protocol-for-acute-chronic-ecotoxicity-testing-turquoise-killifish

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish <em>Nothobranchius furzeri</em>

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

University of Leuven

North-West University

Laboratory ecotoxicity tests and biomonitoring in aquatic systems are currently based on the active component of invertebrate communities. Even though dormant egg banks are crucial for the long term survival and community dynamics of many aquatic organisms, the effects of anthropogenic activities on dormant egg bank dynamics have rarely been studied. In this study we investigated the effects of two pesticides with a different mode of action (carbaryl and fenoxycarb) on hatching of Daphnia magna dormant eggs (ephippia) as well as on survival, growth and reproduction of the hatched neonates. Dormant eggs were exposed to the pesticides simultaneously to incubation under conditions that induce hatching (long daylight and 20 °C). Carbaryl had no negative effects on embryonic development or hatching rate up to concentrations almost 1,000 times the median effect concentration (EC50) of neonate survival in acute tests. Fenoxycarb, however, had a significant dose-related effect by delaying or completely stopping the hatching process and caused severe abnormalities in developing individuals. Both pesticides had significant negative effects on survival and reproduction of the hatchlings. These results indicate that, in addition to inducing mortality of active individuals, pesticides can affect zooplankton communities by altering hatching dynamics and life history traits of hatched individuals. We briefly discuss how such pollution induced changes in the benthic-pelagic coupling could translate into trans-generational effects impacting ecological and evolutionary dynamics. 

Pesticide exposure impacts not only hatching of dormant eggs, but also hatchling survival and performance in the water flea Daphnia magna.

Although Daphnia magna is a key species in many lentic freshwater ecosystems and is commonly used as model organism in ecology and ecotoxicology, very little is known about the effects of chemicals on their dormant life stages. Dormant eggs (ephippia) are produced when environmental conditions deteriorate, and Daphnia switch from clonal to sexual reproduction. Ephippia produced over different growing seasons can accumulate in the sediment of ponds and lakes, where they can be exposed to pesticides and other (anthropogenic) stressors. In the present study, we have investigated the effects of pesticide exposure on dormant eggs at different embryonic developmental stages and evaluated the degree of protection against pollution provided by the ephippial case. We therefore conducted a hatching experiment in which decapsulated and encapsulated dormant eggs were exposed to an insect growth regulator (fenoxycarb) at different stages during their development, both before and after activation of the eggs. In addition, we developed an analytical method to measure fenoxycarb concentrations in the dormant eggs. Fenoxycarb negatively affected development and hatching success and changed the timing of hatching in activated and in dormant eggs. Hatching characteristics as well as fenoxycarb concentrations inside the eggs differed significantly between exposure treatments. Final stages of embryonic development were most sensitive to pesticide exposure and had the highest tissue concentrations of fenoxycarb. Tissue concentrations did not differ significantly between decapsulated and encapsulated eggs, suggesting that the ephippial case offers limited or no direct protection against pesticide exposure. With this study we provide new evidence showing that pesticides can bioconcentrate in and affect D. magna dormant eggs. The severity of the effects on developing embryos depends on the timing of pesticide exposure. Our results stress the importance of considering the full life-cycle of model organisms used in ecotoxicological studies, since these are ultimately aimed at assessing risks of chemical exposure on natural aquatic ecosystems. 

Timing matters: sensitivity of Daphnia magna dormant eggs to fenoxycarb exposure depends on embryonic developmental stage.

Heavy metal toxicity in agro-ecosystems is a global problem. Recently, it has been indicated that the soils used for agriculture and the fresh produce grown on these soils in the Philippi Horticultural Area (PHA) contains heavy metals exceeding the maximum permissible concentrations thereof in South Africa. This study was therefore aimed at evaluating the concentrations of heavy metals in the soils and vegetables produced in the PHA, as well as to determine the major agronomic sources of these metals in this area. Cu, Pb, and Zn concentrations in the soils exceeded the maximum permissible concentrations of 6.6, 6.6, and 46 mg/kg, respectively. Cd, Pb, and Zn concentrations in the vegetables also exceeded the maximum permissible concentrations of 0.1, 0.5, and 40 mg/kg, respectively. The biggest agronomic contributors of these heavy metals to the soils in the PHA were found to be the farmyard manures. Knowing what the major sources of these heavy metals are, it is important to determine ways to mitigate the inputs thereof, as well as to remove existing concentrations from the soils without contaminating the groundwater resources in the area. 

Farmyard manures: the major agronomic sources of heavy metals in the Philippi Horticultural Area in the Western Cape Province of South Africa.

Nothobranchius furzeri is a promising model for ecotoxicological research due to the species' short life cycle and the production of drought-resistant eggs. Although the species is an emerging vertebrate fish model for several fundamental as well as applied research domains, its potential for ecotoxicological research has not yet been tested. The aim of this study was to characterise the acute and chronic sensitivity of this species to copper as compared to other model organisms. Effects of both acute and chronic copper exposure were tested on survival, life history and physiological traits. We report a 24h-LC of 53.93µg Cu/L, which is situated within the sensitivity range of other model species such as Brook Trout, Fathead Minnow and Rainbow Trout. Moreover, in the full life cycle exposure, we show that an exposure concentration of 10.27µg/L did not cause acute adverse effects (96h), but did cause mortality after prolonged exposure (3-4 weeks). Also chronic, sublethal effects were observed, such as a reduction in growth rate, delayed maturation and postponed reproduction. Based on our results, we define the NOEC at 6.68µg Cu/L, making N. furzeri more sensitive to copper as compared to Brook Trout and Fathead Minnow. We found stimulatory effects on peak fecundity at subinhibitory levels of copper concentrations (hormesis). Finally, we found indications for detoxifying and copper-excreting mechanisms, demonstrating the ability of the fish to cope with this essential metal, even when exposed to stressful amounts. The successful application of current ecotoxicological protocols on N. furzeri and its sensitivity range comparable to that of other model organisms forms the basis to exploit this species in further ecotoxicological practices.

Acute and chronic sensitivity to copper of a promising ecotoxicological model species, the annual killifish Nothobranchius furzeri.

This study evaluated Co, Cr, Mn, Ni, Se, Sn and V status in the soils of the PHA, as well as the vegetables produced on these soils. We also determined the agronomic sources of these elements to the soils in the PHA. Farmyard manures applied as fertilizer amendments to the soils in the PHA were found to be the major agronomic sources of the metal and metalloid elements. These elements were however, retained in significantly higher concentrations in the soils compared to the concentrations found in the edible portions of the vegetable crops collected. This, in turn, resulted in these vegetables being poor sources of several of the essential mineral nutrients. It is therefore suggested that: (1) a wider variety of crops are assessed for their mineral nutrient status, (2) to find ways to increase the availability of these mineral nutrients and (3), that the possibilities of micronutrient and trace element deficiencies be assessed in the communities surrounding the PHA.

Essential metal and metalloid elements in the Philippi Horticultural area, and their uptake into selected vegetable crops.

Aquatic organisms of inland waters are often subjected to a combination of stressors. Yet, few experiments assess mixed stress effects beyond a select group of standard model organisms. We studied the joint toxicity of reference toxicants and increased temperature on the turquoise killifish, Nothobranchius furzeri, a promising model for ecotoxicological research due to the species' short life cycle and the production of drought-resistant eggs. The acute sensitivity of the larval stage (2dph) to three compounds (cadmium, 3,4-dichloroaniline and chlorpyrifos) was tested in combination with a temperature increase of 4 °C, mimicking global warming. Dose-response relationships were used to calculate 96h-LC of 0.28 mg/L (24 °C) and 0.39 mg/L (28 °C) for cadmium, 96h-LC of 9.75 mg/L (24 °C) and 6.61 mg/L (28 °C) for 3,4-dichloroaniline and 96h-LC of 15.4 μg/L (24 °C) and 14.2 μg/L (28 °C) for chlorpyrifos. After 24 h of exposure, the toxicity of all tested compounds was exacerbated under increased temperature. Furthermore, the interaction effect of cadmium and temperature could be predicted by the stress addition model (SAM). This suggests the applicability of the model for fish and at the same time indicates that the model could be suitable to predict effects of temperature-toxicant interactions.

Acute sensitivity of the killifish Nothobranchius furzeri to a combination of temperature and reference toxicants (cadmium, chlorpyrifos and 3,4-dichloroaniline).

Article	Year
Pesticide exposure impacts not only hatching of dormant eggs, but also hatchling survival and performance in the water flea Daphnia magna. Ecotoxicology (London, England)\| PubMed ID: 23661096	2013
Timing matters: sensitivity of Daphnia magna dormant eggs to fenoxycarb exposure depends on embryonic developmental stage. Aquatic toxicology (Amsterdam, Netherlands)\| PubMed ID: 25546008	2015
Farmyard manures: the major agronomic sources of heavy metals in the Philippi Horticultural Area in the Western Cape Province of South Africa. Environmental monitoring and assessment\| PubMed ID: 26508018	2015
Acute and chronic sensitivity to copper of a promising ecotoxicological model species, the annual killifish Nothobranchius furzeri. Ecotoxicology and environmental safety\| PubMed ID: 28599128	2017
Essential metal and metalloid elements in the Philippi Horticultural area, and their uptake into selected vegetable crops. International journal of phytoremediation\| PubMed ID: 29053349	2018
Acute sensitivity of the killifish Nothobranchius furzeri to a combination of temperature and reference toxicants (cadmium, chlorpyrifos and 3,4-dichloroaniline). Environmental science and pollution research international\| PubMed ID: 29380199	2018

Luc Brendonck

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