ISBN: 978-1-905046-42-3 © CL:AIRE 2024

Published by CL:AIRE, Reading Business Centre, Fountain House, Queens Walk, Reading, RG1
7QF. Email: enquiries@claire.co.uk

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system,
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Contains public sector information licensed under the Open Government Licence.

Acknowledgements
This guidance has been prepared by a collaborative working group incorporating representatives
from industry, consultancy, and regulators across the devolved administrations. CL:AIRE would
like to gratefully acknowledge the Environment Agency for its seed funding for this project and the
individuals of the working group for their time in authoring sections along with their technical
discussions and review of the guidance. The final document has kindly been reviewed and
supported by the Environment Agency, Natural Resources Wales, Scottish Environment
Protection Agency and the Northern Ireland Environment Agency (NIEA), an agency within the
Department of Agriculture, Environment and Rural Affairs (DAERA). CL:AIRE would also like to
thank its Technology and Research Group for their peer review of the document prior to
publication.

Working Group
George Flower – Arcadis
Thomas Grosjean – BP
Nicola Harries – CL:AIRE
Kirsten Johnstone – Environment Agency
Ian Jones – Natural Resources Wales
Matt Llewhellin – Natural Resources Wales
Brian McVeigh – formerly Northern Ireland Environment Agency
James Rayner – Geosyntec
Matt Riding – WSP
Isla Smail – Scottish Environment Protection Agency
Jonathan Smith – Shell
Rob Sweeney – CL:AIRE
Alan Thomas – ERM
Russell Thomas – WSP
Gary Wealthall – Geosyntec (retired)

Report Citation
It is recommended citation to this report is made as follows:

CL:AIRE, 2024. Guidance on the Assessment and Monitoring of Natural Attenuation of

Contaminants in Groundwater. CL:AIRE, Reading. ISBN 978-1-905046-42-3. Download at
www.claire.co.uk/mna

Disclaimer
The Working Group and Publisher will not be responsible for any loss, however arising, from the
use of, or reliance on, the information contained in this document, nor do they assume
responsibility or liability for errors or omissions in this publication. Readers are advised to use the
information contained herein purely as a guide and to take appropriate professional advice where
necessary.
Executive Summary
Monitored natural attenuation (MNA) can be a sustainable risk management strategy for
a wide range of groundwater contaminants, where environmental data are collected and
assessed that demonstrate natural attenuation will protect receptors from pollution or
harm. Natural attenuation refers to the combination of physical, chemical and biological
processes that act, without human intervention, to reduce contaminant concentrations,
flux or toxicity. Natural attenuation of groundwater contaminants has been extensively
researched over more than four decades. MNA therefore has a long track record of
applications in the UK and elsewhere, either as the sole or primary remediation strategy,
or the final stage following transition from active remediation.

The Environment Agency originally published technical guidance for MNA in 2000 in its
R&D Publication 95. Since then, significant scientific advances have been made in
understanding contaminant behaviour and reactive transport in the subsurface,
alongside ongoing developments in site characterisation, monitoring and predictive
modelling approaches and technologies, that are captured in this updated guidance.
These evolving methods enhance contaminant and process-specific understanding,
required to develop advanced conceptual site models for MNA, addressing complexities
and uncertainties that were previously challenging to deal with. These advancements
further support the development of three lines of evidence typically considered to
demonstrate the effectiveness of natural attenuation for risk management in
groundwater:

• Primary – reduction in contaminant concentration, mass and/or mass discharge in

groundwater;
• Secondary – geochemical data and modelling that provides indirect evidence of the
natural attenuation processes likely causing the observed reductions in
contamination (primary line of evidence); and
• Tertiary – contaminant and/or process-specific evidence (e.g. isotopic,
microbiological) to support the primary and secondary lines of evidence.

MNA viability is considered during remediation options appraisal. The phased approach
described in this guidance supports identification of contaminant plumes for which MNA
is likely feasible, then demonstrates the ability of natural attenuation to protect receptors
now and in the future, and prior to undertaking a monitoring programme to confirm MNA
will achieve remedial objectives within a timeframe suitable for all stakeholders:

• Step 1: MNA Screening – assessing feasibility against technical, practical, economic,

sustainability and regulatory controls;
• Step 2: Field Demonstration – to provide evidence that natural attenuation is
occurring and protective of receptors;
• Step 3: Predictive Modelling – to assure natural attenuation will remain effective in
the future, considering potential adverse effects of changing conditions; and
• Step 4: Implementation of Performance Monitoring and Verification – a programme
of groundwater monitoring confirming progress towards and ultimately meeting
remedial objectives.
i
Understanding natural attenuation and performing MNA requires evolution of an
advanced conceptual site model for relevant processes and associated risks that relies
on quality data collection and analysis.

MNA represents a long-term commitment to groundwater risk management in the order

of years to decades. If circumstances change, and MNA is no longer protective of
receptors or viable to all stakeholders, then contingency measures may be required,
including consideration of remediation alternatives to MNA. Engagement with the
regulator is required throughout the decision-making process regarding MNA as a
risk‑management strategy for contaminated groundwater.

ii
Contents
Executive Summary ....................................................................................................... i
Contents .......................................................................................................................iii
A ‘Lines of Evidence’ Approach to Assessing Natural Attenuation ................................ 1
Step 1: Monitored Natural Attenuation Screening ............................................... 3
Step 2: Field Natural Attenuation Demonstration ................................................ 6
Step 3: Prediction and Modelling Future Natural Attenuation Behaviour ........... 10
Step 4: Implementation – Performance Monitoring and Verification of Monitored
Natural Attenuation Projects ............................................................................. 11
Appendix 1: Summary of Screening Criteria to Assess the Feasibility of Monitored
Natural Attenuation ........................................................................................... 13
Appendix 2: Processes Involved in Natural Attenuation .............................................. 15
A2.1 Introduction............................................................................................. 15
A2.2 Physical Processes................................................................................. 20
A2.3 Geochemical Processes ......................................................................... 25
A2.4 Chemical or Abiotic Degradation............................................................. 28
A2.5 Biochemical Processes........................................................................... 29
A2.5.1 Biodegradation ............................................................................ 29
A2.5.2 Estimates of Contaminant Decay Rates ...................................... 34
A2.5.3 Biodegradation Research on Selected CoPC .............................. 35
Appendix 3: Data Requirements for Lines of Evidence ............................................... 37
Appendix 4: Data Acquisition ...................................................................................... 42
Appendix 5: Methods of Assessment .......................................................................... 46
A5.1 Introduction............................................................................................. 46
A5.2 Primary Lines of Evidence ...................................................................... 46
A5.2.1 Graphical Techniques.................................................................. 46
A5.2.2 Visual Techniques ....................................................................... 48
A5.2.3 Statistical Techniques.................................................................. 50
A5.3 Secondary Lines of Evidence ................................................................. 53
A5.3.1 Natural Attenuation Rates ........................................................... 53
A5.3.2 Biodegradation Indicators ............................................................ 66
A5.4 Tertiary Lines of Evidence ...................................................................... 75
A5.5 Optional Lines of Evidence ..................................................................... 76

iii
 A5.5.1 Demonstration of Assimilative Capacity of an Aquifer.................. 76
A5.5.2 Estimation of the Source and/or Plume Depletion and Longevity. 80
Appendix 6: Implementation – Performance Monitoring and Verification ..................... 81
A6.1 Introduction............................................................................................. 81
A6.2 MNA Performance Monitoring Strategy................................................... 81
A6.2.1 Remediation Objectives and Criteria ........................................... 81
A6.2.2 MNA Monitoring Plan................................................................... 83
A6.3 Ceasing Monitoring ................................................................................ 89
Appendix 7: Groundwater Flow and Transport Models................................................ 90
A7.1 Introduction............................................................................................. 90
A7.2 Model Selection ...................................................................................... 91
A7.3 Calibration and Prediction ....................................................................... 93
Appendix 8: Compound Specific Isotope Analysis (CSIA) ......................................... 101
A8.1 Introduction........................................................................................... 101
A8.2 Applications .......................................................................................... 102
A8.3 Scientific Basis ..................................................................................... 102
A8.4 Quantitative Interpretation of Isotope Data............................................ 104
A8.5 Sampling Technique ............................................................................. 106
A8.6 Supporting Lines of Evidence and Summary ........................................ 107
Appendix 9: Molecular Biological Tools ..................................................................... 108
A9.1 Introduction........................................................................................... 108
A9.2 Molecular Tools for MNA ...................................................................... 108
A9.2.1 Polymerase Chain Reaction and Variants ................................. 110
A9.2.2 16SrRNA Amplicon Sequencing ................................................ 112
A9.2.3 Metagenome Analysis ............................................................... 113
A9.3 Sampling for MBTs ............................................................................... 115
Appendix 10: Selected Literature on Natural Attenuation of key CoPC ..................... 116
References ............................................................................................................... 118

iv
A ‘Lines of Evidence’ Approach to
Assessing Natural Attenuation
Natural attenuation (NA) processes act, without human intervention, to reduce the
concentration, flux or toxicity of contaminants in soil and groundwater. Used as a
remediation approach, Monitored Natural Attenuation (MNA) has a long track record of
research and practical application in the UK and elsewhere.

This document provides guidance for practitioners on the science and practical aspects
of implementing MNA in the UK. It is based on, and supersedes, Environment Agency
R&D Publication 95 (Environment Agency, 2000).

Since the publication of the Environment Agency’s R&D Publication 95, a large amount
of research has been undertaken on NA processes, new monitoring and assessment
methods developed, and site-specific MNA projects completed, which are reflected in
this updated document.

Natural attenuation (NA):

The effect of naturally occurring physical, chemical and biological
processes, or any combination of those processes to reduce the
concentration, flux or toxicity of substances in groundwater.

Monitored Natural Attenuation (MNA):

A risk-management approach that relies on monitoring of groundwater
and technical evaluation to confirm whether NA processes are acting
at a sufficient rate to ensure that unacceptable risks are managed.

A ‘lines of evidence’ approach is recommended for MNA assessment and verification.

This evaluation normally fits within a broader site assessment and management strategy,
following established land contamination risk-assessment and management processes
(Environment Agency, 2023). An MNA lines of evidence assessment should build on an
existing conceptual site model (CSM) and further describe the relevant NA processes
and their effectiveness for relevant constituents of potential concern (CoPC). Three lines
of evidence are typically considered (Rivett and Thornton, 2008):

• Primary – groundwater monitoring data that shows contaminant concentration, flux

or toxicity decreases;
• Secondary – geochemical data and modelling that provides evidence of the
process(es) causing the decreases (e.g. electron acceptor/donor data; aquifer
geochemistry); and
• Tertiary – contaminant and process-specific (e.g. microbiological) evidence to
support the primary and secondary lines of evidence.

MNA may achieve the remediation objectives when applied in isolation or may be used
in combination with other remediation techniques. In the second situation a process-
based technique is typically used to remove significant contaminant mass, and MNA
applied as a secondary polishing step.

1
Consideration of MNA is likely to occur at two points during management of land
contamination (e.g. Land Contamination Risk Management [LCRM] in England &
Wales):

1. An assessment of MNA alongside other potential remediation options (e.g. Options

Appraisal); and
2. Once selected as the preferred solution in the remediation options appraisal, a more
detailed assessment of MNA using a lines of evidence approach is needed to
demonstrate its effectiveness.

This guidance describes a phased approach. The first phase – MNA Screening – is the
typical assessment necessary to support a remediation options appraisal. Once selected
as the preferred risk-management solution, the additional three phases are followed as
part of site-specific assessment and implementation.

In selecting any remediation solution, including MNA, it is recommended that the Options
Appraisal includes assessment of the relative sustainability of feasible remediation
options, for example, by using the SuRF-UK framework (CL:AIRE, 2010a).

The overall process for MNA assessment is illustrated in Figure 1.

A related concept, Natural Source Zone Depletion (NSZD), has also been introduced,
which considers similar intrinsic processes in the depletion of light non-aqueous phase
liquid (LNAPL) sources (Garg et al., 2017; CL:AIRE, 2024). Whilst NA is generally
focused on attenuation processes acting on dissolved phase compounds in groundwater
and monitoring relies on groundwater sampling, NSZD largely focuses on processes
acting on a NAPL source, and monitoring relies on carbon dioxide (CO2) and methane
(CH4) fluxes in the unsaturated zone and proxies for biodegradation, such as
temperature changes. The monitoring approaches for MNA and NSZD are distinct.
Whilst assessment of NSZD is encouraged where appropriate, this document is
restricted to MNA.

2
Figure 1: Step-wise approach to MNA assessment, showing main objectives and
outputs at each step. The diagram shows a linear process, but there may be
iteration.

3
Step 1: Monitored Natural Attenuation Screening
The first step in assessing MNA as a potential remediation option is to consider whether
it is likely to be a feasible and effective remediation solution, which mitigates risks
identified in the CSM and risk assessment process. The initial step is a desk-based
assessment of the evidence to support MNA for the particular CoPC present. The
assessment should consider:

• Technical reliability. Are NA processes likely to be effective in managing risks for all
relevant source-pathway-receptor linkages throughout the duration of MNA?
• Practicability. Is there available time, access to areas of interest on the site and its
surrounding, and access to monitoring points to implement an MNA strategy?
• Economics. Is MNA likely cost-competitive (considering whole-life costs) with other
feasible options?
• Sustainability. Is MNA likely to be more sustainable than other feasible remediation
options, when assessed against the broad sustainability criteria described by SuRF-
UK (CL:AIRE, 2010a)?
• Regulatory and Institutional Controls. Is it compliant with the law and can risks be
adequately controlled throughout the project duration?

When assessing technical reliability, assessors will find that some CoPC have a much
larger research literature on NA processes, and published case studies on MNA
implementation (see Appendix 2). For other CoPC the literature may be less
comprehensive and there may be less evidence for prior investigation of NA processes.
The approach to Step 1 should reflect the existing body of scientific research; for well-
studied CoPC there may be no need for further assessment of the potential for
biodegradation / attenuation at Step 1 other than to provide reference(s) to the published
work.

In the case of MNA, which can take a number of years or even decades to complete, it
is important that the sustainability assessment follows a holistic approach, such as that
described by SuRF-UK. This particularly includes consideration of:

• SuRF-UK Indicator SOC 2 ‘Ethics and equity’, and in particular the effects on
intergenerational equity by bequeathing unacceptable risk to future generations that
might feasibly be addressed sooner; and
• SuRF-UK Indicator ECON 5 ‘Lifespan and flexibility’, and in particular the ability of
an MNA strategy to manage risks in the long term where land-use or ownership might
reasonably be expected to change within the duration of an MNA project, and/or be
impacted by the potential effects of climate change (CL:AIRE, 2022; Environment
Agency, 2023).

NA screening criteria covering a range of relevant considerations are presented in

Appendix 1, and it is recommended that this structure forms the basis for an initial
assessment of MNA viability.

A step-wise approach to MNA screening is provided in Figure 2.

4
Figure 2: Step 1 – monitored natural attenuation screening.

5
Step 2: Field Natural Attenuation Demonstration
Having confirmed that NA processes are likely to occur at the site and that there are no
obvious barriers to selecting an MNA strategy, Step 2 requires site-specific
groundwater/aquifer physical and biogeochemical characterisation to demonstrate:

• whether the NA processes are currently occurring under site-specific field conditions;
• the rate at which NA processes occur;
• whether receptors are currently protected and identified risks are managed; and
• if receptors are not protected, why this might be, and what actions could be taken to
enhance NA processes to overcome the limiting factors.

A lines of evidence approach is taken, and the results are often presented in graphical,
statistical and/or visual form, and may be compared to predictive models (Appendix 5).

Collection of good quality environmental data that meets relevant data quality objectives,
from a suitably designed and constructed monitoring network, is critical to ensure later
interpretation of trends in contaminant concentration, mass discharge and behaviour is
reliable. Characterisation data for MNA assessment should be incorporated into a refined
CSM.

Monitoring of the concentrations and mass flux of the CoPC in relevant and consistent
locations over time, and analysis of trends (to show sufficiently declining plume
concentrations, mass discharge and stability or shrinkage) are the main requirements of
the primary line of evidence. Supporting secondary evidence is provided by analysis of
degradation products/metabolites, other compounds that are consumed or produced
during the degradation of the CoPC (e.g. terminal electron acceptors, or electron
donors), and geochemical parameters that influence attenuation potential including
aquifer organic carbon content (fOC), redox potential, and pH. Statistical analysis and
visualisation can be adopted using spatial-temporal trend analysis and smoothing
techniques, such as GWSDAT and MAROS, which can generate visual images of plume
development over time.

Tertiary evidence includes microbiological and advanced geochemical data that can
further indicate the extent of mineralisation, occurrence of microbial degradation
processes, or other reactive processes, and also provide quantitative estimates of
biodegradation rates.

In all instances the primary and secondary lines of evidence are needed. If these two
lines of evidence are consistent and compelling the tertiary line of evidence may not be
necessary, but if there is ambiguity the tertiary data may be helpful.

Where the evidence is weak or conflicting, or there are novel, unusual, or multiple CoPC
that are subject to different NA processes, all three lines of evidence may be necessary.
Table 1 illustrates typical lines of evidence data for common CoPC.

6
Table 1: Typical lines of evidence data requirements for common CoPC.
CoPC Primary Secondary Tertiary

Petroleum Main CoPC, e.g. Oxidants depleted (e.g. Rarely necessary

hydrocarbons benzene, toluene, O2 [dissolved oxygen],
ethylbenzene, xylene NO3- [nitrate], SO42- Microbial community
(BTEX), total [sulfate]) /by-products analysis (e.g. cell
petroleum generated (e.g. Fe2+ counts) demonstrating
hydrocarbon [ferrous iron], Mn2+ increased biomass
fractions [manganese II], S2- production within
[sulfide], CO2, CH4) plume
during oxidation of
CoPC Compound specific
isotope analysis
pH, redox potential (CSIA) of CoPC and/or
electron acceptors
Inhibitory conditions
(e.g. salinity)

Chlorinated Parent compound Degradation products, Microbial species

ethenes and contaminative e.g. cis-1,2- capable of reductive
degradation dichloroethene, vinyl dechlorination e.g.
intermediates (e.g. chloride, ethene, dehalococcoides
tetrachloroethene, ethane, acetylene, Cl-
trichloroethene, cis- CSIA of CoPC
1,2-dichloroethene, Electron donors –
vinyl chloride) available (labile) organic
carbon, H2 [hydrogen]

pH, redox potential

Reactive FeS [iron

sulfide] mineralogy

Inhibitory conditions
(e.g. O2 [for highly
saturated chlorinated
compounds], SO42-,
chloroform)

Ether Main CoPC, e.g. Oxidants depleted in Rarely necessary if

oxygenates methyl tertiary-butyl oxidation of CoPC, e.g. degradation products
(either as pure ether (MTBE), O2, NO3-, SO42-, or can be detected
product, or tertiary amyl methyl produced by redox
within gasoline) ether (TAME), ethyl process: Fe2+, Mn2+, S2-, Gene sequencing for
tertiary-butyl ether CO2, CH4 known ether
(ETBE) oxygenates
Degradation products,
degradation potential,
e.g. tert-butyl alcohol
e.g. Eth-B
(TBA), tert-butyl formate
(TBF)

pH, redox potential

7
 CoPC Primary Secondary Tertiary

Ammonium Ammonium Oxidants depleted in Rarely necessary

aerobic or anaerobic
oxidation of CoPC, e.g.
O2, NO2- [nitrite]

Transformation
products, e.g. NO2- ,
NO3-, N2 [dissolved
nitrogen] and N-oxide
gases

Aquifer cation exchange

capacity (CEC), and
dissolved major cations

pH, redox potential

Metals Metals Metals speciation Stable isotope analysis

Radioisotopes (if Sorption coefficient, e.g.

applicable) Kd

CEC

Mineralised forms (e.g.

sulfides, oxides,
carbonates etc)

Major ions, redox

chemistry

pH, redox potential,

Fe2+, S2-, TDS [total
dissolved solids], DIC
[dissolved inorganic
carbon] and alkalinity

Guidance on biogeochemical assessment tools (tertiary line of evidence) is presented in

Appendix 8 and Appendix 9.

Alongside specific and targeted data analyses described in Appendix 5, flow and
transport models can be used to integrate and consider variability in complex site
datasets to support demonstration that NA is effective. Modelling can provide a means
to confirm the conceptual model for NA (i.e. whether simulation of the conceptual model
matches observation data) and a rigorous framework for identifying data gaps and
uncertainties. Modelling can be used to quantify attenuation processes and understand
how current conditions arose and may change (Appendix 7).

A step-wise approach to field NA demonstration is provided in Figure 3.

8
Figure 3: Step 2 – field demonstration of natural attenuation.

9
Step 3: Prediction and Modelling Future Natural Attenuation
Behaviour
If the assessment completed at Step 2 shows that NA processes are currently occurring
at a rate that manages any potential risks, Step 3 follows and requires an assessor to
consider how the CoPC will behave in the future under a range of reasonable and
foreseeable scenarios.

Trends in groundwater quality (and supporting lines of evidence) are extrapolated into
the future so that assessors can predict the future performance of MNA, and can
estimate the duration over which NA processes might need to be relied on to reach
project closure.

The future plume predictions should consider the medium to long-term system and how
changes might affect the success of an MNA solution. It is important that any remediation
project is resilient to changing circumstances such as:

• significant water level, flow regime and water chemistry changes (e.g. as a result of
climate change, or flood events);
• foreseeable changes to land use that may change existing or introduce new source-
pathway-receptor linkages, or which restrict access to monitoring infrastructure; and
• foreseeable changes to land ownership that may make long-term access or
accountability for remediation difficult.

Hydrogeological models, including simple tools such as Remedial Targets Worksheet

(RTM) and ConSim, and NA-process models such as CoronaScreen, BioScreen,
BioBalance and BIOCHLOR can be used to predict future concentration trends and
whether MNA remains effective for a range of potential future scenarios. Where more
complex hydrogeological simulation is required, numerical reactive transport models
may provide greater insight (Appendix 7 – models).

The outcome of Step 3 is confirmation (or otherwise) that NA processes can be relied on
to act in the future, and lead to achievement of remediation objectives. Given the
potentially extended timescale for NA processes to reach remedial target concentrations,
wider risk management requirements (i.e. institutional controls to prevent creation of new
exposures, or long-term access rights to monitoring infrastructure) may be helpful.

10
Step 4: Implementation – Performance Monitoring and Verification of
Monitored Natural Attenuation Projects
If an MNA strategy is agreed by the relevant parties at Step 3, the project progresses to
the implementation phase (Appendix 6). Regular groundwater monitoring is undertaken
to collect representative data to confirm MNA effectiveness. The frequency of monitoring
should take account of:

• the range of hydrogeological conditions (e.g. seasonal variation);

• groundwater / CoPC transport velocities (i.e. sample rapidly-moving groundwater
more frequently than slow-flowing);
• distance and travel-time to receptors; and
• provide ability to initiate an alternative course of action in a timely manner if MNA
proves ineffective.

Monitoring is likely to be more frequent in Step 2, reducing in frequency in Step 4 and

ultimately ceasing once trends are established and it has been demonstrated that risks
are being managed.

The duration of monitoring is likely to reflect the potential risks present at site if MNA is
not effective, and also the level of confidence in attenuation of the CoPC (Figure 2).
Monitoring for NA of a moderate concentration petrol (BTEX) plume (which is readily
biodegradable in aerobic conditions) may only require a few years data; whereas a plume
of tetrachloroethene (PCE) from a DNAPL source in a deep aquifer (biodegradable under
anoxic conditions) may require a greater, and perhaps much greater, duration of
monitoring data to confidently demonstrate trends in concentration, plume stability and
footprint towards remedial goals.

As performance monitoring data are collected, assessors will need to review the data
and confirm that the strategy continues to manage potential risks. If it fails to manage
the identified risk adequately, or new and unacceptable risks are identified, an alternative
strategy may need to be put in place. A Contingency Plan should be developed that
identifies alternative approaches if MNA is unsuccessful.

Ultimately, if MNA is successful, a verification report will document the effectiveness of

the works (Environment Agency, 2023). The Environment Agency’s guidance on
verification follows a very similar lines of evidence approach to MNA set out in this
document. Reporting project verification should draw on the existing CSM, NA lines of
evidence, and performance monitoring data to confirm whether MNA has adequately
mitigated risks, and further monitoring is therefore unnecessary.

A step-wise approach to implementation, monitoring and verification is provided in

Figure 4.

11
Figure 4: Step 4 – implementation, monitoring and verification.

12
Appendix 1: Summary of Screening
Criteria to Assess the Feasibility of
Monitored Natural Attenuation
Table A1.1: Summary of screening criteria for assessing the feasibility of MNA.

Feasibility of MNA Strategy

Screening criteria
High Moderate Low

A. Technical factors

Primary source of groundwater Release stopped. Release stopped. Release

contamination1 Soil and Impact not removed continuing. Input
groundwater impact to groundwater
removed or being continuing
removed

Plume delineation Fully delineated Partially delineated, Poorly delineated

including in direction
towards receptors

Contaminant plume (dissolved Shrinking Stable Expanding

in groundwater) status

Non-aqueous phase liquid Absent Residual saturation, Mobile NAPL, and

(NAPL) presence or stable/shrinking expanding NAPL
NAPL footprint footprint

Persistence of CoPC in Readily attenuated Not readily degraded Attenuation

groundwater (degraded) under under conditions processes poorly
conditions present present on site understood
on site

Dominant attenuating Irreversible and Reversible and non-

mechanisms destructive destructive

Mobility of CoPC2 Medium Low High

Pollution potential3 of daughter Less polluting than Equally polluting More polluting than
products parent compound parent compound

Combined effects of multiple No effect - Act as co- Impose inhibitory

contaminants attenuation occurs contaminants effects
independently

Aquifer heterogeneity Homogeneous Moderate Highly

heterogeneity (e.g. heterogeneous (e.g.
layered porous highly fractured /
media) karst)

Rate of groundwater migration Slow Medium Rapid

Receptor No receptors (e.g. Receptors present Receptors present

abstraction wells, (low risk) (high / imminent
surface water) risk)
identified

Groundwater Source Protection Lies outside SPZ Lies within SPZ III Lies in SPZ I or
Zones (SPZ) SPZ II4

Current and foreseeable Low Medium High

groundwater use5

13
 Feasibility of MNA Strategy
Screening criteria
High Moderate Low

Level of confidence in High - Moderate - Low - single set of

monitoring data comprehensive comprehensive monitoring data
monitoring dataset monitoring dataset
spanning multiple spanning multiple
seasons and years seasons

Confidence and understanding High (e.g. dissolved Low (e.g. DNAPLs

of contaminant distribution substances in in deep
shallow heterogeneous
homogeneous aquifer)
aquifer)
B. Regulatory factors

Acceptability to the regulator No policy objection No policy objection Policy objections in

principle
No authorisation Authorisation
required required Authorisation
refused

C. Sustainability, practicability and economic factors

Monitoring locations Access confirmed Access possible for Limited / no access

for on-site and off- on-site and off-site
site monitoring in the monitoring in the
long term long term

Financial provisions Long-term, legally- Long-term, non- No long-term budget

binding budget legally binding in place
secured budget secured

Objectives of landowner Long-term interest in Medium-term interest Short-term

site (>10 years) (3 - 10 years) ownership/
developer (<3
years)

Sustainability More sustainable Less sustainable

than alternative than alternative
options options

OVERALL All high / High, medium and One or more show-

intermediates lows, but no show- stopping criteria
stoppers* present, or
No lows
No factors of high
feasibility rating

* Criteria highlighted in bold italics would normally preclude MNA as a sole remedial option
1 Primary source of contaminants to groundwater, e.g. leaking pipe, sewer, tank, leachable/mobile
contaminants in the deposited materials/soil/unsaturated zone.
2 Medium mobility enables degradation products to be removed, thus driving degradation reactions.
3 Pollution potential is a function of the persistence, mobility and toxicity of the contaminant.
4 Source Protection Zones defined in England Wales. For SPZ II which have been defined by
the Environment Agency using the 25% of total of SPZ III (i.e. low groundwater flow velocity
aquifers), then site-specific factors may increase the feasibility of NA.
5 Groundwater uses that should be considered include: water abstraction (e.g. public water
supply), baseflow to surface waters and groundwater-dependent terrestrial ecosystems.

14
Appendix 2: Processes Involved in
Natural Attenuation
A2.1 Introduction
Natural attenuation (NA) is the reduction of CoPC concentrations in the environment
through three main processes:

1. Physical phenomena (advection, dispersion, diffusion, matrix diffusion, dilution and

volatilisation);
2. Geochemical reactions (sorption and chemical or abiotic reactions); and,
3. Biochemical processes (aerobic and anaerobic biodegradation).

Some of these processes may simply redistribute contaminant mass within the mobile
phase (e.g. dispersion), some transfer the contaminant from the mobile phase to an
immobile phase (e.g. sorption, which results in retardation) and some result in a loss in
contaminant mass (i.e. are destructive, such as degradation).

This section will provide an overview of the processes of NA for common environmental
contaminants such hydrocarbons and chlorinated ethenes. Equations for calculating the
rate of contaminant degradation for MNA are also provided.

A summary of the main processes affecting contaminant transport is provided in

Table A2.1, with further details provided throughout the section.

15
Table A2.1: Summary of important processes affecting solute fate and transport (modified from Wiedemeier et al. 1999).

Category Process Description Dependencies Effect and implications for MNA

Physical processes Advection Movement of solute by bulk Dependent on aquifer Main mechanism driving contaminant
groundwater movement. properties, mainly hydraulic movement in the subsurface and is
conductivity and effective typically calculated and presented as
porosity, and hydraulic average linear groundwater velocity,
gradient. Independent of also termed seepage velocity. It does
contaminant properties. not result in a loss of contaminant
mass.

Dispersion Mixing of fluid and solutes due Dependent on aquifer Causes longitudinal, lateral, and
to groundwater movement and properties (e.g. variation in vertical spreading of the contaminant
aquifer physical pore size and geometry, plume. Reduces solute concentration
heterogeneities layering etc.) and scale of but does not result in mass loss.
observation. Independent
of contaminant properties.
Diffusion Spreading and dilution of Dependent on contaminant Diffusion of contaminant from areas of
contaminant due to molecular and aquifer properties such high concentration to areas of low
diffusion. as grain size variation and concentration. Generally unimportant
contaminant concentration relative to dispersion, except for very
gradients. Described by fine-grained porous media where
Fick’s Laws. advection is very low – in which case
molecular diffusion can be an
important component of hydrodynamic
dispersion. Does not result in loss of
contaminant mass.

16
Category Process Description Dependencies Effect and implications for MNA

Physical processes (cont.) Matrix diffusion Diffusion into a low As above within an aquifer A two-step process; (1) Contaminant
permeability zone within an of heterogeneous diffusion occurs relatively slowly within
aquifer of heterogeneous permeability, for example, low permeability bands in an aquifer,
permeability. bands of silt in a sand and temporarily sequestering a proportion
gravel aquifer. In fractured of the contamination (loading); (2)
dual porosity formations Following a reduction in the
such as a Chalk aquifer, concentration of contamination in
matrix diffusion is important higher permeability zones, the slow
for solute transport and NA. diffusion of contamination out of low
permeability zones results in a gradual
contaminant release into the higher
permeability aquifer over an extended
timeframe, extending the lifetime of
plumes (“back-diffusion”). In fractured
dual porosity aquifers, contaminants
diffuse from the fracture pore water
into the matrix pore water during
loading/plume migration, and diffuse
back into the fracture water when
contaminant loadings decrease. See
Thornton et al. (2006).
Recharge Movement of water into the Dependent on aquifer Causes dilution of the contaminant
saturated zone. matrix properties, depth to plume and may replenish electron
groundwater, depth to acceptor concentrations, especially
contaminant plume, surface dissolved oxygen.
water interactions, and
climate

17
Category Process Description Dependencies Effect and implications for MNA

Physical processes (cont.) Volatilisation Volatilisation of contaminants Only occurs at air-water Removes contaminants from NAPL
dissolved in groundwater into interface. (described by Vp) and groundwater
the vapour phase (soil gas). (described by H) and transfers them to
soil vapour. This is typically more
significant in shallow water tables.
Relative to biodegradation, this is
normally a minor component of MNA.
Further reading is provided in
Technical Bulletin 20 (CL:AIRE,
2019a) and emergent NSZD good-
practice guidance from CL:AIRE
(2024).

Geochemical processes Sorption Reversible partitioning Dependent on aquifer Tends to reduce apparent solute
between aquifer matrix and matrix properties (organic transport velocity or can remove
solute whereby contaminants carbon, clay and mineral solutes permanently from the
become sorbed onto solid content, bulk density, groundwater via sorption to the aquifer
phase, principally organic specific surface area, and matrix, however, it is not considered
carbon and clay minerals, or porosity) and contaminant that solutes are permanently removed
metal oxides / hydroxides. properties (solubility, as desorption may occur. Sorption
hydrophobicity, octanol- does not result in a net loss of
water partitioning contaminant mass.
coefficient for organic
contaminants).

Abiotic degradation Chemical transformations that Dependent on contaminant Can result in partial or complete
degrade contaminants without properties, aquifer and degradation of contaminants. Rates of
microbial facilitation, such as groundwater geochemistry. overall mass destruction are typically
hydrolysis. slower than for biodegradation. Results
in a loss of contaminant mass.

18
Category Process Description Dependencies Effect and implications for MNA

Geochemical processes Partitioning from NAPL Partitioning from NAPL into Dependent on aquifer Dissolution of contaminants from
(cont.) groundwater. NAPL, whether matrix and contaminant NAPL represents the primary source of
mobile or residual, tend to act properties (such as NAPL dissolved contamination in
as a continuing source of composition and effective groundwater. It should be noted that
groundwater contamination. solubility of organic the composition of the dissolved phase
compounds according to plume will vary over time with ongoing
Raoult’s Law), as well as NAPL dissolution, as approximated by
groundwater mass flux the temporal variation in effective
through or past NAPL. solubility of the NAPL constituents.
This can influence the monitoring
priorities over the project duration.

Biochemical processes Biodegradation Microbially mediated oxidation- Dependent on groundwater May ultimately result in complete
reduction reactions that geochemistry and aquifer degradation of contaminants. Typically,
degrade contaminants. geochemical properties, the most important process acting to
microbial population and reduce contaminant mass. It should
contaminant properties. be noted, however, that biodegradation
Biodegradation can occur does not always result in
under aerobic and/or mineralisation. Metabolic intermediates
anaerobic conditions. of contaminants can form, such as cis-
1,2-dichloroethene from the
biodegradation of trichloroethene.

19
Figure A 2 . 1 illustrates the different concentration profiles that would be expected for
advection, dispersion, sorption and degradation.

Figure A 2.1 : Idealised section along a contaminant flow path to illustrate

influence of advection, dispersion, sorption and degradation, after a given
duration of time.

The concentration of a contaminant introduced into an aquifer is shown as Point 1 in

Figure A2.1. At Point 2, processes such as sorption together with the gradual release of
the contaminant from the aquifer matrix result in a lower concentration with distance from
the source, but with greater spreading of the contaminant peak. Point 3 shows the same
active processes as in Point 2, but with degradation occurring simultaneously. This
results in a lower curve peak due to destructive loss of contaminant mass. If advection
occurs alone, the spread of the contaminant peak is limited by the effective solubility of
the contaminating substance, and can mirror the initial contaminant input concentration
if sufficiently water soluble (Point 4). With dispersion and advection together (Point 5),
the solute concentration is reduced due to longitudinal, lateral, and vertical spreading of
the contaminant peak.

A2.2 Physical Processes

Advection is the main process in the migration of contaminants and is driven by the
properties of the media, independent of the molecular physical or chemical properties of
the contaminant. It describes the transport of dissolved substances (solutes) by
groundwater under a hydraulic gradient. Non-reactive (conservative) substances travel

20
at the same rate as water. Reactive solvents may be retarded by other processes and
travel more slowly than the groundwater. The equation for one-dimensional advective
transport is given in Guerrero and Skaggs (2010).

Dilution is a physical process of NA that reduces the concentration of a contaminant,

but does not affect its total mass, toxicity or mobility. It describes the mixing of
contaminated water by clean groundwater. It is likely to be an important mechanism in
reducing concentrations, wherever small quantities of contaminant reach the aquifer with
a comparatively large groundwater flow or throughput. Further dilution can occur by:

• Uncontaminated infiltration (recharge of precipitation and infiltration of surface

waters (lakes, rivers)) away from the source area (this is the only mechanism of
dilution that is strictly applicable to NA);
• Contaminated groundwater discharging to a clean surface water body or mixing with
clean water at an abstraction point.

Infiltration can be an important mechanism in introducing electron acceptors (dissolved

oxygen, nitrate, sulfate) where contaminants are being biodegraded.

Dispersion will reduce contaminant concentrations by spreading the contaminant (in

a longitudinal, lateral and vertical direction) as groundwater flows through the
aquifer. It reduces the concentration of a contaminant, but does not affect its total mass,
toxicity or mobility. Dispersion can facilitate biodegradation by reducing contaminant
concentrations below toxic thresholds and spreading the plume into areas with electron
acceptors. Further information about dispersion and biodegradation can be found in
Wilson et al. (2005).

Dispersion occurs due to mechanical dispersion and molecular diffusion and can be
represented by the following equations:

D = Dd + D* Equation A2.1
and D = ∝Dw + α.v Equation A2.2

where D = hydrodynamic dispersion (m2/s)

D* = mechanical dispersion (m2/s)
Dd = molecular diffusion coefficient through
medium (m2/s)
α = dispersivity (m)
v = groundwater velocity (m/s)
∝ = tortuosity of medium

Dw = molecular diffusion coefficient in water (m2/s)

For many groundwater systems, diffusion is small or negligible compared to mechanical

dispersion and Equation A2.2 reduces to D = α.v

21
Mechanical dispersion is the main process in spreading contaminants and is a
result of variation in the velocity of water movement through pores of different size,
tortuosity (flow path length), and frictional variations within the pore space. Dispersion
has a longitudinal (parallel to the flow direction), transverse (perpendicular to the flow
direction) and vertical component. As the scale of the plume or system increases,
dispersion will also increase (i.e. it is scale dependent). The value of dispersion will
directly reflect the heterogeneity of the system. Further formulas and a more detailed
description can be found in Freeze and Cherry (1979).

Diffusion is observed as the movement of contaminants from regions of higher

concentration to lower concentration but occurs due to random atomic scale
movement of atoms and molecules. It reduces the concentration of a contaminant, but
does not affect its total mass, toxicity or mobility. Diffusion is slow in comparison to
mechanical dispersion, and only becomes significant in no-flow or very low-flow
systems, or over very long timescales. Diffusion is a key process in bringing electron
acceptors and electron donors together with bacteria and transferring solutes to
surfaces. This is because diffusion is highly significant in vertical transport and dispersion
processes and to a lesser extent in horizontal transport and dispersion, thereby allowing
lateral mixing into plumes.

Matrix diffusion occurs in aquifers with variable high and low permeability bands such
as sands and gravels containing silt layers. Diffusion of contamination into a low
permeability zone, temporarily sequesters contamination. Following a reduction in the
concentration of contaminants within the high permeability zones, a concentration
gradient is formed, and diffusion slowly occurs out of the low permeability zone back into
the aquifer, typically extending the lifetime of plumes. The process, is shown in
Figure A2.2, with the consequence of ongoing or renewed contamination of groundwater
commonly referred to as “rebound”.

22
Figure A2.2: Matrix diffusion (Source: WSP).

For dual porosity systems, such as fractured sandstone aquifers and the Chalk,
diffusion of contaminants from the mobile fissure water to the less mobile pore water
can be an important mechanism in retarding contaminant movement, and is referred to
as rock matrix diffusion.

Below is an equation describing the diffusive flux in porous media (based on Equation 3
and Equation 8 in Parker et al., 1994).

23
 𝑅𝑅𝑅𝑅
𝐽𝐽𝐷𝐷 (0, t) = Φ∁𝑠𝑠 � 𝜋𝜋𝜋𝜋𝑒𝑒 Equation A2.3

𝐷𝐷𝑒𝑒
≡ 𝜏𝜏 ≈ Φ𝑃𝑃 Equation A2.4
𝐷𝐷𝑜𝑜

Equation A2.3, where:

JD = diffusive flux at time t [M/T/L2] where M is mass, L is length and T is time

Φ = porosity [L3/L3]
Cs = solute concentration [M/L3] (often taken as pure phase solubility with symbol Sw
[M/L3])
R = retardation factor [-]
De = effective diffusion coefficient [L2/T]
t = time [T]

Equation A2.4, where:

De = effective diffusion coefficient [L2/T]
D0 = diffusion coefficient in water [L2/T]
τ = tortuosity
Φ = porosity [L3/L3]
P = an exponent factor with values between 1.3 and 5.4 depending on the geologic
material

Volatilisation of volatile contaminants to soil vapour occurs at the capillary fringe and
results in removal of contaminant mass from the groundwater, but is not inherently
destructive. Volatilisation is dependent on the physico-chemical characteristics of the
contaminant, and is dependent on site-specific conditions including temperature, depth
to water and porosity. This is generally not a significant mechanism due to the area of
contaminated groundwater exposed to soil gas. Also, as the capillary fringe is quasi-
immobile, transfer across it is dominated by aqueous phase diffusion coefficients, which
are around four orders of magnitude lower for volatile organic compounds (VOCs) in the
liquid phase, than in the gas phase. The limited vertical dispersion across the capillary
fringe is dominated by diffusion, therefore VOCs struggle to transfer. However, once at
the capillary fringe, partitioning of a volatile substance from the dissolved phase into the
vapour phase is described by its Henry’s Law constant:

CV = H × C
Where:
CV = concentration in vapour phase (mg/l)
H = Henry’s Law Constant (dimensionless)
C = concentration in aqueous phase (mg/l)

24
A2.3 Geochemical Processes
Sorption describes the interaction of a contaminant between water and soil. This
process will reduce contaminant concentrations by their removal from solution due to
interaction with the matrix of the aquifer through which groundwater is moving. There
is no mass reduction of the contaminant. Sorption can occur as a result of:

• adsorption, the attachment of a solute to a soil particle’s surface;

• absorption, the movement of a solute (diffusion) into the structure of a porous
particle where it sorbs onto an internal surface; and
• ion exchange, the replacement of a sorbed ion by the contaminant.

Sorption will retard the rate at which contaminants move through the system. The
retardation of a contaminant can be defined as:

𝑣𝑣
𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 (𝑅𝑅𝑅𝑅) = Equation A2.6
𝑢𝑢

Where:
Rf = retardation factor
u = velocity of contaminant or solute (m/d)
v = velocity of groundwater flow (m/d)

Further information and calculations for retardation can be found in Lovanh et al. (2000).

Sorption and desorption kinetics refer to the rate at which a contaminant either attaches
to or detaches from a sorbent. Desorption is generally slower than sorption, such that
contaminant concentrations are reduced, although the sorbed contaminant can
represent a longer-lasting source than those dissolved within groundwater.

Sorption is a function of:

• the nature of the contaminant (conservative contaminants such as chloride are not
sorbed, whereas reactive contaminants, such as metals can be strongly sorbed);
• the contaminant (solute) concentration;
• the nature and concentration of other contaminants (competition with other
contaminants can reduce the number of sites for sorption or competition with other
cations);
• nature of the soil/rock matrix, including surface area;
• presence of clay, organics and oxyhydroxides which can provide sites for sorption;
• environment, the pH and redox potential of the system can influence sorption.
The sorption of some metals is very sensitive to pH and redox conditions; and
• flow rate, in terms of the kinetics of sorption.

For non-polar organic and inorganic contaminants sorption occurs preferentially to soil
organic matter or to clay minerals, and sorption of metals occurs to oxides and
hydroxides. In most aquifers, sorption to organic matter is the dominant process, except
where the organic content is low and then sorption to mineral surfaces is the main
process (Ball and Roberts, 1991).

25
When considering sorption to organic matter as a general process, it is important to
distinguish between Koc (organic carbon-water partitioning coefficient) and Kom
(partitioning coefficient normalised to soil organic matter). The use of these terms will
depend on what is measured in the aquifer material. It is possible to convert Kom to Koc
using a conversion factor of 1.724 (Koc = 1.724 Kom) (Dragun, 1988).

In situations where thermally altered carbonaceous material (TACM) is present it may

produce anomalously high Koc values due to the enhanced sorption to TACM. This could
lead to wrong estimates of sorption by order of magnitude. The issue is described in
more detail in Wang et al. (2013) and Rivett et al. (2019).

The partition coefficient for the sorption of organic contaminants to organic matter can
be calculated as follows:

Partition coefficient for non-polar organic chemicals (e.g. aromatic hydrocarbons

such as benzene, toluene):
Kd = Koc ×foc Equation A2.7
Partition coefficient for ionic organic chemicals (e.g. phenol)

Kd = Koc,n (1 + 10pH-pKa)-1 + Koc,i (1 - (1 + 10pH-pKa)-1) Equation A2.8

Where:

Kd = soil-water partition coefficient (l/kg)

Koc = organic carbon partition coefficient (l/kg)
foc = fraction of organic carbon (fraction)
Koc,n = sorption coefficient for related species (l/kg)

Koc,i = sorption coefficient for ionised species (l/kg)

pH = pH value
pKa = acid dissociation constant

The partition coefficient (Kd) describes the distribution of a solute between groundwater
and the solid and is typically represented by either:

26
 1. Linear isotherm

𝐶𝐶 Equation A2.9
𝐾𝐾𝑑𝑑 =
𝐶𝐶𝑠𝑠

2. Freundlich isotherm
1
𝐶𝐶 𝑁𝑁
𝐾𝐾𝑑𝑑 = Equation A2.10
𝐶𝐶𝑠𝑠

3. Langmuir isotherm

𝐶𝐶𝑠𝑠
𝐾𝐾𝑑𝑑 = Equation A2.11
𝐶𝐶(𝑏𝑏 − 𝐶𝐶𝑠𝑠 )

Where:
= partition coefficient (l/kg)
C = concentration in the aqueous phase (mg/l)
Cs = concentration in the solid phase (mg/kg)

b = maximum amount of contaminant that can be sorbed (g/g)

N = chemical-specific coefficient (values of 1/N typically range from 0.7 to 1.1)

Solutes sorbed onto colloids (colloidal sorption) may be transported through the
aquifer system. Colloidal particles of sub-micron sized organic matter and minerals occur
naturally in soils and groundwater, and have been found to play a role in the transport of
trace metals and radionuclides (Honeyman, 1999).

Complexation. Metal ions in aqueous solution are typically present as complexes. A

complex is an ion in a combination of cations with anions or molecules. Chelating agents
such as humic substances which may be found in landfill leachate can form soluble
complexes with heavy metals such as nickel and zinc which can be highly mobile in the
environment. Soils and aquifer materials components differ greatly in their sorption
capacities, their cation and anion exchange capacities, and the binding energies of their
sorption sites. Polyvalent cations (e.g. Zn, Cu, Ni) may be strongly adsorbed on
phyllosilicates (e.g. clay minerals) due to the presence of –SiOH or –AlOH groups
capable of chemisorbing these ions. Organic matter and variable charge minerals (Mn,
Fe and Al oxides) are much more effective scavengers of polyvalent cations because
complexation processes are the dominant binding mechanisms. Anionic forms of
elements sorb primarily to variable charge minerals, carbonate, and at the edges of
phyllosilicates. They are not typically sorbed on soil organic matter, but certain elements
(e.g. borate, arsenate, arsenite, selenite) can bind to humic substances. As the variable
charge is pH-dependent and varies with pH, anionic sorption to variably charged
surfaces will increase with pH.
27
Oxidation/reduction. A chemical or biological reaction where an electron is transferred
from an electron donor to an electron acceptor and results in a change in the valence
state of the ion. In many cases the solubility of the ion will also be different, giving rise to
precipitation or sorption of the ion. For example, hexavalent chromium (soluble) occurs
under oxidising conditions. If conditions become reducing, this is converted to trivalent
chromium (insoluble) and this metal is precipitated out of solution. Changes to redox
conditions may reverse these reactions.

Solution/precipitation. Contaminants may be precipitated out of solution if

physiochemical conditions change. For example, changes to pH and water chemistry
(e.g. ionic strength and ionic composition) can cause dissolved metals to precipitate out
of solution or become dissolved. This includes (i) precipitation of metal oxyhydroxides
and carbonates under alkaline pH conditions or in the presence of carbonate ions and
(ii) precipitation of metal sulfides under anaerobic sulfate-reducing conditions in the
presence of sulfide ions.

A2.4 Chemical or Abiotic Degradation

Biodegradation is often considered the dominant destructive NA mechanism in
groundwater. However, several common groundwater contaminants can also degrade
through abiotic processes, that, in some cases, may be the primary or only destructive
process occurring (Brown et al., 2007). Abiotic chemical degradation occurs when a
compound reacts in natural conditions without catalysis by microbes or other life forms
(Adamson and Newell, 2014). This section focuses on abiotic degradation processes
for chlorinated solvents, as these are common groundwater contaminants and owing to
the complexity of the reactions potentially occurring. The detoxification or degradation
by abiotic processes of other groundwater contaminants, such as MTBE, chromium (VI)
and uranium (VI), are presented elsewhere in the literature (e.g. Elsner et al., 2007; Hyun
et al., 2012; Lee and Batchelor, 2002).

Since the 1970s, it was understood that trichloroethanes and tetrachloroethanes

underwent spontaneous abiotic degradation in groundwater via hydrolysis to form 1,1-
dichloroethene (1,1-DCE) and trichloroethane respectively (Mabey and Mill, 1978;
Jeffers et al., 1989). Most other chlorinated solvents were assumed to be resistant to
abiotic degradation, so these processes were largely overlooked in MNA protocols
published during 1990s and early 2000s (e.g. Wiedemeier et al., 1998; Environment
Agency, 2000). However, understanding of abiotic degradation reactions, particularly
those associated with catalytic reactions on surfaces of iron-rich minerals (Brown et al.,
2007; He et al., 2009; He et al., 2015), has been advanced over the past 15 years, and
approaches to quantify the contributions of iron-bearing minerals to contaminant
degradation are now available (He et al., 2009; Lebrón et al., 2015; Wiedemeier et al.,
2017).

Initial research focused on carbon tetrachloride, 1,1,1-trichloroethane (1,1,1-TCA),

tetrachloroethene (TCE) and tetrachloroethene (PCE) abiotic degradation processes on
mineral surfaces including iron sulfides (FeS, pyrite), magnetite and so-called ‘Green
Rusts’, that can occur naturally in subsurface anaerobic environments. The significance
of iron-bearing minerals to chlorinated ethene NA is such that the abundances of
magnetite (indicated by magnetic susceptibility measurements) and FeS (iron sulfide)
are amongst five key parameters correlated with TCE, cis-1,2-dichloroethene (cis-DCE)
28
and vinyl chloride (VC) degradation rates in recent industry research (Lebrón et al.,
2015).

It is important to note that these reactive iron minerals are often biogenically formed. For
example, iron-reducing and/or sulfate-reducing bacteria may be responsible for the
formation of iron sulfide, which is involved in the reductive dechlorination of a
contaminant. Consequently, these reactions are often referred to as biogeochemical, or
biologically-mediated abiotic degradation (BMAD), to acknowledge the biological
component (Adamson and Newell, 2014).

Abiotic degradation is typically more favourable for the more chlorinated compounds
(trichloro-, tetrachloro- etc) compared to the less chlorinated compounds ([mono]chloro-,
dichloro-). In general, rates of abiotic degradation are slower than biotic degradation
rates. However, abiotic processes may be important for NA of chlorinated solvents
where high mass loadings of reactive minerals are generated in situ or where the activity
of dechlorinating bacteria is low.

A2.5 Biochemical Processes

A2.5.1 Biodegradation

Biodegradation is the main process in the NA of organic contaminants and results in a

mass loss (destructive) and is typically estimated using a first-order decay model,
although other conceptual models (e.g. instantaneous reaction) can be used to describe
biodegradation processes, if data allows, as emphasised in the suite of numerical models
described in later appendices. Organic compounds are biodegraded via either oxidation
or reduction of the organic contaminant, when electron donors, electron acceptors and
nutrients are combined by microorganisms to produce metabolic by-products and energy
for microbial growth. This can be represented by the following generalised equation.

Microorganisms + electron donor + electron acceptor + nutrients

↓
metabolic by-products + energy + microorganisms

Aliphatic and aromatic hydrocarbons (e.g. BTEX) serve as the electron donor and are
broken down in the process. Electron acceptors, in order of preference for utilisation by
microbes, include oxygen, nitrate, manganese (IV), iron (III), sulfate and carbon dioxide.
Manganese and iron are typically present in the mineral form. Depending on the electron
acceptor used, the metabolic by-products include carbon dioxide, water, nitrogen gas,
manganese (II), iron (II), sulfide, dissolved hydrogen and methane. Specific organic
intermediate compounds may also accumulate or be transiently detected with these
reaction products during biodegradation. The intermediate compounds can be
independent signatures of biodegradation, for example, TBA for ether oxygenate
biodegradation.

Decreases in the concentration of soluble electron acceptors and corresponding

increase in the concentration of metabolic by-products provide indirect evidence for
degradation. Table A2.2 provides a summary of changes in contaminant, electron
acceptor and metabolic by-product concentrations during biodegradation. The
degradation process can vary in different parts of the plume, for example, anaerobic

29
degradation may be occurring at the centre of the plume and aerobic degradation at the
margin of the plume.

Table A2.2: Trends in contaminant, electron acceptor and metabolic by-product

concentrations during biodegradation (modified from Wiedemeier et al., 1998).

Analyte Trend in analyte Terminal electron accepting

concentrations during processes causing trend
2, 3
biodegradation

Petroleum Decrease Aerobic respiration, denitrification,

hydrocarbons and Mn(IV) reduction, Fe(III) reduction,
polycyclic aromatic sulfate reduction, methanogenesis
hydrocarbons
(PAHs)

Highly chlorinated Parent compound Reductive dechlorination and cometabolic

solvents (3 or more concentrations decrease, oxidation
Cl atoms) and daughter products
daughter products increase initially and then
may decrease

Lightly chlorinated Decrease Aerobic respiration and Fe(III) reduction

solvents (2 or less (direct oxidation) and cometabolism
Cl atoms) (indirect oxidation). Also reductive
degradation to ethene, ethane.

Dissolved oxygen Decrease Aerobic respiration

Nitrate Decrease Denitrification

Mn(II) Increase (metabolic by- Mn(IV)1 reduction

product)
Fe(II) Increase (metabolic by- Fe(III)1 reduction
product)
Sulfate Decrease Sulfate reduction
Methane Increase Methanogenesis
Chloride Increase Reductive dechlorination or direct
oxidation of chlorinated compound. In
most cases, a significant difference is
impossible to measure.

Redox Decrease Aerobic respiration, denitrification,

(oxidation/reduction Mn(IV) reduction, Fe(III) reduction,
potential) sulfate reduction, methanogenesis and
halorespiration

Dissolved carbon Increase Aerobic respiration, denitrification, Fe(III)

dioxide reduction and sulfate reduction

Notes:
1. Mineral phase
2. Oxygen is the most favoured electron acceptor for microbes in the biodegradation of organics. Anaerobic
bacteria cannot function if dissolved oxygen concentrations exceed 0.5 mg/l (i.e. if dissolved oxygen levels are
greater than this aerobic degradation is the most likely process). Multiple processes can occur simultaneously
within aquifers due to niche conditions in localised areas.
3. Microorganisms will generally use electron acceptors in the following order of preference: oxygen, nitrate,
manganese, iron, sulfate, CO2

30
Figures A2.3 and A2.4 illustrate the geochemical evolution of a groundwater system
contaminated with petroleum hydrocarbons. There are, however, two theories regarding
the spatial distribution of electron acceptor use; (i) the redox zonation concept; and (ii)
the plume fringe concept. These are shown in Figure A2.4. The redox zonation concept
revolves around microorganisms preferentially and discretely utilising more
thermodynamically-favourable electron acceptors. Recent literature, however, indicates
that biodegradation within a plume of contamination may be better described by the
plume fringe concept, in which the dissolved electron acceptors are depleted in the
plume core, with biodegradation occurring by oxygen, nitrate or sulfate reduction at the
fringes due to replenishment by surrounding groundwater (Meckenstock et al., 2015;
Thornton, 2019).

Figure A2.3: Conceptual section of (a) oxidation/reduction (redox) zones in

groundwater, and (b) changes in distribution of electron acceptor and metabolic by-
products in groundwater, with distance from contaminant source.

31
Figure A2.4: Comparison of redox zonation and plume fringe concepts within a
hydrocarbon plume, both describing the spatial distribution of electron acceptors
and processes of respiration. Reprinted (adapted) with permission from
Meckenstock et al. (2015). © 2015 American Chemical Society.

The degradation of other organics can be more complex. Under anaerobic conditions,
reductive dechlorination is the primary mechanism by which biotransformation of PCE
and TCE occurs, and halorespiration (i.e. microorganisms capable of using chlorinated
ethenes as terminal electron acceptors) is the process by which microorganisms
dechlorinate chlorinated ethenes to ethene. This process is sequential PCE  TCE 
cis-DCE  VC  ethene. The complete chlororespiration of cis-DCE and VC is known
to occur by only a few species of Dehalococcoides, with the dechlorination of VC
occurring most efficiently under highly reducing methanogenic conditions (Thornton et
al, 2016). Complete dechlorination will only occur if sulfate is completely reduced, and a
fermentable source of organic carbon is present to provide hydrogen as the electron
donor (NICOLE, 2005; Xiao et al., 2020).

Under the correct environmental conditions (noting that such conditions often require
human intervention to be achieved and sustained), chlororespiration can play a
significant part in the NA of chlorinated contamination, however, there are several
potential causes of incomplete dechlorination, which frequently result in the
accumulation of cis-DCE and VC (Bradley and Chapelle, 2010):

1. An insufficient supply of electron donors and nutrients/trace elements;

2. Competition for available hydrogen with other species of bacteria;

3. The presence of nitrate, which can act as an alternative electron acceptor;

4. Few or no microorganisms capable of dechlorinating cis-DCE and VC; and

32
5. Inhibitory substances such as chloroform or oxidised chlorinated ethene compounds
present in the groundwater.

Inhibition of chlorinated ethene biodegradation can occur in areas in which high

concentrations of sulfate are present due to sulfate-reducing bacteria out-competing
dechlorinating microorganisms (such as Dehalococcoides spp.) for electron donors
(such as hydrogen) and generating sulfide gas.

Due to the highly oxidised nature of PCE and TCE, neither are considered primary
substrates for aerobic microbial degradation. However, as the number of chlorine
substituents in a chlorinated ethene decreases, the tendency for it to undergo oxidation
increases. Hence, the aerobic degradation of DCE and VC has been demonstrated
(Mattes et al., 2010).

The metabolites formed during the degradation of chlorinated solvents can be used as
an indicator that NA is occurring. However, some daughter products are more toxic than
the parent (e.g. VC produced as an intermediate of TCE reductive dechlorination to
ethene). Metabolites are often susceptible to degradation but may persist if conditions
are unfavourable. Isotopic (e.g. compound specific isotope analysis [CSIA, Appendix 8])
and/or biological analyses (e.g. molecular biological tools [MBTs, Appendix 9]) can
provide supporting evidence to demonstrate when metabolite degradation to benign end
products is occurring.

Cometabolism. Process in which a compound is fortuitously degraded by an enzyme

or cofactor produced during microbial degradation of another compound. Chlorinated
solvents, PAHs and some pesticides can be degraded by cometabolism (Thornton et al.,
2016).

Under oxic conditions, a number of organisms have been shown to be capable of the
cometabolism of chlorinated ethenes to CO2 via a non-specific oxygenase, which
oxidises chlorinated ethenes to CO2 fortuitously (Bradley and Chapelle, 2010). This
process requires the presence of oxygen as well as a primary carbon substrate to
maintain the production of the oxygenase. Plumes containing both chlorinated ethenes
and aromatic compounds are fairly common, and under oxic conditions, the
microorganisms responsible for oxidising aromatic compounds, can co-metabolise
chlorinated ethenes. However, in many field settings, contaminant plumes that contain
high enough concentrations of aromatic compounds for cometabolism to occur, tend to
be anoxic, as oxygen has typically been preferentially consumed during microbial
respiration (Bradley and Chapelle, 2010).

Under anoxic conditions, in situ biotransformation of chlorinated ethene parent

compounds appears to occur primarily by reductive dechlorination (Thornton et al.,
2016).

Fermentation. Microbial metabolism in which a particular compound is used both as

an electron donor and an electron acceptor resulting in the production of oxidised and
reduced daughter products. Fermentation is typically the first step in the breakdown of
complex organic contaminants to simpler organic metabolites which are then used in
respiration reactions (e.g. SO4-reduction, Mn/Fe-reduction, denitrification). The
presence of methane is evidence of fermentation reactions in groundwater.

33
A2.5.2 Estimates of Contaminant Decay Rates

The rate at which many contaminants (such as hydrocarbons and chlorinated

hydrocarbons) transform within the environment is commonly described using first-order
kinetics, often referred to as Single First Order (SFO) (NAFTA, 2015). SFO kinetics
describes reactions in which the concentration of one component is the rate-limiting step
to degradation. If the concentrations of other components are involved in the rate-limiting
step, the order can change to a higher order (e.g. second order), however, this section
will primarily focus on first-order kinetics (Boesten et al., 2006).

Use of SFO kinetics can be useful in the evaluation of attenuation processes (see also
Appendix 5) occurring within groundwater on contaminated sites, such as characterising
trends within contaminant plumes, and providing an estimation of the time required to
reach remediation goals (Newell et al., 2002). They are typically used to estimate bulk
contaminant attenuation rates (sum of all NA processes causing a decrease in
concentration in groundwater) and contaminant biodegradation rates, according to the
focus of the assessment (e.g. single versus multiple well-distance analysis) and specific
calculation method used. Their use can be considered as a primary line of evidence of
the occurrence and rate of NA (Newell et al., 2002). A number of types of rate constants
are available to represent different attenuation processes (Newell et al., 2002):

1) Concentration versus time – used to estimate how quickly remediation goals will
be met;
2) Concentration versus distance – used to estimate plume behaviour through a
combination of attenuation processes through bulk attenuation rate constants; and
3) Biodegradation rate constants – used to characterise the effects of biodegradation
on contaminant migration within models.

Concentration versus Time Attenuation Rate Constants

Concentration versus time attenuation rate constants, or point decay rate constants
(Kpoint), describe the behaviour of the plume at a single point, but cannot be used to
provide an indication of the distribution of contaminant mass within the groundwater
system. Data acquired from a single monitoring location are plotted as the natural log
versus time based on several sampling events. A rate constant is derived from the slope
of the line of best fit. This calculation can be used to estimate the time required (t) to
reach an end goal (Cgoal) at that specific location within the plume using the following
equation (Newell et al., 2002):

𝐶𝐶𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔
−𝑙𝑙𝑙𝑙 �𝐶𝐶 �
𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠
𝑡𝑡 = Equation A2.12
𝑘𝑘𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝

Concentration versus Distance Rate Constants

Concentration versus distance rate constants, or bulk attenuation rate constants (k), are
derived by plotting the natural log of the concentration from several wells downgradient
of a source zone versus the distance, and calculating the rate as a product of the slope
and groundwater seepage velocity (Slope m = k/vel or k = slope m.vel). The resulting
rate is characterised by the distribution of the contaminant in space at that particular time
34
point. However, a single plot cannot provide information on the time required to reach a
remediation end goal. The rate constant derived using this method incorporates all
mechanisms of attenuation for contaminants within groundwater and indicates how
quickly they are attenuating outside of the source (Newell et al., 2002).

The following formula is used to provide an estimate of the amount of time needed for
the contaminants (t) to meet a remediation end goal (Cgoal) as the contaminants move
downgradient (Newell et al., 2002):

𝐶𝐶𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 Equation A2.13

𝑙𝑙𝑙𝑙 �𝐶𝐶 �
𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠
𝑡𝑡 =
𝑘𝑘

To calculate the distance (L) that dissolved contamination will travel over a particular
amount of time (t) as they are decaying, the seepage velocity (Vs) and the retardation
factor (R) can be incorporated in the following equation (Newell et al., 2002):

𝑉𝑉𝑠𝑠
𝐿𝐿 = . 𝑡𝑡 Equation A2.14
𝑅𝑅

Such rate constants do not represent the contaminant biodegradation rate, and should
not be used within solute transport models, as attenuation processes have already been
taken into consideration.

Biodegradation Rate Constant

The biodegradation rate constant can be derived by a number of methods including
calibration of a solute transport model to field data, or comparison of the transport of a
tracer within groundwater to contamination.

A2.5.3 Biodegradation Research on Selected CoPC

The weight of evidence available from the published literature varies, and is likely to
influence the amount of site-specific data required in the early stages of an MNA lines of
evidence assessment. Figure A2.5 presents an assessment of the biodegradation rates
of selected CoPC, and the size of the published, peer reviewed research literature.

A search of the published literature was undertaken using Scopus in May 2020. Search
terms used were the substance name OR common acronyms, AND ‘biodegradation’
(e.g. (mecoprop OR MCPA) AND biodegradation). The number of articles identified by
Scopus is recorded as the number of research publications.

The biodegradation rate is the typical reported biodegradation rate (day-1) in soil or
groundwater, under preferable conditions for the substance in question (e.g. under
aerobic conditions for BTEX compounds, but under anaerobic conditions for chlorinated
ethenes). Biodegradation rate data were collated from Environment Agency (2000),
Aronson and Howard (1997) and Aronson et al. (1999), and should be regarded as
indicative median estimates of biodegradation rate for initial assessment. Site-specific

35
data (i.e. the primary line of evidence) are required to demonstrate MNA at each project
site.

The biodegradation rates are indicative only. CoPC will biodegrade at different rates
depending on site-specific conditions (e.g. electron acceptor/donor supply; microbial
preference to biodegrade more labile CoPC first; toxic effects of high contaminant/salinity
concentrations etc.)

Gridlines are added to separate:

1. CoPC with a small research literature (<50 articles), Moderate (50 – 500) and large
(>500 articles); and
2. CoPC that are rapidly biodegradable in the subsurface environment (equivalent
first-order half-life <100 days), moderately biodegradable (100 – 365 days half-life),
and slowly (or not) biodegradable (>365 days).

Figure A2.5: The size of the published literature on the biodegradation of selected
CoPC, and their illustrative biodegradation rate in the subsurface under conducive
environmental conditions.

Additional Reading
A considerable volume of published material on biodegradation and its role in MNA is
available. Some selected references worthy of further reading include Thornton (2019);
Rivett and Thornton (2008); Thornton et al. (2016); Wilson et al. (2004) and Ottosen et
al. (2019). Recent research by Newell et al. (2021) and Ramos García et al. (2022)
describes the science of MNA to the emerging contaminants perfluoroalkyl and
polyfluoroalkyl substances (PFAS) and 1,4-dioxane respectively.

36
Appendix 3: Data Requirements for Lines of Evidence
Table A3.1: Parameters for MNA site characterisation and conceptual site model development.

Key lines for Applicability Use Potential application to NA evaluation (screening / demonstration / assessment)
assessment
F&T =
fate &
transport
modelling

Inorganic
Organic
General

I; II; III =
lines of
evidence
A. Geological and hydrogeological
Lithology and ✓ F&T Physical and geochemical properties of water-bearing units (aquifers and aquitards). Supports
structure assessment of groundwater flow and plume migration, including preferential pathways.
Porosity ✓ F&T Key property (including effective porosity) in assessing groundwater flow and contaminant transport.

Aquifer hydraulic ✓ F&T Essential for groundwater contaminant plume studies, including estimates of bulk attenuation rate,
conductivity, gradient degradation rate and mass discharge.
& groundwater flow
direction
Seasonal water level ✓ F&T Determines extent of smear zone and whether groundwater velocity and direction vary according to
fluctuations seasons. Note tidal influences and surrounding abstraction points can have impacts on temporal
fluctuations.
Rates of recharge ✓ F&T Factor in groundwater transport and input to numerical models.

B. Chemical
Parent and daughter ✓ I Provides a measure of the type and quantity of parent and biogenic daughter products. Used to estimate
contaminant biodegradation kinetics such as half-life or degradation rate constants.
concentrations
Co-contaminant ✓ ✓ I May indicate that more thermodynamically favourable degradation processes/pathways may occur, either
concentrations by acting as a co-metabolite or as a catalyst.

37
Key lines for Applicability Use Potential application to NA evaluation (screening / demonstration / assessment)
assessment
F&T =
fate &
transport
modelling

Inorganic
Organic
General
I; II; III =
lines of
evidence
C. Geochemical
Dissolved oxygen ✓ ✓ II Highest energy-yielding electron acceptor for biodegradation of organic constituents. Concentrations
typically below ~0.5 mg/l generally indicate an anaerobic pathway.
Nitrate ✓ II Thermodynamically next favourable electron acceptor after oxygen for microbial degradation of organics.
Depletion may indicate (denitrification) anaerobic degradation of organics.
Nitrite ✓ II Product of nitrate reduction, produced only under anaerobic conditions. Generally, a transient reaction by-
product that is rarely detected.
Iron (III) ✓ II Biologically available iron (III) can act as an electron acceptor during anaerobic degradation of organics.
Iron (II) ✓ II Indication of iron (III) reduction during microbial degradation of organic compounds in the absence of
oxygen, nitrate and manganese (IV) and potential for precipitation of reactive iron minerals (e.g. FeS).
Manganese (IV) ✓ II May act as an electron acceptor during anaerobic degradation of contaminants where more
thermodynamically favourable electron acceptors (e.g. oxygen and nitrate) are absent.
Manganese (II) ✓ II Indicator of anaerobic degradation of organics, where manganese (IV) acts as an electron acceptor.
Sulfate ✓ ✓ II Used as an electron acceptor in biodegradation of organic constituents. Reduced to form sulfide.
Sulfide II Reduced form of sulfate indicates reduced conditions and potential for precipitation of reactive iron
minerals (e.g. FeS).
Methane ✓ II Indicator of anaerobic conditions and of degradation of organics by methanogenic bacteria and/or from
biodegradation of acetate. Produced by the microbial reduction of carbon dioxide.
Ethane and ethene ✓ Metabolic end product of reductive dehalogenation of halogenated ethenes and ethane. Provides
evidence of complete dechlorination of these compounds. Indicates activity of methanogenic bacteria.
Dissolved hydrogen ✓ II Provides indicator of redox conditions, since concentrations can be correlated with types of anaerobic
activities (methanogenesis, sulfate reduction) in anaerobic environments.

38
Key lines for Applicability Use Potential application to NA evaluation (screening / demonstration / assessment)
assessment
F&T =
fate &
transport
modelling

Inorganic
Organic
General
I; II; III =
lines of
evidence
Total organic carbon ✓ II A measure of the total concentration of organic material (natural and anthropogenic) in water that may act
as a primary substrate for biological degradation (reductive dehalogenation).
pH ✓ ✓ II Microbial activity tends to be lowered outside of a pH range of 6 to 8.5, and many anaerobic bacteria are
particularly sensitive to pH extremes. Behaviour of metals influenced by pH.
Alkalinity/total ✓ ✓ II Provides an indication of the buffering capacity of the water and the amount of inorganic carbon dioxide
inorganic carbon dissolved in the water. The latter increases due to biodegradation of organic compounds which often is a
clear indicator of previous biodegradation of organic carbon compounds.

Eh (redox potential) ✓ ✓ II A measure of the oxidation/reduction potential of the environment. Typically ranges from +800 mV in
strongly aerobic conditions to -400 mV under methanogenic conditions.
Temperature ✓ ✓ II Affects rates of microbial metabolism. Slower biodegradation occurs at lower temperatures. Also affects
solubility of contaminants involved in reduction – oxidation processes.
Chloride ✓ II Possible indicator of biological dechlorination. Used as a conservative tracer.

Electrical ✓ II General water quality parameter, that can also be used with other water quality data to assess
conductivity groundwater ionic strength, total dissolved solids and salinity.
Phosphorus ✓ II Essential nutrient for microbial growth and biodegradation.

Volatile fatty acids ✓ II Metabolic by-products of the aerobic degradation of BTEX and complex organic matter (e.g. landfill
leachate plumes). Need to be compared to background values.
Abiotic degradation ✓ ✓ III Understand abundance and role of mineral phases in NA of metals, radionuclides, anions and specific
petroleum hydrocarbons and chlorinated solvents. Mineral formation via environmental processes such
as evaporation or degassing or through the presence of reactive minerals.
Carbon dioxide ✓ II Used as an electron acceptor in methanogenic (anaerobic) degradation of organics. Also a product of the
biodegradation of many organics.

39
Key lines for Applicability Use Potential application to NA evaluation (screening / demonstration / assessment)
assessment
F&T =
fate &
transport
modelling

Inorganic
Organic
General
I; II; III =
lines of
evidence
D. Biological
Microbial counts/ ✓ III Demonstrate the indigenous microorganisms are capable of degrading contaminants, and to provide an
biomass indication of degradation potential. Also used to establish nutrient requirements and limitations.
Ribonucleic acid ✓ III Used to detect specific bacteria that degrade contaminants.
(RNA) probes
Compound Specific ✓ III Analyses the relative abundance of various stable isotopes of the component elements of contamination
Isotope Analysis to determine whether contaminant degradation is occurring, investigate the degradation mechanism and
assist in identifying the contaminant source (Appendix 8).
Polymerase Chain ✓ III Amplifies the genetic material of microorganisms to levels that can be further analysed using other
Reaction techniques to detect microorganisms or target genes for contaminant biodegradation and process genetic
material for use in other diagnostic tools.
Quantitative ✓ III Quantifies a target gene based on deoxyribonucleic acid (DNA) or RNA to assess the abundance and the
Polymerase Chain expression of specific functional genes, microorganisms, or groups of microorganisms responsible for
Reaction contaminant biodegradation.
Microbial ✓ III Differentiates, and in some cases identifies, microorganisms by unique characteristics of universal
Fingerprinting biomolecules to provide a profile of a microbial community, identify a subset of the microorganisms
Methods present and quantify living biomass.
Microarrays ✓ III Detects and estimates the relative abundances of numerous genes simultaneously to provide a
comprehensive evaluation of the microbial diversity and community composition.
Stable Isotope ✓ III Detects the presence of an added synthesised form of the contaminant containing a stable isotope (e.g.
Probing 13
C) to determine whether biodegradation of a specific contaminant is occurring and identify
microorganisms responsible for this activity.
Enzyme Activity ✓ III Detects the transformation of surrogate compounds that resemble specific contaminants to quantify the
Probes activity of microorganisms with specific biodegradation capabilities.

40
Key lines for Applicability Use Potential application to NA evaluation (screening / demonstration / assessment)
assessment
F&T =
fate &
transport
modelling

Inorganic
Organic
General
I; II; III =
lines of
evidence
Fluorescence in situ ✓ III Detects the presence of targeted genetic material in an environmental sample to estimate the number
Hybridisation and/or relative activity of specific microorganisms or groups of microorganisms.
Environmental ✓ III Active sampling methods and passive microbial sampling devices in which subsurface microorganisms
Molecular colonise a solid matrix to collect biomass from environmental media to be used in conjunction with
Diagnostics specialised diagnostic methods.
Sampling Methods
Microcosm ✓ III Can be in situ or ex situ tests that allow a variety of amendments to be tested to stimulate bacterial
experiments degradation of contaminants of concern. Also, to examine any potential limitations on biodegradation
activity related to the contaminant mixture (e.g. toxicity effects), environmental conditions in the aquifer
(e.g. nutrient limitations) or obtain data on biodegradation rates (e.g. reaction kinetics).

41
Appendix 4: Data Acquisition
Table A4.1: Data acquisition.

Parameter Main data sources Comments

CoPC • Desk study (source audit) Extent and mass discharge of CoPC and degradation products plumes as
• Site data (sample concentrations, flux required by lines of evidence assessment
meter technology, membrane interface
probe [MIP] etc.)

Porosity • Laboratory measurement Important to differentiate between total and effective porosity and saturated
• Grain size versus partially saturated.
• Hydraulic tests Multiple methods may be required for consolidated versus unconsolidated
• Tracer test materials.
• Rock thin sections
• Literature

Henry’s Law Constant • Literature

Bulk Density • Laboratory measurement Used in estimating Kd and other transport / attenuation factors
• Literature

Clay content • Laboratory measurement Clay size and clay minerals

• Literature

Fraction of organic • Laboratory measurement Soil, aquifer sediment (if unconsolidated), rock core sample
carbon • Literature Used in estimating Kd and other transport / attenuation factors

42
Parameter Main data sources Comments

Sorption/partition • Literature Lithology, bulk density, pH dependent. Note competition between different
coefficient • Laboratory experiments species, chemical reactions, solubility, polarity, changes in media properties.
• Tracers
Hydraulic conductivity • Rising/falling head tests Hydraulic conductivity may vary laterally and vertically (heterogeneity,
• Packer tests anisotropy).
• Pumping tests Unsaturated zone hydraulic conductivity dependent on water saturation.
• Laboratory tests
• Grain size-based estimates Preferable to obtain bulk or horizontal hydraulic conductivity from field
• Hydraulic Profiling Tool (HPT) tests.
• Cone Penetrometer Tool (CPT) Laboratory tests are more appropriate for vertical hydraulic conductivity,
• Literature lower hydraulic conductivity materials, aquitards.
British Geological Survey Aquifer Properties Manuals.

Groundwater • Observation boreholes and/or monitoring Locations should aim to develop CSM, with representative response zones to
levels wells check preferential flow paths. Possibility of more than one hydrogeological
regime (i.e. components of downward, or upward flow, aquitards, semi-
Hydraulic gradient
confined, perched water).

Aquifer thickness • Boreholes Flow may be in discrete zones such that aquifer thickness may differ from
• Geophysical logging the total depth of the formation.
• Packer testing
Mixing depth • CoPC distribution Mixing depth can be estimated using empirical equations.
• Groundwater level variation

Aquifer geometry • Geological maps The hydrogeological framework model is fundamental to developing a
• Boreholes and monitoring wells defensible CSM for NA.
• Geophysical survey
• Core retrieval
• Fracture conditions (aperture size, infill
orientation etc.)

43
Parameter Main data sources Comments
Aquifer geometry (cont.) • Interbedded and stratified depositional
understanding to inform groundwater
regimes
• Sequence stratigraphy

Direct recharge • Climatological data (rainfall, evaporation) Variable recharge through complex depositional environments (including low
• Land-use, land surface permeability drift) and deposits of anthropogenic origin.
• Soil type

Indirect recharge • Flow gauging Include these locations/features in plots of field data to contour hydraulic
(leakage or discharge to • Desk study / utilities mapping gradients. Unusual variation in local gradient may be indicative.
sewers, drains, water
mains)

Receptors • Environment Agency and Natural England, Should include site inspections and walkovers
Scottish Environment Protection Agency
and NatureScot, Natural Resources Wales,
Northern Ireland Environment Agency,
Environmental Health Departments

Abstraction rates • Environment Agency, Scottish Environment Actual abstraction may not equal the licensed abstraction rate.
Protection Agency, Natural Resources
Wales, Northern Ireland Environment
Agency, local authorities

Dispersion coefficient • Empirical values (one tenth of distance The value of the dispersion coefficient is scale-dependent. Values reported in
plume has migrated) field experiments are often several orders of magnitude greater than from
• Model calibration laboratory experiments.
• Tracer studies
• Literature

44
Parameter Main data sources Comments

Aquifer mineralogy • Mineralogical analysis (x-ray diffraction, Distribution and abundance of mineral phases important for understanding
sequential extractions etc.) metal and anion transport, plus reactive minerals involved in biodegradation or
• Literature abiotic degradation (e.g. solid phase electron acceptors [Fe, Mn oxides,
carbonates] and reactive FeS minerals).

Natural Source Zone • Soil gas, vapour and temperature For petroleum hydrocarbon LNAPL, see also Technical Bulletin 20 (CL:AIRE,
Depletion (NSZD) measurement, CoPC source mass 2019a) and emergent NSZD good-practice guidance from CL:AIRE (2024).
discharge in groundwater, LNAPL
compositional change

NAPL properties & • Density, viscosity, interfacial tension, boiling See also the Illustrated Handbook of DNAPL Transport and Fate in the
distribution point, composition analysis (e.g. Subsurface (Kueper et al., 2003), the Illustrated Handbook of LNAPL Transport
fingerprinting, individual components), age and Fate in the Subsurface (CL:AIRE, 2014) and emergent NSZD good-
determination, LNAPL mobility practice guidance from CL:AIRE (2024).
assessments. NAPL dye testing, tracer
tests, bail down tests, Flexible Liner
Underground Technologies (FLUTe) liners,
ultra violet optical screening tool (UVOST),
tar-specific green optical screening tool
(TarGOST), dye-laser induced fluorescence
(Dye-LIF)

Biodegradation • Analysis of observed changes in Breakdown products with different properties. Consideration of the
contaminant concentrations biogeochemical environment. Typically represented as first or second-order
• Microbiological studies decay kinetic reaction. Alternatively, may be linked to available terminal electron
• Literature acceptor process indicator parameters (oxygen, nitrate, sulfate, ferrous iron,
• Lab & in situ microcosms, bio-traps, manganese II, methane).
compound specific isotope analysis,
Geochemical, isotopic and microbiological sampling in groundwater often
microbial cell presence, abundance and
requires specific sample handling and preservation (see BS EN ISO 5667-3
functional gene analysis, polymerase chain
[British Standards Institution, 2018], and laboratory/analytical method specific
reaction (PCR), quantitative polymerase
requirements).
chain reaction (qPCR)

45
Appendix 5: Methods of
Assessment
A5.1 Introduction
This appendix provides supporting information on some of the methodologies, tools and
visualisation techniques that are available to assist with the assessment and
demonstration of NA and degradation rates.

In 2004 the British Geological Survey (Lelliott and Wealthall, 2004) undertook a review
of the qualitative, quantitative and visual means of describing the evidence for NA. This
was organised according to the lines of evidence approach that is central to the
demonstration of NA with additional explanatory discussion and some visualisation
techniques for data. The reader is referred to that report for detailed descriptions of the
methodologies described here. This revised appendix also incorporates advances in
data presentation as appropriate from a number of other sources. The methods
presented are not exhaustive, but are believed to represent the principal methods
employed.

A5.2 Primary Lines of Evidence

A5.2.1 Graphical Techniques

Evidence for natural attenuation can be obtained by comparing contaminant

concentrations or ratios along the groundwater flow path where the change in solute
concentration in the groundwater over time often can be described using a first-order
decay rate constant (Lelliott and Wealthall, 2004; Rivett and Thornton, 2008). Examples
of these type of plots include:

• monitoring well concentration plots (concentration versus time) (Figure A5.1) in which
concentrations of CoPC are analysed over time in a single well to identify trends at
one point in the plume. These data are not representative of the plume as a whole
but can provide a useful indication of temporal behaviour in a particular location and
can be combined with other locations;
• plume centreline concentration plots (concentration versus distance) in which the
change in CoPC concentrations along the centreline of a plume for a given time
period (i.e. specific groundwater sampling event) are plotted (Figure A5.2). Critical in
this instance are a series of monitoring locations that are aligned along the centreline
of a plume and a hydrogeological regime that does not have widely variable flow
directions over the course of monitoring. This method is only really applicable to
analysis of stable or shrinking plumes. The data can also be difficult to interpret where
the geology / hydrogeology is complex and non-uniform along the centreline and the
centreline itself may be difficult to establish; and
• comparison of contaminant ratios where plots can include log-normalised
concentrations of contaminants with distance, and ratios of contaminant
concentrations with distance. Comparison of normalised concentrations for a
conservative contaminant to other contaminants can be used to identify different

46
 rates in migration due to sorption or degradation (Figure A5.3). For these purposes
conservative may be defined as a non-retarded, non-reactive contaminant that is
spilled at the same time or location, or a similar contaminant within the spillage that
is not readily biodegraded but has similar physical characteristics (Lelliott and
Wealthall, 2004). The use of contaminant ratios can be complicated by the presence
of multiple sources and background concentrations requiring subtraction.

It should be noted that concentration data can be mass or molar concentrations. Molar
concentrations are particularly useful for comparing parent with degradation product
(daughter) concentrations.

The above plots can be coupled with calculations of statistical parameters including the
slope of the line of best fit, coefficient of variation (COV), r2 value, and confidence levels.
Such functionality is available in a number of publicly available domain software
packages (e.g. MAROS & GWSDAT).

Examples of each of the three techniques are presented in Figures A5.1 to A5.3 (Lelliott
and Wealthall, 2004).

Figure A5.1: First-order decay for contaminants of concern for a single

monitoring well/location. Reproduced from Lelliott and Wealthall (2004) with
permission from the British Geological Survey © UKRI 2004 (BGS permit no.
CP23/057).

Figure A5.2: Centreline concentration plot for average contaminant of concern

concentrations for a stable plume, or individual monitoring event for a shrinking
plume. Reproduced from Lelliott and Wealthall (2004) with permission from the
British Geological Survey © UKRI 2004 (BGS permit no. CP23/057).
47
 Notes
• This plot is created by normalising the concentration of a solute at successive distances along the
plume flow path to the source concentration of that solute.
• This plot compares the attenuation of reactive solutes (e.g. by sorption or degradation) relative to
that of a conservative species, which is assumed to decrease in concentration along the flow path
by dilution due to dispersion.
• The form of this plot allows (i) different rates of contaminant migration due to sorption to be deduced
for contaminants which are known or assumed to be recalcitrant under the given conditions, (ii)
different rates of degradation to be deduced for contaminants with similar sorption characteristics.
Figure A5.3: Comparison of contaminant concentrations normalised to source
term concentration estimate (i.e. C/Co) (after Environment Agency, 2000).

A5.2.2 Visual Techniques

Plume contour plots

Plume contour plots in either plan view or as a cross-section through the centreline of
the plume are the most common visual method to identify whether a plume is stable,
shrinking, or expanding over time (Lelliott and Wealthall, 2004; Rivett and Thornton,
2008). Contour plots can (and should) also be created to show the spatial-temporal
distribution of biogeochemical indicator species and compared with the contaminant
contour plots for proper interpretation of the plume dynamics and associated degradation
processes. Cross-section contour plots are typically orientated along the centreline of
the plume and are used to give an indication of the vertical variation in contaminant
concentration. The latter is only applicable where suitable multi-level monitoring wells,
or nested groups of monitoring wells with differing depths are positioned in the plume.

In each of the above instances, visual examination over different time intervals can give
an immediate impression of the plume status and can also be used to compare the plume
shape and orientation to the groundwater flow direction and/or modelled predictions, as
shown in Figure A5.4

48
Figure A5.4: Comparison of projected vs actual plume migration (Wiedemeier et
al., 1999). © 1999 John Wiley & Sons, Inc.

2D visualisation or 3D interpolation software is widely available (e.g. Surfer, Earth

Volumetric Studio [EVS], Leapfrog, Spatial Analysis and Decision Assistance [SADA],
GWSDAT and RockWorks) and 2D and 3D contouring and gridding may be used to
construct contour maps and with 3D packages cross-sections along one or numerous
planes to visualise the plume. Visual analysis of plume orientation can also be coupled
with quantitative measures of a number of plume characteristics (Figure A5.5) including
plume area, average concentration, plume mass and centre of mass (Ricker, 2008) and
this functionality is included in GWSDAT (Figure A5.6) and MAROS (Aziz et al., 2000).

Figure A5.5: Decrease in dissolved plume mass over time for a chlorinated solvent
plume undergoing NA (Source: ERM).
49
Figure A5.6: Example of the summary output of plume metrics from GWSDAT:
plume mass (left), plume area (mid) and average concentration (right) (CL:AIRE,
2019b).
The creation of a contour plot or 3D visualisation requires interpolation (e.g. kriging) of
the chemical distribution between monitoring wells (Lelliott and Wealthall, 2004) and care
should be undertaken in interpretation. Contour plots and 3D models should be used
with caution where there is not a high-density monitoring network or where the geology
/ hydrogeology is spatially variable (Wilson et al., 2004), care should be taken on
inappropriate integration of the vertical, with well screens say at different depths / in
different units shown on the same spatial plot that may not always be appropriate and
misleading. In addition, during the course of an MNA study whatever the extent of data
available it is equally or more important the monitoring points and data analysis are kept
consistent (assuming the CSM does not change – e.g. if flow direction changes in
response to abstraction) to ensure that any changes observed are all being measured
to the same baseline and context. Any unavoidable changes should be phased in
gradually wherever possible. For example, if monitoring boreholes require relocation if
neighbouring land ownership changes then new boreholes should be installed ahead of
decommissioning old ones so there is overlap in dataset to allow comparison.

A5.2.3 Statistical Techniques

In addition to or as a supplement to the above, statistical procedures and models can

provide a formal, quantitative method for assessing plume stability (NJDEP, 2012).

The need for application of statistical tests and the nature of the tests will vary as a
function of site-specific conditions and data analysis requirements (UK TAG, 2012). The
methods must be appropriate for undertaking the trend assessment and be applicable
50
to the available data. Groundwater quality data possess unique characteristics that
require specialist approaches to statistical testing. Groundwater data often have
asymmetric or non-normal distributions. These ‘skewed’ datasets may therefore require
use of alternative non-parametric statistical methods where no assumptions are required
about the underlying data distribution (UK TAG, 2012). Alvarez and Illman (2005)
indicate that the Mann-Kendall Test (including the Seasonal Kendall) and the Mann-
Whitney U Test are widely applied.

Mann-Kendall Test

The Mann-Kendall analysis is a non-parametric statistical procedure that is used to

statistically assess if there is a monotonic upward or downward trend of the variable of
interest over time. A monotonic upward (downward) trend means that the variable
consistently increases (decreases) through time, but the trend may or may not be linear.

The Mann-Kendall test neither requires a specific statistical distribution of the data, nor
is the test sensitive to the sampling interval over which the monitoring data are collected.
The outcome of the procedure depends on the ranking of individual data points and not
the overall magnitude of the data points. Therefore, the Mann-Kendall procedure can be
used for datasets that include irregular sampling intervals, data below the detection limit,
and trace or missing data. The approach is particularly advantageous in cases where
outliers in the data could produce biased estimates using parametric trend analysis (GSI,
2012).

The Mann-Kendall test for trend analysis is available within a number of public domain
tools both as a component of broader packages (MAROS, GWSDAT) but also as a
standalone tool (e.g. GSI Mann-Kendall Toolkit [GSI, 2012]). The latter includes three
statistical metrics (GSI, 2012) as follows:

• The ‘S’ Statistic: Indicates whether concentration trend versus time is generally
decreasing (negative S value) or increasing (positive S value).
• The Confidence Factor (CF): The CF value modifies the S Statistic calculation to
indicate the degree of confidence in the trend result, as in “Decreasing” versus
“Probably Decreasing” or “Increasing” versus “Probably Increasing.” Additionally, if
the CF is quite low, due either to considerable variability in concentrations versus
time or little change in concentrations versus time, the CF is used to apply a
preliminary “No Trend” classification, pending consideration of the COV.
• The Coefficient of Variation (COV): The COV is used to distinguish between a “No
Trend” result (significant scatter in concentration trend versus time) and a “Stable”
result (limited variability in concentration versus time) for datasets with no significant
increasing or decreasing trend (e.g. low CF).

The rules applied by the GSI Mann-Kendall Toolkit to classify plume concentration trends
were developed based upon empirical analysis of hundreds of groundwater plumes (GSI,
2012).

An example of the calculation of the S Statistic is provided below in Table A5.1 and Table
A5.2 provides a summary of the statistical approach.

51
Table A5.1: Example of S Statistic calculation (after GSI, 2012).

Sample Event Number 1 2 3 4 5

Benzene concentration Total

13.95 42.08 33.9 33.67 18.05
(mg/l) Points

Comparison to event 1 +1 +1 +1 +1 +4

Comparison to event 2 -1 -1 -1 -3

Comparison to event 3 -1 -1 -2

Comparison to event 4 -1 -1

Apparent Decreasing Trend S= -2

Table A5.2: Example statistical metrics used in GSI Mann-Kendall Toolkit (Aziz et
al., 2003). © 2003 John Wiley & Sons, Inc.

S Statistic Confidence Trend

in Trend
S>0 CF > 95% Increasing

S>0 95% ≥ CF Probably

≥ 90% Increasing

S>0 CF < 90% No Trend

S≤0 CF < 90% No Trend

and COV
≥1

S<0 CF < 90% Stable

and COV
<1

S<0 95% ≥ CF Probably

≥ 90% Decreasing

S<0 CF > 95% Decreasing

Note: CF=Confidence Factor; COV=Coefficient of Variation. The user can identify two other categories of Data:
ND=Dataset where all values are non-detect, and N/A=locations with <4 sample results.

Mann-Whitney U test

The Mann-Whitney U test is another statistical test that may be useful at a site. The
outcome of the test is not influenced by the overall magnitude of the data, but rather is
based on the ranking of individual data points.

The test is conducted by vertically ranking the eight data points from lowest to highest,
with the lowest value on top and greatest value on the bottom. For each individual “A”
concentration, the number of “B” concentrations that occur below the “A” concentration
are counted. The four values (either zero or some positive number) are summed together

52
to obtain the U statistic. All non-detect values are considered zero. If two or more
concentrations are identical, then two vertical columns are constructed. In the first
column, the tying “B” concentration is ranked first, and in the second column the tying
“A” concentration is ranked first. An interim U is calculated for each column, and the
average of the interim U values is used as the final U value. If U = 3 then the null
hypothesis is rejected, and it is concluded with at least 90% confidence that the
concentration for the individual contaminant at that well has decreased over time. If
U > 3, the null hypothesis is accepted, and it cannot be concluded with at least 90%
confidence that the concentration for the individual contaminant has decreased with time
at that well (Wiedemeier et al., 1999).

In many groundwater systems there will be considerable seasonal variability in

parameter concentrations. This variability may introduce problems in the trend analysis
unless it can be corrected for. Where there are sufficient data within a given year, the
best way to do this is to fit a seasonal model to the data and then use this to “de-
seasonalise” the data. The Seasonal Kendall test is a modification of the Mann-Kendall
test that addresses short-term seasonal variability and allows evaluation of overall
trends. In a Seasonal Kendall test, the Mann-Kendall test is applied to each season (e.g.
quarter) separately and then the results are combined for an overall test (NJDEP, 2012).
The alternative, where there are variable or insufficient within year measurements, is to
remove seasonality by calculating the annual means, and then to perform the trend
analysis on the annual means. In this case Sen’s Method has been recommended
(NJDEP, 2012). Both the Seasonal Kendall test and Sen’s Method are robust methods
that allow for some missing data in the time series and are not badly affected by gross
errors or outliers in the data series.

crcCARE (2010) notes that at very low concentrations, these tests may be difficult to
apply, and selection of sampling data is important and to avoid biasing these statistical
tests, the same number of significant figures should be consistently used for a given
contaminant. This ensures that any plume trends are true data trends and not an artefact
of laboratory reporting formats (crcCARE, 2010).

A5.3 Secondary Lines of Evidence

A5.3.1 Natural Attenuation Rates

The use of first-order attenuation rate constants in NA studies has been described in
detail by Newell et al. (2002). Rate calculations based on the graphical methods outlined
in Section A5.2.1 for primary lines of evidence (well concentration plots and centreline
concentration plots) can be used as part of MNA studies to evaluate the contribution of
attenuation processes and the anticipated time required to achieve remediation
objectives. Note that these calculations are most easily applied where contaminant
concentrations are quite high and may be more difficult to interpret at plume margins
where lower concentrations may be at or near the limit of quantification as patterns can
get lost in ‘noise’ in the dataset. Table A5.3 describes each of the rate constants and
summarises the potential uses of each in NA studies. This is followed by a brief
description of each.

53
Table A5.3: Summary of first-order rate constants for NA studies (Newell et al.,
2002).

Rate Constant Method of Significance Use Rate of Constant

Analysis
Plume Plume Plume
Attenuation Trends? Duration?

Point C vs T Plot Reduction in NO* NO* YES

Attenuation contaminant
Rate concentration
over time at a
(Kpoint, time per single point
year)

Bulk C vs D Plot Reduction in YES NO* NO

Attenuation dissolved
Rate contaminant
concentration
(k, time per with distance
year) from source

Biodegradation Model Biodegradation YES NO NO

Rate Calibration, rate for
dissolved
(𝜆𝜆 time per Tracer contaminants
year) Studies, after leaving
Calculations source,
exclusive of
advection,
dispersion,
sorption etc

*Note: Although assessment of an attenuation rate constant at a single location does not yield plume attenuation
information, or plume trend information, an assessment of general trends of multiple wells over the entire plume is useful
to assess overall plume attenuation and plume trends.

Point attenuation rate constant

The point attenuation rate constant (Kpoint) uses contaminant concentrations with time for
a monitoring well located within the plume. The rate constant is calculated by plotting the
natural log of a concentration against time at a particular monitoring point (Figure A5.7).
This rate is the result of the combined effects of dispersion, biodegradation, and other
attenuation processes (Newell et al., 2002).This method is only applicable to shrinking
plumes (ASTM, 1998), if the plume is stable then Kpoint will be very small.

54
Figure A5.7: Determination of concentration versus time rate constant (Kpoint) (after
Newell et al., 2002).

A rate constant derived from a well concentration plot provides information regarding the
potential plume lifetime, or time to reach a remedial target, at that location, but cannot
be used to evaluate the distribution of the contaminant mass within the groundwater
system (Newell et al., 2002). The entire plume can be assessed by determining rate
constants in a number of monitoring wells throughout the plume (Lelliott and Wealthall,
2004).

Bulk attenuation rate constant (k)

The bulk attenuation rate constant (k) uses contaminant concentrations with distance
along the centreline of the plume for a given time period. The constant is derived by
plotting the natural log of the concentration versus distance and (if determined to match
a first-order pattern) calculating the rate as the product of the slope of the transformed
data plot and the groundwater seepage velocity (Figure A5.8). Degradation typically
occurs as a first-order rate reaction and would be expected to plot as a straight line on a
log-linear plot (ASTM, 1998). The rate constant calculated using this methodology is due
to the combined effects of dispersion, biodegradation, and other attenuation processes
(Newell et al., 2002). This technique is only applicable to stable or shrinking plumes.

55
Figure A5.8: Determination of concentration versus distance rate constant (k)
(after Newell et al., 2002).

Biodegradation rate constant (λ)

The biodegradation rate constant (λ) is a component of the bulk attenuation constant
described above and determines the portion of the overall attenuation that can be
attributed to biodegradation. The biodegradation rate constant can be determined by:

• comparing the contaminant concentration along the flow path with a conservative
contaminant (non-degraded), referred to as a conservative tracer;
• using the methodology derived by Buscheck and Alcantar (1995) that identifies
the contribution of biodegradation for a steady-state plume by coupling the
regression of contaminant concentration versus distance downgradient (centreline
concentration plot) to an analytical solution for one-dimensional, steady-state,
contaminant transport that includes advection, dispersion, sorption, and
biodegradation (Figure A5.9); and
• be calculated by calibration of a solute transport model to field data.

The principles of each are briefly described below. Any type of rate constant calculation
should be verified by observed groundwater concentrations during the performance
monitoring period.

56
Figure A5.9: Determination of biodegradation rate constant (λ) (after Newell et al.,
2002).

The use of conservative tracers to calculate biodegradation rate constants

For a tracer to be useful, it will need to be biologically recalcitrant and have similar
Henry’s Law constant and soil sorption coefficients to the contaminant of interest, or be
subject to less retardation than the contaminant(s) of concern. The tracer will also
normally be associated with the original contaminant spill. Examples of a conservative
tracer include:

• chloride or bromide (both examples of non-sorbing and non-degrading tracers), if

released within the original spill;
• trimethylbenzene (TMB) and tetramethylbenzene, (that are sorbing but more
recalcitrant) which are typically present in fuel mixtures, although under certain
conditions these organics can be degraded.

The concentration of a contaminant at a point (B) downgradient of the source (A) can be
corrected for the effect of dispersion, dilution and sorption using Equation A5.1:

𝑇𝑇𝐴𝐴
𝐶𝐶𝐵𝐵 𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜 = 𝐶𝐶𝐵𝐵 � �
𝑇𝑇𝐵𝐵 Equation A5.1

Where:
CBCorr = corrected concentration of contaminant at point B [M/L3]
CB = measured concentration of contaminant at point B [M/L3]
TA = measured concentration of tracer at point A [M/L3]
TB = measured concentration of tracer at point B [M/L3]

57
However, for conservative tracers, the following need to be demonstrated:
• the tracer is recalcitrant; and
• the tracer behaviour is otherwise similar to the contaminant and was released at the
same time and location as the contaminants of concern.

By plotting corrected contaminant distribution on a log-linear plot of corrected

concentration against downgradient travel time along the flow path the degradation rate
can be calculated using Equation A5.2:

1 𝐶𝐶𝐵𝐵
λ=− ln Equation A5.2
𝑡𝑡 𝐶𝐶𝐴𝐴

Where:
λ = first-order degradation rate [t-1]
CB = tracer-corrected contaminant concentration at time t at downgradient point B
CA = measured contaminant concentration at upgradient point A
t = travel time between points A and B where t = x/u (x = distance between A and B, u =
retarded solute velocity due to sorption)

In reality (field conditions) this requires careful planning and execution in all but the
simplest groundwater systems. Calculation of biodegradation rates using the above
method can be difficult in complex groundwater systems but remains a useful technique
to consider.

Determination of degradation rate for a steady state plume

For a steady-state plume, the first-order decay rate is given in Equation A5.3 (Buscheck
and Alcantar, 1995):

Equation A5.3
𝑉𝑉𝑐𝑐 𝑘𝑘 2
λ= �[1 + 2𝛼𝛼𝑥𝑥 ] � � − 1�
4𝛼𝛼𝑥𝑥 𝑣𝑣𝑥𝑥

Where:
λ = first-order biological decay rate [t-1]
Vc = retarded contaminated velocity (due to sorption) in the x-direction [Lt-1]
αx = longitudinal dispersivity [L]
k/vx = slope of line formed by making a log linear plot of contaminant concentration versus
distance downgradient along flow path, and where vx is the groundwater flow velocity

58
Application of analytical and numerical models
The first-order biodegradation rate can also be calculated by calibration of a solute
transport model to field data (Newell et al., 2002). Models that can be used include
BIOSCREEN, BIOCHLOR, BIOPLUME III, and MT3D, however it is necessary to ensure
that the lines of evidence are available to substantiate the derived biodegradation rate
and that the biodegradation rate has not been derived purely to fit the model (other
variables may be wrongly measured or estimated) (Lelliott and Wealthall, 2004). For
chlorinated solvents, models with the ability to stimulate reductive dechlorination as a
sequential first-order decay process should be utilised. Sequential first-order decay
means that a parent compound undergoes first-order decay to produce a daughter
product and that product undergoes first-order decay and so on. An illustration of the
behaviour of TCE and the production of associated daughter products is presented in
Figure A5.10.

Figure A5.10: Reactive transformation of chlorinated ethenes (adapted from Aziz

et al., 2000).

The role of sequential degradation in the MNA of chlorinated solvents is described in

detail elsewhere (Wiedemeier et al., 1998) together with methodologies and approaches
for incorporating the degradation rates of intermediate products (Aziz and Newell, 2002).
The further application of models to demonstrate MNA is addressed in Appendix 7 –
Groundwater Flow and Transport Models.

It is also recommended that:

• In this form of simulation model results should be compared with field data
(Figure A5.11) and the model parameter values adjusted to obtain a model fit with
the observed data.
• The final model parameter values should be assessed for reasonableness. For
example, if the analysis indicates a degradation rate with a half-life of five days, whilst
literature values for similar sites indicate values of 100 to 1000 days are more
appropriate, then the assessment should be critically re-evaluated.

59
• Finally a sensitivity analysis should be undertaken to determine which parameters
have the greatest influence on the model results and assess whether further data are
required.

Overall the analysis should be reviewed in terms of:

• uncertainty in understanding of the system and in the conceptual model;
• uncertainty in parameter values; some may vary by more than an order of magnitude;
• applicability of the model to the site (including model assumptions);
• use and relevance of literature values to define model parameters; and
• whether there is more than one solution, that is whether different combinations of
parameter values can give the same result.

Figure A5.11: Graph adapted from the Cape Canaveral TCE plume case study in
the BIOCHLOR manual (https://nepis.epa.gov/Adobe/PDF/P1000YUW.pdf). r2 is the
coefficient of determination and RMSE is the Root Mean Square Error. The inset
map shows plume concentration contours with observed TCE concentrations
with an overlay showing an inferred plume centreline and monitoring wells used
in the 1D BIOCHLOR model. This example is effectively the same as the bulk
attenuation rate (k) analysis (Figure A5.8).

60
Mass balance methods

The primary line of evidence in NA studies is the documented loss of contaminant mass
in the field (such as historic data showing a reduction in contaminant concentrations with
time). The quantification of reduction in contaminant mass within a plume or across a
defined boundary over time can therefore form an important part of the overall lines of
evidence for NA at a given site.

Calculation of mass in dissolved phase plume

The mass of contaminant in a dissolved phase plume can be estimated using
Equation A5.4:

𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚(𝑀𝑀) = 𝐶𝐶𝑎𝑎𝑎𝑎 𝑏𝑏 𝑛𝑛 𝐴𝐴 Equation A5.4

Where:
M = dissolved mass [M]
Cav = average plume concentration [M/L3]
b = aquifer or plume thickness [L]
n = porosity
A = plume area [L2]

Though a relatively simple equation, care is required in a number of key assumptions:

• determining the area occupied by the plume – this could include the area above
detection limits or the area above regulatory guidance concentrations, which may be
defined as a single area or could include the preparation of contaminant
concentration contours and calculating the area between each contour;
• determining the thickness of the plume, this is ideally based on and defined by, non-
detection of contaminants within the monitored profile and can be established from
multilevel monitoring wells or the results of a high-resolution site characterisation but
can also be estimated from traditional monitoring wells or from calculation of the
mixing zone (crcCARE, 2010).
• determining the average concentration in the plume – this may be calculated from
(crcCARE, 2010):

o the calculated arithmetic or geometric mean of all concentrations inside the

defined overall area of the plume;
o the calculated arithmetic or geometric mean between each contaminant
concentration contour; or
o using kriging or interpolation software.
2D and 3D contouring and gridding using commercially-available packages may be used
to construct contour maps and with 3D packages a volume estimate to allow estimation
of mass (see Figure A5.5 in Section A5.2.2) and are subject to the same assumptions
and limitations as observed for the production of visual graphics (and may be constrained
by the complexity of the hydrogeological setting).

61
Though simple to undertake, the calculation of contaminant mass in the dissolved phase
plume can be subject to considerable uncertainty that may over or underestimate mass
depending on a number of variables including:

• The lateral and vertical delineation of the plume and mixing zone (geological
heterogeneity and resolution, number of wells, spatial distribution, screen length,
single or multilevel monitoring well);
• The need for and consistency in appropriate groundwater sampling methods to
ensure data quality;
• The consistency in monitoring over a period of time (consistency in groundwater flow
direction, water table elevation, groundwater monitoring locations); and
• The methodology used for data interpolation should be consistent for the duration of
the study.

Groundwater plume mass estimates should therefore be interpreted with caution and
may have only order-of-magnitude precision (crcCARE, 2010). Nevertheless, they may
provide a useful relative measure of mass in the context of an MNA study. As a
minimum, a consistent approach that is representative of the plume area should be used
on each occasion to at least allow a relative comparison of mass with time and monitoring
well densities should be encouraged to reduce these uncertainties and investigate the
sensitivity of the mass estimate to new data.

In terms of estimation of a degradation rate from this data then if an estimate of plume
mass is undertaken using the same monitoring well network over a series of time periods
then an overall bulk attenuation rate can be calculated by plotting mass versus time (see
Figure A5.6). Trends of mass decline or stability (where the attenuation rate matches
source mass discharge) should be themselves subject to significance analysis (e.g.
Mann-Kendall, or a graphical treatment).

Calculation of mass flux

An alternative mass balance method is to calculate the contaminant mass flux for a given
plume (Thornton et al., 2016; Thornton, 2019; Farhat et al., 2006). Contaminant mass
flux is the rate at which contaminant mass passes through a defined cross-sectional area
perpendicular to the groundwater plume in an aquifer over time. In the context of MNA
studies then the calculation and documentation of stable or decreasing mass discharge
or flux trends can be a useful secondary line of evidence.

• Mass flux is a rate measurement equal to the contaminant mass moving across a
unit area of aquifer perpendicular to the groundwater flow direction. Units are
mass/area/time.
• Mass discharge is the total mass of contaminant moving across a control plane (or
area of interest) perpendicular to the groundwater flow direction. The area of interest
is generally large enough to contain the entire plume. Units are mass/time.

Within the context of the lines of evidence for NA then the assessment of mass flux or
mass discharge may be used in two ways:

• Over a series of different transects (control planes) drawn perpendicular to the flow
direction of a given plume and the mass flux at each calculated and examined to
indicate evidence of mass flux reduction with distance from the source.

62
• For a single transect and the results repeated over time to indicate reduction in mass
flux at a certain control plane over time.

Figure A5.12 is a schematic of a high-resolution site characterisation where three

transects have been constructed perpendicular to the groundwater plume to enable
detailed characterisation and assessment of mass flux / mass discharge. Figure A5.13
illustrates the calculated mass flux for an example site at four transects at different
distances from the source and over three different time intervals.

Figure A5.12: Use of multiple well transects (control planes) to measure mass
discharge and flux (USEPA, 2021).

63
Figure A5.13: Example mass flux with time (crcCARE, 2010).
The Interstate Technology and Regulatory Council (ITRC) in the USA has produced an
overview of the concepts, practical use and limitations of mass flux measurement (ITRC,
2010). The document describes a number of methods that are used to measure mass
flux and/or mass discharge:

• transects in which individual monitoring points are used to integrate concentration

and flow data;
• transects based on contaminant concentration contours, which rely on concentration
contour maps developed using groundwater monitoring data;
• well capture/pump test methods, which rely on extracting groundwater and
measuring the flow and mass discharge from the wells;
• passive flux meters, which estimate mass flux directly in wells; and
• by using solute transport models that require flow and concentration data as input
parameters.

In a mass transect approach the mass of contaminant flowing across a series of lines
(control planes) drawn perpendicular (normal) to the flow direction is estimated, as
follows:

64
 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷ℎ𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 = �(𝐶𝐶𝑎𝑎𝑎𝑎 𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊)for each depth interval Equation A5.5

Where:
Discharge = summation of flux for each depth increment
Cav = average contaminant concentration for depth increment [M/L3]
W = width of plume [L]
v = groundwater velocity [LT-1]
n = kinematic porosity
D = depth increment for each average concentration, or plume thickness [L]

The calculation is repeated for different lines drawn perpendicular to the flow direction.
This information can be used to compare the change in contaminant flux with time and
distance from the source.

To assist in the calculation of mass flux the Environmental Security Technology

Certification Program (ESTCP) of the U.S. Department of Defense (DoD) funded the
development of the Microsoft® Excel-based Mass Flux Toolkit (GSI, 2006). The Mass
Flux Toolkit is a publicly available software tool with the capability of comparing different
mass flux approaches including individual points or contaminant contours, calculating
the mass flux from transect data and estimating the uncertainty associated with a
calculation.

Uncertainty in mass flux estimates is a key issue in using mass flux as a metric. The
Mass Flux Toolkit describes three main sources of uncertainty in a mass flux estimated
from transect data (GSI, 2006):

• Type 1 Uncertainty in the actual concentration, hydraulic conductivity, and

hydraulic gradient measurements. The calculation of mass flux typically relies on
an adequate monitoring well network (in terms of locations and vertical density) and
ideally would use data from multilevel wells. Data from single long-screened wells
are less useful for this technique. The ITRC notes that the greatest sources of error
and uncertainty in mass flux or mass discharge estimates include estimates of
hydraulic conductivity (K) and contaminant concentrations (ITRC, 2010) and
measurements of specific discharge or Darcy velocity, or mass flux in situ are
recommended.
• Type 2 Uncertainty in the interpolation scheme. Different interpolation schemes
will result in different mass flux estimates. Some interpolation schemes, such as
kriging, provide local estimates of uncertainty.
• Type 3 Uncertainty associated with unmeasured values. This type of uncertainty
is related to Type 2 uncertainty. However, the uncertainty associated with areas of
high mass flux that are missed by the monitoring scheme is difficult to assess. This
may be addressed by installing a dense network of monitoring wells which is
consistent with the known aquifer heterogeneity (geology/stratigraphy) and spatial
variation in preferential flow paths that influence contaminant distribution and
transport (Wilson et al., 2004).

65
A5.3.2 Biodegradation Indicators

Evaluating indicators specific to the biodegradation process is of critical importance

when presenting secondary lines of evidence for NA as it is indicative of contaminant
destruction (Lelliott and Wealthall, 2004; Rivett and Thornton, 2008; crcCARE, 2010).
The microbial processes associated with the degradation of various groups of
contaminants are described elsewhere in this document (Section A2.5) but the major
electron acceptors and anticipated changes during biodegradation are summarised in
Table A5.4.

Table A5.4: Types of biodegradation reactions and preference by energy potential

(after Washington State Department of Ecology, 2005).
Type of Electron Metabolic Geochemical Redox Potential
Microbial Acceptor By-Product Indicator Eh
Respiration Response
(mV@pH 7, 25°C)

Aerobic Oxygen CO2 O2 ↓ CO2 ↑ +820 Most

Preferred
(oxidation)

Anaerobic Nitrate N2 NO3- ↓ CO2 ↑ +720

(reduction) (NO3-)

↓
Manganese Mn2+ Mn2+ ↑ CO2 ↑ +520
(Mn4+)

Ferric Iron Ferrous Iron Fe2+ ↑ CO2 ↑ -50

(Fe )
3+
(Fe )2+

Sulfate H2S SO42- ↓ CO2 ↑ -220

(SO42-)

Carbon Dioxide Methane CH4 ↑ ↑ -240 Least

Preferred
(CO2) (CH4)

Indicators of biodegradation can be identified graphically (contaminant/daughter product

ratios), quantitatively (mass balance and mass flux), or visually (contour/isopleth plots,
radial diagrams).

Contaminant ratio plots

Evidence of biodegradation can be obtained by comparing contaminant and breakdown

product concentrations or ratios along the flow path. A decrease in CoPC concentration
with an associated increase in breakdown product concentration, or an increase in the
ratio of breakdown product to parent contaminant concentration, is indicative of
biodegradation (Lelliott and Wealthall, 2004; Rivett and Thornton, 2008). Examples of
visualisation techniques include:

• plot of contaminant concentrations and breakdown product concentrations with

distance (Figure A5.14)
• plot of the ratio of contaminant concentrations with distance (Figure A5.15).

66
Figure A5.14: Comparison of breakdown products.

Figure A5.15: Comparison of contaminant ratios.

In assessing contaminant ratios, the following should be taken into account:

• the breakdown products may be present in the original contaminant source (for
example, TBA is a breakdown product of MTBE, but this compound is also often a
constituent component in petroleum fuels, and TCE may be present with PCE);
• the breakdown product may have been introduced by other contaminant incidents;
• the analytical technique/sampling method may not be appropriate to identify the
breakdown product;
• the sorption and volatilisation characteristics of the contaminants may not be
identical; and
• the effect of multiple sources or multiple contaminant releases, for example, if the
contaminants have a different history of release.

For chlorinated solvents the effectiveness of MNA can include an evaluation of

contaminant concentration or mass reduction, particularly as reflected in changing molar
concentrations of parent and dechlorination products over time. This includes the
following steps (AFCEE, 2004):

67
Step 1 – Molar Concentration: Calculate the concentration of each compound in mol/L
for each compound in the reaction sequence using the equation:

𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑖𝑖 𝐶𝐶𝑖𝑖 Equation A5.6

=
𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝑀𝑀𝑀𝑀𝑖𝑖

Where:
molesi = moles of compound i
Ci = concentration of compound i (grams per litre)
MWi = molecular weight of compound i (grams per mole)

Step 2 – Total Molar Concentration: Calculate the total concentration in moles per litre
by summing the concentrations of each compound in the reaction sequence.

To illustrate, consider the chlorinated ethenes with PCE as the parent

compound:

𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑒𝑒𝑒𝑒ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝐶𝐶𝑃𝑃𝑃𝑃𝑃𝑃 𝐶𝐶𝑇𝑇𝑇𝑇𝑇𝑇 𝐶𝐶𝐷𝐷𝐷𝐷𝐷𝐷 𝐶𝐶𝑉𝑉𝑉𝑉 𝐶𝐶𝐸𝐸𝐸𝐸ℎ𝑒𝑒𝑒𝑒𝑒𝑒

� = + + + +
𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 𝑀𝑀𝑀𝑀𝑃𝑃𝑃𝑃𝑃𝑃 𝑀𝑀𝑀𝑀𝑇𝑇𝑇𝑇𝑇𝑇 𝑀𝑀𝑀𝑀𝐷𝐷𝐷𝐷𝐷𝐷 𝑀𝑀𝑀𝑀𝑉𝑉𝑉𝑉 𝑀𝑀𝑀𝑀𝐸𝐸𝐸𝐸ℎ𝑒𝑒𝑒𝑒𝑒𝑒

Equation A5.7
Where :
𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑒𝑒𝑒𝑒ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
� = 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑐𝑐ℎ𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 𝑒𝑒𝑒𝑒ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒(𝑚𝑚𝑚𝑚𝑚𝑚/𝐿𝐿)
𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙

Step 3 – Molar Fractions: Calculate the molar fraction (ratio) for each compound.
For illustration, consider PCE. This calculation must also be completed for TCE,
DCE, VC, and ethene.

𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑃𝑃𝑃𝑃𝑃𝑃
𝑀𝑀𝑀𝑀𝑃𝑃𝑃𝑃𝑃𝑃 = 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙
𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑒𝑒𝑒𝑒ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 Equation A5.8
∑
𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙
Where:
MFPCE = molar fraction of PCE (unitless)

An example of a visualisation created using the above is shown in Figure A5.16.

68
Figure A5.16: Change in molar concentration over time in a chlorinated solvent-
impacted monitoring well (Source: ERM).

Mass balance mass flux methods

The mass balance, mass flux method, as detailed in Section A5.3.1, can also be used
to monitor the change in mass of CoPC breakdown products. An inverse relationship is
expected between concentration changes for CoPC and associated breakdown products
(Lelliott and Wealthall, 2004) as indicated in Figure A5.17 for chlorinated solvents and
Figure A5.18 for phenols.

69
Figure A5.17: Relationship between parent compound (TCE), breakdown products
(DCE, VC) and geochemistry over time in a chlorinated solvent-impacted
monitoring well (Source: ERM).

70
Figure A5.18: Relationship between phenol concentration and electron acceptors
over discrete high-resolution vertical intervals within a multi-level monitoring well
(Wilson et al., 2005).

Contour/isopleth plots

Similarly biodegradation can also be assessed visually using contour/isopleth plots for
breakdown products, electron acceptors/donors, and hydrochemical indicators. Contour
plots can be used to indicate the areal extent of indicators, or the vertical distribution and
should be for different time periods to identify temporal changes in indicator
concentrations (Lelliott and Wealthall, 2004; Rivett and Thornton, 2008).

Contour plots of CoPC breakdown products provide visual evidence of where

biodegradation is occurring, and there should be an inverse relationship between CoPC
and breakdown product concentration (Figure A5.19).

It is important to identify if the breakdown product is present in the source zone, or

introduced as a separate incident (multiple sources or contaminant releases), as this
could give a false indication of biodegradation (e.g. road gritting in winter can lead to
erroneously high chloride that can mislead evaluations of chlorinated solvent
degradation). Multiple lines of evidence are required in this case.

71
Figure A5.19: Spatial relationship between parent compound (TCE) and
breakdown products (DCE, VC and ethene) in a chlorinated solvent plume
(Source: ERM).

Radial Diagrams

A radial diagram visualisation approach allows simultaneous comparison of spatial and

temporal trends for multiple chemicals on one map (Lelliott and Wealthall, 2004). In this
approach a radial diagram is constructed with each of the axes assigned to either the
primary source of contamination, degradation products or electron acceptors (Carey et
al., 2003). Concentrations are then plotted on these axes as shown in Figures A5.20 and
A5.21. Variations of radial diagrams include pie charts or bar charts where segments are
defined according to electron acceptor concentrations.

72
Figure A5.20: Relationship between different electron acceptor concentrations in
two monitoring wells with high (>100,000 µg/l) and low (1 µg/l) benzene
contamination, indicating typical changes expected as a result of microbial
activity (Source: ERM).

Figure A5.21: Spatial relationship between benzene and different electron

acceptor concentrations in source monitoring well (shaded area) and
downgradient monitoring wells (Source: ERM).
73
Groundwater at the source or upgradient can be used to provide the initial baseline
graphical shape and this can then be compared spatially with water chemistry from other
parts of the site, or alternatively in the same location over time. Changes in the shape of
the graphic provide visual evidence of changes in concentration of either the
contaminants or electron acceptors.

Radial diagrams can be produced via proprietary software Visual Bio or can be produced
in Excel.

Figure A5.22 presents an example of a Visual Bio radial diagram showing the CoPC and
sequential breakdown products for TCE and how the relative concentrations change
along the contaminant plume. The outer data series (indicated by the blue line)
represents the concentration levels for each of these contaminants as they are measured
at the source of contamination. The inner data series (indicated by the yellow shading)
represents the concentrations of these contaminants measured at the monitoring wells
located downgradient from the contaminant source. This diagram shows decreasing
concentrations of contaminants downgradient of the source (primary lines of evidence),
and the increased concentration of breakdown products (e.g. VC and chloride) provide
evidence of intrinsic biodegradation (secondary lines of evidence) of the CoPC (Lelliott
and Wealthall, 2004).

Scale, in metres

0 150 300

Figure A5.22: Example of output from Visual Bio Software (Carey et al., 2003).
© 2003 John Wiley & Sons, Inc.

The use of radial diagrams allows clear comparison of multiple related chemical
parameters (i.e. parent and breakdown products, sequential redox indicators) at
individual monitoring wells, and also between monitoring wells. Radial diagrams also do
not involve any data interpolation and are based on real chemical concentrations (Lelliott
and Wealthall, 2004).

74
A5.4 Tertiary Lines of Evidence
Tertiary lines of evidence typically use data from laboratory microbiological testing to
show that indigenous bacteria are capable of degrading site contaminants (Rivett and
Thornton, 2008; Thornton, 2019). Conventionally this would have involved microbial
testing such as conducting plate counts, enrichment cultures and microcosm studies. In
the past it was anticipated that this line of evidence would only be required when primary
and secondary lines of evidence are inconclusive as it can be both costly, time
consuming and sometimes inconclusive. However, with the emergence of molecular
biological tools (MBTs) and compound specific isotope analysis (CSIA) on a
commercially-available basis there have been significant advances in the ability to
provide unequivocal evidence of contaminant biodegradation and these tools are being
used on a much more frequent basis to support MNA studies (Thornton et al., 2016).

Recognising the importance of these techniques, a separate dedicated appendix

describing the key features of each is provided elsewhere in this document (Appendix 8
for CSIA and Appendix 9 for MBTs). In summary MBTs are defined as techniques that
target biomarkers (specific nucleic acid sequences, peptides, proteins or lipids) to
provide information about organisms and processes relevant to the assessment or
remediation of contaminants (NJDEP, 2012) and CSIA is the measurement of the
isotope fractionation (typically, the stable isotope ratios of carbon, hydrogen or chlorine)
of individual volatile and semi-volatile compounds extracted from complex environmental
mixtures (Thornton et al., 2016; USEPA, 2008). Data visualisation methods associated
with the results from both MBTs and CSIA typically include means of spatial or temporal
analysis and correlation of results with either changes in contaminant chemistry or
electron acceptors as described above.

Coupled with increasing recognition and use of these techniques has been the
development of additional guidance and tools with which to quantify the potential for NA.
For chlorinated solvents one such tool is called BioPIC (Biological Pathway Identification
Criteria) and was developed by Lebrón et al. (2015) as a quantitative framework to
evaluate whether MNA is an appropriate remedy based on site-specific conditions.
BioPIC consists of a decision flow chart that uses the quantitative relationships between
biotic and abiotic parameters that contribute to the detoxification of chlorinated ethenes
and determine degradation rates. It allows the user to determine if degradation is
occurring and, if it is, to deduce the relevant degradation pathway(s) based on the
assessment of specific analytical parameters. While the document is focused on the
demonstration of MNA in the context of the US regulatory environment it does contain a
number of tools that document the relationship between several biotic and abiotic
indicators and calculated degradation rates (Figure A5.23). These can assist in
answering the question whether the observed degradation can be explained by the
particular indicator as evidence of NA and are relevant in the UK or internationally. These
indicators include:

• Quantification of reductive dechlorination genes to estimate whether biostimulation

is necessary;
• Measurement of the abundance of Dehalococcoides sp. in the subsurface to help
predict the first-order decay rate;

75
• Evaluation of stable isotope analysis to look for evidence of fractionation that is
indicative of degradation; and
• Evaluation of the role of iron sulfide in abiotic reduction.

Figure A5.23: Example of output from relationship between the density of

dehalococcoides and observed first-order decay rates of VC contained in BioPIC
tool. If data plot within the blue zone outlined, then the abundance of
dehalococcoides in groundwater can explain the in situ rate of VC degradation
(Lebrón et al., 2015).

A5.5 Optional Lines of Evidence

A5.5.1 Demonstration of Assimilative Capacity of an Aquifer

An electron mass balance calculation can be used to give an indication of the capacity
of an aquifer to degrade contaminants (its assimilative capacity). The approach relies on
the measurement of changes in groundwater chemistry at a site together with a
stoichiometric relationship describing the amount of contaminant degraded through
oxidation/ reduction reactions.

The amount of a contaminant, such as benzene, that can be theoretically degraded by

an electron accepting process can be estimated from Equation A5.9 as follows:

𝐶𝐶𝐵𝐵 − 𝐶𝐶𝑃𝑃
𝐵𝐵𝐵𝐵 = � = Equation A5.9
𝐹𝐹

76
 Where:
BC = biodegradation capacity (mg/l)
CB = average background concentration of electron acceptor or metabolic by-product
(mg/l)
CP = lowest measured electron acceptor or metabolic by-product concentration within
plume (mg/l)
F = contaminant utilisation factor (mg/mg)
∑ = sum of electron acceptor and metabolic by-products that contribute to degradation

The biodegradation capacity (BC) is the equivalent amount of contaminant that the
electron acceptors can assimilate or degrade based on the observed electron-acceptor
capacity of the aquifer. Note that BC here is based on observations of electron acceptors
consumed (obtained from monitoring data) and may be much less than the total
theoretical BC of the aquifer, based on the unused mass of electron acceptors (e.g. metal
oxide content of aquifer material). This will be a function of:

• Groundwater flow beneath the contaminant source; and

• Recharge/infiltration over the contaminant source area;

The total biodegradation capacity (TBC) of the system can be estimated as:

𝑇𝑇𝑇𝑇𝑇𝑇 = 1000 × 𝑄𝑄 × 𝐵𝐵𝐵𝐵 Equation A5.10

Where:
TBC = total biodegradation capacity (mg/d)
Q = groundwater flow through plume (m3/d)
BC = biodegradation capacity (mg/l)

This calculation can be used to determine whether the BC of the system is sufficient to
have degraded the mass of contaminant. The method can also be used to indicate the
relative importance of different electron acceptor/metabolic by-products to degradation.
The method should be used only as a qualitative tool in assessing the degradation
process, due to uncertainties regarding the cause of the oxidation/reduction reaction
though may be supplemented/supported by other measurements including CSIA and
MBTs. In some circumstances reducing conditions may be natural and for other sites
more than one contaminant may be competing for the electron acceptors.

Examples of electron balance calculations are given in AFCEE (2004) and Wiedemeier
et al. (1999). A more sophisticated electron balance methodology is described for plume-
scale mass balances in Thornton et al. (1998) and Thornton et al. (2001), which shows
that dispersion/mixing at the plume margins can be a significant source of soluble
electron acceptors (e.g. dissolved oxygen, nitrate and sulfate) for contaminant
biodegradation.

Electron balance calculations are included in a number of fate and transport models, for
example, CoronaScreen, RT3D and NAS (see Table A7.1, Appendix 7). They indicate
the capacity of all terminal electron accepting processes to oxidise single or mixed

77
contaminant plumes, integrated with flow and transport processes, and may be regarded
as semi-quantitative or quantitative methods compared to Equation A5.10.

An example of oxidation/reduction processes is given in Table A5.5 for the degradation

of benzene. This table also gives the mass of benzene degraded per unit mass of
electron acceptor consumed and metabolic by-product produced.

Table A5.5: Electron acceptors and metabolic by-products involved in the

degradation of benzene.
Process Acceptor Reaction Mass of Mass of
or benzene benzene
metabolic degraded degraded
by-product per unit per unit
mass of mass of
electron metabolic
acceptor by-
(-) product
produced
(-)

Oxidation Oxygen 7.5O2+C6H6→6CO2+3H20 0.33 -

Denitrification Nitrate 6NO3+6H++C6H6→6CO2+6H20+3N2 0.21 -

Sulfate reduction Sulfate 7.5H++3.75SO42-+ C6H6 →6CO2+3.75H2S+3H20 0.22 -

Manganese Manganese 30H++15MnO2+C6H6→6CO2+15Mn2++18H20 -* 0.094

reduction

Iron reduction Iron 60H++30Fe(OH)3+ C6H6→6CO2+30Fe2++78H20 -* 0.046

Methanogenesis Methane 4.5H2O+C6H6→2.25CO2+3.75CH4 - 1.3

*
The masses of MnO2 and Fe(OH)3 (solid phase electron acceptors) consumed during anaerobic benzene biodegradation
are not shown. In practice, these metal oxide fractions are not measured, whereas the Mn2+ and Fe2+ by-products of these
redox processes are measured in groundwater to estimate the aquifer BC, based on the appropriate utilisation factors of
0.094 and 0.046, respectively.

Similar reactions for toluene, xylene, ethylbenzene and chlorinated solvents reflecting
the stoichiometry of the degradation of these compounds are given in AFCEE (2004)
and Wiedemeier et al. (1999).

Example – Degradation of BTEX

An example of the calculation of the BC for a contaminant plume (with BTEX compounds
as the main contaminants of concern) is given in Table A5.6. In this example the
measured difference in the concentration of electron acceptors and metabolic by-
products would, in theory, be equivalent to the degradation of 13.6 mg/l of BTEX. For a
groundwater throughput of 100 m3/d, then a total of 1.36 kg/d of BTEX could be
degraded. This amount can be compared with the volume of contaminant lost or the
calculated rate of dissolution of BTEX from a NAPL source.

78
Table A5.6: Example calculation (degradation of BTEX)1.
Electron Upgradient Plume Difference (mg/l) Biodegradation
acceptor/ concentration concentration capacity2 (mg/l)
metabolic (mg/l) (mg/l)
by-product

Oxygen 5.2 0.1 5.1 1.63

Nitrate 4.3 0.1 4.2 0.84

Sulfate 34.0 8.0 26 5.46

Manganese 0.01 1.2 1.2 0.11

Iron 0.01 2.4 2.4 0.11

Alkalinity 210 240 30 3.9

Methane 0.01 1.1 1.1 1.4

Biodegradation capacity or quantity of BTEX that could theoretically be 13.5

degraded
1
Utilisation factors (UF) for the BTEX group of chemicals are slightly different than those for benzene alone
(i.e. those shown in Table A5.5). Respective values of UF for the BTEX group have been used to correct the
data in Table A5.6.
2
Biodegradation capacity = difference in concentration of electron acceptors up-hydraulic gradient and within
the plume divided by the Utilisation Factor, (see Table A5.7 below)

Utilisation factors for the electron acceptors and metabolic by-products that are involved
in the degradation of BTEX are given in Table A5.7.

Table A5.7: Utilisation factors.

Electron acceptor/ Utilisation factor1
metabolic by-product

Oxygen 3.14

Nitrate 4.87

Sulfate 4.76

Manganese 6.67

Iron 21.8

Alkalinity 7.69

Methane 0.78
1
mass of electron acceptor consumed per unit mass of BTEX degraded

79
A5.5.2 Estimation of the Source and/or Plume Depletion and Longevity

The effectiveness of NA could be further demonstrated by rates of source and/or plume

depletion and longevity:
a) Predicting a concentration decline (or other performance metric - source mass,
source mass discharge, plume mass, plume area, plume length etc. depending on
what the remedial objectives are) versus time to meet a specific concentration (or
other) objective. For example, if concentration is a relevant metric then the Point
Attenuation Rate (Kpoint, time per year) summarised in Table A5.3 can be
extrapolated to predict when concentrations will meet a remedial objective. This can
be done applying an upper confidence limit on the data to add some
acknowledgement to data variability and uncertainty in predictions.
b) Estimating via a model - a number of models and their use in MNA studies are
described within Appendix 7 (use of Bioscreen, BIOCHLOR, RemChlor, REMChlor
MD, NAS etc.) but there are other models that can provide insights to MNA duration,
for example, SourceDK (Farhat et al., 2011) and the Matrix Diffusion Toolkit (Farhat
et al, 2012).

80
Appendix 6: Implementation –
Performance Monitoring and
Verification
A6.1 Introduction
Implementation is the fourth step in the MNA process (Figure 1). Implementing MNA
involves continuation of groundwater monitoring, termed “performance monitoring”, to
verify remediation objectives have or will soon be achieved. The purpose of performance
monitoring is to demonstrate that NA continues to be an effective remediation strategy
that is protective of identified receptors.

The objectives of performance monitoring are to provide sufficient, reliable data to:

• Demonstrate that there is no impact or imminent risk of impact to downgradient

receptors;
• Confirm compliance with remediation criteria;
• Demonstrate that NA is occurring according to expectations;
• Provide a basis to close out MNA; and
• Identify change, especially reduction, in the effectiveness of MNA due to change in
conditions (e.g. modified groundwater flow direction, increased plume mass
discharge etc.) and provide a basis for effecting the Contingency Plan, if required.

Figure A6.1 summarises the main steps in performance monitoring and verification of
MNA. The feasibility of MNA as a groundwater remediation strategy will depend on
whether the Monitoring Plan required to provide data to verify the remedial objectives
have been achieved can be implemented.

These objectives can be demonstrated through extension of the CSM for flow and
transport of the contaminants, established during Steps 2 and 3 of the MNA process
(Figure 1).

A6.2 MNA Performance Monitoring Strategy

The performance monitoring strategy, that includes a monitoring plan and contingency
plan, is site-specific and based on the conceptual model for MNA. The strategy,
monitoring and contingency plans should be agreed with the regulator and other key
stakeholders. The basic elements of the strategy should consider the steps described in
the following section (A6.2.1).

A6.2.1 Remediation Objectives and Criteria

Remediation objectives that are protective of identified receptors should be set on a site-
specific basis to decide: (i) when monitoring can cease and (ii) when remediation
alternatives to MNA should be considered. Remediation criteria are required to support
either proposition, based on the data collected during performance monitoring.

81
Figure A6.1: MNA performance monitoring and verification.

The principal objective of implementing an MNA strategy is demonstration of the long-

term protection of downgradient receptors due to NA. NA processes are taken into
account in risk-based assessments for groundwater and surface water at Level 3 and
Level 4 of the Environment Agency’s Remedial Targets Methodology (Environment
Agency, 2006). Risk-based target concentrations developed from Level 3 or Level 4

82
assessments performed according to this methodology may be applied as remediation
criteria for MNA, where:

• The risk assessment is representative of the critical NA processes evident in site-

specific data demonstrating MNA viability and associated uncertainties in these data;
• Downgradient points of compliance are appropriately established with consideration
to the contaminant, pathway (aquifer) and sensitivity or use of the receptor (e.g.
statutorily protected wetlands, water resources).

In setting remediation criteria for MNA demonstrating absence of risk to receptors, the
performance monitoring strategy should also define:

• The location(s) at which data indicating compliance with the remediation criteria can
be physically measured over time, for example monitoring well(s), which may be
receptor, contaminant and/or pathway-specific;
• How and when compliance with the remediation criteria will be assessed. Statistical
methods incorporating a pre-determined review period or frequency will typically be
required to demonstrate compliance has been achieved or will be achieved in future,
to an adequate level of confidence.

In some cases, contaminant concentrations may already be below remediation criteria

and performance monitoring is required for confirmation purposes only. Performance
monitoring data demonstrating ongoing compliance with risk-based target
concentrations may support decisions to reduce monitoring frequency or cease
monitoring.

A secondary objective of the MNA strategy is to indicate when NA is no longer sufficiently

effective and contingency measures may be required. Trigger criteria should also be
established to provide advance warning that MNA is not performing as expected and
indicate when the Contingency Plan should be implemented.

Trigger criteria may be based around the following conditions potentially indicating
underperformance of MNA:

• Arrival of contaminants, including contaminative degradation products, at the

receptor(s) or in sentinel monitoring points;
• Adverse change in observed rates or geochemical conditions of key NA processes
(e.g. biodegradation);
• Reversal of mass and/or concentration trends in source and/or performance
monitoring wells;
• Contaminant mass or concentrations not decreasing at a sufficient rate to meet
remediation objectives within the desired timeframe, and/or;
• Other non-technical circumstances potentially triggering the need for contingency
measures, such as insolvency of the operator.

A6.2.2 MNA Monitoring Plan

Monitoring Network and Approach

The location, number and type of monitoring points will depend upon the complexity,
spatial and temporal variability of the groundwater flow regime, the size and stability of
the plume, relative levels of contamination and the location and sensitivity of receptors.

83
Monitoring points will comprise mostly groundwater monitoring wells potentially with
monitoring in abstraction wells, springs and/or surface waters.

A typical performance monitoring network (Figure A6.2) will include:

• Upgradient well(s) to determine changes in background water quality and enable
assessments of MNA performance relative to background conditions;
• Well(s)/well transect(s) immediately downgradient of the source zone(s) to monitor
changes in source mass discharge with time;
• Well(s)/well transect(s) located within the plume(s) to monitor behaviour and
dynamics;
• Well(s) delineating the plume fringes to monitor changes in plume geometry;
• Sentinel well(s) located between the plume(s) and the identified receptor, to provide
early warning of imminent impact(s) at the receptor;
• Monitoring points at the receptor, including abstraction wells, springs and/or surface
waters.

Figure A6.2: Schematic location of performance monitoring well network around

a plume.
84
The configuration of performance monitoring points will be dependent on the distribution
and behaviours of the source(s) and plume(s) indicated by the conceptual model for
MNA and the location and type of receptor (e.g. source protection zone). Monitoring
points used for these earlier stages of MNA may therefore be reused for performance
monitoring.

At most MNA sites, monitoring wells tend to be added sequentially during

characterisation in a process that can take years to decades. The result can be a well
network with tens to hundreds of wells that may have prolific redundancies in spatial
coverage. Depending on the overall stability of the plumes being monitored, there can
be substantial opportunities to streamline both the monitoring network1 as well as the
frequency of monitoring. Geospatial and spatio-temporal modelling techniques (e.g.
MAROS [Aziz et al., 2000]; Geostatistical Temporal-Spatial (GTS) algorithm [Cameron,
2004]; McLean, 2018; Torres, 2019) provide means of assessing the optimal
performance monitoring network, utilising the considerable volume of data typically
collected to demonstrate MNA viability.

The monitoring approach should aim to collect data that are both representative and
comparable with preceding stages of MNA evaluation. Groundwater sampling
technologies are well-established and described in existing guidance (e.g. CL:AIRE,
2008). MNA performance monitoring may be required over a period of months to years.
Repeatable groundwater sampling procedures, that are effective at mitigating sources of
short-term variability in monitoring data (e.g. Kulkarni et al., 2015), are required to
manage noise in monitoring data that may confound evaluation of long-term MNA
performance.

Monitoring Frequency

The monitoring frequency should be designed to detect changes in site parameters that
indicate the potential for MNA to meet remedial objectives, whilst ensuring that the
receptor(s) remain protected. The monitoring frequency should therefore be determined
on a site-specific basis, considering observed plume dynamics, such as velocity,
stability, concentration trends and rate of change, as well as the magnitude and
consequences of risks being managed. The monitoring frequency should be agreed in
consultation with the regulator in advance of implementing MNA.

At many sites, changes in contaminant and biogeochemical systems can be gradual and
take several years to manifest. While there is natural variability in long-term groundwater
monitoring data (Newell et al., 2002), contributions to the observed variability from other
sources 2 can be large (60 to 70% [McHugh et al., 2011]), that mask long-term temporal
concentration trends and limit the ability to understand the performance of MNA
(Figure A6.3). Recent research shows that frequent monitoring (<1 year) serves mostly
to characterise this time-independent variability rather than the long-term time-
dependent trend of interest (McHugh et al., 2015; Kulkarni et al., 2015). The optimal

1
Monitoring wells that are not required for performance monitoring may be decommissioned if
they are unlikely to be reinstated in the event that contingency measures are triggered.
2
Time-independent sources of variability in contaminant concentrations include, but are not
limited to, aquifer and monitoring well dynamics, sample collection and handling procedures,
and sample analysis (McHugh et al., 2015).
85
MNA performance monitoring frequency may therefore be longer (typically >1 year for
most systems) compared to the frequency of data collection previously used to
demonstrate that MNA is a viable risk management strategy (typically <<1 year).

Figure A6.3: Conceptual illustration of monitoring frequency required to

characterise long-term concentration trends (after McHugh et al., 2015).

These findings are supported by other guidance regarding approaches to MNA

performance (Wilson, 2011).

Several tools have been developed that can assist with determining the optimal
performance monitoring frequency in critical plume zones (e.g. MAROS Software [Aziz
et al., 2000]; ITRC, 2013; McHugh, 2015). More advanced spatio-temporal approaches
have recently emerged (e.g. McLean, 2018; Torres, 2019), that utilise geostatistical tools
within a temporal framework to improve interpolation of groundwater concentration
timeseries to identify temporal redundancy in monitoring data. GWSDAT (Jones et al.,
2014) implements the 2D method of McLean (2018), whereas deeper, complex systems
may benefit from the 3D method proposed by Torres (2019).

Analytical Parameters

The analysis of samples collected during the performance monitoring programme should
include laboratory and field parameters necessary to confirm that NA is occurring as
predicted, and to ensure the MNA remedy is protective of receptors. The suite of
parameters may be modified relative to those used during the initial demonstration of the
appropriateness of MNA. As a minimum, the contaminants of concern, any degradation
intermediates and end products should be routinely analysed, adequate to demonstrate
contaminant mass loss or concentration reduction at plume scale.
86
Periodically, geochemical and molecular parameters (e.g. terminal electron accepting
process-indicating parameters, degradation end products, stable isotope fractionation
[CSIA], microbial abundance and functional gene expression [MBTs]) indicating
secondary and tertiary lines of evidence for NA should be determined to confirm
conditions continue to be conducive for principal attenuation processes to be effective,
with no substantial reduction in attenuation rate.

Assessing Long-Term MNA Performance

MNA performance should be evaluated in regular review cycles (typically up to 5 years)

to determine whether:

• Monitoring should continue according to the defined programme or if it needs to be

revised, including change in the frequency of sampling, wells monitored and/or
analytical suite;
• Intervention is required because MNA is not performing as expected; and
• Monitoring can cease.

The cost of the review process needs to be included in financial provisions for MNA.
To assist in the review and assessment of MNA performance, visualising monitoring data
(Appendix 5) should support initial evaluations of spatial and temporal trends,
compliance with remedial targets and potential adverse changes to expected plume
behaviour. Statistical methods provide the most robust means to formally assess
concentration changes and compliance with remediation targets in timeseries data.
Statistical analysis could include (e.g. Wilson, 2011; Jones et al., 2014):

• Re-evaluating attenuation rates with consideration of uncertainty in the estimates

(Figure A6.4);
• Assessing the significance of observed reductions in concentrations and the
probability that reductions are adequate to meet remedial targets within a specified
period.

These methods provide a statistically-based decision criterion, at some predetermined

level of confidence, on which to:

• Cease monitoring
o Contaminant concentrations in the plume have reached background levels; or
o Remedial objectives have been met, and NA can be relied on to further reduce
contaminant levels; or
o Remedial objectives have been substantially met and falling trends in
contaminant concentrations have been defined to the extent that there is a high
degree of confidence that the remedial objectives will be achieved within an
agreed MNA performance review period.
• Continue monitoring
o Reductions in concentrations are statistically significant (i.e. MNA is performing
as expected), but plume concentrations are unlikely to meet remedial objectives
within the predetermined review period, therefore continue monitoring to
establish if plume concentrations will achieve remedial objectives within the
agreed timeframe for MNA.

87
• Trigger contingency planning
o Concentrations are not changing or trends have reversed, MNA is ineffective
and remedial targets will not be met within the agreed timeframe for MNA.
o If rates of NA are significantly slower than expected and MNA is unlikely to
achieve remedial objectives within the time agreed for the MNA programme.

Figure A6.4: Re-evaluating attenuation rates with consideration to uncertainty in

the estimates (Wilson, 2011).

88
Contingency Plan

The performance monitoring strategy should include a Contingency Plan, to govern

additional measures to be implemented if MNA proves to be ineffective or insufficient as
a risk management strategy.

The Contingency Plan should include:

• The decision criteria on which it will be triggered;

• Which stakeholders should be notified and involved in the decision-making process;
• Review of the conceptual model to understand what factors may have caused the
reduction in MNA performance demonstrated in earlier steps of the process; and
• Outline measures that will be implemented and the timescale over which these
measures may be implemented.

Criteria for triggering the Contingency Plan may include the following:

• Imminent risk of impact at the receptor;

• Concentration change relative to remedial target and/or trend reversal in monitoring
wells or at plume scale exceeding a specified threshold value, for example, due to
new releases of contaminants to groundwater and plume expansion;
• Contaminant concentrations are not decreasing at a sufficient rate to meet remedial
objectives within the desired timeframe;
• Changes in groundwater or land use adversely influencing the effectiveness of NA
and/or ability to monitor NA using the monitoring network; and
• Non-technical issues (e.g. operator goes into administration).

A6.3 Ceasing Monitoring

MNA performance monitoring can cease once remediation objectives have been met or
concentration trends are understood with adequate confidence that verify objectives are
expected to be met within an agreed period.

Monitoring data collected to demonstrate MNA may also be used for performance
monitoring to indicate long-term trends. It is therefore plausible that concentration trends
are characterised to an adequate level of confidence within the first review period
following implementation of performance monitoring.

Regulatory approval should be sought to cease monitoring. The case for ceasing MNA
will be provided by the conceptual model demonstrating the long-term effectiveness of
NA at protecting receptors.

Once approval to cease monitoring has been provided, it is recommended that the
performance monitoring network is decommissioned to mitigate risk of recontamination
from the surface via deteriorating, unsealed wells.

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Appendix 7: Groundwater Flow
and Transport Models
A7.1 Introduction
Groundwater flow and transport models are useful tools to assist the demonstration of
NA and evaluate future performance of MNA. A groundwater flow and transport model
can be used in two fundamental ways:

• To understand how current conditions evolved;

• Predict future conditions and MNA performance under these conditions.

With regard to MNA, modelling approaches provide means to integrate and consider
variability in complex and often diverse data collected during characterisation and
performance monitoring stages, quantitatively assess and confirm the main attenuation
processes, then forecast MNA performance with consideration of factors that may
influence its effectiveness and assess whether remedial objectives are likely to be met.

Modelling can provide a means to confirm the conceptual model for NA (i.e. whether
simulation of the conceptual model matches observation data) and provides a rigorous
framework for identifying data gaps and uncertainties. A conceptual model will be
required to develop an initial groundwater model, that then informs refinement and
improvements to the conceptual model. The conceptual model and groundwater models
should work iteratively to develop quantitative understanding of key hydrogeological and
biogeochemical processes on which to base predictions and decision making
(Step 2 Field Demonstration), including:

• Contaminant concentrations at receptors and arrival times;

• Source lifetime;
• Plume extent and dynamics (expanding, stable, shrinking);
• Plume concentration trends and rate of change;
• Quantification of transport and reactive transport parameters (e.g. degradation rates,
sorption coefficients, dispersivity coefficients, and processes influencing contaminant
attenuation);
• Monitoring locations and sampling frequencies; and
• Providing a tool for effective communication with stakeholders, in particular
regulators.

In addition to confirming the effectiveness of NA assuming continuation of current

conditions, scenarios that might be considered to assess the long-term performance of
MNA as part of Step 3 Predictive Modelling could include:

• Reduction in the rate of attenuation due to adverse change in geochemistry, such as

accumulation of cis-DCE or VC during reductive dechlorination stall or saturation of
adsorption sites;
• Change in land use or receptor characteristics, resulting in modification of the flow
regime (e.g. increase in abstraction rate at public water supply well or change in
meteoric recharge rates);

90
• Potential for recontamination of the aquifer due to back diffusion from low
permeability storage zones; and
• Effects of climate change, such as changing water table/flow regime.

Effective application of models to support MNA evaluation will require technical expertise
and comprehensive understanding of governing flow and transport processes.
Groundwater modelling can and must be performed transparently if the results are to be
relied upon. Similarly, modelling applications that support MNA evaluation must be
reported clearly and coherently to instil confidence in stakeholders (including the
regulator), and include explanations of the assumptions and limitations of the modelling
approach(es) applied, how these influence predicted outcomes and inform the Stage 2
and 3 conceptualisation of MNA. The basic steps in the selection, use and reporting of
flow and transport models are provided in UK and international guidance (McMahon et
al., 2001a; McMahon et al., 2001b; McMahon et al., 2001c; Reilly and Harbaugh, 2004;
Barnett et al., 2012)).

This appendix does not aim to provide modelling guidance. Rather it introduces the
types of model currently available for modelling MNA and considerations for transport
model applications.

A7.2 Model Selection

In designing and applying a solute transport model to MNA, the purpose and
expectations of the model need to be defined (i.e. what questions should it answer and
to what level of confidence). The selection of the model should be driven by the
complexity of the site and potential source-pathway-receptor linkages. Inappropriate
selection and/or use of models may give rise to erroneous conclusions, be time
consuming and costly.

Two basic types of model are available – analytical and numerical models:

Analytical models are capable of solving the general transport equation with specific
limitations.

Analytical models such as the Environment Agency’s Remedial Targets Worksheet

(Environment Agency, 2006), BIOSCREEN (Newell et al., 1996), BIOCHLOR (Aziz et
al., 2000; Aziz and Newell, 2002), NAS (Mendez et al., 2004), CoronaScreen (Wilson et
al., 2005), REMChlor (Falta, 2008) and REMChlor-MD (Farhat et al., 2018) have been
established specifically for use in modelling NA and/or MNA (refer to Table A7.1).

The solution technique typically requires assumptions of uniform hydraulic properties

throughout the domain, uniform steady-state groundwater flow (in some cases limited to
one-dimensional advection), simple boundary conditions, simple source geometry, first-
order contaminant transformation with rates constant within a defined area (in some
cases for a single decay pathway) and uniform linear equilibrium partitioning.

Analytical models can be useful in providing estimates of contaminant migration for

plumes where these assumptions can be technically supported based on the site
conditions, for instance a plume with a well-defined contaminant source within a
relatively homogeneous, thin aquifer that is bounded by aquitards or an aquitard and the

91
water table where the aquifer has relatively constant geochemical conditions throughout
the plume.

Analytical models provide valuable assessments of simple sites or screening-level

assessments of more complex conditions. The advantages of these models are that
they are often simpler to use and have fewer data requirements compared to numerical
models. Multiple simulations can be run relatively quickly to evaluate, in broad terms,
the range of potential outcomes. Despite their ease of use, the limitations of analytical
models must be recognised, in particular for insufficiently characterised and/or complex
hydrogeological situations which could generate misleading results. Additional analysis
to explore the sensitivity of analytical models to site data is recommended to better
understand how these variables influence uncertainty in predicted outcomes and
ultimately decisions made concerning MNA.

Multi-dimensional reactive transport numerical models, often capable of simulating

multiple contaminant species simultaneously, discretise the transport equation, which is
solved iteratively within a defined numerical domain. Numerical models allow for more
detailed configuration of the model domain to more closely match site features and,
therefore, have advantages over analytical models for some sites.

Numerical models may be needed when site conditions cannot be described under the
simplified flow, reaction, or adsorption process assumptions required for use of some
analytical models, for example:

• The groundwater flow system at a site may not be uniform spatially and/or temporally
because of a complex distribution of hydraulic conductivity, complex
recharge/discharge elements, or transient flow conditions;
• Sources distributed in multiple locations, multiple contaminant species with multiple
reaction pathways, and multiple oxidation/reduction conditions within the plume area
cause complexities in modelling the reaction processes at a site;
• Linear equilibrium sorption is not appropriate in some cases depending on the nature
of the contaminant and the aquifer solids; and
• NA processes are dependent on contaminant speciation, sensitive to transient
oxidation/reduction conditions, include reactions such as precipitation and ion
exchange and/or can be described by more complex kinetic models (e.g. Monod
kinetics).

Numerical models are more appropriate for site conditions that include any, or all, of the
above complexities. The modular three-dimensional code MODFLOW (McDonald and
Harbaugh, 1988; Harbaugh et al., 2000; Harbaugh, 2005) is used determine
groundwater flow (in 3D), and provides the platform for simulating transport and reactive
transport under these more complex conditions. Examples of transport model codes are
MT3D, MT3DMS and MT3D-USGS (Zheng, 1990; Zheng and Wang, 1999; Bedekar et
al., 2016). Reactive transport codes include RT3D (Clement, 1997) and PHT3D
(Prommer and Post, 2010). Descriptions of these numerical models are provided in
Table A7.1.

Similar to analytical models, numerical models have limitations in how they can be
configured to match site conditions. Equations cannot describe all of the nuances for
each term within the transport equation. Numerical models cannot therefore reproduce

92
reality but can be configured to more closely match the site conditions and processes
than analytical models.

Most analytical and numerical models are deterministic, that is, use a single value to
define each model parameter, and the result is a single number. Although running
deterministic models multiple times can indicate the effects of varying individual
parameters (e.g. variation in hydraulic conductivity) on model predictions, stochastic
approaches can provide more efficient and comprehensive insights on prediction
uncertainty, including parameter covariance. Stochastic models that assess variable
parameters using ranges or probability density functions (e.g. normal, lognormal etc.)
within a Monte Carlo framework include ConSim (Golder, 2018), PREMChlor (Liang et
al., 2010), and, for numerical models (MT3DMS, RT3D etc.), via PEST and some
commercially-available graphical user interfaces (GUIs). More advanced stochastic
approaches aim to reconcile spatial and/or temporal heterogeneity through geostatistical
methods and estimate the uncertainty in predictions (range or distribution) based on
these variables using probability theory (Renard, 2007). Despite recent advances to
consider variability/heterogeneity more in site evaluations, application of these more
advanced stochastic approaches, are not standard practice, but this will undoubtedly
change with time (Konikow, 2011) and increasing accessibility to these methods (e.g.
via the PEST and PEST++ suites).

A7.3 Calibration and Prediction

The reliability of a model may be improved with calibration of variable parameters
(e.g. hydraulic conductivity or degradation rate) to match modelled with observed flow
and concentration data. However, it should be recognised that accurate modelling of
subsurface solute transport processes at plume scale is challenging. A numerically
accurate solution is often expected but all models are a simplification of reality;
conceptual weaknesses in the underlying theory, governing transport equation and
mathematical solutions, plus limitations associated with sufficiency of reliable data on
which to base the model, will inevitably introduce some errors (Konikow, 2011).
Awareness of the sources of error will help model users minimise and account for this
when interpreting model results. Experience indicates that some (if not most) of the
difficulties with transport models arise from errors, inadequacies and uncertainties in the
data used to estimate parameters. As such, transport models should be expected to
reproduce major trends or locally average values rather than be expected to accurately
match all variations observed in the field data (Konikow, 2011).

Groundwater flow is simpler to simulate than solute transport or reactive transport,

although flow and transport are inherently linked. The more accurately and precisely the
flow velocity field can be simulated, the less uncertain transport modelling should
become. There are practical limits to how well heterogeneity and the flow velocity field
can be defined. Good judgment and sensitivity analyses may help in balancing costs
and benefits and in deciding when existing data are sufficiently good.

Solute transport models are often “highly parameterised”. Highly parameterised models
are characterised by having more parameters than can be estimated uniquely for a given
calibration dataset. Non-unique solutions can create calibration difficulties in that there
may be a number of possible combinations of transport parameter values that match the
calibration dataset. “Regularised inversion” is a mathematical approach that provides a
93
means of obtaining a unique calibration from the range of fundamentally non-unique,
highly parameterised model calibrations (Doherty and Hunt, 2010; Doherty et al., 2010a).
“Regularisation” simply refers to approaches that make non-unique problems
mathematically tractable; “inversion” refers to the automated parameter-estimation
operations that use observation data to constrain model input parameters (Hunt et al.,
2007). Regularised inversion problems are commonly addressed by the use of the
parameter estimation codes PEST and PEST++.

While non-uniqueness may be unavoidable in solute transport modelling, performing

multiple calibrations of the model can assist in understanding how prediction uncertainty
is influenced by estimated parameters matching the calibration data. One of the most
significant trends in groundwater modelling over the past two decades has been the shift
in focus from “model calibration” to “calibration-constrained model predictive uncertainty
analysis”. This shift in emphasis recognises the fact that groundwater models are built
to make predictions that support the making of important management decisions, such
as whether to implement MNA. These predictions are often accompanied by a large
amount of uncertainty that should be quantified to allow evaluation of the risks associated
with site management strategies (Doherty, 2015). Procedures for performing parameter
and predictive uncertainty analysis are provided by USGS (Doherty et al., 2010b) and
implemented using the PEST++ suite.

Some reactive transport modelling approaches are capable of integrating data provided
by advanced tools such as compound specific isotope analysis (CSIA, Appendix 8)
and/or molecular biological tools (MBTs, Appendix 9) with more typical observation data
(e.g. groundwater elevations, contaminant concentrations). The application of this class
of models can provide greater confidence in the interpretation of these data and support
the development of combined lines of evidence for NA (primary, secondary and tertiary).

Modified analytical models, such as BIOCHLOR-ISO (Höhener, 2016) and Bioscreen-

AT-ISO (Höhener et al., 2017), have been developed to simulate contaminant transport
in simple hydrogeological systems combining chemical analytical data with isotopic
fractionation data based on the simplifying assumptions of the Rayleigh model
(Appendix 8). CSIA and MBT data can be difficult to interpret at sites with more complex
hydrogeology, biogeochemistry or release histories. More advanced reactive transport
models (e.g. PHREEQC, PHT3D or RT3D) provide the capability to meaningfully
integrate these data through definition of degradation pathways using stable isotope
geochemistry and/or advanced kinetic models describing microbial activity and
population growth.

The research undertaken for prediction of chlorinated solvent bioremediation for the UK
SABRE project provides an indication of the extent to which modelling can extend
understanding of complex problems, incorporating diverse datasets often collected
during MNA (CL:AIRE, 2010b). Table A7.1 identifies analytical and numerical models
with application to MNA studies. This table is not intended to be comprehensive and
other models may be more appropriate in particular situations.

94
Table A7.1: Analytical and numerical models with application to MNA studies.

Model/Code Type Description Reference or Source

BIOCHLOR Analytical- BIOCHLOR is a screening model that simulates NA of United States Environmental Protection Agency (USEPA)
deterministic dissolved chlorinated solvents.
https://www.epa.gov/water-research/biochlor-natural-
Based on the Domenico analytical solute transport model, attenuation-decision-support-system
simulates 1D advection, 3D dispersion, linear adsorption
and sequential degradation assuming first-order decay. It
assumes a homogeneous isotropic aquifer with uniform
regional flow.

BIOSCREEN Analytical- BIOSCREEN is a screening model that simulates NA of USEPA

deterministic dissolved hydrocarbons. The model is designed to
https://www.epa.gov/water-research/bioscreen-natural-
simulate biodegradation by both aerobic and anaerobic
attenuation-decision-support-system
reactions.
BIOSCREEN is based on the Domenico analytical solute
transport model and allows for advection, dispersion,
adsorption, both aerobic decay and anaerobic reactions.
Biodegradation can be modelled as a first-order decay
process or instantaneous reaction with electron acceptors
(dissolved oxygen, nitrate and/or sulfate).

BIOSCREEN-AT Analytical- BIOSCREEN-AT is an enhancement of the standard SS Papadopoulos

deterministic BIOSCREEN program that can implement an exact three-
https://www.sspa.com/software/bioscreen
dimensional analytical solution for solute transport from a
patch boundary condition within a semi-infinite aquifer.
BIOSCREEN-AT simulates advection, dispersion,
adsorption, both aerobic decay and anaerobic reactions.
Biodegradation can be modelled as a first-order decay
process or instantaneous reaction with electron acceptors
(dissolved oxygen, nitrate and/or sulfate).

95
Model/Code Type Description Reference or Source

ConSim 2.5 Analytical- ConSim is a screening level tool that can be used within Golder
stochastic the quantitative risk assessment framework provided by
http://www.consim.co.uk/
the Environment Agency’s Remedial Targets Methodology
(Environment Agency, 2006).
ConSim is used to assess the potential for leaching of
contaminants from multiple sources, migration towards one
or more receptors and attenuation in the unsaturated zone
and an aquifer. Dilution, sorption and
biodegradation/decay may be incorporated.
Prediction uncertainty is taken into account through the
use of parameter input ranges and a Monte Carlo
probabilistic calculation methodology.

CoronaScreen Analytical- CoronaScreen is a package of three spreadsheet-based Groundwater Protection and Restoration Group, University
deterministic analytical screening models for the performance of Sheffield
assessment of NA in groundwater. The models have a
https://www.sheffield.ac.uk/gprg/technology/coronascreen
different conceptual framework and mathematical
formulation for specific contaminant scenarios. The models
simulate the evolution of contaminant plumes in
groundwater in terms of the spatial distribution of (plume
"fringe" and plume "core") biodegradation processes that
occur over time. The models offer the possibility to
estimate the maximum plume length, the time to achieve
this, the plume biodegradation rate and contaminant
concentration at a given compliance point. The models can
be used to evaluate mixed plumes of organic and inorganic
chemicals, using standard site characterisation information
and groundwater quality data collected from a relatively
simple network of single screen monitoring wells and
multilevel sampling wells.

96
Model/Code Type Description Reference or Source

MT3D Numerical- The MT3D family of transport codes were first released in University of Alabama
deterministic 1990 as MT3D for single-species mass transport. Two
MT3DMS http://hydro.geo.ua.edu/mt3d/
updated versions have since been released: MT3DMS and
(stochastic
MT3D-USGS MT3D-USGS. USGS
capability
with some MT3DMS is a numerical multispecies contaminant https://www.usgs.gov/software/mt3d-usgs-groundwater-
GUIs) transport model with a modular structure simulating solute solute-transport-simulator-modflow
transport processes (advection, dispersion, linear and non-
linear sorption, first-order decay/degradation)
independently or jointly. MT3DMS interfaces directly with
the United States Geological Survey (USGS) finite-
difference groundwater flow model, MODFLOW. MT3DMS
contains several transport solution techniques, including
the fully implicit finite-difference method (FDM), the
particle-tracking based method of characteristics (MOC)
and its variants, and a third-order total-variation-
diminishing (TVD) scheme that conserves mass while
limiting numerical dispersion and artificial oscillation.
MT3D-USGS is a USGS updated version of MT3DMS, that
includes additional transport modelling capabilities such as
unsaturated zone processes, surface water interactions,
inter-species and sequential reactions, separate
specification of sorption coefficients in mobile and
immobile zones.

NAS Analytical- Natural Attenuation Software (NAS) is a screening tool to USGS

deterministic estimate remediation timeframes for MNA to lower
Virginia Polytechnic Institute and State University (Virginia
groundwater contaminant concentrations to regulatory
Tech)
limits, and to assist in decision making on the level of
source zone treatment in conjunction with MNA using site- Naval Facilities Engineering Command (NAVFAC)
specific remediation objectives.
https://www.nas.cee.vt.edu/index.php

97
Model/Code Type Description Reference or Source

NAS (cont.) NAS consists of a combination of analytical and numerical

solute transport models. Natural attenuation processes
that NAS models include advection, dispersion, sorption,
NAPL dissolution, and biodegradation. NAS determines
redox zonation, and estimates and applies varied
biodegradation rates from one redox zone to the next.

PHT3D Numerical- PHT3D integrates geochemical and transport modelling by University of Western Australia, Flinders University School
deterministic coupling MT3DMS (Zheng and Wang, 1999) with of the Environment (South Australia) and National Centre
PHREEQC-2 (Parkhurst and Appelo, 1999) for simulation for Groundwater Research and Training
(stochastic
of complex multicomponent reactive transport processes.
capability http://www.pht3d.org
PHT3D is capable of simulating equilibrium-controlled
with some
aqueous complexation / speciation, kinetic reactions of
GUIs)
aqueous components such as biodegradation of organic
compounds, mineral precipitation / dissolution, ion
exchange, and surface complexation reactions.

PREMChlor Analytical- The Probabilistic Remediation Evaluation Model for Strategic Environmental Research and Development
stochastic Chlorinated solvents (PREMChlor) simultaneously Program (SERDP)
evaluates the NA of source and plume considering the
Environmental Security Technology Certification Program
uncertainties in all major parameters, thereby supporting
(ESTCP)
the demonstration of MNA, or selection of remediation
alternatives. https://serdp-estcp.org/projects/details/5bd87a57-f0f7-
4f11-a2ab-dd5213b5bf4a/er-200704-project-overview
PREMChlor simulates plume NA spatially (2D) and
temporally for parent and daughter compounds, based on
advection with dispersion, sorption and sequential first-
order decay.
Probabilistic functionality is provided by coupling
REMChlor with GoldSim, a Monte Carlo modelling
package, and representing variable parameters with
probability density functions.

98
Model/Code Type Description Reference or Source

REMChlor Analytical- REMChlor, or Remediation Evaluation Model for USEPA

deterministic Chlorinated solvents, is an analytical solution for simulating
https://www.epa.gov/water-research/remediation-
the transient effects of groundwater source and plume
evaluation-model-chlorinated-solvents-remchlor
remediation. The contaminant source model is based on a
power-function relationship between source mass and
source discharge, implicitly simulating matrix diffusion in
the source. It can consider partial source remediation at
any time after the initial release. The source model serves
as a time-dependent, mass-flux boundary condition to the
analytical plume model, where flow is assumed to be one
dimensional. The plume model simulates first-order
sequential decay and production of several species. The
decay rates and parent/daughter yield coefficients are
variable functions of time and distance.

REMChlor-MD Analytical- REMChlor-MD is a new version of the REMChlor model GSI Environmental
deterministic with the ability to simulate matrix diffusion processes in the
Environmental Security Technology Certification Program
source and plume.
(ESTCP)
REMChlor-MD is a Microsoft Excel-based management
https://www.gsi-net.com/en/software/free-
tool for addressing contamination in a broad range of
software/remchlormd.html
geological settings, including fractured porous media,
heterogeneous media with low permeability inclusions, and
high permeability zones that are adjacent to low
permeability aquitards. The toolkit allows the accounting of
several types of source and plume remediation activities.

Remedial Analytical- The Remedial Targets Methodology worksheet is an Environment Agency

Targets deterministic Excel-based screening level model that implements the
https://www.gov.uk/government/publications/remedial-
Methodology Environment Agency’s Remedial Targets Methodology for
targets-worksheet-v22a-user-manual
Worksheet v3.2 hydrogeological risk assessment of land contamination
(Environment Agency, 2006).

99
Model/Code Type Description Reference or Source

Remedial This model simulates leaching, dilution, dispersion,

Targets sorption and first-order decay of a polar or non-polar solute
Methodology from a single source within a single aquifer unit. Steady-
Worksheet v3.2 state and transient solutions are simulated to assess
(cont.) concentrations in groundwater at a downgradient point of
compliance and back-calculate remedial target
concentrations in soil or groundwater based on a specified
water quality standard.

RT3D Numerical- RT3D is a 3D multispecies reactive transport model for Pacific Northwest National Laboratory
deterministic solutes in groundwater. RT3D couples MT3DMS (Zheng
and Wang, 1999) with several pre-programmed reaction https://www.pnnl.gov/
(stochastic
modules for common biologically mediated reactions, rate-
capability
limited sorption, NAPL dissolution, kinetic-limited
with some
degradation using multiple electron acceptors, Monod
GUIs)
kinetics and others. Furthermore, RT3D permits users to
add any reaction kinetics desired/suitable to represent
multiple chemical species in aqueous and sorbed phases.

100
Appendix 8: Compound Specific
Isotope Analysis (CSIA)
A8.1 Introduction
The selection of MNA as a viable remedy for a site may require an evaluation of the
contribution of natural biodegradation or abiotic transformation processes within
groundwater. However, demonstrating unequivocally that a contaminant is being
degraded in the environment is challenging. Concentration-based methods
demonstrating primary and secondary lines of evidence for MNA, such as the presence
of intermediates and favourable geochemical conditions, may be confounding,
particularly for those contaminants which degrade slowly and/or whose degradation
pathways produce non-unique or non-persistent by-products or end products (USEPA,
2008). Furthermore, they provide little information about the processes responsible for
removal of a specific contaminant, and cannot distinguish degradation from other
physical processes (e.g. dilution/dispersion), which can reduce concentrations but not
the contaminant mass.

Compound Specific Isotope Analysis (CSIA) is an environmental molecular diagnostic

technique that can assess the ratio of heavy to light stable isotopes of selected elements
within contaminants as well as metals in environmental samples (USEPA, 2008). For
example, 12C is the most common carbon isotope in naturally-occurring organic
compounds but a small fraction of the heavier 13C will also be present. Instrumentation
capable of performing CSIA measurements was introduced in 1997 for chlorinated
ethenes. Between the year 2000 and 2010, the technique has steadily become
applicable to further chlorinated and non-chlorinated hydrocarbons, as well as
nitrobenzenes/anilines and more recently (~2010), pesticides and metabolites, and polar
organic micropollutants. Figure A8.1 shows the timeline of CSIA development, its
maturation and the range of contaminants to which the technique can now be applied.

Figure A8.1: The development of CSIA and range of contaminants to which the
technique can be applied (Source: Geosyntec).
101
A number of reviews have focused on principles and applications of CSIA (Zimmermann
et al., 2020; Cui et al., 2021; Alberti et al., 2017). The elements that make up chemical
compounds have distinct isotopic ratios, which are a function of the manufacturing
process and degradation of the compound after it has been released to the environment.
The ratios in manufactured chemicals are generally well known to within a few percent.
When organic contaminants degrade in the environment, the ratio of stable isotopes
begins to change. CSIA can precisely measure the small changes in isotopic ratios,
providing unequivocal evidence that degradation has occurred and furthermore can
potentially identify the degradation process and estimate the extent and rate of
degradation (USEPA, 2008).

A8.2 Applications
From a NA standpoint, the shift in isotopic ratios measured by CSIA can be useful in
providing the following (USEPA, 2008):

1. Demonstrating that a parent compound is being degraded;

2. Differentiating between destructive and non-destructive attenuation processes;
3. Differentiating between destructive pathways (e.g. anaerobic vs aerobic vs abiotic);
4. Estimating the extent of degradation;
5. Estimating the rate of degradation; and
6. Source identification and differentiation.

The technique can also be applied to environmental forensics and assessing abiotic
degradation as a result of chemical oxidation techniques, however, these are outside the
scope of this document. Abiotic degradation of chlorinated solvents on reactive iron
mineral surfaces also naturally cause measurable fractionation (Liang et al., 2007).

A8.3 Scientific Basis

Isotopes of elements have the same number of protons and electrons, but a different
number of neutrons. For example, carbon has two stable isotopes; 12C which contains
six protons and six neutrons, and 13C, which contains six protons and seven neutrons.
As a result of the additional neutron in 13C, it is heavier, and forms very slightly stronger
chemical bonds. The quotient of heavy to light isotopes is called the isotopic ratio. The
ratios present within chemicals naturally vary according to the source of feedstocks used
in the manufacturing process, and the ratios present in the resulting formulations may
differ from one batch to another.

During the process of biodegradation or abiotic degradation, the lighter isotopes degrade
preferentially over those which are heavier. This is due to slight differences in the
reaction rates of molecules with light isotopes compared to the heavy isotopes (a
phenomenon known as kinetic isotope effect [USEPA, 2008]). Enrichment of the heavier
isotope in the undegraded compound causes a shift in isotopic ratio relative to the
isotopic ratio of the compound source, which becomes more pronounced as
biodegradation or abiotic degradation proceeds (ITRC, 2011). This enrichment is
referred to as isotopic fractionation. Organic metabolites produced during biodegradation
are isotopically lighter than the parent compounds, due to the isotope fractionation. This
enables organic biodegradation products to be linked to specific parent compounds and
102
pathways and is important when interpreting the biodegradation of organic compounds
in mixtures.

An illustrated example how isotopic enrichment occurs along a groundwater flow path in
which biodegradation of TCE is occurring is shown in Figure A8.2.

Figure A8.2: Illustration of 13C enrichment during reductive dechlorination of TCE

with a C-Cl bond. Adapted from ITRC (2011).

Characterisation of the isotopic ratios present within a well-defined source zone will
improve confidence that the relative enrichment of isotopes downgradient are the result
of degradation. If there is more than one source of contamination, it is possible that the
isotopic ratios may differ between plumes, and give a false impression of biodegradation.
Quantification of the isotope ranges from each of the sources is required.
103
In cases where biodegradation or physical destruction of the TCE molecules were not
occurring, no isotopic enrichment would be apparent throughout the plume, despite
reducing concentrations in groundwater with distance from the source due to physical
attenuation processes.

In most instances transport and partitioning of contaminants in groundwater will not mask
the relatively large isotopic fractionation due to biotic degradation (ITRC, 2011). Isotopic
fractionation that occurs during volatilisation, dissolution, diffusion and sorption has been
found to be relatively small and indiscernible in natural systems outside of the typical
analytical uncertainty (USEPA, 2008; Adamson and Newell, 2014).

CSIA measures ratios of one or more stable isotopes in molecules and compounds. The
technique is most commonly applied to carbon isotopes (13C/12C) in organic
contaminants and is generally applied to compounds that contain ten or fewer carbon
atoms, such as BTEX, MTBE, naphthalene, some chlorinated ethenes and ethanes.
However, it can also be applied to hydrogen (2H/1H), nitrogen (15N/14N), oxygen (18O/16O
and 17O/16O), chlorine (37Cl/35Cl), sulfur (34S/32S) and others. The measured isotopic
ratios are normalised with respect to international isotopic standard reference materials
and expressed in delta notation (e.g. δ13C) (USEPA, 2008).

CSIA can also be used to interpret the biodegradation of contaminants at low

concentrations. Bennett (2017) reported that 1,4-dioxane present at 1 µg/l within
groundwater was efficiently sorbed through the addition of a synthetic hydrophobic
carbonaceous material to the sample. The 1,4-dioxane was recovered from the dried
sorbent by thermal desorption within a gas chromatograph, and then analysed for
isotopic ratios of both carbon and hydrogen. It is possible that this method could be
applied to other contaminants that occur at low concentrations within the environment to
extend the applicability of CSIA.

A8.4 Quantitative Interpretation of Isotope Data

For many organic contaminants, the relationship between isotopic fractionation and the
extent of degradation is described by the Rayleigh model. The Rayleigh model states
that isotopic fractionation is proportional to the change in concentration, with the
proportionality constant expressed as the isotopic enrichment factor (ε). If the
degradation pathway is known, literature or laboratory/microcosm-derived ε values can
be used to estimate the fraction of contaminant remaining after degradation (f), and the
extent of degradation (USEPA, 2008).

For the carbon isotope system (13C/12C):

𝑅𝑅𝑡𝑡 = 𝑅𝑅0 𝑓𝑓 (𝛼𝛼−1) Equation A8.1

where Rt is the stable isotope ratio (13C/12C) of the compound at time t, R0 is the initial
isotopic ratio of the compound and f is the fraction of contaminant remaining, where f =
1 at t = 0 and decreases to f = 0 when the reactant compound is fully transformed to
products. The stable isotope fractionation factor (α) is defined as:

104
 (1000 + 𝛿𝛿13 𝐶𝐶𝑎𝑎 )
𝛼𝛼 = 13 Equation A8.2
(1000 + 𝛿𝛿 𝐶𝐶𝑏𝑏 )
where subscripts a and b may represent a compound at time zero (t0) and at a later point
(t) in a reaction.
For degradation in groundwater:

𝑓𝑓 = 𝑒𝑒 (
𝛿𝛿13𝐶𝐶𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 −𝛿𝛿13C 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠)/𝜀𝜀 Equation A8.3

where δ13Cgroundwater is the measure of the isotopic ratio in the organic contaminant in the
sample of groundwater, δ13Csource is the isotopic ratio in the non-degraded organic
contaminant and epsilon (ε) is the stable isotope enrichment factor, defined as:

𝜀𝜀 = (𝛼𝛼 − 1). 1000 Equation A8.4

The Rayleigh equation (Equation A8.5) may be used to predict the extent of
biodegradation based on the changes in isotope ratios. It should be noted that application
of the Rayleigh equation for intermediates in the degradation process (such as those
resulting from reductive dechlorination of chlorinate ethenes) is not strictly possible.
𝜀𝜀
(𝛿𝛿𝑡𝑡 + 1000) 𝐶𝐶𝐵𝐵𝐵𝐵 1000
= � �
(𝛿𝛿𝑜𝑜 + 1000) 𝐶𝐶0
Equation A8.5

Where:
𝛿𝛿t = isotopic signature of the substrate at a time point
𝛿𝛿0 = original isotopic signature of the substrate
CBt/C0 = fraction of substrate remaining at time point t
ε = isotopic enrichment factor in ‰

The extent of biodegradation is often expressed as a percentage (B%) of the initial

concentration using Equation A8.6:
𝐶𝐶𝐵𝐵𝐵𝐵
𝐵𝐵[%] = �1 − � . 100 Equation A8.6
𝐶𝐶0

Use of the above equations enables the quantification of contaminant biodegradation

along a specified flow path or time interval. The amount of a contaminant degraded
between the source, or starting point of observation, and a downgradient location (x) is
described by Equation A8.7 (USEPA, 2008).
1000
𝐶𝐶𝐵𝐵𝐵𝐵 𝛿𝛿𝑥𝑥 + 1000 � 𝜀𝜀
�
𝐵𝐵[%] = �1 − � . 100 = �1 − � � � . 100
𝐶𝐶0 𝛿𝛿0 + 1000
Equation A8.7

105
Based on the changes in isotopic ratios along a groundwater flow path identified by the
Rayleigh equation (Equation A8.5), an in situ zero-order and first-order rate constant can
be estimated.

In addition to demonstrating the extent of biodegradation of certain organic contaminants

in groundwater, it is also possible to use the data to predict the extent and rate of
degradation (USEPA, 2008). The rate of contaminant degradation can often be
calculated from field data by evaluating observed decreases in contaminant
concentrations with travel time along the aquifer flow paths. CSIA can be used to
increase confidence in degradation rate estimates, by providing evidence that the
reduction in concentration is due to destruction of the contaminant, and in many cases
can provide a more reliable estimate of degradation rate (USEPA, 2008). A first-order
rate coefficient is estimated using isotopic ratio data (e.g. δ13C) for a near source and
downgradient location (or early and later time) with the isotopic enrichment factor (ɛ) from
the Rayleigh model. Some analytical transport models are capable of simulating isotopic
ratios in plumes that can assist in determining degradation rates by integrating CSIA with
hydrogeological data and solute transport parameters (e.g. Höhener, 2016; Höhener et
al., 2017).

The amount of degradation that needs to have occurred before CSIA, using 13C/12C
isotopic ratios, can be confident in positively identifying biodegradation and estimating
rates varies between compounds. For example, biodegradation of TCE can be detected
at <20% degradation (USEPA, 2008). In contrast, aromatic hydrocarbons require 60%
degradation to have occurred prior to positively identifying biodegradation, due to the
more subtle carbon isotope fractionation that occurs. For petroleum hydrocarbons,
analysis of both carbon isotope enrichment and hydrogen isotope enrichment can
increase the sensitivity of the analysis to detect biodegradation as fractionation of δ2H is
greater than δ13C for some petroleum hydrocarbons (Fischer et al., 2008; Gray et al.,
2002).

CSIA can be difficult to interpret, especially at sites with complex hydrogeology or release
histories (Kuder et al., 2014). Factors that can confound CSIA data and challenge
application of the Rayleigh model at some sites include:

1. Mixed degradation processes (biotic and abiotic);

2. Contributions from multiple sources;
3. Formation and degradation of sequential transformation daughter products (e.g. cis-
DCE and VC); and
4. Sorption and physical transport processes.

Dual or triple CSIA provides a means to deal with some of these factors. The application
of these data in reactive transport models (e.g. PHREEQC, PHT3D or RT3D –
Appendix 7) provides a way to strengthen the interpretation of CSIA alongside chemical
analytical data and other hydrogeological data to develop clearer evidence for
degradation processes.

A8.5 Sampling Technique

Sample collection for CSIA depends on the compounds of interest. For example, use of
CSIA for the analysis of perchlorate in groundwater, sampling requires the pumping of
106
several hundred litres of groundwater through ion exchange columns to trap perchlorate
(USEPA, 2008). For the analysis of carbon and hydrogen isotopes from volatile organic
compounds, standard low-flow groundwater monitoring techniques can be used, with
collection of samples into standard vials for volatile analysis. Some laboratories forgo the
use of chemicals and rely on chilling of the samples post-collection for preservation,
however, some recommend the addition of a preservative, typically 36% hydrochloric
acid diluted 1:1 in water, resulting in a sample pH of <2. Other preservation methods are
also used and the analytical laboratory should be contacted to ensure observation of
suitable protocols for collecting, handling, and transporting the samples. A
comprehensive guide to sampling is provided in USEPA (2008).

A8.6 Supporting Lines of Evidence and Summary

CSIA is considered a tertiary line of evidence in the assessment of NA (see
Section A5.4). The greatest value from the information can be derived when it is used in
conjunction with hydrogeological, geochemical and microbiological parameters. It is
important to note that use of techniques to study biodegradation in groundwater that use
an artificially labelled isotope (usually 13C) will invalidate the use of CSIA. Small
quantities of the artificially labelled isotope dissolve within the groundwater and alter the
natural abundance of isotopes present within the contaminants. Due to the sensitivity of
CSIA, the addition can skew the ratio of naturally-occurring isotopes sufficiently to result
in inaccurate conclusions (USEPA, 2008).

Advantages of CSIA
• The technique is not dependent on trends in concentrations or daughter product
generation;
• Allows very precise assessment of degradation of specific contaminants across a
site;
• Multiple isotopes in a given molecule can be assessed, for example both 13C/12C,
2
H/1H and 37Cl/35Cl in TCE;
• Possible identification of source provenance; and
• Allows accurate in situ quantification of the extent of degradation and estimation of
contaminant degradation rates.

Limitations of CSIA
• The applicability of the technique is limited for high molecular weight compounds
such as petroleum hydrocarbons. This is because fractionation of an individual atom
at the location of bond breakage due to biodegradation, may be masked by the
presence of multiple copies of that atom at other locations within the molecule.
• For some compounds and degradation pathways, such as aerobic biodegradation of
toluene, fractionation only occurs by reactions that break down the methyl group,
rather than reactions that attack the benzene ring, which may result in an apparent
limited amount of biodegradation. A combination of hydrogen and carbon isotope
fractionation analysis should be performed.

107
Appendix 9: Molecular Biological
Tools
A9.1 Introduction
Molecular biological tools (MBTs) are advanced and evolving techniques that analyse
biological characteristics in soil and groundwater. MBTs provide strong but not definitive
evidence to help understand, quantify and demonstrate the effectiveness of MNA. MBTs
use deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) techniques to identify
organisms potentially responsible for biodegradation and biological processes, their
abundance and function. The theoretical basis and application of MBTs for MNA has
been critically reviewed (Alvarez and Illman, 2005; Illman and Alvarez, 2009; Thornton
et al., 2016).

MBTs are technologies that can be applied to samples of environmental media and have
supplanted microcosms in some cases. They provide an efficient and cost-effective
means to collect spatially and temporally representative data supporting evaluations of
biodegradation and other biologically-mediated processes. MBTs can therefore
complement existing analyses for primary and secondary lines of evidence based upon
comparable chemical, geochemical and biological datasets.

Over the past decade, some MBTs have achieved technological maturity and entered
the commercial market for a broad range of groundwater pollutants. The rise in
significance of MBTs in the MNA toolbox is well demonstrated by industry research that
correlated five key parameters capable of demonstrating MNA for chlorinated ethenes3,
including the abundance of Dehalococcoides sp. (Dhc) (Lebrón et al., 2015), a group of
microorganisms capable of complete dechlorination of PCE and TCE. More recent
research has further highlighted the role of MBTs in the assessment of biodegradation
potential, providing advanced understanding that could not otherwise have been
gathered from conventional analyses alone (e.g. Badin et al., 2016; Ottosen et al., 2020;
Ottosen et al., 2021; Toth et al., 2021). The quality of information afforded by these
technologies has proven invaluable for demonstrating the feasibility of MNA in recent
years and is readily accessible to industry via multiple commercial environmental DNA
sequencing laboratories.

A9.2 Molecular Tools for MNA

An overview of microbiological methods used to interpret microbial communities and
biodegradation processes in environmental samples is provided in Figure A9.1.

3
Lebrón et al. (2015) demonstrated that the following parameters were correlated with the
degradation rates of TCE, cis-DCE and VC: Dehalococcoides sp. [Dhc] abundance; magnetic
susceptibility as a surrogate for magnetite abundance; iron sulfide (FeS); methane (CH4); and
ferrous iron (Fe(II)).
108
Figure A9.1: Overview of methods used to characterise microbial communities in
environmental samples. SIP refers to stable isotope profiling. Reproduced from
Thornton et al. (2016), Springer Nature, with permission of SNCNC.

MBTs using DNA/RNA-based methods are commercially available and are in common
use to support contaminant and process-specific demonstration of biodegradation for
MNA (e.g. ITRC, 2011; Adamson and Newell, 2014; NAVFAC, 2021):

109
• Show that key organisms responsible for biodegradation are present (e.g.
Dehalococcoides for chlorinated ethene reductive dechlorination);
• Show that key enzymes indicating a specific biodegradation process are present and
potentially active (e.g. VC reductase);
• Establish the relative abundance of key microbial populations.

These methods are described further in this appendix.

A9.2.1 Polymerase Chain Reaction and Variants

Polymerase chain reaction (PCR) is a technique that can test for the presence of a
specific organism, family of microorganisms or expressed genes in environmental
samples such as soil or groundwater. This technique can be used to identify
microorganisms capable of degrading contaminants but not provide direct evidence
alone that biodegradation has occurred or is occurring.

PCR utilises DNA from an environmental sample with DNA polymerase, DNA primers
specific to a target 16S ribosomal RNA (16SrRNA) gene and DNA building blocks to
synthesise and selectively amplify sequences of the 16SrRNA genes of interest in new
strands of DNA.

PCR methods have been developed for a wide range of groundwater pollutants such as
petroleum hydrocarbons, fuel oxygenates, phenols, pentachlorophenol, perchlorate,
polychlorinated biphenyls (PCBs), metals, radionuclides and chlorinated solvents. PCR
data for a specific gene or microorganism are usually reported simply as “present” or
“absent”. However, these data can be used with variants of PCR and other MBTs to
provide further insight on the abundance and activity of identified microorganisms.

Quantitative PCR (qPCR) measures fluorescence of specific dyes or “probes” that

adhere to the PCR amplified DNA or genes, quantifying the number of specific
sequences or genes from which the abundance of target microorganisms can be
inferred.

Reverse transcriptase qPCR (RT-qPCR) utilises the production of RNA during

biodegradation (tracked by specific enzyme production) to track the activity of target
microorganisms. 16SrRNA is extracted from environmental samples then converted to
“complementary” DNA (cDNA) that can be analysed by PCR to determine enzyme
presence or measure enzyme abundance by qPCR.

qPCR and RT-qPCR data are usually reported in units of gene copies per litre.
Collectively, there are currently approximately 50 qPCR and RT-qPCR target analyses
in wide commercial use for MNA applications (much fewer than PCR) for chlorinated
solvents and associated compounds (chlorinated ethenes [e.g. Clark et al., 2018],
chlorinated ethanes [e.g. Scheutz et al., 2011], chlorinated methanes [e.g. Puigserver et
al., 2020], chlorobenzenes [e.g. Qiao et al., 2018], chlorophenols [e.g. Mikkonen et al.,
2018], chloropropanes [e.g. Yan et al., 2009], 1,4-dioxane [e.g. He et al., 2017a]),
petroleum hydrocarbons (BTEX [e.g. Beller et al., 2008], PAHs [e.g. Oka et al., 2011]),
fuel oxygenates such as MTBE [e.g. Kuder and Philp, 2008], PCBs [e.g. Liang et al.,
2014] and select metals (e.g. uranium, [Barlett et al., 2012]).

110
Weatherill et al. (2018) cites several studies that show how qPCR methods enhanced
understanding chlorinated solvent biodegradation pathways far beyond what could be
determined from chemical and geochemical groundwater analysis alone. This includes
the combined use of chemical analysis and qPCR to demonstrate co-occurrence and co-
activity of aerobic VC degraders and anaerobic Dhc in riverbed sediments, where sharp
redox gradients are often characterised (Atashgahi et al., 2017). VC degradation studies
in aerobic and anaerobic microcosms provided geochemical evidence for aerobic
mineralisation and reductive dechlorination pathways, yet Dhc and VC reductive
dehalogenase-encoding genes (vcrA and bvcA) were enriched in both microcosms. The
study findings directly influence understanding of VC biodegradation pathways, and
constraints on the performance of MNA.

qPCR and RT-qPCR data indicating abundant and active populations of specific
microorganisms capable of biodegradation could, with primary lines of evidence for
contaminant mass or concentration reduction, support demonstration of MNA.
Conversely, qPCR indicating sub-optimal populations or unacclimated populations of
microorganisms may help explain why observed rates of biodegradation are lower than
expected and the need for intervention.

Threshold values for qPCR data indicating suitable conditions for biodegradation have
been cited for a limited number of specific contaminants. For example:

• Dhc between 104 and 106 gene copies per litre can support MNA and >106 ensures
ethene production (Lebrón et al., 2015);
• Dhc >107 gene copies per litre are cited as associated with high rates of ethene
formation (Lu et al., 2006).

Application of this threshold to Dhc data is further extended by comparing RT-qPCR data
for TCE and VC reductase enzymes (tceA, vcrA and bvcA). VC accumulation is
considered likely where vcrA and/or bvcA concentrations are non-detect or significantly
lower than Dhc and tceA. Where vcrA and/or bvcA concentrations are similar to Dhc
and tceA, complete dechlorination to ethene is much more likely.

qPCR alone can provide sufficient evidence of biodegradation potential for

microorganisms with specific metabolism (e.g. Dhc). However, degradation of some
contaminants (e.g. 1,4-dioxane) is performed by microorganisms with more varied
metabolism. In such cases, RT-qPCR data are required to confirm whether the
abundance of these microorganisms is associated with biodegradation of the target
contaminant, or abundance of organisms expressing suitable functional genes
(Figure A9.2).

111
Figure A9.2: Correlation between dioxane degradation rate and abundance of
propane monooxygenase alpha subunit (prmA) (A) but not 16S rRNA (B) gene
copies. Reprinted (adapted) with permission from He et al. (2017a). © 2017
American Chemical Society.

A9.2.2 16SrRNA Amplicon Sequencing

16SrRNA amplicon sequencing, so-called “Next Generation Sequencing” (NGS),

provides a means to achieve comprehensive microbial community characterisation,
insights into community function and dynamics that are simply not possible with qPCR
methods that target specific organisms (e.g. Badin et al., 2016; He et al., 2017b; Toth et
al., 2021).

NGS provides insights to complex microbial systems, such as those impacted by

contaminant mixtures or contaminants for which degradation is performed by consortia
rather than key, individual species or groups of microorganisms. NGS can indicate
dominant or potential microbial processes, including biodegradation, and assist
identifying conditions which may inhibit biodegradation, for example, related to either the
site or contaminant matrix. Badin et al. (2016) provides intriguing insights to a microbial
community and its function following thermal remediation of a PCE source zone. The
study demonstrated the enhancement of anaerobic biodegradation of PCE due to
release of dissolved organic carbon caused by steam injection and the predominance of
abiotic rather than biotic degradation pathways downgradient of the source with
combined applications of CSIA and qPCR. The role of the microbiological community to
induce abiotic degradation in the plume was evidenced through NGS, that indicated the
abundance of iron-reducing, sulfate-reducing bacteria and pyrite (FeS2) oxidising
bacteria, the potential for abiotic degradation with reactive iron sulfide minerals,
alongside other lines of evidence.

NGS datasets are typically large and complex, indicating the relative abundance of
microbial genus producing 16SrRNA. Novel visualisation techniques facilitate
understanding (Figure A9.3) but multivariate statistical methods are typically required to
interpret these data. NGS does not report to species or strain-level, nor does it provide
information on functional genes. Quantification can be achieved through calibration
against qPCR for total 16SrRNA biomass.
112
Figure A9.3: Maximum likelihood consensus tree showing the affiliation of near-
complete 16SrRNA genes (1231 bp) belonging to anaerobic benzene and PAH-
degrading microorganisms, and select reference strains. Additionally, the
specificity of the Thermincola and ORM2 qPCR primer pairs used in this study is
illustrated. Reprinted (adapted) with permission from Toth et al. (2021). © 2021
American Chemical Society.

A9.2.3 Metagenome Analysis

Metagenome analysis uses PCR to amplify 4 million base pair genes, including
16SrRNA. So-called “shotgun genomics” provides the most comprehensive
characterisation of microbial strains, non-microbial species (e.g. fungi, protozoans), and
their functional genes, from which the activity and potential for sustained contaminant
degradation can be inferred. Dang et al. (2018) applied metagenome sequencing to
quantify chlorinated solvent and 1,4-dioxane biodegradation taxonomy and functional
genes at five sites. The analysis determined the abundance of (1) genera associated
with chlorinated solvent degradation, (2) reductive dehalogenase genes, (3) genes
associated with 1,4-dioxane removal, (4) genes associated with aerobic chlorinated
solvent degradation, and (5) Dehalococcoides mccartyi genes associated with hydrogen
and corrinoid metabolism. The work illustrates the importance of metagenome
sequencing to provide a more complete picture of the functional abilities of microbial
communities and its advantages over simpler MBTs (such as qPCR) because an

113
unlimited number of functional genes can be quantified. Multivariate statistical methods
support a higher level of interpretation that were used to highlight the significant, but
typically overlooked, roles of supporting organisms to Dhc for anaerobic biodegradation
of PCE and TCE, for example (Figure A9.4).

Figure A9.4: Principal component analysis of functional genes (A) and genera (B)
associated with chlorinated solvent and 1,4-dioxane biodegradation in
groundwater. Reprinted (adapted) with permission from Dang et al. (2018). © 2018
American Chemical Society.
114
A9.3 Sampling for MBTs
Sampling for MBT analysis is not onerous. Commonly, groundwater samples are used
for molecular analysis. These can be collected in standard laboratory bottles or by
passing a known volume of groundwater through 0.2 μm filters, which are then analysed
in the laboratory. Such samples can be therefore collected at the same time as routine
groundwater monitoring events to collect data for primary and secondary lines of
evidence for MNA. MBTs can also be applied to samples of soil, bedrock or sediment, to
characterise biofilms attached to aquifer solids.

115
Appendix 10: Selected Literature
on Natural Attenuation of Key
CoPC
Table A10.1: List of selected literature reviews that have critically appraised NA of
key CoPC. This list is not exhaustive but is aimed to help the reader identify key
review papers.

CoPC Literature source

Petroleum Seagren, E.A. and Becker, J.G., 2002. Review of natural attenuation of
hydrocarbons BTEX and MTBE in groundwater. Practice Periodical of Hazardous,
Toxic, and Radioactive Waste Management. Vol. 6, Issue 3.
https://doi.org/10.1061/(ASCE)1090-025X(2002)6:3(156)
Thornton, S.F., Morgan, P.M. and Rolfe, S.A., 2016. Bioremediation of
hydrocarbons and chlorinated solvents in groundwater:
Characterisation, design and performance assessment. In: Protocols
for Hydrocarbon and Lipid Microbiology. McGenity, T.J., Timmis, K.N.
& Nogales, B. (eds), Springer Verlag, Berlin Heidelberg. Series ISSN
1949-2448. pp.11-64, https://doi.org/10.1007/8623_2016_207

Ether oxygenates Thornton, S.F., Nicholls, H.C.G., Rolfe, S.A., Mallinson, H.E.H. and
(petrol additives) Spence, M.J., 2020. Biodegradation and fate of ethyl tert-butyl ether
(ETBE) in soil and groundwater: a review. Journal of Hazardous
Materials. https://doi.org/10.1016/j.jhazmat.2020.122046
Moyer, E.E. and Kostecki, P.T. (eds), 2003. MTBE Remediation
Handbook. Springer Publications. ISBN 978-1-4615-0021-6
Biodiesel additives Thomas, A.O., Leahy, M.C., Smith, J.W.N., Spence, M.J., 2017. The
natural attenuation of fatty acid methyl esters (FAME) in soil and
groundwater. Quarterly Journal of Engineering Geology &
Hydrogeology. https://doi.org/10.1144/qjegh2016-130

Chlorinated Rifai, H.S., Newell, C.J., Wiedemeier, T.H., 2019. Natural attenuation
solvents of chlorinated solvents in groundwater. In: Handbook of Solvents,
Volume 2: Use, Health and Environment (Third Edition).
https://www.sciencedirect.com/book/9781927885413/handbook-of-
solvents

Ammonium Buss, S.R., Morgan, P., Herbert, A., Thornton, S.F., Smith, J.W.N.,
2004. A review of ammonium attenuation in soil and groundwater.
Quarterly Journal of Engineering Geology and Hydrogeology, 37, 347-
359. https://doi.org/10.1144/1470-9236/04-005

Nitrate Rivett, M.O., Buss, S.R., Morgan, P., Smith, J.W.N. and Bemment,
C.D., 2008. Nitrate attenuation in groundwater: A review of
biogeochemical controlling processes. Water Research, 42, 4215-
4232. https://doi.org/10.1016/j.watres.2008.07.020
Rivett, M.O., Smith, J.W.N., Buss, S.R., Morgan, P., 2007. Nitrate
occurrence and attenuation in the major aquifers of England and
Wales. Quarterly Journal of Engineering Geology & Hydrogeology
40(4), 335-352. https://doi.org/10.1144/1470-9236/07-032

116
CoPC Literature source

Metals Gandy, C.J., Smith, J.W.N. and Jarvis, A.P., 2007. Attenuation of
mining-derived pollutants in the hyporheic zone: a review. Science of
the Total Environment, 373, 435-446.
https://doi.org/10.1016/j.scitotenv.2006.11.004

Sulfolane Dinh, M., Hakimabadi, S.G., Pham, A.L-T., 2020. Treatment of

sulfolane in groundwater: A critical review. J. Env. Management, 263,
110385. https://doi.org/10.1016/j.jenvman.2020.110385

Herbicides Buss, S.R., Thrasher, J., Morgan, P. and Smith, J.W.N., 2006. A
review of mecoprop attenuation in the subsurface. Quarterly Journal of
Engineering Geology and Hydrogeology, 39, 283-292.
https://doi.org/10.1144/1470-9236/04-081

Phenols and Thornton, S.F., Quigley, S., Spence, M.J., Banwart, S.A., Bottrell, S.,
cresols Lerner, D.N., 2001. Processes controlling the distribution and natural
attenuation of dissolved phenolic compounds in a deep sandstone
aquifer. J. Cont. Hydrol., 15, 233-267. https://doi.org/10.1016/s0169-
7722(01)00168-1

117
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