ES/RP 532 Applied Environmental Toxicology Page 1 of 14. October 27, Dioxins: Biological Hazards and Risks

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1 ES/RP 532 Applied Environmental Toxicology Page 1 of 14 October 27, 2004 Lecture 16 Dioxins: Biological Hazards and Risks I. Acute Effects A. High exposures of TCDD results in several characteristic syndromes: 1. Chloracne 2. Severe body weight loss known as the wasting syndrome 3. Atrophy of lymphoid tissue 4. Atrophy of gonads B. If pregnant rats are exposed during a specific time in pregnancy, teratogenic effects, most notably cleft palate, are observed in the offspring. C. The acute effects follow weeks of dosing before they appear D. Nearly all toxic effects are now believed to occur following binding to the aryl hydrocarbon receptor (the Ah receptor). E. The Ah receptor is associated with two proteins known as heat shock 90 (hsp90) and another protein known as AIP (AhR interacting protein). (Based on Denison and Heath- Pagliuso 1998, Bull. Environ. Contam. 61:557; and Gonzalez and Fernandez-Salguero 1998, Drug Metabolism Disposition 26:1194) 1. When a ligand (for ex., TCDD or the normal endogenous, but unknown, ligand) binds to the Ah receptor, the hsp90 and AIP are cleaved from the receptor, and the ligand-ah receptor crosses the nucleus and binds with another protein called ARNT (aryl hydrocarbon nuclear translocator).

2 ES/RP 532 Applied Environmental Toxicology Page 2 of The liganded AhR-ARNT complex is the high affinity binding form that binds to the dioxin responsive elements (DRE) upstream of the CYP1A1 gene. The gene is transcribed eventually producing new copies of the enzyme, P4501A1. F. Exactly what happens after induction of P4501A1 synthesis is unknown, but an array of tissue specific and biological responses are noted: (from Denison and Heath-Pagliuso 1998) G. Using null-ahr bred mice, it is now know that the Ah receptor is important in at least several physiological functions. 1. Null-AhR mice develop physiological abnormalities affecting the immune system and the liver. a. One metabolic role of Ah receptor is believed to be induction of P4501A1 to hydroxylated retinoic acid. 1. In the absence of retinoic acid metabolism, it builds up and causes it to be metabolized to retinyl esters, removing it from normal biochemical pathways that utilize the compound. (Fernandez-Salguero 1998) II. Toxicokinetics A. Dermal & Oral Absorption 1. Extent of both dermal and oral absorption are influenced by the vehicle of exposure; (all studies below cited in Olson p. 192 in Dioxins & Health, Schecter & Gasiewiecz, ed. John Wiley & Sons) a. For example, rodent studies have used corn oil as a vehicle for gavage exposures 1. In these studies, oral absorption efficiency (i.e., intestinal absorption in to the blood (as eventually measured by accumulation in organs) is very high (ranging from 66-94% in rats 2. A human volunteer was dosed with TCDD in corn oil, and more than 87% of the oral dose was absorbed from the GI tract. 3. When guinea pigs were dosed with TCDD contaminated soil and compared to TCDD administered in corn soil, TCDD absorption from soil was only 50% of that absorbed from corn oil vehicle.

3 ES/RP 532 Applied Environmental Toxicology Page 3 of 14 b. The extent of oral absorption is also influenced by congener type. 1. Studies with rats show similarly high oral bioavailability of 2,3,7,8- tetracchlorodiobenzofuran, but only 2-15% of the administered oral dose of OCDD (octachlorodibenzodioxin) was absorbed. c. Intraindividual variability has been observed in humans. 1. Schlummer et al. (1998; Toxicol. Appl. Pharmacol. 152: ) observed net absorption of TCDD (ranging from 50% to 87%) among 8 human volunteers eating food with known levels of dioxin residues. 2. However, three volunteers had net excretion of TCDF (2,3,78- tetrachlorodibenzofuran), and five volunteers had a net absorption efficiency of 38-52%. d. When TCDD was applied to rodent skin using acetone solvent, 18% of the dose was absorbed after three days. However, 49% of the administered dose of TCDF was absorbed during the same time interval. In other studies, application of TCDD in a soil-water paste decreased residues showing up in liver to about 2% of the administered dose (i.e., there was a significant reduction in absorption through the skin when compared to TCDD in methanol; in the latter case, the liver contained 15% of the applied dose). B. Metabolism and Excretion 1. It is now known that TCDD is hydroxylated at the 1 and 8 positions; these metabolites were isolated following procedures used to hydrolyze conjugates; excreted dose is mainly in the form of metabolites rather than as parent a. Urinary excretion of metabolites is greater than fecal excretion b. Investigations of TCDD in rats, mice, guinea pigs and hamsters found that >90% of the radiolabelled TCDD excreted in urine and bile represented polar metabolites c. The excretion half-life for a single dose is on the order of about three or four weeks when doses are in the µg/kg range; when doses are in the ng/kg range, excretion half life can be shorter (variable among different rodent species) 2. Storage in the fat and liver; long depuration time after administration of a single dose (most of the administered dose ends up in these two tissues) Half-Life (weeks) of PCDD/Fs After Single Subcutaneous Dose to Marmoset Monkeys (28 µg/kg) Congener Hepatic (Liver) Tissue Adipose Tissue 2,3,7,8-TCDD ,2,3,7,8-PentaCDD ,2,3,4,6,7,8-HeptaCDD OctaCDD ,3,7,8-TCDF ,3,4,7,8-PentaCDF OctaCDF 174 infinity 3. Whether more TCDD ends up in the liver or in the adipose tissue after a single dose depends on the magnitude of the dosage (data from EPA 1992 Draft Dioxin Risk Assessment Document); in the following graph, concentrations were measured 7 days after a subcutaneous injection of TCDD into rats.

4 ES/RP 532 Applied Environmental Toxicology Page 4 of While half-lives for specific organs like liver and adipose tissue can be measured in terms of weeks, half-lives for elimination from the whole body are measured in years a. For example, the Vietnam Ranch Hand study indicated that the half-life of TCDD in the body was 7.1 years; this study was based on 36 subjects b. Other congeners may have a shorter half-life, but the number of individuals studied (many cases 1) is too small to make definite conclusions. III. Carcinogenic Potency of PCDDs A. TCDD is now considered a human carcinogen by the World Health Organization as well as the EPA. 1. EPA estimated that dioxin TEQs exposure within about 3 fold of current background levels may be carcinogenic to humans. 2. However, the evidence is based on high dose (i.e., relative to what humans are exposed to) rodent feeding studies 3. Furthermore, the EPA s epidemiological analysis has been challenged and deemed faulty. (Starr, T. B Significant shortcomings of the U.S. Environmental Protection Agency's latest draft risk characterization for dioxin-like compounds. Toxicological Sciences 64:7-13. & Starr, T. B Significant Issues Raised by Meta-analyses of Cancer Mortality and Dioxin Exposure. Environ Health Perspective 111: B. When unsubstituted dibenzodioxins are tested, they are not very toxic and are not considered carcinogenic C. Higher doses of isomers containing four or more chlorines have been associated with liver tumors D. The table below suggest there are thresholds for tumorigenic responses. Carcinogenic Response Data (Compiled by Kociba, R., 1991, Rodent bioassays for assessing chronic toxicity and carcinogenic potential of TCDD, pp. 3-11, in Banbury Report 35: Biological Basis for Risk Assessment of Dioxins and Related Compounds) Dioxin Congener Species Dose Level Carcinogenic Response unsubstituted rat rat 10,000 ppm in diet 5,000 ppm in diet none none unsubstituted mouse 10,000 ppm in diet none 2,7-dichloro mouse rat rat 5,000 ppm in diet 10,000 ppm in diet 5,000 ppm in diet none none none

5 ES/RP 532 Applied Environmental Toxicology Page 5 of 14 2,7-dichlor 2,3,7,8-TCDD mouse mouse rat (Sprague-Dawley) 10,000 ppm in diet 5,000 ppm in diet 0.1 µg/kg/day suggestive none hepatocellular carcinoma squamous carcinoma of oropharynx & lung 0.01 µg/kg/day hepatocellular nodules µg/kg/day no increase in tumors 2,3,7,8-TCDD rat (Osborne-Mendel) 0.07 µg/kg/day hepatocellular carcinoma, thyroid tumors µg/kg/day questionable increase in thyroid tumors µg/kg/day no increase in tumors 2,3,7,8-TCDD mouse (Swiss) 0.1 no increase in tumors but decreased lifespan 0.01 hepatocellular tumors 2,3,7,8-TCDD mouse (B6C3F1) Males: µg/kg/d no increase in tumors 0.07 µg/kg/day 1,2,3,6,7,8 & 1,2,3,7,8,9 hexachloro mixture 1,2,3,6,7,8 & 1,2,3,6,7,9 hexachloro mixture Females: 0.03 µg/kg/d rat 5, 2.5, or 1.25 µg/kg/wk via gavage mouse Male: 5, 2.5, or 1.25 µg/kg/wk via gavage Female: 10, 5, or 2.5 µg/kg/day 0.3 µg/kg/day none in male rats yes, in female at higher dose yes, at highest dose level E. It is instructive to look at the relationship between dosage, response, and tissue levels of TCDD at termination of a 2-year toxicity study in rats; these studies are used to derive lifetime exposure control limit recommendations; note how large the tissue concentrations are in relation to what we estimate as the body burden; (see the next two tables) Concentrations of TCDD in dosed rats Dose Levels of TCDD Response Terminal TCDD content (ppt, wet weight) µg/kg/day ppt in diet carcinogenicity other toxicity fat liver yes yes 8,100 24, no yes 1,700 5, no no

6 ES/RP 532 Applied Environmental Toxicology Page 6 of 14 Mean TCDD levels (ppt, wet weight basis) from autopsy samples from two humans (from Schecter, A., 1991, Dioxins and related chemicals in humans and in the environment, pp in Banbury Report 35: Biological Basis for Risk Assessment of Dioxins and Related Compounds) Analyte Abdomen Subcutaneous Adrenal Liver Muscle Spleen Kidney TCDD ND 1.3 ND Total PCDDD Total PCDD TEQ % Lipid ND = no detected TCDD (i.e., below detection limit) 1. Note that the ppt above are expressed on a wet weight basis; a lot of tissue analysis for PCB, TCDD, and DDT are based on a lipid basis (normalization procedure); a. To obtain the lipid normalized ppt, divide the wet weight ppt by the fraction of the tissue as lipid (for ex., in the abdomen there is 73% by weight lipid; the lipid based ppt is 6.6/0.73 = ~9 ppt). IV. Potential Carcinogenicity of TCDD A. TCDD is not considered genotoxic, but EPA historically classified it as a B2 carcinogen. 1. According to the currently used system for carcinogen classification, a B2 carcinogen is characterized by the following criteria: a. Sufficient evidence of carcinogenicity from animal studies, with inadequate or no epidemiologic data. b. The next highest category, B1, is characterized as sufficient evidence of carcinogenicity from animal studies with limited evidence of carcinogenicity from epidemiologic studies. 1. The highest category, A, or human carcinogen, is characterized as sufficient evidence from epidemiologic studies to support a causal association between exposure to agents and cancer 2. Currently EPA s classification as a human carcinogen is based on its estimates of excess cancer risks of ~0.001 (i.e., 10-3 ) per picogram TCDD TEQs kg body wt. per day. a. In other words, for every picogram of TCDD TEQs in a body, there is a 1 per 1000 increased risk of contracting cancer. b. The overall epidemiological incidence estimated is 3 excess cancers per 100!!! B. Historically, the International Agency for Research on Cancer (IARC), an international body that deliberates the carcinogenic potential of synthetic chemicals reviewed the evidence for human carcinogenicity of dioxin 1. IARC found the prevailing evidence for humans to be inadequate. However, for animals, the evidence was sufficient (see table above). They concluded-- In the absence of adequate data on humans, it is reasonable, for practical purposes, to regard chemicals for which there is sufficient evidence of carcinogenicity in animals as if they presented a carcinogenic risk to humans. 2. Regarding the animal data, at the highest doses tested, tumors were found in rat and mouse livers (hepatocarcinomas); a. Closer examination of the tissues revealed that dioxin was causing cellular toxicity, which gave a higher count of tumor cells than really existed. b. Furthermore, the evidence of hepatotoxicity could explain the increased incidence of tumors at the high doses (note that the low dose tests gave no indication of tumors)

7 ES/RP 532 Applied Environmental Toxicology Page 7 of According to Brown, W. R., (1991, Implications of the reexamination of the liver sections from the TCDD chronic rat bioassay, pp , in Banbury Report 35: Biological Basis for Risk Assessment of Dioxins and Related Compounds), The toxicity factor is very important with regard to the biology and significance of these liver tumors. Toxicity results in cell death and inflammation of the liver. When it is at a degree in which the animal survives, then there is an attempt to compensate by replacing these cells by regeneration. In this regenerative attempt, the cells continually divide, which contributes to tumor development by allowing increased expression of spontaneous errors in DNA replication and also enhanced tumor promotion and progression. C. During 2001, WHO (United Nations World Health Organization) declared 2,3,7,8-TCDD a known human carcinogen (despite the evidence shown above). D. EPA calculates the carcinogenic potency of chemicals, and has concluded that TCDD is the most potent carcinogen, despite the above hypothesis (which were generated after experimentation, i.e., collection of data that seem to support the epigenetic mechanism of tumor induction) 1. Note however, that the risk model used by EPA (1986) leads to a plausible upper limit to the risk that is consistent with some proposed mechanisms of carcinogenesis. Such an estimate, however, does not necessarily give a realistic prediction of the risk. The true value of the risk is unknown, and may be as low as zero. (This sentence was taken from the EPA guideline as quoted by Gough 1991, see subtopic E below) a. The risk characterization model was further challenged by Starr 2001, 2003 (see citations above) E. Soft tissue sarcoma (STS) is one of the forms of cancer that was initially thought to be linked to TCDD exposure. 1. This is an incredibly rare form of cancer, so any increase in incidence could be cause for concern (even though the numbers would still be low) 2. The possibility of an association came from studies in Sweden by Hardell, Erickson, and coworkers (the original study: Hardell, L., and A. Sandstrom, 1979, Case-control study: soft-tissue sarcomas and exposure to phenoxyacetic acids or chlorophenols. British Journal of Cancer 39: ; other studies followed, for ex. Hardell, L.,1981, On the relation of soft tissue sarcoma, malignant lymphoma, and colon cancer to phenoxy acids, chlorophenols and other agents. Scand. J. Work Environ. Health 7: ); a. Using case-control studies, they showed an odds-ratio of 6-7 for STS in men exposed to phenoxyacetate herbicides, especially 2,4,5-T, which was known to be contaminated with TCDD. 1. Exposure assignments were based on questionnaires mailed to cases and controls or next of kin and sometimes supplemented by telephone interviews 2. Exposure was positive if the men had reported at least one day s exposure at least five years before their cancer was diagnosed 3. Studies of agricultural workers in Kansas showed a link between use of phenoxyacetate herbicides (presumably including 2,4,5-T and therefore TCDD as a contaminant) and increased risk of Non-Hodgkins Lymphoma but not STS (Hoar, S. K. et al., 1986, Agricultural herbicide use and risk of lymphoma and soft-tissue sarcoma. J. Am. Med. Association 256: ) 4. As it turns out, numerous other studies never confirmed the Swedish studies, and even the Swedish authors published a study in 1988 that seemed to contradict their earlier findings of an association between STS and chlorophenols exposure (Hardell, L., and M. Eriksson, 1988, The association between soft-tissue sarcomas and exposure to phenoxyacetic acids. Cancer 62: )

8 ES/RP 532 Applied Environmental Toxicology Page 8 of 14 Odds Ratio (95% C. I.) Hardell & Sandstrom 1979 Eriksson et al Smith et al Hoar et al Vineis et al Woods et al Healty Non-STS Cancer Hardell & Eriksson T phenoxyacetates Eriksson et al (redrawn from Gough 1991) In, (Gough, M., Dioxin: perceptions, estimates, and measures. pp in Phantom Risk: Scientific Inference and the Law, Foster, K. R. et al. (eds.), MIT Press, Cambridge, MA) F. The most scathing report refuting the link between carcinogenicity and TCDD exposure was written by Michael Gough (1991 and others). 1. Reference: Gough, M Human health effects: what the data indicate. The Science of the Total Environment 104: Example table from a study that failed to show a relationship between soft tissue sarcomas (STS) and exposure to phenoxy herbicides (Smith and Pearce, 1986, Chemosphere 15: ) Estimates of Odds Ratios for Exposure to Phenoxy herbicides in New Zealand STS Studies First Study Second Study Combined Studies No. of cases No. of controls No. of exposed cases No. of exposed controls Odds ratios Confidence limits (90%)

9 ES/RP 532 Applied Environmental Toxicology Page 9 of Gough argues that industrial workers in manufacturing plants have had the highest exposures. By comparison, Vietnam War Veterans exposures were much lower. Yet in all these groups, there is generally no excess mortality nor excess incidence of various cancers. a. In known exposed populations, where chloracne (a sure sign of TCDD exposure) has occurred, there have been no excess illnesses or cancers. Exposed Population Number of People Range (ppt) Mean (ppt) U. S. New Jersey Missouri Calculated One- Time Median Dose German workers Total w/ chloracne Seveso children highest level 1 56,000 w/ chloracne ,821 21,873 3,125 no chloracne ,439 4, G. Gough (1993) calculated that the mean dose to the Seveso children (the site of the industrial plant explosion in Italy that spewed kg quantities of TCDD over the town) to be equivalent to 3.1 µg/kg. 1. This dose is about three times as high as the 1 µg/kg dose that is the LD 50 to guinea pigs. 2. So far as is known, there are no illnesses or disease other than chloracne among any of the Seveso population. a. This finding is consistent with the idea that people are not so sensitive to the toxic effects of dioxin as are some animals. 3. In Hardell s studies, dioxin concentrations in cases with soft tissue sarcomas showed no significantly elevated levels. H. Gough (1991) seems almost willing to concede that perhaps there is some relationship between phenoxyacetate herbicide use and Non-Hodgkins lymphoma (NHL), however, increased NHL has not been observed among workers who manufactured the herbicides. 1. B. MacMahon, an epidemiologist, stated to Congress in 1988 that if sarcomas and NHL are associated with pesticide applications, there should be an epidemic of these diseases among industrial populations that have been exposed at much higher levels (Gough 1993). I. Kociba (1991), a Dow Chemical toxicologist who published one of the earlier studies suggesting a link between rat tumors and TCDD in the diet now believes that the prevailing evidence does not support the conclusion that TCDD is carcinogenic (Kociba, R., 1991, Rodent bioassays for assessing chronic toxicity and carcinogenic potential of TCDD. pp. 3-11, Banbury Report 35: Biological Basis for Risk Assessment of Dioxins and Related Compounds, Cold Spring Harbor Laboratory Press) 1. Other researchers at the Banbury Conference agreed with Kociba a. A more realistic assessment would consider dioxin tumorigenicity to be related to hepatotoxicity, and thus there is a threshold for an effect; 1. Studies show that tumors are present in rat and mice only when the TCDD dietary levels caused some liver toxicity

10 ES/RP 532 Applied Environmental Toxicology Page 10 of The prevailing hypothesis is that TCDD is actually a promoter as opposed to a tumor initiator. 3. When a no-threshold hypothesis is used, the acceptable intake is related to the risk of one excess cancer in a million a. For ex., a dose of pg/kg/day is the no-threshold based excess one in a million cancer risk estimate for TCDD 4. By assuming a threshold for tumors, the maximal daily intake can be calculated, and several European countries have determined this value to be 1-10 pg/kg/day J. Starr (2001)conducted a meta analysis using TEQs body burden data that EPA had used to estimated increased incidence risk of cancer 1. In the graph from his 2001 paper in Toxicological Sciences, he showed that there is no slope (and thus no relationship between body burden TCDD and increased incidence of cancer) 2. Furthermore, note how the SMRs (i.e., odds ratios) for a number of the studies had 95% CIs (confidence intervals) that were below 1 (especially note the CI when the body burden was greater than 1000) V. Developmental Effects of Dioxins A. Structural defects following dioxin exposure have been reported in the mouse at doses that do not cause either maternal or fetal toxicity (Birnbaum, L. S., 1995, Development effects of dioxins. Environ. Health Perspectives 103 [Supplement 7]:89-94) 1. The best described malformation is cleft palate in mice a. As with many teratogenic effects, there is a critical window for the induction of this defect, with peak incidence following exposure on day 11 or 12 of gestation (70% incidence in a line of mice known as a dose of 12 µg/kg in 10 ml of corn oil/kg by gavage on gestation days 6, 8, 10, 12, and 14) b. Note that there is a threshold effect with doses from 0-4 µg/kg showing essentially no response c. Note also that potency among PCDD and PCDF congeners differs from TCDD:

11 ES/RP 532 Applied Environmental Toxicology Page 11 of 14 Relative Potency of Chlorinated Dioxin (CDD) and Dibenzofuran (CDF) Congeners in Inducting Teratogenic Effects Congener Relative Potency TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) TBDD (2,3,7,8-tetrabromodibenzo-p-dioxin) TCDF (2,3,7,8-tetrachlorodibenzofuran) TBDF (2,,3,7,8-tetrabromodibenzofuran PeCDF (1,2,3,7,8-pentachlorodibenzofuran) PeBDF (1,2,3,7,8-pentabromodibenzofuran) PeCDF (2,3,4,7,8-pentachlorodibenzofuran) P PeBDF (2,3,4,7,8-pentabromodibenzofuran HCDF 1,2,3,4,7,8-hexachlorodibenzofuran) HBN (1,2,3,4,6,7-hexabromonaphthalene) ,3,4,5,3,4 -HCB (hexachlorobiphenyl) In contrast to cleft palate, the induction of hydronephrosis does not appear to have a peak window of sensitivity during organogenesis and can even be induced lactationally. a. Hydronephrosis is a more sensitive response than cleft palate with thresholds less than 0.5 µg/kg/day VI. Endocrine System Effects of TCDD and Dioxin Congeners A. Safe has shown that dioxins are actually antiestrogenic in experiments similar to those reported for PCB and exposure of mammalian breast tumor cell cultures 1. TCDD inhibits multiple estrogen (E2) -induced responses in rodent uterus and mammary and in human breast cancer cells. E2 induced responses include development or growth of human mammary and endometrial cancer cells, carcinogen-induced mammary cancer in rats, and mammary cancer in mice bearing breast cancer cell xenografts. The mechanisms of AhR-mediated antiestrogenicity are complex; however, studies on the molecular biology of cross-talk between the AhR and estrogen-receptor ER signaling pathways have been initiated using several E2-regulated genes as models. The results indicate that the nuclear AhR complex targets specific genomic core inhibitory dioxin responsive elements (idres) in promoter regions of some E2- responsive target genes to inhibit hormone-induced transactivation. a. Above information from Safe, S., F. Wang, W. Porter, R. Duan, and A. McDougal Ah receptor agonists as endocrine disruptors: antiestrogenic activity and mechanisms. Toxicology Letters : B. One interesting effect noted for dioxins exposure is an altered sex ratio. 1. In cohorts of people exposed to different amounts of dioxin in Seveso, Italy, the site of an industrial plant explosion during the early 1970 s, the parents of offspring exposed to the greatest levels gave birth to an unusually low number of boys compared to the parents further away from the explosion. a. One hypothesis to explain these results is that dioxin exhibits antiandrogenic effects and induces a phenomenon called post-ovulatory over-ripeness pathology. (Jongbloet, P. H., N. Roeleveld, and H. M. M. Groenewoud Where the boys aren't : dioxin and the sex ratio. Environ. Health Perspectives 110:1-3.) 2. A recent study of pesticide applicators in Russia who may have been exposed to dioxin contaminated products also gave birth to a bias of girls (Ryan, J. J., Z. Amirova, and G. Carrier Sex ratios of children of Russian pesticide producers exposed to dioxin. Environ. Health Perspectives 110(11):A699-A701.)

12 ES/RP 532 Applied Environmental Toxicology Page 12 of 14 VII. Potential Developmental Effects in Wildlife (see article by Larson et al. 1996) A. Larson et al. (1996) Reproductive success, developmental anomalies, and environmental contaminants in double-crested cormorants (Phalacrocorax auritus). Environmental Toxicology & Chemistry 15: ) 1. Ecoepidemiology is a relatively new area of study that tries to link some population incidence of a malady in wildlife with exposure to persistent contaminants (or any type of contaminant). a. Focusing on the most persistent contaminants is most promising, because they will be bioaccumulated and stored; 2. In this paper, Larson et al. tried to link the presence of a cormorant bill deformity (crossed bills) and hatching success with the tissue levels of PCBs and TCDD-TEQs (i.e., TCDD equivalents based on their known penchant for inducing the enzyme known as EROD (ethoxyresorufin-o-dethethylase); 3. While their were significant differences in tissue concentrations (i.e., in eggs) collected on a contaminated and pristine island, no correlation could be made with hatching success nor incidence of crossed bill syndrome. B. Jung, R. E., and M. K. Walker Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on development of Anuran amphibians. Environmental Toxicology & Chemistry 16 (2): Many scientists believe that amphibian populations worldwide are in decline. Needless to say, one hypothesis is exposure to chemical contaminants, and those most likely to bioaccumulate are most suspect. 2. However, as implied in the appended abstract to the article by Jung and Walker, exposure to the most toxic dioxin isomer does not appear to be a likely cause. VIII. The Stew Thickens--the EPA Dioxin Reassessment Report A. During 2000, the EPA released its dioxin reassessment report (which was considered a final draft ), many years in the making since its release first draft in ~1992 and second draft in ~1994. The agency concluded that the risk from dioxins exposure is greater than they previously thought, paving the way for more regulation. 1. Naturally, food alone is the largest exposure source. EPA (2000) estimates of residues of chlorinated dioxins (chlorinated dibenzodioxins and dibenzofurans) and PCBs in food items. Food Type CDD/CDFs (pg TEQ/g fresh weight) PCBs (pg TEQ/g fresh weight) Total (pg TEQ/g fresh weight Beef Pork Eggs Chicken Milk Dairy products Marine fish Freshwater fish Marine shellfish Vegetable fats Water (pg/l) NA NA B. The EPA report has not gone unchallenged, however; but environmental advocacy groups agree with its premise.

13 ES/RP 532 Applied Environmental Toxicology Page 13 of 14 C. A main premise of the EPA report is that some health effects are observed at estimated body burden levels close to the average human background body burden level. D. Health Effects of Dioxins in Humans & Animals (after a 1995 ES&T article): The exposure levels at which health effects from dioxin-like compounds are observed vary widely among species. A sampling of the studies presented in the EPA dioxin health assessment indicates that some health effects are observed at estimated body burden levels close to the average human background body burden level. Putative Biological Effects of Dioxin Exposure and Estimated Associated Body Burden Effect Species Estimated body burden of Dioxin (ng/kg or ppt) associated with effects Background level Human 9 Causally associated with Dioxin Exposure Chloracne Human Monkey 1,000 Rabbit 220 Mouse 14,000 Associated with Dioxin Exposure Cancer Human Hamster 500 Mouse 1,000 Decreased testosterone Human 83 Decreased testis size Human 14 Rat 10,200 Altered glucose tolerance Human Low-Dose Effects in Animals Endometriosis Monkey 54 Decreased sperm count Rat 64 Decreased offspring viability Monkey 270 Enhanced viral susceptibility Mouse 7 E. Thus, the issue of daily intake is raised; the EPA estimates daily intake as follows: EPA (2000) estimates of exposure (intake) per day for different age categories. Age Range Intake, mass basis (pg TEQ/day) Intake, body weight basis (pg TEQ/kg/day) 1-5 years years yr Adult 70 1 F. The sources of exposure and estimated intakes are shown next. 1. Note that the intake rates also reflect exposure to PCBs known to have dioxin-like activity (the planar PCBs would be the likely compounds).

14 ES/RP 532 Applied Environmental Toxicology Page 14 of 14 EPA (2000) estimates of contact rates (mass per day) concentrations (as TEQs), and intake for dioxins, dibenzofurans, and PCBs. Chlorinated Dioxins & Dioxin-like PCBs furans Exposure route Contac t Rate Concentrati on (TEQs) Intake (pg TEQ/kg/day Concentrati on (TEQs) Intake (pg TEQ/kg/day Total Intake (pg TEQ/kg/day Soil ingestion 50 mg/d 12 pg/g NA NA Freshwater fish 6 g/d 1.2 pg/g pg/g Marine fish 12.5 g/d 0.36 pg/g pg/g Marine 1.6 g/d 0.79 pg/g pg/g shellfish Inhalation 13.3 m 3 /d 0.12 pg/ m NA NA Milk 175 g/d pg/g pg/g Dairy 55 g/d 0.12 pg/g pg/g Eggs pg/g pg/ g g/kg-d Beef pg/g pg/g g/kg-d Pork pg/g pg/g g/kg-d Poultry pg/g pg/g g/kg-d Vegetable fat 17 g/d pg/g pg/g Water 1.4 L/d pg/l NA NA Total (45 pg/d) 0.35 (25 pg/d) 1.0 (70 pg/d) G. The estimated serum levels in adults (median and 95 Th percentile concentration) are shown below. Thus, comparing the estimated serum levels to the putative relationship between body burdens and several health effects reported in 1995, the EPA has sounded an alarm bell on dioxin TEQ exposure (and echoed loudly as a rallying cry by certain environmental advocacy groups led by Greenpeace). EPA (2000) estimate of distribution of dioxin TEQs in human sera TEQ (pg/g lipid) TCDD (pg/g lipid) Median Mean th Percentile H. Note that the TDI (tolerable daily intake) is now considered (by European guidelines) to be 4 pg TEQ/kg bw. 1. On a fat basis, this would be about 6 pg TEQ/g milk fat.