Tennekes and Sánchez-Bayo, J Environment Analytic Toxicol 2011, S:4
http://dx.doi.org/10.4172/2161-0525.S4-001

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Time-Dependent Toxicity of Neonicotinoids and Other Toxicants:
Implications for a New Approach to Risk Assessment
Henk A. Tennekes* and Francisco Sánchez-Bayo
Experimental Toxicology Services (ETS) Nederland BV, 7201 KN, Zutphen, The Netherlands, and Centre for Ecotoxicology, University of Technology Sydney, Lidcombe,
NSW 4141, Australia

Introduction
A fundamental goal of toxicology is to determine safe levels
of exposure to potentially poisonous substances for humans and
the environment [1]. Traditionally, safe levels have been estimated
in laboratory toxicity bioassays by calculating the non-observable
effect level (NOEL) of a chemical to a variety of organisms which are
representative of certain taxa, i.e. mammals, birds, fish, crustaceans,
algae, etc. There are, however, fundamental problems with the validity
of this approach, both conceptual and statistical in nature, as indicated
by Landis and Chapman [2] and other authors [3]. Thus, the outdated
NOEL concept is being replaced by the no-effect concentration (NEC)
level [4], which assumes that toxic chemicals do not have any effect on
a population of organisms at very low concentrations.
Druckrey and Küpfmüller [5] reasoned that the following processes
determine the complex relationship between exposure levels to poisons
and a toxic effect:
1. Absorption, distribution, metabolism and excretion, which
determine the relationship between dose and the concentration
of the poison at the site of action and its time course in various
parts of the body, commonly referred to as pharmacokinetics.
A linear relationship between dose and effect would require
proportionality between dose (or concentration in a medium) and
the concentration of the poison at the site of action. This may be the
case for many poisons, for as long as the poison does not influence
pharmacokinetics within the considered dose range;
2. The interaction of the poison with critical receptors, which
determines the relationship between the concentration of
the poison at the site of action and receptor binding. A linear
relationship between dose and effect would require proportionality
between the concentration of the poison at the site of action and
the concentration of bound receptors. This is only the case, as will
be shown in section 2, when the proportion of bound receptors
sufficient for an effect is relatively small compared to the initial
level of the receptor; and
3. Receptor binding, which determines the relationship between
the concentration of bound receptors and the actual biological
effect. A linear relationship between dose and effect would require
proportionality between relative receptor binding and the actual
biological effect.
The pharmacokinetics of most pesticides is well known after having
been the subject of intense research in recent decades. For example,
the kinetics of acetylcholinesterase inhibitors such as carbamate and
organophosphorus insecticides was described long ago, and can
be found in most toxicology textbooks (e.g. [6]). The biochemical
interactions between these insecticides and their target enzyme
(acetylcholinesterase), together with the kinetics of uptake, form the
basis of mechanistic models of toxicity such as DEBtox [7]. In a similar
way, the kinetics of narcotic chemicals has been described by models
which consider the time course in their uptake and bioconcentration
by fish [8].
J Environment Analytic Toxicol 				

In this review we will focus on the relationship between the
concentration of the poison at the site of action and the actual biological
effect, which Druckrey has referred to as ergokinetics (Druckrey,
personal communication with Tennekes, 1985). The reason for this is
that recent developments in ecotoxicology suggest that some toxicants
can produce effects at any concentration level provided their exposure
time is sufficiently long [9]. This means that the concept of NEC may not
apply for these toxicants when the life span of the organisms affected
is longer than the theoretical maximum exposure time. Consequently,
risk assessment of these chemicals, which includes neonicotinoid
insecticides and certain metallic compounds, may require entirely new
approaches.

Conceptual model for toxicant effects with time
In their book Dosis und Wirkung (dose and response) [5], Druckrey
and Küpfmüller analyzed ergokinetics in mathematical terms, and
their reasoning leads to fundamentally important conclusions on the
effects caused by interaction of poisons with critical receptors, which
are highly relevant for toxicological risk assessment.
Denoting the initial concentration of critical receptors that a
poison reacts with as R0, the concentration of receptors that a poison
has reacted with as CR, and the poison concentration at the site of
action as C, the velocity of receptor binding (association) is:
K C (R0 - CR)			

		

(1)

where K is the reaction constant for association. The velocity of
dissociation of bound receptors is:
CR / TR					

(2)

where TR is the time constant for dissociation. Therefore, the reaction
kinetics of receptor binding in the case of a bimolecular reaction are:
dCR /dt = K C (R0 - CR) - CR / TR			

(3)

If the effect occurs under circumstances where CR«R0, i.e., with first
order kinetics, then R remains practically constant, in which case
K (R0 − CR) = 1 / TA				

(4)

*Corresponding author: Henk A. Tennekes, Experimental Toxicology Services
(ETS) Nederland BV, Frankensteeg 4, 7201 KN, Zutphen, The Netherlands, Tel:
+31 575 545 500; Fax: +31 575 516 717; E-mail: info@toxicology.nl
Received November 14, 2011; Accepted December 05, 2011; Published
December 07, 2011
Citation: Tennekes HA, Sánchez-Bayo F (2011) Time-Dependent Toxicity of
Neonicotinoids and Other Toxicants: Implications for a New Approach to Risk
Assessment. J Environment Analytic Toxicol S4:001. doi:10.4172/2161-0525.S4001
Copyright: © 2011 Tennekes HA, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.

Toxicology of Pesticides 		

ISSN:2161-0525 JEAT an open access journal

Citation: Tennekes HA, Sánchez-Bayo F (2011) Time-Dependent Toxicity of Neonicotinoids and Other Toxicants: Implications for a New Approach
to Risk Assessment. J Environment Analytic Toxicol S4:001. doi:10.4172/2161-0525.S4-001

Page 2 of 8
where TA can be regarded as the time constant for association. Equation
(3) then simplifies to
dCR /dt = C / TA - CR / TR

			

(5)

In equilibrium, where dCR /dt = 0, Equation (5) simplifies to
CR = C (TR / TA)				

(6)

Replacing the concentration of bound receptors CR by the relative
concentration of bound receptors CR/R0, we obtain
CR / R0 = (C / R0) . (TR / TA)			

(7)

This expression indicates that, with first order kinetics, i.e.
when CR«R0, the proportion of bound receptors is dependent on
the concentration of poison in the body of the organism (exposure
dose) and the relative time for association and dissociation (exposure
duration). Based on this reasoning, Druckrey and Küpfmüller drew
the following conclusions for all poisons that interact with specific
receptors in a first order bimolecular reaction where the toxic effect
is determined by the relative concentration of bound receptors CR/R0:
•

The effect is proportional to the concentration of the poison at the
site of action C (Paracelsus).

•

The effect is inversely proportional to R0. If the concentration of
specific receptors R0 is low, the poison may induce pronounced
toxicity at very low concentrations at the site of action C.

•

The effect is proportional to TR/TA, i.e., to the quotient of the time
constant for dissociation TR and the time constant for association
TA.

•

If TR/TA is high, the poison may induce pronounced toxicity at very
low concentrations at the site of action C.

•

If TR/TA < 1, a compound is innocuous and can only induce toxicity
at relatively high concentrations at the site of action C.

•

If both time constants (TR and TA) are low, i.e. when both association
and dissociation are fast processes, equilibrium between C and
receptor binding (and effect) will be established quickly but the
toxic effect will also regress quickly.

•

The character of a poison is primarily determined by TR, i.e., by the
time constant for dissociation of bound receptors.

•

If TR is low, i.e. when receptor binding is quickly reversible, the time
course of the effect will be the same as the time course of C and the
maximum effect will occur when C is at its maximum. Under these
circumstances, there will be a linear relationship between effect and
the actual concentration of the poison at the site of action C, and
between dose and effect, provided C is proportional to dose (or
concentration in a medium). Such poisons can therefore be termed
“concentration poisons”. The value of TA will only determine the
fraction of the poison that reacts with the specific receptors R.

•

If the time constant for dissociation TR is high, i.e. when receptor
binding is only slowly reversible, the time to maximum effect will
be delayed, and the (toxic) effect will also be slowly reversible. The
higher is TR is, the longer is the time to maximum effect. Upon
repeated exposure in quick succession there will be cumulative
effects. Such poisons can therefore be termed “cumulative poisons”.
With cumulative poisons, a quantitative or even linear relationship
between effect and the actual concentration of the poison at the
site of action C , as seen with concentration poisons, will not exist.
Because equilibrium between C and receptor binding (and effect)

J Environment Analytic Toxicol 				

will be established very slowly, toxicity becomes a process that takes
place in time. There will be a latency period up to a defined effect,
which can be shortened, of course, by increasing the concentration
of the poison at the site of action C.
Thus, according to Druckrey and Küpfmüller, depending on the
time constant for dissociation TR, the toxicity of a compound may
become a process that takes place in time. The traditional approach to
toxicity testing is to consider dose (concentration)-effect relationships
at arbitrarily fixed exposure durations which are supposed to reflect
‘acute’ or ‘chronic’ time scales. This approach measures the proportion
of all exposed individuals responding by the end of specified exposure
times. Toxicological databases established in this way are collections
of endpoint values obtained at fixed times of exposure. As such these
values cannot be linked to make predictions for the wide range of
exposures encountered by humans or in the environment. Thus, current
toxicological risk assessment can be compromised by this approach to
toxicity testing, as will be demonstrated in this paper, leading to serious
underestimates of actual risk.

Time effects of toxicants
In order to overcome this handicap, an increasing number of
researchers are using a variant of the traditional toxicity testing
protocol which includes time to event (TTE) methods. This TTE
approach measures the times to respond for all individuals, and
provides information on the acquired doses as well as the exposure
times needed for a toxic compound to produce any level of effect on the
organisms tested [10]. Consequently, extrapolations and predictions
of toxic effects for any combination of concentration and time are
now made possible [11]. We will demonstrate that this approach is
superior to current toxicological testing procedures, and has important
implications for risk assessment of chemicals.
Following the conceptual model by Druckrey and Küpfmüller,
if receptor binding is virtually irreversible, then TR → ∞ and Eq. (3)
reduces to
dCR /dt = K C (R0 - CR)				

(8)

If the toxic effect occurs when CR « R0, then
dCR / dt = K R0 C				

(9)

If a dose level is kept constant throughout a study, and, as a result, C
remains constant as well, integration yields
CR = K R0 C t					

(10)

This reasoning by Druckrey and Küpfmüller provided a theoretical
explanation for Haber’s rule [12]. Haber’s rule (for a review, see
[13]) states that the product of exposure concentration and exposure
duration produces a constant toxic effect. Haber had noted that
exposure to a low concentration of a poisonous gas for a long time
often had the same effect (death) as exposure to a high concentration
for a short time. The results of Druckrey’s ground-breaking study on
the carcinogenicity of 4-dimethylaminoazobenzene (4-DAB) in BDIII
rats [14] were consistent with Haber’s rule: doubling the daily 4-DAB
dose, and thereby presumably doubling the concentration of the
carcinogen at the site of action, halved the time up to the appearance
of liver cancer.
In the 1960s the molecular biology of this carcinogenic effect was
elucidated and confirmed the theorem of Druckrey and Küpfmüller.
DNA was recognized by Brookes and Lawley [15] as the target for
chemical carcinogens, as recently inferred by Wunderlich [16], and

Toxicology of Pesticides 		

ISSN:2161-0525 JEAT an open access journal

Citation: Tennekes HA, Sánchez-Bayo F (2011) Time-Dependent Toxicity of Neonicotinoids and Other Toxicants: Implications for a New Approach
to Risk Assessment. J Environment Analytic Toxicol S4:001. doi:10.4172/2161-0525.S4-001

Page 3 of 8

It is now apparent that Haber’s rule is highly relevant for
ecotoxicological risk assessment as well. It was recently shown to
describe the toxicity of the neonicotinoid insecticide imidacloprid
to midges Chironomus tentans [18]. The product of exposure
concentration and exposure duration to 50% mortality (t50) for C.
tentans was very similar under acute and chronic exposure conditions.
These observations have also confirmed the theorem of Druckrey
and Küpfmüller and Haber’s rule. Abbink certified in 1991, when
the compound was first introduced, that “imidacloprid is the first
highly effective insecticide whose mode of action has been found to
derive from almost complete and virtually irreversible blockage of
postsynaptic nicotinic acetylcholine receptors (nAChRs) in the central
nervous system of insects” [19]. Imidacloprid mimics the action of
acetylcholine, but unlike acetylcholine, imidacloprid is not deactivated
by acetylcholinesterase and thus persistently activates nAChRs
[20]. Chronic exposure of insects to imidacloprid therefore leads to
cumulative and virtually irreversible blockage of nAChRs in their
central nervous system, which play roles in many cognitive processes.

Exposure time reinforcing the toxic effects
Although equation (10) provided a theoretical explanation for
Haber’s rule, it assumed proportionality between the concentration of
bound receptors CR and the effect. This may not always be the case. As
mentioned earlier, Druckrey and Küpfmüller had pointed out that the
relationship between the poison concentration at the site of action C
and the effect E involves at least 2 steps: the first is the interaction of the
poison with critical receptors leading to receptor binding, the second
is the subsequent biological effect resulting from receptor binding. As
we have seen earlier, if receptor binding is virtually irreversible (i.e. TR
→ ∞), the concentration of bound receptors CR is proportional to the
integral of C over time:
CR ~ ∫ C dt					

(11)

If the subsequent effect is irreversible as well (e.g. death), the
effect E is proportional to the integral of the concentration of bound
receptors CR over time:
E ~ ∫ CR dt					

(12)

In cases of irreversible receptor binding and an irreversible effect,
the effect E is thus proportional to the double integral of the poison
concentration at the site of action C over time, as the combination of
equation (11) and (12) shows:
E ~ ∫ ∫ C dt					

(13)

The implication is that exposure time will reinforce the effect.
Reinforcement of an effect by exposure time was subsequently
demonstrated by Druckrey and co-workers with benchmark
studies of the production of ear duct and liver carcinomas by
4-dimethylaminostilbene (4-DAST) [21] and diethylnitrosamine
(DENA) [22], respectively, in BDII rats (Table 1).
The total carcinogenic dose decreased with decreasing daily 4-DAST
or DENA dose levels, even though the median tumor induction times
increased with decreasing daily dose levels. In a logarithmic system of
coordinates, there was a linear relationship between the median tumor
induction time (t50) and the daily dosage (d):
ln d = ln k – n ln t50				
or
J Environment Analytic Toxicol 				

(14)

d t50 n = constant				

(15)

where the time exponent n was 3.0 and 2.3 for 4-DAST and DENA,
respectively.
The essence of equation (15) is that the total dose required to
produce the same effect decreases with decreasing exposure levels, even
though the exposure times required to produce the same effect increase
with decreasing exposure levels. So, it should be possible, in principle,
to induce cancer with a single dose of a chemical carcinogen. In fact,
this was achieved by Druckrey et al [23] in single dose experiments
with the direct-acting ethylating nitrosamide N-nitroso-N-ethylurea
(ENU) in BD IX rats.This carcinogen is rapidly lost from the blood after
intravenous injection; it has an in vivo half-life of 5-6 minutes.
Upon single dose treatment in early life (1, 10 or 30 days after
birth), the overwhelming majority of animals died from malignancies
of the nervous system [23]. The quantitative relationship (Figure
1) between (post-conception) age at treatment (a), dose (d), and the
median induction period of neurogenic malignancies (t50) can be
described as follows [24]:
a / t50 = K d r					

(16)

Chemical

Daily dose, d
(mg kg-1)

Median tumor
Total dose, D
induction time, t (days) (mg kg-1)

n

4-DAST

3.4

250

850

3.0

2.0

340

680

1.0

407

407

0.5

550

275

0.28

607

170

0.2

700

140

0.1

900

90

9.6

101

963

4.8

137

660

2.4

192

460

1.2

238

285

0.6

355

213

0.3

457

137

0.15

609

91

0.075

840

64

DENA

2.3

Table 1: Induction of ear-duct carcinomas in BDII rats by 4-DAST and of liver
carcinomas by DENA, after Druckrey and Dischler [21], and Druckrey et al. [22].

1
Median age-dependent
velocity of malignant
neurogenic tumor
formation (A/T)

Warwick and Roberts confirmed irreversible receptor binding by
demonstrating covalent binding of a 4-DAB metabolite to DNA [17].

0.1

0.01

1

10

100

1000

Single dose of ethylnitrosourea
(mg/kg-bw)
Figure 1: Median age-dependent velocity of malignant neurogenic tumor
formation (A/T) in BD IX rats versus single dose levels of ethylnitrosourea,
on logarithmic coordinates. Linearity leads to equation (17).

Toxicology of Pesticides 		

ISSN:2161-0525 JEAT an open access journal

Citation: Tennekes HA, Sánchez-Bayo F (2011) Time-Dependent Toxicity of Neonicotinoids and Other Toxicants: Implications for a New Approach
to Risk Assessment. J Environment Analytic Toxicol S4:001. doi:10.4172/2161-0525.S4-001

Page 4 of 8
with K = constant , where r = 0.426
or
d (t50 / a) n = constant				

(17)

where n = 1 / r = 2.35.
Equation (17) indicates that, in this case, the velocity of
carcinogenesis is determined by the initiating dose and the state of the
relevant targets at treatment. This is reminiscent of Wilder’s law of initial
value, which states that the direction of response of a body function to
any agent depends to a large degree on the initial level of that function.
Target cells for ENU in early life are subependymal cells in the brain
and subpial cells in the spinal cord, which are destined to differentiate
into glial cells, i.e., astrocytes or oligodendrocytes. The evidence
suggests that the critical genetic changes induced by the carcinogen in
these target cells lead to inhibited normal cell differentiation processes,
as has been observed in leukemogenesis [25,26]. Yuspa and Morgan
reported that cells resistant to terminal differentiation can be readily
isolated from skin of BALB/c mice exposed to an initiating dose of
carcinogen in vivo but not from control mouse skin [27].

Applications of the Druckrey-Küpfmüller model
The Druckrey-Küpfmüller equation (15) can serve as a basis
for risk assessment of carcinogens. Carlborg pointed out that this
equation is implied by a Weibull model for dose-response functions in
carcinogenesis [28]. The simple form of the Weibull model is a sigmoid
curve defined by four parameters:
(18)

where x is the dose, P is the tumor rate and m, α, β are parameters
to be estimated from the data. The parameter α is determined by the
background tumor probability, β is a scale parameter related to the
units measuring the dose, and m is the important shape parameter. At
very low doses the excess risk over background is approximately β xm.
The virtual safe dose (VSD) corresponding to a one-in-a-million risk
over background is then given by
VSD = (10 -6 / β) 1 / m				

(19)

The extended form of the Weibull model includes the time to a tumor
(t):
P = 1 – e exp , with the exponent = – (α + β x m) t k

(20)

where k is a new parameter. Now suppose that t measures the time
to a tumor. Also, suppose that the background tumor rate is zero (α =
0). For a test group at some dose x, consider the median time to tumor
t50 – that is, the value of t such that P = 0.50. The extended Weibull
model for this dose and time is
0.50 = 1 – e exp , with the exponent = – β x m t50 k

(21)

This reduces to
[ - ln 0.50 / β] 1/m = x t50 k / m = x t50 n 		

(22)

where n = k / m and the left side of the equation is a constant. This is
the Druckrey-Küpfmüller equation (15). The exponent n is given by the
slope of the regression between the logarithms of the dose and that of
the median time to tumor.

Neonicotinoid insecticides
Similar relationships have been demonstrated for the toxicity
J Environment Analytic Toxicol 				

t50= a*C –b

(23)

Accordingly, there was a linear relationship when the logarithms of
the variables C and t were used
ln t50 = a’ – b ln C

(24)

where a’ is the intercept and b is the slope of the regression. Equation
(24) can be transformed to
C b t50 = constant

(25)

or
C t50 1/b = constant

(26)

Equation (26) is very similar to the Druckrey-Küpfmüller equation
(15) for the action of chemical carcinogens such as DENA or 4-DAST.

Genotoxic carcinogens

P = 1 – e exp , with the exponent = – (α + β x m)

of neonicotinoid insecticides to aquatic invertebrates, in particular
imidacloprid to the freshwater ostracod Cypridopsis vidua and to
Daphnia magna and thiacloprid to Gammarus and Sympetrum.
Sánchez-Bayo [29] demonstrated that the relationship between the
concentration of the neonicotinoid insecticides imidacloprid and
thiacloprid in a medium (C) and the time to 50% mortality (t50) of
several species of exposed arthropods followed a hyperbolic curve
described by the equation

Similar to the dose-response characteristics of DENA, exposure
time was found to reinforce the toxicity of imidacloprid and thiacloprid
to the tested arthropod species – Beketov and Liess [30] have referred
to this feature of the neonicotinoids as having “delayed effects”. The C
t50 product, which reflects the total dose required for a lethal effect,
decreased with decreasing toxicant concentration C (Table 2), even
though the times to 50% mortality t50 increased with decreasing
toxicant concentration C.
This toxic behaviour of neonicotinoid insecticides has also been
pointed out by other researchers. For example, Suchail et al. [31] noted
that at concentrations of 0.1, 1, and 10 µg of imidacloprid per liter, the
total cumulate dose ingested by honeybees in chronic intoxication was
about 60 to 6,000 times lower than the doses needed to produce the same
effect in acute intoxication tests. The same effect was also apparent with
two of the metabolites of imidacloprid (5-hydroxyimidacloprid and
olefin). Although the authors could not explain the huge discrepancies
between the results from acute toxicity tests of imidacloprid and its
feeding effects in honeybees, their observations are consistent with the
time-dose model described here. Consequently, a correct understanding
of the ergokinetics of imidacloprid is essential for its risk assessment
in relation to bees. In addition, at sub-lethal doses imidacloprid can
alter honey bee foraging and learning, [32-35]. Imidacloprid has been
detected at levels of 5.7 µg kg-1 in pollen from French hives [36] and
foraging honey bees reduced their visits to a syrup feeder when it was
contaminated with 3 µg kg-1 of imidacloprid [37]. Foraging as well as
hive worker bees and brood are likely to be continuously exposed to
imidacloprid when contaminated food is collected and stored inside
the hive [38]. A honey bee during a foraging flight must learn and
recall many complex visual patterns [39,40]. These cognitive functions
may be perturbed when nAChRs, necessary for the formation of longterm memory and involved in acquisition and retrieval processes, are
persistently blocked [41]. These observations are consistent with the
theorem of Druckrey and Küpfmüller [5]. Both receptor binding and
the effect of receptor binding are virtually irreversible, and exposure
time will therefore reinforce the effect, which may in the course of time
be detrimental to the bee colony, and ultimately cause colony collapse.

Toxicology of Pesticides 		

ISSN:2161-0525 JEAT an open access journal

Citation: Tennekes HA, Sánchez-Bayo F (2011) Time-Dependent Toxicity of Neonicotinoids and Other Toxicants: Implications for a New Approach
to Risk Assessment. J Environment Analytic Toxicol S4:001. doi:10.4172/2161-0525.S4-001

Page 5 of 8
Species

Chemical

Cypridopsis vidua

Imidacloprid

Daphnia magna

Gammarus pulex

Sympetrum striolatum

Concentration (C) in µg L-1

Time to 50% mortality (T50) in days

C x T50 product in µg L-1.days

4

5.2

20.8

16

3.0

48
211.2

64

3.3

250

2.3

575

1,000

2.0

2,000

4,000

0.9

3,600

250

384.7

96,175

750

69.7

52,275

2,220

18.6

41,292

6,700

15.0

100,500

20,000

18.4

368,000

60,000

3.0

180,000

99

63.6

6,296.4

364

16.7

6,078.8

Imidacloprid

Thiacloprid*

Thiacloprid*

988

6.5

6,422

3,100

3.2

9,920

9,520

0.9

8,568

7.2

20.6

148.3

8.0

17.2

137.6

12.7

13.0

165.1

113.3

3.2

362.6

n
4.67

1.35

1.11

1.53

*Original data from Beketov and Liess [30]
Table 2: Mortality of Arthropods Induced by Neonicotinoid Insecticides (After Sánchez -Bayo [29]). Data fit the equation C T50n = constant.

Thus, low environmental concentrations of these insecticides (that
may not be acutely toxic) could be detrimental to many invertebrate
species in the medium to long term, in particular to aquatic
invertebrates because these compounds are quite soluble in water and
their toxicity may be reinforced by exposure time. The results of recent
studies are consistent with this concept. Thus, imidacloprid applied
to rice mesocosms eliminated all zooplankton species for at least two
months when concentrations were above 1 µg L-1 [42], which is 500 to
75,000 times lower than its acute LC50s for ostracods and cladocerans.
By contrast, the insecticide fipronil applied to the same mesocosms
produced only small, non-significant adverse effects on zooplankton,
even if this insecticide is 100–1000 times more toxic to those organisms
than imidacloprid and more persistent in soil [43]. Mayflies of the
genera Baetis and Epeorus showed a reduction in reproductive success
when exposed to concentrations of imidacloprid as low as 100 ng L-1
[44]; whether this is the result of a time-exposure effect or a sublethal
effect is not clear. A single pulse contamination of mesocosms with the
neonicotinoid insecticide thiacloprid resulted in long-term alteration
of the overall invertebrate community structure because some species
did not recover [45]. One species, the stonefly Nemoura cinerea, was
affected at the lowest tested concentration, 70 times below the lowest
known LC50. Imidacloprid can be applied as a systemic insecticide
to trees by direct stem injections or by soil injections and drenches,
in which case it may be indirectly introduced to aquatic systems via
leaf fall or leaching: at realistic concentrations in leaves (18-30 µg g-1
fresh weight) imidacloprid can inhibit leaf litter breakdown through
adverse effects on decomposer invertebrates [46], but it has no effect
on microbial decomposers.
Terrestrial ecosystems have also been reported as negatively affected
by imidacloprid. A study conducted over 3 years on an experimental
home lawn [47] revealed that three consecutive years of imidacloprid
applications to the same field plots suppressed the numbers of total
hexapods, Collembola, Thysanoptera and Coleoptera adults by 54-62%.
However, applications of imidacloprid to an experimental vegetable
patch had only a temporary effect on the arthropod communities of

J Environment Analytic Toxicol 				

the eggplant crop [48], perhaps because immigration of insects from
nearby areas compensated the losses caused by the insecticide. When
imidacloprid is applied as a systemic insecticide to the soil around
trees it may cause sublethal effects on earthworms if concentrations
in the litter reach or exceed 3 mg kg-1 [49,50]. A recent study indicates
high toxicity of imidacloprid to the non-target terrestrial arthropod
Porcellio scaber, at similar levels as the organophophorus diazinon [51].
However, because the blocking of AChRs by diazinon is temporary
(it may last only a few hours), its toxic effect would last as long as it
is present in soil at sufficient concentrations, whereas imidacloprid
effects could last much longer if the nAChRs are permanently blocked.

Other chemicals
Time-dependent effects have also been observed with toxic metals.
Sánchez-Bayo [29] analysed the data provided by other authors and
concluded that the toxicity of copper, zinc, selenium and cadmium
(as CdCl2) to Daphnia magna and that of zinc to the guppy (Poecilia
reticulata) followed the same pattern described by equation (26).
The median times to 50% mortality (t50) decreased with increasing
concentrations of metals, as seen with carcinogens and neonicotinoids,
suggesting that for all metals the time constant for dissociation from the
critical receptor TR is high. However, with the exception of selenium, and
in complete contrast to the action of carcinogens and neonicotinoids,
the total metal dose taken up by and lethal to these aquatic organisms
also decreased with increasing concentrations of metals, as shown here
for Daphnia magna (Table 3). Selenium’s toxicity followed Haber’s
Rule, with n=1, and can safely be assumed to bind irreversibly to the
critical receptor, but the n-values characterizing the toxicity of copper,
zinc, and cadmium were 0.3, 0.47, and 0.60, respectively. So, whereas
with carcinogens and neonicotinoids, lower concentrations are more
effectively poisonous than higher concentrations, it turns out that
lower metal concentrations are less effectively poisonous than higher
metal concentrations. This could reflect some elimination process
which minimizes metal toxicity with time of exposure.

Toxicology of Pesticides 		

ISSN:2161-0525 JEAT an open access journal

Citation: Tennekes HA, Sánchez-Bayo F (2011) Time-Dependent Toxicity of Neonicotinoids and Other Toxicants: Implications for a New Approach
to Risk Assessment. J Environment Analytic Toxicol S4:001. doi:10.4172/2161-0525.S4-001

Page 6 of 8
Chemical

Concentration Time to 50% mortality
(C) in µg L-1
(T50) in days

C x T50 product in
n
µg L-1.days

Copper*

28

32.6

912

32

6.5

208

48

3.2

154

56

2.0

112

64

1.3

80

3.2

292

935

5.6

58

325

10

38

375

18

11

203

32

6

181

56

2

105

158

3.3

514

800

0.7

567

1200

0.5

600

1600

0.4

600

2000

0.3

500

59

163

9617

125

32

4000

250

2

490

500

1.3

667

CdCl2**

Selenium*

Zinc*

0.30

0.60

1.03

0.47

*Original data from Hoang et al. [64]
**Original data from Kooijman [65]
Table 3: Mortality of Daphnia magna induced by metallic elements or compounds
(After Sánchez -Bayo [29]). Data also fit the equation C T50n = constant.

years for inclusion in risk assessments of environmental contaminants
in areas as diverse as agriculture [57], occupational health [58],
engineering [59] and ecology [60], the implications of the timedependent toxicity of some chemicals have not been realised yet. First of
all we need to know what kind of toxicants behave in a time-dependent
manner, which is the same as asking which chemicals bind irreversibly
to specific receptors. So far, the evidence pointed out here indicates
that neonicotinoids, some carcinogens and metal/metalloids fit such
description but other chemical groups may also follow this pattern.
Secondly, if a toxicant has time-dependent effects, the standard risk
assessment procedures would not be valid in situations where there is
exposure to sublethal concentrations of the toxicant for long periods of
time. Traditionally this type of exposure has been considered as chronic
toxicity, and its relationship with standard acute toxicity endpoints
(e.g. LC50) has been studied in many aquatic species [61] ever since
Kenaga introduced a ratio to describe it [62]. However, this traditional
approach ignores the underlying mechanism of toxicity with time that
has been described in this review.
There is no doubt that once ecotoxicologists realise the full potential
and advantages of time-to-event approaches, they will become the
standard tool “for analysis of toxicity from pulse exposures, and the
latent toxic effects emerging after exposure has ceased, because both of
these phenomena are time related” [63]. We already pointed here the
experimental work by Beketov and Liess [30] in this regard, and only
hope that more researchers may follow the same pathway.

Metal toxicity is particularly insidious, as it affects not only
aquatic but also most terrestrial organisms through the food chain.
While more information is necessary to prove that other metallic
and metalloid elements (e.g arsenic) produce time-dependent toxic
effects, the evidence so far obtained from toxicity experiments carried
out with fish (e.g. [52-54]) illustrates a toxicity pattern similar to that
observed with neonicotinoid insecticides. This is likely to result from
metal accumulation in the body, which increases with time until the
organisms reach a sufficient dose to cause a toxic effect (see review in
[55]). At least we know this is the case with mercury contamination in
humans, which leads to the condition known as Minamata disease [56].

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It is apparent that any chemical that permanently binds to a receptor
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whenever the bound receptors remain in the body of the organism. The
examples shown here for carcinogenic substances that bind to DNA,
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metallic elements or compounds, like CdCl2, that accumulate in the
organisms’ tissues, suggest this is probably the case. This suggests that
many other substances may behave in the same way, even though we
may be unaware of their ergokinetics.

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So far we have discussed only toxicants that affect animals, but
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