A simulation study of extrapolation uncertainty in exposure assessment - Use of pilot study results for site investigation.

Accurate characterization of soil contaminant concentrations is often crucial for assessing risks to human and ecological health. However, fine-scale assessments of large tracts of land can be cost prohibitive due to the number of samples needed. One solution to this problem is to extrapolate sampling results from one area to another unsampled area. In the absence of a validated extrapolation methodology, regulatory agencies have employed policy-based techniques for large sites, but the likelihood of decision errors resulting from these extrapolations is largely unexplored. This study describes the results of a simulation study aimed at guiding environmental sampling for sites where extrapolation concepts are of interest. The objective of this study is to provide practical recommendations to regulatory agencies for extrapolating sampling results on large tracts of land while minimizing errors that are detrimental to human health. A variety of site investigation scenarios representative of environmental conditions and sampling schemes were tested using adaptive sampling when collecting discrete samples or applying incremental sampling methodology (ISM). These simulations address extrapolation uncertainty in cases where a Pilot Study might result in either false noncompliance or false compliance conclusions. A wide range of plausible scenarios were used that reflect the variety of heterogeneity seen at large sites. This simulation study demonstrates that ISM can be reliably applied in a Pilot Study for purposes of extrapolating the outcome to a large area site because it decreases the likelihood of false non-compliance errors while also providing reliable estimates of true compliance across unsampled areas. The results demonstrate how errors depend on the magnitude of the 95% upper confidence limit for the mean concentration (95UCL) relative to the applicable action level, and that error rates are highest when the 95UCL is within 10%-40% of the action level. The false compliance rate can be reduced to less than 5% when 30% or more of the site is characterized with ISM. False compliance error rates using ISM are insensitive to the fraction of the decision units (DUs) that are characterized with three replicates (with a minimum of 10 percent), so long as 95UCLs are calculated for the DUs with one replicate using the average coefficient of variation from the three replicate DUs.

Effect of soil particle size and extraction method on the oral bioaccessibility of arsenic.

Recent findings indicate that incidental ingestion of soil by humans primarily involves soil particles <150 µm, rather than <250 µm-sized fraction previously used for most oral bioaccessibility and bioavailability studies. It was postulated that a greater soil surface area in the finer fraction (<150 versus <250 µm) might increase oral bioaccessibility of arsenic (As) in soil. Bioaccessibility and concentrations of As were compared in <150 and <250 µm fractions of 18 soil samples from a variety of arsenic-contaminated sites. The two methods used to measure bioaccessibility were compared - EPA Method 1340 and the California Arsenic Bioaccessibility (CAB) method. Arsenic concentrations were nearly the same or higher in the <150 fraction compared with <250 µm. EPA Method 1340 and the CAB method presented significantly different bioaccessibility results, as well as estimated relative oral bioavailability (RBA) based upon algorithms specific to the methods, but there was no marked difference for <150 and <250 µm soil fractions within either method. When compared with RBA determined previously for these soil samples in vivo in non-human primates, EPA Method 1340 was generally more predictive than the CAB method. Data suggest that soil- or site-specific factors control bioaccessibility under either method and that the test method selected is more important than the particle size fraction (<150 or <250) in using these in vitro methods to predict As RBA for use in risk assessment.

Dose-response assessment for impaired memory from chronic exposure to domoic acid among native American consumers of razor clams.

Domoic acid (DA) is a marine neurotoxin that accumulates in filtering shellfish during harmful algal blooms. A health protection limit of 20 ppm DA in razor clams (RC) has been set based principally upon an episode of acute DA toxicity in humans that included Amnesic Shellfish Poisoning among survivors. The objective of this study was to determine the dose-response relationship between estimated DA exposure through RC consumption and memory loss in Washington state Native Americans from 2005 to 2015. Results from total learning recall (TLR) memory scores were compared before and after the highest DA exposures. A decrease in TLR was related to DA dose (p < 0.01) regardless whether the effect was assumed to be transient or lasting, and whether the dose was expressed as an average daily dose or an average dose per meal. Benchmark dose modeling identified BMDL10 values of 167 ng/kg-day and 2740 ng/kg-meal assuming a transient effect, and 196 ng/kg-day and 2980 ng/kg-meal assuming no recovery of function occurs. These DA dose thresholds for a measurable memory function reduction observed in this study of clam consumers are well below the safe acute dose underpinning the current regulatory DA limit of 20 ppm (ca. 60 µg/kg).

Test-Retest Reliability of a Food Frequency Questionnaire to Assess Seafood Intake Dynamics for High-End Consumers in Coastal Gulf of Mexico Communities.

Background: Estimates for fish and shellfish intake are used to inform communities and healthcare systems about potential health risks and benefits for individuals, communities, and vulnerable populations. A dietary assessment instrument was designed for use in populations of high-end consumers of seafood to examine intake of finfish, shrimp, oysters, and blue crab in coastal communities across the Gulf of Mexico. Objective: To validate the reliability of a novel food frequency questionnaire (FFQ) for seafood intake. Design: Test-retest reliability of the FFQ, which included a species-specific photographic portion guide, was evaluated by the inperson administration and readministration of the instrument with each participant by the same interviewer. Responses from coastal and noncoastal participants were compared to discern FFQ reliability in heterogeneous samples. Participants/setting. A convenience sample of 27 coastal participants from Cedar Key, Steinhatchee, and Apalachicola, Florida, reported data for 101 household members; and 15 noncoastal participants from Gainesville, Florida, reported for 42 household members. Analysis. Repeated measures from the FFQ were evaluated using correlation concordance for continuous variables (age, weight, and height) and kappa coefficient for categorical variables (type, amount, and frequency of seafood consumed). Results: Concordance correlation coefficient (1.00) and kappa coefficient (r = 0.73 to 1.00) for yearly and seasonal seafood consumption indicated substantial to almost perfect reproducibility, i.e., participants provided responses that were reproducible. Test-retest agreement was highest for coastal participants who consumed more seafood, as compared to occasional, noncoastal consumers, based on the intergroup comparison of kappa coefficients for yearly and seasonal seafood consumption (r = 0.69 to 0.99). Conclusions: The seafood FFQ instrument evaluated in this study, included as a supplement to this report, used in tandem with a photographic portion guide, provides a utilitarian tool for assessing fish, shrimp, oyster, and blue crab intake dynamics in adult and youth populations drawn from coastal communities.

Probabilistic risk assessment for high-end consumers of seafood on the northeastern Gulf coast.

The Deepwater Horizon oil spill (April 20, 2010) caused concern regarding Gulf seafood safety. Communities were skeptical of governmental risk assessments because they did not take into account the higher consumption of seafood along coastal areas. The objective of this study was to perform a probabilistic risk assessment based on the consumption rates of high-end consumers of Gulf seafood. We utilized seafood consumption data from five communities across the northeastern Gulf of Mexico. This study collected finfish, shrimp, blue crab, and oysters from these communities and analyzed their tissues for polynuclear aromatic hydrocarbons (PAHs). A probabilistic risk assessment was performed using population-specific seafood consumption rates and body weights for commercial fishers, recreational fishers, and a Filipino-American community. For non-cancer effects, 95th percentile hazard quotients for these targeted populations ranged between 1.84E-04 to 5.39E-03 for individual seafood types. The 95th percentile hazard indices for total seafood consumption ranged from 3.45E-03 to 8.41E-03. Based on total seafood consumption, highest hazard indices were modeled for the Filipino-American community followed by commercial and recreational fishers. Despite higher consumption rates, hazard indices for the high-end consumers targeted in this study were two to three orders of magnitude below the regulatory limit of 1.

Derivation of a chronic reference dose for perfluorohexane sulfonate (PFHxS) for reproductive toxicity in mice.

Perfluorohexane sulfonate (PFHxS) is a six-carbon perfluoroalkyl sulfonic acid that was used as an industrial surfactant, but is now found as an environmental contaminant worldwide. In addition to its use as an industrial surfactant, it is a legacy contaminant from the use of aqueous film-forming foams. Despite its widespread occurrence in the environment and evidence of biological activity associated with PFHxS and similar perfluoroalkyl sulfonic acids in rodents, there is no oral toxicity value currently available from the IRIS Database. To derive an oral reference dose (RfD) for PFHxS, available toxicity studies were reviewed using a weight-of-evidence approach. A 42-day mouse reproductive study was chosen as the critical study for the derivation of the oral RfD. Benchmark dose modeling was utilized to derive a point of departure (POD) for a reduction in litter size. A 95% lower confidence limit on the benchmark dose (BMDL) of 13,900 ng/mL (serum PFHxS) was modeled for a reduction in litter size. An oral RfD for PFHxS of 4.0 ng/kg/d was calculated by conversion of the BMDL to a human equivalent oral dose using a human half-life adjusted dosimetric conversion factor and the application of a total uncertainty factor of 300. Additional research is needed to better characterize the toxicity associated with oral exposure to PFHxS and refine the development of toxicity values.

Bioaccessibility of polychlorinated dioxins and furans in soil from a Superfund site.

Bioaccessibilities of PCDD/PCDF congeners contributing to cancer risk were determined in twelve soil samples from the American Creosote Works Superfund site in Florida. Based upon sample locations, congener profiles (i.e., the same dominant congeners), and total (Toxic Equivalent; TEQ) concentrations, each of these samples has PCDD/PCDF contamination reasonably attributable to the site. Bioaccessibility was determined using a 2-phase in vitro extraction method that included both simulated gastric and intestinal conditions of the human GI tract. Measured congener-specific bioaccessibility values ranged from 34.3 to 62.1%. There was no apparent relationship between the extent of chlorination of PCDD/PCDF congeners and their bioaccessibility. TEQ-weighted bioaccessibility values varied among individual soil samples, which is not unexpected based upon the literature. This variability could not be explained by differences in soil pH, composition, or organic carbon content. The average TEQ-weighted bioaccessibility value of 59% for the twelve samples was accepted as representing site-specific bioavailability of PCDD/PCDFs. This value is higher than most dioxin/furan bioaccessibility values reported in the literature and at the upper end of the range of relative oral bioavailability (RBA) values reported for PCDD/PCDFs from in vivo bioavailability studies. This study used a finer fraction of soil particles (<150 microns versus the more typical <250 microns) to better represent soil that is incidentally ingested. This finer fraction would be expected to have a greater surface area available for extraction of PCDD/PCDFs per unit mass, which might account for the greater than expected bioaccessibility.

Incremental Sampling Methodology: Applications for Background Screening Assessments.

This article presents the findings from a numerical simulation study that was conducted to evaluate the performance of alternative statistical analysis methods for background screening assessments when data sets are generated with incremental sampling methods (ISMs). A wide range of background and site conditions are represented in order to test different ISM sampling designs. Both hypothesis tests and upper tolerance limit (UTL) screening methods were implemented following U.S. Environmental Protection Agency (USEPA) guidance for specifying error rates. The simulations show that hypothesis testing using two-sample t-tests can meet standard performance criteria under a wide range of conditions, even with relatively small sample sizes. Key factors that affect the performance include unequal population variances and small absolute differences in population means. UTL methods are generally not recommended due to conceptual limitations in the technique when applied to ISM data sets from single decision units and due to insufficient power given standard statistical sample sizes from ISM.

Analysis of EPA's endocrine screening battery and recommendations for further review.

EPA's Endocrine Disruptor Screening Program Tier 1 battery consists of eleven assays intended to identify the potential of a chemical to interact with the estrogen, androgen, thyroid, or steroidogenesis systems. We have collected control data from a subset of test order recipients from the first round of screening. The analysis undertaken herein demonstrates that the EPA should review all testing methods prior to issuing further test orders. Given the frequency with which certain performance criteria were violated, a primary focus of that review should consider adjustments to these standards to better reflect biological variability. A second focus should be to provide detailed, assay-specific direction on when results should be discarded; no clear guidance exists on the degree to which assays need to be re-run for failing to meet performance criteria. A third focus should be to identify permissible differences in study design and execution that have a large influence on endpoint variance. Experimental guidelines could then be re-defined such that endpoint variances are reduced and performance criteria are violated less frequently. It must be emphasized that because we were restricted to a subset (approximately half) of the control data, our analyses serve only as examples to underscore the importance of a detailed, rigorous, and comprehensive evaluation of the performance of the battery.

Relevance weighting of tier 1 endocrine screening endpoints by rank order.

Weight of evidence (WoE) approaches are recommended for interpreting various toxicological data, but few systematic and transparent procedures exist. A hypothesis-based WoE framework was recently published focusing on the U.S. EPA's Tier 1 Endocrine Screening Battery (ESB) as an example. The framework recommends weighting each experimental endpoint according to its relevance for deciding eight hypotheses addressed by the ESB. Here we present detailed rationale for weighting the ESB endpoints according to three rank ordered categories and an interpretive process for using the rankings to reach WoE determinations. Rank 1 was assigned to in vivo endpoints that characterize the fundamental physiological actions for androgen, estrogen, and thyroid activities. Rank 1 endpoints are specific and sensitive for the hypothesis, interpretable without ancillary data, and rarely confounded by artifacts or nonspecific activity. Rank 2 endpoints are specific and interpretable for the hypothesis but less informative than Rank 1, often due to oversensitivity, inclusion of narrowly context-dependent components of the hormonal system (e.g., in vitro endpoints), or confounding by nonspecific activity. Rank 3 endpoints are relevant for the hypothesis but only corroborative of Ranks 1 and 2 endpoints. Rank 3 includes many apical in vivo endpoints that can be affected by systemic toxicity and nonhormonal activity. Although these relevance weight rankings (WREL ) necessarily involve professional judgment, their a priori derivation enhances transparency and renders WoE determinations amenable to methodological scrutiny according to basic scientific premises, characteristics that cannot be assured by processes in which the rationale for decisions is provided post hoc.

Derivation of a chronic reference concentration for decalin.

Decalin is found naturally in crude oil and as a product of combustion. It is used commercially as a solvent due to its ability to solubilize oils and fats. Despite its widespread occurrence in consumer products and the environment that lead to inhalation exposures, an inhalation toxicity value is not currently available for decalin. To derive a reference concentration (RfC) for decalin, inhalation toxicity studies were reviewed using a weight-of-evidence approach. A 2-year mouse inhalation study was chosen as the critical study for the derivation of the chronic RfC. Benchmark dose modeling was utilized to derive a point of departure for hepatic necrosis, syncytial alteration, eosinophilic focus, and erythrophagocytosis. A BMDL10 of 44mg/m(3) was modeled for the most sensitive adverse effect, syncytial alteration. A chronic RfC for decalin of 0.08mg/m(3) was calculated by conversion of the BMDL10 to a human equivalent continuous inhalation dose of 7.9mg/m(3) and application of a total uncertainty factor of 100. Future research is needed to better characterize the toxicity associated with the chronic inhalation of decalin and refine the development of toxicity values.

Glucuronidation and sulfonation, in vitro, of the major endocrine-active metabolites of methoxychlor in the channel catfish, Ictalurus punctatus, and induction following treatment with 3-methylcholanthrene.

The organochlorine pesticide, methoxychlor (MXC), is metabolized in animals to phenolic mono- and bis-demethylated metabolites (OH-MXC and HPTE, respectively) that interact with estrogen receptors and may be endocrine disruptors. The phase II detoxication of these compounds will influence the duration of action of the estrogenic metabolites, but has not been investigated extensively. In this study, the glucuronidation and sulfonation of OH-MXC and HPTE were investigated in subcellular fractions of liver and intestine from untreated, MXC-treated and 3-methylcholanthrene (3-MC)-treated channel catfish, Ictalurus punctatus. MXC-treated fish were given i.p. injections of 2mg MXC/kg daily for 6 days and sacrificed 24h after the last dose. The 3-MC treatment was a single 10mg/kg i.p. dose 5 days prior to sacrifice. In hepatic microsomes from control fish, the V(max) value (mean+/-S.D., n=4) for glucuronidation of OH-MXC was 270+/-50pmol/min/mg protein, higher than found for HPTE (110+/-20pmol/min/mg protein). For each substrate, the V(max) values observed in intestinal microsomes were approximately twice those found in the liver. The K(m) values for OH-MXC and HPTE glucuronidation in control liver were not significantly different and were 0.32+/-0.04mM for OH-MXC and 0.26+/-0.06mM for HPTE. The K(m) for the co-substrate, UDPGA, was higher in liver (0.28+/-0.09mM) than intestine (0.04+/-0.02mM). Treatment with 3-MC but not MXC increased the V(max) for glucuronidation in liver and intestine. Glucuronidation was a more efficient pathway than sulfonation for both substrates, in both tissues. The V(max) values for sulfonation of OH-MXC and HPTE, respectively, in liver cytosol were 7+/-3 and 17+/-4pmol/min/mg protein and in intestinal cytosol were 13+/-3 and 30+/-5pmol/min/mg protein. Treatment with 3-MC but not MXC increased rates of sulfonation of OH-MXC and HPTE and the model substrate, 3-hydroxy-benzo(a)pyrene in both intestine and liver. Comparison of the kinetics of the conjugation pathways with those published for the demethylation of MXC showed that formation of the endocrine-active metabolites was more efficient than either conjugation pathway. Residues of OH-MXC and HPTE were detected in extracts of liver microsomes from MXC-treated fish. This work showed that although OH-MXC and HPTE could be eliminated by glucuronidation and sulfonation, the phase II pathways were less efficient than the phase I pathway leading to formation of these endocrine-active metabolites.

Stimulation of transactivation of the largemouth bass estrogen receptors alpha, beta-a, and beta-b by methoxychlor and its mono- and bis-demethylated metabolites in HepG2 cells.

The purpose of this study was to determine the mechanisms by which the pesticide, methoxychlor (MXC), acts as an environmental endocrine disruptor through interaction with the three largemouth bass (Micropterus salmoides) estrogen receptors (ERs) alpha, betaa, and betab. MXC is a less-environmentally persistent analog of DDT that behaves as a weak estrogen. Using transient transfection assays in HepG2 cells, we have previously shown that each receptor is responsive to the endogenous ligand 17beta-estradiol (E(2)) in a dose-dependent manner. The parent compound, MXC, showed dose-dependent stimulation of transcriptional activation through all three ERs. In addition to the parent molecule, each of the metabolites was also estrogenic with all three ERs. The order of potency for ERalpha and ERbetab was HPTE>OH-MXC>MXC, while the opposite order was seen for ERbetaa. HepG2 cells did not substantially metabolize MXC to the active metabolites, thus the activity of MXC was not due to metabolism. When examining the effects of increasing concentrations of MXC at a fixed concentration of E(2), all three ERs show increased activity compared to that with E(2) alone, showing that the effects of MXC and E(2) are additive. However, when this experiment was repeated with increasing concentrations of HPTE at a fixed concentration of E(2), the activity of ERalpha was decreased, that of ERbetab was increased, while that of ERbetaa was unaffected compared to E(2) alone. These experiments suggest that HPTE functions as an E(2) antagonist with ERalpha, an E(2) agonist with ERbetab and does not perturb E(2) stimulation of ERbetaa. While it is clear the ERbeta subtypes are the products of different genes (due to a gene duplication in teleosts) the differences in their responses to MXC and its metabolites indicate that their functions diverge, both in their in vivo molecular response to E(2), as well as in their interaction with endocrine disrupting compounds found in the wild.

Demethylation of the pesticide methoxychlor in liver and intestine from untreated, methoxychlor-treated, and 3-methylcholanthrene-treated channel catfish (Ictalurus punctatus): evidence for roles of CYP1 and CYP3A family isozymes.

Exposure to the organochlorine pesticide methoxychlor (MXC) is associated with endocrine disruption in several species through biotransformation to mono-desmethyl-MXC (OH-MXC) and bis-desmethyl-MXC (HPTE), which interact with estrogen receptors. The biotransformation of [14C]methoxychlor was examined in channel catfish (Ictalurus punctatus), a freshwater species found in the southern United States. Hepatic microsomes formed OH-MXC and HPTE, assessed by comigration with authentic standards. The Km for OH-MXC formation by control liver microsomes was 3.8 +/- 1.3 microM (mean +/- S.D., n = 4), and Vmax was 131 +/- 53 pmol/min/mg protein. These values were similar to those of catfish pretreated with 2 mg/kg methoxychlor i.p. for 6 days (Km 3.3 +/- 0.8 microM and Vmax 99 +/- 17 pmol/min/mg) but less (p < 0.05) than the kinetic parameters for catfish treated with 3-methylcholanthrene (3-MC), which had Km of 6.0 +/- 1.1 microM and Vmax of 246 +/- 6 pmol/min/mg protein. Liver microsomes from 3-MC-treated fish produced significantly more of the secondary metabolite and more potent estrogen, HPTE. Intestinal microsomes formed OH-MXC at lower rates than liver. Methoxychlor pretreatment significantly reduced intestinal metabolite formation from 32 +/- 4 to 15 +/- 6 pmol/min/mg (mean +/- S.D., n = 4), whereas 3-MC treatment significantly increased OH-MXC production to 72 +/- 22 pmol/min/mg. Ketoconazole, clotrimazole, and alpha-naphthoflavone all decreased the production of OH-MXC in liver microsomes, whereas alpha-naphthoflavone stimulated HPTE formation, suggesting that CYP1 and CYP3 family isozymes demethylated methoxychlor. The results suggest that the formation of estrogenic metabolites from methoxychlor would be more rapid in catfish coexposed to CYP1 inducers.