(A) The transmitter generates RFR signal at 3.5 GHz that is amplified by the PA and broadcast in the Faraday cage via antenna. The Faraday cage contains a 6-well plate on a heating pad that maintains temperature of the plate at ~28°C. Each plate is fitted with a temperature probe to measure temperature of the EM. Arrows represent direction of current. An image of the actual setup is included in S1 Fig. (B) View of the 6-well plate from the top (left) and a magnified view of each well from the side (right). Each well contains ~ 3 mL embryo media and 50 embryos. (C) Experimental flow chart, depicting start of exposure (6 hpf), end of exposure (48 hpf) and time of data acquisition (120 hpf).

Embryos were exposed to 3.5 GHz, ~ 30 dBm RFR, at a specific absorption rate (SAR) ≈ 8.27 W/Kg from 6–48 hpf and developmental parameters were measured at 120 hpf. All assays (panels A-C) were sequentially conducted from the same subset of embryos, with a total of 144 embryos from 3 replicate experiments used per treatment condition. (A) Summary of effects observed within our study. Measured parameters include mortality (Mort), 17 morphological parameters and 2 behavioral parameters (larval photomotor (LPR) and startle (LSR) responses). “Any effect” indicates a combination of all 17 morphological parameters. “LSR-All” indicates AUC and Peak measurements combined. (B) RFR exposure does not have any significant effect on photomotor response. (C) RFR exposure results in reduced peak height for startle response. denotes the acoustic signal. * indicates a statistically significant difference at p<0.05.

S1 Fig. Experimental setup for RFR exposure.