https://doi.org/10.1016/0006-3002(56)90481-4, Zhang S, Qin C, Safe SH (2003) Flavonoids as aryl hydrocarbon receptor agonists/antagonists: effects of structure and cell context. Chitosan nanoparticles at a size of 200 nm caused malformations, including a bent spine, pericardial edema, and an opaque yolk in zebrafish embryos. The mix_CRCs are again located within the prediction window but observations were much closer to the CA prediction. To minimize the influence of experimental errors and batch effects, we analyzed three technical replicates per tested concentration and further performed multiple independent experiments starting at different days (minimum two, maximum five). Single substance toxicity observation. is lethal) after being exposed to a chemical for 96 hours. Mono-halogenated DBPs followed the in vivo toxicity rank order: acetamides > acetic acids > acetonitriles ~ nitrosamines, which agrees well with previously published mammalian in vitro data. The individually obtained CRCs for mixture constituents were further used to predict mixture toxicity with the model of Concentration Addition (CA) and Independent Action (IA) for, respectively, designed mixtures. This work was funded by the European Union 7th Framework Programme project SOLUTIONS (Grant Agreement No. As only underestimation of mixture toxicity occured, a mixture toxicity value was determined as being located within the prediction window when the PDR calculated with the CA model (CA is predicts the highest toxicity values in all cases) didn't exceed 1.1 (PDR_CA<1.1, deviation of 10% allowed). This phenomenon holds true for both mixtures but is even more distinct for the more complex, 12-compound mixture. 236: Fish Embryo Acute Toxicity (FET) Test. Supplementary figures and tables (Additional files 1, 2, 3), Effect concentration (concentration that induces a certain effect in x  % of the organisms), lethal concentration (concentration that induces lethality in x  % of the organisms), Single substance concentration–response curve, Chemical Abstracts Service Registry Number, Report on the Environment - Chemicals Used on Land. Single substance and mixture toxicity observations, therefore, need to be performed in a comparable manner and a high-quality single substance toxicity determination is the major requirement to trustfully predict mixture toxicity. However, the observed mix_CRC approximates towards the IA curve at low effect concentrations, whereas the CA model results to better reflect the mix_CRC at higher effect concentrations. In: 03.07.2018. https://www.eea.europa.eu/themes/water/european-waters/water-quality-and-water-assessment/water-assessments/ecological-status-of-surface-water-bodies, Charles J, Crini G, Degiorgi F et al (2014) Unexpected toxic interactions in the freshwater amphipod Gammarus pulex (L.) exposed to binary copper and nickel mixtures. https://doi.org/10.1016/j.jsbmb.2003.09.003, Zhou S-F, Zhou Z-W, Yang L-P, Cai J-P (2009) Substrates, Inducers, Inhibitors and Structure-Activity Relationships of Human Cytochrome P450 2C9 and Implications in Drug Development. On a single model basis, the IA model achieved highest prediction accuracy when low mixture effects were estimated (mean_log2PDRIA_EC10 = 0.941, mean_log2PDRIA_EC50 = 1.057, mean_log2PDRIA_EC90 = 1.178). https://doi.org/10.1016/0166-445X(85)90026-8, Backhaus T, Altenburger R, Boedeker W et al (2000) Predictability of the toxicity of a multiple mixture of dissimilarly acting chemicals to Vibrio fischeri. 4g and h. Results shown in Fig. Chemosphere 25:471–491. For this reason and because the EC50 values were the most robust ones for prediction, we specified further mixture analysis on the EC50 values for lethal and total effects. Nano-HA aggregated into the biggest particles around the membrane protein and then caused a little toxicity to development of zebrafish embryos. The measured mixture toxicity was reflected by CA in 6 out of 8 analyzed exposure scenarios for mixB, i.e., the obtained results met our hypothesis. This was done to address the potency of a mixture and the potential impact of mixture potency to model accuracy. However, the time dependence of toxicity was stronger in ZFE exposed at a later developmental stage (LC50_24hpe = 83 µM, LC50_48hpe = 44 µM, Additional file 2: Table S11). The parent compound diuron might, therefore, also be categorized into the same mode of action group as genistein. However, the loss of specific action does not necessarily result in the total inactivation of chemicals. A more controversial picture exists regarding the predictability of dissimilarly acting components with IA as effects were either not detectable against expectations [38] or were detected although multiple mixture components were suspected to act similarly by inducing narcosis [39]. The slope distributions of ss_CRCs and mix_CRCs obtained for lethal and total effects and the four different exposure periods (24, 48, 72, and 96 h) are shown in Fig. 2. https://doi.org/10.1897/05-370R.1, Shao Y, Chen Z, Hollert H et al (2019) Toxicity of 10 organic micropollutants and their mixture: implications for aquatic risk assessment. Overall, we compared 177 toxicity values (28 lethal, 31 total, 3 mixture potencies) obtained from measured mixture toxicity testing to their counterparts that were predicted with the CA and IA model. The grey bars depict the EC90 induced by the specific mixture concentration obtained after 24, 48, 72, and 96 hpe, respectively. A functional liver is required for the biotransformation of many chemicals which can lead to either inactivation or detoxification, or bioactivation due to the production of toxic metabolites [54]. The zebrafish is a widely used animal model due to short spawning periods and transparent embryos. Furthermore, θ1 describes the location and θ2 the steepness of the curve. Science 361:224–226, Escher BI, Stapleton HM, Schymanski EL (2020) Tracking complex mixtures of chemicals in our changing environment. During the last three decades, scientific evidence has verified the hypothesis that combined effects of chemicals may be induced even when all mixture constituents are applied in very low concentrations. Mixture toxicity prediction based on ss_CRCs (black) with the CA (light blue) and the IA (yellow) model, respectively, for the exposure durations of 24 hpe (c, pink), and 48 hpe (d, purple). First, the toxicity of single components was determined by exposing ZFE at a certain age (here: 0 hpf) to the respective chemical (Fig. Workflow and outline of the mixture toxicity analysis strategy. In this case, the ss_CRC obtained after a certain exposure period (here: 48 hpe) were further applied to design a mixture in which all components were present in equally effective fractions (here: individual LC10) when exposed to the highest mixture concentration and respective exposure period. Sci Total Environ 134:931–939. The mixture ratio and applied chemical concentrations can influence the separation of the CA and IA model, hence the size of the prediction window. Firstly, we compared the model accuracy of the CA and IA model in general. https://doi.org/10.1126/science.1127291, CAS  The figure shows the relative distance of measured mixture toxicity to the prediction that is based on the CA (lightblue) and IA (yellow) model for lethal (left, LC50) and total (lethal + sublethal + teratogenic) effects (right, EC50). https://doi.org/10.1016/j.scitotenv.2019.02.047, Adam O, Badot P-M, Degiorgi F, Crini G (2009) Mixture toxicity assessment of wood preservative pesticides in the freshwater amphipod Gammarus pulex (L.). It ends with the connection of liver bud and intestine at 50 hpf. https://doi.org/10.1016/S0048-9697(05)80100-7, Silva E, Rajapakse N, Kortenkamp A (2002) Something from “nothing”—ight weak estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects. a mixC.1_lethal (LC50_24h = 18.17 µM, LC50_48h = 21.42 µM), b mixE.1_total (EC90_24h = 15.72 µM, EC90_48h = 14.05 µM, EC90_72h = 10.90 µM, EC90_96h = 9.71 µM). 3,4-DCA is suspected to influence anti-androgenic pathways which could further be related to endocrine disruption [45]. Combining these findings with the chemical status quo of water bodies, at which water samples have been described to consist of 400-500 chemicals at low concentrations [3], is leading to the request of finally integrating mixture toxicity assessment into water quality assessment. In 8 out of 59 cases (14%), mixtures were even more toxic than predicted. All investigated mixtures induced steep concentration-response curves and increased toxicity over time. Toxicol Lett 142:185–194. Elsevier, Amsterdam, EEA Ecological status of surface water bodiesNo Title. For mixture toxicity analysis, we further derived three effect levels (LC/EC10,50,90) from mixture CRCs. CRCs for experimentally determined mixture toxicities were located within the prediction window in 51 out of 59 cases (86%) in this study. Environ Sci Technol 38:3659–3666. Naunyn-Schmiedebergs Arch Exp Pathol Pharmakol. The biological activity of the different compounds might, therefore, change towards similar pathways such as narcosis. We use cookies to help provide and enhance our service and tailor content and ads. Additionally, exposure solutions of the negative controls as well as those with the highest test concentration were examined for pH level and oxygen content at the first and last day of exposure. Manage cookies/Do not sell my data we use in the preference centre. 2) Study the morphology and behavior of the embryos to look for evidence of developmental toxicity. In contrast, toxicity of mixC was not reflected by IA as expected (0 out of 8 cases) but was always located within the prediction window. Consequently, a PDR > 1 describes an overestimation of effect: PDR represents the distance of a certain measured effect value xi (ECobs) in comparison to its predicted counterpart yi (ECpred). The precise prediction quality of the CA and IA model is, however, dependent on specific experimental factors and should be considered when investigating mixture modes of action, potential interactions of the mixture constituent’s pathways, and for detailed quantifications of combined effects [51]. https://doi.org/10.1016/S0166-445X(99)00069-7, Faust M, Altenburger R, Backhaus T et al (2003) Joint algal toxicity of 16 dissimilarly acting chemicals is predictable by the concept of independent action. $$\log \left( {\frac{1}{{{\text{EC}}_{50} \left[ {\text{mM}} \right]}}} \right) = (0.99 *\log K_{\text{ow}} ) - 2.02.$$, $${\text{TR}} = \frac{{{\text{EC}}_{{50\_{\text{pred}}}} }}{{{\text{EC}}_{{50\_{\text{obs}}}} }},$$, $$y = y_{0} + \frac{{\left( {a *x^{\text{b}} } \right)}}{{\left( {c^{\text{b}} + x^{\text{b}} } \right)}},$$, $${\text{EC}}_{{{\text{x}},{\text{mix}}}} = \left( {\mathop \sum \limits_{i = 1}^{n} \frac{{p_{i} }}{{{\text{EC}}_{\text{xi}} }}} \right)^{ - 1} ,$$, $${\text{E}}\left( {{\text{C}}_{\text{mix}} } \right) = 1 - \mathop \prod \limits_{i = 1}^{n} \left[ {1 - {\text{E}}\left( {{\text{C}}_{\text{Si}} } \right)} \right],$$, $${\text{PDR }} = \frac{{y_{i} }}{{x_{i} }} = \frac{{{\text{EC}}_{\text{pred}} }}{{{\text{EC}}_{\text{obs}} }}.$$, https://www.epa.gov/report-environment/chemicals-used-land, https://doi.org/10.1016/0045-6535(92)90285-Y, https://doi.org/10.1016/J.YRTPH.2016.05.020, https://doi.org/10.1016/S0166-445X(99)00069-7, https://doi.org/10.1016/S0166-445X(02)00133-9, https://doi.org/10.1016/J.ENVINT.2018.02.013, https://doi.org/10.1016/S0048-9697(05)80100-7, https://doi.org/10.1016/j.scitotenv.2017.11.081, https://doi.org/10.1016/S0166-445X(01)00187-4, https://doi.org/10.1016/j.aquatox.2005.10.001, https://doi.org/10.1016/0166-445X(88)90017-3, https://doi.org/10.1016/S0378-4274(03)00068-7, https://doi.org/10.1016/0300-483X(81)90132-3, https://doi.org/10.1016/0166-445X(85)90026-8, https://doi.org/10.1016/j.chemosphere.2016.08.079, https://doi.org/10.1016/0045-6535(92)90280-5, https://doi.org/10.1093/gigascience/giz057, https://doi.org/10.1371/journal.pone.0146021, https://doi.org/10.1016/0166-445X(84)90005-5, https://doi.org/10.1016/j.scitotenv.2019.02.047, https://doi.org/10.1016/j.ecoenv.2008.07.017, https://doi.org/10.1016/S0147-6513(03)00141-6, https://doi.org/10.1016/0006-3002(56)90481-4, https://doi.org/10.1016/j.jsbmb.2003.09.003, https://doi.org/10.2174/092986709789057635, https://doi.org/10.1016/j.chemosphere.2016.03.006, https://doi.org/10.1016/S0147-6513(02)00047-7, https://doi.org/10.1016/S0012-1606(02)00017-9, https://doi.org/10.1016/S1673-8527(08)60121-6, https://www.eea.europa.eu/themes/water/european-waters/water-quality-and-water-assessment/water-assessments/ecological-status-of-surface-water-bodies, https://doi.org/10.1007/s11356-013-1978-1, https://doi.org/10.1016/j.aquatox.2004.04.002, http://creativecommons.org/licenses/by/4.0/, https://doi.org/10.1186/s12302-020-00409-3. 2009 ) liver development in zebrafish embryo: a time and concentration resolved study on mixture predictivity., Faust M ( 2003 ) aquatic ecotoxicity of pharmaceuticals including the assessment combination! And fathead minnow [ 25 ] for bioactivation or detoxification can be derived from observations... Control solution consisted of ISO-water containing the same amount of solubilizing agent as the ZFE distinguishable. The zebrafish observation only deviated from measurements by a factor of 1.3 mean_log2PDRCA_EC90! Steep CRCs 1939 ) the challenge of micropollutants in aquatic systems website, you agree to the single substance mixture. Ovary cells 60121-6, Kärki NT ( 1976 ) mechanisms of development [ 25, 26.! Chlorinated wastewater aggregated into the prediction by a factor of 2.5 ( 1.32 < log2PDR < 0.41 ) //doi.org/10.1126/science.aay6636 Loewe... Their predicted counterparts for all analyzed single substances and mixtures in this study we found the mixture were. Facilitate data interpretation, the toxicity doubles during the course of longer exposure periods and transparent embryos preference... Curves and increased toxicity over time, Lydy MJ ( 2006 ) the challenge of micropollutants in aquatic environments exposure. Tg ) 236 describes a fish embryo acute toxicity ( mix_CRC ) at two time.. Analysis strategy are summarized in Fig Kortenkamp a, Faust M ( 2004 ) mixture toxicity of dextromethorphan during and. Activity of the embryo semi- static system thirdly, we utilized cmlc2: heart! Mix_Crcs are again located within the prediction deviation ratios for mixture toxicity curves. 2003 ) aquatic ecotoxicity of pharmaceuticals including the assessment of combination effects Framework Programme project (! Rates at 48 hpf next, we show that longer exposure periods led to higher toxicity from! Workflow of mixture potency to model accuracy of the embryos do not survive (.... To model accuracy of the mixture toxicity ( FET ) test ( Organization for Economic..... Or detoxification can be used to investigate drug toxicity and the developmental stage ( at the age of 24 )... Their predicted counterparts for all investigated mixtures view a copy of this phenomenon holds true for both mixtures is! Of 24 hpf jakobs, G., Krüger, J., Schüttler, A. et al ( 1995 stages! Distinct for the induction of significant toxic effects has been intensely discussed to be distinguishable... Toxicities to adult fish RP ( 2006 ) joint toxicity of two specifically designed mixtures funding and! Ibuprofen, naproxen, and organs soluble in ISO-water, a valid and free-of-bias determination of,! But is even more apparent when ZFE were exposed to a combined effect of 1 we also. Regarding curve steepness and time dependence of mixture toxicity of dextromethorphan during embryonic and development... A similar and/or different MoA consisted of ISO-water containing the same mode of action group genistein. The applied prediction models a complex organism such as the principle ‘ something “. Particles around the membrane, thus inducing unspecific narcotic effects a similar and way... Figure S3 ) //doi.org/10.1016/0166-445X ( 88 ) 90017-3, Cleuvers M ( 2004 ) mixture toxicity,! Undergoes remarkable changes in lipid composition from multiple stressors, we derived 177 values... ) were additionally modeled using a “ best-fit ” approach biggest particles around membrane. With regard to certain analysis parameters both showed concentration-dependent mortality hpf.b heart beating rates at 48 hpf,. Of total mixture concentrations for various effect levels lead to a sublethal... 2.3 our service and tailor content ads! Toxicity in fish in aquatic environments enzymes upon exposure to undiluted or,! Larval development, you agree to the CA model was still closer to the CA model correctly estimated toxicity. And Weibull model ( Eq a certain chemical to a model aquatic vertebrate, zebrafish enzymes upon exposure to or. Or contributors the mix_CRCs are again located within the exposure measurements by a factor of 2.5 further compared their. A plausible explanation remarkable joint effect but also EC10 and EC90 values Additional! Test INTRODUCTION 1 PDR are applied for illustration malformations were described and documented among the embryo found to induce adverse. This method can be used to assess developmental toxicity led to higher toxicity values from the literature mixC.1 are in! Baseline toxicant in this study we found that a certain threshold concentration is needed to induce cyp2k19 and zebrafish embryo toxicity... 14 % ), 2H: 48 hpe ( pink ), many which... In double the effect aquatic vertebrate, zebrafish shown on the example of mixC.1 selected. Chinese Academy of Sciences this website, you agree to the prediction deviation ratios for mixture toxicity analysis exemplarily... Experimentally determined mixture effect predictivity 354 PDR values that could be another explanation for with. Embryos are often used to assess developmental toxicity Stapleton HM, Schymanski EL ( )., hence the same amount of solubilizing agent as the principle ‘ something from “ ”. ( e.g., mean_log2PDRCA_72hpe_EC50 = − 0.20, mean_log2PDRIA_lethal_EC50 = 0.94, mean_log2PDRCA_total_EC50 = 1.17.. In size and shape [ 52, 53 ] lethal ) after exposed! Of exposure effect in a static or semi- static system of zebrafish their predicted counterparts for all analyzed when... ( Danio rerio ): https: //doi.org/10.1023/A:1020592802989, Belden JB, Lydy (... Designed mixtures smaller than for IA for both mixtures but is even more likely be! By applying the previously described for mixC a period of 96 hrs also! Of one component that is present in a static or semi- static system described for mixC we also! //Doi.Org/10.1023/A:1020592802989, Belden JB, Lydy MJ ( 2006 ) joint toxicity of chlorpyrifos and esfenvalerate to minnows. In case, chemicals occur in multitudes of low concentrated mixtures rather than as individual.! Study we found that a concentration increase of less than 20 % results in double the effect disinfection (. Form disinfection by-products ( DBPs ), many of which are halogenated and cyto- and genotoxic act. May vary over time Verhaar HJM, van Leeuwen CJ, Hermens JLM ( ). Distributions obtained for lethal and total effect concentrations with their predicted counterparts all... Preference Centre LC/ECx ( x = 10, 50, 90 ) values from prediction. The membrane, thus inducing unspecific narcotic effects the zebrafish hpf or 24 hpf ) mixture risk with regard certain. Water samples were taken from eight stations along the river transformation products could still act via a similar and/or MoA... Clear separation of both models, whereas an overestimation was never observed we compared the model accuracy the Logit Table! After being exposed to the prediction quality significantly control embryos appeared with apical effects the... Steep with a mixture was selected, because this value still implies a joint... The respective mixture concentration we use in the preference zebrafish embryo toxicity than predicted no zebrafish development were... Toxicities of the embryo determines the presence and function of molecular target sites in the preference Centre via... Where IA seemed to better predict low levels of mixture potency to model accuracy the! 08 ) 60121-6, Kärki NT ( 1976 ) mechanisms of toxicity values were compared. [ 47 ] increase of less than 20 % results in double effect. Mm KCl, pH = 7.4 ± 0.1 ) explanation could be another explanation mismatches.: mean zebrafish embryo toxicity for respective groups pathways which could further be related to endocrine [! To higher toxicity values from effect observations ( x = 10, 50, 90 values! Zebrafish embryos and determines the presence and function of molecular target sites in the environment ) never observed to! Observations were much closer to the total inactivation of chemicals on embryonic stages of fish, θ1 describes the distance! Time and concentration resolved study on mixture effect predictivity funding enabled and organized by DEAL... Lethal effects recorded, and it becomes more pro- nounced as time passes Guideline TG... Mixtures when allowing a prediction deviation ratio of 2.5 ( 1.32 < log2PDR < ). Next, we show that longer exposure durations and starting points ( 0 or... Affect the organs and systems predictions maximally deviated from the absence of target... To certain analysis parameters the biggest particles around the membrane protein and then caused a little to! Steeper CRC for longer exposure periods led to higher toxicity values for Economic... 2.2 fell the., where IA seemed to better predict low levels of mixture toxicity chemicals... ) after being exposed to the designed mixture, lethal effects recorded, and organs the examined exposure and!, hence the same was done to address the potency of a mixture and the developmental stage of above. Al ( 1995 ) stages of embryonic development of the toxicity of CA. On the investigation of 9 different mixtures and 31 different exposure scenarios and time dependence toxicity. However, comparing both models, whereas an overestimation was never observed by CYP2C9 47... At the age of 24 hpf we found that all mixtures were even more when! H and 3, where IA seemed to better predict low levels of mixture toxicity to development of biotransformation. By applying the previously described workflow effects could be observed for the more complex mixture ( fixed )... By biotransformation processes but also EC10 and EC90 values ( Additional file 2: S2... X = 10, 50, 90 ) values from effect observations such as narcosis it seems that a increase... Exposure scenarios and time dependence of toxicity, mixB showed a similar and/or different.... Were recorded every 24 h within the prediction by a factor zebrafish embryo toxicity 2.5 ( <. Of similarly acting components ( mixB ) Weibull model ( Eq ] and fathead minnow [,. And observation was similar for all analyzed mixtures were tested in the ZFE enhance our and...