Radiation-induced bystander effects in the Atlantic salmon (salmo salar L.) following mixed exposure to copper and aluminum combined with low-dose gamma radiation

Very little is known about the combined effects of low doses of heavy metals and radiation. However, such “multiple stressor” exposure is the reality in the environment. In the work reported in this paper, fish were exposed to cobalt 60 gamma irradiation with or without copper or aluminum in the water. Doses of radiation ranged from 4 to 75 mGy delivered over 48 or 6 h. Copper doses ranged from 10 to 80 μg/L for the same time period. The aluminum dose was 250 μg/L. Gills and skin were removed from the fish after exposure and explanted in tissue culture flasks for investigation of bystander effects of the exposures using a stress signal reporter assay, which has been demonstrated to be a sensitive indicator of homeostatic perturbations in cells. The results show complex synergistic interactions of radiation and copper. Gills on the whole produce more toxic bystander signals than skin, but the additivity scores show highly variable results which depend on dose and time of exposure. The impacts of low doses of copper and low doses of radiation are greater than additive, medium levels of copper alone have a similar level of effect of bystander signal toxicity to the low dose. The addition of radiation stress, however, produces clear protective effects in the reporters treated with skin-derived medium. Gill-derived medium from the same fish did not show protective effects. Radiation exposure in the presence of 80 μg/L led to highly variable results, which due to animal variation were not significantly different from the effect of copper alone. The results are stressor type, stressor concentration and time dependent. Clearly co-exposure to radiation and heavy metals does not always lead to simple additive effects.     

Exposure‐Based Validation List for Developmental Toxicity Screening Assays

Validation of alternative assays requires comparison of the responses to toxicants in the alternative assay with in vivo responses. Chemicals have been classified as “positive” or “negative” in vivo, despite the fact that developmental toxicity is conditional on magnitude of exposure. We developed a list of positive and negative developmental exposures, with exposure defined by toxicokinetic data, specifically maternal plasma C_max. We selected a series of 20 chemicals that caused developmental toxicity and for which there were appropriate toxicokinetic data. Where possible, we used the same chemical for both positive and negative exposures, the positive being the C_max at a dose level that produced significant teratogenicity or embryolethality, the negative being the C_max at a dose level not causing developmental toxicity. It was not possible to find toxicokinetic data at the no‐effect level for all positive compounds, and the negative exposure list contains C_max values for some compounds that do not have developmental toxicity up to the highest dose level tested. This exposure‐based reference list represents a fundamentally different approach to the evaluation of alternative tests and is proposed as a step toward application of alternative tests in quantitative risk assessment     

The in vitro transformation of a rat liver epithelial cell line with chromium

The transformation of a rat liver epithelial cell line under a wide range of doses of chromium was determined by anchorage-independent growth and tumor formation in syngeneic animals. Chronic exposure to low concentrations and brief exposure to high concentrations of hexavalent chromium (K₂CrO₄) transformed the cells, but one dose (1 mM K₂CrO₄, 2h) was clearly optimal in this regard. The cytotoxicity, effects on cell cycle, rates of chromium uptake, and mutagenic activity under the different treatment conditions were evaluated. The results showed that cells could adapt to the presence of chromium under certain treatment conditions, but this was not the case for the optimal transforming dose. Cells treated with chromium above the optimal transforming dose showed evidence of a transient G2 arrest, whereas all lower levels of treatment did not. A low level continuous exposure to chromate was mutagenic, whereas high level short exposures, including the optimal transforming dose, were not. An increase in the amount of protein complexed with isolated nucleic acids was detected in cells following treatment with the optimal transforming dose of chromate. The results indicate that the effects of chromium on this in vitro system vary with dose; and the identification of those events relevant to metal carcinogenesis will require consideration of treatment conditions.     

A Perspective on the Toxicity of Low Concentrations of Petroleum-Derived Polycyclic Aromatic Hydrocarbons to Early Life Stages of Herring and Salmon

This article presents a critical review of two groups of studies that reported adverse effects to salmon and herring eggs and fry from exposure to 1 μg/L or less of aqueous total polycyclic aromatic hydrocarbons (TPAH), as weathered oil, and a more toxic aqueous extract of “very weathered oil.” Exposure media were prepared by continuously flowing water up through vertical columns containing gravel oiled at different concentrations of Prudhoe Bay crude oil. Uncontrolled variables associated with the use of the oiled gravel columns included time- and treatment-dependent variations in the PAH concentration and composition in the exposure water, unexplored toxicity from other oil constituents/degradation products, potential toxicity from bacterial and fungal activity, oil droplets as a potential contaminant source, inherent differences between control and exposed embryo populations, and water flow rate differences. Based on a review of the evidence from published project reports, peer-reviewed publications, chemistry data in a public database, and unpublished reports and laboratory records, the reviewed studies did not establish consistent dose (concentration) response or causality and thus do not demonstrate that dissolved PAH alone from the weathered oil resulted in the claimed effects on fish embryos at low μg/L TPAH concentrations. Accordingly, these studies should not be relied on for management decision-making, when assessing the risk of very low–level PAH exposures to early life stages of fish.     

Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish

In ecotoxicology, in vitro assays with fish cells are currently applied for mechanistic studies, bioanalytical purposes and toxicity screening. This paper discusses the potential of cytotoxicity assays with fish cells to reduce, refine or replace acute lethality tests using fish. Basal cytotoxicity data obtained with fish cell lines or fish primary cell cultures show a reasonable to good correlation with lethality data from acute toxicity tests, with the exception of compounds that exert a specific mode of toxic action. Basal cytotoxicity data from fish cell lines also correlate well with cytotoxicity data from mammalian cell lines. However, both the piscine and mammalian in vitro assays are clearly less sensitive than the fish test. Therefore, in vivo LC50 values (concentrations of the test compounds that are lethal to 50% of the fish in the experiment within 96 hours) currently cannot be predicted from in vitro values. This in vitro-in vivo difference in sensitivity appears to be true for both fish cell lines and mammalian cell lines. Given the good in vitro-in vivo correlation in toxicity ranking, together with the clear-cut difference in sensitivity, the role of cytotoxicity assays in a tiered alternative testing strategy could be in priority setting in relation to toxic hazard and in the toxicity classification of chemicals and environmental samples.     

Radiotracer Studies on Waterborne Copper Uptake,Distribution, and Toxicity in Rainbow Trout and Yellow Perch: A Comparative Analysis

Rainbow trout (Oncorhynchus mykiss) are often used to estimate important biotic ligand model (BLM) parameters, such as metal-binding affinity (log K) and capacity (B_max). However, rainbow trout do not typically occupy metal-contaminated environments, whereas yellow perch (Perca flavescens) are ubiquitous throughout most of North America. This study demonstrates that dynamic processes that regulate Cu uptake at the gill differ between rainbow trout and yellow perch. Rainbow trout were more sensitive to acute aqueous Cu than yellow perch, and toxicity was exacerbated in soft water relative to similar exposures in hard water. Whole body Na loss rate could account for acute Cu toxicity in both species, as opposed to new Cu uptake rate that was not as predictive. Time course experiments using radiolabelled Cu (⁶⁴Cu) revealed that branchial Cu uptake was rather variable within the first 12 h of exposure, and appeared to be a function of Cu concentration, water hardness, and fish species. After 12 h, new branchial Cu concentrations stabilized in both species, suggesting that metal exposures used to estimate BLM parameters should be increased in duration from 3 h to 12+ h. In rainbow trout, 71% of the new Cu bound to the gill was exchangeable (i.e., able to either enter the fish or be released back to the water), as opposed to only 48% in yellow perch. This suggests that at equal exposure concentrations, proportionally more branchial Cu can be taken up by rainbow trout than yellow perch, which can then go on to confer toxicity. These qualitative differences in branchial Cu handling between the two species emphasize the need to develop BLM parameters for each species of interest, rather than the current practice of extrapolating BLM results derived from rainbow trout (or other laboratory-reared species) to other species. Data reported here indicate that a one-size-fits-all approach to predictive modeling, mostly based on rainbow trout studies, may not suffice for making predictions about metal toxicity to yellow perch—that is, a species that inhabits metal-contaminated lakes around northern Canadian industrial operations.     

Facilitation of electroporative drug uptake and cell killing by electrosensitization

Cell permeabilization by electric pulses (EP), or electroporation, is widely used for intracellular delivery of drugs and plasmids, as well as for tumour and tissue ablation. We found that cells pre‐treated with 100‐μs EP develop delayed hypersensitivity to subsequent EP applications. Sensitizing B16 and CHO cells by splitting a single train of eight 100‐μs EP into two trains of four EP each (with 5‐min. interval) decreased the LD₅₀ 1.5–2 times. Sensitization profoundly enhanced the electroporation‐assisted uptake of bleomycin, a cell‐impermeable cytotoxic agent accepted for killing tumours by electrochemotherapy. EP exposures that were not lethal per se caused cell death in the presence of bleomycin and proportionally to its concentration. Sensitizing cells by a split‐dose EP exposure increased bleomycin‐mediated lethality to the same extent as a 10‐fold increase in bleomycin concentration when using a single EP dose. Likewise, sensitization by a split‐dose EP exposure (without changing the overall dose, pulse number, or amplitude) enhanced the electroporative uptake of propidium up to fivefold. Enhancement of the electroporative uptake appears a key mechanism of electrosensitization and may benefit electrochemotherapy and numerous applications that employ EP for cell permeabilization.     

Phytoremediation of wastewater toxicity using water hyacinth (Eichhornia crassipes) and water lettuce (Pistia stratiotes)

This paper elucidates the phytoremediation potential of water hyacinth and water lettuce on the reduction of wastewater toxicity. Acute toxicity tests were performed in an aquarium with a population of Sarotherodon melanotheron, contaminated by different concentrations of wastewaters before and after phytoremediation with Eichhornia crassipes and Pistia stratiotes. Lethal concentrations (LC₅₀) of the fish's population obtained during 24 hours of exposures were determined. COD, BOD, ammonium, TKN and PO₄^3? concentrations in wastewaters were of 1850.29, 973.33, 38.34, 61.49 and 39.23 mg L^?1, respectively, for each plant. Phytoremediation reduced 58.87% of ammonium content, 50.04% of PO₄^3?, 82.45% of COD and 84.91% of BOD. After 15 days of the experiment, metal contents in treated wastewaters decreased from 6.65 to 97.56% for water hyacinth and 3.51 to 93.51% for water lettuce tanks. Toxicity tests showed that the mortality of fish exposed increased with increase in concentration of pollutants in wastewaters and the time of exposure. Therefore, the highest value of LC₅₀ was recorded for fish subjected to 3 hours of exposure (16.37%). The lowest rate was obtained after an exposure of 20 to 24 hours (5.85%). After phytoremediation, the effluents purified by Eichhornia crassipes can maintain the fish life beyond 24 hours of exposure.     

Enhanced zinc consumption prevents cadmium-induced alterations in lipid metabolism in male rats

It has been investigated, based on a rat model of human exposure to cadmium (Cd), whether zinc (Zn) supplementation may prevent Cd-induced alterations in lipid metabolism. For this purpose, the concentrations of free fatty acids (FFA), phospholipids (PL), triglycerides (TG), total cholesterol (TCh), and high and low density lipoprotein cholesterol (HDL and LDL, respectively) as well as the concentrations of chosen indices of lipid peroxidation such as lipid peroxides (LPO), F₂-isoprostane (F₂-IsoP) and oxidized LDL (oxLDL) were estimated in the serum of male Wistar rats administered Cd (5 or 50 mg/l) or/and Zn (30 or 60 mg/l) in drinking water for 6 months. The exposure to 5 and 50 mg Cd/l resulted in marked alterations in the lipid status reflected in increased concentrations of FFA, TCh, LDL, LPO, F₂-IsoP and oxLDL, and decreased concentrations of PL and HDL in the serum. The concentrations of LDL, LPO, F₂-IsoP and oxLDL were more markedly enhanced at the higher Cd dosage. The supplementation with Zn during the exposure to 5 and 50 mg Cd/l entirely prevented all the Cd-induced changes in the serum concentrations of the estimated lipid compounds and indices of lipid peroxidation, except for the F₂-IsoP for which Zn provided only partial protection. Based on the results it can be concluded that Zn supplementation during exposure to Cd may have a protective effect on lipid metabolism consisting in its ability to prevent hyperlipidemia, including especially hypercholesterolemia, and to protect from lipid peroxidation. The findings seem to suggest that enhanced dietary Zn intake during Cd exposure, via preventing alterations in the body status of lipids may, at least partly, protect against some effects of Cd toxicity, including oxidative damage to the cellular membranes and atherogenic action. The paper is the first report suggesting protective impact of Zn against proatherogenic Cd action on experimental model of chronic moderate and relatively high human exposure to this toxic metal.     

Accumulation,regulation and toxicity of copper,zinc, lead and mercury in Hyalella azteca

Zinc, lead and mercury accumulation in the amphipod Hyalella azteca increases with increasing exposure to metals. During 10 week chronic toxicity tests, metal accumulated at the highest non-toxic/lowest toxic concentration was 126/136 µg Zn g^–1, 7.1/16 µg Pb g^–1 and 56/90 µg Hg g^–1 dry weight. Concentrations of lead and mercyry in control animals were substantially lower (1.3 µg Pb g^–1 and 0.4 µg Hg g^–1), but concentrations of zinc in controls (74 µg g^–1) were about one half those of the lowest toxic concentration. Copper was completely regulated. Accumulated copper concentrations after 10 weeks exposure to all waterborne copper concentrations resulting in less than 100% mortality were not significantly different from controls (79 µg g^–1). Lead and mercury concentrations in wild H. azteca should be useful indicators of potential toxicity. Zinc accumulation may also be a useful indicator of zinc toxicity, but careful comparison with control or reference animals is necessary because of the small differences between toxic and control concentrations. Copper is not accumulated by H. azteca under chronic exposure conditions and body burdens of field animals cannot be used as an indicator of exposure or potential toxic effects. Short term exposures to copper, however, result in elevated copper concentrations in H. azteca, even at concentrations below those causing chronic toxicity. Short term bioaccumulation studies might, therefore, provide a useful indication of potential chronic copper toxicity.     

Amount and distribution of biochemical-genetic variation among wild populations and a hatchery stock of Atlantic salmon, Salmo salar L., from north-east Ireland

The effects of tributyltin (TBT) compounds on gill morphology were examined in the mummichog, Fundulus heteroclitus , in 96-h LC50 and 6-week sublethal exposures. Morphometry was used for the identification and quantification of effects with the light microscope. A 96-h LC50 of 17.2 μg 1 ¹ was determined. Morphometric analysis of gill tissues revealed hypertrophy of the lamellar epithelium in fish exposed to 17.2 μg 1–1. Relative diffusing capacity was significantly decreased (−41 %); ( P <0.05, ANOVA, Bonferroni t -test). At 35.6 μg 1⁻¹, TBT exposure resulted in a significant reduction (− 40%) in the volume of the lamellar blood channels. Both of these observations occurred in fish that showed signs of acute poisoning including loss of equilibrium. In fish exposed to sublethal concentrations of 0.105–2.000 μg TBT 1⁻¹ for 6 weeks, there were no pathological changes in the gill. There were no treatment-related changes in the surface morphology of the gills of fish from both experiments upon scanning electron microscopic examination. Although gill pathology was observed in acutely toxic exposures, it does not appear to be a major mechanism of TBT toxicity.     

Toxicity of γ-hexachlorocyclohexane (Lindane) to Gammarus pulex

SUMMARY. The acute toxicity of the pesticide γ-hexachlorocyclohexane (Lindane) to Gammarus pulex was determined by a standard procedure and by a modification of the conventional toxicity test protocol. Animals were exposed to a range of concentrations for various periods of time from 1 to 1000 min and transferred to clean water. Animals continued to die for up to 3 weeks after the initial exposure to the poison. Toxicity curves were obtained expressing the relationships between concentration of poison and median survival time (LT50) and between concentration of poison and median lethal exposure time, i.e., the duration of exposure required to cause the eventual death of half the animals. For a given concentration of pesticide, the duration of exposure which will cause 50% mortality is far less than is indicated by a conventional toxicity test in which animals are continuously exposed to the poison. It is suggested that the results of conventional acute lethal toxicity tests may have limited predictive value even when applied to field situations involving the discharge of lethal levels of pollutants to receiving waters.     

Fine particle‐induced birth defects: Impacts of size,payload, and beyond

Worldwide epidemiological studies have shown that exposures to particulate matters (PMs), such as PM_2.5 or PM₁₀, during pregnancy cause birth defects in the newborn. Although mechanistic understanding of such effects are not available, recent research using murine models highlights some key progress: (1) toxicity caused by PMs is a combined effects of particles and the adsorbed toxic pollutants, such as heavy metals, persistent organic pollutants, bacteria, and virus. Fine particles may hold on to pollutants and, therefore, reduce their toxicity or enhance the toxicity by carrying pollutants crossing the placental barrier; (2) smaller size, certain particle surface chemistry modifications, early developmental stage of placenta, and maternal diseases all aggravate PM‐induced birth defects; (3) molecular events involved in such toxicity are begin to emerge: induction of oxidative stress, DNA damage, and alteration of molecular signaling or epigenetic events are some possible causes. Despite this progress, a clear understanding of PM‐induced birth defects awaits further breakthroughs on many fronts, including epidemiological studies, animal models, nanotoxicity, and molecular mechanism investigations. Birth Defects Research (Part C) 108:196–206, 2016. © 2016 Wiley Periodicals, Inc.     

Toxic effects of juncusol,a marsh plant phenolic extract,on estuarine fish and shrimp

Juncusol, a phenolic compound, was isolated from the black needlerush, $\text{Juncus roemerianus}$. Different concentrations of this extract were tested for toxicity against selected species of estuarine fish and grass shrimp. Juncusol was ten times more toxic to the various species of fish than to grass shrimp. At 2.0 ppm concentration all fishes tested were killed within 36 hr. One hundred per cent mortality in the grass shrinmp occured at 20 ppm during 95 hr exposure. The LC₁₀, LC₅₀, and LC₉₀ concentration for juncusol were calculated for grass shrimp and for juvenile sailfin molly.     

Evaluation of Acute Toxicity Levels and Ethological Responses Under Tetrachlorocatechol Exposure in Common Carp,Cyprinus carpio (Linnaeus)

Tetrachlorocatechol (TCC) is one of the most toxic chlorinated catechol produced by the chlorobleaching of pulp and frequently found in the kraft pulp mill effluents. Toxicity of TCC to common carp, Cyprinus carpio and their ethological changes were evaluated in the present study. The 24, 48, 72 and 96 h LC₅₀ values of TCC to C. carpio were 4.73, 3.95, 3.20 and 2.43 mg/l respectively. The mortality rate of C. carpio showed significant relationship (p < 0.05) at different exposure times (24, 48, 72 and 96 h) in different treatments of TCC (2, 3, 4, 5 and 7 mg/l). On the other hand, a significant correlation (p < 0.01) was observed between the mortality rate of fish with all the concentrations of TCC. Mean opercular movement in the fish increased significantly with the increasing dose and time of exposure to TCC indicating acute respiratory distress. The jerky movement, somersaulting, circular movement and mucus secretion of fish increased gradually with the increasing concentrations and exposure times of TCC for adapting a compensatory mechanism to derive energy in order to avoid stress due to toxicity.     

Long term health effects of low dose exposure to nerve agent

Possible long-term toxic effcts of nerve agents have been investigated using sensitive toxicological screens and extensive toxicity studies in various animal models. Data on humans have been obtained from controlled studies and accidental exposures. Studies in the area of ‘low dose’ exposure to nerve agents are currently being performed.     

Effects of manufactured nanomaterials on fishes: a target organ and body systems physiology approach

Manufactured nanomaterials (NM) are already used in consumer products and exposure modelling predicts releases of ng to low μg l(-1) levels of NMs into surface waters. The exposure of aquatic ecosystems, and therefore fishes, to manufactured NMs is inevitable. This review uses a physiological approach to describe the known effects of NMs on the body systems of fishes and to identify the internal target organs, as well as outline aspects of colloid chemistry relevant to fish biology. The acute toxicity data, suggest that the lethal concentration for many NMs is in the mg l(-1) range, and a number of sublethal effects have been reported at concentrations from c. 100 μg to 1 mg l(-1). Exposure to NMs in the water column can cause respiratory toxicity involving altered ventilation, mucus secretion and gill pathology. This may not lead, however, to overt haematological disturbances in the short term. The internal target organs include the liver, spleen and haematopoietic system, kidney, gut and brain; with toxic effects involving oxidative stress, ionoregulatory disturbances and organ pathologies. Some pathology appears to be novel for NMs, such as vascular injury in the brain of rainbow trout Oncorhynchus mykiss with carbon nanotubes. A lack of analytical methods, however, has prevented the reporting of NM concentrations in fish tissues, and the precise uptake mechanisms across the gill or gut are yet to be elucidated. The few dietary exposure studies conducted show no effects on growth or food intake at 10-100 mg kg(-1) inclusions of NMs in the diet of O. mykiss, but there are biochemical disturbances. Early life stages are sensitive to NMs with reports of lethal toxicity and developmental defects. There are many data gaps, however, including how water quality alters physiological responses, effects on immunity and chronic exposure data at environmentally relevant concentrations. Overall, the data so far suggest that the manufactured NMs are not as toxic as some traditional chemicals (e.g. some dissolved metals) and the innovative, responsible, development of nanotechnology should continue, with potential benefits for aquaculture, fisheries and fish health diagnostics.     

The Need for Multiple Lines of Evidence for Predicting Site-Specific Ecological Effects

The goal of this paper is to illustrate the value and importance of the “weight of evidence” approach (use of multiple lines of evidence from field and laboratory data) to assess the occurrence or absence of ecological impairment in the aquatic environment. Single species toxicity tests, microcosms, and community metric approaches such as the Index of Biotic Integrity (IBI) are discussed. Single species toxicity tests or other single lines of evidence are valuable first tier assessments that should be used as screening tools to identify potentially toxic conditions in a effluent or the ambient environment but these tests should not be used as the final quantitative indicator of absolute ecological impairment that may result in regulatory action. Both false positive and false negative predictions of ecological effects can occur due to the inherent variability of measurement endpoints such as survival, growth and reproduction used in single species toxicity tests. A comparison of single species ambient toxicity test results with field data showed that false positives are common and likely related to experimental variability or toxicity to selected test species without measureable effects on the ecosystem. Results from microcosm studies have consistently demonstrated that chemical exposures exceeding the acute or chronic toxicity concentrations for highly sensitive species may cause little or no ecologically significant damage to an aquatic ecosystem. Sources of uncertainty identified when extrapolating from single species tests to ecological effects were: variability in individual response to pesticide exposure; variation among species in sensitivity to pesticides; effects of time varying and repeated exposures; and extrapolation from individual to population-level endpoints. Data sets from the Chesapeake Bay area (Maryland) were used to show the importance of using “multiple lines of evidence” when assessing biological impact due to conflicting results reported from ambient water column and sediment toxicity tests and biological indices (benthic and fish IBIs). Results from water column and sediment toxicity tests with multiple species in tidal areas showed that no single species was consistently the most sensitive. There was also a high degree of disagreement between benthic and fish IBI data for the various stations. The lack of agreement for these biological community indices is not surprising due to the differences in exposure among habitats occupied by these different taxonomic assemblages. Data from a fish IBI, benthic IBI and Maryland Physical Habitat Index (MPHI) were compared for approximately 1100 first through third-order Maryland non-tidal streams to show the complexity of data interpretation and the incidence of conflicting lines of evidence. A key finding from this non-tidal data set was the need for using more than one biological indicator to increase the discriminatory power of identifying impaired streams and reduce the possibility of “false negative results”. Based on historical data, temporal variability associated with an IBI in undisturbed areas was reported to be lower than the variability associated with single species toxicity tests.     

Interaction of T-2 toxin and murine lymphocytes and the demonstration of a threshold effect on macromolecular synthesis

Although T-2 toxin intoxications have been described as radiomimetic, we find that T-2 toxin does not preferentially affect multiplying cells. Among the targets of T-2 toxin toxicity, DNA, RNA and protein synthesis inhibition are analysed. All three types of macromolecular syntheses are affected by a threshold dose of T-2 toxin which corresponds to the interaction of approx. 1 X 10(5) T-2 toxin molecules with the same number of T-2 toxin receptors (Gyongyossy-Issa, M.I.C. and Kachatourians, G.G. (1984) Biochim. Biophys. Acta 803, 197-202). Since toxic effects occur faster at higher toxin concentrations than at lower levels, the time-toxic effect relationship may be defined by a constant. Based on these observations, we hypothesize that complete receptor-occupation is the critical first step in the course of T-2 toxin toxicity events.