THE SIGNIFIC.4NCE OF CLAY-OIL FLOCCULATION PROCESSES TO OIL

BIODEGRADATION.

Andréa M. Weise

-4 thesis submitted in conformity with the requirements

for the Degree of Master of Science.

Department of Botany at the University of Toronto.

c3 Copyright by Andréa M. Weise. 1997.

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Canada

THE SIGNIFICANCE OF CLAY-OIL FLOCCULATION PROCESSES TO OIL

BIODEGRADATION.

Andréa iM. Weise

Master of Science. Department of Botan?. University of Toronto. 1997.

ABSTRACT

Clay-oïl tlocculation, involving the interaction of micron-sized mineral tines with oil

droplets. has been showm to be an important natural cleansing process on low-energy

shorelines by enhancing dispersion rates of oil into the sea. While oil may be effectively

removed tiom the shoreline by tlocculation. the significance of this physico-chemical process

on the persistencr of the oil dispersed in the ocean is unknow-n. Our hypothesis is that clay-

oi 1 tlocculation cnhances hydrocarbon degradation rates by preventing the oil from adhering

ris strongiy to solid surfaces and maintaining oil droplets in the water column. thereby

increasing the oil-water interface by several orders of magnitude and making the oil more

accessible to nutrients. oxygen. and bacterial attack. Shaker Hask esperirnents rvere

conducted over a 63 da- period (19°C) and over a 56 day period (10°C). Results indicated

that bacterial activity kvas stimulated and both the rate and estent of hydrocarbon degradation

kvas enhanced.

ACKNOWLEDGMENTS

I wish to thank Dr. Kenneth Lee for supen-king my graduate work and supporting the

project financially. 1 also wish to th& Dr. Czesia Nalewajko. rny co-supervisor. who

diligently read. and corrected my thesis. I express my gratitude to my committcr members:

Dr. J.A. Hellebust. Dr. D.J. Kushner. and Dr. R. Fulthorpe. Valerie Anderson w s a h - a y s

there to answer al1 my "administrative" questions.

This project 1%-ould not have been possible without the financiai support from the

Fonds pcrrrr kr Formttion tic) Ch~.rchrrrrs et ci ii-li~ie N Irr Recherche (FCAR) in the torm of a

scholarship and the Federal Summer Student Employment Program. In addition. the Institut

Maurice-Lamontagne ( Mont-Joli. Québec) proved to b r an ideal environment to conduc t al l

my laboratoq- studiss. I must also thank: Richard Larocque and Denis Guay for their

technical support: Patncia Sto-n-Egli for her wonderful ESEM work: Claude Guay and

Sylvic St-Pierre for hrlping me prrpare the infinite number of columns: and Johanne

Gauthier for hrrr sound insight.

Last. but nor Ieast. rny family and close fnends for their endurinç suppon and love.

TABLE OF CONTENTS

.4 CKiW WL EDGMENTS iii

LIST OF TABLES . . vi1

LIST OF FIGURES vi i i

LIST OF APPENDICES xiii

INTRODUCTION 1

1.1 ENVIRONiMENTAL IMPORTANCE I

1.2 OIL-SUSPENDED PARTICULATE MATTER INTER4CTIONS 4 1 2 . 1 Molecular levrl interactions: adsorption 4 1.7.2 Macroscale level interactions: whoie oil droplets and mineral fines 5

1.3 PROCESS DEFINITION 8 1.3- 1 Flocculation between cIaq-s 8 1.3 -2 Clay-oil flocculation 9

1.4 FACTORS AFFECTING CLAY-OIL FLOCCULATION 1 1 1.4.1 Oil 1 I l.4.Z. Salinity of the \vater 13 1 A.3 Mineral particles 14 1.4.4 Physical parameters 15

1.5 FACTORS AFFECTING HYDROCARBON DEGMDATION 15 I.5.l Hydrocarbon-degrading microbial populations 16 1 3 .2 Physical and chcmical properties of the oil 17 1 -5.3 Temperature 18 1.5.4 Nutrients 19 1 -5.5 Osygen 19

1.6 OBJECTIVES OF THE STUDY 20

L M ~ TERIALS AND METHODS 21

2.1 P R E P A U T I O N OF MATERIAL ? 1 2.1.1 Referencs minera1 fines 2 1

2.1.1.1 Preparation of mineral fines for the descriptive studies on ci--oil flocculation and the tint biodegradation study ( 19°C) 2 1

1 -1 -12 Preparation of minera1 fines used in the second biodegradation study ( 1 O°C) - 24 2.1.3 Test oils 37 2 - 1 3 Seawater 39 2.1 -4 Nutnent solution 39

2.2 ANALYTICAL iMETHODS 30 2.2.1 Microscopy 30

2.2.1.1 U V N isible light microscopy 30 2 2 - 1 2 Environmental Scanning Electron Microscopy 30

7.2.2 Total hydrocarbon concentration 32 2.2.2.1 Oil extraction prior to ultraviolet tluorescence spectroscopy 32 7.2.2.2 Ul traviolet tluorescence spectroscopy 33

2.2.3 Chernical analysis 34 2.2.3.1 Capillary pas chromatography and flame ionization detection 34 2 2 - 3 2 Gas chromatography and mass spectroscopy 37

-.-. 4 Microbiological analysis 39 2.2.4.1 Most-Probable-Number 39 1 -2-42 Bacterial activity as measured by thymidine uptake 4 1 2.2.4.3 Potrntial activity of hydrocarbon-depding bacteria as rneasured by

14 minerdiution of n-[ 1 - C] hesadecane 42 2.2.5 'lutrient analysis 44

2.3 EXPERIMENTAL PROCEDURES -45 2.3.1 Studies on the prxess of clay-oil flocculation 45

2.3.1.1 S haking methods 15 2.3.1 -2 FIocculation of minera1 fines with different oils 46 2 3 ! 3 Clay-clay and clay-oil flocculation 46 2.3.1.4 S i x merisurernents of clay-oil floc aggregatss 47 2.3- 1.5 Association of oil with mineral tines in relation to shaking time 18

2.3.2 Biodsgradation studicss 4 9 3 . 2 1 First biodegradation study ( l9OC) 49 1 3 - 2 2 Second biodegradation study ( 1 O°C) 50

3.1 INTERACTIONS BETWEEN OIL AND FINE MINERAL PARTICLES 52 3.1 - 1 Shaking methods 53 3.1.2 Clay-oil floc formation with various crude oiIs 53 3.1.3 Qualitative assessment of clay-oil floc aggregates 59

3.1 3.1 Effect of mineral fine concentration 59 3.1.3.2 Effect of settling timr 62 1 . 3 Effect of shakinp tirne (duration of turbulence) 62

3.1.4 Analysis of floc aggregates by Environmental Scanning Electron Microscopy 65 3-13 Percentage of oil incorporated into clay-oil floc aggregates 69

3.2 FIRST BIODEGRADATION STUDY (19OC) 73 3 2.1 Chernical analysis 73

3 . 1 1 Hydrocarbon degradation in the saturate fraction 73 3 2 . 1 -2 Hydrocarbon degradation in the arornatic fraction 79 3.2.1 -3 Rates of oil biodegradation 79

3 2.2 Microbiological anaiysis 82 3 -2.2.1 Potential oil-degrading bacteria 82 3.3.2.3 Bacterial productivity 83 3.3.2.3 Microbia1 activity wlthin the first 23 hours 87 3.2.2.4 Estimated numbers of heterotrophic and oil-degrading bacteria 87

3 2.3 Nutrient analysis 89

3.3 SECOND BIODEGUDATION STUDY (lO°C) 92 3.3.1 Chernical analysis 92

33.1. l Total hydrocarbon degradation in the saturate fraction 93, 3.3-1.2 Total hydrocarbon degradation in the aromatic fnction 101 3.3.1.3 Distribution of the aliphatic fraction between the aqueous and solid phase 1 0 9 3.3.1 .-I Distribution of the aromatic fraction between the aqurous and solid phase -1 18

3 . 7 2 !vlicrobiological analysis 123 3 3 1 Potential oil-degrading bactena 123 3 -3.2.2 Bacterial productivity 127 3 -3 2.3 Estirnateci numbers of heterotrophic and oil-degrading bacteria 129

3.3 -3 Nutrient analysis 131

CONCL USIONS 146

R EFER ENCES 149

LIST OF TABLES

Tab

Tab

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 1 O

Percentage minera1 tines inferior to the indicated particle diameters in the 23 mineral fine stock solution used in the descriptive studies on clay-oil tlocculation. as measured by Coulter LS-100 Particle Sizr ha iy s i s .

Percentage minerai fines inferior to the indicated particle diarneters in the 75 rnineral fine stock solution used in the first biodegradation study ( 19°C). as mrasured by Coulter LS- 100 Particle Size Analysis.

Flocculation of a variety of oils with mineral tines following a 20 minute 54 shaking penod.

Wcathering ratios calculated from chrornatographic traces in oiled 76 controls. minera1 fine arnended samples. abiotic oiled controls. and abiotic minrral fine arnended sarnples.

Hrsadecane concentration rernaining in oiled controls and minrral fine 85 amended samples over time. as calculated from GC-FID chrornatographic traces.

Most-Probable-Number ( MPN) and range (95% confidence limit ) of 90 hetrrotrophic and oil-deprading bacteria on Day I and Day 63 in oilrd controls and mineral the. amended samples.

Percentage biodegradation rate for given penods of timr of total n-alkanes 97 ( U - C , ~ to n-C in oiled controls and rnineral fine arnendrd sarnples.

Percentage biodegradation rate for given pet-iods of timr of total aromatics 105 in oiled controls and minerai fine amended samples.

Xromatic weathering ratios in oiled controls and mineral tïne amended samplrs over time. as calculated by GC-MS.

Hesadecane concentration remaining in oiled controls and minera1 fine arnended samples over time.

LIST OF FIGURES

Figure 1 Schematic diagram of a typical ciay-oil floc aggregate. 1 O

Figure 1 Particle diameter distribution and cumulative frequency of mineral fines 26 used in the second biodegradation study ( 10°C). as mrasured by laser eranulometry techniques. C

Figure 3 Ultraviolet fluorescence spectroscopy calibration cunre for weathered 35 Terra Nova Cnide Oil (0.85 m-f i ) .

Figure 4 Photomicrograph of a clay-oil floc aggregate under visible light and LW 56 epi-fluorescence microscope formed with (a) Alberta Sweet Mixed Blend Cnide Oil: and (b) Sable Island Condensate.

Figure 5 Photomicrograph under visible light of (a) a buoyant clay-oil floc 57 aggregate formed with Alberta Sweet Mixrd Blend Cnide Oil ( 1 700 ppm) and mineral fines (1000 ppm) following a 70 minute shaking penod and a 1 -week stationary period: and (b) magnification of the same floc.

Figure 6 Photomicrograph under visible light and UV epi-tluorescence microscope 58 of (a) a non-buoyant clay-oil floc aggregate formed with Alberta Sweer Mised Blend Crude Oil ( 1700 ppm) and mineral fines ( 1000 pprn) Following a 20 minute shaking penod and a 1 -wek stationary period: and (b) magnification of the same floc.

Figure 7 Photomicropraphs under visible light and UV cpi-fluorescence 6 1 microscope of clay-oil tloc aggregates formed with weathered Terra Nova Cnide Oil(60 ppm) and v q i n g arnounts of mineral fines: !O ppm. 60 ppm. 140 ppm. 300 pprn. and 600 ppm.

Figure 8 Frequency of surface area (pn2) of clay-oïl floc aggregates remaining in 63 suspension following a shaking penod of 21 hours and settling times of O. 1 5. 30. and 60 minutes soned by size: 1 (0-200 y?). 2 (?O 1100 3 (401 -600 Pm2). 4 (60 1-800 5 (80 1 - 1000 and 6 (1 00 1 - 1200 pn'): and oii droplets sorted by size: 1 (0-45 2 (46-90 3 (9 1- 135 Pm2). 4 ( i 36- 180 pn'). and 5 ( 1 8 1 - 2 3 pn2).

Figure 9

Figure 1 O

Figure 1 1

Figure 12

Figure 1 3

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Frequency of surface area ( pn2) of clay-oil floc aggregates rernaining in suspension following shaking times of 0.5. 1.5.4.0. and 24.0 hours sorted bp size: 1 (0-100 2 (20 1-100 pn2). 3 (40 1-600 pin2). 1 (60 1-800 pz). 5 (80 1 - 1 000 and oil droplets soned by size: 1 (0-15 pm2). 2 (16-90 pn2). 3 (9 1 - 135 pn'). 1 ( 136- 180 and 5 ( 1 8 1-225 pn2).

Elrctron rnicrograph of a clay-oil floc aggregate (a) wrt: and ( b ) with the surface watsr rernoved.

Electron micrographs comparing tlocculated aggregates containing onl); minera1 fines and clay-oil floc aggregates: (a) clay-clay aggregates (35 ppm mineral fines): (b) clay-clay aggregates ( 150 pprn minera1 fines): c ) clay-oil floc aggregate (3 pprn mineral tines and 75 pprn weathered Tsrn Nova Cnide Oil): and (d) clay-oil floc aggregate ( 150 pprn mineral fines and 75 ppm weathered Terra Nova Cnide Oil).

Electron micrograph of a clay-oil floc aggregate formed with 75 pprn weathered Terra Nova Cnide Oil and 10 pprn minerai iinss.

Percent oil incorporated into clay-oil floc aggregates. as re-coalesced oil. and on glassware vs. shaking time in sarnples arnended with mineral fines.

Distribution of oil os re-coalrscrd oil and on glassware vs. shaking time in oiled controls.

Prrcentagr total 17-alkanes (n-Cl, to n-Cj5) rernaining in oiled controls and mineral fine amendrd samples. relative to Da); 1.

Distribution and concentration of n-altanrs (n-CI? to n-C,5) in abiotic oiled controls and abiotic mineral tine amrndrd sarnples on Day 1 and Day 63.

Distribution and concentration of individual n-alkanes in oiied controls and mineral fine arnended samples on Days 1. 7. 14. 35. and 63.

Percsntage total aromatics remaining in oiled controls and minerai fine amsnded samples relative to Day 1.

Change in total n-alkane (n-Cl? to n-C;,) concentration between Day 1 and Dav 35 in oiled controls and mineral fine arnended sam~les.

Figure 20 Fieniidecane respiration rates in oiled controls and minera1 fine arnrnded 84 sar.iples over time.

Figure 2 1 klethyl-['~lthyrnidine incorporation rates in oiled controls and mineral 86 fine amended samples over time.

Figure 22 ~ e t h ~ l - [ ' ~ ] t h y n i d i n r incorporation rates within the tirst 24 hours in 88 oiled controls and mineral fine amended sarnples.

Figure 3 Percentage n-[ 1 - '"CI henadecane recovered as respired "'CO: in oiled 88 controls and mineral fine arnended samples over time.

Figure 24 Nutrient concentrations in oiled controls and mineral fine amended 9 1 sarnples over time: (a) nitrite and nitrate: (b) arnmonia: and c ) ortho- phosphate.

Figure 25 Prrcentaee - total n-alkanes (n-Cl. to n-Cji) remaining in oiled controls and 93 minera1 fine arnended samples. relative to Day 1.

Figure 26 Change in total n-alkane (n-C,, to n-C ji) concentration between Da- 1 95 and Day 56 in oiled controls and mineral tine amended sarnples.

Figure 27 Percent individual n-alkanes remaining relative to Day 1 in oiled controls 99 and minerai fine amended sarnples on Days 7. 14. 28. and 56.

Figure 28 Distribution and concentration ofn-alkanes on Day 1 and Day 56 in (a ) 100 abiotic oiled controls: and (b) abiotic minera1 fine amended samples.

Figure 29 Tims-series change in the ratio of n-C,,!phytane in oiled controls and 102 minera1 fine amended samplrs.

Figure 30 Percent total aromatics remaining in oiled controls and mineral tine 103 arnrnded samples. reiative to Da- 1.

Figure 3 1 Percent targrt PAH homologues remaining. relative to Day 1. in oilrd 1 O6 controls and mineral fine amended sarnples on Days 7. 14.28. and 56.

Figure 32 Percentage rotal n-alkanes (n-CI- to n-Cj5) remaining in the aqueous 110 phase (water column) and in the solid phase (glassware surfaces) in (a) oiled controls: and (b) minera1 fine amended samples over time.

Figure 33

Figure 34

Figure 35

Figure 36

Figure 3 7

Figure 38

Figure 39

Figure 40

Figure 4 1

Figure 42

Figure 43

Tirne-series change in the ratio of n-CIs/phytane in oiled controls and I l 3 minera1 fine amsnded sarnples.

Change in total n-alkane (n-C,? to n-Cjc) concentration between Day 1 115 and Day 38 in the aqurous phase of oiled controls and minerai fine arnended samples.

Percent individual ît-alkanes remaining. relative to Day 1. in the aqueous 1 16 phase (water column) and solid phase (glassware surfaces) of oiled controls on Days 1 - 7- 14-28. and 56.

Percent individual n-alkanes remaining. relative to Day 1. in the aqueous 1 17 phase (water column) and solid phase (glassware surfaces) of minera1 fine amended sarnples on Days 1. 7. 14.28. and 56.

Percentage total aromatics remaining in the aqueous phase (water column) 1 19 and in the solid phase (glassware surfaces) in (a) oiled controls: and (b ) mineral tine amended sampIes over time.

Percent trugrt PAH homologues remainine. relative to Day 1. in the 121 aqueous phase (water colurnn) and solid phase (glassware surfaces) of oiled controls on Days 1. 7. 14. 28. and 56.

Percent target PAH homologues remaining. relative to Day 1. in the aqueous phase (water column) and solid phase (glassware surfaces) of mineral fine arnended sarnples on Days 1. 7. 14. 28. and 56.

Hssadrcane respiration rates in oiled controls and minera1 fine amended 124 samples over tirne.

Hesadecane turnover time in oikd controls and minera1 fine arnsnded 126 sarnples O\-er time.

~ s t h ~ l - [ ' ~ l t h ~ m i d i n e incorporation rates in oiled controls and mineral 128 fine arnended samples over tirne.

Change in the Most-Probable-Number (MPN) in oiled controls and 130 minera1 fine amended samples of (a) oil-degrading bactena and (b) heterotrophic bacteria.

xii

Figure 4! Nutrient concentrations in oiled controls and minerai fine amended 132 sarnples over timç: (a) nitrite and nitrate: (b) ammonia: and c ) ortho- phosphate.

LIST OF APPENDICES

Appendix .A Characteristics of the five oil groups (National Oceanic and Atrnos- 159 pheric Administration. I 994).

Appendis B Characteristics of the test oils. 160

INTRODUCTION

1.1 ENVIRONMENTAL IiMPORTANCE

Major oil spills rvoke considerable public concem and can result in significant

contamination of ocean and shoreline rnvironments. For esample during the last decade. the

Sea Empre-s~ released 72.000 tonnes of Forties Blend and 370 tonnes of Heavy Fuel Oil in

Milford Haven. UK in 1996 (Lee et al.. 1997a): the Bruer discharged 85.000 tonnes of

Gulltàks Nowegian Cnide Oii into the coastal waters of the Shetland Islands in 1993 (Wolff

et al.. 1993): and the E n o n Cirldez spilled 35.500 tonnes of North Slope Cnide Oil into

Prince William Sound. .AK in 1989 (Wolfe et c d . - 1994). Most spills contaminate substantial

lengths of coastline. posing considerable threats to the environment. For example.

approsimately 2.090 km of coastline ivere oilrd following the EY- on I u l d e ~ spi11 (Bragg rr

trl.. 1992). Such incidents have prompted the development of methods to remove stranded oil

from shorelines. including high pressure washing. in siru buming. dispersants. and chemical

beach cleaning agents. These incidents also stimulated research to obtain a bener

understanding of the "self-cleaning" of oilrd shorelines.

Natural cleaning of oikd shorelines results from a combination of various biological.

chemical. and physical processes. which contribute in varying proportions depending on the

type of oil. the volume of stranded oi1. and the local environmental conditions (Atlas and

Bartha. 1991: Owens rr ul.. 1994b). On esposed coasts. the factors controlling the physical

processes have been w l l documented and include the kvel of wave rnrrgy and the location

of the oil with respect to the zone of wave action. Over the 1989-1990 winter period

following the Ex~on Ciilde= oil spiil in Prince William Sound. Alaska. the estent of surface

oil coverage on the most exposed shorelines decreased to 20% of the initial lsvel. while

intermi ttently esposed shorelines and sheltered shorelines showed smaller reductions. to 30-

40% and 50%. resprcti\,ely (Michel er d. 1991 1. Inputs from mechanical energy. which

include wave and tidal action and various intermittent inputs (storms. ice scour. coastal

erosion. run-off). remain the dominant removal proccsses of stranded oil (Little er ui.. 1993:

Owens. 1978: Orvens et cd.. 1994b). As levels of mechanical energy decrease. othcr

processes which include biodegradation and photo-osidation become more important (Owens

er LI/. . 1994b).

The natural rrcovrp of oiled shorelines in sheltered. lorv rvave-cnergq* snvironmrnts

has rrcently brrn esplainrd b>. a process referred to as "clau-oil flocculation". This procrss

involves the interaction of tïne minrral particles with droplets of bulk oil to form finely

divided oil-in-water rmulsions stabilized by the minrral fines (Bragg and Yang. 1995).

These clay-oil floc aggregates do ncjt ûdhere strongly to sediments and are therefore easily

removed from a shoreline by sven gentle water motion. This process was observed following

the E-non CC~fdez oil spi11 and rxplained the "self-cleaning" of sheltered. low-rnergy oiled

shorelines (Jahns et al,. 199 1 ). Subsequent studies conducted by Bragg and Owens ( 1995)

with sarnples collected from other spiil sites. including the .-lrrow (Nova Scotia. Canada:

1970). .lletirki (Straits of Magellan. Chilr: 1974). BIOS (Cape Hatt. NWT. Canada: 1980).

.\bscrc Forrsr (Tacoma. WA. USA: 1993). and Fred Boz~ch~zrrI (Tampa Bay. FL. L'SA: 1993).

confirmed the ubiquitous occurrence of clay-oil flocculation suggesting that it may play a

significant roie in natural shoreline recovery. Clay-oil Hocculation is now recoçnizrd as an

important natural clransing procrss. capable of accelentinp the removal of stranded oil in

most snvironments. especiall y in low-energy snvironments in the absence of wave action and

abrasion.

Srverai investigators suggest that shoreline cleanup may be accelerated by "surf

washinr" + whcreby oilrd material is moved from the high water mark down to the intertidal

zone using an cixcavator. thersby inducing ciay-oil tlocculation processes to facilitate the

dispersion of the oil into the surf zone (Lee rr ol.. 1997a: Lunel r i d.. 1996: Owens er rd..

19941). Samples collected from field studies on Amroth beach. following the ! k i Emprrss

incident in Milford Haven (L'K) in 1996. suggrst that the oil associated with the mineral

particles susprndrd in the water coiumn \vas biodegraded to a iargrr estent than that found in

the brached oil smulsion (Lee et rd.. 1997a). The process of clay-oil tlocculation is not yrt

fully understood but may influence natural oil biodegradation rates. Since natural rates of

biodegradation are controlled in pan by the availability of esposed oil surfaces. flocculation

and biodegradation are likely to be synergistic processes as the clay-oil floc structure

increases the oil-water interface up to several orden of magnitude. making the oil more

accessible to bacterial attack and providing bttttctr mass transfer of oxygen and dissolved

nutnents required for biodegradation. Therefore. if clay-oil fiocculation c m be induced or

accelented. then the rates of biodegradation may also be accelented whrn other controlling

factors. such as nuirienrs and oxygen. are not limiting.

1.2 OIL-SUSPENDED PARTICULATE :MATTER INTEMCTIONS

Interactions brtween spilled oil and suspended particulate matter (SPM) are important

because they represent a major potential pathway for hydrocarbon transport in coastal

environmrnts (Boehm rr c d . . 198 1: Payne ri cd.. 1989). These interactions may occur at the

molecular or macroscale level. Molrcular scals interactions involve the adsorption by SPM

of individual dissohed mo lecules of the oil while macroscale interactions involve the

interaction ofdiscretc oil droplets with SPM (Payne et cd.. 1989).

1.2.1 Molecular level interactions: adsorption

Early studies on oil-SPM interactions rvaluated the significance of oil transport by

adsorption to SPM. The association of hydrocarbons with clay minerals and SPM has been

quantitativrly esamined in several studies (Button. 1969 : Herbes. 1977 : Meyers and Oas.

1978: Malincky and Shaw. 1979). The adsorption behavior appears to be inversely

proportional to the soiubility of the compound (Meyers and Oas. 1978: Boucher and Lee.

1972) and directly proportional to the percentage of organic rnaner in the particles

(Karickhoff et ul.. 1978; Herbes. 1977: Pierce et al.. 1974). In terms of overall

environmental importance. in a study investigating the adsorption of petroleum hydrocarbons

(radiolabeled decane and biphenyl) with particulate mattrr collected from the water column

of the Gulf of Alaska. Malincky and Shaw (1979) concluded that removing hydrocarbons

solely by adsorption from solution in the water column would not be a major hydrocarbon

transport process. Payne rr al. ( 1989) also concluded that rnolecular scale interactions were

negligible frorn the viewpoint of the overall rnass balance of an oil slick since several

investigators reported that only about 1% of the oil may dissolve in the water column

(biackay and McAuliffè. 1988).

1.2.2 ;Macroscale level interactions: whole oil droplets and mineral fines

Although Payne er ui. ( 1989) confirmed that adsorption of dissolved hydrocarbons

from water ont0 surfaces of mineral fines was not a significant factor in hydrocarbon

transport following a spill. they suggested that discrete droplets of oil associated with

micron-sized mineral particles could be a possible process for dispersing oil floating in the

water colurnn. At hydrocarbon concentrations rxceeding saturation levels. discrets oiI

droplets are involved rather than dissolved fractions of the oïl. Microscopic examinations by

Poirier and Thiel (1941 ) showed depositrd water-oil sedirnent mixtures to be in "a loose

packing with the minerai grains adhering to the oil". The oil (Mid-continent Cnide Oil) in

the oil-sedirnent mixtures was rither in the form of globules or in an irregular. tlaky. stnngy

form held down bu the weight of the adhering mineral grains (> 115 pm). Bassin and Ichiye

( 1977) demonstratsd that dispersed clay particlrs spontaneously interact with oil emulsions in

the presencr of dissolved sdts to form "association colloids". In their study. they

hypothesized that the association colloids consisted of "oil films adsorbed ont0 cl-

particles" with escess oil in the form of "globules" wetted ont0 the tilrned particlrs. This is

contraq to other studies which made no mention of an oil tilm on cl- particles (Bragg and

Yang. 1995: Delvigne PI cil.. 1987: Payne rr al.. 1989). In prior studies by Delvigne ri c l / .

( 1 987) (and Payne et cil. ( 1 989). aggrepated or individual floccules consisting O t' an oil droplet

surroundrd bp minera1 fines were formed when oil droplets were mised with water

containin- SPM. ~1icroscopic esamination indicated the presrnce of discrete oil droplets

incorporated in siIt tlocs. without substantial amounts of oil adsorbed as a thin tilm on

particle surfaces (Delvigne et cd . . 1987). These rsperiments were conductrd with two crude

oils (Prudhoe Bay and Ekofisk) mised in s ramter with two sediment types (kaolinite clay

and Waddensea shoreline silt) and demonstrated that tlocculation readily occurred. OiI-SPM

interactions were measured as a function of oil droplet size. oil composition. and tlocculation

chmcteristics. As \vas O bserved by Payne e l al. ( 1989). the size and buoyancp of the clay-oil

tlocculrs was dependent on a number of parameters including the type and concentration of

oil and SPM. the level of hydraulic energy. and the size of mineral fines.

Oii-SPM interactions in the context of oil removal from shorelines were investigated

by Bragg and Yang (1995) in laborarory studies on oiled shoreline sediments from Prince

William Sound. AK following the Erron C.'i~ldLz oil spi11 in march 1989. Results fiom their

study indicated that fine mineral particles interacted with the oil stranded on shorelines to

t o m solids-stabilized oil-in-\vater emulsions by flocculation of micron-sized mineral fines

with oil droplets. The? concluded that this process allowed the oil to be removed from

subsurface sediments and low-energ shorelines by water rnergy levels less than thosc

needed to move sediment. so sediment abrasion \vas not needed to obtain efficient natural

cleaning of oiled shorelines. This new understanding of how clay-oil flocculation processes

removed stranded oi1 from the intertidai zones of oiled shorelines in the absence of wavs

action and coasral erosion providrd a viable explmation for the obsened natural cleaning of

oil-impacted shorelines (Bragg and Owens. 1 995).

1.3 PROCESS DEFINITION

1.3.1 Flocculation between clavs

Virtually al1 suspensions of fine-gnined particles are to some degree subject to

tlocculation (Johnson er c d . 1990). Flocculation may be defined as the formation of Iarger

particles of a solid phase dispersed in a solution bp the gathering together of smaller particles

(Parker. 1994). Flocculation behavior of clays is a function of the balance between repulsive

rlectrostatic tields of the negativrly chargrd clay flakes and the attractive Van der Waals

forces between them (Bassin and Ichiyr. 1977). Clay platelets normally have a negative

charge due to the anions in the broken chemicai bonds in the cpstal lattice (Edelvang and

Larsen. 1995). In a stable clay solution. the repulsive force predominates. but if a small

amount of electrolyte is added to the solution. the negative charge attracts positive cations

\\,hich f o m a thin film around the particle to produce an electrical double-layr (Bassin and

I c h i ) ~ . 1977: Edelvang and Larsen. 1995). This double-layer reduccs repulsion and allows

panicles to form tlocs.

1.3.2 CIav-oil flocculation

Bragg and Yang ( 1995) suggest that clay-oil floc rmulsions forrn due to the attraction

between the clectrostatic charges on the surfaces of mineral fines. polar hydrocarbon

molecules in the oil. and ions in the seawater. Polar charges in the oil result from

hrteroatoms such as nitrogrn. sulfur. and osygen. Thrse are anracred to the positikSe charges

on cations in semvater. .-\ "cation bridge" is thus formed in the tilm of seawater betwen the

minera1 fines and the oii. Flocculation among the tines themselves and the oil occurs because

minera1 tines c m generate varîed surface charges due to uptake of H- and OH- by osides on

minerais and by isomorphic substitution. rendering surfaces negative whilr caming a

positive charge on thrir rdges (Ives. 1978). These type of interactions form "clay-oil tlocs".

in which small oil droplrts are coated with micron-sized mineral fines and are surrounded by

seawater. In this definition. Bragg and Owens ( 1995) designate "clay" as a particle of small

diameter (< 4 pm) of any composition. and not specifically clay mineral particles. As

illustrated in Figure 1. a t).pical clay-oil tloc ma!. consist of man? oil droplrts. coated with

minera1 tines. associated together to form a larger tloc structure.

, Oil droplets

- L Mineral tines

25 microns

Figure 1. Schernatic diagram of a typical clay-oil floc aggregate. Mineral fines coat and stabilize oil droplets. Numerous bacteria were observed within the floc.

1.4 FACTORS AF'FECTING CLAY-OIL FLOCCULATION

The interactions between minera1 fines and oil may be dependent on a number of

hctors which include the composition of the oil. salinity. chernical and physical proprrties of

the minera1 fines. and other physical parameters such as temperature and turbulence (Bragg

and Owens. 1995: Bragg and Yang. 1995: Delvigne er al.. 1987: Payne rr c d . . 1989).

Bragg and Owens ( 1995) showed that clay-oil flocculation can occur with a variety of

cmdr and refinrd oil types in the presence of mineral tines and seawater from a range of

geographic environments. These conclusions were draw-n following the analysis of archived

oiled sediment samples col lrcted from di fferent oil spills. including the .-lrroicv (Nova Scotia.

Canada: 1970). .I/rrirltr (Straits of Magellan. Chilr: 1974). BIOS (Cape Hatt. NWT. Canada:

1980). .\.bstrc Foresr (Tacoma. WA. USA: 1993). and F r d Boirchtrrd (Tampa Bay. FL. USA:

1993). The oils in the above mentionrd spills included Bunker C Crude Oil. Light Arabian

Cnide Oil. weathered Lago Medio Cnide Oil. Light Bunker Fuel. and No. 6 Fuel Oil.

respective1 y.

Clap-oil tlocculation processes are affected by the physical propenies of the oil such

as the viscosity. degree of weathering. and composition (polar content). The viscosity

influences the ability of the oil to form droplrts for a given amount of energy (Bragg and

Yang. 1995). Delvigne et ai. ( 1987) and Huang and Elliot ( 1977) obsrrvrd the formation of

larger oil droplets with increasing oil viscosity. which indicates that the viscosity of the oil

influences the size of the oil droplets. Bassin and Ichiye (1977) noted that very viscous oils.

such as Bunker C. do not flocculate easily. Several investigators suggest that the degrer of

weathering of the oil intlurnces clay-oïl tlocculation processes. Delvigne rr d. (1987)

observed that 3-day weathered Prudhoe Bay Cnide Oil was associated with SPM to a greater

rxtcnt than unweathered Prudhoe Bay Cnide Oil. Likewise. Bragg and Yang (1995)

concluded from their studies that clay-oïl floc emulsions formed on oiled shorelines primarily

afier the oil had started to weather and had generated increased concentrations of polar

hydrocarbons. On the other hmd. Payne et (11. ( 1 989) tested four types of oil (unweathered

Prudhoe Bay Cnide Oil, 12-day weathered Prudhoe Cnide Oil. unweathered No. l Fuel Oil.

and naturally weathered North Slope Cnide Oil) and concluded that the oil-SPM interactions

u w e esssntially indcpcndent of the type and wrathering state of the oil present.

Laboraton studirs conducted by Bragg and Yang (1995) indicate that polar

hydrocarbons are required for flocculation to occur. .A non-polar oil. consisting of the

recornbined saturate and aromatic fractions of weathered Enon CNidez oil. did not tlocculate

with suspended fines following t-igorous agitation. In addition. no flocculation was observed

following estensivr shaking of hesadrcane [CH3(CH2) & H;]. a pure alkane. with suspended

fines. Thus. the polar fraction of oil seems to play an important role in the cl--oil

tIocculation process.

1.4.2. Salinitv of the water

There is much debate on the salinity needed for flocculation of mineral fines to takr

place (Edelvang and Larsen. 1995). Ichiye ( 1955) observed that intense tlocculation rnay be

eenerated at very low salinity (0.5 %O) if concentrations of fine clays are high (10-10 @LI. C

Kranck ( 1973) reportrd that most fine sediment tlocculates readily in salinities above 3 %O

but Eisma (1986) attached no importance to this threshold. Meade (1977) reported a lack of

direct correlation between flocculation and salinity above 5 460 in estuarine environments.

These differences rnay be explained by variations in the composition of the saline medium.

Ednvald rr trl (1974) found that clays coagulated more rapidly in artiticial seawater than

the? did in NaCl solutions of the same ionic strength. They attributed this difference to the

presence of rnultivalent ions. particularly ~ a ? which provides additional attractive forces.

Results from Payne C r al. (1989) indicate that salinity had a strong controlling

influence on reaction rates for dispersed oil droplets and the sediment types considered. Very

low rates of reaction were observed for SPM types in freshwater. while substantially higher

rates Lvere observed in both 14-15 %O and 28-30 O/OO salinities. Comparable effects of salinity

on the association of dispersed oils and tàny acids uith SPM or minera1 tines have been

s h o ~ i bu othrr investigators (Bassin and Ichiye. 1977: Meyers and Quinn. 1973: Bragg and

Yang. 1995).

1 A.3 Mineral particles

Laboratoq. studies conducted by Bngg and Yang ( 19953 demonstratrd that the type

of mineral tine needed for effective tlocculation was not as important as particle size.

Although weathrred E-Y-Yo~ l X k = oil efictively flocculated with the minera1 types

rvaluated (illite. chlorite. quartz. and feldspar). fine minera1 particles <: prn in size were

more effective in generating very fin+ dispersed spheres of oil coated with mineral fines.

Larger minrral particles. ranging betwren 1 and 5 Fm in size. interacted with the oil but the

oil was not dispersed as effectively and formed larger droplets. Poirier and Thirl (1941)

concludrd that thrre was no obvious correlation betwern the minrral composition of a fine

srdimrnt and its oil-settling capacity but that fine-prained sediments (43 pm) wrre able to

carn; down more oil than coarser grained particles (125-350 prn). Payne et ui. ( 1989)

observed that the particle number density prr unit rnass was correlated with the rate constant

for removal of " free" oil droplets due to reaction with SPM particles. Srnall particles provide

the largest ratio of mineral surface aredparticle m a s . and therefore the largest ratio of

surface clectncal chargeiparticle mass (Bragg and Owens. 1995: Ives. 1978: Poirier and

Thiel. 194 1 ).

The rnergy irnparted to the system also affects the interactions between oil and fine

minera1 particles. Zurcher and Thuer ( 1978) concluded that oil was cither adsorbed to. or

apglorneratsd (as oil droplets) with. suspended solids (kaolinite) depending on stimng sperd.

Delvignr er rd. ( 1987) noted that increasing turbulence levels led to increased numbers of

small oil droplets. increased collision probability. and thus. di fferent tlocculation behavior.

1.5 FACTORS AFFECTING HYDROCARBON DEGRADATION

The fate and prrsistence of petroleum hydrocarbons in the marine environment

drpends on a number of factors which include the microbial population present. physical and

chemical propenies of the oil. :rmperature. nutrient concentrations. oil concentrations. and

availability of osygen.

1.5.1 Hvdrocarbon-de~rading microbial po~ulations

.Microorganisrns capable of degrading a variety of petroleum hydrocarbons are

ubiquitous in the marine environment (Leahy and Coiwell. 1990). The overail ability to

degrade hydrocarbons depends on the composition of the rnicrobial cornmunity and

particularly on the enzymes produced by the hydrocarbon-degrading sprcies (Atlas and

Bartha. 1992). However. no single microbial sprcies appears to be able to completely

degrade any given oil. Thus. a mised comrnunity may be required for significant oil

degradation to occur (Atlas and Bartha. 1992).

Microbial communitirs rxposed to oil become adapted. resulting in increasrd

proportions of hydrocarbon-degrading bacteria (Leahy and Colweil. 1990). The most

prei-alent grnera of h y d r o c b o n d r d i n g microorganisrns in the marine environment are

Psrrlcion~oncts. .-l chrornoh~~c-ter. .-lrthrohct~*rrr, .\ fi~*roc*occ-rrs. .\.oc.nrdicr. Li'brio. .4 ~.inc~tohcrcrc.r.

Brei*ihcrc-rrrirrni. C'orynebcrcreril~nt. FZ~ri.obcrcrc.rizint. Cmditkr. Rhotloror-zikr, and

sporohc~~unyc.es ( Bartha and Atlas. 1977).

1.5.2 Phvsical and chernical properties of the oil

Density play an important role in the fate of spilled oil since the density diffrrence

between oil and water ultimately determines the extent to which the slick is submrrged and

the residence time of oiI droplets in the water cotumn (National Research Council. 1985).

The density of most weathered oils is not great snough for neutral buoyancy. thus significant

amounts remain suspended in the water colurnn. In addition. the densiry of cmde oil is

dependent on temperature and degree of weathering ( Whiticar rr al.. 1993).

Viscosity is a measure of a tluid's resistancs to flow (Whiticar er ul..

increases ivith weathering. and decreases with increasing temperature (National

1993). It

Kesearc h

Council. 1985). This is important in terms of !he degree of spreading of the oil and the

formation of oil droplets. as this determines in part the surface axa of oil available for attack

by hydrocarbon-degrading rnicroorganisms (Atlas and Banha. 1993). If the oil-aater

interface is increased. both biodegradation. and Içaching of componeiits from the oïl. which

accelerate the breakdown of the oil. are enhanced (Bragg and Owens. 1995).

The physical state and composition of the oil will also have a rnarked effect on

hjdrocarbon degradation. Biodegradation occurs at the oil-water interface and will be

enhanced if oil is maintained as discrete oil droplets in solution because the oil-water

interface is increased by several orders of magnitude (Bragg et al.. 1 992). 1 f a n oil-in-water

srnuision with small droplet s i x is formed. there is ample surface area at the oil-water

interface for rapid biodcgradation (Atlas and Bartha. 1992). In trrms of chernical

composition of the oil. 10% biodegradation rates have bren reported when the oil contains a

high proportion of condensed polparomatic compounds. condensed paraffins. asphaltenes.

and resin compounds (Atlas and Banha. 1997: Lee and Levy. 1986: National Research

Council. 1985).

1.5.3 Temperature

Temperature affects bacterial rnetabolism as well as the physical properties of the oil

(Atlas. 198 1 : Leahy and Col\vell. 1990). In general. rates of oil degradation have been

reportrd to decrease Lvith decreasing temperatures due to depressed rates of enzymatic

activity (Atlas and Bartha. 1993: Lee et r d . . 1997b). Howevrr. hydrocarbon degradation has

been obsened to occur over a tvide range of temperatures. Psychrotrophic. mesophilic. and

thrrmophilic hydrocarbon-utilizing microorganisrns have been isolated from the marine

environment (.Atlas and Bartha. 1993: Lee and Levy. 1 986).

Temperature also affects the physicai propenies of the oil. At low temperatures. the

viscosity of the oil increasrs and the solubility of low-molecular-weight hpdrocarbons

increases (Atlas and Bartha. 1992. Walker and Colwell. 1 974).

1.5.4 Nutrients

Microorganisms require nitrogen. phosphoms. and other essential minerai nutrients

for growth. hence the availability of these is critical (Atlas and Bartha. 1991). Srveral studies

have reportrd that the most likely limiting nutrients in aerobic marine environrnents are

nitrogen and phosphorus (Bragg et ul.. 1993: Gibbs and Davis. 1976: Swannell et oz.. 1996).

High concentrations of carbon are made available for microbial prowth following an oil spi11

and some of these nutrients may become relatively more limiting.

Hydrocarbon degradation requires osygen in the initial steps which involves the

osidation of the substrate by oxygenases (Atlas and Bartha. 1992). However. anaerobic

degradation of low-molecular-weight aromatic hydrocarbons has been reponed but proceeds

at very slow rates (Grbic-Gallic and Vogel. 1987: Lee and Levy. 199 1 : Zsyer C r LI/. , 1986).

Osygcn is not a limiting factor in the open ocran as the upper part of the water column.

~vhere oi1 is most likely to be found. is well oxygenated (Chester. 1990).

1.6 OBJECTIVES OF THE STUDY

Rrcent studies ha\.e shown the importance of the clay-oil t1occulation procrss on the

removal and dispersion o f oil stranded on shorelines following an oil spi!l. particularly on

Io~v-energv C . shordines in the absence of uave action and erosion (Bragg and O~vens. 1995:

Bragg and Yang. 1995: Lee rr oz.. 1997a). Howrver. the impact of clau-oil flocculation to

bacterial activity and the naturai n t r s of oii biodegradation has not bren invrstigatrd to date.

The tïrst phase of this study \vas drvotrd to idrntifying the mcchanisms and t'actors

controlling the formation of clay-oil floc aggregates under controlled laboratory conditions.

Anal>.tical procedures included UVivisiblr and Environmental Scanning Elrctron

Microscopy (ESEM) techniques. A varirty of oil types were tested for thsir ability to

flocculate in the presence of minera1 fines. Therrafier. the cffrct of mineral fine

concentration. duration of turbulence. and settling time on the shape and size of ch>--oïl Hoc

aggregatrs \vas evaluated. Finally. the s h d i n g time required for optimal incorporation of oil

droplrts into clay-oil floc aggregatrs \vas rvaluatsd.

Basrd on these esperirnents. the significance of clay-oil flocculation on the rates of

natural oil degradation was investigated in laboratory rsprriments.

MATERIALS AND METHODS

2.1.1 Reference mineral fines

The mineral tines used in the laboraton; ssperiments were recovered with a Shipek

grab - from a water depth of 30-50 m. approximately 10 km ooffshore Rimouski. QuCbec. In

the Laurentian Trough. the glacially dsivzd muds are comprised of quartz. feldspars.

amphiboles and pyrosrnes mised in with the illite dominatrd clay mincrals (Loing and

Yota. 1973). The major clemental composition of samples from this site is as follo~vs: 26 %

Si: 7.5 94 Al: j ?6 Fe; 2.5 Oh Ca; 2.5 ?4 K: 2 O/O Na: 2-3 46 organic rnattsr (Loring and Nota.

1973).

2.1.1.1 Preparation of mineral fines for the descriptive studies on clay-oil flocculation and the first biodegradation study (19OC)

A technique based on Stokes' Law of settling velocities was used to select minerai

tines of desired particle diarnrter (Gagnon. 1990). ..\pprosimately 20 g of sieved ( 170 Pm

nylon mrsh) dry sediment. heated to 500 OC to remove organic matter. was suspended in a

braker containing 400 mL distilled water. Afier mixing with a magnetic stir bar for 15

minutes and sonication in an ultrasound bath for 20 minutes. the suspension %as transferred

to a 4 L graduated cylinder (interna1 diameter of 10 cm) and made up to 4 L with distilled

Lvater. M e r 1 hour of sedimentation. the top 10 cm was transferred to a 250 mL centrifuge

bonle. The graduated cylinder u-as made up again to 4 L with distilled water. and the

rnising. settling and sarnpling procedure was repeated. Once enough suspended matter had

been collectrd. the bottles were crntrifuged (1260 G. 20 O C . 10 min) and the supernatant

discarded. The mineral fines were concentrated in a small volume of distilled water and

srored at room temperature until further use. According to Stokes' Law. the suspended

particles recovered undrr these conditions would have a mean particle diarneter of 5.5 Fm

based on a speciiic gravity of 2.65 at 20 O C (Knimbein and Pettijohn. 1938). Stokes' Law is

based on the senling velocity of spherical particles as a function of size. and affords good

values for sedimentary particles. although it is generally recognized that perfect agreement is

not possible for non-spherical objects. ~Measurement of the p i n size. carried out by Sylvain

Leblanc at the INRS-Oc~anologir (Québec) following the method described in Loizeau el of.

(1994) using a Coulter LS-100 with laser light source (Coulter Electronics Ltd. USA).

revealed that the mean particle diarneter was 10.14 Fm. However. 75% of the minerai fines

had a particle diarneter of less than 4.5 Fm (Table 1 ).

Table 1. Percentage mineral fines infenor to the indicated particle diameters in the mineral fine stock solution used in the descriptive studies on clay-oil tlocculation. as measured by Coulter LS-100 Particle Size Analysis.

Percentage

(%)

To obtain reference mineral fines of srnaller rnean particle diamrter for use in the iirst

biodegradation study ( 19OC). the same procrdure was followed escept that the minenl Anrs

were collected following a 18 hour settlins period. Coulter LS- 100 Particle Size Anal ysis

revealed that the msan particle diameter was 1.5 1 pm and that 7% of the minenl fines had a

particle diarneter of less than 1-45 pn (Table 2).

2.1.1.2 Preparation of minerai fines used in the second biodegradation study (lO0C)

To obtain reference mineral fines of small mean particle diameter with oqanic matter

still present. a third stock solu<ion of minera1 fines was prepared for use in the second

biodegradation study ( 10°C). In order to retain the organic matter. the sedimsnt was not

heated to 500 "C. Approsimately 500 mL of wet sedimrnt \vas added to 70 L distillcd \kater

in a 85 L cylindrical container (30 cm inside diameter). The sediment kvas well mised

throughout the watcr column and then lefi to settle for 18 hours. Thereatier. the first upper

10 cm were removcd and transferred to bottlrs and centrifuged ( 1260 G. 20 "C. 1 O min 1. The

supernatant \vas removed and the minerai tines were concsntrated in a small volume of

distillrd water and stored at room temprnture. Laser-Particle-Sizer anaiysis (Fritsch

Analysette 22)- carried out by André Hébert at the Centre Géoscientifique de Québec

(Quebec) following the method described in the instruction manual (Fritsch. 1991 1. revealed

that the mean particle diameter was of 0.77 pm (Figure 3) and that 90% of the mineral tines

Table 2. Percentage mineral fines inferior to the indicated particle diameters in the mineral fine stock solution used in the first biodegradation study ( 1 9°C). as measured by Coulter LS- 100 Particle Size Analysis.

Percentage

(%)

Particle diarneter (pn)

Figure 2. Particle diameter distribution and cumulative frequency of minerai fines used in the second biodegradation study ( 10°C). as measured by laser granulometry techniques.

consisted of clays (< 1 pm) and the remaining 10% of silt (2-63 pm). X-ray diffraction

analyses were performed by Dr. André Chagnon at the Centre GCoscientitique de QuCbrc

(Qukbec) usine the mrthod described by HCrous and Chagnon (1994) with a Rieaku

difiractometer set at 40 Kv and 20 Ma. a goniorneter speed of 2 degrees/minute, and a

recordinp chart speed of 1 degreekentimeter. The relative abundances of the minrrals were

determined by comparing the intensity of their diffraction retlections. The analysis revealed

that the reference minera1 tinr stock solution was composed of 38% quartz. 22% Feldspars.

20% illite- 1O0/0 chlorite. 6% amphiboles. and 4% inrerstrati fied.

2-1.2 Test oils

Seven cnidr oils were chosen for use in the descriptive studies on clay-oil

tlocculation. Oils c m be dividrd into 5 groups according to their characteristics which

include volati lity . evnpontion. viscosity and speci fic gravity (National Oceanic and

Atmosphet-ic -4dministration. 1994). Group 1 includes gasolinr products. Group I I includes

diesel-likr products and lighr crude oils. Group I I I includrs medium-grade crude oils and

intermediate products. Group IV includes hravy crude oils and residual producrs. and Group

V includes low 4PI oiis (heavier than water). The foliowing 011s were used in the clay-oil

tlocculation tests: Group II: .-ilberta Sweet Mised BIend Cnide Oil. Sable Island Condensats.

and South Louisiana Cnide Oïl: Group III: i inbian Light Cnide Oil. Fuel Oil No. 2. Prudhoe

Bay Cnide Oil. and Terra Nova Cnide Oil: Group IV: Fuel Oil No. 6 (Bunker C Residual).

A1 of the standard reference oils were obtained from the Environmental Monitoring and

Support Laboratory of the US Environmental Protection Agency (Cincinnati. OH) rxcrpt for

Alberta Sweet Mixed Blend Cnide Oit and Sable Island Condensate which were obtained

from the Emergencies Science Division of Environment Canada (Ottawa. Ontario) and Mobil

Oïl Canada Limited. respectively. The characteristics of the test oils are described in

Appendices A and B.

b'rathered Terra Nova Cnide Oil was used in both biodegradation studies. This wauy

crude oil. from an exploraton oil well off Newhundland. Canada. was obtained from Gulf

Oil Canada. The oil \vas wrathcred prior to use in the rxperiments to reflect the natural

procrsses (evapontion. dissolution. osidation. etc.) that occur whtn the oil is spilled in the

marine environment and alter its phyical and chemical properties. A thin layer of oiI

(approsimatrly 1 cm) was poured into a shallow Pyrex baking pan (inside diamrter of 2 1 cm)

and lsfi undrr a ventilation hood at room temprraturr for approsirnately 18 hours to obtain

final densitirs ot'0.90 mg/L and 0.85 mg/L for the biodrgradation studies conducted at 19 O C

and 10 O C . respectively. The a-eathered oil \vas kept in a sealed glass scintillation via1 in the

dark at 4 "C until use.

2.1.3 Seawater

The seawater used in the experiments was collrcted from the St.Law~encr River at a

depth of 15 rneters. 2 km offshore from the Maurice Lamontagne Institutr. Québec (Chenard

and Carter. pers. comm.). The seawatrr was first pre-filtered through sand. pumped through

a filter bcd of sand and quartz (91 5 mm thickness). and then diçtributed to the water basins.

This seawater (salinity 27-28 %O). collected from the water basins. was pre-equilibrated to

experimental temperatures for 48 houn before use.

2.1.4 Nutrient solution

Bushnell-Haas broth (Difco Laboratories. Detroit. MI). a grou-th medium used for the

study of microbial utilization of hydrocarbons. was prepared as speci fied by the manufacturer

(Bushnell and Haas. 1941). The composition of the working solution of the broth \vas as

foliows: 0.20 g/L magnesiuni sulfate: 0.02 g/L calcium chloride: 1.00 g/L monopotassium

phosphate: 1.00 g!L ammonium phosphate dibasic: 1.00 g/L potassium nitrate: 0.05 g/L

ferric chloride.

2.2 ANALYTICAL iMETHODS

2.2.1.1 UVNisible light microscopy

Clay-oil floc aggregates were observrd under an optical microscope (Leitz Aristoplan.

Emst Leitz Wetzlar GmbH) using normal light and UV epi-tluorescence (Leitz tiltsr bloc .A

with an excitation band pass filter 340-380 m. retlection short-pass filter 400 nm.

suppression filter 430 nm) with a 100 Watt rnercury lamp (Emst Leitz Wtzlar GmbH). .A 24

s 36 mm camera back was used to obtain color slides of cl--oil tlocs. A stage micrometer

(smallest division of 0.01 mm) \vas used as a scals reference to determine the surtics area of

the observed tlocs.

2.2.1.2 Environmental Scanning Electron Microscopg

C la)--O il tlocs were esarnined under an environmental scanning electron microscope

(ElectroScan mode1 E3) equipped with an X-ray rnergy dispersive spectrometer (EDS. Noran

Instruments) by Dr. Patricia Stoffyn-Egli using the facilities of the Geological Survey of

Canada (Bedford Institute of Oceanography. Nova Scotia. Canada). The scanning clectron

microscope (SEM) can provide a higher magnification and a larger depth of field than light

microscopes. The remarkablr three-dimensional appearance of the images is due to the large

depth of field and resolution. In the SEM. the electrons do not p a s through the specimen (as

in a transmission rlectron microscope). but rather secondary electrons are collected from the

surface of the specimen. varying as a result of the surface topography as the electron bram is

swept across the specimen. and yielding three-dimensional qualitp micrographs (Goldstein et

uL. 198 1: Kessel and Shih. 1971). However. due to the high-vacuum environment of

conventional SEM'S. viewing wet. oily or slectrically insulating specimens in their natural

States is nearly impossible and has given rise to the routine use of preparation techniques

such as drying. freezing. fracturing. and conductive coating (Baumgarten. 1989). Only

recrntly. since the advent of the environmental scanning electron microscope (ESEM). has it

bern practical to examine vinually any specimen. regardless of composition. Since specimen

temperature and water vapor partial pressure c m be controlled. sarnples under obsrnation

ma? be kept wet for extendrd prriods of time. Consrquently. it is possible to examine clay-

oi 1 tloc aggregates without causing s tnc tural al tentions.

In this study. samples were filtered. but not rinsed. on 0.4 pm pore size filters

(Millipore. HTTP) to rliminate most of the sea salts. The filter was immediately transferred

ont0 a thin laver of distilled water on the pre-cooled SEM stub. which was already on the

cooling stage (set at 1°C). The distilled water did not come in direct contact with the sample

but rnsured that the sample did not dry during the fine tuning of the watrr vapor pressure. A

working distance of 12 to 14 mm and a bearn voltage of 20 krV were used.

2.2.2 Total hvdrocarbon concentration

2.2.2.1 Oil extraction prior to ultraviolet fluorescence spectroscopy

In the shaking tests descnbed in section 2.3.1.4- each sample was transferred to a 500

mL separatory funnel to recover three separate oii fractions: oil incorporated into srdimented

clay-oil floc aggregates- dissolved hydrocarbons in the water column and oïl re-coalesced as

a surface slick. .A laycr of hrsane (50 mL ) was gently poured onto the sample to recover the

oil presrnt as re-coalesced oil. The systrm was Iefi untouched for 74 hours to allow the clay-

oii tloc aggregates to settle. Thereafier. the settled clay-oil floc fraction tvas transfemd to a

50 mL separatory funnel and the oil kvas rxtracted by shaking for 2 minutes with 15 mL

methylrnr chloride (method rnodified from Tremblay Cr cd . . 1992). The methylenr chloride

fraction \vas recovered and the same procedure was repeated twice. The aliquot was

concentrated to 250 pL under nitrogen flow to remove the methylene chlondr and was

resuspended in 50 mL of hesane in a graduated 50 mL glass centrifuge tube. The same

procedure \vas used in a 500 mL separatory fume1 for the watrr fraction containing dissolvrd

hydrocarbons rscept that the oil was successively extracted with 25 mL of methylene

chlonde instead of 15 mL. The hexane fraction. containing the re-coalescrd oil. was directly

transferred to a 50 mL glass centrifuge tube. Sodium sulfate (approxirnatrtly 0.8 g) was

added to al1 tubes to remove residual traces of water hefore analysis by fluorescence

spectrophotometry .

Methylene chloridr was rmployed as an extraction solvent because of its high polarity

yielding p a t e r extraction efficiency of petroleum hydrocarbons in seawater <han pentane

(Law rt d.. 1987) or carbon tetrachloride (Keizer and Gordon, 1973). In addition. methylene

chloide achisved more efficient scavenging of particular-adsorbed hydrocarbons than

pentane due to its higher water miscibility (Law rr ul.. 1987).

2.2.2.2 Ultraviolet fluorescence spectroscopy

Ultra\.iolet tluorescence spectroscopy (UVF) has brsn widely ussd for the

determination of total hydrocarbon concentrations in seawater (Dahab and Al-Adadfa. 1993:

Ehrhardt and Petrick. 1989: Law cr d. 1987: Maher. 1983). The UVF rnethod involves the

escitation of rlrctrons in the UV region of the rlectromagnrtic spectmm and affects onlp

those compounds with excitable rlectrons (Dahab and Al-Madfa. 1993). thereby essentially

rneasuring the concentration of aromatic hydrocarbons in petroleum. Al though the UV F

method c m only yield approsimate concentrations of total petroleum residurs in samplss. it

is useful because it is fast and simple.

The concentration of oil in the extracts uas sstimated by LWF using the method

desct-ibed by Marchand (1983'). Total hydrocarbon concentrations in samples were

drtermined against a standard of weathered Terra Nova Cnide Oil (0.85 mg/L) in the range of

0-10 pg r n ~ - ' with a Perkin-Elmer mode1 LS-50 luminescence spectrometrr using 1-cm

quartz cells. Escitation and emission wavelengths were set at 310 nm and 377 nm.

respectivety. with a slit width of 10 nm. Under these conditions. response was lineariy

related to oil concentration from O to 17 mg'L (Figure 3). Total hydrocarbon concentrations

were intrrpolated tkom the calibration curve.

2.2.3 Chemical anahsis

2.2.3.1 Capillary gas chromatography and flame ionization detection

Changes in the concentration and composition of the aliphatic and aromatic Fraction

of the residual hydrocarbons in the first biodrgradation study as well as the aliphatic fraction

of the residual hydrocarbons in the second biodegradation study w-ere monitored by capillary

cas chromatography and tlame ionization drtection (GC-FID). Seven extraction recovery b

standards of known concentration: perdeuteratrd n-tctradecane (C,,D,,. 125 ng/pg).

O 1 O I 5 20

Concentration (mg/L)

Figure 3. Ultraviolet fluorescence spectroscopy calibration curve for weathered Terra Nova Cnide 0 i l (0 .85 m&).

perdeutrratsd n-hesadrcane ( C ,D3+ 1 3 7 ngpl). perdeutrrated n-nonadecane ( C , ,D,,. 1 27

ngpg). perdeuteratrd n-tetracosane (C2,D,,. 1-2 ng'pl). perdeuteratrd n-dotriacontane

( 130 ng/pl). hexarnethylbenzens (C 12H,8. 80 nglpl). and 9.1 O-dirnethylanthracrns

(CIbH,,. 8 1 nypl) were added to the sarnples before extraction. Samplrs containing no

minerai fines were cxtracted by shaking vigorously for 3 minutes with 50 mL rnethylrnr

chloride (method modified from Tremblay et c d . 1992). The supernatant \vas drawn off and

the procedure reprated twice with 30 rnL methylene chloride. The CH2C12 estracts wrre

combined. reduced in volume to < 1 mL under nitrogen tlow and displaced with hexane. In

samples containing mineral tines. clau-oil flocs w r e collected on pre-extracted Wlatman

GFiF glass tïber tilters (0.7 pm pore size. 47 mm diametsr). The material retainrd on the

filter \vas ultrasonically estracted in 50 mL methylene chloride for a 10 minute penod. and

the procrdure was repeatrd 2 limes (Siron ct c d . . 1993 ). The CH,CI- .. - rstracts were combinrd.

centrifuged. volume-reducrd under nitropn f l o ~ and displaced with hexane. The rstracts

were passed through an alumina-silica gel column to recover the aliphatic and aromatic

fractions (Tremblay et c d . . 1992). The colurnn ( 1.1 s 26 cm) \vas prepared with hesane using

5-42 g silica gel ( 7 0 - 3 0 mesh from EM Science). 10.30 g aluminum oside (70230 rnesh

frorn Eb1 Science) previously activated for 24 hours at 200" C. and 1 . 1 g of sodium sulfate

(Amcrican Chtmicals). Aliphatics wrre eluted with 22 mL hesane and aromatics ai th 30

mL of a hesane/methylene chionde solution (4: 1 v h ) following the method described by

Trembla). et d. (1992). The fractions were injected into a Hewlrtt-Packard 5890 Srries I I

pas chromatograph fitted with a 25 m Ultra-2.0.33 mm i-d.. 0.17 prn film thickness. capillay

column. Conditions for chromatography were as follows: temperature program. 60' to 300'

C at 6" C/min: F.I.D.. 320" C: He flou.. 2.2 mL/min. To determine the concentration of

specific compounds. the peak heights of the extraction recovery standards were compared to

the peak height of the compound.

The following hydrocarbon ratios were caiculatrd from GC-FID chromatographie

analyses: the saturatsd hydrocarbon weathrring ratio (SHWR) and the C,,/phytane ratio. The

SHWR. definrd as the ratio of the surn of n-CIO to H - C , ~ - - over the sum of n-C,, to n-Cz5. is

used as a diagnostic weathrring ratio as the lighter components are lost mainly due to

evaporation and some dissolution (Boehm et ci/. . 1 98 1 : Humphrey et al.. 1 992). The drcrease

in the tr-C,,/phytani: ratio. which is the ratio of easily biodegradable hydrocarbons (linear

alkanes) to the more recalcitrant hydrocarbons (isoprenoids). has bren rxtcnsively used as an

index of bacterial oil degradation (Siron er al.. 1995: Wang rr d. 1993).

2.2.3.2 Gas chromatography and mass spectroscopy

The aromatic fraction of selectcd samples in the second biodegradation study was

maiyzed by gas chromatography and mass spectrometry (GC-MS). Samples were estracted

as describrd in section 2.2.3.1. The estracts of the aromatic fraction (2 PL) were injccted

into a Hrulett-Packard 5890 Series I I -as chromatograph equipped with an HP 5971 mass

selective detector (MSD). The instrument \vas operated in the selectrd ion monitoring mode

( S M ) to quantify specitic polynuclear aromatic hydrocarbons (PAHs). The GC \vas fitted

with a 30 m s 0.25 mm i.d. s 0.25 p m film thicknrss HP-5 MS column and operated in the

splitless mode. Conditions for chromatognphy wrrr as follows: 50' to 300° C at 6' C!min:

Detsctor. 320' C: He tlow. 1 mL/min. The concentration of specific compounds was

deterrnined by comparinç their prak heights to those of the extraction recovery standards.

The ratios of naphthalenes/chrysenrs. phenanthreneskhrysencs. dibenzothio-

phenss/chn;scnes. and tluorenes/chrysenes calculated frorn the GC-MS chromatographie

traces were used to illustrate the overail rxtent and degrer of weatherïng of the aromatic

fraction in samplrs (Wang rr al.. 1995). These ratios are based on the assumption that

chrystines are highly resistant to biodegradation. Howevrr. it is important to note that

althouph chnsenes are highly recalcitrant. it does not mean that rhry do not drgrade.

Nonsthelsss. as the oil becomes more weathered. the relative abundance of chpsenes

increases. resulting in lower ratios of naphthalenesichrysenes. phenanthreneskhrysenes.

di benzothiophenss/c hrysenes. and tl uoreneskhq-senes. These ratios. in conj unction wi th the

n-C , 8 iphpne ratio. are used to drscribe the overall drgrre of weathering of the oil.

2.2.4 Microbioloeical analvsis

.A miniaturized Most-Probable-Number (MPN) method. moditied from Brown and

Braddock ( 1990). \vas used for the enumeration of total heterotrophic marine bacteria and oil-

degrading organisms. The MPN method is a standard microbioIogical technique employed

for the determination of viable organisms in a sample. which uses a statistical analysis based

on a Poisson distribution. Successive dilutions are carried out to the point of extinction. and

replicatr dilutions are scored positive (for growth) and negative (for no growth). The number

of positives and negatives are used in connection with appropriate statistiçal tables in a MPN

Program (Klee. 1993 ) to drtermine the number of heterotrophic and oil-degradin- bacteria.

To reducr the use of numerous test tubes and large quantitirs of media. a miniaturizrd

tik-e-tube MPN method was used (Merlin el d.. 1995). .Marine broth and Bushnell-Haas

broth (both obtained from Difco Laboratories. Detroit. MI). were used for the enumeration of

heterotrophic and oil-drgrading bacteria. respecti~vly. Difco Marine Broth. containing al1 the

nutrients n e c e s s q for the growth of heterotrophic bacteria. was prepared as specified by the

manufacturer. The dehydrated medium (3 7.4 grarnslliter) was suspended in deionized

distillrd water. heated to boiling for 2 minutes. autoclaved for 20 minutes at 12 1 OC. and left

to cool before dispensing. Using a strrile repeater pipette. 1.75 mL of sterile Marine Broth

was dispensed into each dilution well of stenle 24-well tissue culture plates. which consisted

of four rows of six wells. The first ~veii in each row w;ts used for the serial dilution of the

sarnple. and the five remaining wells were used for the inoculation of the diluted sarnple.

Five 100 pL aliquots of the sample were placed in the first row of the 14-well tissue culture

plates. 700 PL of the sample was piprtted into the second dilution well (row 2). Thereafter.

successive 10-fold dilutions were perforrned by taking 100 pL from each dilution well and

adding to the nest well. The five remaining wells in each row were inoculatrd by rrpeatrdly

pipetting 100 pL from the dilution well. Two 24-well tissue culture plates wrre used per

sarnple to obtain 8 dilutions. Al1 samples were incubated at the rsperimental temperature for

3 weeks before scoring for positive or nrgative growth. Wells were scored positive when

turbidity of the medium was evident. To confirm positive groath. a solution of 0.5% (w/v)

p-iodonitrotetrazolium violet dye was added (50 pL) to each well at the end of the incubation

period and allowed to stand at room temperature for I hour (Vsnosa er id. 1993). in the

presence of actively respiring microorganisms. the dye tums from colorless to red.

Di fco Bushnell-Haas broth \vas used for the enurneration of oil-degrading bacteria.

This medium incl udes minera1 salts and is formulated for the dstection of bacteria capable of

hpdrolysing hpdrocarbons (Bushnell and Haas. 194 1 ). The dehydrated medium (3.17

grarns/liter) was suspended in a solution of 3% ( w h ) NaCl in deionized distilled water. +

heated to dissolve the solids. and autoclaved at 1 1 I "C for 30 minutes. The broth was cooled

before dispensing 1.75 mL into each well of the sterile 24-well tissue culture plates.

Approximately 1 O pL of sterilized crude oil (rveathered Terra Nova Cnide Oil) was added to

al1 of the inoculation wells. This producrd a very thin film (sheen) oloil on the surface. The

sarne inoculation procedure and successive 1 0- fold dilutions were performed as described

above. .A11 samples were incubated at the experimental temperature for 3 weeks before

sconng for positive or negative growth. Wells were scored positive whrn the sheen of oil

was disrupted by microbial growth. To confimi positive grouïth. the p-iodonitrotetrazoliurn

violet dye (50 PL) was added to each well. turning red afirr I h o u at room temperature in the

presence of activelp respiring microorganisms (Venosa ef r d . . 1993). Two uninoculated

plates of Bushnrll-Haas broth and Marine broth were incubated with the samples as "control

standards". The MPN was determined from a MPN calculator program (Klee. 1993).

2.2.4.2 Bacterial activity as measured by thymidine uptake

Bacterial activity \vas mcasured bu the incorporation of radioactive thymidine into

dcios)*ribonucleiç acid (DNA) as descnbed in Fuhrmm and Azarn ( 1982). Triplicate samples

(10 mL) were incubated at room ternprrature ( 19 "c) with 5 nM [methyl- '~lth~midine of

high sprcific activity (84 Ci/mmol). purchased frorn NEN Research Products. Two

additional identical samples were used as thymidine adsorption blanks by poisoning with

0.7% formaldehyde. Following 1 hours of incubation. the samples were immersed in an ice

bath to stop metabolic activity prior to cold trichloroacetic acid extraction of soluble pools

(10 rnL 10% K A ) . Afier 30 minutes. the insoluble material was collected by filtration on a

0.22 pm pore-size. 25 mm diarneter blillipore filter and rinsed with 1 mL portions of 5%

TCA. The tilter was dissolved with 1 mL ethyl acetate in a glass scintillation vial. Beckman

Ready-Safe scintillation tluid (10 mL) kvas addrd to the vial which \vas then placed in a

scintillation counter (Beckman LS6000 TA) for the disintegrations-per-minute (DPM) count.

Bacterial activity was estimated as mmoles thymidine incorporated per liter per hour and u-as

calculated as follows (Parsons et al.. 1984) :

U 4.5e-13 1 [methyl -3H]thymidine incorporatrsd (mmoles / L / hr) = - s ,Y -

S I \?

whrre U is the DPM of the tilter tirnes 4.5 x IO-" which is the nurnbrr of curies per DPM. S

is the specific activity in Ciimmole. r is the tirne of the incubation in hours. and L- the volume

of sample incubatsd in liters.

2.2.4.3 Potential activity of hydrocarbon-degrading bacteria as measured by rnineralization of rc-[ l - ' k ] hexadecane

To rneasure the hydrocarbon rnineralization potential of the microorganisms in each

sample. the protocol described by Lee and Levy ( 1 99 1 ) kvas foilowed. Triplicate sarnples ( 1 0

mL) were incubated at room temperature ( 19 O C ) with 0.1 pCi of 60 mCi/mrnole n-[l-

IJ C]hesadecane (Amersham) in 25 mL g l a s tubes. Two additional identical blank samples

were prepared by poisoning uith 1 mL 6N H,SO, prior to isotope addition. Srrum caps

fined with plastic reaction wells (Kontes Scientific Glassware) containing a folded glass fiber

tilter (3 mm. Whatman GFIC) were placed over each tube. Follorving a 48 heur incubation

period. 1 mL of 6N H2S0, \vas injected through the caps to stop rnetabolic activity and to

expel dissolved COz. The CO, was adsorbed with 0.1 mL B-phenethylamine injected ont0

the glass fiber tilters in the reaction wells. The filters were recovered 24 hours later. placed

in 10 mL Beckman Ready-Safe scintillation fluid. and analyzed for radioactivity using a

B e c h a n LS6000 TA liquid scintillation counter. The prrcentage added substrate respired.

rxpressed in units of percent CO2 recovered. was calculated by subtracting the average blank

DPM value from the averaged mran DPM value and dividing by the added disintegrations-

per-minute (Lindstrom et d. rl.. 199 1 ). Respiration rates. expressed in n & ~ r . were calculated

as foIlows:

DPM Y. [Cl n - [ l - l 4 C] hesadecane respiration rate (ng / g I hr) =

T.A. x 48

where DPM is the disintegrations-per-minute of the filter. [Cl is the concentration of

hrsadrcane remaining in the samples as detemined by GC-FID. and TA. is the

disintegrations-per-minute of the total added. Hexadecane turnover time. defhed as the

nurnber of hours required for the existing population to respire a quanti@ of substrate equal

in concentration to the existing in sitir concentration (Atlas md Bartha. 1987). was calculated

by dividing the concentration of hexadecane remaining in the sample by the respiration rate.

2.2.5 Nutrient analvsis

The concentration of onho-phosphate. ammonium. nitrate and nitrite in samples was

measured with a Technicon Autoanalpzer II (Technicon Industrial Systems. 1972: 1973 ).

The determination of inorganic phosphate in samples is based on the reactions o f the ions

with an acidified solution of ammonium moiybdate containing absorbic acid and a small

arnount of antimony to yield a phosphomolybdate cornplex. which is then reduced to a highly

colored blue compound (Koroleff. 1983). The determination of arnmonia in samples is based

on the Berthelot Reaction. whereby a blue colored compound. indophenol. is Formed by

phenol and hypochlorite in the presence of NH, (Koroleff. 1983). The determination of

nitrate and nitrite in sarnples is based on the reduction of nitrate to nitrite bp a copper-

cadmium reductor column (Grasshoff. 1983). The nitrite ion reacts with a sulfanilamide

under acidic conditions to form a diazo cornpound which thrn couples with N-l-

napthylethylenediarnide dihydrochloride to form a reddish-purple azo dye (Technicon

Industrial Systems. 1972).

2.3 EXPERIMENTAL PROCEDURES

2.3.1 Studies on the process of clav-oil flocculation

Expenments on the interactions between oil and tine mineral particies were carrird

out to investigate the formation of clay-oil floc aggregates under a variety of shaking

methods. and with various oil types. oil concentrations. and minera1 fine concentrations.

2.3.1.1 Shaking methods

Several shaking rnethods were investigated to obtain clay-oil floc aggregates. These

included: shaking tlasks by hand. on a reciprocating shaker. on an orbital shaker. and on a

magnetic stirrer using a variety of sperds. Mineral fines (300 pprn) and weathered Terra

Nova Cnide Oil (350 ppm: density of 0.90 mg/L) were added to 500 rnL Erlenmeycr tlasks

tllled with 300 mL seatmter. FIasks were shaken or swirlsd for 30 hours on the orbital

shaker (speeds up to 200 rpm). on a magnetic stirrer (150 rpm and 3 different stir bar

lengths). and on a reciprocating shaker (143 cycles/minute: one cycle consisting of 5.3 cm

travrled in total horizontally). Samples were subsequently viewed under a UVivisible light

microscope (section 2.21.1 ) to detenine whrthrr clay-oil flocculation had taken place.

2.3.1.2 Flocculation of minera1 fines with different oils

To determine whether crude oils possess the ability to flocculate with rnineral fines.

small-scale flocculation tests were conducted with a number of reference test oils. ranging

from light to heavp cnide oils (described in .4ppendices A and B). These included: Type II

(Alberta Sweet blixed Blend Cnide Oil, Sable Island Condensate, South Louisiana Cnide

Oil). Type III (Arabian Light Cnide Oil. Fuel Oil No. 2. Prudhoe Bay Cnide Oil. Terra Nova

Cnide Oil). and Type IV (Fuel Oil No. 6). Mineral fines (1000 pprn: mean particle diameter

of 10.44 pm) and 10 pL of each test oil (approx. 1700 ppm) were added to 25 mL

Erlenmeyer flasks containing 10 mL of seawater. The flasks were shaken on a reciprocatinp

shaker for 20 minutes and lefi standing for 15 minutes before examination by UVhisible

light rnicroscopy (section 2.2.1.1 ) for the presence of clay-oil floc aggregates.

2.3.1.3 Clay-clay and clay-oïl flocculation

.Additional flocculation tests were conducted to differentiate between the aggregates

fonned by the tlocculation of clays among thrmselves and the aggregates fonned through the

interaction of oil droplets kvith rnineral fines. Weathered Terra Nova Cnide Oil (75 ppm:

0.90 m@L) and various concentrations of mineral tines (10 ppm. 25 ppm. 150 ppm: mean

particle diameter of I .j 1 pm) were added to 500 rnL Erlenmeyer flasks containing 300 mL of

seawater. Flasks were shaken for 24 hours to induce flocculation. and sarnples u-ere then

examined using an Environmental Scanning Electron Microscope (ESEM). as described in

section 2.3.1.2.

2.3.1.4 Size measurements of clay-oil floc aggregates

Measurements of clay-oil floc. oil droplet. and mineral fine diameters were made with

a UV/visiblr light microscope (section 2.2.1.1 ). Expenments were conducted to examine the

rffects of minera1 fine concentration. srttling time. and shaking time on the average diameter

of clay-oil tloc aggregates.

A series of 500 rnL g l a s media bottles were prepared to measure the influence of the

quantity of mineral fines present in the water column on clay-oïl ilocculation processes. The

bottles contained 3 0 mL seakvater. weathered Terra Nova Cnide Oil (60 pprn: 0.85 mg/L).

and v q i n g amounts of minera1 fines (mran particle diarneter of 10.14 pm): 10 ppm. 60

ppm. 110 pprn. 300 ppm. and 600 ppm. The bottles were shakrn for 24 hours on a

rrciprocating shaker ( 143 cyclrsiminute) to induce clay-oil ilocculation.

A second expenment was conducted to monitor the effect of settling time on clay-oil

floc aggregatr size. The 500 mL g l a s media bottles contained 350 mL seawater. weathered

Terra Nova Cnide Oil (60 ppm: 0.85 mk@L). and minera1 fines (150 ppm: 10.44 pm mean

particle diameter). The bonles were shaken for 24 hours on a reciprocating shaker (143

cycles/min) to induce tlocculation and left standing for 0. 15. 30. and 60 minutes.

The effect of shaking time on the average diarnrter of clay-oil floc aggregates. oil

droplets. and mineral tines was also monitored following different shaking penods. Clay-oil

tloc aggregates were prepared bp shaking. on a reciprocating shaker (143 cycles/min). 60

ppm weathered Terra Nova Cnide Oil (0.85 mg/L) with 150 ppm mineral fines (mean

diameter of 10.44 pm) in 500 rnL g l a s media bottles containing 350 mL scawater for periods

of 0.5. 13.4. and 74 hours. Samples were not allowed to settle.

2.3.1.5 Association of oil with minerai fines in relation to shaking time

To drtcrmine the time needed for maximum incorporation of oil into clay-oil floc

aggregates. a series of shaking tests were conducted. For the rsperirnent. 60 ppm weathered

Terra Nova Cnide Oil (0.85 m b d ) and 150 ppm mineral fines (mran panicle diameter of

10.44 pm) were added to 500 mL g las media bottles containing 350 mL seawater. The

tlasks werr shaken on a reciprocating shaker at the minimum speed required to break the

surface tension ( 143 cycles/min) to induce clay-oil flocculation. After shaking periods of 1.

1. 12. 24. and 98 hours. the contents of rach bottle was transferred to a 500 mL separatory

fume1 to obtain threr separate fractions following a 24 hour settling penod: sedimented clay-

oil floc aggregates. a water phase containing dissolved hydrocarbons and a surface slick

containing re-coalesced oil. The oil was cxtracted frorn each fraction (section 2.2.2.1) and

the concentration was estimated by ultraviolet fluorescence spectroscopy (Marchand. 1983).

as described in section 3.2.22.

2.3.2 Biodegradation studies

2.3.2.1 First biodegradation study (19OC)

Shaker flask rxperirnents were carried out to dererminc the intluence of the mineral

fines on oil biodegradation. A series of 500 mL Erlenmeyer shaker flasks. with and without

mineral fines ( includine three replicates for eac h experimental treatment ) was rnoni tored over

a 63 day period with sarnpling points on Days 1. 7. 14. 35. and 63. Each tlask contained 300

mL fresh seawater collccted from the S t.Law~ence Estuary (salinity 28Oh0. pre-equil ibrated to

room temperature) and was amended with Bushneil-Haas nutrient broth on a weeklp basis

(calculated arnendments of 20 pM NO;. 15 pM PO,. and 30 pM NH,). Flasks without

mineral tïnes. referred to as oiied controls. contained weathered Terra Nova Cnide Oil (75

ppm: density of 0.90 @mL ). Mineral fines (25 ppm. mean particle diameter 1.5 1 pm) were

added to a set of flasks containing oil to induce clay-oil flocculation. herein referred to as

mineral fine arnended flasks. AI1 flasks were vigorously shaken for 24 hours on a

reciprocating shaker at the minimum speed required to break the surface tension (143

cycles/min: one cycle consisting of 5.3 cm traveled in total horizontally). and transferred to

an orbital shaker ( 100 rpm: 19OC. in the dark) for the remainder of the zxperiment. Abiotic

oiled control tlasks and abiotic mineral fine arnended flasks. to which 3 0 ppm mercuric

chlonde was added to kill microorganisms. were monitored on Days 1 and 63.

Numbers of heterotrophic and oil-degrading bacteria were monitored on Dais 1 and

63 by a modification of the Most-Probable-Number (MPN) method (Brown and Braddock.

1990). Relative heterotrophic activity and potential hydrocarbon degradation rates in

sarnples were monitored on al1 sampling days by rneasunng the microbial uptake of methyl-

['HI thpidine . and the respiration rates of n-[ 1 - i4~]hexadecane (Lee et d . 1995). Changes

in the concentration and composition of the oil were determined by capillary pas

chromatography and tlame ionization detection (Lee et al-. 1993). Weathering ratios were

calculated fiom the GC-FID chrornatographic traces.

2.3.2.2 Second biodegradation study (1 O°C)

A second biodegradation study was conducted at a Iower incubation temperature of 10

OC. Furthemore. to study the impact of clay-oil flocculation on the adhesion of oil on solid

surfaces such as glassware. sarnples destined for chemical analysis were sub-divided into an

aqueous phase ( including dissolved oi 1. re-coalesced oil and oil assoc iated with mineral thes

in the water column) and a solid phase (oil adhering to glassware surfaces). Supplemental

tests were made. which included the estimated number of oil-degrading bacteria and

heterotrophic bactrria by the MPN rnethod on each sampling day: and the analysis of the

aromatic fraction by gas chromatography and mass spectrometry (GC-MS).

Shaker tlasks with and without mineral fines (three replicates for each experimental

treatrnent) were rnonitored over a 56 da. period with sampling points on Days 1. 7. 11. 38.

and 56. Abiotic controis were monitored at the beginning and end of the cspenment. Flasks

without mineral fines (oiled controis) contained wathered Terra Nova Cnide Oil (75 ppm:

density of 0.85 m g L ) and fresh seawater collected from the St-Law~ence Estuary (300 mL.

salinity ?8°/0~) . Mineral tinrs (25 ppm. 0.77 pm mean particle diameter) were added to a set

of flasks (minenl fine amended sarnples) to induce clay-oil flocculation. Al1 flasks were

shaken for 24 hours on a reciprocating shaker ( 1-43 cycledmin) and transferred to an orbital

shaker ( 100 rpm: 1 O°C. in the dark) for the remainder of the esperiment.

Numbrrs of hetrrotrophic and oil-degrading bacteria. relative hetrrotrophic activity

and the potential activity of hydrocarbon-degrading bacteria were measured on each sampling

daÿ. Changes in the concentration and composition of the saturate fraction of the oil was

determined by GC-FID while those of the aromatic fraction were determined by GC-MS.

RESULTS

3.1 INTERACTIONS BETWEEN OIL AND FINE MINERAL PARTICLES

3.1.1 Shakinp methods

No substantial amount of clay-oil floc aggregates was formed by swirling 500 mL

Erlenmryer flasks (tilled with 250 mL seawater: 200 ppm minerai fines: and 350 ppm Terra

Nova Cnide Oil) on an orbital shaker. even at high speeds (300 rprn). The air-water interface

was not broken in those trials. The mineral fines remained suspended in the water column

while the oil rernained as a thin film on the water surface. Similady. no substantial quantities

of clay-oil tloc aggregates were formed using a magnetic stirrer at varying speeds and with a

range of stir bar lengths. I t becarne apparent rhat oil dropiets had to be formed pnor to the

occurrence O i' clay-oii t7occulation.

The reciprocating shaker was deemed to be the most appropriate apparatus for the

formation of clay-oil tloc aggregates. When similar Erlenmeyer flasks were shaken at. or

above. the minimum speed required to break the surfàce tension of the air-water interface

(143 c)-cledmin: one cycle consisting of 5.3 cm traveled in total horizontally). oil droplets

fonned in the water column. Microscope observations showed that mineral tines readily

coated these oil droplets to f o m clay-oit floc aggregates. The reciprocating shaker was

therefore used in subsequent esperiments for the formation of clay-oil tloc aggregates.

3.1.2 Clav-oil floc formation with various crude oils

To investigate the tlocculation ability of the oils with mineral thes. seven

representative crude oils were chosen from Groups II to IV (described in Appendis A).

Visual inspection of the sample and examination of the flocculated emulsion under

UVIvisible lipht microscope were the methods used to confirm the presence of cl--oil

Hocculation. i.s.. the association of oil droplets with minera1 fines. The size of the clay-oïl

tloc aggregates was determined in subsequent experirnents (section 3.1.3). The results are

surnmarized in Table 3 and illustrate that al1 of the tested oils. ranging from the light crude

oils to the heavy crude oils. readily tlocculated with the mineral fines upon shaking for 20

minutes on a reciprocating shaker (143 cyclesimin). Liçht cmde oils from Group 11. such as

Alberta Sweet blixsd Blend Cnide Oil. Sable Island Condensate and South Louisiana Cnide

Oil. flocculated the most readily. producing many clay-oil floc aggregates. Group IV oils.

such as Bunker C Residual. flocculated the least. producing only very few single oil droplets

surrounded by minera1 fines. Medium-grade crude oils From Group III. such as -4rabia.n

Light Cnide Oil. Fuel Oil No. 2. Prudhoe Bay Crude Oil. and Terra Nova Cnide Oil. readily

'A 'LI - .- a

interacted with the minera1 fines. These moderateiy viscous oiis produced rnany clay-oil tloc

aggregates.

Clay-oil floc aggregates were t i n t viewrd by UVivisible liçht microscopy to drscnbe

the interaction between the oil and fine mineral particles. The tluorescence of the oil

increased the visibility of the oil droplets and distinguished oil droplets from mineral fines.

No tluorescencs of the mineral particles was observed suggestinç that very linle oil kvas

adsorbed as a thin film on particles. Rather. microscope observations revealrd the presence

of discretr oil droplets coated with minerai fines. Typical clap-oil floc aggregates formed

with Alberta Sweet Mixrd Blend Cnide Oil and Sable Island Condrnsate are shown in Figure

4. The oil droplets. fluorrscing in blue. are coated with tine mineral particles.

The clay-oil tloc aggregates were stable once formed and did not "de-tlocculate" over

the following werks. However. when flasks were not shakrn. a proportion of the flocs settled

to the bottom of the tlasks. The buopancy of clay-oil tlocs presumably drpends on the

concentration and size of both the mineral fines and oil droplets present in the water column.

Buoyant clay-oil tlocs w r e different from non-buoyant cl--oil tlocs in terms of overall oil

droplet size m d o r quantity and size of minera1 fines surrounding the oil droplet. Figure 5

shows a buoyant tloc formed with Alberta Sweet Mised Blend Cnide Oil while Figure 6

shows a non-buoyant clay-oil tloc aggregate formed under the sarne conditions with the same

oil. Buoyant tlocs typicallg consisted of larger-sized oil droplets (approximate diamrter of

Figure 4. Photomicrograph of a cln)+-oil floc iiggrcgiite under visible liglit and CV epi- rluoresccnct: microscope forrned with ( a > .Albutri Swer hliscd Blcnd Cnide Oil: and i b i Sable Island Condensate. Oil droplets. tlucirescing in biue. arc clearly distinguishcd from the mineral fines.

Figure 5. Photomicrograph undrr visible light microscope of (a ) a buoyant clay-oii floc aegregatr - formed with Alberta Sweet Xlised Blcnd Crude Oi1 ( 1700 ppm) and mineral fines (1000 ppm) following a 20 minute shaking penod and a I-week stationary period; and (b) mapnification of the sarnr floc.

Figure 6. Photomicrograph under visible light and W epi-fluorescence microscope of (a) a non-buoyant ciay-oil floc aggregate formed with Alberta Sweet Mixed Blend Cnide Oil ( 1 700 ppm) and mineral fuies ( 1 000 ppm) following a 20 minute shaking period and a 1 - week stationary period; and (b) magnification of the same floc.

200 pm) and were oniy lightly covered with mineral fines (Figure 5a). Settled. non-buoyant

tlocs usually contained relatively smaller-sized oil droplrts (< 25 pm diameter) surrounded

by a r e a t r r concentration of minera1 tines (Figure 6a). It is interesting to note that larger

grained particles were incorporatcd into clay-oil floc aggregates (Figure 6b). Figures 5b and C

6b clearly show the mineral finrs coatine and surrounding the oil droplets.

3.1.3 Qualitative assessmen t of clav-oil floc aggrepates

3.1.3.1 Effect of minerai fine concentration

Micrographs takcn under visible light and UV cpi- fluorescence microscopy of clay-oi l

tlocs formrd with varying amounts of mineral fines (10 pprn. 60. ppm. 1 JO ppm. 300 ppm.

and 600 ppm) and weathered Terra Nova Cnide Oil (60 ppm: density of 0.85 mCJL) are

s h o ~ w in Figure 7. These suggest that the concentration of mineral fines affected the overall

shape and size of the aggrepates. rspecially oil droplet size. In the presence of 10 ppm

mineral finrs. oil droplets were mainly spherical and consisted of single large oil droplets.

usually measuring bstween 30 pm and 40 pm across (Fisure 7a). With 60 pprn and 1 JO pprn

mineral fines. clay-oil floc aggregates consisted of rnany smaller oil droplets. varying

between 5 and 10 pm in diameter. associated together (Figures 7b and c). Clay-oil floc

Figure 7. Photomicrographs undrr visible light and UV rpi-fluorescence microscope of clay-oil tloc aggregates fomed with weathered Terra Nova Cnide Oi1 (60 ppm) and v-ing arnounts of minera1 fines: I O ppm. 60 ppm. 140 ppm. 300 ppm. and 600 ppm.

10 P P ~

1 JO ppm

300 pprn

600 pprn

20 pm

aggregates were increasingly denser and oil droplet diameters were increasingly smaller in

the presence of 300 to 600 ppm mineral fines (Figure 7e).

3.1.3.2 Effect of sertling time

To assess the reiationship between size and settling time of clay-oil floc aggregates.

size measurernents of tlocs remaining in suspension were made on samples (60 ppm

weathered Terra Nova oil. 150 ppm minera1 fines) that were shaken for 34 hours and then left

to settIe for O. 15. 30. and 60 minutes. Figure 8 illustrates that clay-oil tloc aggregates ranged

in s i x up to 1200 in surface area and that larger sized clay-oil floc aggregates. ranging

in surface area from 400 to 1200 pn'. senled out within the first hour. Most frequently. oil

droplets ranged up to 45 in surface area.

3.1.3.3 Effect of shaking time (duration of turbulence)

Figure 9 suggests that the duration of turbulence in tlasks did not greatly affect the

overall size of clay-oil floc aggregates. Flasks containing 60 ppm weathered Terra Nova oil.

and 150 ppm minenl fines were shaken for 0.5. 1 -5. 4.0. and 24 hours to form clay-oil flocs

and then viewed under a UVhisible light microscope. Most clay-oil floc aggregates

èsarninrd had a surface area of less than 200 with the exception of clay-oil floc

Clay-oil floc aggregates 1 O0 7

l I

60 * o j l t

Oil droplets

IO0 7

Class

O minutes

15 minutes

Figure 8. Frequency of surface area (pz) of clay-oil floc aggregates rernaining in suspension following a shakiyg penod of 24 hours and settiing times of O. 15. 30. and 60 minutes sorted by size: 1 (0-100 pm- ). 1 (20 1-400 ). 3 (30 1-600 ). 4(60 1-800 j (80 1 - 1000 Pm2). and 6 ( 1 00 1-1 200 and oil droplets sorted by size: 1 (0-45 pz). 2 (46-90 Pm2). 3 (9 1- 135 p'). 4 (136-180 pn'). and 5 (181-225 pz).

Clay -O i 1 floc aggregates

C lass

64 Oil droplets

0.5 hours

1.5 hours

'Oo 7 4.0 hourî 80

1 2 3 4 5

C lass

24.0 hours

Figure 9. Frequency of surface area (pn') of clay-oil floc aggregates remaining in suspension following shaking times of 0.5. 1 3. 4.0. and 24 hours sorted by size: 1 (0-200 ). 2 (20 1-400

). 3 (40 1 -600 ). 4 (60 1 -800 and 5 (80 1 - 1 00Opm2) and ail drop[ets ~ 0 ~ t e - j by size: 1 (0-45 jm2). 2 (46-90 pn2). 3 (91-1 35 4 (136-180 and 5 (1 81-225

aggregates formed follorving 4 hours of shaking. The latter had surtàce areas measuring up

to 800 The size of clay-oil tloc aggregates is presumably related to the shear forces

occurring in the tlasks. Oil droplet size in clay-oil floc aggregates \vas not influencrd by

shaking tirne rither. the latter measuring niost frequently up to 15 pn' in surface area.

However. it is interesting to note that both small and large oil droplets. ranging between 1

and 225 pm' in surface area. were incorporated into clay-oit tloc aggregates.

3.1.4 Anaivsis of floc a g g r e ~ a t e s bv Environmental S c a n n i n ~ Electron Microscopv

Clay -oi 1 flocs \vert! also esarnined in an environmental scanning electron microscope

(ESEM). Figure 10 shows an rxarnple of a common cla>.-oil tloc aggregate. produced

throueh t the interaction of 75 ppm weathrred Terra Nova Cnide Oil (density of 0.90 mg/L)

and 10 pprn mineral fines (mean particle diameter 1.51 pm). in a) its wet form: and b)

partiall>. dried fom. In Figure 103- a thin tilm of water is still coating the surface of the

aggregate. Han-sver. dark spherical shapes are present throughout the aggregate. indicative

of interstitial spacrs fomed by oil droplets. Presumably somr oil has leaked out. as evident

tiom the black ring secn around the clay-oil tloc aggregate. As the sample is partially dried

(Figure lob). the loss of definition in these areas is probably due to the oil coating the

cavities. in cornparison. othrr areas of the aggregate are clearly defined.

Figure 10. Electron micrognph of a clay-oil floc aggregate (a) wet: and (b) with the surface water removcd. The smooth dark area around the floc is the oil that has Ieaked out from the aggregate.

To differentiate brtween the natural tlocculation occurring among clays and that

occumng between oil droplrts and clays. samples with and without oil were observed by

ESEM. Figure I I illustrates the difference in appearance of clay-oil tloc aggregates and

clayclay aggregates. Figures 1 l a and b show &y-clay aggregates. without oil. formed in

flasks containing 25 ppm and 150 ppm minera1 fines of rnean particlç diameter of 1.51 pn.

respectively. Thsse micrognphs show the variability in shape and size of clay-cl-

aggregates. In addition. man- unflocculatrd. single mineral fine particles are still present. In

contrast. the aggregates formed in the presence of oil (75 ppm weathered Terra Nova Cnide

Oil: density of 0.90 mg/L). w r e quite different in appearance (Figure 1 l c and d). It is

interesting to note that v e y few single rnineral fine particles were obsenred in the

rnicrographs at rither of the mineral fine concentrations used (25 ppm and 150 pprn).

indicating that most of the rnineral fines were associated with the oil droplets to form clay-oil

floc aggregates. The aggregate in Fisure 1 Ic is clearly a clay-oil floc aggregate as a dari;

black ring surrounds the aggregate. indicative of oil that has Ieaked out from the tloc to block

the pores of the filter. In contrast. no such ring is seen in Figure 1 la. Dark sphrrical shaprs

are sren throughout the clay-oil floc aggregate in Figure 1 lc. suggesting that thesr interstitial

spaces were fomed by oil droplrts. A similar spherical cavitp is seen in Figure 1 ld.

probably also due to the initial presence of oil droplets. Filter pores around the aggregate are

also blockrd. presumably by oil that has leakrd out from the clay-oil floc aggregate.

Figure 11. Electron micrographs cornparhg flocculated aggregates containing only mineral fines and clay-oil floc aggregates: (a) clay-clay aggregates (25 ppm mineral fines): (b) clay- clay aggregates (1 50 pprn minerai fines); c ) clay-oii floc aggregate (75 ppm mineral fines and 75 ppm weathered Terra Nova Cnide Oil): and (d) clay-oil floc aggregate (150 ppm mineral fines and 75 pprn weathered Terra Nova Cnide Oil).

Figure 12 shows a typical spherical clay-oi1 floc formed through the interactions of 75

pprn weathered Terra Nova Cnide Oil with 10 ppm mineral fines. The shape of the aggregate

suggests the presence of two large oil droplcts coated with minera1 tines. Small. dark

spherical shapes are sern on the lower lefi aggregate. which presurnably are smallrr oil

droplets incorporated in the aggregate. As in the othrr micrographs of clay-oil floc

aggregates. it is evident that oil has leakrd out to block the filter pores.

The clay-oil flocs virwed by ESEM were similar to those obscrved by UVivisiblr

light microscope techniques. Under our laboratory conditions. the clap-oil floc aggregates

formed were either in the f o m of single oïl droplets coated with mineral fines or were

aggregates of rnany mineral fine coated oil droplets associated together. forming larger-sized

cIay-oil floc aggregates.

3.1.5 Percentage of oil incorporated into clav-oil floc aggrepates

Figure 13 indicates that the concentration of oil incorporated into clay-oiI floc

aggregates \vas dependent on the duration of turbulence in the flasks. Flasks containing

weathered Terra Nova Cnide Oil (60 ppm: density of 0.85 m g L ) and mineral tines (150

ppm: 10.44 pm mean particle diameter) were shaken on a reciprocating shaker (143

cycles/min) For periods of 1. 4. 12. 14. and 98 hours. Formation of clay-oil floc aggregates

\vas rapid. Within the first hour of shttking. 48% of the weathered oil was associated with

Figure 12. Electron rnicrograph of a clay-oil Hoc aggregate formed with 75 ppm weathered Terra Nova Cnide Oil and L O ppm mineral fines.

1 0O0~o - - 80% -

s w

J 2 6090 . iOlG lassware z - CI L O Re-coalesced oil

4 0 % . L - l a y - o ï l floc aggregates .- d

5 200 / . .

0% -

Shaking time (hours)

Figure 13. Percent oil incorporated into clay-oil floc aggregates. as re-coalesced oil. and on glassware vs. shaking time in samples arnended with mineral fines.

Shaking time (hours)

CllG lassware

0 Re-coalesced oi l

Figure 11. Distribution of oil as re-coalesced oil and on glassware vs. shaking time in oiled controis.

minerai fines. and 74%. 79%. 87%. and 84% of the oil was incorporated aftrr 4. 1 1. 24. and

98 hours of shaking. respectively. In subsequent biodegradaiion srudies. a shaking prriod of

34 hours kvas employed to ensure optima1 clay-oil Iloc formation.

Figures 13 and 14 demonstrate that the presrnce of mineral fines in flasks decreased

the adherence of the oil to the glassware. Between 5% and I l % of the oil remained on

rlassware in sarnples amrnded with mineral thes (Figure 13) while in oiled controls - (samplrs without mineral tines). between 21% and 36% of the oil remained on the çlassware

(Figure 14).

3.2 FIRST BIODEGRADATION STUDY (19OC)

To deterrnine the intluence of oil and mineral fîne interactions on the rates and extent

of oil biodegradation. shaker tlask experiments were c&ed out over a 63 da. period at an

incubation temperature of 19°C. Microbiai activity. nutrient concentrations. as well as

changes in the composition and concentration of the oil were monitored.

3.2.1 Chernical analvsis

3.2.1.1 Hydrocarbon degradation in the saturate fraction

Oil degradation proceeded rapidly in both treatrncnts within the first 7 days sincr 53%

and 39% of the initial total n-alkane (n-CI- to n-C35) concentration remained in oiled controls

and minerai fine amended sampIes. respectively (Figure 15). However- between Day 7 and

Day 14. no additional loss of the n-alkane fraction was noted in oiled controls while the

conce~tration of total n-alkartes decreascd further to 30% of the initial concentration in

sarnptss amended with mineral fines. After 63 days. 30% and 5% of the initial n-alkane

concentration remained in oiled controls and minera1 fine amended samples. respectively.

indicating that the extent of oii degration was greater in the latter sarnple.

Oiled controls Mineral fine arnended

Figure 15. Percentage total n -alkanes (n -C ,- to n -Cj5) remaining in oiied controls and mineral fine amended sarnples. relative to Day 1 . Error bars are standard deviations.

The saturated hydrocarbon weathering ratio ( S H WR) and the n-C ,lphytanr ratio w-ere

calculated tkom GC-FID chromatographie traces to measure the relative contribution of

microbial degradation to the rveathrring process. The SHWR. sum (n-CI2 to n-Gi)/ sum (n-

C,, t~ n-C,<). - - frequently used to monitor the loss of Iow-boiling point. saturated

hydrocarbons by cvaporation (Boehm et d. 1981: Humphrey et r d . . 1992). approached 1.0

over the 9 week period in both sarnple types. suggesting that rvaporative loss occurrrd in

most cases (Table 4). However. the SHWR ratio for abiotic samples rernained constant

between Day 1 and Day 63 indicating that the loss of low-molecular-weight n-alkanes %as

not dur only to rvaporative loss. The distribution of the n-alkanes in the abiotic controls at

the beginning and end of the espenment clearly illustrates this point (Figure 16). Thus. the

decrease in the SHWR c m be attributed to the preferential degradation of low rnolecular

~5-right hydrocarbons by microorganisms. The n-C 8/phytane ratio decreased over time in

both sarnple types. This ratio indicatçs the estent of rnicrobial degndation. as the linear

alkanes are preferentially degraded over their corresponding isoprenoids (Siron et ul.. 1995:

Wang or d.. 1995). In minera1 fins amended sarnplrs. the ratios rapidly drcreased to half

thrir original values within the tirst 7 days while in oiled controls. the ratios decreased only

slightl). within the same period. Phytane was not a tnie consen-ed biomarker as it was no

longer detected in mineral fine amrndrd samples by Day 35 (Table 4).

Figure 17 summarizes the distribution of n-alkanes from n-C,. to n-Cj5 in both oiled

controls and minera1 fine arnended sarnples throughout the esperimental period. In both

treatments. the low-molecular-weight hydrocarbons were preferentially degraded over the

Table 4. Wearhenng ratios calculated from chromatographic traces in oiled controls. mineral fine amended samples. abiotic oiled controls. and abiotic minera1 fine amended samples.

OiIed con trols

Dav i -

SHWR 1.37

n -C,,,'phytane 1.62

Vineral fine amended sam ples

Dav 1 -

ibiotic oiled controls

Dav 1 -

;HWR 1-43

'-C phytane 1.81

Dav 7 -

1.10

1.24

Dav 7 -

1-26

0.63

Dav 7 -

,biotic mineral fine amended samples

Dav I - - Dav 7

HWR 1 -45

-C ,,i'phytane 1.79

Dav 14 Dav 35

Dav 14 Dav 35

Dav 14 Dav 35

Dav 14 Dav 35

Dav 63

1 .O3

1-39

Dav 63

1 .O0

n.d.

Dav 63

1-55

1-84

Dav 63 - 1.48

1.83

Oilcd controls (Da) I ) 0 Mineril the mcndcd (Da! 1 )

Oilcd con~ols (Da> 6 3 )

n-al kanes

Figure 16. Distribution and concentration of n-alkanes (n -C , . - IO n-C,,) in abiotic oiled controls and abiotic minera1 tine arnended samples on Day 1 and Day 63. Error bars are standard deviations.

-

Oiled controls 1-1 Mineral fine amended

Dap 1

?Ooo 1 Day 35

?Ooo 1 Day 63

n-al kanes

Figure 17. Distribution and concentration of individual n-alkanes in oiled controls and mineral fine arnended samples on Days 1 . 7. 14. 35. and 63. Error bars are standard deviations.

high-molecular-weighr hydrocarbons. However. most extensive degradation of the n-

alkanes occurred in minerai fine amended samples. By Da- 63. n-alkanes up to n-C14 were

compirtely degraded while in oiled control flasks. n-alkanes were degraded only up to n-CI6.

3.2.1.2 Hydrocarbon degradation in the aromatic fraction

Due to the resolution of the GC-FID. anaiflical results in this tirst experiment showed

no apparent differencr in hydrocarbon degradation of the arornatic fraction between

treatments (Figure 18).

3.2.1.3 Rates of oil biodegradation

Changes in the concentration of total n-alkanes (n-C ,, to n-Cl i ) - - over time provided

the basis for detrrmining the biodegradation rates in samples amended with minera1 fines and

oiled controls. Since the concentrations declined rsponentially over timr. the logarithrn of

the concentration was plotted vs. tims (Figure 19). Data from Day 63 were not included in

the caiculation of the regression as additional degradation was not observed between Days 35

and 63. For both treatmrnts. the slopes of the linear regression were highlp significant w-ith

correlation coefficients (r') of 0.80 and 0.95 and probabilities showing a

Oiled convois 1-1 Mineral fine amended

Day 1

Aromatics monitored: Naphthalene Acenaphthy lene Acenaphtene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Dibenzo(a.h )anthracene

Day 35 Day 63

Figure 18. Percentage total aromatics remaining in oiled controls and mineral fine amended samples relative to Day 1. Error bars are standard deviations.

Oiled controls

a Mineral fine amended

T i

1 7 14 21 28 35

Time (days)

Treatmsnt Slope Prob>Ttl 1

A-- OiIed controls -0.0 124 0.000 1 0.7987 Minera1 t h e arnended -0.0374 0.000 1 0.9539

Figure 19. Change in total n-alkane (n-C I 2 to tr-C; j) concentration between D- 1

and Day 35 in oiled controls and minera1 fine arnended sampies.

confidence interval of > 99% for oiled contrcrls and minera1 fine amended sarnples.

respectively. .A correlation coefficient of 0.99 means that 99% of the total variation is

accounted for by the mode!. which in this case is a log hnction of concentration vs. time.

Degradarion rates in both sample types were calculated from the regression lines. The

squations are as follows:

OiIed controls: -0.01'4 Log y = 4.5552~

Mineral t h e amended sarnples: -0 0374 Log y = 4.5270~

whrre y is the concentration of total n-alkanes in p&. and x the timr in days.

Figure 19 clearly shows that the ovenll degradation rate of total n-alkanes in tlasks

arnended with mineral fines was significantly greater than in oiled controls.

3.2.2 Microbioloeical analvsis

3.2.2.1 Potential oil-degrading bacteria

Measured respiration rates of n-[l - '4~]hesadecane. were highest on Day 1 and

drcreased in both oiled controls and minera1 fine amended samples following Day 1 (Figure

>O). Calculated respiration rates were highest within the first 24 hours as mineral fine

amended sarnples peaked at 8367 ng/_i$hr. and oiled controls at 6811 ng/z@hr. indicating the

rapid response of the bacterial cornmunity to the presence of oil in the tlasks. Respiration

rates decreaxd in both sarnple types dunng the following days. reaching rates close to zero

by the end of the expenment since letrls of suitable substrate rapidly diminished over time

(Table 5 ) . Respiration rates were higher in oiled controls between Day 7 and Da? 63. most

likely dur to the higher hydrocarbon concentration remaining in those sarnples.

3.2.2.2 Bacterial productivity

Bacterial productivity was rstimated by quantifying the incorporation rate of addrd

methyl-['~lth~rnidine into deosyribonucleic acid (DNA). With the exception of one point on

Da? 14 (oiled controls). incorporation rates remained fair1 y constant throughout the

rsperirnental pcriod in both treatments. ranging betarcn 0.72 s 1 0 - 5 0 2.94 s 1 0 - ~

mmoles/L/hr (Figure 21 1. In abiotic controls. incorporation rates were negligiblr. rançing

between 2.32 s 1 0-12 to 1.2 1 s 1 !Y1 ' rnrnoles/L/lir.

I i-1 Mineral fine amended

7 14 21 28 34 42 49 56 63

Time (days)

Figure 20. Hexadecane respiration rates in oiled controls and mineral fine amended sarnples over time. Error bars are standard deviations.

Table 5. Hexadecane concentration remaining in oiled controls and minera1 fine

arnended samples over time, as calculated from GC-FLD chromatographic traces.

Oiled controls Minera1 fine amended

+ Oiled controls

Time (days)

Figure 21. h4ethyl-['~lth~rnidine incorporation rates in oiled controls and mineral fine amended samples over time. Error bars are standard deviations.

3.2.2.3 Microbial activity within the first 24 hours

Additional bacterial productivity measurements were made to monitor microbial

activity within the first 24 hours. The incorporation rate of rnethyl-['~lthynidine into DNA

indicated that bacterial productivity was tirst suppressed by the presence of the weathered

crude oii but recovered within the tirst 4 hours. and escerded initiai incorporation rates by a

factor of 2 afier 14 hours (Figure 33). Figure 23 suggests that the mineralization of n-[ l -

1-8 14 Clhexadecane to CO2 \vas linear over time. Substrate mineraliwtion tnpled within the

tirst 24 hours in both oilrd controls and minera1 fine arnended samples. No apparent

difference \vas observed between treatments in either tests as the oil was evenly distnbuted in

the watrr column as fine oii droplets dunng the tirst 24 hours due to the vigorous shaking of

the flasks on the reciprocating shaker. The relativeiy high incubation temperature of 19°C

induced a npid bacterial response in both minera1 fine arnendrd sarnples and oiled controls in

the presence of the weathered oil and nutrients.

3.2.2.4 Estimated numbers of heterotrophic and oil-degrading bacteria

Numbers of heterotrophic and oil-dsgrading bacteria were estimated by the Most-

Probable-Number (MPN) method. Unlike total hetrrotrophs. the estimated number of oil-

degrading bacteria increased substantiallp between Da- 1 and Day 63 in both oiled controls

+ Oiicd controls + Mineral fine amendcd -

Time (hours)

Figure 22. ~ e t h ~ l - [ ' ~ ] t h ~ r n i d i n e incorporation rates within the first 24 hours in oiled controls and mineral fine amended sarnples. Error bars are standard deviations.

14 Figure 23. Percentage i l -[ 1 - Clhexadecane recovered as respired '''~0~ in oiled controls and mineral fine amended samples over time. Error bars are standard deviations.

and mineral t h e arnended samples (Table 6 ) . It is interesting to note that the population of

oil-degrading bacteria accounted for less than 1.3O/0 of the total numbers of bacteria present in

samples.

3.2.3 Nutrient analvsis

Nutrient arnendments of Bushnell Haas broth were made on a weekiy basis to al1

samples and monitored throughout the rsperimrnt. Nitrite and nitrate w r e almost

completely dsplrted during the first 14 days in both oiled controls and mineral fine arnended

sarnplss. indicating that enhanced microbial activity resulted in nitrogen limitation (Figure

24). Nitrite and nitrate accumulated from Day 21 on presumablg brcause the wrekly nutrient

amendments esceeded the bacterial metabolic needs. Arnmonia was also consumed during

the tirst 14 days in both oiled controls and mineral fine amendrd samples (Figure 74b).

however. lsss ammonia accumulated in minera1 fine amended samples. Similar to nitrite and

nitrate. ammonia also accurnulated from about Day 21 on. No differenccs were observed in

phosphate concentrations betwrrn oiled controls and mineral fine amended samples over the

esperimental prriod (Figure 3 c ) . Phosphate was presurnably consumed. as recordrd

concentrations were lower than the calculated amounts. Hoivever. phosphate had

accumulated in samples at al1 sarnpling points. indicating that this nutrient was not limiting

for bacteria.

Table 6. Most-Probable-Nurnber (MPN) and range (95% confidence lirnit) of heterotro-

phic and oil-degrading bactena on Day 1 and Day 63 in oiled controls and mineral fine

arnended simples.

m Heterotrophic bacteria

Oiled controls

Mineral fine amended

l Oil-derradin? bacteria

Oiled controls

Mineral fine arnended

Day 1

( MPNs/rnl)

(range)

Day 63

+ Oiled controls + Minerai fine amended + Cumulative concentration of added nuuients

Time (days)

Figure 24. Nutrient concentrations in oiled controls and minera1 fine amended samples over time: (a) nitrite and nitrate: (b) ammonia: and (c) ortho-phosphate. Weekly nutnent amend- ments consisted of 20 pM NO3. 15 FM PO,. and 30 FM NH,. Error barsare standard devia- tions.

3.3 SECOND BIODEGRADATION STUDY (IO0C)

.4 second biodegradation study was conducted at a lower incubation temperature of

10°C. To show the impact of clay-oil tlocculation on the removal of oil from solid

substrates. separate aqurous phase (water column) and solid phase (glassware surfaces)

samples were co llected for chernical anal p i s . The aqueous phase (water column ) contained

the oil dissolved in the water column, re-coaiesced oil and oil adsorbed to, or associated with

minera1 fines. The solid phase represented the oil which adhered to glassware surfaces. An

identical oil to minera1 fine ratio wvs used as in the first biodegradation study: 73 ppm

weathered Terra Nova Cnide Oil (0.85 m-L) with 25 ppm mineral fines (mean particle

diarneter of 0.77 pm).

3.3.1 Chernical analvsis

3.3.1.1 Total hydrocarbon degradation in the saturate fraction

Figure 25 summarizes for both oiled controls and minera1 fine amended samples the

percentage of initiai total n-alkanes (n-CI? to PI-C3c). including those in the water column and

adhering to glassu-are surfaces. remaining over time. In mineral fine arnendrd samples. the

n-alkane fraction was significantly degraded within the first 7 days to 66OA its original

Oiled controls Mineml fine amended

Figure 25. Percentage total n -aIkanes (n -C 12 to n -C34 remaining in oiled controls and mineral fine amended samples. relative to Day 1. Error bars are standard deviations.

concentration (t-test: t=6.87. p=O.Oj). In contrast. no significative degradation was obsewed

in oiled controls (t-test: t=-0.65. p=O.Oj). Degradation of n-alkanes in oiled controls was

obsemed only lbithin the following week. coinciding with the emulsification of the oil within

these flasks. which was probably due to the production of biosurfactants by the

microorganisms. By Day 56. the total concentration of n-alkanes remaining was significantly

lower in mineral fine amended sarnples than in oiled controls (t-test: t4.62. p=O.O5) as 2506

and 48% remained for samples with and without minera1 fines. respectively.

Rates of n-alkane degradation

Rates of n-alkane degradation in oiled controls and mineral fine arnended samples

were calcuiated based on the changes in total n-alkane concentration over time. Since the

concentrations declined exponentially over time. the logarithm of the concentration was

ploned vs. linrar timc (Figure 26). The linear regressions were significant with correlation

coefficients (r2) of 0.79 and 0.76. and probabilities showing a confidence levei of > 99% for

oiled controls and mineral fine amended samples. respectively. Degradation rates were

calculated from the regression lines for both sarnplc types. The equations are as follows:

Oiled controls: Log y = 4.7379s -0.0060

Mineral fine arnended samples: Log y = 4 .5376~ -0.0 IO8

Oiled controls Minera1 fine amended

Time (days)

Treatment Slope Prob>ltl -$- Oiled controls -0.0060 0.000 1 0.7895 Mineral fine arnended -0.0 108 0.000 1 0.7599

Intercept 4.7279 4.5376

Figure 26. Change in total n-alkane (n-C 12 to n-C; j) concentration between Day 1 and Day 56 in oiled controls and mineral fine arnended samples.

The sIopes of the regression lines in Figure 26 clsarly show that the degradation rate

of total n-alkanes in sarnples amended with mineral fines was significantly greater than in

oiled controls.

Another \va- of looking at hydrocarbon degradation rares is to quanti- the loss of

specific hydrocarbon fractions over time. Table 7 shows the O h biodegradation rate of n-

alkanes for given periods of timr for both oiled controls and mineral fine arnended sarnples.

The % biodrgradation rate \vas calculated by dividing the quantity of n-alkanes lost per day

(in a given penod of timr) by the initial quantity and multiplying by 100 to obtain a

percentage. In minera1 fine arnended samples. the n-alkane fraction was degraded at a rate of

-4.78% between Day 1 and Da- 7. -4 similar rrrte of 1 .22% was obsemed in oiled controls.

but only between Day 8 and Da'; 14. illustratinp the "hg" in response in these sarnples.

Table 7. Percentage biodegradation rate for given periods of time of total n -alkanes (n-C ,? to n -C3,) in oiled controls and minera1 fine arnended samples.

Total n -aikanes (n- CI' to n ocJ5)

Timr ( Oiled controls Mineral fine amended (days)

1-7 8-14 15-28 29-56 1-56

( O h Rate) (% Rate) 0.45% -4.78% 4.33% - 1.89% - 1 .Ob% - 1.63% -0.32% -0.18% -0.90% -1.33Y0

Individual analytes in the saturate fraction

Figure 27 sumarizes for both oiled controls and minera1 tinr amended sarnples the

percent individual analytes remaining in the saturate fraction relative to Day 1 over time. In

minera1 fine amrnded samples. n-alkanes up to n-C, were partially degraded within the first

7 days while no apparent degradation was observed in oiled controls. As in the tirst

biodegradation study. low molecular weight hydrocarbons were preferentially degraded over

high molecular weight components in both treatments. Individual analytes were more

extensively degraded by Day 56 in mineral fine amended samples.

Figure 28 illustrates the distribution and concentration of individual analytes in the

saturate fraction on Day 1 and Day 56 for both abiotic oiled controls and abiotic mineral fine

amended samples. No error bars appear on the figure as no replicates were made of the

abiotic controls. With the exception of low-molrcular-weight compounds (< n-CI, ) . Iittle

change in the concentration of the individual analytes was observed. indicating that the oil

loss mcasured in biotic samples was dur to mirrobial degradation and not due to physical

processes.

Day 7

Day 14

Day 28

Day 56 100 7

n-alkanes

Figure 27. Percent individual n-alkanes rernaining relative to Day 1 in oiled controls and mineral fine amended samples on Days 7. 14-28. and 56. Error bars are standard drviations.

. Day 1 Day 56

Figure 28. Distribution and concentration of n-alkanes on Day 1 and Day 56 in a ) abiotic oiled controls: and b) abiotic minerat fine amended samples.

Weathering ratios

The n-C,,/phytane ratio decreased more rapidly in tlasks amendrd with mineral fines

than in oiled controls (Figure 19). Within the tïrst 7 days. the n-C,,iphytane ratio decreasrd

from 1.73 to 0.96 in mineral fine amended samples. thrn remained fairly constant. In oiled

controls. the n-C,,/phytane ratio decreasrd less. dropping from 1.83 to 1.58. indicating that

the sxtent of biodegradation was greater in samples amended with mineral fines.

3.3.1.2 Total hydrocarbon degradation in the aromatic fraction

A similar trend of enhanced degradation in mineral fine amended sarnples was

observed in the aromatic fraction. Figure 30 summarizes the percent total aromatics ranging

tiom naphthalene to brnzo(g.h.i)perylene that rernained. relative to Day 1. in both oiled

controls and mineral tins amrndrd sarnples. Error bars do not appear on the figure because

not al1 replicate sarnples were analyzed. The aromatic fraction was only partially degraded in

mineral fine arnrndrd samplcs within the tirst 7 days since 78 % of the original concentration

remained undegraded. In oiled controls. the arornatic fraction was degraded rven lrss since

94 % of the original concentration remained. However. bp Day 14. a substantial arnount of

the aromatic fraction had been degraded in both treatments. In sarnples amended with

mineral fines. only 10% of the initial total aromatic fraction remained. presumably the most

+Oilrd conuols 4 hlinrnl fine amended

Time (days)

Figure 29. Tirne-series change in the ratio of n -C ,s/phytane in oiled controls and minerai fine amended sarnples.

Oiled controls M i n e n l fine arnended

Figure 30. Percent total aromatics remaining in oiled controls and mineral fine amended sarnples. relative to Day 1.

recalcitrant fraction. In contrast. 10% remained in oiled controls. M e r 56 days.

approximately 20% of the initial concentration of aromatics remained in both treatments.

Rates of aromatic degradation

The biodegradation rates in the aromatic fraction were calculated for given time

intervals (Table 8). Betueen Day 1 and Day 7. % biodegradation values were greater in

mineral fine arnended samples (-3.16%) rhan in oiled controls (-0.79%). Highest %

biodegradation values were obtained between Day 8 and Day 14. which were -7.65% and

-8.39% in oiled controls and minera1 fine arnended samples. respectively. Thereafier. the

rates leveled off in minera1 fine amended samples while remaining somewhat higher in oiled

controls between Day- 29 and Day 56.

Individual analytes in the aromatic fraction

Figure 31 suinmarizes the percent individual analy-tes remaining in the aromatic

fraction in oiled controls and minerai fine amended samples. relative to Day 1. The aromatic

fraction of the weathered oil was composed mainly of ?-ring (naphthalenes). 3-ring

(tluorenes. dibenzothiophenes. phenanthrenes). and 4-ring (pyrenes. c h s e n e s ) compounds.

Table 8. Percentage biodegradation rate for given penods of time of total aromatics in oiled controls and mineral fine amended samples.

Total aromatics

Time (days)

1-7 8-14 15-28 29-56 1-56

Oiled controls Mineral fine amended (% Rate) (% Rate) -0.79% -3.16% -7.65% -8.39% 0.2 1 % -O. f 7% -0.67% O. 16% - 1.33% -1 .-II%

Oiled controls 1-1 Mineral fine arnended

Day I-i

Day 28

Figure 31. Percent target PAH homologues remaining. relative to Day 1. in oiled controls and minerai fine amended samples on Days 7.14. 28. and 56.

accounting for 38%. 50%. and 11% of the total quantified polycyclic arornatic hydrocarbon

(PAH) homologurs. Major compositional changes were observrd in the PAHs. First.

homologues in each P.4H group were drgraded in the following order in both treatrnrnts: CL,

> C, > C? > C3 > C,. Second. as espectrd. there \vas a substantial decrease in the abundance

of the Iow-molecular-weight naphthalenes and their alky lated homologues relative to other

P.4Hs. Following 56 days. 93Oh and 9 1 % of naphthaienes were lost frorn oiled controis and

mineral tke amended samples. respectively. Third. aromatic degradation was dependent on

the number of rings of the molecules. In oilrd controls. 93%. 70%. 32%. 25%. and 27% of

the 2-. 3-. C. 5-. and 6-ring compounds were degraded. respectivelp. by Da' 56. In minera1

fine amended samples. a similar trend was obsrwed: 9 1%. 81%. 3 1%. 1%. and 0% of the 3.

3-. 4-. 5- and 6-ring compounds were degraded. There appears to be a discrepancy in the

obsrlved degradation of the 5- and 6-ring aromatics. based on the rxpected order of

degradation due to the cornplesity of the molecules. The discrepancy is due to analytical

m o r as the concentration of 5- and 6-ring aromatics (accounting for less than 1% of the

aroniûtic fraction) \vas close to the detection lirnit.

Weathering ratios

Tabic 9 sumrnarizes the ratios of naphthalenes/chrysenes. phenanthrenes/chrysenes.

dibenzothiophenes/chrysenes. and tluorenes/chrysencs which were used to describe the

overall extent and degree of weathenng of the aromatic fraction. Generally. these ratios

decrease as the oïl is weathered since the lokv-molecular-weight compounds are more easily

degraded than the high-molecular-weight hydrocarbons. such as the ch-srnes (Wang el c d .

1995). It is important to note that although chrysenss are highly degradation resistant. they

are not necessarily non-degradable. Ratios were consistently lower in mineral tine amrnded

samples throughout the expenmental period. indicating that oil in thrse sarnples was

preferentially degraded.

3.3.1.3 Distribution of the aliphatic fraction bebveen the aqueous and solid phase

Samples dsstined for chernical analysis were sub-divided into an aqueous phasr

(water column) and a solid phase (glassware surfaces) to beaer understand ( 1 ) the impact of

clay-oil tlocculation on the removal of oil from solid substrates such as glassware: (2) the

partitioning of hydrocarbons between the aqucous phase and solid phasr: and (3) the oil

degradation potrntial within rach phase.

Figure 2 summarizss the distribution of the total n-al1

the aqueous phasr and the solid phase for both oiled controls and mineral tine amended

sarnples. This figure illustrates several points: ( 1 ) a greater proportion of the saturate fraction

was found in the aqueous phase in minera1 tine arnended samples (73%) than in oiled

controls (52%) on Day 1: (2) degradation of the n-alkanes srarted within the first 7 days in

Aqueous phase (water column)

Time (days)

Time (days)

molid phase (glassware surfaces)

Figure 32. Percentage total n -alkanes (n -Ci2 to n -CJ5) remaining in the aqueous phase (water column) and in the solid phase (glassware surfaces) in (a) oiled controls: and (b) minera1 fine amended sarnples over tirne.

mineral fine amended samples but only afier Day 7 in oiled controls: (3) n-alkanes were

almost cornpletely degraded in the aqueous phase in both oiled controls and minera1 fine

amended sarnples by Day 28: (4) n-alkanrs were not degraded as cxtensively by Dap 56 in

oilrd controls since a greater proportion of the n-alkanes remained on the glassware in oiled

controls (48%) compared to mineral fine arnended sarnples (25%).

The presence of mineral fines in samples prevrnted the oil from adhering strongly to

the glassware. Within the first 24 hours. 73% of the total n-alkanes were found in the

aqueous phase of minera1 fine arnended samples. In contrast. only 52% of the total n-alkanes

were found in the aqurous phase of oiled controls. Thus. the minera1 fines rfficirntly

scavenged hydrocarbons from glassware surfaces to bring the oil into the aqueous phase.

Once associated with the oil to form clay-oil iloc aggregates. the mineral fines prevented the

oil from re-adhrring strongly to the glassware. The percentage total n-alkanes adhering to the

glassware incrctased by Day 7 to 79% in oilrd controls and to 43% in mineral fine arnended

samples. most likely due to the swirling action of the rotaton; agitator. However. these

values decreased substantially during the following rveeks in both oiled controls and mineral

tinr amended samples. and subsequently reachrd thrir original values by Day 56.

Degradation of the saturate fraction started immediately within the first 7 days in

mineral fine arnendrd sarnplrs but lagged behind in oiled controls. Although the percentage

n-alkanes in the aqueous phase decreased in oiled controls by Day 7. this loss was not due to

degradation but to the displacement of the hydrocarbons towards giassware surfaces (Figure

32). Thus. degradation of the n-alkanes started only afirr Day 7 in oiled controls while

significant degradation of the n-alkanrs had already occurred within the first 7 days in

minera1 fine arnended samples (t-test: t=6.87. p=0.05).

Degradation of the saturate fraction occurred mainly in the aqueous phase since the

percentage total n-alkanes remainine on the solid phase did not vary greatly betwern Day 1

and Da- 56 in both oiled controls and mineral fine amended sarnples. The n-C 181phytane ntio

also indicated that degradation of the hydrocarbons occurred mainly in the aqueous phase as

this n t io did not appear to decrease substantially in the solid phase over the experimental

penod (Figure 33) . Although the n-C,,/phytane ratio decreasrd substantially in the aqueous

phase in both oiled controls and mineral fine amended samples. the decrease was far more

important in the latter. Within the first 7 days. the n-C,,/phytane ratio declined from 1.71 to

0.67 in sarnples amended with minera1 fines. but. in oiled controls. the ratio only decreased

from 1.81 to 1-32. By Day 14. the n-C,s/phytane ratio was similar for both sample types.

decreasing to 0.75 in oiled controls and to 0.65 in sarnples arnended with mineral fines. We

note the degradation of phytane since it was no longer dctectrd in the aqueous phase in both

treatments after 56 days.

The degradation rates of the total n-alkanes in the aqueous phase for both oiled

controls and mineral fine amended samples were obtained from the regression lines

+Oilsd conuols (u atm)

- - ) - - Oilsd conuols (gIasswarr)

-+ Minen1 fint mended (w t r r )

- - 0 - - Mineni fins mondcd (glasswarc)

Figure 33. Time-series change in the ratio of n -Cl dphytane in oiled controls and minerai tine arnended samples.

illustrated in Figure 34. Since the concentrations drclined rsponrntially over time. the

logarithrn of the concentration was pioned vs. time. The sloprs of the linear regressions were

significant with correlation coefficients (r') of 0.92 and 0.93 and probabilities showing a

confidence level of > 99% for oiled controis and mineral fine amended sarnples. respectively.

The slopes of the regression lines in mineral fine arnended samples and oiled controls were

not significantly different. suggesting that the degradation rate of the aliphatic hc t ion was

similx in both treatments. However. it is important to note that degradation of the n-alkanes

started 1 week later in oiled controls, and that the decrease in percentage total n-alkanes in

the aqueous phase on Day 7 is not due to degradation but to a displacement of the oii towards

the solid phase (Figure 32). In addition. the results in Figure 34 refer to only a sprcific

fraction of the total hydrocarbons.

Individual analytes in the saturate fraction

Figure 35 summarizes the distribution and concentration of individual n-alkanrs

remaining in the aqueous phase and in the solid phase of oiled controls on sampling Days 1

through 56. in oiled controls. an approsirnatel\- equal distribution was observrd of each

analyte betwern the aqueous phase and solid phase afrcr 24 hours of shaking. However. in

flasks amended with minrral fines (Figure 36). individual n-alkanes were distributrd

unequally between the two phases. Betkvesn 80°h and 90% of the n-alkancs in the ri-C, to n-

El

Oiled controls Mineral tine amended

1 O' t I

Time (days)

Treatment Prob>[tl 1 Slope - ? F -

Oiled controls -0.05 13 0.000 1 0.9 186 Mineral fine amended -0.0572 0.000 1 0.9306

Figure 34. Change in total n-alkane (n-C 12 to n-C'; j) concentration between Day 1 and Day 28 in the aqueous phase of oiled controls and mineral fine amended samples.

Aqueous phase (water column) 1-1 Solid phase (glassware surfaces)

1 O0 Dav 1

80

60 40

20 O

Day 7

Day 14

100 -, Day 28

n-alkanes

Day 56

II Figure 35. Percent individual n-alkanes remaining. relative to Day 1 . in the aqueous phase (water column) and solid phase (glassware surfaces) o f oiled controls on Days 1 . 7. 14. 28. and 56.

Aqueous phase (water column) r) Solid phase (glassware surfaces)

100 Day 1

8 O 60 40

20 O

1 O0 Day 7

80

60

40

30

O

= - - . -- 100 Day 14 C

80 L

60 - - 30 - - 20

4 - O

Day 28

n-alkanes

Figure 36. Percent individual n-alkanes remaining. relative to Day 1 . in the aqueous phase (water colurnn) and solid phase (glassware surfaces) of minera1 fine amended sarnples on Days 1. 7. 14. 28. and 56.

C,, -- range were preferentially found in the aqueous phase on Day 1. but n-alkanes in the n-C2,

to n-C;; range were eveniy disrributrd between the aqueous and solid phases. The greater

concentration of low-molecular-wcight n-alkanes in the aqueous phase in minera1 fine

arnended samples resulted in p a t e r degradation of these.

3.3.1.4 Distribution of the aromatic fraction between the aqueous and solid phase

The distribution of the total aromatics between the aqueous and solid phase is

sumrnarized in Figure 37 for both oiled controls and mineral fine arnended sarnples. This

figure illustrates sevenl points: ( I ) a greater proportion of the total aromatic fraction kvas

found in the aqueous phase in mineral fine amended samples (89%) than in oiled controls

(3 1%) at the start of the experirnent: (2) the aromatic fraction was degraded more rapidly in

minera1 fine amended sarnplss: and ( 3 ) the aromatic fraction was not fully degraded in the

aqueous phase of rither oiled controls or minera1 fine amendcd sarnples by Day 56.

.As with the aliphatic fraction. the presence o f mineral fines prevented the oil from

adhering strongly to solid surfaces such as glassware. On Day 1. 89% of the total arornatics

\\.ere found in the aqueous phase of mineral fine arnended samples. In oiled controls. only

3 1% was found in the aqueous phase . Degradation o f the aromatic fraction proceeded more

rapidly in mineral fine arnended sarnples. Within the first 7 days. 22% was degraded.

However. by Day 56. the extent of degradation kvas the same for both treatments and the

Time (days)

Time (days)

Aqueous phase (water column)

O Sol id phase (glassware surfaces)

Figure 37. Percentage total aromatics remaining in the aqueous phase (water column) and in the solid phase (glassware surfaces) in (a) oiled controls: and (b) minera1 fine arnended samples over time.

sarne concentration remained in the solid phase. The aromatic fraction \vas not

completelydegraded by the end of the experimental penod in either oiled controls or minera1

fine amended sarnples. By Day 56. 5% of the total aromatics remained in the aqueous phase.

indicating that certain aromatic hydrocarbons were highly recalcitrant.

Individual analytes

Figures 38 and 39 summarize the distribution of individual PAH homologues between

the aqueous and solid phases in oi!rd controls and minera1 tine amended samples.

respectively. With the exception of the low-molecular-~veight naphthalenes and fluorenes in

oiled controls. no fractionation was observed between the aqueous phase and solid phase in

either oiled controls or mineral fine amended samples. In contrast to the n-alkanes. the

aromatic components were not completely degraded in the aqueous phase by the end of the

csperirnental period in both oiled controls and minera1 fine amended samplrs. The more

recalcitrant 4-. 5. and 6-ring aromatics remained undegraded.

Cl\ryst.ne C I Clirysciies C2 Clirysencs

Aqueous phase (water column) r] Solid phase (~ lassware surfaces)

. ,.A Dav 1

Dav 7

Day 14

[O0 , Day 28

100 - Day 56

80 - 60 - -- JO -

- - n. I

Figure 39. Percent target PAH homologues remaining, relative to Day 1. in the aqueous phase (water column) and solid phase (glassware surfaces) in minerai fine amended samples on Days 1, 7. 14.38. and 56.

3.3.2 Microbiological analvsis

3.3.2.1 Potential oil-degrading bacteria

The presence of clay-oil floc aggregates in the water column stimulatrd respiration

n tes within th< first 24 hours in minerai tine amended samples (Figure 10). Respiration rates

reached 1078 n&ihr in mineral fine arnended sarnpies compared to only 187 n & / h r in oiled

control flasks. Respiration rates continurd to increass during the following 7 days. reaching

5 199 ng/g/hr in oiled controls and 3523 n f / g in minera1 fine amended sarnplrs.

Respiration rates were lower in minera1 tine amended sarnpies after Day 7 as the

concentration of hexadrcane remainine in thesc samples had already decreased to about 50%

the initial concentration. In contrast. hesadecane concentration did not decrease substantially

in oiled controls within the first 7 days (Table 10).

Hexadecane turnover timcs for both oiled controls and mineral fine arnended sarnples

are illustrated in Figure 41. The turnover time within the first 24 hours was ciearly lower in

mineral fine amcnded samples than in oiled controls. By Day 7. turnover times were 34.4

days and 19.1 days in oiled controls and in minera1 fine arnended samples. respectively.

Following Dap II. turnover times werc sirnilar in both sarnpie types. and wcre slightly

greater by Da). 56 since the respiration ntes decreased towards the end of the experiment.

Oiled controls 1-1 Mineral fine arnended

3 1 28 49 56

Time (days)

Figure 40. Hexadecane respiration rates in oiled controls and minerai fine amended sarnples over time. Error bars are standard deviations.

Table 10. Hexadecane concentration remaining in oiled controls and mineral fine amended samples over time.

Oiled controls ivl ineral fine amended

Oiled controls 1-1 Mineral fine arnended

O 7 14 SI 28 35 42 49 56

Time (days)

Figure 11. Hesadecane turnover tirne in oiled controls and minera1 fine arnended samples over time. Error bars are standard deviations.

These results indicate that the presence of oil and nutnents induced a considerable and rapid

increase in the microbial population in both oilsd controls and mineral fine arnended

samples. However. the potential for hydrocarbon utilization by microoganisms was far

greater in mineral fine amended samples within the tirst 7 days. suggesting that the presence

of clay-oil floc aggregates in the water colurnn stimulated the activity of potential oil-

degraders.

3.3.2.2 Bacterial productivity

Bacterial productivity. as measured by methyl-['~lth~midinr incorporation. was

enhancrd over the 56 day period in both oiled controls and mineral fine amended samples

(Figure 12). Ho\vr\-er. no differences in incorporation rates were observed between

treatmrnts over the esprrirental period except on Day i-1 and Day 56 where msthyl-

[ '~ l th~midine incorporation rates were greater in oiled controls. It is important to note that

this mrthod estimates bacterial productivity of heterotrophic bacteria and not solely oil-

degrading bac teria.

1 t Oilcd cvntrolr -H- Minera1 fine arnended

Tirne (days)

Figure 42. ~ e t h ~ l - [ ' ~ l t h ~ r n i d i n e incorporation rates in oiled controls and minera1 fine amended samples over time. Error bars are standard deviations.

3.3.2.3 Estimated numbers of heterotrophic and oil-degrading bacteria

Num bers of heterotrophic and oil-degrading bacteria were estimated on each sampl ing

day by the Most-Probable-Nurnber (MPN) method. Results from the MPN method indicated

that both heterotrophic and oïl-degrading bacteria responded more rapidly to oil and nutrient

additions in minera1 fine arnended sarnples than in oiled controls (Figure 43). The estirnated

nurnbers of microorganisms with the potential to degrade weathered Terra Nova Cnide Oïl

were elevated in mineral fine arnended sarnples within the first 7 days. 489 bacteriairnL

compared to 73 bacteridml in oi led controls (Figure 43a). Oii-degrading bacteria were

below the detection Iimit on Day 1. Following Day 7. the situation reversed. and the

estimated number of oil-degrading bacteria was greater in oiled control tlasks. This

corresponds to the onset of oïl degradation in oiled controls. In addition. by Day 14. already

47% of the saturate and 8 1 Oh of the arornatic fraction had been degraded in sarnples amended

with minera1 fines (Figures 25 and 30). As espected. a decrease in the nurnber of potential

oil-degrading bacteria was obsewed in the latter sarnple.

The estimated number of heterotrophic bacteria was also greater in mineral fine

arnended sarnples within the first 7 days. reaching 133.430 bacteridml as compared to

15.056 bacteridml in oiled controIs (Figure 43b). Following Day 14. no dit'ference in

rstimated nurnbers of heterotrophic bacteria was observed between oiled controls and mineral

fine arnrnded samples. I t is worth mentioning that the oil-degrading population accounted

Figure 13. Change in the Most-Probable-Number (MPN) in oiled controls and mineral fine amended sam ples of (a) oil-degrading bacteria and (b) hererotrophic bacteria. Error bars are standard deviations.

for less than 1% of the total heterotrophic population in both oiled controls and mineral fine

amrnded samples during the tïrst 7 daps. By Day 14. oil-drgrading bacteria accounted for

10% of the total hetrrotrophic population in oiled controls. the maximum value reached

throughout the experimental period in both treatments.

3.3.3 Nutrient analysis

Figure 44 surnmarizrs the concentrations of nitrite and nitrate. ammonia. and ortho-

phosphate remaining in both oiled controls and mineral fine arnended samples following

weekly nutrient amendments. Nitrite and nitrate concentrations were lower throughout the

rsperimental period in samplrs arnended with mineral tines. indicating that nutrient demand

(microbial activity) was greatsr in these samples (Figure Wa). Ammonia levels were also

consistentlp lower in sarnples amended with mineral fines (Figure W b ) For esample. on

Day 7. the ammonia concentration in mineral fine amended samplss was 5 pM compared to

26 pM in oilrd controls. Phosphate levels were not different bctween treatments but

phosphate was consumed bu microorganisms. as indicated by loa-sr measured concentrations

than expected concentrations ( Figure 44c ).

+ Oiled controls -3- Minenl fine arnended + Cumulative concentration of added nutrients

Time (days)

Figure 44. Nutnent concentrations in oiled controis and minera1 fine amended samples over time: (a) nitrite and nitrate: (b) ammonia: and (c) ortho-phosphate. Weekly nutrient arnend- rnents consisted of 20 PM NO,. 15 FM PO,. and 30 FM NH,. Error bars are standard devia- tions.

DISCUSSION

.4 number of studies of interactions between oil. minera1 fines. and seawater have

been conducted with respect to adsorption (Kariclchoff rr rd.. 1978: Malincky and Shaw.

1979). sedimentation (Payne et al.. 1989). and dispersion (Delvigne rr al.. 1987) of oil

following a spill in the marine environment. Although previous studies demonstrated that

clay-oil t7occulation accelerated oiI removal from oiled shorelines (Bragg and Owens, 1995:

Bragg and Yang. 1995). these did not investigate its role on biodegradation rates of the

residual oil dispersed in the ocean. This study provides. for the first time. direct information

on oil degradation rates and bacterial activity in clay-oil floc aggregates.

Results presented in this study were derived from shaker tlask rsperiments. Cleari?;.

clay-oil flocculation can take place rapidly in the presence of minerai fines. oil droplets. and

srawatrr with adequatr amounts of rnergy Within the tirst hours of contact. most of the oil

\\-as incorporated into cl--oil floc aggregates. This finding suggests that. in the event of a

catastrophic oïl spill impacting a shoreline. flocculation of oil droplets with mineral fines

ma? occur rapidly. assuming that particles of small diarneter ( c 5 pm) are present in the

water column. In our esperiments. we chose to use Terra Nova Cnide Oil. a wavy cmde oil

tj.pical of that recovered from the continental shelf of Atlantic Canada and the fine fraction

(particle diametrr < 5 pm) of marine sediment collected from the Laurentian Trough. Before

adding the oil to the flasks. it was weathered but not sterilized in order to simulate the natural

processes that occur when the oil is exposed to the rnvironrnent following a spi11

(evaporation. dissolution. osidation. etc.). The source of microorganisms was therefore from

the natural population found in the oil and/or in the seawater. The concentrations used. 75

pprn oil and 3 pprn mineral tines. w r e chosen to reflrct oil spi11 conditions in the nearshore

zone. Delvigne n c d . ( 1987) reportrd that oil concentrations in the water column ranged

from 500 pprn in the nearshore zone during actual oil spills to 0.01 ppm in test spills.

Typical concentrations of suspended-particulate-matter (SPM) were of 1 pprn in coastal

zones. and < 100 pprn for surf zones (Delvigne et al.. 1997). Payne et al. (1989) measured

SPM levels in Prince William Sound. Alaska and noted concentrations < 1 pprn in water

column samples. 4.34.6 pprn in "milky" waters containing glacial tills. and from 63 to 1 10

pprn in the surticial tlocculent layer of sediment samples. Thus. both the oil and minera1 tine

concentrations chosen for the experimental studirs are nithin natural observed ranges.

Several previously reported studies used vrry low concentrations of oil (< 0.3 ppm)

in their studies on oil-SPM interactions. which favored adsorption (Bunon. 1976: Gearing ri

d.. 1980: Karickhoff er rd. 1978: Meyers and Oas. 1978). Karickhoff et trf. ( 1978) and

Meyers and Oas ( 1978) showed that hydrocarbon adsorption to particdate rnatter \vas

proportional to oil concentration. Specifically. Meyers and Oas (1978) reponed that the

moun t of n-alkane (n-eicosane and n-eicosene) adsorbed to smectite clay increased Iinearly

with increasing hydrocarbon concentration in water frorn 0.025 to 0.100 ppm. However.

abovr a hydrocarbon concentration of 0.125 pprn. a sudden increase in the concentration of

hydrocarbon associated with clay was obsened. They attributed this to the onset of oil

droplet formation. The prolonged shaking could have promoted oil droplet formation. and

hencr cl--oil tlocculation. Thus. the type of interaction betwern oil and mineral fines.

either as adsorption ancilor whole oil droplet interaction with the minerai fines depends in

part on the extent of turbulence and on oil concentration. In a laboratory sxperiment

conducted by Zurcher and Thuer (1978). oil was adsorbed ont0 suspendsd solids (kaolinite)

under low stimng intensity on a magnetic stirrer whiie under high stimng intensity. dispersed

oil droplets formed "oiVparticle agglomerates". They noted that 100 times more oil \vas

associated with mineral îïnes under high stimng intensity. indicating that oil droplets were

involved rathrr than the dissolved fraction of the oii adsorbed on the minerai particles.

Clay-oil flocculation involves the formation of solids-stabilized oil-in-water

ernulsions. in which discrete droplets of oil are coatrd with micron-sized mineral fines.

distinctly difkrent from other emulsitication processes such as viscous water-in-oil

ernulsions in which oil is the extemal phase. Bassin and Ichiye ( 1977) desctibed tlocculated

ernulsions consisting of adsorbrd oil films on clay particles: excess oil being in the f o m of

"globules wetted to the films". In contrast. Delvigne et (11. (1987) concluded that no

substantial arnounts of oii was adsorbed in the form of a thin film on particle surfaces. In the

present study. examination by UVivisi ble light microscope showed that ciay -oil %c

aggregatcs consisted of either "single" oil droplets coated with minera1 fines or of many

coated oil droplets associated togerher within the sediment fines matrix. similar to those

reported in recrnt iaboratory and field studies (Bragg and Yang 1995: Bragg and Owens.

1995: Delvigne ar cil. 1987: Lee et al.. 1997a).

The shapr and size of clay-oil tloc aggregates observrd in Our study varied

considerably. ranging frorn 4 to 1100 pn' in surface area. The size distribution of oil droplets

incorporated in clay-oil tloc aggregates varied also. ranging from 1 to 225 Pm2 in surface

area. On the basis of laboratory studies. Delvigne rr al. ( 1987). reported that the sizr

distribution of most of the oil droplets incorporatrd in the aggregates rangcd from 1 to 60 pm

in diameter and did not vary with different oil types (Prudhoe Bay Cnide Oil. Sday

weathered Prudhoe Bay Cnide Oil. Ekofisk Cnide Oil). silt types (kaolinite. Waddrnsea silt).

and turbulence b e l s (440 and 3500 Jls m') tested. Nthough the size of the aggregates

formed in their laboratov study was not tabulated. photomicrographs showed that most were

< 100 Pm in diameter. Clay-oil floc aggregates librrated from column flow tests containing

oiled srdiment tiom Prince William Sound. Alaska following the Erron lirlde- oil spi11 in

1989 also had a similar appearance to those formrd in our laboratory studies. with aggregate

diameters avenging around 100 Pm. and oil droplets ranging from 1 to 10 pm in diametcr

(Bragg and Yang. 1995). Similadp. clay-oil tloc aggregates isolated from near shore

seawater following the Seri Emprrss oil spill in Milford Haven. United Kingdom in 1996

were usually 30 pm across and were composed of one to several oil droplets (Lee et u/..

1997a). Thus. clay-oil flocs formed in Our laboratory studies closrly resembled those

collected from field sites following actual oil spills.

Once clay-oi1 floc aggregates were formed, they remained stable and did not

"de flocculate" over the following weeks. Both buoyant and non-buoyant clay-oil tloc

aggregates were obtained in this study. Buoyant tlocs typically consisted of larger sizrd oil

droplets (approximatr diameter of 200 pm) covered lightly with mineral fines whilc non-

buoyant tlocs contained relativety srnakr-sized oil droplets (< 25 pm in diameter)

surrounded by a greater concentration of mineral h e s . The buoyant or non-buoyant nature of

clay-oil tlocs aggregates may depend on the s i x and concentration of bath oil droplets and

minera1 fines. Delvigne ri cil. ( 1987) determined that oil dropleî size was dependent on the

type of oil. the weathenng state of the oil. and temperature. They also reportrd that droplet

size increased with increasing oil viscosity and decreased with increasing turbulence levels.

Although buoyant ciay-oil tloc aggregates were obsewed in this study. most tlocs settled to

the bottom within several hours if the flasks were not agitated. Other laboratory studirs

which investigated the interactions between oil. suspended particulate matter. and sealvater

also reportrd a settlrd phase containing oil (Bassin and Ichiye. 1977: Huang and Elliot. 1977:

Zurcher and Thuer. 1978). In contrast. Bragg and Yang ( 1995) reponed mostly floating tlocs

with oiled sediments collectrd from the Exxon I i r l r k oil spi11 site. klicroscope observations

of clay-oil tloc aggregates collected from shore waters in Milford Haven. United Kingdom

following the Seo Empress oil spill indicate that clay-oil flocs preferentiall y incorporate finer

material as most of the large (30-100 prn) quartz and feldspar grains present in the lvater

sarnples were not part of any tlocs (Lee et d.. 1997a). Larser grains settle faster and require

more energy to be resuspended. hence may be less available to f o m tlocs in the surf zone

than finer particles. In the field. clay-oil tloc aggregates will not sedirnent as rapidly as in the

iaboratory because the mixing energy in the near shore environment is extensive.

Nonetheless. the buoyancy of ciay-oil floc aggregates requires fiu-ther investigation.

With respect to biodegradation rates. results from this study suggest that claj--oi

tlocculation may accelerate natural oil biodegradation rates and enhance bacterial activity

The presence of mineral fiiies in the expenmental tlasks stimulated bacterial activity bu: 1 )

dispersing and stabilizing oil dropirts in the water column. thereby increasing the oil-water

interface by srveral orders of magnitude and making the oil surface more accessible to

nutrients. oxygen. and bacterial attack: 2) preventing the oil from adhering as strongly to

solid surfaces. thereby introducing a greater concentration of oil into the water column. where

degradation takes place: and 3) prevented the oil Iiom recoalescing and reforming sufiace

slicks. As a result. the number of heterotrophic and oil-degrading bacteria increased more

ripidly in flasks amendrd with mineral fines than in oiled control flasks. In the lO0C

ssperiment. the estimated number of heterotrophic and oil-degrading bacteria was about 7

times greater in minera1 fine arnended samples than in oiled controls within the tirst 7 days.

Since bacterial numbers were greater. degradation of the oil proceeded more rapidly in the

presence of ciay-oil floc aggregates.

Microorganisms were enumerated by the Most-Probable-Number (MPN) method.

The MPN method $ - e s a statistically based estimate of the number of microorganisms using

successive dilutions of the sample to reach a point of extinction (Merlin et d.. 1995). The

MPN method assumes a homogeneous distribution of the microorganisms wi&in each

dilution. Both the number of heterotrophic and oil-degrading bacteria in minerai fine

amended samples rnay have been underestirnated since bacteria rnay be preferentially

associated with clay-oil tloc aggregates. Furthemore. the MPN method is incvitably an

underestirnate of actual bactenal numbers since iess than 1% of a microbial community 1s

detected with viable counts. in comparison to direct counts (Atlas and Bartha. 1987: Fletcher.

1979). This rnay be due to an inability to completely simulate natural natural conditions with

growth media. Although direct count procedures yield the highest estimates of numbers of

microorganisms. this technique rnay not be feasible in this case since microorgnisms rnay br

underestimated due to intrreference caused by high arnounts of background debris including

minera1 fines. In addition. this technique is unable to disringuish between living and dead

microorganisms without selective staining. t g . acridine orange. 4'6'-diamidino-2-

phenylindole (Atlas and Bartha. 1987).

The disappearance of hydrocarbons in the experimental flasks cannot be explained by

physical losses. In both biodegradation studies. with the esception of low-molecular-weight

hpdrocarbons which were presurnably lost by evaporation. hydrocarbon loss in samples was

predominantly attributed to microbial degradation since hydrocarbon concentration and

composition within abiotic control flasks did not v q substantially between the beginning

and end of the experirnental penod. In addition. the n-CIs/phytane ratio. traditionally used to

rvaluate the relative contribution of microbial degradation to the weathenng process (Siron et

d.. 1995: Wang et ai.. 1995). showed important decreases over the experirnental period in

both oiled controls and mineral fine amended samples. which is indicative of microbiai

degradation. However. phytane was not a consenred biomarker as it was complerely

drgraded by Day 56. Recent studies have proposed triterpanes and strranes as biornarker

compounds as these are very resistant to biodegradation (Bragg et ai.. 1991: Butler et dl..

199 1 : Wang and Fingas. 1995). Nonetheless. the n-C, ,lphytane ratio was found to be a valid

indicator of biodegradation. and was useful for the cornparison of oil degradation in oiled

controls and mineral fine arnended sarnples.

This work confirmed the preferential assimilation of n-aikanes over their branched

counterparts as well as over high-rnolecular-weight aromatics (Atlas and Bartha. 1992: Lee

and Le-. 1986: National Research CounciI. 1985: Oudot, 1984). The n-alkanes showed a

consistent decrease in concentration over time and were metabolized at a rate inversely

proportional to their molecular weight. i.e.. low-molecular weight n-alkanes were

preferentially degraded over high-rnolecular weight n-alkanrs. As compared with the

aliphatics whose degradation was very rapid and complete in the aqueous phase of both oiled

controls and mineral fine arnended sarnples. degradation of the aromatics proceeded more

slowly. which confirms that they are less readily attacked by microorganisms. In the 10°C

experiment. the resolved aromatic fraction was only partially degraded within the first 7 days

in both treatments. Hourver. between Day 7 and Day 14. the concentration of the measured

aroma~ics decreased substantially in both treatments. A similar decline in concentration and

subsequent Ieveling off \vas reponed by Walker et ul. ( 1976). The decrease in concentration

of the aromatic fraction WLS mainly due to the degradation of the 2- and 3-ring aromatics.

n e s e results are in good agreement w-ïth other studies which reported that smaller

polpnuclear aromatic hydrocarbons. i-r.. 1-. 2-. and 3-ring arornatics. were degraded more

easiiy and at a more npid rate than larger (> 4-ring) aromatics (Atlas. 198 1: Leahy and

Colwell. 1990: Oudot. 1981). The 4- to 5-ring aromatic compounds were somewhat more

resistant to biodegradation due to their chernical complexity (Mueller et al.. 1994) and were

only partially degraded in both treatrnents by the end of the experiment. Degradation of the

aromatic fraction in the aqueous phase of sarnples was not as complete as for the aliphatic

fraction. and a substantial arnount of the more recalcitrant 4-. 5. and 6-ring aromatics

remained undrgnded. These results are in good agreement with Oudot ( 1984) and Cerniglia

( 1992).

Rates of oil biodrgradation in seawatrr have been the subject of srveral reviews

(Atlas and Bartha. 1993: National Research Council. 1985: Stewart er al.. 1993). Most

degradation rates reported in the literature were expressed as g/m3 day" and based on initial

and final concentrations of oil. These rates have been reported to be as high as 2500- 100.000

dm3 per day under optimal laboratory conditions while in situ. reported rates were in the C

range of 0.00 1-60 d m ' per day (Bartha. 1986: National Research Council. 1985). The tinal

concentration of oil remainine in flasks was not mrasured gravirne<rically in the present

study. Expressing hydrocaïbon degradation rates in this manner. ix.. based on the initial and

h a 1 concentrations of oil. may not adequately reflect reality as componrnts of petrolrum.

although degraded simultaneously. are degraded at different rates over time as the consortium

of residual oil changes (Lee and Levy. 1986: Stewart rr ul.. 1993). Therefore. it is difficult to

ascribe a single rate to what is in reality a compiex mixture of chernicals that degrade at

different rates over time. In addition. hydrocarbon degradation rates cited in the literature are

based on studies conducted under substantially different environmental and laboraton

conditions. using different oil types and concentrations. rnixing snergies. temperature. etc.

Obviously. the rate of hydrocarbon biodegradation is dependent on a combination of factors

which include the concentration of the oil. the physical and chernical properties of the oil. the

biota. and environmental conditions (Atlas and Bartha. 1992: Lee and Lsvy. 1986: Prince.

1993). However. the calculated degradation rates are useful for the cornparison of oiled

controls and minera1 fine amended sarnples. In this study. the n-alkanes were degraded more

rapidly in mineral fine arnended samplrs than in oiled controls. However. when degradation

rates were plotted for on14 the aqueous phase. no signiticant difference in hydrocarbon

degradation rate \vas observed between treatrnents. In reality. degradation of the n-alkane

fraction was occurring in the aqueous phase at a similar rate but out of phase by one week in

the two treatments. Hpdrocarbon degradation proceeded rapidly within the first week in

mineral fine amended sarnples while only beginning the foliowing week in oiled controls

following the emulsification of the oil in these flasks. which is thought to be due to the

production of biosurfactants by rnicroorganisms. Biosurfactants are surface-active agents

that contain hydrophobic and hydrophilic moities. which enablrs them to concentrate on

interfaces and thus reduce surface and intersurface tension (Bertrand el ul.. 1993: Rouse rr

d. 1994). The rmulsification of the oil rendered the oil accessible to microorganisms. much

in the same way as clay-oil tlocculation did. Nonetheless. the oii was degraded more

rxtensively by the end of the expenmental period in mineral fine arnended sarnples since

more oil was made availablr to the bactena than in oiled controls. in which a greater

proportion of oil remained on glassware surfaces.

Hydrocarbon degradation was less extensive and npid at 10°C than at 19°C.

presumably because the lower temperature diminished bacterial activity. causing the oil to be

degraded more slowly. Rates of oil degradation have been observed to decrease with

decreasing temperatures as a result of decreased rates of rnzymatic activity (Atlas and Bartha.

1992: Lee rr ul.. 1997b). The temperature dependence of hydrocarbon biodegradation rates

can be espressed in terms of Q,, values. Q,, describes the increase in process rate per lOoC

change in temperature (National Rrsearch Council. 1985). Although different seawater

sarnples from the same location were used in the studies. the Q,, value was about 1.8 in our

study. which is in çood agreement with other published data. Gibbs et uI. (1975) quoted a

Q ,, value of 1.7 over the 4"- 14°C range under enriched culture conditions. Gibbs and Davies

( 1976) determined a mean Q,, value of 2.7 over the 6*-26OC range. Zobell (1964) quoted a

Q,, value of about 3 for proliferating cells eetting their energy sxc!usively from the oxidîtion

of oil. and slightly highrr than 2 for non-proliferating cells. More recently. Thorpe and

Hellenbrand (1987) rstimated a QI, value of 3.1 for crude oil over the 5"-15°C temperature

range. Temperature affects the physical nature and composition of the oil (Atlas. 1981:

Leahy and Colwell. 1990). At low temperatures. the viscosity of the oil increases and the

solubility of low-molecular weight hydrocarbons increases. factors which affect

bioavailability (Atlas and Bartha. 1997: Walker and Colwell. 1974. This rnay explain why

the n-alkane and aromatic fractions were degraded more extensively by the end of the

experirnental period in the 19°C experiment. For example. only 27% (oiled controls) and 6%

(minera1 tine amended samples) of the original total n-allianes remainrd in the 19°C

experiment compared to 50% and 25 O h in the 10°C experiments. Sirnilar results were

obtained for the aromatic fraction. Thus. lower temperatures drcrease the rate and extent of

hydrocarbon degradation.

The results obtained from the present study provide useful baselinr data on oil

degradation within cl--oil floc aggregates. and show that the process of clay-oil flocculation

stimulates both bacterial activity and hydrocarbon degradation. In terms of in vivo

applications. Lee er cd. (1997) reported that the oil associated with minerai particles

suspended in the water column was biodegraded to a larger estent than in the oil emulsions

on Amroth beach following the Sru Emprrss oil spi11 in the United Kinçdom. They estirnate

that the majority of the oil k a s removed as clay-oil flocs while the remainder was released as

a broken surface slick. In their study. the beach was subjected to repeated "surf washings" in

an attempt to enhance clay-oil flocculation. This technique essentially entails the üsr of

heavy equipment to move matenal at low tide from the oiled zone at the high w t r r mark

dovm towards the intertidal zone. As the tide nses. the snergy imparted to the surf zone is

suffïcient to remove the oil emulsions tiom the oiled cobbles. The mineral fines in the waters

of the surf zones are believed to act as surface active agents to promote the removal of oïl

from the substratum and allow dispersion of the oil into the surf zone (Lunel rr ai.. 1996).

This sediment relocation also esposes the oiled sediments to increased concentrations of

nutnents and degrading bacteria. Acceleration of clap-oil flocculation processrs by surf

washing ma. prove to be a practical method to remove stranded oil on intenidal shorelines.

Research provided by the work in this thesis provides additional Irnowledge on the

factors controlling clay-oit floc formation and on rates of hydrocarbon degradation within

these tlocs.

CONCLUSIONS

Naturai cleaning of oiled shorelines depends on a number of biological. physical. and

chemicai processes. which contribute in v q i n g proportions depending on the oil type. the

quantity of stranded oil. and the environmentai conditions. On exposed coasts. the natural

rates of self-clcaning of oiled shorelines have been correlated to the Ievel of wave energy and

location of the oil. as most oil in the zone of wave action is rapidly removed. However. the

processes goveming the natural recok-ery of low-energy shorelines in the absence of wave

action and abrasion have onlp recently been recognized. One of these processes. referred to

as "clay-oil tlocculation". has been shown to occur with variety of crude oils and in a range

of geographic rnvironrnents. By the interaction with micron-sized minera1 fines. oil droplets

are stabiiized in the w t c r column and no longer adhrre strongly to shoreline sediment. This

facilitates thcir removal and dispersion by waves and tidai currents. Recent studies have

drmonstrated the potential role of ciay-oil tlocculation to remove oil stranded on shorelines.

but not its impact on the environmental persistence of the residuai oii.

To Our knowledge. this is the tïrst studp that has investigated the significance of clay-

oil flocculation processcs on natural oil degradation rates. To obtain a better understanding

of clay-oil floc formation. factors affecting their formation was first esplored. Therrafier. the

influence of clay-oïl flocculation processes on natunl oil drgradation rates and bacterial

activity was investigatrd.

The results of this study demonstrated the following:

( 1 ) Ability to flocculate: a variety of cmde oils. ranging from light cmde oils to heavy crude

oils (e.g. Sable Island Condensate to Bunker C Rcsidual). are capable of interacting with

micron-sized minera1 fines to form "clay-oii flocs" consisting of solids-stabilized oil-in-

water emulsions.

( 2 ) Physical characteristics: the physical characteristics of clay-oil tloc aggregates are

highly variable and are dependent on a number of factors including turbulence. the

concentration of minera1 tines. the size of minera1 fines. oi1 type and viscosity. etc. Oil

droplrts stabilized by mineral fines are stable once formed and do not re-coalrscc as a

surface film over tirne. Clay-oil floc aggregates formed in this study generally remain in

suspension uith minimal mising. %%en no mechanical r n r r p is imparted to the systsm.

they settle out of the water column.

( 3 ) Transfer of oil into the water column: clay-oil flocculation processes mediated the

transtèr of discrete droplets of bulk oil into the water column. prevented the oil frorn

recoalescing as a surface slick and from adhering strongly to solid surfaces.

(4) Hydrocarbon biodegradation: clay-oil flocculation procrsses stimulated bacterial

gro:vth as droplets of oil were stabilized in the rvatrr colurnn by the minera1 fines. thereby

increasing the surface/volurne ratio by several orders of magnitude and making the oi1

more accessible to dissolved nutrients and bacterial attack. Both the overall rate and

extent of hydrocarbon degradation was enhanced in mineral fine amended samples. In

the absence of minera1 fines. significant hydrocarbon degradation \vas only observed

following the rmulsitication of the oïl.

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