Environmental Forensics

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EnvironmentalForensics.pptx

Environmental Forensics

Babatunde Bolaji Benard

[email protected]

The Overview of Pollution in the Niger Delta

Air pollution

Major causes

Industries

Automobiles

Other activities that release carbon monoxide into the atmosphere

Effects

Human health

Soots

Animals

Plants

The atmosphere

Artisanal refining sites and equipment

What is Environmental Forensic

Environmental forensics has emerged as an important area of environmental studies over the past two decades.

There are two basic aspects to any environmental investigation.

The first being a conventional approach where the standard EPA (Environmental Protection Agency) methods are used to determine concentrations of selected compounds released into the environment. These methods are extremely well documented and widely used, but only provide information on specific target compounds. Whilst this information may be useful for monitoring purposes it is of little use when trying to determine the source of a spill or contaminants in the environment.

If the purpose of an investigation is to determine the source of a contaminant, or point of release, then it is necessary to use a wide variety of analytical techniques and integrate all of the resulting data into one comprehensive data set. It may not always be possible to obtain a unique answer, particularly in the case of groundwater contaminants where there might only be one compound, for example PAH. In that case if there are multiple possible sources in the area it may be difficult to narrow it down to a specific source. Furthermore fingerprinting tools that may be useful with complex mixtures may not be directly applicable to single component mixtures.

Historical Perspective

Application

Sources of Pollution

Fate of Contaminants

Contaminant Source Tracking (Food Chain)

Tools

Aerial and satellite Photography

Age dating releases

• Identification of sources and pathways

Remote Sensing Multi-spectral Surveys

• Identify ecological stresses and stressors

• Contaminant identification

• Summary distribution of contaminants

Laboratory Analysis

Identification

Source identification

Cost allocation Cost allocation

Types

GC with various detectors GC with various detectors

Spectrophotometric at various wavelengths Spectrophotometric at various wavelengths

Radiochemistry

Electrochemical

Microscopy

Crystallography

Physical and chemical properties

Toxicological

Tools Cont’d

Products of forensic Investigations

Unique Identifiers

Congener analysis

Biomarkers

Isotopic ratios

Additives

Fingerprinting

Speciation

Current Bacterial indicators of Fecal Contamination

Total coliforms:

drinking, bathing and shellfish water standards

not feces-specific (environmental sources).

Fecal ("thermotolerant") coliforms (FC):

detect by growing at elevated temperature of 44-45oC

ditto total coliforms, but less so

E. coli: the "fecal" coliform

Detect and distinguish from other total and fecal coliforms by Beta-glucuronidase activity

may occur naturally in tropical environments (and possibly elsewhere)

Fecal streptococci (FS):

Mostly Lancefield group D (and some group Q) streptococci and enterococci

not feces-specific.

Enterococci:

More feces-specific sub-set of FS: Enterococcus faecalis & E. faecium

EPA guideline for bathing water quality

Microbial Source Tracking (MST)

MST techniques can be divided into two categories:

1. Molecular (genotypic) and biochemical (phenotypic) techniques rely on the close association of certain microorganisms (generally bacteria or viruses), with a specific host, and genetic or phenotypic differences that allow host-specific microbes to be discriminated from others.

2. Chemical methods generally rely on the detection of chemicals associated with anthropogenic activities.

Molecular and Biochemical Techniques

Molecular and biochemical MST techniques can be divided into two broad categories: library dependent and library-independent.

• Library-dependent techniques identify fecal sources from water samples based on a library or database of bacteria isolated from known fecal sources. The library is developed by collecting microbial isolates from known potential sources. The molecular or biochemical pattern of the individual microbial isolates is sometimes referred to as a fingerprint. These identifying patterns can be discerned by a variety of methods, e.g., ribotyping (molecular) and antibiotic resistance analysis (biochemical).

- Molecular (genotypic) techniques are based on the genetic makeup of a cell or organism, e.g., ribotyping, pulsed field gel electrophoresis, rep- polymerase chain reaction (PCR).

- Biochemical (phenotypic) techniques use observable characteristics or traits of an organism such as biochemical or physiological properties, e.g., antibiotic resistance analysis, carbon source utilization.

Library-Dependent Techniques (LDT)

A library is a database of fingerprints from individual bacterial isolates, obtained from potential pollution sources. Bacteria for a source library are normally recovered from animal feaces, though bacteria from animal waste lagoons, septic tanks, and wastewater treatment plants can be used. Most library-dependent techniques require a cultivation step to obtain the bacterial isolates that will be used to generate the library (knowns) and the water bacterial isolates (unknowns) that will be compared against the library.

Library-dependent techniques rely on either molecular or biochemical discrimination of isolates. A number of different microbes may be used for library-dependent techniques including feacal coliforms, Escherichia coli (E. coli), Enterococcus species (enterococci), or feacal streptococci (essentially enterococci, but includes several additional Enterococcus species).

Recently the trend in MST research has been to move away from library-based techniques. This is due in part to their performance in the Southern California Coastal Water Research Project comparison study (SCCWRP) (SCCWRP, 2003; Hagedorn et al., 2011). In addition, the need to develop large site-specific libraries has decreased the interest in using library-dependent approaches (Santo Domingo et al., 2011).

(LDT)

Molecular (Genotypic) (LDT)

Molecular techniques are based directly on the genetic material of the bacterial or viral organism. Bacteria used for these techniques are usually E. coli or Enterococcus spp.

The theory behind these MST techniques is that unique strains of a bacteria species are adapted to their known specific environment (intestines of a particular host species) and, as a result, differ genetically from other strains found in other host species.

A number of genotypic methods are used to type bacteria for library-dependent techniques including ribotyping, pulse field gel electrophoresis (PFGE), and repetitive (rep) polymerase chain reaction (PCR). These techniques are described below.

It is important to note that (1) the genotypic techniques described below differ in discriminatory ability, and (2) bacterial isolates that are grouped into the same “strain” by one method may be separated into distinct strains by a more discriminatory method. The Bibliography section of this report includes studies that have used these techniques.

Molecular (Genotypic) (LDT) Cont’d

Molecular (Genotypic) (LDT) Cont’d

Ribotyping:

Ribotyping has been one of the most widely used techniques in library-dependent MST applications (EPA, 2011). Ribotyping is based on the detection of genetic differences in the genomic sequences within or flanking the 16S and 23S ribosomal ribonucleic acid (rRNA) genes. These rRNA genes are highly conserved in bacteria (EPA, 2005).

For this method, the chosen bacterial group is cultured from fecal samples using standard techniques. E. coli or enterococci are isolated, and a few isolates are picked for genotypic characterization (generally a percentage of the bacterial count). Genomic DNA is isolated for each E. coli strain. Bacterial DNA is digested into fragments using restriction enzymes.

DNA fragments are separated by size using gel electrophoresis. The fragments are transferred to a gel blot, and a labeled probe is used to hybridize to certain portions of the rRNA genes. Because the genome contains several copies of the rRNA genes dispersed throughout the chromosome, the binding of the probe to the DNA fragments which contain it creates a banding pattern that can be visualized by autoradiography or chemical development. These patterns can be used to discriminate among bacterial strains

Ribotyping Cont’d

The banding pattern is captured using digital cameras. Difference in the size and location of the banding patterns can then be compared to known sources in the library database. Image analysis to compare banding patterns can be performed using commercially available software (Scott et al., 2002; Rees et al., 2010). Variables in ribotyping include the type of fecal indicator bacteria used to form the library, as well as the type and number of restriction enzymes used to fragment the DNA. It has been suggested that two restriction enzymes should be routinely used to increase the technique’s discriminatory ability (EPA, 2005).

Advantages This method can be used to classify isolates from multiple sources. When performed by a skilled technician, it is highly reproducible.

Disadvantages Ribotyping is a demanding procedure that requires multiple steps and specialized equipment.

Also, the need for specialized training, high supply costs, and the time required to complete the procedure are disadvantages. As with many genotypic techniques, lab-to-lab variation, issues of repeatability, gel variability, and analysis techniques often make comparison of results from different laboratories difficult.

Complex statistical analysis is often required to determine which sources are likely present. A good working knowledge of statistics is needed. The database (library) size, geographic distribution of isolated bacteria, and the presence of replicate isolates in the bacterial source library affect the ability of ribotyping to differentiate among bacteria at the host-species level (EPA, 2005). In addition, both genotypic and phenotypic techniques would likely break down in complex watersheds with numerous sources (Rees et al., 2010; Harwood, 2011).

Ribotyping Cont’d

Molecular (Genotypic) (LDT) Cont’d

Pulsed Field Gel Electrophoresis (PFGE)

One of the most common techniques, PFGE, is similar to ribotyping. The difference is the whole DNA genome is used instead of the rRNA portion of the genome. Initial steps for obtaining bacterial isolates are the same as ribotyping.

PFGE uses infrequently cutting restriction enzymes on the entire DNA genome. The procedure for DNA isolation is crucial, as large genomic fragments are generated which must not be broken during sample preparation. The genomic fragments are then separated by alternately pulsed, perpendicularly oriented electrical fields, instead of using standard gel electrophoresis. After electrophoresis and staining of the gels, a banding pattern emerges. Patterns are compared to known sources in the library database.

Pulsed Field Gel Electrophoresis (PFGE) Cont’d

Advantages PFGE can be used to classify isolates from multiple sources, and it is among the most discriminatory genotyping methods. When performed by a skilled technician, the method is highly reproducible.

Disadvantages PFGE requires a high degree of technical skill and specific equipment, is time consuming, and is relatively expensive (EPA, 2011). As with ribotyping, a large, geographically-specific source database (library) is required.

Complex statistical analysis is often required to determine which sources are likely present. A good working knowledge of statistics is needed. Both genotypic and phenotypic techniques would likely break down in complex watersheds with numerous sources (Rees et al., 2010; Harwood, 2011).

Repetitive Palindromic Polymerase Chain Reactions (rep-PCR)

PCR allows for rapid amplification of target DNA sequences. PCR is used both in cultivation dependent and independent approaches. For the rep-PCR technique, intervening sequences between certain repetitive portions of the microbial DNA are amplified using rep-PCR and one primer that targets each end of the repetitive, palindromic sequence.

Repetitive DNA elements are scattered throughout the bacterial genome and are separated by distances which vary according to the bacterial species or strain, which forms the basis for the discriminatory patterns generated by rep-PCR. BOX-PCR is a variant of rep-PCR that uses a different primer in the PCR step.

The amplified DNA fragments are separated in agarose gels, producing a banding pattern or “fingerprint” that discriminates among bacterial strains. Bacteria having the same pattern are considered to be of the same strain.

Advantages

Rep-PCR can be used to classify isolates from multiple sources. Compared to the other library techniques, rep-PCR is quicker, easier to use, less expensive, and potentially has a faster turnaround time.

Disadvantages

Although relatively simple compared to PFGE and ribotyping, rep-PCR results tend to be somewhat less reproducible than PFGE or ribotyping (EPA, 2011).

A highly trained technician is required to obtain reproducible results. Complex statistical analysis is often required to determine which sources are likely present.

A good working knowledge of statistics is needed.

As with the other library techniques, a large source database (library) is required that is geographically specific.

In addition, both genotypic and phenotypic techniques would likely break down in complex watersheds with numerous sources (Rees et al., 2010; Harwood, 2011).

Biochemical (Phenotypic) (LDT)

Biochemical techniques are based on observable physical or biochemical characteristics of an organism, as determined by both genetic information and environmental influences.

Librarydependent biochemical techniques include antibiotic resistance analysis and carbon and nutrient utilization profiling. The Bibliography section includes studies that have used these techniques.

Biochemical (Phenotypic) (LDT) Cont’d

Antibiotic Resistance Analysis (ARA)

ARA uses patterns of antibiotic resistance for identifying sources of fecal contamination. The premise is that humans and animals are exposed to different types of antibiotics, and that this selective pressure will alter the antibiotic resistance profile of their fecal bacteria.

These differences should be useful in discriminating among fecal bacterial sources. For this method to be applied, a source library must be developed, using fecal samples from potential contributors in the watershed (e.g., human, livestock, wildlife).

The known sources are analyzed for antibiotic resistance and patterns of resistance. Discriminant analysis (a form of multiple analysis of variance) or logistic regression (a model used to predict the probability of an occurrence) uses the antibiotic resistance patterns from known sources to generate the predictive equations that are used to classify unknown isolates by source.

Antibiotic Resistance Analysis (ARA)

Advantages ARA is relatively simple and fast, requiring less technical expertise and expensive equipment than genotypic methods. These techniques can distinguish multiple sources including human and domestic animals.

Disadvantages Complex statistical analysis is often required to determine which sources are likely present. A good working knowledge of statistics is needed. A geographically-specific reference database is required because phenotypic techniques are geographic and temporally specific. In addition, both genotypic and phenotypic techniques would likely break down in complex watersheds with numerous sources (Rees et al., 2010; Harwood, 2011).

Biochemical (Phenotypic) (LDT) Cont’d

Carbon Utilization Profile (CUP) and Nutrient Utilization Pattern (NUP).

Both CUP and NUP are based on differences among bacterial uses of a wide range of carbon and nitrogen sources for energy and growth. For CUP and NUP, the BIOLOG system allows the user to rapidly perform, score, and tabulate 96 carbon or nitrogen source utilization tests per isolate. Like ARA, the patterns of known sources can be analyzed using discriminant analysis to generate predictive equations that are used to classify unknown isolates using a source library.

While CUP and NUP work well in the laboratory for pure culture characterization/identification, there are many environmental factors in a watershed that can affect bacterial nutrient requirements that may make this method impractical for field determination (Simpson et al., 2002). Like ARA, the CUP method is relatively simple and allows for the analysis of hundreds of isolates in a short period of time.

Carbon Utilization Profile (CUP) and Nutrient Utilization Pattern (NUP) Cont’d.

Advantages CUP is relatively simple and fast, requiring very little technical expertise. Equipment and supplies are expensive (Harwood, 2011). These techniques can distinguish multiple domestic animal sources. However, the CUP and NUP techniques have been tested on a small scale and therefore require more testing.

Disadvantages Complex statistical analysis is often required to determine which sources are likely present. A good working knowledge of statistics is needed. A geographically-specific reference database is required because phenotypic techniques are geographic and temporally specific. In addition, both genotypic and phenotypic techniques would likely break down in complex watersheds with numerous sources (Rees et al., 2010; Harwood, 2011).

Library-independent techniques do not require the development of a source library database.

These techniques rely on a species-specific genotype or characteristic. A variety of bacteria and viruses have been used for MST. It is important to consider the MST indicator survival rate and abundance in the environment. Survival of microbial indicators depends on a variety of factors including their physiology, exposure to radiation, temperature, salinity, predation and competition, amount of organic matter present, and the type of sediments available (Harwood, 2011). Some indicators more closely correlate with fecal indicator bacteria (e.g., E. coli and fecal coliforms).