Depuration of ingested 14C-labelled polystyrene nanospheres in the Atlantic scallop (Placopecten magellanicus).

The presence of nano-plastics in marine bivalves is well established and may represent a risk to human consumption. The main objective of our work was to study the detailed tissue distribution of 14C-radiolabelled polystyrene nanospheres (PSNP; 325 nm) following their ingestion by commercial-size Atlantic scallop (Placopecten magellanicus) using whole-body autoradiography to assess their translocation, bioaccumulation and depuration mechanisms over a short-term exposure (6h) and a long-term exposure (2 weeks). Results showed that the nanospheres (PSNP) did not accumulate in scallop tissues despite the fact they were ingested and transported all along the digestive system. Elimination of the PSNP was virtually completed within 48 h and no radiolabeling appeared in the edible adductor muscle. This is indicative of the presence of an active depuration mechanism of particles without nutritional value as plastic PSNP. Our preliminary work indicates a rapid translocation of PSNP in scallops minimizing a possible transfer to human consumers.

Radiolabeling of amide functionalized multi-walled carbon nanotubes for bioaccumulation study in fish bone using whole-body autoradiography.

Commercial and medicinal applications of functionalized carbon nanotubes (f-CNTs) such as amidated f-CNTs are expanding rapidly with a potential risk exposure to living organisms. The effects of amidated f-CNTs on aquatic species have received a limited attention. In this work, an easy wet method to prepare [14C]-label amide multi-walled carbon nanotubes (MWNTs) is reported. Labeled carbon nanotubes were prepared by successive reactions of carboxylation, chloroacylation, and final amidation using [14C]-labeled ethanolamine. The f-CNTs were characterized using elemental analysis, electron dispersive X-ray, transmission electron microscopy, thermogravimetric analysis, and Raman and FTIR spectroscopy. An uptake experiment was carried out with juvenile Arctic char (Salvelinus alpinus) using water dispersed amidated [14C]-f-CNTs to assess their biodistribution in fish tissues using whole body autoradiography. The radioactivity pattern observed in fish head suggests that f-CNTs were accumulated in head bone canals, possibly involving an interaction with mineral or organic phases of bones such as calcium and collagen. This f-CNTs distribution illustrates how important is to consider the surface charges of functionalized carbon nanotubes in ecotoxicological studies.

Tissue Distribution of Radiolabeled 110mAg Nanoparticles in Fish: Arctic Charr (Salvelinus alpinus).

This study presents the first whole-body tissue distributions of dissolved (AgI) and 20 nm silver nanoparticles (Ag0NPs20) in fish (Arctic charr, Salvelinus alpinus). The distributions are provided for fish exposed to three different treatments: (i) intravenous (IV), (ii) dietary, and (iii) waterborne. Quantitative whole-body autoradiography (QWBA) analyses obtained on high-resolution images reveal distinct silver distribution patterns according to the treatments. The IV exposures showed that AgNPs20 were mainly located in bile and kidney after 8 d, while AgI was distributed through the whole body and reached particular tissues such as bones, eyes, skin, liver, spleen, kidney, and intestine. The Ag0NPs20 distribution with the dietary exposures suggests that some dissolution occurred within fish organs. We propose that dissolved silver could later precipitate as chloride, sulfide, or selenide and be incorporated in bones during the growth. Consequently, it is yet difficult to state if Ag0NPs20 cross biological barriers. Finally, the waterborne exposures revealed that the gills can capture Ag0NPs20, but in small quantities. This suggests that the stability of Ag0NPs20 in water is critical for the uptake via the gills.

Uptake, Whole-Body Distribution, and Depuration of Nanoplastics by the Scallop Pecten maximus at Environmentally Realistic Concentrations.

Previous studies of uptake and effects of nanoplastics by marine organisms have been conducted at what may be unrealistically high concentrations. This is a consequence of the analytical challenges in tracking plastic particles in organisms at environmentally relevant concentrations and highlights the need for new approaches. Here, we present pulse exposures of 14C-radiolabeled nanopolystyrene to a commercially important mollusk, Pecten maximus, at what have been predicted to be environmentally relevant concentrations (<15 µg L-1). Uptake was rapid and was greater for 24 nm than for 250 nm particles. After 6 h, autoradiography showed accumulation of 250 nm nanoplastics in the intestine, while 24 nm particles were dispersed throughout the whole-body, possibly indicating some translocation across epithelial membranes. However, depuration was also relatively rapid for both sizes; 24 nm particles were no longer detectable after 14 days, although some 250 nm particles were still detectable after 48 days. Particle size thus apparently influenced the biokinetics and suggests a need for chronic exposure studies. Modeling extrapolations indicated that it could take 300 days of continued environmental exposure for uptake to reach equilibrium in scallop body tissues although the concentrations would still below 2.7 mg g-1. Comparison with previous work in which scallops were exposed to nonplastic (silver) nanomaterials of similar size (20 nm), suggests that nanoparticle composition may also influence the uptake tissue distributions somewhat.

Bioconcentration of the anionic surfactant linear alkylbenzene sulfonate (LAS) in the marine shrimp Palaemonetes varians: a radiotracer study.

Uptake and depuration kinetics of dissolved [(14)C]C12-6-linear alkylbenzene sulfonate (LAS) were determined in the shrimp Palaemonetes varians using environmentally relevant exposure concentration. The shrimp concentrated LAS from seawater with a mean BCF value of 120 L kg(-1) after a 7-day exposure. Uptake biokinetics were best described by a saturation model, with an estimated BCFss, of 159 ± 34 L kg(-1), reached after 11.5 days. Shrimp weight influenced significantly BCF value with smaller individuals presenting higher affinity to LAS. To the light of a whole body autoradiography, major accumulation of LAS occurred in the cephalothorax circulatory system (gills, heart, hepatopancreas) and ocular peduncle, but not in the flesh, limiting potential transfer to human consumers. LAS depuration rate constant value of the shrimp was 1.18 ± 0.08 d(-1) leading to less than 1% of remaining LAS in its tissues after 8 days of depuration.

Tissue distribution and kinetics of dissolved and nanoparticulate silver in Iceland scallop (Chlamys islandica).

The fast expansion of the global nanotechnology market entails a higher environmental and human exposure to nanomaterials. Silver nanoparticles (AgNP) are used for their antibacterial properties; however, their environmental fate is yet poorly understood. Iceland scallops (Chlamys islandica) were exposed for 12 h to three different silver forms, dissolved Ag(I) (Agdiss), small (S-NP, Ø = 10-20 nm) and large AgNP (L-NP, Ø = 70-80 nm), labeled with (110m)Ag, and bioaccumulation kinetics and tissue distribution using in vivo gamma counting and whole-body autoradiography were determined. All Ag forms were readily and rapidly accumulated. Elimination process was also fast and bi-exponential, with mean biological half-life ranging from 1.4 to 4.3 days and from 17 to 50 days for fast and slow compartments, respectively. Most of the radioactivity concentrated in the hepatopancreas. Agdiss and S-NP tissue distributions were similar indicating a rapid dissolution of the latter in the tissues, contrarily to L-NP which appeared to form long lasting aggregates in the digestive system. Estimated steady-state bioconcentration factors (BCF), ranging between 2700 and 3800 ml g(-1) for dissolved and particulate silver forms, showed that C. islandica can accumulate significant quantities of Ag in a short time followed by an efficient depuration process.

Distribution of effervescent inhalable nanoparticles after pulmonary delivery: an in vivo study.

BACKGROUND: Effervescent inhalable nanoparticles (NPs) have previously been shown to be a promising alternative to conventional lung cancer treatment in animals. This study investigates the biodistribution of effervescent inhalable NPs after a single dose administration via pulmonary route in lung cancer-bearing mice. METHODS & RESULTS: Whole-body autoradiography and confocal laser-scanning microscopy (CLSM) were used to investigate the distribution of inhalable NPs loaded in an effervescent microcarrier. Inhalable doxorubicin-loaded NPs were tagged with 14C for whole-body autoradiography, or with fluorescein isothiocyanate for CLSM imaging. After pulmonary delivery, NPs were widely disseminated in the lungs with a long retention time (24 h). The heart was radioactivity free at all time points of the study. CLSM images showed that inhalable NPs were taken up by cells and that doxorubicin was released to the cell nuclei. CONCLUSION: This is the first study to investigate the distribution of inhalable NPs in a lung cancer-bearing animal model. Inhalable NPs achieved deep lung deposition, were actively released from microcarrier particles, spread to different parts of the lung and released doxorubicin in vivo. These NP characteristics contribute to the efficacy of effervescent inhalable NPs as a lung cancer treatment.

Radioisotopes demonstrate the contrasting bioaccumulation capacities of heavy metals in embryonic stages of cephalopod species.

Cephalopods play a key role in many marine trophic food webs and also constitute alternative fishery resources in the context of the ongoing decline in finfish stocks. Most coastal cephalopod species of commercial importance migrate into shallow waters during the breeding season to lay their eggs, and are consequently subjected to coastal contamination. Eggs of common cuttlefish Sepia officinalis, European squid Loligo vulgaris, common octopus Octopus vulgaris and the sepiolid Rossia macrosoma were exposed during embryonic development to dissolved (110m)Ag, (109)Cd, (60)Co, (54)Mn and (65)Zn in order to determine their metal accumulation efficiencies and distribution among different egg compartments. Cuttlefish eggs, in which hard shells enclose the embryos, showed the lowest concentration factor (CF) values despite a longer duration of exposure. In contrast, octopus eggs, which are only protected by the chorionic membrane, accumulated the most metal. Uptake appears to be linked to the selective retention properties of the egg envelopes with respect to each element. The study also demonstrated that the octopus embryo accumulated (110m)Ag directly from the dissolved phase and also indirectly through assimilation of the contaminated yolk. These results raise questions regarding the potential contrasting vulnerability of early life stages of cephalopods to the metallic contamination of coastal waters.

Whole-body autoradiography: an efficient technique to study copper accumulation and body distribution in small organisms.

Copepods have been widely used to evaluate toxicity of metals present in marine environments. However, a technical difficulty is to understand the possible routes of metal uptake and to identify in which tissues or organs metals are being accumulated. Traditional techniques are hard to be employed once each organ has to be analyzed separately. Autoradiography is an alternative technique to circumvent this limitation, since metal distribution in tissues can be visualized and quantified, even in small organisms like copepods. In the present study, accumulation and distribution of (64)Cu in the copepod Calanus hyperboreus was studied using autoradiography. Copepods were exposed for 2 h to copper (2.3 mg L(-1); 1.08 MBq (64)Cu mg(-1) Cu) and then allowed to depurate for 2 h in clean seawater. Total (64)Cu was determined by gamma-spectrometry after a metal exposure and a depuration period. (64)Cu distribution was determined based on images generated by autoradiography. Metal accumulation was observed on all external surfaces of the copepods, being accumulated mostly on the ventral region, followed by dorsal, urossoma and internal regions. After depuration, radioactivity levels had a decrease in the sum of external body surface. Our results show that copper uptake by C. hyperboreus is fast and that a non-negligible proportion of the accumulated metal can reach internal tissues, which may lead to detrimental physiological effects. Moreover, whole-body autoradiography was demonstrated to be an efficient technique to study copper accumulation and body distribution in a very small organism such as the copepod C. hyperboreus.

Synthesis and characterization of [(110m)Ag]-nanoparticles with application to whole-body autoradiography of aquatic organisms.

Silver nanoparticles (AgNp) were synthesized using aqueous solution of silver nitrate ([(110m)Ag]NO3) with poly(allylamine) as a reducing agent and a stabilizer of the AgNp suspension. Nanoparticles were characterized by absorption spectroscopy, particle size analysis, atomic force microscopy (AFM), and transmission (TEM) electron microscopy. Different size nanoparticles (10-30 nm and 70-90 nm) were obtained by varying the polymer concentration and reaction time. The application of [(110m)Ag]AgNP to environmental studies, using nuclear techniques such as in vivo gamma counting and whole-body autoradiography, is demonstrated and discussed.

Bioaccumulation and biotransformation of pyrene and 1-hydroxypyrene by the marine whelk Buccinum undatum.

The fates of a phenolic contaminant and its hydrocarbon precursor have rarely been compared, especially in an invertebrate species. Two groups of Buccinum undatum were exposed to equimolar amounts of pyrene and 1-hydroxypyrene over 15 d through their diets. Tissue extracts from the muscle and visceral mass were analyzed by liquid chromatography with fluorescence and mass spectrometry detection. Nine biotransformation products were detected in animals from both exposures. These included 1-hydroxypyrene, pyrene-1-sulfate, pyrene-1-glucuronide, pyrene glucose sulfate, two isomers each of pyrenediol sulfate and pyrenediol disulfate, and one isomer of pyrenediol glucuronide sulfate. These compounds represent a more complex metabolic pathway for pyrene than is typically reported. Diconjugated metabolites were as important in animals exposed to pyrene as in those exposed to 1-hydroxypyrene. Biotransformation products represented >90% of the material detected in the animals and highlight the importance of analyzing metabolites when assessing exposure. A mean of only 2 to 3% of the body burden was present in muscle compared with the visceral mass of both groups. The analytical methods were sufficiently sensitive to detect biotransformation products both in laboratory control whelks and in those sampled offshore. The tissue distribution of [(14)C]pyrene was also studied by autoradiography. Radioactivity was present primarily in the digestive and excretory system of the whelks and not in the gonads or muscle tissue.

2,4-D butoxyethyl ester kinetics in embryos of Xenopus laevis: the role of the embryonic jelly coat in reducing chemical absorption.

The role of the jelly coat in providing a protective barrier to chemical absorption was studied using the embryos of the amphibian, Xenopus laevis. Embryos with or without a jelly coat were water exposed to the butoxyethyl ester of 2,4-dichlorophenoxyacetic acid (2,4-D BEE) and the rates of uptake, metabolism, distribution, and excretion were determined. The water uptake clearance rates were slower for embryos with a jelly coat (1.5-4.5 ml(water).g (embryo) (-1).h(-1) or 0.040-0.022 ml(water).h(-1) per embryo) in comparison to dejellied embryos (14-21 ml(water).g (embryo) (-1).h(-1) 0.0066-0.021 ml(water).h(-1) per embryo). This accounted for the much lower residues in embryos with a jelly coat than in dejellied embryos during 8 h of exposure. Despite quantitative differences in uptake, once 2,4-D BEE had entered the embryos, metabolism and distribution were similar between the two test groups. 2,4-D BEE was metabolized to 2,4-dichlorophenoxyacetic acid (2,4-D) with half-lives ranging from 35 to 42 minutes. The radioactive residues, as determined by whole body autoradiography, appeared throughout the embryo with a slight accumulation in the blastocoel. Furthermore, 35% of the radioactive residues were located in the jelly coat and 65% in the developing embryo. Based on a slower 2,4-D elimination in embryos with a jelly coat, the diffusive properties that decreased 2,4-D BEE uptake appeared to similarly decrease elimination of its metabolite. The common practice of removing jelly coats prior to embryonic amphibian toxicity studies, as in the widely used Frog Embryo Teratogenesis Assay-Xenopus (FETAX), is discouraged based on the kinetic differences observed in this study.

Toxicokinetics of 14C-atrazine and its metabolites in stage-66 Xenopus laevis.

Several recent studies have focused on the toxicodynamic implications of amphibian exposure to the commonly used herbicide atrazine (2-chloro-4-ethylamine-6-isopropylamino-s-triazine). These studies are an important part of the risk assessment process; however, the underlying mechanisms of atrazine toxicodynamics are lacking. In an attempt to more fully describe atrazine exposure, the toxicokinetics of atrazine were studied in stage-66 Xenopus laevis larvae. The absorption, distribution, and excretion capacity of these larvae were found to be comparable to those observed in fish. The calculated bioconcentration factor (BCF) was 1.5-1.6 mLwater/glarvae, and by use of whole-body autoradiography, the radiolabel was found to be concentrated in the gall bladder and gastrointestinal tract. Elimination of atrazine was rapid with a half-life of 48 min. The high metabolic capacity of stage-66 X. laevis larvae was demonstrated where, following 8 h of exposure to 14C-atrazine, the percentages of atrazine and its metabolites deethyldeisopropylatrazine (DACT), deisopropylatrazine (DIA), and deethylatrazine (DEA) in larvae, determined by thin-layer chromatography, were 49.8% +/- 3.3%, 9.8% +/- 2.1%, 16.1% +/- 2.5%, and 15.6% +/- 2.0%, respectively. An unknown metabolite(s) was also produced and accounted for the remaining proportion of the total body radioactive residues. This metabolite(s) is hypothesized to be a conjugate of either atrazine or one of its metabolites. These metabolites, namely, DIA, were responsible for the long elimination half-life (72 h) of the total body radioactive residues. These toxicokinetics data would provide better insights in the interpretation of toxicodynamic data.

Silver accumulation in Daphnia magna in the presence of reactive sulfide.

Previously, we demonstrated a higher silver body burden when Daphnia magna were exposed to silver in the presence of environmentally relevant concentrations (25 nM) of reactive sulfide, but the explanation was unclear. In the present study, D. magna were exposed to AgNO3 (0.93 microg Ag/L=8.6 nM as a mixture of cold Ag and (110m)Ag) in synthetic water in either the presence or absence of 25 nM sulfide as zinc sulfide clusters. After 1-h exposure, daphnids were transferred to clean water for up to 5-h depuration. At different times of Ag exposure and depuration, daphnids were randomly sampled for whole body silver burden. Also, after 1 h, daphnids were sampled for silver accumulation in "gills" (small organs on the thoracic appendages), digestive tract, and carcass. Other groups were exposed to the same silver and sulfide concentrations for 1 h and then sampled for whole-body autoradiography. Silver body burden was about two-fold higher in the presence of sulfide. A two-fold increase in silver burden in "gills" and digestive tract, but not in carcass, was also observed in the presence of sulfide. Absolute differences due to sulfide were greatest in digestive tract and explained most of the difference in whole body burden. Transfer to clean water caused a significant drop in silver concentration in whole body and all compartments to similar levels in the two groups after 5-h depuration. These results indicate that the higher silver body burden observed in the presence of sulfide is mainly due to sulfide-bound silver in the digestive tract of the daphnids. This conclusion is supported by autoradiography, which showed a high concentration of silver in the digestive tract of daphnids exposed to Ag/sulfide.

Transport and subcellular distribution of intranasally administered zinc in the olfactory system of rats and pikes.

Zinc is an essential element, which can act as a neuromodulator and also is bound in zinc proteins in the brain. The olfactory bulb contains high concentrations of zinc. In the present study, 65Zn(2+) was applied on the olfactory epithelium of rats and pikes and the transport of the metal in the olfactory system was then examined. Administration of 65Zn(2+) in the nasal cavity of rats or the olfactory chambers in pikes resulted in an uptake of the metal in the olfactory epithelium and a transport of the metal along the primary olfactory neurons to their terminations in the olfactory bulbs. Low levels of 65Zn(2+) passed these terminals and continued into the interior of the bulbs. In the rats 65Zn(2+) was also detected in the anterior parts of the olfactory cortex. Subcellular fractionations of the olfactory mucosa and olfactory bulb of rats given 65Zn(2+) intranasally showed that the metal is bound both to particulate cellular constituents and to cytosolic components in these tissues. Gel chromatography indicated that some of the zinc in the cytosol is bound to metallothionein in the olfactory mucosa and bulb. Inhalation of zinc-containing dusts or fumes occurs in some work-places and may imply high exposure of the nasal tissues. It is not known whether neurotoxicity may be related to uptake of zinc in the olfactory system. However, this is an issue which deserves attention, since zinc dysregulation has been implied to play a role in Alzheimer's disease. In addition, impairment of the sense of smell and degenerative changes of the olfactory tissues have been seen in early stages of some neurodegenerative disorders.

Uptake of waterborne tributyltin in the brain of fish: axonal transport as a proposed mechanism.

In previous studies, it was shown that waterborne Hg(II), Cd(II), and Mn(II) enter nerves innervating water-exposed sensory organs of fish and are transported to the brain by axonal transport. However, it is not known if organometals, such as tributyltin (TBT), can reach the brain of fish via the same route. In this work, we exposed rainbow trout (Oncorhynchus mykiss) to waterborne [113Sn]-TBT (4.2 kBq/L). Three fish were sampled after a 2-week exposure, and three others were sampled after a 2-week depuration period. Another group of four fish received an intravenous injection of [113Sn]-TBT and were sampled after 2 and 14 d. Distribution of the radiolabel was visualized and quantified by quantitative whole-body autoradiography. The brain accumulated a significant amount of 113Sn, with hot spots being found in parts receiving sensory nerves from water-exposed sensory organs, such as eminentia granulares (lateral lines organs). Labeling of the brain was also seen for i.v.-injected fish, indicating that the blood-brain barrier is not impervious to TBT or its metabolites. Nevertheless, the distribution of radioactivity in the brain was much more uniform, with no evident hot spot. Though the transfer [water --> gills --> blood stream --> blood-brain barrier --> brain] may account for a certain proportion of the radiolabel accumulation in fish brain, exposure to [113Sn]-TBT via water resulted in higher accumulation in some areas of the brain, of which the specific location strongly suggests that it was taken up in different water-exposed sensory nerve terminals and transported directly to the brain by axonal transport, as the parent compound or as a metabolite. The resulting local enhancement of the accumulation of butyltins might jeopardize the integrity of nervous system. Further work is needed to assess the toxicological significance of this process.

Cadmium affects the social behaviour of rainbow trout, Oncorhynchus mykiss.

The present study investigated both the effects of cadmium on the social interactions of rainbow trout and the differential accumulation of waterborne cadmium among social ranks of fish. Fish exposed to waterborne cadmium concentrations of 2 microg l(-1) for 24 h, followed by a 1, 2 or 3 day depuration period in clean water, had a decreased ability to compete with non-exposed fish. However, the competitive ability of exposed fish given a 5 day depuration period was not significantly impaired. Cadmium accumulated in the olfactory apparatus of fish exposed to waterborne cadmium for 24 h and decreased significantly only after 5 days depuration in clean water. Among groups of ten fish held in stream tanks, where all fish were exposed to cadmium, there were significant effects on social behaviour and growth rate. Dominance hierarchies formed faster among fish exposed to cadmium than among control fish, and overall growth rates were higher in the cadmium treatment. In groups of ten fish, social status also affected tissue accumulation of cadmium during waterborne exposure, with dominant fish accumulating more cadmium at the gill. In conclusion, exposure to low levels of cadmium, affects the social behaviour of fish, in part due to accumulation in the olfactory apparatus, and dominant fish accumulate more gill cadmium than subordinates during chronic waterborne exposure.

Cadmium disrupts behavioural and physiological responses to alarm substance in juvenile rainbow trout (Oncorhynchus mykiss).

Alarm substance is a chemical signal released from fish skin epithelial cells after a predator causes skin damage. When other prey fish detect alarm substance by olfaction, they perform stereotypical predator-avoidance behaviours to decrease predation risk. The objective of this study was to explore the effect of sublethal cadmium (Cd) exposure on the behavioural and physiological responses of juvenile rainbow trout (Oncorhynchus mykiss) to alarm substance. Waterborne exposure to 2 microg Cd l(-1) for 7 days eliminated normal antipredator behaviours exhibited in response to alarm substance, whereas exposures of shorter duration or lower concentration had no effect on normal behaviour. Furthermore, dietary exposure to 3 microg Cd g(-1) in the food for 7 days, which produced the same whole-body Cd accumulation as waterborne exposure to 2 microg l(-1), did not alter normal behaviour, indicating that an effect specific to waterborne exposure alone (i.e. Cd accumulation in the olfactory system) results in behavioural alteration. Whole-body phosphor screen autoradiography of fish exposed to (109)Cd demonstrated that Cd deposition in the olfactory system (rosette, nerve and bulb) during waterborne exposure was greater than in all other organs of accumulation except the gill. However, Cd could not be detected in the brain. A short-term elevation in plasma cortisol occurred in response to alarm substance under control conditions. Cd exposures of 2 microg l(-1) waterborne and 3 microg g(-1) dietary for 7 days both inhibited this plasma cortisol elevation but did not alter baseline cortisol levels. Our results suggest that exposure to waterborne Cd at environmentally realistic levels (2 microg l(-1)) can disrupt the normal behavioural and physiological responses of fish to alarm substance and can thereby alter predator-avoidance strategies, with potential impacts on aquatic fish communities.