Table 3.

Troubleshooting guide for neurotoxicity evaluation induced by nanomaterials.

Problem	Possible Cause	Solution
No or few eggs collected after sucrose gradient centrifugation (Basic Protocol 1).	Insufficient release of eggs	Extend the bleaching treatment for 3 more minutes
	The nematodes were at the wrong stage.	“Chunk” plates a day later, in addition to day 1 (Step 1, BP1).
The total number of nematodes (both alive and dead) on the plate is lower than that two days ago (Basic Protocol 2).	Nematodes were dried up in the inner wall of the NGM plate.	Check the inner wall of the NGM Petri dish. Maintain proper humidity conditions.
Locomotion behavior data has a large standard deviation (Basic Protocol 3).	There might be one or two “outliers” in the data pool.	When observing the nematodes, leave out the nematodes moving aberrantly fast or slow (e.g. > 2 fold or < 0.5 fold than the average speed in the same exposure group).
	The sample number is too low.	Increase sample number.
No difference in GFP/RFP signals between groups (Basic Protocol 4).	Dose too low/ exposure time too short	Adjust exposure conditions: extend exposure duration and/or incubate with nanomaterials at higher doses
	Signal below or above the detection limit of the plate reader.	Adjust the number of nematodes in each well until the fluorescent signals of 100, 500, 1000 and 5000 worms are linear.
	Autofluorescence from nanomaterials interferes with the signal.	Check the negative control wells from BP4 step 2 and see whether fluorescent signal is detectable in the serial dilution of nanomaterials.
	The nanomaterial tested at the specific dose induces ROS but does not activate skn-1-dependent pathways.	Examine with other assays, such as H2DCFHDA fluorescent probe (Yoon, Lee, & Cha, 2018; Zheng, Chen, et al., 2020), to verify whether oxidative stress is induced by the nanomaterial.