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soil environments. Applied and Environmental Microbiology 68, 3750–3758. Moreira, B.M., Leobons, M.B., Pellegrino, F.L., Santos, M., Teixeira, L.M., de Andrade Marques, E., Sampaio, J.L., Pessoa-Silva, C.L., 2005. Ralstonia pickettii and Burkholderia cepacia complex bloodstream infections related to infusion of contaminated water for injection. Journal of Hospital Infection 60, 51–55. Pirone, L., Chiarini, L., Dalmastri, L., Bevivino, A., Tabacchioni, S., 2005. Detection of cultured and uncultured Burkholderia cepacia complex bacteria naturally occurring in the maize rhizosphere. Environmental Microbiology 7, 1734–1742. Ramette, A., LiPuma, J.J., Tiedje, J.M., 2005. Species abundance and diversity of Burkholderia cepacia complex in the environment. Applied and Environmental Microbiology 71, 1193–1201. Reik, R., Spilker, T., LiPuma, J.J., 2005. Distribution of Burkholderia cepacia complex species among isolates recovered from persons with or without cystic fibrosis. Journal of Clinical Microbiology 43, 2926– 2928. Salles, J.F., van Veen, J.A., van Elsas, J.D., 2004. Multivariate analyses of Burkholderia species in soil: effect of crop and land use history. Applied and Environmental Microbiology 70, 4012–4020. Speert, D.P., Henry, D., Vandamme, P., Corey, M., Mahenthiralingam, E., 2002. Epidemiology of Burkholderia cepacia complex in patients with cystic fibrosis, Canada. Emerging Infectious Diseases 8, 181–187. Stryjewski, M.E., LiPuma, J.J., Messier Jr., R.H., Reller, L.B., Alexander, B.D., 2003. Sepsis, multiple organ failure, and death due to Pandoraea pnomenusa infection after lung transplantation. Journal of Clinical Microbiology 41, 2255–2257. Vandamme, P., Goris, J., Coenye, T., Hoste, B., Janssens, D., Kersters, K., De Vos, P., Falsen, E., 1999. Assignment of Centers for Disease

Control group IVc-2 to the genus Ralstonia as Ralstonia paucula sp. nov. International Journal of Systematic Bacteriology 49, 663– 669. Vandamme, P., Henry, D., Coenye, T., Nzula, S., Vancanneyt, M., LiPuma, J.J., Speert, D.P., Govan, J.R.W., Mahenthiralingam, E., 2002. Burkholderia anthina sp. nov. and Burkholderia pyrrocinia, two additional Burkholderia cepacia complex bacteria, may confound test results of new molecular diagnostic tools. FEMS Immunology and Medical Microbiology 33, 143–149. Vandamme, P., Holmes, B., Coenye, T., Mahenthiralingam, E., LiPuma, J.J., Govan, J.R.W., 2003. Burkholderia cenocepacia sp. nov. a new twist of an old story. Research in Microbiology 154, 91–96. Vanlaere, E., Coenye, T., Samyn, E., Van den Plas, C., Govan, J., De Baets, F., De Boeck, K., Knoop, C., Vandamme, P., 2005. A novel strategy for the isolation and identification of environmental Burkholderia cepacia complex bacteria. FEMS Microbiology Letters 249, 303–307. Vermis, K., Coenye, T., LiPuma, J.J., Mahenthiralingam, E., Nelis, H.J., Vandamme, P., 2004. Proposal to accommodate Burkholderia cepacia genomovar VI as Burkholderia dolosa sp. nov. International Journal of Systematic and Evolutionary Microbiology 54, 689–691. Wigley, P., Burton, NF., 1999. Genotypic and phenotypic relationships in Burkholderia cepacia isolated from cystic fibrosis patients and the environment. Journal of Applied Microbiology 86, 460–468. Yabuuchi, E., Kosako, Y., Yano, I., Hotta, H., Nishiuchi, Y., 1995. Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov.: proposal of Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb., nov. and Ralstonia eutropha (Davis 1969) comb. nov. Microbiology and Immunology 39, 897–904.

0025-326X/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2007.01.019

The use of a novel Vibrio harveyi luminescence mutagenicity assay in testing marine water for the presence of mutagenic pollution Beata Podgo´rska a, Ksenia Pazdro a, Janusz Pempkowiak a, Grzegorz We˛grzyn a

Institute of Oceanology, Polish Academy of Sciences, Powstan´co´w Warszawy 55, 81-712 Sopot, Poland b Department of Molecular Biology, University of Gdan´sk, Kładki 24, 80-822 Gdan´sk, Poland

1. Introduction Mutagenic chemicals occur in various habitats, including marine waters, and can induce serious diseases, including cancer, due to their genotoxic (mutagenic) activities (El-Bayoumy, 1992; Depledge, 1998; Au et al., 2001; Martin, 2001; Tornqvist and Ehrenberg, 2001; Barton et al., 2005). The germ line of higher organisms may be also affected by these compounds, which may lead to fertility problems and to negative genetic changes in future genera-

*

a,b,*

Corresponding author. Tel.: +48 58 523 6308; fax: +48 58 523 5501. E-mail address: [email protected] (G. We˛grzyn).

tions (Shelby et al., 1993). Currently, mutagenic pollutants appear in the marine environment mostly as side effects or by-products of industrial processes (Heddle et al., 1999; Goldman and Shields, 2003; Vargas, 2003; Jha, 2004). Detection of mutagenic pollutants in the environment is important, however, the procedures used for this purpose are problematic. Because there are thousands of known mutagens that occur in natural habitats at very low concentrations, there are no simple chemical procedures which might be employed for testing the presence of such compounds. On the other hand, mutagens usually reveal genotoxic effects at very low concentrations. Therefore, it appears that for preliminary and rapid detection of mutagenic activities in environmental samples, biological assays

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are more useful than chemical analyses. Among them, microbiological tests are commonly used as they are relatively simple (We˛grzyn and Czy_z, 2003). However, there are also some problems with the commonly used microbiological mutagenicity assays. The Ames test is simple and sensitive, but to obtain results of measurements, one must usually wait as long as two days since sample withdrawal (Mortelmans and Zeiger, 2000). Moreover, survival of Salmonella enterica serovar Typhimurium strains (used in the Ames test) in marine water is poor (Czy_z et al., 2002), which makes the use of this test for assessing marine water samples problematic. The latter problem has been solved by development of a test similar to that of the Ames test, but in which strains of a marine bacterium Vibrio harveyi are used (Czy_z et al., 2000, 2002, 2003; Podgo´rska et al., 2005). Nevertheless, similarly to the Ames test, 48 h is necessary to obtain results, which may be a too long period when rapid assessment of mutagenicity is necessary. More rapid tests, based on bacterial bioluminescence have been developed, including commercially available Mutatox (in which a strain of Vibrio fischeri is used), which requires 16–24 h to obtain results (Ulitzur et al., 1980; Ulit-

809

zur and Weiser, 1981). Recently, a novel bioluminescence mutagenicity assay has been described, which allows to obtain results in 2–4 h from sample withdrawal (Podgo´rska and We˛grzyn, 2006). In this assay, a dim luxE mutant of Vibrio harveyi is employed, and cultures of this strain are induced for light emission upon contact with mutagenic compounds. This assay was demonstrated to work under laboratory conditions, when known amounts of known mutagens were added (Podgo´rska and We˛grzyn, 2006). Therefore, we asked whether this test may be useful for detection of mutagens in samples from natural marine environment. 2. Materials and methods 2.1. Environmental samples of marine water Samples of marine water were collected from Baltic Sea, at stations depicted in Fig. 1 and described in Table 1. For the bioluminescence mutagenicity assay (Section 2.2), water samples (9 ml) were filtered through 0.22 lm bacteriological filters (Osmonics). For the chemical analysis, samples (80–120 l) were prepared as described in Section 2.3.

Fig. 1. A map of the region (Southern Baltic Sea, Gulf of Gdan´sk) from which samples were collected. Numbers correspond to numbers of samples and stations. A map of the Baltic Sea region is shown in upper right corner, with a square indicating a part of the map, which is enlarged.

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Table 1 Stations at which marine water samples were collected (for a map see Fig. 1) Sample/ station

Localization and characteristics

C 1 2 3 4

Artificial marine water Hel Peninsula, open sea side Hel Peninsula, fishery port Puck, sandy beach near a town Gdan´sk (Go´rki Wschodnie), Biological Station of the University of Gdan´sk Gdan´sk (Go´rki Wschodnie), Biological Station of the University of Gdan´sk Gdan´sk (Go´rki Wschodnie), estuary of Vistula River Gdan´sk (Go´rki Wschodnie), estuary lake Gdan´sk (Sobieszewo), estuary of Vistula River Gdan´sk (Sobieszewo), estuary of Vistula River near a bridge (industrial pollution area) Gdan´sk (S´wibno), fishery port Gdan´sk, mouth of Vistula River Gdan´sk, Vistula River Sopot, Swelina River Sopot, Kamienny Potok Stream Gdan´sk (Brzez´no), a pier (tourist area) Gdynia, city boulevard Gdynia, city boulevard Gdynia, city boulevard Gdynia (Orłowo), Kolibkowski Potok Stream Gdynia (Orłowo), mouth of Kacza River Gdynia (Oksywie), navy port Gdynia, Gulf of Gdan´sk, close to navy harbour and shipping channel, near bottom water Gdynia, Gulf of Gdan´sk, close to navy harbour and shipping channel Gdynia, trade port Gdynia, trade port Gdynia, yacht basin Gdynia, yacht basin Gdynia, yacht basin Gdynia, yacht basin Gdynia, yacht basin Gdynia, yacht basin Gdynia, yacht basin Gdynia, yacht basin Mechelinki, close to sewage outflow from the city of Gdynia

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

The samples for both kinds of analysis were collected either using an in situ pumping system (during r/v ‘‘Oceania’’ cruises) or with a stainless can. Each glassware was washed, rinsed with Milli-Q water and heated for 10 h at 400 C or rinsed with a solvent before use. The HPLC grade solvents were used to prepare the extracts. The glass containers were stored in the dark at 4 C for no longer than 5 days. 2.2. Vibrio harveyi bioluminescence mutagenicity assay The assay was performed according to Podgo´rska and We˛grzyn (2006), with the V. harveyi A16 strain (a dim luxE mutant) as an indicator strain. Bacteria were grown in the liquid BOSS nutrient medium (bacto-peptone, 1%; beef extract, 0.3%; glycerol, 0.011 mol l1; NaCl, 0.51 mol l1), described by Klein et al. (1995), at 30 C. Culture (10 ml)

of OD575 = 0.1 was centrifuged and the pellet was resuspended in 9 ml of the filtered water from an environmental sample. Then, 1 ml of the fresh, 10· concentrated BOSS medium was added together with 5 ll of 4% S9 mix (consisting of rat liver microsomal enzymes and cofactors, as described previously by Maron and Ames, 1983). Bacteria were incubated in a water bath shaker for 4 h at 30 C. Bacterial culture (1 ml) was withdrawn, and its luminescene was measured using a Sirius luminometer (Berthold) and expressed as relative light units (RLU). At the same time, A575 of cultures was measured to estimate number of bacterial cells (a correlation between cell number, determined by plating on BOSS plates, and absorbance of the culture was established according to Podgo´rska and We˛grzyn (2006); A575 = 0.1 corresponded to 3 · 107 colony forming units per 1 ml). Results were calculated as relative luminescence (RLU) per cell or per absorbance unit. In control experiments, artificial marine water (MacLeod et al., 1954) was used instead of environmental samples. 2.3. Chemical analysis of marine water samples Selected samples were used for determination of levels of various chemicals (polycyclic aromatic hydrocarbons (PAHs); polychlorinated biphenyls (PCBs); hexachlorocyclohexanes (HCHs), including lindane (c-HCH); and hexachlorobenzene (HCB)), which are potentially mutagenic. Following collection into glass containers, the water was passed through a filtering system with precombusted (450 C, 4 h) GF/F (0.7 lm cut-off) glass fibre filters. Immediately after filtering, the internal standard (octachloronapthalene) was added and the water was passed at a rate of 50–80 ml min1 through teflon columns filled with precleaned Amberlite XAD-2 resin. After passing water, the resin was dried and the adsorbed organics were eluted with methylene chloride. Traces of water were removed with anhydrous Na2SO4 while methylene chloride was replaced with hexane as a solvent, according to Dachs and Bayona (1997). The extracts were concentrated and subjected to clean-up procedures according to Behar et al. (1989), Pazdro (2004), and Tronczyn´ski et al. (2004). Briefly, following removal of elemental sulfur by a treatment with activated powdered copper, silica/alumina columns and solvent mixtures increasing in polarity were used (F1–100% hexane, extracting HCB and PCBs, F2– 90% hexane: 10% methylene chloride, extracting PAHs, and F3–75% hexane:25% methylene chloride, extracting HCHs). The fractions were evaporated and prior to final analysis, dissolved in isooctane. Extracts were analysed by gas capillary chromatography. A Shimadzu 17A GC equipped with a split/splitless injector at 280 C and DB 5 column (60 m · 0.25 mm inner diameter · 0.25 lm film thickness) was used. Flame ionization detector (FID), helium (a carrier gas) and following oven temperatures were used for PAHs analyses: 50 C held for 1 min, followed by 15 C/min increase to 150 C, followed by 30 C/min increase to 310 C, held for 10 min. PCBs and

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respect to individual compounds. The detection limits for individual PCBs, HCHs and HCB, calculated as average blanks plus three standard deviation of the blanks, were at the level of 1 pg l1; 2 pg l1 and 0.5 pg l1, respectively.

organochlorine pesticides (HCB and lindane) were analyzed by applying an electron capture detector (ECD), helium (a carrier gas) and the following oven temperature program: 100 C held for 1 min; 6 C/min to 140 C; 10 C/min to 310 C, held for 20 min. Identifications were performed by means of internal and external standards (LG PROMOCHEM). The individual compounds were quantified by using external five-point calibration curves in the linear range of the response of the detector. The QA/QC procedures included field and procedural blanks, control samples, analyses of replicate samples and the use of internal recovery standards added to each sample prior to extraction. Field blank for each sampling period consisted of 25 L of Milli-Q water in glass container, exposed to the ambient environment during the course of field operation. Procedural samples for each set of samples were prepared by passing the solvent (dichloromethane) through a clean XAD-2 column. Control samples were prepared by spiking Milli-Q water or artificial seawater (S = 7 psu) with known amounts of certified standards (7 PCBs, 16 PAHs, HCB, lindane). Field, procedural blanks and control samples were processed in the same manner as real samples. The field and procedural blanks contained no detectable amounts of target analyses, except for low molecular PAHs (naphthalene, acenapthene and acenapthylene). The identification and quantification was checked by analysing selected extracts using gas chromatography/mass spectrometry techniques in selected ion monitoring mode (HP 5890/ MS HP 5972). Analyses of certified material – IAEA-383 were also used to monitor the efficiency of clean-up procedures. Recoveries in the range of 70–99% and relative standard deviation in the range of 9–22% characterized the method in

2.4. Statistical analysis Statistical analyses were carried out using Statistica for Windows 5.1. The dependence between results obtained in the mutagenicity assay and results of chemical analyses was investigated by fitting linear equations to the original data sets and investigating the Pearson linear correlation (r) and coefficients of determination (R2) values. The level of significance was set at p < 0.05. 3. Results 3.1. Analysis of water samples using the V. harveyi bioluminescence mutagenicity assay Samples of marine water were collected from different habitats (Fig. 1, Table 1) and tested for their mutagenicity. Various signals were detected when measuring luminescence after exposure of the tester bacteria to these samples (Fig. 2). The most intensive luminescence signals (suggesting the presence of relatively high concentrations of mutagens) were detected in samples from stations located close to industrial regions (compare Table 1 and Fig. 2). In a few samples (samples no. 10, 13, 14, 20), extremely low luminescence signals, considerably lower than those measured in a negative control, were observed. We found that this was caused by inhibition of growth of bacterial cultures and a decrease in number of viable (able to form

14

Relative luminescence (RLU)

12

10

8

6

4

2

0 C

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31 32 33 34

sample

Fig. 2. Detection of mutagenic pollution in marine sea water samples. Samples (Table 1) were collected and tested for their mutagenicity using the V. harveyi bioluminescence mutagenicity assay as described in Materials and methods. Average values from three measurements with error bars representing SD are presented. Value = 1 corresponds to 0.0125 ± 0.0022 RLU/cell.

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Table 2 Chemical analysis of water samples (for characteristics of the samples see Table 1) Compounda

CB28 CB52 CB101 CB118 CB138 CB153 CB180 HCB aHCH cHCH F P A Fl Py BAA Chr BbFl BkFl BaPy DBA BghiPe Ipy

Concentration (ng l1) in particular samplesb 1

11

12

22

23

27

30

32

34

0.010 0.030 0.027 0.042 0.015 0.021 0.006 0.008 NT 0.113 1.97 1.62 1.22 7.78 1.69 0.67 1.50 5.76 2.44 0.79 BDL BDL BDL

0.012 0.015 0.067 0.098 0.078 0.079 0.056 0.002 0.866 0.096 5.95 6.59 2.02 38.24 10.31 13.47 3.59 14.26 17.64 21.09 6.52 17.21 0.25

0.297 0.365 0.570 1.161 0.676 0.654 0.504 0.009 0.938 0.145 21.65 78.70 23.13 46.76 6.86 15.32 11.39 22.65 21.16 16.44 15.46 10.72 9.08

1.070 0.500 0.440 0.005 BDL BDL BDL NT NT 0.383 30.54 101.00 11.11 74.20 10.13 4.94 2.11 8.09 1.70 17.35 1.24 1.55 BDL

0.190 0.040 0.030 BDL BDL BDL BDL 0.068 NT 0.188 17.87 58.00 4.41 58.10 12.50 2.73 1.13 4.92 1.10 0.73 2.79 0.82 1.38

0.064 0.060 0.064 0.030 0.049 0.067 0.015 0.044 NT 0.079 0.13 1.34 0.71 1.86 0.55 0.21 0.16 2.32 0.34 0.20 BDL BDL BDL

0.389 0.253 0.185 0.062 0.139 0.107 0.044 0.049 NT 0.019 0.37 0.41 3.06 2.09 0.51 0.45 0.26 8.31 3.54 8.68 BDL BDL BDL

0.015 0.035 0.081 0.017 0.023 0.026 0.007 0.050 0.135 0.494 1.55 1.46 2.41 10.80 2.73 4.07 2.84 17.25 8.66 5.54 5.81 5.64 0.29

0.113 0.115 0.127 0.026 0.026 0.024 0.009 0.068 1.200 0.597 1.11 4.97 1.98 26.68 7.35 2.99 1.01 3.98 6.47 1.06 3.89 BDL BDL

a Abbreviations: CB28 – 2,4,4 0 -trichlorobiphenyl; CB52- 2,2 0 ,5,5 0 -tetrachlorobiphenyl; CB101 – 2,2 0 ,4,5,5 0 -pentachlorobiphenyl; CB118 - 2,3 0 ,4,4 0 ,5 0 pentachlorobiphenyl; CB138 – 2,2 0 ,3,4,4 0 ,5 0 -hexachlorobiphenyl; CB153 – 2,2 0 ,4,4 0 ,5,5 0 -hexachlorobiphenyl; CB180 – 2,2 0 ,3,4,4 0 ,5,5 0 -heptachlorbiphenyl; HCB, hexachlorobenzene; aHCH – a hexachlorocyclohexane; cHCH – c hexachlorocyclohexane; F – fluorene; P – phenanthrene, A – anthracene; Fl – fluoranthene; Py – pyrene; BAA – benzo(a)anthracene; Chr – chrysene; BbFl – benzo(b)fluoranthene; BkFl – benzo(k)fluoranthene; BaPy – benzo(a)pyrene; DBA – dibenzo(a,h)anthracene; BghiPe – benzo(g,h,i)perylene; Ipy – indeno(1,2,3-c,d)pyrene. b Abbreviations: NT – not determined; BDL – below the detection limit. Numbers of samples are the same as in Table 1 and Figs. 1 and 2.

colonies) bacterial cells rather than by an extremely low mutagenicity of the samples (data not shown). Apparently, the deleterious effect of the samples on tester bacteria was caused by their toxicity to V. harveyi A16 cells, according to the explanation proposed previously for impaired viabil-

ity of these bacteria in the presence of very high levels of mutagens (Podgo´rska and We˛grzyn, 2006). Other samples induced various levels of luminescence of V. harveyi A16 cells, suggesting the presence of various amounts of mutagenic compounds (Fig. 2).

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Relative luminescence (RLU)

10

8

6 S 13 PAH

RLU = 0.022*[S 13 PAH] + 4.591 R2 = 0.448

S 7 PCB*100

RLU = 0.013*[S 7PCB*100] + 5.829 R2 = 0.251

BaPy*10

RLU = 0.031*[BaPy] + 4.728 R2 = 0,525

4

2

0 0

50

100

150

200

250

300

350

400

450

Chemical compound concentration (ng L-1) Fig. 3. The relationship between V. harveyi luminescence and concentration of organic contaminants detected in water samples from the Gulf of Gdan´sk (Southern Baltic).

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3.2. Chemical analysis of water samples Selected marine water samples that were found to be either highly mutagenic (samples 11, 12, 22, 23, 27, 30, 32) or of a low mutagenicity (i.e., inducing luminescence comparable to or slightly higher than the control; samples 1, 34) in the V. harveyi bioluminescence assay were tested for the presence of some mutagenic chemicals. Since no information about a chemical nature of mutagens present in these samples was available (which is common in assessment of mutagenic pollution of marine water), presence of only selected groups of chemicals was tested. In each water sample revealing a high luminescence signal in the mutagenicity assay, a relatively high concentration of at least one of potential mutagens could be determined by chemical analysis (Table 2), indicating contamination of the site with the analysed mutagens and, possibly, with other genotoxic compounds. These results corroborate the conclusion about mutagenic contamination of the tested marine water samples. 4. Discussion In this paper, we demonstrate that the recently developed V. harveyi bioluminescence mutagenicity assay (Podgo´rska and We˛grzyn, 2006) may be useful in quick detection of mutagenic pollution of marine waters. Previously, this assay was used only to detect known amounts of mutagens added to a bacteriological medium under laboratory conditions. The presence of increased levels of mutagenic compounds in samples giving the highest signals in the microbiological assay was confirmed by chemical analyses. To establish statistical significance of relationships between the chemical and microbiological indicators of contamination level, correlation factors of the linear dependence of individual contaminants and the luminescence intensity were tested. The major conclusion is that the strongest, statistically significant correlation was found between the V. harveyi luminescence and the concentration of benzo(a)pyrene – a potent mutagenic and carcinogenic agent (Knutzen, 1995; White, 1986). As much as 47% of V. harveyi luminescence changes is caused by the variation in the benzo(a)pyrene concentration (Fig. 3). In the case of other contaminants tested, no statistically significant dependences were found. This can be attributed to one or combination of factors including: limited mutagenic activity, limited concentration, quenching by other factors (as discussed by Podgo´rska and We˛grzyn, 2006). When the analyzed contaminants were grouped into classes (R7 PCB and R13 PAH), no dependence between bacterial luminescence and contaminant concentrations was detected. This is not surprising, since both RPCBs and RPAHs comprise compounds differing strongly in mutagenicity (White, 1986). Obviously, although the chemical analysis of water samples (Table 2; Fig. 3) demonstrated the presence of various

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mutagenic compounds, it did not exclude the presence of other chemical mutagens in these samples. Nevertheless, it indicates that chemical and biological assays used in this work are compatible. The highest signals in the mutagenicity assay were detected in samples from the Vistula River (known to be industrially polluted), a marina port (in which a high pollution level might be expected) and in samples of near bottom water from the station located close to the navy harbour and the shipping channel (a habitat of a high risk of pollution). In contrast, very low luminescence was induced in samples from a relatively clean stream (Potok Kolibkowski) and from an open sea station at Hel peninsula. A water sample from the station located geographically close to the latter one but in a fishery port (suspected to be polluted), induced high mutagenicity signals (compare samples 1 and 2 in Fig. 2). A few water samples caused inhibition of growth of bacterial cultures and a decrease in the number of colony forming units. In these samples, luminescence was extremely low, which was apparently caused by toxicity of compounds present in the water. This problem has been discussed previously by Podgo´rska and We˛grzyn (2006), when inhibition of bacterial growth was observed in the presence of very high concentrations of mutagenic compounds, and such an explanation has been proposed. In summary, results presented in this paper indicate that the V. harveyi luminescence mutagenicity assay is suitable for rapid preliminary detection of mutagenic pollution of marine water. Acknowledgements This work was supported by the Ministry of Science (Project Grant No. 2 P04G 011 26 to B.P.) and by the Institute of Oceanology of the Polish Academy of Sciences (task Grant No. IV.3.2. to G.W.). References Au, W.W., Oberheitmann, B., Heo, M.Y., Hoffmann, W., Oh, H.Y., 2001. Biomarker monitoring for health risk based on sensitivity to environmental mutagens. Rev. Environ. Health 16, 41–64. Barton, H.A., Cogliano, V.J., Flowers, L., Valcovic, L., Setzer, R.W., Woodruff, T.J., 2005. Assessing susceptibility from early-life exposure to carcinogens. Environ. Health. Perspect. 113, 1125–1133. Behar, F., Leblond, C., Saint-Paul, C., 1989. Analyse quantitative des effluents de pyrolyse en milieu ouvert et ferme. Revue de l’Institut Franc¸ais du Petrole 44, 387–411. Czy_z, A., Jasiecki, J., Bogdan, A., Szpilewska, H., We˛grzyn, G., 2000. Genetically modified Vibrio harveyi strains as potential bioindicators of mutagenic pollution of marine environments. Appl. Environ. Microbiol. 66, 599–605. Czy_z, A., Szpilewska, H., Dutkiewicz, R., Kowalska, W., BiniewskaGodlewska, A., We˛grzyn, G., 2002. Comparison of the Ames test and a newly developed assay for detection of mutagenic pollution of marine environments. Mutat. Res. 519, 67–74. Czy_z, A., Kowalska, W., We˛grzyn, G., 2003. Vibrio harveyi mutagenicity assay as a preliminary test for detection of mutagenic pollution

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0025-326X/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2006.12.015

Air–water distribution of hexachlorobenzene and 4,4 0 -DDE along a North–South Atlantic transect Kees Booij b

a,*

, Ronald van Bommel a, Kevin C. Jones b, Jonathan L. Barber

a Royal Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB Texel, The Netherlands Centre for Chemical Management and Department of Environmental Science, Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UK

Concentration data for organic contaminants in remote areas are scarce, particularly for open ocean systems. These data are much needed for the global fate assessment of these compounds, both for water and air. The traditional methods for determining these concentrations, such as high-volume air and water sampling with polyurethane foam or XAD are labour-intensive and – in the case of batch water sampling – require much station time at sea, which makes these methods costly as well. Passive sampling

*

b

Corresponding author. Tel.: +31 222 369 463; fax: +31 222 319 674. E-mail address: [email protected] (K. Booij).

methods like semipermeable membrane devices (SPMDs, Huckins et al., 1990, 2006) have been used as an alternative to the high-volume extraction methods in air (Ockenden et al., 1998; Lohmann et al., 2001; Meijer et al., 2003) and in water (Crunkilton and DeVita, 1997; Luellen and Shea, 2002; Verweij et al., 2004). A ship-based deployment of these samplers during transit time could yield valuable concentration data with little effort and at low cost. However, the sampling rates of SPMDs are in the range 1– 20 m3 d1 for air, and 1–200 L d1 for water, depending on the physicochemical properties of the analytes and on the exposure temperature and flow velocity (Huckins et al., 2006). The very low concentrations in open ocean