Genotype-specific responses of fluctuating asymmetry and of preadult survival to the effects of lead and temperature stress in Drosophila melanogaster

Although fluctuating asymmetry (FA) increases with exposure to certain types of environmental stressors such as temperature extremes, relatively little is known about the effects of interaction (e.g., synergism) between known sources of environmental stress on FA. Knowledge of such interaction effects, and of the magnitude of genotype-by-environment interaction, are of fundamental importance toward predicting the usefulness of FA as a bioindicator of environmental pollution. We tested for synergistic effects on FA between elevated temperature and exposure to lead, and examined FA responses simultaneously in four genetic strains of Drosophila melanogaster known to differ in their degree of developmental instability, and presumably in their buffering capacity. In the absence of heavy metal, bristle FA increased with temperature, but in the presence of lead, FA at high temperature (30 (degrees)C) was reduced to levels similar, or below, that at lower temperature (25 (degrees)C). This temperature by lead interaction was statistically significant, but paradoxical in that the disruptive effects of temperature appeared to be attenuated in the presence of the heavy metal. In no case was there a significant effect of lead on bristle FAs, despite documented assimilation of heavy metal by flies, and in no case was the genotype by environment interaction significant. Whereas lead treatment did not influence survival, survival was reduced at the high temperature, but significantly so only in one genetic strain (Oregon-R). There was no relationship between survival and FA across stress treatments within lines. Thus, any disproportionate stress-induced mortality in developmentally unstable classes (developmental selection) was unlikely to bias the FA results. Our results underscore the need for independent replication of significant findings before FA-based biomonitoring can be responsibly and effectively implemented. The results call for caution in using FA as a biomarker of stress, because stress factors may interact in complex and unpredictable ways, which could result in erroneous conclusions about real levels of stress present in field populations, under the unduly simplistic assumption that stress factors will act additively to increase FA.     

Fluctuating asymmetry in Bobwhite quail chicks (Colinus virginianus) does not follow a predictable dose-response relationship following maternal exposure to four different herbicides

Most biomonitoring studies that have investigated the relationship between fluctuating asymmetry (FA) and anthropogenic stressors have measured organisms from polluted ecosystems and compared them to organisms at reference sites. What has received little attention is whether FA follows a dose-response relationship with stress, a key criterion of a useful biomarker. Using chicks from currently mandated avian reproductive tests we tested whether a composite index of FA (FAC), was related to the dose or duration of exposure of their parents to one of four different herbicides, and if FAC was indeed a more sensitive marker of stress than standard reproductive endpoints measured from this test. We found no consistent relationship between FAC and dose or duration of herbicide exposure in any of the four studies. Exposure to one of the four pesticides did result in significant reproductive toxicity but this was not accompanied or foreshadowed by higher levels of FAC. Our results do not support the hypothesis that FA is a reliable general biomarker of pesticide exposure.     

Insecticide residues in cotton soils of Burkina Faso and effects of insecticides on fluctuating asymmetry in honey bees (Apis mellifera Linnaeus)

Four insecticides (acetamiprid, cypermethrin, endosulfan and profenofos) are used quarterly in the cotton-growing areas of Burkina Faso, West Africa. These insecticides were investigated in soils collected from traditionally cultivated and new cotton areas. Also, the effects of insecticide exposure on the developmental instability of honey bees, Apis mellifera, were explored. In soil samples collected three months after insecticide treatments, endosulfan and profenofos concentrations varied in the range of 10-30 μg kg(-1) in the traditionally cultivated zones and 10-80 μg kg(-1) in the new cotton zones, indicating a pollution of agricultural lands. However, only profenofos concentrations were significantly higher in the new cotton zone than the traditionally cultivated zones. In addition, the index of fluctuating asymmetry, FA1, in the length of second tarsus (L(HW)) was increased for bees when exposed to pesticide treated cotton fields for 82d, and their FA levels were significantly higher than those in the control colony in an orchard. The other studied traits of bees exposed to insecticides were not significantly different from controls. Our results indicate that FA may be considered as a biomarker reflecting the stress induced by insecticide treatments. However, the relationship between FA and stressors needs further investigations.     

Fluctuating asymmetry of invertebrate populations as a biological indicator of environmental quality

Fluctuating asymmetry (FA) of populations of the shrimp, Palaemon elegans, and the blood worm, Chironomus salinarius, was used to assess the environmental impact of a fertilizer manufacturing facility on the surrounding marine and freshwater ecosystems, respectively. Populations of both species were found to have elevated levels of asymmetry compared with control populations, indicating that the facility was having a significant impact on organism development. The use of FA of invertebrate populations as a simple, sensitive and cost-effective biomonitoring system for the detection of environmental stress is discussed.     

Chronic exposure to environmental stressors induces fluctuating asymmetry in shrews inhabiting protected Mediterranean sites

Many ecotoxicological studies have addressed the effects of contaminant exposure at various levels of biological organization. However, little information exists on the effects of toxicants on wildlife populations. Here we examined exposure of populations of the greater white-toothed shrew Crocidura russula (Soricomorpha, Soricidae) occupying two protected Mediterranean sites (a polluted area, the Ebro Delta, and a control site, Garraf Massif). Bioaccumulation of selected elements (Pb, Hg, Cd, Zn, Cu, Fe, Mn, Cr, Mo, Sr, Ba, and B), a body condition index (BCI) and fluctuating asymmetry (FA) were used to assess the chronic exposure to environmental pollution. BCI was correlated neither to metal concentrations nor to FA, suggesting that this fitness measure only reflects environmental disturbances at a local level. However, shrews from the polluted area showed higher concentrations of metals and metalloids (Pb, Hg, B, and Sr) and greater shape FA than specimens from the reference area. A correlation between FA was found for both first and second principal component vectors suggesting that developmental instability increases as a result of exposure to multiple pollutants. Our results corroborate the suitability of C. russula as a bioindicator of environmental quality and show that FA is an appropriate index to examine impact of developmental stressors in populations inhabiting disturbed areas.     

Differential toxicity of silver and titanium dioxide nanoparticles on Drosophila melanogaster development, reproductive effort, and viability: size, coatings and antioxidants matter

Silver and titanium dioxide nanoparticles are known to induce oxidative stress in vitro and in vivo. Here we test if they impact development, mating success, and survivorship in Drosophila melanogaster, and if so, if these effects are reversible by antioxidants. Ingestion of nanotitanium dioxide during the larval stage of the life cycle showed no effects on development or survivorship, up to doses of 200 μg mL⁻¹. Conversely, ingestion of nanosilver had major dose, size, and coating-dependent effects on each of these aspects of life history. Each of these effects was partially or fully reversible by vitamin C. Larvae growing on nanosilver supplemented with vitamin C showed a greater than twofold increase in survivorship compared to flies reared on nanosilver alone, and a threefold increase in mating success. Vitamin C also rescued cuticular and pigmentation defects in nanosilver fed flies. Biochemical assays of superoxide dismutase and glutathione show these markers respond to nanotitanium dioxide and nanosilver induced oxidative stress, and this response is reduced by vitamin C. These results indicate that life history effects of nanosilver ingestion result from oxidative stress, and suggest antioxidants as a potential remediation for nanosilver toxicity. Conversely, the lack of nanotitanium dioxide life history toxicity shows that oxidative stress does not necessarily result in whole organism effects, and argues that nanoparticle toxicity needs to be examined at different levels of biological organization.     

A comparison of the effects of organophosphate insecticide exposure and temperature stress on fluctuating asymmetry and life history traits in Culex quinquefasciatus.

Effects of exposure to a non-chemical (temperature) or chemical (organophosphate insecticide) stressor during larval development were compared in Culex quinquefasciatus mosquitoes. Stress was measured in two ways: using conventional life history traits (survival, development time and a measure of body size) and by calculating the degree of developmental instability from the departure from bilateral symmetry of wing characters (fluctuating asymmetry). Increasing insecticide dose, but not temperature, was observed to elevate wing fluctuating asymmetry in male but not female mosquitoes. Insecticide treatment reduced survival and was associated with a significant reduction in wing trait sizes in both females and males but did not significantly affect development time. Temperature was associated with a significant reduction in all life history traits in both sexes. Therefore wing fluctuating asymmetry in C. quinquefasciatus cannot be used as a general biomonitor of all stress, although it may have potential as a more specific monitor of chemical stress. It needs to be complimented with other measures such as life history and biochemical methods. The significant differences in response between sexes may impact on results of short-term larval exposures to insecticides.     

Toxicity of arsenate and arsenite on germination, seedling growth and amylolytic activity of wheat

Effects of different concentrations of arsenite and arsenate (0-16 mg/l) on seed germination, relative root length and shoot height, arsenic accumulation in young seedlings, alpha-amylase, beta-amylase and total amylolytic activity in wheat were investigated in order to elucidate the toxicity of arsenic in the early developmental stage. Germination percentages of different wheat varieties had different responses to arsenic species and decreased significantly with increasing arsenic concentrations except Duokang 1. Relative root length (RRL) and relative shoot height (RSH) of wheat seedlings decreased with increasing concentrations of arsenite and arsenate. The relative root lengths were correlated with the relative shoot heights for arsenite (r2 = 0.79) and arsenate (r2 = 0.77). Arsenic uptake by seedlings increased with the increasing concentrations of arsenite or arsenate and followed the Michaelis-Menten kinetics function. The average total amylolytic activity and beta-amylase activity had no significant difference comparable to that of controls at the concentration 2 mg/l arsenite or arsenate, but decreased apparently when the concentration was higher than 2 mg/l. Whereas the alpha-amylase activity decreased with increasing concentrations of arsenite or arsenate over the whole concentration range. Arsenite decreased all the endpoints more remarkably than arsenate. In comparison, shoot height and root length were more sensitive to arsenic than other endpoints and might be used as indicators for arsenic toxicity.     

A test of fluctuating asymmetry in Bluegills (Lepomis macrochirus Rafinesque) as a measure of pH-related stress

Fluctuating asymmetry of bilateral anatomical characters, previously proposed as an indicator of stress induced by agents affecting the calcium metabolism of fishes, was measured in bluegills (Lepomis macrochirus) from eleven Wisconsin lakes. The fluctuating asymmetries of three meristic characters-numbers of pored lateral line scales, pectoral fin rays and gill rakers on the outer arch-were either unrelated or only marginally related to either lake pH or waterborne calcium concentration. The asymmetry variance of gill raker counts was negatively correlated with both lake pH and waterborne calcium concentration; however, the overall asymmetry mean of gill raker counts in bluegills from five low pH lakes only slightly exceeded that in bluegills from six lakes with circumneutral pH. Fluctuating asymmetry in adult fishes was judged to be insensitive as a potential measure of pH-related stress.     

The detection of chronic biological effects in the marine intertidal bivalve cerastoderma edule, in model ecosystem studies with pulverised fuel ash: reproduction and histopathology

The cockle Cerastoderma edule was exposed to sediments variously comprised of pulverised fuel ash (PFA) for two consecutive periods of 3 and 9 months. Chronic effects of PFA on this filter feeding bivalve were studied by examining changes in cellular and tissue pathology with the aid of a screening procedure developed for analysing histopathology conditional. The mortality rate was high, up 43% for the 100% PFA sediment and growth of the individual bivalves was low. The accumulation of metals appeared to be low at 1-10 times background tissue levels. Signs of cellular stress were seen in the digestive gland after the first 3 months. However, after 9 months, this same organ was in fairly good condition. Reproduction was delayed due to slow oocyte maturation.     

The accumulation of metals and their toxicity in the marine intertidal invertebrates cerastoderma edule, Macoma balthica, Arenicola marina exposed to pulverised fuel ash in mesocosms

In order to investigate the accumulation of metals and related biological effects from Pulverized Fuel Ash (PFA), three intertidal benthic invertebrates were used in exposure studies with different mixtures of PFA sediments. After the first run of 90 days, high mortality was found in the lugworm Arenicola marina. After intermittent exposure to PFA, high mortality was also found for the cockle Cerastoderma edule. No mortality occurred with the baltic tellin Macoma balthica. Metal accumulation differed widely among the species. A. marina accumulated As to high levels in PFA mixtures, which may be attributed to changes in the redox potential of the sediment. It also appeared from this study that Zn levels in M. balthica tissue, for both control and exposed animals, are apparently normal but extremely high. Disposal of PFA in marine coastal waters will radically affect community structure at the dumping site. Accumulation of certain elements like As will be favoured in the mixing zone at the borders of a dump site due to a higher organic content and consequent higher bioavailability.     

Monitoring behavioural responses to metals in gammarus pulex (L.) (Crustacea) with impedance conversion

An impedance conversion technique was used to study the behaviour of Gammarus pulex (L.) exposed to acutely toxic concentrations of Pb (0.01, 0.05, 0.1 and 0.5 mg Pb 1^-1) and to field concentrations of Cu (≤ 0.05 mg Cu 1^-1). Initial stress responses were studied during short-term exposure (1 h), and sublethal toxic effects were monitored during 7 (Pb) and 35 days (Cu), respectively. Exposure to Pb caused 30 % mortality and resulted in a bioconcentration factor (BCF) of 2700 at 0.5 mg Pb l^-1 after 168 h. Exposure to Cu polluted stream water caused no mortality within 35 days, and uptake was low (BCF 5.8). Gammarus pulex reacted with initial stress responses to metal exposure within 30 min (Cu) or 1 h (Pb). The reactions consisted of increased ventilation and decreased locomotion. Sublethal concentrations of Pb and Cu caused toxic effects on the behaviour of G. pulex after several days of exposure, consisting of increased ventilation and decreased locomotion. Impedance conversion is an appropriate method for detecting stress responses to metals and can be used in “early warning” biomonitoring systems as well as for acute and chronic behavioural toxicity testing.     

Effects of aqueous stable fullerene nanocrystals (nC60) on Daphnia magna: Evaluation of sub-lethal reproductive responses and accumulation

Concerns exist regarding the inadvertent release of engineered nanomaterials into natural systems, and the possible negative ecosystem response that may occur. Understanding sub-lethal effects may be particularly important to determining ecosystem responses as current levels of nanomaterial release are low compared to levels projected for the future. In this work, the sub-lethal effects and bioaccumulation of water stable, nanocrystalline fullerenes as C₆₀, (termed nC₆₀) were studied in Daphnia magna, a globally distributed, parthenogenetic zooplankton. Sub-lethal concentrations were first determined for both mature mother (LD₅₀ = 0.4 mg L⁻¹) and neonate (gestating) daphnids (0.2 mg L⁻¹) in standard 48 h exposure tests. Subsequent experiments focused on the accumulation and effects (at temperatures of 18–28 °C) of nC₆₀, during the D. magna reproductive cycle. The results demonstrate that upon sub-lethal exposure, the mortality rates of gestating daphnids increased with time and developmental stage. The maturation of daughter daphnids was negatively impacted. The mother daphnids were unable to reproduce again after exposure during pregnancy, and differential bioaccumulation occurred as a function of lipid content in the daphnia with the highest accumulation level of 7000 mg kg⁻¹ wet weight. Taken together, these results not only describe the accumulation and sub-lethal effects of nC₆₀ on exposed daphnia, but also highlight the importance of sub-lethal exposure scenarios, which are critical to fully understanding the potential impact of fullerenes and other engineered nanoscale materials on natural systems.     

Growing snails used as sentinels to evaluate terrestrial environment contamination by trace elements

Young garden snails (Helix aspersa) reared in standard conditions (aged two months, mean weight 4.6 +/- 0.5 g) set as sentinels in cages laid on the soil for four weeks, give data for biomonitoring the environmental impact of chemicals on soil ecosystems in the field. The survival and the growth of the snails are influenced by the nature of the biotope and the level of the pollutants. Assay of cadmium, copper, lead and zinc bioaccumulated in the tissues of the sentinel snails provides information on the bioavailability of metals in the environment. The encagement model, little used for terrestrial species, can be useful in monitoring (specific and global endpoints) metal pollution of the environment in reference to the trophic level of the primary consumers. Active biomonitoring is positively compared with the passive biomonitoring.     

Correlating element atmospheric deposition and cancer mortality in Portugal: data handling and preliminary results

This study, framed within geographical epidemiology, presents preliminary findings concerning the association between the concentrations of chemical elements obtained through atmospheric biomonitoring with lichens and cancer mortality in the Portuguese population. Exploratory analyses were performed to identify potential confounders for the relationships between chemical elements and neoplasm mortality and to assess the extent of their interference. The results of this study highlight some methodological and conceptual difficulties inherent to observational and geographical studies, in the specific context of the Portuguese population, and the challenge posed by the large numbers of pollutants considered.     

Effect of copper on growth of an aquatic macrophyte,Elodea canadensis

Elodea canadensis has been proposed as a potential biomonitor due to its wide distribution and apparent ability to accumulate pollutants in aquatic ecosystems. We investigated the effects of copper sulfate on growth in E. canadensis to determine its effectiveness as a biomonitor of copper pollution in aquatic systems and whether growth is a suitable index of sub-lethal stress. Copper sulfate significantly slowed or stopped growth at all concentrations (low: 1 ppm, medium: 5 ppm, high: 10 ppm of copper sulfate) used. Final plant drymass was significantly lower in medium and high copper treatments compared with controls. E. canadensis appears to be very sensitive to copper levels, and may be useful as a biomonitor of copper levels in aquatic systems. However, its utility as a bioaccumulator may be limited, because we observed senescence of most leaves in all copper-treated plants following 4 weeks of treatment.     

Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO

One of the most frequently detected pharmaceuticals in environmental water samples is the anti-rheumatic drug, diclofenac. Despite its increasing environmental significance, investigations concerning the effects of this drug on the early developmental stages of aquatic species are lacking up to now. To determine the developmental toxicity and proteotoxicity of this drug on the growing fish embryos, eggs of zebrafish were exposed to six concentrations of diclofenac (0, 1, 20, 100, 500, 1000, and 2000 microg l(-1)) using DMSO as solvent. Early life stage parameters such as egg and embryo mortality, gastrulation, somite formation, movement and tail detachment, pigmentation, heart beat, and hatching success were noted and described within 48- and 96-h of exposure. After the 96-h exposure, the levels of stress proteins (hsp 70) were determined in both the diclofenac-treated and respective DMSO controls. Results showed no significant inhibition in the normal development until the end of 96 h for all exposure groups. However, there was a delay in the hatching time among embryos exposed to 1000 and 2000 microg l(-1). Late-hatched embryos (108 h) did not differ morphologically from normally hatched embryos. The mortality and average heart rate data did not show significant differences for all embryos in both diclofenac-treated and DMSO control groups. No significant malformations were likewise noted among all developing embryos throughout the exposure period. The levels of heat shock proteins in diclofenac-treated and control embryos did not differ significantly. DMSO control embryos, on the other hand, showed a concentration-dependent increase in hsp 70 levels. We suggest possible modulating effect of diclofenac in DMSO-triggered expression of stress proteins and this might have a possible repercussion on the use of DMSO as solvent in any toxicity assay. Since the present data indicate no significant embryotoxicity and proteotoxicity induced by diclofenac and due to the fact that the concentrations of diclofenac used in the present study is up to 2000-fold higher than the concentrations detected in the environment, it is unlikely that this drug would pose a hazard to early-life stages of zebrafish.     

A new method for testing the sensitivity of active biomonitoring: an example of its application to a terrestrial moss

For the first time, the concepts of limit of detection and limit of quantification, commonly used in analytical chemistry, are applied to the field of active biomonitoring with terrestrial mosses, using the controls as blanks so that the limits indicate the error associated with the transplant technique. The application of these concepts to data corresponding to Hg concentrations in the surroundings of a chlor-alkali plant and a power plant, makes interpretation of the results easier by providing better spatial and temporal coherence. This procedure may allow improvement in the standardization of active biomonitoring techniques as it is applicable to all kinds of biomonitors. One disadvantage of the application of the limit of detection and limit of quantification is that they only take into account the alpha error, or risk of false positives, and do not take into account the beta error, or risk of false negatives.     

Rapid genetic erosion in pollutant-exposed experimental chironomid populations

Few studies have evaluated how effectively environmental contamination may reduce genetic diversity of a population. Here, we chose a laboratory approach in order to test if tributyltin (TBT) exposure at environmentally relevant concentrations leads to reduced genetic variation in the midge Chironomus riparius. Two TBT-exposed and two unexposed experimental populations were reared simultaneously in the laboratory for 12 generations. We recorded several life-history traits in each generation and monitored genetic variation over time using five variable microsatellite markers. TBT-exposed strains showed increased larval mortality (treatments: 43.8%; controls: 27.8%), slightly reduced reproductive output, and delayed larval development. Reduction of genetic variation was strongest and only significant in the TBT-exposed strains (treatments: −45.9%, controls: −24.4% of initial heterozygosity) after 12 generations. Our findings document that chemical pollution may lead to a rapid decrease in genetic diversity, which has important implications for conservation strategies and ecological management in polluted environments.     

H NMR metabolomics of earthworm exposure to sub-lethal concentrations of phenanthrene in soil

¹H NMR metabolomics was used to monitor earthworm responses to sub-lethal (50-1500 mg/kg) phenanthrene exposure in soil. Total phenanthrene was analyzed via soxhlet extraction, bioavailable phenanthrene was estimated by hydroxypropyl-β-cyclodextrin (HPCD) and 1-butanol extractions and sorption to soil was assessed by batch equilibration. Bioavailable phenanthrene (HPCD-extracted) comprised ∼65-97% of total phenanthrene added to the soil. Principal component analysis (PCA) showed differences in responses between exposed earthworms and controls after 48 h exposure. The metabolites that varied with exposure included amino acids (isoleucine, alanine and glutamine) and maltose. PLS models indicated that earthworm response is positively correlated to both total phenanthrene concentration and bioavailable (HPCD-extracted) phenanthrene in a freshly spiked, unaged soil. These results show that metabolomics is a powerful, direct technique that may be used to monitor contaminant bioavailability and toxicity of sub-lethal concentrations of contaminants in the environment. These initial findings warrant further metabolomic studies with aged contaminated soils.