NOAA Technical Memorandum NMFS 

AUGUST 1984 

HISTOPATHOLOGICAL MANUAL FOR 

MONITORING HEALTH OF STRIPED BASS 

IN RELATION TO POLLUTANT BURDENS. 

Jeannette A. Whipple 

Marvin Jung 

R. Bruce MacFarlane 

Rahel Fischer 

NOAA-TM-NMFS-SWFC-46 

U.S. DEPARTMENT OF COMMERCE 

National Oceanic and Atmospheric Administration 

National Marine Fisheries Service 

Southwest Fisheries Center 
NOAA Technical Memorandum NMFS 

The National Oceanicand AtniosphericAdniinistration(N0AA). organized in 1970, hasevolved intoan 

agency which establishes national policies and manages and conserves our oceanic, coastal, and atmospheric resources An organizational element within NOAA, the Office of Fisheries is responsible for 

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In addition to its formal publications, the NMFS uses the NOAATechnical Memorandum series to issue 

informal scientific and technical publications when complete formal review and editorial processing are 

not appropriate or feasible Docunients within this series, however, reflect sound professional work and 

niay be referenced in the formal scientific and technical literature 
NOAA Technical Memorandum NMFS 

This TM series is used for documentation and timely communication 01 preliminary results, imterim reports, or 

special purpose inlormation, and have not received complete formal review, editorial control. or detailed editing 

AUGUST 

HISTOPATHOLOGICAL MANUAL FOR 

MONITORING HEALTH OF STRIPED BASS 

IN RELATION TO POLLUTANT BURDENS. 

Jeannette A. Whipple 

R. Bruce MacFarlane 

Rahel Fischer 

Southwest Fisheries Center 

National Marine Fisheries Service 

Tiburon, California 94920 

Marvin Jung 

State Water Resources Control Board 

Sacramento, California 

NOAA-TM-NMFS-SWFC-46 

U.S. DEPARTMENT OF COMMERCE 

Malcolm Baldrige. Secretary 

National Oceanic and Atmospheric Administration 

John V. Byrne. Administrator 

National Marine Fisheries Service 

William G. Gordon, Assistant Administrator for Fisheries 

1984 
TABLE OF CONTENTS 

Section Pages 

INTRODUCTION .......................................... 1 

HISTOPATHOLOGICAL ASSESSMENT .......................... 3 

Preparations Prior to Autopsy .................... 3 

Coding and Labels ........................... 3 

Data Sets and Supplies ...................... 4 

Examination. Autopsy and Procedures .............. 7 

Exterior Examination ........................ 7 

Measurements ................................ 9 

Color Pattern Determination ................ 11 

Dissection and Interior Abnormalities ...... 13 

Subsampling ................................ 14 

Egg Examination ............................ 20 

Sperm Motility. Hematology and Specimen 

Archiving .................................. 21 

Storage of Samples and Sample Labels ....... 22 

STRIPED BASS HEALTH INDEX ............................ 24 

Introduction ............................... 25 

Objectives ................................. 29 

Methods .................................... 29 

Action Levels .............................. 34 

Recommended Action by Regulatory Agencies .. 36 

Additional Research/Recommendations ........ 37 

Data Analysis .............................. 37 

ii 
TABLE OF CONTENTS 

(CONT . ) 

Section Pages 

LITERATURE CITED ..................................... 39 

ACKNOWLEDGEMENTS ..................................... 42 

APPENDICES ........................................... 43 

I . Variable List for Factor Analysis #3 ....... 43 

I1 . Coding for Data Sets ....................... 50 

I11 . Preparation of Buffered Formalin ........... 63 

IV . Glassware Cleaning Procedure for Trace Metal 

Analysis ................................... 64 

V . Preparation of Clean Foil for PCB Samples .. 65 

VI . Trace Metal Analysis of Fish Liver ......... 66 

VI1 . Processing Tissue Samples for Mono- and 

Polycyclic Aromatic Hydrocarbons by Gas 

Chromatography ............................. 70 

VIII.Procedures for Hematology .................. 72 

IX . Egg Histology .............................. 75 

APPENDIX TABLE ....................................... 78 

APPENDIX FIGURES ..................................... 81 

iii 
ABSTRACT 

Since 1978, the Physiological Ecology Investigation staff at 

the Tiburon Laboratory has been examining the relationship of 

pollutants to the physiological condition or "health" of striped 

bass (Morone saxatilis). During the course of this 

investigation, a series of standard and new techniques were 

developed to quantitatively and qualitatively assess the health of 

prespawning striped bass while simultaneously sampling tissues for 

pollutant residues. Prespawning bass and their gametes were 

selected because they are relatively sensitive to the effects of 

pollutants at low chronic levels (Whipple, Eldridge and Benville, 

1981). 

The autopsy techniques were refined to include the 

measurements and characteristics that were 1) most indicative of 

pollutant stress and 2) also relatively easy to assess in a 

monitoring program. 

This report includes histopathological techniques derived by 

the Cooperative Striped Bass Study (COSBS) and from a report on 

our recommendations to the State Water Resources Control Board for 

monitoring of striped bass in the San Francisco Bay ecosystem. 

iv 
-1- 

INTRODUCTION 

Laboratory experiments performed at Tiburon Laboratory with 

striped bass eggs and larvae (1975-1977) revealed considerable 

variation in viability of eggs obtained from different females. 

We hypothesized that part of the variation might be related to 

pollutant stress on the prespawning females and that this stress 

might be quantified by relating measures of gametic egg condition 

to pollutant burdens in the livers and ovaries of the parental 

female. In an attempt to determine the source of this variation 

and to find out why so many of the eggs were inviable, we 

undertook sampling of prespawning females from the San Joaquin 

River off Antioch in 1978. 

During this research a histopathological autopsy procedure 

was developed to examine the health of striped bass, emphasizing 

measurements of the egg and reproductive condition. Initial 

examinations revealed that many fish were in poor health. For 

example, many fish had skeletal abnormalities, open and healed 

lesions, high parasite burdens and gonads impacted by parasites, 

as well as necrotic eggs and ovaries. Consequently, the autopsy 

examination was expanded to include a number of additional 

measurements and subsamples of organs and tissues in order to 

quantify the relationship of pollutant burdens to parasites and to 

body, liver and egg condition. 
-2- 

An earlier version of this manual was printed in 1981 

(Whipple and Jung, 1981) by the State of California Water 

Resources Control Board (SWRCB) as part of the Cooperative Striped 

Bass Study (COSBS). The present manual has been edited to include 

the most relevant measures and subsamples for indicating the 

health of striped bass. A list of these variables is in Appendix 

I. Selection of variables was based on observations and data 

analyses for more than 500 fish collected from 1978-1983. These 

results are reported in previous COSBS reports (Jung and Bowes, 

1980; Jung et al., 1981; Whipple, Crosby and Jung, 1983) and in a 

paper by Whipple, Benville, Eldridge and MacFarlane (in prep.). 

This manual, therefore, includes the histopathological procedures 

and subsampling recommended to the California Department of Fish & 

Game (CDF&G) for monitoring striped bass during prespawning 

seasons in the future. 
-3- 

HISTOPATHOLOGICAL ASSESSMENT 

I. PREPARATIONS PRIOR TO AUTOPSY 

The following steps should be taken before examining the fish 

to reduce the time spent on each autopsy, There is no 

specific sequence to these steps. 

A. Coding (Appendix 11) 

Post coding for the data sets above the autopsy table. 

Changes should be noted in large print or circled. 

B. Labels (see page 23) 

Complete experiment (EXP.) and location (LOC.) 

codes, FISH NO, and DATE entries on the set of 

labels. EXP. code represents a laboratory experiment 

or a year of field studies; LOC. is the place of 

capture. The FISH NO. is on the label tucked behind 

the gill cover of the fish. 

examination. See Coding for All Data Sets (Appendix 

11). 

DATE is the date of your 

Cut out labels and place them on a clean surface to 

eliminate contamination of samples taken for pollutant 

analyses. 
C. Data Sets (Appendix 

Check to see that D 

DATA (Appendix Fig. 

the sampler. 

-4- 

Figures A- K) 

TA SET - C PTURE OR FIELD SAMPLE 

A) has been completed correctly by 

For the autopsy, you will need Data Sets 2, 3, 4, 5, 6, 

8 and 22 (Appendix Figs. B - K), not necessarily in 

that order. A clipboard for each set is recommended. 

AI1 data are keypunched directly from the data sets as 

entered. Each space on a data set represents one 

computer-card column. Note that some column headings 

have been left blank. It is important that these 

columns remain in the data sets, even though they are no 

longer in use, in order for newly recorded data to be 

compatible with data that have been already keypunched. 

Data are right-justified (entered as far to the right as 

possible in the given spaces). Align numbers with 

decimal points already entered in the columns. Please 

write clearly and align numbers carefully. 

EXAMPLES: 

9.2, 8.7 5 --- 9, --- 2 9, --- ---- 

When entering data codes, do not include letter 

designations in data set blanks. Letters can be written 

in column margins for later reference. 
-5- 

Enter codes for EXPERIMENT, LOCATION and METHOD at 

top of all data sets: use Coding for All Data Sets 

(Appendix 11). Enter FISH NO. on all data sets; 

DISSECTION DATE and TIME (beginning of autopsy) on 

DATA SETS 2 and 6. 

D. Scale Envelopes 

For each fish, label a scale envelope clearly with 

EXPERIMENT code, LOCATION code, FISH NUMBER and 

DATE. 

E. Photo Identification 

Prepare an identification card for each fish--an index 

card with EXPERIMENT, LOCATION, FISH NUMBER and 

DATE written in large, clear numbers. This is used 

when photographing the fish. 

F. Supplies 

Lay out sample jars and lids, plastic freezer bags, 

scale envelopes, 4" x 4" squares of clean aluminum foil, 

and dissecting tools for easy access. 

Check operation of all balances and cleanliness of 

weighing pans. 
-6- 

Check on supply of hexane-rinsed foil for PCB/pesticide 

samples and acid-washed containers for heavy metal 

samples. Procedures for preparing both are in 

Appendices IV & V. 

Supplies for monocyclic aromatic hydrocarbon (MAH) 

analysis are found in Appendix VII. 

Check supply of buffered 10%-formalin solution used as a 

preservative for histology samples. (Appendix 111) 

Filter paper and a Munsell chart for color determination 

should also be nearby. 
-7- 

11: EXAMINATION, AUTOPSY AND SUBSAMPLING PROCEDURES 

The following procedures should be performed in the sequence 

presented. 

A. Exterior Examination 

1. Check for the following (Figure 1: What to Look 

For) and use DATA SET 6 - DISEASE, PARASITES AND 

ABNORMALITIES and Coding for Data Set 6 to 

record your observations. 

a. Skeletal abnormalities--fins, gill rakers, 

vertebral column, skull bones, etc. 

b. Lesions, wounds, scars, reddening -- 

particularly on right ventrolateral surface or 

under pectoral fin. (Run your fingertips 

along these areas to feel for lesions which 

may have healed over; scale pattern is usually 

interrupted.) 

c. Gill erosion, parasites, color 

d. Eyes for opacity, and nares for occlusion 

e. Fins for fin rot 

f. Other 
-8- 

Figure 1. -- What to Look For. 

Check for the following: 

(1) Pugheadedness, scoliosis and 

other skeletal abnormalities. 

(2) Lesions, wounds -- measure size. 

Feel for them with your fingertips. 

(3) Gill erosions, parasites, color. 

(4) Opacity in eyes, occlusion in 

nares. 

(5) Fin rot and other fin abnormalities. 

(6) Other parasites, diseases and 

abnormalities. 

\ 

\ 

\ 

\ 

\\ 
-9- 

2, Record the size of any wound or lesion under 

NOTES on the right-hand side of Data Set 6. 

B. Measurements (Figure 2: What to Count and Measure). 

1. Complete all of DATA SET 2 - MERISTICS AND 

MEASUREMENTS except for AGE, - Record 

measurements for the left side of the fish. 

2. Enter, to nearest 0.5 cm, FORK LENGTH and 

STANDARD LENGTH. DEPTH (width) is optional. 

3. Enter the weight of the fish to the nearest whole 

gram (1.0 g) under WET WT. 

4. Take 8-10 scales from just above the lateral line 

for age determination. Seal them in the prelabeled 

scale envelope. 

-. I- 
-10- 

Figure 2.-- What to Count and Measure. Record Standard and Fork 

Lengths (mm) and Total Wet Weight of Fish (9). Take 8-10 

scales from above lateral line for age determination. 
-11- 

C. Color Pattern Determination 

1. Record your data on DATA SET 3 - COLOR PATTERN. 

Refer to Figure 3: Color Pattern and use the 

Coding for Data Set 3. (Appendix 11) 

2. Record dorsal melanization code under -- COL PAT 

- INT. 

3. Starting with the left side of the fish, assess the 

degree of breakage in the striping pattern for the 

dorsal region. Record breakage codes for 

anterior, mid and posterior sections under 

STRIPING PATTERN BREAKAGE DORSAL A M P. Add 

values for DORSAL A, M and P and record under 

DORSAL TOT. 

EXAMPLE: If dorsal stripes in the anterior 

section are broken, unbroken in the 

midsection, and broken in the posterior, 

record under DORSAL A M P TOT the following 

codes: _--- 2 1 2 5. 

4. Do the same for the ventral striping pattern and 

record under STRIPING PATTERN BREAKAGE VENTRAL 

A M P TOT. 
-12- 

Figure 3.-- Color Pattern. Record (1) degree of breakage in striping 

pattern for dorsal and ventral stripes 

2 = broken stripes) and (2) color pattern intensity and rank. 

(1 = solid stripes; 

vior 

1 

Posterior 

1 

Later 
-13- 

5. Add values for DORSAL TOT and VENTRAL TOT and 

record under ALL STRPS TOTAL. 

6. Enter code for SIDE. 

7. Plhotograph left side of fish, with identification 

card prominently displayed, using black-and-white 

(Kodak Plus-X*, Panatomic*, etc.) or Polaroid* 

film. Record film roll and frame numbers under 

PHOTO ROLL/FRAME. 

8. Note general appearance of dorsal region and enter 

description of overall breakage (1/4, 2/4, or 3/4 

broken) under DORSAL BREAKAGE. 

9. Repeat steps 3 - 8 for right side of fish. 

D. Dissection and Interior Abnormalities 

1. Use DATA SET 6 - DISEASE, PARASITES AND 

ABNORMALITIES. 

* Use of brand name does not imply endorsement by the 

National Marine Fisheries Service. 
-14- 

2. Open fish with ventral incision from vent to lower 

jaw. Lift gonads and check opening to exterior 

with blunt probe for any occlusions. Note on DATA 

SET 6 if blocked. Note any visceral adhesions or 

parasites which press on gonads and/or keep organs 

abnormally bound to the body wall or cause abnormal 

growth. Look for healed lesions in the body wall 

which may not have been visible on the outside. 

Note other parasites. 

3. Tare balance with a pan lined with hexane-rinsed 

foil. Place gonads, stomach, intestines, liver, 

gall bladder and spleen (all organs except heart) in 

the pan. Record total gut weight to nearest whole 

gram (1.0 g) under WET WT VISCERA on DATA SET 

6. 

E. Subsampling 

1. Left Ovary 

a. Use DATA SET 4 - GONADS: MERISTICS AND 

FECUNDITY. Enter codes for - SEX, gonad 

(GND) - and maturity (MTR). - Use one line 

for each gonad. 

b. Record length (TL) - and width (WI) - of each 

gonad to nearest 0.5 cm. 
-15- 

c. Enter weight of each gonad to nearest tenth 

gram (0.1 g) under WET WT. Record combined 

weights of both gonads under TOTAL WW (both 

lines). 

d. Examine for parasites, abnormalities, etc. and 

note on DATA SET 6. 

e. Take small portions of the anterior, mid and 

posterior sections for fecundity. Place in 

small vials with the appropriate labels (see 

page 23). Be sure anterior, mid and posterior 

sections are correctly labelled. 

f. Section remaining ovary for pollutant samples. 

FOLLOW CLEAN TECHNIQUES FOR EACH SAMPLE (Apps. 

IV, VI & VIII). Place POLL-I MAH sample into 

culture tube and process immediately to avoid 

losing the MAHs. Place POLL-I PAH sample for 

polycyclics in glass jar with foil-lined lid, 

shiny side of foil toward samples; freeze 

immediately on dry ice. Place POLL-I1 samples 

for heavy metals (HM) in acid-washed 

conventional polyethylene containers. Wrap 

POLL-I11 samples for chlorinated hydrocarbons 

(DDT, DDD, DDE) in foil prerinsed in nanograde 

hexane. Place labels securely on the outside 

of the containers with strapping tape. 

_I_ -- 
-16- 

2. Left Testis 

Follow procedure for left ovary. No fecundity 

samples will be taken. Use the appropriate labels. 

3. Right Ovary 

a. Weigh, measure, examine and note 

abnormalities, parasites, etc., as with the 

left ovary. Use DATA SET 4 - GONADS: 

MERISTICS AND FECUNDITY and DATA SET 6 - 

DISEASE, PARASITES AND ABNORMALITIES. 

b. Place an anterior sample for color on a piece 

of white filter paper. Match the egg colors 

to the Munsell color chart and enter on DATA 

SET 4 under MUNSELL HUE, VAL. and CHR.. 

Use Munsell codes and Coding for Data Set 4 

to find HUE RANK I and - 11. Use same 

lighting (e.g. fluorescent) throughout 

examinations. 

c. Place an anterior portion in a petri dish with 

fresh water for examination of fresh eggs and 

measurement of egg diameters under microscope. 

See F, Egg Examination, p. 20. Another 

anterior section should be placed in a 

10%-buffered formalin solution for histology. 
-17- 

Make sure sample is no larger than 2 cm 

circumference and is well covered with the 

formalin, Use appropriate label. 

d. (Optional). Take a midsection sample for 

parasitology. Seal in a plastic bag with 

label. 

4. Right Testis 

Proceed as for right ovary. The anterior sample 

for histology is first examined for sperm motility 

(See G., Sperm Motility, p. 21). Use appropriate 

labels. 

5. Liver 

a. Examine liver and gall bladder for parasites, 

abnormalities, etc., and record your 

observation on DATA SET 6 - DISEASE, 

PARASITES AND ABNORMALITIES, using Coding 

for Data Set 6, 

b. Remove gall bladder carefully to avoid 

spilling bile on tissue to be analyzed for 

pollutants (Optional). Place gall bladder in 

a small vial and put into plastic bag to go 

with the liver parasitology sample. 
-18- 

c. Weigh liver to nearest tenth gram (0.1 9). 

Record weight on DATA SET 5 - LIVER, GALL 

BLADDER AND STOMACH under LIVER SIZE WET 

WT . 

d. Rank liver color according to Coding for Data 

Set 5 and record under LIVER COLOR HUE RANK 

- I, If liver is red due to hemorrhaging be 

sure to record on Data Set 6. 

e. Section liver for the following subsamples: 

(1). Take a sample of the right lobe for 

histology. Place in a vial with 

10%-buffered formalin solution with 

label. 

(2). Take four middle samples (POLL-I MAH, 

POLL-I PAH, POLL-I1 HM and POLL-I11 CH) 

for pollutant analyses. Place carefully 

in appropriate containers.(see 1 f., 

ovary pollutant sampling, p.15, and 

Appendix VII). Use the appropriate 

labels. 

(3). Take a sample of the left lobe for 

parasitology, Seal in a plastic bag with 

the gall bladder vial and label. 
-19- 

6. Spleen 

Examine spleen for parasites, abnormalities, etc. 

Record your observation on DATA SET 6 - DISEASE, 

PARASITES AND ABNORMALITIES. 

7. Kidney 

Examine for abnormalities and parasites. Record on 

DATA SET 6 - DISEASE, PARASITES AND 

ABNORMALITIES. 

8. Other Organs 

a. On DATA SET 5 - LIVER, GALL BLADDER AND 

STOMACH, enter code for stomach contents 

(STOMACH CNT); use Coding for Data Set 5. 

On DATA SET 8 - SPLEEN AND OTHER ORGANS use 

Coding for Data Set 8 to rank the amount of 

mesenteric fat and condition of intestines. 

Note any abnormalities on DATA SET 6 - 

DISEASE, PARASITES AND ABNORMALITIES. 

b. (Optional). Seal the stomach and intestines in 

a freezer bag with the appropriate label for 

parasitology. 
-20- 

9. Muscle 

a. Fillet left anterior dorsal muscle for 

pollutant analyses POLL-I MAH, POLL-I PAH, 

POLL-I1 HM and POLL-I11 CH). Use appropriate 

containers and techniques (see 1 f., p. 15 and 

Appendix VII) and the appropriate labels. 

b. Extra samples for pollutant analyses may be 

taken from right dorsal muscle. 

F. Egg Examination 

Record data on DATA SET 22 - GONADS: EGG DIAMETERS AND 

SPERM MOTILITY. Examine sample of eggs taken from 

anterior right ovary under 50X magnification on the 

dissecting microscope. Measure the diameter of five 

individual eggs, picked at random, to nearest 100 

microns; record mean under MEAN EGGS FRESH. Note (1) 

any increase in perivitelline space, (2) if eggs are 

loose or clumped, and (3) if oil drops are dispersed or 

coalesced. Note the approximate maturation stage 

(MATUR) and any abnormalities (NOTES). 
-21- 

G. Sperm Motility (Optional) 

Place a small portion of milt or ripe-running sperm on 

tip of pipette or blunt probe and stir into a 50-ml 

beaker of distilled water. Water temperature should be 

around 18 deg C or higher, but not higher than 25 deg C. 

Allow to sit for at least 5 minutes. Examine for signs 

of motility under 400X magnification on compound 

microscope. Dark-field phase shows spermatozoa very 

well. If spermatozoa are not motile after 5 minutes in 

water, they are probably still immature or abnormal; 

reexamine after 30 minutes. Record motility on DATA 

SET 22 - GONADS: EGG DIAMETERS AND SPERM MOTILITY. 

H. Hematology (Optional) 

Hematocrits and slides of blood smears. See Appendix 

VIII. 

I. Specimen Archiving (Optional). 

Replace fish number tag under gill cover. Place bags 

with parasitology samples (gonad, liver and gall bladder, 

stomach and intestines) in body cavity. Wrap entire 

fish tightly in a large plastic bag (trash bag), seal 

with strapping tape, attach label and freeze. 
-22- 

J. Storage of Samples 

TISSUE 

---------- 

SAMPLE FOR 

--------------- 

CONTAINER 

---------- 

PRESERVATIVE 

------------ 

L. Ovary Glass vial 

L. Gonad 

-Fecundity 

-Pollutants I-MAH 

-Pollutants I-PAH 

-Pollutants I1 

-Pollutants I11 

-Histology 

*-Parasitology 

*-Parasitology 

Freeze 

Culture tube Process 

immediately 

Glass jar, Freeze 

foil-lined lid 

Acid-washed 

polyethylene 

Hexane-rinsed foil 

Freeze 

Freeze 

Glass jar 

Plastic bag 

10% formalin 

Freeze 

R. Gonad 

Gall bladder 

Liver 

Glass vial with 

liver sample 

Freeze with fish 

10% formalin 

Freeze with fish 

Process 

immediately 

Freeze 

-Histology 

-Pollutants I-MAH 

-Pollutants I-PAH 

-Pollutants I1 

-Pollutants I11 

*-Parasitology 

Glass jar 

Plastic bag 

Culture tube 

Glass jar, 

foil-lined lid 

Acid-washed 

polyethylene 

Hexane-rinsed foil 

Freeze 

Freeze 

Stomach and 

Intestine *-Parasitology Plastic bag Freeze with fish 

Muscle -Pollutants Culture tube 

-Pollutants 

-Pollutants 

-Pollutants 

I-MAH 

I-PAH 

I1 

I11 

Process 

immediately 

Glass jar, Freeze 

foil-lined lid 

Acid-washed 

polyethylene 

Hexane-rinsed foil 

Freeze 

Freeze 

10% formalin or 

freeze w/ fish 

Tumors, Parasites (Other) 

(Unusual types) 

Glass jar or 

plastic bag 

*Optiona 1 
-23- 

K. Sample Labels 

............................................................................ 

. EXP. LOC . . . EXP. LOC. .. EXP. LOC . . FISH NO. . . FISH NO. . . FISH NO. 

DATE .. DATE .. DATE . L. OVARY-ANTERIOR . . L. OVARY-MIDDLE . . L. OVARY-POSTERIOR . FECUNDITY .. FECUNDITY . . FECUNDITY . (FREEZE) .. (FREEZE) .. (FREEZE) .. .. ............................................................................ 

............................................................................ 

. EXP. LOC . . . EXP. LOC. .. EXP. LOC . . FISH NO. . . FISH NO. . . FISH NO. 

DATE .. DATE .. . L. GONAD-MIDDLE . . L. GONAD-MIDDLE . . L. GONAD-POSTERIOR . POLL I-MAH . . POLL I-PAH . . POLL 11-HM . (PROCESS IMMEDIATELY). . (FREEZE W/FOIL ON LID). . (FREEZE IN POLYETHYLENE) DATE 

.. .. ............................................................................ 

............................................................................ 

. EXP. LOC . . .EXP. LOC. .. EXP. LOC . . FISH NO. . .FISH NO. . . FISH NO. 

DATE .. DATE .. . L. GONAD-POSTERIOR . . R. GONAD-ANTERIOR . . R. GONAD-MIDDLE . POLL 111-PCB,DDT/DDE . . HISTOLOGY . . PARASITOLOGY . (FREEZE IN RINSED FOIL). . (10% FORMALIN) . . (FREEZE W/FISH) DATE 

.. .. ............................................................................ 

............................................................................ 

. EXP. LOC . . . EXP. LOC . . .EXP. LOC . . FISH NO. . . FISH NO. . .FISH NO. 

. LIVER-R. LOBE .. LIVER-MI DDLE . . LIVER-MIDDLE . HISTOLOGY .. POLL I-MAH . . POLL I-PAH . (10% FORMALIN) .. (PROCESS IMMEDIATELY). . FREEZE W/FOIL ON LID) . 

DATE .. DATE .. DATE 

.. .. ............................................................................ 

............................................................................ 

. EXP. LOC . . .EXP. LOC . . .EXP. LOC . . FISH NO. . .FISH NO. . .FISH NO. 

DATE .. DATE .. . LIVER-MIDDLE . .LIVER-MIDDLE . .LIVER-L. LOBE & GALL- . . POLL 11-HM . .POLL 111-PCB,DDT/DDE . .PARASITOLOGY BLADDER . . (FREEZE IN POLYETHYLENE) .(FREEZE IN RINSED FOIL). .(FREEZE W/FISH) DATE 

.. .. ............................................................................ 
-24- 

STRIPED BASS HEALTH INDEX 

The following document is an example of applications for the 

"Histopathological Manual for Monitoring Health of Striped Bass in 

Relation to Pollutant Burdens." 

The Striped Bass Health Index is extracted from the Guidance 

Document for Regional Effects Monitoring of the Aquatic Habitat 

Program developed for the California Regional Water Quality 

Control Board, Oakland, CA. It is included here to illustrate the 

use of this manual and is currently being used by the Aquatic 

Habitat Program in its 1984 Monitoring Study. 
-25- 

Recommended Components for Monitoring 

the Condition of Striped Bass 

R. Bruce MacFarlane and Jeannette A. Whipple 

May, 1984 

I. INTRODUCTION 

A. ELEMENT 

The striped bass (Morone saxatilis) is a major recreational 

species in the San Francisco Bay-Delta estuary. Mature, 

prespawning females will be sampled annually in April and May at 

two locations downstream of their spawning areas in the San 

Joaquin and Sacramento Rivers. Specimens will be subjected to 

biological and pollutant analyses to determine the health of 

adults and eggs. Analyses will be performed which assess the 

physiological and reproductive conditions of the fish that are 

associated with pollutant exposure. 

B. RATIONALE (For the Element) 

The striped bass is an important member of the Bay-Delta 

community. Assessing the physiological condition, or health, of 

this species can provide valuable information relating to both the 

pollutant status of the ecosystem and the welfare of the animal. 

Considerations that justify monitoring the condition of the 

striped bass include: 
-26- 

(1) It is an ecologically significant species. Since it is a 

tertiary carnivore, the striped bass is at, or near, the 

top of the Bay-Delta trophic structure. Because of this 

position, its life span (to 30 years) (Setzler et al., 

1980), and its relatively high lipid content, (Gadbois 

and Maney, 1983), the striped bass has a high 

bioaccumulation potential for pollutants. In fact, 

pollutant analyses of Bay-Delta striped bass tissues 

performed by several laboratories have shown higher 

concentrations of many classes of pollutants 

(petrochemicals, PCBs, pesticides, metals) than striped 

bass, and several other species, from estuaries on the 

East and West coasts of the United States (Whipple et 

al., 1983; Gadbois and Maney, 1983). 

(2) The striped bass ranges throughout the Bay-Delta, 

thereby acting as an integrator of the pollution status 

of the estuary. However, the population occurs 

predominantely in the northern portion of the bay and in 

the delta, thereby being more affected by water quality 

in these areas than in other portions of the estuary. 

(3) The species is economically and recreationally 

significant. An estimated $20 million are spent annually 

by the recreational fishery for striped bass in the 

Bay-Delta (based on consumer price index projections of 

Stanford Research Institute's 1966 estimate). 
-27- 

Additionally, the species is viewed as an indicator of 

the well-being of the estuary and enjoys high public 

visibility and concern. Within this context, this 

species is of interest from the perspective of public 

health. Studies have shown that Food and Drug 

Administration(FDA) Action Levels have been approached 

or exceeded in striped bass flesh for several 

contaminants (PCB ' s , total DDT , toxaphene, 

aldrin/dieldrin, heptachlor, chlordanes) (Whipple et 

al., 1983). Mercury contamination of the fish prompted 

the State Department of Health to issue an advisory in 

1972 against its consumption by pregnant women and 

children and the warning is still in effect. 

(4) The striped bass population structure is monitored 

annually by the Department of Fish and Game (CDFG). 

Also, the National Marine Fisheries Service (NMFS), the 

State Water Resources Control Board (SWRCB) and the 

University of California have established a five-year 

data base for the physiological and reproductive 

conditions of the population. The data base was produced 

in a Cooperative Striped Bass Study (COSBS), 
-28- 

which also identified impaired condition parameters 

associated with elevated pollutant burdens in the fish 

(Whipple et al., 1983; Whipple et al., in prep.). The 

combination of the previous sampling program with an 

ongoing quantitative assessment of the population will 

provide a better evaluation of the striped bass in the 

Bay-Delta and its recruitment potential. 
-29- 

11. OBJECTIVES 

The primary objective of this element is to provide indices 

of the physiological and reproductive conditions as well as the 

pollutant burdens of the striped bass that reflect the extent of 

anthropogenic contamination in the Bay-Delta system. A secondary 

objective is to provide an assessment of the physiological health 

of the striped bass population that could be used by regulatory 

agencies charged with protecting this valuable resource. 

111. GENERAL METHODS 

We recommend that mature, prespawning females be collected in 

conjunction with the CDFG tagging program in April and May of 

each year in the San Joaquin River at Antioch and in the 

Sacramento River at Clarksburg. If the tagging program should be 

terminated in the future, sampling should still be conducted in 

these areas during April and May even though additional expense 

will be incurred (approximately $10,00O/year). Prespawning females 

provide several advantages for monitoring compared to other life 

stages. First, this stage is among the most sensitive to and 

diagnostic of pollutant effects (Whipple et al., 1981).Secondly, 

measures of reproductive condition can be more easily evaluated 

and used to estimate potential pollutant-related effects to 

recruitment into the population. 
-30- 

Sampling at the specified locations has several advantages 

over other collection strategies. These locations are already 

being sampled by CDFG; therefore, collection is much easier and 

cheaper. Both locations are natural bottlenecks in the rivers, 

immediately downstream of the spawning areas, which makes sampling 

easier and ensures that specimens with ripe gonads will be 

available. Both rivers should be sampled because data indicate 

there may be two distinct (genetic?) "stocks" which spawn in the 

two locations (Whipple et al., 1983). The Sacramento River "stock" 

appears to contain a different pollutant profile qualitatively and 

quantitatively, and exhibits different types and degrees of 

physiological impairments than the San Joaquin River "stock." 

Sampling from only one area may bias the results. Also, both 

rivers provide input into the Bay-Delta and should be represented. 

Mature females, (as a guide, - > 50 cm. standard length), 

provide more information relating to pollutant exposure and 

reproductive potential than males; however, if more funds become 

available, males should also be evaluated since they demonstrate 

pollutant-related impairments that are somewhat different than 

females (Whipple et al., 1983). 

To provide an acceptable evaluation of the species health, we 

recommend that a minimum of 20 females be collected at each 

location, and that the sampling extend over the two month period 

to ensure an adequate temporal representation of the spawning run 
-31- 

and to correct for exposure to episodic pollution (i-e., spills) 

in the migration path, We suggest that four females be sampled per 

week for five weeks during April and May at each site. 

Upon collection, fish should be placed on ice, returned to 

the laboratory and analyzed as soon as possible. Ancillary data 

such as temperature, salinity(conductivity), dissolved oxygen and 

Secchi depth should be recorded at each collection. 

In the laboratory, each fish will be autopsied and subsampled 

according to procedures detailed in the striped bass autopsy 

manual. Briefly stated, each fish will be systematically examined, 

dissected and tissues subsampled for parameters that characterize 

each specimen and have been determined by integrated COSBS field 

and laboratory studies to be associated with pollutant exposure 

(Whipple et al., 1983). The recommended measurements also take 

into consideration time, manpower, expertise, and complexity 

constraints. These analyses are divided between biological 

measurements and pollutant analyses. 

The biological measurements include: 

(1) External examination and meristics -- age (via scales), 

length, weight, body condition, color pattern, 

parasites, deformities, abnormalities, etc. 
-32- 

(2) Internal examination and morphology -- (a) Liver: size, 

weight, color, LSI(Liver somatic index); (b) Ovaries: 

size, weight, eggs/gram, fecundity, egg color, GSI 

(gonadosomatic index), egg stage, ovary maturity stage, 

histology (including degree of egg resorption) and (c) 

Other: mesenteric fat, skeletal deformities. 

(3) Internal parasites and parasitic damage -- roundworm and 

tapeworm larvae, tapeworm lesions, parasite locations, 

severity of parasite damage (adhesions, necrosis, 

scars), host reaction to parasites. 

Factor analysis of data from field specimens revealed a 

correlation between extent of parasite damage and host reactions 

with the concentration of pollutants, primarily monocyclic 

aromatic hydrocarbons (MAH) in tissues (Whipple et al., 1983; 

Whipple et al., in prep.). The cestode larva (Lacistarhynchus 

tenuis) is a common parasite in fish; however, Bay-Delta striped 

bass are heavily infested and exhibit a unique, severe host 

reaction (immune response) (Moser et al., 1984). This response, 

coupled with the parasite damage, may produce extensive 

morphological and physiological trauma to liver, gonad and other 

tissues. 
-33- 

Recommendations for the types and extent of pollutant 

analyses are based on several factors: correlations between the 

contaminants and reduced physiological and reproductive conditions 

determined by data analyses from field specimens; laboratory 

exposure studies; analytical time and complexity, and cost. 

Although other contaminants which have not been as extensively 

measured or evaluated toxicologically may be contributing to the 

reduced health of the species, we believe that the most 

significant classes of pollutants impacting physiological and 

reproductive processes, or reflecting the presence and effects of 

other contaminants, are the MAHs (benzene, xylene isomers, 

ethylbenzene, toluene) and the alicyclic hexanes (cyclohexane, 

methylcyclohexane, dimethylcyclohexane isomers) (Whipple et al., 

in prep.). Perhaps of lesser significance, but thought to 

contribute to physiological decline and found in high 

concentrations in some specimens, are PCBs (Whipple et al., 1983) 

and certain heavy metals including copper, zinc, mercury, cadmium 

and chromium (Whipple et al., in prep.). 

We recommend MAH and alicyclic hexanes be measured firstly in 

liver tissue. Although there is a relationship between 

reproductive condition and concentration of these compounds in 

ovaries, a stronger association exists between reproductive 

fitness and liver concentrations. Also, MAH and alicyclic hexanes 
-34- 

analyses are simpler and less affected by analytical interferences 

in liver. If sufficient funds are available, after these analyses 

are completed, the next priority is heavy metal analysis. 

Pollutant analyses procedures are detailed in previous sections of 

this manual. 

IV. ACTION LEVELS 

Establishing reasonable action levels for environmental 

quality assessment is a formidable task that almost always 

requires subjective decisions and diligent review as new 

information is acquired. Accordingly, the following are suggested 

as initial criteria upon which regulatory action is warranted, and 

are based on the best judgment of COSBS personnel: 

(1) Whenever more than 5% of the mature, prespawning female 

striped bass sampled exhibit resorption or damage of the 

entire ovary. Factor analysis of data from Bay-Delta 

striped bass revealed a strong association between the 

extent of ovary and/or egg resorption and the 

concentration of pollutants in ovary and liver tissues. 
-35- 

(2) Likewise, whenever more than 5% of all the eggs from all 

the mature prespawning females sampled, excluding those 

with complete ovary resorption, are resorbing. 

(3) Whenever there is a 20% or greater differential between 

the percentage of fish sampled with open, unhealed, 

cestode larvae-induced external lesions and fish with 

healed, external lesions (Moser and Sakanari, in prep.). 

An example of this case would be when 10% of the fish 

sampled have healed lesions and 30% of the fish have 

open lesions. The COSBS study determined conclusively 

that the "red sores" and lesions exposing the peritoneal 

cavity are produced in response to cestode larvae 

infestation. Furthermore, data analysis revealed a 

strong correlation between the extent of parasite 

infestation and host reaction with the pollutant body 

burden (Whipple et a]. , in prep.). 

(4) Whenever more than 10% of the liver tissue samples 

subjected to pollutant analysis contain contaminants 

exceeding National Academy of Sciences(NAS) (1973) 

maxima for aquatic life, or FDA (1981) Action Levels. 

(For comparison to FDA Action Levels, pollutant 

concentrations are converted to estimated concentrations 

in muscle using appropriate tissue differential ratio.) 
-36- 

(5) Whenever there is a failure to procure at least 20 

mature, prespawning females at either location that can 

not be explained by lack of effort or climatological 

factors. 

V. RECOMMENDED ACTION BY REGULATORY AGENCIES 

Depending upon which factor or factors "send up the red 

flag", the following regulatory actions may be appropriate and are 

recommended: 

When FDA Action Levels are exceeded, human and/or animal 

health advisories should be announced. Human health 

advisories concerning mercury contamination and 

roundworm infestation (Anasakis sp.), already in place, 

should be maintained until these conditions no longer 

exist . 

When FDA Action Levels or NAS guidelines for aquatic 

life are exceeded, vigorous efforts should be made to 

determine the source and extent of the contamination, 

and to mitigate its occurrence. 

When the cause of the red flag is unknown, but suspected 

to be pollutant-related, a research project should be 

initiated to determine the causative agent and the 

extent of impact to the ecosystem. This may include 

intensive pollutant analysis of the archived samples. 
-37- 

(4) When the causative agent (pollutant) is identified, 

regulatory action should follow to control or eliminate 

the agent. 

VI. ADDITIONAL RESEARCH/RECOMMENDATIONS 

The decline in the abundance and condition of striped bass in 

the San Francisco Bay-Delta community warrants the establishment 

of a concerted, multi-disciplinary research effort to study the 

problem. The study should be maintained until the primary 

contributory factors, pollutant-related and otherwise, are 

definitively resolved and corrected. We also recommend support for 

further research to possibly refine or revise the monitoring 

procedure and data analysis. The proposed monitoring program will 

provide a component of the assessment of water quality; however, 

it is not the only or ultimate program. 

VII. DATA ANALYSIS 

All data will be entered on data sheets provided in the 

autopsy manual. These data should be entered as the fish are 

examined, when appropriate. Data from calculations of derived 

variables (e.g., GSI , LSI) and from subsequent analyses (e.g., 

pollutant analyses, histological analyses, etc.) should be entered 

on the data sheets as soon as available. These data sheets have 

been designed for direct transposition onto computer 
-38- 

cards, computerized data files, or whatever means are used for 

entry into the computer. Prior to, or within, computerized 

statistical analyses (i.e., multivariate analysis, discriminant 

function analysis, etc.) the means and standard errors will be 

calculated and recorded for all variables from individual sampling 

sites as well as for both areas combined. 

The cost for data analyses will depend upon which hardware 

and software are used. If a macrocomputer such as the one at the 

Lawrence Berkeley Laboratory is used, computer analytical costs 

will be minimal. Final recommendations for data analytic 

procedures are in preparation and will be provided to the 

California State Water Resources Control Board when completed. 
-39- 

LITERATURE CITED 

Food and Drug Administration. 

1981. Action levels for poisonous or deleterious substances 

in human food and animal feed. Washington, D.C., 13 p. 

Gadbois, D.F. and R.S. Maney. 

1983. Survey of polychlorinated biphenyls in selected finfish 

species from United States coastal waters. Fish. Bull., U.S. 

81:389-396. 

Jung, Marvin and Gerald Bowes. 

1980. First progress report on the Cooperative Striped Bass 

Study (COSBS). California State Water Resources Control 

Board, Special Projects, Toxic Substances Control Program, 

P.O. Box 100, Sacramento, CA. 95801. March, 1980. 64 p. 

Jung, Marvin and COSBS staff. 

1981. Second progress report: Cooperative Striped Bass Study 

(COSBS). California State Water Resources Control Board, 

Special Projects, Toxic Substances Control Program, P.O. Box 

100, Sacramento, CA. 95801. January, 1981. 69 p. 
-40- 

Moser, M. and J, Sakanari. 

In prep, Parasite-induced host mortality of an introduced 

fish species. 

Moser, M., J. Sakanari, S. Wellings and K. Lindstrom. 

1984. Incompatibility between San Francisco Bay striped bass 

and the metacestode, Lacistorhynchus tenuis. J. Fish 

Disease 7 (In Press). 

National Academy of Sciences. 

1974. Water quality criteria, 1972. EPA-R3-73-033, 

Washington, D.C. 

Setzler, E.M., W.R. Boynton, K.V. Wood, H.H. Zion, L. Lubbers, 

N.K. Mountford, P. Frere, L, Tucker and J.A. Mihursky. 

1980. Synopsis of biological data on striped bass, Morone 

saxatilis, (Walbaum). NOAA Tech. Rep. NMFS Circ. 433, U.S. 

Dept. of Commerce, Washington, D.C, 69 p. 

Whipple, Jeannette A. and Marvin Jung 

1981. Cooperative Striped Bass Study (COSBS) Appendix I: 

Procedures for histopathological examinations, autopsies, 

and subsampling of striped bass. State Water Resources 

Control Board, Special Projects, Toxic Substances Control 

Program. Sacramento, CA. November, 1981. 46 p. 
-41- 

Whipple, J.A., M.B. Eldridge, and P. Benville, Jr. 

1981. An ecological perspective of the effects of monocyclic 

aromatic hydrocarbons on fishes, p. 483-551. In (F.J. 

Vernberg, A. Calabrese, F.P. Thurberg and W.B. Vernberg, 

eds.) Biological monitoring of marine pollutants. Academic 

- 

Press, New York. 

Whipple, Jeannette, Donald G. Crosby and Marvin Jung 

1983. Third progress report: Cooperative Striped Bass Study 

(COSBS). California State Water Resources Control Board, 

Special Projects, Toxic Substances Control Program, P.O. Box 

100, Sacramento, CA. 95801. February, 1983. 208 p. 

Whipple, Jeannette and Marvin Jung 

In Prep. Fourth progress report: Cooperative Striped Bass 

Study (COSBS). California State Water Resources Control 

Board, Special Projects, Toxic Substances Control Program, 

P.O. Box 100, Sacramento, CA. 95801. 

Whipple, Jeannette, Pete Benville, Jr., Maxwell Eldridge and R. 

Bruce MacFarlane. 

In Prep. Impacts of pollutants on striped bass in the San 

Francisco Bay-Delta, California. 
-42- 

ACKNOWLEDGMENTS 

The authors would like to thank Mike Moser, Judy Sakanari 

(University of California-Santa Cruz) and Carol Reilly (U.S. 

Department of Agriculture) for their assistance in preparing this 

manual, with special appreciation to Mike Moser for his work in 

identifying parasites. We would like to acknowledge the 

assistance of Roger Anderson and Jim White of the California 

Department of Fish and Game for reviewing the manual. Finally, we 

thank John Greeley, NMFS, for editing and typing the manual. 

This research was funded by the National Marine Fisheries 

Service, the former Office of Marine Pollution Assessment and the 

California State Water Resources Control Board. 
-43- 

APPENDIX I-- VARIABLE LIST FOR FACTOR ANALYSES #3: MONITORING 

A). AUTOPSY MEASUREMENTS 

VARIABLE NAME DATA SET NUMBER 

EXPERIMENT NO. ALL 

LOCATION ALL 

METHOD OF CAPTURE ALL 

ENVIRONMENTAL DATA: 

FISH NUMBER ALL 

DAY 1 

MEAN TEMPERATURE 1 

MEAN SALINITY 1 

MEASUREMENTS & MERISTICS: 

AGE 2 

FORK LENGTH 2 

STANDARD LENGTH 2 

DEPTH 2 

WET WEIGHT FISH 2 

CONDITION (KFL) * 2 

CONDITION (KSL) * 

CONDITION (K3) * 

2 

2 

*CALCULATED: KFL = (wet weight fish/fork length) x 100; KSL = (wet 

weight fish/standard length) x 100; K3 = (wet weight fish - wet 

weight viscera)/standard length x 100 
-44- 

APPENDIX I--(CONT.) 

VARIABLE NAME 

COLOR PATTERN : 

DORSAL LEFT 

VENTRAL LEFT 

DORSAL RIGHT 

VENTRAL RIGHT 

TOTAL LEFT 

TOTAL RIGHT 

TOTAL DORSAL 

TOTAL VENTRAL 

TOTAL BREAKAGE-ALL 

GONADS: MEASUREMENTS 

SEX 

TOTAL LENGTH-LEFT 

WIDTH-LEFT 

TOTAL LENGTH-RIGHT 

WIDTH-RIGHT 

WET WEIGHT-LEFT 

WET WEIGHT-RIGHT 

TOTAL WET WEIGHT 

EGGS PER GRAM 

DATA SET NUMBER 
-45- 

APPENDIX I-- (CONT. ) 

VARIABLE NAME DATA SET NUMBER 

GONADS: MEASUREMENTS (CONT.) 

FECUNDITY 

COLOR HUE-EGGS 

COLOR VALUE-EGGS 

COLOR CHROMA-EGGS 

EGG COLOR RANK 2 

GONADOSOMATIC INDEX (GSI,)* 

GONADOSOMATIC INDEX (GSI2) * 

LIVER: MEASUREMENTS 

TOTAL LENGTH 

WIDTH 

WET WEIGHT 

COLOR HUE 

COLOR VALUE 

COLOR CHROMA 

LIVER COLOR RANK 2 

LIVER SOMATIC INDEX (LSI I) * 

LIVER SOMATIC INDEX (LSI 2) * 

* CALCULATED: GSI, = (wet weight gonads,Total wet we,ght fish) x 100; 

GSI = wet weight gonads/(Total wet weight fish-wet weight viscera) x 

100; LSI = (wet weight liver / Total wet weight fish) x 100; LSI, = 

wet weight liver / (Total wet weight - wet weight viscera) x 100 
-46- 

APPENDIX I--(CONT.) 

VARIABLE NAME DATA SET NUMBER 

PARASITES: 

ROUNDWORM LARVAE 6 

TAPEWORM LARVAE 6 

TAPEWORM LESIONS 6 

TOTAL LOCATIONS 6 

TOTAL TYPES 6 

TOTAL SEVERITY 6 

TOTAL HOST REACTION 6 

SEVERITY SKELETAL ABNORMALITIES: 

GILL RAKER,FIN RAYS 6 

HEAD BONES 6 

VERTEBRAL COLUMN 6 

OTHER ORGANS: MEASUREMENTS 

RANK MESENTERIC FAT 8 
-47- 

APPENDIX I--(CONT.) 

B.) SAMPLE MEASUREMENTS 

VARIABLE NAME DATA SET NUMBER 

HEAVY METALS (DRY WEIGHT=DW) : 

SAMPLES ARCHIVED 10 

PETROLEUM HYDROCARBONS-** 

MONOCYCLIC AROMATICS, 

ALICYCLIC HEXANES 12 

(LIVER AND GONAD): 

BENZENE 12 

MONOCYCLICS 12 

TOTAL MONOCYCLICS 12 

TOTAL CYCLOHEXANES 12 

TOTAL HYDROCARBONS 12 

CYCLOHEXANE 12 

METHYLCYCLOHEXANE 12 

1, -DIMETHYLCYCLOHEXANE 12 

1, -DMCH2 12 

1, -DMCH3 12 

1, -DMCH4 12 

1, -DMCH5 12 

TOLUENE 12 

........................................ 

**ALL PETROLEUM COMPOUNDS IN PPM WET WEIGHT. 
-48- 

VARIABLE NAME 

ETHYLBENZENE 

- p-xylene 

APPENDIX I--(CONT.) 

DATA SET NUMBER 

12 

12 

12 

12 

13 

- m-xylene 

- o-xylene 

CHLORINATED HYDROCARBONS - PCBS: 

SAMPLES ARCHIVED 

CHLORINATED HYDROCARBONS - DDE, 

DDD, DDT: 

SAMPLES ARCHIVED 

PETROLEUM HYDROCARBONS - 

POLYCYCLIC AROMATICS: 

SAMPLES ARCHIVED 

LIVER: HISTOLOGY: 

SAMPLES ARCHIVED 

TESTES: HISTOLOGY: 

SAMPLES ARCHIVED 

14 

15 

35-36 

37 
-49- 

VARIABLE NAME 

APPENDIX I--(CONT.) 

DATA SET NUMBER 

OVARIES- HISTOLOGY: 

GONAD MATURITY 

STAGE EGG 

MEAN MAX DIAMETER 

% FOLLICLE CELLS 

% PERINUCLEOLUS 

% YOLK VESICLE 

% PRIMARY YOLK 

% SECONDARY YOLK 

8 TERTIARY YOLK 

% MIGRATORY NUCLEUS 

% EARLY COALESCENCE 

% LATE COALESCENCE 

% EGGS RESORBED 

% EMPTY FOLLICLES 

38 

38 

38 

39 

39 

39 

39 

39 

39 

39 

39 

39 

39 

39 
-50- 

APPENDIX II--CODING FOR DATA SETS 

Coding for All Data Sets 

EXPERIMENT 

(Field) 

1 = 1978 

2 = 1979 

3 = 1980 

4 = 1981 

5 = 1982 

6 = 1983 

7 = 1984 

LOCATION 

1 = San Francisco Bay, off Tiburon 

*2 = San Joaquin River, off Antioch 

3 = Sacramento River, off Knights Landing 

4 = Sacramento River, off Clarksburg 

*5 = Sacramento River, off Clarksburg - Traps 

6 = Chipps Island 

7 = Carquinez Strait, Mothball Fleet 

8 = Carquinez Strait, Martinez 

9 = San Joaquin River, off Antioch 

10 = San Pablo Bay, Corte Madera Creek 

11 = Umpqua/Smith River(s), OR 

*Main CDF&G sampling areas 
-51- 

APPENDIX 11--(CONT.) 

12 = Coos River, OR 

13 = Lake Mead, NV 

14 = Cherokee Reservoir, TN 

15 = Montezuma Slough 

16 = Spawned Elk Grove, reared at Steinhart Aquarium and Tiburon 

Laboratory (Exp. 18-20) 

17 = San Pablo Bay, Crockett to Rodeo 

18 = Gill-netted off Tiburon Pier, held 6 months at Tiburon 

Laboratory (Exp. 15) 

19 = Suisun Bay 

20 = West San Pablo Bay 

21 = Spawned Elk Grove, reared at Tiburon Laboratory by Eldridge 

(Exp. 3 - 5) 

22 = Hudson River, NY 

23 = All over San Francisco Bay-Delta (Exp. 61) 

METHOD (of Capture) 

1 = Gill net 

2 = Shocker 

3 = Diversion screen 

4 = Traps 

5 = Trawl 

6 = Hook and line 

7 = Dip net 

8 = Mixed methods 

9 = Combined methods above 
-52- 

APPENDIX II-- (CONT.) 

Codinu for Data Set 2 

DISSECTION DATE = Month/day 

DISSECTION DAY = Julian; refer to perpetual calendar 

DISSECTION TIME = beginning of autopsy; 24-hr clock 

SEX 

1 = Female 

2 = Male 

3 = Predominantly female 

- 

hermaphrodite 

4 = Predominantly male 

hermaphrodite 

COL PAT INT (Color pattern intensity: dorsal melanization) 

1 = Light 2 = Medium 3 = Dark 

STRIPING PATTERN BREAKAGE 

1 = Not broken 2 = Broken 

SIDE 

1 = Left 2 = Right 
-53- 

APPENDIX 11--(CONT.) 

Coding for Data Set 4 

SEX 

1 = Female 

2 = Male 

- 

3 = Predominantly female 

hermaphrodite 

- GND (Gonad) 

1 = Left 

- MTR (Maturity) 

1 = Immature 

2 = Maturing 

3 = Spawning, ripe-running 

4 = Spent 

EGG COLOR RANK 

Munseli Hue = I I1 

2.5 R 1 1 

5.0 R 1 2 

7.5 R 1 3 

10.0 R 1 4 

4 = Predominantly male 

hermaphrodite 

2 = Right 

5 = Resting (recovering) 

6 = Resorbing (abnormal) 

Munsell Hue = I I1 

2.5 YR 2 5 

5.0 YR 2 6 

7.5 YR 2 7 

10.0 YR 2 8 
-54- 

APPENDIX II--(CONT.) 

Coding for Data Set 4 (cont.) 

EGG COLOR RANK 

Munsell Hue = I I1 

2.5 Y 3 9 

5.0 Y 3 10 

7.5 Y 3 11 

10.0 Y 3 12 

Munsell Hue = I I1 

2.5 GY 4 13 

5.0 GY 4 14 

7.5 GY 4 15 

10.0 GY 4 16 

LIVER COLOR HUE I 

1 = Deep red 4 = Yellow 

2 = Deep red with yellow patches 5 = White-yellow 

3 = Yellow with red patches 

STOMACH CNT (Contents) 

1 = Empty 2 = Full 

STOMACH CNT TYPE 

Normally empty during prespawning stage. (Codes not developed, but 

can be added.) 
-55- 

APPENDIX II--(CONT.) 

Coding for Data Set 6 

DISSECTION DATE = Month/day 

DISSECTION TIME = Beginning of autopsy, 24-hr clock 

DISEASE/PARASITE LOC (Location) 

Head Body 

1 = Snout 10 = General on external body, 

2 = Mouth and jaw on all fins 

3 = Operculum 

4 = Upper jaw 

5 = Lower jaw 

6 = Skull bones 

7 = Gill rakers 

8 = Gills 

9 = Eyes 

11 = Dorsal fins 

12 = Pectoral fin 

13 = Pelvic fin 

14 = Anal fin 

15 = Caudal fin 

16 = External body musculature 

17 = External body near vent 

18 = Internal body musculature 

20 = Body cavity 

21 = Vent 
-56- 

APPENDIX I1 (CONT.) 

Coding for Data Set 6 (cont.) 

Organs 

22 = Heart 

23 = Kidney 

24 = Spleen 

25 = Gallbladder 

26 = Intestinal mesenteries, 

fat, or fascia 

27 = Intestine 

28 = Stomach 

29 = Gonad 

30 = Liver 

31 = Brain 

DISEASE/PARASITE TYPE* 

1 = None 

2 = Effects probably due to 

handling, net damage, gaff, 

hooks, etc. 

3 = Blisters (usually inside 

operculum, mouth) 

4 = Isopods 

32 = Swim Bladder 

33 = Adrenal Glands 

34 = Colon, rectum 

35 = Urinary bladder 

36 = Peritoneum 

37 = Pyloric Cecae 

Tissues 

38 = Muscle 

39 = Blood 

40 = Bile 

5 = Copepods 

**6 = Roundworm larvae 

**7 = Cestode larvae, small 

brown cysts 

8 = Roundworm cysts, hyaline 

brown "rafts 

*Refer to Dr. Michael MoSer, University of California-Santa cruz, 

for questions about parasites; ** Most commonly occuring. 
-57- 

APPENDIX I1 (CONT.) 

Coding for Data Set 6 (cont,) 

9 = Acanthocephalans, knobs 16 = Fin rot 

in gut (Hudson River fish) 17 = Cirrhosis of liver(?) 

10 = Lamprey wound 18 = Effect probably due to 

11 = Seal or bird bite/wound benzene exposure 

12 = Possible heavy metal 19 = Papillomas 

effect **20 = Cestode raft (often causes 

13 = Suspected bacterial or lesions) 

viral infection 21 = Adult nematode 

14 = Tumor-unspecified cause 22 = "Pepper spots," dead eggs, 

15 = Unknown cause: individual sacs 

a. Heart fat 23 = Roundworms in Oregon fish 

b. Abnormal ovaries 24 = Roundworm cysts in Oregon 

c. Internal hemorrhaging fish 

d. Abnormal intestinal 25 = Rafts, unknown origin, 

waste in Oregon fish 

e. Wounds 26 = Monogenetic trematode 

f. Clear lumps or eggs 27 = Larval digenetic trematode 

g. Opacity 28 = Leech 

h. Abnormal shape or size 29 = Experimental puncture 

i. Abnormal color 30 = Missing part 

j. Sacs of dead eggs, etc. 31 = Lymphocystis 

k. Abnormal texture 32 = Adult digenetic trematode 

33 = Clam larva (glochidia) 

** Most Commonly Occuring 
-58- 

APPENDIX II-- (CONT.) 

Coding for Data Set 6 (cont.) 

DISEASE/ PARASITE SEV/ABND (Severity/Abundance) 

1 = None 4 = Many 

2 = Few or slight 5 = Very many or heavy 

3 = Average 

DISEASE/ PARASITE HOST RCTN (React ion) 

1 = No reaction obvious 

2 = External hemorrhaging 

3 = Healed external wound 

4 = Open wound 

5 = Excess mucus 

6 = Clear fluid 

7 = Clouding 

8 = Tissue soft 

9 = Bright red 

10 = Reddened 

11 = White, pale or no color 

12 = Greenish or yellowish fluid 

13 = Tissue hardened 

14 = Abnormally green 

15 = Tissue erosion 

16 = Reddened sores 

17 = Hemorrhaging 

18 = Pressure necrosis 

19 = Visceral adhesions 

20 = Fatty; white or yellow 

21 = Generalized necrosis 

(internal organs) 

22 = Black/brownish/white spots 

23a = Not developed 

23b = Underdeveloped 

23c = Abnormal shape 

24 = Dead eggs 

25 = Abnormal tissue growth 

26 = Constriction 

27 = Cracking 

28 = Irregular scale pattern 
-59- 

APPENDIX 11--(CONT) . 

Coding for Data Set 6 (cont.) 

ABNORMALITY LOC (Location) = same as for DISEASE/PARASITE LOC 

ABNORMALITY TYPE-(Skeletal only) 

1 = Abnormal coloration 5 = Pugheaded 

2 = Enlarged size 

3 = Reduced size 

6 = Scoliosis or lordosis 

4 = General skeletal abnormality 

ABNORMALITY SEV/ABND-(Severity/Abundance) 

1 = None 4 = Severe 

2 = Slight 5 = Very severe 

3 = Average 

_____________________-__--__-___-------------------

Coding for Data Set 8 

MESENTERIC FAT 

1 = None 

2 = Slight or sparse 

INTESTINE 

3 = Average 

4 = Abundant 

1 = Empty 2 = Full 
-60- 

APPENDIX 11--(CONT.) 

Coding for Data Set 12 

TISSUE ID: 

1 = Gonad 

2 = Liver 

3 = Blood 

4 = Muscle 

COMPOUND ID: 

5 Cyclohexane 40 1,2 DMCH 

10 Methy lcyc lohexane (MCH) 50 Toluene 

20 Benzene 60 Ethylbenzene 

32 Trans 1,4 & 1,l DMCH* 70 - p-xylene 

33 - Cis 1,4 DMCH 80 - m-xylene 

35 1,4 DMCH 90 - o-xylene 

Totals: 

350 Total MCH-Tissue 

360 Total CH-tissue 

370 Total MCH & CH-tissue 

380 Total MCH-all tissues 

390 Total CH-all tissues 

400 Total MCH & CH-all tissues 

*DMCH = dimethylcyclohexane 
-61- 

APPENDIX 11--(CONT.) 

Coding for Data Set 22 

SEX 

1 = Female 

2 = Male 

3 = Predominantly female 

- 

hermaphrodite 

GONAD 

1 = Left 

MATUR (ity) 

1 = Immature 

2 = Maturing 

3 = Spawning, ripe-running 

4 = Spent 

SPERM MOTIL (ity) 

1 = Immotile 

MEAN FRESH 

(See Text, ignore columns for preserved eggs). 

4 = Predominantly male 

hermaphrodite 

2 = Right 

5 = Resting, (recovering) 

6 = Resorbing (abnormal) 

2 = Motile 
-62- 

APPENDIX 11--(CONT.) 

Coding for Data Set 38: 

MAGNIFICATION: At which eggs were microscopically evaluated. 

MICROMETER: Conversion factor for ocular micrometer determined from 

stage micrometer. 

AREA SCANNED/AREA SUBSAMPLED: Approximate area in square mm. 

AGE: Ages from Data Set 2. 

MAT: Ovary maturity stage(1-6). See Appendix Table A. 

RANK EGG COLOR: Values in Coding for Data Set 4. 

COND: Slide condition. 1= Very good: 2= Average: 3=Poor: 4=Terrible 

AGE TISS: Hours from capture to preservation of ovarian subsample. 

NO. EGGS EXAM.: Number of eggs at max. maturation stage examined. 

EGG STG: See Appendix Table A. 

MEAN EGG DIAM: Mean diameter of 5 eggs at maximum stage and size. 

STAGE W/ VAC: Egg stage that vacuoles are observed. 

RANK OIL: Rank amount of oil in eggs: 

1= lower than average: 2= average: 3= higher than average: 4= 

abnormally large amount. Ranks 3 and 4 indicate egg resorption. 

Oil is bright red from Oil Red-0 lipid stain. 

STAGE RSRBD: Egg stage of resorbing eggs. 

DLY?/NCRT?: First space for delayed code: second for necrotic code: 

l=No: 2= Yes. 

BOX/SLIDE: Box and slide box number. 

PHOTO: Number of roll and exposure number. 
-63- 

APPENDIX I11 --Preparation of Buffered Formalin 

(For Preservation of Eggs and Testes) 

Add 0.340 g potassium phosphate, monobasic (KH2 PO4 ) and 0.355 g 

sodium phosphate, anhydrous, dibasic (Na2 HP04 1 per 100 ml 

formalin. 

Check pH and adjust to 7.0 if necessary with a few drops of 10% 

HC1 or 1N NaOH. 
-64- 

1. 

2. 

3. 

4. 

5. 

6. 

7. 

8. 

9. 

10. 

APPENDIX IV-- 

Glassware Cleaning Procedure for Trace Metal Analyses 

Rinse in Milli-Q (or distilled/deionized) water. 

Place in Micro Bath solution for 24 hours. 

Rinse well with tap water (3 times). 

Rinse with Milli-Q water. 

Soak in 6N HC1 for at least 24 hours. 

Rinse 5 times with Milli-Q water. 

Soak in 2N HN03 for at least 24 hours. 

Rinse three times with Milli-Q water. 

Dry in oven at about 50 deg C (not >70 deg C). 

Store in dust-free area wrapped in assembly wipes. 

Reference: G. A. Knauer, Moss Landing Marine Laboratory (personal 

communication) . 
APPENDIX V 

Preparation of Clean Foil for PCB Samples 

Perform the following under a laboratory hood. 

1. 

2. 

3. 

4. 

5. 

Use nanograde hexane to rinse a metal pan. Put the used 

hexane in a container marked "used hexane". 

Cut pieces of heavy-duty aluminum foil into pieces 

approximately 8" by 10". Handle the foil with rinsed metal 

tongs. 

Dip the foil in the rinsed metal pan that contains fresh 

nanograde hexane. 

Allow the rinsed foil to drip-dry in a covered glass 

cylinder. 

NOTE: A quality control measure of PCB's can be done on the 

used hexane. 

Reference: Dr. Donald Gadbois, NMFS, Gloucester, Mass. 

Modified by Dr. Gerald Bowes, State Water Resources Control Board, 

Sacramento, CA. (personal communication). 
-66- 

APPENDIX VI 

Trace Metal Analysis of Fish Liver 

1. Precleaning procedure 

A. Clean all glassware, plasticware, etc., prior to sample 

preparation as per written procedure. 

2. Sample preparation 

A. Weigh frozen liver sample on tared piece of weighing 

paper. Record as sample wet weight. 

B. Homogenize tissue in Potter-Elvehjem homogenizer for 

three minutes, or until homogeneous, whichever occurs 

first. NOTE: May have to add a small volume (less than 

one ml) of Milli-Q or distilledldeionized water (dd 

water). 

C. Quantitatively transfer to labelled, preweighed 30-ml 

beaker by rinsing residue from homogenizer (including 

pestle) with about 1 to 2 ml Milli-Q or dd water. 

1. Cover with watch glass. 

2. Place in oven at 70 deg C for 72 hours. 

3. Weigh beaker plus dried sample. 

4. After correcting for beaker weight, record as 

sample dry weight. 

D. Under the hood, add 5 ml concentrated HNO cover with 3; 

watch glass. 

E. Under hood, place beakers on hot plate, bring to slow 

boil carefully, and reflux for 3 hours. 
-67- 

APPENDIX VI-- (CONT. ) 

F. 

G. 

H. 

I. 

J. 

K. 

L. 

M. 

N. 

0. 

Reduce heat to below boiling and take samples to 

dryness. 

Place in muffle furnace at 400 deg C overnight; let cool 

to room temperature. 

Dissolve in 5 ml concentrated HNO3. 

Add dropwise %02 until no further oxidation is 

observed. There should be no darkening of solution or 

residue at this point. If there is, bring solution to a 

slow boil again until solution is clear, or slightly 

yellow. 

Heat solution to just below boiling; slowly evaporate 

until residue is only slightly moist. 

Allow to cool. 

Add 20 ml 1% HN03. 

Transfer to 25-ml volumetric flask. 

Make up to volume with 1% HNO . 

If analyzing same day, can aspirate directly from 

volumetric: if analyzing later, transfer into 

acid-cleaned conventional polyethylene vial. 

3 

3. Sample preparation blanks 

A. For each analytical batch of sample, prepare duplicate 

blanks according to sample preparation procedure, except 

no tissue placed in homogenizer or beakers. 
-68- 

APPENDIX VI (Cont.) 

4. Zinc Standards (as example of a trace metal). 

A. Prepare stock solution of zinc at a concentration of 500 

++ 

ppm Zn , freeze in conventional polyethylene, Teflon 

or quartz vials in 1.0 ml aliquots. 

B. Take frozen stock solution of zinc from freezer and let 

++ thaw. Zn = 500 ppm. 

C. Add 0.800 ml stock solution to 200-ml volumetric flask, 

make to 200 ml with 1% (v/v) HN03, mix thoroughly. 

Zn = 2 ppm. 

Take 25.0 ml of 2 ppm Zn++ standard, place in 50-ml 

volumetric flask, make to 50 ml with 1% HN03. Zn++ = 

1.0 ppm. 

++ 

D. 

E. Take 12.5 ml of 2 ppm zinc standard, place in 50-ml 

volumetric flask, make to 50 ml with 1% HN03. Zn++ 

=0.5 ppm. 

F. Take 6.25 ml of 2 ppm zinc standard, place in 50 ml 

volumetric flask, make to 50 ml with 1% HN03 . Zn ++ 

= 0.25 ppm. 

G. Have 0.5 to 1.0 liters 1% HN03 available for standard 

blank determinations. 
-69- 

APPENDIX VI (Cont.) 

Reference: Stephenson, M, D., M, Martin, S. E. Lange, A. R. 

Flegal and J. H. Martin, California mussel watch 1977-1978. 

Volume 11: Trace metal concentrations in the California mussel, 

Mytilus californianus. Water quality monitoring report No. 

79-22. State Water Resources Control Board, Sacramento, CA. 

November 1979. 

Procedure modified by Dr. R. Bruce MacFarlane, NMFS, Tiburon, CA. 
-70- 

APPENDIX VII-- 

Processing Tissue Samples for Mono- and Polycyclic Aromatic 

Hydrocarbon Analyses by Gas Chromatography 

Monocyclies 

1. 

2. 

3. 

4. 

5. 

6. 

7. 

8. 

9, 

10. 

11. 

12. 

Put 10 g tissue (wet weight) on piece of B-2 weighing paper. 

Cut the tissue into 1 cm. pieces and place into a 20 x 150 mm 

screw-cap culture tube with a Teflon liner (33 m3 capacity). 

Add 6 ml of 4N NaOH (0.6 ml per gram sample). 

Add 4 ml of TF Freon that contains a recovery standard of 10 

ppm nonane (0.4 ml/gram sample). 

Tightly cap tube, shake and place in oven or waterbath for 18 

hours at 35 deg C (make sure NaOH has penetrated all of the 

sample). 

Remove from oven and shake tube for 1 minute, 

Centrifuge at 2000 rpm for 15 min. 

Freeze sample for at least 30 minutes. 

Recentrifuge at 2000 rpm for 15 minutes. 

Transfer clear Freon layer to a screw-cap vial. If not 

clear, add dilute H SO and recentrifuge. 

Measure Freon in test tube at room temperature; mark level. 

Sample is ready for injection. If you cannot inject 

immediately, freeze samples. 

24 
-71- 

APPENDIX VI1.--(CONT.) 

Dicyclics or Polycyclics 

1. 

2. 

3. 

4. 

5. 

6. 

7. 

Follow steps 1, 2 and 3 of monocyclics procedures. 

Place in oven for 18 hours at 35 deg C. 

Remove tube from oven and cool. 

Add 15 ml peroxide-free diethyl ether. 

Recap tube, and shake for 1 minute. 

Centrifuge at 3000 rpm for 10 minutes. 

Transfer the ether layer to a 25-ml concentrator tube. Add 

10 ml of peroxide-free diethyl ether and repeat steps 5 and 

6. 

NOTE: If a large emulsion layer forms, try freezing sample and 

recentrifuging as in the monocyclic procedure. 

8. Combine the 15 -ml and 10-ml extracts in the concentrator 

tube. 

9. Concentrate to 2 ml in the Kontes tube heater. Then add 2 ml 

of hexanes and reconcentrate to 1 ml. 

Data Entry: See Appendix I1 and use Appendix Fig. H. 

Reference: Nunes, Peps1 and Pete E. Benville. 1978. Acute 

toxicity of the water-soluble fraction of Cook Inlet crude oil to 

the Manila clam. Mar. Poll. Bull. 9:324-331. 

Warner, J. S. 1976. Determination of aliphatic and aromatic 

hydrocarbons in marine organisms. Anal. Chem. 48:578-583. 

Procedures modified by Pete E. Benville, NMFS, Tiburon, CA. 
-72- 

APPENDIX VI11 

Procedures for Hematology 

1. Obtaining blood specimens 

A. Heart punctures using vacutainers. 

1) Stick the needle (22 gauge 1-1/2") in about 3 or 4 

scales down from the anterior isthmus until you 

feel the heart muscle. 

2) For whole blood, use a 4-cc lavender top tube 

(contains EDTA). 

3) For serum, use a 7-cc red-top tube (sterile). 

B. Heart punctures using a plastic syringe 

1) Use a tuberculin or 3-cc syringe with a 23-26 gauge 

needle (depending on the size of the fish). 

2) For whole blood, use a syringe rinsed in 3% EDTA. 

3) For serum, do not use EDTA. Transfer blood to a 

sterile tube. Spin the blood for 15 minutes at 

4000 rpm. Use a pipette to separate the serum. 

C. Caudal tail cuts 

1) Cut the caudal tail with a scalpel. 

2) Use capillary tubes coated with heparin to 

determine hematocrits or to obtain plasma. 

3) If serum is desired, use noncoated capillary tubes. 
-73- 

APPENDIX VII1.--(CONT.) 

4) Spin the capillary tubes for 7 minutes in a 

microcentrifuge. 

5) Separate the serum or plasma immediately after 

centrifuging. 

2. Hematocrit determinations 

A. If a lavender vacutainer was used, use the plain 

capillary tubes: otherwise use the heparinized tubes. 

B. Fill the tubes, leaving about 0.5 cm of space at one 

end. Seal the tubes with critoseal. 

C. Spin the tubes in the microcentrifuge for 7 minutes. 

D. When determining the hematocrit values, note the buffy 

coat layer and the cherry-red layer of the tubes. 

3. Blood smears 

A. 

B. 

C. 

D. 

E. 

Place a drop of whole blood (from a lavender tube) at 

one end of a clean slide. 

Place another clean slide at a 45-degree angle to the 

first slide. Barely touch the drop of blood, allowing 

the blood to spread across the width of the angled 

slide. Smoothly and evenly push the blood across the 

slide. 

Air dry the slide and place in methanol for 3-5 minutes. 

Stain the slide in Wright's stain for 3-10 minutes. 

Place the slide in sodium phosphate buffer solution (pH 

6.7) for 3-5 seconds. 
-74- 

APPENDIX VII1.--(CONT.) 

F. Wash the slide in distilled water and blot dry with 

Whatman filter paper No. 42. 

G. NOTE: The times of the staining procedure will vary 

with individual fish and with age of the stain. 

Reference: Seiverd, Charles E. 1958. Hematology for the medical 

technologist, second edition. Lea and Febiger, 

Philadelphia. 

Modified by Brian Jarvis, NMFS, Tiburon Laboratory, Tiburon, CA. 
-75- 

APPENDIX IX 

Egg Histology 

A, PREPARATION 

1. SUBBED SLIDES -- Dissolve lg gelatin in 1 litre hot dH20. 

Cool and add 0.lg chromium sulfate. Store in refrigerator. 

Dip slides several times, drain and dry in vertical position. 

Store in dust-free box. 

2. GLYCERINE JELLY--Allow 8.0g gelatin to soak 1-2hr in 52 ml 

H 0. Add 50.0 ml glycerine and 0.lg phenol (or merthiolate). 

Heat 10-15 min. (not above 75 deg. C). Stir until homogenous. 

Store in refrigerator. 

2 

3. OIL RED 0 (C.1. 26125)-- 

Oil Red 0.....................0.7g 

Absolute Isopropanol .......... 2OOml. 

Shake solution well and leave overnight. Filter. Dilute 180 ml of 

Oil Red 0 solution with 120 ml dH20. Leave overnight at 4 deg. C. 

Filter. Let stand at room temperature 30 min. Filter. Stable for 

6-8 months. 

4. SCOTT SOLUTION 

Sodium Bicarbonate ............ 2.0g 

Magnesium Sulfate ............ 20.0g 

dH20 ....................... 1000.0 ml 

Add a pinch (0.lg) phenol or thymol. 
-76- 

APPENDIX 1X.--(CONT.) 

B. CRYOSTAT PROCEDURE 

1. If cryostat has been "down" for a long time, see instruction 

manual for routine maintenance. 

2. Always do daily maintenance (oiling) on microtome before 

starting cryostat. 

Turn machine on and allow chamber to cool to pre-set 

temperature. 

3. 

4. While cryostat is cooling, cut formalin preserved ovary and 

place in water for 10-20 minutes. 

Cover bottom of cryomold with OTC compound and place drained 

tissue on top. Cover tissue with OTC compound and place 

chuck support on top. 

5. 

Place on Cold bar in cryostat. 

6. After tissue is frozen, remove cryomold from frozen block and 

tighten in chuck jaws of microtome. 

7. 10-15 sections are placed on subbed slides and allowed to 

dry. 

8. Follow appropriate staining procedure: 
-77- 

APPENDIX 1X.--(CONTO) 

C. STAINING PROCEDURE 

a.) Mount frozen sections (10-15) on subbed slides. Allow to 

dry 5-10 minutes. 

b.) Rinse in 60% isopropanol ..................... 30 sec. 

c.) Stain in Oil Red O...........................lO-min. 

d.) Rinse in 60% isopropanol for a few seconds. 

e.) Wash in running water 2-3 min. 

f.) Stain in Harris Hemotoxylin .................. 2-3 min.* 

9.) Wash in tap water............................3-min. 

h.) Blue in Scott's Solution.....................3 min. 

i.) Wash in tap water............................3 min. 

j.) Mount in glycerine jelly. 

D. EGG STAGES (See Appendix Table A.) 

E. DATA ENTRY (See Appendix I1 and use Appendix Figs. I,J & K). 

Procedures modified by Michael Bowers, NMFS, Tiburon Laboratory, 

Tiburon, CA. Staining modified from G.L. Humason, Animal Tissue 

Techniques, 3rd edition. W.H, Freeman and Company. San Francisco, 

CA. 641 p. 

*Staining times will vary with age of the stains. 
-78- 

APPENDIX TABLE A.--Working codes for Ovary Maturation and Egg 

Maturation.* For Data Sets 38 and 39 - Histology: Ovaries. 

(Appendix Figs. J - K) 

OVARY MATURATION EGG MATURATION 

STAGE STAGE 

NUMBER DESCRIPTION NUMBER DESCRIPTION 

1 Immature Female 1 Follicle cells 

(never spawned) 2 Perinucleolus stage 

Early 

Late-cytoplasm paler 

3 Yolk vesicle 

2 Maturing Female 1,213 Size and yolk increasing 

(green eggs, but not and 4 Primary yolk stage 

yet ready to spawn) Ova 1 

and Secondary yolk stage 

5 Round 

Or 

6 Tertiary yolk stage 

Rectangular or 

irregular 

* Procedures for analyses of egg condition developed by Jeannette A. 

Whipple, NMFS Tiburon Laboratory. 
-79- 

APPENDIX TABLE A.--(Cont.)Codes for Ovary and Egg Maturation. 

OVARY MATURATION EGG MATURATION 

STAGE STAGE 

NUMBER DESCRIPTION NUMBER DESCRIPTION 

3 Late Maturing or 1,213 Migratory nucleus 

Prespawning Female (irregularly shaped, 

(spawning or near and 7 not in center, can 

spawning) see nucleoli) 

or 

8 

or 

9 

4 Spent Female 1,213 Some resorbing eggs (eggs 

(sometimes degenerating and being 

4) and 10 resorbed). 

11 Mostly collapsed or 

empty egg follicles. 
-80- 

APPENDIX TABLE A.--(Cont.)Codes for Ovary and Egg Maturation. 

OVARY MATURATION EGG MATURATION 

STAGE STAGE 

NUMBER DESCRIPTION NUMBER DESCRIPTION 

5 Resting Mature Female 1,2,3 See above. 

or Recovering Ovary and Sometimes Stage 3 more 

(nonspawning season) 11 less developed than in 

obvious immature fish. 

...................................................................... 

6 Abnormal or 112t3 Often abnormal eggs, 

necrosis and "dark 

Prematurely Resorbing crystals" in eggs. 

Ovary and 10 Resorbing eggs are 

usually stages 415~6; 

rarely 7 or 8 
-81- 

APPENDIX FIGURES 

Appendix Figure A.-- Data Set 1: Capture and Field Samples 

Appendix Figure B.-- Data Set 2: Meristics and Measurements 

Appendix Figure C.-- Data Set 3: Color Pattern 

Appendix Figure D.-- Data Set 4: Gonads-Meristics and Fecundity 

Appendix Figure E.-- Data Set 5: Liver, Gall Bladder and Stomach 

Appendix Figure F.-- Data Set 6: Disease, Parasites and 

Abnormalities. 

Appendix Figure G.-- Data Set 8: Spleen and Other Organs 

Appendix Figure H.-- Data Set 12:Pollutants-Petroleum Hydrocarbons 

Appendix Figure I.-- Data Set 22:Gonads-Egg Diameters and Sperm 

Moti 1 i ty 

Appendix Figure J.-- Data Set 38:Gonads-Histology; Ovaries-Maximum 

Maturity 

Appendix Figure K.-- Data Set 39:Gonads-Histology; Ovaries 

11-Relative Number Stages 

(NOTE: See page 4 for instructions on entering data) 
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I 
RECENT TECHNICAL MEMORANDUMS 

Copies of this and other NOAA Technical Memorandums are available from the National 

Technical Information Service, 5285 Port Royal Road, Springfield, VA 22167. Paper copies vary in 

price. Microfishe copies cost $3.50. Recent issues of NOAA Technical memorandums from the 

NMFS Southwest Fisheries Center are listed below: 

NOAA TM-NMFS SWFC “Recovery of adult green turtles observed or originally tagged 

at French Frigate Shoals, Northwestern Hawaiian Islands.” 

G. BALAZS 

(August 1983) 

“Report of the workshop on long-range planning for the 

North Pacific albacore fishery.” 

D. J. MACKETT, Editor 

(November 1983) 

Distribution of four dolphins (Stenella spp. and Delphinus 

delphis) in the eastern tropical Pacific, with an annotated 

catalog of data sources. 

W. F. PERRIN, M. D. SCOTT, G. J. WALKER, F. M. RALSTON 

and D. W. K. AU 

(December 1983) 

Annotated references to techniques capable of assessing the 

roles of cephalopods in the eastern tropical Pacific Ocean, 

with emphasis on pelagic squids. 

J. B. HEDGEPETH 

(December 1983) 

Summary of environmental and fishing information on Guam 

and the commonwealth of the Northern Mariana Islands: 

Historical background, description of the islands, and review 

of the climate, oceanography, and submarine topography. 

L. G. ELDREDGE 

(December 1983) 

Diving patterns of the Hawaiian Monk Seal, Lisianski Island, 

1982. 

F. V. SCHLEXER 

(February 1984) 

“The Hawaiian Monk Seal of Laysan Island: 1982” 

D. J. ALCORN 

(April 1984) 

Atlas of airborne sea surface temperature observations in 

nearshore California waters, 1980-1983. 

P.N. SUND 

(July 1984) 

Potential impact of deep seabed mining on the larvae of 

tunas and billfishes. 

W.M. MATSUMOTO 

(July 1984) 

Sampling commercial rockfish landings in California. 

A.R. SEN 

(July 1984) 

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44 

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