Toxicity testing' Doseresponse and timeresponse - PowerPoint PPT Presentation

Title: Toxicity testing' Doseresponse and timeresponse


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Toxicity testing. Dose-response and time-response

• Topic 7

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Learning goals

• To learn basic concepts underlying standard
  toxicology models
• To understand and be able to apply dose-response
  and time-response models of toxicology testing
• To learn examples of model test organisms

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Why dose is important?

• All substances are poisons there is none which
  is not a poison. The right dose differentiates a
  poison and a remedy.
• Paracelsus (1493-1541)

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Toxicity testing

• Used to determine acute and chronic effects of a
  toxic compound or mixture of compounds
• Acute typically 96 h or shorter
• Chronic weeks to months (optimally ca. 10 of a
  species life-time)
• Traditionally and most often based on lethality,
  but other endpoints can be used (e.g. growth,
  reproduction)

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Dose-response models
Measure mortality after a certain exposure period
(e.g. after 48 h or 5 days)
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Dose-response models are based on the normal
distribution of biological variables
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Population response to toxicants are usually
skewed to the left
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Individual tolerance concept basis of the
dose-response model

• Individual effective dose (IED) a minimum dose
  required to kill an individual
• Genetically determined for each individual

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IED model results in log normal distribution
(sigmoidal curve)
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Alternative models log logistic model

• Linked to enzyme kinetics, autocatalysis, and
  adsorption
• Probability of death for any individual is a
  consequence of random processes and is determined
  by the probability that a specific sequence of
  events leading to death will occur in a certain
  individual
• These probabilities are equal for all individuals
  in the population
• Which particular individual responds quickly
  (sensitive) or slowly (tolerant) is a matter
  of chance alone

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Log-logistic model predicts similar type of
sigmoidal curve
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So whats the difference?
1st toxic insult 30 died
2nd toxic insult expectations?
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Determination of toxic doses from log normal and
log logistic models

• Data need to be transformed (linearized) a
  sigmoidal curve must be converted into a straight
  line

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Data transformation Step 1Change Dose axis to
log scale
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Data transformation Step 2Convert response to
probit or logit
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Probit

• Derived from normal equivalent deviation (the
  proportion of dying expressed in terms of
  standard deviations from the mean of a normal
  curve)
• Probit(P)NED(P)5

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Alternatively, response can be converted to
logit
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Resulting straight lines are not very different
Probit
Probit or Transformed logit
Logit
Log concentration
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Measures of toxicity derived from dose-response
curves
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Using probit- or logit-linearized dose- response
curve
Probit or logit
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Estimate LC from the linear regression line

• YabX, where Y transformed logit, X- logC, b
  slope of the line, a intercept of the line
• Calculate log(LC50)
• What is the transformed logit value for 50
  mortality?
• Calculate LC5010log(LC50)

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Time-response models
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Time-response LT50

• Mathematical apparatus very similar to that used
  for LC50 determination
• Same methods could be applied
• Probit
• Logit

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Alternatives of LC50 and LD50

• Using endpoints other than mortality
• EC50
• Fixed Dose Procedure (FDP)
• Limit Test
• 5000 mg/kg
• The Up and Down Procedure (UDP)

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Conducting toxicity testing

• Now we know WHAT to measure what about HOW?

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Experimental design for toxicity testing
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Overcoming problems with static design Way 1.
Increasing volume
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Overcoming problems with static design Way 2.
Static-renewal
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Standard Toxicity Tests

• ASTM - American Society for Testing and Materials
• EPA - both federal state agencies
• OECD - Organization for Economic Co-operation and
  Development

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Test organisms are surrogates for natural
populations

• test species not necessarily species at risk
• but are representative of non-target populations
• we assume responses of test species is similar to
  species at risk
• test species chosen for ease of culture, etc.

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Criteria for selection of test (sentinel, model)
organisms

• Widely available - from lab culture, hatchery,
  field collections
• Successfully maintained in the lab, sufficient
  quantities
• Preferably known genetic composition and history
  of culture
• Relative sensitivity to various classes of
  toxicants should be known
• Sensitivity of organism should be representative
  of the phyla that species represents
• In multispecies tests (microcosms field
  studies), the interactions among the component
  species should be understood

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Common organisms used in standard toxicity tests

• Invertebrates
• Lumbriculus variegatus
• (terrestrial worm)
• Daphnia magna (water flea)
• Daphnia pulex (water flea)
• Ceriodaphnia dubia (water flea)
• Chironomus tentans (blood wormmidge larvae)
• Hyalella azteca (scud)
• Gammarus pseudolimnaeus (scud)
• Hexagenia (mayflies)

• Vertebrates
• Pimephales promelas (fathead
• minnow)
• Oncorhynchus mykiss (rainbow trout)
• Lepomis macrochirus (bluegill)
• Fundulus heteroclitus (mummichog)
• Rana sp (frog)
• Rattus (rat)
• Colinus (northern bobwhite)

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Daphnia spp.

• Freshwater crustacean
• Require hard water
• Acute 48h test
• 10 neonates at each conc.
• no food
• static renewal
• endpoint immobility (mortality)
• Chronic test (partial life cycle)
• 10 organisms
• 21 days exposure (3 molts)
• no food
• static renewal
• endpoint survival, growth (length),
  reproduction

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Advantages of Daphnia tests

• easy, inexpensive to rear
• small size rapid test
• can perform partial life cycle tests rapidly
• Disadvantages standard tests are heavily biased
  towards arthropods (insects and crustaceans),
  hardly anything is known about other taxa

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An example of a vertebrate model

• Colinus virginianus (northern bobwhite quail)
• often used to test toxicity of pesticides
• toxicant delivered through food
• birds feed ad libitum
• endpoints mortality, reproduction

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Prokaryotes

• MICROTOX tests

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Factors modifying toxicity

• Acclimation/acclimatization
• Allometry
• Abiotic factors
• Photo-induced toxicity
• pH
• Hardness
• Temperature
• Organic ligands
• Physiological status
• Age
• Gender

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Take-home message

• Formulate three main things you have learned
  about toxicity testing
• Formulate one thing you have learned from this
  topic that was most surprising or unexpected for
  you