Background Information on the ISOFRAC Database

- ISOFRAC parameters
- Monitoring of Natural Attenuation via Isotope Measurements
- Isotope Fractionation Factors
- The ISOFRAC database on biological isotope fractionation
- Calculations

ISOFRAC parameters

Pollutant Group | BTEX, PAH, CKW, fuel additives |

Compound |
benzene, toluene, ethylbenzene, xylene,
cresol naphtalene, 2-methylnaphtalene, 2-methylphenantrene, fluoranthene PCE, TCE, DCE, VC, di-chloromethane, di/tri-chloroethane, trichlorobenzene MTBE, TBA |

Enrichment Factors |
13C/12C (78 factors), D/H (13), 37Cl/35Cl (6) |

Redox Conditions |
oxic/anoxic; nitrate/sulfate/iron(III)-reducing; methanogenic; dehalogenating |

Reference |
ca. 30 references, mostly available as PDF-file |

Field Studies |
ca. 15 references, mostly available as PDF-file |

Monitoring of Natural Attenuation via Isotope Measurements

*In situ* biodegradation in contaminated sites can be assessed
by the enrichment of heavy stable isotopes (13C, D) in the residual
pollutants. For the *quantification* of biodegradation the appropriate
isotope enrichment factor has to be selected, which depends on the compound
of interest, prevailing redox conditions and the degrading microbes.

Isotope Fractionation Factors

Biological degradation is generally proportional to the enrichment of heavy stable isotopes in the substrate. Proportionality factors (α or ε) are specific for environmental conditions and must be determined in laboratory experiments featuring Rayleigh plots. The adequate factor applied on a contaminated site allows quantification of biodegradation from isotope data simply on the basis of increasing isotope signatures.

The ISOFRAC database on biological isotope fractionation

The database provides information to select the appropriate isotope enrichment
factor, necessary for quantification of *in situ* biodegradation in a contaminated
site. Practical application of the monitoring technique in field studies
is noted. Links to references offer more detailed information.

Calculations

The common way to express the stable isotope ratios of a given compound is the deviation δ [‰] from an international standard. For carbon stable isotope 13C the δ13C ratio calculation is defined as

**(1)** δ13C
= (Rs/Rstd - 1) x 1,000

where Rs is the isotope ratio 13C/12C of the sample and Rstd the carbon stable isotope ratio of the standard. The average abundance of the stable isotopes on earth is close to the reference materials used for stable isotope measurements. International standards are the PDB (Peedee Belemnite, a belemnite fossil from the cretaceous Peedee formation in South Carolina) for carbon (13C = 1.1123%), which is equal to the stable isotope proportion of carbon on earth. An increasing 13C δ-value of +10‰ corresponds to a higher percentual amount of approximately 0,011%. For hydrogen and oxygen the Vienna SMOW (Standard Mean Ocean Water, D = 0.0156% and 18O = 0.02%) is used, and sulfur is defined by the CTD standard (34S = 4.21%). Reference for nitrogen isotope analysis is N2 in air (15N = 0.36%), and the standard abundance of the heavier chlorine isotope 37Cl is referred to as 24.47% of total chlorine.

Accuracy of Isotope Measurements

The reproducibility of carbon isotope measurements is commonly in the range of 0.2-0.5‰. Thus an alteration of 2-5 13C-atoms in between 1 million 12C-atoms of a substance is measurable. The reproducibility of hydrogen isotope analysis is typically between 2‰ and 5‰.

Stable Isotope Fractionation

In order to describe isotope fractionation during microbial degradation, the concentrations and isotope ratios of the residual, not yet degraded substrate fraction are analysed. The commonly used mathematical description of isotope fractionation processes is the Rayleigh equation (equation 2 - 4), where Rt is the 13C/12C isotope ratio and δt is the isotope signature at time t, δ0 is the initial isotope signature of the substrate, Ct/C0 is the fraction f of substrate remaining at time t, and α is the isotope fractionation factor.

**(2)** Rt/R0 =
(δt + 1,000)/(δ0 +
1,000)

**(3)** Rt/R0 =
(Ct/C0)(α -
1) = f (α - 1)

**(4)** ln (Rt/R0)
= (α - 1) x ln (Ct/C0)

**(5)** ε = (α -
1) x 100

The kinetic isotope fractionation factor α is a constant for a reaction at given experimental conditions and can be obtained from experiments where ln (Rt/R0) is plotted over ln (Ct/C0) for the time intervals t of the experiment. The slope of the linear regression curve through the data points expresses the kinetic isotope fractionation factor α as (α - 1) according to equation (4). The fractionation factor is also expressed as isotope enrichment factor ε via (equation 5), which generally ranges between 0 and -100.

Biodegradation Derived from Stable Isotope Fractionation

In field studies it might be advantageous to calculate the extent of
biodegradation *B *[%] with

**(6)** *B* = (1 - f) x 100 = (1-
(Rt/R0)(1/α -
1)) x 100

The major advantage using the extent of biodegradation *B* is
that only the isotope ratios of the compound of interest are used for the
calculations. The calculation of *B* is
independent of the contaminant concentrations measured in the field according
to equation (6) and depicts to which degree a certain compound had been
degraded. Since the substrate concentration can be affected by processes
other than biodegradation such as dilution or adsorption the calculation
of *B* might be useful to map zones of
intensive biodegradation without consideration of concentration data.