Establishing Causality between Exposure to Metals and Effects on Fish

This study evaluates causal relationships between chronic exposure of fish to metals and effects at different levels of biological organization based on a weight-of-evidence approach. Criteria for evaluation of causality were strength, consistency, and specificity of the association, as well as biological gradient and plausibility. Field sampling was conducted three times between 1998 and 2000, in Furnas Stream, impacted by an abandoned lead mine, and in three other locations, including two reference and one impacted sites. Levels of Pb, Zn, Cd, and Ag in sediments from the Furnas Stream exceeded background levels, and their concentrations were above sediment quality guidelines. Residual levels of metals in fish tissue were high enough to indicate reduced growth, reproduction and/or survival according to toxicological benchmarks. Lead-induced biochemical changes (ALA-D activity depletion) were observed in two species of siluriform catfish. The condition factor of a predatory catfish was reduced, and the percentage of prey generalists was higher in Furnas than at the noncontaminated sites. Reduction in fish community diversity and density was observed. Integration of data provided supporting evidence that observed effects on fish from the Furnas Stream resulted from long-term exposure to metals, however influences from other stressors cannot be ruled out.     

Forensic Ecotoxicology: Establishing Causality between Contaminants and Biological Effects in Field Studies

Twelve papers in this series were derived from two conference sessions focusing on causality in field studies. Eight of these papers involve case studies examining biological effects of chemical contaminants in field situations. Using a weight-of-evidence approach, these case studies were evaluated against seven proposed criteria for establishing causality. The seven criteria were: strength of association; consistency of association; specificity of association; time order; biological gradient; experimental evidence; and biological plausibility. One of these seven criteria, ‘specificity of association’ was found to be of little utility for establishing causality in these field studies. The case studies are presented in approximate order of increasing levels of biological organization (i.e., going from endpoints at the suborganismal level to endpoints at the population or community level). In case studies examining higher levels of biological organization, it appears that the ‘biological gradient’ criterion was also not useful in establishing causality. These results, together with suggestions from other papers in the series, are used to recommend a set of modified criteria for establishing causality in field studies of the biological effects of chemical contaminants.     

Determinants of Serum PCBs in Adolescents and Adults: Regression Tree Analysis and Linear Regression Analysis

Regression tree analysis, a non-parametric method, was undertaken to identify predictors of the serum concentration of polychlorinated biphenyls (sum of marker PCB ¹ ABBREVIATIONS: BMI: body-mass index, CV: cross validation, ln: natural logarithm, ns: not significant, PCAHs: polychlorinated aromatic hydrocarbons, PCBs: polychlorinated biphenyls, R² _a: adjusted coefficient of determination, VIF: variance inflation factor. View all notes 138, 153, and 180) in humans. This method was applied on biomonitoring data of the Flemish Environment and Health study (2002–2006) and included 1679 adolescents and 1583 adults. Potential predictor variables were collected via a self-administered questionnaire, assessing information on lifestyle, food intake, use of tobacco and alcohol, residence history, health, education, hobbies, and occupation. Relevant predictors of human PCB exposure were identified with regression tree analysis using ln-transformed sum of PCBs, separately in adolescents and adults. The obtained results were compared with those from a standard linear regression approach. The results of the non-parametric analysis confirm the selection of the covariates in the multiple regression models. In both analyses, blood fat, gender, age, body-mass index (BMI) or change in bodyweight, former breast-feeding, and a number of nutritional factors were identified as statistically significant predictors in the serum PCB concentration, either in adolescents, in adults or in both. Regression trees can be used as an explorative analysis in combination with multiple linear regression models, where relationships between the determinants and the biomarkers can be quantified.     

Establishing Causality between Environmental Stressors and Effects on Aquatic Ecosystems

Establishing causal relationships between environmental stressors and observed effects in natural systems is difficult due to the many intrinsic environmental factors that can hinder this process and because there are no widely accepted and proven approaches for determining such relationships. Several types of approaches or combinations of approaches, each with their own sets of advantages and limitations, have been applied in a variety of ecological systems to investigate possible causal relationships between stressors and effects. These include controlled laboratory studies (including acute and chronic bioassays), experimental field manipulations, field studies based on synoptic field surveys, mathematical simulation modeling, statistical associations, various combinations of laboratory, experimental, and field studies, and the ecoepidemiological (weight or evidence) approach. The use of ecoepidemiological (“forensic toxicology”) principles is becoming increasingly attractive as a method to help establish causality because it does not involve the same limitations of other approaches and it can also be used to integrate disparate information within a logical framework so that scientifically and defensible regulatory decisions can be made. The objective of this Commentary series of papers on the issue on causality is to demonstrate the application of the ecoepidemiology approach, using a variety of case history studies, for establishing causal relationships between specific stressors and biological effects. For each case history provided in the following series of papers, the authors describe their study situation, summarize the results supporting a causal relationship, and then compare their study results against seven standard causal criteria.     

Relationship between Measures of Exposure to PCBs/Dioxins and Behavioral Effects in Recent Developmental Studies

This paper does not reflect official EPA policy. Epidemiological studies on the neurodevelopmental effects of exposure to PCBs initiated in the last decade have had the opportunity to take advantage of modern methodologies for the analysis of congeners of PCBs, dioxins, and related orga-nochlorine compounds. Each of these studies is a longitudinal prospective study, in which women were recruited during pregnancy and the children are being followed for at least several years after birth. The study from which the largest body of data has been published to date is being performed in the Netherlands, in which exposure to PCBs and related compounds is through the general food supply. Mother-infant pairs were recruited in two cities. Half of the infants were bottle-fed and half breast-fed in each city. Four PCB congeners (118, 138, 153, 180) were assessed in maternal and cord plasma, breast milk, and plasma of the child at 3.5 years. TEQ in breast milk was calculated based on PCDDs/PCDFs and dioxin-like PCBs. Various measures of in utero exposure were associated with suboptimal neurological status during infancy, whereas maternal plasma PCB concentration was associated with cognitive deficits (Kaufman scores) at 3.5 years of age. The child's concurrent plasma PCB levels and maternal PCB plasma levels independently predicted performance on various aspects of a vigilance task, and maternal and cord plasma levels predicted impairment of complex play behavior. Poor scores on behavioral ratings were associated with concurrent blood PCB concentrations in the child. A study in Oswego in Lake Ontario fish eaters includes mothers who never ate Great Lakes fish and mothers who consumed greater than 40 PCB-equivalent pounds of Lake Ontario fish over their lifetime. Sixty-eight PCB congeners were measured in cord blood. Suboptimal neurological status during infancy was associated with maternal fish consumption and highly chlorinated cord PCB levels, whereas deficits in short-term memory at 6 months and 1 year of age were associated with total chlorinated cord PCB levels. In a study in Germany of 171 mother-infant pairs, PCB congeners 138, 153, and 180 were measured in cord plasma and milk 2 weeks after birth; both measures are considered markers of in utero exposure. Suboptimal neurological status during infancy, decrements in Bayley scores at 30 months and Kaufman scores at 42 months were associated with PCBs in milk but not cord plasma. These studies, combined with data from previous studies, reveal a consistent relationship between PCB exposure and suboptimal neurological status during infancy, and cognitive deficits associated with in utero exposure. Data from the Dutch study revealed effects on other behavioral domains associated with concurrent (postnatal) exposure. Although it is not possible to identify specific congeners or groups of congeners that may be responsible for the neurotoxic effects observed in these studies, the TEQ approach was not particularly predictive for neurotoxic outcomes.     

Establishing Causality between Pollution and Effects at Different Levels of Biological Organization: The VALIMAR Project

The study summarizes the objectives of the VALIMAR project and gives selected examples of biomarker responses that allow causal relationships to be established between exposure and biological effects at different levels of biological organization. In this project, active and passive biomonitoring experiments with brown trout (Salmo trutta f. fario) and stone loach (Barbatula barbatula) were performed in two small streams in southern Germany between 1995 and 1999 in parallel with investigations on contaminant mixtures in the laboratory in order to evaluate the suitability of biomarkers representing different levels of biological organization for the assessment of pollution in small streams. In addition to biomarker studies, the morphology of the test streams was characterized and limnological and chemical parameters were monitored. Early life stage tests and ecological studies of brown trout and stone loach population demography, of the fish assemblages, and the macro- and meiozoobenthos communities in the two test streams were included in the project. Several causality criteria were addressed by means of combined (1) laboratory and field studies, (2) chemical, biological, and statistical investigations, and (3) in vivo and in vitro studies that allowed establishment of cause-effect relationships at different biological levels. The comparison of results obtained at these levels allowed identification of mechanisms responsible for the respective effects (coherence of association, biological plausibility). Finally, individual responses (biomarkers, bioindicators) could be extrapolated to higher biological levels (population, community) thus addressing the criteria of ‘time order’ and ‘coherence of association’.     

Establishing the Causal Relationship between Polycyclic Aromatic Hydrocarbon (PAH) Exposure and Hepatic Neoplasms and Neoplasia-Related Liver Lesions in English Sole (Pleuronectes vetulus)

For almost 25 years our laboratory has studied the impact of PAHs and related industrial contaminants on benthic fish, following an interdisciplinary approach involving chemical exposure assessment linked to synoptic detection of various effects at several levels of biological organization. These data demonstrate a cause-and-effect relationship between neoplastic and neoplasia-related liver lesions in English sole, and exposure to PAHs, and to a lesser degree, chlorinated hydrocarbons such as PCBs. In statistical analyses of data from multiple field studies conducted since 1978, exposure to PAHs measured in various compartments has consistently been identified as a highly significant, major risk factor for neoplasms and related lesions in this species, with PCB exposure shown to be a significant, but less consistent and less strong risk factor for these lesions. A cause-and-effect relationship between PAHs and toxicopathic liver lesions in this species is further supported by the experimental induction of toxicopathic lesions identical to those observed in field-collected fish, in sole exposed in the laboratory to model carcinogenic PAHs such as BaP or to PAH-rich extracts of sediments from Eagle Harbor, a severely PAH-contaminated site in Puget Sound. More recent field studies have identified significant associations between hepatic cytochrome P4501A (CYP1A) induction and xenobiotic-DNA adduct formation, and hepatic lesion prevalences in wild subadult English sole. Field studies in Eagle Harbor subsequent to capping of the most PAH-contaminated region of this harbor with clean dredge spoils have shown a decline in exposure to PAHs as assessed by biliary fluorescent aromatic compounds (FACs) and hepatic xenobiotic-DNA adducts. This decline in PAH exposure has been accompanied by a dramatic decline in risk of occurrence of toxicopathic hepatic lesions in English sole from Eagle Harbor. Further, laboratory studies have induced lesions in English sole by injections of extracts from PAHcontaminated sediments. Overall, these findings relating to exposure to PAHs and chlorinated hydrocarbons and the occurrence of hepatic neoplasms and neoplasiarelated lesions in English sole fulfill the classic criteria for causality in epizootiological or ecological risk assessment studies, including: (1) strength of association, (2) consistency of association, (3) specificity of association, (4) toxicological and biological plausibility, (5) temporal sequence/timing (i.e., exposure precedes disease, effect decreases when the cause is decreased or removed), (6) dose-response or biological gradient, and (7) supportive experimental evidence.     

Association between exudates of brown algae and polychlorinated biphenyls

Exudates from the brown algaeCaepidium antarcticum andDesmarestia sp. were investigated for their ability to associate with hydrophobic pollutants such as polychlorinated biphenyls (PCB s). The percentage of PCB associated with algal exudates ranged from 79% for decachlorobiphenyl to 23% for the pentachlorobiphenyl congener No. 95. Exudates from the tested brown algae may therefore alter the bioavailability of PCBs in natural or artificial ecosystems.     

Using SPMDs to Assess Natural Recovery of PCB-contaminated Sediments in Lake Hartwell,SC: I. A Field Test of New In-Situ Deployment Methods

Results from the field testing of some innovative sampling methods developed to evaluate risk management strategies for polychlorinated biphenyl (PCB) contaminated sediments are presented. Semipermeable membrane devices (SPMDs) were combined with novel deployment methods to quantify the availability of PCBs at the Sangamo-Weston Lake Hartwell Superfund Site in SC. Three locations in Lake Hartwell were examined: a background site (BKG) with little detectable contamination and two contaminated sites (T-M/N and T-O). PCB availability was quantified using sediment surface samplers designed to hold SPMDs in contact with surface sediments, benthic dome samplers designed to enclose and suspend SPMDs at the sediment-water interface, and commercially obtained SPMD cages suspended in the water column. A two-way analysis of variance showed significant effects by sampler type (P < 0.0001) and site (P < 0.0001) for mean time-weighted average (TWA) total PCBs (t-PCBs). Regardless of the SPMD sampler used, mean TWA t-PCBs were directly proportional to the level of contamination present (BKG < T-M/N < T-O). Water column and surface sediment sampler t-PCB uptake patterns were described by a significant linear fit of the data (r² = 0.9625, p < 0.0001, and r² = 0.8188, p < 0.0001, respectively). Dome and sediment surface SPMD samples had a higher percentage of higher chlorinated PCBs compared to water column samples.     

Short- and Long-Term Exposure of Lumbriculus variegatus (Oligochaete) to Metal Lead: Ecotoxicological and Behavioral Effects

Metals are naturally occurring constituents of the environment and although many are essential nutrients for living organisms, at higher concentrations they can be toxic. Some aquatic species can help understand and even predict the impact of those contaminants. Lumbriculus variegatus is a recommended species for use in sediment toxicity tests and is known to have a remarkable ability of segmental regeneration. Short- (10-day) and long-term (28-day) toxicity tests were used to test the effects of a metal on the survival, growth, and behavior of L. variegatus. This work aims to investigate and validate the use of behavior as a new parameter in standard toxicity tests. Worms were exposed to sediments contaminated with different levels of lead and the results indicated a positive relation between lead concentrations and mortality and growth: higher lead concentrations resulted in higher mortalities and strong inhibition of growth. An inhibition of behavior was observed and results suggested that although behavior could not be used in sediment toxicity tests, it proved useful as an addition to short-term tests and helps select sediments. Thus, exposure to sediments contaminated with lead affects the presence of this species in nature, because it interferes with growth and survival.     

Evidence for a Causal Link between Exposure to an Insecticide Formulation and Declines in Catch of Atlantic Salmon

We report a case study showing relationships between historical insecticide applications (1973 to 1990) of an aminocarb formulation (Matacil® 1.8D ) containing 4-nonylphenol (4-NP) and catch data for Atlantic salmon (Salmo salar) populations. To address the hypothesis that Matacil® 1.8D caused reductions in Atlantic salmon catch, seven epidemiological criteria are used to evaluate causality between the stressor and effect. These criteria include: strength of association; consistency of association; specificity of association; time order; biological gradient; experimental support; and biological plausibility. We conclude that the general weight-of-evidence supporting the seven epidemiological criteria is consistent with a causal relationship between declines in salmon catch and Matacil® 1.8D exposure during parr-smolt transformation (PST). If the effects exerted by 4-NP are due to its activity as a weak estrogen, then hormonal activity stemming from other sources (e.g., domestic sewage, agricultural, industrial) might influence present day salmon populations.     

The Lab-to-Field (LTF) Rating Scheme: A New Method of Investigating the Relationships between Laboratory Sublethal Toxicity Tests and Field Measurements in Environmental Effects Monitoring Studies

The Lab-to-Field (LTF) rating scheme is a straightforward method of relating the results of effluent toxicity tests to the field survey measurements and has proven to be a useful tool for interpretation of Environmental Effects Monitoring studies for pulp and paper mills in the province of Ontario. The LTF method uses the same five-level scale (level 1 for no or low response to level 5 for severe response) for rating the toxicity and field survey results. Regression analysis of LTF scores has revealed that the relationship between the Ceriodaphnia reproduction test and benthic invertebrate field survey measurements was significant (p < 0.001, r = 0.79). However, there were not sufficient data to determine if this can be used as a predictive tool. Nonetheless, Ceriodaphnia-to-benthic survey, Selenastrum-to-benthic survey and fathead-to-fish survey relationships were qualitatively rated strong or moderately strong in 94%, 75%, and 60% of the 16 studies, respectively. The LTF rating scheme would benefit from the use of a more sensitive species or life stage of fish to strengthen the sublethal test-to-fish survey relationship. Further validation of the lab-to-field relationship could be accomplished through the synoptic collection of effluent samples for sublethal tests and field measurements.     

Using a stem cell and progeny model to illustrate the relationship between cell cycle times of in vivo human tumour cell tissue populations, in vitro primary cultures and the cell lines derived from them

There is increasing evidence that the growth of human tumours is driven by a small proportion of tumour stem cells with self-renewal properties. Multiplication of these cells leads to loss of self-renewal and after division for a finite number of times the cells undergo programmed cell death. Cell cycle times of human cancers have been measured in vivo and shown to vary in the range from two days to several weeks, depending on the individual. Cells cultured directly from tumours removed at surgery initially grow at a rate comparable to the in vivo rate but continued culture leads to the generation of cell lines that have shorter cycle times (1–3 days). It has been postulated that the more rapidly growing sub-population exhibits some of the properties of tumour stem cells and are the precursors of a slower growing sub-population that comprise the bulk of the tumour. We have previously developed a mathematical model to describe the behaviour of cell lines and we extend this model here to describe the behaviour of a system with two cell populations with different kinetic characteristics and a precursor–product relationship. The aim is to provide a framework for understanding the behaviour of cancer tissue that is sustained by a minor population of proliferating stem cells.