Accumulation and distribution of fluorescent microplastics in the early life stages of zebrafish.Introduction. The bioaccumulation of microplastics play a key role in their toxic effects. However, as particulate, their bioaccumulations are different from many other pollutants.
Described here is a feasible method to visually determine the accumulation and distribution of microplastics in zebrafish embryos or larvae using fluorescent microplastics. The experiments were conducted in compliance with the national guide Laboratory animal Guideline for ethical review of animal welfare. One, embryo collection.
Adult zebrafish are originated from the China Zebrafish Resource Center. Fish are maintained in 20-liter glass tanks with recirculating charcoal-filtered tap water system at a constant temperature, namely, 28 Celsius degree, on a photoperiod of 14 and 10 hour, light:dark. Fish are fed twice daily with Artemia nauplii.
It is recommended that the food is given at maximum 3%fish weight per day, and should be eaten within five minutes every time. Well-developed adult zebrafish are transferred into the spawning tank at a ratio of one male to two females the night before the breeding. The following morning, the fish start to spawn after the onset of the light cycle.
Eggs are collected using a Pasteur pipette, rinsed with 10%Hank's solution several times, and then checked for fertilization using a microscope. Fertilized eggs undergo the cleavage period after approximately two-hour post fertilization and can be clearly identified. The fertilized embryos are incubated in a 500-milliliter beaker containing 200-milliliter of 10%Hank's solution with 1%methylene blue for disinfection at 28 Celsius degree.
The loading rate is not exceeding one embryo in each 2-milliliter solution. Two, preparation of microplastic suspensions. The stock solution of green fluorescently labeled polystyrene beads with nominal diameter of 500 nanometers are sonicated for 10 minutes.
Dilute the stock solution with 10%Hank's solution to produce the desired exposure solutions, namely 0.1, 1, and 10 milligram per liter. The exposure solutions of microplastics are always freshly prepared before exposure. Three, microplastic exposure.
Six newly fertilized embryos are randomly selected, and then transferred into each well of six well plate containing five milliliter of microplastic solutions with different concentrations. The control groups containing 10%Hank's solution are included. Triplicate wells, with a total of 18 embryos, are used for each treatment.
The embryos are incubated under the same light-dark cycle and temperature as adults, and are observed every 12 hours. The dead are removed immediately. The microplastic solutions are 90%renewed every 24 hours.
During the exposure period, the fish are not fed. Generally, the hatching of embryo begins at 48-hour post fertilization and completes at about 72-hour post fertilization. Four, assessment of microplastic distribution.
At 24-48-72-96-and 120-hour post fertilization, the embryos and larvae, one from each of the three replicates, are randomly selected and rinsed with 10%Hank's solution. The embryos and larvae are arranged and prepared for observation. Beforehand, the larvae are transferred into a Petri dish, and exposed to 0.016%tricaine for anesthesia.
The fish are observed with fluorescence microscope, and imaged with imaging software. The fluorescence intensity in fish is further quantified with ImageJ NIH. Representative results.
It is showed in the results that microplastics can bioaccumulate in zebrafish embryos and larvae in a concentration-dependent manner. Before hatching, strong fluorescence is found around the embryonic chorion;while in zebra fish larvae, the yolk sac, pericardium, and gastrointestinal tract are the main accumulated sites of microplastics.Discussion. This result will advance our understanding about the toxicity of microplastics to fish, and the method described here can potentially be generalized to determine the accumulation and distribution of other types of fluorescent materials in the early life stages of zebrafish.
Since the chorion will act as an effective barrier against the particles with large size, the dechorionation process before exposure may be needed. However, the embryo with chorion intact is more recommended to assess the ecotoxicity of pollutants when considering the condition of exposure in the real world. The behavior of microplastics in the solution is critical to the bioavailability as well.
The physicochemical characteristics of microplastics should be tracked over the exposure duration.
