Physical variables and pigment analysis
Physical variables were recorded simultaneously by the CTD rosette and meteorological sensors of the vessel. Oxygen values were calibrated by independent sampling and Winkler titration. Pigments were analysed using a HPLC technique described by Wright et al. (1991). A HPLC system was used from THERMO spectrasystem equipped with a C18 (CLI) inverse column. One litre of sample was filtered at -200mbar onto a 25 mm GF/F filter. Filters were stored at -196°C in liquid nitrogen. Extraction of pigments was conducted with cold methanol (-20°C) for 12 hours. Detection limits range between 0.002 µg/mL for Chl b, Chl c2 and Alloxantin and 0,013 µg/mL for beta-Caroten. Pigments identification and quantification was done by comparing retention times and peaks areas and adsorption spectra obtained using certified standard solutions from DHI Group, Denmark.
Samples for nutrient analysis were taken in 125 ml polyethylene bottles, pre-treated with hydrochloric acid and deionised water, and finally rinsed with the sea water to analyse. These bottles have been kept at -20°C in darkness until the analysis were carried out. For nutrient analysis a semi-automated system was used (TECHNICON, autoanalyser II). The following nutrients were determined:
– Silicic acid (Si(OH)4): following the protocol of Fanning and Pilson (1973).
– Nitrate (NO3-) and nitrite (NO2-): as described by Bendschneider and Robinson (1952) and Wood et al. (1967). Firstly, nitrite concentrations were determined. Then, nitrate was reduced to nitrite and finally the nitrite concentration was measured. The difference between both gives the nitrates concentration.
– Phosphate( PO43-): as defined by Murphy and Riley (1962).
Analysis of volatiles
VHOCs were analysed using a purge-and-trap technique and GC-ECD (Chrompack CP 9000) modified after Pruvost et al. (1999). The purge-and-trap-loop was altered and Valco valves were replaced by highly saltwater resistant Swagelok models. Sampling devices were modified according to Bulsiewicz et al. (1998). These new sampling devices (30 ml) were highly gastight; leak tests showed tightness for over a week. In contrast to Bulsiewicz, sampling devices were directly connected to the Niskin bottle and to the purge-and-trap-loop respectively via Swagelok miniature quick connectors. The connector between Niskin-bottle and sampling device comprises a filter element with 15 µm pore size, to remove larger particles. Samples were stored in the dark at 4°C and finally analyzed within four hours after sampling.
Volatiles were extracted by purging with ultra-pure nitrogen for 20 min at a flow of 90 ml min-1. Purging took place at ambient temperature in a purge chamber, which contained a glass frit (Pyrex 4). The gas flow was dried downstream using a condenser (held at 2 °C) and a magnesium perchlorate trap.
Volatiles were concentrated in a stainless steel capillary tube (150 cm) at -78°C and subsequently injected into a gas chromatographic column by thermodesorption (100°C, backflush). Separation of the compounds was performed using a capillary column (fused silica megabore DB-624, 75 m, 0.53 mm id, 3 mm film thickness, J & W Scientific, flow: 6 ml min-1 ultra pure nitrogen) and a temperature program (10 min at 70°C, rising for 8 min to 150°C and stable for 7 min at 150°C). Quantification of volatiles was performed by external liquid standards (EPA 624 mix standards, AccuStandard; iodoethane, dibromomethane, chloroiodomethane, diiodomethane, bromoiodomethane Carlo Erba). Liquid standards were diluted in seawater and treated like a normal sample.
Multivariate methods such as principal component analysis (PCA) or cluster analysis are useful statistical tools to simplify complex data set by reducing the number of potential correlations between variables (Mudge 2007, see there detailed information about statistical methods). In order to determine physical, chemical and biological variables, which could explain the distribution of VHOC we attempt to visualize possible groupings in our dataset. In a first step, a cluster analysis was performed in order to estimate a possible clustering within the dataset. Various variables were treated with a K-means-cluster analysis, including time of the day, water temperature, sea surface temperature (SST), salinity, density, bottom depth, sampling depth and Chlorophyll a concentration.
In a second step, a PCA was carried out in order to evaluate similarities between variables of our data set. Here, we computed a PCA for a data set of normalized VHOC values. As a result of the PCA, similarities between the sampling stations formed distinct clusters. Hence we estimated the factor, which might be causal for this clustering. For that, we combined results from the cluster analysis with the PCA. It appeared that SST was the crucial factor.
Consequently in a third step variable correlation matrices were performed for each SST-cluster. Data were analyzed performing one-way and factorial ANOVA with subsequent post-hoc Tukey’s honestly significant difference method for unequal sample size. For the comparison of only two data sets, paired t-tests were performed.
Pearson correlation cross tables were calculated in order to determine the extent to which two variables show linear proportionality to each other. For all statistical tests and techniques Statistica (Release 8.0) and Primer E (Release 5) were used.
Detailed information about multivariate exploratory techniques (such as PCA or cluster analysis) are given by Legendre (1998), whereas Sokal and Rohlf (1995) gives a comprehensive introduction about univariate analysis (such as ANOVA or Pearson correlations).
Upwelling during the campaign and sampling strategy
Sampling strategy (for results see Figure 15) was defined by evaluating satellite images (Figure 16). Both Chl-a concentration- and SST-images indicated a proceeding upwelling and phytoplankton bloom along the coast of Portugal.
Figure 16. Satellite image of sea surface temperature (SST) and Chl-a concentration on August 19th 2007.
Source: Data from the MODIS satellite (NASA); plot computed by V. Rossi, CNRS/LEGOS, Toulouse, France. A: Blue reflects low SST (13°C), red high values (23°C). White is cloud cover. B: Red reflects high Chl-a concentration, blue reflects low Chl-a concentration. White is cloud cover.
The north-to-south track followed a 100 m bottom depth isoline and was located within the upwelling. This track was chosen in order to investigate VHOC concentrations within the maximum of phytoplankton density. The west-to-east track followed 40°N degree of latitude from the open ocean toward the upwelling. This track was chosen to investigate differences between coastal influenced upwelled waters and nutrient depleted open ocean waters. A distinct offshore filament was sampled heading westwards at 40.4°N. This track was completed by sampling outside the filament heading eastwards. Additionally, samples were taken at four 30 h stations (ST 1 – 4). Station 1 (40°N, 9.1°W) and 2 (41°N, 9°W) were located within the upwelling. Station 3 (41°N, 10.5°W) was a reference point in the open ocean. Station 4 (40.3°N, 9.2°W) was located just outside the upwelling.
Satellite images indicated that sea surface temperature decreased in the first two weeks of the campaign compared to the previous weeks, indicating that upwelling took place before the start of the campaign. A satellite sea-surface temperature image taken on August 19th showed clear upwelling conditions occurring in the studied area (Figure 16, A). Distinctive upwelling took place along the Iberian Peninsula from Cap Finisterre to 37°N with highest convection about 41°N. A clear temperature gradient is visible from the open ocean (around 21°C) towards the coast (less than 15°C). Filaments were visible at 37.5°N, 40°N and 41.5°N. These cold water bands extend from the Iberian coast up to 12°W and are characterised by temperatures significantly lower than the surrounding water masses. Satellite images of Chl-a concentrations indicated a phytoplankton bloom along the Iberian Peninsula (see Figure 16, B). Concentrations were highest near the coastline (more than 3.5 µg/L) and low in the open ocean (two orders of magnitude lower). Coupled to upwelling and advection, a meandering structure of the phytoplankton density is clearly visible all along the coast. During the last week of the campaign, upwelling conditions were still evident although wind velocity decreased (less than 10 m/s) and sea surface temperatures increased by several degrees along the coast.
Spatial distribution of selected VHOCs
Sea surface concentrations for selected VHOC measured during the MOUTON campaign are presented in Figure 17.
Dibromochloromethane (CHBr2Cl), dibromomethane (CH2Br2), bromoform (CHBr3), chloroform (CHCl3), chloroiodomethane (CH2ClI) and diiodomethane (CH2I2) in pmol L-1. Colour scales with different concentration range.
Generally, concentrations of brominated compounds were high along the coast and low in the filament and in the open ocean. The highest values were found between 41°N and 42°N and around 39.5°N (185.1 pmol L-1 for bromoform, 60.4 pmol L-1 for dibromomethane and 17.5 pmol L-
1 for dibromochloromethane).
Concentrations of iodinated compounds were about the same range than dibromochloromethane (surface mean of 2.7 pmol L-1 for chloroiodomethane and 1.5 pmol L-1 for diiodomethane). The highest surface concentrations of chloroiodomethane were recorded in the open ocean far from the coastline (up to 6.8 pmol L-1). Other sampling sites with elevated chloroiodomethane values were located in the upwelling at 42°N and at the northernmost station (4.5 pmol L-1). Diiodomethane levels were elevated in open ocean waters (up to 4.2 pmol L-1) and at two stations near the coast. Chloroform levels were the highest among the chlorinated compounds (surface mean of 16.9 pmol L-1). Values were elevated in the upwelling between 40.5 and 42° N (up to 23.9 pmol L-1) and south of 40°N. The highest chloroform concentration was recorded at the junction between upwelling and intermediate water masses at 40°N (32.5 pmol L-1). Concentrations were low in the open ocean and in the filament structure.
SST as grouping variable
In order to evaluate VHOC distribution patterns we performed cluster analyses and a principal component analysis (PCA). The result of the PCA was used to visualize relationships among studied VHOCs and their sampling sites (Figure 18).
Figure 18. Principal Component Analysis (PCA) of all normalized VHOC data.
Similarities among VHOC are plotted as vectors, while similarities between the sampling sites are plotted as dots. Samples are grouped by SST-clusters. For abbreviations of VHOC see Table 10. Correlations to the principal component PC1 (x-axis) explain 32 % of the data and correlations to the principal component PC2 (y-axis) explain about 17% of all data.
Diiodomethane, chloroiodomethane and tetrachloromethane are best explained by PC2 and show high similarities among themselves. Dibromomethane, bromoform, dibromochloromethane, bromodichloromethane and perchloroethene formed a second cluster, which is related to PC1. A third cluster is composed of iodoethane, bromoiodomethane, 1,1,2-trichlorethene, methyl chloroform and chloroform. This group however is not well explained by the PCA model. Because of the marginal vector length of perchloroethene and tetrachloromethane, correlations to PC1 and PC2 are low.
Figure 18 illustrates clear similarities between various samples sites. Here we found that sample sites form three clusters. In order to demonstrate the underlying feature of this data spreading, we overlaid the plot with results of a cluster analysis. From the studied physical variables sea surface temperature (SST) was determined as a factor that could best explain the distribution pattern of the sampling sites. Other variables (e.g. salinity or chlorophyll a) did not reflect the clustering of the sampling sites. SST values form three clusters and reflected different water masses: The upwelling water mass reflect stations with a SST-mean of 14.5 °C (see also Figure 15). These stations were located were close to the coast of the Iberian Peninsula. The Intermediate water mass attributed stations with a SST-mean of 16.4°C. Those stations were located either close to the coast line (aged upwelled waters) or in the filament (cold waters mixed with open ocean waters). The Open ocean water mass featured a SST-mean of 19.7°C. All stations were located in the far off Iberian coast.
Relationship Between the Compounds
Similarities between VHOCs were shown in the PCA model (see Figure 18). In order to evaluate correlations in-between gases, we calculated Pearson correlations matrices. In Figure 19 we present those correlations of three representative halocarbons to all other VHOCs. Brominated compounds are well correlated among each other in all water masses. Highest correlations were found between dibromomethane and bromoform in intermediate water masses. Clear correlations between iodinated compounds were remarkable between chloroiodomethane and diiodomethane in the open ocean and less articulated in intermediate water masses. In the upwelling however, no significant correlations were found among iodinated compounds. Correlations between chlorinated compounds were weak. The highest correlations were visible between methyl chloroform and chloroform in the upwelling.
Results of cross-tabulation tables of Pearson r correlation coefficients for all VHOCs. Significant level p<0.05. The correlation cross table was calculated for all samples. Data are clustered in three different water masses: Upwelling (blue), intermediate water (green) and open ocean (red). Remarkable correlations are indicated in yellow.
Vertical distribution of VHOCs compared to environmental parameters
Based on the three water mass types (as defined above), we studied the vertical distribution of VHOCs and environmental parameters in each water mass. Figure 20 shows the distribution of representative halocarbons and synoptic data. Sampling depth was a highly significant factor for most variables. More detailed information is given in Table 11, Table 12 and Table 13. There, VHOCs and environmental variables are grouped by different water mass and depth (mean values of all samples from surface to thermocline and samples below the thermocline).
Mean values of chlorophyll a [µg • L-1], AOU [µmol L-1], N*[µmol • L-1+, density *σ+ and selected VHOC *pmol • L-1] in the three defined water masses at five depth. Row 1: Upwelling waters. Row 2: Intermediate waters. Row 3: Open ocean waters. Values expressed as mean of samples of surface, upper thermocline, maximum of chlorophyll, lower thermocline and bottom. Red lines use primary x-axis (above, red). Blue lines refer to secondary x-axis (below, blue). Note: CIM concentration at open ocean province had different scale from the other provinces.
In the upwelling, σ-values showed a deep mixing without a clear pycnocline (Figure 20, row 1). Maxima of Chl-a were observed in the first 20 m (1.5µg L-1) and reached very low values in the deeper layer below 40 m. The apparent oxygen utilisation (AOU) was negatively correlated to Chl-a: values were low near the surface and in the Chl-a maximum whereras AOU-values increased with depth. N* (a linear combination of nitrate and phosphate; see (Gruber and Sarmiento 1997)) is a benchmark for the marine nitrogen cycle. Low N*-values reflect a nitrate loss whereas high values indicate nitrogen fixation. In the upwelling, N* showed the same pattern as AOU: the lowest values were found in surface waters and in the Chl-a maximum. Bromocarbons showed no clear peak in the water column. While dibromomethane did not vary significantly with depth, bromoform concentrations were significantly lower in the deeper layer compared to the upper layer (factor 2.4, p= 0.001). Variations of chloroiodomethane, diiodomethane and chloroform did not vary significantly with depth in the upwelling.
In the intermediate water mass (Figure 20, row2), the water column was weakly stratified (pycnocline at about 20 m). However, a clear Chl-a maximum was recorded at 20-m depth and corresponded to a minimum N*-value. AOU values were significantly elevated below the pycnocline. For all gases, maximum values were measured just above the Chl a maximum. Values of brominated compounds were up to 6.7 times higher here compared to the deeper layer values. Iodinated compounds and chloroform showed maxima just above and below the Chl-a maximum. However variations of diiodomethane and chloroform did not vary significantly with depth.
In the open ocean (Figure 20, row3), the water column was clearly stratified. The pycnocline (66 m) corresponded with the Chl-a maximum. N*-values were in average 1.5 times higher in the upper layers of the open ocean compared to the upper layer in the identified intermediate water mass, indicating a lower loss of nitrogen in the open ocean. At the Chl-a maximum however, N* values were significantly lower, in average 1.5 times than in all other water layers. Brominated compounds showed no clear maxima throughout the water column. Concentrations were low compared to coastal waters (about 3 times lower for the surface concentrations). However, bromocarbon concentrations were significantly higher at the Chl-a maximum. Iodinated compounds showed maximum values at the Chl-a peak. Values were 2-5 times higher there compared to all other depth, and 2.7 – 3.2 times higher compared with the coastal waters.
In order to investigate the factor time, samples were sorted by the sampling time and divided in four groups: night, day, and intermediates. All samples taken in between 2h after sunset and 2h before sunrise were defined as “night samples”. All samples taken in between 2h after sunrise and 2h before sunset were defined as “day samples”. Intermediate times were defines as morning or evening samples. Results of the ANOVAs indicate that the factor time has significant effects (Figure 21) on bromocarbons. In the upwelling, values were significantly higher after sunset (factor 1.5 to 1.8; p-values between 0.002 and 0.055) and stayed rather low during the rest of the day. Intermediate waters showed higher concentrations between dusk and night, compared to the period between dawn and daytime (factor 1.8 to 2.1). Diurnal variations of bromocarbons in the open ocean were less clear. Variances for dibromomethane and dibromochloromethane are small (factor 1.1 and 1.2) and statistically not significant. Bromoform however showed a significantly elevated concentration during night-time (factor 2, p = 0,05). Time of the day showed no significant influence on iodocarbons and chloroform (data not shown).
In order to investigate the factor tide, samples were sorted by the sampling time and divided in four groups: high tide, low tide, incoming mid tide and outgoing mid tide. Sampling times were compared with tide tables, provides by SHOM (Service hydrographique et océanographique de la marine) for different places along the Iberian coast. Figure 22 illustrates clear effects of tide on VHOC levels within the upwelling. Upwelling values of brominated compounds were significantly elevated when water flowed back from the coast to the open ocean. In intermediate water masses the effect is still noticeable but statistically less significant. Similarly to brominated compounds chloroform and iodinated compounds showed elevated concentrations in the upwelling during the outgoing tide. In intermediate water masses however, values of chloroiodomethane and diiodomethane were significantly elevated at incoming tide. Generally, in intermediate water masses, effects are noticeable and are not significant in the open ocean.
This is the first study of volatile halogenated organic compounds in the Iberian Upwelling, and presents a comprehensive number of brominated, iodinated and chlorinated volatiles in an upwelling system. Our data show that multivariate effects are causal for the distributions of VHOCs. Here we present evidence for different VHOCs sources, which are each causal for the production of a certain group of VHOCs. This study demonstrates for the first time the effect of tide on VHOCs distribution.
Comparison to other studies
Although a wide range of marine regions were investigated for VHOC levels, only a few studies focused on upwelling regions: Class and Ballschmiter (1988) measured bromocarbons and tetrachloromethane near the West African coast (25°N 16°W). The Mauritanian Upwelling was investigated by Quack et al. (2007b) between 17.0 and 20.5 °N in April/March 2005 and by Carpenter et al. (2009) between 16 and 35°N in May/June 2007. Both studies were focused on bromoform and dibromomethane and found similar mean values (see Table 14).
Our values however, were about a factor 2 higher than data reported for the Mauritanian Upwelling, and were rather similar to coastal water values reported for the African Upwelling.
The only study which studied various VHOC along the Iberian Peninsula, were restricted to the shoreline and did not measured in the upwelling(Martinez et al. 2002b). These authors focused on monitoring different anthropogenically produced VHOCs and reported results as class distribution and maximum values. Consequently these results give a broad representation for nonnatural coastal inputs of chlorinated volatiles but are less comparable to our results.
Based on various oceanic data, production of VHOCs has highly localized sources. Saturations are highest in littoral zones, mainly in macro-algae beds. Furthermore, seawater concentrations vary greatly with seasons (Archer et al. 2007) and hence comparisons of different studies might be challenging. Contrary to the assumptions of a strong phytoplankton production in upwelling regions, we report values intermediate between coastal and open ocean values.
On the different origin of VHOCs
Results from the principal component analysis (see Figure 18) showed similarities between three sample sites and between VHOCs. It was shown that sample sites cluster in three groups:
upwelling, intermediate water masses, and open ocean. Moreover we showed high similarities between VHOCs indicating similar sources for three different groups: (1) bromocarbons, (2) two iodocarbons (chloroiodomethane and diiodomethane) and (3) the remaining VHOCs (mostly chlorocarbons).
Similarities between VHOCs (Figure 18) and correlations among them (Figure 19) indicated common sources for brominated compounds. Highest correlations between brominated compounds (seeFigure 19, row 1) were recorded in samples with the highest concentrations (intermediate water masses). In intermediate water masses correlations to other gases were not significant or negligibly low. Hence for this region it can be assumed that bromoform, dibromomethane and dibromochloromethane do have the same origin. Contrary to intermediate water masses, correlations between brominated compounds were less pronounced in the open ocean and the upwelling. Thus an additional and more compound-specific source (and/or sink) can be assumed for both water masses.
Dibromomethane/bromoform ratios were calculated by several authors (Carpenter and Liss 2000; Quack et al. 2007b; Carpenter et al. 2009; Jones et al. 2009). Dibromomethane/bromoform slopes were found to be lower in coastal regions and are caused by different sources: macroalgae-produced bromocarbons cause a lower slope whereas slopes are higher in phytoplankton-dominated regions. Our results supported these findings (Table 11).
Iodocarbons (Figure 19, row 2) show clear correlations among each other: the highest correlation was observed between chloroiodomethane and diiodomethane in open ocean waters, a region where the highest concentrations were measured for both gases. Hence both halocarbons do have the same origin and this origin is located in the open ocean. In a recent study it was shown that both compounds can be formed in the presence of dissolved iodine, dissolved organic matter and ozone (Martino et al. 2009).
The correlations of chlorocarbons (Figure 19, row 3) did not show clear patterns. For example, we present correlations of methyl chloroform to all other VHOCs. The highest correlations (methyl chloroform and chloroform) were observed in the upwelling whereas just one significant correlation was observed in the open ocean. Consequently, a common source for chlorinated volatiles might be connected to the shoreline. Martinez et al. (2002b) reported a high coastal input of anthropogenic chlorocarbons at several places in Portugal.
Evidence for phytoplanctonic production of VHOCs
Correlations between different VHOC groups and chlorophyll (fluorescence sensor) were found for brominated and iodinated compounds (see Figure 23). Moreover, we found a good correlation between both VHOC groups and both biological markers N* and AOU. These correlations were clearly visible for the open ocean and less pronounced in the upwelling or in intermediate water masses. These results indicate that the formation of brominated and iodinated compounds was usually coupled to photosynthetically produced oxygen and nitrogen loss; both caused by phytoplankton activity. The absence of strong correlations in the upwelling and intermediate water could have two explanations: either the main source for brominated and iodinated compounds was non-biological or, and more likely, the formation of those compounds was locally separated. Main sources of bromo- and iodocarbons are likely coastal zones of the Iberian Peninsula. Water masses are transported westwards containing elevated concentrations of those compounds.
Table of contents :
1.1 Thesis Goals
2 VHOC: State of the art
2.1 Chemical properties
2.2.1 Biological sources
2.2.2 Natural, nonbiological formation of VHOCs
2.2.3 Anthropogenic sources
2.3 Lifetimes in the Troposphere
2.4 VHOC contribution to reactive stratospheric halogens
2.5 Air-Sea Exchange
2.5.1 Henry’s law constants
2.5.2 Transfer velocity
2.5.3 Notes on calculations of air mixing ratios
3 Methods and Development of the Analytical System
3.1 Working with Trace Elements
3.2 Sampling Devices
3.3.1 Solid-Liquid extraction
3.3.2 Liquid-Liquid extraction
3.3.3 Gaseous-Liquid extraction
3.4 Purge gas desiccation
3.5 Preconcentration of the analytes and injection
3.6 Separation and Detection
3.7 Limit of Detection
3.8 Implemented GC-ECD-System
3.8.1 Sampling device
3.8.3 Retention times and identification of the compounds
3.8.5 Summary of the PAT-GC-ECD parameters
3.9 Protocols for maintaining the system
4 Distribution of Volatile Halogenated Organic Compounds in the Iberian Peninsula Upwelling System
4.2.1 Study Area
4.2.2 Physical variables and pigment analysis
4.2.3 Analysis of volatiles
4.2.4 Data analysis
4.3.1 Upwelling during the campaign and sampling strategy
4.3.2 Spatial distribution of selected VHOCs
4.3.3 SST as grouping variable
4.3.4 Relationship Between the Compounds
4.3.5 Vertical distribution of VHOCs compared to environmental parameters
4.3.6 Temporal and tide factors in the upper layers
4.4.1 Comparison to other studies
4.4.2 On the different origin of VHOCs
4.4.3 Evidence for phytoplanctonic production of VHOCs
4.4.4 Near shore production: main source for brominated compounds in the upwelling?
5 Annual distribution of reactive halocarbons in a tide influenced estuary: Exchange fluxes between ocean and atmosphere
5.2.1 Sampling area
5.2.2 Sampling strategy
5.2.3 Methods for physical, chemical and biological variables
5.2.4 VHOC measurements
5.3.1 Meteorological variables
5.3.2 Environmental data describing seasonality at ASTAN and ESTACADE point
5.3.3 Environmental data describing seasonality along a salinity gradient in the Bay of Morlaix
5.3.4 Seasonality of VHOC surface concentrations
5.3.5 Formation of halocarbons during a diurnal tidal cycle
5.3.6 Possible input of halocarbons by a sewage treatment plant at Morlaix
5.4.1 Comparison to other costal measurements
5.4.2 Temporal trends
5.4.3 Biogenic sources
5.4.4 Different sources along the gradient
5.4.5 Sea-air Fluxes
6 Physiological function of VHOCs for macroalgae
6.3 Material and Methods
6.3.1 Algal material and elicitation procedures.
6.3.2 Conditioning procedure in the laboratory
6.3.3 Transient transplantation in the field
6.3.4 Aldehydes and volatile halogenated organic compounds (VHOCs) measurements
6.3.5 RNA extraction and RT-qPCR
6.4.1 Wild and laboratory-grown algae display different defense responses
6.4.2 Effect of transient transplantation
6.4.3 Development of a conditioning procedure in the laboratory
6.4.4 The conditioning procedure down-regulated the oligoguluronates-induced release of VOCs
7 Conclusions and perspectives