Monitoring Tools - Isodetect

Eignung verschiedener Methoden zum Nachweis des biologischen Abbaus bestimmter Schadstoffgruppen: 5* = Schlüsselmethode; 3* = Sekundärmethode; 1* = Ergänzungsmethode.

Suitability of different methods to detect the biodegradation of certain contaminant groups: 5 * = key method; 3 * = secondary method; 1 * = supplementary method.

Methods to Prove Contaminant Degradation

Isodetect applies isotope and further analytical tools to investigate natural and enhanced attenuation in contaminated sites. We characterize and quantify degradation processes to enable cost-effective remediation concepts. Some methods are also suitable for forensic assignment of polluters.

Tools to Prove and Characterise Contaminant Degradation

Compound-specific isotope analysis CSIA: ¹³C/¹²C
Compound-specific isotope analysis CSIA: ²H/¹H or CSIA ³⁷Cl/³⁵Cl
Isotope analysis of redox compounds (SO₄, NO₃, CO₂& CH₄)
BACTRAPS (13C-labeled in situ microcosms)
Laboratory microcosms
Metabolite analysis
Quantification of microbial genes (qPCR)
GC/MS screening
Enantiomer analysis (concentration, isotopes)

At our homepage you get insight into sampling protocols, detection limits for isotope analyses, the time frame, and the price ranges for our analytical services.

Gas-chromatoraphy-isotope-ratio-mass-spectrometer with connected upstream purge&trap-unit for online determination of compound-specific contaminant isotopes (e.g. 13C/12C, 2H/1H).

Key Method: Compound-Specific Isotope Analysis (CSIA)

To prove and quantify biodegradation, component-specific isotope analysis of carbon (13C / 12C CSIA, compound-specific isotope analysis) has become the key method for the most important pollutant groups. It is based on the enrichment of heavy isotopes (isotope fractionation) solely by biodegradation. CSIA can be used not only to elucidate pollutant degradation, but also to identify prevalent redox processes and to discriminate contaminant sources.

Features of Compound-Specific Isotope Analysis (CSIA)

In situ degradation is directly and rapidly detected.
Degradation assessment is independent from pollutant concentrations.
Pollutant degradation can be quantified and forecasted.
Attenuation by non-sustainable processes such as dispersion, sorption or dilution is gated out.
Only one sampling campaign is required.
Multiple contaminant sources could be distinguished.
Overall, significantly lower remediation costs.

Basic knowledge on stable isotopes, units of isotope values (δ‰) and isotope fractionation are described here. The feasibility of CSIA in groundwater remediation is highlighted in numerous guidelines. Analytical procedures usually require 2-8 weeks and have their determination limits in the range 1 – 10 μg / L (for carbon and chlorine isotopes). A prerequisite for the isotope analysis is the approximate knowledge of the pollutant concentrations!

Due to the manufacturing process, the isotopic values of most pollutants are inbetween -22 ‰ and -32 ‰ (red lines, range of so-called primary isotope values). Heavier, more positive levels of isotopes (blue lines) in groundwater can only be caused by biodegradation.

– Qualitative Evidence of Degradation

A direct comparison of isotope values in a contaminant plume already provides evidence of natural attenuation. The starting point is the isotope signature of the pollutant source (source isotope value), which always has the most negative isotope value at the site.

Criteria for Detecting Pollutant Degradation from ¹³C/¹²C Isotope Values

13C isotope enrichment > 2 ‰ (US EPA 2008) is found on a flow path between two measuring points (or in a time comparison of two measured values from the same measuring point). Smaller differences can be considered as weak (0.5 ‰) or strong (1.0 ‰) indications.
Isotope values are more positive than they can be according to the manufacturing process. For instance, the primary isotope value for BTEX and PCE / TCE is usually more negative than -22 ‰. Thus, taking into account the analytical uncertainty, isotope values ≥ -20 ‰ have to be considered as an evidence of biodegradation.

For more specific conclusions, the hydrogeological conditions (groundwater flow direction and velocity, thickness of aquifers, porosity, fractures, disturbances) and the environmental conditions (redox ratios) should be regarded. The scientific principles for the qualitative assessment of pollutant degradation and for the allocation of pollutant sources are explained in more detail in the basics section.

Isotopic enrichment (fractionation) and concentration decrease are inversely proportional (e.g. for toluene, see above). The percentual biodegradation of contaminants in the field can therefore be derived from the isotope values of two measuring points along a flow path, and a enrichment factor, which is specific for compounds and degradation processes (below).

– Quantification of Contaminant Degradation via Isotope Enrichment

Isotopic fractionation (isotope enrichment) of pollutants is proportional to degradation. Percentual biodegradation in the effluent of a contaminant source can therefore be calculated from the isotope values of two measuring points along a flow path and adequate isotope enrichment factor ε (see equations in the basics section). Isotope enrichment factors describe for each pollutant and degradation pathway (aerobic, anaerobic) the proportionality between isotopic enrichment and concentration decrease. They are known through laboratory studies and are available in Isodetect’s ISOFRAC database. If certain hydrogeological site properties are known (e.g. groundwater flow velocity), it is possible to derive spatial and temporal degradation rate constants, absolute degradation rates and the future extent of the stationary plume.

Quantitative Degradation Parameters and Required Site Informations

Percentual amount of degradation: two isotope values, isotope enrichment factor
Spatial degradation rate constant (% per m): + groundwater flow direction
Temporal degradation rate constant (% per month): + effective flow velocity
Absolute value of degradation (µg/L per m or µg/L per months) : + pollutant concentration
Future extent of the stationary plume (m): + benchmark concentration for plume front

The quantification of attenuation processes based on the isotopic enrichment involves simplifications of hydrogeological conditions and degradation dynamics. However, it provides valuable insight into the intensity of pollution elimination in certain zones or along certain transects of contaminant plumes. The degradation rates can be particularly useful for the validation of site-specific models of pollutant transport and degradation (e.g., via Modflow).

Due to different levels of isotopic enrichment, the anaerobic or aerobic degradation of benzene can be verified by determining the 13C /12C and 2H / 1H isotope values.

– Degradation Pathways: Two-Dimensional Isotope Monitoring

The distinction of aerobic or anaerobic in situ degradation of pollutants is important for the design and control of enhanced natural attenuation. By analyzing isotopes of two elements of a pollutant, it is possible to determine the dominating pathway of degradation. Generally, anaerobic or aerobic pollutant degradation leads to different data patterns in the dual isotope plot, because the intensity of the isotopic enrichment is very different. It can thus be used to elucidate the success of an oxidative stimulation of BTEX-degradation by oxygen addition (ISCO) as well as a reductive stimulation of chlorinated ethene degradation by molasses. We recommend the parallel determination of 13C/12C and 2H/1H for BTEX and MTBE (Fischer et al. 2009, Zwank et al. 2008), and of 13C/12C and 37Cl/35Cl for chlorinated ethenes (Elsner et al. 2011).

Degradation Pathways that Can be Elucidated by Two-Dimensional Isotope Monitoring

BTEX, MTBE/ETBE: anaerobic or anerobic degradation (¹³C/¹²C und ²H/¹H)
CVOC: anaerobic or anerobic degradation of VC or TCE (¹³C/¹²C und ³⁷Cl/³⁵Cl)

The prevailing redox processes in contaminated sites can be determined by determining the isotope values of nitrate, sulfate and methane/CO2, since the redox partners are also subject to isotopic enrichment during pollutant degradation.

– Redox Processes: Isotope Fractionation of SO4, NO3 and CH4/CO2

Contaminant degradation does not only increase the isotope values of pollutants but also redox partners. Depending on the predominant redox reaction, we find an enrichment of the heavy isotopes of nitrate (15N, 18O) or sulfate (34S, possibly also 18O) in a contaminant plume. With isotope monitoring of methane and bicarbonate, we can also identify different methane production processes (acetoclastic or hydrogenothrophic methanogenesis). These studies are used for MNA concepts or the control of enhanced natural attenuation.

Redox Processes that Can Be Elucidated by Isotope Monitoring

Nitrate reduction
Sulfate reduction
Methanogenesis (acetoclastic or hydrogenotrophic mineralisation)

Isotope values of the most important nitrate sources in groundwater and their alteration by biodegradation (red arrow).

Isotope values of the most important sulfate sources in groundwater and their alteration by biodegradation (red arrow).

Various formation processes of methane from DOC (e.g., acetoclastic, hydrogenotrophic, syntrophic reactions) entail characteristic isotopic levels of CH4 and bicarbonate (HCO3). The elucidation of these processes is important for the establishment of MNA/ENA concepts in contaminated sites or the control of biogas plants (see also Applied Research section).

In situ Microcosms (BACTRAPS)

BACTRAPS are in situ microcosms loaded with a 13C-labeled compound. After exposure to a groundwater well (2-4 months), the microcosms are colonized by microorganisms that degrade and assimilate the adsorbed, isotope-labeled substrate. Subsequent to removal of the BACTRAPs, certain biomolecules (so-called biomarkers) of the colonized microorganisms are extracted (usually fatty acids or amino acids) and respective 13C/12C isotopic values are determined. Significant 13C accumulation in the biomarkers clearly indicates in situ degradation, and a high microbial colonization density is already an indication. The comparison of 13C-labeled microcosms allows a relative quantitative evaluation of degradation intensities at different measuring points. BACTRAPS are a more expensive method than isotope fractionation, but allow for a particularly sensitive investigation of selected target components, especially for PAHs.

Contaminants, Particularly Applicable for BACTRAP Examination

PAH: naphthalene, acenaphthene, acenaphthylene, phenanthrene, fluorene, etc.
Gasoline additives: MTBE, ETBE, TBA
BTEX: benzene, toluene, ethylbenzene, xylenes, ….
Others: all chemicals that can be labeled by 13C

Processing steps of a BACTRAP examination. 1) The isotopically labeled contaminant (e.g., naphthalene) is adsorbed onto a carrier material, 2) exposed to groundwater well in the contaminant plume for 2-4 months (with packers if necessary), 3) colonized by microorganisms which 4) degrade and assimilate the isotope-labeled contaminant. After removal, certain biomolecules (fatty acids, amino acids) are extracted and 5) their concentrations and isotopic signatures are determined.

Laboratory Microcosms with Isotopically Labeled Compounds

The complete metabolisation of a pollutant to CO2 can be reconstructed in laboratory microcosms that contain unlabeled growth material from BACTRAPS and are spiked with an isotopically labeled compound. In contrast to all other methods, the complete degradation and not the first metabolization step is investigated here. The method convinces with its high sensitivity and the quantitative determination of mineralization rates, thereby it compensates for the general disadvantages of laboratory experiments.

Mineralization Rates in 13C Laboratory Microcosms Can Be Determined for

PAH: naphthalene, acenaphthene, acenaphthylene, phenanthrene, fluorene, etc.
Gasoline additives: MTBE, ETBE, TBA
BTEX: benzene, toluene, ethylbenzene, xylenes, …
Chlorinated ethenes and ethanes (CVOC): PCE, TCE, cDCE, TCA, …
Others: chemicals, which can be labelled by ¹³C

As a matter of course, we also carry out laboratory assays without isotope labeling.

Carrier material from BACTRAPS (without isotopic labeling) is used in laboratory for degradation experiments with 13C-labeled pollutants (e.g., fluorene). The increasing 13C isotope value of the resulting CO2 can be converted into a mineralization rate.

BTEX pathways leading to intermittent metabolites (e.g., benzylsuccinate / benzylsuccinic acid).

Metabolite Analysis

The detection of specific decomposition products of a pollutant is a simple qualitative proof of its biodegradation. For BTEX and PAHs, about 20 degradation metabolites have been described so far, which also give indications of dominating degradation processes (aerobic or anaerobic). Isodetect still detects specific degradation metabolites at concentrations below 1 μg / L.

Metabolite Analysis Can Provide Evidence for Degradation of

PAH: naphthalene, acenaphthene, acenaphthylene, phenanthrene, fluorene, ….
Gasoline additives: MTBE, ETBE, TBA
BTEX: benzene, toluene, ethylbenzene, xylenes, ….
Chlorinated Ethenes and Ethanes (CVOC): PCE, TCE, …
Others: certain pesticides, explosives, ….

Note that a lack of metabolites could still be a consequence of significant biodegradation of pollutants. In this case, the metabolites degrade faster than they are formed. Their concentration thus gives no indication of the intensity of biodegradation.

Naphthalene degradation Pathways leading to intermittent metabolites (e.g., naphthoic acid / naphthoic acid).

The population density of Dehalococcoides and their amount in the total microbial abundance indicate the capability for decomposition of chlorinated ethenes in certain zones of a contaminated landfill (top, Rondenbarg). Anaerobic BTEX degradation is indicated by a clear increase of the anaerobic marker gene bssA in the deeper sediment layer (bottom, Gaswerk Augsburg).

Molecular Genetic Analysis (qPCR)

The bacterial group of Dehalococcoides are highly specialized degraders of chlorinated ethenes and the only ones capable of complete anaerobic dechlorination (i.e., VC degradation). Their occurrence in a contaminant plume proves an established capability of intrinsic degradation (US-EPA 2006, Winderl et al., 2008). More recently, quantitative molecular biology methods (qPCR) have been successfully used to determine the abundance of Dehalococcoides species as well as genes for functional degradation enzymes (vcrA, bvcA, tceA). Likewise, microbial attenuation capability of BTEX can be determined by functional genes for aerobic (e.g., tmo) and anaerobic (bssA, bamA) degradation. Provisory, the composition of bacterial populations and the determination of total microbial abundance can indicate qualitatively characteristic potentials and changes of in situ pollutant degradation. More meaningful, however, is the quantification of microorganisms that have specific degradation properties.

Degradation Processes of Major Pollutants that Can Be Detected Semiquantitatively with qPCR

CVOC: reductive dechlorination (dhc, vcrA, bvcA, tceA)
CVOC: aerobic degradaion of cDCE and VC (etnC, etnE)
BTEX: aerobic (z.B. tmo) and anaerobic degraders (z.B. bssA, bamA)
Ammonium: anammox degraders

Dichlorprop and 4-CCP are pesticides that occur as enantiomers. Biodegradation changes both the concentration and the isotope ratios of the mirror-image molecules.

Fractionation of Enantiomeric Pollutants

Some pollutants (e.g., different pesticides) have a molecular structure that allows for mirrored spatial structures (enantiomers). Both enantiomers are structurally identical, but the enzymatic degradation of the pollutant is often preferred to one of the molecules. Thus, there is an enantiomeric fractionation (analogous to isotopic fractionation): with increasing degradation intensity, the ratio of the enantiomeric concentrations changes. At the same time, the isotopic signatures of the two enantiomers also change, allowing additional evidence of pollutant degradation.

Enantiomeric Pollutants

Pesticides (Phenoxyacids): 2,4-D; 2,4-DB; dichlorprop, fenoprop, MCPA, MCPB; 2,4,5-T; a-hexacyclohexane, heptachlor, bromacil, ….
Pharmaceuticals: Ibuprofene, Naproxene, Metoprolole, Venlafaxine, Salbutamole, …