E5080 / E0323 Ecotoxicology Ecotoxicology in Field Studies Jakub Hofman 1 Introduction 2 What is going on? 3 toxic substances (+ other stressors) organisms populations community ecotoxicology aquatic habitat terrestrial complexity interactions ecosytem mixtures Why? §real problems are in real ecosystems ! §lot of problems already happened ! § § § § §how to address ecotoxicity in real situation? §how to find causality between degradation and ekosystem state? § 4 Challenges How? §measurements (observations) directly in the field § §sampling + analyses § §bioindication, biomonitoring § §causality, correlations, weight of evidence, TRIAD approach 5 Bioidication – example of alarming results § 6 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185809 § 7 How to? 8 General scheme 1.site characterization, survey directly in the field 2.assessment parameters selection for the given ecosystem in relation to the stress impact oabiotic components obiotic components -structure parameters (eg species composition – diversity, abundances ...) -functional parameters (eg flows of energy / materials, processes, bilances, resilience/resistence ...) 3.sampling plan (sampling frequency, numbers ...) oabiotic components (water, sediments, soil air) obiotic components (producers – consumers – destruents) 4.sampling campaign + analyses 5.assessment and interpretation, comparison of exposure vs control (!), conclusions 9 1) Site characterization §depending on: oterrestrial ecosystem: terrain influences – slopes, vegetation ... oaquatic ecosystem: flowing – static (lentic / lotic), depth, size, flow speed, fragmentation (macrophyta, benthos ...) § §other properties needed to be recorded: omain weather conditions, wind directions, light intensity ... ospecific parameters (any antrhopogenic activities nearby?, sources of pollution? ...) omap records ... owhat else ? o... § 10 2) Parameters selection abiotic components §where (water, sediment, soil, air) the stressor does occur / act ? §where the residues are expected ? biotic components §which organisms will be evaluated to see the impacts of stressors: orelation to stressor’s influence (eg planktonic – substances dissolved in the water column, ie hydrophilic versus sediments - hydrophobic) oevaluated groups (eg producers – algae, consumers – zooplancton, fish; destruents – planktonic bacteria) okey species, bioindicators ... oparameters evaluated -structural (taxonomic parameters, biomass, abundance ...) -functional (production / respiration, food chains ...) § 11 3) Sampling and analyses A: sampling and analyses of abiotic components § §plan and design of sampling plots / sites oareal, vertical – depth, air sampling §merging and creating mixed samples („average" sample from the site) §assessment of the fundamental chemical and physical parameters (organic carbon, pH, particle sizes ....) §characterization and determination of the contamination oanalytical chemistry and environmental chemistry §ecotoxicological bioassays of the real matrices .... special use of bioassays 12 13 figure4 Water figure4 1 Sediment figure4 figure4 Eckmans sampler 3) Sampling and analyses A: sampling and analyses of abiotic components 14 Air figure4 1 Soil figure4 figure4 figure4 Soil core 3) Sampling and analyses A: sampling and analyses of abiotic components 3) Sampling and analyses B: sampling and analyses of biota § §plan and distribution of the sampling plots / sites §sampling – variable according to organisms... §characterization of defined biotic parameters otechniques of botanical, zoological, microbiological and ecological disciplines §characterization and determination of contamination of biota otechniques of analytical chemistry and environmental chemistry § 15 3B) Sampling - biota §water 16 figure4 Planctonic nets figure4 figure4 figure4 Periphyton – biofilm figure4 3B) Sampling - biota §water 17 Benthic invertebrates figure4 figure4 Fish figure4 figure4 figure4 Obsah obrázku ryba, máloostné ryby, ostnoploutvé ryby Popis byl vytvořen automaticky §different according to type and especially the size of organisms §manual sorting, picking §pitfall traps §extracting methods: Tulgren's extraction, O'Connor's extraction ... 3B) Sampling – biota – soil biota 18 Obsah obrázku země, exteriér, rostlina, zelená Popis byl vytvořen automaticky 3B) Sampling – biota – soil biota 19 3B) Sampling – biota – soil biota 20 3B) Sampling – biota – soil biota §earthworms § 21 3B) Sampling – biota – soil biota §earthworms 22 Malva Hořčice plnotučná 880g - Tesco Potraviny alyl isothiocyanate 3B) Sampling – biota – soil biota §earthworms 23 Obsah obrázku zbraň, boxery Popis byl vytvořen automaticky Obsah obrázku interiér, jídlo, nápoje, snězeno Popis byl vytvořen automaticky Obsah obrázku kontejner, plast Popis byl vytvořen automaticky 3B) Sampling – biota – insects §capture into pitfall traps - those living on the surface of the soil §capture using exhaustor §by sweeping with an entomological net - from vegetation or from air §collection or falling from vegetation §Malaise trap §impact traps (without or with attractants, pheromones) §... and many other methods 24 Obsah obrázku text Popis byl vytvořen automaticky Obsah obrázku text, pracovní stůl, dokument, vizitka Popis byl vytvořen automaticky Obsah obrázku tráva, exteriér, strom, kontejner Popis byl vytvořen automaticky 3B) Sampling – biota – insects 25 3B) Sampling – biota – terrestrial plants 26 Phytocoenological snapshot § §defining area, square or rectangle §units or hundreds of m2 §plants are divided according to height into several vegetation floors: obryophytes and lichens oherbs, seedlings of trees oshrubs and trees with possible epiphytes §estimation of the coverage of individual floors §on each floor, all species, including an estimate of the area they cover (in percent or special scale – 7-point Braun-Blanquet or 11-point Domino) §other information is recorded, of course the exact location and date, but also the slope and its orientation §soil samples can also be taken for later analyzes (eg pH and other chemical analyzes) 1 3B) Sampling – biota – terrestrial plants Quadrat method 27 3B) Sampling – biota – mammals 28 figure4 figure4 Obsah obrázku box, kartotéka Popis byl vytvořen automaticky Direct methods §sampling - capture the representative part of the population §dead-traps (animal is killed) – clap-traps, wire eyes, “pitfall traps” with water and other traps, shooting §alive traps - corridors, fall-doors, baits; Sherman's or Longworth trap; tagging (rings, ears, color ...), release and re -capture (CMR - Catch, Mark, Release) 3B) Sampling – biota – mammals Direct methods §observation – big animals or cameras or phototraps §labelling – bands, collars, telemetry (GPS) 29 3B) Sampling – biota – birds §catching – nets, rings, blood sampling, feathers sampling etc. 30 Obsah obrázku strom, exteriér, osoba Popis byl vytvořen automaticky 3B) Sampling – biota – birds §observation (individuals, nests, singing...) 31 4) Assessment and interpretation §comparision of the exposed and control ecosystem §fundamental parameters of the compared ecosystems should be SIMILAR / COMPARABLE (eg pH values, water hardness, similar geochemical parameters – subsurface ...) §chemical contamination of the environmental compartments versus biota in the compared ecosystems oare there differences in the concentrations of the toxic compounds? ois there any relationship between concentrations in the environment and in biota? §comparing biotic parameters in both compared ecosystems oare there differences in the taxonomic composition of the communities? oare there differences in the coverage – abundance – biomass? oare the food relationships different? owhat about rezistence and resilience (how long the stress has acted and how long it does not act any more?) §correlation is NOT equal to causality ! § 32 Bioindication, biomonitoring 33 Bioindication method, when the Environmental status is assessed on the basis of the properties of biological systems in broader context, we mean all methods when we observe reactions of organisms present in the environment (from individuals to communities) on stress § 34 env. factors biota the environment is forming the living systems living systems provide information about the environment env. factors temperature electromagnetic radiation water chemical composition radioactivity noise Bioindication versus biomonitoring §bio + monitoring §bioindication is an approach §biomonitoring is the use of this approach in the field studies, especially at number of sites and repeatedly in time 35 Bioindication §monitoring of chemicals in the collected biota samples oin anything, preferentially so -called bioacumulators or bioindicator species / samples (eg needles) §tracking biota and its response to the environmental factors obiochemical markers -of effects (stress proteins - HSP - Heat Shock Proteins, chromosome aberations ...) -of exposure (Methalothioneins, EROD – Ethoxyresorufin-O-Deethylase ...) oindicator species - presence/absence indicates a certain feature of the ekosystém -sensitive species (eg stoneflies, mountain Tubellaria, lichens) -oportunist species (eg chironomids, leeches ...) othe condition and function of organisms opopulation - numbers of organisms, distribution, age composition ... ocommunity - species composition and representation, biodiversity ostate of ecosystem or landscape - structure, dynamics, function 36 Accumulation bioindicators - example § 37 SETAC 2020: 1.04.8 Deciphering the molecular mechanisms of pesticide tolerance of the soil engineer biodiversity Indicator species – example: Saprobity index §sapros = rot, blight, decomposition ... §organic "non-toxic" substances (fecal pollution, „nutrients“ for microbes) §many organic chemicals à nutrients for bacteria à degradation of organic substances and consumption of oxygen à impacts on aquatic biota § Increased saprobity §one of the major threats for water quality (and indicator of water pollution / purity) in Europe §not the direct toxicity, rather oxygen depletion (!) §assessment = categorization §polysaprobity / mesosaprobity (alfa-, beta-) / oligosaprobity §(new: catarobity / limnosaprobity / eusaprobity / transsaprobity) § 38 E3 E3 Indicator species – example: Saprobity index Indicator species for saprobity - examples Xeno & oligosaprobity Polysaprobity § Indicator species – example: Saprobity index §Community shift biom170 S = \frac{\sum_{i=1}^n A_i\cdot s_i\cdot g_i}{\sum_{i=1}^n A_i\cdot g_i} Ai – abundance of species i Si – individual saprobity value of species i gi – indikative value of species i How to choose the bioindicator ? Procedures used to monitor biological endpoints in real ecosystems should ideally be: 1.virtually applicable 2.easily interpreted by the executive body 3.ecologically relevant to multiple ecosystems 4.the resulting parameter should be separable from natural fluctuations 5.should give a causal relationship between substance and effect 6.fast and cheap 7.standardizable How to choose the bioindicator ? 42 Kelly J. & Harwell M. (1990). Selection of parameters - pros and contras § 43 EC (2010): Soil biodiversity: functions, threats and tools for policy makers. https://core.ac.uk/display/29245351 Bioindication §example of soil quality bioindicators – are there related to soil ecosystem services? 44 Jensen J. & Mesman M. (2006). Ecological risk assessment of contaminated land. Decision support for site specific investigations. Report 711701047. RIVM, Netherlands Bioindication ISO Logo ISO 14238:2012 Soil quality — Biological methods — Determination of nitrogen mineralization and nitrification in soils and the influence of chemicals on these processes ISO 15685:2012 Soil quality — Determination of potential nitrification and inhibition of nitrification — Rapid test by ammonium oxidation ISO 18187:2016 Soil quality — Contact test for solid samples using the dehydrogenase activity of Arthrobacter globiformis ISO 17155:2012 Soil quality — Determination of abundance and activity of soil microflora using respiration curves ISO/TS 10832:2009 Soil quality — Effects of pollutants on mycorrhizal fungi — Spore germination test ISO/CD 23265 Soil quality — Test for estimating organic matter decomposition in contaminated soil ISO 16072:2002 Soil quality — Laboratory methods for determination of microbial soil respiration ISO 14240-1:1997 Soil quality — Determination of soil microbial biomass — Part 1: Substrate-induced respiration method ISO 14240-2:1997 Soil quality — Determination of soil microbial biomass — Part 2: Fumigation-extraction method ISO 23753-1:2019 Soil quality — Determination of dehydrogenases activity in soils — Part 1: Method using triphenyltetrazolium chloride (TTC) ISO 23753-2:2019 Soil quality — Determination of dehydrogenases activity in soils — Part 2: Method using iodotetrazolium chloride (INT) ISO/TS 29843-1:2010 Soil quality — Determination of soil microbial diversity — Part 1: Method by phospholipid fatty acid analysis (PLFA) and phospholipid ether lipids (PLEL) analysis ISO/TS 29843-2:2011 Soil quality — Determination of soil microbial diversity — Part 2: Method by phospholipid fatty acid analysis (PLFA) using the simple PLFA extraction method ISO 11063:2020 Soil quality — Direct extraction of soil DNA ISO 17601:2016 Soil quality — Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil ISO 20130:2018 Soil quality — Measurement of enzyme activity patterns in soil samples using colorimetric substrates in micro-well plates ISO/TS 20131-1:2018 Soil quality — Easy laboratory assessments of soil denitrification, a process source of N2O emissions — Part 1: Soil denitrifying enzymes activities ISO/TS 20131-2:2018 Soil quality — Easy laboratory assessments of soil denitrification, a process source of N2O emissions — Part 2: Assessment of the capacity of soils to reduce N2O ISO 11266:1994 Soil quality — Guidance on laboratory testing for biodegradation of organic chemicals in soil under aerobic conditions ISO 15473:2002 Soil quality — Guidance on laboratory testing for biodegradation of organic chemicals in soil under anaerobic conditions ISO 14239:2017 Soil quality — Laboratory incubation systems for measuring the mineralization of organic chemicals in soil under aerobic conditions biomass enzyme activity diversity •structural •genetic •functional denitrification Example of available methods to measure soil microbial properties Bioidication Doelman P. & Eijsackers H.J.P. (2004): Vital Soil - Function, Value and Properties. Elsevier. 358 p. ISBN: 0-444-51772-3 Example of available methods to measure soil invertebrates Bioidication Example of available methods to measure soil invertebrates Doelman P. & Eijsackers H.J.P. (2004): Vital Soil - Function, Value and Properties. Elsevier. 358 p. ISBN: 0-444-51772-3 §composition of plant communities – phytocenology §function and condition of plants omeasurement of photosynthesis (oxygen production, fluorescence of photosynthetic pigments) obiochemical markers ogenotoxicity (micronuclei, chromosome aberations) ofunctioning of nitrogen fixation, mycorrhiza §leaf coverage §monitoring the occurrence of indicator organisms omycorrhitic fungi olichens odiseases §pollutants in plants § 48 DSC07503 Bioidication Example of approaches for plants §from practical reasons often focused on „small mammals“ opresence / absence orepeated catch oactivity oabundance odensity orichness odiversity odynamics of the population / community... 49 Bioidication Example of approaches for mammals TRIAD approach 50 TRIAD §long tradition §ISO 19204 (2017): Soil quality - Procedure for site-specific ecological risk assessment of soil contamination (soil quality TRIAD approach) §site-specific risk assessment with 3 lines of evidence (LoE) §their evaluation = „weight of evidence“ - WoE 51 TRIAD §there is scaling step §and finally integration of all results 52 Jensen J. & Mesman M. (2006). Ecological risk assessment of contaminated land. Decision support for site specific investigations. Report 711701047. RIVM, Netherlands TRIAD - příklad 53 Jiang et al. (2015) TRIAD - příklad 54 Jiang et al. (2015) Problems in field ecotoxicology 55 Problems in the field studies §natural fluctuations, large influence of environmental factors §Contamination data in most cases focus on total content obiota, however, reacts only to bioavailable fraction that depends on many factors (cannot be well modeled) oas a result, we often do not see the causality between pollution and the condition of biota, except of very high concentrations §The observed phenomena have a stochastic character oThere is a natural scattering in space and time! oDo we have a sufficiently representative sample? What do we really sample and measure? §Contamination often acts as a selection pressure oLong -term load can lead to creating adaptations and tolerances or even stimulation (especially in microorganisms) oDo we know the history of the locality contamination well? Problems in the field studies §Total interconnection by food and ecological links, continuity of processes oChanges in the activity of one community or population in relation to other communities and functions that are linked oInhibition of one ecosystem component can stimulate another component §Organisms themselves can affect chemical forms of pollutants oFor example, sorbed forms of substances may be mobilized again, or microbial degradation may come §The problem of optimal field study design (biomonitoring) oNeed of a reference state – non-contaminated / non-impacted site (comparison with control) oor a large dataset (correlation, causality) oor time trends (BACI § A reference state is needed BACI = comparing Before and After Control Impact §a control = state of ekosystém before the impact §it needs a monitoring before the impact happens (both biotic and abiotic components must be observed) §ie background values and „natural“ state 58 A reference state is needed Comparison of an exposed ecosystem with another ("control„ – un-impacted) ecosystem §The key is the choice of a control ecosystem: oBoth ecosystems have comparable abiotic properties (terrain, geology, altitude ...) oSimilar biological properties are expected in normal state (ie the same communities, food relations ...) §The derivation of the conclusions in this case is always complicated (there are no two same / equally evolving ecosystems) 59 P8270074 „Normal“ state in the ecosystems stationary state §long term state, no disturbances §this is often not „normal“: ecosystems are naturally „variable“ and „changing“ stable state §surrounding conditions / factors do not change the major features (functions, overal performance ...), but inside there might be changes and fluctuations dynamic stability / ekvilibrium = homeostasis §using action/reaction, positive and negative feedback it keeps long-term stable state succession §ecosystems are never „stationary“ – the go through development in time: so, the Protection should not simply aim on „conservation of the current state“ § 60 „Normal“ state in the ecosystems §regulatory approach – example: water framework directive EU (WFD) §EU WFD aims at good status of all surface waters in EU till 2020 §2 components of quality assessment (“good state”) - „ecological“ and „chemical“ Chemical component §3 lists of defined substances oPriority substances list -good quality = concentration of each individual chemical < EQS (Environmental Quality Standards), AA-EQS – annual average concentration, MAC-EQS – maximum acceptable concentration owatch list – these should be measured for the future assessment, they may become Priority substances ospecific pollutants – according to the plans of the river basins „river basin specific pollutants) § § § 61 „Normal“ state in the ecosystems §regulatory approach – example: water framework directive EU (WFD) §EU WFD aims at good status of all surface waters in EU till 2020 §2 components of quality assessment (“good state”) - „ecological“ and „chemical“ Ecological component § § § 62