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ES/RP 532Applied Environmental Toxicology

Lecture 6Fundamental Principles of Toxicity,Toxicokinetics & Risk Assessment

Toxicity

Toxicity = inherent capacity to cause harmRealization of toxicity is a function of--– Mode of action

• Interactions with receptors, enzymes,ion channels, etc.

– Toxicokinetics• Absorption, distribution, elimination

parameters– Environmental chemodynamics

• Exposure parameters

How Do You Explain--

Variation in response within apopulation to a single doseDifferences among species responsesto a toxicant when MOA is the sameWhy thresholds for physiologicalreactions existWhy toxicity differs for various routes ofadministration

Generation of In VivoToxic Effect

Requires– Absorption from the site of exposure (dose

administration)– Delivery to the target organ, tissue, receptors,

enzymes• Influenced by

–Duration of exposure–Magnitude of exposure–Potential for accumulation of chemical in

target tissues– Initiation of cellular events at the target organ

or tissue

Questions to Be Answered

How does a pesticide cause toxicity?What is the likelihood of toxicity (orharm) from exposure to a pesticide?Why do individuals and species differ intheir responses to a given amount oftoxicant?

Studies Needed

Measurement of Toxicity (bioassays)Elucidation of toxicokineticsElucidation of toxicodynamics

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Conceptual Model ofRelationship

Redrawn from Heinrich-Hirsch et al. 2001

Measuring Toxicity

Determination of relationship between doseor concentration of substance and responseof test organismMust determine toxicological endpoint– Death– Development/Reproductive Effects– Weight Loss– Neurological function– Endocrine function– Enzyme inhibition

Expressing Dose

Dose--mass of substance that anorganism contacts via dermal, oral, orinhalational routesDosage--mass of substance per unitweight of organism (mg/kg)Concentration--mass of substance perunit volume of substrate to whichorganism is exposed (mg/L water;mg/kg soil)

Distribution of IndividualResponses to Increasing Doses

NumbersResponding

50% Response (median)

Dose (mg/kg)

Cumulative ProportionResponding

Dose (mg/kg)

100%

50%

0%

NOEL

Population Response(Cumulative %)

LD50ED50

Slope

Probit Transformation-Linearizationof the Dose-Response Curve

3.724.164.484.755.005.255.525.846.28

102030405060708090

Probit%

Mortality

Log Dose

LD50

7

5

1

3

% M

orta

lity a

sPr

obab

ility

Units

3

The variance about the LD50 is lowerthan at the dose extremes

Importance of Slope

Representsvariability inpopulation responseCharacterizesmargin of safetyResponse ofdifferent speciesexhibiting samemedian response tosame chemical

Importance of Slope

Variation inresponse of twodifferent speciesreacting to the samechemicalVariation inresponse of singlespecies reacting totwo differentchemicals

Monitoring of Resistance Development

Dose, Dosage, Concentration

NumbersResponding

50%

Log Dose

% Response

Utility of the LD50

Only meaningful in context of acomparison– Relative hazard of two or more chemicals– Relative susceptibility of two or more

species or two or more populations of thesame species

– Absolute hazard of a single chemical whenoral and dermal routes of exposure arecompared

Hazardous Compounds HaveOral & Dermal LD50’s <50 mg/kg

DDT phosdrin 2,4-D thifensulfuron1

10

100

1000

10000

LD50

Comparison of Toxicity to Mammals

oral

dermal

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Comparison of Hazard to Different Species

LD50

1

10

100

1000

10000

100000

0.001

0.01

0.1

1

10

100

LC50

DDT Diazinon Permethrin Thifensulfuron

Birds

Fish

For assessing human health andecological hazards, the NOAEL is moreimportant than the LD50.

Warning!

Exposure = Hazard

Must consider toxicokinetics &toxicodynamicsBasic processes– Absorption– Distribution– Elimination

Measure– Extent of process– Rate of process

Absorption (Penetration)

Contaminant or toxicant crosses theoutermost barrier of an organism– Chemical transfers from site of contact into the

cells and eventually into the general circulation• Skin, cuticle, cell wall

Also applicable to crossing integument ofgastrointestinal tract (oral or ingestionexposures)Also applicable to crossing integument oflungs or other ventilatory organs (inhalationalexposures)

AbsorptionControlled bythermodynamicprocesses– Consider nature

of cellmembrane

AbsorptionDiffusion is mainmechanism drivingpartitioning acrossmembranesExtent controlled byKow (hydrophobicityparameter) of chemicalRate controlled byconcentration (first-order process)

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HydrophobicitySurrogate measure is Kow, the octanol:waterpartition coefficientHigher the value, the more the tendency topartition into an “oily” (lipid-dominated) phase(matrix)– Free energy at a minumum– Entropy at a maximum

Thus, hydrophobic compounds cross cellmembranes more easily than hydrophiliccompoundsHowever, extremely hydrophobic compoundsmight be trapped in lipid layers

Distribution

The process of reversible transfer of achemical from general circulation intothe tissues– Animals: blood ---> organs– Plants: xylem/phloem ----> foliage/fruit

Usually very rapid– Rate limited by rate of blood (“sap”) flow– Polarity of the chemical (or hydrophobicity)

DistributionExtent influenced by:– Water solubility (WS)

• Kow and WS are inversely correlated• Patitioning into plasma or interstitial and intercellular

fluids limits uptake by fat tissue and central nervoussystem (CNS)

– Lipid solubility (measured by Kow)• Partitioning into adipose (fat), CNS, or other organs

having high lipid content– Plasma protein binding

• Causes reduction in tissue distribution and retainscompounds longer in circulation

– Tissue protein binding• Causes more extensive distribution among tissues

EliminationMetabolism--Interaction with enzymes

• Detoxification• Activation

– Phase I• Oxidations

– Cytochrome P450 mediated; require NADPHand O2

– Located on endoplasmic reticulum• Hydrolysis

– Mediated by esterases (hydrolases)– Cytoplasmic; plasma– Attack ester linkages

• Reductions– Transfer of electrons to carbon, nitrogen

Elimination

Metabolism– Phase II reactions--Conjugations

• Chemicals usually conjugated to glutathione (atripeptide) or sugar moiety (glucose; galactose)after initial oxidation (or other metabolism)

• Water solubility increased, facilitating filtrationby kidneys and eventual excretion

– Acylanilide herbicides safened by inducersof glutathione-S-transferases

N

ClCl

ClC2H5O

C2H5OP-O

S

N

ClCl

ClC2H5O

C2H5OP-OO

chlorpyrifos chlorpyrifos oxon

P-450 Oxidation

N

ClClHO

ClC2H5O

C2H5O

S

P- OH+

C2H5O

C2H5O

O

P- OHN

ClClHO

Cl+

Oxidativeor

Esteratic

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Herbicide Activation

OCH2COHO

Cl

Cl

Cl

Cl

OCH2C O(CH2)nCH3O

O OHSG

P450 GSH

phenanthrene Phenanthrene 9,10 oxide

Oxidation and Conjugation of aPolyaromatic Hydrocarbon

GSH = glutathione, a three amino acid peptide

SelectivityThe differential toxicity of a compound between apest organism and a nontarget organism– Conferred by unique mode of action or insensitive

biochemical target (pharmacodynamics)• Common among herbicides

– Sulfonylureas, imidazolinones, glyphosate,phenoxys

• Insecticides– Microbial insecticides; insect growth

regulators– Conferred by extent/rate of metabolism

(toxicokinetics)• Pyrethroids

Pyrethroid Metabolism

OOC

O

Cl

Clpermethrin

1R-trans DCCA

1R-cis DCCAPBA

Cl

ClH

H CO

OH

CO

OH

HH

Cl

Cl

OHO

O

EliminationExcretion– Metabolic reactions result in more water soluble

compounds, facilitating excretion– Reduction in amount of parent compound

available to target sites– Clearance

• Volume of blood (or plasma) cleared ofchemical per unit time

– Routes• Expired air, saliva, bile, feces, urine, milk, hair

– Extent less important than rate• All compounds eventually 100% excreted

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StorageType of elimination mechanism– Influenced by rate of metabolism and

hydrophobicity• Example: DDT

– Slow metabolic rate– High Kow

– Temporary mechanism in that chemical isreleased from storage sites• Equilibrium between adipose tissue and blood• Of concern for very recalcitrant compounds

Concept of bioconcentration factor (ecotox.)– Ratio of matrix (or food) concentration to

concentration in an organism

What Do We Know AboutPesticides and Absorption byHumans??

Case Study I

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Toxicokinetics & Plants

All mechanisms & processes same as inanimalsHowever, must consider dose transfer fromthe environment (ditto if considering aquaticand soil dwelling animals)– Other parameters important

• Soil sorption coefficient (Koc)• Air:water transfer coefficient (KH)

Risk AssessmentDetermination of the potentialhazards of a substance and theprobability that it will cause harm tothe environment and/or humanhealth when used in the manner forwhich it is intendedHazard--potential harm; conditionalRisk = Probability of Harm

– Can never be zero

Risk ManagementDetermination of what should be doneto avoid, minimize, or mitigate a risk– Determination of acceptable risk– Evaluation of alternative risk control

actions– Selection among alternatives

• Maybe do nothing– Implement actionsBased on Consideration of Politics,Economics, Ethics & Hopefully Science

Risk Management Devices

Narrative Standards– Statement of desired objective or goal

• “Do Not Drift….”• City of Seattle Pesticide Use Reduction Strategy

– “It is the policy of the City of Seattle to promoteenvironmentally sensitive landscape pest andvegetation management by phasing out the use ofthe most hazardous pesticides and reducingoverall pesticide use while preserving landscapeassets and protecting the health and safety of thepublic and our employees.”

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Risk Management Devices

“Acceptable” Margins of Exposure(MOE)– Hedging your bets with safety factors– “Codified” as numerical standards

• MCLs (Maximum Contaminant Levels)• Ambient Water Quality Criteria for

Protection of Aquatic Organisms• Reference Doses (RfDs)• Population Adjusted Doses (PADs)• Levels of Concern (LOCs)

An “Acceptable”Margin of Exposure (MOE)

Pacific Oyster LC50Glyphosate Concentration Lethal to

50% of Exposed Oysters

3,988 µg/g (ppm), sediment

If MOE = 100Glyphosate in mudflats

Must be < 40 ppm

Don’t Be Confused By the Process--Know Where You Are

Risk Assessment

Scientific

Socio-politicalEconomic

Experimentation(measurement, data analysis)

Risk Management

Elements of Risk Assessment

Hazard CharacterizationDose-Response RelationshipExposure CharacterizationRisk Characterization

Toxicology--Hazard Identification

Chemical characterizationWhat are the relevant toxicityendpoints?Testing starts with high doses todetermine the array of possible effectsOnce an effect is identified, than thebiochemical or physiological causes canbe studied

5 mg/kg/day 75 mg/kg/day 150 mg/kg/day

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Toxicology--Hazard Identification

Endpoints– Death– Systemic toxicity (organ/tissue pathology)– Adverse Developmental Outcomes– Adverse Reproductive Outcomes– Cancer (excess rat tumors)– Subtle Enzyme Changes

• Caution: these may only indicate exposureand not a physiologically adverse effect

Dose-Response AssessmentAfter the hazard is identified,– i.e., the toxicological endpoint of concern is

chosenThen, the next step is to determine how themagnitude of the response varies withincreasing concentrations or doses.Generation of NOAEL’s for most sensitivetoxicological hazard– Relevant for threshold responses (anything but

genotoxic or tumorigenic effects)Generation of slope factor for tumorigenicresponses in chronic assays (2-yr rodentassays)

Exposure Assessment

Environmental Chemistry– What is the distribution of environmental

residues?– How persistent are the residues?– How much does a person contact?

Exposure Calculation:– Magnitude of contact X residue

• Contact: amount of air inhaled, food eaten, soilin mouth, surface area touched, etc.

• Divide by body weight• Yields a dosage or dose rate (or intake rate)

Risk CharacterizationPart Science– Divide the dose observed to cause no effect

by the exposure level– State the ratio (the MOE)

• MOE = NOAEL (mg/kg/day) ÷ exposure (mg/kg/day)

Part Risk Management– Divide the estimated level of exposure by the

dose believed to be “safe” (Exposure/RfD)– Determine if the ratio is acceptable or not

Risk CharacterizationMOE vs. RfD

Margin of Exposure (MOE) =NOAEL (mg/kg/day)

Exposure (mg/kg/day)≥ 100 (EPA not concerned)

Reference Dose (RfD) NOAEL100

=

Risk =( Exposure/RfD) x 100 if < 100, EPA not concerned

Child Sensitivity Is ConsideredIf fetal and newborn rats are more sensitive ata given dose than adult rats, then up to anextra 10-fold safety factor may be applied tothe RfDThe RfD divided by this FQPA Safety Factoris called the– Population Adjusted Dose (PAD)

NOEL100

= Reference Dose (RfD)

RfD10

= Population Adjusted Dose (PAD)