Site Remediation Protocol:
Monitored Natural Attenuation of Contaminated Groundwater
CytoCulture has observed a major advance in the use of Monitored Natural Attenuation (MNA)
to close contaminated sites in California and around the country. We have been attending
conferences and have been directly involved with the leading government agencies, such as
the Air Force Center for Excellence (AFCEE), the US Environmental Protection Agency
(USEPA), and the California Environmental Protection Agency Department of Toxic
Substances Control (DTSC), responsible for the regulation and advancement of
monitored natural attenuation. We would like to pass on some of this information to you,
as our collaborating clients, so that you can benefit from what we have learned.
The natural biodegradation of contaminants has been known for some time, and has often
been used to close contaminated sites. However, there has often been confusion about how
to present a convincing case for monitored natural attenuation (or intrinsic
biodegradation) to agencies such as the USEPA and the CA DTSC. Recent
documents and policies, fortunately, have clarified issues such as the categories of
contaminants susceptible to natural attenuation, ranges of favorable site conditions for
natural attenuation, and the types of monitoring evidence required to demonstrate natural
At the Federal Level
The AFCEE and the US EPA have published the documents that are quickly
becoming the standards for demonstrating natural attenuation. They are the "Technical
Protocol for Implementing Intrinsic Remediation with Long-Term Monitoring for Natural
Attenuation of Fuel Contamination Dissolved in Groundwater (Wiedemeier, et al 1995),"
and the "Technical Protocol for Evaluating Natural Attenuation of Chlorinated
Solvents (Wiedemeier, et al 1996)." The EPA Office of Solid Waste and Emergency
Response (OSWER) has also put out a Draft Interim Final directive titled "Use
of Monitored Natural Attenuation at Superfund, RCRA Corrective Action, and Underground
Storage Tanks (EPA Directive # 9200.4-17, 1997)." These documents describe in depth
the requirements for monitoring natural attenuation of specific contaminants, and provide
guidelines for interpreting the monitoring data. We have summarized some of this
information in the following sections.
At the State Level
The CA DTSC has also taken an aggressive role in setting guidelines for natural
attenuation. Draft checklists of the sampling requirements for demonstrating the intrinsic
biodegradation of fuel contaminants and chlorinated solvents are utilized to close a site
based upon monitored natural attenuation. These requirements are similar to those proposed
by the AFCEE documents, and many of these requirements are included as part of the
site evaluation discussion below. Sampling designs that are intended to meet these
requirements are recommended in making a case for MNA at a particular site. Both the CA DTSC and the AFCEE document specify preferred analytical test methods for the analyses
of biodegradation parameters. In demonstrating natural attenuation, it is best to provide
"multiple lines of evidence for biodegradation activity", and to evaluate
all of the risk factors associated with the contaminants.
First Steps in Site Evaluation
Chemical Indicators of Biodegradation
Strong evidence for intrinsic biodegradation activities can be learned from site
chemical monitoring. The following discussion summarizes site measurement results
interpreted as per the Air Force Center for Excellence and DTSC Guidelines for Intrinsic
Bioremediation and CytoCultures expertise in biodegradation microbiology.
Nutrients: Ammonia-N and o-phosphate are limiting nutrients
required for microbial growth and activity. Ammonia is a preferred nitrogen source and
o-phosphate is a preferred phosphorus source for most soil bacteria. Biodegradation
activity could potentially be limited by low levels of these nutrients even if all other
growth needs (e.g. electron acceptors and carbon sources) are available. Depletion of
these nutrients, as compared to control wells outside of contaminated areas, can also be
used as indicators of biodegradation activity.
DO and ORP (redox): The best overall indicators of anaerobic conditions are
measurements of dissolved oxygen (DO) and redox potential (ORP). In general,
DO measurements of less than 1 ppm suggest that anaerobic conditions may exist. Aerobic CA
groundwater sites with biodegradation activity typically are in the range of 3-5 ppm DO.
Theoretically, aerobic degradative activity occurs at a highly positive redox potential,
while anaerobic microbial processes such as methanogenesis and sulfate reduction will
occur at strongly negative redox potentials. However, caution should be used in the
interpretation of redox potential field data in terms of microbial activity, as these
measurements are due to complex interactions between chemical species present in the
groundwater and microbial byproducts.
Contaminant Biodegradation Theory: Contaminant biodegradation is largely based
upon microbial respiration. In respiration, microbes gain energy from the consumption
(oxidation) of electron donors coupled to the utilization (reduction) of electron
acceptors. Contaminants will either serve as electron donors or electron acceptors. For
example, a common biodegradation activity is the aerobic metabolism of fuel contaminants.
In this case, oxygen is the electron acceptor, while the fuel hydrocarbon is the electron
donor which may be oxidized completely to CO2 by this process. Under anaerobic
conditions, alternative electron acceptors, such as nitrate and sulfate, may be utilized
in contaminant oxidation in the absence of oxygen. In general, rates of biodegradation
follow an order of favorable electron acceptor availability: O2 >MnIV >NO3- >FeIII >SO42- >CO2. In some anaerobic
processes, the contaminant will actually serve as the electron acceptor. This is the case
with the anaerobic reductive dechlorination of contaminants such as PCE, DCA,
chlorobenzenes and PCBs.
Biodegradation activity indicators: It is important to determine the electron
acceptors being utilized during respiration as they will affect the extent and rates of
biodegradation activity. Several chemical species that can be measured in groundwater
samples are specific end or starting products of microbial metabolism. Their presence, or
their absence, in comparison to background levels can therefore be used to infer
biodegradative processes. Nitrate depletion, for example, may indicate
denitrification (the reduction of nitrate to N2) or nitrate reduction. Nitrite,
an intermediate in denitrification, may also be an indicator of this process. Sulfate depletion or the presence of sulfide may indicate sulfate reducing activity. In the
process of iron reduction, Fe(III) is reduced to Fe(II). Therefore, elevated
levels of Fe(II) in the groundwater may be indicative of microbial iron reduction.
Complete biodegradation of hydrocarbons will result in the formation of CO2,
or CH4 in the process of methanogenesis. Elevated concentrations of
these gasses will also indicate microbial activity in groundwater samples. Benzene
biodegradation will also result in the formation of alkalinity in the groundwater
(AFCEE, 1995), and elevated levels of alkalinity can be used as an indicator of benzene
biodegradation both aerobically and anaerobically (except with methanogenesis).
Contaminant-specific biodegradation byproducts: Direct evidence for
biodegradation can also be found by looking for known metabolic byproducts of the
contaminants. For example, Phenol is an intermediate of benzene biodegradation
under methanogenic/ fermentative (anaerobic) conditions, and may indicate anaerobic
benzene metabolism. Ethene is a byproduct of the anaerobic reductive dehalogenation
of chlorinated solvents, such as PCE, TCE, DCE, DCA, and vinyl chloride. Chloride,
as well may be released through dechlorination. Increased chloride concentrations
relative to background levels could be used as an indirect indicator of reductive
dechlorination. Chloride is also often used to determine if groundwater samples are from
the same groundwater flow system.
Biological Indicators of Biodegradation
In developing multiple lines of evidence for intrinsic biodegradation processes,
biological monitoring data can provide strong evidence for microbiological activity.
Biological monitoring can also provide more direct evidence for natural attenuation as
opposed to chemical indicators of biological activity. We have included microbiological
assays routinely used by CytoCulture and others to document natural attenuation (intrinsic
General Microbial Assays There are several means of determining general or
total populations of bacteria at a site. These assays are used to monitor site conditions
favorable for bacteria or population changes relative to site chemistry (natural or
induced). These assays will also provide evidence for microbial activity, and can indicate
zones of microbial inhibition. Total heterotrophic plate counts (anaerobic or
aerobic) determine the total number of bacteria able to grow on a wide variety of carbon/
energy sources. Direct Counts use microscopy to more accurately determine total
bacteria in a sample. Most Probable Number (MPN) assays determine population
numbers of specific groups of anaerobes, such as sulfate, iron, manganese or nitrate
reducers, or methanogens. These groups will have different biodegradative
activities and rates and are active in the absence of oxygen.
Contaminant-Specific Microbial Assays Direct evidence for populations of
contaminant-degrading bacteria can be obtained with specialized enumeration assays. For
fuel and BTEX contaminated sites, hydrocarbon degrading bacterial plate counts can
enumerate populations of bacteria that have the ability to degrade gasoline, jet fuel,
diesel, and/or other hydrocarbons added to the growth matrix. MPN and plate count
techniques have also been adapted for the determination of populations of more specific contaminant-degraders (e.g. benzene, PCB, or TCE degraders). More advanced genetic and
biochemical methods, such as PCR, fatty acid analyses, and genetic probes, have
also been developed for characterizing bacteria capable of specific contaminant
Microcosm Studies in Support of Natural Attenuation
More direct evidence for significant site biodegradation activity may be required for
more recalcitrant contaminants (e.g. non-petroleum compounds or mixed-contaminants), for
contaminants which can biodegrade to more mobile or toxic compounds, or for sites with a
short monitoring history (OSWER, 1997). In cases such as these, the EPA recommends
laboratory microcosm studies in order to demonstrate site specific biodegradation
activity. Microcosm studies (often called treatability studies) are laboratory
simulations using site groundwater, soil, microbes, and contaminants incubated over time.
Information such as microbial byproducts, biodegradation rates, and most favorable
electron acceptor conditions (aerobic vs. anaerobic) can best be gained by these studies.
These microcosm studies are usually performed on a research basis, and are developed using
the information gained from initial monitoring events.
CytoCultures Historic Role in Assisting Clients with Monitored Natural Attenuation
CytoCulture was available to assist clients with changes and developments in the
field of monitored natural attenuation. In addition to our bioremediation
services, we would like to highlight the following services specific to the field of
monitored natural attenuation:
- Technical seminars discussing monitored natural attenuation
- Collaborations for natural attenuation proposals and projects
- Technical consulting to interpret site data and establish monitoring protocols
- Microbiological and chemical laboratory services