- Email: [email protected]
The great lakes fisheries specimen bank
The Science of the Total Environment, 139/140 (1993) 123-138 Elsevier Science Publishers B.V., Amsterdam
123
The Great Lakes Fisheries Specimen Bank W,H. Hyatt, M.J. Keir and D.M. Whittle The Great Lakes Laboratory for Fisheries and Aquatic Sciences, Department of Fisheries and Oceans, 867 Lakeshore Road, P.O. Box 5050, Burlington, Ontario, Canada L7R 4A6 ABSTRACT The Federal Government of Canada, through the Department of Fisheries and Oceans, has been involved in specimen banking since 1977. A collection of archived samples represents more than 12000 fish that have been analysed for total PCBs, organochlorine pesticides, heavy metals and dioxin and furan isomers. Continuing research studies have focused on defining collection, storage and preservation techniques that will maintain the integrity of samples during extended storage periods. Ongoing studies will define the minimal sample aliquot to be stored in order to conserve freezer space yet provide ample tissue to permit future accurate analyses. Proposed studies will utilize archived fish tissue samples to complete an analytical review of contaminant levels to determine the relevance of recently identified contaminants in the Great Lakes ecosystem. Recent advances in analytical chemistry will be used to examine archived samples to determine temporal, spatial and trophic trends of highly toxic (co-planar) PCB isomers.
Key words: fish; specimen banking; homogenized tissue
INTRODUCTION
Specimen banking has been in existence for many years, usually in the form of museum collections. Museums traditionally store whole specimens in a liquid preservative. More recently, specimen banks have also been thought of as collections of samples or whole specimens that may be used in retrospective contaminant analyses. The Federal Government of Canada has been involved in archiving biological samples for several years. One such bank is with the Department of Fisheries and Oceans (DFO), at the Great Lakes Laboratory for Fisheries and Aquatic Sciences (GLLFAS), Burlington, Ontario. This bank was established in 1977 and contains homogenized tissue samples representing more than 12 000 fish and benthic invertebrates. Some plankton samples are also stored in the bank, but to a much lesser extent. Baseline levels of chlorinated hydrocarbon residues, trace metals and lipids have been measured for most of the stored samples. A subset of samples have baseline data for a number of 2,3,7,8 substituted
124
w.n. HYATT ET AL.
dioxin and furan isomers. The baseline contaminant levels or real time contaminant levels may be useful in establishing the accuracy of retrospective analyses. The Great Lakes Fisheries Specimen Bank (GLFSB) is a complementary project to the Great Lakes Contaminants Surveillance Program. The Contaminants Surveillance Program monitors aquatic biota in each of the Canadian Great Lakes to determine spatial and temporal contaminant trends and to investigate emerging problems, as part of the DFO commitment to the Great Lakes Water Quality Agreement (GLWQA) between Canada and the United States. The GLFSB can be used to establish the historical existence of a newly identified contaminant, or the true concentration of a contaminant that has previously been underestimated due to inadequate analytical techniques at the time. The Contaminants Surveillance Program provides a long term data set through the annual monitoring of contaminant levels in Great Lakes aquatic biota and thus may detect an emerging problem. The GLFSB provides the opportunity for analysis of historical samples and the ability to evaluate the stability of samples during storage. Together, the Contaminants Surveillance Program - - an environmental monitoring program and the Fisheries Specimen Bank form the basis for an early warning system for the detection of emerging contaminant problems in the Great Lakes aquatic community. Ongoing experimental studies are concerned with defining collection, preservation and storage conditions and techniques that will allow the optimal use of the stored samples. The GLLFAS also has an extensive collection of preserved larval fish from the Great Lakes. This type of archive may be useful to document morphological, evolutionary and community structure changes. EVOLUTION OF THE G R E A T LAKES FISHERIES SPECIMEN BANK
The Great Lakes Laboratory for Fisheries and Aquatic Sciences established a tissue bank in 1977 as a result of requirements identified by the GLWQA regarding water quality concerns in international waters between Canada and the United States. Since the inception of the GLFSB, it has evolved into an organized, valuable asset that addresses several requirements of the GLWQA, The evolution of the specimen bank has led to many changes to the collection, preparation and storage conditions of the samples collected. The GLLFAS mandate is concerned with the establishment of whole fish contaminant levels in the Great Lakes Basin. As a result, most stored samples are homogenized whole fish-tissue samples. Some fillet samples, as well as a limited number of invertebrate samples are also stored in this archive.
125
THE GREAT LAKES FISHERIES SPECIMEN BANK
The specimen bank will allow the identification and quantifying of chemicals in the future and allows future improvements in analytical techniques to be applied to representative samples from the past. The GLFSB has recently expanded its representation area from the Great Lakes Basin to the whole of Canada, with the inclusion of samples collected during the National Pulp Mill Dioxin Program. The Great Lakes Fisheries Specimen Bank has actually now become a National Fisheries Specimen Bank. Both freshwater (Table 1) and marine (Table 2) species are now represented in the specimen bank.
TABLE 1 Freshwater species represented in the Great Lakes Fisheries Specimen Bank Lake Trout Rainbow Trout Brook Trout Brown Trout Splake Coho Salmon Chinook Salmon Pink Salmon Walleye Northern Pike Yellow Perch Smallmouth Bass Largemouth Bass Rock Bass Lake Whitefish Goldeye Rainbow Smelt Slimy Sculpin Deepwater Sculpin Alewife Sponall Shiner Carp White Sucker Lake Sturgeon Brown Bullhead Burbot American Eel Freshwater Shrimp Amphipods
Salvelinus namaycush Oncorhynchus mykiss Salvelinus fontinalis Salmo trutta Salvelinus fontinalis × S. namaycush Oncorhynchus kisutch Oncorhynchus tshawytscha Oncorhynchus gorbuscha Stizostedion vitreum Esox lucius Perca flavescens Micropterus dolomieui Micropterus salmoides Ambloplites rupestris Coregonus clupeaformis Hiodon alosoides Osmerus mordax Cottus cognatus Myoxocephalus quadricornis Alosa pseudoharengus Notropis hudsonius Cyprinus carpio Catostomus cornmersoni Acipenser fulvescens Ictalurus nebulosus Lota lota Anguilla rostrata Mysis relicta Pontoporeia affinis ( Diporeia hoyi)
126
W.H. HYATT ET AL
TABLE 2 Marine species represented in the Great Lakes Fisheries Specimen Bank Atlantic Salmon
Sockeye Salmon Spring Salmon Chinook Salmon Pink Salmon Silvergrey Rockfish Pacific Red Snapper Ocean Perch American Plaice Winter Flounder English Sole Atlantic Cod Black Cod Haddock Pollock Dogfish Shark American Lobster Dungeness Crab Snow Crab Rock Crab Blue Mussel Pacific Oyster Clam Geoduck Clam Japanese Abalone Sea Scallop Shrimp Prawn
Salmo salar Oncorhynchus nerka Oncorhynchus tshawytscha Oncorhynchus kisutch Oncorhynchus gorbuscha Sebastes brevispinis Sebastes ruberrimus Sebastes marinus Hippoglossoides platessoides Pseudopleuronectes americanus Parophyrs vetulus Gadus morhua Anoplopoma fimbria Melanogrammus aeglefinus Pollachius virens Squalus acanthias Homarus americanus Cancer magister Chionoecetes opilio Cancer irroratus Mytilus edulis Crassostera gigas Mya arenaria Panope generosa Haliotis kamtschatkana Placopecten magellanicus Pandalus danae Pandalus platyceros
RATIONALE FOR THE FISHERIES SPECIMEN BANK
A strong rationale for the justification and establishment of a specimen bank is essential to ensure a continual commitment of support that is adequate to meet financial and personnel requirements of a specimen bank. In the case of the GLFSB, the GLWQA directs that a biological tissue bank be established and maintained to permit the retrospective analysis of toxic substances and calls for the identification of new or undetected problems in the Great Lakes basin ecosystem. A properly maintained specimen bank will provide an early warning system capable of establishing historical trends of previously unrecognized contaminants. Work is also needed to establish the
THE GREAT LAKES FISHERIES SPECIMEN BANK
127
optimum conditions for long term storage, preservation and handling procedures for various specimens. The discovery of previously unknown contaminants in aquatic ecosystems raises questions regarding trends and spatial distribution that cannot be accurately determined without several years of sampling. This type of information can be obtained by analyzing stored samples in a specimen bank collected during previous annual sampling activities. If a strong justification has not been previously established, lobbying and regulatory actions must be used to convince management of the importance of specimen banking as a tool for monitoring contaminant trends and as an early warning system. OPERATIONAL OBJECTIVES OF THE SPECIMEN BANK
Biological materials are stored to permit analyses to establish historical contaminant trends over time. Samples collected for the Contaminants Surveillance Program (CSP) of GLLFAS are analysed shortly after collection for trace metals and organochlorines. Replicate samples are stored in the specimen bank to permit analyses for previously unknown contaminants or for compounds that have inadequate analytical techniques at present. The CSP analyses establish real-time contaminant levels. Current or real-time contaminant levels have been established for all samples stored in the Fisheries Specimen Bank. Real time contaminant levels can be compared to results from future analyses of stored samples to determine if storage conditions were adequate to maintain the original contaminant levels. Current experimental studies are designed to define appropriate preservation and storage conditions required for maintaining the integrity of chlorinated hydrocarbon residues, for extended periods, in a variety of biological tissues. The influence of factors such as field collection and storage techniques, sample volumes, containers, storage temperatures and tissue types on specific residues must be determined and controlled in order to maintain a representative tissue archive. Materials stored in a tissue bank must provide for the accurate analysis of recently identified compounds, in order to establish historical contaminant trends for all trophic levels in the Great Lakes ecosystem. When it is possible, samples for experimental purposes are also taken from the samples processed for pesticides and metals. This gives an extra set of analytical results for a long term storage study and provides baseline contaminant levels for comparison. This also compares laboratories, if different laboratories analyse the pesticide samples and the experimental archive samples.
128
W . . . H Y A T T ET AL.
Management implications Retrospective analyses on tissue bank material for recently identified compounds will provide information of the trend in body burdens of these toxic compounds in the Great Lakes aquatic ecosystem. Therefore, decisions can be made on the necessity of evaluating potential new sources of these materials, drafting additional regulatory legislation or maintaining current monitoring activities. Strong, defensible objectives must be established to ensure the continuation of specimen banks. COLLECTION, STORAGE AND SAMPLE PROCESSING TECHNIQUES
Sample collection The method of sample collection is dependent on the specimen to be collected and the circumstances pertinent at the time of collection. Appropriate methods of sample collection have been developed and modified as situations dictate [1].
Storage methods (I) Upon capture Fresh, whole fish are placed in contaminant-free plastic bags upon capture. These bags have been tested by the United States Food and Drug Administration and by the DFO Ultra-Trace Laboratory at GLLFAS and certified to be contaminant free. It is recommended that these bags be periodically reanalysed to ensure continued quality assurance. The packaged fish are labelled and placed in insulated coolers, on dry ice. Normally the fish are frozen solid within one hour of capture. A supply of dry ice sufficient to last the entire field collection period is taken during trips if a supply near the collection site is not readily available. The fish are kept frozen until arrival at the laboratory, where they are placed in freezers at -20 to -25°C until processed, within 3 weeks of collection.
(2) In the laboratory All homogenized tissue samples are stored in pesticide grade solvent (acetone/hexane) rinsed glass jars. Solvent rinsed aluminum foil is placed over the jar opening before the screw cap is applied. Shrink seals are applied to the jars to prevent any cap movement during frozen storage. This is designed to prevent losses through volatilization in the head space of the sample container. Normally, two or three replicate samples of 20-100 g are stored in
T H E G R E A T L A K E S F I S H E R I E S S P E C I M E N BANK
129
the GLFSB for each sample set analysed for organochlorine residues, trace metals, or dioxin and furan isomers. Archived fish tissue samples, prior to 1982, were stored in regular chest freezers with a temperature o f - 2 0 to -30°C. Since 1982 samples have also been stored in ultra-low temperature freezers, at -80°C. One replicate sample is normally held at -80°C, while other samples are held at -20 to -30°C. Samples used in experimental studies to determine appropriate storage methods have been stored under various conditions. Samples have been oven-dried (+60°C), freeze-dried and frozen at -20, -40, -80, or -196°C. Samples have been prefrozen at -196°C, before being stored at -80°C. Other samples have undergone a stepped rate of freezing. These samples were held at -80°C for 24 h before being stored at -196°C. Samples have been solvent extracted and acid dissolved before storage. The solvent extracted samples were held in sealed glass ampoules at room temperature. The hydrochloric acid dissolved samples were held in sealed culture tubes, at room temperature. Oven-dried and freeze-dried samples were held in sealed glass vials at room temperature. Samples that are frozen at -20 to -80°C are usually held in glass jars. Samples held at -196°C are held in solvent-rinsed tin containers or sealed glass ampoules. These samples are held in liquid nitrogen atmosphere.
Preserved archive material Some fish specimens are preserved, whole, in Davidson's B solution or preserved, cleared and stained. These samples may be used for studies of histological or skeletal anomalies, as possible indicators of environmental stress.
Storage facilities There are seven chest freezers (580 1 each) and one walk-in freezer (14 000 1) that operate at -20°C to -30°C. There are three -80°C freezers (580 1). All freezers are equipped with emergency electrical power supply. All freezers have temperature monitors and audio and visual alarm systems that are activated when the freezer chamber temperature rises to a predetermined alarm set point. The -80°C freezers have temperature recording devices and emergency carbon dioxide back-up cooling systems. Back-up -20°C and -80°C freezers are available on a short term basis in case o f an emergency. The storage capacity of the freezers has been exceeded several times, requiring the periodic culling of stored samples.
130
w n HYATTEl" AL.
Sample processing After capture, all fish are frozen whole. Before processing, all collection and catch data and all morphometric and meristic parameters are entered into the inventory data sheets. Large fish are cut into manageable sections on a stainless steel commercial meat saw. These 'steaks' are passed through a commercial meat grinder five times to ensure adequate homogeneity. Smaller fish may be homogenized whole by passing them through the meat grinder. The homogenized tissue is subsampled into two 50 g samples for chlorinated hydrocarbon residue analysis and trace metal analysis. Where sufficient tissue is available, two 100 g samples are placed into the tissue archive. If the fish are not large enough to provide sufficient tissue for the regular samples, composite samples of five similar-sized fish are prepared. All fish samples for experimental studies are processed by the same method as the regular archive fish samples. Samples for experiments are taken from the bulk fish tissue homogenate used for regular samples when this is possible. This gives real time analytical data that is useful in later comparisons of different storage periods. Sample homogenates that will be stored frozen are usually stored in 20-50-g aliquots. The acid dissolved samples were subdivided into 5-g aliquots. For the solvent extracted samples, 150 g of fish tissue homogenate was extracted, cleaned-up by gel permeation chromatography and evenly divided and sealed in glass ampoules. In most cases five replicate samples are used for each storage condition/period to be examined (i.e. if two storage temperatures were being studied for seven storage periods, there would be 70 replicate samples). Extra samples are taken, where possible, to be used if needed. QUALITY CONTROL/QUALITY
ASSURANCE
Two 100-g samples are stored in the Fisheries Specimen Bank for each fish or composite fish sample analysed for the Contaminants Surveillance Program. Each set of samples analysed for the routine chlorinated hydrocarbon residues and trace metals includes replicate samples. Check samples are inserted randomly. Results from blind check samples produced from the same source are compared over a minimum of 2 years. Duplicate analyses are done at the rate of 10-20%. Quality control analyses for trace metals are conducted on certified reference material obtained from the National Bureau of Standards and the National Research Council. Reference standards are run at the beginning of each set of sample analyses. Data on spike recoveries, blank sample analyses and duplicate analyses are recorded. All samples used for an experimental study are replicate samples taken
THE GREAT LAKES FISHERIES SPECIMEN BANK
131
from the same bulk homogenized tissue sample at the beginning of the study. Five replicate samples are analysed for each storage condition. Duplicate analyses are done at a rate of 10%. Standards are run at the beginning of each set of samples. A method blank is spiked with all compounds to be analysed and processed through each analytical procedure to detect losses. DATA BASE MANAGEMENT
The establishment of data base programs is essential to the concept and usefulness of specimen banks. These data base programs should be developed in concert with other similar specimen banks. Since 1977 various computer data base programs and catalogues have been developed and modified for the Great Lakes Fisheries Specimen Bank. These programs have been developed using a commercial software package and are maintained and updated when required. Future requirements for specimen banking will necessitate the development of commonality among the many data bases now in existence. Data that would be common to all specimen banks should be established for existing banks and for those in the developmental stages. To ensure the compatibility and usefulness of specimen banks, a networking system should be established among the various groups that may have interests in the samples or the data. Data base files have been developed to document the history of a sample from the time of fish capture to the recording of analytical results. Initial capture and storage data When fish are captured, all pertinent information is recorded in the field and transferred to the appropriate data file at headquarters. Upon arrival at the laboratory, data are entered regarding the species, capture site, date, number of fish and location of the stored fish. As fish are removed from the freezer, the number of specimens remaining is adjusted accordingly. This data file is completed for each individual whole fish prior to processing. Contaminants Surveillance Program Capture Data When fish are processed (homogenized), each individual fish, or group of fish, that make up a series of replicate samples is given a Contaminants Surveillance Program Number (CSP No.). This CSP No. is unique to that fish or composite and enables the fish and all the subsamples of that fish to be traced throughout the storage and analytical history of the samples. Various parameters relating to the fish and the samples, such as date processed, age, sex, length, weight, sample weight, sample storage temperature, date ana-
132
w.n. HYATT ET AL.
lysed and analytical laboratory, are recorded. Any additional comments or information may be added to this form.
Sample storage~analytical laboratory information Some of the data from the Processing Sheets are entered into an Archive Sample File that contains data relating to storage time and storage temperature of the samples, number of subsamples left, type of analyses performed and analytical laboratory. From this file we can determine which samples have been analysed for chlorinated hydrocarbon residues and trace metals and what special or non-routine analyses have been done. These analytical results establish the real-time contaminant levels that can be used later for comparison.
Sample and baseline contaminants data file All analytical data, capture data, fish data and sample weights are recorded in a baseline contaminants data file. The trace metal and chlorinated hydrocarbon residue data obtained from the replicate samples analysed for the Contaminants Surveillance Program establish real-time contaminant levels for the tissue archive samples. Provisions are made in this file to record any additional analytical data that may be desired.
Integration of data bases Published data from the GLFSB has been made available to those agencies requesting information. This policy will continue, with reciprocal action requested from other agencies and individuals involved in tissue banking studies. Data base file formats are updated and modified when required. These file formats may be altered to suit those of other agencies. All information is held in an IBM compatible software data base.
Commonality of data All results, for both regular and experimental archive material, are normally reported as t~g/g, on a wet weight basis. Trace metals analysed for the regular CSP samples include: mercury, copper, nickel, zinc, lead, cadmium, chromium, arsenic and selenium. Chlorinated hydrocarbon residues routinely analysed include: heptachlor epoxide, hexachlorobenzene (HCB), o~chlordane, z chlordane, dieldrin, p,p'DDE, o,p'DDT, p,p'DDD, mirex, photomirex and total polychlorinated biphenyls (PCB). Lipid content is reported for samples analysed for chlorinated hydrocarbon residues.
THE GREAT LAKES FISHERIES SPECIMEN BANK
133
Experimental studies have included analyses for the chlorinated hydrocarbon residues listed above, total PCB, PCB isomers, ~4C-HCB, Iac-PCB and lipid. MANAGEMENT POLICY
Criteria for inclusion Fish species collected as part of the Contaminants Surveillance Program and the National Dioxin Program of the Great Lakes Laboratory for Fisheries and Aquatic Sciences are also stored in the Specimen Bank in 100-g aliquots, if sufficient tissue is available. Other biota such as macroinvertebrates and plankton are occasionally stored in the tissue archive when sufficient quantity is available. Fish species that have been archived are listed in Tables 1 and 2. Experimental studies into long term storage conditions have been carried out since 1977. Tissues that have been used in these experiments include lake trout, coho salmon, walleye, Mysis relicta and net plankton.
Access to specimens and data All samples and data collected belong to the Department of Fisheries and Oceans, Great Lakes Laboratory for Fisheries and Aquatic Sciences. All specimens are either collected directly by GLLFAS staff, or by co-operating fisheries agencies in the Great Lakes basin. Specimens are for the express use of the Contaminants Surveillance Program of GLLFAS. Archived samples may be utilized by other agencies or individuals if sufficient need and relevant objectives are demonstrated. Each request will be dealt with on an individual basis. Access to data is free and complete after the data has been published. When samples or data are used by other than the collector, appropriate reference, acknowledgement or joint authorship may be warranted. CURRENT ACTIVITIES OF THE SPECIMEN BANK The majority of samples stored in the Fisheries Specimen Bank represent specimens from the Great Lakes. These are from open water sites, from nearshore areas and from Areas of Concern identified by the International Joint Commission (Canada and the United States). Other samples represent freshwater and marine species that have been analysed as part of the National Pulp Mill Dioxin Program. Two or three 100-g samples are included in the specimen bank for each fish sample analysed for the Contaminants Surveil-
134
WH. HYATTET at.
lance Program, when sufficient material is available. In addition, selected samples of plankton and macroinvertebrates are occasionally stored as archive material for future analyses. The majority of stored samples have realtime levels of chlorinated hydrocarbon residues, trace metals and lipid. The samples from the dioxin program have baseline levels of dioxin and furan isomers, as well as lipid. Numerous studies have been completed or are ongoing that deal with the collection, handling, preservation and storage of biological tissue. Present studies deal with samples stored at -20, -80 and -196°C. Extensive studies have been done and are continuing, regarding the techniques for the extraction of lipids from biological material. The current method of lipid extraction involves the use of a commercial fat extractor, using a chloroform-methanol solvent mixture. FUTURE ACTIVITIES Replicates of all samples routinely analysed for pesticides and metals as part of the Contaminants Surveillance Program and those processed by the laboratory for special projects such as the National Pulp Mill Dioxin Program will be maintained in the GLFSB. All fish samples will be subsampled, where sufficient tissue is available, with two 100-g aliquots being stored in the Specimen Bank. These samples will be held at either -25 or -80°C until they are used or discarded. Studies will continue in order to determine the most appropriate field collection procedures, sample preparation techniques, sample volumes, storage containers, storage conditions, preservation and handling methods to maintain the quality of the samples. The impact of these factors must be determined and controlled in order to maintain a representative tissue archive. Lipid extraction methodology will be updated when appropriate to maintain the highest levels of extraction efficiency. There is the need to review and standardize procedures related to storage, reduction and statistical manipulation of the data held by various banking agencies and to define the conditions under which the samples can be released to other agencies and researchers and the manner of handling any data generated from these samples. The storage of voucher specimens, as well as homogenized tissue samples, would be of benefit to the specimen bank, but the DFO does not have the funding or the mandate to address this aspect of banking. If the storage of whole specimens is deemed to be critical to the future direction of the Specimen Bank, arrangements may be established with museums for this type of specimen storage. Ongoing studies utilizing whole lake trout homogenate samples will docu-
THE GREAT LAKES FISHERIES SPECIMEN BANK
135
ment any changes in the levels of chlorinated hydrocarbon residues and lipid concentrations during multiple thawing and refreezing of samples. If no losses are detected upon multiple thawing and refreezing, the remaining sample could be refrozen and used at a later time. This would allow us to store a 100-g sample and permit four or five different analyses over time. Concern has been expressed by some people regarding the homogenization of whole fish and the validity of the results obtained. Some whole fish samples may contain skin and bone that has not been completely homogenized. A study will be initiated to determine if the analytical result obtained for these samples are truly indicative of whole fish contaminant levels. A study to document any differences in extractability or levels of contaminants in samples of different sizes is ongoing. Results of this study may enable us to store smaller sized samples and extend the storage space available. Studies will be initiated to examine temporal trends in the congener specific levels of PCBs, to determine the relative contribution of coplanar PCBs to total PCB concentrations and to identify major unidentified components in extracts from Great Lakes fish. The concentration of PCBs in Great Lakes fish appears to have declined after the ban on their manufacture and use in 1979, but has stabilized in recent years. Recent analytical data have focused interest on a group of coplanar PCBs. The mode of toxicity of these coplanar compounds may be similar to chlorinated dioxins. Due to the relatively high concentrations of PCBs in fish these congeners may be greater than 10-fold more toxic than the dioxins and furans. Many of the archived samples have been analysed for dioxins and furans, as well as PCBs. Recent advances in analytical chemistry now allow us to re-examine these unique samples. There are limited data available on the distribution of coplanar PCBs in the Great Lakes ecosystem and there is no information available on the temporal trends. Our interpretation of the significance of PCBs and related compounds in the environment and potential impact on human health is limited by a lack of data on the temporal, spatial or trophic trends in specific highly toxic congeners (coplanar PCBs). POTENTIAL SOURCES OF ERROR The first place for sample contamination problems to be encountered is during the capture of the specimens. The use of contaminant-free plastic bags solves many of the contamination problems that can arise when using other types of containers during the field collection. The quality of the specimen must be ensured until it reaches the controlled atmosphere of the laboratory. Dry ice appears to be the best method to ensure immediate preservation of fish during the sequence of capture, field processing, storage and subsequent
136
w.n..yAx-r ET A L
transportation to the holding facility. Other types of biota, such as invertebrates and plankton, must be processed immediately upon collection before the fragile specimens begin to deteriorate. An ongoing problem with many specimen banks is the establishment of continual, long term, adequate funding. It is imperative to establish a strong rationale for a specimen bank and to display the usefulness and application of specimen banking. The Great Lakes Fisheries Specimen Bank has strong justification and rationale established under the Great Lakes Water Quality Agreement, between Canada and the United States. Mechanical problems encountered include freezer failure and electrical power interruptions. These can usually be overcome with some precautions. Physical limitations such as storage space within a freezer and limitations on the number of freezers may dictate the periodic culling or non-collection of samples. Inconsistent analytical results that fluctuate up and down during longterm storage studies are a cause for concern, not only for that particular study, but for the whole concept of accurate analytical results. This may be in part due to changing technology, changing detection limits and the individual chemist performing the analyses. It is sometimes difficult to establish reliable, accurate, baseline contaminant levels for use in storage studies. The major.problem with the long term storage of biological samples for retrospective analysis is the maintaining of adequate storage conditions. The preferred conditions will vary depending on the type of sample and the intended use. The frozen storage of whole specimens would probably allow the greatest latitude for adaptations to future methodologies. This is not practical in most cases, considering the mandate of the particular laboratory and the space and facility requirements for such a venture. Preserved museum specimens are certainly useful for many purposes, but they would be limited in their application for contaminant analyses. For the fish tissue samples we have been involved with, deep freezing appears to be the most appropriate means of sample preservation. The history of a sample must be documented through-out its storage life in order to establish a reliable and accurate paper trail to be used in legal matters. USES O F B A N K E D S A M P L E S
Archived samples have been used for the analysis of dioxins, furans, chlorinated diphenyl ethers, total PCB, PCB isomers, polycyclic aromatic hydrocarbons, pentachlorophenol, toxaphene, mirex, photomirex, organotins and other chlorinated hydrocarbon residues. Archived Lake Ontario fish and sediment samples were retrospectively analysed for mirex, an insecticide and fire retardant [2]. These samples provided information on the history and distribution of this environmental contaminant.
THE GREAT LAKES FISHERIES SPECIMEN BANK
137
An unnamed contaminant was detected in samples of Lake Michigan lake trout. When improved analytical techniques allowed its identification and quantification, analyses of stored Great Lakes fish tissue samples determined that toxaphene had been present in the Great Lakes ecosystem since the early 1970s [3]. Toxaphene was an insecticide that was used almost exclusively in the southeastern United States and was transferred to the Great Lakes through atmospheric deposition. Another example of the usefulness of specimen banking was the discovery of dioxins in the Great Lakes. Analyses of stored frozen eggs from herring gulls demonstrated that levels were significantly higher in the early seventies relative to the year the problem was first evaluated [4]. Faulty, or inadequate analytical procedures have been recognized through comparisons of experimental time study results. Tissue samples presently held in the Great Lakes Fisheries Specimen Bank present a unique opportunity to determine the historical concentration of recently identified compounds in the Great Lakes Basin. The ability to determine historical concentrations and trends of these compounds may permit the development of models relating past environmental stresses to present conditions in the biological community. It may also be possible to determine if previous pollution controls had any as yet undetermined effect on concentrations of non-target compounds and the significance of present day levels to those in the ecosystem years ago. CHALLENGES FACING THE SPECIMEN BANK
Lipid chemistry is important for many of the contaminants we are interested in. Many fish have high lipid content, some in excess of 30%. We need to determine how lipid chemistry is altered under different storage conditions and how these changes may affect the extractability and availability of certain contaminants. Are storage conditions for high lipid content samples the same as for low lipid content samples? Is there variation between species or trophic level components and associated effects on uptake, extraction and long term frozen storage? We have experienced decreases in levels of certain contaminants, followed, at a later time, with increases in the same contaminant. This pattern has been seen in several studies and among different analytical laboratories. Is this phenomenon actually happening, or is there a problem with the analytical methodology of the laboratory? SUMMARY
Based on our fish tissue studies, -80°C is the most appropriate storage temperature to maintain the chlorinated hydrocarbon residues analysed [5].
138
w.~. HYATTETAL.
The concentration of lipid in the tissue is directly related to the potential amount of organochlorine residues that can be accumulated and stored. Therefore the determination of lipid concentrations is an important component of determining the ability of tissues to store organic compounds. Lipid extraction methodology is continually upgraded to improve the efficiency of extraction. The most recent method for lipid extraction involves the use of a commercial fat extractor. Tissue archive studies have resulted in changes in specimen collection techniques, sample processing procedures and storage methods for the different biota studied. Ongoing experimental studies are concerned with further defining of collection, preservation and storage conditions required to maintain sample integrity. The tissue archive data base computer programs have greatly enhanced our ability to record, modify and trace the history of all samples collected. These programs will be updated and modified as needed. The continuing detection of anthropogenic chemicals in the Great Lakes will make the fish tissue samples stored in the Great Lakes Fisheries Specimen Bank more valuable over time. It is important that we have the foresight to maintain and expand this unique archive. REFERENCES 1 W.H. Hyatt, A Compendium of Field Methods Used by the Great Lakes Fisheries Research Branch. A Report to the Surveillance Work Group of the Great Lakes Water Quality Board, International Joint Commission, 1986. 2 K.L.E. Kaiser, The rise and fall of mirex. Environ. Sci. Technol., 12 (1978) 520-528. 3 R.A. Lewis, Guidelines for environmental specimen banking with special reference to the Federal Republic of Germany, US MAB Report No. 12, US Department of the Interior, National Park Service, 1987, p. 182. 4 D.J. Hallet and R.J. Norstrom, TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin) in Great Lakes Herring Gulls. Unpublished Manuscript. Canadian Wildlife Service, 1981. 5 W.H. Hyatt, J.D. Fitzsimmons, M.J. Keir and D.M. Whittle, Biological tissue archive studies, Canadian Technical Report of Fisheries and Aquatic Sciences, 1986, Number 1497.
123
The Great Lakes Fisheries Specimen Bank W,H. Hyatt, M.J. Keir and D.M. Whittle The Great Lakes Laboratory for Fisheries and Aquatic Sciences, Department of Fisheries and Oceans, 867 Lakeshore Road, P.O. Box 5050, Burlington, Ontario, Canada L7R 4A6 ABSTRACT The Federal Government of Canada, through the Department of Fisheries and Oceans, has been involved in specimen banking since 1977. A collection of archived samples represents more than 12000 fish that have been analysed for total PCBs, organochlorine pesticides, heavy metals and dioxin and furan isomers. Continuing research studies have focused on defining collection, storage and preservation techniques that will maintain the integrity of samples during extended storage periods. Ongoing studies will define the minimal sample aliquot to be stored in order to conserve freezer space yet provide ample tissue to permit future accurate analyses. Proposed studies will utilize archived fish tissue samples to complete an analytical review of contaminant levels to determine the relevance of recently identified contaminants in the Great Lakes ecosystem. Recent advances in analytical chemistry will be used to examine archived samples to determine temporal, spatial and trophic trends of highly toxic (co-planar) PCB isomers.
Key words: fish; specimen banking; homogenized tissue
INTRODUCTION
Specimen banking has been in existence for many years, usually in the form of museum collections. Museums traditionally store whole specimens in a liquid preservative. More recently, specimen banks have also been thought of as collections of samples or whole specimens that may be used in retrospective contaminant analyses. The Federal Government of Canada has been involved in archiving biological samples for several years. One such bank is with the Department of Fisheries and Oceans (DFO), at the Great Lakes Laboratory for Fisheries and Aquatic Sciences (GLLFAS), Burlington, Ontario. This bank was established in 1977 and contains homogenized tissue samples representing more than 12 000 fish and benthic invertebrates. Some plankton samples are also stored in the bank, but to a much lesser extent. Baseline levels of chlorinated hydrocarbon residues, trace metals and lipids have been measured for most of the stored samples. A subset of samples have baseline data for a number of 2,3,7,8 substituted
124
w.n. HYATT ET AL.
dioxin and furan isomers. The baseline contaminant levels or real time contaminant levels may be useful in establishing the accuracy of retrospective analyses. The Great Lakes Fisheries Specimen Bank (GLFSB) is a complementary project to the Great Lakes Contaminants Surveillance Program. The Contaminants Surveillance Program monitors aquatic biota in each of the Canadian Great Lakes to determine spatial and temporal contaminant trends and to investigate emerging problems, as part of the DFO commitment to the Great Lakes Water Quality Agreement (GLWQA) between Canada and the United States. The GLFSB can be used to establish the historical existence of a newly identified contaminant, or the true concentration of a contaminant that has previously been underestimated due to inadequate analytical techniques at the time. The Contaminants Surveillance Program provides a long term data set through the annual monitoring of contaminant levels in Great Lakes aquatic biota and thus may detect an emerging problem. The GLFSB provides the opportunity for analysis of historical samples and the ability to evaluate the stability of samples during storage. Together, the Contaminants Surveillance Program - - an environmental monitoring program and the Fisheries Specimen Bank form the basis for an early warning system for the detection of emerging contaminant problems in the Great Lakes aquatic community. Ongoing experimental studies are concerned with defining collection, preservation and storage conditions and techniques that will allow the optimal use of the stored samples. The GLLFAS also has an extensive collection of preserved larval fish from the Great Lakes. This type of archive may be useful to document morphological, evolutionary and community structure changes. EVOLUTION OF THE G R E A T LAKES FISHERIES SPECIMEN BANK
The Great Lakes Laboratory for Fisheries and Aquatic Sciences established a tissue bank in 1977 as a result of requirements identified by the GLWQA regarding water quality concerns in international waters between Canada and the United States. Since the inception of the GLFSB, it has evolved into an organized, valuable asset that addresses several requirements of the GLWQA, The evolution of the specimen bank has led to many changes to the collection, preparation and storage conditions of the samples collected. The GLLFAS mandate is concerned with the establishment of whole fish contaminant levels in the Great Lakes Basin. As a result, most stored samples are homogenized whole fish-tissue samples. Some fillet samples, as well as a limited number of invertebrate samples are also stored in this archive.
125
THE GREAT LAKES FISHERIES SPECIMEN BANK
The specimen bank will allow the identification and quantifying of chemicals in the future and allows future improvements in analytical techniques to be applied to representative samples from the past. The GLFSB has recently expanded its representation area from the Great Lakes Basin to the whole of Canada, with the inclusion of samples collected during the National Pulp Mill Dioxin Program. The Great Lakes Fisheries Specimen Bank has actually now become a National Fisheries Specimen Bank. Both freshwater (Table 1) and marine (Table 2) species are now represented in the specimen bank.
TABLE 1 Freshwater species represented in the Great Lakes Fisheries Specimen Bank Lake Trout Rainbow Trout Brook Trout Brown Trout Splake Coho Salmon Chinook Salmon Pink Salmon Walleye Northern Pike Yellow Perch Smallmouth Bass Largemouth Bass Rock Bass Lake Whitefish Goldeye Rainbow Smelt Slimy Sculpin Deepwater Sculpin Alewife Sponall Shiner Carp White Sucker Lake Sturgeon Brown Bullhead Burbot American Eel Freshwater Shrimp Amphipods
Salvelinus namaycush Oncorhynchus mykiss Salvelinus fontinalis Salmo trutta Salvelinus fontinalis × S. namaycush Oncorhynchus kisutch Oncorhynchus tshawytscha Oncorhynchus gorbuscha Stizostedion vitreum Esox lucius Perca flavescens Micropterus dolomieui Micropterus salmoides Ambloplites rupestris Coregonus clupeaformis Hiodon alosoides Osmerus mordax Cottus cognatus Myoxocephalus quadricornis Alosa pseudoharengus Notropis hudsonius Cyprinus carpio Catostomus cornmersoni Acipenser fulvescens Ictalurus nebulosus Lota lota Anguilla rostrata Mysis relicta Pontoporeia affinis ( Diporeia hoyi)
126
W.H. HYATT ET AL
TABLE 2 Marine species represented in the Great Lakes Fisheries Specimen Bank Atlantic Salmon
Sockeye Salmon Spring Salmon Chinook Salmon Pink Salmon Silvergrey Rockfish Pacific Red Snapper Ocean Perch American Plaice Winter Flounder English Sole Atlantic Cod Black Cod Haddock Pollock Dogfish Shark American Lobster Dungeness Crab Snow Crab Rock Crab Blue Mussel Pacific Oyster Clam Geoduck Clam Japanese Abalone Sea Scallop Shrimp Prawn
Salmo salar Oncorhynchus nerka Oncorhynchus tshawytscha Oncorhynchus kisutch Oncorhynchus gorbuscha Sebastes brevispinis Sebastes ruberrimus Sebastes marinus Hippoglossoides platessoides Pseudopleuronectes americanus Parophyrs vetulus Gadus morhua Anoplopoma fimbria Melanogrammus aeglefinus Pollachius virens Squalus acanthias Homarus americanus Cancer magister Chionoecetes opilio Cancer irroratus Mytilus edulis Crassostera gigas Mya arenaria Panope generosa Haliotis kamtschatkana Placopecten magellanicus Pandalus danae Pandalus platyceros
RATIONALE FOR THE FISHERIES SPECIMEN BANK
A strong rationale for the justification and establishment of a specimen bank is essential to ensure a continual commitment of support that is adequate to meet financial and personnel requirements of a specimen bank. In the case of the GLFSB, the GLWQA directs that a biological tissue bank be established and maintained to permit the retrospective analysis of toxic substances and calls for the identification of new or undetected problems in the Great Lakes basin ecosystem. A properly maintained specimen bank will provide an early warning system capable of establishing historical trends of previously unrecognized contaminants. Work is also needed to establish the
THE GREAT LAKES FISHERIES SPECIMEN BANK
127
optimum conditions for long term storage, preservation and handling procedures for various specimens. The discovery of previously unknown contaminants in aquatic ecosystems raises questions regarding trends and spatial distribution that cannot be accurately determined without several years of sampling. This type of information can be obtained by analyzing stored samples in a specimen bank collected during previous annual sampling activities. If a strong justification has not been previously established, lobbying and regulatory actions must be used to convince management of the importance of specimen banking as a tool for monitoring contaminant trends and as an early warning system. OPERATIONAL OBJECTIVES OF THE SPECIMEN BANK
Biological materials are stored to permit analyses to establish historical contaminant trends over time. Samples collected for the Contaminants Surveillance Program (CSP) of GLLFAS are analysed shortly after collection for trace metals and organochlorines. Replicate samples are stored in the specimen bank to permit analyses for previously unknown contaminants or for compounds that have inadequate analytical techniques at present. The CSP analyses establish real-time contaminant levels. Current or real-time contaminant levels have been established for all samples stored in the Fisheries Specimen Bank. Real time contaminant levels can be compared to results from future analyses of stored samples to determine if storage conditions were adequate to maintain the original contaminant levels. Current experimental studies are designed to define appropriate preservation and storage conditions required for maintaining the integrity of chlorinated hydrocarbon residues, for extended periods, in a variety of biological tissues. The influence of factors such as field collection and storage techniques, sample volumes, containers, storage temperatures and tissue types on specific residues must be determined and controlled in order to maintain a representative tissue archive. Materials stored in a tissue bank must provide for the accurate analysis of recently identified compounds, in order to establish historical contaminant trends for all trophic levels in the Great Lakes ecosystem. When it is possible, samples for experimental purposes are also taken from the samples processed for pesticides and metals. This gives an extra set of analytical results for a long term storage study and provides baseline contaminant levels for comparison. This also compares laboratories, if different laboratories analyse the pesticide samples and the experimental archive samples.
128
W . . . H Y A T T ET AL.
Management implications Retrospective analyses on tissue bank material for recently identified compounds will provide information of the trend in body burdens of these toxic compounds in the Great Lakes aquatic ecosystem. Therefore, decisions can be made on the necessity of evaluating potential new sources of these materials, drafting additional regulatory legislation or maintaining current monitoring activities. Strong, defensible objectives must be established to ensure the continuation of specimen banks. COLLECTION, STORAGE AND SAMPLE PROCESSING TECHNIQUES
Sample collection The method of sample collection is dependent on the specimen to be collected and the circumstances pertinent at the time of collection. Appropriate methods of sample collection have been developed and modified as situations dictate [1].
Storage methods (I) Upon capture Fresh, whole fish are placed in contaminant-free plastic bags upon capture. These bags have been tested by the United States Food and Drug Administration and by the DFO Ultra-Trace Laboratory at GLLFAS and certified to be contaminant free. It is recommended that these bags be periodically reanalysed to ensure continued quality assurance. The packaged fish are labelled and placed in insulated coolers, on dry ice. Normally the fish are frozen solid within one hour of capture. A supply of dry ice sufficient to last the entire field collection period is taken during trips if a supply near the collection site is not readily available. The fish are kept frozen until arrival at the laboratory, where they are placed in freezers at -20 to -25°C until processed, within 3 weeks of collection.
(2) In the laboratory All homogenized tissue samples are stored in pesticide grade solvent (acetone/hexane) rinsed glass jars. Solvent rinsed aluminum foil is placed over the jar opening before the screw cap is applied. Shrink seals are applied to the jars to prevent any cap movement during frozen storage. This is designed to prevent losses through volatilization in the head space of the sample container. Normally, two or three replicate samples of 20-100 g are stored in
T H E G R E A T L A K E S F I S H E R I E S S P E C I M E N BANK
129
the GLFSB for each sample set analysed for organochlorine residues, trace metals, or dioxin and furan isomers. Archived fish tissue samples, prior to 1982, were stored in regular chest freezers with a temperature o f - 2 0 to -30°C. Since 1982 samples have also been stored in ultra-low temperature freezers, at -80°C. One replicate sample is normally held at -80°C, while other samples are held at -20 to -30°C. Samples used in experimental studies to determine appropriate storage methods have been stored under various conditions. Samples have been oven-dried (+60°C), freeze-dried and frozen at -20, -40, -80, or -196°C. Samples have been prefrozen at -196°C, before being stored at -80°C. Other samples have undergone a stepped rate of freezing. These samples were held at -80°C for 24 h before being stored at -196°C. Samples have been solvent extracted and acid dissolved before storage. The solvent extracted samples were held in sealed glass ampoules at room temperature. The hydrochloric acid dissolved samples were held in sealed culture tubes, at room temperature. Oven-dried and freeze-dried samples were held in sealed glass vials at room temperature. Samples that are frozen at -20 to -80°C are usually held in glass jars. Samples held at -196°C are held in solvent-rinsed tin containers or sealed glass ampoules. These samples are held in liquid nitrogen atmosphere.
Preserved archive material Some fish specimens are preserved, whole, in Davidson's B solution or preserved, cleared and stained. These samples may be used for studies of histological or skeletal anomalies, as possible indicators of environmental stress.
Storage facilities There are seven chest freezers (580 1 each) and one walk-in freezer (14 000 1) that operate at -20°C to -30°C. There are three -80°C freezers (580 1). All freezers are equipped with emergency electrical power supply. All freezers have temperature monitors and audio and visual alarm systems that are activated when the freezer chamber temperature rises to a predetermined alarm set point. The -80°C freezers have temperature recording devices and emergency carbon dioxide back-up cooling systems. Back-up -20°C and -80°C freezers are available on a short term basis in case o f an emergency. The storage capacity of the freezers has been exceeded several times, requiring the periodic culling of stored samples.
130
w n HYATTEl" AL.
Sample processing After capture, all fish are frozen whole. Before processing, all collection and catch data and all morphometric and meristic parameters are entered into the inventory data sheets. Large fish are cut into manageable sections on a stainless steel commercial meat saw. These 'steaks' are passed through a commercial meat grinder five times to ensure adequate homogeneity. Smaller fish may be homogenized whole by passing them through the meat grinder. The homogenized tissue is subsampled into two 50 g samples for chlorinated hydrocarbon residue analysis and trace metal analysis. Where sufficient tissue is available, two 100 g samples are placed into the tissue archive. If the fish are not large enough to provide sufficient tissue for the regular samples, composite samples of five similar-sized fish are prepared. All fish samples for experimental studies are processed by the same method as the regular archive fish samples. Samples for experiments are taken from the bulk fish tissue homogenate used for regular samples when this is possible. This gives real time analytical data that is useful in later comparisons of different storage periods. Sample homogenates that will be stored frozen are usually stored in 20-50-g aliquots. The acid dissolved samples were subdivided into 5-g aliquots. For the solvent extracted samples, 150 g of fish tissue homogenate was extracted, cleaned-up by gel permeation chromatography and evenly divided and sealed in glass ampoules. In most cases five replicate samples are used for each storage condition/period to be examined (i.e. if two storage temperatures were being studied for seven storage periods, there would be 70 replicate samples). Extra samples are taken, where possible, to be used if needed. QUALITY CONTROL/QUALITY
ASSURANCE
Two 100-g samples are stored in the Fisheries Specimen Bank for each fish or composite fish sample analysed for the Contaminants Surveillance Program. Each set of samples analysed for the routine chlorinated hydrocarbon residues and trace metals includes replicate samples. Check samples are inserted randomly. Results from blind check samples produced from the same source are compared over a minimum of 2 years. Duplicate analyses are done at the rate of 10-20%. Quality control analyses for trace metals are conducted on certified reference material obtained from the National Bureau of Standards and the National Research Council. Reference standards are run at the beginning of each set of sample analyses. Data on spike recoveries, blank sample analyses and duplicate analyses are recorded. All samples used for an experimental study are replicate samples taken
THE GREAT LAKES FISHERIES SPECIMEN BANK
131
from the same bulk homogenized tissue sample at the beginning of the study. Five replicate samples are analysed for each storage condition. Duplicate analyses are done at a rate of 10%. Standards are run at the beginning of each set of samples. A method blank is spiked with all compounds to be analysed and processed through each analytical procedure to detect losses. DATA BASE MANAGEMENT
The establishment of data base programs is essential to the concept and usefulness of specimen banks. These data base programs should be developed in concert with other similar specimen banks. Since 1977 various computer data base programs and catalogues have been developed and modified for the Great Lakes Fisheries Specimen Bank. These programs have been developed using a commercial software package and are maintained and updated when required. Future requirements for specimen banking will necessitate the development of commonality among the many data bases now in existence. Data that would be common to all specimen banks should be established for existing banks and for those in the developmental stages. To ensure the compatibility and usefulness of specimen banks, a networking system should be established among the various groups that may have interests in the samples or the data. Data base files have been developed to document the history of a sample from the time of fish capture to the recording of analytical results. Initial capture and storage data When fish are captured, all pertinent information is recorded in the field and transferred to the appropriate data file at headquarters. Upon arrival at the laboratory, data are entered regarding the species, capture site, date, number of fish and location of the stored fish. As fish are removed from the freezer, the number of specimens remaining is adjusted accordingly. This data file is completed for each individual whole fish prior to processing. Contaminants Surveillance Program Capture Data When fish are processed (homogenized), each individual fish, or group of fish, that make up a series of replicate samples is given a Contaminants Surveillance Program Number (CSP No.). This CSP No. is unique to that fish or composite and enables the fish and all the subsamples of that fish to be traced throughout the storage and analytical history of the samples. Various parameters relating to the fish and the samples, such as date processed, age, sex, length, weight, sample weight, sample storage temperature, date ana-
132
w.n. HYATT ET AL.
lysed and analytical laboratory, are recorded. Any additional comments or information may be added to this form.
Sample storage~analytical laboratory information Some of the data from the Processing Sheets are entered into an Archive Sample File that contains data relating to storage time and storage temperature of the samples, number of subsamples left, type of analyses performed and analytical laboratory. From this file we can determine which samples have been analysed for chlorinated hydrocarbon residues and trace metals and what special or non-routine analyses have been done. These analytical results establish the real-time contaminant levels that can be used later for comparison.
Sample and baseline contaminants data file All analytical data, capture data, fish data and sample weights are recorded in a baseline contaminants data file. The trace metal and chlorinated hydrocarbon residue data obtained from the replicate samples analysed for the Contaminants Surveillance Program establish real-time contaminant levels for the tissue archive samples. Provisions are made in this file to record any additional analytical data that may be desired.
Integration of data bases Published data from the GLFSB has been made available to those agencies requesting information. This policy will continue, with reciprocal action requested from other agencies and individuals involved in tissue banking studies. Data base file formats are updated and modified when required. These file formats may be altered to suit those of other agencies. All information is held in an IBM compatible software data base.
Commonality of data All results, for both regular and experimental archive material, are normally reported as t~g/g, on a wet weight basis. Trace metals analysed for the regular CSP samples include: mercury, copper, nickel, zinc, lead, cadmium, chromium, arsenic and selenium. Chlorinated hydrocarbon residues routinely analysed include: heptachlor epoxide, hexachlorobenzene (HCB), o~chlordane, z chlordane, dieldrin, p,p'DDE, o,p'DDT, p,p'DDD, mirex, photomirex and total polychlorinated biphenyls (PCB). Lipid content is reported for samples analysed for chlorinated hydrocarbon residues.
THE GREAT LAKES FISHERIES SPECIMEN BANK
133
Experimental studies have included analyses for the chlorinated hydrocarbon residues listed above, total PCB, PCB isomers, ~4C-HCB, Iac-PCB and lipid. MANAGEMENT POLICY
Criteria for inclusion Fish species collected as part of the Contaminants Surveillance Program and the National Dioxin Program of the Great Lakes Laboratory for Fisheries and Aquatic Sciences are also stored in the Specimen Bank in 100-g aliquots, if sufficient tissue is available. Other biota such as macroinvertebrates and plankton are occasionally stored in the tissue archive when sufficient quantity is available. Fish species that have been archived are listed in Tables 1 and 2. Experimental studies into long term storage conditions have been carried out since 1977. Tissues that have been used in these experiments include lake trout, coho salmon, walleye, Mysis relicta and net plankton.
Access to specimens and data All samples and data collected belong to the Department of Fisheries and Oceans, Great Lakes Laboratory for Fisheries and Aquatic Sciences. All specimens are either collected directly by GLLFAS staff, or by co-operating fisheries agencies in the Great Lakes basin. Specimens are for the express use of the Contaminants Surveillance Program of GLLFAS. Archived samples may be utilized by other agencies or individuals if sufficient need and relevant objectives are demonstrated. Each request will be dealt with on an individual basis. Access to data is free and complete after the data has been published. When samples or data are used by other than the collector, appropriate reference, acknowledgement or joint authorship may be warranted. CURRENT ACTIVITIES OF THE SPECIMEN BANK The majority of samples stored in the Fisheries Specimen Bank represent specimens from the Great Lakes. These are from open water sites, from nearshore areas and from Areas of Concern identified by the International Joint Commission (Canada and the United States). Other samples represent freshwater and marine species that have been analysed as part of the National Pulp Mill Dioxin Program. Two or three 100-g samples are included in the specimen bank for each fish sample analysed for the Contaminants Surveil-
134
WH. HYATTET at.
lance Program, when sufficient material is available. In addition, selected samples of plankton and macroinvertebrates are occasionally stored as archive material for future analyses. The majority of stored samples have realtime levels of chlorinated hydrocarbon residues, trace metals and lipid. The samples from the dioxin program have baseline levels of dioxin and furan isomers, as well as lipid. Numerous studies have been completed or are ongoing that deal with the collection, handling, preservation and storage of biological tissue. Present studies deal with samples stored at -20, -80 and -196°C. Extensive studies have been done and are continuing, regarding the techniques for the extraction of lipids from biological material. The current method of lipid extraction involves the use of a commercial fat extractor, using a chloroform-methanol solvent mixture. FUTURE ACTIVITIES Replicates of all samples routinely analysed for pesticides and metals as part of the Contaminants Surveillance Program and those processed by the laboratory for special projects such as the National Pulp Mill Dioxin Program will be maintained in the GLFSB. All fish samples will be subsampled, where sufficient tissue is available, with two 100-g aliquots being stored in the Specimen Bank. These samples will be held at either -25 or -80°C until they are used or discarded. Studies will continue in order to determine the most appropriate field collection procedures, sample preparation techniques, sample volumes, storage containers, storage conditions, preservation and handling methods to maintain the quality of the samples. The impact of these factors must be determined and controlled in order to maintain a representative tissue archive. Lipid extraction methodology will be updated when appropriate to maintain the highest levels of extraction efficiency. There is the need to review and standardize procedures related to storage, reduction and statistical manipulation of the data held by various banking agencies and to define the conditions under which the samples can be released to other agencies and researchers and the manner of handling any data generated from these samples. The storage of voucher specimens, as well as homogenized tissue samples, would be of benefit to the specimen bank, but the DFO does not have the funding or the mandate to address this aspect of banking. If the storage of whole specimens is deemed to be critical to the future direction of the Specimen Bank, arrangements may be established with museums for this type of specimen storage. Ongoing studies utilizing whole lake trout homogenate samples will docu-
THE GREAT LAKES FISHERIES SPECIMEN BANK
135
ment any changes in the levels of chlorinated hydrocarbon residues and lipid concentrations during multiple thawing and refreezing of samples. If no losses are detected upon multiple thawing and refreezing, the remaining sample could be refrozen and used at a later time. This would allow us to store a 100-g sample and permit four or five different analyses over time. Concern has been expressed by some people regarding the homogenization of whole fish and the validity of the results obtained. Some whole fish samples may contain skin and bone that has not been completely homogenized. A study will be initiated to determine if the analytical result obtained for these samples are truly indicative of whole fish contaminant levels. A study to document any differences in extractability or levels of contaminants in samples of different sizes is ongoing. Results of this study may enable us to store smaller sized samples and extend the storage space available. Studies will be initiated to examine temporal trends in the congener specific levels of PCBs, to determine the relative contribution of coplanar PCBs to total PCB concentrations and to identify major unidentified components in extracts from Great Lakes fish. The concentration of PCBs in Great Lakes fish appears to have declined after the ban on their manufacture and use in 1979, but has stabilized in recent years. Recent analytical data have focused interest on a group of coplanar PCBs. The mode of toxicity of these coplanar compounds may be similar to chlorinated dioxins. Due to the relatively high concentrations of PCBs in fish these congeners may be greater than 10-fold more toxic than the dioxins and furans. Many of the archived samples have been analysed for dioxins and furans, as well as PCBs. Recent advances in analytical chemistry now allow us to re-examine these unique samples. There are limited data available on the distribution of coplanar PCBs in the Great Lakes ecosystem and there is no information available on the temporal trends. Our interpretation of the significance of PCBs and related compounds in the environment and potential impact on human health is limited by a lack of data on the temporal, spatial or trophic trends in specific highly toxic congeners (coplanar PCBs). POTENTIAL SOURCES OF ERROR The first place for sample contamination problems to be encountered is during the capture of the specimens. The use of contaminant-free plastic bags solves many of the contamination problems that can arise when using other types of containers during the field collection. The quality of the specimen must be ensured until it reaches the controlled atmosphere of the laboratory. Dry ice appears to be the best method to ensure immediate preservation of fish during the sequence of capture, field processing, storage and subsequent
136
w.n..yAx-r ET A L
transportation to the holding facility. Other types of biota, such as invertebrates and plankton, must be processed immediately upon collection before the fragile specimens begin to deteriorate. An ongoing problem with many specimen banks is the establishment of continual, long term, adequate funding. It is imperative to establish a strong rationale for a specimen bank and to display the usefulness and application of specimen banking. The Great Lakes Fisheries Specimen Bank has strong justification and rationale established under the Great Lakes Water Quality Agreement, between Canada and the United States. Mechanical problems encountered include freezer failure and electrical power interruptions. These can usually be overcome with some precautions. Physical limitations such as storage space within a freezer and limitations on the number of freezers may dictate the periodic culling or non-collection of samples. Inconsistent analytical results that fluctuate up and down during longterm storage studies are a cause for concern, not only for that particular study, but for the whole concept of accurate analytical results. This may be in part due to changing technology, changing detection limits and the individual chemist performing the analyses. It is sometimes difficult to establish reliable, accurate, baseline contaminant levels for use in storage studies. The major.problem with the long term storage of biological samples for retrospective analysis is the maintaining of adequate storage conditions. The preferred conditions will vary depending on the type of sample and the intended use. The frozen storage of whole specimens would probably allow the greatest latitude for adaptations to future methodologies. This is not practical in most cases, considering the mandate of the particular laboratory and the space and facility requirements for such a venture. Preserved museum specimens are certainly useful for many purposes, but they would be limited in their application for contaminant analyses. For the fish tissue samples we have been involved with, deep freezing appears to be the most appropriate means of sample preservation. The history of a sample must be documented through-out its storage life in order to establish a reliable and accurate paper trail to be used in legal matters. USES O F B A N K E D S A M P L E S
Archived samples have been used for the analysis of dioxins, furans, chlorinated diphenyl ethers, total PCB, PCB isomers, polycyclic aromatic hydrocarbons, pentachlorophenol, toxaphene, mirex, photomirex, organotins and other chlorinated hydrocarbon residues. Archived Lake Ontario fish and sediment samples were retrospectively analysed for mirex, an insecticide and fire retardant [2]. These samples provided information on the history and distribution of this environmental contaminant.
THE GREAT LAKES FISHERIES SPECIMEN BANK
137
An unnamed contaminant was detected in samples of Lake Michigan lake trout. When improved analytical techniques allowed its identification and quantification, analyses of stored Great Lakes fish tissue samples determined that toxaphene had been present in the Great Lakes ecosystem since the early 1970s [3]. Toxaphene was an insecticide that was used almost exclusively in the southeastern United States and was transferred to the Great Lakes through atmospheric deposition. Another example of the usefulness of specimen banking was the discovery of dioxins in the Great Lakes. Analyses of stored frozen eggs from herring gulls demonstrated that levels were significantly higher in the early seventies relative to the year the problem was first evaluated [4]. Faulty, or inadequate analytical procedures have been recognized through comparisons of experimental time study results. Tissue samples presently held in the Great Lakes Fisheries Specimen Bank present a unique opportunity to determine the historical concentration of recently identified compounds in the Great Lakes Basin. The ability to determine historical concentrations and trends of these compounds may permit the development of models relating past environmental stresses to present conditions in the biological community. It may also be possible to determine if previous pollution controls had any as yet undetermined effect on concentrations of non-target compounds and the significance of present day levels to those in the ecosystem years ago. CHALLENGES FACING THE SPECIMEN BANK
Lipid chemistry is important for many of the contaminants we are interested in. Many fish have high lipid content, some in excess of 30%. We need to determine how lipid chemistry is altered under different storage conditions and how these changes may affect the extractability and availability of certain contaminants. Are storage conditions for high lipid content samples the same as for low lipid content samples? Is there variation between species or trophic level components and associated effects on uptake, extraction and long term frozen storage? We have experienced decreases in levels of certain contaminants, followed, at a later time, with increases in the same contaminant. This pattern has been seen in several studies and among different analytical laboratories. Is this phenomenon actually happening, or is there a problem with the analytical methodology of the laboratory? SUMMARY
Based on our fish tissue studies, -80°C is the most appropriate storage temperature to maintain the chlorinated hydrocarbon residues analysed [5].
138
w.~. HYATTETAL.
The concentration of lipid in the tissue is directly related to the potential amount of organochlorine residues that can be accumulated and stored. Therefore the determination of lipid concentrations is an important component of determining the ability of tissues to store organic compounds. Lipid extraction methodology is continually upgraded to improve the efficiency of extraction. The most recent method for lipid extraction involves the use of a commercial fat extractor. Tissue archive studies have resulted in changes in specimen collection techniques, sample processing procedures and storage methods for the different biota studied. Ongoing experimental studies are concerned with further defining of collection, preservation and storage conditions required to maintain sample integrity. The tissue archive data base computer programs have greatly enhanced our ability to record, modify and trace the history of all samples collected. These programs will be updated and modified as needed. The continuing detection of anthropogenic chemicals in the Great Lakes will make the fish tissue samples stored in the Great Lakes Fisheries Specimen Bank more valuable over time. It is important that we have the foresight to maintain and expand this unique archive. REFERENCES 1 W.H. Hyatt, A Compendium of Field Methods Used by the Great Lakes Fisheries Research Branch. A Report to the Surveillance Work Group of the Great Lakes Water Quality Board, International Joint Commission, 1986. 2 K.L.E. Kaiser, The rise and fall of mirex. Environ. Sci. Technol., 12 (1978) 520-528. 3 R.A. Lewis, Guidelines for environmental specimen banking with special reference to the Federal Republic of Germany, US MAB Report No. 12, US Department of the Interior, National Park Service, 1987, p. 182. 4 D.J. Hallet and R.J. Norstrom, TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin) in Great Lakes Herring Gulls. Unpublished Manuscript. Canadian Wildlife Service, 1981. 5 W.H. Hyatt, J.D. Fitzsimmons, M.J. Keir and D.M. Whittle, Biological tissue archive studies, Canadian Technical Report of Fisheries and Aquatic Sciences, 1986, Number 1497.