E5080 / E0323 Ecotoxicology Ecotoxicological bioassays Jakub Hofman 1 Content §Introduction – what, why, how, concept §Types of bioassays §Ecotoxicological bioassays’ design and results §Aquatic bioassays - examples §Soil bioassays – examples 2 Introduction – what, why, how, concept 3 Protection of environment / nature §Is and must be primary aim of sustainably developing society §why? How to protect ? §Policy §Legislation §Research §Education § Ecotoxicology – offers knowledge and tools useful for the effective and reasonable environmental protection (these tools = ecotoxicological bioassays) http://s3.amazonaws.com/dfc_attachments/images/3154905/iStock_000004307050Small_web.jpg 4 Ecotoxicology Discipline on the border of ecology and toxicology studying and evaluating direct and indirect effects of man-made or natural harmful chemicals or other stressors on animals (except human), plants and microorganisms at all levels of biological organization 5 Ecotoxicity bioassay, ecotoxicity test §a tool (method, procedure ...) for ecotoxicological research and praxis – for environmental legislation and protection §biota (tissue, organism, population, ecosystem …) is exposed to chemicals (and/or other stress factors), in the lab (controlled conditions) or in the field (less controlled) and effects are evaluated and related to exposure – its experiment (!) §WHY? To understand the cause-effects relationships (causality, dose-response …) obut sometimes also e.g. accumulation, biodegradation ... http://www.teara.govt.nz/files/p15500pc.jpg http://www.ebpi.ca/Rototox%20Kit%20M%20Kit.bmp http://www.ees.ufl.edu/homepp/bitton/images/metpad_metplate.jpg 6 Ecotoxicity bioassay, ecotoxicity test toxicity test versus ecotoxicity test à see toxicology versus ecotoxicology 7 Why ecotoxicological tests ? CHEMICAL ANALYSES ALONE CANNOT show real risk to living organisms: (1) (1)real exposure varies according to bioavailability of toxic substances (2)in real environment, there is always mixture of toxicants that acts differently from individual compounds (3)negative effects of matrix itself, regardless of toxicant content, on organisms or interaction of matrix with effects of toxicants (4)spectrum of analytical methods (i.e. limit values) is limited and un-analysed significantly toxic substances may be present in the sample 8 ADVANTAGES of chemical analyses - Reproducibility, standard-ability - Exact numerical outputs understandable to all people: use in the law Bioassays useful for: §prospective ecological risk assessment ousing bioassays for chemical compounds, pesticides ousing bioassays for materials, mixtures obefore they enter the environment 9 Bioassays useful for: Objectives of the prospective approach: §evaluation of hazards of contaminants (individual and mixtures) and other stressors §analysis of relationships between concentration and effect ("dose-response relationship") §hazard quantification, risk assessment (including legislatively required assessment) and prediction of negative effects of real environmental samples §setting limit values for (legislative) regulation of chemical substances, pesticides and materials that may come in contact with the environment (waste, sludge, fertilizers…) §knowledge of processes and mechanisms related to the effects of contaminants (or other stressors) on biota, fate and bioavailability of contaminants in the environment and exposure of organisms §understanding causes of harmful effects of contaminants on organisms 10 Bioassays useful for: §retrospective ecological risk assessment ousing bioassays for real environmental samples osearching the causalities between pollution and effects ISO 19204:2017 Soil quality — Procedure for site-specific ecological risk assessment of soil contamination (soil quality TRIAD approach) 11 Bioassays useful for: Objectives of the retrospective approach: §knowledge of the links (causality) between the occurrence and fate of contaminants (stressors) and the state of the biota §knowledge of past events and their regularities allows to estimate the development for the future in similar situations (prediction) §evaluation of interventions on real components of the environment in real ecosystems (evaluation of fertilization, remediation, assessment of contaminated sites…) §knowledge of processes and mechanisms related to the effects of contaminants (or other stressors) on the biota, the fate and bioavailability of contaminants in the environment and the exposure of organisms §understanding the consequences of the harmful effects of contaminants, especially at higher levels of the biological organization 12 Bioassays useful for: § 13 INDIVIDUAL TOXICANTS MIXTURES OF CHEMICALS CONTAMINATED ENVIRONMENT RETROSPECTIVE PROSPECTIVE Bioassessment Field assessment Monitoring Bioassessment Field assessment Monitoring Lab studies Lab studies Simulated small ecosystems DISASTERS PREDICTIONS for future Time: NOW ! MIXTURES OF TOXICANTS Bioassays useful for: §Each methodological approach has its limitations and can be interpreted only with regard to its information content and focus § §It is optimal to combine both approaches !!! 14 Types of bioassays 15 Bioassay development §old bioassays – acute, ecologically irrelevant, testing pure chemicals, pesticides §new bioassays – sublethal endpoints, ecological relevancy, chemical mixtures, miniaturization, simple to measure endpoints http://www.ebpi.ca/thamnocephalus%20platyurus%20toxicity%20test%20kit.jpg http://www.ebpi.ca/Rototox%20Kit%20M%20Kit.bmp http://www.ebpi.ca/daphtoxkit%20toxicity%20pulex.jpg http://www.teara.govt.nz/files/p15500pc.jpg 16 Microbiotests §they use some dormant stages of testing organisms §practical = everything in one box §cheap and easy, however, often not very relevant for real situations §http://ebpi.ca/index.php?option=com_content&view=article&id=20&Itemid=50 § § § § § § § § § § §https://www.microbiotests.com/ 17 Obsah obrázku fotka, různé, pták Popis byl vytvořen automaticky Differenciation of bioassays §According to the trophic level of test organisms: otests with producers, consumers, destruents §Depending on the duration of exposure and the nature of the effects: oacute, semiacute (semichronic), sub-acute, chronic -the specific length depends on the generation time of the organism (bacteria <<< trout), the classification is not completely uniform; Division usually into: acute = 24, 48 to 96 hours, usually assessment of lethality chronic - days, weeks to months, evaluation of non-lethal effects §According to the number of species involved: osingle species, two species, multi-species o 18 figure4 Eisenia_fetida scenedesmus_quadricauda_upr http://upload.wikimedia.org/wikipedia/commons/thumb/3/32/EscherichiaColi_NIAID.jpg/210px-Escherichi aColi_NIAID.jpg Differenciation of bioassays §According to the level of the biological system (and complexity): oenzymes, bioprobes, in vitro cell and tissue cultures, intact living organism, population, micro / mesocosm, field experiments o 19 C:\Documents and Settings\Ludek Blaha\Plocha\1.gif Differenciation of bioassays §According to the type of sample: ochemical substance, mixture of substances, natural sample from the environment §According to the tested matrix: owater, soil, air, sediment, waste, chemical §According to the sample modification: oleachate (organic solvent, DMSO, water…), contact (Solid Phase Tests), direct (Direct tests, Whole effluent test), TIE - toxicity identification evaluation §According to the evaluated effect: omortality tests, reproduction tests, escape tests, growth tests, teratogenicity tests, carcinogenicity, xenoestrogenicity, etc. §According to implementation: oin situ and in vitro §+ process bioassays: bioaccumulation, bioconcentration, biodegration 20 Ecological relevance of the bioassays §the tested species should represent the relevant functional group §the test should respect the ecology of the organism §monitored responses should be ecologically relevant and indicate the state and function of the organism (survival, growth, reproduction, food intake and mobility) §when monitoring reproduction, the exposure should cover most of the life cycle §abiotic and biotic factors in the test should be similar to those in the habitat §exposure paths should mimic real exposures §the bioavailability of the contaminant should be similar to that in reality §concentrations should be environmentally realistic 21 Ecological relevance of test species §play a key role in the functioning of the ecosystem §they occur in a number of ecosystems in higher abundance §easy to use in field and laboratory conditions §they come into contact with pollutants §they are sensitive enough to stress § The problem of ecotoxicology in general: I will use organisms A in the tests (for a number of reasons), but the target organisms in the system are B à what is the relationship of the results for A and B? Example: Eisenia fetida - the most famous soil test § 22 Earthworm species Pic1 23 Differenciation of bioassays §limit test / comparison test §concentration – response tests – preliminary, final 24 Standard-ability, legislation … §Law given tests oVery little, especially for new chemicals, pesticides, waste oThe big boom in the use of bioassays in recent years - ecological criteria for environmental quality §Standardized, standardized oMany tests oStandardization ≠ duty, binding oEconomic reasons - accreditation of laboratories §Experimental oA series of tests oSpace for efforts to achieve ecological realism oApplication of new knowledge about mechanisms and effects oEcological studies 25 Norms, standards, guidelines Objective: to reduce interlaboratory variability Over time, standard procedures have been developed for evaluating effects in laboratory tests up to in situ bioindication methods Advantages: oguaranteeing uniformity and repeatability of results ocomparability of results from different laboratories following the procedure ovalidated results suitable for decision making olittle need for optimization Disadvantages: overy specific and limited informative value ("acute lethality for Daphnia crustaceans") ousually suitable only for classification of substances (more - moderately - less toxic ...) olimited number of standardized procedures, usually simple (acute) effects odifficult to apply to other situations or to answer other questions oonly on a few model species - the question of transferability of results oused in inappropriate situations (research, evaluation of cause and effect) oit may not be applicable to a real environment 26 OECD guidelines – water 1 § 27 Organisation for Economic Co-operation and Development https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-2-ef fects-on-biotic-systems_20745761 Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test 2011 Test No. 221: Lemna sp. Growth Inhibition Test 2006 Test No. 238: Sediment-Free Myriophyllum Spicatum Toxicity Test 2014 Test No. 239: Water-Sediment Myriophyllum Spicatum Toxicity Test 2014 Test No. 202: Daphnia sp. Acute Immobilisation Test 2004 Test No. 211: Daphnia magna Reproduction Test 2012 Test No. 231: Amphibian Metamorphosis Assay 2009 Test No. 242: Potamopyrgus antipodarum Reproduction Test 2016 Test No. 243: Lymnaea stagnalis Reproduction Test 2016 Test No. 235: Chironomus sp., Acute Immobilisation Test 2011 Test No. 218: Sediment-Water Chironomid Toxicity Using Spiked Sediment 2004 Test No. 219: Sediment-Water Chironomid Toxicity Using Spiked Water 2004 Test No. 233: Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment 2010 Test No. 225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment 2007 Test No. 224: Determination of the Inhibition of the Activity of Anaerobic Bacteria 2007 Test No. 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) 2010 Test No. 244: Protozoan Activated Sludge Inhibition Test 2017 OECD guidelines – water 2 § 28 Organisation for Economic Co-operation and Development https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-2-ef fects-on-biotic-systems_20745761 Test No. 210: Fish, Early-life Stage Toxicity Test 2013 Test No. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages 1998 Test No. 215: Fish, Juvenile Growth Test 2000 Test No. 234: Fish Sexual Development Test 2011 Test No. 236: Fish Embryo Acute Toxicity (FET) Test 2013 Test No. 203: Fish, Acute Toxicity Test 2019 Test No. 229: Fish Short Term Reproduction Assay 2012 Test No. 204: Fish, Prolonged Toxicity Test: 14-Day Study 1984 Test No. 230: 21-day Fish Assay 2009 Test No. 240: Medaka Extended One Generation Reproduction Test (MEOGRT) 2015 Test No. 241: The Larval Amphibian Growth and Development Assay (LAGDA) 2015 Test No. 248: Xenopus Eleutheroembryonic Thyroid Assay (XETA) 2019 OECD guidelines - soil § 29 Organisation for Economic Co-operation and Development https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-2-ef fects-on-biotic-systems_20745761 Test No. 216: Soil Microorganisms: Nitrogen Transformation Test 2000 Test No. 217: Soil Microorganisms: Carbon Transformation Test 2000 Test No. 207: Earthworm, Acute Toxicity Tests 1984 Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) 2016 Test No. 232: Collembolan Reproduction Test in Soil 2016 Test No. 220: Enchytraeid Reproduction Test 2016 Test No. 226: Predatory mite (Hypoaspis (Geolaelaps) aculeifer) reproduction test in soil 2016 Test No. 228: Determination of Developmental Toxicity to Dipteran Dung Flies(Scathophaga stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae)) 2016 Test No. 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test 2006 Test No. 227: Terrestrial Plant Test: Vegetative Vigour Test 2006 http://upload.wikimedia.org/wikipedia/commons/thumb/0/0b/Redwiggler1.jpg/240px-Redwiggler1.jpg http://www.falw.vu.nl/en/Images/Folsomia%20candida_tcm24-30644.jpg http://www.zuova.cz/sluzby/kontaktni-testy-toxicity2.jpg http://upload.wikimedia.org/wikipedia/commons/0/02/Helix_aspersa-Nl2A.jpg http://beetlespace.wz.cz/druhy/fotky/Oxythyrea_funesta_01.jpg OECD guidelines - other § 30 Organisation for Economic Co-operation and Development https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-2-ef fects-on-biotic-systems_20745761 Test No. 237: Honey Bee (Apis Mellifera) Larval Toxicity Test, Single Exposure 2013 Test No. 213: Honeybees, Acute Oral Toxicity Test 1998 Test No. 214: Honeybees, Acute Contact Toxicity Test 1998 Test No. 245: Honey Bee (Apis Mellifera L.), Chronic Oral Toxicity Test (10-Day Feeding) 2017 Test No. 246: Bumblebee, Acute Contact Toxicity Test 2017 Test No. 247: Bumblebee, Acute Oral Toxicity Test 2017 Test No. 228: Determination of Developmental Toxicity to Dipteran Dung Flies(Scathophaga stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae)) 2016 Test No. 223: Avian Acute Oral Toxicity Test 2016 Test No. 205: Avian Dietary Toxicity Test 1984 Test No. 206: Avian Reproduction Test 1984 ISO standards – aquatic plants § 31 ISO Logo ISO 8692:2012 Water quality — Fresh water algal growth inhibition test with unicellular green algae ISO 14442:2006 Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste water ISO 20079:2005 Water quality — Determination of the toxic effect of water constituents and waste water on duckweed (Lemna minor) — Duckweed growth inhibition test ISO 20227:2017 Water quality — Determination of the growth inhibition effects of waste waters, natural waters and chemicals on the duckweed Spirodela polyrhiza — Method using a stock culture independent microbiotest ISO 16191:2013 Water quality — Determination of the toxic effect of sediment on the growth behaviour of Myriophyllum aquaticum ISO 10253:2016 Water quality — Marine algal growth inhibition test with Skeletonema sp. and Phaeodactylum tricornutum ISO 10710:2010 Water quality — Growth inhibition test with the marine and brackish water macroalga Ceramium tenuicorne http://www.clean-flo.com/wp-content/uploads/pic-1.jpg http://www.microscopy-uk.org.uk/mag/imgoct05/Scenedesmus-quadricauda.jpg http://www.algaebase.org/_mediafiles/algaebase/5B7BE95A076ca2C19Dsxv2CAFF8E/k857fWdJXeJD.jpg http://rostlinna-akvaria.cz/eshop/obrazky/366-Myriophyllum-matogrossense.jpg ISO standards – aquatic invertebrates § 32 ISO Logo http://pcwww.liv.ac.uk/~stewp123/Daphnia%20magna.jpg http://84.244.151.141/~persoone/images/slider/Thamnocephalus_platyurus.jpg http://www.fcps.edu/islandcreekes/ecology/Arthropods/Scud/Hyaellallam1.jpg http://caramelosblog.es/wp-content/gallery/fauna/caenorhabditis_elegans_worm.jpg http://cfb.unh.edu/CFBKey/html/Organisms/PRotifera/GBrachionus/brachionus_calyciflorus/brachionusca lyciflorus1large.jpg http://www.dr-ralf-wagner.de/Bilder/Heterocypris_incongruens-50x_17.jpg ISO 6341:2012 Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) — Acute toxicity test ISO 10706:2000 Water quality — Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea) ISO 10872:2020 Water and soil quality — Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) ISO 14371:2012 Water quality — Determination of fresh water sediment toxicity to Heterocypris incongruens (Crustacea, Ostracoda) ISO 14380:2011 Water quality — Determination of the acute toxicity to Thamnocephalus platyurus (Crustacea, Anostraca) ISO 14669:1999 Water quality — Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea) ISO 20665:2008 Water quality — Determination of chronic toxicity to Ceriodaphnia dubia ISO/TS 18220:2016 Water quality — Larval development test with the harpacticoid copepod Nitocra spinipes ISO 16303:2013 Water quality — Determination of toxicity of fresh water sediments using Hyalella azteca ISO 16778:2015 Water quality — Calanoid copepod early-life stage test with Acartia tonsa ISO 17244:2015 Water quality — Determination of the toxicity of water samples on the embryo-larval development of Japanese oyster (Crassostrea gigas) and mussel (Mytilus edulis or Mytilus galloprovincialis) ISO 20666:2008 Water quality — Determination of the chronic toxicity to Brachionus calyciflorus in 48 h ISO 19820:2016 Water quality — Determination of the acute toxicity to the marine rotifer Brachionus plicatilis ISO 19827:2016 Water quality — Determination of the acute toxicity to the freshwater rotifer Brachionus calyciflorus ISO standards – aquatic vertebrates § 33 ISO Logo ISO 7346-1:1996 Water quality — Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] — Part 1: Static method ISO 7346-2:1996 Water quality — Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] — Part 2: Semi-static method ISO 7346-3:1996 Water quality — Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] — Part 3: Flow-through method ISO 12890:1999 Water quality — Determination of toxicity to embryos and larvae of freshwater fish — Semi-static method ISO 10229:1994 Water quality — Determination of the prolonged toxicity of substances to freshwater fish — Method for evaluating the effects of substances on the growth rate of rainbow trout (Oncorhynchus mykiss Walbaum (Teleostei, Salmonidae)) ISO 15088:2007 Water quality — Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio) ISO 23893-1:2007 Water quality — Biochemical and physiological measurements on fish — Part 1: Sampling of fish, handling and preservation of samples ISO/TS 23893-2:2007 Water quality — Biochemical and physiological measurements on fish — Part 2: Determination of ethoxyresorufin-O-deethylase (EROD) ISO 23893-3:2013 Water quality — Biochemical and physiological measurements on fish — Part 3: Determination of vitellogenin ISO 21115:2019 Water quality — Determination of acute toxicity of water samples and chemicals to a fish gill cell line (RTgill-W1) http://www.fishbase.org/images/species/Darer_m0.jpg ISO standards – aquatic microorganisms § 34 ISO Logo ISO 11348-1:2007/Amd 1:2018 Water quality — Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) — Part 1: Method using freshly prepared bacteria — Amendment 1 ISO 11348-2:2007/Amd 1:2018 Water quality — Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) — Part 2: Method using liquid-dried bacteria — Amendment 1 ISO 11348-3:2007/Amd 1:2018 Water quality — Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) — Part 3: Method using freeze-dried bacteria — Amendment 1 ISO 10712:1995 Water quality — Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test) ISO 15522:1999 Water quality — Determination of the inhibitory effect of water constituents on the growth of activated sludge microorganisms ISO 11350:2012 Water quality — Determination of the genotoxicity of water and waste water — Salmonella/microsome fluctuation test (Ames fluctuation test) ISO 16240:2005 Water quality — Determination of the genotoxicity of water and waste water — Salmonella/microsome test (Ames test) ISO 13829:2000 Water quality — Determination of the genotoxicity of water and waste water using the umu-test ISO 13641-1:2003 Water quality — Determination of inhibition of gas production of anaerobic bacteria — Part 1: General test ISO 13641-2:2003 Water quality — Determination of inhibition of gas production of anaerobic bacteria — Part 2: Test for low biomass concentrations ISO 8192:2007 Water quality — Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation ISO 9509:2006 Water quality — Toxicity test for assessing the inhibition of nitrification of activated sludge microorganisms http://enfo.agt.bme.hu/drupal/sites/default/files/Pseudomonas%20putida_0.jpg http://www.ou.edu/cas/botany-micro/faculty/pictures/vibrio.jpg http://4.bp.blogspot.com/-TN0HBeFL1Pk/T3NrRmy44sI/AAAAAAAABI8/uWCONXl_q18/s1600/salmonella.jpg http://upload.wikimedia.org/wikipedia/commons/thumb/3/32/EscherichiaColi_NIAID.jpg/250px-Escherichi aColi_NIAID.jpg ISO standards – in vitro tests § 35 ISO Logo ISO 19040-1:2018 Water quality — Determination of the estrogenic potential of water and waste water — Part 1: Yeast estrogen screen (Saccharomyces cerevisiae) ISO 19040-2:2018 Water quality — Determination of the estrogenic potential of water and waste water — Part 2: Yeast estrogen screen (A-YES, Arxula adeninivorans) ISO 19040-3:2018 Water quality — Determination of the estrogenic potential of water and waste water — Part 3: In vitro human cell-based reporter gene assay ISO 21427-1:2006 Water quality — Evaluation of genotoxicity by measurement of the induction of micronuclei — Part 1: Evaluation of genotoxicity using amphibian larvae ISO 21427-2:2006/Cor 1:2009 Water quality — Evaluation of genotoxicity by measurement of the induction of micronuclei — Part 2: Mixed population method using the cell line V79 — Technical Corrigendum 1 ISO/CD 24295 Water quality — Determination of the dioxin-like potential of water and wastewater — Method using in vitro mammalian cell-based reporter gene assay ISO standards – biodegradation § 36 ISO Logo ISO 11733:2004 Water quality — Determination of the elimination and biodegradability of organic compounds in an aqueous medium — Activated sludge simulation test ISO 10707:1994 Water quality — Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds — Method by analysis of biochemical oxygen demand (closed bottle test) ISO 7827:2010 Water quality — Evaluation of the "ready", "ultimate" aerobic biodegradability of organic compounds in an aqueous medium — Method by analysis of dissolved organic carbon (DOC) ISO 10708:1997 Water quality — Evaluation in an aqueous medium of the ultimate aerobic biodegradability of organic compounds — Determination of biochemical oxygen demand in a two-phase closed bottle test ISO 11734:1995 Water quality — Evaluation of the "ultimate" anaerobic biodegradability of organic compounds in digested sludge — Method by measurement of the biogas production ISO 14592-1:2002 Water quality — Evaluation of the aerobic biodegradability of organic compounds at low concentrations — Part 1: Shake-flask batch test with surface water or surface water/sediment suspensions ISO 14592-2:2002 Water quality — Evaluation of the aerobic biodegradability of organic compounds at low concentrations — Part 2: Continuous flow river model with attached biomass ISO 14593:1999 Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium — Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test) ISO 16221:2001 Water quality — Guidance for determination of biodegradability in the marine environment ISO 9408:1999 Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium by determination of oxygen demand in a closed respirometer ISO 9439:1999 Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium — Carbon dioxide evolution test ISO 9887:1992 Water quality — Evaluation of the aerobic biodegradability of organic compounds in an aqueous medium — Semi-continuous activated sludge method (SCAS) ISO 9888:1999 Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium — Static test (Zahn-Wellens method) ISO standards – terrestrial plants § 37 ISO 11269-1:2012 Soil quality — Determination of the effects of pollutants on soil flora — Part 1: Method for the measurement of inhibition of root growth ISO 11269-2:2012 Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects of contaminated soil on the emergence and early growth of higher plants ISO 17126:2005 Soil quality — Determination of the effects of pollutants on soil flora — Screening test for emergence of lettuce seedlings (Lactuca sativa L.) ISO 18763:2016 Soil quality — Determination of the toxic effects of pollutants on germination and early growth of higher plants ISO 22030:2005 Soil quality — Biological methods — Chronic toxicity in higher plants ISO 29200:2013 Soil quality — Assessment of genotoxic effects on higher plants — Vicia faba micronucleus test ISO 21479:2019 Soil quality — Determination of the effects of pollutants on soil flora — Leaf fatty acid composition of plants used to assess soil quality ISO Logo http://media-2.web.britannica.com/eb-media/69/10269-004-EEF044DB.jpg http://upload.wikimedia.org/wikipedia/commons/thumb/e/e1/Illustration_Vicia_faba1.jpg/240px-Illustr ation_Vicia_faba1.jpg http://lh3.google.com/luirig/R5yaRJTetsI/AAAAAAAAOL8/WO5-p8bmIQE/s800/sinapis_alba_2.jpg ISO standards – soil invertebrates § 38 ISO 11268-1:2012 Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity to Eisenia fetida/Eisenia andrei ISO 11268-2:2012 Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei ISO 11268-3:2014 Soil quality — Effects of pollutants on earthworms — Part 3: Guidance on the determination of effects in field situations ISO 11267:2014 Soil quality — Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants ISO 16387:2014 Soil quality — Effects of contaminants on Enchytraeidae (Enchytraeus sp.) — Determination of effects on reproduction ISO 21285:2019 Soil quality — Inhibition of reproduction of the soil mite (Hypoaspis aculeifer) by soil contaminants ISO 23266:2020 Soil quality — Test for measuring the inhibition of reproduction in oribatid mites (Oppia nitens) exposed to contaminants in soil ISO 15952:2018 Soil quality — Effects of pollutants on juvenile land snails (Helicidae) — Determination of the effects on growth by soil contamination ISO 17512-1:2008 Soil quality — Avoidance test for determining the quality of soils and effects of chemicals on behaviour — Part 1: Test with earthworms (Eisenia fetida and E. andrei) ISO 17512-2:2011 Soil quality — Avoidance test for determining the quality of soils and effects of chemicals on behaviour — Part 2: Test with collembolans (Folsomia candida) ISO 20963:2005 Soil quality — Effects of pollutants on insect larvae (Oxythyrea funesta) — Determination of acute toxicity ISO 18311:2016 Soil quality — Method for testing effects of soil contaminants on the feeding activity of soil dwelling organisms — Bait-lamina test ISO/DIS 24032 Soil quality — In situ caging of snails to assess bioaccumulation of contaminants ISO 23611-1:2018 Soil quality — Sampling of soil invertebrates — Part 1: Hand-sorting and extraction of earthworms ISO 23611-2:2006 Soil quality — Sampling of soil invertebrates — Part 2: Sampling and extraction of micro-arthropods (Collembola and Acarina) ISO 23611-3:2019 Soil quality — Sampling of soil invertebrates — Part 3: Sampling and extraction of enchytraeids ISO 23611-4:2007 Soil quality — Sampling of soil invertebrates — Part 4: Sampling, extraction and identification of soil-inhabiting nematodes ISO 23611-5:2011 Soil quality — Sampling of soil invertebrates — Part 5: Sampling and extraction of soil macro-invertebrates ISO 23611-6:2012 Soil quality — Sampling of soil invertebrates — Part 6: Guidance for the design of sampling programmes with soil invertebrates ISO Logo http://upload.wikimedia.org/wikipedia/commons/thumb/0/0b/Redwiggler1.jpg/240px-Redwiggler1.jpg http://www.falw.vu.nl/en/Images/Folsomia%20candida_tcm24-30644.jpg http://www.zuova.cz/sluzby/kontaktni-testy-toxicity2.jpg http://upload.wikimedia.org/wikipedia/commons/0/02/Helix_aspersa-Nl2A.jpg http://beetlespace.wz.cz/druhy/fotky/Oxythyrea_funesta_01.jpg Obsah obrázku pes, hnědá, malé, držení Popis byl vytvořen automaticky ISO standards – soil microorganisms § 39 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 ISO Logo Obsah obrázku text Popis byl vytvořen automaticky US EPA - US Environmental Protection Agency OPPTS - Office of Prevention, Pesticides & Toxic Substances Group A - Aquatic and Sediment-dwelling Fauna and Aquatic Microcosms 40 https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-t est-guidelines 850.1010 - Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids (December 2016) 850.1300 - Daphnid Chronic Toxicity Test (December 2016) 850.1020 - Gammarid Amphipod Acute Toxicity Test (December 2016) 850.1025 - Oyster Acute Toxicity Test (Shell Deposition) (December 2016) 850.1035 - Mysid Acute Toxicity Test (December 2016) 850.1045 - Penaeid Acute Toxicity Test (December 2016) 850.1055 - Bivalve Acute Toxicity Test (Embryo-Larval) (December 2016) 850.1710 - Oyster Bioconcentration Factor (BCF) (December 2016) 850.1075 - Freshwater and Saltwater Fish Acute Toxicity Test (December 2016) 850.1400 - Fish Early Life Stage Toxicity Test (December 2016) 850.1730 - Fish Bioconcentration Factor (BCF) (December 2016) 850.1735 - Spiked Whole Sediment 10-Day Toxicity Test , Freshwater Invertebrates (December 2016) 850.1740 - Spiked Whole Sediment 10-Day Toxicity Test, Saltwater Invertebrates (December 2016) US EPA - US Environmental Protection Agency OPPTS - Office of Prevention, Pesticides & Toxic Substances Group B – Terrestrial Wildlife 41 https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-t est-guidelines 850.2100 - Avian Acute Oral Toxicity Test (June 2012) 850.2200 - Avian Dietary Toxicity Test (June 2012) 850.2300 - Avian Reproduction Test (June 2012) 850.2400 - Wild Mammal Toxicity Testing (June 2012) 850.2500 - Field Testing for Terrestrial Wildlife (June 2012) US EPA - US Environmental Protection Agency OPPTS - Office of Prevention, Pesticides & Toxic Substances Group C – Terrestrial Beneficial Insects, Invertebrates, and Soil and Wastewater Microorganisms 42 https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-t est-guidelines 850.3020 - Honey Bee Acute Contact Toxicity Test (June 2012) 850.3030 - Honey Bee Toxicity of Residues on Foliage (June 2012) 850.3040 - Field Testing for Pollinators (June 2012) 850.3100 - Earthworm Subchronic Toxicity Test (June 2012) 850.3200 - Soil Microbial Community Toxicity Test (June 2012) 850.3300 - Modified Activated Sludge, Respiration Inhibition Test (June 2012) US EPA - US Environmental Protection Agency OPPTS - Office of Prevention, Pesticides & Toxic Substances Group D – Terrestrial and Aquatic Plants, Cyanobacteria, and Terrestrial Soil Core Microcosm 43 https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-t est-guidelines 850.4230 - Early Seedling Growth Toxicity Test (June 2012) 850.4100 - Seedling Emergence and Seedling Growth (June 2012) 850.4150 - Vegetative Vigor (June 2012) 850.4800 - Plant Uptake and Translocation Test (June 2012) 850.4300 - Terrestrial Plants Field Study (June 2012) 850.4500 - Algal Toxicity (June 2012) 850.4550 - Cyanobacteria (Anabaena flos-aquae) Toxicity (June 2012) 850.4400 - Aquatic Plant Toxicity Test Using Lemna spp. (June 2012) 850.4450 - Aquatic Plants Field Study (June 2012) 850.4600 - Rhizobium-Legume Toxicity (June 2012) 850.4900 - Terrestrial Soil-Core Microcosm Test (June 2012) Bioassays’ general design 44 1) Prepare the organism Culture media, standardized numbers, age, etc. 2) Prepare the sample Dilution series water/culture media – direct organism exposure Include BLANK (medium only) solvent for organic compounds – minimum to be added Include SOLVENT CONTROL specifics for the SOLID MATRICES 3) Expose of organisms … for appropriate time, number of repetitions, under specified conditions § General scheme of bioassay Schema testu Ps_putida 45 0) culture of organisms 4) Evaluate and report results measure the endpoint / count organisms validity criteria statistical evaluation (means, ANOVA, dose-response …) § Organisms Ideally §good availability (laboratory cultures, commercial availability ...) §easy storage and breeding in laboratory conditions in sufficient quantities for experiments §the biology of the species and the genetics of the respective culture are characterized §the relative sensitivities of the species / culture to different classes of toxic substances are studied §the susceptibility of the species should be a good representative of the relevant group of organisms (Daphnia - crustaceans, Danio rerio - freshwater fish) 46 Organisms Cultures !!! 47 Obsah obrázku stůl, interiér, láhev, brýle Popis byl vytvořen automaticky Obsah obrázku místnost, postel Popis byl vytvořen automaticky IMG_0054 IMG_0061 IMG_3421_1 005 11 Organisms §the result of toxicity determination and interpretation is influenced by a number of other biological parameters ogenetically determined sensitivity of the respective culture / clone / variety ... osize and age of individuals osex odevelopmental stage (eggs, embryos, larvae, adults ...) ophysiological conditions - optimum (diseases, food - antioxidants ...) § §in general, the organisms must be in optimal status before the test è this is checked by the test validity criteria and testing reference substances 48 Exposure in aquatic bioassays §Usually exposure of whole organisms (intake by body surface area, respiratory system, food) less often: single injections (fish, input and dose are not affected by the environment) Distribution according to the arrangement of the exposure ostatic (without exchange of solutions - possible changes of concentrations, oxygen) ostatic with medium change/renewal (change at defined times, ~24 h) orecirculation (medium recirculation, more technically demanding ...) oflow-through (continuous maintenance of concentrations, technically demanding ...) § 49 static static – large volume static – medium change flow-through Exposure of vertebrate animals in bioassays §vertebrates - laboratory rodents, birds § §like in "classical" toxicology: oinjection intramuscular (IM), intraperitoneal (IP), intravenous (IV), subcutaneous (SC) ooral - dosing in food, application of gauze (tube directly into the stomach) ... orespiration - air contamination - closed containers / cells, inhalation ... 50 Exposure in soil bioassays §soil, sediments - bacteria, invertebrates - contact with the whole surface (direct contact tests - solid phase tests) oreal soil / sediment oartificial soil / sediment § §plants - roots - contact with solid or liquid medium, exposure to gaseous pollutants from the air § §often several exposure routes can be realistically assumed at the same time § 51 Exposure in soil bioassays it is specific: §the fate of the contaminant in the soil environment, the influence on the real bioavailability for soil organisms comes into play significantly § 52 Pb: 2 g/kg in soil mortality of earthworms Lanno et al. (2004) Ecotoxicology and Environmental Safety 57, 39-47 Exposure in soil bioassays Bioavailability §soil is heterogenous and there is lot of places available for sorption or sequestration of the pollutants è fate, beahviour, distribution affected è exposure, toxicity, risks affected § 53 bioavailability Ingestion and oral •food and soil particles - organisms consume mineral and organic matter - an important route of exposure for sorbed chemicals •contaminants can be biomagnified - for example in fungi that are consumed by sprintails •important path for arthropods Dermal •from the soil or soil solution - especially organisms drilling in the soil (earthworms and enchytraeids), which have a thin cuticle and are in contact with the soil and pore water •it is also possible to model the results of tests in an aquatic environment by supplementing the model of the distribution of the substance between the soil solution and the sorption on particles = the so-called Equilibrium partitioning theory (EqP) Breathing almost no data Exposure in soil bioassays 54 Potworms (Enchytraeids doerjesi) Exposure in soil bioassays 55 ARTIFICIAL SOIL §10% dry fine peat §20% caoline clay, min 30% calolinite §70% quartz sand fine min. 50% of size 0.05 – 0.2 mm §0.3-1% calcium carbonate à pH of 6 ± 0.5 § Exposure in soil bioassays 56 IMG_4025 §artificial soil is NOT real soil Exposure in soil bioassays 57 §artificial soil is NOT real soil http://lufa-speyer.de Exposure in soil bioassays 58 §the goal is HOMOGENITY of exposure to the test substance water soluble chemicals §in water which is also used to adjust soil moisture insoluble in water §using carrier - non-toxic, water soluble/miscible (acetone, ethanol) §using carrier - non-toxic volatile organic solvent and evaporated rapidly §in both cases, solution can be added to: osmall amount (1-10%) of fine quartz sand; after evaporation of solvent, this is added to soil and mixed odirectly into soil (dry or wet) followed by evaporation and mixing §in all carrier cases, it is necessary to include a control for the carrier/solvent insoluble in water or solvent §mixed directly with quartz sand or whole soil Factors / conditions of the assay §CRUCIAL !!! they affect both the organisms and the tested chemical, they significantly affect the exposure and the final results §must be standardized !!! otemperature olight / photoperiod ooxygen (aquatic consumers) opH owater hardness oclay and organic matter content in soil ofood added/non-added oetc. § 59 1 Results of the bioassays 60 Parameters of evaluation - endpoints §effects ~ response = result of the exposure to toxic chemical (stressor) ohigher/lower with increasing stressor intenzity (except hormesis) §endpoint = measured (measurable) response / effect ooriginal units (numbers, weight, enzyme activity etc.) or relative (% of control) § §acute effects oanimals – letality/mortality, imobilization in case of Daphnia oplants – algae: growth, chlorofyl (fluorescnence); vacular: emergence, growth odestruents – bacteria: growth, activity ... §chronic/sublethal effects oanimals –growth, malformations, reproduction, behavior oplants – growth, reproduction... § 61 Measures of exposure §DOSE versus CONCENTRATION otoxicology – dose - mg/kg b.w. - body weight, mg/kg b.w./day oecotoxicology – usually the concentration in the medium - mg/L, mg/kgsoil etc. o §pure chemicals and defined mixtures oconc. in media - mg/L, mmol/L (= mM), mg/kg etc. § §environmental samples oextracts of the samples and their % dilutions o% of the sample in the reference material § 62 Data from ecotoxicity bioassays § 63 Error bars in the graphs indicate that regardless of the type of response, it is measured in several replicates and the resulting data have some variability Data from ecotoxicity bioassays § 64 Tested factor - qualitative, nominal Contamination (or other stressor tested) in variants (samples) is not quantified, is not quantifiable, or is quantified, but the aim is not to study the influence of its intensity on the effect. Variants (samples) cannot be (or it is not the goal) arranged in any way. It is a comparative test of several variants (samples) each other and / or against the control (e.g. soils from monitoring from different localities, samples of different soil materials - sludge, sediments, waste). The extreme is „the limit test“ - one tested variant (sample) is compared with the control. Tested factor - kvalitative, ordinal Contamination (or other stressor tested) in variants (samples) is not fully quantified, but variants (samples) can be ranked based on some criteria. However, it is not possible to determine how many times the variant is larger or smaller than the previous or next one - the intensity of contamination (stress) cannot be plotted on the axis and no relationship between it and the effect can be modeled. It is a comparative test of several variants (samples) each other and / or against the control (e.g. soils little, medium and very far from the source; soils from little, medium and very damaged ecosystem, etc.). Tested factor – quantitative Contamination (stressor) is quantified to the extent that it is possible to say how many times or by how much its intensity is greater or less than in the previous or next variant. The contaminant concentration (stressor intensity) can be plotted on an axis and the experiment arranged and evaluated as a test of the relationship between concentration and effect, this relationship can then be graphically expressed, modeled and ecotoxicity parameters calculated. Whether the tested factor is quantified by discrete (integer) or continuous (even decimal) data is not very relevant. Data from ecotoxicity bioassays 65 Observed response – qualitative, binary The answer is not quantifiable, it is only possible to determine whether it has occurred or not - a typical example is mortality / survival of organisms, occurrence of some signs (lesions, swelling, mutations,), immobilization, escape reaction, etc. Finding on a number of organisms (biological systems), the binary result can be converted to the frequency or affected fraction and expressed as a percentage of the effect (e.g. mortality, lethality, survival, leakage) or as an affected fraction with values from 0 to 1. In this form, this type of data can be evaluated similarly to quantitative continuous data (test, model, regressions, etc.), but statistical methods designed for binomial data should be used correctly, including, for example, another formula for calculating variance. Observed response – qualitative, ordinal The response is quantified to the extent that the results can be ranked, eg small, medium and large damage. However, it is not possible to determine between the samples (variants) how many times or by how much the result is larger or smaller, and therefore it is not possible to model the relationship between the effect and the concentration. As with binary data, these results can be converted to fraction or frequency and expressed as a percentage in each category. Observed response – quantitative, continuous The response is quantified to the extent that it is possible to say how many times or how much is greater or less in one variant (concentration) than in another (eg weight, size, enzyme activity, production, number of juveniles, biomarker concentration…). The results can be plotted and the relationship between concentration and effect evaluated. Data from ecotoxicity bioassays § 66 Draft Guidance Document for on the Statistical Analysis of Ecotoxicity Data. OECD Environmental Health and Safety Publications, Series on Testing and Assessment, Environment Directorate, OECD, Paris 2003. Dose(concentration) - response relationship AcuteToxScheme_RM No Observed Effect Concentration (NOEC) Lowest Observed Effect Concentration (LOEC) ECx ( x % effects concentration) LCx ( x % lethal concentration) 67 each conc./dose is tested in several replicates !!! § 68 Dose(concentration) - response relationship 69 Aquatic bioassays - examples 70 Aquatic bioassays §huge number of the tests (aquatic ecotoxicology had been for a long time the only one ecotoxicology) §today, standardized bioassays cover the whole range of levels: osuborganismal level -laboratory experiments: study of toxicity mechanisms, in vitro biomarkers, specific types of toxicity (dioxin like toxicity, xenoestrogenicity ...) oindividual species, individuals -laboratory experiments: traditional ecotoxicological bioassays with individual species, comparison of susceptibility of different species ... opopulation effects -laboratory tests - longer-term experiments - lifelong toxicity tests, tests with early developmental stages, plants - reproduction, germination ..., invertebrates - vertebrates - reproductive toxicity tests 71 Aquatic bioassays §today, standardized bioassays cover the whole range of levels (cont): oeffects in communities -laboratory microcosms - artificially established communities of organisms of various species under defined conditions (producers - consumers - destruents) -field multispecies manipulated in situ studies - field studies, aquatic mesocosms (flowing, standing, littoral – coastal ofield manipulated studies (in situ) -studies with individual species - growing plants on contaminated and control areas, cage experiments in aquatic environments (molluscs, fish) oecosystem effects (these are, in fact, not „bioassays“ but „bioindication“) -field observation - evaluation of effects in populations and communities in a real natural situation 72 Aquatic bioassays - producers §cyanobacteria – photosynthetic, nitrogen-fixing eubacteria; colonies, filaments, single cells §unicellular algae – eucaryotic; cells, filamentous, colonies; freshwater or marine §both usually evaluated by the change in the number of cells (growth) – measured often as green color § §vascular plants – aquatic plants §size, length – growth, mortality § §other endpoints: concentration of pigments (eg chlorophyll a), physiological activity (nutrient intake) and metabolic activity (photosynthetic activities, enzymatic activities) 73 Aquatic bioassays – producers - algae §unicellular algae Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Desmodesmus subspicatus (formerly Scenedesmus subspicatus), diatoms (Navicula pelliculosa) or cyanobacteria Anabaena flos-aquae, Synechococcus leopoliensis §cultures or lyophylised stock may be used §equipment needed with high light intensity about 60-120 μE m-2 s-1 (~4500-9000 lux) at 400-700 nm §inicial density of the culture: 74 Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test 2011 ISO 8692:2012 Water quality — Fresh water algal growth inhibition test with unicellular green algae ISO Logo Organisation for Economic Co-operation and Development Aquatic bioassays – producers - algae §defined medium à §72 h exposure at light and 20-24°C §algal biomass determination: cell counts (electronic or microscope or flow cytometer) or fluorescence or color (fluorimeter, spectrophotometer) § 75 Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test 2011 ISO 8692:2012 Water quality — Fresh water algal growth inhibition test with unicellular green algae ISO Logo Organisation for Economic Co-operation and Development Aquatic bioassays – producers - algae validity §controls: oexponential growth by factor of > 16 over 72 h ocoefficient of variation (relative standard deviation, i.e. standard deviation divided by mean) < 35% (each day evaluation) and for average specific growth rate < 7% §reference substance: oe.g. 3,5-dichlorophenol or potassium dichromate should be tested at least twice a year oeffect (EC50) must be within the prescribed range 76 Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test 2011 ISO 8692:2012 Water quality — Fresh water algal growth inhibition test with unicellular green algae ISO Logo Organisation for Economic Co-operation and Development Aquatic bioassays – producers - algae 77 Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test 2011 ISO 8692:2012 Water quality — Fresh water algal growth inhibition test with unicellular green algae ISO Logo Organisation for Economic Co-operation and Development Aquatic bioassays – producers - algae miniaturization 78 Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test 2011 ISO 8692:2012 Water quality — Fresh water algal growth inhibition test with unicellular green algae ISO Logo Organisation for Economic Co-operation and Development http://ebpi.ca/_slideshows_Algaltoxkit%20F%20slide%20show.pdf Aquatic bioassays – producers - plants §„duckweed“, Lemna minor or gibba §start with 10 leaves (fronds) per 1 beaker §several recommended special media §96 h; 6500 - 10000 lux; pH 6.5; 24°C §growth: biomass – weight and no. of fronds (image analysis possible) validity: §control: doubling time < 2.5 days §reference compound: 3,5-dichlorophenol or K2Cr2O7 (EC50: 10-60 mg/L) 79 ISO Logo Organisation for Economic Co-operation and Development Test No. 221: Lemna sp. Growth Inhibition Test 2006 ISO 20079:2005 Water quality — Determination of the toxic effect of water constituents and waste water on duckweed (Lemna minor) — Duckweed growth inhibition test figure4 Aquatic bioassays – producers - plants §miniaturization with Spirodela 80 ISO Logo ISO 20227:2017 Water quality — Determination of the growth inhibition effects of waste waters, natural waters and chemicals on the duckweed Spirodela polyrhiza — Method using a stock culture independent microbiotest https://www.microbiotests.com/wp-content/uploads/2019/07/duckweed-toxicity-test_duckweed-toxkit-f_s tandard-operating-procedure.pdf Aquatic bioassays – producers - plants 81 ISO Logo Organisation for Economic Co-operation and Development ISO 16191:2013 Water quality — Determination of the toxic effect of sediment on the growth behaviour of Myriophyllum aquaticum ISO 10710:2010 Water quality — Growth inhibition test with the marine and brackish water macroalga Ceramium tenuicorne Test No. 238: Sediment-Free Myriophyllum Spicatum Toxicity Test 2014 Test No. 239: Water-Sediment Myriophyllum Spicatum Toxicity Test 2014 Aquatic bioassays – consumers - invertebrates §are very very common - sometimes ecotoxicology is confused with "Daphnia bioassays„ §standard layouts: obeakers/vessels, acute tests 1-few days, extended tests 21 d oevaluation of lethality, growth ... short-term - usually static oevaluation of reproduction etc. ... longer exposures, need for food, well controlled supply of oxygen ... §aquatic planktonic crustaceans - the most common oDaphnia magna, Ceriodaphnia dubia, Artemia salina (marine) §other invertebrates obenthic - Gammarus, Hyallela azteca ooligochaetic worms - Tubifex, Lumbriculus osnails – sand snail oinsects - midges (Chironomus), mayflies ... 82 C:\Documents and Settings\Ludek Blaha\Plocha\figure4.jpg Aquatic bioassays – consumers - invertebrates §also many of them developed to microbiotests 83 https://www.microbiotests.com http://ebpi.ca/_slideshows_Daphtoxkit%20F%20magna%20slide%20show.pdf Aquatic bioassays – consumers - invertebrates Daphnia magna acute test §5 juvenile daphnids per replicate (min 2 ml) §medium = so called reconstituted water 1.CaCl2 · 2H2O 11,76 g/l 2.MgSO4 · 7H2O 4,93 g/l 3.NaHCO3 2,59 g/l 4.KCl 0,23 g/l mix 25 ml of each to 1 L, pH 7.8, aeration §24h, 48h; dark or 16h light / 8h dark; O2 > 80 % (2 mg/l); 20°C; no food §mortality = immobilization validity: §control: mortality< 10 %; §reference compound: K2Cr2O7 (LC50: mg/L) 84 ISO Logo Organisation for Economic Co-operation and Development Test No. 202: Daphnia sp. Acute Immobilisation Test 2004 ISO 6341:2012 Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) — Acute toxicity test C:\DOCUME~1\MARTIN~1\LOCALS~1\Temp\\msotw9_temp0.bmp C:\DOCUME~1\MARTIN~1\LOCALS~1\Temp\\msotw9_temp0.bmp Aquatic bioassays – consumers - invertebrates Daphnia magna acute test § 85 ISO Logo Organisation for Economic Co-operation and Development Test No. 202: Daphnia sp. Acute Immobilisation Test 2004 ISO 6341:2012 Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) — Acute toxicity test other videos: https://www.youtube.com/watch?v=ElENqCeGNSA https://www.youtube.com/watch?v=3AxO36DLjsU Aquatic bioassays – consumers - invertebrates Daphnia magna reproduction test §24 h juveniles, 10 per replicate, 50 ml or more §medium reconstituted water or M4/M7 §21 days; 16/8 light/dark; O2 > 3 mg/l); 20°C; food (algae) §week check, aeration or medium change §mortality + number of juveniles + other parameters (behavior, malformations ...) validity: §control: mortality< 10 %; §reference compound: K2Cr2O7 (LC50: mg/L) 86 ISO Logo Organisation for Economic Co-operation and Development Test No. 211: Daphnia magna Reproduction Test 2012 ISO 10706:2000 Water quality — Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea) Aquatic bioassays – consumers - invertebrates other invertebrates used in the bioassays 87 ISO Logo Organisation for Economic Co-operation and Development Test No. 242: Potamopyrgus antipodarum Reproduction Test 2016 Test No. 243: Lymnaea stagnalis Reproduction Test 2016 ISO 10872:2020 Water and soil quality — Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) ISO 14380:2011 Water quality — Determination of the acute toxicity to Thamnocephalus platyurus (Crustacea, Anostraca) ISO 14669:1999 Water quality — Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea) ISO 20665:2008 Water quality — Determination of chronic toxicity to Ceriodaphnia dubia ISO/TS 18220:2016 Water quality — Larval development test with the harpacticoid copepod Nitocra spinipes ISO 16778:2015 Water quality — Calanoid copepod early-life stage test with Acartia tonsa ISO 17244:2015 Water quality — Determination of the toxicity of water samples on the embryo-larval development of Japanese oyster (Crassostrea gigas) and mussel (Mytilus edulis or Mytilus galloprovincialis) Obsah obrázku jídlo Popis byl vytvořen automaticky Aquatic bioassays – consumers - fish 88 ... Aquatic bioassays – consumers - fish 89 §fish cultures require specific equipment – culture labs with very controlled conditions §need for special approval to work experiments with vertebrates !!! Aquatic bioassays – consumers - fish 90 §fish cultures require specific equipment – culture labs with very controlled conditions Aquatic bioassays – consumers - fish 91 §standard layout: oaquaria smaller or larger, aeration needed ovarious arrangements (static, flow-through ...) oacute tests - 96 h, prolonged tests - days to months olethality, growth, reproduction olot of sublethal endpoints possible: behavior, spasms, food intake, breathing, health, histology, bioaccumulation, teratogenicity, carcinogenicity, xenoestrogenicity è è è Aquatic bioassays – consumers - fish 92 ISO Logo Organisation for Economic Co-operation and Development Acute toxicity test §juvenile fish, acclimatized §medium: reconstituted water, groundwater or clean water §24-96 h §conditions depend on species, e.g. pH, temp, photoperiod §disolved O2 >60% saturation §no feeding §mortality, size, weight validity §controls: mortality < 10%, O2 etc. §dissolved O2 ≥ 60% of air saturation §analytical measurement of test concentrations is compulsory Test No. 203: Fish, Acute Toxicity Test 2019 ISO 7346-1:1996 Water quality — Determination of the acute lethal toxicity of substances to a freshwater fish (Brachydanio rerio ...) - Part 1: Static method ISO 7346-2:1996 Part 2: Semi-static method ISO 7346-3:1996 Part 3: Flow-through method Aquatic bioassays – consumers - fish 93 ISO Logo Organisation for Economic Co-operation and Development Prolonged, chronic toxicity tests §prolonged = longer exposure with mortality endpoint, sublethal endpoints also recorded §from 14 to 28 d §chronic = also other endpoints like reproduction §7-200 d Test No. 210: Fish, Early-life Stage Toxicity Test 2013 Test No. 215: Fish, Juvenile Growth Test 2000 Test No. 229: Fish Short Term Reproduction Assay 2012 Test No. 204: Fish, Prolonged Toxicity Test: 14-Day Study 1984 Test No. 230: 21-day Fish Assay 2009 Test No. 240: Medaka Extended One Generation Reproduction Test (MEOGRT) 2015 ISO 10229:1994 Water quality — Determination of the prolonged toxicity of substances to freshwater fish — Method for evaluating the effects of substances on the growth rate of rainbow trout (Oncorhynchus mykiss Walbaum (Teleostei, Salmonidae)) Aquatic bioassays – consumers - fish 94 ISO Logo Organisation for Economic Co-operation and Development Embryonal, embryolarval tests §fertilized eggs are exposed to chemicals for several days §effects on development = teratogenicity §prolonged to hatching and larvae development §endpoints: hatching, survival, development, behavior, size Test No. 210: Fish, Early-life Stage Toxicity Test 2013 Test No. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages 1998 Test No. 236: Fish Embryo Acute Toxicity (FET) Test 2013 ISO 15088:2007 Water quality — Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio) 1 Aquatic bioassays – consumers - frog 95 FETAX – Frog Embryo Teratogenicity Assay Xenopus §fertilized eggs are exposed to chemicals for several days §effects on development = teratogenicity frog_pict C:\Documents and Settings\Ludek Blaha\Plocha\1.gif C:\Documents and Settings\Ludek Blaha\Plocha\1.gif Aquatic bioassays – destruents - bacteria 96 ISO Logo Vibrio fisheri luminescece test §marine bacteria - bad for samples with high minerals or organic matter - stimulation §very quick - 5-30 min §luminiscence inhibition §problem with particles and colour è flash test § ISO 11348-1:2007/Amd 1:2018 Water quality — Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) — Part 1: Method using freshly prepared bacteria — Amendment 1 ISO 11348-2:2007/Amd 1:2018 Part 2: Method using liquid-dried bacteria — Amendment 1 ISO 11348-3:2007/Amd 1:2018 Part 3: Method using freeze-dried bacteria — Amendment 1 figure4 1 Aquatic bioassays – destruents - bacteria 97 ISO Logo Pseudomonas putida growth inhibition test §... ISO 10712:1995 Water quality — Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test) Aquatic bioassays – destruents - bacteria 98 ISO Logo Mutation and genotoxicity tests §with bacteria, but used also for general testing of mutagenicity and genotoxicity §with liver fraction S9 simulate activation of xenobiotics within de-toxification in vertebrates Salmonella sp. – Ames test §mutants cannot live in medium without histidine §in case of mutagenic chemical the reverse mutation is induces – they grow Umu-C §transgenic bacteria with luciferase gene introduced to operon for DNA reparation §genotoxicity causes activation of reparation and thus luciferase and light ISO 11350:2012 Water quality — Determination of the genotoxicity of water and waste water — Salmonella/microsome fluctuation test (Ames fluctuation test) ISO 16240:2005 Water quality — Determination of the genotoxicity of water and waste water — Salmonella/microsome test (Ames test) ISO 13829:2000 Water quality — Determination of the genotoxicity of water and waste water using the umu-test Aquatic bioassays – destruents - bacteria 99 Testing the effects on activated sludge §important because of biotechnologies – water treatment plants §complex microbial community and its activities: respiration, nitrification §in erlenmeyer flasks etc. §measurements of biological oxygen demand (BOD) or respirometry ISO Logo Organisation for Economic Co-operation and Development Test No. 224: Determination of the Inhibition of the Activity of Anaerobic Bacteria 2007 Test No. 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) 2010 Test No. 244: Protozoan Activated Sludge Inhibition Test 2017 ISO 15522:1999 Water quality — Determination of the inhibitory effect of water constituents on the growth of activated sludge microorganisms ISO 8192:2007 Water quality — Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation ISO 9509:2006 Water quality — Toxicity test for assessing the inhibition of nitrification of activated sludge microorganisms Aquatic bioassays – sediments §sediment = aquatic equivalent of the soil è very heterogenic matrix, contains solid phase (mineral particles and organic matter) and pore water §sediment is often potential long-term reservoir / source of contaminants §distribution of contaminatns between water and sediment (sorption) – sediment organisms are exposed to contaminants in solid and/or liquid components of sediment §benthic organisms 100 Aquatic bioassays – sediments 101 Chironomus tentans Chironomus riparius C:\Documents and Settings\Ludek Blaha\Plocha\figure4.jpg Tubifex tubifex http://www.aquarium-kosmos.de/bilder/streifzuege/Lumbriculus%20variegatus_2.jpg Lumbriculus variegatus Heterocypris incongruens Hyalela aztecaincongruens Aquatic bioassays – consumers - invertebrates Tests with chironomids OECD 235 §same as Daphnia acute test, just with Chironomus larvae OECD 218+219 §10 larvae (cca 10d old) per beaker §OECD artificial sediment §100 ml sediment / 175 ml water §21-28 d; 16/8 h light/dark; controled pH, O2 §food §survival and growth OECD 233 §reproduction = development to midges § 102 Organisation for Economic Co-operation and Development Test No. 235: Chironomus sp., Acute Immobilisation Test 2011 Test No. 218: Sediment-Water Chironomid Toxicity Using Spiked Sediment 2004 Test No. 219: Sediment-Water Chironomid Toxicity Using Spiked Water 2004 Test No. 233: Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment 2010 IMG_0054 IMG_0061 IMG_0059 Soil bioassays - examples 103 Soil bioassays – producents - plants 104 §large number of different tests various endpoints §seed germination and root elongation §emergence §seedling growth §biomass production §life cycle (changes in weight, size, number of flowers, seeds ..) §physiological tests (photosynthesis, respiration) §enzymatic tests §symbiosis – N fixation, mycorhiza Soil bioassays – producents - plants 105 species §usually needed at least monocolyledenous + dicolyledenous §Sinapis alba, Lactuca sativa, Lepidium sativum, Hordeum vulgare, Zea mays § http://media-2.web.britannica.com/eb-media/69/10269-004-EEF044DB.jpg http://upload.wikimedia.org/wikipedia/commons/thumb/e/e1/Illustration_Vicia_faba1.jpg/240px-Illustr ation_Vicia_faba1.jpg http://lh3.google.com/luirig/R5yaRJTetsI/AAAAAAAAOL8/WO5-p8bmIQE/s800/sinapis_alba_2.jpg Hordeum vulgare - Barley - THE WORLDWIDE VEGETABLES botany, corn (Zea mays), field, Germany, Additional-Rights-Clearance-Info-Not-Available Stock Photo - Alamy Soil bioassays – producents - plants 106 Root elongation inhibition §preparation of seeds to 2 mm §15 seeds to 200-300 g soil §3-5 d; soil pH; dark; 24°C §root length ISO Logo ISO 11269-1:2012 Soil quality — Determination of the effects of pollutants on soil flora — Part 1: Method for the measurement of inhibition of root growth P1010052 pokus IMG_0215 IMG_0196 IMG_7021_al IMG_7034 IMG_7022_al Soil bioassays – producents - plants 107 Emergence, early growth, growth, chronic ISO Logo Organisation for Economic Co-operation and Development IMG_0511 IMG_0502 IMG_0819 IMG_0825 plant01.jpg (54.086 Byte) Test No. 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test 2006 Test No. 227: Terrestrial Plant Test: Vegetative Vigour Test 2006 ISO 11269-2:2012 Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects of contaminated soil on the emergence and early growth of higher plants ISO 17126:2005 Soil quality — Determination of the effects of pollutants on soil flora — Screening test for emergence of lettuce seedlings (Lactuca sativa L.) ISO 18763:2016 Soil quality — Determination of the toxic effects of pollutants on germination and early growth of higher plants ISO 22030:2005 Soil quality — Biological methods — Chronic toxicity in higher plants Soil bioassays – producents - plants 108 § https://www.microbiotests.com http://ebpi.ca/_slideshows_Phytotoxkit%20slide%20show.pdf Soil bioassays – consumers - invertebrates 109 Earthworms §earthworms are the most and longest time used representative of soil organisms in ecotoxicology – the oldest test OECD from 1984 §Benefits and reasons: othe whole development cycle takes place in the soil - a typical geobiont oconsume large amounts of soil (high food exposure and accumulation of contaminants) ohave very close physical contact with soil (skin exposure + oral) othey have significant bioaccumulation and bioconcentration character – macroconcentrators overy significant role in soil formation, decomposition processes, soil fertility okey role in the transfer of pollutants in food chains ooccurrence in almost all soils in high numbers and weights owell introduced in laboratory tests (easy breeding) oeasily identified in real samples (thanks to size)... STUPD00Z Lumdiap2 Soil bioassays – consumers - invertebrates 110 Earthworms - acute §Eisenia fetida / Eisenia andrei §culcure in mixture manure/soil/garden-substrate/peat §7,14 days §500 g soil; 50-60 % water holding capacity §artificial soil or LUFA 2.2 or other § ISO Logo Organisation for Economic Co-operation and Development Test No. 207: Earthworm, Acute Toxicity Tests 1984 ISO 11268-1:2012 Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity to Eisenia fetida/Eisenia andrei Eisenia Cycle CE8a-cocoon LR http://t3.gstatic.com/images?q=tbn:ANd9GcR7-3EeXeOV0fHO4s2TOGWEnFANnR74gi6jRAso6-WCvT_OWvmIZlw3FsoM yA 11 Soil bioassays – consumers - invertebrates 111 Earthworms - acute § ISO Logo Organisation for Economic Co-operation and Development Test No. 207: Earthworm, Acute Toxicity Tests 1984 ISO 11268-1:2012 Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity to Eisenia fetida/Eisenia andrei 62 71 019 012 019 012 Soil bioassays – consumers - invertebrates 112 Earthworms - reproduction §2 months: after 1 month adults removed, after 2nd month juveniles counted §soil and conditions same as for acute test but food added (dung) §various techniques how to extract juveniles from soil (usually heat) ISO Logo Organisation for Economic Co-operation and Development Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) 2016 ISO 11268-2:2012 Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei 068 Soil bioassays – consumers - invertebrates 113 Earthworms - reproduction ISO Logo Organisation for Economic Co-operation and Development Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) 2016 ISO 11268-2:2012 Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei WHC vesselsready controlwithworms wormsweighing wahingwormsJPG Soil preparation WHC measurement Water added Soil weighted to jars 10 adults from culture Washed Weighting worms 10 adults to 1 jar drysoils Soil bioassays – consumers - invertebrates 114 Earthworms - reproduction ISO Logo Organisation for Economic Co-operation and Development Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) 2016 ISO 11268-2:2012 Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei wormssearching wormsweighing bottomofvessel Control of the jars, activity markers Mortality assessment Weighting the worms trolleywithworms Temperated room Soil bioassays – consumers - invertebrates 115 Earthworms - reproduction ISO Logo Organisation for Economic Co-operation and Development Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) 2016 ISO 11268-2:2012 Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei waterbathS juvenileszoomedS juvpicking cocoonsievingS cocoonsearchontray cocoonsinthehand After 20 min juveniles appear Water bath, increasing temperature 40ºC - 60ºC Sieving the soil Collecting and counting juveniles Counting Hand sorting of cocoons Soil bioassays – consumers - invertebrates 116 Earthworms - reproduction §... ISO Logo Organisation for Economic Co-operation and Development Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) 2016 ISO 11268-2:2012 Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei Soil bioassays – consumers - invertebrates 117 Enchytraeids §Enchytraeus albidus or Enchytraeus crypticus §28 d; 20-30 g soil; 50-60% water holding capacity §artificial soil or LUFA 2.2 or other ISO Logo Organisation for Economic Co-operation and Development Test No. 220: Enchytraeid Reproduction Test 2016 ISO 16387:2014 Soil quality — Effects of contaminants on Enchytraeidae (Enchytraeus sp.) — Determination of effects on reproduction grindal_worm IMG_3421_1 006 Image14 Soil bioassays – consumers - invertebrates 118 Enchytraeids ISO Logo Organisation for Economic Co-operation and Development Test No. 220: Enchytraeid Reproduction Test 2016 ISO 16387:2014 Soil quality — Effects of contaminants on Enchytraeidae (Enchytraeus sp.) — Determination of effects on reproduction IMG_3360 019 024 027 032 IMG_2981 IMG_2893 pokus IMG_2984 031 Soil bioassays – consumers - invertebrates 119 Enchytraeids ISO Logo Organisation for Economic Co-operation and Development Test No. 220: Enchytraeid Reproduction Test 2016 ISO 16387:2014 Soil quality — Effects of contaminants on Enchytraeidae (Enchytraeus sp.) — Determination of effects on reproduction Soil bioassays – consumers - invertebrates 120 Springtails §Folsomia candida partenogenetic collembola or Folsomia fimetaria §28 d; 20-30 g soil; 50-60% water holding capacity §artificial soil or LUFA 2.2 or other Test No. 232: Collembolan Reproduction Test in Soil 2016 ISO 11267:2014 Soil quality — Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants ISO Logo Organisation for Economic Co-operation and Development 005 008 016 Soil bioassays – consumers - invertebrates 121 Springtails Test No. 232: Collembolan Reproduction Test in Soil 2016 ISO 11267:2014 Soil quality — Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants ISO Logo Organisation for Economic Co-operation and Development IMG_2850 005 http://www.chaloupky.cz/fotografie/foto-150-640.jpg 012 023 024 028 031 NS4 P1010056 IMG_2850 pokus 3 Soil bioassays – consumers - invertebrates 122 Springtails Test No. 232: Collembolan Reproduction Test in Soil 2016 ISO 11267:2014 Soil quality — Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants ISO Logo Organisation for Economic Co-operation and Development Soil bioassays – consumers - invertebrates 123 Avoidance tests §with earthworms or springtails §very quick (1-2 days) - screening ISO 17512-1:2008 Soil quality — Avoidance test for determining the quality of soils and effects of chemicals on behaviour — Part 1: Test with earthworms (Eisenia fetida and E. andrei) ISO 17512-2:2011 Soil quality — Avoidance test for determining the quality of soils and effects of chemicals on behaviour — Part 2: Test with collembolans (Folsomia candida) ISO Logo DSC07739 Soil bioassays – consumers - invertebrates 124 Mites §... Test No. 226: Predatory mite (Hypoaspis (Geolaelaps) aculeifer) reproduction test in soil 2016 ISO 21285:2019 Soil quality — Inhibition of reproduction of the soil mite (Hypoaspis aculeifer) by soil contaminants ISO 23266:2020 Soil quality — Test for measuring the inhibition of reproduction in oribatid mites (Oppia nitens) exposed to contaminants in soil ISO Logo Organisation for Economic Co-operation and Development Hypoaspis aculeifer olog prey predator cont. soil 0471971030 BOLD Systems: Taxonomy Browser - Oppia nitens {species} Soil bioassays – consumers - invertebrates 125 Snails §... ISO 15952:2018 Soil quality — Effects of pollutants on juvenile land snails (Helicidae) — Determination of the effects on growth by soil contamination ISO Logo haas_schnecke 散大蜗牛 Soil bioassays – consumers - invertebrates 126 Nematodes §the most abundant soil invertebrates §in fact aquatic organisms – pore water §very fast tests - short life cycle - screening C. elegans life cycle C. elegans anatomy Soil bioassays – consumers - invertebrates 127 Nematodes §Caenorhabditis elegans §grown on agar plates with E. coli §aseptic techniques and careful handling needed ! §24-48 h exposure ISO Logo ISO 10872:2020 Water and soil quality — Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) ASTM E 2172 – 01 Standard Guide for Conducting Laboratory Soil Toxicity Tests with the Nematode Caenorhabditis elegans J3 stage P.redivivus body1a 082 Soil bioassays – consumers - invertebrates 128 Beneficial arthropods §testing of pesticides §natural enemies of pests Soil bioassays – destruents – microorganisms 129 Whole community testing for microbial activities §real uncontaminated agricultural soil with indigenous microflora of desired properties: osand > 70%; TOC = 1.5%; pH(KCl) = 7–7.5 omicrobial biomass 400-700 µg C . gdw-1; basal respiration 0.5-0.7 µg CO2-C . h-1 . gdw-1 § ISO Logo Organisation for Economic Co-operation and Development Test No. 216: Soil Microorganisms: Nitrogen Transformation Test 2000 Test No. 217: Soil Microorganisms: Carbon Transformation Test 2000 ISO 14238:2012 Soil quality — Biological methods — Determination of nitrogen mineralization and nitrification in soils and the influence of chemicals on these processes Lokalita_04_05b Soil bioassays – destruents – microorganisms 130 Whole community testing for microbial activities ISO Logo Organisation for Economic Co-operation and Development Test No. 216: Soil Microorganisms: Nitrogen Transformation Test 2000 Test No. 217: Soil Microorganisms: Carbon Transformation Test 2000 ISO 14238:2012 Soil quality — Biological methods — Determination of nitrogen mineralization and nitrification in soils and the influence of chemicals on these processes Soil sampling Storage Pre-incubation (7 days) START Substance application Negative control Positive control 7th day 14th day 21st day 28th day Microbial parameters OECD, ISO: respiration and N-mineralization 10 g per replicate aerobic conditions 60% WHC; 22°C; dark ISO 16072:2002 Soil quality — Laboratory methods for determination of microbial soil respiration respiration by: Soil bioassays – destruents – microorganisms 131 Whole microbial community testing §same design of exposure as in OECD 216, 217 §also other parameters can be measured !!! ISO Logo 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 biomass enzyme activity diversity •structural •genetic •functional denitrification Soil bioassays – destruents – microorganisms 132 Whole community testing for microbial activities – quick tests Potential ammonium oxidation §= ammonification, first step of nitrification §3 h oxidation of ammonium sulfate §sodium chlorate added – stops nitrite oxidation to nitrate §nitrite measured by colorimetric test Respiration curves §they indicate the growth of microbial community §respirometry – increase of CO2 or decrease of O2 § ISO Logo ISO 15685:2012 Soil quality — Determination of potential nitrification and inhibition of nitrification — Rapid test by ammonium oxidation ISO 17155:2012 Soil quality — Determination of abundance and activity of soil microflora using respiration curves 92 Soil bioassays – destruents – microorganisms 133 Arthrobacter globiformis test §introduced bacteria species NOT whole community §contaminated soil or other solid materials (waste) or soil spiked with tested chemical(s) §A. globiformis added and dehydrogenase activity measured § ISO Logo ISO 18187:2016 Soil quality — Contact test for solid samples using the dehydrogenase activity of Arthrobacter globiformis Arthrobacter | coryneforms-2 Terrestrial species bioassays - examples 134 Terrestrial species bioassays - polinators §bees or bumblebees §brood development of cells (eggs or larvae) §acute oral or contact tests, prolonged-chronic tests – mortality, behavior §semi-field tests [tunnel tests (~100 m2)] and field tests - mortality, flight density, development of bee colonies (brood and feed), behavior of bee colonies §residue analysis studies in relevant feed matrices § § 135 Obsah obrázku květina, tráva, žlutá, pole Popis byl vytvořen automaticky Obsah obrázku tráva, exteriér, žlutá Popis byl vytvořen automaticky Obsah obrázku osoba, interiér Popis byl vytvořen automaticky Test No. 213: Honeybees, Acute Oral Toxicity Test 1998 Test No. 214: Honeybees, Acute Contact Toxicity Test 1998 Test No. 245: Honey Bee (Apis Mellifera L.), Chronic Oral Toxicity Test (10-Day Feeding) 2017 Test No. 246: Bumblebee, Acute Contact Toxicity Test 2017 Test No. 247: Bumblebee, Acute Oral Toxicity Test 2017 Terrestrial species bioassays - polinators Acute oral test §adult worker honey bees §doses of test substance in sugar solution (100-200 μl, 50% sugar solution) §after consuming food (3-4 hours), the feeding device is removed §mortality is recorded daily for at least 48 h Acute contact test §test substance dissolved in a suitable vehicle §applied directly to anesthetized bees – 1 µl droplets to back area of each bee chest §control for carrier necessary § 136 Organisation for Economic Co-operation and Development Terrestrial species bioassays Birds §dietary toxicity tests §dietary dosage + 14 days of observation §reproduction tests - long-term § 137 Test No. 223: Avian Acute Oral Toxicity Test 2016 Test No. 205: Avian Dietary Toxicity Test 1984 Organisation for Economic Co-operation and Development Japanese quail Bobwhite quail Malard duck Japanese quail - Wikipedia Northern Bobwhite (Colinus virginianus) – Planet of Birds Feral pigeon - Wikipedia Feral pigeon Terrestrial species bioassays Birds §20 weeks of feeding by contaminated feed §reproduction + 14 days observation of juveniles 138 Test No. 206: Avian Reproduction Test 1984 Organisation for Economic Co-operation and Development 1 In vitro bioassays 139 §special cell lines §specific compound è specific cellular or intracellular reporter è reporter gene §dioxin-like toxicity (AhR), estrogenicity (ER), thyroid-hormone-like toxicity ... § In vitro tests 140 ISO 19040-1:2018 Water quality — Determination of the estrogenic potential of water and waste water — Part 1: Yeast estrogen screen (Saccharomyces cerevisiae) ISO 19040-2:2018 Water quality — Determination of the estrogenic potential of water and waste water — Part 2: Yeast estrogen screen (A-YES, Arxula adeninivorans) ISO 19040-3:2018 Water quality — Determination of the estrogenic potential of water and waste water — Part 3: In vitro human cell-based reporter gene assay ISO 21427-1:2006 Water quality — Evaluation of genotoxicity by measurement of the induction of micronuclei — Part 1: Evaluation of genotoxicity using amphibian larvae ISO 21427-2:2006/Cor 1:2009 Water quality — Evaluation of genotoxicity by measurement of the induction of micronuclei — Part 2: Mixed population method using the cell line V79 — Technical Corrigendum 1 ISO/CD 24295 Water quality — Determination of the dioxin-like potential of water and wastewater — Method using in vitro mammalian cell-based reporter gene assay §in vitro special cell lines: mammalian or fish §specific compound è specific cellular or intracellular receptor è reporter gene conected with luciferase è light §dioxin-like toxicity (AhR), estrogenicity (ER), androgenicity (AR), thyroid-hormone-like toxicity (RAR) ... § In vitro tests 141 H295R Microcosms / mesocosms 142 Microcosms / mesocosms §micro < 1 m3, meso > 1 m3 §usually multi-species – big benefit §optimum: less uncontrolled than field but more realistic than lab tests §considers also indirect effects §enable also environmental fate endpoints §many possible desings 143 121 C:\Documents and Settings\Ludek Blaha\Plocha\1.gif Microcosms / mesocosms §Water: large aquariums, tanks, ponds, or artificial stream ecosystems § 144 C:\Documents and Settings\Ludek Blaha\Plocha\1.gif C:\Documents and Settings\Ludek Blaha\Plocha\1.gif Microcosms / mesocosms §Water: large aquariums, tanks, ponds, or artificial stream ecosystems § 145 C:\Documents and Settings\Ludek Blaha\Plocha\1.gif C:\Documents and Settings\Ludek Blaha\Plocha\1.gif C:\Documents and Settings\Ludek Blaha\Plocha\1.gif figure4 C:\Documents and Settings\Ludek Blaha\Plocha\1.gif Microcosms / mesocosms §Soil: usually core/column (made of plastic, wood, metal) of soil with vegetation, contains standard soil or real soil; can be outside (open system) or in the climate chamber (also gas monitoring) § 146 118 118 Microcosms / mesocosms §Soil core microcosm 147 122 122 tme01_b.jpg (66.851 Byte) tme02_b.jpg (57.969 Byte) tme03_b.jpg (52.421 Byte) tme04_b.jpg (57.399 Byte) Microcosms / mesocosms §Soil core microcosm 148 1