Novel techniques for field assessment of copper toxicity on fouling assemblages

Laboratory toxicity tests generally expose organisms to a constant concentration of a toxicant in a uniform environment. The two techniques outlined in this paper assess the impact of a model toxicant, copper, on marine fouling assemblages in the field. These techniques allow natural assemblages to develop under a regime of toxicant dosing. The experiments were conducted using both techniques over time frames ranging from 2 weeks to 6 months. Short term experiments using the techniques assess the effects of toxicant exposure on recruitment. The longer term experiments enabled detection of certain indirect impacts of pollution. These include effects due to competition for space and variations in the susceptibility of organisms with age. Over the course of the experiments, a range of responses to copper was observed for different taxa. Both techniques have the potential to be used for testing the impact of other toxicants. Approaches such as those described are necessary in order to understand how communities react to toxicants in real world situations.     

Consistency and sensitivity of community level endpoints in microcosm tests

We review microcosm toxicity tests with 12 chemical stresses and find that the relative sensitivity of certain endpoints is consistent over toxicant type. Changes in species composition occur at very low levels of chronic stress. Endpoints responding at increasing levels of stress are declines in species numbers relative to expected numbers, followed by decreased oxygen production and decreased total production. Other endpoints are quite sensitive in response to some toxicants but insensitive to others (e.g., autotrophic biomass). In addition, other endpoints respond unpredictably to stress, showing stimulation under some conditions and impairment under others. We compare our observations to the progressions of impact suggested from published whole ecosystem experiments and speculate about a general ecosystem distress syndrome and the implications for choosing endpoints in both toxicity testing and monitoring.     

Delayed fluorescence in algal growth inhibition tests

A series of 72-hour growth inhibition tests with green alga Desmodesmus (Scenedesmus) subspicatus (ISO 8692) has been performed to test the delayed fluorescence (DF) parameters as possible endpoint measurements. Sensitivity to five toxicants with direct and indirect effects on photosynthesis was tested, and the median effective concentration (EC₅₀) values derived from the cell concentration, absorbance and DF were compared. The sensitivity of DF intensity (DFI) was comparable with the two endpoints suggested in ISO 8692 for all five toxicants: potassium dichromate, 3,5-dichlorophenol (3,5-DCP), DCMU, copper and cadmium. In the case of potassium dichromate and copper, DFI was more sensitive than the other endpoints in this study. The analysis of DF relaxation kinetics showed a specific response to the herbicide DCMU. Additionally, a 24-hour test was performed with the same toxicant concentrations (except copper), where DFI was measured 30 minutes, one hour and 24 hours after the exposure. The influence of toxicants on DFI after a 24-hour exposure was comparable with the effects after 72 hours. Only 3,5-DCP influenced DFI after 30 minutes. DF proved to be a simple, reliable and rapid measurement to assess toxicity in algal tests, which can to some extent differentiate among various toxicants.     

Landscape-level toxicant exposure mediates infection impacts on wildlife populations

Anthropogenic landscape modification such as urbanization can expose wildlife to toxicants, with profound behavioural and health effects. Toxicant exposure can alter the local transmission of wildlife diseases by reducing survival or altering immune defence. However, predicting the impacts of pathogens on wildlife across their ranges is complicated by heterogeneity in toxicant exposure across the landscape, especially if toxicants alter wildlife movement from toxicant-contaminated to uncontaminated habitats. We developed a mechanistic model to explore how toxicant effects on host health and movement propensity influence range-wide pathogen transmission, and zoonotic exposure risk, as an increasing fraction of the landscape is toxicant-contaminated. When toxicant-contaminated habitat is scarce on the landscape, costs to movement and survival from toxicant exposure can trap infected animals in contaminated habitat and reduce landscape-level transmission. Increasing the proportion of contaminated habitat causes host population declines from combined effects of toxicants and infection. The onset of host declines precedes an increase in the density of infected hosts in contaminated habitat and thus may serve as an early warning of increasing potential for zoonotic spillover in urbanizing landscapes. These results highlight how sublethal effects of toxicants can determine pathogen impacts on wildlife populations that may not manifest until landscape contamination is widespread.     

Measurement of chronic toxicity using the opossum shrimpMysidopsis bahia

The chronic toxicity of silver and endosulfan to the opossum shrimpMysidopsis bahia was determined using continuous-flow bioassays. The 28-day bioassays measured survival, fecundity, and growth (length and weight measurements). Maximum acceptable toxicant concentrations (MATC) were estimated from measured toxicant concentrations. MATC values were similar using either brood size or growth as a criterion for sublethal effects. As an alternative to the determination of fecundity impairment, measurement of growth reduction in response to exposure to toxicants may provide a useful tool in the assessment of chronic toxicity inMysidopsis life-cycle bioassays.     

Characterizing individual, population and community effects of sublethal levels of aquatic toxicants: an experimental case study using Daphnia

SUMMARY. 1. The relationship between toxicant-induced changes in the feeding behaviour of the cladoceran, Daphnia catawba (Coker), and subsequent effects at population and community levels were experimentally addressed.
2. We adapted a method for measuring the thoracic appendage beat rate of individuals (termed the TAR and positively correlated with daphnid feeding rate) after exposure to toxicant at known levels, for use as a behavioural bioassay for low levels of aquatic toxicants. The TAR declined significantly with an increase in both sublethal and lethal levels of the surfactant, sodium dodecyl sulphate (SDS).
3. We also measured population level effects (i.e. rates of reproduction, survival, and R ₀), and found that reductions in these values occurred at the same concentrations as the effects on individual behaviour.
4. In most cases, the changes in feeding behaviour occurred more rapidly (≤30h) than changes in population parameters (days to weeks).
5. We conclude that toxicant-induced changes in individual feeding behaviour can provide a quicker estimate of effects on individuals and of potential community effects (i.e. effects on their prey populations) than measurements of population parameters. In addition, due to its sensitivity at low levels of toxicants, the behavioural bioassay may be particularly helpful in identifying effects of sublethal levels of aquatic pollutants.     

Combination ecotoxicity and testing of common chemical discharges to sewer using the<Emphasis Type="Italic"> Vibrio fischeri</Emphasis> luminescence bioassay

In order to investigate possible synergistic or antagonistic (more or less than additive) toxicity effects, mixtures of chemicals were tested in water using a microbial bioassay. Ten toxicants (3,4-dichloroaniline, 3,5-dichlorophenol, cadmium, chromium, copper, Lindane, linear alkylbenzene sulphonate, pentachlorophenol, toluene, zinc) were chosen on the basis of their common occurrence in industrial effluents within local waste water treatment plants. These toxicants also cover a wide range of modes of toxic action, namely, polar and non-polar narcosis, membrane disruption, respiratory disruption, uncouplers of oxidative phosphorylation, biochemical disruption and enzyme inhibition. Efficient screening for possible combination toxicity between toxicants involved testing the chemicals both singly and in triplet combinations. The triplets were based on four replicates of a balanced incomplete block design (BIB). A standardised Vibrio fischeri rapid toxicity bioluminescence assay was used. The combinations tested showed that only one mixture was found to be significantly more toxic than expected from the pure single-toxicant results. Two triplets were significantly less toxic. Further tests on the more toxic triplet showed that the effect was due to only one of the 45 pairs originally screened. It is concluded that synergistic effects in combinations of toxicants are rather rare in bioluminescence systems utilising common effluents discharged to sewer. Electronic Publication     

Effects of Environmental Toxicants on Metabolic Activity of Natural Microbial Communities

Two methods of measuring microbial activity were used to study the effects of toxicants on natural microbial communities. The methods were compared for suitability for toxicity testing, sensitivity, and adaptability to field applications. This study included measurements of the incorporation of ¹⁴C-labeled acetate into microbial lipids and microbial glucosidase activity. Activities were measured per unit biomass, determined as lipid phosphate. The effects of various organic and inorganic toxicants on various natural microbial communities were studied. Both methods were useful in detecting toxicity, and their comparative sensitivities varied with the system studied. In one system, the methods showed approximately the same sensitivities in testing the effects of metals, but the acetate incorporation method was more sensitive in detecting the toxicity of organic compounds. The incorporation method was used to study the effects of a point source of pollution on the microbiota of a receiving stream. Toxic doses were found to be two orders of magnitude higher in sediments than in water taken from the same site, indicating chelation or adsorption of the toxicant by the sediment. The microbiota taken from below a point source outfall was 2 to 100 times more resistant to the toxicants tested than was that taken from above the outfall. Downstream filtrates in most cases had an inhibitory effect on the natural microbiota taken from above the pollution source. The microbial methods were compared with commonly used bioassay methods, using higher organisms, and were found to be similar in ability to detect comparative toxicities of compounds, but were less sensitive than methods which use standard media because of the influences of environmental factors.     

How do toxicants affect epidemiological dynamics?

Populations are formed of their constituent interacting individuals, each with their own respective within‐host biological processes. Infection not only spreads within the host organism but also spreads between individuals. Here we propose and study a multilevel model which links the within‐host statuses of immunity and parasite density to population epidemiology under sublethal and lethal toxicant exposure. We analyse this nested model in order to better understand how toxicants impact the spread of disease within populations. We demonstrate that outbreak of infection within a population is completely determined by the level of toxicant exposure, and that it is maximised by intermediate toxicant dosage. We classify the population epidemiology into five phases of increasing toxicant exposure and calculate the conditions under which disease will spread, showing that there exists a threshold toxicant level under which epidemics will not occur. In general, higher toxicant load results in either extinction of the population or outbreak of infection. The within‐host statuses of the individual host also determine the outcome of the epidemic at the population level. We discuss applications of our model in the context of environmental epidemiology, predicting that increased exposure to toxicants could result in greater risk of epidemics within ecological systems. We predict that reducing sublethal toxicant exposure below our predicted safe threshold could contribute to controlling population level disease and infection.     

Are zebra mussels restricted by toxicant levels in the River Meuse? a review

This paper questions if chemical barriers prevent the occurrence of benthic invertebrates in the river Meuse. To this purpose an ecotoxicological analysis is presented, using published observations on the zebra musselDreissena polymorpha, a tolerant species that maintains populations in the river. Zebra mussels collected or exposed at the Belgian-Dutch border contained high levels of several groups of toxicants, and mixture toxicity is likely to occur. A recently developed bio-assay, using the filtration rate of the zebra mussel, demonstrated strong inhibitory effects of water from the river Meuse. To determine which (combination of) toxicants cause such effects, laboratory experiments with toxicant mixtures were carried out. It was demonstrated that in a mixture of five metals (Cu, Zn, Ni, Cd, Pb) the metals contributed to the toxicity of the mixture below the No Observed Effect Concentrations (NOEC) for these metals when tested individually. The average concentration of Cu, Zn and possibly Pb in Meuse water exceed the NOEC values for filtration rate. Thus, it seems likely that joint effects of different (groups of) toxicants in the river Meuse cause the overall toxicity of the water for the zebra mussel, explaining the marginal populations at the test site, while other river species are absent there. It is suggested that toxicants in the river Meuse restrict the recolonisation of the river by invertebrate species more sensitive than the zebra mussel. This will be studied using invertebrates of different sensitivities (molluscs, arthropods), enabling the assessment of toxicity at different stages of water quality improvement of the river Meuse.     

Continuous culture algal bioassays for organic pollutants in aquatic ecosystems

Short-term responses of phytoplankton to organic pollutants are highly transitory. Time-course studies of non-steady state cells in continuous culture showed varying growth or photosynthetic responses such as enchancement, inhibition, adaptation (or development of resistance) or rebound, depending on the direction of changes in the intracellular toxicant concentration and the duration of exposure. However, steady-state cells in a two-stage chemostat system exhibited an increased tolerance to toxicants and subtle physiological effects such as photosynthetic enhancement which was accompanied by a considerable leakage of photosynthesates. It is important to understand such steady-state responses for the prediction and assessment of ecological impact by organic pollution on phytoplankton, since the time scale of changes in the toxicant/biomass ratio in most natural waters is long enough to approximate an equilibrium state.     

Linking proteome responses with physiological and biochemical effects in herbicide-exposed Chlamydomonas reinhardtii

Exposure to a toxicant causes proteome alterations in an organism. In ecotoxicology, analysis of these changes may allow linking them to physiological and biochemical endpoints, providing insights into subcellular exposure effects and responses and, ultimately mechanisms of action. Based on this, useful protein markers of exposure can be identified. We investigated the proteome changes induced by the herbicides paraquat, diuron, and norflurazon in the green alga Chlamydomonas reinhardtii. Shotgun proteome profiling and spectral counting quantification in combination with G-test statistics revealed significant changes in protein abundance. Functional enrichment analysis identified protein groups that responded to the exposures. Significant changes were observed for 149-254 proteins involved in a variety of metabolic pathways. While some proteins and functional protein groups responded to several tested exposure conditions, others were affected only in specific cases. Expected as well as novel candidate markers of herbicide exposure were identified, the latter including the photosystem II subunit PsbR or the VIPP1 protein. We demonstrate that the proteome response to toxicants is generally more sensitive than the physiological and biochemical endpoints, and that it can be linked to effects on these levels. Thus, proteome profiling may serve as a useful tool for ecotoxicological investigations in green algae.     

Ecotoxicological studies with the freshwater rotifer Brachionus calyciflorus II. An assessment of the chronic toxicity of lindane and 3,4-dichloroaniline using life tables

The effects of chronic exposure of the freshwater rotifer Brachionus calyciflorus to the toxicants lindane and 3,4-dichloroaniline (DCA), were evaluated. The parameters used to determine the toxicity on these compounds were the age-specific and fertility, and the demographic parameters: intrinsic rate of natural increase (r), generation time (T), net reproductive rate (R _o), reproductive value (V _x) and life expectancy (e _o). All the demographic parameters studied decreased with increasing toxicant concentrations. The use of life tables techniques with B. calyciflorus as a test method for the determination of chronic toxicity of xenobiotics is discussed.     

Ecosystem health as measured from the molecular to the community level of organization,with reference to sediment bioassessment

The current recognition that chemical measurements are uncertain indicators of biological consequences of pollution has shifted the emphasis away from assessing environmental chemistry alone toward the inclusion of measurements of the health of organisms. Effects of pollutants begin with the individual, have subsequent repercussions on population level processes, and ramifications for community structure and functions. Pollutants act at a molecular level and the biochemical lesions is the first step in the manifestation of effects. Technologies that operate at the cellular level assist in elucidating toxicity. Higher levels of integration include an organism's capacity for growth. Laboratory bioassays andin situ research can monitor physiological incapacities and assist in predicting population level effects. A yet higher level of organization is that of the ecological community.     

The contribution of intra‐ and interspecific tolerance variability to biodiversity changes along toxicity gradients

The worldwide distribution of toxicants is an important yet understudied driver of biodiversity, and the mechanisms relating toxicity to diversity have not been adequately explored. Here, we present a community model integrating demography, dispersal and toxicant‐induced effects on reproduction driven by intraspecific and interspecific variability in toxicity tolerance. We compare model predictions to 458 species abundance distributions (SADs) observed along concentration gradients of toxicants to show that the best predictions occur when intraspecific variability is five and ten times higher than interspecific variability. At high concentrations, lower settings of intraspecific variability resulted in predictions of community extinction that were not supported by the observed SADs. Subtle but significant species losses at low concentrations were predicted only when intraspecific variability dominated over interspecific variability. Our results propose intraspecific variability as a key driver for biodiversity sustenance in ecosystems challenged by environmental change.     

Using elasticity analysis of demographic models to link toxicant effects on individuals to the population level: an example

1. A simple two-stage population model was applied to data from a previously published life-table response experiment (LTRE), which examined the toxicity of 4- n -nonylphenol to life-history traits of the polychaete Capitella sp. I. Population growth rates ( λ ) and the relative sensitivities (= elasticities) of λ to changes in each of the individual life-history traits were calculated.
2. In the present study, the life-history parameters measured in laboratory-reared individuals were manipulated to simulate potential effects of competition and predation on fecundity, time to reproductive maturity and juvenile survival to explore how such factors might influence the sensitivity of population growth rate to toxicant-caused changes in individual life-history traits.
3. Dramatic changes in elasticity patterns among simulations indicate that population growth rates may respond very differently to toxicant exposure depending on the extent to which other demographically limiting factors (e.g. competitors and/or predators) are operating on the population.
4. Effectively predicting the population-level consequences arising from toxicant effects measured on individuals can be improved by exploring the elasticity pattern of λ for the population over a range of realistic ecological situations.     

The Role of Soil Microbial Tests in Ecological Risk Assessment: Differentiating between Exposure and Effects

The use of soil microorganisms in ecological risk assessment is hampered by an unclear dose-response relationship for most contaminants. Establishing dose-response curves for soil microbial communities requires that one have a clear estimate of exposure at the site of toxic action and a response free of confounding environmental factors. It is not clear what methods can estimate toxicant dose at the site of toxic action or determine microbial response to a toxicant. Pollution-induced community tolerance (PICT) is one possible estimate of microbial toxicant exposure. The PICT hypothesis is that the tolerance of a microbial community is proportional to the in situ dose. This method automatically corrects for differences due to differences in soil physical-chemical variables between samples. Various components of the soil nitrogen cycle can act as microbial bioindicators of toxicant impacts. Estimating denitrifica-tion activity presents a number of advantages over other components of the nitrogen cycle. Denitrifying bacteria come from a diversity of habitats, can be autotrophic or heterotrophic, and denitrification is a well-defined enzymatic system, which allows the use of molecular tools. Determining denitrification may be a good estimate of effects of toxicants on microbial communities. However, given the state of our ignorance regarding soil microbial community structure and function, redundant estimates of exposure and effect are necessary to adequately characterize the response of microbial communities to toxicants.     

Sublethal Toxicity Endpoints of Heavy Metals to the Nematode Caenorhabditis elegans

Caenorhabditis elegans, a free-living nematode, is commonly used as a model organism in ecotoxicological studies. The current literatures have provided useful insight into the relative sensitivity of several endpoints, but few direct comparisons of multiple endpoints under a common set of experimental conditions. The objective of this study was to determine appropriate sublethal endpoints to develop an ecotoxicity screening and monitoring system. C. elegans was applied to explore the sublethal toxicity of four heavy metals (copper, zinc, cadmium and chromium). Two physiological endpoints (growth and reproduction), three behavioral endpoints (head thrash frequency, body bend frequency and feeding) and two enzymatic endpoints (acetylcholine esterase [AChE] and superoxide dismutase [SOD]) were selected for the assessment of heavy metal toxicity. The squared correlation coefficients (R²) between the responses observed and fitted by Logit function were higher than 0.90 and the RMSE were lower than 0.10, indicating a good significance statistically. There was no significant difference among the half effect concentration (EC50) endpoints in physiological and behavioral effects of the four heavy metals, indicating similar sensitivity of physiological and behavioral effects. AChE enzyme was more sensitive to copper, zinc, and cadmium than to other physiological and behavioral effects, and SOD enzyme was most sensitive to chromium. The EC50 of copper, zinc, and cadmium, to the AChE enzyme in the nematodes were 0.68 mg/L, 2.76 mg/L, and 0.92 mg/L respectively and the EC50 of chromium to the SOD enzyme in the nematode was 1.58 mg/L. The results of this study showed that there was a good concentration-response relationship between all four heavy metals and the sublethal toxicity effects to C. elegans. Considering these sublethal endpoints in terms of simplicity, accuracy, repeatability and costs of the experiments, feeding is the relatively ideal sublethal toxicity endpoint of heavy metals to C. elegans.     

Cross-generational impact of a male murine pheromone 2-sec-butyl-4,5-dihydrothiazole in female mice

The current understanding of the activity of mammalian pheromones is that endocrine and behavioural effects are limited to the exposed individuals. Here, we demonstrate that the nasal exposure of female mice to a male murine pheromone stimulates expansion of mammary glands, leading to prolonged nursing of pups. Subsequent behavioural testing of the pups from pheromone-exposed dams exhibited enhanced learning. Sialic acid components in the milk are known to be involved in brain development. We hypothesized that the offspring might have received more of this key nutrient that promotes brain development. The mRNA for polysialyltransferase, which produces polysialylated neural cell adhesion molecules related to brain development, was increased in the brain of offspring of pheromone-exposed dams at post-natal day 10, while it was not different at embryonic stages, indicating possible differential brain development during early post-natal life.