since the early 1970s , following the discovery that nutrient fertilizers could enhance the rates of oil biodegradation in marine environments ( atlas and bartha , 1972 ; 1973 ; bartha and atlas , 1976 ) , bioremediation has been a potential treatment for mitigating marine oil spills ( atlas , 1977 ; 1995 ; swannell et  al . ,
as discussed by swannell and colleagues ( 1996 ) , the exxon valdez oil spill ( evos ) has been the most studied case regarding the applicability of bioremediation .
bioremediation based on fertilizer addition was used in prince william sound ( pws ) from 1989 through 1991 with a total of 107000 pounds of nitrogen applied in 2237 separate shoreline applications following physical recovery of oil by shoreline washing and skimming of oil floating on water .
figure  1 shows locations and amounts of nitrogen applied 19891990 : amounts applied in 1991 were significantly less .
diameter of circles scaled to represent pounds of nitrogen . the application of bioremediation to evos followed extensive laboratory and field testing to demonstrate safety and efficacy .
for example , tests conducted for 114 days in large microcosms ( 30.5  cm diameter , 91.4  cm deep ) filled with oiled pws sediments and using rising and falling water to simulate tidal movements showed that 25% of the initial oil mass was lost and that significant degradation of most types of hydrocarbons occurred ( bragg et  al . , 1992 )
these experiments showed that the rate of biodegradation slowed down once the more easily degradable components were depleted even when fertilizer was reapplied .
it is important to understand that bioremediation does not increase the ultimate extent of hydrocarbon degradation , but only the rate of biodegradation while easily degradable hydrocarbons are present ( sugiura et  al . , 1997 ;
changes in composition of oil on sediments from pws in 1990 laboratory microcosm tests of bioremediation as measured by hplc chromatography ( different from more detailed gcms reported for field samples ) .
aromatics ( 24 + rings ) include the 41 target pahs , plus other multiring species .
the polars ( or nso fraction ) include asphaltenes , resins and oxygenated byproducts of biodegradation , and are considered largely inert to biodegradation .
field tests jointly conducted in pws by the united states environmental protection agency ( usepa ) , the alaska department of environmental conservation and exxon ( prince et  al
. , 1993 ) showed , with high statistical significance , that the rate of oil degradation was a function of the ratio of nitrogen / oil , the nonpolar hydrocarbon fraction remaining , and time ( bragg et  al . , 1994 ) .
bioremediation increased the rate of polycyclicaromatichydrocarbon ( pah ) degradation in the relatively undegraded oil found on the shorelines at this time by a factor of 2 , and the degradation rate of total gcdetectable hydrocarbons by a factor of 5 relative to the controls ( bragg et  al . , 1994 ) .
total pah depletion was 44% of that present at the start of the test ( fig . 
depletion of pah groups in subsurface oil residue within fertilized section of kn135b bioremediation field test , pws , 1990 .
it is important to recognize that biodegradation does not remove all of the hydrocarbons in crude oil 
some compounds are recalcitrant to microbial attack such as highermolecularweight pahs and the polars ( nso  molecules containing nitrogen , sulfur and oxygen ) ( oudot et  al . , 1998 ;
further , if the biodegradation rate can not be accelerated significantly  by a factor of at least 2  it has been suggested that bioremediation is not worth considering ( zhu et  al . , 2001 ) . as crude oil weathers the rate of biodegradation declines since hydrocarbon biodegradation follows firstorder kinetics ( e.g. venosa et  al . , 1996 ; wrenn et  al . ,
2006 ) , which means that as the concentration of degradable hydrocarbons decreases so does the potential applicability of bioremediation .
however , some studies indicate that once the easily degraded alkanes and lowermolecularweight aromatics are removed , the rate of biodegradation of the remaining oil residues is no longer limited by nutrients , and the biodegradation rate naturally slows . in bioremediation tests conducted following the prestige spill ( diez et  al . , 2005 ; gallego et  al . ,
2006 ) , for example , the rate of biodegradation of all highermolecularweight components slowed dramatically at a point consistent with the disappearance of the lowermolecularweight alkanes ( fig .  4 ) .
similarly , bioremediation tests conducted by venosa and colleagues ( 1996 ) on a simulated spill in delaware bay showed a decline in the rate of biodegradation once the alkanes were removed .
wrenn and colleagues ( 2006 ) , in a series of microcosm tests , also found that the sensitivity of the oil mineralization rate to nutrient input decreased rapidly as the extent of oil degradation increased , and after about 2 weeks the rate of oil mineralization appeared to be independent of nutrient input .
they concluded that there may be little value in maintaining a longterm supply of nutrients in contact with oilcontaminated sediments .
depletion indices measured at prestige oil spill bioremediation test ( moreira site ) during a 1year sampling period ( gallego et  al . , 2006 ) .
many of the microorganisms capable of attacking highermolecularweight pahs may operate by cometabolism ( cerniglia , 1993 ; kanaly and bartha , 1999 ; van herwijnen et  al . , 2003 ) . when alkanes and lowermolecularweight aromatic substrates have been removed from the oil residues , the enzymes needed to attack the higherweight pahs may not be induced , resulting in decreased rates of biodegradation .
other ratedecreasing factors that may result from the removal of lowermolecularweight compounds are the increase in viscosity of the remaining oil residue and the decrease in mass fraction of degradable hydrocarbons . these slow the diffusion of degradable hydrocarbons to the oil 
uraizee and colleagues ( 1998 ) systematically altered the concentration of nonbiodegradable heterocyclic polars and asphaltenes in crude oil and analysed its impact on transport of biodegradable hydrocarbons to microorganisms .
as the polars content ( and oil viscosity ) increased , the maximum respiratory oxygen uptake decreased .
obviously , the weathering point at which the degradation of pah is no longer nutrient limited will depend on the starting oil composition and the extent of prior weathering , so each case must be evaluated independently . in addition , successful bioremediation in marine environments requires that water containing the applied nutrients must be able to contact the oil .
oil that is sequestered ( buried under layers of sediment that impede free water flow ) may not benefit from applied nutrients . in this case , as well as those where oxygen is limiting , tilling can be considered , but this raises the issue of physical disruption of the environment potentially causing greater environmental harm than benefit .
since bioremediation was previously successful in pws and since subsurface oil residues ( ssor ) remain at some locations , there have been suggestions that bioremediation be applied again ( michel et  al . ,
we examine the extent of weathering that had occurred by 2007 , over the 18 years since the spill , and we estimate bioremediation potentials by examining the composition of the ssor , its distribution by location and extent of depletion , and the current natural nutrient concentrations in pore water in its vicinity .
in 2001 and 2003 the national oceanic and atmospheric administration ( noaa ) conducted random sampling of 4982 pits dug at 114 sites in pws ( short et  al . , 2002 ; 2004 ; 2006 ) , and found that 97.8% of the pits had no oil ( no ) or light oil residues ( lor ) even though these sites had been heavily oiled in 1989 and previously had significant levels of ssor .
separate gridded surveys were also conducted for exxonmobil in 2002 and 2007 . the 2007 survey conducted for exxonmobil ( boehm et  al . , 2008 ) at 22 sites that were found to be most heavily oiled in noaa 's 20012003 surveys found no ssor in 529 pits ( 71% ) .
sediments from an additional 162 pits ( 21.8% ) had light levels ( lor ) or only traces ( tr ) of ssor .
only 34 pits ( 4.6% ) were found with moderate ssor ( mor ) and 19 pits ( 2.6% ) with heavy ssor ( hor ) .
the pits with mor or hor showed a pattern of extreme ssor patchiness , i.e. widely scattered small patches , mostly sequestered under boulder / cobble armour . even where ssor remains it
almost no resolvable alkanes remain in samples collected from 2002 through 2007 ( fig . 
most samples also had lost more than 70% of the original pahs through a combination of biodegradation and other weathering processes .
we believe 70% depletion of total pah is the approximate threshold above which bioremediation is likely to be ineffective .
4 ) that showed dramatic reduction in bioremediation effectiveness upon depletion of light alkanes , the almost total depletion of resolvable alkanes in current pws samples ( fig .  5 ) , and other studies showing lack of biodegradation rate sensitivity to added nitrogen once lighter alkanes and aromatics were depleted ( oudot et  al . , 1998 ;
we have computed bioremediation indices for all analysed samples for which total pah loss could be quantified .
the 70% is meant as a probable threshold , not a specific value above which bioremediation would not work .
figure  6 shows the bioremediation index for all samples collected from 2002 through 2007 .
if the total pah content of a sample was less than 500  ng  g sediment , that sample was not included in plots because the total pah concentration was approaching natural background levels in pws and could not be confirmed as evos ssor .
however , note that the average percentage of depletion of total pah computed for all samples with total pah content <  500  ng  g sediment was 94% .
hor residues were found that contained total pah of less than 500  ng  g sediment ; however , these comprised 8095% polars  they looked like  oil but contained only highly degraded evosderived residues . as shown in fig . 
6 , only about 25% of the total samples from 2002 to 2007 contained ssor that might respond to bioremediation based on the 70% threshold .
depletion of total resolvable alkanes in all oiled sediment samples analysed from 2002 to 2007 as function of total pah depletion .
figure  7 shows further breakdown of the extent of total pah depletion as a function of tidal elevation [ i.e. pit elevation above mllw ( mean low low water tide ) ] .
most samples with less than 70% total pah depletion are located at + 2 and + 3  m ( middle to upper intertidal zone ) .
this is not unexpected since ssor at 0 and + 1  m is exposed to water for much longer times .
bioremediation indices for samples collected in 2007 as function of pit elevation above mean low tide ( mllw ) .
samples least degraded reside in upper intertidal zone ( a and b ) , while few were found that might respond to bioremediation in lower intertidal ( c and d ) .
as expected , the trend in individual analyte degradation demonstrates that percentage depletion decreases as the pah alkyl substitution increases ( fig . 
even at 80% total pah depletion about half of the c3 and c4phenanthrenes remain , probably because the analytes become more recalcitrant and less water soluble as alkyl substitution increases .
adding nutrients would likely have minimal impact on the rates of biodegradation of remaining highly substituted three and fourring aromatics ( oudot et  al .
, 1998 ; wang et  al . , 1998 ) . depletion of c1 to c4alkylated phenanthrenes versus total pah depletion in samples from 2002 to 2007 .
another factor we evaluated was the concentration of nitrogen naturally available within sediment pore water .
the ratio of nitrogen / nonpolar ssor provides a conservative estimate of the ratio of nitrogen / degradable hydrocarbons .
it is a convenient ratio for comparing recent nutrient / ssor ratios with values measured during 1990 bioremediation field tests at knight island shoreline segment 135 ( kn135 ) , considering measured values of oil loading , polars content and nitrateasnitrogen concentration .
figure  9 shows the ratios of nitrogen / nonpolar ssor measured at several sites from 2006 to 2008 normalized to the average value measured over 109 days for the fertilized section of the bioremediation test at kn135b in 1990 .
horizontal lines indicate the ratios for the fertilized and control sections of kn135b in 1990 . clearly , the amount of natural background nitrogen is significantly higher at most sites during 20062008 than at the kn135b control in 1990 , and many sites now approach the same value as realized during fertilizer addition in 1990 . ratio of ( nitrogen concentration / nonpolar hydrocarbons ) in 20062008 pore water samples normalized to same ratio measured in fertilized portion of kn135b during 1990 bioremediation test .
first , the amount of ssor to be degraded ( the ssor loading in mg ssor  g sediment ) is lower , and the polars content of the remaining ssor is higher , resulting in less nonpolar ssor .
second , the average nitrateasnitrogen for 10 segments measured from 2006 through 2008 was 0.36  mg  l water ( with range by site of from 0.1 to 1.18  mg 
the concentrations of nitrogen in the pore water of the sediments where ssor remains today are significantly higher than in the incoming seawater .
the incoming seawater at sites studied had a concentration of approximately 0.014  mg nitrateasnitrogen l. even in 1990 noaa noted higherthanexpected concentrations of nitrogen in oiled sediments during most of the summer . that raises the question as to the nitrogen source .
perhaps it is being generated within the intertidal zone itself by nitrogenfixing cyanobacteria , or it may be coming from groundwater entering the intertidal zone that is possibly enriched in nitrogen by nitrogenfixing frankia species associated with the alder trees that line the upper intertidal zones of many of the shorelines of pws .
we believe that the current presence of relatively high levels of natural nutrients shows that the natural biodegradation is no longer nutrient limited ; the rate of biodegradation is lower , and hence demand for nitrogen is lower .
third , most of the remaining ssor is sequestered in lowpermeability fine sediments under boulder / cobble armour where it may not have good accessibility to the nutrients that flow with the tides .
finally , the small amount of ssor remaining that might respond to bioremediation is scattered in isolated small patches .
figure  10 shows the extent of pah degradation remaining in 2007 at sm006b ( smith island ) , which noaa ( michel et  al . ,
2006 ) identified as the site that in 2001 contained the largest volume of oiled sediment .
this site is typical of the way residual ssor is distributed ; few pit locations contain ssor with less than 70% loss of total pah and most have no detectable ssor . at sm006b
only two pits near the top of the beach ( red crosshatched area ) contain ssor that might be amenable to bioremediation .
however , ssor remains in these locations because it is sequestered under boulders and cobbles .
photo of site sm006b showing the % total pah depletion at pits sampled in 2007 .
only two small patches of oil remain that might respond to bioremediation . as demonstrated by the extensive depletion of total pah in most samples shown in figs  68 ,
however , effective biodegradation requires that flowing water resupply nutrients to microbes in contact with the oil .
the geology ( porosity and permeability ) of pws shorelines is highly heterogeneous , leading to scattered pockets of sequestered oiled sediment that are not fully in communication with flowing seawater .
most of the ssor that is less degraded than 90% resides in such sequestered locations .
fertilizer added to shorelines for in situ bioremediation is unlikely to contact this sequestered oil , but would simply be diluted and washed away by water flowing through more permeable zones / paths .
thus , the degree of sequestration for individual pockets of ssor probably now is the major factor affecting extent of total pah depletion , not nutrient availability .
if microcosm tests were conducted on currently sequestered oiled sediments , results would only indicate the maximum possible increase in biodegradation rate that might be realized if sediments were not sequestered , since contact between oil and added nutrients in microcosms is not impeded by geologic factors .
for this reason , microcosm tests would not be indicative of any actual in situ treatment potential on shorelines , but instead would only show the ideal maximum bioremediation rate increase consistent with limitations of the tested oil composition .
if in situ bioremediation is unlikely to work because the oiled sediments are sequestered , one might consider tilling of the sediments to increase contact with added nutrients .
however , since several studies have shown that pahs from the oil residues in their current sequestered state in pws are not bioavailable to shoreline biota or prey species ( boehm et  al . , 2004 ;
harwell and gentile , 2006 ; neff et  al . , 2006 ) , tilling would cause physical disruption to the environment , disturbance of wildlife and , therefore , greater harm than benefit .
as shown in figs  68 , the highermolecularweight pahs remaining in the oil residue will continue to degrade at rates consistent with adequate natural nutrient content in pore water , but variable extents of sequestration of scattered individual small pockets of ssor . in summary , there is relatively little of the evos oil remaining in pws , it is mostly sequestered under boulders / cobbles , and it will naturally continue to slowly degrade .
therefore , we feel that bioremediation would not be appropriate for this ssor , making natural attenuation the preferable treatment .
in 2001 and 2003 the national oceanic and atmospheric administration ( noaa ) conducted random sampling of 4982 pits dug at 114 sites in pws ( short et  al . , 2002 ; 2004 ; 2006 ) , and found that 97.8% of the pits had no oil ( no ) or light oil residues ( lor ) even though these sites had been heavily oiled in 1989 and previously had significant levels of ssor .
separate gridded surveys were also conducted for exxonmobil in 2002 and 2007 . the 2007 survey conducted for exxonmobil ( boehm et  al . , 2008 ) at 22 sites that were found to be most heavily oiled in noaa 's 20012003 surveys found no ssor in 529 pits ( 71% ) .
sediments from an additional 162 pits ( 21.8% ) had light levels ( lor ) or only traces ( tr ) of ssor .
only 34 pits ( 4.6% ) were found with moderate ssor ( mor ) and 19 pits ( 2.6% ) with heavy ssor ( hor ) .
the pits with mor or hor showed a pattern of extreme ssor patchiness , i.e. widely scattered small patches , mostly sequestered under boulder / cobble armour . even where ssor remains it
almost no resolvable alkanes remain in samples collected from 2002 through 2007 ( fig . 
most samples also had lost more than 70% of the original pahs through a combination of biodegradation and other weathering processes .
we believe 70% depletion of total pah is the approximate threshold above which bioremediation is likely to be ineffective .
4 ) that showed dramatic reduction in bioremediation effectiveness upon depletion of light alkanes , the almost total depletion of resolvable alkanes in current pws samples ( fig .  5 ) , and other studies showing lack of biodegradation rate sensitivity to added nitrogen once lighter alkanes and aromatics were depleted ( oudot et  al . , 1998 ;
we have computed bioremediation indices for all analysed samples for which total pah loss could be quantified .
the 70% is meant as a probable threshold , not a specific value above which bioremediation would not work .
figure  6 shows the bioremediation index for all samples collected from 2002 through 2007 .
if the total pah content of a sample was less than 500  ng  g sediment , that sample was not included in plots because the total pah concentration was approaching natural background levels in pws and could not be confirmed as evos ssor .
however , note that the average percentage of depletion of total pah computed for all samples with total pah content <  500  ng  g sediment was 94% .
hor residues were found that contained total pah of less than 500  ng  g sediment ; however , these comprised 8095% polars  they looked like  oil but contained only highly degraded evosderived residues . as shown in fig . 
6 , only about 25% of the total samples from 2002 to 2007 contained ssor that might respond to bioremediation based on the 70% threshold .
depletion of total resolvable alkanes in all oiled sediment samples analysed from 2002 to 2007 as function of total pah depletion .
figure  7 shows further breakdown of the extent of total pah depletion as a function of tidal elevation [ i.e. pit elevation above mllw ( mean low low water tide ) ] .
most samples with less than 70% total pah depletion are located at + 2 and + 3  m ( middle to upper intertidal zone ) .
this is not unexpected since ssor at 0 and + 1  m is exposed to water for much longer times .
bioremediation indices for samples collected in 2007 as function of pit elevation above mean low tide ( mllw ) .
samples least degraded reside in upper intertidal zone ( a and b ) , while few were found that might respond to bioremediation in lower intertidal ( c and d ) .
as expected , the trend in individual analyte degradation demonstrates that percentage depletion decreases as the pah alkyl substitution increases ( fig . 
even at 80% total pah depletion about half of the c3 and c4phenanthrenes remain , probably because the analytes become more recalcitrant and less water soluble as alkyl substitution increases .
adding nutrients would likely have minimal impact on the rates of biodegradation of remaining highly substituted three and fourring aromatics ( oudot et  al .
, 1998 ; wang et  al . , 1998 ) . depletion of c1 to c4alkylated phenanthrenes versus total pah depletion in samples from 2002 to 2007 .
another factor we evaluated was the concentration of nitrogen naturally available within sediment pore water .
the ratio of nitrogen / nonpolar ssor provides a conservative estimate of the ratio of nitrogen / degradable hydrocarbons .
it is a convenient ratio for comparing recent nutrient / ssor ratios with values measured during 1990 bioremediation field tests at knight island shoreline segment 135 ( kn135 ) , considering measured values of oil loading , polars content and nitrateasnitrogen concentration .
figure  9 shows the ratios of nitrogen / nonpolar ssor measured at several sites from 2006 to 2008 normalized to the average value measured over 109 days for the fertilized section of the bioremediation test at kn135b in 1990 .
horizontal lines indicate the ratios for the fertilized and control sections of kn135b in 1990 . clearly , the amount of natural background nitrogen is significantly higher at most sites during 20062008 than at the kn135b control in 1990 , and many sites now approach the same value as realized during fertilizer addition in 1990 . ratio of ( nitrogen concentration / nonpolar hydrocarbons ) in 20062008 pore water samples normalized to same ratio measured in fertilized portion of kn135b during 1990 bioremediation test .
first , the amount of ssor to be degraded ( the ssor loading in mg ssor  g sediment ) is lower , and the polars content of the remaining ssor is higher , resulting in less nonpolar ssor .
second , the average nitrateasnitrogen for 10 segments measured from 2006 through 2008 was 0.36  mg  l water ( with range by site of from 0.1 to 1.18  mg 
the concentrations of nitrogen in the pore water of the sediments where ssor remains today are significantly higher than in the incoming seawater .
the incoming seawater at sites studied had a concentration of approximately 0.014  mg nitrateasnitrogen l. even in 1990 noaa noted higherthanexpected concentrations of nitrogen in oiled sediments during most of the summer . that raises the question as to the nitrogen source .
perhaps it is being generated within the intertidal zone itself by nitrogenfixing cyanobacteria , or it may be coming from groundwater entering the intertidal zone that is possibly enriched in nitrogen by nitrogenfixing frankia species associated with the alder trees that line the upper intertidal zones of many of the shorelines of pws .
we believe that the current presence of relatively high levels of natural nutrients shows that the natural biodegradation is no longer nutrient limited ; the rate of biodegradation is lower , and hence demand for nitrogen is lower .
third , most of the remaining ssor is sequestered in lowpermeability fine sediments under boulder / cobble armour where it may not have good accessibility to the nutrients that flow with the tides .
finally , the small amount of ssor remaining that might respond to bioremediation is scattered in isolated small patches .
figure  10 shows the extent of pah degradation remaining in 2007 at sm006b ( smith island ) , which noaa ( michel et  al . ,
2006 ) identified as the site that in 2001 contained the largest volume of oiled sediment .
this site is typical of the way residual ssor is distributed ; few pit locations contain ssor with less than 70% loss of total pah and most have no detectable ssor . at sm006b
only two pits near the top of the beach ( red crosshatched area ) contain ssor that might be amenable to bioremediation .
however , ssor remains in these locations because it is sequestered under boulders and cobbles .
photo of site sm006b showing the % total pah depletion at pits sampled in 2007 .
only two small patches of oil remain that might respond to bioremediation . as demonstrated by the extensive depletion of total pah in most samples shown in figs  68 ,
however , effective biodegradation requires that flowing water resupply nutrients to microbes in contact with the oil .
the geology ( porosity and permeability ) of pws shorelines is highly heterogeneous , leading to scattered pockets of sequestered oiled sediment that are not fully in communication with flowing seawater .
most of the ssor that is less degraded than 90% resides in such sequestered locations .
fertilizer added to shorelines for in situ bioremediation is unlikely to contact this sequestered oil , but would simply be diluted and washed away by water flowing through more permeable zones / paths .
thus , the degree of sequestration for individual pockets of ssor probably now is the major factor affecting extent of total pah depletion , not nutrient availability .
if microcosm tests were conducted on currently sequestered oiled sediments , results would only indicate the maximum possible increase in biodegradation rate that might be realized if sediments were not sequestered , since contact between oil and added nutrients in microcosms is not impeded by geologic factors .
for this reason , microcosm tests would not be indicative of any actual in situ treatment potential on shorelines , but instead would only show the ideal maximum bioremediation rate increase consistent with limitations of the tested oil composition .
if in situ bioremediation is unlikely to work because the oiled sediments are sequestered , one might consider tilling of the sediments to increase contact with added nutrients .
however , since several studies have shown that pahs from the oil residues in their current sequestered state in pws are not bioavailable to shoreline biota or prey species ( boehm et  al . , 2004 ;
harwell and gentile , 2006 ; neff et  al . , 2006 ) , tilling would cause physical disruption to the environment , disturbance of wildlife and , therefore , greater harm than benefit .
as shown in figs  68 , the highermolecularweight pahs remaining in the oil residue will continue to degrade at rates consistent with adequate natural nutrient content in pore water , but variable extents of sequestration of scattered individual small pockets of ssor . in summary , there is relatively little of the evos oil remaining in pws , it is mostly sequestered under boulders / cobbles , and it will naturally continue to slowly degrade .
therefore , we feel that bioremediation would not be appropriate for this ssor , making natural attenuation the preferable treatment .
in 2007 , we sampled 22 pws shoreline sites that had also been sampled in 2002 and have been shown to be among the most persistently oiled in earlier surveys ( short et  al .
, 2002 ; 2004 ; 2006 ; michel et  al . , 2006 ; boehm et  al . , 2008 ; page et  al . ,
these 22 study sites included all 10 of the 2001 and 2003 noaa hor sites and 18 of the 23 2001 and 2003 mor or hor sites .
in addition , two sites were included that were both heavily oiled in 1989 and identified as segments having extensive otter foraging . at each shoreline , 100mlong transects
were surveyed either at four elevations ( 0 , + 1 , + 2 and + 3  m ) or just at two elevations ( + 2 and + 3  m ) above mllw .
pits were dug 10  m apart along each transect . using established surveying methods ( owens and sergy , 2000 ; boehm et  al . , 2008 ) , the visual ssor level ( heavy , moderate , light , trace or oil film ) was noted for each pit , and samples of sediment were collected for analysis . to access sediment showing any visible ssor , it was necessary to remove surface boulders and cobbles and then dig to a depth of 3050  cm .
all sediment samples were solvent extracted to determine the total extractable hydrocarbon content ( teh ) .
approximately 25% of all samples showing ssor were then selected for detailed gcms chemistry . however , for sites specifically selected for evaluating detailed bioremediation potential , all grid pits containing a teh of greater than 1  mg ssor  g sediment were analysed by gcms using established protocols ( page et  al . , 1995 ; douglas et  al . , 2004 ) .
concentrations of 41 target pah were measured , including parent and alkyl substituted naphthalene , fluorene , phenanthrene , dibenzothiophene , chrysene and sixring components .
also measured were saturated hydrocarbons including resolvable alkanes ( c9 to c40normal alkanes plus pristine and phytane ) , total gcdetectable hydrocarbons , saturated cyclic hydrocarbon biomarkers and seven c20c28 triaromatic steroids .
the polars ( nso ) fraction was estimated by difference of gravimetric mass of total extract less f1 and f2 fractions ( atlas and bragg , 2007 ) . the analytical and quality assurance and quality control methods for teh , stearane /
triterpane biomarkers , and pah analyses of sediment samples have been published in detail elsewhere ( page et  al . , 1995 ) .
all analyte concentrations were calculated on a dry sample weight basis ( ng  g sediment ) to assess the sediment concentration and on an extract weight basis ( g  g extract ) to calculate analyte losses relative to the original cargo crude oil ( atlas and bragg , 2007 ) .
we evaluated bioremediation potential by computing the extent of depletion of key components relative to their concentrations in the evos cargo crude and comparing that with depletion results from prior bioremediation studies ( atlas and bragg , 2007 ) .
the bioremediation potential index was based on the mass ratio method using c29stigmastane as the most stable ( conserved ) species in evos cargo oil 18 years after the spill ( atlas and bragg , 2007 ) .
the total loss by any oil component , x , from weathering and biodegradation relative to the evos cargo oil was computed by the following equation : 
 where : cx  =  mass concentration of component xcc29r  =  mass concentration of c29rstigmastanes  =  sampleevos  =  evos cargo oil stabilized at atmospheric pressure . 
 
where : cx  =  mass concentration of component x cc29r  =  mass concentration of c29rstigmastane evos  =  evos cargo oil stabilized at atmospheric pressure .
a bioremediation index was calculated from the mass loss data as follows : computing this index , ordering the results in increasing rank and graphing permits a rapid visual determination as to whether the aggregate ssor compositions would respond to nutrient addition .
seventy per cent total pah degradation is used as the normalizing factor above which bioremediation effectiveness is unlikely based on results of prior bioremediation tests where nutrient addition was no longer effective after disappearance of light alkanes . to evaluate the concentration of nitrogen present within the pore water at the surveyed grid locations , stainless steel piezotubes were driven into subsurface sediment to a depth where ssor layers exist ( 3050  cm below boulder
/ cobble armour ) , and in situ measurements were taken of dissolved oxygen , water salinity and temperature using a multimeter .
then 100  ml of samples of pore water were withdrawn by peristaltic pump and stored frozen until analysed for inorganic nitrate / nitrite as nitrogen .
this sampling was completed prior to any pits being dug at the site to prevent contamination of pore water and was usually done 1 day prior to pit sampling .
in 2007 , we sampled 22 pws shoreline sites that had also been sampled in 2002 and have been shown to be among the most persistently oiled in earlier surveys ( short et  al .
, 2002 ; 2004 ; 2006 ; michel et  al . , 2006 ; boehm et  al . , 2008 ; page et  al . ,
these 22 study sites included all 10 of the 2001 and 2003 noaa hor sites and 18 of the 23 2001 and 2003 mor or hor sites .
in addition , two sites were included that were both heavily oiled in 1989 and identified as segments having extensive otter foraging . at each shoreline , 100mlong transects
were surveyed either at four elevations ( 0 , + 1 , + 2 and + 3  m ) or just at two elevations ( + 2 and + 3  m ) above mllw .
pits were dug 10  m apart along each transect . using established surveying methods ( owens and sergy , 2000 ; boehm et  al . , 2008 ) , the visual ssor level ( heavy , moderate , light , trace or oil film ) was noted for each pit , and samples of sediment were collected for analysis . to access sediment showing any visible ssor , it was necessary to remove surface boulders and cobbles and then dig to a depth of 3050  cm .
all sediment samples were solvent extracted to determine the total extractable hydrocarbon content ( teh ) .
approximately 25% of all samples showing ssor were then selected for detailed gcms chemistry . however , for sites specifically selected for evaluating detailed bioremediation potential , all grid pits containing a teh of greater than 1  mg ssor  g sediment were analysed by gcms using established protocols ( page et  al . , 1995 ; douglas et  al . , 2004 ) .
concentrations of 41 target pah were measured , including parent and alkyl substituted naphthalene , fluorene , phenanthrene , dibenzothiophene , chrysene and sixring components .
also measured were saturated hydrocarbons including resolvable alkanes ( c9 to c40normal alkanes plus pristine and phytane ) , total gcdetectable hydrocarbons , saturated cyclic hydrocarbon biomarkers and seven c20c28 triaromatic steroids .
the polars ( nso ) fraction was estimated by difference of gravimetric mass of total extract less f1 and f2 fractions ( atlas and bragg , 2007 ) . the analytical and quality assurance and quality control methods for teh , stearane /
triterpane biomarkers , and pah analyses of sediment samples have been published in detail elsewhere ( page et  al . , 1995 ) .
all analyte concentrations were calculated on a dry sample weight basis ( ng  g sediment ) to assess the sediment concentration and on an extract weight basis ( g  g extract ) to calculate analyte losses relative to the original cargo crude oil ( atlas and bragg , 2007 ) .
we evaluated bioremediation potential by computing the extent of depletion of key components relative to their concentrations in the evos cargo crude and comparing that with depletion results from prior bioremediation studies ( atlas and bragg , 2007 ) .
the bioremediation potential index was based on the mass ratio method using c29stigmastane as the most stable ( conserved ) species in evos cargo oil 18 years after the spill ( atlas and bragg , 2007 ) .
the total loss by any oil component , x , from weathering and biodegradation relative to the evos cargo oil was computed by the following equation : 
 where : cx  =  mass concentration of component xcc29r  =  mass concentration of c29rstigmastanes  =  sampleevos  =  evos cargo oil stabilized at atmospheric pressure . 
 
where : cx  =  mass concentration of component x cc29r  =  mass concentration of c29rstigmastane evos  =  evos cargo oil stabilized at atmospheric pressure .
a bioremediation index was calculated from the mass loss data as follows : computing this index , ordering the results in increasing rank and graphing permits a rapid visual determination as to whether the aggregate ssor compositions would respond to nutrient addition .
seventy per cent total pah degradation is used as the normalizing factor above which bioremediation effectiveness is unlikely based on results of prior bioremediation tests where nutrient addition was no longer effective after disappearance of light alkanes .
to evaluate the concentration of nitrogen present within the pore water at the surveyed grid locations , stainless steel piezotubes were driven into subsurface sediment to a depth where ssor layers exist ( 3050  cm below boulder / cobble armour ) , and in situ measurements were taken of dissolved oxygen , water salinity and temperature using a multimeter .
then 100  ml of samples of pore water were withdrawn by peristaltic pump and stored frozen until analysed for inorganic nitrate / nitrite as nitrogen .
this sampling was completed prior to any pits being dug at the site to prevent contamination of pore water and was usually done 1 day prior to pit sampling .
 summaryin this article we consider what we have learned from the exxon valdez oil spill ( evos ) in terms of when bioremediation should be considered and what it can accomplish . 
 we present data on the state of oiling of prince william sound shorelines 18 years after the spill , including the concentration and composition of subsurface oil residues ( ssor ) sampled by systematic shoreline surveys conducted between 2002 and 2007 . over this period 
 , 346 sediment samples were analysed by gcms and extents of hydrocarbon depletion were quantified . in 2007 alone , 744 sediment samples were collected and extracted , and 222 were analysed . 
 most sediment samples from sites that were heavily oiled by the spill and physically cleaned and bioremediated between 1989 and 1991 show no remaining ssor . where ssor does remain , it is for the most part highly weathered , with 82% of 2007 samples indicating depletion of total polycyclic aromatic hydrocarbon ( total pah ) of >  70% relative to evos oil . 
 this ssor is sequestered in patchy deposits under boulder / cobble armour , generally in the midtoupper intertidal zone . 
 the relatively high nutrient concentrations measured at these sites , the patchy distribution and the weathering state of the ssor suggest that it is in a form and location where bioremediation likely would be ineffective at increasing the rate of hydrocarbon removal . 
Introduction
Results and discussion
Bioremediation potential in PWS 18 years after the spill
Experimental procedures
Distribution and amount of remaining oil residues
Extent of weathering/bioremediation potential index
Measurement of nutrients