activated carbon ( ac ) addition is currently 
 being investigated 
 as in situ technology to remediate polluted sediments and soil . a critical consideration for the application 
 in aerobic soil is the long - term impact of ac on intrinsic microbial 
 community structure and functioning , including the potential to biodegrade 
 pollutants . adding ac to soil will reduce the soil porewater concentration 
 of hydrophobic organic compounds ( hocs ) which will be adsorbed and strongly bound by the ac ( figure 1 , process i ) . pollutants bound in the micropores 
 of ac become less accessible for biouptake by soil - dwelling organisms and plants in comparison with weakly bound or dissolved 
 pollutants . ac amendment may thereby reduce the transfer of hocs from 
 the soil matrix into the terrestrial food - chain and also reduce phytotoxicity ( figure 1 , process iii ) . addition of ac to soil will also reduce hocs leaching ( figure 1 , process iv ) and volatilization 
 risks ( figure 1 , process ii ) . strong binding of hocs by ac will , however , reduce 
 the availability of hocs to microorganisms with the ability to metabolize 
 these compounds . a potential consequence could be the disappearance 
 of hoc degraders from the predominant soil microbial community and/or 
 the down - regulation of hocs metabolism in favor of other carbon substrates . 
 in soils with abundant hocs degrading microorganisms , this may impair 
 the pollution attenuation via biodegradation ( figure 1 , process v ) . illustration of the interlinkage of the various processes 
 affecting 
 the hydrophobic organic compounds ( hocs ) fate in activated carbon 
 ( ac ) amended soil . ( i ) ac amendment increases the sorption of hocs 
 and reduces the concentration of hocs dissolved in porewater . reduced 
 hoc porewater concentrations typically imply ( ii ) less hocs volatilization 
 to the atmosphere , ( iii ) less biouptake 
 of hocs by earth worms and plants , and ( iv ) less 
 hocs leaching to groundwater . reduced 
 hoc porewater concentrations may also change soil microbial community 
 structure and functioning , including ( v ) the hocs biodegradation . secondary effects of ac on other substrates such as dissolved organic 
 carbon and nutrients concentrations , soil 
 wettability , and physical soil structure may also change soil microbial 
 community structure and functioning . there are reports of differing effects of strong 
 sorbent amendments 
 on pollutant biodegradation processes . on the one hand , addition of 
 ac to contaminated soils was shown to reduce spiked c 
 phenanthrene metabolism to co2 in laboratory 
 batch experiments , and karapanagioti et 
 al . showed that slow sorption kinetics 
 limited spiked phenanthrene biodegradation in sediment slurries containing 
 coal particles . on the other hand , vasilyeva et al . suggested that ac helped overcome toxicity of polychlorinated 
 biphenyls to microbes , and payne et al . also reported that addition of ac had a slight stimulatory effect 
 on pcb dechlorination in sediment . bushnaf et al . reported that reduced availability of monoaromatic hydrocarbons 
 in biochar amended soil led to greater biodegradation of linear , branched 
 and cyclic alkanes , and the total petroleum hydrocarbon vapor degradation 
 was comparable in sandy soil with and without 2% biochar . in a direct 
 comparison of ac amendment with biostimulation and bioaugmentation , 
 hale et al . found that 2% ac addition 
 was more effective than biostimulation or bioaugmentation in further 
 reducing the availability of an already strongly sequestered polycyclic 
 aromatic hydrocarbon ( pah ) pollution in river tyne sediment the aim of this paper was to study effects of ac amendment on the 
 predominant bacterial community structure , and its functioning with 
 emphasis on the biodegradation of pahs . to our knowledge , this is 
 the first - ever investigation of the long - term effects of ac amendment 
 on soil microbial communities under realistic field conditions . we 
 report changes in the predominant bacterial community structure over 
 a three year period following amendment of a pah - polluted urban soil 
 with granular or powdered activated carbon ( gac and pac ) . for the 
 samples collected in year 3 , we compared the bacterial communities 
 and their functioning in greater detail using molecular microbiological 
 and chemical methods . we assess the combined effect of sorption and 
 biodegradation on pah availability in soil with and without gac or 
 pac to better understand impacts on environmental risks at contaminated 
 sites . details of the field 
 trials of ac - based soil remediation are reported by hale et al . the soil used in the lysimeter experiment was 
 excavated at a building site in drammen , norway , intermixed using 
 an excavator bucket with 2% wet soil weight of pac ( silcarbon th90 , 
 average 20 m grain size , with 80% < 45 m ) or gac 
 ( silcarbon 0.30.8 , 300800 m grain size ) , and 
 embedded in outdoor lysimeters with 25 m surface area 
 and between 2.5 and 3 m depth . the soil initially had a total organic 
 carbon content of 2.50  0.04% dry weight , and amended soils 
 contained the intended ac dose , although with considerable variability , 
 2.0  1.0% for gac or 2.4  1.9% for pac . two series of soil samples for microbial analysis were 
 taken immediately after the ac amendment of the soil , after 6 months , 
 and after 3 years , and frozen at 20 c with and without 
 the addition of absolute ethanol 1:1 ( v / v ) . for the year 3 samples , 
 additional soil samples were stored in the cold room at 4 c 
 without ethanol addition for the batch experiments . ten l of the year 
 3 sample stored in ethanol was added to 990 l of filter - sterilized 
 phosphate buffer saline ( pbs , oxoid ) and the cells were stained by 
 adding 50 l of sybr gold nucleic acid stain in 100 concentrate 
 in dimethyl sulfoxide ( dmso ) ( invitrogen ltd . , paisley , uk ) , wrapped 
 in foil and incubated at room temperature for 30 min , after which 
 they were filtered using a sterile millipore filter holder and a 0.2-mm - pore - size 
 black polycarbonate filter ( diameter 25 mm ; millipore ) . the filters 
 were transferred to glass microscope slides containing a drop of citifluor 
 ( citifluor ltd . , a further drop of citifluor was placed on top of the 
 filter , and a coverslip was carefully placed on top of the preparation . total bacteria were determined by direct count under an oil - immersion 
 objective ( 100 magnification ) using an olympus bx40 epifluorescence 
 microscope ; 20 randomly chosen fields of view were counted using a 
 dilution that yielded between 30 and 300 fluorescent cells having 
 a clear outline and finite cell shape . the error between samples was 
 estimated using the standard deviation of the mean from three replicates 
 measured per treatment . cell number per g of soil was determined by 
 considering the numbers and the areas of fields of view on the filter 
 membrane , the area of the membrane and the original sample dilution 
 factor . soil respiration was measured for 
 the year 3 samples by monitoring co2 concentrations in 
 50-ml crimp - top vials containing 15 g wet weight of either unamended , 
 pac - amended , or gac - amended soil . for each soil type duplicate batches 
 were monitored for 4 days at room temperature ( 20 c ) . gc - ms 
 analysis of co2 was performed on a fisons 8060 gc linked 
 to a fisons md800 ms with a hp - plot - q capillary column . a fingerprinting method , denaturing gradient gel electrophoresis ( dgge ) , was carried out to 
 determine similarities and differences between the predominant bacterial 
 communities in the experimental treatments , and to identify selected 
 members of these communities . total bacterial dna was extracted from 
 0.5-g ( wet weight ) aliquots of the stored ( without ethanol ) soil samples , 
 taken at times 0 and 6 months and 3 years after the start of the lysimeter 
 experiments . the dna extraction was carried out using fast dna soil 
 kit ( qbiogene ) with a purification step , modified from the method 
 of griffiths et al . , added prior to the 
 nucleic acid extraction in order to prevent the coextraction of compounds 
 such as humic acids and clay minerals , which are known to inhibit 
 pcr amplification . briefly , the pretreatment consisted in the extraction 
 of nucleic acids from the soil matrix by using hexadecyltrimethylammonium 
 bromide ( ctab , sigma - aldrich ) extraction buffer and phenol / chloroform / isoamyl 
 alcohol ( 25:24:1 , sigma - aldrich ) , followed by phenol removal using 
 chloroform / isoamyl alcohol ( 24:1 , sigma - aldrich ) . primers 2 and 3 , 
 targeting the bacteria , were used to pcr amplify the v3 region of 
 bacterial 16s rrna gene fragments , as previously described by muyzer 
 et al . , and the pcr products were analyzed 
 by dgge as described previously . the 
 dgge images were normalized and interpreted using the image analysis 
 software bionumerics ( applied maths nv , st . plymouth , uk ) was used to perform cluster 
 analysis using the pearson product - moment correlation coefficient 
 and analysis of similarity ( anosim ) . dominant dgge bands were excised from 
 the gel , pcr - amplified using primers 2/3 , purified and sequenced with 
 primer 2 or 3 ( 3.2 pmol/es/l ) using the abi prism big 
 dye terminator cycle sequencing ready reaction kit and an abi prism 
 377 dna sequencer ( applied biosystems , usa ) as previously described . sequences were compared against the ribosomal 
 database project ( rdp10 ) and genbank databases using the blast algorithm 
 to determine the closest matching sequence identity . the abundances of specific 
 ring - hydroxylating dioxygenase -subunit ( rhd ) genes were measured in each sample by real - time pcr using the following 
 primer sets : ( i ) p6b ( forward ( f ) : 5-tggcgaactcgtgtcggcac-3 ; 
 reverse ( r ) : 5- cgtccagrcaaccgaadayc 3 ) , targeting 
 a pdoa2/phda clade that includes mostly mycobacterium species and have m. vanbaalenii as a reference 
 strain ; ( ii ) p4 ( f : 5-ccggagacttcctgacgac-3 ; r : 5-gcasacgaaycgacgggt-3 ) 
 targeting a ebda1/etba1/akba1b clade that only includes rhodococcus species and have r. jostii rha1 
 as a reference strain ; and ( iii ) p7b ( f : 5- cacbtgcagctaycacg 
 3 ; r : 5- catgtggtccatgtagaac 3 ) 
 targeting a ipba1/bpha1 clade that includes mostly rhodococcus and some pseudomonas species 
 and , in particular , includes several r. erythropolis strains . real - time pcr experiments were conducted on a biorad cfx96 
 ( hercules , ca ) with a c1000 thermal cycler icycler and software version 
 1.6 ( biorad cfx manager ) . ten l of reaction mixture contained 
 3 l of template dna ( or filtered sterile molecular biology 
 water , sigma - aldrich , as negative control ) , and 15 pmol of each primer , 
 with the ssofast evagreen supermix for the cfx96 ( biorad laboratories 
 ltd . ) . the following temperature profile was used in the amplifications : 
 step one heated to 98 c ( 2 min ) , followed by 40 cycles of 2 
 s of denaturation at 98 c , 5 s at the primer specific annealing 
 temperature ( 58 c for p6b , 60 c for p4 , and 59 c 
 for p7b ) . at the end of the real - time pcr , a melting curve was performed 
 as a final step that consisted of the measurement of the sybr green 
 signal intensities during a 0.2 c temperature increment every 
 10 s from 65 to 95 c . the corresponding plot of change in fluorescence 
 versus temperature shows a single peak for every amplicon at its specific 
 melting temperature . for every set of primers , calibration curves 
 were obtained from standard dna prepared from plasmids containing 
 the cloned target sequences . the plasmid dna concentration was quantified 
 using nanodrop 1000 spectrophotometer ( thermo scientific ) . the copy 
 number of standard plasmids was calculated according to plasmid size , 
 insert lengths , and assuming a molecular mass of 660 da per base pair . stock solutions of standard dna were prepared at a concentration of 
 10 copies of plasmid l . for 
 the calibration curves , dna standards ranging from 10 to 
 10 target gene copies l all the standard curves 
 were linear ( r > 0.99 ) over the 9 
 orders 
 of magnitude ( 10 to 10 l ) of gene copy number . every sample was run in triplicate and each 
 experiment was repeated at least twice . in every run , the accuracy 
 in the detection of the target gene in the soil samples was confirmed 
 by comparing the melt curves and the agarose analysis of the qpcr 
 products to the ones obtained with dna extracted from model organisms 
 ( positive control ) in order to avoid false detection in the environmental 
 samples . a series of batch experiments was set up to study the uptake of 
 available pahs in passive samplers . passive 
 samplers are able to passively accumulate hocs from contaminated water 
 until partitioning equilibrium is established . pollutants taken up 
 by passive samplers are potentially also available for uptake by critical 
 receptors such as plants and earth worms ( figure 1 , process iii ) . to study the effect of sorption alone ( figure 1 , process i ) compared to the combined effect of 
 sorption and biodegradation ( figure 1 , processes 
 i and v ) on pollutant availability , batches were set up with and without 
 sodium azide addition which inhibits pah biodegradation . the microcosms comprised the following : 5 g ( wet weight ) of unamended 
 soil , pac - amended , or gac - amended soil and 40 ml of water . for each 
 soil type triplicate batches were set up with and without 1 g / l sodium 
 azide ( sigma aldrich ) . a 0.15  0.01 g clean polyethylene ( pe ) 
 passive sampler was added to each batch . blank controls were run in 
 parallel and consisted of triplicate batches containing pe samplers 
 and water with and without sodium azide . batches were plugged with 
 cotton balls and mixed at 100 rpm on an orbital shaker ( ika labortechnik , 
 germany ) in a secondary dark containment consisting of a cardboard 
 box with some holes punched into the lid for aeration . pe samplers 
 were removed after 3 weeks and extracted twice for 24 h in 10 ml of 
 50:50 v : v hexane / acetone . a surrogate standard was added to the first 
 extraction to monitor and correct for recoveries , which were 78  
 11% for d10-phenanthrene , 83  6% 
 for d10-pyrene , 92  4% for d12-benzanthracene , and 94  8% for d12-benzperyl . sample cleanup was 
 performed with a silica gel column topped by sodium sulfate before 
 gc - ms analysis with an agilent 6850 gas chromatograph ( db - xlb column 
 length 30 m , i.d . 25 mm , and 1 m film thickness ) coupled to 
 an agilent 5973 mass spectrometer as described by hale et al . a statistically 
 significant difference was observed for the total cell counts 3 years 
 after the amendment , with gac amended soil having the highest cell 
 count of 1.810 cells per gram of dry soil weight , 
 2.5 times higher than the unamended soil , which had the lowest cell 
 count ( figure 2a ) . these values are in the 
 higher range for most typical soil types ( e.g. , ( 19 , 20 ) ) . the soil respiration rate was also a statistically 
 significant factor of 2.6 and 2.7 higher in the gac - amended soil compared 
 to the unamended and pac - amended soils ( figure 2b ) . soil respiration resulted in a linear increase in the co2 batch headspace concentration over the entire 4-day monitoring 
 period for all soils ( r values > 0.98 ) , 
 and soil respiration could be quantified as 0.19  0.02 , 0.18 
  0.01 , and 0.49  0.03 g co2 per g of 
 dry soil per hour for unamended , pac- , and gac - amended soil , respectively . these observations coincide with field observations of better plant 
 growth in gac amended soil , see jakob et al . the soil respiration rates were similar to the values found in another 
 study on a hydrocarbon contaminated site . these results show that ac amendment overall was not detrimental 
 to aerobic microbial activity despite previously reported enhanced 
 doc binding in ac amended soils . ( a ) total microbial 
 cell count for the soil samples taken after 
 3 years and ( b ) soil respiration for the same samples . error bars 
 indicate the standard deviation for replicates defined in the method 
 section . invisible error bars are smaller than the lines or symbols 
 in the figures . the predominant soil bacterial community structure , as assessed by 
 dgge and cluster analysis of amended and unamended soil after 0 , 6 , 
 and 36 months ( figure 3 ) , showed that all the 
 samples from each particular time period clustered closely with other 
 samples from the same time period , regardless of treatment . this indicates 
 that the predominant bacterial community structure shifted more strongly 
 with time than in response to pac or gac amendment ( anosim test for 
 differences between time groups , global r = 0.852 , p < 0.01 ; for differences between treatments , global r = 0.317 , p = 0.97 ) . overall , 
 similarity between predominant bacterial communities , determined by 
 the pearson product - moment correlation coefficient , was greater than 
 65% ( figure 3b ) . duplicate dna samples from 
 the same time point and treatment showed higher similarity ( > 77% 
 overall 
 and average similarity coefficient > 87% , figure 3b ) , indicating good method reproducibility . the most abundant 
 microbial 
 species are important drivers of carbon and nutrient cycling in soil . the fairly high similarities of predominant bacterial community structures 
 in ac amended and unamended soil observed in this study alleviates 
 concerns about strong side - effects of ac amendment . the findings agree 
 with field community surveys of macroinvertebrates in sediments where 
 ac amendment generally had only weak effects on community structure 
 and diversity , although clear effects on species 
 abundance were observed in one field study . ( a ) 
 dgge gel showing the location of excised bands with the correspondent 
 affiliation to three classes of environmental bacteria ( right solid 
 triangle = actinobacteria ;  = proteobacteria ;  = bacteroidetes ; 
  = unknown ) . dna was extracted 
 twice ( dna1 and dna2 ) from the unamended and ac amended soils ( pac , 
 gac ) at time zero , 6 months , and 3 years . ( b ) dendrograms showing 
 cluster analysis of the similarities ( pearson product moment correlation 
 coefficient ) between the different community compositions of the treatments 
 over time , examined by dgge . identification of the nearest 
 matching neighbor to sequenced dgge bands was carried out using the 
 basic local alignment search tool ( blast ) and classification was confirmed 
 using the classifier tool in the ribosomal database project ( rdp 10 ) . twenty - three of the twenty - six most predominant taxa were sequenced 
 and classified into three main phyla , bacteroidetes , actinobacteria , and proteobacteria ( identified by different symbols , figure 3 and table s1 in supporting information ) that harbor common environmental bacteria known to play an important 
 role in the decomposition of organic materials . the three most abundant and prevalent 
 taxa ( bands n6 , n10 , and n17 respectively ) belonged to the order micrococcinaea within the actinobacteria phylum , whose nearest neighbors were identified as an curtobacterium , 
 arthrobacter , and microbacterium species 
 although with varied similarities ( 8999% ; table s1 in supporting information ) . the nearest - matching neighbor of several predominantly 
 abundant ( intense bands ) taxa are of interest because of the reported 
 widespread ability to degrade pahs in members of these taxa ( e.g. , ( 29 ) ) : band n7 was 96% identical 
 to a mycobacterium sp . and 92% similar to the versatile 
 pah - degrading m. vanbaalenii pyr-1 , band n8 was 95% similar to the versatile pah - degrading rhodococcus 
 josttii strain rha1 , band n16 
 95% was identical to an uncultured sphingomonas sp . , 
 and band n23 was 99% similar to a strain of the species rhodococcus 
 erythropolis , members of which are known to degrade biphenyls 
 and isopropylbenzene ( e.g. , refs ( 33 , 34 ) ) . these groups are often present in all soils ( e.g. , ( 35 ) ) , but appear to dominate 
 more frequently in contaminated soils ( e.g. , ( 36 , 37 ) ) , although to our knowledge it is unusual 
 to obtain such a predominance of sequences matching mycolic acid - containing 
 actinomycetes ( e.g. , rhodococci and mycobacteria ) . these bands were 
 present at all times in bacterial communities from unamended , pac- , 
 and gac - amended soil , but their relative intensity varied over time 
 ( see table 1 ) . the relative intensity of bands 
 can provide a rough proxy of the proportional abundance of those taxa 
 in the community . the relative intensities of these bands by year 
 3 were on average stronger than for the beginning of the study , but 
 comparable between treatments , which would indicate a similar relative 
 abundance of these putative pah - degrading taxa in the soils . the r. jostii rha-1-like sequence ( band n8 ) showed a particularly 
 marked increase in intensity between the start and end of the study , 
 where it increased from about 3% to nearly 10% of the predominant 
 bacterial community as expressed by relative intensity in ac amended 
 and unamended soil . phda : iron  sulfur 
 protein large subunit ; pdoa2 : putative pah ring - hydroxylating 
 dioxygenase large subunit ; etba1/ebda1 : ethylbenzene dioxygenase alpha subunit ; akba1b : alkylbenzene dioxygenase ; ipba1 : isopropylbenzene-2,3-dioxygenase 
 iron - sulphur protein subunit ; bpha1 : large subunit 
 of biphenyl 
 dioxygenase real - time pcr is a valuable tool 
 for the quantification of the functional pah ring - hydroxylating dioxygenases . dioxygenase systems add both atoms of molecular oxygen to the aromatic 
 ring as a first step in the aerobic degradation of lower molecular 
 weight pahs , and dioxygenase gene quantification is therefore of interest 
 in the assessment of pah bioremediation potential . the rhd gene copy numbers reported in table 2 indicate that those represented by the reference rhodococcus rha1 species ( from here - on called etbarha1-rhd ) showed the highest concentrations ( 1010 g ) , while those 
 represented by a rhodococcus erythropolis reference 
 species ( from here - on called ipdareryth - rhd ) showed the lowest concentrations ( 1010 g ) for every treatment . those represented 
 by the reference mycobacterium vanbaalenii pyr-1 
 species ( from here - on called pdoa2pyr1-rhd ) had intermediate concentrations ( 1010 g ) . those putative aromatic ring - degrading 
 bacteria ( i.e. , rhd containing bacteria ) analyzed 
 accounted for between 0.003 and 1% of the total bacterial population 
 if each bacterial cell is assumed to contain one gene copy . these 
 values are similar to the absolute and relative abundance values reported 
 by cebron et al . who used quantitative 
 pcr to target general dioxygenase gene populations in contaminated 
 soils , but higher than those in studies that only targeted specific 
 dioxygenase gene populations . the concentrations of each targeted 
 gene fell within an order of magnitude between unamended soil , soil 
 amended with pac , and soil amended with gac . indeed , the predominant 
 etbarha1-rhd gene population increased 
 over the time of the trial , and to a greater extent in ac - amended 
 compared to unamended lysimeters . this confirms that bacteria with 
 the ability to degrade pahs remain in pac- and gac - amended soil at 
 levels comparable to the populations in unamended soil . the real - time 
 pcr method measures gene numbers , or the potential to degrade pahs , 
 not gene expression , which may be down - regulated by some pollutant 
 degraders . in other words , while the 
 results indicate that the ability to synthesize dioxygenases was present 
 in all bacterial communities isolated from the soil samples , regardless 
 of ac amendment , this does not necessarily imply active synthesis 
 of the enzymes . it is interesting to note that there was a significant 
 correlation between targeted rhd gene abundances 
 and the concentration of those taxa found by dgge analysis ( relative 
 intensity  total cell count ) that had close affiliations to 
 the reference species of those rhd genes ( pearson s 
 correlation , r = 0.83 , p < 0.01 , n = 9 ) . however , despite the 
 close affiliation of these 16s rrna gene fragments with the reference 
 species for each targeted clade of rhd , the similarities 
 were often lower ( e.g. , 9296% ) than those cut - offs usually 
 used for species level ( 97% ) and it can not be confidently concluded 
 that these taxa would necessarily contain rhd genes 
 even if they were deemed to be similar taxa . assuming a carbon 
 content of 310 fg per cell and one etbarha1-rhd gene copy per cell , the biomass 
 carbon corresponding to the gene copy numbers in table 2 would be between 40 and 70 g per g of dry soil , which 
 is greater than the solid - phase pah concentration and much greater 
 than the freely dissolved pah concentration of below 1 ng per g of 
 soil . the comparison suggests that microorganisms 
 with dioxygenase genes utilized other main carbon substrates and could 
 therefore grow and persist long - term in the soil with and without 
 ac amendment . the findings alleviate concerns that enhanced pah binding 
 by ac may cause a significant decrease in the abundance of putative 
 pah degraders in ac amended soils . pe passive samplers 
 were employed in batch systems with and without the biocide , sodium 
 azide , and show that both sorption to the ac , and biodegradation , 
 affected the availability of pahs in the soil samples taken 3 years 
 after the start of the lysimeter experiment ( figure 4 ) . pahs taken up by passive samplers are considered available 
 to be taken up by organisms and plants , and tend to provide better 
 correlations with ecotoxity assessments than total soil concentrations . the high standard deviation of the pac - amended 
 sterile soil measurements is likely due to small - scale heterogeneity 
 in the distribution of the pac or pah pollution in 
 soil , as one pe sampler had much higher 
 uptake of pahs than the other two replicates . nevertheless , for the 
 soil slurries with sodium azide , which is normally added to batches 
 to illustrate the effect of sorption only , there was a statistically significant reduction in the pah uptake 
 by pe samplers for the ac amended soils , a 57% reduction for the gac 
 amendment , and a 69% reduction for pac amendment compared to the unamended 
 soil ( t test , one - tailed , p < 
 0.01 for gac and p < 0.05 for pac ) . the effect 
 of biodegradation is illustrated by statistically significant 75% 
 and 46% reductions of the pah uptake by pe passive samplers for unamended 
 and gac - amended soil , respectively , when comparing soil slurries with 
 and without sodium azide ( t test , one - tailed , p < 0.01 for unamended and p < 0.05 
 for gac ) . the pah compound pattern was also indicative of biodegradation ; 
 the uptake of the smaller two- and three - ring pah compounds , which 
 tend to be more readily biodegradable , was on average 99% lower in the unamended soil without , as compared 
 to with , sodium azide ( table s2 in supporting 
 information ) . overall , pah uptake was the lowest for pac - amended 
 soil without sodium azide , and our observations suggest active pah 
 biodegradation despite low pah availability in both ac amended and 
 unamended soil , alleviating concerns that ac amendment may result 
 in down - regulation of pah metabolism . however , the benefit of ac amendment 
 was less clearly pronounced without biocide addition . for soils slurries 
 without sodium azide , only the 44% reduction for pac amended soil 
 compared to the unamended soil was statistically significant ( t test , one - tailed , p < 0.05 ) . therefore , 
 for a realistic assessment of the long - term benefits of ac amendment 
 under field conditions , laboratory pilot - trials should be conducted 
 with both sterile and live soils to account for the pollution attenuation 
 which may occur in control soil due to biodegradation . pah uptake by passive 
 samplers embedded for 3 weeks in slowly mixed 
 slurries of unamended , pac- . and gac - amended soil sampled after 3 
 years . the sum of 11 of the 16 usepa pah compounds is shown ( those 
 with concentrations in pe above the analytical detection limit : acenaphtylene , 
 acenaphthene , fluorene , phenanthrene , anthracene , fluoranthene , pyrene , 
 benz(a)anthracene , chrysene , indeno(123 cd)pyrene , and benzo(ghi)perylene ) . 
 biocide inhibited batches contained 1 g / l sodium azide . to put these results into context , we compare in 
 figure 5 free aqueous pah concentrations cw estimated from the pah concentrations in polyethylene , cpe , using pe - water partitioning coefficients kpe and the relationship cw = cpe / kpe , with free aqueous pah concentrations 
 reported by hale et al . which were derived 
 from passive polyoxymethylene ( pom ) samplers embedded field lysimeters . 
 in - situ and ex - situ assessments are not exactly comparable because 
 of differences in temperature and mixing regimes , which are more optimal 
 under laboratory conditions , and therefore tend to facilitate pollutant 
 mass transfer and biodegradation . nevertheless , 
 the combined data seem to show a continuing trend of decreasing free 
 aqueous pah concentrations in both ac amended and unamended soil over 
 the 3-year remediation trial period , eventually falling to very low 
 levels below eu guidance values for drinking water set out in eu directive 
 200/60/ec in all systems . this overall trend is the combined effect 
 of all attenuation processes which comprise pah sorption , biodegradation , 
 chemical reactions , leaching , and volatilization . the difference between the accelerated pollution attenuation 
 in the ac - amended systems and the natural pollution attenuation become 
 smaller with time , and may eventually converge , when natural attenuation 
 has depleted the readily available pah pool to the same extend as 
 ac amendment . according to our results , the biodegradation of available 
 pahs is a significantly contributing factor to this reduction in free aqueous pah concentrations , even though total pah concentration of 23  15 g per g of untreated dry 
 soil were not measurably changed during 
 the field experiment . ac amendment benefits , 
 such as reduced pah uptake by earthworms and plants and reduced pah leaching , and 
 ac amendment costs , need to be assessed in comparison with the working 
 of intrinsic attenuation mechanisms , which may also reduce risks in 
 the long term , in particular in soils with a high abundance of pollutant 
 degrading microorganisms . comparison of the free aqueous pah concentrations 
 derived from 
 pe sampler concentrations measured in year 3 in ex - situ batch tests 
 with the field data reported by hale et al . , which were derived from pom samplers embedded in the lysimeters 
 17 and 28 months after the start of the experiments . shown is the 
 sum of those compounds of the 16 usepa pahs which could be quantified 
 in both studies : phenanthrene , anthracene , fluoranthene , pyrene , benz(a)anthracene , 
 chrysene , indeno(123 cd)pyrene , and benzo(ghi)perylene .
we collected urban soil samples impacted by polycyclic 
 aromatic 
 hydrocarbons ( pahs ) from a sorbent - based remediation field trial to 
 address concerns about unwanted side - effects of 2% powdered ( pac ) 
 or granular ( gac ) activated carbon amendment on soil microbiology 
 and pollutant biodegradation . 
 after three years , total microbial cell 
 counts and respiration rates were highest in the gac amended soil . 
 the predominant bacterial community structure derived from denaturing 
 gradient gel electrophoresis ( dgge ) shifted more strongly with time 
 than in response to ac amendment . 
 dgge band sequencing revealed the 
 presence of taxa with closest affiliations either to known pah degraders , 
 e.g. rhodococcus jostii rha-1 , or taxa known to harbor 
 pah degraders , e.g. rhodococcus erythropolis , in 
 all soils . 
 quantification by real - time polymerase chain reaction yielded 
 similar dioxygenases gene copy numbers in unamended , pac- , or gac - amended 
 soil . 
 pah availability assessments in batch tests showed the greatest 
 difference of 75% with and without biocide addition for unamended 
 soil , while the lowest pah availability overall was measured in pac - amended , 
 live soil . 
 we conclude that ac had no detrimental effects on soil 
 microbiology , ac - amended soils retained the potential to biodegrade 
 pahs , but the removal of available pollutants by biodegradation was 
 most notable in unamended soil .