Frank Welle
email: welle@ivv.fraunhofer.de

Roland Franz

activation energy of diffusion and migration modelling compared to literature data

Keywords: Exposure modelling, Metals analysis -ICP/MS, Quality assurance Additives/Contaminants: Migration -diffusion, Heavy metals, Packaging -PET Food Types: Beverages, Fruit juices, Drinking water Plastic bottles, migration, polyethylene terephthalate (PET), antimony, diffusion coefficients, activation energy of diffusion, juices, mineral water

Plastics bottles made from polyethylene terephthalate (PET) are increasingly used for soft drinks, mineral water, juices and beer. In this study a literature review is presented concerning antimony levels found both in PET materials as well as in foods and food simulants. On the other hand, 67 PET samples from the European bottle market were investigated for their residual antimony concentrations. A mean value of 224 ±32 mg/kg was found, the median was 220 mg/kg. Diffusion coefficients for antimony in PET bottle materials were experimentally determined at different temperature between 105 °C and 150 °C. From these data the activation energy of diffusion for antimony species from the PET bottle wall into beverages and food simulants was calculated. The obtained value 189 kJ mol -1 was found to be in good agreement with published data on PET microwave trays (184 kJ mol -1 ). Based on these results the migration of antimony into beverages was predicted by mathematical migration modelling for different surface/volume ratios and antimony bottle wall concentrations. The results were compared to literature data as well as international legal limits and guidelines values for drinking water as well as migration limit set from food packaging legislation. It was concluded that antimony levels in beverages due to migration from PET bottles manufactured according to state-ofthe-art can never reach nor exceed the European specific migration limit of 40 µg/kg. Maximum migration levels caused by room temperature storage even after 3 years will never be essentially higher than 2.5 µg/kg and will be in any case below the European limit of 5 µg/kg for drinking water. The results of our study confirm that the exposure of the consumer by antimony migration from PET bottles into beverages and even into edible oils reaches approximately 1% of the current TDI established by WHO. Having substantiated such low antimony levels in PET bottled beverages the often addressed question on estrogenic effects caused by antimony from PET bottles appears to be groundless.

for beverages like soft drinks, mineral water, juices and beer. In 2007 the world-wide consumption of PET was about 15 Mio. tons (Plastics Europe 2008). Most of the PET resin was sold as food grade material for beverage packaging. In comparison to other packaging plastics, PET is one of the most inert polymers with good barrier properties against moisture, oxygen and carbon dioxide and with a very low migration tendency of its constituents. However, migration of substances from PET bottles into beverages is not completely negligible. Polymer additives, catalyst residues, degradation products, polymerisation side-product or residual monomers are potential migrants. Mass transfer of a migrant from PET polymer into food depends on several factors such as storage time and temperature, concentration of the migrant in the polymer, type and nature of the migrant and its solubility in food.

Migration kinetics of several organic impurities and additives in PET have been investigated in systematic migration studies within the last years. Target migrants were for instance acetaldehyde (Ewender et al 2003), the PET cyclic trimer [START_REF] Begley | Evaluation of polyethylene terephthalate cyclic trimer migration from microwave food packaging using temperature-time profiles[END_REF] or UV blocking additives Tinuvin 234 and Tinuvin 1577 [START_REF] Begley | Migration of a UV stabilizer from polyethylene terephthalate (PET) into food simulants[END_REF].

Recently, we investigated the migration of organic model compounds into food simulants and beverages such as soft drinks, flavoured waters and juices to establish the migration determining mass transfer parameters (Franz and Welle 2008). As suitable model migrants, as more volatile compounds toluene and chlorobenzene and as less volatile substances phenyl cyclohexane, benzophenone and methyl stearate were used. The results of this study showed that food simulants like 50% or 95% ethanol which cause a slight swelling of the PET polymer matrix do increase the migration significantly in comparison to usual beverages. Such high alcoholic simulants can therefore be considered as worse case test media for compliance testing of PET beverage bottles. On the other hand, the aqueous food simulants 3% acetic acid, water and 10% ethanol lead in many cases to an underestimation of migration of organic constituents in comparison to beverages. One important conclusion of this study was that the migration of organic compounds from the bottle wall into beverages is largely controlled by the diffusion coefficient in the polymer. The partition coefficient which represents the ratio between the concentration of a migrant in the polymer and in the food at the equilibrium plays a minor role only. Therefore, for the predictive evaluation of compliance of PET bottles knowledge of the diffusion coefficients of potential migrants is highly important since it allows to calculate the concentration of a migrant in the foodstuff under certain storage conditions.

Not only organic components are able to migrate from the PET bottle into the bottled beverages but also inorganic substances, for instance residues from polymerisation catalysts can leach from the polymer. The release of inorganic substances from beverage bottles made of glass versus PET has been investigated in a few studies (see literature review below). Very recently, a comprehensive study was published in which drinking water contamination from bottle materials was investigated for 57 chemical elements [START_REF] Reimann | Bottled drinking water: Water contamination from bottle materials (glass, hard PET, soft PET), the influence of colour and acidification[END_REF]. In this study the only element which was higher concentrated in PET bottled water was antimony with a 21-fold concentration over glass. All other elements were either comparable in PET and glass bottled water or considerably higher in glass bottled water. These parameters were in particular Cer (19-fold higher in glass), Lead (14-fold), Aluminium (7-fold) and other. The higher concentrations of antimony in PET bottle water is expected, because antimony trioxide (Sb 2 O 3 ) or it's reaction product with ethylene glycol is widely used as polycondensation catalyst for manufacturing of PET. The antimony catalyst offers a high catalytic activity and has a low tendency to catalyse side reactions. In addition antimony does not engender undesirable colours [START_REF] Duh | Effect of antimony catalyst on solid-state polycondensation of poly(ethylene terephthalate)[END_REF]. In principle the polymerisation catalyst remains in the PET polymer.

Within the last decade, several studies on the migration of antimony from PET bottles into beverages and on exposure levels in mineral water, soft drinks and juices have been published (see literature review below). In addition antimony, in the form of the chemical species antimony chloride, has been reported to possess weak estrogenicity [START_REF] Choe | Evaluation of estrogenicity of major heavy metals[END_REF] and since then numerous papers have been published in which a link between antimony from PET and possible estrogenic PET trays for ready-to-eat meals consist of crystalline PET. In a second study [START_REF] Alt | Diffusion coefficient of antimony catalysts in polyethylene terephthalate (PET) materials[END_REF] the diffusion coefficients of antimony in PET were determined from 45 °C up to 150 °C, most probably also crystalline PET trays. In comparison to microwave trays, PET beverage bottles have typically a significantly lower crystallinity of about 30% to 40%, which might influence the diffusion coefficients of antimony.

Since measured or predicted values should or need to be compared with established concentration limits the following is a short review of existing regulatory antimony Health Organisation [START_REF] Who | Antimony in drinking water[END_REF]. These drinking water limits are often used for the evaluation of antimony concentrations found in bottled beverages. From a legal point of view, however, these limits are applicable for drinking water itself before it is bottled. In PET packed beverages the specific migration limits according to packaging legislation are applicable. This is in Europe 40 µg/kg (EU 2002) and in Japan 50 µg/kg (JETRO 2009). The US Food and Drug Administration (FDA) has not specified a special migration limit for antimony from PET packaging materials.

Existing toxicological data suggest for humans a tolerable daily intake (TDI) for antimony of 6 µg kg -1 body weight [START_REF] Who | Antimony in drinking water[END_REF] which means that a daily consumption of 360 µg may be tolerable for an adult person with 60 kg body weight.

The objective of this study was: To determine experimentally the diffusion characteristics, i.e. activation energy and diffusion coefficients of antimony in PET

Literature review

Numerous studies have been published either on the antimony content of PET bottle material or on antimony concentrations in beverages like mineral water, soft drinks and juices. According to [START_REF] Duh | Effect of antimony catalyst on solid-state polycondensation of poly(ethylene terephthalate)[END_REF], antimony concentrations in PET plastics range between 190 mg/kg to 300 mg/kg. In the literature, some other studies can be found which confirm this concentration range where antimony has been used as catalyst.

The experimentally determined antimony concentrations found in PET bottles from literature data are summarized in Table 1. Much more papers have been published on investigations to which extend antimony might migrate from the bottle wall into the beverage. A compilation of these literature data for antimony concentrations found in packaged beverages and in food simulants as a result of a migration test is given in Table 2.

Place around here Table 1 Place around here Table 2 Materials and methods

PET bottle and pre-form samples

Altogether 67 PET bottle and pre-form samples were collected from European preform and PET bottle manufacturers, each with different raw materials, additives or recyclate source. The samples were supplied directly after manufacture. Information about the virgin material supplier, the recyclate manufacturer and the recyclate content as well as the use of oxygen or acetaldehyde scavengers was provided by the bottle or pre-form manufacturers in all cases.

Determination of antimony concentration in PET materials

200 mg of the PET bottle wall or pre-form material was treated with 8 ml conc. nitric acid for 15 min at 200 °C in a microwave-assisted digestion (similar to EN 13656).

Subsequently the concentration of antimony was determined quantitatively by ICP-MS according to DIN EN ISO 17294-2 (DIN 2003). Calibration was achieved by external calibration with antimony standards. As internal control standards rhodium and rhenium were used. The limit of detection was determined to 1 mg/kg in the PET material. The antimony concentrations of two bottles were verified by two other testing laboratories.

Determination of antimony migration kinetics into 3% acetic acid

To determine the specific migration of antimony into 3% acetic acid, a commercially available 1.5 l PET softdrink bottle was used. The bottle was supplied from the bottle manufacturer directly after blowing. The antimony bottle wall concentration was determined to 260 mg/kg by ICP-MS. The thickness of the bottle wall was determined to 0.27 mm. The crystallinity of the investigated bottle was determined by differential scanning calorimetry (DSC) to 43%. The bottle was cut into 5.0x5.0 cm pieces. The 5.0x5.0 cm pieces are further cut into 1.0x5.0 cm stripes. Five stripes of 1.0x5.0 cm were placed into a steel cell. Glass pellets of 3 mm diameter were added in order to reduce the dead volume of the extraction cell. The PET material was extracted with a Büchi Speed Extractor E-916 at 30 °C up to 150 °C. The extraction cells were filled with 3% acetic acid at a pressure of 100 bar in the extraction cell.

After an extraction time of 1 h, the extraction cell was purged into a glass vial.

Subsequently the extraction cell was filled and extracted again with 3% acetic acid.

The procedure was repeated every hour up to total extraction time of 4 h. For another part of the samples the extraction intervals were 1 h, 2 h and 3 h instead of 1 h intervals (total extraction time 6 h). Due to the fact, that the extraction volume after the Büchi Speed Extractor was not exactly the same, all migration solutions were filled to exactly 40.0 ml in a volumetric flask before analysing. Quantitative determination of the antimony concentration in the 3% acetic acid migration solutions was achieved by ICP-MS according to DIN EN ISO 17294-2 (DIN 2003). Calibration was achieved by external calibration with antimony standards. As internal standards rhodium and rhenium were used. The detection limit of the applied method was 2 µg/kg of antimony in 3% acetic acid solution.

Determination of diffusion coefficients

The antimony concentrations of the individual solutions at certain time were plotted against the square root of time. From the linear correlation of the diffusion coefficient was determined according to Equation 1. Due to the total immersion of the PET stripes, the surface area was doubled for the calculation of the diffusion coefficients.

Together with the edge surface and active surface of the PET samples of 51.62 cm 2 was calculated.

Place around here Equation 1Migration modelling

In addition to the experimental migration tests, a migration model based on diffusion coefficient estimation of chemical substances in polymers has been used (Piringer 2000). This model has been validated within the EU project SMT-CT98-7513 [START_REF] Piringer | Evaluation of migration models[END_REF][START_REF] Begley | Evaluation of migration models that might be used in support of regulations for food-contact plastics[END_REF]. The calculation of the migration was performed using the AKTS SML software version 4.51 (AKTS AG Siders, Switzerland). The bottles were supplied from pre-form and/or bottle manufactures. In addition to the pre-forms and bottles manufactured from virgin PET materials, pre-forms and bottles with post-consumer PET re-cyclates from eleven different PET recyclers were investigated. In these cases a worst-case situation was evaluated, because preforms and bottles made from 100% re-cyclates were investigated in most cases. The 100% approach was used, because the amount of PET re-cyclates in European softdrink and mineral water bottles varies typically between 10% and about 50% and not in every case, the amount of re-cyclates is known by the filling company. PET bottles and pre-forms manufactured from 100% re-cyclates gave in addition more similar results in comparison to bottles manufactured from virgin PET. The results of the experimentally determined antimony concentrations in the PET bottles and preforms are compiled in Figure 1. As a result, the mean value from all samples was 224 ±32 mg/kg (n = 67). The median was 220 mg/kg. The bottle wall concentrations determined in this study can be assumed as a basis for the migration calculations (see below).

Place around here Figure 1

Determination of the diffusion coefficients of antimony in PET

The diffusion coefficients of antimony in PET were determined with specific migration kinetics into 3% acetic acid as food simulant. In order to establish the diffusion coefficient at temperatures above the boiling point of water (or 3% acetic acid), a ) were introduced into a steel extraction cell. The PET was total immersed with 3% acetic acid at a pressure of 100 bar. The samples were extracted for 1 h at temperatures from 30 °C up to 150 °C. After the first migration phase, the migration cells were emptied and filled again with 3% acetic acid. A second migration phase follows which was either 1 h or 2 h. After the second migration phase, the migrations cells were emptied again. The procedure was repeated up to 4 h or either 6 h. All migrations solutions were analysed by ICP-MS for the antimony concentrations. The antimony concentration in the bottle wall (c P,0 ) was determined by ICP-MS to 260 mg/kg. The results of the migration kinetics are given in Table 3 andFigure 3. As a result of the migration studies Fickian behaviour of the antimony migration from the PET bottle wall into 3% acetic acid was found in all cases. Fickan migration behaviour means that the square root of time versus the migration follows a linear correlation. From the slope of these linear correlations, the diffusion coefficients could be calculated according to Equation 1. The experimentally determined diffusion coefficients are summarized in Table 3. Below a temperature of 90 °C, the diffusion coefficients could not be determined because the antimony concentrations in the migration solutions were below of the analytical detection limit (2 µg/kg) of the applied ICP-MS method. Therefore the determination of the diffusion coefficients was not possible. The diffusion coefficients of antimony in PET were also determined by [START_REF] Alt | Diffusion coefficient of antimony catalysts in polyethylene terephthalate (PET) materials[END_REF] between 45 °C and 150 °C (Table 3 and Figure 3).

Place around here Equation 2From the diffusion coefficients for temperatures between 105 °C and 150 °C the activation energy of the diffusion were calculated according to the Arrhenius approach (Equation 2). As a result, the inverse temperature (in K) and the logarithm of the diffusion coefficient result in a linear correlation (Figure 4). From the slope of this correlation the activation energy of diffusion of antimony from the PET bottle wall into 3% acetic acid was calculated to 189 kJ mol -1 . [START_REF] Alt | Diffusion coefficient of antimony catalysts in polyethylene terephthalate (PET) materials[END_REF] 2). Due to the fact, that antimony is determined in the PET bottle wall after digestion, the total amount of antimony is determined. Antimony might be also bound in the PET polymer chain. Polymer bound antimony, however, will not migrate into beverages. In addition, antimony trioxide itself will most probably also contribute a negligible part to the antimony migration. Therefore, the pre-exponential factor determined by Alt et al (2006) is different compared to the D 0 determined in this study due to most probably a different amount of migration relevant antimony species in the PET materials used for the migration studies.

Place around here Figure 2 Place around here Figure 3 Place around here Figure 4 Place around here Table 3 Modelling of antimony migration

The activation energy of diffusion can be used for the calculation of the migration of antimony from the bottle wall into the beverages. The applied migration model is based on the Equations 3 and 4 [START_REF] Crank | The mathematics of diffusion[END_REF], Piringer 2000). The term (m F,t /A) is the surface related migration. The concentration of antimony in the PET material is c P,0 , the wall thickness is d P and the density of the polymer is ρ P . The packaging geometry is part of the α factor (Equation 4). The kinetic part is represented by the temperature-dependent diffusion coefficient D p . The term q n is a correction factor for the positive square roots of the equation tan q n = -α q n . In conclusion, from the activation energy the diffusion coefficients for antimony in PET can be calculated at any temperature which can be used for the prediction of the antimony migration according to Equation 3.

Place around here Equation 3Place around here Equation 4The experimentally determined activation energy of antimony in PET was used for the prediction of the migration of antimony into contact media. Figure 5 . This is in good agreement with literature data for the antimony migration of 184 kJ mol -1 in a PET microwave tray. The activation energies were subsequently used for the calculation of the migration of antimony from the PET bottle wall into beverages under different conditions (Figure 5). With respect to food safety and consumer protection, the lower and therefore more conservative activation energy of 184 kJ mol -1 was used for the prediction of the antimony migration. In a first scenario, a bottle wall concentration of 224 mg/kg was assumed.

From this a maximum concentration of about 0.9 µg/kg can be calculated after a storage time of 300 d at 23 °C (c P,0 = 224 mg/kg) for a 500 ml PET bottle. In a second scenario, migration was calculated for a bottle wall concentration of As a result, the antimony migration even under worst case considerations is far below the specific migration limits of antimony of 40 µg/kg given by European packaging legislation. Under realistic storage conditions, this migration limit can never be reached. The migration values are calculated for two different bottle volumes (500 ml and 1500 ml). Larger PET bottles will lead to a lower antimony migration due to the more favourable surface/volume ratio, which result in a factor 1.43 higher antimony concentration in a 500 ml PET bottle in comparison to a 1500 ml bottle under otherwise equal contact conditions. Under realistic room temperature storage conditions (e.g. 23 °C), the lowest drinking water limit (5 µg/kg) can also not be exceeded (calculated for storage times up to about 900 d).

The literature data compilation in Table 2 gives an overview of the antimony migration studies from PET bottles beverages published over the last 10 to 15 years.

The data show that the antimony concentrations did not significantly change over the last decade. From the literature data given in Table 2 it is hard to derive, how many different mineral water, softdrink and juices samples were investigated within the last 10 to 15 years. But the amount is definitively several hundreds of beverage bottles.

The samples are coming from many different countries all over the world. However, not in every case it is clear if PET bottles were investigated. On the other hand, most of the literature studies were published from 2000 to 2010. It is therefore most likely that the majority of the investigated mineral water and soft drinks are packaged in PET. Most of the samples were drawn from local markets and the filling date as well as the storage conditions are not or only roughly known. In addition, the bottle wall concentration of antimony was not determined in most cases, which makes a comparison of the results of different migration studies difficult. However, most probably the bottle wall concentrations are in a similar range as found in this study and as given in Figure 1 2010) cannot be related to the packaging material but to the juice itself,. This conclusion which was also taken into consideration by Hansen et al, was supported by the fact, that the concentrations of antimony in cordials (before dilution) were significantly higher than the ready to drink juices. Cordials have to be concentrated which increases also the amount of inorganic compounds in the juice. In conclusion, the antimony contamination found by [START_REF] Hansen | Elevated antimony concentrations in commercial juices[END_REF] occurs in the juices before filling and is due to another contamination way. The migration of antimony from the PET bottle wall plays here a minor role.

Consumer safety considerations

It is very noteworthy that, as a result of the literature study, no single antimony concentration of the reported beverage samples bottled in PET exceeded the lowest drinking water concentration limit of 5 µg/kg. Furthermore, all mean or median values are found below 1 µg/kg. The specific migration limit for antimony of 40 µg/kg for bottled water set by food packaging legislation in Europe is far above any of the reported beverage samples covered by our literature search.

According to the extremely low diffusion coefficients of antimony species in PET at room temperature and somewhat elevated temperature up to 40°C, the resulting migration is extremely low (compare Figure 5). For example, the 5 µg/kg drinking water limit will be reached after about 10 years storage of a PET packed beverage (500 ml, c p,0 = 350 mg/kg, worst case scenario). It is interesting to note, that the equilibrium after such a long room temperature storage is still not established. At 40 °C the 40 µg/kg packaging threshold limit is reached under the above mentioned worst case conditions after about 12 years. At 80 °C the storage time for establishing a migration of 40 µg/kg is significantly reduced to about 33 h, however, still far above typical hot fill conditions of about a only few minutes at 80 °C. The results of our study substantiate as a matter of fact that the European specific migration limit of 40 µg/kg can never be exceeded in PET bottled beverages even when they were stored over years under worst conditions of e.g. 40 °C and even at higher technological relevant antimony levels in PET. This conclusion leads immediately to another consequence: antimony compliance testing on PET bottles for beverages and fatty liquids including edible oils is superfluous. This conclusion is complementary to one of our previous studies [START_REF] Störmer | New Concepts for Food Law Compliance Testing of Polyethylene Terephthalate Bottles[END_REF] on the migration behaviour of PET with regard to PET monomers and concerning overall migration. In this study we concluded that compliance testing on PET with regard to PET monomers and overall migration is unnecessary because the specified migration limits can never be exceeded by PET materials which are today on the market.

Migration modelling considerations can replace here migration testing.

WHO [START_REF] Who | Antimony in drinking water[END_REF] has set a tolerable daily intake (TDI) value for antimony of 6 µg antimony per kg body weight. This would translate into 360 µg intake for an adult with 60 kg body weight. The Swiss BAG (BAG 2005) came to the conclusion that the concentrations of antimony found in bottled mineral water samples can be considered as safe, because a consumption of 3 l of a mineral water per day with 1.2 µg/kg antimony which was the highest value found in the BAG study will result in only 1% of the TDI. Therefore the migrated amounts of antimony relative to the accepted tolerable daily intake (TDI) show that exposure is currently not of toxicological concern [START_REF] Bag | Antimon in Mineralwasser: Beurteilung des Gesundheitsrisikos[END_REF]. The results of our study are not only fully supportive to this conclusion which is based on analytical survey data but allow furthermore the prospective conclusion that as long as PET materials are [a] recycled PET, [b] virgin PET pellets with germanium as catalyst (41 ppm to 80 ppm), [c] virgin PET bottles with germanium catalysts (49 ppm to 82 ppm), [d] additional data from Chinese (n = 72) and Japanese (n = 105) market are given in Figure 1

  from polyethylene terephthalate (PET) are increasingly used

  (see for exampleSax 2010). In conclusion, antimony must be considered as the most important inorganic species which may migrate from the PET bottle wall into the beverages.For prediction of exposure levels, the diffusion coefficients of antimony in PET at different temperatures are helpful. Haldimann et al (2007) determined the migration of antimony into ready-to-eat meals under microwave or oven-cooking conditions.

  levels.. According to the German BfR recommendations the maximum concentration of antimony in PET bottles should not exceed 350 mg/kg (BfR 2010). Limits or guideline values for antimony in drinking water are specified in several countries: 5 µg/kg in Europe (EU 2003), 6 µg/kg in the United States and Canada (US EPA 2009, Canada 2008), 15 µg/kg in Japan (Japan 2003) and 20 µg/kg by the World

  . For this purpose it was also necessary to measure and compile the typical concentration range of antimony present in PET beverage bottles on the European market. Based on the results, the migration of antimony into beverages should be predicted by mathematical modelling for different surface/volume ratios and antimony bottle wall concentrations. Finally these results should be compared with data obtained from a comprehensive literature review on antimony diffusion in and migration from PET materials in contact with packaged beverages.

  the European PET bottle market PET bottles and pre-forms manufactured from materials of 15 different PET raw material suppliers were investigated within this study regarding their antimony concentration in the PET material. All investigated bottles are intended for the use as soft drink or mineral water bottles in Europe. The investigated samples can be considered as a comprehensive overview over the PET bottles available in Europe.

  test was chosen. For that purpose PET bottle wall stipes (surface area of 51.62 cm 2

  V F and volume polymer V P ) as well as the partition coefficient K P,F

  shows the results for the calculation of the migration into beverages from the diffusion coefficients at room temperature(23 °C, 30 °C and 40 °C) up to a storage time of 900 d. Due to the fact, that in our study, the activation energy was only determined above the glass transition temperature (T g ), we assumed the activation energy at temperatures below T g do not change. The data of Alt et al slightly indicate a different slope of the Arrhenius plot below T g , but based on this data a clear conclusion is not possible, because the data fit also in one linear regression over the whole temperature range. As a bottle wall concentration of antimony 224 mg/kg as well as 350 mg/kg was assumed. 224 mg/kg represents the mean value of the antimony bottle wall concentration found in this study. 350 mg/kg of antimony in the PET bottle wall is the worst case of the antimony concentration (BfR 2010). It should be noted here, that due to migration theory (Equation3) the concentration of antimony is directly proportional to the migration at certain contact conditions. E.g. if the antimony concentration in the bottle wall is e.g. by a factor of two lower than 350 mg/kg, the migration at a certain contact time is reduced by the same factor.According to the discussion above, the applied concentration of antimony in the PET bottle can be considered as worse case, because parts of the antimony in the PET is polymer bound and has therefore no contribution to the migration of antimony into the beverages. The partition coefficient was assumed to K = 1, which represents the migrant in the beverage. K = 1 can be therefore considered as the worst case of the antimony migration into beverages.Place around here Figure5Discussion and conclusionsPrediction of the antimony migration from PET bottlesThe most important parameters for the prediction of the migration of a chemical compound are the concentration of the migrant in the packaging material (c p,0 ), the diffusion coefficient (D) of the chemical species in the polymer as well as the partition coefficient (K) between the polymer and the contact media (Equation3). The partition coefficient, however, plays in the case of PET a minor role because the equilibrium is not reached assuming typical a shelf life of beverages. This is due to the low diffusivity of the PET polymer (Franz and Welle 2008), which means that for the prediction of the antimony migration the diffusion coefficients are the most important factors. The diffusion coefficients were determined for temperatures between 105 °C and 150 °C. The results of this study indicate that the migration of antimony follows a Fickian behaviour. From the temperature dependence of the diffusion coefficients, the activation energy of diffusion of antimony in the PET bottle was determined to be 189 kJ mol -1

  kg, which is the maximum concentration of antimony specified according to the German BfR recommendations (BfR 2010).

  levels of antimony, exposure from PET bottles through beverages and edible oils would be less than 1% of the current TDI. Finally, having substantiated such low antimony levels in PET bottled beverages the above addressed question on estrogenic effects caused by PET bottles appears to be groundless. Commission Directive 2002/72/EC of 6 August 2002 relating to plastic materials and articles intended to come into contact with foodstuffs. Last amendment: Commission Directive 2008/39/EC of 6 March 2008. EU 2003. European Commission Directive 2003/40/EC of 16 May 2003 establishing the list, concentration limits and labelling requirements for the constituents of natural mineral waters and the conditions for using ozone-enriched air for the treatment of natural mineral waters and spring waters. Ewender J, Franz R, Mauer A, Welle F. 2003, Determination of the migration of acetaldehyde from PET bottles into non-carbonated and carbonated mineral water. Deutsche Lebensmittel-Rundschau 99(6):215-221. Fiket Z, Roje V, Mikac N, Kniewald G. 2007. Determination of arsenic and other trace elements in bottled waters by high resolution inductively coupled plasma mass spectrometry. Croatica Chemica Acta 80(1):91-100. Fordham PJ, Gramshaw JW, Crews HM, Castle L. 1995. Element residues in food contact plastics and their migration into food simulants, measured by inductively coupled plasma-mass spectrometry. Food Additives and Contaminants 12(5):651-669. Franz R, Welle F. 2008. Migration measurement and modelling from poly(ethylene terephthalate) (PET) into soft drinks and fruit juices in comparison with food simulants. Food Additives and Contaminants 25(8):1033-1046. Güler C. 2007. Evaluation of maximum contaminant levels in Turkish bottled drinking waters utilizing parameters reported on manufacturer's labelling and governmentissued production licenses. Journal of Food Composition and Analysis 20:262-272. Haldimann M, Blanc A, Dudler V. 2007. Exposure to antimony from polyethylene terephthalate (PET) trays used in ready-to-eat meals.

Figure 1 :Figure 3 :Figure 4 :Figure 5 :

 1345 Figure 1: Antimony concentrations in PET beverage bottles and preforms from the European market (n = 67)

  has determined activation energy of diffusion of antimony in PET trays of 184 kJ mol

	the slopes of the logarithm of the diffusion coefficients versus the inverse
	temperature are similar in both studies, the activation energies of diffusion are very
	similar within both studies. However, the pre-exponential factor D 0 determined in our
	study (7.5 10	12 ) is different from that found by Alt et al (1.0 10	14 ). D 0 is mainly
	influenced by the surface area and the concentration of the migrant in the PET bottle
	wall (c p,0 ). Whereas the surface area can be determined very easily, the
	concentration of the migrant in the PET bottle wall for inorganic compounds is not F known exactly. According to Duh (2002), Biros et al (2002) and El-Toufaili et al o (2006) the antimony compound present in PET polymer as antimony glycolate r P complexes (examples are given in Figure
		e
		e r
		R
		e
		v i e
		w
		O n l
		y
			12

-1 which is in good agreement with the activation energy found in this study. Due to the fact, that

  or Table 1. The storage time before analysing is most probably only a few months. Assuming a storage time of only a few months at room

	beverage at 23°C up to one year would lead to a migration below of about 1 µg/kg.
	The calculated migration in this study (Figure 5) is also in good agreement with
	published data (e.g. Figure 2 in Keresztes et al 2009). Keresztes et al found that an
	antimony concentration of about 1 µg/kg in a 1.5 l PET bottle after storage of 950 d
	at room temperature.
	In a very recent study, Hansen et al (2010) found antimony concentrations up to
	about 14 µg/kg in red juices. The authors investigated ready to drink juices or F cordials, which should be diluted with water prior to consumption. It is interesting to o r note, that the highest concentrations (4 µg/kg to 8 µg/kg) found by Hansen et al are
	P coming from one specific brand packed in completely different packaging materials
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

temperature before analysing, the antimony concentrations found in the literature are in good agreement with the calculated data from this study, where a storage of a e.g. PET, cardboard and glass In addition, the highest concentration was found in the glass packed juice. Therefore, the antimony concentrations found by

Hansen et al (

Table 2 :

 2 Literature data for antimony concentrations determined in bottled beverages and food simulants

		Food Additives and Contaminants
	Investigated samples	Determined antimony concentrations	Remarks	Reference
	PET pellets	Individual conc. 3% acetic acid (10 d at	3 g of PET pellets were totally	Fordham et al 1995
		40 °C): 2.7 ppb and 1.2 ppb, 3% acetic	immersed with 20 ml of food
		acid (2 h at 100 °C): 3.9 ppb and 2.6 ppb,	simulant.
		15% ethanol (10 d at 40 °C): 2.3 ppb and
		1.1 ppb, olive oil <0.01 ppb (both
		conditions)
	56 European bottled	Individual conc. 0.003 ppb to 1.06 ppb;	Packing materials were not	Misund et al 1999
	mineral water brands	median conc. 0.17 ppb	specified
	One orange juice	Conc. 2.2 ppb	Packing material was not specified Ulrich 2000
	sample
	42 Mineral water	Mineral water: average conc. 0.32 ppb (n	Packing materials were not	Dabeka et al 2002
	brands from Canada	= 42), spring water: average conc.	specified. Samples drawn
	(199 different	0.30 ppb (n = 102), distilled water:	December 1995 to February 1996
	samples)	average conc. 0.17 ppb (n = 25), soda
		water: average conc. 0.29 ppb (n = 19),
		tap water: average conc. 0.17 ppb (n =
		11), individual conc. 0.03 ppb to 1.31 ppb
	Food simulants 3% acetic acid and 95% ethanol	Individual conc. in 3% acetic acid: 0.15 µg dm 2 to 0.57 µg dm 2 (n = 7, assuming a 1 l bottle with 6 dm 2 surface	simulant Storage for 10 d at 40 °C, surface volume ratio of 2 dm 2 for 100 ml	Störmer et al 2004
		area will result in concentrations between
		0.90 ppb to 3.42 ppb), in 95% ethanol: <0.3 µg dm 2
	Mineral water	Average conc. (PET): 0.43 ppb (n = 49);	Samples were packed in glass and
		average conc. (glass): factor 3.5 lower	PET.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Page 29 of 36 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 3 :

 3 Diffusion coefficients of antimony in PET

	Temperature [°C]	Diffusion coefficient [cm 2 s -1 ]
		this study	Alt et al (2008)
	45		1.4 10 -16
	60		1.0 10 -15
	75		9.1 10 -15
	90		2.7 10 -13
	105	3.8 10 -14	4.7 10 -12
	120	1.4 10 -12	3.0 10 -11
	130	2.7 10 -12

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgement

Thanks are due to Dominik Fiedler, Manuel Huber, Annika Seiler, Veronika Wildgruber and Gerd Wolz (Fraunhofer IVV) for experimental contributions and fruitful discussions. Information from Dr. Yoko Kawamura from the National Institute of Health Sciences in Tokyo on the Japanese PET market is gratefully acknowledged.


Josée Nina Bouchard
email: josee_bouchard@yahoo.com

Duncan Alastair Purdie

3-like protein activity in cultures of the harmful dinoflagellates Karenia brevis and Karenia mikimotoi

Keywords: caspase 3-like protein activity, cell death, harmful dinoflagellate, Karenia spp caspase 3-like protein activity, cell death, harmful dinoflagellate, Karenia spp

come

Temporal variation of caspase 3-like protein activity in cultures of the harmful dinoflagellates Karenia brevis

and Karenia mikimotoi.

INTRODUCTION

The excessive growth of specific microalgae and cyanobacteria are of concern since a number of them can produce potent toxins. Karenia brevis and K. mikimotoi are unarmoured marine dinoflagellates which can form Harmful Algal Blooms (HABs). Karenia brevis is a producer of brevetoxins and is responsible for the occurrence of red tides in the Gulf of Mexico where it is well known to cause fish and marine mammal mortalities [START_REF] Walsh | Red tides in the Gulf of Mexico: where, when and why?[END_REF]. Karenia mikimotoi is common in the waters around Japan and in the Northeast Atlantic region where blooms of this species have been associated with fish and shellfish kills [START_REF] Gentien | Bloom dynamics and ecophysiology of the Gymnodinium mikimotoi species complex[END_REF]. Because of the economic impacts of HABs and their effects on the marine biota and humans, there have been many studies on how environmental factors influence their initiation and the production of toxins [START_REF] Cembella | Molecular physiology of toxin production and growth regulation in harmful algae[END_REF][START_REF] Granéli | Chemical and physical factors influencing toxin content[END_REF][START_REF] Vargo | A brief summary of the physiology and ecology of Karenia brevis Davis (G. Hansen and Moestrup comb. nov.) red tides on the West Florida Shelf and of hypotheses posed for their initiation, growth, maintenance, and termination[END_REF]. Less attention has however been devoted to the study of the factors leading to the demise of HABs. Among some of the different factors known to play a role in the termination of HABs are water mass advection, cyst formation, nutrient limitation, grazing, cell lysis due to lytic bacteria or viruses and cell death [START_REF] Vargo | A brief summary of the physiology and ecology of Karenia brevis Davis (G. Hansen and Moestrup comb. nov.) red tides on the West Florida Shelf and of hypotheses posed for their initiation, growth, maintenance, and termination[END_REF].

The study of cell death processes in microalgae is still in its infancy and is virtually nonexistent in harmful microalgae (Veldhuis and Brussard, 2006). However, several other species of microalgae have been proposed to undergo programmed cell death (PCD), necrosis [START_REF] Dunn | Programmed cell death and cell necrosis activity during hyperthermic stress-induced bleaching of the sea anemone Aiptasia sp[END_REF] as well as a range of intermediate cell death modes [START_REF] Franklin | Mortality in cultures of the dinoflagellate Amphidinium carterae during culture senescence and darkness[END_REF][START_REF] Franklin | What is the role and nature of programmed cell death in phytoplankton ecology?[END_REF][START_REF] Jiménez | Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase[END_REF]. Because it results in the complete loss of the organism, the occurrence of PCD in unicellular cells seems counterintuitive. It is believed that phytoplankton undergo PCD as a strategy to relieve a population of nutrient stress, to remove ageing and/or damaged cells, or to limit the multiplication of viruses during infection (reviewed by [START_REF] Bidle | Cell death in planktonic photosynthetic microorganisms[END_REF]. PCD could also be used to maximise the biological fitness of the community (reviewed by [START_REF] Bidle | Cell death in planktonic photosynthetic microorganisms[END_REF], a strategy suggesting that cells have awareness of the state of their conspecifics/predators and a potential for signalling [START_REF] Toth | Marine dinoflagellates show induced life-history shifts to escape parasite infection in response to water-borne signals[END_REF]. PCD is an autocatalytic process that may lead to the apoptosis of the cell, a process characterized by specific morphological and biochemical requirements such as condensation and margination of the chromatin and nuclear fragmentation [START_REF] Kerr | Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics[END_REF]. This process is orchestrated by caspases, a family of cysteine proteases with unique substrate specificities [START_REF] Kurokawa | Caspases and kinases in a death grip[END_REF]. Alternatively, necrosis is described as a passive and unprogrammed type of cell death characterised by a loss of membrane integrity, 4 cell swelling and lysis of the cell. In contrast to PCD, necrosis has typically been defined as a caspase-independent event, but some evidence suggests that this is not always the case (Van Cruchten and [START_REF] Van Cruchten | Morphological and Biochemical Aspects of Apoptosis, Oncosis and Necrosis[END_REF]. It is particularly challenging to determine how cell death operates in unicellular organisms given the literature's natural bias for multicellular organisms and mammalian cells [START_REF] Franklin | What is the role and nature of programmed cell death in phytoplankton ecology?[END_REF].

In mammalian cells, caspases are divided into two groups: initiator caspases (e.g. caspase-2, -8, -9, -10) which regulate and activate other caspases; and executioner caspases (e.g. caspase-3, -6, -7) which act downstream of the cascade after being activated by upstream caspases [START_REF] Kurokawa | Caspases and kinases in a death grip[END_REF]. Although caspases have never been identified in microalgae, antibodies raised against human caspases have been shown to crossreact with extracts of, among others, marine cyanobacteria and chlorophytes [START_REF] Segovia | Cell death in the unicellular chlorophyte Dunaliella tertiolecta. A hypothesis on the evolution of apoptosis in higher plants and metazoans[END_REF][START_REF] Berman-Frank | The demise of the marine cyanobacterium, Trichodemium spp., via an autocatalyzed cell death pathway[END_REF][START_REF] Moharikar | Apoptotic-like cell death pathway is induced in unicellular chlorophyte Chlamydomonas reinhardtii (chlorophyceae) cells following UV irradiation: Detection and functional analyses[END_REF][START_REF] Zuppini | Heat stress: an inducer of programmed cell death in Chlorella saccharophila[END_REF]. Additionally, caspase activity assays performed using artificial substrates (designed to be specific for mammalian caspases) showed increases in caspase-like protein activities associated with the occurrence of death in a variety of microalgal species under stress conditions [START_REF] Berman-Frank | The demise of the marine cyanobacterium, Trichodemium spp., via an autocatalyzed cell death pathway[END_REF][START_REF] Segovia | Effects of inhibitors of protein synthesis and DNA replication on the induction of proteolytic activities, caspase-like activities and cell death in the unicellular chlorophyte Dunaliella tertiolecta[END_REF][START_REF] Ross | Toxin release in response to oxidative stress and programmed cell death in the cyanobacterium Microcystis aeruginosa[END_REF][START_REF] Zuppini | Heat stress: an inducer of programmed cell death in Chlorella saccharophila[END_REF][START_REF] Bidle | Iron starvation and culture age activate metacaspases and programmed cell death in the marine diatom Thalassiosira pseudonana[END_REF][START_REF] Darehshouri | Cell death upon H 2 O 2 induction in the unicellular green alga Micrasterias[END_REF][START_REF] Affenzeller | Salt stress-induced cell death in the unicellular green alga Micrasterias denticulata[END_REF][START_REF] Bouchard | Implication of nitric oxide in the heat-stress-induced cell death of the symbiotic alga Symbiodinium microadriaticum[END_REF][START_REF] Jiménez | Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase[END_REF]. Finally, the use of mammalian caspase inhibitors has been shown to prevent the occurrence of death in diatoms, dinoflagellates and chlorophytes [START_REF] Vardi | Programmed cell death of the dinoflagellate Peridinium gatunense is mediated by CO 2 limitation and oxidative stress[END_REF][START_REF] Bidle | Iron starvation and culture age activate metacaspases and programmed cell death in the marine diatom Thalassiosira pseudonana[END_REF][START_REF] Segovia | Inhibition of caspase-like activities prevent the appearance of reactive oxygen species and dark-induced apoptosis in the unicellular chlorophyte Dunaliella tertiolecta[END_REF]. The presence of variant forms of caspases, called metacaspases [START_REF] Uren | Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma[END_REF], in protists suggests they could be involved in the PCD process of phytoplankton. However, because metacaspases possess a substrate affinity different from that of caspases [START_REF] Vercammen | Type II metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana cleave substrates after arginine and lysine[END_REF][START_REF] Watanabe | Two Arabidopsis metacaspases AtMCP1b and AtMCP2b are arginine/lysine-specific cysteine proteases and activate apoptosis-like cell death in yeast[END_REF], enzymatic activities measured in protists (and in this study) using the classical aspartatecontaining caspase substrates do not provide any indication of the implication of metacaspases in PCD. Indeed, the actual identity of the proteases responsible for these caspase-like activities still remains to be clearly determined [START_REF] Bonneau | What happened to plant caspases?[END_REF].

Despite the suggested implication of caspase-like proteins in the death of unicellular algae, the actual role of caspase-like proteases in microalgae remains unclear. In mammalian cells, cell death events have been shown to occur with or without the involvement of caspases [START_REF] Leist | Four deaths and a funeral: from caspases to alternative mechanisms[END_REF]) and these enzymes have been proposed to have alternative cellular functions independent of cell death [START_REF] Los | Caspases: more than just killers?[END_REF][START_REF] Yi | The Jekyll and Hyde Functions of Caspases[END_REF]. In microalgae, F o r P e e r R e v i e w 5 caspase-like protein activities have been proposed to be constitutive and to have some housekeeping functions [START_REF] Segovia | Effects of inhibitors of protein synthesis and DNA replication on the induction of proteolytic activities, caspase-like activities and cell death in the unicellular chlorophyte Dunaliella tertiolecta[END_REF] and, increases in caspase-like protein activity have also been reported in cells of Dunaliella viridis undergoing cell death events (e.g. autophagic-like and paraptotic-like deaths) which normally do not require the activation of caspase-like proteases to proceed [START_REF] Jiménez | Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase[END_REF]. Clearly, the implication of caspase-like protein activities in microalgal processes remains to be clarified. Most of the studies performed so far with microalgae have assessed the variations of caspase-like protein activities under stressful conditions. To obtain a better understanding of the role of caspaselike protein activities in microalgal processes, it is important to determine their basal level in microalgae grown in cultures under relatively stress-free conditions. The aim of this study therefore was to monitor the variation of caspase 3-like protein activity along with other physiological variables over more than 50 days in batch cultures of the toxic species of dinoflagellate, Karenia brevis and K. mikimotoi. Two cultures of the harmful dinoflagellates, Karenia brevis (CCMP 2228) and K. mikimotoi (PLY 705) were grown in 500 ml volumes of L1-Si medium [START_REF] Guillard | Stichochrysis immobilis is a diatom, not a chrysopyte[END_REF].

Cells were grown under a 12 h:12 h light : dark cycle at an irradiance of ~ 30 µmol photons m -2 s -1 . Following the respective culture collection's recommendations, K. brevis and K.

mikimoitoi were grown at a temperature of 25 °C and 15 °C, respectively. During the dark phase of day 27, a fault with the incubator set at 25 °C caused the temperature to gradually decline until it reached 18 °C the following morning. The temperature was back to 25 °C at the time of sampling K. brevis on day 28 (at 12:00). Four independent culture flasks were used as replicates for each of the two species considered in this study. Sampling was performed weekly over a 56-d period (excluding the 49 th day for K. brevis) and consistently performed at the same time, i.e., 5 h into the light period. The initial cellular abundance in each culture flask was between 100-300 cells mL -1 .

Cellular abundance, cyst enumeration and growth rate determination

Cell counts were determined on 1-mL of Lugol's fixed samples (kept at 4 °C) using a Sedgwick-Rafter chamber and a Nikon microscope (Eclipse E200). The growth rate (µ) was calculated as µ = ln (N T / N 0 ) / (∆t) where N 0 is the population cell density at the beginning of a time interval, N T the cell density at the end of the time interval and ∆t the length of the time interval (t T -t 0 ). The number of doublings per day (k) was calculated as k = µ / ln2 and the doubling time (T 2 ) calculated as T 2 = ln2 / µ. Cyst-like structures were also observed in K. brevis samples. These cyst-like structures were enumerated using the same method described above for the cellular abundance. The total cellular abundance reported for K. brevis excludes the cyst-like structures. Although a resting cyst stage has never been documented for K. brevis, the presence of a sexual cycle is evident from the existence of planozygotes [START_REF] Walker | Evidence for a sexual cycle in the Florida red tide dinoflagellate, Ptychodiscus brevis (=Gymnodinium breve)[END_REF][START_REF] Steidinger | Bloom dynamics and physiology of Gymnodinium breve with emphasis on the Gulf of Mexico[END_REF][START_REF] Van Dolah | The Florida red tide dinoflagellate Karenia brevis: New insights into cellular and molecular processes underlying bloom dynamics[END_REF]. The cyst-like structures observed in the present study are similar to asexual temporary or ecdysal cysts which have been reported for stressed dinoflagellates and for K. brevis upon exposure to algicidal bacteria [START_REF] Roth | Comparative analysis of two algicidal bacteria active against the red tide dinoflagellate Karenia brevis[END_REF]. To obtain an indication of the physiological condition of the microalgal cells [START_REF] Kolber | Use of active fluorescence to estimate phytoplankton photosynthesis in situ[END_REF], the maximum photochemical efficiency of photosystem II (F v /F m ) was determined by using a fluorescence induction and relaxation (FIRe) fluorometer (Satlantic).

Photosynthetic efficiency -F v /F m

In order to allow for the relaxation of non-photochemical quenching, the cells were darkacclimated for 20 min at the growth temperature. F v /F m was determined as (F m -F 0 )/F m where F 0 is the minimal fluorescence when all the reaction centres are open and F m , the maximal fluorescence measured when all of the reaction centres are closed.

Cell death -SYTOX-labelling

Cell death was quantified using SYTOX-green (Invitrogen), a nucleic acid-specific mortal stain that only passes across the plasma membrane of dead cells, staining the nucleus brilliant green under blue light illumination. Since SYTOX-green only penetrates cells with a compromised membrane it provides an indication of a late stage in cell death. It is important to mention that SYTOX does not label cysts or lysed cells. Stain concentration and incubation time were optimized prior to use and assays were validated using heat-killed cells (heating at 65 °C for 1h) to verify that stain uptake was proportional to the quantity of dead cells. SYTOX-green working stocks were prepared in Milli Q water to a final concentration of 50 µM. After the addition of SYTOX-green to a final concentration of 0.5 µM, 1.5 mLsamples of algal culture were incubated in the dark, at room temperature for 1 h. SYTOXpositive cells were counted using a Zeiss epifluorescence microscope (Ex: 465-495 nm; Em: 515-555 nm) and a glass Sedgwick-Rafter chamber. For each sample, the abundance of SYTOX-positive cells present in four to eight rows of the chamber (50 µl/row) was determined. To estimate the percentage of dead cells, the concentration of SYTOX-positive cells was divided by the concentration of Lugol's fixed cells and multiplied by 100. SYTOXpositive cells were photographed using a Zeiss epifluorescence microscope equipped with a digital camera.

Protein extraction and quantification

Ten mL samples of each culture were centrifuged (Rotanta 460 R, Hettich Zentrifugen, Germany) at 4 °C for 10 min (3800g). The supernatant was then carefully removed and the pellets containing the algal cells snap frozen at -80 °C. Pellets were then suspended in 200 µl of extraction buffer (100 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl, pH 7.4) and sonicated on ice using a microtip sonicator (Vibra-Cell, Sonics and Materials, Inc.) until just thawed

Caspase 3-like protein activity determination

Caspase 3-like protein activity was determined using the Enzchek® Caspase-3 Assay Kit #2 (Invitrogen). Cell extracts (50 µl) were incubated with the caspase substrate Z-DEVD-R110 (25 µM, final concentration) for 3.5 h in the dark, at room temperature. The appearance of fluorescent rhodamine 110 (R-110) upon enzymatic cleavage of the non-fluorescent substrate Z-DEVD-R110 was subsequently assayed using a microplate reader (Ex: 485 nm; Em: 520 nm). Negative controls (samples without the Z-DEVD-R110 substrate) and substrate-only controls (mixture of activity buffer and Z-DEVD-R110 substrate to determine the background fluorescence of the substrate) were also carried out. Pre-incubating the cell extracts for 20 min with the reversible caspase inhibitor Ac-DEVD-CHO (20 µM, final concentration) prior to the substrate addition confirmed that the fluorescence signal observed with the extracts of Karenia cells was due to the activity of caspase 3-like proteases.

DNA fragmentation -TUNEL labelling

Nuclear DNA fragmentation was assessed using the In situ Cell Death Detection Kit, Fluorescein (Roche diagnostics). This terminal deoxynucletidyl transferase-mediated dUTP nick end labelling (TUNEL) assay labels free 3-OH ends of DNA and is used to detect fragmented DNA and to visualise apoptotic cells. Due to the limited culture volume and the length of the experiment, this assay was only performed once, i.e., when the cultures had reached the late decline phase as it was initially hypothesised that the caspase 3-like activity would be highest during this phase. Twenty-five mL of 50-d old cultured cells of K. brevis and K. mikimotoi were fixed with 0.1 % glutaraldehyde, centrifuged for 15 min at 1500g at 4°C. The pellet containing the cells was permeabilized using 0.1% Triton X-100 in PBS for 15 min on ice. Cells were subsequently washed twice with PBS and labelled following the manufacturers' instructions. Samples were resuspended in PBS and the green fluorescence Negative controls were treated as described above but labelled only with the label solution (without terminal transferase). Positive controls consisted of permeabilized cells pre-treated with DNase I (Sigma) for 15 min prior to labelling with the TUNEL reaction mixture. All of the cells present in the positive control samples of K. brevis and K. mikimotoi were stained with the label solution.

Statistical analyses

To assess the differences for the caspase 3-like protein activity in the presence or absence of caspase inhibitor (Fig. 3A-B), repeated measures analyses of variance (rm ANOVAs) (Minitab 15.1.0.0, Minitab Inc.) were performed with time as the repeated factor. When significant differences were found, a t-test was performed for each specific sampling day (except for day 56 for K. mikimotoi due to the lack of replication for the inhibitor treatment).

RESULTS

Cellular abundance, growth rates, cyst-like structure abundance and physiological state of Karenia cells

The cellular abundance of K. brevis increased from ~ 300 cells mL -1 to more than 7,500 cells mL -1 in 21 days (Fig. 1A). On days 28 and 35, the abundance was ~ 4,000 cells mL -1 and after day 35, the abundance gradually decreased until it reached 390 cells mL -1 on the last day of sampling (Fig. 1A). The growth rate calculated for this species was 0.18 d -1 with a doubling time of 3.8 days (Table I). Karenia mikimotoi was characterised by a longer lag phase and slower growth rate compared to K. brevis. The cellular abundance only peaked on day 42

(1,443 cells mL -1 ) after which it declined to reach 212 cells mL -1 on the last day of sampling (Fig. 1A). The growth rate determined for K. mikimotoi was 0.07 d -1 and the doubling time 9.6 days (Table I).

The number of cyst-like structures present in K. brevis samples (Fig. 2B inset) started to increase on day 21 and reached its highest values on day 28 (1,200 cyst-like structures mL - 1 ). Their abundance subsequently declined to reach less then 200 cyst-like structures mL -1 on day 56 (Fig. 1B). The maximum photochemical efficiency of photosystem II, F v /F m, which generally provides an indication of the physiological state of the cells varied between 0.50 and 0.70 for the two studied species (Fig. 1C). For K. brevis, F v /F m values increased from an initial value of 0.55 to a value of 0.65 on day 21 (Fig. 1C). Values subsequently remained relatively high and varied between 0.59 (day 35) and 0.64 (Fig. 1C). F v /F m values for K. mikimotoi, increased from an initial 0.50 to reach 0.70 on day 14, and then remained between 0.66 and 0.69 for the rest of the sampling period (Fig. 1C).

Mortality -SYTOX-positive cells

The DNA marker SYTOX-green was used to discriminate between live cells (SYTOXnegative cells) and cells with a compromised cell membrane (dead cells = SYTOX-positive cells). The abundance of SYTOX-positive cells for K. brevis reached a maximum value on day 21 with 1,273 cells mL -1 (Fig. 2A), then decreased but remained around 300 cells mL -1 up to day 35 before reaching values lower than 110 cells mL -1 for days 42 and 56 (Fig. 2A).

The abundance of SYTOX-positive cells remained variable for K. mikimotoi cells with peaks of ~ 40 cells mL -1 observed on days 21 and 42 (Fig. 2B). The highest value occurred on day 56 with 86 cells mL -1 (Fig. 2B).

The percentage of SYTOX-positive K. brevis cells remained below 10 % up to day 14 (Fig. 2C) and, except for the transient decline observed between days 28 and 35, values subsequently remained near 15 % until the end of the experiment when it increased to 20 % (day 56) (Fig. 2C). The transient decline in the percentage of SYTOX-positive cells observed between days 28 and 35 coincided with the increase in the abundance of cyst-like structures (Fig. 1B). The low initial percentage of SYTOX-positive K. mikimotoi cells increased to reach 12 % on day 14, then subsequently decreased to near zero on day 28 (Fig. 2C), i.e., when the cells were actually growing (Fig. 1A), and increased again on the last day of sampling to reach 44 % (Fig. 2C). Insets in Figures 2A andB In K. brevis cells, a constitutive level of caspase 3-like protein activity was observed throughout the experiment (Fig. 3A). Initial values (on days 0, 7, and 14) were slightly below 4,000 RFU/h/mg protein and the subsequent increase in activity on days 21 and 28 (~ 38,000 RFU/h/mg protein on day 28) coincided with the increase in the abundance of cyst-like structures. On days 35, 42 and 56, the activity remained between 3,140 and 6,664 RFU/h/mgprotein (Fig. 3A). For K. mikimotoi, the caspase 3-like protein activity varied between 1,000 and 1,300 on days 0, 7 and 14 (Fig. 3B). On days 21 and 28 the activity decreased to near detection limits before increasing again on days 35 and 42 (~ 656 and 500 RFU/h/mg protein, respectively), i.e., just before the decline in cellular abundance. Values on days 49 and 56 were also near detection limits (Fig. 3B).

Table II presents the results of the repeated-measures ANOVAs which were performed to verify the presence of significant differences between the caspase 3-like activity measured in the presence and the absence of the inhibitor. Pre-treating the cell extracts with the reversible caspase inhibitor Ac-DEVD-CHO (20 µM, final concentration) before the caspase substrate supplementation efficiently prevented the increase in caspase 3-like protein activity (Fig. 3AB; Table II). The inhibitor was very effective in both K. brevis and K. mikimotoi cells for which it inhibited, on average, 97.6 % and 92.8 %, respectively, of the caspase-like protein activity (Fig. 3A-B, Table II).

DNA fragmentation

Using a TUNEL assay, the presence of DNA degradation was assessed in cells of Karenia which had reached the cultures' decline phase. Figures 4B andF display bright field micrographs of K. brevis and K. mikimotoi, respectively, while figures 4A and E show the same samples with the localized incorporation of the TUNEL fluorescent label, which is indicative of DNA fragmentation in both species of Karenia. Although the total number of TUNEL-positive cells was not counted, the majority (~ 85 %) of K. brevis and K. mikimotoi cells contained degraded DNA. Results obtained for the positive control treatment, which involved the pre-treatment of the samples with DNase (Fig. 4C-G), are comparable to those obtained with Karenia cells from the late decline phase (Fig. 4A-E) and therefore confirm the positive-TUNEL labelling in both species of Karenia. The negative controls show the absence of incorporation of the TUNEL fluorescent label (Fig. 4D-H).

DISCUSSION

Understanding the factors controlling the growth, division and death processes in protists such as dinoflagellates is complex due to the unique nature of these organisms.

Dinoflagellates usually reproduce vegetatively but they can also reproduce sexually, some possessing a thick cellulosic cell wall while others do not, and they can exploit different nutritional modes. The chromosomal structure of dinoflagellates is also unique as they possess a special form of nucleus in which the chromosomes are almost permanently condensed throughout the cell cycle (reviewed by [START_REF] Segovia | Programmed cell death in dinoflagellates[END_REF]. The type of cell death occurring in dinoflagellates and other microalgae is thought to be species-specific and/or to be dependent on a triggering factor [START_REF] Segovia | Programmed cell death in dinoflagellates[END_REF][START_REF] Jiménez | Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase[END_REF]. In mammalian cells, high levels of caspase activation have been proposed to mediate apoptosis while the limited activation of the same enzymes may play a role in other cellular processes such as cell proliferation, cell differentiation, DNA repair, etc. [START_REF] Los | Caspases: more than just killers?[END_REF][START_REF] Algeciras-Schimnich | Apoptosis-independent functions of killer caspases[END_REF][START_REF] Abraham | Death without caspases, caspases without death[END_REF][START_REF] Launay | Vital functions for lethal caspases[END_REF]. Caspase-like activities measured so far in diverse species of microalgae under stress have generally been proposed to play a role in the occurrence of an active cell death process similar to PCD in metazoans [START_REF] Bidle | Cell death in planktonic photosynthetic microorganisms[END_REF]. Recently, however, a form of PCD has been shown to be a caspase-like independent event [START_REF] Affenzeller | Salt stress-induced cell death in the unicellular green alga Micrasterias denticulata[END_REF] while cell death modes other than PCD have been shown to require the involvement of caspase 3-like protein activity to proceed [START_REF] Jiménez | Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase[END_REF]. It is essential to obtain a better understanding of the role of caspase-like proteins in the cellular activities of microalgae. The aim of this study was to follow the temporal variation of the caspase 3-like protein activity in two cultures of harmful dinoflagellates and to verify its possible implication in the cellular processes of Karenia spp.

With a reported optimal growth temperature between 22 and 28 °C [START_REF] Vargo | A brief summary of the physiology and ecology of Karenia brevis Davis (G. Hansen and Moestrup comb. nov.) red tides on the West Florida Shelf and of hypotheses posed for their initiation, growth, maintenance, and termination[END_REF], cells of K. brevis grew well during the first three weeks of the experiment. The growth rate determined for K. brevis (0.18 d -1 ) was in agreement with that reported in the literature for the same species (0.17-0.36 d -1 ; [START_REF] Magaña | The effect of environmental factors on the growth rate of Karenia brevis (Davis) G. Hansen and Moestrup[END_REF]. The percentage of dead K. brevis cells which remained between 3 and 20 % during the whole experimental period, suggests that a fraction of the K. brevis population was constantly undergoing cell death. This observation is in agreement with the constitutive level of caspase 3-like protein activity which was measured throughout the experiment in cells of K. brevis. This constitutive level 13 of caspase 3-like protein activity could well have served some housekeeping functions [START_REF] Segovia | Cell death in the unicellular chlorophyte Dunaliella tertiolecta. A hypothesis on the evolution of apoptosis in higher plants and metazoans[END_REF] and/or played a role in the regulation of the cell cycle [START_REF] Algeciras-Schimnich | Apoptosis-independent functions of killer caspases[END_REF]. The caspase 3-like protein activity generally remained between 3, 000 and 6,665 RFU/h/mg protein throughout the whole sampling period except on days 21 and 28 when it increased to reach its highest value (~ 38, 000 RFU/h/mg protein on day 28). This increase in activity matched the increase of the abundance of cyst-like structures observed on the same days. This suggests a role of caspase-3 like proteases in the cell cycle/encystment progression of K. brevis and provides evidence for their involvement in processes other than cell death [START_REF] Algeciras-Schimnich | Apoptosis-independent functions of killer caspases[END_REF]. Combined together the formation of cyst-like structures and the death of K. brevis cells (revealed by SYTOXlabelling) do not entirely explain the decline in cellular abundance which occurred after day 28 and therefore suggests that a fraction of the community may have undergone lysis and escaped the SYTOX-labelling.

The appearance of cyst-like structures in cultures of K. brevis can cautiously be attributed to the cells entering the stationary phase. However, the presence of such a high abundance of cyst-like structures in other cultures of K. brevis which were grown under the same experimental conditions (i.e., 25 °C, 12:12: L:D cycle at 30 µmol photons m -2 s -1 ) has not previously been observed in our laboratory nor reported in the literature. These cyst-like structures are similar to asexual temporary or ecdysal cysts documented previously for other dinoflagellates under stress conditions [START_REF] Blanco | Cyst production in four species of neritic dinoflagellates[END_REF], such as cold temperatures [START_REF] Hardeland | Induction of cyst formation by low temperature in the dinoflagellate Gonyaulax polyedra Stein: Dependence on circadian phase and requirement of light[END_REF]. The formation of cyst-like structures has also been reported for K. brevis upon exposure to bacteria [START_REF] Roth | Comparative analysis of two algicidal bacteria active against the red tide dinoflagellate Karenia brevis[END_REF]. Whether or not the transient decline in temperature, which occurred on day 27 inside the incubator, contributed to the increased formation of cystlike structures is unclear but remains a plausible explanation.

Compared to K. brevis, lower cellular abundance was reached in K. mikimotoi cultures and the growth rate determined for these cells (0.07 d -1 ) was at the lower end of the values reported in the literature (~0.07 to 0.15 d -1 ; [START_REF] Garcia | The influence of irradiance on growth, photosynthesis and respiration of Gyrodinium cf. aureolum[END_REF][START_REF] Staehr | Detection of Karenia mikimotoi by spectral absorption signatures[END_REF]. The limited growth is not likely to be attributed to the growth temperature selected (15 ºC) since K. mikimotoi is generally grown at temperatures ranging between 11 and 16 °C (as recommended by CCMP). The caspase 3-like protein activity in cells of K. mikimotoi was not as elevated as in cells of K. brevis (Fig. 3AB) but a clear constitutive level F o r P e e r R e v i e w 14 of activity was observed at the beginning of the experiment (values > 1, 000 RFU/h/mg protein on days 0, 7 and 14). This activity may have provided some housekeeping functions and/or played a role in the death of the "less vigorous" K. mikimotoi cells as suggested by the increase of the percentage of dead cells (~ 10 % on days 14 and 21) which followed the caspase 3-like protein activation (on days 0, 7 and 14). Karenia mikimotoi is difficult to grow, and known to be sensitive to agitation [START_REF] Gentien | Motility and autotoxicity in Karenia mikimotoi (Dinophyceae)[END_REF]. To explain the limited growth observed in cultures of K. mikimotoi, [START_REF] Gentien | Motility and autotoxicity in Karenia mikimotoi (Dinophyceae)[END_REF] proposed an autotoxicity mechanism which involves the release of an exotoxin (18 : 5n3 fatty acid) from K. mikimotoi cells. We suggest that the mechanical stress endured by the cells during subculture and/or the inoculation of old cells may have facilitated the release of toxins from K. mikimotoi cells and influenced their growth. Although particular care was taken not to handle the culture flasks between the 7-d sampling intervals, this exotoxin could still have leaked from old/stressed cells and acted as a growth deterrent during the initial part of the study. Interestingly, the subsequent increase, albeit slow, in K. mikimotoi cellular abundance matched the increase in the physiological state (F v /F m ) of the cells, and declines in both the caspase 3-like protein activity (on days 21 and 28) and the percentage of dead cells (on days 28 and 35). The subsequent increase in caspase 3-like protein activity (on days 35 and 42 with values of 656 and 500 RFU/h/mg protein, respectively) observed prior to the decline in cellular abundance suggests a role of caspase 3-like enzymes in the death process of K. mikimotoi.

The high efficiency with which the inhibitor Ac-DEVD-CHO precluded the increase in caspase 3-like protein activity in both species of Karenia strengthens the involvement of caspase-like enzymes, and not of other generic proteases, in the caspase-like activity detected, although the identity of the actual proteases involved in the process is still unclear.

The caspase-3 like protein activity measured in species of Karenia either had some housekeeping functions, played a role in the formation of cyst-like structures (for K. brevis) or in cell death. While the implication of caspase-like protein activity in cyst formation has never been demonstrated before, the existence of a caspase-mediated pathway leading to cell death has been observed in a variety of microalgae. [START_REF] Bidle | Iron starvation and culture age activate metacaspases and programmed cell death in the marine diatom Thalassiosira pseudonana[END_REF] reported an increase in caspase-like activities in an aging culture of the diatom Thalassiosira pseudonana while the occurrence of PCD, with the participation of cysteine proteases, was similarly proposed to explain the sudden termination of a bloom of the freshwater dinoflagellate Peridinium gatunense [START_REF] Vardi | Programmed cell death of the dinoflagellate Peridinium gatunense is mediated by CO 2 limitation and oxidative stress[END_REF]. Because the type of cell death in microalgae can be species-specific and influenced by a triggering factor, it is challenging to identify a [START_REF] Segovia | Programmed cell death in dinoflagellates[END_REF][START_REF] Jiménez | Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase[END_REF][START_REF] Affenzeller | Salt stress-induced cell death in the unicellular green alga Micrasterias denticulata[END_REF]. In cells of the green alga Micraterias denticulate, the occurrence of PCD, associated with an increase in caspase-like activity, has been observed when cells were exposed to H 2 O 2 [START_REF] Darehshouri | Cell death upon H 2 O 2 induction in the unicellular green alga Micrasterias[END_REF] while a caspase-like independent PCD was reported for the same species upon exposure to salt stress [START_REF] Affenzeller | Salt stress-induced cell death in the unicellular green alga Micrasterias denticulata[END_REF]. Similarly, [START_REF] Jiménez | Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase[END_REF] reported the occurrence of a variety of cell death events in the chlorophyte Dunaliella viridis depending on the type of environmental stressors tested. In their study, the increase in caspase-like activity was surprisingly shown to mediate not only apoptosis but also other cell death programmes [START_REF] Jiménez | Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase[END_REF]. Whether harmful dinoflagellates display an ability to undergo a programmed-form of cell death is unclear but seems plausible considering the results obtained from other studies performed with species of non-toxic dinoflagellates [START_REF] Vardi | Programmed cell death of the dinoflagellate Peridinium gatunense is mediated by CO 2 limitation and oxidative stress[END_REF] and coral symbiotic dinoflagellates [START_REF] Dunn | Programmed cell death and cell necrosis activity during hyperthermic stress-induced bleaching of the sea anemone Aiptasia sp[END_REF].

DNA degradation, a generally well accepted marker of apoptosis, can be detected using a TUNEL assay, among other techniques. This assay however has been reported to lack specificity because it can label some forms of non-apoptotic DNA fragmentation [START_REF] Labat-Moleur | TUNEL Apoptotic Cell Detection in Tissue Sections: Critical Evaluation and Improvement[END_REF]. Nervertheless, the positive labelling of degraded DNA was observed in senescent cells of Dunaliella viridis [START_REF] Jiménez | Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase[END_REF]. [START_REF] Zuppini | Heat stress: an inducer of programmed cell death in Chlorella saccharophila[END_REF] also reported increases in caspase 3-like activity, chromatin condensation, DNA fragmentation, cell shrinkage and detachment of the plasma membrane and therefore the occurrence of PCD in heat-stressed cells of Chlorella saccharophila [START_REF] Zuppini | Heat stress: an inducer of programmed cell death in Chlorella saccharophila[END_REF]. Increases in caspase-like activities also coincided with an increase in chromatin margination and DNA fragmentation in darkened cells of Dunaliella tertiolecta [START_REF] Segovia | Cell death in the unicellular chlorophyte Dunaliella tertiolecta. A hypothesis on the evolution of apoptosis in higher plants and metazoans[END_REF] while the occurrence of PCD, defined as the induction of cytoplasmic shrinkage and DNA fragmentation, was proposed for the dinoflagellate Peridinium gatunense upon CO 2 limitation and oxidative stress (H 2 O 2 ) [START_REF] Vardi | Programmed cell death of the dinoflagellate Peridinium gatunense is mediated by CO 2 limitation and oxidative stress[END_REF]. In the present study, a TUNEL assay was used to determine the presence of degraded DNA in cultures of Karenia which had reached the late decline phase. The presence of degraded DNA in cells of K. brevis was associated with a constitutive level of caspase 3-like activity (at least on days 42 and 56) while the presence of degraded DNA in cells of K. mikimotoi followed the activation of caspase 3-like proteases observed on days 35 and 42. While these data are insufficient to claim the occurrence of a specific type of cell death they certainly provide evidence of the cells' deteriorating condition. In the present study, cells of both K. brevis and K. mikimotoi displayed high F v /F m values which are consistent with values previously reported in the literature [START_REF] Schaeffer | A comparison of photoresponse among ten different Karenia brevis (Dinophyceae) isolates[END_REF][START_REF] Vargo | A brief summary of the physiology and ecology of Karenia brevis Davis (G. Hansen and Moestrup comb. nov.) red tides on the West Florida Shelf and of hypotheses posed for their initiation, growth, maintenance, and termination[END_REF]. However, the maintenance of high F v /F m values even during the decline phase of the cultures is unexpected and surprising since F v /F m is commonly used as an indicator of the physiological state of cells, which responds mostly to nutrient limitation and high irradiance [START_REF] Kolber | Use of active fluorescence to estimate phytoplankton photosynthesis in situ[END_REF]. Because large proportions of dead "but intact and pigment-containing" cells are now known to be present in phytoplankton communities [START_REF] Veldhuis | Cell death in phytoplankton: correlation between changes in membrane permeability, photosynthetic activity, pigmentation and growth[END_REF][START_REF] Agustí | Viability and niche segregation of Prochlorococcus and Synechococcus cells across the Central Atlantic Ocean[END_REF]), [START_REF] Franklin | Effect of dead phytoplankton cells on the apparent efficiency of photosystem II[END_REF] assessed the effect of dead cells on the apparent photosystem II efficiency of a variety of microalgae. Their results demonstrated that the presence of photosynthetically non-functional, pigment-containing dead cells had little effect on the photosynthetic efficiency of a number of phytoplankton species, including dinoflagellates. In their study, the efficiency of photosystem II (F v '/F m ') of the dinoflagellate Amphidinium carterae decreased by only 20 % in the presence of 75 % of photosynthetically non-functional cells. Similarly, the presence of 50 % of photosynthetically non-functional cells barely influenced the initial efficiency of photosystem II which was measured in the absence of dead cells. These results are very similar to those obtained in the present study and are in accordance with other results obtained in the field which showed that mass microalgal mortality can be accompanied by relatively constant PSII efficiency [START_REF] Dijkman | Photosynthetic characteristics of the phytoplankton in the Scheldt estuary: community and single-cell fluorescence measurements[END_REF].

Besides the report of increased caspase 3-like activity in stressed cells of toxic cyanobacteria Microcystis aeruginosa [START_REF] Ross | Toxin release in response to oxidative stress and programmed cell death in the cyanobacterium Microcystis aeruginosa[END_REF], to the best of our knowledge, caspase-like protein activities have never been reported for species of harmful microalgae.

We report variations in caspase 3-like protein activity in cultures of the harmful dinoflagellates and suggest that it can have housekeeping functions, play a role in the death of Karenia cells and in the formation of cyst-like structures (in K. brevis). Further research is needed to determine the variation of caspase 3-like protein activity in harmful dinoflagellates under stress and to verify if an active caspase-mediated cell death pathway could contribute to the decline of Karenia blooms in situ.

F

  . This freezing/sonification cycle was repeated three times to ensure maximum extraction of proteins. To remove insoluble material and unbroken cells, a centrifugation (3800g, 5 min) was performed and the supernatant, containing the protein extract, collected and transferred to a clean microtube. The Bio-Rad Protein Assay was used to determine the total protein concentration of samples using bovine serum albumin as a standard protein. The absorbance (750 nm) of the samples was measured using a microplate reader (FLUOstar OPTIMA, BMG LABTECH, Germany).

  with a Zeiss epifluorescence microscope (Ex: 450-490 nm; Em: > 520 nm).

  show bright field micrographs of K. brevis and K. mikimotoi, respectively and cells positively labelled with the SYTOXgreen marker. These pigment-containing cells were considered dead because their nucleus fluoresced bright green when excited with blue light.Caspase 3-like protein activity and effect of the caspase inhibitor

  diverse species of microalgae

Figure 1 .

 1 FIGURE DESCRIPTION

Figure 2 .

 2 Figure 2. Abundance of SYTOX-positive cells for K. brevis (A) and K. mikimotoi (B). Note the different scales between A and B. Micrographs of K. brevis (A inset) and K. mikimotoi (B inset) taken under the bright field (left panel) and blue light fluorescence micrographs of cells with a compromised membrane (dead cells; right panel). Cells were stained with the fluorescence probe SYTOX-green. Scale = 20 µm. Percentage of SYTOX-positive cells for K. brevis and K. mikimotoi (C). Dashed lines show the highest values reached for K. brevis (short dashes) and for K. mikimotoi (long dashes). Each point is the average of samples from four separate flasks ± SE.

Figure 3 .

 3 Figure 3. Temporal variation of caspase 3-like protein activity in K. brevis (A) and K. mikimotoi (B) cultures. Note the different scales between A and B. The white symbols represent the activity of caspase 3-like enzyme measured in the presence of the caspase inhbitor Ac-DEVD-CHO (20 µM). Each point is the average of samples from four (only one sample for the inhibitor treatment at day 56 for K. mikimotoi) separate flasks ± SE. Significant differences at p ≤ 0.05 are indicated by an asterisk. RFU = relative fluorescence units.

Figure 4 .

 4 Figure 4. TUNEL assay results for K. brevis (A-D) and K. mikimotoi (E-H). Fluorescence micrographs of Karenia cells stained with the TUNEL marker (A, E), of the same cells taken under the bright field (B, F), of cells pre-treated with DNase (positive controls) (C, G) and of cells stained only with the label solution, i.e., without terminal transferase (negative controls) (D, H). Scale = 25 µm.

  Figure 1.

Table I .

 I technical support. We also acknowledge Dr. Daniel J. Franklin and two other anonymous reviewers for their constructive comments on ways to improve the manuscript. Comparisons of growth variables for the two dinoflagellate species studied.

		Maximal cell abundance	Day MCA	Growth rate	Division	Doubling time
		(MCA; cells mL -1 )	was reached	(µ µ µ µ; day -1 )	(k; day -1 )	(T 2; days)
	K. brevis	7646	21	0.18	0.26	3.8
	K. mikimotoi	1443	42	0.07	0.10	9.6
		o r F o r F o r F o r F o r F o r F o r F o r
		P P P P P P P P
		e e e e e e e e
		e r e r e r e r e r e r e r e r
			R R R R R R R R
			e e e e e e e e
			v i e v i e v i e v i e v i e v i e v i e v i e
				w w w w w w w w
						23 24

Table II .

 II Results from repeated-measures ANOVAs. The source of variation is partitioned

http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research

Journal of Plankton Research

ACKNOWLEDGMENTS

The authors are grateful to Dr. D. Iglesias-Rodriguez for the use of the microplate reader, to Dr. Thomas S. Bibby for access to the FIRe fluorometer and to M. M. O'Shaughnessy for his

FUNDING

This work was supported by a postdoctoral fellowship awarded to JNB by the Fonds québécois de la recherche sur la nature et les technologies (FQRNT). Consumable costs were provided by a NOCS Interface Fund to D.A.P and Dr. P. Townsend.

Species