Xamples of abnormal phenotypes. (A, D) Normal developing control embyors/fry in o.01 DMSO at 24 hpf (A) and 72 hpf (D); (B) No tail detachment at 24 hpf in 20 mg/L acetaminophen; (C) No somite at 24 hpf in 25 mg/L acetaminophen; (E) Edema at 72 hpf in 20 mg/L lindane; (F) Light pigmentation at 72 hpf in 250 mg/L mefenamic acid; (G) No hatching at 72 hpf in 10 mg/L lindane; (H) Coiled body at 96 hpf in 5 mg/L lindane. Scale bars: 200 mm. doi:10.1371/journal.pone.0055474.gfrom motoneurons in the trunk region. As shown in Figure 4, the larvae in the control group (0.01 DMSO or egg water) had well grown ventral axons. In comparison, the ventral axons were either 12926553 shortened or abolished by treatment with all of the five neurotoxins: acetaminophen, atenolol, atrazine, ethanol and lindane (Figure 4B ). In contrast, the axons were largely unaffected by the neural protectant, mefenamic acid (Figure 4G), indicating the specific response of axon growth to neurotoxins. To further evaluate the toxic effects of these chemicals, lengths of anteiro-posterior body, the central nervous system (CNS) and ventral axons were measured. Among the three lengths, only body length measurement is in wild type larvae. As shown in Figure 5 and Table S1, only high doses of atrazine, ethanol, lindance and mefenamic acid showed measureable difference (P = 0.01?.05) compared to the control groups, but only highest concentration groups of ethanol (2 ) and of mefenamic acid (100, 250 mg/L) showed statistically highly significant difference (P,0.01). For CNS length, only the two highest doses (20 and 25 mg/L) of SC 66 acetaminophen showed highly significant difference (P,0.01) although other four neurotoxins, but not mefenamic acid, also resulted in measurable shortening (P = 0.01?.05) in their high concentration groups. In contrast, by measurement of axon length, we found that even the lowest dose of all of five neurotoxins (2.5 mg/L acetaminophen, 1 mg/L atenolol, 1 mg/L atrazine, 0.1 ethanol, 1.25 mg/ L lindane) caused highly significant (P,0.01) shortening (Figure 5 and Table S1). Compared to the starting concentrations of highly significant changes observed based on standard DarT endpoints examined under a bright-field microscope, the axon length endpoint would increase detection sensitivity by at least 2? foldfor the five neurotoxins. It is interesting to note that there is no observed axon shortening from mefenamic acid treatment except for the highest 1516647 concentration groups (100 and 250 mg/L) while other general toxicological changes (e.g. survival rates, hatching, tail detachment, somite formation, edema etc) were observed at much lower concentration (10 mg/L), suggesting that the shortened axons by mefenamic acid may be a secondary effect resulted from other primary toxicities. These observations suggest that the axon length is a quite sensitive and specific endpoint for testing neurotoxicity. The axon length was generally correlated with the lack of or abnormal touch response (Table S1), which was dosagedependent but an apparently less sensitive trait than axonal length. To further determine the maximum sensitivity of using the axon length as a biomarker for these neurotoxins, another test with lower ranges of neurotoxin concentrations was conducted. As shown in Figure 6, highly significant difference of Hexaconazole site measured axon length (P,0.01) could be detected at the following lowest concentrations: 1 mg/L acetaminophen, 0.5 mg/L atenolol, 0.5 mg/L atrazine, 0.08 ethanol and.Xamples of abnormal phenotypes. (A, D) Normal developing control embyors/fry in o.01 DMSO at 24 hpf (A) and 72 hpf (D); (B) No tail detachment at 24 hpf in 20 mg/L acetaminophen; (C) No somite at 24 hpf in 25 mg/L acetaminophen; (E) Edema at 72 hpf in 20 mg/L lindane; (F) Light pigmentation at 72 hpf in 250 mg/L mefenamic acid; (G) No hatching at 72 hpf in 10 mg/L lindane; (H) Coiled body at 96 hpf in 5 mg/L lindane. Scale bars: 200 mm. doi:10.1371/journal.pone.0055474.gfrom motoneurons in the trunk region. As shown in Figure 4, the larvae in the control group (0.01 DMSO or egg water) had well grown ventral axons. In comparison, the ventral axons were either 12926553 shortened or abolished by treatment with all of the five neurotoxins: acetaminophen, atenolol, atrazine, ethanol and lindane (Figure 4B ). In contrast, the axons were largely unaffected by the neural protectant, mefenamic acid (Figure 4G), indicating the specific response of axon growth to neurotoxins. To further evaluate the toxic effects of these chemicals, lengths of anteiro-posterior body, the central nervous system (CNS) and ventral axons were measured. Among the three lengths, only body length measurement is in wild type larvae. As shown in Figure 5 and Table S1, only high doses of atrazine, ethanol, lindance and mefenamic acid showed measureable difference (P = 0.01?.05) compared to the control groups, but only highest concentration groups of ethanol (2 ) and of mefenamic acid (100, 250 mg/L) showed statistically highly significant difference (P,0.01). For CNS length, only the two highest doses (20 and 25 mg/L) of acetaminophen showed highly significant difference (P,0.01) although other four neurotoxins, but not mefenamic acid, also resulted in measurable shortening (P = 0.01?.05) in their high concentration groups. In contrast, by measurement of axon length, we found that even the lowest dose of all of five neurotoxins (2.5 mg/L acetaminophen, 1 mg/L atenolol, 1 mg/L atrazine, 0.1 ethanol, 1.25 mg/ L lindane) caused highly significant (P,0.01) shortening (Figure 5 and Table S1). Compared to the starting concentrations of highly significant changes observed based on standard DarT endpoints examined under a bright-field microscope, the axon length endpoint would increase detection sensitivity by at least 2? foldfor the five neurotoxins. It is interesting to note that there is no observed axon shortening from mefenamic acid treatment except for the highest 1516647 concentration groups (100 and 250 mg/L) while other general toxicological changes (e.g. survival rates, hatching, tail detachment, somite formation, edema etc) were observed at much lower concentration (10 mg/L), suggesting that the shortened axons by mefenamic acid may be a secondary effect resulted from other primary toxicities. These observations suggest that the axon length is a quite sensitive and specific endpoint for testing neurotoxicity. The axon length was generally correlated with the lack of or abnormal touch response (Table S1), which was dosagedependent but an apparently less sensitive trait than axonal length. To further determine the maximum sensitivity of using the axon length as a biomarker for these neurotoxins, another test with lower ranges of neurotoxin concentrations was conducted. As shown in Figure 6, highly significant difference of measured axon length (P,0.01) could be detected at the following lowest concentrations: 1 mg/L acetaminophen, 0.5 mg/L atenolol, 0.5 mg/L atrazine, 0.08 ethanol and.