Aquatic Ecotoxicolgy PDF

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Session 1: Introduction  Definition  Ecotoxicology:​ According to Newman (2015), Ecotoxicology is the science of contaminants in  the biosphere and their effects on constituents of the biosphere, including humans.  The study of the fate and effects of toxic chemicals on organisms...

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Session 1: Introduction Definition Ecotoxicology:According to Newman (2015), Ecotoxicology is the science of contaminants in the biosphere and their effects on constituents of the biosphere, including humans. The study of the fate and effects of toxic chemicals on organisms and ecosystems. Ecotoxicology considers both direct and indirect effects of toxic substances. Chemical:a substance with specific chemical composition and characteristics Contaminant:a chemical exceeding the natural concentration level Pollutant:a contaminant causing harm to an organism Toxicant:a contaminant causing harm to an organism through interference with its biochemistry or physiology Xenobiotic:a chemical that does not occur naturally (in organisms)

 Ecotoxicology - an interdisciplinary field Chemistry (analytical, organic, biochemistry) Biochemical reactions Pharmacokinetics/pharmacodynamics Environmental physics Molecular/Cellular reactions Ecology  Session 2: Fate of chemicals General processes: Transport:Movement of molecules with an environmental compartment Transfer:Movement of molecules across ecosystem boundaries with changes in their state Transformation:Structural alteration of molecules changes in their state 

What are potential stressors? Heavy metals, Pesticides, Pharmaceuticals and Personal care products, Nanoparticles, Microplastics, Oil spills etc.  Illustrate some potentially problematic groups of compounds. Inorganic toxicants: ● ● ● ●

Organic toxicants: 

Metals Non-metallic salts Acids and bases Nanoparticles (some organic)

● ● ● ● ● ● ●

Pesticides Hydrocarbons and PAHs PCBs, PBBs, PCDDs Organometals Pharmaceuticals and personal care products Polybrominated flame retardants Plasticizers and surfactants

 Sources of pollution: 1. Point sources- direct entry into water Molecules come from a single, well-defined location, e.g. spillages, faulty equipment, washings and waste disposal, drainage, direct entry including overspray, consented discharges etc. 2. Nonpoint (diffuse) sources- indirect entry into water Molecules are introduced into the environment over a large, widespread area, e.g. spray drift, volatilisation and precipitation, surface runoff, leaching, throughflow/interflow, drainflow, base flow seepage etc.  Exposure pattern of chemicals in water: 

Wastewater

Agricultural stream

Chemical composition

Composition depends on local, domestic and industrial usage

Mainly agrochemicals

Duration of exposure

Daily and seasonal fluctuations

Depending on environmental conditions

Concentration of chemicals Dilution depends on receiving stream and weather conditions but rather constant load

Highly variable

 Environmental monitoring of chemicals Point sampling

- episodic events missed

Event-driven sampling Automatic sampler

Simple sampler

- high resolution of

- catches peak

Continuous sampling/ Passive sampling - relies on passive diffusion in

events

concentrations

receiving phase

- recurring monitoring relatively time- and labour intensive

- very high costs for - very economic - available for water, air and soil equipment and analysis alternative to – though majority of automated system applications for water and air

- no sophisticated equipment needed, high quality control

- higher detection limit - rapid sample - exposure time varies from recovery necessary days to months for continuous  monitoring

- Conventional monitoring  relies on grab water sampling



- various receiving phases available for organic and inorganic contaminants

 Passive sampling: Practical application Example: PDMS strips as passive sampler 1. Cutting of rubber strips 2. Conditioning: Removal of impurities before sampling 3. Field deployment and retrieval after defined time, e.g. 3 weeks 4. Extraction in solvent and evaporation → Analysis  Session 3: Fate of chemicals What are the main routes of chemical exposure in surface water? ● Sewage outfalls ● Outfall from commercial premises ● Outfalls of nuclear power stations ● Runoff from land ● From the air (precipitation with rain/snow, direct biocide application, accidental contamination by sprays/dust) ● Dumping at sea ● Release from oil rigs and terminals ● Shipwrecks  Which factors determine exposure? Solubility: Is the chemical repulsed or mix well with water? Partitioning coefficients and vapor pressure: To which compartment does the chemical adhere? Melting point: Does it occur as gas, liquid or solid for a given temperature? Chemical structure: How does it behave in the environment? Persistence: Does the chemical easily degrade and what are the metabolites?  Transfer processes:

 Deposition: Settlement of molecules from the air to a solid or liquid phase Volatilisation: Mobilisation of molecules from a solid/liquid phase Adsorption: Attachment of molecules to a solid phase Desorption: Mobilisation of molecules from a solid phase  Absorption vs. Adsorption Absorption:

Adsorption:

Molecules penetrate the surface of another medium

Adhesion of molecules to a surface

Penetration of a molecules through the surface of another medium





 The higher the Kow the higher its lipophilicity

The higher the Koc the less mobile

 Describe two coefficients that describe the distribution of chemicals in the environment 1. Octanol-water coefficient (Kow) 2. Organic carbon adsorption coefficient (Koc) 

Deposition- Settlement of molecules from the air to a solid or liquid phase Dry deposition: Flux of molecules and particles from air to liquid or solid surfaces Wet deposition: Formation of pollutants in the liquid phase or uptake in the liquid phase during precipitation  Volatilization: Change of a liquid or solid to a gaseous state

 The higher Henry’s constant the less volatile.  Resuspension: Remobilization of contaminants from sediment through biological (bioturbation) and physical processes  Define processes determining the fate of nanoparticles, micro- and nanoplastics Fate of nanoparticles 1. 2. 3. 4. 5. 6. 7. 8.

Fate of micro- and nanoplastics

Dissolution Sulfidation Homo-aggregation Hetero-aggregation Coating with NOM(Natural Organic Matter) Biological surface coating Sedimentation/deposition Persistence

1. 2. 3. 4. 5. 6.

Turbulent transport Settling Aggregation Biofouling Resuspension Burial



 Session 4: Fate of chemicals Name and describe abiotic transformation processes Abiotic processes Hydrolysis

Photolysis

– Electron-rich groups interact with electro-deficit parts of a molecule sites -> formation of new bonds – Not all organic compounds are prone to hydrolysis – Often irreversible – Temperature and pH dependent

– Adsorption of light energy – Light of short wavelength is highly energetic – Direct photolysis • Direct interaction of molecule with light – Indirect photolysis • Energy is transferred from another molecule – Irreversible; not necessarily detoxifying

Assessing hydrolysis according to OECD 111 – Goals: Hydrolysis dynamics depending on pH

Assessing photolysis according to OECD 316 – Goals: Direct photolysis at wavelength >290 nm

 Define biotic transformation processes Biotic processes: Enzymatic degradation – Enzyme inventories of organisms – Different enzymes involved – Aerobic or anaerobic  Define which physico-chemical properties determine fate between and within environmental compartments and into biota

 Exposure modeling FOCUS (FOrum for the Co-ordination of pesticide fate models and their Use) – Tiered approach

– Step 1: simple model accounting for extreme worst-case static ditch; water depth: 30 cm; sediment layer: 5 cm – Step 2: application pattern, degradation, site specific parameters (infiltration etc) – Step 3: 10 realistic worst-case scenarios reflecting approx. 33% of EU agricultural land; reflection of site specific parameters; three water bodies (i.e., pond, ditch, and stream) as relevant – Step 4: mitigation options  Session 5: Bioaccumulation and magnification Define related technical terms Bioaccumulation: Accumulation of a contaminant in an organism from all media (i.e., gaseous, liquid and solid) Bioconcentration: Accumulation of a contaminant in an organism from water Biomagnification: Increase in concentration with trophic level in food webs  What are the cellular uptake mechanisms? Toxic chemicals must pass through cell membrane to exert effect. Membranes consist of bimolecular layer of phospholipids with integral and peripheral proteins. Given the complex and dynamic nature of cell membranes, chemicals pass to and fro across cell membranes in many ways -  - Paracellular transport: Movement through junctions between cells. In case of sulfide in lugworms, that elimination occurred with oxidation to thiosulfate and simple thiosulfate diffusion out of the worm via the paracellular route. - Diffusion:Passive transport of substances through cell wall and ion channels and pores - Endocytosis: Chemicals adsorbed to cell walls are included in cells - Facilitated diffusion:By carrier molecules, faster than simple diffusion - Active transport:Requires energy; might be influenced by competitive inhibition. Energy is used to exchange ions with the surrounding. Metals may be a chemical analog to the actual target ion  Organic contaminants, mostly uptake is by passive diffusion through the lipid bilayer  Biotransformation of organic compounds in organism After entering a gut epithelial cell, organic contaminant is metabolized by Phase I reactions and then conjugated.  – Phase I reaction (often CytochromeP450- dependent – Phase II reaction monooxygenase) • Conjugates are formed • Reactive groups (OH, COOH etc) are added or -> inactivation or faster elimination made available -> large and often polar endogenous -> contaminants become more reactive compounds are covalently added -> enzymatically catalysed

-> more prone to oxidation but also hydrolysis and reduction -> elimination Chemical + O2 + 2e- + 2H+ -> Chemical-OH + H20

– Glutathione (GSH) interacts with electrophilic chemicals (replaces hydrogen, chlorine, nitro-groups) through glutathione S-transferases (GST)

 Define & describe elimination processes Elimination: contaminant loss due to biotransformation or excretion Not to be mixed with Depuration: associated with an experimental design – loss of substance when placed in clean medium Clearance: substance movement between compartments – concentration normalized rate Growth dilution: same amount of contaminant but more biomass in which it is distributed -> body burden remains the same  Elimination processes in - Plants

Animals

– Transformation – Leaching – Evaporation – Leaf fall – Grazing

– Transport across the gills, exhalation, bile secretion – secretion from the hepatopancreas or intestinal mucosa – shedding of granules – loss in feces, molting, excretion through the kidney, egg deposition, or loss in hair, feathers, milk, and skin

 Calculate bioconcentration factors Bioconcentration factor (BCF); estimated at steady-state

 What is a TKTD? Toxicokinetic and Toxicodynamic Model Simulates Toxicokinetic (Uptake, distribution, metabolisation, excretion) processes and Toxicodynamic processes (reactions of the chemical at the target site)

  Session 6: Biological responses Biological responses – sub-organisms (biochemical; physiological responses) – organisms (growth, survival reproduction, behaviour) – populations (population size, structure) – ecosystems (community composition, functions and process)  Define biomarker categories Biomarkers: biological (biochemical, physiological, histological and morphological) indicators measured inside an organism or its products. Biomarkers could help identifying effects on a sub-organismic level. Biomarkers are useful to link external exposure to some internal biological response  According to the WHO (1993), biomarkers can be subdivided into three classes: . Exposure biomarker: Measure the stressor, the metabolite or a reaction product in the organisms Effect biomarker: Biochemical, physiological or other alterations within the organism Susceptible biomarker: Ability (inherent or acquired) of an organism to respond xenobiotic substance  What can we measure through biomarker ? – mRNA levels using e.g., qPCR – CYP1A proteins (isoenzymes) using e.g., ELISA (immunoassays) – Enzyme (catalytic) activity (EROD, AHH) using e.g., spectrophotometry  Downside of CYP450 – Activation of chemicals (e.g., benzo[a]pyrene) – Alteration in endogenous substrate metabolism (e.g., steroids, fatty acids)  Name representative biomarkers for different types of stress 1. Biotransformation enzymes (phase I and II) 2. Oxidative stress parameters 3. Biotransformation products

4. 5. 6. 7. 8. 9. 10.

Stress proteins, metallothioneins (MTs), MXR proteins Hematological parameters Immunological parameters Reproductive and endocrine parameters Genotoxic parameters Neuromuscular parameters Physiological, histological and morphological parameters

 Discuss the benefits and limitations of biomarkers Benefits of biomarkers 1. Responses are more universal on a cellular level than at higher levels of biological organization, so biochemical responses may be similar in a large variety of organisms. 2. Good biomarkers are sensitive indices of both pollutant bioavailability and early biological responses. 3. They can give information on the biological effects of pollutants rather than a mere quantification of their environmental levels. 4. Biomarkers may provide insight into the potential mechanisms of contaminant effects. 5. Biomarkers applied in both the laboratory and the field, can provide an important linkage between laboratory toxicity and field assessment.  Limitations of biomarkers 1. Improper application or interpretation of biomarker responses, may lead to false conclusions as to pollutant stress or environmental quality. 2. Certain responses established for one species are not necessarily valid for other species. 3. Many non-pollution-related variables may have an additional impact on the various enzyme systems, and may thus interfere with biomarker responses 4. The relationships between biomarker responses and field population-level effects are (presently) not well defined. 5. Since various substances may affect the same biomarkers, most biomarker responses are not specific for individual compounds. 6. For mobile species such as fish, the actual duration of exposure is uncertain. 7. In order to avoid potential artefacts, particular care in sampling and handling of samples is required.  OMICS OMICS may allow to follow functional impairments from genome to individuals Omics help to understand how chemicals – affect genomes, – transcription of genes to messenger RNA, – translation of mRNA coded information to proteins and – determine functional implications in organisms 

  • Genomics – Studying the genome of organisms – Informs about DNA sequence

• Proteomics: Information of type and amount of proteins – Protein diversity and quantity – Translation product – Can be done via electrophoresis or mass spectrometry • Analyses peptide signature • Database to identify protein of interest

• Transcriptomics: Do not give information on mode of action unless linked to physiological reactions – Transcription product – Amount of Codes for a protein increases (mRNA) – Quantification by (quantitative) PCR • Reference genes to normalise gene expression – Transcription can be affected by: • Sex • Tissue • Time of day • Season • Developmental state -> need of proper controls – Example: antibiotic resistance genes

• Metabolomics: Consequences on metabolism – Products of metabolic activity (e.g., proteins & enzymes) – Close to a physiological effect – Can be done using mass spectrometry -> metabolites rather independent of the organism

 Session 7: Biological responses Describe principles of standard test procedures ● Highly standardised conditions - Reproducibility ● Few test organisms - many compounds - Comparability ● International guidelines - Uniformity and no duplication

● ●

Quality criteria - Eases decision making Sensitive organisms - all other organisms protected

 Name strength and limitations of testing strategies Standard toxicity test

Non-standard toxicity test (Rapid toxicity testing)

Advantages - Highly standardised conditions - Reproducibility - Few test organisms - many compounds - Comparability - International guidelines - Uniformity and no duplication - Quality criteria - Eases decision making - Sensitive organisms - all other organisms protected Disadvantages - Answer only narrow question: relative toxicity - Test organism selection influences relative toxicity ranking - Ignore ecological aspects of real ecosystems

Advantages - Collection of toxicity data for a representative sample of organisms - Time and resources required for each chemical- species not big - Accept lower quality data - Trade off between precision (validity) and accuracy (bias) - Testing of multiple species concurrently in the same vessel (but no physical contact) - Sequential exposure (first look for critical concentration range, than test this range specifically) Disadvantages - non-standard tests are seldom performed according to GLP - Data not reproducible, uncertainty of toxicity data - low reliability

 Discuss strength and limitations of testing strategies regarding statistical analyses Illustrate some aspects of the ecology/biology of Daphnia magna. Why was Daphnia magnaselected as test organism? Ecology:largest herbivore cladocerans of northern hemisphere, lentic systems (mainly in ponds and pools), prey organism for fish, life-span up to two month, switches between parthenogenetic (clones) and sexual reproduction, several instar stages (molting), feed on algae. Reasons:Easy to culture (cheap), fast generation time, organism of low trophic level, largely herbivore. Evaluate the criticism targeted at NOEC, LOEC and related concepts. Outcome of the tests is directly connected to the tested concentrations since only measured concentrations can emerge as result. Calculation influenced by: - Sample size and replication

-

Number of endpoints observed Number of concentrations tested Variability of endpoint within experimental population Power of statistical test

 Which endpoints are commonly used in toxicity testing? Immobilisation, Mortality, Biomass, Reproduction, Behaviour, Growth, Accumulation, Cellular  Explain the terms EC50, NOEC and LOEC. EC50: concentration at which 50% of test organisms showed an effect (e.g. immobilisation) NOEC (No observed effect concentration): highest observed concentration at which no statistically significant deviation to control is detected LOEC (Lowest observed effect concentration): Lowest observed concentration at which a statistically significant deviation to control is detected Which test conditions can influence the variability of the test outcome? Chemical grade and formulation Org...