Assessment of the effects of phenanthrene and its nitrogen heterocyclic analogues on microbial activity in soil

Soil is a complex microhabitat, supporting diverse microbial populations, which play an important role in breakdown and transformation of organic matter in fertile soils with many species contributing to different aspects of soil fertility (OECD 2000). Microbial uptake and conversion of chemicals is continuously taking place throughout the biosphere and it is widely known that indigenous microflora which utilizes organic contaminants in soil as carbon/energy sources are ubiquitous in the environment (Leung et al. 2007). Any interference with these biochemical processes may potentially affect nutrient cycling and impact the health and fertility of soil (OECD 2000).

Phenanthrene and its nitrogen-containing polycyclic aromatic hydrocarbons (N-PAHs) consist of three aromatic rings containing carbon with one or two atoms of nitrogen. These chemicals are semi-volatile, persistent, toxic, ubiquitously distributed (Švábenský et al. 2009; EC 2011; Anyanwu et al. 2013), and are widely produced by industrial activities (petroleum derived, combustion sources and biological sources) (Švábenský et al. 2009; Hazardous Substance Data Bank 2010; EC 2011; Anyanwu and Semple 2015a). Due to their widespread distribution in the environment, and their physico-chemical properties, they are potentially carcinogenic, mutagenic, teratogenic and genotoxic (Bleeker et al. 2002; EC 2011; IARC 2012; Anyanwu and Semple 2015a, b, c). It has been reported that not only homocyclic aromatic compounds, but also heterocyclic compounds contribute to the changes in microbial activity in soil (Anyanwu and Semple 2015a, b). From both toxicological and epidemiological studies, many heterocyclic aromatics have shown to be highly toxic (Hazardous Substance Data Bank 2010; EC 2011). Although the available published data are limited, there are considerable evidence indicating their toxicity to humans and ecological receptors (Bleeker et al. 2002; Hazardous Substance Data Bank 2010; EC 2011; Brar et al. 2010; IARC 2012; Anyanwu et al. 2013; Anyanwu and Semple 2015a, b, c).

It has been reported that microbes are susceptible to contaminant effects; and high concentrations of chemical in soil can impact on microbial growth, density, viability and development (Welp and Brümmer 1999). As a result of relative sensitivity of microbes to contaminants, toxicity data are important in determining critical loads or safe levels for contaminants in soil; because, protecting the ecosystem community also protects its functions and ecosystem services (SETAC 2012). In assessing the toxicity of chemicals in soil, various procedures must be taken into consideration. A number of bioassays have been developed to assess contaminant toxicity in soil such as: activity of a range of enzymes, C and N mineralisation and nitrification (Welp and Brümmer 1999; Gong et al. 2000; Thiele-Bruhn and Beck 2005; Butler et al. 2011; Pietravalle and Aspray 2013). However, assessing the impacts of a chemical in soil should also focus on the activity and respiration of microbial populations responsible for carbon transformation, since it subjects them to starvation, changes in the community-level physiological profile, chemical stress/inhibition and death (Domsch et al. 1983; Nwachukwu and Pulford 2011; Butler et al. 2011; Fahrenfeld et al. 2013). Microbial respiration therefore is an effective measure of rate of carbon mineralization, since about 70 % carbon added to soil is lost mainly as CO₂ and H₂O, a product of microbial respiration (Usman et al. 2004). Furthermore, evaluating toxicity may be detected in soil in which an easily metabolisable substrate (e.g. glucose) has been added (Meyers et al. 2007; George et al. 2008). Following this, any impact of contaminants may be recorded as changes in the rate and extent of CO₂ production (OECD 2000; Nwachukwu and Pulford 2011).

Thus, substrate induced respiration (SIR) is a measure of the CO₂ production from a soil sample after administering an optimal concentration of an additional readily utilizable carbon source. SIR, using glucose, is an indirect and simple method of estimating microbial activities to chemicals in soil. Although some bioassays use other methods for quantifying respiration rate, the most common criterion is CO₂ released (µg CO₂ h g^?1 soil) or O₂ consumed (µg O₂ h g^?1 soil) (Nwachukwu and Pulford 2011; Pietravalle and Aspray 2013).

Microbes are ecological receptors mentioned in recent reviews as requiring much research attention due to their sensitivity to contaminants. Irrespective of this, studies have focused on metals (Bardgett and Saggar 1994), PAHs (Towell et al. 2010), trinitrotoluene (George et al. 2008; Butler et al. 2011; Fahrenfeld et al. 2013), diesel (Sutton et al. 2013), total petroleum hydrocarbons (Pietravalle and Aspray 2013) and other persistent organic pollutants (Welp and Brümmer 1999). However, there is general lack of information on the impact of N-PAHs on soil microbial respiration. Therefore, the aim of this study was to assess the impact of phenanthrene and its 3-ring N-PAHs on soil microbial respiration. With an automated respirometer, it is possible to obtain real time measurement of the CO₂ production in many samples after the addition of glucose supplement. Thus, SIR using standard laboratory equipment the “Automated Columbus Instrument’s Micro-Oxymax” was used for this study because it is capable of measuring the production of CO₂ from soil over time. In addition, chemical analysis was performed to measure the loss of contaminants in soil over time.