Section VI

EXPERIMENTS WITH AROCLOR 1254

Wide-mouth jars, 30 cm high and 14 cm wide, were used to produce
32 of Aroclor 1254 emulsion per batch. Mechanical considerations
concerning the proper physical agitation of Aroclor 1254 and water
made it necessary to use 250 ml of Aroclor 1254 in the jar to submerge
the blades of the stirrer. Agitation for 0.5 hr at 60°C and 1,800 rpm
produced a cloudy emulsion which was allowed to settle for 48 hrs, when
the range of concentration was found to be 1-20 mg/% and the emulsion
became almost clear. This emulsion is referred to as type-I. A second
homogenization was carried out by transferring to a jar identical to the
one used previously volumes of type-I emulsion to produce emulsions of
10-300 ug/2, and stirring 1 hr at 25°C and 1,800 rpm. This emulsion is
referred to as type-II. Type-III emulsions were prepared by taking an
appropriate volume of type-I emulsion, adding it to a stainless steel
blender jar to make a total volume of 500 ml, and homogenizing at high
speed for 5 min.

All centrifugations were performed in a Beckman Model L3-50 ultra-
centrifuge at 20°C using SW 50.1 and SW 25.2 rotors.

The extraction procedure was that of Schoor (1973), with modifica-
tions of the ratio of water to hexane. Evaporation was carried out by
placing the hexane extracts in a water bath at 35°C and allowing a gentle
stream of air to blow across. This method was found superior to dis-
tillation in percentage recovery and time involved. When the extract
volumes had to be reduced to less than 10 ml, dried, pre-purified nitrogen

was used instead of air.

13


Section KX

REFERENCES

Bagley, G. E., and E. Cromartie. Elimination Pattern of Aroclor 125
Components in the Bobwhite. J. Chromatogr. Sci. 75:219-226, 1973.

Bowman, M. C., F. Acree, Jr., and M. K. Corbett. Solubility of Carbon-14
DDT in Water. J. Agric. Food Chem. 8(5):406-408, Sept. 1960.

Colley, N. R., J. M. Keltner, Jr., and J. Forester. Mirex and Aroclor
1254: Effect On and Accumulation by Tetrahymena pyriformis Strain W.
J. Protozool. 19(4):636-638, 1972.

Duke, T. W., J. Lowe, and A. J. Wilson, Jr. A Polychlorinated
Biphenyl (Aroclom¥ 1254) in the Water, Sediment, and Biota of Escambia
Bay, Florida. Bull. Environ. Contam. Toxicol. 5(2):171-180, 1970.

Hansen, D. J., P. R. Parrish, J. I. Lowe, A. J. Wilson, Jr., and

P. D, Wilson. Chronic Toxicity, Uptake and Retention of Aroclor
1254 in Two Estuarine Fishes. Bull. Environ. Contam. Toxicol. 6(2):
113-119, 1971.

Hutzinger, O., S. Safe, and V. Zitko. Polychlorinated Biphenyls.
Analabs Res. Notes. 12(2):1-11, July 1972.

Iwata, Y., W. E. Westlake, and F. A Gunther. Varying Persistence
of Polychlorinated Biphenyls in Six California Soils Under Laboratory
Conditions. Bull. Environ. Contam. Toxicol. 9(4):204-211, 1973.

Lowe, J. I., P. R. Parrish, J. M. Patrick, Jr.,.and J. Forester.
Effects of the Polychlorinated Biphenyl Aroclor/1254 on the American
Oyster (Crassostrea virginica). Mar. Biol. 17(3):209-214, Dec. 1972.

Nimmo, D. R., R. R. Blackman, A. J. Wilson, Jr., and J. Forester.
Toxicity and Distribution of Aroclo 1254 in the Pink Shrimp (Penaeus
duorarum). Mar. Biol. 11(3):191-197, Nov. 1971(a).

Nimmo, D. R., P. D. Wilson, R. R. Blackman, and A. J. Wilson, Jr.
Polychlorinated Biphenyl Absorbed from Sediments by Fiddler Crabs
and Pink Shrimp. Nature 231:50-52, May 1971(b).

Pauling, L. Nature of the Chemical Bond. Ithaca, Cornell University
Press, 1940. 164 p.

Schoor, W. P. In Vivo Binding of p,p'-DDE to Human Serum Proteins.
Bull. Environ. Contam. Toxicol. 9(2):70-74, 1973.

Walsh, G. E. Insecticides, Herbicides and Polychlorinated Biphenyls
in Estuaries. J. Wash. Acad. Sci. 62(2):122-139, 1972.

Zitko, V. Polychlorinated Biphenyls Solubilized in Water by Nonionic
Surfactants for Studies of Toxicity to Aquatic Animals. Bull. Environ.

Contam. Toxicol. 5(3):219-226, 1970.

30


A REGION OF MAXIMUM c
REGION WHERE SMALL STABILITY REGION WHERE LARGE
AGGREGATES COALESCE AGGREGATES PRECIPITATE

INCREASE
IN
RELATIVE
STABILITY
OF
EMULSION

INCREASE IN AGGREGATE DIAMETER —————————_———_

Figure 1, Theoretical relative stability of different sizes of aggregates in
an emulsion during a given time interval,


Table 1, EFFECT OF STORAGE TIME ON AMOUNT OF AROCLOR 1254 REMAINING
IN THE WATER PHASE

ug/2 Aroclor 1254

Time (days) Type I Type IT
i} 2300 301 50.2
2 286
5 115 23.6
6 113 11.3
8 112
9 123
13 97 98.5
15 502
19 483 87 54.7 6.7
20 48.1
21 44.5
23 78 7.1
26 6.5
28 428 7.7
33 355 7.4
34 350
4l 15.5
43 280 6.8

17


Sections

I

Ir

IIL

Iv

VI

VIL

VIIT

IX

CONTENTS

CONCLUSIONS

RECOMMENDATIONS

INTRODUCTION

THEORY

MODEL

EXPERIMENTS WITH AROCLOR 1254

RESULTS

DISCUSSION

REFERENCES

iii

Page

10
13
15
27

30


of isomers in the water phase shows a loss of the lower chlorinated
biphenyls on standing (Tables 7 and 8). This loss was accounted for
in all cases by adsorption on the stainless steel centrifuge tubes,
the "lost" lower chlorinated biphenyls always being found in the
adsorbed fraction. Thus, at least from water emulsions of Aroclor 1254,
loss of the lower chlorinated biphenyls is due to their relatively greater
affinity for surfaces.

The published values for solubility of Aroclor 1254 in fresh and
salt water of 2-3 mg/g and 1-1.5 mg/g, respectively (Zitko, 1970),
appear much too high. A conservatively high estimate based on my ultra-
centrifugal experiments indicates the average solubility of the isomers
to be less than 0.1 ug/2 for fresh water and approximately 0.04 pg/2
(calculated from Table 6) in water containing 30 g/g NaCl. It is extremely
difficult, in my opinion, to obtain an absolute value for the true solubility
of the average molecular weight isomer of Aroclor 1254. The problem lies
in the fact that at low concentrations, long centrifugation times (in excess
of 12 hrs at 243,000 x g (max.) theoretically are necessary to eliminate
aggregates from the emulsion. At the low concentrations necessary to
eliminate undesirable stirring back after completion of the centrifugation
(Bowman et al., 1960), adsorption on the walls of the stainless steel
centrifuge tubes (67% at 2 yg/2 for 2 hrs, Table 4) makes it all but
impossible to employ ultracentrifugation for extended periods of time.

It appears that at least in the case of type-III emulsions the adsorp-
tion from water emulsions containing 0 and 30 g/% NaCl was the same

(Table 5), although the rate of sedimentation was quite different. The


A Hewlett-Packard Model 5700 gas chromatograph with a linear
electron-capture detector (yi) was used for quantitative determina-
tion of the Aroclor 1254. The linearity of this detector eliminated
use of different standards at each attenuation or reduction in volume
of the sample, either being very time consuming and subject to errors.
An OV-101 column (2% OV-101 on Gas Chrom Q, 100-120 mesh) was operated
at 195°C with the detector at 300°C and the argon-methane (10:1) carrier
gas at a flow rate of 25ml/min. Except where noted, quantitation was
performed by comparing total peak heights of sample and standard.

To determine the amount of Aroclor 1254 adsorbed on walls of
the 34 ml stainless centrifuge tubes, the water phase was decanted and
any adhering droplets removed with a disposable pipet. Since acetone
injected with the sample was detrimental to the chromatographic column,
a sonic probe and hexane were used for removal of Aroclor 1254 from the
walls of the tubes. This was necessary because the thin layer of water
remaining on the walls shielded the Aroclor 1254 and prevented it from
being desorbed into the hexane phase. Sonification emulsified the water
at the boundary layer, thus allowing the hexane to contact the adsorbed

Aroclor 1254.

14


At an 85% total recovery the following distribution was found:

Acetone extract of tubes 66%
Hexane rinses of tubes 18%
Top 50 ml water phase 5%
Bottom 10 ml water phase 11%

The low recovery (85%) was probably due to incomplete extraction
of the tubes in spite of refluxing with acetone.

Polyallomer® centrifuge tubes were tried next. When 180 ml
of 286 ug/% type-II emulsion were centrifuged in 60 ml Polyallomer
tubes for 60 min. at 107,000 x g (max.) the following distribution
was found:

Acetone extract of tubes --

Hexane rinses of tubes 22%
Top 25 ml water phase 5%
Bottom 35 ml water phase 6%

These percentages were based on the total amount of starting material,
i.e., assuming 100% recovery instead of the 85% in the case of
the polyacetate tubes. Extraction of the Polyallomer tubes by reflux-
ing with acetone produced too many interfering peaks on the chroma-
togram, making complete recovery calculations impossible. Direct
adsorption on Polyallomer tubes was achieved by permitting type-II
emulsions to sit undisturbed in the tubes. Table 3 shows the outcome
for two different concentrations.

To permit recovery and study of the material adsorbed on surfaces,

34 ml stainless steel centrifuge tubes were used for static tests,

20


91

AROCLOR 1254
STD-1 (0.4 ng/ul)
5.9ul
X 32

f f AROCLOR 1254
/\ Water Extract

5.4 ul

x4

Vz = 20.0mi

Figure 3. Typical gas chromatograms (see text for

detailed information,


explanation for this lies in the fact that the size of the Aroclor 1254
aggregate is much larger in the presence of salt and, while this is not
apparent at 1 x g, the larger aggregates are removed more quickly from
the salt-containing emulsion during ultracentrifugation. This agrees
very well with my hypothesis that a larger aggregate is more stable

under the given conditions and in the presence of salt, which is conducive

to greater solute-solute (aggregate-aggregate) interaction.

29


The rate of sedimentation during centrifugation is described by:

dx _ 21? (p-p9)u*x (4)

dt 9n

where (t) is time in seconds to reach equilibrium. Integration yields:

_ 242 (p-po)w2t

In x2 - ln x, = (5)
9n
The radius of a spherical particle is then given by:
9n(1n x9-In xq) 1/2
xy =|
2(p-0.,)w2
(p-po)w*t (6)
where w = 0.10472 (rpm) rotor

n = g/cm/sec

p = g/cm3
x = cm
t = sec

Knowing the radius of a particle or assuming a radius, the time necessary
to remove the particle from an emulsion is given by:

9n(1n xg-1n x1)

t
2(p-09) Fw? ”

11


Section II
INTRODUCTION

Laboratory experiments designed to determine the effects of
chemicals on aquatic organisms require that the tests be conducted
under conditions which reproduce those present in nature as closely
as possible. In order to accomplish this in a precise and scientific
fashion, the physical state of a compound in an aqueous dispersion
must be known. Convenience, time and other factors have in the past
often led to the use of techniques in the laboratory which do not take
into consideration that the solubility characteristics of a compound
may possibly affect the toxicity, necessitating extrapolation from an
apparent toxicity established in the laboratory to an expected toxicity
under field conditions. In many instances, the practice of using extra-
polation in scientific investigations is necessary and has proven to be
a valuable tool when certain conditions cannot be met. However, the
range through which the extrapolation is carried out must be chosen
with great care, because without sufficient experimental and theoretical
justification, a resulting extrapolation in this light may well prove to
be unrealistic. Since natural water conditions represent a multi-
component system, any attempt to quantitatively understand it must be
preceded by a study of the system under ideal conditions. While the
knowledge thus gained may or may not be of consequence in direct appli-
cation, it, nevertheless, provides a more precise scientific basis for
choosing valid limits for extrapolation.

The physical state of a compound in water is not a simple and

straightforward phenomenon, even given the idealized conditions of a


Table 8, DISTRIBUTION OF ISOMERS IN THE ABSORBED FRACTION OF AROCLOR 1254
TYPE ILI EMULSION ON STANDING IN STAINLESS STEEL CENTRIFUGE TUBES

% Peak heights 1

Peak number”

NaCl hrs in water phase adsorbed
(g/2) tube (ug/2) (ug) 1 2 3 4 5 6
0 2 47.4 0.122 149 127 135 130 98 100
30 1 46.9 0.075 144 121 129 129 105 100
0 22 39.7 0.190 139 118 113 122 127 100

1¢ompared to standard Aroclor 1254 (Fig. 1).

on the relative heights of peak 6.

Calculations are based

Peak numbers are shown on the chromatogram in Fig. l.

26


It can be seen that even at 50% adsorption from a 3 g/g emulsion only
about 1% (maximum) of the available surface area is occupied, and
surface saturation was not a factor.

The amounts of Aroclor 1254 in the form of emulsions of type-II
and type-III adsorbed on the walls of the stainless steel centrifuge
tubes are shown in Table 5. There is a difference in adsorption of the
two different types of emulsion in the absence of NaCl. At least for
type-III emulsions, the introduction of 30 g/% NaCl appears to have no
effect on the amount of Aroclor 1254 adsorbed. However, centrifugation
reveals a difference in the size of the aggregates formed in the presence
of NaCl, as shown in Table 6.

In comparison with an Aroclor 1254 standard, the relative distri-
bution of the isomers in emulsions of type-II and III is quite different,
as shown in Tables 7 and 8. However, in all cases the adsorbed Aroclor
1254 had a higher percentage of early eluting (gas chromatography) isomers

than did that which remained in solution.

23


ACKNOWLEDGMENTS

The author thanks Messrs. D. Lamb and W. Burgess for assistance with the
analytical work and Dr. Ralph Birdwhistell, Dean, School of Chemistry,
University of West Florida, for reviewing the manuscript.

Aroclo® 1254 is a registered trademark of the Monsanto Company, St. Louis,

Missouri.


TABLES

Effect of storage time on amount of Aroclor 1254 remaining
in the water phase.

Isomer distribution of Aroclor 1254 type II emulsion after
standing for various periods of time in 3% glass bottle.

Adsorption of Aroclor 1254 type II emulsion on Polyallomer
centrifuge tubes on standing.

Adsorption of Aroclor 1254 on stainless steel centrifuge
tubes as a function of time and concentration.

Adsorption of Aroclor 1254 on stainless steel centrifuge
tubes.

Centrifugation of Aroclor 1254 in water of varying salinities
at 69,000 x g (max.).

Distribution of isomers of Aroclor 1254 type II emulsion
on standing in stainless steel centrifuge tubes.

Distribution of isomers in the absorbed fraction of Aroclor

1254 type III emulsion on standing in stainless steel
centrifuge tubes.

iv

21

21

24

24

25

26


The following are particle size limits calculated using equation
(6) for given centrifugation times, with n = 8.94 x 1073 g/sec/cn, R= 6.7 cm,

X_ = 15.3 cm, p - ®, = 0.508 g/cm3 at 25,000 rpm.

Time (hrs) Radius of particle (nm)
1 16.3
2 11.5
3 9.3
4 8.1
6 6.6
8 5.7

The following are particle size limits calculated using equation (6)
for given centrifugation times, with n = 8.94 x 1073 g/sec/cm, x7 6.00 cm,

X) = 10.73 cm, p - p= 0.508 g/cm3 at 45,000 rpm.

Time (hrs) Radius of particle (nm)
1 7.6
2 5.4
3 4.4
4 3.8 (208,000 g/molel;
636 molecules)
6 3.1
8 2.7
12 2.2 (40,000 g/molel;

124 molecules)

average molecular weight Aroclor 1254 = 327 g/mole (Hutzinger et al.,
(1972).

12


Section I]
RECOMMENDATIONS
This study shows, both theoretically and experimentally, that in
so far as physical interactions are concerned, emulsions differing in
degree of dispersion and stability can be formed, depending on the method
of preparation and subsequent treatment. Consequently, the following
questions should be answered before conducting bioassays in disperse
aqueous systems:
(a) What are the solubility characteristics of the compound
under investigation?
(b) To what extent are these characteristics related to
field conditions?
(c) How can the solubility characteristics and field
conditions be best simulated in the laboratory?
Such information would undoubtably result in more precise data on acute
toxicity as well as long-term effects regarding aqueous bioassay of

water-insoluble test compounds.


Table 2, ISOMER DISTRIBUTION OF AROCLOR 1254 TYPE II EMULSION AFTER
STANDING FOR VARIOUS PERIODS OF TIME IN 3% GLASS BOTTLE

Total % Peak Height!
Time conc.

(days) (ug/2)t Peak Numbers”
1 2 3 4 5 6 7
2 286 76 93 95 95 98 104 100
9 123 79 78 89 94 98 99 100
13 98.5 79 79 93 98 99 96 100
19 54.7 72 75 85 93 91 95 100
20 58.1 64 70 80 89 92 94 100
21 44.5 61 65 82 90 99 93 100
41 15.5 37 41 56 70 77 88 100
21 13.4 16 27 44 55 76 87 100
33 - 3.6 12 21 39 45 64 82 100
38 1.6 9 10 31 46 63 100

(3.4 ppm) (41) (80) (87) (100)

loalculations are based on the relative height of peak 7 (see below).

Peak numbers are shown on the chromatogram in Fig. 1.

18


Ecological Research Series

Theoretical Model and Solubility

Characteristics of Aroclor’ 1254 In Water:

Problems Associated With Low-Solubility Compounds
In Aquatic Toxicity Tests

ED S74
ss "5

SZ

aS
74 prot?

nnodlaiys
NV AGENCY

KS
(e)

National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency

Corvallis, Oregon 97330


Section IV
THEORY

To explain and predict the characteristics of water-insoluble sub-
stances at low concentrations, an attempt is made here to redefine the
basic principles underlying a disperse system. No attempts have been
made to include in the definition the somewhat obsolete and often vague
definitions of emulsions, suspensions, colloids, etc. The characteristics
ascribed to each becoming readily apparent as the theoretical treatment
of the proposed model continues.

In this paper, an ideal or true solution is defined as a solute dis-—
persed in a solvent so that any single molecule of solute is surrounded
by enough solvent molecules to insure that at any instant all solute mole-
cules are distributed statistically equidistant, assuming a dilution at
which interactions between solute molecules become negligible.

The ideal solution, under the conditions described, is represented
by the presence of single solute molecules. Solute aggregates consisting
of two or more molecules may represent a deviation from the ideal solu-
tion because, at least theoretically, these aggregates could consist of
any number of molecules whose behavior would not necessarily coincide
with that of a single molecule. For each solute and a single solvent,
there is assumed to exist amongst all aggregates a maximally stable
aggregate which, due to its nature, remains statistically equidistant
from all other aggregates for at least a certain period of time. The
stability of this aggregate depends solely on the molecularly char-
acterized interactions at the solute-solvent interphase and on tem-

perature.


two-component system - a single solute and a single solvent. A
definable system should, however, be the starting point of any
investigation aimed to scientifically arrive at data which lead to
a quantitative understanding of the behavior of a compound in water.
With this data a more precise attempt can be made to extrapolate from
a system employed in the laboratory to the obviously much more complex
system present in natural waters.

The purpose of this work is to provide a working theory on the
behavior of substances of low water solubility and to test this theory

by investigating the solubility characteristics of Aroclor 1254.

&


Table 7, DISTRIBUTION OF ISOMERS OF AROCLOR 1254 TYPE II EMULSION
ON STANDING IN STAINLESS STEEL CENTRIFUGE TUBES
% Peak heights
Peak number”
Storage Hrs in
(days) tube ug/2 1 2 3 4 5 6
1 0 310 water 93 90 98 99 98 100 100
5 0 115 water 53 71 73 91 98 98 100
2 97 water phase 49 67 69 83 100 100 100
2 12 adsorbed 96 106 103 127 119 100 100
8 0 112 water 51 67 71 82 96 97 100
2 102 water phase 48 66 68 79 98 98 100
2 8.0 adsorbed 69 82 85 104 107 100 100
13 0 97 water 47 64 68 81 97 98 100
2 86 water phase 43 59 66 78 92 96 100
2 6.1 adsorbed 47 68 77 94 101 98 100

lcompared to standard Aroclor 1254 (Fig. 1).
on the relative heights of peak 7.

Peak numbers are shown on the chromatogram in Fig. 1.

25

Calculations are based


Table 5, ADSORPTION OF AROCLOR 1254 ON STAINLESS STEEL CENTRIFUGE TUBES

ug Aroclor 12541 adsorbed

Type II Emulsion Type III Emulsion

Time

((hrs) 0 g/% NacL 30 g/% NaCl 0 g/% NaCl
0.5 0.19 0.09

1.0 0.30 0.10 0.10

2.0 0.33 0.14 0.14

4.0 0.42 0.19

19 0.39

22 0.45

Inata adjusted to 4.00 ng total starting amount.

Table 6. CENTRIFUGATION OF AROCLOR 1254 IN WATER OF VARYING
SALINITIES AT 69,000 x g (MAX.).

ug/2 Aroclor 1254

remaining in water phase

g/% NaCl
Time (hrs) 0 15 30
0.5 13.9 7.1 6.0
1.0 12.5 6.6 4.9
2.0 7.2 4.6 2.9

lstarted with 50 ug/2 Type IIL emulsion.

24


Section I
CONCLUSIONS

An extrapolation from the theory presented suggests that the use
of "carriers" be continued with caution, because of two independent
effects that may be present. One effect can most simply be described
as an alteration of the aggregate-solvent interactions by "carriers"
forming transition-like links between aggregates and solvent molecules.
In such a fashion, solute aggregates are surrounded by "carrier"
molecules, thus enhancing the ability of the aggregate to remain in a
stable emulsion by permitting greater solute-solvent interaction. This
can be illustrated graphically in Fig. 1 by enlarging region "B" over
a greater range of aggregate sizes since some aggregates previously
belonging to regions "A" and "C" now become more stabilized. It may
also be visualized by flattening the two curves in Fig. 2, thereby
extending their region of overlap. Thus, when added with a "carrier",
more of an insoluble compound may be introduced into a stable water
emulsion. The other effect may be due to possible interference with the
uptake of a test compound by an organism. Any such uptake must by neces-
sity be preceded by an adsorption to a surface of the organism such as
the gills in a fish. If at this time the "carrier" molecules, which are
located at the surface of the aggregate, affect the actual process of
adsorption in any way, there will be a resultant change in the rate of
transfer of the compound into the organism. If the rate of uptake is

related to toxicity, there will be a concomitant change in toxicity.


Section VIL

RESULTS

A typical chromatogram of an Aroclor 1254 standard in hexane (A)
and a hexane extract of a type-II emulsion (B) is shown in Fig. 3.
Some of the 11 peaks indicated are multiple peaks. Only peaks 1-7
were used to calculate the "total" peak height on which all quantita-
tions were based. Peaks 8-11 were excluded, because they were often
too small to permit accurate calculations.

The effect of storage time on Aroclor 1254 emulsions of type-I
and type-II is shown in Table 1. There is a fairly rapid initial
decrease in Aroclor 1254 in all cases and it appears that a plateau is
reached at around 7 yg/2. This should not be interpreted to mean that
solubility is approached at that point, only that perhaps a stable
emulsion is reached at that point.

The hexane extract of type-II emulsion (chromatogram B) indicates
a relative reduction in peak height for the early eluting peaks. This
phenomenon is better described by the results shown in Table 2. For
comparison peak 7 was arbitrarily assigned a relative value of 100%.
The results indicate that on standing a type-II emulsion shows a reduc~
tion of the individual peaks, with the early eluting components, or less
chlorinated biphenyls (Zitko, 1970), being reduced much more than the late
eluting ones. The degree of reduction depends somewhat on the preparation
and initial concentration of individual type-II emulsions (Table 2).
Type-III emulsions of comparable "total" concentration show a relative

distribution of the isomers identical to that of the standard.


Table 3, ADSORPTION OF AROCLOR 1254 TYPE IL EMULSION ON POLYALLOMER
CENTRIFUGE TUBES ON STANDING

Time (hrs) Aroclor 1254 (ug/2)
in water phase

0 125
3 86
72 3.3
0 45
1 35
3 27

Table 4, ADSORPTION OF AROCLOR 1254 ON STAINLESS STEEL CENTRIFUGE TUBES
AS A FUNCTION OF TIME AND CONCENTRATION

Aroclor 1254 type IT emulsion

Time Total Water S. S. tube % adsorbed
(hrs) (ug) (ug/£) (ug) (ug/£) (ug)

0.5 3.83 113 3.63 107 0.18 5

1 3.83 113 3.31 97 0.30 9

2 3.83 113 3.20 94 0.33 13

16 3.83 113 3.14 92 0.51 16

1 0.48 14 0.35 10 0.08 23

2 0.06 2 0.03 1 0.02 67

stainless steel centrifuge tubes.

21


ABSTRACT

A theoretical model of the behavior of substances having low water-
solubility is presented and discussed with respect to aqueous bioassay.
Ultracentrifugal techniques were used in an attempt to study size distribu-
tions of Aroclor 1254 aggregates in aqueous emulsions. Results indicate
strong adsorption from emulsion by surfaces and a water-solubility at 20°C
of less than 0.lug/& in distilled water and approximately 40% of that value
in water containing 30 g/2 NaCl. Implications with regard to aqueous bioassay
are discussed.

This report was submitted in fulfillment of Program Element 1EA077,
ROAP/Task No. 10AKC/18 by the Gulf Breeze Environmental Research Laboratory
under the sponsorship of the Environmental Protection Agency. Work was com-

pleted as of September, 1974.

ii


Region "A" describes an area in which the aggregates are too small to
exist independently because interactions in the sphere of influence
at that point are such that solute-solute interactions, which have now
become aggregate-aggregate interactions, are more pronounced than the
aggregate-solvent interactions. Therefore, these aggregates are
expected to coalesce, moving them into region "B", which describes a
range of aggregate sizes of maximum stability. The aggregate-aggregate
interactions in this range are weaker than in region "A" for that size
of aggregate. Region "C" described aggregates which are too heavy to
remain in suspension for a given period of time and will settle out or
break into smaller, more stable aggregates. The exact shape of this
curve and especially that of region "B", depends on how tightly the sol-
vent is held within the sphere of influence of the solute aggregate, which
is a function of the molecular interactions between solute and solvent.
The distribution of different aggregate sizes in terms of molecularly
characterized interactions is shown in Fig. 2. The actual equilibrium
reaction taking place is described in a simplified manner at the top of the
figure. The two curves relate the hypothetical strength of interactions
of solute-solvent (aggregate-solvent) type and solute-solute (aggregate-
aggregate) type to aggregate size. The region where the curves cross

corresponds to a distribution of aggregate sizes of maximum stability.


Section V
MODEL

Aroclor 1254 was chosen as a model compound because it has been
extensively used in bioassay at this laboratory (Duke et al., 1970;
Nimmo et al., 1971a; Nimmo et al., 1971b; Hansen et al., 1971; Lowe et
al., 1972; Walsh, 1972; Cooley et al., 1972).

One approach to estimate quantitatively the solubility of Aroclor
1254 in water and the behavior of its aggregates is to use ultracentrifugal
analysis. This technique permits the selective removal of particles of
a certain size. For a spherical particle having a density of (p) and a
radius of (r) the molecular weight (M.W.) is represented by:

M.W. = 4/3nr° N, q@
where Ny is Avogadro's Number.

Two opposing forces (f) which determine the fate of a particle in solution:

sedimentation £ 4/3me (0-0, )85 and (2)
buoyancy f£ = 6mrn, (3)
where (05) is the density of the solvent, (g) is gravity, and (n) is the
viscosity of the solvent.
To remove a small particle from an emulsion at a reasonable rate, a
force larger than gravity must be applied. Using the ultracentrifuge, (g)
in equation (2) is replaced with (wx), the angular velocity of the

centrifuge rotor (w) times the distance of travel (x) of the emulsified

particle.

1 The equations used are normally found in any textbook on physical
chemistry, and their reproduction here is intended merely for the
convenience of the reader.

10


EPA-660/3-74-013
September 1974

THEORETICAL MODEL AND SOLUBILITY CHARACTERISTICS
OF aroc.om® 1254 IN WATER:

Problems Associated With Low-Solubility Compounds
In Aquatic Toxicity Tests

by

W. Peter Schoor
Gulf Breeze Environmental Research Laobratory
National Environmental Research Center
Gulf Breeze, Florida 32561

Program Element 1EA077
ROAP/Task No. 10AKC/18

National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330


TECHNICAL REPORT DATA

(Please read Instructions on the reverse before completing)
VREPORT NO, 2 3. RECIPIENT'S ACCESSION NO,
EPA 660/3-74-013 |
la TiTLE AND SUBTITLE SOSTMS~S~S — ls. REPORT DATE
Theoretical model and solubility characteristics of September 1974
Aroclor” 1254 in water: Problems associated with low- Is. PERFORMING ORGANIZATION CODE

solubility compounds in aquatic toxicity tests.

7. AUTHORIS) Ig. PERFORMING ORGANIZATION REPORT NO.

W. Peter Schoor, Ph.D.

9. PEAFORMING ORG ANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT NO,
U. S. Environmental Protection Agency 1 EA077 / 10AKC / 018
Gulf Breeze Environmental Research Laboratory Ti, CONTRACT/GRANT NO.

Sabine Island
Gulf Breeze, Florida 32561

12, SPONSORING AGENCY NAME AND ADDRESS 13. TYPE i REPORT AND PERIOD COVERED
Fina

14, SPONSORING AGENCY CODE

15. SUPPLEMENTARY NOTES

16, ABSTRACT

A theoretical model of the behavior of substances having low water-solubility is
presented and discussed with respect to aqueous bioassay. Ultgacentrifugal techniques
were used in an attempt to study size distributions of Aroclo: 1254 aggregates in
aqueous emulsions. Results indicate strong adsorption from emulsion by surfaces and a
water-solubility at 20°C of less than 0.l ug’ in distilled water and approximately
40% of that value in water containing 30 g/g NaCl. Implications with regard to
aqueous bioassay are discussed.

KEY WORDS AND DOCUMENT ANALYSIS

DESCRIPTORS D.IDENTIFIERS/OPEN ENDED TEAMS |c. COSATI Field/Group

Solubility Adsorption
Aroclor” 1254

Theoretical Model

Water

Aquatic Toxicity Tests
Low-Solubility Compounds

Emulsion
queous Dispersion
13, DISTRIBUTION STATEMENT 19, SECURITY CLASS (This Report) 21, NO. OF PAGES:
Release to public Unclassified
20. SECURITY CLASS (Tais page) 22. PRICE
Unclassified

EPA Form 2220-1 (9-73) sk U.S. GOVERNMENT PRINTING OFFICE: 1975~698- 089/103 REGION 10


The distribution of isomers in a hexane extract of the gill tissue
of a pink shrimp (Penaeus duorarum) exposed to 2.5 ug/% Aroclor 1254 for
20 days is shown in parentheses at the bottom of Table 2. Because peaks
2, 4 and 7 showed obvious contamination, peak 6 was assigned the arbitrary,
relative 100% value. The "total" concentration of 3.4 mg/kg was based on
the total height of peaks 1, 3, 5 and 6, and on the wet weight of gill
tissue (blotted to remove adhering water).

Filtration of type-I emulsion through 450 nm (0.45u) Millipore®
filters revealed obstructed passage of Aroclor 1254 aggregates smaller
than 450 nm. Starting with a 1 mg/g emulsion and changing filters after
each filtration, less than 0.01 ug/2 of the material remained in the water
after 15 passages. Since aggregates in the starting emulsion were most
likely smaller than 450 nm (calculations using equation 1 lead to roughly
192? times the average molecular weight of Aroclor 1254), the Aroclor 1254
must have been adsorbed on the filter. This was also evidenced by the
fact that the filter paper turned slightly transparent after the first
passage during which about 95% of the material was removed from the
emulsion.

The first centrifugation experiments were carried out by centri-
fuging 180 ml of 42 ug/% Aroclor 1254 type-II emulsion in 60 ml polyace-

tate centrifuge tubes for 60 min at 107,000 x g (max.).

19


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development,

U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:

Environmental Health Effects Research
Environmental Protection Technology
Ecological Research

Environmental Monitoring
Socioeconomic Environmental Studies

aAPwno

This report has been assigned to the ECOLOGICAL RESEARCH STUDIES
series. This series describes research on the effects of pollution

on humans, plant and animal species, and materials. Problems are
assessed for their long- and short-term influences. Investigations
include formation, transport, and pathway studies to determine the
fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living
organisms in the aquatic, terrestrial and atmospheric environments.

EPA REVIEW NOTICE

This report has been reviewed by the National Environmental

Research Center--Corvallis, and approved for publication. Mention
of trade names or commercial products does not constitute endorsement
or recommendation for use.


Section IX

DISCUSSION

The original intent for conducting the work described was to find
the absolute solubility of Aroclor 1254 in fresh and salt water. This,
unfortunately, was not completely accomplished to any accurate degree,
because a series of significant problems occurred at the beginning of
the centrifugation experiments. Recovery of Aroclor 1254 after
centrifugation was low and, hence, led to the discovery that adsorption
occurred on the walls of the polyacetate centrifuge tubes as well as on
Polyallomer and stainless steel centrifuge tubes. Ultimately, only the
stainless steel centrifuge tubes were used in the adsorption and ultra-
centrifugal studies.

The apparent disappearance of early eluting isomers, such as shown
in Table 2, has been observed by others. It was found to occur in the
eggs of the double-crested cormorant and regarded as possibly due to
metabolic breakdown (Hutzinger et al., 1972). Similar behavior in the
carcasses of bobwhite quail after exposure to Aroclor 1254 was observed
and believed to be because of isomeric transformations (Bagley and
Cromartie, 1973). Application of Aroclor 1254 to different types of
soil showed a reduced recovery of the early eluting, lower chlorinated
biphenyls (Iwata et al., 1973), and it was postulated that this may have
been due to evaporation from the soil. My studies did not substantiate the
observations by Zitko (1970) that when Aroclor 1254 emulsions are centri-
fuged the dissolved fraction is richer in the lower chlorinated biphenyls
than is the original preparation. However, the difference could be due
to the method of the preparation of his emulsion, which was similar to my

type-III emulsion. In both type-II and type-I1I emulsions the distribution

27


as well as for ultracentrifugal analysis. Table 4 shows the amounts
of Aroclor 1254 adsorbed on the wall of a stainless steel centrifuge
tube in relation to starting concentration and time. The amounts
adsorbed from the 14 wg/2 and 2 yg/2 emulsions were greater than that
adsorbed from the 113 yg/% emulsion during the same time period. It
should be pointed out that 0.100 yg of Aroclor 1254 adsorbed as a
monomolecular layer per tube represents about 2% of the minimum area
available. The calculated inside area of a stainless steel centrifuge
tube was 60.8 cm. This area must be considered minimum because the
surface was assumed to be ideally smooth, which certainly is not the case.
However, for the approximations involved, this figure was used.

A simple calculation using equation (1) yields 0.613 nm for the
cross-sectional surface area of an average Aroclor 1254 molecule using
the average molecular weight of 327 (Hutzinger et al., 1972), and
ep = 1.505 g/cm (W. B. Papageorge, Monsanto Company, St. Louis,
Missouri, personal communication). Utilizing a molecular model with
the phenyl groups at right angles to each other and bond length
(Pauling, 1940) as the basis for calculations, a cross-sectional area
of 0,643 nm for the fully chlorinated and 0.356 am for the unchlori-
nated or biphenyl molecule was obtained. Values falling between are
not linearly related to amount of chlorination. Using 0.613 an as an
approximate, average cross-sectional area, 0.100 pg of Aroclor 1254
occupies 1.13 cm in the form of a monomolecular layer. This corresponds

to approximately 3 ug/% in a 34 ml stainless steel centrifuge tube.


By definition, a single solute molecule in a disperse system
possesses a certain sphere of influence, the nature of which governs the
fate of the solvent molecules that surround it, which in turn affects the
behavior of the solute molecule, and thus determines the characteristics
of the solute molecule in the system. While precise information is lack-
ing, it is known, nevertheless, that the range of effect of a solute
molecule may extend through several layers of surrounding solvent molecules.
This means, of course, an orderly alignment involving either oppositely
charged polar regions or non-polar regions on the solute and the solvent
molecules. If this interaction between solute and solvent molecules is
of significance, the above defined ideal solution can be visualized, pro-
vided also that there is no competition among the solvent molecules belong-
ing to respective spheres of influence of two separate solute molecules.

The complexity of the situation is increased in cases where the
interactions between solute and solvent molecules (solute-solvent inter-
actions) become less pronounced, and, as a result, the interactions between
solute and solute molecules (solute-solute interactiong become more pro-
nounced. This implies that the sphere of influence around the solute
molecule is diminished with respect to the solvent molecules which are
now no longer attracted to the same degree. As two or more solute mole—
cules start to form aggregates, the factor of size of aggregates versus
their stability in a solvent becomes of utmost importance.

A generalized illustration of the size distribution of aggregates

that one might expect to find in a suspension is shown in Fig. 1.


EQUILIBRIUM BETWEEN
SINGLE MOLECULE (A)
AND AGGREGATES

(B) AND (C)

RELATIVE
STRENGTH
OF
INTERACTION

A B c

a — @
a

SOLUTE - SOLUTE
(AGGREGATE - AGGREGATE)
INTERACTIONS

AREA OF MAXIMUM
STABILITY

|

SOLUTE - SOLVENT
(AGGREGATE - SOLVENT)
INTERACTIONS

AGGREGATE SIZE

>

Figure 2, Theoretical strength of interaction between solute and solvent.