Source: UNIV OF MASSACHUSETTS submitted to
ESTABLISHMENT OF ZEBRAFISH BIOASSAY TECHNOLOGY FOR ASSESSING THE ACUTE, DEVELOPMENTAL & REPRODUCTIVE TOXICITY OF TOXAPHENE & WATER SAMPLES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0194419
Grant No.
(N/A)
Project No.
MAS00862
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2002
Project End Date
Sep 30, 2008
Grant Year
(N/A)
Project Director
Arcaro, K.
Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003
Performing Department
VETERINARY & ANIMAL SCIENCE
Non Technical Summary
Protection of aquatic ecosystems and drinking water supplies is one of the great environmental challeneges facing us and future generations. This project examines the usefullness of zebrafish bioassays for the detection of aquatic pollution.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31408991150100%
Goals / Objectives
There are three major objectives of this proposal: one basic science, one applied science/testing, and one educational/instructional. The first objective is to determine the developmental and reproductive aquatic toxicity of the environmental contaminant, toxaphene. The second objective is to use zebrafish bioassay technology together with whole effluent toxicity (WET) technology to assess the acute, developmental, and reproductive toxicity of various water samples including farm runoff, industrial effluent, residential streams, and flood water. The third objective is to incorporate the knowledge and technology obtained from the first two objectives into a formal undergraduate laboratory course that will include zebrafish bioassay and WET technologies.
Project Methods
A series of zebrafish bioassays will be optimized and used to determine the acute, developmental and reproductive toxicity of toxaphene mixtures and individual congeners. Initial assays will include the following: 1) zebrafish embryo assay to study the developmental effects, 2) measurement of the yolk protein, vitellogenin, in adult male zebrafish to determine the estrogenic activity, 3) induction of ethoxyresorufin O-deethylase activity to assess the dioxin-like activity, and 4) behavioral assays to assess global effects on reproduction. These bioassays will be modified for use in standard whole effluent toxicity (WET) testing. Additionally, we will use transgenic zebrafish with xenobiotic response elements and reporter genes as sentinels for aquatic pollution.

Progress 10/01/02 to 09/30/08

Outputs
OUTPUTS: Results obtained from this project were presented at the national meetings of the Society of Toxicologists, the Society of Environmental Toxicology and Chemistry, the American Water Resources Association, and the American Water Works Association, and at the regional meeting of the Massachusetts Water Resource Research Council. Additionally, the fish bioassays developed and/or optimized as part of this project have formed the basis for two recently funded research projects. The first project, "Establishing Guidelines for the Use of Ozone-GAC for Control of Endocrine Disruptors and Related Compounds in Water" was developed by Professors Arcaro and Reckhow with colleagues at Earth Tech, Inc., MassDEP, and Pawtucket Water Supply Board and was funded by the American Water Works Association Research Foundation as well as by individual drinking water treatment facilities in the northeast. The second project, "Laser Desorption Ionization Mass Spectrometry and Nanoparticle Mass Barcodes: A New Approach for Studying the Environmental Fate, Transport, and Bioavailability of Nanoparticles", was developed by Professors Vachet, Tyson, Rotello, Anderton and Arcaro of the University of Massachusetts and funded by The Center for Hierarchical Manufacturing, an NSF Nanoscale Science and Engineering Center at UMass-Amherst. The third project, using resources in developed through this HATCH project is being prepared by Ethan Clotfelter at Amherst College. Finally, the fish bioassays developed and/or optimized during this project have formed the basis for an undergraduate laboratory course in aquatic toxicology. PARTICIPANTS: Kathleen Arcaro (PI), Lauren Moffatt, Ph.D. student supported in part by this award, and Dr. David Reckhow, professor in Civil and Environmental Engineering at the University of Massachusetts continued to work on this project during the final year of its award. New collaborators included Professors Richard Vachet, Julian Tyson and Vincent Rotello in the Chemistry Department at UMass, Professor Douglas Anderton in the Social and Demographic Research Institute at UMass, and Dr. Ethan Clotfelter in the Biology Department at Amherst College. Additionally, eight undergraduate students worked in the Aquatic Toxicology laboratory. TARGET AUDIENCES: We have identified managers of water treatment and drinking water plants as important target audiences. We are actively enlisting their participation in providing water samples for testing. PROJECT MODIFICATIONS: The major three changes/additions in this project were made during the first year of the award. First, we decided to use the fresh water temperate fishes, Japanese medaka (Oryzias latipes) and fathead minnow (Pimephales promelas) rather than the tropical zebra fish. Second, our focus switched from the environmental toxicant, Toxaphene, to endocrine disrupting compounds found in pharmaceutical and personal care products. Third, we optimized real time RT-PCR to monitor changes in gene expression. Accordingly the title of the project should be changed to: "Fish Bioassays for Measuring the Activity of Endocrine Disrupting Compounds and other Environmental Pollutants" During the final year of this award we have broadened the environmental compounds of interest to include nanoparticles and are using Japanese Medaka fish to assess the pharmacokinetics and pharmacodynamics of nanoparticles in fish.

Impacts
This HATCH award supported the development of an Aquatic Toxicology Laboratory at UMass Amherst and the optimization of several fish bioassays which were used to prepare a proposal entitled "Establishing Guidelines for the Use of Ozone-GAC for Control of Endocrine Disruptors and Related Compounds in Water". This AWWARF-funded proposal will make three major contributions to the field of drinking water treatment. First, it will provide information to decision makers on effectiveness of ozone/biofiltration for the removal of a wide range of EDCs, PPCPs, their daughter products and associated endocrine activity under a competitive scenario where a mixture of these chemicals are spiked prior to the treatment process . Second, it will provide engineers and operators with key information on how to design and run ozone/biofiltration systems for effective control of these compounds. Finally, it will provide utilities with information on likely removals of these compounds under a broad range of water qualities and treatment scenarios.

Publications

  • Moffatt, L.T. and Arcaro, K.F. 2008. Sensitivity of the vitellogenin biomarker for aquatic estrogen detection in Japanese Medaka (Oryzias latipes) to experimental parameter variability Part 1: Time course and dose responses to 3 estrogenic contaminants. Submitted to Environmental Toxicology and Chemistry.
  • Moffatt, L.T., May, C.M., Reckhow, D., Studer, K. and Arcaro, K.F. 2008. Sensitivity of the vitellogenin biomarker for aquatic estrogen detection in Japanese Medaka (Oryzias latipes) to experimental parameter variability Part 2: Exposure volume, depuration time and feeding status. Submitted to Environmental Toxicology and Chemistry.
  • Moffatt, L.T. and Arcaro, K.F. 2008. Diclofenac is not a reproductive endocrine disruptor and does not elicit robust changes in gene expression in the Japanese Medaka (Oryzias latipes). To be submitted to Aquatic Toxicology.


Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: Information developed in this project was disseminated to communities of interest in three ways. First, posters were presented at scientific meetings in which the target audience was researchers and students in similar or related fields. A poster entitled "Quantitative Biomarkers of Estrogenic Exposure in Fish; Gene Expression Bioassays in two Model Species" was presented at the Massachusetts Water Resource Research Council Fourth Annual Meeting on April 9, 2007 in Amherst, MA. The theme of the conference was "Sustainable Waters in a Changing World: Research to Practice". Ms. Lauren Moffatt presented the poster and received "Best Student Poster Award. The same poster was presented at the American Water Resources Association Summer Specialty Conference on June 25-27, 2007 in Vail, CO. The theme of this conference was "Emerging Contaminants of Concern in the Environment: Issues, Investigations, and Solutions". Second, Kathleen Arcaro was an invited speaker at the American Water Works Association Source Water Protection Symposium on January 29 -30, 2007 in Savannah, GA. She presented a talk entitled "The Latest in Measuring Endocrine Disrupting Activity in Source Waters" and the target audience comprised mostly regulators and Water Treatment Plant managers. Third, Kathleen Arcaro and David Reckhow from University of Massachusetts worked with colleagues at Pawtucket Water Supply Board, MassDEP, and Earth Tech, Inc. to develop a proposal entitled "Establishing Guidelines for the Use of Ozone-GAC for Control of Endocrine Disruptors and Related Compounds in Water" that was submitted to the American Water Works Association Research Foundation (AWWARF). The proposal was funded by AWWARF and the medaka bioassay will be used to assess the endocrine disruptor activity in drinking water from a series of participating water treatment plants in the northeast. PARTICIPANTS: Kathleen Arcaro (PI) continued to work on the project as did Lauren Moffatt, the Ph.D. student supported in part by this award. Dr. David Reckhow, professor in Civil and Environmental Engineering at the University of Massachusetts, is a new collaborator on the project. Six undergraduate students and three high school students were trained in the Aquatic Toxicology Laboratory. All of the students learned the basics of fish husbandry. Two undergraduate students also learned to conduct fish exposures, fish dissections, RNA isolation, and real time RT-PCR. TARGET AUDIENCES: We have identified managers of water treatment and drinking water plants as important target audiences. We are actively enlisting their participation in the testing of water samples. PROJECT MODIFICATIONS: The major three changes/additions in this project were made during the first year of the award. First, we decided to use the fresh water temperate fishes, Japanese medaka (Oryzias latipes) and fathead minnow (Pimephales promelas) rather than the tropical zebra fish. Second, our focus switched from the environmental toxicant, Toxaphene, to endocrine disrupting compounds found in pharmaceutical and personal care products. Third, we optimized real time RT-PCR to monitor changes in gene expression. Accordingly the title of the project should be changed to: "Fish Bioassays for Measuring the Activity of Endocrine Disrupting Compounds"

Impacts
The bioassays developed and optimized with the support of this HATCH award were used to prepare a proposal entitled "Establishing Guidelines for the Use of Ozone-GAC for Control of Endocrine Disruptors and Related Compounds in Water". This proposal was recently funded by AWWARF. The AWWARF funded proposal will make three major contributions to the field of drinking water treatment. First, it will provide information to decision makers on effectiveness of ozone/biofiltration for the removal of a wide range of EDCs, PPCPs, their daughter products and associated endocrine activity under a competitive scenario where a mixture of these chemicals are spiked prior to the treatment process . Second, it will provide engineers and operators with key information on how to design and run ozone/biofiltration systems for effective control of these compounds. Finally, it will provide utilities with information on likely removals of these compounds under a broad range of water qualities and treatment scenarios.

Publications

  • No publications reported this period


Progress 10/01/05 to 09/30/06

Outputs
During the tenure of this award we have optimized methods for detecting estrogenic and antiestrogenic compounds in water samples using both Japanese medaka (Oryzias latipes) medaka and fathead minnows (Pimephales promelas). We use real time RT-PCR to detect and quantify changes in the expression of vitellogenin and other estrogen-responsive genes in liver, gonad and brain tissue. We found that after as little as 96-hours of exposure to 1 picomolar 17beta-estradiol, we can reliably detect significant increases in vitellogenin in livers from both male medakak and male fathead minnows. Additionally, we demonstrated that exposure to environmentally relevant (nanomolar) concentrations of the commonly detected estrogenic plasticizer, bisphenol-A can increase the expression of vitellogenin in both male and female fish.

Impacts
Bioassays are valuable tools to detect the presence of bioactive compounds in source water that have a potential to harm human health. Of particular concern are pharmaceuticals with estrogenic activity. The assays we have optimized are ideal for detecting hormonally active compounds in water. We are presently collaborating with a local company to detect estrogenic compounds in effluent.

Publications

  • No publications reported this period


Progress 10/01/04 to 09/30/05

Outputs
During the third year of this project we optimized real time reverse transcriptase-polymerase chain reaction (real time RT-PCR) methods for detecting low levels of endocrine disrupting compounds in waterways. Using the freshwater fish, Japanese medaka (Oryzias latipes) we detected increases in mRNA levels of the egg yolk precursor protein, vitellogenin, after exposure to picomolar levels of 17beta-estradiol (an endogenous estrogen) and nanomolar levels of Bisphenol-A (a plasticizer used in manufacturing, and an estrogenic environmental pollutant). We also completed both time- and dose-response analyses in both male and female fish of varying ages. Surprisingly, female fish, which already express high levels of vitellogenin were sensitive to exposures to Bisphenol-A, and responded with a 20-fold increase in vitellogenin levels after 96 hours of exposure. Additionally, we examined the effects of exposure to Atrazine and Ivermectin on mRNA levels of vitellogenin and cytochrome P450-19 (the aromatase producing gene present primarily in female gonads; aromatase converts testosterone to estradiol). Atrazine, a common herbicide used on a variety of commercial crops in the United States, has been linked to reproductive abnormalities in amphibians. Ivermectin, a veterinary pharmaceutical, is often used on livestock herds and most of the dosage is excreted in the feces. Preliminary results suggest that while low levels of Atrazine had no effect on medaka, Ivermectin increased P450-19 in males. We presented results from our research at the Water Resource Research Conference at UMass, Amherst and at the annual meeting of the Society of Environmental Toxicology and Chemistry in Baltimore, MD. These presentations lead to collaborations with investigators at Tufts University and at Fuss and O'Neill Consulting Engineers Inc. in Manchester, CT.

Impacts
Examining changes in gene expression in medaka fish using real time RT-PCR has proven to be an excellent approach for detecting endocrine disrupting compounds in water because the method is 1) sensitive, 2) reliable, 3) inexpensive, 4) can detect both parent compounds and their metabolites, and 5) can detect both common and novel pollutants. Because changes in gene expression in medaka serves as a sensitive biomarker of both exposure and effect to endocrine disrupting compounds in waterways, we anticipate that this assay will be useful in monitoring and protecting water quality in Massachusetts and beyond.

Publications

  • No publications reported this period


Progress 10/01/03 to 09/30/04

Outputs
During the second year of this project we have made significant progress towards developing inexpensive, sensitive assays for detection of pollutants in water. First, we completed a study comparing two methods of detecting the presence of estrogen-like compounds in water using the temperate freshwater fish, medaka (Oryzia latipes). We measured the production of vitellogenin in male medaka after exposure to the human estrogen, 17beta-estradiol. Expression of vitellogenin, the precursor to the egg-yolk protein, is estrogen-dependent, and therefore normally detected in female fish only. However, vitellogenin has been detected in male fish after exposure to environmental estrogens in the water. We compared the sensitivity and cost-effectiveness of a commercial method for detection of vitellogenin, with a quantitative RT-PCR method. Male fish were exposed to one of three concentrations of 17beta-estradiol (-9, -12, and -15 molar) for three days, livers were excised and RNA extracted. The level of vitellogenin mRNA was assayed 1) using a chemiluminescent-labeled probe (MLT Corp., UK), advertised to be highly sensitive for detection of vitellogenin in medaka, and 2) in a quantitative RT-PCR reaction with primers for medaka vitellogenin. The quantitative RT-PCR assay was determined to be superior to the chemiluminescent-labeled probe assay because the quantitative RT-PCR 1) required less RNA, 2) detected vitellogenin after exposure to lower concentrations of estradiol, and 3) was less expensive to run. Second, we are developing DNA reporter constructs for detection of estrogen-like and dioxin-like compounds. An estrogen or aryl hydrocarbon response element is positioned upstream of a sequence coding for green fluorescent protein (GFP), thus the response element-driven GFP expression may be detected and quantified after fish are exposed to water samples containing either estrogen- or dioxin-like chemicals. The constructs are being tested in cell culture and will be used for injection into fertilized eggs of the see-through medaka to produce stable lines of transgenic medaka for detection of environmental pollutants. We also completed construction of test chambers to be used in field studies with the transgenic medaka. Results from this work were presented in two posters at scientific meetings.

Impacts
We anticipate that the transgenic reporter fish developed in this project will be a benefit to many small communities, both rural and inner city. The reporter fish will provide an inexpensive and easy method for monitoring water quality of local streams, ponds and reservoirs. Additionally, because little sophisticated equipment is needed and the fish are not killed, this method of monitoring pollutants has intrinsic appeal to grammar and high school students, and can be incorporated into an environmental studies curriculum.

Publications

  • No publications reported this period


Progress 10/01/02 to 09/30/03

Outputs
During the first year of this project we have made significant progress on the development of our fish models for toxicity testing and monitoring. The progress includes a few important changes from the original proposal. The first significant change is test species; we have switched from zebra fish to medaka, Orzyias latipes. We have acquired a line of medaka from Japan that lack all pigment, referred to as see-through medaka. The second significant change involves an emphasis on transgenic lines that can be used for 1) developmental toxicity studies and 2) environmental monitoring of pollutants. Specifically, we are using the see-through medaka to generate several transgenic lines of medaka that will respond to estrogens and dioxin-like compounds. The transgenic medaka will contain DNA-response elements that respond to these classes of pollutants and up-regulate an easily assayable reporter gene, green fluorescent protein. These transgenic fish will be a great aid in developmental toxicity studies of toxaphene and other pollutants. Additionally, since medaka are temperate fish and tolerate a wide range of temperatures, we will be able to place these fish in cages in local streams, ponds, lakes, and reservoirs to monitor for specific classes of pollutants. Thus the transgenic "see-through" medaka will serve as sentinels of environmental pollutants, and, because will not need to kill them to detect toxic effects, the fish can be used repeatedly during their lifespan of several years.

Impacts
We anticipate that the transgenic reporter fish developed in this project will be a benefit to many small communities, both rural and inner city. The reporter fish will provide an inexpensive and easy method for monitoring water quality of local streams, ponds and reservoirs. Additionally, because little sophisticated equipment is needed and the fish are not killed, this method of monitoring pollutants has intrinsic appeal to grammar and high school students, and can be incorporated into an environmental studies curriculum.

Publications

  • No publications reported this period