APP I

R BI

TI

w

y

w

IL

ara J.
B.

TH

.,

ni er:ity of

ickin~

orthern

n

ritish

BMITT D IN P RTIAL
THE REQ IR M NTS
MAST R 0

NATURAL RESOURCES AND

OR TH

lumbi a, 200 l

L ILLM NT OF
D GREE

F

S lEN E

NVIRONM NTAL ST DIES

( NVIRONMENTAL SCI NC )

UNIV RSITY 0

NORTH RN BRITISH

April, 2005

© ara J. Dick inson, 2005

OL MBIA

B TR

It ha, r

ntl be n pr p

T

d that th additi on f organic matter may incr as the

phytorem diati n of petr leum h dr

arb o n ~ (PH

) thr ugh th in r ascd av ail ability f

nitr gen ( ) and improv m nt f s il pr perti , . Th additi n of bi so lid ~ t a so il
co ntaminated with w ath red lie. I di d n tin r a~e the
r , idu al PH

tent of PH

concentrati n), but it did increase the rate of d gradati on.

establishm nt did not incr a: th rat

r ex t nt of PH

d grad ati n (i.e.
vera!! , v g tati on

degradati on, relati ve lo th at observed

in non-amended, non-v getated so il s. Veg lati n was, however, an important fac tor within
the bi o. olid -amended treatments as wa, bserved by an in crease in th e ex tent of degradati on
and a decreas in the co ncentration of bios lid ~-de ri ve d mineral

rema ining in th e soil. In

general, the low-amendment rate of 13.34g bio, olids kg-1 so il was the most successful
treatment a determined by th increa ·ed PH

degradation rate, improved s il properti es, and

increased plant growth .

II

T BLE O F

0

TE T

..

II

AB TRA T
TABLE OF

...
Ill

ONTE T

LI T O F T ABLE

v

LIST OF F I GUR E

VII

A KNOWLE DGE M ENT

VIII

1. General Introduction a nd L itera ture R vtew

l
3
3
3
3
5
5
6
7
7
7
9
9

..

...

1. 1. P tr 1 urn H drocarb n ~: our cs, Propcrt i s, at and Impac t on oib
1.1.1. Hydr arb n ontaminati on
1.2 Nature of PH
1.2. 1
ompo, ition f PH s
1.2.2 Phys ical- h mical Pro perti es of PH s
1. 3 Impact of PH C ntamin ati n on oil s and R c ptors
1.4 Behav iour of PH s in oil
1.5 Biodegradation of PH s in oil
1.5.1 Biodegradati on
1.5.2 Factors Influencin g Biodegradation inS il
1.6 Ph ytoremedi ation of PHC
1.6.1 Overv iew of Phytoremediation of Organic ompound s
1.7 Mechani ms of Phytoremedi ati on
1.8 Rhizodegradation
1.8. 1 The "Rhi zo phere ffec t"
1.8.2 Root Exudate,
1.8.3 Mycorrhizae
1.9 Required Re earch fo r the Phytoremediati on of PH s
1.9.1 Plant-Indu ced Influences on Rhizodegradati on
1.9.2 Nutrient Cycling and Amendments in Contamin ated oils
1.9. 2.1 Nutrient Limitati ons in PH -Contaminated oil s
1.9.2.2 Nitrogen Cycling in Contamin ated Soils
1.9.2.3 Impac t of Organic Soil Amendment on Ph ytoremcdi ation
1.10 Thesis Re earch
1.1 0.1 Overview and Research Obj ec ti ves
1. 10.2 Thesis Outline

I1

13
13
14
15
17
17
18
18
19
20
22
22
23

2. T he Applica tion of Biosolids Atnendment in the Ph ytor emediation of Aged , Diesel
24
H ydrocat·bons in a Medium-Textured Soil
2. 1 Introdu ction
24
27
2.2 M Lhods and Mat ri als
2.2. 1
oil am piing and Preparati on
~7
._ 7
2.2.2 Bi oso lids amplin g and Preparati on

Ill

2.2.
Plant p i ,
2.2 .4
perim nta1 tup and re nh us
o nditi ns
2.2 .5
ample Pr c , . ing
2.2. 6 Plant Bi rna,,·
2.2.7 Analyti cal M e th ds
2.2.7. 1 il and Bi . lid s harac teriza ti n
2.2.7.2 PH
nalys i.
2.2.
tati sti al nalysi .
2. R suits
2.3. 1
oi l Pr perti s
2 .3.2 Pl ant Bi ma:s
2.3.3 PH D gradati n
2.4 Di sc u. sion
il Pr perti s
2.4. 1
2.4.2 Pl ant Bi mass
2.4.3 PH
egradati on
2.5 o nclu si n

3. Dynamic of N itroge n in the Pl ant-M icro be-Soil ystem of a Wea thered diesel
contaminated Soil A mend ed with Bi o olids
3. I Introdu cti on
3.2 M ethods and M ate rials
3.2. 1 Soil and Bioso li ds Samplin g and harac teriza ti o n
3.2.2
xperime ntal etup and G reenhouse o ndi ti ns
3.2.3 Sample Process ing
3.2 .4 Soil and Plant N
3.2. 5 Statistical Analysi
3.3 Res ults
3.3. 1 Initi al Soil Properties and oil N Poo L
3.3.2 Pl ant N
3.3.3 Mineral N
3.3.4 Total N
3.3 .5 Soil Microbial Bio mass N
3.4 Di scuss io n
3.4. 1 Soil and Pl ant N
3.4 .2
stimated N Require me nts
3.4.3 Mineral N
3.4.4 Soil Microbial Bi om ass N
3.5 o nclu sio n
4. Sy nth esis of Resea rch F indin gs
4. 1 Rate and xte nt o f PH Degradati on
4 .2 Nutri ent yc lin g and Amendme nts in

2
28
30
31
32
32
3
35
38
38
41
42

49
49
51
53
61

62
62
65
65
66
67

69
70
71
71
71
73
75
75
76
76

78
79

84
4

ontaminatecl

LITERATURE CITED

o il s

87
91

IV

LI T OF TABLE
Table 2.1.

Table 2.2.

Table 2.3.

ppropriatc

ele ted initial . il and am ndm nt hara terizati n pr p rti
pr p rti s pres. d on an o n-dr ba.- is.
il pr p rti , at 2 wk s.
en -dr basis.

h

t and r

ppr priat pr p rti

39

pr ~se d on an

40

t bi m a.· ~ a.- a fun ti n f time and am ndment rate.

41

Table 2.4. P r ent f initial 2 h dr arb ns remaining in a w ath r d, eli se lc ntamin ated so il treat i with ~ m oth bro me and bi s lids. Initi al so il
concentrati n: 14 6 mg kg 1
~o i I.

2

Table 2.5. P rce nt of initi al 3 h y d rocarb o n ~ r mainin g in a wcath red, di e~e l ­
co ntamin ated so iltr ated with ~ m oth br me and bi solids. I niti a l ~ il F3
oncentrati on: 20 6 mg kg 1 D so il.

43

43

Tab le 2.6. Percent r initial total H re mainin g in a weath ered diesel co ntamin ated s il
treated with sm oth bro me and bi ~o lid s. Initi al so il t tal H concentrati on:
3492 mg kg-1 D so il.
44
Table 2.7. Model parameter and R 2 valu es for F2 fracti on d gradati on using Model 2
. 2 .2 ): t= oe -l...t +Yo·
(equ at10n

44

Tab le 2.8. Model paramet r and R '- valu e. for 3 fracti on degradati on usin g Model 2
(equ ation 2. 2): Ct=C 0 e-1..t+Yo·

45

Table 2.9. Model parameter and R 2 values fo r total HC fraction deg radati n using M de l 2
(equ ation 2.2): C 1= 0 e-~ 1 +y 0 •
45
Table 3.1. Selected initial soil and amendment characterizati on pro perti es.
properties expressed on an ove n-dry basis.

ppropri ate
66

Tab le 3.2. Determinati on of biosolids applicati on rate: ava ilable N calculati ons.

68

Table 3.3 . Determin ation of biosolids appli cation rate: low-amendment rate
calculations.

68

Table 3.4. Plant N uptake and % total N re ve r d by sm oth brome from the biosolid~
amendment at 8, 16, 24, and 32 wks.
72
Tab le 3.5.

oil N ' -N , NH4 - N, total mineral N, and total N at 8, 16, 24, 32 wks .

1 ab le 4.1.

ier I lev Is of

M PH

W frac ti ons for soi ls.

v

74
86

Tabl 4.2. PH
nc ntrations at 2 wk, . Initial s il 2 and
• il , res pec ti
1406 and 20 6 mg kg 1
ly.

VJ

ncentrations:

87

LI TOFFIG RE
Figure 1.1. Me hani. m, f ph tor m diati n (m dified fr m unningham t al. 199 ).
Rhi z d gradati n, a, nhanced b th rhiz sph r ff t hibit d around plant
root is nsid r d t b the most imp rtant m hanism in th bi d gradati n
f w ath red PH ~.
11
Figure 1.2. Int rna! c le in th pr s nee of plants (modified from Kay and Hart I 997) .
. mineralizati n· B. nitrifi ali n,
H/ immobilizati n,
1
imm bilizati n: . mi r bial uptak of organic : . mi crobial dc tritu ~ inputs;
. plant uptak f H/ : H. plant uptake of
, : I. plant detritu~ inputs. Note
that rgani c
H +and
, - , may incl ud any amendmen t as w ll as
natural! cc urnn g s il
19
Figure 2.1.

hr matogram~ of v g tated, I w-arn ndm cnt treatm nts ~ h wing the
concentrati n f die ~ I range PH ~ at ( ) t = 0 wks, and (B ) t = 32 wks. otted
lines are the ret nti on time. f r n I 0 16, 34, and 50, rc~pectively, indi catin g
the window to be measured for eac h PH fraction. I ntcn~i ty unit ~ arc gi vc n in
mY .
42

Figure 2.2. Res ults f n n-lin ear regre:sion on q (A) F2, (8) F3, and ( ) total H
remaining for the vege tated, low-ame ndment trea tme nt ~ using eq uation 2.2:
Ct = 0 e-"-t + Yo· Lines indicate the exponential curve fit.
48
Figure 2.3. Chromatogram of bioso li d. oxhlet-ex tracted alone. D tted lines are the
retention times for n 10, 16, 34 and 50, respectively, indicatin g the window t
be mea ured for each PHC fraction. Note the interference meas ur d in the 3
window. Intensity units are given in mY .
58
Figure 3.1. Mi cro bial biomass N. Valu es are reported as the mean of 4 r plicate sa mpl es
where error bar r pre ent the tandard dev iati n. Valu es of zero indicate that a
negative mean valu e was obtained.
76

Vll

A KNOWLEDGEME T
I w uld fir. t like t thank my sup r ~ ~ r Dr. Mi chae l Ruth erford for hi s pati nc and ~ upp o rt o f me
throu gh ut my graduate studi -. at

B . H . p nt man hours di c;;;c uss in g and d

lop in

my ideas,

w rkin g with me thr ugh dirri ulties, and r view in g m pap rs . Hi ~ guidance and wi llin g n es~ to help
m ex pand m se lf intell ctu all
throu h hi s grants

and th e opp rtuniti e-. he aff rded me are trul y appreciated . ll i ~

ith th e B .

nginee rin g Research

ouncil

d anced , -. terns In -. titute (B
f

anada (

, I) and th e

ati nal , ciences and

ER ) th at 1 was abl e to ob tain fin ancial support for thi s

res arch. I w uld aL o like to th ank m com mittee m e mb er~ Dr. Willi am M e ill and Dr. Jose lito
rocena f orth ir guid an e throughout thi ~ research . De'->pite buc;;;y sc hedul es, th ey we re more th an
wi llin g to me t w ith me w hen I ne ded ad ice .
help w ith th e kin eti c m o d e l ~ I pre-.ent in

pec ifi ca ll y, 1 wo uld like to th ank Dr. M e ill for hi -.

hap ter 2, and Dr.

rocena and Dr. Mi chae l Gillingham to

whom I am ve ry grateful for their aid in d ve lop in g my s t a ti ~ ti c~ .

I wo uld like to th ank

orm Gobbi (Prin ce Geo rge W as tewater Treatment Pl an t) and Trevo r

ar l ~o n

(Federated Co-op) f r prov idin g materiab, in -kind support , and intere<.> t in thi ~ projec t. l am al<.>o
grateful to
(UNB

nal yti ca l Ch mi stry L aboratory), A ll en E~ l e r

li ve Dawso n (B .C. Mini stry of Forests

Central Equipment Laboratory), and Tariq

and . oil analy e~ . I would e, pec iall y like to th ank

iddique (

B ) for th eir ass istance with plant

teven torc h and John Orlowsky (U BC

Enh anced Fore, try L aboratory) f or their exce ll ent tec hni cal as. i ~ t a n ce, and W endy Hin es (U

B

hemi stry Stores) for her trernend ou help in obtainin g laboratory materi als and di spos in g f wa~ te~ .

I could not have co mpl eted th e many hour of experimental se tup, sa mpl e pr cess in g, and laboratory
"grunt work" without th e help of my research assistant M ami e Graf and th e many "v luntary"
wo rkers D ean Dickin son, Lauri e Reiffer, Christina Stride, Rob Cimaglia, and M ary Hu ghes whom I
recruited in th e name or sc ience. They not onl y provided an ex tra pair of hands, but much ne dcd
laughter.

Finall y, I wou ld like to th ank my husband and my parents who pro vicl cl un ending emotional support
and never let m forget th e bi gg r pi cture. J ca n honestly say that thi s has been a group effort and I
thank everyo ne for an amazing ex peri ence th at I wi ll ca rry with m throughout m life .

VIII

1.

General Introduction and Literature Review

il

ntaminati n i. a w rldwid pr bl m that has u, t maril b n verlook d. This has

I d t a legacy f hist ricall

ntaminat d . it s that ha

numb r f anthr pog nic ch mi als r l ased into th

y t t be remedial d. As the

n tr nm nt in creases th n d for a

gr ater und rstandin of th tran . port, fat , and remedial i n f thes xenobioti cs also
increa. e . The r sult i ~ a billion d li ar r mediati n indu stry with

x t en~ i ve research

ff rts

being con entrat don th e c ntainmen t and rem val f contami n a nt ~ fr m th e so il before
th y impac t n hum an and ec I gica l r cept r~, r migrat to the groundwat r ( ic ili ano and
Germid a 199 ). In th e past two decades th ere have been a large numb r of new tec hn I og i e~
made available for th e remediation of a variety of anthr poge ni c c ntamin ants in ludin g, but
not limited to , ex plos ive , metals, radionuclid e. , and rganic chemi cals ( usarla et al. 2002).

Some of the most common organic chemicals found in th e environment include polycyclic
aromatic hydrocarbon s (PAH ) and N-heterocyclic aromatic co mpounds, nitr aromati cs,
phenols and anilines, halogenated aromatics, hal og nated aliph atics, pesticides, and
petroleum products (Knox et al. 1999).

f these, petroleum products deserve speci fie

attention as they overl ap many of the above categoric and are released in large quanti tie~ to
th e environment (Kn ox et al. 1999).

oll ec tively, r m diation techn olog ies can be divided into two gro ups with regard to final
product. Th first group involves the d co ntamination of a si t by remo a! or degradation or
the co ntaminant.

enerally the complet r moval or the co ntamin ant from the soil is

preferr d a it n t nly reduce. th ri. k f further e
alu

f th land f r further d

, , t m damag , but al so increas , th

1 pm nt ( unnin gham t al. 19 6 ). The seco nd gr up

provide, tabilizati n r ontainm nt of th

ntami nant ; th es techn 1 gi s ar

when there is an imm diate ri:k f p llutant tran ~ p o rt, r wh n c mpl et

ften used

ntaminant

removal i. n t po. sible ( unningham t al. J 9 6).

The riteri a for u. ing a particular remedi ati on tec hn ology are based n the ri sk to hum an and
environmental health , as well as th c ~ t and time res tri cti ons relevant t the parties inv olved.
me t chnolog ies may be more effe ti ve th an thers give n ~ pec ifi c site c nditions.
urrentl y there is a demand for tec hnolog ies th at prov ide

o~ t effecti ve, tim ely, and

acceptable resu Its. Bioremedi ati n e n co mp asse~ a variety of tec hn ologies th at have bee n
found to fulfill these requirements in many cases. It may be defin ed as th e use f
microorgani sms to degrade pollutants through th e enhanced natural proc ss s of metabo1ism
and mineralizati on (Alexander 1999). In the pas t decade, ph ytoremedi ation has furth ered thi s
co ncept by the in -situ u. e of vegetation to enhance the already ex istin g bi odegradati ve
acti vitie of microorganisms. By definit ion, ph ytoremedi ation is the "use of green pl ants and
their as ociated microbiota, soil amendments, and agronomic techniqu es to remove, co ntai n,
or render harmless enviro nmental co ntaminants" (Cunningham et al. 1996 p. 6 1).

More generall y, these tec hnologies offer cos t effec ti ve, natural, and no n-intru sive method~
for the remediati on of anthropogenic pollutants.

urther sc ienti fic unders tanding of the

factors and process s controllin g these remedi ation meth ods will aid in the de e lopment or
mor effective treatment strateg ies.

2

1. 1

Petroleum Hydrocarbon : ource , Propertie Fate and Impact on oil

1. 1.1

Hydrocarbon

ontamination

It is . timated that anad a g n rat s appr

imat ly . i mi lli n t nnes f hazard us wa. l

p r y ar, leadin g to ten,· f th u sand ~ f

ntaminat d sit s ac r ss th c untr (Hrudey and

Pollard 19 3, i iii an and

rmida 19

,

n ironm nt

rud petroleum and natural gas indu str

1.

ne of th 1 ading ontrihut r~ to thi s was te,

r lea. in g upward .- f 16
Canadian

anada 2000 ,

00 tonnes of pollutant in .... 000 ( PR J 2002).

uncil of Minister: of th e nvir nment (

e ousa 200 1).

he

cc rdin gly, th e

M ) (200 I a) es tim ates th at

app ro imately 60% of th c untry ~ c ntaminatecl sit ~ in vo lv petroleum hydr carbons
(PH

), a pa11icul arl y c mple mi tur of orga ni c co mp o un d~.

Petroleum hydrocarbons are often the by-produ ct of indu strial ac ti vity and can be r leased
into the enviro nment by accident, on purpose, or through ignorance (Pierzynski et al. 2000).
Petroleum hydrocarbon contaminati n in soil often occ urs th ro ugh acc idental spills in clu ding
commercial storage and handling of crude oil , lubrica nt., and fuels. It may also o cur in
fl are pit oil s and drilling mud generated in the wa t

f the upstream oil and gas indu . try.

Engineered landfarming operations often purpo ely incorporate th ese was tes into nonco ntaminated soil to promote biodegradati on.

1.2

Na ture of PHCs

1.2. J

Chemica l Composition of PHCs

Petroleum hydrocarbon mi xtures co ntain th ousand s of indi idual compounds that ma he
foun d in varyin g qu antities. Petroleum products are deri ved pri marily from the proc ssing of

3

rude oil, which when fracti nated int di. tillat cuts, gi

pr ducts that can b d fin d by
f in cr asing b ilin g

boiling point la.. and arb n numb r. The. e ma includ , 10

p int, natural ga. . , r finery gas, liquid p troleum gas, gas lin , ker sen , gas iJ, and
di. tillate re. idu (M

ill tal. 1

I).

. gasoline, di s l fu els,

urther refinem nt pr du

and vari u. iJ s, as well a. a numb r f ther hea i r m le ular weight pr du cts (M e ill ct
al. 1981 , Potter and immons 199 ).

There ar two main cla:ses f PH

~: th e aliphati cs and aro m a ti c~.

there are alkanes (s traight si ngle b ncl cl
(cl uble bonded

), cycl alkane"> (cyc li

), and alkynes (triple bond ed

al. 2000). The aromatic class of PH

Within the aliphatic class,

~ in g l

bonded

), a lk e n e~

) (Pott r and immons 1998 , Pi e rz y n ~ ki et

co mp unci ~ has the benzene rin g as its prim ary

tru ctural blocks and co n. ists of monoaromatics (one rin g), di aro m a ti c~ (two rin g~), and
polynuclear aromatic hydrocarbons (PAH) (three or more ring. ) (Potter and immons 1998).
Generall y, crude oil consi ts predominantl y of cyclic hydrocarbons and n-alkanes th at can b
up to 85% of the total weight of the oil (McGill et al. 198 1).

The elemental compo ition of PHCs i primaril y composed of carbon (C) and hydrog n (H)
molecules, but may also contain small amount of nitroge n (N), sulfur ( ), and xyge n (0).
Generally, crude oils co ntain approx imately 85. 3%
1.0 I% S by weight (McGi ll et al. 198 1).

4

, 12.2% H, 3.6 % 0 , 0.22% N, and

1.2.2

Phy ical- hemical Propertie of PH

he ph ,' ical pr p rti s f individual P I . ar larg 1 d p nd nt n th . ize and . tructur
the m 1e ul .

nera11 , 1arg r m

1 .. : 1ubilit in water (P tt r and

ul s ha

hi gher b iling p inL , 1 wer

1111111 n ~ 199

).

h

f

latili ty, an I

clan !-water partiti n c effici nt

(Kow) normall y in r ases with a deer as in wat r , olubilit , r fl ee ting th e availability f a
ompound in the soil s luti on (Ri se r-Rob rts 19c 8).

he p larit y f the hydr carb n

. tru cture is aL o imp rtant in det-rmining th e water ~o lubilit y of co mp und s.

olubility

ge n rail in r as ~w ith an in r a. e in th p larit y fa mol ul (Poll r and imm n ~ 1998).

1.3

Impact of PH C

ontamination on Soil and Receptors

The tox icological effect of PH . to humans and anim als in clude as ph yx iate di sorders,
dermatiti , irritation, headache ·, dizzines ·, nausea, and nervous sys tem di sorders (

M

2000, Pierzyn. ki et al. 2000 ). More eri ou co nditions can be caused by long-term ex pos ure
or introdu ction of high co ncentration to the body and may lea l to acute poisonin g r death .

In additi on to their tox icity, PHC, can impart a multitude of problems in the soil ecosys tem.
Poor so il ph ys ical properties such as hydrophobicity may dev lop with the introd ucti on or
contamin ant. (Roy et al. 1999). These changes, along with tox icity, may lead t a disruption
in so il microbi al and faun al popu lati ons, nutri ent cyc ling, and many biologica l and chemical
reacti ons (Me ill et al. 198 1,

M 2000). To date, little r s arch h a~ been published on

th e impact of PH son the biological co mmunity and so il bi oc hemi ca l reac tions in nati e
so ils.

5

B haviour of PH

1.4

rgam

10

hemi aL ar subje t t

det rmin d by th

haract ri sti

oil

ral pathwa .

nee the

nter the soil.

f th p llutant: as w 11 as th bi otic an I abi tic factors in

th soil n ir n m nt (Ri s r-R b rts 1

). Petroleum h dr

arb ns, P l-Is in particular, ar

su . pect t s rption n : il compon nt: \uch a~ s il rganic matter (
xt nt , , oil min rals, du l their hy lroph bic pr 1 erti c~ (
and r 19 9).

il . !uti n

h se may be

M) and t a 1 ss r

hr and W bstcr 1996,

ncen trati n ~ of indi vidual hydrocarbo n c mpound s will in

part be dictated by K0 , the so il -wat r partition c cffi ient. Th e alu of Kd for ~ pec ifi c
compounds tend s t in crease with incr asi ng soi l org'mi c matter ontent, but is also
influenced by the aromaticity and polarity f the soi l organ ic matter (Xin g et al. 1994 ). The
carbon-n rma1ized partition coeffi ient, Km: · is a function of b th the sorbat and ~orbc nt.
Sorption i aL o influenced by the pre ence of a residual free product phase (Rutherford eta!.
1997), which can stron gly orb individual hydrocarbon compound:.

Petroleum hydrocarbon may aL o ex peri ence volatilization of small molec ul ar weight
co mpound . This can account f r up to 40% of th e weight of mo t crude oils (McGill et al.
1981 ), and may be important in lowerin g the risk associated with li ghtweight refinement
products . uch as ga oline and di esel fu l.

Finally, onl y a fracti n of the hydrocarbon co mpound s found in crude oil may b subject to
biodegradati on.

he weathering of PHCs in so ils enc mpasses many of th fates discussed

above and deserves sp cial attenti n as it can play a key rol in determining the rat and

6

t nt f PH

biod gradati n (P tt r and imm n. 19

).

h influ nc

f thi s pro . so n

bi d gradati n wi ll be eli . u., d in furth r detail b low.

1.5

Biodegradation of PH

m oil

1.5. 1 Biodegradation

Bi odegradati n as defin ed b

le and r ( 1999 p. ) i')' th bi 1ogi all y catalyzed redu cti n

in c mpl xity of h mi als' .

il

rganism~ may ac

ompli sh thi s l gradati on through th

metaboli ,' m and mineralizati n f rganic co mpou nds u~ed ass u rce~ of
nutrients requir d f r grow th .
in the proces.

, energy, and other

ft n, organic c mp unds are co nv rted to in organi c produ cts

f minerali zati n. This is the mos t efficien t m th od of red uci ng toxici ty and is

therefore mo: t important in sites where risk i~ a co ncern (Alexa nder 1999). Mi croorgani sms
may al o tran. form organi c chemicals into other organic co mpounds by means of metaboli sm
or cometabolism.

ometaboJi m, as defined by Atl as (1984), is th e in adv rt nt

tran fo rmation of a compound whereby th e oil micr organi sm does not gain any benefits
such a energy or nutritional elements as a res ult of the transformati on. This may be an
important area for further research, as little i. known about the implicati ons of co metabolism
on the toxicity and persist nee of xenobiotics in the environment.

1.5.2

Factors Influencing Biodegradati on in Soil

Th re are several co nditions disc ussed by AI xander ( 1999) that must be me t f r
bi odegradati on to occ ur: I) an organi sm(s) th at has the ability to d grade th target
compound mu st xist and be prese nt in the enviro nm nt co nt ainin g the con tami nation; 2) the

7

mp und mu t b availabl t th d grading rga ni. m(.) and nzym (.); and , ) suitabl e
envir nm ntal

nditi n. mu st e i. t forti 1 gical ac tivity.

Petroleum hydr carb n. ar a parti ularl diffi ult cl ass f c ntamin ant. t r medi at du e to
th comple ch mi. try f omp und mi x ture~ that oft n

ntain aliph ati c and ar malic

hydrocarb ns tog ther with p Jar rgani c ompound. . Qu antiti es of th s co mpounds may
vary with s ur e, degree f pr ces~ in g, so i I type, and e t nt f weath rin g (Pi rzynski et aJ.
2000,

M 2001 a).

detailin g th hi ghl y

onf undin g th i~ problem i ~ th e la k fa compr h e n ~ i v database
mple and variable

o mp o~ iti o n of many petrol

urn mi x ture~ (Potter

and immons 199 ).

Generall y the stru cture of PH . affects their degradab ility. A~ di sc u ~sed by Skl adany and
Metting ( 1993 ), there are fo ur generali zati on. th at can be made regardin g the effec t of
. tructu re on the transformation of aliph ati c hydrocarbon. : J ) increasing chain length res ults
in slowed tran fo rmation; 2) increa ed saturation leads to greater degradation potenti al; 3)
branching in the chain stru ture can slow the rate of degradati on; and , 4) compoun ds wi th
high meth ylation tend to resist degradati on. The e generali zations can also be applied to
aromatic hydrocarbon alth ough the number of fu sed rin g structures may also affect their
degradation such that four and fi ve ring PAH. are highl y persistent in the environment
(Alexander 1999).

Petroleum hydrocarbon mi xtures may als bee me "weathered" or "aged" in soils making
them more diffi cult to re medi ate with time. The loss of low molec ul ar weight compounds

8

thr ugh bi d gradati n and v latilization ar , m

f th me hani. m,

w ath ring. Thi . 1 ave. nJ the larger, mor . 1 wl bi d gradable
r f rr d t as th r , idual fra lion ( 1 and r 2000,
individual

mpl

ntributing to
mp n nts oft n

t al. 200 ). In addition,

mp und s rna diffu:e furth r int nanop r s r int a sorbing s lid ph as ,

making de. rpti n

r sl w and r du ing bi a ai lab ilit ( Hat?.inger and

Pignat ll o and Xin g J 96,

and r 2000,

le and r J995,

mplc t al. 2003 ). Thi s ar a of r search has

gained gr ater imp rtanc r cent! as studi s ha e begun to e amin the behavior of aged
chemi als in so il. and , ub. equen tl y th ir avail abi lity for uptak by microorganisms, plants,
animals, and humans (Hatzing rand

Je ander 1995, Loehr and Webs ter 1996, Alexander

2000 ).

1.6

Ph ytoremediation of PHCs

1.6. 1

O verview of Phytoremediation of Orga ni c Compound s

vidence i mounting for the ' ucce. s of phytoremed iatio n a a strategy for enh ancing rgan1c
co ntaminant degradati on in the , oil. Phytoremediatio n itself is not a new co ncept. The
application of plant to treat p !luted water has been employed for hundred, of years through
constru cted wetland ecosys tem. (Sa lt et al. 1998). The , am principles may also be appl i d
to terrestrial ecosys tems. Pl ant , through their ability to chang exi. ting soil conditions and
their intimate co ntact with soil microorgani sms, offer a natural and effective method of
remedi ating a wide vari ty of organi c compounds. To date, there has been considerable
research done on the mi crobial degradation of pes ticides in the agricultura l industr
(A nderso n et al. 1993 ), though the applicati n of these principl s t phytoremediation has
nl y recently bee n the f cus of interes t.

9

, m re u e,, ful trial ar p rf rm d, th li st f plant. kn wn t enh an e th d gradati n
of organic contaminants , uch as PH

gr w, ( rick tal. 1999,

urn r u, plant , p ci ,' in luding gra.. , , 1 gum . , and w

dwin and

hoqJe 2000) .

d speci s hav be n sh wn to

fa ilitat th degradati n f b th indi idu al co mpound. and mi xtur s (Li ste and

lexander

2000, Palmroth et al. 2002).

There i. urr ntl an

paneling li st of ad anlages and disadvantag s to using pl ants as a

remediati on tec hn logy.

s utlined in r

iews by usarl a et al. (2002) and th cr'> , som of

the apparent advantages of ph toremedi ati n in lud : 1) cos t ffe ti ve ness, as
phytor medi ation can o. t half th at f c nve nti onal enginee ring soluti ons du e to the low
maintenance r quirements; 2) phytoremediati n is aes theticall y pleasi ng and therefore hi ghl y
accepted by the public; 3) the proce-. can be p rf rmed in -situ , thu s redu cin g th e di sturbance
to the local ecosy tern ; 4) plant are benefi cial to the soil . tru cture; and fin all y, 5)
phytoremediation i. an ongoin g proce . The la t two points are perh aps th e mos t important
because ph ytoremedi ation i often u ed as a fin al polishing step where plant may be
establi hed on ite and left to c ntinu e the remedi ation undisturbed.

Di sadvantages described in the revi ews include: 1) the spec ificity of ph ytoremediati on to site
co nditions such as soil co nditi ons, weather, and bioavailability; 2) the ex tended time period
needed to reach acceptable endpoints; 3) co ntrasting res ults regardin g the success or
ph ytoremedi ati on in the fi eld; and, 4) the limitatio ns to pl ant and mi crobial grow th such as
tox icity and bioavailability. A fin al di sadva ntage th at appli s to all soi l re m diati on
strategies is the lac k of stand ardi z I tes ting and a cJ finiti on of accc ptabl "ndpoints .

10

1.7

Mechani m of Phytoremediation
onida ( J 9

h r ar thre m d
Plant. can . qu , t r
ontamin ants.

ntaminant., d grad

ntaminantl), r stimuJ at th d grad'lti n f

, . how n in igur 1. 1, th se enc mpas · a an

plant. ma influen

).

f m chani )ms by which

r mcdiati n.

Phytovolatilization

Phytodegradation
. Rhizodegradation
:··, microbial metabolism of
contaminants in the
rhizosphere

Microorgan isms - - - - .

..

..

..

Figure 1.1. Mechanisms of ph ytoremedi ati on (modified from unni ngham et aJ. J996) .
Rhizodegradati on, as enh anced by the rhi zospher ffec t ex hibited aroun d plant roots, i~
co nsidered to be the mo t important m chani. m in th bi d gradation of weathered PH s.

Mechani sms such as ph yto xu·acti on, ph ytovo latilization, and ph tostabi li1.ation ma be
co nsidered forms of plants scqu sterin g contamin ants.

itcraturc on th plant uptake of

PH s from contamin ated so il s has r ported variabl res ults with regards to th succ "~s of

lI

plant. t bioa umulat
L m

rgam

mp und, ( hain au t al. 1 97, Bail

and Me ill 1999,

, tal. 2002). Th ugh th r . ults are g nerall en uragin g, fmih r r s arch n the

fate f PH , in plant uptak

Ph ytod gradati n 1s an

1.· n ed

ampl

d.

f dir

t plant degradati on.

onsidcrahl e kn owl dge ex ists

on th m tab li sm of p . ticid ~ hy plant s and it is sp cul ated th at th sa m principl es c uld
be applied to PH

sci n e ( nderson et al. I 9 3 ).

ph ytor medi ati n are li sted in a re i w arti l b

ve ral enzymes f int res t f r
usarla ct al. (2002). Th es in clud e

d hat genase, p ro idase, nitr r du cta. e, nitrilase, and ph sph atase. Like plant uptake,
further r sear h i: needed to understand th importance of thi s mec hani sm in PH
remediati on.

In additi on to direc t degradati on, plant may also . timul ate the bi odegradati on of
contaminants by microorgani m, in a proce. s know n as rhi zodegradation. The rhizos phere
was fir t de, cribed in 1904 by Lorenz Hiltner and has since become an area of intense
re earch. It is charact rized by a zo ne of increased mi crobial ac ti vity and biomas. at the
root-soil interface whereby plants may influence the degradati on of some contaminants
(Anderso n et al. 1993 ).

ach of the above mechani sms may be res ponsible for part or all of the remed iation, though
it is more likely a combin ati on of mechani sms th at lead to th overa ll reduction in
c ntamin ant I vc ls ( usar! a et a!. 2002).

f those menti oned, however, rhizodegradation is

12

lik 1 th mo. t imp rtant me hani min th d gradation f w ather d PH sand will b
eli u, :eel in furth r d tail.

1.

Rhizodegradation

1.. I

The "Rhi zo phere Effect '

Traditionally, th e plant-micr b relati n~ hip h a~ be n i wed as mutu alistic.
beli v d that plant r
th f rm of

ts pr

nerally it is

id mi roo rga ni s m ~ with a surfac to co loniz and nutriti on in

ud ates while mi ro rga ni sms pr

id rl ants with rrot eli n again st tox ins

through the min rali zati on and metabolism of va ri us compounds (Walton et al. 1994a ).

More pecificall y, the rhi z sph re offers a co ntrolled envir nment th at favo urs mi crobi al
coloni zation. In many cases, re earch rs have bserved in creases in mi crobi al bi o mass by
greater than an order of magnitude when compared with bulk so il popul ati ns (Anderso n et
al. 1993). Thi i known as the "rhi zosphere eff ct" ( igure l . l ). Abiotic factors such as
rhizo phere moi ture, so il tex ture, oxygen suppl y, temperature, and so iJ pH may influence
microbial colonization and affect co mmunity , tru cture (Kenn edy l 998). Plant spec ies and
age may also pa11iall y determine the co mposition of the mi crobi al co mmunity by release of
root ex ud ates, though it is unclear whether pl ants are ac ti ve or passive parti cipants in this
relationship as the degradati on of so il contaminants can occ ur in their abse n e (S ici li ano and
Germida 1998).

13

1. .2

Root E udate

i ilian and

rmida (19

) qu sti n d ur unci rstandin g f th natur

f th plant-

micr b r lati n, hip b h p th , izing that plant. rna ha e both sp ifi and n n-sp cifi c
r 1 in . timul ating mi r bi al d gradati n of s il c ntaminanL . Th

·ummariz d the

pp , ing view, that pl ant.· rna , b pro idin g pol phen lie ompound. , sp
d gradati on f

ntamin ants in the . il, and in

ntras t, th at thi s stimulati on may occ ur

indirec tly thr ugh nutri ents rei as cl fr m th ro L ind p ncl nt

R searchers hav att mpted t
a ail able.

ificall y increase

r th pr se nce f tox ins.

lu cicl atc thi s linkage, though few c ncrele res ults arc

stud y ondu cted by Yo. hitomi and hann (200 I) exa min ed th e impac t f maize

roo t ex ud at so n pyren minerali zati n and f un ci th at the chemica l influcnc

f th e pl ant in

rhi zo phere degradation wa. appare nt , th ough th m chani sm was unclear.

th cr literature

ha. exa mined the ability of pl ants to selec ti vely enhance certain bac terial genotypes kn ow n
to detoxify pecific co mpound . . Again , the resul t are un clear a. the in crease in genotypes
could be the res ult of both plant and co ntamin ant influence, ( icili ano et al. 200 I).

Part of the difficulty in determinin g the mechanism by which plants influence the
rhi zodegradati on of compounds arises from the observati on that many enobiotics pro ide
microorganisms with neither carbon nor en rgy ( in ger et al. 2003 ). N w e ide nee su ggests
a structural conn ection betwee n xenobiotic chemicals, secondary plant metabo lites, and
all elopathi c chemicals. There are surp risin g simil arities betwee n th se chemical structur s
that wo uld ace unt for the ability of many mi cr organi sms to c metabo liz anthropogenic
om pound s ( iciliano and

ermid a 1998, in ger t al. 2003 ).

14

1.. 2

Root Exudate

icilian and

rmida ( 199 ) qu , ti n d ur und rstanding f th natur

f th plant-

mi r b r lati n. hip b h p th , izing that plants rna hav b th sp ifi and non-, p ific
r 1 , in stimulating mi r bial d gradati n of s il
pp sing vi w. that pl ants rna , b pr
degradation f c ntamin a nt~ in th

ntaminants. The , ummariz d the

iding p I ph n li e c mpounds, sp cifically increase

~o iL and in

on tras t, th at thi s stimulati on may c ur

indir ctl thr ugh nutri nts relea~ d fr m the ro t ~ indepe ndent of th e pr sence of tox in s.

Re, earchers ha e attemp ted to lu cid ate thi s linkage, th ugh few co ncre te r suits arc
avail ab le.

stu ly

ndu cted by Yosh itomi and han n (200 1) cxamin d th e impac t of maize

root ex udate. on pyrene mineralization and f und that th chem ical influence of th e plant in
rhizosphere degradation was appare nt, though th mechanism was unclear. Other literature
ha. exa mined the ability of plants to seJ ctiveJy enh anc c rtain bacterial ge n types known
to detoxify pecific compound . Again, the results are unclear as the increase in gen types
could be the re ult of both plant and co ntami nant influence. ( i ili ano et al. 2001 ).

Part of the difficulty in determining the mechanism by which pl ants influence the
rhizodegradation of compound s ari ses from the observation that many xenobiotics pro id
microorganisms with neither carbon nor energy ( in ger et al. 2003). New evidence suggesh
a stru ctural co nn ecti on between enobi tic chemi cals, s ond ary plant metabolit s, and
aJiel pathic chemi cals. There are surpri sin g simil ariti es betw en these chemical structures
that would acco unt for the ability of many micr organisms to cometabolize anthropogenic
compounds ( icilian and G rmich 1998, in ger t al. 2003 ).

14

udat , in par1i u lar, ma act a~ indu

R

h m t als, :ugg . tin g th at pl ant. a ti

r: [ r th
ntr l th

m tat li. m f

~ iL

and

n t i ti

mpo\iti n [ th mi crc bi al

mmunit in th ir rhi z . ph r . Thi \ h p the i\ i\ \upport db \tucli \ ~ u c h a\ th at f
igh t al. (20 2)
ph n li
. ur

and

h

b~

r d th at th r

t turn o

r f mulh rr pl ants r l a~ d th

mp unci ~ m ru\tn , m ru . in I, an I kuwa non
[ gr wth . ub. trat ror

ir ~ t ne (. . . 0

I ), r

; co mpound \ th at

-d gra lin g ha ' tcria. In anoth r \lucl co ndu cted by Mi ya

1t e u l a t e~ we re oh. er d t \limul at th hiod gradatio n o r

phenanthr n b mai ntai nin g a larg r popul ation or phcnan th r ne degrader\ in th e
rhizo: pherc r ~ I nd r at p h nt ~. It i\ imp rtant t not , howe cr, th at \ P c ifi c a\\Oc iati on
d oe~ not impl

cl egradati n ( icili an ) and

and mi cr b s in th

rhi zo~ ph e r

ac ti ve ly redu ce le el. of

erm ida 199 ). Th

~ peci fi c a~\oc i a ti o n of pl ant \

ma onl all ow pl ant \ to urv ivc in co ntamin ated ~o il , not

ntam in ant..

Recent ev idence sugges ts th at root e ucl at ~ m ay ab o in cr a~ th e bi a ail ab ility of
co ntamin ants in soil ( icili ano and Germid a I 98,

haineau et al. 2000, Mi ya and ir . tone

2001 ). Although th r is a lac k of direc t ev idence, roo t
~ u rface~, thu s redu cin g the potenti al for

erm ida 1998).

ud al ~ may bin d to min eral

nt a min a nt ~ to ~orb to th e ~o il matri

( ic ili ano and

he impl icati on of thi s with regard s to contamin ant to i it i\ an ar a that

warrants further research.

1.8.3

Mycorrhizae

h ab ilit y o f mycorrhi za to form sy mbi otic r lati onships with pl ant:-, i:-,

~ 11 h.nown, hut th ~

pot nti al ro l of myco rrhi za l fun gi in t iodcgra lati on has larg -- I h' n ignor --d in the

IS

phyt r m diati n lit ratur . R
fa ilitat enhan

d d gradati n in th rhiz . pher

dire t degradati on f
M harg and

nt . tu li . ha e b gun t

ntami nants (

ect my orrhi za. (

r

M) in ph t rem di ati n.

f fun gi t

f plant through nutri nt acqui . iti n and

nn 1l and

atrne (2000 ) offer an e t nsi

amine th abil it

onnell and ntry 1999 ).
i w hi ghli ghtin g the p t nti al us of

hough th r is limited information avail able

n th me han isms f pollutant d gradati on, th

off r str ng videnc that many

Ms can

directl y degrade a wid range f P H · thr ugh enzymati c m taboli l)m and minerali zati on.

It ha. also been sugges t d that

Ms may have an indi rect innuencc on co ntamin ant

degradati on by means of int ruc ti ons with others il mi cr bcs in the mycorrhi zosphere, an
area analogous to the rhi zosphere (Meharg and

airney 2000 ). A rece nt study by H in nsalo

et al. 2000, examined the influence of the ec tomycorrhi zospher on the bac t ri al c mmunit ylinked C . ource utili zati on and subsequ ent redu cti n in PH s usin g Sco ts pin . It was
ob erved th at the development of rhi zosphere and mycorrhi zospheres pos iti v ly stimul ated
the degradati on of PHCs by micro bial co mmuni ti e , thu lending . upport to the indirect
influence of E M. on PH

degradation.

Th ough the pecific ability of fun gi to degrade PHCs is not well know n, studi es hav shown
positi ve res ults for the degrad ati on of PAH co mpounds in the prese nce of myc rrhizae
(Gramss et al. 1999, Joner and Leyva! 2001 , Joner et al. 2001 ). Jon ret al. (200 1) recently
assessed the influence of arbu sc ul ar mycorrhizas (AM ) on th dissipation of three common
PAHs; anthracene, chrys ne, and dibenz(a, h )anthrace ne. The grow th of clover and r egrass

16

n , pik d , il d m n~trat d that

M ma

th d gradati n f

nh an

1--h in th

rhi z , ph r .

Required Re earch for th e Ph tor m diation of PII ~ s

l.

1.. 1 Plant-Indue d Inlluen

n Rhi zod gradation

Th t rm bi a ail abilit r r r.. , to the "ac
and r 20

po. ~ ibl
to icit

p.

~sc

\ib llit

)r a ch mi ca! fc r a~\ imil a ti n and

). It i.. , p rh ap..., more import ant than th ac tu al

r a c ntamin ant b cau.. , h d finiti on, a om p und mu<.., t b a ail ahl fo r uptak in

rder t be c nsid r d t

1 ,.

le ander (200 ) , ugg ~ t e d th at pl ants and mi croorgani ._, rn . ., ma in crca<..,c the avail ability o f
sorb d co mp unds thr ugh fac ilitated de. rpti on. Pl a nt ~ , b mean<.., of direc t re i a<..,c or
rga ni ~ m ~ in th e rhi zo~ ph

stimul ati on of surfac tant produ ction by mi cr

ava il ability o f aged compound . for degradation.

rc, may increase th

o il di sturbanc and agg regate

fragm ntation by roo ts may fu rther enh ance th e ava il abilit y f co ntamin ant s to bi ~ u r fac t a n t.
and degrading en ymes (Hutchin son et al. 200 I).

Often, du e to the c mpl e nature f hydr carbon mi x ture~, bi degradati on rate ~ tu dies are
carried out usin g ~o il s spiked with spec ific PH

co mp o und ~ in th e laborator

. Thi~ is

imp rtant in understandin g the primary me hani sms b whi ch pl ants ma enhance the
deg radati on of co ntamin ants, but appl yin g th cs d gradati n rates to PH
th fi eld may fail to show signifi cant result s.

mi , turcs found in

s di sc ussed in sc ' li on 1.5.2, th wcath --rin g or

co ntamin ants in so il s may I ad tc d creased bi oa ail abilit .

17

b ' lt 'r undc rstandll1g or the

influ n

f

g tati n , tab li, hm nt n th bi d gradati n fag d
ff cti

f thi,· t

ycling and

mendrnent in

n d d to det rmin th

1.9.2

Nutrient

hn l g f r PH

mpound, 1n . ils is

r m diati n.

ontaminated Soil

1.9. 2. 1 utrient Limitation in PH - ontaminated oil
1 m nts su h a~

, , and pho: ph ru ~ (P) ar

ft n limiting nutri ents f r pl ant and

mi cr organism gr wth in PH -co ntamin at d ~ il ~ .
indu str , many studi s we re quick l

IJ wing the lead of th agri ultur -

b, rv that the additi n [ nutri nts, ~ u c h a~

effec ti vely increa,·ed th ab iI it of p l a nt ~ and micro rgan i~ m ~ to degrade enobi Li e~.
example, Lin and Mend Iss hn ( 199 )

b ~e rve d th at the appli cati

and P,
or

n of N-P-K fertili z r at a

rate of 666 kg N ha-1 , 272 kg P ha-1 , and 5 14 kg K ha-1 signi ficantl y enh anc d oil degradati on
rates in altmar h sods of Spartina a ltern ~flo ra and Spa rtina potens, as well as in creas ing th e
vegetative bi oma,, . It wa, hypothes iz d th at the incr ase in oil degradati n was due in part
to a fertilizer-indu ced increa e in microbi al number.. The res ulting ac ti vity, as well as th e
increa eel plant biomas , may have increased phytoremedi ation (Lin and Mendelssohn 1998).

The positive effec t of added nutrient on phytoremedi ati on is furth er supported by
Hutchinson et al. (200 l ), who observed a , ignificant increase in PHC r ducti on in N and P
fertilized treatments planted with bermud a gra, sand tall fesc ue. Microbial numbers were not
significantl y affected by the f rtilizer rate, how ver, and th e authors specul ated th at the high
level of organi c Nand total P in the co ntamin ant and s il sludge may ha e be n sufficient to
support mi cr bial growth .

Th tran. f rmati n and fat
mpetiti n betw
imp rtant ro l w e

f the. added nutri nl. i. n t w 11 und r. t

n pl ant and mi r

d, h w v r.

rgant. m. f r th , am nutri nt re. ourc s may play an

, stem functi nin g u h as th so il

cy 1 ( igur 1.2) (J acks n t al.

1 89 Ka e and Iart 19 7, H dge et al. 2000 ), and th co mpoun lin g eff cts of ontamin ants

on , il conditi on. and pr p rti s furth r 111 rease th diffi culti s in d t rminin g nutrient
reguirem nts.

Plant N
H

G

Organic N

A

NH 4 . .

B

NO 30

one-vifay

Microbial BioJnass N

E/F

.-- -. Two-w~

Figure 1.2. Internal N cycle in the presence of plants (modi fied from Kaye and Hart 1997).
A. mineralizati on; B . nitrificati on, C. NH/ immobilizati on, D. N0 3 - immob ili zation; .
microbial uptake of organic N; F. n1icrobi al detritu s inpu ts; G. pl ant uptake of NH/ ; H . plant
uptake of N0 3 - ; I. plant detritu s inputs. Note th at organic N, NH/ -N, and N0 1--N, may
include any amendment as well as natu rall y occ urring soil N.

1.9.2.2 Nitrogen Cyclin g in Contaminated Soils

Unders tandin g N cycling in co ntamin ated soils is important as thi s can affect the
biodegradati on of orga nic chemicals. Through the min ralizati on and immobi lization of N,
mi cr organi sms alter th form and ox id ation states f N, and th refore its fate in the soil
sys tem. Bac t ri a, fun gi, and actinomycc tes minerali ze organi c N such as prot in~ . chitins,

19

amm . ugar. , and nu 1 i a id. int in rgan1 ammomum

( H4 - ),

hi h is th n

availabl f r furth r transformati n, int ra ti n with Ia sand th r . oil
n rail

u ptak (Pi rL n. ki t al. 2000 ).
nitrat (

1 )

in a rapid pr

kn

H 4 + i'. furth r co n

n a\ nitrifi 'clli on.

itrat

ll oids, r plant

rt

d t nitrit (

2 ) , th

I\

mor s luhl than

n

amm nium and i. th r for f und at gr at ' r co n ' ntrati on.. , in th . , o il so luti c n. It is thu s
onsid r d t he th e primar form f
Imm hiliLati n is th r

obt ain d h pl ant... (Pi r; yn'. ki t al. .... 000 ).

crse proc . ., . ., or m1n rali; ati on. It occ ur th ro ugh th e as<.., imil ati on

and tran sf rm ati n r in rga ni c

to organ i co mp unci \ h th e m taholi '. m and grow th o f

rga ni sms (Pi rL nski t al. .... 0 0).

itr ge n mine rali za ti on and imm obili zati on i~o, co nt r lied by th e ava il ability of
sys tem (Pi rzynski t al. 2000 ). M icroorgani '. m..., r qui r
which co nsequ entl y di ctates the avail abilit of

in th

fo r m tab Iism and grow th ,

in th e so il. Hi gh

rati os stimul ate

micr bi a1 growth and res ult in incr ased immobiliLati n of N (Pierzynski et al. 2000 ).
Lowering thi. rati by means of inorganic fertili zer additi on, r N-ri ch organic so il
am ndments, can in crease the amount of potenti all y ava il abl e

for mi crobi al bi omass

growth , pl ant growth , and xe nobi otic bi odegradati on.

I .9.2.3 Impacts of Orga ni c Soil Amendment on Ph ytoremediation

ittle research has be n publi shed nth impac t of rga nic so il amendment s in Pll
ph ytor m di ati on efforts. Petroleum hydrocarbons ar a so urce or
thu s increase the e!Tec ts or nutri nt limitati on in a so il s st m.
with a narrc w

for microorganisms and

ddition of )rganic matcnal

:N rati o(< 25: I ) ma incrcas th N 'Ont nt or th soi l (Pi 11 ns k. i ct al.

20

amm . ugar. , and nu 1 ic a id int 1n rgan1 amm mum

( H/- ), whi hi. then

availab l f r furth r tran. [ rmati on int ra Li n with la s and th r ~ il c Jl ids, r plant
uptak (P ierz n ki t al . 20 0).
nitrat (

H/ i. furth r

n rail

,-) in a rapid pr c ss known as nitrifi ali n.

nv rt d l nitrile (

2 ) , th

n

itrat is m r s lubl than

amm nium and i. th r-f r [ und at gr at r co ne ntrati ns in th e~ il sol uti n. ll is thu s
c nsid red to be th pri mar fo rm of
Immobilization i. th r

btained b

r~e p roce~s of min

and tran. f rmati on f in rgam

pl a nt~ (Pierzynski ct al. 2000 ).

ra li zat ion. ll occ urs thr ugh th assimil ati n

to organic c mp und ~ hy the m tabo li sm and gr wth of

oil micr organi: ms (Pi rz nski et al. 2000 ).

Nitroge n minerali zation and immobili zati n JS ontr 11 d by th e ava il ability of
y tern (Pierzyn ki et al. 2000 ). Mi croorganisms req uire
which co n equ entl y dictates th e availability of

in the

fo r metaboli . m and growth ,

in th s il. Hi gh

:N rati os stimul ate

microbial growth and re, ult in increased immobilizati on of N (Pierzynski et al. 2000).
Lowering thi ratio by mean of inorganic fe rtili zer addition, or N-rich organic so il
amendments, can increase the amount of potenti all y availabl e N fo r n1icr bi al bi mass
grow th , plant grow th , and xenobi otic biodegradati on.

1.9.2.3 Impact of Organic Soil Amendment on Ph ytoremediation
Little res arch has been published on the impact of organic so il amendments in PHC
ph ytoremecliati n efforts. Petrol um hydrocarbons are a source of

for microorganisms and

thu s increase the effects f nutri ent limitati on in a soil sys tem. Addi tion or organic material
with a narrow

:N rati (< 25: I ) may incr as lh N content of the soil (PierLynski ct al.

20

2000). B -pr du eL , u h a. animal manure, c mp , Led muni ipal , lid wa, t , municipal
bi , lid, , and ompo. t d pap rmill slud g , ar

ample.

f pol nLial organic ·1m ndments.

The additi n [ -ri h am ndments at appr pri at rat sca n caus significant hang s in th
available in rganic

requir d fo r Jl ant grow th and i~ a pr mising ar a of res arch in

ph t remediati n.

Additi n of organic mat ri al ma als im pr ve the p h y~ i ca l pro perties of the soil such as
bulk d nsity, p r sit y, and wat r holdi ng capa it y, thu s all owi ng forb tter es t ab li ~ h m nl f
pl ant and microb s (Me ill et al. 198 1, L i n d~ay and Logan 1998 , Agge li dcs and Londra
2000). A field stu dy conducted by Lindsay and Logan ( 1998) fo und observable di ffere nces
in oil ph y, ical properties fo ur years aft r th inco rporat i n of anaerob icall y digested sewage
into a Mi ami an silt loam. Bulk de nsity decreased, whil total p rosi ty, moistu r re t nti on,
water table aggregate , and liquid and plas tic limi t ' increa. ed with increas in g slu dge
applicati on. As stated by the author , the. e change, were mo, t lik ly due to an in crease in
stable organi c matter content and co uld have even greater influence in mediu m- to coarsetex tured soils (Lindsay and Logan 1998 ).

The influence of organic substrates on soil ph ysical properties is further . upported by a field
stud y condu cted by Agge lides and Londra (2000 ) in which it was observed that physical
pro perti es improved significantl y in both loam and c lay so il s after the add ition of an rganic
compost (62% t wn wastes, 2 1% sewage slud ge, and 17f'h sawdu st). Though the effects
were greater in the loa m so il , saturated and un saturated hydrauli c cond u tivi ty, water
retenti on capac ity, bulk d nsity, t tal poros it y, pore s ize distri but ion, soi l r sis tance to

21

p n trati n, aggr gati n, and aggr gale , tabilit w r unpr v d pr p rti nal t the
appli ation rate. of th comp . t.

auti n mu. t b applied, h w v r, f r it is p . ," ibl th at PH
partiti n int th add d organic matt r

ander 2000 ).

co mp unds may . orb or
~ tud y by

amk

ng t al.

(2002) b."er d that rati s of rgani c amendment s t . il abov 1: 1 c uld inhibit microbial
a e. s to

ntamin ants, thu s redu cin g th

v rall degradati on of target co mpound s. The

influ nc of am ndment s on th e ph ys ica l chemi stry of hyd r carbons is an area of
ph ytor m di ati n that requi res furth er rc . , arch.

1.10

The is Resea rch

1.10.1 Overview and Resea rch Obj ecti ve
Further research i needed to better und rstand the fate of PHC c mpounds in th e pl antmicrobe-soil y tern . In particular, research i needed to furth er elu cidate th e roles of
vegetation and so il amendment in PH

degradati on and nutrient cyclin g in co ntaminated

soil s. The purpo e of this research wa to inve. tigate the influence of ana robicall y di ges ted
sewage 1udge (biosolids) addition in stimul ating smoo th brome (B romus inennis) grow th
and the subsequ ent increase in : 1) the degradati on of petro leum hydrocarbons [separated into
fracti ons according to the Canadian

ouncil of Ministers of th Enviro nment (C M )

anada Wide Tier 1 Stand ards (

M 200lu , h, c )] ; and 2) the nit roge n ( N ) availab le for

plant and s il microorgani sm gro wth in a weathered di esel co ntamin ated soi l.

22

1.1 0. 2 The i Outline

In hapt r 2, I
,m

amin th influ n

f ana r bi all dig st d s wag sluclg (bi s lids) on

lh bro m (Bromus in ermis) es tabli shmen t and th d gracl ati n of PH s in a w ather cl

di , el c ntaminat d soil. In hapt r _, I ~ p
additi on n th
m

clynami ~o f th plant -mi r b -s il s st m. Fin all y, in chapt r 4 I disc uss

nclu si ns and pr

ummar

ificall inv sti gat th influ nee f bi os lids

ide a ,· nth : i ~ of chapt r~ 2 and _ t pr vide a c mpreh nsiv

f the impac t th at bi .

lid ~ addit io n h a~ on th

ntamin atecl so il.

23

ph yt r m di ali n f thi ~ eli se l-

2.

The Application of Bio olid

mendment in the Phytoremediation of

ged,

Die I Hydrocarbon in a Medium-Te tured oil

2. 1

Introduction

It i. e. timated th at anada g nerat s appr

imat 1 si milli on tonnes of hazar I us was te

p r y ar, I adin g to tens f th usa nd: of co ntamin ated sit ~a ros~ the c untry (Hru l y
P ll ard 1 93,

Moreover, appr

iciliano &

ermid a 19

, n 1ronm nt

imat ly 60gc f these site. in vo l

anada 200 ,

usa 2001 ).

petr !cum hydrocarb n ~ (

M

200 l a).

The

anadi an ouncil of Mini ,' ter.

f th

nvir nment (

M ) has developed

anada-

Wide Standards ( W ) fo r petroleum hydrocarbons (PH s) in :oil s. Th e~e Ti er I . t and a rd ~
are grouped accordin g to the equi valent normal . traight-chain hydr carbon (n ) boilin g point
range and can be defin ed by four fraction , ( CM 2001 a, b , c).

racti on 1 (F 1) ranges

fro m nC6-n 10, fraction 2 (F2), from > n 10-n 16, fracti on 3 (F3 ), from > n 16- nC34, and
fracti on 4 (F4 ), consists of > nC34-nC50 . The use of these frac ti on for determining the
. ucce

of a remediation strategy is valu able because co mplex mixtures of PH s ofte n

ex hibit a wide range of chemical, ph y. ical, and tox icological behavi urs (

M

2000).

ractioning PH s into similar groupin gs all ow researchers to di stin guish the~e behaviours,
and provides a co nnecti on between risk redu cti on and co ntamin ant degradati on .

Bi od gradati on of PH s is an important meth o I of rem di ation. The mineralization and
m tabolism of organi c co ntaminants by soil orga ni sms may reduce to icily and on taminant

24

n ntrati n
bi a ailabilit

t

a

ptabl 1

L in ri. k manag m nl.

rgamsm. will di tate th success f thi s

f th co mp und, t d gradin g mi cr

rem di ati n strat g ( kl adan and M tlin g 1

3,

n rall , th . tru ctur and

le and r 1999).

Ph yt r medi ati n rna b d fi ned as an in-situ r m di ati n ~ tra t gy th at us ~ v g tati on t
nhan e th alread

isting bi d gradati

ac ti iti ,

f soil mi cr

rga ni sms. Th

applicati on p tenti al f thi : t hn ol g to organ ic c nt ami n a nt ~, and more specificall y PH s,
L

nl y now beginning t b reali zed.

um r u ~ sp ci ~ f gra~~e~, legumes, and wo dy

plants have bee n sh wn to fac ilitat th degradati n of both individual PH
mi xture. (Wilt: et al. 1998, chwab

Bank. 1999, Li ~ te &

co mp o und ~ and

1 xancl r 2000, Pichtel &

Liskanen 2001 , Palmr th et al. 2002, Size ki -Robso n et al. 2004 ).

ra~ses, in parti cul ar,

have been used ex tensively in phytor mediati on stu dies du e to their ability to grow under
difficult enviro nmental conditi ns and den. e, fib ro u roo t system that in creases the p tenti al
rhizo phere oil area (Pichtel and Li. kanen 2001 , Adam and Duncan 2002).

Rhi zoclegradation is considered to be the primary mechani sm of PH

co ntamin ant

degradation and ha recei vee! the mos t atten tion in phytoremedi ation . tudies e am ining
PH s. The "rhizosphere effec t" ex hibited by pl ant roo ts increases the nu mber and activities
of so il microorganisms near the root surface clue to a favo urable enviro nment (Anderson et
al. 1993 ). The influence of th e rhi zos phere may in lucie abioti c fac tor. such a · soil moisture,
tex ture, oxygen supply, temperature, and pH, or biotic factors . uch as the rel ase of nutrition
in the form of root exud ates (Kennedy 1998), and has th potenti al to increase the numb r or
so il microo rgani sms with the abilit y to cl grade organ ic 'Ontamin ants such as PH s.

25

P tr 1 um h dr
c nt nt (M

'trh n ~ ar appr

ill t al. 1

I).

im at

art on ( ) i th

5

dditi n of PJ l

~ t

plant a ail ahl amm nium ( H/) and nitrate (

r 1

nitr

n (N)

~

il ma 1 ad t an imm hili a lli n f

1)

lu tc th gro th f th

~o il mi ' rohi al

n 19 7, Pi r; n\k i t al. ~ 000 ).

bi mass n the

ntaminant uh \ tra t ( u and John

P tr I um h dr

arb n ~ ma al\ di \ turh biogeo ' h mica! prop rti c~ in ~o il, I a ling t

di. rupt d . oil ph ~ i ca l prop rti

. u h a bulk cJ n it , poro\ it , wat r-h !ding capac it , and

in r as d h dr ph hi t nd n 1 \ (M
and
as~

~-, ~ u c h , limiting nutri nt \ and th

ndra .... 000 ).
i·lled

ill et al. 19R J, 1nd\a and

ogan JC9R,

gg !ide\

poo r ph \ ical \O il pr p rti \o ft n

co nt amin ati c n can ha e a d irec t impac t on th e ~ u cce\\ ful e~o, t a bli ~ hm e nt

ith PH

of pl a nt ~ in c ntamin at d ~ il . The addi ti on )[ fe rtili; er\ and orga ni c amendment \ h a~ bee n
o b ~e r

edt impr

e th e~e c nditi on\ in non-co ntamin ated \o il \ (Wong and Ho I99 1,

Lindsay and Logan 19

, Zebarth et al. 1999,

gge lid e~o, and

nd ra 2000, Pun \ hon et al.

2002), but a greater understanding of th e tran. f rm a t io n ~ and fate f added nutri nt ~ in th
p re~e n ce of orga nic c

ntamin ants . uch a~ PH s i ~ need d.

The purpose of thi s stud y was to in ves ti gat the influence of anaer bi call y diges ted ~ewage
sludge (hi so lids) addition and sm oth brom (Bromus in ennis) es tabli shm nt on the
degradation f

ME WS Tier I PH

frac ti o n ~ in a weathered di ese l co ntamin at d soil.

pec ifi call y, it was hyp thes ized that I) the prese nce of pl a nt ~ wo uld increase both the rate
and extent of PH

d gradati on, and , 2) the additi on of bi oso lids wo ul d stimul ate plant

growth (including roots) such th at plant-indu ced r ducti on of PH s
th pr sen , of bi oso licls.

oul d be enh anced in

hi s wo rk was a co mponent or a 3~ - wce k ph toremedia tion

p n m nt that aL

plan t-mi r b - ~ il .

~t

Meth od and Ma t ri als

2.2.1

oil

~

I ~ pill\ o cr man

ntaminat db multipl eli

car\ and

n

B

pr

and rh c [,

. It wa\

a c ca atcd an I pl ac d int a

. The pil had rc 'c ive d minim al a ra tio n and no nutri e nt additi on \ in c it was

r ated and pre i u. t stin g h th \ it manag r~ had ho n PH
ab

f th

amplin g and Pr pa rati n

il u~ d in thi . tud wa\ htain d fr ) Ill a b1 ) , -- II I cat d in

hi ce ll in I

d nam 1

m ( hapt r _ ).

2.2

Th

[ bi . I id. a lditi n n th

amtn d th influ n

2 02).

In ial . il stand ards (MWL

ugust I , 2003, appr

co ntamin ant leve l\ to be

imatel 30 kg f

il wa co ll ec ted from th e hi opil e at a depth

of - 50- 100 e m (bel w : urface) and pl aced in 20 L pi a ti c bu c k e t ~. Th \O il wa~ tra n ~ p o rt c d
to

B

where it was parti all y dried to- 5 - I 07c grav imetri c m oi~ ture

ntent on pl a~ ti c

sheets in a shed pri or to ~ i ev in g through a 5 mm sc ree n. Th s iI was the n h m g ni; ed b
hand mixin g and stored in sealed plas tic bu ck ts at 4°

2.2 .2

until required.

Bio olids Samplin g and Preparation

An aerobi call y di gested sewage sludg (bi so lids) from the Prince
Tr atment

ntre wa~ c ll cted in 20

pr s. ing to re move e c ss water.

eorge Was tewater

plasti c bu ck ts on Au gust .... 9, 2003 aft er bc lt -

he bi osolids w r sampl ed at a depth of - 50- I 00 ' Ill

(b I w surfac ) from a larg h !cling pi l ; th is was cl one to nsur "' minim al
amm ni a (Ni h) vo latilization.

loss from

he bios lids wer stor din seal I pl as ti , bu ckets at 4°

27

unti l n d d, at

hand mi tn pn r t additi n t th

hi h tim th

. il.

2.2._

Plant S pecie.

n pl ant ~ p

h a~

in thi .. tud

u g rmin ati n

i ~ fr m a rr

d n it \ h\ n c I Pl I

tole ran ·c.
d\ in

arlt n ) .

2.2.4

penm nt (Rutherford t al. 2005) wa~ u\cd

Ex p e rim e nta l ~ etup a nd ( Jr

gr nh u ~

p rime nt

a.

me oth hrom (B ronws in ermis, cv.
a

rl odg ,

lh rt a.

e nh o use ( o ndition .

nductcd at the

nt

r\ it of

orth crn B r iti ~ h

olum hia

nh an d For str Lab rat r . The c p rim ntal d \ ign CO n\i\ tCd or 6 trea tme nt\ with two
fac tors: vegetati n [planted (P) and n n-pl antcd ( P )] and hio\o lid \ additi on at ra t e~ of zero
(0 ); low (L) r 13.34 g

D bioso lids kg 1

D ~ il ; and hi gh ( H ) or 26.6 g

D hi ~o lid ~ kg

00 soil.

ac h of these trea tm e nt ~ wa~ repli cated 4 tim e~ with 4 d ~ t ru c ti ve ~a mplin g d a t e~

pl anned.

amp! ing dates correspond ed to , 16, 24, and 32 wks afte r th e o n ~c t o f the

experiment with an initial a n a l ys i ~ done on a11 applicahl

m eas ure me nt ~ at time ze ro.

1

A total

f 96 ex perimental units were asse mbled and ~e t up in a co mpletely rand omi zed d e~ig n in the

greenhouse. Day temperatu re was kept at 25°

with a 16 h light p ri od (400 watt hi gh

prcs~ ure sodium supplem ental li ghtin g used); ni ght te mperature wa~ kept at 15°

.

Bas d n the moisture co ntent of th so iL a t tal of 2. 72 kg of fres h ~o iL equ ivale nt to 2.50
kg v n-dry (

) so il , was added t plasti hags for mi ing with the hi o~o lid s amen im nt.

r ~ h hi oso l ids w rc add d to the co ntaminat . . I so iI at th . . two rates d --~c ri hcd aho\ c and

w r th n th r ughl m1

a. unif rm in all tr atm nL durin g th

PH
m1

n ur th p t nti al abi ti 1 ss f

d int th . il b hand .

p nm ntal s tup , n n-am nd I s ils w r

din th . am fas hi n a. bi . li I -am nd d \ iL .

bi s lids

a. th n add d t _ .7

pla\ ti c poh ( 1
nt -fi

p : itioncd in th gr nh ou

m h ight b 17 m di am t r) and

. c d\ wcr pl ac d at a I m d pth in th e

appr pri ·1t tr atm nt poL t g 1v app r) imat 1 I ~-.,
p twa. br ught to t bring th

h entire mi tur of \ il r \ il plu \

1

d p r 7 em-. Wat r co nt nt for ac h

0'£ wat r h !di ng capac it (WH ) b dai l wat ring\ (w ith tap water)

p rim ntal unit to a pr d t rmi ncd w ight. II r WH

i\ dcfin d a~o., the

gra imetri c moi ture co ntent that is rca ' h d wh n a co lumn of \atu ratcd ~o.,o il is all owed to
drain fr

1 f r 24 h unci r co nd it ions that min imi ;:e

f r th e greenh usc

aporati c IO\\C\ . T he waterin g regime

perim nt wa~o., se lc ' ted to ma int ai n optim um plant and ~o.,oi l microb ial

acti vi ty while minimi zing th risk f PII

and nut rien t leachin g from the pot\ . In addi tio n,

the surface . oil in all trea tments wa. we tted wi th tap water (- 20 m ) us ing a garden mis ter
earl y in the ex periment (first 4 wks) to aid . e d ge rmi natio n and plant cs tabl ishm nt. Th
holes in the bottom of eac h p t we re parti all y scaled with tape to preve nt the si
falling out of each pot; this also partiall y redu ced I achin g from eac h pot.

d soi l fr m

n leac hat th at

was produ ced durin g wat rin g was caught by styrofoam pl ates and returned to the s i I
surface within - 30 min of water additi on.

Th bioso lids applicati on rates c rres pondcd to N addi tions of 726 mg total
and 1452 mg of total N kg

1

soil

so il forth low- and hi gh-bioso lids addi tion rat "' S,

respecti ve ly. The low rat was ca l ul atcd to m et th 80 kg

ha 1 plant r 1uircm "'nts for the

sp ci sand reg ion (Z barth t al. 2000 ), as w II as to a ·hie

a ~ 5 : I contaminant

am ndm nt rati

mm nd d b th Phytore111ediution r~F Petroleum ll wlrocarbons in

as r

oil Field rudy Protocol d

p d b th R m d iati n T chn 1 g1 "

Ph t r m di ati n f

ti n T am (RT

rgan1 s

d:

r b th th pl ant. and mi r

thi s rat

n. ur th at th "

n

th at th ca l ul at d rate.

tcm

rga m\111 ~

n

d'>

I c ).

hi ~

orum -

1uld n'>ur th at th

m t. Th hi gh rat

a " I c t I as Io u b Ic

rc n t un d r '> tim at d. It i'> imp rt ant t not

r hi " l id ad liti n vver "' ha

cl on th initi al chara ' t ri; ati on data

f th h mog niL d hi '> Oii d :a '> U ' h it i '> r cog ni ; d th at '>0 111 lo'> c~ o f ga'>co u ~
c ur durin g additi n and m i ing of bio <l id i nto oi l.
and suppl

of a ail ab le

d nam i .

f thi . e p ri mc nt w ill b e paneled upon .

2.2.5

etai l '> of th e prcd i ' t d

wo uld
demand

thr ugh bio o l id'> addition arc prov idec.l in ' hapter] w h --rc the

ample Pr ce in g

t each samplin g date, ra ndom! . el ec ted p t. ( 4 r pi ica te. fr m each tr atm nt) were
de. tru cti el

sampl d for chemical and bi olog ical anal yse'>.

ro t stru cture o f th e sm

ue t th e fibro u ~ and c ten'> i e

th brom e, r ots c uld n t b quanti tati ve ] r m

ed fro m th e . o il

(i .e. f r r ot bi omass determin ati n) w ith out di sruptin g th e so il for other anal yses. Th e so il
plu g was th erefore split in half usin g shoo t pl ac ment as a guide to ensure th at an e en
number o f pl ants occurred on both hal ves .

ne half o f th e t tal so il was subseq uen t!

U'>ecl

for r ot bi omass det rmin ation v ia roo t w ashin g (see b low), w hil e th e oth er hal f wa.
homog ni zed and sub-sa mpl ed for vari ous so il chemi ca l an I bi ologi al anal ses . Non v g tated pots w r processed and handl c.l in the sa me fashi on as vegetated treatm nt~ to
minimi z differ n , s in potenti al abi oti c I H

loss s b ' twe-- n vcge tati 1n t reatment~ .

_ ()

oil

, amp l ,

( I0 m ~h) pri r to m st h m1 al anal '> ~

r pa,', d thr ugh a 2 mm

( c pt PH d t rminali n).

L

r handpi k d fr m

(e.g. PH

tn ti on): th "

R

In additi n, PH anal i'> C
trac tabl PH

~ wer

il '>uh ampl " u d f r '>O il ch mi cal and hi olo i al anal ~c~
ampl

had < I fff ro t hi o ma'>'> pri r t LI'> f r ari U'> anal '>C'>.

h low) n n )t ti ~'> U

found in th e r 10 h .

'> uch, tra e amoun t\ )f r )O t ti '>'> Ue pr '>c nt in

th ~ ,' il . ample. did not int rfc r v. ith " 1i l H

2.2.6

h

ho\\ cl th at n gli gihl co ncentrati on'> of

anal

Plant Bioma

t and r ot bi m a~~ was co ll ec t d at eac h cl e'> truc ti e amplin g elate.

ue to th e

e tensi e and rapid grow th f the ~mo th hr m in the bi o~o lid ~ - am e nd e cltrca tm c nt '.,
intermedi at , non-des tru cti e har es t ~ of . h

t ~ we re aL o perf

added t the total shoot biomas. for th at sa mplin g peri d.
intermedi ate h arves t~, shoo ts were trimm d to th
amendment treatments; i.e., appr

~a m

rmed; thi '> bi o m a\~ wa~

c n ~ ure uniformity durin g

height in b th th low- and hi gh-

imately to the height of the non-amendm ent pl anh (n t

harves ted intermedi ately). At th e specified samplin g da t e~, s h oo t ~ for eac h pot were tri mmed
at the so il surface and put into pre-weigh d br wn paper b ag~ for bi o m a~~ determin at ion.
he bags w re then placed in a dryin g v n at 70 °
Th

m a~s of s h oo t~ (minu s th

overni ght (Kalra and Maynard 1991 ).

mass of the bags) fro m eac h pot i ~ reported in t h i~

study.

-I

R

t bi ma. :

pr

dure. Initial! th r

a. d t rmtn d fr m half th t tal , il in a h p t u\in g a r
t-. il m·t \

a. t wat r

a run thr ugh a~

~

ak in tap wat r o

all w d t
pla

nt )

ass 'lk d in h t ~ ap

utth soil clump · and r ck\ .

a. th n ma~~ ·1 g d t

mt r n (0 .500 mm ) si "'
rni ght.

din pr - e igh d brow n paper hag.s.

t w·1shing
at r; th

h r \Liltin g r ot rna\\ and

, furth r ·Jean I h hand , and

h r ot materi al was sub \ qu ntl

nn ~

d and

h fin al r )O t biomass f r half of th sampl ed p

a: htain d a. desc rih d [ r th sh )O t hie mass abc e: thi \ fi gur was th n multipli d h 2
to gi e th hi

m a~ .

rep )rted f )r th whc Je p ) l. It is stim ated th at < I c1c c r th e roo ts by mass

were n t re

2.2.7

na lytica l Meth ods

2.2. 7 . 1 oil and Bio o lid

haracte ri za ti o n

Water h !ding capac ity was determined h

mea~ urin g th

gra imetri c m o i ~ tu re co ntent of a

column of 5 mm :o il (o r ~o il plu~ bi ~ o lid s mi tur ~)a ft e r ~a tur a tin g with water (ov rni ght )
and all owin g it to drain free ly for 24 hr~ und r co nditi
moisture losses .
at 105°

n ~ th at minimi zed e

aporati v

ravim tri c motsture c ntent was determin ed by dryin g the ~o il ov rni ght

(Kalra and May nard 1991 ). Th foll owin g s il a n a l yse~ we r c ndu cted on e ith er 2

mm sieved so il ~ ampl es (or ~o il plu s bi oso lids mi tur s) or on non-sieved bi oso li d~ sampl es .
Particle size an alysis n so il was determined b th e pip t meth d with pr -treatm nt to
remove carb nates and organi c matter (Kalra and Maynard 1991 ).
a l :4 so il to deioniz d H2 suspension (Kalra and Ma nard 199 1).
and N

1 -N) w a ~ det

rminec.l bye tracti on with 0.5 M K 2

foll owed by co lorimctri N d t rminati on using an

32

oi l pH wa~ m "'<.b ured in
ai lah le

(N H/-

4 ( I :5 soi l:s )Jut io n ratio).

1- nal ti ca l Alpk m

low

stem IV

ut

nal L r (V r n

~ ).

t al. I

n fin I gr und 1 0 m ~ h . amp!
ombu sti n m th d u ~ ing a
6, r mn r l

J ).

i~

tal

and

(g r und in a

111 ~

il and hi ~o lid~

d

rinkman, m de l MM 2 ball mill ) b th dr

n.

nal 1 r (

I ctri cal c ndu cti it

fl11111

a~ d

b on and omm r~

t rmin I in ~a lura t e d pa\l ~ u ~ 1n g

aY IM d I ~ 1

c ndu ' ti vit m

apa ' it (

as d t rmin I h th e umm ati on m th di n 0. 1 M

a b c trac t ~, u ~ in g an

RL 3560 I

(H n I r~ h o t t al. 1 91~ ). Bul k d n ~i t (

~o il (or ~o il plu s

r (Kalra and Ma nard 1

I ).

ff ' ti

h) of th

bi . lids) in th p ts (5 mm ) wa calcul ated accorciJ ng to the equatio n:
: il/t tal

lum

2.2.7.2 PH

A na ly i

h

ati on c ' hang

= m a~~ or

rs il.

Petr 1 um hydr carb n. fo r frac ti n\ F2 ( n I 0- n 16) and 3 ( n 16- n 34) were de termi ned
ace rdin g to the
Method (

M

anada-Wi de tanda rd for Petrol um Hydrocarbo n\ in oil - T ie r I

M 200lh ). Prelimin ary wo rk ~ h we d th at 1 and 4 frac ti o n ~ were neg li gibl e

and c ul d be ignored in th i~ stud y.

Petroleum hydrocarbons wer ex trac ted from 5 mm so il u ~ in g a o hl et ex trac ti on me th od
with a 50:50 (hexane:acc tone) ~ol ve nt mixture (

M 2001/J ). Twe nt -rive g f :-.oi l (wet

we ight ) wa~ mi xed with 2 g or d i a to maceo u ~ earth and e trac ted for 23 hr:-. at 4 c elc\ per hr.
The res ultin g ex trac tant wa~ made up to 250 mL with the he ane:ace tone so l enl.
cl anup procedure as dcsc rib d by th
tor move polar organi c co mpound s.

column

M (200 Ih ) was appli ed to th ' res ulting extractant
lass co lumn. o r appro imatt.: l 16 mm insid'

di am tcr w r plu gg d with glass w ol an d fill ed with appro im at ~ 1 6.. 5 mm of 70-__0

m .h

a ti at d (h at d t 10 l o

nd
imat

b appr

1 pr

di. ard d.

o hi

h

ift m

olumn wa<..

wa~ add

d to th

: tream of
c

0

f th

u b~

r int r

mp und o

anh dr u. I

0 m ~ h ~ dium ~ ulph a t (dri d at 00°

im at 1 15m

r

2 .5 mm f

f r 4 hr. ) ( i h r 54 15- l ).

0

nu ~ h ed

qu nll

f r 12 hr.) ili a g I ( ~ i ~ h r 582 - 1) foll ow d

trac t wa<... quant itati

~ f

a~ run thr

n m of tolu n

ial and th r ~u lt i ng e trac tan t wa~ evaporat cl d wn to I 111

ndeno d air: th i~ wa~ br ught up to ..... 00 mL wi th cyc loh

0

tran. f rred int a 2 m

with a ~ t ad

ane. Tolu en and

n~urc maximum roo m t

r ubseq uent ga~ hro mat graph (

olumn .

n ~ ure all

nt t

II ' t I from th co lu mn in a gla ~ ial.

t

ugh th co lumn and

tra nof rrc I onto th

ith an addi ti nal I') mL of~

I he ane ol e nt ~ were u ~ed atth i ~ t age t
f PH

nt

mp rature ~o lubilit y

) anal ~ i ~. The ~a mpl e wa~ quantitati ve ly

ial, capp d wi th a Ten n-lined li d, and tored at

un til

o

anal i. .
0

olv nt extrac ts wer analyz d n a Vari an M del

P 3 00

a~

hr mat graph eq uipped

with a fl am ionization detec tor ( ID ). The co lumn u ~e d was a 30m MXT- 1 with 0.53 mm
intern al diameter and 0.25 mi cron film thickneos, uppli db
0

Bell efonte, P ,
in to a

A. A 1.0 ~

Re~ tek

v lume wa~ injec ted via a Vari an

plit/s plitl e~s injecti on port (Mod 1 I079 P

rpor·ati on,

P 8400

uto amp l "'r

V) held at J 20° for 2 min .

he injector

port wa~ then in creased at 200° per min to 350° , held for 2 min and was then dccrea~ed to
250° and held for 5 minutes b fore cooling to the start t mperatu re.

he sp lit ratio (5: I),

init iall y off, was a ti vat d after I min . A press ure pul se inje ' li on tcc hn iqu"' was LL --d to
increas the amc unt of sam pi on th

olumn . Pui s pr ssurc was I0.0 p~i (0.~5 min

du rati on). The co lumn wa~ initi all y h ld at 35°

fo r 4 min and was then in 'r'as --d at

15.

, and h ld f r 10 min: t tal run tim

/mint

a 7. 5 m /min.

arn r gas fl

he p ak r t nti n tim
h

ad

h

wa k pt at _

(i .. p ak rna imum

an (n 16 ) an It tratri 'lC ntanc (n _

(final

n ntrati ns f 6_.5, 1_5 and .... 50 ~t g m

r g1 n.

n . amp!

. tand arcl s(

M

nc ntrati on.

a _ 1111n p r sampl . H lium

f

t rn a l~o,t a ncl a rd ~o, d

li~o,~o,

I eel in 50:50 c cl he an :ac t n

1
)

an (n I 0) ,

w r , u~o, I to id entif

M , F2 and F3

hr matogram : ~o, a mpl c~o, we re run co ncurre nt] with th
.... Olb ). P "a k arca v.

re int eg ra te d v. ithinth e F2a nclF 3 rcg i o n ~o,a nd

rc 'alcul ated u~o,in g th av rag

e tern al stand ard s ( 1gma

re~o, p

n c fac t r obtain d from the foll ow in g

ldrich) eac h !iss lvcd in the abo c ~o,o l ve nt ~o, and run at thre

co ne ntrati ns (62. 5, 125 and 250 f,lg m L \ hexad cane ( n 16 ), nonadccane ( n 19 ),
eico. an (n 20 ), d tri ac ntanc (n 32) and tetratri aco ntane (n 34).
included in the cal ulati on of the average res ponse fac tor becaus
minutes bef re the first PH s eluted fr m sample ex trac t
dotri ac ntane extern al stand ard s are not required in the
2001 b). Th y were inc!uded in thi s stud y to all ow for

ecanc wa ~o, not

thi~o, ~o, t a nd a rd

luted sc cral

onade anc, eicosa nc and

M reference meth od (
mpari so n with B

M

so il ~;,t a nd a rd ~o,

(data not shown) .

2.2.8

S tati tical A naly is

tati sti ca1 analys is was p rformcd usin g tati sti ca vers ion 6. 1 so ft ware (2003) and refere nce
icgal ( 1956) and oka l and

ohlf ( 1995). After initi al e pl orator data anal)sis, it v..a.

del rmin d th at non-parametri c sl'lli sti cs were required.

II signifi ca nt rcs ult ~o, were found at

the a $ 0.05 level. Plant biomass data (within eac h samplin g date) and initi al soi l propcrti ".

-5

data

a. anal L d u. ing th n nparam tri

Kru~k a l -W a lli

, . il pr p rti ~d a t a

( i gal 195 ). Within . ub. qu nl . amplin g d·ll
th t

rank data (

kal ·wd R hlf I

t n~i n orth

h ir r-Ra -Har

-wa a1nl . i of arian

n - a anal ~i~ of anan ·

5). Inten li on fr

h

1

Kru~k a l W a lli~ l ~ t ) on

were t e~ t e d , though no ~ i g nifi ·ant

int rac ti on b t e n am ndmcnt an I \ g tatio n wa r und for an da ta. Wh r
~ 0.05 ) : ignifi ·an

d u ~ in g

~ t a ti ~ ti ca l ( P

t ~ l ( i gal I c 56) wa~ perf orm d l t ~ t

a fo un d, a Mann -Wh itn

mp an ~ 11 . or ra n k ~ .

p a 1r - w 1 ~

l a ti ~ ti ca l anal ~I.

on th

ac h :ampl ing dat

u~mg a n

CJc PH

remaining an I degradati on rat data wa~ performed within

nparamc tri c cheir r-Ray-1Iarc two -wa anal ~ i ~ of variance

on rank data ( okal and Rohlf I 95). Interac ti on efT ch we re t c~ ted, th ough no ~ig nifi ca nt
interac tion betwee n amendment and ege tatio n wa~ fou nd for any data. Wh ere ~ t a ti ~ ti ca l ( P
~ 0.05)

ignifi cance wa~ f und , a Mann -Whitney

pair-w is co mpari.

tc t ( iegal 1956) wa~ performed to t ~ l

n ~ of ra nk ~.

Th ree mod Is were examined with res pec t to e~ tim a tin g the d gradati n rate or the PH
fracti ons 2, 3, and total H

(where total H

c nce ntrati n = F2 co ncentrati on + 3

cone ntrati on). They we re a~ fo ll ow~ :

Model I)

First- rder decay:
t-

where

l

oe

1-l

=co n enlrati on or substrate at time t,

[ 2. 1]
0

= init ia l con' "'ntration or

th substrate, k = first-ore.! r d ca co nstant , and t = time (Broo k. tal. 200 I,
Namkoo ng t al. 200 .... ).

36

M difi d fir~t - rd r d

M d 1 2)

a
l-

wh r th additi mal param tcr

0

nC

J..t

[2.2]

+ n

= th r '>idual fracti n not bi oa ailahl

for

d gradati n [ ri gin ~ ft ar v r'>i n 7 , R ~ ( ... 00 ~) ] .

M del

)

M no- ' mponcnt mod I:

l-

h re

n L

a

[2.3 ]

o ('

m tant and th e param tcr'> R and

arc th initi al av rage rate

coe ffi -i nt and ~ p ee I f ag in g o f rc'>idu '> r '> pcc ti vc l ( Y ang and Jan'.'>en
2 00, .... 02). Th a

rage rate coc ffi i nt K at tim e t i'> ca lcul ate I h th

qu ati on: K = Rt ~ .

N onlinear r g re~s i o n was u ~e d to fit th e model t th e PH

data. The coe ffi ci nt of multipl e

2

determin ati n (R ) for each treatm ent w as calcul ated acco rdin g t Bail ey and M e ill (200 I )
by th e equ ation:

f2.4 ]
where the R

= th e res idu al sum of squ ares and

T

= th e co rrec ted total sum

r '> quare'> .

Wh en m re th an one m del fit th e data, th e significance o f th e incr ascd R 2 alu -- wa~
determin ed by th e qu ati n:

= th res idu al sum o f squares or mod I I (s impl er model w ith I '~~ param "t"r:),

w hcr R

1

R

r sidu al sum or squ ar d o r mod I 2 (more ' Ompl

'2 = th

-7

model), PI = th .,. number

or

1n m d I I, p2 = th numb r of param t r~ 111 m d I 2, n = th numb r of lata
p inL fitt din th

ur

( ·1il

n~id

r d slati . ti all (

1·. TR

>

and M

ill 2001 ). M d I\

~ 0.05) b ttcr at

ith m r param l r\

pl ainin g th e ariatic ninth data if

( CX. p2 pi n p2J ·

2.3

Re ult

2. _. I

oil Prop rti es

h r . ult: o f th tr atm nt ff cho n el ctcd \C il prop rt ic\ arc \ how n in T ah le 2. 1 and
Tabl e 2.-. With 5 .... . 17c . an I, _5.5 !( '> ilt , and 12.'-lfk c ia , th tc lu re of thi \ \O il fe ll at th e
int rfa

f a I am and and loa m ( oil

additi on fbi

~ lid ~ t

increa:ed the total

Ia \i ficatio n Worki ng 'roup 199H). Th e initi al

the weath ered dic\e l c nt aminat d oi l \ig nificantl (P ~ 0.05)

, total N, NH/- , pH,

not signifi anti y aff ct

~-

.

, and WH

of th \o il. Bi o~o li d~ additi on d id

l the end of th e ex periment , b i o\o li d~ - a m e nd e d p o t ~ had

signifi cantl y (P ~ 0.05 ) greater total

, total

, and WH

a~ co mpared to non-

amendm nt p L. Bulk den: ity appeared to b lowe r in bioso li cb -amended trea tm e nt ~ a~
compared to n n-amendm nt treatmenb at w ek 32, though no ~ t a ti ~ ti ca J ~ i gn ifi ca n ce ca n be
determined du e to the lac k o f repli cate ~a mpl e~.

y th e end of th e ~ tu dy, th r

significant ff ct due to bi o~o lid ~ additi n for mineral

wa~ no

plu ~

, - ), pH and

Vegetation, however, ~ i g nifi ca ntl y (P ~ 0.05) increased pH , dec reased

an u decrea\ed

(NH/-

min eral N. Vegetati on did not signifi ca ntl y affec t any oth er so il propert at the l a~ t ~amp l ing
elate.

-8

Table 2.1. Selected initial soil and amendment characterization properties.'·x Appropriate properties expressed on an oven-dry basi .....
N0 3 - -N
pH
EC
C EC
Total N
NH/ -N
WHC
Total C
1
1)
1
1))
(g
kg-))
(g
kg-))
(cmol+
kg(dS
m
)
(mg
kg(mg
kg)
(H
2
0
)
(g
H
20
100 g-1 soil
Treatment
0.4 (0.0) a
0.70 (0.62) a
0.04 (0.05) a
6.68 (0.03) a
I .30 (0.03) a
7.5 (0.5) a
23 . 1 (0.8) a
9.94 (0.13)
Soil
0.8 (0.2) h
7.09 (0.06) h
1.77 (0.12) h
87.6 ( 12.0) h
0.05 (0.08) a
9.9 ( 1.7) b
26.7 ( 1.7) h
ND
L Rate
138 (24) c
0.02 (0.02) a
7.74 (0.04) (
2.31 (0.12) l
1.6 (0.5) l
35.5 (0.6) (
16.2 (4 .3) '
ND
H Rate
1.00 ( 1.74)
4758 ( 150)
NO
75.7(1.1)
54.4 (5.1)
Biosolid~ ~
390 (38)
ND
ND
z \alue!:> repre~ent the mean (standard de\iation) of four replicate '>ample'> unle'>'> othcrwt'>e tndtcatcd
} a\erage (')tandard de\ Jation) or three replicate <.,ample'>
' different letter!:> ~1thm a column mdicate ~igntlicant (P:5 0.05) dliTcrence'> between amendment treatment'> 111 '>Oil- Mann Whuney u on patr-wt'>e compart'>Oil">
or ranked data
ND- not determmed

39

Table 2.2. Selected soil properties at 32 wks_z.).\ Appropriate properties expres~ed on an oven-dry ba~i~.
Vegetation
Treatment
p

OJ

p

NP

Lt>

Total C

Total N

(g kg"')

(g kg'')

8 .5
(4.6)
6. 1
(0. 6)
9.5

OJ

0.5 (0.1)

Mineral
N
(mg kg·')

()"

0 .5 (0.1)

Lt>

0.9 (0.1)

La

( I. I )
p

NP
p

plant effect

0 .8 (0.1)

9.5
(0.7)

He

12 .6
( I .2)
12. 1
(3.2)

NS

He

1.2(0. 1)
1.0 (0.2)

NS

H'l

pH
(H 20 )

?, .57
( 1.68)
0.64
(0.?,2)

()"

77.7
(27.9)
0 .77
(0.42)

L'l

45.?,
( 12 .5)
I. I I
(0.59)

H'l

S

6.90
(0.1 H)
7 .45
(0.07)

oa

1.96
(0.51)

CEC
(cmol+ kg-1) '
() "

UN

10 I (0.1)

Db"
(gem-:')

2?, () (0.6)

1.32

NO

1.32

27 5 (2.2)

1.23

NO

1.10

270(1.7)

1.10

ND

I 08

o ·l

10.3 (0.5)

(0.26)

6.60
(0.07)
7.4?,
(0. I 0)

L"

6.74
({l.05)
7.?,6
(0. I I )

H"

S

EC
(dS m-1 )

WHC
(g H 20 100
g·' soil)

4.25

Lt>

(0.64)
I .H6
(0.?,5)
2.66
(0 ?,?,)

12.0 (0 02)

Lt>

II X (0 5 )

Ht>

12 ~ (0.4)

2.40
( 0 61 }

115(02)

S

NS

z NP =no n-planted. P =planted. 0 = 1ero bio"olid'> addition. L = I ?, .?,4 g 00 btO'>OIId'> (..g i()O '>Otl. H = 26 6H g 00 bto'>nltd'> (..g

Ht>

0
1

00 . . otl

~ value-; repre~ent the mean (-;tandard <.leviation) of four rerlicate ~ample'> unk'>'> other'v\t'>e tndtcated
'average (-; tandard devtation) of three rerlicate '>amrte . .
"' based on one -;ample
dtiTerentletter'> vvtlhtn a column tndicate '>Igndicant (P ~ 0 .05) dtllercnce'> between amendment treatment.... tn the ran(..cd data of the column
to the Immediate nght- Mann Whitney U on pair-V\i'>e comran . . on . . or ran"-ed data
S- '>tgntficant at P ~ 0.05 ( Kru'>kai -Waiii'> on rank.cd data). NS - not . . igntlicant
NO - not determmed

-W

ND

2.3.2

Plant Biorna

Bi , lid, additi n , ignifi antl (P $ 0.05) in r a. ed b th r

t and , h

t bi nn. sal v ry

. ampJing date; t tal bi ma .. was gr at , t in th hi gh-am ndm nt rat lr atm nt ( ab l 2. _ ).
R

t t sh

th

t bioma:: rati was not . ignifi antl aff cted b th amendm nt tr atm nt, with

pti n f th z r am ndment rat at

wk~ .

This re~ult i~ m st lik 1 du to Lh p or

gr wth f th plants in the contaminated~ iJ , which 1 d to

tremely low hi mass. Th 1 w

plant bioma. s

mpared t the mass of the dryin g v ~s 1 mak this r ~ult suspect.

Table 2.3.

t and r

h

nt

Time

Plant Bi oma\s
hoot..,
Roots
(g _eot

R:,

)

0

L
H
0

L
H
24

IN

1

(wks)

16

t bioma~s a~ a function [tim and amendm nt rate.I ·y·

0

L
H

0. I 0 (0.04) a
5.84 ( 1.06) b
12.0 (0.7) (

0.29 (0.04) a
3.26 ( I .29) h
7.42 (3.46)

3.67 (2 .22) a
0.55 (0.16) h
0.61 (0.27) h

0.01 (0.0 1)a
14.8 (0.6) h
25.4 ( 1.5) (

BDL

BDL

12.6 (0.5)"
I 0.5 (6.3) x

0.83 (0.0 I )~
0.43 (0.25) X

0.22 (0.1 0) a
17 .9(1 .7)b
34.4 (5.0) c

0.20 (0.05) a
17 .0(6.9)h
34.4 (5.6) c

0.99 (0.32) a
0.93 (0.31) a
I .00 ( 0. I I ) a

L

32

0
0.62 (0.1 I) a
0.39 (0.22) a
0.65 (0.38) a
24 .8 (0.7) b
17 .8 (7.0) b
0.72 (0.29)''
L
36.5 ( 4.7) c
0.85 (0.13) a
H
43.0 (2.2) c
1
0 = 1ero bi oso lid ~ additi on, L = 13.34 g 00 biosolids kg- OD so il, H = 26.68 g 00 bi oso lid s kg 1 OD <.,oil
Y va lues represe nt the mean (s tandard dev iati on) of four repli cate sa mple s unless otherwise indicated
x average (standard dev iati on) of two replica te sa mples- stati sti cs not determined
w different letters within a ampling time and co lumn indi cate sig nifi cant diiTeren ccs (P ~ ().05 ) on ranked data Mann Whitney U on pair- wi~c co mpari so ns o r ranked data
BOL - below detec tion limit
R:S - root to shoot ratio
1

41

2. _·-

PHC D gradation

I nitial p tr I um h dr
ontaminantl
fra ti

n~ , r sp

nfirm~ that

d

b 140 mg kg

ls l

f th h m g 1111 d ~ il del nnll1 d th

arb n anal . 1
1

s il an I ....

mg k g

1

ti
I and

4 h lr

lind 1n alltr atm nh

arh on

ere n gli gible in thi\ \o il.

<.; oil f r

M _,

2 and -. 3

eight ).

igure 2. 1

rail, PJI

Jc eh

\ r th co ur\e < f th c e p riment a\ indi ' ate I b th e cl crea\e 111

th ar a und r th indi idu al p ak\ an I th g n ral unrc\o l eel . co mpl e mi turc (
hump n th e in li idu al

M frac ti n.
0

hro matogra m\ ( igur .... . I ).

a bl e~ .... .4-2.9).

~~

500

l

IJ

"

0

~

500

- ---· - - -·:

... :--~

F2

j
,,. . .,_.,.J./

J~,J,~
r

I
I

--~

F3

\J.
' .. ,J

- "ll

I

.I.

•

0

F4

~~~~ I ~

I
I

Vl

c
(I)
......
c

B

mi lure into

eel differ nt d gradati m ra te\ an I rc\ idu al co nce ntrati on\

.... and F3 \h

r th 3_ wk r ri d (

i iding the PH

M)

_,_

--- "1
-.

I

Time

1~ igure 2. 1.

hromatogram s o f v getated, low -Lun n lm nt trca tm "nts show ing th ..
co ne ntrati on of di sc i range PII sat ( ) t = 0 w ks, and ( ) t = _. . w ks. ott --d lines ar-- th ..
r l nti on tim e~ for n I 0, 16, 3 , and 50, r ~ p 'c ti cl , indi ca tin g the wi ndow to b m a\urcd
for a ' h PI l frac ti on. Int nsity units ar gi -- n in mY .

2

ath r d di , I ntaminat d
nt f initial 2 h dr arb ns r mmmn in a
1
ith , m th br m and bi . lid~ .' · " Ini tial , oi l 2 n ntrati n: 1 06 mg kg

Table 2.4. P r

mendment
Tr atment'\
0"

nt

L"
p
p

H 'l
p

8

32

.5( )
(. )

9 (I)
;J(2)

19 ( ~)
I (I)

7(I)

. () ( 4)

~ I

I7 ( I )

(I)

(5)
7 (I)

9 (I)
6 (0)

.... 0 ( 6)
7 (I)

p = no n- planted. r = planted.() = /Cro h10\0IId\ additiOn , L = 111-+ g 01 hiO\OIId\ kg I ()[ ~ H = 26 .6X
1
g 00 hio...,oltd..., k g 00 "oil
\ va lue" represent th e mean (<..,tandard de\tation) ol four replicate \ C.IInple..., unlc<.,<, other\\t<.,e tndtcated
' m ea n (<.,ta nd ard de\tallon) or three repltcate . . amp le"
" dtffere nt lett er<., \\lthtn ame ndment treatment co lumn tndt c ate \ tg nlft c ant (f $0 05) dilference..., 111 the rankl:d
d ata c (It- F2 rem a1n1ng at .32 wk..... ann
httney
on pa1r \\1\e compan .... on<.., of ranked data

r

, - <.,ig nlflca nt at P $ 0 .05 (. c hetrer- Ra - H are on ranked datal

0 - no t determtned

Table 2.5. Percent of initial

3 hydro arb ns remainin g in a weath red di s I co ntaminated
, il treat d with smooth brome and hi s lids .1 · 1 Initials il F3 c ncentrati n: 20 6 mg kg 1
OD . il.
Amendment
Treatment'~

Vegetation
Treatment
NP

45 (3)
37 (6)

31 (2)
31 (6)

p

51 ( 4)
50 (5)

39 (2)
35 (3)

43 (8)
30 (2)

NP

74(5) '
61( )

60 (4)
32 (4)

49 (5)
34 (4)

L ah
p

p

plant effec t
I

32

64 (4)
64 (6)

p

Hh

8

N

NP = no n- pl anted . p = pl anted . () = ; ero hi O\O iid <., addition . L = 11.1-l g or
1
g 00 hi o<.,O itd <., kg 00 '>O i l

kg I ()L) '>OIL H = 26 6

Y va lu es represe nt th e mea n (s tandard de v iatton) or four repli ca te <..,a mple " unlc..., . ., o th en\ I'>C tn Liicatcd
X

Ill 'a n (standard dcv ial1011 )

or three I'Cpltcatc <.,a f1lpl '<.,

amendment trea tm ent co lumn tndt ca tc srg nlfl ca nt ( I $ () 05) diiTcrcnce..., 111 the ranked
data o f (;,( F1 remainin g at 12 wb - Mann Wl11tn ·
o n pair- wtse co mpan '>O il '> o l ranked data
S <., ig ni fi ca nt at P $ {).05 (Scheirer- Ra - Hare on ranked data)
Nl - not d etermin ed

w dlfTere ntl ettet s w tthin

Table 2.6. P r nt f initial total H v r mammg in a w ath r d di . I c ntaminat d so il
1
at d ith . m th br m and bi . olid . .' ·' Initi al . il t tal H co ne ntrati n: 3492 mg kg
' il.
lin g date (wk )

Amendment
T rea tmen t"

p

p

8

24

32

53 (4)
52 (4)

32 (2) '
27 (4)

22 ( J )
22 (4)

40 (3)
37 ( )

28 ( I )
25 (2)

31(
2J ( I )

6 (4) \

40 (4)
25 (3)

p

47 ( )

47 (4)
24 (3)

plant effe ct

0

D
1

7

P =non -plan ted. P = planted. 0 = ;cro hio'>olid.., addition . L = 13 .i4 g OD hio..,olid" kg OD '>o il . H = 26.6R
1
g 00 biosolids kg OD soi l
) alues repre ent th e mean (stanuard dcvwlion) or four replicate '>ample'> unlc..,.., otherwi<;e ind1 catcd
' mean ( ·tandard deviation) or three repl1cate samp le'>
" different lelters w ithin amendmen t treatment column indicate -,i gnilicant (P :5 0.05) diiTeren ce'> in the ranked
data of C!( tota l H remaining at 32 wb. - M ann Whitney
on pair- wise compari'>On'> of ranked data
' H co nce ntration = F2 concentration + F3 concentration
- sig nifica nt at P :5 0.05 (Scheirer-Ray-H are on ranked data )
NO - not determined

Table 2.7. Model parameter and R values for F2 fraction d gradation u~in g Model 2
(eq uati on 2.2): ,=Coe-"'+Yo·' ·>·"
2

Vegetation
Treatment
NP
p

NP
p

plant effec t

Initial
degradable
fraction (Co)

Degradation
rate (k)

Reca lcitrant
fraction (y 0 )

(o/o)

(%)

Rz

I I (6)

89 (9)
9 1 (5)

0. I 6 I ( 0.04 7)
0. 15 1 (0.024)

9 (3)

0 .99
0.99

9 l (3)
93 (2)

0.274 (0.042)
0 .322 (0.033)

9 (2)
7 (I)

0.99
0.99

79 ( I 0)
93 (2)

0.27 0 (0. 147)
0.275 (0.029)

2 1 (6)
7 (I)

0.99
0.99

NO

N

Ha

N

' N P = non-planted, P = planted, 0 = ;,ero bioso lid s addi ti on, L = 13.34 g 00 hiosolids kg 00 soil. H = 26 . 6~
1
g 00 bioso lid s kg 00 s )i l
Y va lues represe nt th e mea n (s tandard dev iation) or no nlinear regress ion
x one less number of sa mpl es used in nonlin ear regress ion ( n = 15)
w different letters within a co lumn indi ca te sig nifi ca nt (P :5 0.05) diiTcrencc s hetwecn amendment treatments in
the ranked data of th e co lumn to the immediate r ight - M ann Whitn c U on pair- w 1sc ·omparisons
NS - not signifi ca nt (Sc heirer-Ra y-H arc on rank ed data )
NO - not determined
1

44

1

Table 2.8. M d 1 param t rand R- al u . f r F _ fra ti n I gradati n using M d l 2

( quati n 2.2):

Vegetati n
Treatment

,-

~'+
0

O·

l .'y \\

Initial
d gradab l
fraction
o)

R2

(

)
70 ( 15 )
74( 16 )

p

p

oa

0 ''

_9( 1_)
69 ( )

L ''

0._ 19 (0 . 14 2)
0.160 (0 .060 )

p

5_ ( 20)

H 'l

0 .07 - (0.0 0)
0 . 107 (0.065)

p

70 ( 15 )

0 .98
8

o.c

p
p

Re aJcitrant

Degradati n
k

,]

Il 'l

I (7)
- I ( )

0 .97
0.9C

7 ( 20)
30 (I 2)

0.95
0.97

p = non - rlanteu. p = rlanted. 0 = /ern htO'-tOitd'-t addttton . L = I~ ·q g 01
gOD htO'-tOIIU'-t 1-.g I ()[ '-tOtl

'-tO ll. H

26 6X

' value\ rerre..,entthe mean ('-ttandard de\tatton) o! nonltnear regre'-t'-tton
one le. '-t numher or..,amrk.., u'>ed tn nonl111ear regre'>'>ton (n = 15)
" dtrferent letter'> V\tthtn a column tnlltcate '> tgntftcant (P :50 05) dtrlerence'> hetween amendment tn:atment'> 111
th e ranl-.eu data or th e column to the tmmedtate n g ht • - not '>ignt!icant (. c he trer- Ra; - H arc on ranked data)

ann Whttnc;

on ratr- '-"'t'>e ·omra tt '>Oil'>

D - no t determ111ell
1

Table 2.9. Model parameter and R- valu ~ for total H ' fraction degradati on u~in g Model 2
(equation 2.2): ,- oe ~'+Yo· '·}· ''

Vegetation
Treatment

Initial
degradable
fraction ( o)
77 (9)
79 (I 0)

NP

0"

71 (8)
77 (6)

p

58 (I I )
76 (9)

I

Deg radation
rate (k)

H ''

Recalcitrant
fra ction (y 0 )

0 . I I 7 (0.04 I )
0. 1 12 (0.042)

( c)

R2

23 (7)

0.99
0 .99

21 ( )

0.243 (0. 103)
0.209 (0 .054)

L 'l

0 . 142 (0.084)
0 . I 52 (0 .054)

H"

29 (5)
23 (4)

0.99
0 .99

42 ( )
24 (6)

0 .97
0 .99

plant errect
Nl
N.
NP = non- rlant ed . p = rlanted . () = / ero htmo liU '> addition , L = I ~ - ~-~- g ()I) htO'-tOitd'-t 1-.g I ()[)'-,{)II. H = 26 ()
1

g OD hio..,olich kg OD so il
>' values rerre <.,e nt the mean (standard de iatton) or nonlinear re gress ton
x one I e..,.., numher or samrles used tn nonltnear r ·gres'>ion (n = 15)
"" dif! erent letter'> wtthin a co l umn indt ca te '>tgntricant (P :5 0.05) dt!lcrcn "C'-t hct\.'vl'L'tl amendment trcatmcnh 111
th e ranked data or the co lumn to th e immedtate n g ht Mann Whttnc
on patr "" t'-tc compatt'-tOth
v II 'co nce ntratton - F2 co nce ntration+ F~ co nce ntration
S stgnt!icant at P :5 0 .05 (Schetrer Ra
Nl
notlletermtn ed

Hare on ranked data).

5

S - not '-tt g ntfi cant

he gr at , t d 1in in th

2 h dr

arb n 1

L o

urr d luring th fir. t 8 wks of th

periment wh r th v g tated, low-am n lm nt tr atm nl had th great st d cr ase of 86
(Tabl 2.4). Th rat

f PH

degradati n in mo. tlr atmenL' , how ver, sl w d as th

p riment pr gr , : d. B week 32, 6-20\k of th

2 fra tion r main d with no signifi ant

eli ff renee bet we n the am ndment tre a tm e nt ~. th ough v ge tat d tr atments had signifi ant ly
(P ~ 0.05 ) lower 2 h

lrocarb o n ~ remai nin g a,'

mpared to non-veg tat d tr atments within

the 1 w and hi gh bi s lid~ application rate~ ( ab l 2.4 ).

Fra ti n 3 hydrocarbons and total H sh wed a general trend simi lar to th at of th e 2
hydrocarb ns, th ough there wa~ less of an ove rall decrease in PH

leveb (Tab les 2.5 and

2.6, re, pectively ). Ma imum rem va l ccurred during the first 8 wk~, where the vegetated,
low-amendment treatm nt again had the greatest decrease (i.. 507£ and 63 % for th e F3
fraction and total HC, re. pectively). By week 32, 30-49% of the F3 hydrocarbon fraction
remained with ignificantly (P ~ 0.05) lower 3 hydrocarbons remaining in th e nonamendment treatment a compared to the high-amendment treatme nt (Table 2.5). No
ignificant difference in the percent 3 hydrocarbon fraction remaining between th e non- and
low-amendment treatmen ts, or betwee n the low- and high-amendme nt treatments occurred at
the end of the 32 wk period. Additionally, 2 1-40% of the total H

remain ed at 32 wks,

where the non-amended and low-amendment treatment. had signifi cantl y (P $ 0.05) less total
H

remainin g as compared to the high-amendment treatment (Table 2.6) . On av rag ,

vegetated treatments showed signifi canlly (P ~ 0.05) lower F3 and total H remaining than
n n-vegetated treatments within the low and hi gh bi oso licls application rates (Tables 2.5 and
2.6) .

6

')

11 thr e m d L ( qu ati n. 2.1 , 2.2 and 2.3) fit th data r a. nabl well with R ~ alu s
( quati n. 2.4 and 2.5) foil win g the patt rn 2 > H

>

(data not . h wn ). Model 2
')

xplained . ignifi ant! mor

f th

ari ati n in the data th an mod 1 1. Th R ~ val u s f r

model 2 and m d I 3 w r simil ar and rang d bet ce n 0.95 and 0. 9, depe nding on the
fra ti n in

stigated.

tali . ticall , m del 3 wa<., f un d t e pl ain more f th vari ati n with

fewer param t e r~ (data not sh wn), th ough m del 2 wa~ chosen a~ the bes t fit for thi ~ data
du t its bi oi gical . ignifi canc in w ath red, co ntaminated s ib : thi ~ will bee panded
upon in the di sc uss ion se ti n.

or thi ~ rea~o n , nl y th res ults of mode l 2 arc prese nted.

An e ampl e of the curv -fitted data can be f und in

igur 2.2. Th e R2 va lu es for all curve

e. timati ons were hi gh and rang db twe n 0.95 and 0 .99 d p ndin g o n th e frac ti n
examined. It i · important to note that the

P-H treatment in all frac ti ons had one l c~s sampl e

than expec ted and the NP-0 treatment had an outlier in F3. Th ese treatments therefore had
fewer ampl e · than expec ted. In pa1ticul ar, removin g th e outli er in the 3 and total H

data

2

for the NP-0 treatment inc rea ed the R valu e by> 0.10 and made bi ologica l sense to
re move, as it is unlikely to find an increased concentration of PHCs in a closed sys te m ver
time.

The results for the nonlinear regress ion of fracti ons F2, F3, and total H

ca n be fo un d in

Tables 2.7, 2.8, and 2.9, respectively. In all cases, the general tre nd was fo r the degradation
rat , k, to be greater in the bi oso lids-amended treatments, though onl y the low- and high am ndment rates in 2, and the low-am ndment rat in total H , w re fo und to be
significantl y ( P~ 0.05) greater as co mpar d to the non-am nded treatm nt s. Biosol ids

7

•

100·

I • F2, P·L I

80

''.fl~llh '" }
, h1 ~.: p .. r

Ol

~

R'

60·

c:

iii
E
Q)

\f'l ,·u

\1

.

(lr.r,uv.
\I
1

7\b

4it,l(

~11"111

\f,JI

~lfi'NJH

'\

I

a:

,,

0-l:.'~f>ll

~IIJif'<lt,oJ

40

N
LL
0

0

20

I

•

I

0
T

5

~

T

T

0

T

10

5

~

15

I

T

r

20

25

30

-1
35

T

T1me (weeks)

B

110

•

100
90

I • F3 , P-L

80

IA/Uo•ill~:>n

Ol

E

Cll

70

E
Q)
a: 60
0
0

'"·

,,

11

\1

'~9.!('1)4\

.

)J ~~k

1~.4;

t"''M

16

•
•

C')

LL

I k'\lf,..,

qu"' llol
k

c:

I

\I• 'J'f\11 .. ,1

,

"'-1\if

•

50
40

••

30

•

I

I

20
5

0

5

10

15

20

25

'35

30

T 1me (wee ks)

•

100

80

I • HC , P·L I

Ol

c:

c

iii
E

Q)

I qu..1.1o"n ~

··-

a:
u
I

0
0

\I

( tll"'!'l'o<·f

60

40

'~ \1)1 .. \0

6 l'il""i
11"1111\

'"\1

2.! Ill I~,.,
7., l.l11"1

-.-j'i(,(ll!\

II

!T'Ir1lJ

~·I It l'iM I

~J<)_J<.III_

!

.

.

I

20

5

0

5

10

r

I

15

20

I

-,
25

30

-,
35

T1me (wee ks)

Figure 2.2. Res ults of non-linea r regr ss i n on lk ( ) 2, (B) F3, and (C) total HC
remaining [or th vegetated, low-amendment treatments using eq uation 2.2: ct = n e J.. t +
ines indi cat the exponenti al curve fit.

48

d gradati n rat , th ugh th 1 w-am ndm nt

am ndm nt had n

ignifi ant effect on

tr atm nt did ha

a high r k alu than b th th z r and hi gh-am ndm nt tr atm nL .

Y g tali n had n . ignifi ant ff ct n the d gradati on rate in an PH

2.4

Di cu ion

2.4 .1

oil Properti e

Initi all y, bi s lids additi on signifi cant]
with thee

pti on f

1-

,

fraction .

< .OS) in cr as d all meas ur d so il pr perti s

f whi ch th bi solids had a very low conce ntrati on (Tabl e

2. 1). The initi al ff t f bi o: lids additi ons n so il properti es was m st likely clu e to the
high organic matter c ntent and hi gh co ncentrati n of NH/- N in th bi os lid s (Stev nso n
1986). Thi s improve ment in soil pr perti s, specificall y WH

in medium - t coarse-tex tured

oils, is c nsi tent with other stu dies fo und in the lit rature (Wong and Ho 199 I , Zebarth et
al. 1999, Aggelides and Londra 2000, Punsh n et al. 2002) . Tt is important to note th at
though the addition of bio, oUds raised the EC of the . oil to a point where so me plants can be
affected by (i.e. an EC of 2 dS m-1 or greater), the initi al in crease in E

to a max imu m of

2.3 1 ciS m-1 in the high-amendment treatment of thi study did not appear to affec t the
germination or growth of smooth brome (Table 2.1 ). An E

of greater than 4 d m-1 in the

soil olution is considered saline (Punshon et al. 2002).

At week 32, onl y total , total N,

, and WH

were significantl y (P ~ 0.05) greater in the

biosoJids-amendecl treatments as omparecl to the non-amended treatment (Table ........... ). The
general trend was for bu lk density ( b) to be lower in the bioso lids-amendcd treatments a~
co mpared to the non-amended tr atment (Table 2.2). Aga in , these resu lts are consistent with

9

. tudi . fbi , lid additi n t . il s b Z barth t al. ( 1999 ),
and W nb and H ( 1991 ), that b: r
ears foll

gg lid , and

ndra (2000),

d , il bulk d n. iti , w re signifi antly r due d up t 3

ing bi . lid. additi n. Thi. r sult i. ft n du to th additi n of th organic

materi al, whi h i hi gh! p rous and ha. a 1 w parti le densit .
to a oars -t
increa:ed WH

tured s il ,

dditi n of organi c materi al

hi ch t pi call ha. low water retenti n apabil iti ~. likely I d t

through lower bulk d nsity and an in r ased prop ttion f mi cropores

( tevens n 19 6). It i. interes ting to note that at 32 wks,

and WH

w r n t affec ted

by an in reased rate of hi s lids add ition, where the low-amendment rat was not
. ignifi cantl y diff r nt than th high-amendment rat

f addit i n. Thi s wou ld ~ u ggcs t th at

although initiall y significa nt , increas in g the bioso lids additi on rate does not lead to
. ignificantl y improved so il pro perti s vcr tim e.

The lack of amendment effects on mineral N (NH/-N and No ~ - - ), pH, and E

valu es at

week 32 were likely du e to the signifi cant ( P~ 0.05) effect of v getati on on th ese properties
(Table 2.2). Soil pH and water potential generally decrease in the pre. enc of plants, whi ch
may increase the nutrient and ion transport in the rhi zosphere ·oil (W alto n et al. 1994/?, Paul
and Clark 1996 ). During the roo t uptake of N03-, however, alkaline conditi ons may d ve lop
du e to an increased soil so lu tion concentration of HC0 3 - ions ex uded by th e roo t (Bolton

t

aJ. 1993 ). This i, con is tent with the res ults of the current stud y, which found the pH of the
v getated soil s to be significantl y (P ~ 0.05) greater than that of th non-vegetated soi l.

so

2.4.2

Plant Bioma

n rail PH

c ntaminati n r du

, plant 0 rminati n and gr wth ith r lir ctl , du t

r indir ctl , du t di , rupti n in s il mi r bial and faunal p pulati ns, nutri ent
cling and bi 1 gical and h mt al reacti n,· (M
200 I, iddiqui and

dams 20 2). Man : tudi , ha

with the additi n f rgant

'lm

ill et al. 1

1, i htel and

i, kanen

shown an impr v m nt in plant vi gour

ndments. In a simil ar stud rep rted by Punsh n tal.

(2002), additi n of coal fl a: h and p ultry bi so lids to seve rely er ded land Jed t a 26%
increa, e in grass pr du li on. Wh n bi ,' lids w re added as a so il and fertili zer am endment
in thi , . tudy, ther was a significant (P ~ 0. 5) in r asc f at leas t an rd r of magnitud in
the , moo th brom bi rn a,·, at all ,· ampling dat s as c mparcd t th non-am nded treatm ent
(Table 2.3).

Plant in the bio ·olid:-amended treatments were not onl y bi gger and mor dense, but did not
, how the di co!ouration ob erved in the non-amended, vegetated treatm ents. This may be
du e to several rea on, . Fie t, organic amendment such as bi osolid have been , hown to
improve poor soil properties often associated with contamin ated so il s. These may include
the bulk density, porosity, water holdin g capacity, and hydrophobicity of so il (McGill et al.
198 1, Lindsay and Logan 1998, Agge!ides and Londra 2000 ). Th ough it was not m as ured
directly , th e oil showed hydrophobic tend ncies and was ex tremely difficult to we t at the
onset of the experiment. The additi on of bi solids at both rates appeared to reduce this
ten l ncy and in creased the wettability of th s il wh n compar d t th non-am nded
treatment. The increased water availability to the pl ant as a res ult of the increased

51

f th bi , lids lik I I d t unprov d

and high water h !ding apacit

mi

germinati nand gro th f th . m

c nd , a. dis u. sed pre i us! ,
In additi n t

th br m (Pun. hon et al. 2002).

ts ften the limiting nutri nt in PH c ntaminated . oils.

ntr !ling the min ralizatio n f rgan tc

are th ught t b b tt r c mp titor: f r pl ant ava il abl
(J a k. n t al. 19
mineral

to in rgani c f rm s, mtcr
such as

rgan1 sms

H +-N and

, Kaye and Hart l 97, Hodge et al. 2000). The I w pl ant ava il abl e

in th e initi al n n-amend d . oil ( able 2. 1), sugges ts th at

was limiting. A <:, can

Ht

Th in creased pl ant

b s en in Tabl 2. 1, th e add d biosoli d<:-, w re a source hi gh in

biomas. in bi so lids-am nded tr atm nts wo ul d sugges t th at bio<:-, lid s addit io n reduc d th e
co mpetition between mi croo rga ni sms and sm oth br me for min eral

. Thi \ will be

expanded upon in Ch apter 3.

Though eli colouration of pl ant . hoo ts can indi cate tox icity as we ll as several nutri ent
defi ciencies, the , hoot discolouration in the non-amended treatment of thi s study was most
likely due to a nutrient deficiency, considerin g th e low mineral N leve ls of the initi al nonamended oil (Table 2. 1) (Stevenso n 1986). Toxicity is often attributed to F 1 frac ti on PH s
with le s than n 10 carbon atoms (Adam and Duncan 2002, iddiqui and Adams 2002) and
although diesel range hydrocarbons include this frac tion, the soil us d in thi s stu dy was
weathered. Data prov ided by the site manager co nfirmed low co nce ntrati ons of volati le (i.e.
F 1) co mponents. ln addition, so il processing in this study mos t li ke ly led to th volatili zation
of any res idu al li ght end co mpounds before the onse t of thee p riment (Figure :2 . 1).

52

Th bi , lid. rna , h wev r, ha

dilut d r . orbed th PH : thu s redu cing th t

the, e

dis u. s d b

mpound , to th plant .

n. id r d t be minimal as each

uilhm z and Milk (200 1), thi s ma b

perim ntal unit in thi: study had th same am unt f

contaminat d . il, r gardl ss of th treatment appli d.
am ndm nts in a suitabl n noutlin d b the

i ity of

urther in es ti gati n usin g bi s lids

ntaminated c ntrol s il, and

men an

'O t

i it y t sts . uch as those

sting and Materi als ( T M 199 ) on the

co ntamin ated , oil in qu sti on w uld b b n fi ia l in dct rmin ing the innu nee of tox icant
, rption n succes. ful plant stab lishmcnl. F r the purpos s of thi s e peri mcnt , however, it
is ufficient to say th at bi . olids am ndm nt signifi antly (P :5 0.05) in creased the smoo th
brome bioma.. .

2.4.3

PHC Degradation

Many tudies appl y Model 1 (equ ati on 2. 1) to th e micro bi al minerali zati n of
evoluti on a a mean of determining PH

and use

02

degradation rates. The rate th at is calc ulated is

ba ed only on the bioavailable PHC co mpound s if the C0 2 evolved is ass um d to be fro m th e
micro bial mineralization of available

in the PHC compounds. Appl yin g equ ati on 2. 1

direc tly to PHC co ncentrati on data may overe. timate the actu al PHC degradati on rat
because it doe not consider the Jess bioavail able res idu al frac ti on th at occ urs due to the
agin g processes ac tin g upon co ntaminants (Brook et al. 200 I). This residu al fraction is oft n
cited in many studi es (Alexander 2000, em1J e et al. 2003) and attempts to increase the
ex tent and rate of degradati on of thi s fracti on have oft en fail ed (Pinelli et al. 1999).

53

M d 1 ( quati n 2. ) in r a. . th fit fth data
. ub. trat und r w
3 pr p

rate

sti gation be

m . 1 .. a

.. ibl

r M d 1 l b cau. e it a.. ume. that th
r more re al itrant with tim . M del

that degradati n can be d . ribed as a m, n -

mp n nt who! , where th a

ffici nt (K) change. with tim (Yang and Jans: n 2000) . Th param t r

rag

r pr s nts

the . peed f aging and a

unt. f r th deer as

t . orption on th : il matri

r re al itran

increases due to biochemical transformati on

and nrichm nt f the rc. idu

111 c

r incr a\ecl siz and co mrl

mpound\

f K a. resi lu bioavailability de r as s du

ity (Brow n ct al.

1998, Yan g and Jansse n 2000).

Thi . tudy , how v r, us d a so il th at had been co ntam in at d ve r many years and had been
virtually untouched in a biocell f r aim st 5 yrs prior to samplin g. The effec t ~ of weathering
in thi s oil would presumabl y already be pres nt due to the age f the co ntam in ants within
the oil. Thou gh th re. idu al fraction may in fact be degrading, the degradation rate would
be expected to be so low that degradation would approach zero in thi s hort-term (8 mo)
experiment. Modifying the first ord er decay equation to include the parameter Yo (i.e. Model
2), which i an e timate of there idual PHC fraction already pres nt in the soi l (Noce ntini et
al. 2000), is likely a closer approximation of the characteris tics of the soi l used in th is study.

xamination of the d grad ati on rate co nstants given in Tables 2.7 and 2.8 suggest that 2
hydrocarbons were degraded faster than 3 hydrocarbons in thi s particular soil. In general,
2 fracti on degradation rates of 0.15 1-0.322 wk-1 w re 2-3 times greater than F3 fracti n
1

d gradati on rates of 0.07 3-0.2 19 wk ,
tr atm nt where th

pl in the non- g tated, low-amendment

2 fracti n degradati on rat was onl y sli ghtly higher at 0 . ~ 74 w" 1 than

54

th

3 d gradati n rat of 0.2 19 wk 1 •

h

r , ulL agr

with th w rk f

(2002) wh found diff r nt degradati n rat , f r diff rent fracti n.
mp : ting , tud

n : pik d di , el c ntaminant:. Though th

, ugg . t d fracti nati n th

amk

f PH ,

ng t al.

r a 30 d

did not u, e the

M

w re able t di s rn d grad ali n rat s f 0.37 -0.54 d 1 and 0.18-

0.24 d-1 f r n-alkan chain. di ided into carbon chain 1 ngth s f n I 0-n 15 and n 16n 20, resp cti

1 ; th s frac ti m. appr

imate the 2 and 3 r a n ge~ rcco mm nded by the

M.

The greater d gradati n rate for 2

r~ u .

3 in the current ~ tud y is c n s i ~ t e nt with res ult s

report d for crud oil degradation in we~ te rn

anadian fi eld ·oib (Vi sser et al. 2003). A

le , r degradati on rate fo r the 3 frac tion is expec ted, give n th e larger ~ i z

f th e c mponent

molecule a. compared to th F2 frac tion. The 3 fraction has a lower water solubility and
greater Koc th an the F2 fr action (

ME 2000 ), likely co ntributing to stronger sorpti on to

native- or amendment-deri ved oil organic carbon. It has been es tablished that ~o rpti o n
proce es reduce bioavailability of noni onic hydrophobic molec ules to potentiall y degrading
microorganism. (Alexander 1999).

The low-amendment treatment signifi cantl y increased the degradation rate of th total HC in
this study by approximately 2 fold over the untreated co ntrol. In co mparison, the omposting
study by Namkoong et al. (2002), observed that the addition of sewage slu dge to a spiked,
diesel-co ntamin ated soil at a rati o of 1:0.1 (w t-weight) so il to sewage slu dge, which is
equivalent to the hi gh rate in this stud y, incr ased the degradati on rate of PHCs almost 2 fo ld
over th e untreated co ntrol. A degradati on rate of 0.505 wk 1 was dcmo nstrat "db Namkoon g

55

t a!. (2002) a. c mpar d t th high-am ndm nt rat
tr atm nt f thi, , tud y.
sh rter tim peri od

f 0. 142 wk~ 1 in th non-v getat d

h cliff r n e in d gradati n enhan

m nt 1na b attribut d t th

amined and more bi ac tiv ~ c mp sting co nditi ons used in the

amko ng et a!. (. . . 002) stud , as well as the ag

f the c ntamin ants under study in ach

perim nt. D gradation rates in spik d so il:, .· uch as that f Namko ng t al. (2002), are
p ted t b high r th an in w ath er d so ils du e to th
(Hatzin g r and

ander 1995,

ffe ts of agin g on th c ntaminants

le ander 2000 ).

Br ok et a!. (200 1) e a mined the bi remed iati n of a weathered di esel contamin ated so il
(2 .2<fl PH

by dry we ight ) and fo un d degradation rates close r to th ose found in this study.

When inorganic

was added t the s il t ac h iev a

:N ra ti o of 20: I, PH

degradati on rate

valu e corre ·pondin g to 0.027-0.224 wk-1 were observed over 60 d dep ndin g on the N
ource applied. Degradati on rate value. were I .5- 12 times the rate of th e untreated co nt ro l.
Thi. would ugge t that care mu st be taken when appl yin g degradation rate fo und on spiked
soil to aged co ntaminants in the fie ld.

Generally the res idu al fraction of PHCs re ulting from bi oremedi ati on efforts is estimated to
be 20-30% ( haineau et al. 2000), though this valu e may vary depending on so il conditions
such as tex ture, oxygen and nutrient co ntent, and PH

·ource, co mpos iti n, concentration,

and age (Hatzin ger and Alexander 1995 , Loehr and Webster I 996, Rise r-Roberts 1998,
Alexand er 2000, Nocentini et al. 2000, Ri vera-Espinoza and D ndo v n 2004 ). Th results
of thi s ex periment roughl y ag ree with thi s estimate where the average % remaining over all

56

tr atm nt. wa. f und t b 10, 6, and 2 ~ f r 2,

, and

mbin d H

fracti n ,

r sp ti el (Tabl s 2.4-2. 6).

The : ignifi ant (P::;; 0.05) increase in d gradati n rat du e t bios lid s additi on (Tab! 2.7),
with ut a significant de rea~e in th <7r F ... frac tion r maining at 32 wk. (Tab! 2.4), would
sugges t th at the bi ~ lid ~ additi on incr a~e cl th rate f d gracl ati n of th bi oavailabl e
mp unds in thi ~ frac ti n, but di d n t ~ i g ni fi 'antl y affec t th r sidu al frac ti on. Thi ~ ag ree~
with th findin gs f th r s tu d i e~ (Pin lli et al. 1999,

c ntini et al. 2000, Ri vera- ""' ~ pin oza

and Dend ov n 2004) where the re~ i d u a l co nce nt ra tion wa~ fo und t be re~ i ~ t a nt t furth er
treatment eff cts aim d at in r a~ in g th e degradati n rate.

The lack of a ignificant effec t f the bi oso lid ~ additi n on the F3 degradati on rate (Tabl e
2.8), and the signi ficant (P::;; 0.05 ) increase in the 7l

3 frac tion re maining at 32 wks in th

high-amendment treatment a · co mpared to the non-a mended treatm ent (Tabl e 2.5 ), may be
partly du e to the interf renee of the bio olids in the gas chro matograph y analys i:. As can be
. een in Figure 2.3, which shows the gas chromatographi c profile of th e biosolids ex trac ted
alone, there is a definite interference in the F3 frac ti on wind ow. Th ugh th e am nd ment
treatments in this stud y contained a much lower conce ntration of biosolids (- 1%) mi ed in
with the soil, a correspondin g unresolved co mplex mix tur hump was fo und in all the lowand hi gh-amendment rate chromatograms. This inter[ rene is mos t likely due to organic
co mpounds found in the biosolids, whi ch are difficult to different ial fro m the PHC
co ntamin ants in the so il , and may have incr ascd the apparent % 3 frac ti on re mai ning.

57

~·

2000

~

... F2

('"

... !' ...

I
I
I

I
I
I
I
I
I
I
I
I
I
I
I
I
I

.. I

I '

0

~

l ·~

u

I
I

-

l

~

: 1\ ..

..

I ~

F3

F4

I
I
I
I
I
I
I
I
I
I

~\ I I

))C~ ~ 1,~L[ ,I \~ :
I

-,... '

-

I

I
I

I

I

-·

'·

-

I
I
I

...

-· ~ r

I

...

... ... .

I

"
"'

.. -

Figure 2.3. hromat gram of hi s lids
hlet- tra ted alone .
tted lines are th e
retenti n time: fo r n 10, 16, 34, and 5 , re'> p eli v I indicatin g th wind ow t he meas ured
f r ach PH frac ti n.
t th interfere nce meas ured in th e 3 wi nd ow. Intensit y units are
giv n in mY .

R ger. ( 1996) rev iews th diffi cul ties in e trac tin g bioso lids mixture\ , part icul arly th e Jack
of specificit y of Sox hlet ex trac ti n in redu cin g co-ex trac tab le analyti ca l interferences and th e
need for effecti ve clean-up procedure. to redu ce th e interferences of co-elutin g pea ks,
detector saturati on, and capillary co lumn overl oading in gas chro matograph y analys is.
Though column chromatography was used in this experiment to clean sample ex trac ts b f re
analy is, the choice of olvents and materi als were based on procedures d signed for PH
contaminated soil, not sewage slud ge mixtures (C M 200lb ). Littl e is know n about th e
sorption of hydrophobic co mpounds on organic matter or the degradation of biosolids
organic (Rogers 1996) . A suitable non-co ntamin ated co nt rol so il was not ava il able for this
stud y, but a biosolid s treatment added to such a soil may all w for the es ti ma tion of the
influence of the bi osolids in the gas chromatograph y over time. Thi s approac h would ass um "
that the ch mical, ph ys ical, and bi olog ical tra nsformations of the bioso li ds in a non contamin ated so il are the same as in a PH -c ntaminated so il.

58

Th ugh there wa, n , ignifi ant ff ct of

Yo alu

g tati n n th degradati n rat , k, th

, timal

in Tab1 , 2.7 2. , and 2.9 w uld sugg , t that eg tated tr atm nts did in fa ct

incr a. e th

t nt f PH

bi d gradati n. This i~ reO ct d in Tab1 , 2.4, 2. 5, and 2. 6,

wh r vegetated treatm nts in the bi so lids-am nd d :oiL had signifi cantly (P:::; 0.05) less%
PH , remainin g in all fra tions at we k _2. Plant bi mass in th bi s lids-am nd d s ils
was , ignifi antl y (P:::; .05 ) greater than in the non-amend ed ~o il (Tab1 2.3); thi s would
. ugges t that th rhi zosph r enh anc ~ the bioremedi ati on of PH

co ntaminated soils.

haineau t aJ. (2000 ), f und a 6- 19% increas in the earl y stages of PH

bi odegradati on in

the rhi zo. phere of maize planL as compared to non-vegetated so ils. Thi s study observed
roughl y the sa me range of cliff renee in th % 2, 3, and total H

remainin g after th e first 8

wk in the vegetated a compared to the non-vege tated bi osolids-amended so il s (T a bJe~ 2.4,
2. 5 and 2.6). The hi gh-amendment treatments had a greater di ffe rence between th e
vegetated and non-vegetated oil · than the low-amendment treatment . In co ntrast to the
re ult found by Chaineau et al. (2000), however, thi. pattern remained throughout th
current study; this may be du e to the lack of a growth plateau imposed by the intermedi ate
shoot harvest .

Care mu st be taken, however, in interpreting the. e res ults. A stud y by Palmroth et al. (2002)
found that the removal of di esel fu el hydrocarbons under a mi xtu re of grass sp ci s (red
f s ue, smoo th meadowgrass, and perenni al ryegrass) was similar to that wit h no vegetation .
The signifi cant effect of veg tati on on th % PH

remainin g in thi s stu dy may t "due to

differ nces in the moisture co ntent betwee n the vege tated and non- cge tat --d soi ls. Despite

59

an int n 1

waterin g r gime f r the v g tated pot , the fibr u. root sys t m f the gra. s and

warm, dr c nditi n. of th gr nhou . e led t dri r . ib in the vegetated tr atm nts (-713 ~) a. c

mpared to th n n-

content at harv st ma

a!~

getat d tr atm nts (- 12-279£ ). This diff r nc in water

ha e led to a differ nee in , ample h mog nization as drier soil

. ample. , such as th . e found in th v getat d tr atmcnt~, w re

a~ i

r t mi and sub-sample.

It i. interestin g t note th at in rea~ in g th e amo unt of bio~o!ids additi on did not necessaril y
lead to an increase in th e degradation rat of PH

~. th ough thi ~ would be expec ted ba~cd on

the increased root biomas. and immediate, increased availability of mineral N for PH
degrading microbes (Tab les 2.:i and 2. 1, respectiv ly). This was es pec iall y obvious in th e
degradation rates of total H

wher the low-am ndment rat

wa~ signifi cantl y (P ~ 0.05)

greater than both the non-amendment and hi gh-amendmen t treatments, which were n t
ignificantl y different from each other (Tab le 2.9). Namkoong et al. (2002) found simil ar
re ult and ob erved that PHC degradation wa inhibited when sewage sludge amendment
rates were much hi gher than those used in this tudy. There has been so me sugges ti on that
PHC compounds may so rb or partition into the added organic matter depending on the
octanol-water partition coefficient (K0 w), thu s reducing PHC bioavailabi!ity to
microorgani sms (Haigh 1996, Rogers 1996, Al xander 2000, Vouillamoz and Milke 200 I).
Further study on the properties of the biosolids would be needed to determine the effect of
biosolids on the bi oavail ability of PH s in a so il environ ment.

60

2. 5

Conclu ion

h additi n [ rgani am ndm nL , u h as bio. li L ma hav b th direc t and indir ct
b n fit. t : il r m diati n such a. in cr a. d c ntamin ant d gradati n, r si n c ntr 1, and
unpr

d , it a , th ti ~. In thi s

p rim nt , bi . o li d~ additi on to di se l-c ntamin at d s il

. ignifi antl y incr as d th pl ant bi mass. B w k 32, eg t'll d treatm nt. in the bi os lid samend d so iL had I w r r sidu al F .... , F3, and total H

conce ntrati ons than non-veg tated

tr atm nt s.

The greates t chang in PH

I vels occ urr d duri ng the firs t

wks f th e ex perim ent after

which the degradati n slowed. Thi.', ugges ts red u d bioava il ab ility f th e remainin g PH
compound. to d grad ing so il micr organisms, but may also be a fac tor f the initi al so il
proces. in g, which in trod uced oxyg n and water to the sys tem. F2 frac ti on PH s had greater
degradation rate and lower res idual frac ti ons than F3 frac ti on PH s ver th e 32 wk period.
Thi. may refl ect a greater bioavail ability of smaller and generall y more so lu ble co mpo un ds
to microorgani sm, in the oil enviro nment. In general, the low-amendment rate of 13.34g
biosoJids kg-1 soil appears to have been the mos t successful treatment a. de term ined by th
increased PHC degradation rate and improved so il properti s when co mpared to th nonamended and hi gh-amend ment treatm nts.

61

3.

ontaminated oil

.I

tern of a Weathered Die el

Dynamic of Nitrogen in the Plant-Microbe- oil
mended with Bio olid

Introduction

The impac t f petr leum h dr

arb ns (PH . ) n hum an and e

1 gical recept r~ 1s an

en vir nm ntal c nc rn in many

untri . where th indu stri al

ntamin ati n f so il s has

hi . tori all y b n un regul ated . In additi n to to i ants, P

s may caus po r so il ph ys ical

prop rti . such as d grad d so il stru ctu re and d crease l wettab ility (M e ill et al. 198 1, Roy
et al. 1999) and pr vid an abu ndant . uppl y of n rgy f r s il rgani sms th at metabolize
PH

(M

ill et al. 198 1). Th e~e fa tors may change so il microbial and faunal p pul ati ons,

nutrient cyc ling, and nu m r us biolog ical and chemical reac ti ons (M e ill et al. 198 1, Vi sser
et al. 2003 ).

Of particular concern in PHC-contaminated so il s, are the nutri ent limitati ons created by
microbial bioma s production th ro ugh decomposition of PHC co mpound s. Th elemental
co mpo ition of PH s i. primaril y carbon ( ) and hydrogen (H), but may also c ntain small
amounts of nitrogen (N), sulfur (S), and oxygen (0 ). GeneraJJ y crud e oils co ntain
approximately 85.3 %

, 12.2% H, 3.6% 0 , 0.22o/c N, and 1.01 %

198 1). Hi gh co ncentrati ons of available

by we ight (M Gi ll el al.

in PH -co ntaminated so ils may lead to the net

immobilizati on of N as indigenous so il mi croorga ni s ms utili ze co ntam inan t

and s il

mineral N for grow th and metabolism (X u et al. 1995) .

fertili zers and

s such, in rgan1c

nutri ent-rich organic substrates such as municipal sew 'lg and farm ard manures ma be
added to so il to in cr ase N avail abilit y to bo th pl ants and so il microorganisms.

62

The value f bio
and

lid, , in particu lar, a. a

fertilizer and rgani amendm nt for legracl ed

ntaminated . oil ha: re entl r cei v cl increa. eel attenti n.

ad antag

h r ar s veral

t u: ing rganic amendments, rath r than in rganic fertilizers, in bi remediation

, trategi s. In additi on to being a readil a ailabl and renewabl r s urce, th e hi gh N
c ntent low

rati

and sl w nutri ent releas pr perti s of bio. lids (clu to net N

minerali zati on) make it a parti cul ar! us ful amendment f r the remediati on of PH ntaminat d . ils ( Pi erzy n ~ ki et al. 2 00 ). Bi ~o licl ~ can also impr ve oth r soil chemi al
and physi al prop rti , su h a~ d

reas cl bulk cl e n ~ it y, in reased p ro~ it y, and increased

water-h lding capac ity (Wong and H 199 1, Zcbarth et al. I 999, Aggelidcs and Londra
2000).

Public perception of organi c was tes is often negati ve, howeve r, often limitin g the use of
biosolid in land applicati ons (Kashmani an et al. 2000 ). Primary and seco ndary treatments
of raw ewage by means of aerobic and anaerobi c di ges tion, as well as treatm ent in hi gh pH
condition , i regulated in North Ameri ca to remove mos t or all path ogens ( SEPA 1999).
In additi on to health concern -, the presence of trace element and heavy metals (As,

d, Cr,

Cu , Hg, Ni , Pb, Se, Zn ) in this product are also of co ncern (Sims 1995, Sim. and Pi erzynski
2000). These co ntamin ant co ncentrations are often low, howev r, and it is th nutrient
loadin g, rather than the trace element loading, that usuall y dictates the agro nomic app lication
rate of biosolids ( ogger et a1. 200 I ).

The appli cati on of bi oso licls at th e agronomi c rate, or the rat th at prod uces the maximum
crop yield whil minimi zin g the excess N left in the so il , is often ca lcul ated in North

6.

Am n a a

rding l

Tal tal. 2004).

n 1ronm ntal Pr t cti n Ag n

r gulati n: (

J 999 , Bar-

auti n . h uld b appli cL h wev r wh n d t rmining th application rat s

fbi , lid, f r r m diati n ,' trateg ie. , a: PH
d c mpositi n rate and th e c clin g f

c ntamin ati on rna influenc th bi solid s

in the plant-micr be-so il sys t m ( ims 1995).

treme so i I ch mi al, ph ,· ical, and bi 1 gical pr pc rti e~ are ftcn assoc iat d with
ontaminat d sit sand littl

h a~ bee n publi. hed on

dynami cs in PH - ontaminated 'Oils.

This has made it di ffi cult t predi t the am unt f N th at will b avail ahl for
phyt r m di ati n in biosol i d~ - a me nd e d soib .

~s u c h , many studies use forage yields and

the 0'£. t tal N rec ve ry from the amendment to indicate if N was sufficient for pl ant grow th at
ariou, bi o olids rates ( ghball et al. 2002). Thi ~ is based on th e ass umpti on th at N is the
limiting nutri ent in mos t s il s and added N may b particul arl y useful in PH -contamin ated
oil .

The purpo, e of thi , greenhouse study was to in vesti gate th e influence of anaerobica ll y
dige ted . ewage sludge (biosolids) addition on the N dynami c. of the pl ant-micro be-so il
sy tern within a weathered diesel co ntaminated soil pl anted to smoo th bro me (B romus
inermis) . It was hypothe ized that bi osolids addition wo ul d increase available soil N, leading

to increased plant and microorgani sm growth and N content as compared to non-ame nded
controls. Max imum N uptake from the mineral N poo ls should corresp nd t the p riod of
maximum PHC removal and to the peri od of greates t grow th of so il microbial biomass
Detail s of the PH

degradati on in thi s 32-we k ex p riment are presen ted in

6

hapter 2.

.2

Method and Material

.2.1

oil and Bio olid

ampling and

haracteri za tion

etails of , oil and bi . lids . amplin g and charact rizati n ar pr s nl din
Bri fl , am dium-l
c urs

raJ

Yand rh

[, B .

hapt r 2.

tur d 1 am!sand loam so il c ntaminat d with di . el ru 1 over th
ars

as oll

t d n

ugust 18, 2003 fro m an indu strial si le in

pp ro imatel 300 kg [ so iI was tran.· p rted to

N

in 20

plas tic

buck ts and was parti al! air- lri d (- 5- 1 <7r gra imetri c moistur content ) on pl as tic shee ts
pri r t hom g ni zin g and sie ing thr ugh a 5 mm scree n.

mpari son of total

co ntent or

contamin at d s il ver. us co ntaminated so il that was so lvent-ex trac t d (w ith ace tone: hexanc)
d t rmin d th at co ntamin ant

cont nt in th s i1 was 0 . 357 ~ on a dry we ight bas is (data not

shown ). Further analysis f PH s in thi s so il by G -FID usin g th e
Soil - Tier 1 Meth od (

M

W

f r PH s 111

ME 2001 h), determined the F2 (nC 10-nC 16) and 3 (> n 16 1

nC34) PHC co ncentration. to be 1406 mg kg-' OD s i1 and 2086 mg kg- OD so il ,
re pecti vely. The concentration. of F 1 and F4 PHC fracti ons were n gli gibJe in thi s so il
(Chapter 2). The pH ( 1:4 . oil: deionized H 20 ) and electri cal co nducti vity ( aturated pas te
extract) of thi . . oil were 6. 68 and 1.30 dS nf

1
,

re pec ti vely.

ffec tive cati on exchang

capacity (C C) was 9.94 cmol+ kg-1 • Initi al c nce ntrations of total soil

, total soi l N, so il

amm onium (NH/-N), and so il ni trate (NO,- -N) are given in Table 3.1. Methods r r N
determinations are described below.

Anaerobi call y di ges ted sewage sludge (bi so lids) fr m the Prince George Wast water
Treatment

entre was co il ctcd on August 29, 2003 after be lt-press ing to remov c. c ss

wat r. Bi osolids wc r stored in seal d pl as ti · bu ·k ts at 4° ani 3.90 g H 2

65

g1

D

bi . lid, ra im tri m i. tur

nt nt pri r t u, in thi. study .
, and

on

ntrati n. f t tal

s1

d bio ' lid . . M th ds arc d . cribed b l w.

, t tal

H 4+_

~

-

Tab l

.1 f r

in th initi al h m g nt z d, n n-

Ta ble 3.1. el t d initial soil and am ndm nt haracterization properti s. 1.x.•
pr p rti s e pr . . d n an
T rea tment
0 i1
L Rate
H Rate

7

Total C
(g k -I)
7. 5 (0.5 ) a
9.9 ( 1.7) h
16.2 (4. _ ) c
390(_)

Total
( k -I)
0.4 (0. 0) a
0.8 (0.2) h
1.6 (0.5)c
54.4(5.1)

H/-N
(m g k g-1)
0.70 (0.62) a
87.6 ( 12.0) 11
138 (24) L
475 (150)

ppropriate

N0 3 --N
(1ng k -l)
0.04 (0. 0 5) a
0.05 (0.08) a
0.02 (0.02)a
1.00(1.74)

Bi oso lid ~'"
1
1
L = I :L"4 g 00 hiosolic.J-, kg OD '>oil. 1-1 = ~6 . 6R g 00 hio<,olid-, kg 00 -,oil

7

1

summari;ed from c hapter 2
'va lu es reprc-,cntthe mean ('>tandard deviation) of four replicate sample.., unle<.,<, otherwi<.,e Indi ca ted

" average( . landard devialion) or three replicate <.,ample..,
' diff'crentleLLer<., within a column indicate <.,Ignificant (P ~ 0 .05) dilTerenccs between amendment treatment<.,
Mann Whilne y
on pair- wi<.,c compamon<.,
ranked data

or

3.2.2

Experimental etup and Greenh ouse Condi tion

A greenhouse experiment was conducted as detailed in

hapter 2. Briefly, 3.7 L plastic pols

were filled with 2.50 kg (OD equivalent weight) of 5 mm sieved, mixed and partially airdried, diesel-contaminated oil. The experimental design consisted of 6 treatments with two
factors: vegetation [planted (P) and non-planted (NP )] and amendment at rate. of zero (0);
low (L) or 13.34 gOD biosolids kg-1 OD . oa; and high (H ) or 26.68 gOD biosolid~ kg-1
soi l. S1nooth brome (Bro mus in ermis, cv.
demonstrated PH

D

arlton) was se lected for use based on its

tolerance (Rutherford et al. 2005 ).

ach of the 6 treatments were

repli cated 4 times with 4 destructive samplin g dates planned. A total of 96 pots were placed
in the green house in a completely randomized design and wer maintained at - RO% wat ..,.r
holdin g apaci ty th rough daily waterings (water addition d termined h weight). Da
te mp rature was k pt at 25 °

with a 16 h li ght period (400 watt high pressure sodium

66

. uppl m ntallighting u. d) ; night t mperature was k pt at l 5°
kg-1

rr . p nd d t 726 mg t tal

D : il and 1452 mg t tal

Bio. lids additi n
kg 1

D s il in th I w-

and high-a m ndm nt rat , . Th se rates were es tim at d to meet (L) r e e d (H) th
ne ds of b th th plants and mi roor 0 anisms based on the initi al mineral N content f th
bio. lids and an

~timat

bios lids (RT

1999,

d first

ar

-. pA 1 99).

min ralizati n rat

f 209( o f th e t tal N add d by th e

etail ed calculati ons are pr sented in Tabl es 3.2 and

3.3. Th se alc ulations assume that net N min raliL.ation fr m the nativ so il organic matter
was negli gible c mpar d to that fr m the bio~ lids.

3.2.3

Sample Proce ing

After initial characteri zatio n, pots w re rand mly selected and de~tructivcly sampled for
chemical and biological analyses (as de cribed in

hapter 2) on dates corr sp nding to 8, 16,

24, and 32 wks. Due to the fibrous and extens ive root stru cture of the smoo th brome, r ots
could not be quantitatively removed from the soi l with ut disrupting the soi l for oth er
analyse .. The so il plu g was therefore split in half using shoot placem nt a a guide to ensure
that an even number of plant occ urred on both halve . One half of the total so il was
subsequ ently used for root biomass determination via root washing (see

hapter 2), while the

other half was homogeni zed and sub-sampled for various soil chemical and bi logical
analyses (roots were removed by hand from soil samples th at were to be used for subsequent
anal yses). Non -vegetated pots wer processed and hand! din the same fashion as vegetated
treatments to insure that abiotic losses of volatile PH s or NH 1 w "'r" simi lar betw en plant d
and non-planted treatments.

oil sampl es were pass d through a 2 mm sieve prior to most

chemical anal yses .

67

Table 3.2. Determination of biosolids application rate: available N calculations.L
kg N Mg· 1 OD bioso1ids
54.41

1. Total N in biosolids (Table 3.1)
2. Initial mineral N in biosolids (Table 3.1)

[4.76 kg N Mg-1 + 0.001 kg N M g-1]
[54.41 kg N Mg-1 - 4.76 kg N M g-1]
( 49.65 X 0.20]

4.76
0.001
4.76
49.65
9.93

[4.76 kg N Mg-1 + 9.93 kg N M g-1]

14.69

NH.t+-N
N03--N
Total mineral N
3. Organic N in biosolids
Mineralization of organic N in bioso1ids in
151 year 20% organic N (USEPA 1999)
4. Total avail N in biosolids over 1st year

=

z all concentrations expressed on an oven-dry (00) weight basis

Table 3.3. Determination of biosolids application rate: low-amendment rate calculations.'
1. Plant requirement in 1st year
1
1
80 kg N ha- y( (Zebarth et al. 2000))

=

2. Biosolids needed to meet 1st year plant
requirement
3. Microbial requirement [to achieve 25:1
contaminant C:avail N ratio
(RTDF 1999)f
4. Biosolids needed to meet microbial
requirement
5. Required biosolids application rate

0.053 kg N Mg"I ~oil

80. ke, . N. lw - I

[ 15 oOMg .~QD . soil ·lw 1 f

3.63 g biosolids kg- 1 soil

0.053. kg. N. Mg - I . soil
[
[

14.69 ·kg· N · Mg

-1

.

.

· bwsoltds
3.57. g. c. kg 1 . soil
? ')

]

1

0.14 g N kg- soil

]

-[

1

0.14·g·N·kg - ·soil

]

9.71 g biosolids kg-1 soil

0.0 1-+69 · g · N · g - I · hiosolids
[3.63 g kg- 1 + 9.71 g kg- 1]

13.34 g biosolids kg- 1 soil

all concentration<, expre-,sed on an oven-dry (OD) weight basis
) assuming a "olume of hectare furrow '-.lice= I500 m1 and a bulk density= 1.00 Mg m'
1
' contaminant C = 3570 mg kg OD o.;oil: estimated from total C in contaminated soil minus total C tn '-.Ghent-extracted contaminated soil
l

68

.2.4

oil and Plant N

tal

and

LL ing a

f oil and t tal

iso n.' 1500

(

f plant sampl . , wer det rmin d by dr

L'On and omm rs 199 ).

mbu stion

ir-dri d s il (2 mm ) an I

en-dri d

(70° ) plant mat riaL w r gr und to 100 m sh (u. in g a Brinkman , Mod el MM 2 stain! ss

. teel ball mill) prior t instrumental analysi~.
wa. perf rmed in a co mm rcial co ff

Per ent total

oars -grindin g and mi in g of plant mat rial

grinder pri r t gri ndin g in th e ball mill.

ry fr m th bi ~ lids by smo th brom was alculated ace rdin g t

re

Zebarth et al. (2000 ):
C7£ bio~olid .

wh r , A= plant

N recovery= [(A-8 )/ ] x 100

[ 3. 11

uptake f r treatment of intere~t (mg N pot 1 ), B =plant N uptake for non-

amended treatment (mg N pof\ and C =to tal N applied from bi solids (mg N pot

1
).

Available N (NH/ -N and N03--N) wa determined by ex trac tion with 0.5 M K 2 0 4 ( 1:5
oil:. olution ratio), followed by colorimetric N determination using an OI-Analytical Alpkem
Flow Sy tern IV Auto Analyzer (Voroney et al. 1993 ). Micro bial biomas: N was determined
by th chloroform fumigation-extraction m thod with 0.5 M K 2 0 4 ( 1:5 soi l:so lution rati )
(Voroney et al. 1993); colorimetric N determinati on was performed using an OI-Analytical
Alpkem Flow System IV Auto Analyzer following alkalin persulfate oxidati on to onvert all
extractable N (organic and inorga ni c) to N03--N as described by

abrera and Beare ( 1993 ).

A K r:N factor of 0.18 was used to calc ulate mi crob ial biomass N from the flush of total
ex tractabl e N foll wing fumi gation (Voron --y ct al. 1993 ).

69

ac h microbial bioma~~ N

m a. urem nt wa. p rform din duplicat (i .. 2 fumi gat d . ub. ample. and 2 n n-fumi gat d
. ub. ample. p r . il . ampl ).

3.2.5

tati tica l Analy is

tati. tical analys i ~ w a~ p rf rm d u. in g tati sti ca version 6. 1 . oft ware (2003) and r f renee
i gal ( 195 ) and

kal and Rohl f ( 1995).

ft er initi al

pl oratory data analys is, it was

d t rmined th at n n-parametri c s t a ti s ti c~ wcr rcq uir d. All significant r sults wer f unci at
th a:::; 0.05 I

el. Du t th large va riation in th e microb ial biomass N data, s t a ti ~ ti cs wcr

deemed n t prac ti all y applica bl e.

~a res ult ,

nl y the tre nds f r mi cro bi al bi omass N will

be prese nt d and eli. cuss d.

Initial . oil and bi solids charac teri zati on data and plant N data (w ithin eac h sampiing date)
were analyzed using the nonparametric Kru skai-Wallis one-way analysis of variance on rank
data (Siegal 1956). Wh ere tatistica1 (P:::; 0.05 ) . igni ficance was fo und, a Mann -Whitney U
te t (Siegal 1956) was performed to test pair-w ise co mpari sons of ranks. Stati stical analys is
on the oil N data was performed within each sampling date usin g a nonp ara metric cheirerRay-Hare two- way analys is of variance on rank data (Sokal and Rohl f 1995). Interac tio n
effects were tes ted, th ough onl y one signi ficant (P:::; 0.05) interac tio n betw en ame ndment
and vegetati on was found as noted in th res ults sec ti on. Where stati st ical (P :::; 0.05)
signifi cance was found , a Mann-Whitney U tes t ( iegal I 956) was p rform d to test pairwi se co mpari so ns of ranks.

70

.3

Re ult

_. . I

Initial oil Prop rtie and oil

Pool

dditi n fbi , lids am ndment . ignifi canll (P ~ 0.05 ) incr ased t tal
NH/-

, t tal

, and

a. ompared to th n n-am nd d tr atment at tim z ro (Tabl 3. 1). Increasin g the

bi s lid s additi n rat fr m I w t hi gh : ignifi anti (P ~ 0.05) incr ased ach f th abov
pr p rti , by 1.5-2 tim s.

itrat -

treatments. It was n t d in

hapt r 2 th at bi solids additi on signifi cantl y i ncr ased th pH

c nce ntrati ons were not significantl y differ nt betw en

from 6.68 in the n n-amended tr atm nt to 7.09 and 7.74 in th e low and high amendm nt
treatm nt, r sp cti v ly.

lec trical

nducti it y was als signi ficantl y increased from 1.30

d m-1 in th n n-amend d to 1.77 and 2.3 1 d m 1 in the low and hi gh amendment
treatment ( hapter 2).

3.3 .2

Plant N

Total N uptake by smoo th bro me sh ots and roots was signifi cantl y (P ~ 0.05 ) increased by
bi o olids addition a co mpared to the non-amended treatment ; shoo t uptake was
approximately 2-3 fo ld greater than root N uptake (Table 3.4). The hi gh-amendm nt rate
significantly (P ~ 0.05 ) increa. eel total N uptake as compared to the low-amendment nte.
Similarl y, the high-amendment rate significantl y (P ~ 0.05 ) increa. ed the plan t total

uptake

from the biosolids as co mpared to the low-amendm =- nt rate (Table 3.4 ). Tt is importan t to
note th at sh ot N in th e non-amended treatment was meas ured to be near zero at 16 wks .
hi s r suJt is m , t likely du to the poo r growth of the pl ants in th co ntaminat d soiL which
led to ex tr mely low biomass weights. The low w ight of the plant bi omass compar d to the
weight of the dryin g v sse! make thi s res ult suspe ' l. Ma imum % total N re over from th "

71

Table 3.4. Plant N uptake and % total N recovered by smooth brome from the biosolids amendment at 8, 16, 24, and 32 wks. '-y.w

Time
(Vv ks)
8

Treatment

0
L
H

16

0
L
H

24

0
L
H

32

0
L
H

Smooth brome total N UJ2take
Shoots
Roots
T otal J21ant
( mg N par ')
0.8 (0.3) a
1.9 (0.3) a
2.7 (0.5) a
199 (49)h
146 (32) b
53.4 (20. 1) b
131 (87) c
255 (9) c
386 (85) c

Smooth brome N
uptake from
biosolids' ~
Total ~! a nt N
1
(mgNJ2of )

N recove ry by
smoo th brome
from biosolidsu
(%)

197 ( 49) a
384 (85) h

J0.8 (2.7) a
I 0.6 (2.3) a

395 (40) \
489 (92) \

21.7 (2.2) ..
13 .5 (2.5) ..

0.03 (0.04) a
287 (22) b
45 I (99) c

BDL
108 (3) X
I 18 (56)'

0.03 (0.04) \
395 (40) \
489(92) '

2.3(1.5) a
3 19 (4)b
535(47) c

1.9 (0.5) a
136 (27)b
306 (59) c

4. 1 (2.0)'1
455 (27) h
84 1 (98)c

451 (27) a
837 (98) h

24.8(1 .5) a
13
- -. I ( ?
_.7) a

3.3 (0.9) a
34 1 (40) b
577(42) c

3.2 ( 1.3)a
145 (49)h
299(42)(

6.5 ( I. I ) a
486 (30) h
875(40)(

480 (30) a
869 (40) h

?6
_ .4 ( I .7) a
23 .9(1.J) b

'0 = Lero bio-,olids addition. L = 13.34 g 00 bio-,olids kg·' 00 soil, H = 26.68 g 00 biosolids k.g 00 soil
} values repre'>ent the mean (standard deviation) of four replicate samp les unless otherwtse indicated
x average (<.,tandard deviation) of two replicate <.,amples- statis ti cs not determined
~different letter<., within a sampling time and column indicate significant (P :S 0.05) dil'ferences between amendment treatments- Mann Whitney U on pair-wise
compan'>ons of ranked data
estimated from the difference between total plant N in amended treatments versus total plant N in the non-amended treatment
u calculated U'>mg equatJOn (3.1 ): <..,ee text in Methods and Materials (3.2.4)
BOL- heJov.., detection limit

72

bio. lid. wa. at 2 wk. ; significant! (P ~ 0.05 ) J w r C7£ total
th high-amendm nt tr atm nt (24%) a.

recov ry was b. erv d in

mpar d t th low-am ndm nt treatm nt (26 )

(Tabl :).4) . P rcent t tal N r cov ry wa. n t signifi cantly diH rent betw

n the low- and

hi gh-amendm nt tr atm nt: prior t 32 wks.

3.3.

M inera l N

. umm ary f th e~ il

H/-N ,

1 -N ,

and total mineral N in th tr atments for eac h

samplin g dat is pres nted in Tab! 3. 5. In general, there wa~ a larg

d ec rea~e in total

mineral N fr m time 0 to ti m 8 wks in the bi ~ Jid ~-am e nc.l e d trea tme nt~ ( T a bl e~ 3. J and
3.5); m st of thi s d crease was attribut d to redu cti on of NH 4 +-N conce ntrati ons. A
, ignifica nt (P ~ 0.05 ) int racti on eff ct bet ween the vegetati n and amendment treatm ents
wa. found for NH/- N at 8 wks. Vegetated trea tm e n t~ had a greater initi al decrease and had
significantly (P ~ 0.05 ) less total mineral N and NH 4 +-N as co mpared to n n-vegetated pots
after week 16. T otal min raJ N and NH/ -N in th e biosolids-amended treatments remained
relatively co nstant after 8 wks. Nitrate in the bi osolids-am nded pots increased between 0
and 8 wks and remained , ignifi cantly (P ~ 0.05) greater in the non-v getatedtreatments as
con1pared to the vegetated treatments fo r the remainder of the 32 wk peri od.

Non-amended treatments also showed a sli ght reducti on in total mineral N between 0 and 8
wks (Tables 3. 1 and 3.5); these levels remained relati vely co nstant thro ughou t mos t of the
e periment and increased onl y sli ghtl y at wee k 32. Vegetati n di d not have an appar nt
effect on th e mineral N in non-amended so ils until 32 wks, where

ge tat d treatm "'nts had

signifi cantl y (P ~ 0.05) l e~~ N H/ -N and N ~ -N as 'O m pared to non-vcgetat "d tr ~atnPnts.

73

Table 3.5. Soil N0 3--N, NH/-N, total mineral N, and total N at 8, 16, 24, 32 wks.L.y.x
Time
(\\kS)
....

Minera l N

Vegetation
Treatment
NP
P
NP

Oa
Lb

p

Hb

p
plant effect
NP

0

Lb

P
NP

Hb

P
24

plant effect
NP

Oa

p

32

1

NP
P
NP
P
plant effect
NP
P
NP
p
NP
p
plant effect

Oa
Lb

Hab

S
3

p
NP

0.02(0.01)
0.0 I (0.0 I)
-+5.4 ( 16.1)
1.01(1.39)
41.6(11.8)
0.13(0.21)

Lb
H ao

0.01 (0.02)
0.03 (0 .02)
48.8 ( 17 .6)
0.06 (0.03)
50.5 ( 16.7)
0.37 (0.66)
NS
0.10(0.07)
0.06(0.03)
84.0(22.9)
0.28 (0.24)
54 .2(15.1)
0. 10 (0.08)

0

La
Hb

Oa

La
Ha

S

Oa
La
Ha

2.84(1.64)
0. 13 (0.20)
76.5 (27.5)
0.40 (0.28)
40.5 (9.8)
0.53 (0.30)
S

OJ

0.23(0.1 1)
0.23 (0.13)
1.80 (0.88)
1.14(0.3 1)
5.69( 1.90)
0.24(0.15)

Lb

Hh

NS
3

0

La
Ha

1

00 soil)
0.25(0. 10)
0.25 (0.12)
47.2 ( 16.9)
2.15(1.39)
47 .3(10. 1)
0.37(0.20)

Oa
Lb

He

NS

0.18(().14)
0.21 (0.25)
0.90 (0.72)
0.10 (0.08)
6. 13 (3.39)
0.38 (0.22)
S
0.3 1 (0.09)
0.2-+(0. 16)
1.33(0.72)
0.34 (0.27)
6.49(2.90)
0. 13 (0.09)

0"

L '10
Ho

0. 19(0.1-+)
0.24 (0.26)
49.7 (I R.3)
0. 16 (O.OR)
56.6 ( 16.2)
0.76 (0.79)

OJ

Lb
H

l

S
Oa

L '1
Ha

S
3

Total N

To tal M ineral
(mg N kg

NP

16

NH.1+-N

N03--N

0.41(0. 11)
0.30(0. 15)
85.3(23 .6)
0.62 (0.28)
60.7(17.5)
0.23 (0.13)

Oa

Lb

0'

0.73(0. 19)
0.5 I (0.12)
1.22 (0.S:3)
0.37 (0.18)
-+ .74 (3.82)
0.57 (0.33)
S

La
HJ

3.57(1 .68)
0.64 (0.32)
77.7 (27.9)
0.77 (0.42)
45 .3 ( 12.5)
1. 11 (0.59)
S

1.08xl0 ~( 152)
744 (I 17)

Hh

1.78xl0~(540)

806 (I 58)

S
1

527(12-+)
427 (I 0)
847 (69)
921(75)
1.34xl0~(359)
984(95)
NS
471 (27)
450 (23)
905 ( 174)
796 (60)
1.49x I 0 ~ ( 195)
1
1.05x 10 H04)
NS
404(10)
-+16(31)

NS
Oa

Lo
Hc

454(64)
45 I (86)
873 (93)
752 (79)
l.l6x I 0 3 ( 125)
957 (250)
NS

NP =non-planted. P =planted. 0 = ;ero hio..,olid.., addition. L = 13 .34 gOD bioso l ids 1-.g 1 00 <>oil. H = 26.68 g 00 hiosolids kg 1 00 -;oil

- -..alueo., repreo.,ent the mean (standard devtallon) or four replicate samples
'different letter<, wtthtn a <,ampling time and column indicate significant (P $ 0.05) differences between amendment treatments in the ranked data of the column
to the tmmedtate nght- Mann Whitney U on pair-wise comparisons of ranked data
"'o.,ignificant ( P $ 0.05) Interaction between plant and amendment for N H/-N
S- o.,igntllcant at P $0.05 (Kru'>kai-Wallto., on ranked data). NS - not -;ignillcant

74

. .4

Total

T tal , il

r mam d r lati

ly

n, tant o er th

2 wk , tudy p ri d ( ab l

am nded treatments had significant! (P $ 0.05 ) gr ater t tal
tr atm nts.

nerall

in cr a, d th t tal s il

.5). Bi , olids-

as c mpar d to n n-amend ed

incr a~ i ng th rate f bios lid, additi n signifi cantl y (P $ 0.05 )
I thou h th e t tal soil

did n t sh w a significant v g tati on

effe t, the general trend wa~ f r the v gelated treat me nt ~ t hav I wer total so il N
ncentrati n: th an n n-vcb tated tr atm nts.

3.3. 5

oil Microbial Biomass

Th re. ults of mi ro bi al biomass N are shown in Figure 3.1.

!though the res ults were

hi ghl y vari able (a. , hown by err r b a r~ in Fi gure 3.1 ) and n further stati sti ca l treatment was
deemed appr priate, th e ge neral trend was for bi osol ids-amend cl so il s t have hi gher mean
valu e, th an non-amended so il ; thi i , particularl y apparent at 8 wk~. Max imum mi cr bi al N
occurred at 8 wk followed by a gradu al dec!in ov r tim . Initi ally, non-vege tated
treatment had greater microbial biomas N than vegetated treatment , at 8 wks, though thi s
trend was reversed in the bio, olids-amended treatments by w ek 16. By 32 wks, little
apparent difference occ urred between treatments.

75

WA NP-Zero

140

-

~ N P - Low

-·aen 120
- Ol
.Y

P-Low
~ NP-High

100

c=J P-High

z
Ol

E

P-Zero

80

+

I

+

~

z

en
en
Cil
E

60

0

(6

co
.0
0\....

S:?

40
20

2

0
0

16

8

24

32

T1me (weeks)

Figure 3. 1. Micr bi al bioma ' S N. Valu es are reported as the mean of 4 replicate . ampl es
wh re err r bars represent the ~tandard deviation. V a lu e~ of zero indi cate that a negati ve
m an valu wa btained.

3.4

Discussion

3.4.1

Soil and Plant N

Nitrogen is an essential nutrient for pl ant and . oil microorganism growth.

Jth ough plants

can utilize NH/ -N, N0 3- -N is the primary form of N taken up by pl ant roots du to
inc rea ed mobility in the soil environmen t (S teven:on 1986 ). Because soil micr

rgamsm~

are primaril y responsible for the mineralization of organic Nand nitrifi ation of

H/-

into

plant available No ~ - - N, it is thought that th ey are bett r competitors for avai labl N than
plants in the soil environment (Jackson et al. 1989, Kaye and Hart 1997, Hodge et al 2000) .
Thi s may lead to reduced plant grow th in aN limited environmen t, such as that oh:erved in
th non-amended treatment o f thi s stud y. Th addition of biosolids, high in available

H-1 t -

Nand potentiall y minera li zable N, may hav r du

d the 'o mp tition in this e. perim nth

incr asing th plant-available N in the so il.

Is or minera l N in the vegetated

ow le

76

tr atm nt thr ugh ut th , tud indi at d that min ral

wa. b in g u, d by the plant as fast

a, it wa. b ing min rali z d (Tabl _ .5). The . ignifi cantl (P $ 0.05) gr ater plant uptake of
in th bi . lid -amended treatm nts a.
indicat . that a aiJ able

mpared to the n n-am ndment tr atment

met both th r quirement: f PH -degrading micr

that of plant: growing in th ese am nded so il s (Tabl

rgani sms and

.4).

itrog n r covery b f rage gn sses fr m bi so lid. -amendment in non-co nt amin at d field
so iL typi call y ranges fr m 1 4-4 0 ~ in th lit ratur , depending on soil pro perti es, fi ld
conditions, and the typ of bi osolids used (Warman 1986, Kiemn ec et al. 1987, ulli van et al.
J 997,

ogger et al. 1999, mith and Tibbett 2004). Little is publi shed on th e additi on of

bio olid. N to PH -contamin ated so il ·, th ough sli ghtl y lower valu s of J 5-23 % amendment
recovery have been reported for anaerobicall y di ge. ted sewage sludge. used in gree nh ouse
experiment in non-contamin ated . oils (Kin g 198 1, Amund so n and Jarrell 1983 ). The ~24
and 26% N recoveri e. ex hibited by smoo th brome in th e low- and hi gh-amendment
treatment , respec ti vely, of this study are typical of non-co ntamin ated so ils reported in the
literature and would suggest that the N suppli ed by the bi osolids, along with the oth r so il
improvements, were sufficient to overcome any detrimental eff cts of the PH
contaminati on on the pl ants.

Increasin g the rate of biosolids additi on signi ficantl y (P $ 0.05) increased th e plant uptake of
N from the bi osolids, th ough its did not lead to an increase in ~ total N recovery b the
pl ants from th e amendment (Table 3.4). Th

/'o N r cov ry by the plants from the hi gh-

amendment rate was either th sam as, or signifi 'antl y ( P $ 0.05) less than th ., low-

77

am ndm nt rat at ea h , ampling dat . It i, 1mp rtant t n t that th 1 w-amendmenl
tr atm nt g n rall had th , am

r gr ater

I

-

and t tal min raJ

c n entrati ons a,

ompar d t th hi gh-amendm nt treatment in the n n-v g tat d so il s (TabJ 3.5).
Inc n, i, tent

reco

r fr m , iJ 'lm ndm nts i ,· n t unc mm n in th literature. Palazzo

( 1977), obs rved hi gh r N rec vcries fr m low r appJi ati on rat s f was tewat r

am ndm nts as ompared t hi gh r applicati n rat s in agri cultural s il s. Thi s tr nd was also
ob erved in a fi eld stud y by Barb arick et al. ( I
accumul ati on, and gr ater

6) th at observed less

ss, greater so il N

ge lati on r mova l of N from an agri cultural s iJ amended at 6.7

Mg bi os lid · ha 1 as compared t 26. Mg bi solids ha-1 • As di sc ussed by Ki cmnec l a!.
( 19 7), thi s effec t may b th re, ult of increased vo latilizati on of NH 1 , due to excess N

pre, ent in the oil , or du e t r du e d min erali zati on relati ve to the increased rate of bioso lids
additi on. The low conce ntrations of so il NH/-N and N0 3--N in the vegetated treatments
(Table 3.5) and greater pl ant bioma s in biosolid, -amended treatments of thi s experiment
(Chapter 2) indi cate that the plant uptake of mineral N may ha e been limited by th e rate of
biosolids-derived N mineralization and not the effi ciency of the pl ants in ass imil ating th e
ava il able N (Jans-Hammermeister et a!. 1994).

3.4.2

Estimated N Requirements

The low-amendment treatment in thi s stud y w ~L calcul ated in part to meet a 25:1
(contaminant

:so il N) ratio as reco mmended in the literatur (RTD

1999). The amount of

ntamin anl C was determined from the differenc betwee n the total C in the PH co ntamin ated so il (nati ve so il

+co ntamin ant

) and the total

in the same sc il Collowing

oxhlct ex tracti on with a 50:50 h xane:ace tone solve nt (nati e soi l

78

onl y, assuming

n gligibl

lra ti n f humu.

am unt f

n d d to achi ve ptimal

) ( hapter 2).

di, cu .. ed in Jut au et al. (2003), the

:N ratios f r th bi remediati n f PH -

ntaminat d soiL i. ft n ov r stimal d fr m t tal c ntaminant

s me PH

value, du e to th e fact that

mp n nts are quit r cal itrant and may n t be present in forms that are

bi a ailabl t p t ntiall degrad ing rgani. ms.

Adding v g tati n t th s ~ t m
the n ar-zero l ve ls f mineral
3.5).

mg

mpensated for p tcnti al cxc ss N addition as indicated by
remaining in the v getated treatments at 32 wk~ (Tabl e

er th e 32 wk per iod, the ~moo th brome t

k up - J 95 mg N kg 1 0

s il and - 350

kg-1 OD s il for the 1 w- and high-amendment tr atmen ts, respec tive ly; thi s was well

above the 50 mg N kg-1 OD so il stimat d to be r quired in the first year (Tab le 3.3). Thi s
may b du e to the warm and moist condition~ kept in the greenhouse, as opposed th e field
itu ati on upon which the estimated N req uirements were based on (Zebarth et al. 2000), or
may be the re ult of the multiple shoot harve ·ts imposed on the plants that kept them in an
ac tively growing vegetati ve state.

3.4.3

M inera l N

In addition to increasi ng the net immobilization of inorganic N in th soi l, it has been
sugges ted th at PH

compound s may decrease th e nitrification of NH ~+ to N0 1 . In a study

by Xu et al. ( J 995 ), the nitrifi cati on rates in seve ral soil s with varying oil contents (0-60 mg
kg-1 OD so il) , were decreas din the presence of hydr carbons, suggesting that nitrifying
mi croo rga nisms were s nsitive to PH

co mpounds. Th increas d concentrations of N

79

1

-N

in lh bio. lid, -am nd d n n-

g tat d treatm nts b tw

indicat an activ nitrifi r population

le~pite th

n 0 and 8 wk. of this study

eli , 1 ontaminati n (Table 3.5).

There ar s veral p ssible reaso ns f r th lack of a n gati v PH

effect n th

,--N level s

in thi . e p riment. In th study by Xu t al. ( 1995 ), nitrificati on rates were lowes t in the
I a. t c ntaminated, but solv nt e tracted soil (< 5 mg kg

nitrifyin g micr

rga ni~nb may ha ve been more se n ~ iti v

rath r than th total PH

1

D soil) . It was concluded that

to th e r siduaJ oil and solv nt ,

once ntrati n in the ~o il. Allh ough the current . tudy used a eli sci-

contaminated so il with a higher initi al total H

~o il c

nt amin ant c ncentrati n (-3 500 mg

kg-' OD soil ), it c ntained n res idu al so lve nts and minim al h avy petroleum co mpounds
( hapter 2).

L o, as eli, cussed by Williams et al. ( 1999), the p tenti al for xiclati on of

ammonia to nitrate would be hi gh unci r th e aerobic and near n utral so il pH co nditi ons
occurring in this experiment.

It i. aL o important to note th at the biosoJids were a so urce hi gh in immedi ately ava il able
NH/-N (Table 3. 1). Ammonium is the preferred so urce of in organi c N by soil

microorgani sms (Stevenson 1986, Broo k et al. 199 1) and has been shown to increase nitrifi r
growth in non-contamin ated soils at co nce ntrati ons gr ater th an 50 mg kg-1 OD so il (Yang et
al. 2004). The Jack of a large increase in the NO,--N co ne ntrati ons of the non-ame nded
treatments would indicate th at PH s might limit th e avail ability o r NH/-N to nitrif ing
rgani sms throu gh net N immobilizati on, rath r than by being to ic. lt is also recognized
that the total N content of th non-am nd ed so il was quite low (0.04% ), therefore litt le n "'I
minerali zati on would be c pect d from thi s so il.

80

.4.4

oil Microbial Bioma

It i. difficult t

m

t

an pr

N

nclu , i n. ab ut th mi r bi al bi rna s N in this study;

thi. i. du t th unr liab1 data btained fr m the mi r bial bi ma., measurements. Th
flu sh f

tractabl

following fumi gati on was s metim s very , mall , or negativ ,

mpared t n n-fumi ga t d c nt ro l ~ ( igur 3. 1), sp cially in non-veg tat d tr atments.
Th large ariati n in the data was Iike!y du e to a c mbi nati on f fact rs in luding l) th
difficult in h m ge ni zing the small am unt of bioso lids thr ugh ut the soil , leadin g to "h t"
and "co ld" , p ts f mi robial bi ma:s; and 2) the 1arg diff rene

in ~

il moisture co ntent

betwe n the veg tat d and n n-v ge tat d treatments th at may have led to ineffi cient
fumi gation ( parlin g and Wes t 1989). Des pit multipl e dail y waterings, the vegetated
treatments were drier th an the non-vegetated treatments; thi s was due to the hi gh water
requirement of th e ac ti vely grow in g smoo th bro me.

There does, however, appear to be an increase in microbi al biomass N from time 0 to 8 wks,
fo ll owed by a gradu al decline (Figure 3. 1); thi s is more pronounced in th e biosolids-a mended
treatments. Thi s flu sh co incides with the large decrease of so il mineral N (Table 3.5), and
the large decrease (40 - 6 3~) of total H in the bio olids-amended treatments from 0 to 8 wks
(Chapter 2). In a stud y by Chaineau et al. (2000), it was observed that the addi tion of 3300
mg PH

kg-1 to an elu viated brown so il (Mollie eutrochrept ) res ulted in an increase in

microbi al bi omass.

hough the so il used in thi s study co ntained weathered PH s. th-. initial

so il processing may have increased the bioavail ability of PH

compoun ds pre iousl

inaccess ible to soil mi croorgani sms, res ultin g in an initial net immob il ization of inorgani N
by the mi crobi al biomass. It is also import ant to nc te that the appli ·ation of biosolids, which

81

hav be n f und t c ntain larg microbial populati n. and pr vide a r adily availabl
. urc , may hav , timul ated micr bial a tivit and led to th initial incr ase in micr bial
bioma..

(Banerj e

t al. J 997 ,

!though th mtcr bia1 bi mass
valu es btain d ( L to 63 mg

amk

ng t al. 2002).

valu , were quit vari abl in thi s experiment , th e ran ge f
kg 1 s il ) was simil ar t th ose reported by th rs f r PH

contaminated . oib . F r e ampl , Xu and John son ( J 997) f und mi crobi al biomass N in an
ag d, il -c ntamin ated (55 g PH
mg

kg-1) Bl ack

h rn zemi c so il t ran g between 2 1 and 32

kg-1 • oil when seeded to barl ey. Mi cr bial bi o m a~~ N in the non-e ntamin ated control

ranged between 4 and 13 mg N kg-1 soi l. In co mp ari ~o n , Banerjee et al. ( J 997) observed
1

micr bi a1 bi ma. s N valu es rangin g between 59 an d 93 mg N kg and 70 and 395 mg N kg
in a non-co ntamin ated Lake land clay loam s il amended with 50 and I 00 Mg ha 1
anaerobicall y di gested ewage sludge, respec ti vely. Microbi al bi omass N in the non1

amended control soil ranged between 17 and 3 11 mg N kg- •

3.5

Conclusion

The relati onship between th e ac ti ve biodegrading m icrobi al popul ation, v g ration, and
available nutrients is of part ic ul ar co ncern in contamin ated soib as competition bc tw en
plants and mi croorganisms for the same nutri ent resources may lead to red uced contaminant
degradati on and pl ant growth ( adowsky and Turco 1999). The add itio n or biosolids to th
PH -co ntamin ated so il , whi ch had a low initi al c nc ntration of min ra l N, 1"d to increased
pl ant bi omass. Thi s was the result of increased total N uptak fro m the hioso lids h smooth
bromc shoo ts and roo ts as co mpared to th e non-ame nded treatment. M icrohial biomass N

1

aL

111

r a, d initially, foll wed b a gradual d clin

tr atm nts t nded t ha e gr ater mi r bial bi rna ..

itrification of

H4 -

v r lim . Bi

th an non-amended treatm nts.

wa~ un affec ted by PH

,-

co ntamination and in reased in

bi s lid. -amended treatm nL' with hi gh initial on ntrati o n ~ of
nc ntrati n [

1 i~

f on

olid. -amend d

H4 +.

he incr as d

rn be ause in crea~ in g th bi s lids addition rate did not

1 ad to an in reas d % t tal N r c

er by the plants fr m th e am ndment. Thi s may be

imp rtant in soi ls hi hl y ~ u sceptib l to

vo latili zati n and leachin g and should be

con, idered when d termining the rat of bi so lid ~ add iti on in phytoremediation strategies.

8.

4.

ynthe i of Re earch Findin g

, di , cu, ,·ed in

hapter l , th bi d gr'ldati n f PH s i. great] y affec t d by th pr p rti s f

the h dr ca rb n: pre. ent , th

nvir omental c nditi ns in the

ntamin ant t d grading organisms.

il , and th availability of th

s such, th successful applicati n f a micr bial-

ba. ed r medi ati n strateg to a co ntamin at d s il requires an understanding f the
interacti ns between the

ntaminant, so il microorganisms, and . urr undin g nvironment

( adowsk and Turc 1999). In a ph yt rem di ati n strat gy, th e interac tion of plants with
th abov co mponents must als b c n. idered.

4.1

Rate and Extent of PHC Degradation

The bioavailability of aged or w athered PH s is parti cul ar]y important to th e success f
bioremediati on trategie becau. e res idu al co mp o und ~ may redu ce the rate and overall xtent
of contaminant degradati on. Modify in g th e firs t order decay equ ati on to:
C t = oe

-kt

[2.2 ]

+ Yo

where the additi onal parameter Yo is an es timate of the res idu al fracti on already pre. en t in the
soil (Noce ntini et al. 2000), improved the estimation of the degradatio n rat by a. suming
only a porti on of the total co ntamin ant co ncentration was ava il able to , oi I microorganism~
for degradati on over the study peri od. Generall y the res idual frac ti on of PHCs res ul ting from
bioremedi ation efforts is es tim ated to be b twe n 20-30CJO ( haineau et al. ... 000).
r sidual frac ti n of 26% was found for the total H

frac ti on (

ME fracti ns F2 + F3)

remaining at the end of thi s stud y, suggesting th at the modified fi rs t order de ' a
(i.e. Model 2) co uld be applied to stimat the rat

84

n overall

quation

or biod gradation in field situation: .

urth r t ting f thi. m d 1 und r var in g envir nm ntal and , oil conditi n, will b tt r
a -,, its r 1 vance.

ll i. also inter stin g t n te th at the fit R 2 , f th m difi d first -ord er d cay equation n th e

t tal H

data was qually as effec ti eat e plaining th e variation in the data as fittin g th

mod I to th F2 and

fractions s parately ( hapter 2). This may be particularly important

in studi s that are interested nly in the

rall d grce f contamination .

how ver, has begun to focus on th e variabl e pr p rties and t xiciti s of PH

urr nt research,
mi turcs nee

intr du ced int the soil nv1ronm nt (Hatzinger and Alexander l 995, oehr and Webster
1996, Al xander 2000). The

M fracti nation of PH s, based on th strai ght -chain

hydrocarbon b ilin g poi nt ranges, reflects thi s need for a greater unci rstandin g of th e effects
of PH

contaminants on human and environmental recept rs (

M 2000 ). Th ab ility t

e. timate the degradati on rate. of different PHC fractions is therefore r levant and may lead to
more efficient bioremediation strategies.

As discu sed in Chapter 2 vegetation in the biosolids-amended treatments significan tl y (P ~
0.05) reduced the % of initial F2 and 3 fraction hydrocarbons remaining as compar d to the
non -vegetated treatments. The deer ased residual fraction in vegetated treatments wa. most
likely due to an in creased PH

degrad ing '>o il microb ial biomas. res ulting from the

"rhizospherc effect" indu ced by an increased root biomass in the biosolids-amended
treatm nls. [n addition to increas in g the so il mi cro bial activity in the rhi?osph re, th
ex t nsivc root sys tem in the bioso lid s-amcndccl so il s ma ha . . in ' reased the bioavailabilit
of the PH s through fa cilitat d d sorpti on or ph ysica l so il disturbance (Ale . ander ... 000,

85

Hutch in. n tal. 2001 ).
bi ma.-., thu. n eff

n-am nd d treatm nL had 1 w mineral N and little plant
f v g tati on n th

t

PH

r maining wa. found ( hapter 2).

Th lack of an vera!I direc t effe t fbi s lid . additi on on th s1z of the res idual PH
fracti nat 32 wks (Tabl s 2.7-2.9) i: co nsist nt with other studi es that have bserved
re. idu al P I fracti ns res i ~ t ant to funh r tr atm cnt ffec ts aim d at increas ing the
d gradation rate (Pin lli et al. 1999, N ce ntini et al. 2000, Ri vera- spin oza and Dendooven
2004 ). Alth ugh bi ~ lids additi n did not decreas the r ~ idu a l PHC fra li on, it is

imp rtant t not th at all trcatm nts res ulted in F2 and 3 hydrocarbon co ntamin ant levels
b l ow

M PH

Tier l

:oil and 1700 mg 3 kg-1

anada Wide tandards (

M 200 l c) of 760 mg F2 kg-1 OD

D s il req uired f r c mmerc ial/industri al coarse-grained surface

, oils (Table 4. 1). In additi on, the 2 frac tion, with the excepti n of th non-vegetated, hi ghamendment rate, also fe ll below the mos t stringe nt res identi al/parkland land use standard of
450 mg F2 kg-1 OD soil (Table 4.2).

Table 4.1. Tier 1 levels of C M
Coarse-grain ed
(median > 75 f:Lm )

Surface
Sub-s urface

F raction 1
(nC6-nC10)

130-330
350-700

PHC CW frac tio ns fo r soi ls.'·Y·"'
F raction 2
(> nC10-nC 16)

F r·action 3
(> nC16-nC34)

( mg kg-1 OD soil )
450-760
400- 1700
1500-2000
2500-3500

Frac ti on 4
(> nC34-n 50)

2800-3300
10000

's ummari 1cd from C M E 200 1c
Y va lues represe nt the ceo soi l contac t ex pos ure pathwa
w range in va lue represents the land usc where : rc~iuc n tia l / p ar kl and <agricultural< indu . . trial/commcrc Jal

86

Table 4.2. PH
1
20 6 mg kg
Treatment

P-0
P-0
P-L
P-L
P-H
P-H
'
1

nc ntrati n at 2 wk, .z Ini tial , oil 2 and
. oil, r , pe li v 1y.

nc ntrati ons: 1406 and

F2 fraction
F3 fraction
(m k - )
13 1 (1 3)
654 (37)
123(3 1)
63 (11 )
l 02 ( 17)
873 ( 173)
7 ~ (3)
606 (46 )
223 70)
1143( 11 8)
~ (7)
792 ( 8)

aluc<., rcprc'>e nt the mea n (<., tandanJ dn 1a1ion ) or· four re pl icate <., ampl e<.,
1
P = non-planted, P = planted. 0 = ; cro hio<.,o li d'> add it ion . L = 13. ~4 g 00 hio'>o li d'> kg 00 '>o il .
1
H = 26.6R g 00 bi mo lich kg OD oil

4.2

N utrie nt Cycling a nd A m endment in C onta min a ted Soil s

Th r lati onship betwee n the ac ti ve bi odegradin g micr bi al popul ati on, vege tati on, and
avail abl nutri ents is of parti cul ar co ncern in c ntamin ated so il s as co mpetiti on betwee n
plant and microorgani sms for th e sa me nutrient r so urc s may 1 ad to reduced contamin ant
degradation and pl ant growth (Sadowsky and Turco l 999). Nitroge n is oft en the Iimitin g
nutrient in PH

contamin ated , oiL du e to net immobili zati on during de omp siti on of PH

compound by , oil microorganisms (Xu et al. 1995). As such, the additi on of N to so iIs with
high

:N ratios has bee n hown to increase th e biodegradati on rate of PHCs (Lin and

Mendelssohn 1998, Brook et al. 200 l , Hutchinson et al. 2001 , Namkoo ng et al. 2002) .

The res ults of thi s study are consistent with the literatur . In Ch apter 2, it was obser eel that
bi osolicls additi on ge nerall y increased the first-order degradati on rate co nstan t of PH . in the
so il as co mpared t the non-amended treatments. Thi s increased degradation rat

W<.L' mo.' t

likely du e to th increased lev Is o f r aclil y ava il ab1 min ra l N in the bio., olids -am -- nded
soil s. As di scussed in

hapte r 3, min raJ so il N le

Is in bioso lids-amend --d treatment~ had

the greates t drop betwe n 0 and 8 wks, during which the greates t decrease in PII

87

on

ntrati n.

curr d ( hapt r 2). Th ugh it mu t be acknowledg d that part f th

mm ral N w uld b tak n up b the veg tati on during thi. peri d, a large p rti n would have
b n immobiliz d by , oil micr
flu . h in micr bial

rgani . ms during gr wth n contaminant carbon ( ). Th

fr m - 13 mg

kg-' so il to betwe n -27 and 63 mg N kg 1 s il in the

vari us tr atments durin g this peri d w uld ~ upp o rt thi . s nari .

It i. intere ting t n t , how ver th at n n-amended treatments, b th vegetated and n nveg tat d had fin al res idu al fra ti ns similar t those of th e bi osolids-am nded treatments.
Th

3 fra ti n, and the f_ non-veg tated, hi gh-amendm nt treatment , had larger res idual

frac tion th an those of th e non-amend ed treatments. In addit ion, increasing th e biosol ids
addition rate from 726 mg total N kg-' OD so il (25: l C:N) to 1452 mg total N kg-1 OD so il
(1 2.5: 1 : ) did not ignifica ntl y increase the PHC degradati on rate.

or total H , th e first

order degradati on rate constant was ob. erved to be signi ficantl y (P $ 0.05 ) low r in th e non1

vegetated , hi gh-amendment treatment (0.142 wk- ) as co mpared to the non-vegetated, lowamendment treatment (0.243 wk-1 ) over 32 wks. These res ults may have occ urred for several
reasons.

irst, the - 0.35% contaminant co ne ntration prese nt in the so il at the onset of the

pcnm nt

may not have been hi gh enough to demon"ltrate the benefit s of bioso lids-ame ndment on PH
degradati on. Nitroge n minerali zation of the nati v so il organic matter, wh ich ma have be "n
in r as din th warm, moist so il co nditions of the greenhouse, co ul d have been sufficient to
stimul ate the bi odegrad ati on of PH

co mrounds in th e non-amended soils of this

ex periment. Many of th papers cit ed 10 thi ~ stud have found significant effects of N

88

fertili zati n n PH

d gradati n at , oil onlaminant cone ntration, b twe n 1 and 6%

( madi et al. 1993 , Xu and John , on 1 97 , Br wn et al. 1998 , Namk ong et al. 2002).

ls , as dis u, s d in

hapt r 2, PH

d grad ati on may be inhibit d by se wag slud ge

am ndm nt du e to s rpti n or partiti nin g of th PH

co mpound s int the organi fracti n

( amkoo ng t al. 2002).

rganic compound s found in the bi osolids, whi ch are diffi cull to

differenti ate from th PH

ontamin ants in the so il , may also hav in reased the apparent

res idu al fra ti n due t i nterfer nee in the gas chr matograph y analys is (Rogers 1996 ).
urther stud on the degradati on f PH s and rga ni c matter substrates in bi osolids would
aid in determinin g the influence of this so il amendment in bi odegradati on applicati ons.

Smoo th brome es tablishment did not increase the ex tent of PH

d gradati on; the

ignificantl y (P ~ 0.05 ) greater pl ant bi omass in the bi oso lid:-amended so ils did not
signifi cantl y degrade more PH s th an the non-vegetated, non-amended so ils. Vegetati on
wa , however, an important fac tor within the bi osolids-amended so iL by increasing the
ex tent of PHC degradati on and decreasin g the concentrati on of bi osolids-deri ved mineral N
remaining in the so il at the end of the 32 wk ex periment. The pos iti ve effec t of planh on the
retenti on of add ed N is espec iall y important in so il s hi ghl y susceptibl toN olatili zation and
leachin g.

Jn g neral, the low-a mendment rate or 13.34 g OD bioso lids kg 1 OD soi l was the most
success ful treatment as determined by the increased PH

deg radation rate, improved soi l

properti es, and increased pl ant growth when co mpared to th .,. non-ame nded and high -

89

am ndm nt treatm nL .

h ugh th

f PH

d gradati on wa. not significantl y aff cted

by th bi ,' lid. additi n, th increa. drat of d gradati n and fa. t r . tabll. hment of
g tati n would b ec n mi all ad antageo us in a fi ld situ ati n wher gr wings asons
ar . h rt r and tim 1 r med iati on . trategies are preferred.

90

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