{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Dr. Steve Ayobahan"],"organism":["Danio rerio"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14544"],"description":["This research provides a thorough ecotoxicological evaluation of natural biopolymers (BP) and naturally modified polymers (NMP). The study focuses on biopolymers such as chitosan, xanthan, alginate, and carboxymethyl cellulose (CMC), alongside NMPs like Jelucel HM 200 and Emwaxy Jel 100. Each polymer was individually tested on zebrafish embryos using a zebrafish embryo toxicity (zFET) test. Natural polymers, although prevalent in agrotechnical products, remain largely unregulated under REACH due to their natural origins. There is a critical lack of data regarding their potential impact on closed ecosystem cycles, raising concerns about environmental risks. To assess the sublethal effects of these polymers, a modified zebrafish embryo toxicity test (ZFET) was utilized, incorporating transcriptomics as an additional evaluation endpoint. This modified methodology adheres to the OECD 236 guidelines but includes specific adjustments for gene expression analysis, such as changes in the number of embryos used, test setup volume, and sample processing procedures. Zebrafish embryos were exposed for 96 hours to each polymer's filtrate at a maximum concentration of 100 mg/L. After exposure, the embryos underwent assessments for survival rates, hatching rates, and morphological malformations. The study identified differentially expressed genes (DEGs) for each polymer exposure, shedding light on the transcriptomic changes induced by both biopolymers and NMPs. The primary objective is to understand how these substances influence gene expression profiles in zebrafish embryos, aiming to identify transcriptomic biomarkers that could assist in environmental risk assessment. This research contributes valuable insights into the potential ecological impacts of natural and modified polymers, highlighting the need for further regulatory consideration."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Collection - The test solution of these natural biopolymers (BP) and naturally modified polymers (NMP) were prepared by first preparing a highly concentrated stock solution in water, stirring for approximately 30 minutes and adjusting the pH to 7-8.5. The test solutions were prepared from these stock solutions immediately before the start of the test to avoid any degradation of the substance. All test solutions were prepared in copper reduced tap water. They were constantly aerated and used over the course of the 96 hours exposure experiment. The glass wells for incubation of the embryos were pre-saturated with the respective test solution overnight and renewed at the beginning of the experiment. Approximately 40-50 fertilized fish eggs were pre-picked in glass Petri dishes (10 cm diameter) filled with 30 ml of the test solution. Using a binocular optical microscope, 15 embryos of the same early blastula stage were transferred from the pre-picking plate to the test glass wells filled with 8 ml of test solution. At test termination, 10 embryos were randomly selected and pooled in a 1.5 ml Eppendorf tube. All tubes were then immediately and simultaneously placed on ice to kill the larvae prior to RNA extraction.","Nucleic Acid Extraction - Total RNA and Protein were extracted from the tissue lysate using the NucleoSpin RNA Protein kit from Macherey-Nagel following the manufacturer's protocol. The RNA concentration was measured using a Nanodrop 2000 spectrophotometer from Thermo Scientific, with a minimum concentration of 100 ng/µl required. The overall RNA quality and RIN value were determined using the Agilent 2100 Bioanalyzer system and the Agilent RNA 6000 Nano Kit, respectively. Only samples with a RIN value greater than 8 were used for downstream analysis. The samples were stored at -80°C until they were sent to the sequencing facility on dry ice. For each sample, 10 zebrafish larvae were collected and transferred to a low binding Eppendorf tube. The larvae were euthanized simultaneously by placing the tubes on ice for 10 minutes. The supernatant was carefully removed without injuring the larvae, and RP1 buffer from the NucleoSpin RNA Protein kit (Macherey-Nagel 740933.250) was added for tissue homogenization. The RP1 buffer was prepared with TCEP as a reducing agent. Detailed information about embryonic incubation conditions, husbandry of adult broodstock, exposure duration, and conditions can be found in the \\\\\"Growth protocol\\\\\" and \\\\\"Treatment protocol\\\\\" respectively.","Library Construction - Adapter sequences were removed using trimmomatic (v0.39) and library’s sequence quality was assessed with FastQC (v0.12.0). Reads were checked for potential contaminations with FastQ Screen (v0.15.3) using bowtie2 (v2.5.3) against the following genomes: Daphnia magna (ftp://ftp.ensemblgenomes.org/pub/metazoa/release-48/fasta/daphnia_magna/dna/Daphnia_magna.daphmag2.4.dna.toplevel.fa.gz), Homo sapiens, Mus musculus, Drosophila melanogaster, Saccharomyces cerevisiae, Eschericia coli as well as custom build human rRNA database. With the exception of D. magna, pre-built Bowtie2 indices for those genomes were directly downloaded from Babraham Bioinformatics with the built-in function ‘fastq_screen --get_genomes’.","Growth Protocol - Adult animal husbandry: Wild-type zebrafish (Danio rerio, Strain AB) broodstocks were maintained under flow-through conditions in 150 L tanks at 26 +/- 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® (Tetra Werke, Melle, Germany) main feed ad libitum and Artemia salina nauplii. One day before the start of the test, glass spawning trays containing artificial substrate (green glass beads strung on stainless steel wire) were placed at the bottom of each tank. After mating and spawning in the morning hours eggs were rinsed with clear Cu-reduced water and placed into glass dishes for pre-picking.","Sequencing - Poly(A)+ RNA was purified and fragmented. It was then transcribed into cDNA using the TruSeq RNA library Prep Kit v2 from Illumina. After adapter ligation and PCR amplification, the RNA fragments were sequenced using an Illumina NovaSeq 6000 system. The sequencing was done in paired-end mode with a read length of 150 base pairs. A minimum read depth of 30,000,000 read pairs per sample was ensured. Adapter trimming and sequencing quality control were performed using fastp and FastQC, respectively. Contamination assessment was done using FastQScreen. The overall quality report was generated using MultiQC.","Sample Treatment - Fish embryo incubation: For each sample 15 fertilized eggs in the early blastula stage were placed in glass petri dishes (diameter 6 cm) filled with 8 ml of the respective testing solution. Embryos were incubated at 27 ± 1°C on a 14:10 h light/dark cycle. At 24 hours post fertilization (hpf), eggs were inspected visually and single coagulated eggs were recorded and removed. At 48 hpf, overall survival, hatching rates, morphological malformations and physiological changes were recorded and aged solutions were replaced by fresh, aerated test solutions. At 96 hpf again, overall survival, hatching rates, morphological malformations and physiological changes were recorded before RNA extraction."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Gene level raw counts from the experiment’s libraries were bind into a single count matrix in R (version 4.2.2) where rows correspond to genes and columns to the sample (CountMatrix.txt). Only genes with a minimum sum of N counts across N samples (rowSums >= N) were kept for further downstream analysis. After low read counts were removed, the remaining feature mapped gene counts in the matrix were normalized with the DESeq2 package (1.42.1) using a parametric fit type model for dispersion estimates (default). The resulting normalized count matrix (DESeqNormCounts.txt) was used for differential gene expression analysis with DESeq2 and further downstream data analysis.","Sequence Alignment - Sequences were aligned to the reference genome Danio rerio GRCz11 (version 91, https://www.ensembl.org/Danio_rerio/Info/Index) using STAR aligner (v2.7.11) allowing for 2 mismatches. Subsequently, feature mapped reads were directly counted by STAR’s ‘-- quantMode GeneCounts’ function which produces counts coincide with those produced by HTSeq’s ‘htseq-count’ with default parameters (union mode)."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA"],"species":["Danio rerio"],"pubmed_authors":["Dr. Sebastian Eilebrecht","Dr. Steve Ayobahan"],"additional_accession":[]},"is_claimable":false,"name":"Transcriptomic Responses of Zebrafish Embryos to Sublethal Exposure of Natural Biopolymers (BP) and Naturally Modified Polymers (NMP): A 96-Hour RNA-Seq Analysis","description":"This research provides a thorough ecotoxicological evaluation of natural biopolymers (BP) and naturally modified polymers (NMP). The study focuses on biopolymers such as chitosan, xanthan, alginate, and carboxymethyl cellulose (CMC), alongside NMPs like Jelucel HM 200 and Emwaxy Jel 100. Each polymer was individually tested on zebrafish embryos using a zebrafish embryo toxicity (zFET) test. Natural polymers, although prevalent in agrotechnical products, remain largely unregulated under REACH due to their natural origins. There is a critical lack of data regarding their potential impact on closed ecosystem cycles, raising concerns about environmental risks. To assess the sublethal effects of these polymers, a modified zebrafish embryo toxicity test (ZFET) was utilized, incorporating transcriptomics as an additional evaluation endpoint. This modified methodology adheres to the OECD 236 guidelines but includes specific adjustments for gene expression analysis, such as changes in the number of embryos used, test setup volume, and sample processing procedures. Zebrafish embryos were exposed for 96 hours to each polymer's filtrate at a maximum concentration of 100 mg/L. After exposure, the embryos underwent assessments for survival rates, hatching rates, and morphological malformations. The study identified differentially expressed genes (DEGs) for each polymer exposure, shedding light on the transcriptomic changes induced by both biopolymers and NMPs. The primary objective is to understand how these substances influence gene expression profiles in zebrafish embryos, aiming to identify transcriptomic biomarkers that could assist in environmental risk assessment. This research contributes valuable insights into the potential ecological impacts of natural and modified polymers, highlighting the need for further regulatory consideration.","dates":{"release":"2025-05-30T00:00:00Z","modification":"2024-10-18T14:54:55.837Z","creation":"2024-10-18T14:54:55.837Z"},"accession":"E-MTAB-14544","cross_references":{"ENA":["ERP165295"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184","EFO_0003969"]}}