Bioaccumulation of heavy metals in Liza saliens from the Esmoriz-Paramos coastal lagoon, Portugal

Heavy metal (Cu and Zn) concentrations in liver, gills, and muscle of leaping grey mullet, Liza saliens, from the Portuguese Esmoriz–Paramos coastal lagoon were measured to evaluate their bioaccumulation as a function of sediment contamination. The highest metal concentrations were observed in the liver (254 mg Cu kg⁻¹) and gills (114 mg Zn kg⁻¹). Bioaccumulation factors (BAFs) were found to follow the order: Cu-liver>Cu-gills>Cu-muscle and Zn-gills>Zn-liver>Zn-muscle. The highest BAFs were observed in the organs mainly implicated in metal metabolism and a significant positive relationship was found between BAFs and fish age. These results suggest the loss of homeostatic capacity of L. saliens under chronic metal exposure leading to bioaccumulation. Furthermore, Cu-liver and Zn-gills accumulation can be good environmental indicators of metal stress in L. saliens.     

Long-term exposure effects in vitellogenin, sex hormones, and biotransformation enzymes in female carp in relation to a sewage treatment works

In the present study, the estrogenic response in the reproductive cycle of female carp, Cyprinus carpio, in relation to a sewage treatment works (STWs) was measured as: alterations in plasmatic and hepatic vitellogenin (VTG) content, plasma levels of testosterone (T) and estradiol (E2), and effects on key enzymes of phases I and II biotransformation, ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST). Low plasma VTG content in females collected 4 km downstream of an effluent discharge suggested possible estrogenic disturbances; however, hepatic VTG content and sex hormone levels did not mirror this. In fact, E2 and T largely fluctuated among fish, with individual variations even greater than among sampling stations or periods. This suggests that at moderately polluted sites, biological variations of female carp and genetic particularities are prevalent. Hepatic biotransformation enzymes, such as EROD, increased with increasing water temperature while, in contrast, GST was maximal at the lowest water temperature.     

Effects of salinity on biomarker responses in Crassostrea rhizophorae (Mollusca, Bivalvia) exposed to diesel oil

Crassostrea rhizophorae is a euryhaline oyster that inhabits mangrove areas, which are widely distributed along the Brazilian coast. The aim of this study was to investigate the effects of salinity (9, 15, 25, and 35ppt) on the activities of glutathione S-transferase (GST), glucose 6-phosphate dehydrogenase (G6PDH), catalase (CAT), and acetylcholinesterase (AChE) in the digestive gland of this species after exposure to diesel oil for 7 days at nominal concentrations of 0.01, 0.1, and 1mlL(-1) and after depuration for 24h and 7 days. GST activity increased in a diesel oil concentration-dependent manner at salinities 25 and 15ppt and remained slightly elevated even after depuration periods of 24h and 7 days. No changes were observed in the activities of G6PDH, CAT, and AChE in the oysters exposed to diesel and depurated. Based on these results, GST activity in the digestive gland of C. rhizophorae might be used as a biomarker of exposure to diesel oil in sites where the salinity is between 15 and 25ppt, values usually observed in mangrove ecosystems.     

Toxicokinetics and biotransformation of p-nitrophenol in white sturgeon (Acipenser transmontanus)

White sturgeon (Acipencer transmontanus) were exposed to 7.2 microM (1.0 ppm) 14C-labeled p-nitrophenol (PNP) in brackish water for 24 h and then allowed to depurate in clean brackish water for another 24h. Absorption, conditional uptake clearance, and conditional elimination rate constants were 0.08+/-0.04 h(-1), 8.1+/-3.6 mL g(-1) h(-1), and 0.46+/-0.21 h(-1), respectively. A whole-organism total concentration factor of 18.7+/-2.6 was determined from equilibrium tissue and water concentrations. Sturgeon depurated 89.4% of absorbed PNP within 24h, of which 53.0+/-8.3% was unmetabolized parent compound, 9.6+/-3.6% was p-nitrophenyl-beta-d-glucuronide, and 39.1+/-8.3% was p-nitrophenylsulfate.     

Health status of Mya arenaria bivalves collected from contaminated sites in Canada (Saguenay Fjord) and Denmark (Odense Fjord) during their reproductive period

The purpose of this study was to examine the health status and gametogenetic activity in Mya arenaria clams collected at various sites in the St. Lawrence Estuary (Quebec, Canada) and in the Odense Fjord (Denmark). Clam soft tissues were analyzed for metals/metalloids and organotin compounds to confirm their exposure to these contaminants. Their health status was assessed by a test battery of biomarkers designed to measure the early biological effects of contaminants, which include expression of defence mechanisms such as xenobiotic conjugation (glutathione S-transferase), expression of stress proteins (i.e., heme oxygenase and metallothioneins), changes in gametogenetic activity, and individual morphometric characteristics. Clam tissues were also examined for the presence of oxidative damage to lipids, formation of DNA strand breaks, and alterations in heme metabolism. The results showed that clams sampled from sites with either ferry activity or intensive boat traffic in marinas were contaminated by metals/metalloids such as Ag, Al, As, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Sn, V, and Zn. The clams also contained relatively high amounts of tributyltin (TBT) in their tissues (in the ng TBT/g range for both areas), with digestive glands containing more organotins than did gonadal tissues. Moreover, clams collected from TBT-contaminated sites had higher amounts of tin-heme adducts and lower total heme in their digestive glands. Condition factor, age distribution, and sex ratio were significantly altered in clams from impacted sites in the Saguenay Fjord and accompanied by an increased male/female sex ratio. Gametogenetic activity was also negatively affected, as revealed by reductions in gonadosomatic index, maturation index, aspartate transcarbamylase activity, and vitellogenin-like proteins. The Saguenay Fjord clams displayed a complex pattern of stress responses and damage such as increased heme oxygenase activity, phase 2 conjugation enzyme activity, lipid peroxidation, and altered DNA strand breaks. The integration of biomarker response data into a biomarker index at the whole-individual level (morphometric characteristics) and for various organs (gill, digestive gland, and gonad) revealed that, relative to the control site, morphological characteristics and gonadal activity were most affected at the most contaminated site, while the effects were more pronounced in the digestive gland and gill at moderately impacted sites. We conclude that the health status of M. arenaria clams at these contaminated sites is compromised, with obvious disruption of reproductive activity.     

Effects of removing symbiotic green algae on the response of Hydra viridissima (Pallas 1776) to metals

Hydra viridissima is distinctively green due to symbiotic algae within the endodermal cells. The current investigation was designed to see if these algae influenced the response of Hydra to pollutants, by comparing the toxicity of copper, cadmium, and zinc to both symbiotic and aposymbiotic (free of their endosymbiotic algae) H. viridissima. The results demonstrated that the toxicity of the metals was generally similar for both groups of Hydra. However, at the lowest copper concentrations there was a difference between the two group of polyps, with aposymbiotic animals dying at concentrations where symbiotic Hydra survived. The lowest observed effect concentrations were 0.0068 and 0.016 mg/L for aposymbiotic and symbiotic Hydra, respectively. It is suggested that the symbiotic Hydra derive benefits from the association that enable them to better tolerate the toxicant. This work demonstrated that experimental manipulation of symbionts can help to explain their complex interactions and the ways in which they respond to pollutants.     

Effects of dietary methylmercury on liver and kidney histology in the neotropical fish Hoplias malabaricus

Methylmercury is a potent toxic present in Amazonian fish species due to gold mining activities. In the present work, we investigated the morphological effects of methylmercury in liver and kidney of Hoplias malabaricus feeding contaminated prey fish over 70 days. Two groups of nine mature fish (tested and control) were acclimatized for four weeks to laboratory conditions and then the tested group fed prey fish previously contaminated at an additional level of 0.075 microg MeHg g(-1) at 5-day intervals and over 14 successive intervals whereas control group fed uncontaminated fish. H. malabaricus specimens were then dissected for chemical and morphological analyses. The low and realistic level of MeHg in the prey fish induced a low increase of total mercury in liver (1.8-fold) and muscle (2.2-fold). The biomagnification factor (Hg in predator/Hg in prey) reached 142 in liver and 21 in muscle and was indicative of a relatively fast contamination of internal organs by dietary exposure. The liver of exposed individuals presented leukocyte infiltration, increased number of melano-macrophage centers, necrotic areas and lesions in Disse's space. Evident disorder and chaos in cytoskeleton organization suggest a strong toxic effect in hepatocytes, such as organelles positioning and movement, vesicles traffic and secretion. Head kidney showed large necrosis areas, increased number of melano-macrophages centers, phagocytic areas, intercellular space among parenquimal cells and atypical cells. Injuries and damages to tissues suggest too slow defense mechanisms to immobilize or eliminate ingested methylmercury, demonstrating that the sensitivity of fish cells to methylmercury exposure is higher than it has been previously described in the literature.     

Effects of heavy metals on production of thiol compounds and antioxidant enzymes in Agaricus bisporus

In a pre-experiment, Agaricus bisporus mycelia grown in PDL medium were found to have a substantial ability to tolerate and accumulate heavy metals. In the study, we investigated changes in the contents of soluble protein and thiol compounds as well as the activities of antioxidant enzymes caused by copper, zinc, lead, and cadmium (nitrate salts) in mycelia of A. bisporus during short-and long-term exposure. Results showed that high-level metal concentrations significantly decrease the contents of soluble protein after long-term exposure, Cu and Zn concentrations significantly increase the thiol compounds levels after long-term exposure, while high-level Cd significantly decrease thiol compounds after long-term exposure. Additionally, SOD activities were significantly increased after long-term exposure to metals, especially to Cd. The CAT activities were enhanced after long-term exposure to low-level Cu and high-level Zn, and enhanced after short-and long-term exposure to high-level Pb. The POD activities were significantly increased after long-term exposure to metals, and increased after short-term exposure to Cd and high-level Pb.     

Effects of Sublethal Zn<Superscript>++</Superscript> and Cd<Superscript>++</Superscript> Concentrations on Filtration Rate,Absorption Efficiency and Scope for Growth in <Emphasis Type="Italic">Donax trunculus</Emphasis> (Bivalvia; Donacidae)

Physiological parameters (filtration rate measured as clearance rate; absorption efficiency measured as Conover index) and energy balance [scope for growth (SFG)] where determined under Zn++ (essential metal) and Cd++ (nonessential metal) at different concentration levels and timing. The toxic effect observed on Donax trunculus was higher for Cd++ than for Zn++ in almost all the parameters studied. The SFG is proposed as an integrated environmental biomarker to be used like a tool with others from different organization levels to obtain a more comprehensive vision of the ecological consequences of pollutants.     

Use of two biomarkers (CYP450 and acetylcholinesterase) in zebra mussel for the biomonitoring of Lake Maggiore (northern Italy)

The use of zebra mussel Dreissena polymorpha as a bioaccumulator for lipophilic compounds is nowadays standardized, but its employment in early warning systems by the biomarker approach is much less frequent. One of the main problems with the biomarker approach is due to natural variation of abiotic factors such as temperature that influence the activity of several enzymes. In this study, we investigated the influence of this environmental parameter on the activities of two different biomarkers: acetylcholinesterase (AChE) (inhibited by organophosphorus compounds) and CYP450 (inversely influenced by planar compounds and heavy metals). We used these two biomarkers to evaluate the environmental pollution of Lake Maggiore (northern Italy). Results showed a strong AChE inhibition in mussel specimens collected in some sampling sites of the lake, indicating heavy pollution by neurotoxic compounds. We also found a twofold effect on CYP450 activity, probably due to the activating effect of planar compounds and the inhibiting effect of trace metals.     

Toxicity and removal of heavy metals (cadmium, copper, and zinc) by Lemna gibba

Effects of cadmium, copper, and zinc on the aquatic plant Lemna gibba were determined under controlled conditions; in parallel their removal from the growth medium was followed. The results showed that the three heavy metals affected growth, a physiological stress index defined as the ratio of Chlorophyll to phaeophytin (D665/D665a), and the contents of proline. After 4 days, 10⁻³–10⁻¹ mg/L Cd inhibited growth by 25–100%, reduced D665/D665a by 35–89%, and increased proline content by 44–567%. Under the same conditions, 10⁻⁴–10⁻¹ μg/L Cu inhibited growth by 36–75%, reduced D665/D665a by 19–81%, and increased proline content by 67–288%. Comparable concentrations of Zn had little effect. However, higher concentrations (4, 30, and 50 mg/L) inhibited growth by 50–79%. Also, 0.1 and 30 mg/L induced a small reduction of D665/D665a (−3.8% and −22%) and an increase in proline contents (+144% and +177%). When it was observed, proline accumulation was always transient and the maximum was reached after 4 days. Monitoring metal concentration in the medium showed that L. gibba was able to remove metals from the medium. Zn and Cu removal was biphasic, it was rapid during the first 2 days (> 60% reduction) and slow (10–20%) during the following 8 days. For Cd, removal was linear and depended on the initial concentration. It reached approximately 90% after 6 or 8 days for initial concentrations of 10⁻¹ and 10⁻³ mg/L, respectively.     

Effects of carbofuran on the sea bass (Dicentrarchus labrax L.): study of biomarkers and behaviour alterations

The objective of this study was to investigate the acute effects of the pesticide carbofuran on the sea bass (Dicentrarchus labrax) using parameters at different levels of biological organisation (swimming behaviour and several biomarkers) and possible relationships between alterations found in different effect criteria. In a bioassay, sea bass juveniles were individually exposed to different doses of carbofuran (31, 63, 125 and 250 μg/L) for 96 h. At the end of the bioassay, the swimming performance and 11 biomarkers were determined. Biomarkers were: hepatosomatic index (HSI), lipid peroxidation (LPO), reduced glutathione and the activities of the enzymes ethoxyresorufin O-deethylase (EROD), glutathione S-transferases, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, brain acetylcholinesterase (AChE) and muscle cholinesterases (ChE). After 96 h of exposure, carbofuran induced a decrease of the swimming velocity and inhibition of EROD activity at all concentrations tested, and inhibition of muscle ChE and brain AChE activities at 250 μg/L. No relevant alterations in any of the other tested parameters were found. These results show that carbofuran induced adverse effects on fish by interfering with neurofunction, capability of detoxication and swimming velocity. In addition, positive and significant correlations between the swimming velocity and (i) brain AChE activity, (ii) muscle ChE activity and (iii) EROD activity suggest that the inhibition of these enzymes may somehow be related to the behavioural changes observed. Since these functions are determinant for the survival and performance of the fish in the wild, the findings of the present study suggest that adverse effects may occur in populations exposed to carbofuran if a sufficient number of animals is affected.     

Chronic accumulation of cadmium and its effects on antioxidant enzymes and malondialdehyde in Oxya chinensis (Orthoptera: Acridoidea)

The accumulation of cadmium (Cd) and its effects on antioxidant enzyme activities and malondialdehyde (MDA) levels of Chinese rice grasshopper (Oxya chinensis) were evaluated under the laboratory conditions. Our results showed that Cd accumulation in O. chinensis exhibited a concentration-dependent increase in both males and females under Cd pollution. Environmental Cd can lead to the absorption of large quantities of Cd, which induces oxidative damage in insects by altering antioxidant defense enzyme systems. Our results demonstrated that Cd stress caused a significant decrease in glutathione peroxidase (GPx) levels and a significant increase in superoxide (SOD) dismutase and catalase (CAT) activities. In the grasshoppers, the MDA content was also enhanced, with an increase in Cd concentrations and a positive correlation between them; for females from second instar nymphs to the adult stage, R² was 0.6467, 0.9136, 0.6516, 0.942 and 0.7182, whereas for males, it was 0.6467, 0.8239, 0.9302, 0.7861, 0.8632, respectively. We also observed differences in the effects of Cd between grasshoppers of different developmental stages and genders, which suggested that the insect’s developmental stage and sex should be considered when studying enzyme activity.     

CYP1A2-mediated biotransformation of cardioactive 2-thienylidene-3,4-methylenedioxybenzoylhydrazine (LASSBio-294) by rat liver microsomes and human recombinant CYP enzymes

We describe herein the metabolic fate of cardioactive 1,3-benzodioxolyl N-acylhydrazone prototype LASSBio-294 (4) and the structural identification of its major phase I metabolite from rat liver microsomal assays. Our results confirmed the hard-metabolic character of N-acylhydrazone (NAH) framework of LASSBio-294 (4). The development of a reproducible analytical methodology for the major metabolite by using HPLC-MS and the comparison with an authentic synthetic sample, allowed us to identify 2-thienylidene 3,4-dihydroxybenzoylhydrazine derivative (7), formed by oxidative scission of methylenedioxy bridge of LASSBio-294, as the main metabolite formed by action of CYP1A2 isoform. The identification of this isoform in the LASSBio-294 in the clearance of LASSBio-294 (4) oxidation was performed by the use of selective CYP inhibitors or human recombinant CYP enzymes.     

Metal effects on fathead minnows (Pimephales promelas) under field and laboratory conditions

The consequences of low-level metal exposure to early life stages of fathead minnows (Pimephales promelas) were investigated along a contamination gradient near Sudbury, Canada. Field exposures resulted in elevated hatching time and increased mortality in metal-contaminated lakes, in contrast to laboratory exposures where no effects were observed. Dissolved and ionic Cd and Ni were associated with changes in hatching time and larval mortality under field conditions, though other potential contaminants were not examined and may also have had an influence. The increased biological response of field-exposed fish, relative to fish exposed to the same water in laboratory conditions, may be the result of higher stress in natural environments, which could sensitize fish to contaminants. Analysis also indicated that, as contamination increases, the discrepancies between laboratory and field estimates of effect also increase. A temperature versus hatching time relationship was also quantified for fathead minnows.     

Effects of anionic surfactant and salinity on the bioenergetics of juveniles of Centropomus parallelus (Poey)

The acute and sublethal chronic effects of sodium dodecyl sulphate (SDS) on the survival, metabolism, and growth of juveniles of Centropomus parallelus were investigated at three different salinities. Results of 96 h LC50 test showed that juveniles of C. parallelus were very sensitive to SDS in comparison to other species investigated. For each group of exposure to nominal concentrations of SDS (0.10 and 0.25 mg/L) and the control group (0.0 mg/L), at the different salinities (5, 20, and 30) there were significant differences in the specific growth rate, oxygen consumption, and ammonia excretion rates, O:N atomic ratio at the different exposure periods (15 and 30 days). There were also interactions between factors for the parameters investigated. The present results show a pronounced effect of SDS, mainly at the highest concentration and salinity, as well as after a long time of exposure.     

Effects of ghrelin on the feeding behavior of Lymantria dispar L. (Lymantriidae) caterpillars

Ghrelin is a 28-amino acid peptide which has significant effects on animal appetite, thus influencing body mass. The aim of our study was to examine the effects of ghrelin on the feeding behavior and physiology 4th instar caterpillars of the pest insect, Lymantria dispar L. Treatment of 4th instar caterpillars with four subpicomolar amounts of ghrelin had a positive influence on daily food intake, frass elimination, body mass. Also, locomotor activity increased, while stadium duration decreased in treated caterpillars. The similarity between the effects of ghrelin on caterpillar physiology and those in mammals suggests that using this model system for further studies of neuroendocrinological processes underlying feeding could lead to essential information about more complex organisms.     

Effects of low levels of lead on growth and reproduction of the Asian earthworm Perionyx excavatus (Oligochaeta)

The effects of sublethal concentrations of lead nitrate on growth and reproduction of the Asian composting earthworm species Perionyx excavatus was studied experimentally by exposing worms in an organic substrate to lead nitrate-contaminated food over a period of 76 days. The results revealed that growth was affected negatively by the presence of lead while maturation rate and cocoon production was not affected. In agreement with other studies on Eisenia fetida, cocoon viability was affected negatively by lead, making this a sensitive toxicity endpoint. Lead accumulation was of the same order of magnitude as for other previously studied species.     

Effects of zinc exposure on the reproduction of Spodoptera litura Fabricius (Lepidoptera: Noctuidae)

Reproductive toxicity of Zn to insects was investigated in this study. By exposing phytophagous insect Spodoptera litura Fabricius to Zn in artificial diets of larvae, we investigated the effects of Zn on reproduction at ecological and molecular levels. A significantly shorter period of laying eggs was observed in S. litura exposed to 300–750 mg Zn/kg. The oviposition rate, fecundity and hatchability of female adults treated with 750 mg Zn/kg were significantly lower than those of the controls (31.43%, 20.95% and 52%, respectively, compared to the control). The Zn accumulation and vitellin (Vn) content in eggs were tested by inductively coupled plasma-atomic emission spectrometry and Bradford combining Western-blot, respectively. The results showed that Zn accumulated in the eggs, which has affected the weight and Vn content of eggs with significant negative correlations. The down-regulated expression levels of vitellogenin (Vg) mRNA were detected by real-time polymerase chain reaction (RT-PCR): the relative quantity of Vg mRNA was less than half of the controls at higher than 450 mg Zn/kg wet weight. These results indicated that excess Zn made expression of Vg gene down-regulated and caused poor accumulation of egg yolk, which led to a reduction in egg numbers and failure of eggs to hatch.     

Linking lysosomal biomarker and population responses in a field population of Aporrectodea caliginosa (Oligochaeta) exposed to the fungicide copper oxychloride

The effects of the fungicide copper oxychloride on a field population of the earthworm Aporrectodea caliginosa were investigated. Changes were monitored in earthworm numbers and biomass, copper concentrations in soil and earthworms, and neutral red retention times (NRRTs) of coelomocytes. The aim was to establish whether the cellular responses could be linked to changes at the population level. Results indicated that there was a significant (P<0.05) impact on worm density and biomass on the treated plots 6 months after spraying with the fungicide stopped. NRRTs of treated earthworms were significantly lower after one spraying when earthworms had elevated concentrations of copper in their body tissues. Six months after spraying stopped, copper levels in both soil and earthworm tissues declined significantly, but the earthworm numbers and biomass were still significantly lower in the treated plots. This indicated that the effects of the copper oxychloride exposure manifested at a much later stage at the population level, than on NRRT. We conclude that the decrease in NRRTs, which preceded the decrease in both earthworm biomass and numbers, may have predictive value as a biomarker of impending population changes.