Sublethal Changes Research Articles

Contaminants and their effects on estuarine and coastal organisms in the United Kingdom in the late twentieth century

The biological effects of contaminants in British estuaries and coastal waters have been studied for over 100 years. Until the 1970s, the major pollution impact on estuarine organisms was probably caused by poorly treated sewage which led to severe oxygen deficits and consequent asphyxiation of many water-breathers. However, since the introduction of improved sewage treatment in the last 30 years, a number of continuing impacts have come to light which represent true toxic effects of micro-contaminants. Sublethal changes observed in various bioassays and biomarkers are widespread in both fish and invertebrates, and in the most urbanised and industrialised estuaries, these effects are probably having impacts at the population and community levels. For example, there is good evidence to show that tributyltin from some antifouling paints has not only affected the sexuality and reproductive success of individual estuarine and coastal molluscs, but has also damaged some benthic communities of which molluscs are but a part. Although there are data to show that some contaminant concentrations are now declining, we do not yet have enough data in most cases to decide whether organisms have also begun to recover. This paper reviews the entire field of contaminant effects in UK estuarine and marine waters, focusing especially on the years 1985–2000, and makes some recommendations for future research and monitoring programmes.

Automated Biomonitoring Using Real Time Movement Analysis of Euglena gracilis

An automated biomonitoring system for early warning of pollutants in aquatic environments is described and characterized. The system uses sublethal changes in the movement behavior of the flgellate Euglena gracilis as biological endpoints. The movement is determined by real time image analysis. All parameters describing motility, velocity, orientation, and form of the cells are calculated during measurement, and changes of these parameters are interpreted as effect. By automatic dilution of the water sample, dose-effect relationships can be recorded automatically. A total measurement procedure, including control and sample measurement and filling and rinsing of the system, typically requires 8 min. Measurements with different organic and inorganic toxic compounds were performed and the calculated EC50 values compared with literature data for the bioluminescence test with Vibrio fischeri. Also, measurements with waste water samples from different industrial plants were performed. The fast response time, the small size, the reliable image analysis system, the calculation of several endpoints, and the automatic measuring procedure are major advantages compared to other biological test systems.

Supply-Side Ecology Works Both Ways: The Link between Benthic Adults, Fecundity, and Larval Recruits

“Supply-side” ecology recognizes the potential role that recruitment plays in the local population dynamics of open systems. Apart from the applied fisheries literature, the converse link between adults and the production of cohorts of recruits has received much less attention. We used a hierarchical sampling design to investigate the relationships between adult abundance, fecundity, and rates of larval recruitment by acroporid corals on 33 reefs in five sectors (250–400 km apart) stretching from north to south along the length of the Great Barrier Reef, Australia. Our goal was to quantify patterns of recruitment at multiple scales, and to explore the underlying mechanisms. Specifically, we predicted that large-scale patterns of recruitment could be driven by changes in the abundance of adults and/or their fecundity, i.e., that corals exhibit a stock–recruitment relationship. The amount of recruitment by acroporids in each of two breeding seasons varied by more than 35-fold among the five sectors. Adult density varied only twofold among sectors and was not correlated with recruitment at the sector or reef scale. In contrast, fecundity levels (the proportion of colonies on each reef that contained ripe eggs) varied from 15% to 100%, depending on sector, year, and species. Spatial and temporal variation in the fecundity of each of three common Acropora species explained most of the variation (72%) in recruitment by acroporids, indicating that the production of larvae is a major determinant of levels of recruitment at large scales. Once fecundity was accounted for, none of the other variables we examined (sector, reef area, abundance of adults, or year) contributed significantly to variation in recruitment. The relationship between fecundity and recruitment was nonlinear, i.e., rates of recruitment increased disproportionately when and where the proportion of gravid colonies approached 100%. This pattern is consistent with the hypothesis that enhanced fertilization success and/or predator satiation occurs during mass-spawning events. Furthermore, it implies that small, sublethal changes in fecundity of corals could result in major reductions in recruitment.

SUPPLY-SIDE ECOLOGY WORKS BOTH WAYS: THE LINK BETWEEN BENTHIC ADULTS, FECUNDITY, AND LARVAL RECRUITS

“Supply-side” ecology recognizes the potential role that recruitment plays in the local population dynamics of open systems. Apart from the applied fisheries literature, the converse link between adults and the production of cohorts of recruits has received much less attention. We used a hierarchical sampling design to investigate the relationships between adult abundance, fecundity, and rates of larval recruitment by acroporid corals on 33 reefs in five sectors (250–400 km apart) stretching from north to south along the length of the Great Barrier Reef, Australia. Our goal was to quantify patterns of recruitment at multiple scales, and to explore the underlying mechanisms. Specifically, we predicted that large-scale patterns of recruitment could be driven by changes in the abundance of adults and/or their fecundity, i.e., that corals exhibit a stock–recruitment relationship. The amount of recruitment by acroporids in each of two breeding seasons varied by more than 35-fold among the five sectors. Adult density varied only twofold among sectors and was not correlated with recruitment at the sector or reef scale. In contrast, fecundity levels (the proportion of colonies on each reef that contained ripe eggs) varied from 15% to 100%, depending on sector, year, and species. Spatial and temporal variation in the fecundity of each of three common Acropora species explained most of the variation (72%) in recruitment by acroporids, indicating that the production of larvae is a major determinant of levels of recruitment at large scales. Once fecundity was accounted for, none of the other variables we examined (sector, reef area, abundance of adults, or year) contributed significantly to variation in recruitment. The relationship between fecundity and recruitment was nonlinear, i.e., rates of recruitment increased disproportionately when and where the proportion of gravid colonies approached 100%. This pattern is consistent with the hypothesis that enhanced fertilization success and/or predator satiation occurs during mass-spawning events. Furthermore, it implies that small, sublethal changes in fecundity of corals could result in major reductions in recruitment.

Anthropogenic contaminants and fish health along an urban waterway

AbstractField studies were conducted along an urban/industrial river to investigate the potential relationship between anthropogenic contaminants, including those related to oil refining, and fish health. The channel catfish, Ictalurus punctatus, served as the benthic sentinel species for these studies. At least 15 aromatic hydrocarbons known to be associated with oil refining and/or other industrial activities were detected at impacted stations including known carcinogens and mutagens [e.g., anthracene and benzo(a)pyrene]. At an appropriate reference station, no anthropogenic contaminants were identified in the waters and only 2 were observed in the sediments. Catfish collected from the reference station were significantly heavier, longer, and had higher relative weights. Morphological deformities such as clubbed, split, and missing barbels were observed on 37.8% of the fish collected from the impacted stations, while none were observed on fish from the reference station. Livers of catfish collected from the impacted stations exhibited a variety of histological changes consistent with contaminant exposure including vacuolated hepatocytes, pigment deposits, and other hepatoxic changes. These sublethal changes appear to be associated with chemical contamination and represent compromised health of individual organisms and, potentially, populations. © by John Wiley & Sons, Inc.

Evaluation of a biomonitor based on the phototactic behaviour of Daphnia magna using infrared detection and digital image processing

A biological early warning system based on the phototactic behaviour of Daphnia magna has been evaluated in relation to the reproducibility of its response and sensitivity towards potential pollutants. In addition to the infrared detection system of the monitor, video recording and digital image processing have been used in order to obtain more information on the behaviour of the Daphnia. The biomonitor performed better with Daphnia clones selected on positive phototactic behaviour than with non selected organisms. The variations in response over a five day period were too large to allow a reliable use of the monitor. The biomonitor was not able to detect sublethal changes in behaviour. Digital image processing of video recordings yielded more information on the behaviour of the Daphnia that the infrared detection system. Using this technique it was possible to detect sublethal changes in Daphnia behaviour as well as conceptual shortcomings in the existing behaviour.

Effects of contaminated sediments on viability, length, DNA and protein content of larval surf smelt, Hypomesus pretiosus

Mortality, abnormal morphology, growth, DNA and protein content of larval surf smelt (Hypomesus pretiosus) were evaluated after 96-h exposures to contaminated sediments from Puget Sound, Washington. Radiolabeled polynuclear aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) added to sediment were accumulated by exposed larvae, providing evidence of an exposure route for hydrophobic compounds from the sediment to the larvae. Sediment mixtures with high concentrations of contaminants were often acutely toxic, while exposure to diluted sediments resulted in sublethal changes—decreases in growth and DNA content. The DNA content of larvae was found to be the most sensitive of the sublethal measures. Both DNA content and the proportion of normal larvae were inversely related to PAH and PCB concentrations of test sediments. The results suggest that measurement of DNA content in conjunction with determination of the proportion of normal larval surf smelt can provide better discrimination for assessing toxicity due to contaminants in this sediment bioassay system than measurement of mortality alone. Because some larval fish may be at risk to sediment-associated contaminants, this bioassay may be useful for assessing the effects on larval marine fishes exposed to contaminants.

Oxidative Injury Induces Influx-Dependent Changes in Intracellular Calcium Homeostasis

Understanding of the mechanisms of cell injury and cell death is fundamental to the understanding of both protection against and initiation of cell injury and cell death. We subjected primary cultures of proximal tubular epithelium (PTE) from adult rats to an exogenous oxidative stress, generated by xanthine/xanthine oxidase (X/XOD), and studied its effect on the concentration of cytosolic ionized calcium ([Ca2+]i) by means of digital imaging fluorescence microscopy (DIFM) using a cytosolic calcium probe, fura-2. Exposure to 25 mU/ml X/XOD caused notable increases in [Ca2+]i detectable within 15 sec and increasing to micromolar levels with time. Experiments with Ca(2+)-free medium containing ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) showed that the increase of [Ca2+]i was due to influx from the extracellular space. Smaller and slower increases in [Ca2+]i were seen after exposure to lower concentrations of X/XOD (5 and 10 mU/ml). PTE injury and killing were assessed by measuring the release of cytosolic lactate dehydrogenase (LDH), exclusion of trypan blue, and observation of morphologic changes. Exposures to the 25 mU/ml concentration of X/XOD caused significant LDH release after 2 hr and correlated with trypan blue staining of exposed cells. Again, lesser concentrations of X/XOD resulted in a slower release of smaller amounts of LDH, and thus delayed trypan blue staining. Cytoplasmic bleb formation was seen by phase microscopy within minutes of exposure to 25 mU/ml, followed by cell rounding, retraction, and disintegration. Transmission electron microscopy revealed a progression of changes characteristic of lethal cell injury, beginning with dilatation of the endoplasmic reticulum, detachment of ribosomes, condensation of mitochondria, and chromatin clumping and terminating with mitochondrial swelling and formation of intramitochondrial flocculent densities. These studies clearly show that notable increases of [Ca2+]i precede both sub-lethal and lethal changes in rat PTE. These results indicate that interventions designed to minimize or to accelerate calcium entry could be of importance in cell preservation or cell killing, respectively, and therefore to therapeutic strategies for myocardial infarction, stroke, or shock in the former instance and for cancers in the latter.

Metabolic response of the grass shrimp Palaemonetes kadiakensis Rathbun, to acute exposure of sublethal changes in pH

The effects of sublethal changes in environmental pH on the grass shrimp Palaemonetes kadiakensis were determined using changes in metabolic rate and critical oxygen concentration as indicators of stress. Oxygen consumption was measured using a computer automated intermittent flow respirometer which allowed multiple sampling of a single individual without exposing it to prolonged oxygen stress. Metabolic rate and critical oxygen concentration were determined using a quadratic model with plateau nonlinear regression procedure. Sublethal changes in pH (6.5, 9.0) did not have a significant effect on metabolic rate. Temporary disturbances in hemolymph acid-base status caused by acute pH stress, may not elicit a metabolic response when not accompanied by necrosis of the gill epithelium. There was an effect of pH on critical oxygen concentration, with pH 6.5 significantly higher than the control (pH 7.8), and pH 9.0 significantly lower than the control. Shifts in critical oxygen concentration were attributed to changes in blood oxygen affinity caused by hemolymph acidosis or alkalosis.

A Chimera Embryo Assay Reveals a Decrease in Embryonic Cellular Proliferation Induced by Sperm from X-Irradiated Male Mice

Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as "mean ratio") was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group). However, cell numbers of singly cultured experimental embryos differed from those of singly cultured control embryos for just Week 7 for the 0.29 and 1.73 Gy dose groups, even though the mean ratios of heterologous chimeras had differed significantly from those of homologous chimeras for 3 weeks prior to and 1 week following Week 7. We conclude that sublethal changes sustained by sperm in vivo from only 0.05 Gy of X irradiation can be inherited by the embryo as a proliferative disadvantage that becomes expressed if challenged by direct cell contact with an unirradiated embryo in an aggregation chimera.

An Assay Using Embryo Aggregation Chimeras for the Detection of Nonlethal Changes in X-Irradiated Mouse Preimplantation Embryos

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.

Regeneration of hamster tracheal epithelium after mechanical injury. IV. Histochemical, immunocytochemical and ultrastructural studies.

All stages of regeneration in hamster tracheal epithelium were studied following a denuding mechanical injury. At 1 h all the cells had sloughed from the wound site leaving a bare and sometimes disrupted basal lamina. Viable cells at the wound margins rapidly changed shape, flattened and migrated to cover the denuded lesion by 12 h. In addition, epithelial cells that remained viable demonstrated sublethal changes that included the rapid discharge of mucous granules from secretory cells, internalization of cilia by ciliated cells and evidence of heterophagy in both cell types. By 24 h a wave of epithelial cell divisions occurred, primarily by secretory cells. This produced a multilayered epidermoid metaplasia that was best developed at 48 h. The metaplastic epithelium was largely composed of cells with both secretory (mucous granules) and epidermoid (tonofilament bundles and numerous desmosomes) characteristics. The peroxidase-antiperoxidase (PAP) method demonstrated a few keratin-positive cells in the wound as early as 12 h post-wounding and keratin was demonstrated in more cells by 24 h. All cells in the metaplastic wound epithelium were keratin-positive by 48 h. Following 48 h some of the most superficial keratinized cells sloughed from the epithelium and the keratin content of the remaining cells began to decline. At 72 h pre-ciliated and pre-secretory cells were seen in the wound. Pre-ciliated cells were characterized by an abundant electron-lucent cytoplasm, large pale nucleus, filiform apical microvilli and evidence of ciliogenesis, similar to that seen during fetal development. Pre-ciliated cells often contained apical mucous granules, apparently carried over from the parent secretory cells. With the appearance of these columnar cells the normal mucociliary morphology was restored in small wounds by 120 h, but some persistent epidermoid metaplasia remained in the large wounds through 168 h post-wounding. These data provide further evidence for the important role of secretory cells in the histogenesis of epidermoid metaplasia and the regeneration of normal morphology following injury. The implications of these findings in understanding the histogenesis of other lesions in the tracheo-bronchial epithelium are discussed.

Development, survival, and structural alterations of embryos and alevins of Atlantic salmon, Salmo salar L., continuously exposed to alkaline levels of pH, from fertilization

Embryos of the Atlantic salmon, Salmo salar L., were incubated continuously from fertilization at pH 6.8 (control) and pH 9.0 and 9.5, initially at 6.7 °C but with a gradual decline in the first 5 weeks to 5.0 °C for the remaining 10 weeks of exposure. Subsequently, the alevins were maintained in these environments for 50 days after hatching. Developmental processes and hatching were not affected by these levels of pH. Percentage cumulative mortality of treated embryos, 8%, was approximately that in the controls. Alevin mortality in the control lots was 1.2 and 1.3%. At pH 9.0, cumulative mortality was 0.4%, but at pH 9.5 there was an accelerating increase to 18%, at the termination of observation.Sublethal changes in embryos were confined mostly to cell necrosis and sloughed rudimentary epidermis. Some metaplasia of the brain stem occurred at pH 9.5. Sites and intensity of alterations increased in alevins at pH 9.5, following the loss of the zona radiata. In addition to ongoing injury of epidermis, including mucous cells, deleterious alterations occurred in branchial epithelium, erythrocytes, myocardium, blood vessels of the viscera, liver, brain, and optic lenses. In general, sublethal changes caused by hydroxylions are similar to those caused by excessive hydrogen ions but are somewhat less extensive in the structures affected or in their degree of severity.

Effect of zinc sulphate on the growth of mono- and multispecies cultures of some marine plankton algae

The effect of zinc sulphate has been investigated in mono- and multispecies cultures of the dinoflagellatesScrippsiella faeroense, Prorocentrum micans andGymnodinium splendens and of the diatomsSchroederella schroederi andThalassiosira rotula. Multiplication rate, in vivo chlorophyll fluorescence, maximum cell densities and Zn-conditioned disturbance of the species equilibrium of the multispecies cultures were used as criteria of sublethal toxic inhibition. In monocultures, the first effect became manifest after addition of 0.01 to 0.1 mg Zn++ · l−1. Diatoms proved to be more sensitive than dinoflagellates. In multispecies cultures, the growth of each species depended on the ratio of the inocula. Interrelation between interspecific competition and Zn-caused decrease in the number of algal cells regulated further growth of the cultures. Algal sensitivity to zinc increased with the number of species combined in the test medium: in a 5-species culture sublethal changes appeared already after addition of 0.005 to 0.01 mg Zn++ · l−1. In a few cases, interspecific competition depressed the growth of some species to an appreciable extent, even in the control cultures. At the resulting low cell numbers, the effect of zinc became apparent only in higher concentrations from 5 to 10 mg Zn++ · l−1. Morphological aberrations became manifest inScrippsiella faeroense and in the diatoms in concentrations from 1 and 0.01 mg Zn++ · l−1 respectively. The results show that multispecies experiments are a more sensitive test method for investigating the influence of zinc on plankton algae than are monoculture experiments. In natural plankton communities, however, the toxicity of heavy metals may become effective at considerably lower limit concentrations; this is suggested by the simplified model investigations in the laboratory.

Microbicidal mechanisms of human granulocytes: synergistic effects of granulocyte elastase and myeloperoxidase or chymotrypsin-like cationic protein.

The antibacterial activity of a myeloperoxidase (MPO)-glucose oxidase system was found to be greatly increased by granulocyte elastase, present in azurophil granules of human neutrophils. The MPO-H2O3-mediated killing of both Escherichia coli and Staphylococcus aureus was potentiated by granuocyte elastase at an acid pH, whereas at pH 7.4 only killing of E. coli was potentiated. The potentiating effect of elastase was not dependent on the enzymatic properties of the protein since it was not abolished by heating, which destroys the enzymatic activity. A peptide chloromethyl ketone elastase inhibitor abolished both elastolytic activity and the pctentiating effects on MPO-H2-O2-mediated bacterial killing. The antibacterial activity of chymotrypsin-like cationic protein of human neutrophils was also potentiated by elastase. Other degradative enzymes isolated from human granulocytes, e.g., collagenase and lysozyme, did not potentiate MPO-H2O2-mediated or cationic protein-dependent bacterial killing. The present study indicates that a neutrophil constitutent, elastase, which is not microbicidal by itself, can initiate sublethal changes that render some microorganisms more susceptible to the action of microbicidal agents like MPO and chymotrypsin-like cationic protein.

Effects of three pesticides on respiration, coughing, and heart rates of rainbow trout (Salmo gairdneri Richardson).

Short-term, sublethal changes in respiration, heart, and coughing rates in juvenile rainbow trout (1–1.5 years) exposed to the organochlorine pesticides p,p′-DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) and HEOD (dieldrin) and the methyl carbamate insecticide carbaryl (1-naphthyl N-methyl carbamate) were monitored at 13 ± 2 °C to investigate the effects of these pesticides on fish in fresh water. Sublethal responses to carbaryl and HEOD on physiological parameters were difficult to measure because dosages required to elicit them resulted in 30% to 60% mortality. DDT in various concentrations gave graded sublethal responses, and it is suggested that the coughing response in particular might be useful as a water-quality index. The DDT threshold for the coughing response was between 52 and 140 parts per billion.

II. Sublethal effects and changes in ecosystems. Assessment of the effects of pollutants on physiology and behaviour.

Many pollutants, even when present in the water in concentrations well below lethal levels, may cause marked changes in the physiology and behaviour of fish. The work reported deals mainly with salmonids. The responses to insecticides are particularly interesting. Of fundamental interest is the suggestion that DDT seems to act by interfering with the normal thermal acclimation mechanism(s), probably within the central nervous system. The results are not without practical significance. Some responses, including those induced by heavy metal ions from mining wastes, may cause long-term ecological changes of consequence.

Interphase death and relaed biochemical radiation effects in murine leukaemia cells.

WITHIN a few hours after X-irradiation with sub-lethal doses, cytological and biochemical changes become demonstrable in the lymphocytes of the thymus and other lymphatic tissues.