Sublethal Effects to Individuals

EcotoxicologyBiology 5868

2009

Sublethal Effects

Sublethal effects: changes in physiological processes, growth, reproduction

behavior, development, etc.

- common theme: adverse effect on individual’s fitness

Ecological context and concepts:

- sublethal effects may have lethal consequences

- individuals must compete for food, avoid predation, mate, etc.

- ecological mortality v. somatic mortality

- toxicant-related diminution of fitness may equal somatic mortality

(effects expressed in the next generation)

General Adaptation SyndromeSeylean stress - response- specific suite of responses to generalized stress

1) Alarm reaction - short-term

- catecholamine release; epinephrine, norepinephrine)

- glucocorticosteroid release; e.g. cortisol, corticosterone

- increased blood pressure

- increased blood sugar

- immune suppression

2) Adaptation or resistance – mid/long term

- tissue-level response

- hypertrophy, atrophy

3) Exhaustion - long term

- failure to compensate

- eventually leads to death

- depletion of reserves

General Adaptation Syndrome

Exposure Duration

“Cost”

Alarm Adaptation Exhaustion

deathrapid

heart

rate

adrenal

hypertrophy

Immediate Long-Termlow

high

Sublethal Effects - Growth

Growth - common response variable for sublethal effects

- often related to individual fitness

Toxicant-influenced growth – potential biomarker

- often shows dose-response effect of toxicants

- caution – be aware of threshold effects; i.e. no effect at low dose

Nomenclature:

Lethal – L

Sublethal Effect – E

Dose – D

Concentration – C

Inhibitory - I

50% mortality – LD50, LC50

50% response – EC50, ED50

50% reduction in normal response – IC50, EC50

by definition, 50% mortality (response) = median dose

test duration: e.g. 48, 96 h

- 48 hour LC50, 96 h EC50

- integrates a suite of biochemical and physiological effects

- easy to measure

Sublethal Effects - Growth

Gro

wth

Hormesis

[toxicant]

Hormesis may also be evident

- biphasic response

- usually not toxicant-specific

- may reflect regulatory overcompensation; e.g.

- detoxification enzymes

- immune response

- heat stress proteins

- metallothioneins

- antioxidants

Growth - common response variable for sublethal effects

- often related to individual fitness

Toxicant-influenced growth – potential biomarker

- often shows dose-response effect of toxicants

- caution – be aware of threshold effects; i.e. no effect at low dose

- integrates a suite of biochemical and physiological effects

- easy to measure

Developmental ToxicityDevelopmental stages are generally more sensitive to toxicants. (Why?)

bill deformity

Toxicant exposure:

- egg stage

- contaminants deposited in yolk

- some contaminants cross placenta

- many vulnerabilities

- embryonic/larval stage

lordosis (flounder)

Critical periods or windows for developmental toxicity; e.g.

- Hg effects (Minamata disease)

- thalidomide

- endocrine disruptors

- malformations (teratogenesis)

- developmental mortality

Effects (e.g.)

- functional deficiencies

- slow growth

- behavior

Teratogenesis Assays

FETAX assay (frog embryo teratogenesis assay – Xenopus)

- 96 h exposure to contaminants

- compare concentrations producing 50% death (LC50)

and 50% abnormalities (TC50)

- mortality and abnormal development scored

- teratogenic index (TI) = LC50/TC50

- reflects developmental hazard of a contaminant

FETAX

Developmental Toxicity

Behavioral teratology – behavioral abnormalities arising after

embryonic exposure to toxicant; e.g.

- mummichogs exposed to Hg as embryos had decreased ability to capture prey

polymelia (Rana pipiens)

- green frogs exposed to pesticides as embryos had decreased

predator avoidance response

Developmental stability – capacity of an organism to

develop into a consistent phenotype

- correlated to fitness

- deviations measured from norm; e.g.

- bilaterality: fluctuating asymmetry (FA)

- FA thought to reflect perturbations in

normal developmental processes

Sexual CharacteristicsEndocrine Disrupting Chemicals (EDCs)

- xenobiotic estrogens (xenoestrogens) mimic estrogens

- regulate activity of estrogen-responsive genes by binding to estrogen receptor (ER)

- disrupt hormonal signaling

- affect sex organ development, brain development, behavior, fertility, physiology; e.g.

- male vitellogenisis (fish)

- male gulls ignore nesting colonies

- female gulls pair, lay infertile eggs (DDT/DDE)

- imposex (mollusks)

- intersex (fish, amphibians)

- sex reversals (fish)

- abnormal sex ratios

Imposex in the dogwhelk

testicular oocytes (leopard frog)

ReproductionEffect: lowered fitness of individuals due to reproductive impairment; e.g.

- DDT/DDE induced eggshell thinning (inhibition of Ca-dependent ATPase

in eggshell gland)

- imposex in mollusks

- various fish:

- reduced egg size

- reduced egg and larval survival

- reproductive failure

- lower fry survival

- lowered fertilization rates

- lower embryo success

- lab experiments; e.g.

- Daphnia – reduced number of young

- Gambusia – low number of embryos

Sublethal Effects - Physiology

Physiological biomarkers: deviations from homeostatic state

- may reflect threshold effects

- may be used to infer mode of action

- e.g. AChE inhibitors may affect feeding, respiration, etc.

- imply lowered fitness

Physiological biomarkers include:

- impaired performance; swimming speed, stamina

- changes in respiration

- excretion

- ion regulation

- osmoregulation

- bioenergetics (i.e. food conversion efficiency)

- immunological capabilities/disease resistance

- etc.

Physiological Biomarkers

Respiratory activity:

- e.g. fish “coughs” (gill purges)

Adenylate energy charge (AEC)

- reflects balance between anabolic and catabolic activities

AEC =ATP + ½ ADP

ATP + ADP + AMP

Respiration (oxidative phosphorylation)

- O2 consumption under minimal and maximal activity levels

- compare with N excretion rate (protein degradation) to determine

CHO v. protein catabolism

Behavior

Contaminants/toxicants cause a wide variety of behavioral changes.

- often measured in lab settings; occasionally in the field

- difficult to objectively score

- considerable “normal” variability

- difficult to extrapolate accurately from structured lab

experiments to field situations

- preference/avoidance

- e.g. light, temp, salinity

- movement towards or away from stimulant

- activity levels; e.g. fatigue, hyperactivity

- feeding; e.g.

- diminished or cessation (lab)

- deviations from predictions from optimal foraging theory (field)

Behavioral biomarkers include:

Behavioral Biomarkers

- performance

- swimming against current

- maintaining proper orientation

- critical swimming speed

- learning

- memory impairment (humans)

- memory loss (humans)

- predation

- avoidance

- sub-optimal foraging behavior

- reproductive behavior

- social interactions

Behavioral biomarkers, cont.

Toxicant Delivery and Exposure

Measured Exposures – Identical to nominal exposure, except that actual

exposures are quantified by analytical chemistry methods.

Nominal Exposures – Toxicant is weighed/diluted/mixed as necessary to

theoretically match the target exposures.

Aquatic organisms are typically exposed to a Concentration of toxicant;

i.e. toxicant is in solution with the water.

Terrestrial organisms are typically administered a Dose of toxicant;

i.e. toxicant is injected into or fed to the test organism.

Nomenclature:

Lethal – L

Sublethal Effect – E

Dose – D

Concentration – C

Inhibitory - I

50% mortality – LD50, LC50

50% response – EC50, ED50

50% reduction in normal response – IC50, EC50

by definition, 50% mortality (response) = median dose

test duration: e.g. 48, 96 h

- 48 hour LC50

Detecting Toxic Effects

Experimental Designs

Sublethal – no death to the test organism; however, effects to its

behavior and/or biochemical & physiological processes occur.

Chronic Lethal – death to the test organism following a prolonged

low-intensity exposure to a toxic substance.

Acute Lethal – death to the test organism following a brief, and

often intense, exposure to a toxic substance.

Effects detected and quantified in tests involving different exposures,

replicates, time series (intervals or single time points)

Proportion of Life Span and System Complexity

Toxicity Tests

Acute

Chronic

Microcosms

Mesocosms

Field studies

Aquatic

Toxicity

Study

Methods

Toxicity Tests

Acute (lethal) - short period of life span

- fish, daphnids, rats, birds

- 24, 48 h

Chronic (lethal) - significant portion of lifespan

- must include gestational period (female)

- must include portion of spermatogenesis (male)

Test SystemsAcute - Aquatic single species tests

toxicant concentrations and O2 returned to original values

animals fed; waste products removed

small amount of waste products

Static: one-time addition of toxicant (test compound), water, food, etc.

O2 content and toxicant decrease over time

- high COD or BOD can be dangerous

metabolic waste products increase

least sensitive; chemical degradation may reduce apparent sensitivity

simple, cost effective

small amount of chemical, waste+

-

Static renewal: periodic addition/removal of toxicant, water, food, etc.; or,

organisms are periodically placed in new solutions

more handling of test vessels and organisms

- increased chance of accidents

- increased stress on animals

less sensitive than flow-through; chemical degradation, absorption

+

-

Test Systems

Flow-through (recirculating, intermittent flow, continuous flow): continual

replacement of toxicant, O2, etc. removal of waste

Acute - Solid Phase (terrestrial, sediment) – cages, pens, soil (flats or plots),

sediments, controlled-environment chambers. • use contaminated substrates

• use spiked substrates

provides more representative evaluation of acute toxicity; sample

can be administered directly from source

higher loading factor (biomass) may be used

maintains water quality

constant toxicant exposure; loss due to volatilization, absorption,

degradation, uptake, reduced

large volumes of sample and dilution water required

increased complexity; space, equipment

uncertainty about effect of water treatment on toxicant concentration

opportunities for mechanical failure

large increase in waste water and toxicant

+

-

Acute - Aquatic single species tests

Test Organisms

Terrestrial – earth worms, quail, honey bees, and plants

Semi-aquatic – amphibians and mallard duck

Aquatic – freshwater & marine fish, invertebrates and algae

- a few of many examples:

Test Considerations and Parameters

Dilution water properties – e.g. D.O., hardness, alkalinity, pH, conductivity, salinity

Test substance properties – e.g. solubility, volatility and stability

Test organisms must be subjected to an acclimation period where

all key environmental parameters match the actual test conditions

in the absence of toxicant.

Test organism considerations

- source, age, condition, handling, acclimation or acclimatization

Acclimatization – same processes applied to natural or field settings

Acclimation – (controlled environments or laboratory settings)

Physiological adjustment to a change in environmental conditions;

maintains or minimizes deviations from homeostasis

Test Considerations and Parameters

Feeding – generally avoided, unless organisms are cannibalistic or test duration

significantly overlaps organism’s life span

Environmental variables – temperature and photoperiod

Loading – minimize biomass per unit volume of test solution

Concentration or Dose Units

Aquatic

mg / L = ppm

μg / L = ppb

ng / L = ppt

Terrestrial

mg / kg = ppm

μg / kg = ppb

ng / kg = ppt

ppm – part per million

ppb – part per billion

ppt – part per trillion

Experimental Design

exposure environment

replicates

experimental units

various concentrations = dose/response

[a] [b] [c] [d]

[a] [b] [c] [d] [a’] [b’] [c’] [d’] [a’’] [b’’] [c’’] [d’’]

Design Considerations

Physical Design

air flowair flow

[a][d] [c]

[d]

[a][b] [c]

[b][a]

[b]

[c]

[d]

Randomization schemes; e.g.

- Latin square

- modified block

Results and Analysis

Resp

on

se

Concentration

10

50

100

- how potent is the toxicant?

- what is a “safe” concentration

- at what concentration can we expect

lethal effects?

March 19, 2007

FixedFixedOriginalOriginalDateCumulative

LengthWeightLengthWeightStageEuthanizeGrowoutIndividualTubN

200.6411230.81314508/0207/311221

190.5107220.67464508/0208/031292

180.423220.60624508/0808/03123

180.3994210.55494508/0808/04144

180.5047220.68154508/1008/061435

230.9364251.25744508/2808/02136

220.8834231.24234509/0409/011107

220.7950251.21444509/0408/301138

231.0923261.27624509/0809/01119

220.9822261.30244509/0809/042110

241.0814281.44984509/0809/043111

241.2044271.43684509/0809/041812

200.6842230.96404509/081913

231.0118251.25414509/0809/0412414

251.0983271.47974509/0809/0113215

220.7700251.10164509/0809/0414216

241.2381281.65524509/0809/0424317

220.9307251.28644509/1009/042418

220.9640261.26624509/1009/042819

220.8503241.07534509/0809/043820

190.6440230.89094509/1009/0611121

220.7479251.02054509/1009/0411522

230.9425271.20004509/1009/0621523

220.9258261.21084509/1009/0611624

241.0554261.29264509/1009/0413325

230.9516261.23094509/1009/0414126

220.8469251.14054509/1009/0424227

261.1196291.55104509/1009/0434328

250.8391261.06154509/1509/062229

230.7794250.99784509/1509/041530

240.8452241.14274509/1509/0811731

230.8782251.12684509/1509/0821732

261.1169281.59124509/1509/0611833

240.9794271.34294509/1509/0621834

220.7293240.94924509/1509/0612535

210.5885230.81294509/1509/1122536

240.8653261.21804509/1509/0812637

230.7477240.98174509/1509/0813638

240.7812261.11814509/1509/0813839

271.2711291.58664509/1509/0844340

220.7941251.05434509/1509/0814441

230.6952240.97734509/1509/0824442

220.6480240.87124509/1609/1112043

240.8189261.08564509/1609/1122244

240.8290261.08714509/1609/1132245

230.8460261.10954509/1609/1124146

230.7544250.90614509/209/8,11,122347

230.7349250.95624509/209/8,11,123348

240.9958261.23364509/209/8,11,124349

220.6836240.86994509/2009/153450

220.6610230.86274509/209/11,152551

230.7129250.84134509/209/11,153552

230.8777251.06934509/209/14,151653

200.5687230.77334509/209/14,152654

220.8767261.06884509/209/14,153655

210.7083240.95044509/2009/151756

220.8678251.15084509/2009/144857

220.7147250.93404509/2009/1121358

210.7155240.90044509/2009/1511459

240.9278261.19534509/2009/1123360

220.7230230.92364509/2009/0814061

210.7473241.04524509/2009/1534262

220.6897230.86384509/284163

Independent variable – concentration

Dependent variables – response

(e.g. growth, reproduction, behavior,

development, etc.)

Analysis

Hypothesis Testing: Which of the treatment groups is significantly different

from the others? Includes:

1. ANOVA (analysis of variance)

- ANOVA compares variance within treatments (i.e. replicates)

to variance among treatments

-variance within treatments assumed to reflect sampling

- or error variance

- variance among treatments includes error variance plus

any additional variance associated with the treatment

- Null hypothesis states that there should be no difference between,

within, and among variance

- F statistic tests the null hypothesis of equal means among treatments

Analysis

- ANOVA assumptions:

1) equal variances among treatments

2) normally distributed data (Shapiro-Wilk’s test)

- Also, observations must be independent

- depends on good experimental design

- random assignment of treatments

NOTE – data can be transformed (e.g. arcsine, log, square root)

to achieve normality/linearity

concentration log concentration

Hypothesis Testing: continued

**

*

**

Post-ANOVA Tests

ANOVA - differences in variances indicate different means

e.g. Dunnett’s test, students t-test, Williams’s test

- assign a significance to the response means for different treatments

- null hypothesis rejected (differences in variances) - post-ANOVA tests

show which treatments differ significantly from each other

Resp

on

se

Concentration

- level of significance (α) set; most often 0.05 (i.e. 0.95)

10

50

100- various biological effects can be predicted

EC50

However: statistical hypothesis testing only

demonstrates that something that is present

in the data set differs significantly from

the null hypothesis

- null hypothesis accepted (no difference in variances) - no further analysis

Non-Parametric Post-ANOVA Tests

Some data sets will not qualify for ANOVA

- various tests can be used to establish significant differences

- Steel’s many-one rank test

- Wilcoxon rank sum test

- ANOVA assumptions:

1) equal variances among treatments

2) normally distributed data

Sublethal Effects Concepts/Terminology

MATC – maximum acceptable toxicant concentration:

- undetermined concentration within the

interval bounded by the NOEC and

LOEC that is presumed safe

NOEC (NOEL) - no-observed-effects concentration:

- highest test concentration for which there was no statistically

detectable difference of the control response

NOAEC - no observed adverse effects concentrations

LOEC (LOEL) - lowest-observed-effects concentration:

- lowest concentration in a test with a statistically significant difference

from the control response

LOAEC - lowest observed adverse effects level

NOEC < MATC < LOEC

log concentration

NOEC

LOEC

Sublethal Effects Concepts/Terminology

- “safe” MATC is dependent on species, exposure duration, etc

also:

- NOEC and LOEC totally dependent on test concentrations

- can be considered artifacts of experimental design

- process can produce higher than optimal NOEC and LOEC with

suboptimal experimental design

- therefore, poor technique can be “rewarded” with high NOEC and LOEC

Note:

Regression Methods

Data fit to a concentration-effect model by various regression methods; e.g.

- least-squares

- maximum-likelihoodR

esp

on

se

Concentration

50

10

- concentrations and their associated confidence intervals having biologically

significant effects can be calculated by interpolation

- e.g. IC10, LC5, etc.

EC50

EC10