INHSBull14No9


. -

ST.A':'l!.i OF ILT .!NOJS 
DBP - P..NT O UJ.GISTRA.l'ICN .\ND r.l.DUCA'f!ON 

DIVISION Ot.' ':11."E 
NATURAL HISTORY SURVEY 

Sl'llt'l.lE.N' A. •OlIBll.S, ( •~ 

·vol. ~,..... • BULLETIN 

:: ne Dcterm.h.'1.ation of Iiyc:rol!,en -o:i 
Concentration in Co:r.mecrl.01..! 

With 3:resh. .. Water Bio-
lo~,ic1d Studies 

BY 
VICTOR E. SHEWOR:) 

PaINTED BY A~fB:ORrJ."!' CI' '.M£ S'i.A'li', OF IL!..:NO:Ib 

tffi.BANA, ILLINOIS 

Fc1Mll1.."T, 1923 



STATE OF ILLINOIS 

DEPARTMENT OF REGISTRATION AND EDUCATION 

A, M, SHELTON, Di.rector 

BOARD OF 

NATURAL RillSOURCES AND CO SERVATION 

A. M. SHELTON, Chairman 

WILUA;I[ TnELEASE, Biology 
JOHN M. CoULTER Forestry 
EosoN S. BASTIN, Geology 
WJLLIAJ\r A. NOYES, Chem,istry 

JOHN w. A.LVORD, Engineering 
KENOa1c C. BABCOCK, Representing the 

President of the University of Illi-
nois 

THE NATURAL HISTORY SURVEY DIVISION 

STl'CPllEN A. FORBE , Chief 

PHILLIP• • IIOa ,• PIIINT •~J 8fl'fllNQFl&l.0 , 1'.\.INOI& 

71'548-1200 



II 

CONTENTS 

Introduction 
PAGE 

379 
Methods of determining H ion concentration (pH) . . . . . . . . . . . . . . . . . . . . . . 380 
The influence of Hydrogen ion concentration on fertilization, development, 

growth, and survival in water deficient in dissolved oxygen . . . . . . . . . 381 
Reactions of fish to differences in hydrogen ion concentrations.......... 385 
An examination of certain Illinois waters with special reference to hydro-

gen ion concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386 
General relations of hydrogen ion concentration to other factors. . . . . . . . 388 
Discussion and summary of results............ ........... ..... ..... .. .. 391 
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 
Acknowledgments .. ! . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394 
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394 



ARTICLE IX.-The Determination of Hydrogen Jon Concentration 
in Connection with Fresh-T·Vater Biological Stu,dies. BY VICTOR E. 
SHELFORD. 

INTRODUCTION 

Generally speaking, the significance of hydrogen ion concentration 
and its determination are among the most complicated problems con-
fronting tuclent of fre h-·water ecology. Detem1ination of carbon 
dioxid, "alkalinity," and dissolved oxygen have long been among the 
means used for ascertaining the uitability of a water for organisms, as 
well as its suitability for domestic and industrial purposes. but the rela-
tions of these properties of a water to each other chemically or to organ-
isms have never been fully investigated. 

There is great confusion as to methods of measuring and express-
ing so-called "alkalinity." It is expressed (a) in terms of a carbonate 
of a predominating alkaline metal-for e..--carnple, as CaC0:1 ; ( b) as bound 
and half-bound CO 2 ; (c) as alkali reserve; and (d) as buffer value. 
It is from time to time confused with CO2 tension. total CO2, and the 
normal acid used. It is not possible to straighten out this confu ion. 
The investigator can only ascertain the methods used by different writers 
and carefully translate their records and statements into the terms he 
himself has elected to use. 

There is almost a great confusion in the use of terms and methods 
in connection with hydrogen ion concentratior.. This grows out of the 
fact that the turning points of various standard indicators have been 
referred to as "neutral." notably in the case of phenolpbthaJein, the 
turning point of which is decidedly alkaline. There are now improved 
methods of determining hydrogen ion concentration. including true neu-
trality. These have been used in the work here reported, which has con-
si. teJ chieAy of ( I ) observation on the hydroo-en ion concentration over 
fish breeding-grounds in everal Illinoi localities, July to June, 1919-20 ; 
(2) tudie of the reactions of fishes to hydrogen ion concentration; and 
( 3) tu dies of the effect of different hydrogen ion concentrations on the 
urvival of fi he in water of low oxygen-content. This work has been 

supplemented by studies by Fenner Stickney on the re istance and re-
action of a dragon-fly nymph to ariou hydrogen ion concentrations and 
by experiments l y Ii s da Hall on the effect of hydrogen ions in the 
development of toad and whitefish egg , carried on under the auspices 
of the Department of Zoology and of the Graduate School of the Uni-
versity of Illinois. The author' acquaintance with the que tions dis-
cu. e<l has also I een extended by a serie of determination of hydrogen 
ions in river and lakr in the follo\\'ing drainage ystem : Puget Sound, 
Columbia River, Interior Basin. olorado Riv r, ancl Arkansa River. 
These determination \\ ill be puhlishe<l elsewhere. 



380 

These studies have been made by the author largely with a view to 
ascertaining whether or not hydrogen ion determinations give indica-
tions of value in the field of animal ecology. The author's answe!" is in 
the affirmative. It is accordingly the purpo e of this paper to show ( 1) 
that hydrogen ion concentration is one of the factors governing the 
movement and distribution of fishes, its importance on the whole probably 
equaling that of well-known factors; (2) that breeding places of fish 
are characteri2ed by differences in hydrogen ion concentration; (3) that 
hydrogen ion concentration has marked influence on the survival of 
fishes under adverse conditions. 

METHODS OF DETERMINING H loN CONCENTRATION (pH) 
The methods in common u e are the colorimetric and the electro-

metric. The former is based on the colors assumed by solutions of 
various dyes in the presence of various hydrogen ion concentrations. 
The latter is based on measurement of the differences of the electric po-
tential between the solution to be tested and a hydrogen electrode. In 
this process it is necessary to bubble hydrogen through the sample, and 
as this withdraws CO2 it is not practicable as a field method. It is de-
sirable to dip the apparatus into the mud or water and measure while 
in situ. Thi might be po sible except for the additional fact that the 
pressure of the hydrogen (gas) must be maintained at one atmosphere, 
or corrected to this pressure, which renders the use of the electrometric 
method impracticable in the field. Colorimetric standards may be color 
charts for rough work or for work on soils- those published in Clark's 
book ('20) may be purchased separately-but for use in fresh water 
greater accuracy is necessary. For this purpose "buffer" solutions of 
known hydrogen ion concentration are placed in hard glass tubes, a 
standard amount of indicator added, and the tubes sealed up and used 
for comparison with water samples with the same amount and kind of 
indicator added. everal firms make such sets of standards, but the 
range at present is limited, and others must be added for some waters. 
These additional buffer may be made by a competent chemi t. Standard-
ization of buffers electrometrically is quite essential; also the purchased 
set must be checked electrometrically at the beginning and end of each 
series of readings. 

I 

I 

' 



381 

Hydrogen ion concentration is expressed as pH-the logarithm of 
the reciprocal of the normality of free hydrogen ions. The pH scale 
and relative amounts of free hydrogen ions are as follows: 

Q) - .s 
'C f ..... ..., 
,:.) ::, < ctl Q) 

z 
pH 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

I I I I I I I I I I l l I l I 
0 0 0 0 0 0 0 .-1 .-1 .-I .-I .... 
0 0 0 0 0 0 M 0 0 0 0 0 0 0 

0 0 0 0 0 .-1 0 0 0 0 0 0 

0 0 0 0 M 0 0 0 0 0 

0 0 0 M 0 0 0 0 

0 0 r-i 0 0 0 

0 .-I d C! 
.-1 0 

The range to be expected in Illinois waters is about 8.5 to 6.7 for 
most streams and lakes not contaminated. Streams receiving mine seep-
age may have much higher hydrogen ion concentrations and still support 
certain species of fish. 

THE INFLUENCE OF HYDROGEN loN CONCENTRATION ON FERTILIZATION, 
DEVELOPMENT, GROWTH, AND SURVIVAL IN WATER DEFICIENT 

IN DISSOLVED OXYGEN 

1. Fertilization of fish eggs.-Miss Hall found that when oxygen 
is 4 c.c. per liter fertilization of the eggs of the whitefish takes place 
equally well in acid, neutral, and alkaline waters. When oxygen is 
lower (2.9 c.c. per liter) pH 6.2-6.6 is more favorable to fertilization 
than 7.0-8.4. This relation is quite inexplicably the reverse of what 
would be expected by comparison with other relations to oxygen. 

2. Developn-ient.-Miss Hall showed that in the toad and whitefish 
the early cleavage and gastrulation period is most sensitive to hydrogen 
ion concentration and low oxygen. She further showed that early death 
or retarded development occurred in both toad and whitefish embryos 
with pH 6.2 and 0 2 about 1 c. c. per liter. In the case of the whitefish, 
still lower oxygens gave little difference correlated with pH. The experi-
ments with sufficient oxygen indicate that pH 7.0 is near the maximum 
H ion concentration which permits best development. Even so hlgh a 
concentration is probably not common on fish breeding-grounds in Illinois 
waters. Higher concentrations, to pH 6.2, show interference with de-
velopment, hatching, or length of life. That acid interferes with de-
velopment was demonstrated by Loeb in 18981 and this having been 
found generally true, of marine animals at least, the demonstration has 
become a class experiment with marine forms. 

3. Survival.-The influence of hydrogen ion concentration on the 
survival of fishes in low-oxygen water is shown in Tables I-IV. 



382 

TABLE I 
1101d11.g th (feet of hydrogen i.on concentration 011 the swrvwal of rock 

bass in low-oxygen, warter, A.itgiist. 191 

Number of I 
fish 

a 

b 

C 

d 

e 

t 
{} 

h 

i 

1 

Wei ht 
gm. 

10 

10 

11.6 

14.5 

16 

18 

18 

21 

2 

13 

Sur vival 
in min. 

43 

55 

50 

1500+ 

71 

350 

60 

55 

45 

124 

TABLE II 

pH 

6.0 

6.5 

6.2 

6.5 

6.1 

6.5 

6.2 

6.5 

6 . 3 

6.0 

0, in C. C. 
per l iter 

.42 

?" . .,., 
./;8 

.79 

.70 

.79 

.68 

.42 

.23 

.72 

Temp., deg. 
C. 

16 

16 

16 

16 

16 

16 

16 

16 

16 

16 

S11owing the effect of hyclrogen ion cone ntrati.on on tlle survival of large-
1no11,thed blaclc bass i.11 low-oxygen water. Janua1·y, 19;?1 . 

Number of I Av. weight I 
fish gm. 

a{ 6 346 

6 2 'i 

b{ 7 275 

6 2 5 

c{ 1: 255 
328 

10 320 

Survival 
in min. 

4 

62 

64 

47 

70 
63 
50 

pH 0,, c. c. Temp .. deg. 
C. 

8.6 .18 13 . 5 

6.5 .18 13.5 

8.5 . 055 13.5 

6.2 .06 13.5 

8.5 

I 
.06 

I 
13.5 

7.6 .06 l~.5 
6.2 .06 13.5 

Comparing individual a, b. and c (Table I) it is evident that a de-
crea e of .5 on the pH scale is more important than one of .20 c.c. in 0:: 
and that a d crease of .3 on the pH cale equals in effect a difference 
of .46 c.c. in oxygen. Furthermor , fi h c was heavier than a or b, and 
the larger fi h usually live longe t. Fi hes f and g show a triking differ-



383 

ence in survival when both pH and :i are moved in the unfavorable direc-
tion. Fishes '1 and i are imilar to f and g, but the 28-gram fi h ( i) should 
have lived longer considering its larger size. 

Turning to Table II, group a shows that the average time-survival 
is greater at pH 8.6 than at 6.5, but the difference in weight would oper-
ate to exaggerate this. However, the same result is shown in group b, 
where both oxygen and size of fish would operate to reverse the result. 
Furthermore, group c, with fi h-weight standing on the whole so as to 
reverse the result, shows a progressive decrease in survival-time with 
increase of hydrogen ion concentration . 

TABLE III 
Showing the etrect of hyd,rogen ion concentration on the survival of bluegills 

i1i low-oxygen water. Decemb r, 1920, and Jan1tary, 1921. 

Number of I Av. weight I Av_. suryival I 
fish gm. m mm. pH 

a{: 11± •21+ 7.25 

11± t21+ 8.86 

b{: 11± t60 8.0 

11± §47 8.8 

c{: 14.6 16 6.4 

16 30 8.8 

d { ! 11 31 8.8 
11 23 6.6 
11.5 18 6.2 

e{ 13 12.3 8 8.5 

15 11.8 27 6.5 
• 0 . K. nt 21 minutes. t .tlnne, lbetlzed al 21 mlnute~. 

0 ~, C. C. 
per l. 

.1.2 

.12 

.12 

.12 

• .12 

.12 

.12 

.12 

.12 

.06 

.06 

i ..lll anaesthetized al 21 minute . * Two anne tbetized al 21 mlnute2. 

I 

Temp., deg. 
c. 

13 

13 

13 

13 

13 

13 

13 
13 
13 

13.6 

13.5 

Table III shows results with bluegills. Groups a and b are self-
explanatory. Group c shows survival one-half as long at the low pH 
as at the high. Group d shO\vs a progrc sive hortening of life a pH 
is lowered. Group e suggests that with ery low o.·ygen, pH 6.5 is more 
favorable than 8.5. The work of \Veil and Hall (Table IV shows 
similar result , ith di:ffcrenl pecie . 

• 



3 4 

TABLE IV 
Showing the relation of hyd,rogen ions (OOi added,) and, oxygen to the 

s1trvi'Val of fishes. Taken fro1n the work of authors indicated.. 

Author , I Species 0., c: c. time I I Survival 
per hter per gram 

A.mbloplites +2.0 22 6.6* .1-.15 10.9 
Wells ......... :±:2. 0 46 7.0* .1-.15 20.2 rupestris +2.0 140 7.7* .1-.15 62.5 

Wells ......... Notropis .6-.8 15 6.6* .1-.15 25 
corn11t1ts .6-.8 195 7.8* .1-.15 246 

Ooregonu,~ ......... 148 6.3 .1 
Hall .......... cl1ipeif orm is . ........ 225 9.0 .1 

at ......... 240 6.3 .1 
hatching ......... 300 9.0 .1 

• pH calculated. 

Powers ('22) has shown that marine fi hes require more 0 2 at high 
hydrogen ion concentrations. Thus, in general. the unfavorable effect 
attributed to low oxygen is partly due to the pH which accompanies it, 
and this depends largely on the amount of carbonates in the water. The 
hydrogen ion concentrations which many workers have considered that 
they were using when acid was added to disti11ed water have not been 
attained becau e of the carbonates present. It has been customar to 
regard distilled water as essentially free from salts, especially carbonates. 
Wells ('15) stated that he conducted experiments in distilled water which 
contained no salt . Later he showed that it had a conductivity of 600X 
10-7 which suggests + .0005 N solution of carbonates, which is now 
known to be approximately the arbonate content of distilled wate,r 
from the source where he obtained his. He did not have anything like 
the H ion concentration he a sumed, either in the killing or gradient 
experiments. If no salt had been present the pH of his '' .000075 
H~ ·o,· would have been between 4.0 and 5.0, which would have 
quickly killed the £ h. An unfortunate loo e tatement (115, p. 253) 
that "fresh-water fishes * * * cannot live normally in neutral [he 
u ed the term neutrality for the turning point of phenolphthalein, 
which a he u ed it is pH 8.0] or alkaline water caused misunderstand-
ing. In fact, most fresh waters of the Eastern nited tates are alka-
line, and mo. t ire h-water animals live in such waters. The facts 
were that in water with pH 8.0+ the particular sp cies with which 
he worked becam slucrgi b and were un uitable for behavior studies, 
or died in rugher hydroxyl ion concentrations. The concentrations 
in which he killed the fish were probably only titrated with standard 
acid, and ther for it i not po ible to tate what the pH may have 
been. 

• 



TAill.E I'' 

SbolrtD& the TaDRe or b)'drogi?n Ion ronccntrallons entered and s~lee!led br slwernl 6})edes ot fiS:h. The fis hes were confined lo n long uor row tank t.ltffc:rlng In hydrogen loo concentrattoo e.t the t 'llro en~:, 
Ordlnarilr they did not s~im t.be cnlire hmgth or tbe tank, bul mo\'efl baok and ro rth , turnin~ a.t de.fl11 llt\ r10tnt1, :iml 6nall)' rumln,; lo rest, ThNs._. t.ur11io,:: and resting- polnt,i iJltrered in dlf!trenl erperimeot.: 
ISeic column headings) or Lb@ !ame series. 'l'he upper osures of eat! h c.:ouolet abow tbe pH range within which the flsbes came to rest : thf: lower figure:B of each couplet give tbe mean Polnta o[ tnllllDg 
bticll: when beaded toward bigbeT or lower pH, and the mean columns gh•e the average pH content at the rHHns- points tt JP<"ttd. The tbiTd column Crom the rl s:ht s bowe for an experiments the meao 
pH of the selectlooi; made. The 1iecond oohunn from the right gives. merely tho maximum and minimum or the upper flgurl'i:°I or lbo couplets OJJflORita. APrated W}lter was deet) we11·water aerated (Spr<.ie:. 
ncunenclature a-ttet Forbes and Richardson.) 

Su.mbeT or expe.rtments 
'.!ipecles 11~d 

!l•l 
J.rtitllrJ it h.u,,um., 

(1 21 
'11croptt rn ,alm•idu 

I Acrntcd water 
and H:80, 

I 
l~fean 

; , )l - j 0 

7 ~-1-1.3 

I 1 . 2 
7 G- 7 1 

7 :; 

I Atrnc.d water I 
HCI 

Dolled wa ter I Bolled water I 
<'O. FICI 

NaiC.'O, N:1P01 

~lean Meon M ean 

·····:: -~ jt l 
I ;,s I ; ·:: 1.. . 1·:· :· '1 ·····1. . ..... I 

- ----(-1,--------,.--8-0---6.-9-,- ~, ; .H -6---, 

Ui, rt1f1tt'r11s dol,1mtPU 7, I i. t . O 
7.87li 81-71 

i . j 

I lj) 
" 111,1n111ir,·.rr n1p~s1rls 

IU) 
.-4 l,ru,,, it C'"rJJtolcucot . • 

113) 
l.tpfjm1t 1u~oalatJ1. 

(0) 
l.t[utmit c,·yan tlllll• 

l 

·····I 

l . , 

7 . 2 

; 31 

1 S-1 l 

7 :t· 7 2 

1 3-j 1 

7 2-6.8 

7.~J o I 
' '4- 7 

I i 8-G.B 
1 r 1 6 

7 r.-7 . 4 

'i , G FL!I 

Uts tUletl wa.ier 
Nn .,CO.a 

IIC'I 

:-.t~a.a l 
7 I 

Ill L111ed water 
N~co. 

D~~:•:d "~~'lt:: /Me-an 
mium and or I EI:tre llU 

~ndlnm cblorhle~ ls:~1ec-_ sdtct@d 
Fa tal lloni.. 

\fen.It 

il- j , ,4 

l-1 . ti I 

Extreme range, 
mean in pa• 

reothesls 
Summary 

O.> 

s., (j ii 

s , ,r s.i 



385 

It eem abundantly demon trated that various fresh-,vater fishes 
will live in pll 9.0. This is shown by the work of Miss Hall on white-
fish and by the observation of Ganey ('16) in connection with the St. 
Loui city water. The re ·istance of forms other than fish, especially 
stagnant water bottom-forms may be great. Stickney ('22) found 
that a dragon-fly nymph, Libell1ila pulchella, lived in very high H ion 
concentrations. It tolerated pll 1.0 for 12 hours or more. Miss Hall 
found toad ' eggs very resistant as compared with those of whitefish. 

REACTIONS OF F1sn TO DIFFERENCES IN HYDROGEN IoN 
(ON CENTRATIONS 

A large series of experiments was conducted by the author* with 
ten specie of fish and a number of types of water; aerated well-water, 
boiled water. distilled water, rain-water, etc. The results are given h1 
Table V and in Figure 1. In nearly all cases the fishes reacted definitely 
to differences. "\ Vith any given temperature, salt content, etc., they 
usually behaved consistently, and with any one set of conditions could 
be depended on to select a given hydrogen ion concentration; but as 
conditions were varjed and the number of readings increased the results 
varied and were as represented in Table V and Figure 1. The selections 
tend to fall in two or three places, which with a larger number of read-
ings would probably be reduced to one ma.,imum. Each species will 
be seen to have a definite range which differs from every other species-
as shown by the polygons. All figures run higher with the N a2COa· 
An average of approximately twelve experiments were run with each 
species ( the exact number is given in Table V). Fishes accustomed to 
live in clear open waters, especially the minnows, select the lower hydrogen 
ion concentrations. It is evident from the range of concentrations se-
lected by them that aII these species might be found in the ame mall 
stream during non-critical periods. The order in which the species 
arrange themselves corresponds to the frequency of their occurrence in 
the bodies of \'vater mentioned (Fig. 1). 

The range selected by each species is rather wide, though with a few 
exceptions the fi hes show unmistakable evidences of reacting. ome 
of the reasons for the broken character of the curves, irregularitie , wide 
range selected, and variation from time to time are as follows: ( 1) differ-
ence in alt content of the water, both e ·perirnental differences and 
those due to differences in aeration in storage reservoirs; (2) difference 
in steepness of the gradient-a difference of 6.5 to 8.2 would not be en-
countered in so short a distance in nature. 

The following additional reactions have been estimated from pub-
lished graphs and by calculations by one of the equations of Greenfield 
and aker ( '20), which are presented on page 388. 

• 1-•ol' m i;- tbvd '! •we lhll. 111 . • ·tnl • f.al1. _·n1 . IIi t. Yo) 11 (Art. 'VT) , (l8g' :?93. 



3 6 

pH selected• 
Bluegills (Lepomis pallid,·us), Wells ('15).. . . . . . . . . . . . . . . . . . . . . . . . . . . 7, 7-7. 9 
Bullhead (Ameiurus melas), Wells ('15) . ............... . ........... , 7. 4-7. 7 
Crappie (Ponwxis annularis), Wells ('16) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 . 3-7. 4 
Roel-: bass ( Amolo1.)lites rn.pe.stris). Shelford and llee ( '11) . . . . . . . . . . 7. 9 
Golden shiner ( bram.is cryso7eiicas), Shelford and AI1ee ('11)........ 7. 7 

•pa: calculat d b.v G1·eenfi 1d aod Bak 1· equation mentioued on p. 3 5. 

M &a 1D l2 t• 1.E 1Jj 8'l !7 

Lepomis humil is 
I L-~-~Ai/.:-~~, l I I - --- --:7 ----! _J__J_,~Li~_ 

::::,. I I I_L I _!__J_LJ __ , __ ~_!_J_ s ----- ----......:.. 

1 ~--L ., 
I I I l I I I I I I I I I _! ___ /_ -------- -- LC 

'--~J__l_~j 
I J-1~-J.JJ CI 

,J__l_L!~ I .L-1__1_ - f• 

M icropterus sal mo ides 

Micropterus dolomieu 

Ambloplites rupest ri s 

Abramis cryso l eucas 

Lepomis megalot is 

Lepomis cyanell us 

Pimephales notatus 

Notropis whipplii 

Notropis cornutus 

! JJ-L.,Sl) JJ _ _L l 
j _ I I I I I I I I I I cs 

e• 

I 
:::. 
I 

...__ 

Fie. 1. • howing lhe rnng of hydrogen ion concentrations selected 
by ten spcr: ies of fish (about 12 e. periments per s pecie, . The actual 

lections an• plott u. and a broken line indicates the author's impression 
a to the probable range in which they would be fouud. Tile polygon fall 
opp1~ximately in the ord r of occunenc in swift water. Th letters 
indi ·ale occurrence in creek ( ) . mall rivers ( ), aud in lake and 
pond ( I, 1. \Yhen two 01· three letter are u ed in connection with a 
polrgon. rhe fli- t one inclic:i tes th typl' ot wat r in whi. h the p cies 
ts moi;t numHous, ond a single letter signifie thal the species is commonly 
found only in that one type of water. 

AN EXAMINATION OF CERTAl ILLINOI 
REFERENCE TO HYDROGEN lo 

\i\f ATER \ ITH 
CoNCENTRA TIO 

PECIAL 

• 

study of fish breeding-grounds was conducted by the author in 
1919-20, and the pH values observed are shown in Table VI; the oxygen, 
in Table II. There was a marked difference between the carp and 
ba s breeding-grounds; in no case were they the same either at the top 
or bottom. T he ba s breeding-grounds were characteri zed by clean sand 
bottom; the carp grounds, by dark mud. Table VI hows ha rp differ-
ences in oxya-en content at the bottom at all dates on which they were 
examined. 



3 7 

TABLE VI 
howino OJ.'YOeH i11 c.c. 11er liter and per cent. of sa.turation 011 fish breedin!}-

grounds, 1919-.W: Round Prairie, carp breedJno-grou1uls: Warner's Cut, bass 
breedi11g-grounds: Hiokory Creek. a typical un·1>0Uttted stream,; ponds at the 
head of Lake Michigan. 

July 16 Sept. 4 INov. 28 Apr. 2 June 3 Mean 
Locality 

c.c. ] % C. C. % C. c.j o/o c.c. j % c.c. j % C. C. 1 % 
I 

14.6• 1 n• 1 3.45 1 Round Prairie .. 9.75 12 1 dry dry I 5. 5 58 4.1 52 44 
Warner's Cut ... 8.0 150 dry dry l9.2 95 5.4 70 <t> .... I 7.5 105 
Illinois River ... 3.7 61 2.5 40 ... . . 6.0 77 4.8 78 4.2 64 
Pond I . . . . . . . . .... 1 ... 14.7 235 7. 8 94. .... . ... 6.9 I 110 I 9.8 146 
Pond V ....... . ... .. . 7.3 135 7.5 8 Ii:~ 70 6.4 98 
Pond VII, W ... ....... 117.8 330 8.1 96 120 11.1 182 
Pond XIV ..... . . . . . .. 12.4 230 1 6.8 1 1 . . . . . ... 6.9 1112 8.7 141 

• Not included ill the m an. t lligh on• bore wind prevented our reaching this station. The strong waves should 
have given a. 100% saturation. 

TABLE VII 
Showing the hydrogen ion concentration in different fi,S1vwaters, July, 1919, 

to Jtme, 19ZO. 

Bottom Top 
<O ..,,. 00 <O '<I' 00 

Locality rl CN C-1 M rl c-1 ~l CQ 

..; > Q) Cl ..; > Q) c:I 
>, C. s::: >, Q, A s Q) 0 C. .; Q) ;::, Q) 0 Q. ::s Q) 

rn z < "") r:n z < 
I -

Lake Micbigan .. 1 
•••• I .... ... ... 1 ... ~I .... 7.9 1.9 7.9 7.8 7.9 1.9 

Round Prairie ... 7.15 dr}' 7.3 7 .3 7. 3 7. 27 7.2 dry 7.3 7.4 7.6 7.4 
Warner's Cut ... 17.95 dry 7.9 7.4 . . . , 7.75 8.2 dry 7.9 7.5 ... 7.8 
Ill. Riv., Jefferson I 

St., Havana ... 7.58 . . . . ... 1 · .. . .... . . . . 7.7 7.4 I 1 .6 7.5 '7. 4 7.5 
Spoon River . . . . 7.4 . . . . .. ..... .... . ... 7.5 7 .'5 7 .9 . ... '7. 3 7.65 
Hickory Cr ...... 7.6 7.6 7 .5 7 .5 7.8 I 1. 6 7.9 7 .8 7 .5 7.6 '7.8 7.7 
Pond I ......... 7.55 7.4 7.9 . .. 8.0 7.7 7.6 7.8 7.9 . ... 7.9 7.8 
PondV ......... 7.7 7.3 7.5 . .. 7.8 7.6 7.0 7 .7 , 7 .6 8.0• 7.9 7.5 
Pond VII, W ..... 8.5 7.85 7.9 . . . 7 .4 7.9 8.2 8.2 7 .9 .... 7.9 8.05 
Pond XIV ...... 7.86 7.5 7.6 . . . 7.3 7.6 7.9 8.2 7.9 . ... 7 .9 17 .in 
Pond VII, E ..... 6. 6.8 7.3 . . . 7.6? 7.1 7.2 .. ·• . 7.3 . ... 7. 7 7 .4 

• 'ot included In the mean. 

The series of pond wa quite fully studied in 1909-1911 (see Bio-
logical Bulletin, volumes 21 and 22). 

Pond I at that time contained ba s, unfish, and perch. Bare sand 
breeding-bottom. 

Pond V contained perch and chub-suckers. Bare sand breeding-
bottom. 



388 

Pond VII contained chub-suckers golden shiner, bullhead, and mud 
minnow. No bare breeding-bottom. Pond VII, E, is much older than 
VII, W. 

Pond XIV contained black bullheads. No bare breeding-bottom. 
A few observations were made on pH at bottom under certain types 

of vegetation: Under white water-lilies, 6.9-7.2; under yellow water-
lilies, 7.7-7.9; under duckweed and smartweed, 6.8-7.3. 

GENERAL RELATIONS OF HYDROGEN foN CONCENTRATION 
TO OTHER FACTORS 

l. Relations of pH to dissolved oxygen.-There are nearly always 
correlations between CO2 content and dissolved oxygen, at least with a 
fairly constant 'alkalinity." A fairly constant alkalinity exists in the sea. 
Relative to the sea, McClendon ('17a) tate that "in so far as the sea 
is a closed system 0 2 varies inversely with CO2, due to the action of 
organisms, the possible error being 30 percent." He presents a chart 
showing the amount of oxygen to be expected in sea water of various 
"alkalinities, ' etc. Below the thermocline a lake in summer is a closed 
system, and in so far as alkalinity remains constant there is an in verse 
relation of 0 2 and CO2 , which is, however, by no means constant. ln 
some cases the sum of 0 2 and CO2 in c.c. per liter is a constant, but the 
relation is always an inverse one. This stability of the 0 2 and CO2 values 
probably depends upon circulation, diffusion, changes in alkalinity, etc., 
but in all the work described herein there is a direct relation between 
pH and 0 2 ( cf. Table VII and Table VI). When hydrogen ions content 
decreased, as indicated by higher pH figures, oxygen increased. 

2. Relations of pH lo ca1·bon dioxid and "alkalinity".- ln waters 
of about the same alkalinity the amount of free CO2 is as good an index 
of its suitability for fishes as hydrogen ions ( Shelford and Allee, '10). 
The amount of CO 2 means nothinCT, however, unless alkalinity be meas-
ured. The work of Greenfield and Baker ('20) shows that the H + 
ions may be calculated. The equation is 

4 CO2 X 10-7 

H+ =----+I X 10-s 
(HCQ3-) 

when CO2 is expressed in p.p.m. and bicarbonate as p.p.m. CaCOs 
but when both bicarbonate and free COi are expressed in c.c. per liter* 
the equation is 

3.5 X 10-7 CO2 

(H+) =-----+ 1 X 10-8 
(HC03-) 

"To check the accuracy of these calculations, several samples of 
water, from a variety of sources and varying widely in mineral an<t 
organic content, were examined. Bicarbonate and free carbon dioxide 

• Total must be used, I. e. bound and half-bound. 



389 

were determined according to ' tandard Ivlethods of Water Analysis'. 
The free carbon dioxide titrations were continued to a faint pink which 
was persistent for 3 min. The hydrogen ion concentration was 
determined colorimetrically, using standard buffer solutions which had 
been checked by means of the hydrogen electrode. * * * 

"In only one case [ out of 63] was the difference between the deter-
mined and calculated P 11 + greater than 0.3, and the mean variation is 
about 0.1. It will also be noted that the wider variations occurred in the 
cases of low bicarbonate content, which is to be expected from the 
assumptions made in the development of the equation. * * * 

"From their experience with the colorimetric P0+ determinations, 
the authors <lo not feel that -determination can be made much more ac-
curately than 0.2 ph+, using open tubes and ordinary methods of trans-
ferring the test sample to the tube. The effect of aeration of such un-
stable solutions as natural waters should amount to this much or more. 
It is advisable in all cases to make several determinations. 

"The formula cannot be applied to waters which are alkaline to 
phenolphthalein. An attempt was made to develop such an equation, 
but no waters naturally a1ka1ine to phenolphthalein were available for 
checking the cakulat1ons. For unusual cases of this kind and cases of 
low bicarbonate content, it may be better to use some more complete and 
more complex equation, such as has been developed by Prideaux (Proc. 
Roy. Soc. London (A) 91, 535) ." 

"Equations are developed for calculating H+-ion concentration, in 
which the carbon dioxide and bicarbonate are expressed in the manner 
in which they are ordinarily determined. These equations are less ac-
curate with low bicarbonate concentrations, and do not apply to waters 
alkaline to phenolphthalein." 

TABLE VIII 
Comparison of calculated H ion concentr-attons with those determ,ined- color£. 

·1netrical1y. (Excerpts froni a table by Greenfield, and Baker.•) 

Res. Free Bicarb. 
pb+ Ph + on CO: as Error Source of sample p.p.m. det. calc. evap. p. p. m. CaCO, I 

375 
I 

0.0 154 7.9 I 8.00 I -.10 Vermilion River 
376 7 . 0 162 7.5 7.64 .04 Vermilion River 
375 15.6 152 7.2 I 7.28 .08 Vermilion River 
375 16.5 152 

I 
7.1 7.29 .19 Vermilion River 

376 29.6 162 6.9 7.06 .15 Vermilion River 
3 4 I 6.0 100 7.3 7.47 .17 Kankakee River 
384 14.0 100 7.1 7.1 .08 Knnlcakee Rtver 
384 26.0 96 6.7 I 6.94 .24 Kankakee River 
206 15.0 44 6.9 6.83 -.07 Ohio River 
206 29.0 44 6.5 6.52 .02 

I 
Ohio River 

206 30.0 H 6.6 6.52 I .02 Ohio River 
226 11.0 90 7.3 7.23 - .07 Miss. River 

• Jour. loduetr. and Eng. Chem., Vol. 12 (No. 10), p. 991. 1920. 



390 

TABLE IX 
alues of pH corresponding to varying concentrations of free carbonic acid, and, 

bicarbonates cazc-,tlated by the 'l'1tass-law quation. (Ta,ble prepared by G1·eenfield, a,11a 
Baker.) Near-neutrality in boid,..face figures. V. E. . 

HC00 

p.p.m. 
CaC03 

10 
20 
40 
60 
70 
80 
90 

100 
120 
140 
160 
180 

0 
--
8.00 
8.00 
8.00 
8.00 
8.00 
8.00 
8.00 
8.00 
8.00 
8.00 
8.00 
8.00 

1 --
7.30 
7.52 
7.70 
7.78 
7.80 
7.82 
7.84 
7.85 
7.87 
7.89 
7.90 
7.91 

2 4 --,_ 

7 .05 6.77 
7.30 7 05 
7.52 7.22 
7.63 7.43 
7.67 7.48 
7.70 7.52 
7.72 7.56 
7.74 7.58 
'7. 78 7.63 
'7 .80 7.67 
7.82 7.70 
7.84 7.72 

co!,, parts per million 

6 8 10 12 14 16 20 30 40 ----,_ -- --
6.60 6.48 6.39 6.31 6.24 6.19 6.09 5.92 5.79 
6 89 6.77 6.68 6.60 6.54 6.48 6.39 6.21 6.09 
7.15 7 05 6 96 6.89 6.82 6.79 6.68 6.51 6.39 
7.30 7.20 7.12 7 04 6.98 6.93 6.84 6.68 6.56 
7.35 7.25 7.17 7.10 7 04 6.99 6.90 6.74 6.62 
7.40 7.30 7.22 7.15 7.10 7 05 6.96 6.80 6.68 
7.43 7.34 7.26 7.20 7.14 7.09 7 00 6.84 6.73 
7.47 7.38 7.30 7.24 7.18 7.13 7.05 6.89 6.77 
7.52 7.44 7.36 7.30 7.25 7.20 7.11 6.96 6.84. 
7.57 7.48 7.41 7.35 7.30 7.25 7.17 7 .02 6.91 
7.60 7.52 7.46 7.40 7.35 7.30 17 .22 7.07 6.96 
7.63 7.56 7.49 7.43 7.39 7 .34 7 .26 7.12 7 00 

3. Relations of pH to pntrescibility and pollution.- nder ordinary 
conditions putrescibility accompanies high free CO2• This is determined 
partially, however, by alkalinity but with a constant alkalinity, CO2 

usually varies directly with the putrescibility. In the Illinois River, with 
an alkalinity of 100 to 140 p.p.m. of CaC03 , samples show pH value 
up to 7.2 with 0 2 at p.p.m. several days after being shipped from 
point above Chillicothe. Here the calculated values were about 3 points 
too high, which was probably because the formula gives incorrect results 
in the presence of free ammonia. The low CO2 values are surprising for 
the 111 inoi River.* Farther clown the tream, at Chillicothe, eptember 
6, 1919, I found pH 6.8 at the surface, and 6.9 at the bottom with green 
algae in evidence, and 0 2 .9 c.c. per liter at the bottom, 20 feet from the 
west hore. On eptember 3, at Liverpool we found pH 6.9 at the 
bottom and 0 2 .96 c.c. per liter, with the bottom fauna destroyed. The 
fact that CO2 values appear to be small in this stream makes the calcu-
lated pH values too high. In this ca e, therefore, pH determinations 
would appear more significant than CO2 determinations. Calculated 
values should not be used except where determinations are not practi-
cable-e. g., in working over old data. Too many waters contain ammonia 
or other disturbing substances. 

• ·w eston and Turner· '17) fonud bigh O!l in n polluted stream, but fail to give the 
alkalinity. 



391 

D1scu s10 A. D Su.n.IARY oF RESULTS 

Present-day methods of determining hydrogen ion concentration in 
detailed manner have been developed by biochemists, bacteriologi ts. and 
oceanographers; not by physical chemi t , as one might expect. Its im-
portance in the study of fishes came to the author's attention in cour e 
of experiments performed in 1911 (Shelford and llee). Thi work 
was followed by that of several students. The important papers by 
Palitzsch and orensen-see bibliography in Clark ('20)-\ ere over-
looked by the writer until 1916. These important papers appear not to 
have been appreciated until 1915. The aid and advice of physical chemists 
was sought, but these inve tigator · regarded differences in pH of les 
than unit alue-e. g. between 8.0 and 9.01-of little importance, though 
they made important contributions to the subject. The turning-point of 
phenolphthalein ,va stated by \Vashburn ('15) to be pH 9.0; its turning-
point in , ater analysis ( faint pink lasting 3 minute ) i pH 8.0; all 
depends upon the method employed. 

The swmnary of Wells' paper ('15) previou ly referred to, hould 
read about a follows: 

1. Hydrogen ion concentration is an important factor in determin-
ing the reaction and resistance of fishes. 

2. Jo t £re h-water fishes select slightly alkaline .water in a gra-
dient, but when offered a gradient from the turning-point of phenol-
phthalein to higher hydroxyl ion concentrations (pH 8.0-9.0) the more 
alkaline end is selected. 

3. The optimum CO2 varie from O (pH 8.0) for bluegills to 6 c.c. 
per liter in water with about 200 part per million alkalinity (pH 7.4-7.6) 
for . unfi he and crappie~. Optimum ulphuric acid wa not determined 
because of carbonates in the di tilled water u ed. 

4. The distribution of plankton in \Viscon in and ew York lakes 
shows fewe t animals in the stratum at the turning-point of phenol-
phthalein pH 8.0), uggesting a negative reaction to water with pH 8.0. 

- _To good results were obtained with once-di tilled water becau e 
of carbonate pre ent. 

The chief occa ion for critici m of \Veil · paper is the con fu ion 
relative to neutrality. A r petition of hi experiments, which wa one of 
the purpo es of the pre ent paper, and a close study of his work shows 
that he regarded the turning-point of phenolphthalein (pH 8.0) a neutral, 
or "near enough" to it. The avoidanc of pII 8.00 when this accom-
pani cl hi,e-hcr alkalinity. a foun I by Vvell , ha been confim,ed by 11iss 
Hall in case of young whitefish. The table con tructed by \Vell showing 
the avoidance of pH 8.0 by plankton ( r ew York and Wisconsin lakes) 
is correct in thi re pect, but the column to the right and left of the 
phenolphthalein neutrality are by no means comparable in hydrogen ion 
concentration. The statements by \\Tell relative to CO:i, together with 
tho e in the paper of 1911 by helforcl ;mcl _\llee, are correct only for 
the "alkalinitjes" u en, which varied around 100 p.p .m. of CaCO3 or 
120 p. p. m. of HCOa. everal tatement quoted from \Velis ('l 5) and 



392 

Washburn ('15) in my chapter in Ward and hippie's "Fresh Water 
Biology" are erroneous. Shull's "Principles of nimal Biology" (page 
277) includes statements of a similar nature which should be corrected. 
These various statements relative to CO2 optima, etc., are not to be re-
garded as errors when made; later evidence has merely shown that hy-
drogen ion concentration is a better index than CO2 • 

There are a number of general reasons for determining hydrogen 
ions, such as the close regulation of their concentration in the animal 
body and their apparent great physiological importance in vertebrates. 
The writer does not, however, wish to give the impression that he means to 
advocate the use of pH determinations to displace any other determina-
tions now in common use unless it be CO2, and not even in this ca e until 
further investigations have been conducted. 

CONCLUSIONS 

Hydrogen ions should be determined for the following specific 
reasons: 

1. The effect of various concentrations of oxygen are modified by 
the hydrogen ions present, particularly the ill effect of low oxygen on 
survival and development, which is increased by the high hydrogen ion 
concentration which accompanies it in waters of low alkalinity. In a 
general way the hydrogen ion concentration varies inversely with the 
oxygen content in bodies of water with stable alkalinity ( e. g., see Tables 
VI and VII and Graph 1 ) . 

2. With the same oxygen content, different hydrogen ion concen-
trations have definite effects on the rate of development and time of 
survival of aquatic animals. 

3. Many animals react definitely to hydrogen ion concentration 
( fishes, crayfishes, Entomostraca) in a manner similar to their reactions 
to temperature. Each species tolerates a rather wide range with a 
fairly de.finite optimum. 

4. The determination of hydrogen ions is to be preferred to CO2 

determinations because it is the free hydrogen ions which are most effec-
tive, and, as shown in Table IX column 6, for example, while the CO2 

remains constant ( 6 p.p.m.), hydrogen ions concentration changes by 
tenfold (pH 6.6 to 7.63) owing to difference in alkalinity. 

5. Colorimetric determinations of pH are about as accurate as CO2 

determinations and can be made on the spot very quickly. With some 
simple device for collecting ( see Richardson's Fig 61 page 372, Vol. 
XIII of this series), aeration can be reduced and very small quantities 
of water used as contrasted with the usual 250 c.c. samples. 

6. The distribution of plants and animals is correlated with hydro-
gen ion concentration. 



c,c. ppm 
c0i caco, ·-120 272 

110 · 249. 

100 221 

90 204. 

80 181 

70 1sa 

60 136. 

50 Ill 

40 go.a 

30 68.1 

20 45.4 

10 221 

0 51) 

10~ L 100 

! 

5.5 
3.20 

. 

393 

-

)( ., 
/ 

I~ 
k{r1 

6.0 
1.00 

6.5 
320 

)( 

X 

I 

lO 
.100 

/4 

)t 

')( )t' ..... 
X ·, 

X 
)C 

)( 

J 

X 

)(')( )C 
X 

X 

)( 

:,( 
,< 

I 
I 

T " X 

I 
x I)( 

)(;& 

-
)t' 

ZS 
.032 

X 

ao 
.010 

GJ!.U>B. 1. Showing the ron~e or hydrogen ion concentration accompanying a 
trace or oxyf{en In WI ·com•ln hikes. ulculated from data obtained by 

Birge and Judny ( '11). At the Jett, alkalinity ts shown a co~ o.nd CaCOa: below, hydrogen tons, as pH and ns 
grnms per million liters. 



394 

CK OWLEDC 1E TS 

The writer especially indebted to Mr. R. E. Greenfield, of the 
Illinois State V ater urvey for numerous suggestions throughout the 
course of the inve tigation. The paper would have been deficient at 
several points but for his a sistance. cknowledgments are also due 
Me r . R. E. Richardson and G. C. Baker for advice in variou con-
nections. I am al o indebted to fr. Ralph Bradford, Chief State ish 
and Game Warden, for the blacl- bass used; to the nited tates Fisheries 
Station at Fairport; Iowa, for bluegills furnished me; and to Dr. F. W. 
Mahlman, who kindly gave me the r ult of determinations of shipped 
Illinois River water. 

Birge, E. 
'11. 

BIBLIOGRAPHY 

., and Juday, Chaunc 
Inland lakes of Wisconsin. The dissolved gases of the water 

and their biological significance. Bui. XXII ( ci. er. 7), Wis. 
Geol. and Nat. Hist. Surv. 

Clark, W. M. 
'20. The determination of hydrogen ions. Baltimore. ( Contains 

an extensive biblioaraphy.) 
Forbes, . and Richard on, R. E. 

08. The fi he of Illinoi . ol. III, at. Hist. urv. Ill. Sec. ed., 
1920. 

Garrey, W. E. 
'16. Resistance of fre h-water fish to changes of osmotic and chem-

ical conditions. m. Jour. Physiol. 39: 313-329. 

Greenfield, R. E., and Baker, G. C. 
'20. Relation hip of hydrogen i n c ncentration of natural waters 

to carbon di :xide content. Jour. Indu tr. and Eng. hem. 12: 
989-992. 

Hall <la R. 
'21. The effect of oxygen and carbon dioxide on the development 

of certain cold-blooded vertebrates. (To be published.) 
Je'> ell, M. E. 

120. The quality of water in the angamon River. Bul. Ill. tate 
\i ater urv. 16: 230-246. 

'22. The fauna of an acid stream. Ecology, 3: 22-26. 
J uday Chauncy 

'20. Behavior of the larvae of Corethra pltlzctipennis Say. Anatom-
ical Record, 17: 340. 



395 

l\IcClendon, J. F. 
'17. The use of the an Slyke CO2 apparatus for determination of 

total CO2 in sea water. Jour. Biochem. 30: 259-263. 
'1,a. The tandardization of a new method for the deternunation 

of hydrogen ion concentration, C 2 tension and 0 2 content of 
ea water. Ibid., 265-288. 

Matthews, . P. 
'15. Physiological chemistry. New York. 

Powers, E. B. 
'21. The variation of the condition of sea water, especially hydro-

gen-ion concentration, an<l it relation to marine organisms. Pu-
get ound Biol. tation Pub. 2: 369-385. 

'21 a. Experiments and observations on the behavior of marine fishe 
toward hydrogen-ion concentration of sea wate1· in relation to 
their migratory movements and habitat. Ibid., 3 : 1-22. 

'22. The physiology of the respiration of fishes in relation to the 
h~drogen ion concentration of the medium. Jour. Gen. Phys., 
3. 305-317. 

Richardson. R. E. 
'21. The mall bottom and bore fauna of the middle and lower 

Illinois River and its connectino- lakes, Chillicothe to Grafton; 
it valuation; its sources of food supply; and it relation to the 
fishery. Bul. Ill. Nat. Hist. urv., 13 : 372, Fig. 6. 

Stickney, F. 
'22. The relations of nymphs of Libel!ula pnlchella to acid and 

temperature. Eco)oo-y, 3 : 250-254. 
\V'ashburn, E. V\T. 

'15. Introduction to the principles of physical chemistry. New 
York. 

\\Tells, M. 1. 
'15 Reaction and resistance of fishes in their natural environment 

to acidity, alkalinity, and neutrality. Biol. Bui. 29: 221-257. 
Weston, R. S., and Turner, C. E. 

'17. tudies on the dige tion of a sewage-filter effluent by a small 
and otherwise unpolluted stream (Coweeset Cr.). Contr. San. 
Res. Lab. and ewage Exper. tation, 10: 1-96. 

Wherry, E. T. 
'21. Soil acidity and a field method of measuring it. Ecology, 1: 

160-173.