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| Status |
Public on Feb 22, 2024 |
| Title |
Transcriptomics-based points of departure for Daphnia magna exposed to 18 per- and polyfluoroalkyl substances. |
| Organism |
Daphnia magna |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Per- and polyfluoroalkyl substances (PFAS) represent a large group of contaminants of concern based on their widespread use, distribution and persistence in the environment, and potential toxicity. Many of the traditional models for estimating toxicity, bioaccumulation, and other relevant toxicological properties are not well suited for PFAS. Consequently, there is a need to generate hazard information for a large number of PFAS in an efficient and cost-effective manner. In the present study, Daphnia magna were exposed to multiple concentrations of 22 different PFAS for 24 h, in a 96-well plate format. Following exposure, whole body RNA was extracted and pooled extracts, each representing five exposed individuals, were subjected to RNA sequencing. Following analytical measurements to verify PFAS exposure concentrations in-well, and quality control on processed cDNA libraries for sequencing, concentration-response modeling was applied to the data sets for 18 of the tested compounds, and the concentration at which a concerted molecular response occurred (transcriptomic point of departure; tPOD) was calculated. The tPODs, based on average measured concentration of PFAS in the exposure wells, generally ranged from 0.03-0.58 µM (9.9-350 µg/L; interquartile range). In most cases, these concentrations were two orders of magnitude lower than similarly calculated tPODs for human cell lines exposed to PFAS. They were also lower than apical effect concentrations reported for seven PFAS for which some crustacean or invertebrate toxicity data were available, although there were a few exceptions. However, despite being lower than most other available hazard benchmarks, the Daphnia magna tPODs were, on average, four orders of magnitude greater than the maximum aqueous concentrations of PFAS measured in Great Lakes tributaries. Overall, this high throughput transcriptomics assay with Daphnia magna holds promise as a component of a tiered hazard evaluation strategy employing new approach methodologies.
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| Overall design |
Test chemicals were sourced through contract with Evotec (Branford, CT, USA; Contract #EP-D-12-034). Substances were provided solubilized in dimethylsulfoxide (DMSO) at two stock concentrations, 30 mM and 9.5 mM. Stock solutions were shipped frozen at -20° C, and stored at -80°C until thawed for use in the bioassays. A dilution series for each assay was prepared using a Biomek i5 liquid handler (Beckman Coulter, Brea, CA, USA). Stock solutions of two PFAS at 30 mM and 9.5 mM (nominal) were pipetted into designated wells of a 96 well plate (300 µl; catalog #650101; Greiner Bio-One, NC, USA). Ten-fold serial dilutions in DMSO (or methanol) were then prepared from each stock solution to yield a total of 8 concentrations with ½ log spacing for each chemical. Stocks from the dilution plate were then diluted to 0.33% in sand-filtered and UV-treated Lake Superior Water amended with 94 µM Na2SO4; 24 µM NaCl; 23 µM KCl; 15 µM CaCl2; 48 µM MgCl2 (hereafter amended LSW) yielding a nominal concentration range from 100-0.03 µM (1/2 log spacing) and distributed to a dosing plate (2 ml deep-well plates; catalog # 503162; NEST Scientific, NJ, USA), such that each concentration of an individual PFAS to be tested was represented by five wells on each plate, with two wells for each concentration on the perimeter of the plate (edge wells) and the remaining three wells for each concentration positioned on the interior. A concentration response series for two PFAS, with a shared set of controls, was present on each 96-well plate and three replicate plates were prepared for each pair of two PFAS tested. Control wells contained the same concentration (0.33%) of either DMSO or methanol as the wells treated with PFAS. Daphnia magna were obtained from an onsite culture unit at the U.S. EPA Great Lakes Toxicology and Ecology Division, Duluth, MN. For use in the assays, Daphnia magna were reared in mass culture in amended LSW and held at 20° C until they were 72 hours old. They were fed a diet of yeast-Cerophyll-trout chow and algae (USEPA 2016d) until ready for use in the assays. All procedures involving animals were approved by a local animal care and use committee. Bioassays were conducted using the same experimental design. All assays were conducted in 96-well plate format using 1 ml volume deep-well plates (Catalog # 502162, NEST Scientific). Uniform age (72 ± 6 h) Daphnia magna were manually loaded to each well of three replicate 96-well plates using a P1000 pipet with the end of the tip cut off to a one cm bore diameter. The volume in each well was reduced to a uniform volume of 50 µl using the Biomek i5 liquid handler. Assays were initiated by adding 550 µl of exposure solution from the dosing plate to the three replicate exposure plates for each assay. Immediately after dosing, each plate was covered with a silicone sealing mat (catalog #506065; NEST Scientific) and then placed in a 20°C incubator for 24 h. At test termination, each well was inspected and all mortalities (immobile with gentle prodding) were recorded and removed. Live daphnia were also examined for phenotypic abnormalities, although none were noted at sublethal concentrations. The Biomek i5 was then used to transfer 100 µl of exposure solution to an “exposure verification plate”, where the n=5 samples for each treatment concentration were subsequently pooled together into a total volume of 500 on a per-plate basis. The pooled exposure solution for each treatment, per plate, was diluted 1:1 in acetonitrile (with the exception of PFTeDA, PFUdA, PFTrDA, and PFNA which were diluted 3:1 in acetonitrile and FHxSA, N-EtFOSA-M, 8:2 FTS, and PFTDoDA diluted 3:1 in methanol) for subsequent use in exposure verification. The remaining volume of exposure medium in each well was replaced with 60 µl of Trizol (Zymo Research, CA, USA). A 2.3 mm stainless steel bead was added to each well, then the plate was homogenized at 30 Hz for two minutes using an Omni-Bead Ruptor bead mill. Homogenates of individual Daphnia magna from the same treatment group were pooled together, on a per plate basis to generate one pooled sample per treatment, per plate (three biological replicates per treatment). Only live daphnia were included in the homogenate pools.
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| Contributor(s) |
Villeneuve DL, Blackwell BR, Bush K, Harrill J, Harris F, Hazemi M, Le M, Stacy E, Flynn KM |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| BioProject |
PRJNA1018977 |
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| Submission date |
Oct 12, 2023 |
| Last update date |
Feb 22, 2024 |
| Contact name |
Daniel L. Villeneuve |
| E-mail(s) |
villeneuve.dan@epa.gov
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| Organization name |
US EPA
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| Department |
Mid-Continent Ecology Division
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| Lab |
National Health and Environmental Effects Research Laboratory
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| Street address |
6201 Congdon Blvd
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| City |
Duluth |
| State/province |
MN |
| ZIP/Postal code |
55804 |
| Country |
USA |
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| Platforms (1) |
| GPL28557 |
Illumina NovaSeq 6000 (Daphnia magna) |
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| Samples (392)
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| Supplementary file |
Size |
Download |
File type/resource |
| GSE245268_DM_4-2FTS_counts.filtered.+1.normFinal.txt.gz |
2.6 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_6-2FTS_counts.filtered.+1.normFinal.txt.gz |
2.5 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_8-2FTS_counts.filtered.+1.normFinal.txt.gz |
2.9 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_FC10diol_counts.filtered.+1.normFinal.txt.gz |
2.4 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_FC10diol_counts.filtered.+1.normFinal_QA_2687trt_removed.txt.gz |
1.6 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_FC8DoD_counts.filtered.+1.normFinal.txt.gz |
2.8 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_FC8diol_counts.filtered.+1.normFinal.txt.gz |
2.7 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_FHxSA_counts.filtered.+1.normFinal.txt.gz |
2.9 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_N-EtFOSA-M_counts.filtered.+1.normFinal.txt.gz |
2.9 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_PF4OPeA_counts.filtered.+1.normFinal.txt.gz |
2.8 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_PFHxS_counts.filtered.+1.normFinal.txt.gz |
2.8 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_PFNA_counts.filtered.+1.normFinal.txt.gz |
2.8 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_PFOA_counts.filtered.+1.normFinal.txt.gz |
2.8 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_PFOS_counts.filtered.+1.normFinal.txt.gz |
2.9 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_PFOS_counts.filtered.+1.normFinal_QA_contaminated_910_trt_removed.txt.gz |
1.8 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_PFPB_counts.filtered.+1.normFinal.txt.gz |
2.3 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_PFPeA_counts.filtered.+1.normFinal.txt.gz |
2.7 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_PFTDoDA_counts.filtered.+1.normFinal.txt.gz |
2.9 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_PFTP_counts.filtered.+1.normFinal.txt.gz |
2.6 Mb |
(ftp)(http) |
TXT |
| GSE245268_DM_PFTrDA_counts.filtered.+1.normFinal_5_concentrations.txt.gz |
2.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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