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Appendix IV.

FISH TISSUE ANALYSIS AND WORK PLAN

Appendix IV.1     Fish Tissue Work Plan:
Collection of Fish Samples for Metal Analysis

1. INTRODUCTION

Alternatives for fish tissue analysis were discussed at the April 29, 1999 Steering Committee meeting. The lack of sufficient numbers of benthic invertebrates at the reference and exposed sites required that an alternative be developed for assessing the biological uptake of metals from stream sediments. Golder was asked to prepare recommendations regarding alternatives for tissue analysis, including a full rationale plus costs. It was requested that a discussion of whether the sex of fish affects metal uptake be included in the memo. In addition, specifics such as sample size per location were provided.

This document provides the rationale for and the details of the recommended scope of work.

2. RATIONALE

2.1 Suggested Changes to the Tissue Analysis Presented in the Golder Proposal

Metals and radionuclides accumulate to different degrees in different fish tissues. The literature suggests that some metals can accumulate to greater amounts in the kidney than in the liver. The literature also suggests that other tissues, such as gill, are often the primary sites of accumulation, depending upon the metal and that radionuclides usually collect primarily in bone tissue. Hence, we suggested in our proposal (Golder Associates 1999) that we re-visit the fish tissue component of the program as the RFP required that only the livers be analyzed for metals uptake.

The Golder proposal also suggested that specific radionuclides be analyzed instead of gross alpha and gross beta in order to provide more relevant information with respect to possible radiotoxicity.

2.2 Recommended Changes Based Upon Consultation with the Steering Committee and a More Thorough Review of the Literature

Radionuclides

Analysis for individual radionuclides does not appear to be warranted because of the very low levels of radioactivity detected in samples to date. We were under the impression when we prepared our proposal, that radionuclide levels could be substantially higher than background. Thus we suggested that analysis of individual radionuclides might provide a more accurate basis for screening of potential radiotoxicity risks to human health. We now suggest that individual radionuclide analysis would only be triggered if exposed fish have significantly higher levels of gross alpha and gross beta activity than reference fish. We note that in the unlikely event that such differences occur, laboratories equipped to perform neutron activation analysis for uranium and thorium can perform the analyses for a reasonable cost (e.g., Saskatchewan Research Council performs these analyses for about $25 per sample).

If analysis of individual radionuclides is warranted, bone is the recommended tissue to sample, particularly for radionuclides such as radium-226, which are analogous to calcium (Whicker and Schultz). Levels of uranium-series radionuclides in fish tissue are generally highest in bone, followed by skin, liver, gonad and muscle (Swanson 1985, Eisenbud 1987). Radionuclide uptake does not vary with fish age, size or sex (Swanson 1985).

Metals and Arsenic

There were two main objectives for the analysis of metals and arsenic in fish tissue:

1. Obtain levels in fish flesh for use in the human health risk assessment; and
2. Obtain exposure information for the sentinel species (i.e., to assess the degree of metals uptake in white suckers and longnose dace.)

The first objective is being met by the ongoing sport fish sampling conducted by the MOE, which we understand includes the analysis of muscle tissue for metals, arsenic and radionuclides. The second objective is the issue addressed in the paragraphs below.

Metals Uptake in Sentinel Fish Species

Development of quantitative links between exposure and effects is one of the most challenging aspects of effects monitoring and ecological risk assessment. Traditionally, researchers have used indirect indications of exposure (by measuring contaminant levels in water, sediment and food organisms). These indirect measures can be misleading when dealing with contaminants with poorly understood and/or complex bioavailability. In other words, it is often extremely difficult to predict the actual uptake of a contaminant from water or sediment data. Thus the analysis of tissue residues is highly desirable.

Although logical, the application of a tissue residue approach to exposure characterization is complex. First, there should be an understanding of the mechanisms or modes of action for the chemical of concern, such that appropriate tissues are sampled. For example, in order to define robust residue-based effects relationships for cationic metals, it may be necessary to focus upon tissues or sites of action (e.g., gill) that are not routinely sampled (Wood et al. 1997). Alternatively, even though such relationships may not allow determination of mechanistic processes, it might be possible to derive useful correlations between some internal chemical residue for metals and observed effects (Wood et al. 1997). Therefore, the challenge for the Moira River Study is to be sure that we select the appropriate tissue in order to allow a reasonably confident analysis of relationships between tissue residue levels and observed effects.

Selection of Appropriate Tissues for Analysis

Selection of appropriate tissues can be specific to the contaminant. For example, the appropriate tissue for arsenic may be different than that for copper or zinc. Existing water quality data for the Moira River system indicates that the primary contaminant of concern is arsenic.

The majority of studies linking tissue level with effects use whole-body analysis of arsenic (Jarvinen and Ankely 1999). For example, McGeachy and Dixon (1990, 1992) reported whole body levels of sodium arsenate along with survival or growth endpoints for rainbow trout. Cockell and Hilton (1988) reported "Carcass" residues of arsenic trioxide and survival and growth endpoints. Sorensen (1976) reported whole body, liver, gut, and muscle residues of sodium arsenate (As⁺⁵) in green sunfish along with survival endpoints. Gilderhus (1966) reported whole body levels of sodium arsenite (As⁺³) in bluegill, along with survival and growth endpoints.

On the basis of the existing literature linking tissue residues with effects, we recommended that whole body analyses be conducted for arsenic in both white sucker and longnose dace. This allowed for a comparison of results with a longer list of literature values and a more comprehensive database linking residues with effects than for liver analysis only.

Metals Analysis

Metals analyzed in fish tissue included: Ag, Al, Co, Cu, Ni, Pb and Zn (Kilgour et al. 1999). Copper accumulates to a greater extent in liver than in most other tissues, and linkages between liver residues and effects on growth and survival have been made by several authors (Jarvinen and Ankley 1999). However, linkages between whole-body residues of copper and effects are also available in the literature (Jarvinen and Ankely 1999). Whole-body, liver and gill residues of zinc appear to be similar in several studies of fish (Jarvinen and Ankley 1999). Linkages between zinc residues in whole body, liver, gill and kidney and growth, reproduction and survival endpoints are available (Jarvinen and Ankley 1999). Nickel appears to accumulate primarily in gill tissue and in kidney, although liver, muscle and whole body residues are also significant (Jarvinen and Ankley 1999). The only linkage between nickel tissue residue and effect is for survival. Whole body appears to be a reliable predictor of other tissue levels of aluminum (Jarvinen and Ankely 1999). Linkages are available between whole body residues of aluminum and survival. The only data on the linkage between silver tissue residues and effects are for whole body, gill and "internal organs" data (Coleman and Cearley 1974). Lead appears to accumulate preferentially in kidney and spleen, although liver and gill tissues also can contain significant amounts (Jarvinen and Ankley 1999). Linkages between tissue residues and effects have been made for liver, kidney, gill, spleen, and whole body data. There are no data linking tissue residues of cobalt and effects.

As can be seen by the above review, literature values for linkages between whole body analysis and effects are available for all of the metals of concern in the Moira system except for cobalt. Other tissues are often analyzed, but not necessarily the same tissues across several metals. Furthermore, whole body residues will be appropriate for arsenic.

The original study design called for tissue residue analysis in white sucker and in composite invertebrate samples. As recorded in the Minutes of the April 29, 1999 meeting, it will not be possible to analyze invertebrate samples because of the lack of sufficient numbers of invertebrates at the sampling stations. Small-bodied fish can be considered as an alternative to invertebrates because they are in close contact with sediments (this point is also recorded in the Minutes). Furthermore, information about exposure to the chemicals of concern in the other sentinel fish species will be very valuable.

Based upon the above review, we recommended that:

1. Whole body residues of metals and arsenic are determined for the Moira River Study; and,
2. residues are determined in longnose dace as well as in white sucker.

Influence of Fish Age, Size, and Sex

Rate of arsenic uptake is greatest in young fish; residues in both liver and muscle decline with size in almost all species (Moore and Ramamoorthy 1984). Depuration of most arsenic compounds from fish tissues is rapid. Barrows et al. (1980) reported that the half-life of As₂O₃ in bluegill muscle was only 1 day. Similarly, there was an 80% reduction in arsenic residues in whole rainbow trout within 96 hours of administration of an oral dose of⁷⁴ As (Oladimeji et al. 1979). Based upon this information, arsenic residues in white sucker and longnose dace from the Moira River system are not expected to be significant and may be higher in the longnose dace than in sucker.

Residues of copper in muscle tissue frequently decline with age and size of fish, but residues in liver have been shown to be positively correlated with fish age (Moore and Ramamoorthy 1984). There is usually little accumulation of lead in marine and freshwater species of fish, except in cases of extreme pollution (Moore and Ramamoorthy 1984). Furthermore, there is usually no correlation between lead residues and age or size of fish. There are few data relating the age/size of fish to nickel levels in fish; however, muscle residues either increase or remain constant as the fish grows (Moore and Ramamoorthy 1984). Zinc residues in freshwater and marine fish are generally much lower than those found in algae and invertebrates. Although there is often no correlation between residues in muscle and age/size of fish, uptake of zinc by young fish is generally greater than that of old fish. Most species show a seasonal variation in zinc levels in muscle and organs, with peaks during the spring and early summer (Moore and Ramamoorthy 1984). Aluminum, cobalt and silver uptake in fish is not well studied. Concentrations of silver in white sucker liver were correlated with sediment concentrations in the Lower Susquehanna River Basin (Lindsey et al. 1998). Liver tissue of smallmouth bass had higher concentrations of aluminum and cobalt that white sucker liver. White sucker liver had higher concentrations of silver than smallmouth bass (Lindsey et al. 1998). Therefore, there may be significant differences among fish species and metals with respect to relative uptake of aluminum, cobalt and silver from sediments. No information on uptake with age or size of fish is available.

The above literature suggests that there is a relationship between body burden of some of the contaminants of concern in the Moira River system and fish size or age. Therefore, analysis of body burden versus size/age was recommended in this study.

There are no data to suggest that there is a difference in uptake of arsenic or metals with sex of fish.

3. SCOPE OF WORK

The fish tissue analysis program was undertaken by collecting white sucker and longnose dace from sites located throughout the Moira River system.

Longnose dace is a small-bodied riverine species and as such was collected from the following river sites:

• Moira River half way between the mine site and Bend Bay;
• Moira River above the mine site; and
• Black River.

White sucker was collected from lakes and larger sections of the Moira River including:

• Moira River at Bend Bay;
• Moira River (3 stations downstream of Stoco Lake);
• Moira Lake;
• Stoco Lake;
• Consecon Lake; and
• Round Lake.

The collection of these fish species at these locations corresponds to the requirements of the fish population survey, which was conducted at the same time. The MOE collected the white sucker samples, as presented in the original RFP, and Golder collected the longnose dace samples.

Ten specimens of white sucker and 10 composite samples of longnose dace were taken and analyzed for arsenic, metals and gross alpha/gross beta activity. The sample size of 10 has been shown to be adequate to distinguish "exposed" from reference sites in other studies (radionuclides and metals) (Swanson 1985, U.S. EPA 1993). This sample size has been shown to distinguish differences of approximately 30-35% for uranium-series radionuclides (Swanson 1985) and approximately 10-20% for metals (assuming a mean: variance ratio of 0.5-1.0) (U.S. EPA 1993).

The 10 samples of white sucker were individual fish. Each of the longnose dace samples consisted of a 9 to 10 individuals. Whole-fish samples (gutted) were analyzed, rather than specific tissues (see above rationale).

Two size-classes of white sucker were selected for analysis (5 specimens from each size class). The first size-class represented the younger, faster-growing fish (with potentially greater arsenic or metal uptake). The first size-class was 10-30 cm (fork length). The second size class was > 30 cm. Nine to 10 individuals were collected from each sample class. This provided greater statistical power within each size class.

Supporting information accompanying the fish samples taken for analysis included:

• Full documentation of the sample location (UTM coordinates);
• Water depth, approximate water velocity, water temperature;
• Site name/number;
• Fish species and sample type (whole or composite, size-class);
• Fresh weight and fork length;
• External condition of the fish (any lesions, injuries, deformities will be noted);
• Internal condition of the fish, including sex and reproductive stage, presence of parasites, and any abnormal tissues or lesions;
• Age (aging materials will be taken from each specimen);
• Name of person taking the sample; and
• Habitat mapping at the longnose dace collection sites.

A detailed Technical Procedure for sampling fish for metals and radionuclides is provided in this Appendix (see attached Technical Procedure 8.16-0).

Samples will be frozen and shipped to the analytical laboratory for analysis.

PROTOCOL FOR PREPARATION OF WHOLE FISH SAMPLES FOR METALS ANALYSIS: MOIRA RIVER STUDY

1. Thaw specimens. Start with reference fish (Round Lake and Consecon Lake). Thaw only as many as you have time to process that day.
2. Knives to be used: high-quality, corrosion-resistant stainless steel knives must be used. Initial wash: wash with a detergent solution, rinse with tap water, soak in 50% HNO3 for 24 hours at room temperature, and rinse with distilled water. All knives must be washed with a detergent solution, rinsed with tap water, rinsed in 50% HNO3 for 15 minutes and rinsed with distilled water between each fish. All washed knives must be wrapped in heavy plastic wrap during storage.
3. Cleaning of the food processor blades: as above for knives.
4. Cleaning of the food processor plastic bowl, stainless steel bowls used during homogenization, spatulas used to transfer from processor-to-bowls and from bowl-to-glass vials and the glass vials used to store aliquots of the homogenized fish: Initial wash – wash with a detergent solution, rinse with tap water, soak in 50% HNO₃ for 24 hours at room temperature, and rinse with distilled water. Between each fish (food processor bowl, stainless steel bowls and spatulas) – wash with a detergent solution, rinse with tap water, rinse in 50% HNO₃ for 15 minutes at room temperature, and rinse with distilled water.
5. Preparation of fish for processing: cut the thawed fish into equally-sized pieces that will fit into the food processor bowl without jamming. The fish should be cut horizontally (crosssections). If the fish is too big to process in one batch, place the processed portion in a stainless steel bowl and proceed with the next portions until done. Stir the contents of the stainless steel bowl thoroughly with a plastic or stainless steel spatula. Re-process the contents of the bowl 5 times.
6. Once the entire fish has been homogenized at least 5 times, place a 50 g aliquot of the homogenized fish into an acid-washed glass container.
7. Label with an indelible glass marker which has ink that is resistant to freezing. Also place a label inside each vial on waterproof paper labelled with indelible ink. The outside and inside labels must contain the following information: Species (WHSC); Site Code; Specimen Number; Date of Capture; Project Number; Project Name (Moira R. Study); and, "Golder Associates".
8. Freeze the sample. Freeze the remaining homogenized fish in a heavy-duty plastic bag with internal and external labels.
9. Enter the information on the chain-of-custody form (Len Rozon has these forms) as required: (sign the sample in for processing and out again for sending to the MOE lab).
10. Once all homogenates have been prepared, ship to MOE laboratory in a cooler with dry ice, properly labelled and packaged to comply with TDG regulations.

References

Barrow, M.E., S.R. Petrocelli, K.J. Macek and J.J. Carroll. 1980. Bioconcentration and elimination of selected water pollutants by Bluegill Sunfish (Lepomis macrochirus). In: R. Haque (Ed.), Dynamic exposure and hazard assessment of toxic chemicals. Ann Arbor Science, Ann Arbor, pp. 379-392.

Cockell, KA and JW Hilton. 1988. Preliminary investigations on the comparative chronic toxicity of four dietary arsenicals to juvenile rainbow trout. Aquat. Toxicol. 12: 73-82.

Coleman, RL and JI Cearley. 1974. Silver toxicity and accumulation in largemouth bass and bluegill. Bull. Environ. Contam. Toxicol. 12:53-61.

Eisenbud, M. 1987. Environmental radioactivity: from natural, industrial and military sources (3rd ed.). Academic Press Inc., FL.

Golder Associates. Proposal: Impact study of the Moira River system downstream of the former Deloro Mine site, Tender Number 004604. March 15, 1999.

Gilderhus, PA. 1966. Some effects of sublethal concentrations of sodium arsenite on bluegills and the aquatic environment. Trans. Am. Fish. Soc 95: 289-296.

Jarvinen, A.W. and G.T. Ankley. 1999. Linkage of effects to tissue residues: development of a comprehensive database for aquatic organisms exposed to inorganic and organic chemicals. SETAC Technical Publication Series. SETAC Press. Society of Environmental Toxicology and Chemistry, Pensacola, FL.

Kilgour, B.W., C.B. Portt and D.G. Dixon. 1999. Moira River Impact Study: Detailed design to determine the impact of the former Deloro Mine site on the Moira River System. Final Report submitted to Ontario Ministry of Environment.

Lindsey, B.D., K.J. Breen, M.D. Bilger and R.A. Brighthill. 1998. Water quality in the Lower Susquehanna River Basin, Pennsylvania and Maryland, 1992-1995. U.S. Geological Survey Circular 1168.

McGeachy, SM and DG Dixon. 1990. Effect of temperature on the chronic toxicity of arsenate to rainbow trout. Can. J. Fish Aquati. Sci. 47: 2228-2234.

McGeachy, SM and DG Dixon. 1992. Whole-body arsenic concentrations in rainbow trout during acute exposure to arsenate. Ecotoxicol. Environ. Saf. 24: 301-308

Moore, J.W. and S. Ramamoorthy. 1984. Heavy metals in natural waters: Applied monitoring and impact assessment. Springer-Verlag, New York.

Oladimeji, A.A., S.U. Qadri, G.K.H. Tam and A.S.W. DeFreitas. 1979. Metabolism of inorganic arsenic to organoarsenicals in rainbow trout. Ecotoxicology and Environmental Safety 3: 394-400.

Sorensen, EMB. 1976. Thermal effects of the accumulation of arsenic in green sunfish. Arch Environ. Contam. Toxicol. 4: 8-17.

Swanson, S.M. 1985. Food-chain transfer of U-series radionuclides in a northern Saskatchewan aquatic system. Health Physics 49(5): 747-770.

U.S. EPA 1993. Guidance for assessing chemical contaminant data for use in fish advisories. Environmental Protection Agency Report EPA 823-R-93-002.

Whicker, F.W. and V. and Schultz 1982 Radioecology: nuclear energy and the environment. CRC Press, Boca Raton, FL

Wood, C.M., WS Adams, GT Ankley, DR DiBona, SN Luoma, RC Playle, WC Stubblefield, HL Bergman, RJ Erickson, JS Mattice and CE Schlekat. 1997. Environmental toxicology of metals. Pp. 31-51 In Bergman, HL, Dorward-King EJ (eds.) Reassessment of metals criteria for aquatic life protection. SETAC Press, Pensacola, FL.

Appendix IV.2     MOE Laboratory Services Branch Method
References for Fish Tissue Analysis

MOE LABORATORY SERVICES BRANCH (LSB)
METHOD REFERENCES FOR FISH TISSUE
ANALYSES

E3072
THE DETERMINATION OF HEAVY METALS IN BIOMATERIALS BY ATOMIC ABSORPTION SPECTROPHOTOMETRY
(AAS)

E3416
THE DETERMINATION OF TRACE METALS IN FISH TISSUE BY ICP-MS

LSB Detection Limits

E3416 – Detection Limits (µg/L)(1999)

Appendix IV.3     Concentrations of Metals of Concern in Fish Samples and Graphs Not Included in Main Report

Mean Fish Tissue Concentration of Metals the 1999 Sentinel Fish Sampling Program [ug/g-wet weight]

Parameter	Longnose Dace			Average	White Sucker								Average	Total Average
	F1	F2	F3		CL	ML	MR11	MR17	MR14	MR15	RL	SL
Arsenic	0.094	0.11	1.08	0.41	0.034	0.39	0.56	0.24	0.31	0.12	0.070	0.50	0.27	0.31
Cobalt	0.32	0.052	0.36	0.24	0.024	0.12	0.14	0.062	0.097	0.040	0.027	0.055	0.070	0.11
Copper	0.79	0.65	1.15	0.86	0.55	0.63	0.83	1.42	0.73	0.76	0.54	0.60	0.76	0.78
Lead	0.014	0.0056	0.0052	0.0081	0.11	0.0070	0.012	0.023	0.012	0.0020	0.020	0.040	0.028	0.023
Nickel	0.29	0.25	0.42	0.32	0.25	0.24	0.20	0.22	0.32	0.30	0.18	0.25	0.25	0.26
Silver	0.0074	0.0077	0.041	0.018	0.0023	0.0030	0.0080	0.0061	0.0041	0.0027	0.0031	0.0025	0.0040	0.0077
Uranium	0.0014	0.00071	0.00071	0.00095	0.00067	0.00067	0.00073	0.0018	0.0015	0.00083	0.0011	0.0026	0.0012	0.0011

Average Sediment Grab Concentration of Metals in Moira Water Shed 1999 [ug/g-dry weight]

Parameter	Station									Grand Total
	BR	CL	ML	MR	RL	SK	SL	SR	YC
Arsenic	4.7	1.34	158.49	232.27	3.014	2.3	34.6	2.2	3000	143.9
Cobalt	7.1	1.95	308.79	251.13	8.07	6.3	42.53	8.5	1600	162.0
Copper	6	5.25	58.14	52.75	21.57	6	15.73	19	3200	81
Lead	4	12.38	36.71	25.19	43.86	6	24.2	33.67	56	28
Nickel	8.5	3.39	225.86	201.76	11.8	9.3	31.39	17	1000	122
Silver		0.1	3.46	6.35	0.4	0.2	0.33	0.2	4	3
Uranium	0.37	0.41	1.10	0.87	0.91	1.23	1.24	1.39	19	1

Average Sediment Grab Concentration of Metals in Moira River 1999 [ug/g-dry weight]

Parameter	Moira River Station #																Grand Total
	MR-1	MR-10	MR-11	MR-12	MR-13	MR-14	MR-15	MR-16	MR-2	MR-3	MR-4	MR-5	MR-6	MR-7	MR-8	MR-9
Arsenic	1.4	260	660	17	50	21	11	29	2.9	14	140	170	280	500	1300	260	232
Cobalt	5.6	740	1100	45	150	55	33	38	7.5	9	25	140	240	450	440	540	251
Copper	6	100	190	10	40	13	4	35	12	11	23	39	78	110	75	98	53
Lead	3	28	66	11	51	26	9	69	5	8	11	25	25	22	20	24	25
Nickel	92	470	680	24	73	34	14	26	15	13	33	77	250	550	520	440	202
Silver		11	19		0.6	0.2		0.2			0.5	10	4.6	6.3	6.5	11	6
Uranium	0.26	0.89	1.2	1.03	3.91	1.7	0.43	0.51	0.30	0.39	0.48	0.47	0.52	0.54	0.41	0.89	0.9

Concentration of Metals in Moira River and Tributaries [mg/L-wet weight]

PDESC	Year	UNITS	Station
			Black R	Clare R	Deer Cr	ML	MR-1	MR-10	MR-11	MR-2	MR-5	MR-7	PC	Skootamatta R	SL
Arsenic	1999	mg/L	0.0005	0.00063	0.00075	0.020	0.0005	0.0041	0.00067	0.059	0.0047	0.0032		0.0005	0.0052
Cobalt	1999	mg/L	0.00031	0.00013	0.00038	0.0012	0.00040	0.00058	0.00030	0.0016	0.00037	0.00034	0.00033	0.00023	0.0005
Copper	1999	mg/L	0.00055	0.00094	0.0012	0.00044	0.00022	0.00030	0.00043	0.0012	0.00071	0.00031	0.00048	0.00054	0.0006
Lead	1999	mg/L	0.0011	0.0018	0.0026	0.0015	0.0032	0.0023	0.0027	0.0027	0.0019	0.0027	0.0039	0.0020	0.0026
Nickel	1999	mg/L	0.00031	0.00057	0.00048	0.0039	0.00087	0.0011	0.00020	0.0020	0.0024	0.00085	0.00068	0.00022	0.0025
Silver	1987	mg/L			0.0002
Uranium	1991	mg/L	0.00024

Bio-Accumulation Factor (BAF) for Fish Tissue Concentration of Metals in 1999 [Fish/Sediment]

Parameter	Longnose Dace			Average	White Sucker								Average	Total Average
	MR1	MR2	MR3		CL1	ML1	MR4	MR5	MR6	MR7	RL1	SL1
Arsenic	0.020	0.011	0.023	0.018	0.0076	0.00074	0.0012	0.00042	0.00033	7.04E-05	0.0070	0.0041	0.0027	0.010
Cobalt	0.017	0.0021	0.012	0.010	0.0037	0.00011	0.0017	0.00013	0.00012	2.634E-05	0.00099	0.00039	0.00090	0.0056
Copper	0.04	0.016	0.031	0.029	0.031	0.0032	0.011	0.011	0.0028	0.0021	0.0075	0.011	0.010	0.019
Lead	0.0014	0.00034	0.00019	0.00063	0.0026	5.7E-05	0.00033	0.00028	0.00014	2.771E-05	0.00013	0.00049	0.00050	0.00057
Nickel	0.0093	0.0050	0.0096	0.0080	0.022	0.00032	0.0018	0.00087	0.00038	0.00017	0.0046	0.0024	0.0040	0.0060
Silver					0.0070	0.00026	0.0048	0.00018	0.00027	0.00013	0.0023	0.0023	0.0022	0.0022
Uranium	0.0017	0.00072	0.00054	0.00097	0.00049	0.00018	0.00046	0.0011	0.00085	0.00046	0.00035	0.00064	0.00057	0.00077

Sediment Moisture Content    70%

Bio-Accumulation Factor (BAF) for Fish Tissue Concentration of Metals in 1999 [Fish/Water]

Parameter	Longnose Dace			Average	White Sucker								Average	Total Average
	MR1	MR2	MR3		CL1	ML1	MR4	MR5	MR6	MR7	RL1	SL1
Arsenic	189.00	1.81	18.41	69.74		19.72	9.50	4.07	5.34	2.01		90.52	21.86	45.80
Cobalt	800.83	33.20	967.87	600.63		97.87	88.78	40.01	62.32	25.36		119.90	72.37	336.50
Copper	3598.64	552.66	1612.50	1921.27		1435.61	700.71	1203.95	620.71	645.95		1017.75	937.44	1429.36
Lead	4.26	2.02	2.70	2.99		4.53	4.41	8.34	4.18	0.73		15.32	6.25	4.62
Nickel	329.52	121.19	174.30	208.34		60.98	98.64	109.02	155.95	148.08		101.37	112.34	160.34
Silver	37.14	38.71	202.75	92.87		15.10	40.02	30.33	20.72	13.73		12.64	22.09	57.48
Uranium	6.02	2.97	2.94	3.97		2.81	3.055	7.37	6.12	3.44		11.03	5.64	4.80

Moisture Content of Dace    0%

Moisture Content of Sucker    0%

Figure IV-1

Average (with Standard Error) of Lead Concentration in White Sucker Tissue Samples Taken at Each Location

SITE

Figure IV-2

Average (with Standard Error) of Nickel Concentration in White Sucker Tissue Samples Taken at Each Location

SITE

Figure IV-3

Average (with Standard Error) of Cobalt Concentration in Longnose Dace Tissue Samples Taken at Each Location

SITE

Figure IV-4

Average (with Standard Error) of Copper Concentration in Longnose Dace Tissue Samples Taken at Each Location

SITE

Figure IV-5

Average (with Standard Error) of Lead Concentration in Longnose Dace Tissue Samples Taken at Each Location

SITE

Figure IV-6

Average (with Standard Error) of Uranium Concentration in Longnose Dace Tissue Samples Taken at Each Location

SITE

Appendix IV.4 Analysis of Fish Metal Content Versus Size of Fish

Concentration of contaminants of concern in fish tissue (µg/g dry weight) plotted against white sucker length (mm) for exposed and reference sites; 1999 Sentinel Fish Sampling Program.

Exposed site indicates the pooling of data from the following sampling stations:

• Bend Bay
• HWY-37 / Moira River
• Latta
• Moira Lake
• Stoco Lake
• Thomasburg

Reference site indicates the pooling of data from the following sampling stations:

• Consecon Lake
• Round Lake

Concentration of Arsenic in White Sucker Tissue (µg/g dry weight) and Fork Length (mm) for Exposed and Reference Locations

Exposed

REF

Concentration of Cobalt in White Sucker Tissue (µg/g dry weight) and Fork Length (mm) for Exposed and Reference Locations

Exposed

REF

Concentration of Copper in White Sucker Tissue (µg/g dry weight) and Fork Length (mm) for Exposed and Reference Locations

Exposed

REF

Concentration of Lead in White Sucker Tissue (µg/g dry weight) and Fork Length (mm) for Exposed and Reference Locations

Exposed

REF

Concentration of Nickel in White Sucker Tissue (µg/g dry weight) and Fork Length (mm) for Exposed and Reference Locations

Exposed

REF

Concentration of Silver in White Sucker Tissue (µg/g dry weight) and Fork Length (mm) for Exposed and Reference Locations

Exposed

REF

Concentration of Uranium in White Sucker Tissue (µg/g dry weight) and Fork Length (mm) for Exposed and Reference Locations

Exposed

REF

Appendix IV.5 Golder Technical Procedure for Collection of Fish Samples for the Study of Metal Effects

GOLDER TECHNICAL PROCEDURES

Golder Technical Procedure 8.16-0 Fish Health Assessment – Methods

TABLE OF CONTENTS

1. PURPOSE
2. APPLICABILITY
3. DEFINITIONS
   • 3.1 Ageing Structures
   • 3.2 Archive Samples
   • 3.3 Bile
   • 3.4 Biomarker
   • 3.5 Chain-of-Custody Forms
   • 3.6 Contaminants
   • 3.7 Electroshocking
   • 3.8 Fecundity
   • 3.9 Gonads
   • 3.10 GSI
   • 3.11 LSI
   • 3.12 Lesions
   • 3.13 Metallothionein
   • 3.14 Necrosis
   • 3.15 Reference Site
   • 3.16 Sampling Error
   • 3.17 Secondary Sex Characteristics
   • 3.18 Sex Determination (Lethal)
   • 3.19 Sex Determination (Non-Lethal)
   • 3.20 Standard Deviation
   • 3.21 SWI
   • 3.22 TDG
   • 3.23 WHMIS
4. REFERENCES AND SUGGESTED READING
5. DISCUSSION
   • 5.1 General Safety
   • 5.2 Sampling Procedures for Fish Health Assessment
     • 5.2.1 General Considerations
     • 5.2.2 General Preparations for Sampling
     • 5.2.3 Special Precautions to Prevent Contamination of Samples Taken for Metals Analysis
     • 5.2.4 Specific Steps Involved in Sampling for Fish Health Assessment
   • 5.3 Assigning Fish Sample Numbers
   • 5.4 Instructions for Preparing a Composite Tissue Sample
   • 5.5 Sample Identification Label
     • 5.5.1 Labelling for Individual Samples for Contaminant Analysis
     • 5.5.2 Labelling Composite Samples for Contaminant Analysis
     • 5.5.3 Labelling for Liver Metallothionein, Blood and Bile Samples
     • 5.5.4 Labelling for Histology and Egg Samples
   • 5.6 Documentation
     • 5.6.1 Field Record Keeping
     • 5.6.2 Chain-of-Custody Form
     • 5.6.3 Field Records and Logbook
   • 5.7 Change of Procedures
   • 5.8 Shipping of Samples
6. RESPONSIBILITY
7. EQUIPMENT AND MATERIALS.
   • 7.1 General Supplies
   • 7.2 Record Keeping
   • 7.3 Biomarking Equipment (to be stored in waterproof, sealable equipment containers)
   • 7.4 Sample Preservation and Shipping Supplies

LIST OF TABLES

• Table 1 Recommended Fish Ageing Structures
• Table 2 Maturity Codes and Definitions

LIST OF FIGURES

• Figure 1 Protocol for Fish Health Sampling at Metals Sites

LIST OF EXHIBITS

• Exhibit A    Fish Health Assessment Form
• Exhibit B    Biomarker Data Form
• Exhibit C    Variables Form
• Exhibit D    Chain-of-Custody Form

1 PURPOSE

The purpose of this technical procedures document is to:

• describe sampling methods for fish health assessment;
• provide standardization of these methods amongst personnel, sites, sample times, and studies; and,
• document the standard fish health sampling methods employed by Golder Associates Ltd.

The following methods are covered in this technical procedure document:

• special precautions for samples taken for metal contaminant analysis;
• general sampling procedures for fish health assessment;
• sample packaging, preservation, labelling, and shipping procedures; and,
• field documentation.

2 APPLICABILITY

These technical procedures are applicable to all personnel involved in fish health surveys. These procedures are to be used only at sites where there is potential exposure of fish to metals. At other sites, where potential fish exposure to organic compounds, or mixtures of organics and metals are an issue, refer to TP 8.15 and 8.17, respectively. These technical procedures assume that fish have been captured according to methods outlined in TP 8.1-3 Fish Inventory Methods.

3 DEFINITIONS

3.1 Ageing Structures

Parts of the fish that are taken for ageing analyses. These structures contain bands (annuli) that delineate seasonal variation in growth; these bands can be counted to estimate age. Primary examples of these structures are scales, fin rays, otoliths, cleithra and opercula. The appropriate ageing structures to collect vary according to fish species and lifestage and include lethal and non-lethal sampling measures (Table 1).

3.2 Archive Samples

Extra samples which are taken and kept in storage for possible later analysis.

3.3 Bile

An alkaline secretion of the vertebrate liver, which is temporarily stored in the gall bladder. It is composed of organic salts, excretion products and bile pigment. It is responsible primarily for emulsifying fats in the small intestine.

3.4 Biomarker

Biomarker refers to a chemical, physiological or pathological measurement of exposure or effect in an individual organism from the laboratory or the field. Examples include levels of: liver detoxification enzymes (e.g. metallothionein); metabolized contaminants in bile; sex steroids in serum.

3.5 Chain-of-Custody Forms

Standardized forms used as a means of keeping close track of samples that are taken from the field and transported to laboratories for analysis. Whenever the samples are transported from the field, the custody is relinquished from the delivery person to the receiver by signatures on the forms. These forms substantially decrease the risk of losing samples because they provide a clear record of the chain of transport and handling of the samples.

3.6 Contaminants

A general term referring to any chemical compound added to a receiving environment in excess of natural concentrations. The term may include chemicals not generally regarded as "toxic", such as nutrients, colour and salts.

3.7 Electroshocking

The use of electricity to stun and capture fish. An electrical current is passed between electrodes placed in the water; this current causes passing fish (galvanotaxis) to be attracted toward the positive electrode (anode). Once fish pass close to the anode the current acts as a narcotic and stuns the fish (galvanonarcosis), allowing them to be easily netted. Once captured, the fish may be identified, weighed, measured, tagged and then returned to the water. Fish taken by electrofishing revive quickly when returned to the water. Effort is automatically recorded by the electrofishing unit as the number of seconds of active electrofishing (i.e. time current is applied to the water.).

3.8 Fecundity

The most common measure of reproductive potential in fish. It is the total number of eggs in the ovary of a gravid female fish. Fecundity normally increases with the size of the female within a given species.

3.9 Gonads

Organ that are responsible for producing haploid reproductive cells in multicellular animals. In the male, these are the testes and in the female, the ovaries.

3.10 GSI

Gonad-Somatic Index. The proportion of reproductive tissue in the body of a fish. It is calculated by dividing the total weight of the gonad by the total body weight and multiplying the result by 100. It is used as an index of the proportion of growth allocated to reproductive tissues in relation to somatic growth.

3.11 LSI

Liver-Somatic Index. Ratio of liver versus total body weight. Expressed as a percentage of total body weight.

3.12 Lesions

Pathological change in body tissue.

3.13 Metallothionein

Metallothioneins (MT) are a group of proteins present in fish that are responsible for binding and maintenance of metals, including heavy metals. MTs are inducible, that is, exposure to metals causes increase MT production. Therefore, MT levels in fish tissue are a useful indicator of exposure to heavy metals.

3.14 Necrosis

The death of a tissue due to injury or disease.

3.15 Reference Site

A site used for comparison with a site exposed to the discharge being studied. Ideally, reference sites should be as similar as possible to the exposed site, but without the discharge.

3.16 Sampling Error

Sample inaccuracy caused by bias or imprecision in sampling; e.g., bias towards large fish because of the type of sampling gear. In statistics, sample error is expressed by the standard deviation, which expresses the variability of results around the mean.

3.17 Secondary Sex Characteristics

External sexual characteristics displayed by fish, particularly during spawning season. Examples are tubercles on fins or body coloration.

3.18 Sex Determination (Lethal)

Sex can be determined by examining the gonads during the internal examination. Ovaries appear whitish to greenish to orange and have a granular texture. Testes appear creamy white and have a smooth texture (Texas Water Commission, 1990).

3.19 Sex Determination (Non-Lethal)

For some species, sex may be determined from external secondary sexual characteristics, observable either during the spawning season (e.g. suckers – tubercles in males) or at any time of year (e.g. goldeye – anal fin morphology). For most fish species, sex can be determined during the spawning season by forcing extrusion of the sexual product (milt/roe)

3.20 Standard Deviation

A measure of the variability or spread of the measurements about the mean. It is calculated as the positive square root of the variance.

3.21 SWI

Specific Work Instructions

3.22 TDG

Transport of Dangerous Goods

3.23 WHMIS

Workplace Hazardous Materials Information System

4 REFERENCES AND SUGGESTED READING

Anderson, R.O. and S.J. Gutreuter. 1983. Length, Weight and Associated Structural Indices. In: Fisheries Techniques. L.A. Nielsen and D. Johnson (eds.). American Fisheries Society, Bethesda, MD. Pp. 283-300.

Carlander, K.D. 1969. Handbook of Freshwater Fishes of the United States and Canada. 3rd Ed. Iowa State University Press, Ames, I.A.

Environment Canada. 1993. Technical Guidance Document for Aquatic Environmental Effects Monitoring Related to Federal Fisheries Act Requirements. Department of Fisheries and Oceans. Ottawa, Ontario.

Environment Canada. 1995. Further Guidance for the Adult Fish Survey for Aquatic Environmental Effects Monitoring Related to Federal Fisheries Act Requirements. Department of Fisheries and Oceans. Ottawa, Ontario.

Hayes, M.L. 1983. Active Capture Techniques. In: Fisheries Techniques. L.A. Nielsen and D.L. Johnson (eds.). American Fisheries Society, Bethesda, M.D. pp. 123-146.

Jearld, A. 1983. Age determination. In: Fisheries Techniques. L.A. Nielsen and D. Johnson (eds.). American Fisheries Society, Bethesda, MD. Pp. 301-324.

MacKay, W.C., Ash, G.R., and H.J. Norris (eds.). 1990. Fish Ageing Methods for Alberta R.L.&L. Environmental Services Ltd. in assoc. with Alberta Fish and Wildlife Div. and Univ. of Alberta, Edmonton. 113 p.

Puget Sound Estuary Program. 1990c (revised). Recommended Protocols for Fish Pathology Studies in Puget Sound. In: Recommended Protocols and Guidelines for Measuring Selected Environmental Variables in Puget Sound. Prepared by PTI Environmental Services, Bellevue, WA. Region 10, U.S. Environmental Protection Agency, Seattle, WA.

Rohif, F.J., H.R. Akcakaya and S.P Ferraro. 1991. Optimizing Composite Sampling Protocols. Contract 68-CO-0051. Prepared for the U.S. Environmental Protection Agency. Applied Biomathematics, Corvallis, OR.

Scott, W.B. and E.J. Crossman. 1973. Freshwater Fishes of Canada. Bulletin #184, Fisheries Research Board of Canada, Fisheries and Oceans, Ottawa, Ontario. 966 p.

Smith, R.L. 1985. Guidance on Sampling Aquatic Organisms for Tissue Analyses during FY 1986. Environmental Services Division, Region 3, U.S. Environmental Protection Agency, Philadelphia, PA.

Stober, Q.J. 1991. Guidelines for Fish Sampling and Tissue Preparation for Bioaccumulative Contaminants. Environmental Services Division, Region 4, U.S. Environmental Protection Agency, Athens, G.A.

Texas Water Commission. 1990. Texas Tissue Sampling Guidelines. Texas Water Commission, Austin, TX.

USEPA. 1993. Guidance for Assessing Chemical Contaminant Data for Use in Fish Advisories; Volume 1: Fish Sampling and Analysis. United States Environmental Protection Agency. Office of Science and Technology and Office of Water. Washington, D.C.

WDNR (Wisconsin Department of Natural Resources). 1988. Fish Contaminant Monitoring Program -- Field and Laboratory Guidelines. Report No. 1005.1. Madison, WI.

5 DISCUSSION

5.1 General Safety

Refer to Golder Associates Ltd. Safety Manual for general safety procedures.

All solvents and preservatives required for field work must be packaged, labelled, shipped, and used according to WHMIS and TDG regulations.

All used "sharp" dissecting/sampling equipment (needles, scalpel blades, etc.) must be placed in a designated "sharps" disposal container.

5.2 Sampling Procedures for Fish Health Assessment

5.2.1 General Considerations

The procedures outlined here assume that fish have been captured according to methods outlined in TP 8.1-3, and that associated supporting receiving environment measurements have been recorded. These measurements may include: water temperature, pH, dissolved oxygen, conductivity, secchi reading and current weather conditions (e.g., cloud cover, air temperature, approximate wind speed, precipitation, etc.). Also, relevant data for fish population status assessment may be recorded according to TP 8.1-3. These data may include: species identification, weight (g), length (mm), life history staging (fry, juvenile, adult), sex (refer to Section 3.27) and sexual maturity (if possible), and presence of abnormal external pathology (e.g., fin erosion, ulcers, skeletal anomalies, neoplasms). A fish sample number may be assigned according to TP 8.1-3; and aging structures may be collected. Ageing materials to be collected for each fish species are summarized in Table 1 (MacKay et al. 1990).

Concerns regarding the effect of capture and holding stress on fish, particularly on sensitive physiological biomarkers, may require that fish are captured using specific techniques, such as electroshocking. Refer to specific work instructions (SWI) for instructions.

Prior to sampling for fish health, fish can be held temporarily in a live holding facility, such as a live well, holding pen, or tub. If necessary, fish can be marked at the time of capture using temporary tags (floy tags or fin clipping) for later identification during sampling.

For fish selected for biomarker processing, record time (24-hour clock) of capture. Large fish that are moribund or dead should be fully processed for biological data (sex maturity, internal pathology) unless time limits between capture and processing for specific parameters (e.g. histopathology) have been established.

The full set of procedures outlined in this document may not be required for a particular project. Always refer to Specific Work Instructions (SWI) for specific instructions.

Samples must not be allowed to thaw once frozen. Protect sample integrity by ensuring adequate dry ice levels in cooler and then take measures to expedite shipping to the analytical laboratory.

Ensure that histology samples are properly preserved in 10% neutral buffered formalin. In order for total preservation to take place, 10% neutral buffered formalin should be added so that there is a formalin to tissue ratio of 10 to 1. This is best accomplished by having one nalgene container dedicated to all the histology samples from one fish. Place all tissue cassettes for an individual fish into a single nalgene container, and fill the container with 10% neutral buffered formalin.

5.2.2 General Preparations for Sampling

All new personnel must read the technical procedures for Fish Inventory (TP 8.1-3), and Fish Health Assessment (TP 8.15-0, 8.16-0, or 8.17-0 - whichever is applicable). Personnel must understand the protocol for fish health assessment, have it demonstrated and then practice the procedures on at least 2 practice fish.

Battery operated balances are to be checked daily. Level balance at work area and check calibration using standard weights. A vial, weigh paper; anything that has been weighed on a calibrated lab balance may be used. Shield from wind if necessary.

All biomarker data are to be recorded in waterproof field notebooks, Biomarker forms (Exhibit A), External and Internal Examination forms (Exhibit B and C).

5.2.3 Special Precautions to Prevent Contamination of Samples Taken for Metals Analysis

Special care is required to minimize the chance of contaminating samples for metals analysis.

If you are collecting contaminant samples, use a fresh filleting knife and dissecting equipment for each fish. Fresh syringes must also be used for each bile and blood sample.

During the dissection process, take care that the tissues designated as metal samples are dissected on a washable plastic surface covered with a plastic sheet which is changed after each dissection. The fish is to be placed on the plastic dissection surface and kept there while the fillet for metals are taken. The fillet for metals must be placed in a labelled plastic bag. If other organs are also being taken for contaminants analysis (e.g. liver, kidney) special care will have to be taken to isolate the section of the organ to be used for metals and take it while still on the plastic and then carefully remove the other section of the organ without contacting the plastic.

All samples to be analyzed for metals must be placed in plastic packaging, not foil.

Stainless steel dissecting instruments are made predominantly of chromium and nickel. If these metals are not of concern (refer to SWI), the use of high-quality, corrosion-resistant stainless steel sample processing equipment is acceptable (USEPA 1993). Knives with titanium blades and PTFE handles are recommended for performing tissue resections (Lowenstien and Young 1986, USEPA 1993) but clean plastic handles can also be used. Following use, dirt and tissues should be removed from the instruments with distilled water before washing. For washing, utensils and containers should be cleaned thoroughly with a detergent solution, rinsed with tap water, soaked in acid, and then rinsed with metal-free water. Quartz, PTFE, glass, or plastic containers should be soaked in 50% HNO3 for 12 to 24 hours at room temperature (USEPA 1993). Stainless steel parts may be cleaned as stated for glass or plastic, omitting the acid soaking step (Stober 1991, in USEPA 1993).

All dissecting equipment is to be wrapped in heavy plastic wrap during storage; and all dissecting equipment, sample containers, sample wrapping and wash equipment must be shipped and stored in clean waterproof containers with leak-proof lids.

5.2.4 Specific Steps Involved in Sampling for Fish Health Assessment

This section provides detailed step-by-step instructions for sampling for fish health assessment; these steps are summarized in the flow chart entitled "Protocol for fish health sampling at metals sites" (Figure 1). Throughout these procedures, refer to specific work instructions (SWI) for project applicable procedures.

1. Insure the dissection surface (i.e. cutting board) is covered in clean, heavy plastic wrap and that all instruments are clean and readily available to you. Check with the data recorder that all is in order before dissection begins (e.g. sample numbers, labels, dry ice supply, etc.).
2. Assign a biomarker number to fish based on instructions in Section 5.3
3. Put on a clean pair of non-chlorinated, non-powdered latex examination gloves.
4. The next step taken is governed by whether fish is alive or dead, and if dead, for how long. If fish is alive, fish will be processed for the full series of biomarkers and tissue sampling called for in the SWI; in this case proceed to step 5. If fish has been dead for less than 4 hours, proceed to step 8. If fish has been dead for more than 4 hours, then the fish must be rejected for histological sampling, but other measurements may be taken; in this case, proceed to step 8, but disregard instructions for collecting histological samples.
5. Select live fish from holding facility. Excessive handling of fish and stress is to be avoided. Ensure that the skin on the specimen has not been lacerated during sampling. If there has been laceration and loss of fluids, reject the specimen.
6. Collect blood: Specific blood sample collection and treatment methods will depend on the objectives of the work; refer to SWI for specific project applicable methods. Special requirements for serum steroid or other types of analyses may require that the fish be sampled within 1 hour of capture; refer to SWI for project specific instructions. Generally, blood collection must be done as quickly as possible, while minimizing stress to the fish. Blood collect on proceeds as follows: place fish on dorsal surface into a foam block; withdraw blood from caudal vein using a clean, disposable syringe (size of syringe depends on size of fish – 1 to 5 ml may be suitable); label vial; and, place blood on wet ice for later preparation according to SWI.
7. Sacrifice the fish with a blow to the head. Note sacrifice time (24 hour clock) on log sheet.
8. Rinse the fish in ambient water to remove any foreign material from the external surface.
9. Weigh and measure the fish (to nearest gram and mm) and record results. Use fork length measurement except for species with no anal fin indentation (e.g. burbot) which should be measured for total length.
10. Place the fish on the cutting board.
11. Collect gill samples for histopathology: The gill tissues begin to degrade almost immediately after death, and must be removed as quickly as possible. Open the opercula and find the second gill arches on both sides of the fish. Cut out the second gill arch with a pair of scissors taking care to cut as close as possible to the dorsal and ventral insertions. Place the gill arches in a labelled histology cassette and immerse in a nalgene container filled with 10% neutral buffered formalin.
12. Examine the fish for external abnormalities and note on External Examination Form (Exhibit B).
13. Collect fillet samples for metals analysis: Remove two approximately 100 g fillets with a filleting knife that has been cleaned according to instructions outlined above. Remove skin from fillets and ensure that no part of the fillet touches a surface that has not been covered in plastic. Place each fillet on a piece of plastic and record weight. Wrap each fillet in heavy plastic wrap, insert a label in the plastic (between wrapping) taking care not to touch the fillet. Securely tape a second label onto the outside of the plastic. Label one fillet per fish as an archive sample for possible later analysis. Place one fillet sample from each fish in a cooler with dry ice and store there until shipment to a lab. Place the archive fillet sample per fish in a cooler with dry ice clearly marked "archive samples or QC samples". Note: an alternative packaging for contaminant samples is a plastic bag.
14. Collect skeletal muscle sample for histology: After removing a fillet, locate the spinal cord just posterior to the dorsal fin. Remove a piece of skeletal muscle approximately 3 cm × 1 cm × 0.5 cm. Place the skeletal muscle in a labelled histology cassette and immerse in a nalgene container filled with 10% neutral buffered formalin.
15. Open body cavity by making an incision on the ventral surface of the body from a point immediately anterior to the anus toward the head to a point immediately posterior to the pelvic fins; cut intestinal tract at both ends; remove intestinal tract and set aside; remove gonads and set aside. Throughout this procedure and the procedures to follow, examine and record the internal condition of the fish, including the presence of parasites, on the Internal Examination Form. Preserve any abnormal tissues and parasites in 10% neutral, buffered formalin for later analysis. Ensure that histology samples contain both internal labels (waterproof paper and pencil) and external labels. Record tissues taken on the Internal Examination Form (Exhibit C) and in field notebook.
16. Collect gall bladder: Remove liver from body cavity, taking care not to puncture gall bladder. Identify the gall bladder, clamp bile duct using hemostat forceps, and remove gall bladder. Collect bile from gall bladder using a clean syringe, place in a cryovial, and freeze on dry ice. Alternately (depending of size of the organ), you may place the entire gall bladder intact into a cryovial and freeze on dry ice.
17. Measure and record weight of entire liver. Place liver onto a tared piece of plastic on scale, record weight.
18. Collect sample for liver histology: Make a longitudinal section through the middle of the liver and remove a strip of tissue approximately 3 cm × 1 cm × 0.5 cm. Place the sample into a tissue cassette; and preserve in 10% neutral buffered formalin in a nalgene container. For histology samples, the liver tissue should originate from a portion of the liver away from the bile duct.
19. Collect sample for liver metallothionein analysis: Collect an approximately 1 gm. sample of liver for metallothionein analysis. Record the exact weight, wrap the sample in plastic wrap with internal (placed between wrappings, not touching the sample), and external labels, and freeze on dry ice.
20. Collect sample for liver metal contaminant analysis: Take the remainder of the liver, weigh it, and wrap in heavy plastic wrap for contaminants analysis. Insert label in the plastic (between wrapping) taking care not to touch the tissue; and securely tape a label onto the outside of the plastic. Freeze the sample on dry ice.
21. Collect sample for spleen histology: The spleen is a dark purple or wine coloured organ at the distal end of the intestines. It is usually housed in a thin, clear mesentery which is sometimes surrounded in adipose tissue. Remove the spleen in its entirety and place it in a tissue cassette and preserve in 10% neutral buffered formalin inside a nalgene container.
22. Remove and record weight of entire kidney. The kidney is difficult to remove without disrupting the integrity of the organ; usually, the organ has to be scraped from the fish. However, histological analysis must be performed on intact undisturbed tissue. To sample the kidney, perform the following steps: place a piece of clean plastic on the scale, and place a tissue cassette onto the plastic; tare the scale; using a scalpel, obtain two 1 cm thick portions of kidney (one near anterior end, one near posterior end) for histological analysis, place these portions into the cassette on the scale; remove the remainder of the kidney from the fish by scraping with a scalpel, placing the pieces of tissue next to the histological samples on the scale; record total kidney weight.
23. Collect sample for kidney histology: Preserve the histological samples previously placed in the tissue cassette in 10% neutral buffered formalin in a nalgene container.
24. Collect sample for kidney metal contaminant analysis: Wrap the remaining kidney tissue in heavy plastic wrap for contaminants analysis. Insert label in the plastic (between wrapping) taking care not to touch the tissue; and securely tape a label onto the outside of the plastic. Freeze the sample on dry ice.
25. Collect sample for gonad histology: Examine gonads, remove and weigh to nearest 0.1 g. Note sex and assign maturity rating (refer to Table 2 for maturity codes). Make a crosssectional cut through the middle of the organ after weighing and determining state of maturity. Take the cross-section of testes or ovary and place it in a tissue cassette and preserve in 10% neutral buffered formalin inside a nalgene container.
26. Collect samples for fecundity and egg diameter determination (perform this step only if fish is a pre-spawning female): Remove approximately 1 gram of eggs from the midregion of the ovary and place them in round histology cassettes that are lined with foil. Tare the balance to the weight of any empty cassette and then weigh the samples. Label the cassette. A minimum of 50% of the total sample size per species per site must have fresh measurements of egg diameter. Measure 30 individual fresh eggs/female using a micrometer for egg diameter. Record, label and store each egg individually in a histology cassette with 10% neutral, buffered formalin. After the minimum sample size for fresh measurements has been reached, store 30 eggs/female together in a histology cassette with 10% neutral, buffered formalin for analysis at the lab. At the lab, the individual eggs measured fresh in the field will be remeasured and calculated for % shrinkage. The % shrinkage will be used to correct measurement of the samples that were not measured fresh in the field. Volumetric determinations of egg size are made by counting 100 eggs and placing in a graduated cylinder with a pre-measured volume of water. Measure the new volume after the eggs have been added and record. Eg. Pre-volume = 5 ml. Volume with eggs = 5.5 ml. Volume of eggs = 0.5 ml. Precision of volumetric measurements will be dependent upon the graduated cylinder used. NOTE: The precision required for volumetric measurements of egg size in Environmental Effects Monitoring studies is ± 1%. (E.g. if a 10-ml cylinder is used, measurements are expected to be precise to 0.1 ml. This may not be achievable with 100 very small eggs. If not achievable, make note in the field notes and record the actual precision – e.g. 0.5 ml).
27. Collect sample for heart histology: Cut away the pectoral girdle that protects the heart. Grasp the anterior portion of the heart, pull caudally and very gently separate the connection to the ventral aorta. The heart can be pulled far enough out of the body to expose and sever the sinus using a scalpel. For histology samples, make a sagittal cut through the heart, place in a tissue cassette and preserve in 10% neutral buffered formalin inside a nalgene container.
28. Collect sample for thyroid histology: Cut away the ventral insertions of the gill arches, anterior to the heart. The isthmus of tissue in which the gill arches insert contains the thyroid gland although there is no obvious structure. Cut out this triangle of and place it in a tissue cassette and preserve in 10% neutral buffered formalin inside a nalgene container.
29. Open and examine intestinal tract: Observe and record qualitative stomach contents on internal examination form. Qualitative measurement of stomach contents is to be done by estimating the % of total volume of contents taken up by each food item. Be as specific as possible. For example, mayflies, stoneflies, caddisflies, water boatmen, water striders, beetles, not just "insects". Include % sediment or detritus and % plant material. Identify fish to species if possible, e.g., longnose sucker. Identify amphibian, bird or mammal prey as accurately as possible. If stomach contents are to be retained (refer to SWI), then place the stomach contents into a prelabelled whirlpak bag, and preserve by adding 10% neutral buffered formalin.
30. Collect ageing structures: Collect two different ageing structure materials (i.e., scales, pectoral fin ray, otoliths) as per species requirements in Table 1, unless otherwise specified in the Specific Work Instructions. Place ageing materials in an "ageing materials" envelope and label.
31. Discard the remains of the specimen into a sealed bag for later disposal in an appropriate manner (e.g. temporary storage in a freezer, or disposal at a landfill). Discard latex gloves.
32. Rinse off cutting board with ambient water. Put on a clean pair of latex gloves and place a fresh piece of washed foil on the board. Proceed to the next fish.

Once all samples have been taken from one site, ensure adequate dry ice levels in the cooler, attach a Chain-of-Custody (Exhibit D) to the inside lid of the cooler and then seal the cooler using duct tape. Do not mix samples from different sites in one cooler.

5.3 Assigning Fish Sample Numbers

All fish that are selected for fish health surveys are to be given an individual biomarker number. This is in addition to the fish number assigned at the time of capture. The biomarker number is to be recorded on all individual sample labels. The biomarker number is a unique number which identifies the fish by project, species type, site, season and year.

The following format is to be used for biomarker numbering:

e.g.,

WLD/Project    95/Year    P/Season    2A/Site    LNSC/Species    013/Fish No.

Project – a unique 3 character code relating to the project.
Year – use the last two numbers of the year e.g., 1986 = 86.
Season – a one character code relating to season.

P – Spring
U – Summer
F – Fall
W – Winter

Site – a one or two alphanumeric code relating to the site the sample was caught.

Species – a four character abbreviation for species, based on the following rules (MacKay et al. 1990):

1. use a four letter base abbreviation
2. for a one word name – use the first four letters

   e.g., GOLD for goldeye

3. two word names – use the first letter in each word plus the next consonant in each word

   e.g., ARGR for Arctic grayling,
   LKWH for lake whitefish, and,
   WHSC for white sucker

4. three word names – use the first letter in the first two words and the first letter and next consonant in the last word

   e.g., NRDC for northern redbelly dace

Fish No. – a three digit consecutive number. Individual numbering scheme for each species are to be used.

This labelling scheme may be superseded by labelling requirements specific to a project. However, a special labelling scheme may only be used at the authorization of the project manager and QA officer.

5.4 Instructions for Preparing a Composite Tissue Sample

All fish fillet samples for each composite will be placed in a labelled bag. The bag containing the samples to be composited will be labelled in indelible ink with the following information:

• name of the composite
• type of sample (i.e. bile, fillet etc.)
• project number
• name of the collecting company (i.e. Golder)
• analysis requested

Upon arrival at the laboratory all samples should be kept in the labelled bag and returned to it upon completion of preparation of the composite.

For fish fillets the following procedure should be used to prepare the composite:

• unwrap the individual samples that are to form one composite
• take a portion of fillet from each individual sample to use in the composite
• make sure to retain a portion of each individual sample and rewrap it
• the remaining portion of each individual sample should be returned to the labelled composite bag and archived (frozen to -25° C)

For bile samples the following procedure should be used to prepare the composite:

• take a small amount of bile from each cryovial that is to form part of the composite
• if possible, leave some of the bile in each cryovial to be archived for future analysis. In some circumstances when the volume of bile is small (< 0.1 ml), the whole sample may have to be used
• the remaining portion of each individual sample should be returned to the labelled composite bag and archived (frozen to -25° C)

For serum samples the following procedure should be used to prepare the composite:

• take a small amount of serum from each cryovial that is to form part of the composite
• leave some of the serum in each cryovial to be archived for future analysis
• each remaining portion of the individual samples should be returned to the labelled composite bag and archived (frozen to -25° C)

5.5 Sample Identification Label

The use of pre-printed labels is strongly encouraged.

5.5.1 Labelling for Individual Samples for Contaminant Analysis

Each label must be completed in indelible ink for each sample. For contaminant samples, the following information must be included on the label:
Project number
Collecting Agency or Firm-Golder
Biomarker number
Sampling date/time (24 hour clock)
Sample type: F = fillet, W = whole, ungutted, L = liver, B = bile, G = gonad, S = stomach, K = kidney.

Time-frame for analysis – immediate or archive

A completed sample identification label must be taped securely onto each foil-wrapped or bagged sample.

5.5.2 Labelling Composite Samples for Contaminant Analysis

Each label must be completed in indelible ink for each sample. For contaminant samples, the following information must be included on the label:

Project number
Collecting agency or firm
Sampling date/time (24 hour clock)
Sample Site
Sampler (name and signature)
Composite number
Species abbreviation
Sample type: F = fillet, W = whole, ungutted, L = liver, B = bile, G = gonad, S = stomach, K = kidney.
Chemical analysis requested – or refer to an accompanying Chain-of-Custody Form or Analysis Request Form

Time-frame for analysis – immediate or archive

A completed sample identification label must be taped securely onto each wrapped or bagged sample. An additional label identifying the composite sample must be placed on each plastic bag containing the wrapped or bagged samples. The same type of label may also be used for archive samples; simply indicate on the label that the samples are to be archived.

5.5.3 Labelling for Liver Metallothionein, Blood and Bile Samples

Each label must be completed in indelible ink for each sample; and, the following information must be included on the label:

Biomarker number
Sampling date/time (24 hour clock)

Then place a label on outside of the dewar, bag or cooler containing several bile, blood or liver metallothionein samples and including the following information on the label:

Project number
Collecting agency or firm
Sampler (name)
Time frame for analysis – immediate or archive
General sample type (e.g., bile, liver, etc.)

5.5.4 Labelling for Histology and Egg Samples

Each label must be completed in indelible ink for each sample. For histological and egg samples, the following information must be included on the label:

Project number
Sampling date/time (24 hour clock)
Biomarker number
Tissue type:

O = ovary     T = testes
L = liver     S = spleen
H = heart     K = kidney
G = gill     I = intestine
ST = stomach     SK = skin
F = fin     AB = air bladder

An additional label must be placed on the jar or plastic bag identifying the several cassettes or jars of preserved specimens contained within. The label must include the following:

Project number
Collecting agency or firm
Time frame for analysis – immediate or archive
General sample type (e.g., eggs for fecundity, histology samples)

5.6 Documentation

5.6.1 Field Record Keeping

For proper interpretation of field survey results, thorough documentation of all field sample collection and processing activities is required. All logbooks should be perfect-bound and waterproof, forms should be preprinted on waterproof paper, and only indelible ink and pencil (if form or paper is wet) should be used.

To document field activities, sample identification labels, field logbooks, Biomarker Forms (Exhibit A), External (Exhibit B) and Internal (Exhibit C) Examination Forms, and Chain-of- Custody forms (Exhibit D) should be used, in addition to forms described for fish capture records (see TP 8.1-3). This will serve as an overall "Chain-of-Custody", documenting all field samples and field events beginning with sample collection through biomarker processing and preservation and shipment to the laboratory.

5.6.2 Chain-of-Custody Form

Sample possession and proper handling of samples must be traceable from the time of sample collection, through laboratory and data analysis. A Golder Chain-of-Custody form must be completed and signed in indelible ink for each shipping container (e.g. ice cooler) used. Two copies of the Chain-of-Custody form must be sealed in a plastic bag and taped to the outside cover of the cooler. Ensure that the carrier responsible for delivering the samples also signs and dates all Chain-of-Custody forms.

5.6.3 Field Records and Logbook

All pertinent information on field activities and sampling efforts must be recorded in an appropriate (i.e., waterproof) bound logbook. The field crew leader is responsible for ensuring that sufficient detail is recorded in the logbook. The logbook must be complete enough to enable someone unfamiliar with the project to completely reconstruct field activity without relying on the memory of the field crew. All entries must be made in indelible ink, with each page numbered, signed and dated by the author, and a line drawn through the remainder of any partly used page. All corrections are made by a single-line cross-out of the error, entering the correct information, dating and initialing the change. Upon return to the office, all field notes must be photocopied and placed in the appropriate project files.

Entries in the field logbook must include:

• Purpose of proposed sampling effort.
• Date and time (24 hour clock) of sampling.
• Names of field crew leader and team members.
• Description of each sampling site, including information on any photographs that may be taken.
• Location of each sampling site, name and number, applicable navigational coordinates, waterbody name/segment number.
• Details of sampling method and effort, particularly deviations from Specific Work Instructions.
• Clear identification of site names and sample numbers.
• Field observations.
• Field measurements taken (e.g., pH, temperature, flow, dissolved oxygen, secchi, weather conditions).
• Sample shipping information.

The field logbook should also be used to document any additional information on sample collection activities, hydrologic conditions, boat or equipment operations, or any unusual activities observed or problems encountered that would be useful to the project manager when evaluating the quality of the monitoring data.

A biomarker logbook should also be kept. All pertinent information on fish biomarkers must be recorded in an appropriate (i.e., waterproof) bound logbook. The field crew leader is responsible for ensuring that sufficient detail is recorded in the logbook. The logbook must be complete enough to enable someone unfamiliar with the project to completely reconstruct fish biomarker field activity without relying on the memory of the field crew. All entries must be made in indelible ink, with each page numbered, signed and dated by the author, and a line drawn through the remainder of any partly used page. All corrections are made by a single-line cross-out of the error, entering the correct information, dating and initialing the change. Upon return to the office, all field logbooks and notes must be photocopied and placed in the appropriate project files.

Entries in the fish biomarker field logbook must include:

• Date and time (24 hour clock) of sampling.
• Names of field crew leader and team members.
• Site name and number.
• Secchi, water temperature, conductivity,
• Fish number and Biomarker number.
• Capture time/sacrifice time (24 hour clock)
• Length/ Total Weight.
• Sex and stage
• Liver: total weight, sample weights, sample time (24 hour clock), canister storage number, colour
• Fillet: sample weights
• Bile: colour, volume, canister storage number
• Gonads: total weight (testes or ovaries), egg weights, total fecundity count
• Abnormal tissues collected and preserved
• Ageing structures collected

Biomarker Forms, Catch Records, Fish Sample Records, External/Internal Examination Forms and Photo-Log Sheets are to be filled out, dated and signed. All forms should be cross-referenced to the appropriate field record via the fish number and/or composite number.

5.7 Change of Procedures

Variations from the established procedure requirements may be necessary due to unique circumstances in the field. All variations from established procedures shall be documented on Procedure Alteration Checklists (Exhibit G) and reviewed by the Project Manager and the QA Manager.

The Project Manager may authorize the individual Field Crew Members to initiate variations as necessary. If practical, the request for variations shall be reviewed by the Project Manager and the QA Manager prior to implementation. If prior review is not possible, the variation may be implemented at the direction of the Field Biologist, provided that the Project Manager is notified of the variation within 24 hours of implementation and the Procedure Alteration Checklist is forwarded to the Project Manager and the QA Manager for review within 2 working days of implementation. If the variation is unacceptable to either reviewer, the activity shall be repeated or action shall be taken as indicated in the Comments section of the checklist.

All completed Procedure Alterations Checklists shall be maintained in project records.

5.8 Shipping of Samples

Samples are to be shipped by the fastest possible means to the analytical laboratory. The primary QA consideration in shipping samples is protecting sample integrity. Preserve sample integrity by ensuring adequate ice levels in coolers before shipment to laboratory. Coolers are to remain sealed throughout shipment. Weigh-bill numbers are to be noted on the copies of the Chain-of- Custody form retained after sealing the coolers. Each transfer of custody is to be noted and signed for. The coolers should be labelled as Perishable/Keep Cold/Time-Sensitive. Clearly indicate the analytical laboratory address as well as a Golder contact person and phone number. The crew leader is to telephone the processing laboratory and inform them of the upcoming delivery. The crew leader is also required to phone the processing laboratory to confirm arrival of the shipment and that analysis instructions are clear.

6 RESPONSIBILITY

All aquatic field crew members engaged in conducting fish inventories or fish biomarking studies are responsible for compliance with this procedure.

7 EQUIPMENT AND MATERIALS

7.1 General Supplies

First aid kit (including emergency phone numbers of local hospitals, family contacts for each crew member)
Topographical maps of sampling sites
Flagging material
Tool box
Fish tubs

7.2 Record Keeping

Field logbook (perfect-bound, waterproof)
Labels
Chain-of-Custody forms
Fish Sample Records
Unique Catch Records (boat, backpack, gillnet, seine net, etc.)
Indelible pens
Pencils
Applicable MSDS sheets and TGD placards

7.3 Biomarking Equipment (to be stored in waterproof, sealable equipment containers)

Specific Work Instructions
20 Litre pails for transfer and holding of fish
Fish measuring board (metric)
Balance (metric), calibration weights, balance levels and 9 volt batteries
Stainless steel forceps
Stainless steel filleting knives
Stainless steel dissecting scissors
Stainless steel scalpels
Stainless steel scalpel blades
Centrifuge (if taking blood samples)
Small whirlpacks
Nalgene bottles for histology samples
Histology cassettes
Hemostats for clamping off gall bladder
5 ml Cryovials
Blood tubes
Tube rack
Paper towels
0.15 M KCl
Non-chlorinated, non-powdered latex surgical gloves
Plastic wrap
10 ml syringe
18 g needle
5 ml syringe
27 g needle
Pipettes (if taking blood smears or serum samples)
Pipette Bulbs
Goggles
Gloves
"Sharps" disposal containers
Wash-tubs for field-washing of dissecting equipment
Detergent solution
Metal-free water
HNO₃
Distilled water
Used washing solution containers
Plastic cutting boards (washable)
Fish bonker
Folding tables (for biomarking stations)
Biomarker tent
Teflon wash bottle with distilled water
Medical tape
String
Several sizes of plastic bags including garbage bags
Cage material for holding fish in situ, if live-wells or fish tubs not available or too small

7.4 Sample Preservation and Shipping Supplies

Ice (wet ice and/or dry ice)
10% neutral buffered formalin
0.15% KCl
Scale envelopes
Ice chests
Duct tape
Clear shipping tape for Chain-of-Custody forms
Pre-printed labels

TABLE 1:

RECOMMENDED FISH AGEING STRUCTURES (FROM MACKAY ET AL. 1990).

SPECIES	AGEING STRUCTURE (most preferred structure in bold)
	LETHAL		NON-LETHAL
	Preferred	Secondary	Preferred	Secondary
lake sturgeon	otoliths	none	first pectoral fin rayA	none
Arctic grayling	sagittal otoliths	none	scalesB	pectoral fin rays
cisco	sagittal otoliths	none	scalesB (if fast growing)	none
lake whitefish	sagittal otoliths	undetermined	scalesB (if fast growing)	pelvic fin rays
mountain whitefish	sagittal otoliths	none	scalesC	undetermined
lake trout	sagittal otoliths	none	first three pelvic fin raysA	scales (for immature fish)
bull trout	sagittal otoliths	none	none (scales not suitable)	none
brook trout	sagittal otoliths	none	scalesC (if < 3 yrs. old)	none
brown trout	sagittal otoliths	none	scalesD (if < 3 yrs. old)	none
rainbow trout	sagittal otoliths	none	scalesE (if fast growing)	none
cutthroat trout	sagittal otoliths	none	scales (unreliable)	none
northern pike	cleithrum (freeze)	opercular bonesand vertebrae	first three pelvic fin raysA	scalesD (fish up to 3 yrs. old)
goldeye	operculum	none	first three pelvic fin raysA	scalesC (fish up to 5 yrs.)
mooneye	operculum	none	first three pelvic fin raysA	scalesC (fish up to 5 yrs.)
yellow perch	opercular bone	none	pelvic spine and first twofin raysA	two anal spinesA
walleye	opercular bones	otoliths	pelvic spine and first twofin raysA	dorsal spine
sauger	opercular bones	otoliths	pelvic spine and first twofin raysA	dorsal spine
burbot	sagittal otoliths	none	none	none
suckers spp.	none	none	pectoral fin raysA	scales (if <5 yrs.)
trout-perch	otoliths	none	none	none
sculpin spp.	otoliths	none	length-freq. analysis	none
cyprinids	otoliths	none	scales	length-freq. analysis
flathead chub	otoliths	none	pectoral fin rayA	scales
sticklebacks	otoliths	none	length-freq. analysis	none

TABLE 2:

MATURITY CODES AND DEFINITIONS

UNKNOWN (UN): This category is used when state-of-maturity cannot be determined. This will most often occur for fish which have only been examined externally, where no examination of the gonads has been conducted. It may also be used following internal examination of the gonads when the observer cannot definitely determine the maturity of the fish. The gonads have been examined but the observer is unsure which maturity category to use, or the conditions of the gonads do not appear to match any of the maturity categories. If this is the case, record a complete description of the gonads and, if possible, collect a sample for microscopic examination.

IMMATURE (IM): This category is for immature fish (fry or juvenile life stages); defined as fish which have never spawned before and will not spawn in the coming spawning season. The gonads will be undeveloped and will be small and largely transparent. They will be string-like organs situated on the dorsal surface of the body cavity (dorsal to other internal organs) and will lie close under the vertebral column. In very young or small fish, determination of sex from examination of the immature gonads may be difficult or impossible.

Male: The testes will typically be smooth in texture and yellow, pink or white in colour. In suckers and percids, immature male testes can be identified by the position of the testicular artery. The artery is usually totally or partially imbedded in the organ.

Female: The ovaries will typically have a granular texture and will be yellow or pink in colour. In suckers and percids, immature female ovaries can be identified by the position of the ovarian artery. The artery is usually completely outside the organ, resting on top of the surface tissue and attached with connective tissue.

MATURING (MA): A maturing fish is a fish which has not spawned before but will spawn in the coming spawning season. This category refers to a fish whose gonads are developing for the first time. Fish in the maturing category are, for the first time, considered adult fish as they are hormonally similar to sexually mature individuals. Since the gonads are developing for the first time, development may not be complete at the time the fish is examined. The gonads may be developed (enlarged and showing sperm or egg development) primarily at the anterior end. The posterior end of the gonad may still be undeveloped and appear thinner (similar to an immature gonad). This category can be difficult to interpret in the field, being difficult to tell from the Green category, and examination of the gonads by microscope may be required. In general, the gonads of a maturing fish will be smaller than those for a Green fish.

Male: In the field, maturing testes will be smaller and paler than those of fully developed males but considerably larger than immature testes. If unsure, take a sample for histological analysis and designate the fish as Green (GN).

Female: In the field, maturing ovaries will be smaller and paler than those of fully developed females but considerably larger than immature ovaries. If unsure, take a sample for histological analysis and designate the fish as Green (GN).

SEASONAL DEVELOPMENT (SD): Fish in this category are sexually mature adults which have spawned in one or more previous spawning seasons and will spawn in the coming spawning season. The gonads are undergoing their seasonal development following the last spawning season. This is the longest of the sexually mature stages as it extends from just after the post-spawning period until the next pre-spawning period, as the fish utilizes its resources to produce new gametes. For spring spawning fish (e.g. walleye, northern pike, longnose sucker, rainbow trout, etc.), this category would last from late May to early April of the next year. For fall spawning fish (e.g. lake whitefish, mountain whitefish, bull trout, brook trout, etc.) this category would last from the end of the fall spawning season one year (September to November) through to the fall of the next year. However, for most fish, gonadal development occurs primarily during the growing season with only limited gonadal development during the winter months.

Male: The testes will vary greatly in size and colour within this category depending on the time of year the fish is examined. Early in development (i.e. after the post-spawning period), the testes will be small and yellow to light orange in colour. By early fall (i.e. after the primary gonad development period in the summer), they will have grown to nearly mature size and be white in colour. At this point, the testes will be large and distinct. Note: Suckers have a black coloured testicular membrane which may mask the white colour of the testes.

Female: The ovaries will vary greatly in size and colour within this category depending on the time of year they are sampled. Early in development (i.e. after the post-spawning period), the ovaries will be small and yellow to light orange in colour. Developing oocytes will be small and dark orange in colour and will give the ovary a granular appearance. By early fall (i.e. after the primary gonad development period in the summer), the ovaries will have grown considerably to nearly mature size and be bright yellow to orange in colour. The individual eggs will be readily apparent.

PRE-SPAWNING (PR): Fish in this category are sexually mature adults which have spawned in one or more previous spawning seasons and will spawn in the coming spawning season. The Pre-spawning category follows right after the Seasonal Development category, with respect to both time and stage of gonadal development, and occurs when the gonads have completed their seasonal development prior to the spawning season. This is a short term condition which extends from time the gonads are fully developed until the start of spawning activity.

Male: Externally the abdomen will be slightly distended. Semen can sometimes be extruded with pressure to the abdomen. If this is the case, small amounts of loose semen will be extruded followed by more viscous semen if pressure is re-applied. Internally, the testes will be large and white and will fill much of the body cavity. Pre-spawning condition can also be inferred by the capture location of the male. Males will usually only enter spawning condition once they are on the spawning grounds and around mature females. Thus a male caught away from the spawning grounds as the spawning season approaches is most likely still in pre-spawning condition, even if some sexual products can be extruded. Note: Semen can be extruded from sexually mature males as early as February in spring spawning species.

Female: Externally the abdomen will be noticeably distended. Sometimes a few eggs can be extruded with strong pressure to the abdomen. Care must be taken when applying pressure as the eggs are difficult to extrude and injury to the female can occur. The abdomen will feel tight and hard. Internally, the ovaries will be large and bright yellow to bright orange in colour. The size can be up to 25% of the total body weight and the gonads will fill much of the body cavity. Individual eggs will be large, round and obvious, some eggs will be translucent. Pre-spawning condition can also be inferred by capture location. Females will usually only enter spawning condition once they are on the spawning grounds and around mature males. Thus a female caught away from the spawning grounds as the spawning season approaches is most likely still in pre-spawning condition, even if some sexual products can be extruded.

RIPE (RP): Fish in this category are sexually mature adults. Ripe is the term for the spawning condition. The Ripe category follows right after the Pre-spawning category, with respect to both time and stage of gonadal development, and occurs when the gametes (semen and eggs) have become loose in the gonads. This is a short term condition which extends from start to the end of spawning activity. Externally the fish will appear as they do during the Pre-spawning stage but extrusion of the gametes will occur in response to slight pressure on the abdomen.

Male: Externally the abdomen will be slightly distended. Semen can be extruded with light pressure to the abdomen. Internally, the testes will be Large amounts of loose semen will be produced if pressure is applied. large and white.

Female: Externally the abdomen will be greatly distended. Eggs immersed in ovarian fluid can be extruded with light pressure to the abdomen. Large amounts of loose eggs will be produced if pressure is applied. Internally, the ovaries will be large and yellow or orange. The eggs will be large and translucent and some will appear to be loose as the ovarian tissue is weak (i.e. the ovarian sac will be transparent and thin). Eggs will be loose inside the sac and they will be immersed in clear ovarian fluid.

SPENT (SP): Fish in this category are sexually mature adults. Spent is the term for the post-spawning condition. The Spent category follows right after the Ripe category, with respect to both time and stage of gonadal development, and occurs following spawning activity when the gametes (semen and eggs) have been largely extruded during spawning. This length of time a fish will spend in this category depends on how long it takes for the fish to begin the next cycle of seasonal gonadal development, at which time the fish will again be classified as Green.

Male: Externally, the abdomen will be slightly flaccid, especially ventrally. Some semen can still be extruded with pressure to the abdomen but it will most likely be watery (i.e. not as intense a white colour as in spawning males). Internally, the testes will be reduced in size and gray to creamy-white in colour. Hemorrhaging and distended blood vessels on the surface of the organ are common. Post-spawning males are known to stay on the spawning grounds for some time (up to 2 weeks) so capture location is not always a reliable indication of whether the fish has finished spawning.

Female: Externally, the abdomen will be noticeably flaccid, especially ventrally. The surface of the abdomen may be red or roughened with abrasions and the urogenital opening may be extended or swollen. Some eggs can still be extruded with pressure but will be few in number and they will be associated with watery ovarian fluid. Internally, the ovaries will be greatly reduced in size and dark orange to brown in colour. Hemorrhaging and distended blood vessels on the surface of the organ as well as within it are very common and normal. Some residual eggs (from a few up to 25% of the ovary volume) are common. It is not common for post-spawning females to stay on the spawning grounds, most spawn and leave the area immediately. However, capture location is not always reliable indicator.

REABSORBING (RB): Fish in this category are sexually mature fish which have developed to some extent for the coming spawning season but, instead of completing gonadal development or instead of spawning after completing gonadal development, these fish are reabsorbing materials from the gonads back into the body. This category represents arrested gonadal development or interrupted spawning activity. There are several reasons why a fish may terminate gonadal development or decide not to spawn after completing gonadal development. These include the condition of the fish with respect to nutrition and/or health, aspects of population dynamics or environmental cues such as improper water temperatures, poor water quality conditions or adverse water level conditions. Interrupted gonadal development can occur at any stage of development and prior to entering the reabsorbing category the fish may have been Maturing, undergoing Seasonal Development or in Pre-spawning condition.

Male: This condition is extremely rare in males and difficult to observe as reabsorption of the semen by the testes is usually a rapid process. Very rarely will a case be observed of a male actually retaining the entire contents of the testes for re-absorption. Should you suspect this condition the testes should be preserved and stage verified by a qualified biologist.

Female: This condition is primarily observed in females. Reabsorption of the eggs by the ovary is usually a lengthy process which can take up to a full year. Some females may retaining the entire contents of the ovaries for re-absorption. Identification of this stage is not always easy. Externally, the female will still have a distended abdomen if caught within a few months of the spawning season. The abdomen will feel unusually hard as compared to normally developing females. Later in the season, it will be impossible to distinguish a normally developing female from a reabsorbing one without an internal examination. Internally, reabsorbing ovaries go through a series of distinct stages. Early in the reabsorption process, the ovary is dark orange to brown in colour. The eggs are dark and flaccid. Heavy amounts of watery ovarian fluid collect at the posterior of the ovary. This fluid most often is ejected readily if the fish is handled. Later, the ovary becomes smaller and hard. The colour becomes darker and the eggs become atritic. Atritic eggs are easily identified as they are small, hard and white. Ovaries in the later stages of eggs reabsorption have few new oocytes. The remnants of the old eggs collect in the middle of the organ. New oocytes production is restricted to the periphery of the ovary. Should you suspect this condition the ovaries should be preserved and stage verified by a qualified biologist. Occasionally, females have been observed which aborted spawning activity after they had became Ripe. Functionally speaking, eggs at this stage are no longer connected to the ovaries and cannot be reabsorbed. Instead they remain in the body cavity. Internal examination of a fish in this condition will show the newly developed gonad as well as residual (brown, desiccated) eggs which could not be reabsorbed in the posterior portion of the body cavity.

RESTING (RS): Fish in this category are sexually mature adults which have spawned in one or more previous spawning seasons but will not spawn in the coming spawning season. These fish are different from Reabsorbing fish in that their gonads are either not developing or are developing too slowly to be ready for the upcoming spawning season.This is a common condition for fish which do not spawn every year (alternate year spawners).

Male: This condition is extremely rare in males. It can only be used as an alternative to the Green category. A few cases of males in the resting condition have been observed. They are most common in northern latitudes where the growing season is short or in ultra-oligotrophic lakes. Testes will appear flaccid and dirty-white to yellow in colour. They will be larger in size than the testes of immature fish.A good indication is the size of the testicular artery in relation to the organ. In immature fish this artery is very thin whereas in resting males the testicular artery is much larger because of prior testicular development. Should you suspect this condition the testes should be preserved and stage verified by a qualified biologist.

Female: This condition is primarily observed in females but is still relatively infrequent, affecting usually only 0.5 to 1% of the population. This stage can only be used as an alternative to the Green category. It is most common in northern latitudes where the growing season is short or in ultra-oligotrophic lakes. The ovaries will appear to have some oocytes but they will be few in number and arrested in their development. The colour of resting ovaries varies greatly with fish species but most often they are a light orange. They will be larger in size than the ovaries of immature fish. A good indication is the size of the ovarian artery in relation to the organ. In immature fish this artery is very thin whereas in resting females the ovarian artery is much larger because of prior egg development. Should you suspect this condition the ovaries should be preserved and stage verified by a qualified biologist.

FIGURE 1:

PROTOCOL FOR FISH HEALTH SAMPLING AT METALS SITES

FISH HEALTH ASSESSMENT

Waterbody:_______________________ Site:_______________________Date: _____________________

Fish Species:_________ Biomarker Fish # ____________________Fisheries Inventory # ______________

Fork length (mm):____________Total Length (mm)_____________Total Weight (g):_____________________

Carcass Weight (g): __________Capture Method:___________ Aging Structure Taken: FR    Sc    Ot    Op    Cl

Internal and External Examination
Eyes:
N	B1	B2	E1	E2	H1	H2	M1	M2	OT (other):____________________
Gills:
N	F	C	M	P	OT(other):_________________________
Pseudobranchs:
N	S	L	I		OT (other):_________________________
Thymus:
0	1	2	3	comments:_________________________
Skin:
0	1	2	3	comments:_________________________
Bodyform Deformities
	Description:--------------------------------------------------------------------------------------------------------------------
Fins
0	1	2	3	comments:_________________________
Opercles:
0	1	2		comments:_________________________
Hindgut:
0	1	2	3	comments:_________________________
Sex:
M	F	U		Maturation stage: IM MA SD PR RP SP RS RB UN
Mesenteric Fat
0	1	2	3	4	comments:_________________________
Liver:
A	C	D	E	F	OT(other):_________________________
Spleen
B	G	D	E	OT (other):_________________________
Gall Bladder:
0	1	2		comments:_________________________
Kidney:
N	S	M	G	U	OT(other):_________________________
Parasites:
0	1	2	3	comments:_________________________

General Comments:_________________________________________________________________________

________________________________________________________________________________________

________________________________________________________________________________________

________________________________________________________________________________________

________________________________________________________________________________________

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BIOMARKER DATA     Project No.______________ CUTTER:______________
(Full name)

---

Waterbody:____________________ Site:________________________Species:_________________________

Biomarker Fish #:__________________________ Fisheries Inventory #:----------------------------------------

Fork length (mm)----------------: Total length(mm):------------------- Total Weight (g):---------------------

Carcass Weight (g)------------------Sex:------------------- Maturity:-------------------------

Capture Time: Date ____________________ Hr. _____________ Capture Method: ___________

Sacrifice Time: Date ___________________ Hr. ________________Sacrifice Method: __________

---

COLLECTION DETAILS:

STOMACH:

Observed: _________

Collected: _________

% Fullness: _________

Description/% of Contents:____________________

Other Observations:_________________________

HISTOLOGY EXAMINATION:

      Abnormality     Preserved (g)

Liver _________   __________________

Spleen _________   _________________

Heart _________   ____________________

Gill _________   ____________________

Kidney _________   ___________________

Other _________   ___________________

BLOOD: Time Taken ________

Total Volume: _____ mL

Time of Centrifugation: ________

Plasma Volume: _____ mL

Plasma Colour: ________

Hematocrit: ______ %

______ %

Leucocrit: ______ %

______ %

Plasma Protein: _____g/dL

LIVER: MFO / MT: _______ g

Contaminants: _______ g

Histology: _______ g

Archive: _______ g

Total Liver Weight: _______ g

Time MFO Sample Taken: ________

KIDNEY: MT: _______ g

Contaminants: _______ g

Histology: _______ g

Total Kidney Weight: _______ g

FILLETS: Organics: _______ g

Metals: _______ g

Other: _______ g

BILE: Gall Bladder Fullness (%): ________

Colour: ________

Volume Collected: _____ mL

GONADS: Contaminants: _______ g

Histology: _______ g

Region Collected From: ________

Fecundity Subsample Weight: _______ g

Region Collected From: ________

No. of Eggs in Subsample:

Ave. Egg Diameter: _____ mm

No. of Eggs Measured: ________

Total Gonad Weight _______ g

Recorder (Full Name) ________

VARIABLE	VARIABLE CONDITION	ORIGINAL FIELD DESIGNATION
Eyes	No aberrations; good “clear” eye	N
	Blind; an opaque eye (one or both)	B
	Swollen, protruding eye (one or both)	E
	Hemorrhaging or bleeding in the eye (one or both)	H
	Missing one or both eyes	M
	Other; any manifestation not fitting the above	OT
Gills	Normal; no apparent aberrations	N
	Frayed; erosion of tips of gill lamellae resulting in “ragged” gills	F
	Clubbed; swelling of the tips of the gill lamellae	C
	Marginate; gills with light, discolored margin along tips of the lamellae	M
	Pale; very light in color	P
	Other; any observations not fitting above	OT
Pseudobranchs	Normal; flat, containing no aberrations	N
	Swollen; convex in aspect	S
	Lithic; mineral deposits, white, somewhat amorphous spots	L
	Inflamed; redness, hemorrhage, or other	I
	Other; any condition not covered above	OT
Thymus	No hemorrhage	0
	Mild hemorrhage	1
	Moderate hemorrhage	2
	Severe hemorrhage	3
Skin	Normal; no aberrations	0
	Mild skin aberrations	1
	Moderate skin aberrations	2
	Severe skin aberrations	3
Fins	No active erosion	0
	Light active erosion	1
	Moderate active erosion with some hemorrhaging	2
	Severe active erosion with hemorrhaging	3
Opercle	No shortening	0
	Mild shortening	1
	Severe shortening	2
Hindgut	Normal; no inflammation or reddening	0
	Slight inflammation or reddening	1
	Moderate inflammation or reddening	2
	Severe inflammation or reddening	3
Mesenteric	Fat None	0
	< 50 %	1
	50 %	2
	> 50 %	3
	100 %	4
Liver	Normal; solid red or light red color	A
	"Fatty" liver; “coffee with cream” color	C
	Nodules in the liver; cysts or nodules	D
	Focal discoloration; distinct localized color changes	E
	General discoloration; color change in whole liver	F
	Other; deviation in liver not fitting other categories	OT
Spleen	Normal; black, very dark red, or red	B
	Granular; rough appearance of spleen	G
	Nodular; containing fistulas or nodules of varying sizes	D
	Enlarged; noticeable enlarged	E
	Other; gross aberrations not fitting above categories	OT
Gall Bladder	Normal	0
	Enlarged	1
	Parasites	2
Kidney	Normal; firm dark red color, lying relatively flat along the length of the vertebral column	N
	Swollen; enlarged or swollen wholly or in part	S
	Mottled; gray discoloration	M
	Granular; granular appearance and texture	G
	Urolithiasis or nephrocalcinosis; white or cream-colored mineral material in kidney tubules	U
	Other; any aberrations not fitting previous categories	OT
Parasites	No observed parasites	0
	Few observed parasites	1
	Moderate parasite infestation	2
	Numerous parasites	3

Maturity Codes:   IM = Immature   MA = Maturing   SD = Seasonal Development   PR = Pre spawning

  RP = Ripe   SP = Spent   RS = Resting   RB = Reabsorping   UN = Unknown

GOLDER ASSOCIATES LTD.
CHAIN-OF-CUSTODY RECORD
AND ANALYTICAL REQUEST FORM

Page______ of_______

Field Sampler: (Signature)________________________

Phone No.:_____________________________________

Shipment Date:_________________________________

Carrier:_______________________________________

Waybill No.:______________________________________

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Ship To:

Send Results To:

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Project Name: __________________________________________

Project No:_______________________________________________
P.O. No.:______________________________________________________

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Relinquished by: (Signature)

Received by: (Signature)

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Time:

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Relinquished by: (Signature)

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Received at lab by: (Signature)

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Date:

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Time:

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Relinquished by: (Signature)

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Received at lab by: (Signature)

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Date:

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Relinquished from lab by: (Signature)

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Received by: (Signature)

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ANALYSIS REQUEST

Sample ID No.	Sample Description	Date/Time Sampled	Analysis Requested	Sample Condition Upon Receipt

Special Instructions/Comments:

Rush (surcharge):_________________________Standard Turnaround Time:___________________________

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WHITE COPY      RETURN TO GOLDER ASSOCIATES LTD.
YELLOW COPY     LABORATORY COPY
PINK COPY      RETAINED BY FIELD CREW LEADER

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^A proximal end

^B collect 10-15 scales from the left side between the front edge of the dorsal fin and the lateral line

^C collected between the dorsal fin and the lateral line

^D collected from above the lateral line just posterior to the dorsal fin

^E collected from immediately dorsal to the lateral line, between the posterior edge of the dorsal fin and origin of the anal fin