Experimental design. A 2D simplex axial design and randomized Design of Experiments (DoE) design grid representing the composition for each solution. B Exposure plan for larval zebrafish from 6 h post fertilization (hpf) to 6 days post fertilization (dpf) followed by the high throughput behavior testing, proteomics analysis, and endpoints measurements for these studies

Viability assessment. A Kaplan–Meier cumulative survival plots comparing percent survival between four treatment groups: Control (n = 108), BMAA (n = 162), AEG (n = 162), and 2,4-DAB (n = 108) (P < 0.001, Mantel-Cox log-rank test). B Bar graph representing percent viability across all treatment groups. C 2D contour plot representing zebrafish percent viability in response to each mixture of BMAA, AEG, and 2,4-DAB (Linear regression model, P < 0.0001)

Spontaneous Locomotion Response. A–C Bar graphs represent the total distance traveled during the 18.5 min spontaneous movement assay for each larva and average speed across the same assay for BMAA, AEG, and 2,4-DAB alone, their binary mixtures, and three-component mixtures, respectively. Levels not connected by the same letter are significantly different (Tukey–Kramer HSD, Alpha 0.05)

BMAA and AEG augment the short-latency c startle (SLC) response. A–C Bar graphs display the distribution of the short-latency C-bend (SLC) sensitivity indices for each tested larva after exposure to individual cyanotoxins, binary mixtures, and three-component mixtures, respectively. SLC sensitivity index is determined for each fish by calculating the area under the curve of SLC frequency vs. stimulus intensity (n = 108 siblings; mean ± SEM). Levels not connected by the same letter are significantly different (Tukey–Kramer HSD, Alpha 0.05). D 2D contour plot for the SLC sensitivity at different points in the design space, including each individual cyanotoxin (BMAA, AEG, and 2,4-DAB) and their seven different mixture ratios (Linear regression model, P = 0.0257)

2,4-DAB modulates kinematics of the C startle response. A Three representative images of the peak SLC curvature in 6 days post fertilization (dpf) larvae exposed to vehicle (control, top) and to 2,4-DAB at 500 μM (bottom). B and C Bar graphs quantifying the responses latency and curvature, respectively. D and E 2D contour plot for both the latency and curvature responses at different points in the design space, including each individual cyanotoxin (BMAA, AEG, and 2,4-DAB) and their seven different mixture ratios (Linear regression model, P < 0.05). F and G Bar graphs quantify responses maximum angular velocity and duration, respectively. Levels not connected by the same letter are significantly different (Tukey–Kramer HSD, Alpha 0.05)

A Venn diagram shows no overlap in the differentially Expressed Proteins (DEPs) between 2,4-DAB (500 µM) zebrafish larvae exposed groups at 2 dpf (left) and 4 dpf (right). B Top 10 DEPs found to be up or downregulated in the zebrafish larvae groups that were exposed to 2,4-DAB from 6 hpf until 4 days post fertilization (4 dpf). C Pie chart showing the percentage of enriched GO Biological Processes derived from each set DEPs time points: 2- and 4-day post fertilization. D Top 20 canonical pathways found to be activated or inhibited in the zebrafish larvae groups that were exposed to 2,4-DAB until 4 days post fertilization (4 dpf). Red shading indicates predicted activation (z-score > 2), and blue shading indicates predicted inhibition (z-score < − 2). This bar graph width represents enrichment values in the form of minus log of p value that were greater than 1.3

Systematic proteomics analysis of developmental changes in protein expression in control and 2,4-DAB (500 µM) treated zebrafish larvae. A DEPs were identified in by comparing 4 dpf and 2 dpf samples in control and 2,4-DAB-treated larvae. Venn diagram showing DEP overlap between control and the 2,4-DAB treatment. B Canonical pathways impacted during development from 2 to 4 dpf in control and 2,4-DAB samples. C Predicted upstream regulators. D Diseases and biological functions. The red- or blue-colored rectangles in each column indicate the z-score activities for each analysis. Red shading indicates predicted activation and blue shading indicates predicted inhibition. This heat map represents the activated of inhibited z-scores that were greater than the absolute value of 2