The PDR describes the relative distance of an observed toxicity value to its predicted counterpart. 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 developmental stage of the embryo determines the presence and function of molecular target sites, tissues, and organs. These are just examples for the induction of the biotransformation system and the list of metabolites could easily be extended. In gene expression studies with ZFE, diclofenac was found to induce cyp2k19 and cyp2c9 [32, 48]. https://doi.org/10.1021/es015844c, Klüver N, Vogs C, Altenburger R et al (2016) Development of a general baseline toxicity QSAR model for the fish embryo acute toxicity test. https://doi.org/10.1016/0166-445X(88)90017-3, Cleuvers M (2003) Aquatic ecotoxicity of pharmaceuticals including the assessment of combination effects. Cyprodinil, another component of mixC, was the only assumed baseline toxicant in this mixture. Due to time dependence of single substance toxicity, the impact of a certain chemical to a combined effect may vary over time. Incubation was conducted at 26 °C with a 12:12 h light:dark photoperiod and vials shaken at 75 rpm using a horizontal agitator (Edmund Bühler GmbH, SM–30 control). Expose zebrafish embryos to at least 1,000 chemicals. This phenomenon is considered as the principle ‘something from “nothing”’ (Fig. is lethal) after being exposed to a chemical for 96 hours. Although acute toxicity disappears more and more off the monitor of concern, the environmental status of European surface waters is still in very poor condition [55]. This work was funded by the European Union 7th Framework Programme project SOLUTIONS (Grant Agreement No. Comparison of observed mixture toxicity to predicted mixture toxicity. c further refers to the LC50 or EC50 value, b to the slope of the tangent at the inflection point and x to the related concentration. An overview of time dependent mortality for all analyzed single substances and mixtures in this study is given in Table 4. 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. Terms and Conditions, The parent compound diuron might, therefore, also be categorized into the same mode of action group as genistein. the zebrafish embryo toxicity and teratogenicity assay Jordan Ferdin A. Halili and Jonas P. Quilang Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon 6 indicate that a significant mixture effect was induced even though individual chemicals were applied in concentrations below their individual effect thresholds (EC20, see “Methods” section). Figure 4a depicts the distribution of all 354 determined PDRs (grey bars), whereas the colored density plots show the distributions of PDRs for CA (light blue) and IA (yellow), respectively. One mixture contained only similarly acting substances (mixB), hence mixture toxicity expected to be predictable with the CA model, and another mixture contained only suspected dissimilarly acting constituents (mixC), hence the IA model was assumed to best predict mixture toxicity. This was done to address the potency of a mixture and the potential impact of mixture potency to model accuracy. The data shown in Fig. Visualization: GJ. Recalling the main principle of the IA model at which a combined effect is induced through interaction of mixture components with independent target sites by different modes of action, leads to the hypothesis that an independent action of chemicals may not be exerted in complex organisms [16]. The present study was designed to use zebrafish as a model to investigate the potential toxicity of dextromethorphan during embryonic and larval development. Gene expression analysis in ZFE also revealed an induction of CYP1 enzymes upon exposure to diuron [32]. Surveying the entirety of results revealed that for a remarkable number of 90% (53 out of 59 cases) a combined effect of 90% is caused by a mixture constituted of very low concentrated chemicals (Additional file 2: Figure S11–21). From effects of nanosized HA, SiO(2) and TiO(2) particles on the zebrafish embryos development, they were adsorbed on the membrane surface confirmed by the electronic scanning microscopy. © 2017 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Predictions appeared to be slightly more robust when the totality of effects were inspected (lethal, sublethal, and teratogenic) but the inspected effect type seems to be less relevant concerning the performance of both models, in general. Project administration: WB. https://doi.org/10.1016/J.ENVINT.2018.02.013, Grimme LH, Altenburger R, Backhaus T, Bödeker W, Faust M, Scholze M (1998) Vorhersagbarkeit und Beurteilung der aquatischen Toxizität von Stoffgemischen: Multiple Kombinationen von unähnlich wirkenden Substanzen in niedrigen KonzentrationenNo Title. Hence a concentration increase of less than 20% results in double the effect. Sci Total Environ 134:931–939. All authors read and approved the final manuscript. Google Scholar, Busch W, Schmidt S, Kühne R et al (2016) Micropollutants in European rivers: a mode of action survey to support the development of effect-based tools for water monitoring. The zebrafish embryo offers an inexpensive system that combines many features that are desirable for the development of new approaches to drug development (Bowman and Zon, 2010).As a vertebrate, the zebrafish shares a high degree of conservation with mammalian systems: the genomes of zebrafish and humans are highly … Carbendazim, cyprodinil, and genistein show no time dependent toxicity, whereas lethal concentrations of diclofenac and diuron are decreasing over time (LC50_diclofenac_0to24hpf = 133.15 µM, LC50_diclofenac_0to48hpf = 15.84 µM). This did not consolidate the trend described in Figs. The utility of fish embryos for pesticide hazard assessment was investigated by comparing published zebrafish embryo toxicity data from pesticides with median lethal concentration 50% (LC50) data for juveniles of 3 commonly tested fish species: rainbow trout, bluegill sunfish, and sheepshead minnow. 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. Environ Sci Technol 38:3659–3666. To facilitate data interpretation, the log2 values of PDR are applied for illustration. Figure 2g and h show the observed mix_CRCs (solid, black line) vs. the predicted CRCs calculated with the CA (dashed, yellow line) and IA model (dashed, light blue line), respectively, for the two different exposure durations (Fig. 2g: 24 hpe (pink), 2H: 48 hpe (purple)). This workflow is shown on the example of mixC.1 and selected results for mixC.1 are depicted in Fig. For detection of effects induced by controls, six technical replicates were used, whereas three technical replicates were conducted to analyze the effects induced by treatment solutions. The total molarity is calculated from the entirety of all single components present in their specific concentrations. (Submitter supplied) Zebrafish is an ideal model for the toxicity studies on medicines and environmental genetic toxicants.Different development defects were observed in zebrafish embryos exposed to -ray and heavy ion (carbon or iron) irradiation We used microarrays to detail the … © 2021 BioMed Central Ltd unless otherwise stated. In Gammarus pulex [40], mixture toxicity was underestimated by both models, whereas the combined effect of either similarly or dissimilarly acting components was proven to be predictable by the appropriate model in freshwater algae Scenedesmus vacuolatus (CA: [19, 20, 41], IA: [13]), luminescent bacteria Vibrio fischeri (CA: [21], IA: [12]) and Daphnia magna (CA: [22, 41]). By the definition of the concepts, CA predicts the toxicity of mixture containing similarly acting substances, whereas the IA model predicts the toxicity of mixtures that are composed of dissimilarly acting components. We compared the experimentally determined mixture effect concentrations with their predicted counterparts for all tested mixtures. 8). According to the mathematical formalizations of these two concepts, the CA concept implies that every toxicant in any concentration contributes, in proportion to its toxic unit, to the overall combined effect of a mixture [9, 10]. The IA model revealed its highest model accuracy for low effect concentrations and short-term exposures (mean_log2PDRIA_EC10 = 0.941, mean log2PDRIA_24hpe = 0.978), but was on average always less accurate in comparison to the CA model. 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. https://doi.org/10.1021/es0101227, Thrupp TJ, Runnalls TJ, Scholze M et al (2018) The consequences of exposure to mixtures of chemicals: something from ‘nothing’ and ‘a lot from a little’ when fish are exposed to steroid hormones. J Steroid Biochem Mol Biol 87:285–299. 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. bbmle: Tools for General Maximum-Likelihood Estimation. Mixture toxicity analysis in zebrafish embryo: a time and concentration resolved study on mixture effect predictivity. The test method described in this Test Guidelineis inteneded to determine the acute or letal toxicity of chemicals on embryonic stages of fish (Danio rerio). No significant difference between the 24 h and a 48 h exposure duration was observed for the early treatment period (mixB.1, LC50_24hpe = 35 µM, LC50_48hpe = 34 µM), whereas the late exposure start resulted in an increase in toxicity over time (mixB.2, LC50_24hpe = 108 µM, LC50_48hpe = 77 µM). In a developing system such as the ZFE, the time point of exposure, i.e., the age of the embryo at exposure start, is an additional determining factor for toxicity. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. statement and J Biophys Chem 03:334–340. 6. The parameter of all concentration–response curves are summarized in Additional file 2: Tables S2–5. The increase in toxicity over time and a more pronounced time dependency of toxicity in older embryonic stages can be explained with toxicokinetic (e.g., chemical uptake kinetics and metabolism) and toxicodynamic (e.g., presence of certain target sites and functionality of pathways) processes. For both, single substance and mixture toxicity determination, 23 integral effects, classified in lethal, sublethal, and teratogenic (see Additional file 2: Table S1), were recorded using a dissecting microscope (5×, Olympus IX70). In this study, we investigated the accuracy of two mixture toxicity prediction models, the model of Concentration Addition (CA) and Independent Action (IA). https://doi.org/10.1016/J.YRTPH.2016.05.020, Kortenkamp A, Faust M (2018) Regulate to reduce chemical mixture risk. Ecotoxicol Environ Saf 54:139–150. The MR describes the proportion of one component that is present in a mixture relative to the total molarity of this mixture. For that purpose, two non-linear models, the Logit (Table 3, Eq. Figure 4 illustrates the distributions of the log2 of these PDRs with regard to certain analysis parameters. Furthermore, we show that the detection of this phenomenon is not dependent on the examined exposure scenario and considered phenotype. The different regression models were fitted using the software R (version 3.4.4) and the package bbmle (Ben Bolker and R, Development Core, Team (2017). Environ Toxicol Chem 24:324. https://doi.org/10.1897/04-032R.1, Field HA, Ober EA, Roeser T, Stainier DY (2003) Formation of the digestive system in zebrafish. Subsequently, ZFE were exposed to the designed mixture, lethal effects recorded, and mixture toxicity observed (Fig. 4b, where the PDR distributions obtained for lethal and total effects were plotted separately. 2a). Data analysis. Dev Biol 253:279–290. https://doi.org/10.1016/S1673-8527(08)60121-6, Kärki NT (1976) Mechanisms of toxicity and metabolism. For example, a mixture that induces an effect in 10% of examined organisms is less potent than a mixture that induces effects in 90% of the organisms. In this study we found that all mixtures induce steep CRCs. This Test Guideline (TG) 236 describes a Fish Embryo Acute Toxicity (FET) test with the zebrafish (Danio rerio). Although the highest mixture toxicity prediction accuracies were achieved for longer exposures with high concentrations, CA-based predictions deviated from measurements by a factor of 2.5 only, even for mixtures with only suspected dissimilarly acting components and short-term exposures with low concentrations. Distributions are grouped by a prediction models, b prediction models and phenotype, c mixture potency, d mixture potencies and prediction models, e EC50 and phenotype, f by EC50 and phenotype and prediction model, g EC50 and exposure duration, and h EC50 and exposure duration and prediction model. https://doi.org/10.1016/S0166-445X(02)00133-9, Altenburger R, Scholze M, Busch W et al (2018) Mixture effects in samples of multiple contaminants—an inter-laboratory study with manifold bioassays. The authors declare that they have no competing interests. In this study, lethal describes all effects that imply the death of an organism (e.g., coagulation, missing heartbeat [33]), whereas sublethal and teratogenic effects are defined by effects which lead to a change in development and fitness of the organism or to malformations of the ZFE (e.g., change in blood circulation or frequency of heartbeat, formation of edema, or malformation of head, yolk, or tail, etc. https://doi.org/10.1126/science.aay6636, Loewe S, Muischnek H (1926) Über Kombinationswirkungen1 Mitteilung: Hilfsmittel der Fragestellung. Silva et al., for example, showed that a mixture of eight weak estrogenic compounds were able to evoke a significant combined effect in yeast although the mixture components were present below their individual NOEC [17]. 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. Zebrafish embryo toxicity test. For mixture toxicity prediction, the CRC of single substances (ss_CRC) were additionally modeled using a “best-fit” approach. Meeting the requirements of high-quality and consistent toxicity observation as well as distinctive separation of toxicity predictions allowed us to reliably compare measured and predicted mixture toxicity values and evaluate the respective comparisons. For mixture toxicity analysis, we further derived three effect levels (LC/EC10,50,90) from mixture CRCs. 4e, f, h). This resulted in 354 PDR values that could be considered (177 for each model, Additional file 2: Table S6). This became even more apparent when total effect concentrations are considered (Fig. https://doi.org/10.1371/journal.pone.0146021, Grimme LH, Altenburger R, Backhaus T, Boedeker W, Faust M, Scholze M (1998) Vorhersagbarkeit und Beurteilung von Mischungen, Hermens J, Canton H, Janssen P, De Jong R (1984) Quantitative structure-activity relationships and toxicity studies of mixtures of chemicals with anaesthetic potency: acute lethal and sublethal toxicity to Daphnia magna. Chemosphere 25:471–491. Chemicals with a time dependent toxicity have a varying impact on the toxicity of a respective mixture over time, when the mixture ratio is kept constant. The results are exemplarily shown for a rather simple (mixC.1) and more complex mixture (mixE.1) in Fig. Sci Total Environ 619–620:1482–1492. Slopes of single substance and mixture concentration–response curves (CRCs). Here, we demonstrate for the first time that mixture toxicity can be robustly estimated with the prediction window for a diversity of mixtures and exposure scenarios. a Survival rates throughout 24–120 hpf.b Heart beating rates at 48 hpf. Biochim Biophys Acta 19:548–549. A further detailed resolution is shown in Fig. Initially, we investigated the predictability of the toxicity of two specifically designed mixtures. I. liver morphogenesis. We further calculated LC/ECx (x = 10, 50, 90) values from each mixture CRC. The PDR distributions according to exposure times are shown in Fig. This phenomenon holds true for both mixtures but is even more distinct for the more complex, 12-compound mixture. The values are expressed as the mean ± SE. Embryos at approximately endpoints used to assess developmental toxicity were pericardial at 3 hpf (hours post fertilization) were used for the exposure. However, comparing both models, the CA model was still closer to the observations than the IA predictions. While we found both, increased and decreased toxicity over time, for the single compounds, mixture toxicity increased over time in all cases. For three exposure scenarios no lethal effects could be observed for the tested concentrations (0–24_mixE.2, 24–48_mixE.2, and 24–48 h_mixF). 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. 4c (all data) and Fig. We analyzed whether certain experimental and analysis parameters, such as the mixture design, exposure duration, phenotype, and mixture potency influence the predictability of mixture toxicity. Aquat Toxicol 49:49–61. Inspecting the predictability of different effect levels revealed that the highest model accuracy was obtained when high effect concentrations were predicted with the CA model. OECD, Ritz C, Baty F, Streibig JC, Gerhard D (2015) Dose-response analysis using R. PLoS ONE 10:e0146021. Finally, we studied the induction of combined toxic effects in ZFE after exposure to mixtures in which the components were present in concentrations that failed to evoke individual toxicity. Where θmin is the minimum effect of 0 and θmax the maximum effect of 1. The Fish Embryo Acute Toxicity (FET) test with the zebrafish (Danio rerio) embryo, the OECD test guideline (TG) 236, has been designed as an alternative for acute fish toxicity testing such as the OECD Acute Fish Toxicity Test (TG 203). a Exposure of ZFE to different concentrations of single substances and effect determination at different time points. Environ Toxicol Chem 24:2665. https://doi.org/10.1897/04-639R.1, Kienzler A, Bopp SK, van der Linden S et al (2016) Regulatory assessment of chemical mixtures: requirements, current approaches and future perspectives. https://doi.org/10.4236/jbpc.2012.34041, Zhou S (2018) Cytochrome P450 2D6, 1st edn. Science 367:388–392. 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. Regarding usually standard errors of ecotoxicological studies and concentration response determinations, this could be seen as reasonable prediction accuracy. Environ Int 114:95–106. On average we found that a concentration increase of approximately 15% resulted in twice the effect in the zebrafish embryo toxicity test (see Table 5) based on the LC/EC 50. Experimental workflow of mixture analysis, exemplarily shown for mixC.1. 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]. In total, 149 out of 177 (84%) inspected mixture effect concentration values were located within the prediction window, whereas in 28 out of 177 cases, mixture toxicity was underestimated with both models. However, genistein was also identified to interfere with the aryl hydrocarbon receptor (AhR), e.g., as agonist in HepG2 cells [43] and rat liver [44], resulting in the induction of monooxygenases of the CYP1 family. All mixtures were tested in the zebrafish embryo acute toxicity assay (ZFET) and obtained measurements were compared with respective predictions. The mix_CRCs also occurred to be steep (LC75/LC50 (24 hpe) = 1.10, LC75/LC50 (48 hpe) = 1.15, Additional file 2: Table S8). Similar findings were obtained when comparing not only EC50 but also EC10 and EC90 values (Additional file 2: Figure S10). Another test that uses zebrafish embryos is the OECD’s fish embryo toxicity test (FET), the final draft of which became available in July 2013.2 Both the fish embryo test, which exposes fish embryos to various chemical compounds to determine LC50 and toxicity, and the zebrafish embryo toxicity test rely on parallel methodologies. It has been reported that combined toxic effects were even observable when chemicals were applied in concentrations below their individual effect thresholds. Every 24 hrs. Aquat Toxicol 76:93–110. https://doi.org/10.1016/S0166-445X(01)00187-4, Junghans M, Backhaus T, Faust M et al (2006) Application and validation of approaches for the predictive hazard assessment of realistic pesticide mixtures. We also investigated whether the developmental stage (0 or 24 h post fertilization (hpf)) at which the ZFE were exposed to the mixtures influenced the predictability of mixture toxicity. Manage cookies/Do not sell my data we use in the preference centre. Open Access funding enabled and organized by Projekt DEAL. Chemosphere 25:531–542. The slopes of all analyzed mixtures were steep with a trend towards steeper CRC for longer exposure durations. 1d). https://doi.org/10.1021/es0351591, Verhaar HJM, van Leeuwen CJ, Hermens JLM (1992) Classifying environmental pollutants. The fish embryotoxicity test (FET) in zebrafish was used to identify toxicological hotspots along the Atoyac River, Mexico, to determine the presence of chemical substances with teratogenic effects for a possible monitoring program. A test was classified valid if less than 20% of control embryos appeared with apical effects. Jakobs, G., Krüger, J., Schüttler, A. et al. pearing in the development of zebrafish, Danio (Brachydanio) rerio, embryos fertilized simultaneously in vitro (C. Walker and G. Streisinger, in Westerfield, 1994) and incubated at an optimal temperature with- out crowding (28.S0C, 5-10 embryos/ml). We estimated the toxicity of all mixtures with two models, the concept of Concentration Addition (CA) and Independent Action (IA). 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]. Newly fertilised zebrafish eggs are exposed to the test chemical for a period of 96 hrs. The PDR distributions respecting the three effect levels (EC10: light blue, EC50: yellow, EC90: pink) are depicted in Fig. Gigascience. The light blue (CA) and yellow bars (IA) indicate the effects predicted by the models for the respective mixture concentration. https://doi.org/10.1002/etc.5620190927, Kimmel CB, Ballard WW, Kimmel SR et al (1995) Stages of embryonic development of the zebrafish. $$\log \left( {\frac{1}{{{\text{EC}}_{50} \left[ {\text{mM}} \right]}}} \right) = (0.99 *\log K_{\text{ow}} ) - 2.02.$$, $${\text{TR}} = \frac{{{\text{EC}}_{{50\_{\text{pred}}}} }}{{{\text{EC}}_{{50\_{\text{obs}}}} }},$$, $$y = y_{0} + \frac{{\left( {a *x^{\text{b}} } \right)}}{{\left( {c^{\text{b}} + x^{\text{b}} } \right)}},$$, $${\text{EC}}_{{{\text{x}},{\text{mix}}}} = \left( {\mathop \sum \limits_{i = 1}^{n} \frac{{p_{i} }}{{{\text{EC}}_{\text{xi}} }}} \right)^{ - 1} ,$$, $${\text{E}}\left( {{\text{C}}_{\text{mix}} } \right) = 1 - \mathop \prod \limits_{i = 1}^{n} \left[ {1 - {\text{E}}\left( {{\text{C}}_{\text{Si}} } \right)} \right],$$, $${\text{PDR }} = \frac{{y_{i} }}{{x_{i} }} = \frac{{{\text{EC}}_{\text{pred}} }}{{{\text{EC}}_{\text{obs}} }}.$$, 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. The control solution consisted of ISO-water containing the same amount of solubilizing agent as the treatment solutions (0.1% V:V).