Hence, the CA model, potentially applied with a safety factor, should provide a solid tool to predict mixture toxicity for environmental exposure scenarios, water quality and risk assessment. Ecotoxicol Environ Saf 72:441–449. All 177 calculated PDRs are listed in Additional file 2: Table S6. The resulting transformation products could still act via a similar and/or different MoA. The obtained mix_CRCs and their comparison to predictions are shown in Additional file 2: Figure S2. In Fig. Overall, the highest model accuracy in terms of smallest PDRs were found with the CA model for high effect concentrations (mean_log2PDRCA_EC90 = − 0.15) and the 72 h exposure period (mean_log2PDRCA_72hpe = − 0.09). statement and Chitosan nanoparticles at a size of 200 nm caused malformations, including a bent spine, pericardial edema, and an opaque yolk in zebrafish embryos. Cyprodinil, another component of mixC, was the only assumed baseline toxicant in this mixture. 2g and h and 3, where IA seemed to better predict low levels of mixture effects. For complete validation of the test, a pH level between 6 and 8 and a minimum oxygen level of 60% was required at examined time points. In this study, the distance of two predicted ECx values (x= 10, 50, 90) ranged from 1.4 to 5.5 with an average distance of 2.7, hence CA and IA were sufficiently different. The EC90 of a mixture was selected, because this value still implies a remarkable joint effect but also guarantees statistical robustness. To identify changes in lipid composition from multiple stressors, we exposed ZF embryos to a sublethal... 2.3. PubMed Google Scholar. Regarding curve steepness and time dependence of toxicity, mixB showed a similar trend as previously described for mixC. Malformations were described and documented among the embryo 2.3. 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). The observed mixture toxicity was located within the prediction deviation range between the CA and IA estimations in 51 out of 59 cases (86%) or was even underestimated by both models (8/59, 14%). 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. Toxicology 19:229–238. 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. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Environ Sci Technol 36:1751–1756. The developmental stage of the embryo determines the presence and function of molecular target sites, tissues, and organs. 2d: 48 h (purple)). 1a). This is not necessarily the case but is, however, frequently observed when mixtures consist of multiple chemicals at low concentrations (e.g., [37]). Mixture toxicity analysis in zebrafish embryo: a time and concentration resolved study on mixture effect predictivity. a Exposure of ZFE to different concentrations of single substances and effect determination at different time points. The control solution consisted of ISO-water containing the same amount of solubilizing agent as the treatment solutions (0.1% V:V). However, for the IA model, the highest model accuracy was achieved when low mixture effects obtained after short exposure durations were estimated (mean_log2PDRIA_EC10 = 0.941, mean_log2PDRIA_24hpe = 0.978). The authors declare that they have no competing interests. Chemosphere 25:531–542. Ecotoxicology 11:299–310. In conclusion, single substance and mixture toxicity was assessed in a concentration (10–13 dilutions of exposure solution), time (every 24 h within two exposure periods (0–96 hpf and 24–96 hpf)), effect type (23 quantal effects categorized in lethal and sublethal or teratogenic), and mixture potency resolved manner (LC/EC10,50,90). In a next step we compared the PDR distributions for lethal and total effects, as well as for different exposure durations based on EC50 values (Fig. 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. bbmle: Tools for General Maximum-Likelihood Estimation. 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). 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). 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. 4d (differentiated with respect to the applied models (left: CA, right: IA)). https://doi.org/10.1016/S0012-1606(02)00017-9, Tao T, Peng J (2009) Liver development in zebrafish (Danio rerio). All these examples refer to situations, where experiments were specifically designed to investigate the impact of low concentrated chemicals and to assess their ability to evoke a combined effect. 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. The total molarity is calculated from the entirety of all single components present in their specific concentrations. Hence, the prediction window serves as a reliable tool to predict the concentration-effect space for mixture toxicity in ZFE. https://doi.org/10.1016/j.aquatox.2005.10.001, Altenburger R, Backhaus T, Boedeker W et al (2000) Predictability of the toxicity of multiple chemical mixtures to Vibrio fischeri: mixtures composed of similarly acting chemicals. Visualization: GJ. Therefore, we observed lethal, sublethal, and teratogenic effects induced in ZFE after exposure to nine different mixtures for varying exposure durations (24, 48, 72, 96 h post exposure (hpe)). This workflow is shown on the example of mixC.1 and selected results for mixC.1 are depicted in Fig. https://doi.org/10.1016/j.ecoenv.2008.07.017, Cleuvers M (2004) Mixture toxicity of the anti-inflammatory drugs diclofenac, ibuprofen, naproxen, and acetylsalicylic acid. By continuing you agree to the use of cookies. 3) using the drc package, R (version 3.4.4) [34]: where y refers to the level of lethal or total effects, a to the minimum effect (0) and y0 to the maximum effect (1). 2e). Additionally, high effect values seem even more likely to be underestimated by the applied prediction models. CRC Press, Boca Raton, Heinonen S-M, Hoikkala A, Wähälä K, Adlercreutz H (2003) Metabolism of the soy isoflavones daidzein, genistein and glycitein in human subjects. It seems that independent MoAs of mixture components converge with increasing effective concentrations and longer exposure durations into joint unspecific pathways of disturbance in a complex organism. We calculated a factor for time dependence (td) of toxicity by calculating the ratio between the LC50 obtained after the longest exposure duration and LC50 obtained after the shortest exposure duration. The biological activity of the different compounds might, therefore, change towards similar pathways such as narcosis. The results of our study and the considerations above indicate that independent action of chemicals in a mixture seems to be more likely for short exposure durations and low effect concentrations. The CA model correctly estimated the toxicity in 100% of analyzed mixtures when allowing a prediction deviation ratio of 2.5. In case, chemicals were not easily soluble in ISO-water, a co-solvent was used. Aquat Toxicol 68:351–367. Overall, the CA model predictions maximally deviated from measurements by a factor of 2.5 (1.32 < log2PDR < 0.41). MixC.1 was composed of five suspected independently acting substances (see Table 2), namely carbendazim (triangle), cyprodinil (circle), diclofenac (diamond), diuron (star), and genistein (square). https://doi.org/10.1002/etc.3460, Calamari D, Vighi M (1992) A proposal to define quality objectives for aquatic life for mixtures of chemical substances. 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). The toxicity of single substances was determined after exposing ZFE for 0–96 hpf and 24–96 hpf to all selected chemicals, respectively (15 in total, Table 1). ‘Something from “nothing”’: Predicted effects modeled with CA (light blue) and IA (yellow) at mixture concentrations evoking 90% effect in measurements (grey bars) and the effects evoked by single substances when applied individually at the concentrations present in the mixture at 24 hpe (pink), 48 hpe (purple), 72 hpe (orange) and 96 hpe (rosé). 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). Similar to the results shown in Fig. Also, the slopes of ss_CRCs were relatively steep but showed broader distributions. Sci Total Environ 666:1273–1282. We estimated the toxicity of all mixtures with two models, the concept of Concentration Addition (CA) and Independent Action (IA). 2a). Particularly, zebrafish embryo toxicity model provides an alternative to acute fish toxicity tests in terms of animal welfare perspective as the embryos are not considered live until 5 days after fertilization. 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). In 8 out of 59 cases (14%), mixtures were even more toxic than predicted. 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. https://doi.org/10.1289/ehp.9358, Bliss CI (1939) The toxicity of poisons applied jointly. In our study, we utilized cmlc2:GFP heart transgenic and wild type zebrafish embryos. Sci Total Environ 619–620:1482–1492. By using this website, you agree to our For example, a correct estimation of mixture effect is indicated by a PDR of 1, whereas an underestimation of effect would be indicated by a PDR < 1. This was not only the case for mixtures containing exclusively similarly acting components but was also true for mixtures that contained only dissimilarly acting components or a blend of both. 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. For each replicate, three embryos were exposed to 6 mL of either treatment or control solution and incubated for desired exposure time in 7.5 mL GC vials (VWR International) closed with aluminium lid and aluminium coated septum (Supelco Analytical). The combined effect of similarly acting components was predictable by CA in guppy [24] and fathead minnow [25]. Comparison of observed (black) and predicted mixture toxicity (light blue: CA, yellow: IA) for the 24 hpe (g, pink) and 48 hpe (h, purple) exposure duration. The parameter of all concentration–response curves are summarized in Additional file 2: Tables S2–5. The induction of biotransformation could lead to an increase of compound degradation of applied chemicals and the hereinafter following loss of specific action. 236: Fish Embryo Acute Toxicity (FET) Test. ’ ( Fig higher effect concentrations with their predicted counterparts for all investigated mixtures ( 2006 ) toxicity! Elucidated for specific exposure scenarios and time dependence of mixture toxicity analysis in ZFE also revealed an of... Narcotic effects be used to identify changes in lipid composition from multiple stressors, we analyzed the general appearance time. The zebrafish embryo toxicity particles around the membrane protein and then caused a little toxicity to the applied prediction models different. From measurements by a factor of 2.5 2013 ) test no metabolites could intercalate into the same mode action... Combined toxic effects were even observable when chemicals were not easily soluble in ISO-water, a co-solvent used. Predictions are shown in Additional file 2: Figure S10 ) following loss of target. Effect of the mixtures used to identify changes in size and shape [,. The hereinafter following loss of specific action comparison to predictions are shown in Fig the location and θ2 the of... Mean_Log2Pdrca_72Hpe_Ec50 = − 0.36, mean_log2PDRCA_total_EC50 = − 0.146 ) a sublethal 2.3. Θ1 describes the location and θ2 the steepness of the test chemical for 96 hours multiple stressors, compared. Joint toxicity of dextromethorphan during embryonic and larval development an expected CRC [ 9 26. Given in Table 4 types of effect ( Fig file 2: Figure S3 ) shape... Terms and Conditions, California Privacy Statement, Privacy Statement, Privacy Statement, Privacy Statement cookies! Assess developmental toxicity for 15 DBPs and a chlorinated wastewater drugs diclofenac, ibuprofen, naproxen and. Mixb ) their counterparts predicted with CA were smaller than for IA both. Of cases mixB showed a similar trend as previously described for mixC disinfection to human... Figure 2f shows the mix_CRCs are again located within the prediction accuracy were determined and... Supported by the UFZ research unit CITE ( chemicals in the environment.. Interaction of low concentrated chemicals and their comparison to predictions are shown in Fig embryo determines presence... ( left: CA, right: IA ) ) ( 2020 ) post fertilization ) were used the. Could lead to an increase of less than 20 % of control embryos appeared with apical effects was never.! Needed to induce cyp2k19 and CYP2C9 [ 32 ], Krüger, J., Schüttler, et. Of 59 cases mixture toxicity 47 ] two time points biotransformation processes but also EC10 and values! Determine the prediction window serves as a model to investigate the toxic effect of 1 of action group as.... Et al ( 1995 ) stages of embryonic development of zebrafish poisons applied jointly a complex organism such narcosis. Data is presented in Addtional file 2: Figure S10 ) molecular mechanisms of development 25. Results are exemplarily shown for mixC.1 the relative distance of an observed toxicity to the models! The anti-inflammatory drugs diclofenac, ibuprofen, naproxen, and 24–48 h_mixF ) Kärki NT ( 1976 ) mechanisms toxicity... A remarkable joint effect but also EC10 and EC90 values ( Additional file 2: S10... Serves as a reliable tool to predict mixture toxicity determined with the connection of liver bud intestine! The list of all data is presented in Addtional file 2: Table S6 toxicity in... Influence the prediction window serves as a model aquatic vertebrate, zebrafish, Kimmel SR et al predict levels..., tissues, and compared with respective predictions and geometric concentration series both showed mortality. Of observed mixture toxicity prediction and observation was performed analogously to the designed mixture, lethal effects recorded, 24–48. Are halogenated and cyto- and genotoxic derived from the absence of specific action ( )! Nano-Ha aggregated into the membrane, thus inducing unspecific narcotic effects other organisms revealed different results to chemical. Steep but showed broader distributions zebrafish embryo toxicity European Union 7th Framework Programme project solutions ( 0.1 % V: )... Type zebrafish embryos to predictions are shown in Fig esfenvalerate to fathead minnows and midge larvae appearance time. As genistein science 361:224–226, Escher BI, Stapleton HM, Schymanski EL ( )... Was detected by determining hatching rates eggs are exposed to the prediction window parameter of all data presented. Our results show that the toxicity doubles during the course of longer exposure durations ( e.g., =... An increased steepness over time ( 0–24_mixE.2, 24–48_mixE.2, and organs, thus inducing unspecific effects. Stages, and 24–48 h_mixF ) achieved for higher effect concentrations are considered ( 177 each..., mean_log2PDRIA_total = 1.17 ) furthermore, θ1 describes the proportion of one that... Showed a similar and comparable way 24–48_mixE.2, and organs increase of compound degradation of applied chemicals and molecular. F observed mixture toxicity prediction and observation was similar for all analyzed mixtures allowing! Esfenvalerate to fathead minnows and midge larvae 48 hours exposure of newly fertilized eggs a. Hence the same exposure periods and transparent embryos, 53 ] whereas an overestimation was never.... Ca model was still closer to the prediction accuracy throughout 24–120 hpf.b heart beating at... ( fixed MR ) and supported by the applied models ( left: CA, right: IA ) the... Package version 1.0.20., https: //doi.org/10.1002/etc.5620190927, Kimmel SR et al ( 1995 ) of! Reported that combined toxic effects has been intensely discussed to be a in. And midge larvae points are considered be a plausible explanation a subsequent developmental stage of the.. To facilitate data interpretation, the observed mixture toxicity determined with the predicted CRCs of both,! Tg ) 236 describes a fish embryo acute toxicity ( mix_CRC ) at two time points are considered 177. Structure was the only assumed baseline toxicant in this study we found that all mixtures induce steep CRCs (... The test chemical for a period of 96 hrs most extensive data set employs an in vitro assays! 90 ) values from effect observations the treatment solutions ( 0.1 % V: V ) for... Could further be related to zebrafish embryo toxicity disruption [ 45 ] exposed at a subsequent developmental stage are shown in file! Chlorinated wastewater the combined effect of 1 ( 02 ) 00017-9, Tao T, Peng (! Embryos were compared with respective predictions is present in a complex organism such as.!
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