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CALIFORNIA

FISH-GAME




California Fish and Game is a journal devoted to the conserva-
tion of wildlife. Its contents may be reproduced elsewhere pro-
vided credit is given the authors and the California Department
of Fish and Game.

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scribers are asked to report changes in address without delay.

Please direct correspondence to:

CAROL M. FERREL, Ed/for
Department of Fish and Game
722 Capitol Avenue
Sacramento 14, California

Individuals and organizations who do not qualify for the free
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Printing Division, Documents Section, Sacramento 14, California.
Money orders or checks should be made out to Printing Division,
Documents Section.



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VOLUME 44



OCTOBER, 1958



NUMBER 4




Published Quarterly by the

CALIFORNIA DEPARTMENT OF FISH AND GAME

SACRAMENTO



STATE OF CALIFORNIA

DEPARTMENT OF FISH AND GAME



GOODWIN J. KNIGHT
Governor



FISH AND GAME COMMISSION

WELDON L. OXLEY, President
Redding

T. H. RICHARDS, JR., Vice President WILLIAM P. ELSER, Commissioner

Sacramento Son Diego

CARL F. WENTE, Commissioner JAMIE H. SMITH, Commissioner

San Francisco Los Angeles



SETH GORDON
Director of Fish and Game



CALIFORNIA FISH AND GAME
Editorial StafF

CAROL M. FERREL, Editor-in-Chief.— Sacramento

JOHN E. FITCH, Editor for Marine Fisheries Terminal Island

ELTON D. BAILEY, Editor for Inland Fisheries Sacramento

MERTON N. ROSEN, Editor for Game Sacramento



TABLE OF CONTENTS

Pass
The Control of pH by Buffers in Fish Transport

William N. McFarland and Kenneth 8. Norris 291

An All-plastic Dart-type Fish Tag

Daniel T. Yamashita and Kenneth D. Waldron 311

Conditions of Existence, Growth and Longevity
of Brook Trout in a Small, High-altitude Lake
of the Eastern Sierra Nevada Norman Reimers 319

Responses of Brush Seedlings to Fertilizers

A. M. ScMiltz, H. H. Biswell, and /. Vlamis 335

Note

Southerly Occurrences of Three Northern Species
of Fish During 1957, a Warmwater Year on
the California Coast J. B. Phillips 349

Note

Intestinal Flukes as a Possible Cause of Mortality in Wild Trout

J. II. Wales 350

Reviews 353

Index to Volume 44 359



( 280



THE CONTROL OF pH BY BUFFERS IN
FISH TRANSPORT' '

WILLIAM N. McFARLAND
Department of Zoology, University of California, Los Angeles

and

KENNETH S. NORRIS
Curator, Marinelond of the Pacific, Marineland, California

TABLE OF CONTENTS

Page

Introduction 291

Acknowledgments 292

The Pi-cactical Aspects of Buffer Application to Fish Transport 292

Application of Tris-buffer 292

Theoretical and Experimental Background 294

Causes of Mortality During Transport 294

Mortality in Closed-system Transport 296

Use of Buffers to Control pH 296

Use of Tris-buffer in Oix-n System Transjiort 301

Tests Performed With Tris-buffer in the Laboratory 302

Discussion 305

Summary 808

References 308

INTRODUCTION

The heavy fishing pressure on our Nation's streams and lakes which
has developed in recent years has required large-scale propagation and
planting of fishes, particularly trout, and has led to the development
of the complex modern fish tanker.

One of the major problems confronting the fisheries biologist has
been the development of better means for transporting fishes. Confine-
ment of fishes in transport apparatus can produce critical conditions in
the water which necessitate control if mortality is to be avoided. Several
of these conditions have been investigated. However, one of these fac-
tors, that of increasing acidity, resulting from waste products and
respiration, has not been adequately studied. We feel that an under-
standing of increasing acidity is not only of theoretical importance
but also of great i^ractical value. Such information should lead to
more effective transport by serving as a basis for the development of
better means for controlling water quality.

In this investigation several chemicals, capable of stabilizing acidity
levels, have been studied. One of these, Tris-hydroxymethyl-aminome-
thane, has promise both for marine and fresh water application (this
compound will henceforth be referred to as "tris-buffer"). This chem-
ical can increase the acid-absorbing capacity of sea water by as much

1 Submitted for publication May, 1958.

- Contribution Number 3, Marineland of the Pacific Biological Laboratory.

( 291 )



292 CALIFORNIA FISH AND GAIME

as 50 times Avitliout deleterious effects upon fishes. Tlic application of
this buffer has been studied in the laboratory and on several occa-
sions successfully used duriiipf transport operations.

This study lias been separated into two divisions: first, ilic i)i'actical
aspects of buft'er application to fisli li-ansport, and second, the theoreti-
cal and experimental ])acls<ji'onn(l underlying the practical methods.

ACKNOWLEDGMENTS

We wish to thank Dr. IJoyd W. AValker of 1h(^ Tniversity of Cali-
fornia, Los Aufjeles. for his suwigfestions (lui'int:' Ihe coufsc of iliis re-
search and for his lielj)ful review of the manuscrijjt. AVe also thank
the staffs of the Steinliart Aqnarinni, the "Waikiki Af|narinm and mem-
bers of the Territoiy of Hawaii, Division of Fish and Game, for their
assistance durino- the transport operations reported here. We wisli to
thank Muriel Johnson for her help during preparation of the manu-
script.

THE PRACTICAL ASPECTS OF BUFFER APPLICATION
TO FISH TRANSPORT

Transport operations which involve heavy loads, long distances, or
poor water supply are apt to be limited by increasingly acid waters
which may result in debilitation or death of fish. Under these circum-
stances it is desirable to stabilize pH in some w^ay. Tris-bnffcr has
proved to be the most effective for this purpose of several ('om]ionnds
tested. It was nontoxic to all fish species examined.

Application of Tris-bufFer

Tris-buffer is a highly stable, soluble white crystalline compound. •"'
It is applied by dissolving in the transport water. Tests indicate that
fishes can stand high concentrations (to 20 grams per gallon) of buffcn*
with no ill effects. Light dosages (two to five grams per gallon) will
generally be adequate to stabilize pH during transport of fish for
moderate distances and while carrying moderate Aveights per unit
volume.

One undesirable feature, but one not difficult to overcome, is the
high initial pH produced by the buffer. This pH varies between levels
of 9.2 and 9.8. depending upon the amount used. It is necessary to
back titrate solutions to desii-ed j)!! levels prioi- to use. We have
employed concentrated hydrochloric acid for this purpose since chlo-
ride ion is already present in sea water in high concentration. Other
acids, such as sulfuric or citric could be used. A portable ]ill meter
can be used to measure final ])II in the field. Tlow(»ver. by adding
knowai amounts of acid and buffer, the desired pll can be closely
approximated without a pll measuring device. Both li(|uid and dry
acids were tested fllCl and citric). This method of pll adjustment
has j)roven reliable; the error of back titration amounting to ;i maxi-
mum of 0.05 pH units in our tests. Buffer and dry acid could be pre-
mixed for field use and i)ackaged in known nmonnfs (Table 1). It is

3 Obtainable from the Siffina Chemical Co., 3500 DeKalb St., St. Louis IS, Mo.



BUFFERS IN FISIT TRANSPORT



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204 CALIFORNIA FISH AND GAIME

recommended that an cud point bot^v(M■M pi I of s.'Jd jind >.:]{) Ix" sdn^^'lit
for normal salt water transjiort. Tins will allow cnou-zli lalitude to
absorb small measurinp,- errors without dan«>'er to the tisli, while main-
taiiung high buttering eapaeity. Table 1 indieates the amounts of IKM
(concentrated) or citiric acid (di\i i'c(prn-cd io riducc pll in \arious
levels in one gallon of sea water after addition ol' ;i known amount of
trisdmtfer. By nndtiplying this tigure by the number of gallons in a
tran.sj)oi-t tank and ad(liug the resulting amount of acid and butl'er the
desired pH can be approximated. Ex]ici'ience has shown that the allow-
able ]dl range dnring salt water trauspoi-t lies between 7.5 and 8.5.
The pll lauge for fresh water is nnu-h widci- than this, as uornuil
lluct nations are greater and fresh water fish are generally hardier to
lower pH.

The bntfer should be added and back titrated before fish are intro-
duced to insure final adjustment of pll. If the compomid is to be nsed
in tanks with a recirculation system, the buffer and acid shouhl be
added and the w'ater recycled to ])roduce a stabilized pH before the
fish are introduced.

When fish are to be shipped by air in plastic bags, concentrations of
five grams per gallon will generall)- produce sufficient buffering. The
actual amount of buffer which will lie consumed during any transport
operation is dependent upon the weight of fish per unit volume of
water, the total metabolic activity, the initial pi I, the natural buffering
of the transport water, the temperature, and the time during trans-
port. It is felt that the major factor contributing to pH decline result-
ing from this complex is carbon dioxide. Tris-buffer readily "absorbs"
carbon dioxide and therefore should prove efficient in stabilizing pll
in any system which tends to accumulate this gas. Because of the
complex nature of pH change it is best to rely upon periodic measure-
ment during transport.

The data indicate that tris-buffer is nontoxic to 29 species of fishes
(Table 3). Other forms should be tested prior to transport.

THEORETICAL AND EXPERIMENTAL BACKGROUND
Causes of Mortality During Transport

Understanding the factors which cause death or distress to fishes
dui'ing transport is basic to improving present transpon methods. Sev-
eral variables can become lethal agents during transportation. These
can act individually, or more likely, in combiimtion, and thus witli
increased possibilities of causing mortality. The most obvious factors
which ma}^ become lethal are: temperature, oxygen tension, w^aste
product levels, carbon dioxide tension, and pll.

Temperature

In general, increasing temperatures produce difficulties dining ti-aiis-
port primarily because rates of metabolism ai-e iuci-eased and other
parameters thus tend to become critical.

Oxygen

Most species of fi.sh do well when the tensions are above 2.0 cc Oo/
liter. Under extremely crowded conditions however, Haskell (1940)



BUFFERS IN FISH TRANSPORT 295

has shown that tensions above 8-10 cc Oo/liter are required by trout.
Such high requirements are possibly related to the existing level of
COo, in addition to possible effects of increased activity.

Waste Products

The accumulation of wastes from the metabolism of fishes can be-
come a serious problem. Brockway (1050) points out that most wastes
excreted through the gills consist of NH.3, urea, and amino oxides,
while creatine and uric acid are excreted by the kidneys. In addition
to these materials the production of mucus and its liberation into the
water and regurgitated food material can contribute to ])ollution. Ellis,
Westfall, and Ellis (1946) have shown the extreme toxicity of ammonia
to certain fish species even at levels as low as 1.0 part per million
(p.p.m.). In his experiments upon survival of Tikipia niossamhica in
sealed jerry cans, Vaas (1952) found that the bacterial counts of the
medium tended to increase exponentially with time. This increase in
bacteria was supposedly caused by an increase in the organic content
of the water from metabolic wastes. Phillips and Brockway (1954)
found that the ammonia content rose to 25 p.p.m. during 12 hours
in acpiaria containing brook trout at high density. Placing the fish
in cooler water, prolonged starving and treating starved fish with
sodium amytal, all caused signifieant declines in the accumulation of
ammonia during 12 hours. The results from these experiments are
inconclusive, but indicate that accumulation of waste products may
reach lethal levels in transport under extreme conditions. As yet no
completely adequate agent has been developed to control ammonia,
but Saha, Sen, and Mazumdar (1956) report that several resins have
the ability of absorbing it. A product called Amberlite proved to be
the most useful. Nemoto (1957) has also tested resins. His results show
Amberlite-IR-120 to be useful in the absorption of nonprotein nitro-
gen (NH3).

Carbon Dioxide and pH

The anaesthetic effects of COo have long been known. Its accumu-
lation during transport has been controlled by providing adequate
gas exchange, through aeration. Fish and Hanavan (1948) indicate
that during transport of salmon around Grand Coulee Dam, the pH
fell from 7.8 to 6.4, while the CO2 rose from three to four p.p.m. to
a high of 17-18 p.p.m. during the four hours of hauling. These figures
are important in that the transport water was continually aerated
and the Oo concentration, after an initial decline, returned to a noimial
level of seven p.p.m. We have noticed similar changes during transport
of marine fishes over distances as great as 1,000 miles. During one
such trip (Guaymas, IMexico, to Los Angeles, California) the pH
declined during the first hour from 8.20 to 7.40. The addition of a
second circulation pump was only sufficient to maintain this lower pH.
Oxygen was always at normal levels. It is evident that even with
aerating systems capable of maintaining ample quantities of dissolved
O2, COo may accumulate to high levels and result in a marked de-
cline in pH.



296 CALIFORNIA PISH AND GAME

In addition to the narcotic action of COo, a fall in pH can produce
otlier serious effects. Townsend and Cheyne (li)44j dcinoiistrated that
increasing acidity (from several acids) can cause mortality. Further
changes in both COo and pll have serious effects on fish liaemoglobins.

Mortality in Closed-system Transport

In recent years pure O^ has been employed in the iiiiportation of
both fresh water and marine fishes. It is usuallj^ api)lied by placing
the fisli in a polyethylene plastic bag and displacing tlic ;iii' over the
water with O^.. The bag is sealed, cartoned, and shipped to its destina-
tion by air.

The use of oxygen in fish ti'aiisport was apparently first employed
in Eurojx' in the last i)art of the lOth century (Shebley, 1927). Osburn
(1910) reports the use of ()o in fish transport and since that time
several papers have appeared regarding its use (Wiebe and McGavock,
1932; Haskell, 1940; Mitra, 1943; Khan, 1946; Sundara and Cor-
nelius, 1949; Basu, 1949, 1952; Vaas, 1952; and Saha, Sen, and Mazum-
dar, 1956). Of these papers only the Avorks of Basu, Vaas, and Saha.
et al. make any attempt to relate the use of O2 to mortalitA' and the
weight of fish per unit volume which can be transported.

At Marineland of the Pacific Ave have employed this method to
imjiort fishes from the HaAvaiian Islands. Because of the air freight
charges, resulting almost entirely from the poundage of shipping
Avater Avhich must be used, the method is expensive. Therefore it is
necessary to keep the Aveight of fish per unit A'olume as higli as pos-
sible. As a result mortality Avas often high on arrival and many bags
contained fish in poor condition, exhibiting scA^ere upsets in equilib-
rium. It Avas decided to determine, if possible, the causative agent of
mortality and loss of equilibrium. Subsequent shipments Avere analyzed
for dissolved Oo and pH.

Oxygen analyses revealed that the oxygen tensions in shipping
Avater upon arri\'al Avere more than ample to maintain metabolism
under normal conditions. Oxygen tensions never fell beloAv a value of
6.60 cc 02/liter even though in a few instances mortality of all fish
in the bag had occurred. Oxygen Avas detei'mincd by the sodium nzide
modification of the Winkler method.

Seventy-one bags were analyzed for |)11 upon an'i\;d. luitinl pll
A'alues in HaAA'aii Avere grouped around a A'alue of 8.20. A mortality of
31.9 percent of all fish occurred. In contrasting the pH values for bags
containing live fishes against those in whicli mortality had occurred
(mortality considered as 25 percent or more dead animals in a bag),
it was found that a significant difference in pll existed (Table 2).
Measurements of pll Avere madi^ Avith a Beckman ])IT meter f^lodel X).

Use of Buffers to Control pH

The results suggest that increasing acidity and CO^ could have been
the major factors involved in mortality. If these factors Avere not the
actual causative agents, they are correlated Avith mortality. The in-
creases in acidity are attributable to the accumulation of COo and
Avaste products. Since pH in most instances fell drastically, AA'hether
death occurred or not, it Avas felt that the addition of a buffer to the



BUFFERS IX FISH TRANSPORT



297



TABLE 2
Analysis of pH of Plasfic Bag Transport Water Containing Fishes Sfiipped From Hawaii



Condition of fish


Number of bags


Mean pH upon arrival


pH range


Alive

Dead*


48
23


7.03
6.78


6.00-7.92
6.42-7.15



(Difference between means: to.99 = 2.37, t = 58.1, P < 0.01)
* Bags containing more than 25 percent dead specimens are considered in this group.

medium would be beneficial. Since O2 never sank to normal lethal levels,
control of pH was expected to result in an increase of time during
which fish could survive in the bags.

Inorganic Buffers

Several attempts have been made to stabilize pH during fish trans-
port by use of inorganic buftering compounds. The results from the
Hawaiian shipments agree with the findings of Vaas (1952) Avho showed
that mortality of Tilapia niossaniMca in sealed jerry cans, was asso-
ciated with increasing COo, and not with falling O2 tensions. He found
that the addition of sodium monophosphate (NaoHP04) to the water
at concentrations from 1.5-3.0 grams per liter increased the time to
mortality by an average of about 40 percent. Srinivasan, Chacko, and
Valsan (1955) report that the mortality of carp fry, after 48 hours
in sealed cans was 2 to 4 percent in NaoHP04 buffered water, Avhile
it was 10 percent in nonbuffered water. In open containers sodium
monophosphate did not produce differences in mortality. Saha et al.
(1956), report opposite findings using eyprinid spawn. They found a
decrease in the time to mortality of 37 percent upon the addition of
sodium monophosphate. They suggest that the addition of a single
component cannot explain the increase in time to mortality reported
by Vaas. They point out that in buffer theory the equilibrium relation-
ship existing between a salt and a weak acid or base determines the
buffering capacity. Although only one component was added by Vaas
(op. cit.), other ions were present as indicated by the initial alkalinity
figures, and these could give the necessary weak acid-salt ratio required
by theory. Further, the negative results reported by Saha et al.. might
reflect a sensitivity of eyprinid spawn to the buft'er. Xevei'tlieless, the
rate of pH decline in Vaas's bufl^ered groups was lower than in con-
trols. Further, the nonbuffered groups could not utilize the available
oxygen by reducing its tension as low as the buttered grou]is. This
suggests that the presence of sodium moiiophos])hate considerably de-
creased the CO2 concentrations. The relationship of CO2 to mortality
may be correlated with its effect on the Go loading tensions of the
blood. The results of Vaas and Srinivasan et al. (op. cit.), are at vari-
ance with those of Saha et al. (oj). cit.) ; therefore the value of using
phosphate buffers in the transport of fresh water fishes remains in
doubt. Nemoto (1957) reports doubtful utility of Na2HP()i in trans-
port of fishes.



298



CALIFORNIA FIRTI AND OAMF



Sea "water, in comparison to most fresh waters, has a hiulu'i- IjufOM-ing
capacity (Sverdnip, Johnson, and FUnninj;, 1942, pp. 19.1 and 202).
This action is due to the jiresence of carbonate, phosphate, and hoi-ate
salts. Sea water absoi-bs abont seven times as mncli liydrogen ion as
distilled water when the pll is back-titrated to 4.r)(). The buttering
capacity of sea water seems insufficient to absorb the (juantity of acidic
substances given off by fishes during normal transpoi-t. Thei-cfore. it
was attempted to increase this capacity by utilizing ihc salts known
to be ])i'('scn1 ill sea walcr. Sodium carbonate and sodium hicarbmiate
were tried at various concentrations. Sodium carbonate, above a con-
centration of five m^I (two gi'ams per gallon), caused preci])itation
of calcium and magnesium and was therefore considered undesii'al)le.
This precipitate became (piitc lu'avy at concentrations above 10 nuM
(five gi'ams per gallon). Sodium bicai'bonate did ]U)t ('aus(> prcci|)iia-
tion at concentrations np to 63 dvM (20 grams ])er gallon). It showed
marked buffering at concentrations from 15 m^I to 63 mJ\I. The amount
of acid necessary to reduce the pH to a valtie of 4.r)() was 7.4 and 30
times as great respectively, as the amount i'(M|uii'e(l in untreated sea
water (Figure 1).




ml. of 0.01 N. HCI per 50 ml. sample

FIGURE 1. Titration curves for 50 ml. samples of sodium bicarbonate, tris-buffered, and
untreated sea water. A, untreated sea water; B, tris-bufFer, 5 gms. per gallon; B', tris-buffer,
20 gms. per gallon; C, sodium bicarbonate, 5 gms. per gallon; C, sodium bicarbonate, 20
gms. per gallon. The stippled area between pH of 7.5 and 8.5 represents a recommended
range for transport of marine fishes.



BUFFERS IN FISH TRANSPORT 21*9

In Figure 1, curves C and C represent the amount of 0.01 N HCl
required to reduce the pll to 4. .10 in two 50 ml. samples, when the
concentrations of sodium bicarbonate in sea water were increased by
15 and 63 niM respectively. Curve A shows the titration curve for
untreated sea water. The curves show clearly that the buffering capac-
ity is markedly increased, but the pH range at which the greatest
hydrogen ion absorption occurs is low. This pll range, lying between
6.0 and 7.0, has already been associated with mortality of marine fishes
in ])lastic bag transport.

The recommended pH range (8.5-7.5) is represented by the stippled
area in Figure 1. The buffering capacity of sodium bicarbonate is very
low in this area. For this reason we turned our attention to other
buffers with high capacities, but in the desired range. Several concen-
trations of sodium monophosphate and sodium phosphate were tried.


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