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From Last Week Another Type of Envl EpidemicInvestigation: Cancer Clusters An epidemic with unusual spatial patterns Only a handful of cases (of many thousands) of reportedcancer clusters have been considered to be likely due toenvl exposures (Woburn MA [TCE,PCE]; Toms River NJ[TCE and others]; Camp LeJeune NC; Hinckley CA [Cr(VI)])

These were all related to contaminated ground water Why so few?

High background rates of cancer Random occurrences of spatial clustering must be exceeded Long latency periods of cancer requires long retrospective exposure

assessment (So which bias happens?) Workplace clusters are more common due to higher exposuresand greater ease of accurately estimating exposures

Which Spatial Samplings are Random?

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1

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Example: Incidence of Childhood Leukemiahttp://serc.carleton.edu/woburn/overarching/cancer_clusters.html

Childhoodleukemia fairlyrare.

Incidence inWoburn aboutdouble whatwas expected,but 7x higherin EasternWoburn.

Another Kind of Clustering? Cancer Cluster Simulation UsingBirthdays

We plotted your birth dates on a calendargrid

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Birthdays to Simulate Cancer ClustersWillOnly 50 Birth Dates Cluster and Match on a

Grid of 365 Dates?

Birth dates are rather rare and nearly random, similar tocancer (which is random in both space and time) butclusters in time are a kind of snap shot.

When birthdates plotted in calendar format, this simulatesspatial randomness quite well

How many matches should we see? How many clustersof three birthdays in three days?

ANY GUESSES?

RESULTS SHOW EXCEL SHEET

The Problem with Drawing Boundaries After the Fact

5.5x4.9x

2.4x

4.6x

Overall gridrate is asexpected

Clustersshown arehighlyelevated ratescompared toexpected rates

Immigrantsrates of cancerstart out withlarge disparitiesthat disappearafter twogenerations in anew country

Nevertheless, Evidence for EnvironmentalImpact on Cancer Rates is Strong

Birthday Odds: Easiest to MultiplySuccessive Odds of Each New Birthday

Not being a MatchReason for this is that there are many combinations(and separate odds) of matching but only a singlecalculation for never matchingThis also applies to any strange coincidences of rareevents, but it is impossible to add up all the possiblecoincidences that dont happen.Equation:

If you Want to Win a BetThe 50-50 even odds break point is only 23 people(Collect any bet money before you win and be prepared toexplain the math)

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Determining the causes in any increased rates ofrare events of long latency requires a very largesample size or an extremely large increase overexpected rates for any geographic geographiclocationunlike acute events such as the LondonSmog or the Barcelona asthma epidemics

They also require lots of evidence beyond merelyassociation, e.g., Hills Criteria

When it comes to rare illnesses

Intro To Toxicology: How Do WeKnow What Toxins to Worry About?

Scott FruinPM529, Jan 28, 2015

Slides marked RS courtesy of Theresa RyanStueve, Sept 23, 2014

Examples of Policy and Health Questions Addressedby Toxicology (and Exposure Assessment)

Is my tap water safe?

Are there harmful pesticides in myvegetables? Should I buy organic?

Are plastic food containers or toysunhealthy for me or my children?

If I am pregnant, should I stop eatingfish?

Examples of Policy and Health Questions Addressedby Toxicology (and Exposure Assessment)

Is my tap water safe? Yes, especially if you filter it, but withone possible exception: arsenic

Are there harmful pesticides in my vegetables? Should Ibuy organic? Yes but depending on the type of vegetable or fruit, usuallynot of health concern. Buying organic helps, but some F & V more importantto buy organic than others

Are plastic food containers or toys unhealthy for mychildren? Sometimes yes; varies by plastic type, use andcondition

If I am pregnant, should I stop eating fish? No, butimportant to choose low Hg fish (non-predator fish)

The Role of Toxicology and ExposureAssessment

Toxicology (the science of poisons) tells you whatlevels of contaminants are harmful

Exposure assessment tells you if you are likely to exceedthese levels or not (which helps prioritize behaviorchoices and guide policy making and regulation).

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Another Way to Look at it

AmbientMeasurementsand Modeling

ExposureAssessment

Toxicology

Outline Principles of toxicology

Mechanisms of toxicityhow andwhere do the most important toxicharms occur?

Dose-responsewhat is the relationshipbetween amount (dose), risk and harm? How do we determine this?

Example toxicants: endocrine disruptors

Trends in toxicity: are concerns gettingbetter or worse? What you can do

What Makes a Substance Toxic? The Dose Makes the

Poison is commonly citedas a fundamentaltoxicological concept Dose is a singleadministration of a cpd

Major assumption is: Health effects higher as doseincreases (monotonic)

The problem is, this maynot be true for allchemicals!

http://www.nanotortlaw.com/2012/05/31/

Toxicity: Large Differences between CpdsCompared to Beneficial or Non-Toxic Range

Toxic tonon-toxiclimit ratio

2x

2-3x

7x

15x

3.5x

Range

100s ofgrams

ppm inair

mg

g

0.1 g

Factors Affecting Toxicity Chem Properties

Chemical form, e.g., Cr(VI) vs Cr(III) Ability to be absorbed:

Fat soluble cpds can cross cell membranes Amount and timing

Total dose / dosage over time: Longer time per dose or between dose is less toxic

Distribution within the body: target organs? Species, age, sex:

Very young and old can be more vulnerable Ability to metabolize a cpd.

Route of exposure: skin < gastrointestinal tract < lungs

Lung exposures bypass the liver and go directly to blood, unlike GI Skin a relatively good barrier

The Major Routes of Exposure to Toxins Inhalation: lungs have huge surface area and thin

epidermal layer where gas exchange occurs Water soluble, high vapor pressure cpds can readily enterblood stream

Particle pollution deposits in lungs; the smaller the particle,the deeper in the lung and the more difficult to remove

Ingestion: food or water Gut lining: fat soluble cpds quickest to adsorb, but adsorptionaided by active processes (see later slide)

Dermal: skin a good barrier but can absorb fat-solublecpds

No contact: Ionizing (highest energy) radiation canpenetrate skin and damage DNA

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A. Passive Transport

no energy required;down gradient.

Fat-soluble

2) FacilitatedDiffusion

(B) open pores (urea)or

(C) saturable proteincarrier-mediatedtransport, downgradient(glucose).

3) Active transport

up gradients throughsaturable proteincarriers

Types of Cross-Membrane Transport

Adapted from 2013 Biology with Miss Buchheit, http://biologywithmissbuchheit.com/cell-transport.php

A) small polarB)fat-soluble polarC)

O2, benzene water, urea glucose MeHg-L-cysteine

saturatedand

inhibitedsaturated

andinhibited

porecarrier

D)

Very Simply

Biological Processes that Affect ToxicityExposure to chemical

Absorption

Free form Bound form

translocation & Distribution

Storage sites(Fat)

Metabolism(Biotransformation)

sites

Toxic sites

Excretion toxicity

Toxicaction

Protein binding

UCB NST110, D. Nomura

Fat Soluble Also referred to as lipophilic (fat loving) or

hydrophobic

Fat soluble means low solubility in water

Allows easy absorbing across cell membranes(membrane wall is two layers of phospholipids)

Tend to be non-polar compounds

What Makes a Compound Polar? Uneven distribution of electrons

and orbitals give a molecule anaturally uneven chargedistribution or polarity Not as strong as an entire missingor extra charge

Because water is very polar,other polar compounds mix well(hydrophilic) and are watersoluble These compounds are not soreadily absorbed (except

through lungs) and are rapidlyfiltered from the blood by thekidneys

Non-Polar Compounds Symmetrical and tightly-held electrons, often halogen

group (Cl, Br, F) Tend to be very stable bonds, often environmentally persistent

Not very water soluble More easily absorbed through skin and gut Harder for body to removeneed to be converted to

more soluble cpds Tend to build up in fat; tend to bioaccumulate in food

chains The worst of these are called POPs, Persistent Organic

Pollutants and many are banned, such as organochlorinepesticides like DDT

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Mechanisms of Toxicity (a few of many) Direct cell death Disruption of protein synthesis or enzyme function

(metal ions such as Cd, Hg, Pb) Disruption to mitochondria fxn and ATP synthesis

(DDT and other OC pesticides, CCl4, Hg, Acetaminophen) DNA damage

(ionizing radiation, nitrosamines from cured meats) Lipid peroxidation (CCl4) Carbohydrate metabolism (TCE) Nerve signal transmission (organophosphate pesticides) Hemoglobin binding to oxygen (CO) Interfering with hormone fxn (endocrine disruptors) Interference with nutrient uptake or utilization

Injuries Range from Temporary to Permanent Skin Inflammation organ birth deathIrritation damage defects

The body can and will heal itself if the toxic dose is lowenough: Dose is a single administration of a toxin;

Generally the more spread out a dose is in time, the less toxic it is(exception is some carcinogens)

Temporary Permanent

Categorizing Toxicities by Organ Liver a common site since responsible for majority of

detoxification including drugs and alcohol* Kidneys vulnerable as the bodys primary blood filtering

system, esp. proximal tubules E.g., cadmium very toxic to kidneys

Lungs vulnerable due to air pollution, esp. particles Particles irritating, cause inflammation; metals can cause oxidative

stress DNA also a type of system since vulnerable to mutations

that can propagate and potentially turn cancerous Nerve cells: vulnerable since slow to regenerate

*(Liver metabolism of fructose and alcohol essentially the same; highfructose intake during weight gain appears to increase liver /visceral fatgain);

Other Ways to Categorize Toxicity By broad, related systemic effects

Mutagenicity Via in vitro tests

Carcinogenicity: Initiators: direct damage to DNA Promoters e.g., hinder tumor suppression genes

Neurological Reproductive:

Rapidly dividing cells vulnerable ; also, EDC effects on hormones Immunological Developmental: fetal growth very sensitive

Toxins Have Always Been with Us Products of incomplete combustion from fire (particulate

matter, polycyclic aromatic hydrocarbons [PAHs], carbonmonoxide [CO], dioxinsvery long list) Similar to cigarettes Cooked food: e.g., PAHs from burned fat, acrylamide in toastedstarches

Naturally occurring pesticides in plants, e.g., caffeine! Byproducts of mold, bacterial breakdown

One major worry now is that many of the man-madetoxins behave differently, as we will discuss

How the Body De-Toxifies Itself Water soluble cpds generally filtered from the blood by the

kidneys Less water soluble cpds are converted to soluble via Phase I

and Phase II metabolism, mostly in the liver. Phase I adds or exposes functional groups to make more polar, soluble,

but mostly to allow further Phase II is conjugation of larger molecules(see next slide)

Direct storage of lipophilic cpds in body fat, e.g., DDT, PCBs,etc. results in slow removal from the body

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Phase I and II Metabolism

http://ocw.jhsph.edu/courses/PublicHealthToxicology/PDFs/Lecture2_Trush.pdf

Phase I Metabolism - addition of small chemical hook (-OH) to relativelyinert hydrophobic toxicants by oxidative cytochrome p450 enzymes

Phase II Metabolism - conjugation of new hydroxyl groups with highly watersoluble glutathione, sulfate, and glucuronic acid groups (tags) forexcretion via urine

http://toxicology.usu.edu/660/html/metabol.html

can sometimeslead to

bioactivation, orthe the formationof more reactive

toxicintermediates

Metabolism/Biotransformation

Chemical

Lipophilic Hydrophilic

Phase I Phase II

Bioactivation: The Downside of ToxinMetabolism

Occasionally a cpd is bioactivated by transformation intoa more toxic cpd, such as benzo[a]pyrene [B(a)P], a potentPAH carcinogen B(a)P isolated from coal tar in 1933 Coal tar one of first demonstrated carcinogens One of the major reasons cigarettes are carcinogenic

PAHs common in soot, wood burning, charred fat, tobaccosmoke, etc. Low MW cpds are vapors e.g., naphthalene

Why B(a)P is ToxicMany productsresult frommetabolism ofBaP, but onecpd, BPDE, adiol epoxide,covalentlybinds to DNA

DNA adduct

Irreversible

43

Can We Predict How Chemicals Will Act?Key Chemical Properties they never taught you in

Organic Chemistry!

UNECE.ppt

Kow: Octanol Water Partition Coefficient, the tendency toadsorb to particles, soil, sediment or fat Generally inversely proportional to water solubility (S) Also reflects tendency to bioaccumulate

VP: Vapor Pressure, the tendency to evaporate Volatile vs semi-volatile vs non-volatile

H: Henrys Coefficient, the tendency to go from water toair Ratio of partial pressure of solute above solution to

solution concentration

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Envl FateWhere to Look in the Environment

UNECE.ppt

AirVP

Water

Soil, Sediment, FatLog Kow > 3 or 4

Higher Henrys

Higher S

Higher Kow

Higher VP

45

Bodily Fate

UNECE.ppt

Lung uptake rapid,as is removal

Carried in blood tokidneys forexcretion

Crosses cell membranes;stored in body fat

Log Kow > 3 or 4

Higher Henrys

Higher S

Higher Kow

Higher VP

Metabolism

46

Toxin Groupings

UNECE.ppt

VOCs like benzenePolar molecules,biotransformed

metabolites

POPS: Organochlorinepesticides; halogenated cpds

like flame retardants

Higher Henrys

Higher S

Higher Kow

Higher VP

Metabolism

How is Toxicity Quantified? Toxins suspected or known to have harmful effects at

low concentrations are tested to determine DoseResponse, usually on animals because thoseexposures can be measured accurately.

Dose is the amount of a chemical that comes incontact with an organism or some part of anorgansim at one time. E.g., the amount you breathe (conc x vol) would be an

exposure, but the amount that does not come out is thedose.

Measured as toxin mass/body mass

Types of Dose-response RelationshipsRadiation

Metabolite is toxic

Types of Dose Response3. Hormetic or non-monotonic:

J curve: small doses better than nodose e.g., micronutrients and vitamins; anti-inflammatories like aspirin; alcohol;

Inverted U curve: some endocrinedisruptors have stronger effects atlower concentrations(!),presumably because that moreclosely mimic low dose hormonalsensitivity

http://www.epa.gov/ord/endocrinedisruption/non-monotonic.htm

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How is Dose Response Tested? Usually animals, but they are given high doses to

guarantee a high rate of adverse effect Allows the use of fewer animals Increases uncertainty about relevance to low dose effects

Use of animals usually involves factors of safetyof 100 to 1000 when extrapolating to humans 10x for inter-species differences 10x for inter-human variability 10x for vulnerable human such as children

How are Safe Thresholds Determined? LD50 is dose where 50% death rate

occurs Lowest Observable Adverse Effect

Level (LOAEL) No Observable Adverse Effect Level

(NOAEL)

http://toxlearn.nlm.nih.gov/htmlversion/module1.html

http://www.epa.gov/ord/endocrinedisruption/non-monotonic.htm

Weakness in the Toxicity Testing System ~80,000 registered chemicals, only 200-300 have been

tested for safety Animal testing very expensive, difficult to adequately cover lowexposures (e.g., EDCs)

In U.S., chemicals are innocent until proven guilty Mixtures rarely tested

De facto assumption is that risks are additive, but synergisticeffects are possible

Attempts Underway to Improve theToxicity Testing System

High throughputstructure activityscreening(moleculartoxicology) E.g., Tox21

Graphic to rightshows recent resultsfor mitochondrialtoxicity

Endocrine Disrupting Chemicals (EDCs):A Game Changer in Toxicology

What happens if lowdoses not tested anddose-response curve isinverted U shape?

This is the centralconcern with EDCstheymay have missedimportant health effectsat the lower real-worldenvironmentalconcentrations or eventypical concentrations inpeople

From Vandenberg et al., 2012. Endocrine Reviews

http://www.epa.gov/ord/endocrinedisruption/non-monotonic.htm

What Are EDCs? Hormonally active agents that interfere with the endocrine system

including hormone production, transport, signaling, or elimination Endocrine system is basically an exquisitely sensitive communication system Any interferences are especially critical during times of development from

fertilized egg to infant Ecological effects on fish and amphibians at very low concentrations such as

downstream of water treatment plants Concerns first raised in early 1990s

Most common worrisome sources are related to plastics,pesticides and some drugs

Most environmental EDCs are estrogenic (related to estrogen), asex hormone much higher in women than men, but can also beandrogenic or related to thyroid hormones, primarily a metabolicconcern

(Nice overview in Wikipedia)

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High Volume EDCs1. BPA: in food can linings, thermal paper receipts

In July 2014, FDA declared "BPA is safe at the current levelsoccurring in foods

2. Pthalates: plasticizers that soften plastic; also infragrances

3. Perfluoro-octanioc acid and other teflon like cpds4. Flame retardants (chlorinated and brominated

organics), e.g., PBDE (Polybrominated diphenylether)

Additional EDC worries besides non-monotonic D-R: Recent introduction to society means short history of use, novel

physiologically Commonly lipophilic and some environmentally persistent,

bioaccumulating, and slow to leave the body (BPA an exception)

Example # 1: BPAUses: lightweight and hard polycarbonate plastics and epoxy resins

Sources: food packaging and canned food lining, water and sports drink bottles, old babybottles, plastic dinnerware, toys, medical devices, canned foods, thermal paper.

High exposure groups: infants & children. FDA banned BPA from kids sippy cups/bottles2012/13. Detected in the urine of > 95% of US adults, primariiy from canned foods.

Health effects & associations: BPA mimics estrogen.Chapel Hill Consensus Statement: "BPA at concentrations found in the human body isassociated with organizational changes in the prostate, breast, testis, mammary glands, bodysize, brain structure and chemistry, and behavior of laboratory animals. Average BPA levelsin people were above those that cause harm to many animals in laboratory experiments.Associated with obesity and metabolic syndrome risk in NHANES participants;

RS

Example # 2: PhthalatesUses: to soften plastics, esp. PVC. Not chemically bound to plastics, so come out. LowerMW types being replaced with more stable higher MW types.

Sources: cosmetics, perfume, soap, shampoo, nail polish, and skin moisturizers. Flexibleplastic and vinyl toys, shower curtains, wallpaper, minibinds. Food packaging and plasticwrap. Wood finishes, adhesives, plastic plumbing, medical tubing/fluid bags, PVC flooringand fragrances

High exposure groups: Infants from baby care products, children chewing on soft vinyltoys; dialysis patients, hemophiliacs, premature newborns.Most exposure from fatty foods, but vapor inhalation from low MW types, house dust.

Health effects and associations: DEP is "reasonably anticipated to be a humancarcinogen (NTP). Many studies of endocrine disruption in animals. Links to human ratesof obesity and metabolic outcomes.

RS

Example # 3: PFOA and Fluorinated TelomersUses: PFOA (Perfluorooctanoic acid ) is a breakdown product of fluorinated telomers used for

fire resistance and oil, stain, grease, and water repellency. EPA investigating alternatives forphase-out in 2015

Sources: non-stick cookware, outdoor clothing, food, drinking water. Unlike other POPs, PFOA iswater-soluble, does not bind well to soil/sediments, and bioaccumulates in serum rather thanin fat. In 2006 EPA asked 8 companies to phase-out by 2015. PFOA present in Patagonia fabricsat 100 ppb.

High exposure groups: PFOA persists in humans with a half-life of several years and is found inthe serum of almost all U.S. residents (99%) and in populations worldwide.

Health effects & associations: PFOA classified as "likely to be carcinogenic in humans" by theUS EPA. Adverse effects on mammary gland development in mice (EPA). Thyroid disease inNHANES (2010). Altered puberty timing in human females (PMID: 24129614, 21534542).Infertility in couples and decreased semen quality (PMID: 22197512)

Gore-Tex phase out 2013 RS

Example # 4: PBDE and Brominated Flame RetardantsUses: carpets, upholstery fabric, cushions, plastics. Penta-BDE banned but mostly used in

polyurethane foams; Octa-BDE also banned, mostly used in electronics. Deca-BDE still usedalthough banned in Maine and Washington

Sources:

High exposure groups: PFOA persists in humans with a half-life of several years and is found in theserum of almost all U.S. residents (99%) and in populations worldwide.Most exposure from fish, fatty foods and breast milk, but dust-bound and vapor phase exposures alsooccur

Health effects & associations: Animals show liver and thyroid toxicity; also a developmental andneurological toxicity. Humans: altered thyroid hormone levels in pregnant women and infants,reduced birth weights, reduced motor and mental development

Californians have higher levels in their house dust and body fluids thanresidents of other states

Kellyn S. Betts et al. (2008) Environmental Health Perspectives 116, A202 208 .

Source: Elevated House Dust and Serum Concentrations of PBDEs in California: Unintended Consequences of Furniture Flammability Standards? Zota, Ami R.,Rudel, Ruthann A., Morello-Frosch, Rachel A., and Brody, Julia Green, Environ. Sci. Technol., (2008) Environ Sci Technol. 42(21):8158-64

PBDE Exposure

ng/g

PBDE Flame Retardant Concentration in Household Dust

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www.ewg.org

2003restrictionsimplementedin 2008

Parting Observations on Toxicity Trendsand Related Risk Reduction Advice

(with more next week)

Emphasize whole, plant-based foods. Choose frozen vegetables over canned. Pop your own popcorn on the stove. Use glass or stainless steel containers, cups, commuter mugs, silverware. Opt for foods packaged in alternatives such as Tetra Pak cartons or glass jars. Never microwave food in plastic containers. Avoid non-stick cookware (restaurants dont). Avoid canned: tomatoes, coconut milk, soup, meat. Limit exposure to plastics of all kinds when pregnant and in early life.

Choose synthetic fragrance-free laundry detergents, shampoo/conditioner, body-wash. Avoid nail polish and nail salons After handling receipts, wash your hands before eating. Limit dryer sheets, synthetic perfume, and cologne. Look for flame retardant free foam in new furniture or replacement foam/padding

What to do about EDCs?when you can....

RS

You can find saferalternatives inpersonal careproducts and

home furnishingshttp://www.ewg.orgVisit the EnvironmentalWorking Groups ConsumerGuide

RS

Taking Good Care of Plastics Choose polyethylene or polypropylene where plastics

desirable (#2,4,5) Intact, smooth, clear plastic is the safest

Scratches from wear and tear are more susceptible to release ofcomponents

Never heat food in plastic containers in microwaves! Avoid hot water, detergent (soap OK), abrasives, acidic

foods (citrus, tomato, etc.)

Plastic Types by Label

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General Trends in Toxicity: Pesticides Pesticide toxicity peaked with heaviest organochlorine

use in the 1950s(?) High persistence and bioaccumulation issues (Silent Spring) Most now banned by individual nations or by Stockholm convention

(e.g., aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, mirex, toxaphene (manybanned)

Organophosphate alternatives still very toxic but much lesspersistent Problems still persist with highly soluble Atrazine, the #1 pesticide water

contaminant in US (Atrazine banned in Europe)

Produce with Highest Residues(Envl Working Group, 2013)

Apples Celery Cherry tomatoes Cucumbers Grapes Hot peppers Imported nectarines Peaches Potatoes Spinach Strawberries Sweet bell peppers Kale Summer squash

These bestto try to buyorganically

General Trends in Toxicity: Herbicides The herbicide Glyphosate (Round Up) developed in 1974

and began widespread use since mid-1990s inconjunction with GMO seed Tocxicity is plant-specific Glyphosate-resistant corn and soy developed (the largest GMOfraction of agriculture and 90% of US production)

Overuse has created resistant super-weeds that are now agrowing problem

2,4-D combo just approved by EPA, so we just took a giant stepbackwards towards greater toxicity

How to Choose the Right FishWant young fishlow in the foodchain, to avoidbioaccumulationof Hg, PCBs, etc.,e.g., swordfish,tuna,

Wikipedia

Also Good to Avoid Over-Fished Varieties Avoid:

Chilean sea bass Freshwater eel Atlantic halibut and sole (Pacific OK) Trawl-caught cod, snapper (hook and line OK) Farmed salmon (wild OK) Imported shrimp and tiger prawns Dredged scallops (farmed OK) Long-line caught tuna and ahi

More detailed list athttp://www.enature.com/articles/detail.asp?storyID=509

Nice pocket guide at:http://www.montereybayaquarium.org/cr/cr_seafoodwatch/content/media/MBA_SeafoodWatch_WestCoastGuide.pdf

Part of Pocket Guide