Ecotoxicology• Ecotoxicological study is a multi-step proces

s, involving:– The entry, distribution and fate of pollutant

s within the environment;– The entry and fate of pollutants in living (b

iota) organisms within an ecosystem; and – The harmful effects of the chemical polluta

nts on the constituents (biotic & abiotic) of ecosystems (which include man).

Beyond our toxin trailIs the grave deeper than we

thought?

Toxinemitted

Transportand fate

Ingested

Contactshuman

Reachesan organ

Metabolizedand/or stored

Physiologicalchain of eventsPopulation

effectsCommunity

effects

Ecosystemeffects

Toxicology• Host defense

mechanisms• Individual

susceptibility states

• Single effects• Cumulative

exposure

Ecotoxicology• Bioaccumulation• Bioconcentration (i

n water)• Biomagnification• Never single effects• Movement between

media (air, water)

• The basis for determining the effects of contaminants on ecosystem is at organism level

• At organism level, response can be:– Acute toxicity causing mortality– Chronically accumulating damage ultimately c

ausing death– Sublethal impairment of various aspects of ph

ysiology and morphology– Sublethal behavioral effects– Measurable biochemical changes

Ecological bases of Ecotoxicology

•At population level, response can be:–Size and dynamics (based on birth rates, death rates, gains, from immigration and losses from emigration)–Cause a reduction or an increase in the natural flowchart of numbers, in the biomass, sex ratio, etc.

•At community level, response can be:–species diversity–predator prey relationship, etc

•Change in ecosystem –nutrient cycling rates, patterns of nutrient flow, –physical-chemical conditions etc.

Assessment of Structural Changes

    changes in species / population  structure

                  -  appearance/disappearance of an indicator species

                  -  number of individuals of a species                   -  biomass of a species                   -  presence or absence of a species

Biomass-a quantitative estimate of the total mass of living plant or animal materials

       

changes in community/ecosystem structure

                        -  biomass

                        -  abundance

                        -  biotic indices (e.g. trophic types)

                        -  species richness / diversity

                        -  dominance

                        -  food chain length/complexity

Chemicals of ecotoxicological interest

• They are toxic and in many cases their metabolites are also harmful e.g. DDT & DDE (metabolite of DDT)

• They are very stable both chemically and environmentally

• Their stability has lead to their persistence and ubiquitous nature in the environment

• Almost all chemicals of ecotoxicologigal interest are bioavailable and in most cases undergo bioaccumluation and biomagnification (food chain)

Bioavailabiltiy The fraction of a chemical that is in an available form to an organism e.g. fish: food, absorption from water

Bioconcentration - where the chemical concentration in an organism exceeds the concentration in the surrounding media (i.e. aquatic environment) as a result of exposure through the respiratory surfaces (i.e. gills/dermal surfaces) - not food!

Bioconcentration Factor = conc. in organismconc. in ambient medium (usually water)

Bioaccumulation - where the chemical concentration in an organism achieves a level that exceeds that in the water/media as a result of chemical uptake through all routes of exposure. Bioaccumulation factor = Conc. in organism

Conc. in food (or ingested water)

•Bio-accumulation of Cd is higher than most metals as it is assimilated rapidly and excreted slowly

•depends on the rate of excretion

Biomagnification - where the chemical concentration in an organism achieves a level that exceeds that in the organism’s diet due to dietary absorption. i.e. higher trophic levels accumulate more chemical

Biomagnification Factor = Conc. in predator

Conc. in prey

Factors that influence bioaccumulation-Environmental persistence-Lipophilicity-Biotransformation

Toxic Effects• The biochemical (molecular in nature) or

physiological (observed at organ and whole organism levels) changes which adversely affect individual organisms’ birth, growth or mortality rates.

• Both biochemical and physiological changes could lead to behavioral (whole organism level) changes.

• Toxicant binding: – Reversible vs. Irreversible binding

• Irreversible binding (covalent) causes harmful effects.– Types of bonding: – Covalent > ionic > Hydrogen binding > Vanderwaals > hy

drophilic

• Biochemical responses:– Biochemical response could be protective or non-protec

tive (may or may not cause harmful effect).

• Non-protective biochemical responses have carcinogenic, mutagenic, teratogenic and neurotoxic potentials.

• Protective biochemical responses

– Monoxygenase (OCs and PAHs)

– Induction and binding to metalothionein (Cu, Cd, Zn and Hg)

– Binding to blood plasma, bones and hair (Metals and xenobiotics)

– Dissolving in fat (organics- e.g. OCs)

– Mineralization ( e.g. MeHg to Hg 2+)

– Demineralization (As to MeAs)

Protective biochemical response

• Heavy metals for example can be stored and detoxified by organisms either by binding to specific proteins e.g. metallothioneins (-SH proteins)

• In some cases it is mineralized to inorganic form, which is less toxic: e.g. Hg bound to Se is a mineralized Hg (detoxified Hg: MeHg to Hg). On the other hand, the inorganic form, which is more toxic can be methylated to a less toxic form e.g. As.

Protective biochemical response

PHASE 1 REACTION. • Organic pollutants could also be metabolized and d

etoxified by Cytochrome P450 enzymes (Microsomal Monoxygenase; MMO).

PHASE 2 REACTION • The metabolites undergo conjugation with endogen

ous molecules e.g. GSH.

• For some chemicals the metabolites/conjugated form are more toxic than the parent compound and can lead to cancer formation.

– Binding to DNA (DNA adduct)

– DNA structural damage (strands break) induced by genotoxic compounds

– Binding to SH-Protein (Protein adduct); enzymes and proteins

– Neurotoxicity: prolongation of K and Na flow and inhibition of AChE activity in the brain

Non-protective response

Non-protective response

– Mitochondrial Poison (lost of proton gradient)

– Inhibition of vitamin K cycle (competition with vit K binding site)

– Inhibition of Thyroxine (competition with thyrosine binding site)

– Inhibition of ATPase (enzymes for transport of ions e.g. K, Na, Ca)

Non-protective response

• Environmental estrogens (eg DDT) and androgens (tributhyl Tin)

• Endocrine disrupters (binding to endocrine receptors)

• Photosystems of Plants (interruption of electron flow)

• Plant growth regulation

Physiological changes Non-protective biochemical responses lead to

Physiological changes which could be observed at organ and organism levels

• Organ level:– accumulation of Cd in kidney, which could cause

cell death (cytotoxicity), resulting in dysfunction of the kidney

– PAHs and Lung cancer

• Organism level:– decrease in production (growth and reproduction)– changes in gene frequency– decrease in resources acquisition and uptake

Behavioral Changes

– Either or both physiological and biochemical effects could lead to behavioral effects at organism level-

– migration, – intraspecific attraction, – aggregation, – aggression, – predation, – vulnerability, – mating – caring for young ones and avoidance of predator.

.

Population Changes

• Changes in population may come about as a result of direct changes in numbers of individual organism and gene frequency

• By indirect means (decrease in population of predators due to toxic chemicals could lead to increase in numbers of its prey).

Diclofenac residues as the cause of vulture population decline in Pakistan.

Nature. 2004 Feb 12;427(6975):630-3.• Diclofenac cause

s kidney damage, increased serum uric acid concentrations, visceral gout, and death.

• Changes in community structure– change in pyhtoplankton assemblage due to

eutrophication– acid rain affecting microorganisms in the soil, aquatic life

• Changes in Ecosystem level (earth as an ecosystem)– carbon dioxide increase – ozone depletion

What is an Endocrine Disruptor ?

““An exogenous agent that interferes with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body that are responsible for the maintenance of homeostasis, reproduction, development and/or behavior. “

Mechanisms of endocrine disrupting compounds

1) Binding and activating the estrogen receptor 2) Binding but not activating the estrogen

receptor (therefore acting as an anti-estrogen)

3) Binding other receptors4) Modifying the metabolism of natural

hormones5) Modifying the number of hormone receptors

in a cell6) Modifying the production of natural hormones

Hormone regulation and feedback controlEstrogen levels depend on

Estrodiol sulfateEstrodiol serum-binding proteins-fetoprotein (AFP)Testosterone-estradiol binding globulin

Xenoestrogens (ex. DES)100-fold lower affinity than E2 to these binding proteinBioavailability increased

Non-genomic mechanisms of ED action

• Compounds of the azole type, such as ketoconazole and the fungicide fenarimol, inhibit CYP isoforms and consequently can also affect steroid synthesis while the now-banned anti-fouling agent tributyltin and its metabolites, which have strong ED potential, are thought to act by the same mechanism, probably by inhibition of aromatase.

Genomic mechanisms of ED action

• bind to oestrogen receptors and so act as pseudoestrogens in vivo, giving feminizing effects

• tamoxifen and diethylstilbestrol and industrial chemicals (e.g. octylphenol and bisphenol-A

• fungicide vinclozolin binds competitively to the androgen receptor, blocking the cellular actions of testosterone on androgen-dependent tissue growth and behaviour patterns

• chlordecone, inhibit binding to the oestrogen and progesterone receptors, whereas bisphenol-A can block ligand binding to the thyroid receptor

Timing, duration, and amount of exposure. Organization vs. activationTiming, duration, and amount of exposure are each important determinants of the outcome. There are windows of vulnerability during fetal development in which small exposures to endocrine disruptors may have profound effects not observed in adults.Studies of the intrauterine position of mice during fetal development show that slight fluctuations of steroid hormone levels influence genital morphology, timing of puberty, sexual attractiveness, sexual behavior, aggressiveness, and activity level of offspring.

Various Classes of EDCsFlame Retardants

Fungicides

Herbicides

Insecticides

Metals

Pharmaceuticals

Phenols

Plasticizers

Polyaromatic Hydrocarbons

Soy Products

Surfactants

Polybrominated diphenyl ether

Vinclozolin

Atrazine

Methoxychlor

Tributyltin

Ethynyl Estradiol

Bisphenol A

Phthalates

PCBs, dioxins

Genistein

Alkylphenol

Ethoxylates

PBDEs （多溴二苯基醚） • Polybrominated diphenyl ethers (PBDE

s) are a class of recalcitrant and bioaccumulative halogenated compounds that have emerged as a major environmental pollutant. PBDEs are used as a flame-retardant and are found in consumer goods such as electrical equipment, construction materials, coatings, textiles and polyurethane foam (furniture padding). 　　

Found in animalsIncrease in fishIncrease in whalesSewage sludgePCBs Found in Lake Washington

Fish (PBDEs next?)

Found in human (breast milk)

Bioavailability of PBDEs

PBDEs Breast Milk - Sweden

(Norén and Mieronyté, 1998)

Similar to PCBs (Polychlorinated biphenyls)

Persistent Bioaccumulative Toxicant

No human dataAnimals studies indicate

Changes in thyroid hormone levelsNeurobehavioral toxicity

Development effects- alters BehaviorImpairs memory and learning

Delays sexual development

Health Effects of PBDEs

Vinclozolin

• Vinclozolin is a fungicide that has been shown to cause Leydig cell tumors and atrophy of the accessory sex glands in adult rodents. In addition, exposure of rats during pregnancy causes a pattern of malformations in the male urogenital tract .

• Androgen receptor antagonist

Atrazine• A chlorotriazine herbicide, is used to control annu

al grasses and broadleaf weeds. • Suppression of the luteinizing hormone surge duri

ng the estrus cycle by atrazine leads to the maintenance of elevated blood levels of 17beta-estradiol (E2) and prolactin.

• The mechanism for tumor development may include one or more of the following: the induction of aromatase (CYP19) and/or other P450 oxygenases, an antagonist action at the estrogen feedback receptor in the hypothalamus, an agonist action at the mammary gland estrogen receptor or an effect on adrenergic neurons in the hypothalamic-pituitary pathway.

BPA is used in the manufacture of polycarbonate plastics and epoxy resins from which food and beverage containers and dental materials are made. Perinatal exposure to environmentally relevant BPA doses results in morphological and functional alterations of the male and female genital tract and mammary glands that may predispose the tissue to earlier onset of disease, reduced fertility and mammary and prostate cancer.

雙酚ＡBisphenol-A

聚氯乙烯（ PVC ）製的嬰兒固齒器、玩具讓長牙的嬰兒咬玩的固齒器、洗澡玩的軟性玩具、價格不貴的流行卡通玩具常常是 PVC 製品，在使用中可能釋出鄰苯二甲酸（ phthalates ）這類有致癌性的環境荷爾蒙。【安全替代品】仔細查看成分標示，凡是嬰幼兒可能放到口中把玩的玩具一定選購 PE （聚乙烯）製品。

苯乙烯 alkylphenol （烷基酚）攤販、自助餐店、速食店的熱飲杯（裝湯、茶、咖啡）、泡麵的碗麵及杯麵絕大多數都使用聚苯乙烯（ polystyren ）的塑膠容器，簡稱為 PS ，被國人稱為保麗龍。其原料單体叫苯乙烯，是已知致癌物，且製造過程所添加的塑化劑 alkylphenol （烷基酚）也是會干擾內分泌的環境荷爾蒙，二者在使用過程很容易溶出到食物中。

化妝品中的環境荷爾蒙

多數的化妝品、卸妝用清潔用品含有幾類的環境荷爾蒙：•壬基苯酚乙烯（一種非離子界面活性劑）•鄰苯二甲酸（ phthalates ）•烷基酚（ alkylphenol ）

Phthalates 鄰苯二甲酸酯 • 廣泛存在於化粧品、兒童玩具、食品包裝中 。• 香水中可當作溶煤或香料固著劑使用 (已禁用 )。

• 常用於塑膠類製品的塑化劑，可能遇熱溶出。PVC 或 PVDC 保鮮膜； PVC 塑膠手套。

• male infertility • Interfere with cholesterol uptake and androgen biosynthesis

Tributyltin (TBT)

三丁基錫是一種有機錫化合物，常被添加於船舶油漆中，以防止貝類及藻類附著於船身，由於具有殺菌效果，

所以也可以作為殺菌劑使用

受到三丁基錫或三苯基錫污染的雌岩螺，因生殖孔阻塞受精卵無法排出，堆積在生殖管道內變紅變黑形成壞死組織，此時長出陰莖的雄化作用也同時被引發

TBT

Yucheng ( 油症 ) of Taiwan, 1979

• ~2000 people exposed to PCB-contaminated rice oil

• with chloracne, fatigue, skin and nail pigmentation, polyneuropathy, and abnormal liver function

• Long-term follow-up study was established for 1st and 2nd generation (prenatal exposed)

Likely Kanechlor 500 (a Japanese PCB mixture)

Semen Analysis40 Yucheng men (37-50 yr old)

compared with 28 controls

• Volume and count: no difference• Oligospermia: 23% vs. 4% • Morphology: 28% vs. 23% abnormal (~18%

increase)• Motile sperm: no difference• Speed by CASA: no difference • Chinese hamster oocyte penetration after o

ne re-thaw cycle: 16% vs. 32% (50% drop)

(Hsu et al., JAMA 2003)

Children Prenatally Exposed

• More stillbirth (Yu et al., 2000)

• Called Yucheng children• Born with darker skin, nail pigmentati

on and deformity, and developmental delay (Rogan et al., 1988)

• Reduced neurocognitive functioning up to ages of 12 years (Chen et al., 1992)

• Behavioral problems (Lai et al., 2002)

*Measured by “Blinded” physicians

Penile length (cm)* in Yucheng and Control Boys by Age

6 to 7 8 to 9 10 to 11 12 to 13 14 to 150

1

2

3

4

5

6

7

8

9

6 to 7 8 to 9 10 to 11 12 to 13 14 to 15

Yucheng Controls

Hormones in prenatal PCB/PCDF-exposed boys and unexposed controls after puberty

Sex Hormones Control Yucheng

Testosterone (TT, ng/mL) 4.2 ± 2.2 3.0 ± 2.4

Estradiol (E2, pg/mL) 21.3 ± 13.2 48.6 ± 53.9

√(TT/E2) 0.5 ± 0.2 0.3 ± 0.2

Follicle-stimulating horm

one (FSH, mIU/mL)

3.4 ± 0.8 4.6 ± 2.2

√(TT/FSH) 1.1 ± 0.3 0.8 ± 0.5

√(E2/FSH) 2.4 ± 0.8 3.1 ± 1.4

No difference: luteinizing hormone prolactin

>

<

>

<

>

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(Hsu et al., JTEH 2005)

（一）殺蟲劑或其代謝中間產物：計有 26種，（二）殺菌劑：計有 9種（佔 13％）。（三）除草劑：亦有 9種（佔 13％）（四）塑膠之塑化劑：亦有 9種，（佔 13% ）。禁用鄰苯二甲酸鹽作為玩具塑膠之塑化劑。（五）醫藥、化工原料合成之中間產品：計有 6種。（六）有機氯化物之污染副產品或菸煙中之芳香族烴計有 3種，其中以戴奧辛夫喃廣布於空氣、土壤、底泥、甚至於食品、乳製品中最為令人憂心。（七）熱媒及防火材料：有 2種，其中多氯聯苯，惡名昭彰，雖已禁用多年，但在環境介質中，仍時常檢出。（八）界面活性劑之代謝分解中間產物：非離子界面活性劑廣用於各種民生日用清潔劑、乳化劑中（九）有機錫：計有 2種，作為魚網之防腐劑及船上抗腐蝕油漆。（十）重金屬：計鉛、鎘、汞三種，亦列為內分泌干擾之疑似物質

EDSTAC Tier 1 AssaysConcerned with detecting

• Receptor binding assays (ER and AhR)Receptor binding assays (ER and AhR)

• UterotrophicUterotrophic

• HershbergerHershberger

• Pubertal femalePubertal female

• SteroidogenesisSteroidogenesis

• Frog metamorphosisFrog metamorphosis

• Fish reproductive screenFish reproductive screen

EDSTAC Tier 2 dose-response relationship

• Mammal development and reproductiMammal development and reproductionon

• Bird development and reproductionBird development and reproduction

• Mysid shrimp life cycleMysid shrimp life cycle

• Fish reproduction and development Fish reproduction and development

• Amphibian development and reproduAmphibian development and reproductionction

Species-dependent sex determination

Mammal XY/XX

synthesis of testosterone/functional androgen receptors

estrogen receptor in the brain

Birds WZ/WW

The ability to synthesize and recognize 17-estradiol is necessary for female CNS and gonadal sexual development to occur

Reptile

temperature-dependent sex determination (aromatase related)

thermosensitive period (TSP)

Temperature-dependent sex determination

Temperature determines their sex. A nest temperature of 73.5 degrees would develop males. If it heats up to 83.5, hormones would trigger changes causing the embryonic cells to differentiate as females.