Kaouthar Kessabi

Anna Navarro

Marta Casado

Khaled Saïd

Imed Messaoudi

Benjamin Piña

EVALUATION OF ENVIRONMENTAL IMPACT ON NATURAL POPULATIONS OF THE MEDITERRANEAN KILLIFISH Aphanius fasciatus BY QUANTITATIVE RNA BIOMARKERS

Keywords: Cyprinodontiformes, brackish waters, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), pollution markers, mRNA quantitation, gene expression, Western Mediterranean Sea, sentinel species

The Mediterranean killifish, Aphanius fasciatus (Valenciennes, 1821) represents an excellent sentinel species for analysis of environmental impact in coastal areas.

Quantitative changes on liver mRNA levels of five stress-related genesmetallothionein (metal exposure), vitellogenin, (estrogenic effects), cytochrome P4501A (CYP1A, dioxin-like compounds), superoxide dismutase (oxidative stress) and HSP70 (general stress)-were tested as markers of exposure to pollutants both in natural populations and in experimental treatments with model effectors. Analysis of fish from a metal-contaminated site in the Tunisian coast (Sfax) indicated that fish presenting spinal deformities (eight times more frequent at this site than in reference sites) showed increased levels of hepatic CYP1A mRNA levels compared to non-deformed fish from the same site or from the reference Louza site, suggesting a combined impact by metals and organic pollutants. Due to the strategy used in their design, these quantitative RNA markers will likely be useful to analyze environmental impacts on other related fish species.

Introduction

The Mediterranean killifish, Aphanius fasciatus (Valenciennes, 1821) is a small cyprinodont fish inhabiting brackish waters in coastal habitats of the central and eastern Mediterranean Sea. It tolerates a wide range of physico-chemical parameters, such as temperature (5-39ºC) and salinity (0-180 g/L). Although migration of individuals from one habitat to another is possible, it is considered a non-migratory species, as populations from relatively close habitats show evidences for genetic isolation [START_REF] Triantafyllidis | Phylogeography and genetic structure of the Mediterranean killifish Aphanius fasciatus (Cyprinodontidae)[END_REF] and references therein). Therefore, A. fasciatus represents an excellent sentinel species for analysis of environmental impact in coastal lagoons, estuaries and river deltas in the highly anthropized Mediterranean Sea.

The use of small animals as sentinels for environmental pollution has to overcome the difficulty of obtaining enough biological material for biochemical analyses. In this context, markers based on RNA quantification methods, such qRT-PCR, provide a suitable alternative, as they only require minute amounts of sample [START_REF] Piña | Analysis of gene expression as a new tool in ecotoxicology and environmental monitoring[END_REF][START_REF] Rotchell | Molecular markers of endocrine disruption in aquatic organisms[END_REF][START_REF] Valasek | The power of real-time PCR[END_REF]. These techniques have proved their usefulness for monitoring of different types of environmental pollutants, like estrogens [START_REF] Barucca | Analysis of vitellogenin gene induction as a valuable biomarker of estrogenic exposure in various Mediterranean fish species[END_REF][START_REF] Garcia-Reyero | Use of vitellogenin mRNA as a biomarker for endocrine disruption in feral and cultured fish[END_REF][START_REF] George | A comparison of pollutant biomarker responses with transcriptional responses in European flounders (Platicthys flesus) subjected to estuarine pollution[END_REF], metals [START_REF] Cheung | Regulation of Tilapia metallothionein gene expression by heavy metal ions[END_REF][START_REF] Navarro | Physiological responses to mercury in feral carp populations inhabiting the low Ebro River (NE Spain), a historically contaminated site[END_REF][START_REF] Tom | Quantifying fish metallothionein transcript by real time PCR for its utilization as an environmental biomarker[END_REF], and organic substances [START_REF] Eljarrat | Distribution and biological impact of dioxin-like compounds in risk zones along the Ebro River basin (Spain)[END_REF]Quirós et al., 2007a;Quirós et al., 2007b). It is clear that biomarkers based on mRNA levels only reflect the initial steps of a cascade of events linking environmental insults to ecological impact [START_REF] Van Der Oost | Fish bioaccumulation and biomarkers in environmental risk assessment: a review[END_REF]. However, they are very useful when the specific nature of the pollutants (or of their primary effects) is unknown, or when dealing with very small animals or tissue portions [START_REF] Piña | Analysis of gene expression as a new tool in ecotoxicology and environmental monitoring[END_REF]. However, gene expression results should be further supported by parallel biochemical, immunological and/or morphological/pathological data, when possible.

The main disadvantage on the use of mRNA markers for environmental monitoring is the requirement for the sequence of test and reference genes in the impacted species. In this work, we develop mRNA markers for monitoring A. fasciatus exposure to different kinds of pollutants, using known DNA sequences from evolutionarily related species (mainly Cyprinodonts and Percomorphs). Genes used in this analysis included the main metal-responsive metallothionein (MT) gene [START_REF] Cheung | Regulation of Tilapia metallothionein gene expression by heavy metal ions[END_REF][START_REF] Navarro | Physiological responses to mercury in feral carp populations inhabiting the low Ebro River (NE Spain), a historically contaminated site[END_REF][START_REF] Schlenk | Expression of hepatic metallothionein messenger RNA in feral and caged fish species correlates with muscle mercury levels[END_REF][START_REF] Tom | Quantifying fish metallothionein transcript by real time PCR for its utilization as an environmental biomarker[END_REF][START_REF] Van Cleef-Toedt | Killifish metallothionein messenger RNA expression following temperature perturbation and cadmium exposure[END_REF], the egg-forming, liver-expressed vitellogenin (Vtg), a known marker for estrogens [START_REF] Barucca | Analysis of vitellogenin gene induction as a valuable biomarker of estrogenic exposure in various Mediterranean fish species[END_REF][START_REF] Denslow | Multiple responses in gene expression in fish treated with estrogen[END_REF][START_REF] Garcia-Reyero | Use of vitellogenin mRNA as a biomarker for endocrine disruption in feral and cultured fish[END_REF], the dioxin-like responsive Cytochrome P4501A (CYP1A, [START_REF] Chung-Davidson | betanaphthoflavone induction of CYP1A in brain of juvenile lake trout (Salvelinus namaycush Walbaum)[END_REF][START_REF] Craft | Measurement of cytochrome P4501A induction in dab (Limanda limanda) and other teleosts with species-specific cDNA probes: isolation and characterisation of dab cDNA and its use in expression studies with beta-naphthoflavone-treated fish[END_REF]Quirós et al., 2007a;Quirós et al., 2007b), the oxidative stress-related gene Superoxide dismutase (SOD, [START_REF] Cho | Differential expression of Cu/Zn superoxide dismutase mRNA during exposures to heavy metals in rockbream (Oplegnathus fasciatus)[END_REF][START_REF] Gonzalez | Comparative effects of direct cadmium contamination on gene expression in gills, liver, skeletal muscles and brain of the zebrafish (Danio rerio)[END_REF][START_REF] Pandey | Biomarkers of oxidative stress: a comparative study of river Yamuna fish Wallago attu (Bl. & Schn.)[END_REF], and the protein chaperone HSP70, which are involved in the processing of missfolded proteins due to different kinds of stress [START_REF] Carnevali | Exposure to xenobiotic compounds: looking for new biomarkers[END_REF][START_REF] Maradonna | Vitellogenin, zona radiata protein, cathepsin D and heat shock protein 70 as biomarkers of response to xenobiotics[END_REF][START_REF] Schroder | Correlation between the level of the potential biomarker, heat-shock protein, and the occurrence of DNA damage in the dab, Limanda limanda: a field study in the North Sea and the English Channel[END_REF][START_REF] Varo | Bioaccumulation of chlorpyrifos through an experimental food chain: Study of protein HSP70 as biomarker of sublethal stress in fish[END_REF]. In addition, probes for two housekeeping genes (ß actin and the elongation translation factor EF1) were also designed and tested for suitability as reference genes [START_REF] Olsvik | Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon[END_REF].

These quantitative RNA markers were used to monitor environmental impact in two natural A. fasciatus populations from the coast of Tunisia, one close to the industrialized coast of Sfax and a second one in the undisturbed Luza area (Kessabi et al., 2009;Messaoudi et al., 2009b). The Sfax population presents eight times higher incidence of spinal deformities (including scoliosis, lordosis, and kyphosis from the head to the caudal fin) than reference sites [START_REF] Kessabi | Involvement of Cd Bioaccumulation in Spinal Deformities Occurrence in Natural Populations of Mediterranean Killifish[END_REF]Messaoudi et al., 2009b). Deformed fish from Sfax accumulate more Cd than the non-deformed ones, suggesting a direct correlation between Cd accumulation and susceptibility to spinal deformations.

The purpose of this work is to develop and analyze mRNA markers of pollutant exposure in A. fasciatus, and use them to explain the high proportion of deformed fish in the Sfax population. We consider that this may be a general approach to study environmental impacts in natural populations of nonstandard species for which no appropriate DNA sequences are available.

2.Materials and Methods

Fish sampling

Sampling sites were selected in the Gulf of Gabès, in the South Eastern coast of Tunisia (Fig. 1). The industrialized coast of Sfax (S1), surrounded by important industrial activities, mainly crude phosphate treatments and chemical industries, and contaminated with Cd and other heavy metals, was chosen as a polluted site. The coast of Luza (S2), located 50 km north of S1 and apparently unaffected by human activities, was used as reference site.

A. fasciatus specimens were captured in coastal waters (0.5-1m depth) by hand nets during May 2009, anesthetized in ice and its length and weight measured.

Condition factor was calculated as the ratio of weight in grams to the cube of the length in cm X 100. Sex determination was performed by visual inspection of the bands along the body (colour pattern). As the number of male specimens captured in the current sampling effort was very low (a total of 3 fish), the study was performed using only females. Both normal (8 animals from Luza and 8 animals from Sfax) and deformed fish (10 animals from Sfax) were dissected and liver samples were placed in a cryogenic vial with 1 mL RNAlater (Sigma-Aldrich, St. Louis, MO), transported to the laboratory on ice, and stored at -80ºC for mRNA analysis.

Induction experiments

Normal Aphanius specimens (females, mean total length 3.61 ± 0.61 cm; mean body weight 0.54 ± 0.42 g) were captured from the reference Luza area (S2). The specimens were placed in 15L fish tanks in groups of 10, and acclimatized for 2 weeks in clean seawater under natural conditions of photoperiod and temperature and fed once per day with tetramin. Treatments with cadmium (10mg/L), estradiol (100nM) and beta naphthoflavone (1µM) were performed by adding appropriate amounts of each compound in water. No mortality occurred during exposure. Animals (10 fish per time and treatment) were sacrificed after 0h, 24h and 48 h of exposure, and liver samples were removed and stored in RNA later.

Sequence analysis and primer design

DNA sequences from ß Actin, elongation factor 1 (EF1), metallothionein (MT), Vitellogenin (Vtg) Cytochrome P4501A (CYP1A), Superoxide dismutase (SOD), and heat-shock protein (HSP70) were obtained using the SRS tool from the European Bioinformatics Institute (http://www.ebi.ac.uk/). Conserved sequence regions were derived from the alignment of known Cyprinodonta sequences when available, and supplemented with Percomorpha sequences when necessary, using the EMBOSS EMMA tool [START_REF] Rice | EMBOSS: the European Molecular Biology Open Software Suite[END_REF]. Gene specific primer sets were designed from consensus sequences using the Primer Express 2.0 software (Applied Biosystems, Foster City, CA)

RNA extraction, sequencing, and qRT-PCR analysis

Total RNA was isolated from the tissues using Trizol reagent ® protocol (Invitrogen).

RNA concentration was measured by spectrophotometric absorption at 260nm in a NanoDrop ND-8000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE) and the quality checked by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA). RNA was treated with DNAseI to remove genomic DNA contamination.

Primers for seven genes were deduced from existing sequences in other cyprinodonts (ß Actin, EF1, MT, Vtg, CYP1A, SOD and HSP70) and used to amplify the corresponding cDNA fragments. The PCR products (amplicons) were sequenced in an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems), and searched for homologous genes in GenBank by the BLAST algorithm at the NCBI server (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi, see primer sequences in Table 1). The sequences obtained in this process (minimum, three independent clones) were deposited in GenBank (see Table 1 for accession numbers). Quantities from 1µg to 100ng of DNAseI-treated RNA were reverse transcribed to cDNA using First Strand cDNA Synthesis Kit from Roche (F Hoffmann-La Roche, Basel, Switzerland) and stored at -20ºC. Aliquots of 100 ng of total RNA were used to quantify specific transcripts in LightCycler ® 480 Real-Time PCR System (Roche) using SYBR ® Green Mix (Takara Bio INC, Siga, Japan) and the primer pairs listed in Table 1. Relative mRNA abundances of different genes were calculated from the second derivative maximum of their respective amplification curves (Cp, calculated by triplicates). Cp values for stressrelated target genes (tg) were compared to the corresponding values for a reference gene (ref) to obtain ∆Cp values (∆Cp = Cp ref -Cp tg ). ß Actin was selected as reference gene after examination of its variability among all available samples (section 3.1). PCR efficiency values for reference and tested genes were calculated using at least five serial 1:3 dilutions for each gene [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF]. When necessary, primers were redesigned to obtain efficiency values better than 95% for all tested genes. To facilitate reading of tables and graphs, mRNA abundance values are represented as mRNA copies of target gene per 1000 copies of the reference gene mRNA (‰ of reference gene, 1000x2 ∆Cp ).

Statistical analysis

Statistical tests were performed using the SPSS 17 (SPSS Inc., Chicago, Ill) package.

All statistical calculations were performed using ∆Cp values, as this parameter followed normal distributions, as assessed by the Kolmogorov-Smirnov test.

Results

3.1.-Cloning and Sequence of stress-related genes from A. fasciatus

DNA fragments from putative ß actin, EF1, Vtg, CYP1A, SOD and HSP70 genes were amplified by PCR from A. fasciatus liver cDNA (Table 1). BLAST analysis confirmed the close sequence homology of these amplicons with the corresponding genes in different fish species, including representatives from different Cyprinodontiformes families (Cyprinodontidae, Fundulidae, Poeciliidae, etc.) and some Perciformes (Sparida, among others). This result indicates the high evolutionary conservation of the amplified fragments.

3.2.-Validation of stress genes as exposure markers.

Translation elongation factor EF1 and ß-Actin were tested for mRNA abundance stability in a large set of A. fasciatus RNA samples (84 independent qRT-PCR reactions). ß-Actin (average Cp = 19.31±0.72) showed a significantly lower variation than EF1 (average Cp = 17.82±1.22), and it was therefore selected as reference gene for all subsequent experiments. Exposure of control A. fasciatus individuals to model substances (Cd for metals, 17-ß estradiol for estrogens, and ß-naphthoflavone as a surrogate for dioxin-like substances) resulted in an increment of liver mRNA abundance of the target genes for each substance. MT mRNA levels were increased approximately 200-fold upon Cd exposure after 24 h of exposure, but this response faded by 80% after a further 24 h (Figure 2A). In contrast, Vtg mRNA levels remained essentially unchanged in liver during the first 24 h of treatment with estradiol, whereas a 48 h-length exposure resulted in a more than 100x induction of the transcript (Figure 2B). ß-naphthoflavone acted as a mild CYP1A inducer, increasing mRNA levels of this gene twofold in 24h and by an extra twofold after a further 48 h of exposure (Figure 2C). These behaviours are typical for these inducers in fish species [START_REF] Piña | Analysis of gene expression as a new tool in ecotoxicology and environmental monitoring[END_REF] and corroborate the cloned fragments as encompassing bona fide MT, Vtg and CYP1A gene sequences from A.

fasciatus. The different induction kinetics for the tested mRNAs is likely related to regulatory processes affecting transcription of the corresponding genes; it is also likely that the protein products codified by those genes remain active for longer periods of time than mRNA.

3.3.-Biometric parameters and mRNA abundance of stress genes in natural A. fasciatus populations.

A. fasciatus females were captured in Luza (control site, n=9) and in Sfax (polluted site, n=18). Ten out of the 18 animals captured in Sfax showed apparent spinal deformities, a phenotype long associated to this site (Messaoudi et al., 2009b). Animals from Luza were longer (6.0±0.7 cm versus 4.5±0.7 cm) and heavier (3.0±1.2 g versus 1.4±0.7 g) than animals from Sfax, either normal or deformed (Figure 3A). No difference in condition factor (CF) was observed between normal fish of both sites, suggesting that the size variability did not alter the animal proportions. The increased CF value observed for deformed animals (1.6±0.2 versus 1.3±0.2, Figure 3A) is likely related to the deformation itself, which shortens the animal without changing its weight. Two of the five analyzed genes showed significant variations in mRNA abundance among the three fish populations. Animals from Luza showed elevated (about 4-fold, Figure 3B) levels of MT transcript relative to normal or deformed fish from Sfax, whereas the CYP1A transcript was 10 times more abundant in deformed fish for Sfax than in normal animals from either site (Figure 3B). Very high and uniform levels of Vtg was observed in all three sites, which is consistent with the period of capture, close to the spawning season (June-July), and suggests that differences in size and ecological impact between the two sites did not affect the female reproductive cycle (Figure 3B), although more extensive studies are required to corroborate this hypothesis.

Bivariate correlation analyses between mRNA transcript levels for different genes and biometric parameters showed that SOD and CYP1A were the only two genes whose transcript levels appeared mutually correlated (Figure 4A). In addition, CYP1A mRNA levels in different fish correlated with the corresponding CF values (Figure 4B). This correlation applied especially to samples from Sfax, either deformed and normal (grey and black circles in Figure 4B), suggesting a link between the extent of deformity of impacted fish and their exposure to CYP1A-inducing pollutants (dioxins, dioxin-like PCBs, PAHs, etc.) in the environment.

The strong correlation between MT transcript abundance and size and weight of the animal shown in Figure 4C was surprising, but consistent with the high MT mRNA levels found in the Luza population (Figure 3A). Figure 3C shows estimates of mean transcript levels for each gene and population after suppressing the effect of the differences in weight, calculated by ANCOVA. This calculation indicates that all the differences in MT transcript abundance between fish groups can be explained by differences in fish size. Although statistical correlation cannot be taken as an indicator of cause-effect relationship, the results do suggest that MT transcript levels in this population were related to some unknown physiological parameter linked to size and weight rather than to the presence of Cd or other metals in the environment. In contrast, the 10-fold higher CYP1A transcript abundance in deformed fish appeared unrelated to fish weight (Figure 3C).

Discussion

Use of markers based on mRNA quantification techniques has many advantages for environmental monitoring of natural populations. These techniques require very small amounts of samples (less than 100 mg of tissue is often enough for performing hundreds of tests [START_REF] Piña | Analysis of gene expression as a new tool in ecotoxicology and environmental monitoring[END_REF]), permitting the use of small species or small pieces of tissue [START_REF] Fossi | Skin biopsy of Mediterranean cetaceans for the investigation of interspecies susceptibility to xenobiotic contaminants[END_REF][START_REF] Quiros | A noninvasive test of exposition to toxicants: quantitative analysis of cytochrome P4501A expression in fish scales[END_REF]. In combination with RNA stabilizing agents, samples can be stored and shipped with minimal or no refrigeration, allowing sampling in remote sites. Total mRNA contains information of the whole transcriptome of the sample; as it can be stored indefinitely at -80ºC, this allows re-evaluation of old samples if a new potentially interesting marker is developed. And last, comparison of multiple markers (theoretically, the whole transcriptome) from a single sample allows a complete analysis of the effects of environmental changes at the transcriptional level to define regulatory networks of genes involved in different stress responses.

The main disadvantage on the use of mRNA markers for environmental monitoring is the requirement for the exact DNA sequences of test and reference genes in the impacted species. In most cases, species abundant in interesting sites are poorly known, and our current genetic knowledge of many species of high environmental relevance is still scarce. To apply these techniques to A. fasciatus, we used known DNA sequences from evolutionarily related species (mainly Cyprinodontiformes and Perciformes) to find conserved regions of the desired genes and to derive qRT-PCR primers out of them. Due to the high conservation of the gene regions used in the process, these primers are likely useful for other related Cyprinodont species.

The response of the putative A. fasciatus MT, Vtg and CYP1A genes to the presence of model effectors was similar to the one observed for these genes in other better known fish systems [START_REF] Piña | Analysis of gene expression as a new tool in ecotoxicology and environmental monitoring[END_REF]. Estradiol induced Vtg mRNA some 100fold after a lag period, whereas MT transcript peaked at 24 h of Cd exposure and reduced its abundance afterwards. ß-naphthoflavone appeared as a weak, but steady inducer of CYP1A. The transient effect of Cd (and other heavy metals) in liver MT transcript abundance has been observed in different systems [START_REF] Gonzalez | Comparative effects of dietary methylmercury on gene expression in liver, skeletal muscle, and brain of the zebrafish (Danio rerio)[END_REF]Quirós et al., 2007b). In natural fish populations, metal chronic exposure usually results in elevated MT mRNA levels in kidney and other tissues, but not necessarily in the liver [START_REF] Hayes | Cloning of a chub metallothionein cDNA and development of competitive RT-PCR of chub metallothionein mRNA as a potential biomarker of heavy metal exposure[END_REF][START_REF] Navarro | Physiological responses to mercury in feral carp populations inhabiting the low Ebro River (NE Spain), a historically contaminated site[END_REF]Quirós et al., 2007b;[START_REF] Tom | Monitoring of hepatic metallothionein mRNA levels in the fish Lithognathus mormyrus -Evaluation of transition metal pollution in a Mediterranean coast[END_REF]. This may well be the case for A. fasciatus, as we did not observe an increment of MT mRNA levels in the Cd chronically exposed Sfax fish population. Rather, we found a direct correlation between MT transcript levels and fish size and weight, for which we do not have any explanation. The smaller size and weight of Sfax animals could in principle be related to pollution, although some aspects of our data indicate otherwise. A surprisingly conclusion of our data is the relatively good health status of fish in Sfax, including deformed ones. They showed CF values similar to, if not higher than (see below) those from Luza sites, they seem to spawn normally, and HSP70 transcript levels, a marker for general stress [START_REF] Schroder | Correlation between the level of the potential biomarker, heat-shock protein, and the occurrence of DNA damage in the dab, Limanda limanda: a field study in the North Sea and the English Channel[END_REF], were identical in both sites.

These data suggest a specific etiology for spinal deformities, rather than a massive toxic effect.

The fish sampling coincided with the pre-spawning period, a circumstance reflected in the very high levels of Vtg transcript present in all three fish groups. These levels were not affected by the approximately two-fold differences in weight between Luza and Sfax fish populations. This suggests that the smaller fish in the impacted area, including deformed ones, were spawning synchronously with the larger Luza fish. The likely conclusion is that, whatever the pollution impact that determines the deformed phenotype may be, it did not affect the Vtg synthesis in liver. Possible effects in other reproductive functions in the impacted animals remain to be elucidated.

The only mRNA biomarker clearly differentiating deformed from normal fish is their 10 times higher CYP1A mRNA levels. This gene, and the enzymatic activity associated to its protein product, EROD, are classic markers of exposure to dioxin-like pollutants, like dioxins, other organochlorine compounds and polycyclic aromatic hydrocarbons [START_REF] Van Der Oost | Fish bioaccumulation and biomarkers in environmental risk assessment: a review[END_REF][START_REF] Whyte | Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure[END_REF]. Hepatic CYP1A mRNA abundance has been directly correlated to the presence of organic pollutants in different fish species [START_REF] Jarque | Altitudinal and thermal gradients of hepatic Cyp1A gene expression in natural populations of Salmo trutta from high mountain lakes and their correlation with organohalogen loads[END_REF]Quirós et al., 2007b;[START_REF] Roberts | Gene expression in caged fish as a first-tier indicator of contaminant exposure in streams[END_REF][START_REF] Roling | Differential gene expression in mummichogs (Fundulus heteroclitus) following treatment with pyrene: comparison to a creosote contaminated site[END_REF]. In contrast, metals tend to suppress, rather than induce, CYP1A expression [START_REF] Bruschweiler | Inhibitory effects of heavy metals on cytochrome P4501A induction in permanent fish hepatoma cells[END_REF]. Therefore, our data suggest that the elevated CYP1A transcript levels were not related to the known metal pollution in Sfax, but to still uncharacterised organic pollutan(s). The correlation between individual levels of CYP1A and SOD transcripts may also be related to this putative environmental impact, as SOD is a classical marker for oxidative stress affected by many pollutants, both organic and inorganic [START_REF] Bainy | Oxidative stress in gill, erythrocytes, liver and kidney of Nile tilapia (Oreochromis niloticus) from a polluted site[END_REF][START_REF] Hansen | Induction and activity of oxidative stress-related proteins during waterborne Cd/Zn-exposure in brown trout (Salmo trutta)[END_REF][START_REF] Peters | 7-Etoxyresourfin odeethylase (EROD) and antioxidant enzyme activities in larvae of sardine (Sardina pilchardus) from the nord coast of Spain[END_REF][START_REF] Porte | Assessment of coastal pollution by combined determination of chemical and biochemical markers in Mullus barbatus[END_REF].

High CF values are usually associated to healthy, well-fed fish, under the assumption that heavier fish of a given length are in better condition than leaner ones [START_REF] Bervoets | Metal concentrations in water, sediment and gudgeon (Gobio gobio) from a pollution gradient: relationship with fish condition factor[END_REF][START_REF] Fulton | The rate of growth of fishes[END_REF]. However, spinal deformation decreases the animal length; therefore, deformed fish would have higher CF than normal ones, provided deformity does not impair feeding capacity. Surprisingly enough, this seems to be the case, as normal and deformed fish from the same area were very similar in weight. Under these circumstances, CF can be considered as a measure of the extent of the deformity, and its correlation with CYP1A transcript is consistent with a possible link between abnormal animal shape and exposure organic pollutants.

Spinal deformities have been reported in natural fish populations [START_REF] Ayed | Incidence of kyphosis deformities in natural population of Atherina lagunae (Trabelsi et al. 2002) from the Tunis North Lake, Tunisia[END_REF][START_REF] Kessabi | Involvement of Cd Bioaccumulation in Spinal Deformities Occurrence in Natural Populations of Mediterranean Killifish[END_REF]Messaoudi et al., 2009a;Messaoudi et al., 2009b;[START_REF] Sun | Morphological Deformities as Biomarkers in Fish from Contaminated Rivers in Taiwan[END_REF][START_REF] Tutman | Incidence of spinal deformities in natural populations of sandsmelt, Atherina boyeri (Risso, 1810) in the Neretva river estuary, middle Adriatic[END_REF] as well as in farmed fish [START_REF] Aunsmo | Association of spinal deformity and vaccine-induced abdominal lesions in harvest-sized Atlantic salmon, Salmo salar[END_REF][START_REF] Aunsmo | Modelling sources of variation and risk factors for spinal deformity in farmed Atlantic salmon using hierarchical-and cross-classified multilevel models[END_REF][START_REF] Bardon | What is the heritable component of spinal deformities in the European sea bass (Dicentrarchus labrax)?[END_REF][START_REF] Silverstone | Spinal deformities in farmed Atlantic salmon[END_REF]. Whereas skeletal malformations in aquaculture appear to be related to a complex interaction of genetic, procedural and infectious/immunological factors [START_REF] Aunsmo | Association of spinal deformity and vaccine-induced abdominal lesions in harvest-sized Atlantic salmon, Salmo salar[END_REF][START_REF] Aunsmo | Modelling sources of variation and risk factors for spinal deformity in farmed Atlantic salmon using hierarchical-and cross-classified multilevel models[END_REF][START_REF] Bardon | What is the heritable component of spinal deformities in the European sea bass (Dicentrarchus labrax)?[END_REF][START_REF] Lall | Role of nutrients in skeletal metabolism and pathology in fish -An overview[END_REF], the presence of deformities in natural populations are most likely related to pollution [START_REF] Kessabi | Involvement of Cd Bioaccumulation in Spinal Deformities Occurrence in Natural Populations of Mediterranean Killifish[END_REF][START_REF] Lemly | Symptoms and implications of selenium toxicity in fish: the Belews Lake case example[END_REF]Messaoudi et al., 2009a). Spinal deformities can be experimentally induced in fish upon exposure to different organic and inorganic substances, including metals, metalloids and organic substances like PAHs, OC and dioxins [START_REF] Cachot | Characterization of toxic effects of sediment-associated organic pollutants using the lambda transgenic medaka[END_REF][START_REF] Gagnon | Diuron increases spinal deformity in early-lifestage pink snapper Pagrus auratus[END_REF][START_REF] Holth | Differential gene expression and biomarkers in zebrafish (Danio rerio) following exposure to produced water components[END_REF][START_REF] Miller | Effects of acute and subchronic exposures to waterborne selenite on the physiological stress response and oxidative stress indicators in juvenile rainbow trout[END_REF][START_REF] Yamauchi | Toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing red seabream (Pagrus major) embryo: An association of morphological deformities with AHR1, AHR2 and CYP1A expressions[END_REF]. In the case of A. fasciatus Tunisian populations, deformed animals show a high contend in different heavy metals, especially Cd [START_REF] Kessabi | Involvement of Cd Bioaccumulation in Spinal Deformities Occurrence in Natural Populations of Mediterranean Killifish[END_REF]. Whereas the effect of heavy metals in fish bone density and malformations is well reported [START_REF] Bengtsson | Vertebral deformities and physiological effects in fourhorn sculpin (Myoxocephalus quadricornis) after long term exposure to a simulated heavy metal-containing effluent[END_REF][START_REF] Cheng | Cellular and molecular basis of cadmium-induced deformities in zebrafish embryos[END_REF], our data suggest that the observed high incidence of adult, deformed fish in the Sfax area results from a combination of organic and metal pollution. We are aware that the present study is only a first approach on the characterization of the physiological processes linked to the appearance of spinal deformities in the examined populations; additional information on either activity and or protein levels would further support and possibly clarify RT-PCR data.

We consider that the interest of the analysis performed here is not restricted to a specific problem in the Tunisian coast. Brackish water bodies, like deltas, estuaries, marshes and coastal lagoons are severely endangered in the Mediterranean Sea and elsewhere, both by the increasing population pressure and by the impending raise of sea levels. Cyprinodontiformes are often found in these environments, and constitute therefore excellent sentinel species of environmental impact. With qRT-PCR probes likely adaptable to any Ciprinodontiform species and RNA transport and quantification procedures that allow the preservation and precise analysis of milligram-size tissue samples, the markers and the techniques shown here cope with the three main problems intrinsically present in this type of studies: largely unknown fish species of very small size and captured in remote areas.

Figure 1 .

 1 Figure 1.Localization of sampling sites in the Gulf of Gabès (Tunisia).

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3

Table 1 .

 1 qRT-PCR Primers and GenBank accession numbers of the corresponding A. fasciatus sequences developed in this work.

	Gene	Acronym	Accession #	Forward primer (5'-3')	Reverse primer (5'-3')
	Metallothionein	MT	HM017068	GCGAGTGCTCCAAGACTGG	TGGGCAGCATGAGCAGC
	Vitellogenin	Vtg	HM017070	GACTGGATGAAAGGAAAGACCTGT ACGCGTCCGTTGGGAGT
	Heat-shock protein 70	HSP70	HM017074	TTTTATCCTGGTGGTGGC	GCCCTCTTGTTGTCTCTGAT
	Superoxide dismutase	SOD	HM017073	AGAGCATGGTTTCCATGTCCAT	CCAACTCACCAGGTCTCCAA
	Cytochrome P4501A	CYP1A	HM017071	ACATCTCTGAATGGCTACTT	TTCTCTCCATCTAGCTTGTT
	ß-Actin	ß-actin	HM017072	AGATCATTGCCCCACCAGAG	ACTCATCGTACTCCTGCTTGCTG
	Translation elongation factor 1EF1	HM017075	AGGAAATCCGTCGTGGATACG	TGACCTGAGCGTTGAAGTTGTC

Acknowledgements

This work has been supported by the Spanish Ministry of Research, Science and Innovation (CGL2008-01898) and the Spanish Agency of Cooperation for Development AECID (AECI A/019101/08). We thank also the Tunisian Ministry of Higher Education, Scientific Research and Technology.


W G Graham
email: b.graham@qub.ac.uk

K R Stalder

Plasmas in Liquids and some of their Applications in Nanoscience

Keywords: 52.77.Fv, 52.50.-b, 52.80.Wq, 64.70.F-, 81.07.-b

The range of applications for plasmas in liquids, plasmas in contact with liquid surfaces and plasmas containing liquid drops is growing rapidly across a range of technologies. Here the focus is on plasmas where the electrodes are immersed in liquids and their applications in nanoscience. The physical phenomena in both high voltage (~10's kV) and low voltage (few hundred V) plasmas in liquid are described together with a discussion of the plasma induced chemistry. Studies show that in water the plasmas are formed in water vapour created by Joule heating as either channels in the liquid or as layers on the electrodes. The chemistry in these water vapour plasmas and at their interface with the liquid is discussed in the context of the highly reactive radicals produced, such as H and OH. The current use of a variety of plasmas-in-liquid systems in the area of nanoscience is discussed, with an emphasis on nanoparticle growth.

Introduction

Plasmas produced by electrical discharges in liquids are finding wide applicability in a number of modern technology areas. Whereas conventional gas discharges operate in environments where the electrodes and insulating structures are fixed and operate with gas pressures typically at or below one atmosphere, discharges in liquids have the added complexity that the liquid can vaporize-which leads to discontinuous liquid-vapour boundaries-and the liquid-vapour boundary is not fixed so that the discharges can exhibit much more complex hydrodynamic behaviour. Moreover, these discharges typically operate at or above atmospheric pressure, so the vapour densities are usually quite high compared to low-pressure discharges; consequently these discharge regimes provide a plethora of chemical reactions whose unique products can be exploited for the intended application. Many of the experiments on plasmas in liquids produce strongly non-equilibrium plasmas, where the electron temperatures are much larger than the vapour temperature and the liquid temperature. Earlier reviews of research on non-equilibrium plasmas in liquids have been compiled, for example, by Yerokhin et al. 1999, Malik et al. 2001, Locke et al. 2006 and more recently by Bruggemann and Leys 2009a. Combinations of high density and strongly non-equilibrium plasma conditions can produce many interesting and technologically-useful chemically-active environments. For example, high-voltage discharges in low-conductivity water produce short-lived gas-phase and liquid-phase transient species such as hydroxyl (OH) and atomic hydrogen (H) radicals, as well as more stable chemical species such as hydrogen peroxide (H 2 O 2 ) and ozone (O 3 ); all of these species are chemically active and can be used to cleanse the water of unwanted pollutants.

In this review we restrict ourselves to plasmas formed by electrical discharges employing Confidential: not for distribution. Submitted to IOP Publishing for peer review 14 December 2010 electrodes immersed in the liquid. Broadly speaking, and for the purposes of this review, we separate discharge regimes into two main classes: those at kilovolt or higher voltages in relatively low conductivity liquids and those below kilovolt voltages in relatively high conductivity liquids. Applications in the high-voltage regime include pollution remediation [e.g. Lukes et al. 2004, Sunka et al. 2004, Kirkpatrick and Locke 2006, Yan et al. 2008], dielectric insulation [e.g Jones and Kunhardt 1994], pulse-power applications and switches [e.g. Martin et al.] and sources of UV radiation [e.g. Robinson et al. 1973]. These high voltage discharges produce some features also seen in gaseous discharges, such as streamers and strong acoustic effects [e.g. Devins and Rzad 1982, Chadband 1988, Beroual et al. 1988[START_REF] Massala | [END_REF], Touya et al. 2006, An et al. 2007, Kolb et al. 2008, Ceccato et al. 2010] Applications in the low-voltage regime include some plasma-based electrosurgery [e.g. Stalder et al. 2001, Woloszko et al. 2002, Stalder et al 2005, Stalder and Woloszko 2007], sterilization [e.g. Sakiyama et al. 2009], glow discharge electrolysis for a variety of applications [e.g. Sengupta et al. 1997, Sengupta et al. 1998, Kobayashi et al. 2000, Gao et al. 2001, Gao et al. 2003a, Gao et al. 2003b, Gao et al. 2004, Mizuno et al. 2005, Gao et al. 2006, Yan et al. 2006, Liu and Jiang 2008, Wang et al. 2008, Wang 2009], and deposition of nanocrystalline films [e.g. Paulmier et al. 2007a, Paulmier et al. 2007b]. Recent basic studies of low-voltage discharges include those by Schaper et al. 2010a andSchaper et al. 2010b. A noteworthy feature of many of the low-voltage discharges in highly conducting liquids is an approximately linear (ohmic) current-voltage behaviour at low-voltage which transitions to a higher resistance regime as the voltage is increased, thus indicative of the formation of a lowconductivity vapour layer over the electrode. As the voltage is increased further the resistance decreases due to the initiation of electrical discharges in the vapour layer and subsequent formation of an ionized plasma in the vapour layer. Some experiments have been conducted in hybrid regimes that don't fit easily into our discharge classification scheme, that is, for example, high-voltage discharges in high-conductivity water [Boguslavsky et al. 2001, Skibenko et al. 2006] and high-voltage DC discharges in lowconductivity water [Bruggeman et al. 2009b] and high-voltage AC capillary discharges in lowconductivity water [De Baerdemaeker et al. 2007] . It should also be noted that plasmas may be formed in liquid by other means, including laser heating [e.g. [START_REF] Vogel | [END_REF]Vogel 1996], and even through focused sound waves [e.g. Flannigan and Suslick 2005]. In addition, there have been numerous studies of plasmas formed externally to liquids and made to interact with them [e.g. Janca et al. 1999, Baba et al. 2006, Kitano et al. 2006[START_REF] Sugama | [END_REF][START_REF] Richmonds | [END_REF], Shirai et al. 2009, Chang et al. 2010, Chiang et al. 2010].

In addition, by looking at voltage pulses with durations from tens of nanoseconds to dc, we restrict our discussion to situations where a liquid-vapour phase transition is involved in the discharge mechanism. We therefore exclude the recent work with ultrashort, intense voltage pulses, which intriguingly suggests direct ionization in the liquid phase [START_REF] Starikovskiy | Proc. 20 th ESCAMPIG[END_REF].

Although discharges in liquids can be produced in a number of different types of liquids, water-and solutions derived from it-clearly has received the most attention, primarily because it is ubiquitous and it is one of the most versatile solvents. Since salts and other ionic compounds are easily dissolved in water, thus forming an electricallyconducting medium through ionic conduction, its conductivity can be tailored over a wide dynamic range, simply by adjusting the concentration of the salt. For example, the difference in ionic conductivity between ultrapure water and water with 1% NaCl by weight dissolved in it is over 6 orders of magnitude. Water also has a fairly large temperature coefficient of electrical conductivity; the conductivity of 1% NaCl nearly doubles from 10 o C to 40 o C. Finally, as is well known, at atmospheric pressure water boils at approximately 100 o C, which is easily achieved in many electrical discharges in water. In a boiling process, the density of the water vapour decreases by nearly 3 orders of magnitude from that of the liquid.

Since this topical review is in the area of nanoscience there will be an emphasis on nanoparticles which can be produced or treated in plasmas produced by electrical discharges in liquids. We note that nanoparticles may also be produced in plasmas produced externally of the liquid but impinging on the liquid [e.g. [START_REF] Richmonds | [END_REF], Chang et al. 2010, Chiang et al. 2010], and in spark discharges in liquid-free environments [e.g. Messing et al. 2009].

Physics

The study of the physics phenomena in the initiation, maintenance and termination of plasmas or discharges created in liquids is very challenging involving plasma physics, thermodynamics, hydrodynamics, cavitation and interactions at the gas -plasma -electrode -liquid interfaces. As mentioned above, the discussion is confined to a description of the underlying phenomena associated with two diverse systems which both have potential use in the nanosciences. These are the high-voltage (~10's kV), large-gap breakdown in water and the relatively low-voltage breakdown (few hundred volts) in a higher conductivity, saline solution.

High voltage breakdown in water

In recent reports by Ceccato and co-workers [Ceccato 2009a, Ceccato et al. 2009, Ceccato et al. 2010] there are excellent summaries of the temporal development of a high voltage, large gap plasmas in water from a positively biased pin to a grounded plane electrode in water based on their measurements which enhance but are largely consistent those of others [Beroual et al. 1988, Beroual 1993, Lesaint and Gournay 1994, Fofana and Beroual 1998[START_REF] Massala | [END_REF][START_REF] Vasily | Impulse Breakdown of Liquids, Power Systems[END_REF]]. The spatially and temporally resolved evolution of the system is recorded by imaging of the light emission from the plasma and the refraction of backlighting light using shadowgraphy. The latter allowing the density variation of the water and hence vapour production to be observed. In the results from that publication, reproduced here, the peak applied voltage was between 30 and 40 kV, the gap was 40 mm and the water conductivity was between 7 and 50 µS/cm.

The typical discharge current behaviour, shown in figure 2.1, indicates that the electrical breakdown process can be described in terms of four phases or modes. The initial current is initially almost constant, at a few hundred mA, producing Joule heating of the liquid. Shadowgraphs reveal a micro-bubble growing on the metal surface of the tip of the electrode to some tens of microns in diameter (Figure 2.2). The length of this initiation phase-Region 1 in Figure 2.1-before the first light emission is observed, is not reproducible from shot to shot but is typically a few microseconds. The discharge current then slowly rises (Region 2) and a luminous region forms and grows around the microbubble. This mode is difficult to resolve since it is composed of a series of weakly emitting, small filaments propagating less than a mm from the pin electrode to form bushlike filaments. This is the primary discharge mode (Figure 2.3 a). The emission intensity is higher at the tip of the primary filaments. This indicates that a high field region is concentrated in the filament tip and is responsible for the emission intensity. This is a well known phenomenon in gas-phase streamers where the propagation and the emission are entirely driven by a space charge separation in the filament tip [START_REF] Raizer | Gas discharge Physics[END_REF], Kulikovsky 1997, Kulikovsky 1998].

The increase in current at the beginning of Region 3 marks the onset of secondary mode. One, or sometimes two, of the primary phase filaments emerges from the expanding bush of primary mode filaments and branches into other filaments, which all propagate simultaneously and develop into a tree-like pattern (figure 2.3c). .

Figure 2.3

The transition between the primary modes (a) to the secondary mode (c) captured in time resolved images (2ns). (From Ceccato 2009b) In low conductivity water some filaments which have ceased growing or emitting light will subsequently re-illuminate and continue to grow one filament at a time. This is in Region 4 in Figure 2.1. It is indicative of charge deposition and electric field separation at the plasma-liquid interface inducing dielectric-barrier-discharge-like behaviour.

In both the primary and secondary modes shadowgraph indicates the presence of shock waves at the tips of the filaments indicating that the growth of the filaments is stepwise rather than continuous.

The length of propagation of the filaments into the electrode gap depends on the applied voltage duration. If the filament reaches to the other electrode an arc is created. If the filaments fail to reach the other electrode the gas channels left behind expand and fragment into a string of micro bubbles.

The negatively-biased pin electrode discharges are also filamentary but the propagation mechanism is clearly different since the electrons come to the interface from the liquid in the positive case and from the vapour with negative polarity. The light emission, which is significantly less than that in positive polarity, starts almost immediately after the application of the high voltage and propagate more slowly than with positive polarity.

Low voltage breakdown in saline

The production of plasmas in saline solution at low voltages has been studied for some time by Stalder and co-workers [Stalder et al. 2001, Woloszko et al. 2002, Stalder et al 2005, Stalder and Woloszko 2007]. It was shown that the plasma production was associated the initial production of a vapour layer on the electrode surface. Recently this work has been extended to specific experimental measurements of vapour layer and computer simulation [Schaper et al. 2010a, Schaper et al. 2010b].

In Schaper et al.'s experiments, the plasma was created in the vicinity of a 0.5 mm diameter tungsten tip electrode insulated so that only the last 0.5 mm is immersed in 0.9% NaCl solution (w/w). The return electrode is a grounded plate in the liquid. A 20 ns risetime high-voltage switch supplies a single DC voltage pulse to the powered electrode with a duration of up to 10 ms.

The time dependence of the plasma produced light emission was monitored using a fast photomultiplier tube and the time integrated spectra over the period of the pulse were measured using an imaging spectrograph. An ICCD was used to obtain time resolved images of the plasma emission and shadowgraphs of the electrode tip region. Finite element multiphysics software, coupling thermal and electrical effects, is employed to model the vapour layer formation [Schaper et al. 2010a].

The typical electrical behaviour of the system when a 9 ms, -225 V applied voltage pulse is applied to the tip electrode shown in Figure 2.4. The first 0.7 to 1.1 ms of the electrical behaviour of the system is very reproducible from pulse to pulse and is seen to be associated with the growth of a vapour layer over the electrode. The vapour layer production process is reproducible and can be simulated in the finite element multiphysics based simulation of the vapour layer formation. [Schaper et al. 2010a].

. For positive polarity the final layer depth and the depth at full coverage are similar to those with negative polarity pulses, the time scales to reach those stages are about 30% longer.

For negative polarity discharges the exact time of the first light emission varies from pulse to pulse but 95% of the voltage pulses produce first light emission-originating from plasma production in the vapour layer-between 0.7 and 1.1 ms after their start. By this time the vapour layer has grown to a thickness of about 250 µm over the electrode surface. This implies electric fields of ~10 6 Vm -1 in the vapour layer, close to the values of 2.5 x 10 6 to 7.5 x 10 6 Vm -1 predicted by the simulations of vapour growth [Schaper et al. 2010a].

The images in Figure 2.5 show that the intensity and volume of the plasmas produced vary greatly and are unpredictable. However they can be broadly classified as follows: Short lived (~few µs), low intensity plasmas, connected to the electrode surface and occurring through out the voltage pulse (Figures 2.5 a-c), Long lived, high intensity plasmas, persisting for up to 500 µs and spreading from the surface more extensively into the vapour layer (figure 7d). The first plasmas can be either short or long lived but have a maximum duration of about 100 µs. The location of the discharges is also unpredictable however, from the statistical spatial distribution of first discharges, it is clear that they are predominantly produced at the rim of the electrode where the electric field strength would be at a maximum. For positive polarity the final layer depth and the depth at full coverage are similar to those with negative polarity pulses, the time scales to reach those stages are about 30% longer and higher voltages are required to initiate the discharges, In summary, these measurements confirmed that plasma production occurred in vapour layers formed on the electrode and this happens after the vapour layer has completely covered the exposed electrode tip. The vapour layer is approximately 0.3 mm thick when the first plasma occurs. The high electric fields in the vapour layer, approach 10 7 Vm -1 , the presence of a sodium and chlorine on the electrode surface and sodium in the vapour layer. There are generally multiple plasmas of different character in each pulse. They can be broadly classified as short (few µs) or long (up to 500 µs) lived.

The reproducible vapour layer formation make these systems particularly conducive to fundamental studies of the physics and chemistry of plasmas in liquids.

Chemistry

As noted above, electrical discharges in liquids and vapours above them frequently produce plasmas that are highly non-equilibrated in the sense that the electron temperature is significantly higher than the ion and neutral molecule temperatures. This condition leads to numerous electron-collision-dominated reactions that can fragment molecules into neutral and ionic products. Subsequent ion-molecule reactions subsequently produce additional chemical species not formed in the initial collision. Since the neutral vapour densities are usually quite large at typical atmospheric pressures and temperatures, the chemical kinetics drives the production of new species on rapid time scales.

Aqueous solutions are dominated by water, with minor concentrations of added solutesfrequently easily dissolved salts-providing means by which the conductivity of the liquid can be adjusted. Electrical discharges dissipate heat through Joule heating of these conductive solutions, and, if the local heating rate is sufficiently large compared to the rate at which the energy is dissipated through heat conduction or convective processes the liquid can vaporize. Due to water's large dipole moment the salts are strongly bound to the water in the liquid phase and do not volatilize easily. So the vapour is dominated by water molecules in the form of steam. Energetic electrons in non-equilibrium plasmas formed in water vapour produce a number of primary species through reactions such as:

e + H 2 O H + OH +e, e + H 2 O 2H + O +e, e + H 2 O H 2 O + + 2e,
Hydrogen atoms [H] and hydroxyl radicals [OH] are extremely reactive chemical species and can interact strongly with nearby materials, especially organic molecules containing significant hydrogen-terminated groups. The primary species in the vapour phase can also subsequently react with one another to produce a number of secondary species, usually influenced strongly by the presence of third bodies [M] to conserve energy and momentum in the association reactions:

OH + OH + M H 2 O 2 + M [M=third body] O + O + M O 2 + M O + O 2 + M O 3 + M
Here, the third body M is typically H 2 O, because water comprises the bulk of the vapour in which the plasma is formed. ], and protonated water cluster ions H + •H 2 O can also be produced in the vapour phase. Chemical kinetics software can be used to follow the evolution of all the relevant species once the rate constants and initial species concentrations are specified. [e.g. Gao et al. 2001, Gao et al. 2003a, Gao et al. 2003b, Gao et al. 2004, Stalder et al. 2005, Gao et al. 2006, Wang et al. 2008, Wang 2009] Many of the species produced by the reactions outlined above are water soluble and thus can react with solutes in the liquid phase. Hydrogen peroxide and ozone are notable in this regard. In fact these are the species most sought after for electrical discharges in liquids used for pollution remediation.

The energetic free electrons in the vapour phase continuously react with the vapour species and lose energy. Their ultimate fate depends on whether they react to form negative ions, such as the superoxide ion O 2 -, or whether they enter the liquid phase and become bound to water molecules to form so-called aqueous electrons, e aq . Aqueous electrons can also react with solute particles. Studies relating to the radiation chemistry induced by high-energy electron beams interacting with liquid-phase and vapour-phase water provide significant data on the chemistry of aqueous electrons [Jonah 1995, Pimblott andLaVerne 1998]. There have been some studies of the chemistry of aqueous electrons and hydroxyl radicals reacting with biological molecules, including DNA [e.g. LaVerne and Pimblott 1993 andFalcone et al. 2005]. Although these studies usually involve the impact of very high energy electrons (>1 keV) on liquid water, there is significant energy loss through linear energy transfer (LET) mechanisms so that the electrons eventually degrade to energies that produce low-energy electrons, aqueous electrons and radicals relevant to the low-temperature nonequilibrium plasmas of interest in this review.

Chemically-active species produced in the vapour phase plasma can also diffuse to the vapour-liquid interface and enter the liquid; conversely species in the liquid phase can diffuse to the liquid-vapour interface and enter the vapour. The net transport of species across the interface is called the uptake coefficient. Studies on the atmospheric chemistry of radicals and liquid droplets provide information on many of these processes for species related to water. For example, the uptake coefficient for hydroxyl radicals [OH] entering liquid water has been measured to be greater than 2 x 10 -3 . [Takami et al. 1998] Some relatively high-energy (~10 eV) electrons in the high energy tail of the electron energy distribution of the low-temperature nonequilibrium plasmas can impinge on the liquid and induce chemistry directly at the interface. These can leave the vapour phase discharge and deposit their energy within microns of the water interface. The ionization threshold in water is ~ 13 eV and the excitation threshold is ~7.4 eV. Since the O-H bond energy is ~5 eV water molecules will break up to give a series of reactive radicals (OH, H, HO 2 ) and possibly a small yield of positive and negative ions (H 3 O + , OH -). The electrons will rapidly become thermalised and hydrated on sub-nanosecond timescales to form aqueous electrons e aq . Typically the radical and aqueous electron lifetimes will be of the order of microseconds.

Experimental studies of the products and rates of bimolecular and termolecular chemical reactions of plasma-produced species of the sort described above are relatively tractable using conventional chemical techniques, including chromatography, optical absorption spectroscopy and mass spectroscopy. Studies of the interactions of plasma-produced species with solid targets are much less amenable to basic chemical methods. In this case, modelling can be particularly helpful to provide insight and sometime. For example, it is well known from organic chemistry that hydrogen atoms and hydroxyl radicals are particularly reactive towards alkanes. Attempts to understand how plasmas interact with collagen triple helix molecules in moist environments borrow ideas from these types of reactions, plus hydrolysis, to propose a chemical model that might explain the observed efficient ablation in plasma-based electrosurgery [Stalder et al. 2005]

Spectroscopy

Plasmas produced in liquids usually produce some observable optical emissions. In the case of high energy discharges in highly purified water the radiation can be quite intense and significant in the UV wavelength range [Robinson et al. 1973]. In low-voltage discharges the radiation is also frequently clearly visible, but is usually dominated by the salt cations used to increase the conductivity of the water. For example, electrosurgical plasmas produced by electrical discharges in NaCl-based saline solutions produce sodium D-line emission at 589 nm, which gives rise to their characteristic orange glow [Stalder et al. 2001, Woloszko et al. 2002]. These types of lowvoltage discharges also produce UV and visible emissions associated with excited state OH * and H * species, thus indicating that even low-energy discharges can produce electron energies sufficient to fragment water molecules into excited products. Recently Bruggeman and colleagues [Bruggeman, et al. 2010] have pointed out that OH* may also be produced by dissociative recombination of H 2 O + ions and electrons. This process is likely to occur at low pressures and may also occur at atmospheric pressure. However, clustering reactions of H 2 O + with water molecules at atmospheric pressure are rapid, so this species is usually not the dominant positive ion in these discharges. The reactions are numerous, and many of the relevant ones have been tabulated by Bychkov and Yurovskii in their studies of electron-beam-induced ionization in water vapour [START_REF] Bychkov | Institute of High Temperatures[END_REF]. .A very nice optical emission study of DC-excited discharges in low conductivity water was recently reported [Bruggeman et al. 2009b] Plasmas produced by contact glow discharge electrolysis also produce spectra characteristic of the salts in the liquid [Kobayashi et al. 2000].

Applications in Nanoscience

Nanoparticle growth in plasmas in liquids has been explored by a number of groups and here we have selected a few examples to illustrate the type of nanoparticles that can be produced and some of the techniques that have been used. Jwo et al. 2005 prepared TiO 2 nanoparticles using a pulsed arc system in deionized water maintained at a reduced pressure of 50 torr. The voltage required to produce an arc under these conditions was only 100 volts, with positive polarity, and a maximum current of 2.5 A was developed. The voltage was pulsed on for 10 µs and pulsed off for 30 µs, thus using a 25% duty cycle, for a working time of 10 minutes. Particle diameters ranging from about 5-15 nm were reported. Sergiienko et al. 2006 andShibata et al. 2007 have reported on the use of an electrical discharge in liquid ethanol to synthesis of iron and cobalt carbide-filled carbon nanocapsules. They used a low current plasma discharge, generated between an immersed Fe (or Co) anode and cathode. The cathode is fixed to the top of an ultrasonic horn allowing bubbles to be introduced between the electrodes. A DC voltage difference of 55 V between the anode and cathode is sufficient to create the plasma. A carbonaceous powder is formed in the liquid ethanol which after treatment if found to contain carbon nanocapsules with spherical, crystalline carbide (orthorhombic Fe3C and monoclinic -Fe2.5C structures) cores about 5 nm in diameter and covered with graphite layers. Whole samples showed a ferromagnetic behaviour, attaining saturation at fields of ±1200 kAm -1 . Toriyabe et al. 2007 produced spherical metallic nanoparticles, which they refer to as "nanoballs," in a plasma electrolysis cell. Their composition include Ni, Ti, Ag, and Au, and they have a rather broad size distribution with a mean size of between about 100-400 nm, depending on the cell voltage. The trend in the data suggests that they might achieve sub-100-nm mean diameters if the cell voltage exceeded 200 volts. They report the typical I-V characteristic of these types of discharges, and show evidence of a glow discharge formed in the thin vapor layer covering the biased electrode.

Gold nanoparticles around 10-50 nm in size have been synthesized [Hieda et al. 2008 andTakai 2008] using a pulsed DC discharge. The typical preparation conditions used a chloraunic acid (HAuCl 4 ) stabilized by the addition of sodium dodecyl sulfonate to prevent particle agglomeration. The applied voltages were between 1.6 and 3.2 kV, and pulses at 15 kHz, 2µs pulse widths were used. They believe hydrogen atoms are essential as reducing agents for the particle growth to proceed. Hydrogen peroxide was also detected. Although the starting solution was highly acidic to begin with (pH ~3.1), the discharge reduced the pH even further to about 2.6. The gold nanoparticles had average diameters of 10.5 ± 3.6 nm. In addition it was possible to synthesis of topological ( e.g. trigonal, pentagonal, and hexagonal) gold nanoparticles.

Cabanillas et al. 2010 were able to fabricate spherical iron particles in liquid nitrogen using what they call a spark erosion process. They employed a commercial electric discharge machining (EDM) system. Arc currents of 9.5 to 25 amps were used. They used a pulsed voltage source, with an on time of 3000 µs and an off time of 340 µs. The particle size distribution was rather broad, ranging from about 5 microns to greater than 1 micron in diameter. [START_REF] Burakov | Proc. 29th Int. Conf. Phys. Ioniz. Gases[END_REF] made copper-based nanoparticles in either ethanol or distilled water by applying 3.5 kV pulses at 100 Hz. They claim two distinct modes of operation; either an arc discharge, or a pulsed spark discharge, a local thermodynamic equilibrium (LTE) conditions were achieved under either mode. The pulsed arc discharge developed currents as large as 9 amps with a rather long pulsed duration of 4 ms. The pulsed spark discharge developed a current up to 60 amps but for a much shorter duration of 30 µs. They performed optical spectroscopic measurements and focused attention on the broadening of the H emission line. The electron temperatures thus derived by them ranged from about 1.0 to 1.4 eV, depending on discharge model and liquid. The electron density was about 3-4 x 10 16 cm -3 in the arc discharge, but ranged from about 30-60 x 10 16 cm -3 in the pulsed spark discharge, depending on whether water or ethanol was used at the dielectric liquid. Some of the other nanoscience related applications of plasmas in liquids include the deposition of graphite films and surface modification. Paulmier et al. 2007a,b reports on the deposition of nanocrystalline graphite films in 96% ethanol with 4% phosphate buffer and 0.2 M KCl added to provide conductivity to the solution. They reported that the conductivity of this solution was 16.7 mS/m. A ramped negative DC voltage (cathodic) was applied to the central electrode, composed of either Cu, Al or Ti, starting at 0 and ending at -1000 volts after typically 400 seconds. The I-V curves showed characteristics typical of these kinds of discharges, with impedance starting to increase at a voltage of about -200 volts, indicative of vapour layer formation. Plasma discharges began to appear at about -400 volts. A carbon film was deposited, and Raman analysis confirmed both diamond-like and graphite-like characteristics of the disordered film. Interestingly, the optical emission spectrum at relatively high voltages, 700 and 1000 volts, showed a rather broad continuum ranging from about 400 nm to 900 nm along with the expected peaks characteristic of the Titanium electrode, and water dissociation products. Nomura et al. 2006 have also demonstrated the synthesis of carbon-related materials with a microwave-generated plasma in a hydrocarbon liquid but under vacuum. The vacuum prevents the oxidation of products such as graphite, silicon carbide, diamond-like carbon, and carbon nanotubes. The most attractive feature of this process is that the liquid phase plays an important role as a plasma reactor. However, this method is limited to nonpolar solvents such as hydrocarbons, because polar solvents adsorb microwaves and prevent plasma formation.

Oxidation, nitriding, and carburizing are also of nanoscience interest. These processes been investigated mainly with the gas-phase plasma however Takai 2008 also reports that plasma formation can also be used to enhance electroplating, in this case pulsed (60Hz) electroplating with 2 g/L NaOH in deionized water as the electrolyte. The applied voltage was made high enough (175V) to have short-lived and randomly moving microarcs develop on the surface of the aluminium anode. The growth rates of aluminium and titanium oxide films increased with discharge current densities. A high growth rate of 1.7 µm/min was obtained for both aluminium and titanium oxide. The aluminium oxide film consisted of two layers, as revealed by SEM observation of the cross-section of an aluminium substrate after modification. The external layer had low strength and high porosity, and could be removed easily. The internal layer was hard and adhered well to the substrate, believed to be because of locally developed transient high temperature melted the growing oxide layer on the substrate and improved the adherence.

Conclusions.

Plasmas or discharges produced in liquid have been studied for some time. As the understanding of their underlying physics and chemistry has progressed their exploitation into areas such as biomedicine and nanoscience has created much interest and excitement. In much of the fundamental plasma research the liquid has been water. These studies show that in water the discharges or plasmas are formed in water vapour created by Joule heating either as channels in the liquid or as layers on the electrodes.

The plasmas created are often highly non-equilibrated i.e. the electron temperature is significantly higher than the ion and neutral atom and molecule temperatures. The result is that numerous electron-collision-dominated reactions occur in such plasmas producing dissociation of molecules into reactive and charged species. In the highly collisional atmospheric pressure environment chemical kinetics produces further species on rapid time scales. In water vapour this produces species OH, H, HO 2 and H 2 O 2 and smaller yields of positive and negative ions and their hydrated clusters.

Plasmas in liquids are already being used in nanoscience for nanoparticle, film growth and surface functionalisation. The pressing issue in optimizing and extending the use of plasmas in liquids is to understand the plasma-induced physical and chemical phenomena exhibited in current, well defined applications. This understanding can then identify and inform the implementation of other potential applications. The complexity of these environments is challenging but the synergistic use of the multi-physics and plasma chemistry computer-based simulations and advanced imaging and spectroscopic tools, as described above, is already providing new insights. Important to interpreting the results of such measurements and as additional input to the simulations will be learning from and using the large knowledge and data base generated for many years by the radiation chemistry community.

In addition the full potential from using other liquids, liquid mixtures and dissolved precursors will allow the creation of a plethora of reactive species. This will lead to an enhanced ability to predetermine the liquid-plasma device combinations best tuned for specific applications, in a similar manner to the approaches that have been successful in the microelectronics industry. It will create many more opportunities to exploit plasmas in liquids across the whole technological landscape, including nanoscience. The immediate challenge now is to get plasma and materials scientists working closely together to understand the key processes and phenomena.

.

Figure 2 . 1 .

 21 Figure 2.1. A typical current and voltage waveform in a pin to plane configuration in water (from Ceccato et al 2010)

Figure 2

 2 Figure 2.2. A typical shadowgraph taken in the pre-plasma phase showing the formation of a microbubble. The tip is 400 µm in diameter [from Ceccato 2009b]

  Figure 2.4, The measured current and voltage time dependence of the system during a 9 ms, -225 V pulse with liquid at room temperature (~20°C). Also shown is the response of a fast photomultiplier tube (PMT) viewing the electrode tip region [from Schaper et al. 2010b].

Figure 2 . 5 .

 25 Figure 2.5. ICCD images of typical discharges. The dark areas are vapour. For a, b and c the ICCD gate width was 100µs, delayed 1ms from the pulse initiation. For d the ICCD gate width was 10 µs, delayed 1.1 ms from the pulse initiation [from Schaper et al. 2010b].

  Thus, more stable but still chemically active species such as hydrogen peroxide [H 2 O 2 ] and oxygen [O 2 ] and ozone [O 3 ] are formed. It should also be noted that long lived metastable species such as singlet oxygen [O 2 ( 1

g )], and superoxide ions [O 2 -