Richard Saint-Louis

Toxic effects of tributyltin and its metabolites on harbour seal (Phoca vitulina) immune cells in vitro.

The widespread environmental contamination, bioaccumulation and endocrine disruptor effects of butyltins (BTs) to wildlife are well documented. Although suspected, potential effects of BTs exposure on the immune system of marine mammals have been little investigated. In this study, we assessed the effects of tributyltin (TBT) and its dealkylated metabolites dibutyltin (DBT) and monobutyltin (MBT) on the immune responses of harbour seals. Peripheral blood mononuclear cells isolated from pup and adult harbour seals were exposed in vitro to varying concentrations of BTs. DBT resulted in a significant decrease at 100 and 200 nM of phagocytotic activity and reduced significantly phagocytic efficiency at 200 nM in adult seals. There was no effect in phagocytosis with TBT and MBT. In pups, the highest concentration (200 nM) of DBT inhibited phagocytic efficiency. A reduction of tumor-killing capacity of adult natural killer (NK) cells occurred when leukocytes were incubated in vitro with 50 nM DBT and 200 nM TBT for 24h. In adult seals, T-lymphocyte proliferation was significantly suppressed when the cells were exposed to 200 nM TBT and 100 nM DBT. In pups, the proliferative response increased after an exposure to 100 nM TBT and 50 nM DBT, but decreased with 200 nM TBT and 100 nM DBT. The immune functions were more affected by BTs exposure in adults than in pups, suggesting that other unsuspected mechanisms could trigger immune parameters in pups. The toxic potential of BTs followed the order of DBT>TBT>MBT. BT concentrations of harbour seal pups from the St. Lawrence Estuary (Bic National Park) ranged between 0.1-0.4 ng Sn/g wet weight (ww) and 1.2-13.4 ng Sn/g ww in blood and blubber, respectively. For these animals, DBT concentrations were consistently below the quantification limit of 0.04 ng Sn/g ww in blood and 0.2 ng Sn/g ww in blubber. Results suggest that concentrations measured in pups are considered too low to induce toxic effects to their immune system during first days of life. However, based on our in vitro results, we hypothesize that BTs, and DBT in particular, could pose a serious threat to the immune functions in free-ranging harbour seal adults.

Potential link between exposure to fungicides chlorothalonil and mancozeb and haemic neoplasia development in the soft-shell clam Mya arenaria: a laboratory experiment.

The aetiology of haemic neoplasia (HN) is unknown, so far but many causative factors are suggested such as viral, pollution and genetics. The aim of this study was to determine if, under chronic exposure, two major pesticides (chlorothalonil and mancozeb) which are used in potato production could induce HN in soft-shell clams (Mya arenaria). Short-term experiments with acute exposure were also performed. Clams were collected from an epizootic site (North River, PEI) and from a site free of the disease (Magdalen Islands, Quebec). The tetraploid level of haemocytes was assessed by flow cytometry for each clam to determine the HN status. The bioaccumulation of pesticides in tissues was quantified by gas chromatography/mass spectrometry (GC/MS) for chlorothalonil while mancozeb and manganese were quantified by inductively coupled plasma-mass spectrometer (ICP/MS). Long term exposure to fungicide Bravo 500((R)) did not induce high tetraploid levels on negative calm from North River and the analysis of the digestive gland and the mantle did not reveal any detectable level of chlorothalonil. In the Manzate 200 DF((R)), some clams revealed high level of tetraploid cells but no difference were observed between the treatments and the control. The analysis of the digestive gland and the mantle for manganese did not highlight any significant difference in tissue concentration (p=0.05). For the acute exposure, chlorothalonil analysis showed that the active ingredient is distributed between four chlorinated compounds: 99.5% for chlorothalonil isomers, 0.4% for pentachlorothalonil and 0.1% for trichlorothalonil isomers. For a 72 h experiment, the accumulation was within 4h; the higher tissue concentration of chlorothalonil was 59.2 microg g(-1) in the mantle after 48 h, following by a decrease to an undetectable level at the end. For the manganese, the accumulation was detected after 4h; the higher tissue concentration was 48.8 microg g(-1) in the mantle after 24h and, over the following 48 h, the accumulation decreased until the end of the trial. Based on the data, the accumulation of these fungicides seems to be transitory. Chlorothalonil and mancozeb are both oxidative-stress promoters and could have induced cell dysfunction while in the tissue. Study on the effect of these fungicides on the p53 protein system is an example of strategy that would provide information on cellular events promoting neoplasia.

High Risk of Lead Contamination for Scavengers in an Area with High Moose Hunting Success

Top predators and scavengers are vulnerable to pollutants, particularly those accumulated along the food chain. Lead accumulation can induce severe disorders and alter survival both in mammals (including humans) and in birds. A potential source of lead poisoning in wild animals, and especially in scavengers, results from the consumption of ammunition residues in the tissues of big game killed by hunters. For two consecutive years we quantified the level lead exposure in individuals of a sentinel scavenger species, the common raven (Corvus corax), captured during the moose (Alces alces) hunting season in eastern Quebec, Canada. The source of the lead contamination was also determined using stable isotope analyses. Finally, we identified the different scavenger species that could potentially be exposed to lead by installing automatic cameras targeting moose gut piles. Blood lead concentration in ravens increased over time, indicating lead accumulation over the moose-hunting season. Using a contamination threshold of 100 µg.L−1, more than 50% of individuals were lead-contaminated during the moose hunting period. Lead concentration was twice as high in one year compared to the other, matching the number of rifle-shot moose in the area. Non-contaminated birds exhibited no ammunition isotope signatures. The isotope signature of the lead detected in contaminated ravens tended towards the signature from lead ammunition. We also found that black bears (Ursus americanus), golden eagles and bald eagles (Aquila chrysaetos and Haliaeetus leucocephalus, two species of conservation concern) scavenged heavily on moose viscera left by hunters. Our unequivocal results agree with other studies and further motivate the use of non-toxic ammunition for big game hunting.

Distribution et effets du chlorure de tributylétain et de ses produits de dégradation sur la croissance de l'algue marine Pavlova lutheri en culture continue

Organotin compounds have been used as biocides, wood preservatives, plastic stabilizers and antifouling agents for many years (Maguire, 1991). The acute toxicity as well as the sublethal effects of tributyltin (TBT) derivatives have been tested for numerous freshwater and marine species (Alzieu, 1989; Kelly et al., 1990). The biodegradation of TBT in dibutyltin (DBT) and monobutyltin (MBT) is well established for some phytoplankton species (Lee et al., 1989) but little is known about the physiological adaptation of microalgae to a long term sublethal contamination by TBT. The objective of this study was to evaluate the ability of marine alga Pavlova lutheri, cultured in “chemostat mode” under axenic conditions, to accomodate increasing cocentrations of TBTCl in the culture medium and to determine its capacity to degrade TBT in DBT and MBT. Chemostat culture of P. lutheri were exposed to contaminated nutrient supplies containing 18.5, 74, and 185 nmol l−1 of TBT chloride (expressed as TBT+) respectively. For comparison purposes, a batch culture of P. lutheri was exposed to a concentration of 13 nmol l−1 of TBTCl for a 48 h-period. Organotin species (BuSn) were monitored by GC/ITD in three different fractions: dissolved in the culture medium, adsorbed on the external cellular walls, and dissolved in the cellular fluid. Within a 24 h-period, TBT was observed in the cellular fluid indicating a rapid uptake of the contaminant. The ability of P. lutheri to degrade TBT was confirmed and found more effective in a continuous culture than in a batch culture under similar contamination conditions. MBT was observed in both cellular fractions of all cultures (Tables 2 and 3) but P. lutheri seems particularly efficient to form MBT at low TBT concentration. The amount of BuSn adsorbed onto the cell was directly related to TBT concentration in the nutrient supply but intracellular BuSn decreased from 150 nmol g−1 (dry weight) to less than 60 nmol g−1 (dry weight) when the TBT concentration increased from 74 to 185 nmol l−1 (Fig. 2). The cell density and the growth rate of a continous culture of P. lutheri (Figs 3 and 4) were not affected by a contamination level of 18.5 nmol l−1 of TBT. In a continuous culture receiving 74 nmol l−1 of TBT P. lutheri suffered a toxic impact in the first few days of the experiment, loosing 40% of its cell density, However, the culture recovered its initial growth rate in the next 10 days in spite of an uninterrupted contamination. Finally, a severe toxic shock was observed for a culture receiving 185 nmol l−1 of TBT leading to a major dysfunction of the culture in only three days. The toxic effects observed in culture productivity (Fig. 5) for treatments at 74 and 185 nmol l−1 are also illustrated by a significant decrease of chlorophyll α concentration in culture [Fig. 6(A)] but not in the individual cells [Fig. 6(B)] which was an indication of the preservation of life functions of cells in all experiments. These results evidence the ability of P. lutheri, cultured in chemostat mode, to resist and accommodate to a continuous input of a high level of TBT to the culture medium degrading TBT into much less toxic species MBT and DBT. As a chemostat culture is comparable in many aspects to an open natural environment (Rhee, 1980; Wangersky and Maass. 1991). The behavior of P. lutheri observed in this study might be extended to the natural coastal environment where this alga is present. If such an assumption is correct at least regarding the general processes of bioaccumulation and degradation, this study clearly indicates that P. lutheri might play an important role in the food uptake of BuSn by filter feeders and other herbivorous species due to its ability to bioaccumulate large quantities of BuSn [Fig 2(A) and 2(B)] without being irreversibly affected. Fortunately, the natural environment as simulated by our continuous cultures seems to strongly accelerate the degradation rate of TBT into DBT and MBT, two much less toxic tin species. Results of previous studies on the toxicity effects of TBT on micro-algae, all conducted in batch cultures, should be interpreted with some caution since the ability of P. lutheri and possibly other marine algae to survive and grow in TBT contaminated media seems to have been grossly underestimated.

Lipase-catalyzed synthesis of structured phenolic lipids in solvent-free system using flaxseed oil and selected phenolic acids as substrates.

Structured phenolic lipids (PLs) were obtained by lipase-catalyzed transesterification of flaxseed oil, in a solvent-free system (SFS), with selected phenolic acids, including hydroxylated and/or methoxylated derivatives of cinnamic, phenyl acetic and benzoic acids. A bioconversion yield of 65% was obtained for the transesterification of flaxseed oil with 3,4-dihydroxyphenyl acetic acid (DHPA). However, the effect of the chemical structure of phenolic acids on the transesterification of flaxseed oil in SFS was of less magnitude as compared to that in organic solvent system (OSS). Using DHPA, the APCI-MS analysis confirmed the synthesis of monolinolenyl, dilinolenyl, linoleyl linolenyl and oleyl linolenyl dihydroxyphenyl acetates as phenolic lipids. A significant increase in the enzymatic activity from 200 to 270 nmol of PLs/g solid enzyme/min was obtained upon the addition of the non-ionic surfactant Span 65. However, upon the addition of the anionic surfactant, sodium bis-2-ethylhexyl sulfosuccinate (AOT), and the cationic one, hexadecyltrimethylammonium bromide (CTAB), the enzymatic activity was decreased slightly from 200 to 192 and 190 nmol of PLs/g solid enzyme/min, respectively. The results also showed that the increase in DHPA concentration from 20 to 60 mM resulted in a significant increase in the volumetric productivity (P(V)) from 1.61 to 4.74 mg PLs per mL reaction mixture per day.

Biosorption of thorium on the external shell surface of bivalve mollusks: The role of shell surface microtopography

External shell surface (ESS) of bivalve mollusks is known to adsorb various metals dissolved in ambient water in high concentration. It is hypothesized here that the surface microtopography of the thin organic coating layer, periostracum, or calcareous shell (if periostracum was destroyed) plays a major role in the adsorption of actinides on ESS. Thorium (natural alpha-emitter) was used in short-term biosorption experiment with shell fragments of five bivalve mollusks. After a 72 h exposure to Th (~6 kBq L(-1)), thorium concentration was measured on ESS using laser ablation inductively coupled plasma mass spectrometry; the distribution and density of alpha tracks were subsequently visualized by α-track autoradiography. A trend in reduced Th concentrations on the ESS was observed depending upon the species tested: (group 1 ~4000 μg g(-1)) Chlamys islandica (M.), Mercenaria mercenaria (L.), Dreissena polymorpha (P.)>(group 2 ~1200 μg g(-1)) Crassostrea virginica (G.)≫(group 3 ~150 μg g(-1)) Mytilus edulis L. The microtopography of ESS was characterized by scanning electron microscopy revealing the high porosity of the calcareous surface of C. islandica and M. mercenaria, lamellate surface of periostracum in D. polymorpha, uneven but a weakly porous surface of periostracum of C. virginica, and a nearly smooth surface of the periostracum of M. edulis. This work has demonstrated, for the first time, the presence of a strong correlation between concentration of adsorbed Th and ESS microtopography, and the role of the periostracum in this process is discussed.