Monique Boily

Combined effects of agricultural activity and parasites on biomarkers in the bullfrog, Rana catasbeiana.

Agricultural contaminants can have devastating impacts on amphibian survival and development, particularly considering their sensitivity to environmental perturbation. However, it is commonly overlooked that amphibians are infected with various parasites that can influence the overall health of the animal when exposed to a stressful environment. We investigated the interaction of agriculture and parasitism on the health of bullfrogs (Rana catesbeiana) in the field. Nine physiological and immunological biomarkers were related to naturally acquired parasite infections, along a gradient of agricultural activity. Most health biomarkers were affected by agriculture, parasitism, or both. Although bullfrogs residing in agricultural areas were infected with fewer parasite species, reflecting environmentally compromised ecosystems, certain persistent parasites interacted with agricultural disturbance to alter the physiology and immune competence of bullfrogs. The consequences of the combination for animal health highlight the importance of parasitism in ecotoxicological studies. Consideration of parasitism is warranted when evaluating the influence of anthropogenic disturbance on amphibian declines and environmental health.

Hepatic retinoids of bullfrogs in relation to agricultural pesticides

Agricultural pesticides often have been cited as a factor affecting indigenous amphibian populations, but possible effects of pesticides and other factors associated with agricultural practices are understood poorly. Adult bullfrogs (Rana catesbeiana) were collected within the Yamaska River basin (Quebec, Canada) in subwatersheds representing low, medium, and high agricultural activities and 53 pesticides were analyzed in surface water. More pesticides were detected in subwatersheds associated with high agricultural activities like Rivière Noire and Rivière à la Barbue and pesticide concentrations were higher compared to the other study sites. Female and male body weights differed between sites. In the case of males, body weight was significantly less at Rivière à la Barbue. Liver retinol stores were decreased significantly in male bullfrogs from Rivière Noire, although total retinyl esters concentrations varied between sites having the highest concentration at Yamaska-Nord where the agricultural activity was considered low. The ratio of hepatic retinyl palmitate to retinol tended to be higher for male bullfrogs from Rivière Noire and Rivière à la Barbue. These results suggest that factors associated with intensive agricultural practices may affect the body weight and retinoid stores in male bullfrogs living in these agroecosystems.

β-carotene and retinoids in eggs of Great Blue Herons (Ardea herodias) in relation to St Lawrence River contamination.

The potential use of retinoids and β-carotene as biomarkers in the eggs of the Great Blue Heron was investigated. In the spring of 1991, 65 eggs were collected from nine heronries (seven along the St Lawrence River and two reference sites). A method was specifically developed for the extraction and analysis of β-carotene and the retinoids in heron egg yolks by reversed-phase HPLC. When results were expressed either as the molar ratio of retinol: retinyl palmitate or as retinyl palmitate concentration, significant differences were found between colonies; however, retinyl palmitate concentration was deemed the better biomarker because it was not significantly influenced by embryonic stage of development. Retinyl palmitate concentrations in freshwater colonies were negatively related to PCB congeners Nos 105 and 118 as well as their TCDD-EQ values (p < 0.02, r2=0.78). Egg tetrachloro-mono-ortho biphenyl concentrations were also negatively related to retinyl palmitate (p < 0.005, r2=0.90). With the exception of the two mono-ortho co-planar congeners detected in the present study, the contamination levels found in heron eggs were well below those found for other bird species in the Great Lakes area and, so far, no detrimental effects have been reported in Great Blue Heron populations in Quebec. These results suggest that retinyl palmitate may be useful as a sensitive and non-invasive biomarker for monitoring organochlorine contaminant effects in the Great Blue Heron in freshwater sites.

Assessment of Contamination and Biomarker Responses in Two Species of Herons on the St. Lawrence River

This study was undertaken to validate potential biomarkers of exposure and effects due to chemical contaminants in breedingcolonies of the Great Blue Heron and the Black-crowned Night-Heron on the St. Lawrence River. Eggs and fledglings from both species were collected from many colonies along theRiver. The fledglings from colonies in freshwater and brackishwater were more contaminated by mercury and PCBs than those from estuarine and gulf colonies. With respect to fledglings ofthe two heron species, some morphometric and blood biochemicalmeasurements, including plasma thyroid hormones and retinol, were significantly different among colonies. Significant differences were also observed in liver retinoids, EROD and porphyrins among colonies. The results of this study suggestthat plasma retinoids and thyroid hormones are good biomarkersof exposure and effects, and are sufficiently sensitive to reflect local and regional variations in contamination. Along with the measure of contaminants in egg and plasma, they constitute non-invasive biomarkers which represent an importantcriteria for long term monitoring of wildlife species. It is concluded that the Great Blue Heron is an appropriate sentinelspecies in the surveillance network for the St. Lawrence River.

Retinoid metabolism (LRAT, REH) in the liver and plasma retinoids of bullfrog, Rana catesbeiana, in relation to agricultural contamination

Retinoids have been extensively studied in birds, fish and mammals where their imbalances are associated with adverse effects on growth and reproduction along with decreased embryo survival and deformities. Organochlorine compounds may alter the retinoid system but little is known about the effects of agricultural contaminants on their metabolism. In the Yamaska River project, the retinoid system in bullfrogs is monitored to investigate the possible impact of agricultural contaminants on retinoid homeostasis. Retinoids were measured in liver and plasma of male bullfrogs collected from six locations subject to increasing agricultural activity in the Yamaska River watershed. Bullfrogs living in medium and high agricultural activity areas demonstrated lower hepatic retinyl palmitate and higher hepatic retinol levels when compared to frogs associated with low contaminated sites. Changes in the concentration of hepatic esters could be related to an altered activity of REH or LRAT, enzymes respectively linked to the hydrolysis of retinyl palmitate and the esterification of retinol. A partial characterization and the analysis of liver microsomial REH and LRAT showed significantly higher hydrolysis and lower esterification activities in highly contaminated sites. Enzymatic activities seemed to be influenced by plasma DROH but not by plasma retinol. Bullfrogs from the most contaminated sites showed altered retinoic metabolism that should increase concern for frogs living in intensive agricultural areas.

Low dietary levels of Al, Pb and Cd may affect the non-enzymatic antioxidant capacity in caged honey bees (Apis mellifera)

Several hypotheses have been proposed to explain the abnormally high mortality rate observed in bee populations in Europe and North America. While studies based on the effects of pesticides are paramount, the metals present in agroecosystems are often overlooked. Sources of metals are linked to the nature of soils and to agricultural practices, namely the use of natural or chemical nutrients as well as residual materials from waste-water treatment sludge. The aim of this study was to investigate the effects of metals on honey bees exposed for 10 days to environmentally realistic concentrations of Al, Pb and Cd (dissolved in syrup). The monitoring of syrup consumption combined with the quantification of metals in bees revealed the following order for metal bioconcentration ratios: Cd > Pb > Al. Alpha-tocopherol, metallothionein-like proteins (MTLPs) and lipid peroxidation were quantified. When bees were exposed to increasing amounts of Cd, a marked augmentation of MTLPs levels was found. Lead (Pb) and Cd caused an increase in α-tocopherol content, while alteration of lipid peroxidation was observed only with Al exposure. These findings raise concerns about the bioavailability and the additional threat posed by metals for pollinators in agricultural areas while providing new insights for potential use of the honey bee as a sentinel species for metal exposure.

Retinoids, LRAT and REH Activities in Eggs of Japanese Quail Following Maternal and in ovo Exposures to 3,3′,4,4′-Tetrachlorobiphenyl

Environmental monitoring for the effects of organochlorine contaminants has identified changes in egg yolk retinoid concentrations, however underlying mechanisms are unknown. Breeding female Japanese quail, Coturnix coturnix japonica, received 3 bimonthly injections of a low dose (5 µg/g) of the coplanar polychlorinated biphenyl (PCB) 3,3′,4,4′-tetrachlorobiphenyl. No overt maternal toxicity or embryotoxicity was detected. For the exposed females, oocytes (yolk deposition nearly completed) and freshly laid eggs contained higher concentrations of retinol. For PCB-exposed female quail, freshly laid eggs and fertile eggs incubated for 6 days contained higher concentrations of retinyl palmitate. These results suggest that the PCB congener caused greater deposition of retinoids in yolks during late oogenesis or greater net esterification in the yolk-sac membrane. Retinyl ester hydrolase (REH) activity, assayed in the microsomal fraction of the yolk-sac membrane, was significantly inhibited in the PCB-exposed group. Therefore, the higher retinyl palmitate concentrations may be explained by less retinyl palmitate conversion to retinol. In a second experiment, fertile quail eggs were injected with 2, 10 or 20 µg of the PCB congener and incubated for 6 days. In contrast to the maternal exposure, yolk retinol decreased and retinyl ester hydrolase increased in the PCB-injected eggs. The difference in the results of adult versus egg injections may be related to several factors including adult transformation of the PCB to toxic metabolites and differences in post-transcriptional regulation of REH expression.

P19 neuronal differentiation and retinoic acid metabolism as criteria to investigate atrazine, nitrite, and nitrate developmental toxicity.

Atrazine and nitrogenous fertilizers are agrochemical contaminants frequently detected in water systems in North America. Several studies reported their ability to affect amphibian and mammalian development. Retinoids, supplied in the diet or synthesized by cells, are essential to embryogenesis. Disturbance of their homeostasis may lead to teratogenic effects. Retinoic acid (RA) is a major retinoid regulator of cell proliferation and differentiation. Previous studies reported alterations of retinoid stores in bullfrogs of Yamaska River subwatersheds (Québec, Canada), a region of intensive agricultural activities associated with atrazine, nitrate, and nitrite contaminants. These contaminants could affect RA metabolism and RA-mediated processes. Mouse P19 embryonic stem cells, which can differentiate to neurons in response to RA, were used to test this hypothesis. Cells were cultured in the absence or presence of contaminants during neuroinduction with RA and assayed by flow cytometry for expression of stage-specific embryonic antigen-1 (SSEA1) (embryonic marker) and betaIII-tubulin (neuronal marker). Cell cultures were also analyzed for RA metabolism by high performance liquid chromotagraphy (HPLC). Downregulation of SSEA1 paralleled betaIII-tubulin upregulation in an RA concentration-dependent manner. Atrazine, nitrate, and nitrite did not affect differentiation at environmentally encountered micromolar concentrations. However, low molar nitrite prevented RA-induced SSEA1 downregulation and decreased betaIII-tubulin appearance. Decreased cell viability/proliferation accompanied these differentiation effects. P19 cells metabolized RA to polar retinoids. RA metabolism was not affected at any concentration of atrazine, nitrate, or nitrite. Environmentally relevant levels of these contaminants, thus, had no gross effect on neurodifferentiation and RA catabolism of embryonic stem cells. P19 cell-based bioassays may provide valuable tools in monitoring developmental toxicity.

Mixtures of herbicides and metals affect the redox system of honey bees

The increasing loss of bee colonies in many countries has prompted a surge of studies on the factors affecting bee health. In North America, main crops such as maize and soybean are cultivated with extensive use of pesticides that may affect non-target organisms such as bees. Also, biosolids, used as a soil amendment, represent additional sources of metals in agroecosystems; however, there is no information about how these metals could affect the bees. In previous studies we investigated the effects of environmentally relevant doses of herbicides and metals, each individually, on caged honey bees. The present study aimed at investigating the effects of mixtures of herbicides (glyphosate and atrazine) and metals (cadmium and iron), as these mixtures represent more realistic exposure conditions. Levels of metal, vitamin E, carotenoids, retinaldehyde, at-retinol, retinoic acid isomers (9-cis RA, 13-cis RA, at-RA) and the metabolites 13-cis-4-oxo-RA and at-4-oxo-RA were measured in bees fed for 10 days with contaminated syrup. Mixtures of herbicides and cadmium that did not affect bee viability, lowered bee α- and β-carotenoid contents and increased 9-cis-RA as well as 13-cis-4-oxo-RA without modifying the levels of at-retinol. Bee treatment with either glyphosate, a combination of atrazine and cadmium, or mixtures of herbicides promoted lipid peroxidation. Iron was bioconcentrated in bees and led to high levels of lipid peroxidation. Metals also decreased zeaxanthin bee contents. These results show that mixtures of atrazine, glyphosate, cadmium and iron may affect different reactions occurring in the metabolic pathway of vitamin A in the honey bee.

Integrated spatial health assessment of yellow perch (Perca flavescens) populations from the St. Lawrence River (QC, Canada), part B: cellular and transcriptomic effects

Multi-biological level assessments have become great tools to evaluate the health of aquatic ecosystems. Using this approach, a complementary study was designed to evaluate the health of yellow perch (Perca flavescens) populations in the St. Lawrence River (Quebec, Canada). In the present study, stress responses were compared at the transcriptomic, cellular, and tissue levels in yellow perch collected at six sites along the river: Lake St. François, Lake St. Louis (north and south), Beauregard Island and Lake St. Pierre (north and south). These results complement the physiological and chemical parameters as well as pathogen infection investigated in a companion paper published in the present issue. Thiobarbituric acid reactive substance (TBARS) analyses indicated the presence of oxidative stress in fish collected in the southern part of Lake St. Louis and at the downstream sites of Lake St. Pierre. High lipid peroxidation levels were found in the muscle of yellow perch caught at Beauregard Island, located downstream of the Montreal’s wastewater treatment plant, suggesting an impact of the municipal effluent on redox homeostasis. Transcriptomic results indicated the down-regulation of genes related to lipid, glucose, and retinoid in southern Lake St. Pierre as well as a decrease in retinoid storage. Overall, biochemical and molecular markers indicated that the health status of yellow perch followed a decreasing gradient from upstream to downstream of the St. Lawrence River. This gradient is representative of the cumulative negative impacts of human activities on water and habitat quality along the river.