BIOACTIVE COMPOUNDS IN THE AQUATIC

ENVIRONMENT: UPTAKE AND LOSS OF DDT AND

DIELDRIN BY FRESHWATER MUSSELS
J. W. BEDFORD
Bureau of Water Management
Department of Natural Resources
Lansing, Mich. 48914
and
M. J. ZABIK
Departm en t of En tom ology
Michigan State University
East Lansing, Mich. 48823

Freshwater mussels were exposed to several concentrations of DDT [ 1, 1, 1 -

t r i c h l o r o - 2 , 2 - b i s - ( p - c h l o r o p h e n y t ) ethanel and HEOD ( 1 , 2 , 3, 4, 10, 1 0 - h e x a -
c h l o r o - 6 , 7 - e p o x y - I , 4, 4a, 5, 6, 7, 8 a - o c t a h y d r o 1, 4 - e n d o - e x o - 5 , 8-di-
methanonaphthalene principal component of dieldrin) in natural lake water and in
reconstituted distilled water, under continuous flow and constant temperature con-
ditions. The mussels concentrate DDT approximately 2400 fold and HEOD 1200
fold in lake water; they concentrated DDT about 1000 fold in distilled water. The
concentration of the insecticide chemicals in the mussels reaches equilibrium with
the level in the water faster in lake water than in distilled water and the insecticide
chemicals have a shorter h a l f - l i f e in the mussels than in lake water. The h a l f - l i f e
of HEOD is 4.7 days in lake water compared to 13.6 days for total DDT residues in
lake water. The insecticide chemical residue concentrations are highest in the di-
gestive and reproductive tissue, and lowest in the muscle, mantle, and gill tissues.
The residue concentrations are very low in the marsupia (abominal pouch), in the
tests made in distilled water, but they are almost as great as those in the digestive-
and reproductive tissue, in lake water.

Aquatic organisms have been used to considerable extent as biological monitors of

pesticide chemicals. The most widely used group has been the bivalves (Class Lamelli-
branchia). The eastern oyster (Crassostrea virginica) has been used extensively as a pesticide
chemical monitor in the marine environment (Casper 1967, B u g g e t al. 1967, U. S.
D e p a r t m e n t o f I n t e r i o r 1967). Several species of freshwater mussels (Unionidae) have been
used in various freshwater situations (Miller et al. 1966, Goodsit and Johnson 1968,
Bedford et al. 1968, F e t t e r o t f and Willson, 1970). The freshwater mussel has been
used by state agencies in Michigan, Wisconsin, Minnesota, and Indiana to monitor
pesticide chemicals in tributaries of the Great Lakes.

97

Archives of Environmental Contamination and Toxicology,

Vol. 1, No. 2, 1973, © 1973 by
Springer-Verlag New York Inc.
98 J.W. Bedford et al.

Little is known about the uptake, metabolism, and elimination of insecticide chemi-
cals by freshwater mussels or, even about what significance certain concentrations of
these chemicals, in the mussels, have in terms of the concentration in the environment
from which they are removed. Thus, the purpose of this study was to increase the un-
derstanding of this potentially important biological tool in the monitoring of insecticide
chemical pollution by determining 1) the time required for the pesticide residue concen-
tration, in the mussel, to reach equilibrium with the concentration in the water, 2) the
half-life of the pesticide chemical in the mussel after its introduction is terminated, and
3) the primary locations in the mussel where pesticide residues (including metabolites)
are stored, and correlating the concentration of pesticide residue stored in the mussel with
the concentration in the water.

General methods and materials

Mussels were collected by hand from local streams and lakes. Anodonta grandis and
Elliptio dilatartus were collected from the Red Cedar River, Ingham County and the
Looking Glass River, Clinton County, Michigan, and Lampsilis sitiquoidea from Gun
Lake, Barry County, Michigan. The mussels were held in a recirculating tank (Frigid
Units, Inc.) at 9°C until tested.

Two methods were used to provide a continuous flow of a known concentration of

insecticide chemical. The first involved the daily mixing of the insecticide chemical solu-
tion in a 190-liter stainless steel tank and siphoning of the solution into 35-liter test
aquaria maintained at a 20-liter level. The second method utilized a Beckman solution
metering pump (Model 756), using a filter flask as a mixing vessel into which the in-
secticide chemical was metered, and the water was siphoned from a constant head
source. The constant head was provided by continuously overflowing a 190-liter stainless
steel tank. In both methods, the insecticide chemical was dissolved in absolute ethanol
before addition to the water. (The ethanol concentration in the water never exceeded 10
ppm.)

The mussels (20 per aquaria) were exposed to the insecticide chemicals in aquaria
partially submerged in constant-temperature water baths. Water containing the in-
secticide chemical was preconditioned to the temperature of the bath before addition to
the aquaria, by flowing through stainless steel coils submerged in the bath. The water was
siphoned out of the aquaria into a second filter flask, thus maintaining a constant level in
the aquaria. Either glass or Teflon @ tubing was used throughout the system. Sterile silica
sand was used as a substrate for the mussels in all experiments.

Mussels from the test aquaria were prepared for insecticide chemical analysis, as follows:
The living mussels were removed from their shells, drained, and weighed to the nearest mil-
ligram. The mussels were blended in a Sorvall Omni-Mixer for three minutes at 10,000
 Uptake and Loss of DDT and Dieldrin by Freshwater Mussels 99

rpm with 50 ml of hexane-acetone mixture (2:1 by volume). The solvent mixture was
decanted and the sample was blended twice more with 50-ml portions of fresh solvent
mixture. The combined extract was washed with a 10-percent NaC1 solution to remove the
acetone. The extract was dried over anhydrous Na2SO 4 and a t 0 - m l aliquot was removed
for determination of percent of fat, by evaporation of the solvent in a vacuum oven at
60°C. The remaining extract was concentrated to approximately 5 ml in a water bath, at
80 ° C, for introduction onto a clean-up column.

Pyrex columns (2.4 ID x 50 cm), fitted with a fritted-glass disk, were packed with
10 g of Florisil--Celite mixture (5:1 by weight), with a layer of anhydrous Na2SO 4 above
and below the packing. The Florisil, activated at 650°C by Floridin, Inc., was deactivated
with approximately 10 percent distilled water and the chromatographic mixture was cali-
brated before use to ensure its conformation to the elution procedure used. Each sample
was etuted with 200 ml ofn-hexane and then reconcentrated to a volume of 5 ml. (The ex-
traction and clean-up procedures used generally follow those recommended by Shell
Development Co. (1964) with several modifications.)

One-liter water samples, taken periodically (4 to 7 times per week) from the outlets
of the test aquaria, were extracted successively in 2-liter separatory funnels with 100-,
5 0 - , 5 0 - , 5 0 - , and 50-mt portions ofhexane. The combined extract was dryed over anhy-
drous Na2SO 4 and concentrated to 5 ml for introduction into a gas chromatograph. All
solvents were redistilled before use.

A Beckman GC 4 chromatograph, equipped with a discharge electron capture detector,

was used for the determination of the insecticide chemical. It was fitted with a pyrex
glass column [6 ft (1.83 m) x 1/16 in (1.59 mm) ID], packed with 11 percent Q F - 1 and
3 percent DC 200, by weight on Gas Chrom Q (60/80 mesh) and was operated at a column
temperature of 220°C and a 30 ml/min helium (99.995% pure) flow. The injection tem-
perature was 250°C and the detector temperature was 275°C. Quantitations were based
on peak height and the concentrations were based on the weight of the mussel.

The identities of the insecticide chemicals, and their metabolites, were confirmed gas
chromatographically, using columns packed with 5 percent by weight of DC t 1 on Gas
Chrom Q (60/80 mesh) and I 1 percent by weight of Q F - 1 - OV-17 mixture (1.3: t by
weight) on Gas Chrom Q. Selected samples were also spotted on Brinkman pre-made
silica gel thin layer plates, developed with hexane-diethyl ether mixture (4:1 by volume)
and detected with Rhodamine B.

The insecticide chemicals used were DDT [1, 1, 1-trichloro-2, 2-bis-(p-chloro-

phenyl) ethane], obtained from City Chemical Corporation, New York, N.Y., and HEOD
(1,2, 3, 4, 10, 10-hexachloro-6, 7 - e p o x y - I , 4, 4a, 5, 6, 7, 8 a - o c t a h y d r o - 1 , 4 - e n d o -
exo-5, 8-dimethanonaphthalene, principal component of dieldrin), obtained from Shell
100 J.W. Bedford et al.

Chemical Co., N. Y. Both compounds were recrystallized and were more than 99 percent
pure. The DDT metabolites (DDE, TDE) used as standards for GLC and TLC confirmatory
tests were obtained from the Perrine Primate Laboratory.

Recovery of DDT from lake water, ranged from 87 to 95 percent and, from tapwater,
75 to 84 percent. HEOD recovery, from lake water, ranged from 66 to 72 percent and,
from tapwater, 66 to 72 percent. The efficiency of the extraction and cleanup methods
on mussels ranged from 89 to 95 percent for DDT and from 77 to 84 percent for HEOD.
The values reported were not adjusted on the basis of percent recovery. All water con-
centrations were reported as the mean value along with their standard deviations.

Experimental methodology. Two series of experiments were made in which freshwater

mussels were exposed to known concentrations of DDT or HEOD (the principal compo-
nent of dieldrin). In one series, mussels were exposed to various concentrations of DDT
or HEOD in lake water to ascertain the uptake and loss of the insecticide chemicals under
natural conditions. In the other series, mussels were exposed to various concentrations of
DDT in distilled water with 50 ppm Ca++ (as CaC12) added for shell maintenance. (The
purpose of the latter series was to determine the uptake and loss of the insecticide chemi-
cal under conditions where there are no stimuli to feed. This condition can occur in small
cold streams which have very little plankton or suspended material and do not naturally
support mussel populations.)

In all experiments, three mussels were analyzed for insecticide content prior to the
start of the experiment and each sample consisted of three mussels, unless otherwise
noted.

Results a n d discussion

Lake Water

HEOD (dieldrin) exposure. Lake Lansing, a shallow, warm water, eutophric lake in
Ingham Co., Michigan, provided the water for this series of experiments. The water con-
tained DDT (0.01-0.05 ppb) but not any detectable amounts of HEOD (dieldrin),
DDE, or PCB's. The lake water was pumped, periodically, to a stainless steel storage tank
which fed a constant-head tank. This provided a constant flow of lake water to the
mixing flask into which the insecticide chemical solution was metered.

Mussels (Lampsilis siliquoidea) were exposed to a mean water concentration of

0.57 + .12 ppb HEOD for three weeks and, immediately afterwards, to a concentration of
1.16 + .13 ppb for one week. At the end of four weeks, introduction of HEOD was ter-
minated and unfortified lake water was allowed to flow through the system containing
the mussels, for three additional weeks. One week following the termination of the
 Uptake and Loss of DDT and Dieldrin by Freshwater Mussels 10t

dieldrin introduction, the insecticide chemical level in the water was found to be 0.11
ppb, in the tank overflow, and, at the end of the experiment, it was 0.04 ppb.

The control mussels contained a low level of HEOD residue (Table I). After one week,
the mussels attained their highest concentration of HEOD residue during the initial ex-
posure, decreasing slightly (statistically not significant) during the next two weeks
(Table I). When the HEOD input was doubled, the HEOD residue concentration in the
mussels also doubled. With the cessationof HEOD introduction, the residues in the mussels
declined rapidly for the first two weeks, remaining constant in the following week.

A semi-tog plot of the HEOD residue values during the post-exposure period, is
linear with a negative slope (Fig. 1). The equation for this relationship is:
Y -= (A) ( - B x) (I)
where: Y = concentration of HEOD in the wet mussel, in ppm; A = regression inter-
cept, in ppm; X = time following cessation of HEOD introduction, in weeks;
B = relative rate of residue toss.

For the rectified data, the equation is:

Log Y = log A - (log B) X. (H)

Table I. Mean HEOD (Dieldrin) Residue Content o f Wet Lampsilis Siliquoidea

Exposed to 0.5 7 +-.12 ppb HEOD in Lake Water at 20°C for Three Weeks and
to 1.16 +-.13 ppb tlEOD for an Additonal Week

Fat HEOD
Exposure, content, content,
weeks % ppm

HEOD concentration in water: 0.57 + .12 ppb

0 1.01 0.010
1 1.08 0.699
2 0.89 0.672
3 1.08 0.612

HEOD concentration in water: 1.t6 +- .t3 ppb

4 [ 0.91 1.355

HEOD introduction stopped

5 ] 0.83 0.301
6 t .06 0.088
7 1.54 0.073
102 J.W. Bedford et al.

Log B, the regression coefficient, estimates the logarithmic rate of HEOD residue
loss. In this case, the log o f the HEOD concentration in the mussel decreases at an
estimated uniform rate of 0.437 and the residue half-life, calculated from equation II, is
4.7 days (solid line, Fig. 1). If one eliminates from the regression analysis the third week
values, where the mussels appear to be in equilibrium with the dieldrin that still remained
in the water, the log of the concentration in the mussels decreases at an estimated rate o f
0.607 (dashed line, Fig. 1), and the half-life value decreases to 3.5 days.

Mussels (L. siliquoidea) were also exposed to 0.06 -+ .01 ppb HEOD in lake water for
three weeks. As at the higher HEOD exposures, the highest level was reached after one
week and the average residue concentration, for the three weeks, was 0.054 ppm HEOD
in the wet mussels.

\
o\
1.0 \

0.5
E
0.4

~ 0.3-
E
o.2.

.E

o.1

a
o
,,, 0.05
"1"
0.04

0.03

d a' 2 5
Time after HEOD introduction stopped, weeks

Fig. 1. The dissipation of HEOD (dieldrin) residues from mussels (Lampsilis siliquoidea)
in unfortified lake water, after exposure to HEOD--containing lake water (Table t); solid
line: T½ =4.7 days,log B= 0.437;dashed line: T% = 3.5 days, log B = 0.607
 Uptake and Loss of DDT and Dieldrin by Freshwater Mussels 103

Under equilibrium conditions, there is a linear relationship between the HEOD residue
in wet mussels and the HEOD content of the lake water in which they grow (Fig. 2). The
slope of the least-square line or regression coefficient is 1.19 when the HEOD concen-
tration in the mussel is expressed in ppm and that in water is in ppb. Thus, the mussels
concentrate the HEOD content to a value about 1200 times that in water. The correlation
coefficient (r) is 0.976, indicating a very close fit and a highly significant correlation
between the concentrations (Fex p. = 376.8, F.995 = 10.1). However, the assumptions of
variance homogeneity, and independence of means and variances are not met due primarily
to the small mean and variance of the residue content of mussels exposed to 0.06 ppb
HEOD.

DDT exposure. Mussels (Anodonta grandis) were exposed to three levels of DDT in
lake water, using the same method and apparatus utilized in the HEOD study (above).
Initially, mussels were exposed to a mean concentration of 0.62 +- .13 ppb of DDT for

1 . 5 . - -

1,4,
1.3 ¸
1.2-
E 1.1-
o..
0..
1.0-

'E 0.9-
0.8-
0.7-
0.6-
0,5-
O
o 0.4-
LLI
'1-
0.3-
0.2-
0.1
0.0
0.'1-- 6.2--" 013 0',4 6.5 016 0'.7 018 0'.9 110 1'.1 1.2

HEOD content of water, ppb

Fig. 2. The relationship of HEOD (dieldrin) residues in mussels (Lampsilis sitiquoidea) to

the HEOD content of fortified take water in which they were grown, under equilibrium
conditions
104 J.W. Bedford et al.

three weeks and, at the end of that time, to unfortified lake water for five weeks more.
The DDT concentration in the overflow of the test aquaria dropped to 0.24 opb at the
end of one week, after DDT fortification stopped, and to 0.1 t ppb three weeks after the
termination of the DDT fortification.

Before determination of the DDT residue, the mussels were dissected into parts con-
sisting of 1) the"viscera" comprising the combined intestinal tract, the associated digestive
gland, and the gonad, 2) the "muscle", comprising the muscle, mantle, and gill tissues,
and 3) the marsupia from specimens that were gravid. In mussels exposed for one week,
the total DDT residues in the whole animal increased to over 1 ppm, remained at that
approximate level for a week, and increased again to approximately 2.5 ppm at the end
of the third week (Table II). The marked increase in DDT residue content, after three
weeks, was the result of excessive concentration of DDT that developed in the test tanks
during a 2 - 1 / 2 day period (in the third week) when the dilution water line became
plugged with debris and periphyton. Unfortunately, water samples were not taken when
this problem was discovered. Later in the week, after the line was cleaned, water samples
showed the DDT concentration to be only slightly higher than the mean for the three
weeks.

The total DDT-derived residue contents are much higher in the viscera than in the
"muscle" (Table ll). [h similar distribution was found in oysters by Butler (1966).] The
marsupia contain high concentrations of insecticides, in most cases nearly as high as'the
viscera. On a fat-~-weight basis, the difference in insecticide concentration in the muscle
and viscera portions is much reduced (Table II). The DDT residue in the marsupia, when
placed on a fat-weight basis, are, in some cases, considerably higher than the DDT
residue content of the viscera as a result of the relatively low fat content of the marsupia.
In the post-exposure period, the mussels show a steady decline in DDT residue content
(Table II) and, on a semi-log plot against time, the residue levels in the mussels decrease
in a linear fashion as occurs with HEOD (dieldrin) (see Fig. 1). When equations.I and II
(above) are applied, the regression coefficient (log B) for total DDT residue content
(including metabotites) is found to be 0.148 and the half-life is 13.6 days (13.0 days for
DDT as such). Thus, in mussels, the half-life of DDT is between three and four times
longer than the half-life of HEOD. Gakstatter and Weiss (1967) also report that fish
eliminate DDT residues more slowly than they eliminate HEOD and lindane, and they
conclude that the u p t a k e - and elimination rates are related to the solubility of the
insecticide chemical in water. Grzenda et al. (1970) report a logarithmic rate of loss of
0.0725 and a half-life of 29.5 days for DDT residues in goldfish; this is about half the
elimination rate in mussels. However, the fact that the initial body residue burden was
produced in the fish by feeding them contaminated food rather than by exposure to con-
taminated water, and that the fish had a greater fat content (5 to 29 percent) and a greater
initial body residue burden (1.2 to 18.4 ppm) can explain the different rates of residue
elimination found for fish and mussels.
 Uptake and Loss of DDT and Dieldrin by Freshwater Mussels 105

Mussels (A. grandis) were also exposed to two other concentrations of DDT in lake
water for two-week periods. In one test, the mussels were exposed to a mean concentra-
tion of 0.42 + .12 ppb DDT. The DDT residue levels increased greatly after one week and
then leveled off with an average concentration of 0.792 ppm in the mussels. In the
second test, a mean concentration of 0.14 -+ .02 ppb DDT was maintained in the water.
The DDT residue content in the mussels again increased above that in the controls,
after one week, and then did not change the second week. The average DDT residue con-
tent in the mussels was 0.106 ppm.

The equilibrium concentrations for DDT in the mussels were plotted in a manner
similar to the HEOD results and fitted with a least square line. The regression coefficient
or slope of the line is 2.36 when the concentration of DDT in the mussels is expressed as
ppm and the concentration in water as ppb. Thus, the mussels concentrate the DDT
residue to a level about 2400 times the DDT concentration in the water or approximately
twice the degree of concentration found for HEOD (dieldrin).

The correlation coefficient (r) is 0.880 a value that is not quite as high as the one for
HEOD but that is high enough to indicate a close fit. Again, the correlation between t h e
concentrations is highly significant ( F e x p . = 54.76, F.995 = 10.58) but, as in the case of
HEOD, the assumptions of variance homogeneity, and independence of the means and
variances are not met because of the relatively low variance of the DDT residue in mussels
exposed to 0.14 ppb DDT in lake water.

In the course of the experiments with DDT and HEOD in lake water, several samples
of the fortified water were filtered through Whatman No. 1 filter paper, and both the
filtrate and the residue were analyzed for insecticide chemical content in order to deter-
mine the relative distribution of soluble- and insoluble portion of insecticide chemical
into the lake water. The majority (60 to 65 percent) of the HEOD was found in the sus-
pended matter while the DDT was almost equally distributed between the water and
suspended matter. This result is somewhat at odds with the water solubilities of the two
compounds. Robeck et al. (1965) report that HEOD is several times more soluble than
DDT in water but, in the experiments reported here, a larger proportion of DDT was
found in the filtrate than was found for HEOD.

Distilled Water

Mussels were exposed in three separate experiments, to approximately the same con-
centrations of DDT (0.27 to 0.38 ppb) in distilled water containing 50 ppm Ca÷ + (added as
CaC12). tn the first experiment (designated as I), mussels (Elliptio ditatatus), which had
been held without food for three months at 9°C, were exposed to 0.38 -+ .11 ppm DDT
for three weeks. The mussels were then exposed to pure distilled water for one week.
[Considerable mortality (50 percent) occurred during the experiment and, for this reason,
 Table 11. Mean DDT Residue Cbntent (Including DDT Metabolites) o f Anodonta Grandis Exposed to
O.62 +-. 13 ppb DDT in Lake Water at 20°C for Three Weeks

Residue content, ppm

Fat
Exposure, content, W e t - s a m p l e basis Fat--wt basis
weeks Sample a % DDE d TDE e DDT Total Total

0 Viscera b 0.75 0.013 0.005 0.013 0.031 4.13

Muscle c 0.54 0.008 0.002 0.007 0.017 3.15
Whole mussel 0.63 0.010 0.004 0.010 0.024 3.81
1 Viscera 1.23 0.055 0.119 1.649 1.823 148.21
Muscle 0.70 0.030 0.071 0.923 1.024 146.29
Marsupium ( 1) 0.44 0.015 0.036 0.563 0.614 139.55
Whole mussel 0.85 0.039 0.091 1.239 1.368 160.94
2 Viscera 1.04 0.052 0.145 1.251 1.448 139.23
Muscle 0.76 0.038 0.071 0.592 0.701 92.24
Marsupium ( l ) 0.77 0.062 0.198 1.498 1.758 228.31
Whole mussel 0.90 0.047 0.114 0.941 1.102 122.56
3 Viscera 1.03 0.093 0.218 3.073 3.384 223.79
Muscle 0.70 0.039 0.102 1.597 1.738 248.28
Marsupium ( 1) 0.71 0.058 0.213 2.219 2.490 350.70
Whole mussel 0.85 0.063 0.156 2.255 2.474 290.94
DDT introduction stopped
4 Viscera 0.85 0.086 0.296 2.359 2.741 322.47
Muscle 0.67 0.025 0.109 0.847 0.981 146.42
Marsupium (2) 0.64 0.063 0.237 1.629 1.929 301.41
Whole mussel 0.73 0.053 0.198 1.536 1.787 246.03
 Viscera 0.97 0.061 0.225 1.744 2.030 209.28
Muscle 0.77 0.027 0.115 0.847 0.989 128.44
Whole mussel 0.85 0.041 0.157 1.194 1.392 t63.76
Viscera 0.98 0.059 0.191 1.474 1.724 t75.92
Muscle 0.77 0.022 0.074 0.624 0.720 93.51
Marsupium (2) 0.46 0.034 0.096 1.196 1.326 288.26
Whole mussel 0.77 0.036 0.115 0.915 1.066 138.44
Viscera 1.08 0.055 0.159 0.835 1.049 97.13
Muscle 0.84 0.024 0.081 0.429 0.534 63.57
Marsupium ( 1) t .37 0.041 0.082 0.773 0.896 65.40
Whole mussel 0.95 0.036 0.1t0 0.594 0.740 77.89
Viscera 1.20 0.039 0.122 0.490 0.651 54..25
Muscle 0.86 0.021 0.070 0.242 0.333 38.72
Whole mussel 1.00 0.028 0.092 0.345 0.465 46.50

aMean of data from three mussels, unless otherwise noted (as number 1 or 2, in parenthesis) in the sample column.
bCombined reproductive and digestive tissue.
Clncludes gills and mantle.
dDDE -- 1, 1 - d i c h l o r o - 2 , 2 - b i s - ( p chlorophenyl) ethylene. ?,
eTDE = 1, 1-dichloro 2, 2 - b i s - ( p - c h l o r o p h e n y l ) ethane.
108 J.W. Bedford et al.

the experiment was terminated after the fourth week.] The total DDT residue content
increased during the first two weeks and remained the same the third week (Table III).
The concentration of DDT in the mussels decreased slightly (10 percent) after the intro-
duction of DDT was halted.

In the second experiment (designated as II), two changes were made to reduce the
mortality. The temperature was lowered from 20 to 15°C and the flow rate was in-
creased from 30 to 120 ml/min. Freshly collected mussels (A. grandis) were exposed to
0.27 + .08 ppb DDT for five weeks and the experiment continued for four weeks after
the introduction of DDT was terminated. The control mussels contained at least three
times as much total DDT as any other used in this study (Table III). The DDT residue
concentration in the mussels increased during the first two weeks, then it leveled off ex-
cept for a decrease four weeks after DDT introduction was begun (Table III). The maxi-
mum DDT residues in the mussels were considerably higher, than in the first experiment (1),
even though the mussels were exposed to a slightly lower mean concentration of DDT at
a lower temperature. When the introduction of DDT was terminated (at the end of the
fifth week), the mussels showed a sudden increase in insecticide content and then
gradually decreased (Table IlI). This increase cannot be explained as it did not occur in
the first experiment (/) and in the third experiment (IH).

Table 1II. Mean Total DDT Residue Content Including Metabolites in Wet Mussels
(Anodonta Grandis) Exposed to 0.27 to 0.38 ppb DDT in Distilled Water
Containing 50 ppm Calcium Ion for Three to Five Weeks
DDT residue, ppm
Exposure Expt I Expt II Expt III
weeks (0.38 -+ .11 a) (0.27 -+ .08 a) (0.34 -+ .07 a)

0.062 0.220 0.027

0.238 0.386 0.084
0.408 1.080 0.541
0.392 b 0.818 0.387
0.356 0.484 0.415 b
0.806 b 0.447
1.259 0.434
1.144 0.329
t .006
0.853

aMean DDT content, ppb, of water in period of DDT introduction.

bDDT introduction halted at end of indicated period.
 Uptake and Loss of DDT and Dieldrin by Freshwater Mussels 109

A similar DDT concentration (0.34 -+ .07 ppb) was used for the third experiment (111)
in order to ascertain why there was such a large difference in the concentrations reached
in the mussels in the first two experiments (I and ID. Mussels (A. grandis), held at 9°C for
one month prior to testing, were exposed to DDT for four weeks. The experinaent was
continued for three weeks after the DDT introduction was stopped. As in the first and
second experiments (1 and I/), the concentration of DDT in the mussels increased for two
weeks, then leveled off; when the introduction of DDT was stopped the concentrations
in the mussels declined very slowly (Table II1).

The large difference between the equilibrium concentrations in the mussels of the
second experiment (II) and the other two experiments (I and II1), despite the sim-
ilar mean water concentrations of DDT in the three experiments, is difficult to
explain. The main differences were in the initial "physiological state" of the mussels and
the species of mussel. The mussels in the second experiment (11) contained higher initial
concentrations of DDT and were placed in test aquaria a few days after collection while
the others were held for one to three months at 9°C before use. In theory, the initial
tissue concentration should not affect the equilibrium concentration unless it exceeds the
equilibrium concentration. In distilled water containing calcium ions, DDT was slowly
eliminated, suggesting that a high initial concentration in the mussel could affect the
equilibrium concentration. Perhaps, the freshly collected nmssels used in the second ex-
periment (II) filtered more actively than did mussels conditioned to no food and cold
temperatures prior to exposure, thereby concentrating the insecticide chemical to a
greater extent.

The difference in species did not correspond with the difference in equilibrium con-
centrations as Elliptio in the first experiment (I) and Anodonta in the third experiment
(III) reached similar concentrations while the concentration of DDT in Anodonta in the
second experiment (11) was much higher. The absence of differences between species of
freshwater mussels in concentrating pesticides was also noted by Bedford et aL, 1968.

The mussels in the third experiment (111) were dissected into combined digestive and
reproductive systems (viscera), muscle, mantle, and gills ("muscle"), and marsupia before
analysis. As occurred in the mussels exposed to DDT in take water, the highest DDT
residues were found in the viscera with the "muscle" usually containing 1/2 to 2/3 of the
concentration of DDT found in the viscera. However, the marsupia from mussels exposed
to DDT in distilled water fortified with calcium ion contained very low levels of DDT in
contrast to the relatively high levels found in the marsupia of mussels exposed to DDT in
lake water. In the manner of mussels exposed to DDT in lake water, there was a much
smaller variation in DDT residues between tissues of mussels grown in the DDT-contain-
ing reconstituted distilled water when based on the fat content of the tissues, indicating
little selective storage in the mussel.
110 J.W. Bedford et al.

Mussels (A. grandis), held for three months at 9°Cbefore testing, were also exposed to
0.08+-.02 ppb DDT in distilled water containing 50 ppm calcium ion for four weeks. The
control mussels contained 0.048 ppm total DDT and, after two weeks, the mussels con-
tained 0.066 ppm; thus, the residue concentration factor was less (circa 800 vs 1000) than
for the other experiments using reconstituted distilled water.

Conclusions
This study reveals three general conditions which must be considered when using fresh-
water mussels as monitors of insecticide chemical levels in water. First, the previous con-
ditioning and insecticide chemical residue burden of the mussel can influence the concen-
tration of the insecticide chemical attained by the mussel. This seems to be especially im-
portant when they are placed in waters which contain little or no natural food. [The mus-
sel's filtering rate is dependent on the quality and quantity of food in the water (Wilbur
and Yonge, 1966).] Second, the type of water into which the mussels are placed is a factor.
A large difference is found in both uptake and elimination of insecticide chemical in ex-
periments in which there is no food (reconstituted distilled water) and essentially un-
limited food (Lake Lansing water). Thus, mussels placed in cold, clear streams can be ex-
pected to yield different bioassay results from those in streams which have native mussel
populations. It is also possible to have a load of suspended material that is too great, result-
ing in decreased filtering (Loosanoff and Engle, 1947). Poor water quality conditions,
which cause the mussel to close and not filter for periods of time, can yield l o w e r - t h a n -
representative concentration of insecticide chemicals in the mussel. This condition ap-
parently occurred in previous field studies (Bedford et al., 1968). Finally, the type of in-
secticide chemical being monitored must be considered. Although both DDT and dieldrin
are classed as chlorinated hydrocarbon insecticide chemicals, the degree to which they
concentrate in the mussels is considerably different. Most of these limitations apply to any
organism used for monitoring pesticides. Therefore, in comparison with other aquatic or-
ganisms, the mussel's trait of feeding by filtering large quantities of water, moving very
little, and having a long life span (up to 20 years) makes them especially well adapted as
monitors of pesticide chemicals that store in their tissues.

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Manuscript received August 11, 1972; accepted October 11, 1972