Neurotoxic pesticides change respiratory parameters in early gill-breathing, but not in skin-breathing life-stages of zebrafish (Danio rerio).

Neurotoxic compounds can interfere with active gill ventilation in fish, which might lead to premature death in adult fish, but not in skin-breathing embryos of zebrafish, since these exclusively rely on passive diffusion across the skin. Regarding lethality, this respiratory failure syndrome (RFS) has been discussed as one of the main reasons for the higher sensitivity of adult fish in the acute fish toxicity test (AFT), if compared to embryos in the fish embryo toxicity test (FET). To further elucidate the relationship between the onset of gill respiration and death by a neurotoxic mode of action, a comparative study into oxygen consumption (MO2), breathing frequency (fv) and amplitude (fampl) was performed with 4 d old skin-breathing and 12 d old early gill-breathing zebrafish. Neurotoxic model substances with an LC50 FET/AFT ratio of > 10 were used: chlorpyrifos, permethrin, aldicarb, ziram, and fluoxetine. Exposure to hypoxia served as a positive control, whereas aniline was tested as an example of a narcotic substance interfering non-specifically with gill membranes. In 12 d old larvae, all substances caused an increase in MO2, fv and partly fampl, whereas effects were minor in 4 d old embryos. An increase of fv in 4 d old embryos following exposure to chlorpyrifos, aldicarb and hypoxia could not be correlated with an increased MO2 and might be attributed either to (1) to the successfully postponed decrease of arterial partial pressure of oxygen (PO2) through support of skin respiration by increased fv, (2) to an unspecific stimulation of the sphincter muscles at the base of the gill filaments, or (3) to the establishment of oxygen sensing for later stages. In gill-breathing 12 d old zebrafish, a concentration-dependent increase of fv was detected for aniline and chlorpyrifos, whereas for aldicarb, fluoxetine and permethrin, a decline of fv at higher substance concentrations was measured, most likely due to the onset of paralysis and/or fatigue of the gill filament sphincter muscles. Since alterations of fv serve to postpone the decrease in arterial PO2 and MO2 increased with decreasing fv, the respiratory failure syndrome could clearly be demonstrated in 12 d old zebrafish larvae. Passive respiration across the skin in zebrafish embryos could thus be confirmed as a probable reason for the lower sensitivity of early life-stages to neurotoxicants. Integration of respiratory markers into existing testing protocols with non-protected developmental stages such as embryos might help to not underestimate the toxicity of early life-stages of fish.

A novel automated method for the simultaneous detection of breathing frequency and amplitude in zebrafish (Danio rerio) embryos and larvae.

Stress responses of fish to disruption of oxygen homeostasis include adjusted oxygen consumption rate (MO2) as well as the hyperventilation consisting of changes in breathing frequency (fv) and amplitude (fampl). However, studying the HVR in very small organisms such as zebrafish (Danio rerio) embryos and larvae is challenging, and breathing movements (i.e., fv) are usually manually counted, which is time- and human resource-intense, error-prone and does not provide information on the amplitude of breathing movements of the response, the breathing amplitude (fampl). Hence, in the present study, a new automated method was developed to simultaneously measure fv and fampl in small zebrafish embryos and larvae with the computer software DanioScope™. To compare HVR strategies at different life-stages of zebrafish and the physiologically linked MO2, hatched 4 d old embryos and early gill-breathing 12 d old larvae were treated with the HVR-inducing neurotoxic compound lindane (γ-hexachlorocyclohexane; γ-HCH) as a model substance. Comparison of manually counted fv with fv data measured by DanioScope™ at both life-stages showed high to moderate agreement between the two methods with respect to fv in control fish and in fish treated with lower lindane concentrations (3 - 18% deviation at 25 µg/L γ-HCH). With increasing lindane concentrations (100 and 400 µg/L γ-HCH), however, manual counts showed an average underestimation of fv by up to 30%, mainly due to very fast, rapidly successive, and indistinct movements of the fish, which cannot be properly detected by manual counts. Automated measurement thus proved significantly more sensitive, although several pre- and post-processing steps are needed. The improved automated detection of fv and the first reliable estimation of fampl in small fish embryos and larvae, as well as the inclusion of MO2, may provide new insights into different respiratory strategies and may, thus, represent a tool to lower the detection limit for reactions of different life-stages of fish to environmental stressors. In the present study, this became evident, as early gill-breathing 12 d old zebrafish larvae showed symptoms of respiratory failure (i.e., increase in fv, fampl and MO2, followed by subsequent lethargy) after exposure to lindane, whereas skin-breathing in 4 d old embryos proved mainly insensitive to the paralytic effects of lindane.