cytoculture logo    Monitored Natural Attenuation


CytoCulture Home


Site Remediation Protocol:

Monitored Natural Attenuation of Contaminated Groundwater

Technical Update


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 attenuation.

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 CytoCulture’s 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 bioremediation).

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 degradation activities.

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.

CytoCulture’s 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

Figure 1: Typical MNA site

Table 1: Example of MNA results



For more information about CytoCulture and services,