MIREX INCORPORATION IN THE ENVIRONMENT:

UFI'AKE IN AQUATIC ORGANISMS AND

EFFECTS ON THE RATES OF
PHOTOSYNTHESIS AND RESPIRATION
A. A. DE LA CRUZ and S. M. NAQV1
Department of Zoology
Mississippi State UniversiO,
Mississippi State, Ms'. 39762

Exposure of selected aquatic invertebrates to mirex reveals species variability in

uptake which is generally a function of exposure period and mirex concentration.
The metabolic effects examined relate to photosynthesis and respiration rates.
Dissolved oxygen changes, in light and dark bottles containing naturally occurring
plankton populations previously exposed to 1 ppb mirex, show progressive
photosynthetic inhibition in time from 16% at 6 days exposure to 33% at 18 days
exposure. Pure culture of the phytoplankton, Chlamydomonas sp., exhibits a 55%
photosynthetic reduction after exposure to 1 ppm mirex for 168 hours. Respiration
rates of plankton, Physa gyrina (pond snail), Gambusia affinis (mosquitofish), and
Lepomis macrochirus (sunfish) demonstrate an initial increasing trend to a
maximum of 62%, at low concentration of mirex or during early incubation period,
but decrease by as much as 50%, at elevated concentration or extended exposure at
low concentration.

Mirex is a polycyclic chlorocarbon (dodecachlorooctahydro-1, 3, 4-metheno-2H-cyclo-

buta [cd] pentalene, CloC1~2) which is used as the toxic ingredient in the formulation of
the oil-impregnated corncob grit bait applied in Mississippi, and other southeastern states,
for the control of fire ants. The bait is aerially sprayed, according to USDA prescribed
dosage, on infested areas and becomes available to foraging fire ants which are attracted
by the soybean oil in the bait. Non-target species get in contact with mirex by direct
consumption of unattended baits and/or by scavenging on contaminated carcasses of
killed ants. Among aquatic organisms, contamination can result primarily by exposure to
the active ingredient which leaches out of the carrier bait into water courses. Finley et al.
(1970), Lowe et. al (1972), and de la Cruz and Naqvi (1973) have found that mirex does
leach out of the formulated bait when suspended in water or exposed to the elements in
the field. Thus, the pesticide chemical gets incorporated into standing bodies of water or
transported elsewhere, downstream. In monitoring studies reported elsewhere, the
authors have detected mirex residue in aquatic organisms collected from a lake and from
an estuarine bay which never received any form of mirex bait application (Naqvi and de la
Cruz 1973).
255

Archives of Environmental Contamination and Toxicology,

Vol. 1, No. 3, 1973, © 1973 by
Springer-Verlag New York Inc.
256 A.A. de la Cruz and S. M. Naqvi

The ecological importance of mirex incorporation in the aquatic environment does not
necessarily depend on the amount of residue that gets into the water. Indeed, Alley
(1973) state that the residue level (about 0.0001 ppb) detected in natural waters does not
have any significant impact in the ecosystem. Furthermore, the solubility of mirex in
water does not exceed 1 ppb (Dollar 1973). What is biologically significant is the manner
by which mirex is taken up by aquatic organisms and the effects which the pesticide
chemical has on the biota chronically exposed to low concentrations of the chemical. In
this paper, the authors report their preliminary findings on mirex uptake of selected
freshwater invertebrates and its effects relative to photosynthesis and respiration rates.

Methods
Mirex residue uptake. Healthy specimens of 3 species of free-living freshwater leeches
(Placobdella rugosa, Glossiphonia sp., Erpobdella punctata), one shrimp (Palaemonetes
kadiakensis), one crayfish (Orconectes mississippiensis), an amphipod (Hyallela azteca),
and a dragonfly naiad (Maeromia sp.) freshly collected from the field were acclimatized
for 24-hours in separate glass containers containing clean pond water, in the laboratory. A
stock solution of mirex was prepared from technical mirex powder (98-99% pure)
dissolved in acetone and appropriate concentrations of test solutions were made by
dilution with clean pond water. The different species of acclimatized freshwater animals
were transferred into the aqueous test solutions made up to contain a range of mirex
concentrations (0.001-2.0 ppm) and the animals were held in the test solutions for
various lengths of time (24-672 hr) in order to allow interspecific comparison as well as
intraspecific comparison relative to these two factors. The total concentration of the test
solutions equals the maximum amount of mirex dissolved in the water (about 1 ppb) and
the mirex residues incorporated in the suspended particulates.

At the end of each residue-uptake experiment, the organisms were removed from the
holding containers, washed thoroughly, and rinsed by briefly dipping them in acetone to
remove any mirex residue absorbed to the external surfaces of the animal body. The
specimens were either temporarily stored frozen or immediately prepared for mirex
extraction and gas chromatographic analysis. The extraction procedures employed in
determining mirex in animal tissues, as well as water samples in the metabolism
experiments, were based on those which were used by Markin et al. (1972), and which are
described elsewhere (Naqvi and de la Cruz 1973). Mirex uptake is determined on the basis
of mirex residue (ppm) found in whole body extracts, with nannograde hexane, by means
of a 5360 Barber-Colman pesticide analyzer.

Hankton metabolism. Four holding tanks (aquaria) with the following dimensions
were used: length = 77 cm, width = 32 cm, and depth = 31 cm; each was filled with 50
dm3 of water containing naturally occurring plankton populations collected from a
nearby pond. The aquaria were left in the environmental chamber at constant
temperature and illumination. (The pond water was slightly greenish indicating high
 Mirex Incorporation in the Environment 257

density of phytoptankton suspended in the water.) After 24 hours, 5 ml of water from

two aquaria marked "experimental" was replaced with 5 ml of a 10 ppm solution of
mirex in acetone to make a 1 ppb test solution. In the two control aquaria, 5 ml water
was replaced with 5ml of the solvent (acetone). The water was thoroughly mixed and
lO-ml aliquots were removed from each holding tank and analyzed by gas chromatog-
raphy to confirm the mirex concentrations. The tanks were briefly aerated daily to
circulate the suspension.

At intervals of 0, l, 5, 10, 15, and 20 days, metabolic activity of the biota in both
experimental and control tanks was determined by measuring dissolved oxygen changes in
12 replicates contained in light and dark B.O.D. bottles. The bottles were incubated for
12 hours at exactly the same environmental conditions existing in the chamber. Dissolved
oxygen was detected by means of a YSI Oxygen Meter Model 54 provided with a
B.O.D.-bottle self-stirring probe-electrode.

In between biological oxygen demand (B.O.D.) experiments, the water level in the
aquaria was kept constant by the addition of filtered pond water. (The high humidity in
the environmental chamber prevented appreciable evaporation of water.) At the
conclusion of the experiments, 10-ml samples of water were again analyzed for mirex
concentration.

Pure cultures of Chlamydomonas sp. were exposed to water, containing 1 ppm mirex,
held for one week in aquaria kept in the environmental chamber. The same procedure
used for the pond plankton experiment described above was utilized for Chlamydomonas
except that smaller glass aquaria were used to hold the pure culture. After about 168
hours, metabolic activity was determined in 12 replicates contained in light and dark
B.O.D. bottles incubated for 7 hours at 19°C under constant illumination.

Metabolic activity was estimated in terms of gross photosynthesis (light bottle) and
respiration (dark bottle) for natural plankton and net photosynthesis (light bottle minus
dark bottle) and respiration (dark bottle) for Chlarnydomonas culture according to a
simplified formula suggested by Darnell (I 97 t).

Animal respiration. Oxygen consumption of the common pond snail, Physa gyrina,
and two common pond fish, the mosquitofish Gambusia affinis and the green sunfish
Lepomis macrochirus, was determined after they have been exposed in water containing
mirex leached from toxic fire ant baits. The snails, collected from a nearby pond, were
acclimatized in batches of 10 individuals of similar size in finger bowls containing 300 ml
of clean pond water. After 24 hours, duplicate batches of 10 snails each were transferred
to a series of mirex solutions with concentrations ranging from 0.003 to 1.0 ppm. The
snails were kept in the mirex solution for 3 days and then transferred into 300 ml B.O.D.
bottles containing aerated water of exactly the same nature and mirex concentrations.
The B.O.D. bottles were incubated at ambient temperature (about 21°C) in the dark for 3
258 A.A. de la Cruz and S. M. Naqvi

hours. The snails were recovered from the bottles after measurement of respiration,
killed, and dried at 103°C overnight. The experiment was repeated and, in both instances,
a control was included consisting of clean pond water inoculated with a suspension
leached from nontoxic fire ant bait prepared in exactly the same manner as the 1-ppm
mirex test solution. The toxic fire ant bait consisted of corncob grits impregnated with
soybean oil carrying the active ingredient, mirex, while the nontoxic bait was formulated
in the same manner as the toxic bait except that the toxicant was omitted. Aliquots of
the water, in which the snails were held for 3 days, were extracted and analyzed for mirex
content by gas chromatography.

Several small specimens of Gambusia affinis (mosquitofish) and Lepomis macrochirus

(sunfish) were collected from a pond with dip nets. The fish were divided into two groups
consisting of equal numbers of each species and held in separate tanks containing 50 dm 3
pond water. Prior to the introduction of the fish, the water was allowed to stand for 2
weeks in the test tanks; in one tank, small bags constructed from fine bolting silk and
containing collectively about 50 g mirex fire ant bait were suspended; in the other tank,
equal number of silk bags containing collectively about 50 g of nontoxic bait were placed.
The tanks were aerated sufficiently to cause agitation and circulation in the tank. At the
time the fish were introduced and throughout the period the fish were held in the tanks,
the experimental tank contained about 0.89 ppm mirex. After 3 weeks exposure to
mirex, respiration rates of G. affinis and L. macrochirus were determined weekly, for 5
consecutive weeks. Individual fish were carefully transferred into B.O.D. bottles which
were incubated for 2 hours in the dark at room temperature.
In all respiration experiments, the initial dissolved oxygen content in the experimental
water was determined and changes in oxygen tension of the water in the B.O.D. bottles
were measured by means of an oxygen membrane electrode and recorded by a YSI Model
54 Oxygen Meter.

Results a n d discussion
Uptake of mirex. The mirex concentration of the test solutions, the durations of
exposure, and the uptake of the pesticide by the test animals, on the basis of parts per
million whole body residue, are given in Table I. Generally, there is species variability in
the rate of uptake. As can be seen in Table I (Group A), tests conducted at 2 ppm level of
mirex for 48 hr show a mirex uptake in the crustaceans of 10.00 (Palaemonetes
kadiakensis) to 11.0 ppm (Orconectes mississippiensis) which is more than twice the level
of residue detected in the 3 species of free-living leeches, namely Erpobdella punctata
(1.9 ppm), Placobdella rugosa (4.9 ppm), and Glossiphonia sp. (5.1 ppm). The uptakes, at
0.5 ppm mirex test concentration conducted for 120 hr, in Placobdella and Orconectes
(Table I, Group B), as well as that, at 0.001 ppm for 672 hr, in Orconectes and the
amphipod Hyatlela azteca (Table I, Group D), are indicative of species differences. Mirex
uptakes in Orconectes, Placobdella, and the dragonfly naiad Macromia sp. show
differences in residue levels with respect to concentration and duration of exposure
 Mirex Incorporation in the Environment 259

Table I. The uptake o f mirex by selected freshwater invertebrates after exposure to

different concen trations o f mirex for various periods o f time.

Mirex Mirex
concentration Period of residue in
in water, exposure, animala
Species ppm hr ppm

Group A
Erpobdella punctata (leech) 2.0 48 1.92
Placobdella rugosa (leech) 2.0 48 4.91
Glossiphonia sp. (leech) 2.0 48 5.07
Palaemo netes kadiakensis (shrimp) 2.0 48 9.95
Orconectes mississippiensis (crayfish) 2.0 48 1 t.01
Group B
Orconectes mississippiensis (crayfish) 2.0 120 21.20
Orconectes mississippiensis (crayfish) 0.5 120 7.12
Plaeobdella rugosa (leech) 0.5 120 1.95
Group C
Macromia sp. naiad (dragonfly) 0.5 24 10.37
Macromia sp. naiad (dragonfly) 1.0 24 13.68
Macromia sp. naiad (dragonfly) 1.0 168 92.90
Group D
Hyallela azteca (amphipod) 0.001 672 2.53
Oreonectes mississippiensis (crayfish) 0.001 672 1.06

aValues represent mean of 2-3 analyses of whole-body residue pooled from 200 individuals for
Hyallela, and from 5-10 animals for all others.

(Table I, Groups B and C). The leeches survive indefinitely in 2.00 ppm Mirex but the
crustaceans and Macrornia naiad suffer 100% mortality after 168-hr exposure, at all test
concentrations. The high toxicity of mirex to crustaceans has been previously reported by
Ludke et al. (1971) and Lowe et al. (1971). [A brief report of our bioassay tests on
selected freshwater species has been reported previously (Naqvi and de la Cruz, 1972).]
The uptake of mirex by, and its toxicity to, non-target aquatic organisms at
concentrations greater than 1 ppb (the solubility of mirex in water) represent only a
trend in model systems and indicate what would occur in nature in mirex concentrations
of the environment were indeed above 1 ppb. Mirex residues in natural waters can
possibly exceed 1 ppb on account of the residues incorporated in the seston.

Effects on plankton. Photosynthesis, measured as gross primary production of oxygen

in light bottles, and respiration, in terms of oxygen consumption in dark bottles, of
naturally occurring plankton populations exposed to I ppb mirex in water are illustrated
in Fig. 1. No immediate effect of mirex on plankton metabolism is observed but, after 5,
10, 15, and 20 days exposure, photosynthesis is inhibited by 16, 10, 33, and 19%,
respectively. Moore (1972) reported that mirex is not significantly toxic to marine
260 A.A. de la Cruz and S. M. Naqvi

phytoplankton populations exposed for 24-48 hr to a range (.5 ppb to 1 ppm) of mirex
concentrations. This supports our observations and that of other workers, e.g., Ludke et
al. (1971) on the delayed effect exhibited by mirex. Butler (1963), on the other hand,
reports 46% decrease in phytoplankton productivity at 1-ppm concentration of mirex
even after only a 4-hr exposure. Similarly, effects on respiration rates is observed after
about 5 days exposure; respiration increases by 37% on the fifth day. Mirex stimulation
of respiration is observed up to 10 days exposure but increases only to approximately
17%. Measurements taken on the fifteenth and twentieth day, however, reveal respiration
inhibition of 10% and 50%, respectively. The mirex concentration in the water at the
beginning and at the conclusion of the experiment was about 1.0 ppb and 0.89 ppb,
respectively.

The light and dark B.O.D. bottles experiments similarly conducted on an unialgal
culture of Chlamydomonas sp. demonstrate inhibition of photosynthesis and respiration
rates after 168-hr exposure to water containing 1 ppm of mirex (Table II). Net
photosynthesis (calculated as carbon from oxygen consumption) is reduced from 122 mg
C m -3 hr - l in the control to 55 mg C m -3 hr - l in the mirex-treated culture; respiration
rate is reduced from 362 to 259 mg C m -3 hr -1. Experiments by Tumipseed (1973) also
reveal reduction in photosynthesis of Chlamydomonas exposed to mirex.

Production Respiration

30

7. E

"It
20
7
E
C.)

F 10
N
i

0
0 1 10 15 20 0 1 5 10 15 2O
Number of days

Fig. 1. Gross production of oxygen by photosynthesis and consumption of oxygen by

respiration of naturally occuring plankton populations exposed to water containing 1
ppb mirex for various periods of time. Light and dark bottles were incubated for 12 hr at
19-21°C. Values in mg C m -3 hr -1 are mean of 2 experiments consisting of 12 replicates
each. Light bars: control; dark bars: mirex treatment.
 Mirex Incorporation in the Environment 261

Table II. Metabolic activity o f C h l a m y d o m o n a s sp. after exposure to water containing

1 ppm mirex f o r 168 hours. Light and dark B.O.D. bottles were incubated
for 7hours at 19°C

mg C m - 3 h r - la

Metabolic Control Mirex-con-

activity water taining water

Net photosynthesis 122.21 54.54

(1 1.0) (6.7)

Respiration 361.79 258.88

(17.0) (6.9)

avalues are mean of 12 replicates; numbers in parentheses

are standard deviations.

Our findings on the effect of mirex on plankton metabolism is in agreement with what
have been reported b y a number of workers regarding the inhibition of photosynthesis by
various kinds of pesticides and fungicides (Butler 1963; Wurster 1968, Menzel et al. 1970,
Moore 1972. Harriss et al. 1970).

7
3.0
e-

1.5-
" i.i. ml m~, J i J J J i l i l i i

O O
d ~5 c5 d

Mirex concentration, ppm

Fig. 2. Respiration rates relative to control of pond snail Physa gyrina after 3 days of
exposure to a range (0.003-1.00 ppm) of mirex concentrations in water.
262 A.A. de la Cruz and S. M. Naqvi

Effects on animal respiration. Respiration rates of the pond snail Physa gyrina and
two species of pond fish Gambusia affinis and Lepomis macrochirus, exposed to mirex
leached from mirex fire ant baits, are shown in Fig. 2 and Fig. 3. In comparison with
controls treated with leachates of nontoxic bait, oxygen consumption of Physa, exposed
for 3 days at very low concentrations of mirex (0.008-0.07 ppm), increases to a
maximum of 62%. The decrease observed at the lowest concentrations (0.003-0.005 ppm)
used is insignificant. At 1 ppm centration of mirex, oxygen consumption drops by 44%.
Both Gambusia and Lepomis exhibit insignificant increases in oxygen consumption after
3 weeks exposure in water containing 1 ppm mirex. From the fourth week of exposure,
the oxygen consumption of mirex-treated fish decreases slightly but to a substantial level
beginning the fifth week for Lepomis and on the seventh week for Gambusia. The initial
increase in oxygen consumption, at very low mirex concentrations in Physa and during
the early incubation period in fish at 1 ppm, seems to be a normal physiological response
to the toxic stress. Naqvi and de la Cruz (1972) report similar stimulation of respiration
rates of certain freshwater invertebrates exposed for 24 hrs to 1 ppm mirex. The reduced
oxygen utilization is presumably due to inhibition of the aerobic respiratory enzyme
system as has been reported for rotenone (Yarbrough 1973) and for organochlorine
compounds (Johnson 1951, Colvin and Phillips 1968).

2.4- Garnbusia affinis Lepomis macrochirus

1.8
T
.E

1.2
v
E

o~ 0.6

3 4 5 6 7 3

Number of weeks
4 5 6
M
7

Fig. 3. Respiration rates of Gambusia affinis and Lepomis macrochirus exposed to water
containing 1 ppm Mirex for various periods of time. Values in mg 0 2 hr - I gm -1 are
mean of 3-6 replicates. Light bars: control; dark bars: mirex treatment.
 Mirex Incorporation in the Environment 263

Acknowledgments

We gratefully thank J. D. Yarbrough for the use of the glc facility in his laboratory,
Glynn Tumipseed for providing the Chlamydomonas culture, Allen Dearing for assistance
in the field work, and Ronald Altig for editorial suggestions during the preparation of the
manuscript. This study is a part of our continuing investigation on the physiological and
ecological effects of mirex under Cooperative Agreement No. 12-14-100-10,935-33 with
the USDA Agricultural Research Service.

References

Alley, E. G.: The use of Mirex in control of the imported fire ant. Jour. Environ. Quality,
2, 52 (1973).

Butler, P. A.: Commercial fisheries investigations. U. S. Fish Wildl. Serv. Circ. 167, 11
(1963).

Colvin, H. J., and A. T. Phillips: Inhibition of electron transport enzyme and

cholinesterase by endrin. Bull. Env. Contam. Toxicot. 3, 106 (1968).

Darnetl, R. M.: Organism and environment, a manual of quantitative ecology. San

Francisco: W. H. Freeman and Co. (1971).

de la Cruz, A. A., and S. M. Naqvi: Unpublished data (1973).

Dollar, D. A.: Mississippi State Chemical Laboratory, Mississippi State, Ms., personal
communication (1973).

Finley, M. T., D. E. Ferguson, and J. L. Ludke: Possible selective mechanism in the

development of insecticide-resistant fish. Pest. Monitor. J. 3,212 (1970).

Harris, R. C., D. B. White, and R. B. MacFarlane: Mercury compounds reduce

photosynthesis by plankton. Science 170, 736 (1970).

Johnson, C. D.: The in vitro effect of DDT and related compounds on the succinoxidase
system of rat heart. Arch. Biochem. 30, 375 (1951).

Lowe, J. I., P. R. Parrish, A. J. Wilson, P. D. Wilson, and T. W. Duke: Effects of mirex on

selected estuarine organisms. Trans. 36th N. Amer. Wildl. and Nat. Res. Conf.,
Portland, Oregon (1971).
264 A.A. de la Cruz and S. M. Naqvi

Ludke, J. L., M. T. Finley, and C. Lusk: Toxicity of mirex to crayfish, Procarnban¢s

blandingi. Bull. Em4ron. Contam. Toxicol. 6, 89 (1971).

Markin, G. P., J. H. Ford, J. C. Hawthorne, J. H. Spence, J. Davis, H. L. Collins, and C. D.

Loftis: The insecticide mirex and techniques for its monitoring. U.S.D.A. Anim.
and Plant Health Inspect. Serv. 18-3, 1-19 Hyattsville, Md. (1972).

Menzel, D. W., J. Anderson, and A. Randtke: Marine phytoplankton vary in their

response to chlorinated hydrocarbons. Science 167, 1724 (I 970).

Moore, S. A.: Some effects of selected organochtorine compounds on radiocarbon uptake

by marine phytoplankton populations in St. George Sound, Florida. Abstracts,
35th Annual Meeting Amer. Soc. Linmol. and Oceanogr., Tallahassee, Fla. (1972).

Naqvi, S. M. and A. A. de la Cruz: Effects of mirex insecticide on selected freshwater

invertebrates. ASB Bull. 19, 87 (1972).

_ _ Mirex incorporation in the environment: Residues in non-target organisms. Pest.

Monitor. J., in press (1973).

Turnipseed, G. D.: Department of Botany, Mississippi State University, Mississippi State,

Ms., personal communication (1973).

Wurster, C. F., Jr.: DDT reduces photosynthesis by marine phytoplankton. Science 159,
1474 (1968).

Yarbrough, J . D . : Department of Zoology, Mississippi State University, Mississippi State,

Ms., personal communication (1973).

Manuscript received January 22, 1973; accepted April 30, 1973