Monitoring of environmental pollutants by ...

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of pentachlorophenol [28] have been studied using a battery of ecotoxicological model systems: immobilization of D. magna, bioluminescence inhibition in the ...
a n a l y t i c a c h i m i c a a c t a 6 0 8 ( 2 0 0 8 ) 2–29

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Review

Monitoring of environmental pollutants by bioluminescent bacteria Stefano Girotti ∗ , Elida Nora Ferri, Maria Grazia Fumo, Elisabetta Maiolini Department of Metallurgic Science, Electrochemistry and Chemical Techniques, University of Bologna, Via S. Donato 15,40127 Bologna, Italy

a r t i c l e

i n f o

a b s t r a c t

Article history:

This review deals with the applications of bioluminescent bacteria to the environmental

Received 11 September 2007

analyses, published during the years 2000–2007. The ecotoxicological assessment, by bioas-

Received in revised form

says, of the environmental risks and the luminescent approaches are reported. The review

6 December 2007

includes a brief introduction to the characteristics and applications of bioassays, a descrip-

Accepted 9 December 2007

tion of the characteristics and applications of natural bioluminescent bacteria (BLB), and a

Published on line 31 December 2007

collection of the main applications to organic and inorganic pollutants. The light-emitting genetically modified bacteria applications, as well as the bioluminescent immobilized sys-

Keywords:

tems and biosensors are outlined. Considerations about commercially available BLB and BLB

Environmental pollutants

catalogues are also reported. Most of the environmental applications, here mentioned, of

Bioluminescent bacteria

luminescent organisms are on wastewater, seawater, surface and ground water, tap water,

Water

soil and sediments, air. Comparison to other bioindicators and bioassay has been also made.

Soil

Various tables have been inserted, to make easier to take a rapid glance at all possible

Genetically modified bacteria

references concerning the topic of specific interest.

Biotoxicity assays

© 2007 Elsevier B.V. All rights reserved.

Contents 1. 2.



Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eco toxicological assessment of environmental risk: the bioassays and the luminescent approaches . . . . . . . . . . . . . . . . . . . . 2.1. Characteristics and applications of bioassays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Characteristics and applications of natural bioluminescent bacteria (BLB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Applications to organic pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Applications to inorganic pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Analysis by cellular components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Genetically modified bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +39 051 2095660; fax: +39 051 2095652. E-mail addresses: [email protected], [email protected] (S. Girotti). URL: http://biocfarm.unibo.it/∼girotti/ (S. Girotti).

0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.12.008

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3.

4.

1.

2.5. Bioluminescent immobilized systems and biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Commercially available BLB and BLB catalogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. How to measure the light emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Sea water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Surface and ground water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. Tap water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.5. Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Soil and sediments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Sediments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Air . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction

Increasing need for effective tools to estimate the risks derived by the large number of organic and inorganic noxious substances, both of natural origin and released in the environment by human activities, leads to the development of very sensitive detectors of harmful substances. It is an accepted assumption that the simple measurement of chemicals concentration, with reference to established regulatory rules, will not give an accurate account of the environmental noxiousness. Therefore, much attention has been paid to biological sensors, markers or detectors. Organisms from different trophic levels have been used: bacteria, nematodes, cladocerans, fishes, amphiphods, algae, plants, cultured cell lines. Toxicological animal-based methods employed in the identification of hazard chemicals are sometimes expensive, time consuming, require large sample volumes, and rise ethic problems. In vitro methods, also commonly used for screening and ranking chemicals, must be included in battery tests for risk assessment purposes. Their major promise is to supply mechanism-derived information, considered pivotal for adequate risk assessment. Nevertheless, results from this kind of assays are prone to the criticisms concerning the deep differences existing between the real conditions in the ecosystems and those of the in vitro assay. Since several years, all studies have emphasized the benefits of using rapid, sensitive, reproducible and cost-effective bacterial assays for toxicity screening and assessment. Bioluminescent bacteria (BLB) can offer the further advantage of an easy record of the effects produced on a living organism. A bioluminescence (BL) inhibition assay is often chosen as the first screening method in a test battery, based on speed and cost considerations. The BLB tests protocol is usually simple, especially when applied to aqueous samples or extracts. Therefore, it becomes unnecessary to perform costly chemical analysis of all samples: this will be done only if the bioluminescent sensor signals an alarm. The versatility of this kind of tests has been increased by subsequent modifi-

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cations, for example, the introduction of the kinetic assay for turbid and coloured samples, of the solid phase test for analyzing samples without extraction. Researchers have reported the bioluminescence assay based on Vibrio fischeri, the first and most employed luminescent strain, as the most sensitive, across a wide range of chemicals, compared to other bacterial assays such as nitrification inhibition, respirometry, ATP luminescence and enzyme inhibition. Moreover, the BL assays show good correlation to toxicity bioassays like those on algae, crustacean, fishes, etc. [1]. The bioluminescent assays gained increased attention during the last years thanks to the advancements in the genetic manipulation techniques, which offered the possibility to change the no emitting organisms, isolated from different habitats, into both luminescent and specifically responsive ones. Therefore, there is a growing interest, from regulatory and research institutions other than biomedical, in employing highly sensitive and specific bioluminescent techniques for analytical purposes. The fundamental characteristics and analytical applications of luminescent organisms and techniques have been previously extensively reviewed, referring to the researches published until the end of the nineties [2]. This review deals with the analytical applications of bioluminescent bacteria developed in the past seven years just in the environmental field. The information, collected from the consulted literature, have been divided in two main groups. The first (paragraph 2) reports on those papers concerning general features, useful information, and/or analytical methods based on both natural and genetically modified bacteria, but lacking of significant applications on real samples. All papers reporting the actual applications on environmental samples are cited in the third paragraph, divided according to the analysed matrix (water, soil, air). Tables have been inserted to make easier to take a rapid glance at all possible references concerning the topic of specific interest. Moreover, papers that can be of interest according to different aspects, for example as biosensors or as water monitoring systems, have been reported in more than one of the summarizing tables.

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2. Eco toxicological assessment of environmental risk: the bioassays and the luminescent approaches 2.1.

Characteristics and applications of bioassays

Traditionally, the impact of pollutants discharged into the environment by human activities has been assessed using chemical assays or evaluating physical parameters. HPLC or gas–MS techniques have been widely used. Limitations to their use include: high costs, long analysis times, the requirement of an experienced operator, information limited to the concentration of the pollutants, no one on their toxicity. To overcome these limitations, biological analyses have been introduced. These analyses are important because of the complexity of natural environment, in terms of organisms and of differences in physiological status, and because of the need to establish cause/effect relationships between concentration of pollutants and consequent environmental damages, as well as to measure the possible synergistic effect of complex mixtures of chemicals. The use of effect-based screening tools has the advantage of indicating the real impact of all chemicals present in a given sample or ecosystem. Rapid biological tests are playing their major role in hazard and risk assessment, especially at the screening level. Bioassays measure changes in physiology or behaviour of living organisms resulting from stresses induced by biological or chemical toxic compounds, which can cause disruption of the metabolism. In particular, the in vivo luminescence is a sensitive indicator of xenobiotics toxicity, because it is directly coupled to respiration, via the electron transport chain, and thus reflects the metabolic status of the cell. If noxious substances are present, the luminescence decreases (Fig. 1). This kind of assays can reveal any unusual condition in the ecosystem, since the organisms respond with rapidity and sensitivity to all factors that contribute to stress. Fast acting toxins asso-

ciated with acute effects are most quickly detected. Long time exposures (chronic toxicity tests) reveal slower acting toxicants, or those with chronic effects. Bioassays have been usually performed employing bacteria (prokaryotic cells) and eukaryotic cells, as well as organisms such as Daphnia, mussels, algae, plants and their seeds, fishes, such as fathead minnow and rainbow trout. Concerning the aquatic environment, risk-assessment studies have used macro invertebrates extensively. One of the most common invertebrate toxicity tests uses Daphnia magna and Ceriodaphnia, both freshwater species pertaining to Cladocera. The use of Daphnia has many advantages, such as high sensitivity and short reproductive cycle. The mortality (the acute lethality test for 21 days is well established and standardized) or the reproduction parameters are measured. Organisms, or cells, that have been genetically modified to contain specific response elements and reporter constructs, can be designed to respond to specific contaminants. The most frequently used reporter genes include the lux genes from BLB, or Firefly (Photinus pyralis), or the gfp gene from Aequorea victoria. Concerning biological tests, it is known that organisms at the same or at different trophic level respond differently to a range of toxicants and there is the need to develop multiple toxicity bioassays using various organisms. [3] Bioassays using higher organisms (fish, crustacean, etc.) are characterized by a long reproductive period, a long sensing time, and high costs. Bioassays using bacterial cells have been developed just to reduce the cost and duration time of the experiments, behind to improve the sensitivity. Bacteria based bioassays can be useful employed as early warning screening methods, thanks to their rapid response to biological or chemical toxins. A further property of whole-cell bacterial biosensors is to provide measurements just of the bioavailable fraction of toxic compounds. The ease of use of bacterial bioassays represents another advantage, when compared with traditional methods.

Fig. 1 – Naturally bioluminescent bacteria reduce or stop light emission in presence of toxic compounds that impair their metabolism.

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2.2. Characteristics and applications of natural bioluminescent bacteria (BLB)

FMNH2 + RCHO + O2

Luciferase

−→

FMN + RCOOH + H2 O + LIGHT (2)

Luminous bacteria are ubiquitously distributed. The major part is marine forms, free living in water or parasitic to marine or terrestrial animals [4]. The intense, uniform, and sustained glow that extends to the horizon in all directions, reported by mariners many times over the centuries, the so-called “milky seas”, are manifestations of unusually strong bioluminescence produced by bacteria in association with a micro algal bloom. Very recently has been recorded the first satellite observations of the phenomenon: an about 15,400-km2 area of the north western Indian Ocean was observed to glow over three consecutive nights [5]. Bioluminescence is also characteristic of numerous marine and few land organisms (about 666 genera from 13 phyla) other than bacteria, extending from dinoflagellates, to marine vertebrates, not higher than fishes. Most studies have been carried out on American firefly (Photinus pyralis), crustacean (Cypridina hilgendorfi), coelentrates (sea pansy Renilla reniformis, and Aequorea), dinoflagellates (Gonyaulax polyedra) [6]. The origin of bioluminescence was considered a problematic question of the Charles Darwin theory of evolution. The problem arose from the fact that it is generally believed that the function of bioluminescence is directly associated with the visual behaviour of organisms. However, results of recent studies revealed that bacterial bioluminescence might have a role in stimulation of DNA repair and detoxification of the deleterious oxygen derivatives. It has been proposed that these processes could be evolutionary drives at early stages of development of light-emitting biological systems, until emission of photons became sufficiently high to ensure to the luminescent organisms evident ecological benefits [7]. Experimental evidences for the physiological role of luciferase in protection of cells against oxidative stress have been demonstrated only in Vibrio harvey, raising the question whether this is a specific or a more general phenomenon. In presence of various oxidants (hydrogen peroxide, cumene hydroperoxide, t-buthylhydroperoxide and ferrous ions), the growth of dark mutants of Vibrio fischeri and Photobacterium leiognathi is impaired, to various extents, with respect to wild-type bacteria. These deleterious effects could be reduced by addition of antioxidants. Significantly, different efficiencies of oxygen derivatives detoxification are characteristics for various luciferases [8]. Recently, studies have been reported on the isolation and characterization of bioluminescent bacteria showing significant hydrocarbons degrading capacity, able to utilize the hydrocarbons as the sole source of carbon and energy, and then interesting candidates to the role of bioremediation organisms [9]. The bioluminescent enzyme system consists of a NAD(P)H:FMN oxidoreductase and a luciferase, that emits light at 490 nm in the presence of FMN, NAD(P)H, a long chain aliphatic aldehyde, and molecular oxygen, according to reactions (1) and (2) [10]:

NAD(P)H + FMN + H+ +FMNH2

NAD(P)H:FMN oxidoreductase

−→

+

NAD(P)

(1)

Bacterial luciferase is highly specific for FMNH2 , but the enzyme also shows weak activity toward other flavins. Tu [11], published a review on the biochemistry of bacterial bioluminescence, focusing on the mechanism and structure of luciferase. Only the aliphatic aldehydes with a chain length of eight or more carbon atoms are effective in the luminescent reaction. Various substances of biological interest and enzyme activities can be analyzed by coupling luciferase and oxidoreductase to a third reaction, which produces or consumes NADH or NADPH. To exploit for analytical purposes the inhibition, by noxious substances, of naturally light-emitting bacteria was first proposed over 27 years ago, as a method for monitoring the toxicity of aquatic samples. Nowadays, the biotests based on natural BLB are used extensively and the most suitable species include V. fischeri, V. harvey, P. leiognathi and Pseudomonas fluorescens. V. fischeri has been reclassified as Photobacterium fisheri [12], to underline its phylogenetic distance from other vibrios, but only few authors have adopted the new name, and then in the major part of the papers, even recently published, the old denomination V. fisheri can be found, and only few times the new, correct, classification as Photobacterium fischeri. Luminescent methods, such as time-resolved fluorescence, steady-state techniques, and BL kinetics study, have been applied to understand the mechanism of action of, for example, fluorescent dyes, organic oxidizers, organic and inorganic heavy-atom containing compounds, and metallic salts. Five mechanisms of toxicity, produced by exogenous molecules on living organisms, have been proposed: (1) change of electron-excited states’ population and energy transfer; (2) change of efficiency of singlet–triplet conversion in the presence of external heavy atom; (3) change of rates of coupled reactions; (4) interactions with enzymes and variation of enzymatic activity; (5) non specific effects of electron acceptors [13]. To obtain, from these organisms, data with a good reliability, the stability and uniformity of their characteristics and metabolism are fundamental. Continuously cultivated bacteria represent a good source of stable bio indicators. Optimization of light emission necessitates careful choice of growth medium and culture operating conditions. The optimized conditions for continuous cultivation of the bioluminescent bacterium, V. fischeri NRRL-B-11177, in a fermenter has been reported by Scheerer et al. [14]. The system provided a reliable long-term (more than 1 month) continuous culture facility for the reproducible measurement of perturbation of V. fischeri metabolism by monitoring changes in its luminescence. Concerning the toxicity assays, the luminescent bacteria system can be used both for short- and long-term tests. Short-term tests are based on the change of light intensity, while long-term tests can examine also changes in viability or growth rate. The sensitivity of the bioluminescence test systems, when compared to those of other bioassays is, usually, sufficient to

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reveal any compound toxic to humans and other mammals. In some cases, the BL systems can have not enough sensitivity to detect a toxic substance at its Maximum Permissible Concentration. but they result the quicker, or more convenient, bioassays. This is the case, for example of deltamethrin determination. The sensitivity of the BLB test was higher than that of mice test, but lower of sensitivity of fish test. However, the fish test lacks fast response (testing time is 7 days) and it is very laborious to perform [15]. Bioluminescent tests can be applied to assess toxicity in water, sediments, and soils. These assays employ the original, marine, BLB, or organisms isolated from the environment to test, and transformed in luminescent strains by genetic manipulation. These organisms, perfectly adapted to their habitats, will offer precise response to the presence of unusual, dangerous xenobiotics. During remediation processes, the application of luminescent tests allows to monitor in a rapid, simple and not expensive way the progress and the completion of the treatment. To monitor effectively an ecosystem a battery of bioassays has to be used. This general principle is true also in case of bioluminescent assays. They must be combined to other bio tests and/or chemical analysis. In case that only a battery of luminescent assays is employed, several of these tests must be used simultaneously, since they differ in their sensitivity to the various contaminants. The best combination of assays, i.e. those that will offer all possible information on a certain sample or area, avoiding redundances, cannot be decided in theory. A battery of tests can be proposed, within certain limits, a priori, but the most suitable one will be defined later, according to the preliminary results, for example analysing them by a multivariate statistical method [16]. A full acceptance by Regulatory Institutions and Environment Protection Agencies of toxicity bioassays as effective analytical tools in environmental controls needs guarantees of standardization and validation. These goals can be achieved by realization of ring studies among several laboratories. In one of these studies [17], involving ten European laboratories investigating on luminescence inhibition of V. fischeri assay, more than 70% of the laboratories showed a satisfactory performance in terms of reproducibility and stability. This study also proved that, in case of luminescence, the use of different commercial devices does not represent an additional source of variation. Before to mention some of the more recent application of BLB assays to the study of environmental problems, it must be underlined that the well-known stimulatory effect produced by sub-lethal or low concentration of toxic chemicals on organisms metabolism, referred to as hormesis [18,19], has been found to be common in the luminescence bioassay. This phenomenon must be carefully take into account determining the environmental protection guidelines, since it is necessary to protect natural biota without imposing excessive penalties to dischargers and, at the same time, not relaxing environmental standards. This can only arise from a deep investigation and understanding of the role of hormesis in toxicity data used for risk assessment [20].

2.2.1.

Applications to organic pollutants

When real samples are tested by BLB, the responses obtained by these assays are usually compared with those supplied by different biotests, and vice versa. As an example, the sensitivity of an Early Life Stage (ELS) test studying survival, growth and histopathological changes in seabream (Sparus aurata) yolk sac larvae in determining simazine, a s-triazine herbicide, was compared with that of the commercial kit “Microtox” [21]. Simazine did not exert any significant toxicity to the marine bacterium V. fischeri at the tested concentrations, while the survival of the larvae was significantly reduced. On the other hand, luminescent data are often confirmed by instrumental analysis. BLB assays, using various strains of Vibrio sp., were developed as microplate format to evaluate the impact of the residues of veterinary pharmacological treatments on environmental and human health [22]. The bacterial responses, to pure antibiotic solutions and to residues extracted from excreta of treated animals, were compared with the direct quantification by HPLC analysis, obtaining good correlation values. Quinolones are one of the most important groups of synthetic antibiotics used in aquaculture. The toxicity of a single of such molecules and of a mixture of ten quinolones was studied using a long-term inhibition assay on V. fischeri, an organism very sensitive to these substances. The EC50 values ranged from 14 ␮g L−1 for ofloxacin to 1020 ␮g L−1 for pipemidic acid [23]. The photoinduced toxicity of various kinds of compounds, by photosensitization or by photomodification, is a phenomenon that requires to be directly evaluated in the environment. A method is available for measuring photoinduced short- and long-term toxicity of polycyclic aromatic hydrocarbons (PAHs), based on inhibition of luminescence and growth of V. fischeri [24,25]. The short-term assay detects toxicity of chemicals that are taken up rapidly and/or whose photosensitization activity is immediate. The long-term assay identifies chemicals whose rate of assimilation is slow and/or time is required for photoinduced effects to be realized. For several PAHs, the short-term assay did not reveal photoinduced toxicity that is evident only in the long-term assay. Ecotoxicity tests [26] on photolytic degradation derivatives of chlorinated paraffins showed that by the green algae test no inhibition of cell growth was observed. On the contrary, a significant acute toxicity for Daphnia as well as a clear chronic toxicity for luminescent bacteria were detected [26]. Again, the need for application of multiple bioassays is evident. Information useful to design sustainable, alternative, solvents can be obtained performing detailed biological studies. Methyl- and some ethylimidazolium ionic liquids have been tested on luminescent bacteria as well as on the IPC-81 (leukaemia cells) and C6 (glioma cells) rat cell lines [27]. The Effect Concentrations were generally some orders of magnitude lower than for acetone, acetonitrile, methanol and methyl-tert-buthylether, pointing out and quantifying the clear influence of the alkyl chain length on toxicity. The effects of pentachlorophenol [28] have been studied using a battery of ecotoxicological model systems: immobilization of D. magna, bioluminescence inhibition in the bacterium V. fischeri, growth inhibition of the alga Chlorella vulgaris, micronuclei induction

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in the plant Allium cepa, inhibition of cell proliferation and MTT reduction in Vero cells, neutral red uptake, cell growth, MTT reduction, lactate dehydrogenase leakage and activity in the salmonid fish cell line RTG-2. The sensitivity of the different system resulted: micronuclei induction in A. cepa > D. magna immobilization > bioluminescence inhibition at 60 min > cell proliferation inhibition of RTG-2 cells. Inhibition of cell proliferation and MTT reduction on Vero monkey cells showed intermediate sensitivity [28]. Toxicity of a new energetic substance, hexanitrohexaazaisowurtzitane (or CL-20), was tested by Microtox and by 96-h algae (Selenastrum capricornutum) growth inhibition test [29]. CL-20 showed no adverse effects on both systems up to its water solubility. Its biotransformation products did not inhibit seed germination and early seedling (16–19 d) growth of alfalfa (Medicago sativa) and perennial ryegrass (Lolium perenne) up to 10,000 mg kg−1 , confirming the negligible toxicity of this compound. Synthetic ester lubricants and their additives have been tested for their ecotoxicological effects by various standardized assays [30]: bacterial growth of V. fischeri and Pseudomonas putida, luminescence inhibition assay of V. fischeri, survival assay of D. magna, and algal growth inhibition assay of Scenedesmus subspicatus. The results showed that some of the substances that are normally added to base fluids in order to enhance the applicability of the oils may possess a high toxicological potential. The classic applications of luminescent assays, i.e. the assessment of the toxicity in polluted environments or the monitoring of remediation processes, are still of great topical interest. Environmental hazard of sites contaminated by coal tar and its product, is usually ascribed to pollutants such as the 16 polycyclic aromatic hydrocarbons (PAHs) prioritized by the U.S. Environmental Protection Agency (U.S. EPA). Such hazard was evaluated by the Lumistox test, a luminescent bacteria commercial kit [31]. Pure naphthalene, acenaphthylene, acenaphthene, fluorene, and phenanthrene revealed inhibiting effects, but elutriates of PAH-contaminated soils produced a negligible inhibition of the light emission, since the amount of PAHs was very low. In this case, the solubility problems impaired the BLB test applicability. Lumistox 300 has been again employed to test the toxicity of 11 reactive dyestuffs and 8 auxiliaries from a textile dyeing and finishing mill [32], as well as of the degradation products of azo reactive dyes treated by photo-Fenton and Fenton-like oxidation reactions [33]. In both cases, the results demonstrated that the toxicity assessment with luminescent bacteria is effective and of practical use, with the only exception of samples from deep dark-coloured dye bath samples and from the related effluents. The radiolytic degradation effectiveness of 2-, 3- and 4chlorophenol, 2,4-di- and 2,4,6-trichlorophenol, as well as their products toxicity can be obtained by testing the inhibition of V. fisheri bioluminescence [34,35]. The results showed an increase of toxicity, for all compounds, after low doses of radiation. The toxicity was completely suppressed at high doses (10–20 kGy for pure 2,4-DCP). The toxicity, pre- and post-biotreatment, of nitrocellulosebased materials has been evaluated by using bioluminescent bacteria. Results showed the biodegradation effectiveness in

7

reducing toxicity and removing cellulose-based compounds [36]. A BLB assay has been also employed in the evaluation of non-ionic surfactants (Triton [i-octylphenolethoxylates], Tergitol [2,6,8-trimethyl-4-nonanoloxethylates], Symperonic [n-nonylphenol-oxethylates], Brij [fatty alcohol ethoxylates]) toxicity, with the aim to relate their rather low biodegradability, measured by the BOD5:COD ratio, to their toxicity [37]. Another study, on the effects of surfactants on the mineralization of phenanthrene by Pseudomonas aeruginosa in batch reactors, indicated that the presence of solubilised phenanthrene increased the toxicity of the surfactant by a 100-fold, suggesting that the toxicity of solubilised substrates needs also to be considered in the application of surfactant-amended remediation treatments [38]. The effects of noxious molecules of biological origin have been also frequently determined by bioluminescent assays. The commercial kit “ToxAlert 100” has been used in determining the toxicity of crude and purified cyanobacterial extracts containing microcystin [39]. The crude samples inhibited the luminescence of bacteria at higher degree than the pure substance, indicating the influence of other components such as pigments, acids or salts. The acute and chronic effects of another biological toxin, the mycotoxin T 2, have been studied on Photobacterium phosphoreum Sq3 and V. fischeri F1, that resulted both suitable to develop a biosensor technology for testing this toxin in the environment [40].

2.2.2.

Applications to inorganic pollutants

The assessment of mercury toxicity was performed comparing three microbial test systems: the Microtox, the motility test by using Spirillum volutans, and the growth zone inhibition test on B. cereus. Out of these three tests, Microtox was found to be the most sensitive [41]. Again, Microtox test was used to assess the toxicity of the solid and dissolved corrosion products of galvanostatic polarization: Co–Cr–Mo corrosion products were found to be more toxic than those of stainless steel, and the most toxic ions were Cr(VI), Ni2+ , and Co2+ [42]. The biological effects of Arsenic, in solution under the arsenate or arsenite forms—As(V) and As(III) [43] have been also investigated by the Microtox assay. The short-term and long-term effects of Cd(II) and Cr(VI) were studied observing the growth rate and viability of V. fisheri [44,45]. The recorded effects confirmed that the viabilityinhibition assay appeared to be more sensitive than the acute light inhibition. Interestingly, it was possible to observe a clear hormesis phenomenon, especially for Cd(II), under the conditions of both viability- and growth-inhibition assays [44,45]. Data showing a hormesis effect of toxic compounds must be carefully evaluated, since it is not rare that false hormesis effects have been observed when data from environmental, complex, samples have been determined with reference to a blank sample that did not contained exactly all components, but the analyte, present in the tested samples. For example, at the Author’s laboratory this phenomenon has been observed analysing soil samples extracts. Waiting for a suitable blank sample, from a not contaminated area, the inhibitory effects of the extracts have been compared to those of the extracting solvents. The less contaminated samples showed a stimulatory effect on light emission, since their extracts contained

8

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Fig. 2 – Recording a light emission from samples higher than from blank, caution must be used to define it a hormesis phenomenon. The same situation can be observed when the reference blank does not contain exactly the same components, but the analyte, present in the samples, like in the case showed here, in which soil sample extracts have been compared to the extracting solvents solution, as the reference blank.

substances useful for bacterial metabolism, and not present in the pure solvents (Fig. 2). The application of bioluminescent test based on P. phosphoreum B7071 light inhibition, coupled to a semicondutor or to optic fibers is under study to develop a biosensor to determine Au, both in ionic and colloid form, in gold-recovery wastewater [46]. Extracts from the mollusc Scapharca inaequivalvis have been prepared to monitor Cd and Cu in seawater by BLB inhibition. The degree of inhibition decreased as the time of molluscs exposure to metals increased, suggesting a reduction of the “bioactive” metals [47]. The biotoxicological assay results slightly correlated with the biochemical parameters. Two mathematical approaches, applied to predict the toxicity of all the possible binary equitoxic mixtures of Co, Cd, Cu, Zn and Pb, were compared to the data of their inhibition effect on V. fischeri, revealing a complex pattern of possible interactions [48]. Moreover, Cd appears much less toxic to the bacterial model than to animal cells. The synergistic effect of the Co–Cu combination and the strong reduction of Pb toxicity in presence of Cd deserve much attention when establishing environmental safety regulations.

2.2.3.

Miscellaneous

This section deals with assays that were developed and/or employed to determine, in the same experiment, both organic and inorganic compounds. A luminescent bacterial strain, Vibrio sp., isolated from Mediterranean sea has been employed in BL assays of heavy metals, organic chemicals (benzene, toluene, ethylbenzene, xylene), and a wide range of pesticides. The use of this strain represents an improvement, with respect to the main part of commercial and non-commercial BLB assays, since it allows to work at room temperature and to employ simple, not-thermostated microplate reader. [49]. By the light inhibition assay it is possible to reveal low amounts of residues from cleaning chemical, in liquids and on surfaces. The total cleanliness of the process facilities can be determined in a more complete way than the traditionally used bacterial

load determination [50]. Microtox bioassay (15 min), rotifer (Brachionus plicatilis) mortality (24 h) and population growth inhibition (48 h), and chemical analyses have been simultaneously applied to assess the ecotoxicological effects of sewage sludges dumped in oceans. All tests clearly revealed different toxicity levels depending on the sludge sources. However, there was no significant correlation between pollutant concentration levels and the toxicity values of the sludge. This confirms once again that for a correct ecological risk assessment the bioassays of potential toxicity must be always combined to chemical analysis [51]. A particular approach to the assessment of anthropogenic contamination on ecosystems is represented by the bioluminescent to total bacteria ratio determination (bioluminescent ratio, BLR). It has been employed in the study of estuarine systems, showing that the BLR of natural bacterioplankton communities was proportionally reduced in the presence of contaminants like diesel fuel, Hg, and polychlorinated biphenyls (PCBs). This reduction depended on a physiological, rather than a population, response of native microbial communities [52]. Another unusual application of light inhibition tests has been the evaluation of the compost maturity [53]. Results from the acute toxicity test on V. fischeri correlated well with those of the plant growth assays. The immature composts studied were toxic in both assays when processed for less than 3 months, but non-toxic after maturing during six months of composting. The inhibitory effects of cyanide and tetramethylene disulfotetramine (tetramine) on Vibrio qinghaiensis luminescence have been compared with the response of a PbO2 electrochemical sensor. The electrochemical method offered the lower detection limits and the faster response [54]. At the end of this report on the applications of bioluminescent systems in toxicity assays, just few comments are necessary to underline the slight variations in the results obtained, for the same sample, from the different luminescent systems. For example, to compare the light inhibition to the growth inhibition of the same strain can results in not concordant data. Chronic inhibition of luminescence tests and growth inhibition of V. fischeri by chemicals like: Cu2+ , Cr6+ , Zn2+ , Hg2+ , Cd2+ , Pb2+ , cetyltrimethylammonium bromide, 3,4-dichloroaniline, acetone, DMSO, ethanol, nitrobenzene, methanol, and 3,5-dichlorophenol have been compared, revealing that the growth inhibition indicate more reliably the presence of substances with chronic toxic properties than the loss of bioluminescence [55]. On the other hand, growth inhibition responded weaker to the majority of the analyzed toxicants than bioluminescence inhibition. The sensitivity of the growth inhibition assay is competitive when a poor medium is employed [55]. More pronounced differences could be observed when the isolated luminescent reagents are employed instead of the whole bacterial cells. Bacterial cells have higher threshold sensitivity to chlorophenols in comparison to the sensitivity of the bioluminescence enzyme system [56]. This can be expected because the different accessibility of the two systems and the possible different mechanisms of action. Results suggest that bacterial bioluminescence system is not the primary target of the chlorophenol-induced effect on photobacteria. Neutral phenols inhibit luciferase by competing with decanal, whereas a mixed mechanism of inhi-

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9

bition with this substrate is typical of phenoxyacetic acids. With respect to FMNH2 , all chlorophenols are uncompetitive inhibitors [56].

luminous bacteria, coupled enzyme system NADH:FMNoxidoreductase–luciferase and triple enzyme systems with alcohol dehydrogenase and trypsin [60,61].

2.3.

2.4.

Analysis by cellular components

Isolated luminescent systems (see Section 2.2) have been employed, alone or in combination with whole BLB and/or other enzymatic systems, as effective toxicity monitoring tools. It was shown that the elongation of the enzymatic reactions chain, by coupling various reactions, results in enhancing of the sensitivity of bioluminescent tests [11,13]. Methods to determine NAD(H)-, NADP(H)- and NAD(P)dependent enzymes and substrates using the bacterial luciferase and the NAD(P)H:FMN oxidoreductase, alone or coupled to other enzymatic reactions, have been available since several years. The high redox potential of the NAD+ /NADH couple tends to limit the applications of dehydrogenases in coupled assay, since the equilibrium does not favour the NADH formation. Nevertheless, studies on the isolated components of luminescent reactions continued in the direction of basic research on the mechanisms of interaction among luminescent systems and xenobiotics, as well as in the application to environmental monitoring systems. Moreover, to study the influence of xenobiotics on enzymes provides a basis for prediction and interpretation of their effects on bacterial metabolic processes. The direct effects of water-miscible organic solvents on the spectra of P. leiognathi luciferase were studied [57], and the changes observed must be taken into account when working in presence of these compounds. The light intensity of luciferase dropped sharply with increasing concentration of solvent, but the max values, in vitro 506 nm, were almost identical after addition of glycerol, formamide, and methanol. On the opposite, in the presence of 4.76% (v/v) acetone the luciferase produced emission spectra with increasing maximal emission wavelengths, from 506 to 516 nm [57]. Kudryasheva reported the results of the interactions between the luminescent enzymes and different compounds: fluorescent dyes, organic oxidizers, haloid compounds [58]. The compounds binding to the enzymes were tracked through time-resolved fluorescence techniques. Three main mechanisms of xenobiotics’ influence have been distinguished (1) change of electron-excited states population in a bioluminescent emitter; (2) change of rates of the coupled reactions; (3) interactions with the enzymes. Correlations between physico-chemical characteristics of exogenous compounds, such as hydrophobicity or atomic weight of haloid substituents, and efficiency of their interactions with the enzymes, have been demonstrated [58]. The fluorescence characteristics of luciferase and NAD(P)H:FMN-oxidoreductase endogenous flavin, fluorophore lifetime and rotational correlation time have been applied to study the interactions of luciferase from P. leiognathi and NAD(P)H:FMN-oxidoreductase from V. fischeri with non-fluorescent compounds like quinones [59]. These studies can lead to the creation of an universal system of biosensors, based both on bioluminescent organisms and on their enzymes. For example, a proposed set of bioluminescent tests to monitor water quality, in natural and laboratory ecosystems, consisted of four bioluminescent systems:

Genetically modified bacteria

Bioluminescence, emerged as an extremely useful and versatile reporter technology, provides a sensitive, non-destructive, and real-time assay that allows for temporal and spatial measurement. Over the years, many researchers have studied the physiological, biochemical and genetic control of bacterial bioluminescence. These discoveries have revolutionized the area of Environmental Microbiology using luminescent genes as biosensors for environmental studies. The ability to introduce the lux phenotype, coupled to specific promoters allowing its expression only in presence of the proper analyte, into different bacterial species, provides a convenient method to multiply the possibilities for rapid, simple and sensitive screening of the environmental conditions. The lux operons, employed in these genetic manipulations, include those encoding for the bacterial luminescent system, the lux genes of firefly luciferase as well as those of gfp, the green fluorescent protein from A. victoria. A number of recombinant plasmids, carrying the lux operon expressed constitutively in many Gram− and Gram + bacteria, have been constructed and used to transform a wide variety of bacteria. A promoter is usually required on the plasmid to obtain a high expression. The possibility to transform in luminescent organisms the bacteria previously isolated from the media under analysis allows to produce biosensors with the maximum of the suitability, since the vitality of the transformed organisms in their environment will be optimal, as well as its response to the presence of extraneous, noxious compounds [62]. In Fig. 3 a simple scheme of the principles of genetically modified bacteria employment is reported, and some of the more recent and significant environmental applications of the genetically modified bacteria (GMB) will be presented in the following pages. A large number of procedures, testing the wastewater toxicity, are based on the GMB Shk1. A quick estimation of the toxicities of organic chemicals with different functional groups has been proposed, developing quantitative structure–activity relationship (QSAR) models, based on the logarithm of the octanol–water partition coefficient, as well as an overall QSAR model without discriminating the functional groups [63]. Model predictions were compared to experimental data and the model accuracy was found to be one order of magnitude from the experimental values. The performance of the construct obtained by fusing the reporter genes of green fluorescent proteins (A. victoria gfp) and bioluminescence (V. fischeri luxCDABE) genes, to either SOS (recA) or heat shock (grpE) promoters, were compared [64]. In both cases, bacterial bioluminescence allowed faster and more sensitive detection. The fluorescent (FL) reporter proteins were much more stable and in long-term tests allowed detection at levels similar to that of the bioluminescence. To combine the advantages of both reporter functions, representatives of both types were jointly encapsulated in a sol–gel matrix and immobilized onto a glass surface, to generate a BL toxicity and

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Fig. 3 – Genetically modified bacteria, originally non-emitting species, become luminescent after introduction of a plasmid including a lux gene and appropriate promoters. In this way, the presence of a toxicant is revealed by the inhibition of the light production.

a FL genotoxicity sensor, with no interference from the coimmobilized member [64]. To evaluate the nitrification inhibition activity in wastewaters, a recombinant, tryptophan-dependent Nitrosomonas europaea the luciferase gene, has been employed. In case the ammonia monooxygenase activity of Nitrosomonas has been inhibited, a rapid drop in NADH or ATP concentration occurred. This reduction in the overall energy level of the cell was revealed by the reduction of the light emission intensity [65]. The most suitable strain, among the different transformed ones, derived from the same parental strain, must be selected according to the specific priorities of each application, since they can show different performance. Various Escherichia coli strains, containing plasmid-borne fusions of V. fischeri lux to the recA promoter-operator region, have been created: (1) modifying the host cell’s toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E. coli host [66]. When compared, fastest responses were exhibited by Sal94, a S. typhimurium strain harbouring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by an E. coli strain in which the same fusion was integrated into the bacterial chromosome (DPD3063), and by a plasmid-bearing tolC mutant (DPD2797) [66]. Bacterial biolumi-

nescence can be combined with other techniques to enhance its response in specific conditions. A bioluminescent Campylobacter jejuni organism, generated from the fusion of luxCDABE genes from Xenorhabdus luminescens to the flaA promoter of C. jejuni, was used in conjunction with two-dimensional gradient gels to map the responses of this organism to pH, NaCl concentration, temperature, and to various concentrations of l-fucose, d-fucose, and sodium desoxycholate [67]. To monitor the bio-active fraction of Hg, instead of the total Hg, bioluminescent sensor systems were developed by fusion of a Hg-resistant operon (mer operon) from Pseudomonas sp. K-6y4 with the lux operon from V. fischeri. The resulting recombinant plasmids were cloned in E. coli cells for environmental applications [68]. An analogous whole-cell bacterial sensor, restricted to the detection of bioavailable Hg2+ , has been constructed by gene fusion between a mercury resistance (mer) operon from pMR26 of Pseudomonas strain K-62 and a promoterless luxAB gene from V. harvey [69]. Cadmium, lead, chromium, and zinc ions did not interfere with the assay, even at the same concentration of Hg2+ . Methyl mercury, phenyl mercury, and mercuric sulphide also did not affect the biosensor [70]. Employing luminescent sensors to evaluate the heavy metals toxicity, it must be take into account that their response depends not only on the metal concentration but also on pH and on concentrations of any complexing ligand in the environment, including other ions. Significantly higher light output was obtained when 0.1 M KCl was employed, instead of 0.1 M KNO3 , in lyophilized bacteria re-hydratation.[70], demonstrating that

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Cl− has a direct effect on the bioluminescence response of E. coli biosensor. Increasing concentrations of Cl− ions increased the toxicity of Hg, apparently because of the formation of HgCl3 − , and increased the toxicity of Pb because of PbCl+ formation. The toxicity of Cu decreased at high Cl− concentrations as free Cu2+ decreased, in accordance with the free ion model. Concentrations of Cl− had no significant effect on the toxicity of Cd [70]. A remote, near real-time monitoring of estrogenic compounds in the environment have been achieved by constructing an estrogen-inducible bacterial lux-based bioluminescent reporter, designated S. cerevisiae BLYES. It contained the constitutively expressed luxA and luxB, as well as the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) [71]. The endocrine disrupting chemicals (EDCs) genotoxicity can be detected by a recombinant E. coli (DPD2794), containing the recA promoter region fused to luxCDABE from V. fischeri [72]. Other recombinant bioluminescent bacteria, including TV1061, sensitive to protein damage (grpE::luxCDABE), DPD2511, sensitive to oxidative damage (katG::luxCDABE), and DPD2540, sensitive to membrane damage (fabA::luxCDABE), can be used for evaluating other possible modes of toxicity [72]. Different engineered strains are effective in classify the toxicity of azo dyes [73]. They include constitutive luminescent bacteria, like P. phosphoreum and E. coli GC2 (lac::luxCDABE), as well as stress-inducible sensors like the DNA damage sensitive E. coli, DPD2794 (recA::luxCDABE), the membrane damage sensitive DPD2540 (fabA::luxCDABE); the oxidative damage sensitive DPD2511 (katG::luxCDABE); and the protein damage sensitive TV1061 (grpE::luxCDABE) [73]. P. fluorescens OS8 (pDNdmpRlux), containing luxCDABE operon under the control of the phenol-inducible Po promoter from Pseudomonas sp. CF600, has been developed to detect phenols in the environment. Expression of lux genes initiates in the presence of phenolic compounds [74]. The toxicity of phenolic compounds has been determined also by employing the bioluminescent bacteria Shk1, already known as effective tool for heavy metal determination in wastewaters [75]. The bacteriostatic, bactericidal and bacteriolytic activities of antibiotics such as: ampicillin, erythromycin, nalidixic acid, polymyxin B, tetracycline, and trimethoprim have been readily determined observing the bioluminescence, fluorescence, and real-time kinetic data, based on optical detection, of E. coli containing the green fluorescent protein and the firefly luciferase (lucFF) genes [76]. A bioluminescent E. coli K-12 strain for the specific detection of the tetracycline family was optimized to work on fish samples [77]. The extraction of oxytetracycline from rainbow trout (Oncorhynchus mykiss) tissue did not need for centrifugation of homogenized tissue, nor for use of organic solvents. The assay was able to detect oxytetracycline residues below the European Union Maximum Residue Limits [77]. The antibacterial activity of some dyes was also assessed by using a genetically modified strain, the E. coli TG1 (pXen7). Bioluminescence emission has been efficiently suppressed by white light. This test is an effective tool for assaying the antibacterial activity of dyes and compiling optimal schemes and regimes of photosensitization treatment [78]. The toxicity of mono-, di- and tri-chlorophenols and of their degradation by-products has been assessed following the decrease in bioluminescence of: Burkholderia species RASC and

11

P. fluorescens marked with lux genes; P. phosphoreum and GC2, stress-inducible bioluminescent bacteria (DPD2540, TV1061, DPD2794, and DPD2511) [79,80]. By correlating EC50 values, from lux marked RASC c2 and P. fluorescens, to toxicity values from Pimephales promelas (fathead minnow), Tetrahymena pyriformis (ciliate) and V. fischeri, it was apparent that lux marked RASC c2 correlated well with the freshwater aquatic species (P. promelas and T. pyriformis). These data confirmed that the lux marked RASC c2 could be utilized as a rapid and suitable surrogate to predict the toxicity of organic xenobiotic compounds to higher organisms [79,80]. The use of several, recombinant, bioluminescent bacteria provides a quantitative analysis of the different modes of pesticide toxicity, according to the various stress-promoters inserted in modified bacteria [81]. To describe the different mechanisms of toxicity of dibenzo-p-dioxins and dibenzofurans an array of five different recombinant bioluminescent strains of E. coli has been employed [82]. They contained, respectively, the recA (responsive to DNA damage related stress), the fabA (membrane damage), the katG (oxidative damage), the grpE (protein damage), and the lac (constitutive expression, general toxicity) promoters fused to the bacterial lux operon from V. fischeri or P. luminescens [82]. An immobilized, recombinant luminescent E. coli strain, harbouring a lac::luxCDABE-fused plasmid, was able to distinguish between the effects of the pericondensed polycyclic aromatic hydrocarbons and those of the catacondensed ones. Only the latter type was found to cause cellular toxicity, resulting in a dose-dependent decrease in the bioluminescent output [83]. The S. typhimurium strain TA1535/pTL210 generates luminescence when induced by the expression of SOS gene, caused by DNA damaging agents [84]. This genotoxicity test did not use any reagents, and from the comparison with the conventional umu test, using nine kinds of typical mutagens, it showed satisfactory dose–response curves. The sensitivity was 1.6–33 times higher than those of the conventional test [84]. A biological sensor, to distinguish the organomercury from inorganic mercury, was created by transforming E. coli into two similar bacterial strain lines: organomercurials can induce the bioluminescence only in one of these lines, inorganic mercury in both. The system was capable to detect bio-affecting inorganic mercury from several hundred nanomolar to several ten micromolar [85]. The evaluation of organotin compounds effects on living organisms has been done using an E. coli strain specific for tributyl tin monochloride (TBT) and dibutyltin chloride (DBT) (with Cl, Br or I as the halogen group) with the central tin atom important for light production [86]. Organic compounds like alkanes, alcohols, and aldehydes can be quantitatively determined exploiting their transformations into one of the substrates of the bacterial luciferase, i.e. an aldehyde. By introducing the genes encoding for bacterial luciferase, alcohol dehydrogenase and alkane hydroxylase, into a plasmid for simultaneous expression in an E. coli host cell line these compounds were detected within seconds, with sensitivity in the micromolar range [87]. Two recombinant E. coli strains, DPD2794 and GC2 were used to demonstrate the quantitative stress responses, in terms of DNA damage, and the general

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Table 1 – Assays based on bioluminescent, genetically modified bacteria, also in the biosensor formata Reference Air Enzymatic degradation

[91,92*,93*,94*,95*] [96–101]

Soil General toxicity Bioremediation Inorganic pollutants Organic pollutants

[102,103*,104,95*] [105*,106] [107,108,109*,110] [84,111,112*]

Wastewater General toxicity Inorganic pollutants Organic pollutants

[65,66,113*,114*,115*] [70,116*,117*,118,119,120*] [63,75,86,121,122*,123*,124*,125*,126,127]

Water General toxicity Inorganic pollutants Organic pollutants Genotoxicity Marine water

[88,90,128*,129*,130,131*,132*,133,134,135,95*,136,137*,138*,139*,140*,141,142*,143*] [68,69,85,144,145] [54*,73,74,78*,79,80,81,82,83,87,128,146,147*,148,149,150,151,152*,153,154,155*,156*,157*] [64*,71,72,89*,91,122,158,159,160*,161*,162*] [163]

a

Bioluminescent biosensors using genetically modified bacteria are indicated by asterisk (*).

toxicity produced by gamma-ray irradiation [88]. Another two stress-responsive E. coli strains, individually or concurrently responding to oxidative and genotoxic conditions, have been constructed by including the genes for the green fluorescence protein, the Xenorhabdus luminescens luciferase operon and the promoters for the recA and katG genes [89]. The two strains carried the fusion genes oriented divergently or in a tandem orientation. This latter type demonstrates its ability to detect specific stress responses, both genotoxic and oxidative, within a multiple toxicity environment [89]. The luxAB genes from V. harveyi, functionally expressed in Streptomyces lividans exposed to heavy metals, chlorinated phenols, or pesticides, allowed observing a bioluminescence decrease proportional to the concentration of toxic compounds in the assay mixture [90]. The degree of sensitivity and specificity pattern characterized in this recombinant bioluminescence streptomycetes were unique when compared with previously reported bacterial bioluminescence systems. Those above reported and more papers, concerning the application of genetically modified bacteria, are listed in Table 1.

2.5. Bioluminescent immobilized systems and biosensors Bacterial luciferase immobilized systems can offer a unique and general tool to analyse many chemicals and enzymes in the environment, exactly like they are already employed in other fields like research and clinical laboratories. When the luminescent reagents and enzymes, or the whole cells, are immobilized on solid supports their peculiarities changes, usually in a positive way. The sensitivity, specificity, and stability of the light-producing systems may be improved by using immobilized enzymes and they can be used for several analyses, reducing costs. The sensitivity is generally increased owing to the creation of a microenvironment with locally high concentrations of the involved reagents.

Several solid supports and procedures are available to immobilize macromolecules or whole cells. The chemical methods give better yields, in terms of active luciferase, than the physical ones. Nevertheless, chemical procedures involve covalent coupling, and usually lead to some protein inactivation. Agarose, collagen, epoxy methacrylate and nylon have proved to be the most effective among the different solid supports that have been investigated. Gel entrapment technique has the advantage of better protein stability and enables the co-immobilization of luciferase and other enzymes with their substrates through an easy process [164]. An important, general, feature of these immobilized enzymes is the possibility to incorporate them into flow cells, used for multiple assays, recycled and reused in automated devices. The continuousflow format offers greater possibilities than a single-batch system, and it leads to rapid and sensitive assays. Flow assays are characterized by extremely accelerated kinetics: a very high surface-area-to-volume ratio is obtained and the reactions do not have to rely on passive diffusion to bring reagents together. Many analytes can be detected at pmol levels, with good precision and a wide range of linearity [165]. The immobilization of biological components on a solid support is quite regularly the preliminary step to the creation of a biosensor. In fact, biosensors are generally described as probe-type devices made up of a selective biological layer, with very sharp molecular-recognition capacity, and of a physicochemical transducer, often an optical system. Among these transducers, the (bio- or chemi-) luminescent ones have the advantages that they do not require light sources and monochromators [2]. Any kind of luminescent bacteria can undergo immobilization, included the genetically modified ones. A review, showing how the recombinant bioluminescent bacteria can be utilized as environmental biosensors is that of Gu [95]. Examples are reported on phenanthrene toxicity in soil samples, of benzene in gaseous form. With further findings and developments of new non-specific stress promoters, the potency and

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extent of the information that can be obtained using these environmental biosensors is immense [95]. A systematic review on the methods to immobilize the different luminescent enzymes and microorganisms (natural and genetically engineered), to use them in biosensors and bioinvestigations is that of Kratasyuk and Esimbekova [166]. Stability, sensitivity, precision, and effects of interfering substances and microenvironment are outlined. Advantages and limitations of immobilized enzyme biosensors in bioluminescent analyses are also highlighted in this review. Microbial biosensors allow rapid measurement, no need for complex sample preparation or specialized personnel and easy handling, and their managing can be enhanced by miniaturization and increased stability [167]. An immobilized film of P. phosphoreum has been used to prepare a sensor based on the determination of acute toxicity effects of toxicoids, molecules that are difficult or even impossible to be measured by traditional analytical chemistry methods [128]. The film should be stored at 4 ◦ C and the stability of the sensor exceeds one month with no measurable deterioration of the signal [128]. The recombinant strain S. typhimurium TA1535 (a SOS promoter fused to a promoterless luxCDABFE operon from P. leiognathi), has been immobilized on microtiter plates to permit its immediate utilization after long storage periods and on field experiments [168]. After 4 weeks of storage, the bioluminescence kinetics and intensity, in response to different concentrations of inducer (mitomycin C), were not significantly different from those of freshly prepared samples. Genotoxic agents, such as mitomycin C, have been also revealed by a stable dark variant of P. phosphoreum (A2), fixed in agar-gel membranes immobilized onto the exposed end of optic fibers, linked with a bioluminometer [131]. A bio-MEMS based cell-chip can be fabricated by patterning and immobilizing bioluminescent bacteria in a microfluidic chip. The pattern of the recombinant E. coli strain GC2 was successfully generated by photolithography, utilizing the PVA-SbQ (polyvinyl alkyl-styrylpyridinium) polymer, as the immobilization material [138]. A hydrogen peroxide detection system was obtained simply immobilizing bioluminescent bacteria, the DK1 strain, which increased bioluminescence in the presence of oxidative damage in the cells [140]. Bioluminescent strains have been immobilized in a chip assembly, in which a silicon substrate is placed between two poly(dimethylsiloxane) (PDMS) substrates. Microchannels fabricated on the two separated PDMS layers are connected via perforated microwells on the silicon chip, creating a three-dimensional, microfluidic, network [132]. Bacteria, mixed with agarose, have been injected into the channels of one of the two PDMS layers, and immobilized in the microwells by gelation. Induction of the luminescence emission occurs when channels on the other layer are filled with samples containing the mutagen [132]. A BOD monitoring system was prepared developing a bio-chip. Luminous bacteria (P. phosphoreum IFO 13896) have been immobilized, with 3 or 15% Na alginate gel, in micrometer-order holes arrayed and fabricated by micromachine techniques. The acrylic chip (3 cm × 3 cm) comprised nine micro-holes (diameter: 700 ␮m or 1 mm, depth: 100 ␮m) arranged in a 3 × 3 array [169]. Bioluminescence from the each hole was gray-scaled and measured by a chemi-imager or a digital camera and a personal computer. The obtained BOD

values showed a good correlation with those of the conventional method [169]. P. fluorescens HK44, that emits light when in contact with naphthalene and its metabolites, has been immobilized into a silica matrix, on glass slides, by the sol–gel technique [153]. The test slides could be used for multiple determinations, since the bacteria responded to the inductor at least 8 months after immobilization, and to more than 50 induction cycles [153]. A series of two-stage minibioreactor systems, connected by a fiber optic probe to a luminometer, have been assembled to set up a multi-channel system for continuous monitoring and classification of toxicants. Each channel was used for cultivating different recombinant bacterial strains: TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2540 (fabA::luxCDABE), which are induced by protein-, DNA-, and cell membrane damaging-agents, respectively. GC2 (lac::luxCDABE) is a bacterium expressing bioluminescence constitutively [123]. Each channel showed a specific bioluminescent response according to the chemicals contained in wastewater samples, while GC2 showed a general response to cellular toxicity [123]. A portable format of the previously described biosensor consists of three parts, a freeze-dried biosensing strain within a vial, a small lightproof test chamber, and an optic-fiber connecting the sample chamber to a luminometer. It can be used for field sample analyses and monitoring on-site of various water systems [141]. A different arrangement of genetically engineered luminescent organisms has been used in a bacterial cell chip, creating the possibility to emit light of different colours from each well, by using quantum dots. Quantum dots have several advantages such as broad absorption spectrum, narrow emission spectra and stability [139]. Each well gives off the specific signal (light) according to the recombinant strain exciting the quantum dots. A multi strain bacterial cell array chip has been developed also by Lee et al., as a revealing test for the presence of reactive oxygen species (superoxide radical and hydrogen peroxide), generated by different chemicals in the sample [156]. The previously described multichannel system has been successfully implemented in the form of computerbased data acquisition. The bioluminescent signatures are delivered from four channels by switching one at once, while the data are automatically logged to a personal computer [162]. Improvements of the system have been the manipulation of the dilution rate and the use of thermo-lux fusion strains. The system is now being implemented to a drinking water reservoir and river for remote sensing as an early warning system [162]. Those above reported and more papers concerning the biosensors based on luminescent bacteria are listed in Table 2.

Table 2 – BLB Biosensors without genetically modified bacteria Reference Air Soil Wastewater Water

[170] [171,172,173] [169,174] [167,172,173,175–179]

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2.6.

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Commercially available BLB and BLB catalogues

Researchers interested to employ bioluminescent organisms probably will meet some difficulties to find in commerce, or ready-to-use, most of the generically modified organisms reported in literature. In fact, most of these organisms have been usually created by a research group, just to perform the works in which those organisms are mentioned. Only the necessary information about the genetic materials and the procedure to repeat the transformation are made available. Any laboratory that will need such organisms must start preparing again, and cultivating, their own transformed strain, even in case that the transformed bacteria are the fundamental components of well-known tests, such as the SOS-LUX-Test [91], based on the S. typhimurium TA1535 transformed by the promoterless luxCDABFE operon of P. leiognathi. This test, able to identify compounds producing DNA damages, is the precursor of another widely used assay, the Lux-Fluoro-test: cells respond to DNA damage by induction of the promoter and the subsequent production of bioluminescence [159]. In a similar way, the Lac-Fluoro-test, based on the constitutive expression of the Green Fluorescent Protein, detects the cellular response to cytotoxins by evaluating the reduction of fluorescent emission [91,145]. It could happen that a laboratory is able to supply to the scientific community their specific transformed organisms. This occurs, to our knowledge, in a limited number of cases, and mainly through direct contacts between the researchers. For example, the Shk1 strain (parental strain P. fluorescens), widely used in wastewater monitoring, can be obtained from the culture collection of the Center for Environmental Biotechnology at the University of Tennessee, Knoxville [117]. Transformed organisms are available on the market when they are the components of commercial kits, such in the case of VITOTOXTM system, a SOS-test produced by Gentaur, (Brusselles, Belgium). It is a geno and citotoxicity test based on two strains of S. typhimurium, SOS-bioluminescent, genetically modified by the promoterless V. fisheri luxCDBAE operon. The test detects any DNA damage and gene mutation [180]. Definitely different is the situation concerning the naturally occurring bacteria, especially those belonging to strains isolated and characterized from long time. A typical example is the V. fischeri strain, which has been introduced in 1999 as the BS EN ISO 11348-3 Standard Method for water quality control. Now, it represents the component of most commercial kits employed in this field. Moreover, classical strains of BLB can be supplied by different institutions, like the Microbiological Laboratories of the PASTEUR Institute (Paris, France), the DSM (Braunschweig, Germany), the American Type Culture Collection, (Rockville, MA, USA), etc. A special case of “bacterial catalogue” is the unique collection of natural and transgenic luminous bacteria stored in the Culture Collection of the Institute of Biophysics, Siberian Branch of the Russian Academy of Sciences (CCIBSO) [181,182]. It consists of about 700 strains of luminous bacteria, with specific properties, that have been isolated from different regions of the world’s ocean. They are mesophiles and psychrophiles, free-living or associated with various marine inhabitants. The CCIBSO collection also include genetically modified E. coli

strains with a marker lux gene. The culture collection is a source of lux genes and biologically active substances. A database, named “BiolumBase”, has been designed for the selection, systematization and distribution via the global network of information on microorganisms containing bioluminescent systems; it includes the two sections of “natural” and “transgenic” luminous microorganisms [181,182]. At present, logic schemes of divisions, classification of the objects, presentation of characteristics, and the inputs of relative information, as well as the necessary program modules including links to the database, are developed. The database has been constructed collecting published data and experimental results of researchers belonging to the Institute of Biophysics. The subsequent linkage of the database to Internet is envisaged. Users will be able to obtain: the catalogues of the available strains; the information concerning the properties and functions of the known species of luminous bacteria, the structure, regulatory mechanisms, and application of bioluminescent systems and genetically engineered constructions. On the database it will be possible to find references and to search strains by using any set of attributes. The database will provide information of interest for the development of microbial ecology and biotechnology, in particular for the prediction of biological hazard from the application of transgenic strains [181,182]. The separated components of the light-emitting systems, enzymes and substrates, are commercially available from long time. They are supplied as both different, commercially available, single reagents and kits. These latter, ready-to-use, systems are well known and widely applied not only in the environmental field. Only few of the mostly used commercial systems will be mentioned below, just because it is impossible to prepare a surely complete list of these products, and of their suppliers, because the continuous variations during time, of their presence on the market and of their names. These frequent changes cause confusion both among the different luminescent tests and between the luminescent reagents and other, completely different, products. Microtox and its portable field version, Deltatox, (Strategic Diagnostic Inc., Carlsbad, CA, USA), are usually employed as early warning systems for drinking water quality, thanks to their sensitivity and rapidity of response (30 and 15 min, respectively). Both are based on the reduction of luminescence of V. fisheri. In addition, the MUTATOX REAGENT is produced by Strategic Diagnostic Inc, and it is based on a dark variant of Vibrio fisheri. Genotoxic damages induce the recovery of luminescence and it is able to detect frameshift and base-pair mutations, intercalating agents, DNA-damaging agents and DNA-synthesis inhibitors (Fig. 4). The analogous MUTATOX TEST is distributed by AZUR Environmental (Carlsbad, CA, USA), produced by employing the dark variant M169 of V. fischeri. Other test systems are based on Vibrio fisheri, like the “BioToxTM Rapid Toxicity Testing System” and its improved version, the “Biotox Flash Test”, that automatically corrects for turbidity/colour interference (Hidex Oy, Turku, Finland). The average time of measurement lasts few seconds. The measurement of the influence of toxic compounds on bacterial metabolism, indicated by changes in biolumines-

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Fig. 4 – Dark strains of naturally BLB are affected by mutations of their genome that concern the luminescence system. Toxic compounds able to act on genetic material can reverse those mutations and the light emission activity is restored.

cence emission, is also the basis of the “Tox Screen II” assay (CheckLight Ltd., Qiyrat Tiv’on, Israel, and under its licence by other companies) but the employed organism is P. leiognathi and it is a field system which emission is detected by a portable luminometer. Because human cells are eukaryotic, cells of this kind could be better models for toxicity to humans than bacterial ones. Following this hypothesis, has been developed the only one eukaryotic cell-based biomonitor system (dinoflagellatesbased), the LUMITOX® , a field portable system (Lumitox Gulf L.C. River, Ridge, LA, USA). The luminescent dinoflagellate mutants detect toxins in the ppb range and it can test both marine and non-marine fluids, soils and chemicals, either water-soluble or lipophylic. The testing instrument is the TOX BOX system. Under a very similar name, LUMISTOX, the Hach-Lange ¨ GmbH (Dusseldorf, Germany) produces kits to perform the Lumitox assay according to the DIN/EN/ISO 11348 Standardized Method. Behind these, new kits are continuously developed, an example is that announced by the ChromaDex Analytics, the BioLuminex. It is a kit system designed to support material identity, detect adulterations, identify potential bioactive compounds, and control manufacturing procedures. It couples directly the TLC separation of complex mixtures with the bioluminescent bacteria identification of toxic compounds. In presence of a toxicant, a dark zone can be documented by CCD camera, X-ray, polaroid or 35 mm film, reaching limits of detection in the picomol range [183].

2.7.

How to measure the light emission

The measurement of the light emission intensity and/or kinetics can be easily performed in laboratory, by using simple instruments, available on the market such as: tube luminometers, luminescence plate-readers and imaging devices. Frequently the devices are homemade. In the first case, the photomultiplier is able to detect the intensity of light emit-

ted in a more or less wide spectrum of wavelengths. The more recent 96-well microplate readers are usually equipped with one or more automatic injection systems and software that allow to choose the frequency and duration of repeated measurements. Most of these instruments can work as luminometer and/or spectrophotometer or fluorimeter. The high number of data that can be obtained by these devices is collected and processed by a computer. Charge-coupled device (CCD) cameras are the most required digital-imaging detectors. Another recently developed technology is the complementary metal–oxide semiconductors (CMOS) image detectors. Like a CCD camera, they are based on semiconductor materials and convert light into electrons using an X–Y array of photodiodes. The quality is not so high like for CCD cameras, but their low energy consumption offer advantages, mainly in hand-held products. The increasing request and employment of biosensors, mainly for on-field applications, caused the most active development of new devices to be used for light emission measurements directly on field. Among these, Picart et al. [184,185] developed an original opto-electronic bioreactor, performing simultaneously the measurement of bioluminescence and optical density of V. fischeri, maintained under continuous culture condition in a fully autoclavable module. A modulated laser diode dedicated to optical measurement and calibrated to measure optical density up to 2.5, a detection head for the acquisition of both bioluminescence and light signals, a bifurcated fiber bundle to detect the light without any sampling, were also included. The bioluminescence was measured through a highly sensitive photomultiplier unit, able to detect a light flux of only a few pW. The device could be applied to the realization of biosensors with any bioluminescent cells [184,185]. Another device to measure toxicity in solid or liquid samples has been developed by Hart and Bainton [173]. It is based on a container, comprising one or more modular inspection trays, each one having an array of cells with a transparent base, holding bioluminescent bacteria and receiving the samples.

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The luminescent emissions are detected by an opto-electronic unit, which includes an array of light sensors arranged in a configuration complementary to that of the inspection trays. A portable optical biosensor, the Lumisens 2, has been described by Horry et al. [179], for the online detection of pollutants. Lumisens 2 features three main parts: (I) a central unit, (II) a disposable card where bacteria are immobilized, and (III) an acquisition unit to control the device. Because any bioluminescent bacteria can be immobilized in the card, Lumisens 2 could become a multipurpose optical biosensor, either for the detection of the overall toxicity of a sample, or for the identification of one particular pollutant [179]. A hand-held luminometer was designed, as reported by Philp et al. [125], for laboratory or field uses, like the in situ continuous monitoring of wastewater treatment plants influents and effluents. Genetically modified bacteria have been immobilized on thin films of poly(vinyl alcohol) (PVA) cryogels, according to: the geometry of the instrument, the need for containment of GM bacteria, the maximization of the bioavailability of the wastewater to the biosensor. The biosensor was tested on phenols-containing industrial wastewater and it proved to be able to discriminate among the different toxicity levels of the various compartments within the wastewater treatment plant [125]. A “microluminometer”, realized by using a standard CMSO process and including integrated photodiodes and a signal processor has been developed by Vijayaraghavan et al. [94]. It is able to detect accurately, at low concentrations, a wide range of toxic substances in both gas and liquid environments. The bioreporter organisms are genetically modified luminescent bacteria, like the P. fluorescens 5RL strain. A monitoring system for the continuous, real-time, monitoring of contaminants in water, has been recently patented by Gibson and Jones [186]. The system includes a samples supply system and a detecting system, that measures the light emitted by a culture of BLB. The appropriate method has been also patented. A novel, handheld, field-deployable Water Toxicity Analysis (WTA) device is under development at the Physical Electronics Department of the Tel-Aviv University. The research started from a previously optimized sensor, based on a disposable

plastic biochip, with micro-chambers and micro-fluidic channels on which different luminescent modified E. coli strains have been immobilized. The changes in bioluminescent signal from the biochip, received by a motorized photomultiplierbased analyzer, are interpreted by signal processing software [155].

3.

Environmental applications

3.1.

Water

Water samples represent the easier environmental matrix to be tested by bioluminescence assays, which, for the main part, are based on marine bacteria. To apply these assays in the assessment of toxicants has a great importance for the wellness and effective protection of ecosystems and of human health. New researches are continuously carried out on this topic, a large number of them concerning the control of wastewaters and treatment plants efficiency. The toxicity of the influents from industries into urban wastewaters, of the effluents from treatment plants, as well as of the water in the various compartments, must be continuously monitored. This is necessary to avoid damages to the activated sludge, to check, at any moment, the effectiveness of the treatments and the quality of water released to the environment. The sensitivity, selectivity and robustness required to the bioassays in the mentioned situations are greatly different, and then several organisms, often isolated from this special environment and genetically engineered, have been tested and developed to comply with the needs of that extreme conditions. Table 3 (modified from [28]) is an example of the various bioassays, and of their respective sensitivity, that can be combined to perform a toxicity assay. Table 4 (modified from [187] shows how the different organisms can result more or less sensitive, according to the compounds contained in the sample. Studies employing BLB assays in wastewater monitoring will be reviewed as first, and then the applications to the various aquatic environments will follow.

Table 3 – Toxic effects of pentachlorophenol on different model systems according to their indicators Model system Daphnia magna Vibrio fischeri Chlorella vulgaris Allium cepa A. cepa Vero cell line (Monkey) Vero cell line RTG-2 cell line (Rainbow trout) RTG-2 cell line RTG-2 cell line RTG-2 cell line RTG-2 cell line a

b

Indicator

Exposure period

EC50 a (␮M)

Immobilization Bioluminescence Growth Mitotic index Micronuclei rate Cell proliferation MTT* reduction LDH leakage Neutral red uptake Cell proliferation LDH activity MTTb reduction

24–48–72 h 5–15–60 min 24–48–72 h 24 h 24 h 24–48–72 h 24–48–72 h 24 h 24 h 72 h 24 h 24 h

2.1–2.0–1.5 3.2–2.3–1.9 59–38–29 >10 1.2 34–24–6 37.6–10.3–8.6 >200 90 2 200 >200

Mean effective concentration: concentration of test chemical that modified each biomarker by 50% in comparison with appropriate untreated conrols. Thiazolyl blue.

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Table 4 – Sensitivity ranking of three bioassays according to their sensitivity frequency (number of times a bioassay was most sensitive) for wastewater effluents from different lines of production, as described in the footnote Effluenta A B C D E a

Rank Microtox > Algae > Daphnids Daphnids > Algae = Microtox Daphnids > Microtox > Algae Algae > Microtox > Daphnids Microtox > Daphnids > Algae

Main compounds present in the effluents: (A) photographic chemicals, colours, phosphorous organic compounds, phenol derivatives, chemicals for plastics manufacturing. (B) Caoutchouc, plastics, explosives, polyvinylchloride, chlorinated aromatic compounds for fuel additives. (C) Mineral oil processing, aromatic compounds, substances for pharmaceutical and pesticides, chemicals for plastics manufacturing. (D) plastics manufacturing, inorganic substances, materials for detergents. (E) Acetic acid, chlorinated hydrocarbons and inorganic salts.

3.1.1.

Wastewater

Obvious candidates for monitoring toxicity in wastewater systems are bioluminescent bacteria. However, the natural bioluminescent bacteria are too much sensitive to some wastewaters, and therefore their response in these operational conditions cannot reflect the status of the more resistant microbial community, responsible for wastewaters treatment. Wastewaters, especially of industrial origin, can contain so high concentration of strong toxicants, that it is impossible, for the natural BLB, to survive and then to be useful in the monitoring of the overall toxicity, presence of specific compounds, detection of any variations on toxicity levels. Industrial wastewaters often overload the treatment plants by toxic influent, sometimes so intensively to destroy treatment activities for extended periods. Organisms isolated from activated sludge are more resistant to wastewater toxicants and can respond to the appearance of a specific toxicant or to a significant increase of its concentration. Their transformation in light-emitting organisms is the basis for their use in monitoring of treatment plants efficiency and influents toxicity. An important example of this approach has been the development of a continuous influent wastewater monitoring system based on the bioluminescent bacterium Shk1, a genetically modified P. fluorescens, containing the lux genes. P. fluorescens has been isolated from the activated sludge in an industrial wastewater treatment plant [117]. The comparison of Shk1 toxicity data with the Microtox ones showed that the Shk1 assay is less sensitive than the Microtox assay and could therefore be more suitable for influent wastewater toxicity monitoring [188]. A protocol for production, storage, and use of Shock 1 (Shk1) bioreporter cells was developed by Lajoie et al. [126], examining the factors affecting Shk1 growth and bioluminescence, including: growth medium, tetracycline concentration, storage conditions, and test media. Effective use of this approach requires standardized time intervals for cell growth, storage, activation and exposure in the test media. Bioluminescence from Shk1 cells was measured in nutri-

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ent broth, influent wastewater and activated sludge mixed liquor from a municipal wastewater treatment plant [126]. The effectiveness of the developed toxicity monitoring method was also evaluated in batch experiments, and in a benchscale activated sludge system exposed to heavy metals (Cu, Zn, Ni, and Cd) by using influent wastewater (primary clarifier supernatant) and activated sludge from a municipal wastewater treatment plant [119]. The same pattern of sensitivity was observed in batch and bench-scale evaluations. The adsorption of metals to activated sludge and reduction in bioavailability, due to chelation by soluble organic compounds or by precipitation in wastewater, was found to be an important effect in mediating differences in toxicity response between bioluminescence and respirometry. The activated sludge adsorption capacity was highest for Cu, followed by Cd, Ni, and then Zn [119]. The appropriateness of Shk1 strain as a surrogate for wastewater treatment facilities was examined further by comparing its results with V. fischeri luminescence inhibition assay and activated sludge respiration inhibition assay [154]. Nearly all the narcotic chemicals (11 nonpolar, 9 polar) tested in batch mode exhibit higher toxicity to V. fischeri than to Shk1. Shk1 data were more similar to activated sludge respiration inhibition data for these compounds, confirming the appropriateness of the use of Shk1 assay for assessing wastewater toxicity to activated sludge [154]. Ren and Frymier [118] have performed a kinetic study of heavy metals toxic effects on Shk1 cells. At sufficiently high concentration this organism turns to sensitive to these toxicants, showing bioluminescence repression. The kinetics of heavy metals toxic effects to Shk1 can be mathematically described in a manner similar to the non-competitive inhibition of enzymes, and the inhibition coefficients Ki , of seven heavy metals has been determined. Under appropriate conditions, a prediction on toxicity can be based on EC50 values, which contain kinetic information similar to that contained in predictions using Ki [118]. The construction of whole cell, genetically modified, bioluminescent biosensors and their immobilization on thin films of poly(vinyl alcohol) cryogels has been carried out by Philp et al. [125]. The biosensor was designed for use in monitoring the toxicity of industrial wastewaters containing phenolic materials. It has been proved that they operate predictably with pure toxicants, within the wastewater treatment plant [125]. Several recombinant bioluminescent bacteria have been employed to set up a multi-channel, continuous, water toxicity monitoring system. That one developed by the research group of Kim was based on channels, each one hosting a different recombinant bacterial strain, and was composed by two mini-bioreactors, to enable a continuous operation, i.e., without system interruption due to highly toxic samples [115]. The luminescent strains were: DPD2540 (fabA::luxCDABE), DPD2794 (recA::luxCDABE), and TV1061 (grpE::luxCDABE), induced by cell membrane, DNA-, and protein-damaging agents; GC2 (lac::luxCDABE) was a constitutive strain. Field samples were waters discharged from a nuclear power plant and a thermo-electronic power plant. Each channel showed specific luminescent response profiles and by comparing the BL signals of the standard chemicals with those of discharged water samples, the equivalent toxicity of the field water could be estimated [115].

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The total hazard existing in effluent water is greatly reduced with respect to the influent wastewater, and then different bioassays must be employed in this case. An in vitro cytotoxicity test, using a human cell line, an inhibition test using luminescent bacteria, and an algal growth inhibition test have been all applied to landfill leachate samples, by using 96-well microplates and a microplate fluorimeter [189]. The total scores of the samples indicate that the total hazard, existing in raw leachate, was significantly reduced by a wastewater treatment process. Thus, this type of scoring of the total hazard existing in landfill leachate may be useful for priority setting for the risk management of landfill sites [189]. Fractionation schemes that combine sample preparation, chemical analysis, and biological measurements have been presented, and reviewed, by Farre and Barcelo [174]. Emphasis has been placed on the use of combined approaches involving chemical analysis and bioassays. The protocols included solid-phase extraction (SPE) followed by chromatographic techniques, such as liquid chromatography–mass spectrometry (LC–MS) or gas chromatography–mass spectrometry (GC–MS). Toxicity testing is carried out by either bioluminescence inhibition or whole-cell bacterial biosensor. Examples of using different bacterial acute toxicity assays are presented for phenols, polyethoxylate surfactants, linear alkyl benzene sulfonates, naphthalene and benzene sulfonates, polycyclic aromatic hydrocarbons, pesticides, and pharmaceutical drugs [174]. The photo-Fenton process can be used, in case of hospital wastewater, as a pre-treatment to improve the overall biodegradability. The COD, BOD, total organic content (TOC) and V. fischeri assays have been selected as the environmental sum parameters to follow the performance of this process [190]. The luminescent test indicated, by the reduction of the inhibition percentage, when a level of toxicants safe for microorganisms degrading the residual organic substance, has been reached [190]. The toxicity of industrial wastewater, treated by Fenton’s reagent, was measured using V. fischeri NRRL B-11177. In this case, a high efficiency of organic components degradation was not always followed by an equivalent reduction of toxicity without an increase of both the H2 O2 dose and reaction time [191]. Toxicity tests were developed using also freshwater recombinant bioluminescent bacteria. The groundwater-borne bacterium Janthinobacterium lividum YH9-RC, was modified by inserting the luxAB gene and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals [113]. LuxAB-marked YH9-RC cells were much more sensitive (on average 7.3–8.6 times) to tested chemicals than marine V. fischeri. The relationship between chemical components of industrial wastewaters and their biological effects has been often assessed using a combination of toxicity tests, like those with algae, Daphnia and luminescent bacteria. Strongest correlations were found in case of absorbable organic halogens in wastewaters, in order from strongest to weakest correlation: microtox bioassay > algae bioassay (S. subspicatus) > daphnid bioassay (D. magna). No correlations were found between these three bioassays and detection sensitivity for total organic carbon or total bound nitrogen in wastewaters [187]. The efficiency of ferric sulphate, used as coagulant in the chemical treatment of raw wastewaters,

was evaluated also by bacterial bioluminescence inhibition [192]. In effluents collected after ferric sulphate treatment, the inhibition percentage was reduced to 99% of Fe in marine systems is complexed to organic chelates of biotic origin. The papers by C.E. Mioni et al. [163,207] explore the use of a heterotrophic, halotolerant, bacterial reporter that quantitatively responds to the concentration of bioavailable Fe by producing light. The BL reporter has been tested in a defined seawater medium, and then in marine surface waters. The papers describe how the bioreporter detected the changes in Fe bioavailability. The results demonstrated the potential utility of this tool in elucidating the relationship between Fe bioavailability and Fe chemistry in complex marine systems.

3.1.3.

19

Surface and ground water

Freshwaters, which content of toxicants is usually low, would require the use of sensitive assays, like the marine BLB ones. However, the not salted waters are not the ideal environment for the marine bioluminescent bacteria, which need salts concentration comparable to that of the seawater. These salts must be added to freshwater samples, introducing any case a new variable in the chemical composition of the specimen to test. To avoid this problem several organisms have been isolated from the specific environment and genetically manipulated, to include the luminescence genes and to develop BL assays. A set of bioluminescent tests consisting of: luminous bacteria, coupled enzyme system NADH:FMN-oxidoreductase– luciferase, triplet enzyme systems with alcohol dehydrogenase, and trypsin has been applied to monitor the water quality of a small forest pond and of a laboratory microecosystems, polluted with benzoquinone and blue-green algae (batch colture) [208]. The performance and the response of the biotests were not affected by the natural, seasonal variability of pond water quality, but revealed the heavy pollution and the bloom of blue–green algae. Such a set of tests can function as alarm system to detect a real, toxic event in natural water bodies. An integrated monitoring approach, comprising chemical analysis and biological toxicity tests by V. fischeri and by D. magna, was applied to evaluate the pollution level of groundwater samples collected near a large, not water-tight, city landfill [209]. The GC–MS analysis indicated the presence of several substances, and the most polluted samples were also found to be the most toxic ones. However, the identity of potential problematic chemicals cannot be revealed by biotests and, therefore, chemical analyses and biotests should be, as usual, applied in combination. A bioluminescence-producing, arsenic-inducible, bacterium based on E. coli has been used as reporter organism to measure arsenic concentrations, in natural water resources, in association with atomic absorption spectroscopy (AAS) [158]. Specific protocols have been developed to avoid the influence of iron ions on arsenic availability to the bioreporter cells. Comparisons between the biological and chemical assays gave an overall average of 8.0% false negative and 2.4% false positive identifications by the bioreporter, at the WHO recommended acceptable arsenic concentration of 10 ␮g L−1 . These results are far better than the performance of chemical field test kits. Another genetically engineered microorganism, P. putida mt-2 KG1206, has been constructed to monitor toluene analogues in groundwater samples collected from petroleum hydrocarbon contaminated sites [152]. The protocol of this simple bioluminescent assay consisted of mixing one volume of groundwater sample with four volumes of broth culture, followed by bioluminescence measurement, after 30 min. A bioassay, utilizing a highly sensitive variant of the P. leiognathi, allowed to detect in water, at levels below mg L−1 , diverse toxicants: heavy metals, pesticides, PCBs, polycyclic aromatic hydrocarbons, and fuel traces [210]. For most toxic agents the new assay was markedly more sensitive than the Microtox assay. Additional features of the new bioassay include: the ability to discriminate between cationic heavy metals and

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organic toxicants and the option of being run at ambient temperatures (18–27 ◦ C), thereby enabling on-site testing by low-cost luminometers.

3.1.4.

Tap water

The interest in online drinking water quality monitoring has increased significantly in the last years. The quality control methods for water intended for human uses must be the more sensitive, reliable and complete among all those available in this field. The aim is to ensure the consumers, and the control authorities, that any compound whit possible adverse effects on human health is absent, or under the safety levels. This lead to the introduction of new monitors, which can provide (near) real-time information on water quality, for example in continuous river water quality control as well as in drinking water protection against intentional contamination. The combination of complementary assays, like a chemical analytical technique and a bioassay, into a single integrated monitoring platform, would greatly enhance the performance of the control systems. Where a chemical analysis identifies and quantifies specific contaminants, biomonitoring gives an indication of the total quality, including the effects of unknown, toxic substances. Such a combination has been realized in the TOX control, a biological toxicity monitor, which include luminescent bacteria and a submersible UV–vis spectrophotometer probe, the scan spectroyserTM . It has been applied in the evaluation of drinking water safety. The alarm signals from one instrument can be verified with the signal from the other, reducing false alarm rates [176]. Another combined system has been used to evaluate the effectiveness of innovative drinking-water treatments, designed to remove toxic and mutagenic organic micropollutants from lake waters used for human consumption. Lake water samples were analyzed for mutagenic activity using Ames assay, for toxicity using bioluminescent bacteria, and for the presence of organic compounds using the GC–MS technique [211]. The work by Haddix describes the design, and initial development, of an AOC assay that uses bioluminescent derivatives of AOC test bacteria [149]. P. fluorescens P-17 and Spirillum sp., strain NOX, have been mutagenized with luxCDABE operon fusion and inducible transposons, and then selected on minimal medium. Independent mutants were screened for luminescence activity and predicted AOC assay sensitivity. All tested mutants were able to grow in tap water under AOC assay conditions, retaining a full range of AOC measurement capability. Peak bioluminescence and plate count AOC were linearly related for tested bacteria. Bioluminescence results were obtained 2 or 3 days post-inoculation, a quite rapid assay in comparison with the 5 days for the ATP luminescence AOC assay and the 8 days for the plate count assay. This bioluminescent method is amenable to automation in a microplate format, with programmable reagents injection [149].

3.1.5.

To control water of effluents deriving from purification plants and marine sediments, BLB Acute Toxicity Test and the Algal Growth Inhibitory Test (on sediments only) were used [202]. Samples were also characterized by microbiological parameters. From the 29 samples from effluent waters, 6 showed an inhibition of bacterial luminescence higher than 20%, whereas 9 exhibited a hormesis effect. Microtox and S. typhimurium TA 1535 pSK1002 umu-assay have been employed to estimate the cytotoxic and genotoxic potential of water samples from rivers, primary, and secondary effluents of some sewage treatment plants. Rainbow trouts (O. mykiss) were exposed to different concentrations (20–40%) of secondary effluents [200]. The toxic potential of water samples has been determined by the acetylcholinesterase activity in the muscle, and the DNA unwinding assay in the liver of the fish. Based on the results obtained during this research it is possible to affirm that both bacterial methods can be successfully used to analyze the cytotoxicity and genotoxicity of industrial and domestic wastewater and to estimate the effectiveness of sewage treatment units. However, because of their low sensitivity and high susceptibility, they are not reliable as a single test for the detection of cytotoxicity and genotoxicity in surface water. The Microtox test and a bacterial luminescence bioassay have been applied to evaluate the seawater quality in areas close to wastewater pipe discharges of small-rate municipal sewers. These assays appeared to be less effective than the ecotoxicological test based on the luminescent fraction of epibacteria [204]. An array of bioluminescent bacterial strains (DPD2794, DPD2540, TV1061 and GC2) has been employed to investigate the toxicity of field waters, using a multi-channel continuous monitoring system [130]. The system showed easy and long-term monitoring use, without any system shut down due to pollution overloading. The light emission of the bacteria, which respond respectively to DNA-, cell membrane-, proteinand general cellular-damaging agents, increased for DPD2794, DPD2540 and TV1061 strains, and decreased for the GC2 strain. This result demonstrates the presence of chemicals that inhibited cellular metabolism, and affected the integrity of the cellular membrane, leading to either protein denaturation or to cell death [130]. Two bioluminescent bioassays, one based on lyophilized marine luminous bacteria, the Microbiosensor-B17-677F, and the other on genetically modified luminous strain of E. coli, the Microbiosensor-ECK, have been employed to reveal areas of impaired water quality in the river and sewage waters of different regions of Siberia, showing the same dependence on the concentration of the toxicants [136]. Nevertheless, the sensitivity to phenol compounds of the Microbiosensor-ECK was higher, and corresponded to that determined on intact cells of P. phosphoreum and of various hydrobionts. All papers reported in Sections 3.1.2, 3.1.3, 3.1.4 and 3.1.5 and other concerning the applications in aquatic environments different from wastewater are listed in Table 6.

Miscellaneous

In this section are included papers dealing with assays performed in at least two different aquatic environments, for example, sewage water and seawater, or sewage water and rivers, etc.

3.2.

Soil and sediments

Biological assays, or sensors, are valuable complements to traditional chemical analysis also in the evaluation of contam-

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21

Table 6 – Application of BLB assays to aquatic environments Drinking water Groundwater

[176,211] [133,209]

Surface water Cyanobacterial bloom toxicity Inorganic pollutants Organic pollutants Surfactants PHA Phenols Multianalyte (organic and inorganic) Sea water

[39] [41,45,48,68,70,85,120,144,138,139,158,163,186,212,213] [21,23,26,27,33,38,40,50,61,95,96,123,141,152,155,161,162,200,214] [37,175,215] [24,25,31,83,216] [28,34,60,74,136,123,141,147,148,157,208,212,217] [1,49,52,55,90,104,113,114,130,135,140,143,160,173,210,218,219] [9,47,201,207]

inated soils and sediments. They have the unique property to respond to the presence of any kind of compound with noxious effects on living organisms, included possible synergic effects. Chemical analysis, necessary to quantify or to characterize the compounds present in a sample, are more expensive and time consuming, and then not perfectly suitable for large contaminated site characterization and monitoring. Moreover, any kind of analysis on soil samples requires, usually, an extraction procedure, a step that further increases the time required for the analysis. The investigation, by using bioassays, on the contamination and toxicity of soils or sediments can be complicated by the contemporary presence of compounds with different solubility features. The whole sample rarely can be tested as it is, extracts must be prepared and measured, but an aqueous extract would not contain the hydrophobic organic components and vice versa. The measured effect could be not corresponding to the real, total amount of contaminants in the sample. This can be the reason because, despite the widespread and successful use of luminescence-based bioassays in water testing, their application to soils and sediments is less proven. Preliminary information about the most probable contaminants is of fundamental importance to make the correct choice about the solvents and the organisms to employ. A partial solution could be to employ a water-miscible co-solvent, or a transfer solvent, used either to extract the soil directly, with a single procedure, or to take up the residues after a previous extraction with another solvent. A large number of studies have used DMSO as the co-solvent with contradictory results. In some cases, its addition did not affect the BLB assay [106,111,220], in many others the researchers have been advised against using DMSO and other solvents in BLB assays [221]. Terrestrial bacteria, genetically modified to contain the bacterial lux genes, have been the most often used for assessment of contaminated soils and, since the promoters that control the lux genes expression can be induced by different chemicals, the corresponding sensitive bacteria can be used for specific tests.

3.2.1.

Soil

Taking into account also the difficulties above described, it is easier to assess separately the presence of organic or inorganic compounds. Nevertheless, attempts have been made to arrange assays able to analyse, in one-step, heavy metals, polar organic contaminants, and hydrophobic organic contaminants (HOCs). This goal was reached, for example, using a

range of luminescence-based bioassays (V. fischeri, E. coli HB101 pUCD607 and P. fluorescens 10586r pUCD607) [222]. Concerning heavy metals and polar organic compounds, the assays were highly reproducible, when an optimized extraction procedure has been applied. For the HOCs, the response of the bioassays was poor. The bacterial bioassays were significantly less sensitive than the sub lethal tests for T. pyriformis. There is no doubt that the wide range of bioluminescent-based bioassays offers complementary applications to traditional testing techniques, but the extraction efficiency is a problem to study carefully [222].

3.2.1.1. Assays of organic pollutants. The evaluation and monitoring of remediation treatment on contaminated soils can be another, but not less important, application of bioassays in this field. Frequently, it can be the unique application, when the presence of surely dangerous pollutants is well known, and has been already quantified by different techniques, for example chemical analysis. Remediation processes, on the opposite, would need a long monitoring period to assess their effectiveness, and low costs for this activity is usually desired. Apart from physico-chemical procedure, various can be the degrading organisms, mainly fungi and bacteria, natural or genetically modified, employed in these remediation treatments. Any case, it can result very useful to monitor the effectiveness of the treatment by a set of bioassays. The 3and 4-ring unsubstituted aromatic hydrocarbons (PAH) degradation, by means of white-rot fungal cultures of Irpex lacteus and Pleurotus ostreatus, has been studied simultaneously by an ecotoxicological evaluation of fungal-treated and non-treated soil: fungus-treated soil indicated a decreased inhibition on the V. fischeri bioluminescence and an increased inhibition of mustard (Brassica alba) seed germination [223]. V. fischeri light inhibition has been employed to investigate many other aspects of remediation processes, for example to evaluate the efficiency of the composting technique applied to an industrial soil, originating from a former tarcontaminated site [224]. Bacterial tests showed a decrease in toxicity both after composting and maturation phases, while toxicity tests on mustard-seed germination did not reveal any significant changes during composting and maturation phases. Biotoxicity and genotoxicity assays, using bioluminescent bacteria E. coli K12, earthworms, and plant seeds, have been performed to evaluate a different compost-assisted remediation of a soil, contaminated by polycyclic aromatic

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hydrocarbons [225]. After composting, performed using mushroom compost (wheat straw, chicken manure, and gypsum), inhibition of bioluminescence decreased, whereas no significant change in toxicity was observed for earthworm survival and seed germination. Bacterial genotoxicity tests showed that genotoxicity decreased only in the upper part of the composted pile [225]. Analogous toxicity tests (earthworm survival and bioluminescence inhibition in V. fischeri) were employed, together with clover and ryegrass grown, on soil samples spiked with anthracene, chrysene, and dibenz(a,h)anthracene [226]. It has been demonstrated, for the first time, that dissipation of condensed PAH may be enhanced in the presence of Arbuscular mycorrhiza, which reduced the toxicity of PAH and/or their metabolites and counteracted a temporary enhanced toxicity mediated by surfactant addition. The classic Microtox test, combined with physico-chemical (IR spectrometry, gravimetry, gas chromatography and thermodesorption) and biological (worms survival, seeds germination, growth of plants, and photosynthesis inhibition) assays has been applied to assess fate and effects of hydrocarbons (HC) on a polluted clayey soil [227]. The worms survival and seeds germination tests did not recorded toxic effects at the day 480. However, growth of plants was reduced in treated soils and a potential residual toxicity was observed based on the inhibition of photosynthesis and of bacterial bioluminescence that resulted once more a very sensitive bioassay. Behind the naturally luminescent bacteria, several engineered bacteria can be used during bioremediation monitoring. Bundy et al. [106] evaluated the performance of five genetically modified bacteria, compared to V. fischeri, in monitoring the changes in toxicity of crude oil and diesel-spiked soils. Three GMB contained the lux genes linked to promoters for hydrocarbon degradation pathways, two expressed the genes constitutively. Octane induced an appreciable response, while the heavy oils elicited a much weaker response. The metabolic (lux constitutively expressed) bioassays showed that there was a general increase in toxicity over the course of the experiment [106]. Organic contaminants can function also as substrates for degrading, luminescent bacteria. P. fluorescens HK44, harbouring the bioluminescent reporter plasmid pUTK21 (a nahG-luxCDABE gene fusion in a salicylate inducible operon), emits light when degrading naphthalene and other polycyclic aromatic hydrocarbons. In the first, EPA approved, environmental release of a genetically engineered microorganism for bioremediation purposes, P. fluorescens HK44 has been introduced into semi-contained subsurface soil lysimeter structures [131]. One of the aims was to show that bioluminescent light emission could be utilized as an in situ tool for online bioremediation process monitoring and control. HK44 population dynamics were tracked using classical plate count, while light emissions were measured using fiber optic guides and a portable photomultiplier tube. HK44 cells provided, in real-time, a general assessment of bioremediation effectiveness. Up to 1150 days after initial release HK44 cells were still active and could be induced to emit light upon exposure to naphthalene [131]. Natural or genetically modified luminescent bacteria can be successfully employed to test environmental samples,

especially when there is the need of a sensitive measure, able to distinguish minimal differences. Aqueous extract of soils have been tested by the lux-marked bacterial biosensor E. coli HB101, to reveal the presence and toxicity of four commonly used herbicides (atrazine, diuron, mecoprop and paraquat) [112]. Toxic responses, for all four herbicides, were stronger in the extracts than in the corresponding spiked water samples, suggesting that intrinsic soil factors may be altering the bioavailable fraction of herbicides, making them more toxic than equivalent concentrations in water.

3.2.1.2. Assays of inorganic pollutants. Among inorganic pollutants that can affect soil and sediments the heavy metals are undoubtedly the most important and dangerous, both for their mechanisms of action and for their typical property to accumulate in living organisms. Then, the major factor governing the toxicity of heavy metals in soils is their bioavailability. Traditionally, chemical analysis have been used with the aim to determine the biologically available fraction of metals in soils, but the direct transfer of these results to biological systems is certainly questionable. While chemical analysis of heavy metals proved essential in defining the extent of contamination, environmentally relevant ecotoxicology tests complemented these data by demonstrating the environmental impact of the pollution. Sometime, the bacteria can be used as an alternative method for different types of samples, as it was suggested by Petanen and Romantschuk [110] for mercury and arsenite detection in humus, mineral and clay extracts in water, ammonium acetate, hydrogen peroxide and nitric acid. In this case, P. fluorescens OS8 (pTPT11) was used for mercury and P. fluorescens OS8 (pTPT31) for arsenite detection. The biosensors detection range was similar, or considerably better, than those of chemical methods [110]. The ecotoxicological approaches, studying both the bulk soil and pore water, represent the key to understanding the fate of heavy metals in soil. Several bacterial biosensors, developed by fusing transcriptionally active components of metal resistance mechanisms to lux genes, have been applied, and compared to chemical methods [109]. Biosensors showed certain advantages, such as selectivity, sensitivity, simplicity, and low cost. Despite certain inherent limitations, bacterial bioluminescent systems have proven their usefulness in soils under laboratory and field conditions. Moreover, also the green fluorescent protein-based bacterial biosensors are suitable for determining with high sensitivity the bioavailability of heavy metals in soil samples [109]. Another important factor to assess in the best way the toxicity of heavy metals contaminated soils is the knowledge of their speciation and its relationship with biological responses. Vulkan et al. [107], determined the pore water Cu concentration and the free Cu2+ activities. To evaluate the biological importance of these data the pore water samples have been analyzed by two bioluminescent biosensors: E. coli HB101 pUCD607 and P. fluorescens 10586r pUCD607. P. fluorescens responses correlated more closely with pore water pCu2+ , whereas both values for Cu fitted the responses of E. coli equally well [107]. Bacterial and yeast biosensors have been confirmed very sensitive, in comparison with respiration and nematode populations, in a large historical study on the copper and nickel contaminations of Kola Peninsula forest soil [171]. Samples

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have been analyzed both by chemical and by a set of ecological, biological tests: soil respiration, total nematode count, microbial heterotrophic numbers and minimal inhibitory concentrations to copper and nickel on bulk soil. Bioluminescent bacterial and fungal biosensors were applied to the pore water. The overall information obtained from these assays is that there were considerable impacts on some microbial parameters but other measures were insensitive to pollutant level. On the other hand, it can happen that the biosensors are not suitable to obtain all requested information on specific samples. In a study on samples of urban soil with a wide range of Pb (14–5323 mg kg−1 ) and Cu (8–12987 mg kg−1 ) contents, bioluminescent Pb (RN4220(pTOO24)) and Cu (MC1061(pSLcueR/pDNPcopAluc)) specific bacterial biosensors and a Cu specific yeast sensor were used to determine the bioavailability of the heavy metals [108]. By using the yeast sensor, 12–20 samples were below the detection limit, however, the yeast sensor was capable of detecting Cu at high concentrations. The biosensors used in this study were not capable of detecting the natural soil concentrations of Pb and Cu in the studied area.

3.2.2.

Sediments

The most different kind of toxic wastes derived from human activities reach the sea and the estuaries, most of them separating from water and accumulating in the sediments. As it has been already demonstrated for soil analysis, ecotoxicological assessment of sediments quality must be performed by a battery of bioassays, luminescent organisms included. This means to use an integrated, hierarchical approach, combining toxicological, chemical, and ecological information. Acute and chronic bioassays have been coupled to chemical methods to assess the contamination of sea and internal water sediments, and some of the more recent applications are reported below, to offer further examples of the endless possibilities to combine these tests. A bioassay-battery for the ecotoxicological assessment of brackish and marine sediment samples (dredged material) was established: bacteria bioluminescence test and marine algae test for the water phase (elutriates), amphipod test as a whole-sediment test [228]. Acute and chronic toxicity have been performed and a broad spectrum of endpoints, physiological, behavioural and integrating parameters (reproduction and mortality) have been considered. For the first time, the amphipod Corophium volutator has been continuously reproduced over a period of 1 year under laboratory conditions, allowing the development of a chronic amphipod test. To evaluate the toxicology of superficial saltwater sediments, amphipods have been exposed to whole sediments in survival and reburial tests. Sea urchin fertilisation and larval development tests have been conducted on pore water, and Microtox test on organic solvent extracts and pore water [229]. The order of test sensitivity resulted: amphipod survival < Microtox test of pore water < amphipod reburial < sea urchin larval development < sea urchin fertilisation < Microtox test of solvent extracts. Concordances between tests, in classifying samples toxicity, ranged from 47 to 79%. This indicated some similarities between test results, but not complete equivalence.

23

Periphyton colonization and sediment bioassessment were used to compare the relative environmental condition of different sampling sites, located in sea bay and slough areas [230]. Toxicities of whole sediments and pore waters were detected for two species of rooted macrophytes, an epibenthic invertebrate and bioluminescent bacteria. The results provide an initial indication of differences in the role of several slough areas as possible sources of bioavailable contaminants. On sediments containing organics/heavy metals mixtures, field toxicity tests such as DeltaTox, to qualitatively identify polluted areas, and Microtox, to quantifies toxicity, have been applied [231]. Samples have been also analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and gas chromatography/mass spectrometry (GC/MS). Results indicate that lead was the primary toxic metal, and polycyclic aromatic hydrocarbons are the most abundant group of organics, which contributed to the overall toxic response. Toxicity results from both assays agreed, but were well correlated with concentration measurements only for certain sediment fractions. Four bioassays were tested on sediment-elutriates of three flowing waters to determine the most sensitive one to employ for sediment routine screening procedure [221]. Whereas the sensitivity of the D. magna assay was relatively low, the S. subspicatus chlorophyll fluorescence test denoted the presence of high loads of nutrients at all sites. The results obtained using the V. fischeri short-term bioassay suggested that an ecotoxicological risk could be excluded. The long-term assay (24 h) should track down subacute toxicities, thus leading to a greater degree of sensitivity. However, this assay is not suited for routine deployment without major improvements. The use of DMSO as solvent caused a decrease of bioluminescence in both bacterial assays. The inhibition was caused by toxic metabolic products of DMSO, formed by autochthonous bacteria from the sediment samples during the elutriation procedure. Thus, the use of DMSO is not recommended for examinations of environmental samples when applying a luminescent bacteria assay [221]. Sediments contaminated by pulp and paper mill effluents were investigated by V. fischeri assay [232]. The measured amounts of resin acids and retene correlated well with the toxicity of some sediment-water elutriates, but it was obvious that other chemical, and also physical, factors were behind the strong toxicity observed in some investigated areas. Results showed that, historically, massive concentrations of wood extractives have been buried in the industrial sediments. Serious consideration of the potential ecotoxicological risks should be made when planning any dredging or remediation actions at areas contaminated in this manner. The chemical quality and biotoxicitiy of whole sediments and pore waters of coastal areas receiving wastewater outfalls were assessed during a two years period [203]. Rooted plants, invertebrates, and fishes were used to assess the acute and chronic toxicities of sediments. Inhibition of bacterial light emission, early seedling biomass, survival, reproduction, fertility, and growth were determined in bioassays, ranging from 30 min to 28 days in duration. Toxicity to either the plant or animal species was observed occasionally, but its detection was dependent on the type of bioassay and the frequency of use. Consequently, a suite of bioassays conducted on multiple

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Table 7 – Soil and sediments analysis by luminescent bioassays References Soil Biodegradation bioremediation Inorganic pollutants Organic pollutants Pesticides PHA Multianalyte (organic and inorganic)

[53,105,106,227,225,226] [102,103,107–110,171,203] [22,29,38,74,84,111,95,175] [112,188,233] [31,216,223,224] [49,51,188,218,222]

Sediments Marine sediments

[221,230,234,232] [52,202,230,228,229,231]

occasions appears to be necessary for toxicity assessments of these sediments, to ensure relevancy of the results. This is particularly true for low to moderately contaminated sediments, where acute toxicity is uncommon, like it was the case in this study [203]. All papers reported in the above Sections 3.2.1 and 3.2.2 and other concerning the applications of BLB assays to the analysis of soil and sediments are listed in Table 7.

3.3.

Air

In spite of the high number of contaminants released into the air by industrial and urban activities, only few are the luminescent methods, based on biosensors that have been developed to detect toxic compounds in this environmental compartment. The importance to perform a rapid detection of air pollutants, or to set up a remote monitoring of toxic sites, is surely out of discussion. Nevertheless, when the idea is to employ BLB the difficulties in the realization of an effective contact of air pollutants with the bacterial cells, in maintain them in good conditions of culture, in control factors such as the appropriate cell growth media, oxygen and cell concentrations, cannot be underestimated. An attempt has been made by proposing a biosensor that measured the light emission inhibition of V. fischeri, immobilized on polyvinyl alcohol gels and placed inside a miniature flow cell [170]. The biosensor could function either as a single unit mounted on an exploratory robot or as numerous units spatially distributed throughout a contaminated environment for remote sensing applications. More recently, genetically engineered BLB were employed to develop whole cell biosensors for the detection of toxic gaseous chemicals. A solid agar medium was employed as immobilization support, to measure toxicity through direct contact of the cells with the gas [93]. The sensitivity has been enhanced by using glass beads, which facilitated gas diffusion through the solid medium, and by the reduction of the agar layer thickness. The biosensor kit was connected to a luminometer by a fiber optic probe. The test assays concerned the evaluation of the effects produced by controlled vapours of BTEX (benzene, toluene, ethylbenzene, and xylene) gases. A different approach, recently patented (in Russian) [235], consists of a bioassay designed to determine quantitatively the gases, prepared in the form of equilibrium solutions in water or other suitable solvents. The biological activity of the gas components is determined by the responses of the bioassay,

in dependence of the concentration of dissolved components. The biological activity of the entire gas medium is calculated from the biological activities of its components. The toxicity of benzene in air was determined using as luminescent bacteria, the Ecolum-5.

4.

Conclusions

From the data reported in the wide collection of papers composing this review, it is possible to conclude that BLB tests are, on average, enough sensitive to detect compounds that can be toxic to humans and to the whole environment. The continuous development, and application, of bioluminescent alarm-tests support this assertion. The sensitivity, easy of use, rapidity, flexibility and low costs of the BL test systems, when compared to the features of other bioassays, suggest luminescent assays as the better choice, at least concerning some of the cited characteristics. Genetic manipulation techniques, transforming in luminescence emitters the native bacteria from the environmental matrix to analyse, increased further the applicability and reliability of GM BLB assays, many of them cited in this review. The final decision about which kind of method, chemical, biological, or a combination, is better to apply in each specific situation will depends on the most important features required in that case. For example, in developing countries two of the most important parameters to take into account are the technical possibilities of the local institutions, and the economic impact of the project, depending also on the starting situation and on the goals to reach. Any case, and once again, the principle that to monitor effectively an ecosystem a battery of bioassays, luminescent or not, is necessary has to be always followed. The com-

Table 8 – Papers applying a set of bioassays, BLB included Reference Soil Control/assessment of toxicity Bioremediation Inorganic pollutants Organic pollutants Multianalyte (organic and inorganic)

[53,112,216,222,227] [26,225,226] [171] [223] [188]

Sediments Sediment quality assessment Marine sediments

[232] [51,52,202,203,225,229–231]

Wastewater Control/assessment of toxicity Organic pollutants Inorganic pollutants

[187–189,196,236,237] [17,154,174,194,197,198] [204,206]

Water Control/assessment of toxicity Surface water Groundwater Drinking water Seawater Multianalyte (organic and inorganic)

[21,27,28,30,41,76,149,157, 214,216,210,238–240] [60,79,200,208] [135,209] [211] [201] [188]

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bination to chemical analysis can be fundamental in some cases, very useful in others. Even in the case that only luminescence-based assays are employed, several tests must be used simultaneously, as they differ in their sensitivity to diverse contaminants. A list of papers reporting the contemporary application of diverse bioassays is reported in Table 8. A last comment concerns the groups involved in this research on BL assays application, and their papers. Having a glance at the references list, it is easy to see that several scientists, worldwide, are interested to this topic, and that some groups can boast of a great number of published papers, even during few years. New genetically modified bacteria, and biosensors, are continuously produced in those laboratories; nevertheless the new commercially available products are actually few. During these years, various companies and products remained, at least for short periods on the market, but up to day only the classical tests have been, and can be, regularly supplied to researchers and Environmental Agencies laboratories. It remains an open question if the new systems can be applied only in research laboratories, or if not all possible efforts have been made to transform them in commercially suites of bioindicators.

Acknowledgements This work was supported by grant from the University of Bologna (Fundamental Oriented Research). L. Bolelli is gratefully acknowledged for helpful discussion and editing work.

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