a comparison of treatments for anthracene uslng … · i gratefully acknowledge enzo barresi, pat...
TRANSCRIPT
A COMPARISON OF TREATMENTS FOR ANTHRACENE USlNG TVVO
BENCH-SCALE TREATABILITY TEST METHODS
A Thesis
Presented to
The Faculty of Graduate Studies
of
The Unkersity of Guelph
by
KEVlN R. HOSLER
In partial fuifillment of requirements
for the degree of
Master of Science
January, 1999
O Kevin Robert Hosler, 1999
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ABSTRACT
A COMPARISON OF TREATMENTS FOR ANTHRACENE USlNG TWO
BENCH-SCALE TREATABILITY TEST M t H O D S
Kevin R. Hosler University of Guelph
Advisor: Professor R. P. Voroney
Two bench-scale treatability methods were investigated for assessing the
degradation of anthracene - a three-ringed polycyclic aromatic hydrocarbon (PAH)
frequently discovered in soils contaminated with petroleum hydrocarbons, creosote, mal
tar, and other hazardous wastes. Solid-phase and slurry-phase test methods were used
to evaluate six treatments and a control, chosen from previously published
bioremediation research. The treatments included DARAMEND" amendment, IgepalB
and TweenQ surfactants, ORCO and Fenton's oxidation, and bioaugrnentation. The use
of radio-labelled anthracene, spiked into a previously uncontaminated soil, allowed for
detailed monitoring of anthracene mineralization over a 91-day study period. Five
treatments (DARAMEND " , Fenton's, bioaugmentation, Igepal@, and O RC@)
mineralized significantly more anthracene than did the CO ntrols for the solid-phase
method whereas only three treatments (bioaugmentation, DARAMENDN and IgepaiO)
did so for the slurry-phase method. The solid-phase rnethod was superior to the slurry-
phase method regarding the recovered amount of radio-labelled anthracene, and
especially the amount that completely rnineralized.
ACKNOWLEDGEMENTS
Numerous individuals have played a part in the performance of this research and 1
would be remiss for failing to acknowledge them.
I gratefully acknowledge Enzo Barresi, Pat Failetta, Eric Vale, and Ayman Youssef
for their tutelage and support in analyod and radio-chemistry and other technicd portions
of the research.
Dr. Ken Mullen (retired professor; Department of Mathematics and Sta?istics,
University of Guelph) providec! valuable technical support for the statistical anaiysis of the
data, which was financially supported by the Environmental Science and Technology
Alliance Canada (ESTAC).
The management at the Wastewater Technology Centre W C ) allowed me the
opportunity to pursue part-time graduate studies while continuing rny employment, and
Environment Canada funded the rnajority of this research, for which 1 am very thankful.
I am especially grateful for the enthusiasm, advice, and academic guidance and
support provided to me by the members of my supervisory cornmittee: Mr. Rob Booth,
Dr. Jack Trevors, Dr. Pieter Groenevelt, and most especially the chair - Dr. Paul
Voroney.
Finally, 1 wish to thank my parents for their constant love, encouragement, and
understanding.
TABLE OF CONTENTS
............................................. ABSTRACT ........... ....................................................... I
.................... .................. ACKNOWLEDGEMENTS ..................................................... I
TABLE OF CONTENTS ................................................................................................. Il
LIST OF TABLES ......................................................................................................... N
LIST OF FIGURES ................... .................................................................................. V
. CHAPTER 1 INTRODUCTION ..................................................................................... 1
........................................................................ 1.1 BACKGROUND .......................... 1 1.2 STATEMENT OF THE PROBLEM .................... ... ......................................m. 3 1.3 OBJECTIVES AND APPROACH OF THE RESEARCH ...................................... 4
CHAPTER 2 . LITERATURE REVIEW ............................................................................ 7
2.1 SITE REMEDIATION TECHNOLOGIES .................... .............................. 7 2.1.1 Bioremediation Technolo~ies ........................................................................ 8 . 2.1.2 PhysicaVChemical Technologies ............................................................. 10
.......................... .........m.................................... 2.2 TREATABILIN TESTING ... 12 2.2.1 Definitions and Theory ............................................................................... 12 2.2.2 Methods and Guidance ............................................................................... 14
2.3 SOL TREATMENT TECHNOLOGY APPARATUS ............ .. .......................... 16 2.4 PAHS AS ENVIRONMENTAL CONTAMINANTS ......................... ...............m.... 17
2.4.1 Anthracene ................................................................................................ 18 2.5 SELECTION OF TREATMENTS FOR STUDY ................... ... ...........m.....9.... 19
CHAPTER 3 . METHODS AND MATERIALS .......... ....................... ....................m..... 26
......................................... 3.1 EXPERIMENTAL DESIGN ....................................... 27 ............................................................. ................... 3.2 SAMPLING DESIGN ... 28
3.3 TREATMENT DESIGN ...................................................................................... 29 3.3.1 Solid-Phase Treatments .......................................................................... 29 3.3.2 Slurry-Phase Treatments ............................................................................ 30
3.4 EXPERIMENTAL SET-UP ................................................................................. 31 3.4.1 Soil Properties ........................................................................................... 31 3.4.2 Solid-phase apparatus ................................................................................ 33 3.4.3 Slurry-phase apparatus ............................................................................ 35 3.4.4 Soil Spiking with Radio-Labelled and Unlabelled Anthracene ..................... 36
................... .............*................................. 3.5 SAMPLING AND MONITORING .. 38 ................................................................................................ 3.5.1 Solid-Phase 38 ............................................................................................... 3.5.2 Slurry-Phase 39
...................................................................... 3.6 ANALmlCAL.. ......................... 39 3.7 STATISTICAL DATA ANALYSlS .....................~................................................ 40
CHAPTER 4 . RESULTS AND DISCUSSION ........m.m..................m............................. 41
4.1 SOLID-PHASE STUDY . CO^ ............ ......... ...................~........................ 41 4.2 SLURRY-PHASE STUDY - ' ' ~ ~ O z ................ ................m......................... 47
............................. 4.3 SOLID-PHASE SOL 14c RECOVERY ................... .... 53 4.4 SLURRY-PHASE SOIL 14c RECOVERY.. ............... ................................... 54 4.5 SLURRY-PHASE LIQUID 14c RECOVERY ............. ........................... 56 4.6 SOLIDIPHASE TOTAL 14c RECOVERY.. ..................m.................... 57
....................................................... 4.7 SLURRY-PHASE TOTAL 14C RECOVERY 58
CHAPTER 5 . CONCLUSIONS AND RECOMMENDATIONS ................................. 59
5.1 SIGNIFICANCE CF THE RESEARCH ............................................................... 61 5.2 RECOMMENDATIONS FOR FUTURE RESEARCH ......................................... 62
REFERENCES ............................................................................................................. 64
APPENDM A . ANNOTATED BIBLIOGRAPHY OF SITE REMEDlATlON TECHNOLOGY OVERVIEW PUBLICATIONS ....................................................... 74
APPENDM B O PROPERTIES OF ANTHRACENE ................................................. 77
............... APPENDÏX C . TUKEYS HONESTLY SIGNIFICANT DIFFERENCE TEST 78
APPENDIX D O GLOSSARY ................. ... ................................................................ 79
APPENDM E = FENTON'S OXlDATlON REACTIONS ..................... .. .................. 86
APPENDIX F = PAH DEGRADATION MECHANISMS ................... ................ . 89
APPENDIX G = CHEMICAL PROPERTIES OF THE TESTED SURFACTANTS ......... 89
APPENDM H O STANIER'S LlQUlD MINERAL SALTS MEDIA INGREDIENTS ......... 90
APPENDfX I O SOIL CHARACTERIZATION; DETAlLED RESULTS .......................... 91
APPENDIX J -TREATABILlTY TESTING REGULATORY GUIDANCE (U.S.) ............ 93
APFENDIX K O DATA ..................... ... .................................................................... 96
iii
LIST OF TABLES
Table 1 . Soil Textual Analysis Data for Duplicata Samples (%) .......................... 32 Table 2 . Soil Carbon Analysis Results for Triplicate Samples (%) ...................... 32 Table 3 . Soil Nutrient Analysis ResuRs for Triplicate Samples (mglkg) .............. 32 Table 4 . Soil Metal Analysis Resutts for Triplicate Samples (rnmg) ................... 33 Table 5 . Soil CEC and pH Analysis Data for Triplicate Samples ..... .... ................. 33 Table 6 . Initial Amounts of Radio-labelled and Unlabelied Anthracene .............. 37 Table 7 . Cumulative ' 4 ~ ~ ~ Z Recovered (?6 of Added) from Solid-phase
Treatments; Triplicate Averages ......................................................... 41 Table 8 . Cumulative 14c-Co2 ~ecovered (% of Added) from Slurry-phase
Treatrnents; 1 riplicate Averages ........................................................ 48 Table 9 . 'h Recovered (96 of Added) from Solid-phase Treatrnent Soils; . . Triplicate Averageô ................................................................................ 53 Table 10 . 14c Recovered (96 of Added) from Slurryphase Treatrnent Soils;
Triplicate Averages ................................................................................ 55 Table 1 1 . Total 14C Recoveries from Solid-phase Treatments; Triplicate Averages .
...... ...........................m... ............................................................ 57 Table 12 . Total 14c Recoveries from Slurry-phase Treatments; Triplicate
Averages ............................................................................................... 58 Table 13 . Chemical and Physical Properües of Anthracene ........ ...... .......... ...... 77 Table 14 . Soil Textural Analysis Data for Duplicate Sarnples (%) .... ...................... 91 Table 15 . Soil Carbon Anaiysis Resufts for Triplicate Samples (%) ...................... 91 Table 16 . Soil Nutrient Analysis Resufts for Triplicate Samples (ppm) ................. 91 Table 17 . Soil Metal Analysis Resuîts for Triplicate Samples (mglkg) ................... 91 Table 18 . Initial Arnounts of Radio-tabelled and Unlabelled Anthracene .............. 92 Table 19 . Elements of the Approach to Performing Site Remediation .................... 94 Table 20 . Solid-P hase Method; Summary Table of Recovered '% Radio-label (% of
Added) - Raw Data . ......................... ... ............................................ 97 Table 21 . SolidPhase Method; Summary Table of Recovered 14C Radio-label (% of
Added) - Edited Data ...................... ..... ................................................ 98 Table 22 . Slurry-Phase Method; Summary Table of Recovered '% Radio-label (%
of Added) - Raw Data ..................... ..... ........ .............................. 99 .... Table 23 . Slurry-P hase Method; Summary Table of Recovered 1 4 C Radio-label (%
of Added) - Ediied Data ................................................................... 100 Table 24 . Solid-Phase Data and Tukey's Honestly Significant Difference Test .. 101 Table 25 . Slurry-Phase Data and Tukey's Honestly Significant Difference Test . 102
LIST OF FIGURES
Figure 1. Figure 2.
Figure 3.
Figure 4. Figure 5. Figure 6. Figure 7. Figure 8.
lwchart for the BR1 Biotreatability Protocol. ................... ..-................. 15 hematic of the Biometer@ Flask used in the Solid-Phase lethodology. ...................... ..............~............................................. 34 hematic of the Flask Apparatus used in the Slurry-Phase lethodology. ................... ...............~.....mm...~............m.m......m.~~.......... 35 lid-phase Study - Cumulative Recovery of 14c-C02 (96 of Added) ...... 45 lid-phase Study - Periodic Recovery of CO^ (% of Added). .......... 46 irry-phase Study - Cumulative Recovery of 1 4 ~ C ~ 2 (% of Added). .. 51 rry-phase Study - Peiiodic Recovery of 14c-C02 (% of Added). ....... 52 rematic of the Role of Treatability Studies in the RVFS and RD/RA rocesses. ...mm.mm.m~.....*.. .......*m.... -...m.. . . ..*....~.........*.... . . . . Q 5
CHAPTER 1. INTRODUCTION
1.1 BACKGROUND
Since the 1970 '~~ when ecologists and environmentai activists heraided warnings
that the world's ecosystems were at nsk due to contamination from anthropogenic
sources, hundreds of thousands of contaminated sites have been identified or
inventoried worldwide (e.g., U.S. Environmentai Protection Agency [US. EPA], 1997).
An entirely new discipline of environmental science and engineering - known as site
remediation - has developed to address issues related to assessing areas of
contamination, researching and developing clean-up technologies, implementing these
technologies, and reducing or removing environmental risks.
Over 325 innovative treatment technologies for site remediation - 70% of which
are commercially available - are listed in the U.S. EPA's VIS177 pendor Information
System for hnovative Treatment Technologies] computer database (U.S. EPA, 1995). A
more recent and comprehensive computer database - the REMTEB (i.e., Remediation
Technologies) database cornpiled by Water Technology International Corporation (WTI,
1996) - lists over 500 remediation technologies. Comrnonly, these technologies are
classified as physicai, chemical, or biological, according to the nature of the treatment
processes that are dominant in that specific technology. Often, some combination of
these technology types is required to completely remediate contaminated environrnents
or environmental matrices (Toronto Harbour Commissioners, 1 993).
Biologically-mediated remediation - or biorernediation - technologies ernploy
cost-effective, environmentally-friendly techniques for decontaminating various matrices
(Le., soils, sediments, air, and groundwater) in both in situ and ex situ technology
applications (Sims, Sims and Matthews, 1989; WASTECH, 1995). Bioremediation has
been variously defined as:
a. "a managed, dernonçtrated active treatment process that uses microorganisms
to degrade and transfomi organic chernicats in wntaminated soil, sludges and
residues" (Loehr, 1991);
b. "a managed or spontaneous process in which biological, especiaily
microbiologiml, mtalysis acts on pollutant compounds, thereby remedying or
eliminating environmental contamination" (Madsen, 1991); or,
c. ''the use of organisms to irnprove environmental quaMy by taking advantage of
their ability to treat toxic, hazardous or rnereiy offensive wmpounds at
contaminated sites" (Interdisciplinary Bioremediation Working Group, 1991).
Numerous physical and chernicd remediation technologies have also been
developed, and occasionaily are combined as physico-chernical technologies. These
technology types rnay also be applied in conjunction with bioremediation technologies - often configured as sequential treatment processes or treatment trains. For instance,
the initial oxidation of certain chemicai bonds in specific contaminants that resist
microbial attack may permit subsequent biodegradation of the remaining contaminant
molecules (Martens and Frankenberger, 1 994).
An annotated bibliography of sorne important oveiview publications describing
established and innovative site remediation technologies appears in appendix A. This
bibliography permits the reader to find more detailed information about specific site
remediation technologies and their applications.
Polycyclic (or polynuclear) aromatic hydrocarbons (PAHs), are unsaturated cyclic
hydrocarbons containing two or more fused benzene rings. Many PAHs are toxic,
carcinogenic and/or rnutagenic, and have thus been characterized as environmental
pollutants or contaminants-of-concem (International Agency for Research on Cancer,
1973). PAHs occur in significant quantities in creosote, coal tar, and petroleum
hydrocarbons - substances that are practicaily ubiquitous in modem society. The
2
remediaion or treatment of PAH-contaminated environments has received much
attention by the scientific community since the early 1980's (e.g., Sims and Overcash,
1983; Bulman et al., 1985; Pollard et al., 1994).
1.2 STATEMENT OF THE PROBLEM
The selection, design, and irnplementation of appropnate site remediation
technologies should include treatabilrty testing as a prelirninary step (US. EPA, 1989;
l992b). Treatability testing typically involves several tiers of increasingly complex test
procedures, culminating in appropriate treatment methods for use in full-scale remedial
actions or site clean-ups. Bench-scaie testing to screen potential treatments is the
recommended first step in the overail process, followed by more detailed bench- to full-
scale remedy selection tests. Unlike the EPA in the United States, Environment Canada
has not provided regulatory guidance for treatability testing in support of site remediation
(Environment Canada - personai communication, 1998). Thus, if testing is perforrned at
al1 in Canada, commercial technology vendors or developers will frequently perform only
a single bench-scale treatability - or more accurately a feasibility test - to support
subsequent full-scale technology applications (Hosler & Booth, 1995). The importance
of performing sufficient testing to support remediation technology implementation has
however been frequently substantiated, and is thus highly recommended (Dupont, 1991 ;
McFarland, Sims and Blackburn, 1991). Testing that is performed with well-designed,
scientifically-defensible methodologies will likely yield the most useful results - ailowing
more effective evaluations and/or cornparisons of technology performance.
Due to the high cost of working with large-volume, specially-engineered or
custom-fabricated apparatus, technology research and treatability testing are often
performed using bench-scale microcosms or srnall-scale testing apparatus (Gillett,
1989). Further, the choice of apparatus and method of treatability testing directly affects
3
the type of data collected and it's relevance to a scaled-up application of that particular
technology (Dupont, 1991). It is therefore imperative for the remediation technology
researcher to carefully choose the bench-scale methodology and to attempt to simulate
as many of the biological, chernid and physical processes that may ultimately occur in
a scaled-up treatment apparatus.
A review of the published literature covering up to 1995 reveaied that relatively
few authors had sufficiently described their treatability testing apparatus and overall
methodologies, or had performed cornparisons or evaluations of different testing
methods. Further, there were fewer than ten published protocols or guidance
documents for aiding technology researchen and developers to follow appropriate
treatability testing approaches (Hosler and Booth, 1 995).
A wide variety of treatments have been described in the literature for rernediating
polycyclic aromatic hydrocarbon contarninants in various environmentai matrices
however the methods used to screen or select these treatments are not welI
documented (Hosler and Booth, 1 995).
The problem for the researcher or remediation technology developer becomes
how to select or design appropriate apparatus and useful test methodologies for
developing or testing potentiril treatments applicable for one or more site contamination
scenarios.
1.3 OBJECTIVES AND APPROACH OF THE RESEARCH
There were two main goals for this research. One goal was to compare and
evaluata two distinct bench-scaie soi1 treatability testing methodologies, referred to as
either the solid-phase, or the sluny-phase methodology. A second goal was to screen
several specific treatments for degrading anthracene - a three ringed polycyclic aromatic
hydrocarbon in soil.
4
The evaluation of the two testing methodologies involved assessing the amounts
of the target contaminant that were completely degraded (i.e., mineralized) in each of the
solid-phase versus slurry-phase experiments. Contaminant degradation would be
indirectly quantified by capturing and measuring radio-labelleci carbon dioxide evolved
from the experimental units in each experiment. Further, the ability to close a
contaminant mass balance by accounting for as much of the total radio-labelled
contaminant initially spikad into the experimental units (or incubation flasks) - upon
cornpletion of the study - was an important facet of the research. Use of radio-labellad
contaminant analogue chernicals in environmental research - including treatability
studies - has aIlowed researchers to monitor the fate or environmental partitioning of the
contaminant, and to more accurately model fate mechanisms such as degradation,
sorption, volatilization, and recalcitrance.
The solid-phase incubation method em ployed static incubation Biometer@ flasks,
and was intended to simulate or represent ex-situ soi1 treatrnent technologies (e.g., land-
treatment unls, bioreactors, biopiles). The slurry-phase incubation method used custom-
built flasks and a common shaker platform apparatus and was intended to simulate single
batch-mode processes that mig ht occur in commercial bioslurry reactors (e-g., ECOVA
Bioslurry Reactor - U.S. EPA, 1992% EIMCO BioliftTM Reactor - U.S. EPA, 1993). Very
few head-to-head cornparisons of solid-phase versus slurry-phase remediation
technologies exist in the literature, aithough authon of single-method studies often attempt
to compare their resuts with those achieved by othen under different conditions (e.g.,
Ross, 1991).
The six bioremediation or combined physico-chernical-plus-bioremediation
treatments tested were:
1) bioaugmentation (i.e., with microbiai inoculum);
2) biostimulation with a physical amendment (Le., DARAMEND");
5
3) Fenton's (reagent) oxidation;
4) rnagnesium peroxide oxidation (Le., oxygen release compound or ORC@);
5) the commercial surfactant IgepaI@ CA-720; and,
6) the commercial surfactant Tween@ 80.
These treatments were selected based on their previous successful use in
treatability work as described in the literature and on their potential for future success in
full-scaie site remediation.
The research was intended to further develop the treatability testing capabilities
at the Wastewater Technology Centre (Environment Canada, Burlington, ON) and to
corroborate or expand-upon related work published by Bulman, Lesage, Fowlie and
Webber (1 985), Bulman, Hosler, Fowlie, Lesage and Camilleri (1 988), Aziz and Melcer
(1991), Hosler, Bulman and Booth (1992), Cotton and Aziz (1993), Aziz, Andersen and
Melcer (1 994), and Hosler and Booth (1 995).
CHAPTER 2. LITERATURE REVIEW
2.1 SITE REMEDIATION TECHNOLOGIES
As stated in section 1.1, technologies that presently exist for decontaminating
environmental matrices found at hazardous-waste-contaminated sites are commonly
classified as physical, chemical, or triological technologies, according to the nature of the
dominant treatment processes. Combinations of many differing technology classes may
be applied in treatment trains, in which sequential remediation proœsses are used. For
example, contaminated soils or sediments may be treated first by size separation (i.e., a
physical treatment process), followed by metal chelation / removal (a chemical treatment
process). and lastly by a biologicai treatment such as a bioslurry reactor to degrade
organic contarninants (e.g., Toronto Harbour Commissioners, 1 989).
Numerous different biological treatment technologies have been researched
andlor implemented for full-scale site remediation worldwide during the past five years
and the U.S. EPA's A'ITIC', CLU-IN" and ORD 66s3 databases represent a significant
source of project listings. Severai international symposia are presently devoted to the
subject of bioremediation technologies, which have likely gained popularity because of
their reputed mst-effectiveness and the public's acceptance of them as being natural or
environmentally-friendly.
The combination of physical and/or chemical treatment processes with biological
processes has resulted in some unique and potentially useful treatment technologies - which are described following a detailed discussion of bioremediation processes.
1 Aiternative Treatment Technologies Information Center ( A m C) . 2 Cleanup Information Bulletin (CLU-IN). 3 Office of Research and Development (ORD) Bulletin Board Service (BBS).
2.1.1 Bioremediation T 'no log ies
Bioremediation normaily involves either biostimulation, or bioaugmentation
processes - but in some cases rnay involve both.
Biostimulation generally refen to the enhancement or stimulation of the growth
and metabolism of microorganisms that are native to the contarninated matrix or site.
These indigenous microbiai populations are often capable of producing contaminant-
degrading enzymes, if provided with required metabolic substrates, under certain
environmental conditions. For aerobic degradation of contaminants, facilitated by
heterotrophic bacteria, oxygen is required as the terminal electron acceptor in cellular
metabolism and the bacteria use the carbon-based contaminant molecules as growth
substrate (i.e., food). Biostimulation can often be facilitated by one or more of the
following strategies (Hosler and Booth, 1995):
0
i i)
iii)
iv)
VI
adding nutrients required for microbial growth and function;
adding CO-metabolites (i.e., substances which are not primary growth
substrates but which are CO-metabolized with the contaminants);
adding altemate electron acceptors for cellular respiration, or increasing the
amounts of prirnary electron acceptors (e.g., adding oxygen by aerating a
soil);
improving physical factors (e.g., matrix temperature, moisture conditions, soi1
porosity), that enhance the growth and function of the contaminant-degrading
microbes; and,
improving contaminant availability (e.g., by using a surfactant to enhance
contaminant solubilization).
Occasionally, the removal or reduction in certai'n restrictions to microbial growth
andfor function rnay be viewed as an indirect method of biostimulation. Examples would
include the removal of toxic metAs, or a reduction in contaminant concentrations beiov~
some threshold Ievel.
A product known as DARAMEND" is an example of a speciaiized soi1
amendment that has been shown to facilitate the biodegradation of recalcitrant organic
soi1 contaminants such as polycyclic aromatic hydrocarbons (PAHs) and chlorinated
phenols (e.g . , pentachlorophenol, or PCP) . This commercial amendment is incorporated
into contaminated soils and facilitates the growth of contaminant-degrading
rnicroorganisms (Seech, Marvan and Trevors, 1 994).
Bioaugmentation generally refers to addition of speciaiized contaminant-
degrading microbial populations to matrices where these microorganisms do not
presently exist, or do not exist in sufficient quantities to significantly degrade
contaminants. Rittman et al. (1992) defined bioaugrnentation as the introduction of
selected bacteria containing genes that encode for enzymes that degrade xenobiotic
contaminants. Two possible mechanisms for augmented contaminant degradation were
suggested. In the first, bacteria containing the necessary gene pool for producing
enzymes capable of degrading the contaminant are added to the contaminated matrix.
This method generally requires a sufficient amount of readily biodegradable
contamination such that growth of the added bacteria (inoculum) is supported. The
second mechanism involves transfer of the genes responsible for controlling the creation
of degradative enzymes, from the added bacteria to the indigenous or native bacterial
population. This transfer usually takes place through conjugation of bacterial cells; a
process that involves the transfer and subsequent replication of DNA-containing
plasmids from the donor bacterial ceIl to the receptor bacterial cell, after direct cell-to-cell
contact. Conjugation must occur rapidly, so that should the introduced bacteria die off,
the introduced gene pool will have become viable within the indigenous population and
the necessary degradative enzymes wiIl be sufficientiy produced.
9
Other researchers have included the addition of indigenous bacterial populations
in their definition of bioaugmentation, however the distinction is made that enrichment
culturing techniques are used to increase the quantities of the indigenous
microorganisms prior to their re-inoculation into the contarninated soi1 on-site. For our
purposes, bioaugrnentation will include augmentation with exogenous populations,
indigenous populations, or both. While being less popular than biostirnulation
techniques4, bioaugmentation has nonetheless proven effective in certain specific site
remediation applications (e.g., pp. 493-551, Volume 4, Battelle Press, 1997).
2.1.2 Physical/Chemical Technologies
Whereas biological treatment technologies exploit the ability of certain
microorganisms to degrade hazardous organic contaminants, physicai/chemical
treatments exploit the physical characteristics of the contaminated matrix (or matrices),
andjor the chemicai properties of the contaminant(s).
The American Academy of Engineers identifies the most common physicai
treatment technologies for soils as:
1) thermal desorption; and,
2) thermal destruction (WASTECH / AAEE, 1 995).
Other physical treatment processes include:
3) separation, including screening, filtration, and/or sedirnentation.
Chemical treatment technologies generally encompass the "use of reagents to
destroy or chemically modify target contaminants by means other than pyrolysis or
combustion." (WASTECH / AAEE, 1995). Examples may be classified as:
4 There is a continuing debate in the scientific cornmunity about the utility of bioaugmentation
strategies, especially regarding economic and technicai aspects (i.e., costs and overall effectiveness)(see Battelle Press, 1997).
1) substitution processes (e.g., dechlorination of polychlorinated biphenyls
[PCBsl);
2) chemicai oxidation processes;
3) chemicai precipitation processes.
Examples of combined physicai-chernical technology processes include:
1) soi1 washing / soi1 flushing;
2) stabilization / solidification;
3) ultra-violet / hydrogen peroxide oxidation; and
4) vacuum vapour extraction (WASTECH / AAEE, 1995)'.
Fenton's Reagent (Le., hydrogen peroxide and ~ e ~ ' catalyst which is added as
ferrous suifate) and other chemical oxidizing agents (e-g., hydrogen peroxide,
magnesium peroxide) have proven successfui for remediating certain chemical
contaminants in the past (e.g., Watts et al., 1990). A patented / comrnercialized product
called ORC@ (i.e., Qxygen Blease Ompound; REGENSIS Bioremediation Products,
San Juan Capistrano, CA), is made from magnesium peroxide which supplies
supplemental oxygen as an electron acceptor for microbiai respiration. It has apparently
been used successfully in several remediation applications (Koenigsberg et ai., 1995).
The use of chemical surfactants (i.e., surface-active agents) for solubilizing
contaminants bound to soi1 particles, thus rnaking them available for subsequent
bioremediation, has been researched or used for numerous cleai-up projects, including
in situ subsurface remediations (West and Harwell, 1992). Hundreds of commercial
surfactants presently exist, and research has shown the potential of using surfactants in
the remediation of specific contaminants (Ellis et al., 1985; Laha et al., 1990; Abdul et
- - - - - - - - - - - - - - -
5 For more detailed descriptions of each of these technologies, the reader is referred to the 8-
volume series of books entitled lnnovative Site Remediation Technology published by WASTECH
and the American Academy of Environmentai Engineers (WASTECH / AAEE, 1995).
al., 1990; Liu et al., 1990). There is generally a lack of reliable data correlating the
physico-cherniml properties of commercial surfactants with their ability to desorb or
solu bilize specific soi1 contarninants (Selvakumar et al., 1 995).
2.2.1 De finitions and Theory
Treatability testing is an important wmponent of m y prudent site remediation
prograrn. While it is generally acknowledged that treatability testing to validate vendor
technologies and verify site-specific applicability is a scientifically rational procedure, there
often appears to be limits to the levels of testing that vendon think should be performed
(Le., size, scope, and costs of the treatability testing prograrn).
There is usually a distinction made between feasibillity and treatability studies or
testing, although the definitions used are occasionally researcher-specific. The US. €PA
defines a feasibility study (FS) as:
"The analytical part of the RIIFS [remedial investigation/feasi bility study]
process, the FS serves as the mechanism for development, screening, and
detailed evaluation of potentially applicable treatment technologies. The success
of the FS is highly dependent on the data generated in the RI [remedial
investigation]. "
A treatability study is defined as:
'The testing of a remedial aItemative6 in the laboratory or field to obtain
data necessary for a detailed evaluation of its feasibility." (U.S. EPA report 540/2-
89/058, 1989).
6 Alternative is likewise defined as ''A potentially applicabie remedial treatment technology or treatment train. Alternatives are developed and screened during scoping of the RIIFS and throughout the RIIFS process. Alternatives are investigated by performing treatability studies and selected as remedies after a detailed analysis of each alternative is conducted."
In a Canadian context, without the existence of regulatory definitions, the
distinction between feasibility and treatability has become somewhat obscure, and
generally seems to focus on the Ievel-of-effort involved. Feasibility studies can refer to
simplified testing (i.e., at laboratory scale), to determine whether or not a particular
technology approach is likely to be successful when tested with adequate controls. One
research group designed a "Fiowchart for Evaluation of Bioremediation Feasibility"; the
word "feasibility" was used to refer to the entire process of assessing whether specific
contamination was " bioremediable", including the performance of treatability studies
(Major and Cox; Beak Consultants, 1992).
The term treatability shrdies has also been variously used, atthough generally it
refers to the studies performed at either bench or pilot-scale for assessing the affect that
different treatment technologies, or variations of one or more technologies, have on the
degree of bioremediation for a particular contarninated matrix. Treatability studies often
involve an element of process optimization (e.g., optimal levels of nutrient addition), to
provide data for deterrnining contaminant degradation kinetics under specific test
conditions.
In generai, feasibility studies are commonly performed to:
asses the feasibility of a remedy (i.e., the effects that the treatrnent
technologies have on the fate of selected target chemicais or chernical
wastestreams within specific matrices such as soil, groundwater, air, etc.).
Conversely, treabbility studies are commonly performed to:
provide performance criteria (i.e., fundamental data on the specific
conditions and the time required to remediate contaminated matrices to
pre-determined treatment goals or criteria, for example, a determination of
contaminant degradation haif-lives) ;
assess risk to human heaith and environmental protection that may occur
as a result of both the presence of the contarninants and the remedial
action (e.g., possible formation of toxic metabolic intermediaries during
remediation) ;
provide basic process scale-up information or criteria, and,
provide basic cost information for the remedy (but not likely accurate cost-
estimates for scaled-up remediation scenarios).
2.2.2 Methods and Guidance
The federal govemment (Le., Environment Canada) and other agencies have
provided guidance on the requirements, procedures, and/or recommendations for bench-
scale feasibility / treatability testing supporting technology use (Samson, Greer and
Hawari, 1992; Hoster and Booth, 1995). The Biotechnology Research lnstitute (BRI;
National Research Council of Canada) in Montreal published a Demonstration of a New
Biotreatabi1.W Protocol to Monitor a Bioprocess for the Treatrnent of Contaminated Soils
(Samson et al., 1992). This publication introduces the results of the first application of a
recommended biotreatability protocol that was used for remediation of a petrdeum-
contaminated site. A flowchart representation of the protocol - adapted from Samson et
al. (1 992) - appears as Figure 1.
Phvsical analvsis to determine whether the structure of the soi1 permits the development of acceptable biological activity,
4 4 1 1 - 1
Chemical analvsis to determine whether environmental conditions are favourable to biodegradation and ensure there are no pollutants present which could cause inhibition,
- 1 . 1 1 4 . 5
Gene probinq to verify whether the microorganisms present in the soi! have the genetic potential to biodegrade pollutants,
J- l i l i
. Microcosm Studies usina ''~-labelled pollutants to verify whether the pollutants are in fact degraded by the microorganisms,
1 s . J J . s .
. Respirometry to assess the biological activity of the soi1 microorganisms in relation to a control soil, and,
i ls. l i
Bioassays to determine whether the biotreatment has succeeded in detoxifying the soi1 and verrfy that toxicity has not been transferred to groundwater.
Figure 1. Flowchart for the BR1 Biotreatability Protocol.
The BR1 protocol provides specific examples of bench-scale laboratory studies or
activities that should be used to support full-scale applications of bioremediation. The
protocol publication (referenced earlier) deais with petroleum contamination, however, the
protocol can be adapted for other types of biodegradable organic contamination. Creation
of this protocol was based upon techniques developed or modified by BR1 personnel.
Many of the techniques have been extensively published; cost information is available
upon request from the BRI.
15
Many govemment funding programs and departrnentç, boM provincial and federal
(e.g., DESRT', GA SR^^, P.E.I. MO^ have historicaily evaluaded proposais for
bioremediation projects on an ad hoc basis. While the managers of these funding
programs norrnally ensured mat a technical review process was completed prior to
selection of a suitable technology vendor, the criteria for selecting vendors was often
detenined by the vendors' commercial capabilities or limitations, and not by a minimum
technid quaiity standard. Imposition of such a standard for performing bioremediation
research and applications muld easily have been introduced if it had been shown to be
cost effective, involve reasonable demands on technology vendors, and resuit in improved
performance of bioremediation treatability support studies and follow-up operations.
Dupont (1 991) emphasized the performance of bench-scale treatability studies.
even if highly successful in achieving contaminant remediation, should not in any way
eliminate the inherent requirement for pilot-scale studies pnor to full-scale technology
implementation. Pilot-scale work provides process parameten for full-smle remediation
that cannot possibly be learned from bench-scale work atone. In fact, many researchers
believe that bench-scale studies should often be forgone in favour of pilot-scale work that
more closely reflects the uttirnate full-scale remediation process (Hosler and Booth, 1995).
2.3 SOlL TREATMENT TECHNOLOGY APPARATUS
Hundreds of technology apparatus or treatment systems have been developed
for decontaminating soils, for both in situ and ex situ applications. For ex situ
7 Development and Dernonstration of Site Rernediation Technology Prograrn; Environment
Canada. e Groundwater and Soil Remediation Program; Environment Canada. 9 Province of Prince Edward Island Ministry of the Environment.
treatments, soils can generally be processed in either slurry-phase or solid-phase.
Examples of solid-phase, ex situ remediation systems include:
enhanced land treatment - referred to as landfarming in the petroleum
industry (API, 1983);
biopiles or composting-type systems; and,
bioreactors, biopads, or biocells.
For slurry-phase contaminated soi1 remediation, engineered slurry bioreactors (or
bioslurry reactors) have been developed. These reaction vessels vary considerably in
size and configuration depending upon the technology developer / commercial vendor.
Bioslurry reactors have reportedly demonstrated superior rates and arnounts of
degradation for certain contarninants, when compared to results from conventional solid-
phase remediation technologies (U.S. EPA, 1990; Zappi et al., 1991). Bioslurry reactors
typically operate with contarninated soi1 that is slurried with up to 85% iiquid, thus
relatively small arnounts of contarninated soi1 are handled in relation to the overall
volume of waste. This is especially true for batch-type as opposed to flow-through
reactor systems.
2.4 PAHS AS ENVIRONMENTAL CONTAMINANTS
Polycyclic (or polynuclear) aromatic hydrocarbons (PAHs) are a group of organic
chemicals produced by both natural (e.g., g ras and forest fires) and man-made (e.g.,
industrial) processes. Many PAHs have been characterized as environmentai pollutants
(Mackay et al., 1 992) and sixteen PAHs appear on the U.S. EPA's Priority Pollutant List
(Sims et al., 1988). Creosote, coal tar, and petroleum hydrocarbons - substances that
are practically ubiquktous in modern society - contain large proportions of PAHs. Tables
showing the typicai relative weight fractions of PAHs in creosote refined from coal tar,
and in vhole creosote, were published by Mueller et al. (1989) and McGinnis et al.
17
(1991), respectively. The environmental fate and exposure of these substances to
humans are of major concem, as discussed by Menzie et al. (1992).
Significant research or review publications describing the environmentai fate of
PAHs can be found in works by: Battelle Press (1976 to-date), Ann Arbor Science
(1 980), Brown and Weiss (1978), Sims and Overcash (1 983), and Enzminger and Ahlert
(1 987). Wilson and Jones (1993) published a good relatively recent review of the
bioremediation of PAH-contaminated soils.
The microbial degradation of aromatic hydrocaxbons, including polycyclic
aromatics, was discussed by Gibson (1971) and Gibson and Subramanian (1984).
Schematics of proposed pathways for the bacteriai oxidation of several of the
predominant PAHs, including anthracene, were also provided in these publications.
Major and Cox (1 992) reviewed the literature and presented biodegradation pathways
and metabolic products of some major contaminant classes (e.g., aliphatics, aromatics,
polycyclic aromatics, and halogenated organics) in tabular form. Bouwer and Zehnder
(1 993) provided general descriptions of aerobic versus anaerobic, and baderial versus
fungal degradation pathways for both aliphatic and aromatic substances.
2.4.1 An thracene
Anthracene is one of the 16 PAHs on the United States Environmental Protection
Agency's Priority Pollutant List- a list of pollutants that are monitored at US. Superfund
designated sites. Anthracene is also presently listed in Ontario's surface soil,
groundwater, and sediment criteria, for use at contaminated sites (MOEE, 1997).
Anthracene has been s h o w to be biologically degraded by certain species of
soil-inhabiting microorganisms, with haIf-life estimates for its degradation in soil varying
between 3.5 and 175 days (Sims and Overcash, 1983); a 45-day haif-life was reported
by Bulman et al. (1 987). Specific physico-chernical properties of anthracene are
presented in appendix B.
Aside from being a pn'ority pollutant, anthracene was chosen as the target
contaminant in this study mainly because:
it has sornewhat intermediate characteristics of al1 the 16 EPA-priority PAHs;
a suffioient quantity of radio-labelled anthracene was readily available;
previous research had demonstrated its biodegradability using sirnilar
methods to the ones proposed for this research (e.g., Buiman et al., 1985);
andl
it is less hazardous to work with than some other PAHs.
2.5 SELECTION OF TREATMENTS FOR STUDY
A review of the literature was undertaken to identrfy potential treatments and/or
methodologies that have been used to remediate polycyclic arornatic hydrocarbons
(PAHs) - particularly anthracene - in contarninated soils. In particular, publications that
described bench-scale solid-phase and/or slurry-phase (e.g., bioslurry reactor)
bioremediation treatability testing methodologies were noted. Previous research that
had been performed by the author was aiso included.
Bulman et al., (1985) described the fate of PAHs in two types of soil incubation
tests. In the first test, a mixture of eight PAHs was spiked into unacclimated agricultural
soil, at two rates of addition, in static incubation flasks. Contaminant concentrations
were monitored / measured over time using subsampling techniques. Contaminant
disappearance curves were plotted and kinetic parameters (i.e., reaction orders and rate
constants) were caiculated. In the second test, '4~-labelled benzo(a)pyrene (BaP) and
anthracene were added to unacclimated agricultural soi1 and the distribution of the "C
label as volatile, adsorbed, or degraded products was determined in sterilized and
biolog ically-active soil. Volalilization and adsorption to soi1 solids were apparently
predominant loss mechanisms for anthfacene whereas adsorption was the predominant
loss mechanism for BaP.
In follow-up bench-scale research to that described above, Bulman et al., (1 988)
assessed the fate of PAHs in landfmed or oily waste-arnended soils. In this work, the
fates of spiked t4~-labelled BaP, naphthalene, and anthracene were individuaily
monitored over approxirnately 4- to &month periods. Contaminant losses via
voiatilization, adsorption, partial degradation, and total degradation (Le., mineralization)
were measured for two rates of nutrient addition (600 and 1,000 mg N / kg soil) and soil
poisoning (O and 2% HgCI). Factors such as bioactivity, nutrient addition, and soil pH
were statistically characterized for their influence on contaminant fate. For anthracene,
total recoveries of the added 14c-label ranged from 87.5 - 11 1.3% in the flask apparatus;
a maximum of only 28.9% of the added 14C-label was rewvered as evolved CO2 (Le.,
indicative of cornpiete contaminant mineralization) over a 120-day incubation period.
There was no statistically significant amount of volatilized anthracene in this study.
Seech, Marvan and Trevors (1992) and Hosler, Booth and Seech (1992)
described the use of a specific particle-sized solid-phase soi1 amendment that has since
been commercialized as the DARAMEND" treatrnent technology (GRACE
Bioremediation Technologies). This amendment was effective in biodegrading PAHs
and pentachlorophenols (PCPs) in various contaminated soils, and has also been
independently evaluated and approved by the US. EPA's SITE (Superfund lnnovative
Technology Evaluation) Program (1 995).
Hosler, Bulman and Booth (1992) described fate studies for '4C-labelled 2-
methyl-naphthalene in soi1 column microwsms containing oily-waste-arnended soils.
Five treatments - physical aeration, fertilizer, H202, ww manure, and chernical
20
surfactant (Igepai@ CO 71 0, an ethoxylated nonyl-phenol) - were assessed for possible
enhancements to contaminant biodegradation over a 95-day study period. Total
recoveries of the radio-label ranged from 69.3 to 87.1%. Amounts of mineraiized
contaminant - detemined by scintillation counting of ' 4 ~ - ~ ~ 2 trapped in NaOH - ranged
from 38.5 to 59.5% of the total radio-label added to the soil, and accounted for the
greatest portion of recovered label. For the surfactant treatment, 73.3% of the added
radio-label was eventualiy recovered. Of this amount, 50% was recovered as 14C-CO2
trapped in NaOH, 14.7% as nonextractable or soit-bound residue, 3.9% as volatile
compounds, 2% as leachate, and 2.6% as extractable soi1 residues. These values were
not significantly different from the control treatment.
Cotton and Aziz (1993), in a report prepared for the Burlington Environmental
Technolog y Office (Environment Canada, Burlington, ON) described an investigation of
Fenton's reagent for treating PAH-contaminated soils in slunies. Two types of studies
were performed. The firçt was a fate study that used 27 g of soi1 in approximately 130 mL
of distilled water incubated in a 250 mL flask wkth flow-through aeration impingers and
volatile traps containing methanol (for organics) and NaOH (or KOH)(for carbon dioxide).
Radio-labelled benzo(a)pyrene ( B e ; Sigma Chemicals) was spiked at a concentration of
1.8 x 10E+05 dpdg of soi1 (1 6.2 mCi/mrnol) into a soil previously-contaminated with coal
tar. After a 24 hour incubation period, combustion analysis of the biological component of
the soils using a biological materials oxidizer (BMO) combined with liquid scintillation
counting analysis of soil solids and filtered liquids after centrifugation, and Iiquid
scintillation counting of volatile trap and carbon dioxide trap contents were performed.
Resutts indicated no significant volatilization or degradation of the radio-labelled BaP as
compared to controls. The second study assessed transformation of the radio-labelled
BaP using GC/MS analysis as well as monitoring before and after Total Organic Carbon
(TOC) vaiues - to see if total organics were being degraded - and assessing toxicity by
21
using solid-phase Microtox testing. No significant rernovai of TOC or change in the ring
distribution of PAHs was observed. Treatments (two) using 25 mM of Fenton's reagent
showed reduced final toxicity with the Microtox test. It was suggested that the pH of 5-6
that was used in the study was too high to aflow for adequate oxidation of the PAH
contarninants and that a pH closer to 3 would have been more appropriate.
Black, Ahlert, Kosson, and Brugger (1991) used 2.0 L working-volume reacton to
investigate the biodegradation of distillate bottoms from a benzene, toluene, and xylenes
(BTEX) production process, wtiich consisted of cracking naptha in the presence of
sulphuric acid. The reactors were initiaily charged with 50% soi1 slurry, 33% mixed
culture inoculum (i.e., an aerobic activated sludge from a sewndary treatment basin of a
wastewater treatment plant - a bhugmentation treatment), and 17% mineral salts
medium. A special soi1 pre-treatment process consisting of phys id particie size
separation effectively reduced the amount of contarninated material. Losses attributed
to volatilization were significant - especially for toluene and xylenes - however
biodegradation appeared to account for almost haif the removal of benzene and ethyl
benzene.
Jones, Brinkmann, and Mahaffey (1 992) studied Pseudomonas fluorescens, P.
stutzeri, and Alcaligenes sp. as inoculants (concentration of 9.3 x 1 o7 per gram of soil),
for ElMCO bioslurry reactors (ive., continuously-stirred tank reactors, or CSTRs) . The
degradation of PAHs in cresosote-mntaminated soils by specific PAH-degrading
bacterial populations was monitored over time. Bacteria were grown on mineral salt
agar and salicylate-amended minerai salt agar with phenanthrene or pyrene as carbon
sources. Salicylate-arnended media was used to screen strains capable of co-oxidizing
PAH's while growing on an alternate carbon source. The authors stated that:
"Often biological reactions c m be accelerated in a slurry system because of the
increased contact efficiency between wntarninants and microorganisms due to
22
higher sustained levels of bacterial populations in the aqueous phase (e.g., 10' -
1 og colony-forming units /milliliter (du/mL)). In a 30% slurry this translates to 1 og
- 10'' cfdgrarn of soi1 whicn 1s 10 - 100-fold higher than typicaliy attainable in
solid-phase treatment processes."
In addition to the bacteriai inoculum, the chemicai surfactant Tween 80@ was
used. An overall PAH reduction of 93.4 +/- 3.2% was measured over the 12-week study
period. The authors observed an appreciable thickening (viscosity) in the slurry over the
study period, and aiso an increase in the extraction efficiency of the PAHs from soi1
particles over time. Between weeks 3 and 9 of the study, the levels of PAHs actually
increased, especially in the 4- to 6-ringed PAHs. Pre-treatment of soi1 particles by soi1
milling yielded a greater portion of A 0 0 mesh-sized particles (i.e., fine sands and
smaller-sized particles) than existed in the original soil. Cornminution of soi1 particles
was believed to occur during the study period. Factors contributing to a lack of fuPher
PAH degradation may have been:
1. bacterial utilization of metabolic degradative intermediates as preferential
carbon sources;
2. reductions in the more readily degradable PAHs to levels below that which
sustain an acclimated biomass;
3. the low bioavailability of recalcitrant 4-, 5-, and 6-ring PAHs; or,
4. the generation of inhibitory metabolic end-products that repress catabolic
act ivity .
A shake-flask treatabilrty test was petforrned for biodegrading PAHSs in sediments
from a western Canadian water retention pond. The flask test was followed by pilot-scale
biosluny reactor tests using 60 L batch CSTRs; either singly, or two in series with one
operated in a semi-continuous mode. This research by Simkin and Giesbrecht (1994) aiso
examined three treatments - nutrients, a biomass inoculant, and an organic supplement -
23
aithough no information was provided about the succeççfulness of these treatrnents. The
conclusions published in this technical note were that sluny-phase bioreactors were
effective in degrading PAHs and that the semi-continuous mode of reactor operation was
the most successful.
Berg, Nesgard, Gundersen, Lorentsen, and Bennett (1994) described both bench-
and pilot-scaie washing and slurry-phase biotreatment of creosote-contarninated soils.
Soi1 washing, perforrned as a pre-treatment, involved a rod mil1 for crushing and surface
washing as well as a spiral classifier and air flotation ceIl where particle sizing and
separation occurred. At bench-scale, two types of treatability tests were performed; an
initial screening test in which nutnents were added to slurried soils in Micro Oxymax
respirometers (5% solids in 100 mL), and a subsequent test using 2 1 aerated slurry
reactors with 20% soil sk ies. The nutrients included: (NH4)2SO~i NH4NOa, KH2P04,
NA2HP04 at concentrations of 1500, 750, 150, and 25 mg/L respectively. Pilot-scaie work
used 454 L EIMCOB reactors. Overall PAH reduction of 95% from an initial 5,700 rngfkg
concentration was achieved after only 8 days in the pilot reactor.
Both soil-free and soil systems were used in a study by Martens and
Frankenberger (1994) that evaluated the degradation of para-chlorophenoxyacetic acid.
A 10 g aiiquot of moist soil was placed in a 125 mL Erlenmeyer flask and treated with a
chlorinated aromatic cornpound. Three millilitres of O - 2.79 M H202 plus 3 mL of O - 0.1
M Fe2S04 were mixed together and added to the soil as a treatrnent. It was noted that
only the more water-soluble species of organic contaminant degraded significantly
(water solubility of the contaminant being an important characteristic that is necessary
for OH radical attack) . Urea peroxide as a source of H202 was not as effective as H202
and Fe2S04.
Tyre, Watts and Miller (1991) treated four biorefractory contaminants
(pentachlorophenol, trifluralin, hexadecane, and dieldrin) in four soils having different
24
organic carbon contents, using catalyzed hydrogen peroxide (H202), or Fenton's
Reagent as a chemicai oxidation treatment. Their bench-scaie test systems used 2.5 g
of contaminant-spiked soi1 (ail four contaminants at 200 mgkg each) in 40 m l glass vials
treated with 12.5 mL of I30,OOO mg/L &O2. Reactions were initiated with 1 mL of
Fe&04 and slurry pHs were adjusted to 3. Rates of H202 consumption and treatment
Miciencies were determined. Greater than 99% loss - presumed to be decomposition -
of trifluralin and pentachlorophenol occuned over the 5-day study period but the
degradation rates decreased as a function of soi1 organic carbon content. For
hexadecane and dieldrin, the soi1 organic carbon content had no effed on the
degradation rates.
Other researchers who have shown promising contaminant degradation using
Fenton's oxidation include: Watts (1990; chlorophenols), Barbeni, Minero, and Peliuetti
(1 987; chlorophenols) , Sedlack and Andren (1 991 ; chlorobenzene), and Salameh,
Friday, and Nakles (1 994; manufactured gas plant PAH contaminants).
Phagoo and Skog (1996) described research using a pilot-scale "ATAR'~"
bioreactor for treating PAH-contaminated soils. The microbiai population (Le., biomass)
produced during operation of the bioreactor was shown to be capable of degrading
significant quantities of PAHs and could be used as a bioaugmentation treatment. Use
of a previously-acclimated microbiai consortia as a starter inoculum in organic
contaminant remediation systems has been recognized as a useful bioremediation
methodology, especially when native microbial populations are likely to piovide Iittle or
no cornpetition for organic substrates (e-g., Barbeau et al., 1 997).
--- --
1 O ATAR = Autothermal themophilic aerobic reactor.
25
CHAPTER 3. METHODS AND MATERIALS
This research had the following objectives:
1. to evaluate and compare two distinct methods for bioremediation
treatability testing - solid-phase versus sluny-phase soi1 incubation; and,
2. to assess various physico-chernical and/or biological treatments for
enhancing anthracene degradation in uncontaminated / unacclimated
garden soil.
The research involved monitoring and assessing contaminant fate - especially
mineralization - by using a radio-labelled or 14Gspiked contaminant (Le., anthracene), thus
facilitating a contaminant mass balance monitoring approach.
The successfulness of each of the treatments was based on the respective
contaminant mineralization or biodegradation / bioremediation rates, as determined by
the arnounts of radio-labelled carbon dioxide ("C-CO2) captured from the incubation
flasks.
The use of spiked, radio-labelled contaminants in treatability studies to represent,
mimic, or model actual contaminant degradaiion that might occur when particular
treatments are applied in soil remediation scenarios, is a recognized research method
(e.g., Bulman et al., 1985 & 1988; Voroney et al., 1991). There are however certain
assumptions andior limitations for using such methods. They include, but are not limited
to, the following:
the spiked, radio-labelled chemical (contaminant) may be preferentially
degraded as compared to the same chemical that may exist in previously-
contaminated soi1 as a bound, or weathered contaminant - either individually or as
part of a chernical mixture; and,
methodç for quantifying, applying, and recovering the radio-labelled
chemicai are sufficiently controlled by the use of adequate QNQC procedures (i.e.,
use of sunogate spikes and testing spike recoven'es, use of duplicate samples,
blanks, and other checks).
3.1 EXPERIMENTAL DESIGN
The research study employed a cornpletely randornized design (CRD) with six
treatments (or five treatments plus a control), each replicated three times for both the
solid-phase and slurry-phase incubation set-ups, resulting in thirty-six experimental
units. Additionally, three initial and three final spike check flasks - to ascertain initial and
final recoveries of radio-labelled spikes - were prepared for eacb of the solid-phase and
slurry-phase set-ups. These flasks did not receive any treatments. Thus, twenty-four
flasks were used for each set-up methodology, or a total of forty-eight flasks were used
for the entire study.
AI1 procedures pertaining to experirnental set-up (Le., assignment of treatments
to experimental units) and sampling, used randomization techniques".
Use of randomizing in this study involved greater logistical effort, and thus had
the potential to introduce more transcriptional or data errors than would likely occur in
non-randornized treatability tests, however randomizing was necessary to control
potentially serious systematic sources of variation (Le., experimental errors).
11 Randomization is the process of using a randomiùng device to avoid biases or reduce or
eliminate sources of variation. Cochran and Cox (1957) descnbe in greater detail how
randomization should be used to ensure that a treatment is not either continually favored or
handicapped by known or unknown sources of variation.
3.2 SAMPLING DESIGN
There are normally two distinct approaches to the experimental sampling design
- sacrifice or destructive sampling of experimental units versus subsampling or non-
destructive sampling of everirnental units. Previous contarninated soi1 treatability
experirnents have yielded acceptable results using elher approach (see Hosler and
Booth, 1995). However, the sacrifice sarnpling approach requires more individual
experimental units and alsa. in this case, more incubation space (i.e., a large enough
shaker unit that could be placed in a fumehood enclosure) - which was a limiting factor
for this study.
The subsampling approach requires fewer experimental units, but for slurried
soils it introduces the difficulty of trying to obtain representative subsamples of both
liquid and solid slurry fractions.
For this study it was decided to perform only one - a final - destructive sampling
of the experimental units for solid-phase soils and slurry-phase solids and liquids. Initial
soi1 and liquid 14c-anthracene concentrations were represented by spike check flasks
which were set-up at the same time and in the same manner as the other experimental
units but which were immediately sacrifice-sarnpled and analyzed. Additionally, frequent
monitoring and sampling of evolved radio-labelled carbon dioxide (Le., CO^)
provided data for rates of contaminant mineralization. A twice-weekly sampling
schedule was originally maintained for evolved '4C-COp but subsequently changed to
less frequent penods due to a lower-than-anticipated rate of contaminant mineralization
(i.e., evolved 14C-CO2).
The durations of biorernediation treatability tests, as reported in the literature, are
highly variable. Typical endpoints are reflective of the study budget or available
resources, and the purpose of the study (e.g., long-terni venus short-term remediation
scenarios). Endpoints may be when a majority (Le., >50%) of the target contaminant
28
has been shown to have degraded or can be accounted for via metabolic products or
tosses (e.g., volatilization, leaching), or when target clean-up criteria have been met
(Hosler and Booth, 1 995).
3.3 TREATMENT DESIGN
3.3.1 Solid-Phase Treatments
The solid-phase experimental units each received 20 g (dry weight basis) of soil.
The experimental treatments, and the rates at which they were applied for the solid-
phase rnethodology, were (in alphabetical order):
Bioaugmentation (a microbial inoculum, previously acclimated to PAHs,
added as a sludge)[lQ mL @ ca. 25% suspended solids; based on the
work of Phagoo and Jones, 19971.
Control (no additives or arnendments; air-dried soil) [based on the
unpublished work of Hosler, 1996bl.
DARAMEND" 6382 soi1 amendment (Environment Canada; exclusively
licensed to GRACE Bioremediation Technologies Inc., Mississauga,
ON)[2% application rate based on unpublished work by Hosler, 1994 and
communication with Dr. A. Seech of GRACE BTI].
Fenton's reagent i2.1 mL of 5.6 M hydrogen peroxide + 2.1 mL of 0.2 M
ferrous sulfate as per Martens and Frankenburger (1 99511.
IgepalB CA 720 surfactant (Rhône Poulenc, Cranbury, NJ)[1%
application rate as per Vigon and Rubin, 19891.
ORCB - oxygen release compound (Regenesis Bioremediation Products,
San Juan Capistrano, CA)[1% application rate as per manufacturer's
recommendations; personal communication with Dr. S. Koenigsberg].
3.3.2 Slurry-Phase Treatments
The sluny-phase experimental units each received 10 g (dry weight basis) of soi1
and a maximum of 50 mL of Stanier's liquid minerai saits (formulation appears in
appendix H) was used to create a 20% solids:liquid ratio. If water was added as part of
a specific treatrnent (e.g., in sludge, for the bioaugrnentation), then this amount of water
was subtracted from the amount of liquid media required so that al1 flasks were of equal
1iquid:solid ratios.
The experimentai treatments, and the rates at which they were applied for the
slurry-phase methodology, were (in alphabetical order):
Bioaugmentation (a microbial inoculum, previously acclimated to PAHs,
added as a sludge)[lO mL @ ca. 25% suspended solids; rate based on the
work of Phagoo and Jones, 19971.
Control (no additives or amendments) .
DARAMEND" soi1 amendment [2% application rate based on unpublished
work by Hosler, 1994 and communication with Dr. A. Seech of GRACE BTI].
IgepalB CA 720 surfactant (#1) [1% application rate, based on the work of
Cotton and Aziz, 19931.
ORCB - oxygen release compound)[l% application rate as per
manufacturer's recommendations; personal communication with Dr. S.
Koenigsberg].
Tweena 80 surfactant (#2) (Fisher Scientific, Fair Lawn, NJ) [1% application
rate based on the work of Cotton and Aziz, 19931.
3.4 EXPERIMENTAL SET-UP
3.4.1 Soil Properties
The soi1 used in this research was taken from an urban garden that had not
received any pesticide or inorganic nutrient additions for at least eight years prior to
sampling. Characterization of the soil for significant properties previously shown to
impact bioremediation processes (Sims et al., 1989), was performed on soi1 subsarnples
taken after physicai preparation of the bulk soil. This preparation involved air-drying
followed by manual sieving using a No. 10 U.S. standard testing sieve to obtain the Q
mm particle size fraction.
The significant properties that were characterized, and references for the
analytical methods used, included:
soil textural anaiysis [the hydrorneter method of Day (1965)l;
amounts of inorganic, organic, and total soil carbon [Leco Instruments
Corporation, Mode 1 CS-34-41;
soi1 nutrient analysis [NO2-N, NO3-N, TKN, and TP as per Standard Methods
using a Technicon TRAACS 800 - Method Manual No. 787-86T (Technicon,
1986)l;
quantification of potentially-toxic metals (As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb,
Zn) [Methods of Soil Analysis, 2nd Edition, 19821;
cation exchange capacity [Chapman (1 965)];
pH analyses [2: 1 distilledldeionized watf3r:soil ratio; CSSS (1 993)l; and,
rnoisture content and water holding capacity (Soit Microbiology 65-414
Laboratory Manual; J.T. Trevors, University of Guelph, 1984).
Results of the soi1 particle anaiysis performed by the Soil Testing Laboratory
(University of Guelph) appear in Table 1. This soi1 belongs to a sandy loam textural
class, although it is very close to being either a loam or a sandy clay loam. Soil texture
31
is one of the more important parameters to consider when assessing the suitability of
bioremediation as a treatment technology. A loam soi1 is generally considered to be
arnenable to biorernediation, as opposed to excessively clayey, or silty, soils (Sims et al.,
1989; U.S. EPA, 1993).
Table 1 . Soil Textural Analysis Data for Duplicate Samples (%).
When the carbon and nutrient analyses are considered together (Table 2 and
Table 3), they indicate that a C:N:P ratio of approximately 120:lO:l was also satisfied
Sarnple Mean Standard Error
(i.e., an optimum level for bioremediation as suggested by Sims et a/.,l989).
Sitt 30.3 0.57
Sand 52.4 0.92
Table 2. Soil Carbon Analysis Results for Tripkate Samples (%).
Clay 17.1 O
Table 3. Soil Nutrient Analysis Results for Triplicate Samples (mg/kg).
Sample Mean Standard Error
Using the Canadian Council of Ministers of the Environment recommended soi1
quality guidelines as a guide, none of the metal species that were anaiysed (Table 4)
occurred at concentrations that would be considered to pose a potential risk to the
environment - especially microbiota (CCME, 1 997).
Summary statistics for the anaiysis of three (3) samples for cation-exchange
capacity (CEC; Chapman, 1965) and pH analyses (2:l distilled/deionized watensoil
ratio; CSSS, 1993) appear in Table 5.
Total lnorganic Carbon 1.90 0.50
Total Organic Carbon 3.10 0.30
Sample Mean Standard Error
Total Carbon (TIC + TOC) 5.00 N/A
NOTN 0.60 0.04
NO3-N 369 44
TKN 2503 64
TP . 1623 1 45
Table 4. Soil Metal Analysis Resutts for Triplicate Samples (mg/kg).
- - - - -
Table 5. Soil CEC and pH Analysis Data for Triplicate Samples.
1
Both the CEC and pH numbers fall within optimum ranges for contaminated soi1
bioremediation (Sims et al., 1 989).
The mean soi1 moisture content determined for triplicated samples of the initial
soi1 was 6.47% on a dry weight basis - before treatments were applied. The mean water
holding capacity (Le., 100% WHC) of triplicated initial soi1 samples was 0.316 mUg
(oven-dried soil). A WHC value equai to 25-85% of the maximum, or a value of
approximately -0.01 MPa, was suggested by Sirns et a/. (1989) as being optimal for
bioremediation. For this study, soi1 moisture contents of approximately 75% of the
maximum WHC (or 24% rnoisture on a dry weight basis) were maintai-ned for the solid-
phase incubation rnethod.
In 131
10
Sample Mean Standard E rro r
Sample Mean Standard Error
3.4.2 Solid-phase appara tus
The solid-phase set-up used Bellco Biometer0 flasks as experimental apparatus,
As 4.88
0.07
- -
CEC (cmoVkg) 23.3 0.21
but without the stoppered Ascarite@ holder or the side-arm stopper and syringe, as
- pH 6.83 0.33
shown in Figure 2. Aeration was performed passively &ter opening the flasks to the
atmosphere during periodic sampling of the NaOH (15 mL of 2.0 M) in the COn traps.
Scintillation vials, placed inside the side-am of the flasks, were used to hold the NaOH
solutions for trapping evolved CO2 and these vials - with fresh NaOH - were periodicaily
33
Pb 7.5
2.9
Cd 0.0
NIA
Cr 22.5
1.3
Cu 23.6
2.8
Ni 13.2
0.6
Fe 19133
1803
Hg 0.07
0.00
Mn 533
40
replaced as opposed to placing NaOH directiy in the flask side-ans and removing via
syringe. Although actuai oxygen concentrations were not deterrnined during the course
of the study, the arnount of aeration was deemed to be sufficient based upon previous
studies using similar contaminated matrices (e.g., Bulman et aL, 1988), and supported
by periodic use of BBL@ GasPakB anaerobic indicator pads. The flasks were incubated
at a constant 22.5 +/- 2°C temperature. The study method was based upon the initiai
work of Bartha and Prarner (1 965); subsequently used by Bulman et al., (1 988).
Figure 2. Schematic of the Biometer@ Flask used in the Solid-Phase Methodology.
3.4.3 Siurry-phase apparatus
The sluny-phase set-up used custom-fabricated 250 mL flasks with speciaiized
screw-on top-caps that accommodated Viton" tubing connections to ffitted gas-
washing-impingers, contained within screw-capped test tubes (20 mL capacity; filled wlh
15 rnL of 2.0 M NaOH to trap 14C-CO2). The flasks were secured on top of a shaker tray
(Labline" rnodel 3525), whereas the test tubes were held in adjoined, non-shaken, test
tube holders. A schematic of the flask apparatus and COn trap appears in Figure 3.
Figure 3. Schematic of the Flask Apparatus used in the Slurry-Phase Methodology.
The flasks were randomly assigned to locations within the shaker tray and were
incubated inside a fumehood enclosure in a room with a 22.5 +/- 2°C temperature. It
was later discovered that the shaker plafform created sufficient heat as to raise the
temperature of the flask contents by approximately 34°C.
3.4.4 Soi1 Spiking with Radio-Labelled and Unlabelled Anthracene
The radio-labelled (14c) anthracene used in this study was obtained from Sigma
Chernical Co. and prepared in a solution of ethanol; based on previous success with
ethanol as a carrier solvent for anthracene. Its specific activity was 0.865 mCi/mMot.
The anthracene was labelled at the number 9 carbon position of the molecule.
A target arnount of '%-anthracene spike was mathematically determined, based
upon calculations of the soi1 sarnple sizes, the activity of the spike, the minimum
detection limits for the liquid scintillation counter and previous experience with this radio-
tracer technique.12
The target amount of "C-anthracene was subsequently verified using the
triplicated initial test or spike check flasks for both solid-phase and slurry-phase
methodologies (Le., the triplicated soi1 sarnples for both solid-phase and slurry-phase
methodologies were prepared, spiked, immediately sampled, extracted and analyzed for
recovery of the 14c radio-label). This procedure was intended to validate the actual
spike levels for the experimental units, which could not be sacrifice sampled. The
replicate averages of the recovered arnounts of spike were thus used to represent the
baseline radio-labelled contaminant concentrations for ail experimental units.
Additionaily, non-radioactive (i.e., 12C) anthracene was added to al1 experimental
units as a carbon source for the metabolism of heterotrophic microorganisms.
Target spike levels were 2,670 dpm/g soil for 14c-anthracene - based on the
known activity of the spike solution and the amount being added to the flasks - and 55.5
pg/g for I2C-anthracene. Replicate averages and standard deviations for the recovered
arnounts of labelled and unlabelled spikes from tnplicated spike check flasks are
reported in Table 6.
Table 6. Initial Amounts of Radio-labelled and Unlabelled Anthracene.
'*C (pgm 53.2 2.2
#
Methodology Solid-phase; soi1
S lurry-phase; solids
The measured spike values displayed slightly higher activity than the predicted
Sluny-phase; liquids
target amounts. The arnount of total spike recovered from the liquid extract of the sluny-
Triplicate Statistics , Sample mean Standard Error
Sample mean Standard Enor
phase test flasks was <1 .O% of the total added to the flask. This extract was obtained
I4C (dpmfg) 3,136 4.7
N.B. The values appearing in this table have been background-corrected.
Sample mean Standard Error
after adding water to the spiked soil, gently swirling the flask contents a few times and
3,066 304
then centrifuging and removing the supernatant (Le., the extract). The extract was
48.1 0.6
237 (Total dprn) 131
analyzed imrnediately using the liquid scintillation wunter for '4~-anthracene and gas
5.0 (p9/L) 1.8
chromatography with a mass selective detector for '*c-anthracene.
There are various different methods for adding a radio-labelled spike to soil. One
method involves adding the spike to a batch of soil that would then be divided and
distributed to the individual experimentai units (i.e., the batch technique). Alternatively,
individual quantities of spike could be added to soils that have already been apportioned
to individual experimental units. In the first method, a concern arises over the ability to
distribute a small quantity of radio-labelled spike to a large quantity of soil, and then
- - -
12 Detailed methods for detemining the quantity of 14~labelled tracers for use in soi1 organic
37
distribute this spike equaily amongst experirnental units. This concem may usuaily be
alleviated by thoroughly rnixing the spiked sail prior to distribution to units. With the
second method, the concem is the potential difficulty in reproducing or replicating the
distribution of small quantities of liquid radio-labelled spike via syringe to many
experirnentai units. For our study, we chose to employ the first method described; the
batch technique. However due to the large arnount of soi1 that required mixing, we used
two batches; one batch for al1 of the soi1 used in the experimental units being rnonitored
for evolved CO2, and one batch for the soi1 distributed to the six initial and final spike
check or test flasks.
After the spike check was performed, the incubation study was initiated by
distributing the spiked soi1 to the flasks for both the solid-phase (20 g of soi1 per flask on
a dry wt. basis) and slurry-phase (10 g of soi1 per flask on a dry wt. basis) methods.
Treatments were applied, NaOH was placed in the carbon dioxide traps and the flasks
were closed and the study began. For the slurry-phase method, the platform shaker
table was tumed on to initiate the slurrying process.
3.5 SAMPLING AND MONITORING
3-51 Solid-Phase
The solid-phase treatment fksks (or experimental units) were sampled according
to a pre-determined schedule designed to allow for sufficient aeration of the incubation
flasks and to provide enough data for accurate assessment of periodic and cumulative
amounts of evolved 14c-co2 - based on previous experience using similar treatability
systems (Hosler, 1 996b).
carbon research were discussed by Voroney, Winter and Gregorich (1 991).
38
Moisture contents of the incubation flasks were rnaintained at initial levels, with
periodic water additions made to flasks as required.
No interirn nutrient additions were made to any of the flasks.
3.5.2 SIurry-Phase
The slurry-p hase treatment f lasks were sampled and aerated according to the
same schedule as the solid-phase flasks. No interirn water or nutrient additions were
made to any of the slurry-phase flasks during the study period.
The shaker apparatus rnaifunctioned at two periods in the study. The first
breakdown occurred after 26 days of incubation and lasted for approximately 24 hours
before the shaker was fied. The second breakdown occurred after 76 days of
incubation and lasted for a maximum of 76 hours before being corrected. The possible
implications of these breakdowns are discussed in Chapter 4.
3.6 ANALmlCAL
The anaiytical methods used in this study are standard operating procedures
developed for use at the Wastewater Technology Centre and accredited by the
Canadian Association for Environmental Analytical Laboratories (CAEN). Since these
methods are however considered proprietary material, detailed descriptions cannot be
provided. Instead, brief descriptions of the analyticai methods appear below.
For the solid-phase methodology, there were two matrices that were sampled
and anaiyzed for the fate of the radio-label; the soi1 solids and the liquid NaOH-CO,
traps.
For the slurry-phase methodology, three matrices were sampled and analyzed
for the fate of the radio-label; the slurry solids (i.e., soils) and slurry liquids as separated
by centrifugation (5 minutes at 3,590 rpm), and the liquid NaOH-CO2 traps.
39
The solids from ail experirnental units for both methodologies (Le., a portion of
the 20 g from the solid-phase flasks and the 10 g from the slurry-phase flasks), were
Soxhlet-extracted for 16 hours using standard laboratory Soxhlet apparatus and 350 mL
of 59% acetone: 41% hexane solvent mixtures. The liquid samples from the sluny-
phase methodology (i.e., supernatants; approximately 35-40 mL were recovered) were
1iquid:liquid extracted using dichloromethane and a separatory funnel.
Liquid scintillation counting to quantify the ' 4 ~ label in NaOH samples, Soxhlet
extracts of solids, and 1iquid:liquid extracts of sluny liquids, was performed on a liquid
scintillation wunter (Rackbeta brand), using ScintiVerse II@ cocktail (Fisher Scientfic,
Fair Lawn, N J) .
3.7 STATlSTlCAî DATA ANALYS IS
The data for the percentages of spiked 14c-anthracene recovered as 14c-co2
represent the wmplete degradation (i.e., minerakation) of the radio-labelled anthracene
and as such directly meaçure the rernediation of the contaminated matrix (Le., soi1 solids
or slurry supernatant). The data was analyzed using a combination of Excelm
(Microsoft@ Corporation) and STATISTICA" (StatSoft@, Tulsa OK) software packages.
Replicate means and variances (i.e., mean squared errors - MSE - or s2), were
calculated and one-way analysis of variance (ANOVA) procedures were performed for
each tirne period for the rates of evolved '4C-C~,. Paiiwise comparisons among
treatment means were then performed using the Tukey Honestly Significant Difference
(HSD) test (Tukey, 1949); described in Appendix C (as reproduced from Kuehl, 1994).
Summary results of the analyses are presented in the following sections; data sheets
and modfied summary ÇTATISTICAm printouts appear in appendix K.
CHAPTER 4. RESULTS AND DISCUSSION
4.1 SOLID-PHASE STUDY - 14c-~on Graphicai results for the cumulative amounts of evolved 14C-CO2 appear in
Figure 4 and periodic rates of evolved 14C-COr appear in Figure 5; both figures display
the replicate means or average values13.
Cumulative 14c-co2 results show that the bioaugmentation treatrnent displayed
the most consistent pattern of anthracene degradation for the majority of the 91-day
incubation period. However, after approximately 37 days of incubation, the Fenton's
reagent, IgepalB surfactant, and DARAMEND" amendment treatrnents ail exhibited
rapid contaminant degradation, as reflected by increased levels of evolved '4C-C02.
The replicate average cumulative amounts of 14C-co2 that were measured by the end of
the study (i.e., 91 days) are shown in Table 7. The statistical anaiysis revealed the
treatments deemed to be significantly different from each other, using the HSD test with
a 5% experiment-wise Type I error rate (Le., cc, = 0.05).
Table 7. Cumulative ' 'C-~02 Recovered (96 of Added) from Solid-phase Treatments; Triplicate Averages.
13 Note that the x-axis tirne scales are not proportionai in the figures.
41
Treatment 1 'C-CO2 (%) DARAMEND" Fenton's Reagent
1
Bioaugmentation IgepalB surfactant ORC@ Control
45.8 a* 44.2 a 43.7 a ,
36.8 b . 13.8 c 0.0 d
Legend: Treatments that were not significantly different from each other have the same letter designation in the second column of the table whereas treatments that were significantly different have different letter symbols (e.g., a = a, a t b, b t cl*.
The anaiysis reveals that the DARAMEND", Fenton's Reagent and
Bioaugmentation treatments were not significantly difkrent from each other for
mineraking 14c-anthracene when the entire period of the study is considered.
All treatments were significantly better than the controI treatment which was
intended to be abiotic and which in fact did not show any evolved ' 4C -~02 .
The 91-day period required to achieve the near-to-50% recovery of the radio-
label as 14c-co2 for the DARAMEND" treatment fails within the generai range of values
reported in the literature. For example, Sims and Overcash (1 983) reported a range of
half-Iife (Le., degradation) values for anthracene of 3 to 195 days. A haif-Iife of 134 days
(with lower and upper confidence limits of 106 and 182 days, respectively) was reported
for anthracene biotransformation (not necessari ly degradation) in an unaccl imateci
Kidman sandy loam soi1 by Park et al., (1 990)14. Bulman et al. (1 985) reported a 17-day
half-life for anthracene at a concentration of 5 mg/kg, and a 45-day haff-life for
anthracene at 50 mg/kg in unacclimated agricultural soi1 incubated under similar
conditions to the ones used here. A similar soi1 moisture content - approximately 29%
(dry weight basis) - was also used. An increase in contaminant degradation haff-life, as
the contaminant concentration increased, was also evident for phenanthrene - a three-
ringed PAH having an equal rnolecular weight to anthracene. However, hat-lives for the
other PAHs studied (Le., fluoranthene, pyrene, benzo(a)anthracene, chrysene and
benzo(a)pyrene) decreased when the PAH concentration increased. In another, similar
research study by Bulman et al. (1 988), mineralization of radio-labelled anthracene
added to acclimated, oily-waste-arnended soils resulted in a maximum of onty 28.9% of
added radio-label being captured as 14c-c0, after a 120-day incubation period. The
14 Various soi1 degradation rates values for anthracene are published on page 150, Volume I f , of
Mac kay et al. (1 992).
treatrnents studied were two soil pH levels (4.5 and 7.5) in combinaiion with two nutrient
additions.
For the ORC@ treatment, one anomalous flask displayed 38% of added 1 4 ~
asI4c-CO2 whereas the other two replicates showed no evolved 14c-CO2. A review of
the order of sampling showed that a DARAMEND" treatment flask preceded this
anomalous ORCB treatment flask at just the point in the study (period #15 or day 37)
when the DARAMEND" treatment was beginning to show accelerated contaminant
mineralization. A g l a s stir rod was used to mix all flask soils after sampling of the
NaOH, to ensure both unifonn contaminant distribution and rnicrobial growth. Although
the stir rod was washed and wiped after soi1 mixing, it is plausible that some microbial
inoculum was transferred from the acdimated DARAMEND" treatment flask to the
previously unacclimated ORC@ treatment flask, thus resulting in microbial growth and
contaminant mineralization. No other treatment showed a single, apparently anomalous
result for evolved I4c-co2.
The results indicate that bioaugmentation was the most effective treatment in
providing immediate and consistent mineralization of the anthracene. An approximate
40-day acclirnation period - during which a microbial population capable of degrading
anthracene Iikely developed - was in evidence for the remaining treatments, excepting
the controls. The ffintrols involved contaminant-spiked, but dry soil, intended to prevent
microbial growth and metabolism, and thus no contaminant biodegradation was
anticipated. The controls were however intended to quantify any non-biological
contaminant mineralization (e.g., by photolysis or other phyçico-chemical mechanisms).
Since no 14c-Co2 was recovered and measured from any of the control flasks, and since
al1 of the radio-label was recovered (discussed in detail Iater), apparently no abiotic
mineralization occurred with this methodology.
The approxirnate 40-day acclimation period for this research compares with a 27-
day period reported by Bulman et al. (1988) who used a soi1 that previously received
PAH contamination and would thus be expected to contain some microorganisms
capable of anthracene degradation.
Comeau et al. (1994) discussed the role of inoculum preparation and densrty on
the biorernediation of a herbicide (2.4-D)-contarninated soi1 using bioaugmentation. It
was apparent in our study that the bioaugmentation inoculum cuntained a sufficient
population of anthracene-degrading microorganisms due to the immediate and steady
mineralization of anthracene that was measured.
4.2 SLURRY-PHASE STUDY - '%CO2
Graphitai results for the cumulative total amountç of evolved I4c-co2 appear in
Figure 6 and periodic rates of evolved 14c-co2 appear in Figure 7; both figures display
the replicate average values.
The bioaugmentation treatment displayed the most consistent and the largest
cumulative degradation of radio-labelled anthracene - although a plateau was reached
afier 27 days - a point that corresponded with the first breakdown of the shaker unit.
The temporary decrease of sluny aeration may have terminated the growth phase of the
microbiai population responsible for degrading anthracene. After 34 days of incubation,
the IgepalGD surfactant treatment began to display slow but consistent contaminant
degradation. The DARAMEND" treatment exhibited relatively rapid contaminant
degradation after 44 days had elapsed but the curve then reached a plateau after 76
days. The Tweena surfactant treatment exhibited a similar, but significantly less
pronounced ' 4 ~ C - ~ ~ 2 recovery curve to the DARAMEND" treatment. The ORC@
treatment did not exhibit any contaminant degradation during two-thirds of the study
period, but some contaminant degradation occurred in the final one-third of the study.
The controis behaved the same as the ORCB treatment and began to display
contaminant degradation only near the final one-third of the study (i.e., beginning at day
62). Incubation conditions had not been changed, nor were any other modifications
made to the flasks at that point in the study. The controls were the only flasks however
to exhibit significant evoived I4C-CO2 during the last monitoring period of the study (days
76 to 91). The cumulative amounts of 14C-CO2 measured by the termination of the study
at 91 days are shown in Table 8. As with the solid-phase results reported above, use of
Tukey's HSD test revealed which treatments were deemed to be significantly different
from each other, with a 5% experiment-wise Type I error rate (Le., cr, = 0.05).
Table 8. Cumulative '%-Co2 ~ecovered (% of Added) from Slurry-phase treatments; Triplicate Averages.
Control 1 2.8 d 1
Treatment Bioaugmentation DARAMEND" IgepalB surfactant Tweenm surfactant
ORC@ 1 1.3 e 1 Legend: Treatments that were not significantly different from each other have the same letter designation in the second colurnn of the table whereas treatments that were
14C-CO2 (%) 25.9 a ,
8.4 b m
5.5 c 3.4 d
significantly different have different letter symbols (e.g., a = a, a t b, b # c)*.
In Table 8 we see that the bioaugrnentation treatment was superior for
contaminant rnineralization, aithough the amount of recovered 14C-CO2 was only
moderately more than one-hatf of the amount achieved using the solid-phase
methodology (i.e., in Table 7). The statistical analysis revealed that al1 treatments but
the TweenB treatment and the control differed significantly from each other in the slurry-
phase methodology. The amounts of contaminant remediated were less than amounts
suggested in the literature (e.g., Zappi, 1991) using bioslurry methods but not radio-
labelled mntaminants. Two of three control treatment flasks evolved 14C-CO2 in the final
monitoring period, a time when contaminant mineralization in al1 but one of the other
treatment flasks (an IgepalcB flask) had ceased. The ORC@ treatment exhibited the
least arnount of recovered ' 4 ~ - ~ ~ i . Unlike in the solid-phase method where the controls
were dry and abiotic, the controls for this method were slurried and thus some biological
activity was anticipated - however less evolved 14C-C02 was anticipated than for the
other treatments.
It appears that a similar 40-day acclimation period to that occuning with the solid-
phase method aiso occurred with the sluny-phase method - for al1 but the
bioaugmentation treatments. The bioaugrnentation treatment involved inoculation of a
known PAH-degrading microbial species or consortium and thus immediate and
consistent contaminant degradation was anticipated, and did in fact occur.
The plateaus in the contaminant degradation curves (i.e., evolved I4c-co2 graph
curves) for the DARAMEND", TweenB, IgepalB, ORC@ and control treatments for the
final two sampling periods are likely attributable to the second breakdown in the shaker
unit, which ocwrred after approximately 76 days of incubation. As occurred with the
bioaugrnentation treatment after 27 days, a lack of aeration likely suspended or
terminated the microbiai growth and activity that had been previously occurring.
The arnounts of contaminant mineralization for al1 treatments were higher than
values obtained in a previous treatability study using this methodology (Cotton and Aziz,
1993). However, bioslurry methods are often reported to be superior to solid-phase
methods (e.g., Zappi, 1991; Salameh et al., 1994) and thus more contaminant
rnineralization was expected. The low initial contaminant concentration (approximately
100 pg total anthracene /g dry soil), and potentially low levels of contaminant
solubilization are possible reasons for the results obtained. Recent research by Karimi-
Lotfabad, Pickard and Gray (1996) suggests that soil catalyzes the formation
(oligomerization) of higher molecular weigM aromatic compounds ( e . starting with
anthracene) which are not bioavailable to active microbial populations in liquid
bioreacton. Further, water apparently competes with anthracene for active sites on soi1
mineral surfaces, and thus the recovery of anthracene from soi! depends upon the
moisture content of the soil.
For our slumed system in which the loarn soi1 had approximately 3% organic
carbon content, sorption of anthracene ont0 soi1 minerais and / or incorporation into soi[
organic material may have minimized contaminant bioavailability (i.e., for subsequent
microbial metabolism) .
Time Elapsed (days)
+ Bioaugmentation - - + - Control -.- DARAMEND - + - lgepal CA-720 -t ORC -c Tween 80
Figure 6. Slurry-phase Study - Cumulative Recovery of ' 4~ -C02 (% of Added).
4.3 SOLID-PHASE SOIL 14C RECOVERY
Soxhlet extractions of flask soils and liquid scintillation counting of the extract for
recoverable radio-label ("c), yielded somewhat vansable results, as displayed below.
The final spike checks displayed slightly greater than 100% recovery of the 14c on
average (12.2% coefficient of variation), although the values are close to the range of
variation seen in similar tests (e.g.; see Bulman et al., 1988). The initial 14c-anthracene
concentration was based on the average value from the triplicated spike check flasks
set-up in the same manner as these final spike checks.
Table 9. '% Recovered (% of Added) from Solid-phase Treatrnent Soils; Triplicate Averages.
1 82.3: corrected to 108.2 1 49.0: conected to 28.0 1 Treatrnent Spike Check
Three treatments (wntrol, ORC@ and IgepaI@ surfactant) each displayed
Triplicate Average (%) 104.8
ORC@ IgepaI@ surfactant Bioaugmentation DARAMEND" Fenton's Reagent
anomalies concerning recoverable I4c. The results displayed in the table include both
Std. Error (%) 7
12.9
the before-correction (i.e., anomalous values included) and the der-correction values
(Le., anomalous values excluded) .
57.2; corrected to 85.8 10.2; corrected to 0.0 0.0 0.0 0.0
For the control treatment, one of the three replicate flasks had a lower than
49.6; wrrected to 1.2 A
17.7; wrrected to 0.0 l
0.0 J
0.0 0.0
expected recovery of 14c that can be attributed to a fault in the Soxhlet extraction
procedure, thus the wrrected value displayed in the table is likely more representative of
reality. The controls were meant to display only abiotic contaminant losses and thus it
would be expected that ail of the original spike would be recovered if no evolved I4c-co2
had been measured - as it was for this method.
For the ORC@ treatment, one replicate showed no recoverable 14c from soil,
aithough it had showed a significant arnount of evolved 14c-cop, whereas the other two
replicates were nearly equal in the high amount of radio-label recovered, aithough they
had not shown any evolved I4C-CO2. From a perspective of the overall mass balance of
radio-label, this result appears to make sense. It may be that the magnesium peroxide
ingredient in ORC@ was effective in oxidizing the contaminant, but only in the one flask
up to that point in the study.
The Igepal@ surfactant was the only treatment that had just a single replicate
flask displaying significant Soxhlet-remverable "C from soil. If this result is considered
an anomaly and removed from the replicate average calculation, then this treatment
behaved similarly to the bioaugmentation, DARAMEND", and Fenton's Reagent
treatments in that there was no recoverable radio-label from the soils at the conclusion
of the study. Conversely, if the result is not an anomaiy, then it rnay be that the
surfactant was effective in solubilizing the contaminant in two of the three flasks - making
it available for biodegradation. In the third flask for which much less '%-CO2 evolved, it
is reasonable that the anthracene rnight be bound and thus non-extractable from the soi1
- at least up to that point in the study.
4.4 SLURRY-PHASE SOIL 14c RECOVERY
Soxhlet extractions of flask soils - after centrifugation and removal of the
supernatant - and liquid scintillation counting of the extract for recoverable radio-label
(14C), yielded results that are displayed in Table 10.
Table 10. 14C Recovered (96 of Added) from Slurryghase Treatment Soib; Triplicate Averages.
1 Treatment 1 Triplicate Average (%) 1 Standard Enor (%) I Spike Check ORCB
1
Tween 80@ surfactant IgepalB surfactant DARAMEND"
The final spike checks again displayed slightiy greater than 100% recovery of the
14 C on average, aithough the variation amongst replicates was almost nil.
No measurable I4c was remvered from any of the control treatment flask soils
and this result suggests a contaminant loss mechanism for which there were no
experimental wntrols in this study. The technical difierence between the control
treatment flasks and the spike check flasks provides a possible explanation for the
seemingly contradictory results regarding 14c recoverable from the soil. The spike check
flask soils were slurried - as were the control soils - however the check flasks were not
shaken for 91 days but instead remained stationary; due to a lack of shaker platform
space. Further, the check flasks were sealed, non-aerated and not intended to support
a microbial population - so as to provide a control for non-biological 14c IOSS
mechanisms. Thus it is possible that microbial processes were responsible for reducing
the Soxhlet-extractability of soil-bound anthracene via sorption or other processes. A
recent publication by Eschenbach, Weinberg and Mahro (1 998) supports this possibility.
Three treatments (ORCB, IgepalB surfactant and Tween 80@ surfactant) each
displayed possible anomalies concerning recoverable I4C after soi1 Soxhlet extractions.
The results displayed in fable 11 include bdh the before-correction (i.e., anomalous
values included) and the after-correction values (i.e., anornalous values excluded).
- - -
Bioaugmentation Control
102.8 27.5; corrected to 0.0 21 -6; corrected to 0.0 17.3; corrected to 0.0 0.0
1.9 1
47.6; wrrected to 0.0 I
37.5; corrected to 0.0 30.0; wrrected to 0.0
1
0.0 1
0.0 0-0
0.0 I
0.0
For ail three treatrnents mentioned, a single replicaïe flask displayed a significant
arnount of recoverable I4c. None of the values could be attributed to errors in the
Soxhlet extraction procedure thus the uncorrected values might represent reaiity.
However, there is strong agreement amongst the results from the remaining replicates
for each of these three treatments, and this seems to provide some evidence upon
which to disregard the possibly anornalous values. These three treatments, perhaps not
coincidentally, are the only physiw-chernical treatments used with the slurry-phase
method. It is thus possible that chemicai reactions such as oligomerization - as
poçtulated by Karimi-Loffabad et al. (1 996) - somehow affect the recoverable amounts
of PAHs (anthraœne included) from soils. It is known that chemical surfactants may
interfere with the recovery and analysis of various organic contaminants from soils (E.
Barresi - WTI/CPET; personal communication, 1 998).
4.5 SLURRY-PHASE LlQUlD 14C RECOVERY
The arnount of 14c recoverable from the slurry-phase method flask liquids (Le.,
the supernatants after centrifugation of the slurries), from al1 of the treatment and spike
check flasks - considered together - averaged 0.81% of the total 14c-anthracene added,
with a standard deviation of 0.22%. This result implies there was no significant radio-
label recoverable from any of the sluny flask liquids.
Previous research assessing the biodegradation of PAH's in slurried soils
suggests that biological reactions can be accelerated in slurry systems (e.g., Jones et
al., (1 9W), although an acclimated microbial population is a necessity.
Anthracene has been shown to have a rnoderate Henry's Law Constant (i.e., kH;
approxirnately 6.39 Pa rn3 / mol; Mackay and Shiu, 1981). and thus rnay be sufficiently
volatile from an aqueous phase - suggesting a possible contaminant loss mechanism
that was not monitored.
56
4.6 SOLID-PHASE TOTAL I4C RECOVERY
For the solid-phase methodology, the total mass balance for the radio-labelled
anthracene involves the cumulative arnount of '"c trapped by the NaOH during the
course of the study and the arnount of '% extracted from the soi1 subsamples at the
conclusion of the incubation period (i.e., 91 days).
Table 11 displays the wrrected (Le., without the anomalous values) mass
balance recoveries of "c-anthracene for the solid-phase treatments as replicate
(triplicate) averages with their associated standard errors (treatments are listed in
alphabetid order).
Table 11. Total 14c Recoveries from Solid-phase Treatments; Triplicate Averages.
Treatment CO^ Soil 14C Total 14C Standard (%) (%) (%) Error (%)
DARAMEND" 45.8 0.0 45.8 1.4 Fenton's Reagent 44.2 0.0 44.2 5.1 IgepalB surfactant 36.8 0.0 47.3 1 4.2 ORCoù 13.8 85.9 87.6 1 1.3
The mass balance recoveries of 14c without the anomalous data points showed
relatively low experimental variation (Le., good agreement amongst replicates) for al1 but
the controls - where the replicate recoveries of '"C ranged from 88.5 to 128.0% of
added spike.
Previous research with radio-labelled anthracene added to soi1 showed similar
14 C-CO2 recoveries but higher total recoveries, due to the inclusion of a biological
oxidation or combustion analysis that accounted for bound or non-Soxhlet-extractable
PAH (Bulman et al., 1988).
4.7 SLURRY-PHASE TOTAL 14C RECOVERY
The total recoveries of '4~-anthracene (without the anomaious results) for the
slurry-phase treatrnents, as replicate (triplicate) averages with their associated standard
errors, are presented in Table 12.
The recovery values are much more variable amongst treatment replicates than
for the solid-phase method, however one would usually expect this higher variability as
the analytical method detection limits are approached.
Table 12. Total I4C Recoveries from Slury-phase Treatments; Triplicate Averages.
Total 14C
(%) 26.5 3.4 9.2 6.5 2.1 4.2
Treatment
Bioaug mentation Control DARAMEND" IgepaiB surfactant ORC@ TweenB surfactant
Soil I4C (%) 0.0 O. O 0.0 0.0 0.0 0.0
Standard Enor (%) 2.8 1.3 5.9 4.5 1.4 2.8
14C-CO2 (%) 25.8 2.8 8.5 5.5 1.3 3.4
Liquid I4C ( O h )
0.7 0.6 0.7 1 .O 0.8 0.8
CHAPTER S. CONCLUSIONS AND RECOMMENDATIONS
The bioaugmentation treatment used sludge obtained from Water Technology
International Corporation's pilot-scale ''ATAR'~" bioreactor, which had previously been
shown to possess a PAH-degrading bacteriai population (Phagoo and Skog, 1996). Use
of a previously-accfimated microbial wnsortia as a starter inoculum in organic
contaminant remediation systems has been recognized as a useful bioremediation
methodology, especially when native microbial populations are likely to provide Iittle or
no competition for organic substrates (e.g., Comeau et al., 1993; Barbeau et al., 1997).
In this study, the indigenous soi1 microbes from the garden soi1 had likely never been
exposed to anthracene, and thus there was no significant population of PAH-degraders
present at the outset of the experiment. Apparently, an acclimation period of 30-37 days
was required before native microorganisms evolved the capacity (i.e., enzymes) to
degrade anthracene.
One can make the following conclusions regarding the treatments used in both
study methods:
The bioaugmentation treatment displayed the most immediate and
consistent rate of contaminant degradation, as reflected by evolved 14C- CO^, for both
solid-phase and slurry-phase incubation methods. This treatment used sludge obtained
from the pilot-scale WTI "ATAD" bioreactor unit, which had previously been shown to
possess a PAH-degrading bacterial population (Phagoo and Skog, 1996) and this fact
was supported here for anthracene.
The DARAMEND" treatment achieved the highest total contaminant
degradation, as ref lected by evolved ' 4 ~ - ~ ~ 2 , and this occurred for the solid-phase
incubation regime. Similar results were not however obtained for this treatment in the
15 ATAR = Autothemal thennophilic aerobic reactor.
59
slurry-phase incubation regime. This product was however onginaily developed for, and
has proven effective in solid-phase applications (Seech et a!., 1994).
The control treatments perforrned as expected in the solid-phase method
and displayed no biotic or abiotic degradation of the contaminant. For the slurry-phase
method, the controls were biologicaIly active, and were intended to provide a reference
against which to compare the treatment effects. Evolution of 14c-CO2 from control flasks
in the final few monitoring periods was somewhat anomaious, but was attributed to
biologicai degradation of the contaminant.
The spike check flasks provided useful and reliable data for establishing
the initiai and final radio-labelled contaminant concentrations for both methods. As well,
the final spike check flasks for the slurry-phase method, which were incubated under
anaerobic conditions, provided a useful cornparison to the control flasks which were
incubated under aerobic conditions.
Overall, the solid-phase incubation methodology proved to be superior to
the slurry-phase methodology, when comparing evolved 14C-CO2 - indicative of
contaminant mineralization - and total recoverable ' 4 ~ results frorn treatments that were
used in both regimes. This may be due to purely scientific reasons, experimental
technique (i.e., technical / mechanical aspects of the methodologies), or some
combination of these. The addition of a liquid fraction to the slurry-phase method flasks
resulted in a significant reduction in both the amount of CO^ evolved during the study
period, and the arnount of radio-label (14c) that could be recovered at the conclusion of
the study.
Considering the length of time this study operated, it is much greater than the
typical or average study period for feasibility / treatability studies commonly performed
by commercial treatability vendors, where most operate for no more than 6 to 8 weeks
(Hosler and Booth, 1995). If the solid-phase study had been concluded any earlier than
62 days, no significant contaminant degradation would have been detected in any but
the bioaugmentation treatment.
The results suggest that bioaugmentation cm be a useful strategy for short-tenn
contaminant degradation, but it may not necessarily yield the most degradation over the
long-term.
5.1 SlGNlFlCANCE OF THE RESEARCH
The use of bench-scale treatability tests to support pilot- andlor full-scale site
remediation technology implementation is highly recommended by researchers (e.g.,
Dupont, 1991; McFarland et ai., 1991) and is supported by regulatory groups in the
United States (U.S. EPA, 1992 and 1993). Treatability testing practices are ako
supported - in principle - by Environment Canada (Hosler and Booth, 1995). Akhough
nurnerous examples of treatability testing methodologies or protocols presently exist, few
well-documented head-to-head cornparisons of methodologies have been published.
Further, although a multitude of biological, physiml, and chernical treatments potentially
exist for remediating contaminated soils, relatively few research publications describe
experiments that assess several treatments sçimultaneously and formulate conclusions
based on statistical data-evaluation techniques. In fact, there is presently a notable lack
of use of statistical procedures supporting site remediation research, technology
development, and technology implementation in general (Mullen, Hosler and Chapman,
1 998).
This research is significant in that it involved a head-to-head cornparison of two
treatability testing methodologies supporting the remediation of anthracene-
contaminated soils, while at the same tirne assessing several potential biological or
combined physico-chemical and biological treatments. The study used a 14c radio-
61
labelled contaminant tracer technique that allowed for a reasonably effective partial
m a s balance of the contaminant, and proof of contaminant mineralization. Few
literature publications can be found describing simitar experimentai methods to those
performed in this study, especiafly regarding radio-labelled contaminant fate in slurfied
soils.
Statistical evaluation of the data using accepted procedures supported the study
conclusions.
It is recomrnended that if cornpansons between the results obtained in this study
and those from another study are attempted, one should consider the specific conditions
under which the results were obtained. The important conditions and factors include the
anthraœne concentration levels, treatment application rates, and specific incubation
conditions (i.e., soi1 type, temperature, apparatus, aeration status, etc.) in effect for both
study methods. Providenti, Lee and Trevors (1 993) provided a good review of significant
factors that limit microbial degradation of recalcitrant cornpounds such as PAHs.
The true economic cost of perforrning a treatability test using one of the methods
described here - assuming that a commercial analytical laboratory is used, capital
equipment wsts are factored in, and the operating technician is at least an intermediate
scientist - is estimated to range from $20,000 to $25,000 (CDN). This amount is
substantially higher than what most rernediation technology vendors or site owners are
willing to pay for treatability support of a site clean-up (Hosler and Booth, 1995). The
resuIts of this study will hopefully provide useful evidence for designing and performing
more effective treatability studies.
5.2 RECOMMENDATIONS FOR FUTURE RESEARCH
The described work was performed as one part of an overall program to develop
and subsequently evaluate various bench-scale rnethodologies for supporting physico-
62
chemicai andlor biologicd (Le., bioremediation) treatment technologies that could
potentially be implemented at pilot- and/or full-scale.
While this research did not positively prove the feasibility of using both of the
tested rnethods, possible errors in the techniques used for the sluny-phase methodology
might be eliminated if different apparatus or contaminant fate monitoring and detection
methods are used.
The use of a biological materials oxidizer (BMO; e-g., Harvey Instruments
Corporation) or similar instrument as reported by Bulrnan et al. (1 985; 19881, Sims et al.
(1987), and Eschenbach et al. (1998), is recornmended to account for the non-soxhlet-
recoverable fraction from soils. This fraction may be either reversibly or irreversibly
bound and this may have a direct bearing on the remedial status of an apparently
contarninated soil.
It is reiterated that researchers use either a radio-tracer or some other
conservative tracer technique for treatability testing to permit either a partial or full mass
balance approach to contaminant fate.
Since the bioaugmentation treatment showed such promise in this work, it is
recommended that in future, the joint use of bioaugmentation with other physico-
chernical treatments should be more thoroughly evaluated. It may also be valuable to
assess other treatment combinations to detenine if synergies occur.
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Karickhoff , S. W. 1 98 1 . Semiempirical Estimation of Soption of Hydrophobic Pollutants on NaturalSediments and Soils. Chemosphere, Vol. 10, pp. 833-846.
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Mackay, D., and W.Y. Shiu. 1981. A Critical Reviewof Henq'ç Law Constants for Chernicals of Environmental Interest. J. Phvs. Chem. Ref. Data, 10, 1 175-1 199.
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Major, D., and E. Cox. 1992. A Sunrev of MicrobiaI lnoculants for Bioremediation and Identification of Information Requirements Suitable for the Feasibilitv Evaiuation and Validation of Bioremediation. Report prepared for the Hazardous Contaminants Branch, Ontario Ministry of the Environment. by BEAK Consultants Ltd. Copyright: The Queen's Printer for Ontario - P I E 2152 - November, 1992.
Martens, D.A., and W.T. Frankenberger Jr. 1 995. Enhanced Degradation of Polycyclic Aromatic Hydrocarbons in Soil Treated wiai an Advanced Oxidative Process - Fenton 3 Reagent. Journal of Soil Contamination, 4(2): 175-1 90.
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McFarland, M.J., R.C. Sims, and J.W. Blackburn. 1991. Use of Treatabilify Studies in Developing Remediation Strategies br Con tamina ted Soils. Environ m entai Biotechnoloav for Waste Treatment, G.S. Sayler, R. Fox, and J.W. Blackburn (editors), pp. 163-174. Plenum Press, New York, NY.
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Menzie, C.A., B. P. Potocki, and J. Santodonato. 1992. Exposure to Carcinogenic PAHs in the Environment. Environmental Science and Technoloqv, Vol. 26, No. 7. American Chemical Society, Columbus OH 4321 0.
Ministry of Environment and Energy of Ontario (MOEE). 1997. Guideline for Use at Contaminated Sites in Ontario. The Queen's Printer for Ontario. Pl BS 31 61 EO1. ISBN 0-7778-61 1 4-3.
Mueller, J.G., P.J. Chapman, and P. H. Pritchard. 1 989. Creosote-Cantaminated Sites. Environmentai Science and Technology, Volume 23, No. 10, pp. 1 lW-l2Ol. American Chemical Society.
Mullen, K., K.R. Hosler, and GR. Chapman. 1998. Hmdbook for Statisticai Procedures Supportincl Site Remediation. Prepared for the Environmental Science and Technology Alliance Canada (ESTAC), Sarnia, ON, and the Nakiral Sciences and Engineering Research Council of Canada (NSERC), Ottawa, ON. August 1998.
Muke jee, P., and K.J. Mysels. 1971. Critical Micelle Concentrations of Aqueous Surfactant Svstems. NSRDS-NBS 36, U.S. Government Pnnting ûffice, Washington, D.C.
Park, JS., Y.J. Kim, and I.S. Kim. 1998. Modeling he E&ct of Nonionic Suhctants on the Biodegradation of Polycyclic Aromatic Hydrocarbon in Soil Slurry Using Respirometric Technique. - 7. Physicochemical Effect Bioremediation and Phytorernediation; Chlorinated and Recalcitrant Compounds. G.B. Wickramanayake and R. E. Hinchee (editors) . Battelle Press, Columbus OH, 43201 -2693. ISBN 1-57477-0594.
Phagoo, D., and S. Skog. 1996. Thermophilic Slurrv-Phase Bioremediation of Hamilton Harbour Çediment us in^ BenchScale ~ioreacton. Report prepared for the Great Lakes 2000 Cleanup Fund (GLCUF) and the Burlington Environmental Technology ûffice (BETO) - Environment Canada, Burlington, ON. Report prepared by Water Technology International Corporation, Burlington, ON.
Pollard, S.J.T., S.E. Hsudey, and P. M. Fedorak. 1994. ûioremediation of Petroleum- and Creosote-Contamha ted Soil: A Revie w of Constrainfs. Waste Manage ment & ~esearch, Vol. 12, pp. 173-1 94.
Providenti, M.A., H. Lee, and J.T. Trevors. 1993. Degradation of Recalcitrant Compounds. 12, pp. 379-395.
Rittrnann, B.E., A.J. Vaiochi, E. Seagren, C. Ray, Critical Review of In Situ Bioremediation,
Selected Factors Limiting the Microbial Journal of Industrial Microbiolo~, Vol.
B. Wren, and J.R. Gallagher. 1992. A Topicai Report GRI-92/0322, prepared
for the Gas ~ese&ch Institute and t h a s . Department of Energy, August 1992.
ROSS, D. 1991. Slurry-Phase Bioremediation: Case Studies and Cost Comparisons. Remediation, Vol. 1, No. 1, Winter 1990/91. Executive Enterprises Publications Co., Inc. New York NY 1001 0-6990. ISSN 1051-5658.
Salemeh, M.F., D.D. Friday, and D.V. Nakles. 1994. Economic Evaluation of the Biological Treatment of MGP Soils in a Liquids/Solids Slurry Reactor. Prepared by Remediation TechnoIog ies, lnc. for the Gas Research Institute, Chicago, Illinois. Topicai report #GW-94/0395.
Samson, R., C.W. Greer, and J. Hawari. 1992. Demonstration of a New Biotreatability Protowl to Monitor a Bioprocess for the Treatment of Contaminated Soils. National Research Council final report of the collaborative project between the Biotechnology Research Institute, SheIl Products Canada Limited, Groundwater Technology Canada Limited, and the St. Lawrence Centre (Environment Canada), Montreal, PQ.
Seech, A.G., 1. J. Marvan, and J.T. Trevors. 1 994. On-SiteB Situ Bioremediation of Industria/ Soils Containing Chlorinated Phenols and Polycyclic Ammatic Hydrocarbons. Bioremediation of Chlorinated and Polvcvclic Aromatic H~drocarbon Compounds. R.E. Hinchee, A. Leeson, L. Semprini, and S.K. Ong (symposium co-chairs). Pp. 451 -455. Lewis Publishers, Boca Raton, FL 33431.
Sedlack, D L , and A.W. Andren. 1991. Oxfdation of Chlorobenzene with Fenton's Reagent. Environmental Science and Technoloqv , Vo1.25:777.
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Tyre, B.W., R.J. Watts, and G.C. Miller. 1991. Treatment of Four Biorefractory Con taminan ts in Soils Using Catalyzed Hydrogen Peroxide. JO u rn al of Environmental Quaiitv, Vol. 20332.
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U.S. EPA. 1993. Pilot-Scaie Demonstration of a Slurrv-Phase Biolo~ical Reactor for Creosote-Contarninated Soil; Applications Anaiysis Report. Office of Research and Development, Washington, DC 20460. EPN540/A5-91/009, January 1993.
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U.S. EPA. 1992b. Seminar on the Use of Treatability Guidelines in Site Remediation. Office of Research and Development, Washington, DC 20460. EPA/GOO/K- 92/003, July, 1992.
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U.S. EPA. 1989. Guide for Conductincr Treatability Studies Under CERCLA; lnterim Final. EPA/54û/2-891058. -
Vigon , B.W., and A. J. Ru bin. 1 989. Practical Considerations in the Surfactant-Aided Mobilization of Contaminants in Aquifers. Journal of the WPCF, Volume 61, No. 7, JUIY 1989, pp. 1233-1240.
Voroney, R.P., J.P. Winter, and E.G. Gregorich. 1991. Microbe / Plant/Soil Interactions; Chapter 5 in Carbon Isotope Techniques. D.C. Coleman and D. Fry (eds.). Acaâemic Press.
WASTECH / AAEE. 1995. lnnovative Site Remediation Technology: Bioremediation. Volume 1 of an 8-volume sefies. W.C. Anderson (ed.). WASTECH and the Arnerican Academy of Environmental Engineers. lS BN 1 -883767-01-6 (v. 1). 288 pp.
Wastewater Technology Centre. 1994. Analvticai Methods used in the Environmental Analvtical Chemistrv Laboraton/. P. J.A. Fowlie (editor) .
Watts, R.J., M.D. Udell, and P.A. Rauch. 1990. Treatment of Pentachlorophenol- Confaminated Soils Using Fenton's Reagent Hazardous Waste and Hazardous Materiais, Vol. 7,No. 4.
West, C.C., and J.H. H m e Il. 1 992. Surfactants and Subsurface Remediation. Environ. Sci. Technol., Vol 26, No. 12. Pp. 2324-2330. American Chernical Society, Washington DC 20036.
Wilson, S.C., and K.C. Jones. 1993. Bioremediation of Soil Contarninated with Polynuclear Aromatic Hydrocarbons (PA Hs): A Revie W. Environmental Poil ut ion, Vol. 81, pp. 229-249. Elsevier Science Publishers Ltd., England.
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Yeom, 1.-T., M.M. Ghosh, C.D. Cox, and K.-H. Ahn. 1996. Dissolution ofPolycyclic Aruma tic Hydrocarbons from Weathered Con taminated Soil. Waî. S ci. Tech. Vol. 34, No. 7-8, pp. 335-342. Elsevier Science Ltd., Great Britain.
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APPENDIX A - ANNOTATED BIBLIOGRAPHY OF SITE REMEDIATION
TECHNOLOGY OVERVIEW PUBLICATIONS
The arnount of information about site remediation technologies has increased
almost exponentially in recent years. Numerous databases containing descriptions of
various technologies, and examples of their applications, have been developed by both
government and private organizations. These databases often allow a user to efficiently
review possible rernediation options for specific contaminants, under various
contamination scenarios. Economic or cost information is sometimes included. The
following annotated bibliography describes overview publications or information sources
for current, emerging, andlor innovative environmental remediation technologies.
American Academy of Environmental Engineers (WASTECH / AAEE). 1995. lnnovative Site Remediation Technology. Volumes 1-8; W.C. Anderson (ed.). AAEE, Annapolis, MD, 21401.
Each volume in this eight-volume set of monographs describes an innovative site remediation technology in detail, including individual treatment processes, process summaries and descriptions and applications. The innovative treatment technologies described include: Siorernediation (in situ and ex situ); Chernical treatment; Soil washing / soi1 flushing; Stabilization / solidification; Solvent / chernical extraction; Thermal desorption; Thermal destruction; and, Vacuum vapor extraction.
Armishaw, R., R.P. Bardos, R.M. Dunn. J.M. Hill, M. Pearl. T. Rampling, and P.A. Wood. 1 992. Review of lnnovative Contaminated Soil Clean-Up Processes. Prepared by Warren Spring Laboratory, the Executive Agency of the Department of Trade and Industry. Warren Spring Laboratory, Gunnels Wood Road, Stevenage, Hertfordshire, Great Britain, SGf 2BX.
A review of the innovative treatment technologies available for cleaning-up contaminated soil. Fie generic categories of technologies are listed, including: Physical; Chemicai; Biological; Thermal; and, Solidification.
Environment Canada 1 997. REM- Site Remediation Treatment Technology Database. Prepared by Water Technology International (WTI) Corporation. Avaiable from WTI, P.O. Box 5068, Buriington, ON, L7R 4L7, Tel. (905) 336-4665.
This user-friendly cornputer software product provides a cornprehensive collection of over 500 bench-, pilot-, andior commercial-scale technologies for the treatment of sediment, soil, groundwater, and off-gases. Powerful search options and a unique problem-solving approach are provided.
Environment Canada 1997. SEDTEC Sediment Treatment Technologies Database - fd Edition. Prepared by Water Technology International (WTI) Corporation, Burlington, ON, L7R 4L7, Tel. (905) 336-4665.
This hardcopy daabase provides comprehensive information on over 213 technologies for the treatment of contaminated sediments. The database was compiled from vendor-supplied responses to a detailed questionnaire.
Government of Canada - Contaminated Sites Management Working Group (CSMWG). 1997. Site Remediation Technoloqies: A Reference Manual. Prepared by Water Technology International Corporation, Burlington, ON. 120 pages. Available from ~nvi ronrn~nt Canada, ~azardous Waste ranch, Place vinceni Massey, 351 St. Joseph Blvd., Hull Quebec, KIA OH3, Tel. (81 9) 953-0458.
This publication provides descriptions of technologies for: ln situ treatment of soi1 and groundwater; Treatment of extracted groundwater; Off-gas treatment; In situ containment of soi1 and groundwater; Ex situ treatment of excavated material; and, Monitoring methods.
Martin, l., and P. Bardos. 1 996. DraR Final Report: Sumrnary Report on the NA TOICCMS Pilot Study on Research, Development, and Evaluation of Remedial Action Technologies for Contaminated Soi1 and Groundwater. Technical Stahrs May 1996.
This report provides detailed information on internationa1 technology demonstrations for remediation of contaminated soils and groundwater.
United States Environmental Protection Agency. 1975-to-date. Annual RREL Research Symposia Proceedings. 2 1 st Symposium (EPA/600/ R-95/012) April 1 995. Office of Research and Development, Washington, DC 20460.
The proceedings from the annual Risk Reduction Engineering Laboratory (RREL) Research Symposia, held in Cincinnati OH present the findings of ongoing and recently completed research projects funded by the US. EPA's RREL. Subjects include pollution prevention demonstrations and life cycle analysis; remediation technologies from the SITE Program, RREL technologies, oil spills remediation technologies; drinking water and wastewater technologies; municipal solid waste technologies; and hazardous waste technologies.
United States Environmentai Protection Agency. 1995. VlSI77 (Vendor Information System for lnnovative Treatment Technologies). EPA 542-N-95-008. Solid Waste and Emergency Response (51 02W). Washington, DC.
This user-friendly database provides data on 325 innovative treatment technologies - 70% wmmercially available - provided by 204 vendors.
United States Environmentai Protection Agency. 1988-to-date. Innovative Treatrnent Technologies: Annual Status Report. 5th Edition, €PA 542-R-93-003, Num ber 5, September 1993. Office of Solid Waste and Ernergency Response, Washington, DC 20460.
These annual reports (semi-annual prior to 1993) document and analyze the selection and use of innovative treatment technologies in the US. EPA Superfund Program, and at some non-superfund sites under the jurisdiction of the Departments of Defense (DoD) and Energy (DoE). The following innovative treatment technologies to clean ground water (in situ), soils, sediments, sludge, and solid- matrix wastes, are documented: ln situ and ex situ bioremediation; Chernical treatment; Dechlorination; In situ flushing; ln situ vitrification, Soil vapor extraction; Soil washing; Solvent extraction; Thermal desorption; Other technologies (air sparging, contained recovery of oil wastes, limestone barriers and furning gasification).
United States Environmental Protection Agency and U.S. Air Force. 1993. Remediation Technologies Screening Matrix and Reference Guide. EPA 542-6-93-005. Tech nology Innovation Office, Washington, DC.
This report "summarizes the strengths and limitations of innovative, as well as conventional, technologies for the remediation of soils, sediments, sludges, groundwater, and air emissions 1 off-gases."
APPENDM B - PROPERTIES OF ANTHRACENE
The basic diemical and physical properties for anthracene, the three-ringed
polycyclic arornatik hydrocarbon used as the contaminant-of-concern in mis research,
are listed below, as adapted from Mackay, Donald, Wan Ying Shiu, and Kuo Ching Ma
(1 992); lllustrated Handbook of Physical-Chernical Properfies and Environmental Fate
fororganic Chernicals - Volume Il. Pp. 143-152. Lewis Publisherç, Chelsea MI.
Table 13. Chernical and Physical Properties of Anthracene.
Synonym (s) : parmaphthalene, green oil, tetra olive NZG CAS Registry No.: 1 20- 1 2-7 Molecular Formula: Ci4h0
Molecular Weight: 1 78.24 (daltons) Melting Poht (OC): 216 (API) '~ Boiling Point 0: 340 Densiîy (g/cd a t 20 OC): 1.283 Molar Volume (cm' /mol): 139 - 197 Water Solubility ( g / d or mgh at 25°C): 0.075 Vapour Pressure (Pa at 25OC ' 0.001014 Henry's Law Constant (Pa nfbmol): 6.59 Octanolmter Partfiion Coefficient (log &) : 4.45
The anthracene moleaile - with the positions of the carbon atorns numbered - appears below.
7
6
-
16 API = Amencan Petroieum lnstitute
APPENDIX C - TUKEY'S HONESTLY SIGNIFICANT DIFFERENCE TEST
Tukey's Honestly Significant Difference Test can be described as follows (from Kuehl, 1 994) :
For any pair of observed treatment means. Sand n, in a group of k treatment rneans the Honestly Significant Difference is
HSD (k, q) = dsZ/r
Where is the studentized range statistic for a range of k treatment rneans in an ordered array. Critical values for an experiment-wise Type I error rate and v degrees of freedorn can be found in Appendix Table VI1 of Kuehl (p. 61 3).
The nuIl hypothesis Ho: = ~ 4 . is rejected if:
Abiotic
Absorption
Acclimation
Nonbiologicai; without biology. In reference to mechanisms or processes not caused by living organisms. In treatability studies and experimental research, use of an "abiotic corttrot" allows assessrnent of possible contributions to contaminant fate attributed to p hotolysis, hydrolysis, vofatilization, leaching, and/or other non-biological (Le., physico-chemicai) processes.
The process of absorbing (e.g., a liquid, gas or solid substance). In biology, the uptake, drinking, or imbibing of a substance (e.g., the rnovernent of substances into a d l ) . In chemistry, the t r a d e r of substances from one medium to another (e-g., the dissolution of a gas in a liquid). In physics, transfer of energy f rom electromagnetic waves to chernical bond and/or kinetic energy (e.g., the transfer of light to chlorophyll) . (Atlas, 1984)
The process of "acclimatizing" to environmental change. In reference to microrganisms' abilities to adapt physiologically/biochemically to changes in their environment (e.g . , temperature, moisture, nutrients, contaminant concentrations, etc.).
Activated soi1 process "A biotreatment concept in which the degrading organisms present in contaminated soi1 are cultured in a soiVslurry bioreactor. m e contaminated soiCJ is then bioaugmented with this acclimated, resistant, and active biomass to enhance the efficiency of biodegradation." (Ott et al, 1994)
Activated sludge
Adsorption
Aerobe
Aerobic
Allochthonous
Anaerobe
In wastewater treatment, the use of previously acclimated microbial populations, taken from sludge digesten, and used as "starter" cultures for "seeding' new batches of untreated wastewater.
The process of adsorbing. "A surface phenomenon involving the retention of solid, liquid, or gaseous molecules at an interface." (Atlas 1984)
Organisms (esp. microrganisms) that live in the presence of air or more specifically, oxygen.
"having molecular oxygen present; growing in the presence of air." (Atlas, 1 984)
"an organism or substance foreign to a given ecosystem." (Atlas, 1 984)
Organisms (esp. microrganisms) that live in the absence of air or more specifically, oxygen.
Anaerobic respiration "the use of inorganic eiectron acceptors other than oxygen as terminai electron acceptors for energy-yielding oxidative metabolism. " (Atlas, 1 984)
Autochthonous
Autotrophs
Bacteria
Bioaugmentation
Biodegradation
Biomass
Biostimulation
"microorganisms and/or substances indi~enous to a given ecosystem; the tnre inhabitants of an ecosystern; often used to refer to the mmmon microbiota of the body or those species of soi1 microorganisms that tend to remain constant despite fluctuations in the quanti@ of fermentable organic matter in the soil." (Atlas, 1984)
"organisms whose growth and reproduction are independent of extemal sources of organic compounds, the requirement for cellular carbon being accomplished by the reduction of COa and the need for cellular energy being met by the conversion of ligM energy to ATP or the oxidation of inorganic compounds to supply the free energy for the formation of ATP. " (Atlas, 1984)
"memben of a group of diverse and ubiquitous prokaryotic, single-celled organisms." (Atlas, 1984)
The addition of exogenous / ailochthonous microorganisms (typically) to a particular site or treatment process for the purpose of enhancing bioremediation of the target contaminant(s); has also been used to refer to the addition of indiaenous microorganisms that were initially removed from the site soil, externally cultured to increase biomass, and then re-introduced to the contaminated matrix.
"the process of chernid breakdown of a substance to srnaller products caused by microorganisms or their enzymes." (Atlas, 1984)
"the dry-weight, volume, or other quantitative estimation of organisms; the total m a s of living organisrns in an ecosystem." (Atlas, 1984)
The addition or favourable modification of the various physico-chernical and/or biological requirernents (e-g., nutrient, co-factor, heat, moisture levels), for enhancing biorernediation of the target contarninant(s).
Bioremediation
Bioreclamation
Bi~restoration
Biotransformation
Bioventing
Carcinogenic
Catabolism
Chemoautotrophs
Contaminant
Variously defined as: i) "a managed, demonstrated active treatment process that uses rnicroorganisms to degrade and transfomi organic chernicals in contarninated soil, sludges and residues." (Loehr, 1991); ii) "a managed or spontaneous process in which biological, especially microbiological, catalysis acts on pollutant compounds, thereby remedying or eliminating environmental contamination." (Madsen, 1991); iii) "biological degradation that resuits in detoxification of a parent compound to a product or products that are no longer hazardous to human h e m and/or the environment." (adapted frorn S ims et a/., 1 989) ; iv) the applied use of biological (predominantly microbiological) processes for the degradation of contaminants in soil, sediment, water, air, or mixed matrices, to end-products that are l e s hamiful than the original contaminants.
Basically analogous to bioremediation.
BasicaJly analogous to bioremediation.
"The conversion of a compound or its intermediates to the next product in the biochemical pathway." (McFarland et al., 1991). Biotransformation does not necessarily result in complete remediation.
The process by which in situ bioremediation of a contarninated unsaturated soi1 zone (i.e., above the water table), is performed using auxiliary aeration and, possibly, moisture and nutrient enhancement.
cancer causing; often used in reference to either a thing (e.g., a contaminant), or a process (e.g., ultra-violet irradiation).
"reactions involving the enzymatic degradation of organic compounds to simpler orgmic or inorganic compounds with the release of free energy." (Atlas, 1 984)
"microorganisms that derive their energy source from the oxidation of inorganic compounds; organism that obtain energy through chernical oxidation and use inorganic compounds as electron donon; aiso known as chemolithotrophs." (Atlas, 1984)
a chernical (typically), that is undesirable in a given environment, due to the deleterious effects that it has imparted, imparts, or may impart, on one or more biological receptors; a poflutant.
Endogenous Native to a particular site; from internai origins (refer to Bioaugmentation)
Ex situ away frorn the natural location or environment; out-of-place
Not native to a particular site; from extemal origins (refer to Bioaugmentation)
Facuîtaüve anaerobes "microorganisms capable of growth under both aerobic and anaerobic conditions; bacteria capable of both fermentative and respiratory metabolism. " (Atlas, 1984)
Fungi
Gene probe
Genetic engineering
Heterotrophs
Henry's Law
lndigenous
lnoculum
ln situ
"a group of diverse and widespread unicellular and rnukicellular eukaryotic organisms, Iacking chlorophyll, usually bearing spores, and often filamentous." (Atlas, 1 984)
"a probe consisting of part or al1 of one or more distinct genes, used to identify homologous or sirnilar genes in a target nucleic acid ." (Samson et al., 1 992)
"the deliberate modification of the genetic properties of an organism either through the selection of desirable traits, the introduction of new information on DNA, or both; the application of recombinant DNA technology." (Atlas, 1984)
"organisms requin'ng organic compounds for growth and reproduction, the organic compounds serving as sources of carbon and energy." (Atlas, 1 984)
"a relation stating that the solubility of a gas in a liquid is proportional to the pressure of the gas above the liquid." (Masterton and S lowinski, 1 977). Henry's Law Constant ( k~ ; in Pa m3 I mol) provides an indication of the extent to which a chemical will volatilize from liquid or an aqueous solution. The kH of a chemical is directly proportional to the vapour pressure of the chernical and inversely proportional to the water solubility of the chemiml. The greater the kH of a chemical, the greater the tendency will be for the chernical to volatilize from an aqueous solution. (U.S. EPA, 1 99Ob)
Native to a particular site; occurring naturally in an area (see also Biostimu lation)
'Wie material containing viable microorganisrns used to inoculate a medium." (Atlas, 1 984)
in the natural location or environment; in-place
Lithotrophs
Mesophiles
Metabolism
Metabolites
Methodology
"microorganisms that utilize and obtain energy from the oxidation of inorganic matter; autotrophs." (Attas, 1 984)
"organisms whose optimum growth is in the temperature range of 20-&O." (Atlas, 1984)
"the sum total of al1 chemicai reactions by which energy is provided for the vital processes and new ce11 substances are assimilated." (Atlas, 1 984)
"chernids participating in metabotism; nutn'ents." (Atlas, 1984)
"A system, or body of procedures, methods, and rules used in a particular field or discipline" (Gage Canadian Dictionary).
Obligate anaerobes "organisms that grow only under anaerobic conditions, Le., in the absence of air or oxygen; organisms that cannot carry out respiratory metabolism. " (Atlas, 1 984)
OctanoCWater Partition Coefficient (Kow) "The octanoi-water partition coefficient Kow provides a direct estimate of hydrophobicity or of partitioning tendency from water to organic media such as lipids, waxes, and natural organic matter such as humin or humic acid. It is invaluable as a rnethod of estimating Koc, the organic carbon-water partition coefficient, the usual correlation invoked being that of Kanckhoff (1981); Koc = 0.41 Kow (Mackay et al., 1992). Octanol is used as a surrogate for organic media.
Organiearbon Partition Coefficient (Koc) The Koc provides a measure of the extent of chernical partitioning behiveen solid-phase organic carbon and water at equiiibrium. The larger the K&, the more likely a chemical will bind to organic carbon (e.g., in soit or sediment) than rernain in water (refer to Kow above). Koc is the ratio of the amount of chernid adsorbed per unit weight of organic carbon to the concentration of the chemical in solution at equilibrium. (U.S. EPA, 1 99Ob)
P hotoautotrophs
P hotoheterotrophs
"organisrns whose source of energy is light and whose source of carbon is carbon dioxide, characteristic of some aigae and some prokaryotes." (Atlas, 1 984)
"organisms that obtain energy from light but require exogenous organic compounds for growth." (Atlas, 1984)
P hototrophs
Plasmid
"organisms whose sole or principal primary source of energy is light; organisms capable of photophosphorylation." (Atlas, 1 984)
extra-chromosomal genetic structures (D NA) that c m replicale independently within the bacterial cell, can be transferred from one microbiai population to another, and that usually contain information for speciaiized processes such as antibiotic resistance, degradation of xenobiotic chemicals, etc.
Polynucleai Aromatic Hydrocarbons (PAHs) (Also polycyclic aromatic hydrocarbons) . A distinct class of unsaturated cyclic hydrocarbons with two or more fused rings, severai of which are carcinogenic.
P rotocol "The rules for any procedure" (Gage Canadian Dictionary).
Psychrophiles
Recalcitrant
"an organism that has an optimum growth temperature below 20°." (Atlas, 1984)
resistant to degradation. m e n in reference to a chemicai / contaminant that cannot be degraded - within a reasonable timeframe - by naturai physico-chernical, or especially, microbiological processes.
Site Remediation the application of environmental sciences and engineering technologies for cleaning-up or demntaminating hazardous waste-contaminated sites.
Soil
Solubility
Technology
Therrnophiîes
the unconsolidated material compriçed of air, water, mineral and organic matter phases that resides in the lithosphere - the immediate surface of the earth.
"The ability or tendency of one substance to blend uniformly with another, e.g., solid in liquid, liquid in liquid, gas in liquid, gas in gas. Solid Vary from O to 100% in their degree of solubility in liquids, depending on the chernical nature of substances; to the extent that they are soIubIe, they lose their crystalline form and become molecularly or ionically dispersed in the solvent to form a true solution." (Hawley, 1977)
"Scientific knowiedge applied to practicai uses; applied science." (Gage Canadian Dictionary, 1 980)
"organisms having an optimum growth temperature above &O." (Atlas, 1984)
Treatability Tdng
Ubiquity Principle
Vapour Pressure
Xeno biotic
the process of bench- or pilot-scale testing or dernonstrating the efficacy of specific clean-up or remediation technologies prior to their full-scale application at contaminated sites.
a principle of rnicrobiology that States that microorganisms are virtually ubiquitous (i.e., present, in some form, aimost everywhere on Earth) .
"The pressure (usuaily expressed in millirneters of mercury) characteristic at any given temperature of a vapor in equilibrium with its liquid or solid form." (Hawley, 1977). The vapour pressure of water at 20°C is 17.5 mm Hg (US. EPA, 1990b).
a foreign substance or poison; a substance not formed by naturai biosynthetic processes. (Atlas, 1984). Often used in reference to environ mental contaminanfs.
APPENDIX E - FENTON'S OXIDATION REACTIONS
Primary Reactions in Fenton's (Reagent) Oxidation Process (Tyre, Watts and Miller; 1991)
HpOz + ~e*+ - OH. + O H + ~ e * Cl]
H2Q2 + ~ e ~ + HO2* + H+ + Fe2+ P I
OH. + Fe2+ 4 O H + Feh Pl
Where OH. = hydroxyl radical and HO,* = perhydroxyl radical. In the presence of organic substrates (e.g . , PAHs) the reactions include:
RH + OH. -. R.+ H20 Pl
R. + ~e~ -. ~ e ~ ' + products m W here: R- = organic radicals.
APPENDIX F - PAH DEGRADATION MECHANISMS
Laboratory research of the degradation of PAHs wth pure cultures of microbes
has reveaied that there are three possible types of degradation rnechanisms (Mahro,
Schaefer and Kastner, 1994):
1. Compfete PAH mineraiization;
2. Cometabolic degradation; and,
3. Unspecific radical oxidation.
The first step in the degradation of a PAH is oxidation Le., incorporation of an
oxygen molecule into the aromatic ring. This reaction is catalyzed by a dioxygenase
enzyme and leads to formation of cisdihydrodiol intermediates. In complete
mineraiization processes, the microorganisms use the PAH as the sole carbon and
energy source.
For cometabolic degradation, the microorganisms do not use the PAH as a sole
source of carbon and energy; instead using another organic compound as the primary
substrate. Often the aromatic rings are not split open and phenolic, carboxylic, or
chinoic derivatives of the PAHs accumulate as dead-end products. Investigation of the
degradation of PAHs by fungi has revealed that the PAHs may be cornetabolically
transformed into transdiol intermediates. Fungi use monooxygenases to catalyze the
initial oxidation of PAHs.
The unspecific radical oxidation of PAHs by white-rot fungi was described by
Bumpus et al. (1 985). A wide variety of metabolic products rnay be created, including
phenolic oxidation products, polymerizates, and conjugations to other organic molecules.
Reactions cataiyzed by oxygenases (Gibson, 1989) can be characterized as
follows:
S+O2+XH2+SO+X+H2O
Monooxygenase
S +O2 +son Dioxygenase
Where:
S = substrate
XH2 = Electron donor
The degradation or metabolism of anthracene by Pseudomonads was deduced
by W.C. Evans, H.N. Fernley, and E. Griffiths and published in the Biochemistry Journal,
Volume 95, p. 81 9 (1 965). The reaction products and order of formation were described
as follows (N.B. a schematic diagrarn appeared in the original publication):
Anthracene -, anthraœne cis l,2-dihydrodiol: -c 1,2-dihydroxy anthracene
cis 4-(2'-hydroxynaphth-3-yl) -2-oxobut-3-enoic acid -+ 4-(2'-hydroxynap hth-3-yl) -
2-0x0-4hydroxybutyric acid - +pynrvate + 2-hydroxy-3-naphthaldehyde -, 2-hydroxy-
3-naphthoic acid -r 2,3-dihydroxy-naphthalene -. salicylate etc.
APPENDIX G - CHEMICAL PROPERTIES OF THE TESTED SURFACTANTS
Surfactant #1:
lgepal CA-720 (Rhhe Poulenc Ltd.; Cranbury, NJ)
Nonionic surfactant (Octylphenyl polyethoxylate - with 12 ethoxylate units) - Liquid; density = 1 .O4
Formula: CaHirCsHrO(CH2CH20)12H
Molecular weight = 735
Critical micelle concentration = -6 x 104 M [from Mukejee and Mysels (1 971) as quoted
in Laha et al. (1 990)]
Surfactant #2:
TweenB 80 (Fisher Scientific; Fair Lawn, NJ)
Nonionic surfactant (Polyoxyethylene-20-Sorbifan MonoOleate)
Formula: C64H 124026
Molecular weight = 1309.68
Critical micelle concentration = -2.5 x IO-' M [from Park, Kim, and Kim (1998)l
CAS 9005-65-6
APPENDIX H - STANIER'S LIQUID MINERAL SALTS MEDIA INGREDIENTS
ln 1 .O L of distilled water, add the following ingredients:
a) 1.0 g of NH4CI;
b) 1 .O g of &HP04;
c) 0.2 g of MgS04 7 H20;
d) 0.01 g of FeS04 H20;
e) 0.01 g of CaCI
APPENDIX I - SOlL CHARACTERIZATION; DETALED RESULTS
Table 14. Soil Textural Analysis Data for Duplicate Samples (%).
1 1 Sand 1 Siit 1 Clav 1
Table 15. Soil Carbon Analysis Results for Triplicate Samples (%).
I
17.1 17.1
I
1 Totai inorganic 1 Total Organic 1 Total Carbon 1
17.1 O
Sarnple Mean Standard Error
29.9 30.7
Sample #1 Samole #2
53.0 51.7 52.4 0.92
Samole #1
Table 16. Soil Nutrient Analysis Results for Triplicate Samples (ppm).
30.3 0.57
Sarnple #2 Sample #3 Sample Mean Standard Error
I I NO,-N I NOS-N I TKN I TP I
- Carbon 1.36 2.17 2.27 1 .90 0.50
- Carbon 3.09
Sample #1 Sam~le #2
Table 17. Soit Metal Analysis Resutts for Triplicate Samples (mgtkg).
(TIC + TOC) 4.45
3.45 2.90 3.10 0.30
L
Sample #3 Sampie Mean Standard Error
5.62 5.17 5.00 N/A
0.63 0.55 0.60 0.60 0.04
Sample #1 Sarnple #2 Sample #3 Sample Mean Standard Error
346 341 421 369 44
As 4.94
4.80
4.91
4.88
0.07
2550 2530
1520 1790
2430 2503 64
1560 1623 1 45
Cd 0.0
0.0
0.0
0.0
N/A
Hg 0.069
0.078
0.070
0.07
0.00
Cr 22.7
21.1
23.6
22.5
1.3
Mn 564
488
548
533
40
Cu 21.7
22.2
26.9
23.6
2.8
Ni 13.8
12.6
13.3
13.2
0.6
Fe 17400
19000
21000
19133
1803
Pb [Zn 6.72
5.07
10.8
7.5
2.9
124
128
143
131
10
Table 18. Initial Amounts of Radio-labelled and Unlabelled Anthracene.
Methodology Solid-ohase: soi1
Slurry-phase; sotids
#3 Average
12c (pg/g) 52.6
Sarnple/Replicate #1 #2 #3 Average Std. Error #1 #2
Slurry-phase; liquids
''C (dpmlg) 3138
2875 3065
#2 #3 Average
3130 3138 3135 4.7 341 6 2905
47.7 48.1
Std. Error #1
51.3 55.7 53.2 2.2 47.8 48.8
1 74 150 237
304 388 (Total dpm)
4.5 3.5 5.0
Std. Error 1 131
0.6 7.0 (Total dpm)
1.8 N.B. The values appearing in this table have been background-corrected.
APPENDIX J - TREATABILITY TESTlNG REGULATORY GUIDANCE (U.S.)
In 1988, the US. Environmental Protection Agencyts (EPA) Office of Emergency
and Remedial Response (OERD - Washing!on, D.C.) and Office of Research and
Development (ORD - Cincinnati, OH), through the Risk Reduction Engineering Laboratory
(RREL - Cincinnati, OH), contracted PEI Associates Inc. to prepare a document providing
information on conducting treatability studies. A Guide for Conductin~ Treatabilm Studies
Under CERCLA'^ - lnterim Final was published in December 1989 as report number
EPA/540/2439/058. This document described a three-tiered approach to conducting
treatability studies in support of remedy selection (i.e., pre-Record of Decision (ROD)).
Studies suppoiting remedy implementation or post-ROD were discussed in a final
document entitled Guide for Conducting Treatabilitv Studies under CERCLA -
Biodeqradation Remedv Selection (1 nterim Guidance), US. EPN54ûIR-93/519a, August
1 9931''. The three-tiered approach consisted of:
i) laboratory screening,
i i) bench-scaie testing, and,
iii) pilot-scde testing [N.B. any order of testing may be possible, and some
tiers may in fact not be necessary].
The Guide for Conducting Treatability Studies Under CERCLA - Interim Rnal
provided a logistical protowl for conducting treatability studies in support of any
- - -
17 Comprehensive Environmental Response, Compensation, and Liability Act
l 8 For definfiions and a summary of pertinent U.S. govemment environmental legislation related to cleanup of contaminaied soils (e.g., specific acts such as CERCLA, RCRA, SARA), refer to the article by C.R. Schraff (1988) in "Soils Contaminated by Petroleum: Environmental & Public Health Etkcts", E. J. Caiabrese and P.T. Kostecki, editors. For a brief discussion of the remedial investigation / feasibility study (RIIFS), record of decision (ROD), and rernedial design / remedial action (RD/RA) processes, refer to uAppIications of Treatability Studies h Management of Fuels/Peiroleum Waste lmpacteâ Soils" by R.R. Dupont (1991), in the Air 8 Waste Management Association Proceedings from the 84th Annual Meeting 8 Exhibition, Vancouver, B.C.
technological process (e.g., chernical, physicai, biological, etc.). The protocol was not a
specific guide to study methodologies but rather a suggested approach for use by
remediai projed managers, responsible parties, and contractors, for ail phases of the
remediation evaluation process. The elements of the suggested approach to perfoning
remediai work appears below.
Table 19. Elernentî of the Approach to Perfoirning Site Remediation.
. Establishing data quaiity objectives
l a Selecting a contracting rnechanisrn l a Issuing the Work Assignment l a Preparing the Work Plan
O Preparing the Sarnpling and Anafysis Plan Preparing the H e m and Safety Plan Conducting communrty relations activities Cornpl ying with regulatory requirements Execuüng the study Anaiyzing and interpreting the data
O Reporting the results
N.B.: Adapted from EPA/540/2-891058, 1 989.
Science Applications international Corporation (SAIF) uf Cincinnati was
contracted by the Office of Research and Development (ORD - Cincinnati, OH) and the
Risk Reduction Engineering Laboratory (RREL - Cincinnati, OH), to prepare both a report
and a fact sheets respectively entitled Guide for Conducting Treatability Studies Under
CERCLA: Aerobic Biodeçlradation Remedy Screening (EPA/540/2-91 /O 1 38 - published in
July 1991), and Conductina Treatability Studies Under RCRA (9380.3-09FS - published in
July of 1992). The SAC report provides guidance for performing aerobic biodegradation
studies during remedy screening; required in the U.S. remediai investigation / feasibility
study (RI/FS) and remediai design / remedial action (RD/RA) processes (see footnote 3).
A schematic of the role of treatabilty studies in the RI/FS and RD/RA processes appears
below.
The SAIC fact sheet "provides a summary of information to facilitate the planning
and execution of aerobic biodegradation remedy screening treatability studies in support of
the RIIFS and the remediai designlremediai action (RDIRA) processes" (page 1, 9380.3-
09FS, Juiy 1992). Remedy selection guidance was published in a separate document
(Guide for Conducting Treatability Studies Under CERCLA: Biodearadation Remedy
Selection llnterim Guidance) (EPAl5401R-931519a - August 1 993).
-c- Remedial investigation / -.-. Feasibility Study (RIIFS)
Record of Decision Remedial Design/ Rernedial Action (RDIRA) +-+
Identification of Alternatives Remedy Selection
CC*
Scoping the RIIFS 44-
+++Site Characterization and Technology Screening +-.+
tCt
Evaiuation of Alternatives +-.+
C t C
lmplernentation of Remedy +--.
Lie rature S creeni ng and Treatability Study Scoping
REMEDY SCREENING to Detemine Technology Feasibility
REMEDY SELECTfON to Develop Performance and Cost Data
RD/RA to Develop Scale-Up, Design, and Detailed Cost Data
1 1 I
J. B. Adapted from EPA/54û/R-93/519a, U.S. EPA 1 993c.
Figure 8. Schematic of the Role of Treatability Studies in the RVFS and RDIRA P rocesses.
APPENDIX K - DATA
Appended Data Tables:
Solid-Phase Method; Summary Table of Recovered 14C Radio-label (% of Added) - Raw Data.
Solid-Phase Method; Summary Table of Recovered 14c Radio-label (% of Added) - Edited Data.
Slurry-Phase Method; Summary Table of Recovered ' 4 ~ Radio-label (% of Added) - Raw Data.
Slurry-Phase Method; Surnrnary Table of Recovered "C Radio-label (% of Added) - Edited Data.
Solid-Phase Data and Tukey's Honestly Significant Difference Test
Slurry-Phase Data and Tukey's Honestly Significant Difference Test
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8'bO 1 9'6 1 1 9'6 1 C SIN Y W 3 W ! ~ S L8 9'88 L'98 8'1 3HO 26 9'98 0'98 9'1 3UO 82
Ira
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, Z'H 1 V88 10'0 #8€ I luefieeu s,uo)ued 22
'OZ @lWl
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8'96 8'96 VIN Y3043 W ! ~ S 8E 9'611 9'61I V/N Wl(3 W ! ~ S LC 9'88 L'98 8'1 3UO Z€
9'L8 9'98 038 9'1 3H0 82 1' CL 0'8E V/N 0'8E 380 kZ
E'bb 0'0 Eabk OZL-V3 PdeBl, SE E 'L* 6'09 0'0 C'OS OZL-V3 Pd061 OZ - 8'9E 8'9 5 WIN 8'9 1 OZL-V3 IBdeDl 6 1
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L'W 0'0 L'bb uo!~~~u)uew6n~o!q g~
- --
9% L'O 0'0 6'b 08 Ue0M.L
L'bE 6'ÇZ 0'99 0'1 6'b9 0'0 08 uafJMl 6'201 0'1 6' CO 1 VIN Y 3 3 4 3 ~ Y I ~ s 1'901 1'1 O'ÇO 1 VIN YW3 W ! ~ S
0'2 6'€0 1 l'€O 1 O'C L'ZO1 VIN Y=Y3 W ! ~ S
9'6Z L ' l L'O 0'0 0'1 3UO b ' l l L'O 0'0 LbO 1 O Z L W PdQBl S'Z 8'0 0'0 9'1 OZ&-V3 lede61
Qe€Z Ç'LÇ 9'5 O'ZÇ O'b OZL-V3 Pd061 9'L L'O 0'0 6'9 QN3WVUVCi 8'9 1 &'O 0'0 0'9 1
2'6 G'tr L'O 0'0 9% ON3 WVUVU 9'tr 9'0 0'0 0% lOJlUO3
Ç'E 9'0 0'0 0'6 l04UO3
E'C
'ml 'PIS
BP pal!pa sluase~da~ VIN fpaldw~s 1ou sueew SIN :pue6al SIN lOJlUO3 X zz
b'Ç O8 UaeMl 9 1 1'9 L'O 0'0 8'9 9'9 L'O 0'0
€'ZOC 0'1 E' CO C 1'901 C'C 0'90 1
L'Z l O 10'1 I
8'ÇC L'O 0'0 2'6 E ' t L'O 0'0
O'b lOJlUO3 BC Ob& l O W J 0 3 6 5'6 9'0 0'0
O'Z &'O 0'0
Table 24. Solid-Phase Data and Tukey's Honestly Significant Difference Test
lime Period Fenton's Daramend lgepal ORC Bioaug. Control Variance Time 1 Time 2 Time 3 Time 4 TIme 5 Time 6 Time 7 Time 8 Time 9 Time 10 Time 11 Time 12 Time 13 Time 14 Time 15 Tirne 16 Time 17 Time 18 Time 19 Time 20 SUM 1-20
Using Tukey's Honestly Significant Difference Test Degrees of Freedom for Error = 20x12=240; n.60; k=6; q-4.03; s squared-82.373 Critical difference = q * s /(square root of n); where s = 9,075 Critical difference = 4.721 9552
Treatments: Daramend Fenton's Bioaug. lgepal ORC Control Treat, Surris: 45,8 44.2 43.7 36.8 13,8 O Signif. Diff.: a a a b c d
o o o m 0, 9
= C (II
(II CI
P
Q)aala E E E E i=i=i=i=
E E E FFi=
aa>a>a> E E E E F F F F