Institute of Biological Sciences

EcotoxicologyErik BaatrupMark Bayley

EcotoxicologyEnvironmental medicine

Toxicology Pharmacology

Biochemistry

Histochemistry

Occupational medicineEcologyMicrobiology

Hydrobiology

Pathology

Chemistry

Bioavailability

Uptake

Metabolism

Toxicity

Molecular changesPhysiological changes

Structural damage

Changed behaviour

Reduced fitness

PopulationGrowth, mortality, development time, fercundity

Communitydensity, diversity

Ecosystembalance, production

Discharge of xenobiot-amount-organic / inorganic-vapour pressure-degradability-solubility-Kow Dispersal

-air-water-soil

Elimination

Tolerance-physiological-geneticResistence

Exposure / effectbiomarkers

Effect / healthbiomarkers

Monitoring

Mat

hem

atic

al m

odel

ling

http://www.aula.au.dk/

Ecotoxicology

Introduction to xenobiotics

All substances are poisons; there is none which is not a poison. The right dose differentiates a poison from a remedy.

Paracelsus (1493-1541)

TOXICITY

Compound Median lethal dose mg kg-1

Ethanol 10000DDT 100Nicotine 1 Tetrodotoxin 0.1Dioxin 0.001Botulinus toxin 0.00001

Only the dose makes the poison

Dose - response

The receptor concept

- Toxicity is substance specific

- The response is proportional to the concentration at the receptors

- The concentration at the receptors depends on the dose (absorption)

- The concentration in the animal relates to the concentration in the environment

DOSE - RESPONSE

CONCENTRATION - RESPONSE

. .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

..

Linear Concentration

Response frequency distribution

In real life response frequencies are usually impossible to measure

0 5 1510 2520 30

Linear Concentration

0

10

20

30

40

50

Response frequency distribution

2.5 5 10 20 40

Log Concentration

0

10

20

30

40

50

Response frequency distribution

Toxicological endpoints are often log-normally distributed

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

. . . .

..

..

..

..

..

...

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.. .

.

.

.

.

..

.

.

.

..

.

..

.

.

.

.

.

.

.

..

.

.

..

.

..

.

..

.

.

.

.

.

..

.

.

..

.

..

.

..

.

.

.

.

.

.

..

.

.

..

.

..

.

..

.

.

.

.

.

.

..

.

.

..

.

..

.

..

.

.

.

..

.

.

..

.

.

..

.

..

.

..

.

.

.

.

.

.

..

.

.

..

.

..

.

..

.

.

.

..

1 10 100 1000 10000 100000

Concentration

Num

ber d

ying

in

sub-

popu

latio

n

Cumulative response

1 10 100 1000 10000 100000

Concentration

%cu

mul

ativ

em

orta

lity

Cumulative response

0

50

100

Transformation of data

Sigmoid curves difficult to treat mathematically

Wanted: A Linear relationship between dose and response

Dose/concentration is often log-transformed

The response is PROBIT-transformed

The Normal distribution is characterized by its mean

and scatter

Concentration

Freq

uenc

y of

resp

onse

0-1 +1 +2-2

FROM FREQUENCY TO PROBIT

Res

pons

e %

-4 -3 -2 -1 0 +1 +2 +3 +4

50

100

2 (0.1)

3 (2.3)

4 (15.9)

5 (50.0)

6 (84.1)

7 (97.7)

8 (99.9)

0.12.3

15.950

84.197.799.9

Normal Equivalent Deviation (NED):

The proportion of responding animals in terms of standard deviations from the mean of the normal distribution

ex: 15.9% respond: NED = -1

Probit(P) = NED + 5

Dose-response for two chemicals

Log concentration10-4 10-3 10-2 10-1 10 10+1 10+2 10+3 10+4

0.1

2.3

15.9

50.0

84.1

97.7

99.9

Res

pons

e (%

)

The two chemicals have the same LC50

Chem. A: Slowly absorption, fast detoxification

or excretion, delayed toxic effect.

Chem. B: Fast absorption and toxic effect

1 10 50 100 500

Log Dose (mg/kg)

% m

orta

lity

Assessment of toxicity

0

50

100

LD 50= 40 mg/kg

LOED

NOED

Assessment of toxicity

LD50/LC50 Median lethal dose/concentration

ED50/EC50 Median effective dose/concentration

EC10 Concentration affecting 10% of the population

LOEC(D) Lowest observable effect concentration (dose)

NOEC(D) No observable effect concentration (dose)

NEC(D) No effect concentration (dose)

ADI Acceptable daily intake

Safety factor Usually 100. 10 to account for human response variation, 10 for the extrapolation from experimental animals to humans

Essential and non-essential elements

Optimum

Window of essentiality Conc

P No effect

Conc

P

Ex: C, H, O, N, Ca, Na, K, P, Mg, Cl, STrace: Fe, I, Cu, Mn, Zn, Co, Mo, Se, Cr, Ni, V, Ar

Ex: Hg, Cd, Pb

METALS

Hg, Cd, Zn, Pb

PESTICIDES

Insecticides

Organochlorines (DDT, Lindan, Dieldrin)

Organophosphates (Dimethoat, Diazinon)

Carbamates (Methiocarb, Carbofuran)

Pyrethroides (Deltamethrin, Cypermethrin)

Rodenticides (Warfarin)

Herbicides (“Agent orange”, diquat, paraquat, dinitrophenoles)

Fungicides (Metylbromid, Dazomet, Borax)

POLYCHLORINATED BIPHENYLES (PCB)

Stable, non-reactive fluids (hydraulic- and coolant fluids, plastic paints)

POLYKLOREREDE DIBENZODIOXINER (PCDD)

By-products of burning PCBs and pesticide manufacturing

OIL PRODUKTS

Polyaromatic hydrocarbons (PAHs) - naphtalene, benzo(a)pyrene

ENDOCRINE DISRUPTORS

Alkylphenolpolyethoxylates, Bisfenol A, DDE, PCB, farmaceutikals, 17α-ethinylestradiol

CHLORIDE PHENOLS

Polychlorinated phenols (wood preservatives, bleaching agents)

TRIBUTYLTIN

Ship paints

DETERGENTS

Washing powder

Important xenobiotics

Cl

Cl Cl

Cl

Cl

Cl

3,3,4,4,5,5- HexachlorbiphenylPCB

p,p,DichlorDiphenyl TrichlorethaneDDT

ClCl C

ClCl Cl

C

H

Cl

Cl Cl

Cl

O

O2,3,7,8-tetra-chlorodibenzodioxin

Dioxin

N CH3CH3 N

Paraquatherbicide

Cl

ClOH

Cl2,4,5-Trichlorphenol

Bleaching agent

N

N

CH3

CH3

CH3

CH

OPS

C2H5OC2H5O

DiazinonOrgano phosphate

3,4-Benzo(a)pyrenePAH

OH

OH

CH3

17β- EstradiolHormone

p,p,DichlorDiphenyl Trichlorethane

DDTH

Cl

Cl C

Cl

Cl

Cl

C

HISTORICALSynthetized first time in 1874Insecticide properties discovered in 1939

- alter ion transport in axons - convulsionStable in nature – low mammal toxicity – cheap productionControl of malaria started in 1944

Ceylon 1946: 2.8 mill. cases - 13.000 died1963: 17 cases - 0 died

Saved about. 5 mill lives (100 mill serious cases)Now forbidden in most countries

Evolution of resistance and persistence in natureAccumulation of DDT and metabolites in food chainsEgg shell thinning

Half-lives for Cl-pesticides

0 2 4 6 8 10 12

Years

1

10

100% remaining in soil

DDT

Dieldrin

EndrinLindanAldrin

Cl

Cl

Cl

ClOClCl

Dieldrin

DDT/DDE in terrestrial systems

0 5 10

Year

0

5

10

15m

g D

DE

kg-1

Farming land treated with 9 kg DDT ha-1

DDE i earthworm

Reduction in Scandinavian Peregrine falcon populations

1940 1950 1960 1970 19800

1

2

Migratory birds/dayNumbers of migratory birds counted at Falsterbo in southern Sweden

Egg shell index of the British peregrine falcon

Egg shellindex

Peregrine falcon populations inCanada and North America

I=weight/LxB

DDT is highly lipid soluble

103 104 105 106 107

n-octanol:water distribution coefficient

10

100

1000

104

105

BC

F

2,2',4,4',5,5'-PCB

2,3,4'-PCB DDD

DDT

DieldrinEndrin

HCH

DDT biomagnification

Loss of organic matter through respiration

Fat solublepersistent chemical

DDT spreads globally in the atmosphere to water and soil

High lipophility ⇒ accumulates in adipose tissue

Air 0.000004

Attmospheric dust 0.04 mg/kg

Rain water

0.0002

Untilled soil ? Fresh water

0.0002

Salt water

0.0002

Agricultural soil 0.000004

Insects ?

Plants 0.05

Invertebrates in soil 4

Water plants 0.01

Invertebrates in water 0.001

Plankton 0.0003

Plant- & Insectivorous birds 2

Plant- & Insectivorous mammals 0.5

Fresh water

Fish 2

Salt water

Fish 0.5

Carnivorous mammals 1

Carnivorous Birds 10

Vegetables 0.02

Meat 0.2

Humans 6

Resistance against DDT

strain 1 strain 2 strain 3

LD50 (mg DDT/kg) 12-15 93-100 2500-3500

DDE produced (ug DDE/flue/time) 0 1.0 -1.5 3.8 - 5.2

Resistance in 3 fly strains

Genetic activation of the enzymatic metabolism of

DDT to DDE and faster elimination

DDT DDD

ClCl CH

Cl ClCCl

ClCl CH

H ClCCl

Metabolism of DDT

Non-reactive chlorine atoms - DDT og DDE degrade slowly

Degraded photochemically and microbially in nature

Partly metabolized in higher organisms (depending on enzyme systems)

DDT and DDE accumulate in adipose tissue

DDA (and DDD) are excreted by the kidneys

ClCl C

Cl ClC

H

Cl

ClCl CH

ClCl CHDDT

H ClCCl

DDD

Cl ClCH

CO OH

DDA

Cl ClC

DDE

PCBs, Dibenzofurans and Dioxins

5

2' 3'

4'

5'6'

1

23

4

6

1'

O

1

7

82

3

4 6

9 O2

34 6

91

7

8

O

DioxinsPCBs Dibenzofurans

Cl

Cl Cl

Cl

O

O

2,3,7,8-tetra-chlorodibenzodioxin

DIOXIN

Dioxins are generated as unwanted by-products by:

The production of herbicides - ex 2,4,5-T (Seveso accident)

Heating of the fungicide pentachlorphenol

OH

Cl

Cl

Cl

ClCl

Cl

Cl

Cl

Cl

OH

Cl

Cl

Cl

OCH2COOH

Cl

Cl

Cl

NaOH

180ºC

CICH2COOHNaOH

Cl

Cl Cl

Cl

O

O230ºC

Tetraklorbenzene 2,4,5-Trichlor phenol2,3,7,8-tetra-chlorodibenzodioxin

Waste combustion of e.g. phenol compounds, PCB and PVC at too low temperature (< 1200ºC)Exhaustion gasses from leaded petrol (halogenated hydrocarbons)Chlorine bleaching of paper (e.g. Trichlor phenol)

Dioxines’ distribution, metabolism and harmful effectsAtmospherically and aquatic distribution leads to global pollution

3 10 25 50 100 150 km

7000

6000

5000

4000

3000

2000

1000

pg T

CD

D/g

glø

deta

b)

Concentration of 2,3,7,8 TCDD in sediment-transect at a Swedish paper mill

Dioxins are present in most aquatic organisms (pg)Very lipophilic – accumulate in adipose tissue2,3,7,8-TCDD is considered the most toxic synthetic compound

LD50 µg/kgGuinea pig 0.6Rat 22Chicken 25-50Monkey 70 .

Toxicological mechanism partly unknown

Loss of weight and death after 1-7 weeks

Teratogenic (embryonic injury)

Carcinogenic

Environmental data for dioxin

Log Kow 6.8

Log BCF’s fish 3.2-4.4

snail, dafnia 4,3-4.4

algae 3.6-3.95

Volatisation1/2 life soil, sediment >50 YEARS

Source: EPA (USA)

2,3,7,8-TCDD ‘acute’ toxicity

Experimentalanimal

LD 50

(µg/kg)

rat 22 ♂rat 45 ♀Guinea pig 0.6♂Guinea pig 2.1 ♀rabit 10-115chicken 25-50dog 100-3000monkey 70mouse >150hamster 1157-5000frog >1000

Acute symptoms: liver damage, thymus damage, significant weight loss, death occurs between 1 and 7 weeks after intake

Current views on Dioxin effects

No effectsUrinary systemInconsistent evidenceImmunological findings

Compelling evidence but not completely consistent

Thyroid function

Inconsistent findings, but reports from Vietnam and Seveso. Not depression

Neurological, Psychological findings

InconsistentRespiratory system effects

Positive association in high dose studies, not entirely consistent

Cardiovascular disorders

Inconsistent but found in agent orange studies

Diabetes

Inconsistent but compelling evidenceReproductive disorders including outcome

Accepted?Increase in cancer incidence, including reproductive cancers

Temporary effect but provenInduction of liver enzymes (AHH)Proven associationChloracne

Consistency of evidenceEffect

A Japanese chloracne patient

Many sp. High dosesOvarian dysfunctionRatsReduced circulatory

androgensRatsReduced spermatogenesisMonkeys, Rats, Mice Low concentrations, chronic exposure

Endometriosis (Uterus epithelium cysts outside the uterus)

Rats, High dosesAnovulation, suppression of oestrous cycle

Many sp. High dosesInfertility and fetal lossSpecies, commentsEffect

Reproductive effects seen in experimentalanimals after dioxin exposure

A cancer promoting pathway for dioxin

TCDD + AHH Activated product

(Adduct)

Unwinding enzymes

Excision

ResynthesisRepair

Mistake Mutation

Cancer

Ahh activity can be used to determinerelative toxicity in complex mixtures

TCDD

TCDD

+

TCDD

mRNA

AHH

DNA

Ah

Aryl hydrocarbon hydroxylase

TCDD

TCDD

TCDD Ah

Ah

Relationship between AHH-inductionand general toxicity

2 3 4 5 6 7 8

Weight loss (-log ED 50)

4

5

6

7

8

9

10

11AH

H-in

duct

ion

(-log

EC

50)

DioxinFuran

Substitution pattern Conversion factor ng/g fat TEQ

PCDD2,3,7,8-tetraTCDD 1 2,3 2,31,2,3,7,8-pentaTCDD 0,5 4,8 2,41,2,3,6,7,8-hexaTCDD 0,11,2,3,7,8,9-hexaTCDD 0,1 6,4 0,641,2,3,4,7,8-hexaTCDD 0,1 35 3,51,2,3,4,6,7,8-heptaTCDD 0,01 41 0,41octaTCDD 0,001 132 0,13

total PCDD 9,3

TCDD-equivalents(Toxic equivalent quotient)