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Journal: Aquatic toxicology (Amsterdam, Netherlands)


In ecotoxicology, it is continuously questioned whether (nano)particle exposure results in particle uptake and subsequent biodistribution or if particles adsorb to the epithelial layer only. To contribute to answering this question, we investigated different uptake routes in zebrafish embryos and how they affect particle uptake into organs and within whole organisms. This is addressed by exposing three different life stages of the zebrafish embryo in order to cover the following exposure routes: via chorion and dermal exposure; dermal exposure; oral and dermal exposure. How different nanoparticle sizes affect uptake routes was assessed by using polystyrene particles of 25, 50, 250 and 700nm. In our experimental study, we showed that particle uptake in biota is restricted to oral exposure, whereas the dermal route resulted in adsorption to the epidermis and gills only. Ingestion followed by biodistribution was observed for the tested particles of 25 and 50nm. The particles spread through the body and eventually accumulated in specific organs and tissues such as the eyes. Particles larger than 50nm were predominantly adsorbed onto the intestinal tract and outer epidermis of zebrafish embryos. Embryos exposed to particles via both epidermis and intestine showed highest uptake and eventually accumulated particles in the eye, whereas uptake of particles via the chorion and epidermis resulted in marginal uptake. Organ uptake and internal distribution should be monitored more closely to provide more in depth information of the toxicity of particles.

Concepts: Epithelium, Stomach, Tissues, Adsorption, Eye, Skin, Epidermis, Activated carbon


The mean value of any parameter and its changes are usually discussed, when ecotoxicological studies are carried out. However, also the variation of any parameter and its changes can be important components of the responses to environmental contamination. Although the homogeneity of variances is commonly tested, testing is done for the use of correct statistical methods, not because of exploring the possibility that variability and its changes could be important components of environmental responses. We evaluated recent aquatic toxicological literature and found that in the majority of articles indicating that homogeneity of variances was tested and giving the result of testing, the assumption of homogeneity was not fulfilled. Further, it was observed that in some studies experimental treatment clearly affected the variability. In this commentary we discuss the reasons for variability: measurement errors, experimental design, genetic heterogeneity and phenotypic plasticity, and conclude that even after accounting for experimental design and genetic makeup significant variability remains. This plasticity may change in environmental responses as suggested by a hypothetical example, and as confirmed by experimental data. As a consequence, the changes of variability can be significant, even when the means do not differ. Because of this, variability and its changes should always be analysed and reported. This will be easy, since the datasets are exactly the same for comparing the variances and means, and as normally variances are tested for homogeneity. It is likely that much new information about the responses of organisms to environmental contamination will be obtained. However, the present journal practises tend to discourage one from concentrating on anything but the mean. In contrast, we think it is imperative that variability is always included as an endpoint in data analysis in the future.


Dispersants are commonly used to mitigate the impact of oil spills, however, the ecological cost associated with their use is uncertain. The toxicity of weathered oil, dispersed weathered oil, and the hydrocarbon-based dispersant Slickgone NS(®), to the diatom Phaeodactylum tricornutum has been examined using standardized toxicity tests. The assumption that most toxicity occurs via narcosis was tested by measuring membrane damage in diatoms after exposure to one of the petroleum products. The mode of toxic action was determined using microarray-based gene expression profiling in diatoms after exposure to one of the petroleum products. The diatoms were found to be much more sensitive to dispersants than to the water accommodated fraction (WAF), and more sensitive to the chemically enhanced WAF (CEWAF) than to either the WAF itself or the dispersants. Exposure to dispersants and CEWAF caused membrane damage, while exposure to WAF did not. The gene expression profiles resulting from exposure to all three petroleum mixtures were highly similar, suggesting a similar mode of action for these compounds. The observed toxicity bore no relationship to PAH concentrations in the water column or to total petroleum hydrocarbon (TPH), suggesting that an undescribed component of the oil was causing toxicity. Taken together, these results suggest that the use of dispersants to clean up oil spills will dramatically increase the oil toxicity to diatoms, and may have implications for ecological processes such as the timing of blooms necessary for recruitment.

Concepts: Gene expression, Petroleum, Toxicity, DNA microarray, Diatom, Phaeodactylum tricornutum, Diatoms, Exxon Valdez oil spill


The major thiol-containing molecules involved in controlling the level of intracellular ROS in eukaryotes, acting as a nonenzymatic detoxification system, are metallothioneins (MTs), glutathione (GSH) and phytochelatins (PCs). Both MTs and GSH are well-known in the animal kingdom. PC was considered a prerogative of the plant kingdom but, in 2001, a phytochelatin synthase (PCS) gene was described in the nematode Caenorhabditis elegans; additional genes encoding this enzyme were later described in the earthworm Eisenia fetida and in the parasitic nematode Schistosoma mansoni but scanty data are available, up to now, for Deuterostomes. Here, we describe the molecular characteristics and transcription pattern, in the presence of Cd, of a PCS gene from the invertebrate chordate Ciona intestinalis, a ubiquitous solitary tunicate and demonstrate the presence of PCs in tissue extracts. We also studied mRNA localization by in situ hybridization. In addition, we analyzed the behavior of hemocytes and tunic cells consequent to Cd exposure as well as the transcription pattern of the Ciona orthologous for proliferating cell nuclear antigen (PCNA), usually considered a proliferation marker, and observed that cell proliferation occurs after 96h of Cd treatment. This matches the hypothesis of Cd-induced cell proliferation, as already suggested by previous data on the expression of a metallothionein gene in the same animal.

Concepts: DNA, Protein, Bacteria, RNA, Caenorhabditis elegans, Animal, Model organism, Ciona intestinalis


Cadmium-caused head and eye hypoplasia and hypopigmentation has been recognized for a long time, but knowledge of the underlying mechanisms is limited. In this study, we found that high mortality occurred in exposed embryos after 24hpf, when cadmium (Cd) dosage was above 17.8μM. Using high-throughput in situ hybridization screening, we found that genes labelling the neural crest and its derivative pigment cells exhibited obviously reduced expression in Cd-exposed embryos from 24hpf, 2 days earlier than head and eye hypoplasia and hypopigmentation occurred. Moreover, based on expression of crestin, a neural crest marker, we found that embryos before the gastrula stage were more sensitive to cadmium toxicity and that damage caused by Cd on embryogenesis was dosage dependent. In addition, by phenotype observation and detection of neural crest and pigment cell markers, we found that BIO and retinoic acid (RA) could neutralize the toxic effects of Cd on zebrafish embryogenesis. In this study, we first determined that Cd blocked the formation of the neural crest and inhibited specification of pigment cells, which might contribute to the molecular mechanisms underlying the phenotype defects of head and eye hypoplasia and hypopigmentation in Cd-exposed embryos. Moreover, we found that compounds BIO or RA could neutralize the toxic effects of Cd.

Concepts: DNA, Gene, Embryo, Organism, Toxicology, Cadmium, Toxicity, Germ layer


We analyzed the effect of exposure to 25% 96h-LC50 of copper at low (24.5μgL(-1) Cu, pH 4.5), neutral (7.25μgL(-1) Cu, pH 7.0) and high pH (4.0μgL(-1) Cu, pH 8.0) at 20°C on antioxidant defenses and oxidative stress in the liver, gills and white muscle of the fish Prochilodus lineatus. Water at pH 4.5 and 8.0 affected the enzymatic and non-enzymatic antioxidant systems of the liver and gills, but not of the white muscles of P. lineatus, when compared to water at pH 7.0. After Cu exposure, SOD (superoxide dismutase), GPx (glutathione peroxidase), GR (glutathione reductase) and GST (glutathione S-transferase) activities increased and CAT (catalase) activity decreased in the liver at water at pH 4.5 and 8.0. Meanwhile, the activities of SOD, CAT, GPx, GR and GST increased in the gills at these pHs. SOD and CAT activities increased in the white muscle after Cu exposure at pH 8.0 and GPx, GR and GST activities decreased after Cu exposure at pH 4.5 and 8.0. LPO levels decreased in the liver and gills of fish that were exposed to water at pH 4.5 and 8.0 and, after Cu exposure, the LPO level increased in the liver, gills and white muscle of fish that were exposed to water at pH 4.5 and 8.0, when compared to the control group at pH 7.0. The metallothionein (MT) concentration increased in the liver of fish in water at pH 4.5 and 8.0 and the gill of fish in water at pH 8.0. After Cu exposure, MT in the liver and gills was significantly elevated in fish exposed to water at pH 4.5 and 8.0, but remained at levels similar to the control group in the white muscle. These results indicate a differing sensitivity of fish organs and tissues to essential metals, such as copper, and that toxicity may be relevant at environmental concentrations. These results indicate that the effect of Cu on the response of antioxidant defense systems is determined by water pH.

Concepts: Antioxidant, Oxidative stress, Superoxide dismutase, Liver, Copper, Glutathione, Glutathione peroxidase, Antioxidants


Invertebrates typically carry out detoxification of accumulated metals. There is, therefore, no threshold total body concentration of accumulated metal initiating toxicity, the onset of toxic effects rather being related to a critical concentration of metabolically available (MA) accumulated metal. The challenge remains as to whether any particular combination of subcellular fractions of accumulated metal can be identified to represent this theoretical MA component. One candidate combined fraction is the so-termed metal sensitive fraction (MSF), consisting of metal bound to organelles and non-detoxificatory soluble proteins. In this study, we used laboratory zinc accumulation and toxicity data for four populations of the oyster Crassostrea hongkongensis with different histories of zinc exposure in the field to address the challenge. We conclude that in a ‘control’ population of the oyster, the MSF does approximate to the theoretical metabolically available zinc concentration. In populations with a history of field exposure to raised zinc bioavailabilities, however, the MSF would include more zinc detoxified in the lysosome component of organelle-bound metal, and the MSF in such populations would deviate more from the theoretical MA metal concentration.

Concepts: Organelle, Metal, Toxicology, Toxicity, Poison, Fraction, Eastern oyster, Rational function


The physiological and toxicological effects of Cd and Pb have been thoroughly studied, but relatively little work has been done to determine how mixtures of these metals affect fishes in soft (<100μmolL(-1)Ca(2+)) slightly acidic (pH ∼6) waters typical of many lakes in the Canadian Shield and other regions. Recently, it has been suggested that acute exposure to Cd plus Pb mixtures (3h) had greater than additive effects on both Ca(2+) and Na(+) influx, which could potentially exacerbate disturbances to ion balance and result in greater toxicity in rainbow trout (Oncorhynchus mykiss). The goal of the present study was to test this hypothesis by assessing the physiological and toxicological effects of Cd plus Pb mixtures over longer time periods (3-5 days), but at relatively low, more environmentally relevant concentrations of these metals. Accordingly, toxicity and measurements of blood acid-base regulation (PaO2, pHa), hematology (Ht, Hb, MCHC, and Protein), ionic composition (body ions and plasma Ca(2+), Na(+), Cl(-), osmolality), unidirectional Na(+) fluxes and branchial Na(+)/K(+)-ATPase activity were measured in rainbow trout exposed to Cd plus Pb mixtures. Experiments on rainbow trout, implanted with dorsal aortic catheters for repetitive blood sampling, demonstrated that exposure to Pb alone (26nmolPbL(-1)) was less toxic than Cd alone (6nmolCdL(-1)), which was much less toxic to the fish than a Cd plus Pb mixture (7nmolCdL(-1) plus 45nmolPbL(-1)), which led to greater than additive 80% mortality by 5d. Both Cd and Pb inhibited Na(+) influx over 3d exposure to the metals, which was partially offset by decreases in the diffusive efflux (outflux) of Na(+) across the gill. Despite an absence of detectable effects of Pb alone on plasma ion balance, Cd plus Pb mixtures exacerbated Cd-induced reductions in plasma Ca(2+) concentration, and resulted in pronounced reductions in plasma Na(+), Cl(-), and osmolality. No effects on Na(+)/K(+)-ATPase activity were noted following exposure to Cd, Pb or Pb plus Cd mixtures. We conclude that the greater than additive toxicity of Cd plus Pb mixtures observed in the present and previous studies is because these metals not only have common, but also independent binding sites and mechanisms of action, which could exacerbate the pathophysiological effects caused by each metal alone.

Concepts: Toxicology, Salmon, Oncorhynchus, Salmonidae, Toxicity, Poison, Rainbow trout, Cutthroat trout


Omics technologies have long since promised to address a number of long standing issues related to environmental regulation. Despite considerable resource investment, there are few examples where these tools have been adopted by the regulatory community, which is in part due to a focus of most studies on discovery rather than assay development. The current work describes the initial development of an omics based assay using 48h Pimephales promelas (FHM) larvae for identifying aquatic exposures to pyrethroid pesticides. Larval FHM were exposed to seven concentrations of each of four pyrethroids (permethrin, cypermethrin, esfenvalerate and bifenthrin) in order to establish dose response curves. Then, in three separate identical experiments, FHM were exposed to a single equitoxic concentration of each pyrethroid, corresponding to 33% of the calculated LC50. All exposures were separated by weeks and all materials were either cleaned or replaced between runs in an attempt to maintain independence among exposure experiments. Gene expression classifiers were developed using the random forest algorithm for each exposure and evaluated first by cross-validation using hold out organisms from the same exposure experiment and then against test sets of each pyrethroid from separate exposure experiments. Bifenthrin exposed organisms generated the highest quality classifier, demonstrating an empirical Area Under the Curve (eAUC) of 0.97 when tested against bifenthrin exposed organisms from other exposure experiments and 0.91 against organisms exposed to any of the pyrethroids. An eAUC of 1.0 represents perfect classification with no false positives or negatives. Additionally, the bifenthrin classifier was able to successfully classify organisms from all other pyrethroid exposures at multiple concentrations, suggesting a potential utility for detecting cumulative exposures. Considerable run-to-run variability was observed both in exposure concentrations and molecular responses of exposed fish across exposure experiments. The application of a calibration step in analysis successfully corrected this, resulting in a significantly improved classifier. Classifier evaluation suggested the importance of considering a number of aspects of experimental design when developing an expression based tool for general use in ecological monitoring and risk assessment, such as the inclusion of multiple experimental runs and high replicate numbers.

Concepts: Gene, Gene expression, Bacteria, Chemistry, Experiment, Insecticide, Pyrethroid, Pyrethroids


Nitrite (NO2(-)) is commonly present as contaminant in aquatic environment and toxic to aquatic organisms. In the present study, we investigated the effects of nitrite exposure on haematological parameters, oxidative stress and apoptosis in juvenile turbot (Scophthalmus maximus). Fish were exposed to various concentrations of nitrite (0, 0.02, 0.08, 0.4 and 0.8mM) for 96h. Fish blood and gills were collected to assay haematological parameters, oxidative stress and expression of genes after 0, 24, 48 and 96h of exposure. In blood, the data showed that the levels of methemoglobin (MetHb), triglyceride (TG), potassium (K(+)), cortisol, heat shock protein 70 (HSP70) and glucose significantly increased in treatments with higher concentrations of nitrite (0.4 and/or 0.8mM) after 48 and 96h, while the levels of haemoglobin (Hb) and sodium (Na(+)) significantly decreased in these treatments. In gills, nitrite (0.4 and/or 0.8mM) apparently reduced the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione (GSH), increased the formation of malondialdehyde (MDA), up-regulated the mRNA levels of c-jun amino-terminal kinase (JUK1), p53, caspase-3, caspase-7 and caspase-9 after 48 and 96h of exposure. The results suggested caspase-dependent and JUK signaling pathways played important roles in nitrite-induced apoptosis in fish. Further, this study provides new insights into how nitrite affects the physiological responses and apoptosis in a marine fish.

Concepts: Gene, Gene expression, Antioxidant, Oxidative stress, Superoxide dismutase, Hydrogen peroxide, Glutathione, Heat shock protein