Specific detection of organotin compounds with a recombinant luminescent bacteria

Chemosphere 52 (2003) 103–111 www.elsevier.com/locate/chemosphere

Specific detection of organotin compounds with a recombinant luminescent bacteria Marie Jos
e Durand a, G
erald Thouand a,*, Tania Dancheva-Ivanova a, Patricia Vachon a,b, Michael DuBow c a

D
epartement G
enie Biologique, Laboratoire de Microbiologie, Universit
e de Nantes, IUT, 18 Bd G. Defferre, La Roche sur Yon 85000, France b Biolumine SA, Plate-Forme Vend
ee Recherche, parc d’activit
e de la croix verte, Bouff
er
e 85600, France c Institut de G
en
etique et de Microbiologie, B^ atiment 409, Universit
e Paris Sud, Orsay, Cedex 91405, France Received 1 August 2002; received in revised form 5 February 2003; accepted 19 February 2003

Abstract Organotin compounds are widely used as biocides in marine and terrestrial environments. Several currently used techniques allow either the measurement of the chemicals or their effects on living organisms. Our current research focuses on the development of a complementary method based on a bacterial bioluminescence-based bioassay for the specific detection of organotin compounds. The performance of the bioassay was assessed. The Escherichia coli bacterial strain used in this study is specific for TBT and DBT (with Cl, Br or I as the halogen group) with the central tin atom important for light production. The assay is conducted after overnight culture of the bacterial strain, followed by 60 min of contact time with the organotin compound for significant light production. The detection limits were found to be 0.08 lM for TBT (26 lg l
1 ) and 0.0001 lM for DBT (0.03 lg l
1 ) with a linear range of one logarithm. The repeatability of the bioassay is 8% and the reproducibility for TBT and DBT was approximately 14%. Lyophilization of the strains did not significantly modify the detection limit as well as the range of detection. Applications of the bioassay to environmental samples are discussed. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Organotins; Biocides; Bioassay; Bioluminescence; Bacteria; Detection

1. Introduction In 2001, the European community published a list of prioritary substances in the field of water policy (EEC no. 2455/2001/EC, 2001). In this list, the organotin compounds are classified as ‘‘dangerous priority substances’’ along with 10 others. The triorganotin com-

* Corresponding author. Tel.: +33-2-51-47-84-41; fax: +33-251-47-84-51. E-mail address: [email protected] (G. Thouand).

pounds such tributyl tin chloride (TBT) are the most widely used as biocides in marine, wood, industrial cooling system, paper mills (Cooney and Wuertz, 1989; Alzieu, 1998). Its toxicity at the ng 1
1 level in sea water led for example to the settling of oyster spat and caused oyster shell thickening and growth inhibition at concentrations as low as 1–10 parts per trillion (p.p.t.) (Alzieu, 1998). Although TBT use is currently regulated, the compound is still found in both marine and freshwater ecosystems (Wuertz et al., 1991). In order to assess the impact of TBT on living organisms, the OSPAR Joint Assessment and Monitoring Program Guidelines proposes the use of Nucella lapillus or Littorina littorea for TBT-specific biological effects monitoring in the

0045-6535/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0045-6535(03)00225-X

104

M.J. Durand et al. / Chemosphere 52 (2003) 103–111

inshore coasts of the North-East Atlantic (Barroso and Moreira, 2002). The detection of low concentration of organotin compounds in water requires sensitive methods as chromatographic flame photometric detection (GCFPD, Carlier-Pinasseau et al., 1997; Bancon-Montigny et al., 2001) or liquid chromatography inducible coupled plasma mass spectrometry (LC-ICP-MS). Though those methods allow the detection of less than 1 ng Sn l
1 in water, they are time consuming, expensive and extensive technical competence is required. They also have the disadvantage that they do not take into account the bioavailability of the compounds. Complementary to conventional detection methods, new methods are required by government associations and industrial partners in order to measure environmental pollutants such as the organotin compounds. Among them, bacterial or microbial bioassays and biosensors play an increasing role in the development of new methods (approximately 400 articles in the PubMed data-base were found from 1990 to 2002, containing the words biosensor/bioassay and bacterial/bacteria/microbial in the title or the abstract). These microbial bioassays offer quick, cheap and easy to use measurements as well as the possibility to measure the bioavailable amount of the pollutant in the environment. The first microbial bioassay to report the presence of a metal or toxic chemical substance measured bioluminescence from the marine bacteria Vibrio harveyi or Vibrio fischeri. Baldwin et al. (1984) were the first to apply the luxAB genes of Vibrio harveyi as a reporter of transcription in Escherichia coli. In these bacteria, the emitted light (mainly at 490 nm) is generated after a complex biochemical reaction involving the luciferase enzyme, oxygen, a long chain aldehyde and a reduced flavine mononucleotide (Hastings, 1996; Blum, 1997 for reviews). From these results, the luxAB reporter gene has been applied to the detection of bacteria (Thouand et al., 2001) or to the detection of chemical compounds (K€ ohler et al., 2000). In the latter case, the luxgene, minus its promoter, is inserted downstream of the promoter of a gene involved in a resistance to a metal, the biodegradation of organic compound or a stress (Billard and DuBow, 1998). When the compound is present, the promoter of the gene is induced and luciferase is produced. Luciferase expression is thus under the control of the particular gene and its expression is generally proportional to the concentration of the compound of interest. This strategy has been applied to the study of (i) overall environmental stress detection (Van Dyk et al., 1994; Belkin et al., 1996; Vollmer et al., 1997; Gu and Chang, 2001); (ii) detection of specific organic molecules such as benzene (Kobatake et al., 1995); naphtalene (Heitzer et al., 1994); fluorene (Bastiaens et al., 2001) and (iii) metals, Cu (Corbisier et al., 1993); Hg (Ras-

mussen et al., 2000; Tauriainen et al., 2000). A second application of luciferase reporter gene insertion is to find new, previously uncharacterized genes (Guzzo and DuBow, 1991). In this case, the luciferase reporter gene is randomly inserted at a single copy into the chromosome of a host bacterium (E. coli, for example). Several luminescence-inducible strains were discovered that were able to detect aluminium (Guzzo and DuBow, 1994) or arsenic (Cai and DuBow, 1997). Few studies have reported the use of bacteria for the detection of organotin compounds. Bundy et al. (1997) constructed a luminescent Pseudomonas fluorescens based on a decrease of luminescence in the presence of the organotin compounds. The EC50 value after 15 min contact time was 2.62 lM but this assay was found to be less sensitive than the Microtoxâ assay (which uses bioluminescent Vibrio fischeri bacteria). Cha and Kim (2002), fused a promotorless lacZ operon to a DNAdamage inducible promoter (recA, dinD, uvrA). The corresponding plasmid was transformed in an E. coli recipient and the reporter gene was induced with a relatively high level of TBT (from 0.01 to 1 mg l
1 depending on the genetic construction). Nevertheless, the above-mentioned strains lacked specificity for organotins. In 1996, Briscoe et al. reported the isolation of an E. coli strain containing a chromosomale luxAB transcriptional fusion using luxAB from Vibrio harveyi as the reporter gene inserted at random into the chromosome embedded within a modified Tn5 element. The research presented here presents a simple and quick luminescence bioassay incorporating this strain for the specific detection of organotin compounds.

2. Materials and methods 2.1. Bacterial strains E. coli strain TBT3 was obtained after random insertion of the luxAB genes from Vibrio harveyi into E. coli DH1 (Guzzo and DuBow, 1991; Briscoe et al., 1996). The strain was cultivated in batch condition in LB medium to an A620 nm ¼ 0:5 and then washed with 10 ml MgSO4 0.01 M prior to storage via the addition of 1.8 ml of a sterile glycerol solution to reach a final concentration of about 15% and frozing at )80 °C in cryotubes (Simport, ref. T310-2A). 2.2. Growth medium LB medium (Atlas, 1997) was prepared with one liter of distilled water containing: NaCl (5 g, MERCK, ref. 1.06400.1000), yeast extract (5 g, Biokar Diagnostics, ref. 112002) and tryptone (10 g, Biokar Diagnostics, ref. 104001).

M.J. Durand et al. / Chemosphere 52 (2003) 103–111

Glucose medium with a C/N/P ratio (w/w/w) of 100/ 10/1 was prepared with one liter of tape water filtered through a 0.45 lm membrane filter (Sartorius): D (+)glucose monohydrate (1.376 g, Merck, ref. 1.04074. 0500), NH4 Cl (0.1919 g, Merck, ref. 1.01145.0500), K2 HPO4 (0.028 g, Merck, ref. 1.05101.1000), NaCl (5 g), yeast extract (0.5 g) and tryptone (1 g). The pH was adjusted to 7 with a pH meter (WTW) and the medium was sterilized by autoclaving for 30 min at 100 °C. After cooling, 100 ll of a sterile oligoelement solution (SL7, Mergeay et al., 1985) was added. SL7 medium was prepared as follows for 1 l of distilled water: HCl 25% (13 ml, Merck, ref. 1.00317.1000), ZnSO4  7H2 O (1.44 g, Labosi, ref. A4930701), MnCl2  4H2 O (1 g, Carlo-Erba, ref. 351507), H3 BO3 (620 mg, Labosi, ref. A4703751), CoCl2  6H2 O (1.9 g, Labosi, ref. A4774101), CuCl2  2H2 O (170 mg, Carlo-Erba, ref. 364507), NiCl2  6H2 O (240 mg, Aldrich, ref. N6136), Na2 MO4  2H2 O (360 mg, Labosi, ref. A4895857). Appropriate antibiotics were added after sterilization (final concentration): ampicillin (40 lg ml
1 , Sigma, ref. A-6140), tetracycline (10 lg ml
1 , Fluka, ref. 87128). Antibiotics and the SL7 solutions were sterilized by filtration through a 0.22 lm membrane (GVWP, Millipore). 2.3. Chemical substances All chemicals were purchased at a purity exceeding 95%. Tributyl tin monochloride (TBT, ref. T5,020-2), Dibutyltin chloride (DBT, ref. 20,549-4), Butyltin trichloride (MBT, ref. 20,105-7), Bis(tributyl tin) oxide (TBTox, ref. B5,338-3), Tributyl tin iodide (ref. 33,3034) were purchased from Sigma Aldrich. Triphenyltin chloride (ref. 93 191) was purchased from Fluka. Tributylgermanium monochloride (ref. 40,917-0), Dibutylgermanium dichloride (ref. 44,758-7) and Dimethylmercury (ref. 593-74-8) were purchased from Aldrich. Organotin stock solutions (600 lM) were prepared with 60 ml ethanol 70% adjusted to 100 ml with pure water. Dimethylmercury solutions (300 lM) were prepared in DMSO (Riedel de Haen, ref. 34869). From stock solutions, serial dilutions were prepared in synthetic sea water pH 8.2 (Instant Oceanâ aquarium systems, Sarreboug, France) or in spring water pH 7.4 (Prystelâ , Vend
ee, France). N-Decyl aldehyde solution (Sigma, ref. D-7324) 210 lM was prepared in water with 1.2% isopropanol. 2.4. Experimental conditions and luminescence measurements E. coli strain TBT3 was grown overnight (14 h) in glucose medium at 37 °C with appropriate concentration of ampicillin and tetracycline. The cells were diluted to

105

an A620 nm of 0.075 in fresh medium. Culture aliquots (100 ll) were added to each well of an opaque white 96 microwell plate (Nunce, ref. 236108) containing dilution series of the compound to be tested in 50 ll sea water or spring water. In all experiments, each dilution of compound was tested in triplicate. After 1 h incubation (30 °C), luminescence was followed using a temperature controlled (30 °C) microtiter plate luminometer (Microlumate L96V, EGG Berthold) after automatic injection of decanal to reach a final concentration of 30 lM. Light production was monitored in repeated mode for 15 min (0.5 s counting time, 7 measurement cycles per well). The peak RLU s
1 of the measurement was normalized to the measured absorbance (A620 nm ) of the bacterial suspension. The Induction Ratio (IR) was calculated as follows: IRi ¼ ðRLU s
1 A
1 620 nm Þi =
1
1 ðRLU s
1 A
1 A620 nm Þi is the lumi620 nm Þ0 where ðRLU s nescence after induction with organotin and ðRLU s
1 A
1 620 nm Þ0 is the background luminescence. 2.5. Bacterial growth measurement Batch growth was performed with 100 ml of glucose medium in a 500 ml flask with 1–2% of an over-night culture grown in the same medium. The flask was maintained at 37 °C. Every hour, 1 ml was used to measure optical density (A620 nm ) with a spectrophotometer (Secomam BP106, Anthelie Advanced, France) in spectrovette (Evergreen, ref 201-3166-010, 1 cm wide). A second milliliter was retreived in order to follow the luminescence output as described above. 2.6. Lyophilization The protocol was adapted from Wagner and Van Dyk (1998). One liter of glucose medium contained in a 2 l bioreactor (New Brunswick, Discovery 100) was inoculated with 10% of an over-night preculture of E. coli strain TBT3. Cells were grown for 14 h to the stationary phase (A620 nm ¼ 0:8) at 37 °C. Oxygen (50%) was regulated by feedback regulation with aeration and agitation. The pH was regulated to 7 via the addition of 0.2 M HCl or NaOH solutions sterilized by autoclaving. Cells were then centrifuged (10 000  g, 10 min, 4 °C) and resuspended in a mixture containing one volume of fresh glucose medium plus one volume of a sucrose solution at 24% (W/V). The final optical density (620 nm) was adjusted to 0.1. One hundred microliters were then added in each well of a 96 well-microtiter plate (Nunce, 236108), frozen for 18 h at )80 °C and then lyophilized for 24 h in a Christ Alpha 1–2 lyophilizer (Bioblock Scientific). Before the bioassay, the lyophilized bacteria were reconstituted with 100 ll of pure water for 30 min at 30 °C. The assay was performed as described above.

106

M.J. Durand et al. / Chemosphere 52 (2003) 103–111

3. Results 3.1. Influence of growth phase on light production Cells were grown over night in glucose medium for 14 h and then bacteria were inoculated in a fresh batch culture in the same conditions as above. The cells were harvested at different culture times from latency to late stationary phase, and TBT was then added. Fig. 1 shows the influence of growth phase on the induction ratio of the strain exposed for 1 h to 1.25 and 2.5 lM TBT. The induction ratio was maximal (IR ¼ 25) just after dilution in fresh medium of the overnight culture. From latency to early exponential phase, luminescence dropped and then increased during exponential phase, with a maximum reached after 120 min. 3.2. Influence of the induction time on light production An overnight culture of strain TBT3 was used to assess the induction time when TBT is diluted either in synthetic sea water or in a 0.01 M MgSO4 solution used for bacteria dilution. Three different concentrations of TBT (1, 0.5 and 0.1 lM) were tested for a period ranging from 15 to 150 min (Fig. 2). For all TBT concentrations diluted in synthetic sea water, the trend was similar. The bacteria began to luminesce 18 min after TBT addition, which increased up to 30 min and then became steady for 60 min (i.e. the 30–90 min period). Light decreased when the TBT concentration was high (1 lM), perhaps due to a toxic effect. Light production was also found to be concentration dependant. The dilution of TBT in magnesium solution had a dramatic effect on light production that decreased from IR ¼ 25 to IR ¼ 7 with 1 lM of TBT. For the following experiments, bacteria

30 Growth rate Induction with 2.5 µM of TBT Induction with 1.25 µM of TBT

Growth rate (h-1)

0.8

25

0.6

20

0.4

15

0.2

10

0.0 0

100

200

300

400

500

600

Induction ratio

1.0

5 700

Culture time (min)

Fig. 1. Influence of growth stage on the induction ratio of E. coli TBT3 strain induced when exposed for 1 h with TBT. Each point shown are the averages of data from four replicates. Standard errors are below 1% and hence are not shown. For comparative assays, two other experiments were repeated and the trends were found to be identical.

Fig. 2. Time course of light emission in the presence of different TBT concentrations. Each point shown are the averages of data from four replicates. Standard errors are below 1% and hence are not shown. For comparative assays, two other experiments were repeated and the trends were found to be identical.

were exposed to TBT for 60 min before bioluminescence measurements were undertaken. 3.3. Sensitivity of the bioassay The TBT3 strains were induced with several TBT concentrations from 0.01 to 10 lM either in sea water or in freshwater. The shape of the bioluminescence curve was found to be almost gaussian whatever the media were used for TBT dilution, with a maximum at 1 lM (IRSea water ¼ 45 and IRFreshwater ¼ 15). In addition, less light was produced when TBT was diluted in freshwater. This result was not attributable to a difference of pH between the sea water and the freshwater (results not shown). The same results were observed for all tested organotin compounds DBT, MBT, TPT and TBTox (results not shown). Fig. 3 presents three zones characteristic of the TBT3 behavior for several TBT concentrations. The first zone started from 0.01 to 0.1 lM of TBT, followed by the second zone (above 0.1–1 lM) for which light production is proportional to TBT concentration. From 1 lM, TBT was toxic (third zone) and the induction ratio dropped dramatically from 45 (TBT in sea water) to 0 when the TBT concentration was 10 lM. 3.4. Specificity All the commercial formulations of organotin compounds were tested in order to assess the specificity of the TBT3 strain. E. coli strain TBT3 was incubated with the chloride form of TBT, DBT, MBT, TPT and with TBTox (Fig. 4). As can be seen, light induction is most marked by DBT and TBT. Induction with DBT led to increased light output than with TBT (at the most

M.J. Durand et al. / Chemosphere 52 (2003) 103–111 50

107

80 Sea water Freshwater

45

TBT Br TBT Cl TBT I

70 60

35

Induction ratio

Induction ratio

40

30 25 20

50 40 30

15

20

10

10

5 0 0.001

0.01

0.1

1

10

100

Concentration (µM)

Fig. 3. Induction of strain TBT3 with TBT in sea water or in freshwater. Each point shown are the averages of data from four replicates. Standard errors are below 1% and hence are not shown. For comparative assays, two other experiments were repeated and the trends were found to be identical.

Fig. 4. Specificity of strain TBT3 for several organotin compounds. Each point shown are the averages of data from four replicates. Standard errors are below 1% and hence are not shown. For comparative assays, two other experiments were repeated and the trends were found to be identical. (TBT: tributyl tin monochloride; DBT: dibutyl tin dichloride; MBT: monobutyl tin trichloride; TBTOx: Bis (tributyl tin) oxide; TPT: triphenyl tin monochloride.)

inducible organotin concentration, IRDBT ¼ 70 and IRTBT ¼ 40). The detection limit was 0.0001 lM (0.1 nM) DBT and 0.08 lM TBT. For the other form, MBT, TPT and TBTox, a decrease of the induction ratio and a shift of the detection limit to the higher concentrations was observed, with maximum light output recorded for

0 0.0001

0.001

0.01

0.1

1

10

100

Concentration (µM)

Fig. 5. Influence of the halogen group on the induction of strain TBT3. Each point shown are the averages of data from four replicates. Standard errors are below 1% and hence are not shown. For comparative assays, one other experiments was repeated and the trends were found to be identical. (TBT Br: tributyl tin monobromide; TBT Cl: tributyl tin monochloride, TBT I: tributyl tin monoiodide.)

5 or 10 lM of the organotin form. The strain had much less sensitivity with the MBT compounds. The induction ratio was not found to be proportional to the number of butyl groups bound with tin. The substitution of the butyl group with a phenyl group led to a loss of sensitivity of the strain, as well as for the TBTox compound (Fig. 4). The importance of the halogen group (X) was then tested (Fig. 5). The strain was exposed to TBT bound with bromide, chloride or iodide. Fig. 5 presents the results for TBT-X concentrations ranging from 0.001 to 20 lM. The three halogen forms of TBT induced the strain (maximum, IRTBTBr ¼ 70; IRTBTCl ¼ 40; IRTBTI ¼ 60). TBTBr is the most potent inducer of the strain, with a detection limit of 0.02 lM. For the two other forms, the detection limit was the same (0.08 lM). After the minimum inducible concentration, the trend was the same but the toxic effect was stronger for TBTCl than TBTI, as induction ratio began to decrease from 1 lM for TBTCl versus 8 lM for the iodide form. Finally, the tin atom seems to play a role in the inducibility of the strain for the organometallic compound. Indeed, when tin was substituted with germanium (an atom in the same column as Sn in the Periodic Table), the strain was not induced whatever the concentration added. Moreover, the same results were obtained when tin was substituted with mercury (results not shown). 3.5. Repeatability and reproducibility The repeatability and reproducibility of the bioassay were also evaluated. Forty assays were performed with

108

M.J. Durand et al. / Chemosphere 52 (2003) 103–111 70

Induction ratio

50

Induction factor

50

60

former (13%). The variation between each independent experiment is more important in the linear zone of the nontoxic or toxic part of the curve (24% and 32% respectively). The main difference between each experiment concerned the induction ratio (from 30 to 60 units). The latter varied mainly due to little variation of the background luminescence level of the control (results not shown).

60

Experiment 1 Experiment 2 Experiment 3 Experiment 4

B

40 30 20 10

40

A

0 0.01

0.1

1

Concentration (µM)

30 20

3.6. Conservation of the strains 10 0 0.001

0.01

0.1

1

10

In order to simplify the bioassay, several forms of strain conservation were tested (freezing at )80 °C, immobilization in alginate beads, and lyophilization). The latter was the most promising. Cells were lyophilized directly in 96 microwell plates after batch growth in glucose medium in a bioreactor. Fig. 7 shows the results of TBT3 strain induction with TBT for two different lyophilization batches after 10 days of conservation at )20 °C. The response of the lyophilized strains were very close for the two experiments and in comparison to the strains before lyophilization. Detection limits were virtually the same (0.05 lM before lyophilization; 0.07 lM for the first and 0.08 lM for the second experiment). Moreover, the maximum induction ratio was comparable and was obtained for 1 lM of TBT in each case (IRbefore lyoph ¼ 35; IRlyoph 1 ¼ 30, IRlyoph 2 ¼ 28).

100

Concentration (µM)

Fig. 6. (A) Evaluation of the reproducibility of the bioassay in four independent experiments. (B) Representation of the linear part of each curve in the nontoxic part. Each point shown are the averages of data from four replicates. Standard errors are below 1% and hence are not shown.

TBT or DBT under the same conditions using the same inoculum. In these conditions, the coefficient of variation between these 40 experiments was 8–9% (results not shown). The reproducibility was also assessed after several expositions of the strain to TBT or DBT. Fig. 6 shows a typical response of the strain after induction with TBT for four independent experiments performed on different days. The reproducibility was calculated either from the detection limit (DL) or the slope of the linear curve in the nontoxic (NT) or toxic (T) part of the response curve. From Table 1, the lowest TBT concentration that the strain was able to detect occured at a mean value of 0.076 lM, with a coefficient of variation of 14%. At the opposite end of the curve, in the toxic part, the highest TBT concentration that eliminated the light was 7.25 lM with a reproducibility very close to the

4. Discussion The present research focused on the development of a bioassay for organotin detection, the performance of which was found to be dependent on the physiology of the bioelement. The E. coli TBT3 strain was isolated after a random insertion of the luxAB (promoter-less) genes of Vibrio

Table 1 Comparison of the detection limit and the slope of four independent experiments in the nontoxic and the toxic parts after induction of the E. coli TBT3 strain with TBT Experiments

Nontoxic part DL (lM)

Equation of the linear zone

R (n ¼ 4)

DL (lM)

Equation of the linear zone

R2 (n ¼ 3)

1 2 3 4 Mean CVe (%)

0.09 0.08 0.07 0.065 DLNT ¼ 0.076c 14

IR1b ¼ 12.7  log10 [TBT] þ 39.1 IR2 ¼ 15.8  log10 [TBT] þ 44.7 IR3 ¼ 21.8  log10 [TBT] þ 65.2 IR4 ¼ 14.7  log10 [TBT] þ 47.3 SlopeNT ¼ 16.25 24

0.94 0.99 0.99 0.99

6 8 7 8 DLT ¼ 7.25d 13

IR1 ¼ )18.3  log10 [TBT] þ 33.8 IR2 ¼ )24.9  log10 [TBT] þ 43.9 IR3 ¼ )38.9  log10 [TBT] þ 64.2 IR4 ¼ )25.9  log10 [TBT] þ 49.3 SlopeT ¼ )26.9 32

1 0.99 0.95 0.97

a

a

Toxic part

DL: detection limit. IR: induction ratio. c NT: data calculated in the nontoxic zone of the curve. d T: data calculated in the toxic zone of the curve. e CV: coefficient of variation. b

2

M.J. Durand et al. / Chemosphere 52 (2003) 103–111 40 35

Before lyophilization After lyophilization (Experiment 1) After lyophilization (Experiment 2)

Induction ratio

30 25 20 15 10 5 0 0.001

0.01

0.1

1

10

Concentration (µM)

Fig. 7. Induction of the strain before and after lyophilization with TBT in sea water (10 days conservation at )20 °C). Each point shown are the averages of data from twelve replicates. Standard errors are below 5% and hence are not shown.

harveyi into the E. coli DH1 chromosome (Guzzo and DuBow, 1991; Briscoe et al., 1996). The strain is specific for bioluminescence induction by TBT and DBT (with Cl, Br or I as the halogen group). The presence of a central tin atom was also found to be important for light induction. So far, the induction mechanism of luminescence at the gene level after organotin addition is not currently know. The mechanism by which the TBT3 strain reacts with organotins as TBT and DBT remains to be elucidated. The organotin compounds may act as both cationic metal and as organic compounds. Hence, TBT and DBT could adsorb on some electronegative part of the cell wall and be dissolved in the membrane and enter the cytosol of the TBT3 strains, a result previously reported in the literature for other bacterial strains (White et al., 1999; Gadd, 2000). The physiological state of the strains plays an important role in light production (Neilson et al., 1999). The relationship between growth and luminescence is well described for Vibrio harveyi and Vibrio fischeri that emit light in stationary phase (Hastings et al., 1985; Meighen, 1999). Recombinant E. coli strains harboring a luxAB insertion either in the chromosome or in a plasmid, produce light mainly in the early or middle exponential phases of growth (Kobatake et al., 1995; Rozen et al., 1999; Thouand et al., in press). In the present conditions (37 °C, growth in Glucose medium), strain TBT3 emits light after exposure to TBT (or DBT) during batch growth but mainly at the end of the overnight and middle exponential growth phases. Future research could explore the effects of relatively low nutrient media (e.g. minimal salts media) on the growth, light induction and response patterns of the strain TBT3. This could lead to toxicity response data with improved applications to water and sediments in aquatic ecosystems (freshwater and marine).

109

The production of light (for cells in the same growth phase) was also enhanced when cells were induced with organotins diluted in sea water rather than in freshwater with no change in detection limit observed (Fig. 3). White et al. (1999) reported that the effects of organotin contamination will vary between freshwater and marine environments. The presence of Naþ can reduce interactions with the cell surface by competing for binding sites or interacting with the organotin compound itself, as well as a change in the lipid level and cell surface (Rozen and Belkin, 2001). A decrease of organotin solubility was also noticed with Cl
anion associated to the cation to form covalent organotin chloride. Hence, considering these results, the detection limit of E. coli TBT3 in sea water should be less than in freshwater. However, this was actually not observed in our conditions. The nature of the ions in the sea water solution and their ionic strength could influence the luminescence reaction in the cytoplasm leading to the observed light output. The performance of a sensor or a bioassay include the determination of the specificity, sensitivity, the time to detection, its repeatability and its reproducibility. The bioassay presented here allows the detection of two important organotin compounds, TBT and DBT, present in the environment. The detection limits (DL) ranged respectively from 0.08 lM (26 lg l
1 ) and 0.0001 lM (0.03 lg l
1 ). The induction time of 60 min (the delay from the addition of the chemical to light production) allows a rapid response time for the bioassay. The repeatability of our bioassay (approximately 8%) is a strong point of the assay. The reproducibility of results between different assays is also good. However, it is recommended to repeat the standard curve with known organotin concentrations for each assay series. Lastly, we demonstrate that lyophilization of the bacteria did not modify the response of the bioassay, as previously observed by Tauriainen et al. (1998). The application of the bioassay to environmental samples is still under development and will depend on the contamination levels in relation to the DL of the bioassay for TBT and DBT. Water analyses carried out in Arcachon Bay in 1992 showed that TBT concentrations ranged from 0.2 to 16 ng Sn l
1 (0.55 to 44 ng TBTCl l
1 ) in harbors and marina (Ruiz et al., 1996). Higher contamination levels of 10–199 ng TBTCl l
1 were reported in a Mediterranean marina by Michel and Averty (1999). The military harbor of Toulon on the French Mediterranean coast is particularly contaminated, with TBT concentration reaching 237 ng TBT-Cl l
1 . From these data, only the higher organotin concentrations could be detected by the bacteria in water samples. The main application of the bioassay reported here may likely be the screening of sediments where very high levels of TBT are reported (from 1 to 10  103 lg BT kg
1 , Hoch, 2001).

110

M.J. Durand et al. / Chemosphere 52 (2003) 103–111

Acknowledgements This research is supported by a contract/grant CER 2000-2006, Action no. 15 (Section I), research program no. 18035 (Ville de la Roche sur Yon, Conseil G
en
eral de Vend
ee, Conseil R
egional des Pays de la Loire, Ministere Francßais charg
e de la Recherche). We also thanks the firm Biolumine SA. References Atlas, R.M., 1997. Handbook of Microbiological Media. In: Parks, L.C. CRC Press, Boca Raton, New York, p. 744. Alzieu, C., 1998. Tributyltin: case study of a chronic contaminant in the coastal environment. Ocean Coast. Manag. 40, 23–26. Baldwin, T.O., Berends, T., Bunch, T.A., Holzman, T.F., Rausch, S.K., Shamansky, L., Treat, M.L., Ziegler, M.M., 1984. Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli. Biochem. 24, 3663–3667. Bancon-Montigny, C., Lespes, G., Potin-Gautier, M., 2001. Optimisation of the storage of natural freshwaters before organotin speciation. Wat. Res. 35, 166–170. Barroso, C.M., Moreira, M.H., 2002. Spatial and temporal changes of TBT pollution along the Portuguese coast: inefficacy of the EEC directive 89/677. Mar. Poll. Bul. 44, 480–486. Bastiaens, L., Springael, D., Dejonghe, W., Wattiau, P., Verachtert, H., Diels, L., 2001. A transcriptional luxAB reporter fusion responding to fluorene in Sphingomonas sp.LB126 and its initial characterization for whole-cell bioreporter purposes. Res. Microbiol. 152, 849–859. Belkin, S., Smulski, D.R., Vollmer, A.C., Van Dyk, T.K., LaRossa, R.A., 1996. Oxidative stress detection with Escherichia coli harboring a katG0 ::lux fusion. Appl. Environ. Microbiol. 62, 2252–2256. Billard, P., DuBow, M.S., 1998. Bioluminescence based assays for detection and characterization of bacteria and chemicals in clinical laboratories. Clin. Biochem. 31, 1–14. Blum, J.L., 1997. Bio- and Chemi-luminescent sensors. World Scientific Publishing, Singapure, London. Briscoe, S.F., Diorio, C., DuBow, M.S., 1996. Luminescent biosensors for the detection of tributyltin and dimethyl sulfoxide and the elucidation of their mechanisms of toxicity. In: Moo-Young, M., Anderson, W.A., Chakrabarty, A.M. (Eds.), Environmental biotechnology, principles and applications. Kluwer Academic Press, Dordrecht, Boston, London, pp. 645–655. Bundy, J.G., Wardell, J.L., Campbell, C.D., Killham, K., Paton, G.I., 1997. Application of bioluminescence-based microbial biosensors to the ecotoxicity assessment of organotins. Lett. Appl. Microbiol. 25, 353–358. Cai, J., DuBow, M.S., 1997. Use of a luminescent bacterial biosensor for biomonitoring and characterization of arsenic toxicity of chromated copper arsenate (CCA). Biodegradation 8, 105–111. Carlier-Pinasseau, C., Lespes, G., Astruc, M., 1997. Determination of butyl- and phenyltin compounds in sediments by GC-FPD after NaBEt4 ethylation. Talanta 44, 1163–1171.

Cha, J.-M., Kim, B.-W., 2002. Bioassay of environmental endocrine disruptors TBT and DMSO using recombinant Escherichia coli. Bioprocess Biosyst. Eng. 24, 405–410. Cooney, J.J., Wuertz, S., 1989. Toxic effects of tin compounds on microorganisms. J. Indust. Microbiol. 4, 375–402. Corbisier, P., Ji, G., Nuyts, G., Mergeay, M., Silver, S., 1993. luxAB gene fusions with arsenic and cadmium resistance operons of Staphylococcus aureus plasmid pI258. FEMS Microbiol. Lett. 110, 231–238. EEC No. 2455/2001/EC, decision of the european parliament and of the Council of 20 November establishing the list of prioritary substances in the field of water policy and amending Directive 2000/60/EC, Official Journal, L331, 44, 15 December 2001. Gadd, G.M., 2000. Microbial interactions with tributyltin compounds: detoxification, accumulation and environmental fate. Sci. Total Environ. 258, 119–127. Gu, B., Chang, S.T., 2001. Soil biosensor for the detection of PAH toxicity using an immobilized recombinant bacterium and a biosurfactant. Biosens. Bioelectron. 16, 667–674. Guzzo, A., DuBow, M.S., 1991. Construction of a stable, single copy luciferase gene fusions in Escherichia coli. Arch. Microbiol. 156, 444–448. Guzzo, A., DuBow, M.S., 1994. Identification and characterization of genetically programmed responses to toxic metal exposure in Escherichia coli. FEMS Microbiol. Lett. 14, 369–374. Hastings, J.W., Potrikus, C.J., Gupta, S.C., Kurf€ urst, M., Makemson, J.C., 1985. Biochemistry and physiology of bioluminescent bacteria. Adv. Microb. Phys. 26, 235–291. Hastings, J.W., 1996. Chemistries and colors of bioluminescent reactions: a review. Gene 173, 5–11. Heitzer, A., Malachowsky, K., Thonnard, J.E., Bienkowski, P.R., White, D.C., Sayler, G.S., 1994. Optical biosensor for environmental on line monitoring of naphtalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium. Appl. Environ. Microbiol. 60, 1487–1494. Hoch, M., 2001. Organotin compounds in the environment: an overview. Appl. Geochem. 16, 719–743. Kobatake, E., Niimi, T., Haruyama, T., Ikariyama, Y., Aizawa, M., 1995. Biosensing of benzene derivatives in the environment by luminescent Escherichia coli. Biosens. Bioelectron. 10, 601–605. K€ ohler, S., Belkin, S., Schmid, R.D., 2000. Reporter gene bioassays in environmental analysis. Fresen. J. Anal. Chem. 366, 769–779. Meighen, E.A., 1999. Autoinduction of light emission in different species of bioluminescent bacteria. Luminescence 14, 3–9. Mergeay, M., Nies, D., Schlegel, H.G., Geritz, J., Charles, P., Van Gijsegem, F., 1985. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid bound resistance to heavy metals. J. Bact. 162, 328–334. Michel, P., Averty, B., 1999. Distribution and fate of tributyltin in surface and deep waters of the northwestern Mediterranean. Environ. Sci. Technol. 33, 2524–2528. Neilson, J.W., Pierce, S.A., Maier, R.M., 1999. Factors influencing expression of luxCDABE and nah genes in Pseudomonas putida RB1353(NAH7, pUTK9) in dynamic systems. Appl. Environ. Microbiol. 65, 3473–3482.

M.J. Durand et al. / Chemosphere 52 (2003) 103–111 Rasmussen, L.D., Sorensen, S.J., Turner, R.R., Barkay, T., 2000. Application of a merlux biosensor for estimating bioavailable mercury in soil. Soil Biol. Biochem. 32, 639–646. Rozen, Y., Nejidat, A., Gartemann, K.H., Belkin, S., 1999. Specific detection of p-chlorobenzoic acid by Escherichia coli bearing a plasmid borne fcbA::lux fusion. Chemosphere 38, 633–641. Rozen, Y., Belkin, S., 2001. Survival of enteric bacteria in seawater. FEMS Microbiol. Rev. 25, 513–529. Ruiz, J.M., Bachelet, P., Caumette, P., Donard, O.F.X., 1996. Three decades of tributyltin in the coastal environment with emphasis on Arcachon Bay, France. Environ. Pollut. 93, 195–203. Tauriainen, S., Karp, M., Chang, W., Virta, M., 1998. Luminescent bacterial sensor for cadmium and lead. Biosens. Bioelectron. 13, 931–938. Tauriainen, S.M., Virta, M.P.J., Karp, M., 2000. Detecting of bioavailable toxic metals and metalloids from natural water samples using luminescent sensor bacteria. Wat. Res. 34, 2661–2666. Thouand, G., Durand, M.J., Picart, P., Daniel, Ph., Mass
e, J., DuBow, M.S., 2001. Detection of bacteria in the food industry with bioluminescent sensors. Recent Res. Develop. Microbiol. 5, 79–93. Thouand, G., Daniel, Ph., Horry, H., Picart, P., Durand, M.J., Killham, K., Knox, O.G.G., DuBow, M.S., Rousseau, M., in press. Comparison of the spectral emission of lux recombinant and bioluminescent marine bacteria. Luminescence. Van Dyk, T.K., Majarian, W.R., Konstantinov, K.B., Young, R.M., Dhurjati, P.S., LaRossa, R.A., 1994. Rapid and sensitive pollutant detection by induction of heat shock gene bioluminescence gene fusions. Appl. Environ. Microbiol. 60, 1414–1420. Vollmer, A.C., Belkin, S., Smulski, D.R., Dyk, T.K., LaRossa, R.A., 1997. Detection of DNA damage by use of Escher-

111

ichia coli carrying recA0 ::lux, uvrA0 ::lux or alkA0 ::lux reporter plasmids. Appl. Environ. Microbiol. 63, 2566– 2571. Wagner, L.W., Van Dyk, T.K., 1998. Cryopreservation and reawakening. In: LaRossa, R.A. (Ed.), Bioluminescence Methods and Protocols. Humana Press, Totowa, New Jersey, pp. 123–127. White, J.S., Tobin, J.M., Cooney, J.J., 1999. Organotin compounds and their interactions with microorganisms. Can. J. Microbiol. 45, 541–554. Wuertz, S., Miller, C.E., Pfister, R.M., Cooney, J.J., 1991. Tributyltin-resistant bacteria from estuarine and freshwater sediments. Appl. Environ. Microbiol. 57, 2783–2789. Marie Jos
e Durand received her Ph.D. degree in Biology from the University of Metz in 1993. She is assistant Professor in Ecotoxicology at the University of Nantes (University for Technology, IUT, La Roche sur Yon, France). Her research interests includes environmental toxicology bioassay and risk assessment in marine and terrestrial environments. G
erald Thouand received his Ph.D. degree in Microbiology from the University of Nancy in 1993. After a Postdoctoral Fellowship in the VITO center (Belgium) in 1995, he became assistant Professor in Microbiology at the University of Nantes (University for Technology, IUT, La Roche sur Yon, France). His research interests includes environmental monitoring of biodegradation and biotechnology. He is leading a French governmental program for the development of biosensors for the detection of pollutants in environment and bacteria in food industry. Michael DuBow received his Ph.D. degree in Microbiology from Indiana University (USA) in 1977. After a Postdoctoral Fellowship at the Cold Spring Harbor Laboratory, he became a Professor in the Department of Microbiology and Immunology of McGill University (Montreal, Quebec, Canada). He is currently a Professor in the Institute of Genetics and Microbiology of the Universit
e Paris-Sud, Centre Scientifique dÕOrsay.