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Organophosphate Insecticide Toxicity in Neural Development, Cognition, Behaviour and Degeneration: Insights from Zebrafish

Organophosphate Insecticide Toxicity in Neural Development, Cognition, Behaviour and... Journal of Developmental Biology Review Organophosphate Insecticide Toxicity in Neural Development, Cognition, Behaviour and Degeneration: Insights from Zebrafish Jeremy Neylon, Jarrad N. Fuller, Chris van der Poel, Jarrod E. Church and Sebastian Dworkin * Department of Microbiology, Anatomy, Physiology and Pharmacology, La Trobe University, Melbourne, VIC 3086, Australia * Correspondence: s.dworkin@latrobe.edu.au Abstract: Organophosphate (OP) insecticides are used to eliminate agricultural threats posed by insects, through inhibition of the neurotransmitter acetylcholinesterase (AChE). These potent neuro- toxins are extremely efficacious in insect elimination, and as such, are the preferred agricultural insec- ticides worldwide. Despite their efficacy, however, estimates indicate that only 0.1% of organophos- phates reach their desired target. Moreover, multiple studies have shown that OP exposure in both humans and animals can lead to aberrations in embryonic development, defects in childhood neu- rocognition, and substantial contribution to neurodegenerative diseases such as Alzheimer ’s and Motor Neurone Disease. Here, we review the current state of knowledge pertaining to organophos- phate exposure on both embryonic development and/or subsequent neurological consequences on behaviour, paying particular attention to data gleaned using an excellent animal model, the zebrafish (Danio rerio). Keywords: organophosphate; insecticides; zebrafish; neurodevelopment Citation: Neylon, J.; Fuller, J.N.; van der Poel, C.; Church, J.E.; Dworkin, S. 1. The Need for Insecticides Organophosphate Insecticide Toxicity Insecticides are a class of pesticides used to eliminate insects and are employed in areas in Neural Development, Cognition, such as crop development, animal husbandry, agriculture and household environments [1]. Behaviour and Degeneration: On a global scale, insecticides account for 20% of the pesticide market [2], where their Insights from Zebrafish. J. Dev. Biol. 2022, 10, 49. https://doi.org/ widespread use in agriculture improves crop quality and yield, and optimises the revenue 10.3390/jdb10040049 and economic yield of harvests [3–5]. Additionally, the use of insecticides protects con- sumers against vector-borne diseases [6,7], forming a crucial front-line defence to safeguard Academic Editor: Simon J. Conway the agricultural sector. However, these pesticides do not only eliminate the insects they Received: 5 October 2022 target, but, as we will discuss here, also cause significant off-target effects to livestock, Accepted: 16 November 2022 aquatic species and humans. Published: 21 November 2022 Whilst insects constitute an integral component of terrestrial ecosystems [8,9], their control requires significant expenditure from the agricultural sector. In 2012–2013, usage in Publisher’s Note: MDPI stays neutral Australian agriculture totalled approximately $350 billion on insecticides [10,11], demon- with regard to jurisdictional claims in strating the economic burden of pests in agrarian settings. Despite this, the alternative published maps and institutional affil- iations. (that is, non-use) is financially unviable owing to the limitless potential for insect-mediated agricultural destruction, indicating that insecticides are necessary to safeguard and main- tain food production. With an increasing population and limited agrarian land available, improving production and productivity on existing land is integral for safeguarding long- Copyright: © 2022 by the authors. term food security [12]. Therefore, insect management is critical in commercial agriculture, Licensee MDPI, Basel, Switzerland. yet the dangerous neurotoxic activity of the most commonly used insecticides must be This article is an open access article strictly modulated, in order to ensure both efficacy and safety to non-insect species. distributed under the terms and conditions of the Creative Commons 2. Insecticides—The Move to Organophosphates (OPs) Attribution (CC BY) license (https:// Among the earliest implementation of synthetic compounds for insecticidal purposes was creativecommons.org/licenses/by/ the development of organochlorine (OC) insecticides, such as dichlorodiphenyl-trichloroethane 4.0/). J. Dev. Biol. 2022, 10, 49. https://doi.org/10.3390/jdb10040049 https://www.mdpi.com/journal/jdb J.J. De Dev.v Bi . Biol.ol. 2022  2022, 10 , 10, x, x FOR  FOR PE PEER ER REVIEW  REVIEW    2 2of  of  21  21    J. De J. De v. vBi. Bi ol.ol. 2022  2022 , 10 , 10 , x,  FOR x FOR PE PE ERER REVIEW  REVIEW    2 2of  of  21  21        2.2. I n Insec sectic ticidideess—The —The Move  Move to to Organo  Organoph phosphates osphates (OPs)  (OPs)   2.2. In Isec nsec tictic idid ese—The s—The Move  Move to to Organo  Organo phph osphates osphates (OPs)  (OPs)    J. Dev. Biol. 2022, 10, 49 2 of 22 Among Among the  the e eaarlie rliestst implem  implementation entation of of synthetic  synthetic comp  compounds ounds fo for rins  inseectic cticididalal purposes  purposes   Among Among the the ea erlie arlie stst implem  implem entation entation of of synthetic  synthetic comp  comp ounds ounds fo fo r ins r ins ectic ectic idid alal purposes  purposes    was was the  the deve  development lopment of of or orggaannoocchhlolorirne ine (OC)  (OC) in insec sectitcicides ides, ,su such ch as as dich  dichlorod lorodiphenyl iphenyl‐tr‐tri‐i‐ was was the  the deve  deve lopment lopment of of or or gagnaoncohclholroine rine (OC)  (OC) in in sec sec tictiides cides , su , su chch as as dich  dich lorod lorod iphenyl iphenyl‐tr‐tr i‐i‐ chloroethane chloroethane  (DDT (DDT),),  cyclodienes cyclodienes  incl incluudding ing  alaldri drinn, , dieldrin dieldrin, , hep heptach tachlor lor  an andd  ch chlolord rdaannee   chloroethane chloroethane  (DDT   (DDT ), ),cyclodienes   cyclodienes  incl   incl uduing ding  al  al dri dri n,n  dieldrin ,  dieldrin ,  hep ,  hep tach tach lorlor  an  an d dch  ch lolrodradnaen e  (DDT), cyclodienes including aldrin, dieldrin, heptachlor and chlordane [7,13]. These com- [7[7,1,13]3]. .These  These compounds  compounds are  are neurotox  neurotoxicic and  and exer  exert tth their eir ef effefecctsts on  on ta target rget or orga ganinissmmss by  by   [7,13]. These compounds are neurotoxic and exert their effects on target organisms by  [7,13]. These compounds are neurotoxic and exert their effects on target organisms by  pounds are neurotoxic and exert their effects on target organisms by rapidly opening rapid rapidlyly opening  opening sod  sodium ium (N  (Naa) )ch channels annels in in neurons,  neurons, which  which re results sults in in the  the con  contin tinuous uous st stim im‐‐ + + rapid rapid lyl yopening  opening  +sod  sod ium ium (N (N a a) ch ) ch annels annels in in neurons,  neurons, which  which re re sults sults in in the the con  con tintin uous uous st st imim‐ ‐ sodium (Na ) channels in neurons, which results in the continuous stimulation of cellular ululation ation of of cellu  cellulalar rrecep  receptor torss [1 [14,15 4,15].]. Al Althou thougghh rem  remaarkrkably ably ef effic ficacacioious us ag agains ainst tinsects,  insects, the  the   ulul ation ation of of cellu  cellu lala r recep r recep tor tor s s[1 [1 4,15 4,15 ]. ].Al Al thou thou ghg hrem  rem arakrably kably ef ef ficfic acac ioius ous ag ag ains ains t insects, t insects, the the    receptors [14,15]. Although remarkably efficacious against insects, the use of OC insecti- use use of of OC  OC ins  inseectic cticide idess in in agri  agricult culture ure is is now  now lar  larggelelyy banned  banned wor  worldldwwide, ide, ow  owing ing to to the  the si sigg‐‐ use use of of OC  OC ins ins ectic ectic ide ide s in s in agri  agri cult cult ure ure is is now  now lar lar gel gel y ybanned  banned wor  wor ldlw dw ide, ide, ow ow ing ing to to the the si si g‐g‐ cides in agriculture is now largely banned worldwide, owing to the significant teratogenic nificant teratogenic and carcinogenic effects observed in wildlife and livestock, as well as  nificant teratogenic and carcinogenic effects observed in wildlife and livestock, as well as  nifi nifi can can t ter t ter ataotgenic ogenic and  and ca ca rcinogen rcinogen icic ef ef fefe ctcst observed s observed in in w w ildild life life an an d dli vest livest ock, ock, as as well  well as as    and carcinogenic effects observed in wildlife and livestock, as well as humans. Further humans. humans. Fu Furthe rther roff off‐ta‐targe rget teffec  effectsts inc  includ ludiningg carc  carcinoge inogennesis esis, ,hy hype pertrtrophy rophy and  and red  reduuced ced fer fer‐‐ humans. Further off‐target effects including carcinogenesis, hypertrophy and reduced fer‐ humans. Further off‐target effects including carcinogenesis, hypertrophy and reduced fer‐ off-target effects including carcinogenesis, hypertrophy and reduced fertility in rodent titililityty in in rode  rodentnt models,  models, as as well  well as as geno  genotoxic toxicity, ity, red  reduuced ced f efertrtililitityy and  and neuro  neurotoxicity toxicity [1 [16]6], ,  tilitity lity in in rode  rode ntnt models,  models, as as well  well as as geno  geno toxic toxic ity, ity, red  red uced uced fe frtert ilitilyit yand  and neuro  neuro toxicity toxicity [1 [1 6]6] ,  ,  models, as well as genotoxicity, reduced fertility and neurotoxicity [16], have dictated the have have di dicctat tateedd the  the rap  rapidid deve  developme lopmentnt of of al altern ternatative ive in insect secticid icides. es.   have have di di ctcattat ede dthe the rap  rap idi ddeve  deve lopme lopme ntnt of of al al tern tern atat ive ive in in sect sect icid icid es.es.    rapid development of alternative insecticides. Today, Today, the  the vas  vast tma  majojorirtiyty of of insec  insectic ticides ides us used ed are  are organ  organoophosphates phosphates (OP)  (OP), ,which  which, ,al alt‐t‐ Today, Today, the the vas  vas t ma t ma jojroitryit yof of insec  insec tictic ides ides us us eded are are organ  organ ophosphates ophosphates (OP)  (OP) , which , which , al , al t‐t‐ Today, the vast majority of insecticides used are organophosphates (OP), which, al- hough hough al also so n neeurotoxic, urotoxic, are  are gener  generaally lly co consider nsidered ed a a “s “safe afer”r” al alternati ternativvee [17  [17],] ,as as th thee leve  levelsls of of   hough hough al al soso n enurotoxic, eurotoxic, are are gener  gener ally ally co co nsider nsider eded a  a“s “s afe afe r”r ”al al ternati ternati vev e[17 [17 ], ]as , as th th e eleve  leve lsls of of    though also neurotoxic, are generally considered a “safer” alternative [17], as the levels of bioacc bioaccumul umulation ation (a (accu ccummula ulatitoionn of of chem  chemicals icals ins  inside ide an an organism  organism th throrough ugh di direrectct or or ind  indirec irect t  bioacc bioacc umul umul ation ation (a (a ccu ccu mm ula ula tiotino nof of chem  chem icals icals ins ins ide ide an an organism  organism th th rorugh ough di di rerctect or or ind ind irec irec t t  bioaccumulation (accumulation of chemicals inside an organism through direct or indirect uptake uptake) )are  are fa far rlower  lower for  for OP  OP rat  rathheer rth thaann OC  OC insec  insectic ticides ides [18  [18].] .Mo  Moreover, reover, OP  OP in insect secticid icideses   uptake) are far lower for OP rather than OC insecticides [18]. Moreover, OP insecticides  uptake uptake) ) are ar fa erfar lower lower for for OP OP rat rather her th than an OC OC insec insecticides ticides [18 [18]].. Mo Morreover, eover, OP OP insecticides insecticidesar  e are are fu furtrther her fa favoured voured in in ag agricu ricultltur uralal setti  settings ngs du  duee to to thei  their rcost  cost e effe ffectivene ctivenessss [1 [1] ]as as well  well as as   are are fu fu rtrt her her fa fa voured voured in in ag ag ricu ricu ltur ltur alal setti  setti ngs ngs du du e eto to thei  thei r rcost  cost e ffe effe ctivene ctivene sss s[1 [1 ] as ] as well  well as as    further favoured in agricultural settings due to their cost effectiveness [1] as well as their their their ra rapid pid mo  moddee of of ac action tion (immediat  (immediatee n neeurotoxic urotoxicity) ity) ag again ainsst ta a wi wide de variet  varietyy of of ta target rget or or‐‐ their their rapid  ra ra pid pid mode  mo  mo ded of eof action of ac ac tion tion (immediate  (immediat  (immediat neur e en enurotoxic otoxicity) eurotoxic ity) ity) against  ag ag ain ain asts wide  at  awi wi de variety de variet  variet ofy tar yof of get ta ta rget or rget ganisms,  or or‐ ‐ ganisms, ganisms, lea  leadding ing to to broad  broad‐spect ‐spectru rumm su succes ccesss in in pest  pest e elilm imina ination tion [19  [19].] .The  The most  most commonly  commonly   ganisms, ganisms, leading  lea lea d toing dbr ing oad-spectr to to broad  broad‐spect um ‐spect success rurm um su su cces incces pest s in s in elimination  pest  pest e leim lim ina ina [19 tion t].ion The [19 [19 ].most  ]The . The most commonly  most commonly  commonly used OPs   used OPs are shown in Table 1, and include chemicals to safeguard livestock in both ter‐ used OPs are shown in Table 1, and include chemicals to safeguard livestock in both ter‐ are shown in Table 1, and include chemicals to safeguard livestock in both terrestrial and used used OPs  OPs ar ar e eshown  shown in in Tab  Tab lel e1, 1, and  and inc inc lude lude chem  chem icals icals to to sa sa fefgeugauradr dli vest livest ock ock in in bot bot h hter ter‐ ‐ restrial restrial and  and aq aqua uatiticc‐ba‐basesedd farm  farming ing a approa pproaches. ches.   restrial aquatic-based  and aquaticfarming ‐based farm appring oaches.  approaches.  restrial and aquatic‐based farming approaches.  Table Table 1. 1. Commonly  Commonly us used ed orga  organophosphate nophosphate (O (OPP) )inse  insectic cticide ides sin in agriculture  agriculture and  and aquaculture.  aquaculture. Shown  Shown   Table Table 1.  1. Commonly  Commonly us us eded orga  orga nophosphate nophosphate (O (O P)P inse ) inse ctic ctic ide ide s in s in agriculture  agriculture and  and aquaculture.  aquaculture. Shown  Shown    Table 1. Commonly used organophosphate (OP) insecticides in agriculture and aquaculture. Shown here here are  are approvals  approvals us usiningg the  the Pu  Public blic Chemical  Chemical Registration  Registration Inform  Information ation Sy System stem Se Searc archh (Pub  (PubCR CRISIS),) ,  here here are are approvals  approvals us us inign gthe the Pu Pu blic blic Chemical  Chemical Registration  Registration Inform  Inform ation ation Sy Sy stem stem Se Se arc arc h h(Pub  (Pub CR CR ISI)S, ),  here are approvals using the Public Chemical Registration Information System Search (PubCRIS), molecu molecular lar for  formmula ula and  and general  general us uses. es.   molecular formula and general uses.  molecular formula and general uses.  molecular formula and general uses. Molecu Molecular lar Formu  Formula: la:     Molecu Molecular lar Formu  Formula: la:   Molecular Formula:   Molecular Formula:  Molecular Formula:   Molecular Formula:  Molecular CC 9H 9H 1010CIN CIN Formula 2O 2O 5PS 5PS  : CC 1010HH 1212NN 3O 3O 3PS 3PS 2 2  CC 9H 9H 10CIN 10CIN 2O 2O 5PS 5PS    CC 10H 10H 12N 12N 3O 3O 3PS 3PS 2  2  Molecular Formula: C H CIN O PS GGeene ne 9rara 10l lUses  Uses 2 of 5of OP  OP: :  GGeene neraral lUses  Uses of of OP  OP: :  C H N O PS GG ene ene rara l Uses l Uses of of OP OP :  :  GG ene ene 10 rara l 12 Uses l Uses 3  of 3 of OP 2 OP :  :  General Uses of OP: Used Used in in Aquatic  Aquatic Farm  Farm‐‐ Used General Used on on Uses  Orch  Orch ofard ard OP  : Used Used in in Aquatic  Aquatic Farm  Farm‐ ‐ Used Used on on Orch  Orch ard ard    Used in Aquatic Farming Used on Orchard Fruits and ing ing (A (Atltlant anticic Sa Salmon) lmon) to to   Fruits Fruits an andd Nu  Nut tCrops  Crops   (Atlantic Salmon) to Control ing ing (A (A tlant tlant ici Sa c Sa lmon) lmon) to to    Fruits Fruits an an d dNu Nu t Crops t Crops    Nut Crops to Control Moths. Cont Control rol Pa Pararasisitetess. .  toto Control  Control Mot  Mothhss. .  Parasites. Cont Cont rolrol Pa Pa rarsiasi tetse. s.  toto Control  Control Mot  Mot hsh. s.                  Molecu Molecular lar Formu  Formula: la:   Molecu Molecu larlar Formu  Formu la:la:    Molecu Molecular lar Formu  Formula: la:     Molecu Molecu larlar Formu  Formu la:la:     CC 1212HH 2121NN 2O 2O 3PS 3PS   Molecular Formula: Molecular Formula: CC 12H 12H 21N 21N 2O 2O 3PS 3PS    CC 9H 9H 1111Cl Cl 3NO 3NO 3PS 3PS   CC 9H 9H 11Cl 11Cl 3NO 3NO 3PS 3PS    C H Cl NO PS GGeene ne Crara H l lUses  Uses N O of of PS OP  OP: :  9 11 3 3 12 21 2 3 GG ene ene rara l Uses l Uses of of OP OP :  :  GGeene neraral lUses  Uses of of OP  OP: :  General Uses of OP: General Uses of OP: GG ene ene rara l Uses l Uses of of OP OP :  :  Used Used on on crop  cropss   Used Used on on crop  crop s s  Used Used broa  broadly dly (crops  (crops/a/anini‐‐ Used broadly Used on crops Used Used broa  broa dly dly (crops  (crops /a/ni ani‐ ‐ (fr (fruiuitsts/vege /vegetata‐‐ (crops/animals/buildings) to (fruits/vegetables/nuts/field (fr(fr uiui tsts /vege /vege tat‐a‐ mals mals/bu /builidldinings) gs) to to con  con‐‐ mals mals /bu /bu ildilin din gs) gs) to to con  con‐ ‐ bles/n bles/nuts/field uts/field crops)  crops)   control roundworms, crops) to control ants, fleas bles/n bles/n uts/field uts/field crops)  crops)    trol trol roun  roundwo dworms, rms, mos  mos‐‐ trol trol roun  roun dwo dwo rms, rms, mos  mos‐ ‐ toto con  control trol an antsts, ,fle fleasas   mosquitos and termites. and cockroaches. toto con  con trol trol an an tst, sfle , fle asas    qu quito itoss and  and te term rmite itess. .  ququ itoito s and s and te te rm rm iteite s. s.  and and coc  cockkroac roaches. hes.   and and coc coc kroac kroac hes. hes.                           J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  3  of  21  J. Dev. Biol. 2022, 10, 49 3 of 22 J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  3  of  21  J. De   v. Biol. 2022, 10, x FOR PEER REVIEW  3  of  21  J. De J. De v. vBi. Bi ol.ol. 2022  2022 , 10 , 10 , x,  FOR x FOR PE PE ERER REVIEW  REVIEW    3 3of  of  21  21        J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  3  of  21  J.  Dev. Biol. 2022, 10, x FOR PEER REVIEW  3  of  21      Table 1. Cont. Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecu Molecu larlar fo fo rmu rmu la:la:     Molecu Molecu larlar fo fo rmu rmu la:la:     C4H7Cl2O4P  C9H12NO5PS  Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   CC 4H 4H 7Cl 7Cl 2O 2O 4P4 P  CC 9H 9H 12NO 12NO 5PS 5PS    C4H7Cl2O4P  C9H12NO5PS  C4H7Cl2O4P  C9H12NO5PS  General uses of OP:  General uses of OP:  CC 4H 4H 7Cl 7Cl 2O 2O 4P 4P    CC 9H 9H 1212 NO NO 5PS 5PS    General uses of OP:  General uses of OP:  General uses of OP:  GeMolecular neral uses Formula  of OP::  GG Molecular ene ene rara l uses l uses Formula  of of OP OP ::  :  GG ene ene rara l uses l uses of of OP OP :  :  Used broadly (house‐ Used broadly (public  General uses of OP:  General uses of OP:  General uses of OP:  Gene Cra Hl uses NO PS of OP:  Used Used broa  broa dly dly (hou  (hou sese‐ ‐ Used Used broa  broa 9 12dly dly (publ 5 (publ ici c  C H Cl O P Used  4broa 7 dly 2 4 (house‐ Used broadly (public  Used broadly (house‐ Used broadly (public  hold/  health/agriculture) to  General Uses of OP: Used Used broa  broa dly dly (hou  (hou sese‐‐ Used Used broa  broa dly dly (publ  (publ ici c  hold/  health/agriculture) to  General hold/ Uses  of OP: health/agriculture) to  hold/ hold/    heal heal thth /a/a gric gric ulutultre) ure) to to    Used broadly (public agriculture) to control  control beetles, grubs,  hold/  health/agriculture) to  Used broadly hold/ (household/   health/agriculture) to  agr agr icu icu lture lture ) to ) to contro  contro l l  control control beetle  beetle s,s, gr gr ubus, bs,    health/agriculture) to control agriculture) to control  control beetles, grubs,  agriculture) to control  control beetles, grubs,  flies, caterpillars, thrips  locusts, flies, mosqui‐ agriculture) to control flies, agr agr icu icu ltlt ure ure ) )to to contro  contro l l  control control beetle  beetle s,s, gr gr uu bs, bs,    flies, caterpillars, thrips  locusts, flies, mosqui‐ flies, caterpillars, thrips  locusts, flies, mosqui‐ beetles, grubs, locusts, flies, flifli es,es, caterp  caterp illars, illars, thrip  thrip s s  lolcu ocu stst s, s,fl ies, flies, mosq  mosq uiui‐ ‐ caterpillars, thrips and mites. and mites.  tos, etc.  flies, caterpillars, thrips  locusts, flies, mosqui‐ flies, caterpillars, thrips  locusts, flies, mosqui‐ mosquitos, etc. and and mi mi tetse.s .  tos, tos, et et c.c .  and mites.  tos, etc.  and mites.  tos, etc.      and and mi mi tetse.s .  tos, tos, et et c.c .                          Molecular formula:   Molecular formula:   Molecu Molecu lar lar fo fo rmu rmu la:la:     Molecu Molecu lar lar fo fo rmu rmu la:la:     Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   C10H19O6PS2  C8H10NO5PS  Molecu Molecu lar lar fo fo rmu rmu la:la:     Molecu Molecu lar lar fo fo rmu rmu la:la:     C10H19O6PS2  C8H10NO5PS  C10H19O6PS2  C8H10NO5PS  Molecular Formula: C10H19O6PS2  C8H10NO5PS  C10H19O6PS2  C8H10NO5PS  Molecular Formula: General uses of OP:  General uses of OP:  CC 1010 HH 1919 OO 6PS 6PS 2 2  CC 8H 8H 1010 NO NO 5PS 5PS    GG ene ene Crara lH uses l uses O of PS of OP  OP : :  GG ene ene rara l uses l uses of of OP  OP : :  10 19 6 2 General uses of OP:  Gene C ra Hl uses NO PS of OP:  General uses of OP:  General uses of OP:  8 10 5 Used broadly (landscap‐ Used in open fields  GG General ene ene rara l luses   Uses uses of of of OP OP  OP :: :  GG ene ene rara l luses  uses of of OP  OP : :  Used broadly (landscap‐ Used in open fields  Used broadly (landscap‐ Used in open fields  General Uses of OP: Used Used broa  broa dly dly (l and (land scap scap‐ ‐ Used Used in in open  open fie fie lds lds    Used broadly ing/public health/agri‐ (cotton, soybean, veg‐ Used broadly (landscap‐ Used in open fields  Used broadly (landscap‐ Used in open fields  ing/p ing/p ubl ubl icic health/agri  health/agri‐ ‐ (cot Used (cot ton, ton, in open  soyb  soyb fields ean, ean, veg (cotton,  veg‐ ‐ ing/p (landscaping/public ublic health/agri‐ (cotton, soybean, veg‐ ing/public health/agri‐ (cotton, soybean, veg‐ culture) to control mos‐ etable) to control boll  ing/p ing/p uu blbl icic health/agri  health/agri‐‐ soybean, (cot (cot ton, ton, vegetable)  soyb  soyb ean, ean, to veg  veg contr ‐‐ ol culture) to control mos‐ etable) to control boll  culture) to control mos‐ etable) to control boll  health/agriculture) to control culture) culture) to to co co ntrol ntrol mos  mos‐ ‐ etab etab le)le) to to con  con trol trol boll  boll    quitos, fleas and ants.  boll weevils, weevils, et etc. c.  culture) to control mos‐ etable) to control boll  culture) to control mos‐ etable) to control boll  qu mosquitos, qu itoito s,s fl, ea flea fleas s sand  and and an an ants. tsts . .  weevils, weevils, et et c.c .  quitos, fleas and ants.  weevils, etc.  quitos, fleas and ants.  weevils, etc.  qu qu ito ito s,s fl, fl eaea s sand  and an an tsts . .  weevils, weevils, et et c.c .                              Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   C11H12NO4PS2  Molecu Molecu lar lar fo fo rmu rmu la:la:     Molecu Molecu larlar fo fo rmu rmu la:la:     CC 11H 11H 12NO 12NO 4PS 4PS 2 2  C10H14NO5PS  Molecular Formula: Molecular Formula: Molecular formula:   Molecular formula:   C11H12NO4PS2  C11H12NO4PS2  CC 10H 10H 14NO 14NO 5PS 5PS    General uses of OP:  CC 1111 HH 1212 NO NO 4PS 4PS 2 2  C10H14NO5PS  C C10H H14NO NO5PS PS  Gene C ra Hl uses NO PS of OP:  General uses of OP:  Gene 10ral 14 uses 5 of OP:  11 12 4 2 CC 1010 HH 1414 NO NO 5PS 5PS    General uses of OP:  General uses of OP:  General uses of OP:  General uses of OP:  Used broadly  General Uses of OP: General Uses of OP: GG ene ene rara l luses  uses of of OP  OP : :  GG ene ene rara l uses l uses of of OP OP :  :  Used Used broa  broa dly dly    No longer used (banned  General uses of OP:  General uses of OP:  Used broadly  Used broadly  No No lon  lon ger ger us us eded (b (b anned anned    No longer used (banned (plan Used ts/anim broadly als) to  Used Used broa  broa dly dly    No longer used (banned  No longer used (banned  (plants/animals) to  (plants/animals) to  largely worldwide) due  No No lon  lon ger ger us us eded (b (b anned anned    largely worldwide) due to its (plants/animals) (plan (plan tst/sanim /anim ato las)lcontr s) to to   ol largely worldwide) due  largely worldwide) due  control moths, mites,  (plants/animals) to  (plants/animals) to  larlar gel gel y ywor  wor ldlw dw ide) ide) due  due    control control mot  mot hs, hs, mit  mit es,es,    to its high toxicity.  high toxicity. moths, mites, flies and aphids. largely worldwide) due  largely worldwide) due  control moths, mites,  control moths, mites,  toto it sit shigh  high tox  tox iciici ty.ty.    flies and aphids.  control control mot  mot hh s,s, mit  mit es, es,    to its high toxicity.  to its high toxicity.  flies and aphids.  flies and aphids.  toto it it s shigh  high tox  tox iciici ty. ty.    flifli eses and  and ap ap hihds. ids.    flies and aphids.  flies and aphids.                              Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   C10H9Cl4O4P  Molecular Formula: Molecu Molecu lar lar fo fo rmu rmu la:la:     CC 10H 10H 9Cl 9Cl 4O 4O 4P4 P  C10H9Cl4O4P  C10H9Cl4O4P  C H Cl O P General uses of OP:  10 9 4 4 CC 1010 HH 9Cl 9Cl 4O 4O 4P 4P    General uses of OP:  General uses of OP:  General Uses of OP: General uses of OP:  General uses of OP:  Used on animals (cattle,    GG ene ene rara l luses  uses of of OP  OP : :  Used Used on on an an imim als als (c (c atattle, tle,        Used on animals (cattle, hogs, Used on animals (cattle,    Used on animals (cattle,    hogs, goats, chickens,  Used Used on on an an im im als als (c (c atattlte,le,        goats, hogs, chickens,  goatsand , chihorses) ckens, to hogs, goats, chickens,  hogs, goats, chickens,  hogs, goats, chickens,  and horses) to control  control flies and mites. hogs, hogs, goats  goats , ch , ch icikceknen s,s ,  and and horses)  horses) to to contro  contro l l  and horses) to control  and horses) to control  flies and mites.  and and horses)  horses) to to contro  contro l l  flies and mites.  flies and mites.  flies and mites.  flies and mites.  flifli eses and  and mit  mit es. es.            3. OPs—Mode of Action In Vivo  3. OPs—Mode of Action In Vivo 3.3. OPs—M  OPs—M odoed eof of Ac Ac tion tion In In Vivo  Vivo    3. OPs—Mode of Action In Vivo  3. OPs—Mode of Action In Vivo  The primary neurotoxic action of organophosphate (OP) insecticide exposure is the  3.3. OPs—M  OPs—M od od e eof of Ac Ac tion tion In In Vivo  Vivo    The primary neurotoxic action of organophosphate (OP) insecticide exposure is the The primary neurotoxic action of organophosphate (OP) insecticide exposure is the  The primary neurotoxic action of organophosphate (OP) insecticide exposure is the  The primary neurotoxic action of organophosphate (OP) insecticide exposure is the  The primary neurotoxic action of organophosphate (OP) insecticide exposure is the  irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons  irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons The The prima  prima ryr yneurotoxic  neurotoxic ac ac tion tion of of organ  organ ophosphate ophosphate (OP)  (OP) in in sect sect iciici dd e eexposure  exposure is is the  the    irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons  irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons  irrever irrever sible sible inhibition  inhibition of of acety  acety lcholinesterase lcholinesterase (AChE  (AChE ) in ) in the the syn  syn aptic aptic ju ju ncntciotino nof of neurons  neurons    (Figure 1), leading to the hyperstimulation of post‐synaptic cells [7,20]. At a biochemical  (Figure 1), leading to the hyperstimulation of post-synaptic cells [7,20]. At a biochemical irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons  irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons  (Figure 1), leading to the hyperstimulation of post‐synaptic cells [7,20]. At a biochemical  (Figure 1), leading to the hyperstimulation of post‐synaptic cells [7,20]. At a biochemical  (Fi(Fi gure gure 1) 1) , le , le adaing ding to to the the hypersti  hypersti mula mula tion tion of of post  post‐syna ‐syna ptipti c cce ce llslls [7, [7, 202]0. ]At . At a  abiochemica  biochemica l  l  level,  OPs  bind  to  the  hydroxyl  group  of  AChE  through  phosphorylation,  preventing  level, OPs bind to the hydroxyl group of AChE through phosphorylation, preventing (Fi (Fi gure gure 1) 1) , le , le adad ing ing to to the  the hypersti  hypersti mula mula tion tion of of post  post‐syna ‐syna pti pti c cce ce llslls [7, [7, 202]0.] At . At a  abiochemica  biochemica l l  level level , ,OPs   OPs  bin   bin d dto  to  the   the  hydroxyl   hydroxyl  gro   gro up u pof  of  AChE   AChE  th  th rorugh ough  phosphoryla   phosphoryla tion, tion,  pr  pr eventing eventing    level,  OPs  bind  to  the  hydroxyl  group  of  AChE  through  phosphorylation,  preventing  level,  OPs  bind  to  the  hydroxyl  group  of  AChE  through  phosphorylation,  preventing  AChE from hydrolysing ACh [21]. In turn, the reduction of AChE activity results in an  AChE from hydrolysing ACh [21]. In turn, the reduction of AChE activity results in level level , ,OPs   OPs  bin   bin dd  to  to  the   the  hydroxyl   hydroxyl  gro   gro uu pp  of  of  AChE   AChE  th  th rorugh ough  phosphoryla   phosphoryla tion, tion,  pr  pr eventing eventing    AChE AChE fr fr om om hy hy drdorloylsyisnign gACh  ACh [ 21] [21] . In . In turn,  turn, the  the re re duc duc tion tion of of AChE  AChE act act iviivi tyty results  results in in an an    AChE from hydrolysing ACh [21]. In turn, the reduction of AChE activity results in an  AChE from hydrolysing ACh [21]. In turn, the reduction of AChE activity results in an  abnormal build‐up of ACh in the synaptic junctions, leading to hyperstimulation of the  an abnormal build-up of ACh in the synaptic junctions, leading to hyperstimulation of AChE AChE fr fr om om hy hy dd rorloylsyisnin g gACh  ACh [ 21] [21] . .In In turn,  turn, the  the re re duc duc tion tion of of AChE  AChE act  act ivi ivi tyty results  results in in an an    abab norm norm ala build l build‐up‐up of of ACh  ACh in in the  the syn  syn apatpictic ju ju ncnticotinos, ns, lea lea ding ding to to hyperstimulatio  hyperstimulatio n nof of the  the    abnormal build‐up of ACh in the synaptic junctions, leading to hyperstimulation of the  abnormal build‐up of ACh in the synaptic junctions, leading to hyperstimulation of the  musca thermuscarinic inic and nico and tini nicotinic c receptor receptors s involved involved  in cholin inergic choliner  patgic hwa pathways ys [22]. Ther [22efore, ]. Ther in efor ‐ e, abab norm norm ala lbuild  build‐up‐up of of ACh  ACh in in the  the syn  syn apap tictic ju ju nn cticti on on s,s, lea  lea dd ing ing to to hyperstimulatio  hyperstimulatio nn of of the  the    muscarinic and nicotinic receptors involved in cholinergic pathways [22]. Therefore, in‐ muscarinic and nicotinic receptors involved in cholinergic pathways [22]. Therefore, in‐ musca musca rin rin icic and  and nico  nico tintin ici cre re cece ptpt oror s sinvo  invo lved lved in in cho  cho linergic linergic pat  pat hw hw ays ays [22 [22 ]. ]Ther . Ther efore, efore, in in‐ ‐ creased increased  OP expos OP exposur ure is li ekis ely likely  to suto bstant substantially ially impac impact t upon upon  neural neural  func function, tion, andand  the the riskrisk   muscarinic and nicotinic receptors involved in cholinergic pathways [22]. Therefore, in‐ muscarinic and nicotinic receptors involved in cholinergic pathways [22]. Therefore, in‐ creased OP exposure is likely to substantially impact upon neural function, and the risk  creased OP exposure is likely to substantially impact upon neural function, and the risk  creased creased OP OP expos  expos uruer eis is li kliekly ely to to su su bstant bstant iaia llyll yimpac  impac t upon t upon neural  neural func  func tion tion , an , an d dthe the ris ris k k  of OP-dependent toxicity can be quantitated through measuring AChE levels and activity of OP‐dependent toxicity can be quantitated through measuring AChE levels and activity  creased OP exposure is likely to substantially impact upon neural function, and the risk  creased OP exposure is likely to substantially impact upon neural function, and the risk  ofof OP OP‐depen ‐depen dent dent tox tox iciici tyty can  can be be q uqan uan tita tita ted ted th th rough rough measuring  measuring AChE  AChE leve  leve lsls an an d dac ac tiv tiivtyity    of OP‐dependent toxicity can be quantitated through measuring AChE levels and activity  of OP‐dependent toxicity can be quantitated through measuring AChE levels and activity  ofof OP  OP‐depen ‐depen dent dent tox  tox iciici tyty can  can be be q u qu anan tittaita ted ted th th rough rough measuring  measuring AChE  AChE leve  leve lsls an an dd ac ac titviivty ity            J. Dev. Biol. 2022, 10, 49 4 of 22 J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  4  of  21  in blood [23] and pesticide metabolites, such as dialkyl phosphate (DAP) compounds in in blood [23] and pesticide metabolites, such as dialkyl phosphate (DAP) compounds in  urine [24,25] and plasma [26]. urine [24,25] and plasma [26].  Figure 1. The physiological action of acetylcholine (ACh; purple triangles) at the neuronal cell syn‐ Figure 1. The physiological action of acetylcholine (ACh; purple triangles) at the neuronal cell apse, the breakdown of ACh through acetylcholinesterase (AChE; orange diamonds), and the phos‐ synapse, the breakdown of ACh through acetylcholinesterase (AChE; orange diamonds), and the phorylation of AChE through organophosphate insecticide (OP; red hexagons) exposure.  phosphorylation of AChE through organophosphate insecticide (OP; red hexagons) exposure. AChE is integral for regulating neurotransmission [27], achieved through the hydro‐ AChE is integral for regulating neurotransmission [27], achieved through the hy- lysing of acetylcholine (ACh) into its two structural products, acetyl CoA and choline, the  drolysing of acetylcholine (ACh) into its two structural products, acetyl CoA and choline, latter of which is returned to the pre‐synaptic neuron via the sodium (Na ) choline trans‐ the latter of which is returned to the pre-synaptic neuron via the sodium (Na ) choline porter, enabling regeneration of ACh in the pre‐synaptic neuron. This process occurs in  transporter, enabling regeneration of ACh in the pre-synaptic neuron. This process occurs the central nervous system (CNS) within the brainstem, striatum and basal forebrain [28],  in the central nervous system (CNS) within the brainstem, striatum and basal forebrain [28], as well as in the peripheral nervous system (PNS) at both the neuroglandular and neuro‐ as well as in the peripheral nervous system (PNS) at both the neuroglandular and neuro- muscular junctions [29]. ACh from pre‐synaptic neurons plays important roles in initiat‐ muscular junctions [29]. ACh from pre-synaptic neurons plays important roles in initiating ing skeletal, smooth and cardiac muscle contraction, and more recently have been impli‐ skeletal, smooth and cardiac muscle contraction, and more recently have been implicated cated in the development and maturation of the brain [30–32].  in the development and maturation of the brain [30–32]. As a result of acute and chronic exposure, OPs can cause extensive damage to cells  As a result of acute and chronic exposure, OPs can cause extensive damage to cells including  cytotoxicity,  apoptosis  [33,34],  genotoxicity  and  subsequent  DNA  mutations  including cytotoxicity, apoptosis [33,34], genotoxicity and subsequent DNA mutations [35]. [35]. In previous studies, pesticides have been shown to produce covalent adducts with  In previous studies, pesticides have been shown to produce covalent adducts with DNA, DNA, and as a result form interstrand cross‐links which inhibit cellular replication and  and as a result form interstrand cross-links which inhibit cellular replication and tran- transcription [36]. Given that DNA adducts are destructive compounds that cause cellular  scription [36]. Given that DNA adducts are destructive compounds that cause cellular damage, they are nonetheless useful biomarkers for identifying oxidative stress and gen‐ damage, they are nonetheless useful biomarkers for identifying oxidative stress and geno- otoxicity [37].  toxicity [37]. Other Other cellular cellular irr irre egularities gularities iden identified tified inc include lude inc incrrea eases ses in in cytokine cytokine secre secretion tion (p (partic- artic‐ ul ularly arly tumour tumour necrosis necrosis fac factor tor  and and interleukin interleukin  6) 6) thr thro ough ugh neur neuroin oinflammation, flammation, improper improper  clearance clearance of of rre eactive activeoxygen  oxygen species,  species, and  and pathologically-induced  pathologically‐induced alterations  alteration ins gene in gene expr ex es-‐ pression sion [38– 40 [38– ]. 40].  4. OPs—Occupational, Household and Waterway Exposure 4. OPs—Occupational, Household and Waterway Exposure  Although OP insecticides are highly effective, it is estimated that only 0.1% of these Although OP insecticides are highly effective, it is estimated that only 0.1% of these  pesticides reach their target organisms [41,42], with the majority being lost in soil, food pesticides reach their target organisms [41,42], with the majority being lost in soil, food  and drainage [22,43,44]; in fact, OPs are the most common synthetic material found in and drainage [22,43,44]; in fact, OPs are the most common synthetic material found in  waterways, soil and animal tissues [5]. OP exposure is generally considered to be most prob- waterways, soil and animal tissues [5]. OP exposure is generally considered to be most    J. J. Dev Dev.. Biol. Biol. 2022 2022,, 10 10,, 49 x FOR PEER REVIEW  5 5of of  21 22  lematic in agricultural environments, and indeed occupational settings, such as farms and problematic in agricultural environments, and indeed occupational settings, such as farms  factories, contribute a significant avenue of OP exposure. However, OP exposure frequently and factories, contribute a significant avenue of OP exposure. However, OP exposure fre‐ occurs also in the household environment through food contamination and oral/epidermal quently  occurs  also  in  the  household  environment  through  food  contamination  and  exposure [27]. Although less common than occupational exposure, residential OP exposure oral/epidermal exposure [27]. Although less common than occupational exposure, resi‐ can occur from the use of these insecticides in the household or garden (Figure 2), and dential OP exposure can occur from the use of these insecticides in the household or gar‐ usually occur from improper storage and spills [45]. Additionally, the infiltration of OPs in den (Figure 2), and usually occur from improper storage and spills [45]. Additionally, the  the diet, where OPs are consistently detected in foods at low levels [46], is considered to be infiltration of OPs in the diet, where OPs are consistently detected in foods at low levels  a significant route of exposure and subsequent poisonings [47]. Children are particularly [46], is considered to be a significant route of exposure and subsequent poisonings [47].  vulnerable to OP exposure through diet due to the fact that they eat 2.8–4.8 times more Children are particularly vulnerable to OP exposure through diet due to the fact that they  food per unit of body mass, and the types of food that they eat (fruits and vegetables) eat 2.8–4.8 times more food per unit of body mass, and the types of food that they eat  contain higher levels of OP residues [47,48]. Although techniques do exist for monitoring (fruits and vegetables) contain higher levels of OP residues [47,48]. Although techniques  environmental OP exposure (surrogate skin, fluorescent tracers, air sampling pumps, etc.), do exist for monitoring environmental OP exposure (surrogate skin, fluorescent tracers,  most OPs are only detected once they have entered the body [49]. Therefore, greater un- air sampling pumps, etc.), most OPs are only detected once they have entered the body  derstanding of the lifelong effects of OP exposure are necessary in order to better govern [49]. Therefore, greater understanding of the lifelong effects of OP exposure are necessary  their use and mandate appropriate safety measures for use where the likelihood of human in order to better govern their use and mandate appropriate safety measures for use where  ingestion is high. the likelihood of human ingestion is high.  Figure 2. The primary direct and indirect routes of organophosphate (OP) exposure on target and  Figure 2. The primary direct and indirect routes of organophosphate (OP) exposure on target and non‐target organisms in agricultural, household and aquatic environments.  non-target organisms in agricultural, household and aquatic environments. Waterway exposure can occur unintentionally via agricultural surface run‐off, creat‐ Waterway exposure can occur unintentionally via agricultural surface run-off, creating ing a significant risk not only directly to aquatic ecosystems, but indirectly to humans as  a significant risk not only directly to aquatic ecosystems, but indirectly to humans as well  [50].  OPs  can also  be  intentionally  released  into  waterways,  a  phenomenon  com‐ well [50]. OPs can also be intentionally released into waterways, a phenomenon commonly monly observed in commercial fish farms and fishing sports that use these substances to  observed in commercial fish farms and fishing sports that use these substances to eliminate eliminate water‐borne pests [51] such as flat worm parasites [52]. Assessing chronic OP  water-borne pests [51] such as flat worm parasites [52]. Assessing chronic OP exposure exposure in waterways is challenging owing to high OP solubility, relatively short half‐ in waterways is challenging owing to high OP solubility, relatively short half-lives, and lives, and relatively low bioaccumulation [53]; however, data suggest that OP contamina‐ relatively low bioaccumulation [53]; however, data suggest that OP contamination is of tion is of global concern. With 95% of urban streams in the US showing detectable levels  global concern. With 95% of urban streams in the US showing detectable levels of OP of OP contamination [54], one can assume that aquatic species are consistently exposed to  contamination [54], one can assume that aquatic species are consistently exposed to OP OP insecticides for prolonged periods.  insecticides for prolonged periods.      J. Dev. Biol. 2022, 10, 49 6 of 22 5. Major Findings: The Zebrafish as Model for Testing Organophosphate (OP) Insecticides The use of mammalian models such as mice for screening experiments is both expen- sive and, owing to the relatively lengthy gestation periods, time consuming. Therefore, alternate animal models of environmental susceptibility, which are low cost and high- throughput, are desirable for toxin-screening, as these would provide rapid functional data that could narrow down OPs of interest and allow for subsequent targeted testing in mammalian models. One such model is the zebrafish (Danio rerio), which has become a favoured model for developmental research [55]. The zebrafish is an excellent model of human diseases, as the zebrafish and human genome share more than ~80% similarity [56]. This well-established genetic conservation of the zebrafish is one of the reasons why it is supported as a model for environmental toxicology studies, specifically in relation to vertebrate embryogenesis [57]. The rapid rate in which the structures of the zebrafish develop, coupled with optical clarity and ease of access, makes it a model of choice for observing embryo development. The toxic effects of OP insecticides at early developmental stages of zebrafish embryo- genesis have been investigated in multiple studies, which for the first time we have collated here (Table 2). From an embryological toxicity standpoint, a substantial number of these studies employed an acute exposure period from 0–5 h post-fertilization (hpf), allowing early phenotypes to be investigated. These timepoints coincide with the initial critical stages of cellular proliferation, migration (epiboly) and the onset of gastrulation, processes essential for the establishment of the three germinal layers and subsequent patterning of tissue primordia. The consequences of OP exposure in these acute early-stage studies are diverse; however, various morphological (spinal, yolk sac, body length, pigment and eye surface area), physiological (heart rate, AChE levels, genetic), and behavioural (locomotor activity, anxiety) impairments are commonly identified. Exposure to OPs at zebrafish adult stages led to predominately behavioural (anxiety, startle response) and physiological (ATP, AChE, GSH, MDA, etc.) irregularities, with fewer concomitant morphological impairments, highly consistent with lifelong morbidity as a consequence of acute exposure primarily in the early stages of development. While symptoms and defects present in zebrafish models do not always accurately predict human disease, the broad effects of OP on development appear consistent across both fish and humans. The neurological effects of OP in development appear largely consistent across species and correlate with the known mechanism of action of OP on the cholinergic system. Further, the >80% commonality in genome between zebrafish and humans indicates that zebrafish studies are valuable for identifying the molecular changes that may be common to harmful OP exposure in humans and zebrafish. To this end, we believe our summary table will serve as an invaluable resource for future continued implementation of the zebrafish in determining consequences for human OP-dependent disease, as indicated in the sections below. J. Dev. Biol. 2022, 10, 49 7 of 22 Table 2. Studies utilising zebrafish (Danio rerio) to determine developmental and neurotoxic effects of organophosphate insecticides. dpf; days post-fertilisation. hpf; hours post-fertilisation. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference - increased impaired spatial discrimination - both decreased (10 ng/mL) and increased (100 ng/mL) response latency in adult Chlorpyrifos (CPF) CPF: 10 & 100 ng/mL - 0–5 dpf [58] zebrafish - decreased swimming activity Chlorpyrifos (CPF) CPF: 100 ng/mL - 0–5 dpf [59] - increased mortality - decreased hatching rates - decreased body length Malathion (MAL) MAL: 2.5 & 3 mg/L - 3 hpf–5 dpf [60] - and decreased surface area of eye. - decreased Adenosine Di-Phosphate (ADP) and Adenosine Tri-Phosphate (ATP) Malathion (MAL) MAL: 0.25, 0.5, 1, 3 & 5 mM - Adult (sexually mature) [61] levels - increased heart rate - increased mortality - increased morphological irregularities (axial and tail deformities, yolk sac/heart Diazinon (DZN) DZN: 2000 & 3000 g/L - 8 hpf–96 hpf [62] oedema, eye irregularities - reduced pigmentation - decreased hatching rate - Acute—increased (0.25 mg/L) and decreased (0.75 mg/L) locomotor activity. Acute: 5 dpf for 2 h Chlorpyrifos (CPF) CPF: 0.25, 0.5, 0.75 & 1 mg/L - [63] - Sub-chronic—increased behavioural irregularities Sub-chronic: 1 hpf–11dpf - increased mortality - increased phenotypes (axial curvature, reduced body size and reduced Rohon-Beard Develop- pigmentation) ment/Axonogenesis: 3 hpf–27 hpf/51 hpf/72 - decreased functioning AChE Chlorpyrifos (CPF) CPF: 300, 1500 & 3000 nM [64] agrin#, cntn2#, ntf3#, hpf/4 dpf - increased average chevron angle (somites) sema3d# - decreased HNK-1-positive cells - decreased axonogenesis-related genes J. Dev. Biol. 2022, 10, 49 8 of 22 Table 2. Cont. Gene(s) Organophosphate(s) Dosage(s) * Exposure Period Observations Reference Involved ** - significantly increased startle response - increased transmitter turnover in larvae Chlorpyrifos (CPF) CPF: 0.29 M - 0–5 dpf [65] - decreased dopamine/serotonin levels in adults Chlorpyrifos-oxon - Defective peripheral neuron development CPF: 300 nM 0.1 g/L, 3 g/L 3 hpf–75 hpf [66] (CPF metabolite) - decreased hatching rates - increased pericardial oedema Dichlorvos (DCV) DCV: 20.81, 25 & 66.78 mg/L - 0 hpf–96 hpf [67] - increased spinal irregularities - decreased swimming activity - DCV: Dichlorvos (DCV) DCV: N/A - low toxicity (determined by LC ) PHO: 0.469, 0.513, 0.700 & 1.28 - Adult (sexually mature) [4] - PHO: Phoxim (PHO) mg/L - intermediate and high levels of toxicity (determined by LC ) - decreased swim rates - increased freeze response Chlorpyrifos (CPF) CPF: 0.6 M - 1 ypf for 24 h [68] - decreased AChE in muscle - decreased functioning AChE - increased TCPy (trichloro-2-pyridinol) Chlorpyrifos (CPF) CPF: 0.01, 0.1 & 1 M - 6 hpf–24/48/72 hpf [69] - decreased functioning primary/secondary motor neurons, axonal growth and sensory neurons. - Both CPF and DZN: - increased mortality Chlorpyrifos (CPF) - decreased functioning AChE CPF: 0.3, 3 & 30 M - decreased locomotor activity Diazinon (DZN) DZN: 10 & 30 M - 6 hpf–5 dpf [70] - PA: PA: 10 & 30 M - increased mortality Parathion (PA) - decreased functioning AChE J. Dev. Biol. 2022, 10, 49 9 of 22 Table 2. Cont. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference - decreased swim speed - decreased anxiety-like behaviour Chlorpyrifos (CPF) CPF: 0.01 & 0.1 M - 0–7 dpf [71] - increased behavioural irregularities - shortened body lengths and tail defects - increased proportion of females Sexual Differentiation: cyp19a1a", Monocrotophos (MCP) MCP: 0.001 & 0.100 mg/L 72 hpfV–16 dpf [72] - alteration in expression of sexual differentiation genes cyp19a1b", foxl2", dmrt1#, B-actin, ef1-a - CPF: - increased mortality - increased kyphosis - decreased spine length - increased spontaneous movement Chlorpyrifos (CPF) CPF: 1, 10, 100 & 1000 M - and decreased heart rate Dichlorvos (DCV) DCV: 100 & 1000 M - 1 hpf–5 dpf [73] - DCV: Diazinon (DZN) DZN: 100 & 1000 M - increased mortality - increased spontaneous movement. - DZN: - increased mortality - increased pericardial oedema - significantly decreased hatching rates - increased spine and yolk sac abnormalities Chlorpyrifos (CPF) CPF: 30, 100 & 300 g/L Gfap, Mbp#, Elavl3", Ngn1", Nestin", Shha" 0–5 dpf [50] - significantly decreased heart rates - significantly decreased swim speed/distance - decreased AChE activity - increased AChE gene expression Chlorpyrifos (CPF) CPF: 200 & 400 g/L - 2 hpfV–72 hpf [74] - increased glutathione (GSH) levels J. Dev. Biol. 2022, 10, 49 10 of 22 Table 2. Cont. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference - decreased cholinesterase (ChE) levels in the heart/brain - increased myo-degeneration Oxidative Stress: Nrf2 (many other - increased testis degeneration Dichlorvos (DCV) DCV: 6, 19, & 32 mg/L associated genes within the Nrf2 6–12 mpf [75] - increased pancreas zymogen granule depletion pathway also examined) - decreased glycogen in liver - altered expression of genes involved in Nrf2 signalling - moderate toxicity (determined by LC ) - decreased body length Monocrotophos (MCP) MCP: 10, 20, 30, 40, 50 & 60 mg/L - 4 hpf–96 hpf [1] - decreased heart rate - decreased functioning AChE levels - decreased whole-body cortisol HPI Axis: Crf, Gr#, POMC#, Adult (sexually - increased/decreased hypothalamic-pituitary-inter-renal (HPI) Monocrotophos (MCP) MCP: 100 g/L P450 #, 11B-HSD2, StAR, mature)— [76] 11b axis associated genes 20B-HSD2", MC2R#, TAT, PEPCK 21 d exposure - increased oxidative stress Adult (sexually - decreased neurotransmitter metabolism Chlorpyrifos (CPF) CPF: 2 & 5 M - [77] mature) - increased energy exhaustion Embryo (1 hpf), - CPF was determined to be more toxic than PHO (determined Chlorpyrifos (CPF) CPF: 0.28- 13.03 mg/L larvae (72 hpf) and - [78] byLC ) Phoxim (PHO) PHO: 0.89–26.48 mg/L juvenile (1 mpf)— 96 h exposure - Moderate toxicity (determined by LC ) Diazinon (DZN) DZN: 6.5 mg/L - 6 hpf–5 dpf [2] - increased levels of malondialdehyde (MDA) in liver/kidney - increased glutathione (GSH) in liver/kidney/brain Adult (sexually - increased superoxide dismutase in liver Dichlorvos (DCV) DCV: 15 mg/L - mature) 4–5 m— [79] - decreased levels of superoxide dismutase in brain 24 h exposure - decreased catalase in kidney/brain. J. Dev. Biol. 2022, 10, 49 11 of 22 Table 2. Cont. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference - low toxicity (determined by LC ) HPG Axis: vtg1, vtg2, era", erB1, - upregulation of gene expression within the Malathion (MAL) MAL: 250, 500 & 1000 g/L 6 dpf–10 dpf [80] erB2, cyp19a1a, cyp19a1b" hypothalamic-pituitary-gonadal (HPG) axis - CPF: - increased mortality - decreased hatching rates - increased spinal lordosis Chlorpyrifos (CPF) CPF: 1, 10, & 25 M - reduced activity - 6 hpf–102 hpf [18] Diazinon (DZN) DZN: 10 & 100 M - DZN: - increased mortality - increased pericardial oedema - decreased mitochondrial bioenergetics - DNA damage observed in peripheral blood Monocrotophos (MCP) MCP: 0.125, 0.625 & 1.25 uL/L 24–72 hpf [81] - decreased functioning AChE - decreased carboxylesterase (CaE) Phosalone (PSL) PSL: 86–505 g/L - 8 wpf–96 h exposure [82] - increased glutathione (GSH) Oxidative stress: Mn-Sod"/#, Cu/Zn-Sod#, Gpx#, Cat#, Ucp2#, - increased levels of gut mucus bc12, Cox1# Glycolysis/Lipid: Gk#, - decreased y-Protobacteria in gut Adult (sexually Chlorpyrifos (CPF) CPF: 30, 100 & 300 g/L HK1, Pk#, Pepckc#, Aco#, CPt1#, [56] - decreased oxidative stress genes in gut and liver mature) Ppar-A#, Acc1#, Srebp 1a#, Ppar-y#, - and decreased glycolysis and lipid metabolism-related genes Fas#, Fabp6, Apo#, Dgat#, LDLR#, HMGCR, Fabp5 Cardiovascular: Mef2c#, Bmp4#, - decreased lipid accumulation in heart VEGFR-2, JunB", Tbx2 - decreased triglyceride and total cholesterol Chlorpyrifos (CPF) CPF: 30, 100 & 300 g/L Lipid: Ppar-a, Ppar-y#, Srebp 1a, 2 hpf–7 dpf [83] - increased cellular apoptosis of heart tissue Acc1, Fas#, Cpt1#, Aco, Apo#, Fabp5, - decreased lipid metabolism genes Fabp6#, Dgat#, LDLR J. Dev. Biol. 2022, 10, 49 12 of 22 Table 2. Cont. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference - DZN: - decreased swimming distance - decreased velocity - increase in AChE associated gene expression inhibited functioning AChe - increased carboxylesterase activity - DCV: Diazinon (DZN) DZN: 0.1 & 100 g/L - increased AChE associated genes. Cholinergic: AChE"/# Dichlorvos (DCV) DCV: N/A - MAL: Neurodegeneration: 5 hpf–5 dpf [84] Malathion (MAL) MAL: 100 g/L - decreased swimming distance c-Fos, lingo-1b", grin-1b# Parathion (PA) PA: 0.1 g/L - decreased velocity - increase in AChE associated gene expression - increase in neurodegenerative associated gene expression - increased carboxylesterase activity - PA: - decrease in AChE associated gene expression - and decrease in neurodegenerative associated gene expression - decreased body length - decreased heart rates - decreased surface area of eye Dichlorvos (DCV) DCV: 1, 5 & 10 mg/L - 1 hpf–7 dpf [85] - decreased escape responses - decreased speed - decreased mobile time - increased blood glucose levels Adult (sexually - increased frequency of micronucleus in erythrocytes Sumithion (SMT) SMT: 1 mg/L - [86] mature)—96h exposure - increased erythrocyte cellular and nuclear abnormalities - increased anxiety related activity (Novel Tank Diving Test) Adult (sexually mature) - increased approach response in shoaling assay Chlorpyrifos (CPF) CPF: 1 & 3 M - [87,88] 6–8 m—2/5 w exposure - increased predator avoidance activity (predator avoidance assay) J. Dev. Biol. 2022, 10, 49 13 of 22 Table 2. Cont. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference Oxidative Stress: Cat, CuSod, - Both CPF and MAL: MnSod CPF: 0.019, 0.077, 0.31, 0.41, 1.01, - severe toxicity at larvae, juvenile and adult stages Chlorpyrifos (CPF) Immunity: Cxcl#, IL", Tnf"/# 1.53 & 6.15 mg/L (compared to embryo stage) Malathion (MAL) Apoptosis: Cas8"/#, Cas9, 1 hpf–96 hpf [57] MAL: 0.039, 0.16, 0.62, 2.90, 8.04, - significant changes in expression of immunity, apoptosis, P53, Bax 8.54 & 12.45 mg/L and endocrine related genes Endocrine: TRa, TRb#, ERa, Tsh#, Crh, cyp19a" - increased mortality in embryos and larvae - decreased hatching rates - increased morphological irregularities in embryos (damaged/underdeveloped and darkened yolk sac, broken chorion, and aberrant notochord formation) Sumithion (SMT) SMT: 0.1, 0.2, 0.4, 0.8 & 1.6 mg/L - Embryo/larvae [89] - increased morphological irregularities in larvae (yolk sac ulcerations/swelling and oedema, heart damage, lesion at caudal region, uninflated swim bladder, head malformation, jaw irregularities, and notochord abnormalities). - decreased brain cholinesterase (ChE) activity Adult (sexually Chlorpyrifos (CPF) CPF: 1 M - (Hawkey, 2021) - increased fleeing score. mature)—5 w exposure - Changes in Mitochondrial oxygen utilization in the brain Diazinon (DZN) DZN: 0.4, 1.25 & 4.0 M 5–120 hpf [90] and testes - increases in reactive oxygen species Malathion (MAL) MAL: 5, 50 ug/L 0–14 dpf [91] - induction of oxidative stress Chlorpyrifos (CPF) CPF: 0.1 & 3 ug/L - Significantly elevated ROS levels Adult—8–12 months old - Elevated Reactive nitrogen species levels in high CPF Chlorpyrifos (CPF) Caspase 3#, Bcl-2#, [92] 14 day exposure dosage groups * All dosages listed are associated with the observations summarised here, other dosages in the individual studies may have been used, but did not impact on development, behaviour or gene expression. **" and# arrows indicate where there has been a significant increase or decrease in a particular gene as a response of organophosphate (OP) exposure—where no arrow shows, no significant change was noted. J. Dev. Biol. 2022, 10, 49 14 of 22 6. Organophosphate Toxicity—Acute Cholinergic Syndrome (ACS) Although having conserved function as a neurotoxin, the phenotypic consequences of OP exposure are nonetheless extremely variable, and symptoms may manifest following either acute (high dose) or chronic (typically lower dose) exposure (Figure 3). The earliest stage of OP toxicity is referred to as acute cholinergic syndrome (ACS), which is a result of the effects of AChE inhibition [93]. ACS can occur within minutes of OP exposure, and impairs both muscarinic and nicotinic receptors found in the nervous system [94]. The consequences of hyper-stimulated post-synaptic receptors (hyperstimulation) vary depending on their locations. In the CNS, hyperstimulation more commonly occurs at muscarinic receptors, resulting in heart irregularities, gastrointestinal issues including stomach cramps, diarrhoea and vomiting, respiratory complications including bronchor- rhea and bronchospasms, as well as neurological effects including seizures, agitations and anxiety [95,96]. Comparatively, hyperstimulation in the PNS is more commonly associated with nicotinic receptors, and this is often expressed as muscle weakness, cramps or paral- J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  14  of  21  ysis [7,97]. Symptoms of OP exposure are quite complex in that they are not limited to a localised area and are a result of the diverse effect of OPs on both CNS and PNS pathways. Figure 3. The major consequences of OP exposure in humans.  Figure 3. The major consequences of OP exposure in humans. 7. 7. Organop Organophosphate hosphate T Toxicity—Intermediate oxicity—Intermediate Syndrome Syndrome (IMS) (IMS)  The The inte intermediate rmediate sy syndr ndrome ome (IMS (IMS) ) fofollows llows 1–4 1–4  days days  afteafter r ACS ACS  (Figure (Figur  4), eand 4), is and  char is‐ characterised acterised as the as the onset onset  of muscle of muscle  we w ak eakness, ness, papart rticula icularly rly inin the the proximal proximal limbs limbs,, neck neck and and  rrespir espiratory atory system system [[98 98]. ]. If If untr untreated eated,, fr from om 14–21 14–21 days days after after acute acute exposur exposure, e, weakness weakness in in  the the peripheral peripheral muscles muscles becomes becomes evident evident [[4 499] ].. It It is is estimate estimated d that that only only ~20% ~20% of of humans humans  exposed to OP will have symptoms that progress from ACS to the IMS stage [99]. The IMS exposed to OP will have symptoms that progress from ACS to the IMS stage [99]. The IMS  is commonly associated with respiratory failure as a result of nicotinic receptor paralysis. is commonly associated with respiratory failure as a result of nicotinic receptor paralysis.  Respiratory failure from prolonged OP exposure is primarily linked to the CNS, specifically Respiratory failure from prolonged OP exposure is primarily linked to the CNS, specifi‐ the depression of the pre-Botzinger complex (glutaminergic and muscarinic fibres) located cally the depression of the pre‐Botzinger complex (glutaminergic and muscarinic fibres)  in the ventrolateral medulla in the brainstem [100]. This has resulted in respiratory failure located in the ventrolateral medulla in the brainstem [100]. This has resulted in respiratory  being recognised as a significant comorbidity in OP mortality [94]. failure being recognised as a significant comorbidity in OP mortality [94].  Figure 4. The symptoms of organophosphate (OP) exposure at progressive time points; acute cho‐ linergic syndrome (ACS) occurs within minutes of OP exposure, symptoms of intermediate syn‐ drome (IMS) display at 1–4 days after OP exposure, and symptoms of organophosphate‐induced  delayed neuropathy (OPIDN) occur ~2–3 weeks after OP exposure.    J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  14  of  21  Figure 3. The major consequences of OP exposure in humans.  7. Organophosphate Toxicity—Intermediate Syndrome (IMS)  The intermediate syndrome (IMS) follows 1–4 days after ACS (Figure 4), and is char‐ acterised as the onset of muscle weakness, particularly in the proximal limbs, neck and  respiratory system [98]. If untreated, from 14–21 days after acute exposure, weakness in  the peripheral muscles becomes evident [49]. It is estimated that only ~20% of humans  exposed to OP will have symptoms that progress from ACS to the IMS stage [99]. The IMS  is commonly associated with respiratory failure as a result of nicotinic receptor paralysis.  Respiratory failure from prolonged OP exposure is primarily linked to the CNS, specifi‐ cally the depression of the pre‐Botzinger complex (glutaminergic and muscarinic fibres)  J. Dev. Biol. 2022, 10, 49 15 of 22 located in the ventrolateral medulla in the brainstem [100]. This has resulted in respiratory  failure being recognised as a significant comorbidity in OP mortality [94].  Figure 4. The symptoms of organophosphate (OP) exposure at progressive time points; acute cho‐ Figure 4. The symptoms of organophosphate (OP) exposure at progressive time points; acute linergic syndrome (ACS) occurs within minutes of OP exposure, symptoms of intermediate syn‐ cholinergic syndrome (ACS) occurs within minutes of OP exposure, symptoms of intermediate drome (IMS) display at 1–4 days after OP exposure, and symptoms of organophosphate‐induced  syndrome (IMS) display at 1–4 days after OP exposure, and symptoms of organophosphate-induced delayed neuropathy (OPIDN) occur ~2–3 weeks after OP exposure.  delayed neuropathy (OPIDN) occur ~2–3 weeks after OP exposure. 8. Organophosphate Toxicity—Organophosphate-Induced Delayed Neuropathy (OPIDN) OP toxicity does not solely result in AChE inhibition, and depending on the physical structure of the compound, OPs can target other secondary hydroxyl sites on enzymes other than AChE [101]. OP insecticide exposure can also give rise to another type of toxicity, referred to as organophosphate-induced delayed neuropathy (OPIDN), which, depending on dose and chemical structure, occurs ~2–3 weeks after ACS [94,102]. This pathology is characterised as the degeneration of distal axons in the CNS and PNS, is expressed as sensory loss in both hands and feet, weakness in distal muscles and coordination issues [7] and is associated with the inhibition of neuropathy-target-esterase (NTE) [103]. NTE is an integral enzyme employed at the neurite initiation stage of neuronal morphogenesis, with these neurites maturing into axons and dendrites to form part of the nervous system [104]. Importantly, the consequences of OPIDN are only present when 70% of NTE is inhibited [105]. Additionally, in order for irreversible inhibition of NTE, there must be a secondary chemical reaction, where there is a displacement of an R-group (aging) [103], and, therefore, NTE inhibition and OPIDN is thought to contribute to diseases characterised by axonal degradation such as Alzheimer ’s disease, Parkinson’s disease and motor neuron diseases (MND) that include amyotrophic lateral sclerosis (ALS) and progressive bulbar palsy [106]. As these diseases are primarily associated with aging in humans, it is interesting to note that animal models show that adults are both far more susceptible to, and recover far more poorly from, OPIDN than juveniles [106]. The inhibition of NTE itself is not responsible for axonal degeneration, as has been demonstrated with non-OP inhibitors (organophosphinates, sulfonyl fluorides and carba- mates) that covalently react with NTE, but do not undergo the enzyme ageing process [107]; this indicates that R-group displacement confers a “gain” of neurotoxicity that is damaging in its own right. 9. Organophosphate Toxicity—Effects on Embryogenesis Pre-natal OP exposure is of particular concern, as developing babies are highly sus- ceptible to chemical injury [108,109]. This is partly a result of their immature detoxification mechanisms, i.e., reduced expression of OP specific detoxifying enzymes such as paraox- J. Dev. Biol. 2022, 10, 49 16 of 22 onase and chlorpyrifos-oxonase, compared to adults [110,111], and also as the cholinergic system (which is targeted by OPs) is heavily involved with placental processes including amino acid uptake and nitric oxide signalling [112]. Pre-natal OP exposure has been associ- ated with shortened gestational periods [113], reduced birth weight and birth length [114], as well as impaired reflexes [115] and neurobehavioral irregularities [116]. While the influence of OPs on the mature blood brain barrier (BBB) is unclear, the developing and newborn BBB is “leaky”, allowing toxins, in particularly pesticides in the fetal circulation, to cross and have negative effects on the developing brain [117,118]. Over- all, the neurotoxic properties of OPs, and the resultant syndromes have been reasonably well documented (Figure 4), with AChE inhibition and hyperstimulation of postsynaptic neurons being key contributors to these impairments [119]. 10. Organophosphate Toxicity—Effects on Neurodevelopment and Early Behaviour The timing of prenatal OP exposure plays a critical role in fetal development and postnatal behaviour. OP exposure during the 1st and 2nd trimester of pregnancy has been shown to be associated with delayed cognitive performance at both 2 and 6 months of age, whereas OP exposure in the 3rd trimester of pregnancy is associated with delayed communication and motor performance at 6 months of age [120]. In terms of neurodevelopmental impairments, OPs such as chlorpyrifos and diazinon have been shown to cause decreased DNA synthesis in neuronotypic PC12 and gliotypic C6 neural cell lines, the latter of which continues to develop into the postnatal period [121]. Additionally, children aged ~3 years have been identified as having increased risk of displaying developmental delays and a higher incidence of behavioural disorders such as ADHD [108,122], and prenatal exposure to OP insecticides was associated with poorer intellectual development in seven year old children [123], as well as poorer motor skills and cognitive recall when compared to non-exposed children [124]. Taken together, these studies show that not only does pre-natal exposure to OPs affect embryogenesis, but also cognitive development in young children. 11. Organophosphate Toxicity—Effects in Adulthood and Neurodegenerative Diseases Whilst infants and children are highly susceptible to OP insecticide toxicity, these chemicals have also been associated with impaired health at later stages of life, particu- larly neurological disorders such as Alzheimer ’s Disease (AD) and Parkinson’s Disease (PD) [125]. Recent studies have shown that chronic exposure in agricultural workers is associated with neural irregularities including neurodegenerative diseases, attention im- pairment and short-term memory loss [11]. The cholinergic system, which is affected by OP insecticides, has long been associated with neurodegenerative diseases, with ACh one of the key neurotransmitters involved in cellular signalling in the brain [125]. The reduction of ACh is a critical element in memory loss diseases such as AD, where ACh in the basal forebrain is known to play an integral role in memory and learning; as OP insecticides promote an imbalance of ACh at cellular junctions in the brain, these chemicals are, therefore, linked to impaired memory diseases [126]. Although PD involves a depletion of dopaminergic cell bodies, it is symptomatically dissimilar to AD in that it is characterised by motor impairments such as tremor at early onset and posture/gait issues at later stages [127]. Along with genetic predisposition, pesticides are widely acknowledged as an environmental risk factor for PD, with OPs having been implicated in some studies of the disease [128–130]. Variability in the PON1 gene (when exposed to various OPs—diazinon/chlorpyrifos) has been shown to correlate with a greater than two-fold increase in PD risk [131]. Despite the causal relationship being still largely unknown, an epidemiological analysis of 23 case-control studies found that 13 of the studies reported a statistically significant risk of PD with pesticide exposure, with both chlorpyrifos (OP) and organochlorines (OC) being key contributors to the study [132]. However, one limitation of pesticide research on neurodegenerative diseases such as PD is J. Dev. Biol. 2022, 10, 49 17 of 22 that the study of pesticides does not encompass the lifespan, making it difficult to analyse the long term effects of these chemicals [133]. 12. Conclusions Rapid population growth and changing diets in developing countries have increased the demand for food, to the point that food production must increase by 70% to meet the estimated food demands in 2050 [134]. Pesticides will play an essential role in achieving this production increase. While the economic and societal benefits of pesticides are inarguable, the effects of pesticide exposure on non-target animals and humans are a continuing concern. Of note is the growing use of OP insecticides, which are linked to poor neurodevelopment in both developed and developing countries worldwide. While our understanding of the relationship between OP exposure and poor health outcomes is growing, it remains unclear the extent to which AChE inhibition and OP exposure lead to developmental abnormalities. However, addressing this relationship between OP exposure and neurodevelopment and behaviour will encourage improvements in the regulation of use and handling of OPs in agricultural, industrial and domestic environments around the world. Author Contributions: Conceptualization, J.N. and S.D.; writing—original draft preparation, J.N., J.N.F., C.v.d.P., J.E.C. and S.D., writing—review and editing, J.N., J.N.F., C.v.d.P., J.E.C. and S.D.; project administration, S.D., funding acquisition, S.D. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a La Trobe University Securing Food, Water and the Environ- ment, Research Focus Area Grant Ready Grant #3.2509.07.48. Institutional Review Board Statement: Not Applicable. Informed Consent Statement: Not Applicable. Data Availability Statement: Not Applicable. Conflicts of Interest: The authors declare no conflict of interest. References 1. Pamanji, R.; Bethu, M.S.; Yashwanth, B.; Leelavathi, S.; Venkateswara Rao, J. Developmental toxic effects of monocrotophos, an organophosphorous pesticide, on zebrafish (Danio rerio) embryos. Environ. Sci. Pollut Res. Int. 2015, 22, 7744–7753. [CrossRef] [PubMed] 2. 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Organophosphate Insecticide Toxicity in Neural Development, Cognition, Behaviour and Degeneration: Insights from Zebrafish

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Abstract

Journal of Developmental Biology Review Organophosphate Insecticide Toxicity in Neural Development, Cognition, Behaviour and Degeneration: Insights from Zebrafish Jeremy Neylon, Jarrad N. Fuller, Chris van der Poel, Jarrod E. Church and Sebastian Dworkin * Department of Microbiology, Anatomy, Physiology and Pharmacology, La Trobe University, Melbourne, VIC 3086, Australia * Correspondence: s.dworkin@latrobe.edu.au Abstract: Organophosphate (OP) insecticides are used to eliminate agricultural threats posed by insects, through inhibition of the neurotransmitter acetylcholinesterase (AChE). These potent neuro- toxins are extremely efficacious in insect elimination, and as such, are the preferred agricultural insec- ticides worldwide. Despite their efficacy, however, estimates indicate that only 0.1% of organophos- phates reach their desired target. Moreover, multiple studies have shown that OP exposure in both humans and animals can lead to aberrations in embryonic development, defects in childhood neu- rocognition, and substantial contribution to neurodegenerative diseases such as Alzheimer ’s and Motor Neurone Disease. Here, we review the current state of knowledge pertaining to organophos- phate exposure on both embryonic development and/or subsequent neurological consequences on behaviour, paying particular attention to data gleaned using an excellent animal model, the zebrafish (Danio rerio). Keywords: organophosphate; insecticides; zebrafish; neurodevelopment Citation: Neylon, J.; Fuller, J.N.; van der Poel, C.; Church, J.E.; Dworkin, S. 1. The Need for Insecticides Organophosphate Insecticide Toxicity Insecticides are a class of pesticides used to eliminate insects and are employed in areas in Neural Development, Cognition, such as crop development, animal husbandry, agriculture and household environments [1]. Behaviour and Degeneration: On a global scale, insecticides account for 20% of the pesticide market [2], where their Insights from Zebrafish. J. Dev. Biol. 2022, 10, 49. https://doi.org/ widespread use in agriculture improves crop quality and yield, and optimises the revenue 10.3390/jdb10040049 and economic yield of harvests [3–5]. Additionally, the use of insecticides protects con- sumers against vector-borne diseases [6,7], forming a crucial front-line defence to safeguard Academic Editor: Simon J. Conway the agricultural sector. However, these pesticides do not only eliminate the insects they Received: 5 October 2022 target, but, as we will discuss here, also cause significant off-target effects to livestock, Accepted: 16 November 2022 aquatic species and humans. Published: 21 November 2022 Whilst insects constitute an integral component of terrestrial ecosystems [8,9], their control requires significant expenditure from the agricultural sector. In 2012–2013, usage in Publisher’s Note: MDPI stays neutral Australian agriculture totalled approximately $350 billion on insecticides [10,11], demon- with regard to jurisdictional claims in strating the economic burden of pests in agrarian settings. Despite this, the alternative published maps and institutional affil- iations. (that is, non-use) is financially unviable owing to the limitless potential for insect-mediated agricultural destruction, indicating that insecticides are necessary to safeguard and main- tain food production. With an increasing population and limited agrarian land available, improving production and productivity on existing land is integral for safeguarding long- Copyright: © 2022 by the authors. term food security [12]. Therefore, insect management is critical in commercial agriculture, Licensee MDPI, Basel, Switzerland. yet the dangerous neurotoxic activity of the most commonly used insecticides must be This article is an open access article strictly modulated, in order to ensure both efficacy and safety to non-insect species. distributed under the terms and conditions of the Creative Commons 2. Insecticides—The Move to Organophosphates (OPs) Attribution (CC BY) license (https:// Among the earliest implementation of synthetic compounds for insecticidal purposes was creativecommons.org/licenses/by/ the development of organochlorine (OC) insecticides, such as dichlorodiphenyl-trichloroethane 4.0/). J. Dev. Biol. 2022, 10, 49. https://doi.org/10.3390/jdb10040049 https://www.mdpi.com/journal/jdb J.J. De Dev.v Bi . Biol.ol. 2022  2022, 10 , 10, x, x FOR  FOR PE PEER ER REVIEW  REVIEW    2 2of  of  21  21    J. De J. De v. vBi. Bi ol.ol. 2022  2022 , 10 , 10 , x,  FOR x FOR PE PE ERER REVIEW  REVIEW    2 2of  of  21  21        2.2. I n Insec sectic ticidideess—The —The Move  Move to to Organo  Organoph phosphates osphates (OPs)  (OPs)   2.2. In Isec nsec tictic idid ese—The s—The Move  Move to to Organo  Organo phph osphates osphates (OPs)  (OPs)    J. Dev. Biol. 2022, 10, 49 2 of 22 Among Among the  the e eaarlie rliestst implem  implementation entation of of synthetic  synthetic comp  compounds ounds fo for rins  inseectic cticididalal purposes  purposes   Among Among the the ea erlie arlie stst implem  implem entation entation of of synthetic  synthetic comp  comp ounds ounds fo fo r ins r ins ectic ectic idid alal purposes  purposes    was was the  the deve  development lopment of of or orggaannoocchhlolorirne ine (OC)  (OC) in insec sectitcicides ides, ,su such ch as as dich  dichlorod lorodiphenyl iphenyl‐tr‐tri‐i‐ was was the  the deve  deve lopment lopment of of or or gagnaoncohclholroine rine (OC)  (OC) in in sec sec tictiides cides , su , su chch as as dich  dich lorod lorod iphenyl iphenyl‐tr‐tr i‐i‐ chloroethane chloroethane  (DDT (DDT),),  cyclodienes cyclodienes  incl incluudding ing  alaldri drinn, , dieldrin dieldrin, , hep heptach tachlor lor  an andd  ch chlolord rdaannee   chloroethane chloroethane  (DDT   (DDT ), ),cyclodienes   cyclodienes  incl   incl uduing ding  al  al dri dri n,n  dieldrin ,  dieldrin ,  hep ,  hep tach tach lorlor  an  an d dch  ch lolrodradnaen e  (DDT), cyclodienes including aldrin, dieldrin, heptachlor and chlordane [7,13]. These com- [7[7,1,13]3]. .These  These compounds  compounds are  are neurotox  neurotoxicic and  and exer  exert tth their eir ef effefecctsts on  on ta target rget or orga ganinissmmss by  by   [7,13]. These compounds are neurotoxic and exert their effects on target organisms by  [7,13]. These compounds are neurotoxic and exert their effects on target organisms by  pounds are neurotoxic and exert their effects on target organisms by rapidly opening rapid rapidlyly opening  opening sod  sodium ium (N  (Naa) )ch channels annels in in neurons,  neurons, which  which re results sults in in the  the con  contin tinuous uous st stim im‐‐ + + rapid rapid lyl yopening  opening  +sod  sod ium ium (N (N a a) ch ) ch annels annels in in neurons,  neurons, which  which re re sults sults in in the the con  con tintin uous uous st st imim‐ ‐ sodium (Na ) channels in neurons, which results in the continuous stimulation of cellular ululation ation of of cellu  cellulalar rrecep  receptor torss [1 [14,15 4,15].]. Al Althou thougghh rem  remaarkrkably ably ef effic ficacacioious us ag agains ainst tinsects,  insects, the  the   ulul ation ation of of cellu  cellu lala r recep r recep tor tor s s[1 [1 4,15 4,15 ]. ].Al Al thou thou ghg hrem  rem arakrably kably ef ef ficfic acac ioius ous ag ag ains ains t insects, t insects, the the    receptors [14,15]. Although remarkably efficacious against insects, the use of OC insecti- use use of of OC  OC ins  inseectic cticide idess in in agri  agricult culture ure is is now  now lar  larggelelyy banned  banned wor  worldldwwide, ide, ow  owing ing to to the  the si sigg‐‐ use use of of OC  OC ins ins ectic ectic ide ide s in s in agri  agri cult cult ure ure is is now  now lar lar gel gel y ybanned  banned wor  wor ldlw dw ide, ide, ow ow ing ing to to the the si si g‐g‐ cides in agriculture is now largely banned worldwide, owing to the significant teratogenic nificant teratogenic and carcinogenic effects observed in wildlife and livestock, as well as  nificant teratogenic and carcinogenic effects observed in wildlife and livestock, as well as  nifi nifi can can t ter t ter ataotgenic ogenic and  and ca ca rcinogen rcinogen icic ef ef fefe ctcst observed s observed in in w w ildild life life an an d dli vest livest ock, ock, as as well  well as as    and carcinogenic effects observed in wildlife and livestock, as well as humans. Further humans. humans. Fu Furthe rther roff off‐ta‐targe rget teffec  effectsts inc  includ ludiningg carc  carcinoge inogennesis esis, ,hy hype pertrtrophy rophy and  and red  reduuced ced fer fer‐‐ humans. Further off‐target effects including carcinogenesis, hypertrophy and reduced fer‐ humans. Further off‐target effects including carcinogenesis, hypertrophy and reduced fer‐ off-target effects including carcinogenesis, hypertrophy and reduced fertility in rodent titililityty in in rode  rodentnt models,  models, as as well  well as as geno  genotoxic toxicity, ity, red  reduuced ced f efertrtililitityy and  and neuro  neurotoxicity toxicity [1 [16]6], ,  tilitity lity in in rode  rode ntnt models,  models, as as well  well as as geno  geno toxic toxic ity, ity, red  red uced uced fe frtert ilitilyit yand  and neuro  neuro toxicity toxicity [1 [1 6]6] ,  ,  models, as well as genotoxicity, reduced fertility and neurotoxicity [16], have dictated the have have di dicctat tateedd the  the rap  rapidid deve  developme lopmentnt of of al altern ternatative ive in insect secticid icides. es.   have have di di ctcattat ede dthe the rap  rap idi ddeve  deve lopme lopme ntnt of of al al tern tern atat ive ive in in sect sect icid icid es.es.    rapid development of alternative insecticides. Today, Today, the  the vas  vast tma  majojorirtiyty of of insec  insectic ticides ides us used ed are  are organ  organoophosphates phosphates (OP)  (OP), ,which  which, ,al alt‐t‐ Today, Today, the the vas  vas t ma t ma jojroitryit yof of insec  insec tictic ides ides us us eded are are organ  organ ophosphates ophosphates (OP)  (OP) , which , which , al , al t‐t‐ Today, the vast majority of insecticides used are organophosphates (OP), which, al- hough hough al also so n neeurotoxic, urotoxic, are  are gener  generaally lly co consider nsidered ed a a “s “safe afer”r” al alternati ternativvee [17  [17],] ,as as th thee leve  levelsls of of   hough hough al al soso n enurotoxic, eurotoxic, are are gener  gener ally ally co co nsider nsider eded a  a“s “s afe afe r”r ”al al ternati ternati vev e[17 [17 ], ]as , as th th e eleve  leve lsls of of    though also neurotoxic, are generally considered a “safer” alternative [17], as the levels of bioacc bioaccumul umulation ation (a (accu ccummula ulatitoionn of of chem  chemicals icals ins  inside ide an an organism  organism th throrough ugh di direrectct or or ind  indirec irect t  bioacc bioacc umul umul ation ation (a (a ccu ccu mm ula ula tiotino nof of chem  chem icals icals ins ins ide ide an an organism  organism th th rorugh ough di di rerctect or or ind ind irec irec t t  bioaccumulation (accumulation of chemicals inside an organism through direct or indirect uptake uptake) )are  are fa far rlower  lower for  for OP  OP rat  rathheer rth thaann OC  OC insec  insectic ticides ides [18  [18].] .Mo  Moreover, reover, OP  OP in insect secticid icideses   uptake) are far lower for OP rather than OC insecticides [18]. Moreover, OP insecticides  uptake uptake) ) are ar fa erfar lower lower for for OP OP rat rather her th than an OC OC insec insecticides ticides [18 [18]].. Mo Morreover, eover, OP OP insecticides insecticidesar  e are are fu furtrther her fa favoured voured in in ag agricu ricultltur uralal setti  settings ngs du  duee to to thei  their rcost  cost e effe ffectivene ctivenessss [1 [1] ]as as well  well as as   are are fu fu rtrt her her fa fa voured voured in in ag ag ricu ricu ltur ltur alal setti  setti ngs ngs du du e eto to thei  thei r rcost  cost e ffe effe ctivene ctivene sss s[1 [1 ] as ] as well  well as as    further favoured in agricultural settings due to their cost effectiveness [1] as well as their their their ra rapid pid mo  moddee of of ac action tion (immediat  (immediatee n neeurotoxic urotoxicity) ity) ag again ainsst ta a wi wide de variet  varietyy of of ta target rget or or‐‐ their their rapid  ra ra pid pid mode  mo  mo ded of eof action of ac ac tion tion (immediate  (immediat  (immediat neur e en enurotoxic otoxicity) eurotoxic ity) ity) against  ag ag ain ain asts wide  at  awi wi de variety de variet  variet ofy tar yof of get ta ta rget or rget ganisms,  or or‐ ‐ ganisms, ganisms, lea  leadding ing to to broad  broad‐spect ‐spectru rumm su succes ccesss in in pest  pest e elilm imina ination tion [19  [19].] .The  The most  most commonly  commonly   ganisms, ganisms, leading  lea lea d toing dbr ing oad-spectr to to broad  broad‐spect um ‐spect success rurm um su su cces incces pest s in s in elimination  pest  pest e leim lim ina ina [19 tion t].ion The [19 [19 ].most  ]The . The most commonly  most commonly  commonly used OPs   used OPs are shown in Table 1, and include chemicals to safeguard livestock in both ter‐ used OPs are shown in Table 1, and include chemicals to safeguard livestock in both ter‐ are shown in Table 1, and include chemicals to safeguard livestock in both terrestrial and used used OPs  OPs ar ar e eshown  shown in in Tab  Tab lel e1, 1, and  and inc inc lude lude chem  chem icals icals to to sa sa fefgeugauradr dli vest livest ock ock in in bot bot h hter ter‐ ‐ restrial restrial and  and aq aqua uatiticc‐ba‐basesedd farm  farming ing a approa pproaches. ches.   restrial aquatic-based  and aquaticfarming ‐based farm appring oaches.  approaches.  restrial and aquatic‐based farming approaches.  Table Table 1. 1. Commonly  Commonly us used ed orga  organophosphate nophosphate (O (OPP) )inse  insectic cticide ides sin in agriculture  agriculture and  and aquaculture.  aquaculture. Shown  Shown   Table Table 1.  1. Commonly  Commonly us us eded orga  orga nophosphate nophosphate (O (O P)P inse ) inse ctic ctic ide ide s in s in agriculture  agriculture and  and aquaculture.  aquaculture. Shown  Shown    Table 1. Commonly used organophosphate (OP) insecticides in agriculture and aquaculture. Shown here here are  are approvals  approvals us usiningg the  the Pu  Public blic Chemical  Chemical Registration  Registration Inform  Information ation Sy System stem Se Searc archh (Pub  (PubCR CRISIS),) ,  here here are are approvals  approvals us us inign gthe the Pu Pu blic blic Chemical  Chemical Registration  Registration Inform  Inform ation ation Sy Sy stem stem Se Se arc arc h h(Pub  (Pub CR CR ISI)S, ),  here are approvals using the Public Chemical Registration Information System Search (PubCRIS), molecu molecular lar for  formmula ula and  and general  general us uses. es.   molecular formula and general uses.  molecular formula and general uses.  molecular formula and general uses. Molecu Molecular lar Formu  Formula: la:     Molecu Molecular lar Formu  Formula: la:   Molecular Formula:   Molecular Formula:  Molecular Formula:   Molecular Formula:  Molecular CC 9H 9H 1010CIN CIN Formula 2O 2O 5PS 5PS  : CC 1010HH 1212NN 3O 3O 3PS 3PS 2 2  CC 9H 9H 10CIN 10CIN 2O 2O 5PS 5PS    CC 10H 10H 12N 12N 3O 3O 3PS 3PS 2  2  Molecular Formula: C H CIN O PS GGeene ne 9rara 10l lUses  Uses 2 of 5of OP  OP: :  GGeene neraral lUses  Uses of of OP  OP: :  C H N O PS GG ene ene rara l Uses l Uses of of OP OP :  :  GG ene ene 10 rara l 12 Uses l Uses 3  of 3 of OP 2 OP :  :  General Uses of OP: Used Used in in Aquatic  Aquatic Farm  Farm‐‐ Used General Used on on Uses  Orch  Orch ofard ard OP  : Used Used in in Aquatic  Aquatic Farm  Farm‐ ‐ Used Used on on Orch  Orch ard ard    Used in Aquatic Farming Used on Orchard Fruits and ing ing (A (Atltlant anticic Sa Salmon) lmon) to to   Fruits Fruits an andd Nu  Nut tCrops  Crops   (Atlantic Salmon) to Control ing ing (A (A tlant tlant ici Sa c Sa lmon) lmon) to to    Fruits Fruits an an d dNu Nu t Crops t Crops    Nut Crops to Control Moths. Cont Control rol Pa Pararasisitetess. .  toto Control  Control Mot  Mothhss. .  Parasites. Cont Cont rolrol Pa Pa rarsiasi tetse. s.  toto Control  Control Mot  Mot hsh. s.                  Molecu Molecular lar Formu  Formula: la:   Molecu Molecu larlar Formu  Formu la:la:    Molecu Molecular lar Formu  Formula: la:     Molecu Molecu larlar Formu  Formu la:la:     CC 1212HH 2121NN 2O 2O 3PS 3PS   Molecular Formula: Molecular Formula: CC 12H 12H 21N 21N 2O 2O 3PS 3PS    CC 9H 9H 1111Cl Cl 3NO 3NO 3PS 3PS   CC 9H 9H 11Cl 11Cl 3NO 3NO 3PS 3PS    C H Cl NO PS GGeene ne Crara H l lUses  Uses N O of of PS OP  OP: :  9 11 3 3 12 21 2 3 GG ene ene rara l Uses l Uses of of OP OP :  :  GGeene neraral lUses  Uses of of OP  OP: :  General Uses of OP: General Uses of OP: GG ene ene rara l Uses l Uses of of OP OP :  :  Used Used on on crop  cropss   Used Used on on crop  crop s s  Used Used broa  broadly dly (crops  (crops/a/anini‐‐ Used broadly Used on crops Used Used broa  broa dly dly (crops  (crops /a/ni ani‐ ‐ (fr (fruiuitsts/vege /vegetata‐‐ (crops/animals/buildings) to (fruits/vegetables/nuts/field (fr(fr uiui tsts /vege /vege tat‐a‐ mals mals/bu /builidldinings) gs) to to con  con‐‐ mals mals /bu /bu ildilin din gs) gs) to to con  con‐ ‐ bles/n bles/nuts/field uts/field crops)  crops)   control roundworms, crops) to control ants, fleas bles/n bles/n uts/field uts/field crops)  crops)    trol trol roun  roundwo dworms, rms, mos  mos‐‐ trol trol roun  roun dwo dwo rms, rms, mos  mos‐ ‐ toto con  control trol an antsts, ,fle fleasas   mosquitos and termites. and cockroaches. toto con  con trol trol an an tst, sfle , fle asas    qu quito itoss and  and te term rmite itess. .  ququ itoito s and s and te te rm rm iteite s. s.  and and coc  cockkroac roaches. hes.   and and coc coc kroac kroac hes. hes.                           J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  3  of  21  J. Dev. Biol. 2022, 10, 49 3 of 22 J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  3  of  21  J. De   v. Biol. 2022, 10, x FOR PEER REVIEW  3  of  21  J. De J. De v. vBi. Bi ol.ol. 2022  2022 , 10 , 10 , x,  FOR x FOR PE PE ERER REVIEW  REVIEW    3 3of  of  21  21        J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  3  of  21  J.  Dev. Biol. 2022, 10, x FOR PEER REVIEW  3  of  21      Table 1. Cont. Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecu Molecu larlar fo fo rmu rmu la:la:     Molecu Molecu larlar fo fo rmu rmu la:la:     C4H7Cl2O4P  C9H12NO5PS  Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   CC 4H 4H 7Cl 7Cl 2O 2O 4P4 P  CC 9H 9H 12NO 12NO 5PS 5PS    C4H7Cl2O4P  C9H12NO5PS  C4H7Cl2O4P  C9H12NO5PS  General uses of OP:  General uses of OP:  CC 4H 4H 7Cl 7Cl 2O 2O 4P 4P    CC 9H 9H 1212 NO NO 5PS 5PS    General uses of OP:  General uses of OP:  General uses of OP:  GeMolecular neral uses Formula  of OP::  GG Molecular ene ene rara l uses l uses Formula  of of OP OP ::  :  GG ene ene rara l uses l uses of of OP OP :  :  Used broadly (house‐ Used broadly (public  General uses of OP:  General uses of OP:  General uses of OP:  Gene Cra Hl uses NO PS of OP:  Used Used broa  broa dly dly (hou  (hou sese‐ ‐ Used Used broa  broa 9 12dly dly (publ 5 (publ ici c  C H Cl O P Used  4broa 7 dly 2 4 (house‐ Used broadly (public  Used broadly (house‐ Used broadly (public  hold/  health/agriculture) to  General Uses of OP: Used Used broa  broa dly dly (hou  (hou sese‐‐ Used Used broa  broa dly dly (publ  (publ ici c  hold/  health/agriculture) to  General hold/ Uses  of OP: health/agriculture) to  hold/ hold/    heal heal thth /a/a gric gric ulutultre) ure) to to    Used broadly (public agriculture) to control  control beetles, grubs,  hold/  health/agriculture) to  Used broadly hold/ (household/   health/agriculture) to  agr agr icu icu lture lture ) to ) to contro  contro l l  control control beetle  beetle s,s, gr gr ubus, bs,    health/agriculture) to control agriculture) to control  control beetles, grubs,  agriculture) to control  control beetles, grubs,  flies, caterpillars, thrips  locusts, flies, mosqui‐ agriculture) to control flies, agr agr icu icu ltlt ure ure ) )to to contro  contro l l  control control beetle  beetle s,s, gr gr uu bs, bs,    flies, caterpillars, thrips  locusts, flies, mosqui‐ flies, caterpillars, thrips  locusts, flies, mosqui‐ beetles, grubs, locusts, flies, flifli es,es, caterp  caterp illars, illars, thrip  thrip s s  lolcu ocu stst s, s,fl ies, flies, mosq  mosq uiui‐ ‐ caterpillars, thrips and mites. and mites.  tos, etc.  flies, caterpillars, thrips  locusts, flies, mosqui‐ flies, caterpillars, thrips  locusts, flies, mosqui‐ mosquitos, etc. and and mi mi tetse.s .  tos, tos, et et c.c .  and mites.  tos, etc.  and mites.  tos, etc.      and and mi mi tetse.s .  tos, tos, et et c.c .                          Molecular formula:   Molecular formula:   Molecu Molecu lar lar fo fo rmu rmu la:la:     Molecu Molecu lar lar fo fo rmu rmu la:la:     Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   C10H19O6PS2  C8H10NO5PS  Molecu Molecu lar lar fo fo rmu rmu la:la:     Molecu Molecu lar lar fo fo rmu rmu la:la:     C10H19O6PS2  C8H10NO5PS  C10H19O6PS2  C8H10NO5PS  Molecular Formula: C10H19O6PS2  C8H10NO5PS  C10H19O6PS2  C8H10NO5PS  Molecular Formula: General uses of OP:  General uses of OP:  CC 1010 HH 1919 OO 6PS 6PS 2 2  CC 8H 8H 1010 NO NO 5PS 5PS    GG ene ene Crara lH uses l uses O of PS of OP  OP : :  GG ene ene rara l uses l uses of of OP  OP : :  10 19 6 2 General uses of OP:  Gene C ra Hl uses NO PS of OP:  General uses of OP:  General uses of OP:  8 10 5 Used broadly (landscap‐ Used in open fields  GG General ene ene rara l luses   Uses uses of of of OP OP  OP :: :  GG ene ene rara l luses  uses of of OP  OP : :  Used broadly (landscap‐ Used in open fields  Used broadly (landscap‐ Used in open fields  General Uses of OP: Used Used broa  broa dly dly (l and (land scap scap‐ ‐ Used Used in in open  open fie fie lds lds    Used broadly ing/public health/agri‐ (cotton, soybean, veg‐ Used broadly (landscap‐ Used in open fields  Used broadly (landscap‐ Used in open fields  ing/p ing/p ubl ubl icic health/agri  health/agri‐ ‐ (cot Used (cot ton, ton, in open  soyb  soyb fields ean, ean, veg (cotton,  veg‐ ‐ ing/p (landscaping/public ublic health/agri‐ (cotton, soybean, veg‐ ing/public health/agri‐ (cotton, soybean, veg‐ culture) to control mos‐ etable) to control boll  ing/p ing/p uu blbl icic health/agri  health/agri‐‐ soybean, (cot (cot ton, ton, vegetable)  soyb  soyb ean, ean, to veg  veg contr ‐‐ ol culture) to control mos‐ etable) to control boll  culture) to control mos‐ etable) to control boll  health/agriculture) to control culture) culture) to to co co ntrol ntrol mos  mos‐ ‐ etab etab le)le) to to con  con trol trol boll  boll    quitos, fleas and ants.  boll weevils, weevils, et etc. c.  culture) to control mos‐ etable) to control boll  culture) to control mos‐ etable) to control boll  qu mosquitos, qu itoito s,s fl, ea flea fleas s sand  and and an an ants. tsts . .  weevils, weevils, et et c.c .  quitos, fleas and ants.  weevils, etc.  quitos, fleas and ants.  weevils, etc.  qu qu ito ito s,s fl, fl eaea s sand  and an an tsts . .  weevils, weevils, et et c.c .                              Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   C11H12NO4PS2  Molecu Molecu lar lar fo fo rmu rmu la:la:     Molecu Molecu larlar fo fo rmu rmu la:la:     CC 11H 11H 12NO 12NO 4PS 4PS 2 2  C10H14NO5PS  Molecular Formula: Molecular Formula: Molecular formula:   Molecular formula:   C11H12NO4PS2  C11H12NO4PS2  CC 10H 10H 14NO 14NO 5PS 5PS    General uses of OP:  CC 1111 HH 1212 NO NO 4PS 4PS 2 2  C10H14NO5PS  C C10H H14NO NO5PS PS  Gene C ra Hl uses NO PS of OP:  General uses of OP:  Gene 10ral 14 uses 5 of OP:  11 12 4 2 CC 1010 HH 1414 NO NO 5PS 5PS    General uses of OP:  General uses of OP:  General uses of OP:  General uses of OP:  Used broadly  General Uses of OP: General Uses of OP: GG ene ene rara l luses  uses of of OP  OP : :  GG ene ene rara l uses l uses of of OP OP :  :  Used Used broa  broa dly dly    No longer used (banned  General uses of OP:  General uses of OP:  Used broadly  Used broadly  No No lon  lon ger ger us us eded (b (b anned anned    No longer used (banned (plan Used ts/anim broadly als) to  Used Used broa  broa dly dly    No longer used (banned  No longer used (banned  (plants/animals) to  (plants/animals) to  largely worldwide) due  No No lon  lon ger ger us us eded (b (b anned anned    largely worldwide) due to its (plants/animals) (plan (plan tst/sanim /anim ato las)lcontr s) to to   ol largely worldwide) due  largely worldwide) due  control moths, mites,  (plants/animals) to  (plants/animals) to  larlar gel gel y ywor  wor ldlw dw ide) ide) due  due    control control mot  mot hs, hs, mit  mit es,es,    to its high toxicity.  high toxicity. moths, mites, flies and aphids. largely worldwide) due  largely worldwide) due  control moths, mites,  control moths, mites,  toto it sit shigh  high tox  tox iciici ty.ty.    flies and aphids.  control control mot  mot hh s,s, mit  mit es, es,    to its high toxicity.  to its high toxicity.  flies and aphids.  flies and aphids.  toto it it s shigh  high tox  tox iciici ty. ty.    flifli eses and  and ap ap hihds. ids.    flies and aphids.  flies and aphids.                              Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   Molecular formula:   C10H9Cl4O4P  Molecular Formula: Molecu Molecu lar lar fo fo rmu rmu la:la:     CC 10H 10H 9Cl 9Cl 4O 4O 4P4 P  C10H9Cl4O4P  C10H9Cl4O4P  C H Cl O P General uses of OP:  10 9 4 4 CC 1010 HH 9Cl 9Cl 4O 4O 4P 4P    General uses of OP:  General uses of OP:  General Uses of OP: General uses of OP:  General uses of OP:  Used on animals (cattle,    GG ene ene rara l luses  uses of of OP  OP : :  Used Used on on an an imim als als (c (c atattle, tle,        Used on animals (cattle, hogs, Used on animals (cattle,    Used on animals (cattle,    hogs, goats, chickens,  Used Used on on an an im im als als (c (c atattlte,le,        goats, hogs, chickens,  goatsand , chihorses) ckens, to hogs, goats, chickens,  hogs, goats, chickens,  hogs, goats, chickens,  and horses) to control  control flies and mites. hogs, hogs, goats  goats , ch , ch icikceknen s,s ,  and and horses)  horses) to to contro  contro l l  and horses) to control  and horses) to control  flies and mites.  and and horses)  horses) to to contro  contro l l  flies and mites.  flies and mites.  flies and mites.  flies and mites.  flifli eses and  and mit  mit es. es.            3. OPs—Mode of Action In Vivo  3. OPs—Mode of Action In Vivo 3.3. OPs—M  OPs—M odoed eof of Ac Ac tion tion In In Vivo  Vivo    3. OPs—Mode of Action In Vivo  3. OPs—Mode of Action In Vivo  The primary neurotoxic action of organophosphate (OP) insecticide exposure is the  3.3. OPs—M  OPs—M od od e eof of Ac Ac tion tion In In Vivo  Vivo    The primary neurotoxic action of organophosphate (OP) insecticide exposure is the The primary neurotoxic action of organophosphate (OP) insecticide exposure is the  The primary neurotoxic action of organophosphate (OP) insecticide exposure is the  The primary neurotoxic action of organophosphate (OP) insecticide exposure is the  The primary neurotoxic action of organophosphate (OP) insecticide exposure is the  irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons  irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons The The prima  prima ryr yneurotoxic  neurotoxic ac ac tion tion of of organ  organ ophosphate ophosphate (OP)  (OP) in in sect sect iciici dd e eexposure  exposure is is the  the    irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons  irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons  irrever irrever sible sible inhibition  inhibition of of acety  acety lcholinesterase lcholinesterase (AChE  (AChE ) in ) in the the syn  syn aptic aptic ju ju ncntciotino nof of neurons  neurons    (Figure 1), leading to the hyperstimulation of post‐synaptic cells [7,20]. At a biochemical  (Figure 1), leading to the hyperstimulation of post-synaptic cells [7,20]. At a biochemical irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons  irreversible inhibition of acetylcholinesterase (AChE) in the synaptic junction of neurons  (Figure 1), leading to the hyperstimulation of post‐synaptic cells [7,20]. At a biochemical  (Figure 1), leading to the hyperstimulation of post‐synaptic cells [7,20]. At a biochemical  (Fi(Fi gure gure 1) 1) , le , le adaing ding to to the the hypersti  hypersti mula mula tion tion of of post  post‐syna ‐syna ptipti c cce ce llslls [7, [7, 202]0. ]At . At a  abiochemica  biochemica l  l  level,  OPs  bind  to  the  hydroxyl  group  of  AChE  through  phosphorylation,  preventing  level, OPs bind to the hydroxyl group of AChE through phosphorylation, preventing (Fi (Fi gure gure 1) 1) , le , le adad ing ing to to the  the hypersti  hypersti mula mula tion tion of of post  post‐syna ‐syna pti pti c cce ce llslls [7, [7, 202]0.] At . At a  abiochemica  biochemica l l  level level , ,OPs   OPs  bin   bin d dto  to  the   the  hydroxyl   hydroxyl  gro   gro up u pof  of  AChE   AChE  th  th rorugh ough  phosphoryla   phosphoryla tion, tion,  pr  pr eventing eventing    level,  OPs  bind  to  the  hydroxyl  group  of  AChE  through  phosphorylation,  preventing  level,  OPs  bind  to  the  hydroxyl  group  of  AChE  through  phosphorylation,  preventing  AChE from hydrolysing ACh [21]. In turn, the reduction of AChE activity results in an  AChE from hydrolysing ACh [21]. In turn, the reduction of AChE activity results in level level , ,OPs   OPs  bin   bin dd  to  to  the   the  hydroxyl   hydroxyl  gro   gro uu pp  of  of  AChE   AChE  th  th rorugh ough  phosphoryla   phosphoryla tion, tion,  pr  pr eventing eventing    AChE AChE fr fr om om hy hy drdorloylsyisnign gACh  ACh [ 21] [21] . In . In turn,  turn, the  the re re duc duc tion tion of of AChE  AChE act act iviivi tyty results  results in in an an    AChE from hydrolysing ACh [21]. In turn, the reduction of AChE activity results in an  AChE from hydrolysing ACh [21]. In turn, the reduction of AChE activity results in an  abnormal build‐up of ACh in the synaptic junctions, leading to hyperstimulation of the  an abnormal build-up of ACh in the synaptic junctions, leading to hyperstimulation of AChE AChE fr fr om om hy hy dd rorloylsyisnin g gACh  ACh [ 21] [21] . .In In turn,  turn, the  the re re duc duc tion tion of of AChE  AChE act  act ivi ivi tyty results  results in in an an    abab norm norm ala build l build‐up‐up of of ACh  ACh in in the  the syn  syn apatpictic ju ju ncnticotinos, ns, lea lea ding ding to to hyperstimulatio  hyperstimulatio n nof of the  the    abnormal build‐up of ACh in the synaptic junctions, leading to hyperstimulation of the  abnormal build‐up of ACh in the synaptic junctions, leading to hyperstimulation of the  musca thermuscarinic inic and nico and tini nicotinic c receptor receptors s involved involved  in cholin inergic choliner  patgic hwa pathways ys [22]. Ther [22efore, ]. Ther in efor ‐ e, abab norm norm ala lbuild  build‐up‐up of of ACh  ACh in in the  the syn  syn apap tictic ju ju nn cticti on on s,s, lea  lea dd ing ing to to hyperstimulatio  hyperstimulatio nn of of the  the    muscarinic and nicotinic receptors involved in cholinergic pathways [22]. Therefore, in‐ muscarinic and nicotinic receptors involved in cholinergic pathways [22]. Therefore, in‐ musca musca rin rin icic and  and nico  nico tintin ici cre re cece ptpt oror s sinvo  invo lved lved in in cho  cho linergic linergic pat  pat hw hw ays ays [22 [22 ]. ]Ther . Ther efore, efore, in in‐ ‐ creased increased  OP expos OP exposur ure is li ekis ely likely  to suto bstant substantially ially impac impact t upon upon  neural neural  func function, tion, andand  the the riskrisk   muscarinic and nicotinic receptors involved in cholinergic pathways [22]. Therefore, in‐ muscarinic and nicotinic receptors involved in cholinergic pathways [22]. Therefore, in‐ creased OP exposure is likely to substantially impact upon neural function, and the risk  creased OP exposure is likely to substantially impact upon neural function, and the risk  creased creased OP OP expos  expos uruer eis is li kliekly ely to to su su bstant bstant iaia llyll yimpac  impac t upon t upon neural  neural func  func tion tion , an , an d dthe the ris ris k k  of OP-dependent toxicity can be quantitated through measuring AChE levels and activity of OP‐dependent toxicity can be quantitated through measuring AChE levels and activity  creased OP exposure is likely to substantially impact upon neural function, and the risk  creased OP exposure is likely to substantially impact upon neural function, and the risk  ofof OP OP‐depen ‐depen dent dent tox tox iciici tyty can  can be be q uqan uan tita tita ted ted th th rough rough measuring  measuring AChE  AChE leve  leve lsls an an d dac ac tiv tiivtyity    of OP‐dependent toxicity can be quantitated through measuring AChE levels and activity  of OP‐dependent toxicity can be quantitated through measuring AChE levels and activity  ofof OP  OP‐depen ‐depen dent dent tox  tox iciici tyty can  can be be q u qu anan tittaita ted ted th th rough rough measuring  measuring AChE  AChE leve  leve lsls an an dd ac ac titviivty ity            J. Dev. Biol. 2022, 10, 49 4 of 22 J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  4  of  21  in blood [23] and pesticide metabolites, such as dialkyl phosphate (DAP) compounds in in blood [23] and pesticide metabolites, such as dialkyl phosphate (DAP) compounds in  urine [24,25] and plasma [26]. urine [24,25] and plasma [26].  Figure 1. The physiological action of acetylcholine (ACh; purple triangles) at the neuronal cell syn‐ Figure 1. The physiological action of acetylcholine (ACh; purple triangles) at the neuronal cell apse, the breakdown of ACh through acetylcholinesterase (AChE; orange diamonds), and the phos‐ synapse, the breakdown of ACh through acetylcholinesterase (AChE; orange diamonds), and the phorylation of AChE through organophosphate insecticide (OP; red hexagons) exposure.  phosphorylation of AChE through organophosphate insecticide (OP; red hexagons) exposure. AChE is integral for regulating neurotransmission [27], achieved through the hydro‐ AChE is integral for regulating neurotransmission [27], achieved through the hy- lysing of acetylcholine (ACh) into its two structural products, acetyl CoA and choline, the  drolysing of acetylcholine (ACh) into its two structural products, acetyl CoA and choline, latter of which is returned to the pre‐synaptic neuron via the sodium (Na ) choline trans‐ the latter of which is returned to the pre-synaptic neuron via the sodium (Na ) choline porter, enabling regeneration of ACh in the pre‐synaptic neuron. This process occurs in  transporter, enabling regeneration of ACh in the pre-synaptic neuron. This process occurs the central nervous system (CNS) within the brainstem, striatum and basal forebrain [28],  in the central nervous system (CNS) within the brainstem, striatum and basal forebrain [28], as well as in the peripheral nervous system (PNS) at both the neuroglandular and neuro‐ as well as in the peripheral nervous system (PNS) at both the neuroglandular and neuro- muscular junctions [29]. ACh from pre‐synaptic neurons plays important roles in initiat‐ muscular junctions [29]. ACh from pre-synaptic neurons plays important roles in initiating ing skeletal, smooth and cardiac muscle contraction, and more recently have been impli‐ skeletal, smooth and cardiac muscle contraction, and more recently have been implicated cated in the development and maturation of the brain [30–32].  in the development and maturation of the brain [30–32]. As a result of acute and chronic exposure, OPs can cause extensive damage to cells  As a result of acute and chronic exposure, OPs can cause extensive damage to cells including  cytotoxicity,  apoptosis  [33,34],  genotoxicity  and  subsequent  DNA  mutations  including cytotoxicity, apoptosis [33,34], genotoxicity and subsequent DNA mutations [35]. [35]. In previous studies, pesticides have been shown to produce covalent adducts with  In previous studies, pesticides have been shown to produce covalent adducts with DNA, DNA, and as a result form interstrand cross‐links which inhibit cellular replication and  and as a result form interstrand cross-links which inhibit cellular replication and tran- transcription [36]. Given that DNA adducts are destructive compounds that cause cellular  scription [36]. Given that DNA adducts are destructive compounds that cause cellular damage, they are nonetheless useful biomarkers for identifying oxidative stress and gen‐ damage, they are nonetheless useful biomarkers for identifying oxidative stress and geno- otoxicity [37].  toxicity [37]. Other Other cellular cellular irr irre egularities gularities iden identified tified inc include lude inc incrrea eases ses in in cytokine cytokine secre secretion tion (p (partic- artic‐ ul ularly arly tumour tumour necrosis necrosis fac factor tor  and and interleukin interleukin  6) 6) thr thro ough ugh neur neuroin oinflammation, flammation, improper improper  clearance clearance of of rre eactive activeoxygen  oxygen species,  species, and  and pathologically-induced  pathologically‐induced alterations  alteration ins gene in gene expr ex es-‐ pression sion [38– 40 [38– ]. 40].  4. OPs—Occupational, Household and Waterway Exposure 4. OPs—Occupational, Household and Waterway Exposure  Although OP insecticides are highly effective, it is estimated that only 0.1% of these Although OP insecticides are highly effective, it is estimated that only 0.1% of these  pesticides reach their target organisms [41,42], with the majority being lost in soil, food pesticides reach their target organisms [41,42], with the majority being lost in soil, food  and drainage [22,43,44]; in fact, OPs are the most common synthetic material found in and drainage [22,43,44]; in fact, OPs are the most common synthetic material found in  waterways, soil and animal tissues [5]. OP exposure is generally considered to be most prob- waterways, soil and animal tissues [5]. OP exposure is generally considered to be most    J. J. Dev Dev.. Biol. Biol. 2022 2022,, 10 10,, 49 x FOR PEER REVIEW  5 5of of  21 22  lematic in agricultural environments, and indeed occupational settings, such as farms and problematic in agricultural environments, and indeed occupational settings, such as farms  factories, contribute a significant avenue of OP exposure. However, OP exposure frequently and factories, contribute a significant avenue of OP exposure. However, OP exposure fre‐ occurs also in the household environment through food contamination and oral/epidermal quently  occurs  also  in  the  household  environment  through  food  contamination  and  exposure [27]. Although less common than occupational exposure, residential OP exposure oral/epidermal exposure [27]. Although less common than occupational exposure, resi‐ can occur from the use of these insecticides in the household or garden (Figure 2), and dential OP exposure can occur from the use of these insecticides in the household or gar‐ usually occur from improper storage and spills [45]. Additionally, the infiltration of OPs in den (Figure 2), and usually occur from improper storage and spills [45]. Additionally, the  the diet, where OPs are consistently detected in foods at low levels [46], is considered to be infiltration of OPs in the diet, where OPs are consistently detected in foods at low levels  a significant route of exposure and subsequent poisonings [47]. Children are particularly [46], is considered to be a significant route of exposure and subsequent poisonings [47].  vulnerable to OP exposure through diet due to the fact that they eat 2.8–4.8 times more Children are particularly vulnerable to OP exposure through diet due to the fact that they  food per unit of body mass, and the types of food that they eat (fruits and vegetables) eat 2.8–4.8 times more food per unit of body mass, and the types of food that they eat  contain higher levels of OP residues [47,48]. Although techniques do exist for monitoring (fruits and vegetables) contain higher levels of OP residues [47,48]. Although techniques  environmental OP exposure (surrogate skin, fluorescent tracers, air sampling pumps, etc.), do exist for monitoring environmental OP exposure (surrogate skin, fluorescent tracers,  most OPs are only detected once they have entered the body [49]. Therefore, greater un- air sampling pumps, etc.), most OPs are only detected once they have entered the body  derstanding of the lifelong effects of OP exposure are necessary in order to better govern [49]. Therefore, greater understanding of the lifelong effects of OP exposure are necessary  their use and mandate appropriate safety measures for use where the likelihood of human in order to better govern their use and mandate appropriate safety measures for use where  ingestion is high. the likelihood of human ingestion is high.  Figure 2. The primary direct and indirect routes of organophosphate (OP) exposure on target and  Figure 2. The primary direct and indirect routes of organophosphate (OP) exposure on target and non‐target organisms in agricultural, household and aquatic environments.  non-target organisms in agricultural, household and aquatic environments. Waterway exposure can occur unintentionally via agricultural surface run‐off, creat‐ Waterway exposure can occur unintentionally via agricultural surface run-off, creating ing a significant risk not only directly to aquatic ecosystems, but indirectly to humans as  a significant risk not only directly to aquatic ecosystems, but indirectly to humans as well  [50].  OPs  can also  be  intentionally  released  into  waterways,  a  phenomenon  com‐ well [50]. OPs can also be intentionally released into waterways, a phenomenon commonly monly observed in commercial fish farms and fishing sports that use these substances to  observed in commercial fish farms and fishing sports that use these substances to eliminate eliminate water‐borne pests [51] such as flat worm parasites [52]. Assessing chronic OP  water-borne pests [51] such as flat worm parasites [52]. Assessing chronic OP exposure exposure in waterways is challenging owing to high OP solubility, relatively short half‐ in waterways is challenging owing to high OP solubility, relatively short half-lives, and lives, and relatively low bioaccumulation [53]; however, data suggest that OP contamina‐ relatively low bioaccumulation [53]; however, data suggest that OP contamination is of tion is of global concern. With 95% of urban streams in the US showing detectable levels  global concern. With 95% of urban streams in the US showing detectable levels of OP of OP contamination [54], one can assume that aquatic species are consistently exposed to  contamination [54], one can assume that aquatic species are consistently exposed to OP OP insecticides for prolonged periods.  insecticides for prolonged periods.      J. Dev. Biol. 2022, 10, 49 6 of 22 5. Major Findings: The Zebrafish as Model for Testing Organophosphate (OP) Insecticides The use of mammalian models such as mice for screening experiments is both expen- sive and, owing to the relatively lengthy gestation periods, time consuming. Therefore, alternate animal models of environmental susceptibility, which are low cost and high- throughput, are desirable for toxin-screening, as these would provide rapid functional data that could narrow down OPs of interest and allow for subsequent targeted testing in mammalian models. One such model is the zebrafish (Danio rerio), which has become a favoured model for developmental research [55]. The zebrafish is an excellent model of human diseases, as the zebrafish and human genome share more than ~80% similarity [56]. This well-established genetic conservation of the zebrafish is one of the reasons why it is supported as a model for environmental toxicology studies, specifically in relation to vertebrate embryogenesis [57]. The rapid rate in which the structures of the zebrafish develop, coupled with optical clarity and ease of access, makes it a model of choice for observing embryo development. The toxic effects of OP insecticides at early developmental stages of zebrafish embryo- genesis have been investigated in multiple studies, which for the first time we have collated here (Table 2). From an embryological toxicity standpoint, a substantial number of these studies employed an acute exposure period from 0–5 h post-fertilization (hpf), allowing early phenotypes to be investigated. These timepoints coincide with the initial critical stages of cellular proliferation, migration (epiboly) and the onset of gastrulation, processes essential for the establishment of the three germinal layers and subsequent patterning of tissue primordia. The consequences of OP exposure in these acute early-stage studies are diverse; however, various morphological (spinal, yolk sac, body length, pigment and eye surface area), physiological (heart rate, AChE levels, genetic), and behavioural (locomotor activity, anxiety) impairments are commonly identified. Exposure to OPs at zebrafish adult stages led to predominately behavioural (anxiety, startle response) and physiological (ATP, AChE, GSH, MDA, etc.) irregularities, with fewer concomitant morphological impairments, highly consistent with lifelong morbidity as a consequence of acute exposure primarily in the early stages of development. While symptoms and defects present in zebrafish models do not always accurately predict human disease, the broad effects of OP on development appear consistent across both fish and humans. The neurological effects of OP in development appear largely consistent across species and correlate with the known mechanism of action of OP on the cholinergic system. Further, the >80% commonality in genome between zebrafish and humans indicates that zebrafish studies are valuable for identifying the molecular changes that may be common to harmful OP exposure in humans and zebrafish. To this end, we believe our summary table will serve as an invaluable resource for future continued implementation of the zebrafish in determining consequences for human OP-dependent disease, as indicated in the sections below. J. Dev. Biol. 2022, 10, 49 7 of 22 Table 2. Studies utilising zebrafish (Danio rerio) to determine developmental and neurotoxic effects of organophosphate insecticides. dpf; days post-fertilisation. hpf; hours post-fertilisation. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference - increased impaired spatial discrimination - both decreased (10 ng/mL) and increased (100 ng/mL) response latency in adult Chlorpyrifos (CPF) CPF: 10 & 100 ng/mL - 0–5 dpf [58] zebrafish - decreased swimming activity Chlorpyrifos (CPF) CPF: 100 ng/mL - 0–5 dpf [59] - increased mortality - decreased hatching rates - decreased body length Malathion (MAL) MAL: 2.5 & 3 mg/L - 3 hpf–5 dpf [60] - and decreased surface area of eye. - decreased Adenosine Di-Phosphate (ADP) and Adenosine Tri-Phosphate (ATP) Malathion (MAL) MAL: 0.25, 0.5, 1, 3 & 5 mM - Adult (sexually mature) [61] levels - increased heart rate - increased mortality - increased morphological irregularities (axial and tail deformities, yolk sac/heart Diazinon (DZN) DZN: 2000 & 3000 g/L - 8 hpf–96 hpf [62] oedema, eye irregularities - reduced pigmentation - decreased hatching rate - Acute—increased (0.25 mg/L) and decreased (0.75 mg/L) locomotor activity. Acute: 5 dpf for 2 h Chlorpyrifos (CPF) CPF: 0.25, 0.5, 0.75 & 1 mg/L - [63] - Sub-chronic—increased behavioural irregularities Sub-chronic: 1 hpf–11dpf - increased mortality - increased phenotypes (axial curvature, reduced body size and reduced Rohon-Beard Develop- pigmentation) ment/Axonogenesis: 3 hpf–27 hpf/51 hpf/72 - decreased functioning AChE Chlorpyrifos (CPF) CPF: 300, 1500 & 3000 nM [64] agrin#, cntn2#, ntf3#, hpf/4 dpf - increased average chevron angle (somites) sema3d# - decreased HNK-1-positive cells - decreased axonogenesis-related genes J. Dev. Biol. 2022, 10, 49 8 of 22 Table 2. Cont. Gene(s) Organophosphate(s) Dosage(s) * Exposure Period Observations Reference Involved ** - significantly increased startle response - increased transmitter turnover in larvae Chlorpyrifos (CPF) CPF: 0.29 M - 0–5 dpf [65] - decreased dopamine/serotonin levels in adults Chlorpyrifos-oxon - Defective peripheral neuron development CPF: 300 nM 0.1 g/L, 3 g/L 3 hpf–75 hpf [66] (CPF metabolite) - decreased hatching rates - increased pericardial oedema Dichlorvos (DCV) DCV: 20.81, 25 & 66.78 mg/L - 0 hpf–96 hpf [67] - increased spinal irregularities - decreased swimming activity - DCV: Dichlorvos (DCV) DCV: N/A - low toxicity (determined by LC ) PHO: 0.469, 0.513, 0.700 & 1.28 - Adult (sexually mature) [4] - PHO: Phoxim (PHO) mg/L - intermediate and high levels of toxicity (determined by LC ) - decreased swim rates - increased freeze response Chlorpyrifos (CPF) CPF: 0.6 M - 1 ypf for 24 h [68] - decreased AChE in muscle - decreased functioning AChE - increased TCPy (trichloro-2-pyridinol) Chlorpyrifos (CPF) CPF: 0.01, 0.1 & 1 M - 6 hpf–24/48/72 hpf [69] - decreased functioning primary/secondary motor neurons, axonal growth and sensory neurons. - Both CPF and DZN: - increased mortality Chlorpyrifos (CPF) - decreased functioning AChE CPF: 0.3, 3 & 30 M - decreased locomotor activity Diazinon (DZN) DZN: 10 & 30 M - 6 hpf–5 dpf [70] - PA: PA: 10 & 30 M - increased mortality Parathion (PA) - decreased functioning AChE J. Dev. Biol. 2022, 10, 49 9 of 22 Table 2. Cont. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference - decreased swim speed - decreased anxiety-like behaviour Chlorpyrifos (CPF) CPF: 0.01 & 0.1 M - 0–7 dpf [71] - increased behavioural irregularities - shortened body lengths and tail defects - increased proportion of females Sexual Differentiation: cyp19a1a", Monocrotophos (MCP) MCP: 0.001 & 0.100 mg/L 72 hpfV–16 dpf [72] - alteration in expression of sexual differentiation genes cyp19a1b", foxl2", dmrt1#, B-actin, ef1-a - CPF: - increased mortality - increased kyphosis - decreased spine length - increased spontaneous movement Chlorpyrifos (CPF) CPF: 1, 10, 100 & 1000 M - and decreased heart rate Dichlorvos (DCV) DCV: 100 & 1000 M - 1 hpf–5 dpf [73] - DCV: Diazinon (DZN) DZN: 100 & 1000 M - increased mortality - increased spontaneous movement. - DZN: - increased mortality - increased pericardial oedema - significantly decreased hatching rates - increased spine and yolk sac abnormalities Chlorpyrifos (CPF) CPF: 30, 100 & 300 g/L Gfap, Mbp#, Elavl3", Ngn1", Nestin", Shha" 0–5 dpf [50] - significantly decreased heart rates - significantly decreased swim speed/distance - decreased AChE activity - increased AChE gene expression Chlorpyrifos (CPF) CPF: 200 & 400 g/L - 2 hpfV–72 hpf [74] - increased glutathione (GSH) levels J. Dev. Biol. 2022, 10, 49 10 of 22 Table 2. Cont. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference - decreased cholinesterase (ChE) levels in the heart/brain - increased myo-degeneration Oxidative Stress: Nrf2 (many other - increased testis degeneration Dichlorvos (DCV) DCV: 6, 19, & 32 mg/L associated genes within the Nrf2 6–12 mpf [75] - increased pancreas zymogen granule depletion pathway also examined) - decreased glycogen in liver - altered expression of genes involved in Nrf2 signalling - moderate toxicity (determined by LC ) - decreased body length Monocrotophos (MCP) MCP: 10, 20, 30, 40, 50 & 60 mg/L - 4 hpf–96 hpf [1] - decreased heart rate - decreased functioning AChE levels - decreased whole-body cortisol HPI Axis: Crf, Gr#, POMC#, Adult (sexually - increased/decreased hypothalamic-pituitary-inter-renal (HPI) Monocrotophos (MCP) MCP: 100 g/L P450 #, 11B-HSD2, StAR, mature)— [76] 11b axis associated genes 20B-HSD2", MC2R#, TAT, PEPCK 21 d exposure - increased oxidative stress Adult (sexually - decreased neurotransmitter metabolism Chlorpyrifos (CPF) CPF: 2 & 5 M - [77] mature) - increased energy exhaustion Embryo (1 hpf), - CPF was determined to be more toxic than PHO (determined Chlorpyrifos (CPF) CPF: 0.28- 13.03 mg/L larvae (72 hpf) and - [78] byLC ) Phoxim (PHO) PHO: 0.89–26.48 mg/L juvenile (1 mpf)— 96 h exposure - Moderate toxicity (determined by LC ) Diazinon (DZN) DZN: 6.5 mg/L - 6 hpf–5 dpf [2] - increased levels of malondialdehyde (MDA) in liver/kidney - increased glutathione (GSH) in liver/kidney/brain Adult (sexually - increased superoxide dismutase in liver Dichlorvos (DCV) DCV: 15 mg/L - mature) 4–5 m— [79] - decreased levels of superoxide dismutase in brain 24 h exposure - decreased catalase in kidney/brain. J. Dev. Biol. 2022, 10, 49 11 of 22 Table 2. Cont. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference - low toxicity (determined by LC ) HPG Axis: vtg1, vtg2, era", erB1, - upregulation of gene expression within the Malathion (MAL) MAL: 250, 500 & 1000 g/L 6 dpf–10 dpf [80] erB2, cyp19a1a, cyp19a1b" hypothalamic-pituitary-gonadal (HPG) axis - CPF: - increased mortality - decreased hatching rates - increased spinal lordosis Chlorpyrifos (CPF) CPF: 1, 10, & 25 M - reduced activity - 6 hpf–102 hpf [18] Diazinon (DZN) DZN: 10 & 100 M - DZN: - increased mortality - increased pericardial oedema - decreased mitochondrial bioenergetics - DNA damage observed in peripheral blood Monocrotophos (MCP) MCP: 0.125, 0.625 & 1.25 uL/L 24–72 hpf [81] - decreased functioning AChE - decreased carboxylesterase (CaE) Phosalone (PSL) PSL: 86–505 g/L - 8 wpf–96 h exposure [82] - increased glutathione (GSH) Oxidative stress: Mn-Sod"/#, Cu/Zn-Sod#, Gpx#, Cat#, Ucp2#, - increased levels of gut mucus bc12, Cox1# Glycolysis/Lipid: Gk#, - decreased y-Protobacteria in gut Adult (sexually Chlorpyrifos (CPF) CPF: 30, 100 & 300 g/L HK1, Pk#, Pepckc#, Aco#, CPt1#, [56] - decreased oxidative stress genes in gut and liver mature) Ppar-A#, Acc1#, Srebp 1a#, Ppar-y#, - and decreased glycolysis and lipid metabolism-related genes Fas#, Fabp6, Apo#, Dgat#, LDLR#, HMGCR, Fabp5 Cardiovascular: Mef2c#, Bmp4#, - decreased lipid accumulation in heart VEGFR-2, JunB", Tbx2 - decreased triglyceride and total cholesterol Chlorpyrifos (CPF) CPF: 30, 100 & 300 g/L Lipid: Ppar-a, Ppar-y#, Srebp 1a, 2 hpf–7 dpf [83] - increased cellular apoptosis of heart tissue Acc1, Fas#, Cpt1#, Aco, Apo#, Fabp5, - decreased lipid metabolism genes Fabp6#, Dgat#, LDLR J. Dev. Biol. 2022, 10, 49 12 of 22 Table 2. Cont. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference - DZN: - decreased swimming distance - decreased velocity - increase in AChE associated gene expression inhibited functioning AChe - increased carboxylesterase activity - DCV: Diazinon (DZN) DZN: 0.1 & 100 g/L - increased AChE associated genes. Cholinergic: AChE"/# Dichlorvos (DCV) DCV: N/A - MAL: Neurodegeneration: 5 hpf–5 dpf [84] Malathion (MAL) MAL: 100 g/L - decreased swimming distance c-Fos, lingo-1b", grin-1b# Parathion (PA) PA: 0.1 g/L - decreased velocity - increase in AChE associated gene expression - increase in neurodegenerative associated gene expression - increased carboxylesterase activity - PA: - decrease in AChE associated gene expression - and decrease in neurodegenerative associated gene expression - decreased body length - decreased heart rates - decreased surface area of eye Dichlorvos (DCV) DCV: 1, 5 & 10 mg/L - 1 hpf–7 dpf [85] - decreased escape responses - decreased speed - decreased mobile time - increased blood glucose levels Adult (sexually - increased frequency of micronucleus in erythrocytes Sumithion (SMT) SMT: 1 mg/L - [86] mature)—96h exposure - increased erythrocyte cellular and nuclear abnormalities - increased anxiety related activity (Novel Tank Diving Test) Adult (sexually mature) - increased approach response in shoaling assay Chlorpyrifos (CPF) CPF: 1 & 3 M - [87,88] 6–8 m—2/5 w exposure - increased predator avoidance activity (predator avoidance assay) J. Dev. Biol. 2022, 10, 49 13 of 22 Table 2. Cont. Organophosphate(s) Dosage(s) * Gene(s) Involved ** Exposure Period Observations Reference Oxidative Stress: Cat, CuSod, - Both CPF and MAL: MnSod CPF: 0.019, 0.077, 0.31, 0.41, 1.01, - severe toxicity at larvae, juvenile and adult stages Chlorpyrifos (CPF) Immunity: Cxcl#, IL", Tnf"/# 1.53 & 6.15 mg/L (compared to embryo stage) Malathion (MAL) Apoptosis: Cas8"/#, Cas9, 1 hpf–96 hpf [57] MAL: 0.039, 0.16, 0.62, 2.90, 8.04, - significant changes in expression of immunity, apoptosis, P53, Bax 8.54 & 12.45 mg/L and endocrine related genes Endocrine: TRa, TRb#, ERa, Tsh#, Crh, cyp19a" - increased mortality in embryos and larvae - decreased hatching rates - increased morphological irregularities in embryos (damaged/underdeveloped and darkened yolk sac, broken chorion, and aberrant notochord formation) Sumithion (SMT) SMT: 0.1, 0.2, 0.4, 0.8 & 1.6 mg/L - Embryo/larvae [89] - increased morphological irregularities in larvae (yolk sac ulcerations/swelling and oedema, heart damage, lesion at caudal region, uninflated swim bladder, head malformation, jaw irregularities, and notochord abnormalities). - decreased brain cholinesterase (ChE) activity Adult (sexually Chlorpyrifos (CPF) CPF: 1 M - (Hawkey, 2021) - increased fleeing score. mature)—5 w exposure - Changes in Mitochondrial oxygen utilization in the brain Diazinon (DZN) DZN: 0.4, 1.25 & 4.0 M 5–120 hpf [90] and testes - increases in reactive oxygen species Malathion (MAL) MAL: 5, 50 ug/L 0–14 dpf [91] - induction of oxidative stress Chlorpyrifos (CPF) CPF: 0.1 & 3 ug/L - Significantly elevated ROS levels Adult—8–12 months old - Elevated Reactive nitrogen species levels in high CPF Chlorpyrifos (CPF) Caspase 3#, Bcl-2#, [92] 14 day exposure dosage groups * All dosages listed are associated with the observations summarised here, other dosages in the individual studies may have been used, but did not impact on development, behaviour or gene expression. **" and# arrows indicate where there has been a significant increase or decrease in a particular gene as a response of organophosphate (OP) exposure—where no arrow shows, no significant change was noted. J. Dev. Biol. 2022, 10, 49 14 of 22 6. Organophosphate Toxicity—Acute Cholinergic Syndrome (ACS) Although having conserved function as a neurotoxin, the phenotypic consequences of OP exposure are nonetheless extremely variable, and symptoms may manifest following either acute (high dose) or chronic (typically lower dose) exposure (Figure 3). The earliest stage of OP toxicity is referred to as acute cholinergic syndrome (ACS), which is a result of the effects of AChE inhibition [93]. ACS can occur within minutes of OP exposure, and impairs both muscarinic and nicotinic receptors found in the nervous system [94]. The consequences of hyper-stimulated post-synaptic receptors (hyperstimulation) vary depending on their locations. In the CNS, hyperstimulation more commonly occurs at muscarinic receptors, resulting in heart irregularities, gastrointestinal issues including stomach cramps, diarrhoea and vomiting, respiratory complications including bronchor- rhea and bronchospasms, as well as neurological effects including seizures, agitations and anxiety [95,96]. Comparatively, hyperstimulation in the PNS is more commonly associated with nicotinic receptors, and this is often expressed as muscle weakness, cramps or paral- J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  14  of  21  ysis [7,97]. Symptoms of OP exposure are quite complex in that they are not limited to a localised area and are a result of the diverse effect of OPs on both CNS and PNS pathways. Figure 3. The major consequences of OP exposure in humans.  Figure 3. The major consequences of OP exposure in humans. 7. 7. Organop Organophosphate hosphate T Toxicity—Intermediate oxicity—Intermediate Syndrome Syndrome (IMS) (IMS)  The The inte intermediate rmediate sy syndr ndrome ome (IMS (IMS) ) fofollows llows 1–4 1–4  days days  afteafter r ACS ACS  (Figure (Figur  4), eand 4), is and  char is‐ characterised acterised as the as the onset onset  of muscle of muscle  we w ak eakness, ness, papart rticula icularly rly inin the the proximal proximal limbs limbs,, neck neck and and  rrespir espiratory atory system system [[98 98]. ]. If If untr untreated eated,, fr from om 14–21 14–21 days days after after acute acute exposur exposure, e, weakness weakness in in  the the peripheral peripheral muscles muscles becomes becomes evident evident [[4 499] ].. It It is is estimate estimated d that that only only ~20% ~20% of of humans humans  exposed to OP will have symptoms that progress from ACS to the IMS stage [99]. The IMS exposed to OP will have symptoms that progress from ACS to the IMS stage [99]. The IMS  is commonly associated with respiratory failure as a result of nicotinic receptor paralysis. is commonly associated with respiratory failure as a result of nicotinic receptor paralysis.  Respiratory failure from prolonged OP exposure is primarily linked to the CNS, specifically Respiratory failure from prolonged OP exposure is primarily linked to the CNS, specifi‐ the depression of the pre-Botzinger complex (glutaminergic and muscarinic fibres) located cally the depression of the pre‐Botzinger complex (glutaminergic and muscarinic fibres)  in the ventrolateral medulla in the brainstem [100]. This has resulted in respiratory failure located in the ventrolateral medulla in the brainstem [100]. This has resulted in respiratory  being recognised as a significant comorbidity in OP mortality [94]. failure being recognised as a significant comorbidity in OP mortality [94].  Figure 4. The symptoms of organophosphate (OP) exposure at progressive time points; acute cho‐ linergic syndrome (ACS) occurs within minutes of OP exposure, symptoms of intermediate syn‐ drome (IMS) display at 1–4 days after OP exposure, and symptoms of organophosphate‐induced  delayed neuropathy (OPIDN) occur ~2–3 weeks after OP exposure.    J. Dev. Biol. 2022, 10, x FOR PEER REVIEW  14  of  21  Figure 3. The major consequences of OP exposure in humans.  7. Organophosphate Toxicity—Intermediate Syndrome (IMS)  The intermediate syndrome (IMS) follows 1–4 days after ACS (Figure 4), and is char‐ acterised as the onset of muscle weakness, particularly in the proximal limbs, neck and  respiratory system [98]. If untreated, from 14–21 days after acute exposure, weakness in  the peripheral muscles becomes evident [49]. It is estimated that only ~20% of humans  exposed to OP will have symptoms that progress from ACS to the IMS stage [99]. The IMS  is commonly associated with respiratory failure as a result of nicotinic receptor paralysis.  Respiratory failure from prolonged OP exposure is primarily linked to the CNS, specifi‐ cally the depression of the pre‐Botzinger complex (glutaminergic and muscarinic fibres)  J. Dev. Biol. 2022, 10, 49 15 of 22 located in the ventrolateral medulla in the brainstem [100]. This has resulted in respiratory  failure being recognised as a significant comorbidity in OP mortality [94].  Figure 4. The symptoms of organophosphate (OP) exposure at progressive time points; acute cho‐ Figure 4. The symptoms of organophosphate (OP) exposure at progressive time points; acute linergic syndrome (ACS) occurs within minutes of OP exposure, symptoms of intermediate syn‐ cholinergic syndrome (ACS) occurs within minutes of OP exposure, symptoms of intermediate drome (IMS) display at 1–4 days after OP exposure, and symptoms of organophosphate‐induced  syndrome (IMS) display at 1–4 days after OP exposure, and symptoms of organophosphate-induced delayed neuropathy (OPIDN) occur ~2–3 weeks after OP exposure.  delayed neuropathy (OPIDN) occur ~2–3 weeks after OP exposure. 8. Organophosphate Toxicity—Organophosphate-Induced Delayed Neuropathy (OPIDN) OP toxicity does not solely result in AChE inhibition, and depending on the physical structure of the compound, OPs can target other secondary hydroxyl sites on enzymes other than AChE [101]. OP insecticide exposure can also give rise to another type of toxicity, referred to as organophosphate-induced delayed neuropathy (OPIDN), which, depending on dose and chemical structure, occurs ~2–3 weeks after ACS [94,102]. This pathology is characterised as the degeneration of distal axons in the CNS and PNS, is expressed as sensory loss in both hands and feet, weakness in distal muscles and coordination issues [7] and is associated with the inhibition of neuropathy-target-esterase (NTE) [103]. NTE is an integral enzyme employed at the neurite initiation stage of neuronal morphogenesis, with these neurites maturing into axons and dendrites to form part of the nervous system [104]. Importantly, the consequences of OPIDN are only present when 70% of NTE is inhibited [105]. Additionally, in order for irreversible inhibition of NTE, there must be a secondary chemical reaction, where there is a displacement of an R-group (aging) [103], and, therefore, NTE inhibition and OPIDN is thought to contribute to diseases characterised by axonal degradation such as Alzheimer ’s disease, Parkinson’s disease and motor neuron diseases (MND) that include amyotrophic lateral sclerosis (ALS) and progressive bulbar palsy [106]. As these diseases are primarily associated with aging in humans, it is interesting to note that animal models show that adults are both far more susceptible to, and recover far more poorly from, OPIDN than juveniles [106]. The inhibition of NTE itself is not responsible for axonal degeneration, as has been demonstrated with non-OP inhibitors (organophosphinates, sulfonyl fluorides and carba- mates) that covalently react with NTE, but do not undergo the enzyme ageing process [107]; this indicates that R-group displacement confers a “gain” of neurotoxicity that is damaging in its own right. 9. Organophosphate Toxicity—Effects on Embryogenesis Pre-natal OP exposure is of particular concern, as developing babies are highly sus- ceptible to chemical injury [108,109]. This is partly a result of their immature detoxification mechanisms, i.e., reduced expression of OP specific detoxifying enzymes such as paraox- J. Dev. Biol. 2022, 10, 49 16 of 22 onase and chlorpyrifos-oxonase, compared to adults [110,111], and also as the cholinergic system (which is targeted by OPs) is heavily involved with placental processes including amino acid uptake and nitric oxide signalling [112]. Pre-natal OP exposure has been associ- ated with shortened gestational periods [113], reduced birth weight and birth length [114], as well as impaired reflexes [115] and neurobehavioral irregularities [116]. While the influence of OPs on the mature blood brain barrier (BBB) is unclear, the developing and newborn BBB is “leaky”, allowing toxins, in particularly pesticides in the fetal circulation, to cross and have negative effects on the developing brain [117,118]. Over- all, the neurotoxic properties of OPs, and the resultant syndromes have been reasonably well documented (Figure 4), with AChE inhibition and hyperstimulation of postsynaptic neurons being key contributors to these impairments [119]. 10. Organophosphate Toxicity—Effects on Neurodevelopment and Early Behaviour The timing of prenatal OP exposure plays a critical role in fetal development and postnatal behaviour. OP exposure during the 1st and 2nd trimester of pregnancy has been shown to be associated with delayed cognitive performance at both 2 and 6 months of age, whereas OP exposure in the 3rd trimester of pregnancy is associated with delayed communication and motor performance at 6 months of age [120]. In terms of neurodevelopmental impairments, OPs such as chlorpyrifos and diazinon have been shown to cause decreased DNA synthesis in neuronotypic PC12 and gliotypic C6 neural cell lines, the latter of which continues to develop into the postnatal period [121]. Additionally, children aged ~3 years have been identified as having increased risk of displaying developmental delays and a higher incidence of behavioural disorders such as ADHD [108,122], and prenatal exposure to OP insecticides was associated with poorer intellectual development in seven year old children [123], as well as poorer motor skills and cognitive recall when compared to non-exposed children [124]. Taken together, these studies show that not only does pre-natal exposure to OPs affect embryogenesis, but also cognitive development in young children. 11. Organophosphate Toxicity—Effects in Adulthood and Neurodegenerative Diseases Whilst infants and children are highly susceptible to OP insecticide toxicity, these chemicals have also been associated with impaired health at later stages of life, particu- larly neurological disorders such as Alzheimer ’s Disease (AD) and Parkinson’s Disease (PD) [125]. Recent studies have shown that chronic exposure in agricultural workers is associated with neural irregularities including neurodegenerative diseases, attention im- pairment and short-term memory loss [11]. The cholinergic system, which is affected by OP insecticides, has long been associated with neurodegenerative diseases, with ACh one of the key neurotransmitters involved in cellular signalling in the brain [125]. The reduction of ACh is a critical element in memory loss diseases such as AD, where ACh in the basal forebrain is known to play an integral role in memory and learning; as OP insecticides promote an imbalance of ACh at cellular junctions in the brain, these chemicals are, therefore, linked to impaired memory diseases [126]. Although PD involves a depletion of dopaminergic cell bodies, it is symptomatically dissimilar to AD in that it is characterised by motor impairments such as tremor at early onset and posture/gait issues at later stages [127]. Along with genetic predisposition, pesticides are widely acknowledged as an environmental risk factor for PD, with OPs having been implicated in some studies of the disease [128–130]. Variability in the PON1 gene (when exposed to various OPs—diazinon/chlorpyrifos) has been shown to correlate with a greater than two-fold increase in PD risk [131]. Despite the causal relationship being still largely unknown, an epidemiological analysis of 23 case-control studies found that 13 of the studies reported a statistically significant risk of PD with pesticide exposure, with both chlorpyrifos (OP) and organochlorines (OC) being key contributors to the study [132]. However, one limitation of pesticide research on neurodegenerative diseases such as PD is J. Dev. Biol. 2022, 10, 49 17 of 22 that the study of pesticides does not encompass the lifespan, making it difficult to analyse the long term effects of these chemicals [133]. 12. Conclusions Rapid population growth and changing diets in developing countries have increased the demand for food, to the point that food production must increase by 70% to meet the estimated food demands in 2050 [134]. Pesticides will play an essential role in achieving this production increase. While the economic and societal benefits of pesticides are inarguable, the effects of pesticide exposure on non-target animals and humans are a continuing concern. Of note is the growing use of OP insecticides, which are linked to poor neurodevelopment in both developed and developing countries worldwide. While our understanding of the relationship between OP exposure and poor health outcomes is growing, it remains unclear the extent to which AChE inhibition and OP exposure lead to developmental abnormalities. However, addressing this relationship between OP exposure and neurodevelopment and behaviour will encourage improvements in the regulation of use and handling of OPs in agricultural, industrial and domestic environments around the world. Author Contributions: Conceptualization, J.N. and S.D.; writing—original draft preparation, J.N., J.N.F., C.v.d.P., J.E.C. and S.D., writing—review and editing, J.N., J.N.F., C.v.d.P., J.E.C. and S.D.; project administration, S.D., funding acquisition, S.D. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a La Trobe University Securing Food, Water and the Environ- ment, Research Focus Area Grant Ready Grant #3.2509.07.48. Institutional Review Board Statement: Not Applicable. Informed Consent Statement: Not Applicable. Data Availability Statement: Not Applicable. Conflicts of Interest: The authors declare no conflict of interest. References 1. Pamanji, R.; Bethu, M.S.; Yashwanth, B.; Leelavathi, S.; Venkateswara Rao, J. Developmental toxic effects of monocrotophos, an organophosphorous pesticide, on zebrafish (Danio rerio) embryos. Environ. Sci. Pollut Res. Int. 2015, 22, 7744–7753. [CrossRef] [PubMed] 2. 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Journal

Journal of Developmental BiologyMultidisciplinary Digital Publishing Institute

Published: Nov 21, 2022

Keywords: organophosphate; insecticides; zebrafish; neurodevelopment

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