LBL Researchers Evaluate Ecological Hazards

Fall/Winter 1990

By Jeffery Kahn ([email protected])

Gauging the health of an ecosystem is a lot like asking a sick whale just what ails it. The dimensions of your patient are leviathan, yet you have only a crude ability to interrogate and diagnose it.

In a world at environmental risk, ecological health customarily is estimated by measuring the concentrations of contaminants. Whether the concern is pollutants blackening the air around Mexico City or sewage gushing into Boston Harbor, we measure potential ecological harm in parts per million.

Chemical testing of exposure levels actually is only one aspect of ecological risk assessment. Hazard evaluation -- ascertaining the biological effects of exposures to organisms -- is another approach. Up until now, hazard evaluation has been virtually neglected.

San Francisco Bay's Hayward Shoreline marsh is a prime example of why current environmental evaluations are inadequate. Five hundred acres of marsh within this park, once commercial salt evaporation ponds, have been transformed into an ecological showcase for birds and aquatic life. Touted as a model marsh, the wetlands is fed a flow of fresh water from the Union Sanitary District sewage treatment plant, a facility which earns regulatory raves for the quality of its effluent. Evaluated on some 30 separate chemical standards, the wastewater comes up smelling like a rose. It is in compliance with all state standards.

But what about the chain of life -- the resident and migratory water fowl and shorebirds, the invertebrates, fish, and marine mammals -- which live in the marsh or that partially depend on this aquatic nursery for sustenance? How do they react to the wastewater? Is what is clean enough for the water chemists viable for aquatic life, too?

LBL researchers led by Applied Science's Susan Anderson recently asked these unconventional questions, collecting water from the Hayward marsh and monitoring aquatic species cultured in these samples. They documented declines in the reproductive rates of some species and saw other organisms go belly up. The water chemistry results not withstanding, the bioassays indicate the water may be acutely toxic to aquatic life.

Environmental hazard evaluation -- assessing not the exposures but their effects -- is a field of science that has been slow to develop. Anderson, an aquatic toxicologist, and Applied Science Environmental Research Group faculty ecologists John Harte aim to change that. They are deploying biological assays to assess hazards to the biota in disappearing habitats such as the San Francisco Bay estuary. And, they are developing a new generation of genotoxicity assays to identify hazards to future as well as current generations anywhere on the globe.

The new assays being developed at LBL will assess genotoxic effects -- chemically-caused, sublethal damage which can be transmitted from one generation to the next. The assays are being designed to evaluate the hazards in a wide range of environments including contaminated air, water, soil, and sediments. Practical and versatile, LBL's new screening tools should allow the field of environmental hazard evaluation to take root and grow.

Despite an undisputed need, genotoxic damage resulting in effects such as cancer, decreased reproductive success, and genetic mutations is rarely assessed in the environment.

"Up until now," says Anderson, "genetic toxicologists have evaluated the effect of manmade chemicals on human health, but not on wildlife and the environment. Scientists monitor the Alaska oil spill, for instance, and can report that fish are dying, but not how much genetic damage is being transmitted to future generations."

The first step in predicting future effects is developing a more precise understanding of how contaminants are harming present wildlife populations. Often, scientists know what is lethal. But, they do not understand the sublethal biological effects resulting from typical exposures, which are chronic, low-level, and often involve a mix of toxicants.

San Francisco Bay provides a classic example. The bay has been the subject of environmental attention for several decades. Yet, the most basic of questions remain unanswered about the health of this major west coast ecosystem.

For instance, while there is ongoing concern about potential harm to bay life from toxicants, specific scientific evidence of biological harm has been rare. Anderson and Harte recently completed a year's research investigating the bay's toxicity. Their San Francisco Bay Project, which also involved Research Associates Erika Hoffman and David Steward, substantiates the potential effects to aquatic life from exposure to bay waters. Conducted with extant assay techniques, the project includes the first comprehensive study of the biological effects of effluent discharge into the bay's marshes. In addition to marshes, the San Francisco Bay Project also surveyed the toxicity of the bay's open waters.

Researchers report that toxic effects were evident in four of the five marshes surveyed. The most toxic conditions observed -- widespread death of fish and decreased reproduction of other species -- were at the Hayward Shoreline.

Bioassays conducted in open water site samples indicate that moderately toxic water may be widespread in the bay. The cause of this toxicity -- whether a pollutant or natural substances -- has not been identified.

Previously, only a few biological assessments of the bay's toxicant levels have been performed. Relying on adult striped bass as an indicator species, these studies have been the subject of widespread debate among aquatic toxicologists.

"Rather than focus on just one species," says Anderson, "we attempt to estimate the risk of adverse effects for the broad community of species in the bay. We want to know what is the level of toxicity at which there is little likelihood of hazard to the aquatic life. Predictions of toxicity based on just one species during its adult stage are very limited. Granted, you can't test all the resident species, or even the groups. But, we can test a genetically diverse range of species, and test during different stages of their life spans."

LBL's bay monitoring project involves three diverse groups of species that fill different niches in an ecosystem -- silverside minnow larvae, marine mollusk embryos, and sea urchins and sand dollars (echinoderms) gametes -- and examines different stages of their life spans. Fathead minnows, mysid shrimp and water fleas are used at some freshwater marsh sites. No such family of bioassays has ever been applied to San Francisco Bay.

The bioassay for each group of species differs, but all species are cultured in water that is brought to the laboratory from specific open water or marsh sites. Individual bioassays variously assess the effects of bay water on fertility, growth, survivorship, and number of offspring.

In the fish assay, Hoffman and Steward cultured silverside minnow larvae in beakers of bay water. Each day, they exchanged the beaker water with new water drawn from the sample site and fed the larvae to saturation. After a week, they recorded the number of survivors and weighed the biomass of the fish and that of a control group, providing a comparative measure of the growth rate in bay water.

The bioassay performed on mollusks is designed to detect developmental anomalies. To induce spawning, researchers place the mollusks in a pan of warm sea water, and fertilize the eggs with sperm. Then, they expose the embryos to bay water samples for 48 hours, preserve them, and examine the specimens under a microscope for normal and abnormal development.

The echinoderms track the effect of bay water on fertility, recording its toxicity to sperm and egg cells. Sand dollars or sea urchins are spawned, then their eggs and sperm are added to bay water samples for an hour. Following that, researchers use a microscope to ascertain whether fertilization has been successful.

In characterizing the toxicity of the bay's open waters, researchers chose sample sites they believe typify the overall quality of bay waters. Twelve sites were tested ranging from the Carquinez Strait in the north bay to below the Dumbarton Bridge in the south bay, each some distance from any known pollutant discharge point so as not to skew the open water study.

The battery of bioassays was repeated four times, recording the cycle of seasonal conditions in the open bay waters over the course of a year. Minnows and mollusks thrived in the waters taken from all 12 stations, but the same waters caused a pattern of fertility decreases in the sea urchins and sand dollars that ranged up to 100 percent.

"Studies elsewhere demonstrate that, just as we found in San Francisco Bay, ambient toxicity often can be widespread, yet only detectable in assays with one species. We know some effluents are very, very toxic to one species, and totally innocuous to others. Again," says Anderson, "this variability reinforces the need to use a diverse range of species when assessing an ecosystem's toxicity."

Researchers do not know what toxicant(s) in the bay cause the fertility declines in the echinoderms. When the bioassay was repeated using day-old water samples rather than water fresh from the bay, no toxicity was observed, indicating the substance(s) degrade rapidly.

In addition to the open water studies, researchers looked at the bay's marshes. Marshes are thought to be the Earth's most productive nurseries of life, yet over the past 150 years, diking, draining and filling have reduced the tidal marshes on the margins of San Francisco Bay until only about five percent of its marshes remain. Almost all are affected by effluent from among the 43 municipal wastewater plants and the 19 large industries that discharge into the bay.

Four of the five marshes surveyed are in the south bay in the vicinity of discharges from Silicon Valley. Said to be the most contaminated area of the bay, the south bay is polluted with levels of cadmium, copper, mercury, lead, nickel, silver, selenium and other toxicants that periodically exceed state and federal laws. To make matters worse, the south bay has poor tidal flushing and the biological effects of toxicants are not as easily diluted there.

The Hayward Shoreline marsh was surveyed twice. Scientists observed acute toxicity in several of the species assayed with the toxicity evident in over half of the basins sampled in the marsh.

Subsequently, a followup evaluation attempted to identify the cause of the toxicity. Chemical tests indicate that at least a portion of the toxicity is attributable to inadequate treatment for ammonia at the Union Sanitary District wastewater plant which discharges into the marsh, even though the plant's effluent complies with environmental regulations.

At the Mountain View Sanitary District marsh near Martinez, also a marsh reclamation project, researchers observed minor levels of toxicity. Likewise, minor toxicity was detected at the San Francisco Bay National Wildlife Refuge. At the marsh near the San Jose municipal wastewater plant, scientists found no toxicity even though some 80 million gallons of effluent are discharged daily into the marsh. However, in water taken from the marsh near the Sunnyvale wastewater plant, bioassays recorded decreased reproduction of water fleas and decreased fertilization of sea urchins.

Researchers caution that the marsh studies were restricted to assaying conditions during a period of less than one week, and that toxicity, much like effluent quality, can vary over time. Additionally, they note that contaminants often settle and concentrate in muddy sediments, and their assays did not evaluate the toxicity of sediments.

While the battery of bay bioassays focus on short-term effects, the new genotoxic assays under development at LBL are tools with which to look into the future.

Currently, environmental scientists and policy makers must resort to crystal-ball gazing or guesswork when it comes to predicting the effects of chronic, low-level exposure to a mix of toxicants on future generations of fish and wildlife. Anderson, Harte, and Research Associate Gillian Wild's multi-year effort to develop practical new genotoxic assays for the environment could help change that. If successful, these assays will allow questions to be asked that today remain not only unanswered but unaddressed. Will a marsh that is next to a sewage plant and that is alive with birds, fish, and invertebrates remain healthy generations into the future? Is chronic exposure to the contaminated soil and water around a landfill site causing mutations or lowered birth and survival rates that won't show up for a decade? Will removing the heavy metals from an effluent stream be sufficient to stop a decline in fish populations? And, how clean is clean enough in an environmental remediation effort? The new genotoxic bioassays are part of the Superfund Program Project, a nationwide program of basic research to aid in the cleanup of hundreds of the most contaminated waste sites in America. Sites targeted for cleanup by the federal Superfund program include landfills, mines, manufacturing sites, as well as Department of Energy nuclear weapons facilities. LBL and the University of California at Berkeley play an important role in the research aspect of the cleanup. The work, which aims at developing better means to characterize health and environmental risk, is directed by Applied Science Division Berkeley faculty researcher Martyn Smith.

Anderson's group is evaluating and developing various bioassays using a species of nematode (a cylindrical worm) called Caenorhabditis elegans. Biologists have assembled an archive of genetic information about this species including a partial though extensive map of its genome. Current sequencing of the C. elegans genome -- the effort is the invertebrate equivalent of the Human Genome Project -- and the fact that it can live in air, water, or soil makes the animal an ideal subject for assaying.

Scientists rely on a repertoire of existing genotoxic assays to predict the effects of industrial chemicals, food additives, pesticides, and other toxicants on humans. But genetic damage to forests, fisheries, and wildlife is not being assessed because of the inherent limitations of current assays. Existing genotoxic assays include yeast cultures, mammalian cell tests, and the Ames (salmonella bacteria) test developed by the Cell and Molecular Biology Division's Bruce Ames.

Various limitations impede the ability of existing assays to model risk to aquatic and terrestial species. The Ames test, for example, examines the response of bacteria, members of a simple family of life forms called prokaryotes which do not have cell nuclei and which are fundamentally different from the more complex forms of life called eukaryotes which have membrane-bound nuclei, like birds, fish, and mammals. The mammalian cell test is ostensibly more applicable to higher life. But, single cells do not easily model multi-generational effects.

Anderson's group hopes the nematode assays will avoid these shortcomings. They involve whole, eukaryotic organisms. Adult nematodes can reproduce another adult in 3 1/2 days, so studies of multi-generational effects can be accomplished quickly. Unlike existing genotoxicity assays which can take months and years to complete, these new assays can provide results within a period of a few weeks. Scientists have been seeking such short term assays for two decades.

Additionally, nematode assays offer the unique ability to model native environments.

Says Anderson, "The assays we are developing are unique in that we hope to test air, water, effluent, sediments, sludge, and soil. Other assays lose value because they lack this versatility. For instance, you can't put Drosophila (fruit flies) under water and expect them to live. Our nematodes are ubiquitous in nature, and can survive in all kinds of media."

Safety to laboratory personnel is another advantage of the nematode assay. Existing assays for aquatic life can involve buckets of toxicants, whereas LBL's nematode assays use four milliliters of solution.

Caenorhabditis elegans -- researchers call them "the worm" -- are among the most widely studied organisms in genetics. Consequently, over a thousand genetically-distinct strains or "mutants" have been identified. Many have readily identifiable characteristics which visibly distinguish them. Toxicants can induce mutations that can alter these characteristics, allowing researchers to detect mutations.

Among the many strains isolated and studied by geneticists are strains in which "crossing over" is suppressed over a large region of the genome. Crossing over is the normal process by which new combinations of genes are passed on to progeny. Suppressing this process greatly reduces spontaneous mutations. Working with crossover-suppressed strains which are exposed to toxicants, scientists can be confident that the vast majority of mutations that occur in the targeted region of the genome are induced. This approach increases the sensitivity of an assay to toxicants, and allows testing for very low concentrations.

Currently, researchers are developing a battery of bioassays, comparing the virtues of different test strains.

One prospective assay involves a mutant worm, called unc54, that is partially paralyzed, or nonmotile.

Nonmutant nematodes that move normally have a gene called unc54. When this gene is disabled as in the case of the mutant worm used in this assay, the nematode is partially paralyzed.

The assay records mutations to a targeted region of DNA that plays a role in disabling the unc54 gene. Exposing these worms to mutagenic agents can alter these targeted genes, short-circuiting the genetic activity responsible for disabling the unc54 gene. Because the unc54 gene is no longer disabled, such mutations result in progeny worms that are no longer paralyzed.

"It's very simple to distinguish the motile from the nonmotile worms," says Anderson. "Looking through a microscope at a sample plate with both, it's like staring down on a slow-moving crowd. One character can be seen zipping through the crowd, like a purse- snatcher in Manhattan."

Like the unc54 test, all of the nematode assays use mutants that have targets which code for visibly evident characteristics. This allows researchers to determine the frequency of mutations in offspring worms simply by looking through a microscope.

"By using a microscope to examine the progeny, we can tell by the appearance that they either have or do not have induced mutations," Anderson says. "We don't need to analyze the DNA because we are looking for visible mutations. These new assays are not just interesting laboratory experiments, but may become practical environmental tools. The genetics are difficult to comprehend, but the assay itself is relatively simple to perform. To determine the mutation frequency, you just look through a microscope and count the mutants and nonmutants. "

In the unc54 assay, the potential for induced mutations is relatively high because the target region is commensurately large. Despite this, researchers must pinpoint the frequency with which unc54s spontaneously revert to moving worms in order for the assay to have a high degree of accuracy or sensitivity in measuring toxicant-induced mutations. Tests are underway to determine the rate of spontaneous reversions. Optimally, less than one worm in ten million will revert spontaneously.

If the unc54 meets this criterion for sensitivity, the assay will allow researchers to accurately predict a dose-dependent increase in mutation frequency. Thus, they can correlate the dose of an environmental contaminant to the number of mutants produced when a given population is exposed.

Four other prospective worm assays also are being developed. Researchers are assessing the respective sensitivities of these screens, determining the frequency of both spontaneous and induced mutations.

Quantifying the rate of induced mutations from specific toxicants is a prelude to the studies of ecosystems and Superfund sites planned for the near future. Various doses of individual contaminants, many that are energy-related and known to be genotoxic such as PCBs and ionizing radioactive waste, are being assayed. Prior studies have defined the doses that kill, but the doses that cause sublethal effects remain unknown. LBL's assays gradually will help fill this major void of knowledge about the long-term effects of pollution.

Anderson and Applied Science researcher David Littlejohn, for example, are investigating the genotoxic effects of ultraviolet light.

Exposures to ultraviolet light are increasing for most life forms due to the depletion of stratospheric ozone. Scientists know that the consequent incidence of human skin cancer will increase, but many potential ecological ramifications have not been explored.

Certain mutant strains of nematode are known to be sensitive to the levels of ultraviolet light exposure which can occur in the region beneath the Arctic ozone hole. Anderson is examining the use of these ultraviolet-sensitive strains as environmental monitors. Sunburn is not the concern here. Rather, the worms can help define how much exposure is necessary before ultraviolet light begins to cause mutations.

Chronic exposures can induce mutations that can surface not only in current but in future generations. Through ultraviolet light studies, researchers are documenting the transmission of mutated traits to 2nd and 3rd generation progeny. From this, new wildlife population models are being developed to estimate the frequency with which specific mutant traits and genes will recur in future generations, and to project changes in population levels.

Next year, the group's research will evolve from assessing single toxicants to characterizing the effects of low levels of a mix of toxicants. Mixed toxicants are the rule rather than the exception at waste sites. Yet, they remain a realm about which little is known. Conventional chemical analysis provides scant insight into the potential genotoxic effects from the contaminants mixed in effluent from a wastewater treatment plant, or in an underground plume from a landfill, or in the water from a contaminated aquifer.

Versatile and practical, LBL's new generation of genotoxicity bioassays are designed to help resolve the complex toxicological and genetic mysteries posed by 20th century environmental contamination. Whether it's the air in our cities, the soils in agricultural belts, or the waters of San Francisco Bay, these screens can document the hazards and define the long-term biological consequences.

What is more, the bioassays can assist in the cleanup of the environment. Environmental remediation is a notoriously inexact art. Defining a pollution problem does not guarantee that the solution chosen will be successful. But with the feedback that will be available from bioassays, engineers can model or track a remediation effort, gauge its effectiveness, and guide it to success.

Funding statement:

The San Francisco Bay Project was supported by the Environmental Protection Agency and the State Water Resources Control Board. The Superfund Basic Research Program is funded by the National Institute of Environmental Health.