Chesapeake Ecotox Research Program: Research - Biomarkers of Experosure and Effect

Biomarkers of Exposure and Effect

Responses at molecular and cellular levels can be sensitive biomarkers of environmental exposure and biological effect (Stegeman et al. 1992). They provide linkage between the chemical environment and biological effects expressed at higher levels. Cytochrome P4501A (CYP1A) and metallothionein will be used as biomarkers of exposure; measures associated with endocrine and immune functions, which are indicative of reproductive fitness and disease susceptibility, will be used as biomarkers of effect. Changes can be related to the exposure module through the estimation of critical body residues (Kane Driscoll et al 1997).

Induction of cytochrome (Van Veld et al. 1990) and metallothionein (Roesijadi 1992) is responsive to environmental concentrations of organic pollutants and metals. Tissue specific expression of both can be used to identify exposure routes; for example, both are expressed in gill and gut. Activation of PAHs to carcinogenic metabolites and biotransformation of steroid hormones link cytochrome to carcinogenic and reproductive effects. Metallothioneins are believed to play a role in metal regulation and detoxification; induction confers increased metal tolerance. Genes for cytochrome and metallothionein may be under coordinate regulation in higher animals.

When an aquatic organism is exposed to environmental estrogens, altered levels of reproductive hormones and other abnormalities are often observed. Thus, vitellogenin, a precursor of yolk protein, and steroid reproductive hormones such as estradiol, testosterone, and 11-keto-testosterone, which are known to be altered by exposure, will be used as biomarkers of endocrine and reproductive dysfunction. For example, pollutant exposure alters vitellogenin levels in females and causes inappropriate vitellogenin expression in males (Gimeno et al. 1997). Changes in these biomarkers can be related to higher-order, organismal-level effects on reproductive parameters such as fecundity and viability of offspring.

Exposure to chemicals can trigger enhanced progression and intensity of parasitic disease (Anderson et al. 1996). Impaired macrophage functions of phagocytosis, production of reactive oxygen intermediates (ROI), and antimicrobial activity occur in fish and invertebrates (Roszell and Anderson 1994; 1996). These measures will be used to assess immunotoxicity associated with sediment exposure in mesocosms. Changes can be related to increased risk of infection; diminished health, growth and reproductive capacity; and increased mortality. Organisms exposed to contaminated sediments in the mesocosm experiments will be analyzed for the above responses.

Impact of exposure on individual and population level processes. The deleterious effects of contaminant exposure on physiological processes and behavior within individual organisms is ultimately expressed in the patterns of growth, reproduction and survival at the population level. Even in the absence of lethal effects, the consequences of individual-level responses to the subsequent dynamics of the population remain of fundamental importance. The need is to fully understand and predict the consequences of contaminant exposure to patterns of growth, reproduction and survival if population level responses are to be understood. Various lethal and sub-lethal responses by individuals that can lead to higher order effects will be examined including behavioral changes and modified energy allocation strategies. This suite of responses can then be directly related to the results of biomarker investigations.

Impact on bioenergetics and behavior. Individual-level processes that both are impacted by contaminant stress and, at the same time, are responsible for phenomena that influence fitness, are most likely candidates to produce higher-level effects. If an individual organism is to contribute to the next generation it must be able to: (1) derive an adequate supply of resources from the environment, (2) allocate assimilated energy to basic survival requirements (maintenance), and (3) allocate energy that remains after maintenance requirements have been met to production of new tissue (growth, energy storage, and reproduction). The relationship between net energy assimilated and allocation processes can be expressed simply as N = M + P, where

: N = net energy assimilated (a finite quantity based upon resource availability, harvesting, and assimilation efficiency)
M = allocation to maintenance (supporting basic physiological costs)
P = allocation to production of new tissue.

The production budget (P) supports the processes of growth (G), energy storage (S), and reproduction (R). Given a finite amount of assimilated resources, the maintenance and production budgets can be viewed as competing processes. Clearly an environmentally- induced change in expenditures for one portion of the budget must detract from energy available to support the remaining portion.

The maintenance pathway, supporting basic survival costs, is the most energetically-demanding allocation pathway. Congdon et al. (1982) determined that maintenance costs accounted for greater than 80% of the annual total energy budgets of several reptiles. Thus only a small proportion of the total energy acquired (less than 20%; Congdon et al. 1982) can be allocated to processes that are not essential to maintenance, but nonetheless influence individual fitness traits and population dynamics. Maintenance costs may be slightly less for invertebrates (i.e., 60-80%) but remain a substantial portion of the energy budget (Valiela 1995). Any change in maintenance costs, therefore, would be expected to produce a greater proportional gain or loss in energy available for the pathways supporting growth, energy storage, and reproduction.

Extensive research has shown increases in maintenance expenditures in vertebrates and invertebrates that are chronically exposed to mixtures of various organic and inorganic compounds (Bayne et al. 1979; Moore et al. 1987; Koehn and Bayne 1989; Sibly and Calow 1989; Calow and Sibly 1990; Calow 1991; Widdows and Donkin 1991; Forbes and Depledge 1992; Weber and Spieler 1994; Rowe 1998; Rowe et al. 1998; Hopkins et al. in press). Elevated maintenance costs may result from energetic expenses incurred via cellular repair, excretion of toxicants, and elevated protein turnover rates in pollution-stressed individuals (see Hawkins et al. 1986; Koehn and Bayne 1989; Hawkins 1991). The relative roles that these and other processes play in modifying maintenance costs are unknown, and likely to vary with conditions of the particular habitat. Yet from an ecological perspective, the cause for the increased maintenance costs is not as important as the potential effects of the elevated costs: an associated decrease in energy available for production, influencing processes such as growth and reproduction.

Scientists will examine maintenance expenditures of both Leptocheirus and Fundulus by measuring standard metabolic rates. Concentrations of non-polar (storage) lipids will be measured (Gardner et al. 1985). The measures of maintenance, growth, and lipid storage will be supplemented with measurements of egg production and egg quality (lipid content) in reproductive females. Effects of conditions with regions of concern on specific portions of the energy budget will thus allow an examination of the effects on individual organisms (maintenance budget) as well as on those processes that can influence population-level features (production budget).

Changes in behaviors, particularly those associated with feeding and maintenance activities, have direct implications for an organism's energy balance: decreased energy uptake and increased maintenance expenditures in the presence of chemical contaminants can detract from energy available for growth and reproduction, thereby potentially influencing fitness (Calow and Sibly 1990; Forbes and Depledge 1992; Weber 1997; Rowe 1998; Rowe et al. 1998; Hopkins et al. in press). For amphipods, feeding behaviors and burrow irrigation rates have significant implications for organism energy balance. To develop indices of sub-lethal effects, these behaviors will be evaluated using a non-invasive, computer-aided monitoring system. The video cameras will allow direct observations of organism behaviors, either in the mesocosms or, more likely, in smaller laboratory "ant-farm" type microcosms. The flow thermistors are placed at the amphipod burrow openings in order to directly record water flow velocities and periodicity of flow through the burrow.

Observations will be made of multiple individuals with age class, sex and density as additional factors (neonates vs. juveniles vs. adults; males vs. females for adults only). Video imagery during selected time intervals will be used to verify changes in behaviors and to categorize specific behaviors which may influence energy uptake (e.g., feeding), energy expenditure (normal activity level, hypoactive, hyperactive, no activity) or increase predation risk (e.g., time spent at burrow opening or on sediment surface). Amphipods retained at the conclusion of each trial will be sexed and measured (length and weight).

Recording behavioral responses of fish in estuarine mesocosms is problematical (E. D. Houde, UMCES, personal communication). A high resolution video camera with an underwater housing will be employed to estimate swimming speeds of fish in the experimental mesocosms. Trials will be conducted on each mesocosm individually. Video recordings will be taken for 1 hr periods at 06:00, 12:00, 18:00 and 24:00. Swimming speeds of individual fish will be estimated from PC-based imaging of successive frames.

Contaminant-mediated behavior changes associated with reproduction and predator avoidance also have significant implications for fitness. For example, altered swimming behaviors or escape reflexes may indirectly lead to mortality via elevated predation risks (Jung and Jagoe 1995; Weis and Weis 1995, Drewes 1997; Raimondo et al. 1998). For Fundulus, swimming trials will be used to examine swimming speeds and responses to predation-related stimuli (Atchison et al. 1987; Weis and Weis 1995; Weber et al. 1997). The swimming speed and latency of response will be quantified for larval, juvenile and adult Fundulus that have been exposed to contaminants to a predation threat (looming silhouette or sound pulse) in experimental arenas. Trials will be conducted in an 80-L experimental arena that permits video recordings to be made in two orthogonal planes.

The ability of Leptocheirus plumulosus to re-burrow following removal from the sediment is sensitive to chemical contaminant exposure (Mcgee and Schlekat 1992) and will be used as an indicator of predator avoidance ability. Amphipods obtained from mesocosms at selected time intervals during the exposure period will be tested for their ability to re-burrow into "clean" control site sediment.

Because chemical contaminants can disrupt the complex precopulatory behavior necessary for successful mating in some amphipods (Linden 1976; Davis 1978; Lyes 1979; Pascoe et al. 1994), the utility of a precopulatory behavior assay (Pascoe et al. 1994) for Leptocheirus plumulosus will be evaluated. The reproductive behavior of most amphipods (and other crustaceans such as the blue crab) involves a complex behavior in which the male holds and carries the female during a precopulatory guarding phase (Hynes 1955). This insures that insemination can occur as soon as the female molts and is ready to release eggs into the brood pouch. For Gammarus pulex, a well-studied species, the strength of this coupling is highly dependent on exposure to sublethal concentrations of contaminants such as cadmium, copper, lindane, atrazine and 3,4-dichloroaniline. Male and female Leptocheirus show strong sexual dimorphism (Schaffner pers. obs.) and are likely to exhibit similar precopulatory behaviors. A precopulatory separation test will be performed for 20 precopulatory pairs of Leptocheirus plumulosus for all experimental units.

Impact on population processes. In a contaminated habitat, organisms can experience reduced allocation of energy to the production pathway because of increased costs of maintenance. The relationship between the maintenance pathway, supporting short-term survival of the individual, and the production pathway, supporting processes that influence population-level processes, illustrates a potentially strong link between toxicological responses occurring at the individual level and effects at the population level.

The specific processes supported by the production budget are clearly interdependent, such that effects on one process can have an important influence on other processes. For example, female size (growth history) and provisioning of stored resources influence reproductive traits via effects on egg production and egg quality. Furthermore, changes in the production budget can have ramifications for ecological processes such as competition and predation, which are strongly size-dependent in aquatic systems. Smaller individuals are often inferior competitors and more vulnerable to predation than larger individuals. Growth and reproductive investment are a compounding process; small changes in growth rates or reproduction can have dramatic impacts when summed across an entire life cycle (Gotelli 1995).

Long term changes in growth can lead to delays in the onset of maturity, affecting rates of population growth by reducing the number of lifetime reproductive events, or increasing the likelihood of mortality prior to reproduction (Sibly 1996). At the same time, energetic restrictions can affect the investment strategy by females, influencing the trade-off between the number of offspring produced and the per capita energetic investment made for each offspring (see Sinervo 1990; Bridges and Heppell 1996; Reznick et al. 1996). Reduced production of offspring (fecundity) clearly can have population-level consequences. Yet, even in the absence of an effect on offspring number, reductions in the energy invested per egg (offspring quality) may reduce offspring survival or performance. Thus, the sub-lethal responses can have a variety of important ramifications for population dynamics. Implications of individual level responses to contaminant stress on population dynamics will be explored the through controlled mesocosm experiments and modeling.

Using whole life-cycle exposures in mesocosms and field-enclosures, researchers will examine relationships between chronic pollutant exposures and traits related to reproductive fitness, energy storage, growth and survival. Repeated sampling of individual organisms from the series of experiments will provide measures of growth, maintenance expenditures, and storage of energy throughout the life-cycle. Further, time to first reproductive event will be scored for each treatment, and gravid females will be analyzed for egg number, size, and egg quality (lipid content). A sub-sample of eggs from females in each treatment will be hatched and raised under optimal conditions throughout the juvenile period to examine effects of maternal exposure on offspring survival.

The strength of the relationship between sub-lethal responses by individuals and reproduction/ recruitment will be examined. Results of this work should be important in developing predictive models of population-level changes related to specific responses by individuals in polluted environments.