Levels and risk assessment of hydrocarbons and organochlorines in aerosols from a North African coastal city (Bizerte, Tunisia).

The aim of this study was to assess, for the first time, the concentrations, sources, dry deposition and human health risks of polycyclic aromatic hydrocarbons (PAHs), aliphatic hydrocarbons (AHs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) in total suspended particle (TSP) samples collected in Bizerte city, Tunisia (North Africa), during one year (March 2015-January 2016). Concentrations of PAHs, AHs, PCBs and OCPs ranged 0.5-17.8 ng m-3, 6.7-126.5 ng m-3, 0.3-11 pg m-3 and 0.2-3.6 pg m-3, respectively, with higher levels of all contaminants measured in winter. A combined analysis revealed AHs originating from both biogenic and petrogenic sources, while diesel vehicle emissions were identified as dominant sources for PAHs. PCB potential sources included electronic, iron, cement, lubricant factories located within or outside Bizerte city. The dominant OCP congeners were p,p'-DDT and p,p'-DDE, reflecting a current or past use in agriculture. Health risk assessment showed that the lifetime excess cancer risk from exposure to airborne BaP was negligible in Bizerte, except in winter, where a potential risk to the local population may occur.

Natural and anthropogenic particulate-bound aliphatic and polycyclic aromatic hydrocarbons in surface waters of the Gulf of Gabès (Tunisia, southern Mediterranean Sea).

Particulate-bound aliphatic and polycyclic aromatic hydrocarbons (AHs and PAHs) were investigated in the surface waters of the Gulf of Gabès (Tunisia, southern Mediterranean Sea). Samples were collected off the Sfax and Gabès-Ghannouch coasts. Concentrations in total resolved n-alkanes ranged from 0.03 to 3.2 µg L-1, and concentrations in total parents + alkylated PAHs ranged from bdl to 108.6 ng L-1. The highest concentrations were recorded in the southern Sfax. AHs were mainly of biogenic origin with odd n-alkane predominance, although an anthropogenic contribution was also detected. The PAH molecular patterns revealed a mixed origin with the presence of low molecular weight and alkylated compounds, characteristic of uncombusted oil-derived products, and the presence of high molecular weight compounds, typical of combustion residues. Rainfall events induced an increase in PAH concentrations by a factor 1.5-23.5. The particle-water partition coefficients (Koc) suggest that the partitioning of PAHs between the particulate and dissolved phases is driven by hydrophobicity and organic matter composition.

Remobilization of polycyclic aromatic hydrocarbons and organic matter in seawater during sediment resuspension experiments from a polluted coastal environment: Insights from Toulon Bay (France).

Polycyclic aromatic hydrocarbons (PAHs) and organic matter contents were measured in seawater during resuspension experiments using sediments collected from Toulon Bay (Northwestern Mediterranean Sea, France). The studied sediments were very highly contaminated in PAHs, especially in 4-ring compounds emitted from combustion processes. The sediments used for resuspension experiments were collected at 0-2 cm (diagenetically new organic matter, OM) and 30-32 cm depths (diagenetically transformed OM). They were both mostly composed of fine particles (<63 µm), enriched in organic carbon (8.2 and 6.3%, respectively) and in PAHs (concentration of Σ34 PAHs: 38.2 and 35.7 × 103 ng g-1, respectively). The resuspension of these sediments led to an increase in concentrations of dissolved Σ34 PAHs, dissolved organic carbon (DOC) and dissolved humic- and tryptophan-like fluorophores in seawater up to 10-, 1.3-, 4.4- and 5.7-fold, respectively. The remobilization in seawater was higher for 4-6 ring PAHs, especially benzo(g,h,i)perylene, whose concentration exceeded the threshold values of the European Water Framework Directive. This noted the potential harmful effects of sediment resuspension on marine biota. From these sediment resuspension experiments, we determined OC-normalized partition coefficients of PAHs between sediment and water (Koc) and found that during such events, the transfer of PAHs from sediment particles to seawater was lower than that predicted from octanol-water partition coefficients (Kow) (i.e., measured Koc > Koc predicted from Kow). The results confirmed the sequestration role of sedimentary OC quality and grain size on PAHs; the OM diagenetic state seemed to impact the partition process but in a relatively minor way. Furthermore, differences were observed between 2-4 ring and 5-6 ring PAHs, with the latter displaying a relatively higher mobility towards seawater. These differences may be explained by the distribution of these two PAH pools within different OM moieties, such as humic substances and black carbon.

Spatio-temporal variability of fluorescent dissolved organic matter in the Rhône River delta and the Fos-Marseille marine area (NW Mediterranean Sea, France).

The spatio-temporal variability of fluorescent dissolved organic matter (FDOM) and its relationships with physical (temperature, salinity) and chemical (nutrients, chlorophyll a, dissolved and particulate organic carbon, nitrogen and phosphorus) parameters were investigated in inland waters of the Rhône River delta and the Fos-Marseille marine area (northwestern Mediterranean, France). Samples were taken approximately twice per month in two inland sites and three marine sites from February 2011 to January 2012. FDOM was analysed using fluorescence excitation-emission matrices (EEMs) coupled with parallel factor analysis (PARAFAC). In inland waters, humic-like components C1 (λEx/λEm: 250 (330)/394 nm) and C3 (λEx/λEm: 250 (350)/454 nm) dominated over one tryptophan-like component C2 (λEx/λEm: 230 (280)/340 nm), reflecting a background contribution of terrigenous material (~67% of total fluorescence intensity, in quinine sulphate unit (QSU)) throughout the year. In marine waters, protein-like material, with tyrosine-like C4 (λEx/λEm: <220 (275)/<300 nm) and tryptophan-like C5 (λEx/λEm: 230 (280)/342 nm), dominated (~71% of total fluorescence intensity, in QSU) over a single humic-like component C6 (λEx/λEm: 245 (300)/450 nm). In inland waters of the Rhône River delta, humic-like components C1 and C3 were more abundant in autumn-winter, very likely due to inputs of terrestrial organic matter from rainfalls, runoffs and wind-induced sediment resuspension. In marine sites, intrusions of the Berre Lagoon and Rhône River waters had a significant impact on the local biogeochemistry, leading to higher fluorescence intensities of humic- and protein-like components in spring-summer. On average, the fluorescence intensities of FDOM components C4, C5 and C6 increased by 33-81% under lower salinity. This work highlights the complex dynamics of FDOM in coastal waters and confirms the link between marine FDOM and the Rhône River freshwater intrusions on larger spatial and temporal scales in the Fos-Marseille marine area.

Influence of PAHs among other coastal environmental variables on total and PAH-degrading bacterial communities.

We evaluated the relative impact of anthropogenic polycyclic aromatic hydrocarbons (PAHs) among biogeochemical variables on total, metabolically active, and PAH bacterial communities in summer and winter in surface microlayer (SML) and subsurface seawaters (SSW) across short transects along the NW Mediterranean coast from three harbors, one wastewater effluent, and one nearshore observatory reference site. At both seasons, significant correlations were found between dissolved total PAH concentrations and PAH-degrading bacteria that formed a gradient from the shore to nearshore waters. Accumulation of PAH degraders was particularly high in the SML, where PAHs accumulated. Harbors and wastewater outfalls influenced drastically and in a different way the total and active bacterial community structure, but they only impacted the communities from the nearshore zone (<2 km from the shore). By using direct multivariate statistical analysis, we confirmed the significant effect of PAH concentrations on the spatial and temporal dynamic of total and active communities in this area, but this effect was putted in perspective by the importance of other biogeochemical variables.

'Rare biosphere' bacteria as key phenanthrene degraders in coastal seawaters.

By coupling DNA-SIP and pyrosequencing approaches, we identified Cycloclasticus sp. as a keystone degrader of polycyclic aromatic hydrocarbons (PAH) despite being a member of the 'rare biosphere' in NW Mediterranean seawaters. We discovered novel PAH-degrading bacteria (Oceanibaculum sp., Sneathiella sp.) and we identified other groups already known to possess this function (Alteromonas sp., Paracoccus sp.). Together with Cycloclasticus sp., these groups contributed to potential in situ phenanthrene degradation at a rate >0.5 mg l(-1) day(-1), sufficient to account for a considerable part of PAH degradation. Further, we characterized the PAH-tolerant bacterial communities, which were much more diverse in the polluted site by comparison to unpolluted marine references. PAH-tolerant bacteria were also members of the rare biosphere, such as Glaciecola sp. Collectively, these data show the complex interactions between PAH-degraders and PAH-tolerant bacteria and provide new insights for the understanding of the functional ecology of marine bacteria in polluted waters.

Identification and quantification of known polycyclic aromatic hydrocarbons and pesticides in complex mixtures using fluorescence excitation-emission matrices and parallel factor analysis.

Polycyclic aromatic hydrocarbons (PAHs) and pesticides are among the most widespread organic contaminants in aquatic environments. Because of their aromatic structure, PAHs and pesticides have intrinsic fluorescence properties in the ultraviolet/blue spectral range. In this study, excitation-emission matrix (EEM) fluorescence spectroscopy and parallel factor (PARAFAC) analysis were used to characterise and discriminate fluorescence signatures of nine PAHs and three pesticides at the µg L(-1) level in the presence of humic substances (0.1-10 mgCL(-1)). These contaminants displayed a diversity of fluorescence signatures regarding spectral position (λEx: 220-335 nm, λEm: 310-414 nm), Stokes shift (39-169 nm) and number of peaks (1-8), with detection limits ranging from 0.02 to 1.29µgL(-1). The EEM/PARAFAC method applied to mixtures of PAHs with humic substances validated a seven-component model that included one humic-like fluorophore and six PAH-like fluorophores. The EEM/PARAFAC method applied to mixtures of pesticides with humic substances validated a six-component model that included one humic-like fluorophore and three pesticide-like fluorophores. The EEM/PARAFAC method adequately quantified most of the contaminants for humic substance concentrations not exceeding 2.5 mg CL(-1). The application of this method to natural (marine) samples was demonstrated through (1) the match between the Ex and Em spectra of PARAFAC components and the Ex and Em spectra of standard PAHs, and (2) the good linear correlations between the fluorescence intensities of PARAFAC components and the PAH concentrations determined by GC-MS.

Spatial and seasonal variabilities of dissolved hydrocarbons in surface waters from the Northwestern Mediterranean Sea: results from one year intensive sampling.

Dissolved aliphatic hydrocarbons (AHs) and polycyclic aromatic hydrocarbons (PAHs) were analysed from surface water collected in continental, harbour and off-shore marine sites from Marseilles coastal area (Northwestern Mediterranean Sea) from February 2011 to February 2012. AH and PAH concentrations were in the range of 0.04-0.53 µgl(-1) and 8.1-405 ngl(-1), respectively. They both displayed seasonal and spatial variations in their concentrations and molecular composition. The lowest AH concentrations were found in summer and the highest PAH concentrations in winter. Both natural and anthropogenic (pyrogenic and petrogenic) hydrocarbon sources were identified. In winter, concentrations and composition patterns highlighted an increase in the signature of unburned and combusted fossil fuels, while they suggested an enhancement of weathering processes in summer months. Hydrocarbon inputs to the dissolved phase seemed to originate mainly from the atmosphere and the Rhône River. Hydrocarbon additional sources were identified only at the harbour site, emphasising the intense shipping traffic and industrial activities occurring in one of the most important Mediterranean harbours. This study underscores the strong dynamics of dissolved hydrocarbons and the uncoupling of the sources, transport and removing processes affecting AHs and PAHs. It also demonstrates the pertinence of taking this dynamics into account for the budget assessments of organic pollutants in coastal environments.

Fast growth may impair regeneration capacity in the branching coral Acropora muricata.

Regeneration of artificially induced lesions was monitored in nubbins of the branching coral Acropora muricata at two reef-flat sites representing contrasting environments at Réunion Island (21°07'S, 55°32'E). Growth of these injured nubbins was examined in parallel, and compared to controls. Biochemical compositions of the holobiont and the zooxanthellae density were determined at the onset of the experiment, and the photosynthetic efficiency (Fv/Fm ) of zooxanthellae was monitored during the experiment. Acropora muricata rapidly regenerated small lesions, but regeneration rates significantly differed between sites. At the sheltered site characterized by high temperatures, temperature variations, and irradiance levels, regeneration took 192 days on average. At the exposed site, characterized by steadier temperatures and lower irradiation, nubbins demonstrated fast lesion repair (81 days), slower growth, lower zooxanthellae density, chlorophyll a concentration and lipid content than at the former site. A trade-off between growth and regeneration rates was evident here. High growth rates seem to impair regeneration capacity. We show that environmental conditions conducive to high zooxanthellae densities in corals are related to fast skeletal growth but also to reduced lesion regeneration rates. We hypothesize that a lowered regenerative capacity may be related to limited availability of energetic and cellular resources, consequences of coral holobionts operating at high levels of photosynthesis and associated growth.

Occurrence and distribution of hydrocarbons in the surface microlayer and subsurface water from the urban coastal marine area off Marseilles, Northwestern Mediterranean Sea.

Aliphatic (AHs) and polycyclic aromatic hydrocarbons (PAHs) were analyzed in dissolved and particulate material from surface microlayer (SML) and subsurface water (SSW) sampled at nearshore observation stations, sewage effluents and harbour sites from Marseilles coastal area (Northwestern Mediterranean) in 2009 and 2010. Dissolved and particulate AH concentrations ranged 0.05-0.41 and 0.04-4.3 µg l(-1) in the SSW, peaking up to 38 and 1366 µg l(-1) in the SML, respectively. Dissolved and particulate PAHs ranged 1.9-98 and 1.9-21 ng l(-1) in the SSW, amounting up 217 and 1597 ng l(-1) in the SML, respectively. In harbours, hydrocarbons were concentrated in the SML, with enrichment factors reaching 1138 for particulate AHs. Besides episodic dominance of biogenic and pyrogenic inputs, a moderate anthropisation from petrogenic sources dominated suggesting the impact of shipping traffic and surface runoffs on this urbanised area. Rainfalls increased hydrocarbon concentrations by a factor 1.9-11.5 in the dissolved phase.

Characterization of dissolved organic matter in a coral reef ecosystem subjected to anthropogenic pressures (La Réunion Island, Indian Ocean) using multi-dimensional fluorescence spectroscopy.

La Saline fringing reef is the most important coral reef complex of La Réunion Island (southwestern Indian Ocean; 21°07'S, 55°32'E). This ecosystem is subjected to anthropogenic pressures through river inputs and submarine groundwater discharge (SGD). The goal of this study was to characterize the pool of fluorescent dissolved organic matter (FDOM) in different water bodies of La Saline fringing reef ecosystem using excitation-emission matrix (EEM) spectrofluorometry. From EEMs, we identified the different fluorophores by the peak picking technique and determined two fluorescence indices issued from the literature: the humification index (HIX) and the biological index (BIX). The main known fluorophores were present within the sample set: humic-like A, humic-like C, marine humic-like M, tryptophan-like T1 and T2, and tyrosine-like B1 and B2. In some samples, unknown fluorophores ("U") were also detected. The surface oceanic waters located beyond the reef front displayed a typical oligotrophic marine signature, with a dominance of autochthonous/biological material (presence of peaks: T1>B1>A>T2>M>C; HIX: 0.9±0.4; BIX: 2.3±1.1). In the reef waters, the autochthonous/biological fingerprint also dominated even though the content in humic substances was higher (same relative distribution of peaks; HIX: 1.6±0.6; BIX: 1.0±0.1). Sedimentary and volcanic SGD showed very different patterns with a strong terrestrial source for the former (A>T1>C>B1 and A>C>B1; HIX: 9.8±2.0; BIX: 0.8±0.0) and a weak terrestrial source for the latter (A>B1>U3>B2>C and A>U4>C; HIX: 2.4±0.3; BIX: 0.9±0.0). In the Hermitage River, both humic substances and protein-like material were abundant (T1>A>U5>B1>C>B2; HIX: 2.3; BIX: 1.4). We provide evidences for the presence of anthropogenic DOM in some of these water bodies. Some oceanic samples (presence of peaks U1 and U2) were likely contaminated by oil-derived PAHs from ships navigating around the reef front, whereas the Hermitage River was highly impacted by sewage effluents, numerous in this coastal area of La Réunion Island. We conclude that multi-dimensional fluorescence spectroscopy (EEM) coupled to the determination of HIX and BIX is a good tool for assessing the origin and distribution of DOM in the coral reef ecosystems submitted to anthropogenic impacts.

Hydrostatic pressure affects membrane and storage lipid compositions of the piezotolerant hydrocarbon-degrading Marinobacter hydrocarbonoclasticus strain #5.

A new piezotolerant alkane-degrading bacterium (Marinobacter hydrocarbonoclasticus strain #5) was isolated from deep (3475 m) Mediterranean seawater and grown at atmospheric pressure (0.1 MPa) and at 35 MPa with hexadecane as sole source of carbon and energy. Modification of the hydrostatic pressure influenced neither the growth rate nor the amount of degraded hexadecane (approximately 90%) during 13 days of incubation. However, the lipid composition of the cells sharply differed under both pressure conditions. At 0.1 MPa, M. hydrocarbonoclasticus #5 biosynthesized large amounts ( approximately 62% of the total cellular lipids) of hexadecane-derived wax esters (WEs), which accumulated in the cells under the form of individual lipid bodies. Intracellular WEs were also synthesized at 35 MPa, but their proportion was half that at 0.1 MPa. This lower WE content at high pressure was balanced by an increase in the total cellular phospholipid content. The chemical composition of WEs formed under both pressure conditions also strongly differed. Saturated WEs were preferentially formed at 0.1 MPa whereas diunsaturated WEs dominated at 35 MPa. This increase of the unsaturation ratio of WEs resembled the one classically observed for bacterial membrane lipid homeostasis. Remarkably, the unsaturation ratio of membrane fatty acids of M. hydrocarbonoclasticus grown at 35 MPa was only slightly higher than at 0.1 MPa. Overall, the results suggest that intracellular WEs and phospholipids play complementary roles in the physiological adaptation of strain #5 to different hydrostatic pressures.

Utilization of a submersible UV fluorometer for monitoring anthropogenic inputs in the Mediterranean coastal waters.

We evaluated the performances of a submersible ultraviolet fluorometer (EnviroFlu-HC, TriOS Optical Sensors) dedicated to the real time measurement of polycyclic aromatic hydrocarbons (PAHs) in the aquatic media. We conducted calibration experiments and in situ measurements in the coastal Mediterranean Sea. We found that the EnviroFlu-HC was not strictly specific to PAHs, even though it exhibited the highest sensitivity for phenanthrene, but could response to tryptophan-like material as well, and in a much less extent, to humic substances. The sensor signal showed great spatial and temporal variations in clean and polluted sites, with likely a high contribution of PAHs in the harbors, and a high contribution of tryptophan-like and humic-like materials in the sewage effluent. We conclude that the EnviroFlu-HC is a good tool for monitoring anthropogenic inputs in the coastal waters, although its utilization should be combined to other fluorescence measurements to improve the information about the nature of the aromatic compounds detected.

Determination of lipid degradation by marine lipase-producing bacteria: critical evaluation of lipase activity assays.

With the aim of obtaining a better understanding of lipids-lipases interactions in bacterioplankton communities in oceans, we used different methods for measuring lipase activities in pure cultures of the marine strain Alteromonas macleodii. The decay of tripalmitate added to cultures was followed chemically over time. In an enzymatic approach, lipase activities were measured using the fluorogenic lipid analogs MUF-palmitate and ELF-palmitate. When hydrolyzed by lipase, the non-fluorescent substrates release MUF and ELF Alcohol (ELFA) which are fluorescent. As shown by spectrofluorometry, ELF-palmitate was an efficient competitor for MUF-palmitate. However, the activities reached using these two fluorogenic substrates were different, but still much higher than the tripalmitate hydrolysis rate, measured chemically. MUF- and ELF-palmitate would not be hydrolyzed by lipase sensu stricto (defined as triacylglycerol acylesterase E.C. 3.1.1.3) but rather reflects lipolytic activities in a broad sense. ELFA is also water-insoluble and theoretically precipitates in the external membrane of bacteria causing its hydrolysis, which would allow microscopic identification of active cells. By epifluorescence microscopy, the accumulation of ELFA fluorescence over time was detected (as large, diffuse halos), but no precipitates were clearly associated with bacteria on slide preparations, neither for pure cultures of Alteromonas macleodii nor for natural samples from the Bay of Marseille, France. Among possible biases, those related to the hydrophobic/hydrophilic conditions required for precipitation are discussed.

Application of a specific and sensitive radiometric assay for microbial lipase activities in marine water samples from the lagoon of nouméa.

Marine microbiologists commonly assay lipase activities by using a synthetic fluorescent analog, 4-methylumbelliferyl (MUF)-oleate. The technique is convenient, but it is considered to be unspecific because of the structure of this analog. This study reports the design of a new specific and sensitive lipase assay based on the use of a radiolabeled triglyceride, [3H]triolein. Free fatty acids (FFA) resulting from its hydrolysis are isolated as a function of time in a one-step liquid-liquid extraction and then radioassayed. MUF-oleate and [3H]triolein techniques were compared by measuring lipase activities at similar substrate concentrations along a trophic gradient in the Southwest Lagoon of New Caledonia, near Nouméa. Hydrolysis rates decreased from the nearshore station to the offshore station and showed similar trends regardless of the technique used. Rates decreased from 5.83 to 0.88 nmol of FFA. liter-1. h-1 and from 0.76 to 0.23 nmol of 3H-FFA. liter-1. h-1, respectively. These results appeared to be consistent with bacterial production results, which also decreased similarly (from 0.59 to 0.26 micrograms of C. liter-1. h-1). However, the ratio of MUF-oleate activities to [3H]triolein activities, which was constant at the offshore stations (3.8 +/- 0.1), gradually increased at the nearshore stations (from 4.1 to 7.6). This result shows that the two assays respond in different ways to changes in environmental conditions and validates the need to set up more specific enzymatic assays.