CHAPTER 17 • Luminescence for the Analysis of Organic Compounds in Natural Waters
411
lac-Z, encoding the enzyme f3-galactosidase (Routledge and Sumpter 1996). The
activity of the analyte-induced enzyme was detected by a chemiluminescent
l,2-dioxetane-f3-D-galactopyranoside substrate. The CL detection proved to be much
more rapid than the colourimetric one, allowing for the achievement of a detection
limit of 10 pmol rl oestradiol after incubating the cells with the analyte for 24 hours
compared to at least 72 hours required with the colourimetric detection. The applicability of the chemiluminescent biosensor to the analysis of natural water samples was
preliminarily checked by assessing samples from influent and effluent of an activated
sludge sewage treatment plant (Pasini et al. 2001).
A different receptor-mediated, chemical-activated luciferase reporter gene-expression bioassay (CALUX) was developed for the detection of aryl hydrocarbon receptor-active compounds, such as polyhalogenated aromatic hydrocarbons, which include
polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofuranes (PCDFs) and
polychlorobiphenyls (PCBs). These substances either derive from combustion or industrial processes as by-products, or are used in productive processes (e.g. plastics
manufacture) and are included in a list of 16 persistent organic pollutants (POPs) identified to be monitored at international level. A rat hepatoma cell line, which naturally
expresses the aryl hydrocarbon receptor (AhR), was stably transfected with a plasmid
containing the dioxin-responsive elements sequence and the luciferase luc reporter
gene. The biosensor had a detection limit of 1 pM 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) and was applied to the analysis of sediments and pore water. In the case of
pore water, only a simple and rapid extraction procedure was needed, without additional clean-up (Murk et al. 1996).
In addition to the previously described specific or group-selective luminescent reporter gene bioassays, a number of total toxicity luminescent tests have been developed. The most widely accepted microbial toxicity test system is based on the wildtype luminescent bacterium Vibrio fischeri. Bacterial bioluminescence has proved to
be a convenient measure of cellular metabolism and consequently a reliable sensor
for measuring the presence of toxic chemicals in aquatic samples (De Zwart and Sloof
1981). Sample toxicity is assessed from the decrease in luminescence following a short
exposure to several concentrations of the sample. This system is commercially established and marketed as Microtox. It is commonly used to assess the general toxicity of
potentially contaminated water samples, e.g. in wastewater treatment plant effluent
testing for protection of receiving waters, in surface water monitoring for identification of pollution sources, and in monitoring raw drinking water for contamination due
to spills or pollution sources. A recombinant bacterial sensor, in which the DNA damage-inducible promoter recN from Escherichia coli was fused to the lux operon of
V. fischeri and introduced into Salmonella typhimurium, was developed for genotoxicity
assessment (Verschaeve et al. 1999). Also this system is now commercially available as
the Vitotox test. A different approach for toxicity assessment was based on a set of E. coli
strains harbouring different plasmids, each carrying a different stress promoter:lux
fusion. The panel included an oxidative stress sensor, a DNA damage sensor (genosensor) and two general stress sensors. The system allowed for the evaluation of different toxic effects in industrial wastewater samples (Belkin et al. 1997). These luminescent biosensors, though rapid, well-standardized and often ready-to-use, provide
information on the sample total toxicity only, giving no indication on the chemical
nature of contaminants.
411
lac-Z, encoding the enzyme f3-galactosidase (Routledge and Sumpter 1996). The
activity of the analyte-induced enzyme was detected by a chemiluminescent
l,2-dioxetane-f3-D-galactopyranoside substrate. The CL detection proved to be much
more rapid than the colourimetric one, allowing for the achievement of a detection
limit of 10 pmol rl oestradiol after incubating the cells with the analyte for 24 hours
compared to at least 72 hours required with the colourimetric detection. The applicability of the chemiluminescent biosensor to the analysis of natural water samples was
preliminarily checked by assessing samples from influent and effluent of an activated
sludge sewage treatment plant (Pasini et al. 2001).
A different receptor-mediated, chemical-activated luciferase reporter gene-expression bioassay (CALUX) was developed for the detection of aryl hydrocarbon receptor-active compounds, such as polyhalogenated aromatic hydrocarbons, which include
polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofuranes (PCDFs) and
polychlorobiphenyls (PCBs). These substances either derive from combustion or industrial processes as by-products, or are used in productive processes (e.g. plastics
manufacture) and are included in a list of 16 persistent organic pollutants (POPs) identified to be monitored at international level. A rat hepatoma cell line, which naturally
expresses the aryl hydrocarbon receptor (AhR), was stably transfected with a plasmid
containing the dioxin-responsive elements sequence and the luciferase luc reporter
gene. The biosensor had a detection limit of 1 pM 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) and was applied to the analysis of sediments and pore water. In the case of
pore water, only a simple and rapid extraction procedure was needed, without additional clean-up (Murk et al. 1996).
In addition to the previously described specific or group-selective luminescent reporter gene bioassays, a number of total toxicity luminescent tests have been developed. The most widely accepted microbial toxicity test system is based on the wildtype luminescent bacterium Vibrio fischeri. Bacterial bioluminescence has proved to
be a convenient measure of cellular metabolism and consequently a reliable sensor
for measuring the presence of toxic chemicals in aquatic samples (De Zwart and Sloof
1981). Sample toxicity is assessed from the decrease in luminescence following a short
exposure to several concentrations of the sample. This system is commercially established and marketed as Microtox. It is commonly used to assess the general toxicity of
potentially contaminated water samples, e.g. in wastewater treatment plant effluent
testing for protection of receiving waters, in surface water monitoring for identification of pollution sources, and in monitoring raw drinking water for contamination due
to spills or pollution sources. A recombinant bacterial sensor, in which the DNA damage-inducible promoter recN from Escherichia coli was fused to the lux operon of
V. fischeri and introduced into Salmonella typhimurium, was developed for genotoxicity
assessment (Verschaeve et al. 1999). Also this system is now commercially available as
the Vitotox test. A different approach for toxicity assessment was based on a set of E. coli
strains harbouring different plasmids, each carrying a different stress promoter:lux
fusion. The panel included an oxidative stress sensor, a DNA damage sensor (genosensor) and two general stress sensors. The system allowed for the evaluation of different toxic effects in industrial wastewater samples (Belkin et al. 1997). These luminescent biosensors, though rapid, well-standardized and often ready-to-use, provide
information on the sample total toxicity only, giving no indication on the chemical
nature of contaminants.