Zebrafish Embryo Neonicotinoid Developmental Neurotoxicity in the FET Test and Behavioral Assays

The need for reliable, sensitive (developmental) neurotoxicity testing of chemicals has steadily increased. Given the limited capacities for routine testing according to accepted regulatory guidelines, there is potential risk to human health and the environment. Most toxicity studies are based on mammalian test systems, which have been questioned for low sensitivity, limited relevance for humans, and animal welfare considerations. This increased the need for alternative models, one of which is the zebrafish (Danio rerio) embryo. This study assessed selected neonicotinoids at sub-lethal concentrations for their effects on embryonic development and behavior. The fish embryo acute toxicity test (OECD TG 236) determined the lowest observable effect concentrations, which were used as the highest test concentrations in subsequent behavioral assays. In the FET test, no severe compound-induced sublethal effects were seen at <100 µM. In the coiling assay, exposure to ≥1.25 µM nicotine (positive control) affected both the burst duration and burst count per minute, whereas ≥50 µM thiacloprid affected the mean burst duration. Exposure to ≥50 µM acetamiprid and imidacloprid induced significant alterations in both mean burst duration and burst count per minute. In the swimming assay, 100 µM acetamiprid induced alterations in the frequency and extent of movements, whilst nicotine exposure only induced non-significant changes. All behavioral changes could be correlated to findings in mammalian studies. Given the quest for alternative test methods of (developmental) neurotoxicity, integration of zebrafish embryo behavior testing proved beneficial for future tiered testing scheme.


kyphosis (K), scoliosis (S) pericardial edema (PE) and craniofacial deformation (CD) in the FET test observed in zebrafish (Danio rerio) embryos between 72 and 120 hpf
In vitro mice: Adult 5-6 wks ♀ mice were mated and sacrificed to isolate the embryos at 2-cell stage.
Method 3: HH4 embryos exposed to 500 µM imidacloprid through injection into windowed egg in vivo and incubated for another 4.5 or 14 days.

Morphology (at < 66 hpf): body length
Behavior: escape and swimming on tactile stimulus of tail bud Nervous system development: immunocytochemistry, GFP visualization and motoneuron innervation assessment • Reduced overall growth from 42 hpf onwards • 33 µM induced muscular response, but lack of swimming at 42 hpf, remained paralyzed until 120 hpf • Partial recovery when exposed between 22 and 66 hpf and allowed to recover by 120 and 168 hpf • > 66 hpf only few GFP-expressing motoneurons in spinal cord, with increased expression in rescued embryos at 120 hpf • 15 and 33 µM reduced% ventral myotomes with GFP-expressing axons • Continued expression of zn5 indicated delay in normal downregulation program • 33 µM reduced% of innervated dorsal segments at 66 hpf, with recovery potential Svoboda et al., 2002 Wildtype (TL, AB and WIK) and TG (isl1:gfp, fli1:gfp, and nbt:maptgfp) zebrafish Reared at 28°C until 13 hpf, then at 25°C. Some embryos were dechorionated via enzymatic digestion exposed to 1-30 µM nicotine from 22 hpf, with daily renewal. For coiling response, control and dosed individuals were examined, as well as all groups after 3 min acute exposure to 5-30 µM, and after a recovery phase. Some embryos decapitated to determine tail movement alone.
Behavior: motor output (spinal musculature bend), and percentage of full movements (doublets)

Immunohistochemistry: [ 3 H]-nicotine uptake: measuring -(-)-[N-methyl-3 H] nicotine activity via liquid scintillation counting
Influx and efflux: radioisotopes • 33 µM exposure of non-dechorionated embryos induced paralysis by 66 hpf, with brief transient period of increased motor output • 5-30 µM produced increased muscle bends in dechorionated embryos at 27-28 hpf, but reduced percentage of doublets to almost zero, which recovered during washing • 30 µM caused 4-fold increase in musculature bend in dechorionated embryos at 22, 24, 25 and 26 hpf, but failed to induce the same response to 30 µM after a 2 h wash period, desensitizing the receptors • Tails from decapitated embryos exhibited increased musculature bends when placed in 5-30 µM nicotine • Exposure to high, followed by low concentration nicotine did not induce increased musculature bends after washing • Steady state of exposure for nicotine accumulation in embryos reached after 10 min, increasing with medium concentration, but always being less than Thomas et al., 2009 Model organism Methodology Endpoint(s) assessed Significant finding(s) Reference water concentration Wildtype (EkkWill) and TG (isl2b:gfp) zebrafish Reared at 28°C with 14/10h light/dark cycle, exposed to 3-300 µM nicotine. For some assays, embryos reared in 0.002-0.0045% PTU for 24 h. Embryos placed in 100 mm petri dished for microinjection with morpholino antisense oligonucleotides (MOs). RT-PCR performed at 24 and 48 hpf. In vivo: Continuous exposure of embryos to tobacco smoke (TS), aerosol (AE) extracts (generated from cigarettes and ecigarettes, containing 1 e-cigarette cartridge or 22 cigarettes) or nicotine until 72 hpf, with daily renewal. 14/10 h light/dark cycle, at 27.5°C.

In vivo:
Morphology at 72 hpf: heart malformation, heart rate Gene expression at 24 hpf

Model organism Methodology Endpoint(s) assessed Significant finding(s) Reference
In vitro: 1.7, 3.4, 6.8 and 13.7 µM nicotine from extracts from differentiation onset, and renewed daily.

In vitro:
Gene expression, flow cytometry, immunofluorescence, cell stress assay • 13.7 µM TS extract induced decreased hatching and pigmentation • TS and AE extracts induced heart defects, but only TS extracts reduced the heart rate • Only TS significantly affected gene expression (of cmlc2, tnnt2, nkx2.5, mef2ca, and cx43) In vitro: • TS extract > 6.8 µM altered beats per minute and cardiomyocyte maturity, whilst 13.7 µM reduced cardiomyocyte yield and purity • TS affected gene expression more significantly than EA extracts Virgin Sprague-Dawley rats Experiment 1: Pregnant ♀♀ dosed with 0.05 mg/mL nicotine as source of drinking water. Dosed for the last 14, 6 or 4 d of pregnancy.
Experiment 2, dosing continued post-delivery; both adult and fetal rats sacrificed at 21 or 22 PND. Experiment 1: fetal body weight was measured, and brain and liver lipid and nitrogen determination on pooled organs) Experiment 2: body weight Experiment 1: • Significant reduction in fetal body weight • Brain weight slightly increased, liver weight not affected • Significant differences between treated and control rats in liver lipid/tissue, lipid/nitrogen, and cholesterol/lipid ratios Experiment 2: • Nicotine during weaning led to rougher fur and reduced fetal mean body weight Mosier and Armstrong, 1964 Sprague-Dawley rats Dosing of ♀♀ via drinking water. High dose: 20 µg/mL until parturition, 10 µg/mL during weaning. Low dose: 20 µg/mL for 1 week, 40 µg/mL until parturition, 20 µg/mL during weaning. When dosed with highest concentration, mating proceeded, and litters were reduced to 8 pups. Litters from dosed ♀♀ either remained with original mother or were switched with control litter 1 d after delivery. All pups weaned and sacrificed on PND 20, 30 or 40.
Plasma LH analysis • Prepubertal ♀ and ♂ offspring exposed to low dose of nicotine during lactation showed significant variation in LH levels from control • ♀ offspring of rats dosed during pregnancy or lactation showed significantly reduced body weight Meyer and Carr, 1987 Sprague-Dawley rats Pregnant ♀♀ on gestation day 1 to implant subcutaneous minipump with 1.5 mg/kg/day saline or nicotine for 28 d. On PND 1, litter examined and saline-and nicotine-exposed pups cross-fostered to drug-free females. Maternal plasma levels of nicotine and cotinine (nicotine metabolite) determined after birth. Behavioral assessment with pups conducted on PND 5, 9, and 14. Striatal levels of neurotransmitter examined in 14 d pups.
Upon delivery: Number, viability, sex ratio, birth weight and body length Behavior: Position reflex, surface righting and negative geotaxis Biochemistry: DA and its metabolite 3,4dihydroxyphenylacetic acid (DOPAC).
• Effective nicotine administration shown by nicotine and cotinine in maternal blood • Number of pups of nicotine treated ♀♀ reduced, as well as affecting pup body weight and length Fung and Lau, 1989 Sheep and Sprague-Dawley rats Sheep: Pregnant ewes with ♂ fetuses fitted with catheters in fetal and maternal femoral veins on GD 130. After acclimatization, 10 or 25 µg/kg nicotine intravenously infused via the maternal vein in 5 min.
Rat: From GD 3 to delivery, treated subcutaneously with Sheep: Maternal and fetal heart rate and blood flow, Fetal blood analysis (pH, PO 2 , PCO2, lactic acid, hematocrit, Na + and K + ) Rat: Electrocardiogram in 4-5 mo ♂ rat offspring Sheep: • Fetal PO2 decreased and PCO2 increased with ewe dosing • Intravenous infusion of 10 and 25 µg/kg into ewes induced reduced heart rate within 15 min, followed by fetal heart rate Feng et al., 2010 Model organism Methodology Endpoint(s) assessed Significant finding(s) Reference either 0.3 mL saline solution or 1.5 mg/kg nicotine hydrogen tartrate twice daily. Pups born naturally and allowed to wean. ♂ offspring removed after weaning and examined at 4-5 months of age, assessing heart rate of control and exposure groups after acclimatization and after injection of 2 mg/kg nicotine. increase • Various types of fetal arrythmia only after maternal nicotine infusion Rat: • Heart rate of nicotine exposed rats higher during immobilization period • After nicotine injection, rats of un-dosed and dosed maternal rats showed decreased heart rate • Offspring of exposed rats showed less of a decrease • Nicotine injection increased arrythmia in exposed offspring more than in control offspring S-strain mice 5-15 d post mating, 0.1% aqueous solution nicotine injection (either subcutaneous or intraperitoneally) 1, 2 or 3 times (on consecutive days). Most were sacrificed at term, whilst some were sacrificed mid-pregnancy.
At term and mid-pregnancy observations: total litter, average litter, fetal death, congenital abnormalities • Dosing induced fetal death and complete resorption at different time points of dosing (exposure at d 9, 10 and 11 most severely) • Most malformations linked to the skeletal system, predominantly affecting the limbs, as well as spinal curvature and cleft palate Nishimura and Nakai, 1958 Swiss-Webster mice ♀♀ dosed with nicotine for 5 weeks (dose increases as follows: Days 1 to 7 20 µg/mL; from day 8 60 µg/mL. For one group: from day 21 100 µg/mL). Breeding conducted after 2 weeks after final dosing. Pregnant ♀♀ were injected with 1.3 mg/kg nicotine either once or twice daily, from GD 12. On GD 17, mice sacrificed 20 min after receiving the final dose.
• Nicotine reduced fetal weight in concentration dependent manner • Dose-related inhibition of intracellular concentration of AIB when dosed via water • Nicotine injection 20 min prior to sacrifice induced similar intracellular AIB reduction, but not when injected 5 d prior to sacrifice Rowell and Clark, 1982 CD-1 mice 30-35 d old ♂ mice housed 6 per cage. Nicotine dissolved in 0.9% saline, injected intraperitoneally in doses of 0, 0.05, 0.4, or 0.8 mg/kg in 0.0075 mL/g 5, 15 or 25 min before assessment. Activity was simultaneously assessed as horizontal and vertical activity of two animals.  TC  TC TC  TM   TC  TM   A  N  TC  TM   A  C  N  TC  TM   A  N  TC  TM   A  C  D  TC  TM   A  C  D  TC TM 1-5: Lowest to highest exposure concentrations: 6.25, 12.5, 25, 50 and 100 µM; A, Acetamiprid; C, Clothianidin; D, Dinotefuran; I, Imidacloprid; N, Nicotine; TC, Thiacloprid; TM, Thiamethoxam. ↓: reduced; ↓↓: severely reduced; ↓↓↓: not detectable; ↑: increased; K: kyphosis; L: lordosis. Areas shaded in blue: time points during which this endpoint cannot be observed.