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Biogenic Toluene

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What is biogenic toluene?

Toluene is a petroleum hydrocarbon that is common in crude and refined products. It is often detected and reported along with benzene, ethylbenzene and xylene isomers. A word of caution about toluene as well, it is highly ubiquitous and is found in tape, pen ink, and adhesives which commonly cause incidental contamination of environmental samples.

Toluene can also be formed naturally, as part of a decomposition pathway within specific ecosite types. The presence of natural toluene in these ecosystems was problematic when petroleum releases were also in these areas. Since toluene has environmental guidelines that need to be met and would be present in both the natural ecosystem as well as from the release, environmental practitioners required a method to distinguish and quantify natural toluene. CMI produced a peer-review scientific paper discussing the formation of toluene in natural environments, and the forensic approach to definitively determine the source of toluene as either petrogenic or biogenic (Richards and Sandau 2017).

Toluene from a petrogenic source will be present with other alkylated benzenes, such as ethylbenzene and xylenes, and many other more complex alkylated benzenes. Toluene from a biogenic source will not be present with these other alkylated benzenes, except for para-cymene, which is formed as part of the same biological decomposition process from terpenes, in particular alpha-pinene, a major chemical of pine resins. Benzaldehyde may also be present, which is the decomposition product of toluene. This pathway, as well as a possible pathway from phenylalanine, is presented in Figure 1.

Figure 1: Potential mechanisms for the formation of toluene
Figure 1. Potential mechanisms for the formation of toluene.

Where do we find biogenic toluene in the environment?


We have detected biogenic toluene in air, water and soil samples, including peat samples. This is because toluene has a high volatility and high water solubility for an organic chemical, meaning detectible concentrations can readily be found in all matrices.

Ecosite type

The detection of toluene in uncontaminated samples is most obvious in ecosites that are anaerobic, acidic, highly organic and contain a low mineral content. We have observed highest concentrations of toluene in treed poor bog (type i1). Toluene was also detected in treed poor fen (type j1), however at much lower concentrations. Based on most likely formation mechanisms, the detection of toluene in a j1 fen, is most likely due to migration from adjacent i1 bog. Figure 2 shows typical concentration ranges of toluene from sampling locations in treed poor bog and treed poor fen.

Figure 2: Range of toluene concentrations for samples collected from treed poor bog and treed poor fen
Figure 2. Box-whisker plot showing the range of toluene concentrations for samples collected from treed poor bog and treed poor fen.


Detection of toluene in peat and water samples from northern Alberta shows a strong spike in July. This is indicative of a biological response to seasonality and an indicator for a biogenic source. A petrogenic source would be either unresponsive to seasonality, or even show reduced concentrations in summer due to dilution from melt-water and increased rates of loss by weathering mechanisms. Figure 3 shows a typical profile of toluene concentrations from sampling locations in northern Alberta, with a clear strong increase in early summer.

Figure 3: Toluene concentrations from an investigation site
Figure 3. Toluene concentrations from an investigation site. Eight locations were sampled through the spring, summer and fall. The results show a strong seasonality.
  • Legal Sampling
  • Chain of Custody
  • Study Design
  • Data Analysis and Visualization
  • Data Wrangling
  • Multivariate Statistical Analysis
  • Principal Component (PCA); Hierarchical Cluster (HCA)
  • Science Communication
  • Data Science/Big Data
  • Multidimensional Gas Chromatography (GC×GC)
  • Source Apportionment
  • Chemical Fingerprinting
  • Diagnostic Ratios
  • Fate and Transport
  • Arsonous Wildfires

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Environmental Forensics

We developed and published environmental forensics methodologies for the defensible determination of biogenic toluene in any environmental sample ( Richards and Sandau 2017 ). These methodologies comprise validated analysis processes that remove interfering chemistry and concentrate sample extracts to provide low levels of detection as well as forensic chemistry data interpretation. The environmental forensics interpretation comprises two main lines of evidence, in addition to information relating to ecosite and season. These are diagnostic ratios and fingerprint chromatograms.

Diagnostic ratios

The construction of diagnostic ratios are most powerful when they clearly differentiate source types, especially where two ratios can be plotted on a double ratio graph. This type of graph presents regions that are indicative of different source types. In this case, ratios using clear differences within the BTEX and cymene groups between biogenic and petrogenic toluene sources are suitable.

Toluene ratio = toluene / (benzene + toluene + ethylbenzene + xylenes)

Cymene ratio = p-cymene / (o-cymene + m-cymene + p-cymene)

Example petroleum sources and example biogenic toluene sources from our library are presented on a graph shown in Figure 4. In this plot samples showing biogenic toluene plot in the top right corner, and in general petrogenic toluene sources plot towards the bottom left, with different petrogenic source types collecting into clusters, but all are separated from the biogenic source type.

Figure 4: Double ratio graph for the toluene ratio and cymene ratio in a variety of petroleum products and natural sources
Figure 4. Double ratio graph for the toluene ratio and cymene ratio in a variety of petroleum products and natural sources, showing clear distinction of biogenic toluene and petrogenic toluene origins.

Fingerprint chromatograms

Chromatograms of alkylated benzenes are produced from GC-MS analysis. These present the family of chemicals similarly associated with toluene in a sample. A biogenic origin for toluene is shown with a specific pattern of alkylated benzenes, which is restricted to toluene, p-cymene and possibly benzaldehyde. Examples of both of these source types from reference samples are presented in Figure 5 and Figure 6.

Figure 5: Chromatogram for a reference petrogenic toluene sample
Figure 6: Chromatogram for a reference biogenic toluene sample
Figure 6. Chromatogram for a reference biogenic toluene sample.

Expert Witness

CMI have constructed these forensic lines of evidence from standard chemistry principles following environmental forensics processes. CMI have been involved in litigious cases where toluene was present in air samples during monitoring events, drinking water well samples due to electrical tape on pumps, surface water samples due to tape on bottles and insect repellant from samplers. It is important to know potential alternative sources and provide defensible conclusions regarding the variety of potential sources of toluene in environmental monitoring samples. CMI personnel have extensive experience in the collection, analysis, identification and chemical fingerprinting of biogenic toluene in relation to crude and refined product releases, including the defence of this process with regulatory bodies.

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