Biological degradation of certain contaminant groups

General significance (quality and quantification) of the most important methods for investigating the degradation of pollutants.

Detection methods for specific contaminant groups

According to our long-year experience, there is no site-independent strategy for characterizing biodegradation of contaminants. The selection of exploration methods is primarily based on the objective, e.g. providing evidence for natural attenuation or preparing in situ enhancement of degradation. Further aspects are contaminant patterns as well as the historical, hydrogeological and remediation conditions. Depending on the contaminant profile, specific methods can be recommended.

Suitable detection methods for the degradation of different pollutant groups

PAK: Metabolite analysis – BACTRAPS – Laboratory tests
MTBE, ETBE, TBA: CSIA ¹³C/¹²C – CSIA ²H/¹H – BACTRAPS – repeated CSIA – laboratory tests

BTEX: CSIA ¹³C/¹²C – Metabolite analysis – BACTRAPS – repeated CSIA ¹³C/¹²C – CSIA ^2H/¹H – CSIA redox partner – qPCR – < a href="http://wp1010791.server-he.de/altlasten/Methods/#labormikrokosmen">Laboratory tests
LCKW: CSIA ¹³C/¹²C – qPCR – ³⁷Cl/³⁵Cl CSIA< /a> – CSIA redox partners – Laboratory tests

We interpret the analytical results in the context of the pollutant concentrations, environmental factors, and hydrogeological conditions.

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

Suitability of various methods to demonstrate the biological degradation of certain groups of pollutants: 5* = key method; 3* = secondary method; 1* = complement method.

Degradation processes of chlorinated ethenes (above) result in isotopic enrichment of the primary contaminants and the resulting metabolites (when further degraded). In the initial stage of degradation, the metabolites have particularly light isotope values (below, high 12C content), since the 13C-containing molecules of the starting materials are converted more slowly.

CFC

Component-specific isotope monitoring of carbon (¹³C/¹²C-CSIA) is a key method for demonstrating CE degradation. It can even detect and quantify the complete dechlorination of the pollutants. The isotope values of the primary contaminants (e.g. PCE, TCE), the metabolites (e.g. cDCE, VC) and the sum isotope value (¹³C/¹²C_CFC ) provide important evidence on the initial pollutant degradation, the degradation stage and the VC degradation (complete dechlorination). At certain locations, we can also describe the CE degradation with analytical models that fit concentration and isotope data and thus allow more precise statements (Hoehener et al. 2015).

In addition to isotope signatures, we determine the abundance of Dehalococcoides (qPCR) at CFC-contaminated sites and draw independent, semi-quantitative conclusions on the importance of anaerobic, reductive dechlorination at the site. Certain degradation pathways (reductive dechlorination, oxidation, cometabolic degradation) can be better identified with the additional analysis of chlorine isotopes in a two-dimensional isotope monitoring. In addition, the chlorine isotope analysis is a very good, additional tool for differentiating sources of pollutants (see Forensik).

Key statements of CFC degradation monitoring

Isotope values of the primary contaminants → proof of degradation, quantification, sources of pollutants
Metabolite isotopic values (TCE, cDCE, VC) → Degradation stage
Total isotope value → Detection and quantification of VC degradation
two-dimensional monitoring (³⁷Cl/³⁵Cl) → secondary sources of pollutants, Degradation proof, quantification, paths (reductive, oxidative, cometabolic)

Laboratory tests (¹³C), redox reactions → preparation of an ENA measure

qPCR (Dehalococcoides, vcrA, etnC) → importance of certain degradation processes

In the case of in situ measures, detailed tests on the degradation of pollutants are necessary to determine the effectiveness of degradation stimulants (e.g. molasses, iron nanoparticles, oxygen, …) on the microbial in situ to examine community. For the composition of the optimal injection solution, we carry out degradation tests in the laboratory. In this context, a detailed investigation of the prevailing redox reactions at the site using isotope analyzes of NO₃, SO₄ or CH₄/HCO₃ beneficial.

Frequency of supplementary methods for detailed investigation of CE-contaminated sites, where 13C/12C isotope monitoring was always carried out at the same time.

The cumulative isotope signature summarizes the isotope values of the CE individual components by weighting them with the respective concentration fractions. The value only changes when complete dechlorination (VC degradation) occurs.

Isotope enrichment occurs in parallel to degradation rate in each individual conversion step. All enrichment processes can be summarized in an integrated enrichment factor that enables the quantification of complete dechlorination.

Reductive dechlorination modeling shows exponential changes in CE concentrations associated with linear increases (enrichment) in 13C isotope levels (PCE=red, TCE=turquoise, cDCE=green, VC=yellow; ΣCE=blue).

13C/12C isotope values of BTEX (here benzene) often provide evidence of degradation at the edge of pollutant plumes (above, Rondenbarg landfill). The repetition of the monitoring campaign after 4 years (below) largely confirmed the degradation, but showed a decrease in degradation dynamics in certain areas (red fields, more negative isotope values than before).

BTEX and MTBE/ETBE (gasoline additives)

BTEX and gasoline additives such as MTBE/ETBE are primary contaminants whose degradation intensity can be easily detected and quantified by component-specific carbon isotope analysis (¹³C/¹²C-CSIA). The limit of the quantitative proof of degradation is in the range of 20 – 60% percentage degradation. With the two-dimensional isotope analysis of hydrogen (²H/¹H-CSIA), the degradation can be determined more sensitively and the dominant degradation process can also be identified.

Key evidence of BTEX or MTBE degradation monitoring with different tools

¹³C/¹²C CSIA → quantitative proof of degradation for all components (limited sensitivity)

Metabolite analysis: → qualitative proof of degradation, wide range of components

BACTRAPS → sensitive proof of degradation, semi-quantitative, for single target compounds

²H/¹H CSIA → degradation processes, additional quantification (high limit of quantification)

qPCR (bss) → Quantification of the degradation potential

Laboratory experiments (13C label) → to prepare ENA

Another evidence of degradationcan be provided by metabolite analyzes that cover a comprehensive spectrum of pollutants (BTEX, PAH), but do not allow any quantitative statements. With standardized BACTRAPS, on the other hand, a semi-quantitative proof of degradation is possible (comparison between several measuring points). The method is recommended for single, dominant contaminants (e.g. benzene, MTBE, TBA, naphthalene, phenanthrene, fluorene) and is also particularly sensitive. Another tool, qPCR analysis of taxonomic or functional genes (e.g. bssA, tmo) reflects the abundance and thus the degradation potential of microorganisms that are specialized in certain degradation pathways.

In preparation for in situ measures (e.g. addition of oxygen, sulphate, peroxidases), the natural redox and degradation processes at the site should be carefully checked. In laboratory tests, we investigate the effect of several potential injection solutions.

Details on the work flow of various monitoring tools can be found under Procedures.

Application frequency of supplementary methods for detailed investigation of BTEX-contaminated sites, where 13C/12C isotope monitoring was always carried out.

Chromatogram of 13C/12C isotopic measurement of BTEX in an environmental sample. Subsequent to the three measurements of the reference standard, a number of peaks from BTEX derivatives appear.

Detailed delineation of a benzene degradation pathway. Hydroxylation and carboxylation leads to the intermediate product benzoate, which can be detected in situ.

Marker genes for aerobic and anaerobic degradation of toluene.

BACTRAPS are directly exposed in monitoring wells (above), soil or coastal sedimtents. They are excellently suited for the semi-quantitative detection of PAH degradation. The 13C label of the pollutants is assimilated in situ and finally appears in biomolecules (e.g. amino acids). The relative comparison of the results reveals higher degradation intensities in certain plume areas (below). Unmarked BACTRAPS can be used as growth material for subsequent laboratory experiments.

PAH and MKW (diesel and heavy oils)

Carbon isotope analysis (¹³C/¹²C-CSIA) for demonstrating the degradation of pollutants is only suitable for PAHs for naphthalene. For molecules with more than 12 carbon atoms isotope enrichment of ¹³C is generally not detectable. Therefore, PAH/BTEX metabolite analysis is recommended to prove degradation. Since certain PAHs dominate at many sites, substance-specific BACTRAPS are an excellent tool for a single-compound PAH degradation monitoring. Parallel laboratory tests are a particularly sensitive and quantitative detection method for the complete mineralization of the pollutants (formation of isotope-marked ¹³CO₂).

MKW (diesel and heavy oils)

Degradation assessment for middle distillates (diesel, heating oil) can be carried out using fingerprint analyzes (FID or MS). The relative ratio of pristane/phytane, n-heptadecane/n-octadecane or n-alkanes to iso-alkanes indicates the so-called biological aging of the oils. Depending on the distillate, other groups of substances (e.g. isoprenoids, alkylbenzenes, terpanes) are used to assess (and also roughly date) oil damage – preferably in comparison to pure-phase reference samples. Isotopic values of ¹³C/¹²C or ²H/¹H are for the total mixture (EA analysis of the phase) and for individual components (CSIA) can be determined. The proof of the biodegradation from the component-specific isotope enrichment is best derived from the comparison with corresponding pure phases. For special questions (e.g. on mineralization) laboratory tests can be carried out with isotope-marked components.

Key statements of a PAH or MKW degradation monitoring with different methods

BACTRAPs → sensitive proof of degradation for individual ones that can be marked with ¹³C Components

FID fingerprinting → MKW composition for subsequent detailed analysis

GC-MS fingerprinting → Aging assessment (relation of certain components)

EA isotope analysis → Distinction of pollutant sources, only for pure phases< /li>
Laboratory tests (also with ¹³C) → Mining and mineralization under defined conditions

Amino acids in the microbial Aufwuchs of BACTRAPS show the different biological dynamics within a PAH pollutant plume (Gaswerk Augsburg).

BACTRAP biofilms (without 13C label) can be used for degradation experiments with 13C labeled pollutants.

Laboratory tests with 13C-labeled fluorene show different mineralization activities (→ ¹³C-CO₂) in different plume areas.

Degradation stages of aliphatic and polyaromatic hydrocarbons can be derived from GC and GC/MS fingerprints by comparison of compound-specific peak height ratios.

Isotopic chromatogram of a BTEX- and naphthalene-contaminated groundwater sample including further pollutants (small peaks).

Micropollutants (pesticides, drugs, explosives, …)

The in situ degradation of various pesticides (lindane/HCH, atrazine, glyphosate, AMPA, phenoxy acids, …), drugs (diclofenac, ….) or explosives (nitroaromatics) can be detected by isotope fractionation of carbon (¹³C/¹²C CSIA) or nitrogen (¹⁵N/¹⁴N CSIA). However, the isotope approach is limited by the ¹³C/¹²C concentrations below 1 µg/L.

Micropollutants whose decomposition can be verified with isotope analysis

Pesticides: lindane/HCH, atrazine, glyphosate, AMPA, benzotriazoles, phenoxy acids, ….

Medicines: Diclofenac, ….

Explosives: Nitroaromatics, RDX, TNT, NT, NB, ….

Other: ….

For some pesticides that form enantiomers (e.g. phenoxy acids), concentrations and isotope values of the two steric molecular forms can be used to demonstrate degradation. The biodegradation here leads to a fractionation of left/right rotating forms similar to molecules with heavy/light isotopes. Complementary, laboratory tests can be carried out with ¹³C-labelled substances for almost all trace pollutants, which have the advantage of complete mineralization (formation of ¹³CO_{2 or ¹³CH₄).}

Nitrate inputs and outputs in groundwater (top) and source-specific isotope values (bottom). Biodegradation leads to isotopic enrichment (red arrow).

Nitrate & Ammonia

Nitrate pollution is currently one of the greatest challenges in groundwater and drinking water protection. At some locations, the high ammonium content is also a major problem. With the help of isotope fractionation of ¹⁵N/¹⁴N or ¹⁸O/¹⁶O, characteristic sources can be identified and degradation processes can be quantified for both components.

Applications of ¹⁵N/¹⁴N isotope analysis in groundwater remediation

Agricultural land: nitrate sources and nitrate removal

Legacy: dominating redox processes, potential for stimulating ENA

Landfill: ammonium and nitrate removal

The investigations provide information on large-scale protection or rehabilitation concepts. They can also show whether certain protective measures are successful in the medium term.

In a two-isotope plot (³⁴S vs. ¹⁸O) of sulfate, samples (plume areas) with strong sulfate reduction can usually be clearly distinguished, because 34S is isotopically enriched in proportion to degradation. ¹⁸O, on the other hand, can undergo side reactions (exchange with H₂O) that have a disruptive effect.

Sulfate & Sulfide

In groundwater, sulfur conversion processes (sulphate reduction, sulfur precipitation, sulphide formation, sulphide oxidation) can be localized and characterized with isotope investigations, since sulfur is always subject to isotope fractionation. Geogenic or anthropogenic sulphate inputs are also recognizable due to their isotopic fingerprint. We therefore analyze sulfur isotopes for projects the remediation of contaminated sites, geothermal energy and subsurface energy storage.

In the remediation of contaminated sites, sulphate is examined in order to characterize the natural pollutant reduction in more detail or to control in situ remediation measures. Depending on the remediation concept, sulfate reduction (and subsequent secondary reactions such as sulfide formation or FeS precipitation) can be a desirable or disruptive reaction at a remediation site. It is reflected in a significant isotope enrichment of ³⁴S in the remaining sulfate (in most cases also of ¹⁸O). If only sulfide is formed, the resulting H₂S has the same isotope signature as the SO₄ initially (closed mass balance).

Applications of ³⁴S/³²S isotope analyzes for hydrogeological questions

Legacy: dominating redox processes, potential for stimulating ENA

Geothermal energy: colmation through redox processes, corrosion of plant components, deposits

Natural gas storage: geogenic/artificial redox processes, corrosion, deposits

Other Chemicals

According to REACH regulations (Rregistration, Evaluation, Authorisation and Restriction of Chemicals) the biological and chemical degradability of chemical substances must be documented in safety reports. In addition, traceability to the manufacturer should be guaranteed. Additional documentation requirements apply to pesticides and pharmaceuticals, which are aimed in particular at degradability in groundwater and soil.

Isotope methods for chemical characterization under REACH or for authentication

Isotopic fingerprint → product authentication, Traceability

Laboratory tests with ¹³C label → standardized degradation tests according to OECD methods

The traceability of a chemical product is guaranteed by the specific isotopic fingerprint of the formulation or individual, easily separable components. A significant advantage of this approach is product authentication and thus protection against piracy for chemicals or pharmaceuticals. For this purpose, a targeted labeling with stable isotopes can also be carried out.

According to the OECD recommendation for chemical tests, laboratory tests with ¹³C-labeled substances are very well suited to clarifying the quantitative conversion and the reaction paths in soil (Method 307) and aquatic sediments (Method 308).

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