111.972.66

14 th day depuration0.030.651.353.170.020.650.931.010.0313.665.887.070.0110.781.091.15

Sheet2

Sheet3

Findings & Conclusion

All the clams exposed to lethal concentration exhibited increase in protein content of muscles during the initial exposure period of 4 days followed by marked decline in protein level throughout the exposure period.

Protein levels in muscle and hepatopancreas were unaffected by exposure of clams to acutely stressful hydrocarbon concentrations for 48 hr. This was also true of clams exposed for 7 days to the lower chronically sublethal concentrations (5, 10, and 25 percent WSF dilution).

The protein values of exposed animals showed an increase over control values during initial exposure period of 96 h in (whole body tissue) all the sublethal concentrations. However, the same concentrations indicated reduced protein levels in muscle in comparison to hepatopancreas.

The control value of the protein in muscle, hepatopancreas and gill showed lower values than the exposure values during 96 h.

Cont----The values indicated a decrease in values from control after prolonged exposure (14 days) in all concentrations. In the adductor muscles the highest percentage variation was observed after 28 days of exposure.

The significant increase of protein values at 96 hr of recovery in the digestive gland may be due to the resumption of normal synthetic activity.

Reason of significant initial higher levels of proteins in exposed clams may be due to the synthesis of polynuclear aromatic binding protein (PBP) as reported by Willett et al., (1999) in a similar study with mussels. Metal binding proteins have been reported from a number of mollusks (Engel and Brouwer, 1982).

Significant reduction in the protein levels in digestive gland, adductor muscle and gills was observed in clams which could have been caused by increased proteolysis due to the effect of PAH on the various tissues of clams.

Conclusion----

There was a significant increase of protein values at 72h of recovery in digestive gland. Levels of adductor muscle protein were found to be higher than control at 96 h of exposure in all concentrations.

It is also suggested that there may be a limit to the synthesis of PAH binding proteins and spill over of toxicant on saturation of these proteins may be found on other ligands.

Decline in the protein content of Dosia fibula exposed to WSFs of crude oil brings marked changes in tissues enzyme activities, which in turn affect the normal physiological behavior during prolonged exposure period.

Willett et al., (1999) demonstrated elevated levels of Aryl hydrocarbon hydroxylase (AHH) and glutathione S-transferase (GST) activities in the hepatopancreas and gill respectively, in mussels collected from PAH contaminated site.

Plate no: 1.1- Normal gill of control (7th day): showing cavity between the lamellae divided by several partitions, the interlamellar junctions (ILJ), and a number of vertical compartments called water tubes (WT). Filled blood sinuses (BS) are very promenient.

Plate no: 1.2 Exposed gill of 5% WSF dilution (7th day): showing incomplete interlamellar junctions (ILJ), Widening of water tubes (WWT), Necrotic junction (NJ) and loss of epithelium (LE). Vacuolization and desquamation in lamellae is seen.

Plate no: 2.1 - Magnification 100X) Normal digestive diverticula of control (7th day): Showing folding in epithelium (FE), inner lumenar space (ILS) and basement membrane (BM).

Plate no: 2.5 - (Magnification 100X) Exposed digestive diverticula of 50% WSF dilution (7th day): showing loss of cilia (LC) in border of typhlosole, disruption of inner lining of tubule (DLT), and infiltration of haemocytes (IH). Atrophy of diverticula, disintegration of epithelial cells, necrotic epithelium with sloughing and vacuolization.

Plate no: 3.2 - (Magnification 100X) Exposed mantle of 5% WSF dilution (28th day): showing loss of basal membrane (LBA) and damage to fibrous connective tissue (DEC). disintegration of mucus sereting cells, total loss of structural integrity, disruption of hemocoelic spaces, and intermingled cells.

Plate no: 3.1 - (Magnification 100X) Normal mantle of control (7th day): section showing outer columnar epithelium (OE), middle fibrous connective tissue (FCT) and inner highly folded ciliated epithelium (ICE).

Plate no: 4.1 - (Magnification 100X) Normal adductor muscle of control (7th day): showing striated and smooth muscle fibres with extended inter muscular spaces (EIMS) structural integrity of the tissue is seen Plate no: 4.2 - (Magnification 100X) Exposed adductor muscle of 5% WSF dilution (7th day): section showing reduced intermuscular space (RIMS) and few necrotic fibres (LST). wide gap between the inter fibrous spaces, infiltration of hemocytes near thick band of tissues

Future strategy

The research study is conducted in cooperation of NIO, versova & Zoology Department, GM Momin College, Bhiwandi. Research project with awareness programme are further planned as follows:

Conducting regular surveys.

Public awareness programs.

Collaborative work in direction of bioremediation. (Microbially mediated removal of contaminants) Oil-Eating Enzyme pseudomonas bacteria contained enzymes that enabled them to break down hydrocarbons

Research in efficacy and effects Oil spill dispersants

Publication of data

References:

Anderson, J.W., J.M. Neff, B.A. Cox, H.E. Tatem, G.M.Hightower 1974. Mar. Biol. 27,75-88.Cohen. 1997. Environ. Pollut. 12: 173-189.Farrington, J.W., A.C. Davis, N.M. Frew, K.S. Rabin 1982. Mar. Biol. 6:15-26.Gilek, M., M. Bjoerk, C. Naef. 1996. Mar Biol. vol. 125 no.3: 499-510.Lee, R.F., R. Sauerber, A.A. Benson 1972. Science 177, 344.Moles,A. 1998. Environ. Contam. Toxicol. 61: 102-107. Metcalf, J.L., M.N. Carlton 1990. Sci. Tot. Environ. 97/98: 595-615.Neff, J.M., B.A. Cox, D. Dixit and J.W. Anderson 1976. Mar. Biol. 38(3): 279-289.Neff,J.M. and J.W. Anderson 1975. In 1975 oil spill conference proceedings. American Petroleum Institute. Washington D.C. pp. 469-472.Rice, S.D., D.A. Moles, T.L. Taylor, J.I. Karinen 1979. In Proceeding of the 1979 oil spill conference, American Petroleum Institute.Shaw,G.David, Thomas.E.Hogan, Douglas, J. McIntosh 1986. Estuarine. Coastal and Shelf Science. 23: 863-872.Stainken, D.M. 1976. Environ. Cont. Toxicol. 16: 730-738. Stegeman, J.J. and J.M. Teal 1973. Mar. Biol. 22: 37-44.

Websites/search enginemarinebiotech.org

http://edac_ecowatch.northerngulfinstitute.org/activity/survey.html (my paper)

http://perdurabo10.tripod.com/storagej/id96.html

http://www.bowdoin.edu/faculty/d/dpage/html/oilspill.shtml

http://www.nap.edu/catalog.php?record_id=11283

http://wiki.ask.com/Deepwater_Horizon_oil_spill