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Table 1 Behavioral effects of the anatoxin-a toxin in various model systems. Only behavioral data from studies using the individual synthetic antx-a or extracted antx-a from culture were used. This excludes data from organisms exposed to antx-a producing cyanobacterial cells. Missing data were denoted with NA (not available)

From: Differential influences of (±) anatoxin-a on photolocomotor behavior and gene transcription in larval zebrafish and fathead minnows

Enantiomer Toxin purity Purified toxin extracted from culture? Organism Age Treatment levels Analytically verified? Exposure method Exposure duration Study duration Behavior type Response References
(+) NA No Non-nicotine-tolerant male hooded rats NA 10–200 µg/kg NA Subcutaneous injection NA 60 min immediately after dosing Locomotion Rats showed decreased cage crosses (movement from one infrared beam to another across the cage) and repeated moves (successive interruptions of the same beam of light) compared to saline controls at 100 and 200 µg/kg (+) antx-a Stolerman IP, Albuquerque EX, Garcha HS (1992) Behavioural effects of anatoxin, a potent nicotinic agonist, in rats. Neuropharmacology 31:311–314. https://doi.org/10.1016/0028-3908(92)90182-O
(+) NA No Nicotine-tolerant male hooded rats NA 10–200 µg/kg NA Subcutaneous injection NA 60 min immediately after dosing Locomotion Rats showed decreased repeated moves and a tendency toward a reduced number of cage crosses at 200 µg/kg (+) antx-a from saline control Stolerman IP, Albuquerque EX, Garcha HS (1992) Behavioural effects of anatoxin, a potent nicotinic agonist, in rats. Neuropharmacology 31:311–314. https://doi.org/10.1016/0028-3908(92)90182-O
(+) NA No Male hooded rats trained to discriminate nicotine from saline NA 10–200 µg/kg NA Subcutaneous injection NA 60 min immediately after dosing Nicotine discrimination stimulus Rats showed decreased rates of operant responding in nicotine discrimination procedures and showed partially nicotine-like discriminative stimulus effects at 100 µg/kg (+) antx-a compared to saline controls Stolerman IP, Albuquerque EX, Garcha HS (1992) Behavioural effects of anatoxin, a potent nicotinic agonist, in rats. Neuropharmacology 31:311–314. https://doi.org/10.1016/0028-3908(92)90182-O
(+) NA No Male CD-1 mice NA 30–50 µg/kg Yes Slow intravenous injection 15 min  > 1 min Motor coordination (+) Antx-a-treated mice showed clinical signs of cholinergic stimulation and CNS effects before death. 2 of 6 exposed to 50 µg/kg and 1 of 6 exposed to 30 µg/kg died. Surviving mice recovered and rota-rod testing was comparable to control Fawell JK, Mitchell RE, Hill RE, Everett DJ (1999) The toxicity of cyanobacterial toxins in the mouse; II anatoxin-a. Hum Exp Toxicol 18:168–173. https://doi.org/10.1177/096032719901800306
Unknown NA No Zebrafish (Danio rerio) 55 h 400 µg/L NA Immersion NA NA Heart rate Fish heart rate decreased 9% temporarily in antx-a treatment compared to control Oberemm A, Becker J, Codd GA, Steinberg C (1999) Effects of cyanobacterial toxins and aqueous crude extracts of cyanobacteria on the development of fish and amphibians. Environ Toxicol 14:77–88. https://doi.org/10.1002/(SICI)1522-7278(199902)14:1<77::AID-TOX11>3.0.CO;2-F
Unknown NA No Zebrafish (Danio rerio) 80 h 400 µg/L NA Immersion NA NA Heart rate Fish heart rate increased 12% temporarily in antx-a treatment compared to control Oberemm A, Becker J, Codd GA, Steinberg C (1999) Effects of cyanobacterial toxins and aqueous crude extracts of cyanobacteria on the development of fish and amphibians. Environ Toxicol 14:77–88. https://doi.org/10.1002/(SICI)1522-7278(199902)14:<77::AID-TOX11>;3.0.CO;2-F
Unknown  ≥ 90% No CD-1 mice Adult 100–250 µg/kg NA Intraperitoneal injection NA 5–10 min Abnormal behavior Decreased motor activity, altered gait, difficulty breathing, and convulsions in antx-a treatment mice Rogers EH, Hunter ES, Moser VC, Phillips PM, Herkovits J, Muñoz L, Hall LL, Chernoff N (2005) Potential developmental toxicity of anatoxin-a, a cyanobacterial toxin. J Appl Toxicol 25:527–534. https://doi.org/10.1002/jat.1091
Unknown  ≥ 90% No CD-1 mice Pre-weaning 125–200 µg/kg NA In utero Exposure from mother (intraperitoneal injection) 30–60 s Neurological tests No antx-a-related changes to righting reflex, negative geotaxis time, nor hang time Rogers EH, Hunter ES, Moser VC, Phillips PM, Herkovits J, Muñoz L, Hall LL, Chernoff N (2005) Potential developmental toxicity of anatoxin-a, a cyanobacterial toxin. J Appl Toxicol 25:527–534. https://doi.org/10.1002/jat.1091
(+) NA No Male Long Evans rats Adult 75–225 µg/kg NA Injection NA 30 min Locomotion (+) Antx-a dose dependent decreased horizontal and vertical activity, no tolerance was developed over weeks MacPhail RC, Farmer JD, Jarema KA (2007) Effects of acute and weekly episodic exposures to anatoxin-a on the motor activity of rats: Comparison with nicotine. Toxicology 234:83–89. https://doi.org/10.1016/j.tox.2007.02.001
(±) NA No Male Long Evans rats Adult 200–950 µg/kg NA Injection NA 30 min Locomotion (±) Antx-a dose dependent decreased horizontal and vertical activity at higher doses, no tolerance was developed over weeks MacPhail RC, Farmer JD, Jarema KA (2007) Effects of acute and weekly episodic exposures to anatoxin-a on the motor activity of rats: Comparison with nicotine. Toxicology 234:83–89. https://doi.org/10.1016/j.tox.2007.02.001
(+) NA No Male Long Evans rats 3 month 50–200 µg/kg NA Subcutaneous injection 5 min Variable Operant performance Rats were trained to respond under a multiple variable ratio 30-response variable-interval 60 s schedule of food reinforcement. (+) Antx-a-exposed rats initially decreased in response and reinforcement rate. Though some tolerance occurred over 4 weeks of injections Jarema KA, Poling A, MacPhail RC (2008) Effects of weekly exposure to anatoxin-a and nicotine on operant performance of rats. Neurotoxicol Teratol 30:220–227. https://doi.org/10.1016/j.ntt.2008.02.001
Unknown 98% No Rainbow trout (Oncorhynchus mykiss) 3 month 129–499 µg/L Yes Immersion 96 h 5 min–3 h Abnormal behavior In all antx-a treatments fish showed irregular/erratic swimming, jaw spasms, air gulping at surface, difficulty in maintaining equilibrium after 5 min with fish recovering by 3 h Osswald J, Azevedo J, Vasconcelos V, Guilhermino L (2011) Experimental determination of the bioconcentration factors for anatoxin-a in juvenile rainbow trout (Oncorhynchus mykiss). Proc Int Acad Ecol Environ Sci 1:77–86
(±) NA No Cladocera (Daphnia magna) NA  > 4000 µg/L NA Immersion 24 h, 48 h NA Free swimming 24 h EC50 was 2090 µg/L (±) antx-a and 48 h EC50 was 1700 µg/L (±) antx-a daphnia were unable to swim freely Sierosławska A (2013) Evaluation of the Sensitivity of Organisms Used in Commercially Available Toxkits to Selected Cyanotoxins. Pol J Environ Stud 22:1817–1823
(±) NA No Rotifer (Brachionus calyciflorus) NA  > 4000 µg/L NA Immersion 24 h NA Free swimming 24 h EC50 was > 4000 µg/L (±) antx-a rotifers were unable to swim freely Sierosławska A (2013) Evaluation of the Sensitivity of Organisms Used in Commercially Available Toxkits to Selected Cyanotoxins. Pol J Environ Stud 22:1817–1823
(±) NA No Male Wistar strain albino rats 5–7 weeks 1250–2500 mg/kg, 1,250,000–2,500,000 µg/kg NA Subcutaneous injection Variable Variable Abnormal behavior Extreme seizures, tremors, tachycardia, gasping, fasciculation, acute asphyxiation, latency followed up by twitching, decrease in locomotor activities, coma, before death Banerjee S, Chattopadhyay P, Ghosh A, Pathak MP, Gogoi J, Veer V (2014) Protection by a transdermal patch containing eserine and pralidoxime chloride for prophylaxis against (±)-Anatoxin A poisoning in rats. Eur J Pharm Sci 56:28–36. https://doi.org/10.1016/j.ejps.2014.01.013
Unknown NA No Wild-type roundworms strain N2 (Caenorhabditis elegans) L4 larvae .1–100 µg/L NA Added to agar 24 h or 72 h 20 s Locomotion Antx-a exposure led to dose dependent decreased body bend frequency at 24-h and 72-h exposure and lowered move length at all concentrations in both 24-h and 72-h exposure Ju J, Saul N, Kochan C, Putschew A, Pu Y, Yin L, Steinberg C (2014) Cyanobacterial Xenobiotics as Evaluated by a Caenorhabditis elegans Neurotoxicity Screening Test. Int J Environ Res Public Health 11:4589–4606. https://doi.org/10.3390/ijerph110504589
Unknown NA No Wild-type roundworms strain N2 (Caenorhabditis elegans) L4 larvae .1–100 µg/L NA Added to agar 24 h or 72 h 3 times over 60 s Food intake Decreased pharyngeal pumping 10–100 µg/L antx-a in 24-h exposed worms and 1–100 µg/L antx-a in 72-h exposed worms Ju J, Saul N, Kochan C, Putschew A, Pu Y, Yin L, Steinberg C (2014) Cyanobacterial Xenobiotics as Evaluated by a Caenorhabditis elegans Neurotoxicity Screening Test. Int J Environ Res Public Health 11:4589–4606. https://doi.org/10.3390/ijerph110504589
Unknown NA No Wild-type roundworms strain N2 (Caenorhabditis elegans) L4 larvae .1–100 µg/L NA Added to agar 24 h or 72 h 50 s Defecation assay Lowered defecation period interval at 100 µg/L antx-a in 2-h exposed worms Ju J, Saul N, Kochan C, Putschew A, Pu Y, Yin L, Steinberg C (2014) Cyanobacterial Xenobiotics as Evaluated by a Caenorhabditis elegans Neurotoxicity Screening Test. Int J Environ Res Public Health 11:4589–4606. https://doi.org/10.3390/ijerph110504589
Unknown NA No Wild-type roundworms strain N2 (Caenorhabditis elegans) L4 larvae .1–100 µg/L NA Added to agar 24 h or 72 h 1 h Chemotaxis (NaCl) Lowered chemical index .1–100 µg/L antx-a-exposed worms after 24- and 72-h exposure Ju J, Saul N, Kochan C, Putschew A, Pu Y, Yin L, Steinberg C (2014) Cyanobacterial Xenobiotics as Evaluated by a Caenorhabditis elegans Neurotoxicity Screening Test. Int J Environ Res Public Health 11:4589–4606. https://doi.org/10.3390/ijerph110504589
Unknown NA No Wild-type roundworms strain N2 (Caenorhabditis elegans) L4 larvae .1–100 µg/L NA Added to agar 24 h or 72 h 1 h Thermotaxis Lowered fraction of worms in 20 C category for 1–100 µg/L antx-a after 24-h exposure and lowered fraction of worms in 20 C and movement between 20 and 25C category for .1–100 µg/L antx-a-exposed worms for 72 h Ju J, Saul N, Kochan C, Putschew A, Pu Y, Yin L, Steinberg C (2014) Cyanobacterial Xenobiotics as Evaluated by a Caenorhabditis elegans Neurotoxicity Screening Test. Int J Environ Res Public Health 11:4589–4606. https://doi.org/10.3390/ijerph110504589
Unknown NA No Wild-type roundworms strain N2 (Caenorhabditis elegans) L4 larvae .1–100 µg/L NA Added to agar 24 h or 72 h 1 h Mechanical sensory stimulus No nose touch response change from control for any antx-a concentration or exposure duration Ju J, Saul N, Kochan C, Putschew A, Pu Y, Yin L, Steinberg C (2014) Cyanobacterial Xenobiotics as Evaluated by a Caenorhabditis elegans Neurotoxicity Screening Test. Int J Environ Res Public Health 11:4589–4606. https://doi.org/10.3390/ijerph110504589
(±) 98% No Zebrafish (Danio rerio) 1 year 800 µg/kg NA I.p. injection Immediate observation After 5 min Abnormal behavior (±) Antx-a-exposed fish showed rapid respiration as evidenced by opercular movement, frenetic swimming or complete lack of swimming with some moving backward, abnormal body position, gulping for air Carneiro M, Gutiérrez-Praena D, Osório H, Vasconcelos V, Carvalho AP, Campos A (2015) Proteomic analysis of anatoxin-a acute toxicity in zebrafish reveals gender specific responses and additional mechanisms of cell stress. Ecotoxicol Environ Saf 120:93–101. https://doi.org/10.1016/j.ecoenv.2015.05.031
(+)  ≥ 98% Dolichospermum flos-aquae (prev. Anabaena flos-aquae) Cladocera (Daphnia magna) Neonate 500–50,000 µg/L NA Immersion 10 s, 5 min, 15 min, 30 min, 2 h, 24 h  ≥ 1 min Swimming speed 500, 2500, 50,000 µg/L (+) antx-a-treated Daphnia showed some increased movement before 24 h, while all (+) antx-a concentrations showed roughly 5 times lowered swimming speed at 24 h compared to controls Bownik A, Pawlik-Skowrońska B (2019) Early indicators of behavioral and physiological disturbances in Daphnia magna (Cladocera) induced by cyanobacterial neurotoxin anatoxin-a. Sci Total Environ 695:133,913. https://doi.org/10.1016/j.scitotenv.2019.133913
(+)  ≥ 98% Dolichospermum flos-aquae (prev. Anabaena flos-aquae) Cladocera (Daphnia magna) Neonate 500–50,000 µg/L NA Immersion 10 s, 5 min, 15 min, 30 min, 2 h, 24 h  ≥ 1 min Abnormal circular movements 2500–50,000 µg/L (+) antx-a-treated Daphnia showed increased circular movements from 10 s to 30 min of exposure, though all concentrations were similar to control at 24 h Bownik A, Pawlik-Skowrońska B (2019) Early indicators of behavioral and physiological disturbances in Daphnia magna (Cladocera) induced by cyanobacterial neurotoxin anatoxin-a. Sci Total Environ 695:133,913. https://doi.org/10.1016/j.scitotenv.2019.133913
(+)  ≥ 98% Dolichospermum flos-aquae (prev. Anabaena flos-aquae) Cladocera (Daphnia magna) Neonate 500–50,000 µg/L NA Immersion 2 h or 24 h  ≥ 1 min Heart rate While 500 and 2500 µg/L (+) antx-a-treated Daphnia showed slightly lowered heart rate compared to control, 10,000 and 50,000 µg/L treated Daphnia showed highly decreased heart rate. All exposed Daphnia showed time-dependent decreases between 2- and 24-h exposure Bownik A, Pawlik-Skowrońska B (2019) Early indicators of behavioral and physiological disturbances in Daphnia magna (Cladocera) induced by cyanobacterial neurotoxin anatoxin-a. Sci Total Environ 695:133,913. https://doi.org/10.1016/j.scitotenv.2019.133913
(+)  ≥ 98% Dolichospermum flos-aquae (prev. Anabaena flos-aquae) Cladocera (Daphnia magna) Neonate 500–50,000 µg/L NA Immersion 2 h or 24 h  ≥ 1 min Thoracic limb activity 500 µg/L (+) antx-a treated Daphnia showed slightly higher thoracic limb activity at 2 h while 2500–50,000 µg/L (+) antx-a treated Daphnia showed lowered limb activity with 50,000 µg/L (+) antx-a leading to 0 beats per minute at 2 h and 10,000 µg/L leading to 0 beats per minute after 24 h Bownik A, Pawlik-Skowrońska B (2019) Early indicators of behavioral and physiological disturbances in Daphnia magna (Cladocera) induced by cyanobacterial neurotoxin anatoxin-a. Sci Total Environ 695:133,913. https://doi.org/10.1016/j.scitotenv.2019.133913
(+)  ≥ 98% Dolichospermum flos-aquae (prev. Anabaena flos-aquae) Cladocera (Daphnia magna) Neonate 500–50,000 µg/L NA Immersion 2 h or 24 h  ≥ 1 min Post-abdominal claw movement 500–2500 µg/L (+) antx-a treated Daphnia showed increased claw movement while 10,000–50,000 µg/L (+) antx-a treated Daphnia showed no claw activity for either time point Bownik A, Pawlik-Skowrońska B (2019) Early indicators of behavioral and physiological disturbances in Daphnia magna (Cladocera) induced by cyanobacterial neurotoxin anatoxin-a. Sci Total Environ 695:133,913. https://doi.org/10.1016/j.scitotenv.2019.133913
(±) NA No Female Japanese medaka (Oryzias latipes)  > 6 month 200–20,000 µg/kg Yes Oral gavage   Immediate observation after dosing Abnormal behavior  < 6670 µg/kg (±) antx-a no apparent symptoms of toxicosis, at 20,000 µg/kg (±) antx-a within 5 min of exposure stop or lowered opercular movement, abnormal swimming, muscle rigidity. All but one fish at 10,000 µg/kg (±) antx-a still breathing with cessation at 15 min Colas S, Duval C, Marie B (2020) Toxicity, transfer and depuration of anatoxin-a (cyanobacterial neurotoxin) in medaka fish exposed by single-dose gavage. Aquat Toxicol 222:105,422. https://doi.org/10.1016/j.aquatox.2020.105422
(±)  > 98% No Zebrafish (Danio rerio) Embryo 4–6 h post-fertilization 11–3490 µg/L Yes Immersion 96 h 50 min Larval photomotor response/locomotion Consistent larval photomotor response to control. Stimulatory trend in movement in 11–1950 µg/L (±) antx-a-exposed fish showing more locomotion at highest speed (> 20 mm/s), then lowered movement at all speeds at 3490 µg/L (±) antx-a. Both findings more pronounced in light periods vs. dark Current study
(±)  > 98% No Fathead minnow (Pimephales promelas) Larvae < 48 h post-hatch 12–1960 µg/L Yes Immersion 96 h 50 min Larval photomotor response/locomotion Consistent larval photomotor response to control. Refractory movement in 145–1960 µg/L (±) antx-a-exposed fish showing less locomotion at highest speed (> 20 mm/s). Fairly consistent results in light and dark periods Current study