In situ analysis of cadmium uptake in four sections of the gastro-intestinal tract of rainbow trout (Oncorhynchus mykiss).

This study links results from past in vitro and in vivo experiments, by implementing an in situ experiment in order to determine the relative importance for cadmium (Cd) uptake of different sections of the gastro-intestinal tract (GIT) of rainbow trout. Transport of Cd from four sections of the GIT of adult rainbow trout (~220 g) was individually examined by infusing ligated sections of the GIT in live, free-swimming fish with 50 µM Cd spiked with radiolabelled (109)Cd (0.5 µCi ml(-1)). Fish were exposed for an 8-h period. The percentage of the total injected (109)Cd which was internalized from the different segments was only between ~0.1 and ~7%, indicating low uptake efficiency. The stomach is the most important GIT segment for Cd transport into the internal compartment of the animal, while the posterior intestine also plays a significant role. The majority of (109)Cd recovered at the end of the flux period was detected within gut material (ranging from 28 to 95%); the portion of Cd which was internalized was largely found in the carcass (32 to 60%). Distribution between the measured organs varied with uptake from the various GIT sections. Our results also confirm that the GIT acts as a protective barrier against Cd uptake from dietary exposure.

In vitro assessment of the genotoxicity and immunotoxicity of treated and untreated municipal effluents and receiving waters in freshwater organisms.

Municipal wastewater effluent is one of the largest sources of pollution entering surface waters in the Laurentian Great Lakes. Exposure to wastewater effluent has been associated with impaired immune systems and induction of genotoxicity to aquatic animals. Due to habitat degradation and environmental pollution linked to industrial development and population growth, several regions of the Great Lakes have been designated Areas of Concern (AOCs). In this study, we assessed the effect of extracts of sewage influent, (treated) effluent and receiving surface waters from the Hamilton Harbour AOC and the Toronto and Region AOC (Ontario, Canada) on the phagocytic immune response of rainbow trout (Oncorhynchus mykiss) kidney leukocytes and the genotoxicity (DNA strand breaks) of these extracts on freshwater mussel (Eurynia dilatata) hemocytes. We identified and quantified numerous chemicals present in the various samples extracted for exposure. In freshwater mussels, extracts from Hamilton Harbour AOC induced DNA damage with the most frequency (12 out of 28 samples) regardless of sample type, reflecting past and present industrial activities. In contrast, extracts from Toronto and Region AOC induced DNA damage infrequently (2 out of 32 (summer) and 5 out of 32 (fall) samples, respectively) and from different WWTPs at different times. None of the extracts induced any significant effect on phagocytosis of rainbow trout kidney leukocytes. The present study indicates that despite overall improvements to effluent quality, treatment of influent by WWTPs may not result in a corresponding improvement of the genotoxicity of effluents. In vitro bioassays are useful and cost-effective rapid-screening tools for preliminary assessments of contamination of aquatic ecosystems.

Effects of using synthetic sea salts when measuring and modeling copper toxicity in saltwater toxicity tests.

Synthetic sea salts are often used to adjust the salinity of effluent, ambient, and laboratory water samples to perform toxicity tests with marine and estuarine species. The U.S. Environmental Protection Agency (U.S. EPA) provides guidance on salinity adjustment in its saltwater test guidelines. The U.S. EPA suggests using commercial sea salt brands, such as Forty Fathoms (now named Crystal Sea Marinemix, Bioassay Grade), HW Marinemix, or equivalent salts to adjust sample salinity. Toxicity testing laboratories in Canada and the United States were surveyed to determine synthetic sea salt brand preference. The laboratories (n = 27) reported using four brands: Crystal Sea Marinemix (56%), HW Marinemix (22%), Instant Ocean (11%), and Tropic Marin (11%). Saline solutions (30 g/L) of seven synthetic sea salts were analyzed for dissolved copper and dissolved organic carbon (DOC) content. Brands included those listed above plus modified general-purpose salt (modified GP2), Kent Marine, and Red Sea Salt. The synthetic sea salts added from < 0.1 to 1.2 microg Cu/L to the solution. Solutions of Crystal Sea Marinemix had significantly elevated concentrations of DOC (range = 5.4-6.4 mg C/L, analysis of variance, Tukey, alpha = 0.05, p < 0.001) while other brands generally contained < 1.0 mg C/L. The elevated DOC in Crystal Sea Marinemix was expected to reduce copper toxicity. However, the measured dissolved copper effective concentration 50% (EC50) for Crystal Sea Marinemix was 9.7 microg Cu/L, similar to other tested sea salts. Analysis indicates that the organic matter in Crystal Sea Marinemix differs considerably from that of natural organic matter. On the basis of consistently adding little DOC and little dissolved copper, GP2 and Kent Marine are the best salts to use.

Toxicity and bioconcentration of the pharmaceuticals moxifloxacin, rosuvastatin, and drospirenone to the unionid mussel Lampsilis siliquoidea.

Pharmaceuticals and personal care products (PPCPs) and their metabolites are continually released from wastewater treatment plants into the aquatic environment; however, their impact on aquatic biota is poorly understood. This study examined the toxicity and bioconcentration of three pharmaceuticals: moxifloxacin, rosuvastatin, and drospirenone to the unionid mussel Lampsilis siliquoidea. Effects of moxifloxacin and rosuvastatin were assessed through aqueous 21-d static-renewal tests using 2-year-old mussels, at 0.01, 0.1, 1, 10 and 100mg/L (nominal concentrations). Following exposure, survival, behavior, algal clearance rate, hemocyte viability and density, and glutathione S-transferase (GST) activity were assessed. In addition, the acute (48 h) toxicity of moxifloxacin (0-100mg/L) and drospirenone (0-3mg/L) to glochidia (larval mussels) were examined. In 21 day exposures (2-yr old mussels), there were no differences in survival, oxygen consumption, hemocyte density, or GST activity over the range of concentrations examined; however, the proportion of time mussels spent filtering, and consequently the algal clearance rate, decreased at the higher moxifloxacin and rosuvastatin concentrations. Bioconcentration factors (BCFs) ranged between 0.03 and 70 for moxifloxacin, and between 0 and 0.05 for rosuvastatin for exposures up to 100mg/L. The BCF for moxifloxacin at the highest exposure concentration was lower than that at the mid-level concentrations, likely due to decreased filtering activity at the higher exposure levels. The feeding rates declined and the amount of time the subadult mussels spent with their valves closed increased at the higher moxifloxacin and rosuvastatin exposures. Glochidia viability did not vary with exposure to drospirenone, but declined at the highest moxifloxacin concentration, resulting in an EC50 of 120 mg/L. Overall, observed sublethal and lethal effects occurred at concentrations which exceed expected environmental concentrations through aqueous exposure, suggesting a low risk to freshwater mussels from these particular PPCPs.

Gastro-intestinal transport of calcium and cadmium in fresh water and seawater acclimated trout (Oncorhynchus mykiss).

Transport of calcium (Ca) and cadmium (Cd) was examined along the gastro-intestinal tract (GIT) of freshwater and seawater Oncorhynchus mykiss irideus (FWT and SWTies respectively) using in vitro and in vivo experiments. Based on known physiological differences between FWT and SWT which aid in regulating ion levels and osmolarity, we hypothesized that SWT would have lower rates of Ca uptake. Also, we predicted that Cd rates would also be lower because Cd is known to share a common transport mechanism with Ca. Kinetics of Ca and Cd transport were determined using mucosal salines of varying concentrations [1, 10, 30, 60, and 100 (mmolL(-1) for Ca, µmolL(-1) for Cd)]. Linear and saturating relationships were found for Ca for FWT and SWT, but overall SWT had lower rates. Linear and/or saturating relationships were also found for Cd uptake, but rates varied little between fish types. Elevated Ca had no inhibitory effect on Cd transport, and Ca channel blockers nifedipine and verapamil had little effect on Ca or Cd uptake. However, lanthanum reduced Ca transport into some compartments. A 21 day in vivo feeding experiment was also performed where FWT and SWT were exposed to control diets or Cd-spiked diets (552 µg Cd g(-1) food). Whole body Cd uptake between fish types was similar, but the majority of Cd in SWT remained in the posterior intestine tissue, while FWT transported more Cd through their gut wall. Overall it appears that large differences in Ca and Cd uptake between FWT and SWT exist, with SWT generally having lower rates.

In vitro characterization of cadmium transport along the gastro-intestinal tract of freshwater rainbow trout (Oncorhynchus mykiss).

An in vitro gut sac technique was used to examine the mechanism(s) of cadmium (Cd) uptake along the gastro-intestinal tract (GIT) of rainbow trout (Oncorhynchus mykiss). The spatial distribution of Cd between three compartments (mucus-binding, mucosal epithelium, and transport into blood space) was determined using a modified Cortland saline containing 50µM Cd (as CdCl(2)) labeled with (109)Cd radiotracer. Taking into account total surface areas, the order of relative importance for total Cd uptake rate was: posterior intestine>anterior intestine>stomach>mid intestine. Cd transport was not inhibited by experimentally reducing fluid transport rates by manipulation of osmotic gradients using mannitol, but was sensitive to internal luminal pressure changes, suggesting a mechanosensitive pathway. Q(10) values (1, 11, and 19°C) indicated a facilitated transport of Cd in the anterior- and mid-intestine. The effects of 10mM Ca on the kinetics of Cd uptake suggest the presence of a common uptake pathway for Cd and Ca in the stomach, anterior-, and mid-intestine. Further evidence of a shared route of entry was found using three Ca channel blockers, lanthanum, verapamil, and nifedipine: both voltage-insensitive and voltage-sensitive Ca channels appear to be present in either some, or all portions of the GIT. Elevated Fe (500µM), Mg (50mM), and Zn (500µM) showed varying degrees of inhibition of Cd transport depending on the compartment and segment of the GIT. Overall it appears that there are multiple sites, and mechanisms, of Cd uptake along the GIT of rainbow trout.

Does dietary Ca protect against toxicity of a low dietborne Cd exposure to the rainbow trout?

We examined the toxicity of Cd, provided in a natural diet and at an environmentally relevant concentration ( approximately 12microgg(-1) dry wt.), to the juvenile rainbow trout (Oncorhynchus mykiss). In addition, we tested the protection by elevated dietary Ca against both the accumulation and toxicity of dietary Cd from this natural diet (background Ca approximately 1mgg(-1) dry wt.). Food pellets were made from blackworms (Lumbriculus variegatus), and spiked with Cd and either no additional Ca or elevated ( approximately 60mgg(-1) dry wt.) concentrations for each of the treatment diets. Survival was unaffected for trout fed diet with 12microgg(-1) dry wt. Cd for a month, but growth was potentially reduced. Tissue burden analysis revealed that the stomach, liver and kidney accumulated the most Cd, with concentrations progressively increasing in the liver and kidney over the whole exposure period. Cd concentrations in the plasma and red blood cells were unaffected by the different treatments, but subcellular fractionation analysis indicated that a higher concentration of Cd was associated with the metal-sensitive fractions of red blood cells of the fish that were exposed to the dietborne Cd. Dietary Cd exposure also caused potential toxicity to cells of the stomach in that they bound more Cd to heat-denaturable proteins. However, detoxification appeared to take place in the Cd-exposed fish because more Cd was bound to metallothionein-like proteins by week 4 of exposure. Elevated Ca in the Cd diet generally protected against accumulation and toxicity of dietborne Cd. The protection against Cd accumulation was almost complete at the gills, robust in the stomach and whole body (> or =50% reductions), but not significant in the liver, kidney, carcass, plasma, or red blood cells. Elevated dietary Ca also reduced Cd accumulation in the organelles of the fish stomach and red blood cells. In addition, dietborne Ca not only reduced the uptake of Cd by the cells, but also altered how the cells handled Cd intracellularly. In general, our results have demonstrated the need to use diets with natural compositions for dietary toxicity studies.

Cadmium accumulation and in vitro analysis of calcium and cadmium transport functions in the gastro-intestinal tract of trout following chronic dietary cadmium and calcium feeding.

Juvenile rainbow trout (Oncorhynchus mykiss) were fed diets made from Lumbriculus variegatus containing environmentally relevant concentrations of Cd (approximately 0.2 and 12 microg g(-1) dry wt) and/or Ca (1, 10, 20 and 60 mg g(-1) dry wt) for 4 weeks. Ten fish per treatment were removed weekly for tissue metal burden analysis. In all portions of the gastro-intestinal tract (GIT) (stomach, anterior, mid, and posterior intestine), chronic exposure to elevated dietary Ca decreased Cd tissue accumulation to varying degrees. At week five, the GITs of the remaining fish were subjected to an in vitro gut sac technique. Pre-exposure to the different treatments affected unidirectional uptake and binding rates of Cd and Ca in different manners, dependent on the specific GIT section. Ca and Cd uptake rates were highly correlated within all sections of the GIT, and the loosely binding rate of Cd to the GIT surfaces predicted the rate of new Cd absorption. Overall, this study indicates that elevated dietary Ca is protective against Cd uptake from an environmentally relevant diet, and that Ca and Cd uptake may occur through both common and separate pathways in the GIT.