Pharmacology & Pharmacy, 2012, 3, 381-387
http://dx.doi.org/10.4236/pp.2012.34051 Published Online October 2012 (http://www.SciRP.org/journal/pp) 1
Isobolographic Method and Invertebrate (Planarian)
Model for Evaluating Combinations of Waterways
Pollutants
Robert B. Raffa1*, Deborah A. Gallo2, Christopher S. Tallarida1,3, Scott M. Rawls3,
Ronald J. Tallarida3
1Department of Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, USA; 2United States Public Health
Service, Tsehootsooi Medical Center, Fort Defiance, USA; 3Department of Pharmacology, School of Medicine, Center for Substance
Abuse Research, Temple University, Philadelphia, USA.
Received July 5th, 2012; revised August 13th, 2012; accepted September 14th, 2012
ABSTRACT
Agricultural, pharmaceutical, and other biologically active substances are emptied or leach into waterways and ground-
water, where they can dose-relatedly cause pharmacologic or toxic effects on the resident or dependent animal species.
Standard methods can be used to evaluate the effects of individual substances, but evaluation of combinations of sub-
stances is more difficult. The mathematically rig orous method of isobolographic analysis was coupled with a simple in
vivo invertebrate model. Planarians were selected because they are the lowest extant species with a centralized nervous
system. Neostigmine bromide and monopotassium phosphate (KH2PO4) were selected as representative of two types of
potential pollutants. Neostigmine bromide and KH2PO4 individually produced dose-related lethality over a 60-minute
observation period with LD50 values of 122 an d 70 mM, respectively. The LD50 value of a 1:1 combination of the two
was significantly different (p < 0.05) from the isobologr aphic line of additivity. We used planarian s as a representative
fresh-water species and joint-action (“isobolographic”) analysis to examine possible interaction between pollutants. In
the demonstrative example reported here, there was a subadditive interaction between a 1:1 fixed-ratio combination of
neostigmine bromide (as a representative acetylcholinesterase inhibitor used in pesticides) and potassium phosphate
(used in fertilizers and detergents).
Keywords: Combinations; Isobolographic Analysis; Methods; Pollutants; Planarians
1. Introduction
The extent to which active pharmaceutical ingredients
(APIs) are present in aquatic environments was revealed
in 2004 during the first nationwide survey of pharmaceu-
tical compounds detected in surface waters (rivers, lakes,
and marine waters), groundwater, and drinking water [1].
The problem is widespread throughout the world [2-15].
APIs can enter waterways by several routes, including
excretion following therapeutic use, discharge of treated
wastewater from manufacturing facilities, or disposal of
unused medications [16]. Agricultural substances, deter-
gents, and a host of other biologically active chemicals
are dumped into or leach into these same waterways
[5,17-29]. Unfortunately, there is very minimal amount
of information regarding potential effects on human and
aquatic ecosystems from exposure to combinations of
APIs and other chemicals.
The nature of the interaction between the components
of a combination can lead to additive, sub-additive, or to
supra-additive (synergistic) pharmacological or toxico-
logical effects. A mathematically rigorous method to
evaluate combinations (known as joint action analysis)
has been developed and has been applied to pharmacol-
ogical systems [30-40].
We report a convenient model for the measurement
and quantitative assessment of the toxicity of water pol-
lutant combinations using a fresh-water species that has a
primitive nervous system, including neurotransmitter and
2nd messenger systems [41-50] and that are useful for
study of drug action and physiological processes associ-
ated with drug abuse, such as physical dependence and
withdrawal [51-61]. We cho se for illustrative p urposes of
the method the combination of a representative of sub-
stances used as insecticides (an acetylcholinesterase in-
hibitor, neostigmine bromide) and a representative of
substances used in detergents and fertilizers, potassium
*Corresponding autho
r
.

Isobolographic Method and Invertebrate (Planarian) Model for Evaluating Combinations of Waterways Pollutants
382
monophosphate.
2. Materials and Methods
2.1. Animals and Chemicals
The planarians (Dugesia dorotocephala) were purchased
from Carolina Biological Supply Co. (Burlington, NC).
They were acclimated to laboratory conditions, at room
temperature (21˚C), and tested within 48 h. Neostigmine
bromide and potassium monophosphate (KH2PO4) were
obtained from commercial sources and prepared at the
desired concentration in tap water.
2.2. Testing
Planarians were placed individually into a four-quadrant
plastic Petri dish (diameter = 100 mm) containing 10 mL
of neostigmine bromide (6 doses), KH2PO4 (6 doses), or
1:1 combination of neostigmine bromide and KH2PO4 (6
doses). Percent lethality at the end of a 60-min exposure
was determined.
2.3. Isobolographic Analysis
Isobolographic analysis and pharmacologic applications
have been described (for a comprehensive review, see
monograph [39]). An isobolograph is a plot of dose (or
concentration) pairs in the combination that produce the
same effect, which is often selected as the half maximal
effect (ED50 value). If drug B of combination A and B
acting alone gives effect in concentration B, pairs (a, b)
are related to b plus the b-equivalent of a such that b +
beq = B. This may be written baR B and rear-
ranged to the form 1bB aA. If substances A and B
produce equal maximal effects, the concentration pairs (a,
b) are points that constitute a straight line. If the experi-
mental combination yields a result that plots as a point on
this line, it is additiv e. If it plots as a point below the line
of additivity (i. e ., a lower dose of each is needed), the
combination is greater than additive (synergistic). If it
plots as a point below th e line of additivity (i.e., a higher
dose of each is needed to produce the same effect), the
combination is sub-additiv e. Numerous studies have used
the isobolographic approach [62-69]. In the illustrative
example used here, constituent doses were used in fixed
ratio, which allows simple determination of combination
doses that produce the specified level of effect (ED50 in
the example). This is acco mplished by fitting dose-effect
data using an appropriate regression procedure and the
intersection of this line with the additive isobole gives
the dose pair that is additive. It also allows quantitative
assessment and statistical testing of any departure from
additivity.
2.4. Statistics
The linear isobole of additivity, applicable in the present
study because of constant relative potency, is convenient
for estimating the variance of the additive total dose. All
points (a, b) on this line can be expressed as fractions (f
and (1 – f )) of the respective potencies A and B, that is, a =
fA and


1bf B, and thus any combination with
constituent amounts chosen such that


dose fBfAdose 1BA has a total quantity given
by


1TfA fB. The variance of the additive total
T is therefore given by ,
where f and
 
2
21VTfVAf VB


1
f
 are reasonably estimated from the
mean A and mean B. The total additive variance calcu-
lated from the above allows a comparison with the ex-
perimentally determined total dose variance.
3. Results
3.1. Substances Alone and in Combination
Planarians (N = 18 per dose) were placed individually in-
to each of the four quadrants of the Petri dish. At 60 min,
the number of planarians dead was counted and % lethal-
ity was determined. Neostigmine bromide by itself pro-
duced dose-related lethality (% lethality = 1617.4 dose –
146.7). The LD50 value for neostigmine bromide alone
was 122 mM. KH2PO4 also produced dose-related lethal-
ity alone (% lethality = 1572.5 dose – 61.5). The LD50
value for KH2PO4 by itself was 70 mM. The fixed-ratio
combination (1:1) of neostigmne bromide plus KH2PO4
produced dose-related lethality (% lethality = 1 535. 2 d ose
– 76.2). The LD50 value for the 1:1 combination was 82
mM. The data are plotted in Figure 1.
Figure 1. Dose-related lethality at 60 minutes produced by
monopotassium phosphate alone, neostigmine alone, or a
1:1 combination of monopotassium phosphate plus neostig-
ming bromide. N = 18 planarians.

Isobolographic Method and Invertebrate (Planarian) Model for Evaluating Combinations of Waterways Pollutants 383
3.2. Isobolographic Analysis
An isobologram was constructed using the percent le-
thality data and is displayed in Figure 2. The LD50 value
for neostigmine bromide alone (i.e., 122 mM) is plotted
on the ordinate and the LD50 value for KH2PO4 alone
(i.e., 70 mM) is plotted on the abscissa. A straight line
connecting the LD 50 values of the individual agents is
the line of additivity. The LD50 value of the fixed-ratio
combination of neostigmine bromide plus KH2PO4 is
plotted as the point. The LD50 value of the combination
is significantly (p < 0.05) above the line of additivity.
This is indicative of a sub-additive interaction.
4. Discussion
Pharmaceutical and agricultural substances increasingly
are being found in waterways in concentrations that
produce deleterious biological effects [70-72]. Evaluation
of the toxicity of individual substances is a common
practice, however evaluation of the contribution of
potential interactive effects in combinations of pollutants
is increasingly being appreciated and investigated
[73-77]. In combinations, the possibility of non-additive
interaction arises. Thus three outcomes are possible:
additive, sub-additive, and supra-additive (synergy). For
the non-additive interactions, the determination of statistical
significant difference from additivity requires a thorough
evaluation using rigorous proc edu res (re view ed in [39] ).
Figure 2. Isobologram of the LD50 values obtained from the
data shown in Figure 1. The LD50 values for potassium
monophosphate or neostigmine exposure alone are plotted
on the ordinate and on the ordinate and abscissa. The LD50
value of the combination is significantly different (p < 0.05)
(the error bars are within the dimension of the circle) from
the line of additivity.
The present work investigated the use of a simple in
vivo planarian model. Planarians have a simple nervous
system and mammalian-like neurotransmitter systems
(e.g., [41,48,50]). Thus, they are the lowest form of
animal that would display relevant neurotoxicity. They
respond with quantifiable dose-related behavioral ch anges
to drug exposure and withdrawal (e.g., [41,43,45,46,49-
51,54,57,58,61]). And receptor-mediated mechanisms
can be verified using receptor-selective antagonists. We
have previously used planarian models for investigating
drug action and the physiological processes involved in
physical dependence (for review, see monograph [55]).
Like-wise, we have previously developed and used iso-
bolographic analysis for a variety of biological endpoints
(for review, see [39]).
In the present study, the two substances were selected
for illustrative purposes: one (neostigmine bromide) as
representative of acetylcholinesterase inhibitors, a class
of substances that have been common ingredients of
insecticides; the other one (potassium monophosphate) as
a representative of chemicals that have been common
ingredients in fertilizers and detergents. Both substances
produced dose-related lethality (reaching 100%) when
they were tested alone. The dose-response curves were
parallel, thus relative potency was constant throughout
the range of doses. The straight-line isobole of additivity
applies if and only if the relative potency is a constant, as
was the case in this study.
The 1:1 fixed-ratio combination of neostigmine bromide
and KH2PO4 produced dose-related and maximal let h al i t y.
Compared to the toxicity of exposure to the agents tested
individually, the toxic effect of the combination was
sub-additive. This was a surprising finding. We had anti-
cipated an additive or possibly even synergistic inter-
action. Thus the importance of actually testing combina-
tions was unintentionally emphasized. It should be noted
that the demonstration of sub-additivity in the present
study applies to the conditions used. It is possible that the
use of other fixed-ratios would have yielded additive or
supra-additive interactions. Likewise, lethality is only
one adverse effect and should not be used as the only
measure of safety. Multiple more subtle sub-lethal adv er s e
effects of combinations might result from particular com-
binations of pollutants. Each case requires careful and
rigorous ev aluation. The present study offers an example
of rigorous mathematical joint action analysis applied in
a convenient in vivo model.
5. Acknowledgements
The authors thank Timothy Shickley, Ph.D., for sug-
gesting Planaria as a model. This work was supported by
NIDA grants DA15378 (RBR), DA022694 (SMR) and
DA09793 (RJT).

Isobolographic Method and Invertebrate (Planarian) Model for Evaluating Combinations of Waterways Pollutants
384
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