Methyl Mercury and Heavy Metal Contaminant Levels in Alaskan Halibut

Methyl mercury and heavy metal contaminant levels in Alaskan halibut

Claude L. Dykstra

Abstract

During the setline surveys in 2002, the International Pacific Halibut Commission (IPHC) collected halibut muscle and liver tissue samples from locations within Alaska as part of a larger study on environmental contaminants in fish being conducted by the Alaska Department of Environmen- tal Conservation. In 2003, the principal samples were analyzed for heavy metals and the preliminary data have been released and are reported here. Initial results support the Alaska Division of Public Healths belief that all Alaskans, including pregnant women and children, are not in danger when consuming fish from Alaskan waters.

Introduction

Recent reports from health officials and media have raised the profile of mercury contamination in fish. In 2002, the Alaska Department of Environmental Conservation (ADEC) in conjunc- tion with the Environmental Protection Agency (EPA), launched an environmental contamination study looking into levels of organochlorine pesticides, dioxins, furans, polybrominated diphenyl ethers, PCB congeners, methyl mercury and heavy metals (arsenic, selenium, lead, cadmium, nickel, chromium) within 13 Alaskan fish species, including halibut. During the setline surveys in 2002, the IPHC collected 60 halibut muscle and liver tissue samples from eight locations within Alaska for the principal ADEC study and 58 flesh samples for additional methyl mercury analyses. In 2003 the principal samples were analyzed for heavy metals and methyl mercury. A commercial lab is currently in the process of analyzing a subset of these halibut samples for levels of organochlorine pesticides, dioxins, furans, polybrominated diphenyl ethers, and PCB congeners and these data will be reported, as they become available.

Methods

Halibut samples where collected from standard grid survey vessel operations during the summer of 2002. Sampling focussed on stations in the following general locations: southeast of Ketchikan, southeast of Juneau, Prince William Sound, Cook Inlet, Kodiak, Dutch Harbor and the Bering Sea. The goal at each site was to collect samples from four fish in the 20 to 40 pound category and four fish in the 40 to 100 pound category. All samples came from fish targeted for an age sample (otolith) and were free from any gross abnormalities or disease. After processing the fish in the usual manner and collecting an otolith from it, samplers collected a three to five pound fillet from behind the head while wearing nitrile gloves, and stored the sample in a food grade plastic bag. The liver was then removed, placed in a separate food grade plastic bag, sealed and labeled. Care was taken in all cases to avoid any gross contamination of the fish with bilge, waste- 323 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 water, fuel or exhaust emissions. Upon landing, all samples where shipped to ADECs Seafood and Food Safety Lab in Palmer, AK. As methyl mercury is currently a topical issue in seafood, ADEC agreed to analyze some additional samples we provided. An additional 58 samples of muscle tissue (approximately 1/3 of a cup in size) where collected from six of the locations to be sampled for methyl mercury alone. ADEC prepared the samples for analysis using a standard protocol outlined in the Quality Assurance Project Plan established for the study (Gerlach, 2002). All samples where prepared from skinless fillets, and fatty tissue located between the skin and muscle was removed. The Seafood and Safety Lab analyzed the samples for arsenic, cadmium, chromium, lead, methyl mercury and nickel. Those results are reported here. A commercial lab has been contracted to analyze the samples for pesticides, selected PCB congeners, dioxins, and furans following EPA approved methods. The results from the commercial lab will be published, as they are available.

Results

While 64 samples where targeted for collection, a total of 60 samples where successfully collected from the target areas. Heavy metal concentrations for the samples are listed in Table 1. FDA Action levels exist only for crustacea and molluscan bivalves, and are listed as a base reference. A large percentage of the samples registered non-detectable levels of cadmium and chromium. Nickel readings came out below the detectable range (0.02 ppm at 95% confidence interval) for all of the samples tested. A total of 118 samples were tested for methyl mercury. Mean methyl mercury levels by region are listed in Table 2. All analyses were performed on skinless muscle tissue. The average methyl mercury level found in these samples was 0.2054 ppm. The FDA level of concern is 1.00 ppm, and the EPA action level is 0.50 ppm. The average levels of methyl mercury in these halibut are less than 50% and 25% of these reference levels, respectively.

Discussion

This joint study with ADEC shows that average levels of heavy metals and methyl mercury in Alaskan Pacific halibut are well below levels of concern of both the FDA and EPA. Currently there is discrepancy between the levels of concern of these two agencies for methyl mercury (1.00 ppm and 0.5 ppm respectively) as well as how the two standards are calculated. The FDA is expected to update their reference level of concern in December of 2003. According to the Alaska Division of Public Health, the concentrations of heavy metals and methyl mercury detected in these samples are not a public health concern. The data support the Divisions belief that all Alaskans, including pregnant women and children, are not in danger when consuming halibut from Alaskan waters. These data appear to support the findings of Hall et al. (1976) in which the methyl mercury concentration increased in fish of the same size from the northern to the southern part of the geographic range studied. The IPHC and ADEC are continuing to qualify the data with physical param- eters (age, size, and weight) and additional analyses will be done on the samples. This joint project was continued in 2003 with a more focussed effort on three areas (Bering Sea, Gulf of Alaska, southeast Alaska). The data for these samples will add to our understanding of contaminant levels

324 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 in Pacific halibut, and how it relates to the commercial fishery. The IPHC anticipates working cooperatively with ADEC for the foreseeable future on this project.

Acknowledgements

We appreciate the cooperation and lab analysis performed by Bob Gerlach and his team at ADEC on this joint project.

References

Gerlach, R., Grimm R., Patrick-Riley, K., Beelman, J. 2002. Quality Assurance Project Plan: Fish Safety Monitoring Plan. Alaska Department of Environmental Conservation, Seafood and Food Safety Laboratory, Palmer, Alaska.

Hall, A. S., Teeny F. M., Lewis, L. G., Hardman, W. H., and Gauglitz, E. J. Jr. 1976. Mercury in fish and shellfish of the northeast Pacific. I. Pacific Halibut, Hippoglossus stenolepis. Fishery Bulletin. Vol. 74: 783-789.

325 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 Table 1. Average heavy metal concentration (ppm) from Pacific halibut sampled in 2002.

Number of Number FDA action levels Samples of non- Minimum Maximum Mean Standard (ppm) Analyzed detects Value Value Value Deviation Arsenic 76 (for Crustacea)1 60 0 0.2000 5.3000 1.5692 0.9860 Cadmium 3 (for Crustacea)1 60 38 <0.0020 0.0050 0.0017 0.0011 Chromium 12 (for Crustacea)1 60 49 <0.0060 0.0260 0.0046 0.0042 Lead 1.5 (for Crustacea)1 60 5 <0.0200 0.0500 0.0307 0.0086 Nickel 70 (for Crustacea)1 60 60 <0.0200 <0.0200 N/A N/A Selenium N/A 60 0 0.1000 0.6100 0.2607 0.1193

* The FDA Action/Guidance Levels exist only for Crustacea and molluscan bivalves.

Table 2. Average methyl mercury concentration (ppm) by region from Pacific halibut sampled in 2002. Number of Mean Standard Region Samples Value* Deviation Cook Inlet 180.1009 0.0789 Aleutian 18 0.1036 0.0757 Kodiak 18 0.1551 0.0805 Prince William Sound 13 0.1559 0.1026 Bering Sea 18 0.1583 0.1539 Juneau 8 0.2309 0.1571 Cordova 70.3284 0.3356 Ketchikan 18 0.4854 0.3772 Total 118 0.2054 0.0211

*The FDA level of concern for methyl mercury is 1.0 ppm. This level is currently being reassessed, and may be adjusted down in December. Health Canadas guideline for total mercury is 0.5 ppm.

326 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 Project report: Understanding chalky halibut

Charles Crapo, Robert Foy, and Donald Kramer University of Alaska Fairbanks, Fishery Industrial Technology Center (Kodiak)

Abstract

The International Pacific Halibut Commission, on behalf of the industry, contracted with Uni- versity of Alaska Fairbanks Fishery Industrial Technology Center in Kodiak to examine the causes of chalk, examining and describing the actual biochemical process that generates the chalky condition, and the potential for mitigation or treatment of the condition. Their results of a laboratory simulation of catch-struggle and increased ambient temperature on 27 halibut failed to produce chalk. The inability to produce chalkiness was likely due to several factors. The fish were collected in late fall 2002 and had a high moisture, low protein and fat content, indicating perhaps recovery from spawning was one element. The test fish also had higher muscle pH and many of the females had well developed roe sacs. The authors suggest the experiments be repeated with fish collected at a different time of year.

Introduction

In the fall of 2002, IPHC contracted with the University of Alaska Fairbanks Fishery Industrial Technology Center in Kodiak to conduct research into the causes of chalk, examining and describing the actual biochemical process that generates the chalky condition, and the potential for mitigation or treatment of the condition in Pacific halibut. The project was jointly funded by IPHC and the Halibut Association of North America (HANA), on behalf of the industry. The summary below is excerpted from the draft project report submitted to IPHC in late August, 2003. A final report has not yet been accepted.

Project summary

Laboratory simulation of catch struggle and increased ambient temperature failed to produce chalky halibut. Of the 27 fish subjected to three, six or 12 hours of struggle on a longline, only three exhibited chalkiness. The remainder showed an initial drop of muscle pH as the result of the struggle and then a gradual decline over five or six day refrigerated storage periods. Final muscle pH of treated fish was similar to the control fish. Fish muscle had low glucose levels that limited their production of lactic acid. In addition, the high initial muscle pH, often above 7.3 was considered unusual. In the experience of the researchers, muscle pH of normal halibut usually is around 6.7 to 6.9. Raising the ambient temperature to 12ºC for three days had no effect on the development of chalkiness. It was notable that the temperature-abused fish continually swam in the tanks during the test period and were completely exhausted. However, pH decrease over subsequent storage time did not approach values needed to produce chalkiness. Lactic acid and glucose levels were low. The inability to produce chalkiness was likely due to several factors. Using late fall halibut that had high moisture, low protein and fat indicating perhaps recovery from spawning was one element. The test fish also had higher muscle pH and many of the females had well developed roe sacs. 327 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 328 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 A project to test the use of digital video for monitoring the compliance of seabird avoidance devices and seabird mortality in halibut longline fisheries

Robert N. T. Ames and Gregg H. Williams International Pacific Halibut Commission

Shannon M. Fitzgerald National Marine Fisheries Service

Abstract

Under contract to National Marine Fisheries Service (NMFS), the International Pacific Halibut Commission investigated the ability of an electronic monitoring system (EMS) to perform two functions: (1) detect and monitor seabird avoidance devices behind a vessel setting halibut gear; and (2) monitor the incidental catch of seabirds. The project was conducted on two stock assessment survey vessels in 2002. Comparing 106 paired vessel and video observations on halibut gear being set demonstrated that EMS was successful in detecting streamer line deployment and relative position on 100 percent of daytime sets when two stern cameras operated in tandem. Examination of the images of retrieved dead seabirds at the roller using two cameras showed improved species recognition with a higher recording speed. Full monitoring of all setting and haul backs in the halibut fishery off Alaska was estimated at $8.5 million for an on-board observer program; an EMS was estimated at $2.7 million A final report was submitted to NMFS on September 3, 2003 and will be distributed as a NOAA Technical Memorandum in late 2003 or early 2004.

Project summary

The incidental take of seabirds, including rare takes of the endangered short-tailed albatross (Phoebastria albatrus), is known to occur in the Alaskan longline fishing fleet. Current fishery regulations have no requirements for observer coverage in the halibut fishery unless a vessel is above 60 ft length over all (LOA) and participating in other federally managed fisheries. The lack of at-sea observations has resulted in little information on seabird bycatch numbers and on the level of compliance with seabird avoidance measures within the halibut fishery. The National Marine Fisheries Service (NMFS) contracted with the International Pacific Halibut Commission (IPHC) for a project examining the feasibility of electronic monitoring systems (EMS) in the Pacific hali- but longline fleet operating off the state of Alaska. The project was conducted on two of the IPHC stock assessment survey vessels fishing in Alaska during 2002. The objectives of the project were to: 1) examine the ability of an electronic monitoring system to provide images that would allow an analyst to monitor seabird avoidance devices for regulatory compliance; 2) determine the feasibil-

329 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 ity of using video images for detecting and identifying incidentally-caught seabirds; and 3) discuss options for the future use of electronic monitoring as a fishery management tool. Vessel and video observations were compared on 106 setting events. Video observation results determined that the EMS was successful in detecting streamer line deployment and relative position on 100% of the daytime sets when two setting cameras were operating together. The streamer line performance evaluations suggest that accurate performance recognition was positively related to the increase in image recording speed and an analysts ability to distinguish measured interval markings on the streamer lines. The ability of a video analyst to recognize and identify the species of retrieved seabirds was examined by deliberately setting previously caught frozen seabirds on the fishing gear. The results of the first examination using 63 seabirds showed a positive relationship between correct seabird species identification and EMS recording frame rates. A fast recording speed resulted in 91% being correctly identified as seabirds and 64% were identified accurately according to species. The results also revealed that nine of 12 albatross (Diomedea spp.) specimens were correctly identified to species; one was determined to be an unidentified albatross, and two were identified improperly. A second examination had 14 NMFS North Pacific Groundfish Observer Program staff examining the video images of six frozen seabirds. The results indicated that correct seabird identification is related both to the analysts knowledge of distinguishing species characteristics, and to the size of the seabird. A third independent evaluation showed that an analyst was capable of detecting 96% of the seabirds deliberately set with the gear, and that 79% were correctly identified to species. The cost of two monitoring programs were estimated for the halibut fishery off Alaska, at two levels of coverage. Full monitoring of all setting and haul backs was estimated at $8.5 million for an on-board observer program; an EMS was estimated at $2.7 million. Coverage levels of 100% for >125 feet LOA, 30% for 60-124 feet LOA, and no coverage of <60 feet LOA vessels was estimated at $0.41 million for an on-board observer program, whereas EMS was estimated at $0.22 million.

330 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 The efficacy of electronic monitoring systems: A case study on the applicability of video technology for fisheries management

Robert N. T. Ames

Abstract

Electronic monitoring (EM) systems were installed aboard two International Pacific Halibut Commission (IPHC) stock assessment survey vessels to collect fisheries data during the summer of 2002. The precision of EM technologies was contrasted to the traditional human method of collecting data on fishing locations, dates, effort, and catch. Quantitative analyses were preformed to determine whether EM technologies could provide precise and verifiable fisheries data for management.

Introduction

The IPHC in collaboration with the National Marine Fisheries Service (NMFS) conducted a project to examine the feasibility of EM technologies for use in the Alaskan halibut longline fleet. EM systems are used to monitor fishing activities and are composed of automated computing devices with data loggers linked to video cameras and a Global Positioning System (GPS). The EM project was conducted aboard two IPHC stock assessment survey vessels during the summer of 2002. The project evaluated the use of EM technology to address several critical information needs concerning endangered seabirds (Ames et al. 2003). Furthermore, data collected by the EM systems allowed for additional investigations. The second study investigated the precision of EM technologies in contrast to the traditional human method of collecting data on fishing activities. Three primary objectives were tested to determine if EM technology could provide: 1) an accurate and unbiased record of fishing locations and dates, 2) an accurate record of fishing effort and piece counts of target and non-target species, and 3) images of sufficient resolution and clarity to allow an analyst to identify species for management.

Materials and methods

Tracking vessel locations The vessel skippers recorded the longitude and latitude coordinates of the start and end position of each survey station during setting operations. The geographic coordinates from 83 survey stations recorded on the F/V Heritage were compared with the coordinates provided by the EM system. Each coordinate was entered into geographic information system software. Overlying the data points from the paired data sets onto a geographic chart provided a comparison of these setting and hauling locations. Measuring the differences between the paired data sets provided a means of quantifying data set consistencies. Inconsistencies in the recorded coordinates were categorized 331 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 into: 1) resolution deficiencies from the recording method, 2) resolution deficiencies from GPS model types, and 3) error from the vessel captain or EM system. The findings of this evaluation will determine the precision and reliability of EM technology for tracking individual vessels in large and remote areas.

Monitoring fishing effort and catch Fisheries data were collected simultaneously by the at-sea samplers and by the EM cameras during gear retrieval. The sea-samplers and video analysts record of catch composition by station were matched and aligned in the IPHC database. The two methods of species identification and counts as well as hook totals were compared. The comparison was performed on a total of 82 stations from the F/V Heritage and 21 stations from the F/V Pacific Sun. Species identification categories and procedures used in this analysis were similar to the species and species groups outlined in the 2003 North Pacific Groundfish Observer Sampling Manual (NMFS, 7600 Sand Point Way N.E., Building Four, Seattle, WA 98115, unpublished). Therefore data quality comparisons could be made with the IPHC survey data and other data collected by NMFS observers. For the survey data, Cohen Kappas coefficient measured the agreement between the sea samplers and video analysts data sets for all species identified and counted from each vessel. A second examination, using the McNemars test, focused on individual species groups. The McNemars test determined whether the proportions between the two observed species frequencies were equal. Fishing effort was evaluated to determine the differences in the number of hooks counted at each station by the two observations. A one-sample t-test was conducted on the differences in the hook totals to determine if the mean differences were zero. Fisheries data provided by the North Pacific Groundfish Observer Program established the degree of precision in which at-sea observers identified species on longline vessels in Alaska. The degree of species resolution was compared between the NMFS observer, IPHC sea sampler, and the video analyst. These analyses will quantify the differences between the methods of collecting fisheries data and the precision of the data collection processes. The conclusions reached by this study will assist in the evaluation of the applicability of EM technologies, whether EM technologies can provide precise and verifiable fisheries data on longline vessels for management.

Results

The analysis of this project is currently in progress. A draft of the report will be completed by February 2004 with final submission anticipated by May 2004.

References

Ames, R. T., Williams, G. H., and Fitzgerald, S. M. 2003. A project to test the use of digital video for monitoring the compliance of seabird avoidance devices and seabird mortality in halibut longline fisheries. U.S. Dep. Commer. NOAA Tech. Rep: in review.

332 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 Analysis of gonad staging on the IPHC setline survey

Sara Wilson Simon Fraser University 1

Abstract

This paper reviews the methods currently used by the International Pacific Halibut Commis- sion (IPHC) to determine female gonad maturity staging of Pacific halibut, Hippoglossus stenolepis. The maturity staging pilot study was conducted in southwest Alaska during three weeks of the annual IPHC setline survey. This was designed to collect and compare qualitative and quantitative data that distinguish features of the gonads and identify problematic issues around maturity staging. Aside from summarizing and reporting general measurement results, this paper focuses on the identification and characterization of stages assigned during the summer setline survey. A second method of maturity staging derived from the analysis of qualitative characteristics is compared here with the results of staging assigned at sea during the setline survey. The paper discusses the rel- evance of each stage and the potential sources of misidentification of stages one, two and four in the ovary and gives recommendations for future studies.

Introduction

Background The International Pacific Halibut Commission (IPHC) collects maturity staging data during its summer setline surveys. The Commission scientists use these data to estimate the biomass of hali- but contributing to the following years spawning population in order to determine the appropriate harvest rate for the fishery. These data are also part of an ongoing effort to understand the life cycle of the Pacific halibut from a purely biological perspective. There has historically been some uncer- tainty in the classification of maturity stages for both female and male Pacific halibut. A seven- stage system of classifying female halibut was used up until 1994 when it was simplified to four stages. This system was refined further in 1999 in an attempt to eliminate staging ambiguities and to limit the variation in the stage designation assigned by the IPHC biologists (IPHC unpub)2. Currently IPHC biologists identify female halibut as belonging to one of the four following stages: F1 is immature, F2 is mature, F3 is currently spawning and F4 is spawned out where F stands for female and the number refers to the timing or progression of egg development.

1 The Commission provides training opportunities for student interns and encourages them to prepare reports of their projects conducted at the IPHC. Reports by interns are included here with only minor editing. 2 IPHC 1995 & 2000 Stock Assessment Survey Manual. Unpub. International Pacific Halibut Commission. P.O. Box 95009, Seattle, WA, 98145-2009.

333 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 Initially this survey was conceived as a pilot study to gather information and to identify problematic issues around maturity staging. It was hypothesized that a misclassification trend was oc- curring and has persisted despite the re-working of the protocols for staging, particularly of immature (F1) and resting (F4) females, but possibly among other stages as well. The hope was that this study would yield enough information to determine how future research might address maturity issues and become part of a continuing effort to collect and compare winter and summer staging.

Study objective The objective of this survey was to explore the methods currently used to establish maturity staging during the setline survey. By collecting and comparing qualitative and quantitative data that distinguish features of the gonads, we hope to better understand halibut maturity and development. The fieldwork portion of this study involved identifying, describing, and distinguishing the differences within female and male maturity staging. In describing the size, shape, and appearance of the halibut gonad it was hoped that groups of characteristics particular to the pre-determined stages would be confirmed. In addition it was expected that patterns of ambiguous combinations of characteristics would be identified and sources of misidentification recognized.

Methods

Data collection Gonad data were collected from fish caught during the IPHC standard stock assessment survey from June 11 to June 26, 2003. The F/V Pacific Sun caught the fish during two trips in IPHC Regulatory Area 4B around Adak in the Aleutian Islands of Southwest Alaska (Fig.1). Halibut were targeted using longline gear consisting of five, 1800-foot skates per station. Each skate had 100 hooks set at 18-foot intervals. On average three stations were fished per day. All captured halibut were sampled by two survey biologists, referred to in this paper as biologists 1 and 2, and a third biologist (referred to here as biologist 3) conducted gonad measurements. Biologists 1 and 2 measured halibut length, sex, gonad maturity as well as collecting otoliths for later determination of age. After this sampling was done the gonad pairs were passed along to biologist 3 for closer examination and measurements. Initially all the female gonads were collected. However, both storage space and time available for processing often limited the numbers collected. An attempt was made to process as many ovaries as possible during a fishing day, but many were not measured and in some cases ovaries were measured but their corresponding otolith numbers were not recorded. Occasionally gonad pairs from the last string of the day were kept in sealed bags until the following day and processed before or during the first haul. When there was sufficient time the male gonads were collected and the corresponding measurements were recorded.

Collection protocol Once the gonad pairs had been staged by the survey biologists 1 or 2, they were examined, measured, and weighed. An attempt was made to match the gonads with the assigned otolith number so that a corresponding length and age could eventually be determined for each set of gonads collected. If time permitted biologists 1 or 2 lined the gonads up in order of collection and they were then processed in that order. The fish order was double-checked to confirm otolith number. At times

334 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 the gonads were placed into a divided plastic box (Fig. 2) and the otolith numbers were written into a grid where each number in the grid corresponded to the gonad location in the box. When time was short, and gonads could not easily be matched with the otolith number, the standard measurements were still taken. There was occasionally the opportunity to put the gonads into plastic bags for processing later in the day or early the next day.

Measurement protocol Quantitative data included gonad length, width, depth, average weight of right and left sides, and average volume of right and left sides. Length and width numbers are the average of both gonads. These numbers were estimated to the nearest 0.5 cm by laying the gonad on a measuring strip. Width was from the edges of the wide end of the gonad, and length was from the center of the wide end to the tip (Fig.3). Depth was read from a ruler held behind the gonad at the thickest point. The weight was taken by placing each gonad into a sealed bag clipped to a 1000 g hanging balance, and weighed to the nearest 5 g. Gonads larger than 1 kg were divided into several pieces for weighing. Volumes were measured by water displacement in a graduated beaker, estimating to the nearest 10 ml. Since the majority of the weight and volume measurements were taken at sea on an unsteady platform the estimates have high variability and low precision. Each ovarys appearance was recorded using the seven categories as follows: egg development and color, gonad texture, membrane thickness and color, and capillary development and color (Table 1). For the male gonads, milt extrusion, average number of crenulations, average length and width, and average weight were recorded for each set of gonads. These data and qualitative observations, along with additional notes were recorded on water-resistant forms. These criteria were adapted from the 2003 survey manual (unpub)3 and expanded upon during field observations.

Analysis Qualitative ovary data were examined for patterns. The categories were given a ranking order (Table 1) which allowed the gonads to be staged according to the four classifications of maturity currently used in IPHC surveys. This also allowed comparisons between staging assigned at sea and those assigned from the analysis of combinations of characteristics. The maturity staging derived from the analysis of qualitative characteristics is hereafter referred to as the qualitative staging or qualitative method of staging. These data are derived using Table 1 as a maturity key that is similar to the chart found in the 2003 survey manual (unpub)². Depending on the combination of sub category characteristics, the ovary fell into one of the four maturity stages. Maturity staging, halibut fork length and age data, collected by IPHC biologists 1 and 2 were aligned by otolith number with the qualitative data in order to compare the characteristics and the staging. Using the qualitative method, a fish was ultimately designated as an F1 if there were no eggs visible in the gonad tissue; an F2 if it was swollen and contained homogenous opaque eggs; an F4 if it contained

3 IPHC 2003 Stock Assessment Survey Manual. Unpub. International Pacific Halibut Commission. P.O. Box 95009, Seattle, WA, 98145-2009.

335 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 resorbing clear eggs or if it had a thick membrane and flaccid texture (F4 was assigned to any fish that appeared to have recently spawned). There were no spawning halibut, (F3) encountered on this survey trip.

Results

Quantitative analysis A total of 226 ovary pairs were sampled although in some cases the information collected was incomplete. For example not all gonads sampled had weight and volume measurements taken due to the inability to correctly measure during rough sea conditions. The quantitative data are summa- rized in Table 2, which reports the number of observations taken (n) for each measurement, average with standard error, and minimum and maximum values. These data show a wide range of gonad size and variability. A total of 168 male gonads were sampled, but these data have not yet been analyzed. Results show that there is a good probability of a difference between the weight of the right and the left gonad but further analysis and data are required to confirm this trend. There were also slight staging differences within pairs of gonads, particularly between the F2 and F4 gonads.

Qualitative staging characteristics The following descriptions combine general observations of those characteristics most commonly found together. These may be thought of as a re-description of the IPHC stage classes one through four. The immature ovaries (F1) are most commonly gray to pink in color and firm to touch with a very thin membrane of less than ¼ mm. The length ranges five to 16 cm. The capillaries are usually thin and hair-like; covering only part of the surface of the gonad and tended to be red to pink in color. There are no visible eggs inside and the tissue is firm and grayish. The pre-spawning mature ovary (F2) is a white to pink color and appears swollen or puffy. The membrane is also very thin and translucent so that the eggs are visible through the membrane. The gonad size ranged from ten to 16 cm in length. The capillaries are thick (up to two mm) at the point of origin (inside and top of the gonad), but grow thin towards the edges and tip and are usually red and well developed. The eggs inside are a uniform size, approximately one mm in diameter, with a grainy or cream of wheat appearance. When the gonad is opened the eggs fall apart rather than sticking together (Fig. 4). There were no spawning halibut, (F3) encountered on this survey trip. The post-spawning (F4) ovary is flaccid and the outside is opaque with a dirty yellow-pink colored membrane that is thick at the top end of the gonad and thinner towards the tip. Occasionally it is possible to see though the membrane towards the tip, but the top is opaque. The size varies widely from 20 to 60 cm in length. The capillaries are large and extensive, sometimes looking deflated. The capillary color range from red to purple, often outlined by white, and sometimes appears to have burst or bled into the tissue. Eggs are visible inside and have a grainy appearance similar to that observed in the F2 halibut but are smaller (less than one mm in diameter). They are often bloodied or contain a pinkish hue, and are more bound to the tissue and to one another. Resorbing, clear eggs, which range from two to five mm in diameter, are sometimes found inside the folds of the egg tissue, towards the tips of the gonad. Scarlet red blood clots may also be found inside these gonads (Fig. 4).

336 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 Comparison of staging methods It was most difficult to differentiate between the F2s and the F4s. This was reflected in the high discrepancy between designations of F2 and F4 using the two methods of staging. Table 3 shows the number and proportion of each designation of maturity staging for each method. There were few discrepancies between the F1 and F2 ovaries and no discrepancies between the F1 and F4 ovaries. This is presented again in Figure 5 which clearly shows the discrepancy between the stages determined by the sea samplers and those determined from qualitative characteristics. The sea sampler maturity designations were determined aboard the fishing vessel, and the qualitative stages were designated after analysis of qualitative data collected at sea. The F1 is an immature fish, the F2 is a mature fish that will spawn the upcoming year and the F4 halibut has recently spawned. A quick visual assessment was more likely to result in an F2 instead of an F4 and a closer look was most likely to designate a greater number of F4s in the older fish (Fig. 6). This was shown by the large number of discrepancies in classification for halibut aged 18 years and older. Halibut fork length was not closely correlated with maturity (Fig. 7). A similar trend is seen when the last five years data were compared (Fig. 8). Overlap in the maturity classifications with size is seen.

Discussion

It appears that the immature gonad (F1) is easily distinguished. Although it might be confused with the spawned-out (F4) gonad, this is probably not a significant source of error because the membrane is much thicker on the F4 ovary. The age range of immature halibut observed from this study were similar to those observed in previous studies throughout the coast (St-Pierre, 1984). Based on data collected by Gilbert St-Pierre (1984) it is probably safe to assume that the fish observed as F1s were immature. Histological confirmation of this is needed since some of these gonads came from larger fish of up to 130 cm in length. Further investigation of size at maturity might reveal patterns of changes in growth rate. There was a large range of ovary characteristics that were considered indicative of F2 and F4 and these clearly overlap. Resting or spawned-out tissue characteristics range from a nearly cream of wheat texture inside to one with no egg structures visible. The latter are observed mostly during winter charters. The common characteristics among all F4s in the summer were a thick membrane and a flaccid or loose texture. These characteristics should be noted first and foremost when desig- nating stages. The perfect F2 was easily distinguished by its swollen appearance, thin membrane, and the cream of wheat textured eggs. In general the F2 was cleaner with no blood, resorbing eggs or clots. However if these characteristics are found with resorbing eggs then the halibut has recently spawned and should therefore considered an F4. Several explanations may account for the misidentification of F2 and F4 gonads. The most obvious is that the appearance is similar for some F2 and F4s, as illustrated by the discrepancies in staging. Figure 4 shows a set of gonads classified as F2 and a set as F4 both open and closed. Without cutting open the gonad, the membrane thickness is the only way to tell the two apart and this alone is not likely to be a very reliable method of staging. Where the differences are subtle, close attention to detail by IPHC biologists would be suggested to improve the accuracy of the

337 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 staging results. In addition it is suggested that both ovaries be cut open in case of differing characteristics within a pair and that the designation of an F4 in one be assigned instead of F2. Staging between the F2 and F4 gonads appear to be relatively independent of fork-length. The overlap of F2 and F4 staging with size may reconfirm the assumption that once mature, female halibut cycle in a similar manner regardless of size. However, based on the qualitative data, the gonads from younger halibut were most likely to show the characteristics of an F2, which leads to questions about the connection between the spawning cycle of a halibut and the age. It is possible that the first spawning event or events is not the same as subsequent spawning events in terms of cycle timing or gonad appearance. These fluctuations may be the source of some of the classification problems. In trying to determine the source of the misidentification of the F2 and F4, it has become apparent that there may be problems with the conceptual framework of the IPHC survey staging system. The progression of ovary staging from one through four is illustrated in Figure 9 and summa- rized below from descriptions made by W.F. Thompson (1917) and report on egg production in Kolloen (1934). This is presented here in order to review the current understanding of the ovary cycle. The immature gonad passes into the mature phase, where indistinct oocyte tissue becomes visible and opaque described as the F2 stage. In the late fall the mature gonad enters the spawning phase through a hydration process which occurs quickly according to Thompson (1917). This inter- mediate should not be observed during spring or summer surveys and would be expected only during the late fall and winter, just before spawning season. The next generation of ova is visible and becomes uniformly opaque by the end of the spawning season, which occurs around March (Thompson 1917). This is the F4 described in the results above. For a period, currently undeter- mined, the remainder of the full sized translucent eggs are resorbed. Although Kolloens (1934) account of Thompsons work does not mention membrane thickness, it may be assumed from our observations that the membrane remains thick, possibly thinning half way through the resting phase as it enters the pre-spawning phase. It is possible that the thick membrane helps to aid the process of resorption by concentrating the enzymes needed for this process and maintains the integrity of the ovary while it is in the resting phase. The clots and blood observed in the F4 ovaries could be the result of incomplete resorption of last seasons eggs, which will disappear as the ovary progresses towards the next stage. According to Thompson (1917) the mass of opaque or semi-opaque eggs, characterized by the cream of wheat texture, is the next generation of eggs. Several years of ova are thought to be continuously present, only becoming visible and opaque, shortly after spawning is finished (Kolloen 1934). There is some disagreement between the assumptions about the time frame of the halibut spawning cycle. A yearly spawning cycle is used in the IPHC harvest policy simulations, and this is the general understanding under which analysis are conducted at the Commission. However, when the fish is staged as an F2 in June, the assumption is made that this fish has not recently spawned. If this were the case and a one-year cycle is assumed then this fish is primiparous, or reaching its first spawning year after moving from an F1, or was an early spawning fish. If the F2s are primiparous, the F2s observed would be a small proportion of the total number of mature fish, depending on the region and the season. If the halibut is an early spawning fish, perhaps spawned in December rather than during peak spawning in February (Thompson 1915), that may give the gonad time to recover and become clean looking again. There is also the possibility is that not all fish spawn each year and the F2 fish represents those that will spawn in the coming winter but did not spawn the previous 338 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 winter. Under the assumption of the yearly spawning cycle, the ovary observed in early summer as an F4 may be functionally the same as the F2. Both groups of fish would have spawned last season, except for the small proportion of new spawners, and both will spawn in the upcoming season. If halibut do spawn each year and this occurs at and continues from first maturity, then there may be no practical reason to stage the F2 and F4 as separated categories unless it could be shown that each contributes differently to the upcoming years spawning population. It may be possible that halibut follow a two-year spawning cycle with resting and spawning on alternate years. In that case a snapshot of the current maturity stages, such as this study should show approximately equal proportions of F2 and F4 individuals. The staging results did not show this when the qualitative method of staging was used. In a two-year cycle the F2s would represent next years spawners (which would include new spawners) and the F4s would represent last years spawners. The age data (qualitative method of staging) suggest that younger halibut spawn less fre- quently than older halibut and thus are more likely to look like they have not recently spawned which is characterized by the clean F2 look (Fig. 6). This pattern would be easily explained through allocation of energy. We may expect that newly mature halibut would have less energy to allocate to reproduction and that it may take a smaller halibut longer than a year to fully recover and be able to spawn again. More data and analysis are required to support this theory, but the small number of data points currently available does appear to support it.

Summary This study has raised many questions about the consistency of current staging methods. Al- though it was initially suspected that there would be problems differentiating the F1 from the F4, this was not observed in this study. It may be that a comparison of winter with summer data would show the F1 and F4 similar in appearance. The short time frame and small scope of this study would not be expected to show seasonal effects and the only basis for this suggestion is historical photo data. The main differentiation problems observed during this survey were between the F2 (mature) and the F4 (spawned out) stages. This study also brought into question the current understanding of the Pacific halibut spawning cycle and some of the assumptions which it makes. Although there were many more questions raised than answers, this pilot study was successful in that it shed light on potential avenues of future research.

Recommendation for future studies Overall the gonad investigation should be more comprehensively developed with respect to both observational and histological data collection. An investigation of time frame for egg production and maturation would be helpful in clarifying the current assumptions used in the models. Fall, winter and spring links and transitions would probably fill in ambiguous staging questions and reconfirm or refute current assumptions. Histological analysis of each of the stages would be useful in determining the true stage of each ovary. This could then be compared with the visually determined maturity staging. It would be useful to have ongoing data, including both qualitative data and physical samples for histological analysis, from the same region throughout the year. If this were collected on a monthly basis (for example) egg production and maturation could be tracked throughout the cycles. 339 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 A region by region comparison of maturity would be easily conducted using the current data available from the setline surveys. It would be interesting to see if gonads mature at different rates depending on environmental conditions particular to each area.

Acknowledgements

Many thanks to Lauri Sadorus and Bruce Leman for their support of the internship program, to Tracee Geernaert and Kelly Van Wormer for initiating the gonad project and for their ongoing support, to Tim Loher for his support and helpful comments, to Tom Kong for graphics help, and to Aaron Ranta for support accessing and organizing data. Thanks to Steven Hare and Bill Clark for application and background information, to Mike Larsen for planning advice, to Andy Vatter for providing sampling gear, and to Din Chen for statistical help in preparation for data collection. Special thanks to Suzanne Sullivan, Dean Gaidica and to the captains and crew of the F/V Pacific Sun for helping me collect the gonads and keep them in order at sea.

References

St-Pierre, G. 1984. Spawning locations and season for Pacific halibut. Int. Pac. Halibut Comm. Sci. Rep. 70.

Kolloen, L. 1934. Egg production in the Pacific halibut Hippoglossus hippoglossus correlated with length, weight and age. University of Washington. Masters Thesis: 36-42.

Thompson, W. F. 1915. A preliminary report on the life-history of the halibut. Province of British Columbia. Report of the Commissioner of Fisheries: 76-79.

Thompson, W. F. 1917. The egg production of the halibut of the Pacific. Province of British Colum- bia. Report of the Commissioner of Fisheries: 35-38.

340 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 Table 1. The ranking order of gonad characteristics with sub-categories, used to determine the staging codes for female halibut. The resulting data are used to compare staging with sea sampler data.

Rank Category Sub-category order 1 Membrane 0.25: very thin, not measurable Thickness 0.5: thin, but estimated as ½ of a mm thick 1, 1.5 or 2: approximate thickness in millimeters Note: since the thickness was not precisely measured, these are considered qualitative estimates. 2 Gonad Texture Firm Swollen Flaccid or baggy 3 Egg 1: none Development 2: homogenous, small opaque eggs (0.5-1mm in diameter) which have a ‘cream of wheat’ look 3: eggs barely visible, mostly translucent with yolks starting to turn opaque 4: large hydrated fully developed eggs 5: large resorbing eggs visible among small opaque eggs 4 Egg Color Creamy Pink or Red-Pink Grey or Translucent 5 Membrane Clear: translucent or see-through Color Opaque: white, yellow or pink 6 Capillary Tiny: hair-like Development Thin: a bit larger than hairs Large: larger than 0.5 mm across in any part of the gonad Deflated: usually large, with white lines tracing the veins Note: all capillaries observed were also branched 7 Capillary Color Red: applied to gonads with only red capillaries Purple: these may also have red capillaries Note: many of the capillaries were also white

341 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 Table 2. Summary of quantitative ovary measurements where length, width and depth are estimated in centimeters, weight is in grams and volume is in millimeters. The standard error is calculated from the standard deviation divided by the square root of n. A 95% confidence interval for the average values can be calculated by adding and subtracting two times the standard error.

Minimum Maximum Average Standard error n Length (cm) 5.5 59 20.1 0.695 222 Width (cm) 3.25 30 10.6 0.290 222 Depth (cm) 1 4.25 2.1 0.053 220 Average weight (grams) 8 2525 246.9 28.566 106 Right wt. (g) 15 2450 264.3 29.141 106 Left wt. (g) 15 2600 342.6 37.370 107 Average volume (ml) 8 2375 268.5 52.055 63 Right volume (ml) 2 2250 295.2 87.758 29 Left volume (ml) 20 2500 352.8 7.486 31

Table 3. The number and proportion of F1, F2 and F4 ovaries as identified by the biologists on the setline survey, and those determined by the qualitative ranking order developed from closer observations.

At sea maturity staging Maturity staging by qualitative designation characteristics number proportion number proportion F1 63 0.33 65 0.34

F2 91 0.48 39 0.21 F4 36 0.19 86 0.45 Total 190 1 190 1

342 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 Figure 1. The Adak charter region of Regulatory Area 4B. Dots are survey station locations.

Figure 2. Divided box used for storing the gonads between collection and measurement tak- ing. This photo also illustrates the difficulty of weighing gonads, particularly small ones at sea aboard a moving vessel.

343 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 TIP

Figure 3. The orientation of length and width measurements taken on female and male gonads: A is the length taken from the top of the middle of the gonad to the tip, B is the width from one side of the wide end to the other.

Figure 4. The above photos show the same two pairs of gonads: left is open and the right is closed. The top pair is from a recently spawned fish (F4) and the bottom pair is from a fish which will be spawning in the upcoming season (F2). 344 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 100% 90% 0.19

80% 0.45 70% F4 60% 0.48 50% 0.21 F2 40% 30% F1 20% 0.33 0.34 10% 0% sea sampler data qualitative data

Figure 5. This histogram shows the percentage of halibut designated by each staging method into each of the three maturity categories observed during this survey.

1 qualitative Maturity code (two estimates) code Maturity sea samplers 0 0 5 10 15 20 25 30 35 40 Halibut Age

Figure 6. Both methods of staging are shown here against age of fish. The sea sampler maturities were determined aboard the fishing vessel, and the qualitative stages were designated after analysis of qualitative data collected at sea. 345 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 5

1 Sea Sampler

Maturity code (two estimates) code(two Maturity Qualitative 0 50 70 90 110 130 150 170 190 210 230 fish forklength (cm)

Figure 7. Both methods of staging are shown here against size of the fish. The sea sampler maturities were determined aboard the fishing vessel, and the qualitative stages were designated after analysis of qualitative data collected at sea.

2 Maturity code Maturity

0 50 100 150 200 250 Fork length

Figure 8. The maturity classifications given to ovaries between 1998 and 2002 in Adak. These are plotted against the halibut fork length to show the historical overlap of staging and size. 346 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 Figure 9. The progression of the ovary staging is shown by this diagram. The F1 is immature, F2 will spawn during the upcoming winter, F3 is currently spawning (seen only during winter except in rare cases), and F4 is recovering and resting.

347 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003 348 IPHC REPORT OF ASSESSMENT AND RESEARCH ACTIVITIES 2003