Khudur et al. 2015; Wang et al. 2016). The uncertainty of the results in this case may
be caused by the selection of the primers as well as the extraction step and PCR
running (Bundy et al. 2004). Other possibility to measure soil bacteria diversity is the
use of qPCR, which allows for quantification of active and total bacteria as well as
distinction of functionally important phylogenetic groups, e.g., Pseudomonas and
assessment of the proportion of Gram-negative and Gram-positive bacteria (Shahi
et al. 2016). Molecular methods, which do not employ DNA, like phospholipid fatty
acid (PLFA) profiling also do not ensure full overview on soil microbiota as Archaea
are not reported to be active in TPH degradation (Bundy et al. 2004).
Community-level fingerprinting may also be performed on physiological basis,
e.g., taking advantage of differences in preferred carbon source between bacterial
strains. Community level physiological profiling employs special microplates, e.g.,
Biolog
® , divided into several partitions filled with different carbon sources, designed
to distinguish spatial and temporal changes in microbial communities. However, the
resolution of the plates is lower than molecular methods especially in case of Grampositive bacteria and fungi (Heuer and Smalla 1997).
5.4.3 Bioassays
Different ecotoxicological bioassays standards have been developed by ISO, OECD,
US EPA, and individual researchers (Masciandaro et al. 2013). The most common
organisms used for soil toxicity assessment are bacteria, plants, and invertebrates
(Płaza et al. 2005; Masciandaro et al. 2013). Evaluation of soil ecotoxicity should be
based on tests employing organisms representing different trophic levels (Hubálek
et al. 2007). Depending on test specificity, bioassays can estimate acute or chronic
toxicity.
Bioassays are based on the response of living organism after direct exposure to
contaminated soil or indirect to soil solution. Tests performed on soil eluates give
faster response in comparison with direct tests on soil (Bundy et al. 2004). However,
organic contaminants are often characterized by low water solubility and high
hydrophobicity, which may affect the sensitivity of indirect tests (Bundy et al.
2004). Moreover, bioassays on soil leachates are often performed in diluted medium
what may additionally affect the results (Hubálek et al. 2007). Despite a number of
available methods for measurement of ecotoxicity, assays dedicated for soil samples
as well as soil amended with organic matter still need to be improved.
5.4.3.1 Bacterial Assays
Common methods applied by researchers are still often based on soil leachates. An
example may be the Ames test, a chronic toxicity bioassay based on the detection of
genotoxicity in Salmonella typhimurium. Although, many literature studies
employed Ames test to evaluate soil toxicity (Genouw et al. 1994; Pagnout et al.
270
A. Gielnik et al.
be caused by the selection of the primers as well as the extraction step and PCR
running (Bundy et al. 2004). Other possibility to measure soil bacteria diversity is the
use of qPCR, which allows for quantification of active and total bacteria as well as
distinction of functionally important phylogenetic groups, e.g., Pseudomonas and
assessment of the proportion of Gram-negative and Gram-positive bacteria (Shahi
et al. 2016). Molecular methods, which do not employ DNA, like phospholipid fatty
acid (PLFA) profiling also do not ensure full overview on soil microbiota as Archaea
are not reported to be active in TPH degradation (Bundy et al. 2004).
Community-level fingerprinting may also be performed on physiological basis,
e.g., taking advantage of differences in preferred carbon source between bacterial
strains. Community level physiological profiling employs special microplates, e.g.,
Biolog
® , divided into several partitions filled with different carbon sources, designed
to distinguish spatial and temporal changes in microbial communities. However, the
resolution of the plates is lower than molecular methods especially in case of Grampositive bacteria and fungi (Heuer and Smalla 1997).
5.4.3 Bioassays
Different ecotoxicological bioassays standards have been developed by ISO, OECD,
US EPA, and individual researchers (Masciandaro et al. 2013). The most common
organisms used for soil toxicity assessment are bacteria, plants, and invertebrates
(Płaza et al. 2005; Masciandaro et al. 2013). Evaluation of soil ecotoxicity should be
based on tests employing organisms representing different trophic levels (Hubálek
et al. 2007). Depending on test specificity, bioassays can estimate acute or chronic
toxicity.
Bioassays are based on the response of living organism after direct exposure to
contaminated soil or indirect to soil solution. Tests performed on soil eluates give
faster response in comparison with direct tests on soil (Bundy et al. 2004). However,
organic contaminants are often characterized by low water solubility and high
hydrophobicity, which may affect the sensitivity of indirect tests (Bundy et al.
2004). Moreover, bioassays on soil leachates are often performed in diluted medium
what may additionally affect the results (Hubálek et al. 2007). Despite a number of
available methods for measurement of ecotoxicity, assays dedicated for soil samples
as well as soil amended with organic matter still need to be improved.
5.4.3.1 Bacterial Assays
Common methods applied by researchers are still often based on soil leachates. An
example may be the Ames test, a chronic toxicity bioassay based on the detection of
genotoxicity in Salmonella typhimurium. Although, many literature studies
employed Ames test to evaluate soil toxicity (Genouw et al. 1994; Pagnout et al.
270
A. Gielnik et al.