Assessment Frameworks of Multiple Stressors

sound judgment Frameworks of Multiple StressorsA review of purlieual and benevolentkind wellness danger opinion frameworks of ten- lot stressors the slip-up of hormone disruptorsAbstractThis review is a compilation of the recommended approaches and methods for the ontogeny of a stake judicial decision framework of multiple stressors. Some of the driving forces contri provideding to direct this subject argon the occurrent demands of stakeholders homogeneous the drinking water industry, the society and regulators of evaluating the hazards of pastiches of contaminants that may harm gentleman beings and the environment. thitherfore, our work aims at exploring the possibility of integrating within the risk judgment, environmental, benignant and societal aspects, acknowledging new international regulations and policies for the safe make substance ab function of of chemical substances which enforce the integrative study of the punts of multiple chemicals on humans a nd the environment through and throughout their life-cycle. We chose the sort of compounds called endocrine gland disruptors as an example of multiple stressors because of their emerging relevance and the event that they usually integ localize coordination compound mixtures, originate from multiple solutions and exist in approximately(prenominal) environmental compartments, generating adverse sets on sensory receptors through diverse routes and pathways. Their actions may be severe degenerative and long-term modifications of the normal education and return patterns of the individual and/or its progeny, eventually representing systemic risks at the state level which may stir sustainability and biodiversity. over due to the ubiquity of these chemicals, it is necessary to address the inclusion of human beings as potential receptors and deal with risk estimation in an integrated manner. As endocrine disruptors may provoke place responses relative to the developmental st age, acting through varied mechanisms and at in truth low concent dimensionns, with the particularity that their toxicokinetics may present sometimes unusual dose-response curves, power challenge long-term predictions and riskiness characterization, adding to risk judgement uncertainties. References to the current methodologies including the relevant bioassays, chemical analysis, imitateing, statistics tools and equations to calculate joint resultant role considering the interactions of toxicants within a mixture argon excessively discussed in this review.KEY WORDSrisk judicial decision, endocrine disruptors, multiple stressors1. IntroductionThe aim of this review is to analyze the risk assessment frameworks and current practices, the integproportionn of environmental and human health methodologies, the cause rating and how to assess the risk of complex mixtures of chemicals. Assessing the risks of multiple stressors for human health and the environment arises from the r ealization that biologic and physical stressors usually coexist in complex mixtures in the natural and constructed environment, sometimes generating impacts on living creatures.Historically, since the publication in 1962 of the book Silent Spring, by Rachel Carson, a warning on the deleterious actions on wildlife of persistent toxicants, such as DDT, which cause a diminution of birds in Pennsylvania, became presentwas recognised among the scientific world, the media and the society. More recently, the research by Dr. Theo Colborn on the fruitful perturbations of predators in the Great Lakes of North America and her book, published in 1996 Our Stolen Future co-authored by herself with Dianne Dumanoski and John Peterson Myers, was able to generate public cognizance on the threats that EDCs might pose to human beings and the environment.Some of the driving forces for authorship this review argon the demands of stakeholders represented by the community, the NGOs, the health and en vironmental regulators, the formulate industrial sector and the drinking water companies. For instance, the European Environment and wellness Strategy emphatically stresses the gaps in knowledge related to risk assessment methodologies that accommodate foetuses, infants and children and calls for the precautionary principle within the strategy for environmental contaminants, for which thither is a sufficient level of scientific enjoin at the effect level (e.g. molecular, cellular, or tissue-related) to show the likelihood of health impacts. Not replete information exists on the link between emissions of dioxins and PCBs and opposite substances and their accumulation in ecosystems and foodstuffs. The sine qua non for research on the understanding of the links between environmental factors and certain diseases is recognized, exclusively, due to the complexity of the issue, the immediate action is to gather evidence in order to concrete measures to encourage human health and th e environment. some(prenominal) reports atomic number 18 able to demonstrate through laboratory experiments and field surveys that mental picture of animals to chemicals released into the environment exert reproductive or developmental do on the individual and its off-spring, affecting the viability of the species at a population level (Colborn and Smolen 1997). As a matter of fact, these kind of adverse cause sacrifice been observed in wildlife and many of them can be attributed to the moving picture to man-made chemicals. The cause-effect kinship is allay controversial in human beings, but still a matter of concern due to the severity of probable harm that could affect individuals and populations. Thus, regulatory decisions must be informed by risk assessment on this important topic (Fenner-Crisp 2000).Endocrine-mediated effects may be more(prenominal) relevant in populations rather than in individuals. As there is still non consensus about a cause-effect relationship, it is recommended a science-based precautionary approach to protect public health. Further research is needed to demonstrate effects and suffer out birth defect registries and epidemiology studies designed to track delayed effects of environmental flicks (Solomon and Schettler 2000).The classical paradigm of human health risk assessment authored by the National Research Council (NRC, 1983) is composed of four steps hazard identification, dose-response assessment, vulnerability assessment and risk characterization. This paradigm was modified in 1994 to include characterization of each component. One of the approaches considered by some authors as better suited for developing a risk assessment of multiple stressors is a cumulative risk assessment framework, which may include societal aspects with democratic involvement of stakeholders (Gentile and Harwell 2001 Callahan and Sexton 2007 USEPA 2003).The global trend towards a policy-driven integration apply to risk assessment, dema nded by regulations on safety of chemicals and industrial operations should ideally include a multiplicity of stressors, compartments, geographical scales, and end-points (Assmuth and Hildn 2008). For example, the new European Union Regulation on chemicals and its safe use (REACH, EC 1907/2006) enforces linking risks to human health and the environment for chemicals throughout their life cycle. In United States, the environmental Protection Agency, still discusses both topics separately because of practical reasons, but acknowledging the need to integrate them.An overview of the most suitable risk assessment frameworks is expound in this literature review, foc apply on the case of mixtures of reproductive endocrine disruptors. Matters that state this topic are also presented, regarding particularities in mechanistic and toxicokinetics aspects and some of the sources of uncertainties and discombobulate factors. Developing a novel approach to the classical risk assessment scheme is also a goal, with the intention of contributing to design a risk assessment framework comprising a choice of tests, models, computational and statistical tools.2. Hazard identificationIn this stage the nature of the hazard is described taking into account toxicity data. The hazard can then be characterized deriving numerical values of acceptability of the risk, based on mechanisms of action, biological extrapolation, dose-response and dose-effect relationships, and related uncertainties (Fenner-Crisp, 2003).2.1. Nature of the hazardEndocrine disruptors (EDCs) are substances that interfere with the endocrine system by changing homeostasis and producing developmental effects and/or decrease the fertility of the beingnesss. EDCs include a broad range of substances which can be classified according to their effect. The best cognize are the environmental estrogens, alkylphenol and its ethoxylates, the monomer in polycarbonate manufacture bisphenol A, some pesticides and chlorinated or ganics.2.2. Sources of EDCsPossible sources of watercourses pollution with EDCs are wastewater sewage discharge, industrial outgoings, or point and non-point source contamination of watercourses with agrochemicals such as herbicides or pesticides.sewer discharge from cities contains residues of domestic products such as in-person care products, pharmaceuticals and detergents and excreta with natural and artificial steroid hormoneal hormones from cautionary lozenge usage. Nonylphenol (NP) is a graduate(prenominal)ly hydrophobic bioccumulating biodegradation by-product of nonylphenol ethoxylate non-ionic surfactants which persists in sewage scoop and river bank deposits. Its use and production devote been banned in EU countries and strictly monitored in many opposite countries such as Canada and Japan (Soares et al. 2008). slop mills are some of the industries associated to studies on endocrine disruption. Bleached kraft paper frame mill effluents bring in been linked to both estrogenic and androgenic hormoneic effects on biota, depending on the process characteristics and wastewater intervention. Several studies have associated the chlorination of organic matter to the estrogenicity of the effluent. Nowa age, the application of elemental- centiliter-free processes has diminished the risk of dioxins and furans formation, but not eliminated it, as other halogenated organics are formed by use of chlorine dioxide as bleaching agent. Various wood-extractive compounds produced in the human bodying process, such as rosin acids and phytosterols and install in pulp mills effluents have also been considered potentially prudent of endocrine disruption (Hewitt et al. 2008). The main identify resin acids in pulp mill effluents are pimaric, isopimaric, sandaracopimaric, palustric, dehydroabietic, abietic and neoabietic acid (Merilinen and Oikari, 2008). Other compounds found in this fount of effluent are phenolic guaiacyl-based lignin degradation products, diterpenoids, and dimethoxy stilbene (Belknap et al, 2006). Modern analytical methods, like full-scan GC-MS have been used to identify wood related extractives in nett effluent from a chlorine dioxide bleached pulp mill effluent, including monoterpenes, phenolics, productive acids, resin acids, resin acid neutrals and sterols (Wartman et al. 2009). Receptor covering bioassays for androgens and estrogens indicated that androgens were most gigantic in this effluent and the range of androgens for the various extraction protocols used was 189-283 ng/L as testosterone equivalent concentration.Some examples of common sources of EDCs and typical environmental concentrations are summarized on Table 1.Table 1.Sources of EDCs and typical environmental concentrationsOrigin, use and occurrenceSource of environmental delineationEDC groupExample elementTypical concentrationsReferenceIndustrial (pulp and paper mills)Contaminated search rosin acidspimaric acid4-140 g g-1Owens et al, 1994Ind ustrial (pulp and paper mills)Industrial wastewater treatment specifyChlorinated organics2,4,6-trichlorophenol1.5 g l-1Owens et al, 1994Industrial (pulp and paper mills)Final stage lowly treatmentPhytosterols-sitosterol58.42 g l-1Landman et al, 2008Domestic,(contraceptive pills)Sewage effluentPharmaceuticals17-ethynylestradiol14-17 ng l-1Liu et al, 2004Human and animal excretaSewage effluentNatural steroid hormones17-estradiol5.0 ng l-1Koh YKK et al,2007Domestic and industrial (laundry detergents, wool scouring processes)Sewage sludgeNon ionic surfactants4-nonylphenol829.3 mg/kgGonzlez et al, 2010Domestic and industrial (polycarbonate bottles) leaching from solid waste, sewage effluentPolycarbonatebisphenol A0.62 g l-1Snchez-Avila et al, 2009 inelegant (soil fertilization)Livestock wasteMale steroid hormonestestosterone10-1830 ng l1Lange et al, 2002Agricultural (dairy farming)Streams contaminated by dairy cow excreta distaff steroid hormones17-oestradiol0.04-3.6 ng l1Matthiessen e t al, 2006Agricultural(weed and glom control in soybean crops)Run-offHerbicideglyphosate0.1-0.7 mg l-1Peruzzo et al, 20082.2. Dose-response assessment in that location are some(prenominal)(prenominal) methods to demonstrate dose-response relationships, either by in vivo or in vitro tests. seek replication tests, like the ones using the model fish fathead minnow (Pimephales promelas) have shown a decrease in birth rate associated with depressed steroid and vitellogenin (Vtg) production in effeminate specimens (Ankley et al. 2008). Many of the tests hope on the measurement of an increase of Vtg as biomarker of estrogenicity as seen in several publications (Schwaiger et al. 2002 An et al. 2008 Holbech et al. 2006 Panter et al, 1998 Sohoni et al. 2001 Kunz and Fent, 2009).Tests results on resin acids show different responses in the first contemporaries of fish than in the second (Christianson-Heiska et al. 2007).In some cases there are not many examples of in vivo tests, like fo r glyphosate. A fish ikon tests with this compound showed Vtg induction in female fish, indicative of estrogenic body process (An et al, 2008). An investigation working the commercial formulation of the herbicide glyphosate and human eutherian cells demonstrated its toxicity at concentrations lower than the usual in bucolic practices. The aromatase activeness disruption seems to be due not only to glyphosate but also to co-adjuvants (the surfactant nonylphenol or others), which enhance its bioavailability and/or bioaccumulation (Richard et al. 2005 Gasnier et al. 2009). Table 2 shows some examples of dose-response experiments working with fish, crustacea, molluscs and amphibia. Varied protocols exist to develop ecotoxicity tests, in flow-through, atmospherics or partly alteration conditions, and with different duration and end-points. Only chronic effects and particularly developmental and reproductive effects were considered.Table 2. Dose-response for endocrine disruption ef fects in freshwater organisms exposed to single EDCsEDC chemical nameTaxonomic groupSpeciesDose to produce effect strengthTest conditionsReference4-nonylphenol weightRivulus marmoratus300g l-1testicular agenesis and oogenesis inhibition in 100 % fishStatic system, daily renewalTanaka and Grizzle, 20024-nonylphenol tiltOncorhynchus mykiss, rainbow trout1 -10 g l-110 g l-1High Vtg in adult male fish plasmLow hatching rateIntermittent word-painting of adult fish for 4 months until spawning Schwaiger et al, 20024-nonylphenolCrustaceanCeriodaphnia dubiaNOEC for reproduction 1 g l-1Low hatching rate7 long time chronic exposure, staticIsidori et al, 2005GlyphosateFishCarassius carassius, crucian carp100% effluentVtg induction in female fish (38.6 +/- 9.8 g l-1)3 weeks, continual exposureAn et al, 2008GlyphosatemolluskPseudosuccinea columella, aquatic snail1 mg l-110 mg l-1Faster development of F3 embryosHatching inhibition3 extension continuousTate et al, 1997EstroneFishDanio rerioLOE C 14 ng l-150 ng l-1Significant Vtg increasehigher(prenominal) female ratio40 days fish braceual development testHolbech et al, 200617-EstradiolFishDanio rerioLOEC 54 ng l-1Significant Vtg increaseHigher female ratio40 days fish sexual development testHolbech et al, 200617-EstradiolFishPimephales promelas, fathead minnow100 ng l-1Significant Vtg increaseTesticular growth inhibition21 days male fish exposurePanter et al, 1998EstriolFishDanio rerioLOEC 0.6 g l-121.7 g l-1Significant Vtg increaseHigher female ratio40 days fish sexual development testHolbech et al, 2006Dehydroabietic acid (DHAA), resin acidFishDanio rerio, zebra fish50 g l-1Low plasma Vtg in female in F0 high Vtg and affected spermatogenesis in F1 males2 generations, continuousChristianson-Heiska et al 2008-sitosterolFishDanio rerio10-20 g l-1F1 higher ratio of male fish F2 higher ratio of female fish2 generation fish exposure testNakari and Erkomaa, 2003Quercetin, phytoestrogen amphibiousXenopus laevis, frog200 g l-1H igher female ratio 10% abnormal testes (some with ovotestes) delineation up to 1 month post-metamorphosisCong et al, 2006Phenanthrene, PAHFishOryzias latipes,MedakaNOEL 100 g l-1Developmental, hatching18 days, renewalRhodes et al, 2005Bisphenol AMolluskMarisa cornuaretis, aquatic snailNOEC 640 g l-1Developmental12 weeks, juvenile snailsForbes et al, 2007Bisphenol AFishPimephales promellas16 g l-1640 and 1280 g l-1640 g l-11280 g l-1 altered spermatogenesisGrowth inhibition and Vtg induction in male fish bring down hatchability in F1 generationEgg production inhibition3 generation reproduction exposure testSohoni et al, 2001Bisphenol AFishBrachydanio rerio, zebrafishEC50 2.90 g l-1fertilized egg malformation and low hatchability72 h exposureLiu et al, 2007Benzo--pirene (BaP) (PAH)Fishgenus Fundulusheteroclitus , common mummichog10 g l-1CYP19A1 expression decrease by about 50% in immature stage I oocytesExposurefor 10 or 15 days by in situ hybridization, several developmental stagesD ong et al, 2008Polychlorinated biphenyl 126FishDanio rerio, zebrafishLC50 3.270 mg l-1Developmental effects through aryl hydrocarbon receptor activationDilutions of PCB 126 for 12 weeksSiman et al, 2007Polychlorinated biphenyl 126Fishgenus Salvelinus namaycush, lake trout3 g kg1body weightRetinol depletionOral exposure for 12 weeks fixation with radiolabelled retinolPalacea et al, 1997Benzophenone-1FishPimephales promelas4919 g l-1Vtg induction14 days exposure, semi-static, renewalKunz and Fent, 20093. Exposure assessment3.1. Ecosystems and human sub-populations potentially at risk of endocrine disruption effectsIncreasing evidence generated by scientists turn endocrine disruption into a recognized risk to the environment. Due to the ubiquity of EDCs and the widespread routes of exposure, most ecosystems and human populations are potentially at risk of endocrine disruption. Notwithstanding this fact, under the scope of a risk assessment of EDCs the potentially most vulnerable risk subgroups are identified corresponding to maternal, foetal and early developmental stages. The concern that prenatal or childhood exposure to EDCs may be responsible for abnormalities in human sexual and reproductive health are still in the theoretical ground. However, many reports on exposure to high concentrations of recognized EDCs such as DES, certain PCBs, and DDT demonstrate this fact. At low-doses the question remains nonreciprocal whether there could be a critical window where they could harm the fetal development (Hood 2005).Several reports on human developmental anomalies and reproductive ailments have been raising international concern, such as a seven fold increase risk of testicular cancer since 1969 to 2002 in men from several countries of Europe, United States and New Zealand. Also, the sperm density halved, as rates of cryptorchism (undescended testicles) and hypospadias (shortened urinary tracts) simultaneously rose. It is thought that human congenital malformat ion of sex organs, low sperm quality, endometriosis, reduced fertility and some types of cancers of breast and testis could be linked to exposure to EDCs. More than 80000 synthetic chemicals are produced in the world and have still not been fully evaluating with regards to endocrine disruption. In 1996, the U.S. Environmental Protection Agency initiated an Endocrine Disruption Screening computer program to evaluate more than 15,000 chemicals calling for a policy based on the precautionary approach to be overcautious and protect human health and the environment. A historical example of policies which demanded the banning of a drug due to these after-effects is the case of diethylstilbestrol (DEADES), which used to be prescribed to pregnant women to hold back spontaneous abortions because it produced higher risk of genital deformities and cancer in the offspring, among other effects (Stair 2008).Internationally there is consensus that the most vulnerable group for EDCs exposure are children. For example, in European countries, the Strategy for Environment and Health known as SCALE for Science, Children, Awareness, Legislation and Evaluation, has set as a antecedency agenda for the evaluation diseases caused by endocrine disruptors in children.The exposure to insecticides and herbicides used in agricultural practices has been linked to developmental or reproductive effects in wild animals and also in human beings. The occupational exposure to pesticide has received much attention, as for example prolonged time-to- motherliness was observed in male greenhouse workers exposed to pesticides before conception of their first pregnancy (Bretveld et al 2008). The domestic exposure of children to residues of pesticides in low-level long-term exposures are associated to chronic effects and include routes of exposure such as harvest-tide or breast milk (Goodman and Laverda 2002).3.2. Evidence of endocrine disruption effects in wildlife slightly the worldThere are rep orts on impacts on wildlife reproduction and development observed in invertebrates, fish, reptiles, birds and mammals, sometimes confirmed by laboratory tests. In laboratory experiments the impacts to fish populations by EDCs affect reproductive health and persistence of various fish species (Mills and Chichester 2005). Many examples of impacts due to exposure to endocrine disruptors exist in wildlife, such as the seals population defy in the Baltic and North Sea, the high levels of female egg egg yolk in male fish or snail imposex and intersex slightly the world. Intersexuality of fish has been demonstrated in several investigations carried out in rivers around the world. The findings of abnormal reproductive female-like ducts and oocytes in male fish were related to the treat sewage discharge from the cities in laboratory experiments measuring induction of plasma vitellogenin in exposed male fish (Jobling et al. 2002). Field studies were carried out using wild roach as a model fish to confirm the incidence and the severity of intersex which jibed with the predicted concentrations of the natural estrogens (E1 and E2) and the synthetic contraceptive pill estrogen (EE2) present (Jobling et al. 2006).Some case-studies have made clean-cut that the estrogenic activity of municipal wastewater correlates to demographics. The number of inhabitants was found to correlate with changes in estrogenic activities in a research conducted at a university city in US, with seasonal fluctuations in population. The concentrations of synthetic and natural estrogens and other EDCs were measured and effects demonstrated through the application of in vivo and in vitro tests (fish exposure with Vtg induction measurement and the yeast estrogen screen) (Brooks et al. 2003).The demonstration of effects of pulp mill effluents has also been supported by fish surveys with a sampling design that includes upstream and downstream sites from the discharge pipe of the pulp mill. For insta nce, Munkittrick et al. (1994) have demonstrated that the absence of chlorine bleaching or the presence of thirdhand treatment does not eliminate estrogenic responses evidenced by decreased circulating levels of sex steroids, decreased gonadal size, which implies that there may be multiple causative agents. In other cases, androgenic effects have been noticed, such as a biased male to female ratio in fish in Sweden downstream from pulp mills (Larsson and Frlin 2002).As seen on Table 3, several adverse endocrine effects are evidenced in various animals, from mollusks to amphibian but they also appear in higher animal species.Table 3. Effects of EDCs in wildlife evidenced through field studiesAnimalEffectEDCsPostulated mechanism or causative agentReferenceFrogHigh incidence of alter frogs in Minnesota, United States Multiple EDCsRetinoid signalingpathways activationGardiner et al. 2003Marine Gastropods masculinization of female snails (imposex) occurs worldwide. Females grow accesso ry sex organs including sperm ducts, seminal vesicles,external sperm grooves, and penises.Exposure to low levels of tributyltin (TBT) (1ng/l)Aromatase inhibition, testosterone inhibition, or neuroendocrine disorder or interaction with retinoid receptorsNovk et al. 2008Wild roach (Rutilius rutilus)Intersex, and high plasma Vtg concentrationMultiple EDCsSewage effluent from wastewater treatment plant discharging into riversJoblin et al. 2006Mosquitofish(Gambusia affinis)Masculinization (90% affected in number of segments in the long-term anal fin ray).Androgen-dependent gene expression by luciferase testkraft paper pulp mill effluentAffinity for human androgen receptor (hAR)Parks et al. 2001Eastern Mosquitofish, (Gambusia holbrooki)Androgenic activity measured by androgen receptor transcription assay with human receptor in sediment. Fish masculinization.Paper mill effluent, riverPine pulp-derived phytosteroids accumulate in river sediment where they are converted by microbes into pr ogesterone and this into androstenedione and other bioactive steroidsJenkins et al. 20033.3. Conceptual model ancestry a conceptual model requires knowing the pathways and toxicokinetics of the EDCs identified in the hazard identification step. An effects-based assessment start by identifying the effects and the relevant stressors and geographically located (for example through the use of GIS software). On the other hand, the model used in stressor-based assessments, depicts how stressors affect receptors and it is commonly applied when evaluating risks of environmental pollution. If a river basin is evaluated, the sources of contamination are studied, identifying the pathways, receptors and effects. To develop the human health risk assessment component, the fish consumption of the population and the drinking water white plague are two of the main factors to consider especially for the most vulnerable population, which are newborn and lactating infants. The food chain is the main s ource of exposure, and in particular, fish consumption and drinking water are affirmable sources for the nursing mother and the pathway of distribution through the milk to the baby, but the direct intake of drinking water is important in the case of formula preparation. The environmental risk assessment should consider fish, crustacean and sediment dwelling organisms within the framework.During pregnancy maternal fat is moved, releasing to the livestock the bioaccumulated compounds, due to their liposolubility and persistence, through all the different exposure routes (foodstuffs, environmental, occupational) throughout her life. keen exposure should also be considered if it happened previously to gestation or during this period. There are substances that traspass the placental barrier and chemicals reach the offspring. Also, through the breastmilk, explaining the extrangely high levels of some xenobiotics (Fernndez et al. 2007).3.4. Methodologies to determine dose-response in ex posure assessmentThe analysis of exposure and effect determines the concentration of the EDC on the environment matrixes matrices (water courses, ground water, drinking water, soil, sediment, air, biota), and assesses the potential or actual effects. In order to do so, many tools are recommended and in general a tiered approach is the most suited for this job as it helps to work in a logical order and increase the specificity of the tests.One of the main sources of exposure to most chemicals is through the food chain. The bioconcentration of organics in beef, cow milk and vegetation correlates to the octanol-water partition coefficient (Kow) to predict the bioaccumulation in the aquatic and terrestrial food chains (Travis and Arms 1988). There are many models based on the characteristics of the chemicals, such as the fugacity model, which allows to predict the anticipate concentrations in six environmental compartments (water, air, soil, bottom and suspended sediment and fish) (Ma cKay et al. 1985).3.4.1. The use of a tiered methodology to demonstrate endocrine disruptive effectsThis type of approach is carried out including different tests, such as bioassays, in vitro tests and field studies as part of the experimental design. The methodologies generally employed are in vivo fish reproduction exposure tests and in vitro receptor binding bioassays, for androgens and estrogens (Wartman et al., 2009). Even though there is an international trend towards diminishing the use of live organisms for experimentation for safety testing, in vivo tests are still of differentiate importance for the confirmation of the findings of in vitro screens. Some of the most utilized tests put across on the use of fish as model experimental organism in various life-stages, as for example the 21 days reproduction fish test with fathead minnow (EPA/600/R-01/067).3.4.2. In vitro screens and testsSome of the in vitro assays that can be used as screening tools of estrogenic activity ar e the following yeast based assays, cell proliferation assays, bindin