Structure activity toxicity of different micro-nanoplastics on developing Zebrafish (Danio rerio) based on individual polymer chemistry

Plastics and their smaller products are ubiquitous in our ecosystem with limited knowledge as to their impacts on organisms. Very little is known of the toxic effects of micro-nanoplastics (MNPs) on eukaryotic organisms. Extensive research is well underway determining the fate and effects of some of these compounds on biochemical pathways. There are fewer studies of the effects of individual MNPs based on polymer chemistry. The studies described in this dissertation examine, the toxicity associated with exposure to specific plastic polymers on Zebrafish (Danio rerio) as a model eukaryotic organism. Until the complexities inherent in plastics on a chemical level are determined and ranked, a foundation to the resolution of MNPs will not be achieved. MNPs’ toxicities need to be determined sans plasticizer, or persistent organic pollutants (POPs), or microbiota, which is examined in the research presented below. Plastic is an umbrella term for many polymers with very different monomeric chemical structures. The assumption that all the different MNP types would behave the same biochemically. That the size of the MNP is the determining toxic factor giving all MNPs similar responses in biological systems and that their chemical structure has no bearing on toxicity is unlikely.A series of studies using the Zebrafish as a toxicity model system for interrogating the effects of different MNPs on morphological endpoints and altered gene expression are presented in the chapters below. I have hypothesized that structurally different plastics would result in different target systems and organ toxicity, different potencies between chemical based plastic types, and differential gene expression. These differences in endpoint toxicities could be used to determine which MNPs pose the greatest risk to developing eukaryotic organisms. Based on this ranking the MNPs that pose the greatest risk could be limited in their production and use by substituting the less toxic plastics.Fifteen different plastic polymers including weathered field samples and virgin/pure plastics purchased from Sigma-Aldrich® were tested for toxicity in a morphometric study using the Zebrafish embryo larval test (ZELT) protocol. The Zebrafish is a well-established eukaryotic animal model for comparison across vertebrate species. Early development is a critical life-stage to assess embryonic impacts and those alterations that may be manifested later in life. During early embryogenesis cell proliferation is rapidly occurring and leads to the formation of endodermal mesodermal and ectodermal layers that form the organ systems in early juvenile and adult Zebrafish. The process of embryonic development, from 1 cell stage to hatched yolk-sac larvae, occurs at approximately 3 days post fertilization (dpf). The Zebrafish embryos were treated during the Mid-Blastula period from 512 cell to high cell stage, approximately 3 hours post fertilization (hpf). During this time rapid metasynchronous cell cycles occur ahead of lengthened, asynchronous cycles; epiboly stages then begin, at approximately 4 hpf. Morphometric data were obtained from images of the MNP treated larvae and were found to have differing total mean length and size of organ regions compared to the control larval group at protruding mouth stage, 96 hpf. This indicates that the MNPs affect the normal development of Zebrafish larvae and that differing MNP polymers resulted in significantly different morphologies. Polymethyl methacrylate (PMMA) and polyethylene terephthalate (PET) were two MNP types determined to morphometrically cause altered growth compared to the control at 96 hpf and were used for the next series of studies. Yolk sac area (YSA) was measured when treated with PMMA and PET and were found to significantly decrease in size compared to the control, additionally yolk lipid density (YLD) significantly increased compared to the control at 96 hpf. Further, gene expression of bactin, a protein coding gene and cardiac transcription factors: gata4, hand2, nkx2.5 and tbx5a, were analyzed with gapdh as an endogenous control and while no significance was found, the profiles of relative quantification (RQ) varied among the MNP types indicating differing effects on expression. Based on the findings in this dissertation it can be concluded that different MNPs can be ranked on their biological effects observed in the Zebrafish model. This ranking could be useful in setting risk management decisions and limits based on structure activity toxicity of MNPs.